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PMC11698355 | Gastrointestinal tract (GI) cancers, comprising oral, gastric, and colorectal malignancies, impose a huge burden on global health. These cancers are responsible for one-quarter of oncological diagnoses and approximately one-third of cancer-related deaths. 1 Screening and surveillance pathways have helped reduce the incidence rate of certain forms of these cancers. For example, the use of colonoscopy to remove precancerous polyps has become the paradigm of an effective cancer-reducing intervention. 2 However, interventional endoscopy isn’t a ‘magic bullet’ as it does not necessarily prevent cancer development in certain GI organs such as the stomach. 3 In addition, issues such as poor patient adherence to endoscopy screening (∼ only 75%), financial cost , and accessibility to adequate infrastructures in remote and deprived regions, hinder the optimization of screening programes. As a consequence of the aforementioned factors, only about 10% of GI cancers are diagnosed via screening programes. Therefore, evaluation of accessible and economical cancer prevention strategies remains the mainstay of current clinical aspiration. Environmental factors such as a low-quality diet (i.e., dietary habit comprising of high saturated fats/trans-fats, highly processed, low fiber, high salt, and/or high sugar content meals) and smoking significantly influence the development of GI cancers. 4 These risk factors bring about a chronic inflammatory state that creates the conducive microenvironment which enables the multi-step process preceding the development of malignancy in mucosal tissues. Consequently, the use of anti-inflammatory agents has garnered attention for a potential role in cancer prevention, particularly chemoprevention. Chemoprevention entails the use of chemical agents (natural or synthetic) to inhibit or delay cancer development. Jankowski and Hawk summarized all key chemoprevention agents in their review, ranging from aspirin, acid suppression, hormone replacement therapy (HRT) to fish oil. 5 These agents have the potential to impair dysfunctional protein activities that lead to initiation, promotion, and progression of tumorigenesis. As chemoprophylactic agents reduce malignant properties and tendencies, they could ultimately play a significant role in anticancer and systemic cancer treatment strategies. 6 Importantly, aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) have chemoprotective properties against cancer via multiple pathways. Grancher and colleagues have illustrated these pathways ; most importantly, they demonstrated the ability of aspirin to irreversibly inhibit cyclooxygenase-2 (COX2) enzyme and the subsequent production of prostaglandin E2 (PGE 2 ). COX-2 and PGE 2 are inflammatory mediators released by tumour cells, and they induce apoptosis, cell migration, invasion, and angiogenesis, thus the progression of carcinogenesis. 7 The aim of this paper is to discuss existing data that promotes the use of aspirin as a prophylaxis for GI cancers. English-language studies dating back to year 2006 were identified manually by the lead author. Publication search engines used in this search were Cochrane and PubMed. Key terms included in the search were gastrointestinal, esophageal, gastric, colorectal, and hepatobiliary cancer, and aspirin chemoprevention. A manual search of articles within the reference lists of publications was also conducted. This paper explores data which covers the use of aspirin as a chemoprophylaxis for GI and digestive tract cancers, the data reviewed are from randomized control trials (5), meta-analysis (3), cohort studies (2), and population-based studies (1), and the studies excluded are i) research that assesses non-aspirin anti-inflammatory agents as a chemoprophylaxis and ii) research that did not stratify the types of GI or digestive tract cancers. Existing guidelines on aspirin use as a chemoprophylaxis were identified via manual search. Ren et al.’s UK Biobank cohort study with over 300,000 participants illustrated that regular use of COX-PGE 2 inhibiting NSAIDS lowers the incidence of CRC by 36%. 9 Aspirin has been the preferred NSAID due to its safer toxicity profile, and the vast knowledge base it has compared to other agents like celecoxib. A dose of aspirin as low as 81 mg daily inhibits COX-PGE 2 mechanism by 45%, whereas other agents such as ibuprofen required much higher doses, such as 800 mg four times daily for a 63% decrease in PGE 2 synthesis. 10 Interestingly, regular aspirin use has been associated with protective changes in the gut microbiota. Particularly, aspirin intake prevents the growth of some pro-inflammatory bacteria that promote neoplastic transformation in the gut. Prizment et al.’s randomized control trial (RCT) supports this; they found a difference in the diversity and proportionality of varied bacteria within the gut microbiota of patients taking aspirin compared to the placebo cohort. For example, a larger proportion of Akkermansia bacteria was seen in patients taking aspirin. Akkermansia is known for its beneficial impact in colonic health. 11 Rothwell and colleagues also reported that the prophylactic effects associated with aspirin were stronger with tumors within the proximal colon, the burdensome site where tumors are usually missed during colonoscopy. 12 Similarly, Hull et al.’s ‘seAFOod Polyp Prevention Trial’ which evaluated the effect of omega-3 and aspirin on the reduction of colorectal adenomas, found that aspirin was the only chemoprotective agent that decreased the number of adenomas in the proximal colon. Colorectal adenoma was explored, given that it’s a well understood pathological predictor for CRC development, but they were unable to conclude that both drugs significantly reduced risk of CRC development in high-risk individuals. Nevertheless, both aspirin and omega-3 decreased the number of colorectal adenomas in participants at year 1 of colonoscopy surveillance. 13 It is probable that significant benefit was not deduced as the research was run for only a short time (1 y). It is therefore likely that the study was underpowered as the benefits of chemopreventative measures are typically delayed by at least several years. In addition to CRC, other GI cancers have had favorable associations with low dose aspirin (LDA) therapy, Garcia et al showed that the use of LDA as low as 75 mg daily for at least 1 y was associated with a 41% and 54% risk reduction for esophageal and gastric cancer, respectively. Even though they found an increasing benefit with the duration of LDA use, there was no significant difference when comparing the duration of 1 to 3, 3 to 5 or >5 yrs. 14 On the contrary, Rothwell’s meta-analysis showed that the benefit was strongest after 10 to 20 yrs follow up. 12 This suggests the benefits of LDA use are much delayed than expected and it would ideally benefit individuals who are able to take aspirin for a lengthened duration of time. Thus, more research is required to help form an informative guideline which incorporates factors such as patient age and duration of aspirin use. The ‘AspECT’ trial by Jankowski and colleagues evaluated the benefits of aspirin and proton pump inhibitors (PPIs) in precancerous metaplasia i.e., Barrett’s esophageal adenocarcinoma, and the mortality caused by both diseases. They found both drugs protected against all-cause mortality. However, high-dose PPI was the most protective of the two; it significantly prevented the progression of Barrett’s’ to cancer. The most impactful finding from this study was the additive chemoprotective effect of both drugs; the greatest protection and survival was found with the concurrent use of high-dose PPIs and LDA. 15 Furthermore, serious adverse events from concurrent use of both agents were exceptionally low, less than 1%. There is also some evidence that aspirin might be beneficial in other digestive tract cancers. However, research here is not as extensive as that of GI luminal gut cancers. The meta-analysis conducted by Bosetti et al acknowledged that aspirin’s chemoprotective effect is present in HPB and pancreatic cancers. They found a stronger relative risk (RR) in liver cancers e.g., hepatocellular (HCC) than cholangiocarcinoma (Liver; RR of 0.71, 95% confidence interval (CI) 0.46 – 1.09, Cholangiocarcinoma; RR of 0.53 95% CI 0.24 – 1.14). Regarding pancreatic cancers an inverse relationship with aspirin was seen; a 20% deduction in pancreatic cancer risk was reported, which contradicts their previous study that found no significant reduction. 8 In similarity to benefits seen in GI cancers, the protective effects seem to also increase with longer duration of aspirin use. 8 Although liver cancers have the highest impact from aspirin (amongst HPB subgroup), the reports of chemo-preventative benefit are inconsistent and has been identified mainly in hepatitis B-virus related HCC. 16 Controversial findings seen in HPB, and pancreatic cancers could be due to the more diverse etiological factors that progress into malignancy. In correspondence to GI cancers, risk factors such as advancing age, alcohol and smoking predispose the chronic inflammatory changes that lead to neoplastic transformation. However, HPB and pancreatic cancers have a wider range of risk factors such as diabetes mellitus, cholelithiasis, and infections such as hepatitis and liver fluke. 17 These all potentially have varied mechanisms which may or may not incorporate COX-2 pathway. Therefore, more research is still required to review the potential of chemoprophylaxis in this subgroup. Other chemical agents, that have been associated with reduced risk of these cancers include statins and metformin. 18 , 19 So far, the main hesitancy towards the consideration of aspirin as a prophylaxis for GI cancers is the risk of hemorrhage. Ingestion of aspirin leads to the inhibition of thromboxane-A2 platelet aggregation; as a result, bleeding time is prolonged, and this could last hours to days as this inhibitory process could persist throughout the platelet cells’ life span. 20 Major hemorrhagic events come with significant complications such as bleeds which require transfusions, intracranial hemorrhage that can lead to neurological deficit or death. Thus, the benefit of regular LDA use is becoming increasingly questioned in day-to-day clinical practice, especially its use as a primary prevention for cardiovascular diseases (CVD) or strokes in the older population. Aspirin and other NSAIDS also cause GI wall damage, leading to ulcers and predisposition to GI bleeds. In fact, it has been advised that routine prescription of doses higher than 81 mg should be avoided due to dose-dependent adverse effects. 21 However, in the aforementioned AspECT Trial, the combined use of LDA and PPIs had an exceptionally low GI bleed rate ∼ <0.1% over 9 yrs. 15 The use of aspirin in GI cancer prevention has been incorporated in some countries, albeit with some variations in recommended dosage and target populations. Australia implemented LDA use for CRC prevention in their general population in 2017. There, Cancer Council Australia released a guideline recommending daily use of 100 to 300 mg aspirin for 2.5 to 5 yrs amongst people aged 50 to 70 yrs. 22 Thereafter, RCTs such as the ‘Should I take Aspirin trial’ (SITA) trial were initiated to guide patients’ decision on aspirin use. Although, the SITA trial revealed that exposure to the decision aid they designed prompted participants to discuss aspirin with their care provider, there was no statistical evidence to show that it led to an increase in the uptake of regular aspirin. 23 This suggests that clinical knowledge is not enough to guide patients’ informed choices, and these clinical encounters could benefit from the addition of a clinical scoring system. Whereby, clinicopathological risk factors (such as nutritional status or predisposition to chronic GI inflammation) that estimate patients’ likelihood of developing colorectal cancer are used alongside decision aids. In England, the National institute for health and Care Excellence (NICE) released a new guideline for an off-label use of daily aspirin for people living with lynch syndrome to decrease their risk of CRC. However, their recommended dose has not been decided and current practice is to use 150 to 300 mg aspirin. 24 The United States proposes aspirin prophylaxis in a multi-systemic fashion. They have initiated a dual recommendation where individuals aged 50 to 59 yrs with a ≥ 10% likelihood of developing CVD in 10 yrs, and who also have an inherited risk of colorectal cancer are advised to take up LDA for at least 10 yrs for primary prevention of CVD and CRC. 25 Within the world of oncology, the impressive benefits of aspirin have also been noted in non-GI cancers such as breast and prostate cancer, as its use have led to better cancer outcomes. 26–28 Coyle et al are currently running a phase III trial, the ‘ADD-aspirin trial. This trial is assessing the use of 100 and 300 mg aspirin for prevention of cancer recurrence. Patients treated for early-stage breast, colorectal, gastric, esophageal, and prostate cancers have been recruited and will be revaluated after ≥ 5yrs of aspirin use. 29 Such an integrative trial is promising, as it can help weigh risk and benefits, and thus help with a much distinct guideline, as people who have previously had cancer have a substantial risk of relapse or developing new cancers. The 5-year survival rate for localized and distant CRC is 91% and 13% respectively. 30 The sizeable difference in the estimated outcome between these CRC extremes illustrates the significant burden posed by disease progression. On this account, it would be worthwhile to generate further guidelines where prognostics factors such as the manner of surgery (radical or non-radical), genetic mutations such as TP53, and/or basic blood tests such as albumin level are incorporated to recommend aspirin prophylaxis to improve disease outcome. Several scoring systems such as Gustave Roussy Immune (GRIm-Score), MD Anderson Cancer Centre (MDACC), Royal Marsden Hospital (RMH) scores that use diverse clinicopathological parameters have been generated and are currently being used to guide prognostication and patient selection for clinical trials. 31 , 32 Aspirin is readily available and is one of the most used drugs globally. Its potential as a preventative measure against GI cancers is supported by a growing body of evidence. Aspirin’s beneficial association is not limited to chemoprevention; its preventative effects are well known in ischaemic diseases such as stroke and myocardial infarction. 33 Thus, regular aspirin use benefits overall morbidity and mortality, and possibly lengthens an individual’s lifespan. The main limitation with the use of aspirin is its link with hemorrhage. However, there are many factors to bear in mind when understanding the bleeding risks. Some of these risks, such as GI hemorrhage are curbed with the use of PPIs; their synergistic uptake has been proven to enhance chemoprevention in cancers such as esophageal cancer. Even though aspirin inhibits coagulation cascades, its use is usually not the primary cause of bleed, and research has shown that other factors, such as Helicobacter pylori exposure and chronic hypertension, exacerbate the clinical impact of hemorrhaging arterioles and capillaries. 34 So, we know that aspirin works; we can appreciate that a dose as low as 75 mg daily is effective; and furthermore, we know that the longer a patient uses aspirin, the more likely they will gain the chemoprotective benefits over time. As seen in current studies, a 10-year duration is associated with the reduction of GI cancer risk; thus, the ideal population would be middle-aged patients who have been deemed to have low bleeding risk following a risk assessment by their care provider. Regarding recommendations for HPB and pancreatic cancers, there are some benefits, but more research is still required to deduce considerable conclusions. On a molecular level aspirin impacts on multiple cancer signaling pathways and studies have also illustrated the benefits of aspirin across many digestive tract cancers. Although for some cancers the benefits are more modest than others. The existing aspirin-GI chemoprevention guidelines discussed earlier have different recommendations for aspirin use. For example, in Australia it is recommended for a specific age bracket, whilst in the UK it is recommended for patients with lynch syndrome. This further emphasizes the need for more research on how to best implement aspirin chemoprophylaxis. All aspects considered it remains clear that aspirin use makes sense. As deduced from the SITA study more measures are also required to reinforce patient decision aids and thus, successfully implement aspirin introduction for GI cancer prevention. Additionally, benefits of aspirin in established cancer patients are also being uncovered. Whilst the facts discussed in this paper are from published sources, there are some limitations that should be acknowledged. Firstly, no statistical analysis was performed and the discussions in this paper are based on previously published research. Secondly, a few of the reports were from case control studies which are prone to biases such as recall, selection, or interviewer bias. Also, similar to any research that looks at the relationship between factors such as lifestyle or medication history to a specific disease, there is usually a risk of healthy population bias where participants are possibly healthier than the general population. Therefore, the presence of other external factors could potentially augment the good results seen from regular aspirin use. In conclusion, the use of LDA as a chemoprophylaxis has a huge role in the future of gastrointestinal cancer prevention schemes. Low-cost cancer prevention schemes are readily used in other medical fields e.g., the use of retinoids and sunscreen in dermatology, for skin cancer incidence. 35 , 36 Therefore, application of these chemoprophylaxis findings into day-to-day clinical practice is promising, especially for patients who are at increased risk or who have previously had GI cancers. There are still some loopholes in the interpretation of the existing studies. Firstly, an agreed dose needs to be determined; more work is still required in the future to help determine the lowest effective dose that would inhibit micro-oncogenic inflammatory processes without causing harm. Secondly, the complexity of drug history and existing comorbidities in middle aged adults’ results in some level of uncertainty regarding the exact effect of aspirin. For instance, patients who are on regular aspirin, tend to be on statins also, which both have an influence in GI chemoprevention. Thus, these confounding factors need to be considered in future research. | Review | biomedical | en | 0.999995 |
PMC11698378 | Sentinel lymph node biopsy (SLNB) is the established technique for staging clinically node-negative breast cancer. SLNB, first described by Giuliano et al. in 1994, has superseded the need for axillary lymph node dissection (ALND) as the standard procedure . The minimally invasive nature of SLNB reduces the morbidity associated with ALND . The use of a radiotracer in combination with blue dye has resulted in sentinel node detection rates of up to 97% . Therefore, SLNB using a radioisotope with blue dye is the gold standard for intraoperative axillary node assessment . Technetium-99m (99mTc), a metastable nuclear isomer of technetium-99, is the most commonly used radiotracer for SLNB . Subareolar or peritumoral radioisotope dye injection is performed preoperatively . 99mTc decays into its ground state through gamma emissions permitting the intraoperative assessment and identification of positive nodes using a gamma probe . Blue dye also may be used to stain positive lymph nodes . 99mTc is generally considered safe for both patients and staff; however, the radioactive nature of the isotope results in the strict regulation of its use, management, and disposal to prevent unintended contamination . Furthermore, 99mTc is not easily accessible in most parts of the world, and the increased costs associated with the special handling required of the radiotracer limit the expansion of conventional SLNB . Superparamagnetic iron oxide nanoparticles (SPIO) have been studied as an alternative to radioisotope tracers for SLNB . The superparamagnetic properties of the nanoparticles allow for high spatial resolution and sensitivity through the detection of the net magnetization of the particles when an external magnetic field is applied . Magtrace® (Endomagnetics Limited, Cambridge, United Kingdom) has been introduced as an SPIO-based tracer which can be detected using the SentiMag® handheld probe (Endomagnetics Limited, Cambridge, United Kingdom) . The use of SPIO-based agents in staging the axilla, which has been studied in different trials, has shown non-inferiority to the 99mTc for axillary SLN detection in early-stage breast cancer . Given the equivalence of SPIO-based tracers to the current gold standard, it may be preferential to the 99mTc-based radiotracer due to the logistical benefits associated with SPIO. Firstly, since SPIO-based tracers are not radioactive, there is no requirement for the special handling and disposal of the dye itself and any consumable equipment contaminated by the radioactive dye . Furthermore, Magtrace® can be injected up to 30 days prior to the procedure, unlike the radiotracer which can only be injected up to a maximum of 24 hours prior to the procedure owing to the six-hour half-life of the isotope . This allows for increased flexibility for the patient. Additionally, the increased buffer time allows for redundancy in case of operating list delays or cancellations which is not possible with the radiotracer . The aim of this study was to evaluate the potential equivalency of the new Magtrace® and SentiMag® techniques in comparison to the "gold standard" technique using 99mTc-based radiotracer. The primary outcome was the node detection rate per patient and the number of lymph nodes retrieved. The secondary outcome was the postoperative length of stay and postoperative complication rates . We conducted a retrospective cohort study at the Rotherham NHS Foundation Trust, UK. The study period was from October 1, 2021, to January 31, 2022. The study data was collected from physical and digital patient medical records. A master list of all adult female patients who underwent breast surgery with SLNB for invasive breast cancer was compiled. This was then stratified into two groups: the "control" group using the 99mTc radiotracer and the "intervention" group using Magtrace. Thirty patients were randomly assigned to each group using simple random sampling. Ethical approval was obtained from the Local Ethics Department of the Rotherham NHS Foundation Trust . The inclusion criteria for this study were as follows: primary breast cancer stages T1-T3 and >18 years of age of any gender. Patients who underwent neoadjuvant chemotherapy were excluded as well as any metastatic disease at the diagnosis. Additional exclusion criteria were pregnancy, previous axillary surgery, radiation therapy to the axilla, or impaired axillary lymphatic function. The primary endpoint for this study was the nodal detection rate. The number of nodes retrieved, postoperative complications, and the total length of stay were measured as the secondary outcomes. We analyzed a total of 60 patients, of which 30 received SLNB using Magtrace® and 30 received SLNB using 99mTc. The mean age of all patients was 64.25±11.6 (33-85) years. Between both groups, the mean ages were slightly dissimilar, but not statistically significant (Magtrace®: 66.9±11.8 vs. 99mTc: 61.6±11.1; p=0.077). The mean BMI of all patients was 28.8±6.2 (16-47). BMI was similar in both groups (Magtrace®: 29.8±6.7 vs. 99mTc: 27.9±5.8; p=0.189). Other patient characteristics such as tumor lateralization, tumor and nodal stage, tumor histopathology, and estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER-2) status were similar and are shown in Table 1 . We found the total operative time matched for patients who only had wide local excision (WLE) with SLNB to be statistically significantly higher in the Magtrace® cohort than the 99mTc cohort (Magtrace®: 89.0±36.9 (30-146) minutes vs. 99mTc: 69.4±17.6 (36-101) minutes; p=0.012). We also found the surgery type between the two groups to be significantly variable with a p-value of 0.049. The breakdown of surgery type can be seen in Table 2 . Nodal detection was similar in both cohorts; however, the 99mTc cohort had a 100% nodal detection rate compared to 93.3% with Magtrace®. Nonetheless, the results were statistically comparable in our study. Additionally, lymph node retrieval and SLN detection were similar with both techniques. The detailed characteristics have been documented in Table 2 . The median length of stay was identical in both groups at postoperative day 1 (p=1). We found an overall complication rate of 20% (n=12): eight complications in the Magtrace® group and four complications in the 99mTc group. Axillary seroma was the most common complication (n=10), with six axillary seromas developing in the Magtrace® cohort and four seromas developing in the 99mTc cohort. Additionally, there was one instance of breast superficial skin necrosis in the Magtrace® group. There was no statistical significance between complication rates in the two cohorts with a p-value of 0.333 as shown in Table 2 . Our study aimed to evaluate the role of Magtrace® at our district general hospital by comparing its efficacy with the established gold standard 99mTc radioisotope-based SLNB. This novel technique has already demonstrated its efficacy and equivalence in trial settings. However, our aim was to determine whether these results can be replicated in everyday practice. Our primary outcome measure was the nodal detection rate. We found no statistical significance in the nodal detection rate which supports the findings in published literature. The SentimagIC trial established Magtrace® as non-inferior to 99mTc radioisotope-based SLNB by undertaking nodal detection with Magtrace®, followed by confirmation using the dual tracer method. In this study, the dual tracer method identified nodes in 98.6% of patients, while the magnetic tracer identified nodes in 99.3% of patients . Furthermore, there was a 100% concordance between dual tracer and magnetic tracer node detection . Our study reflects similar findings with a 93.3% node detection rate in the Magtrace® arm and a 100% node detection rate in the 99mTc arm. The number of lymph nodes retrieved was similar in both cohorts. Nevertheless, SPIO-based tracers have been associated with higher nodal retrieval rates in a meta-analysis conducted by Karakatsanis et al. in 2016 . It has been hypothesized that increased nodal retrieval may be associated with increased postoperative morbidity; however, no such association has been proven. Conflicting results have been reported in the literature following the 2016 meta-analysis with Shams et al. also finding a significant association between magnetic tracers and an increased node retrieval rate, whereas the SMART study found no difference between their cohorts. Our study found the total operative time to be significantly higher in the Magtrace® cohort versus the 99mTc tracer group (89.0±36.9 minutes vs. 69.4±17.6 (36-101) minutes; p=0.012, respectively). As this was a retrospective study, we were unable to acquire data for lymph node detection time alone. Furthermore, due to there being a significant variation in the procedures performed between the groups, an accurate conclusion cannot be drawn in this regard due to the possible confounding effect. There is a relative paucity in the literature regarding the differences in the mean operative time between both techniques; however, Shams et al. did report no difference in the mean operative time in their study. With an identical postoperative length of stay in both groups and no significant difference in postoperative complications, this was a secondary outcome measure that we had specified at the beginning of this study. Given the comparability of results between the study groups, within our study, the postoperative complication rate and postoperative length of stay associated with Magtrace® were equivalent to the established gold standard technique using 99mTc. The 99mTc-based tracer must be administered 3-24 hours preceding the procedure owing to its short half-life of <6 hours. Furthermore, the administration of this must take place within a nuclear medicine department. Careful coordination between the nuclear medicine department and operating department is essential, to ensure adequate 99mTc during the procedure. SPIO-based tracers afford both clinician and patient flexibility, unlike the 99mTc-based tracer as Magtrace® can be administered on the operating table not only 20 minutes prior to the procedure but also up to 30 days prior to the procedure as licensed by the National Institute for Health and Care Excellence (NICE). This not only mitigates the unnecessary radiation exposure to the patient in the event of the cancellation of a procedure but allows for patients to have adequate time and flexibility prior to the procedure, unlike the restrictive timing necessitated by 99mTc-based tracer injection. Our study demonstrates that Magtrace® can successfully be implemented in our institution and similar district general hospitals with outcomes equivalent to the current established gold standard practice of using 99mTc-based tracers to facilitate SLNB in breast cancer. Furthermore, not only were detection rates equivalent in the Magtrace® cohort, but postoperative length of stay and complication rates were similar in both groups as well. Therefore, Magtrace® can be feasibly and safely implemented in a district general setting with equivalent efficacy. | Review | biomedical | en | 0.999996 |
PMC11698379 | Erythrodermic psoriasis (EP) is a rare and severe form of psoriasis vulgaris, with a 1-2.25% prevalence among psoriatic patients . It is defined by considerable erythema that covers at least 80-90% of the body surface and is frequently associated with fever, chills, headache, and general discomfort . EP often mimics a burn-like appearance of the skin due to the intense inflammation, and the associated systemic symptoms can make it difficult to differentiate from other severe skin conditions. The onset might be abrupt or gradual and can be triggered by a range of factors, such as mental stress, infections, or new medications . The pathophysiology of EP requires additional research; however, certain immunological biomarkers, like interleukin-4, interleukin-10, IgE antibodies, and T-helper 2 lymphocytes, are suspected to be implicated. EP is a medical emergency because it disrupts the skin’s normal barrier functions, which are crucial for regulating body temperature, preventing infections, and maintaining fluid and electrolyte balance. The loss of these protective functions can lead to severe fluid loss, multiorgan failure, and an increased risk of infections. The management of EP can be very challenging, and if left untreated, the patient could be at risk of experiencing multi-system organ failure and high-output heart failure due to cutaneous volume loss . We present a case where EP is evident in a patient, serving as a manifestation of recent psychological stress on a background of multiple treatment failures. The extent of the disease in this report was measured using the Dermatology Life Quality Index (DLQI). DLQI is a questionnaire comprising 10 questions aimed at assessing the influence of skin conditions on an individual's quality of life. Each question receives a score between 0 and 3, resulting in a total score range from 0 (indicating no effect of the skin condition on quality of life) to 30 (suggesting the highest impact on quality of life) . During the clinical assessment, psoriasis was observed to involve the entire body surface area, accompanied by a low-grade fever. The skin was warm to touch, dry, and thickened with prominent silvery-white scaling . Notably, there was substantial edema in the lower extremities with localized cellulitis, resulting in restricted mobility . In the hospital setting, the patient was initially treated with intravenous clindamycin at a dose of 600 mg four times daily for two days, after which therapy was switched to oral clindamycin, and the patient subsequently remained afebrile. Methotrexate was withheld while treating the acute infection, and the patient was started on adalimumab following a short course of intravenous hydrocortisone. The results of the blood tests during admission indicated an improvement in C-reactive protein and normalization of serum creatinine following fluid management (Table 1 ). Mild thrombophilia had also improved after discharge from the hospital, and neutrophilia was observed to be related to steroid initiation. Psoriasis (psoriasis vulgaris) is a chronic (long-term) autoimmune condition characterized by extensive scaling caused by epidermal cell hyperproliferation. It typically presents with large oval-circular plaques over the scalp, trunk, and extensor body surface . More commonly, EP arises as a complication of psoriasis vulgaris . Although EP is classified as a subtype of psoriasis, the pathogenic processes are distinct from those seen in plaque psoriasis , IL36RN mutations have been linked to pustular psoriasis, and the class I antigens HLA-Cw6, HLA-B57, HLA-B13, and HLA-B17 have been linked to psoriasis vulgaris. However, very little is known about the genetic foundation of EP . Several studies suggest that the disease is associated with a predominantly T-helper 2 phenotype. In this report, the patient’s symptoms were classic EP, including widespread erythema, desquamation, and systemic involvement. Notably, the patient also developed cellulitis of the lower limbs . Given the severity of the infection, the cellulitis was treated with antibiotics to prevent further complications, such as sepsis, which can occur if left untreated. The co-occurrence of an infection with EP underscores the delicate nature of the skin’s integrity in this condition. The mechanism by which psychological stress contributes to the onset or worsening of psoriasis is not yet fully understood. Psychological stress activates the hypothalamic-pituitary-adrenal axis, leading to the release of stress hormones (cortisol, adrenaline, and noradrenaline) that interfere with immune regulation and amplify inflammatory responses, fostering a pro-inflammatory environment . Although cortisol typically exerts anti-inflammatory effects, prolonged stress may diminish its regulatory capacity, allowing unopposed inflammatory pathways . EP, in particular, is propelled by an immune response dominated by T-cells, particularly Th1 and Th17 subsets, along with dendritic cells that produce potent pro-inflammatory cytokines such as IL-17, IL-22, TNF-α, and IFN-γ . Chronic stress further amplifies T-cell activation, significantly intensifying the immune response. This excessive cytokine production may result in a “cytokine storm,” a widespread and severe inflammatory reaction that manifests as extensive skin redness, scaling, and edema . Additionally, EP compromises the skin’s barrier function, heightening the risk of infection and fluid loss. In cases triggered by stress, dysregulated cortisol levels may further undermine skin barrier integrity. Together with the high systemic inflammation, these factors render the skin highly susceptible to infections, while stress-related immune suppression increases the body’s vulnerability to opportunistic infections . Nonpharmacological treatments, such as effective stress management techniques, mindfulness meditation, cognitive-behavioral therapy, relaxation techniques, and physical exercise, can help patients reduce their physiological stress response. In five out of six randomized control trials, participants demonstrated improvements in self-administered psoriasis area and severity index scores following eight or 12 weeks of meditation and/or mindfulness interventions. Additionally, two studies indicated psychological benefits for psoriasis patients after engaging in these practices. Collectively, these findings imply that meditation may serve as an effective method for enhancing both psoriasis severity and the quality of life for patients . Pharmacological treatment usually includes oral hydration, topical steroids, vitamin D analogs, and a trial of biological therapy. In this instance, due to the severity of the condition and the risk of further systemic complications, the decision was made to initiate treatment with adalimumab, a TNF-alpha inhibitor. Adalimumab was chosen because of its potent anti-inflammatory properties, which specifically target the overactive immune response seen in psoriasis. It effectively neutralizes TNF activity by impeding its interaction with TNF receptors on the cell surface. This inhibition curtails the migration of leukocytes, subsequently reducing the proliferation and differentiation of keratinocytes . This case report represents a rare presentation of EP associated with psychological stress. When patients present critically ill with an erythrodermic rash, it is important to consider EP in the differential diagnosis, especially with recent psychological stress. The mechanisms through which psychological stress interferes with psoriasis onset or exacerbations are not completely understood. However, psychoneuroimmunology studies have shown that acute and chronic stress can affect immune function, leading to a worsening of psoriasis by inducing keratinocyte proliferation and incomplete maturation. | Other | biomedical | en | 0.999998 |
PMC11698405 | Visual methods represent a novel approach in qualitative evidence synthesis (QES) by introducing another dimension to the synthesis process and contributing to the understanding and generation of knowledge . The terminology “qualitative data or evidence” broadly refers to findings from primary qualitative studies (e.g., analysis of data from interviews, focus groups, and the production of new theories or theoretical insights), or qualitative data (such as narrative responses to open ended questions). Numerous methods can be used for data synthesis in a QES including meta‐ethnography , thematic synthesis , and framework synthesis . Irrespective of the method used for analysis and synthesis, additional visual methods can play a crucial role in aiding review authors and readers to comprehend, organize and display qualitative data collected from included studies . There is a tendency for review authors to underutilize the diverse array of visual display methods, tools, and techniques available to enhance a chosen synthesis method . Some review authors may assume that their selected synthesis method includes all necessary supplementary methods, tools, and processes, while others may lack the skills or confidence to generate alternative formats for their synthesis. This paper provides an overview of accessible visual methods supporting the synthesis stages of a QES, with examples and their application in the development of a Cochrane QES using meta‐ethnography. The paper also addresses the role of stakeholders, considerations of equity, diversity, and inclusion, and reflexivity in selecting and employing additional visual methods. Although the focus is on QES, the visual methods described here can also be used to facilitate communication of complex and sensitive topics in primary qualitative research during data collection and analysis . To illustrate the use of visual methods to support a synthesis, a worked example of a recent QES using meta‐ethnography is used . Meta‐ethnography is one of the most complex QES methods designed to synthesize mainly rich data from primary qualitative studies in a series of steps to develop new theoretical insights and theory . The review authors investigated how children and young people with chronic noncancer pain and their families experience and understand their condition, pain services and treatments . The whole team was involved in conducting the analytic synthesis with two members leading on and carrying out the majority ofthe analytic synthesis. They produced three lines of argument, a model and a theory of chronic pain management. The combination of their lines of argument was named “The journey of living with chronic pain” which expressed the experiences of children and young people with chronic pain and their families from the onset of chronic pain; their struggle to navigate health services seeking a cure, and to have their needs and expectations met; and the outcome, moving on either to prioritize living well with pain or give up hope . This method was used among the review team only between phases 5 and 6 as part of the analytic synthesis process (to translate the studies into one another and in synthesizing translations). The review authors used paper labels to initiate the synthesis process and start developing novel insights . In a previous step, at least two review authors interpreted the meaning of every relevant finding, concept, or theme from the studies using NVivo version 12. The authors then compared the meanings within and across studies to identify common or unique concepts. Where possible, the common concepts were then matched, merged, and further interpreted by two review authors and discussed with the wider team of review authors to develop new interpretations. The common concepts and new interpretations were summarized on paper labels. Unlike NVivo 12 , paper labels provided the necessary visual and textual components needed to allow a larger number of researchers to work together during in‐person analysis. For instance, labels included a title and a short summary explaining the specific findings and contributing studies. All labels were color‐coded according to health condition and whose interpretation was presented (i.e., that of the primary study author or the review team). Paper labels were also numbered to match the structure of an accompanying detailed Word document which gave the full details of the primary study data underpinning the short summary of the findings. This strategy of visually displaying all findings helped the review authors to iteratively test different ways of thematically grouping the findings. It also helped to conduct a thematic analysis with the creation of new themes signposted using Post‐it notes. Photos were taken to record different versions (e.g., version 1, version 2, etc.) so that the review authors could follow the development of their analysis and subsequent synthesis. When working remotely, the review authors adapted the method by recreating all paper labels virtually using Padlet , a real‐time collaborative web platform. Padlet virtual labels were color‐coded according to whose interpretation was presented (i.e., whether it was the interpretation of the primary study author or of the review team) and included a title and description of the construct. Both physical and virtual labels were used together during the team meeting. The idea was that members joining remotely via Microsoft Teams could participate in the thematic synthesis in real time using the virtual labels. However, the review authors learned that constantly updating Padlet to match the thematic groupings in real time was challenging and time‐consuming. This process could have been more efficient with the involvement of a dedicated facilitator, who could have taken responsibility for regularly updating the Padlet . As a result, the review authors that were joining the meeting online were updated verbally regarding the changes in the configurations of labels and Padlet was used only as a visual aid. At the end of the meeting, photos showing the labels that were used to create “new constructs” or understandings were uploaded on Padlet to facilitate discussion with the whole team and to provide a record of the analysis (Appendix S1 ). A digital interactive whiteboard, Google Jamboard, was used during phase 6 of the meta‐ethnography to display data and develop analytic categories remotely . Google Jamboard is composed of different “frames,” similar to pages or slides. The authors used each frame to analyze a specific cluster of related themes, which were grouped together into a broader “analytic category,” for instance, as shown in the frame in Figure 4 . All findings were recreated as notes that were color‐coded according to the “analytic category” to which they belonged. All notes included a title, the contributing studies, the health condition, and whose interpretation was presented (i.e., that of the primary study authors or the review team). The “analytic categories,” themes, and their constituent findings and all notes were numbered to match the same structure as the textual synthesis (i.e., a Word document containing the full details of the primary study data underpinning the findings, themes, and analytic categories). This strategy allowed the authors to easily transfer any changes or new interpretations into the textual synthesis document. Google Jamboard also facilitated the tracking of how the themes were organized according to the different interpretations from the team and facilitated team discussions of the different interpretations. This method was used during phase 6 of the meta‐ethnography to visualize and further develop the synthesis. Microsoft Whiteboard is a multiplatform application which simulates a virtual whiteboard and enables real‐time collaboration. The review authors used Microsoft Whiteboard to express and understand how findings were connected to one another to create a coherent “storyline” [line of argument] (see Section 1.3 ). Initially, the authors included all themes under their respective category as text boxes on Whiteboard . All text boxes were color‐coded according to context (i.e., different colors were used to indicate starting points, potential links with other categories, and findings representing a positive impact). The authors used arrows to indicate which findings/themes were related, and the result was a large diagram linking all five categories (Appendix S2 ). Short descriptions for each analytic category were created based on the diagrams and these were discussed during an analysis meeting with the research team. At this point, the review authors focused on creating a better understanding of each analytic category. Subsequently, the diagram was further developed incorporating different interpretations and perceptions from the multidisciplinary team, resulting in major modifications to allow a more in‐depth exploration of these data (Appendix S3 ). At this stage, the visual representation of all analytic categories in the form of diagrams allowed the team to develop their understanding of and start developing the initial “overarching storylines” or lines of argument. The initial interpretations and hypotheses were inserted in the diagram as virtual notes. The final step was the creation of a further simplified version of the diagram that displayed how all four final analytic categories and findings were connected. The researchers used this last version of the diagram to further develop the description of the diagram to include how all categories and themes/findings were related which was used to create the textual synthesis. Diagrams were used by the review authors during phases 6 and 7 to further understand and express how the three lines of argument they developed were related. They used the final diagram they had produced using Microsoft Whiteboard , data from the PPI workshop, and the textual synthesis, to create an initial version of a visual model to refine and represent the findings of the synthesis connecting all lines of argument in Microsoft Word . The initial synthesis model was developed following feedback from the whole research team and depicted the nonlinear nature of the phenomenon of interest (i.e., families' journeys living with chronic pain and how they are affected by services). Subsequently, the researchers used Drawio to draw and refine the model with the inclusion of more context and nuance. This process of further refining the model consisted of rich interpretative discussions among the core members of the research team until an intuitive final version was constructed . The synthesis model expressed the concept of a journey families are navigating while they deal with chronic pain and access services. To express the concept of the journey and time, the researchers used rounded arrows to create an illusion of a cycle and described (text in red) where families might stay “stuck.” Two text boxes between both pathways indicated how families might navigate between these distinct pathways. Diagrams were used during phases 6 and 7—synthesizing the translations and expressing the synthesis. The review authors produced a theory explaining their phenomenon of interest (i.e., theory of good chronic pain management). This process included multiple analysis meetings with the core review authors and it also integrated insights from PPI lived experience and the key findings from the synthesis of the studies included in the review data. The authors used two of their main analytic categories (related to family life and their social relationships and their experiences navigating health services) to construct the initial structure for the theory in the center of the diagram on Microsoft Whiteboard . The review authors then placed all factors that had a positive impact on family life on the right side of the diagram, and factors with a negative impact on the left side. They used arrows to indicate when an aspect could be modified by the factors placed on each side of the diagram. The final version of the diagram mapped all factors that had the potential to “modulate” families' experiences with chronic pain (Appendix S4 ). Figure 9 shows the simplified version of the diagram. We have shown that the use of additional visual methods in a QES facilitated better data visualization, remote analysis group meetings, interpretation of large amounts of data, and meaningful PPI during synthesis. Visual methods varied in complexity, costs, and required expertise, allowing flexibility to adapt to different contexts, whether virtual or face‐to‐face. For instance, certain methods such as paper labels worked better in face‐to‐face settings and facilitated group work involving multiple people. This method was essential to allow group work when dealing with large amounts of data. In contrast, the use of labels in virtual platforms such as Padlet was time‐consuming and demanded the presence of a facilitator and could only cope with moderate amounts of data. It is imperative to carefully consider equity, inclusion, and diversity in the development and application of visual methods to ensure their accessibility and relevance across diverse populations. In addition, when developing and tailoring all virtual methods or outputs, the review authors need to carefully consider their personal biases and professional perspectives and positioning concerning what they would choose to present visually and how they interpret it. As such, it is essential that visualizations are either informed by or created with those they represent so they are inclusive and relatable to their audience. For example, in France's meta‐ethnography the authors co‐developed cartoons with members of the public and were careful to ensure these were representative of different ethnicities, and genders and did not promote an idealized context. The review authors also carefully considered the scenario and context of each cartoon ensuring these were appropriate and inclusive (e.g., a plain doctor's surgery and non‐descript hospital settings). Visual methods should also use accessible and engaging language and include accessibility features such as subtitles, image, and audio descriptions. To the best of our knowledge, this is the first study to date showing the application of additional visual methods in a published QES. Visual methods are currently underused and underreported in QESs. QES authors should consider making use of available visual methods, particularly when involving members of the public during synthesis. Selecting the appropriate visual method for synthesis should be guided by its ability to enhance analysis and align with the study's objectives. Methods must suit the content and stage of synthesis, whether for idea development or presenting findings. Practical factors, including available resources and the team's technical skills, are crucial, especially for remote work. Additionally, group‐based methods may require significant preparation, facilitation, and specialized skills. Careful consideration of these aspects will ensure the effective and efficient use of visual methods. | Other | biomedical | en | 0.999997 |
PMC11698477 | Unlike large-scale commercial fisheries, which typically take place at a greater distance from shore, recreational anglers often access coastal environments that are critical habitat for many fish species at multiple life stages and can be especially important for juveniles . While shallow waters may offer young teleost fishes and sharks protection from natural predators or access to coastal resources, proximity to land increases the probability of recreational fishing interactions and exposes them to capture stress during a critical life stage [ 19 – 21 ]. Additionally, species with nearshore distributions and strong site fidelity, like the nurse shark ( Ginglymostoma cirratum ), are more accessible to recreational anglers, including the probability of repeated captures, and in turn, are potentially more susceptible to angling-related impacts. Nurse sharks are a coastal species found in the eastern Pacific and western Atlantic Oceans with neonates and juveniles remaining close to shore in seagrass flats and mangrove islands . Tagging studies have revealed a high degree of site fidelity in adults, including long-term mating site fidelity , and particularly limited movement patterns compared to other shark species [ 22 – 23 , 25 ]. The Everglades National Park Creel survey reported that between 1972 and 2002, nurse sharks were the second most frequently caught species from sport fishing trips . Although nurse sharks are common in South Florida and are often encountered in recreational fisheries, and their population there is believed to be increasing, the western Atlantic population overall is listed as “Vulnerable” by the IUCN with a decreasing population trend due to ongoing fishing pressures, habitat degradation, and the impacts of climate change . As a resident, largely non-migratory species, the nurse shark is especially susceptible to exposure to repeated capture stress in frequently fished environments. A study by Hammerschlag et al. found nurse sharks in Biscayne Bay did not avoid urbanized areas, even during periods of higher human activity. Furthermore, Whitney et al. reported nurse sharks have the lowest mass and temperature-adjusted metabolic rate known for any shark species, and suggested they may incur a much higher relative metabolic cost of activity in switching from rest to movement compared to more active elasmobranch species. Perhaps as a result of these characteristics, nurse sharks exhibit a relatively passive “fight” during capture compared to other species . However, these studies captured adults and used commercial or scientific fishing methods that have relatively long standardized soak times. Therefore, they lack key data on how juveniles respond to capture and the behavioral and physiological responses that this species exhibits as a result of recreational angling methods . With a relatively high cost of movement and low metabolic rate, nurse sharks are energetically suited for, and perhaps dependent on, a minimally active lifestyle and may experience negative physiological consequences if forced to actively ‘fight’ on a line. Identification of these angling-related consequences and their impact is essential for the development of best management and conservation practices for this species as they are often captured by shore-based recreational anglers and they are commonly perceived as a robust shark species by both scientists and recreational anglers in South Florida . The purpose of this study was to collect data on the physiological stress response of juvenile nurse sharks ( Ginglymostoma cirratum ) to catch-and-release recreational angling and the potential influence of biotic and abiotic factors in shaping that response. Specifically, we sought to measure the physiological status of juvenile nurse sharks at multiple points in time during an active fight on hook and line. To assess these impacts, lactate, glucose, hematocrit and osmolality were measured, and the relationship between these parameters and fight time, shark size, and water temperature were evaluated. This is the first study to examine the physiological disturbance caused by recreational angling in nurse sharks and therefore provides information on a data limited species. Understanding these physiological impacts could have applications for species-specific management of recreational fisheries and better support conservation initiatives for nurse sharks. Sharks were captured via hook and line using 8-foot recreational rods equipped with 50 lb. test monofilament, a stainless steel leader, and size 13/0 barbed circle hooks to reduce the risk of foul hooking ( i . e ., hooking in any part of the body other than the jaw) and increase the probability of successful landing. The time was recorded from the first sign a shark was detected on the line until the initial blood sample was taken (“time 0”), based on the methods of Hoffmayer and Parsons . Once hooked, sharks were retrieved as quickly as possible (within 3 min) to allow for the best estimate of pre-disturbance blood parameters . After the initial blood sample, each shark remained on the hook-and-line and was returned to the water where it was angled for the additional time between blood samples. Between blood draws the sharks were continuously engaging in active swimming against the line and not allowed to rest. Over a period of 30 min, 4 small blood samples (2 ml each) were drawn from the caudal vein of each animal. After each blood sample, the shark was returned to the water and the fight continued with each subsequent blood sample taken with variable fight time intervals between sampling ( i . e ., randomized ordering of an interval of 5, 10, and 15 min between each sampling event to control for the influence of shark handling and the blood draw procedure; Table 1 ). During the final landing, the sharks were also rapidly measured (pre-caudal length, fork length, total length, and girth), sexed (visual survey of cloaca), and tagged with small, plastic Floy-brand spaghetti tags (individuals < 1 m) or NOAA cooperative tags (individuals > 1 m; S1 Dataset ). Lastly, small fin clip samples were collected to be used in future genetic studies, and surface water temperature was recorded. At each time interval, individuals were retrieved from the water and whole blood samples (<2 ml) were drawn with 18-gauge 3-inch needles within 1 min of landing the shark via caudal venipuncture. Lactate and glucose were immediately measured by adding approximately 10 μl of blood each to a glucose meter (Accu-Chek glucose meter; Roche Diagnostics, Basel, Switzerland) and to a lactate meter . The remainder of the blood was then added to blood collection tubes spray-coated with lithium heparin (BD Vacutainer® Heparin Tubes) and stored on ice until returning to the lab. Hematocrit was immediately determined upon returning to the lab through centrifugation (13,000 g x 5 min) of plain capillary tubes and visual measurement of the packed red blood cell volume (% Hct). The remainder of the heparinized blood was transferred into vials and spun via centrifugation (13,000 g x 5 min) for plasma extraction. Vials containing plasma were frozen at -20°C for later lab-based analysis using a Westcor Vapor Pressure Osmometer to measure osmolality. Linear mixed models (LMM) were used to explore how total fight time (the total amount of time a shark has been on the line) and fight time (the amount of time the shark fought on the line between blood draws, i . e ., 5, 10, or 15 min) relate to physiological parameters (lactate, glucose, hematocrit and osmolality). Total length, sex, water temperature, and fishing site were also included in the analysis as covariates. To control for the influence of shark handling and the blood draw procedure, the order of the fight time intervals (5, 10, and 15 min) varied randomly. Additionally, two variations of the model were used for each physiological parameter. The first model for each physiological parameter explored total fight time while the second model included blood draw number as a variable to investigate the potential influence of the blood draw procedure on the model output while also investigating relationships between physiological parameters and fight time. A separate LMM was used to determine the relationship between initial levels of each physiological parameter and the time it took to obtain the initial blood sample. For all the models, a random intercept (shark ID number) was included to account for between-subject variation. The sum of squared residuals was calculated for each model. Relative effect sizes were computed following Cohen with d = 0.2 representing a small effect size, d = 0.5 a medium effect size, and d = 0.8 a large effect size. Individual models were run using the “lme4” and “EMAtools” packages in R version 3.6.3 and statistical significance was set a priori at p < 0.05 ( S2 Dataset ). Based on Giesy , the Lactate Pro meter overestimates lactate concentrations, but in a predictable way. Therefore, all lactate concentrations were converted using a calculated adjustment curve equation (y = 1.79x – 0.08). There were no differences in statistical significance for the variables in either model between the original Lactate Pro values and the adjusted lactate concentrations. The Accu-Chek glucose meter has not been validated with laboratory analysis. Therefore, the data presented were not converted to laboratory-based values. Between November 2019 and October 2020, a total of 27 nurse sharks (15 females, 12 males), with a total length ranging from 63 to 153 cm (mean ± SD = 101.8 ± 25.0 cm), were caught and sampled ( Table 2 ). Four sharks were recaptured and resampled (31 total angling events) one of which was caught three separate times and another of which was recaptured one day after being caught for the first time. The remaining two sharks were recaptured after 14 days and assumed to be fully recovered; therefore, data from all capture events were pooled for analysis. Additionally, one shark was able to break free from the line before the final blood draw could be performed. This individual had a NOAA cooperative tag and total length was estimated based on previous capture data from seven months prior linked to the tag. It was found that sex had no significant (all p > 0.05) effect on the stress response across any physiological parameter, so it was excluded from the models. Water temperature at the time of capture ranged from 21.1 to 33.9°C (mean ± SD = 30.5 ± 3.4°C). Fishing site was found to only be significant for osmolality (all other p > 0.05), so it was excluded from the other physiological parameter models. All initial blood samples in this study were collected within 3 min (mean ± SD = 1.84 ± 1.02 min) with the exception of two of the largest sharks. There were no statistically significant relationships between initial levels of lactate, glucose, hematocrit or osmolality and the time (0.55 to 2.58 min for all other sharks, 4.25 and 5.33 min for the two largest sharks) it took to obtain the initial blood sample (Lactate: p = 0.195, Glucose: p = 0.297, Hematocrit: p = 0.469, Osmolality: p = 0.519). The average initial lactate value was 1.22 ± 0.64 mmol/L ( Table 3 ). Lactate increased by an average of 611% (6.7 ± 2.17 mmol/L) over the 30-min fight . Furthermore, the LMM established a significant linear relationship between lactate, total fight time, and temperature . When blood draw number was added as a variable, model results indicated that blood draw number, fight time, and temperature were all significantly related to lactate ( Table 4 ). Glucose values increased by 72% (2.2 ± 0.87 mmol/L) , but only significantly changed with blood draw number ( Table 4 ). Hematocrit decreased by 0.7 ± 1.9% on average over the 30-min fight . Model results indicated increasing total fight time and blood draw number were inversely related to hematocrit and total length was positively related to hematocrit values ( Table 4 ). Osmolality decreased by 1.2% (17.9 ± 196.8 mOsm/kg) over the 30-min fight time , but fishing location was the only variable to have a significant correlation to osmolality ( Table 4 ). The stress from a capture event elicits a complex physiological response in fish. Although this physiological response has been relatively well-studied for some elasmobranchs [ 15 , 42 – 44 ], little research has been done to understand this response in nurse sharks, and what is known focuses on adults and scientific fishing methods . Their strong site fidelity and low metabolic rates make them particularly susceptible to repeated captures at recreational fishing sites and chronic exposure to localized stressors . Indeed, in this study, a total of four angling events were of recaptured sharks, with one shark caught three separate times and one caught only one day after it was first captured. It has been well-documented in previous studies that other species of elasmobranchs use shallow, subtidal zones as refuges from predators, and it is likely that juvenile nurse sharks are using these fishing sites around shallow, highly impacted urban areas ( e . g ., areas near marinas and sea walls) for protection from predation . Recaptures in this study suggest they may be site attached with small home ranges, which is supported by existing literature on other species . For example, juvenile lemon sharks ( Negaprion brevirostris ) use subtidal mangrove habitat to decrease the likelihood of encounters with predators even under conditions of decreased habitat quality . Although nurse sharks appear to be generally more resilient to capture stress than many other species , their strong site fidelity increases the risk of repeated captures and subsequent physiological responses. This potential chronic stress has been shown in other species to suppress growth, reduce reproductive success, and limit foraging energy, and can lead to increased predation risk and reduced survival . The results of the LMM for lactate revealed a significant linear relationship with fight time, although this and other physiological studies have been unable to distinguish between the stress response to an initial hooking event and the response to the “fight”. The relationship between fight time and lactate is reflected in existing literature including for juvenile sand tiger sharks . Similarly, Gallagher et al. found lactate to be the sole blood parameter to be significantly affected by increased time on the line for several shark species. The increase in lactate over time indicates that the “fight” on the line caused a shift to anaerobic respiration, which resulted in the accumulation of lactate as a byproduct. The elevated lactate levels (6.7 mmol/L on average) measured after 30 min in this study are consistent with previous research on other species, and similar increases in lactate are known to have negative physiological effects . Therefore, lactate values can serve as a proxy for assessing the immediate condition or health of a shark . Fuller et al. found lactate was able to significantly predict a shark’s behavior upon release with higher lactate values corresponding to more sluggish behavior post-release. Higher lactate values were also significantly correlated with moribund shortfin mako , blue , and blacktip sharks . Although our study did not observe post-release behavior or record post-release mortality rates, the literature suggests the observed increases in lactate represent a considerable physiological cost that could impact short-term health, potentially rendering juveniles more susceptible to predation or disease. Nurse sharks have regularly exhibited low behavioral and physiological response to capture stress. For example, Jerome et al. found nurse sharks consistently had the lowest relative levels of physiological disturbance among studied species, including lactate levels and reflex impairment. Likewise, nurse sharks caught on longlines exhibited low-intensity exercise in response, with maximum lactate levels only reaching 3.25 mmol/L , compared to a maximum of 22.1 mmol/L in this study. However, previous studies reporting lower lactate results used commercial or scientific fishing methods ( e . g ., drumlines or longlines) where the nurse sharks are able to settle on the bottom for the duration of the soak time (1 hour). This is the first study to examine the effects of active recreational angling on nurse sharks, and the first to explore physiological stress in juvenile nurse sharks. A period of hyperglycemia is expected after stress or exhaustive exercise in fish as utilization of glucose mobilized through the stress response or depletion of hepatic glycogen stores is suggested to occur [ 55 – 57 ]. Manire et al. observed a substantial decrease in glucose for both bonnethead and bull sharks caught with gillnets. Conversely, significant increases in glucose levels were detected in both Atlantic sharpnose and blacktip sharks caught using hook-and-line . In this study, a weak relationship between glucose and fight time was measured. Results from the initial model showed a significant, positive relationship existed between glucose and total fight time for nurse sharks. However, in the final model, blood draw number was significant and fight time was nearly significant, which suggests juvenile nurse sharks may exhibit a more pronounced stress response from handling and air exposure than from short periods of exhaustive exercise. Longer periods of air exposure were found to increase blood-based disturbances and mortality rates in bonefish , and significantly disrupt physiological parameters in little skates ( Leucoraje erinacea ) with acute thermal stress exacerbating these impacts . Previous work on elasmobranchs has also documented no meaningful change in glucose as a result of capture stress . The combination of these results suggests changes in glucose values may be species-specific, a finding that is supported in the literature . It should be noted that the relationship and accuracy of the Accu-Chek glucose meter compared to values obtained with a colorimetric assay in the laboratory has not been validated for nurse sharks, due to insufficient laboratory data . Inconsistencies between values obtained by the glucose meter versus a colorimetric assay could explain why there is only a weak relationship between glucose and fight time. Further investigation is needed to understand the accuracy of the Accu-Chek glucose meter. Several previous studies have found that capture stress either causes significant increases in elasmobranch hematocrit values or no significant changes . Conversely, the final model for hematocrit in this study found blood draw number decreases hematocrit values. These lower hematocrit levels suggest that blood loss, potentially from the venipuncture site or hooking location, may result in hemodilution in small nurse sharks. This has been measured in other studies of elasmobranchs, and given that many studies engage in serial sampling including of small sharks , should be considered in analysis. Repeated blood sampling of Port Jackson sharks and gummy sharks resulted in a significant decrease in hematocrit values, suggesting that the decline in hematocrit over the serial blood sampling protocol used here was likely a consequence of repeated blood extraction . Similarly, Mohan et al. measured significantly reduced hematocrit levels with increased handling times in blacktip sharks that were not related to the physiological stress associated with a capture event, and hypothesized the decrease was due to blood loss. Capture stress can lead to disruption in the ability to osmoregulate in aquatic animals due to water shifting out of vascular compartments in response to increased intercellular lactate in the blood . This is consistent with recent data showing an increase in osmolality with fight time in blacktip sharks and Atlantic sharpnose sharks . Interestingly, there was no evidence of a significant difference in osmolality in juvenile nurse sharks due to fight time. Similar results were observed in another study on blacktip sharks . It is possible that the decrease in hematocrit, which in theory could also have resulted from plasma water moving into the circulation from the tissues, may have offset any increase in osmolality that occurred during the 30-min fight . Additionally, it may take longer than 30 min for the effects of capture stress on osmolality to be clearly detectable. It is well known that many shark nurseries are susceptible to environmental fluctuations in salinity due to their shallow, coastal nature . In this study, osmolality varied significantly by fishing site with lower osmolality levels corresponding to sharks caught at a location near water outflows from canals that affect the salinity of surrounding habitats. Therefore, for species that typically have high site fidelity, local abiotic conditions may matter and proximity of the stress event to an outflow could affect the capture stress response. Additionally, coastal locations like the fishing sites in this study provide essential habitat for many elasmobranch species but are subjected to various anthropogenic disturbances. Aquatic animals are exposed to pollutants via the delicate respiratory surface of the gills and these environmental toxins are known to impair overall fish health and cause both acute and chronic stress . Some studies have found that sharks residing in wastewater-impacted areas are exposed to and can accumulate measurable quantities of human pharmaceuticals . Furthermore, Rangel et al. found that juvenile nurse sharks sampled in highly impacted urban areas of Biscayne Bay in Miami had higher concentrations of plasma-saturated and bacterial fatty acids, suggesting they consumed lower-quality food resources than conspecifics in less urban-impacted areas. Therefore, these juvenile nurse sharks may already be under environmental stresses that could affect their long-term health and ability to respond physiologically to capture stress, which may pose a greater threat to these animals than previously believed. Additional research is needed to understand the relationship between environmental factors, especially in highly-human-impacted areas, and capture stress. Although fishing effort for this study largely focused on the summer months, precluding concrete comparisons, the estimated Catch Per Unit Effort (CPUE) was approximately three times higher in this study in summer than in winter. This suggests juvenile nurse sharks may not be utilizing these popular fishing locations during the cooler months or may not be feeding as frequently, and therefore are likely caught less frequently during the winter months. In addition, model results revealed that higher lactate levels corresponded to warmer water temperatures; a relationship that has been previously noted for several other elasmobranch species . This indicates juvenile nurse sharks may experience increased capture stress during summer months when water temperatures are higher. Increased likelihood of being caught by recreational fishers and the amplified physiological response to capture during the summer months could represent a meaningful stressor to juvenile nurse sharks around South Florida, but this warrants additional research. This study suggests juvenile nurse sharks exhibit a greater physiological stress response when exposed to recreational angling than some other shark species or their adult counterparts captured using other fishing methods. This provides insight into nurse shark stress responses that was not previously available, which can help to inform age- and practice-specific management and conservation strategies around shore-based recreational fisheries. Furthermore, results revealed blood draw number to be significant for lactate, glucose, and hematocrit, suggesting that handling and air exposure can cause substantial physiological stress for juvenile nurse sharks. These results and others suggest air exposure and handling by recreational anglers should be limited whenever possible, in line with other findings from recreational fisheries for teleosts . Therefore, policy decisions should be centered around reducing fight time as well as handling and air exposure in order to moderate the physiological stress experienced by juvenile nurse sharks caught on hook-and-line. Future studies should assess the primary endocrine stress response to capture as well as additional physiological variables (e.g., fatty acids and electrolytes) to gain a better understanding of the entirety of the capture stress response. | Study | biomedical | en | 0.999996 |
PMC11698478 | Anemia is one of the most commonly prevalent nutritional deficiency disorders globally. It is characterized by decreased hemoglobin (Hb) concentration in relation to age, sex, altitude, and smoking status. Anemia can result from physiological factors such as pregnancy or pathological factors like increased loss or decreased production of blood or its components . Globally, about half a billion women are anemic. In the developing world, such as in a low to middle-income country like India, anemia is a major public health issue . Alarmingly, the prevalence of anemia in children, adolescent girls, and pregnant women is in excess of 50.0%, according to the National Family Health Survey-5 (NFHS-5) . The prevalence is higher in rural areas compared to urban areas . Adolescence is a period of rapid growth and development characterized by major physiological and biochemical changes in the body. In terms of nutrient requirement, it is a phase of increased demand. In both adolescent girls and boys, increased iron uptake is required to meet a rapid growth spurt; also, in girls, further supply is required to compensate for menstrual blood loss. Worldwide, one in four individuals aged 10-24 years suffers from anemia, which amounts to 430 million individuals. India is considered a nation of youth, and it is home to 253 million adolescents, out of which 72 million were anemic in 2018. Uttar Pradesh has the largest adolescent population in India and has twice the number of anemia cases compared to other Indian states . According to WHO, anemia is defined as a Hb concentration of less than 13 g/dL in males (15 years and above) and less than 12 g/dL in non-pregnant females (15 years and above) . Anemia in adolescents may lead to decreased growth and productivity and low scholastic performance, and, in the long term, can cause cardiomegaly and left ventricular failure. Additionally, anemia also causes complications related to pregnancy and childbirth in young females. This study was planned to assess the prevalence of anemia in the adolescent age group, as well as to examine the association between the prevalence of anemia and the sex of the participants, and the role of deworming tablets in preventing anemia. A community-based cross-sectional study was conducted in a rural area of Meerut district from September to December 2023. The study population involved adolescent beneficiaries of the Anemia Mukt Bharat program in the Behsuma area. The inclusion criteria were as follows: all adolescent health beneficiaries of the Anemia Mukt Bharat program. Those not willing to participate or those suffering from any chronic debilitating illness were excluded from the study The sample size was calculated based on the following formula: N = \begin{document}\frac{Z^2_{\alpha/2} \times p \times q}{d^2}\end{document} ; based on the NFHS-5 data for the prevalence of anemia in Meerut district (45.1%), and at 7.5% absolute precision, the required sample size came out to be 170. Data were collected using a pre-designed and pre-tested questionnaire (see Appendices). Consent and assent were obtained from the guardians and beneficiaries, respectively, after explaining the nature of the study. A capillary blood sample was taken after cleaning and drying the index or ring finger of the non-dominant hand. Hb estimation was performed using a True-Hb hemoglobinometer (Wrig Nanosystems, New Delhi, India). WHO classification of anemia was used to classify beneficiaries. Data were tabulated and analyzed using Microsoft Excel. Chi-square was used as the test of statistical significance, and a p-value less than 0.05 was considered significant. Ethical approval was obtained from the institutional ethical committee . Out of the 170 respondents, 60 (35.3%) were males, while 110 (64.7%) were females. Of note, 102 (60.0%) of the total respondents were found to be anemic, while 68 (40.0%) were non-anemic. Among males, 27 (45.0%) were anemic, whereas among females, 75 (68.2%) had anemia, as shown in Table 1 . There was a significant association between the sex of the participant and the prevalence of anemia, as females were more anemic compared to males . Among anemic individuals, moderate anemia was the most common entity, affecting 69 (67.6%) participants, followed by mild anemia in 20 (19.6%) and severe anemia in 13 (12.8%) individuals. Amongst anemic males and females, moderate anemia was present in 14 (51.9%) and 55 (73.3%) participants, respectively. Mild and severe anemia was present in eight (29.6%) and five (18.5%) anemic males, and 12 (16.0%) and eight (10.7%) anemic females respectively. The association between the sex of the participant and the severity of anemia was not significant (p=0.123), as indicated in Table 2 . Among the 170 participants, 110 (64.7%) had consumed deworming tablets in the last six months, while 60 (35.3%) had not. As shown in Table 3 , we observed a slightly lower prevalence of anemia (n=65, 59.1%) in participants who had consumed deworming tablets as compared to those who had not consumed deworming tablets (n=37, 61.6%). The findings were not significant (p=0.74). Among 102 anemic participants, 65 (63.7%) had consumed deworming tablets in the last six months, while 37 (36.3%) had not. Among anemic individuals who had consumed deworming tablets, mild, moderate, and severe anemia was present in 13 (20.0%), 45 (69.2%), and seven (10.8%), respectively; in anemic individuals who had not taken deworming tablets, seven (18.9%), 24 (64.9%) and six (16.2%) had mild, moderate, and severe anemia respectively. Although the findings were not significant (p>0.05), severe anemia was more common in those who had not consumed deworming tablets in the last six months, as demonstrated in Table 4 . The present study revealed the overall prevalence of anemia in the adolescent age group to be 102 (60.0%), which is slightly higher compared to NFHS-5 data for rural areas of Uttar Pradesh. This study has shown a higher prevalence of anemia among females (n=75, 68.2%) compared to males (n=27, 45.0%). There was a significant association between the sex of the participant and the prevalence of anemia (p<0.05). Similarly, NFHS-5 data for rural areas of Uttar Pradesh revealed a higher prevalence of anemia in non-pregnant females (15-49 years), i.e., 50.7% as compared to males (15-19 years), in whom the prevalence of anemia was 29.9%. Kishore et al.'s study conducted in Rishikesh (Uttarakhand) showed that the overall prevalence of anemia was 53.2%, out of which 45.1% were males and 54.6% were females. The increased prevalence of anemia in females can be due to increased physiological demand, menstrual blood loss, poor eating habits in females, as well as social neglect. In the present study, moderate anemia was the most common type, affecting 69 (67.6%) of the anemic individuals, whereas mild and severe anemia was present in 20 (19.6%) and 13 (12.8%), respectively. Similar findings were reported by Bajaj et al. from Pune, Maharashtra, where moderate anemia was the most prevalent form, affecting 55.0% of the study participants. This was followed by mild anemia at 40.7% and severe anemia at 4.3%. Among anemic males, mild, moderate, and severe anemia was present in eight (29.6%), 14 (51.9%), and five (18.5%), respectively; among anemic females, the prevalence was 12 (16.1%), 55 (73.3%), and eight (10.7%) respectively. Moderate anemia was more common in females, while mild and severe anemia was common in males. Kishore et al. reported that moderate and severe anemia were more common in females than males, whereas mild anemia was common in males. The reason for this prevalence of moderate and mild anemia could be that moderate anemia can go unnoticed for long periods with nonspecific symptoms like weakness and fatigue while severe anemia causes more pronounced symptoms like dyspnea on slight exertion or even at rest and hence is treated promptly in clinical settings. In this study, 110 (64.7%) children had consumed deworming tablets in the last six months and they exhibited a slightly lower prevalence of anemia: (n=65, 59.1%) as compared to those who had not consumed (n=37, 61.6%). However, the difference was not statistically significant (p>0.05). This shows that deworming alone is not effective in anemia prevention. The most probable reasons for ineffective deworming could be low nutritional intake, increased iron demand in the adolescent age group, recurrent worm infestations in rural settings, and poor service utilization. Scott et al. reported that 25.8% of children in their study had consumed deworming tablets in the last six months and that deworming tablets are an effective intervention in anemia prevention in southern India (OR: 1.77). Kamble et al. reported that anemia prevalence among adolescent girls who received albendazole tablets for deworming on National Deworming Day was significantly lower as compared to those who did not undergo deworming (p<0.001). Alamneh et al.'s study from Ethiopia among different age groups (children aged 6-59 months) reported that deworming was effective in reducing moderate anemia by 21.0%, but its effect on mild and severe anemia was not significant. Severe anemia was reported in a lower proportion of participants (n=7, 10.8%) who had a history of deworming in the last six months compared to those who had not undergone deworming (n=6, 16.2%), although the difference was not significant (p>0.05). There is a paucity of research on the effects of deworming in reducing the severity of anemia. The present study included adolescent beneficiaries (aged 10-19 years) of the Anemia Mukt Bharat program in a rural area of Meerut, Uttar Pradesh. About two-thirds of the study population were females. More than half of the study participants were anemic, and anemia was significantly higher in females compared to males. This can be attributed to increased menstrual blood loss, selective food habits among females, and, perhaps, ignorance due to gender discrimination. Among anemic participants, more than two-thirds were moderately anemic. The association between deworming and the prevalence of anemia was not significant. Among the anemic population, severe anemia was more common in those who had not consumed deworming tablets, but the results were not significant. Deworming alone can neither effectively prevent anemia nor decrease its severity significantly. Dietary patterns, nutritional deficiencies, vitamin C supplementation, and other non-nutritional causes of anemia need to be addressed promptly and extensively. | Review | biomedical | en | 0.999999 |
PMC11698479 | Diabetes mellitus (DM) is a chronic endocrine disorder caused by insulin deficiency or resistance. Insulin is a hormone produced by β-cells in the pancreas that helps maintain normal blood glucose levels . A large portion of the world's population is affected by diabetes and its associated complications. Dental problems should not be ignored in patients with DM. Poor oral health can be a major barrier to maintaining normal blood glucose levels in diabetic patients. DM is a metabolic disorder with long-term complications, often accompanied by comorbidities such as obesity, hypertension, cardiovascular problems, and renal diseases. Emergencies such as cardiac failure, cerebrovascular disease, pneumonia, and infections are common among diabetic patients and can be fatal if left untreated. Dental practitioners can assist in diagnosing DM based on early oral symptoms. Preventative measures to control metabolic health and maintain oral health pave the way for a healthier lifestyle. Therefore, dental practitioners play a significant role in improving the quality of life for diabetic patients . According to a World Health Organization (WHO) report, more than 400 million people are affected by this chronic disease, with global prevalence increasing each year. Diabetes has become a major cause of blindness, kidney failure, heart attacks, stroke, and lower limb amputation. In 2016, it was identified as the seventh leading cause of death by the WHO . Consequently, dental patients with DM require comprehensive care to avoid disruptions in metabolic homeostasis. Both diagnosed and undiagnosed diabetic patients may seek various dental treatments. Proper precautions are essential while treating diabetic patients to avoid life-threatening risks . Therefore, clinicians must have a thorough understanding of the symptoms and management strategies for diabetes to minimize the risks associated with dental treatment . Additionally, patients need to be educated on diabetes-related oral health issues and the importance of maintaining oral hygiene through preventative care routines to minimize dental complications . A quantitative study was conducted at the College of Dentistry, Jouf University, involving dental students from the third to fifth year and interns working in the clinical setting. A self-reported survey was used to assess the knowledge and attitudes of these participants regarding the management of diabetic patients in dental clinics. The survey instrument was developed based on validated tools and relevant literature in diabetes management within dental settings to ensure content validity. Expert input from faculty members specializing in diabetes management and dental education was incorporated to finalize the survey questions. To refine the instrument further, a pilot test was conducted with a small subset of dental students and interns. Also, Zα/2 is the critical value of the normal distribution at α/2 (for a confidence level of 95%, α is 0.05, and the critical value is 1.96), MOE is the margin of error (5%), and p is the sample proportion (50%). The Microsoft Excel data (Microsoft Corporation, Redmond, WA, USA) collected from the Google spreadsheet was exported to SPSS Statistics version 21 and encoded. Initially, a Wilk-Shapiro test was performed to assess the normality assumption of the collected data. A skewed distribution was observed, and non-parametric tests, Spearman's test, Mann-Whitney test, and Kruskal-Wallis test were performed based on the independent variables considered for the study. A p-value of <0.05 was considered statistically significant. A significant positive correlation was observed between the knowledge and attitude of the dental students (Table 4 ). As knowledge increased, attitudes also improved. The open-ended inquiries revealed participants' knowledge and attitudes toward diabetic patient management techniques. Most participants suggested scheduling early appointments, while a few recommended providing proper counseling before treatment to alleviate diabetic patients' anxiety about dental procedures. Additionally, participants were aware of the possible complications associated with diabetic patients during dental treatment, including the risks of syncope, hypoglycemia, and hyperglycemia. However, most considered delayed healing to be an immediate complication during dental treatment. Most importantly, many students were unaware of how to manage medical emergencies involving diabetic patients during dental treatment. However, those who had previously attended educational programs on diabetic patient management provided appropriate responses. They suggested stopping dental treatment if symptoms of hypoglycaemic shock occurred. They also recommended checking the blood glucose level and administering fast-acting carbohydrates if necessary to prevent further complications. If the patient was unconscious or had difficulty swallowing or breathing, they advised seeking immediate medical attention and administering subcutaneous glucagon. Oral health problems, such as periodontal diseases, dental caries, and xerostomia, are common in people with uncontrolled or poorly controlled diabetes. Diabetes and periodontal disease have a bidirectional relationship: periodontal disease can worsen glycemic control, and poorly managed diabetes can lead to periodontitis. Additionally, uncontrolled diabetes and certain medications used for diabetes management can cause xerostomia by impairing the normal function of the salivary glands. Elevated blood glucose levels also increase glucose in the saliva, creating an environment that promotes microbial accumulation and biofilm formation on the tooth surface, thereby increasing the risk of dental caries and gum disease. Therefore, it is essential to educate diabetic patients on maintaining oral hygiene. Dental practitioners should have a thorough understanding of diabetes-related oral health problems and risk factors . A study conducted among future dentists at Jouf University, Saudi Arabia, observed a positive correlation between knowledge and attitude toward diabetic patient management. Similar results were found in studies among oral health professionals in Australia and India . As shown in Table 1 , participants' age, gender, and level of education were considered to assess their knowledge and attitudes toward diabetic patient management in dental clinics. Age and gender differences did not affect participants' knowledge. However, academic level showed a significant variation in the knowledge score, with interns having lower knowledge compared to other study groups (Table 2 ). This may be due to the structure of the dental curriculum, where medical courses are integrated in the initial years to build a strong foundation for understanding diabetes and its systemic implications. Additionally, problem-based learning sessions are incorporated to enhance the application of knowledge in clinical contexts. As students progress, particularly in the later years, the focus shifts toward dental specialties, which may reduce the emphasis on medical subjects. In the fifth year, the "Problem Solving in Dentistry" course specifically addresses the management of medically compromised patients, reinforcing both practical and theoretical knowledge relevant to dental practice. These findings align with those of Komerik et al., who reported that dental students showed negative enthusiasm toward learning medical subjects in their curriculum. The dental students in their study felt that certain course content, including aspects of the human body unrelated to dentistry, was unnecessary and that they quickly forgot this information . Furthermore, attitudes toward managing diabetic dental patients varied based on gender and academic level. As shown in Figure 3 , males displayed more positive attitudes than females despite there being no significant difference in knowledge. This difference may be attributed to higher levels of confidence in managing diabetic patients, which could result from prior exposure to and participation in workshops specifically focused on diabetic patient management. These workshops likely provided them with a stronger foundation of both knowledge and practical experience, enhancing their comfort levels and promoting a more proactive approach to patient care. A similar trend in knowledge scores was observed for attitudes based on academic level . The least positive attitude was observed among interns (Table 3 ), which is consistent with Mussalo et al.'s findings. They reported that students' interest in biomedical sciences declined as they progressed through their dental program and entered the clinical phase . The current study supports these findings, confirming that positive attitudes toward managing patients in clinical settings are closely linked to both knowledge and confidence levels . The responses related to participants' knowledge and attitudes highlighted the need for educational programs on diabetic patient management for dental students. Although some participants demonstrated basic knowledge of diabetes, most were unaware of the proper management of diabetic emergencies during dental treatment. Clinicians must be efficient in managing hypoglycemic shock, which is associated with symptoms such as confusion, shakiness, tremors, agitation, anxiety, sweating, dizziness, tachycardia, a feeling of impending doom, seizures, and loss of consciousness . If any of these symptoms arise, it is advisable to stop dental treatment immediately. Administering 15 grams of rapid-acting carbohydrates is recommended to prevent further complications. Additionally, monitoring glucose levels is essential to determine if the patient requires further treatment. If the patient is unconscious or has difficulty swallowing or breathing, immediate medical attention should be sought, and glucagon should be administered subcutaneously . The participants in the current study were not confident in recognizing hypoglycemic shock symptoms and indicated that they would seek advice from supervisors, reflecting their anxiety about managing such patients in the dental clinic. However, students who had attended a previous workshop on diabetic patient management showed a more positive attitude toward managing medical emergencies. The participants in this study represent the future of dental practice. Therefore, the findings suggest that special training in diabetic patient management should be provided to dental students during their internship years. To build on these findings, it is recommended to integrate hands-on workshops, role-playing scenarios, and practical resources such as clinical posters or digital guides into the dental curriculum to strengthen the application of skills. Additionally, conducting regular evaluations, including periodic surveys, can help assess retention and ensure the ongoing effectiveness of the training. This would help dental graduates recall the basic medical knowledge learned in the early years of their graduate programs and apply that knowledge in practice. The study found that education level significantly influenced dental students' knowledge and attitudes toward managing diabetic patients, with fifth-year students performing better than interns. Gender differences impacted attitudes, though knowledge remained unaffected, while age showed no influence on either. A positive correlation between knowledge and attitude was observed, particularly among students with prior training. These findings highlight the need for specialized training, interactive workshops, regular assessments, and resources like posters to enhance students' confidence and preparedness in managing diabetic patients. | Review | biomedical | en | 0.999996 |
PMC11698481 | Hemosuccus pancreaticus (HP) is a rare yet recognized cause of upper gastrointestinal (GI) bleeding, with delayed diagnosis often contributing to significant mortality . Chronic pancreatitis is the most common risk factor (60-65% of cases), while less common causes include peripancreatic arterial pseudoaneurysms (10-20%), pancreatic pseudocysts (10-20%), and acute pancreatitis (5-10%) . The pathophysiology of HP involves chronic enzymatic autodigestion of peripancreatic vessels, which leads to aneurysmal formation and rupture, resulting in bleeding through the pancreatic duct . Rupture often involves major peripancreatic arteries, including the splenic, pancreaticoduodenal, and gastroduodenal arteries . Notably, HP accounts for only 1 in 1,500 cases of upper gastrointestinal bleeds . In most cases, bleeding is usually intermittent and presents as abdominal pain, GI bleeding, and anemia, but severe cases can cause hemorrhagic shock and rapid bleeding into the duodenum, with mortality rates around 10%, higher in males (7:1 ratio) . Diagnosis is challenging due to coexisting conditions common in GI bleeding, such as cirrhosis, critical illness, and alcohol use . Despite the low index of suspicion for HP due to its rarity, HP should be considered in individuals with pancreatic disease and unexplained or recurrent GI bleeding . Acute GI hemorrhage should prompt suspicion for HP in cases with peripancreatic arterial pseudoaneurysms, pancreatic hematomas, or acute necrotizing pancreatitis, with alcohol use adding further complexity given its overlap with other etiologies of upper GI bleeding . We present a case of HP and hemorrhagic shock in a 34-year-old with acute necrotizing pancreatitis and decompensated alcoholic cirrhosis. Initially, the patient presented with altered mental status and melena. Computed tomography (CT) angiography revealed a pancreatic tail hematoma and pancreatic head calcification, without evidence of aneurysm or necrosis. After several days, further imaging revealed a peripancreatic pseudoaneurysm and enlarging pancreatic tail hematoma amid acute onset necrotizing pancreatitis. Esophagogastroduodenoscopy (EGD) showed large clots and bleeding through the ampullary duct, leading to hemorrhagic shock and a worsening prognosis. This case highlights HP as a severe complication of acute necrotizing pancreatitis, demonstrating the diagnostic challenges in patients with cirrhosis and GI bleeding, as well as the importance of a multidisciplinary approach in managing HP-associated hemorrhagic shock. The case also addresses the prognostic implications in patients with pancreatitis-related vascular complications, particularly in those with end-stage liver disease. A 34-year-old male with a history of alcoholic cirrhosis (MELD 3.0 score of 30) presented after being found unconscious following heavy alcohol use, along with intermittent episodes of melena. The initial laboratory evaluation revealed elevated aspartate aminotransferase (AST), alanine transaminase (ALT), and total bilirubin with associated leukocytosis, normocytic anemia, elevated lipase, lactic acidosis, and acute kidney injury. On exam, he had encephalopathy, jaundice, scleral icterus, and mild, non-tender abdominal distention. A CT angiogram of the abdomen revealed a 6.2 cm hematoma anterior to the pancreatic tail, extending into the splenic hilum and abutting the greater curvature of the stomach, along with possible pancreatitis and splenic vein thrombosis . He required multiple packed red blood cell (pRBC) transfusions for suspected ongoing GI bleeding and treatment for alcohol withdrawal. Initial EGD demonstrated LA grade D esophagitis, no esophageal varices, mild portal hypertensive gastropathy, and mild extrinsic compression of the greater gastric curvature from the hematoma. Magnetic resonance cholangiopancreatography (MRCP) revealed a 6.9 cm pancreatic tail mass with possible pancreatic duct dilation. A follow-up CT confirmed a 6.5 cm pancreatic tail hematoma, an 18 mm splenic artery pseudoaneurysm, nonocclusive splenic vein thrombosis, and suspicion of necrotizing pancreatitis. Antibiotics were initially withheld due to the absence of signs of active infection. A repeat CT on day 13 of hospitalization confirmed necrotizing pancreatitis, an enlarged 2.5 cm splenic artery pseudoaneurysm, and a significantly increased pancreatic tail hematoma (10 cm x 19 cm x 13 cm) . The patient developed worsening ascites, confirmed as portal hypertensive ascites without spontaneous bacterial peritonitis (SBP), followed by an episode of hematochezia. Urgent CT angiography showed no active extravasation but noted a stable lesser sac hematoma and persistence of splenic artery pseudoaneurysm. On day 18 of hospitalization, during preparation for a colonoscopy, the patient developed fever, tachycardia, and hypotension, requiring intensive care unit (ICU) transfer and vasopressor support. He was started on vancomycin and piperacillin-tazobactam for suspected septic shock secondary to necrotizing pancreatitis, alongside hemorrhagic shock from GI bleeding. Repeat EGD revealed a small distal esophageal varix, moderate portal hypertensive gastropathy, and blood with clots in the duodenum. A side-viewing duodenoscope revealed a large clot extruding from the ampulla of Vater, consistent with HP . Compression of the greater curvature of the stomach by the expanding pancreatic tail hematoma was also noted. Interventional radiology (IR) was consulted and diagnostic angiography was performed on the peripancreatic vasculature, identifying a pseudoaneurysm involving an unnamed pancreatic perforator artery from the splenic artery, anastomosed with the pancreatica magna artery. Initial attempts to embolize were unsuccessful due to prominent arterial spasms; however, a repeat attempt showed active arterial extravasation seen from a small pancreatic arterial branch that was successfully treated with coil embolization . The antibiotic regimen was escalated to meropenem for three days for suspected ongoing infection. Despite the resolution of septic and hemorrhagic shock, persistent hematochezia necessitated near-daily pRBC transfusions. A repeat CT angiography revealed a 7 mm residual pseudoaneurysm. IR advised against repeat angiography or embolization due to the development of multiple aneurysms. General surgery was consulted, and he was deemed a poor surgical candidate due to his decompensated cirrhosis. Given the patient's deterioration and risks of complications, a goals-of-care discussion held with the patient and family led to the decision to not escalate medical care. This case presents a unique presentation of HP secondary to acute necrotizing pancreatitis, resulting in hemorrhagic shock in a patient with decompensated cirrhosis. HP with coexisting cirrhosis is exceptionally rare, with only a few reports documenting this combination . In a retrospective study at a single center, only 1 out of 17 patients treated for HP had cirrhosis . Most documented cases attribute intermittent GI bleeding in HP to a pseudoaneurysm from chronic pancreatitis, with rare instances of severe bleeding or hemorrhagic shock . Notably, no previous reports associate HP with acute necrotizing pancreatitis, making this case significant as the first to link HP, severe hemorrhagic shock, and death with end-stage liver disease. Our patient’s severity was compounded by a pancreatic pseudocyst, arterial pseudoaneurysm, and a pancreatic tail hematoma, emphasizing the complexity of managing HP in patients with cirrhosis, where treatment options are often limited. This case contributes to the growing literature on HP, emphasizing the need to promptly recognize and manage comorbidities to enable timely diagnosis and intervention, potentially preventing fatal outcomes. Pathophysiological links between hemosuccus pancreaticus, cirrhosis, and acute necrotizing pancreatitis In this case, the patient’s decompensated cirrhosis (MELD 30, Child Pugh C) significantly worsened the prognosis, as cirrhosis-mediated portal hypertension and coagulopathy amplified the risk of severe hemorrhage . In cirrhosis, elevated pressures in the portal venous system, including the splenic and mesenteric veins, drive collateral formation, indirectly promoting the formation of pseudoaneurysms . Splenic vein congestion or thrombosis increases stress on local vasculature, including the splenic artery . Splenomegaly, resulting from venous congestion, places extrinsic pressure on the splenic artery, raising the likelihood of aneurysms, as shown in studies linking enlargement of the splenic vein with splenic artery aneurysm . Chronic elevation in portal pressure, leading to the formation of collateral circulation, further increases hemodynamic demand on the arterial system. This compensatory hyperdynamic circulation places increased strain on arterial walls, resulting in localized vessel weakening and dilation . Cirrhosis-associated coagulopathy also worsened the patient’s condition, as impaired clotting factor synthesis, vitamin K deficiency, and platelet sequestration from splenomegaly elevated the risk of bleeding . Overactive fibrinolytic pathways, in addition to vessel fragility, increase susceptibility to severe hemorrhage in acute necrotizing pancreatitis . Vascular involvement occurs early in acute pancreatitis in up to 25% of cases, increasing to 43% of cases in necrotizing pancreatitis, manifesting as venous phlebitis, arteritis, venous thrombosis, or pseudoaneurysms . Hemorrhage occurs in 3-6% of necrotizing pancreatitis cases, with over half due to pseudoaneurysmal rupture . In this case, we propose that acute necrotizing pancreatitis triggered a rupture in a pancreatic arterial branch of the splenic artery, leading to hemorrhagic via HP. Bleeding typically occurs in the retroperitoneum, but in cases like this, blood may flow through the pancreatic duct into the gastrointestinal tract . Necrotizing pancreatitis results in HP through mechanisms such as vascular erosion, tissue necrosis, and pseudocyst formation . Pancreatic enzymes induce autodigestion, eroding vessels and promoting pseudoaneurysms, which develop in approximately 4% of necrotizing pancreatitis cases, with a mortality of up to 14% . The splenic artery is most affected (38%), followed by the gastroduodenal and pancreaticoduodenal arteries . Pancreatic tissue necrosis compromises the integrity of surrounding vasculature, facilitating hemorrhage in the acute setting . Pancreatic pseudocysts may also exacerbate HP by compressing and rupturing nearby vessels, where compromised vasculature leads to rapid GI hemorrhage . HP is a challenging diagnosis due to its anatomic complexity and poor visualization of the pancreaticobiliary system during endoscopy . Intermittent bleeding further complicates detection, as endoscopy may fail to detect HP if active bleeding is absent . Studies report that upper endoscopy identifies HP in only ~30% of cases, with improved success using a side-viewing duodenoscope for ampulla visualization . Negative endoscopy does not rule out HP, as other causes of bleeding like hemobilia and ampullary tumors remain in the differential diagnosis . In our case, the initial EGD demonstrated esophagitis and portal hypertensive gastropathy, without ampullary bleeding. Subsequent imaging revealed a pseudoaneurysm and an enlarging pancreatic tail hematoma, leading to repeat endoscopy and observation of clots protruding from the ampulla using a side-viewing duodenoscope. Diagnosis often requires multi-modal imaging, with contrast-enhanced CT used to detect pseudoaneurysms, pseudocysts, and extravasation . Angiography of peri-pancreatic vessels enables both diagnosis and treatment through embolization . Angiographic embolization is currently the first-line treatment for HP from pseudoaneurysmal bleeds and has seen technical success rates of 97%, with long-term success rates around 82-88% . Technological advances have improved outcomes considerably over the past decade . In cases of concurrent cirrhosis, the management of HP is multidisciplinary, requiring collaboration amongst gastroenterology, interventional radiology, surgery, and critical care physicians. For initial stabilization, crystalloid fluids, blood transfusions, and vasopressors are crucial for achieving hemodynamic stability until definitive intervention is performed . In cirrhotic patients, management includes coagulopathy correction using vitamin K, fresh frozen plasma (FFP), and platelets . Vasoactive agents such as octreotide or terlipressin reduce portal pressure, alleviating strain on collateral vessels, while antibiotics prevent bacterial translocation and spontaneous bacterial peritonitis . When coil embolization is unsuccessful, surgical hemostasis may be necessary to prevent exsanguination, with procedural options including distal or central pancreatectomy, splenectomy, or aneurysm ligation, depending on the bleeding location . However, in cirrhotic patients with high Child-Pugh scores, surgical intervention has high operative mortality, especially in patients with Class C cirrhosis (up to 80%) . For this patient, distal pancreatectomy might have been an option if embolization had failed but was contraindicated due to advanced cirrhosis . Effective long-term management, which includes nutritional support, pancreatic enzyme replacement, and management of pancreatic pseudocysts or vascular abnormalities, remains critical for improving patient outcomes and long-term survival. Recent advancements in endoscopic technology have highlighted the utility of contrast-enhanced endoscopic ultrasound (EUS) for diagnosing and managing HP. EUS, particularly with contrast enhancement, provides a detailed view of vascular structures surrounding the pancreas, allowing for precise localization of pseudoaneurysms responsible for bleeding . While non-contrast EUS is valuable in delineating pancreatic anatomy, it lacks the sensitivity needed to identify small, bleeding culprit vessels within the pancreatic tissue . Contrast enhancement overcomes this limitation, making EUS a suitable option even for patients with renal impairment, as it avoids nephrotoxic contrast agents used in CT angiography. A recent case report describes successful EUS-guided treatment in two cases of HP using cyanoacrylate injection into the pseudoaneurysm, resulting in hemostasis and no recurrence over follow-up periods of three and six months . This outcome highlights the potential role of EUS not only for diagnosis but also as a minimally invasive therapeutic intervention. Although underreported, EUS-guided management of HP may emerge as a critical intervention for managing pseudoaneurysmal bleeding in HP, particularly when traditional imaging and treatments pose greater risks or fail to control bleeding effectively. To summarize, acute necrotizing pancreatitis can lead to severe vascular complications, including pseudoaneurysmal rupture, ultimately resulting in HP. The mortality associated with pseudoaneurysmal rupture is high in cases of massive hemorrhage and hypovolemic shock. In patients with decompensated cirrhosis, underlying coagulopathy and hemodynamic changes exacerbate bleeding and further worsen outcomes. Therefore, early identification and intervention through endovascular coiling or surgical intervention is paramount for survival, alongside fluid resuscitation, and blood transfusion. While coil embolization remains the gold standard for endovascular intervention, newer therapies such as contrast-enhanced endoscopic ultrasound (EUS) are emerging as promising diagnostic and therapeutic tools. Ultimately, a multidisciplinary approach is essential for improving outcomes, particularly in navigating complex cases. | Review | biomedical | en | 0.999994 |
PMC11698532 | Glioblastoma is the most common primary malignant brain tumor, accounting for 48.6% of all malignant central nervous system tumors . Despite advancements in treatment over the past two decades, patient outcomes remain dismal, with a median overall survival (OS) of only 15 months following initial diagnosis . The standard treatment for glioblastoma includes maximal safe resection followed by radiotherapy with concomitant temozolomide . Typically, external beam radiotherapy (EBRT) is administered five days a week over six weeks, delivering a total dose of 60 Gy in 30 fractions. However, even with aggressive adjuvant chemoradiotherapy, tumor recurrence is nearly universal. Salvage treatment options for recurrent disease remain limited, with bevacizumab being the primary standard option. Approximately 85% of recurrences occur within the margins of the prior resection cavity , making intracavitary adjuvant brachytherapy an attractive strategy. This approach offers potential advantages, including the immediate initiation of radiation therapy, the elimination of multiple visits required for EBRT, and intensified radiation delivery to areas most prone to disease progression. Intracranial brachytherapy has been previously investigated using iodine-125 (I-125), but it was associated with high rates of adverse radiation effects (14-33%), likely due to I-125’s long half-life of 59.4 days . GammaTile ® (GT) brachytherapy, incorporating Cesium-131 (Cs-131), may provide a better therapeutic ratio due to Cs-131’s shorter half-life of 9.7 days and higher dose rate of 0.342 Gy/hr . GT received FDA approval in 2018 for recurrent brain tumors and in 2020 for newly diagnosed malignant brain tumors . GT utilizes a bioresorbable collagen matrix embedded with four Cs-131 seeds. This design allows for precise dosimetry and delivers 88% of the therapeutic dose (80-120 Gy to the first few millimeters from the implantation site) within 30 days, and over 95% within six weeks (equivalent to four half-lives of Cs-131), during which the collagen matrix fully dissolves . This fixed-seed configuration offers more predictable radiation distribution compared to the earlier “loose-seed” implantation methods. While GT appears to reduce the incidence of adverse radiation effects , its impact on improving progression-free survival and OS in recurrent glioblastoma patients remains an active area of investigation. Furthermore, survival outcomes in patients treated with repeat brachytherapy are rarely reported in the literature. This report presents a case of two separate GT implantations in the management of multiple recurrent glioblastoma. The patient achieved more than three years of survival from the initial diagnosis while maintaining good functional status, without evident treatment-related toxicity or surgical wound complications. The patient was a 62-year-old right-handed male who presented to the emergency department of an outside hospital with several weeks of worsening headaches and gait instability. MRI with and without contrast revealed a large right temporal mass accompanied by significant vasogenic edema . During that hospital admission in November 2020, the patient underwent partial resection of the mass with Gliadel wafer implantation. Histopathological analysis of the tumor sample confirmed a high-grade glioma consistent with glioblastoma, characterized as IDH-wild type, MGMT-unmethylated, and BRAF V600E-mutated. Following the resection, the patient received standard-of-care treatment at our institution, consisting of temozolomide chemotherapy (140 mg) in combination with radiation therapy (60 Gy in 30 fractions). Four months after the initial resection and following one cycle of pembrolizumab, the patient presented with blurry vision, and MRI confirmed tumor recurrence . Given his relatively good performance status, the patient was offered and underwent repeat resection, with placement of seven GTs and a prescribed dose of 60 Gy to a depth of 0.5 cm. Postoperative MRI showed near-total resection of the previously noted enhancing lesion. Representative dosimetric images are presented in Figure 5 . The patient was discharged home a few days later without postoperative complications or the need for steroids. Final surgical pathology confirmed a diagnosis of recurrent glioblastoma. However, three days after discharge, the patient returned to the emergency department with low-grade fevers and fatigue. A CT head was negative for acute findings. The patient was discharged with symptomatic management and presented to the clinic three days later with persistent fevers, mild headache, and neck pain. Lumbar puncture revealed a normal opening pressure, but elevated protein and white blood cell count (total nucleated cells of 1,159 per µL, with normal percentages of neutrophils and lymphocytes) and decreased glucose. A diagnosis of aseptic meningitis was made, and the patient was treated with dexamethasone, resulting in symptomatic improvement. The patient subsequently restarted temozolomide at a dose of 380 mg daily. The patient’s clinical status and surveillance MRIs remained stable until December 2022 (25 months following the initial resection), during which time temozolomide had been discontinued. The patient then reported a new left-sided peripheral visual field deficit, and MRI findings were consistent with tumor progression. A third resection was performed, followed by a second GT implantation involving eight tiles, with a prescribed dose of 60 Gy to a depth of 0.5 cm. A cumulative dose, including external beam treatment and the two GT implants, was calculated in the treatment planning system. The optic apparatus (both nerves and chiasm) received a maximum point dose of 81 Gy, with a 0.1 cc dose of 74 Gy. The brainstem received a maximum point dose of 141 Gy, and the doses of 0.1 cc, 1 cc, and 10 cc were 133 Gy, 114 Gy, and 49 Gy, respectively. These accumulated doses represent algebraic sums, with no correction for biologically equivalent doses. Vemurafenib was initially started but discontinued shortly afterward due to poor tolerance and negative BRAF V600E testing on the updated surgical specimen. The patient was then started on bevacizumab and lomustine approximately three months following his third resection. The patient was later hospitalized for a perforated colon and entered a period of observation, during which chemotherapy treatment was paused. A surveillance MRI in December 2023 (37 months following the initial resection) showed enhancement along the posterior wall and right ventricular margin of the prior resection cavity . At the time of the last follow-up in our clinic, the patient had a good neurological status but elected to enter hospice care in January 2024 due to his refusal to undergo further abdominal surgery. The use of brachytherapy in glioblastoma patients presents a potential alternative or complement to existing therapeutic approaches, such as EBRT and radiosurgery, particularly in cases of recurrent disease. To our knowledge, this report represents the first published case of a patient treated with two separate GT implants. The patient survived for 10 months following the second brachytherapy treatment and approximately 37 months from the initial diagnosis. Brachytherapy has an established role in the treatment of other malignancies, such as cervical, uterine, and prostate cancers, among others . However, its effectiveness in treating intracranial neoplasms remains less clear. Two prior randomized controlled trials did not show significant survival benefits when I-125 implants were combined with EBRT or with EBRT and carmustine in patients with high-grade glioma. These trials were conducted before the routine use of neuronavigation to aid in achieving gross-total resection and before the widespread use of sensitizing chemotherapy . Additionally, there may be advantages to using Cesium-containing GT compared to traditional iodine-based seeds, given its higher dose rates and more predictable dosimetry. While GT has shown promise in treating recurrent meningiomas and metastatic brain tumors , its use in glioblastoma cases is less well documented. In a study of 22 patients with IDH-wild type recurrent glioblastoma treated with GT, the median OS for the treatment group was 24.4 months, compared to 17 months in the control group who did not receive GT implantation . Importantly, the study reported no differences in length of hospital stay or postoperative complication rates between the two groups . Another report involving 20 patients with recurrent glioblastoma found the median post-recurrence OS to be nine months for those treated with Cs-131, with no cases of radiation necrosis in patients treated concurrently with bevacizumab . However, it is important to recognize that the patients eligible for intensified therapy may have had clinical characteristics that predisposed them to improved survival, and thus, selection bias must be considered when interpreting these results. Ongoing trials are expected to provide further insight into the role of GT in the treatment of newly diagnosed and recurrent glioblastoma. Two randomized trials, the GammaTile and Stupp in Newly Diagnosed GBM (GESTALT) trial and NCT04427384, are currently enrolling patients. The eventual clinical decline of our patient was likely multifactorial, including the challenges of multiple recurrences requiring repeat resections and other health complications, which ultimately led to the family’s decision to pursue hospice care. Nonetheless, survival of 37 months in a case of MGMT-unmethylated glioblastoma is significantly above the expected median survival for this patient population. Further research is needed to better understand the role of GT brachytherapy in recurrent high-grade glioma and whether repeat implantation is a safe and viable treatment option. Despite advances in chemotherapy, EBRT, and tumor-treating fields over the past two decades, outcomes for patients with glioblastoma remain poor, and there is currently no established standard of care for recurrent disease. Repeat resection and GT brachytherapy offer a potential treatment option for patients with recurrent high-grade glioma, and studies are ongoing to evaluate its use in newly diagnosed glioblastoma. This report highlights a case in which a patient diagnosed with glioblastoma underwent two separate GT implantations during their treatment course and survived for 37 months without evident treatment-related toxicity. | Review | biomedical | en | 0.999997 |
PMC11698533 | Until recently, it was common practice to categorize musculoskeletal disorders as a single entity . As our knowledge of the human body has improved, etiopathogenetic differences between disorders affecting various components of the musculoskeletal structure have emerged . Researchers used to focus on the physiology and pathology of certain muscle components and the therapeutic interventions targeting them . However, research results from the past 20-30 years suggest it is a mistake to treat contractile muscle tissue as a standalone entity separate from connective tissue, even if the relationship is not yet clear . Recent findings indicate that what has traditionally been identified as muscle is, in fact, largely composed of connective tissue, which we now refer to as fascia, and which plays a fundamental role . As stated in a recent article , an examination of the literature available on PubMed shows that the earliest mention of fascia in the medical and clinical field dates back to 1814 , while the term "fasciæ" appears in a journal from 1824 . According to the American Heritage Stedman’s Medical Dictionary , fascia is defined as "a sheet or band of fibrous connective tissue enveloping, separating, or binding together muscles, organs, and other soft structures of the body." Therefore, only well-defined fibrous connective tissue layers should properly be referred to as fascia, and it is inaccurate to use this term to describe all connective tissues in the body. Fascia is a viscoelastic tissue that forms an uninterrupted, three-dimensional collagen matrix . This tissue permeates the entire body, surrounding, supporting, protecting, connecting, and dividing the multiple muscular and visceral components of the organism . Fascia performs several physiological and functional roles related to joint stability, general movement coordination, proprioception, and nociception . Most importantly, it is responsible for transmitting mechanical forces (mechanotransduction) . Due to its presence in every tissue at multiple hierarchical levels, fascia represents the main structural interconnective element, both externally and internally, between the different constituents of the human body. Huijing et al. found that only 70% of muscle tension is transferred through tendons, which confirms their mechanical role. However, the remaining 30% of muscle force is conveyed to the connective tissue surrounding muscles, underscoring the deep fasciae's function in coordinating agonist, antagonist, and synergistic muscles at the peripheral level. In the literature, various classifications of fascia exist . For example, in an attempt to organize the nomenclature for fascial structures provided by the Federative International Committee on Anatomical Terminology (FICAT) , a functional classification system was developed that includes four categories of fascia: connecting, fascicular, compressive, and separating fascia. Each category was developed from descriptions in the literature on macroscopic anatomy, histology, and biomechanics; the category names reflect the function of the fascia. More recently, the Foundation of Osteopathic Research and Clinical Endorsement (FORCE), an organization that brings together various scientific figures from a multidisciplinary perspective, tried to find a common nomenclature that can be shared, starting from the scientific notions currently available . Additionally, the Fascia Nomenclature Committee has proposed the following definition for the term fascia: "A fascia is a sheath, a sheet, or any other dissectible aggregations of connective tissue that forms beneath the skin to attach, enclose, and separate muscles and other internal organs” . Instead, the final definition of the Fascial System proposed by a subcommittee of five members from the Fascia Nomenclature Committee is: "The fascial system consists of the three-dimensional continuum of soft, collagen-containing, loose and dense fibrous connective tissues that permeate the body. It incorporates elements such as adipose tissue, adventitiae and neurovascular sheaths, aponeuroses, deep and superficial fasciae, epineurium, joint capsules, ligaments, membranes, meninges, myofascial expansions, periostea, retinacula, septa, tendons, visceral fasciae, and all the intramuscular and intermuscular connective tissues including endo-/peri-/epimysium” . For the purpose of this work, we will consider the simplified subdivision by Stecco et al. , an Italian researcher who has significantly contributed to the study of fascial systems. He classifies the simple fascial component involved in mechanotransduction into three main areas: (a) Superficial: This fascial layer is closest to the body’s surface and includes tissues such as the superficial fascia of the body and the subcutaneous fascia. (b) Deep: This fascial layer is located deeper than the first layer and includes structures such as the deep muscle fascia and the epimysial fascia that envelop the muscles. (c) Visceral: This is the innermost of the three fascial layers and surrounds the internal organs, providing structural support and facilitating organ movements within the body. The deep fascia is a continuous layer running from the trunk through to the upper and lower limbs and is considered the key element for transmitting loads in parallel, bypassing the joints . The focus of this work will primarily be on the fascia involved in the transmission of tension essential for body movement, such as that produced by muscles, as it provides the insertion and/or origin for all muscle fibers, with an estimated percentage of approximately 30% . Fascia is an uninterrupted structure composed of layers of dense connective tissue (type I and III collagen) interfaced with loose connective tissue. It exhibits the typical properties of viscoelastic structures . In addition to fibers and cells, connective tissue systems also comprise an extracellular matrix (ECM) . This ECM surrounds cells, protects them, holds them together, and also provides physical and biochemical signals that play a key regulatory role in determining the shape and activities of a cell. It can take various forms in different tissues but is generally composed of similar fibrous (non-globular) macromolecules secreted into the extracellular space, where they self-assemble. The macromolecules that make up the ECM are mainly glycoproteins; in animals, collagen is by far the most abundant protein. In addition to collagen, the matrix also contains proteoglycans, fibronectin, and laminin. According to Bordoni et al. , it is important to include the liquid component (liquid fascia) as part of the fascia's composition. The "motor unit" or "muscle" has traditionally been seen as the fundamental unit in movement control. However, recent research has highlighted the significant interaction between muscle fibers and intramuscular connective tissue, as well as between muscles and fasciae, indicating that muscles alone cannot be regarded as the sole organizers of movement. Additionally, muscle innervation and blood supply are closely linked with intramuscular connective tissue . This realization led Luigi Stecco, in 2002 , to coin the term "myofascial unit" to emphasize the interconnected relationship, both anatomically and functionally, between fascia, muscles, and their associated structures. The stiffening of connective tissue is a feature of various painful syndromes . Indeed, localized increases in fascial rigidity are observed in certain "muscular" contractures - an abnormal increase in the passive stiffness of muscles - which are marked by dense connective tissue abundant in myofibroblasts and often linked to ongoing inflammatory processes . Some studies have clarified the mechanisms by which stretching, applied to conditions of myofascial stiffening, can improve the range of motion (ROM) and reduce pain, as reported by a review study . The painful syndromes most frequently associated with the stiffening of localized connective tissue include compartment syndrome of the lower leg induced by exercise, runner's knee (iliotibial friction band), tennis/golfer's elbow, frozen shoulder, plantar fasciitis, and Dupuytren's disease . Other examples of conditions caused by localized stiffness that can manifest even without pain include scoliosis and high-arched feet. General conditions of increased stiffness include spastic paralysis such as after a stroke, neuromuscular diseases such as Parkinson's disease, and autoimmune diseases such as rheumatoid arthritis or scleroderma. In these pathological conditions, histological studies reveal accumulations of fibroblasts or contractile myofibroblasts . Although hypotheses have been proposed, it is still not entirely clear if and how these cells contribute to the development of connective tissue-induced contractures . Recent findings also indicate that the fascial epimysium is pivotal in the pathogenesis of delayed onset muscle soreness (DOMS), which occurs following intense physical activity without adequate preparation . In addition, increased ECM density may contribute to myofascial pain . Painful contractures are especially associated with an increase in fascial tissue thickness, resulting in increased relative stiffness . It has also been suggested that nonspecific back pain may be mediated, at least in part, by fascial structures ; the thoracolumbar fascia, in particular, absorbs a significant portion of the mechanical force transmitted during lumbar flexion and is rich in nociceptive nerve endings . Tears, microlesions, and mechanical irritations of the thoracolumbar fascia can cause malfunctions and painful contractures. The same applies to the musculo-connective structures of the hip, where a significant number of nerve endings have been found not only in the skin (64.0 ± 5.2/cm 2 ), where they are most concentrated, but also in the subdermal tissue (24.0 ± 1.4/cm 2 ), superficial fascia (33.0 ± 2.5/cm 2 ), and deep fascia (19.0 ± 5.0/cm 2 ) . It is interesting to note that in men with chronic back pain, there is a tendency toward increased thickness of the thoracolumbar fascia compared with healthy subjects . Furthermore, a reduction in shearing movement, or the ability to glide during passive lumbar flexion, has been documented in this fascial structure in relation to the underlying musculature in both sexes . According to Stecco et al. , one of the issues with fascial system gliding, which causes densification and pain, is partly due to an alteration in the production of hyaluronic acid, an important lubricant located between fascial layers. Although experts in the field have not reached a consensus on the etiology and pathogenesis of myofascial pain syndrome , primarily due to the lack of specific laboratory markers and imaging evidence that would enable a unified diagnostic criterion, in general, the term myofascial pain syndrome describes sensory, motor, and autonomic symptoms attributed to myofascial trigger points (MTrPs) . MTrPs, which consist of numerous contraction knots, are defined as highly sensitive areas within taut, discrete bands of hardened muscle, leading to localized and referred pain along with other symptoms. Each contraction knot appears as a section of muscle fiber characterized by markedly contracted sarcomeres and an increased fiber diameter. The integrated MTrP hypothesis suggests that, in cases of myofascial pain, motor endplates release excessive acetylcholine, histopathologically indicated by shortened sarcomeres . These zones of intense focal sarcomere contraction have been documented in both animals and humans. Although MTrPs are thought to be quite prevalent, there is limited quality literature available on their exact prevalence . MTrPs can be divided into active or latent types. An active MTrP causes a clinical pain disturbance. It is always tender and produces recognizable pain upon compression. It inhibits the full extension of the muscle, weakens the muscle, and mediates a local contraction response of muscle fibers when adequately stimulated . A latent MTrP is clinically asymptomatic and only painful when palpated with some force. A latent MTrP may have all the other physical characteristics of an active MTrP and always presents within a tight band that increases muscle tension and limits the ROM . The underlying mechanisms of MTrPs have only recently started to be elucidated. Jarvholm et al. report that elevated intramuscular pressure in the supraspinatus substantially restricts local muscle blood flow, likely resulting in hypoperfusion and ischemia in the area. The partial oxygen pressure (pO 2 ) within regions of muscle hardening, termed myogeloses - presumed clusters of multiple MTrPs - has been found to be extremely low (near zero) at the center of an MTrP region . This notably low pO 2 is likely associated with a deficiency in adenosine triphosphate (ATP), a factor that may contribute to the localized hypercontractions (contractures) observed in these areas. ATP is essential for breaking myofilament bonds and stopping muscle contraction. Current data on myofascial pain indicate that the discomfort linked to active MTrPs may be indirectly related to reduced blood supply in the affected region. Hypoperfusion, for instance, can lead to low pO 2 and contractures. Additionally, it is known that low pO 2 significantly promotes the release of bradykinin, which sensitizes muscle nociceptors. Since capillary pressure ranges from around 35 mmHg at the beginning (arterial side) to approximately 15 mmHg at the end of the capillary network (venous side), capillary blood flow is transiently obstructed during muscle contractions . Blood flow immediately restores with relaxation, consistent with its normal physiological mechanism. In dynamic rhythmic contractions, intramuscular blood flow is improved by this contraction-relaxation rhythm, also known as the muscle pump. However, during sustained muscle contractions, muscle metabolism is highly dependent on oxygen and glucose levels, which are reduced due to altered vascular dynamics. Contractions performed at 10% to 25% of maximal voluntary contraction are sufficient to produce high intramuscular pressures that significantly compromise intramuscular blood flow . According to Bron and Dommerholt , these increases in pressure gradient, even during low-level efforts, producing hypoxia, can contribute to the formation of MTrPs and ultimately to the development of pain symptoms . Often referred to in the literature as myofascial pain syndrome , its symptomatology involves the muscular, sensory, motor, and autonomic nervous systems, caused by the stimulation of MTrPs . In the functional classification of the fascial system , as previously outlined, one of the main roles is the transmission of forces. To understand how the fascial system performs the task of force transmission, in terms of both stability and movement, we must refer to Hill's model. One of the most common and simplified versions of the muscle model proposed by Hill consists of three elements: a contractile (active) element in series with an elastic (passive) element and both in parallel with another elastic (passive) element . It is the contractile element that is responsible for the change in muscle length and the generation of force when the muscle is activated. Although this simplified interpretation is not unanimously accepted, as it is considered one-dimensional , it remains a useful model to explore this topic in greater depth in this article. Its basic structure is schematically composed of (a) contractile component, typically identified with the sliding filaments of actin and myosin, where the force generated depends on the number of active cross-bridges between these filaments; (b) connective component, often linked to the intrinsic elasticity of the myofilaments and cross-bridges, as well as the tendons; and (c) parallel component, associated with the elasticity of connective tissues such as the epimysium, perimysium, and endomysium, along with the sarcolemma . According to Huijing , the real situation becomes further complicated because these individual units in turn combine in series and in parallel. While the role of the in-series connective systems is to transfer, usually to the bone, the tension developed by the shortening of the muscle contractile component, the parallel component is responsible for energy recycling. Indeed, many movements in animals, including humans, are cyclic in nature and are associated with a stretch-shortening cycle of the myofascial complex . Many myofascial systems are built to take mechanical and energetic advantage of the stretch-shortening cycle through their parallel elastic elements, influencing the rate of change of the muscle’s contractile elements , storing and releasing potential energy , and increasing work output during the shortening phase through mechanisms of residual force enhancement . The effectiveness of energy storage in fascial tissue is further exemplified by animals such as gazelles and kangaroos, which use the fascial tissue in their lower limbs as an elastic spring . Another example can be observed in athletes with below-knee amputations, who, using a passive system made of elastic metal blades that essentially replace the parallel myofascial component, are able to reach high speeds during running . The hypothesis we propose is that myofascial pain syndrome, caused by the formation of MTrPs, may be linked to an altered load of connective systems, as schematized by Hill's model . In the case of incorrect use of parallel connective systems, which are crucial for joint stability, the serial fascial system may be compensating through muscle activation. This activation is constant, not for dynamic purposes (i.e., for producing movement), but for static purposes related to stabilization. Muscle activation within a non-extensible connective sheath may increase internal pressure, reducing blood flow as proposed by Jarvholm et al. in the supraspinatus muscle. This mechanism seems plausible in cases of lower back pain (LBP) where the absence of the flexion relaxation phenomenon (FRP) represents the compensation of serial connective systems, with associated muscle activation observed electromyographically during trunk flexion postures. FRP, as studied by Floyd and Silver in maximum trunk flexion during upright standing, has also been observed in subjects during slumped sitting postures . These postures are commonly adopted during daily life, especially given the time many people spend sitting in chairs, sofas, and armchairs. Such compensatory muscle contraction may trigger a vascular crisis, which may in turn lead to the formation of MTrPs, as identified via ultrasound in the thoracolumbar fascia and erector spinae muscles . The results of a study focusing on ultrasound measurements of the thickness of the thoracolumbar fascia and multifidus muscle indicate that individuals with chronic LBP exhibit thickening of the thoracolumbar fascia and thinning of the lumbar multifidus muscle compared with healthy control groups. In particular, an increase in thoracolumbar fascia thickness was correlated with pain intensity, while a reduction in multifidus muscle thickness was associated with decreased lumbar flexion capacity. The authors emphasized the importance of incorporating tailored rehabilitation regimens for LBP patients that target both fascial and muscular components. Another study confirms the reduction in multifidus thickness in patients with LBP. Many studies suggest treatments aimed at stretching the fascia , including manual techniques and stretching exercises . The goal of these therapies is to modify the mechanical properties of the fascia, such as density, stiffness, and viscosity so that it can more easily adapt to physical stress . In fact, manual therapists report a localized release of tissues following the application of slow manual force to abnormally tense fascial areas . These outcomes have been explained as a disruption of fascial cross-links, a transition from a gel state to a sol state in the ECM, as well as other passive viscoelastic changes in the fascia . In the early 1900s, Sherrington defined the underlying concepts of neuromuscular facilitation and inhibition. These later led to the development of clinical stretching techniques described by Kabat as proprioceptive neuromuscular facilitation (PNF). PNF is based on neurophysiological phenomena, particularly reciprocal inhibition: the process wherein, when a muscle group is activated, its antagonist is inhibited. Initially, PNF techniques were used for the rehabilitative treatment of patients with spasticity and paresis, facilitating muscle stretching. Soon after, the therapeutic use of PNF was extended to patients with conditions of non-neurological origin , including more common complaints treated in sports medicine . Two commonly used PNF-stretching techniques are contract-relax (CR) and contract-relax followed by agonist contraction (CRAC). During the CR technique, the therapist passively brings the target muscle group of the patient - the group to be stretched - to the point of maximal resistance, referred to as the barrier , where further stretching or ROM is limited. In this position, the treated muscles are contracted isometrically for a few seconds and then stretched to a new barrier. Some authors further differentiate between CR techniques and the hold-relax (HR) method, depending on the type of contraction used before stretching . Theoretically, CR involves an isotonic contraction resisted by the therapist, while HR requires a resisted isometric contraction . Both methods, rooted in the PNF approach, are applied with the intention of stimulating sensory receptors that provide information about body position and movement to facilitate the desired movement . In both cases, the joint or body part is actively or passively repositioned to the new limit of the ROM following contraction . The primary mechanism underlying this technique is postulated to be the inverse myotatic reflex, also known as autogenic inhibition, due to the action of Golgi tendon organs (GTOs) . In autogenic inhibition, maximal muscle contraction activates the GTOs - structures sensitive to force - thereby inhibiting the alpha motor neurons of the same muscle via type-Ib inhibitory interneurons. A 2006 review reports that these techniques often vary in their descriptions and are referred to by different names. For example, the CRAC technique consists of two phases: the first is identical to the CR technique, while the second phase adds a contraction of the antagonist muscle group as the therapist stretches the target muscle group. The CRAC technique appears to exploit the myotatic reflex, meaning that the increased discharge frequency of the spindles in the antagonist muscle (due to isometric contraction) stimulates Ia inhibitory interneurons, which in turn inhibit the alpha motor neurons of the antagonist muscles . This should lead to a relaxation of the activated muscles and/or a reduction in the amplitude of the muscle stretch reflex response . Kabat attributed this to the induction resulting from the second phase and used it to develop a PNF strengthening technique, which became known as "antagonist reversal" . Several studies have reported that PNF stretching techniques led to greater increases in ROM compared with static or ballistic stretching. This response seems to be due to the relaxation of the muscle being stretched, as a result of reflex inhibition . The hypothesis that PNF exerts its effects through muscle activity inhibition has been challenged by several researchers. Using surface electromyography (EMG), Moore and Hutton studied the relative level of relaxation of the hamstring muscles achieved during different types of stretching. They found that the CRAC technique not only produced the greatest increase in ROM but also significantly higher EMG activity compared with the static or CR technique. Subsequent surface EMG studies evaluating various stretching techniques confirmed, as defined by Etnyre and Abraham , the apparent paradox that, when using PNF stretching techniques, the greatest ROM gains coincide with increased EMG activation of the stretched muscle, rather than a reduction. Furthermore, several years ago, Chalmers questioned the neurophysiological foundations of CR, particularly the role of GTOs. After this article appeared, several other authors also expressed doubts about the actual mechanisms underlying PNF techniques and stretching methods in general. Traditionally, it has been considered that GTOs serve as "safety devices" that help prevent excessive force during muscle contraction . When the forces of muscle contraction and external factors combine to a point where injury to the muscle, tendon, or bone becomes possible, GTOs generate inhibitory postsynaptic potentials on the cell bodies of the agonist motor units . According to Moore , recent research has demonstrated that these concepts regarding GTO physiology are inaccurate. The new evidence can be summarized as follows: (a) GTOs respond throughout the entire ROM, even to weak active and passive contractions, although they are much less sensitive to passive contractions than active ones. (b) GTO impulses likely reach the cerebral cortex, informing the spatial position of the limbs. (c) GTOs and their autogenic inhibition reflex reduce, but do not deactivate, the excitability of the motor neurons and the innervated muscle. (d) During CR stretching, the GTO autogenic reflex induces a momentary inhibition that persists only for the duration of the active muscle contraction. Therefore, it is unlikely that GTO activity significantly influences the subsequent relaxation phase, as proposed in earlier literature . The cause of the hypothesized change in stretch perception with PNF stretching remains unknown. Increased stretch tolerance has been proposed to explain the acute increases in ROM observed with static stretching and as the basis for the greater ROM in individuals with greater relative physiological flexibility, particularly in the hamstring muscles . However, if we simply attribute the increased ROM induced by stretching to greater pain tolerance during the stretch, this cannot explain how, in addition to increased joint mobility, there is also a reduction in "muscular stiffness" immediately after exercise, as assessed by ultrasound shear-wave . Other studies using the same method have confirmed the acute reduction in muscle stiffness at rest following stretching . The global relationship between the nervous system and the fascial system is highly complex. Starting with the simple constitution of what is referred to as nervous tissue, it actually involves a significant participation of supporting fascial tissue (endoneurium, perineurium, and epineurium). Further complicating the picture is the response of these fascial structures to mechanical stress in relation to the activity of the autonomic nervous system (sympathetic and parasympathetic systems), which certainly plays a role in the determination of baseline tension states. Based on the current information available, there is insufficient data to fully define these relationships. For this reason, we will limit our discussion to the hypothesized afferent responses of fascial system receptors, specifically regarding force transmission. According to Schleip , the immediate fascial plasticity observed after manual treatment cannot be explained solely by the mechanical properties of the connective tissue system. The fascia is densely innervated by mechanoreceptors, which include Ruffini endings and a rich network of interstitial receptors with a high reactivity to tangential pressure. The manual stimulation of these sensory endings by the therapist’s manipulation likely leads to changes in the tone of motor units mechanically linked to the tissue involved. Therefore, it is not just mechanical action that is seen but also profound changes in the autonomic nervous system triggered by the stimulation of these receptors. Supporting Schleip's proposal is the work of Guissard and Duchateau , whose title “Neural aspects of muscle stretching” clearly reflects the authors’ stance. The idea that stretching interacts not only with the muscle component but also with the nervous component is not a recent discovery. To our knowledge, the earliest data date back to the work of Robinson et al. in 1982 , when the authors used the Hoffman neurological reflex evaluated with EMG to investigate the effect of stretching, which was erroneously termed muscular, referring to muscle as the tension-producing system. Unlike rapid muscle stretching, static stretching does not enhance reflex activity in the stretched muscle but instead reduces spinal reflex excitability. This inhibitory effect can be evaluated in the soleus muscle by measuring the Hoffmann reflex (H-reflex) and the tendon reflex (T-reflex) through EMG. The H-reflex is triggered by electrically stimulating the Ia fibers of the tibial nerve in the popliteal fossa , whereas the T-reflex is elicited by tapping the Achilles tendon with a reflex hammer. Traditional weight training loads the muscle within its typical ROM, thereby reinforcing the fascial tissues aligned in series with the active muscle fibers. Additionally, the transverse fibers within the muscle sheath are also stretched and activated. However, minimal effects can be expected on extramuscular fascia and on those intramuscular fascial fibers arranged parallel to the active muscle components, as proposed by Huijing . On the other hand, classical stretching, in which the muscular contractile component is inactive, will show minimal effects on those fascial tissues arranged in series with the active muscle component. The reason is that relaxed myofibers, being much more elastic than their fascial/tendinous extensions arranged in series, can absorb most of the stretch . However, slow, light, and constant stretching provides good stimulation for tissues like the extramuscular fascia and the intramuscular fascia arranged parallel to the myofibers. The classic method proposed in the 1980s by Anderson involves passively stretching the myofascial structures by separating the insertion points . In this situation, the first barrier to stretching is the parallel fascial component. Indeed, the muscular contractile component, due to the greater elasticity of the relaxed myofibrils, reduces the tension induced by the stretch on the series fascial component. Therefore, this method might stimulate the elongation only of the fascia arranged in parallel. The CR stretch, by activating the contractile component of the muscular system while in the stretched position produced by the previous method, may tend to primarily load tension onto the series fascial component in the Hill model, while simultaneously reducing the tension on the parallel system . On the other hand, the CRAC technique, especially in the second phase , which involves the contraction of the antagonist anterior muscles, further separates the insertion points of the posterior fascial system, involving the parallel fascial component. This model is novel because, instead of relying on the neurophysiological principles outlined by Sherrington (particularly considering the reconsideration of the function of the Golgi tendon organ previously discussed), it is primarily based on the effect of mechanical load on fascial components arranged in series and parallel. This model should also be considered when applying certain manual therapy techniques, such as muscle energy technique (MET) , which are directed toward the fascial system. This article is the first, to our knowledge, to address fascia dysfunction in two modalities: excessive stiffness and reduced stiffness. This first part of an investigation of physical exercise and the fascial system provides an introduction to the composition of fascia and an overview of the methods proposed in the literature to address dysfunctions caused by excessive fascial rigidity. In addition, the neurophysiological principles underlying traditional muscle stretching are questioned, exploring a model that shifts the focus more toward the fascial component rather than the muscle's contractile component. By revisiting models that schematize the fascial system, such as Hill's finite element model, it attempts to apply these to the techniques most commonly proposed in the literature. This article presents a new hypothesis on how MTrPs may develop as a result of imbalanced tension loading on the fascial components arranged in series and parallel to the contractile muscular component. If future research were to confirm these hypotheses, it would follow that, for an effective and complete treatment of fascial system stretching, all three methods should be used in sequence. This model should also be considered when applying certain manual therapy techniques. | Review | biomedical | en | 0.999997 |
PMC11698535 | Precision medicine, an approach that considers individual variability in genes, environment, and lifestyle to allow for more precise prediction and treatment, has significantly influenced various medical specialties . Integrating genomic data and advanced diagnostics has led to personalized therapies, particularly in fields such as oncology and cardiology . However, applying precision medicine principles in emergency medicine (EM) remains relatively underexplored. EM is characterized by the need for rapid assessment and immediate decision-making to manage acute illnesses and injuries. Traditional EM practices often rely on generalized protocols designed for the average patient, which may not account for individual variations that can impact clinical outcomes . The heterogeneous nature of emergency department (ED) presentations underscores the potential benefits of adopting precision medicine approaches to tailor interventions more effectively. Incorporating precision medicine into emergency care, termed precision emergency medicine (PEM), holds promise for enhancing diagnostic accuracy and optimizing therapeutic strategies. By leveraging patient-specific data such as genetic profiles, biomarkers, and point-of-care diagnostics, PEM could transform emergency care from a protocol-driven practice to one that is more personalized and outcome-focused . For example, pharmacogenomics could guide medication selection and dosing in the ED, reducing adverse drug reactions and improving efficacy. Several studies have begun to explore the integration of precision medicine in emergency settings, highlighting both opportunities and challenges. Kingsmore et al. demonstrated the potential of rapid whole-genome sequencing in critically ill infants, enabling timely and precise diagnoses that can significantly alter clinical management . Additionally, point-of-care ultrasound (POCUS) has been identified as a tool for delivering precision care in pediatric emergencies, allowing for individualized assessment and intervention . Despite these advancements, the literature on PEM is limited, with only a handful of studies directly addressing the topic. A recent search identified only a limited number of papers specifically focused on PEM, indicating a significant gap in research . This paucity of data underscores the need for comprehensive evaluations of how precision medicine principles can be effectively implemented in emergency care to improve patient outcomes. Moreover, challenges such as limited time for data analysis in acute settings, the need for rapid diagnostic tools, and ethical considerations related to genetic testing remain significant barriers to widespread adoption . This systematic review aims to critically evaluate the existing literature on PEM, focusing on the limited number of studies available. Specifically, it will analyze how precision medicine principles have been applied in emergency care settings, assess preliminary impacts on clinical outcomes, and identify reported barriers and facilitators to implementation. By synthesizing findings from these studies, the review seeks to provide a foundational understanding of PEM, highlight gaps in current knowledge, and propose directions for future research to advance the field. This systematic review was conducted to evaluate the application of precision medicine principles in emergency care settings. The review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines to ensure a transparent and replicable methodology . A comprehensive literature search was conducted across multiple electronic databases, including PubMed, Embase, Cochrane Library, Scopus, and Google Scholar, covering articles published from January 2010 to October 2024 to capture the latest advancements in PEM. The search, completed in October 2024, employed a strategy that combined keywords and Medical Subject Headings (MeSH) terms related to precision and emergency medicine. Example search terms included phrases such as "precision medicine", "personalized medicine", "individualized medicine", "genomic medicine", "machine learning", and "artificial intelligence", combined with terms specific to emergency settings such as "emergency medicine", "emergency department", "acute care", and "emergency services". Additionally, the reference lists of relevant articles were manually reviewed to identify further studies that met the inclusion criteria. The inclusion and exclusion criteria for this study were defined to ensure relevance and rigor. The inclusion criteria encompassed studies involving patients in emergency care settings where precision medicine principles, such as genomics, machine learning (ML), point-of-care diagnostics, biomarkers, or personalized therapeutic strategies, were applied. Eligible study types included original research articles, reviews, editorials, consensus statements, and meta-analyses, focusing on English-language publications from January 2010 to October 2024. The exclusion criteria ruled out studies not directly related to precision medicine in emergency care, including case reports, conference abstracts, letters to the editor, non-peer-reviewed articles, and research focused on non-emergency settings or specialties outside EM. All identified articles were imported into EndNote (Clarivate, Philadelphia, Pennsylvania) reference management software, and duplicates were removed. Two independent reviewers screened the titles and abstracts of the retrieved articles for eligibility based on the inclusion and exclusion criteria. Discrepancies between reviewers were resolved through discussion or consultation with a third reviewer. Additionally, full-text articles were obtained for studies that met the initial screening criteria. The same two reviewers independently assessed the full-text articles for final inclusion. The study selection process is documented in Figure 1 . Data extraction and management were conducted using a standardized form to capture essential information from the included studies. The extracted data encompassed study characteristics, including author(s), year of publication, and study design; focus areas, detailing specific precision medicine applications in emergency care settings; and key findings, highlighting the application of precision medicine principles, impacts on clinical outcomes, and identified barriers and facilitators to implementation. Two reviewers independently performed the data extraction, with any discrepancies resolved through discussion or consultation with a third reviewer to ensure accuracy and consistency. The quality of the included studies was assessed through a general appraisal of their relevance, credibility, and contribution to the topic of PEM. Given the diversity of study designs, including original research articles, reviews, an editorial, and a consensus conference report, and the exploratory nature of this review, a formal quality appraisal using standardized tools was supplemented with a detailed bias assessment. Each study was evaluated based on its relevance to the research question, ensuring that it directly addressed the application of precision medicine principles in emergency care settings. To provide a transparent evaluation of study quality, we examined potential biases specific to each study type, categorizing risks as low, high, or unclear. Table 1 presents a summary of the risk of bias across the included studies, focusing on factors such as selection, performance, detection, reporting, confounding, and conflict of interest biases. Additionally, the clarity and transparency of objectives, methodologies, and findings were considered to determine the reliability of the information presented. Finally, the contribution of each study to the field was assessed by examining the significance of its findings in advancing the understanding of PEM. A qualitative synthesis of the findings was conducted due to the heterogeneity of the studies in terms of interventions and outcomes measured. The studies were grouped based on the focus area of precision medicine applications in emergency care, such as genomic applications, ML, point-of-care diagnostics, and education. The initial database search yielded 218 records. After removing duplicates and screening titles and abstracts, 35 articles were selected for full-text review. Of these, 25 articles were excluded for not meeting the inclusion criteria, such as not focusing directly on PEM or lacking relevant data. Ultimately, 10 studies were included in the systematic review. The 10 included studies comprised original research articles (n = 5), reviews (n = 3), an editorial (n = 1), and a consensus conference report (n = 1). The studies were published between 2015 and 2024. The characteristics of the included studies are summarized in Table 2 . Genomic and molecular applications: Several studies highlighted the use of genomic data in emergency settings. Limkakeng et al. described how systematic molecular phenotyping, including genomics, proteomics, and metabolomics, can transform emergency care by enabling precise diagnoses based on individual patient profiles . They emphasized that while this approach holds great promise, challenges such as cost, turnaround time, and the need for rapid interpretation of complex data in acute settings are significant barriers that need to be addressed. ML and data science: Lee et al. explored the role of ML and artificial intelligence (AI) in EM, discussing how these technologies enhance precision medicine by enabling predictive analytics and personalized care plans . They highlighted that ML is transforming diagnostic processes, especially in predicting conditions such as sepsis, thus improving real-time clinical decision-making, risk stratification, and personalized treatment. Salhi et al. developed a framework for integrating big data and technology to improve healthcare delivery and access in EM . They identified core domains such as personalized ED care, expedited prehospital care, and prediction tools for system readiness, underscoring the importance of data science in advancing PEM. Point-of-care diagnostics and biomarkers: POCUS was identified as a valuable tool for delivering precision care. Kessler et al. reviewed cutting-edge applications of POCUS in pediatric emergency care, suggesting that it allows for personalized assessment and intervention . Shaban et al. conducted a systematic review and meta-analysis demonstrating that POCUS is highly effective in diagnosing abdominal aortic aneurysm (AAA) in the ED, supporting its role in PEM . Both Kessler et al. and Sanz-García et al. emphasized the importance of using biomarkers and early warning scores (EWS) to create individualized diagnostic pathways in emergency settings . Biomarkers and EWS help identify patients at risk for clinical deterioration, adding specificity to diagnostics and optimizing treatment timing and choice, thereby improving outcomes. Biomarkers and EWS: Sanz-García et al. highlighted the need for incorporating biomarkers and EWS to enhance diagnostic accuracy and patient outcomes in acute settings . The use of these tools facilitates high-precision EM by enabling clinicians to identify subtle changes in a patient's condition that may indicate impending deterioration. Health professions education: The integration of PEM into medical education was addressed by Chan et al. , who emphasized the existing gap in precision medicine education among emergency physicians . They proposed a research agenda for educating future emergency physicians, stressing the importance of comprehensive training programs that include structured education in genomic medicine, ML, and AI. By equipping clinicians with these skills, the EM workforce can be better prepared for future precision medicine applications. While the studies suggest potential improvements in diagnostic accuracy and personalized care, direct evidence of improved clinical outcomes is still emerging. Kingsmore et al. reported that rapid genomic diagnostics could alter clinical management in critically ill infants, potentially improving outcomes by enabling timely, targeted interventions . Shaban et al. found that the use of POCUS in diagnosing AAA led to accurate detection and management, which could translate into reduced morbidity and mortality . Barriers: Common barriers identified across the studies include time constraints, technological limitations, education and training gaps, and ethical and privacy concerns. The acute nature of emergency care limits the time available for comprehensive data analysis , making it challenging to integrate complex genomic data into fast-paced emergency environments. Technological limitations are evident in the need for rapid and accurate diagnostic tools that can be feasibly implemented in the ED, as the cost and turnaround time for technologies like whole-genome sequencing can be prohibitive . Additionally, there is a noted lack of familiarity among emergency physicians with precision medicine tools and techniques , highlighting the need for comprehensive training programs. Ethical and privacy concerns related to genetic testing and data security further complicate the adoption of precision medicine, with issues such as informed consent, potential for genetic discrimination, and data security being significant . Facilitators: Facilitators for implementing PEM include advancements in technology, interdisciplinary collaboration, and education initiatives. The development of rapid sequencing technologies and portable diagnostic tools supports the practical application of precision medicine in EDs . Interdisciplinary collaboration among data scientists, clinicians, educators, and policymakers is essential for integrating precision tools into EM , as it helps address technological and logistical challenges. Furthermore, incorporating precision medicine into medical curricula prepares future physicians to utilize these tools effectively , with structured training in genomic medicine, ML, and AI being crucial for advancing PEM. The existing literature on PEM is limited but growing. The studies reviewed provide insights into various aspects of precision medicine in emergency care, including genomic applications, ML, point-of-care diagnostics, biomarkers, and educational needs. While there is enthusiasm for the potential of PEM, significant barriers, such as time constraints, technological limitations, ethical concerns, and training gaps, must be addressed to realize its full impact on patient care. These findings illustrate the current applications and challenges of PEM. Advances in ML, genomic profiling, point-of-care tools, and education are driving the future of this field. Addressing ethical and logistical barriers is crucial for the full implementation of precision medicine in emergency settings, with the goal of enhancing patient outcomes in real-life clinical settings. This systematic review critically evaluated the existing literature on PEM, focusing on the application of precision medicine principles in emergency care settings, their impact on clinical outcomes, and the barriers and facilitators to implementation. The inclusion of additional in-depth analyses in our results has enriched our understanding of how precision medicine is currently being integrated into EM and the potential it holds for transforming patient care. The findings demonstrate that precision medicine applications in emergency settings can significantly enhance diagnostic accuracy and personalize patient care. An illustrative example is provided by Kingsmore et al. , who utilized rapid whole-genome sequencing (STATseq) in critically ill infants, resulting in timely and precise diagnoses that significantly influenced clinical management and outcomes . This breakthrough underscores the practical feasibility and clinical impact of integrating genomic profiling into acute care settings. Similarly, the use of POCUS has been shown to facilitate personalized assessment and intervention. Kessler et al. and Shaban et al. demonstrated the efficacy of POCUS in pediatric emergencies and AAA diagnosis, respectively, highlighting how point-of-care diagnostics can contribute to precision care by providing immediate, patient-specific information that guides clinical decisions . Another promising development is the incorporation of ML and AI into EM. Lee et al. discussed how ML algorithms are transforming diagnostic processes, particularly in predicting conditions such as sepsis, thereby improving real-time clinical decision-making and risk stratification . These technologies enable the analysis of large datasets to identify patterns and predict outcomes on an individual level, aligning with the goals of precision medicine. Despite these advancements, the integration of precision medicine into emergency care is not yet widespread. Several barriers contribute to this gap between potential and practice. Time constraints inherent in emergency care limit the feasibility of complex data analysis and interpretation required for genomic and molecular applications . Technological limitations, including the cost and turnaround time of advanced diagnostics such as whole-genome sequencing, further impede adoption . Insufficient education and training among emergency physicians is another critical barrier. Chan et al. emphasized the need for comprehensive training programs to equip clinicians with the necessary skills in genomic medicine, ML, and AI . Without adequate education, clinicians may be hesitant to adopt new technologies or may not utilize them effectively, limiting the potential benefits of precision medicine. When comparing our findings with previously published work, there is a consensus on both the potential benefits of PEM and the challenges that impede its adoption. Rizos et al. highlighted that while precision medicine has revolutionized fields like oncology, its application in EM is limited due to the acute nature of care and the need for rapid decision-making . This aligns with the barriers identified in our review and underscores the unique challenges of integrating precision medicine into EM. The theoretical implications of these findings suggest that PEM could significantly enhance patient outcomes by tailoring interventions to individual needs. Practical applications are vast, ranging from the use of ML algorithms to predict patient deterioration to employing genomic data for precise pharmacotherapy in acute settings . As discussed by Sanz-García et al. , the use of biomarkers and EWS further illustrates how precision tools can aid in early detection and intervention, potentially reducing morbidity and mortality . Despite the promise, the path to implementing PEM is fraught with challenges. Addressing these barriers will require a multifaceted approach. First, investment in rapid, cost-effective diagnostic technologies suitable for emergency settings is essential, including the development of portable devices and point-of-care tools that provide immediate results to mitigate time constraints and enhance feasibility. Second, collaboration among clinicians, data scientists, educators, and policymakers is crucial, as such interdisciplinary partnerships can drive innovation, address technological limitations, and develop practical solutions tailored to the emergency care environment . Third, integrating precision medicine into medical curricula will prepare current and future emergency physicians to adopt these approaches effectively, with training programs focusing on genomic literacy, data interpretation, and ethical considerations . Fourth, establishing standardized protocols and ethical guidelines is imperative to address concerns related to data privacy, informed consent, and genetic discrimination, necessitating prioritization by policymakers and professional organizations . Lastly, advocacy for policies that support the integration of precision medicine into EM, including funding for research and infrastructure, can facilitate widespread adoption. By pursuing these strategies, the implementation of PEM can overcome existing barriers and realize its potential to transform emergency care. Our review highlights several key findings. First, the evidence base for PEM is limited but growing, with only 10 studies directly addressing the topic. This underscores a significant gap in research and emphasizes the need for more studies to build a robust evidence base. Second, the application of precision medicine principles in emergency settings shows promise for enhancing diagnostic accuracy and personalizing patient care, potentially leading to better clinical outcomes. Third, significant barriers to implementation have been identified, including time constraints, technological limitations, lack of education and training, and ethical concerns, all of which need to be addressed to facilitate the integration of PEM into clinical practice. Fourth, advancing PEM will require interdisciplinary collaboration and education, necessitating cooperation between clinicians, data scientists, educators, and policymakers, as well as the incorporation of precision medicine into medical education to prepare future emergency physicians. Lastly, the development of standardized protocols and guidelines is crucial for the safe and effective implementation of PEM. This review has limitations. The small number of studies included reflects the limited research available on PEM, which may affect the generalizability of the findings. Additionally, the heterogeneity of the studies in terms of focus areas and methodologies makes it challenging to draw definitive conclusions. Publication bias may also be present, as studies with negative findings are less likely to be published. To advance PEM, several key directions should be pursued. Large-scale, multicenter studies are needed to assess the effectiveness of precision interventions in emergency settings, providing robust evidence for their impact on patient outcomes. Investment in technological innovations is crucial to developing rapid and accurate diagnostic tools that align with the fast-paced nature of emergency care . Additionally, integrating precision medicine into EM curricula will equip clinicians with the necessary skills to utilize these tools effectively. Establishing ethical guidelines and policies that address data privacy and consent issues is essential to ensuring patient trust and compliance with regulatory standards. Finally, fostering interdisciplinary collaboration will help bridge the gap between technological capabilities and clinical application, enabling a more seamless integration of precision medicine into emergency care . PEM is an emerging field with the potential to significantly improve patient care in emergency settings. These advanced diagnostic tools, including genomic profiling, ML algorithms, and point-of-care technologies, highlight the significant potential of PEM to transform emergency care. While the current literature is limited, the studies reviewed highlight promising applications and underscore the importance of addressing barriers to implementation. By fostering interdisciplinary collaboration, investing in education and technology, and developing supportive policies and ethical guidelines, the integration of precision medicine into emergency care can be realized, ultimately enhancing patient outcomes in acute settings. | Other | biomedical | en | 0.999994 |
PMC11698537 | Hepatoblastoma is the most common primary malignant hepatic tumor in children. Hepatoblastoma accounts for 79% of liver cancers in children under the age of 15 but only makes up one percent of all pediatric cancers . Hepatoblastoma typically presents asymptomatically with an enlarging abdominal mass, elevated alpha-fetal protein (AFP), and less commonly with anorexia, weight loss, and pain . Some cases have reported the production of ectopic adrenocorticotropic hormone (ACTH), parathyroid hormone-related peptide (PTHRP), and beta-human chorionic gonadotropin (hCG), presenting with a paraneoplastic presentation of precocious puberty, which is speculated to be related to the ectopic beta-hCG secretion . Precocious puberty is defined as early signs of sexual development in children. The age of onset of precocious puberty is defined as eight in female patients and nine in male patients. In male patients, signs and symptoms of precocious puberty include penile and testis enlargement, pubic or axillary hair growth, deepening of voice, and development of body odor . In female patients, this precocious puberty would present as early breast development, axillary hair growth, menstruation, and rapid height growth . The exact mechanism by which hepatoblastoma causes precocious puberty is still unknown. However, reports of this rare presentation have been increasing over the last three decades as indicated by the increased number of reported cases. However, delays in diagnosis as with our patient are still prevalent, which indicates a need to highlight this presentation for diagnostic application. We present a two-year-old male patient with persistent abdominal and genital pain for three months. His weight was 25.4 kg, and his height was 105 cm, both of which are above the 99th percentile for his age group. A physical exam revealed Tanner stage three pubic hair, a mature-appearing phallus, a large rugated scrotal sac, and deepened voice. Laboratory studies revealed normal liver function, ACTH, and cortisol levels. Testosterone was noted as 666 ng/dL (normal <9 ng/dL), dehydroepiandrosterone (DHEA) sulfate was 16 mcg/dL (normal <15), androstenedione was 50 ng/dL (normal <22), 17-hydroxyprogesterone was 256 ng/dL (normal <134), and beta-hCG was 72 mIU/mL (normal <50). An abdominal X-ray revealed course calcifications within the right upper quadrant . Abdominal ultrasound revealed a right upper quadrant soft tissue mass measuring 8.1 x 7.9 x 7.2 cm, which contained hyperechoic shadowing calcifications and internal vascularity . CT of the chest, abdomen, and pelvis revealed a large heterogeneously enhancing right suprarenal mass measuring approximately 10.1 x 8.7 x 8.4 cm with calcifications and surrounding the intrahepatic inferior vena cava (IVC) and abutting the right hepatic vein . A definitive diagnosis of hepatoblastoma was made via ultrasound-guided biopsy. The tumor was staged as a Pretext 1. It encompassed the right posterior section of the liver, without caudate or additional hepatic foci, that partially encased the right hepatic vein and intrahepatic IVC. There was no evidence of tumor rupture, portal vein involvement, or distant metastasis. At this time, AFP was measured as 113,601 ng/mL (normal 0.5-7.9 ng/mL). Histological evaluation demonstrated epithelial hepatoblastoma with three subtypes, which included fetal subtype, embryonal subtype, and small-cell undifferentiated cells . Furthermore, membranous staining highlighted the fetal and embryonal subtypes, while the nuclear was positive in the embryonal subtype in beta-catenin staining . Lastly, microphotographs demonstrated that the mesenchymal portion of the tumor consisted of osteoid and osteoid-like tissue and that the calcifications had melanocytes as part of their composure . The tumor was determined to be resectable, so the patient underwent a right hepatic lobectomy with right retroperitoneal nodal sampling. Final pathology identified a 10.5 x 8.5 x 5.0-cm hepatoblastoma that consisted of mixed epithelial (fetal) and mesenchymal (small-cell undifferentiated pattern (5%)) types and mitotically active with teratoid features. Parenchymal margins were negative for tumor, and lymphovascular invasion was not identified. Post-operatively, the patient recovered well and was discharged home on post-operative day nine. After resection, AFP levels decreased to 8,563.0 ng/mL. The patient went on to receive two cycles of cisplatin therapy. After his first round of cisplatin, his AFP dropped to 28.7 ng/mL, and after the second round, his AFP was within normal limits at 5.5 ng/mL. Beta-hCG remained within normal limits after treatment, and a repeat CT scan showed no evidence of pulmonary metastasis or local disease recurrence. The patient was determined to be in remission, and therefore, his port was removed. There have been approximately 40 reported cases of hepatoblastoma presenting as precocious puberty . While the true mechanism by which hepatoblastoma leads to the presentation of precocious puberty is still unknown, there are several theories that speculate on the etiology of this relationship. The two potential hypotheses for the paraneoplastic manifestation of precocious puberty secondary to hepatoblastoma are the following: (1) beta-hCG produced by the tumor stimulates Leydig cells to produce testosterone and (2) beta-hCG’s structural similarity to luteinizing hormone and follicle-stimulating hormone leads to direct testosterone secretion in a gonadotropin-releasing hormone-independent manner . It is imperative to note that all reported cases of virilizing hepatoblastoma were seen in patients who presented with elevated beta-hCG levels, highlighting the central role that it has in the development of this presentation . Early studies suggested that virilizing hepatoblastoma carried worse outcomes compared to non-virilizing, but more recent studies report positive prognosis with long-term survival following surgery and chemotherapy, as seen in our patient . The majority of reported cases were recorded over three decades ago and therefore fail to reflect the progress that has been made in diagnostic, imaging, and treatment modalities, which have the potential to facilitate better outcomes within this population. In the 1970s, the cure rate was around 30%, but the development of better neoadjuvant chemotherapy and surgical resection has increased this value to 70% . This highlights a need for further studies that evaluate potential differences in prognostic outcomes and mortality between virilizing and non-virilizing hepatoblastomas. The surgical management of hepatoblastomas remains complex and subject to ongoing debate. While upfront resection via laparotomy at the time of diagnosis has historically been the standard approach, recent advancements have led to the development of risk stratification systems to guide treatment decisions. Leaders of the four cooperative trial groups (SIOPEL, Children’s Oncology Group, the German Society for Paediatric Oncology and Haematology, and the Japanese Study Group for Paediatric Liver Tumours) developed a risk stratification system that divides patients into four risk groups: very low risk (group A), low risk (group B), intermediate risk (group C), and high risk (group D) . These groups are determined with consideration of the PRETEXT group, a radiologic system that describes the extent of a liver tumor before treatment, age at diagnosis, AFP level, and the presence of a PRETEXT annotation factor . Tumors that are characterized as low risk are resected at the time of diagnosis. However, for tumors that do not fall into this category, the determination of surgery is less straightforward. Surgical intervention is determined after a thorough evaluation of imaging and after neoadjuvant therapy, with general recommendations suggesting that upfront resection be performed only when segmentectomy or nonextended hemihepatectomy with at least 1 cm margins is possible on middle hepatic vein and/or main portal vein division, and there is no concern for macrovascular involvement . However, there is still much debate on whether complete resection is the gold standard, what margins are acceptable, and how/when to manage metastasis. The presentation of precocious puberty in hepatoblastoma remains an intriguing paraneoplastic phenomenon, with elevated beta-hCG levels being a key factor in its manifestation. Although virilizing hepatoblastoma is a rare presentation, the growing frequency of reports indicates that there is a need for the incorporation of this presentation as part of the work-up and diagnosis of precocious puberty presentations. There is an increased need to share this presentation with multidisciplinary providers, such as general pediatricians, endocrinologists, and oncologists, to prevent delays in diagnosis, as was seen in this patient, and to treat the underlying cancer. As surgical management continues to evolve, the adoption of sophisticated risk stratification systems, like those pioneered in the Paediatric Hepatic International Tumour Trial, represents a significant step forward in personalizing treatment strategies. The debate surrounding optimal surgical approaches, whether upfront resection or the timing and extent of surgery following neoadjuvant therapy, remains active. Continued refinement of these protocols, alongside comprehensive studies comparing outcomes across risk categories, will be crucial in enhancing the quality of life for affected patients. | Study | biomedical | en | 0.999996 |
PMC11698539 | In recent decades, women have achieved substantial progress in entering the medical profession. Notably, in 2017, women medical school matriculants surpassed their men counterparts for the first time . However, gender disparities persist in science and medicine. Numerous studies have identified a persistent achievement gap between men and women faculty in areas such as research productivity, career advancement, leadership roles, compensation, and scientific recognition . Women researchers often offer distinct viewpoints, such as addressing health concerns specifically related to gender or prioritizing the inclusion of a diverse range of populations. This can result in outcomes that are more equitable and pertinent. The absence of their representation can lead to the continuation of biases and neglect of essential research areas, which ultimately restricts the significance and relevance of the results produced. Clinical trials are integral to evidence-based medicine and critical for advancing new medical therapies and devices. Leading a clinical trial as a principal investigator (PI) provides physicians with enhanced visibility, national recognition, and opportunities for career progression, including promotions, tenure, and future funding . Despite these benefits, women remain underrepresented as PIs in clinical trials. For example, a study of hip and knee arthroplasty clinical trials found that only two of 192 (1%) PIs were women from January 2015 to December 2021 . Similarly, Nguyen et al. reported that from December 2014 to August 2019, just six of 266 (9.5%) cardiac surgery clinical trials were led by women . Although these disparities are well documented in other specialties, the representation of women in genetic clinical trial leadership remains largely unexplored. Investigating the gender distribution of PIs in genetic clinical trials can provide valuable insights into the extent of this gender gap. This study aimed to address the following questions: (1) What proportion of genetic clinical trial PIs are women, and how has this changed over time? (2) Are certain trial characteristics (such as phase, status, funding source, or intervention) associated with women PIs? (3) What is the geographic distribution of genetic clinical trials led by women PIs within the United States? We performed a thorough analysis of clinical trials using the publicly accessible ClinicalTrials.gov database, managed by the National Institutes of Health (NIH). This platform provides comprehensive data on registered trials, including information such as trial phase, sponsor, submission date, and location. In the United States, it is legally required to register all Phase II, III, and IV trials on ClinicalTrials.gov . To focus on genetic-related clinical trials, we conducted a targeted search using specific Medical Subject Heading terms and keywords such as "genetics", "genetic testing", "gene therapy", "genomic sequencing", and similar terms. All trials found with these search terms were included in the screening process. On March 4, 2024, data from clinical trials were extracted in XML format from ClinicalTrials.gov and imported into Microsoft Excel (Microsoft Corporation, Redmond, Washington) for further processing. Our analysis focused on studies initiated in 2007 or later, aligning with the implementation of the US Food and Drug Administration Amendments Act. This legislation requires the registration of all Phase II, III, and IV trials conducted in the United States on ClinicalTrials.gov . Collected information included the trial phases (I, II, III, IV, or unspecified), sources of funding (industry, federal, NIH, or other), the year the trial started, types of interventions (behavioral, drug, device, procedure, etc.), the lead investigator's first name, and details of the trial's location (city, state, country). The US Census Bureau's definitions of Northeast, Midwest, South, and West were used for regional categorization. We compiled a list of investigators' first names using their full names and titles. Drawing from methods used in prior studies, we utilized Genderize.io ( https://genderize.io ), a well-known tool for determining gender from first names . Genderize.io predicts gender based on a large dataset of names and their associated probabilities. Each first name was analyzed to provide a predicted gender with a probability value. In accordance with previous studies, names were categorized as man or woman, with a 60% threshold for gender assignment to ensure a balance between accuracy and inclusivity . Mapping PI names to Genderize.io posed several challenges, particularly ambiguous or unisex names. Before entering names into Genderize.io , we manually extracted the first name of each PI from our XML file, which was a time-intensive process. The tool itself relies on probabilistic assignments based on name patterns, leading to inaccuracies for uncommon, culturally specific, or unisex names. Additionally, names with regional or linguistic variations often resulted in low confidence scores. Names with lower probabilities or inconclusive results were excluded. Our study initially included 9,133 genetic trials conducted in the United States, drawn from the ClinicalTrials.gov database. To narrow our analysis, we selected only trials that had a PI listed and had a defined trial phase as per FDA standards (ranging from Phase I to Phase IV). Trials without a defined phase, those with organizations as PIs, or those with incomplete or unclear PI names were excluded as per previous studies . We focused on trials with a defined phase to ensure consistency and facilitate meaningful comparisons. Phase designation reflects the structured progression of clinical trials, aligning with FDA standards. Including non-phase-based studies could introduce variability, making analysis less reliable, so narrowing the scope helped maintain clarity and comparability. After applying these criteria, 4,112 genetic trials were retained for the final analysis . We presented the basic characteristics of the trials using frequencies and percentages for categorical variables such as geographic region, study phase, funding source, study status, and trial year. Fisher's exact test was used to assess the association between gender and categorical trial attributes, such as geographic region, study phase, funding source, and study status. This method was chosen because it provides accurate results for categorical data, even when some categories have low frequencies, as observed in certain subsets of our data. By applying this test, we ensured a robust analysis of gender differences across trial characteristics, maintaining the validity of our findings. Drawing from methodologies used in prior research, we employed univariate linear regression to analyze trends in the annual percentage of woman PIs . We used heat maps with color gradients to visualize geographic variation. All statistical analyses were conducted using RStudio software version 2023.12.1 (Posit, Boston, Massachusetts), with statistical significance set at p < 0.05. Mapping was performed using QGIS 3.36.0. The distribution of trial phases showed significant differences between men and women PIs . Trials with no applicable phase had the highest representation of women leaders, with 604 out of 1,552 (38.9%), compared to 666 out of 2,560 (26%) for men leaders. For trials with specified phases, Phase II had the highest representation of women leaders, with 393 out of 1,552 (25.3%), whereas Phase IV had the lowest, with 82 out of 1,552 (5.3%) of women PIs. Similarly, the highest percentage of men PIs was in Phase II, with 821 out of 2,560 (32.1%) (Table 2 ). There was also a statistically significant difference in trial status between men and women PIs . A higher proportion of women PIs led active or recruiting trials, with 516 out of 1,552 (33.2%) compared to 709 out of 2,560 (27.7%) for men. Conversely, more men PIs led discontinued trials, with 439 out of 2,560 (17.1%) compared to 193 out of 1,552 (12.4%) for women PIs (Table 1 ). There was a significant difference in the distribution of intervention types between men and women PIs . Women PIs led a larger proportion of studies involving behavioral interventions, with 240 out of 1,552 (15.5%), and other interventions with 218 out of 1,552 (14%), compared to their men counterparts, who had 196 out of 2,560 (7.7%) and 222 out of 2,560 (8.7%), respectively. On the other hand, men PIs had a higher proportion of studies involving genetic interventions, with 111 out of 2,560 (4.3%), and drug interventions, with 1654 out of 2,560 (64.6%), compared to 44 out of 1,552 (2.8%) and 841 out of 1,552 (54.2%) for woman PIs (Table 2 ). There was no significant difference in the geographic distribution of trials led by men and women PIs (p = 0.062). The proportion of women PIs was relatively similar across US Census regions to that of their male counterparts. The states with at least 10 trials that had the highest proportions of women PI were New Mexico (7 of 10, 70%), the District of Columbia (33 of 66, 66%), Virginia (15 of 30, 50%), and Nebraska (7 of 14, 50%). Conversely, the lowest proportions of woman PIs were found in Kentucky (2 of 16, 13.3%), Utah (4 of 26, 15.4%), and Arizona (30 of 160, 18.9%) (Table 3 ) . Clinical trial leadership is a prestigious role for academic physicians, offering opportunities for increased visibility, career advancement, and access to future funding. It also allows physicians to make substantial contributions to the medical field and improve patient outcomes. The representation of women in leadership roles within genetic clinical trials has not been thoroughly studied or characterized. Our findings revealed that while the proportion of women leading clinical trials fluctuated over time, there was no consistent upward or downward trend. Representation ranged from a low of seven out of 24 (29.2%) in 2008 to a peak of 17 out of 29 (58%) in 2010 before leveling off to 573 out of 1,712 (33%) in 2024. Notably, women surpassed men as PIs during only two years of the study period, making up only 1,552 out of 4,112 (37.7%) of the entire PIs of clinical trials. Women PIs were more likely to lead studies focusing on behavioral and other interventions, whereas men PIs led a higher proportion of genetic and drug trials. Additionally, women PIs received more funding from other sources, while men PIs were more likely to secure industry funding. These findings underscore the need for continued efforts to address gender disparities in clinical trial leadership and highlight the importance of developing strategies to promote equity in this field. Achieving sustained progress will require targeted policies, mentorship opportunities, and collaboration to create an inclusive research environment that supports diverse leadership. Since 2007, there have been more than two times as many clinical trials led by men as women in genetics. The proportion of women leading clinical trials showed fluctuations over time but remained stagnant between 2007 and 2024, with women comprising 33% of PIs in both years. This is not consistent with women's representation as PIs in other medical specialties . For instance, Burkhart et al. reported that the proportion of women leading orthopedic clinical trials increased from 13% in 2007 to 22% in 2022 . One possible explanation for this discrepancy is that genetics began with a greater baseline of female PIs in 2007, which lessened the perceived urgency of equity initiatives. This early lead may have resulted in fewer measures focused on improving representation, as opposed to other fields that benefitted from focused campaigning to address disparities. Furthermore, genetics may have received less institutional push to enhance leadership diversity, resulting in stagnation over time. Even though there was a higher proportion of women PIs in genetic trials as compared to other medical specialties, the lack of growth is concerning. Certain specialties have demonstrated substantial growth in women's representation, with women comprising the majority of clinical trial PIs in some cases. For example, the proportion of women PIs in Phase III gynecologic oncology trials increased markedly from 10% in 2010 to 60% in 2020 . This suggests there is still substantial room for improvement. Women exceeded men as PIs in genetic clinical trials only in 2008 and 2010, suggesting isolated advances rather than a sustained trend toward gender parity in this discipline. These brief periods of female predominance might be attributed to external influences such as specific regulations, financial campaigns, or cultural transformations that emphasized women's leadership in science during those years. However, their brief existence shows that such attempts were either not sustained or insufficient to overcome larger systemic obstacles. To increase the number of women PIs in genetics, institutions and professional organizations should implement mentorship programs focused on career development and provide networking opportunities tailored to their needs. Continuous evaluation of clinical trial leadership is crucial as the presence of women in academic leadership roles within genetics evolves. Understanding these trends can guide the development of effective strategies to reduce gender disparities and promote equity in the field. Our analysis revealed that women were more likely to receive federal or other funding, whereas men were predominantly funded by industry sources. This aligns with previous studies indicating that men receive a disproportionately larger share of industry funding. For instance, a 2016 study found that women academic orthopedic surgeons received only 29% of industry payments, even after adjusting for factors such as faculty rank, H-index, and subspecialty selection . These funding disparities are not isolated to orthopedics but are widespread across medical specialties for women surgeon-scientists . A potential explanation for this inequity is that 75% of physicians with financial ties to biomedical companies are men . Funding differences between male and female PIs may impact trial scope and results. Women PIs' dependence on federal and "other" funding sources indicates a preference for fundamental or exploratory research, which may have less immediate economic appeal but contributes to scientific progress. Men PIs, on the other hand, may receive more industry funding, allowing them to conduct larger, commercially motivated investigations. These disparities may influence the types of research conducted and how findings are translated into clinical practice, emphasizing the importance of fair financing to promote a balanced research environment. Interestingly, our findings differ slightly from those of Burkhart et al., who reported no significant differences in the distribution of men and women across trial phases, statuses, or funding sources in orthopedics . The distinctions from Burkhart et al. might stem from the distinctive characteristics of genetics research, encompassing a variety of interventions such as gene therapy and diagnostics, which draws a wider array of researchers and funding opportunities. Genetics trials may additionally provide greater chances for exploratory or federally supported studies, where there is a higher representation of women, in contrast to orthopedics, which is generally more influenced by industry and consistent in trial attributes. Furthermore, our results showed an increased number of female PIs in Phase II trials, which might be attributed to the collaborative character of these investigations, which involve input from interdisciplinary teams and are consistent with leadership styles that emphasize cooperation and communication. Furthermore, Phase II trials frequently obtain funding from smaller organizations or agencies that encourage diversity, possibly increasing prospects for women-led research. These trials are usually carried out in academic or research-focused institutions, where women are more likely to be represented than in industrial settings, and they benefit from mentorship and support networks. Furthermore, the combination of clinical practice and research in Phase II studies may appeal to women who want to combine patient care with medical advancements. Additionally, our analysis demonstrated that women PIs led a higher proportion of studies focused on behavioral and other interventions, while men PIs were more involved in drug-related trials. This observation is consistent with other studies showing that women are more likely to lead observational studies . Understanding the distribution of PIs across various trial phases and intervention types is critical for informing research strategies, guiding funding allocation, and shaping policy decisions to address gender disparities in clinical trial leadership. The lack of women in higher-phase trials, such as Phase III and IV, may stem from obstacles related to resources and career growth. These trials demand significant funding and leadership experience, which tend to be more readily available to men due to systemic issues such as implicit bias in funding, limited mentorship, and fewer opportunities for leadership. Such challenges restrict women's capacity to oversee extensive, resource-heavy studies, thereby sustaining the gender disparity in advanced clinical research. A diversified strategy is needed to address the differences in clinical trial PIs between men and women in the field of genetics. Mentorship programs should be established by institutions and professional associations to assist and guide women who wish to pursue careers as PIs in genetics. To ensure fair representation, department heads must aggressively advance women faculty members to prominent roles in leadership and research. Grant programs aimed at women researchers should be created and extensively publicized in order to further address these inequities. Making successful women academics more visible through prizes, press attention, and high-profile conference speaking engagements can motivate and inspire others to follow in their footsteps. Equal funding distribution should also be required by institutional and governmental policy reforms, guaranteeing that women researchers have the tools necessary to conduct and complete clinical studies. These could also be integrated with efforts to minimize implicit bias in hiring and funding choices, guaranteeing women equal chances. Ultimately, instituting transparency standards regarding gender representation in leadership roles could ensure accountability for institutions while offering metrics for assessing progress toward equity. When these strategies are used together, they can foster a more equitable and inclusive research setting. Our analysis has several limitations that should be considered when interpreting the findings. First, our reliance on data from ClinicalTrials.gov , a global registry for clinical trials, introduces a potential bias, as trials not requiring FDA regulation are not mandated to register, potentially underrepresenting international studies. Second, the use of Genderize.io to determine the gender of PIs carries inherent risks of misclassification, particularly for names with ambiguous or culturally diverse origins. Although we excluded names with lower prediction probabilities to improve accuracy, this approach may have inadvertently removed some correctly classified individuals, potentially affecting the representativeness of our findings. Additionally, our analysis was limited to gender as the sole diversity variable, excluding other important dimensions of social identity such as race, ethnicity, and socio-economic background. Finally, we did not provide a detailed breakdown of subspecialties within genetic trials or account for other potential confounding variables, such as trial initiation year, that may influence gender representation. Future studies should address these limitations by incorporating broader datasets, expanding diversity metrics, and utilizing software that can more accurately determine the gender of a name or directly contacting PIs to determine accurate gender to capture the intersectionality of social identity factors and their impact on clinical trial leadership. Our findings highlight significant gender disparities in clinical trial leadership within genetics, with women underrepresented as PIs and showing limited progress over time. While women were more likely to receive federal or other funding, men predominantly secured industry funding, reflecting broader inequities seen across medical specialties. Despite some fields achieving notable growth in women's representation, the standstill in genetics underscores the need for targeted interventions. Addressing these disparities will require a comprehensive approach, including mentorship programs, equitable funding policies, increased visibility for women researchers, and active promotion of women into leadership roles. By fostering an inclusive research environment and addressing funding imbalances, we can work toward achieving greater equity in clinical trial leadership, ultimately enhancing the diversity and impact of genetic research. | Review | biomedical | en | 0.999997 |
PMC11698540 | The World Health Organization (WHO) defines cigarette addiction as a chronic illness characterized by progression and recurring relapses . This epidemic, spanning centuries, has severe consequences in terms of morbidity and mortality . Annually, more than seven million people worldwide and upwards of 100 thousand people in Turkey die as a result of smoking-related diseases . Considering variables such as the age of smoking initiation, duration of use, daily consumption volume, and quality of the product used, it has been observed that as exposure to the dose increases, smoking-related diseases and mortality increase . The toxins in cigarette smoke permeate the bloodstream and harm nearly every organ in the body . Smoking is linked to the onset of chronic obstructive pulmonary disease (COPD), as well as chronic respiratory diseases, such as asthma and interstitial lung disease . Smoking is critical due to its status as the leading preventable factor associated with cardiovascular diseases . The risk of myocardial infarction is directly correlated with the quantity of smoking. Over half of the deaths attributed to coronary artery diseases (CADs) are sudden in nature, and the cessation of tobacco can lead to a decreased risk of sudden death . Quitting smoking can diminish smoking-related diseases and fatalities, decelerate the rate of disease progression, and augment life expectancy . Smoking cessation therapies are extensively utilized, and smokers now have more access to smoking cessation methods than ever before. Smokers who receive smoking cessation treatment, a combination of behavioral interventions and medication, are five times more likely to succeed in quitting compared to those who attempt to quit independently . Numerous studies report disparate findings regarding the factors influencing the success of smoking cessation. Prominent factors include motivation and determination, sociodemographic attributes, addiction severity, psychological and environmental influences, and comorbidities . Approximately 70-80% of tobacco users desire to quit, with approximately one-third attempting to quit independently each year. However, less than 10% succeed in their cessation endeavors . Two approaches have proven beneficial in the endeavor to quit smoking. These approaches encompass motivational interviewing and pharmacotherapy, incorporating behavioral therapy techniques . In their routine clinical practice, physicians ought to inquire of every patient, irrespective of the reason for the visit, regarding their smoking status, the quantity of smoking, and their contemplation of cessation. Additionally, healthcare professionals should support those wishing to quit and conduct motivational interviews with those not yet ready. Research has demonstrated that the mere act of physicians delicately inquires patients' smoking habits and issuing admonitions prompts contemplation toward cessation, culminating in a success rate of 1-3% in quitting . Motivational interviewing is based on identifying and exploring the underlying issues pertinent to smokers, aiming to effect behavioral change. The dynamic is grounded in a collaborative and amicable partnership, diverging from the traditional consultant-client paradigm . Randomized controlled trials have shown that motivational interviewing for smoking cessation yields superior efficacy compared to dispensing brief advice . In our study, we administered motivational interviews for smoking cessation to a specific cohort comprising both patients and healthy smokers. Our objective was to demonstrate the impact of motivational interviewing on smoking cessation. Furthermore, we investigated the influence of medical conditions and individual traits on the efficacy of motivational interviewing for smoking cessation. This prospective study was conducted with smokers who presented at the pulmonology and cardiology outpatient clinic. The study encompassed 100 participants each from asthma, COPD, and healthy individuals with smoking (HIWS) habit cohorts who were admitted to the pulmonary outpatient clinic, along with 100 individuals diagnosed with CAD who attended the cardiology outpatient clinic during the period spanning from April 20, 2023, to July 20, 2023. Our study enrolled healthy individuals of any gender, aged 18 years or older, who had smoked for at least 15 years. Individuals with deficient medical documentation, immune system disorders, malignancy, a second chronic disease other than COPD/asthma/CAD, and a smoking history of less than 15 years were not included in the study. Routine laboratory tests, electrocardiography (ECG), pulmonary function testing (PFT), chest radiography, Fagerstöm nicotine dependence test (FTND), and hospital-scale anxiety-depression test (HAD-A/HAD-D) were performed on patients. In addition, low-dose chest computed tomography (CT) was administered for lung cancer screening among individuals aged over 50 years of age and with a smoking history exceeding 20 pack years. FTND serves as a standardized instrument for evaluating the degree of physical nicotine dependence. Uysal et al. adapted the FTND into Turkish, confirming its validity and reliability through a study conducted in 2004 . The assessment comprises six items, yielding a cumulative score from 0 to 10. In this study, an FTND score of 9 or higher defined very high dependence, with categorizations ranging from very low (0-2), low (3-4), moderate (5-6), and high levels of dependence levels. The HAD scale was devised to detect anxiety and depression among patients grappling with physical diseases . The scale comprises a total of 14 items. Of these, seven items measure symptoms indicative of anxiety, while the remaining seven assess symptoms indicative of depression. Following studies conducted in Turkey, the cut-off score for the anxiety subscale (HAD-A) was set at 10, and for the depression subscale (HAD-D), it was established as 7. Interviews were conducted by pulmonologists. The pulmonologists gave a concise lecture covering topics such as nicotine addiction, nicotine withdrawal syndromes, the adverse effects of smoking, the advantages of quitting, strategies to manage weight gain, and techniques to navigate trigger situations. Patients were scheduled for outpatient clinic follow-up appointments at the conclusion of the first month, the third month, and the sixth month. In addition, brief support and behavioral interviews were conducted with the patients via telephone weekly for the first month, followed by sessions every 15 days after that. During the follow-up visit, the patient's smoking cessation status was assessed. Patients who were unable to quit smoking received another brief support session. During each visit, discussions averaging about 10 minutes centered on motivational factors, identification of triggers, management of weight gain, and utilization of medications. The statistical analysis utilized the IBM SPSS Statistics software, version 17.0 (IBM Corp., Armonk, USA). The Kolmogorov-Smirnov test was used to assess the normal distribution of the data, while the ANOVA test was employed to evaluate the homogeneity of the data. Medians with 25-75th percentiles were reported as continuous variables, discontinuous variables were presented as frequencies and percentages, and a chi-square test was applied. Mann-Whitney U and Kruskal-Wallis H tests evaluated median comparisons. Statistical significance was determined at a P-value below 0.05. The study included 400 smokers, comprising 100 individuals with asthma, 100 with COPD, 100 healthy smokers, and 100 patients with CAD, without gender-based differentiation. The median age of the patient cohort was 52 (44-58) years. Among the 400 patients, 177 (44.2%) were female, with a median age of 50 (42-58) years, and 223 (55.8%) were male, with a median age of 53 (46-59) years. The median body mass index was 29.75 (26.51-31.23) kg/m 2 . According to smoking cessation, 85 (21.3%) patients quit smoking at the end of the first month. It was 55 (13.8%) at the end of the third month and 42 (10.5%) at the end of the sixth month. Within all subgroups, the smoking cessation ratios were higher at the end of the first month than in the third and sixth months, with the asthma subgroup exhibiting a particularly notable increase. Although, by the end of the sixth month, smoking cessation rates declined across all subgroups, the most notable smoking cessation rate was observed among the asthma subgroup, with a ratio of 3.5%, but it was statistically insignificant (Table 3 ). At the end of the sixth month, all variables within the study population, encompassing age, age of smoking initiation, smoking quantity, FTND, HAD-A, and HAD-D, were examined concerning smoking cessation. Upon analyzing these variables, no statistically significant difference was identified between individuals who had ceased smoking and current smokers. Conversely, females (26, 61.9%) demonstrated superior outcomes in smoking cessation compared to males (16, 38.1%). In addition, the majority of individuals who quit smoking had a high dependence on nicotine, but it was statistically insignificant (Table 4 ). A linear correlation was observed between the first month of smoking cessation (p=0.007) and smoking pack-year history (p=0.025) (Table 5 ). Furthermore, a significant correlation was found between the third month of smoking cessation and HAD-D (p=0.027). A difference was found between the third month of smoking cessation in median values of smoking pack-year history, but it was not statistically significant (p=0.053) (Table 6 ). Furthermore, a relationship was found between the sixth month of smoking cessation and gender (p=0.015) and smoking pack-year history (p=0.022) (Table 4 ). Females exhibited a higher propensity to cease smoking. As the smoking rate decreased, the smoking cessation rate increased. However, according to smoking cessation, no difference was found between subgroups in the first month (Table 3 ). Numerous studies document disparate findings regarding the determinants influencing the efficacy of smoking cessation efforts . Approximately 70-80% of tobacco users desire to quit, with approximately one-third endeavoring to do so independently each year. Nevertheless, the success rate is less than 10% . In a study conducted in Taiwan, it was demonstrated that augmenting one's comprehension regarding the deleterious ramifications of smoking within the context of transtheoretical model (TTM)-based smoking cessation interventions markedly enhanced the endeavor to cease smoking . In an alternative study, the efficacy of the health belief model and the TTM model in smoking cessation was evaluated, determining that both methodologies were effective, albeit with the TTM model demonstrating superior effectiveness . In the study by Sharifirad et al., the smoking cessation rate after a six-month follow-up period was revealed to be 3.3% in the control group and 46.0% in the intervention group. Notably, all components of the TTM model yielded significant outcomes . A meta-analysis study concluded that motivational interviews are effective in quitting smoking . Numerous additional studies have corroborated the effectiveness of motivational interviews as a valuable method for facilitating smoking cessation efforts . In our research, following the smoking cessation interview and regular follow-up sessions, the success rate for smoking cessation was 21% at the end of the first month. At the end of the sixth month, our smoking cessation rate declined to 10.5%. The main reason for this decline could potentially be attributed to a dilution in patient follow-up after the initial month. In our study, during the initial evaluation at the outpatient clinic, comprehensive inquiries were made regarding the demographic profiles of individuals and smoking histories. The patients were also assessed using respiratory function tests, FNBT, and the HAD-A/HAD-D scales. The data were compared with those at the end of the sixth month. Studies have demonstrated that certain sociodemographic attributes, including male gender, older age, higher education attainment, elevated socioeconomic status, employment status, and having a stable partnership (such as being married or cohabiting), positively influence the success of quitting smoking . Conversely, there are also publications asserting that no discernible gender disparity exists in smoking cessation outcomes . Studies have indicated that varenicline treatment exhibits greater efficacy in facilitating smoking cessation among females compared to males. Conversely, nicotine replacement therapy and bupropion treatment demonstrated a lower success rate in smoking cessation among females relative to males . Enhancing smoking cessation rates among women could be achievable through the implementation of gender-specific studies . One study from China observed variations in smoking behaviors based on gender and rural-urban distinctions . Monsó et al. demonstrated that age, gender, and housing conditions exert a notable influence on smoking cessation outcomes among European participants engaged in smoking cessation programs . Our study gave results that were diverged from those reported in previous studies. In our study, after the sixth month, females exhibited higher smoking cessation rates compared to males. Females had a twofold propensity to quit smoking compared to males. Within the disease categories, the most notable smoking cessation rate was observed within the asthma cohort. This trend could be attributed to the elevated presence of females in the asthma cohort. Several studies indicate that the duration of tobacco use serves as a pivotal factor in the efficacy of cessation efforts. In the study conducted by Górecka et al., smoking duration emerged as a significant variable influencing the likelihood of successful cessation . In a study conducted in Turkey, it was discovered that individuals who commenced smoking prior to the age of 15 exhibited a markedly reduced likelihood of smoking cessation compared to those who initiated tobacco use later . Likewise, in our study, a correlation was observed between the quantity of tobacco consumed and the cessation status of smoking. However, no significant association was detected between the age of smoking initiation. Numerous studies in the literature assess the correlation between smoking cessation status and FNBT, HAD-A, and HAD-D scores. While some studies have reported a significant relationship, others have not. In our study, a statistically significant relationship was identified between smoking cessation status and HAD-D (in the first and third months of control), but not with FNBT and HAD-A scores. Study outcomes vary regarding the impact of chronic disease presence on the efficacy of smoking cessation. The study by Salepçi et al. found that the presence of comorbidities among smokers did not influence their success in quitting . Conversely, Uzaslan et al. found that the participants' health problems positively influenced the success of smoking cessation endeavors . Komiyama et al. reported that the success rate of smoking cessation among COPD patients, psychiatric patients, females, and minors was low . In addition, Perret et al. elucidated the relationship between asthma and smoking in their review . Furthermore, Gratziou et al. demonstrated the importance of an intensive smoking cessation program with regular and prolonged follow-ups for smokers with asthma or COPD. Regular and frequent follow-up visits, particularly within the initial three months, were positively correlated with cessation success . Ludvig et al., in their review, compared smoking cessation success rates among patients with CVD and noted that the success rate during treatment reached up to 25%. However, they also noted that the relapse rates were notably high . A meta-analysis by Taylor et al. highlighted that the success rate could increase to 45% with diligent follow-up and cardiac rehabilitation for patients following acute coronary syndrome . In our study, no difference was observed among disease subgroups (asthma, COPD, HIWS, CVD) regarding smoking cessation. The highest rates of smoking cessation were observed during the initial month of intensive follow-up. Consistent with numerous studies, the smoking cessation rates at six months were 10%. When we evaluated our study together with other studies, we thought that motivational interviewing was effective in smoking cessation treatment. We interviewed our patients for smoking cessation support, and at the end of the first month, we achieved success in about a quarter of them. At the end of the sixth month, this success rate decreased to around 10%. Considering that patient interviews were made more frequently in the first months, it is suggested that these rates may not change if patient follow-ups are made frequently in other months. Smoking cessation success was higher in the female population than in males. In addition, the smoking cessation rate was higher in the first months in groups with respiratory disease. Throughout the entire study, the asthmatic patient group showed a higher smoking cessation success than the other groups. It is evident that supporting motivational interviewing with psychosocial and quality-of-life support tests would increase smoking cessation. | Review | biomedical | en | 0.999997 |
PMC11698543 | Hypokalemia-induced rhabdomyolysis caused by primary hyperaldosteronism is a rare occurrence. Primary hyperaldosteronism, also known as Conn's syndrome, is typically due to bilateral adrenal hyperplasia (BAH) or, less commonly, an adrenal adenoma. Hyperaldosteronism leads to sodium retention and potassium excretion. It mostly presents as hypertension, although a minority of patients may experience symptoms of hypokalemia, such as muscle weakness, cramps, and fatigue . A severe decrease in serum potassium levels can disrupt the skeletal muscle vascular dilation system, which results in ischemia and necrosis of the muscle fibers, leading to rhabdomyolysis. Rhabdomyolysis classically manifests as progressive weakness, fatigue, and dark urine due to the release of myoglobin and other toxic intracellular elements such as creatine kinase (CK) and lactic acid. Myoglobin has the potential to cause acute kidney injury (AKI), and in severe cases, rhabdomyolysis can also be fatal. More common causes of rhabdomyolysis include traumatic muscle injuries, exertion due to exercise or seizures, and alcohol abuse . In this case, the patient used a massage gun to relieve the soreness in her legs, the use of which has been reported to result in rhabdomyolysis . However, further investigation revealed a left-sided adrenal adenoma, which, after being surgically removed, led to the resolution of her symptoms, suggesting that the causative factor in this case was hyperaldosteronism, leading to hypokalemia. We present the case of a 37-year-old female with a prior history of hypertension, diabetes, and schizoaffective disorder. She was also a known case of chronic hypokalemia secondary to primary hyperaldosteronism. She presented to the Emergency Department (ED) with complaints of bilateral lower extremity weakness for seven days, along with fatigue. She had been using a massage gun extensively to alleviate the discomfort in her legs. A day before her presentation, she experienced extreme weakness and was unable to hold up her weight and fell on her lower back. She denied other symptoms like fever, dizziness, abdominal pain, cough, shortness of breath, or headache. However, a few nights ago, she experienced chest discomfort without pain and was unable to fall asleep. A year ago, during her pregnancy, she had multiple presentations with similar symptoms of weakness. Her lab results showed raised aldosterone levels (113 ng/dL, reference values < 3.0-23.2 ng/dL), decreased plasma renin levels (0.59 pg/mL/hour, reference values ≤33.2 pg/mL/hour), and decreased serum potassium levels (3.0 mmol/L, reference values 3.5-5.1 mmol/L), for which she received both IV and oral potassium chloride (KCl). She was discharged with a prescription for KCl supplements, which she had run out of a week prior to her ED visit. She had not obtained a refill due to her upcoming appointment with an endocrinologist. Blood work at her current presentation showed an initial serum potassium level of 2.6 mmol/L and a raised CK level (9,306 U/L, reference values 20-170 U/L). Blood pressure was 153/80 mmHg. EKG showed U waves . A computed tomography (CT) lumbar spine scan was done in the ED to evaluate the bilateral lower extremity weakness, which revealed a 3.3 cm low-density cystic mass arising from the left adrenal gland versus the superior pole of the left kidney . She received 3 L of normal saline (NS) in the ED and KCl three times. Her potassium level dropped to 1.9 mmol/L, and she received IV KCl twice and 80 mEq orally; however, her potassium further dropped to 1.7 mmol/L, and she was admitted to the stepdown unit (SDU). In the SDU, endocrinology was consulted, and the patient was started on D5-half NS with 40 mEq KCl and 100 mg spironolactone. Serum potassium remained low despite aggressive repletion, and CK levels continued to rise despite aggressive hydration and no further massage gun usage. CK levels rose to a maximum of 22,000 U/L and began to downtrend when potassium started to normalize in a sustained manner (Table 1 and Table 2 ). Her erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were minimally elevated, ruling out inflammatory myopathy. The patient was discharged once stable and had a follow-up with an endocrinologist and endocrine surgeon. A month later, she had a dedicated CT abdomen with contrast, which showed a 3.2 cm left adrenal nodule with precontrast, dynamic, and delayed phase imaging as 16, 81, and 36 Hounsfield units, respectively. The relative percent washout value was greater than 40% and was compatible with adenoma . The right adrenal gland was unremarkable. Her latest potassium level was 4.8 mmol/L with potassium supplementation and spironolactone. Laparoscopic adrenalectomy was then performed, after which her potassium and aldosterone levels returned to normal. Histopathology report further supported the diagnosis of an adrenal adenoma due to positive melanin A and calretinin, focal weak positive synaptophysin, and negative chromogranin and paired-box gene 8 (PAX8). After her discharge, she had no recurrence of her symptoms. Rhabdomyolysis is a pathological condition in which skeletal muscle breakdown leads to the release of muscle-breakdown products such as CK, myoglobin, lactic acid, and lactate dehydrogenase (LDH) into the bloodstream . The causes of rhabdomyolysis are typically categorized as physical and non-physical. Physical causes include crush syndrome in approximately 37% of cases, intense physical activity, e.g., strength training, which makes up about 38% of cases related to physical activity, and muscle exertion due to seizures . Non-physical causes include multiple factors such as chronic alcoholism, drugs like cocaine and amphetamines, medications like statins, infections, inflammatory myopathies, heat stroke, neuroleptic malignant syndrome (NMS), and genetic disorders of glycogen storage like McArdle's disease . During pregnancy, women are at risk of hypokalemia-induced rhabdomyolysis due to causes such as hyperemesis-induced hypokalemia and underlying distal renal tubular acidosis, which can be unmasked in pregnancy . There has also been a reported case of rhabdomyolysis following intravenous iron sucrose administration during pregnancy . Meanwhile, in non-pregnant adults, iron-dextran and ferric gluconate supplements have led to rhabdomyolysis . In this case, the patient had no history of anemia or iron supplementation during or after her pregnancy. She reported using a percussion massage gun to alleviate cramps and muscle weakness, the use of which has been attributed as a cause of rhabdomyolysis . However, the discovery of an adrenal adenoma and the subsequent resolution of her symptoms suggest that the adrenal pathology was most likely the causative factor. Rhabdomyolysis, the breakdown of muscle tissue, releases potassium into the bloodstream, leading to hyperkalemia; it also releases toxic substances like myoglobin, which can result in multi-organ failure, with AKI being the most serious complication. Increased levels of myoglobin can cause up to 40% of patients with rhabdomyolysis to develop acute renal failure . A commonly used clinical definition for rhabdomyolysis is CK levels almost five times the normal limit, and typically, CK levels >5,000 are correlated with severe muscle injury and AKI . This patient was diagnosed as having rhabdomyolysis due to extremely high CK levels (9,306 U/L) on presentation, which further rose to a maximum of 22,000 U/L. Rhabdomyolysis associated with hypokalemia is a rare presentation, as hyperkalemia is generally the expected outcome of muscle breakdown. Typically, rhabdomyolysis occurs when potassium levels fall below 2.0 mmol/L . Hypokalemia can be due to several causes; these include medications such as diuretics and laxatives, diarrhea, renal diseases like types I and II renal tubular acidosis, inadequate nutrition, and insulin overdose . Low potassium levels lead to ECG findings like T-wave and ST segment depression, QT-prolongation, and the appearance of a U-wave - potentially causing fatal cardiac arrhythmias . Other complications may include intestinal and respiratory paralysis . Hypokalemia usually presents as progressive muscle weakness, more commonly in the lower limbs, as seen in this patient, along with other symptoms such as fatigue, constipation, and palpitations . Another cause of hypokalemia is hormonal imbalance, particularly that of aldosterone. This can result from primary hyperaldosteronism, also known as Conn Syndrome, which may be caused by either an adrenal adenoma (30%) or BAH (60%) . Secondary hyperaldosteronism is usually due to hyperactivity of the renin-angiotensin-aldosterone system (RAAS), usually due to a renin-producing tumor. Excessive aldosterone causes sodium retention and potassium excretion due to the activation of mineralocorticoid receptors on renal cells, which then leads to sodium reabsorption via the epithelial sodium channels (ENaC) on the luminal side of the cortical collecting duct and potassium excretion via the sodium-potassium exchange pumps. Sodium reabsorption leads to water retention, which, alongside aldosterone's direct action of vasoconstriction, causes hypertension . In the majority of cases, potassium levels are within the normal range. Because hypokalemia is observed in only about 28.1% of patients with hyperaldosteronism, this case report highlights how unusual it is for hypokalemia to be severe enough to result in rhabdomyolysis . One proposed explanation for the connection between hypokalemia and rhabdomyolysis is that potassium plays a role in dilating arterioles in skeletal muscles, particularly during physical activity. This dilation is impaired when serum potassium levels are low, resulting in reduced blood flow and relative ischemia in the muscle fibers . Another theory suggests that hypokalemia inhibits the production and storage of glycogen within cells and disrupts normal ion movement across cell membranes . Given the vital role potassium plays in maintaining normal physiological functions like muscle contraction and normal heart rhythm, inpatient management of such patients is crucial to replete total body potassium stores and correct serum potassium levels to prevent them from developing critical illnesses such as muti-organ failure and AKI. Primary hyperaldosteronism leading to hypokalemia is a rare but important cause of rhabdomyolysis and should be considered as a differential when patients present with muscle weakness and lab findings consistent with rhabdomyolysis. Therefore, physicians must maintain a high degree of clinical suspicion for this diagnosis, especially in the setting of muscle weakness, elevated CK levels, and low serum potassium. Recognizing the potential link between hypokalemia and rhabdomyolysis is essential for guiding further investigation and ensuring appropriate management of affected patients. Early recognition and intervention can prevent serious complications, ultimately improving patient outcomes. | Clinical case | biomedical | en | 0.999997 |
PMC11698544 | Craniosynostosis is a congenital condition in which one or more of the sutures in the skull fuse prematurely, restricting skull growth and leading to an abnormal head shape. These sutures are normally flexible joints that allow the skull to expand as the brain grows; however, when they fuse too early, the skull cannot grow in the affected areas, resulting in compensatory growth in other regions. Oxycephaly is a rare form of craniosynostosis that causes the skull to have a cone or tower-shaped appearance. It occurs due to the premature fusion of the coronal and sagittal sutures, which prevents the skull from growing normally in width . This condition is typically identified shortly after birth and is often treated with surgical intervention aimed at reducing pressure on the brain and correcting the abnormal skull shape. The management of craniosynostosis has seen significant advancements in recent years, with innovations in both surgical techniques and supportive care. Traditional open cranial vault remodeling, once the primary approach, has been refined to improve outcomes and reduce recovery time. If left untreated, oxycephaly can lead to developmental delays and blindness . We present this case to highlight key aspects of late-presenting multi-suture craniosynostosis and its management. A five-year-old girl, born preterm at 35 weeks via lower-segment cesarean section (LSCS) from a non-consanguineous marriage, had a birth weight of 1.7 kg. She presented with a history of frequently rubbing her right eye. Although her mother did not observe any signs of visual impairment, she had noted an abnormal head shape since birth. The child had no significant systemic history, and her developmental milestones were achieved on time without any delays. General examination revealed alert child with GCS 15. Physical examination revealed a high, peaked forehead and a shortened, pointed head shape indicative of oxycephaly . Notable craniofacial features included maxillary recession, a trapezoid-shaped skull with right facial scoliosis, bony nasal septal deviation to the left, and right brow asymmetry. The recorded anthropometric measurements included a head circumference of 44 cm (normal range: 49-51 cm), a weight of 26 kg (normal range: 15-23 kg), a height of 136 cm (normal range: 105-115 cm), and a calculated body mass index (BMI) of 14.1 kg/m² (normal range: 14-17 kg/m²). The ocular assessment revealed a best corrected visual acuity (BCVA) of 1/60 in the right eye and 6/6 in the left eye, with bilateral proptosis. The right eye exhibited defective color vision and generalized field depression. Fundus examination revealed a pale disc with peripapillary infarction in both eyes . CT brain imaging with 3D reconstruction confirmed pan-sutural craniosynostosis, showing a flattened forehead, prominent pre-coronal hump, and inner calvarial table scalloping, all indicative of raised intracranial pressure (ICP). Contrast-enhanced MRI (CEMRI) with MRV and MRA revealed normal brain architecture but severe optic nerve sheath edema. MRV demonstrated partial transverse sinus hypoplasia with collateral circulation & venous shunts, suggesting long-standing raised ICP . Exposure keratitis was identified as an early postoperative complication, with signs observed on the first postoperative day, including mild conjunctival hyperemia, punctate epithelial erosions, and mild lagophthalmos. Management included the use of lubricating eye drops and ointment, along with a temporary tarsorrhaphy, which was removed after one month. These interventions effectively addressed the ocular surface concerns during the early postoperative period. The follow-up schedule for the study included weekly evaluations for the first month, followed by monthly visits for three months, and subsequently monitored every six months till date. Post-surgery, the patient's vision improved to 6/24 in her right eye at the end of one year follow-up . Craniosynostosis is a relatively rare condition that affects approximately 1 in 1000 infants . Craniosynostosis is a developmental disorder characterized by the premature fusion of cranial sutures, which restricts normal skull and brain growth. Typically, the sutures in an infant’s skull remain open to allow the skull to expand as the brain grows. However, early fusion results in restricted growth perpendicular to the fused sutures (Virchow's law), causing cranial deformities, increased intracranial pressure, and potential neurological deficits . This condition is due to abnormal development of the primitive mesoderm. Recent studies have highlighted a concerning rise in the occurrence of craniosynostoses, a medical condition where the skull sutures fuse prematurely. The increase in craniosynostosis cases is thought to result from both genetic and environmental factors . Environmental risks include fetal head constraint, abnormal fetal position, low amniotic fluid, exposure to teratogens, maternal smoking, and specific medications during pregnancy . About 20% of craniosynostosis cases have a genetic basis, often due to autosomal dominant FGFR (Fibroblast growth factor receptor) mutations-FGFR1 in Pfeiffer's syndrome and FGFR2 in Apert’s and Crouzon’s syndromes . Non-inherited mutations, as observed in our case, account for approximately 50% of cases. Craniosynostosis primarily affects the sagittal suture (60%), followed by the coronal (25%), metopic (15%), and lambdoid sutures (2%) . In our case, the patient presented with premature pansutural craniosynostosis, leading to a tower-shaped skull due to restricted transverse brain growth. The absence of developmental delay, focal neurological deficits, or feeding problems made this condition challenging to detect during infancy. Furthermore, measuring head circumference during routine pediatric vaccination visits can aid in the early detection of craniosynostosis. Regular vision screenings conducted by school teachers play a crucial role in identifying any problems at an early stage and preventing complications like amblyopia. Interdisciplinary team care is important for diagnosis and treatment. Ophthalmic manifestations of craniosynostosis include optic atrophy, exophthalmos, exotropia, nystagmus, and mental deficiency . Optic atrophy is one of the most concerning ocular consequences of craniosynostosis. It occurs due to increased intracranial pressure, mechanical traction, compression, and narrowing of the optic foramen . The occurrence of asymmetric vision loss may stem from disparate sizes of optic canals, as depicted in our case. The primary goals of treatment are to ensure normal brain development and achieve a cosmetically acceptable appearance. The optimal time for surgery is debated, but it is generally considered between six and twelve months of age . The type and extent of surgery depend on the patient's age and presentation. The options for surgical intervention include open craniotomy and reconstruction or an endoscopic procedure. Endoscopic intervention is more suitable until the age of 6 months when cranial bones are still flexible. After surgery, additional corrections with helmets may be needed for four to six months. After six months, open surgery is preferred due to bone stiffness as done in our case. In particular, frontal advancement open surgery is an effective surgical method for expanding the cranial vault, regardless of age . This procedure involves removing the fused sutures and reshaping the forehead, which can significantly reduce the pressure on the optic nerve. Ideally, surgery should be performed in the first year of life . In craniosynostosis surgery, specifically frontal orbital advancement combined with posterior vault remodeling, several factors regarding its benefits and potential complications must be considered. This combined approach addresses both anterior and posterior cranial vaults, facilitating balanced expansion to accommodate normal brain growth while significantly improving craniofacial aesthetics by correcting deformities and enhancing facial symmetry. Additionally, it reduces the risk of elevated intracranial pressure, thereby preventing neurodevelopmental complications. Despite the improved safety of craniosynostosis surgery, complications remain a concern . A study conducted by Esparza et al. analyzed the complications associated with the surgical treatment of craniosynostosis and craniofacial syndromes involving 306 transcranial procedures . The most frequent complication was postoperative hyperthermia, occurring in 13.17% of cases, followed by infections (8.10%), subcutaneous hematomas (6.08%), dural tears (5.06%), and cerebrospinal fluid leakage (2.7%). Notably, endoscopic-assisted osteotomies reported the lowest complication rate at 2.5%, while complete cranial vault remodeling had the highest rates of complications. Despite these complications, all cases were resolved without permanent deficits, highlighting the effectiveness of surgical interventions . Endoscopic correction offers certain advantages over open cranial vault reconstruction, including shorter surgery times, reduced blood loss, and quicker recovery periods, with fewer ICU and hospital stays. However, endoscopic techniques often require postoperative use of helmet-molding orthoses for several months to guide proper bone alignment and prevent suture refusion as the brain continues to grow. Both surgical approaches require long-term follow-up to monitor for late complications such as irregular bone contour, incomplete bone healing, or refusion of the sutures, which may necessitate reoperation. These considerations highlight the importance of tailored surgical planning and diligent postoperative care to optimize outcomes and minimize risks. However, even if the diagnosis is made at a later stage, the prognosis can be improved through intervention as seen in our case. The study by Gutierrez-Pineda F et al. demonstrated a 1% reoperation rate, a 95% success rate for achieving excellent aesthetic outcomes, and an 86% requirement for transfusions during procedures. Furthermore, the pooled complication rate was 2%, underscoring the minimal morbidity and highlighting the overall safety and efficacy of these surgical techniques . Fortunately, our patient experienced no serious complications post-surgery. Regular follow-ups are essential to monitor head growth, detect increased ICP, and identify any potential issues. | Clinical case | biomedical | en | 0.999997 |
PMC11698545 | Nephrogenic bladder adenoma is a rare benign metaplastic lesion of the urothelium, also called nephrogenic adenoma, mesonephroid metaplasia, or adenomatous metaplasia . Although it is most often observed in the urinary bladder (68.6%), it can also affect other parts of the urinary tract, such as the urethra (13.3%), ureter (8.2%), renal pelvis (8.2%), and in rare cases, the prostate (2%) . Research shows that environmental factors and specific lifestyle behaviors, such as cigarette smoking, can contribute to its development . This condition is often associated with previous inflammatory processes in the bladder, such as recurrent infections, inflammation caused by bladder stones, foreign bodies, intravesical therapy, chemical exposure, radiotherapy, or previous bladder surgery . Nephrogenic adenoma is of particular importance due to the differential diagnosis, which can favor clear cell adenocarcinoma, endocervicosis, papillary urothelial carcinoma, prostatic adenocarcinoma of the bladder, and nested variant of urothelial carcinoma. Nephrogenic adenoma manifests clinically with nonspecific symptoms of the urinary tract, with irritative symptoms such as urgency and frequent urination being the most common. Hematuria is uncommon . An altered mucosal appearance, which is typically characterized by vesicles and bullae, is typically revealed by cystoscopic examination. This appearance can be closely resembling that of papillary carcinoma of the urinary bladder . Histologically, nephrogenic adenoma is defined by tubular and papillary formations lined by low cuboidal to columnar epithelial cells. Its immunohistochemical profile can vary but is often marked by positivity for paired box (PAX) 2, PAX8, and cytokeratin 7 (CK7), along with negativity for p63 and prostate-specific antigen (PSA) . An 84-year-old female patient presented for examination with a history of hematuria and stated that she had been passing sandlike granules in her urine for the past few months. It also provides anamnestic data on recurrent urinary infections. Urinalysis revealed a significant number of leukocytes and 5-10 erythrocytes per high-power field. Echosonographic examination of the urinary tract revealed bilateral microcalculosis up to 6 mm as well as hyperechoic thickening with a diameter of about 10 mm on the right side wall of the urinary bladder . Rigid cystoscopy, performed based on prior sonographic findings, showed bullous and hyperemic changes on the right lateral wall, partially covered by fibrin deposits, which were suggestive of a tumorlike lesion . Transurethral resection of the lesion was subsequently performed, along with electrocauterization of the surrounding mucosa. Histopathological examination, including hematoxylin and eosin (H&E) and immunohistochemical staining (AE1/AE3, CK7, CK20, CD34, p63), confirmed the diagnosis of nephrogenic adenoma vesicae urinariae, characterized by chronic inflammatory infiltrates and metaplastic mucosa forming papillary, cystic, and tubular structures lined by a single layer of cuboidal cells with minimal atypia . At the first follow-up, one month after surgery, the patient reported no symptoms. Urinalysis, which included checking the appearance of color, clarity/turbidity, pH, specific gravity, glucose, ketones, nitrites, leukocyte esterase, bilirubin, urobilinogen, blood, protein, erythrocytes, leukocytes, squamous epithelial cells, crystals, and bacteria, was normal. Microbiological analysis, urine culture, was also performed, which was free of bacterial growth, while urine cytology did not show the presence of abnormal cells. Follow-up with rigid cystoscopy was planned every three months, followed by periodic urinalysis and culture. Although significant bacteriuria was occasionally detected, the patient remained asymptomatic and was treated with non-antibiotic preparations. Nine months after the first surgery, cystoscopy revealed hyperemic bullous-cystic changes on the posterior wall and roof of the bladder as well as near the scar from the previous surgery. These lesions measured up to 10 mm and bled easily when manipulated. A second transurethral resection was performed, and histopathological findings were consistent with the previous diagnosis of nephrogenic adenoma vesicae urinariae. This case highlights the importance of accurate histopathological diagnosis and careful follow-up of patients with nephrogenic adenoma due to the lack of standardized treatment protocols. An accurate diagnosis reduces potential diagnostic confusion, as changes that mimic other lesions such as urothelial carcinoma or chronic inflammation can be seen. It reduces the need for additional diagnostic procedures such as imaging and invasive biopsy. It reassures patients and allows them to focus on an appropriate treatment plan, thereby ensuring the preservation of urinary tract function. Misdiagnosis can lead to delays in appropriate treatment and can cause significant psychological distress, leading to anxiety and uncertainty about the prognosis. However, overtreatment of a benign lesion could result in unnecessary surgical risks, prolonged recovery time, and potential complications. Vemulakonda et al. reported a case of recurrent nephrogenic adenoma of the bladder in a 10-year-old boy with a history of plum syndrome. They stated in their opinion that their reported case was the first reported case of recurrent nephrogenic adenoma in a patient with a history of plum belly syndrome. While most cases of nephrogenic bladder adenoma have a benign and unrepeatable biological behavior, the conclusion drawn from this report indicates that regular follow-up cystoscopies would be necessary in order not to miss a recurrent nephrogenic bladder adenoma. Boscolo-Berto et al. described a case in which a patient experienced three relapses within the same diverticulum, with an identical pathohistological finding of a nephrogenic adenoma that was treated with transurethral resections. Since they considered the nephrogenic adenoma to be a benign lesion without any direct evidence of possible evolution into malignancy, they conducted several years of cystoscopic follow-up. Their work is also one of the first reports of a nephrogenic adenoma showing recurrence, but the first case of a highly recurrent nephrogenic adenoma in the same diverticulum that was treated conservatively with routine transurethral resection. Hartmann et al. documented the case of a 70-year-old woman who experienced multiple recurrences of nephrogenic metaplasia in the bladder, which eventually led to the development of clear cell adenocarcinoma. The results of the molecular studies they conducted suggested the clonal evolution of nephrogenic metaplasia to the appearance of cellular adenocarcinoma in this case. Hungerhuber et al. reported a 25-year-old man who had trauma to the urinary bladder caused by a traffic accident. After the patient recovered from the accident, he developed a nephrogenic adenoma and recurrent urinary tract infections. Since the accident, he had a nephrogenic bladder adenoma for 18 months. The adenoma was treated several times with transurethral resections. Initial pathological findings were benign; however, the last resection revealed that the previously benign adenoma had transformed into a moderately differentiated bladder adenocarcinoma. Dhaliwal et al. present a rare case of clear cell adenocarcinoma arising in the area of nephrogenic metaplasia. This is an unusual case because the morphology of nephrogenic metaplasia has been seen to change over time with the subsequent development of clear cell adenocarcinoma. Nephrogenic adenoma, though benign, presents notable clinical challenges due to its rare but documented risk of malignant transformation and potential for recurrence. Its unpredictable nature combined with the lack of standardized treatment protocols underscores the need for careful, long-term monitoring. While transurethral resection remains the main treatment option, it is not always definitive, as recurrence can still occur even after complete resection. To improve management strategies that address both its benign and possible malignant aspects, further research is needed to better understand the underlying pathogenesis of nephrogenic adenoma. | Review | biomedical | en | 0.999996 |
PMC11698547 | Carotid body tumors (CBTs), also known as paragangliomas or chemodectomas, are uncommon neuroendocrine neoplasms that develop in glomus cells generated from the embryonic neural crest close to the carotid bifurcation. CBTs are reported to occur in 1-2 individuals per 100,000 people . Most of these tumors are asymptomatic and are typically discovered incidentally during radiological imaging or clinical examination. However, in symptomatic cases, discomfort, dysphagia, and autonomic dysfunction are the most commonly reported symptoms. The diagnosis of paragangliomas is primarily based on imaging, with computed tomography angiography (CTA) being the examination of choice. If a paraganglioma is suspected, a biopsy is contraindicated due to the high risk of hemorrhage . The preferred treatment is surgical excision, although embolization may also be performed to reduce tumor size and minimize bleeding during surgery . In cases where surgery is not feasible or necessary, radiotherapy may be considered . Through this article, we aim to contribute to the collective understanding and knowledge surrounding the incidental discovery and diagnosis of this rare, slow-growing CBT. A 75-year-old woman with a 34-year history of diabetes and hypertension presented to the emergency room (ER) after experiencing multiple episodes of nausea and vomiting over the past 24 hours. She also reported a severe headache but denied any chest pain, abdominal pain, diarrhea, or dysuria. Upon admission, her blood pressure was 196/134 mmHg, and her heart rate was 140 beats per minute (bpm). The electrocardiogram (EKG) revealed atrial fibrillation (AF) with a rapid ventricular rate. She denied any prior history of AF and reported being compliant with all her medications with regular follow-ups with her primary care physician. She also reported no history of fever, cough, weight loss, anorexia, night sweats, diaphoresis, palpitations, dizziness, lightheadedness, or syncopal episodes. Physical examination showed elevated jugular venous pressure (JVP) with normal cardiac auscultation, and the rest of the systemic examinations were unremarkable. Initial blood tests, including a complete blood count, renal function tests, liver function tests, and thyroid function tests (Table 1 ) were all within the normal limits. Additionally, a urine toxicology screen was negative for cannabinoids, amphetamines, or cocaine. The chest X-ray revealed no acute abnormalities. Similarly, the contrast-enhanced CT scan of the thorax and abdomen was normal. The CT scan of the head with contrast also indicated no acute abnormalities, except for an unclear submandibular mass. Further evaluation with a contrast-enhanced CT scan of the neck and an MRI of the neck with and without contrast revealed a stable mass measuring 38 x 53 x 15 mm on the left side, consistent with a carotid body paraganglioma (CBP) . This mass had remained stable in size compared to the previous examination seven years ago. Given the patient's newly diagnosed AF and hypertension, concern arose about a functionally active tumor. The hormonal evaluation showed elevated levels of spot plasma and urine metanephrines, while an abdominal CT scan showed no adrenal tumors. Her home medications of lisinopril 40 mg and amlodipine 10 mg were resumed. Despite being on two antihypertensives, her blood pressure remained at 180/94 mmHg, with a heart rate of 140 bpm. She was initially treated with intravenous (IV) metoprolol, and once her blood pressure and heart rate stabilized, she was switched to oral metoprolol. A conservative dosage of 12.5 mg metoprolol was given twice daily, with gradual increases based on her tolerance. Nausea was managed appropriately, and her electrolytes were optimized, allowing for diet advancements as tolerated. Given her age and associated comorbidities, radiotherapy was planned; however, the patient declined the treatment. A paraganglioma is a type of tumor that arises from paraganglia, which are clusters of neuroendocrine cells derived from neural crest cells. These tumors commonly arise in areas with abundant paraganglia, such as the carotid body, the vagus nerve in the head and neck, the jugular foramen, the middle ear, the aortopulmonary window, and the organ of Zuckerkandl . Among these, the carotid body is the most significant as it contains the largest concentration of paraganglia in the head and neck. This structure functions as a chemoreceptor, primarily monitoring oxygen levels in the blood. The carotid body helps regulate vital autonomic functions by initiating a sympathetic nervous system response to detect arterial hypoxemia. Typically, the carotid body measures around 3-5 mm, though it can grow over 8 cm in response to persistent hypoxic conditions . As a chemoreceptor, the carotid body responds to acidosis, hypoxia, and hypercapnia, playing a vital role in regulating blood pressure, heart rate, respiration, and blood temperature through increased sympathetic nervous system activity . CBTs account for approximately 65% of all paragangliomas in the head and neck . These tumors can manifest at any age, though they are most commonly seen in individuals aged 50-70 years, with a range from 18 to 94 years. Women are slightly more affected than men, with a male-to-female ratio of 1:1.9 . CBTs are typically sporadic, with around 25% having a hereditary basis due to mutations in succinate dehydrogenase genes, including SDHD, SDHB, and SDHC . Genetic mutations are the only identified risk factor for the development of CBTs, aside from hypoxic stimulation, and familial cases often present with bilateral CBTs at a higher frequency . CBTs and paragangliomas in the head and neck are typically painless, slow-growing tumors that often remain undetected for years before patients seek medical attention. The tumors can grow to significant sizes and may exhibit infiltrative growth, with local recurrence posing a risk of fatality. Five percent of paragangliomas secrete catecholamines (dopamine, epinephrine, and norepinephrine), with hypertension being the most common manifestation. This hypertension can be continuous, intermittent, or paroxysmal . The classic triad of headache, sweating, and palpitations occurs in 40% of cases . Dopamine-producing paragangliomas, which are rare, can present with normal blood pressure or even hypotension. Limited case reports suggest that CBTs, like pheochromocytoma, may present with AF. In cases of elevated catecholamine levels, additional imaging is recommended to exclude intra-abdominal tumors. Malignancy rates range from 10% to 50%. Histologic features indicating malignancy include necrosis, extensive capsular or vascular invasion, increased mitotic activity, atypical mitotic figures, loss of a well-differentiated zellballen pattern, reduction in the S-100 positive sustentacular cell population, and tumor cell spindling . CBTs are most commonly discovered incidentally during imaging studies or through clinical assessments. The most effective screening method for CBTs is a color-flow carotid duplex, where CBTs are identified by a well-defined hypoechoic mass causing the splaying of the internal carotid artery (ICA) and external carotid artery (ECA). Color Doppler imaging reveals hypervascularity within the lesion with a low-resistance flow pattern. Surgical removal depends on the tumor's relationship with the artery bifurcation and its proximity to cranial nerves, which can be accurately assessed through CTA or MR angiography (MRA). MRI is often more precise than CT due to its lack of ionizing radiation. Dynamic contrast-enhanced MRI and diffusion-weighted imaging (DWI) are particularly effective in distinguishing paragangliomas from other benign neoplastic tumors . Radical surgical resection is performed to prevent malignant transformation . The preoperative adrenergic blockade is recommended, while preoperative embolization remains controversial due to potential risks . Although embolization may be considered for large tumors, it increases the risk of transient ischemic attacks and ischemic strokes . Radiotherapy, recommended for patients with various medical conditions at risk during general anesthesia, has been shown to reduce tumor size or halt its growth. This case involves a 75-year-old woman with carotid body paraganglioma (CBP), complicated by new-onset AF and severe hypertension. While Hoang et al. focused on the presentation and surgical management of CBTs, this case offers novel insights into the association between paragangliomas and cardiovascular complications like AF, which are rarely documented . This case discusses the successful management of AF and severe hypertension using IV and oral metoprolol, illustrating the importance of careful cardiovascular management in patients with functionally active paragangliomas. This approach, particularly in an older patient with multiple comorbidities, adds value to the literature by focusing on the nonsurgical management of cardiovascular symptoms in CBT patients. Compared to the case review by Hoang et al. , which focused on more typical symptoms of CBTs (e.g., neck mass, dysphagia, and hoarseness), this case demonstrates an atypical presentation without overt neck swelling, hoarseness, or swallowing issues. This challenges the assumption that CBTs present predominantly with local symptoms, expanding the understanding of the disease’s clinical spectrum. This report contributes to the literature by documenting the occurrence of AF, which has not been commonly emphasized in previous case studies of paragangliomas, including the study by Hoang et al. . The link between paragangliomas and secondary cardiovascular complications, particularly in the absence of adrenal tumors, is an emerging area that this case explores, offering an update on potential complications that should be monitored. The detailed hormonal workup and the discovery of elevated plasma and urine metanephrines underscore the importance of assessing functional activity in suspected paragangliomas. This reinforces the need for comprehensive hormonal evaluation, an aspect that may not have been emphasized in earlier reports. This finding is particularly relevant for clinicians when considering differential diagnoses for patients with new-onset AF and uncontrolled hypertension. This article shares our insights and experiences in managing CBTs, which are rare, slow-growing, hypervascular neuroendocrine tumors with the potential for malignant transformation. Diagnosis typically involves a thorough assessment, including patient history, physical examinations, and radiological studies. This report presents an older patient with CBP and new-onset AF, highlighting the need for further research into the cardiovascular manifestations of paragangliomas. | Review | biomedical | en | 0.999997 |
PMC11698549 | Clostridium difficile is a spore-forming, Gram-positive, anaerobic bacillus ubiquitously found in the intestinal tracts of humans and animals, as well as in various environmental settings. Transmission occurs via the fecal-oral route through the ingestion of spores . Healthcare environments often facilitate transmission through contaminated surfaces and the hands of healthcare workers . C. difficile infection (CDI) is a leading cause of antibiotic-associated diarrhea, predominantly acquired as a nosocomial infection . Since the late 20th century, CDI rates have risen markedly, particularly among individuals with recent or ongoing hospitalizations, with prevalence rates ranging from 3% to 26% . In India, CDI has emerged as a significant cause of antibiotic-associated diarrhea, with reported prevalence rates of 5-20% in the western and central regions . CDI is a toxin-mediated gastrointestinal disease caused by the exotoxins A and B, produced by C. difficile . The clinical spectrum ranges from mild, watery diarrhea and abdominal pain to severe conditions like life-threatening colitis . Potential complications include toxic megacolon and intestinal perforation . Risk factors for CDI include broad-spectrum antibiotic use, advanced age, prolonged hospitalization, and colonization by C. difficile . CDI imposes a substantial burden on healthcare systems by prolonging hospital stays, increasing ICU admissions, and raising treatment costs. It also contributes significantly to morbidity and mortality, with all-cause mortality rates estimated between 13% and 30% . While some patients recover with prompt treatment, others face challenges such as recurrent infections . Effective prevention of CDI requires timely case detection and the implementation of comprehensive prevention strategies . Case detection involves identifying clinical symptoms, particularly diarrhea defined as three or more loose or unformed stools within 24 hours , alongside diagnostic methods like stool culture, enzyme immunoassays, or PCR to detect C. difficile toxins or their genes . Treatment typically involves antibiotics such as vancomycin, fidaxomicin, and metronidazole, although recurrence rates remain high, necessitating novel approaches like fecal microbiota transplantation for recurrent cases . CDI represents a significant global health challenge, with increasing rates of community-acquired infections. Its impact on healthcare systems, particularly through heightened mortality, underscores the importance of understanding its burden in both hospital and community settings. However, studies on CDI in the eastern region of India remain limited, despite its unique sociodemographic and healthcare dynamics. The 2021 ICMR national surveillance study reported that less than 5% of its data originated from eastern India, highlighting significant knowledge gaps regarding CDI's epidemiology and burden in this region . Determining the incidence of CDI and identifying the factors associated with infection among hospitalized patients can provide valuable insights for healthcare workers and stakeholders. These findings can guide the implementation of effective diagnostic methods, optimize therapeutic strategies, and strengthen infection control practices to mitigate the burden of CDI in healthcare settings. A prospective observational study was conducted over six months, from October 2022 to March 2023, after obtaining Institutional Ethics Committee approval . The study was carried out at the Kalinga Institute of Medical Sciences in Bhubaneswar, India. All consecutively admitted patients aged over 18 years, regardless of clinical condition or gender, and who consented to participate, were enrolled. A total of 200 patients were included in the study. Patient demographic data, including age and sex, as well as medical history, were collected via a case record form. This included information on underlying diseases such as irritable bowel syndrome, inflammatory bowel disease, type 2 diabetes mellitus, and other immune-related disorders. Clinical variables, including provisional diagnoses at admission, broad-spectrum antibiotic usage during hospitalization (such as fluoroquinolones, third- and fourth-generation cephalosporins, and carbapenems), and hospital-related variables for gastrointestinal symptoms like diarrhea, dysentery, and abdominal pain occurring after 72 hours of hospitalization, were also recorded. The duration of hospital stay was noted as well. Two stool samples were collected from each patient: the first within 24 hours of admission and the second on the day of discharge or after one week, whichever was longer. Patients were provided with wide-mouth sterile containers with lids for stool collection and were instructed not to take any laxatives the night before collection. Fresh stool samples of 10-15 grams, either formed or unformed, were placed in leakproof containers and transported to the laboratory in ice packs to maintain a temperature of 4-8°C, then stored in a refrigerator for further testing. Stool samples were processed in the microbiology laboratory. A routine microscopic examination was performed to detect fecal leukocytes or pus cells. The samples were tested for C. difficile toxins A and B using the enzyme-linked immunosorbent assay (ELISA) method in a multi-step process to confirm CDI cases. The Epitope Diagnostics ELISA kit for C. difficile Toxin A and B was used for toxin detection. The kit included all required reagents and instructions specific to toxin detection. The procedure was performed according to the manufacturer’s guidelines. At the end of the ELISA procedure, the absorbance (optical density, OD) of each microwell was measured using a Bio-Rad ELISA reader at 450 nm, with duplicate readings for each well. The average OD value for each well was calculated. Results were interpreted according to the manufacturer's guidelines: a positive result was defined if the OD value exceeded the positive cutoff (0.168), negative if the OD was below the negative cutoff (0.138), and equivocal if the OD was between the two cutoffs (0.139-0.167). For equivocal results, the test was repeated with a different sample. Clinical and laboratory data were entered into Microsoft Excel. Statistical analysis was performed using IBM SPSS Statistics for Windows, Version 26.0 . Descriptive statistics such as mean, standard deviation, and median were calculated for continuous variables. Proportions were determined for categorical variables. The Mann-Whitney U test was used to assess associations between continuous variables, while the chi-square or Fisher’s exact test was used for categorical variables. A p-value of less than 0.05 was considered statistically significant. The present study enrolled a total of 200 patients admitted to the medicine wards of a tertiary care hospital. The incidence of CDI during the study period was found to be 9% (18/200). The median age of the participants was 45.5 years, with a range from 18 to 80 years. Male patients (60%) outnumbered female patients (40%). A total of 38 patients (19%) had a history of prior hospitalization, and 22% had underlying diseases. The median duration of hospitalization among participants was four days. Broad-spectrum antibiotic exposure during hospitalization was noted in 60% of patients. After 72 hours of hospitalization, 21% of the participants developed loose stools. At the time of admission (when the first stool samples were collected), none of the samples showed the presence of pus cells on routine microscopic examination or C. difficile toxin on the ELISA test. However, stool samples collected at discharge or after one week (second stool sample) tested positive for both stool pus cells and C. difficile toxins by ELISA in 9% of cases. Table 1 provides the sociodemographic and clinical parameters of the study participants. Table 2 presents the sociodemographic and clinical profile of the C. difficile -infected cases. The mean age of the C. difficile toxin-positive patients was 50.77 ± 16.12 years, with a median age of 49 years. CDI was detected in 10% (12/120) of male patients and 7.5% (6/80) of female patients. Among participants with and without a history of prior hospitalization, CDI was found in 4/38 (10.5%) and 14/162 (8.6%), respectively. Fourteen of the 18 toxin-positive cases had no underlying comorbidities. There was no significant association between CDI-positive cases and prior hospitalization or underlying comorbidities. The median duration of hospital stay was significantly longer in ELISA-positive cases (eight days) compared to ELISA-negative cases (three days). All CDI cases (100%) were exposed to broad-spectrum antibiotics during hospitalization. After 72 hours of hospitalization, 42/200 patients developed loose stools, 28.6% of whom were C. difficile toxin ELISA positive. Stool pus cells were detected in all CDI-positive cases. The development of CDI was significantly associated (p < 0.001) with the mean duration of hospitalization, exposure to broad-spectrum antibiotics, the development of gastrointestinal symptoms, and the presence of stool pus cells. In Figure 3 , the frequency of antibiotic use among C. difficile toxin ELISA positive and negative cases is presented. Among the 200 study participants, the majority (32%) were treated with a combination of various antibiotics, followed by third-generation cephalosporins. Of the 18 CDI cases, the majority (six cases) were treated exclusively with third-generation cephalosporins, followed by a combination of antibiotics (four cases). CDI is a significant healthcare concern, primarily due to its association with antibiotic use and nosocomial transmission. This study aimed to investigate the incidence of CDI among patients attending a tertiary care teaching hospital and explore the associated risk factors and clinical characteristics. Our findings revealed that the incidence of CDI among hospitalized patients during the study period was 9%, aligning with previous studies reporting incidence rates ranging from 3% to 29% . Notably, this rate was slightly higher than that reported by Ozaki et al. , but consistent with studies by Chaudhry et al., which reported a 6% incidence rate . Interestingly, studies from India have shown considerable variation in CDI prevalence, with some reporting rates as high as 25-30% among hospitalized patients with diarrhea. In contrast, Segar et al. reported a prevalence rate of 4% in their study . These differences can be attributed to variations in population characteristics, diagnostic methods, and hospital settings. The relatively low prevalence observed in our study could be due to several factors, including selective prescribing of probiotics to patients on prolonged and broad-spectrum antibiotic therapy, stringent surveillance with an improved antibiotic policy, and the active involvement of the hospital infection control team. Additionally, we did not include ICU-admitted patients in our study, who are typically exposed to higher antibiotic use and have longer hospital stays, increasing the likelihood of CDI occurrence. Although age was not found to be a significant factor for CDI development in this study, the mean age of the C. difficile toxin-ELISA positive cases was numerically higher (50.77 ± 16.12 years) than that of the negative cases. This finding is consistent with previous studies by Vishwanath et al. and Segar et al. , which reported a higher prevalence of CDI among older patients. Advanced age (≥60 years) is a recognized risk factor for CDI, likely due to increased healthcare exposure, antibiotic use, and the presence of underlying comorbidities. However, this observation was not statistically significant in our study and should be interpreted with caution. Regarding gender distribution, our study showed a higher proportion of C. difficile toxin-positive cases among males (60%), consistent with findings by Chaudhry et al. . This finding contrasts with reports by Vaishnavi et al., suggesting variability in CDI gender distribution across different studies . Gender differences in CDI incidence may result from biological factors such as differences in immune responses and sociocultural factors like variations in healthcare-seeking behavior or antibiotic exposure. Further studies are needed to understand the factors contributing to gender disparities in CDI prevalence. In this study, cases of acute febrile illness, pneumonia, and enteric fever were associated with CDI development, likely due to the widespread use of broad-spectrum antibiotics and prolonged hospital stays. Remarkably, the majority of toxin-positive cases in our study lacked underlying comorbidities, indicating that CDI can affect individuals regardless of their health status. The mean duration of hospitalization among ELISA-positive cases was significantly longer (8.0 ± 1.53 days) than that of ELISA-negative cases (3.75 ± 1.25 days). The strong association between hospitalization duration and CDI positivity highlights the role of prolonged hospital stays as a risk factor for CDI acquisition, which is consistent with research conducted by Choudhury et al., emphasizing the importance of hospital infection control measures and antimicrobial stewardship programs . Our study highlighted antibiotic exposure as a critical risk factor for CDI, as all toxin-positive cases (100%) were exposed to broad-spectrum antibiotics during hospitalization. Third-generation cephalosporins were the most frequently implicated antibiotic class, in line with findings from studies showing an association between cephalosporin use and CDI incidence . This reinforces the need for cautious and judicious antibiotic prescribing practices to mitigate CDI risk in hospital settings. The development of gastrointestinal symptoms after 72 hours of hospitalization was significantly associated with CDI in our study, consistent with findings from previous research . Additionally, the presence of fecal leukocytes in stool samples from CDI-positive patients indicated an inflammatory response, which is commonly associated with CDI. However, studies by Reddymasu et al. have noted the poor predictability and low sensitivity of fecal leukocyte counts for CDI detection . Our study had a few limitations. The study duration was short, and the sample size was relatively small. Only ELISA was employed for CDI detection, and we only included patients from indoor wards. Including patients from all hospital wards and ICUs could provide a more comprehensive analysis of CDI trends. Furthermore, molecular testing, such as PCR for toxin gene detection, is more sensitive and specific, and using this method might yield a more accurate CDI rate. The present study provides valuable insights into the incidence rate, risk factors, and clinical characteristics of CDI among hospitalized patients. The incidence of CDI was found in one-tenth of the study population. Patients admitted with provisional diagnoses of acute febrile illnesses and pneumonia were more likely to develop CDI. Significant factors for CDI development included the use of third-generation cephalosporins and longer hospitalization durations. These findings suggest that targeted interventions are needed to reduce CDI incidence and associated morbidity and mortality. The results emphasize the importance of implementing effective infection control measures, including hand hygiene, contact precautions, surface disinfection, antimicrobial stewardship programs, and diagnostic strategies to mitigate the CDI burden in healthcare settings. Further research is needed to explore CDI diagnostics, risk factors, pathogenesis, and community-acquired CDI to develop targeted diagnostic, therapeutic, and preventive strategies. | Other | biomedical | en | 0.999998 |
PMC11698572 | Feeding practices and growth patterns during the early months of life are linked to future body mass index levels . Rapid weight increase in the initial months is a predictor of obesity in later years . Current recommendations suggest exclusive breastfeeding for the initial six months, followed by the gradual introduction of specific solid foods, which is associated with reduced fat mass in children . Introducing solid foods earlier than recommended can lead to quick weight gain during infancy, potentially impacting the risk of obesity in childhood . Infants who followed dietary guidelines closely, consuming high amounts of fresh fruits and vegetables, meals prepared at home, and breast milk, experienced more significant weight and skin fold thickness increases between six and 12 months compared to their peers, regardless of the type of milk consumed, when solids were introduced, and maternal influences . Bioelectrical impedance (BIA) is a painless, non-invasive, and easily portable technique that may clarify how the human body is operational. Body composition (BC) assessment is commonly established as a clinical technique for evaluating and estimating disease status . Strong debates are still present about the effect of timing and type of introduction of complementary food on metabolic programming and later on obesity, so this study aimed to assess the effect of feeding practices in the first two years of life on the body composition of preschool-age children and to highlight the importance of using Bioelectrical Impedance Analysis (BIA) in clinical settings for identifying individuals at risk of overweight and obesity. This is a cross-sectional study that was carried out on preschool-aged children over one year from the 1st of January 2022 to the end of December 2022. The sample size was calculated using OpenEpi, Version 3, open source calculator-SSPropor, for proportion with the prevalence of exclusive breastfeeding of 28% and a total population of 350, and a confidence limit of 5%. The calculated sample was 165. A total coverage of all eligible children yielded only 160 children (with a dropout rate of 3%). Data were analyzed using IBM SPSS software package version 25.0 (IBM Corp., Armonk, NY, USA). Qualitative data were described using numbers and percent, using the Chi-square test as well as the Fisher's exact test for variables with small expected numbers. The Kolmogorov-Smirnov test was used to verify the normality of the distribution. Quantitative data were described using range (minimum and maximum), mean, and standard deviation, and then compared using an independent t-test or Mann-Whitney test for the analysis of data between two groups. One-way ANOVA if the comparison between more than two groups, significant level when the p-value <0.05. Receiver operating characteristic curve (ROC) was generated by plotting sensitivity (TP) on the Y axis versus 1-specificity (FP) on the X axis at different cut-off values. The area under the ROC curve denotes the diagnostic performance of the test. An area of more than 50% gives acceptable performance, and an area of about 100% is the best performance for the test. The study included 160 full-term children; 91 of them were males (56.9%) with a mean birth weight of 3.00±0.48 kg. Breastfeeding was the most common feeding method in 83 children (51.9%) and the first food introduced to the children was vegetables and fruits in 98 children (61.3%) followed by yogurt in 35 children (21.9%). Regarding the time of the first food introduced, 42 children i.e. more than a quarter of children (26.25%) began complementary feeding early (before 17 weeks) while the majority of them (96 children, 60%) received complementary feeding at the optimum time. The average body composition for the subjects included 9.57 ± 1.83 kg of water, 2.54 ± 0.48 kg of protein, 0.88 ± 0.22 kg of minerals, 4.98 ± 2.07 kg of fat, and 5.67 ± 1.48 kg for skeletal muscle mass. The mean height, weight, and BMI of children were 105.23 ± 7.2 cm, 17.97 ± 3.50 kg, and 16.56 ± 1.79, respectively with 39.4% of children being overweight/obese (Table 1 ). Regarding factors affecting body composition, formula-fed children showed significantly higher body fat mass and mean BMI (p-values of 0.002 and 0.001, respectively). Neither the type nor timing of the first food introduced showed a significant difference in the body composition of children. As regards BMI classification, the percentage of infants with a normal BMI was significantly lower in the formula feeding group (40.9%) vs. in the breastfeeding (71.1%) and mixed feeding groups (50.9%). The percentage of infants who were obese or overweight was higher in the formula feeding group than in the other two groups with a significant association between being overweight/obese and formula feeding/mixed (p-value 0.003) (Table 2 ). With regards to the prognostic performance of BIA to discriminate overweight patients (n = 97) from normal BMI (n = 63), it was found that BIA has significantly excellent discrimination power for fat mass (p-value < 0.001 & AUC = 0.916). The cut-off point for body fat mass is ≥5.2, and it has a high sensitivity of 85.71% and specificity of 89.69%. The positive predictive value (PPV) is 84.4%, while the negative predictive value (NPV) is 90.6%. These results suggest that body fat mass is a good predictor for discriminating between overweight and normal BMI individuals . Breastfeeding has been observed to have a modest but consistent protective effect against obesity in children . It is a well-established fact that traditional formula milk contains higher levels of protein compared to breast milk. Protein-rich formulas are considered a factor that can accelerate plasma insulin levels and lead to the release of insulin-like growth factor-1, consequently resulting in weight gain and later obesity . Aligning with findings from the present study, Malekzadeh et al. detected that despite similar weight for age and weight-for-length Z-score (WLZ) at birth in the studied groups, WLZ was significantly higher in the formula-fed group at six months of age, indicating that formula feeding can lead to higher weight gain than breast milk in infants . Li et al. found that predominantly formula-fed infants weighed more than the exclusively human milk-fed group at term-corrected age, and their greater change in weight Z-score throughout the study was accompanied by higher non-adipose tissue mass deposition . When comparing the first food introduced (dairy product group, vegetable/fruit group, and fortified cereals group), the results indicated no significant differences in body composition measures between the three groups. A randomised trial studied the effect of feeding on body weight composition and insulin resistance and found that decreased weight was most associated with increased intake of legumes and decreased intake of total meat, fish, and poultry . Those consuming a low-fat vegan diet also increased their intake of carbohydrates, fibre, and several micronutrients and decreased fat intake. Reduced fat intake was associated with reduced body weight (r = +0.15; P = 0.02) . In the current study, the BMI of infants did not show significant differences based on the timing of food introduction. Similarly, a meta-analysis by Pearce et al. concluded that there is no clear association between the timing of introducing complementary foods and childhood overweight or obesity, although a very early introduction of solid foods (≤4 months of age) may result in an increase in childhood BMI . However, two systematic reviews did not find a clear association between the timing of CF introduction and measures of adiposity, such as skin-fold thicknesses, fat mass, and percentage fat mass measured by dual-energy X-ray absorptiometry and/or bioelectrical impedance . In the current study, the prognostic performance of BIA to discriminate overweight children from normal BMI was evaluated; the AUC for skeletal muscle mass is 0.655, indicating that it has a fair statistically significant discrimination power (P = 0.043). The cut-off point for skeletal muscle mass is ≥5.7, and it has a sensitivity of 65.08% and specificity of 52.58%. The PPV is 47.1%, and the NPV is 69.9%. These results suggest that skeletal muscle mass is a less reliable predictor than body fat mass in discriminating between overweight and normal BMI individuals. Aligning with this study, Khan et al. found that BIA machines vary in relative accuracy when measuring body composition in children who are obese and severely obese . However, two other studies in obese/severely obese children demonstrated an underestimation of body fat percentage using an SF4 BIA device (Omron Portable Fat Analyser; Omron, Dongguan, China) or an alternate MF4 BIA (Tanita MC-180MA; Tanita, Dongguan, China) device compared to DXA, with similarly wide limits of agreement (−15 to +5%). de Silva et al., using their MF4 BIA machine-generated resistance data with both published paediatric-specific and their own derived equations for estimating body fat and free fat mass, were able to reduce systematic bias to <1%, but the limits of agreement remained wide (−9 to +9%) . Breastfeeding has a protective effect against the risk of obesity in children where formula-fed children have higher body fat mass and BMI than breastfed. A greater proportion of infants who started food with cereals had a normal BMI, in contrast to groups that started with dairy products and were mostly obese. Body fat mass as detected by BIA is a good predictor for obesity, and skeletal muscle mass is less reliable for discriminating between overweight and normal BMI individuals than body fat mass in obese. | Study | biomedical | en | 0.999998 |
PMC11698603 | The temporomandibular joint (TMJ) is among the most complex joints in the human body and serves as a crucial growth site within the craniofacial complex due to its unique anatomical, histological, and biomechanical properties. Its cartilaginous tissue is capable of remodeling in response to external forces, even after growth has ceased. Therefore, the condyles are not only primary targets in orofacial orthopedics, but also key determinants of long-term stability in orthognathic surgeries. TMJ morphology can vary significantly among individuals and may even differ between the right and left sides of the same individual. Given the close relationship between form and function, orthodontists often anticipate a correlation between condylar characteristics and craniofacial morphology . The morphology and position of the TMJ are critical for the stability of orthodontic treatments and can be influenced by several factors, including age, gender, growth patterns, muscular activity, and occlusal changes . The type of skeletal pattern can impact the morphology and position of the condyle and the glenoid fossa . The ANB angle is a recognized cephalometric measurement used to evaluate the anteroposterior relationship between the maxilla and mandible. Clinically, this angle is crucial for diagnosing skeletal relationships. Its measurement provides insight into whether a patient has a Class I, Class II, or Class III skeletal pattern, which in turn influences the therapeutic approach in both orthodontics and maxillofacial surgery. The ANB angle is a key factor in clinical treatment decision-making. Some studies found significant correlations between ANB and masticatory function, including masseter muscle activity and abnormal chewing patterns. These results highlight the ANB angle's role in determining treatment type, helping clinicians decide between camouflage orthodontics and surgical intervention . Some studies have explored the correlation between serological variables and temporomandibular disorders (TMDs) in patients with systemic and chronic autoimmune diseases, such as rheumatoid arthritis (RA), which involve inflammation of the synovial joints. Certain serological factors may serve as predictive markers for specific TMDs . Imaging plays a crucial role in accurate diagnosis. Conventional radiographic techniques used in orthodontic treatments, such as cephalometry and panoramic radiography, do not provide a clear, multidirectional view of the TMJ . Cone beam computed tomography (CBCT) has proven valuable in detecting significant changes in condyle position in patients with TMJ disorders . CBCT allows for the assessment of TMJ anatomy without the superimposition of structures, thereby, facilitating the analysis of bone structure, joint space, and dynamic function in three dimensions. This technology surpasses the limitations of other imaging techniques like panoramic radiography and traditional computed tomography . Various studies have examined the relationship between skeletal malocclusions and TMJ morphology, highlighting significant differences across Angle's classifications. For instance, some studies found that patients with Class III malocclusion exhibited significant changes in joint space, with a more anteriorly displaced condyle compared to those with Class I or II malocclusions . Differences in condylar morphology, joint space, and fossa dimensions between skeletal patterns, suggesting that these variations could play a role in the development of TMDs across different skeletal classes. In particular, class III subjects were characterized by a wider mandibular fossa and a more superior condylar position, whereas Class II patients showed more pronounced changes in joint space and condylar height . The morphology and position of the condyles are significant in orthodontics as they can influence the outcomes and stability of orthodontic treatments . The ideal position of the condyle within the glenoid fossa remains a fundamental question in dentistry . There is ongoing debate regarding the clinical importance of condylar positioning in the TMJ. The position of the condyle is the result of various dynamic processes, including growth, remodeling, and responses to functional and occlusal changes [ 15 – 17 ]. The impact of occlusion on the condyle–fossa relationship continues to be a subject of research. Some studies have recognized the role of occlusion in the relationship between the condylar process and the mandibular fossa [ 18 – 20 ]. Beyond the position, occlusion may also be associated with the vertical height, anteroposterior, and mediolateral thickness of the condylar head . The condyle–fossa relationship may or may not be associated with variables such as sex, age, and laterality (left and right sides), as suggested by various studies . Orthognathic surgeries and growth modification treatments can affect the position of the condyle and the dimensions of the joint spaces. Numerous studies have indicated that orthognathic surgeries can alter the position of the condyle within the joint space, though these changes are often not significant in the long term . However, other studies have reported that such changes can be significant even over the long term . Changes in condylar position and joint spaces can impact the recurrence of surgical outcomes [ 27 – 29 ]. Growth modification treatments can also alter the position of the condyle, potentially causing its displacement . Furthermore, the correction of Angle's Class I malocclusions using fixed orthodontic appliances has been associated with significant posterior movement of the condyle. The posttreatment position of the condyle is crucial as it can aid orthodontists in predicting the condylar position during treatment planning . Significant variations in the dimensions and spatial orientation of the mandibular condyles within the glenoid fossae have been observed between male and female subjects with different sagittal skeletal relationships. These morphological differences, as demonstrated through CBCT analysis, are likely to correlate with specific malocclusion classifications. Consequently, these anatomical variations may influence the efficacy and long-term stability of orthodontic interventions. Therefore, understanding the extent of joint spaces and the condylar position across different anterior–posterior skeletal patterns is essential for optimizing outcomes in orthognathic surgery and growth modification treatments, aiding orthodontists and surgeons in treatment planning. Additionally, there is still a gap in comprehensive knowledge regarding the relationship between TMJ morphology and various types of malocclusions, particularly within the Iranian population. The aim of this study is to evaluate and compare the size and position of the right and left condyles in the glenoid fossa among male and female patients with different skeletal patterns in the anterior-posterior dimension using CBCT images. From March 2019 to April 2023, a total of 153 CBCT images were collected from two oral and maxillofacial centers. The inclusion criteria required appropriate image quality, coverage of the examined area, symmetrical skeletal appearance, complete dentition (with or without the third molars), and participants aged over 16 years. Exclusion criteria included evidence of trauma or surgery, crossbite or open bite, a history of orthodontic or orthopedic treatment, congenital syndromes, and craniofacial deformities. Ultimately, 119 images (33 males and 86 females) met the criteria and were analyzed in the study. Based on the ANB angles, the samples were categorized into three groups: Class I ( n = 49), Class II ( n = 52), and Class III ( n = 18). The classifications were defined as follows: ANB < 0 for Class III, 0 < ANB < 4 for Class I, and ANB > 4 for Class II. For borderline cases, an additional Wits analysis was performed. In this analysis, Points A and B were projected perpendicularly onto the functional occlusal plane in the midsagittal section of the three-dimensional view, and the distance between these points was measured. Individuals were categorized as Class I if the measurement ranged from −1 to 1, Class II if greater than 1, and Class III if less than −1. In cases where discrepancies existed between Steiner and Wits analyses, the Wits analysis was used as the primary reference. The CBCT images were acquired using a CBCT device (HDX-WILL, South Korea) with a field of view (FOV) of 14.5 cm × 16 cm and exposure settings of 110 kVp, 17 s exposure time, voxel size of 300 µm, and a current of 4 mA. Participants were instructed to remain still, refrain from swallowing, and maintain centric occlusion with relaxed lips and tongue during imaging. The images were analyzed using the OnDemand3D software (version 10.1). The Frankfurt plane was aligned parallel to the horizontal plane. The horizontal reference axis of the condyle was established by selecting the innermost and outermost points of the condyle head in the axial section . Based on this reference, the condyle and articular eminence were examined in three planes: axial, coronal, and sagittal, for both the right and left joints. In the sagittal plane, the upper joint space was determined by measuring the distance between the SG point (located at the top of the glenoid fossa) and the SC point . Additionally, in this plane, a line tangent to the anterior edge of the condyle head was drawn from the SG point, intersecting at a point designated as AC. A perpendicular line from AC to the anterior wall of the glenoid fossa was measured as the anterior articular space . Similarly, a tangent line from the SG point to the posterior limit of the condyle head identified a point called PC. A perpendicular line from PC to the posterior wall of the glenoid fossa was measured as the posterior articular space . In the coronal plane, from the SC point, a line was drawn perpendicular to a line connecting the MC point (the most medial part of the condyle head) and the LC point (the most lateral part of the condyle head), measuring the height of the condyle . The width of the condyle was determined by measuring the distance between the MC and LC points in the coronal plane . In the sagittal plane, the length of the condyle was measured as the distance between the AC and PC points . The sample size for this study was determined using PASS15 software for a one-way ANOVA design. The parameters used for calculation were a type I error rate ( α ) of 0.05 and a type II error rate ( β ) of 0.2 (80% power). The following parameters were considered for the main dependent variable: • Mean for Group 1 ( μ 1 ): 18.48 • Mean for Group 2 ( μ 2 ): 17.01 • Mean for Group 3 ( μ 3 ): 18.55 • Standard deviation ( σ ): 2.78 Based on these parameters, the sample size calculation for each group was conducted using the formula for a one-way ANOVA: n = 2 σ 2 Z α / 3 + Z β 2 μ 1 − μ 2 2 , where Z α /3 is the critical value of the standard normal distribution at α /3 to adjust for multiple comparisons (for three groups). The position (superior–anterior–posterior articular space) and size (width–height–length of the condyle) were measured for each individual on both the right and left sides using OnDemand software. The average measurements were recorded. Statistical analyses were performed using SPSS software (version 24.0 for Windows). The Shapiro–Wilk test was utilized to assess the normality of data distribution, while Levene's test was applied to evaluate the equality of variances. A T -test was conducted to compare gender differences across various skeletal patterns. The Bonferroni method was employed for pairwise comparisons of the dependent variables across different skeletal patterns. An analysis of covariance (ANCOVA) was used to examine the effects of variables such as gender and different skeletal patterns on the dependent variables, adjusting for the impact of age. Statistical significance was set at p < 0.05. Different skeletal patterns did not show a statistically significant effect on the superior space (SS; p = 0.392). However, a statistically significant difference in SS was observed between genders, with men exhibiting a higher SS compared to women ( p ≤ 0.001). The interaction between gender and skeletal patterns was not statistically significant ( p = 0.337), but SS values were consistently higher in men than in women. The analysis indicated that the impact of different skeletal patterns was not significant in women ( p = 0.443) or men ( p = 0.367; Table 2 ). Similarly, different skeletal patterns ( p = 0.481), gender ( p = 0.386), and the interaction between them ( p = 0.257) did not significantly affect the anterior space (AS). The influence of different skeletal patterns was not significant in women ( p = 0.236) or men ( p = 0.196; Table 2 ). The evaluation of gender effects within each skeletal pattern group revealed a statistically significant difference only in the Class I group ( Table 3 ). For posterior space (PS), different skeletal patterns ( p = 0.027) and gender ( p = 0.001) had statistically significant effects. However, the interaction between them did not significantly impact PS ( p = 0.556). PS values were consistently higher in men than in women, with the skeletal pattern differences ranked as follows: Class III > Class II > Class I. A significant difference was noted only between the Class I and Class III groups, with the PS value being lower in Class I compared to Class III; no other group comparisons showed statistically significant differences ( Table 4 ). There were no significant effects of different skeletal patterns ( p = 0.367), gender ( p = 0.100), or the interaction between them ( p = 0.825) on the height of the condyle ( Table 5 ). However, gender had a significant effect on condyle width, with men consistently showing a larger condyle width than women ( p ≤ 0.001). The interaction between gender and skeletal patterns did not significantly affect condyle width ( p = 0.720; Table 5 ). Different skeletal patterns significantly influenced condyle length ( p = 0.002), though gender ( p = 0.198) and the interaction between gender and skeletal patterns ( p = 0.851) did not ( Table 5 ). Significant differences in condyle length were observed between Class I and Class II and between Class II and Class III, with Class II exhibiting the lowest values ( Table 6 ). Our study investigated the relationship between the morphology and position of the condyle and different skeletal patterns in the sagittal dimension using CBCT images. The morphology and position of the condyle are critical features in diagnosing and planning treatment for issues related to the TMJ. Various factors, such as gender, erosion, hyperplasia, and different syndromes, can affect the morphology of the condyle. The impact of condylar morphology and position on the function of the masticatory system and the development of malocclusions has been extensively researched. However, comprehensive knowledge of the relationship between TMJ morphology and various types of malocclusions, especially in the Iranian population, remains limited. Systemic diseases, such as RA, can significantly alter the dimensions and positioning of TMJ structures. Youssef Mohamed et al. identified that patients with RA exhibited significantly reduced condylar height and increased radiographic osteoarthritic changes compared to a control group. Additionally, there was a significant inverse correlation between anticitrullinated protein (ACCP) levels, disease activity score 28 (DAS28), and high to moderate disease activity with condylar anteroposterior dimensions. Consistent with these findings, our study demonstrated that individuals with a Class II skeletal pattern had significantly shorter condylar lengths compared to other skeletal groups. Moreover, our results corroborated Youssef Mohamed et al.'s observation that ACCP levels did not significantly correlate with mediolateral dimensions. Furthermore, we found no statistically significant differences in condylar height and width across various skeletal patterns. Previous research has provided important insights into how TMJ morphology is clinically relevant in individuals with different skeletal malocclusions. Variations in condylar position and joint space across skeletal classes may contribute to an increased risk of TMDs in specific populations. For instance, Song et al. found that Class III malocclusions were linked to more anteriorly positioned condyles and reduced joint space, which could lead to higher stress on the TMJ and worsen TMD symptoms. In contrast, Alhammadi, Fayed, and Labib found that Class II patients exhibited larger condylar height and anterior joint space, potentially presenting different biomechanical challenges. Additionally, they highlighted the importance of understanding the three-dimensional aspects of TMJ morphology, as both Class II and Class III patients showed notable differences in condylar and fossa dimensions, which may influence orthodontic treatment decisions and TMD management. In alignment with Chae et al. , the current study found no statistically significant differences between the size of the upper joint space and different skeletal patterns. Akbulut and Kılınç reported that the superior joint space, defined as the distance from the condyle to the fossa, had the highest values in Class II subjects on both sides, with statistical significance on the right side but not on the left. Tariq and Jan observed that the upper joint space was smallest in Class III subjects and largest in Class I subjects. Arieta-Miranda et al. and Song et al. also reported the highest values in Class I individuals, but contrary to their findings, did not find significant differences between Class II and Class III individuals. In our study, there was no statistically significant difference between the size of the anterior joint space and malocclusion, consistent with studies conducted by Chae et al. and Song et al. . Arieta-Miranda et al. used different landmarks from our study to measure the anterior space and concluded that Class I individuals have the largest amount of anterior space compared to Class II and Class III individuals. Posterior joint space values in Class I individuals were significantly lower than those in Class III subjects, aligning with the findings of Tariq and Jan . Contrary to our results, studies by Chae et al. , Akbulut and Kılınç , and Arieta-Miranda et al. found no statistically significant differences in the size of the posterior joint space across different skeletal patterns. Akbulut and Kılınç and Arieta-Miranda et al. used the external auditory canal as the posterior border of the posterior joint space. Discrepancies in joint space size between our study and others can be attributed to differences in sample size and racial variations. This study found no statistically significant differences in condylar height across different skeletal patterns. In contrast, Song et al. reported that Class I individuals had the highest condylar height values, using the same measurement method as ours. This difference may be attributed to racial variations. Hasebe et al. and Santander et al. , who used different landmarks than the present study to define condylar height, concluded that condylar height was greatest in Class III individuals. There was no statistically significant difference in condylar width across different skeletal patterns, similar to the findings of Chae et al. . However, other studies have reported different results. Our study found a statistically significant relationship between condylar length and different skeletal patterns, with Class II individuals exhibiting lower condylar length values than Class III and Class I individuals. Hasebe et al. and Tariq and Jan measured condylar length using the AC and PC points, selected on the anterior and posterior limits of the condyle head at a distance of 4 mm from the uppermost point of the condyle head in the sagittal dimension, and found no statistically significant difference between the three groups. In contrast, Song et al. , using the most anterior and posterior points of the condyle head in the axial dimension, concluded that Class I individuals have the highest values. Santander et al. and Chae et al. used methods similar to ours, connecting the most anterior and posterior points of the condyle head in the sagittal dimension. Santander et al. concluded that Class III individuals have the lowest values, whereas Chae et al. found no statistically significant differences between the three groups. The differences in results between our study and others may be due to variations in measurement methods and inclusion and exclusion criteria. For example, Chae et al. excluded cases with discrepancies between CO-CR and underlying diseases affecting metabolism, which were not considered in the present study. A limitation of this study is the unequal distribution of male and female participants across skeletal classes, with an overrepresentation of Class II in females. This imbalance may affect the generalizability of the findings and limiting their interpretability. Future studies with a more balanced gender distribution are recommended for more reliable conclusions on the relationship between TMJ morphology and skeletal patterns. The limited number of Class III individuals and the lack of access to patient histories to account for other potential factors, such as TMD, in the development of skeletal malocclusions were another limitations of this study. Future studies should include larger sample sizes from various regions and consider additional factors that may influence the development of different skeletal patterns in the sagittal, vertical, and transverse dimensions. Based on the results of this study, Class I individuals had smaller posterior joint space compared to Class III individuals, while Class II individuals had shorter condyle length than both Class I and Class III. These findings emphasize the role of skeletal patterns in TMJ morphology, with potential implications for orthodontic treatment and TMJ disorder management. | Review | biomedical | en | 0.999994 |
PMC11698605 | Complications, such as arterial puncture, pneumothorax, hemothorax, and infections, are reported to occur less frequently with the placement of peripherally inserted central venous catheters ([CVCs] PICCs) than with the placement of other CVCs . The use of PICC has increased in clinical settings, including during the perioperative period. Consequently, more patients undergo surgery under general anesthesia with PICCs inserted presurgically. PICC use has become more widespread due to newly introduced and more convenient insertion techniques, such as those adopting passive magnet tracking and the electrocardiogram (ECG)-guided tip confirmation system (TCS) , which facilitates catheter placement at the bedside without confirmatory imaging, and insertions performed by trained nurse practitioners. It is well known that guidewires used during PICC placement may cause cardiac arrhythmias if they progress too far into the right atrium (RA) . There are limited case reports on positional change–related arrhythmias as the insertion of PICCs is usually performed in awoken patients . Moreover, arrhythmias that occur during general anesthesia and are induced by a catheter inserted with the TCS-guided technique have not been reported. Arrhythmias related to PICCs are rare. In three cases , nonsustained ventricular tachycardia (VT) occurred in awoken patients when their body positions were changed following PICC placement and confirmation of the tip position in the lower superior vena cava (SVC) by fluoroscopy or chest radiography. We presented a case of nonsustained VT that developed after a change in the patient's position under general anesthesia. We also discussed the factors affecting positional change–related arrhythmias in patients with preoperatively inserted PICCs using TCS and suggested possible measures for their prevention and treatment. The patient was a 44-year-old woman (height 155 cm and weight 62 kg) scheduled for laparoscopic right adrenalectomy for primary aldosteronism. She had no previous history of arrhythmia and had not taken any medications that cause arrhythmias. On the day before surgery, a PICC (PowerPICC, Becton, Dickinson and Company, NJ, USA) was inserted via the right brachial–basilic vein and the Sherlock 3CG TCS (Becton, Dickinson and Company, NJ, USA) was used to confirm the position of the catheter tip. It was placed 41 cm from the insertion site based on the maximal P -wave on the ECG, observed after confirmation of P -wave inversion. The correct position of the tip was then confirmed using chest radiography . After induction of anesthesia, when the patient was placed in the left lateral decubitus position with adduction followed by internal rotation of her right shoulder and flexion of the elbow, monomorphic nonsustained VT occurred . With further, almost maximal, internal rotation of the shoulder, the arrhythmia ceased, and the surgery was commenced as planned. Intraoperatively, the right atrium (RA) and right ventricle (RV) pressures were measured using a PICC lumen . The data indicated respiration-related movements of the catheter in the RV; however, no further arrhythmias were observed. When the patient was returned to the supine position after surgery, arrhythmia did not occur, and the postoperative course was uneventful. “The Practical Guidelines for Safe CVC Placement and Management” issued by the Japanese Society of Anesthesiologists recommend Zone B , an area around the junction of the left and right innominate veins and the upper SVC, as the optimal position of the catheter tip following insertion ; these guidelines pertain to all CVCs and do not particularly focus on PICC tips. The European Society for Clinical Nutrition and Metabolism guidelines also pertain to all CVCs, presumably including PICCs, and accordingly, the adequate site for catheter placement is when “the tip is in the lower third of the SVC, at the cavoatrial junction (CAJ), or in the upper portion of the RA” . Similar recommendations are presented in “Safe Vascular Access ” (the United Kingdom), where the lower SVC or the upper RA position is considered optimal ; however, some American authors exclude the upper RA and limit the optimal position to the lower SVC and in the vicinity of the CAJ . Hence, adequate placement of the catheter, as recommended in the Japanese Society of Anesthesiologists guidelines, is slightly lower ; however, these guidelines have been established to reduce the frequency of CVC-related complications (vessel or myocardium perforations, thromboses, occlusions, catheter-related infections, pneumothorax, and arrhythmias) and are based on reported data where CVC tip positions were confirmed either with fluoroscopy or portable chest radiography. With technological advancements in magnetic tracking and intracavity ECG, PICCs have recently been inserted at the bedside without postinsertion imaging. PICCs placed with the Sherlock 3CG TCS, where the tip position at the CAJ is considered correct according to the observed inversion of the electrocardiographic P -wave (NICE Medical Technology Guidance), tend to be inserted too far into the RA. Malpositioning of the catheter tip with the Sherlock 3CG occurs in 56.1% or 20.5% of patients, depending on the definition of the adequate tip position (low SVC/CAJ or mid SVC/low SVC/CAJ/high RA, respectively) . According to Johnston et al., malpositioning might be due to specific characteristics of the 3CG technology (targeting the CAJ, which is sometimes impossible to achieve in clinical settings), difficulty in defining the CAJ position on chest radiographs, and/or inconsistent CVC placement guidelines . Thus, even if the catheter is inserted and fixed in an optimal position, complications may still occur. In our case, PICC was inserted with the Sherlock 3CG TCS one day before surgery, and arrhythmia did not occur until the patient was positioned for the operation. Movements of the catheter tip (caudally up to 5.3 cm [or 2.2 rib spaces]) due to shoulder adduction have been reported, but only rarely induce VTs. The catheter tips constantly move according to the blood/injection fluid flow changes in the vicinity of the CAJ (lower risk of thrombosis) , or the upper RA might occasionally contact the atrioventricular (AV) node/RV wall; however, if this contact is only momentary, arrhythmia will presumably not occur. In contrast, in the lateral decubitus or prone position, contact with the AV node/RV wall may be sufficient to induce VT, which is not observed immediately after PICC insertion (usually in the supine position). A minor change in the body position (in our case, further adduction of the shoulder) might terminate the arrhythmia, but respiration- and blood flow–related movements would not cease . In an awoken patient, arrhythmia may produce symptoms such as shortness of breath, chest pain, or palpitations, and thus provoke a timely response . In contrast, in patients under general anesthesia, suspicion and vigilant observation of the ECG tracing when changing body position remain the only means of early recognition and treatment of PICC-induced arrhythmias. Simulation of the intraoperative position immediately after PICC insertion might allow detection of positional change–induced arrhythmias and prompt early correction of the catheter position (usually pulling back the catheter); however, this is not always feasible in clinical settings. Notwithstanding inconsistencies in CVC placement guidelines , TCS-guided PICC insertions seem to produce fewer instances of tip malpositioning and PICC-related complications. However, PICCs inserted with the Sherlock 3CG TCS tend to be extended further into the RA or RV than in conventionally placed PICCs under fluoroscopic or radiographic guidance and might induce arrhythmias on changing the patient's position. In patients under general anesthesia, such arrhythmias should be suspected and vigilant ECG monitoring should be performed. Placing the patient in the final intraoperative position immediately after PICC insertion may be reasonable, but further studies are required to assess the validity of its routine implementation. | Study | biomedical | en | 0.999996 |
PMC11698606 | Recently, foodborne-related diseases are the most common health concern for humans worldwide. Microbial deterioration and oxidative rancidity are the two major factors for spoilage of foods . During the 20th century, several new technologies and chemical compounds were developed for food preservation. However, pathogenic microorganisms in the food system continue to create multiple human diseases and cause substantial economic losses to food processors . At the same time, the indiscriminate use of antibiotics in human medicines, animal husbandry, and food industries against pathogenic bacteria has led to antimicrobial-resistant (AMR) development . AMR continues to develop more powerful mechanisms than ever and will be the major challenge shortly . Globally, AMR gets much attention among the research communities since it is widespread, reduces the therapeutic effect of these drugs within a shorter period, and leads to severe problems in the entire healthcare system . Therefore, AMR has been categorized as one of the top 10 public health threats to humans . Fatality rates due to AMR infections are increasing drastically, and it is expected to reach nearly 10 million per year by 2050. If no more effective and unique therapeutic methods are identified to contain AMR, there will be tremendous global economic losses . This situation forced the research communities to look back into traditional medicines or herbal products, which may provide appropriate and acceptable alternative solutions to maintain human health . Oxidative rancidity is another factor for food spoilage during raw material handling, processing, and storage, which results in loss of nutritional quality, flavour, taste, and colour and leads to consumer rejections . Oxidized molecules in food systems are the causative agents for degenerative diseases such as arthritis, cancer, diabetes, multiple sclerosis, and cardiovascular-related diseases . Moreover, oxidation and rancidity are the biggest problems for the food industries, as they are directly involved in food waste generation and economic losses . To avoid these situations, synthetic additives have been widely used in industries due to their low cost, purity, and high activity at low concentrations. However, some harmful effects such as allergies, poisoning, and metabolic disorders have been reported when used in high doses . Therefore, it is necessary to find new antioxidant compounds that can be added to foods without affecting consumers' health. Recently, natural antioxidants have received greater attention in the research communities to prevent oxidative rancidity in food products. It could be one of the best options for food industries to replace synthetic antioxidants or preservatives, due to its bioavailability and health-beneficial effects. In addition, consumers' awareness of quality food products processed with natural preservatives has forced the food industries to focus on new natural preservatives in recent times. As a result, finding a new preservative with antioxidant and antimicrobial properties from natural sources is a primary goal for several researchers to benefit food processors and consumers. India is the land of spices and herbs and has made a major contribution to world production. Wide ranges of spices are produced in India, with varying tropical climate conditions that make almost all spices grow splendidly. During 2021–2022, spices/spice products export reached around 1,531,154 tons, valued at $4102.29 million . Most spice extracts are in the form of essential oils (EOs). They are extensively used in folk medicines, food flavourings, and pharmaceutical applications. In addition, food industries use them as a natural preservative to extend storage stability without affecting the organoleptic properties [ 18 – 20 ], due to their unique properties, including antimicrobial, anti-inflammatory, antioxidant, and antitumour [ 21 – 23 ]. Some EOs are branded as generally recognized as safe (GRAS) by the Food and Drug Administration (FDA) and are considered potential alternatives to conventional antibiotics . However, a wide range of spices are cultivated in India, and their biofunctional properties and composition may vary due to several factors, such as variety, geographical conditions, maturity stage, and extraction methods, compared to worldwide production. In addition, the information on the antioxidant and antimicrobial component(s) of different Indian-origin EOs and their chemical compositions is minimal. Therefore, the current study is aimed at profiling the phytochemical composition and dual preservative properties, namely, antioxidant and antimicrobial properties, of four important commercial Indian-origin EOs, including clove, ginger, garlic, and eucalyptus. The chemical profile of EOs was carried out using GC–MS analysis at ICAR-Indian Spice Research Institute, Kozhikode, India. For analysis, Restex 5MS capillary column having a length of 30 m × 0.25 mm inner diameter (ID), 0.25 μ of film thickness was used. The injector temperature was set at 250°C. GC oven temperature was programmed as follows: Initial holding temperature was kept at 60°C for 5 min, followed by heating temperature at 200°C for 3°C/min, and finally kept the constant temperature at 220°C for 7 min. The running time was set for 50 min, and Helium gas was used as a carrier gas with a steady flow speed (1.0 mL/min), and EOs (0.20 μ L) were injected into the instrument. An electron ionisation system with 70 eV ionisation voltage and a mass scanning range of 40–650 m/z was used for MS detection. The EO components were identified by comparing them with the NIST library or mass spectra from the literature . The DPPH radical scavenging activity of EOs was measured according to Yen and Wu , with certain modifications. Briefly, an aliquot of 100 μ L EOs with various concentrations (20%–100% of ginger, garlic, and eucalyptus EOs and 1%–5% in clove EO) was added into 2 mL of methanolic DPPH (6 × 10 −5 M). Afterward, the mixtures were vortexed (Neuation Digital Vortex Mixer, iSwix VT, India) and kept in a dark place for 30 min at room temperature. Finally, absorbance was measured at 517 nm using a UV-VIS spectrophotometer . Methanolic DPPH solution was used as a control. The percentage inhibition was calculated using the following formula: % I = A control – A sample A control × 100 where I is the inhibition percentage of DPPH, A control is the absorbance of DPPH solution at 0 min, and A sample is the absorbance of DPPH solution with EOs after 30 min. The ferric-reducing capacity of different EOs was determined according to Benzie and Strain . Briefly, 20 μ L of EO with various concentrations (20%–100% of ginger, garlic, and eucalyptus EOs and 1%–5% in clove EO) were mixed into freshly prepared FRAP reagent (2 mL). Then, the mixture was mixed well and kept in a dark place for 4 min. Subsequently, the absorbance at 593 nm was measured using a UV-VIS spectrophotometer . FRAP reagent without EO was used as control. The obtained absorbance values were interpolated in a standard curve developed using iron (II) sulfate solution . The results were expressed as micromole Fe (II)/grams. The TPC of EOs was calculated according to Singleton and Rosy . Briefly, 30 μ L of EOs with various concentrations (5%–25% ginger, garlic, and eucalyptus EOs and 1%–5% clove EO) were added into 3 mL distilled water and vortexed. Subsequently, the Folin–Ciocalteu reagent (0.5 mL) was mixed into the diluted EOs. After 3 min of incubation, 20% sodium carbonate (2 mL) was poured into the mixture and placed in a water bath for 1 min. Finally, absorbance at 650 nm was measured using a UV-VIS spectrophotometer . Catechol is used as a standard to plot the standard curve. The phenolic content was catechol equivalents in mg phenols/100 g of EOs. The bacterial strains— Staphylococcus aureus , methicillin-resistant Staphylococcus aureus (MRSA) , Bacillus cereus (lab isolate), Salmonella enterica typhi , Vibrio cholera , Escherichia coli , Pseudomonas aeruginosa , and Aeromonas hydrophila —were procured from Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh, India. All the cultures were conserved at 4°C and kept alive by continuous subculture on a nutrient agar medium. For the antimicrobial study, brain heart infusion (BHI) broth was inoculated with test bacterial culture, which was then cultured for 24 h at 37°C. Normal saline was used to adjust the bacterial culture turbidity (1.5 × 10 8 CFU/mL) with 0.5 McFarland standard turbidity. The antibacterial properties of EOs were employed with the agar well diffusion method against food-borne and fish-borne bacteria such as S. aureus , MRSA, B. cereus , S. typhi , V. cholera , E. coli , P. aeruginosa , and A. hydrophila . Briefly, the bacterial cell suspension (1.5 × 10 8 CFU/mL) was spread over Mueller–Hinton agar (MHA) (HiMedia, India) plates and followed by 6 mm diameter wells prepared using sterile cork borer. Subsequently, 100 μ L of EOs and the control were poured into each empty well. Then, the plates were incubated at 37°C for 24 h. Finally, the inhibition zone ( n = 4) was measured using a standard antibiotic zone scale (PW 096, Hi Media). Resazurin microdilution assay was carried out to measure the MIC values of EOs against food-borne and fish-borne bacterial strains such as S. aureus , MRSA, B. cereus , S. typhi , V. cholera , E. coli , P. aeruginosa , and A. hydrophila . In summary, an aliquot of 100 μ L Mueller–Hinton broth (MHB) (HiMedia, India) was filled into 96 well plates and followed by 200 μ L of EOs and 20 μ L of Tween 20 as an emulsifying/solubilizing agent was added into the first well. Further, serial dilution (100% to lower level) was carried out until the last well. At last, 30 μ L (approx. 3.0 × 10 6 CFU/mL) of bacterial suspension (0.5 McFarland standard adjusted) was added to each well and mixed thoroughly. After overnight incubation at 37°C, resazurin solution (30 μ L) was added into each well and incubated further at 37°C for 1 h. The MIC values are defined as the lowest concentration before the colour change. To determine MBC, a loopful of inoculum was taken from wells before the colour change and streaked on MHA plates. Then, the plates were incubated for 24 h at 37°C. The lowest concentration, which has no bacterial growth on the MHA plates, was considered MBC. The results are expressed in mean ± standard deviation ( n = 3). The variance among the samples was analyzed using a one-way analysis of variance (ANOVA), and a comparison of mean values was performed by Duncan's multiple range test ( p ≤ 0.05). The chemical constituents of ginger, clove, eucalyptus, and garlic EOs with retention time and percentage share in the sample are represented in Tables 1 and 2 . About 31 components were identified from ginger EO, comprising more than 94% of the EO composition ( Table 1 ). The most abundant compounds included α -zingiberene (28.01%), β -sesquiphellandrene (18.09%), β -bisabolene (9.72%), α -farnesene (7.40%), and β -citral (5.48%), which represents about 68.70% of the total EO compounds. The findings agree with previous studies in ginger EO containing α -zingiberene and β -sesquiterpene as principal components (10%–60%) responsible for different bioactivities such as antioxidant and antimicrobial properties . They can be used as a natural, long-lasting food preservative. Similarly, citral is another precarious compound identified from ginger EO, which inhibits much bacterial and fungal growth by reducing ATPase interactions, mitochondrial structural alteration, and cell wall and membrane deterioration [ 34 – 36 ]. The chemical composition and its variations can be influenced by several factors, such as varieties, agroclimatic conditions, stage of maturity, the part where the EO is derived, and distillation conditions . The presence of 18 compounds was identified from clove oil, which covers 98.64% of the total EO composition ( Table 1 ). Eugenol (70.12%) was identified as a major constituent, followed by trans- β -caryophyllene (12.56%) and eugenol acetate (11.32%). Similarly, other studies revealed that eugenol is the prime component in clove oil [ 39 – 41 ] and is used in food, medical, cosmetics, and agricultural industries as an antioxidant, antimicrobial, antiviral, and antifungal agent . The amount of eugenol in the EO can be directly related to the different geographic regions where the plant has grown, influencing biotic and abiotic factors such as seasonality, developmental stage, plant age, and climatic conditions . In addition, the extraction method of EO can also influence the content of its volatile compounds , these differences in chemical composition can be directly related to pharmacological properties . GC–MS results reveal that 12 components in eucalyptus EO are identified, accounting for 99.54% of the total EO composition ( Table 2 ), which mainly consisted of oxygenated and nonoxygenated monoterpenes as well as oxygenated sesquiterpenes. Of these, 1,8-cineole, also named as eucalyptol (52.95%) and terpinen-4-ol (2.63%) were the main oxygenated monoterpenes, while α -pinene (20.75%), p-Cymene (10.10%), and β -myrcene (8.70%) were the main nonoxygenated monoterpenes. These results are similar to EOs obtained from Tanzania, Iran, and Tunisia [ 47 – 49 ]. Most of these compounds are commercially used in the food, pharmaceutical, agricultural, and chemical industries as flavourants, drugs, pesticides, and industrial feedstocks. Particularly, eucalyptol is used in flavourings, fragrances, and cosmetics due to its pleasant aroma and taste. It is also the main ingredient in mouthwashs, cough suppressants, insecticides, and insect repellents . In earlier studies, Tyagi and Malik and Kumar et al. reported 45.4% and 33.6% of eucalyptol, respectively, from eucalyptus EOs from India, which is less than our results. This reveals that the chemical composition of EOs could vary within India, due to the different geographic regions, which can influence the EO composition. In garlic oil, 13 compounds were detected in GC–MS analysis, covering more than 96.15% of the total EO composition ( Table 2 ). The chemical composition showed that allyl polysulfides and terpenes, such as diallyl disulfide (22.18%), diallyl trisulfide (43.25%), allyl methyl trisulfide (9.32%), and 2-vinyl-4H-1,3-dithiine (4.80%), were the most predominant components, and the similar result was observed by Herrera-Calderon et al. . Most of these components are responsible for the characteristics of odour and flavour, antioxidant , antimicrobial , antifungal , anti-inflammatory , and antiobesity properties . DPPH values of EOs with different concentrations showed a significant difference ( p < 0.05) ( Table 3 ). The inhibition percentage has gradually increased with the increasing concentration of EOs. Based on the results, it can be inferred that eucalyptus EO showed maximum antioxidant activity (93.36 ± 0.30%) followed by ginger (87.52 ± 2.84%) and garlic (67.14 ± 1.21%) at 100% concentration. At the same time, the clove EO showed 70.84 ± 3.95% inhibition even at a 5% level, which showed the predominant radical scavenging activity at lower concentrations than the other EOs. The chemical composition ( Table 1 ) displayed the high contribution of eugenol (70.12%), indicating that the total antioxidant activity of the polyphenol mixture is a reflex of the individual components' activity . It allows hydrogen atom donation, stabilizes the phenoxyl radical generation, and creates stable compounds. Generally, eugenol reduces two or more DPPH radicals despite having only one hydrogen atom in the hydroxyl group. Further development of dehydrodieugenol (dimers) with two phenolic hydroxyl groups originating from eugenol intermediate radicals has also been proposed as a mechanism between eugenol and DPPH radicals . Similarly, the level of antioxidant activity of other EOs, including eucalyptus, ginger, and garlic, are influenced by their principal constituents such as 1,8-cineole , α -zingiberene , and diallyl trisulfide , respectively. These compounds are responsible for stabilizing the free radicals by transferring hydrogen atoms or electrons, which can prevent the oxidation process . The reducing power of EOs from ferric iron (Fe 3+ ) to ferrous iron (Fe 2+ ) was measured using the FRAP method, and the results are listed in Table 3 . Among the EOs, ginger possesses maximum reducing potential (561.87 ± 8.13 μ mol Fe (II) g −1 ) followed by garlic (155.45 ± 1.12 μ mol Fe (II) g −1 ) and eucalyptus (87.62 ± 2.71 μ mol Fe (II) g −1 ) at 100%, whereas clove EO demonstrated powerful reducing ability (142.29 ± 1.05 μ mol Fe (II) g −1 ) even at a 5% level, surpassing the 100% eucalyptus EO. This evidence shows that the electron-donating properties of clove EO neutralize free radicals and form a stable product. Similarly, other research studies revealed that eugenol and other phenolic components are the predominant components in clove EO, responsible for its potential antioxidant properties . Phenolic compounds generally have a positive correlation with antioxidant activities due to their electron-donating capability during free radical reactions, and results have shown an increasing trend with increasing concentrations ( p < 0.05) ( Table 3 ). The results indicated that clove EO contains high TPC (375.91 ± 14.21 mg/100 g) at a 5% level, directly correlated with high antioxidant activities. Other EOs showed less TPC than clove EO, even at 25% concentration. Among the other EOs, ginger showed high TPC (124.66 ± 6.03 mg/100 g), followed by garlic (102.33 ± 5.39 mg/100 g), and eucalyptus EO had the least TPC (19.41 ± 12.99 mg/100 g). Generally, TPC may be attributable to intrinsic and extrinsic factors, such as cultivars, soil types and growing conditions, maturity state, and harvesting conditions . The results revealed that the TPC of EOs is directly correlated with antioxidative properties, which may help the cells protect against the oxidative damage produced by free radicals . Moreover, these phenolic compounds are responsible for the defense mechanisms of EOs against microbial attack or environmental stress . It is used to study the antimicrobial properties of eucalyptus, clove, garlic, and ginger EOs against foodborne and fishborn bacteria . Ginger EO displayed antibacterial activity against Gram-positive bacteria such as S. aureus (10.33 ± 0.57 mm), MRSA (12.33 ± 0.57 mm), and B. cere us (10.33 ± 0.57 mm). Still, it failed to inhibit the Gram-negative bacterial growth, possibly due to the thick outer lipopolysaccharide layer of Gram-negative bacteria structure, which provides additional protection against ginger antibacterial compounds and reduces the ginger EO activity . In contrast, Gram-positive bacteria have a monopetide layer , which may be easily ruptured by the ginger EO. Garlic EO exhibited the maximum zone of inhibition against both Gram-positive ( S. aureus , 48.33 ± 0.57 mm) and negative bacteria ( V. cholerae , 49.16 ± 0.28 mm). However, it showed no inhibition zone against S. typhi and E. coli . Organosulfur compounds such as allicin and other aliphatic sulfides are responsible for the antimicrobial properties of garlic oil . These compounds protect the gut microbiome and prevent food poisoning . Similarly, eucalyptus EO indicated the highest inhibition zone against Gram-positive bacteria S. aureus (44.66 ± 0.57 mm) followed by Gram-negative bacteria V. cholerae (40.16 ± 0.28 mm). However, it did not exhibit antimicrobial activity against P. aeruginosa . This result agrees with a previous study , where the EO of eucalyptus showed better antimicrobial properties against gram-positive bacteria than gram-negative bacteria. The antimicrobial properties of eucalyptus EO have been due to their prime compounds, such as 1,8-cineole, α -pinene, p-Cymene, and β -myrcene , which affect the fatty acids in the bacterial cell membrane and cytoplasm, as well as proteins, ATP, cell morphology, and antiquorum sensing activities . Moreover, clove EO exhibited a zone of inhibition against all the tested microorganisms. It showed the highest zone of inhibition against Gram-negative bacteria V. cholerae (42.16 ± 0.28 mm), followed by Gram-positive bacteria, MRSA (41.33 ± 0.57 mm). Several researchers have found clove effective against gram-positive and negative microorganisms [ 79 – 82 ]. Among the EOs, clove EO demonstrated the highest zone of inhibition against MRSA in the agar well diffusion method, revealing the potential antimicrobial effect against multidrug resistance organisms. In the present study, the higher concentration of eugenol, eugenyl acetate, and other phenolic compounds in clove EO is a significant attribute of its antimicrobial activity . These compounds can denature proteins and change the cell permeability by reacting with cell membrane phospholipids, which cause the inhibition of both Gram-negative and Gram-positive bacteria growth . Based on the antibacterial activity, the hierarchy of the oils' has been observed as follows: garlic > eucalyptus > clove > ginger EO for the Gram-positive bacteria, garlic > clove > eucalyptus EO for Gram-negative bacteria. Moreover, the antimicrobial activity of EOs has correlated with the diffusion abilities such as diffusion coefficient, zeta potential, and droplet size of EOs through bacterial cell membranes . Similarly, different factors, including microbial cultures, geographical origin, plant part, extraction methods, and harvest period, affect the antimicrobial activity of EOs . Overall, the study demonstrated that EOs, except ginger, demonstrated broad-spectrum antibacterial properties against Gram-positive and Gram-negative bacteria. In particular, clove EO displayed a prominent antimicrobial effect against all the tested pathogens and was proposed as a broad-spectrum antimicrobial agent in pharmaceutical industries or as a biopreservative in the food or cosmetic industries. The antibacterial effect of EOs was determined using MIC and MBC methods, and the results are given in Table 4 . There was a high correlation between the zone of inhibition and the MIC and MBC results of clove EO, which showed the largest zone of inhibition ( S. aureus , MRSA, and V. cholerae ) and lowest MIC and MBC values, ranging from 0.000089 ± 0.0002 to 2.94 ± 0.90 and 0.00018 ± 0.00005 to 2.94 ± 0.90 mg/mL, respectively. Other EOs showed little correlation between the zone of inhibition and MIC and MBC values, which showed the largest inhibition zone but not the lowest MIC and MBC values compared to clove EO. Eucalyptus EO showed the MIC and MBC values ranged from 1.33 ± 0.40 to 85.50 ± 0.80 and 5.34 ± 1.70 to 85.50 ± 0.80 mg/mL, respectively. Garlic oil displayed MIC and MBC values ranging from 0.005 ± 0.001 to 515.70 ± 0.00 and 5.14 ± 1.70 to 386.70 ± 12.80 mg/mL, respectively but did not show antimicrobial activity against E. coli . Similarly, ginger oil exhibited MIC and MBC values ranging from 5.34 ± 1.70 to 439.15 ± 0.00 mg/mL and 5.34 ± 1.70 to 439.15 ± 0.00 mg/mL, respectively, and did not show microbial resistance against salmonella and E. coli . A similar range was confirmed in another study against S. aureus , B. subtilis , E. coli , and Penicillium spp. . This could be due to the EOs' relatively more water-soluble components that can diffuse further through the agar during overnight incubation. The lack of correlation between the results of EOs from the two assays suggests that the two methods are not necessarily comparable. The agar well diffusion method has been regarded as problematic for the antimicrobial testing of natural products, qualitatively, and useful for screening purposes only. Quantitative testing can be conducted using the broth microdilution method to study the antimicrobial activity . Based on the MIC values, the hierarchy has been made for the EOs, that is, clove oil > eucalyptus oil > garlic > ginger (based on lower MIC value). In particular, clove oil demonstrated potent antibacterial activity against all the tested microorganisms. Also, it exhibited the maximum MIC and MBC values against MRSA, which indicates the antimicrobial potential against multidrug-resistant bacteria. The current study demonstrated different EOs' antioxidant and antimicrobial properties and could be a potential natural antioxidant and antimicrobial source to replace the synthetic compounds. Regarding antimicrobial activities, eucalyptus, clove, and garlic EOs demonstrated their antimicrobial properties against gram-positive and gram-negative bacteria and could be used as natural preservatives to delay the bacterial spoilage of various food products. Among them, clove EO exhibited better antimicrobial activity against all the tested microorganisms, mainly showing a maximum zone of inhibition against MRSA, which reveals the potential against multidrug-resistant bacteria and makes these oils a possible alternative to synthetic antibiotics. Similarly, based on the antioxidant assay, clove oil possesses a high antioxidant capacity with the least concentration and can readily be used as a natural preservative to delay the oxidation process, maintain nutritional quality, and prolong the food product's shelf life. Further comprehensive research should be conducted to understand the underlying mechanism behind these major components' antioxidant and antimicrobial properties in EOs. | Review | biomedical | en | 0.999997 |
PMC11698608 | Cytoreductive surgery with hyperthermic intraperitoneal chemotherapy (CRS-HIPEC) has become the standard of care for patients with primary peritoneal cancers and peritoneal metastasis from gastrointestinal and ovarian carcinomas . The procedure involves the removal of intraperitoneal tumor masses, which may include the excision of affected organs and, in some cases, stripping of the diaphragm. During the HIPEC phase, chemotherapeutic agents heated to temperatures between 41°C and 43°C are instilled into the abdominal cavity to enhance the cytotoxic effects of the chemotherapy . The Peritoneal Carcinomatosis Index (PCI) is a scoring system used to assess tumor burden in patients with peritoneal metastasis. It ranges from 0 to 39, based on the extent and distribution of the tumor in 13 abdominal regions, with each region assigned a score of 0 (no visible tumor), 1 (tumor less than 0.5 cm), 2 (tumor between 0.5 and 5 cm), and 3 (tumor larger than 5 cm) . The PCI score is a crucial prognostic tool, as higher values correlate with more extensive disease and are associated with longer surgeries, increased blood loss, and significant fluid shifts. These factors contribute to significant hemodynamic instability, posing a challenge to perioperative management and leading to an elevated risk of postoperative morbidity and mortality. CRS-HIPEC is known to cause considerable physiological stress. The combination of extensive organ resection and hyperthermic chemotherapy leads to fluid shifts, blood loss, and increased intra-abdominal pressure due to the infusion of 3-4 L of heated carrier solution. This rise in intra-abdominal pressure impairs venous return, reduces cardiac output, and can precipitate hemodynamic instability [ 2 – 6 ]. In addition, hyperthermia increases the risk of end-organ ischemia and renal dysfunction, especially in vulnerable patients. The procedure is associated with significant morbidity (20%–40%) and mortality (1%–5%), largely increased by these physiological challenges [ 2 – 4 ]. Intraoperative management during CRS-HIPEC requires careful attention to fluid balance and hemodynamic stability. Large volumes of fluid, often ranging from 8 to 12 mL/kg/h, may be necessary to maintain homeostasis due to the significant blood loss and fluid shifts. Hemodynamic monitoring plays a key role in guiding fluid therapy and the use of vasoactive agents to maintain cardiac output and adequate end-organ perfusion. Advanced hemodynamic parameters such as cardiac index (CI), Stroke Volume Index (SVI), and stroke volume variation (SVV) are essential for monitoring cardiovascular function and ensuring stable hemodynamics during surgery. Given the complexity of CRS-HIPEC, the PCI score is a crucial factor influencing intraoperative hemodynamic management. Higher PCI scores often correlate with more severe intraoperative hemodynamic fluctuations, increased need for fluid resuscitation, and advanced hemodynamic monitoring to optimize patient outcomes. Despite its potential therapeutic benefits, the physiological stress induced by CRS-HIPEC necessitates careful perioperative management to reduce the risk of postoperative complications and mortality . This study aims to evaluate the relationship between tumor burden, perioperative hemodynamic management, fluid therapy, and postoperative outcomes in patients undergoing CRS-HIPEC. We hypothesize that effective hemodynamic management during and after the procedure is associated with improved outcomes, including a reduction in complications, shorter hospital stays, and lower postoperative morbidity and mortality. This prospective observational study was conducted from April 2016 to December 2022 after approval from Institutional Ethics Committee (IEC). The inclusion criteria for the study were primary peritoneal cancers, gastrointestinal and gynecological cancer patients posted for CRS-HIPEC, aged between 18 and 70 years, and belonging to the American Society of Anesthesiologists physical status class I–III. Waiver of consent was granted by IEC in view of the observational nature of the study. Patients in whom HIPEC was canceled after CRS because of change in surgical plan were excluded from the study. The study was conducted as per principles of the Declaration of Helsinki and its subsequent amendments. In the operating room, standard monitoring included continuous measurement of heart rate, blood pressure, oxygen saturation, and temperature, with documentation in the anesthesia chart. Anesthesia induction was performed using intravenous propofol and fentanyl, along with muscle relaxants such as atracurium or vecuronium, as appropriate. An arterial catheter was placed in all patients for invasive blood pressure monitoring and advanced hemodynamic monitoring. Central venous catheter placement was also performed for administering vasopressors if needed. Hemodynamic monitoring was enhanced using the FloTrac system with the EV1000 monitoring platform (Edwards Lifesciences) connected to the arterial line. Parameters such as CI, cardiac output (CO), stroke volume (SV), SVI, and SVV were recorded and utilized to guide goal-directed fluid therapy (GDFT). The objective of fluid management during the perioperative period was to maintain CI, SVV, SVI, and pulse pressure variation (PPV) within 20% of the baseline values, established after connecting the FloTrac system. The main objective of anesthesia management during CRS-HIPEC included a balanced anesthetic technique to ensure optimal hemodynamic stability, oxygenation, and normothermia. The FloTrac system with the EV1000 platform was used as a key tool for hemodynamic monitoring, providing real-time measurements of dynamic parameters such as CI and SVI. This allowed precise adjustments in fluid administration and vasopressors. The anesthesia team adhered to GDFT protocols, which emphasized maintaining parameters within a 20% range to reduce intraoperative hypotension and maintain organ perfusion. The use of low dose norepinephrine (often referred to as “baby norepinephrine”), was adjusted as needed to support hemodynamic goals. Data on patient characteristics, anesthetic management, intraoperative fluid and blood transfusion, coagulation management, body temperature, and delta temperature (defined as the difference between maximum and minimum temperatures measured throughout the CRS-HIPEC procedure), as well as arterial and venous blood gas measurements, were collected. At the conclusion of the procedure, the PCI score, the number of organs resected, the number of anastomoses performed, diaphragmatic stripping (if performed), the completeness of cytoreduction (CC) score—where CC-0 indicates no visible residual tumor, CC-1 indicates residual tumors up to 2.5 mm, CC-2 indicates residual tumors between 2.5 mm and 2.5 cm, and CC-3 indicates residual tumors larger than 2.5 cm, and the chemotherapeutic agent(s) administered during HIPEC were documented. The duration of each phase of the surgery, along with the total surgical time, was also recorded. The HIPEC protocol involves the use of mitomycin-C (15 mg/m 2 of body surface area [BSA]) and doxorubicin (15 mg/m 2 of BSA) for pseudomyxoma peritonei, mesothelioma, colorectal, and appendiceal cancers. For gastric and ovarian cancers, cisplatin (75 mg/m 2 of BSA) is used. HIPEC is performed for 90 min using the open “Coliseum” technique, with peritoneal dialysis solution (3 L) serving as the carrier solution. Sodium thiosulfate was not used in any of the patients due to its unavailability. Temperature control during CRS was managed using fluid warmers and warming blankets to maintain normothermia. During the HIPEC phase, warming devices were discontinued, and cold fluids along with cool air blankets were used to keep the core body temperature below 39°C. In the postoperative period, if a patient developed hypothermia, fluid warmers and warming devices were used as needed to restore and maintain normothermia. The postoperative course, including complications classified according to the Clavien–Dindo (CD) classification ( Table 1 ), was tracked. The postoperative intensive care unit (ICU) stay and morbidity were assessed using the Sequential Organ Failure Assessment (SOFA) score ( Table 2 ). In addition, the length of stay in both the ICU and hospital, as well as 30- and 90-day mortality, were recorded. We classified patients based on their PCI score into three groups: PCI < 10, PCI 11–20, and PCI > 20, representing the extent of disease. This classification was used to assess intraoperative hemodynamics, fluid therapy, and perioperative outcomes. The primary outcome of this study was to evaluate the impact of PCI scores on hemodynamic monitoring parameters, including CI, CO, SVI, delta SVI, PPV, SVV, and fluid management. Secondary outcomes included postoperative complications according to the CD classification, 30- and 90-day mortality, and the length of ICU and hospital stay. Demographic, clinical, and disease-related variables were presented as frequencies (percentages), means (standard deviations), or medians (interquartile ranges), as appropriate. Parametric or nonparametric statistical tests were used based on the distribution of the data (normally distributed or not). The Kolmogorov–Smirnov test was used to assess the normality of baseline characteristics among patients classified into different PCI groups. For non-normally distributed data, the Mann–Whitney U test and Kruskal–Wallis test were employed, while ANOVA was applied for normally distributed data to evaluate associations among various perioperative parameters and related outcomes. Fisher's exact test was used to compare expected and observed outcomes and to assess associations between categorical variables. Univariable and multivariable logistic regression analyses were performed to identify significant associations between perioperative parameters and postoperative outcomes. A p value of less than 0.05 was considered statistically significant. A total of 203 patients underwent CRS-HIPEC during the study period. The age, gender, primary malignancies, and duration of surgery for different PCI groups are detailed in Table 1 . Information on chemotherapeutic drugs used, total fluid administered, blood transfusions, CC score, number of organs resected, number of anastomoses, delta-temperature, complications, and duration of hospital stay are provided in Table 2 . Patients with a greater burden of disease who underwent extensive resections (PCI > 20) had a significantly longer mean duration of surgery (796.2 ± 158.3 min) compared with those with a PCI between 0 and 10 (551 ± 127 min) and a PCI between 11 and 20 (661.78 ± 137.7 min) ( p ≤ 0.01) ( Table 3 ). The mean volume of fluids administered, excluding blood and blood products, in different PCI groups is detailed in Table 3 . The volume of intraoperative fluids administered was found to be statistically significant across all three PCI score-based patient groups (Pearson's Chi-square: 48.315; p ≤ 0.01) ( Table 3 ). The correlation between IV fluid given during CRS and various perioperative parameters are mentioned in Appendix table 2 . Baseline mean CI values were significantly different among patients with PCI scores of 0–10 , 11–20 , and > 20 (ANOVA: sum of squares: 79.028; p ≤ 0.01). The mean SVI values were significantly different at 10 and 60 min after starting HIPEC in patients with PCI scores of 0–10 (SVI-10 min: 48.29; SVI-60 min: 47.97), 11–20 (SVI-10 min: 41; SVI-60 min: 42.11), and > 20 (SVI-10 min: 39.5; SVI-60 min: 39.48) (ANOVA: SVI CRS beginning: sum of squares: 16,493.679, p =0.002; HIPEC 10 min: sum of squares: 28,393.892, p ≤ 0.01; SVI HIPEC 60 min: Sum of squares: 25,613.411, p ≤ 0.01) . Baseline mean CI values were significantly different among patients with PCI scores of 0–10 , 11–20 , and > 20 (ANOVA: sum of squares: 79.028; p ≤ 0.01). However, these differences were not observed in mean CI values during the intraoperative or postoperative periods . Mean SVV values were significantly different 60 min after starting HIPEC in patients with PCI scores of 0–10 , 11–20 , and > 20 . However, there were no statistically significant differences in SVV trends during the CRS or postoperative periods . We found that the mean body temperature did not significantly change during CRS from the initial temperature. However, body temperature decreased by about 0.6°C–0.8°C 10 min after commencing HIPEC, followed by an increase of 1.3°C–1.4°C during HIPEC, compared with the beginning of CRS. There was no significant difference in mean temperature at any corresponding time point based on PCI scores during the perioperative period (Appendix Table 1 ). The mean temperature after patients' arrival to the ICU was 35.6°C (SD: 1.91). Of the 203 patients we studied, 145 patients had a recorded temperature on shifting to the ICU. Of these, 44 patients (30.3%) were hypothermic (< 35°C core body temperature) on arrival to the ICU. Primary diagnosis (disease), the number of organs resected during CRS, and the number of anastomoses done were analyzed for their impact on postoperative complications. Primary diseases significantly affected postoperative complications ( p ≤ 0.01), while the number of organs resected ( p =0.258) and anastomoses done (0.431) did not affect the postoperative complications ( Table 4 ). Interestingly, diaphragmatic stripping did not independently increase ICU stay length or in the regression model. However, diaphragmatic stripping was significantly associated with the need for mechanical ventilation for 24 h or more . Total blood loss, need for mechanical ventilation for 24 h or more, duration of surgery, CC score after CRS, blood loss during CRS, total number of PRBCs transfused during the procedure, and PCI score were highly significant on univariable logistic regression. Renal function was assessed using serum creatinine values. The mean (SD) of creatinine levels on postoperative days (PODs) 1, 3, and 5 were recorded ( Table 3 ). On POD 1, two patients had serum creatinine levels greater than 1.5 mg/dL (2.06 and 1.60 mg/dL), and in both patients, serum creatinine levels normalized by POD 3. On POD 3, one patient had a serum creatinine level of 1.80 mg/dL, which increased to 3.00 mg/dL by POD 5, and this patient required renal replacement therapy. Another patient had a serum creatinine level of 1.5 mg/dL on POD 3, which normalized by POD 5. These differences between the various PCI groups were statistically significant on POD 3 ( p ≤ 0.01). The median duration of ICU stay varied across patients with different PCI scores. Patients with PCI > 20 had a median ICU stay of 4 days (IQR: 3–7 days), compared with 3 days (IQR: 2–3 days) for those with PCI scores between 0–10 and 3 days (IQR: 2–4 days) for those with PCI scores between 10 and 20. (Kruskal–Wallis H = 22.495; p ≤ 0.01). The 30-day survival rates were 99.1% for patients with PCI 0–10, 95.5% for those with PCI 11%–20%, and 93.3% for those with PCI > 20. While this difference is clinically significant, it was not statistically significant (Pearson's Chi-square: 2.354; p =0.308). The 30-day readmission rates were comparable across all PCI groups (Pearson's Chi-square: 0.849; p =0.654) ( Table 3 ). We analyzed the effect of these seven parameters viz., total blood loss, need for mechanical ventilation for 24 h or more, duration of surgery, CC score, blood loss during CRS, total number of PRBCs transfused during the procedure, and PCI score on ICU stay length using multivariate logistic regression and found that mechanical ventilation for 24 h or more and CC score at the end of CRS predicted an ICU stay longer than 8 days (reference outcome: ICU stay of up to 7 days) ( Table 5 ). On univariable logistic regression, SVI at 60 min after starting HIPEC, ICU stay longer than 7 days, and readmission within 30 days of surgery significantly predicted 30-day survival. However, ICU stay longer than 7 days and maximum SVV during HIPEC phase were predictive of 30-day survival on multivariable logistic regression ( Table 6 ). On multivariable analysis, average heart rate and average CO during CRS and end-tidal carbon dioxide during reconstructive phase were predictive of postoperative complications after CRS-HIPEC ( Table 7 ). A total of 21 out of 203 patients (10.34%) were readmitted within 30 days after surgery. The reasons for readmission included sepsis (3/21; 14.28%), electrolyte imbalances such as hyperkalemia (3/21; 14.28%), medical conditions (e.g., dengue fever and acute exacerbation of asthma) (3/21; 14.28%) patients, intestinal obstruction (2/21; 9.52%), nutritional support (1/21; 4.76%), further management of residual disease (2/21; 9.52%), perforation peritonitis (1/21; 4.76%), anastomotic leaks (2/21; 9.52%), and short bowel syndrome (1/21; 4.76%). In 3 patients, the reason for readmission was not found on record (14.28%). This study focuses on highlighting major perioperative concerns in CRS-HIPEC, including duration of surgery, blood loss, delta temperature, PCI, and fluid management. To the best of our knowledge, this is one of the largest cohorts studied for hemodynamic parameters in CRS-HIPEC. Large fluid shifts are common during cytoreduction, and intraoperative fluid losses can reach up to 8–12 mL/kg and require adequate crystalloids and colloids to ensure adequate perfusion pressure and urine output without causing fluid overload . Patients with lower cardiopulmonary reserve may not tolerate high volumes of intravenous fluids and may need vasopressors/inotropes based on the extent of their ability to tolerate fluids, as also their fluid responsiveness. Liberal fluid strategy, with fluid as high as 1500 mL/h given to patients described in some studies, has been shown to cause several complications, including fluid overload, surgical site edema, end organ dysfunction, and greater length of ICU and hospital stay [ 11 – 15 ]. Conversely, restrictive fluid strategies may compromise end-organ perfusion, particularly during the hemodynamic fluctuations during CRS-HIPEC, leading to renal dysfunction reported in few studies . However, a retrospective study has showed that restrictive fluid strategy decreased the length of hospital stay (11.5 vs. 9.7 days; p ≤ 0.01) and 60-day postoperative complications (28% vs. 45%; p =0.02), with incidence of postoperative renal dysfunction and the highest serum creatinine levels not significantly different in the restrictive versus permissive fluid therapy groups . CRS-HIPEC procedure is known to cause acute kidney injury (AKI) in the postoperative period, and apart from the decreased renal perfusion, use of cisplatin chemotherapy alone or in combination is an important cause of AKI . GDFT approach which involve fluid administration based on CI, SVI, or SVV is well-suited for CRS-HIPEC, where physiological challenges of major surgery and fluid shifts, and hyperthermic environment leads to difficulty in maintaining hemodynamic stability and adequate tissue oxygenation . In our study, fluid administration in patients with three different PCI groups showed a highly significant difference ( p ≤ 0.01), suggesting that patients with higher PCI scores need aggressive fluid management. In addition, significant variations were observed in the mean CI and mean SVI at various stages of the CRS-HIPEC, particularly at the beginning of CRS and during HIPEC, which indicates the impact of PCI on cardiac function. Mean SVV at 60 min of HIPEC phase also showed significant difference ( p ≤ 0.01), highlighting the hemodynamic instability in patients with higher PCI scores. The use of FloTrac system with the EV1000 platform ensured optimal hemodynamic stability and organ perfusion during the procedure. While some groups focus on the duration of mean arterial pressure (MAP) below 60 mmHg, our protocol prioritized maintaining hemodynamic parameters within the defined range to minimize hypotensive episodes. Emerging technologies, such as machine learning-based early warning systems (e.g., HYPE Randomized Clinical Trial) and predictive models for intraoperative hypotension (e.g., Hypotension Prediction Index (HPI)) were shown to have good clinical outcomes, but their application in our study was not implemented except for 4 cases where we used HPI in addition to the FloTrac . As per the international expert consensus, the PCI score is crucial in decision-making for CRS-HIPEC. In patients with colorectal cancer, a PCI score ≤ 16 is acceptable for CRS-HIPEC, whereas a PCI scores > 20 may indicate a need for alternative treatments and supportive care . However, optimal PCI score cutoff is not yet determined for some cancers like gastric cancer and pseudomyxoma peritonei . Esteve-Perez et al. studied 92 adult patients with colorectal and ovarian malignancies undergoing CRS-HIPEC and found significant interindividual variation in the fluid requirement. They also observed that PCI and duration of surgery, fluid therapy, and intraoperative transfusion percentage were highly correlated ( p < 0.02). Similar to these findings, we found that all three of the PCI score-based patient groups showed a statistically significant variation in the volume of intraoperative fluids given. This was found to be clinically relevant, as larger burden of disease necessitates more extensive and technically complex surgical resection, entailing greater blood loss and thus need for more fluids to be administered during surgery. Higher PCI values are a major predictor of postoperative morbidity, especially for scores > 20. High volume diseases (greater PCI) lead to increased blood loss and higher fluid requirements during CRS-HIPEC . Multivariable analyses from previous studies underscore age, intraoperative fluid rates, and blood loss as independent predictors of postoperative morbidity in CRS-HIPEC . Specifically, higher fluid infusion rates (> 15.7 mL/kg/h) have been associated with increased comprehensive complication indices and prolonged ICU stays . On multivariable analysis, we found that higher PCI scores were associated with prolonged mechanical ventilation (> 24 h), increased 30-day hospital readmission rates, and reduced 30-day survival. We also found a positive correlation between total fluid administered and ICU stay, but there was no significant association between total fluid administered and 30-day survival, suggesting that other factors may influence survival outcomes. The identification of predictors for prolonged ICU stays and 30-day survival enhances risk stratification and prognostication in CRS-HIPEC. The significance of mechanical ventilation for 24 h or more and the CC score underscores their utility as key determinants of postoperative recovery and overall survival, as found in our study. These findings are similar to what were found by Macrì et al. in their retrospective analysis of outcomes of CRS-HIPEC in patients with ovarian malignancy . In this study, 30.3% patients had a core body temperature < 35°C on arrival to ICU (rebound hypothermia). The hypothermia in postoperative period may be because of failure to restart the warming devices after completion of HIPEC phase and because of the higher delta temperature. The emphasis of our study on tailoring patient care based on disease extent (PCI scores) and identified perioperative predictors allows for a more individualized approach to the perioperative care of patients undergoing CRS-HIPEC. Our findings align with the existing literature on the challenges associated with the perioperative management of CRS-HIPEC. The prolonged duration of surgery in patients with extensive disease corroborates studies highlighting the intricacies of extensive resections and their subsequent impact on procedural timelines. The pattern of hemodynamic parameters observed in this study underscores their critical role in the perioperative management of CRS-HIPEC patients. Our study has certain limitations. First, as a single-center study, the generalizability of the findings to different geographical and healthcare settings is limited. Second, variations in clinical practices, surgical techniques, and postoperative care protocols may lead to differences in outcomes. In addition, our study lacked a predefined sample size, as we enrolled consecutive CRS-HIPEC patients. Our study highlights significant variations in CI, SVI, and fluid requirements across different PCI groups. A higher PCI score is significantly associated with increased duration of surgery, fluid requirements, the necessity for invasive hemodynamic monitoring, postoperative complications, and ICU stay. Tailoring perioperative strategies based on PCI scores has the potential to optimize these outcomes. | Other | biomedical | en | 0.999997 |
PMC11698610 | Linell and Norden first described Mycobacterium marinum in humans in 1954. The mycobacteria were isolated from the skin lesions of swimmers who had bathed in a contaminated pool . M. marinum can infect many fish species, leading to contamination of water and fish products . Inoculation to humans occurs through injured skin, resulting in the formation of a solitary nodule known as the “fish tank granuloma” . The bacteria can spread via the lymphatics, in a sporotrichoid-like fashion, leading to further verrucous plaques and nodules, typically involving the hands . Disseminated infection has been reported in the immunocompromised and can present with extracutaneous manifestations such as arthritis and osteomyelitis . A 63-year-old Caucasian woman presented with a 6-week history of low-grade fever and widespread pustules that were resistant to treatment with flucloxacillin. Bacterial and viral swabs were performed and found to be negative. Blood tests showed mildly raised inflammatory markers but were otherwise unremarkable. The patient had a history of rheumatoid arthritis and bronchiectasis. Her medications included long-term methotrexate and intermittent courses of prednisolone. A skin biopsy was performed and was found to exhibit numerous pustules and intraepidermal neutrophils. The entire dermis and superficial subcutis showed heavy neutrophilic infiltration and necrotic debris within an apparent abscess. A diagnosis of a pustular variant of Sweet's syndrome was considered and treatment with prednisolone was commenced with some initial improvement of the symptoms. Despite that, the patient continued to deteriorate, and a third skin biopsy was performed. This time, histological examination demonstrated a necrotic abscess with deep dermal neutrophilic infiltration . The amount of infiltration and degree of fatty involvement was beyond that expected for Sweet's syndrome. Ziehl-Neelsen (ZN) staining was performed which revealed abundant atypical acid-fast bacilli (AFB). Infection with M. marinum follows contact with infected water or via contact with an infected fish tank or shellfish . The two conditions thought to be required for M. marinum infection are thought to be injury or abrasion to the skin and exposure to a contaminated aqueous environment. In one study, almost half (49%) of M. marinum infections were aquarium-related, 27% were related to fish or shellfish injuries, and 9% were related to injuries associated with saltwater or brackish water . The histology of M. marinum infected tissues often reveals the development of noncaseating granulomata from a neutrophilic abscess . Few bacilli can be observed except in the immunocompromised host . The surrounding dermis shows a lymphohistiocytic infiltrate, whilst the overlying epidermis can be ulcerated, parakeratotic, or acanthotic . Overall, features are rather nonspecific with pseudoepitheliomatous hyperplasia accompanied by a poor yield of positive isolates from culture specimens . We argue that a differential diagnosis of M. marinum infection should be considered in patients with indicative clinical and histological features, especially in the background of immunocompromise, despite the absence of an obvious aquatic source of infection. Awareness of this entity could lead to earlier diagnoses and reduced morbidity and mortality. | Study | biomedical | en | 0.999999 |
PMC11698612 | In recent years, the increase in irregular use of antibiotics around the world has become unstoppable. Antibiotics have become commonplace, routinely used in medical practice. Consequently, resistance mechanisms in microorganisms have developed, enhancing their ability to survive by disrupting the action pathways of antibiotics. As a result, antibiotics used to treat many diseases have become insufficient, leading to a search for alternative treatments. Many scientists are now exploring innovative treatment areas and new molecules. Our environment harbors numerous microorganisms, many of which are pathogenic and highly dangerous to human health. These pathogens have developed significant resistance mechanisms against antibiotics, and at this point, antimicrobial peptides (AMPs), potent and naturally and synthetically occurring structures, are emerging as a defense against these detrimental consequences . AMPs are naturally and synthetically occurring structures that are produced and synthesized by many living organisms, including humans, which act against microbial sources. These structures play a crucial role in the innate immune system, contributing to nonadaptive defense mechanisms against various unsanitary microorganisms. In addition, another advantage of AMPs is that they exhibit broad-spectrum antimicrobial activity. Thus, they could be innovative therapeutic molecules with high effectiveness against many microorganisms [ 3 – 5 ]. Examples of AMPs found in nature include cathelicidins and defensins. These peptides are produced by mammals. Cathelicidins are positively charged peptides . Their mechanism of action involves direct effects on the cell membrane. Specifically, they interact electrostatically with the cell membrane and create pores, leading to death of the microbe . LL-37, AMP, and cathelicidin derivative is produced in humans. This peptide, which has an α -helical structure, penetrates the cell membrane and directly kills the microorganism by disrupting the cell membrane . In previous studies by Polat et al. , LL-37-like peptides were designed, their amino acid sequences were synthesized, and their antimicrobial activity and cytotoxicity results were published. These NET peptides were rendered resistant to protease and their activity was tested on different biofilm forming bacteria . Determination of the effect of these designed peptides on macrophages are revealed for the first time in our study. Understanding the role of peptides in the immune system is crucial for the future development of these peptides as new drug molecules. In the literature, AMPs are generally described as hydrophobic and positively charged . In our previous study, we designed four hydrophobic and positively charged peptides of 10–20 amino acids in length using d- and l-form peptides with a similar structure as LL-37, which we found to be effective against bacterial biofilms . In this study, we examined the effects of these four peptides on macrophages to clarify their effects on the immune system ( Table 1 ). The peptides were synthesized using a Liberty Blue peptide synthesizer (CEM, Matthews, NC, USA). After the synthesis was complete, the peptides were cleaved from the solid support using a Razor device, as specified in the manufacturer's manual. The synthesized AMP samples were first prepared at a concentration of 2 mg/mL for analysis by reversed-phase HPLC. For HPLC analysis, a C-18 analytical column was used, with Buffer A (dH 2 O + 0.05% TFA) and Buffer B (acetonitrile + 0.025% TFA) as the mobile phases. After equilibrating the column at 5% Buffer B, the peptide was injected and eluted using a gradient protocol (%20–%80 Buffer B). The peptides were run on an analytical HPLC column, and their approximate hydrophobicity was calculated according to the retention times of the chromatographic peaks. Next, the peptides were prepared at a concentration of 10 mg/mL and run on a semipreparative C-18 HPLC column. The purified peptide peaks were collected, and the samples were subsequently lyophilized (freeze-dried). The concentrations of the lyophilized AMP samples were determined using the Pierce Quantitative Fluorometric Peptide Assay kit (Thermo Fisher Scientific, Waltham, MA, USA). The TIB-71 RAW 264.7 macrophage line, which was obtained from Mus musculus , was used in this study, and this cell line was supplied by ATCC (Manassas, VA, USA). The MTT Cell Proliferation Kit (Sigma–Aldrich, St. Louis, MO, USA), a colorimetric method, was used to evaluate the damage caused by the peptides in eukaryotic cells. TIB-71 RAW 264.7 macrophages were revived under appropriate culture conditions (37°C, 5% CO 2 ) to prepare for the experiment. The final concentration of peptides ranged 0.125–64 μg/mL. Samples were examined in triplicates for each concentration. Cells incubated without peptide treatment under the same conditions were used as the control. Magainin 2 (Mag2), a known natural AMP with no cytotoxic effects, was used as a positive control . The results were obtained using a 96-well plate reader at 550 and 690 nm. In this study, a cell line that was not treated with AMPs and LPS, a cell line treated only with AMPs, a cell line treated only with LPS, and a cell line treated with both LPS and AMPs were used. To find the changing TNF- α ratio after incubation, the kit named Mouse Tumor Necrosis Factor α ELISA Kit-RAB0477 (Sigma–Aldrich) was applied according to the protocol. Graphpad Prism (9.0.0) was utilized for performing a one-way ANOVA test ( α = 0.05) for the statistical analysis. Similarly, ELISA was applied to measure the change in IFN- γ levels in response to the peptides and LPS. In this experiment, the Mouse IFN- γ ELISA Kit-E-EL-M0048 was applied according to the specified kit protocol, and the results were evaluated. Protein was extracted from TIB-71 cells for use in western blotting. For this purpose, the supernatant of the cells grown in the appropriate environment was separated for ELISA, whereas protein was extracted from the cellular pellets. Protein obtained using the Protein Extraction Kit (Abcam, Cambridge, UK) was prepared for western blotting. The concentrations of protein samples were determined using the Pierce BCA protein assay kit (Thermo Fisher Scientific). The results were analyzed by reading them on a 96-well plate reader at 565 nm. IL-6 was separated by 12% (w/v) SDS-PAGE and transferred onto a PVDF membrane using the wet transfer technique to compare the expression levels of the markers. Proteins were measured using horseradish peroxidase (HRP)-based clarity western ECL substrate and read using ChemiDoc devices. The area of the bands obtained in the western blotting results was computed using Image Lab software. Specifically, the volume occupied by the bands on the membrane was determined, and the band volume of IL-6 was normalized to that of β -actin to assess protein expression in a semiquantitative manner. The Liberty Blue peptide synthesizer was used for the chemical synthesis of peptides. In each synthesis, the amounts of amino acids used and the amount of dimethylformamide (DMF) dissolved in milliliters were calculated by the software of the peptide synthesizer. The peptide peaks appeared at 50%–55% hydrophobicity. Purity levels were determined by the ratio of peak areas to the total peak areas, and they were above 95%. In the analytical HPLC results, a single peak was observed for each peptide . The concentrations of the peptides were measured using the Pierce Quantitative Peptide Assays kit. Once the peptide concentrations were determined, the peptides were stored under appropriate conditions for use in the experiments ( Table 2 ). The cytotoxicity of the synthesized peptides was determined in TIB-71 cells using the MTT method. The toxic effects of peptides at varying concentrations are extremely important for the vital activities of macrophages, which are members of the immune system. The MTT assay was performed separately with and without LPS. Figure 2 presents the results of cytotoxicity assays for various concentrations of the AMPs and Mag2. This experimental group was first tested in an LPS-free environment. NET1, NET2, and Mag2 did not cause significant cytotoxicity at concentrations up to 4 μ g/mL, and they were further evaluated in the presence of different LPS concentrations. The LPS-free wells were used as a control. Figure 3 presents the percent cytotoxicity in the presence of different concentrations of LPS and the peptides. ELISA was employed to assess IFN- γ secretion into the TIB-71 cell supernatant in the presence of LPS (50 ng/mL) in and increasing concentrations of the peptides. The significance of the change in expression was determined by one-way ANOVA. p < 0.05 indicated statistical significance. The graph showing the amount of IFN- γ in the samples is shown in Figure 4 . The symbol “ ∗ ” means p ≤ 0.05, the symbol “ ∗∗ ” means p ≤ 0.01, and the symbol “ ∗∗∗ ” means p ≤ 0.001. TNF- α secretion from TIB-71 macrophages in the presence of LPS (50 ng/mL) and various concentrations of the peptides was examined by ELISA. One-way ANOVA was used to determine whether the changes in TNF- α responses were statistically significant. Figure 5 displays the concentrations of TNF- α in the samples. IL-6 expression was normalized to β -actin expression and compared with the control (unstimulated cells). The relationship between different experimental groups was examined statistically by one-way ANOVA. In Figure 7 , the fold changes in IL-6 expression in the presence of AMPs and LPS are presented. This research examined the antimicrobial activities of peptides based on helical structure, a crucial component of the immune system that is found in a wide variety of organisms. These peptides have important roles in natural defenses, and they simultaneously participate with the secondary immune response. The emergence of AMPs and their increasing importance in recent years are also related to the resistance of microorganisms to antibiotics, which has led to a need for alternative drug or molecule candidates. The AMPs used in this study include peptides previously developed and used by Polat et al. . The peptides were resynthesized by solid-state synthesis and used in our experiments. The 16-amino acid peptides used in this study were composed of two distinct amino acid types, namely the d- and l-forms. TIB-71 mouse macrophages were used in various tests to examine the effects of the peptides, the minimal inhibitory concentrations (MICs) of which in microorganisms were previously determined , on the immune system. We examined the interactions of the peptides with IL-6, IFN- γ , and TNF- α , which are important components structures of the immune system, through cytotoxicity assays, ELISA, and western blotting. First, the peptides were synthesized and purified by HPLC, as presented in Figure 1 . The appearance of a single peak indicated that the peptide was pure and confirmed the absence of external molecules. Fluctuations observed before purification indicate impurities. These contaminants can be attributed to the fact that the purification and analysis arms of the device were not completely washed. In addition, the rate at which the peptide is introduced into the system during HPLC also causes contamination. To eliminate these impurities, we collected the peptides injected into the system after analyzing the impurity. Each peptide gave a single peak in the system, and it was collected back in pure form . For cytotoxicity analysis, assays were conducted using macrophages, an immune system cell type that plays an effector role in many infectious diseases. In this context, TIB-71 mouse macrophages were chosen. The selectivity/safety index was calculated as a ratio of the MIC at which each AMP affects the microorganism to the concentration of AMP that causes 50% toxicity in the cells . The important finding of this assessment was that AMPs exhibited both low inhibitory effects and low toxicity . To conduct a broad screening, treatment with the peptides was started at a concentration of 64 µg/mL and reduced to 0.125 µg/mL. In this context, Mag2, which served used as a reference, exhibited low toxicity at almost all concentrations. NET1 and NET2 displayed similar cytotoxicity, starting at a concentration of 4 µg/mL. In particular, NET2 exhibited lower toxicity than the other peptides, even at high concentrations. Because of low concentrations of NET1 and NET2 compared with the other peptides, subsequent studies continued with these two peptides, and the concentration range was 0.5–4 µg/mL. LPS, which had a highly toxic effect alone, as expected, exhibited lower toxicity when applied in combination with the AMPs. At a concentration of 4 µg/mL, NET1 displayed the lowest toxicity. The toxicity of NET1 was increased threefold compared to that in the absence of LPS. The level of toxicity of NET2 was increased by tenfold compared to that in the absence of LPS. There were no significant changes in the cytotoxicity depending on the LPS concentration. The cytotoxicity of NET2 and Mag2 increased as the LPS concentration decreased. The results were similar when the peptide concentration was 2 µg/mL. The cytotoxicity of NET2 and Mag2 peptides increased with as the LPS concentration decreased. On average, NET1 displayed lower cytotoxicity than NET2 regardless of the LPS concentration. NET1 also exhibited lower cytotoxicity in samples in which the peptide concentration was 1 µg/mL. Similarly, in this analysis, the cytotoxicity of the peptides increased as the LPS concentration decreased. Finally, in the analysis in which the peptide concentration was 0.5 µg/mL, decreased NET1 cytotoxicity was observed in response to decreasing LPS concentrations, whereas increased cytotoxicity was observed for NET2. Mag2 displayed a constant effect at all concentrations. This might be attributable to the slight mitogenic response to low levels of the peptide and LPS . After cytotoxicity studies, ELISA was performed for NET1 and NET2 to assess IFN- γ secretion. In this context, the effects of the peptides both with and without LPS on IFN- γ secretion were investigated. The presence of LPS resulted in greater IFN- γ secretion than observed in unstimulated cells. When cells were treated with NET1 at three different concentrations in LPS-free medium, IFN- γ secretion increased significantly. IFN- γ secretion decreased as the peptide concentration decreased. When cells were treated with NET1 in the presence of LPS, a decrease IFN- γ expression occurred. Consequently, the secretion of IFN- γ also decreased. The expression of IFN- γ in cells stimulated with 8 µg/mL NET1 increased significantly at three different concentrations compared with the control level. Meanwhile, NET2 induced lower IFN- γ expression than NET1. Moreover, with increasing concentrations of NET1, IFN- γ secretion increased, whereas the opposite was observed for NET2. Furthermore, in contrast to NET1, the IFN- γ response was stronger when NET2 and LPS were used as costimulators. In effector cells stimulated with NET2 and LPS, IFN- γ secretion was significantly increased. Normally, the major sources of IFN- γ expression are T lymphocytes and NK cells. However, some studies revealed that this situation can change, and IFN- γ can be secreted by macrophages or different structures . This study illustrated that NET1 and NET2 significantly increased IFN- γ secretion, and NET2 displayed strong stimulatory properties in the presence of LPS. This situation, in which the effect changed in the presence and absence of LPS, was conspicuous in terms of its pro-inflammatory effect. Cytokine secretion, which becomes evident during acute inflammation, is effective against many infections. Studies should also be conducted with different cytokines to observe the immune effect of these peptides against various diseases. IFN- γ is involved in immune response modulation, including host defense against intracellular infections and tumors. IFN- γ facilitates Th1 differentiation, enhances macrophage activity, promotes leukocyte migration to the infection site, and increases major histocompatibility complex expression to improve T-cell identification of cancerous or infected cells. Nonetheless, uncontrolled IFN- γ expression in mice induces autoimmunity, and unchecked IFN- γ protein synthesis is likewise harmful to the host. Consequently, maintaining a balance between immunological tolerance and inflammation requires strict control over IFN- γ expression [ 17 – 19 ]. Cytokine secretion, which comes to the fore during acute inflammation, is effective against many infections. Studies should also be conducted with different cytokines to observe the immune effect of these peptides against diseases. Although their main job is to control inflammation, cytokines are essential for controlling the immune system in both health and illness. Cytokines are classified as either pro- or anti-inflammatory . In this study, we examined the effects of NET1 and NET2 on TNF- α expression at the cellular level in the presence and absence of LPS. There was no change in its expression in cells exposed only to LPS compared with the control. Different concentrations of NET1 in LPS-free medium significantly increased TNF- α secretion by 50%. However, there was no significant difference according to the peptide concentration. In addition, the TNF- α response did not differ between cells simultaneously stimulated with the peptide and LPS and cells treated only with the peptide. Similar results emerged in studies conducted with NET2. TNF- α secretion decreased with decreasing peptide concentrations in LPS-free medium. In the presence of LPS, the TNF- α response is stronger than that observed in the NET1 group. TNF- α secretion increased significantly, especially in the presence of 2 or 4 µg/mL NET2 combined with LPS. Studies conducted using lower peptide concentrations in an LPS-free environment have the potential to reveal anti-inflammatory effects [ 21 – 23 ]. The findings illustrated that NET1 and NET2 might have anti-inflammatory effects. The results were evaluated on the basis of the amount of IL-6 released from the cells into the medium alone. In this context, the amount of IL-6 released from LPS-treated cells increased by 50%, consistent with the data in the literature . The long-term presence of IL-6 can cause chronic inflammation. Because of cellular treatment with NET1, the amount of IL-6 released into the medium was decreased. It is predicted that the peptides have anti-inflammatory effects. However, no concentration-dependent change was observed. The same result was obtained when the cells were treated with LPS alone, namely, decreased IL-6 expression. An important point is that at the lowest concentration of 2 µg/mL, IL-6 expression was threefold lower than observed in the other groups. Similar results were obtained for NET2. When NET2 is used alone, IL-6 expression was 50% lower than that observed in NET1-treated cells. However, there was no significant change in IL-6 as the peptide concentration was decreased. When cells treated with LPS were then treated with NET2, an anti-inflammatory effect was recorded at a peptide concentration of 8 µg/mL. However, as the concentration was decreased, this effect disappeared, and IL-6 expression increased significantly. Both peptides induced the highest IL-6 expression at a concentration of 4 µg/mL in both the presence and absence of LPS. IL-6 expression was significantly increased by both 2 and 4 µg/mL NET2 in combination with LPS in comparison to the findings in the other groups. This might have been caused by the aggregation of peptides at low concentrations or disruption of peptide structures upon stimulation with LPS. Detailed studies should be conducted using a wide range of concentrations. The aims of the study were to compare the effects of our synthesized peptide structures on the immune system and evaluated their effectiveness. Many AMPs are promising structures for the treatment of various diseases with two-way effects on the immune system, as they exhibit both pro-inflammatory and anti-inflammatory effects, especially in response to inflammation. In this context, NET1 and NET2 exhibited anti-inflammatory effects by decreasing IL-6 and TNF- α at certain concentrations, whereas pro-inflammatory effects were induced through IFN- γ . This situation is compatible with the literature, and by increasing the diversity of experiments and improving the peptide structures, new therapeutic structures could be generated. In addition, the use of some AMPs alone or in combination gave better results on immune system elements. For example, it has been observed that LL-37 peptide, used in combination as an innovative approach in ovarian cancer, has an antitumor effect and increases the activity of NK cells . In another example, macrophage transformation and effects of monocytes were examined using various stimuli in the presence of LL-37. Monocytes cultured with M-CSF were directed to macrophages with pro-inflammatory properties . A further instance of macrophage turnover and the balance of pro-inflammatory and anti-inflammatory cytokines, LL-37 peptide induced anti-inflammatory IL-10 while reducing TNF- α and IL-17. It also affected the production of TGF- β . Inflammatory effects are also observed in peptides that are not natural but produced in mutant form. For example, mutated chensinin-1 peptide blocked LPS-induced pro-inflammatory cytokines . In another example of structurally changed peptides, pseudin-2 peptide isolated from Pseudis paradoxa was structurally changed and its anti-inflammatory effects were examined . Also full Ala-scan can also be performed to reveal the effect on cells. Performing these tests can provide detailed insights into the effects of AMPs against pathogenic cells. The interactions between AMPs and cells are crucial for modifying the pathways needed to neutralize pathogens, including chemical, biological, and electrostatic interactions. Changes and improvements in the primary and secondary structures of peptides directly influence their effectiveness against pathogenic cells. Additionally, investigating the D-Ala-D-Ala structures, which are vital components of bacterial peptidoglycan and play a role in antibiotic resistance mechanisms, is essential. Modifying synthetically produced AMPs based on their mechanisms of action is also critical for developing innovative treatment products against pathogenic microorganisms. Furthermore, conducting a full Ala-scan could further elucidate the impact of these peptides on cells. In this context, the NET1 and NET2 peptides we synthesized cause anti-inflammation at certain concentrations against IL-6 and TNF- α , while IFN- γ also causes pro-inflammation. This situation is compatible with the literature, and by increasing the diversity of experiments and improving the peptide structures we have obtained, it can lead to new generation therapeutic structures. Based on the experimental results, we believe it is important to apply these peptides over a wider concentration range and determine their effects on different immune system elements. Investigating the effects on the AMPs on different IL molecules working together simultaneously or evaluating their effects on different cytokine groups could expand their applicability. Animal studies should also be conducted to investigate the effects and toxicity of the peptides at different doses. | Review | biomedical | en | 0.999996 |
PMC11698615 | Cystic artery pseudoaneurysms are a rare entity causing hemodynamically significant and life-threatening hemorrhages that are usually secondary to acute cholecystitis . Other etiologies for the development of cystic artery pseudoaneurysms are pancreatitis, cholelithiasis, and trauma during cholecystectomy . The incidence of vascular injury during cholecystectomy is low (0.2-0.5%) and pseudoaneurysms associated with laparoscopic cholecystectomy generally arise from the right hepatic artery. The pathogenesis of cystic or hepatic artery pseudoaneurysms is thought to be related to inflammatory damage and weakening of the adventitia caused by cholecystitis or pancreatitis, which may be exacerbated by manipulation, clip application, or thermal injury . Complications of cystic artery pseudoaneurysms are serious and can include bleeding into the biliary system or peritoneum necessitating resuscitation and requiring prompt intervention . We present a case of an iatrogenic cystic artery pseudoaneurysm arising as a complication of transjugular liver biopsy (TJLB). TJLB is a widely performed and useful technique in patients with severe coagulopathy and/or severe ascites . TJLB is performed via a needle introduced through a hepatic vein, without causing a transcapsular injury, so any bleeding related to the procedure will drain back into the venous system. Since it was initially described in 1964, many large studies have shown it is a safe technique with complications reported between 0.6% and 20%, the most common major complications being intraperitoneal bleeding, vessel injury, or ventricular arrhythmia . In the present case, due to patient-specific anatomy, the biopsies were taken from an accessory hepatic vein resulting in a traumatic cystic artery pseudoaneurysm. Pre-procedural lab work showed an international normalized ratio (INR) of 3.6, fibrinogen of 81 mg/dL, 67 platelets/µL, and hemoglobin of 11.6 g/dL. After the transfusion of two units of fresh frozen plasma and cryoprecipitate, the INR and fibrinogen improved to 2.7 and 152 mg/dL, respectively. At the start of the procedure, the right internal jugular vein was accessed under ultrasound guidance utilizing a micropuncture system (Bloomington, IN: Cook Medical), and a 10-french Check-Flo sheath (Bloomington, IN: Cook Medical) was placed. Through the sheath, a multipurpose angled catheter (Shibuya, Japan: Terumo) was used to access a hepatic vein. Multiple attempts were made to access the main right hepatic vein; however, this was not possible due to sharply angulated anatomy at the venous origin. Selective free hepatic venography was performed showing the catheter within an accessory right hepatic vein . At this point, a Fogarty balloon catheter (Irvine, CA: Edwards Lifesciences) was advanced into the accessory hepatic vein, and pressure recordings were obtained with a portosystemic gradient of 20 mmHg. Next, the Rösch-Uchida transjugular liver access set and Quick-Core Biopsy Needle (Bloomington, IN: Cook Medical) were placed within the accessory hepatic vein and two core needle biopsies were then taken through the system . Multiple venograms throughout the procedure, before and after the biopsy, showed that the sheath was persistently within the hepatic vein. After the removal of the sheaths, hemostasis was obtained at the internal jugular vein using manual compression. Post-procedurally, the patient’s hemoglobin steadily trended down from pre-procedural values of 11.6 g/dL to 6.4 g/dL despite transfusion of multiple units of packed red blood cells. Multiphasic computed tomography angiogram of the abdomen was performed which showed arterial phase contrast extravasation into the gallbladder lumen with pooling of contrast in the lumen on delayed phases . The patient was then taken back to the fluoroscopy suite for a mesenteric angiogram with possible embolization. The micropuncture kit was used to access the right common femoral artery and a 6-french access sheath (Shibuya, Japan: Terumo) was placed. The celiac and superior mesenteric arteries were catheterized using a C2 catheter (Bloomington, IN: Cook Medical). Angiograms from the celiac artery showed a faint vascular blush in the gallbladder. The proximal gastroduodenal artery was accessed through a microcatheter system using a Progreat microcatheter (Shibuya, Japan: Terumo) and Fathom microwire (Marlborough, MA: Boston Scientific). Superselective angiograms from the proximal cystic artery showed a 3 x 3 mm pseudoaneurysm with contrast blush into the gallbladder lumen arising from the distal superior division of the cystic artery . Irregular traumatized vessels were also seen at the superior aspect of the gallbladder. The microsystem was advanced to the superior division of the distal cystic artery, and embolization was performed using Nester metallic coils (Bloomington, IN: Cook Medical). Post-embolization angiogram from the cystic artery demonstrated continued filling of the pseudoaneurysm from the inferior division of the cystic artery . The inferior division was similarly embolized with metallic coils with post-embolization angiography from the cystic artery demonstrating no filling of the previously seen pseudoaneurysm as well as no active extravasation . Hemostasis of the right common femoral arteriotomy site was achieved using a StarClose SE closure device (Chicago, IL: Abbott Laboratories). The patient tolerated the procedure well and required no further blood products during admission and was discharged on post-operative day six. The patient has been listed on the liver transplant waiting list at this time and is awaiting transplantation. He has undergone a repeat multiphasic computed tomography angiogram for hepatocellular carcinoma screening without evidence of pseudoaneurysm recurrence, non-target embolization, or complication secondary to endovascular embolization. Cystic artery pseudoaneurysms are a rare entity that generally arise from complications associated with acute cholecystitis. We present a case of an iatrogenic cystic artery pseudoaneurysm after TJLB with biopsy samples taken from an accessory right hepatic vein. Given the unfavorable right main hepatic vein anatomy in this patient, TJLB through the accessory vein was thought to be less risky for the patient than conversion to a percutaneous approach. Sampling from the accessory right hepatic vein is an accepted approach; however, it has been reported to be associated with an increased risk of complications, including direct arterial injury, liver capsule perforation, or even accidental renal sampling . In the present case, biopsies taken from this position likely caused trauma to the superior division of the distal cystic artery and resultant hemorrhage and hemobilia. Transarterial embolization has been shown to have an approximately 95% success rate in treating post-laparoscopic cholecystectomy cystic and hepatic artery pseudoaneurysms, with less morbidity and mortality than repeat surgery . However, approximately 88% of pseudoaneurysms associated with laparoscopic cholecystectomy arise from the right hepatic artery. Only a few cases have been reported to be secondary to TJLB, and of these cases, the traumatized artery is a branch of the hepatic artery . The present literature review did not identify the case of cystic artery pseudoaneurysm after TJLB. Cystic artery pseudoaneurysms are rare complications usually associated with acute cholecystitis and laparoscopic cholecystectomy. We discussed a rare case of iatrogenic cystic artery pseudoaneurysm after TJLB. This rare yet life-threatening complication impacts patient outcomes. This case raises the idea that close post-procedure monitoring for hemodynamic instability or changes in laboratory parameters after TJLB may be warranted in patients with risk factors for hemorrhage, such as baseline coagulopathy to rule out rare yet morbid complications such as iatrogenic cystic artery pseudoaneurysm. | Clinical case | biomedical | en | 0.999996 |
PMC11698687 | A healthy placenta is essential for fetal well-being because it delivers oxygen and nutrients and removes carbon dioxide and other waste products 1 . The placenta also functions as an intermediary between the maternal immune system and the fetus and protects the fetus from pathogens 2 . Recent placental research has focused on diverse aspects of placental function, maternal-fetal health, and potential therapeutic applications. Studies have explored the impact of environmental factors on placental health, investigated the levels of non-essential trace metals and their effects on placental function 3 . Research has also emphasized the critical role of timely medical intervention in placental complications, which comes from a study linking maternal late hospital arrival with adverse fetal outcomes in placental abruption cases 4 . In a normal human pregnancy, the intrauterine immune response starts as soon as implantation 5 . The placental immune response evolves across the gestation, and each trimester is characterized by a unique inflammation environment 6 . The accumulation of immune response could trigger the labor onset 7 . Several types of immune cells function in the placenta 8 , 9 . In a healthy placenta, uterine or decidual natural killer (NK) cells, maternal/fetal macrophages, dendritic cells, and regulatory T cells participate in spiral artery remodeling and trophoblast invasion. In pathological scenarios, additional immune cells often infiltrate the placenta. For example, neutrophils enter the placenta in cases of chorioamnionitis, chorionic vasculitis, and acute villitis 10 ; eosinophils enter the placenta in cases of intervillous thrombosis, cysts, and chorionic vasculitis 11 ; basophils enter the placenta in cases of acute placental infarction 11 ; increased macrophages enter the placenta in cases of listeria, tuberculosis, syphilis, acute atherosis, and villitis of unknown etiology 12 – 14 ; and CD4 + T cells significantly increase in cases of villitis of unknown etiology 15 . However, most of our knowledge of these processes in humans comes from immunohistochemistry of the ex vivo placenta after delivery. To longitudinally monitor immune infiltration of the placenta in pregnancy, we need a safe, non-invasive, and accurate imaging modality. Current imaging techniques for monitoring placental conditions, such as ultrasound and computed tomography (CT), have significant limitations. Ultrasound is widely used for placental assessment due to its cost-effectiveness and real-time imaging capabilities. However, its accuracy can be compromised by factors like maternal obesity and bladder fullness. Additionally, ultrasound resolution diminishes in the later stages of pregnancy, making image interpretation challenging due to low contrast. Moreover, ultrasound is not sensitive to detecting placental inflammation. While CT can be valuable for assessing trauma during pregnancy, its use is restricted by the risks associated with ionizing radiation exposure, limiting its broader application for routine placental monitoring. As an emerging clinical imaging tool in the field of Obstetrics and Gynecology, MRI forms high-quality and multi-contrast 3D images using water molecules, which are naturally abundant in the human body. Therefore, MRI can be employed repeatedly in longitudinal studies to assess human pregnancy. Latest advancements in MRI, such as diffusion-relaxation MRI and T2*-weighted MRI, have become an important tool in placental research. Diffusion-relaxation MRI have been used to detect altered placental development in pregnancies affected by congenital heart disease (CHD). A study found significant differences in placental microstructure and perfusion between CHD-affected pregnancies and controls, particularly after 30 weeks’ gestation 16 . T2*-weighted placental MRI has been investigated as both a basic research tool and a potential clinical test for placental dysfunction. This imaging technique shows promise in directly reflecting placental function, which could significantly improve antenatal screening for placental dysfunction 17 . Especially, diffusion MRI (dMRI) enables the researcher to image and quantify the great complexity and heterogeneity in human placental microstructure 18 . In hemodynamic studies, dMRI enabled the exploration of the blood flow in the healthy placenta 19 and the mal-perfusion in pre-eclampsia 20 . dMRI was also used to track the myometrium alternation induced by placenta accrete 21 . In addition, merging dMRI with other MR contrasts brings new insight into the compartment-wise oxygenation property 22 . Although dMRI was successfully applied to detect inflammation in brain and other organs 23 – 25 , its placenta application has not yet yield imaging biomarkers specific to placental inflammation. To overcome a similar challenge of measuring neuro-inflammation in vivo in human brain, DBSI was successfully developed 26 , 27 . This advanced dMRI method is able to disentangle the highly restricted intracellular diffusion signal associated with cellularity from the diffusion associated with other microstructural components. DBSI has been extensively validated in the brain to detect increased cellularity during inflammation demyelination 26 and other forms of neuroinflammation in tumors 28 . Here, we customized DBSI based on the unique placental microenvironment to image immune cell infiltration in the human placenta. We performed ex vivo and simulation experiments to develop and validate the method and then used it for in vivo longitudinal analysis of the placenta. We found that our in vivo placental DBSI method reveals an increasing immune response after the first trimester, particularly in the placenta tissues compartment. We also discovered greater immune cellularity in placentas with pathology-defined signs of inflammation than in placentas without signs of inflammation at as early as 11–13 weeks’ gestation. A total of 91 patients were enrolled in the study, and 239 MRI scans were scheduled, ranging from 12 to 38 weeks. MRI scans were scheduled on the same day as the patients’ regular obstetric visits at approximately 12, 20, 32, and 36 weeks’ gestation. A few patients were dual enrolled in other studies and thus underwent additional MRI scans. 202 scans from 82 patients were analyzed after excluding unfinished scans and poor-quality scans which are not sufficient to be analyzed by DBSI. At delivery, placentas from all participants were collected for pathologic and single-nucleus RNA sequencing examination. We used the pathological diagnosis to divide the cohort into two groups. The placentas of 12 patients were defined as the inflammation group (Table 1 , Supplemantary Table 1 ). The placentas from 70 patients did not have any of these diagnoses and were defined as the non-inflammation group. There were no significant differences in age (29.17 ± 4.15 years vs. 29.28 ± 4.98 years) or body mass index at delivery (27.42 ± 5.89 kg/m 2 vs. 28.04 ± 5.79 kg/m 2 ) between the inflammation and non-inflammation groups. A subset of the non-inflammation group (50 scans from 17 patients) who have additional T2* scans was used to conduct intra-placental segmentation. To perform ex-vivo validation, we obtained the ex-vivo MRI and immunohistochemistry (IHC) from five placenta after delivery. Among the five IHCs, we built 3D geometry on 400 0.25 × 0.25 mm 2 regions and conducted a Monte Carlo simulation. Table 1 Demographic and clinical information Age BMI Gestational age Pathology Non-inflammation group ( n = 70) 29.28 ± 4.98 28.04 ± 5.79 266.58 ± 20.62 Inflammation group ( n = 12) 29.17 ± 4.15 27.42 ± 5.89 265.83 ± 18.12 Pt #1 36 23.16 281 Multifocal villitis with associated intervillositis Fetal membrane with pigmented amniotic macrophages. Pt #7 31 28.34 274 Fetal membranes with numerous pigment-laden amniotic macrophages Pt #14 20 18.54 280 Fetal membrane with moderate acute chorioamnionitis Trivascular cord with modera funisitis Pt #35 27 25.97 263 Acute chorionitis Trivascular cord with umbilical arteritis Pt #42 26 33.80 278 Fetal membranes with numerous pigmented amnionic macrophages Chronic lymphohistiocytic villitis, non-necrotizing Pt #44 29 28.30 277 Chronic lymphohistiocytic villitis Increased perivillous fiberinoid deposition, approximately 10% of volume Pt #48 32 22.04 260 Chronic lymphohistiocytic villitis, mild/focal, non-necrotizing Pt #50 29 35.98 243 Acute chorionitis Pt #60 27 20.45 220 Fetal membrane with acute chorioamnionitis Pt #109 34 25.14 264 Incidental septal cyst (2 cm in greatest dimension) Pt #112 28 32.01 273 Intervillous fibrin Peripheral placental cyst with 0.9 cm greatest dimension. Pt #116 31 35.35 277 Fetal membranes with pigmented amniotic macrophages Mild acute chorioamnionitis p -value 0.88 0.76 0.90 In DBSI, “dictionaries” are used to disentangle diffusion characteristics of water molecules in and around multiple cellular components within an imaging voxel . For this study, we were most interested in distinguishing immune cells from other cells in the placenta. To do so, we took advantage of the large difference in the sizes of placental cells. Using the placenta collected in this study, single-nucleus RNA sequencing (snRNA-seq) was conducted to determine the composition of cell types in the placenta . Fig. 1 Schematic demonstration of diffusion basis spectrum imaging (DBSI) methodology. A Schematic of a Magnetic Resonance Imaging (MRI) voxel that contains several microstructures. B Schematic of measured diffusion signals from the voxel. C Diffusion dictionary assembled from prior knowledge. D DBSI-derived spectrum. E The dictionary corresponds to placental cells of different radii: T cells, 2–3 µm; natural killer (NK) cells, 3–4 µm; B cells, 4–5 µm; neutrophils, 6–7 µm; dendritic cells (DC), 7–10 µm; macrophages, 10–11 µm; cytotrophoblasts (CTB), syncytiotrophoblast (STB), extravillous trophoblasts (EVT), and decidua stromal (dS) cells, and other placental resident cells, >10 µm. F t-distributed stochastic neighbor embedding (tSNE) map of variant types of placental cells and their percentage, which were annotated (and labeled from blue color to red color, correlated to Diffusion Dictionary) by single-cell RNA sequencing. The decidua is largely composed of round decidual and trophoblast giant cells, and the chorionic plate is mainly composed of fetal blood vessels embedded in fetal stromal tissue, lined by trophoblastic cells. Those decidual and trophoblast cells are usually over 40 µm in the largest dimension, and many have irregular shapes. The chorionic villi are mainly composed of cytotrophoblasts, which have a radius over 20 µm, and syncytiotrophoblasts, which are formed by the fusion of many cytotrophoblasts. In contrast, immune cells are generally less than 8 µm in radius. Thus, we conducted a Monte-Carlo simulation on cell structures with a radius of 8 µm (Supplementary Material 3 ). The computed cell apparent diffusion coefficient (ADC) was 0.6 mm 2 /msec. Therefore, our DBSI analysis method extracted placental immune cells from the isotropic spectra by using ADC values between 0.01 and 0.6 mm 2 /msec . To determine whether DBSI could be used to assess immune cell infiltration in the placenta accurately, we performed ex vivo validation. We obtained samples from five placentas after delivery, embedded them in agar, and performed ex vivo MRI. We then bisected the samples, fixed them, embedded them in paraffin, cut sections, and performed IHC with an anti-CD4 antibody . We quantified the CD4-positive area and used landmarks to register the IHC-derived immune cell density maps with DBSI-derived immune cell density maps . Qualitatively, we found similar patterns in all samples in the DBSI-derived and IHC-derived maps . Linear regressions of the data from all five samples showed significant correlations (r 2 between 0.38 and 0.71, p < 0.001). Moreover, the estimated slopes from the linear regressions were all close to 1 . Fig. 2 Correlation between DBSI- and immunohistochemistry-derived immune cell densities. A Raw immunohistochemistry (IHC) images of CD4 staining. B Positive stain area ratio on CD4 IHC, registered to the corresponding DBSI image. Images at the far right are high-magnification views of indicated regions in raw CD4 staining images. C DBSI-estimated immune cell density. The IHC- and DBSI-derived maps share the same heatmap keys. D Correlation between the DBSI- and IHC-derived immune cell densities. Each point represents the mean value in a 5-by-5 pixel patch in ( A ) and ( B ). The 95% confidence intervals of the linear regressions are shaded green. The dashed line has a slope of 1 and is shown as a reference. To assess our method’s sensitivity and noise resistance, we built a Monte Carlo simulated three-dimensional microstructure geometric model derived from histology images collected in this study . We then simulated diffusion MRI signals with different signal-to-noise ratios consistent with the ranges observed in ex vivo and in vivo MRI data (58.6 ± 7.9 and 37.4 ± 3.7, respectively). We simulated 400 0.25 × 0.25 mm regions on histology images. There was a significant linear correlation between the simulated MRI-derived cell density and the IHC-derived cell density under signal-to-noise ratios of 75 to 25 . Fig. 3 Monte Carlo simulation of diffusion MRI based on histology images. A CD4 staining of a placenta specimen. Four representative regions were selected (boxes). B Hexagonal prisms were used to model placental resident cells. C A tube surrounding the entire villus was used to model the syncytiotrophoblast layer. D Water molecules were placed outside the villous model to simulate diffusion in the intervillous space. E Spheres of radius 4–8 µm were placed where CD4 positive stain was detected to represent immune cells. F Diffusion trajectories from the example random seeds in the four structures. G Built a 3D model based on the histology image in ( A ). The entire 3D model covers a 5 × 5 mm region and is the input for the Monte Carlo simulation. The model was divided into 400 0.25 × 0.25 mm regions, one of which is illustrated in the dashed-line box. Hexagonal prisms representing resident cells are marked grey, models representing syncytiotrophoblasts are marked blue, and spheres representing immune cells are marked red. H Diffusion MRI signals were simulated in 400 0.25 × 0.25 mm regions with various signal-to-noise ratios. The simulated MRI- and IHC-derived immune cell densities were compared by linear regression. The green line shows the linear regression and 95% confidence range. The red dashed line has slope of 1 and is shown as a reference. We first used DBSI to identify the placental regions that had the most immune cell infiltration. To do so, we used our previously developed and published intra-placental segmentation method based on T2* and diffusion MRI to divide the placenta into three major compartments: intervillous space, placental vessels, and placental tissue 29 . We combined this method with the placental DBSI analysis on 50 MRI scans from 17 patients in the non-inflammation group who have both T2* and diffusion MRI scans. Figure 4A–D shows images from a representative patient (29 years old, viable male infant delivered at 39 weeks and 4 days) imaged at 36 weeks’ gestation. The highest voxel-wise cellularity ratio was in the placental tissue region . We then combined all the data from the 17 patients and analyzed them in two ways. First, we used linear regression to examine the cellularity ratio in each placental region at multiple time points in pregnancy . This analysis revealed that cellularity was significantly greater in the placental tissue region late in pregnancy than early in pregnancy . Second, we compared cellularity between the compartments at each time point . This analysis showed that cellularity was significantly greater in the placental tissue region than in the placental vessels or intervillous space regions at 32 weeks’ gestation ( p < 0.05) and 36 weeks’ gestation ( p < 0.01) . Fig. 4 Cellularity ratio in each placental compartment. A – D Representative cellularity maps in the intervillous space (IVS), placental vessels (PV), placental tissue (PT), and total placenta are illustrated in heat maps. E Boxplots of voxel-wise cellularity ratio in the representative placental regions in ( A – D ). F – I Linear regressions of the mean cellularity ratios in each compartment and entire placenta. The colored region indicates a 95% confidence interval. J – L Comparison of mean cellularity in each visit (20/32/36 weeks). In the box plots, the horizontal line indicates the median, the + indicates the mean, the edges of the box indicate the 25th and 75th percentiles and the whiskers indicate 95% confidence intervals. * p < 0.05, ** p < 0.01, **** p < 0.0001 by Wilcoxon Rank-Sum test. Next, we compared our placental DBSI data from the two groups of placentas: non-inflammation and inflammation. To visualize and compare the immune cell density in the two groups, we plotted the color-coded immune cell density maps on top of the anatomical reference T2 weighted images. Figure 5A–F shows four axial views of different MRI slices from one representative non-inflammation placenta and from one representative inflammation placenta (patient #109, see Supplementary Table 1 for details) at 20, 32, and 36 weeks’ gestation. Qualitatively, immune cell density appeared higher in the inflammation placenta than in the non-inflammation placenta . In both placentas, voxel-wise cell density increased over pregnancy . Fig. 5 Longitudinal progress of placental cellularity in two representative placentas. A – F Placental cellularity map at 20, 32, and 36 weeks’ gestation from ( A – C ), a patient in the non-inflammation group, and ( D – F ), a patient in the inflammation group. Background gray scaled images are T2WI. Cellularity ratios >0.01 are depicted in heat maps. G , H Boxplots of voxel-wise placental cellularity ratio across three-time points in the two patients. In the box plots, the horizontal line indicates the median, the + indicates the mean, the edges of the box indicate the 25th and 75th percentiles and the whiskers indicate 95% confidence intervals. **** p < 0.0001 by Wilcoxon Rank-Sum test. We asked whether there were group-wise differences in immune cellularity between non-inflammation and inflammation placentas. We performed linear regressions on the mean placental cellularity ratios for each of the two groups . Cellularity was significantly greater later in pregnancy than earlier in the non-inflammation group but not in the inflammation group . Throughout pregnancy, the cellularity ratio was significantly higher in the placentas from the inflammation group than in the placentas from the non-inflammation group . This difference was significant at each of the four time points ( p < 0.01) . Fig. 6 Longitudinal placental mean cellularity. A , B Mean cellularity ratio in the ( A ) non-inflammation group and ( B ) inflammation group. C Combined plot of ( A ) and ( B ). D Mean placental apparent diffusion coefficient (ADC) in both groups; blue indicates non-inflammation, and red indicates inflammation group. In ( A – D ), colored regions indicate 95% confidence intervals. E – H Comparison of cellularity ratio between the two groups at the indicated weeks’ gestation. In the box plots, the horizontal line indicates the median, the + indicates the mean, the edges of the box indicate the 25th and 75th percentiles and the whiskers indicate 95% confidence intervals. ** p < 0.01; *** p < 0.001 by Wilcoxon Rank-Sum test. Finally, to compare DBSI to another MRI method, diffusion tensor imaging, we examined mean ADC in non-inflammation and inflammation placentas. Diffusion tensor imaging-derived ADC values did not differ between non-inflammation and inflammation placentas at any time point (Supplementary Material 5 ). Moreover, in a linear regression of ADC values, the non-inflammation and inflammation groups did not significantly differ from each other . This finding suggests that diffusion tensor imaging failed to distinguish between the non-inflammation and inflammation groups compared to DBSI. Herein, we developed and validated a DBSI method to image and quantify immune cell density in the human placenta across gestation. Additionally, we used our method to reveal placental differences between non-inflammation and inflammation cases, as defined by the placental pathology reports after delivery. Our method could detect significant differences in immune infiltration as early as 11–13 weeks’ gestation. With further clinical validation, this DBSI method could be used clinically to identify pregnant patients with excess placental inflammation. Early detection would allow for timely interventions, such as enhanced monitoring or anti-inflammatory treatments, potentially preventing complications like preeclampsia or preterm birth. This approach could enable more personalized management of high-risk pregnancies, significantly improving both maternal and fetal outcomes. The DBSI metric of cellularity revealed significant differences between the non-inflammation and inflammation placentas, whereas the standard diffusion tensor imaging metric of ADC did not differentiate between the two groups. This difference is likely because diffusion tensor imaging cannot distinguish different diffusion components within an imaging voxel, so ADC reflects the average water diffusion across all placental microstructure components within the same voxel. In contrast, DBSI can specifically detect diffusion of water molecules within cells of a defined size range to reflect placenta immune cellularity. Specifically, the DBSI method can successfully image the immune cellularity in the human placenta by taking advantage of the fact that immune cells are generally much smaller than other cells in the placenta. To strictly establish DBSI in accurately imaging and quantifying placenta immune cells, we conducted a careful validation study using the placenta collected in this study. Based on the RNA-sequencing results , we found the majority of placental immune cells were CD4 positive. We also conducted and tested other IHCs, including CD3, CD68, and CD79a, but none of them showed significant enough positive stains, similar to previously published studies 15 , 30 , so we did not include the data and results from other IHCs. Hence, we decided to validate the accuracy of the DBSI method by comparing it to IHC with an antibody specific to CD4. Our landmark-based voxel-to-region registration method allows accurate alignment between IHC and DBSI images, which enables quantitative, voxel-wise comparison between DBSI- and IHC-derived cellularity maps over the entire placenta tissue sections. In addition to IHC validation, our Monte Carlo computer simulation revealed that the DBSI method is accurate with signal-to-noise ratios as low as 25 dB on non-diffusion weighted b0 images. This is much lower than the typical signal-to-noise ratios of 50–75 dB in ex vivo MRI. Given our ex vivo and simulation data, we concluded that our DBSI method can sensitively and accurately image and quantify immune cell density in the placenta. Taken together, the validation measures all suggest that DBSI can accurately reflect in vivo inflammation in the placenta. Our finding that inflammation significantly increased across pregnancy in the placental tissue compartment is consistent with previous reports that immune cells reside in the placental parenchyma and accumulate during pregnancy. Additionally, recent studies show that NK and T cells increase and differentiate in late gestation 31 , and neutrophils and macrophages infiltrate the placenta from the myometrium in late pregnancy to prepare for labor 32 , 33 . The immune cellularity in the placental vessel compartment was consistently low (around 5%) over pregnancy. Work from others suggests that immune cells in this compartment help to protect the fetus from infections 34 and maintain uterine homeostasis 35 . The immune cellularity in the intervillous space was also consistently low (around 5%) over pregnancy. Mucosal-associated invariant T cells have been identified in the intervillous space and shown to accumulate upon bacteria stimulation 36 . In this study, we are especially intrigued by our observation of localized cellularity in the inflammation placenta in Fig. 5D–F . On pathology, this placenta was noted to have a 2.5 cm cyst, but its location was not noted in the pathology report, so we were unable to determine whether it corresponded to the location of high cellularity. By conducting ex vivo DBSI of whole placentas in future studies, we may be able to identify DBSI features that reflect pathological observations. In group-wise analysis, we found that immune cell density increased as pregnancy progressed in the non-inflammation group. This finding is consistent with previous observations that placental inflammation increases from second to third trimester 6 , 37 and can trigger uterine contractions, cervical ripening and dilation, and placental separation 38 – 40 . Hence, placental inflammation has been suggested as a mechanism responsible for both term and preterm labor onset 41 , 42 . In future work, we will perform DBSI at more time points near the end of pregnancy to determine whether we can predict the onset of labor. This study has serval limitations. First, immune cells have more complex geometries than the isotropic models we used in DBSI. As we defined any cell of radius less than 8 µm to be an immune cell, we could not assess the dynamic composition of different types of immune cells during pregnancy. In addition, relying solely on CD4+ cells as a marker for DBSI validation could overlook the presence of other immune cell types, potentially limiting the scope and comprehensiveness of the analysis. In future work, we can overcome these limitations by combining high-resolution immune cell 3D geometries with Monte Carlo simulations to develop more realistic diffusion signal models of various placental immune cells in DBSI. Additionally, in future studies, we plan to utilize more specific immune cell markers in IHC beyond CD4+ to enhance the accuracy and depth of the analysis. And we will also include more pathological data of patients for more comprehensive analysis. Second, we were unable to perform intra-placental segmentation in inflammation placentas in this study due to the significant motion of the fetus, uterus, and placenta during the imaging session. In future work, we aim to enroll more patients from both non-inflammation and inflammation placentas and conduct more high-quality compartment-based analyses, especially for the inflammation group, to determine whether particular regions of the placenta are more prone to immune cell infiltration based on the different pathologies behind it. Third, we did not account for potential confounders such as comorbidities, medication use, multiple gestations, and fetal anomalies in this study. In future research, we will incorporate these factors, as they may influence placental inflammation in human subjects. This study was approved by the University’s Institutional Review Board, and all participants signed informed consent forms. Potential participants were included if they had a singleton pregnancy with no identified fetal anomalies at recruitment (before 12 weeks’ gestation) and intended to receive obstetric care and deliver at our institution. To make our study cohort homogenous, potential participants were excluded if they had multiple gestations, had a major fetal anomaly or had contraindications to MRI. MRI was performed on a 3 T Siemens Vida scanner (Siemens, Erlangen, Germany). Diffusion MRI of the entire uterus was acquired by a 2D EPI sequence in 86 different weightings (directions are the same as those in ex-vivo), with a max b-factor of 900 s/mm 2 . Eight b0 images were placed across the entire diffusion MRI scans. Other acquisition parameters were: 12.8 s repetition time, 62 ms echo time, in-plane resolution of 3 mm by 3 mm, and slice thickness of 3 mm. The total scan time was approximately 22 min. The Echo train of additional T2* MRI were: [3, 5.11, 7.2, 9.3, 11.4, 13.5, 15.6, 17.7, 19.8, 21.9, 24] (msec). The in-plane resolution was 4 mm by 4 mm, and the slice thickness was 4.2 mm. The FOV of Both T2* and diffusion MRI covers the entire placenta. This was a retrospective study, and we sorted the dMRI data into two groups based on post-delivery pathology. For all enrolled patients, the placenta was collected post-delivery for routine gross and histological exams performed by a board-certified pathologist with expertise in placental pathology (I.H.). Patients were divided into two groups (inflammation/non-inflammation) based on the findings on the pathology report: placenta noted to have villitis, chorioamnionitis, funisitis, chorionitis, or infarction and were defined as the inflammation cases. A subset ( N = 13) of placenta samples were collected for single-nucleus RNA sequencing test. Placenta specimens were cut from a subset ( N = 5) of placenta for ex-vivo validation between MRI and IHC and simulation validation. snRNA-seq was performed with 10X Genomics technology using the placental tissues collected in this study ( www.10Xgenomics.com ). Placental cell types and their relative percentages were annotated and visualized with t-distributed stochastic neighbor embedding (tSNE) (32), as presented in Fig. 2F . The size of variant types of cells was labeled, and the density of cell clusters was marked blue and red. Both cell size and density were correlated to the Diffusion Dictionary detected with diffusion basis spectrum imaging (DBSI). Specimens were collected from five fresh placentas after delivery to conduct ex vivo MRI scans. The samples were approximately 1 cm in width and length and were the entire height (thickness) of the placenta. Samples were stored at 4 °C, and within 12 h after delivery of the placenta, the specimens were embedded in a customized container filled with 2% agar. The ex vivo MRI was conducted on an 11.7 T Varian MRI scanner at 20 °C. A 2D EPI sequence was used, with TR = 1000 msec, TE = 32 msec, voxel size = 0.25 × 0.25 × 1 mm 3 , diffusion time = 16 msec, diffusion interval = 7 msec, 86 different diffusion weightings, and 5x diffusion intensity . After MRI scans, the samples were bisected, fixed in 10% neutral-buffered formalin for one week, and processed into paraffin. Then, slide-mounted 5 µm histologic sections underwent immunohistochemistry with an anti-CD4 antibody (CONFIRM anti-CD4(SP35), Pre-dilute, Ventana, USA) and hematoxylin counterstain. Finally, the stained sections were scanned in a Hamamatsu NanoZoomer at 200x magnification. The details of quantification, co-registration, and correlation analysis between ex-vivo data can be found in Supplementary Material 2 A customized preprocessing pipeline for placental diffusion MRI data is shown in Supplementary Material 1 . In DBSI analysis 26 , the diffusion MRI data are separated into a linear combination of signals across known physiological and pathological ranges (Eq. 1 ) . By solving the linear equation, the mixture of diffusion compartments inside a single imaging voxel can be revealed. The solved linear coefficients are the diffusion basis spectrum . 1 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${S}_{k}=\mathop{\sum }\limits_{i=1}^{{N}_{{aniso}}}{f}_{i}{e}^{-\left|\vec{{b}_{k}}\right|{\lambda }_{{\perp }_{i}}}{e}^{-\left|\vec{{b}_{k}}\right|\left({\lambda }_{{\parallel }_{i}}-{\lambda }_{{\perp }_{i}}\right){cos }^{2}{\phi }_{{ik}}}+\mathop{\sum }\limits_{j=1}^{{N}_{{iso}}}{f}_{j}{e}^{-\left|\vec{{b}_{k}}\right|{D}_{j}}$$\end{document} S k = ∑ i = 1 N a n i s o f i e − b k → λ ⊥ i e − b k → λ ∥ i − λ ⊥ i c o s 2 ϕ i k + ∑ j = 1 N i s o f j e − b k → D j Where \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${S}_{k}$$\end{document} S k and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\vec{{b}_{k}}$$\end{document} b k ⃗ are the signal and b -value of the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${k}^{{th}}$$\end{document} k th diffusion gradient; 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Additionally, conventional diffusion tensor imaging was performed to compute the ADC. The DBSI-derived ‘restricted’ isotropic diffusion fraction ( D j ≤ 0.6 μm 2 /ms) is defined to reflect cellularity ratio in placenta. | Review | biomedical | en | 0.999996 |
PMC11698692 | Chronic otitis media (COM), a common condition in otorhinolaryngology, is characterized by chronic, intermittent, or persistent discharge through a perforated tympanic membrane (TM) . COM is clinically characterized as an inflammatory condition associated with otorrhoea and tympanic membrane perforation in some cases . In COM, tympanic membrane perforation and ossicular chain erosion cause defective middle ear function leading to hearing loss. The proposed mechanism for erosion is chronic middle ear inflammation resulting from the overproduction of cytokines TNF alpha, interleukin-2, fibroblast growth factor, and platelet-derived growth factor, which promote hypervascularization, osteoclast activation, and bone resorption causing ossicular damage . The human ear can recognize sound waves in a frequency range of 20-20,000 Hz. Human speech frequencies are in the range of 500-4000 Hz, and the decibel level for a normal conversation is between 45 and 60 dB. The degree of hearing loss depends on the size of perforation in the tympanic membrane, ossicular chain, and inner ear. Pure tone audiometry helps to identify hearing thresholds of an individual based on the intensity and pitch of the sound waves. Since hearing loss is the most common complication associated with COM, which affects day-to-day activity and the quality of life of an individual, an attempt is made to analyze the hearing loss by audiometry in patients with COM . The goals of any surgery for COM include the creation of a dry, safe ear and the maximal preservation or restoration of hearing . The management of chronic otitis media, with or without cholesteatoma, is cortical mastoidectomy with TM perforation repair and/or ossicular chain repair, which is considered the treatment of choice for active chronic otitis media . The aim of this study was to evaluate the postoperative audiological outcomes in middle-aged patients compared to younger patients who underwent tympanoplasty with mastoidectomy via post-auricular approach for the treatment of COM. Additionally, we compared these postoperative results with those of younger patients, providing insights into the effectiveness and safety of the procedure across different age groups. This study was conducted at the Department of Otorhinolaryngology, Jawaharlal Nehru Medical College and Hospital, Aligarh Muslim University, Aligarh, after obtaining ethical clearance from the Institutional Ethics Committee . A total of 70 patients, admitted in wards from August 2017 to January 2019, suffering from chronic otitis media with or without cholesteatoma who underwent tympanoplasty with mastoidectomy were included. The patients were divided into two groups: Group A (middle-aged COM group) that included patients 41 to 60 years of age and Group B (younger COM group) with patients 21 to 40 years of age. The sample size was calculated using the formula, n = 2(SD) 2 (Zα+ Zβ) 2 /d 2 , for comparison of means (audiological gain) between two groups (middle-aged COM group and younger COM group). Based on this calculation, the sample size was determined to be 35 in each group. Here, the pooled standard deviation (SD) was taken as 3.10 from a previous study, Zα was 1.96 (corresponding to a 95% confidence interval), Zβ was 0.84 (indicating 80% power), and the minimum detectable difference (d) was 2 dB based on previous research findings . The study aimed to compare the audiological gain between the two groups, i.e., the middle-aged COM group and the younger COM group. All the patients were subjected to detailed history taking, a general as well as systemic examination, which included clinical examination of the ear, nose, and throat, and a complete otological evaluation done to assess the exact nature and extent of the disease. Patients with chronic otitis media with isolated conductive hearing loss undergoing tympanoplasty with mastoidectomy, patients between 21 to 60 years of age, and patients with appropriate cochlear reserve were included in the study. Patients with medical contraindications to surgery, COM with intracranial complications, and cases requiring revision surgery for residual disease were excluded. To compare the postoperative results of tympanoplasty with mastoidectomy between middle-aged and younger-aged COM groups, we compared the hearing improvements in the two COM groups. We calculated mean hearing levels by averaging the hearing thresholds at 500, 1000, 2000 and 4000 Hz (to yield the four-tone pure tone average) after three, six, and nine months of surgery. Then we compared the preoperative and postoperative air-bone (AB) gaps between the middle-aged and younger COM groups. Additionally, we compared postoperative complications between the two groups. Postoperative results of tympanoplasty with mastoidectomy were evaluated in terms of average hearing gain. Ear discharge and decreased hearing were the most common symptoms in both groups. Specifically, ear discharge was reported in 82.86% of middle-aged patients and 91.43% of younger patients, while decreased hearing was reported in 74.28% of middle-aged patients and 65.71% of younger patients. Other symptoms, such as postauricular swelling, earache, tinnitus, facial weakness, dizziness, and fever, were less frequently observed and showed no statistically significant differences between the groups (Table 2 ). In both middle-aged and younger patients, canal wall up mastoidectomy was more common than canal wall down mastoidectomy. However, compared to younger patients, canal wall down mastoidectomy was more common in middle-aged patients, and canal wall up mastoidectomy was more common in younger patients compared to middle-aged patients. The difference in mastoidectomy procedures was not statistically significant, with a p-value of 0.192 (Table 3 ). The postoperative audiological gain in younger patients after three, six, and nine months is presented in Table 5 . The middle-aged patients showed a larger preoperative AB gap (32.36 ± 3.36) than the younger patients (27.90 ± 2.43). This difference was statistically significant with a p-value of <0.00001. After three, six, and nine months post-surgery, the overall improvement in the AB gap (mean audiological gain) was higher in middle-aged patients compared to younger patients. However, the difference in the mean audiological gain was not statistically significant, with p-values at 0.3034, 0.3271, and 0.2923, respectively. The audiological gain demonstrated a slight decrease over time in both groups. In this study, we aimed to compare the outcomes of tympanoplasty with mastoidectomy in middle-aged and younger patients diagnosed with chronic otitis media, with or without cholesteatoma. The primary focus was on evaluating differences in audiological improvement between the two age groups. The clinical symptoms, particularly, ear discharge and decreased hearing were the most common presentations in both groups. The high prevalence of ear discharge in both groups (82.86% in middle-aged patients and 91.43% in younger patients) highlights the chronic nature of the disease and its impact on quality of life. These findings are consistent with previous studies that identified ear discharge as a primary symptom of COM. Despite the slight difference in percentages, the statistical analysis showed no significant difference between the groups . The overall postoperative mean audiological gain in the middle-aged patients was 7.59 ± 3.28 dB at three months, 7.32 ± 3.42 dB at six months, and 7.38 ± 3.43 dB at nine months; in younger patients, it was 7.23 ± 2.53 dB at three months, 6.97 ± 2.77 dB at six months, and 6.90 ± 2.86 dB at nine months. Although the postoperative mean audiological gain was slight more in the middle-aged patients, the difference was not statistically significant with p-values of 0.30, 0.33, and 0.29 at three, six, and nine months, respectively. This is in concurrence with the study of Ahn et al. who also found that there was no significant difference in the improvement of the AB gap between the two groups, although both preoperative and postoperative AB gaps in the elderly patients were significantly larger than those in the younger patients . In both groups, the postoperative mean audiological gain was more in those patients who underwent tympanoplasty with canal wall up mastoidectomy. The results were consistent with those of Hirsch et al., who reported better hearing outcomes with canal wall up mastoidectomy . Albu et al. also demonstrated that postoperative auditory results significantly improved in canal wall up mastoidectomy cases when compared to open technique cases . Wetmore et al. found in a series of 161 patients with cholesteatoma that the mean pure tone average remained unchanged after surgery. They concluded that canal wall up and canal wall down mastoidectomy procedures had no influence on the hearing outcome . The results of hearing reported in the literature are contrasting. In fact, Paparella et al. reported better results with canal wall up mastoidectomy . Karmarkar et al. did not find significant differences between canal wall up and canal wall down mastoidectomy procedures . Tos and Lau were unable to demonstrate a significant difference in the hearing outcomes of canal wall up and canal wall down mastoidectomies . Audiological improvement, measured as the mean audiological gain, demonstrated a slight decrease over time in both groups. In younger patients, the mean audiological gain reduced from 7.23 dB at three months to 6.90 dB at nine months. In middle-aged patients, the mean audiological gain decreased from 7.59 dB at three months to 7.38 dB at nine months. These findings suggest that while initial improvements in hearing are achieved, maintaining these gains over time can be challenging. The research highlighted that patients aged 21-40 years exhibited the best outcomes in terms of graft uptake and hearing improvement. In contrast, patients younger than 20 years and older than 40 years experienced poorer outcomes. This trend aligns with previous studies that also noted age as a pivotal factor influencing tympanoplasty results. For instance, Tos and Lau reported that the youngest patients, particularly those under 10 years, demonstrated better results in dry ear conditions compared to older patients with discharging ears. Similarly, Emmett found that age significantly affected the success rate of tympanoplasty, with older patients generally having poorer outcomes. However, in our study, we did not find any significant difference in audiological outcomes . This study has certain limitations that should be considered. Firstly, the sample size was relatively small, which may limit the generalizability of the findings to a broader population. Additionally, the postoperative follow-up period was short, restricting the ability to assess long-term audiological outcomes and potential complications. Future studies with larger sample sizes and extended follow-up durations are recommended to provide more robust and comprehensive insights into the audiological outcomes of tympanoplasty with mastoidectomy in middle-aged and younger patients with chronic otitis media. Middle-aged patients with COM showed a significantly larger preoperative AB gap than younger patients. Audiological improvement, measured as the mean audiological gain, demonstrated a slight decrease over time in both groups. However, there was no significant difference in the improvement of the AB gap between middle-aged and younger patients. These findings suggest that tympanoplasty with mastoidectomy is an equally effective surgical intervention for improving hearing outcomes in both age groups, irrespective of the initial AB gap. Further studies with larger sample sizes and longer follow-up periods are needed to validate these results and explore additional factors that may influence long-term audiological outcomes. | Study | biomedical | en | 0.999998 |
PMC11698696 | As students take on more leadership roles, collaborative efforts between faculty and students are becoming increasingly commonplace . Student involvement in medical education is particularly fruitful as medical students are highly motivated and can provide a unique perspective often not apparent to medical educators . Moreover, faculty working collaboratively with students allows for a better learning environment as students help the College of Medicine modify the curriculum and extracurricular activities to stay up to date with the evolving culture of medicine . Medical students are eager and interested in engaging in the transformation and innovation of medical education; however, their voices are underrepresented in the medical education scholarship . In a commentary from two medical student leaders who summarized student-led initiatives in medical education, they concluded that “…we should move beyond simple engagement and instead empower trainees to lead and co-create initiatives that can ultimately benefit the entire medical education community” . In this article, we describe how a co-leadership dyad involving a faculty member and medical student was used to advance inclusion in medical education. In this model, of co-leadership, a faculty member and a medical student work together as equal co-chairs. This model was chosen because it brought together faculty and students working toward a common goal, with each co-chair bringing to the table a unique set of skills and perspectives. These diverse skills allowed the co-chairs to benefit and learn from each other. Here, we detail the structure and logistics of this co-leadership model and present not only the advantages but also the lessons learned from this faculty-student co-leadership model. | Review | biomedical | en | 0.999997 |
PMC11698704 | The use of multiple-choice questions (MCQs) to assess higher order learning is standard practice for assessing clinical knowledge in the health professions . However, the creation of such questions is not straightforward, often requiring ‘Item Writing Workshops’ to train staff in the creation of clinical scenarios for such questions . There is a need to expand the use of higher order MCQs, beyond the evaluation of clinical scenarios and into medical science education more generally, where assessments of ‘higher order learning’ may be currently given in the form of written coursework such as essays and dissertations. These assessments tend to show limited coverage of the curriculum and are vulnerable to a number of forms of misconduct, such as plagiarism , outsourcing to commercial writers , and the use of Chatbot AI tools such as ChatGPT . Despite these criticisms, it is still common for Bloom’s taxonomy to be used as the reference point when defining higher order assessment items. One approach is to ask participants (e.g. academics or students) to assign assessment items to the relevant level of Bloom’s taxonomy, or to identify relevant action verbs when writing the items . These sorts of validations were undertaken in the studies which were reviewed to develop the higher-order item writing guidance being tested here . However, this sort of validation normally requires collapsing the six tiers of the taxonomy into just two or three, and then asking subject experts to make subjective ratings about the level of learning which is being assessed by the question. Even then it has been repeatedly shown that academics find it difficult to do this reliably (e.g. the ratings of an assessment item as ‘higher’ or ‘lower order’ will vary between academics) [ 24 – 27 ]. Other taxonomies of learning have also been used to classify learning by cognitive level; for example, the ‘Structure of Observed Learning Outcomes’ (SOLO) Taxonomy, popular in higher education in the UK, has been used to design MCQs which aim at assessing ‘higher order’ learning , while in North America, clinical question banks may be classified into 1st, 2nd, and 3rd orders, with 1st-order questions assessing factual recall, while 3rd-order questions require critical thinking and problem-solving . Again though, while there are guidelines for the creation of questions which map to these levels, there is a paucity of objective evidence showing that the assigning of questions to these levels is reliable. This guidance has been published previously, in a detailed form , based on a number of papers describing the use of MCQs to assess higher order learning [ 10 , 12 , 14 , 31 – 46 ]. A summary of the guidelines is shown in Table 1 . Examples of higher order questions written using the guidance, and their lower order equivalents, are given in ESM Appendix 1 . Table 1 Summary of principles for the creation of higher-order bridge MCQs. Derived from • Start with a lower order MCQ that assesses factual knowledge. Identify an existing question, or write a simple MCQ that tests factual knowledge • Problem scenario . (Re)write the question stem into a problem that needs to be solved rather than a question to be answered. One simple way to do this is to write a scenario that describes the correct answer, but in non-technical terms • Identify assumed knowledge (bridge). The student should have some prior knowledge in order to answer the question, without which the question cannot be answered. This prior knowledge must be relevant to interpreting both the question and the answer options. One way to identify assumed knowledge is to identify a sequence of steps/knowledge needed to solve a problem, and take out those in the middle • Use common language . Using everyday language, as a more authentic problem presentation. Avoid technical terms, as these can signpost correct answers. Where technical terms are unavoidable, try to put them in only one of either the scenario, or the answer options • Use active answers . Having the answer options as actions, or describing actions, makes them less likely to be a list of facts • Use annotated images. (optional) • Number of answers . Increase these to ~ 8, to allow for more granularity in the answer options The first experiment used both lower and higher order questions written by the authors, which potentially leads to a conflict when we also designed the research. Experiment 2 was designed to test the guidance in a more general context that reflects the practical application of the guidance, by starting with lower-order questions that had not been written by the authors, but which subject novices could obtain the correct answer through simple Googling. These questions were identified as described below, and then rewritten by the authors following the guidance in Table 1 . We hypothesised that, once rewritten subject experts would still be able to answer the questions, but novices would not, even in the open book condition. In Experiment 1, participants were also not provided with any specific motivation to find the correct answer in the open book condition. We did not want participants to simply give up, especially since this seemed more likely for novices and so could artificially bias the data. This was addressed in Experiment 2 by giving participants a small financial incentive for answering correctly under the open book condition. A final difference between the two studies was that, in Experiment 1, the answer options were different between some of the lower and higher order formats of the same question, in accordance with the guidance (e.g. to make the answer options active). Here we retained the same answer options with the lower and higher order versions, including keeping the same correct answer. This was to ensure that the revised questions were assessing the same learning outcome as the original lower-order question, although it did present a challenge with utilising some aspects of the guidance (e.g., to make the answer options active, and to present them in plain language). There was a clear effect of condition, where an average of 53.4% of participants were able to answer the questions in the open-book condition when the questions were written in the lower order format, compared to 18.6% when the questions where in the higher order format. In the closed book condition, an average of 24% of participants answered correctly in the lower order format with 13.2% answering correctly in the higher format. The percentage of participants able to answer each question in the lower order, open-book format was significantly higher than for every other format/condition when analysed using a two-way repeated measures ANOVA, with percentage of participants answering correctly as the dependent variable, and test format (closed book vs open book) and question format (higher order vs lower order) as the conditions. There was a significant effect of question format ( F (1, 16) = 25.03, P = 0.0002), and test format ( F (1, 16) = 14.59, P < 0.0001) and a significant interaction between the two ( F (1, 16) = 6.95, P = 0.0018). Post hoc Bonferroni tests revealed a significant difference between the lower-order, open-book condition, and all other conditions. No other significant differences were observed. The results are shown in Fig. 3 . Fig. 3 Rewriting lower order questions into higher order makes it harder for subject matter novices to answer. Different groups of participants were given the same question in two different formats, lower order and higher order, and under two different conditions, open book and closed book. Participants were able to answer the lower order questions in the open-book condition but were not able to answer the questions in the closed-book condition or when they were rewritten into the higher order format, even under the open-book condition. * P , 0.05 when compared to all other conditions by post hoc Bonferroni tests following two-way repeated measures ANOVA (see text for details) The results are shown in Fig. 4 . Novices were again able to successfully answer lower order questions by Googling the answers, an average of 73.3% answering correctly but this dropped to 23% when the questions were rewritten into a higher order format. A two-way repeated measures ANOVA was conducted, with percentage of participants answering correctly as the dependent variable, and test format (closed book vs open book) and expertise (novice vs expert) as the conditions. For lower order questions , there was a significant effect of question format ( F (1, 9) = 163.0, P < 0.0001) and expertise ( F (1, 9) = 17.09, P = 0.0025), with a significant interaction between these two conditions ( F (1, 9) = 61.91, P < 0.0001). A post hoc Bonferroni multiple comparisons test showed a significant effect of expertise in the closed-book condition but not the open-book condition . For the higher order questions , a two-way repeated measures ANOVA again showed a significant effect of question format ( F (1, 9) = 6.553, P = 0.0307) and expertise ( F (1, 9) = 18.40, P = 0.0020), but no interaction between the two conditions ( F (1, 9) = 0.2687, P = 0.6167). Fig. 4 Rewriting publicly available lower order questions into higher order questions makes it harder for novices to answer them, but experts can still answer under closed-book conditions. Different groups of participants were given the same question in two different formats, lower order and higher order, and under two different conditions, open book and closed book. Novices were able to answer the lower order questions in the open-book condition but not when the questions when rewritten into a higher order format using the guidance tested here. Experts showed good ability to answer questions under all conditions. See text for statistical analysis | Other | biomedical | en | 0.999998 |
PMC11698806 | There is evidence in the literature of temporal trends in attitudes toward immigration, which could be period or cohort effects. For example, McLaren and Paterson identified cohort effects in European data, Wilkes and Corrigall-Brown found a strong period effect and only a small cohort effect in Canadian data, while Beller found cohort and period effects when analysing German data. However, the linearity between age, period, and cohorts requires special consideration in the modelling strategy. Therefore, following the statistics and migration literature, we address the potential confounding influence of age and periods by applying hierarchical age-period-cohort regression analysis (HAPC) . More specifically, we apply a hierarchical cross-classified random effects model . This model allows us to account for the hierarchical structure of the unbalanced repeated cross-sectional data, with individuals being cross-classified and nested in both country-period and country-cohort while clustered in 15 European countries. Thus, we estimate the variability across periods and cohorts while testing the role of individual education and contextual (country-period) factors. In our approach, we enter age as a level one variable, country-period and country-cohort as cross-classified level two units, and country as a level three variable. Moreover, we add individual-level variables to the participants’ characteristics, such as education, gender, place of residence, native population group membership, left–right party affiliation, satisfaction with the economy and country-period variables, and contextual variables, such as migrant inflow rate and unemployment rate. Our results from the hierarchical three-level cross-classified null model show that the smallest proportion of variance in cultural attitudes is explained by the period effect (country-period: 1.2%), followed by the cohort effect (country-cohort: 2.6%) (Model 0; Table 2 ), confirming the not too prominent role of the period already emphasised in our descriptive results. However, in the Eastern European countries, the period effect explains 4.2%, while in the non-Eastern European countries, it explains only 0.7% of the variance (Model 0; Tables 4 and 5 ), meaning that period effects manifest stronger in Eastern European countries than in their non-Eastern counterparts. This is different for economic attitudes. While the variance between country-cohort explains 1.1% and country-period 2.15% of the variance in economic attitudes in the pooled sample, the cohort effect only contributes to a small extent to explaining the variance in economic attitudes among the non-Eastern European countries (country-cohort: 0.9% and country-period: 2.2%) (Model 0; Tables 3 and 7 ). The contribution of the cohort and period effects is of a similar magnitude among the Eastern European countries (country-cohort: 2.5% and country-period: 2.0%) (Model 0; Tables 6). The random period effect illustrated in Fig. 3 only shows statistically significant negative changes in cultural attitudes after 2012 in Hungary and Poland and, to a smaller extent, in Slovenia. However, there have been significant changes over time in economic attitudes in most countries . For example, in Germany, Ireland, Portugal, and the United Kingdom, economic attitudes became significantly more positive after 2010 and 2012. Table 2 Hierarchical, three-level, crossed-classified random effects models of cultural attitudes toward immigration in 15 European countries Model 0 Model 1 Model 2a Model 3a Model 2b Model 3b Intercept 5.767 (0.162) *** 5.962 (0.146) *** 6.148 (0.158) *** 5.960 (0.159) *** 5.61 (0.153) *** 5.652 (0.155) *** Individual level Education (ref: ISCED I) ISCED II 0.347 (0.019) *** 0.347 (0.019) *** 0.524 (0.035) *** 0.347 (0.019) *** 0.333 (0.038) *** ISCED III 0.654 (0.018) *** 0.654 (0.018) *** 0.846 (0.032) *** 0.654 (0.018) *** 0.538 (0.038) *** ISCED IV 1.088 (0.022) *** 1.088 (0.022) *** 1.427 (0.040) *** 1.087 (0.022) *** 1.037 (0.045) *** ISCED V 1.656 (0.019) *** 1.656 (0.019) *** 1.862 (0.034) *** 1.656 (0.019) *** 1.665 (0.037) *** Country-period level Migrant inflow rate − 0.244 (0.087) ** 0.019 (0.093) Unemployment rate 0.045 (0.009) *** 0.040 (0.010) *** ISCED II x migrant inflows − 0.261 (0.042) *** ISCED III x migrant inflows − 0.289 (0.040) *** ISCED IV x migrant inflows − 0.453 (0.045) *** ISCED V x migrant inflows − 0.306 (0.041) *** ISCED II x unemployment 0.001 (0.004) ISCED III x unemployment 0.014 (0.004) *** ISCED IV x unemployment 0.006 (0.005) ISCED V x unemployment − 0.002 (0.004) Random effects: var Country (level 3) 0.337 0.282 0.272 0.270 0.238 0.237 Country-period (level2) 0.083 0.101 0.095 0.095 0.085 0.085 Country-cohort (level 2) 0.178 0.024 0.024 0.023 0.025 0.025 Residual 6.323 5.529 5.529 5.527 5.529 5.529 N 245,291 219,299 219,299 219,299 219,299 219,299 Higher scores indicate a more positive attitude; *p<0.05, **p<0.01, ***p<0.001; sampling weights are considered; model 0 represents the null model, model 1 presents the coefficients (S.E.) of education with ISCED I as reference category and individual characteristics as control variables; model 2 adds contextual variables migrant inflow rate (model 2a) and unemployment rate (model 2b); model 3 adds the interaction of education and contextual variables (migrant inflow rate in model 3a and unemployment rate in model 3b); the complete table with a list of all individual-level control variables can be found in Table A2 in the appendix Table 3 Hierarchical, three-level, crossed-classified random effects models of economic attitudes toward immigration in 15 European countries Model 0 Model 1 Model 2a Model 3a Model 2b Model 3b Intercept 5.038 (0.146) *** 4.529 (0.122) *** 4.446 (0.133) *** 4.333 (0.135) *** 4.592 (0.141) *** 4.682 (0.143) *** Individual level Education (ref: ISCED I) ISCED II 0.388 (0.019) *** 0.388 (0.019) *** 0.507 (0.034) *** 0.388 (0.019) *** 0.317 (0.037) *** ISCED III 0.636 (0.018) *** 0.636 (0.018) *** 0.736 (0.031) *** 0.636 (0.018) *** 0.498 (0.036) *** ISCED IV 1.062 (0.021) *** 1.061 (0.021) *** 1.240 (0.039) *** 1.062 (0.021) *** 1.018 (0.044) *** ISCED V 1.619 (0.018) *** 1.619 (0.018) *** 1.763 (0.033) *** 1.619 (0.018) *** 1.484 (0.036) *** Country-period level Migrant inflow rate 0.109 (0.075) 0.267 (0.081) ** Unemployment rate − 0.008 (0.008) − 0.018 (0.009) * ISCED II x migrant inflows − 0.174 (0.040) *** ISCED III x migrant inflows − 0.152 (0.038) *** ISCED IV x migrant inflows − 0.243 (0.043) *** ISCED V x migrant inflows − 0.210 (0.039) *** ISCED II x unemployment 0.008 (0.003) * ISCED III x unemployment 0.016 (0.004) *** ISCED IV x unemployment 0.004 (0.004) ISCED V x unemployment 0.015 (0.003) *** Random effects: var Country (level 3) 0.287 0.192 0.186 0.185 0.203 0.203 Country-period (level2) 0.139 0.070 0.069 0.069 0.069 0.069 Country-cohort (level 2) 0.073 0.018 0.018 0.018 0.018 0.018 Residual 5.974 5.108 5.108 5.107 5.108 5.108 N 244,946 218,672 218,672 218,672 218,672 218,672 Higher scores indicate a more positive attitude; *p<0.05, **p<0.01, ***p<0.001; sampling weights are considered; model 0 represents the null model, model 1 presents the coefficients (S.E.) of education with ISCED I as reference category and individual characteristics as control variables; model 2 adds contextual variables migrant inflow rate (model 2a) and unemployment rate (model 2b); model 3 adds the interaction of education and contextual variables (migrant inflow rate in model 3a and unemployment rate in model 3b); the complete table with a list of all individual-level control variables can be found in Table A3 in the appendix Fig. 3 Period random effect estimates on cultural attitude from the hierarchical, three-level, cross-classified null model Fig. 4 Period random effect estimates on economic attitude from the hierarchical, three-level, cross-classified null model | Study | other | en | 0.999995 |
PMC11698880 | One of the most influential biological associations is the interrelationship between insects and plants. The evolutionary interplay between insects and plants, either as friends or foes, has shaped terrestrial ecosystems for at least the last 400 million years . Of the myriad ways in which these titans of biodiversity interact, none is more intimate than that of pollination, a mutualism, with some specialised exceptions, that has aided the proliferation of both lineages . Indeed, today insects are the primary pollinators for nearly 90 % of all flowering plants . Perhaps among all of these insect pollinators, none are more iconic than the bees, a specialised lineage of phytophagous stinging wasps that feed their larvae pollen and nectar . With over 20,500 species, bees are critical to the success of many terrestrial ecosystems through their pollination services and especially vital for agriculture and food security where there is a particular overreliance on honey bees (genus Apis Linnaeus). Over the last 120 million years, bees have evolved many specialisations for the gathering and processing of pollen, nectar, floral oils, and plant exudates . While most bees are polylectic, numerous lineages have evolved various degrees of host-plant specialisation (oligolecty) and, in rare instances, outright monolecty . While much intensity is devoted to the study of bee pollination, an understanding of the evolutionary history of this association has been restricted mainly to indirect inferences either through phylogenetic estimation or from isolated fossil occurrences. For example, the inference of orchid pollination in Miocene Hispaniola based on the presence of orchid bees in Dominican amber even in the absence of attached orchid pollinia . The history of insect pollination as documented by the fossil record is gaining momentum , particularly through the exploration of pollen grains preserved on or within insect fossils belonging to pollinating lineages and this has been especially true for the bees. Some of these exceptional fossils provide direct evidence of pollen collected by the bees during life and a glimpse into ancient insect-plant interactions . Here, we report the discovery of a large carpenter bee (genus Xylocopa Latreille) from the middle Eocene of Messel, Germany. Species of Xylocopa have been previously reported as compression fossils from various localities, nearly all of which are from the Miocene . A notable exception was the fragmentary Xylocopa gabrielae Engel from the Eocene-Oligocene boundary of Florissant, Colorado , hitherto the earliest occurrence of the genus. The new species reported here now supersedes X. gabrielae in age. Of significance, the new fossil has adhering pollen preserved on the body, allowing for an investigation into the floral visits of this individual immediately prior to death and providing a snapshot of bee-flower relationships during the Eocene. Direct evidence of floral visitation by fossil insects is exceptionally limited. We describe the xylocopine bee and its adhering pollen and explore the occurrences of the floral families represented and their modern insect visitors in relation to the discovery of their association with the bee documented herein. This is the first documentation of pollen from a fossil carpenter bee and only the third genus of bees from the Eocene in which adhering pollen has been recorded. The fossil bee was examined and sampled for pollen according to the method described in Grímsson et al. . A stereomicroscope equipped with epifluorescence illumination was used to examine and photograph the fossil. Pollen grains were extracted from the head, metasoma, and legs of the bee and processed for combined light microscopy (LM) and scanning electron microscopy (SEM) analysis by bleaching followed by acetolysis . Pollen grains were investigated using the “single-grain method” by Zetter . Morphological terminology for the description of the bee follows Engel and Michener , and the classification of Xylocopa follows Michener . The classification of plants follows that of the APG IV , with pollen of Theaceae described first, followed by the Araliaceae. Pollen descriptions include all features observed with both LM and SEM. The pollen terminology follows Punt et al. and Halbritter et al. . Taxonomic actions made herein are registered in ZooBank ( www.zoobank.org ) with the article LSID: urn:lsid:zoobank.org:pub:3B66630B-F719-4A16-ABC6-FF2DB08907CE Diagnosis: It is challenging to compare the current fossil with modern relatives in many characters traditionally employed in the classification of Xylocopa (e.g. facial carinae, form of metabasitibial plate, sternal and tergal graduli, pygidial plate). Nonetheless, there are sufficient details to make some important distinctions, most notably in the forewing venation. The new subgenus differs from all other groups of Xylocopa by the much shorter and broader marginal cell that tapers away from the anterior wing margin near its base and ends at about the apex of the third submarginal cell, rather than that of the living subgenera in which the cell is noticeably slender, elongate, extends well beyond the third submarginal cell, and tapers away from the anterior wing margin in its apical third or quarter. In addition, the second submarginal cell, while having the usual asymmetrical trapezoidal shape of most Xylocopa species, is quite exaggerated in its proximal extension and narrow anterior margin, with the posterior margin over 10× greater than the anterior margin (this is usually over 2× in most extant Xylocopa but never exceeds 4.5× the length). Additional features of the subgenus include: 2rs-m prominently bowed apically (as in most extant subgenera); given the orientation of the longitudinal axis of the compound eye relative to the vertex it would seem that the eyes do not converge above or below, as in the subgenera Xylocopoda Hurd and Moure and some species of Xylomelissa Hurd and Moure ; the mesal margin of the compound eye is weakly incurved, as in many subgenera of Xylocopa ; the vertex is straight before arching laterally above the compound eye, as is the case in various subgenera; the median ocellus is slightly smaller than the antennal torulus, as is the case in most Old World subgenera; the mesoscutellum is not flattened nor projecting over the metanotum. Description: ♀: Total body length (as preserved, apical metasomal segments missing so length in life would have been much longer) 17.68 mm; forewing length ~15.08 mm, maximum width 4.66 mm; integument dark brown to black as preserved, sculpturing not discernible as preserved. Head broader than long, medial length 3.92 mm, width (as preserved) 3.99 mm; compound eye length 2.94 mm, mesal margin of compound eye weakly incurved; vertex straight. Mesosoma length 5.62 mm (as preserved, apical portion of propodeum missing), maximum width 5.34 mm; mesoscutum length 3.66 mm, mesoscutellum length 1.11 mm; mesoscutellum not flattened nor extending posteriorly over metanotum (i.e. not as in Koptortosoma Gribodo or Mesotrichia Westwood), seemingly low and slightly rounded. Forewing prestigma virtually absent; pterostigma virtually absent owing to absence of abscissa of R between r-rs and anterior wing margin; r-rs elongate, angling posteriorly from apex of R to meet 2Rs; R nearly immediately diverging from anterior wing margin (i.e. in proximal portion of marginal cell) to point near tangent with apex of third submarginal cell, then steeply angling posteriorly to meet Rs; apex of marginal cell feebly appendiculate; marginal cell comparatively broad, nearly as wide as third submarginal cell (presumed plesiomorphy; in extant subgenera apical portion of marginal cell 0.5× width or less of third submarginal cell), marginal cell not extending well beyond apex of third submarginal cell (unique apomorphy; marginal cell extending well beyond apex of third submarginal cell in extant subgenera); 1Rs and 1M forming a straight line; 1M nearly confluent with 1cu-a; 1cu-a orthogonal to M+Cu and A; Rs+M comparatively short, slightly less than one-third length of 2M; 2Rs elongate, meeting r-rs close to 1rs-m; 1rs-m straight, approximately orthogonal to Rs, slightly basad 1m-cu; second submarginal cell strongly asymmetrically trapezoidal, with posterior margin more than 10× anterior margin (unique apomorphy); 1m-cu and 2m-cu both entering third submarginal cell, former near base of cell, latter slightly distad cell midlength; third submarginal cell large, much longer than wide; 2rs-m strongly bowed outward; 2Cu with enigmatic posterior bend before origin of 2m-cu (unclear whether this may be an artifact); wing membrane beyond veins papillate. Hind wing with 1Rs straight, 2Rs elongate and weakly arched; rs-m slightly less than 0.5× length 1Rs; 1M slightly longer than 1Rs, slightly more than 2× as long as 2M; 2M comparatively short, slightly longer than rs-m; 1Cu shorter than 1M; 2M+Cu about as long as 1M. Metabasitarsus elongate (as is typical for Xylocopa ), distinctly longer than combined lengths of remaining tarsomeres and probably longer than metatibia, length 3.75 mm, densely setose. Metasoma broad, wider than mesosoma, width 7.72 mm (as preserved across tergum II). Fig. 3 Light microscopy (LM) ( a, d, g, j, m, p ) and scanning electron microscopy (SEM) ( b, c, e, f, h, i, k, l, n, o, q, r ) micrographs of fossil Theaceae and Araliaceae pollen extracted from the fossil carpenter bee ( Xylocopa primigenia sp. nov.). a–l Theaceae pollen extracted from the head ( a–f ) and the metasoma ( g–l ); m-r Araliaceae pollen extracted from the mid leg; a Theaceae pollen in polar view; b same gain as in a; c detail of colpus membrane; d Theaceae pollen in polar and equatorial view; e same grain as in e; f detail of polar area and adjacent colpus; g Theaceae pollen in polar view; h same gain as in g; i detail of mesocolpium; j Theaceae pollen in polar view and equatorial view; k same grain as in j; l detail of the bridge covering the porus; m Araliaceae pollen in polar view and equatorial view; n same grain as in m; o detail of the aperture; p Araliaceae pollen in polar view and equatorial view; q same grain as in p; r detail of polar area. Scales = 10 µm ( a, b, d, e, g, h, j, k, m, n, p, q ), 1 µm ( c, f, i, l, o, r ) Description: Pollen, monad, prolate, outline convex-triangular to circular in polar view, elliptical in equatorial view; polar axis 20–24 µm in LM, 17–22 µm in SEM, equatorial diameter 15–24 µm in LM, 15–21 µm in SEM; tricolporate, endoporus lalongate and weakly outlined, colpi 3/4 of polar axis long (SEM), nexine thickened along colpi towards endoapertures, colpus membrane granulate to microareolate, bridge over porus; exine 1.1–1.7 µm thick (LM), sexine and nexine equally thick; sculpture scabrate in LM, microreticulate, rugulate, fossulate, and perforate in SEM, occasional granulate suprasculture (SEM). Remarks: The current circumscription of Theaceae comprises approximately 367 species in eight genera . To our knowledge there currently exists no comprehensive palynological work on pollen from extant Theaceae and only a small portion of the species have been investigated palynologically. Still, selective taxa from all genera have been published, either using only LM or SEM, combined LM and SEM, or combined SEM and TEM . Based on the combined features observed in the fossil pollen grains we are able to assign this pollen type to Theaceae, but not with certainty to any extant genus. The general size, outline and shape, as well as the aperture configuration in LM is similar between pollen of extant genera. The thickening of the nexine around the apertures observed with LM, as observed in the fossil pollen, is prominent in some species of Camellia L. . The sculpture of the fossil pollen observed with SEM is most comparable to that of pollen from extant Camellia , Schima Reinw. ex Blume, and Stewartia L. . Based on the available information from pollen morphology and ultrastructure of extant Theaceae it is most conservative to state that the fossil pollen is an morphological intermediate type between the three aforementioned genera and could be affiliated with any of them, but see fossil record below. Table 1 Numbers of extant genera and species of Theaceae and Araliaceae. Note: Generic names and species numbers compiled from POWO Theaceae Species Araliaceae Species Apterosperma Hung T.Chang 1 Hedera L. 19 Camellia L. 230 Heptaleurum Gaertn. 321 Franklinia W. Bartram ex Marshall 1 Heteropanax Seem. 9 Gordonia Ellis 22 Hydrocotyle Tourn. ex L. 177 Polyspora Sweet 49 Kalopanax Miq. 1 Pyrenaria Blume 27 Macropanax Miq. 18 Schima Reinw. ex Blume 16 Merrilliopanax H.L.Li 3 Stewartia L. 22 Meryta J.R.Forst. and G. Forst. 27 Metapanax J.Wen and Frodin 2 Araliaceae Species Motherwellia F.Muell. 1 Anakasia Philipson 1 Neocussonia Hutch. 16 Aralia L. 73 Neopanax Allan 5 Astropanax Seem 15 Oplopanax (Torr. and A.Gray) Miq. 3 Astrotricha DC. 20 Oreopanax Decne. and Planch. 146 Brassaiopsis Decne. and Planch. 46 Osmoxylon Miq. 61 Cephalaralia Harms 1 Panax L. 14 Cephalopanax G.M.Plunkett, Lowry & D.A.Neill 2 Plerandra A. Gray 33 Cheirodendron Nutt. ex Seem 6 Polyscias J.R. and G.Frost 180 Chengiopanax C.B.Shang & J.Yhuang 2 Pseudopanax K.Koch 7 Crepinella Marchal 33 Raukaua Seem. 6 Cussonia Thinb. 20 Schefflera J.R.Frost. and G.Frost. 13 Dendropanax Decne. & Planch. 95 Sciodaphyllum P.Browne 145 Didymopanax Decne. & Planch. 38 Sinopanax H.L.Li 1 Eleutherococcus Maxim. 29 Tetrapanax (K.Koch) K.Koch 1 Fatsia Decne. & Planch. 3 Trachymene Rudge 59 Frodinia Lowry & G.M.Plunkett 2 Trevesia Vis. 8 Gamblea C.B.Clarke 4 Seemannaralia R.Vig. 1 Harmsiopanax Warp. 3 Woodburnia Prain 1 Description: Pollen, monad, prolate, outline triangular to tri-lobate in polar view, lens-shaped to elliptic in equatorial view; polar axis 19–31 µm in LM, 23–27 µm in SEM, equatorial diameter 16–30 µm in LM, 17–25 µm in SEM; tricolporate, endoporus large and lalongate, colpus 2/3 of polar axis (SEM), massive nexine thickening along apertures (up to 3 µm thick in LM); exine 1.1–1.8 µm thick (LM), sexine as thick or thicker than nexine; sculpture reticulate in LM and SEM, reticulum gradually decreasing in size from polar areas and mesocolpium towards colpi, forming a perforated and psilate margin along colpi (SEM). Remarks: Araliaceae are a large family, currently comprising about 1680 species in 46 genera . There are several publications on the pollen morphology (LM, SEM) and ultrastructure (TEM) of Araliaceae pollen . Based on the combined morphological traits (incl. size, shape, reticulate sculpture, exine thickness, thickened nexine around apertures, and lalongate endopori) observed with combined LM and SEM we assign this pollen type to the Araliaceae. However, pollen of species and genera of Araliaceae often overlap in most diagnostic morphological LM and SEM based features. Members of Dendropanax Decne. and Planch. , Eleutherococcus Maxim. , Heteropanax Seem. , Macropanax Miq. , Merrilliopanax H.L.Li , Metapanax J.Wen and Frodin , Neopanax Allan , Polyscias J.R.Forst. and G.Forst., and some of Schefflera J.R.Forst. and G.Forst. and Tetrapanax (K.Koch) K.Koch are more likely to be the parent plants of the fossil araliaceous pollen. Others, including Aralia L., Didymopanax Decne. and Planch., Dendropanax , Kalopanax Miq. , and some members of Panax L. and Schefflera have ambivalent morphologies. Only a few genera, including Brassaiopsis Decne. and Planch. , Fatsia Decne. and Planch. , Hedera L., Oplopanax (Torr. and A.Gray) Miq. , Osmoxylon Miq. , Panax L., and Trevesia Vis, exhibit differences in pollen morphology that allow for them to be excluded as the potential parent plant of the fossil araliaceous pollen type. Therefore, this fossil pollen type could consequently belong to several extant genera, but see fossil record below. The fossil record of Theaceae dates back to the Upper Cretaceous with scarce findings of Pollenites pollen ( Gordonia Ellis and Schima type) from Alabama, U.S.A. ; Palaeoschima seeds ( Schima like) from Germany, Austria, and Czechia ; and Schimoxylon wood (similar to Schima ) from Egypt . Most other Cretaceous records have been rejected or are considered doubtful by Grote and Dilcher . Cenozoic records are numerous, including both extinct genera like Andrewisocarpon and Gordoniopsis as well as extant representatives of Camellia , Gordonia , Polyspora Sweet, Schima , and Stewartia . Even though Paleocene fossils of Theaceae are more or less absent, other Paleogene records, especially of Eocene age, show that the family had already diverged and dispersed across the globe during that time. Camellia (and alike) fossils have been reported from the middle Eocene of Germany ; the late Eocene of Honshu, Japan ; the early Oligocene of Washington, U.S.A and Bulgaria ; and the late Oligocene of Nanning, South China . Fossils of Gordonia are documented from the middle Eocene of Kentucky and Tennessee U.S.A ; the middle to late Eocene of Germany ; the late Eocene of England ; and the Oligocene of Japan . Fossils of Polyspora have been reported from the middle to late Eocene of Germany and the late Eocene of Czechia . Schima (and alike) fossils are known from the middle Eocene of Germany , the middle Eocene of Myanmar , and the late Oligocene of Guangxi, China . The Theaceae pollen type reported herein is not the first representative of this family from the Messel pit. Wilde described from this locality leaves of Polyspora and Collinson et al. fruit of Camelliacarpoidea (similar to Camellia ). Considering these two records as well as other Eocene fossils of Theaceae reported from Germany , it is likely that the fossil theaceous pollen belongs to one of the following genera: Camellia , Gordonia , Polyspora , or Schima . The early fossil pollen records of Araliaceae extend back to the Upper Cretaceous (lower Campanian) of Wyoming, U.S.A.; Upper Cretaceous (Senonian) of China; the Paleocene and Eocene of Greenland; the Eocene of western North America; and the Paleocene (France) and Eocene (Germany) of Europe . Early macrofossil records include fruit/seeds of Acanthopanax (Decne. and Planch.) Witte (now within: Eleutherococcus Maxim.) and Aralia from the Upper Cretaceous (Maastrichtian) of Germany , flowers of Tetraplasandranthus from the Upper Cretaceous/Eocene of India , araliaceous wood ( Plerandreoxylon ), fruits ( Paleopanax ; similar to Pseudopanax ), and leaves from the middle Eocene of Oregon, U.S.A. , and leaves of Dendropanax from the middle Eocene of Tennessee . As with the Theaceae, the fossil record of Araliaceae suggests that the family had already dispersed around the globe prior to the end-Eocene and was represented by a number of extinct and extant genera by that time. Oligocene records include mostly fossils assigned to extant genera, such as Aralia (now incl. Pentapanax Seem.) and Schefflera from Germany . Fossils of Araliaceae from Messel are rare and restricted to cuticle remains of cf. Fatsia and two Araliaceoipollenites pollen taxa . Dispersed pollen assigned to the larger subspecies of A. euphorii from Messel is probably the same pollen type (shared parent plant/taxon) as the Araliaceae pollen extracted from the fossil bee. As to date, no flower containing this pollen type is described from Messel, leaving a more precise affiliation pending. Considering the morphology of the fossil pollen and extant Araliaceae genera that have been documented in Europe in the Eocene-Oligocene, it is possible that the araliaceous pollen type from the bee originates from Schefflera . Knowledge about the pollination biology of Theaceae is meagre and only a few species have been investigated thoroughly. Among all genera, Camellia is the most studied due to its economic importance for both tea and oil production . Camellia sinensis (L.) Kuntze shows floral morphologies of a generalist pollination syndrome and is visited and pollinated by various animals including short-tongued flies, bees and wasps, beetles, moths, butterflies, and true bugs . For C. pubipetala Y.Wan and S.Z.Huang the primary pollinators are sun birds ( Aethopyga christinae Swinhoe) and honey bees ( Apis cerana Fabricius). During nectar feeding both sunbirds and bees come into contact with male and female reproductive organs of the flowers and are covered with pollen. Observations show that birds have pollen on their beaks and heads, while bees carry pollen on their entire body including mouthparts, metasoma, and legs . Flowers of C. oleifera C.Abel are also visited by a number of insects, especially bees and flies but also wasps. Bee visitors include cultivated honey bees ( A. cerana and A. mellifera L.) as well as wild bees ( Colletes gigas Cockerell , Andrena striata Fabricius , A. camellia Wu , A. hunanensis Wu , A. chekiangensis Wu). Flies that visit flowers of C. oleifera belong to the Calliphoridae, Muscidae, Sarcophagidae, and especially hover flies (family Syrphidae). The only wasp observed visiting C. oleifera flowers was Vespa velutina Lepeletier . Camellia japonica L. is also visited by song birds (among others, Zosterops erythropleura erythropleura Swinhoe), honey bees ( A. cerana and A. mellifera ), syrphid hover flies, and even a squirrel has been observed visiting the flowers . Flowers of C. sasanqua Thunb. are visited by birds, wasps, syrphid flies, moths, butterflies, and colletid bees . Gordonia lasianthus (L.) Ellis is visited by small beetles, ants, and halictid bees , while flowers of Schima superba Gardner and Champ. are visited by A. cerana and scarab beetles ( Protaetia brevitarsis (Lewis) and Popillia mutans (Newman)) . Schima wallichii (DC.) Korth. primary pollinators are honey bees ( Apis L.), carpenter bees ( Xylocopa ), and butterflies ( Sinthusa nasaka (Horsfield) , Cabera pusaria (L.), and Dysphania militaris (L.)) . Based on the currently available literature on flower visitations and pollinators of Theaceae it is clear that the family is animal pollinated and insects are the most common pollinators. Bees and flies seem to be the most frequent visitors/pollinators, followed by beetles and butterflies. Extant species of Xylocopa are important generalist pollinators in tropical, subtropical, and warm temperate regions and visit species of Theaceae, especially the flowers of Schima . Xylocopa ( Nyctomelitta ) tranquebarica (Fabricius) is known to visit Theaceae, and X. ( Biluna ) nasalis Westwood collects pollen of Schima that is fed to the larvae . Extensive studies on the pollination ecology of Araliaceae are lacking. In general, Araliaceae are pollinated primarily by insects, which are attracted by both nectar and pollen as rewards. Pollinators include flies (Bombyliidae, Calliphoridae, Muscidae, Syrphidae), bees and wasps (Apidae, Formicidae, Halictidae, Ichneumonidae, Vespidae), butterflies (Nymphalidae, Tortricidae), and beetles (Scarabeidae). Bird and bat pollination are also encountered, but are rare . Aralia hispida Vent. is probably pollinated by bumble bees . The pollinators of Eleutherococcus trifoliatus (L.) S.Y.Hu include flies of the families Syrphidae and Tachinidae, as well as honey bees ( Apis ) . Hedera helix L. is visited by about 20 taxa of insects. Wasps seemed to be important pollinators, next to honey bees, bumble bees, bristle flies, as well as small and large hover flies . Both Panax quinquefolius L. and P. wangianus S.C.Sun are visited by syrphid flies and halictid bees . Flowers of Polyscias pancheri (Baill.) Harms are visited by nomiine bees ( Austronomia sicheli (Vachal)), wasps (unidentified), scarab beetles ( Heteronyx caledoniae Fauvel), as well as ants that feed on nectar ( Polyrhachis guerini Roger) . Schefflera heptaphylla Z.H.Tsi floral visitors comprise primarily flies ( Chrysomya Robineau-Desvoidy and Syrphinae Latreille) and wasps ( Vespula Thomson and Eumenes Latreille) . The limited data on floral visitors and pollinators of Theaceae and Araliaceae are certainly consistent with the observation of bee visitation during the Eocene. The genus Xylocopa belongs to the same family (Apidae), as honey bees and bumble bees, albeit quite distantly related within that clade, and includes largely polylectic bees, some of whose species are today known to visit at least theaceous flowers, and likely also those of the Araliaceae. It is clear from the areas of localization of pollen on the fossil bee that early large carpenter bees were potential pollinators of flowers for both of these plant families in the local environment of Messel during the Eocene . Today species of Xylocopa transport pollen in often small loads on their legs (principally the metatibia and metabasitarsus), moving the pollen to the hind legs from the fore- and midlegs, and therefore with pollen occurring on all legs to some degree. In addition, species of Xylocopa are capable of buzz pollination whereby the sonicate flowers to dislodge pollen on to their venter, including the metasoma, and then use the legs to redistribute the pollen to their legs . Poricidal anthers necessitating buzz pollination are rare in Theaceae and Araliaceae and it is unlikely the Eocene species necessitated sonication to dislodge pollen. The heads of bees are also often covered with pollen when entering the corolla. Thus, the locations of the pollen on the fossil are consistent with modern species of Xylocopa visiting flowers for pollen and nectar . Since we discovered pollen from two completely different angiosperm families we assume a polylectic behaviour for the fossil Xylocopa bee. The family Apidae is well represented at Messel, with various species of the extinct genera Electrapis Cockerell and Protobombus Cockerell , both of which were also found to have visited a variety of flowers, including Araliaceae from Messel . However, it is possible that some were oiligolectic as suggested by the eucerine bee (Eucerini; a tribe with numerous oligolectic species) record from Messel . Unfortunately, it is not yet possible to make a comparison of pollen collection by fossil xylocopine bees as X. primigenia is the first extinct species of its subfamily to have been documented with adhering pollen. Fossils of Xylocopinae are rare and most were described long ago necessitating the location of historical type specimens to explore for potential pollen. For at least those species of Xylocopa described from the mid-Miocene of Öhningen, Germany no pollen was located on material preserved at ETH Zürich, Switzerland (M.S.E. pers. obs.). Only through greater sampling of potential pollinating insects from Messel and documentation of their associated pollen will permit a fuller understanding of the ecology of this ancient ecosystem. Fortunately, the exceptional preservation of fossils at Messel makes this an ideal locality from which to build such a comprehensive picture of flower-pollinator interrelationships from the Eocene. | Study | biomedical | en | 0.999995 |
PMC11698920 | Subject-specific finite element (FE) analysis of the mandible bone is one of the most recent advancements in biomechanics and craniomaxillofacial field and has increasingly gained attention, specifically in the field of implant design and bone-implant interaction analysis [ , , , , ]. Alongside geometry preparation, mesh generation, and boundary conditions, the material properties are one of the main parameters influencing the accuracy of FE analysis, as they are an integral part of the stiffness matrix . Despite the common use of homogeneous material properties for the mandible bone , a critical step for subject-specific FE modeling is the heterogeneous material modeling . This refers to the process of assigning appropriate physical and material properties, density and Young's modulus, to different anatomical areas of the bone within a computational model. Incorporating heterogeneous material properties of bone is particularly crucial in cases where bone quality should be respected e.g., for modeling osteoporotic mandibles , age-related changes in bone properties , tumors-related surgery , bone fractures and osteosynthetic treatment , bone reconstruction , and temporomandibular joint replacements . In this context, the primary method employed revolves around mathematical equations that correlate Hounsfield units from computed tomography (CT) and digital imaging and communications in medicine (DICOM) databases to both bone density and Young's modulus. However, different material models proposing a relationship between density and Young's modulus show considerably varied material behavior. In this context, there has been no consensus on the reliability of such density-modulus relationships for the mandible bone , which makes comparison of different FE studies difficult . Hussein et al. used four different material models for mandible material properties and studied bone-implant interaction. Their comparative findings underscore the unreliability of results when employing heterogeneous bone material properties derived from different density-modulus relationships. Additionally, most of the available material models are primarily proposed for other bones e.g. femur or tibia and may not accurately model the relationship of density and Young's modulus of the mandible bone . Therefore, the present study aims to review studies employing heterogeneous material modeling of the human mandible bone, to synthesize these models, investigate their origins, and assess their quality. This may help researchers in the craniomaxillofacial field in more accurately selecting material models for constructing subject-specific FE models of the mandible bone. A systematic review was conducted on FE studies of the mandible that used heterogeneous material to model bone mechanical properties. This systematic review identified the most frequently applied material models. Furthermore, an extended search was performed to summarize and review all original studies referenced by these FE studies that proposed a density-modulus relationship. This involved examining the original studies to understand the methodologies and assumptions underlying the heterogeneous material models and assessing the risk of bias. The systematic literature review was done according to the preferred reporting items for systematic reviews and meta-analyses guidelines . Literature searches were conducted on studies published prior to 11 th October 2023 across electronic databases, including Scopus, PubMed, and Web of Science. The search strategy was constructed by combining relevant terms and Boolean operators. These terms were derived from the titles, abstracts, and keywords indexing of 10 known and relevant studies and categorized into the following three fields of interest. This search string was examined by authors several times in a back-and-forth manner to find the optimum studies in terms of number of the studies and range of the field of studies and was used to search within article title, abstract, and keywords. The Boolean operator AND was chosen to combine the search terms and create a search string. • Anatomical site of interest : (mandibular OR mandible OR temporomandibular OR "masticatory system") • Modeling strategy : "finite element" AND biomech∗ • Metrics of interest : (Hounsfield OR density OR ((young∗ OR elastic) AND modu∗) OR elasticity OR heterogeneous OR ((patient OR subject) AND (specific OR personali∗)) OR ((material OR mechanical) AND propert∗)) Several criteria were considered to include studies in this systematic review: (A) studies that explicitly stated their density-modulus relationship. This was done either by proposing the relationship or by clearly citing the original study describing the material model, and (B) only FE studies conducted on the mandible bone were included and considered valid for this review. Also, five criteria were considered for excluding the studies from this review: (a) non-English studies, (b) studies with a homogenous definition for the bone, (c) studies proposing density-modulus relationship, which require extra information on bone tissue that cannot be provided by conventional CT or cone beam computed tomography scanners, (d) animal studies, because of differences in the mechanical properties of the bone between human and animal mandibles, and (e) studies with no full text available for review. Three independent scientists (authors A-K. B., I. S., J-O. S.) first screened the abstract and title and afterward carefully reviewed each study to ensure its comprehensiveness and eligibility and extracted the density-modulus relationships explicitly mentioned in the respective study. All relevant articles were evaluated, and those that did not fit the study's inclusion criteria were excluded. Inconsistencies over the inclusion or exclusion of a study were debated by authors until a decision was reached by consensus. In addition to the material model used, the following information was extracted from the studies: the aim of the study, material assignment toolboxes, year of publication, working group/university, and whether a full mandible bone or a block of mandible bone was used in their FE analysis. The included FE studies can be synthesized according to the employment of various density-modulus relationships. The origin of these material models was investigated, and the test conditions, determination coefficient (R 2 ), and sample characteristics (bone site and domain, sizes, and geometries) were evaluated and tabulated for the original studies. As per PRISMA guidelines, assessing the quality of the original studies, representing the study syntheses, gives insight into the study's credibility by identifying potential biases . Because the original studies included two distinct types of research, separate evaluation scales, tailored to each type, were employed. The Quality Assessment Tool for In vitro Studies, the so-called QUIN Tool offers a standardized method for evaluating the individual risk of bias for in vitro studies . This was carried out for all original studies that conducted in vitro experiments. The risk of bias for original studies based on FE simulations was assessed using the risk of bias tool for the use of finite element analysis in dentistry, the so-called ROBFEAD . The aspects of both tools can be scored as either adequately/high specified = Two points, moderate specified = One point, poorly/not specified = Zero points. To provide a quantitative measure of the overall risk of bias in each study, the following equation (Equation (1) ) was used for both QUIN and ROBFEAD tools . (1) Final score = Total score × 100 2 × number of criteria applicable With the described literature search strategy, 1787 studies were retrieved . Following eliminating duplicates, 1055 studies underwent the abstract and title screening and 382 studies were deemed eligible for a thorough analysis. Following the full-text analysis, 77 studies were extracted that met the inclusion criteria [ 1 , 12 , 18 , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ]. Despite our extensive efforts, 23 reports were not retrieved. While we cannot definitively assure whether these studies used homogeneous or heterogeneous material models, we note that the inability to review them does pose a risk of missing some material models relevant to our study. Unfortunately, we have no control over this matter. Fig. 1 The systematic review flowchart of the study selection process (PRISMA flowchart ). n stands for the number of publications and CT for computed tomography. Fig. 1 The reviewed papers cover a wide spectrum of FE analysis within the craniomaxillofacial research field. Topics include bone-implant interaction (17 studies) , fracture fixation (13 studies [ 12 , 29 , 31 , 48 , 49 , , , , , , , 86 , 95 ]), mandible reconstruction (10 studies [ 32 , 38 , 41 , , , , , , 93 , 97 ]), osteotomy techniques (eight studies ), implant-retained overdentures (six studies ) and motion/temporomandibular joint dynamics (seven studies ). Less frequent topics, represented by less than five publications each including dental bridges (four studies ), general investigations on healthy mandibles (three studies ), condylar prosthesis (three studies ), periodontal ligament (two studies ), influence of CT imaging on FE studies (two studies ), tumor pathology (one study ), and third molar tooth orientation analysis (one study ). Among the 77 papers, 54 studies used the full geometry of the mandible bone for FE analysis, while 23 studies employed just a block of mandible bone in the region of interest of their studies. The process of attributing specific material properties to individual elements or nodes within a FE model based on information obtained from medical images and established material models is called “material assignment”. This procedure is referred to by various names across different studies, such as material mapping based on Hounsfield Units, and heterogeneous or inhomogeneous material modeling . This can be done by various approaches including voxel-based , element-based or node-based methods . There are several commercial software packages and in-house developed toolboxes available to perform the material assignment. Among the 77 reviewed studies, 20 used Mimics software package (Materialise NV, Leuven Belgium), one used mechanical finder (Research Center for Computational Mechanics, Tokyo, Japan), four used in-house developed Abaqus subroutines, and the remaining 52 studies did not clearly mention which toolboxes they used for material assignment to the mandible bone. While reviewing the 77 FE studies and their employed density-modulus relationships, 12 different material models for the relationships between apparent ( ρ app ) or ash density ( ρ ash ) and Young's modulus ( E ) were identified and listed in Table 1 [ 10 , 15 , , , , , , , , ]. The Mimics empirical default formula (first cited by Xin et al. ) is the most frequently cited density-modulus relationship (Equation (2) ), considering solely the number of citations (18 times). However, when counting research groups using heterogenous material models, the Mimics formula is quoted only by three out of 24 research groups. Both material models proposed by Keyak et al. (Equation (3) ) and Rho et al. (Equation (4) ) emerge as the most used density-modulus relationships with each cited by six out of 31 research groups. (2) E [ GPa ] = − 0.3888 + 5.925 ρ a p p [ g / cm 3 ] E [ GPa ] = 33.9 ρ a s h 2.2 [ g / cm 3 ] , ρ a s h ≤ 0.27 (3) E [ GPa ] = 5.307 ρ a s h [ g / cm 3 ] + 0.469 , 0.27 < ρ a s h < 0.6 E [ GPa ] = 10.2 ρ a s h 2.01 [ g / cm 3 ] , ρ a s h ≥ 0.6 (4) E [ GPa ] = 0.51 ρ a p p 1.37 [ g / cm 3 ] Table 1 12 heterogeneous material models used in the reviewed finite element studies for the mandible. Table 1 Despite conducting an extensive literature review, the original sources for the Mimics empirical default formula could not be identified. Nevertheless, this density-modulus relationship has gained widespread recognition and become well-established since 2013 . The remaining 11 material models originate from 10 studies with two employed density-modulus relationships proposed by an individual study from Rho et al. . In five studies, the material model was proposed based on in vitro experiments, investigated with bone samples, typically through uniaxial compression or tension tests . Two studies used a complete femur for their experiments and adapted previously proposed density-modulus relationships . However, most recent studies employed a mathematical approach, where the material models were derived based on theoretical values for density and elastic modulus or involved cone beam computed tomography-based estimations . Taking into account the mathematical characteristics behind the material models, these 12 density-modulus relationships presented in Table 1 include 18 sub-functions. Among these, there are 11 power functions with exponents ranging from 1.16 to 7.4 [ 10 , 15 , , , , , , 116 , 117 ] and one sigmoid function . Additionally, six linear relationships were determined , one of which serves as an interpolation between two power functions . The origin of the density-modulus relationship varies among different bones. Two utilize data from the femur , one from the tibia , one from the pelvis , one employs a pool of various bone sites , and one model is derived from the femoral and tibial diaphysis of the steer . Among these, five density-modulus relationships were specifically proposed based on data for the human mandible bone [ 10 , 15 , , , ]. Considering the constitutive modeling of the bone tissue, three material models were initially developed to accommodate the orthotropic nature of bone, addressing the directional variations by separated sub-functions . Rho et al. proposed the two density-modulus relationships as orthotropic material models for the trabecular tibia bone and cortical mandible bone. Also, O'Mahony et al. proposed an orthotropic material model consisting of three directional sub-functions. The differences between the material models proposed by original studies were also compared qualitatively in Fig. 2 A and 2B. Prior to this comparison the material models defined in ash density ( ρ ash ) by Cong et al. and Keyak et al. for femur bone were converted to apparent density ( ρ app ) using the correlation ρ ash = 0.6 ρ app . According to Schileo et al. , this correlation is valid for the whole density range of the femur bone. In Fig. 2 A each curve presents the proposed density-modulus relationships which allows a comprehensive understanding of how different material models relate to one another. Fig. 2 B shows numerical calculations of Young's modulus for two representative apparent density values. Fig. 2 Comparison of density-modulus relationships used by finite element studies for material modelling of the human mandible bone. A: The heterogeneous material models are presented on a logarithmic scale, with the corresponding bone domain and type. Two vertical lines in light grey indicate the representative apparent density values in relation to bone domains used for the calculations in Fig. 2 B B: Numerical calculation of Young's modulus for two representative apparent density values of 1.3 g/cm³ for trabecular (blue bars), and 1.6 g/cm³ for cortical (orange bars) bones . In both subfigures A and B, dashed horizontal lines represent the most frequently used homogeneous Young's modulus for trabecular (1.37 GPa) and cortical (13.7 GPa) bone . Fig. 2 Table 2 summarizes the quality assessment results of the risk of bias with the QUIN and ROBFEAD tools for each original study. Studies were characterized by a low to medium risk of bias. The highest risk in the in vitro studies was found in Carter and Hayes , Dalstra et al. , and O'Mahony et al. each with 54 % score, and the highest risk among the FE studies was found in Ramos et al. with 57 % score. The density-modulus relationship known as Mimics empirical equation is characterized by an unknown/high risk of bias as the origin and methodology remained unclear. Table 2 Quality assessment of original studies. A: Quality Assessment Tool for In vitro Studies , B : risk of bias tool for the use of finite element analysis in dentistry (ROBFEAD). Table 2 To achieve precise results in FE analysis, in addition to various other considerations, e.g. loading scenarios, boundary conditions, and the geometric details of the bone, ensuring the accurate integration of material properties stands out as a critical factor . Bone is inherently a heterogeneous structure, composed of various materials arranged in complex patterns. Using heterogeneous material models in FE analysis allows for a more accurate representation of the actual mechanical behavior of bones compared to assuming homogeneity and enables more realistic calculations of localized stress and strain distributions . The consideration of heterogeneous material models in comparison to homogeneous models facilitates the FE models to have a greater energy dissipation and a more realistic prediction of the apparent stiffness and transition between the bone domains, which avoids numerical singularities at the interface of cortical and trabecular domains . Furthermore, integrating CT-based bone properties into FE analysis enables subject-specific modeling, allowing the comparison of individuals with different pathologies in biomechanical analysis and clinical applications. The increasing interest in the topic of density-modulus relationships within biomedical research on the mandible is evident from the notable concentrations of publications in recent years. However, given the challenge posed by the multitude of existing material models from the literature and the increasing complexity of heterogeneous material modeling, which requires more effort, knowledge, and computational resources, yet a tendency to use homogeneous material models prevails . This persists despite the multiple benefits associated with heterogeneous material modeling, as previously discussed. The review revealed a lack of consensus among these studies regarding heterogeneous material modeling and assigning methods. Also, some studies were found which used the material model outside their intended density ranges or applied the density-modulus relationship uniformly in all directions (isotropic) despite them being primarily proposed for orthotropic materials. Such assumptions, which are common in FE analysis, may impact the accuracy of the modeling and should therefore be examined through sensitivity analysis . Careful attention must be given to the correct unit conversion of the parameters of the density-modulus relationship e.g., density and elastic modulus, as this may have a significant influence on the results. Additionally, it was observed that after selecting the appropriate heterogeneous material model, researchers used various software toolboxes for the material assignment process. Next to the toolboxes employed by the 77 reviewed studies (Mimics software package and mechanical finder) there are additional toolboxes such as Bonemat , Bonemapy , Py_bonemat_abaqus , Simpleware ScanIP (Synopsys Inc., United Kingdom) , CTPixelMapper , and in-house developed python plugins . Based on authors experience, using a unique material model with various toolboxes, each developed using distinct assignment methods, results in considerably different Young's modulus distributions . Notably, 52 out of the 77 studies did not specify which toolboxes or assignment strategy (node-wise or element-wise) they used, making it difficult to compare their findings. In order to provide a better insight into the origin and reliability of density-modulus relationships and to sort the existing approaches, the second goal was to conduct an extended search identifying original studies that initially introduced heterogeneous material models. In this context, Helgason et al. and Knowles et al. published review studies of heterogeneous material models for human bones and discussed influences on the Young's modulus and outcomes of FE modeling . Through the present review, 12 material models from 10 original studies were identified in total. The absence of a consensus on the material model and the considerable large deviation among them may yield to serious implications for modeling accuracy . Several factors were identified as sources of this deviation: 1) difference in study type including in vitro experiments, mathematical-based approach or combined simulation-experimental-based approach, 2) differences in experimental mechanical test types e.g., tension or compression test or test setups, such as constrained or uniaxial loading, and sampling rates, 3) differences in the methodology of the numerical approaches, such as different boundary condition or use case, 4) different bone samples or donors in terms of anatomical sites, geometries (cubes/cylinders or full bone), dimensions, bone domains (cortical or trabecular), the quantity of samples, sex, and even specimen-specific differences. Irrespective of the study approaches, there is significant variation in the anatomical sites of the specimens studied in the original research. Studies by Ciarelli et al. and Morgan et al. revealed considerable variability in the density-modulus relationship depending on the anatomical site [ 22 , , , , , ]. This becomes apparent, for instance, in samples of vertebral bones, which result in a weaker relationship between elasticity and density than the values derived from long bone samples . The quantity and characteristics of bone samples differ in original studies. Despite Cong et al. claiming that complete bones are more clinically relevant, most in vitro studies use cubic or cylindrical bone samples. Those are generally employed in a sizable cohort of samples, but it is notable that these samples often originate from a limited number of donors. For instance, Dalstra et al. tested 57 samples taken from only two donor pelvic bones. This methodology restricts the ability to explore population-based differences in bone characteristics. Additionally, the potential influence of sex, age and ethnic factors on bone properties raises questions, especially when the ethnicity of the bone samples is not specified or if studies do not select a cohort of equal size in terms of their sex . Since the trabecular and cortical bone domains cover a wide range of density, dividing the material model into sub-functions based on density ranges, known for both bone domains, is an option to better estimate the Young's modulus. Therefore, Pinheiro et al. created separated sub-functions for the cortical and trabecular domains. However, this approach poses a challenge of discontinuity in material properties at the interface of bone domains, leading to complications in mesh convergence in FE analysis. An alternative strategy is provided by Keyak et al. , who addressed this issue by considering the transitional domain between cortical and trabecular bone within a linear sub-function, which ensures continuity at the boundaries . Additionally, several FE studies were identified that employed a density-modulus relationship proposed as orthotropic sub-functions for an isotropic behavior of the bone. For instance, all 15 FE studies that used the orthotropic material model from O'Mahony et al. applied the superior-inferior sub-function as an isotropic material model for all directions. Bone anisotropy is crucial for accurately estimating the elastic modulus since the relationship between mineral content and modulus varies with the direction of loading [ , , ]. According to the quality assessment analysis, all assessed studies have a low to moderate risk of bias ( Table 2 ). Nevertheless, the density-modulus relationship proposed by Mimics software lacks transparency regarding its development and reliability and should therefore be used with caution. Considering the focus of this review, several recommendations can be made for using heterogeneous material models for subject-specific FE models. – When using a material model based on in vitro experiments, special attention should be paid to the type of experiments and the variation in samples. In the studies in which the proposed material model is generated based on mathematical calculations, caution is required due to the potential issues with missing validation and mesh convergence, which can compromise the accuracy of the FE analysis. – The range of applicability of the proposed density-modulus relationship in terms of bone domain and directional behavior should be respected and researchers are advised to check in advance if the density-modulus relationship they intend to use is valid only for specific bone domains, density ranges or direction . For instance, the material model from Dalstra et al. is proposed only for the trabecular bone, covering the range of 0.109–0.959 g/cm 3 , and should not be used to model the bone out of this range. – We also recommend that researchers carefully consider the material assignment method of the toolbox they employ and explicitly report it in their studies. This will enhance the reliability, comparability and reproducibility of the results for future researchers. – Furthermore, if original studies have proposed several density-modulus relationships, for example for different anatomical sites proposed by Rho et al. , the specific employed material model from the original study should be cited in the scientific papers. Although best effort was made to comprehensively gather available information, this study has some limitations. Initially designed to focus on reviewing FE analysis of the mandible using density-modulus relationships to model bone mechanical properties, it did not specifically target the material models themselves. Consequently, we may have missed some material models developed for mandible bone that have not yet been identified or cited. However, we attempted to overcome this issue through a randomized hand search. Additionally, 23 reports could not be accessed, and the inability to review them represents a potential risk of overlooking important material models that are relevant to our systematic review. Further experiments and validations have to be conducted to: (1) provide recommendations on existing heterogeneous material models, (2) analyze the characteristics of the density-modulus relationships such as the sex-dependency, (3) investigate the influence of the employed heterogeneous material model through a comparative FE study on field variables such as stress and strain and (4) propose new models exclusively designed for both domains of the mandible bone. Prior to this, a systematic review is necessary to design a standard experimental protocol. It is also advised to use a validation approach by combining simulation and experiments such as the method presented by Cong et al. to validate the material model. They estimated both power and sigmoid functions parameters, which are commonly used for bone material models, by means of optimization techniques. They iteratively updated the parameters of these functions, aiming to align the predicted femur stiffness derived from FE analysis with the experimentally determined bone stiffness acquired by them through mechanical testing . Future research needs to go beyond isotropic heterogeneous material models and explore orthotropic heterogeneous material models in detail . This approach provides a more accurate representation of the anisotropic nature of bone tissue, improving the precision of stress and strain predictions, and leading to better-informed clinical decision-making and more effective treatment planning. Furthermore, orthotropic material modeling offers the potential for even more precise subject-specific analysis by accounting for the directional dependencies of bone mechanical properties. Of the 77 reviewed scientific papers, 75 FE studies applied isotropic heterogeneous material models. While these models are more realistic than isotropic homogeneous ones, they still fail to capture the anisotropic nature of bone. Furthermore, a systematic study is required to investigate the influence of the material assignment methods and toolboxes on the results and stress distribution within the bone as inter-method differences were observed in the results produced by various material assignment toolboxes. This systematic review aims to assist researchers in the craniomaxillofacial field in selecting more accurate heterogeneous material models for building subject-specific FE models of the mandible. This was achieved by conducting a systematic review on density-modulus relationships used for material modeling of the mandible bone, providing an overview of the material models that have been used and discussing the origin of the models and their quality. The lack of consensus on the optimal material model for estimating Young's modulus from mineral density for the mandible bone remains a significant challenge. The presented review highlights the variability in relationships in terms of bone domain, the methodology based on which the relationships are created, different experimental setups, isotropy or anisotropy structure, and anatomical site. This reveals the importance for researchers to carefully consider the characteristics of a relationship in accordance to the objective of their study before employing it. Future research should focus on developing new heterogeneous material models that more accurately and exclusively reflect the biomechanical properties of the mandible bone and its anisotropic structure based on a standardized experimental protocol and validating the models through methods combining simulation and experiment. Additionally, a comparative FE analysis is needed to investigate the significance of the available material models and different material assignment approaches on the resulting field variables of the bone. By addressing these challenges, researchers can improve the accuracy of subject-specific FE analysis, leading to better clinical decision-making and treatment outcomes in the craniomaxillofacial field. | Study | biomedical | en | 0.999996 |
PMC11698924 | At the core of living tissue, the cell serves as a functional unit , coordinating a vast range of biochemical processes essential to life. This entity harbours a dynamic world of subcellular structures and molecular mechanisms, within which energy metabolism operates. It defines the set of pathways, known as metabolic pathways, corresponding to a complex network of enzymatic reactions necessary for the renewal of the cellular needed energy, continuously changing according to the demand , . Within these metabolic pathways, glucose degradation initiates within the cytoplasm, leading to the synthesis of adenosine triphosphate (ATP) and pyruvate, subsequently diverging into lactate or acetyl-CoA depending on cellular demand. Acetyl-CoA, which is also produced from fatty acids via the β -oxidation pathway and from the breakdown of amino acids, plays a crucial role in the tricarboxylic acid cycle, facilitating energy production in the mitochondria. Among the possible known factors impacting energy metabolism, physical exercise causes major changes , , requiring the body to adapt to supply the energy needed for muscular contraction. Depending on the intensity, duration, and type of exercise, the amount and rate of energy consumption changes. This leads to the activation of complementary pathways, and the tune control of the enzymatic activities through cross-reaction between the different metabolites of the cells as well as of the organs. In this context, fatty acids, through the β -oxidation, represent the most efficient cellular energy source but are constrained by production rates. Increased energy demand leads cells to resort to alternative sources, such as more powerful sources like glucose. Interestingly, because all of the energy pathways are interconnected and finely regulated by common co-activators and co-inhibitors, the overuse of glycolysis and carbohydrate oxidation in case of high energy demands, will limit the use of fatty acids. Energy metabolism is, therefore, a complex system and it is difficult to predict its behaviour simply by knowing its elementary components. It is therefore necessary to model it to facilitate its understanding and study . Health condition is also a major factor influencing changes in energy metabolism, notably by affecting the way the body produces, stores, and uses energy. Multiple chronic and inherited diseases, such as cancer, diabetes, and cardiovascular diseases, lead to metabolic alterations, particularly in the mitochondria, which can participate in the disease progression by limiting cell functioning and/or favouring inflammation and pathogenic functions. Among these diseases, neuromuscular diseases represent a critical clinical challenge by their multiplicities in terms of origin and symptoms. While metabolic defaults can be a direct consequence of disease, certain neuromuscular disorders, such as calpainopathy, present metabolic defaults as a secondary effect, making it even more challenging to understand and possibly anticipate. Given the involvement of various enzymes, traditional experimental approaches would face limitations as it would be complicated to test each enzyme. Therefore, the need to develop new tools aimed at modelling the energy metabolism and its adaptation to different contexts of demand is of paramount importance and could be used as a lever for the development of new therapeutic approaches. Calpainopathy , also known as Limb Girdle Muscular Dystrophy type 2A (LGMDR1), is an autosomal recessive genetic disorder affecting the proximal muscles of the shoulder and pelvic girdles. It is characterised by muscular atrophy leading to a progressive loss of mobility in affected patients. Unlike other dystrophies, calpainopathy is caused by mutations in the CAPN3 gene, which codes for a calcium-dependent protease, calpain-3, involved in calcium homeostasis, and in the remodelling of sarcomeres, the multiprotein structures responsible for contraction. In the case of muscle damage, persistent calcium influx activates calpain-3, enabling the muscles to repair and adapt. Mutation of the CAPN3 gene induces a loss of expression of the protein, leading to a disorganisation of sarcomeres, a deregulation of calcium flux and, as a secondary consequence, oxidative stress and energy metabolic alterations. Among these alterations, the loss of calpain-3 leads to the dysfunction of mitochondria, the alteration of β -oxidation, and a loss of equilibrium between the different energy pathways ultimately leading to a decline in ATP production rate , . While the exact mechanisms by which this occurs are not yet fully understood, the understanding of its exact disturbance among the disease heterogeneity is of definite interest at preclinical and clinical levels. To this date, there is no cure for this disease, and clinical care strategies remain based on symptom and pain relief. With the idea of studying the potential of physical exercise as a complementary approach to improve the clinical care of patients with this disease, previous research made the hypothesis that an exercise protocol, tailored to each patient's metabolic state, could lead to improvements in motor function and be considered as a clinical care strategy , . Modelling in biology is usually used to decipher biological processes, to reproduce experimental results or, conversely, to predict experimentally unstudied events. In the case of metabolism studies, we can predict which pathway will be favoured by given conditions (initial conditions of metabolites, pathways voluntarily blocked) or, on the contrary, which pathways will be altered by changes in enzymatic activity. The main powerful methods for analysing and predicting the properties of genome-scale metabolic models are Flux Balance Analysis (FBA) and related approaches , , . The fundamental assumptions underlying the FBA framework for computing solutions are the steady state condition and the principle of optimality. The goal of FBA is to identify a flux distribution that achieves the maximisation of an objective function, such as the rate of biomass production or energy production. An approach to enhance the quantitative accuracy of FBA predictions involves imposing constraints on uptake fluxes. In absence of measurements of these fluxes, FBA computations depend on setting bounds for exchange reactions to simulate specific cellular environments. Various techniques have been employed to approximate uptake fluxes, leveraging intricate details of regulatory networks , transporter saturation kinetics , transcriptomic data , and training datasets for machine learning-based methodologies . We chose to use MitoCore , a constraint-based metabolic model specifically designed to capture mitochondrial processes. Constraint-based models such as MitoCore work by imposing physiological constraints to simulate steady-state metabolic fluxes. This approach enables us to anticipate key aspects of energy metabolism in healthy and pathological conditions, and to understand how cells balance pathways such as oxidative phosphorylation and glycolysis. MitoCore's focus on mitochondrial pathways makes it particularly useful for understanding energy dysregulation, specifically in diseases such as calpainopathy where mitochondrial dysfunction plays a central role. In addition, the model captures reversible reactions, such as those catalysed by enzymes like lactate dehydrogenase, enabling us to examine changes in metabolic fluxes under variable cellular conditions. While not dynamic in the traditional sense, constraint-based modelling allows for a snapshot of the metabolic state under specific conditions such as those defined by energy demand, substrate availability, and environmental factors (nutrient intake) and the addition of constraints allows us to mimic energy production for multiple energy demands and its defects with altered metabolic pathways instead of blocked ones. It is in this context, at the interface between physiology, pathophysiology and metabolic modelling, that this study takes place. Using Flux Balance Analysis (FBA) , we have constructed a healthy constraint-based model of the energy metabolism of a murine muscle cell that we can adapt to different pathological or individual situations, providing an instantaneous view of metabolic fluxes. First, we simulated on this model the multiple energy demands of physical exercise, expressed at 4 different intensities of maximum oxygen consumption, called V O 2 m a x . These intensities correspond to the extremes of energy demand from rest (25%) to 100% of V O 2 m a x , and including the intensities of the transitions between pathways: 65%, corresponding exclusively to the use of the aerobic pathway, and 85% corresponding to a combination of the aerobic and anaerobic pathways. This allowed us to ensure that the healthy model had a proper adaptation in the balance between the main energy pathways. Secondly, as a case study, we constrained this model with the known metabolic disorder in the most common limb-girdle dystrophy, calpainopathy. By applying predetermined values of key fluxes, we can compare the healthy to the calpainopathy-like model and highlight potential compensatory pathways and key regulators of the altered metabolism. The method developed in this study is intended to be adapted to other diseases with dysregulation of energy metabolism and should open up new potential therapeutic approaches. In order to model muscle energy metabolism and to adapt it to different contractual conditions while allowing to anticipate compensatory mechanisms and/or potent modulators, we decided to adapt the well described MitoCore model , a mitochondrial metabolic model containing 485 reactions, including 155 mitochondrial reactions, 168 cytosolic reactions and 162 transport reactions of a human cardiac cell, to a healthy murine muscle cell used as control. To do this, we automatically checked for the presence of murine enzymes using the Brenda and Unitprot databases, suppressing only one enzyme for which no clear information supporting its existence in murine were found (see Method for more details). Since the model relies on FBA with an objective function focused on biomass production, the fluxes of nutrient uptakes have to be settled. The original model has been defined with human cardiac glucose, fatty acids and amino acids fluxes entry, which do not correspond to murine skeletal muscle cell. Since these fluxes of nutrient uptakes, notably the glucose and fatty acids, are not well quantified for the murine skeletal muscle cell, we attempted to predict them using a proper FBA combined with Flux Variability Analysis (FVA) approach, which optimises energy production with respect to the proportions of muscle energy pathways known from the literature (see Method for more details), using experimental data of ATP production associated to oxygen consumption as constraints. Under resting conditions (25% of maximal oxygen consumption) we obtained ranges of glucose and fatty acid fluxes allowing us to determine the exact value for each nutrient flux matching the expected proportion of 40% of carbohydrate oxidation (CHO) and 60% of β -oxidation. We then performed the same calculation but at different levels of exercise intensity with increasing ATP production rate, dioxygen consumption rate and different proportions of energy pathways . At 65, 85 and 100% of maximum oxygen consumption, FVA provides ranges of fluxes for each nutrient allowing us to determine these optimal values for both glucose and fatty acids ( Table 1 ). Table 1 Fixed constraint from experimental data of O 2 inputs and ATP outputs fluxes set to compute the input values of glucose and fatty acids in the control model, and expressed in μmol.gDW −1 . min −1 . Each set of O 2 and ATP is used to simulate 4 conditions of energy demands, expressed as 4 percentages of maximum oxygen consumption: 25, 65, 85 and 100% of VO 2 max . Glucose and fatty acids uptake fluxes were computed through a FVA method, allowing to respect constraints of energy demand, associated to carbohydrate oxidation (CHO) and β -oxidation proportions physiologically consistent . Table 1 Intensity of exercise (% of V O 2 m a x ) 25% 65% 85% 100% Fluxes fixed O 2 -3.7 -7.9 -11.1 -14 ATP 14.6 36.52 54.8 75 Fluxes computed by FBA Glucose -0.03 -0.13 -0.6 -2 Fatty acids -0.1 -0.27 -0.3 -0.08 This approach allowed us to determine that the needed fluxes of glucose in a single cell increase continuously from 25% to 100% of V O 2 m a x ( Table 1 ) reaching a fold of 66. However, concerning the fatty acids entry in a cell, we noted an increase by 3 fold from 25% to 85% of V O 2 m a x then followed by a limitation of fatty acids entry due to excess of glucose consumption. Once the constraints on O2 consumption, ATP production, glucose and fatty acids had been applied, the fluxes obtained in the FBA solution had to be checked to validate the behaviour of our model. For this purpose, we selected key enzymatic reactions from the main energy pathways involved in the ATP production to evaluate the biological consistency of our model and the change in their balance with the intensity of energy demand . Figure 1 Fluxes expressed in absolute value (A) and proportions (B) of selected key enzymatic reactions from the main metabolic pathways used for ATP production, for each energy demand of physical exercise in a control muscle cell model. The reactions chosen as representative are phosphoglycerate mutase for glycolysis, acetyl-CoA acyltransferase for β -oxidation, 3-hydroxybutyrate dehydrogenase for ketone body degradation and leucine and isoleucine transaminase. The computed proportion of the main metabolic pathways are physiologically consistent, validating the behaviour of the control model. Figure 1 For each exercise intensity simulation, the instant fluxes and proportions of the ATP producing pathways changed as expected. We observed an increase by 57 fold in glycolysis flux from 25% to 100% of V O 2 m a x . If the Isoleucine, Leucine and ketone bodies degradation was noticeable at 65% and 85%, their contribution to the total ATP produced dropped with the increase in intensity, whereas the fatty acids increased from 25% to 85% of V O 2 m a x and then totally dropped at 100% . Looking at the proportion of metabolic pathways used to produce ATP, the results are consistent with the literature , and confirm that there is around 60% of fatty acids degradation (Fatty Acid metabolism) at rest, 50% at medium exercise intensity, 20% at high exercise intensity and around 2% at maximal intensity. Thus, we have built and validated a control murine model of single-cell energy metabolism under multiple constraints of demands, which we can now adapt to any pathological condition. To simulate the disease in the metabolic network model, we then gathered all reported mitochondrial consequences and metabolic dysfunctions associated with calpainopathy described in the literature from murine models , . It is important to note that, to date the experimental data available are limited and only two studies provide quantitative or semi-quantitative evaluations of muscular metabolic alterations in calpainopathy. Most of these disease-induced regulations come from the analysis of differential gene expressions (RNAseq data) and few come from direct measurements of enzymatic activity. Regarding the studies reporting gene expression changes, the match between the list of enzymes in our control model with the RNAseq results gives us 18 altered enzymes of interest in the murine calpainopathy model (see Supplementary Table S4). These enzymes are mainly related to β -oxidation, the Krebs cycle, the mitochondrial respiratory chain, and amino acid degradation. Since it's known that a given amount of enzyme can give rise to different levels of activity depending on the cellular context and co-factors, we assumed that only the maximal enzymatic activity is proportional to the total gene expression. Concerning the data obtained from direct enzymatic activity measurements in the muscle of the calpainopathy murine model, only two enzymes from glycolysis matched with the enzyme included in our control model . The activity of aldolase was reported to increase by 133%, while the activity of lactate dehydrogenase increased by 287% when compared to non-mutated animals. Because the reported enzymatic activities were measured under controlled conditions in vitro , we also assumed that these activities referred to their maximum. Thus, to build the calpainopathy-like model, we weighted the maximum fluxes calculated by FBA under all conditions of intensity from the control model for each selected enzyme, with their corresponding correction factor. The resulting fluxes are then used as an upper bound for the model, allowing variations in enzyme activity but limiting their maximum. Our developed calpainopathy-like model, with disease-specific constraints, was then run with FBA calculation using the same nutrient entry fluxes than those calculated for the control model. Finally, we compared the capacity of the calpainopathy-like model to produce ATP at each intensity of V O 2 m a x and the balance between energy pathways with the control model . Figure 2 Comparison of the ATP production fluxes (A) and the proportions (B) of its main contributing pathways, for each intensity of energy demand, in control and calpainopathy-like models. The reactions chosen as representative are phosphoglycerate mutase for glycolysis, acetyl-CoA acyltransferase for β -oxidation, 3-hydroxybutyrate dehydrogenase for ketone bodies degradation, leucine and isoleucine transaminase and glutamate dehydrogenase. Figure 2 In both models, the total flux of ATP production increased with intensity level, but less so in the calpainopathy-like model . Although the disease-like model was able to produce the necessary ATP at 25% of V O 2 m a x , as effort increases, the model became more and more deficient in terms of ATP production, dividing the required ATP by 1.8 at 100% of V O 2 m a x . Associated with these intensity-induced decreases in ATP production, we noted an imbalance between the energy pathways when compared to the control model overall to the detriment of β -oxidation (FA metabolism). If at 25% of V O 2 m a x , we failed to observe any change in the proportion between the main energy pathways, we noted a marked decrease in β -oxidation compensated by an increase in ketone bodies and amino acid degradation at 65% of V O 2 m a x . However, With the increase in intensity until 85% and then 100% of V O 2 m a x , the calpainopathy-like model induced a marked switch to the glycolysis when compared to the control model with the decrease and then disappearance of all other sources of fuel . Taken as a whole, our calpainopathy-like model highlights that, with an equivalent source of nutrients when compared to the control model, energetic metabolism is no longer able to produce large amounts of ATP at a proper rate despite a switch in energy pathways proportion, suggesting the need to improve the nutrient uptake to compensate. To better understand the disease-induced adaptation, we then ran a FVA analysis on selected enzymatic reactions from the main energy pathways at each intensity of energy demand that we plotted with the values of flux given by FBA results . This allows us to evaluate the adaptability of the system and its capacity to modulate its flux level by showing the range of values that the flux of the enzyme of interest could take without changing the optimal value of the objective function (ATP production). This FVA-given range enables the solution to be adjusted and highlights possible compensations. Interestingly, our calpainopathy-like model showed a complete disappearance of any variability in enzymatic fluxes when compared to the control model from 65% of V O 2 m a x , suggesting that under disease condition and common nutrient entry, we have only one solution to produce the highest amount of biomass. This unique solution per intensity highlighted an equal level of glycolysis between calpainopathy-like and control models over the different intensities in energy demand, noted by the aldolase flux, followed by a global limitation in oxidation power: limitation in TCA, β -oxidation, and PDH fluxes. In compensation, at 100% of V O 2 m a x , the lactate production (lactate dehydrogenase) has increased from 0.3 to 1.5 μ m o l . g D W − 1 . m i n − 1 in the calpainopathy-like model compared with the control model. This result suggests that the limitation of the oxidation capability of the model attempts to compensate through an increase in the anaerobic lactic metabolism but failed due to limited nutrient entry for a less powerful energy pathway. Taken as a whole, this modelling can not only anticipate the energy capabilities of a pathological condition, but can also highlight potent needed adaptations to compensate, leading to potential compensatory mechanisms to test. Figure 3 Plot of FBA solutions (dots) with the range of variability given by FVA solutions (blurred colour) for fructose biphosphate aldolase (aldolase, glycolysis), pyruvate dehydrogenase (PDH, glycolysis), lactate dehydrogenase (LDH, glycolysis), 3-hydroxyacyl-CoA dehydrogenase (HADH, β -oxidation), acetyl-CoA acyltransferase 2 (ACAA2, β -oxidation) and citrate synthase (CS, TCA cycle). Values are given according to the energy demand expressed as a percentage of maximal O 2 consumption, in the control (left) and calpainopathy-like (right) models. Figure 3 Our pathological model relies on disease-induced enzymatic flux constraints and the set of nutrient influx upper-bounds. When we applied the same upper-bound of the different nutrients for the calpainopathy-like model as the control model, we observed a limitation in ATP production from 65% of V O 2 m a x , without any glycolysis increase to compensate despite the elevation of aldolase upper-bound known in the literature. This suggests that the calpainopathy-like model could partly compensates its failure in producing energy by increasing its nutrient uptake. To demonstrate this hypothesis, we experimentally tested to remove the intake upper-bound of nutrients, nutrient per nutrient, for the calpainopathy-like model only, and we compared their capacity to compensate for the ATP production failure over each intensity of energy demand . As expected, removing the upper-bound of fatty acids uptake didn't improve ATP production as the β -oxidation was already at its maximum capacity , with an identical Root Mean Square Error (RMSE) when compared to the control model than without removing the fatty acids upper-bound . However, if removing the upper-bound of ketone bodies didn't affect the ATP production (data not shown), removing them for glucose uptake allowed to fully correct the ATP production at 65% of VO2max, and enhanced the ATP production at higher intensity without matching the control model. This adaptation decreased the RMSE to 13 . When we removed upper-bounds of glucose and glycerol, we allowed the calpainopathy-like model to quietly fully recover the ATP production capacity, with RMSE at 2 , while adding the remove of amino acid upper-bound finished to correct the metabolic state . Our modelling suggests that despite enzymatic constraints, the calpainopathy-like model can compensate for its defect in ATP production through the combined increase of nutrient uptake, a new approach for this disease that should be evaluated in vivo . Figure 4 Plot of ATP production fluxes of the control model (grey dotted lines) compared to the calpainopathy-like model (red dotted lines) depending on the energy demand from 25 to 100% of VO 2 max . Evolution of the calpainopathy-like model fluxes of ATP production under control model nutrient uptake (A), by removing upper-bound of fatty acids (B), of glucose (C), of glucose + glycerol (D) and of glucose + glycerol + amino acids (E). The root mean square error (RMSE) is calculated to estimate the distance of computed values of calpainopathy-like model with those obtained with the control model. Figure 4 When we focus on this restored situation to better understand the underlying mechanisms ensuring this correction, we ran an FVA analysis on the main enzymes of interest . The results showed that increasing the possibility of uptake, restored a possible variability for each flux. Confirming previous results and the necessity to increase other sources of energy, we can see here that glycolysis activity increases over time reaching its maximum activity illustrated by aldolase and lactate dehydrogenase, as well as the β -oxidation. Interestingly, not only enzyme activity of β -oxidation are limited by the regulation applied on the calpainopathy-like model, but the FVA results also show a change in reactions orientation meaning the model could produce fatty acids instead of oxidising them. When looking at the activity of the TCA cycle, we can see a decrease in citrate synthase activity from 85% of V O 2 m a x , while oxygen transport and Complex V activities are still increasing, an behaviour enabled by the increase of amino acids uptake, allowing the production of ATP to reach its objectives. Figure 5 Plot of FBA solutions (dots) with the range of variability given by FVA solutions (blurred colour) for fructose biphosphate aldolase (aldolase, glycolysis), pyruvate dehydrogenase (PDH, glycolysis), lactate dehydrogenase (LDH, glycolysis), 3-hydroxyacyl-CoA dehydrogenase (HADH, β -oxidation), acetyl-CoA acyltransferase 2 (ACAA2, β -oxidation), citrate synthase (CS, TCA cycle), ATP synthase (complex V, TCA cycle) and oxygen transport (oxygen, TCA cycle) of the calpainopathy-like model when we fully restored the ATP production rate by removing the upper-bounds of glucose, glycerol and amino acids. Values are given according to the energy demand expressed as a percentage of maximal O 2 consumption. Figure 5 In this study, we built a constraint-based metabolic model of a murine healthy skeletal muscle under different energy demands that we can adapt to various pathological or individual situations. We adjusted the upper bounds of the uptake fluxes of main nutrients (glucose and fatty acids) to match the known evolution of the proportions of energy pathways with an increase in O2 consumption and ATP production mimicking a range from 25 to 100% of maximal O2 consumption. The values for ATP production and O2 consumption were extracted from experimental values, while we determined adequate nutrient uptake. Based on this correspondence between energy production and nutrient consumption, we have a model of muscular energy production ready to be adapted to disease contexts to evaluate metabolic changes and possibly anticipate compensatory mechanisms. To this end, we focused on calpainopathy disease, the most common limb-girdle muscular dystrophy, in which complex metabolic alterations have been reported as indirect defects of the CAPN3 gene mutation. To model this pathology, we combined published dysregulations of muscle metabolism in a calpainopathy murine model and used these alterations to constrain enzyme fluxes by calculating dedicated bounds. The tune analysis of this calpainopathy-like model, not only confirmed a loss of ATP production capacity but also revealed for the first time that increasing the uptake of a combination of nutrients (glucose + glycerol + amino acids) can overcome the energy production defects. Our study not only developed mitochondrial bioinformatic models but also showed that we can use such models to orientate preclinical and clinical research, creating an adaptive tool for personalised medicine. Our study is the first attempt to model dystrophic disease with secondary metabolic alterations using a constraint-based method. This unique approach allowed us to mimic in vivo energy production defects with altered metabolic pathways instead of blocked ones. The increase and decrease of enzymatic activity related to a specific disease context is of paramount challenge, knowing that for an equivalent enzymatic quantity, its activity can be multiplied under specific cellular contexts, such as a decrease in ATP content and/or a change in the amount of cofactors. This leads us to consider two priorities for the model: 1. a drastic change in energy demand to simulate changes in muscle fate under different conditions of use; 2. the use of FBA and FVA allowing a range of enzymatic fluxes. The combination of these two parameters opens new avenues for further research in the field. As a proof of concept of the interest of such an original approach, we used calpainopathy as a pathological context to constrain the model. Interestingly, thanks to the modelling of a gradual increase in energy demand, we were able to objectify the muscular defect in ATP production while at rest the modelled cell did not show any defect. Moreover, this approach also highlighted the imbalance between pathways that change for each percentage of energy demand, revealing the complexity of cellular adaptation and providing a precise guide for experimental research. However, of the three enzymes known to be up-regulated in calpainopathy, two were represented in the model (lactate dehydrogenase and fructose biphosphate aldolase), and only lactate dehydrogenase flux was used at its potential compared to the control at 100% of V O 2 m a x (see supplementary table S2), suggesting a compensatory overuse of the anaerobic lactic acid pathway in this disease context. The value of fructose-biphosphate aldolase flux, an enzyme that catalyses the first steps of glycolysis, remained the same as control at all levels of energy demand, consistent with fixed glucose uptake and thus limiting potential compensations. If this could highlight the limitations of the FBA method due to its dependence on fixed nutrient uptake, it also demonstrates its ability to focus on blocking steps of energy metabolism, opening up compensatory mechanisms to decipher. This hypothesis is also confirmed by the FVA analysis , which shows little variability for the resting state and none for the other states with higher energy requirements. Disease-induced regulations we used to build the model, therefore allowed only one solution and no alternative pathway, meaning there is no possible adjustment with the first set of parameters fixed. The calpainopathy-like model is therefore a constrained model offering little to no flexibility compared to the control model. However, it's well known that the nutrient uptake capacity of a cell can vary under different conditions, pathological or under treatment or exercise training. Importantly, when we modulated the nutrients uptake fluxes of the model, to observe compensation and reduce the current ATP deficit, FVA showed a return of enzymatic flexibility allowing the diseased model to produce more ATP and to adapt differently to a change in ATP consumption rate. This result not only pushes the preclinical research to focus on muscle uptake capacity but also open avenues for therapeutic approaches to improve glucose uptake, including physiotherapy. By first increasing the glucose and FA inputs, we were able to confirm that β -oxidation had already reached its maximum activity, as the increase in FAs didn't change the lack of ATP production, illustrating the inability of the muscle to trigger the expression of genes associated with fatty acid metabolism after exercise . Since β -oxidation and the mitochondrion are limited by regulation, the model should be able to produce more ATP via the anaerobic-lactic pathway to compensate. This is indeed the case at all intensities, allowing greater ATP production than with the baseline glucose value and even reaching ATP demand at 65% of V O 2 m a x . However, glycolysis is limited both by the constraints applied to fructose-biphosphate aldolase (maximum flux of 2.66 μ m o l . g D W − 1 . m i n − 1 ) and by those applied to lactate dehydrogenase (maximum flux of 2.05 μ m o l . g D W − 1 . m i n − 1 ), values that are reached for a glucose input flux of 3 μ m o l . g D W − 1 . m i n − 1 at 85% and 100% of V O 2 m a x . It might be tempting at first to try to remove these limitations in order to improve the anaerobic lactic pathway. However, increasing lactate production may not be a viable solution, as several diseases are characterised by an excess of lactate production, which we generally try to avoid as it may lead to lactic acidosis . So although these results showed a corrective potential through glycolysis, increasing glucose uptake flux alone is not enough to compensate for the lack of ATP production. Therefore, in addition to the variation in glucose, we also allowed variation for other energy sources by increasing their possible uptake. Increasing amino acid uptake alone allowed compensation, but their flux values were well above what could be found in the literature, with a 5 fold . By combining ketone bodies, glycerol and amino acid uptake, we were able to address this problem and show ATP production compensation within a more consistent range. Our study has demonstrated the potential of multiple pathway combinations to compensate for disease alterations and to reinforce the interconnection of the metabolic pathways, especially in the context of mitochondrial dysfunction, a common feature of numerous neuromuscular disorders. To characterise oxidative phosphorylation activity, we analysed key metrics such as the rate of oxygen consumption and ATP synthesis, which are essential indicators of mitochondrial function. Concerning the design of the control model and its functioning under gradual energy demands, knowing the ATP consumption rate over different O2 consumption rate was not sufficient to mimic physiological muscular function. The FBA-based model failed to produce the required ATP or balance the metabolic pathways at the published nutrient limits. As O2 and ATP consumption increases, muscle cells use different metabolic pathways according to their respective “power”, i.e. their respective ability to produce a certain amount of ATP per unit of time. If β -oxidation is defined as the most efficient pathway, the number of subsequent enzymatic steps to produce ATP slow down their capacity which then has to be compensated by glycolysis, less effective but much faster. Unfortunately, our model failed to mimic this physiological adaptation pushing us to search for proper nutrient boundaries ensuring the match between energy demand and the known proportions of the main metabolic pathways. This manual adjustment of the control model highlight FBA approach limitations which should be addressed by combining other computational calculation to properly anticipate the special cases we would model. To enhance our approach, integrating genome-scale metabolic models with transcriptomics data, facilitated by tools such as COBRApy, could offer a more comprehensive view of cellular metabolism. This integration would allow for more accurate predictions of metabolic fluxes, leading to better constraints on nutrient uptake rates and more reliable metabolic behaviour predictions. Recently, a machine learning method has been proposed to learn and generalise statistical patterns between input conditions and output reference fluxes. However, these models still suffer from the lack of experimental data set to train. Moreover, it is worth noting that our method does not take into account the concentration of either the metabolites or the enzymes. Knowing the kinetic parameters could allow us to find more accurate uptake values, in particularly the glucose uptake, and to predict the variation of enzymatic activities for possible therapeutic targets. However, calculating the concentration of metabolites and enzymes in metabolic networks is a challenging task due to the non-linear nature of enzyme kinetics. When including non-linear enzyme kinetics into models that contain alternative pathways, the solution space is no longer convex, making such problems extremely difficult to solve computationally. One approach to address this challenge is the Enzyme Cost Minimization algorithm, which identifies the set of metabolite concentrations that minimise the total enzyme mass required to sustain a pre-determined flux through a single pathway. To find the optimal pathway in metabolic networks , all elementary flux modes (EFMs) must be computed. However, the number of EFMs increases combinatorially for large-scale networks. Even if the computation of relevant EFMs that respect diverse linear and boolean constraints is possible with a large set of constraints , the identification of all the kinetic parameters is challenging , . To validate and refine our model predictions, several experimental approaches can be employed. One key method involves combining oximetry measurements with ATP production rate assessments on isolated mitochondria from human biopsies or animal model muscles . Additionally, direct enzymatic activity measurements of key reactions in the muscular energy pathways of interest can be performed. For instance, assessing the activities of phosphofructokinase and hexokinase would provide valuable data on glycolytic flux. These experimental approaches would not only serve to validate our model's predictions but also provide new data to further refine and improve the model's accuracy. Furthermore, these experimental validations could be extended to compare healthy and calpainopathy-affected tissues, allowing us to verify the predicted metabolic alterations and potentially identify new therapeutic targets. By iteratively integrating experimental data and model predictions, we can develop a more comprehensive understanding of energy metabolism dysregulations in calpainopathy and potentially other neuromuscular diseases. In conclusion, our pioneer approach intends to be a generic model of murine muscle metabolism, making it suitable for any pathology with available data on regulatory enzyme activities. Our method operates automatically, and can be continuously optimised with experimental enrichment of clinical data, pointing out metabolic blockage, compensatory mechanisms and opening the way to new therapeutic approach as well as to personalised medicine. It is important to acknowledge that this study is based on data from only two studies, with different kind of cells, and in various kinds of experiments (RNAseq and enzymatic activities). This reflects the current scarcity of quantitative data on metabolic alterations in calpainopathy. Thus, the computed calpainopathy-like model may not reflect all cases of the different variations of the disease, but despite this limitation, our work serves as a prof-of-concept, showing how constraint-based modelling can integrate available experimental data to generate testable hypotheses about disease mechanisms and potential therapeutic approaches. As this is meant to be an upgradeable model, designed to guide the biology researcher, it can then easily be supplemented with new enzymatic activity, or expression data, by adding the type of regulation to be taken into account to the existing list. Therefore analyses were carried out in this study to validate similar behaviours of the metabolism of a calpainopathy muscle cell, but this approach could be adapted to other neuromuscular diseases, providing a valuable tool for understanding metabolic dysregulations and guiding therapeutic strategies. This study was carried out using the MitoCore model , which was originally built to represent the metabolism of a human heart cell mitochondrion and therefore does not correspond exactly to the model needed. MitoCore includes 485 reactions covering all parts of central carbon metabolism. The first step to adjust this model to our study was to assure each reaction represented in MitoCore existed in mice metabolism. In order to do that, we took the KEGG ID associated to each reaction in our model, requested through the KEGG API the EC-number associated to the enzyme catalysing the reaction and checked the presence in murine in the BRENDA database. In the set of reaction of MitoCore, 7 didn't get an entry in BRENDA and 39 didn't exist in murine, but after manually checking in literature, only one of them did not have clear evidence to support its existence, and was therefore removed of the model: the 4-acetamidobutyrate deacetylase. In this study, we chose to build our healthy skeletal muscle cell model using only a constraint-based approach, without employing additional pipelines . This decision allowed us to simulate the metabolic activity of skeletal muscle under varying energy demands, capturing key pathways such as oxidative phosphorylation and glycolysis. By applying generalised physiological constraints, we were able to explore core aspects of energy metabolism relevant to both healthy and disease conditions, without relying on tissue-specific omics data, which is required to build highly specialised models like those automatically generated. Several studies discuss the effects of physical exercise on skeletal muscle metabolism, but a smaller number include precise measurements of metabolite production and consumption , . These measurements describe the flux of ATP produced and the proportion of pathways contributing to this production for different percentages of V O 2 m a x . The proportions of glycolysis and fatty acids degradation expected for the healthy model are summarised in the Table 2 . Table 2 Requirements of metabolic pathway percentages between glucose and fatty acid sources, and sets of ATP production fluxes associated with O 2 absorption fluxes for all energy demands of physical exercise. The fluxes are expressed in μmol.gDW −1 . min −1 . The literature-based constraints were used to run the FBA, while the pathways percentages were used to validate the model behaviour. Table 2 Intensity of exercise (percentage of V O 2 m a x ) 25% 65% 85% 100% Pathways percentages Glycolysis 40% 50% 80% 100% β -oxidation 60% 50% 20% 0% Literature-based constraints O 2 absorption -3.7 -7.9 -11.1 -14 ATP production 14.6 36.52 54.8 75 Among the flux values of the absorbed metabolites, only oxygen values could be found , describing maximum oxygen consumption as a function of different exercise intensities in untrained rats. The values used as constraints on the models will therefore be those for oxygen consumption and ATP production also summarised in Table 2 . To determine the uptake of glucose and fatty acids for a given ATP production and O2 absorption respecting the proportions of carbohydrate oxidation (CHO) and β -oxidation in the Table 2 , an optimisation problem has to be solved, computing a flux distribution for a maximal ATP production. We then computed the maximal uptake of glucose with FVA and decrease this value until the wanted proportions were reached. As the glucose uptake decreases, the fatty acids uptake increase to reach the maximal ATP production, allowing to find the right balance of pathways proportion. Metabolic dysfunctions were gathered from different studies. The first part of the regulations was measured on C57BL/6 mice , five of those consist of differential gene expression in plantaris muscle cells, between the control and the calpainopathy mice. For RNAseq data, it was assumed that only the maximal enzymatic activity was proportionally linked to the total gene expression to be used for model construction. Two regulations were taken from the same study but consist of frozen muscles glycolytic enzymes activities at 25 °C and particularly the difference of activity between the control and the calpain-3 knock-out cell. The second part of the regulations was measured on immortalised muscle cell cultures from C57BL/6 mice grown at 33 °C and consisted of differential gene expression in myoblasts and myotubes. The temperature difference comes from the fact that the first study focused on measuring enzyme activities, while the second study set the temperature solely for cell growth, which did not influence gene expression. And finally there are some reactions that are known to be subject to metabolic defaults such as the oxidation of fatty acids (Hydroxyacyl-Coenzyme A dehydrogenase) and their use for ATP production , and mitochondrial dysfunction. Although there are multiple cases regarding the activity of Citrate Synthase in calpainopathy (increase, decrease or no significant change), as mitochondrial dysfunctions are known to exist, a warning has been placed on the enzyme which translates as a limitation of its activity. The flux distributions to optimise energy production are carried out using Flux Balance Analysis (FBA) , , a mathematical approach that involves creating a mathematical model of the metabolic network and then using this model to predict the flux of metabolic molecules within the cell. Using COBRApy , an object-oriented Python library that provides the main COBRA methods and facilitates the representation of metabolic pathways and gene expression, the strategy is to maximise the ATP production, while constraining the dioxygen fluxes associated to each exercise intensity, and to infer glucose and fatty acid fluxes absorption that couldn't be found in the literature. The results relying on FBA solutions are deterministic regarding the constraints. Thus, repetition of the calculation always gives the same values for identical constraints, as optimal qualitative values. In the case of multiple solutions that maximise the objective function, FBA offers one that may not be the one previously expected. To compute an alternative solution, Flux Variability Analysis (FVA) is a method that provides a range of possible flux values for each reaction of the model, while maintaining the state of the network and thus satisfying the core optimisation problem of FBA. FVA offers a more complete view of the behaviour of the metabolic network, enabling variability and potential regulation points to be identified. The reaction bounds can then be modified accordingly to adjust the optimal solution, to approximate the characteristics of the different conditions studied. | Other | biomedical | en | 0.999997 |
PMC11698930 | Gastric ulcer is a lesion in the mucosa of the stomach lining that penetrates through the submucosa and sometimes to the muscularis propria and extends more than 5 mm in diameter. When alterations occur to the defense mechanisms of the stomach, it can cause changes in the gastric mucosa which will eventually result in erosion and then ulceration . Gastric ulceration is a benign lesion on the mucosal epithelium upon exposure of the stomach to excess acid and aggressive pepsin activity. It is the most prevalent gastrointestinal disorder ever known, accounting for an estimated 15 mortality out of every 15,000 complications yearly in the world . The use of Nonsteriodal Anti-inflammatory Drugs (NSAIDs) is the second most common cause of Peptic Ulcer Disease (PUD) with H. pylori infection being the first. The secretion of prostaglandin normally protects the gastric mucosa. NSAIDs block prostaglandin synthesis by inhibiting the COX-1 enzyme, resulting in decreased gastric mucus and bicarbonate production and a decrease in mucosal blood flow. Epigastric pain usually occurs within 15–30 min following a meal in patients with a gastric ulcer . As a result of indiscriminate use of NSAIDs by people without prescription and not taking cognizance of what they eat, it accounts for over 90 % of all ulcers and approximately 25 % of NSAID users will develop peptic ulcer disease. Aspirin users are also twice as likely to develop peptic ulcers as the general population . Despite the availability and use of most orthodox and conventional drugs, gastric ulcer still remains a global health challenge because they do not total undo the effect of NSAIDs. Also, not everybody likes taking the conventional drugs and not every patient can afford them. That is why gastric ulcer is still prevalent in most developing countries in which Nigeria is one of them . Hence, the search for newer and efficacious approach to its management continues. Yogurt is abundant in calcium, zinc, B vitamins, and probiotics; it is a good source of protein; and it may be supplemented with vitamin D and additional probiotics associated with positive health outcomes . Probiotics are known to have beneficial roles in curing antibiotic associated diarrhea, constipation, traveler's diarrhea, food allergies and cancer . According to Momoh et al. , the consumption of unsweetened yogurts (containing probiotics) may improve glucose metabolism to a degree, but is more effective in treating gastrointestinal complications. The benefits of yogurt consumption to gastrointestinal function are most likely due to effects mediated through the gut microflora, bowel transit, and enhancement of gastrointestinal innate and adaptive immune responses . We therefore seek to look at the antioxidative and anti-inflammatory effects of yoghurt in gastric ulceration. We also seek to determine the effective percentage concentration of the yoghurt on gastric ulceration. Thirty (30) rats weighing (100–150g) each were purchased from Mctemmy concepts laboratory animals for research and a disease-free animal farm in Osogbo, Osun state, Nigeria. The animals were kept in polypropylene plastic cages and maintained at normal and standard laboratory conditions of temperature (28 ± 2°C) and relative humidity (46 ± 6 %) with 12-h light-dark cycle and adequate ventilation for two weeks to allow for acclimatization to their new environment. The rats were weighed to ensure that none of them was outside their initial weight of 100–150g was lost. The entry point weight range was chosen to ensure that all the rats were matured enough for the laboratory experiment. The rats were fed with commercially available standard rat feed (Ladokun feeds Nig. Ltd) purchased from a commercial branch depot in Ondo city, Ondo state. They were given water ad libitum during the period of acclimatization. They were cared for and maintained in the animal facility in accordance with regulations, guidelines and policies governing the use of animals in research as described in public health service policy on human care and use of laboratory animals and approved by the University of Medical Sciences Animal Research Ethics Committee (UAREC) with approval number/ethical consent code UNIMED-AREC/Apv/2023/029. The rats were divided into 5 groups of 6 animals each and pre-treated as stated below: Group 1 (Control, n = 6): pre-treated with distilled water for 14 days Group 2 (Indomethacin, n = 6): pretreated with distilled water for 14 days and thereafter administered with single dose of indomethacin (40 mg/kg) . Group 3 (50 % yoghurt, n = 6): pretreated with 50 % diluted yogurt for 14 days and thereafter administered with single dose of indomethacin (40 mg/kg). Group 4 (100 % yogurt, n = 6): pretreated with 100 % yogurt for 14 days and thereafter administered with single dose of indomethacin (40 mg/kg). Group 5 (Omeprazole, n = 6): pretreated with omeprazole (20 mg/kg) for 14 days and thereafter administered with single dose of indomethacin (40 mg/kg). Gastric ulceration was induced in the animals according to the procedure described by Akpamu et al., 2013. Rats were administered with a single oral dose of indomethacin (40 mg/kg body weight). They were deprived of food but had free access to water 24 h prior to ulcer induction. Various degrees of ulceration have manifested 4 h after indomethacin administration. After the period of the experimental procedures, the animals were euthanized by cervical dislocation after being fasted for 24 h with free access to water. Retro-orbital blood collection was done and kept in plain sample bottles to obtain serum for anti-inflammatory assays and the stomach was carefully dissected out. The stomach was thereafter divided into two, a part for histological evaluation and the other part for biochemical assays. It is a complex process that involves evaluating the size, depth, tissue type, presence of necrosis, exudate, and surrounding skin condition of the ulcer, among other factors. The USI is based on several factors that are known to affect the severity and healing of gastric ulcers. These factors include the size of the ulcer, the depth of the ulcer, the presence of necrotic tissue, the type of tissue in the wound bed, the presence of exudate or drainage, and the surrounding skin condition. Each factor is assigned a score based on its severity, and the scores are added together to give an overall USI score. The USI scoring system is as follows: • Size: Measure the length and width of the ulcer in centimeters. Multiply the length by the width to get the ulcer area. Assign a score based on the area of the ulcer: ✓ Less than 1 cm: 1 point ✓ 1–5 cm: 2 points ✓ 6–10 cm: 3 points ✓ More than 10 cm: 4 points • Depth: Determine the depth of the ulcer. Assign a score based on the depth of the ulcer: Once each category is evaluated and scored, the individual scores are added up to determine the total ulcer score. A higher total score indicates a more severe gastric ulcer . A part of the stomach tissue was kept in 10 % buffered formalin and thereafter embedded in paraffin. The sections (5 μm) were and stained with routine Hematoxylin and Eosin (H&E) protocol. The slides were observed under the light microscope and a semiquantitative scoring was done by modifying the scoring system of Turkyilmaz et al., 2019. The histological changes that occurred at the gastric mucosa were scored by observing the following; loss and architectural disruption in crypts, edema, vascular congestion and infiltration of polymorphonuclear cells and lymphocytes modified using a grading of normal, mild, moderate and severe and was given a score of 0, 1, 2 and 3 respectively. Determination of Malondialdehyde (MDA), Superoxide dismutase (SOD), Catalase and Glutathione peroxidase in stomach tissues. The stomach tissues prepared for biochemical assays were homogenized in Tris KCl buffer (pH 7.4) and thereafter centrifuged to obtain a clear homogenate. Lipid peroxidation assay was done by determining Malondialdehyde (MDA) activity according to the method of Varshney and Kale . This method is based on the reaction between 2-thiobarbituric acid and MDA resulting in a pink solution which can be read at absorbance of 532 nm. Catalase (CAT) activity was performed according to the method of Sinha, 1971. The method is based on the reduction of dichromate in acetic acid to chromic acetate when heated in the presence of H 2 O 2 . The end product gives a deep green color which is read at 560 nm. Superoxide dismutase (SOD) activity was measured using the method described by Misra and Fridovich . The method is based on the ability of SOD to inhibit the auto-oxidation of epinephrine at pH 10.2. Total antioxidant capacity was determined according to the method described by Rubio et al., 2016. The method employed the use of ferric reducing antioxidant power (FRAP) assay and the principle is based on the reduction of ferric-tripyridyltriazine complex (Fe 3+ -TPTZ) reagent to ferrous tripyridyltriazine (Fe 2+ -TPTZ) by the antioxidants present in the sample at low pH. Ferrous tripyridyltriazine (Fe 2+ -TPTZ) has a blue color and can be read at 593 nm. Tumor Necrosis Factor-alpha (TNF-α), Interleukin 10 (IL-10) and Interleukin 1 Beta (IL-1β) were quantified using enzyme-linked immunosorbent assay (ELISA) kits purchased from Elabscience, Houston, Texas, United state and the manufacturer's instruction followed appropriately. The control group showed normal mucosa with no visible lesion. The administration of Indomethacin resulted in severe ulcer in indomethacin group compared resulting in a very high ulcer scoring index the control group. The ulcer scoring index was significantly decreased in both Yoghurt groups compared to indomethacin group as the both showed mild ulceration . Histological sections revealed normal architecture of the crypts in the control group from the mucosa to the serosa layers. However, severe crypt disruption, edema and infiltration of inflammatory aggregates were observed in indomethacin (40 mg/kg) group following indomethacin administration. These changes were mild in yoghurt groups and moderate in omeprazole group as shown in Fig. 2 . Fig. 3 furthermore showed an increase in histology score in indomethacin (40 mg/kg) group compared to control group. This score was significantly reduced in both yoghurt groups and omeprazole group. Fig. 1 Effect of Yoghurt on gross morphology and ulcer score A: Control; B: Indomethacin; C: 50 % Yoghurt; D: 100 % Yoghurt; E: Omeprazole (40 mg/kg). Values are expressed as Mean ± SEM (n = 6). ∗significant when compared to Control. # significant when compared to Indomethacin (40 mg/kg). Fig. 1 Fig. 2 Photomicrograph of stomach for respective groups X100. A (Control) All tunics appear intact and normal. Submucosa (SM) and Mucosa (M). B (Indomethacin 40 mg/kg) Severe architectural disruption of crypt. Severe inflammation and infiltration of inflammatory aggregates up to the submucosa (Black arrow). Edema in the submucosa (Red arrow) and vascular congestion (green arrow). C (50 % Yoghurt) Moderate erosion of epithelium of mucosa (yellow spanning arrow). Other tunics appear normal. Mild infiltration of inflammatory aggregates at the mucosa layer (black arrow). D (100 % Yoghurt) Mild erosion of epithelium of mucosa (yellow spanning arrow). Other tunics appear normal. Mild infiltration of inflammatory aggregates at the mucosa layer (black arrow). E (Omeprazole 20 mg/kg) Mild erosion of epithelium of mucosa (yellow spanning arrow) and vascular congestion (green arrow). Other tunics appear normal. Mild infiltration of inflammatory aggregates at the mucosa layer (black arrow). Fig. 2 Fig. 3 Effect of Yoghurt on Histological score. Values are expressed as Mean ± SEM (n = 6). ∗significant when compared to Control. # significant when compared to Indomethacin (40 mg/kg). Fig. 3 Effect of yoghurt on biochemical parameters is depicted in Fig. 4 . Malondialdehyde (MDA) was significantly increased in indomethacin grouped compared to control group. Treatment with yoghurt and omeprazole significantly reduce MDA compared to indomethacin group. Superoxide dismutase (SOD), Catalase (CAT) and Glutathione peroxidase (GPx) where all significantly decreased after indomethacin administration in the indomethacin group compared to control. Yoghurt treatment as well as omeprazole induction significantly reverse CAT and GPx but not SOD. Fig. 4 Effect of Yoghurt on biochemical assays in indomethacin induced gastric ulceration. A: Malondialdehyde (MDA); B: Superoxide dismutase (SOD); C: Catalase (CAT); D: Glutathione Peroxidase. Values are expressed as Mean ± SEM (n = 6). ∗significant when compared to Control. # significant when compared to Indomethacin (40 mg/kg). Fig. 4 Inflammatory markers are shown in Fig. 5 . Interleukin 1- beta, interleukin 10 and tumor necrosis factor alpha were all significantly increased in indomethacin (40 mg/kg) group compared to control group. Yoghurt groups significantly decreased these parameters compared to indomethacin (40 mg/kg) group. Furthermore, total antioxidant capacity was significantly higher in 100 % yoghurt group and control group compared to indomethacin (40 mg/kg) group. Omeprazole (20 mg/kg) and 50 % yoghurt groups showed no significant difference compared to indomethacin (40 mg/kg) group. Fig. 5 Effect of Yoghurt on inflammatory markers in indomethacin induced gastric ulceration. A: Total antioxidant capacity; B: Interleukin 1β; C: Tumor necrosis factor-α; D: Interleukin 10). Values are expressed as Mean ± SEM (n = 6). ∗significant when compared to Control. # significant when compared to Indomethacin (40 mg/kg). Fig. 5 Our study has shown that consumption of pasteurize yoghurt for 2 weeks was able to boost the immune system by decreasing inflammatory markers and decrease oxidative stress via enhancement of antioxidant enzymes in the stomach against indomethacin induced gastric ulceration. Peptic ulcer is the most prevalent gastrointestinal disease and has long been reported to occur when the gastro-aggressive factors overwhelm the gastroprotective factors . Administration of indomethacin successfully induce gastric mucosa lesion evident in gross observation of the stomach and high ulcer index of the indomethacin group. Pre-treatment with both concentrations of yogurt (50 and 100 %) resulted in a remarkable decrease in gastric mucosa damage and ulcer index that is comparable to the standard drug administered (omeprazole). Previous report has shown similar effect of yogurt in acetic acid-induced gastric ulcer where yogurt containing lactobacillus gasseri promote gastric ulcer healing . Also according to Momoh et al. , it was observed that administration of the yogurt caused cell infiltrations of the stomach wall, distinct ductile without aberrant superior or inferior diverticular of the stomach interlobular lining and well-formed stomach blood vessels. Histological observations from this study also showed deep ulcer and severe inflammation as a result of increase infiltration of inflammatory cells into the stomach. Yogurt exert cytoprotective effect on indomethacin induced gastric ulcer by decreasing gastric mucosa erosion and reducing infiltration of inflammatory aggregates. This action was more effective in the 100 % yogurt compared to the other treated groups. Yogurt has been reported to have cytoprotective effects on the gastric gland against HCl-induced acute gastric lesions and this action was exerted via their ability to promote prostaglandin production . Prostaglandins are important in the stomach to enhance blood flow and for the production of mucus and bicarbonates. Indomethacin is a potent cyclooxygenase blocker thus; the administration of indomethacin will inhibit COX which is the enzyme that catalyzes the production of prostaglandin. This will lead to decrease in prostaglandin which in turn will lead to reduced gastric mucosa blood flow, mucous and bicarbonate production as well as increase acid production thereby promoting ulcer formation . Consequently, the gastroprotective action of yogurt observe in this study can be linked to its ability to promote prostaglandin production. Oxidative stress occurs as a result of imbalance between oxidants (free radicals) and antioxidant enzymes in favor of the former, which then causes damage to tissues. It is usually associated with indomethacin induced gastric ulcer model . One major end product of lipid peroxidation is malondialdehyde (MDA) which has been widely used as a marker of oxidative stress in several disease conditions such as cardiovascular, gastrointestinal and neurodenerative disease . In this study, the level of MDA was significantly higher in the indomethacin group compared to control indicating that the administration of indomethacin resulted in lipid peroxidation and this is consistent with other reports . There was a corresponding decrease in antioxidant enzymes; superoxide dismutase-SOD, catalase- CAT and glutathione peroxidase- GPx. Yoghurt pretreatment resulted in a significant restoration of MDA level to control value and increase in the antioxidant enzymes which suggest that yoghurt has antioxidant properties. It has been reported by several studies that probiotics have direct antioxidative activities and can also modify the immune system . The effect of yoghurt on MDA could be as a result of its probiotic properties. The lactic acid bacteria present in yogurt has direct ability to scavenge free radicals and also suppress their generation . Thus, preserving the body's antioxidants defense mechanism as noted in the significant increase in antioxidants enzymes compared to indomethacin group. Superoxide dismutase (SOD) which serve as first line defense against reactive oxygen species by converting the highly reavtive O2- to a less reavtive H2O2 which is further mopped up by the actions of catalase (CAT) and glutathione peroxidase- (GPx). Furthermore, the total antioxidant capacity which measures the activities of the non-enzymatic component of the antioxidant defense mechanism was also enhanced in the yogurt treated groups. The infiltration of inflammatory aggregates such as macrophages as well as the generation of reactive oxygen species have been reported to activate pro inflammatory cytokines especially tumor necrosis factor alpha (TNF-α) and Interleukin 1 beta (IL1β) . These inflammatory cytokines are closely associated with gastric ulcer indicating the severity of the disease condition. TNF-α and IL1β production are very high at the inflammatory phase of gastric ulcer which usually decreases as healing progress . Interleukin 10 is usually considered as an anti-inflammatory cytokine. It was initially noted to be a product of the T helper 2 cells which inhibits the activation of T helper 1 cell. However, it is now known to be produced by several immune cells . Interleukin 10 regulates immune responses and maintains immune homeostasis and its reduction indicates a decrease in inflammatory process . In conclusion, this study has observed the effect of pasteurized yogurt on gastric ulcer by assessing oxidative stress parameters, pro- and anti-inflammatory cytokines as well as histological appearance. The study demonstrated that pasteurized yogurt exerts anti-oxidative and anti-inflammatory effect in indomethacin-induced ulcerated male Wistar rats, as indicated by the decrease in IL-10, IL-1B, TNF-a, and increase in TAC level and a significant difference in the anti-oxidative parameters. These findings support the potential of pasteurized yogurt as a dietary intervention for oxidative and inflammation-associated conditions. | Other | biomedical | en | 0.999997 |
PMC11698932 | Ticks (Parasitiformes: Ixodida) are obligate hematophagous ectoparasites of all terrestrial vertebrate classes and are vectors or reservoirs of human and animal pathogenic virus, bacteria, protozoa and fungi . Two tick families encompassing numerous genera hold considerable significance in public health and veterinary matters . Currently, there are 996 known tick species worldwide, divided into three families: approximately 774 hard tick species (Ixodidae), around 221 soft tick species (Argasidae), and the monotypic family Nuttalliellidae, which exhibits features of both families . The identification of tick fossils in amber has led to the description of two novel families: Deinocrotonidae and Khimairidae, alongside several unique genera, including Compluriscututla , Cornupalpatum , Deinocroton , and Khimaira . Most protozoan pathogens carried by ticks that affect mammals of medical or veterinary interest are classified under the order Piroplasmida (e.g., Babesia , Theileria , and Cytauxzoon ) or as haemogregarines within the Hepatozoon genus (Adeleorina: Hepatozoidae) . The Trypanosomatidae family consists of unicellular eukaryotes, including pathogens of humans and animals from the genera Trypanosoma and Leishmania , as well as Endotrypanum and Porcisia . The life cycle of these organisms is primarily characterized by the involvement of arthropod vectors from the Hemiptera and Diptera orders, with some trypanosomes involving vectors from the Siphonoptera order, and fish trypanosomes from the Arhynchobdellida (Hirudinidae, leech) order. Two subspecies of Trypanosoma brucei (i.e., Trypanosoma brucei gambiense and T. brucei rhodesiense ), along with T. cruzi and T. rangeli , as well as at least 23 species of Leishmania , including the recently described Leishmania (Mundinia) chancei, are pathogenic to humans. These pathogens cause human African trypanosomiasis (sleeping sickness), Chagas disease, and cutaneous, mucocutaneous, or visceral leishmaniasis [ , , , , , , ]. These diseases also affect domestic, feral, and wild animals. Canine visceral leishmaniasis is primarily caused by L. infantum infection, with occasional cases attributed to L. donovani or L. tropica . Trypanosoma congolense , T. evansi, T. b. brucei , T. vivax , T. simiae , T. suis , T. theileri , and, more rarely, T. godfreyi infect livestock. Trypanosoma equiperdum infects equids during mating . Additionally, Trypanosoma theileri , considered non-pathogenic to humans, is found in cattle, buffalo, and antelope worldwide . It has been infrequently linked to disease resembling nagana in specific cases involving a calf , cattle , or a cow , and it can cause illness in cattle under severe stress due to concurrent diseases or poor nutrition . Trypanosoma caninum was described in 2014 as a new species infecting dogs, typically in asymptomatic cases with low humoral immune responses . Furthermore, T. lewisi , a parasite of Rattus , is also an opportunistic parasite in humans and shares common vertebrate hosts with T. cruzi . Altogether, more than 30 million people are infected, and over 48 million cattle are at risk of contracting animal trypanosomiasis in Africa, resulting in approximately 3 million cattle deaths annually . Since the early 20th century, the transmission of protozoan parasites from Leishmania and Trypanosoma has been investigated, and more recently, molecular biology techniques (e.g., next-generation sequencing) have been employed to revisit this hypothesis . There remains an ongoing debate within the scientific community regarding the ability of ticks to transmit Trypanosomatidae parasites of medical or veterinary interest. To provide an updated perspective on this debate, we conducted a systematic review and meta-analysis with the following objectives: (i) to gather published field and experimental data on the detection of Trypanosomatidae with medical and veterinary relevance in ticks, including T. theileri , T. lewisi , and T. caninum , and to assess their capacity to act as vectors for these pathogens; and (ii) to explore factors associated with the transmission of these pathogens by ticks. A set of keywords was used: “Trypanosomatidae” and all species of “ Trypanosoma ” or “ Leishmania ”, known to be pathogenic for humans or animals and having medical or veterinary interest, including T. theleiri , T. lewisi and T. caninum , in combination with “ticks” “Ixodidae”, “Argasidae”, and selected tick genus (e.g., “ Rhipicephalus ”, “ Hyalomma ”, “ Ornithodoros ”). Database search was done between January 1900 and June 30 , 2023, without language restrictions. Articles reporting “Trypanosomatidae” in ticks by direct examination (e.g., culture, microscopy, immunohistochemistry “IHC”) or molecular method (e.g., conventional Polymerase chain reaction “PCR”, real-time PCR “RT-PCR”, capillary or next generation sequencing “NGS”) were included in the study. We exclude studies not in the scope of our study and reporting, i.e., those whose scope and objective are “Vaccine”, “Virus”, “Drug”, “Treat”, “Pest”, “Acaricide”, “Immunology”, “Serology”, “Serum”, “ELISA”, “Antigen”. We conducted the review in accordance with the current recommendations established in 2015 and reported our findings following the PRISMA guidelines, specifically addressing the remarks for “biological” meta-analyses . Two authors independently performed a preliminary review of the articles by examining their titles and abstracts. Articles selected by at least one reviewer were retrieved and duplicated (same bibliographical record) papers were excluded. The two reviewers then performed a second selection based on full-text analysis, resolving any disagreements through discussion with a third reviewer. Studies were considered eligible based on established inclusion or exclusion criteria. The selection of eligible articles was based on the following criteria: (1) articles addressing with the detection or transmission of Trypanosomatidae parasites of medical or veterinarian interest, including T. theleiri , T. lewisi and T. caninum , in ticks; (2) field or experimental studies; (3) no restrictions on host origin; and (4) no language restriction. The exclusion criteria included (1) certain literature categories (letters, books, and reviews); (2) studies that did not focus on ticks as vectors for Trypanosomatidae (e.g., cellular immune responses of ticks, host immunogenetic influences on tick resistance); (3) studies that did not focus on Trypanosomatidae of medical or veterinary interest; (4) studies lacking information on tick collection locations and origins; and (5) studies that did not specify the identity of tick and Trypanosomatidae species (6) studies not stating the sample size and the number of positive cases where excluded from the meta analysis. The quality of the selected publications was assessed using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) methodology . The total score for each article was calculated based on the following seven criteria: (1) the tick family and species provided; (2) the identified Trypanosomatidae species; (3) the detection method used; (4) the number of tested ticks reported; (5) the number of positive ticks reported; (6) information on the prevalence of Trypanosomatidae parasites in ticks; and (7) the identity of the host from which the positive tick(s) originated. Items 1–5 were scored at 2 points each, while items 6 and 7 were scored at 1 point each. Based on the total score, each publication was classified as high quality (score = 8–12), medium quality (score = 5–7), or poor quality (score = 0–4). For data related to field studies, we conducted a meta-analysis of proportions using the ‘meta’ and ‘metafor’ packages in R software version 4.3.1. A Freeman-Tukey transformation with double arc sine (PFT) was applied to convert the proportions before meta-analysis. This transformation standardizes and stabilizes the distribution variance (dat<-escalc(measure="PFT", xi=xi, ni=ni, data=dat). Due to the high heterogeneity expected in the meta-analysis, a random-effects model was employed to combine the overall effect size perform and subgroup analyses. Cochrane statistics I 2 and Q (expressed as X 2 and P , respectively) were used to assess and quantify heterogeneity. An I 2 value < 50 % indicates low heterogeneity, whereas I 2 value > 50 % signifies high heterogeneity. Meta-analysis statistics were visualized using Forest plots. The Egger test and Funnel plots were utilized to assess publication bias, and results underwent stability analysis, which evalues the impact of excluding data from any single article on the results of the remaining studies. We also carried out subgroup analyses of potential risk factors, including sampling year , continent (Europe, Asia, Africa, South America, and Iceland), host family (Canidae, Bovidae, etc.), tick genus, tick species, detection methods, parasite type, and the location in the tick's organs. Additionally we performed a meta-regression using the studied parameters as covariates to address possible sources of heterogeneity. Regarding the experimental studies, many papers lack quantitative data, such as the number of ticks used in the research. To address this, we performed a meta-analysis using semi-quantitative data, specifically the detection or absence of pathogens in ticks (0: absence, 1: presence). Through this methodology, we analyzed the detection of pathogens in ticks after blood feeding, the transmission of Trypanosomatidae to uninfected hosts during blood feeding, infection following the injection of infected tick materials, vertical transmission, and transstadial passage. Statistical tests were conducted, and subgroup analyses of associated factors were performed based on tick family, tick species, donor host, receiving host, and parasite family and species. A total of 29,592 articles were retrieved during the systematic search on multiple databases, including PubMed, Google Scholar, and WOS, . After the removal of duplicate paper (n = 21,007) and ineligible papers (n = 8,423), 166 published studies were selected . After analysis of their titles, abstracts, and detailed contents, out of those 166 articles, 94 satisfied the eligibility criteria to be included in the systematic review (84 in English and 10 in other languages i. e Portuguese, Russian …), 86 articles were eligible for the meta-analysis, of which 49 dealt with field studies and 37 with experimental studies. In addition, 46 focused on the transmission of Leishmania , while 40 are focused on Trypanosoma . Full-text Portable Document Format (PDF) files not freely accessible online were obtained through the French Development Research Institute (IRD) library. Publications in French, German, Spanish, Portuguese, Russian, and Turkish languages were handled by authors and/or native language colleagues. Fig. 1 PRISMA flowchart illustrating the process of the systematic review and meta-analysis. Fig. 1 The phylum Arthropoda hosts both monoxenous and dixenous Trypanosomatidae . Sand flies (Diptera; Phlebotominae) are known biological vectors of Leishmania , with some exceptions . Other biological vectors of Trypanosoma include Diptera (e.g., tsetse flies, tabanids, and Stomoxys ), Hemiptera (e.g., triatomine ‘reduviid’ bugs), and Siphonaptera (e.g., fleas) . Although most Trypanosomatidae colonize members of the class Insecta, presumably monoxenous trypanosomatids also develop in ticks (class Arachnida) [ , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ]. Since the late 19th and early 20th centuries, field trials and experiments have been conducted to detect these parasites in ticks . The ability of ticks to transmit Leishmania parasites has been documented for quite some time . In recent years, advancements in molecular detection methods for Trypanosomatidae DNA in ticks have renewed interest in the role of ticks in the transmission of both Trypanosoma and Leishmania . Specifically, since 2010, the number of studies investigating this topic has increased significantly. Fig. 2 Temporal distribution of collected and selected scientific papers. Fig. 2 The hypothesis that ticks may act as reservoirs and/or vectors for human and animal trypanosomes has been investigated since the early 20th century [ , , , , , , , , , , , , , , ]. In 1972, Hoare reviewed existing data on ticks as potential vectors and concluded that ticks could not transmit trypanosomes due to the absence of infective trypomastigote forms . However, subsequent research documented the presence of various trypanosome developmental stages in ticks, including amastigotes, sphaeromastigotes, epimastigotes, and trypomastigotes, suggesting a more complex relationship between ticks and trypanosomes . The hypothesis that ticks may contribute to the transmission of Leishmania emerged in the 20th century. Early experiments suggested that ticks could act as vectors for Leishmania species responsible for human and canine kala-azar ( L. donovani/L. infantum complex) in the Mediterranean region. The observation of Leishmania surviving for extended periods in the tick gut raised questions about the possibility of direct transmission . However, this hypothesis has been challenged, with conflicting evidence regarding the involvement of ticks in the L. infantum transmission cycle . In the mid-1980s, further doubt was cast on this theory, although one study demonstrated transmission during a tick's blood meal . Subsequent field and laboratory studies have detected Leishmania RNA or DNA in various tick organs, including salivary glands and ovaries, across different developmental stages (larvae, nymphs, and adults), providing additional support for the potential role of ticks in Leishmania transmission [ , , , , , , , , , , , , , , , , , , , , , , , , ]. As detailed in the following sections, numerous field studies have been conducted to investigate the prevalence of pathogenic Trypanosomatidae in ticks. Detection of the parasite has been achieved through various methods, including direct examination (e.g., culture, microscopy), molecular tools such as Polymerase Chain Reaction (PCR), quantitative PCR (qPCR), capillary sequencing, and next-generation sequencing, as well as immunological techniques like immunochromatography. Traces of Trypanosoma species of medical and veterinary importance have been reported in ticks, including T. cruzi , T. vivax , T. evansi , T. theileri , T. theileri -like, T. congolense , and T. caninum [ 49 , , , , , , , , , , , , , , ]. All these studies focused on hard tick species (Ixodidae). Most of the positive tick species belonged to four genera: Amblyomma , Hyalomma , Ixodes , and Rhipicephalus , including A. cajennense , A. longirostrum , A. variegatum , H. detritum , H. marginatum , I. ricinus , R. (Boophilus) microplus , R. (Boophilus) spp. , R. sanguineus , R. sanguineus s.l. , and Rhipicephalus sp . Conversely, other species tested negative for Trypanosoma spp. ( Table 1 ) [ 89 , 90 , , , , , ]. Ticks collected from dogs and cattle were more frequently infected by Trypanosoma compared to those collected from other domestic animals (e.g., sheep, goats, camels) or wild animals (e.g., foxes, boars). • Trypanosoma cruzi Table 1 Detection of Trypanosoma species of medical and veterinary importance in field-collected ticks. Table 1 Species Sampling year/ Publication year§ Trypansoma species Tick infection status (P/N)& Localization in the tick Detection method Host Country Continent Refs A. cajennense 2013 T. cruzi N H-Gr Microscopy, PCR Canis lupus Brazil South America 1905 T. vivax N H Microscopy Bos taurus Cuba South America 1905 T. vivax P G Microscopy Bos taurus Cuba South America 2021 T. vivax P Gr PCR, Sequencing buffalos Brazil South America A. longirostrum 1941 T. cruzi P NS NS Cercolabidae ( Arboreal porcupine ) Venezuela South America Amblyomma spp. 2013–2019 T. cruzi N Gr Metagenomics ∗ Wild ungulates Carnivores Kenya Africa A. tigrinum 2013 T. cruzi N Gr PCR, Sequencing Canis lupus Chile South America A. variegatum 2016 T. congolense P Gr Parasitological analysis, PCR Cattle Nigeria Africa Haemaphysalis spp. 2017–2018 T. evansi N Gr PCR, Sequencing Cattle India Asia H. detritum 1982 T. theileri P H Microscopy Bos taurus ; Canis lupus Algeria Africa H. dromedarii 2015–2016 T. evansi N Gr PCR ∗∗ Wild ruminants Tunisia Africa H. excavatum 2015–2016 T. evansi N Gr PCR ∗∗ Wild ruminants Tunisia Africa 1982 T. theileri; T. evansi N H Microscopy Bos taurus ; Canis lupus Algeria Africa H. lusitanicum 1982 T. theileri; T. evansi N H Microscopy Bos taurus ; Canis lupus Algeria Africa H. marginatum 2015–2016 T. evansi N Gr PCR ∗∗ Wild ruminants Tunisia Africa 1990 T. theileri P H Microscopy Bos taurus Portugal Europe 1982 T. theileri; T. evansi N H Microscopy Bos taurus ; Canis lupus Algeria Africa Hyalomma spp. 2017–2018 T. evansi N Gr PCR, Sequencing Cattle India Asia I. ricinus 2013 T. caninum P Gr Culture, PCR, Sequencing Vegetation Slovakia Europe 1979–1982 T. theileri P H Microscopy Bos taurus ; Canis lupus Switzerland Europe 2015–2016 T. evansi N Gr PCR ∗∗ Wild ruminants Tunisia Africa R. (Boophilus) microplus NS T. theileri-like P H ND Bos taurus Brazil South America 1905 T. vivax N H Microscopy Bos taurus Cuba South America 1905 T. vivax P G Microscopy Bos taurus Cuba South America NS T. vivax P Gr PCR Bos taurus Venezuela South America 2021 T. vivax P Gr PCR, Sequencing buffalo Brazil South America R. (Boophilus) spp. 2017–2018 T. evansi P Gr PCR, Sequencing Cattle India Asia R. bursa 2015–2016 T. evansi N Gr PCR ∗∗ Wild ruminants Tunisia Africa R. sanguineus 2013 T. cruzi N H-Gr Microscopy, PCR Canis lupus Brazil South America 1982 T. evansi P H Microscopy Bos taurus , Canis lupus Algeria Africa 2013 T. evansi P Gr PCR Canis lupus Brazil South America 2013 T. vivax P Gr PCR Canis lupus Brazil South America R. sanguineus s.l 2013 T. cruzi P Gr PCR, Sequencing Canis lupus Chile South America 2015–2016 T. evansi P Gr PCR Wild ruminants Tunisia Africa Rhipicephalus spp. 2013–2019 T. cruzi P Gr Metagenomics ∗ ∗ ∗ Wild ungulates Carnivores Regular domestic Kenya Africa R. turanicus 1982 T. theileri; T. evansi N H Microscopy Bos taurus ; Canis lupus Algeria Africa §: When the sampling date was not specified in the publication, the year of publication was added in italics ; & information on positivity is available in the data collection sheet in the supplementary material ; P: positive; N: negative; F: faeces ; G: gut ; Gr: whole body ; H: hemolymph ; O: ovaries ; SG: salivary glands; PCR: conventional Polymerase chain reaction, Sequencing: Capillary sanger sequencing. ∗ Wild ungulates [ Black rhinoceros ( Diceros bicornis ), white rhinoceros ( Ceratotherium simum ), buffalo ( Syncerus caffer ), elephant ( Loxodonta africana ), giraffe ( Giraffa camelopardalis ), Grévy's zebra ( Equus grevyi ), plains zebra ( Equus quagga ), hartebeest ( Alcelaphus buselaphus ), impala ( Aepyceros melampus ) ] ; Carnivores [ leopard ( Panthera pardus ), lion ( Panthera leo ), spotted hyena ( Crocuta crocuta ), wild dog ( Lycaon pictus ) ] ; Regular domestic [ farmed Boran ( Bos indicus ) and cattle ( Bos taurus ) ]. ∗∗Wild ruminants: Scimitar-horned oryx; Addax antelope; Barbary red deer; Dorcas gazelle. ∗∗∗ Wild ungulates [ plains zebra ( Equus quagga ) ] ; Carnivores [ lion ( Panthera leo ), spotted hyena ( Crocuta crocuta ) ] . Since the 1940s, the prevalence of T. cruzi in field-collected ticks has remained uncertain. The presence of T. cruzi in A. longirostrum , an ectoparasite of porcupines, was documented during a survey of ectoparasites conducted in the state of Yaracuy, Venezuela . Between 2013 and 2015, ticks collected from 148 dogs in the urban area of Campo Grande, Mato Grosso do Sul, Brazil, revealed that R. sanguineus and A. cajennense were not infected by T. cruzi . More recently, the presence of T. cruzi was documented in dogs and their ectoparasites in a rural area of central Chile, where 57 % of blood samples were infected by T. cruzi , and 5.4 % of ticks tested positive by PCR. Specifically, R. sanguineus s.l. (5/82) was positive, while all A. tigrinum specimens (0/11) were negative . The same year, a study focused on using ticks as xenosurveillance sentinels to monitor circulating pathogens in the Kenyan drylands. Ticks were collected from wild ungulates, carnivores, domestic animals, and Boran cattle and screened using metagenomics. T. cruzi DNA was detected in 3 out of 46 (6.5 %) pools of Rhipicephalus spp., but not in Amblyomma spp . However, the examination of the sequences could not definitively confirm the presence of T. cruzi . • Trypanosoma vivax Trypanosoma vivax infects wild and domestic ungulates and is transmitted mechanically via tabanids and other blood-sucking insects in the Americas . An analysis of the gut contents of A. cajennense (Fabricius) sensu stricto (s.s.) and R. (Boophilus) microplus (Canestrini) (Acari: Ixodidae), collected from cattle ( Bos taurus ) on two farms in Cuba, revealed the presence of living T. vivax forms 96 h after repletion, though not in the hemolymph . Of the 285 R. (Boophilus) microplus ticks collected from cattle in livestock areas of Merida, Venezuela, 7.7 % tested positive using PCR . That same year, an examination of 63 R. sanguineus ticks collected from dogs in Campo Grande, Brazil, revealed the presence of Trypanosoma spp. via PCR, with 15 (23.8 %) testing positive for T. vivax . Lastly, in a search for T. vivax in A. cajennense s.s. and R. (Boophilus) microplus collected from cattle, 6.25 % (3/48) of A. cajennense s.s. and 4.5 % (2/45) of R. (Boophilus) microplus were positive. The sequences obtained were 99 % identical to those of bovine T. vivax from northeastern Brazil . • Trypanosoma evansi Trypanosoma evansi is primarily transmitted by tabanid flies and Stomoxys spp. It affects a wide range of hosts, including livestock, camelids, equids, carnivores, rodents, and humans, producing variable clinical symptoms depending on the host . In a study on the seasonal dynamics of R. sanguineus in dogs in urban areas of western Algeria, hemolymph smears were examined for potential microorganisms. Five out of 250 R. sanguineus ticks (four females and one male) were found to be infected with trypanosomes, representing 2 % of the total. Two infected females showed pathological reactions (globular bodies and milky hemolymph) related to the infection. Based on the observed form and small size of the protozoan, it was hypothesized to be T. evansi (referred to as T. berberum in the manuscript), a parasite of camelids that causes deadly trypanosomiasis in dogs . This hypothesis was further supported by a report that 15 out of 63 dogs in Campo Grande, Mato Grosso do Sul, Brazil, tested positive for T. vivax and 7 for T. evansi using PCR . In 2021, 0.2 % of the 352 ticks collected from the environment and wild ruminants in Tunisia were also reported positive for T. evansi by PCR . Finally, in India, the sequencing of 240 pooled tick-DNA samples collected from two regions revealed no prevalence of T. evansi in Karnataka but a prevalence of 8.3 % in Kerala . • Trypanosoma theileri and T. theileri-like Multiple studies have documented the presence of trypanosomes in tick hemolymph . Most of these studies identified T. theileri , which is typically transmitted by tabanid flies . T. theileri was first reported in field-collected ticks in a 1979 study conducted in Switzerland, where 0.19 % of I . ricinus were found to carry trypanosomes morphologically similar to T. theileri . Between 1979 and 1982, the presence of T. theileri in the hemolymph of ticks from Switzerland and Algeria was confirmed, with 0.24 % of H. detritum and I. ricinus ticks collected from bovines testing positive . In 2008, Martins reported the presence of Trypanosomatidae epimastigote forms in the hemolymph of the cattle tick R. (Boophilus) microplus in Rio Grande do Sul, Southern Brazil, with microscopic examination suggesting T. theileri . • Trypanosoma congolense Animal trypanosomosis is a complex disease caused by one or more species of pathogenic trypanosomes, including T. congolense . Clinical symptoms are characterized by intermittent fever, parasitemia, anemia, lymphadenopathy, jaundice, progressive emaciation, weakness, and reduced productivity . Trypanosomes transmitted by tsetse flies ( Glossina spp.) are responsible for animal African trypanosomosis in sub-Saharan Africa . In 2016, trypanosomes were detected in engorged adult A. variegatum collected from cattle in the Unguwan Rimi and Kaduna state areas of northwestern Nigeria. Of the 33 samples examined microscopically, 14 tested positive for T. congolense , representing a prevalence rate of 42.4 %. On the first day of parasitemia follow-up, 10 (30.3 %) samples were microscopically positive for T. congolense , while 23 (69.7 %) were negative . • Trypanosoma caninum Trypanosoma caninum , the most recently identified species within the Trypanosomatidae family, has been reported to infect dogs in Brazil . Since its description, 67 cases of natural infection in dogs have been documented in areas where canine visceral leishmaniasis is endemic . In Slovakia, a novel trypanosome was isolated and partially characterized from I. ricinus . Sequence analysis suggests that this trypanosome, referred to as Trypanosoma sp. Bratislava1, is a new species closely related to several trypanosomes isolated from or detected in ticks in South America and Asia, including T. caninum from Brazil the most recent species within the Trypanosomatidae family, was reported to infect dogs in Brazil . Despite its recent identification, 67 cases of natural infection in dogs have been documented in areas where canine visceral leishmaniosis is endemic . In Slovakia, a novel trypanosome was isolated and partially characterized from I. ricinus . The resulting sequences support this trypanosome, referred as Trypanosoma sp. Bratislava1, as a new species closely related to several trypanosomes isolated from, or detected in, ticks in South America and Asia, including T. caninum isolated in Brazil . The presence of Leishmania in ticks is well documented, including species such as L. infantum/L. donovani , L. major , L. chagasi (syn. L. infantum ), L. braziliensis , L. guyanensis , and L. martiniquensis [ 64 , , , , , , , , , , , , , , , , 97 , , , , , , , , , ]. Most field studies focus on ticks from domestic animals in urban areas, particularly R. sanguineus [ 60 , 64 , , , , 70 , 71 , 75 , , , , 87 , , , , , 108 , 109 , 111 ]. Ticks collected from more wild ecosystems have highlighted the diversity of tick species positive for the presence of Leishmania , including A. sabanerae , Amblyomma spp. , A. tigrinum , A. variegatum , Hyalomma aegyptium and H. dromedarii , Ixodes ricinus , I. spp. , I. ventalloi , R. (Boophilus) microplus , R. pusillus , R. sanguineus s.l., and R. turanicus . ( Table 2 ). • Leishmania infantum and L. chagasi (Syn L. infantum) Table 2 Detection of Leishmania species of medical and veterinary importance in field-collected ticks. Table 2 Species Sampling year/Publication year§ Leishmania species Tick status(P/N)& Localization in the tick Detection method In tick Host Detection method in the host Country Continent Ref A. cajennense 2013 L. chagasi (Syn L. infantum ) N H-Gr Microscopy, PCR Canis lupus ND Brazil South America A. ovale 2008 L. infantum N Gr qPCR Canis lupus IFAT Brazil South America A. sabanerae 2022 Leishmania sp. P Gr qPCR Pecari tajacu ; Chelonoidis denticulata ND Peru South America Amblyomma spp. 2012 L. guyanensis P Gr PCR; HRM-PCR Tapirus terrestris ; Pecari tajacu ND Peru South America A. tigrinum 2010–2013 Leishmania sp. P Gr PCR Pseudalopex griseus PCR, qPCR Argentina South America A. variegatum 2014–2015 L. martiniquensis P Gr HT-qPCR Cattle ND Guadeloupe Island D. marginatus 2007–2008 L. infantum N Gr PCR Wild boars ND Italy Europe 2007 L. infantum N Gr qPCR Canis lupus ELISA, PCR Italy Europe 2014 Leishmania sp. N Gr PCR Human ND Turkey Asia Ha. longicornis 2012 L. infantum N Gr PCR Sheep, cattle and dog ND China Asia Ha. parva 2014 Leishmania sp. N Gr PCR Human ND Turkey Asia Ha. punctata 2014 Leishmania sp. N Gr PCR Human ND Turkey Asia Ha. sulcata 2007–2008 L. infantum N Gr PCR Sheep and Goat ND Italy Europe 2014 Leishmania sp. N Gr PCR Human ND Turkey Asia H. aegyptium 2015–2018 L. infantum P Gr PCR; Sequencing Testudo graeca ; Camelus dromedarius ND Israel Asia 2014 Leishmania sp. N Gr PCR Human ND Turkey Asia H. dromedarii 2015–2018 L. infantum P Gr PCR, Sequencing Testudo graeca ; Camelus dromedarius ND Israel Asia H. excavatum 2014 Leishmania sp. N Gr PCR Human ND Turkey Asia H. marginatum 2007–2008 L. infantum N Gr PCR Cattle ND Italy Europe 2014 Leishmania sp. N Gr PCR Human ND Turkey Asia Hyalomma spp. 2014 Leishmania sp. N Gr PCR Human ND Turkey Asia I. ricinus 2007 L. infantum P Gr qPCR Canis lupus ELISA, PCR Italy Europe 2007–2008; 2010 L. infantum P Gr PCR Dog, horse, cat, bovine, human ND Italy Europe 2011 and 2013 L. infantum P Gr PCR Felis catus ND Italy Europe I. ricinus 2012–2013 L. infantum P Gr qPCR Felis catus PCR, qPCR Italy Europe 2010 L. infantum P Gr PCR; Sequencing $Flagging ND Italy Europe 2014 Leishmania sp. P Gr PCR Human ND Turkey Asia Ixodes spp. 2011 and 2013 L. infantum N Gr PCR Felis catus ND Italy Europe 2012–2013 L. infantum P Gr qPCR Felis catus PCR, qPCR Italy Europe I. ventalloi 2011 and 2013 L. infantum P Gr PCR Felis catus ND Italy Europe 2012–2013 L. infantum P Gr qPCR Felis catus PCR, qPCR Italy Europe R. (Boophilus) microplus 2012 L. guyanensis P Gr PCR; HRM-PCR Tapirus terrestris ; Pecari tajacu ND Peru South America 2012 L. infantum N Gr PCR Sheep, cattle and dogs ND China Asia Leishmania sp. P Gr qPCR Pecari tajacu; Chelonoidis denticulata ND Peru South America 2014–2015 L. martiniquensis P Gr HT-qPCR Cattle ND Guadeloupe Island 2014–2015 L. martiniquensis P Gr HT-qPCR Cattle ND Martinique Island R. bursa 2014 Leishmania sp. N Gr PCR Human ND Turkey Asia 2007–2008 L. infantum N Gr PCR Sheep, goat, cattle, horse, deer ND Italy Europe R. pusillus 2011 and 2013 L. infantum P Gr PCR Felis catus ND Italy Europe 2007–2008 L. infantum N Gr PCR Hedgehog ND Italy Europe 2012–2013 L. infantum P Gr qPCR Felis catus PCR, qPCR Italy Europe R. sanguineus 2012–2013 L. infantum P Gr qPCR Felis catus PCR, qPCR Italy Europe 2016–2017 L. major P Gr PCR, Sequencing Rhombomys opimus ; Nesokia indica ND Iran Asia 2013 L. chagasi (syn L. infantum ) P H-Gr Microscopy, PCR Canis lupus ND Brazil South America NS L. braziliensis P Gr PCR, qPCR Canis lupus ND Brazil South America 2007 L. infantum P Gr PCR; qPCR Canis lupus IFAT, PCR, qPCR Brazil South America 2007 L. infantum P Gr-SG PCR; qPCR; sequencing Canis lupus ND Italy Europe 2008 L. infantum P Gr-SG PCR; qPCR; sequencing Canis lupus ND Brazil South America 2007–2008 L. infantum P Gr PCR Canis lupus ELISA Brazil South America 2007–2008 L. infantum N Gr PCR Canis lupus ND Italy Europe 2008–2009 L. infantum P Gr PCR, RT-PCR, Sequencing Canis lupus ELISA, PCR Brazil South America R. sanguineus 2007 L. infantum P Gr qPCR Canis lupus ELISA, PCR Italy Europe 2006–2007 L. infantum P Gr qPCR Canis lupus ELISA, PCR Italy Europe 2011–2012 L. infantum N NS Infestation Canis lupus IFAT, ELISA Brazil South America 2013 L. infantum P Gr PCR, qPCR, seuqencing Canis lupus PCR, qPCR Brazil South America 2012 L. infantum P Gr PCR Canis lupus IFAT, ELISA Brazil South America 2013 L. infantum P Gr PCR, Sequencing Canis lupus PCR, Sequencing Brazil South America 2011 L. infantum P G PCR, RFLP, seuqencing, Parasit culture Canis lupus DPP, IFAT, ELISA, PCR Brazil South America 2011 L. infantum P SG PCR, RFLP, Sequencing, Parasit culture Canis lupus DPP, IFAT, ELISA, PCR Brazil South America 2011 and 2013 L. infantum P Gr PCR Felis catus ND Italy Europe 2002 Leishmania sp. P G Microscopy Canis lupus IFAT Brazil South America 2009 Leishmania sp. N NS present of tick or no Canis lupus IFAT Brazil South America 2012–2014 Leishmania sp. N Gr qPCR Canis lupus qPCR China Asia 2015 Leishmania sp. P G; O; SG IHC, qPCR, IHC Canis lupus ND Brazil South America 2016 Leishmania sp. P G; O; SG IHC, qPCR, IHC Canis lupus ND Brazil South America 1934 £L. kala azar P NS NS Human NS France Europe 2008 L. infantum N Gr qPCR Canis lupus IFAT Brazil South America R. sanguineus s.l. NS L. infantum P Gr PCR, Sequencing $Flagging & ∗Animals ND Israel Asia R. turanicus 2007–2008 L. infantum N Gr PCR Sheep and Goat ND Italy Europe 2014 Leishmania sp. N Gr PCR Human ND Turkey Asia 2022 L. infantum P Gr PCR, Sequencing $Flagging & ∗Animals ND Israel Asia §: When the sampling date was not specified in the publication, the year of publication was added in italics ; & information on positivity is available in the data collection sheet in the supplementary material ; NS : Not specified; P: positive; N: negative; F: faeces ; G: gut ; Gr: crushing ; H: hemolymph ; O: ovaries ; SG: salivary glands; PCR: conventional Polymerase chain reaction, RFLP: restriction fragment length polymorphism; qPCR: real time PCR; RT-PCR : reverse transcriptase PCR; HRM-PCR : High Resolution Melting PCR; HT-qPCR : High-throughput microfluidic qPCR; Sequencing: Capillary sanger sequencing, IFAT: Indirect Fluorescent Antibody Test; ELISA: Enzyme Linked Immunosorbent Assay; DPP: rapid immunochromatographic test; IHC : immunohistochemistry ∗ Animals: dogs ( Canis familiaris ); Tortoise ( Testudo graeca ); hedgehogs ( Erinaceus concolor ); badger. £ L. kala azar: refers to members of the L. donovani complex ( L. infantum and L. donovani ) without any other information on parasite typing at the time of the study. We therefore use the term proposed by the author. $ Flagging : refers to the use of a method to collect questing ticks. The hypothesis that ticks could serve as vectors for Leishmania was first proposed in the early 20th century . Domestic dogs play a central role as reservoirs of L. infantum in the peridomestic zoonotic transmission cycle. The brown dog tick, R. sanguineus , has been the subject of extensive research due to its prevalence among urban dogs . The overall prevalence of L. infantum in R. sanguineus collected from dogs or cats ranges from 2.5 % to 70.3 % in Brazil and Italy. Significant L. infantum presence has been recorded in various Rhipicephalus species, including 9.2 % of R. sanguineus s.l. ticks collected by flagging (questing ticks) and 23.5 % of R. turanicus collected from dogs, hedgehogs, and tortoises in Israel . Additionally, infections were reported in R. pusillus from cats in Italy, with 10.9 % and 17.6 % positivity. Studies have also detected L. infantum in I. ricinus (1.5 %–50 %) , I. ventalloi (19 % and 62 %) , Ixodes sp. (10.9 %) , H . aegyptium (38.7 %), and H. dromedarii (55.6 %) , collected from various hosts, including dogs, horses, cats, bovines, tortoises, camels, and humans in Italy and Israel. In most of these studies, L. infantum DNA was detected in whole ticks, and in some cases, specifically in the gut or salivary glands . Leishmania chagasi (syn. L. infantum ), which causes American visceral leishmaniasis, is widely distributed across Latin America . It is believed that L. infantum was introduced to South America by the conquistadors' dogs . In Campo Grande, Mato Grosso do Sul, Brazil, two tick species, R. sanguineus and A. cajennense , were collected from dogs in urban areas. Tick samples from 36 dogs tested positive for L. infantum , all of which were R. sanguineus . • Leishmania major American cutaneous leishmaniasis (ACL) is primarily caused by L. braziliensis . The challenges in controlling ACL may be linked to the disease's complex epidemiology, which involves various vector species, including ticks. Two rural areas in Pernambuco, northeastern Brazil, where ACL is endemic, were investigated for canine ectoparasites. Genomic DNA was extracted from 75 R . sanguineus ticks, 32 of which (42.67 %) tested positive for L. braziliensis using both conventional PCR and real-time PCR (RT-qPCR) . • Leishmania guyanensis Leishmania guyanensis is a causative agent of American tegumentary leishmaniasis. In 2017, DNA belonging to the Viannia subgenus was detected in 81 R . (Boophilus) microplus and Amblyomma ticks collected from three Tapirus terrestris and three Pecari tajacu in Madre de Dios, Peru, following amplification of kinetoplast DNA (kDNA) . Additionally, Leishmania (Viannia) kDNA was detected in three R. microplus ticks collected from a P. tajacu hunted in the forests of Madre de Dios. High-Resolution Melting PCR (HRM-PCR) identified one positive sample with a kDNA melting curve compatible with L. (V.) guyanensis . • Leishmania martiniquensis Leishmania martiniquensis was first isolated in 1995, its taxonomical classification was established in 2002, and it was officially named in 2014 [ 11 , , , ]. The vectors and reservoirs remain unidentified, although biting midges are suspected to be involved in the transmission of Leishmania species belonging to the Mundinia subgenus . In 2020, a high-throughput microfluidic real-time PCR system designed for genus-level parasite screening was applied to 132 adult specimens of Amblyomma variegatum and 446 specimens of R . microplus collected in Guadeloupe and Martinique. It was found that 0.7 % of the R. microplus ticks from Martinique tested positive for Leishmania spp., with sequences identifying L. martiniquensis . • Other/ unidentified Leishmania parasites In 2010, the presence of leishmania promastigote forms in ticks parasitizing domestic dogs was reported in the Municipality of São Vicente Férrer, located in the northern agreste of Pernambuco, Brazil . In 2015 and 2016, leishmania was detected in the intestines, ovaries, and salivary glands of R. sanguineus from dogs in Brazil, using both immunohistochemistry (IHC) and real-time PCR. IHC revealed Leishmania in 98 % of intestines, 14 % of ovaries, and 8 % of salivary glands, while real-time PCR confirmed these organs as the most positively tested sites . Additionally, Leishmania DNA, likely belonging to the L. donovani complex, was found in 11 out of 17 pools (64.7 %) of A . tigrinum ticks collected from eight foxes (six grey foxes, Pseudalopex griseus, and two culpeo foxes, P. culpaeus) in Argentine Patagonia . Although sandflies are typically found 2000 km and 750 km north of the study area, this finding suggests either a wider distribution than currently believed or the persistence of Leishmania in another vector. In 2022, 95.7 % of R. (Boophilus) microplus and 90 % of A. sabanerae collected from Pecari tajacu and Chelonoidis denticulata in leishmaniasis-endemic zones of the Peruvian Amazon were found to carry Leishmania , with parasite loads of 34.1 and 5428.6 per arthropod, respectively . In the early 20th century, laboratory experiments were conducted to assess the vector competence of ticks for transmitting trypanosomes. Several studies demonstrated the presence of various Trypanosoma or Leishmania forms—including trypomastigotes, amastigotes, epimastigotes, promastigotes, and sphaeromastigotes—in both ixodid and argasid ticks. Transmission routes were investigated, including transmission through tick bites and blood meals, as well as alternative routes such as ingestion of infected ticks, transstadial passage (between different life stages of ticks and their hosts), and transovarial transmission (ovarian infection). The role of ticks as vectors of trypanosomes has been increasingly supported by recent studies, which provide additional evidence of the transmission of distinct trypanosome clades to various mammalian species by ticks . Several reports document the detection of Trypanosoma in ticks after blood-feeding on an infected animal in experimental settings . Trypanosomes of medical and veterinary interest have been detected in both hard and soft ticks from three genera ( Rhipicephalus , Hyalomma , and Ornithodoros ) and four species: R. sanguineus , H. a . anatolicum , O. crossi , and O. lahorensis . Experimental infections following a bite or blood meal have been reported for T. cruzi , T. evansi , and T. theileri . • Trypanosoma cruzi The earliest reports of dog infection by T. cruzi -infected R. sanguineus ticks date back to 1913 . However, attempts to infect rats using infected soft ticks of the genus Ornithodoros were unsuccessful . Several other studies have also failed to demonstrate the transmission of T. cruzi by soft ticks in laboratory settings, including O. moubata , O. talaje , and O. turicata . • Trypanosoma evansi Investigations into the transmission of T . evansi by ticks began in the early 20th century. Some studies suggested that the soft tick Or . crossi , infected with T. evansi , could transmit the parasite to non-infected animals after a blood meal . However, in 1924, Yorke and Macfie did not confirm the transmission of this protozoan by O. crossi . Similarly, in 1976, Taylor-Lewis reported the unsuccessful transmission of T. evansi by H . dromedarii to rodents after blood feeding . More recently, Mahmoud et al. also failed to detect T. evansi DNA in uninfected rodents after blood feeding by infected O. savignyi . • Trypanosoma theileri and Trypanosoma theileri-like To date, sandflies (Phlebotominae) are recognized as the primary biological vectors of Leishmania , though the potential role of ticks, fleas, and leeches as secondary vectors has been suggested . Five laboratory studies have attempted to investigate transmission after collecting ticks from vertebrates experimentally infected with Leishmania . All experiments were conducted on hard ticks (Ixodidae), with only ticks from the Rhipicephalus genus involved. Ticks were fed on either dogs or rodents, and two species were used: R . turanicus and R. sanguineus . R hipicephalus . sanguineus was the only species shown to be infected by Leishmania , specifically L. infantum and Leishmania sp . • Leishmania infantum The transmission of L . infantum by ticks was first reported in 1930 by Blanc and Caminopétros, who infected squirrels after exposure to R . sanguineus ticks infected with what they referred to as ‘Leishmania kala azar,’ which most likely corresponds to parasites of the L. donovani complex today . However, this finding was not confirmed by later studies . In contrast, a report of successful transmission to 14 hamsters by R. sanguineus infected with L. infantum was documented , though this transmission method has not been further supported by subsequent research . Documented infection by trypanosomes following the ingestion of an infected invertebrate suggests that trypanosomes may also be transmitted through the ingestion of infected ticks . To date, nine studies on the Argasidae family and five on the Ixodidae family have investigated the transmission of trypanosomes in the laboratory, either through ingestion of infected ticks or by injection of infected material [ 41 , 42 , 45 , 47 , 48 , , , , , , 98 , 128 , 129 , 131 ]. Of these 14 studies, nine confirmed infections through the injection of infected tick material, while and only one confirmed infection through ingestion of infected ticks. However, drawing clear conclusions from these results remains challenging. Seven tick species were tested, O. moubata, O. crossi, O. venezuelensis, O. turicata, O. furcosus, R. pulchellu s, and H. a. anatolicum excavatum . Five Trypanosoma species were tested. • Trypanosoma cruzi Experimental transmission of T. cruzi through ingestion or injection of tick-infected material began in the 20th century. The injection of O. moubata -material infected with T. cruzi successfully caused rodent infection . Later studies confirmed rodent infection by T. cruzi -infected O. venezuelensis, O. turicata, O. furcosus, and O. moubata ticks . • Trypanosoma theileri and T. theileri-like Pioneering studies conducted in 1973 documented the inability to infect rodents by injecting T. theileri -infected hard tick material collected from cattle . In contrast to this observation, successful infections has been reported using O. moubata or H. a. anatolicum material infected with T. theileri . • Other Trypanosoma As early as 1924, Yorke and Macfie attempted to transmit T. rhodesiense to rodents by injecting them with material from infected O. crossi ticks . In 1948, Packchanian was unsuccessful in infecting animals with T. brucei using similar protocol . Finally, in 1976, Taylor-Lewis also failed to infect rodents by injecting material from H. a. excavatum ticks infected with T. lewisi . Several studies have reported the presence of L. infantum in R. sanguineus and I. ricinus , as well as the transmission of other protozoa, such as Hepatozoon canis , to dogs after ingestion of ticks . Six studies have experimentally investigated the transmission of Leishmania via the oral route and the infective potential of injecting material from infected ticks [ , , , 61 , , , ]. Among these, five confirmed infections were documented in seven reports-four after injection and one after ingestion. All these studies focused exclusively on ticks from the Ixodidae family, specifically R. sanguineus , which were infected with various Leishmania species and fed on rodents or dogs. • Leishmania infantum and Leishmania chagasi ( Syn: L. infantum) Experimental infection of L. infantum by injecting infected R. sanguineus material was reported in 2010 . This finding was further supported by Coutinho and colleagues demonstrating infection with L. chagasi (syn: L. infantum) in rodent from infected dog ticks . • Leishmania In 1984, a study assessed infection by injecting material from crushed, infected R. sanguineus ticks into two hunting dogs, crossbred between a German Shepherd and a Labrador Retriever. Following the injection, Leishmania species were detected in the recipient dogs using several diagnostic methods, suggesting the possibility of infection through the injection of tick-infected material . The ability of a microorganism to survive the shedding process and persist for an extended period within the tick is crucial for successful transstadial transmission. For T . thylacis such transmission was documented in the tick I. tasmani , and then to the Australian short-nosed bandicoot ( Isoodon macrourus ) . Transstadial passage of T . theileri in H . a. anatolicum ticks was reported in larvae and nymphs that fed on infected calves and tested positive after molting to the adult stage . This finding was corroborated in more recent studies . Transstadial transmission of L . infantum in R . sanguineus has been documented since the 1930s . Recent studies have once again demonstrated the persistence of L. infantum kDNA in R. sanguineus nymphs and adults that fed on infected dogs before molting . However, the presence of live parasites could not be confirmed through microscopic examination or culture from the infected tick material . The detection of flagellates and trypanosomes in tick ovaries was first reported in 1961 , leading to the proposal of transovarial transmission as a potential alternative route for maintaining the parasite in the wild. The earliest experimental reports of transovarial transmission of Trypanosoma in ticks date back to the early 1900s with T. cruzi , though further evidence was lacking for O . moubata or Rhipicephalus ticks . Transovarial transmission was also investigated for T. theileri in Hyalomma a. anatolicum , but no conclusive evidence was found . It wasn't until 2004 that a laboratory study demonstrated pathogenic trypanosomes being transmitted via tick ovaries . No conclusive evidence of transovarial transmission of T. evansi in R. sanguineus has been reported . Recent reports have confirmed the transovarial transmission of Leishmania in R . sanguineus , with the first evidence of this mode of transmission emerging in the early 1980s . Experimental studies have demonstrated the presence of L. infantum kDNA in R. sanguineus larvae four months after experimental infection of females . However, controversies still exist , and the only experiment conducted with L. major produced no conclusive evidence . Significant associations between tick infestation prevalence and the presence of anti- Leishmania antibodies in dogs from endemic areas of canine leishmaniasis have been reported [ 63 , 116 , , , ]. The prevalence of R . sanguineus infestation was significantly higher (p = 0.04) among seropositive dogs (38.5 %) compared to their seronegative counterparts (29.0 %). The probability of leishmania seropositivity was found to be 1.5 times higher in tick-infested dogs than in non-infested animals . Conversely, the odds of infection did not significantly differ between non-infested and R. sanguineus -infested dogs in one study . The near-perfect correlation between leishmania detection in dogs through fine-needle aspiration biopsy of the lymph nodes and the detection of leishmania in the tick's intestine via immunohistochemistry provides additional circumstantial evidence . For the field studies, we identified 42 publications for meta-analysis and meta-regression . The use of a random-effects model was justified by the P and I 2 statistics, which showed significantly high heterogeneity . We identified publication bias in the selected studies based on visual inspection of the graph's asymmetry , which was confirmed by Egger's test ( Supplementary File , Table S1 ). The elimination, one by one or simultaneously, of the four publications showing bias did not affect the cumulative prevalence recorded. Fig. 3 Forest plot showing the prevalence of Trypanosomatidae detection in field-collected ticks. The horizontal lines represent the 95 % confidence intervals, while the diamond indicates the pooled effect size. Fig. 3 Fig. 4 Funnel plot with 95 % confidence intervals for assessing publication bias. Fig. 4 The 42 studies selected included approximately 12,000 ticks collected worldwide. However, in some publications, it was not possible to determine the exact number of ticks used for the experiment. Therefore, the data we used for our analysis was the number of pools. Our results depicted an overall cumulative prevalence for Trypanosomatidae detection of 15.48 % (95 % CI: 7.99–24.61 %) . We then examined factors associated with the detection of Leishmania and Trypanosoma having medical/veterinary interest in ticks. Factors examined included the sampling decade, study area (continent), animal host (family), tick genus or species, the detection method, parasite species, and location in the tick's organs ( Table 3 ). We recorded high heterogeneity in all subgroups; therefore, the pooled seroprevalence estimate for each subgroup was calculated using the random-effects model. Table 3 Pooled prevalence of parasite detection in ticks, categorized by sampling year, continent, host, tick genus/species, detection method, parasite genus/species, and localization. Table 3 Category Variable No. of studies No. of tested No. of positive % [95 % CI] Heterogeneity Univariate meta regression ꭕ2 P -value I 2 (%) P -value R 2 (%) I 2 -res (%) Sampling year 2000 or before 2 6470 19 0.22 [0.16–0.45] 1.14 <0.0001 12.0 0.000∗∗∗ 0.00 98.74 2001–2010 5 480 130 12.19 [0.87–32.08] 126.37 <0.0001 96.8 2011–2019 23 3043 518 21.13 [7.7–23.77] 818.31 <0.0001 97.3 2020 or after 12 1721 96 11.65 [0.3–30.84] 289.5 <0.0001 96.2 Continent Africa 4 4392 22 2.44 [0.00–9.54] 23.24 <0.0001 85.8 0.0056∗∗ 23.77 97.78 Asia 7 1165 44 3.11 [0.00–20.83] 131.61 <0.0001 95.4 Europe 10 3693 69 4.36 [1.49–9.06] 158.52 <0.0001 94.3 Island 1 478 2 0.42 [0.01–1.26] 0.000 <0.0001 – South America 20 1986 626 33.35 [14.27–37.83] 828.68 <0.0001 96.1 Host (Family) Several# 6 4894 43 2.83 [0.0–8.60] 80.46 <0.0001 93.8 0.0007∗∗ 0.00 98.34 Bovidae 6 1192 44 3.92 [1.20–7.85] 24.26 <0.0001 88.2 Canidae 20 2184 595 27.92 [14.51–43.57] 1071.56 <0.0001 98.2 Felidae 2 221 19 8.47 [5.07–12.60] 0.81 <0.0001 0.0 Other 7 722 57 21.23 [0.0–62.24]0 252.480 <0.0001 97.6 Tick Genus Amblyomma 10 328 27 8.78 [0–29.41] 100.12 <0.0001 91.0 0.0001∗∗∗ 2.20 97.09 Dermacentor 3 23 0 0.00 [0–5.25] 0.41 <0.0001 0.0 Haemaphysalis 4 360 00 0.00 1.58 <0.0001 0.0 Hyalomma 6 639 17 1.20 [0–14.55] 70.55 <0.0001 92.9 Ixodes 9 6906 42 1.91 [0–10.06] 71.41 <0.0001 88.8 Rhipicephalus 33 3271 668 19.06 [9.64–30.39] 1412.59 <0.0001 97.7 Tick species A. cajennense 1 131 0 0.00 [0–7.04] 3.86 <0.0001 0.0001∗∗∗ 3.29 97.50 A. ovale 1 10 0 0.00 [0–16.52] 0.00 <0.0001 – A. sabanerae 1 10 9 90.00 [61.91–100] 0.00 <0.0001 0.0 A. tigrinum 2 28 0 24.86 [0–95.91] 16.89 <0.0001 – A. variegatum 2 165 11 3.09 [0–24.26] 0.43 <0.0001 94.1 Amblyomma spp. 2 43 4 0.00 [0–0.64] 11.54 <0.0001 91.30.0 D. marginatus 3 21 0 0.00 [0–5.25] 0.59 <0.0001 0.0 H. aegyptium 2 32 12 32.69 [11.66–56.75] 0.46 <0.0001 0.0 H. detritum 1 1 1 100 [0–100] 0.00 <0.0001 – H. dromedarii 2 45 5 16.56 [0–88.80] 17.10 <0.0001 94.2 H. excavatum 3 291 0 0.00 1.45 <0.0001 0.0 H. lusitanicum 1 3 0 0 [0–50] 0.00 <0.0001 – H. marginatum 4 171 0 0.00 2.04 <0.0001 0.0 Hyalomma sp. 2 81 0 0.00 [0–2.36] 0.01 <0.0001 0.0 Ha. longicornis 1 308 0 0.00 [0–0.58] 0.00 <0.0001 – Ha. parva 1 41 0 0.00 [0–4.15] 0.00 <0.0001 – Ha. punctata 1 6 0 0.00 [0–26.80] 0.00 <0.0001 – Ha. sulcata 2 4 0 0.00 [0–78.74] 0.07 <0.0001 0.0 Haemaphysalis. sp. 1 13 0 0.00 [0–12.82] 0.00 <0.0001 – I. ricinus 8 6580 32 1.36 [0–10.39] 64.66 <0.0001 87.6 I. ventalloi 1 62 3 6.45 [1.42–14.19] 0.00 <0.0001 – Ixodes sp. 1 5 0 0.00 [0–31.73] 0.00 <0.0001 – R. (Boophilus) 6 1025 62 0.00 [0–3.92] 0.51 <0.0001 0.0 R. bursa 3 23 0 13.54 [0–47.12] 185.13 <0.0001 22.2 R. pusillus 2 18 3 17.65 [2.57–39.97] 0.02 <0.0001 0.0 R. sanguineus 21 2222 583 25.15 [11.67–41.19] 1092.57 <0.0001 97.9 R. turanicus 4 158 10 2.48 [0–1467] 23.70 <0.0001 87.3 Rhipicephalus sp. 1 43 3 6.98 [0.9–16.93] 0.00 <0.0001 – Detection method Microscopy 3 6533 20 0.1 [0.0–0.23] 3.47 <0.0001 42.4 0.000∗∗∗ 5.37 98.84 Molecular 39 5181 743 17.55 [9.31–27.44] 1937.17 <0.0001 98.1 Parasite genus Leishmania 30 4569 719 18.87 [9.12–30.75] 1642.76 <0.0001 98.294.6 0.0046∗∗ 7.10 98.88 Trypanosoma 12 7739 93 4.62 [1.14–9.79] 203.03 <0.0001 Parasite species L. braziliensis 1 75 32 42.67 [31.64–64.66] 0.00 <0.0001 – 0.000∗∗∗ 9.25 98.97 L. chagas ( syn L. infantum) 1 444 107 24.10 [20.23–28.19] 0.00 <0.0001 – L. guyanensis 1 81 3 12.12 [2.79–25.84] 0.00 <0.0001 – L. infantum 18 2124 323 15.44 [7.18–25.89] 679.35 <0.0001 97.5 L. major 1 1 1 100 [0–100] 0.08 <0.0001 – L. martiniquensis 1 578 2 0.35 [0–1.04] 0.00 <0.0001 – Leishmania sp. 6 683 194 51.82 [8.42–93.57] 0.00 <0.0001 94.4 T. caninum 1 9 1 11.11 [0–41.77] 0.00 <0.0001 – T. congolense 1 33 4 3.70 [0.47–9.18] 0.00 <0.0001 – T. cruzi 3 583 8 2.39 [0–10.25] 895.52 <0.0001 91.2 T. evansi 4 892 38 6.44 [0–23.33] 13.00 <0.0001 95.9 T. theileri 2 6220 14 0.20 [0.12–0.36] 73.71 <0.0001 0.0 T. vivax 3 441 42 10.88 [2.91–22.73] 22.82 <0.0001 84.6 Localization in ticks H 2 6470 19 0.29 [0.16–0.45] 1.14 <0.0001 12.0 0.000∗∗∗ 0.00 98.89 G 3 209 164 74.32 [28.48–99.99] 312.09 <0.0001 97.3 GR 35 4532 478 13.45 [6.62–21.87] 1234.08 <0.0001 97.2 GR-SG 1 95 11 11.58 [5.82–18.88] 0.00 <0.0001 – H-GR 1 444 107 24.10 [20.23–28.19] 0.00 <0.0001 – SG 2 110 35 31.79 [23.31–40.91] 0.00 <0.0001 0.0 O 2 165 74 44.48 [37.27–52.53] 0.67 <0.0001 0.0 G: gut ; Gr: whole body ; H: hemolymph ; O: ovaries ; SG: salivary glands ∗0.05; ∗∗0.01; ∗∗∗0.001, # In these studies, various tick species were allowed to feed on diverse infected host (Swiss mice, Meadow voles, Rats, Mice, Rabbits, Camel, Dog, Guinea pig or White rat). In these papers, it was not possible to retrieve the information on the host origin of the ticks tested. Molecular biology yielded the highest detection rates, reaching 17.55 % (with a confidence interval of 9.31–27.44 %), surpassing the rates detected by microscopic methods, which stand at a mere 0.1 % (confidence interval: 0–0.23 %). Despite the specificity of microscopy in identifying Trypanosomatidae they demand skilled personnel for accurate parasite detection. The efficacy of these methods varies with the type of sample and generally falls short of the sensitivity and specificity offered by PCR and cell culture methods . Due to their superior sensitivity and specificity, molecular techniques are regarded as more effective . There were statistically significant differences across the collection decade ( Table 3 ). The highest annual prevalence recorded was 14.92 % between 2011 and 2019, and the lowest at 0.29 % was reported before the 2000s. This increasing prevalence trend since 2000 is likely due to advances in the performance of molecular detection methods and/or the impacts of climate change on vector populations and behaviors . We also assessed differences according to geographical origin (continent) ( Table 3 ). The highest prevalence, 25.17 % was recorded in South America, while the lowest was in Africa . The cumulative prevalence statistics by host animal on which ticks were collected, displayed a significant difference between animal hosts, with the highest cumulative prevalence being on Canidae . The prevalence of tick infection significantly varies according to the tick's taxonomic status , from 19.06 % in Rhipicephalu s to 8.78 % (95 % CI: 0–29.41 %, 27/328) in Amblyomma or 1.91 % and 1.20 % in Ixodes and Hyalomma , respectively, to 0.0 % in Dermacentor and Haemaphysalis ( Table 3 ). We recorded statistical differences according to the detection method (p > 0.05). The prevalence was higher at 17.55 % (95 % CI: 9.31, 27.44) with molecular methods and lowest at 0.1 % (95 % CI: 0–0.23) using microscopy. Moreover, the infection rate was significantly higher at 18.87 % [9.12–30.75] for Leishmania than for Trypanosoma at 4.62 % [1.14–9.79]. Infection rate also varied significantly (p = 0.000) by organ in ticks: the highest rate is 74.32 % (95 % CI: 28.24–99.99, 164/209) in the digestive tract, followed by 44.48 % [37.27–52.53] in the ovaries, 31.79 % [23.31–40.91 %] in the salivary glands, and the lowest rate of infection is 0.29 % [95 % CI: 0.16–0.45 %] in the hemolymph ( Table 3 ). For the experimental studies, 37 publications were selected. Data were extracted from these research papers. Quantitative data such, as the number of ticks used for experimental infections, were not considered in the meta-analysis. They were transformed into semi-quantitative ones (presence or absence of parasites following experimental infection or transmission). Since no heterogeneity was detected in the data set, the common-effect model was chosen for the meta-analysis. The meta-analysis was performed on 36 studies. The analysis showed the presence of parasites in ticks after their blood meal in 91 % [72–100 %] of studies . The factors associated with the detection of Leishmania and Trypanosoma in the blood meal were analyzed taking into account each actor involved in the transmission cycle, tick, animal host and parasite ( Table 4 ). No heterogeneity was detected in these subgroups. Rate estimates pooled for each subgroup were calculated using a common-effects model. • Tick's family and genus Table 4 Pooled rate of parasite detection in ticks from experimental transmission studies. Table 4 Acquisition of pathogens via tick blood feeding Category Variable No. of studies No. of positive % [95 % CI] Heterogeneity Univariate meta regression ꭕ2 P -value I 2 (%) P -value R 2 (%) I 2 -res (%) Ticks Family Argasidae 16 16 100 [81.65–100] 0.00 0.9966 0.0 0.0976 0.00 0.00 Ixodidae 21 15 78.01 [48.91–98.34] 15.86 0.9966 0.0 Ticks Genus Amblyoma 1 1 100 [0–100] 0.00 0.9938 – 0.3905 0.00 0.00 Hyalomma 4 3 82.18 [16.67–100] 2.78 0.9938 0.0 Ornithodoros 15 15 100 [80.68–100] 0.00 0.9938 0.0 Rhipicephalus 16 11 74.57 [40.89–98.60] 12.72 0.9938 0.0 Ticks species A. americanum 1 1 100 [0–100] 0.00 0.9989 – 0.6118 0.00 0.00 B. decoloratus 1 1 100 [0–100] 0.00 0.9989 – D. andersoni 2 2 100 [21.62–100] 0.00 0.9989 0.0 H. a . anatolicum 3 2 72.14 [2.45–100] 2.47 0.9989 18.9 H. a . excavatum 1 1 100 [0–100] 0.00 0.9989 – H. dromaderii 1 1 100 [0–100] 0.00 0.9989 – H. impressum 1 1 100 [0–100] 0.00 0.9989 – O. amblus 1 1 100 [0–100] 0.00 0.9989 – O. crossi 4 4 100 [48.72–100] 0.00 0.9989 0.0 O. furcosis 1 1 100 [0–100] 0.00 0.9989 – O. hermsi 2 0 0 [0–78.74] 0.00 0.9989 0.0 O. lahorensis 2 2 100 [21.26–100] 0.00 0.9989 0.0 O. moubata 7 7 100 [65.46–100] 0.00 0.9989 0.0 O. parkeri 1 1 100 [0–100] 0.00 0.9989 – O. savignyi 1 1 100 [0–100] 0.00 0.9989 – O. talaje 1 0 0 [0–100] 0.00 0.9989 – O. turanicus 2 2 100 [21.62–100] 0.00 0.9989 0.0 O. venzualensis 1 1 100 [0–100] 0.00 0.9989 – R. sanguneus 13 11 91.99 [58.62–100] 6.26 0.9989 0.0 R. sanguneus s. l . 2 0 0 [0–78.74] 0.00 0.9989 0.0 R. pulchellus 1 1 100 [0–100] 0.00 0.9989 – Parasite genus Leishmania 14 9 69.10 [62.64–97.17] 11.90 0.9974 0.0 0.0794 0.00 0.00 Trypanosoma 22 21 99.11 [80.73–100] 3.53 0.9974 0.0 Parasite Species L. donovani 1 1 100 [0–100] 0.00 0.9928 – 0.7192 0.00 0.00 L. chagasi (syn L.infantum) 2 2 100 [21.26–100] 0.00 0.9928 0.0 L. infantum 5 4 87.58 [29.26–100] 2.96 0.9928 0.0 L. kala azar £ 4 1 17.82 [0–83.24] 2.78 0.9928 0.0 L. major 1 1 100 [0–100] 0.00 0.9928 – Leishmania sp. 1 1 100 [0–100] 0.00 0.9928 – T. bruci 1 1 100 [0–100] 0.00 0.9928 – T. cruzi 8 8 100 [68.71–100] 0.00 0.9928 0.0 T. evansi 7 6 92.94 [46.20–100] 3.17 0.9928 0.0 T. gambiense 1 1 100 [0–100] 0.00 0.9928 – T. lewisi 2 2 100 [21.26–100] 0.00 0.9928 0.0 T. rhodesciense 2 2 100 [21.26–100] 0.00 0.9928 0.0 T. theileri 4 3 82.18 [16.67–100] 2.78 0.9928 0.0 T. theileri like 1 1 100 [0–100] 0.00 0.9928 – Donor host family Bovidae 4 3 82.18 [16.67–100] 2.78 0.9471 0.0 0.9455 0.00 0.00 Canidae 13 9 75.34 [37.81–99.71] 10.25 0.9471 0.0 Camelidae 2 2 100 [21.26–100] 0.00 0.9471 0.0 Rodentia 15 14 93.73 [63.97–100] 42.76 0.9471 0.0 ND 2 2 100 [21.26–100] 0.00 0.9471 0.0 Other 1 1 100 [0–100] 0.00 0.9471 – Ticks 1 1 100 [0–100] 0.00 0.9471 – Infection via the injection of ticks infected material Ticks Family Argasidae 9 7 85.25 [41.62–100] 5.76 0.6062 0.0 0.2884 0.00 0.00 Ixodidae 13 7 55.23 [18.49–89.66] 11.96 0.6062 0.0 Ticks Genus Hyalomma 4 2 50.0 [0–100] 3.70 0.5378 18.9 0.5954 0.00 0.00 Ornithodoros 9 8 85.25 [41.62–100] 5.76 0.5378 0.0 Rhipicephalus 7 4 59.68 [11.06–99.18] 6.34 0.5378 5.4 Ticks species B. decoloratus 1 0 0 [0–100] 0.00 0.8803 – 0.2229 0.00 0.00 D. andersoni 2 0 0 [0–78.74] 0.00 0.8803 0.0 H. a. anatolicum 3 2 72.14 [2.45–100 2.47 0.8803 18.9 H. a. excavatum 1 0 0 [0–100] 0.00 0.8803 – H. dromaderii 1 0 0 [0–100] 0.00 0.8803 – H. impressum 1 0 0 [0–100] 0.00 0.8803 – O. amblus 1 1 100 [0–100] 0.00 0.8803 – O. crossi 1 0 0 [0–100] 0.00 0.8803 – O. furcosus 1 1 100 [0–100] 0.00 0.8803 – O. hermsi 2 0 0 [0–78.74] 0.00 0.8803 0.0 O. moubata 4 4 100 [48.72–100] 0.00 0.8803 0.0 O. perkeri 1 1 100 [0–100] 0.00 0.8803 – O. talaje 1 0 0 [0–100] 0.00 0.8803 – O. turanicus 1 0 0 [0–100] 0.00 0.8803 – O. turicata 2 1 50.0 [0–100] 1.85 0.8803 46.0 O. venzualensis 1 1 100 [0–100] 0.00 0.8803 – R. pulchellus 1 0 0 [0–100] 0.00 0.8803 – R. sanguineus 6 5 90.82 [38.83–100] 3.08 0.8803 0.0 R. sanguineus s.I. 1 0 0 [0–100] 0.00 0.8803 – Parasite genus Leishmania 7 5 78.10 [27.34–100] 5.29 0.5774 0.0 0.7666 0.00 0.00 Trypanosoma 14 9 69.10 [32.64–97.17] 11.9 0.5774 0.0 Donor host Family Bovidae 4 2 50.0 [0–100] 3.70 0.5180 18.9 0.7557 0.00 0.00 Camelidae 1 1 100 [0–100] 0.00 0.5180 – Canidae 5 4 87.58 [29.26–100] 2.96 0.5180 0.0 Rodentia 11 8 68.26 [27.33–98.66] 9.42 0.5180 0.0 Receiver host Family Bovidae 3 2 72.14 [2.45–100] 2.47 0.5387 18.9 0.5494 0.00 0.00 Canidae 1 1 100 [0–100] 0.00 0.5387 – Other 1 0 0 [0–100] 0.00 0.5387 – Rodentia 15 11 63.49 [28.31–93.43] 13.32 0.5387 0.0 Ticks 2 2 100 [21.26–100] 0.00 0.5387 0.0 Parasite species L. chagasi (Syn L. infantum) 1 1 100 [0–100] 0.00 0.8397 – 0.2614 0.00 0.00 L. infantum 1 1 100 [0–100] 0.00 0.8397 – L. kala azar £ 4 2 50.0 [0–100] 3.70 0.8397 18.9 Leishmania sp. 1 1 100 [0–100] 0.00 0.8397 – T. brucei 1 0 0 [0–100] 0.00 0.8397 – T. cruzi 6 6 100 [61.37–100] 0.00 0.8397 0.0 T. evansi 1 0 0 [0–100] 0.00 0.8397 – T. lewisi 1 0 0 [0–100] 0.00 0.8397 – T. rhodeseinse 1 0 0 [0–100] 0.00 0.8397 – T. theileri 4 3 82.18 [16.76–100] 2.78 0.8397 0.0 T. theileri like 1 0 0 [0–100] 0.00 0.8397 – Transmission through blood feeding Ticks Family Argasidae 10 3 23.71 [0–66.41] 7.77 0.5314 0.9 0.4962 0.00 0.00 Ixodidae 11 5 34.33 [0.0–94] 10.09 0.5314 0.0 Ticks Genus Hyalomma 4 2 50.0 [0–100] 3.70 0.4677 18.9 0.7745 0.00 0.00 Ornithodoros 10 3 23.71 [0–66.41] 7.77 0.4677 0.0 Rhipicephalus 7 3 40.32 [0.48–88.94] 6.32 0.4677 5.4 Parasite genus Leishmania 5 2 36.51 [0–92.81] 4.44 0.9952 9.9 0.5011 0.00 0.00 Trypanosoma 16 8 33.22 [5.25–67.40] 13.88 0.9952 0.0 Donnor host family Bovidae 4 2 50.0 [0–100] 3.70 0.9017 18.9 0.0405∗ 0.00 0.00 Camelidae 2 2 100 [21.26–100] 0.00 0.9017 0.0 Canidae 7 4 59.68 [11.06–99.18] 6.34 0.9017 5.4 Rodentia 8 0 0 [0–31.29] 0.00 0.9017 0.0 Receiver host family Bovidae 3 2 72.14 [2.45–100] 2.47 0.8461 18.9 0.0678 0.00 0.00 Canidae 3 2 72.14 [2.45–100] 2.47 0.8461 18.9 Other 2 2 100 [21.26–100] 0.00 0.8461 0.0 Rodentia 13 4 8.01 [0–41.38] 6.26 0.8461 0.0 Ticks species B. decoloratus 1 0 0 [0–100] 0.00 0.6280 – 0.6077 0.00 0.00 D. andersoni 1 0 0 [0–100] 0.00 0.6280 – H. a. anatolicum 3 2 72.14 [2.45–100] 2.47 0.6280 18.9 H. a. excavatum 1 0 0 [0–100] 0.00 0.6280 – H. dromaderii 1 0 0 [0–100] 0.00 0.6280 – H. impressum 1 0 0 [0–100] 0.00 0.6280 – O. crossi 4 3 82.18 [16.67–100] 2.78 0.6280 0.0 O. hermsi 1 0 0 [0–100] 0.00 0.6280 – O. lahorensis 2 2 100 [21.26–100] 0.00 0.6280 0.0 O. moubata 3 0 0 [0–61.92] 0.00 0.6280 0.0 O. savigny 1 0 0 [0–100] 0.00 0.6280 – O. talaje 1 0 0 [0–100] 0.00 0.6280 – O. turanicus 1 0 0 [0–100] 0.00 0.6280 – O. turicata 1 0 0 [0–100] 0.00 0.6280 – O. venzualensis 1 0 0 [0–100] 0.00 0.6280 – R. pulchellus 1 0 0 [0–100] 0.00 0.6280 – R. sanguineus 6 3 50.0 [2.78–97.22] 5.55 0.6280 9.9 Parasite species L. infantum 2 1 50.0 [0–100] 1.85 0.5302 46.0 0.5529 0.00 0.00 L. kala azar £ 2 0 0 [0–78.74] 0.00 0.5302 0.0 Leishmania sp. 1 1 100 [0–100] 0.00 0.5302 – T. cruzi 6 1 9.18 [0–61.17] 3.08 0.5302 0.0 T. evansi 6 3 50.0 [2.78–97.22] 5.55 0.5302 9.9 T. lewisi 1 0 0 [0–100] 0.00 0.5302 – T. theileri 3 1 27.86 [0–97.55] 2.47 0.5302 18.9 T. theileri like 1 1 100 [0–100] 0.00 0.5302 – Vertical transmission Ticks Family Argasidae 1 0 0 [0–100] 0.00 0.4606 – 0.2809 0.00 0.00 Ixodidae 12 7 61.27 [22.41–94.58] 10; 79 0.4606 0.0 Ticks Genus Hyalomma 2 1 50.0 [0–100] 1.85 0.3787 46.0 0.5422 0.00 0.00 Ornithodoros 1 0 0 [0–100] 0.00 0.3787 – Rhipicephalus 10 6 63.49 [21.05–97.58] 8.88 0.3787 0.0 Ticks species H. a. anatolicum 2 1 50.0 [0–100] 1.85 0.4143 46.0 0.4394 0.00 0.00 O. moubata 1 0 0 [0–100] 0.00 0.4143 – R. pulchellus 1 0 0 [0–100] 0.00 0.4143 – R. sanguineus 9 7 72.14 [27.07–100] 7.40 0.4143 0.0 Parasite genus Leishmania 8 7 82.18 [35.16–100] 5.55 0.6668 0.0 0.0634 0.00 0.00 Trypanosoma 5 1 12.42 [0–70.74] 2.96 0.6668 0.0 Donor host family Bovidae 3 1 27.86 [0–97.55] 2.47 0.8446 18.9 0.0425∗ 0.00 0.00 Canidae 7 6 92.94 [46.20–100] 3.17 0.8446 0.0 Rodentia 3 0 0 [0–61.92] 0.00 0.8446 0.0 Receiver host family Bovidae 2 1 50.0 [0–100] 1.85 0.4748 46.0 0.3582 0.00 0.00 Canidae 1 1 100 [0–100] 0.00 0.4748 – ND 4 3 82.18 [16.67–100] 2.78 0.4748 0.0 Rodentia 5 1 12.42 [0–70.74] 2.69 0.4748 0.0 Ticks 1 1 100 [0–100] 0.00 0.4748 – Parasite species L. chagasi ( Syn L. infantum) 2 1 50.0 [0–100] 1.85 0.5046 46.0 0.3655 0.00 0.00 L. infantum 3 3 100 [38.08–100] 0.00 0.5046 0.0 L. major 1 0 0 [0–100] 0.00 0.5046 – L. kala azar £ 1 1 100 [0–100] 0.00 0.5046 – Leishmania sp. 1 1 100 [0–100] 0.00 0.5046 – T. cruzi 1 0 0 [0–100] 0.00 0.5046 – T. evansi 1 0 0 [0–100] 0.00 0.5046 – T.theileri 3 1 27.86 [0–97.55] 2.47 0.5046 18.9 ∗0.05; ∗∗0.01; ∗∗∗0.001 £ L. kala azar refers to members of the L. donovani complex ( L. infantum and L. donovani ) without any other information on parasite typing at the time of the study. We therefore use the term proposed by the author. # ticks (receiver host) were infected via the inoculation of A. americanum -infected hemolymph (donor host). For tick families, positivity rates were 100 % (95 % CI: 81.65–100) for Argasidae and 78.01 (95 % CI: 48.9–98.84 %) for Ixodidae, with no statistical difference according to tick family (p > 0.05). Slight variations were recorded for the genus, with no statistically significant difference. For the genera Ornithodoros and Amblyomma , all publications reported positive detection after the blood meal; for the genera Rhipicephalus and Hyalomma , detection rates were 74.57 % and 82.18 %, respectively. A large variation in prevalence is recorded at the species level, related to the low number of studies published ( Table 4 ). • Parasite genus The highest detection rate was 99.11 % (95 % CI: 80.73–100) for Trypanosoma , whereas Leishmania was detected in 69.10 % (95 % CI: 62.64–97.17) of the studies. However, this difference is not statistically significant (P > 0.05) ( Table 4 ). • Host family The infection rate elicited via the injection of Argasidae-infected material was statistically higher than of Ixodidae-infected ones (rate of 85.25 % vs. 55.23 %). The highest rate, 85.25 %, was recorded for Ornithodoros , followed by Rhipicephalus and Hyalomma . However, if all these observations were not statistically significant, they interestingly point to some specificity according to the tick genus ( Table 4 ). • Parasite's genus and species These analyses disclosed that tick material collected from infected Camelidae was more likely to initiate infection when injected into a noninfected recipient and Canidae appears to be more susceptible to infection when injected with infected tick material. Tick-borne pathogen transmission can occur via mechanical or biological means. In mechanical transmission, ticks act as carriers, transferring pathogens between hosts without mandatory pathogen development within the tick. In contrast, biological transmission involves the pathogen undergoing necessary biological changes or replication within the tick, completing part of its life cycle before infecting the next host. We performed a meta-analysis on data extracted from 21 publications dealing with experimental transmission by ticks via blood feeding ( Table 4 ). This analysis showed 34 % (95 % CI: 8–64 %) of studies confirming transmission . The common-effects model was used to analyze associated factors. • Tick's family The Ixodidae appeared to be more able to transmit Trypanosomatidae of medical or veterinary interest than Argasidae, with rates of 34.33 % (95 % CI: 0–94 %) and 23.7 % (95 % CI: 0–66.4 %), respectively ( Table 4 ). Regarding the genus, minor variations were recorded without significant differences (p > 0.05). Regarding tick species, R. sanguineus , O. lahorensis , O. crossi , and H. a. anatolicum seemed more able to transmit Trypanosomatidae parasites of medical and veterinary interest. However, the sample size is too small to get insight into the statistical significance of these observations. • Parasite genus Although the success of transmission attempts is higher with Leishmania (82.18 % CI:35.16–100 %) than with Trypanosoma (12.42 % CI: 0–70.74 %), the meta-analysis does not record a statistically significant difference (p > 0.05) ( Table 4 ). • Donor and receiver host family Surprisingly, the donor host appears to be a factor influencing the subsequent transmission of the pathogen during tick blood feeding (p < 0.05) Table 4 . The infection rate for ticks varied from 100 % to 95 % (CI: 21.26–100 %) in Camelidae, to 59.68 % for Canidae and 50.0 % for Bovidae. The eight studies focusing on ticks collected on infected rodents reported no transmission. The host recipient also showed heterogeneity, with Canidae and Bovidae being more frequently infected at a rate of 72.14 % [2.45–100 %] than rodents (8.01 %, [0–41.38 %]). Our analysis includes only 13 studies focusing on the vertical transmission of Trypanosomatidae of medical or veterinary interest by ticks, with 55 % supporting the vertical transmission, as detailed in the supplementary file . The factors associated with this transmission were evaluated using the common-effect model. Despite the small sample size, vertical transmission is recorded at 61.27 % [22.41–94.58] for ticks of the Ixodidae family and 0 % for the Argasidae, although only one study deals with this issue. No significant difference exists according to genus and species ( Table 4 ). Concerning genus and parasite species, parasites belonging to the Leishmania genus were better adapted to vertical transmission (82.18 [35.16–100]) than those of the Trypanosoma genus (12.42 [0–70.74]). Parasites from the L. donovani complex ( L. donovani or L. infantum ) appear to be adapted to vertical transmission. Also, the host family from which ticks feed plays a significant role in variability . Ticks feeding on dogs (Canidae) have the highest vertical transmission rate at 92.94 % [46.20–100 %], followed by those feeding on Bovidae at 27.86 % [0–97.55], and lastly, rodents with no vertical transmission of Trypanosomatidae in ticks following blood meal on rodent infected host. Biologists have shown particular interest in insects that serve as vectors for Trypanosomatidae due to the significant impact these parasites have on various animal species, including humans . While nearly all Trypanosomatidae are transmitted by insects, a single publication documented an avian trypanosome transmitted by a non-traditional vector: an arachnid from the subclass Acari . This anomaly spurred further investigations into the presence of Trypanosomatids within ticks . Consequently, there has been an increased focus on understanding the potential role of ticks in harboring and transmitting these pathogenic protozoans. To determine the prevalence of Trypanosomatidae of medical and veterinary interest in wild-caught ticks from endemic areas, both molecular (PCR, qPCR, PCR-HRM) and parasitological/immunological (microscopic examination, IHC) methods were employed. Molecular techniques that amplify genomic DNA, which can persist after parasite death, are more sensitive than parasitological/immunological methods that target living parasites or their immunological determinants, which degrade rapidly upon the death of the parasite. Statistical differences in detection rates were observed depending on the method used. Specifically, the highest prevalence (17.55 %) was recorded using molecular methods, compared to only 0.1 % for microscopic detection of parasites. The presence of DNA or parasites in the digestive systems of blood-fed ticks collected from hosts has limited predictive value for inferring a vectorial role. Most publications included in our systematic review and meta-analysis focused on ticks collected directly from hosts, concentrating on detecting pathogens from host blood deposited in tick bodies. DNA detection alone is insufficient, particularly in the case of ticks, due to their digestive capabilities and other metabolic peculiarities. To further analyze the prevalence of Trypanosomatidae in field-collected tick samples, it will be necessary to isolate the parasites themselves from these ticks and/or detect parasite-specific mRNA, which serves as a better indicator of parasite survival in the digestive tract and other tick organs. Data on field tick infections reveal that the detection of Trypanosomatidae of medical or veterinary interest in ticks varies significantly, ranging from 0.01 % to 1.26 % in some regions to as high as 14.27 %–37.83 % in others, depending on the country or continent ( Table 3 ). Many studies utilize pooled samples to ascertain prevalence, which can greatly overestimate the results. Nonetheless, the meta-analysis provides initial evidence of a high frequency of contact between infected hosts and ticks, a crucial factor for facilitating transmission by arthropod vectors. Unfortunately, in many regions where Trypanosomatidae infections are common, there is a lack of available information regarding the infection rates of these pathogenic agents in their proven vectors and/or in ticks. For instance, while the Mediterranean area has a high incidence of leishmaniases , there is no data on the carriage of Leishmania by ticks in countries such as Egypt, Libya, and Morocco. Similarly, Latin America and the Caribbean are affected by Chagas disease, caused by T. cruzi , yet no information on its presence in ticks is available. This lack of data complicates efforts to conduct a global analysis of the seasonal activity patterns of ticks in relation to the incidence of Trypanosomatidae pathogen infections in hosts, as well as the geographical overlap between tick populations and human or animal infections caused by Trypanosomatidae pathogens. If 996 species of ticks (774 hard ticks and 221 soft ticks) are described worldwide , with 25 species acting as vectors of major diseases, the presence of Trypanosomatidae pathogens of medical or veterinary interest has been investigated in twenty tick species. In South America, 137 species of hard ticks from five genera and 87 species of soft ticks have been reported . All studies collected for this review focused on the Ixodidae family, with no field data available on soft ticks from the Argasidae family. The detection rate among members of the Rhipicephalus genus is the highest at 17.49 %, followed by Amblyomma (11.47 %), Hyalomma (2.68 %), and Ixodes (1.87 %), while Dermacentor and Haemaphysalis ticks were negative. Variability in field studies regarding the detection of Trypanosomatidae in ticks can often be attributed to host effects. Specifically, ticks harvested from Canidae—predominantly non-questing ticks belonging to the Rhipicephalus genus—exhibit a higher detection rate of pathogens compared to ticks collected from other mammals . This discrepancy may partly result from the close relationship between dogs and humans, coupled with dogs' heightened susceptibility to T. cruzi , which could increase the risk of tick infection . Additionally, dogs serve as significant reservoirs for several Leishmania species, including L. infantum , L. peruviana , and L. donovani . The extensive geographical distribution of ticks belonging to the genus Rhipicephalus , their wide range of animal hosts, known vector competence, and diverse morphology provide strong arguments for this meta-analysis on their potential role in the transmission of Trypanosomatidae . R hipicephalus sanguineus is a representative species of this genus, particularly due to its close association with domestic dogs, which are known reservoirs for Leishmania . This connection highlights the likely involvement of this tick species in the persistence and transmission of these parasites in natural habitats . The concept of vector competence refers to the innate ability of an arthropod to harbor and transmit microbial agents . Establishing the vector capacity of a tick involves confirming its ability to become infected during a blood meal on a host, facilitating the multiplication of the pathogen prior to transmission through saliva, and maintaining the pathogen throughout the tick's developmental stages for potential vertical transmission . Regarding parasite acquisition, the meta-analysis reveals an efficient tick infection following blood feeding in experimental settings, with 31 successful infections out of 37 attempts ( Table 4 ). While no significant factors were associated with parasite acquisition from an infected host (donor host), the analysis does indicate some trends worth discussing. Firstly, Argasidae ticks appear more likely to become infected compared to Ixodidae ticks when feeding on an infected host . The nature of the host does not seem to influence tick infection rates; however, the family of the parasite does impact infection rates. Specifically, ticks have a higher infection rate when feeding on a Trypanosoma -infected host compared to a Leishmania -infected host, with tick infection being more efficient when they take a blood meal from a host infected with Trypanosoma ( Table 4 ). Trypanosoma cruzi and T. evansi are particularly likely to infect ticks following blood feeding. These observations may relate to the intrinsic ability of trypanosomes to multiply in the blood of the infected host, increasing their availability to ticks during the blood-feeding process. Additionally, the disparity in infection rates might be attributed to differing feeding behaviors between the two tick families: Ixodidae ticks attach to the host's skin and feed slowly over several days, whereas Argasidae ticks rapidly ingest large volumes of blood in a short time (20–70 min), facilitating a more efficient uptake of infectious agents, especially under experimental conditions . The rapid feeding behavior of Argasidae ticks enhances their efficiency at acquiring blood-circulating parasites like T. brucei or T. evansi , compared to the slower-feeding Ixodidae ticks, which may be less effective at acquiring tissue-located parasites. Furthermore, the prolonged feeding process of Ixodidae ticks, lasting up to two weeks, exposes engorged pathogens to the full spectrum of host defense mechanisms, including specific acquired immunity that may impact the survival of Trypanosomatidae . Information gathered on the efficiency of infection through the injection of contaminated tick material provides insight into the presence of infectious parasitic stages in infected ticks. Our meta-analysis did not identify any significant factors related to the donor or receiver host, ticks, or parasites. While Argasidae ticks appear more efficient in such transmission than Ixodidae (7 successes out of 9 attempts compared to 7 successes out of 13 attempts), these differences are not statistically significant . Since ticks feed only once at each life stage, vertical transmission of pathogens is a crucial factor to consider when addressing the vectorial status of ticks for Trypanosomatidae parasites. The literature survey indicates that vertical transmission occurs in experimental settings, and only in Ixodidae ticks. Although not statistically significant, Leishmania seems more likely to be vertically transmitted in R. sanguineus than Trypanosoma species . Interestingly, Babesia can undergo transovarial transmission in ticks, suggesting potential similar behavior for Trypanosomatidae . However, the limited number of studies on vertical transmission restricts further discussion on the influence of tick identity or parasite species on vertical transmission. Trypanosoma evansi and T. vivax are mechanically transmitted pathogens, meaning they are spread from host to host without undergoing biological replication within their vector. This mode of transmission does not align well with the feeding habits of ticks, except in instances of interrupted feeding . While there is limited data on T. vivax , a relatively good success rate of 3 out of 6 attempts was recorded for the transmission of T. evansi via tick blood feeding. Therefore, the transmission of T. evansi or T. vivax following interrupted blood feeding warrants further investigation. Salivarian trypanosomes, such as T. brucei , T. evansi , T. congolense , T. vivax , and Leishmania parasites, are transmitted through blood-feeding via the injection or regurgitation of saliva during the feeding process. In contrast, stercorarian trypanosomes, like T. cruzi , infect their hosts through the deposition of the pathogen on the host's skin during blood-feeding, subsequently entering the host through scratching of the infected blood onto mucosal surfaces. Infection via ingestion is also documented for T. cruzi . Considering this aspect, we observed more successful attempts to transmit Trypanosomatidae pathogens through the blood-feeding of infected ticks harboring salivarian trypanosomes, such as T. evansi , T . lewisi , and T. theileri , compared to those with stercorarian pathogens. Specifically, there were 11 attempts with 5 successes for salivarian trypanosomes, and 6 attempts for T. cruzi . The data indicate a general trend of higher infection rates for salivarian Trypanosomatidae during tick blood meals. However, these differences are not statistically significant, preventing us from drawing definitive conclusions about the impact of the transmission route. Overall, this systematic review and meta-analysis provide an updated overview of the vector status of ticks for Trypanosomatidae. This perspective highlights the limited information available regarding the presence of Trypanosomatidae infecting humans and animals of veterinary importance in field-collected specimens or during experimental studies. Specifically, data are available for 20 species out of the 774 recognized hard ticks and 221 soft ticks. Of the 23 Leishmania and 11 Trypanosoma species of medical or veterinary interest, we gathered information on 6 species from the Leishmania genus and 9 from the Trypanosoma genus. Notably, we also collected data on the presence of trypanosomes associated with human African trypanosomiasis ( T. b. gambiense and T. b. rhodesiense ) in field-collected ticks. However, we could not provide conclusive quantitative evidence regarding the vectorial role of ticks for Leishmania and Trypanosoma parasites in medical or veterinary contexts. For R. sanguineus, the documentation of Trypanosomatidae infecting humans and animals is the most extensive, with 21 studies. The capacity of this tick species to acquire parasites during blood feeding on infected hosts has been successfully demonstrated in 11 out of 15 experiments. Additionally, the presence of the infective parasite stage has been assessed through the injection of tick-infected material (7 attempts with 5 successes), as well as re-transmission via infected tick blood feeding (6 attempts with 3 successes). Importantly, data on vertical transmission have also been collected (9 attempts with 7 successes). While most of these collected data pertain to L. infantum , they collectively support the vectorial competence of R. sanguineus , which now needs to be more thoroughly demonstrated using advanced molecular methods in field-collected specimens alongside additional experimental evidence. Furthermore, the presence of Trypanosomatidae parasites with medical or veterinary significance in ticks is not uncommon, and ticks from the Argasidae family may also play a role in the transmission of these pathogens. The precise role of ticks in sustaining both the parasitic developmental and epidemiological cycles of Trypanosomatidae requires further investigation and continuous scrutiny. | Study | biomedical | en | 0.999996 |
PMC11698937 | Atherosclerosis is the dominant cause for major cardiovascular events and premature death, with coronary artery disease (CAD) responsible for 10 % of all deaths in Australia . The Australian government recommends routine cardiovascular risk assessment for all individuals over the age of 40, with the goal of implementing lifestyle changes and/or therapeutic treatment in susceptible individuals to reduce the burden of CAD . The Framingham equation, originally devised in 1998 using traditional risk factors to predict cardiovascular risk in asymptomatic individuals , has been reported to be used in 44 % of online risk calculators and used by 42 % of general practitioners to inform disease management . However, cardiovascular risk scoring (CVRS) is an imperfect tool. Between 14 and 27 % of first-time myocardial infarction (MI) patients have no standard modifiable risk factors (SMuRFs) . Albarqouni et al. found that the Framingham equation and the pooled cohort risk equation for atherosclerotic cardiovascular disease (PCE-ASCVD) incorrectly predicted 16 % of males and 31 % of females as non-high-risk despite them experiencing a cardiovascular event. Cardiovascular risk estimation errors have been the subject of considerable debate, with the potential to lead to overtreatment (in the absence of significant disease in apparently high-risk individuals) or no treatment (in the presence of severe disease in apparently non-high-risk individuals). This mismatch between apparent risk and actual atherosclerosis burden may increase the risk of a potentially fatal, but preventable, coronary event . Evaluation of CAD plaque burden can be enhanced by coronary artery calcium score (CACS) identified by non-contrast CT , as increasing Agatston-units of coronary calcium are associated with adverse survival . Further, non-invasive coronary computed tomography angiography (CCTA) adds important prognostic information by demonstrating the presence and extent of CAD , and predicts cardiovascular events after adjustment for CVRS . However CCTA is not risk-free and has financial implications, and as such its role in preventative cardiology has not been entirely defined . There is a significant knowledge gap as to whether cardiovascular risk scoring accurately predicts the presence and extent of CAD in asymptomatic, but potentially at-risk, individuals. The aim of this study was to examine asymptomatic individuals (and no prior history of cardiovascular disease (CVD)) undergoing CCTA for cardiovascular risk assessment, to explore the predictive accuracy of CVRS in detecting the presence and extent of pre-clinical CAD. The initial cohort consisted of all consecutive patients undergoing CCTA at Perth Radiological Clinic (PRC) in Perth, Western Australia, between January 1, 2018 and December 31, 2019. Patients were excluded if they had a Medicare indication for CCTA (symptoms suggestive of CAD, for exclusion of a coronary anomaly, and/or prior to non-coronary cardiac surgery) or a prior CVD diagnosis, if they were outside of the required age range of 35–74 years old for CVRS calculation, or if any required information to calculate CVRS was missing . The radiologist report from the PRC CCTA results was transcribed by AI-based Natural Language Processing (NLP) through the Australian Institute for Machine Learning (AIML) at the University of Adelaide into “Database 2”. The NLP extracted information on CACS, and radiologist-reported coronary artery findings including presence, extent and severity of plaque, calcification, and location (left anterior descending, circumflex, right coronary and left main arteries). The NLP was trained using manually extracted examples and tested on a separate dataset not previously seen by the AI, and two cardiologists manually verified the output in 200 randomly selected scans. For CACS, NLP accuracy was 99.5 %, recall 100 % and precision 99.3 %; for the presence of atherosclerosis, accuracy was 99.5 %, recall 100 % and precision 99.3 %; and severe vs non-severe stenosis had 99.5 % accuracy, recall 98.7 % and precision 95.7 %. Three categorical variables were obtained from the CCTA and CACS results and classified according to the Coronary Artery Disease-Reporting and Data System 2.0 (CAD-RADS 2) : CACS in Agatston units (classified as no calcium (0), mild (1−100), moderate (101−300) and high (>300) calcium burden), plaque extent (number of abnormal arteries with ≥1 coronary segment involved) and stenosis severity (either severe (≥70 %) or non-severe (<70 %) from the most severe artery). These numerically categorized variables were added together, with the resultant sum categorized as no atherosclerosis and low plaque-burden, moderate plaque-burden and extensive plaque-burden atherosclerosis. A binary categorical variable was created to record atherosclerosis presence (plaque present and/or non-zero CACS) and atherosclerosis absence (no zero plaque and zero CACS). Three pathology providers (Clinipath Pathology, Australian Clinical Laboratories and Western Diagnostic Pathology) extracted the most recent blood test result prior to the CCTA scan for each patient, and these databases were merged to form “Database 1” (linked by their Medicare ID). Sex, medical and cardiac history (smoking status, diabetes diagnosis and previous CVD diagnosis), current medications, and at least three systolic blood pressure readings (mmHg) taken during the CCTA visit were obtained from three patient questionnaire forms in PRC records . This was transcribed into “Database 3” using a combination of manual imputation for the handwritten information, and NLP for the tick-box information . These identifiable databases were merged on a patient-level (linked by a randomly generated study ID) into an SPSS datafile, and then all identifying information was destroyed after manual verification and data cleaning (merging duplicate cases into single patient rows). Only the Principal Investigator and honors student had access to the identifiable information (protected by a16-character alphanumeric password) and take responsibility for its integrity, and additional researchers only had access to the de-identified master database. See Supplemental Fig. 3. Each medical record was examined for lipid-lowering medications, as use of these medications can cause underestimation of CVRS . When present, LDL adjustment was undertaken using composite scores from population data on each dose and type of statin , and corrected according to The Dutch Lipid Clinic Network Score . For CVRS calculations, the measured total cholesterol was used in patients not taking lipid-lowering therapy, and adjusted LDL concentration was used to calculate the total cholesterol used for those taking lipid-lowering therapy. A CVRS was calculated for each eligible individual using the Australian Absolute Cardiovascular Risk Calculator , using information on the individual's sex, age, systolic blood pressure, diabetes and smoking status, total cholesterol, and HDL cholesterol levels . Risk scores were classified as high risk (>15 %), moderate risk (10–15 %), low risk (1–9 %) or “zero” risk (>1 %) of experiencing a cardiovascular event in the next five years, with a maximum of 35 % . A binary categorical variable was created with a zero-low risk score (≤9 %) and moderate-high risk score (>9 %). The number of traditional risk factors for each individual was counted and categorized as low (0–2), moderate (3–4) or high (5–6). Risk factors included diabetes, diagnosed hypertension (≥140 mmHg systolic blood pressure), current smoking status or having ceased within the last 12 months, male sex, age 35 to 74 years old at the time of the CCTA scan, and hypercholesterolemia (total cholesterol level of ≥5.5 mmol/L) . Due to the possibility of a white-coat effect influencing measured blood pressure readings , only diagnosed hypertension was used as a risk factor. All analysis was performed with IBM SPSS, version 28.0.1.1, and statistical significance was set at p < 0.05. Normality was assessed using the Shapiro-Wilk statistic, and the distribution of CVRS was compared in individuals with and without atherosclerosis through the independent t -test or Mann-Whitney U test (95 % CI), as appropriate. Non-parametric continuous variables were reported as median and interquartile range (25th percentile, 75th percentile), and categorical variables were reported as frequencies and percentages. The primary analysis of this study was to determine the predictive accuracy of risk scoring in detecting pre-clinical CAD. Degree of atherosclerosis was compared to CVRS using the Fisher-Freeman-Halton or Monte-Carlo tests (95 % CI) to determine the significance of the relationship, and a weighted Cohen's kappa analysis for the level of agreement. The same analysis was undertaken to compare degree of atherosclerosis and risk factor count, and to compare CVRS to plaque extent, stenosis severity and CACS. The proportion of the cohort with a match, and mismatch, between their CAD and CCTA results was determined to calculate the percentage of overestimation and underestimation. A correct match was defined as any direct match between the categories of CVRS and degree of atherosclerosis (e.g., low plaque-burden and low risk) and a mismatch was any other combination (e.g., extensive plaque-burden and low risk). The relationship strength was visualized by relationship maps and statistically tested. Proportions of the cohort with or without atherosclerosis, and with a moderate-high (>9 %) or zero-low (≤9 %) CVRS, was determined to calculate the sensitivity and specificity of the calculator and presented in a scaled rectangular diagram . With CCTA as the reference group, the potential to improve classification of patients at non-high-risk using CVRS was examined by calculating the proportion of individuals who would be reclassified as high risk. The risk calculator accounted for the potential confounding variables of sex and age, and males and females were analyzed separately. For a sensitivity analysis, variables were compared between the groups included and excluded from analysis. An independent t -test or Mann-Whitney U test was done for age, and patient frequencies were compared for the categorical variables of gender, presence of atherosclerosis and degree of atherosclerosis. The study flow chart is summarized in Fig. 1 . 828 patients met the inclusion criteria for this study. Of the total cohort of 8430 patients, 5519 had known CVD diagnosis or symptoms suggestive of CAD, were being evaluated for non-coronary cardiac surgery, or were missing at least one patient questionnaire form. A further 248 participants were outside of the required age range for CVRS calculation, and 1850 had at least one missing required blood measurement required. Another one patient was excluded because of the absence of a recorded CACS. Therefore, the analysis cohort consisted of 828 asymptomatic individuals with no prior history of CAD and sufficient data to calculate their CVRS. Fig. 1 Final cohort extraction. PRC indicates Perth Radiological Clinic; CCTA indicates coronary computed tomography angiography; CVD indicates cardiovascular disease. Fig. 1 Cohort characteristics are summarized in Table 1 . The median age was 58.6 years (IQR = 52.0, 65.3), with 358 (43.2 %) females and 470 (56.8 %) males. Low CVRS (1–9 %) was the most common subgroup of calculated 5-year risk (239 (50.9 %) males and 244 (68.2 %) females). Atherosclerosis to any extent was identified in 78.5 % (369) of males and 50.0 % (179) of females. A non-zero CACS was found in 386 (46.6 %) patients in the whole cohort (66.8 % male), with a median Agatston Unit of 2 (IQR = 0, 112) for males and 0 (IQR = 0, 15) for females. At the time of the CCTA, 144 (17.4 %) patients were prescribed lipid-lowering medication. Table 1 Cohort clinical characteristics. Table 1 Males ( n = 470) Females ( n = 358) Age, y, median (IQR) 57.35 (5, 18) 60.18 (3,11) Hypertension, n (%) 183 (38.9 %) 140 (39.1 %) Hypercholesterolemia, n (%) 212 (45.1 %) 120 (33.5 %) Diabetes, n (%) 38 (8.1 %) 31 (8.7 %) Smoking, n (%) 193 (41.1 %) 120 (33.2 %) Blood pressure (mmHg), mean (SD) 131.84 (15.45) 130.64 (17.94) Total cholesterol (mmol/L), mean (SD) 5.90 (1.64) 6.03 (1.53) HDL cholesterol (mmol/L), mean (SD) 1.33 (0.32) 1.67 (0.46) Risk factor count a , n (%) Low 82 (17.4 %) 216 (60.3 %) Moderate 344 (73.2 %) 139 (38.8 %) High 44 (9.4 %) 3 (0.8 %) Cardiovascular risk score (%), median (IQR) 9 (5, 18) 5 (3, 11) Cardiovascular risk score category b , n (%) Zero 1 (0.2 %) 12 (3.3 %) Low 239 (51.0 %) 244 (68.0 %) Moderate 84 (17.9 %) 29 (8.1 %) High 145 (30.9 %) 74 (20.6 %) Degree of atherosclerosis category, n (%) Low plaque-burden 147 (31.3 %) 72 (20.1 %) Moderate plaque-burden 85 (18.1 %) 39 (10.9 %) Extensive plaque-burden 137 (29.1 %) 68 (19.0 %) No atherosclerosis 101 (21.5 %) 179 (50.0 %) IQR indicates interquartile range, expressed as 25th percentile, 75th percentile; SD indicates standard deviation; HDL indicates high-density lipoprotein. a Low (1–2 risk factors), moderate (3–4 risk factors), high (5–6 risk factors). b “Zero” (<1 %), low (1–9 %), moderate (10–15 %) and high (>15 %) risk of experiencing a cardiovascular event in a 5-year period. The continuous variable of CVRS was not normally distributed for males (W = 0.83, p < 0.001) or females (W = 0.69, p < 0.001), and homogeneity of variance was not assumed for males (F(1,468) = 8.69, p = 0.003) but was assumed for females (F(1,365) = 3.70, p = 0.055). The median risk score for males and females was, for the atherosclerosis group, 11 % and 7 %, and for the no atherosclerosis group, 5 % and 4 %, respectively. Significant differences in CVRS distribution were found between those with atherosclerosis and those without, in males (Mann-Whitney U 95 % CI: U = 26,533.00, n 1 = 369, n 2 = 101, p < 0.001) and females (U = 20,579.00, n 1 = 179, n 2 = 179, p < 0.001), shown in Fig. 2 . Fig. 2 Distribution of cardiovascular risk score in individuals with and without atherosclerosis. Evidence of atherosclerosis indicates a detected abnormality in coronary computed tomography angiography and/or a non-zero coronary artery calcium score. Fig. 2 The mismatch between CVRS and degree of atherosclerosis is shown in Fig. 3 . Of those with extensive atherosclerosis (137 males, 68 females), 47 (34.3 %) males and 38 (55.9 %) females had a low CVRS (1–9 %), and of those with no atherosclerosis (101 males and 179 females), 15 (14.9 %) and 27 (15.1 %) respectively were classified as high risk (>15 %). Additionally, of those with a low CVRS (238 males, 245 females), 47 (19.7 %) males and 38 (15.5 %) females had extensive atherosclerosis and of those with a high CVRS (147 males, 72 females), 15 (10.2 %) and 27 (37.5 %) respectively had no atherosclerosis. For males ( n = 470), CVRS overestimation occurred in 43.0 % (202), underestimation in 25.7 % (121) and correct estimation of atherosclerosis risk in 31.3 % (147). In females ( n = 358), overestimation occurred in 55.9 % (200), underestimation in 21.5 % (77) and correct estimation in 22.6 % (81). Fig. 3 Proportion of individuals in each comparison group between cardiovascular risk score and degree of atherosclerosis categories. Category of risk score is “zero” (<1 %), low (1–9 %), moderate (10–15 %), high (>15 %) risk of experiencing a cardiovascular event in 5 years. Fig. 3 A significant relationship (Monte-Carlo 95 % CI: male, p < 0.001; female, p = 0.024) but little to no agreement (Cohen's kappa: male, κ = 0.149; female, κ = 0.096) was found between degree of atherosclerosis and CVRS for both sexes, with low data accuracy (male, 2.2 %, κ 2 = 0.022; female, 0.9 %, κ 2 = 0.009). For CACS, a significant relationship (male, p < 0.001; female, p = 0.008) but little to no agreement (male, κ = 0.108; female, κ = 0.084) was found with CVRS for both sexes. Similarly for plaque extent, a significant relationship was found with CVRS for males ( p < 0.001), but an insignificant relationship for females ( p = 0.121), with little to no agreement for males ( κ = 0.110) and females ( κ = 0.080). Finally for stenosis severity, a significant relationship (male, p < 0.001; female, p = 0.012) but little to no agreement (male, κ = 0.035; female, κ = 0.030) was found with CVRS for both sexes. The relationship between the degree of atherosclerosis and CVRS is shown in Fig. 4 , with line thickness indicating relationship strength and color representing a match or mismatch. In males, a strong relationship was observed between low risk and no atherosclerosis as well as low burden atherosclerosis (count = 75, 78), and between extensive atherosclerosis and high risk (count = 54). However, a moderate relationship was observed between extensive atherosclerosis and low risk (count = 47) and between low burden atherosclerosis and high risk (count = 45). For females, the only strong relationship was between no atherosclerosis and low risk (count = 132). The relationship between low risk and extensive atherosclerosis (count = 38) and high risk and no atherosclerosis (count = 27) was stronger than between high risk and extensive atherosclerosis (count = 19). As shown in Fig. 5 , for both sexes, there is a substantial proportion of low-risk individuals with demonstratable atherosclerosis. Of the 369 males and 179 females with atherosclerosis, 44.2 % (163) and 65.4 % (117) were classified as zero or low risk, respectively. The sensitivity of the risk calculator for males and females was determined to be 89.2 % and 61.4 %, and the specificity 31.8 % and 54.5 %, respectively. Fig. 4 Relationship maps comparing degree of atherosclerosis categories and cardiovascular risk score. The red and orange lines represent a mismatch (red = severe, orange = not severe), and the green lines represent a match, between the categories. The line thickness represents the relationship strength (relationship count, the number of patients in that comparison group) and the circles represent categories of each variable, with the size correlating to patient count. Fig. 4 Fig. 5 Proportions of individuals classified as high risk and low risk, and who had detected atherosclerosis. CVRS indicates cardiovascular risk score, and high-risk is moderate-high (>9 %) and low-risk is zero-low (≤9 %). Created using custom srd software (Roger Marshall, PhD). Fig. 5 Assuming the 147 males and 72 females with a high CVRS remain in this category even if CCTA did not demonstrate atherosclerosis, we calculated the potential for CCTA to reclassify the 239 males and 257 females with a CVRS of ≤9 % according to their actual observed plaque-burden. Identifying a moderate to high plaque-burden would reclassify 84 males (35.1 %) and 63 females (24.5 %) to a CVRS of >9 %. Identifying any atherosclerosis would reclassify 163 men (68.2 %) and 117 women (45.5 %) to a CVRS of >9 %. The risk factor count and degree of atherosclerosis mismatch is observed in Fig. 6 . Of the 137 males and 68 females with extensive atherosclerosis, 16 (11.7 %) and 32 (47.1 %) respectively had only one or two risk factors. Of the 101 males and 179 females without atherosclerosis, 76 (75.2 %) and 55 (30.7 %) respectively had more than three risk factors. A non-significant relationship between risk factors and atherosclerosis was found for males (Monte-Carlo 95 % CI: p = 0.101) and a significant relationship for females ( p < 0.001), but little to no agreement in both sexes (Cohen's kappa: male, κ = 0.027; female, κ = 0.044), indicating low data accuracy (male, 0.1 %, κ 2 = 0.001; female, 0.2 %, κ 2 = 0.002). Fig. 6 Comparison of risk factor count and degree of atherosclerosis categories. Risk factor count is categorized as low (1–2 risk factors), moderate (3–4 risk factors) and high (5–6 risk factors). Fig. 6 Of the total cohort with missing data , 2447 (44.8 %) were female and 3016 (55.2 %) were male, with a median age of 59.7 years (IQR = 52.4, 66.3). The age distribution was similar to the analysis cohort for both sexes (males, U = 748,426.00, p = 0.065; females, U = 466,714.50, p = 0.081). In this cohort, no atherosclerosis was reported in 1051 (43.0 %) females and 739 males (24.5 %). To our knowledge, this is the first study to demonstrate a substantial mismatch between predicted CVRS and the extent of CAD identified using CCTA in asymptomatic individuals with no prior CAD history. Although subclinical CAD was detected more frequently in those with higher risk scores, agreement between degree of atherosclerosis and CVRS was poor. Importantly, a low CVRS was calculated in 34 % of males and 56 % of females found to have extensive CAD. At least some mismatch was identified in 69 % of males and 77 % of females, highlighting that CVRS is not reliable in predicting CAD. This has important implications in clinical practice, particularly in apparently low risk individuals who may be falsely reassured against the presence of CAD. Pen et al. used a similar methodology to investigate the discordance between Framingham risk score (FRS) and CCTA-defined atherosclerosis burden, however most patients in their US-based cohort had chest pain or other symptoms suggestive of CAD. Consistent with our findings, they found a weak association between plaque-burden and FRS, with 47.6 % of low FRS-risk patients having detected atherosclerosis. Their analysis compared FRS to total plaque score (TPS) obtained by CCTA, and did not consider plaque location, stenosis severity or the number of affected arteries, as addressed in the present study. Most prior studies assessing CVRS reliability have investigated subsequent cardiovascular event rates instead of the presence and burden of atherosclerosis in asymptomatic individuals. It is consistently demonstrated that untreated high-risk individuals have high event rates , highlighting the importance of aggressive risk factor reduction. Conversely, relatively few CV events occur in low-risk individuals, however due to the high proportion of apparently low-risk individuals in the community, the largest total number of events occur in this group . This highlights a significant unmet need to identify those individuals not traditionally considered high-risk, but who will subsequently have a cardiovascular event . Vernon et al. reported that, of those hospitalized with an MI, 25 % did not have any traditional risk factors and had a higher mortality risk than those with at least one risk factor . Similarly, we demonstrated that 12 % of males and 47 % of females with extensive atherosclerosis had only one or two traditional risk factors, with poor agreement between CVRS and degree of atherosclerosis for both sexes. Although better than chance alone, CVRS used in isolation would leave many non-high-risk patients with undetected CAD, including apparently low-risk individuals with extensive disease, potentially leading to a fatal coronary event. Additionally, we observed CVRS overestimation in approximately half of the cohort, leading to potential over-treatment in the absence of disease. This overestimation tendency has been reported by others , however we found a larger proportion of overestimated patients, suggesting that CVRS may have a more important role in prediction of a future cardiovascular event risk, than that of subclinical CAD. Therefore, mechanisms other than atherosclerosis may contribute to cardiovascular events, although identifying asymptomatic CAD may be seen as an opportunity to detect-and-treat prior a fatal event. Recent findings by Medina et al. demonstrated reasonable performance of CVRS in predicting future cardiovascular events, with only 2.3 % of non-high-risk individuals experiencing an event, although CCTA or CACS were not examined in this study. Outside of the potential benefits of CCTA, CACS has emerged as the single best predictor of subsequent coronary events in asymptomatic individuals . Hoffman et al. demonstrated that in those with a low CVRS (<6 %), 32 % of men and 23 % of women had a non-zero CACS. Further, Venkataraman et al. identified 77 % of patients with CACS>100 that were classified as low-risk. Hence, CACS has been recommended to form part of standard cardiovascular risk assessments in middle aged and older non-high-risk individuals . Beyond this, Hadamitzky et al. reported that CCTA provides better prediction of future cardiovascular events than CVRS and reinforces that identification of subclinical atherosclerosis may have important prognostic implications in otherwise low-risk patients. The mismatch between CVRS and presence and extent of CAD is likely explained by the combination of environmental influences and non-traditional risk factors that can influence atherosclerosis development, such as additional lipoproteins not considered in CVRS [such as Lp(a)], genetic loci , psychosocial factors , and exercise and diet . However, it is not currently viable to assess all potential non-traditional risk factors across a population. Similarly however, although CCTA may reveal the final atherosclerotic result of exposure to risk factors, it is also not feasible to deploy such imaging across a population of apparently low-risk individuals. Additional research is needed to identify which apparently low-risk individuals would benefit from a more intensive search for atherosclerosis including CCTA. It is important to note that we excluded patients with known CVD and symptoms suggestive of CAD. In addition, this study has limitations to its applicability to a broader population. A selection bias is likely since patients were referred for CCTA presumably because of clinical uncertainty by the referring doctor. Further, since CCTA for a cardiovascular risk assessment in asymptomatic individuals is not Medicare-funded in Australia , the scan cost could be a potential source of bias. While the proportion of individuals with atherosclerosis in this cohort is likely larger than that of the general population, the study still illustrates the limitations of current CVRS techniques compared with the gold standard of CCTA for atherosclerosis assessment. Finally, we cannot exclude the possibility that the missing data, due to the retrospective nature of this study, may have influenced the measured outcomes. However, a sensitivity analysis confirmed that the excluded sample had similar characteristics to the final analysis cohort. In asymptomatic individuals with no CVD history undergoing CCTA, CVRS does not reliably predict the presence or extent of CAD. A mismatch between CVRS and the degree of CAD (identified by CCTA and CACS) was found in majority of the cohort, and extensive CAD was observed in a significant proportion of individuals who otherwise appeared to be at low-risk. This highlights a potential role for CCTA in non-high-risk individuals with clinical uncertainty, where reclassification of risk based on cardiovascular imaging may influence treatment decisions (i.e., initiation of lipid-lowering therapies). Randomized prospective studies are required to address whether such an approach will improve outcomes in Australia. | Review | biomedical | en | 0.999995 |
PMC11698939 | The modification of feedstocks or pristine biochar can be classified into three different types: activated biochar (AB), engineered biochar (EB), and functionalized biochar, based on the methods used to alter the biochar. AB is produced by exposing biochar to high temperatures in the presence of certain gases (such as CO 2 or steam), which enhance its physicochemical properties , while EB is modified by changing the feedstock or adding compounds like metal ions (MgCl 2 and ZnCl 2 ) . Biochar modified by adding functional groups to enhance its chemical properties is referred to as functionalized biochar . However, the term B generally distinguishes between pristine and altered biochar. Given the limitations mentioned earlier associated with the utilization of pristine biochar in soil, there is a notable shift toward the adoption of MB . Thus, by adjusting the physicochemical properties of biochar, it becomes feasible to amplify its beneficial effects while concurrently mitigating any adverse impacts on plant growth and soil health . However, before choosing any modification technique, it is imperative to have a comprehensive understanding of the various physicochemical properties of biochar that have been discovered and subjected to modification, as well as their respective advantages and limitations. Thereby, this review article provides an in-depth discussion of recent advancements in the understanding of physicochemical characteristics of MB, including surface area, porosity, alkalinity, pH, elemental composition, and functional groups . Additionally, it also highlights the factors that can influence the efficacy of MB in soil . Fig. 1 The number of published articles per year for a period from 2010 to 2023 retrieved using keywords “activated biochar,” “functionalized biochar,” “engineered biochar,” and “pristine biochar.” MB: modified biochar Fig. 2 Fate and limitation of biochar in agricultural soils. Fig. 3 Physicochemical characteristics and analytical techniques of modified biochar. Fig. 4 Factors impacting the efficacy of modified biochar following soil applications. (For color interpretation, the reader is referred to the Web version of this article) Fig. 5 Factors impacting the efficacy of modified biochar following soil applications. (For color interpretation, the reader is referred to the Web version of this article) Several researchers have reported numerous findings regarding the fate and beneficial aspects of biochar. Apropos of soil physical properties, biochar amendment has been shown to improve soil pore structures and aggregate stability, which apparently upgrades soil water holding capacity. However, soil texture contributes a critical role in acquiring such benefits; for instance, biochar amendment is more noticeable in sandy soils than in clay soils. Hence, the positive feedback of biochar is not similar for all soil types . Similarly, depending on pH and EC, different soil types react distinctively to the ash content delivered by biochar. The high levels of ash content can pose detrimental impacts on plants and may produce reactive oxygen species in soil . Similarly, the agronomical traits of plants can also be significantly improved by biochar application through upgrading nutrient cycling and soil environment . However, such positive effects may vary depending on the plant species and the targeted part of the plant because different plant species have distinct nutrient requirements, and biochar may not necessarily provide all the required nutrients. Biochar may also affect soil environmental conditions and the microorganism's diversity by disturbing the decomposition of organic matter, also known as the priming effect . The impact may arise from changes in microbial communities induced by biochar, potentially resulting in either heightened or diminished decomposition of native soil organic carbon (SOC), consequently influencing its stability . For example, microorganisms, specifically fungi species such as Ascomycota and Basidiomycota , are more effective at breaking down biochar and releasing nutrients for plant uptake, while others may be less efficient . Similarly, Dai et al. found a significant decrease (11%) in the relative abundance of Basidiomycota by easily mineralizable C from biochar. The aforementioned limitations can be managed by modifying biochar to enhance specific chemical properties such as pH, cation exchange capacity (CEC), surface area, functional groups, and nutrient content that are beneficial for the targeted plants or specific plant parts. For example, the modifying process introduces new substances such as nitrogen, phosphorus, and potassium, which can minimize the adverse effects on SOM (soil organic matter) decomposition and soil microbial communities by interrupting C:N:P ratios . Additionally, increasing the pH and CEC of biochar by activation and/or engineering methods can enhance its ability to retain nutrients and prevent leaching . Previously, Xu et al. reported that the combined application of biochar and phosphorus fertilizer in saline-sodic soil depicted a considerable decline in available P levels by accelerating phosphate precipitation/sorption processes owing to its high surface area and adsorption capacity . Hence, it can act as a sink rather than a P source for plants. Biochar modification by altering its surface area, pore size distribution, and functional groups, can increase or decrease its adsorption capacity for specific nutrients and help to regulate its selectivity for specific soil nutrients while reducing its affinity for others . Previous studies enable us to refer that the capacity of biochar to adsorb essential nutrients such as nitrogen (N) or iron (Fe), sometimes surpasses the limits that are conducive to plant growth . If biochar adsorbs nutrients exorbitantly, it can lead to a reduction in their availability for plants, which can result in stunted growth or even plant death . Therefore, it is essential to carefully manage certain physicochemical characteristics to ensure that it does not have a negative impact on plant growth by competing with the plants for these essential nutrients. This can be achieved by using MB that has been specifically tailored to the needs of the soil and the plants being grown . The physicochemical characteristics of MB (Table 1 ) vary based on various alteration techniques, pyrolysis temperature, and the kind of feedstocks and modifying compounds (Table 2 ). This section discusses how different modification methods and agents improve the physical and chemical characteristics of MB . i. Surface Area and Pore spaces Table 1 Overview of essential characteristics of biochar Property Definition Reference Anion exchange capacity (AEC) The potential of biochar to adsorb anions Ash content These residues are non-combustible byproducts resulting from pyrolysis, originating from mineral and inorganic constituents of biochar Cation exchange capacity (CEC) The potential of biochar to adsorb cations Density Density refers to the mass of a substance divided by its volume, factoring in any spaces between particles. A lower density indicates a lighter weight per unit volume Electric conductivity It signifies the conductivity of a material, indicating its capacity to conduct electric current Elemental composition It represents the mole ratios of oxygen (O), carbon (C), hydrogen (H), nitrogen (N), and sulfur (S). Typically, the ratios of O/C and H/C moles are used as indicators of the degree of carbonization, where low ratios often indicate a higher stability of biochar Fixed carbon content The extent of carbonization can be inferred from the fixed carbon content, which is derived by subtracting the percentages of moisture, volatile matter, and ash from a given biochar sample. This calculation is expressed by the formula: FC (%) = [100 − (VM + Ash)] Heating value A metric denoting the utmost thermal energy accessible from complete combustion, often referred to as energy content, is defined as the heat produced per unit mass or per unit volume Hydrophobicity The attraction or affinity of biochar toward the water Mass yield An indicator of biochar production efficiency, denoting the ratio of the mass of pyrolyzed products to the mass of raw biomass pH-value An indicator of the alkalinity or acidity of biochar, expressed as pH, which is calculated using the formula pH = − log[H +] Pore volume and pore size distribution The cumulative volume of pores and voids within biochar defines its pore space. The distribution of pore sizes signifies the proportional occurrence of each pore size within the structure of biochar Porosity The ratio of the volumes of voids or pore space within a substance divided by the total volume of that substance is known as the porosity Specific surface area (SSA) The SSA of a substance, calculated as the total surface area per unit mass, serves as an indicator of both adsorption capacity and water retention ability in biochar Stability The percentage of original carbon content that remains following both abiotic and biotic degradation signifies the recalcitrance of the material in specific applications, such as carbon sequestration, under different conditions and time frames Surface functional group The functional groups found on the surface of biochar, such as carboxylic (-COOH), hydroxyl (-OH), amine, amide, and lactonic groups, enhance its sorption properties. These groups indicate the biochar's capacity to adsorb organic compounds and pollutants effectively, as well as its catalytic performance The functional groups present on the surface of biochar, including hydroxyl (-OH), carboxylic (-COOH), amine, lactonic, and amide groups, play a crucial role in enhancing their sorption properties. These groups are indicative of the biochar's ability to effectively adsorb organic compounds and contaminations, as well as its catalytic performance Water holding capacity The capacity of biochar to absorb and retain water Table 2 Overview of specific physicochemical characteristics of modified biochar used as a soil amendment Raw material Feedstock Activation Pyrolysis Temperature (°C) Modification Treatment SA (m 2 g −1 ) pH CEC (cmolc kg −1 ) C H O N Reference Plant Water hyacinth O 450 Post-treatment 32.48 10.3 3.53 46.7% – – 2.53% Fe 155.91 9.4 6.35 50.3% – – 3.57% Mn 34.34 9.4 3.73 47.6% – – 3.05% Zn 95.65 8.8 6.04 50.4% – – 3.14% Cu 62.11 9.4 3.93 49.1% – – 3.46% Pine chips HCl 400 Post-treatment - 2.5 17.4 608 g kg −1 – – 1,372 µg g −1 Peanut hull - 2.5 15.7 625 g kg −1 – – 1,8 µg g −1 Rice straw FeOS 300 Post-treatment 37.4 - - 37.4% 3.9% 0.8% 34.6% FeCl 3 40.9 - - 40.9% 4.7% 0.96% 30.8% Fe 38.5 - - 38.5% 3.4% 0.76% 23.6% Fe 3+ ( 1%) 500 Post-treatment 23.3 9.9 - 46.4% 2.8% 16.2% 2.23% Fe 3+ ( 5%) 26.4 5.7 - 43.8% 2.7% 15.8% 2.27% Fe 3+ ( 10%) 7.3 3.1 - 3.25% 2.5% 15.9% 1.72% FeCl 3 (1%) 14.6 10.2 - 45.5% 2.0% 11.2% 2.23% FeCl 3 (5%) 5.9 3.4 - 44.2% 2.1% 12.0% 2.32% FeCl 3 (10%) 5.7 3.1 - 44.1% 2.0% 13.8% 2.40% Corn stem Fe–Mn 600 Post-treatment 60.67 - - 72.6% 2.2% 4.7% 1.2% Platanus orientalis L Fe 650 Post-treatment 74.5 4.4 - 59.9% 0.9% – 2.2% Corn straws FeCl 3 600 Pre-treatment 4.1 2.5 - 38.0% 1.4% 27.7% 1.0% Rice hull NaOH 450 Post-treatment 396 6.5 3.2 77.9% 3.4% 15.7% 1.7% Cracking crop straws Commercial 500 - 22.9 8.3 – 414.7 g Kg −1 – – 14.36 g Kg −1 Rice straw Thiol 500 Post-treatment 0.34 2.3 – 43.7% 0.6% – – Wheat straw Mg 600 Post-treatment 292.5 – – 54.5% 2.3% 15.4% 0.5% Al 169.6 – – 46.8% 3.1% 16.9% 0.3% Mg–Al 268.5 – – 43.0% 2.0% 12.9% 0.4% Microbial 600 Pre-treatment 3.77 9.7 – 48.5% 5.8% – 0.6% Corn straw KMnO 4 600 Post-treatment 3.18 10.7 – 73.0% 0.7% 10.9% 0.3% Rice husks FeCl 3 600 Pre-treatment - 7.8 – 34.5% 1.0% 11.3% 0.3% Platanus orientalis L FeCl 3 650 Post-treatment 74.5 10.6 59.9 2.2 0.9 Carrot pulp Thiourea CH 4 N 2 S 550 Post-treatment – 9.1 59.18 47.2% 4.1% 23.2% 8.9% Cotton straws H 3 PO 4 500 Pre-treatment – – – 67.8% 4.1% 14.3% 0.11% NaOH – – – 68.2% 3.9% 6.5% 0.18% Corn straw Fe (NO₃)₃ 600 Post-treatment 207 9.8 1.0 68.0% 18% – 2.4% Animal Poultry manure Chitosan 450 Post-treatment 3.7 10.4 – 11.3% 1.2% – – Sheep manure 5.2 10.4 – 88.6% 1.0% – – Pig carcass FeCl 3 650 Post-treatment 18.4 10.6 30.8 1.3 2.1 CEC Cation exchange capacity, SA Surface area The specific surface area (SSA) and pore structure of biochar play a significant role in adsorption and regulating the nature of biochar as hydrophilic or hydrophobic . The general purpose of modification in pristine biochar is to expand its surface area, which ultimately modifies its functional groups and enhances its magnetic performance and catalytic capacity . Many studies showed that pyrolysis temperature and functionalization with innovative materials can enhance the SSA and porous structure of biochar . Rong et al. observed a significant surge in surface area in pre-mixed banana peel biochar with Fe 2 O 3 after hydrothermal carbonization. Their results illustrated an increment of 407, 504, 451, and 446 m 2 g −1 by combining with 0.05–1.0 g of the precursor solution, respectively. Similarly, Park et al. reported that during high pyrolysis temperatures (500–600 °C), the volume of pores in sesame straw increased from 0.0716 to 0.1433 cm 3 g −1 . Furthermore, in another research, it is depicted that the surface area of Saccharina japonica- derived biochar was positively influenced by the temperature, resulting in a notable increase (2.9–175 m 2 g −1 ), with the highest surface area achieved at 500 °C noted that ZnCl 2 − EBcan enhance the pore size up to 0.2–0.9 cm 3 g −1 . The BET (Brunauer, Emmett, and Teller) approach is frequently used to determine the SSA of biochar. In this method, the amount of N 2 adsorbed on the surface of the biochar is evident at low temperatures (77 K) . Apart from modification by nano-scale zero-valent iron and the effects of pyrolysis temperature, the subsequent impregnation of biochar with Mn or Mg enhances the surface area from 209.6 to 463.1, 12.68 to 174.29, and 244 m 2 g –1 , respectively . On the contrary, several studies have documented a decrease in the SSA of functionalized biochar. The decline in surface area could potentially be attributed to the pore openings or pores being obstructed by chitosan . Recently, manganese oxide activation revealed a considerable decrease in SSA ranging from 60.97 to 3.18 m 2 g −1 . Another research found that functionalized biochar through sulfur significantly affected surface area in contrast to pristine biochar. The highest surface area was observed in biochar generated at 500 °C and 700 °C (382 and 404 m 2 g −1 ), whereas functionalization with sulfur led to a significant reduction in surface area to 10.06 m 2 g −1 and 5.10 m 2 g −1 . Hence, it is crucial to thoroughly evaluate the characteristics of the final product concerning the impact of the modification process. ii. Elemental Composition Generally, the pristine biochar comprises 2–5% H, 45–60% C, and 10–20% O. Although the individual components employed to develop the product vary widely and rely on the feedstock. In order to assess the degree of hydrophobicity and carbonization in biochar, researchers commonly employed the O/C and H/C molar ratios . Additionally, it includes minerals, such as Si, P, Ca, Al, and K, that are prominent inorganic components of biochar . Many studies highlighted that type of feedstock, pyrolyzing procedures and functionalization may modify the properties of pristine biochar . In one study, Mn-oxide-MB illustrated a surge in O (10.9%) and Mn (7.41%) concentrations. Meanwhile, similar research observed that C, H, and N levels declined from 85.3–73.0%, 1.75–0.33%, to 0.80–0.72% . This reduction was attributed to the activation process wherein previously MB was exposed to additional high temperatures, leading to the further breakdown of C, H, and N and their conversion into ash . Furthermore, Wu et al. also reported a decline in H and C contents when biochar was magnetically modified with FeOS, Fe, and FeCl 3 . In FeOS biochar, C and H contents were the highest at C at 12.65% and 1.23%, while the lowest was in FeCl 3 -engineered at 9.16% and 0.28%, respectively. In addition to the mentioned modifications and their effects on the elemental constitution of biochar, various additional substances, such as chitosan and alkali/acid, have also demonstrated beneficial outcomes. Chitosan amendment resulted in a lower C percentage and increased H, O, and N ratios, confirming the inclusion of treated material on the surface of biochar . iii. Alkalinity and pH The pH of typical biochar usually varies from neutral to alkaline and is highly dependent on the type of feedstock, thermochemical process, and functional material, while studies about acidic biochar are also present . During the high pyrolysis temperature, the acidic functional groups (bionic acid) decompose, elevating the pH of biochar and causing an increase in inorganic alkali metal ions . Additionally, several organic functional groups, including –COOH, –COO, –O, and –OH, can similarly raise the pH of biochar . In another study, Zhou et al. recorded pH values of different biochars prepared from bamboo (7.9), sugarcane bagasse (7.5), hickory wood (8.4), and peanut husk (6.9), but after chitosan modifications, pH values changed to more alkaline 8.2, 8.1, 8.6, and 7.3, respectively. Similarly, the pH of corncob biochar produced at 600 °C was shown to be neutral (7.17), but after being modified by Mg-oxide, it reduced considerably to 10.4 . On the contrary, FeCl 2 -impregnated biochar exhibited an acidic characteristic (4.87) contrasted to conventional biochar (10.7) . Moreover, hydrophilicity, hydrophobicity, sorption, and adsorption can be influenced by organic groups and linked with the buffering action of acid and base. Additionally, the organic groups present on the biochar surface carry negative charges, increasing the CEC . As the CEC of biochar increased, it ultimately surged in adsorption capacity . Biochar modification by surface oxygenation via dry ozonization proved a promising technique for enhancing the CEC 10 times, in contrast to conventional biochar . For instance, biochar derived from the limb of a pine tree and subjected to ozone gas for 1.5 h, the CEC of the biochar considerably elevated from 15.39 cmol kg −1 to 32.69 cmol kg −1 . Although this technique decreased the pH and demonstrated the oxygenic functional group formation of the biochar surface . iv. Functional Groups and Aromaticity Several functional groups are associated with the surface of biochar, such as carboxylic, hydroxyl, and phenolic functional groups that contribute significantly to the remediation of contaminated soils . Among them, the most prevalent are O-containing functional groups, which can additionally be classified into neutral, and alkaline groups according to their inherent characteristics. The carboxyl, lactonic, and phenolic groups are examples of acidic groups, while the chromene and pyrone groups are known as basic active sites . Their characteristics may be related to the carbon’s surface basic nature, which is more evident in carbon atoms without oxygen because of the existence of delocalized electrons . The pyrolysis temperature significantly influences the functional groups present on the biochar surface. It is found that C=C and –CH 2 functional groups could be successfully retained in pyrolysis, although C–O–C and –OH, C=O functional groups reduced with increasing pyrolysis temperature . Meanwhile, biochar’s water affinity, CEC, and polarity are regulated by oxygen-containing surface functional groups . Generally, the yield of biochar declines, while on the contrary, alkaline functional groups, pH, and ash concentration increase with increasing temperature . In a recent study, the adsorption capacity of MB derived from rice straw was observed to be higher than that of cotton straw-MB, likely due to the presence of additional functional groups . The aromatic π-system is known to be involved in significant types of noncovalent specialized engagements termed electron donor–acceptor (EDA) interactions . The aromatic structures of biochar can significantly enhance pollutant adsorption, as they act as electron donors or acceptors and create bonds with the pollutants in soil . Usually, the carboxyl functional groups on the surface of carbonaceous adsorbents serve as electron acceptors, whereas the hydroxyl groups behave as electron donors . The aromaticity of biochar, determined using O/C and H/C ratios is highly influenced by pyrolysis temperature. The biochar derived from plant residues contains higher aromaticity due to high carbon material concentration. The cellulose and lignin decompose into small molecules that ultimately reduce H/C and O/C ratios as a result of depolymerization or dehydration. On the other hand, biochar from animal fecal and sludge does not possess any lignocellulosic molecules and hence avoids the depolymerization process . In a recent sulfamethoxazole structural analysis research, it was observed that the N-heteroaromatic ring, unprotonated sulfonamide group, and amino functional group act as significant π -electron acceptors, although they possess a high capacity to donate electrons . Similarly, Zhao and Zhou also reported π − π EDA interactions between biochar aromaticity and sulfamethoxazole. However, potential electron-donating groups identified include the phenolic −COOH−C−O and −OH groups . v. Hydrologic Properties The ability of MB to resist water is a crucial quality that has a significant impact on soil water retention . But the water retention property of biochar is highly dependent on the source of feedstock, synthesis technique, wetting characteristics, and particle size . The temperature during pyrolysis significantly influences the hydrologic characteristics when biochar is applied to soils . The most favorable hydrologic qualities were reportedly found in acid-functionalized biochar generated at higher temperatures (400–600 °C); in such amendment, water holding capacity was 18.45–22.45% higher than the control. This surge in water retention can be a result due to (i) high surface area and pore spaces and (ii) more hydrophilic functional groups that aid functionalized biochar . Besides, Zn (23.85%) and Fe (22.45%), impregnated biochars also exhibited a considerable increase in water-holding capacity in soil against soil gravity losses —the mineral formation of Fe and Zn aids in water retention by increasing chemisorption and physisorption. For instance, hematite surfaces possess the capacity to sorb water 23–24 A 2 per molecule, allowing it to adsorb the polar water molecules . In contrast, the hydrologic properties of biochar are found to be independent of soil types, such as coarse-grained (non-cohesive) soil or fine-grained (cohesive) soil . The mechanical resilience of aggregates at both micro and macro-scales was enhanced in biochar-amended soils, leading to notable improvements in cohesion and compressive behavior . There are possibilities that the hydraulic characteristics of soil that have been treated with EB extensively correlate with the quality and content of biochar, but these interactions are inadequately understood . vi. Complexation and Van der Waals forces Complexation is a special ability of biochar for adsorption that indulges ligand exchange, and bridging ligands are used to transport electrons . The electron donors and acceptors engage to produce different compounds during the surface complexation. During the formation of these surface compounds, several polyatomic structures significantly improve the sorption potential of biochar . Besides, chelation is an exceptional kind of complexation that is described as an equilibrium process and distinguished by the development of a complex between organic molecules with more than one functional group (multiple bounds) and a single central atom . The functionalized biochar contains a larger surface area and more functional groups than pristine biochar, such as carboxylic, phenolic, and lactonic functional groups that act as electron acceptors while the product of low pyrolysis possesses more electron-donating functional groups including amino –NH 2 and hydroxyl –OH . The complexion potential of biochar can improve if it is pyrolyzed in the presence of oxygen because of more surface oxidation. It is observed that plant-derived engineered biochars have higher complexion capacities for heavy metals in soils as compared to poultry litter and dairy manure-derived biochars . The van der Waals forces, also known as hydrophobic interactions, refer to intermolecular interactions that bind molecules together. These interactions are divided into two types: (i) weak London dispersion forces and (ii) stronger dipole–dipole forces . The van der Waals forces possess relatively modest energy about 0.4–4 kJ mol −1 ), while most of the sorbates are associated with biochar nonspecifically (weak London dispersion forces) . These forces specifically contribute to interactions between carbonaceous sorbents and ionic organic compounds because of the larger molecular size of IOCs as van der Waals forces increase with increasing contact area, and the high van der Waals coefficient of activated carbon (graphite) . It is noted in a study that ZnCl 2 -MB enhanced the pore size up to 0.2 cm 3 g −1 to 0.9 cm 3 g −1 and the adsorption capacity of biochar via van der Waals forces . Similarly, montmorillonite-biochar composites pyrolyzed at 400 °C were also found to be highly effective for ammonium adsorption via Van der Waals interaction . Usually, the adsorption of organic pollutants on biochar occurs via weak physical adsorption without any strong chemical bonding. These weak bonds include hydrogen bonding, van der Waals forces, hydrophobic interactions, and electrostatic forces . The flexibility and interaction of biochar with soil are highly influenced by its various physical and chemical characteristics. Hence, a deep structural and physical analysis via appropriate methods has been proven beneficial . If left unaddressed, the presence of a non-carbonized layer may lead to fluctuations caused by soil contaminants or other materials, potentially yielding inaccurate results . A detailed summary of the physicochemical attributes of the MB employed in soil, along with a comprehensive overview of the advantages and disadvantages of the analytical techniques, is presented in Table 3 . Table 3 Summary of various analytical techniques of modified biochar Technique Utilization Advantages Limitation Evaluated Biochars Reference Scanning Electron Microscopy (SEM) • It is used to determine morphological differences between biochar surfaces and pores arrangement • Limited need for conductive coatings due to high gas pressure • High magnification can be obtained in a range between 300000x and 500000x • Not applicable for organic contaminants • Magnetic biochar (γ-Fe 2 O 3 ) • Magnetite (Fe 3 O 4 ) biochar • MgO biochar nanocomposites Energy Dispersive X-ray (EDX) • It can evaluate the biochar surfaces and mapping of abundant elements • Have the potential to precisely identify and assess the arrangement and existence of elements in the scanned domain • High-speed data collection and processing • Easy to handle • Only detect the elements that have higher atomic numbers than boron • Highly dependent on probe current and voltage • Not applicable for organic contaminants • S, Mn and Fe MB • Magnetic biochar (γ- Fe 2 O 3 ) Transmission Electron Microscopy (TEM) • TEM is used to determine the surface morphologies like SEM • It can also analyze the lattice constant and chemical composition of biochar • Very High magnification can be obtained in the range between 200000 and 1000000x • A multifunctional instrument that can be used for spectroscopy and nanoscale imaging • It requires a high degree of vacuum to restrict the electron scattering they travel from the electron source to the electron optics • Fe 3 O 4 Biochar • Biochar nanoparticles (wheat straw) X-ray Diffraction (XRD) • It can analyze the composition of biochar, such as crystalline C or other materials • It is also used to determine organic compounds, including cellulose, hemicelluloses, and lignin, as well as inorganic compounds, such as oxides and sulfides carbonates • A non-destructive technique that can produce 3D characterization of interface structure • It can be used with different combinations of in-situ methods • In-situ methods and Time resolution is possible • Requires high-intensity X-ray beam • The usage is limited to access time to synchrotron source and single crystal surfaces • S and Fe 3 O 4 -MB X-Ray Absorption Near Edge Structure (XANES)/ Near Edge X-Ray Absorption Fine Structure (NEXAFS) • XANES/NEXAFS used to investigate the surface chemistry of highly complex types of C materials, for instance, charcoal • It can be used to identify the C species and stability with several structures at different pyrolysis time intervals (100–700 °C) • Direct structural determination of any matter and isotope • Oxidation state and spin state direct determination • Spectroscopy carried out in bulk gives an average structure • Very little information about the angle of the structure determines • Synchrotron X-ray source required • Fe- EB • KOH steam–activated pecan shell biochar X-ray photoelectron spectroscopy (XPS) • It is effective for surface characterization of biochar (surface elemental composition) • It can analyze chemical bonds, chemical, and the presence of distinct species of elucidated compounds on the surface of biochar • It can carry sensitive surface quantitative analysis such as material composition and empirical formula (without hydrogen) • Have the potential to analyze the material up to the depth of 1–10 nm • This technique is only applicable to solids since a high vacuum is needed • Highly time-consuming and cannot recognize hydrogen and helium atoms • FeCl 3 , FeSO 4 , Fe/Ca, and Mn EB • MnO/NiO biochar composite Fourier‐transform infrared spectroscopy (FTIR) • It can investigate mineralogy and chemical functional groups of biochar • Variation in the carbonation degree also makes it possible to determine • The analysis can be performed on any matter (solid, liquid, or gas) • Fast and non-destructive data acquisition and processing • Mapping with a good special resolution of large surface samples is possible • The interpretation of data is difficult, especially when working with a complex matter • Aqueous mixtures are complicated to investigate because water has a high infrared absorption capacity • DOM/Cu MB • Fe/Mn EB Raman spectroscopy • This technique is effective in quantifying the structural characteristics, especially graphite structures of biochar • Functional groups and crystalline C structures can be evaluated as well • A precise single-point assessment carried out with an excellent special resolution • Spectra possess quantitative and qualitative information • It is feasible to acquire insight into the functional groups in the polymer • Measurement parameters variation may affect the signal (e.g., laser wavelength) that negatively affects data interpretation • Fe/Ca-EB • (NH 4 ) 3 PO 4 impregnated biochar • Magnetic biochar (γ-Fe 2 O 3 ) X-ray fluorescence spectroscopy (XRF) • Commonly utilized to evaluate the compounds of biochar • Determination of inorganic compounds present on the surface of biochar • Very effective in quantifying the composition of biochar • More powerful and precise for inorganic materials determination as compared to XRD • Very costly • Fe-EB • N and O activated Solid-state nuclear magnetic resonance (NMR) • The structural composition of biochar and carbonization degree and stability can be determined by this method • It can be used to evaluate the contents of functional groups such as aromatic hydrocarbons, phenolic, methoxyl, and aliphatic in a biochar • Findings for stability have strong correlations with several other techniques • It gives deficient signals/noise ratio when encountering with high temperature pyrolyzed biochar • Signals obscured in the presence of ferromagnetic materials • NaOH- MB • CO 2 and steam-activated biochar • N and O activated biochars The presence of hydrogen (H + ) and aluminum (Al 3+ ) ions in the soil exchangeable sites causes acidity, severely affecting crop yield . Pristine biochar is primarily alkaline in nature . The pyrolysis temperature (> 400 ℃) produced biochar with alkaline pH . When applied to soil, organic compounds on the biochar surface will dissolve in water entering the pore spaces, increasing soil pH . Also, biochar increases the sorption of nutrients and decreases acidity in acidic soils . However, the effectiveness of biochar in changing soil pH in acidic soils depends on the composition, properties of feedstock, and pyrolysis temperature . Thus, several modifications of biochar have been developed. The activation of rice hull biochar by immersing with dimethyl dithiocarbamate sodium solution effectively decreased pH from 10.28 to 6.53 and improved soil properties . The application of Fe-MB in Cd-contaminated soils slightly increased pH units from 7.83 to 7.93 and helped to immobilize Cd in soil . In the case of red soils, the manganese-oxide-MB increased soil pH by 1.4 units while only 0.4 units increased in pristine biochar, thus preventing acidification in red soils . On the other hand, the pH of As-contaminated paddy soils (pH 7.12) were markedly decreased by Fe–Mn MB by around 0.75–1.16 units compared to the control . Similarly, It was observed that soil resistance to acidity was enhanced through the application of HNO 3 /H 2 SO 4 -MB . Furthermore, thiourea-modified poplar-bark biochar was applied to Cd-contaminated soil, resulting in a 17% increase in soil pH as compared to the control, which led to an increase in base cations in the soil . In multi-polluted soils, the magnetic biochar derived from eucalyptus wood and poultry litter increases soil-surged pH by 0.2–0.3 units . Likewise, the porous magnetic biochar from wheat straw was added to the soil and increased soil pH by approximately 6% . But in calcareous soils, steam-AB (acidic biochar) effectively decreases soil pH by 0.2–0.4 units , while in As-contaminated soil, Ca-MB causes an increase in pH . In contrast, rice straw MB with a 1:1 mixture of HNO 3 /H 2 SO 4 and 15% H 2 O 2 effectively improved pH buffering capacity and resistance to soil acidification compared to HCl-treated and unmodified biochar. The enhanced resistance was attributed to surface functional groups that increased soil resistance to acidification by generating protonation of organic anions, which retarded the decline in soil pH. However, HNO 3 /H 2 SO 4 MB showed a higher number of carboxyl functional groups compared to the 15% H 2 O 2 -modified biochar, resulting in greater resistance to soil acidification. The application of HNO 3 /H 2 SO 4 MB in paddy soil increased pH after wet-dry cycles, suggesting that this modification is an effective solution for remediating acidic soils. The underlying mechanism involves weak acid functional groups on the biochar surface that exist as organic anions in alkaline and neutral soils. Under acidic conditions, these anions protonate with H + and convert to neutral molecules, inhibiting soil acidification and preventing a decline in soil pH , Biochar application with a high surface area increases pore space and enhances soil moisture, which helps boost water retention capacity. Applying P-laden biochar considerably increased soil moisture by 93% over ordinary biochar when applied to acidic sandy soil planted with lettuce and improved seed germination and higher yield . Besides, soil moisture is enhanced by the EB derived from wood, switchgrass, and swine manure . It is claimed that nano-biochar contains larger pores and increases the number of small pores. Thus, it could increase the soil moisture content more than pristine biochar . Nano-biochar application enhanced soil water infiltration and soil moisture content . Similarly, nano-biochar was found to decrease soil water loss due to high surface area . Another, AB from the microwave catalytic process also increases water holding capacity by 98% more than ordinary biochar due to higher porosity, which shows a high correlation between WHC and micropore area . Similarly, An et al. studied biochar made from peach shells and pig manure, modified with H 3 PO 4 and KOH, and applied in four dosages (0%, 2%, 3%, and 8%). They found that H 3 PO 4 -modified biochar had superior water retention compared to KOH-modified and pristine biochar, while KOH modification reduced the hydraulic functional groups on the biochar surface. Pig manure biochar demonstrated higher crack suppression intensity than that of other functionally activated biochars. However, the study generally recommended a 5–8% dosage for enhancing water retention and minimizing cracks . iii. Dosage of application The majority of biochar studies focused on application rates between 5 and 50 tons ha −1 , resulting in increased crop yield and soil properties and remediating pollutants in soil . However, this is not applicable in some cropping systems due to the high cost of production and transportation . However, several studies reported that biochar modification and application to the soil at certain rates effectively improve soil characteristics . Magnesium-oxide-derived biochar applied to saline rice paddy soils at 4.5 Mg ha −1 increases rice yields and adsorbs more phosphates . Similarly, applying Mn-oxide-MB at an increasing rate (0.5%, 1%, and 2% wt/wt) surged rice biomass and decreased As concentration in the root and rice grains compared to conventional biochar . In a similar investigation, the application of 1% magnesium-impregnated biochar to soil (wt/wt) increased available P content in the surface soil by around 50% . On the other hand, Fe-MB applied to saline paddy soil at 4.5 Mg ha −1 increased P adsorption via co-precipitation of P to iron oxides, thus increasing P availability by 79–90% over control . The yield of Cd and As-contaminated rice enhanced by Goethite-MB at the rate of (0.5%, 1.0%, and 1.5% wt/wt), the shoot biomass and root biomass increased around 56–88% and 55–98%, respectively. Also, goethite-MB decreases Cd and As concentration in rice tissues, improves photosynthesis capacity, and reduces oxidative stress . Likewise, acidified biochar derived from various raw materials, including rice husk, sugarcane-bagasse, and wheat straw, applied in the soil at 1.5% wt/wt, increased plant nutrient uptake and biomass by around 40 and 30%, respectively . Nano-biochar applied to salt-affected soils at different rates (0, 0.10, 0.20, and 0.50% wt/wt) increased P adsorption capacity . In another study, it was observed that nano-Fe-MB applied to soil at 0%, 0.05%, 0.1%, 0.2%, 0.4%, 0.8%, and 1.6% wt/wt concentrations reduced soil pH, Cd concentration and enhanced soil CEC. The application of lower rates (0.2–0.4%) of nano-Fe-MB effectively decreased Cd toxicity in the soil and rice. However, high nano-iron-MB application rates (0.8–1.6%) stimulated Cd toxicity in leaves. Thus, finding the optimum application rates should be taken into consideration to avoid risk and toxicity . Nano-hydroxyapatite applied at different levels (1%, 5%, 10%, and 20%) in Pb-contaminated soils effectively immobilized Pb by around 44–57% after 28 days of incubation, effectively decreasing Pb toxicity in soils and accumulation in plants . iv. The ratio of functional material saturation The ratio of functional material to modify biochar was one of the primary gauges for the adsorption of pollutants . Biochar activation was done by soaking raw biochar in solution (30% H 2 O 2 , 1 M HCl, and 1 M KOH) (1:100 ratio) for 24 h to effectively adsorb Cr (VI). The ratio of Fe to nano-Fe-modified-biochar was 19.5%, effectively improving soil properties (CEC) and reducing Cd toxicity . Similarly, Fe and Al-MB, at the ratio of 1 mol L −1 in FeCl 3 and AlCl 3 , effectively increased soil pH from 6.93 to 7.30 and enhanced P retention capacity . Furthermore, magnetic biochar prepared by mixing biomass with a solution containing (a 1:1 molar ratio of FeCl 2 and FeCl 3 ) effectively remediates soil and adsorbed heavy metals, including As, Cd, and Pb . In a recent study, Mg–Al impregnated biochar at 1:20 g ml −1 (solid to liquid ratio) was observed to be adequate in adsorbing phosphate in the soil and contained a higher amount of oxygen functional groups (MgO and AlO) as compared to pristine biochar . Similarly, MB nanoparticles exhibited a maximum adsorption capacity of 147 mg g −1 , significantly higher than the 67.8 mg g −1 capacity of unmodified biochar . This enhancement is attributed to the nanoparticles increasing the surface area and the number of functional groups on the MB surface. Moreover, KOH modification of biochar resulted in a substantial increase (approximately 2.4 times) in specific surface area and more than a 50% improvement in the adsorption capacity for Cd 2+ and Cu 2+ ions. Specifically, pristine biochar had a specific surface area of 189 m 2 g −1 , whereas KOH-MB had a surface area of 455 m 2 g −1 . The Cd 2+ adsorption capacity of pristine biochar was recorded as 4.48 mg g −1 , while KOH-treated biochar achieved a capacity of 6.81 mg g −1 , representing a 50.7% significant increase. However, the Cu 2+ adsorption capacity of unmodified biochar was 2.64 mg g −1 , whereas the KOH-modified biochar showed a significantly enhanced capacity of 4.03 mg g −1 . v. Soil types The physicochemical properties of soil, including texture, structure, pH, and organic matter content, can influence the interactions between MB and soil, consequently affecting the availability and mobility of nutrients and contaminants. . It is worth noting that sandy soils with low CEC and organic matter content may have limited capacity for MB adsorption and nutrient retention. This, in turn, leads to an increased adsorption of MB and an improvement in nutrient retention . MB also increases the presence of organic matter. For instance, Kun et al. found a significant increase in soil C/N ratios and soil organic carbon after the application of Cd-binding biochar. Furthermore, observed a higher accumulation of soil organic matter (SOM) and organic carbon with the application of rice husk biochar modified with NaOH, HNO 3 , and dimethyl dithiocarbamate sodium (3% w/w). Similarly, Moradi and Karimi noted increased SOM and organic carbon with Fe-modified biochar (2% w/w). Higher organic matter was also observed with the application of 2% (w/w) Fe–Mn biochar , 1% (w/w) S-biochar , 1% (w/w) S-Fe biochar, 3% (w/w) , iron-modified biochar, 8% (w/w) thiourea-modified biochar , 8% (w/w) carrot pulp biochar, and 3% (w/w) iron-zinc oxide composite modified corn straw biochar, compared to pristine biochar . This variation can be attributed to the slower decomposition of biochar depending on the soil type. The formation of soil aggregates or organo-mineral complexes provides substantial physical protection to SOM, potentially preventing its decomposition. Additionally, the temperature sensitivity varying with soil type limits short-term decomposer access to SOM, while the majority of decomposition is carried out by slower microbial metabolism . For instance, in a study by Feng , it was observed that the temperature sensitivities of biochar vary in soils with different textures and mineralogy. The temperature coefficient ( Q 10) values of biochars ranged from 1.93 to 2.20 in Oxisols and from 2.74 to 2.77 in Vertisols, within a temperature range of 20–40 °C. Additionally, the presence of biochar was found to alter the Q 10 values of native SOM as well, depending on the soil type . Following the advancements and findings outlined in earlier sections, future research on MB should prioritize several key areas to address existing gaps and enhance our understanding. These research efforts can be categorized into four main segments. However, to achieve optimum results, collaborative efforts among experts from different domains are strongly recommended. Analytical advancement Developing Advanced Techniques : Employ more sophisticated techniques such as Pyrolysis Gas Chromatography-Mass Spectrometry (Py-GC–MS) and Thermogravimetric-Fourier Transform Infrared Spectroscopy-Mass Spectrometry (TG-FTIR-MS) to elucidate biochar formation mechanisms more accurately. Advanced Spectroscopy : Apply advanced spectroscopic methods to gain deeper insights into the functionalization of MB, allowing for better control over functional groups and porosity. Integrated Approaches : Use a combination of unique spectroscopic investigations and theoretical modeling to enhance the understanding of factors impacting biochar efficiency after soil amendment. Future perspectives in fundamental and material research Mechanism Investigation : Explore the mechanisms behind changes in biochar properties due to different pyrolysis conditions or modification procedures. Prioritize sustainable feedstock materials and their functionalization for specific contaminants to promote a green and sustainable environment. Synergistic Integration : Investigate the potential for synergistic integration with other treatment modalities, such as combining MB with biofilters, to enhance biochar's adsorption capabilities and address environmental challenges. Regeneration Processes : As biochar research advances, it brings forth new challenges and dilemmas. A primary concern revolves around regeneration processes. Addressing how to efficiently desorb pollutants adsorbed on biochar for subsequent safe treatment and optimizing the recycling of the adsorbed biochar stands as paramount issues requiring meticulous consideration. Environmental Risks : A critical aspect under scrutiny is the risk of secondary pollution arising from MB. For instance, there is a pertinent question regarding the possible interaction of metal-MB with other environmental compounds following its application in soil and leaking into groundwater. Understanding the possibility of antagonistic effects with different compounds and developing effective methods for separating treated biochar from soil and groundwater are pressing issues that demand immediate attention in future research endeavors. Future research in aging and stability Long-Term Effects : Investigating the long-term effects of MB on soil functions and its behavior across diverse soil types necessitates immediate attention. A recent study illustrated the direct influence of MB on soil biological communities' composition and abundance, as well as affects the retention of applied pesticides . Consequently, weed control in MB-amended soils may pose challenges, as the efficacy of preemergence herbicides could diminish. Environmental Behavior: MB proved to facilitate the formation of soil aggregates, enhance the physical protection of organic carbon, and increase yield production, with other positive environmental impacts such as the decrease in methane and gross greenhouse gas emissions. However, these initial positive effects might shift to negative over time, necessitating attention to the long-term environmental behavior of MB. It is particularly crucial to investigate how biochar interacts with minerals and how such interactions affect the performance of soil and water remediation over short-term and long-term aging. Addressing these issues in future research is imperative for a comprehensive understanding of MB's role and impact on soil. Simulated Aging Methods : There is a need to develop simulated aging methods that integrate multiple artificial aging processes. Specifically, considering the aging time scale in natural environments, it is crucial to quantitatively explore response models of MB properties using various artificial aging methods. This approach will enhance our understanding of how biochar evolves over time under environmental conditions and improve the accuracy of predictions regarding its behavior and efficacy in practical soil applications and its implications for different crop systems. Geochemical Behavior : In the context of pollutant management by MB, there exists a pressing need for further exploration into the geochemical behavior of emerging contaminants under long-term application MB. Understanding how MB aging interacts with and influences the fate of emerging contaminants across diverse environmental contexts is crucial for devising effective pollutant remediation and management strategies. For instance, investigating potential scenarios such as the movement of MB to groundwater and the release of metals in the case of metal modification. However, the impacts of such interactions are contingent upon soil types, with sandy soils often exhibiting more significant benefits compared to others . As a result, there is an urgent need for extensive research on the long-term effects of MB across various soil types. In conclusion, this article explores the physicochemical characteristics of biochar altered through various techniques, emphasizing the significance of comprehending the impact of these alterations on biochar properties. This insight will assist readers in discerning effective methods to modify biochar for specific applications. Our assessment suggests that MBs have significant potential for improving soil quality and health, and mitigating greenhouse gases. Furthermore, environmentally-friendly modifications can enhance energy and carbon storage in soils. Additionally, this review will offer comprehensive insights into advanced analytical techniques developed to analyze modifications necessary prior to application, enabling precise targeting of specific objectives. However, further investigation is required to analyze the relationships between material composition, structure, and energy storage. Overall, this article contributes to our understanding of the potential of MB as a versatile and sustainable material with significant environmental and economic benefits. This comprehensive review thoroughly explores the recent advances in utilizing Modified biochar (MB), specifically in soils. Beyond mere synthesis, it delves into the evolved physicochemical characteristics of MB, providing invaluable insights into its efficacy and limitations. Indeed, comprehending the unique properties endowed by each modifying agent is paramount, as they directly shape the characteristics of the final product. Therefore, it is imperative to fully grasp the attributes of both MB and the modifying agent prior to production, enabling targeted applications within soil environments. However, in the rapidly advancing world, it is essential to ascertain the exact nature and properties of processed MB. Hence, our review underscores the significance of embracing advanced analytical methodologies to characterize modified biochar and gauge its effectiveness precisely. By doing so, we aim to shed light on the crucial role these techniques play in ensuring biochar quality and performance in modern applications. Furthermore, this paper contains a comprehensive discussion of the mechanisms behind the factors influencing the efficiency of MB post-soil application. Additionally, we have highlighted the need for future research to explore the mechanisms behind alterations in biochar properties due to different modifications. Our future research perspective section is delineated into four segments. Firstly, analytical advancement advocates for leveraging sophisticated techniques, such as Pyrolysis Gas Chromatography-Mass Spectrometry (Py-GC–MS) and Thermogravimetric-Fourier Transform Infrared Spectroscopy-Mass Spectrometry (TG-FTIR-MS) to deepen our comprehension of biochar formation mechanisms. Additionally, it underscores the significance of advanced spectroscopic methods in elucidating modified biochar functionalization for precise control over functional groups and porosity. Secondly, future research in fundamental and material research underscores the importance of synergistically integrating MB with other treatment modalities, and investigating the mechanisms underlying changes in biochar properties resulting from modification procedures. | Other | other | en | 0.999997 |
PMC11698997 | Objective structured clinical examinations (OSCEs) are widely used in Health Professions’ Education globally as a tool to test specific skills relevant to clinical practice. Whilst in some countries they have been used for decades, in others they have been introduced relatively recently. In the UK for example, the implementation of integrated OSCE stations where multiple tasks are combined in a single practical station is commonplace . In comparison, for many educational programmes and institutions elsewhere in the world, this integrated OSCE style is still novel and faculty members are unlikely to be familiar with the operational approach to this style compared to the more traditional long case or viva styles of practical assessment . International partnerships in medical education are an increasingly popular tool to enable new and existing organisations to raise standards with assessment being a particularly important area in these partnerships. The UCL Medical School’s Centre for International Medical Education Collaborations (CIMEC) works collaboratively with institutions around the world to develop tailored medical educational programmes by co-developing and embedding high-quality education and assessments . Collaborating medical institutions are located in various countries and are often at the early stages of developing and establishing a medical educational curriculum and assessment structure. Thus, they often value the knowledge and expertise that the UCL Medical School CIMEC team can provide. All collaborations aim to co-create high-quality, sustainable and locally customised education programmes . Assistance with the development of assessments has encompassed various areas of the partnership both in assessment design and delivery of high-quality written and practical assessment items including OSCEs, where feedback and experiential knowledge are often highly valued by collaborators . NGU and UCL worked collaboratively to identify some of the key challenges posed by this methodology. Firstly, the feedback was not always timely as the method of writing OSCE stations; their dissemination for review and duration for turn around of feedback can be time consuming. Moreover, feedback provided was unidirectional in delivery, which we know is certainly a historically conceptualised process in medical education , and maybe perceived as less effective in the spirit of collaboration. This approach has also posed difficulties for the UCL team to confirm whether the feedback has been fully understood or can be readily applied to enable the changes to be implemented accurately. This lack of a two-way dialogue in provision of feedback also failed to provide NGU with an opportunity to voice any adaptations that they required to follow through with advice on an OSCE station to make it relevant to the local community. For example, there may be differences in the availability of certain resources such as specific prosthetic models or access to simulated patients with relevant training to deliver the OSCE station. | Other | biomedical | en | 0.999996 |
PMC11699018 | Globalization leads to an increasing number of students studying and working abroad, necessitating proficiency in the local language for effective communication with colleagues and patients in the medical field. Medical interns face challenges in hospitals due to language barriers and ineffective communication. Fluency in Dutch is essential for international medical students in the Netherlands to effectively communicate with Dutch-speaking patients and colleagues in their future careers. Accordingly, courses teaching medical Dutch need to be introduced, and course designs needs to support efficient medical second language (L2) learning. Although medical simulations enhance students’ proficiency, having native speakers as learning partners offers experiential examples, feedback, and corrections for sentences and phrases. These approaches aim to equip students with the language skills necessary for successful medical practice in a Dutch-speaking environment . In medical L2 learning, study partners are often nonnative peers rather than native speakers of the language . In this study, the term nonnative indicates a person who is not a native speaker of the language in which the medical terms are studied. To assess the effects of nonnative or native study partners on language learning, we conducted a comparative study examining students’ levels of confidence in their language skills, their collaborative strategies, and learning experiences in homogeneous (study pairs of nonnative students) and mixed (study pairs comprising of a native and a nonnative student) groups. Varying proficiency levels between learning partners can result in differential impacts on students, and our study aimed to address this issue . Drawing from Kolb’s experiential learning theory , and considering relevant literature, we incorporated simulated consultations into the medical Dutch course to compare the effects of learning partners . Kolb’s experiential learning theory proposes a learning cycle involving concrete experience, reflective observation, abstract conceptualization, and active experimentation . Simulated patient consultations have proven effective for medical consultation and L2 learning, aligning with concrete experience and active experimentation. Pairing students for consultations fosters reflective observation and abstract conceptualization, offering valuable benefits through observational feedback and deep thinking [ 17 – 20 ]. Integrating these course design methods offers students’ opportunities to engage in real-life scenarios and reflective discussions with their partners. This fosters improved communication skills, language learning confidence, and collaborative benefits during consultations, enhancing professionalism in using medical Dutch [ 21 – 23 ]. Fig. 1 Model of Kolb’s experiential learning This mixed-methods study utilized a pre- and post-test comparative design with 12 third-year international medical students (mean age = 22.3 years, range from 20 to 28 years) enrolled in a medical Dutch course at Maastricht University . Four exceptionally proficient native Dutch-speaking students, exempted from the course based on a rigorous language evaluation, were invited back as native partners to assist the medical Dutch learning of the other students. The mixed group consisted of these four native partners randomly paired with four of the 12 students, while the remaining eight students formed four homogeneous groups. All students attained a proficiency level of at least B2 in Dutch, according to the Common European Framework of Reference for Languages (CEFR), which signifies their ability to engage in intricate social dialogues, comprehend and analyze sophisticated Dutch texts and books, and articulate complex thoughts and viewpoints in writing. Level B2 is the standard for foreign diploma holders who aspire to pursue higher professional education in the Netherlands. Additionally, the course was conducted in Dutch, and they possessed a minimum of 2 years of practical experience in simulated patient consultation sessions conducted in English, which allowed them to focus on Dutch language learning during the course. The nonnative participants were German (4), French (2), Spanish (2), Finish (1), Irish (1), Romanian (1), and Italian (10) students. Fig. 2 Background information on the quasi-experimental groups. Native facilitators are indicated in blue, and nonnative participants in green. Gender, student number, and nationality are indicated in the image. Mean age in both groups was similar (22 years) The individual semi-structured interviews underwent content and thematic analyses to uncover the collaborative strategies and learning experiences underlying both the mixed and homogenous groups . The collaborative strategies were independently identified by two researchers from the field of education (H.Y. and M.A.) and were summarized into six categories, available as supplemental digital appendix 4 . Two researchers (H.Y. and F.J.) independently coded the interview data, compared and discussed the resulting codes and themes, and resolved any discrepancies in two team meetings. The thematic findings underwent a code co-occurrence analysis, which allowed the generation of nodes and edges for subsequent network analysis [ 27 – 29 ]. The network analysis was based on data from the code co-occurrence matrix and coefficients of codes and was available as supplemental digital appendix 3 . A force-directed graph was provided to visualize the structure of the networks. We analyzed the NSCS results to determine the confidence of the participants in their medical Dutch at the beginning and the end of the study (Table 1 ). The Mann–Whitney U test result and the normalized gain score revealed that the confidence of the mixed group decreased marginally ( U = 3.85, Z = − 0.55, p = 0.58, r = 0.28) resulting in a small negative normalized gain (N-gain = − 10.7%, SD = 0.25; Table 1 ) while the confidence of the homogenous group improved substantially ( U = 15.50, Z = − 2.21, p = 0.03, r = 0.38) and showed a positive normalized gain (N-gain = 11.3%, SD = 0.08). Table 1 Confidence of nonnative students in the mixed and homogenous groups Confidence Mixed ( n = 4) Homogenous ( n = 8) Median IQR Median IQR Pre-test 5.53 1.33 4.67 1.41 Post-test 5.33 2.67 5.83 1.17 N-gain − 10.7% ± 0.25 (SD) 11.3% ± 0.08 (SD) Research question 2 first investigated collaborative strategies in mixed and homogeneous groups using content analysis. Responses to the interview question “How did your partner affect your medical Dutch learning?” yielded a total of 238 responses on strategies. Among these, mixed groups reported 58 times on strategies (24% of 238), while homogeneous groups reported 180 times on strategies (76% of 238), indicating higher strategy exchange in the homogeneous groups. Available as supplemental digital appendix 4 , collaborative strategies: Language use in SPC, covering grammar and word usage during sessions. Communication skills, including tone, facial expressions, empathy, questioning style, and other communication-related aspects. Partner comparison, involving feedback or observations comparing one’s performance with their partner. Interaction and feedback, concerning the nature of feedback received, including positivity, constructiveness, warnings or criticisms, and knowledge sharing. Personal growth, referring to flexible thinking, enhanced learning effectiveness, and self-recognition of personal growth. Drawbacks, encompassing challenges like language communication difficulties, anxiety, lack of feedback, and ineffective feedback. The network properties of nodes and edges were presented using code concurrence and code coefficients (Table 2 ). The weighted in-degree (wd) indicates a node’s connectivity and importance within the network, while the PageRank (PR) value measures network centrality, indicating the significance of a specific node. Table 2 Network metrics of the homogenous and mixed groups Network metrics Homogenous group Mixed group Nodes ( N ) 5 5 Edges ( N ) 10 9 In degree ( M ± SE) 4.0 ± 0 3.6 ± .49 Weighted in-degree .24 ± .05 .12 ± .03 PageRank ( M ± SE) .14 ± .02 .14 ± .02 Diameter 1 2 Density 1 .90 Modularity 0 0 Communities ( N ) 1 1 Path length ( M ) 1 1.1 Consistent PR values were observed for the “themes” at both mixed group and homogenous group levels, indicating similar centrality and importance throughout the networks. Additionally, all networks showed a single prominent community with comparable density and similar diameter, and the average path length indicated similar interconnectedness within the networks. For research question 1, our findings indicate that the presence of supportive and encouraging partners boosts students’ confidence, playing a significant role in contributing to a notable improvement in their performance . Students in mixed groups initially exhibited high self-confidence during the pre-test but showed a decrease in confidence after the course, reflected in a negative normalized gain score. This drop in confidence was likely due to their reliance on positive feedback from native partners and the collaborative strategy of comparing themselves to their partners. In contrast, the homogeneous group likely did not experience such a drop in confidence as they were not directly comparing themselves to students with high performance levels. It is worth noting that there was an initially high confidence level in the nonnative students in the mixed group, which we infer was influenced by the initial encounters and assistance from native partners. The native partners consistently outperformed the nonnative students throughout the learning process, leading to a basis for comparison or appreciation among the students . The stark contrast in performance could have led to feelings of inadequacy and self-doubt, ultimately undermining their self-confidence . Constantly being overshadowed by their native partners could have eroded the students’ belief in their own abilities, resulting in a decrease in confidence over time . The similar proficiency levels in medical Dutch within the homogenous group likely facilitated their use of collaborative strategies by enabling better mutual understanding and support. Our study identified six collaborative strategy domains. Both groups emphasized language use, progress monitoring, and feedback. However, the homogenous group focused more on communication skills, likely due to their shared linguistic proficiency and understanding of challenges, enhancing intra-group communication. Moreover, the mixed group focused more on personal growth as collaborative strategies, which included self-improvement, reflection, analytical thinking, and confidence building (available as Supplemental Digital Appendix 4 ). We posit that this can be attributed to the role modeling effect, as documented in previous studies [ 35 – 37 ]. In the mixed group, native peers serve as valuable learning models, inspiring students to reflect on their own skills and qualities to emulate the successful behaviors and characteristics exhibited by these role models . Inspired by their native partners, students in the mixed group aimed to reach the level of competence demonstrated by these role models. Consequently, the homogenous group faced more challenging drawbacks than the mixed group . To understand the theme construct, we used network analysis to model relationships in both the homogenous and mixed groups in line with previous research that thoroughly investigated these themes to validate their construct [ 38 , 40 – 44 ]. In the homogenous group, confidence was central and closely linked to other themes, while motivation was central in the mixed group . These findings align with Kolb’s theory, which emphasizes the importance of a motivational environment and building students’ learning confidence to enhance their learning experiences . The use of native or nonnative partners in the course, as guided by Kolb’s theory and thematic analysis outcomes, effectively accounts for the differences in network themes observed between the mixed and homogeneous groups. Our study had limitations that may have influenced the interpretation of results and discussions. The small sample size, particularly in the mixed group (4 pairs), led to higher standard deviation in the normalized gain score for research question one, making it difficult to definitively establish a decrease in the gain score. Future studies could replicate our design with a larger sample size to strengthen and expand our conclusions. The gender distribution in both groups was imbalanced, with a higher number of female students ( n = 13) participating compared to male students ( n = 3). In the international track of medicine program, more women (80%) enlisted than men. In our study, there were more women than men in both groups, especially in the homogeneous group. It is unclear whether gender could play a role in the learning outcomes. We hope future research will explore similar contexts with a balanced gender composition. The students and native-speaking partners were all familiar with each other since they were enrolled in the same program with 50 students/year at the university and have studied simulated patient consultation in English for 2 years. For other designs, students could become familiar with each other in small groups before pairing them up. Additionally, the unique nature of each network in the network analysis, along with differing parameters used to describe network attributes across groups, made direct comparisons challenging. Lastly, there was a discernible tendency to emphasize the advantages of native partners, although our study primarily did not involve interviewing native partners. We recommend that future study also conduct interviews or surveys on the native partners’ perceptions for gaining further understanding of near-peer teaching and mentoring. | Other | biomedical | en | 0.999998 |
PMC11699050 | The right heart catheterization (RHC) remains an important diagnostic tool, providing data on hemodynamics, cardiac output, and cardiopulmonary pressures to help clinicians make decisions in a variety of disease processes. With the development of the balloon floatation catheter (Swan-Ganz catheter) by Drs. Swan and Ganz in 1970, it attained widespread use without significant critical review due to its ability to obtain hemodynamic measurements at the bedside . Several studies in the 1990's and early 2000's investigating the use of pulmonary artery catheters (PAC) suggested that it led to detrimental effects including increased length of stay, inconsistent interpretation of data, and increased mortality. Ultimately this resulted in a substantial decline in its use . Despite such a dramatic change in utilization, it continues to play a key role in the diagnosis and management of pulmonary hypertension (PH), cardiogenic shock (CS), valvular heart disease, and evaluation prior to organ transplant, particularly of the heart and lungs. In this review, we discuss the indications, contraindications, complications, and interpretation of waveforms and measurements of RHC. The RHC was revolutionary with the availability of a balloon tipped, flow-directed catheter, giving clinicians the ability to place one at bedside and leave it in place for continuous monitoring. It achieved widespread use without empirical studies looking into the benefits or risks, and by the mid-1980s, up to 40 % of critically ill patients underwent RHC . Concerns over this widespread utilization led to several studies which called the safety into question. Furthermore, several meta-analyses, observational studies, and randomized controlled trials have also shown no survival benefit from PAC use in critically ill patients. Ikuta et al. studied the national trends in PAC use and found a 67.8 % decline from 1999 to 2013. Specifically, there was a significant reduction in PAC placed for hospitalizations involving respiratory failure (92.3 % reduction) and acute myocardial infarction (74 % reduction). HF admissions on the other hand, demonstrated a 56 % reduction until 2009 .. Hernandez et al. demonstrated similar trends with a decrease in PAC use in HF from 2004 to 2007, followed by an increase in 2014. The authors also showed that use of PAC in CS has declined from 2004 to 2014. With the ability to provide data on right and left sided filling pressures, CO, vascular resistance, and other hemodynamic parameters, the RHC provides valuable insight for the diagnosis and management of CS. Invasive hemodynamic monitoring can guide the use of vasoactive pharmacologic therapies and help determine when mechanical circulatory support (MCS) is necessary. Proponents state that invasive monitoring can guide therapy and optimize hemodynamics, however, several early studies showed a lack of benefit with use of the PAC in critically ill patients . Several studies that investigated use of RHC or PAC in CS are summarized in Table 1 . Cooper et al. set out to develop a noninvasive criterion for the diagnosis of CS and validated it against the PAC standard. Their noninvasive criteria utilized hypotension, hypoperfusion state, predisposition to CS, and echocardiographic findings and reported a sensitivity of 96.9 %, specificity of 90.8 %, positive predictive value of 64.6 %, and negative predictive value of 99.4 %. The authors suggested that these criteria be used to noninvasively exclude CS, while PAC be reserved to confirm CS in patients who meet criteria . Table 1 Summary of studies investigating use of RHC in CS. Table 1 Study name and author Primary indication Outcomes Comments “SUPPORT”, Connors et al. Critically ill patients in the ICU: acute respiratory failure, multiorgan system failure, CHF Patients with RHC vs no RHC: - Increased 30-day mortality (OR 1.24) - Increased LOS (14.8 days vs 13.0 days) - Increased resource utilization “ESCAPE”, Binanay et al. Severe symptomatic heart failure despite recommended therapies Use of PAC: - No difference in mortality (OR 1.26, 95 % CI 0.78 to 2.03) - Increased in-hospital adverse events (47 vs 25, p = 0.04) - No deaths related to PAC use Excluded patients in cardiogenic shock requiring inotropes, vasopressors, or MCS “PAC-Man”, Harvey et al. ICU patients; acute respiratory failure, multiorgan dysfunction, decompensated heart failure Patients with PAC: - No difference in hospital mortality (68 % vs 66 %, p = 0.39) - Similar hospital and ICU LOS No deaths related to PAC; Insertion related complications = 10 % O'Neill et al. Acute myocardial infarction complicated by cardiogenic shock with Impella use Use of PAC: - Improved survival rate of 63 % versus 49 % in patients with PAC use versus patients without PAC use “CardShock”, Sionis et al. CS Use of PAC: - Mortality was 42 % compared to 34 % in non-PAC patients ( P = 0.6) Most common complication was VT during PAC insertion (5.6 %, 4/71) “CSWG”, Garan et al. CS Complete PAC hemodynamic data: - lowest in-hospital mortality compared to no PAC assessment (OR 1.57; 95 % CI 1.06 to 2.33) and to incomplete PAC data (OR 1.71; 95 % CI 1.29 to 2.25) - No significant difference between no PAC compared to incomplete PAC data Complete PAC was defined as having all the following: PA systolic and diastolic pressure, PAWP, pulmonary artery saturation and right atrial pressure Hernandez et al. CS and HF without CS PAC use in CS - Lower mortality (35.1 % vs 39.2 %) PAC use in HF without CS: - Increased mortality (9.9 % vs 3.3 %) National Inpatient Sample, Retrospective cohort study In addition, PAC in patients with CS can help tailor management by identifying their unique hemodynamic profile e.g., a patient with a low cardiac index and high systemic vascular resistance (SVR) may benefit from afterload reduction strategies with agents such as Sodium Nitroprusside as opposed to utilizing an inotrope only strategy. This was further exemplified by Ranka et al. who showed that in patient's admitted with acute CS, RHC use was associated with a significant reduction in mortality in index admission by 31 %, a reduction in 30-day readmission rate, and a six-fold increase in left ventricular assist device (LVAD)/orthotopic heart transplantation during rehospitalization. The authors hypothesized that RHC allowed for early and accurate classification of CS and increased MCS or advanced therapy in cases of severe CS, resulting in improved outcomes . Together these data suggest that RHC should be considered in CS that remains refractory to initial therapy or when advanced heart failure therapies are being considered. In recent years, SCAI (Society of Cardiovascular Angiography&Interventions) definition and stages of cardiogenic shock and have been increasingly adopted in both research and clinical practices . Through stage A (at risk) to stage E (extremis), this new definition highlights the dynamic nature of the CS as well as the importance of timely recognition and interventions to improve the outcome of patients with CS. Right heart catheterization is helpful in both evaluation and prognostication of CS as described above. When there is concern for full blown CS, invasive hemodynamic measuring with cardiac index <2.2 L/min/m2 and pulmonary artery wedge pressure (PAWP) >15 mmHg remains to be gold standard for diagnosis. In addition, Cardiac power output (CPO) and Pulmonary artery pulsatility index (PAPI) are also useful hemodynamic index to identify the severe LV dysfunction and RV failure, respectively . Right heart catheterization and invasive hemodynamic monitoring have been recommended for guidance of utilization of MCS when treating CS, as clinical assessment of PAWP and CVP are often inaccurate and unreliable . All cardiogenic shock has decreased cardiac output, but they may have different hemodynamic profiles. In the setting of predominantly elevated left side filling pressure, an unloading device such as percutaneous LVAD can help decrease PAWP and improve CVP by reducing RV afterload. However, when there is commitment right sided congestion that is not responding to the LV unloading, diuresis or CRRT might be indicated to further improve the cardiac output. When there is more predominant RV failure evidenced by marked elevated CVP and RV dysfunction, pRVAD might be indicated for RV unloading. In the face of severe and rapid cardiopulmonary collapse, VA-ECMO is often used to restore organ perfusion. However, its use among CS patients with severe LV dysfunction may further increase left side filling pressure and cause pulmonary edema. pLVAD such as Impella can be added to the VA-ECMO to further unload the LV and improve the hemodynamics . Invasive hemodynamic monitoring has been shown to be associated with improved outcome of CS patients who received MCS. In a large retrospective study of 1414 patients with CS, the use of complete PAC-derived hemodynamic data prior to MCS initiation is associated with improved survival from CS . RHC remains the gold standard to diagnose, risk stratify and classify PH. The European Society of Cardiology (ESC) and European Respiratory Society (ESR) guidelines recommend use of RHC to investigate when echocardiography findings demonstrate high probability of PH to determine the etiology and confirm the diagnosis, and to support treatment decisions . Additionally, RHC is recommended in patients with intermediate probability of PH by echocardiography and unexplained signs or symptoms of PH, particularly if they have co-morbidities that increase their risk of PH. Echocardiogram has been shown to have good correlation but only moderate precision of estimating the Pulmonary artery systolic pressure (PASP) value calculated from TR velocity . The errors in measurement of peak TRV signal can lead to under or overestimate of PASP. In a large study of 1695 patients who had both echocardiogram and RHC, echocardiogram was found to have sensitivity of 87 % and specificity of 79 % for PASP cut off value of 36 mmHg . Due to this limitation, echocardiographic information can only grade the probability of PH being present rather than making the diagnosis . The World Health Organization (WHO) classification categorizes PH into 5 groups based on the specific pathophysiology and response to treatment: Group 1: pulmonary arterial hypertension [PAH], Group 2: PH due to left heart disease; Group 3: PH due to lung disease and/or hypoxia; Group 4: PH due to pulmonary artery obstructions; Group 5: PH with unclear and/or multi-factorial mechanisms . Table 2 provides an overview of the classifications. Prior to 2019, PH was defined as a mean pulmonary artery pressure (mPAP) ≥ 25 mmHg at rest which was arbitrarily defined. A retrospective analysis of all normative published RHC data by Kovacs et al. found that supine mPAP at rest in healthy individuals was 14.0 ± 3.3 mmHg and two standard deviations above the mean (20.6 mmHg), would represent the upper limit of normal . According to ESC 2022 Guidelines, PH is defined as a mPAP > 20 mmHg. The Task Force included a pulmonary vascular resistance (PVR) ≥ 2 Wood units (WU) and a PAWP ≤ 15 mmHg to define all forms of pre-capillary PH . The hemodynamic definitions can help with determining the diagnostic groups as outlined in Table 3 . Currently, there are no treatments approved for mPAP between 20-24 mmHg and PVR between 2-3 WU. Table 2 Overview of the groups of pulmonary hypertension . Table 2 Classification of Pulmonary Hypertension Group 1: Pulmonary Arterial Hypertension (PAH) 1.1 Idiopathic 1.2 Heritable PAH 1.3 Drug- and toxin-induced PAH 1.4 PAH associated with 1.4.1 connective tissue disease 1.4.2 HIV infection 1.4.3 portal hypertension 1.4.4 congenital heart disease 1.4.5 schistosomiasis 1.5 PAH long-term responders to CCB 1.6 PAH with overt features of venous/capillaries (PVOD/PCH) involvement 1.7 Persistent PH of the newborn syndrome Group 2: PH due to left heart disease 2.1 Heart failure with preserved LVEF 2.2 Heart failure with reduced LVEF 2.3 Valvular heart disease 2.4 Congenital/acquired cardiovascular conditions leading to post-capillary PH Group 3: PH due to lung diseases and/or hypoxia 2.1 Obstructive lung disease 2.2 Restrictive lung disease 2.3 Other lung disease with mixed restrictive/obstructive pattern Group 4: PH due to pulmonary artery obstructions 2.1 Chronic thromboembolic PH 2.2 Other pulmonary artery obstructions Group 5: PH with unclear and/or multifactorial mechanisms 2.1 Hematological disorders 2.2 Systemic and metabolic disorders 2.3 Others 2.4 Complex congenital heart disease Table 3 Hemodynamic findings to categorize PH . Table 3 Type of PH mPAP PAWP PVR Pre-capillary (groups 1, 3, 4 and 5) >20 mmHg ≤15 mmHg ≥2 WU Isolated post-capillary (groups 2 and 5) >20 mmHg >15 mmHg <2 WU Combined pre- and post-capillary (groups 2 and 5) >20 mmHg >15 mmHg ≥2 WU Key hemodynamic measurements obtained from RHC that categorize PH. PH = pulmonary hypertension, mPAP = mean pulmonary artery pressure, PAWP = pulmonary artery wedge pressure, PVR = pulmonary vascular resistance, WU = Wood units. In patients with newly diagnosed PAH, vasoreactivity testing is recommended in those with idiopathic, heritable or drug/toxin induced PAH only to assess for possible treatment response with calcium channel blockers . Inhaled nitric oxide (10–20 ppm) or intravenous epoprostenol are the two preferred agents which can be utilized in this setting. Based on data by Sitbon et al. a positive vasodilator response was defined as a reduction in mPAP ≥10 mmHg to reach a mPAP ≤ 40 mmHg with an increased or unchanged CO . Only 10 % of idiopathic PAH patients will be vasoreactive. RHC is an important part of mortality risk stratification in PAH . Elevated right atrial pressure and reduced cardiac index have repeatedly been demonstrated to predict increased mortality risk in PAH. Elevated right atrial pressure is included in the REVEAL 2.0 risk score , a highly validated risk score that can help guide therapy. In patients with PAH, repeat right heart catheterizations are often utilized to determine the response to therapy. Even with hemodynamic measurements, it can be challenging to distinguish pre- and post-capillary PH in the setting of HF with preserved ejection fraction (HFpEF) or optimal volume status. Evocotive manuevers, such as exercise and fluid challenge, can be very useful to distinguish HFpEF for PH as a cause of dyspnea. The ratio of change in PAWP per change in CO >2 mmHg/L/min can be used to identify HFpEF . The saline challenge protocol can help differentiate Group 1 PAH from Group 2 PH and involves intravenous administration of a 500 mL 0.09 % saline bolus over 5 to 10 min . Although normal subjects can have an increase in filling pressures with a saline bolus, those with HFpEF had a steeper rise in PAWP comparatively . Several studies have demonstrated that a PAWP >18 mmHg after saline challenge is suggestive of post-capillary PH [ , , , ]. D'Alto et al. utilized the saline challenge to correctly reclassify five out of 66 patients without PH and eight out of 124 with pre-capillary PH into post-capillary PH . Left ventricular end-diastolic pressure (LVEDP) and PAWP are often used interchangeably to estimate left-sided filling pressures. However, several studies have demonstrated discrepancies between these two measurements leading to misclassification of PH group. PAWP underestimates LVEDP in older age and overestimates LVEDP in atrial fibrillation, rheumatic valve disease, or those with a larger left atrial diameter. If RHC and PAWP were used to classify PH group in the above situations, it would misclassify nearly 30 % of patients . Therefore, ESC/ESR guidelines recommend use of LVEDP when PAWP is unreliable . Some authors have advocated for earlier use of RHC in the diagnostic schema of PH than some societal guidelines have recommended . Invasive cardiopulmonary exercise testing (iCPET) can be a valuable tool in evaluating unexplained dyspnea, especially when non-invasive workup is inconclusive. iCPET allows for the measurement of physiological data that facilitates diagnosis of exercise-induced PAH, exertional HFpEF, or preload dependent limitations to CO . It is also performed to measure the contribution of cardiac and lung pathology to a patient's symptoms in multifactorial disease and to determine the severity of cardiac limitations in heart failure with reduced ejection fraction . When performing iCPET, RHC and sometimes radial artery catheterization (vrs pulse oximetry) are obtained to measure cardiac hemodynamics and peripheral tissue oxygen consumption. Caution needs to be used in patients with significant arterial insufficiency or Raynaud's phenomenon as radial artery catheterization can induce acute limb ischemia. An upright or semi-supine cycle ergometer, treadmill, or arm ergometer are utilized until the resistance is increased and the patient is exhausted. The pulmonary arterial pressure, right atrial pressure, and mean arterial pressure are measured continuously, while PAWP, arterial blood gases, and lactate are measured every minute. Baseline ventilation and pulmonary gas exchange measurements are obtained and repeated throughout exercise testing . Herve et al. investigated the accuracy of mPAP alone compared to mPAP combined with peak exercise total PVR (TPVR) and supine dynamic exercise testing and found that the combination of maximal mPAP >30 mmHg and TPVR >3 WU corresponded to a similar sensitivity (0.93) but an improved specificity from 0.77 to 1.0 when distinguishing healthy subjects from ones with exercise-induced PH. The authors, however, were unable to accurately differentiate if PH was caused by left heart disease or pulmonary arterial disease . While exercise PH has been reintroduced in the 2022 PH guidelines, there remain no proven therapies. In patients with PAH and HFpEF, Santos et al. hypothesized that exercise upright RHC would provide additional information for the diagnosis of HFpEF and PAH in patients with unexplained exertional dyspnea. Patients had same day supine resting RHC followed by iCPET. 212 patients had normal supine resting RHC, however, 46 (22 %) of these patients had abnormal iCPET results. The use of iCPET reclassified diagnosis for 16 (43 %) of patients. 12 (40 %) patients with HFpEF had normal cardiac profile and 4 (31 %) of patients with PAH had no intrinsic pulmonary vascular disease found with iCPET . This study highlighted the hemodynamic changes in relationship to position during iCPET which may alter the interpretation of the result . Although diagnostic criteria for exercise-induced PAH have been attempted, they remain controversial as normal ranges for mPAP are highly variable and dependent on age . Kovacs et al. found that there were no significant association with age and resting mPAP, however, mPAP during exercise was independently linked to age with nearly half of the subjects ages ≥50 years exceeding an exercise mPAP >30 mmHg. The authors also found that nearly 20 % of patients younger than 50 years of age had mPAP >30 with maximal exertion, making it difficult to define normal ranges during exercise . Cardiopulmonary exercise testing has proved to be useful in differentiating cardiopulmonary causes of dyspnea from others. Elevated PAWP/CO slope during exercise (>2 mmHg/L/min) is common in patients with dyspnea on exertion with normal wedge pressure at rest and predicts exercise capacity and heart failure outcomes .Single measurements at rest might be insufficient and exercise measurements may refine the HFpEF diagnosis. Due to the complex procedures and requirement of muti-specialty collaboration. The iCPETs are usually performed at larger referral centers. In a retrospective study of iCPET center at Brigham and Women's hospital, 530 of 864 referrals were investigated with iCPET, over a span of 3 years . Exercise PAH, HFpEF, dysautonomia, oxidative myopathy accounted for 16.6 %, 17.7 %, 21.1 % and 24.5 % of the final diagnoses, respectively . Constrictive pericarditis is associated with equalization of diastolic pressures in all chambers, impaired ventricular filling, and signs or symptoms of right heart disease. As restrictive cardiomyopathy can present with similar findings, it is important to differentiate these entities to determine whether pericardial stripping may be indicated. The diagnosis can be challenging, and differentiation between the two requires a comprehensive evaluation with physical examination, computed tomography, magnetic resonance imaging, echocardiographic, and hemodynamic findings. In constrictive disease, rapid ventricular filling occurs early in the cycle followed by a rapid increase in pressure due to the noncompliant pericardium. This results in a rapid “y” descent on the atrial pressure waveform and a “square root” sign on ventricular pressure waveform, which may also be noted on pressure tracings of restrictive cardiomyopathy . The most reliable hemodynamic signs of constrictive pericarditis are those related to respiratory variation in RV versus LV filling. Constrictive physiology is demonstrated by significant ventricular discordance during the respiratory cycle, whereas concordance of ventricular pressures is the rule in restrictive cardiomyopathy . There have been several criteria to help differentiate constrictive and restrictive disease, but they have poor sensitivity and specificity. The systolic area index is a ratio of the RV area to LV area in inspiration versus expiration, and a value >1.1 is 97 % sensitive and 100 % specific for identification of constrictive pericarditis . Other findings that differentiate the two can be found in Table 4 . Table 4 Comparative Findings of Constrictive Pericarditis and Restrictive Cardiomyopathy . Table 4 Constrictive Pericarditis Restrictive Cardiomyopathy Traditional findings End-diastolic pressure equalization LVEDP-RVEDP ≤5 LVEDP-RVEDP ≥5 Dip and plateau morphology Prominent rapid diastolic filling waves on LV tracings (≥5 mmHg; square-root sign) LV rapid filling wave <5 mmHg Right Ventricular Systolic Pressure < 55 mmHg > 55 mmHg Right ventricular end-diastolic pressure (RVEDP) RVEDP ≥1/3 RV systolic pressure RVEDP <1/3 RV systolic pressure Kussmaul's sign Failure of RA pressure to drop upon inspiration. Normal respiratory variation in mean RAP Dynamic findings Dissociation of intrathoracic-intracardiac pressures A difference ≥ 5 mmHg between expiratory and inspiratory gradients between the PAWP and LV early diastolic pressures Concordant variation with respiration yielding to a difference between gradients of <5 mmHg Ventricular interdependence An inspiratory/expiratory systolic area index ≥1.1 An inspiratory/expiratory systolic area index<1.1 RHC is an essential part of the evaluation for heart transplant. The International Society for Heart and Lung Transplantation (ISHLT) Guidelines recommend that RHC should be performed in all adult candidates prior to listing for cardiac transplant (Class 1, level of evidence C). Once patients are listed, RHC should be repeated at 3-to-6-month intervals, most notably in the setting of reversible PH or worsening HF symptoms . The presence of elevated PVR or PH that is deemed irreversible with vasodilators remains a contraindication to isolated heart transplant, as the donor right ventricle may fail in the operating room or immediately postoperatively in the face of high PA pressures . Candidates with PVR > 5 WU and transpulmonary pressure gradient (TPG) > 15 mmHg have an increased 30-day mortality, and RHC remains crucial to recognizing these individuals . In patients with systolic PAP ≥ 50 mmHg and either PVR > 3 WU or TPG ≥ 15 mmHg, a vasodilator challenge with nitroprusside, IV prostanoids, or inhaled nitric oxide is indicated. Patients deemed to be reasonable candidates for transplantation are able to achieve a PVR of ≤3 WU while keeping systolic blood pressures above 85 mmHg . If the vasodilator challenge fails and the patients significantly decompensate, as evidenced by elevated PAWP and central venous pressure (CVP), they should be hospitalized for continuous hemodynamic monitoring and optimized with diuretics, inotropes, and vasoactive agents for 24 to 48 h. If medical therapy fails to reverse PH, consideration can be given for a trial of termporary MCS or intra-aortic balloon pump although having a clear endpoint for support is needed. Durable LVAD may be considered in patients otherwise felt to be candidates (including careful attention to RV function) and in some cases longterm LV unloaded may result in improved PVR [double check literature on this]. It is also reasonable to trial milrinone for 3 to 6 months and then reevaluate hemodynamics. Hemodynamics should be measured at 3 to 6 months after LVAD according to ISHLT guidelines (Class IIA, Level of Evidence: C). When both medical therapy and MCS fail, these patients are categorized as irreversible PH and isolated heart transplant remains contraindicated . Klotz et al. attempted to reduce PVR < 2.5 WU and TPG < 13 mmHg in patients being evaluated for heart transplant using prostaglandin E1 (PGE-1). The 1-year mortality after heart transplant was 22 % in the PH group treated with PGE-1, while it was 14 % in patients without any PH. There was no statistical difference in survival between these groups . One of the indications for combined heart-lung transplant includes secondary pulmonary hypertension (in many cases related to congenital heart disease) that does not respond to therapy, inotropes, and/or MCS, however combined heart-lung transplant is only performed in a limited number of centers and organ scarcity has prevented any considerable volumes of this procedure. The RHC also plays an important role prior to listing for lung transplantation. The Lung Allocation Score is utilized to waitlist patients based on mortality and posttransplant survival .. While the LAS takes some hemodynamic metrics of PH into consideration, PAH patients remain at a disadvantage in getting organs. Right heart catheterization has been also utilized in solid organ transplantation other than heart and lung for pretransplant evaluation. For orthotopic liver transplantation (OLT), guideline recommends mandatory screening for portopumonary hypertension (PoPH) with echocardiogram in all OLT candidates . For patients with elevated PASP (≥40 mmHg), RV dysfunction, LV dysfunction or significant valvular disease, RHC is indicated for evaluation degree and the type of pulmonary hypertension to further assess the candidacy of liver transplant . Brachial vein might be a preferred access site for RHC for patients with severe coagulopathy . For renal transplantation, the echocardiogram is indicated for patients who have been on dialysis for >2 years, has risk factors for pulmonary hypertension . For those who have an estimated pulmonary systolic pressure >45 mmHg should be referred for further evaluation with right heart catheterization . All patients being considered for pancreas should be evaluated with echocardiogram. For candidates with right ventricular systolic pressure (RVSP) >50 mmHg, a right heart catheterization is also recommended . An assessment of oxygen saturation of the superior vena cava (SVC) and the main pulmonary artery (PA) should be performed during RHC to detect presence of a shunt. A significant step-up in blood oxygen is defined as an increase in blood oxygen content or saturation that exceeds the normal variations of the right heart in the absence of a shunt [ , , ], and a step-up in oxygen saturation of >8 % should prompt further investigation with stepwise assessments of oxygen saturation, also known as detailed sampling . Upon confirming the presence of a significant step-up, measurements are obtained with fluoroscopic guidance and pressure measurement from the SVC (low and high), inferior vena cava (IVC) [low and high], right atrium (RA) [high, mid, and low], right ventricle (RV) [outflow tract, mid, apex], right ventricular outflow tract, main PA, left and right PA, and left ventricle (LV) as well as distal aorta in selected patients . The criteria for a significant step-up in oxygen saturation or oxygen content to signify a shunt are displayed in Table 5 [ , , ]. The magnitude of the shunt is then provided by the ratio of pulmonic flow to systemic flow (Qp/Qs), such that a Qp/Qs < 1 is managed conservatively, while a Qp/Qs > 1.5 warrants surgical or percutaneous repair . Table 5 Oxygen saturation associated with significant shunts [ , , ]. Table 5 Criteria for Step-Up Level of Shunt Atrial (SVC or IVC to RA) Ventricular (RA to RV) Great Vessel (RV to PA) Δ Mean O2 %Sat (distal and proximal chamber samples) ≥7 % ≥5 % ≥5 % Δ Highest O2 %Sat (distal and proximal chamber samples) ≥10 % ≥5 % ≥8 % The evaluation of valvular heart disease using invasive hemodynamics has fallen out of favor largely due to improvements in non-invasive imaging. When echocardiography and other noninvasive imaging is inconclusive, RHC can be considered per the ESC guidelines . Below are the following instances where RHC may prove to be useful in valvular disease: In aortic stenosis, coronary angiography in addition to RHC is often utilized to evaluate for existing coronary artery disease and PH as part of the pre-transcather aortic valve replacement evaluation . RHC is indicated in patients with mitral stenosis who have symptoms of PH or when non-invasive measurements of the mitral valve are inconsistent. Simultaneous measurement of the PAWP and LV pressure are used to evaluate the trans-mitral gradient . The PAWP will frequently overestimate the trans-mitral gradient by 30 % to 50 %, as it does not account for a delay of transmission of pressure through the pulmonary circulation . Stocker et al. showed that evaluation by invasive hemodynamic monitoring plays an important role in identifying patients with severe tricuspid regurgitation who would benefit from transcatheter tricuspid valve repair (TTVR). Patients were stratified into 3 groups by hemodynamic measurements to examine post-operative outcomes and mortality after TTVR. Expectedly, patients without any PH (mPAP <30 mmHg) and post-capillary PH (mPAP >30 mmHg, TPG ≤17 mmHg) demonstrated better post-operative outcomes and 1-year survival rate compared to patients with pre-capillary PH (mPAP >30 mmHg, TPG >17 mmHg). PAP, TPG, PVR, RV stroke work were predictors of all-cause mortality after valve repair, while PAWP, RAP, CO, and pulmonary artery pulsatility index had no significant association. The authors suggest invasive hemodynamic assessments should be used to stratify patients to select for TTVR . The use of blood drawn from a PAC in the wedge position may be useful in evaluating for lymphangitic carcinomatosis in patients who are unable to undergo invasive procedures, such as a lung biopsy. Masson et al. collected blood drawn through a wedged PAC and examined the cytologic characteristics in eight patients with lymphangitic carcinomatosis and 40 control patients divided into two groups (23 without cancer but various pulmonary disorders and 17 with cancer but without pulmonary metastases). Seven out of eight patients with lymphangitic carcinomatosis were found to have malignant cells on pulmonary microvascular cytology. The authors suggested this as an alternative method when a RHC is being performed for hemodynamic assessment or lung biopsy is too risky . Mechanical tricuspid valve has been traditionally thought to be absolute contraindication for RHC due to the risk of catheter entrapment, prosthetic vale damage or thrombosis formation, but it has been increasingly reported it can be safely performed in selected cases with precautions . Coagulopathy: There are limited recommendations in the setting of coagulopathy which can also be corrected with transfusion. In a study by Betz et al., the bleeding complications were compared between patients with an INR < 1.8 and INR ≥1.8 (range 1.8–4.0). The overall bleeding complications were extremely rare occurring in 0.6 % (2/312) of patients, both who developed a hematoma requiring prolonged manual compression and belonged in the INR < 1.8 group . RHC is an invasive procedure, and as with any procedure there are complications associated with each step. In the ESCAPE trial, only a total of 9 (4.2 %) procedural complications occurred, and none were associated with in-hospital deaths. These included infection (4 patients), bleeding (2 patients), catheter knotting (2 patients), pulmonary infarction (1 patient), pulmonary hemorrhage (1 patient), and ventricular tachycardia (1 patient) . PAC-Man (Pulmonary Artery Catheters in Patient Management in Intensive Care) demonstrated a complication rate of 9.5 % (46 patients) which included hematoma (4 %), arterial puncture (3 %), and arrhythmias (3 %). Similar to the ESCAPE trial, these complications were not fatal . A combined 5-year retrospective and 6-month prospective analysis looked at the number of adverse events and complications associated with RHC. Out of a total of 7218 RHC performed, there were 76 serious adverse (1.1 %) events during the study period. Seventy-two of these serious adverse events were non-fatal and twenty-one (28 %) of these non-fatal events resulted in a hospital admission or prolonged stay. The most common complications were related to venous access followed by arrhythmias. There were only four (0.055 %) procedure-related deaths during the study period . With the widespread use of point of care ultrasound in nearly all critical care settings, the rate of complication associated with insertion has improved compared to when PAC-Man and the latter study were published-2005 and 2006 respectively. Complications with venous access include hematoma at insertion site, arterial puncture, pseudoaneurysm formation, pneumothorax, thoracic duct injury, or thrombosis. The rate of thrombosis varies on the insertion site, with subclavian access having the lowest thrombotic risk at 1.9 % and length of time left indwelling. The rate of pneumothorax is lower with the use of the internal jugular veins for insertion compared to the subclavian veins and obviously, non-existent with a femoral vein insertion . The next set of complications are related to insertion of the PAC and include arrhythmias, catheter knotting, and PA rupture. Cardiac arrhythmias from PAC are mostly transient and include premature atrial or ventricular contractions, although sustained ventricular and superventricular arrhythmias may occur. Right bundle branch block can occur in up to 5 % of PAC insertions, therefore, patients with pre-existing left bundle branch block (LBBB) are at a risk of a complete heart block making LBBB a contraindication for RHC unless immediate pacing is available during the procedure [ 7 , , , ]. The most feared complication of RHC is PA rupture, and although the incidence remains low at 0.03 % to 0.2 %, it has a mortality rate of 70 %. When a patient with a PAC develops new onset hemoptysis, rupture of the PA should be suspected. It can be complicated by hemothorax or endobronchial hemorrhage and require emergent surgical approach such as a thoracotomy . PA rupture occurs from over-inflation of the PAC balloon or migration of the catheter tip , and risk factors include severe pulmonary hypertension, age > 60, use of anticoagulation, cardiopulmonary bypass, and mitral valve disease . In a study by Kearney and Shabot, PA rupture was seen in ten patients out of 32,442 (0.03 %) who required hemodynamic monitoring with PAC. All ten patients had hemoptysis and seven died. The authors found that emergent thoracotomy was essential for survival in the presence of hemothorax . In order to minimize risk, a chest x-ray should always be obtained before and after adjustment of the PAC, and fluoroscopy may be preferrable for PAC insertion in certain situations. Weinberg et al. showed that insertion under video fluoroscopy allows for positioning of the catheter with less time, fewer attempts, and decreased rate of complications compared to PAC floated by real-time pressure waveform monitoring . It is also important to adopt safe techniques when using PAC. Avoid inflating the ballon in the distal pulmonary artery or against pressure. In case of suspected PA vascular injury due to right heart catheterization that resulted in significant bleeding, it is important to first secure the airway, often with endotracheal intubation. Acute hemostasis can be achieved by inserting a new Swan-ganz catheter into the same PA branch and inflating the ballon proximally to the bleeding site . The PA rupture can be treated endovascularly with stents, coil embolization or vascular plug or surgically with lobectomy . Indwelling PAC increased the risk of catheter site infection, bacteremia/sepsis, or right-sided endocarditis. Rowley et al. studied the frequency of right-sided endocardial lesions in catheterized patients. One or more right-sided endocardial lesions were found in 29 of the 55 catheterized patients, with four (7 %) of these lesions representing infective endocarditis while the remaining were uninfected. The authors observed infective and non-infective vegetations were in similar locations, including the pulmonic valve and right atrium, which would suggest it was a consequence of direct endocardial damage from the PAC. The length of time the catheter was in place was not significantly related to the frequency of the lesions occurring . Al-Hijji et al. studied the major adverse events from left heart catheterization (LHC) and LHC combined with RHC using the electronic medical record and ICD-9 codes. The combined risk of death, myocardial infarction, stroke, coronary dissection, pericardial effusion, or cardiac tamponade was 8.2 out of 10,000 procedures. Furthermore, adding a RHC to a LHC procedure did not increases risk of these complications . Pulmonary infarction was described by Foote et al. to be a result of thrombi formed around the catheter or catheter occlusion of the small pulmonary artery. Pulmonary infarction was found in 9 % of patients, and they recommended frequent chest x-ray to monitor for distal migration of the catheter tip . In another study, pulmonary infarction was found in 2 out of 112 RHC and both resulted from distal migration and pulmonary artery occlusion by the catheter . This necessitates monitoring with daily chest x-ray to lessen the risk. Overall, the rate of complications of PAC and RHC is low in the hands of an experienced provider. Invasive hemodynamic monitoring with a pulmonary artery catheter should be performed to guide therapy in patients who have respiratory distress or clinical evidence of impaired perfusion in whom the adequacy or excess of intracardiac filling pressures cannot be determined from clinical assessment. (Level of Evidence: C) Class IIa: Invasive hemodynamic monitoring can be useful for carefully selected patients with acute HF who have persistent symptoms despite empiric adjustment of standard therapies and: 1. whose volume status, perfusion, or systemic or pulmonary vascular resistance is uncertain; 2. whose systolic pressure remains low, or is associated with symptoms, despite initial therapy; 3. whose renal function is worsening with therapy; 4. who require parenteral vasoactive agents; or 5. who may need consideration for MCS or transplantation. (Level of Evidence: C) Class III: No Benefit Routine use of invasive hemodynamic monitoring is not recommended in normotensive patients with acute decompensated HF and congestion with symptomatic response to diuretics and vasodilators. (Level of Evidence: B). Class I: 1. Patients with unexplained exertional dyspnea, syncope, and/or signs of right ventricular dysfunction should be screened with transthoracic echocardiography followed by confirmed diagnosis with RHC 2. RHC is required when treatment for PH is being considered. RHC is required to confirm diagnosis of pulmonary hypertension. It is required for diagnosis of PAH and Chronic thromboembolic pulmonary hypertension. 3. RHC is recommended in all cases of suspected PAH due to connective tissue disease 4. RHC is recommended for patients with congenital cardiac shunt to consider surgical correction 5. RHC is recommended in patients with PH due to left-sided heart disease (Group 2 PH) or chronic lung disease (Group 3 PH) if being considered for organ transplantation 6. High doses of CCBs are recommended in patients with idiopathic PAH, heritable PAH, and drug-induced PAH who respond to acute vasoreactivity testing. Reassessment after 3–4 months of therapy with RHC is recommended after treatment Class IIa: 1. RHC should be considered in pulmonary arterial hypertension (Group 1 PH) to assess the treatment effect of medications Class I, Level of Evidence C: 1. RHC should be performed on all candidates in preparation for listing for cardiac transplantation and annually until transplantation 2. RHC should be performed at 3- to 6-month intervals in listed patients, especially in the presence of reversible pulmonary hypertension or worsening of heart failure symptoms) 3. A vasodilator challenge should be administered when the pulmonary artery systolic pressure is ≥50 mmHg and either the transpulmonary gradient (TPG) is ≥15 or the pulmonary vascular resistance (PVR) is >3 Wood units while maintaining a systolic arterial blood pressure > 85 mmHg 4. When an acute vasodilator challenge is unsuccessful, hospitalization with continuous hemodynamic monitoring should be performed, as often the PVR will decline after 24 to 48 h of treatment consisting of diuretics, inotropes and vasoactive agents such as inhaled nitric oxide. Internal jugular vein access is commonly used for patients in the critical care setting for continuous hemodynamic monitoring, and it is easier to manipulate into the PA compared to femoral vein access. Ultrasound is utilized for cannulation and a double Seldinger technique allows for lower risk of bleeding complications . Brachial vein access can also be utilized. This can easily be done by swapping out a 20G venous catheter and over the wire exchanging it for a 5–8 F sheath. Consensus statements have recommended measurement of blood oxygen saturation from the distal PAC tip in the wedge position if PAWP is elevated or if its accuracy is questionable . A arterial oxygen saturation level would indicate true wedge position, while a lower than expected value would indicate incomplete occlusion of the PA. Viray et al. performed a prospective trial to determine the differences in PAWP values and PH classification utilizing oxygen saturation. Oxygen saturation was measured when PAWP measurements were >15 mmHg and occlusive saturation was defined as SaO2 > 90 % or within 5 % of systemic arterial saturation. If these criteria were not met, two additional attempts were made. The authors found that even with confirmation of PAWP by fluoroscopy and appropriate waveform appearance, an occlusive SaO2 was only obtained 50 % of the time. They found that 29 out of 45 (64.4 %) had significantly lower PAWP obtained with position confirmed by SaO2 compared to initial PAWP measurement, and 10 patients were reclassified - 6 changed from post-capillary to pre-capillary PH and 4 changed from post-capillary PH to combined pre- and post-capillary PH . Measurements and calculations can be referenced in Table 6 , Table 7 , and waveforms obtained from RHC are in Fig. 1 , Fig. 2 , Fig. 3 , Fig. 4 , Fig. 5 , Fig. 6 , Fig. 7 , Fig. 8 , Fig. 9 , Fig. 10 , Fig. 11 , Fig. 12 . Table 6 Direct hemodynamic measurements obtained from RHC. Table 6 Hemodynamic measurement Normal Range Comments Central Venous Pressure 2–6 mmHg Right atrial pressure 2–6 mmHg Right Ventricular Pressure (systolic and diastolic) Systolic: 15–25 mmHg Diastolic: 1–8 mmHg Pulmonary artery pressure (systolic/diastolic/mean) Systolic: 15–25 mmHg Diastolic: 4–12 mmHg Mean: 9–18 mmHg Pulmonary Artery Wedge Pressure 6–15 mmHg Recorded at end expiration, mean of three to five measurements Cardiac Output 4–6 L/min Determined using Thermodilution or Fick Mixed Venous Oxyhemoglobin Saturation (SvO 2 ) 60–75 % Table 7 Hemodynamic Measurements calculated from RHC measurements: Table 7 Hemodynamic measurement Calculation Normal values Systemic Vascular Resistance (SVR) mean systemic artery pressure – CVP CO 700–1600 dynes- sec /cm 5 Pulmonary Vascular Resistance (PVR) mPAP – PAWP CO 20–120 dynes-sec/cm 5 Cardiac Index (CI) CO BSA 2.5–4.0 L/min/M 2 Stroke Volume Index CI HR × 1000 ≥ 35 mL/m 2 Right Ventricular Stroke Work Index (RVSWI) mPAP – RAP × CI × 0.0136 HR 5–10 g/m 2 /beat Cardiac Power Output CPO = (mean arterial pressure − RAP) × cardiac output 1.0 W Transpulmonary Gradient (TPG) mPAP – PAWP < 12 mmHg Diastolic Pulmonary Gradient (DPG) Diastolic PAP – PAWP < 7 mmHg Pulmonary Artery Pulsatile Index systolic PAP – diastolic PAP RAP PAPi <1.85 RV failure after LVAD PAPi <1.0 RV failure in acute MI Oxygen Delivery CO × CaO 2 = CO × [(Hgb × SaO 2 × 1.34) + ] 1000 mL of O 2 /min Oxygen Uptake 1.3.4 × [CO × Hgb × (SaO 2 – SvO 2 )] CVP = central venous pressure, CO = cardiac output, mPAP = mean pulmonary artery pressure, PAWP = pulmonary artery wedge pressure, BSA = body surface area, CI = cardiac index, HR = heart rate, RAP = right atrial pressure, PAP = pulmonary artery pressure, CaO 2 = arterial O 2 content, Hgb = hemoglobin, SaO 2 = oxygen saturation, PaO 2 = partial pressure of arterial oxygen, SvO 2 = mixed venous oxygen saturation Fig. 1 Normal Waveform RA Pressure (Normal RA pressure waveform is described by the “a”, “c”, and “v” waves and “x” and “y” descents. The a wave represents atrial systolic. The c wave occurs with closure of the tricuspid valve. The x descent shows atrial relaxation with atrial diastole. The v wave is associated with rise in RA pressure due to RA filling during ventricular systole. Finally, the y descent occurs during RA emptying in early diastole when rapid ventricular filling occurs). Fig. 1 Fig. 2 Cannon A Waves (The cannon a wave are seen with with atrioventricular dissociation when the RA contracts against a closed tricuspid valve. These can be seen with ventricular tachycardia or complete heart block.). Fig. 2 Fig. 3 Loss of A Wave (The loss of a wave is seen with a loss of atrial contractions, primarily in the setting of atrial fibrillation.). Fig. 3 Fig. 4 Blunted Y Descent (The blunted y descent occurs in the setting of elevated RV pressures and in the following conditions: tamponade or tricuspid stenosis). Fig. 4 Fig. 5 Prominent X and Y Descent (A prominent x descent is found in constrictive pericarditis or cardiac tamponade. The y descent is prominent in found in constrictive pericarditis.). Fig. 5 Fig. 6 RV Pressure Waveform (The RV pressure waveform is shown above. Normal pressure ranges of the RV during systole are 15–25 mmHg while pressures during diastole range from 1 to 8 mmHg.). Fig. 6 Fig. 7 PA Pressure Waveform (The PA diastolic pressure will be representative of the PAWP. The dicrotic notch is when the pulmonic valve closes.). Fig. 7 Fig. 8 PAWP Waveform (This tracing represents the PAWP. Normal pressure ranges from 6 to 15 mmHg. The a wave represents left atrial contraction, and the v wave is associated with ventricular systole causing a rise in LA pressure. The x descent represents atrial relaxation, and passive filling of the LV is represented by the y descent.). Fig. 8 Fig. 9 Overwedge PAWP (Overwedging of the PAWP leads to falsely elevated pressures. The pressure waveform may become non-pulsatile as well). Fig. 9 Fig. 10 Underwedge PAWP (Under-wedging occurs in the setting of incomplete occlusion of the PA and leads to falsely elevated PAWP resulting in misdiagnosis of PH.). Fig. 10 Fig. 11 Waveform of Restrictive Cardiomyopathy (Restrictive cardiomyopathy shows concordance of ventricular pressures during respiratory cycle. The rapid y-descent can also be noted.). Fig. 11 Fig. 12 Waveform of Constrictive Disease (Ventricular discordance is found during the respiratory cycle in constrictive physiology. The square root sign can be found as a result of the rapid y-descent.). Fig. 12 PVR ( Table 7 ) is a useful measurement to guide diagnostics and decision making for PAH, subtypes of PH, and heart transplantation. It provides important prognostic information about mortality in PH and heart transplantation patients. Pre-capillary PH is defined as mPAP >20 mmHg, PAWP ≤15 mmHg, and PVR ≥ 2 WU, while post-capillary PH consists of mPAP >20 mmHg, PAWP >15 mmHg, and PVR < 2 WU . Some data have recommended a PVR cut-off of 2.2 WU to differentiate subtypes, and values greater or equal to 2.2 have shown increased mortality . Once diagnosis is established, pulmonary vasoreactivity testing is recommended for idiopathic PAH, heritable PAH, or drug-induced PAH. A positive response is defined as a reduction of mPAP ≥ 10 mmHg to reach an absolute value of mPAP ≤ 40 mmHg with an increased or unchanged CO. Responders are suitable for calcium channel blocker (CCB) therapy and long-term response is defined by New York Heart Association Functional Class I or II and sustained hemodynamic improvement after at least 1 year on CCB therapy alone . Pulmonary effective arterial elastance (Ea) is regarded as a measure of total right ventricular afterload and represents both resistive and pulsatile components. In a study by Tampakakis et al., both Ea and pulmonary artery compliance were better predictors of mortality and right ventricular dysfunction than PVR or TPG in patients with PH due to left heart disease. The authors suggested these parameters could be utilized for therapies targeted towards RV load . Although RHC provides data and insights that cannot be obtained from clinical exam, it is subject to variability and errors in measurement and interpretations. These errors can lead to misclassification of PH group resulting in inappropriate therapy in patients. The first step to avoid errors in measurement is to ensure the correct set-up of pressure lines and transducers. The pressure transducer should be zeroed at the mid-thoracic level, halfway between the anterior sternum and bed surface while the patient is lying supine. This represents the location of the left atrium, and deviations from can lead to alterations in measurement . PAWP should be measured at the mid a-wave of end-expiration with normal breathing (reference Tedford and/or others). Pressures are usually recorded as an average of three to five measurements . The PAWP will accurately reflect LA pressure when the catheter is located in West Zone III (define) where arterial pressure > venous pressure > alveolar pressure in the lung. This position allows for the best indicator of the LA pressure as it creates a continuous column of blood from the catheter directly to the LA. The alveolar pressure will be measured if the catheter ends up in Zone I or II, often signified by absence of a- and v-waves or PA diastolic pressure exceeding PAWP . In patients with chronic obstructive pulmonary disease, increases in intrathoracic pressure during end-expiration can lead to errors in mPAP and PAWP, and measurements should be averaged over the entire respiratory cycle . This may also be the case in obesity, although this should be interpreted with caution as obesity is strongly associated with HFpEF . While these observations were made using an esophageal pressure-transducing catheters as a surrogate of intrathoracic pressures, this technique is unlikey to be routinely employed . Positive end-expiratory pressure (PEEP) and mechanical ventilation affect RHC hemodynamic values. PEEP leads to increase in pleural and transpulmonary pressures causing reduced RV filling and increased RV afterload. This contributes to an elevated PAWP and overestimation of the LA pressure. Measurement for patients on positive pressure ventilation should be at end-inspiration. Over-wedging or under-wedging can lead to erroneous PAWP measurements. Over-wedging occurs from excessive inflation of the balloon and makes values falsely low or high. When a catheter balloon is over-wedged, deflating the balloon usually fixes the issue. Over-wedging can lead to increased risk of PA rupture. Under-wedging occurs when the balloon is not completely occluding the PA and provides an artificially elevated measurement. This can be solved by deflating and then inflating the catheter until it wedges completely or sending it into a different PA branch . Cardiac output (CO) can be measured by either Thermodilution (Td) or Fick methods. Td is obtained by injecting a fluid bolus into the right atrium, then measuring the temperature change of the fluid once it reaches the PA. This change in temperature is plotted against time, and the area under the curve is inversely related to CO . Inaccuracies with Td can occur in patients with severe tricuspid regurgitation, intracardiac shunts, arrythmias, and low CO states . The estimated oxygen uptake Fick (eFick) method estimates oxygen consumption (V̇O 2 ) using body surface area, age, gender, and heart rate, but it can be altered in the setting of HF, PH or abnormal body habitus . In a study comparing measured to estimated V̇O 2 , Narang et al. found that estimated V̇O 2 is inaccurate leading to a proportional error when deriving CO . Opotowsky et al. studied the correlation between eFick and Td in 15,000 patients undergoing RHC and found that CO estimates differed by >20 % in one-third of patients. Low CO/CI measured by Td was a stronger predictor of mortality compared to eFick. Therefore, Td remains the preferred method for CO assessment as it is a better predictor of all-cause mortality and prognosis in HF and PAH. Furthermore, even in the setting of tricuspid regurgitation, Td measurements were associated with mortality while eFick was not . When the type of shock remains undetermined or there are signs to suggest multi-factorial shock, invasive hemodynamic data can be critical in timely diagnosis and monitoring therapy. Although routine use of PAC after ST segment elevation myocardial infarction (STEMI) is not recommended, it is recommended in patients with CS or hypotension after infarction to determine additional therapy and response. Overall 30-day mortality for CS associated with STEMI remains around 40–50 %, therefore recognition of those at risk is of utmost importance . Multi-factorial shock is particularly challenging to identify without hemodynamic data, and analysis of the patients in the SHOCK (Should we emergently revascularize Occluded Coronaries for cardiogenic shocK) revealed that 18 % of patients with CS showed signs of severe systemic inflammation manifesting as distributive shock. A reduced SVR was found to occur early during shock, prior to the diagnosis of sepsis, which suggested that vasodilation plays an important role in CS . In the SHOCK Trial Registry, cardiac power output (CPO) and cardiac power index (CPI) were independently associated with in-hospital mortality. CPO is calculated as the mean arterial pressure x CO/451, and a CPO ≤0.53 W had a sensitivity and specificity of 66 % for in-hospital mortality . Therefore, the use of RHC can provide additional data if patients do not present or respond to therapies in a classical pattern ( Table 8 , Table 9 , Table 10 ). Table 8 Calculations of Ea and PAC : Table 8 Hemodynamic Measurement Calculation Pulmonary effective arterial Elastance (Ea) End-Systolic PAP Stroke Volume Pulmonary Arterial Compliance (PAC) Stroke Volume Pulmonary Artery Pulse Pressure Table 9 Cardiogenic shock data interpretation [ 14 , , , ]. Table 9 Right Ventricular Dominant Left Ventricular Dominant Biventricular Dominant Variables CVP > 15 mmHg < 15 mmHg > 15 mmHg PAWP < 18 mmHg > 18 mmHg > 18 mmHg CVP/PAWP > 0.8 < 0.8 > 0.8 PAPI < 1.5 > 1.5 < 1.5 CI < 2.2 L·min −1 ·m −2 < 2.2 L·min −1 ·m −2 < 2.2 L·min −1 ·m −2 CPO < 0.6 < 0.6 < 0.6 Table 10 SCAI classification of cardiogenic shock (adapted) . Table 10 Description Typical Physical exam/bedside findings Typical Biochemical markers Typical Hemodynamics Stage A (at risk) A patient who is not currently experiencing signs or symptoms of CS, but is at risk for its development Normal JVP Warm and well perfused Normal lactate Normotensive B (beginning CS) A patient who has clinical evidence of hemodynamic instability (including relative hypotension or tachycardia) without hypoperfusion Elevated JVP Warm and well-perfused Normal lactate Hypotension (SBP < 90 mmHg, MAP<60 mmHg or > 30 mmHg drop from baseline) Tachycardia (HR > 100 bpm) C (classic CS) A patient who manifests with hypoperfusion and who requires one intervention(pharmacological or mechanical)beyond volume resuscitation Volume overload Lactate ≥ 2 mmol/L Cardiac index<2.2 L/min/m2 PAWP>15 mmHg D (deteriorating/doom) A patient who is similar to category C but is getting worse. Failure of initial support strategy to restore perfusion as evidenced by worsening hemodynamics or rising lactate Any of the C and worsening (or not improving) signs/ symptoms of hypoperfusion despite the initial therapy Any of the stage C and lactate rising and persistently ≥ 2 mmol/L Any of stage C and requiring escalating doses or increasing numbers of pressors or addition of a mechanical circulatory support device to maintain perfusion E (extremis) Actual or impending circulatory collapse Typically unconscious Lactate ≥ 8 mmol/L Profound hypotension despite maximal hemodynamic support | Review | biomedical | en | 0.999997 |
PMC11699068 | Listeria monocytogenes is an important food-borne pathogen causing life-threatening disease in immunocompromised populations . The main vehicle of infection is ready-to-eat food , which is often contaminated from the processing environment [ , , ]. Except for a decrease in the number of cases to 1887 in 2020 (the COVID-19 pandemic period), the general trend for listeriosis did not show any significant increase or decrease in the last decade. However, the mortality rate in EU increased to 18.1 % in 2022, higher than in 2021 and 2020 (13.7 % and 13.0 %, respectively) . L. monocytogenes is a Gram-positive, facultatively anaerobic, non-spore-forming, motile rod bacterium (in the range of 20–25 °C). Its cardinal growth parameters referring to the main intrinsic and extrinsic food environmental factors are well known ( T min = −1.5 °C, T opt = 37 °C, pH min = 4.39, pH opt = 7.0, pH max = 9.6, a w min = 0.92 μ opt = 2.0 h −1 ) and are successfully used in growth predictions . However, variability in growth should be expected and can be quantified considering experimental, biological, and strain differences . Recently, the term microbial ‘persistence’ was defined as the ability of a given organism to be established in niches (or harbourage sites) within the food and feed processing environment for a long term, despite the regular application of cleaning and disinfection . Long-term presence means months or years accompanied by growth or survival in specific places of the processing environment. Despite extensive research, the persistence mechanism of L. monocytogenes in the food processing environment remains a topic of interest . The main candidate phenotypic traits responsible for persistence include increased biofilm formation , formation of mixed biofilms , higher resistance to sanitisers and better growth in suboptimal conditions . While the contribution of biofilm formation and resistance to sanitisers has been extensively discussed for L. monocytogenes persistence, this study aims to add information on the contribution of growth rate to this phenomenon. Primary mathematical modelling enables the analysis of the behaviour of microorganisms involving spore germination, growth, inhibition, inactivation, or survival over time. It provides kinetic models with parameters that are, however, sensitive to changes in food environmental factors . The mathematical relationships between the primary model parameters and the independent variables, such as intrinsic and extrinsic factors, such as a w , pH , temperature or modified atmosphere, are usually the subject of secondary modelling. Combining primary and secondary models, even more sophisticated tertiary models or expert systems can be created to predict the fate of microorganisms in specific foods with known environmental factors. This approach may help food microbiologists and manufacturers ensure the microbiological quality and safety of food throughout the food chain. For these reasons, mathematical modelling and predictive microbiology approach are integral parts of microbiological exposure assessment and, in combination with dose-response models, are also an inevitable part of risk assessment . The present study focused on growth quantification and modelling using classical primary and secondary modelling to characterise the growth of a persistent and a sporadic L. monocytogenes strain. The main goal was to compare their growth responses in two media, Tryptic Soy Broth (TSB) and semi-synthetic cheese medium (SCM), and to assess the variability as well as the statistical significance of potential differences in their behaviour. L. monocytogenes LM9611-19 (LM-P) and L. monocytogenes LM120/5 (LM-S) were used as representatives of persistent and sporadic potentially non-persistent strains, respectively. LM-P was isolated from ewes' milk stored in a cooling tank at a farm that produces raw milk cheese and identified by the State Veterinary and Food Institute, Dolný Kubín, Slovakia. It was classified as molecular serogroup IIa, clonal complex 14, sequence type 14 and was considered persistent based on <10 allelic differences in cgMLST (39 isolates during 18-month period) in our previous study . Based on testing according to Stepanović et al. and Di Ciccio et al. , the strain was classified as a moderate biofilm producer (data not shown). Upon testing substrate utilization with API Listeria (Biomérieux, Marcy l'Étoile, France), the strain gave typical results with a numerical profile 6510. LM-S was isolated from the milking equipment filter in the same facility as LM-P and characterised in our laboratories. It was classified as molecular serogroup IIa, clonal complex 14, sequence type 91 and considered sporadic (single isolate for 18 months) in our previous study . Based on testing according to Refs. , the LM-S strain was classified as a weak biofilm producer (data not shown). Upon substrate utilization testing with API Listeria, the strain gave atypical results of positive fermentation of D-xylose and D-tagatose, resulting in a numerical profile 6711. For growth experiments, freeze-dried L. monocytogenes strains were revitalised by incubation in TSB (Merck, Darmstadt, Germany) at 37 °C for 24 h with mild shaking (1.7 Hz). The culture was streaked on TSA (Merck) and incubated at 37 °C for 48 h. Plates with cultures were stored at 5 °C with re-inoculation every 14 days for a maximum of 5 times. Cultures preserved in this way were used to prepare the basic suspension by picking a well-isolated colony into 10 ml of TSB, mixed with vortex, and incubated at 37 °C with mild shaking (1.7 Hz). After 18 h, microbial counts were determined by spreading 200 μl of decimal dilutions in peptone saline (containing 8.5 g NaCl (Mikrochem, Pezinok, Slovakia) and 1 g peptone (Lab M, Heywood, United Kingdom) per 1000 ml) on the surface of TSA plates in duplicate. Counts were calculated and expressed according to ISO 4833–2 . The numbers determined for the basic suspension were used to adjust the inoculum so that, after adding 1 ml–300 ml of growth medium, the concentration at the beginning of each experiment was within the range of 10 2 –10 3 CFU/ml. The semi-synthetic cheese medium (SCM) was prepared according to Kagkli et al. and Schrama et al. to imitate the nutritive potential of cheese. The following components were dissolved in 275 ml deionised water: 0.06 g CaCl 2 ·6H 2 O (Sigma-Aldrich, St. Louis, USA), 0.30 g yeast extract (HiMedia, Mumbai, India), 0.31 g MgSO 4 ·6H2O (Lachema, Brno, Czech Republic), 1.8 g L-methionine (Merck, Darmstadt, Germany), 2.04 g of KH 2 PO 4 (Centralchem, Bratislava, Slovakia), 3 g NaCl (Centralchem), 4.5 g casein (bioreagent for cell culture grade, Sigma-Aldrich) and 11.4 ml sodium DL-lactate (Sigma- Aldrich). The lactose stock solution was prepared by steam sterilisation at a temperature of up to 100 °C for 20 min. After cooling, 25 ml of the solution was added aseptically to the cheese medium base which finally contained 8.4 g of lactose (Slavus, Bratislava, Slovak Republic) in 300 ml. Using a 10 % NaOH solution (Mikrochem) or lactic acid (Lachema), the pH value of the complete medium was adjusted to pH 7.2 before sterilisation to obtain pH 7.0 ± 0.1 after sterilisation. The a w value of the medium was 0.974 ± 0.003. A volume of 1 ml of the appropriate dilution of the basic bacterial suspension was aseptically added to a flask with 300 ml of TSB or SCM and thoroughly mixed. Inoculated growth media were incubated at temperatures of 6 °C, 10 °C, 16 °C, 22 °C, 25 °C, 30 °C, 37 °C, 40 °C and 43 °C. For quantification, 1 ml of the culture from each flask was taken at predetermined time intervals over a period until the bacterial population reached a stationary phase or for up to 14 h. Subsequently, 200 μl of certain set dilutions were plated on the TSA surface in duplicate. The agar plates were incubated at 37 °C for 24–48 h. Duplicate growth curves were constructed within each experiment in TSB at each temperature, except for 6 °C, 30 °C and 43 °C, at which two independent experiments were carried out. In SCM, two independent experiments were carried out in duplicates at each temperature. A) Inspired by Aryani et al. , who defined the experimental, biological, and strain variabilities for growth and inactivation rates of 20 strains of L. monocytogenes in detail, indices such as MSE and RMSE were estimated in this study. Indices were calculated for the growth parameters of LM-P and LM-S strains at each temperature. According to the mentioned authors, experimental variability was defined as the difference between duplicate experiments conducted in parallel at the same time on the same experimental day. Biological variability was defined as the difference between independently reproduced experiments of the same strain performed on different experimental days from new pre-cultures and newly prepared media, and strain variability means the difference between strains of the same species. B) Alternatively, experimental RMSE values were also calculated from primary growth curve data (experiments with two replicates). Then, biological RMSE resulted from the combined data of 2 experiments, each with 2 replicates (4 replicates altogether), and analogously, the strain RMSE was calculated from all combined growth data for both strains in each medium used. These data sets involved data from 4 experiments (8 replicates; Table 1 and Table 2 for 6 °C and 30 °C, respectively). The mentioned temperatures were chosen because they generally represent low and optimal growth temperature ranges, and also because the highest number of replicates was available at these temperatures. This approach can provide an additional perspective to the evaluation of experimental, biological, and strain variabilities. Table 1 Growth data of L. monocytogenes strains in TSB and SCM at 6 °C. Table 1 Strain n rep / n exp Medium Model parameters Statistical indices n λ (h) rate log N 0 log N end RMSE RMSE ave Variability R 2 (log CFU/h) (log CFU/mL) LM-P 2/1 TSB 45.0 0.022 3.58 9.24 0.063 0.072 experimental 0.999 15 42.6 0.022 3.55 9.43 0.084 0.998 15 70.0 0.022 3.22 8.97 0.072 0.998 12 67.0 0.022 3.19 9.00 0.069 0.999 12 LM-S 78.7 0.020 3.48 9.71 0.109 0.080 0.997 12 59.4 0.022 3.31 9.49 0.095 0.998 13 87.7 0.022 3.14 8.98 0.042 0.999 12 89.6 0.021 3.05 9.15 0.075 0.999 13 LM-P 2/1 SCM 98.9 0.020 3.18 7.65 0.110 0.121 experimental 0.996 11 107.9 0.021 3.23 7.52 0.211 a 0.987 11 103.9 0.021 3.36 7.63 0.072 0.998 13 108.8 0.023 3.27 7.58 0.091 0.997 13 LM-S 116.3 0.025 2.53 7.49 0.361 a 0.264 0.970 11 116.0 0.021 2.52 7.63 0.333 a 0.971 11 127.5 0.024 2.94 7.30 0.264 a 0.979 16 95.9 0.020 2.99 7.55 0.100 0.997 17 LM-P 4/2 TSB 42.3 0.021 3.33 9.21 0.388 0.289 biological 0.964 54 LM-S 78.9 0.021 3.28 9.45 0.371 0.968 50 LM-P SCM 104.0 0.021 3.28 7.60 0.133 0.994 48 LM-S 105.4 0.020 2.89 7.55 0.266 0.978 55 LM-P + LM-S 8/4 TSB 51.9 0.020 3.25 9.35 0.487 0.394 strain 0.945 104 LM-P + LM-S 8/4 SCM 105.0 0.020 3.10 7.56 0.301 0.971 103 a Declination of the numbers observed during the lag phase. n r ep - number of replicates within the experiment, n exp - number of experiments. Table 2 Growth data of L. monocytogenes strains in TSB and SCM at 30 °C. Table 2 Strain n rep /n exp Medium Model parameters Statistical indices n λ (h) rate log N 0 log N end RMSE RMSE ave Variability R 2 (log CFU/mL/h) (log CFU/mL) LM-P 2/1 TSB 2.6 0.469 3.58 9.45 0.106 0.069 experimental 0.998 16 2.5 0.464 3.55 9.43 0.075 0.999 16 2.0 0.452 3.63 9.34 0.041 0.999 15 1.7 0.446 3.57 9.38 0.054 0.999 16 LM-S 3.3 0.492 3.07 9.56 0.113 0.084 0.998 15 3.2 0.493 3.06 9.52 0.107 0.998 15 2.5 0.464 3.16 9.38 0.048 0.999 16 2.5 0.475 3.17 9.45 0.068 0.999 16 LM-P 2/1 SCM 3.6 0.383 3.80 7.93 0.107 0.085 experimental 0.996 14 4.1 0.439 3.77 7.82 0.103 0.997 12 4.0 0.363 3.75 8.03 0.081 0.998 18 3.4 0.344 3.73 7.97 0.049 0.999 18 LM-S 4.4 0.315 3.27 8.01 0.130 0.085 0.995 18 3.5 0.308 3.21 8.00 0.089 0.998 18 3.2 0.351 2.68 7.94 0.057 0.999 21 2.8 0.331 2.68 8.10 0.066 0.999 21 LM-P 4/2 TSB 2.3 0.475 3.49 9.25 0.181 0.157 biological 0.994 63 LM-S 2.8 0.476 3.10 9.48 0.150 0.996 62 LM-P SCM 3.7 0.372 3.76 7.93 0.131 0.994 62 LM-S 3.5 0.326 2.96 8.00 0.167 0.992 78 LM-P + LM-S 8/4 TSB 2.3 0.475 3.49 9.25 0.181 0.312 strain 0.985 125 LM-P + LM-S 8/4 SCM 3.5 0.341 3.36 7.79 0.444 0.937 140 n r ep - number of replicates within the experiment, n exp = number of experiments. The density of the cultures of both L. monocytogenes strains was evaluated as a function of time in the two growth media according to Baranyi's D-model using the Excel tool DMFit v. 3.5 (Institute of Food Research, Norwich, United Kingdom). To evaluate the effect of temperature on specific growth rate and lag phase (secondary modelling), non-linear regression using Excel Solver (Microsoft, Redmont, USA) was applied. Baranyi's model is described by the following equations: (1) y ( t ) = y 0 + μ max ⋅ A ( t ) − 1 m ⋅ ln ( 1 + e m ⋅ μ max ⋅ A ( t ) − 1 e m ⋅ ( y max − y 0 ) ) (2) A ( t ) = t + ln ( e − μ max ⋅ t + e − h 0 − e − μ max ⋅ t − h 0 ) μ max where y (t) = ln N (t), y 0 = ln N 0 , y max = ln N max , m is the parameter of the curvature of the growth line when the growth of microorganisms slows down from the exponential phase to the stationary phase. A (t) is an integral form of the adjustment function α ( t ) whose value increases with increasing time, t is time and h 0 is a dimensionless factor that quantifies the initial physiological state of cells and is related to lag phase ( λ ) by the equation h 0 = μ · λ . Using log 10 N in the DMFit tool, the main outputs of primary modelling were the growth rate k max (log CFU/mL/h) and the duration of lag phase λ (h). Before secondary modelling, k max was converted to μ max ( h −1 ) according to the equation μ max = k max ⋅ ln ( 10 ) . To describe the effects of temperature on the growth of L. monocytogenes strains in TSB and SCM at a w values of 0.995 and 0.974, respectively, the square root Cardinal model (sqrtCM) of Rosso et al. was used in the following form: (3) μ max = μ o p t ⋅ C M ( T ) (4) 1 λ = 1 λ o p t ⋅ C M ( T ) where (5) C M ( T ) = ( T − T max ) ⋅ ( T − T min ) 2 ( T o p t − T min ) ⋅ [ ( T o p t − T min ) ⋅ ( T − T o p t ) − ( T o p t − T max ) ⋅ ( T o p t + T min − 2 T ) ] In this work, a square root transformation of the whole cardinal model was used for both growth parameters μ max and reciprocal λ . The secondary model includes five parameters with biological meaning, namely, μ opt , λ , T min (theoretical minimum temperature), T opt (optimum temperature) and T max (maximum temperature above which growth is not likely). Except for normalising residuals, the modification allowed the estimation of parameters μ opt and 1/ λ opt in the original non-squared form, which is more practical particularly for μ opt (Eqs. (3) , (4) )). Specific growth rates of the two strains at 6–37 °C were used for internal cross-validation, in which a set of μ max calc values for LM-P was validated with LM-S μ max exp values and vice versa. Due to statistically significant differences in μ max values, the data at 40 °C and 43 °C were excluded from this validation. External validation of secondary rate models for both strains in cheese medium was performed using growth rates acquired from the ComBase Predictor (CBP) at a w 0.974, pH 7. For CMs in TSB, the experimental μ max values estimated by Bajard et al. in Mueller-Hinton broth (MHB) were used for validation. Data are collected in Suppl. Table S1 . To evaluate the accuracy of fitting, the coefficient of determination ( R 2 ) and the root mean square error ( RMSE ) were calculated: (6) R 2 = ∑ i = 1 n ( y i o b s − y i c a l ) 2 ( y i o b s − y ‾ i c a l ) 2 (7) R M S E = ( y i o b s − y i c a l ) 2 n − p where y i o b s , y i c a l and y ‾ i c a l are the observed (experimental), calculated (predicted) and averaged μ max or λ data, respectively; n is the number of observations; and p is the number of model parameters . The CMs for growth rate were externally validated using independent data excerpted from the Combase database at aw of 0.974 and pH 7 as well as the data from Ref. . The following equations were used to calculate validation indices bias and accuracy factors , : (8) B f = 10 ∑ i = 1 n log μ m a x i o b s - log μ m a x i p r e d n (9) A f = 10 ∑ i = 1 n | log μ m a x i p r e d − log μ m a x i o b s | / n According to te Giffel and Zwietering , the bias factor ( B f ) provides information on the distribution of the observed values towards the identity line between the observed (experimental) and predicted (calculated) μ max values. In this case, a value of B f lower than 1 indicates a ‘fail-safe’ prediction and a value of B f greater than 1 means a ‘fail-dangerous’ model. The accuracy factor ( A f ) describes how close are the predicted μ max to μ obs values. The statistical differences between μ max values of the two L. monocytogenes strains at each temperature were statistically evaluated using the Analyse-it Method Validation package ver. 6.15 (Analyse-it Software, Leeds, United Kingdom). Data were treated using Student's t-test followed by Tukey's test with the least significant difference of 95 %. In this work, 63 independent cultivation experiments were carried out with duplicate trials for the two strains, persistent LM-P and sporadic – potentially non-persistent LM-S, in TSB and SCM. TSB represented a theoretically optimal culture medium ( a w of 0.995, pH 7.0), while the semi-synthetic cheese medium simulated conditions that more closely resembled the internal environment of cheese ( a w of 0.947 ± 0.003, pH 7.0). The nine cultivation temperatures used (6–43 °C) covered almost the entire range of L. monocytogenes growth. In total, more than 120 growth curves were provided with growth parameters and statistical indices. Experimental, biological and strain variability was evaluated according to Aryani et al. using specific growth rates and were also based on the RMSE values of the growth curves from individual experiments or combined data from 2 to 4 experiments (4–8 replicates) performed at different times. To quantify the internal experimental, biological and strain variability of μ max according to Aryani et al. , we used μ max data recalculated from growth rates of all individual curves carried out in SCM at each temperature. In this way, we could evaluate the MSE values and compare RMSE values for our persistent and sporadic strains with those of 20 strains by Aryani et al. . The variances of μ max for our strains were independent of temperature. The following RMSE exp , RMSE biol , and RMSE str of 0.016 h −1 , 0.022 h −1 , and 0.017 h −1 were calculated, respectively, for both strains LM-P and LM-S in this study. Aryani et al. observed almost identical variability and the same trend between the values as we did in the current study, as RMSE biol was the highest, followed by RMSE str and RMSE exp . However, in the second case the RMSE values of growth curves, which are log 10 N -based and express the goodness of fit for the primary growth model, can also provide an opportunity to evaluate experimental, biological and strain variability. The RMSE average values (RMSE ave ) may come not only from individual trial duplicates conducted at the same time (experimental variability) or from experiments with duplicates performed at different times (biological variability) but also, in our case, from data combined from experiments with LM-P and LM-S (strain variability). Together with other growth parameters, the RMSE values are presented in Table 1 , Table 2 for temperatures of 6 °C and 30 °C, respectively. The experimental RMSE values fluctuated between 0.063 and 0.130 log CFU/mL for both strains, temperatures, and media with almost the same mean RMSE exp of 0.082 and 0.081 for 6 °C and 30 °C, respectively. Similarly, the RMSE ave values representing biological variability (2 experiments performed at different times containing data from 4 individual replicates) were 0.289 and 0.157 log CFU/mL for 6 °C and 30 °C, respectively. Finally, the highest average RMSE values of 0.394 and 0.312 log CFU/mL described the strain variability in both media (TSB and SCM) at 6 °C and 30 °C, respectively. In addition, the growth curves fitted with the Baranyi D-model using experimental data for both strains (LM-P exp + LM-S exp ; 8 replicates) and representing strain variability in each medium are shown in Fig. 1 (TSB in blue; SCM in yellow). We suppose that similar values could be found in the suboptimal temperature range. However, it could not be properly demonstrated as only one experiment with two replicates was performed in TSB at other temperatures within the study. Fig. 1 Growth curves of L. monocytogenes in TSB (blue) and SCM (yellow) at 6 °C and 30 °C. Merged data for LM-P and LM-S strains (LM-P exp + S exp ) come from 4 experiments and 8 replicates in total. Dashed lines represent fitted values ± standard error (SE) of the fit and the solid red line in the right button figure represents the recalculated growth curve using equalized N 0 of 2.96 log CFU/mL. Fig. 1 The comparison of variability based either on specific growth rates or fitting errors of growth curves needs to use relative indices. To do so, the ratios between the average RMSE values and μ -values were calculated for replicates, experiments, and combined strain data. Similarly, for relative indices based on growth curves, the ratio of RMSE and growth increases (log N end - log N 0 ) averages was used. The experimental, biological and strain variabilities based on μ data were represented with relative errors of 5.2 %, 4.0 % and 8.3 %, respectively. On the other hand, the relative indices based on the log N data showed slightly lower values of 1.6 %, 2.9 % and 6.1 %, respectively. It seems that both methods of evaluation provided acceptable outputs and, in this case, also certain relations between them could be defined. Except for one case, the relative indices doubled from experimental to biological and from biological to strain variability. The cardinal temperature model with inflection (CM) is one of the most widely used secondary models that reliably describes the effect of temperature in the full range of growth on microbial growth parameters. The model is often preferred as it provides four parameters that have biological interpretation. In contrast, the extended square root model contains two regression coefficients and parameters T min and T max , which are only theoretical minimum and maximum temperatures for growth and may differ significantly from the actual cardinal temperatures . Graphic presentations of the dependences of sqrt μ max and sqrt 1/ λ on the temperature for LM-P and LM-S in TSB (a w 0.995) and SCM (a w 0.974) are shown in Fig. 2 , Fig. 3 , respectively. The dashed lines represent the calculated data using standard errors of model parameters, which are summarised in Table 3 . Fig. 2 Modelling the effect of temperature on sqrt μ max of LM-P and LM-S strains in Tryptone Soy Broth (TSB) and semi-synthetic cheese medium (SCM). Fig. 2 Fig. 3 Modelling the effect of temperature on sqrt 1/ λ of LM-P and LM-S strains in Tryptone Soy Broth (TSB) and semi-synthetic cheese medium (SCM). Fig. 3 Table 3 Parameters of the square root CM model and statistical indices of fit for LM-P and LM-S strains in TSB and SCM. Table 3 Parameters CM sqrt 1/λ LM (TSB) CM sqrt μ max LM (TSB) CM sqrt 1/λ LM (SCM) CM sqrt μ max LM (SCM) LM-P SE LM-S SE LM-P SE LM-S SE LM-P SE LM-S SE LM-P SE LM-S SE T min (°C) −1.151 0.012 1.076 0.008 0.197 0.001 0.821 0.003 0.675 0.003 1.252 0.007 0.086 0.001 −1.115 0.005 T opt (°C) 40.820 0.134 36.895 0.133 37.203 0.030 36.376 0.066 38.923 0.105 38.811 0.103 38.026 0.091 36.367 0.063 T max (°C) 43.223 0.045 43.180 0.015 44.303 0.021 43.580 0.018 43.356 0.017 44.233 0.097 49.518 0.466 43.890 0.029 1/ λ opt (h −1 ) 0.704 0.024 0.495 0.016 – - 0.392 0.009 0.387 0.010 – – μ opt (h −1 ) – – 1.282 0.009 1.259 0.020 – – 0.944 0.018 0.894 0.013 RMSE 0.040 0.038 0.011 0.030 0.018 0.021 0.026 0.022 R 2 0.971 0.970 0.999 0.991 0.989 0.987 0.991 0.992 The solid lines representing the sqrtCM model with sqrt μ max for both strains in both media agreed with the observed μ max in the suboptimal temperature range. Differences were found in the region beyond T opt . The μ max exp values for LM-P and LM-S strains in TSB and SCM did not show significant differences in the suboptimal temperature range (6 °C–30 °C) and at 37 °C in both media. At the 5 % significance level, this was confirmed by ANOVA, Student's t-test, and Tukey's test at each temperature. However, in the region between optimal and maximum temperature, which contained two experimental temperatures (40 °C and 43 °C), the differences between μ max exp values from repeated trials were statistically confirmed for both strains and both media . Interestingly, the values of sqrt μ max were higher for LM-P than for LM-S at the three highest incubation temperatures, but they were statistically significant only at the last two, 40 °C and 43 °C. Furthermore, both cardinal parameters T opt and T max determined by the sqrtCM model for sqrt μ max were also higher for LM-P than for LM-S in both media ( Table 3 ). In terms of growth rate, Magalhães et al. found that persistent and non-persistent strains showed different responses to NaCl and acidic conditions (pH 5). Average rates were significantly higher (p < 0.05) for persistent than for non-persistent isolates when grown at 22 °C, 2.5 %, 4 %, 8 % NaCl, and at pH 5. In our case, the situation was different as significantly higher growth rates were observed for the persistent strain in both media only at temperatures higher than 37 °C (at 40 °C and 43 °C). A similar trend could be observed in Fig. 3 , which is the output of the sqrtCM model application to the lag phase. In this case, higher values of sqrt 1/ λ, which means a shorter lag phase, could be seen more clearly in the region of temperatures beyond T opt . Likewise, the T opt values resulting from secondary modelling of sqrt 1/ λ against temperature were higher in the case of LM-P in both media ( Table 3 ). Magalhães et al. estimated similar lag times for persistent and non-persistent isolates when grown at 37 °C, 22 °C and 4 °C but significantly shorter (p < 0.05) for persistent strains grown at 2.5 %, 4 % and 8 % NaCl, and at pH 5. In our case, significantly shorter (p < 0.05) lag times were observed for persistent strain (in both media) only at temperatures 40 °C. and 43 °C. However, the estimated T max values from the lag phase modelling were within the range of 43.2–44.2 °C for both strains and the media. The sqrtCM model fitted well all the data obtained from our experiments, as documented not only by the standard error of the parameters but also by the values of other statistical parameters such as RMSE and coefficient of determination R 2 ( Table 3 ). Associated with the minimal errors of estimation, all cardinal parameters for L. monocytogenes strains in this study were consistent with those mentioned by Bajard et al. , te Giffel and Zwietering and Jia et al. . Regardless of whether calculated on μ max or λ basis, in TSB or SCM, for a persistent or non-persistent strain, the most discussed cardinal parameter T min , here with estimation errors from 0.0003 °C to 0.012 °C, ranged from −1.2 °C to 1.3 °C. For LM-P strain, the range of T min values determined with the CM model in the two media was −1.2 to 0.7 °C with an average of 0.0 ± 0.8 °C (mean ± SD; n = 4). As in Table 3 , strain LM-S showed a slightly higher average T min of 0.5 ± 1.1 °C. A similar estimated minimum growth temperature of 0.6 ± 0.2 °C was reported for L. monocytogenes in pasteurised milk by Jia et al. . Even with sound statistical indices of the sqrtCM and model parameters shown in Table 3 , the results should be validated using independent data. In this work, growth data for two strains with different genetic and physiological characteristics offered an option of internal cross-validation, within which the sqrtCM model for LM-P could be validated with μ max exp values of LM-S and vice versa. As statistically significant differences were found between the μ max model and experimental data at 40 °C and 43 °C for the two strains, these data were excluded from internal cross-validation. Fig. 4 shows the comparison of two sets of predicted and observed μ max values (recalculated from sqrtCM) in SCM at a w of 0.974 for the suboptimal temperature range (6 °C–37 °C). Fig. 4 Comparison of the observed and predicted values at internal cross-validation with LM-P and LM-S in SCM. Fig. 4 Table 4 provides accompanying linear regression data for the predicted and observed μ max values with low parameter errors and acceptable RMSE 0.027 and 0.029, respectively. Table 4 Comparison of predicted and observed μ max values as a part of internal cross-validation. Table 4 Comparison between Linear regression and statistical parameters Intercept SE Slope SE RMSE R 2 predicted LM-P and observed LM-S μ max values −0.0047 0.0093 1.0544 0.0181 0.0270 0.9927 predicted LM-S and observed LM-P μ max values 0.0121 0.0097 0.9322 0.0176 0.0287 0.9912 The values predicted using the CM model for the LM-P strain slightly overestimated the experimental data for the LM-S strain. On the other hand, the calculated data for LM-S underestimated the experimental data for LM-P. However, the B f values of 1.12 and 0.98 for the LM-P and LM-S CM models, respectively, were close to 1.0. Furthermore, Fig. 4 showed that, on average, the experimental values for LM-S were higher than for LM-P in the range of 6 °C–37 °C. However, the opposite situation could be seen in the temperature range between T opt and T max . Square root CMs for growth rate were externally validated using independent data from Combase at a w of 0.974, pH 7 and from Bajard et al. . For Combase data, the bias factors for the LM-P and LM-S models in SCM were 0.93 and 0.96, respectively, while the accuracy factors were 1.09 and 1.07, respectively. This means that the sqrtCM model for both L. monocytogenes strains provided a ‘fail-safe’ prediction to Combase data with 9 % and 7 % accuracy. On the other hand, the validation of LM-P and LM-S models based on data from this study in TSB with experimental growth rates by Bajard et al. performed in Mueller-Hinton broth revealed ‘fail-dangerous’ predictions with B f 1.06 and 1.07, respectively. Both accuracy factors were equal to 1.15. In this work, the question of whether persistent or sporadically L. monocytogenes strains can grow better was dealt with by a quantitative study using two strains in two media in response to various temperatures. In the suboptimal temperature range, the growth parameters of persistent and sporadic, potentially non-persistent strains were almost indistinguishable, and it seems that growth predictions of persistent strains may be performed using the data of non-persistent strains (and vice versa), e.g., in exposure assessment. However, the models revealed differences in the behaviour of the strains at 40 °C and 43 °C. The results suggest that secondary modelling is suitable for testing a panel of L. monocytogenes strains regarding the possible correlation of their growth characteristics with persistence in food processing environments. The approach used in this study proved to be effective in revealing different growth responses in the range of higher temperatures than T opt . It can be extended also for mild inactivation temperature range as some similar differences in behaviour between persistent and non-persistent strains might also be expected. | Study | biomedical | en | 0.999997 |
PMC11699074 | An interview is a required part of nearly all medical school admissions processes. With a few exceptions, prior to the COVID-19 pandemic, most interviews were conducted in person and consisted of either an unstructured one-on-one interview or a structured interview such as the multiple mini-interview (MMI). To protect both applicants and medical school staff during the COVID-19 pandemic, all medical school interviews went to a virtual format, starting either mid-cycle in 2019–2020 or at the beginning of the 2020–2021 admissions cycle. The transition required either the use of existing video conferencing technologies (e.g., Zoom, Microsoft Teams), the creation of an in-house system, or the purchase of a virtual interview system from a third-party vendor. Virtual interviews for medical school admissions were not unknown prior to the COVID-19 pandemic. A study published in 2018 placed the number of virtual interviews at US allopathic medical schools at 10% . Virtual interviews for graduate medical education (i.e., residency and fellowship) were already being used by many programs prior to the pandemic . In May of 2022, the Association of American Medical Colleges (AAMC) recommended the continuation of virtual residency interviews . Several studies performed by residency and fellowship programs reported satisfaction with the virtual interview format [ 6 – 8 ]; however, some surveys found that in-person fellowship interviews were preferred by a majority applicants since they thought they could better evaluate a program’s facilities and meet faculty . Few studies in the pre-COVID-19 era directly compared virtual and in-person medical school interviews, and only one used virtual MMI (vMMI) as the interview format . The University of New Mexico School of Medicine described their experience using a video-based platform in the pre-COVID-19 era but used a semi-structured interview format . Several medical schools, both in and outside the USA, have published their experience of switching from in-person MMI (ipMMI) to vMMI, and none of them commented on the differences between the URiM applicants and the rest of the interview pool [ 11 , 22 – 27 ]. Cork looked at Zoom vMMI and ipMMI and found the delivery of the MMI had comparable scoring . Hammond and colleagues compared a synchronous vMMI using Zoom and their pre-COVID ipMMI for pharmacy school admissions and found that the vMMI was a reliable alternative to the ipMMI . These forementioned studies did not specifically investigate race or gender as potential differences in the vMMI and ipMMI performance. This lack of comparison between vMMI and ipMMI performance across demographic factors is surprising given well-established literature on interview and rater bias and several articles describing how bias may manifest differently in the virtual setting [ 17 , 18 , 29 – 31 ]. This study describes a completely automated, synchronous MMI system using the same rater user interface for both vMMI and ipMMI with special attention paid to differences between different populations of interviewees. The vMMI was developed using .NET (.NET Foundation, Richmond, WA) and integration with Microsoft Teams web version (Microsoft Corporation, Richmond, WA). An in-house developed student information system, MedOneStop, was used as an admissions interface for applicants, admissions staff, and raters. Both the vMMI and ipMMI had applicants rotate through eight stations, each with a different rater (interviewer) . The same bank of MMI scenarios were used in both the ipMMI and vMMI. Scenarios that required interaction between two applicants were not used in the vMMI. Actors, either members of the community or medical students, were used in the ipMMI, but during the vMMI, the rater assumed the actor’s role. The raters were recruited primarily from the faculty, staff, and medical students at the University of Cincinnati College of Medicine (UCCOM) and Cincinnati Children’s Hospital Medical Center. First-time raters were required to attend a 1-hour online training session in addition to a general admission’s training session. Fig. 1 Comparison of in-person and virtual multiple mini-interview day sequence Virtual adjusted annual MMI scores from medical school applicants in the 2021 and 2022 admissions cycles ( N = 627) were compared with ipMMI scores from 2017 to 2020 (Table 1 ). Additional comparisons were made between applicant subgroups including reported gender applicant-reported gender identity (male and female) and minority status (URiM and non-URiM). We used the AAMC definition of URiM as “any U.S. citizen or permanent resident who self-identified as one or more of the following race/ethnicity categories (alone or in combination with any other race/ethnicity category): American Indian or Alaska Native; Black or African American; Hispanic, Latino, or of Spanish Origin; or Native Hawaiian or Other Pacific Islander” . The applicants select their race/ethnicity when completing the American Medical College Application Service® (AMCAS®) application. Table 1 Number of interviewed applicants by MMI method, gender, and URiM status Year MMI Method Female Male Total URiM URiM Female URiM Male 2017 ipMMI 271 296 567 94 46 48 2018 ipMMI 301 325 626 123 52 71 2019 ipMMI 303 329 632 163 74 89 2020 ipMMI 322 296 618 153 86 67 Total ipMMI 1197 1246 2443 533 258 275 2021 vMMI 275 275 550 165 92 73 2022 vMMI 352 316 668 170 89 81 Total vMMI 627 591 1218 335 181 154 Total 1824 1837 3661 868 439 429 MMI multiple mini-interview, ipMMI , in-person MMI, vMMI virtual MMI, URiM underrepresented in medicine Comparisons were made between applicant subgroups including reported gender (male and female) and minority status (URiM and non-URiM). To analyze the data, we conducted a three-way analysis of variance (ANOVA) with gender, URiM status, and application year group (in-person vs. virtual) as independent variables and the adjusted MMI score as the dependent variable. This approach allowed us to examine main effects and interactions between these factors. While Likert scale data are ordinal, the large sample size and the tendency for scores to be distributed around the mean justify the use of parametric statistics in this case. Levene’s test was used to assess the homogeneity of variances across groups. The UCCOM institution review board approved this research , including waiver of consent and the minimal risk to participants. The three-way ANOVA revealed several significant findings regarding the effects of gender, URiM status, and application year group on MMI scores (Tables 2 and 3 ). Examining main effects, we found a significant influence of gender ( F = 25.707, p < 0.001), with females ( M = 0.066, SD = 0.417) scoring higher on average than males ( M = − 0.066, SD = 0.428). However, neither URiM status ( F = 0.063, p = 0.802) nor application year group ( F = 0.139, p = 0.710) showed significant main effects, indicating no overall differences between URiM and non-URiM applicants or between in-person and virtual MMI scores. Table 2 Descriptive statistics for adjusted MMI scores by gender, URiM status, and application year group Gender URiM App Yr (group) Mean Std. seviation N F N In-person 0.065 0.419 939 F N Virtual 0.048 0.393 446 F Y In-person 0.078 0.424 258 F Y Virtual 0.100 0.448 181 M N In-person − 0.057 0.421 971 M N Virtual − 0.055 0.419 437 M Y In-person − 0.092 0.444 275 M Y Virtual − 0.102 0.467 154 Overall − 0.001 0.428 3675 F (Overall) 0.066 0.417 1824 M (Overall) − 0.066 0.428 1837 N (Overall) 0.001 0.419 2803 Y (Overall) − 0.003 0.452 868 In-person (Overall) 0.000 0.428 2450 Virtual (Overall) − 0.002 0.428 1225 Table 3 Three-way ANOVA results for adjusted MMI scores Source Type III sum of squares df Mean square F Sig Corrected model 17.905 11 1.628 9.111 .000 Intercept 0.787 1 0.787 4.406 .036 Gender 13.778 3 4.593 25.707 .000 URiM 0.011 1 0.011 0.063 .802 App Year group 0.025 1 0.025 0.139 .710 Gender * URiM 0.834 1 0.834 4.667 .031 Gender * App year group 0.031 2 0.016 0.087 .917 URiM * App year group 0.028 1 0.028 0.155 .694 Gender * URiM * App year group 0.102 1 0.102 0.570 .450 Error 654.421 3663 0.179 Total 672.327 3675 Corrected total 672.326 3674 R -squared = .027 (adjusted R -squared = .024) We observed a significant interaction between gender and URiM status ( F = 4.667, p = 0.031). URiM females tended to score higher ( M = 0.087, SD = 0.434) compared with non-URiM females ( M = 0.060, SD = 0.411), while URiM males tended to score lower ( M = − 0.096, SD = 0.452) compared with non-URiM males ( M = − 0.056, SD = 0.420). It’s worth noting that Levene’s test indicated unequal variances across groups ( F = 1.990, p = 0.026). While this suggests some caution in interpreting the results, the large sample size and the robustness of ANOVA to moderate violations of homogeneity of variance lend credibility to our findings. Second, Levene’s test indicated unequal variances across groups. While ANOVA is generally robust to moderate violations of homogeneity of variance, especially with large and balanced samples, this should be considered when interpreting the results. The transformation from in-person multiple mini-interviews (ipMMI) to virtual multiple mini-interviews (vMMI) in medical school admissions amid the COVID-19 pandemic offered an unanticipated opportunity to examine biases and differences within a complex evaluation framework. Our study, conducted at the University of Cincinnati College of Medicine, stands as the first to describe and compare both vMMI and ipMMI modalities, with an emphasis on their effect on different demographic groups. Our study demonstrates that the transition from in-person to virtual MMIs did not introduce significant biases or alter performance patterns across demographic groups. The consistent gender effect and the complex interaction between gender and URiM status highlight the need for continued research into factors influencing MMI performance. These findings support the use of virtual MMIs as a viable alternative to in-person interviews, potentially offering a more accessible and equitable admissions process. Future research should focus on understanding the underlying mechanisms of these demographic differences and exploring ways to ensure fair assessment across all applicant groups. | Study | biomedical | en | 0.999997 |
PMC11699083 | A novel optical fiber with micro-structured architecture, PCF, shows a longitudinal periodic pattern of air holes. Due to its unique architecture, the PCF exactly manages its optical properties, such as photon incarceration, variation, and nonlinear behavior. Because of its unique laser-guiding qualities, with features of configurable architecture, the PCF provides advantages such as superior light instruction, customizable dispersion features, and the possibility of a range of diversified uses like detecting telecommunications, and nonlinear optical technologies . There are very many applications of PCF across various disciplines (M. ) (S.K. ). The communication sector benefits from PCF by allowing faster data transmission while reducing signal distortion and dispersion. For sensing applications, PCF can be very useful because of its excellent reactivity to changes in the external environment, like temperature, pressure, and chemical concentrations. PCF provides accurate light delivery and imaging capabilities in healthcare imaging and laser-induced surgery. PCF finds applications in quantum physics, specifically in photon manipulation for quantum information processing and nonlinear optics for the generation of ultrafast pulses (J. ). THz radiation, in PCF, deals with the guiding and handling of electromagnetic waves in the frequency range of 0.1–10 THz. THz transmission is highly restricted along with low loss due to micro-structured air pores in PCFs (H. ; [null]). Due to the given property, the potential of THz spectral analysis, scans, and communication-related fields is huge with PCFs (H. ) (F.M. ). PCFs can be designed to produce effective and compact THz generators and sensors, due to which the potential of these new approaches in noninvasive material testing, healthcare diagnosis, and safety inspection has become very high (A.Y. ) (L. ) (J. ). A gas analyzer uses the special qualities of PCF to measure the amount of gas present by examining how illumination interacts with its molecules inside the fiber. Zhang Zhi-guo et al. proposed, "Gas sensing characteristics of the index-directed PCF using an air core" (Z. ). Simon alongside other fellows demonstrated, "Ideal PCF narrows for super continuum emitters boosted by blue" . To precisely monitor temperature swings, a PCF temperature gauge makes use of the fundamental properties of the PCF or heat-dependent fluctuations in its RI. Guolu as well as his mate innovated, "Refractive index and temperature measurements simultaneously using liquid-filled PCF and LPFG" (G. ). A pair parallel cores under each PCF framework make up a dual-core PCF detector, which enables asymmetric assessment for improved perception. Shanyong et al. proposed, "Transformation of Spatial Modes using Hybrid Dual-Core Photonic Crystal Fiber" (S. ). A PCF velocity tracker uses the relation amongst radiation and the turbulent media inside the PCF to estimate the motion of liquids or things that are moving. Khurram among his fellows suggested, "Two-dimensional bending Velocity Detector with Excellent Sensitivity Using an Elliptic Two-Core PCF" (K. ). Once placed under illumination, an object's surface, usually made of Au or Ag, and may vary its RI. This can be measured using surface plasmon resonance (SPR), a type of monitoring approach. SPR are produced whenever polarized radiation strikes the metallic layer at an exact point and causes its free ions to vibrate. Jian-Ying et al. performed, "Refractive index detecting properties of a photonic crystal fiber SPR sensor coated with carbon nanotubes" (J.Y. ). Two distinct radiation waves are used by a dual-wavelength enabled PCF device to improve sensitivity. It may assess several variables either linearly or in tandem by using numerous wavelengths, which increases the instrument's flexibility along with applicability. Inaki plus his fellows invented, "A Modular Dual-Wavelength Locked Fiber Laser Source with Switch ability for In-PCF Parametric Frequency Modulation in CARS Microscopy" (I. ). A hollow-core PCF (HC-PCF) monitor makes use of a special construction in which the centre of a fiber is encircled by a regular pattern of air gaps. The radiation can go across the HC in this layout. Nikoleta alongside fellows found, "Rapid Molecule Spectroscopy Employing a Hollow-Core Photonic Crystal Fiber Light" (N. ). A PCF biosensor detects biomolecular contacts by taking advantage of the special qualities of PCF. They enable exceptional responsiveness, label-free recognition, including immediate tracking by analyzing shifts in optical conveyance or resonant throughout the fibre induced by adsorption interactions. As such, they are essential resources in bioengineering along with healthcare. Iddrisu et al. preferred, "PCF-based SPR refractive index biosensor assaying: from new setups to exceptional detection limits" (I. ). The exact regulate of how photon travels is made possible by a hexagonal structure that contains air spaces encircling the center of a hexagonal PCF device. The accuracy and versatility of the scanner to determine different chemical alongside physical variables are improved by this construction. Shuvo et al. innovated, "An optical sensor based on hexagonal photonic crystal fibers (H-PCF) with minimal confinement loss and high relative sensitivity for the terahertz (THz) regime" (S. ). Mohammad as well as his partner suggested, "High-sensitivity gold-coated PCF biosensor with surface plasmon resonance technology" (M.R. ). The RI is a key factor in PCF since it controls and directs photons. The radiation may be contained alongside steered inside the interior of a fibre thanks to the sequential configuration of air spaces that either produces a bandgap of photons or alters the effective RI. Xili Jing alongside his mates performed," A PCF coated in silver that serves as the basis for a broadband SPR dual-channel sensor for temperature and refractive index measurements" (Z. ). Petrochemicals are crucially processed goods that utilize oil from petroleum, including gasoline, petrol as well as diesel. In several areas of the earth, gasoline is a necessity for both home and commercial use since it is often utilized as aviation fuel along with warmth and illumination purposes in lanterns alongside burners. Petroleum, often known as petrol, is an essential component of the automotive sector since it is largely utilized as oil for the engines with combustion in automobiles. In addition to powering a variety of vehicles, such as automobiles, buses, and trucks, diesel is a necessary component of commercial along with farming equipment. Diesel-powered vehicles run on this fuel. A. H. M. Iftekharul with his fellows demonstrated, "Using terahertz spectrum analysis to reveal new information about oil derivatives: the hybrid refractive index rectangle core photonic crystal fiber viewpoint sensing" . Khalid et al. offered, "Terahertz spectrum petrochemical sensing: a hybrid structure photonic crystal fiber refractive index method" (K.S. ). Md. Asaduzzamzn among fellows proposed, "Fuel quality assurance using terahertz region operation management and hybrid hexagonal circular hollow core PCF sensing" (M.A. ). R. Kanmani et al. suggested, "The terahertz spectrum effectiveness of providing core components for slotted core Q in PCF" (R. ). The distinctive characteristics of the fiber composition are used by a PCF chemical analyzer to identify and evaluate chemical elements. Such gadget provide excellent responsiveness, effectiveness, along with immediate tracking skills by taking advantage of variations in the movement of light or resonant inside the fiber brought about by connections to particular molecules. They enable quick and precise chemical ingredient analysis in a variety of domains, including process manufacturing oversight, clinical tests, along with surveillance of the environment. Kawsar with his partner established, "Creation of an optical sensor utilizing terahertz spectroscopy for chemical detection" . Diponkar Kundu et al. innovated, "Analyzing the Terahertz photonic crystal fiber sensor's performance in chemical detection" (D. ). Methanol is sometimes referred to as wood alcohol or methyl alcohol. It smells slightly pleasant and is a colorless, uncertain, burning solvent. It is extremely harmful to living things and may trigger serious illnesses, like as cataracts, organ harm, or even death, if consumed, inhaled, or absorbed through skin . Ethanol, sometimes referred to as alcohol or ethyl alcohol. It is a dynamic, ignited, transparent solution having a distinct flavour and smell that burns . Ethanol is harmless for humans to consume in moderation, compared to methanol. Because methanol and ethanol have rather distinct impacts on people's health and are used for very distinct purposes, it is important to distinguish between the two because methanol is extremely harmful that may trigger major health problems . In recent years, various techniques have been explored for methanol detection, including surface plasmon resonance (SPR) sensors, fiber optic sensors, and electrochemical methods. While these approaches offer some advantages, they often suffer from limitations such as lower sensitivity, reduced accuracy, or limited operational range. Photonic Crystal Fiber (PCF) sensors have gained significant attention due to their high sensitivity, low material loss, and ability to operate in the THz range, making them a promising solution for methanol detection in industrial and commercial applications where precision and safety are crucial. The structural properties of PCF sensors, such as their unique core design and air-hole arrangement, significantly enhance performance in the THz domain for alcohol detection (Z. ). These features improve light confinement and reduce loss, allowing efficient propagation of THz waves through the fiber. The high porosity of the PCF structure increases interaction between the guided light and the analytes (methanol and ethanol), boosting sensitivity to refractive index changes. This structural optimization is critical for achieving high detection accuracy and low material loss in the THz range. THz PCF sensors offer several advantages over traditional alcohol detection methods. They provide higher sensitivity and selectivity by leveraging the unique absorption characteristics of alcohols in the THz range, enabling precise identification of methanol and ethanol. The photonic crystal structure enhances light-matter interaction, improving detection accuracy. Additionally, PCF sensors operate non-invasively and require minimal sample preparation, making them faster and more efficient than conventional chemical or chromatographic methods. Their compact design also allows for real-time monitoring in various industrial and safety applications. To address the critical need for distinguishing between methanol and ethanol, we propose a Photonic Crystal Fiber (PCF) sensor designed to operate in the THz range. This sensor leverages the unique properties of PCF, such as enhanced light confinement and low-loss propagation, to achieve high sensitivity in detecting refractive index variations associated with different alcohols. With its tailored heptagonal core structure, the sensor is optimized for real-time alcohol detection, making it suitable for both industrial and commercial applications where precise chemical identification is essential. This design not only improves detection accuracy but also enhances safety monitoring, offering a robust solution for environments where methanol contamination or misidentification can pose significant health risks. Fig. 1 illustrates the block diagram of the setup used to test the sensor's performance in detecting alcohol. This figure provides a schematic of the PCF sensor experimental setup, essential for detecting methanol and ethanol in controlled conditions. The setup enables a systematic investigation into how the sensor performs under varying alcohol concentrations and frequencies. Fig. 1 Schematic of experimental setup for proposed PCF sensor. Fig. 1 The first process of developing the PCF sensor using COMSOL Multiphysics involves determining as well as characterizing the size along with components of the PCF layout. Recent advancements in photonic crystal technology and THz source development have significantly improved the efficiency of PCF sensors by enhancing light confinement and reducing losses. Innovations in THz generation and detection have also lowered costs, making the sensors more affordable for practical applications. These developments have broadened the scope of PCF sensors for various industries, including alcohol detection, with better performance and accessibility. This model tries to simulate the energy transmission procedure through chemical action comprising alcohol (Methanol, Ethanol) and the exterior operation of the PCF using COMSOL Multiphysics for alcohol identification through PCF. We construct this new device and examine it with COMSOL Multiphysics. This PCF has an unusual heptagonal core, a unique heptagonal clad surface, with 14 airways (R 1 & R 2 ) that originate within the cladding. The heptagonal core is designed considering polygonal function of COMSOL Multiphysics alongside desired angle of 51°. Whereas 14 airways are created in two cycles. R 2 be the radius of outer circle that we have use to create the outer layer airholes by using difference function. Similarly, the inner airways cycle consists of 7 similar airways that are created in similar manner but instead of circle we consider heptagon with R 1 radius for this portion and also use difference function here to achieve this shape. Zeonex is the base material used for the suggested system. Suggested THz PCF sensors can distinguish between different types of alcohols by detecting their unique refractive index and absorption properties in the THz range. This capability enhances practical applications, such as preventing methanol contamination in industrial settings and ensuring public safety by accurately identifying hazardous alcohols in real time. Zeonex is a material with a pre-calculated Refractive Index (RI) of 1.53. But its core (C) uses RI to detect Alcohol, where RI for methanol and ethanol are 1.329,1.361 respectively. A Perfectly Matched Layer, or PML for short, is a computing borders that simulates proper instrument behaviour by absorbing outbound pulses as well as preventing rebounds. Here, Fig. 2 (a) Displays the cross-sectional structure, showing the heptagonal core and air holes that facilitate alcohol detection. The cross-sectional view of the heptagonal core structure reveals how air holes are arranged around the core, which enhances light confinement. This layout is crucial for achieving high sensitivity in detecting refractive index differences between methanol and ethanol. Fig. 2 (b) Depicts the stages in designing the heptagonal core structure of the PCF sensor. This figure illustrates the construction process of the heptagonal core, showing the layered assembly of air holes that optimize sensitivity. Each step in the construction enhances the sensor's ability to differentiate between analytes based on their refractive indices. Fig. 2 a): Cross-sectional view of the proposed heptagonal core PCF sensor. Fig. 2 (b): Step-by-step construction of proposed heptagonal core. Fig. 2 (c): Mesh representation of the proposed PCF sensor. Fig. 2 (d): Power density distribution for (I) methanol (II) ethanol Fig. 2 (e): Power confinement analysis in core and cladding for (I) methanol (II) ethanol Fig. 2 (f): Workflow for material selection, sample preparation, and analysis. Fig. 2 The discretized description of detector topology used in calculations is known as a mesh representation. Such a mesh consists of minor elements like triangular and quadrilaterals, which when combined roughly, may represent the intricate framework inside the PCF. The use of triangular and quadrilateral elements in meshing is vital for accurate simulation of our heptagonal core PCF sensor. Triangular elements are applied to irregular regions, while quadrilateral elements cover more structured areas, ensuring an optimal balance between precision and computational efficiency. This hybrid meshing improves convergence and reduces numerical errors, enhancing the accuracy of key parameters like Relative Sensitivity (RS) and Effective Material Loss (EML). As a result, the mesh ensures reliable analysis and performance of the sensor for methanol and ethanol detection. The principles regarding the transmission of light can be solved by techniques like the FEM, working by breaking the detector down to such functional components. There are 74 apex factors, 1309 border factors, and 11228 items in total. The minimal grade for a substance is 0.5861. Upon which Fig. 2 (c) Shows the meshing layout used in the simulation, illustrating triangular and quadrilateral elements for accuracy. The meshing diagram used in simulations shows how triangular and quadrilateral elements cover the PCF structure. This meshing improves computational accuracy, particularly in simulating the interaction between light and analytes within the core. A PCF sensor's power distribution explains how the power of light is dispersed evenly throughout a fibre's topology. Because of its cladding with sequential patterns of air pores, radiation within a PCF is firmly constrained inside its core and improves guidance via adjusted TIR, or optoelectronic bands. Extreme reactivity to fluctuations in the outside world is created via an evanescent area that extends through the panelling and primarily operates in a basic pattern. The spacing is often analyzed through computer modeling and testing, so as to optimize layout as well as functioning. It is of prime importance to the effectiveness of the sensor. Upon which Fig. 2 (d) depicts Visual representation of the power density distribution across the sensor when detecting methanol (I) and ethanol (II). showing how the design effectively confines light, which is critical for enhancing sensitivity to each analyte. The PCF sensor density distribution is explained by the changes of RI in the core and the surrounding. Energy is mostly confined by the higher RI of the core rather than the cladding. This configuration affects the guiding of illumination, modal area confinement, and the susceptibility to external perturbations of importance to the optimization of detector performance for a wide range of applications. It is often periodic due to the designs of air vents in the flow. Upon which Fig. 2 (e) Shows power confinement in the core and surrounding cladding for methanol (I) and ethanol (II), demonstrating the sensor's sensitivity. emphasizing the sensor's capability to guide light with minimal leakage, a feature essential for precise detection. The simulation results indicated differences in Effective Area (EA) and spot sizes (SP), with EA values of 3.92 × 10⁻⁸ m 2 for ethanol and 3.54 × 10⁻⁸ m 2 for methanol, and SP values of 2.28 × 10⁻⁴ μm for ethanol and 2.12 × 10⁻⁴ μm for methanol. While these numerical differences suggest distinct optical properties for each analyte, a formal significance analysis was not performed since the data is derived from deterministic simulations rather than experimental iterations. In future work, we plan to incorporate sensitivity and uncertainty analyses to evaluate the significance of these differences. This will help us better understand how small parameter variations could influence the sensor's performance and ensure reliable detection of methanol and ethanol in real-world applications. The design process for a heptagonal core Photonic Crystal Fiber (PCF) sensor begins with defining the problem, specifically the need for detecting methanol and ethanol based on their refractive indices to ensure safety and monitoring in various industries. The sensor is then designed with a focus on the heptagonal core structure, shape, and arrangement of air holes. Appropriate materials for the core and cladding, such as Zeonex or silica, are selected based on their refractive indices to ensure compatibility with THz sensing. The next step involves setting up the sensor simulation in COMSOL Multiphysics, configuring parameters like pitch, air hole diameter, and the operational THz range. The refractive indices of methanol and ethanol are inputted into the simulation to analyze their effects on light propagation through the sensor. The sensor's performance is evaluated based on key metrics, including Relative Sensitivity, Effective Material Loss, Confinement Loss, and Numerical Aperture. Finally, the sensor design is optimized by tweaking the geometrical parameters to achieve maximum sensitivity and low loss for both methanol and ethanol detection. Fig. 2 (f) Outlines the procedural flow from material selection to sample analysis for the PCF sensor. Outlines the comprehensive process from material selection to sample analysis, ensuring that each phase is optimized for high-performance detection in commercial and industrial settings. Finite Element Method (FEM) is a computational method applied in science and technology for the solution of awkward problems. Simulation of optical characteristics and light transmission in PCFs utilizes FEM. Special visual features of PCF include adjustable divergence and strong ambiguity due to the sequential placement of ventilation pores throughout their length. FEM enables the segmentation of longitudinal shapes into PCF pieces, where the characteristics can be analyzed with accuracy; this way, it becomes easy to research electrical field dispersion, together with the general effectiveness of the fiber. Relative Sensitivity (RS) is a property that gauges the capacity of PCFs to limit alongside direct light in response to variations in the RI of their surrounding environment. On PCFs, there is a repeating pattern of air vents that influences the characteristics of guiding. Photon containment and the fiber's acoustic characteristics are measured by RS, which also measures changes in these holes or the RI difference. High RS in PCFs is essential for purposes such as recognition and telecommunications since it assures proper light direction alongside the responsiveness of environmental changes, giving the fiber fitter the ability to identify and quantify the impacts in the surroundings. Firstly, we measured the RS about both P and FR at the same time. The vertical axis to be considered for RS and horizontal axis considered for FR or P. As we made variation in FR or in P then corresponding change will occur in the RS. Here pink curve for ethanol and brown curve for methanol represent the RS. Fig. 3 (a) Shows the relationship between frequency and relative sensitivity, demonstrating that the sensor achieves peak sensitivity at 3.2 THz, which is key for reliable detection of methanol and ethanol. Where the max RS for methanol and ethanol is 95.72 % and 97.55 % at f = 3.2 THz. By contrast, Fig. 3 (b) Examines how pitch affects sensitivity, revealing that certain pitch values enhance the sensor's ability to detect subtle differences in refractive indices, optimizing performance for each analyte. Fig. 3 Demonstrate the influence of the Relative Sensitivity (a) Frequency [THz] (b) Pitch [μm]. Fig. 3 In PCF, Effective Material Loss (EML) describes light retardation resulting from the fiber element's natural acceptance, silica, for example. The EML in PCFs depends on light restriction in the core and its interaction with the surrounding micro-structured cladding. Light interaction intensity with the fiber material depends on several things, which include the wavelength used and the air-hole layout concept. EML is one of the most important factors when it comes to determining the total loss for PCFs. It also affects how suitable and efficient they are for various applications, from sensors to telecommunications. Implement those computational equations to determine EML . (3) α e f f = ( ε 0 μ 0 ) 1 2 ∫ A m a x n α m a t | E | 2 d A 2 ∫ A L L S z d A Whereas the electric field is represented by E and the zeonex loss coefficient is α m a t . We are now exploring the EML of our proposed PCF. The relationship between EML, pitch, and operational frequency is highlighted in Fig. 4 . The vertical axis to be considered for EML and horizontal axis considered for FR or P. As we made variation in FR or in P then corresponding change will occur in the EML. Here pink curve for ethanol and brown curve for methanol represent EML. Where at f = 3.2 THz the EML finds 0.0066 cm⁻ 1 and 0.0044 cm⁻ 1 for methanol and ethanol respectively. To be more specific , Fig. 4 (a) Analyzes Effective Material Loss (EML) across frequencies, demonstrating the sensor's efficiency in retaining light within the core for both methanol and ethanol, which directly influences detection accuracy, while Fig. 4 (b) Illustrates the influence of pitch on EML, showing that an optimal pitch configuration minimizes loss, thereby enhancing light confinement and the sensor's overall sensitivity. Fig. 4 Demonstrate the influence of the Effective Material Loss (a) Frequency [THz] (b) Pitch [μm]. Fig. 4 An indicator of a PCF's capability of keeping photons within its core is Numerical Aperture (NA). It measures the spectrum of orientations at which the fiber can support photons and is dependent on the RI difference between the cladding and core. The microstructure configuration of air holes surrounding the core of PCFs determines the NA, and these holes can be tailored for delivering certain light-guiding characteristics. An increased NA, for systems that require precise modal containment and efficient transmission of light, indicates a stronger ability to capture and steer photons. As seen in Fig. 5 , the NA of the suggested PCF is examined by varying the pitch and frequency. The vertical axis to be considered for NA and horizontal axis considered for FR or P. As we made variation in FR or in P then corresponding change will occur in the NA. Here pink curve for ethanol and brown curve for methanol represent NA. Where at f = 3.2 THz the NA finds 0.270 and 0.257 for methanol and ethanol respectively. Fig. 5 (a) Shows How Numerical Aperture (NA) varies with frequency, indicating that the sensor captures light most effectively at specific THz frequencies, which is important for maximizing detection reliability. In the meantime, Fig. 5 (b) Examines the effect of pitch on NA, highlighting that a suitable pitch value enhances the sensor's photon capture, improving its responsiveness to changes in refractive index. Fig. 5 Demonstrate the influence of the Numerical Aperture (a) Frequency [THz] (b) Pitch [μm]. Fig. 5 The Effective Area (EA) of PCFs is a measure of the cross-sectional region over which light is efficiently directed and contained in the fiber core. The EA is a parameter that impacts some of the important characteristics, such as dispersion and nonlinearity. The higher light intensity inside the core due to a small EA would thus enhance nonlinear effects and hence be useful for applications like supercontinuum generation. At the same time, a larger EA is preferred when high-power transmission is required since it reduces nonlinearity. The EA is defined by the core size and the configuration of air holes in the cladding, which can be modified to tailor the optical characteristics of the fiber for specific applications. The present experiment investigates the link between EA and changes in the PCF's operating frequency and pitch. The vertical axis to be considered for EA and horizontal axis considered for FR or P. As we made variation in FR or in P then corresponding change will occur in the EA. Here pink curve for ethanol and brown curve for methanol represent EA. Where at f = 3.2 THz the EA finds 3.54 × 10⁻⁸ m 2 and 3.92 × 10⁻⁸ m 2 for methanol and ethanol respectively. The dependence of EA on frequency is shown in Fig. 6 (a), Investigates the effective area as a function of frequency, illustrating that controlling the core area is essential for achieving optimal light confinement and detection sensitivity. In addition, Fig. 6 (b) Shows the effect of pitch variation on the effective area, revealing that pitch adjustments help optimize the core's ability to contain light and increase the sensor's sensitivity to analytes. Fig. 6 Demonstrate the influence of the Effective Area (a) Frequency [THz] (b) Pitch [μm]. Fig. 6 In PCF, the Spot Size (SP) is the effective diameter of the optical mode enclosed in the fiber core. It is an important factor that influences the efficiency of coupling, bending losses, and modal dispersion of the light transmission. The core size and the configuration of the air pores in the cover define the spot size. In asymmetric photonic programs, smaller spot sizes lead to higher intensity and better phase confinement. However, in applications that need significant power, larger spot sizes minimize discontinuity, thereby improving the excellence of the beam and, consequently, decreasing the intensity. Using the PCF layout can modify the spot size to meet certain requirements regarding performance. Illustrated As in Fig. 7 , this section explores the relationship between pitch, operating frequencies, FR, and spot size. The vertical axis to be considered for SP and horizontal axis considered for FR or P. As we made variation in FR or in P then corresponding change will occur in the SP. Here pink curve for ethanol and brown curve for methanol represent SP. Where at f = 3.2 THz the SP finds 2.12 × 10⁻⁴ μm and 2.28 × 10⁻⁴ μm for methanol and ethanol respectively. Fig. 7 (a) Illustrates the variation of spot size with frequency, demonstrating that smaller spot sizes improve light confinement within the core, critical for achieving high detection accuracy. Conversely, Fig. 7 (b) Examines the effect of pitch on spot size, showing that fine-tuning the pitch results in an ideal spot size that maintains the balance between confinement and light intensity. Fig. 7 Demonstrate the influence of the Spot size (a) Frequency [THz] (b) Pitch [μm]. Fig. 7 This section explore relation between light dispersion with the operating frequency of this suggested PCF sensor. The vertical axis to be considered for DP and horizontal axis considered for FR or P. As we made variation in FR or in P then corresponding change will occur in the DP. Here pink curve for ethanol and brown curve for methanol represents DP. Where at f = 3.2 THz the DP finds 0.2 ps/THz/cm and 0.14 ps/THz/cm for methanol and ethanol respectively. Fig. 8 Provides an analysis of dispersion in the THz range, showing how frequency impacts light speed and, consequently, the sensor's ability to distinguish between different analytes based on dispersion properties. Fig. 8 Demonstrate the influence of the Dispersion of light due to Frequency [THz]. Fig. 8 This section's Table 1 compares the envisioned sensor's functionality to that of several already in consideration. We have mentioned the sensing analytes, operating frequency, RS, EML, NA and EA of those sensors with ours suggested sensor. From the table you can see that the sensing ability or RS of our intended detector is much better than that of others. It also having minimal EML that the mentioned sensors. Table 1 Performance study among currently available detector with our invented detector. Table 1 Ref. Analytes FR (THz) RS (%) EML (cm −1 ) NA EA (m 2 ) (R. ) Benzene 1.0 74.61 0.0138 – – Ethanol 1.0 74.53 0.0148 – – Water 1.0 74.17 0.0172 – – Water 1.0 73.20 – – 1.43 × 10 −7 Ethanol 1.0 76.44 – – 1.36 × 10 −7 Benzene 1.0 77.16 – – 1.35 × 10 −7 (S. ) Benzene 1.0 81.46 – – – (M.S. ) Ethanol 1.6 85.7 – 0.372 69800 (μm 2 ) (M.A. ) Benzene 1.7 89 0.028 0.42 – (D. ) Ethanol 2.0 95.21 0.0078 0.314 6.52 × 10 −8 Benzene 2.0 94.67 0.0083 0.307 6.84 × 10 −8 (M.E. ) Water 1.0 96.25 – – 6.84 × 10 −6 (μm 2 ) Present Research Methanol 3.2 95.72 0.0066 0.270 3.54 × 10 −8 Ethanol 3.2 97.55 0.0044 0.257 3.92 × 10 −8 Our planned chemical sensing to existing PCF monitors is the topic of a structured discussion that we are having. Table 1 provides additional background on our PCF research by detailing the main components of the current PCF type. The proposed methanol detection method using a heptagonal core PCF sensor demonstrates impressive performance when compared to existing works based on key factors such as Relative Sensitivity (RS), Effective Material Loss (EML), Numerical Aperture (NA), and Effective Area (EA). Achieving a maximum RS of 97.55 % for ethanol and 95.72 % for methanol at 3.2 THz, the sensor significantly outperforms other methods in the literature, such as those in (S. ) (M.S. ), where RS values range between 74.17 % and 85.7 % at lower frequencies (1.0–1.6 THz). These high RS values reflect the sensor's superior ability to detect slight variations in refractive index, which is critical for accurate chemical identification. Moreover, the low EML of 0.0066 cm⁻ 1 and high NA of 0.270 demonstrate superior signal transmission and improved light confinement compared to other techniques that report higher EML or lower NA values, such as in (M.A. ) (D. ). These advancements not only enhance the sensitivity and optical performance of the proposed sensor but also make it highly suitable for hazardous industrial environments, ensuring accurate methanol detection and reducing the risk of poisoning. Achieving such high sensitivity at 3.2 THz further underscores the sensor's advanced performance in the terahertz range, making it a highly effective solution for industrial monitoring and safety. The previous type is generally composed of a mixture of silicon dioxide and related materials and was particularly developed for constructing PCF measurement devices. Generally, fibers are shaped by the stack-and-draw process, which involves heating the mold before dragging it over a fiber. For specific purposes, perforations can be carved or engraved to assist with scanner engagement. A general function to enhance selectivity and sensitivity is to add functional coatings. Moreover, for individual sensors, detection mechanisms are essential such as microscopic particles present in cell walls (A.I. ) (C.G. ). Our study is based primarily on simulations conducted using COMSOL Multiphysics, which involve complex numerical calculations that are highly precise. Unlike experimental data, these simulations are deterministic and do not contain the random measurement errors commonly encountered in experimental work. As a result, the use of error bars, typically used to reflect experimental uncertainties, is not as relevant in the context of our simulation-based research. However, we have incorporated numerical equations that allow for the estimation of potential errors for various parameters such as relative sensitivity, confinement loss, effective material loss, numerical aperture, spot size, and effective area. These equations provide a theoretical basis for approximating uncertainties in these parameters. The probable error can be calculated using statistical techniques, particularly during sensor calibration or testing. The process for calculating probable error involves the following steps. (1) Data Collection: Gather multiple readings from the sensor. (2) Mean Calculation: Compute the average (mean) value by summing all the readings and dividing by the total number of readings (K.S. ): (9) x ‾ = ( 1 / n ) ∑ i = 1 n X i (3) Standard Deviation (SD) Calculation: Measure the variation of the readings from the mean using the standard deviation formula (K.S. ): (10) σ = ( 1 / n ) ∑ i = 1 n ( X i − x ‾ ) exp ( 2 ) (4) Probable Error (PE) Calculation: Finally, determine the probable error by multiplying the standard deviation by a constant (K.S. ): (11) P E = 0.6745 × σ To apply the heptagonal core PCF sensor for detecting alcohol in real commercial food samples, the process begins with collecting a variety of samples, such as alcoholic beverages or food with ethanol-based preservatives. After proper sample preparation, the sensor is calibrated using known concentrations of methanol and ethanol. The prepared samples are then analyzed with the sensor to detect and differentiate the alcohols based on their refractive index variations. The data gathered from the sensor is compared with conventional methods like gas chromatography to validate its accuracy and reliability in real-world applications shown in Fig. 9 . Finally, the results are documented to highlight the sensor's effectiveness and potential for improving alcohol detection in commercial food products. Fig. 9 Illustrates the workflow for applying the PCF sensor in detecting alcohol in food products, from sample preparation to data analysis. Fig. 9 Detection steps for analyzing alcohol in commercial food samples. Fig. 9 The limitations of THz PCF sensors in alcohol detection include challenges in precise fabrication, which can impact sensitivity and performance. Additionally, maintaining consistent sensor calibration in real-world conditions, such as varying temperatures and environmental factors, can be difficult. High costs of THz components and limited availability of commercial THz sources further restrict widespread adoption in practical applications. While this study demonstrates the sensor's high sensitivity and specificity in detecting methanol and ethanol under controlled conditions, future work could involve validating its performance with real commercial food samples, such as alcoholic beverages or foods preserved with ethanol-based ingredients. To ensure accurate readings in complex matrices, additional calibration may be necessary to account for potential interferences from food constituents, including sugars, proteins, and other additives. Experimental testing on real samples would provide valuable data on the sensor's robustness in practical applications, as well as insights into any sensitivity adjustments required. Key challenges include maintaining sensor calibration in variable environmental conditions and addressing the high costs and limited availability of commercial THz sources, which may affect widespread adoption. Incorporating such real-world data in future studies would further solidify the sensor's utility for alcohol detection in food safety applications. The primary objective of this research is to develop a novel heptagonal core photonic crystal fiber (PCF) sensor capable of detecting both methanol and ethanol simultaneously with high sensitivity. Methanol is extremely harmful to living organisms, potentially causing serious illnesses such as cataracts, organ damage, or even death, while ethanol is generally safe for human consumption in moderation. Misidentifying methanol as ethanol can lead to severe toxicity and fatal consequences, making accurate detection vital for public safety. The proposed sensor, designed using COMSOL Multiphysics, Key metrics include a high relative sensitivity (RS) of 95.72 % for methanol and 97.55 % for ethanol, along with low effective material loss (EML) values of 0.0066 cm⁻ 1 and 0.0044 cm⁻ 1 , respectively. Furthermore, the sensor features optimized Effective Areas (EA) of 3.54 × 10⁻⁸ m 2 for methanol and 3.92 × 10⁻⁸ m 2 for ethanol, and small spot sizes (SP) of 2.12 × 10⁻⁴ μm and 2.28 × 10⁻⁴ μm, respectively. This high level of precision and performance in the THz range ensures reliable detection, which is critical for improving safety in industrial and commercial environments. The ability to detect both methanol and ethanol simultaneously at such high sensitivity distinguishes this sensor from existing technologies, contributing to enhanced public health protection and industrial monitoring. The proposed heptagonal core PCF sensor offers significant real-world applications, particularly in industrial monitoring and public safety. Its high sensitivity and precision in detecting methanol and ethanol make it crucial for preventing methanol poisoning in industries that handle alcohol-based substances. Additionally, it enhances safety by ensuring accurate chemical identification in environments where the risk of contamination or accidental exposure is high. For future research, efforts should focus on optimizing the fabrication process to make the sensor commercially viable. Investigating materials with higher thermal and mechanical stability could enhance durability, while scaling up production methods would enable widespread industrial use. Additionally, exploring integration with real-time monitoring systems could further expand its practical applications. | Other | biomedical | en | 0.999996 |
PMC11699085 | In addition to the issues which arise during life transitions, medical training brings a unique set of challenges, which include high academic expectations, rigid course structures and exposure to a complex, sometimes distressing, clinical environment . As such, medical students may experience a negative impact on well-being, interpersonal relationships, quality of life and academic performance during their degree [ 4 , 6 – 8 ], especially when transitioning from pre-clinical into clinical years . One possible method to provide additional support for medical students is through mentoring programs [ 11 – 14 ]. In such programs, relationships are typically established between more experienced individuals (‘mentors’), and those more junior (‘mentees’), with the aim of promoting personal and professional development, as well as psychosocial support . Within medical schools, mentors for medical students can be faculty, senior or junior clinicians, or other students. Mentor selection may include factors such as academic performance, specialty, or sociodemographic backgrounds (e.g. cultural or religious ). Whilst faculty and clinician mentors bring more experience, peer mentors are typically considered more available and approachable . Peer mentors may be peers at the same level in their training, or near-peers, who have progressed academically beyond the mentee. In this review, we use the term peer mentoring to include both peers and near-peers for conciseness. Peer mentoring programs, especially where both mentors and mentees are medical students, are thought to benefit from the high level of ‘cognitive and social congruence’ that exists between students in similar educational environments . The shared understanding of academic frameworks, knowledge and interpersonal roles allows for the development of complex and comfortable mentor–mentee relationships . Furthermore, elements exclusive to student mentoring, such as the use of familiar language, similar social roles and an empathic understanding of the ‘student experience’, may provide benefits that faculty mentoring does not. A diverse student cohort, moreover, means that social congruence can extend to students from culturally and linguistically diverse backgrounds . Accordingly, one review found that the ‘comfortable’ learning environment established by peers has been highlighted as an important advantage over formalised teaching . Similar principles apply for ‘peer-assisted learning’ (or peer teaching). However, the difference is peer-assisted learning primarily targets academic goals, whilst peer mentoring prioritises wellbeing and support . Whilst mentoring in the medical profession is relatively common, there is also evidence to support mentoring of medical students. A 2020 qualitative study evaluating different university initiatives to reduce stress found that peer mentoring ‘effectively reduce(d) stress in medical students, and facilitate(d) their transition into medical school’ . A 2018 systematic review included five studies on near-peer mentoring programs, focusing on outcomes for first-year health professional (medical, allied health and nursing) students and reported personal development and psychosocial benefits for these mentees. In 2019, a large narrative review with 82 included studies on medical student mentoring also reported similar findings. This study showed mentees benefited from attainment of skills, knowledge and personal/professional development, whilst mentors improved in leadership and communication skills. However, the mentors in these studies were predominantly junior doctors, not medical students. Another systematic review focusing on the impact of mentoring on medical students from under-represented minority groups identified benefits for academic, research and career pathways. Finally, a systematic review on peer mentoring, specifically in Iranian medical schools, found benefits for both mentees and mentors. Though the evidence for peer mentoring programs for medical students is promising, and several systematic reviews have been conducted to evaluate the utility of mentoring for medical students, none to date have specifically reviewed the benefits of near-peer mentoring by medical students, for medical students. This scoping review aims to determine the benefits and challenges of medical student peer mentoring programs, and identify elements for program success, accounting for differences in curriculum, program structure and mentor/mentee composition. Findings may inform future research and discourse and assist medical schools in the organisation and optimisation of their peer mentoring programs to benefit their students. An initial limited search of Ovid MEDLINE, EMBASE and ERIC was undertaken using a search strategy developed with the assistance of a librarian. The text words contained in the titles and abstracts of relevant articles and the index terms used to describe the articles were used to develop a full search strategy with further librarian assistance. As ‘mentoring’ is not a MeSH term, and various phrasing is used to describe mentoring programs, the two-step development of the final search strategy was required. The following databases were searched: Medline, ERIC, EMBASE, Web of Science, WorldWideScience and the British Library on 22/1/2024. Keywords and MeSH terms used included medical student*, peer mentor* and medical education. Boolean operators (AND, OR, NOT) were used to refine search results. The search strategy was then adapted for other databases (see Appendix). The reference list of included studies and relevant reviews were screened for additional studies. Grey literature was searched using OpenGrey ( http://www.opengrey.eu ) and GreyNet ( https://www.greynet.org/ ). Inclusion criteria were as follows: Primary studies of any design published in English. Participants: Medical students undertaking a primary medical degree (undergraduate or postgraduate)—applicable to both mentor and mentee participants. Intervention: Peer or near-peer mentoring programs. Comparison: No program or different types of mentoring programs. Outcomes: Any that related to mentors or mentees including satisfaction with program, academic skills, professional skills and health and wellbeing, elements for peer mentoring program success and challenges of these programs. Two reviewers (AG/MK/CP and MR) independently screened titles and abstracts obtained from the search, excluding irrelevant citations, using Covidence. Full-text articles were subsequently retrieved and reviewed for final inclusion. Disagreements were discussed amongst three reviewers (AG/MK/CP, MR and LN) and resolved through consensus. The selection process was reported via Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow diagram . Fig. 1 PRISMA flowchart outlining search results, screening and study inclusion process The research question was ‘What are the benefits and challenges of medical student peer mentoring programs and what are the elements associated with their success?’ This question was derived from the authors’ experience as medical educators and medical students, namely, the challenge to develop and optimise a peer mentoring program for medical students which would best support their psychosocial, professional and academic needs. Therefore, the search strategy was developed in light of the authors’ joint interests. The two authors who primarily undertook the data extraction (AB, CP) were medical students at the time and participants of a peer mentoring program in their clinical school, and the extraction was verified by two other authors (MR, LN), one of whom (LN) had developed the clinical school mentoring program. The inclusion of a non-medical practitioner who had not been directly involved in the peer mentoring program (KF) ensured a level of rigour in data analysis and interpretation. A total of 1676 citations were identified through the initial search . Of these, 48 full-text studies were assessed for eligibility and 20 (two of the same program) met the inclusion criteria. Of the included studies, seven were cohort studies [ 13 , 14 , 17 – 19 , 27 , 28 ], seven mixed-methods [ 26 , 29 – 34 ], three qualitative and three cross-sectional [ 37 – 39 ] (Table 1 ). There was a total of 4591 participants with a range of 9–2362 participants in each study. When including only studies which specified year level of mentees/mentors, most mentees were in their first clinical year ( n = 898), whilst most mentors were near peers in either their ultimate or their penultimate year ( n = 224). Three programs had mentors and mentees from the same year level . In general, mentors and mentees were randomly matched, typically following a selection process for mentors. Six of the included studies considered specific characteristics (such as international students, being taught by the same faculty, personal relationships) when matching mentors and mentees. Mentoring groups were generally small, with one notable exception where a social media platform was used for large group mentoring . Twelve studies reported a component of peer teaching as part of the mentoring program [ 13 , 14 , 16 , 26 , 28 – 31 , 36 ]. Table 1 Summary of included studies Study (Country) Design Participants Intervention Assessment time points Measure Outcome Longitudinal programs Abdolalizadeh et al. 2017 (Iran) Qualitative Mentors—fourth year medical students (clinical, n = 6; non-clinical, n = 6) Mentees—first year medical students randomly selected from cohort (n = 36) Recruitment of mentors : volunteers Allocation of mentees to mentors : N/A; 2 mentors (one clinical, one preclinical) and 6 mentees per group Contact logistics : Contact made via telephone calls, email and face-to-face in varying frequency Training and orientation provided : Yes, were trained on purpose of mentoring and communication skill development Activities organised : Weekly inter-mentor sessions for idea sharing Duration of program : Academic year Conclusion of academic year Four focus groups administered to mentors and mentees Questions were regarding experiences in the program, perceptions of development, strengths and weaknesses of the program, and the quality of each component Length of each focus group was ~ 60 min. All meetings were recorded and transcribed Data analysis : Conventional content analysis • Mentors and mentees agreed on several characteristics that made a good mentor: patience, active contact and persistence • Mentees commented that their mentor played a role in their academic progress, mainly via discussions regarding study methods/techniques • Mentees benefitted from having both clinical and non-clinical mentors • The perceived independence of the program from the medical school was seen as a bonus for mentees and mentors • Diversity in communication methods was seen as a strength of the program, however most participants preferred face-to-face meetings • Mentors reported both professional and social development Altonji et al. 2019 (USA) Cohort study Mentors—2nd year medical students ( n = 112) Mentees—1st year medical students ( n = 190) Recruitment of mentors : Volunteers Allocation of mentees to mentors : Pairing done by student leaders (learning representatives in their second year, elected by their peers), no guidelines on pairing but was guided by personal relationships the leaders had to both mentors and mentees Contact logistics : Casual meetings at least once a month Training and orientation provided : Yes, large group training received once Activities organised : None Duration of program : Academic year Conclusion of academic year One survey and one focus group administered to mentees Mentorship Effectiveness Scale (MES)—twelve Liker-scale items to assess effective mentor behaviours, including perceptions of the benefits of the program, overall satisfaction and accessibility of contact of the mentor to the mentee A focus group was used to modify the survey to suit the needs of the study, and was non-evaluative ( n = 9, first years) Data analysis : Stepwise linear regression • Items that scored higher than 3.5 on the MES included the following: mentor was accessible (4.2 ± 0.99), approachable (4.41 ± 1.16), supportive and encouraging (4.36 ± 1.08), answered questions satisfactorily (4.13 ± 1.19), and suggested appropriate resources (4.09 ± 1.21), demonstrated expertise in an area of need (3.85 ± 1.16), provided useful and constructive critique (3.68 ± 1.43), provided guidance on how to succeed in medical school (3.83 ± 1.41), and provided useful advice on how to study (3.84 ± 1.26) • Overall satisfaction with the mentoring program was high (7.47 ± 2.45, out of 10) • Predictors to perceived satisfaction and benefit included the following: mentor knowledge, mentor advice on handling medical school, mentor providing guidance on professional matters, there was no statistically significant benefit to either male or female mentors; however, regular meetings and involvement were seen as a positive predictor • Results of focus group were used to modify the survey only and hence not separately reported Andre et al. 2017 (USA) Mixed methods Mentors—fourth year medical students (‘MiMs’) ( n = ~ 60) Mentees—first, second and third year medical students participating in the ‘Veritas’ mentoring program ( n = ~ 220/year level) Recruitment of mentors : Volunteer Allocation of mentees to mentors : All students randomly assigned to one of 20 Veritas groups upon enrolment; each group consisted of 11 students from MS1-MS4. 3 volunteer mentors allocated to each ‘Veritas’ group randomly Contact logistics : Group and one-on-one meetings, minimum once a month Training and orientation provided : None Activities organised : Weekly mentor meetings with Veritas director Duration of program : Academic year Conclusion of academic year with each new cohort Two self-developed surveys (piloted and refined prior to use) measuring effectiveness of peer-mentoring and perception of improved experience of medical school, administered to mentors and mentees Survey 1 (Veritas All Students Survey) asked mentees Likert scale questions regarding MiMs engagement with getting to know their mentees, assisting with rotation planning, stress management, guidance and advice, emotional support, the mentees preference for approaching MiMs over faculty mentors, and if the mentees saw MiMs a useful resource Survey 2 (Mentors in Medicine, or MiMs survey) asked the mentors questions about their perceived preparation in running meetings, providing guidance, personal and professional development, and their perceived success in their mentoring role Data analysis : Mantel–Haenszel Chi square • The findings showed that in general, peer mentors were able to provide advice about academic planning, psychosocial support, promoted interpersonal relationships, peer support and a safe space for mentees to discuss personal issues • Level of satisfaction varied year to year • In 2012 and 2013, 100% of mentors said they would volunteer again the following year • 71% of mentors felt sufficiently prepared in their role • Among first- and second-year mentees, there was an overall perceived benefit in performance year to year from 2011 to 2014 • Over the years there is a visible trend in improvement in satisfaction with the medical school experience via post hoc testing • Other up trending components of the survey included: discussion of professionalism questions, academic planning, future year planning, networking, peer support, relationships between classes, social supports and discussions of emotional issues that relate to the patient experience Behkam et al. 2022 (Iran) Cross-sectional Mentors: third/fourth year medical students Mentees: first year medical students Non-mentees: first year medical students Recruitment of mentors : Volunteers Allocation of mentees to mentors : Assigned based on gender, ethnicity, living place, scientific background. One-to-one allocation Contact logistics : Face to face meetings on a weekly basis Training and orientation provided : 3 day workshop on concept of mentoring, communication, program rules/expectations Activities organised : Weekly sessions centred around assisting new students with transition to medicine and aimed at improving their perception of educational environment Duration of program : Academic year During first year DREEM (Dundee Ready Education Environment Measure) tool (Persian translation) used to assess students’ overall perception of their learning environment Student responses were compared across the mentored and non-mentored groups and tested for significant differences Data analysis: T -test, ANOVA to compare means of quantitative variables. P < 0.01 used for statistical significance • 169 out of 190 study recruits (87 mentees, and 82 non-mentees) participated in data collection • Total DREEM score not different based on demographics • Mean DREEM scores of students with mentors not statistically different from those without • No DREEM item was significantly different across the two groups • More mentees stated that ‘there is a good support system for students that get stressed’ than non-mentees, P = 0.009 Chatterton et al. 2018 (UK) Cohort study Mentors—selected second and third year medical students ( n = 49) Mentees—first year medical students Recruitment of mentors: Initial year organised by Senior Tutors in collaboration with two student volunteers. Subsequent years was carried out by group of experienced peer mentors from the previous year Allocation of mentees to mentors: Certain groups matched accordingly (e.g. international student with international student peer mentor) Contact logistics: Peer mentors encouraged mentees to make contact when required, and contact made by peer mentors at various ‘high-stress’ points Training and orientation provided: 3 training sessions in total amounting to 6 h formal training over an 8-month period Activities organised: Informal welcome event Duration of program: Academic year Post second and third training session (mentors) Five-point Likert scale questionnaire evaluating peer mentor’s perception of whether the training sessions met their expectations, application of knowledge learned, confidence in their roles, perceived support, knowledge about where to sign-post mentees when indicated and overall questions about the quality of the trainers and materials/course content Post-third session: similar to post-second training session questionnaire, but was amended to incorporate questions eliciting specific feedback on new issues discussed and not covered in former session (resilience, mental health and revision/ study skills) • Both sessions were rated highly with a mean (± standard deviation) session of 4.37 (± 0.21) after the second training session from 29 responses, and 4.33 (± 0.38) following the third session from 32 responses • Following the second session, the question rated highest was ‘I feel that I will be supported in my role as peer mentor’ with a mean score of 4.72 and the question rated lowest was ‘the training met my expectations’ with a mean score of 4.03 • Following the third session, the question rated the highest was ‘The trainer was knowledgeable’ with a mean score of 4.72, and the question rated the lowest was ‘My own revision strategy will change in light of the station on revision/study skills’ with a mean score of 3.13 Cho and Lee 2021 (Korea) Mixed methods study Mentors—either in the same year level as their mentee, or two academic years above Mentees—preclinical second year (PY2), first year (Y1) or second year (Y2) medical students Total n = 67 participating in peer-mentoring program; breakdown unspecified Recruitment of mentors: Volunteer Allocation of mentees to mentors: Choice to pair themselves or school to pair mentor and mentee Contact logistics: Meeting approximately 4 or more times per semester Training and orientation provided: At least 2 h of education on mentoring, rules to follow and appropriate communication and feedback skills Activities organised: None Duration of program: 2 years Conclusion of program Quantitative: survey assessing general information regarding program implementation (how often students participated; how mentors and mentees would meet; how many times, where, and at what times mentors and mentees would meet) and appropriateness (satisfaction with the program, purpose for participation and mentors’ role). Program satisfaction and appropriateness were answered using the Likert scale of 1–5 (with 1 being ‘not at all’ and 5 being ‘very high’) Qualitative: open-ended survey questions (regarding benefits and outcomes gained by both) and focus group interviews (1–2 h of probing questions: (1) why and how students participated in the mentoring program, as well as how they felt or what they thought about the program; (2) the personal experiences of students; and (3) the significance of their participation. Also included discussion regarding what more was needed to improve) Data analysis: Mann–Whitney U -test was used to compare differences in continuous variables between groups due to small sample size • Program implementation: 84% face-to-face meetings, 74% used text messages, 64% had meetings for 1–2 h, 21% had meetings for less than an hour • Participant satisfaction and evaluation of program: mentors rated 3.6 (± 0.5) and mentees rated 3.8 (± 1.0) • Purpose of students’ voluntary participation and role of mentors: most common answer for both mentors and mentees were ‘teaching assistance for test-taking strategies and guidelines’ followed by emotional support • Qualitative outcome and mutual benefits: improved grades for mentees’, an increased willingness and motivation to study, improved lifestyle patterns, emotional support. Specific to mentees’: improving interpersonal relationships and obtaining support. Specific to mentors: better understanding of previously learnt content, sense of responsibility • Qualitative negative experiences: one mentee response ‘feeling pressure’, and mentors ‘not improving mentees’ learning habits or attitudes nevertheless continuous asking improvement’, ‘not doing homework’ and ‘not contacting or requesting in advance’ • Suggestions for improvement from both mentees and mentors: ‘specific guidelines on how to do’, ‘training and education for mentees’ and ‘appropriate matching between mentor and mentee’. Mentors specifically suggest knowing the characteristics and tendencies of both mentee and mentor, the mentee must have a clear purpose when they participate, and the mentee’s voluntary participation is essential Fleischman et al. 2019 (USA) Cross-sectional study Mentors—77 fourth year medical students in the formal mentoring program (‘Cicerone’) Mentees—first and second year medical students; number not specified Recruitment of mentors: Not specified Allocation of mentees to mentors: Defined by programs that ‘formally and thoughtfully assign mentor to mentee’. 4th year medical students assigned to 2nd years, and 3rd year students to 1st years Contact logistics: Not specified Training and orientation provided: Not specified Activities organised: Not specified Duration of program: Not specified Conclusion of academic year Survey including categorical options and open-ended questions. Respondents who participated in Cicerone peer mentoring program were specifically compared to those who did not participate in any formal mentoring A second purely descriptive analysis was performed including only those who did not participate in any mentoring to understand their motivation for not participating A third analysis of only Cicerone (formal peer mentoring) to assess their experience Data analysis: Simple statistics used, including Fishers exact and chi-squares for categorical variables, and t tests and Kruskal–Wallis for continuous variables • 60% response rate (107/178); 42 participated in Cicerone program (77% response rate), 17 mentored in other programs, and 48 non-mentors responded • Non-mentors did not participate in the mentoring program due to: not knowing about program (69%), not having time (26^) and not believing it was useful (23%). Men were more likely than women to cite ‘not knowing about the program’ and ‘did not believe it would be useful’ • Cicerone mentors were twice as likely to believe being a mentee was beneficial in medical school. 75% of mentors felt it was a worthwhile experience • Mentors were 4 times more likely to believe that being a mentor in an SSM program was valuable and 83% stated they would mentor again • Career choice was discussed by 79% of pairs, and 42% mentors felt more positively about their career choice Nebhinani et al. 2020 (India) Cohort study Mentors—undergraduate medical students in their 3rd, 4th and first half of 5th year, or faculty members Mentees—undergraduate first and second year medical students ( n = 162) Recruitment of mentors: Volunteer Allocation of mentees to mentors: Either peer or faculty mentor randomly assigned to 2–4 mentees Contact logistics: Encouraged at least one mentorship meeting every fortnight Training and orientation provided: Guidelines for mentees and mentors Activities organised: Discussions Duration of program: 8 months Not specified; questionnaire distributed to ‘receive feedback for ongoing mentorship program’ Semi-structured questionnaires, which involved survey items and questions assessing perceived benefits and qualities of mentors, barriers in mentorship meetings, commonly discussed topics in mentorship meetings, and the role of faculty mentor and their relationship Data analysis: Comparison between the groups have been done using chi-square test and t -tests for categorical and continuous variables respectively • Over the 8-month period, mean number of meetings were 4 with faculty mentor and 11 with peer mentor • For both the faculty member and peer mentors, mentees commonly reported that their qualities included being understanding, being supportive and having a helping attitude • The most common barriers faced with faculty members include difficulty synchronising meeting times, students’ lack of time or no interest from students • The most common barriers faced with peer mentors include difficulty synchronising meeting times, mentors’ lack of time and then students’ lack of time Commonly discussed topics with both faculty and peer mentors include education in general, life as a medical student and work-like balance related issues *Neufeld et al. 2020 (Canada) Cohort study Mentors—second year medical students as ‘instructor-mentors’ n = 17 Mentees—first year medical students as ‘learner-mentees’ n = 38 Recruitment of mentors: Voluntary Allocation of mentees to mentors: 2 s year mentors matched with 2 or more first year students weekly Contact logistics: Weekly from October to April with exception for holidays and exams Training and orientation provided: Mentors received only general instructions prior to mentoring without any specific prerequisite training Activities organised: ‘PULSE’ (Peers United in Leadership and Skills Enhancement) sessions; topics coincided with students’ curriculum and progressed from basic history taking and physical exams, to procedural (e.g. otoscope) and interpretative skills (e.g. chest x-ray), to problem solving (e.g. formulating differentials, investigations and management plans) and presenting cases Duration of program : 6 months Within 3 months (April–June) post-PULSE (Peers United in Leadership & Skills Enhancement) sessions General feedback form, and adapted 6-item Learning Climate Questionnaire which measures perceived autonomy support • First year medical students attended an average of 2–3 sessions • PULSE helped reduce performance anxiety around OSCE’s, not only providing ‘a safe and relaxing learning environment, but comradery and professional relationships as well’ • Mentors ‘were incredibly helpful and had a different perspective than instructors, which made it easier to ask questions’ and ‘supportive feedback and non-judgmental environment’ *Neufeld et al. 2021 (Canada) Mixed methods study Mentors—second year medical students as ‘instructor-mentors’ n = 37 Mentees—first year medical students as ‘learner-mentees’ n = 53 Recruitment of mentors: Voluntary Allocation of mentees to mentors: 2 s year mentors matched with 2 or more first year students weekly Contact logistics: Weekly from October to April with exception for holidays and exams Training and orientation provided: Mentors received only general instructions prior to mentoring without any specific prerequisite training Activities organised: ‘PULSE’ (Peers United in Leadership and Skills Enhancement) sessions; topics coincided with students’ curriculum and progressed from basic history taking and physical exams, to procedural (e.g. otoscope) and interpretative skills (e.g. chest x-ray), to problem-solving (e.g. formulating differentials, investigations and management plans) and presenting cases Duration of program : 6 months Conclusion of academic year 3 validated Likert-type surveys: • LCQ (Learning Climate Questionnaire): 6-item scale measuring learners’ perspective of the autonomy supportiveness of their mentors • W-BNS (Basic Need Satisfaction at Work Scale): 21-item scale measuring workplace need satisfaction • PCS (Perceived Competence Scale): 4-item scale assessing feelings of competence in a given task, referring to learning or teaching clinical skills in PULSE sessions Data analysis: Cronbach’s alpha values for each scale to calculate reliability Linear regression used to assess learner perception of how PULSE’s learning climate impacted need satisfaction and how need satisfaction impacted perceived competence in learning. Further analysis of how each of the autonomy, competence and relatedness satisfaction subscales predicted their perceived competence in learning as separate independent variables Linear regressions used to assess mentor perceptions of how overall need satisfaction in PULSE impacted perceived competence in teaching, followed by how need satisfaction subscales predicted perceived competence in teaching • Both groups’ mean scores for autonomy, competence and relatedness supported high levels of need satisfaction • PULSE’s learning climate positively related to learners’ overall need satisfaction ( R 2 = .39, p = .01) • When overall need satisfaction was entered into the regression as the independent variable, it positively related to perceived competence in learning the material ( R 2 = .32, p < .05) • Mentors’ overall need satisfaction in PULSE positively related to their perceived competence about teaching the material • Both mentors and mentees found PULSE sessions highly supportive to their autonomy, competence and relatedness; this corresponded with greater perceived competence about learning and teaching of clinical material Shafiaai et al. 2020 (Malaysia) Mixed methods study Mentors—‘senior year medical students’ ( n = unspecified) Mentees—undergraduate second year pre-clinical medical students Recruitment of mentors: Elected through an interview for their excellent communication and inter-personal skills and have achieved excellent results in all course-related examinations with a ranking of upper third quartile in their cohort of students Allocation of mentees to mentors: Not specified Contact logistics: Weekly basis Training and orientation provided: Two-day Peer-Assisted Study Sessions (PASS) leaders training is conducted by a certified trainer-supervisor for PASS program Activities organised: 1 hourly weekly based integrated learning session through problem-solving and active discussion regarding various topics anatomy, biochemistry, physiology and clinical skills, etc Duration of program: One 12-week semester Baseline and at the end of 12-week program (questionnaire) Focus group discussion 1 year after completion of 12-week program Validated questionnaire (5-point Likert scale) which included 12 items with focus on establishing level of communication skill and pedagogical skill Structured focus group interview exploring whether or not participation in the PASS program as peer leaders helped in developing skills required to become a successful doctor in the future from participant’s point of view • On completion of PASS program, peer leaders reported an increase in oral and written skills to engage with junior students, and skills to develop interaction and collaboration • Peer leaders reported a significant increase in leadership skills and teamwork, stress and time management skills, interpersonal and critical thinking skills, ability to create an effective learning environment, ability to learn new skills, problem-solving and innovative thinking, facilitate teaching sessions and ability to develop independent learning amongst their students • Qualitative analysis revealed 2 main themes: personal growth and professional growth • Within the theme of Personal Growth, 4 skill subthemes were identified: communication, leadership, learning and pedagogical skills Within the theme of Professional Growth, 4 subthemes were identified: administrative, experience, responsibility and time management Singh et al. 2014 (India) Cohort study Mentors —a mix of second, third, and fourth year volunteer medical students ( n = 57). Plus, faculty supervisors ( n = 52) Mentees —first year medical students ( n = 148) Recruitment of mentors : Volunteers Allocation of mentees to mentors: Randomly to 52 groups; one mentor and one faculty supervisor for to three mentees Contact logistics: Meetings, frequency not specified, method of contact not specified Training and orientation provided: None Activities organised : 2 ‘open house’ meetings with entire mentoring cohort Duration of program: Academic year Conclusion of academic year Two self-developed questionnaires were administered to mentors and mentees Questionnaire 1: to mentees, questions focused on the amount and quality of the contact with mentors Questionnaire 2: to mentors, focused on what they gained from the program, and the barriers that affected them Data analysis : Chi-square for categorical variables, student unpaired t -test for continuous variables • Average contact time between peer mentors and mentees increased when compared with contact time between mentees and faculty mentors • Peer mentors reported benefits to them from engagement in the program; particularly improvement in problem-solving, responsibility, confidence and counselling, dual-way mentoring Taylor et al. 2013 (USA) Cohort study Mentors—second year medical students, in a ‘teaching academy fellow’ role ( n = 28) Mentees—first year medical students ( n = 96) Recruitment of mentors : Volunteers Allocation of mentees to mentors : One mentor to four mentees; mentors assigned to groups taught by the same faculty as in their first year when possible Contact logistics : Meetings twice a week Training and orientation provided : Yes, 2-h orientation Activities organised : Group discussions, wellbeing checks Duration of program : Academic year Academic years ending mid 2009 mid 2010 Immediately post orientation (mentors) 2-months post orientation workshop (mentors) Conclusion of academic year (mentees) Non-validated surveys administered to mentors and mentees The first after the orientation workshop received by the mentors, and then 2 months into the program. Surveys aimed to examine the application of skills taught to mentors in orientation workshops, as well as student satisfaction as mentors Data analysis : Not specified • Evaluation of orientation: Reported increase in confidence in ability to provide adequate and constructive feedback ( p < .001) • Evaluation of whole academic year program: 94% of TA mentors suggested the orientation workshop be continued in years to come, with suggestions on how to improve made • Majority of mentees were satisfied with the quality of feedback, reported confidence in performing medical interview increased after mentoring sessions Yang et al. 2021 (USA) Qualitative Mentors—third and fourth year medical students randomised to Education Centered Medical Home (ECMH—a team-based clerkship program consisting of students across all 4 year levels) Non-mentors: third year students randomised to the Individual Preceptorship stream (IP; one-on-one clerkship program pairing a student with a physician) Mentees—students from all four year levels participating in the ECMH program Recruitment of mentors: Students randomly assigned to ECMH stream Allocation of mentees to mentors : students randomly assigned to the ECMH stream. Group size not specified Contact logistics: Students attended ECMH clinic 26 times throughout third and fourth years Training and orientation provided: None Activities organised : None Duration of program: 4 years 3 months into the semester of third year One-to-one semi-structured interview with M3 mentors using an interpretivist paradigm. Interview questions included: What opportunities have you had to teach your peers, what opportunities have you had for your peers to teach you, can you tell me about a time when you have served as a role model or mentor for other medical students and in general, how does it feel to be an M3 in clinic now, thinking back to when you were an M1? Data analysis: two cycle-based inductive approach to coding and analysis • Derived three main themes: (1) diversity of peer teaching and mentoring opportunities, (2) transitioning one’s role from learner to teacher and (3) personal and professional development • Whilst participants from both clerkships participated in peer teaching and mentoring experiences, ECMH students described more opportunities to interact with students across all years of medical school training, noting that ‘getting that guidance and in turn being able to teach is a valuable experience’ • ECMH students further perceived the responsibility of creating a comfortable learning environment for others • Students from both clerkships (ECMH and IP) reflected on ‘learning through teaching’, that teaching served as a reaffirmation of the knowledge they gained, and that teaching experience contributed to their personal and professional growth • ECMH students further described a heightened sense of self-confidence and fulfillment Yusoff et al. 2010 (Malaysia) Cross-sectional study Mentors—select second year medical students—‘BigSibs’ Mentees—first year medical students n = 449 students; breakdown unspecified Recruitment of mentors : Selection based on academic performance, ‘BigSib’ Allocation of mentees to mentors : Not specified Contact logistics : Monthly 2-h meetings, method of contact not specified Training and orientation provided : None Activities organised : Meetings, indoor games, workshops, treasure hunts, hiking, public speaking, picnics, outings Duration of program : Academic year Conclusion of program Validated questionnaire administered to mentors and mentees, with two domains measuring perception of the program in promoting campus life, mental health and wellbeing and confidence and respect of peers Data analysis : Chi-square goodness of fit test for categorical variables, Pearson chi-square to establish relationships and multiple logistical regression for individual effects of targets • Majority of mentees felt as though the program did little in aiding communication between students and lecturers, and that the aims and objectives of the program were not made clear to them • Mentees (60%) felt that the program improved soft skills, professionalism • Less than half (45.9%) perceived the program as effective, but overall, it was perceived in a positive light • Program (from mentees perspective) was seen to aid in study skills, stress reduction, improve confidence and assist in adjustment to campus life Focused programs Barker et al. 2012 (UK) Mixed methods study Mentors—fifth year medical students ( n = not specified) Mentees—first year medical students ( n = not specified) Recruitment of mentors : Volunteer Allocation of mentees to mentors : Random Contact logistics : Mentees expected to contact their mentor prior to the day Training and orientation provided : None Activities organised : Hospital orientation day (HOD) for early clinical exposure. Pre-visit planning sheet (mentees), orientation to the hospital environment, mentees to join fifth years in a teaching session Duration of program : One day Immediately post intervention (survey) One-year post intervention (focus group) One questionnaire and a focus group administered to mentees Non-validated questionnaire, consisting of questions regarding the organisation of the HOD. The answers were reported, and further, used to guide the focus groups the following year Focus groups (number of participants ranged from 4 to 5), semi-structured, digitally recorded and transcribed Data analysis : Not specified • Mentees reported little to no fear regarding entering clinical placement, which was attributed to work experience, pre-visit contact and following the fifth year as key factors • Mentees found positive interactions with clinical staff and patients reassuring • Mentees saw the day as an insight into what their future in clinical medicine would look like and was associated with improved motivation • Mentees gained an understanding of the culture of the hospital and clinical learning and commented on the self-directed and autonomous learning styles of the fifth years • Mentees saw the day as valuable in facilitating discussions with clinical students Perception of the day was deeply affected by the ‘quality of the fifth year’. Characteristics that had made a quality mentor in this context included: ‘friendly’, ‘helpful’, ‘interested’, ‘proactive’ and ‘cheerful’ Choudhury et al. 2014 (USA) Cohort study Mentors—fourth year medical students ( n = not specified) Mentees—first year medical students ( n = 77) Recruitment of mentors : Volunteer Allocation of mentees to mentors : Random Contact logistics : Mentees joined Mentors at one of four student run clinics Training and orientation provided : None Activities organised : Interview skill practice, patient interactions, examination practice, case presenting, clinical note writing Duration of program : Duration of clinical placement; not specified Immediately post intervention Questionnaire administered to mentees, consisting of questions regarding frequency of volunteering, improvement in clinic-associated skills, and the role fourth year mentors played in improvement. General questions inquired about the fourth-year presence, role and impact on mentee comfort Data analysis : One-sample Wilcoxon sign-ranked median test and ordered logistic regression • Mentees reported increased comfort with patients in clinic, and increased level of mentorship due to mentor presence • Mentees did believe the role played by fourth year mentors was different to the role played by attending physicians but found that fourth year presence enhanced mentee interactions at a comparable level • The perceived improvement was independent of which clinic the mentees attended, as well as the number of clinics volunteered at, frequency of volunteering or the number of mentors at the clinic • Logistic regression showed significant association between mentor presence and self-reported improvement in physical exam skills, and case presentation skills at one clinic site (however, no association with patient interviewing and note writing skills), but not at the other three sites Lynch et al., 2022 (US) Mixed methods Mentors—‘clinical-level students’ who had already taken the USMLE step 1 (M3, M4) n = 75 (breakdown unspecified) Mentees—pre-clinical students or M3s yet to sit the USMLE step 1 (M2, M3); n = 125 (breakdown unspecified) Recruitment of mentors : Volunteers Allocation of mentees to mentors : Random; opt-out program Contact logistics : Mentors to contact mentees via email and explain program, offer frequency of meetings Training and orientation provided : Mentors attended a mandatory 1-h training session outlining the ‘Step Sibling’ program aims—told NOT to provide academic support Activities organised : Mentors encouraged to organise wellbeing checks monthly Duration of program : October 2020–March 2021 (beginning of year to USMLE Step 1 test) Immediately post USMLE Step 1 exam Questionnaire aimed at ‘elicit[ing] student reflections on the program’s effect on their stress levels and overall wellness and to seek feedback for future improvements to the program’ Data analysis: Response data evaluated for trends in agreement/disagreement Qualitative thematic analysis for comments • 91% of mentees took exam after participating in program • 77% of mentees felt they were being contacted sufficiently by their mentors; 56% of mentees agreed that they found the program helpful; 53% of mentors agreed that they felt they were helpful • Majority of mentors (70%) and mentees (62%) agreed that ‘sharing perspective and experience was helpful’; sharing resources described as least helpful role of mentors • Most mentees and some mentors expressed desire to offer academic support. Some mentees reported being confused about the aims of the program • Majority of mentees ‘found the program helpful in decreasing stress’. A minority did not find peer mentors helpful, citing ‘already having existing mentors or supports in place.’ • Future program would involve information session for mentees to outline aims of program and the intention to provide wellbeing support only Pinilla et al. 2015 (Germany) Mixed methods study Mentors/mentees—First and second preclinical year medical students in Facebook groups for their respective year level First years n = 1149 Second years n = 1213 Recruitment of mentors: No formal recruitment of mentors, who were existing Facebook users present in year-level groups Allocation of mentees to mentors: To be accepted into the Facebook group by people already in the group Contact logistics: no formal contact organised; posts and comments as needed in group Training and orientation provided: None Activities organised: Posts and comments in two preclinical year Facebook groups Duration of program : 5 months At the beginning of each semester, time periods directly correlating to critical written and oral exams, and between examination periods Analysis of all posts and comments in the two preclinical year Facebook groups using a social constructivist perspective to identify emerging peer-mentoring themes that are relevant for undergraduate medical students and reflect social norms, values and needs of medical students in this context. Anchoring examples were defined for each peer-mentoring subcategory and the final coding scheme was applied to three critical weeks of each preclinical year with particularly high or low posting frequency, as well as at the time of high-stakes exams Frequency of peer-mentoring related elements in posts and comments was reported Two focus groups with medical students enrolled in different semesters were conducted • The preclinical year 1 (PCY1) Facebook group and preclinical year 2 (PCY2) Facebook group had significant number of individuals being members of both Facebook groups, with enrolled PCY1 and PCY2 students being 950 and 966 respectively • Most common categories for PCY1: social activities, study related topics and resources. Most common categories for PCY2: social activities, study related topics and knowledge/skills • More complex peer-mentoring elements such as empowering and fostering personal development seem to be underrepresented in these groups From the focus groups, 3 major themes emerged: similarity in experience and rank, pool of skills/experiences/resources within a group and empowerment and emotional support Prunuske et al. 2019 (USA) Qualitative study Mentors—second year Native American and Alaskan Native medical students ( n = 9) Mentees—First year Native American and Alaskan Native medical students ( n = 40) Recruitment of mentors : Selected based on interpersonal skills and academic performance. Mentors were paid an hourly salary Allocation of mentees to mentors : 1 mentor to 4.5 mentees, allocation method not specified Contact logistics : Daily interaction via activities coordinated by program organisers Training and orientation provided : None Activities organised : Clinical skills, problem-based learning, microbiology labs and faculty lectures Duration of program : 4 weeks for each year however program ran for 5 years with 5 different cohorts 6 months after program conclusion Semi-structured interview administered to mentors, comprising 19 questions regarding what they gained, what could be improved and what sort of guidance was provided to the mentees Data analysis : Not specified • Role was described by mentors as beneficial in several ways: emotional/social support, academic support and give an insider view of medical experience • Social congruence was a common theme, under comments such as ‘you remember what it feels like’ etc • Mentors noted that small cohorts and frequent meetups strengthened the relationship between mentee and mentor • Mentors also found the experience beneficial to consolidating their knowledge and skills, as well as reinforcing a commitment to rural First Nations health * Two reports of the same program Peer mentoring programs could broadly be divided into two types. Fifteen studies (two describing the same program) were ‘longitudinal’ programs, aimed at establishing mentor–mentee relationships to offer ongoing support over a longer period, such as an entire academic year. The remaining five studies were more ‘focused’—generally shorter-term programs with the intent of providing mentorship during significant moments for mentees, such as transition to clinical placement, medical school orientation or exam periods. Accordingly, where programs were named, these names generally reflected the goals—‘BigSibs’ and ‘Mentors in Medicine’ for longitudinal programs, compared to ‘Step Sibling’ , which focused on providing (non-academic) support for the USMLE Step 1 exam. In terms of outcome measurements, 17 studies used quantitative tools, mostly self-developed (piloted or validated in four studies ). Three studies used standardised and validated tools: Mentorship Effectiveness Scale , DREEM and LCQ, W-BNS and PCS . Assessment timepoints were short-term (start and end of the programs). Only one study assessed additional longitudinal outcomes (12 months post-program). Results were analysed for statistical significance in 12 studies [ 18 , 19 , 26 – 30 , 34 , 36 – 39 ]. Studies assessed the impact of their programs on either the mentors, mentees or both. Seven studies assessed outcomes measures for both, whilst three assessed mentee outcomes and three mentor outcomes. Of the seven studies which assessed outcomes for both, all studies reported positive outcomes in confidence, perceived learning environments and psychosocial wellbeing for both mentees and mentors. Where peer mentors or physician mentors were compared, there was no difference in reported mentee benefits . Positive outcomes for mentees were academic development, psychosocial wellbeing and communication skills. Five studies reported mentees felt more confident with their academic skills due to skills gained from mentors, such as academic planning and study techniques . Mentees also perceived the learning environment more positively and reported improvement of social, interpersonal and clinical communication skills . Two studies found improved psychosocial and emotional peer support for mentees, and one study showed mentees were more likely to feel they had a good support system in times of stress. The provision of a ‘safe space’ for mentees was highlighted in three studies . Another study emphasised that mentees felt more competent and autonomous as learners in the learning climate fostered by near-peer mentors . Our scoping review included 20 peer mentoring studies where both mentors and mentees were medical students. Of these, most (15) were longitudinal programs, aimed at first year students or the pre-clinical to clinical transition. All programs reported academic (development of clinical confidence, skills in patient care, procedural skills, medical knowledge, interviewing and examination skills) and non-academic (confidence, social support, peer relationships and communication skills) benefits for both mentors and mentees. Further, many studies identified elements for success such as orientation and training for mentors and challenges such as avoidance overload of mentors and logistics. This review identified several elements that were important for a successful program. Firstly, a number of included studies highlighted the importance of matching mentors and mentees based on additional shared characteristics such as clinical experiences or demographics . These shared traits could facilitate the initiation of a mentoring relationship. A recent systematic review suggested that sharing of specific socio-demographics may be particularly important for more vulnerable mentees such as underrepresented groups . Secondly, an important element of program success was the orientation and training provided to mentors which clarified their role and the objectives of the program and this was reinforced as a recommendation by programs where this was lacking . Orientation was also important for mentees . Interestingly, two studies reported that training was not necessary, given students had spent time as a mentee prior to being a mentor . However, these exceptions would require further evaluation. A related consideration was the balance between training and overload of mentors as ‘extensive, uncompensated training (could) discourage volunteer mentors’ . As part of the training, consideration could be given to ensuring mentors understand the role of greater support systems, especially since perceived independence of the program from the medical school was seen as a bonus for mentees and mentors . This would mitigate concerns which have been raised where the independence of peer mentoring programs from faculty staff could potentially result in the loss of other, formalised support systems . Bias awareness training on diversity and inclusion could also be considered for a more inclusive environment . Limitations of this study included the challenges of developing a search strategy in the absence of ‘mentoring’ as a MeSH term and the heterogeneous use of terminology (such as ‘buddy’) to describe mentoring programs. Several keywords were used in the search strategy for comprehensiveness; however, it is possible these did not capture all available studies. It is also possible that peer mentoring programs were missed in the exclusion of peer-assisted learning studies; however, there was a low threshold for full text reviews to be conducted when uncertain. Further, restrictions to English language only and to the listed databases meant that it is possible that studies in other languages and/or other databases could have been missed. All included studies had significant methodological weaknesses, such as lack of randomisation, blinding and control groups. There was also heterogeneity of the study designs and outcome measures which made comparisons challenging and all included studies reported at least one positive outcome, raising the possibility of publication bias although a grey literature search was conducted. This scoping review has highlighted significant gaps in the current literature, and hence, further research should consider the following: Using robust methodology with validated outcome measures and performing statistical analysis for significant differences. Comparing different types of interventions (given the ethical difficulties of having controls). Including delayed outcome measures to determine longitudinal outcomes. Our review found that peer mentoring programs where medical students are mentored by medical students provide benefits, including improving psychosocial wellbeing and academic development. Challenges in development and implementations included workload concerns for mentors, logistical concerns for face-to-face programs and financial limitations. | Other | biomedical | en | 0.999996 |
PMC11699105 | Successful biofuel feedstock crops necessitate a high germination rate, emergence, and stand establishment . The efficient establishment of new feedstock species in a novel environment relies on its capacity to emerge and establish uniformly and rapidly , as well as to yield under diverse environmental conditions. Climate change is progressing rapidly, resulting in variations in abiotic stresses, including frequent flooding, extended drought periods, and increased occurrences of both low and high-temperature episodes . Hence, identifying adaptable genotypes and appropriate management practices is essential for optimizing yield . The establishment of a crop stand is influenced by internal factors such as seed viability, maturation, genotype, and dormancy, as well as external factors, including water, light, temperature, and oxygen . Brassica carinata A. Braun, known as Ethiopian mustard or carinata, is an oilseed crop recognized for its potential in biofuel production , attributed to its high erucic acid content . Carinata, due to its area of origin, exhibits superior adaptation to semi-arid regions , demonstrating enhanced tolerance to moisture stress, elevated temperatures , seed shattering , drought , and diseases . Carinata is the product of interspecific hybridization between black mustard ( B. nigra L.) and wild cabbage ( B. oleraceae L.), both of which are genetically related to Brassica species . Carinata is an amphidiploid demonstrating high survival and adaptability [ , , ] yet exhibiting low cold tolerance . Genetic characteristics among genotypes may influence the adaptability range of a genotype to specific regions . The Southeast Partnership for Advanced Renewables from Carinata (SPARC), led by the University of Florida, FL, USA, is engaged in efforts to eliminate physical, environmental, social, and economic barriers to its adoption and output while also addressing risks across the supply chain . Temperature significantly influences seed germination, emergence, growth, and development . It affects both the percentage of seed germination and the germination rate in most crop species [ , , ]. Elevated temperatures can lead to restricted availability of photosynthetic assimilates during seed development [ , ], potentially causing physiological damage and resulting in either lower or no germination . Successful establishment of commercial crops requires knowledge of optimum growing conditions and effective establishment techniques . In addition, the environmental conditions during growth and developmental stages influence the yield of crops, including Brassica species . The process of seed germination typically occurs within a defined temperature range known as cardinal temperature . This range includes minimum (T min ), optimum (T opt ), and maximum (T max ) temperatures, which are essential for constructing models that predict seed germination and developmental processes . Various mathematical models have established the correlation between germination rate and temperature . For instance, quadratic, linear, and nonlinear regression models were employed to quantify the response of seed germination to temperature stress [ , , , ]. However, the estimation of germination curves based on nonlinear regression leads to inappropriate standard errors, instead a log-logistic function will better fit the time-to-event data . To date, no attempts have been made to document the seed germination and its cardinal temperatures in carinata species. The assessment of temperature adaptability in genotypes has primarily relied on screening nurseries, field performance, and visual evaluations focused on survival . Interactions that are difficult to control complicate the separation of water, heat, and biotic factors from germination potential. Consequently, an efficient, reliable, and straightforward approach is necessary to evaluate numerous genotypes for thermotolerance in controlled environments . Numerous studies have employed parameters such as seed germination rate and potential to assess the tolerance of crop genotypes to abiotic stresses across various species . This evaluation encompasses both vegetative and reproductive, as well as physiological and biochemical factors during the germination stage . Multiple studies made use of the cumulative temperature response index (CTRI) [ 40 , , , ] to categorize different crop genotypes into tolerance groups for temperature stresses. The study aimed to (a) quantify the impact of temperature on carinata maximum seed germination (MSG) and seed germination rate (SGR), (b) identify the cardinal temperatures for MSG and SGR, and (c) screen carinata genotypes for temperature tolerance. The study evaluated eleven advanced carinata genotypes, including three breed types: inbred, double haploid, and hybrid, alongside one commercial check entry, Avanza 641( Table 1 ). The seeds were collected in Florida (8 genotypes) and Canada (4 genotypes) from Agrisoma Biosciences Inc., Canada (now Nuseed). Prior to the experiment, the seeds were treated with Helix Vibrance, consisting of fungicides (difenoconazole, metalaxyl-M, fludioxonil, and sedaxane) and insecticides (difenoconazole and metalaxyl thiamethoxam). All the seeds were stored in a refrigerator at 4 °C for equal time to maintain optimum quality until time for further use. The seeds with uniform size, shape, and maturity were randomly selected from a single seed lot for each genotype to study the germination behavior of B. carinata genotypes across a range of temperatures. The seed weight (SWGT) of individual genotypes was recorded before starting the temperature treatments and was expressed in mg seed −1 . Table 1 Type and origin of Brassica carinata genotypes sourced from Agrisoma Biosciences, 2019 (now Nuseed). Table 1 Genotype Type a Justification AX17001 Inbred Selection from SE16-17 AYT (Avanza family selection) Florida AX17002 Inbred Selection from SE16-17 AYT (Avanza family selection) Florida AX17004 Inbred High shatter tolerance family, good potential in a winter environment AX17005 Inbred High shatter tolerance family, good potential in a winter environment AX17006 Inbred High shatter tolerance family, good potential in a winter environment AX17007 Double haploid Among the highest Sclerotinia incidence, Jay and Quincy, FL AX17008 Double haploid Selection from SE16-17 PYTB Florida AX17009 Double haploid Selection from SE16-17 PYTA Florida AX17010 Double haploid Selection from SE16-17 PYTB Florida AX17014 Hybrid Top 2016-17 Quincy test hybrid Florida AX17015 Hybrid Promising test hybrid from 2017, frost-tolerant female Avanza 641 Check Commercial check a Genotypes are classified into three types (I = inbred, DH = double haploid, and H = hybrid). Seed trials (SE - Southeast, AYT - advanced yield trial, PYT - preliminary yield trial). The germination of carinata seeds was evaluated under various temperatures from May to September 2019. The study was conducted in vitro at the Environmental Plant Physiology Laboratory, Mississippi State University, Mississippi State, MS, USA. The study was performed following the guidelines established by the Association of Official Seed Analysts without humidity control. The experiment followed a completely randomized, two-factorial design with genotypes and temperatures serving as the primary source of variation. A total of 12 carinata genotypes were subjected to eight temperature treatments, with each treatment replicated four times using 100 seeds per replication. The eight levels of germination temperatures ranged from 8 to 37 °C in 5 °C increments; however, the actual temperatures recorded during the experiment were 8.2, 12.73, 15.65, 19.93, 23.8, 29.28, 34.22, and 36.96 °C. Each experimental unit consisted of 100 counted and weighed seeds, which were uniformly arranged on sterilized plastic trays measuring 31 cm in length × 24 cm in width. The trays were lined with double layers of sterilized paper towels (Scott Shop towels, Kimberly-Clark, Irving, TX, USA). Paper towels were wetted with sterile distilled water, and trays were covered to reduce moisture loss and arranged vertically in a germination chamber (Fisher Scientific, Inc., Suwanee, GA, USA), maintained at designated treatment temperatures. The chamber's internal temperature was measured using data loggers (WatchDog Model 100, Spectrum Technologies, Inc., Aurora, IL, USA) positioned uniformly on the top, middle, and bottom shelves. After incubation, the trays were examined at 2-h intervals to record seed germination. Seeds were counted as germinated when the radicle reached a length of at least 50 % of the seed's total length. The germinated seeds were counted, recorded, and subsequently discarded. The seed germination experiment was concluded after observing no germination for five consecutive days or eight days post-incubation. Temperature and seed germination time course data were analyzed using a 4-parameter Weibull function (Eq. (1) ) in Sigma Plot 13 (Systat Software, Inc., San Jose, CA, USA). (Eq.1) Y = a ∗ [ 1 − e − [ x − x 0 + b ln 2 1 c b ] c ] This function estimates the total seed germination percentage (Y) using the maximum cumulative seed germination percentage (a) at a specific time (x), the shape (c) and steepness (b) of the curve, and the time required to achieve 50 % of the MSG (x 0 ). The reciprocal of the time to 50 % of the cumulative MSG (x 0 ) was used as the rate of development or the seed germination rate (SGR). The responses of MSG and SGR to temperature treatments were analyzed using best-fitted linear and nonlinear regression models to ascertain the cardinal temperatures for all tested genotypes. The optimal curve fitting model was determined based on the highest coefficient of determination ( r 2 ) value. A quadratic model effectively described the relationship between MSG and SGR response to temperature, evidenced by a mean r 2 of 0.85. In contrast, the modified bilinear model test resulted in an overestimation of T max and an underestimation of T min for SGR. The quadratic model was employed to estimate MSG (%; Eq. (2) ) and SGR (d −1 ; Eq. (3) ). The cardinal temperatures (T min , T opt , and T max ) were determined using Equations (Eq. 4) , (Eq. 5) , (Eq. 6) ). The temperature adaptability range (TAR) for each genotype was determined using Equation (7) . (Eq. 2) M S G = a + b T + c T 2 (Eq. 3) S G R = a + b T − c T 2 (Eq. 4) T o p t = − b ( 2 c ) (Eq. 5) T min = − b + b 2 − 4 a c 2 c (Eq. 6) T max = − b − b 2 − 4 a c 2 c Where: The high-temperature response indices were computed based on the standardized vigor index . The individual temperature response index (ITRI) for each of the six parameters (P), specifically, MSG, SGR, T opt , and T max of MSG and SGR, was assessed for high-temperature tolerance across each genotype. The computation involved dividing the value for each genotype by the maximum value observed across all studied genotypes. The cumulative high-temperature response index (CHTRI) was determined by summing the six ITRI derived from MSG, SGR, and T opt , and T max of MSG and SGR. The ITRI for low-temperature tolerance was derived from MSG, SGR, T min and T opt temperatures for MSG and SGR, which are analogous to the determination of CHTRI. The ITRI from T min from MSG and SGR was calculated by dividing the minimum value by the values for each genotype observed across all the genotypes. The cumulative low-temperature response index(CLTRI) was determined by summing all six ITRI values. Regression procedures in Sigma Plot 13 were used to estimate MSG with time and to fit Weibull and polynomial functions for cumulative time series and germination rate data. The MSG, SGR, and SWGT data were analyzed using two-way factorial ANOVA and the ‘doebioresearch’ package in R studio. The data for T min, T opt , T max , and TAR were analyzed using the PROC GLM (one-way ANOVA) procedure in SAS to determine the effect of the germination temperature treatments on MSG and SGR together with their respective cardinal temperatures. Means were separated using Fisher's protected least significant difference (LSD) at p < 0.05. Temperature and time to germination were considered as independent variables, while germination factors (MSG and SGR) were dependent variables. The ITRI for each of the six parameters, i.e ., MSG, SGR, and T min , T opt , and T max of MSG and SGR of individual genotypes from high- and low-temperature tolerance response index were used to obtain the principal components (Dimension; Dim.) for high- and low-temperature response. The factor vectors were identified using the loadings of each trait at individual principal components. Depending on the genotype loading values, a scatter plot was created for high- and low-temperature tolerance. The analysis was performed in R studio using ‘factoextra’ package . Temperature is a critical factor in the regulation of crop growth and development. This study is the first attempt to address the impact of temperature on seed germination traits across multiple B. carinata genotypes. This functional algorithm between seed germination and temperature derived in the study will be instrumental in developing models for carinata in field applications. The thermotolerance capacity observed among carinata genotypes will assist breeders in developing new germplasm suitable for both low and elevated temperatures during seed germination. The impact of temperature on cumulative MSG and SGR, as well as variations in germination time, was examined across all carinata genotypes . The impact on four different breed types, viz., cultivar , inbred , double haploid , and hybrid , are shown in Fig. 1 . A four-parameter Weibull function exhibited a good fit across all tested carinata genotypes ( Table 2 ; mean r 2 = 0.86). All genotypes exhibited variability in their response to various temperature treatments. At the maximum temperature tested (36.96 °C), fewer than 10 % of germination was recorded for over 50 % of the genotypes. The cumulative MSG for all genotypes declined below 23.80 °C. The MSG was highest at 23.80 °C and lowest at 36.96 °C . Dawadi et al. indicated that the carinata seed MSG exceeded 80 % at a constant temperature of 25 °C, taking approximately 60 h to achieve this MSG, with germination onset occurring after 12 h. The duration required for the initiation of germination (≤24 h) exhibited variability among carinata genotypes and treatments in this study . Seed emergence occurred most rapidly at temperatures of 23.80 °C and 29.28 °C, within 0.5 days. The two lower temperatures (8.20 and 12.73 °C) and the highest temperatures (36.96 °C) resulted in a delay of seed emergence exceeding one day. Fig. 1 Seed germination time courses of four Brassica carinata genotypes representing various groups, (a) cultivar – Avanza 641, (b) Inbred – AX17005, (c) Double haploid – AX17008, and (d) Hybrid – AX17014, germinated at eight temperatures (8.2, 12.73, 15.65, 19.93, 23.8, 29.28, 34.22, and 36.96 °C); the symbols indicate the observed cumulative germination data, and the lines indicate the germination time courses fitted using a 4-parameter Weibull function. Data are means of four replications. Fig. 1 Table 2 Quadratic equation constants (a, b, and c), coefficients of determination ( r 2 ), cardinal temperatures (T min , T opt , and T max ), maximum seed germination (MSG), temperature adaptability range (TAR) for MSG, and mean individual seed weight (SWGT) of 12 Brassica carinata genotypes evaluated under eight temperature treatments. Table 2 Genotype Equation constants r 2 Cardinal temperatures (°C) TAR (°C) d SWGT (mg seed −1 ) c MSG (%) c a b c T min d T opt d T max d AX17001 96.97 bc −39.76 12.96 −0.3073 0.85 3.33 bc 21.09 c 38.86 b 35.53 cd 0.426 e AX17002 59.01 f −66.19 11.08 −0.2452 0.77 7.08 a 22.60 b 38.11 b 31.03 d 0.372 f AX17004 100.0 a −82.47 14.34 −0.2770 0.93 6.59 ab 25.89 a 45.18 a 38.59 b 0.343 g AX17005 96.48 b 14.88 8.39 −0.2159 0.76 −1.70 d 19.44 de 40.58 b 42.28 b 0.506 ab AX17006 73.12 e 13.32 5.38 −0.1208 0.84 −2.35 cd 22.25 b 46.85 a 49.21 ab 0.378 f AX17007 98.20 b 16.61 9.03 −0.2498 0.95 −1.75 d 18.07 f 37.90 b 39.65 c 0.500 b AX17008 88.00 ed 8.56 8.43 −0.2237 0.92 −0.99 d 18.85 f 38.68 b 39.67 c 0.408 e AX17009 100.0 a 43.09 6.12 −0.1536 0.64 −6.11 e 19.92 d 45.94 a 52.05 a 0.514 a AX17010 93.00 c −14.53 10.58 −0.2601 0.82 −1.42 cd 20.33 d 39.24 b 37.82 c 0.384 ef AX17014 92.8 c 14.10 8.42 −0.2254 0.94 −1.60 d 18.69 ef 38.98 b 40.58 c 0.493 bc AX17015 96.37 bc 9.70 9.02 −0.2346 0.91 −1.05 d 19.22 d 39.49 b 40.54 c 0.470 d AVANZA641 77.71 e 10.04 7.27 −0.1952 0.93 −1.33 d 18.62 f 38.57 b 39.91 c 0.428 e Mean 89.30 ˗ ˗ ˗ 0.86 −0.14 20.41 40.70 40.57 0.4355 LSD 4.7 b ˗ ˗ ˗ ˗ 4.43 b 0.89 b 3.74 b 7.97 a 0.0203 b The letters near the values are the results of Fisher's protected least significant difference (LSD); values with different letters are significantly different (p < 0.05), and the same letters are not significantly different (p > 0.05). a Significant at the 0.05 probability level. b Significant at the 0.001 probability level. c Two-way ANOVA was performed, taking temperature and genotype as main factors. The effect of temperature, genotype, and interaction were significant at p < 0.001 for MSG and SWGT. d One-way ANOVA was performed, taking genotype as the main factor. Fig. 2 Temperature effects on maximum seed germination (MSG) of 12 Brassica carinata genotypes. (a) Genotypes, AX17001, AX17002, AX17004, AX17005, AX17006, and AX17007, (b) AX17008, AX17009, AX17010, AX17014, AX17015, and Avanza 641. The lines are fitted with quadratic equations. Data are means of four replications. The symbols observed the maximum seed germination percentage. Fig. 2 A significant genotype × temperature interaction was observed on MSG (p < 0.001). The response of MSG to temperature was modeled using a quadratic regression, yielding a mean r 2 of 0.86. The response of carinata genotypes to temperature for MSG exhibited variability, with values ranging from 59.01 % to 100 % , resulting in a mean of 89.30 % across genotypes . The variations in response patterns contributed to the interaction effect. Parameters derived from the germination-time course data were used to calculate T min , T opt , and T max for MSG. The cardinal temperatures varied significantly among ( p < 0.001; Table 2 ). The estimated T min values varied from −6.11 to 7.08 °C , yielding a mean of −0.14 °C. The mean T opt was 20.41 °C. Genotype AX17004 exhibited the highest T opt value at 25.89 °C, whereas genotype AX17007 recorded the lowest at 18.07 °C. The mean T max was 40.70 °C, with genotype AX17007 exhibiting the minimum T max value of 37.90 °C, whereas genotype AX17006 and AX17009 displayed the maximum values of 46.85 and 45.94 °C which are statistically non-significant ( Table 2 ). The TAR (T max -T min ) indicated genotype-specific germination capacities across different temperature conditions. Carinata genotypes exhibited variations in TAR (p < 0.05; Table 2 ), with a mean value of 40.57 °C, ranging from 31.03 °C to 52.05 °C . Research on the germination response to temperature in various plant species indicates that while there is variability in seed germination, utilizing this parameter for genotype screening may be impractical due to the influence of multiple factors on MSG . These factors include seed traits , the duration of storage between ripening and planting , and the environmental conditions during maternal seed production . The variation in TAR among genotypes suggests that differing genetic traits influence their adaptation and performance under varying temperatures . The optimal temperatures for MSG differed among genotypes, exhibiting a range of responses with varying MSG levels across all genotypes. Genotype AX17002 exhibited the lowest percentage of MSG at 59.01 %. The cardinal temperatures for canola, as indicated by a comparable model, show a T min range of 0 to 3 °C, T opt of 29 to 33 °C, and T max of 35 °C . Our study indicated that the values for T min and T opt ranges were lower, whereas T max was higher. In Canada and the northern tier states of the United States, carinata and canola are grown in similar areas. Establishing the cardinal temperatures for carinata genotypes aids plant breeders and producers by enhancing the understanding of crop species and their necessary conditions for establishment. A quadratic regression model effectively characterized the relationship between SGR and temperature (mean r 2 = 0.85; Table 3 ). A significant genotype × temperature interaction was observed on SGR (p < 0.001). Among genotypes, SGR increased with a temperature rise from 8.2 to 23.8 °C, followed by a gradual decline as the temperature reached 36.69 °C. The estimated SGR was highest at 0.98 d −1 at 19.93 °C and lowest at 0 d −1 at 36.96 °C . The findings align with similar results obtained from eight rapeseed cultivars assessed within a temperature range of 3 to 23 °C . Table 3 Quadratic equation constants (a, b, and c), coefficients of determination ( r 2 ), cardinal temperatures (T min , T op t, and T max ), seed germination rate (SGR), and temperature adaptability range (TAR) for SGR, of 12 Brassica carinata genotypes evaluated under eight temperature treatments. Table 3 Genotypes Equation constants Cardinal temperatures (°C) SGR (d −1 ) c a b c r 2 T min d T opt d T max d TAR d AX17001 0.63 f −0.3342 0.0799 −0.0017 0.88 4.63 c 22.04 c 43.45 ab 38.82 bc AX17002 0.52 g −0.339 0.0779 −0.0018 0.75 4.90 c 21.98 d 39.06 c 34.17 c AX17004 0.97 b −0.8306 0.1305 −0.0024 0.85 7.34 a 27.65 a 47.96 a 40.62 a AX17005 0.97 b −0.7459 0.1355 −0.0027 0.86 6.28 a 25.38 b 44.49 ab 38.20 bc AX17006 0.71 e −0.2518 0.072 −0.0013 0.82 3.76 d 26.71 ab 49.65 a 45.89 a AX17007 0.76 d −0.6386 0.1261 −0.0028 0.85 5.83 abc 22.20 c 38.57 c 32.74 c AX17008 0.70 e −0.2767 0.0798 −0.0016 0.89 3.75 d 24.55 b 45.35 ab 41.60 a AX17009 0.99 b −0.7696 0.1417 −0.0029 0.89 6.21 a 24.82 b 43.42 b 37.22 bc AX17010 0.74 d −0.3717 0.0888 −0.0018 0.93 4.61 c 25.08 b 45.56 ab 40.95 ab AX17014 0.93 c −0.6156 0.1283 −0.0027 0.86 5.41 abc 24.11 b 42.81 b 37.41 bc AX17015 1.08 a −0.7178 0.1433 −0.0029 0.90 5.64 abc 25.14 b 44.64 ab 39.00 bc AVANZA 641 0.66 f −0.4614 0.0999 −0.0022 0.71 5.23 b 22.40 b 39.57 c 34.34 c Mean 0.81 ˗ ˗ ˗ 0.85 5.30 24.51 43.71 38.41 LSD 0.04 b ˗ ˗ ˗ ˗ 2.03 a 1.73 b 4.58 a 6.58 b The letters near the values are the results of Fisher's protected least significant difference (LSD); values with different letters are significantly different (p < 0.05), and the same letters are not significantly different (p > 0.05). a Significant at the 0.05 probability level. b Significant at the 0.001 probability level. c Two-way ANOVA was performed, taking temperature and genotype as main factors. The effect of temperature, genotype, and interaction were significant at p < 0.001 for SGR. d One-way ANOVA was performed, taking genotype as the main factor. Fig. 3 Temperature effects on seed germination rate (SGR) of 12 Brassica carinata genotypes. (a) Genotypes, AX17001, AX17002, AX17004, AX17005, AX17006, and AX17007, (b) AX17008, AX17009, AX17010, AX17014, AX17015, and Avanza 641. Data are means of four replications. The symbols are observed seed germination rates. Fig. 3 The cardinal temperatures for SGR varied significantly across the genotypes ( p < 0.05; Table 3 ). The T min ranged from 3.75 °C to 7.34 °C , with a average of 5.30 °C. The estimated mean of T opt was 24.51 °C, with a range from 21.98 °C to 27.65 °C . The two genotypes, AX17007 and AX17002, exhibited the lowest T max value of 38.57 °C and 39.06 °C, respectively. The highest T max was 49.65 , resulting in an estimated mean of 43.71 °C ( Table 3 ). The TAR for SGR varied between 32.74 °C and 45.89 °C with an average of 38.41 °C ( Table 3 ). Unlike reports on other crop species, the SGR for carinata varied across a range of temperatures, lacking a clearly defined optimum . The rate decreased linearly and rapidly with rising temperatures. Soltani et al. reported that in most plant species, the growth rate increases from minimum to optimum temperatures and declines between optimum and maximum temperatures, consistent with the quadratic model predictions and actual data for the carinata genotypes studied. In our study, the SGR cardinal temperatures exceeded those of MSG, consistent with findings on ornamental peppers and switchgrass . This aligns with the findings of Schimpf et al. , indicating that the MSG exhibits lower temperature sensitivity compared to SGR. Seed germination is a temperature-dependent process; however, variation in cardinal temperatures among genotypes exists due to intra-specific differences related to genetic diversity, area of origin, or adaptation of these entries. Comparable results were observed for switchgrass . Kiniry et al. proposed that cardinal temperatures may vary based on genotype and specific processes. The PCA is an efficient dimension reduction technique widely used in data analysis . It simplifies the data into a set of uncorrelated variables called principal components (Dim.) based on the original data points. The method has been extensively used in the screening and selection of varieties and hybrids in multiple crops . The PCA analysis of carinata genotypes using six parameters (MSG, SGR, T opt, and T max of both MSG and SGR) resulted in six dimensions (Dim) with a major contribution from Dim.1 (55.9 %) and Dim.2 (26.7 %) . The correlation between parameters and Dim.s suggests that T opt of SGR (r 2 = 0.90) is highly contributing for Dim.1, followed by T max for SGR (r 2 = 0.77), T max for MSG (r 2 = 0.65), and SGR (r 2 = 0.59) . On the other hand, Dim.2 was highly explained by MSG (r 2 = 0.59). The fourth, fifth, and sixth Dimensions were only slightly correlated with the tested parameters from the studied genotypes. Fig. 4 The principal component analysis of 12 carinata genotypes for high-temperature tolerance, (a) Scree plot of six principal components with their respective variance (%), (b) Correlations of principal components with the variables (MSG, SGR, and T opt, and T max of both MSG and SGR), and (c) The individual parameter loadings towards the principal components, Dim.1 and Dim.2. The red to green color indicates the least to highest loading values of parameters in each dimension, (d) Variables as factor vectors at first two principal components (Dim.1 and Dim.2), and (e) Scatter plot of 12 carinata genotypes on the Dim.1 and Dim.2 factor planes based on the factor scores. Fig. 4 Depending on the loadings on Dim.1 and Dim.2, the parameters were placed on the vector planes . The Dim.1 was negatively related to all the high-temperature tolerance parameters, featuring the first quadrant as a low-temperature tolerant plane. The MSG and SGR had positive loadings on Dim. 2, while the other parameters were negatively associated with Dim.2 . The T max for MSG and SGR was located in the third factor quadrant, indicating that the genotypes placed in this quadrant will be tolerant to high temperatures. In addition, the MSG and SGR were placed in the second quadrant, indicating this will accommodate genotypes with high-temperature tolerance. Meanwhile, the first and fourth quadrants harbor high-temperature sensitive genotypes . Based on the loadings of the carinata genotypes, a scatter plot was created revealing their temperature tolerance levels . The genotype AX17004 was the most high-temperature tolerant one due to its high loading value in the third quadrant, followed by AX17006. In the second quadrant, genotype AX17009 had the highest loading, showing its high-temperature tolerance. The second also accommodates AX17005 and AX17015 genotypes, expressing their moderate high-temperature tolerance compared to other genotypes. However, AX17007 and AX17002 were located in the first and fourth quadrants with a high loading value, revealing their notable high-temperature sensitivity among all the studied genotypes. The Avanza 641 was also located in the fourth quadrant, expressing its high-temperature sensitivity along with other sensitive genotypes. As with the high-temperature tolerance, the PCA was performed for low-temperature tolerance using six factors derived from the seed germination assay (MSG, SGR, T min, and T opt for both MSG and SGR), where each parameter varied in their input based on its relation to the maximum or minimum constant for that particular factor measured across all the genotypes. The PCA analysis generated six principal components (Dim.s) to explain 100 % variance among the genotypes tested. The first two components collectively accounted for 76.9 % of the total variance . The SGR was highly correlated with Dim.1 (r 2 = 0.90), followed by MSG (r 2 = 0.71) . While Dim.2 was highly correlated with T opt for MSG (r 2 = 0.92). All the parameters were negatively associated with Dim.1 . In contrast, Dim.2 was positively associated with MSG, SGR, and T opt for MSG . This contributed to the separation of the parameters in the four-factor quadrants . The Tmin for MSG and SGR was located in the second and third quadrant, respectively. Thus, genotypes in this quadrant will have low-temperature tolerance. Fig. 5 The principal component analysis of 12 carinata genotypes for low-temperature tolerance, (a) Scree plot of six principal components with their respective variance (%), (b) Correlations of principal components with the variables (MSG, SGR, and T opt, and T min of both MSG and SGR), and (c) The individual parameter loadings towards the principal components, Dim.1 and Dim.2. The red to green color indicates the least to highest loading values of parameters in each dimensions, (d) Variables as factor vectors at first two principal components (Dim.1 and Dim.2), and (e) Scatter plot of 12 carinata genotypes on the Dim.1 and Dim.2 factor planes based on the factor scores. Fig. 5 The first and fourth quadrants accommodated genotypes such as Avanza 641 and AX17002 with low-temperature sensitivity. From the scatter plot using Dim.1 and Dim.2 components, the genotype AX17009 was observed to have a better low-temperature tolerance capacity, followed by AX17005 and AX17015. However, the genotypes harboring in the third quadrant exhibited a profile of being moderately low-temperature tolerant. A crucial goal for most plant breeding programs is to identify genotypes with essential traits that make them resilient to extreme thermal conditions , as well as to develop new genotypes with high field stress tolerance that can thrive under variable weather conditions . Several studies have used identical parameters to screen crops for temperature tolerance . Evaluating a crop genotype's thermal adaptability range is typically limited to visual field observation, field performance, and nursery screening. In these conditions, separating different abiotic and biotic stress factors is challenging. Hence, there is an urgent need for an environment where these stress factors are controlled. Setimela et al. suggested that evaluating the thermal adaptability range has paved the way for developing faster, more reliable, and low-cost methods to screen a large batch of plant materials for thermotolerance characteristics. In our study, genotype AX17009 was grouped as the most low-temperature tolerant. Also, genotypes AX17005, AX17014, and AX17015 appear to have good germination at the low-temperature treatment. The genotypes AX17002 and Avanza 641 were categorized as low-temperature sensitive. Data concerning thermotolerance screening for carinata genotypes and intraspecific variation in the establishment under different regions and thermal conditions are limited in the literature; however, there is a need for further testing. The cumulative indices for high- and low-temperature tolerance responses were calculated to assess the overall reaction of 12 genotypes based on factors obtained from the germination assay. The CHTRI identified genotypes that are both high-temperature tolerant and sensitive. The genotype AX17004, with a CHTRI value of 5.82, demonstrated significant high-temperature tolerance relative to other genotypes . The genotypes AX17009 (5.43), AX17015 (5.32), and AX17005 (5.26) exhibited moderate tolerance to high temperatures. Conversely, AX17002 was recognized as a sensitive genotype, exhibiting a CHTRI value of 4.31. In contrast, AX17009 is identified as a low-temperature tolerant genotype, exhibiting a CLTRI value of 6.23. AX17005 achieved this with a value of 5.45, AX17015 at 5.23, and AX17007 at 4.99. The lowest CLTRI values were observed in AX17002 (2.86) and AX 17001 (3.88), indicating their sensitivity to low temperatures relative to the other carinata genotypes examined. The PCA analysis and bubble plot results complement each other, thereby reinforcing the reliability of the identified genotypes for high- and low-temperature tolerance. Fig. 6 Bubble plot of carinata genotypes for cumulative high-temperature tolerance response index (CHTRI) and cumulative low-temperature tolerance response index (CLTRI). Fig. 6 A weak positive linear relationship was identified ( r 2 = 0.43) between the cumulative low-temperature response index (CLTRI) and the cumulative high-temperature response index (CHTRI) for 12 evaluated carinata genotypes evaluated . This relationship suggests that low- and high-temperature tolerance responses among these genotypes are distinct traits. Consequently, identifying genotypes exhibiting both low- and high-temperature tolerance is challenging. Thus, when developing tolerant genotypes, selection for low and high-temperature tolerance must be conducted separately. Fig. 7 The relationship between cumulative low- and high-temperature response index (CLTRI; CHTRI) of 12 Brassica carinata genotypes. Fig. 7 A weak linear relationship exists between MSG T opt and T max , whereas a poor inverse relationship is observed between MSG T min and T max . As the minimum temperature for carinata genotypes increased, the optimal temperature also increased . Furthermore, SGR T max exhibited an increase corresponding to the rise in T opt . A weak inverse relationship was observed between SGR T min and T max , as well as a weak linear relationship between T min and T opt (r 2 = 0.043) . The study revealed that as the optimum temperature increased, the maximum temperature varied across genotypes but showed a weak correlation with T opt , suggesting that each genotype exhibited distinct T max values necessary for achieving MSG. There was a weak correlation between T min and T opt . As the minimum temperature for MSG increased, the optimum temperature also rose. The optimal temperature for SGR exhibited a linear increase with maximum temperature; however, T min , T max , and T opt were more specific to genotypes for SGR, as indicated by the observed weak relationships. Fig. 8 Relationship between the cardinal temperatures for maximum seed germination (MSG) of 12 Brassica carinata genotypes, the relationship between (a) minimum (T min ) and optimum or (T opt ) and maximum ( Tmax ) temperatures, (b) optimum (T opt ) and maximum (T max ) temperatures, and the relationship between (c) seed germination rate (SGR) and minimum (T min ) and optimum (T opt ) and maximum (T max ) temperatures, and (d) seed germination rate optimum (T opt ) and maximum ( Tmax ) temperatures of 12 Brassica carinata genotypes. Fig. 8 Research indicates that seed weight was not utilized as a criterion for classifying crop species into thermotolerance groups, owing to variations in origin and the influence of the parental environment . Individual seed weight varied among genotypes (p < 0.001; Table 2 ) and exhibited a weak correlation with MSG and SGR , suggesting that seed weight did not influence either MSG or SGR. A weak positive linear relationship was identified between MSG and seed weight as well as between SGR and seed weight . Fig. 9 The relationship between maximum seed germination (MSG), seed germination rate (SGR), and seed weight of 12 Brassica carinata genotypes. Fig. 9 This study assessed the impact of temperature on the MSG capacity and SGR of 12 advanced carinata genotypes through in vitro analysis. Genotype-specific cardinal temperatures and thermal accumulation rates were estimated using best-fit regression models. The mean minimum, optimum, and maximum temperatures of the MSG for carinata genotypes were −0.14, 20.41, and 40.70 °C, respectively. The mean minimum temperature for SGR was 5.30 °C, the mean optimum temperature was 24.51 °C, and the mean maximum temperature was 43.71 °C. The average TAR for the genotypes was 40.57 °C for MSG and 38.41 °C for SGR. There was a variability among the genotypes concerning MSG, SGR, TAR, and cardinal temperature. Carinata genotypes were categorized into distinct groups based on their seed germination vitality traits, specifically regarding low- and high-temperature tolerance using principal component analysis and temperature response indices. The genotypes AX17004 and AX17009 were identified as the most tolerant to high and low temperatures, respectively. The double haploid and hybrid carinata breeding groups exhibited stable thermotolerance during the seed germination stage. The inbred group exhibited a more extensive cluster in response to both minimum and maximum temperatures. The research highlighted the necessity of a distinct selection of genotypes exhibiting tolerance to low or high temperatures. The data collected in this study indicate that the identified cardinal temperatures will enhance the development and application of carinata crop models in field production systems. Additionally, identifying genotypes that are tolerant to low and high temperatures will aid in delineating cultivation regions. Future evaluations of these genotypes under field conditions are necessary to identify and quantify those that exhibit tolerance to both low and high temperatures. | Study | biomedical | en | 0.999998 |
PMC11699107 | Patient-centered care is becoming the most valued medical care approach worldwide. It focuses on patients’ preferences, needs, and values in medical procedures . It has been defined through a multidimensional concept, consisting of five dimensions: the biopsychosocial perspective, patient-as-person, sharing power and responsibility, therapeutic alliance, and doctor-as-person . This theoretical framework translates into a clinical practice in which the doctor, through his own communication and relational skills, is able to catch the patient’s issues, concerns, needs, beliefs, and meanings related to health and disease, in addition to symptoms and medical anamnesis. In this such way, it comes to be built a common ground of knowledge that allows for a concordance on disease etiology and treatment chances, and a sharing clinical decision-making, prevention, and health promotion . Patient-centered care is proven to contribute to positive medical outcomes by promoting treatment adherence , reducing the occurrence of complications linked to poor prognosis and adverse events , and enhancing self-management . Moreover, it aims to improve patients’ emotional wellbeing and satisfaction for care , and contributes to reduce healthcare costs through reductions in expenses related to diagnostic testing and specialist referrals . Therefore, promoting and sustaining patient-centered care attitudes in future doctors has become a gold standard in medical education. However, a recent systematic review found that healthcare students have low attitudes toward patient-centered care . As a result, evaluating the level of such attitudes among students is a pressing and foremost priority, followed by the development of educational interventions able to fill the gap. Among the instruments built to measure patient-centered care attitudes to date, the Patient-Practitioner Orientation Scale (PPOS) is one of the most specific . The PPOS is an 18-item instrument in Likert format developed to capture healthcare students and professionals’ attitudes toward patient-centeredness . The 18-item version of the PPOS is the result of multiple studies which lack some clarity as a whole. The initial PPOS version was developed using a pool of 35 potential items, drawn from an extensive search of the literature about patient-practitioner relationship, administered to a sample of undergraduate medical students. All the selected statements were written to refer to one of the two a priori postulated dimensions underlying the patient-centeredness construct, namely Sharing and Caring. The first reflects responders’ beliefs about sharing information and decision-making power with the patients. The seconds corresponds to the disposition to take care of them as “people,” respecting their expectations, feelings, and beliefs. The initial pool of items was progressively modelled eliminating one statement at a time based on item-total correlation value, calculated for each dimension separately, up to a 20-item scale . A second study has been performed based on a different pool of 61 potential items administered to a sample of undergraduate psychology students. By using procedures similar to the above, 33 items were retained and the exploratory factor analysis (EFA) confirmed the scale construct as conceptually composed by the dimensions of Sharing and Caring . Both the studies explored basic psychometric properties of the scale and claimed the tool was reliable based on good total internal consistency, with Cronbach’s alpha ranging from .80 to .90. In a third study performed on a mixed sample of physicians and patients, the PPOS was presented as an 18-item instrument, notwithstanding this further shortening was not documented in that nor in other studies available in the literature. In this latter study, the internal consistency observed was acceptable for the total scale and poor for the items pertaining to the Caring dimension. No other assessment of psychometric properties of the tool had been carried out . Despite this questionable and poor process of scale modelling and validation, the 18-item version of the PPOS began to be used in the medical education literature. It has been translated into various languages and the validity and reliability of the different adaptations have been tested in multiple studies [ 15 – 21 ]. Some of these found the two-factor solution unstable and questioned the two-dimensional model . Further studies suggested three-factor and four-factor solutions . In summary, patient-centeredness construct dimensionality, as measured by the PPOS, is far from clear and the distinction from other basic related constructs (e.g., empathy, attitude toward care) has not already been ascertained. This study aims to begin to fill this gap by investigating the PPOS psychometric properties, factor structure, and construct validity on a large sample of undergraduate medical students. In particular, this study aims to answer the following research questions: (Q1) Is the PPOS a psychometrically valid tool for assessing the patient-centeredness among first-year medical students? (Q2) Is the PPOS factor structure two-dimensional? (Q3) Is the construct measured by the PPOS significantly different between groups distinguished by gender, preferential interest, and setting toward medical practice? This cross-sectional study was conducted at the beginning of a lectures cycle on doctor-patient relationship and healthcare environment features. It targeted first-year students at the University of Turin Medical School during the initial half of the academic year between October and November. All the questionnaires used in the study (i.e., the Patient-Practitioner Orientation Scale, the Jefferson Scale of Empathy, the Frommelt Attitude Toward the Care Of the Dying Scale, and the Perceived Stress Scale) were administered two times during the first and the third lecture, with an interval ranged between 15 and 17 days. Students accessed online platform via tablets or computers in the classroom to complete the questionnaires. They were informed about purposes and methods of the study; participants voluntarily agreed to participate without any reward, and informed consent was obtained before they started filling out the questionnaire. Prior to questionnaires’ completion, students accessed the online platform through their university accounts. To maintain anonymity, they were required to enter a personal identification code. This procedure was implemented to ensure anonymity and facilitate questionnaire cross-matching. The study met the principles of the Declaration of Helsinki and obtained the ethical approval by the University of Turin Ethical Review Committee . Data gathering was repeated for three consecutive academic years with the same procedures in order to enlarge the sample size. The PPOS is a self-report questionnaire consisting of 18 items scored on a 6-point Likert-type scale (from 1 = “strongly agree” to 6 = “strongly disagree”) assessing attitudes toward patient-centeredness of healthcare students, physicians, and patients through two dimensions (9 items each): Sharing and Caring . Items are written in physician-oriented style, except items 6, 13, and 17. Scores of the scale and subscales are obtained by summing the items and dividing them by their corresponding number, so as the scores range between 1 and 6. The higher the score, the higher the attitude toward patient-centeredness. The 18-item Italian version of the PPOS developed by Ardenghi and colleagues was used in this study . The JSE is a self-report questionnaire consisting of 20 items on a 7-point Likert-type scale (from 1 = “strongly disagree” to 7 = “strongly agree”) assessing empathic attitude in clinical practice of healthcare students and professionals . The Italian translation provided by Jefferson Medical College, previously validated by the authors , was used in this study. JSE scores range from 0 to 140; the higher the score, the greater the empathic attitude. The FATCOD scale is a self-report questionnaire consisting of 30 items on a 5-point Likert-type scale (from 1 = “totally disagree” to 7 = “totally agree”) assessing the attitude toward the care of dying patients . The Italian shortened version validated by the authors (FATCOD-9IT) was used in this study . FACTOD-9IT scores range from 9 to 45; the higher the score, the greater the aversion toward the care of the dying. Data were analyzed with SPSS software v. 25.0 for Mac (IBM). Floor and ceiling effect for each PPOS item was investigated through the inspection of frequency tables, with a percentage ≥ 15% of respondents having scored the lowest or the highest point on the Likert-type scale considered indicative. PPOS factor structure was investigated through exploratory factor analysis (EFA) using the principal axis factoring (PAF) estimation method with orthogonal rotation and Kaiser-Meier-Olkin (KMO) measure of sampling adequacy > 0.70 (middling value) considered acceptable. Factors were extracted using the Kaiser-Guttman criterion (eigenvalues ≥ 1) and through the inspection of Cattell’s scree plot and the percentage of variance explained by the eigenvalues in the first round. Two fixed factors were extracted in the second round to simplify the factor structure (i.e., each variable should have one factor that clearly loads on it and each factor should clearly load on at least three variables), and improve its interpretability and understanding. Internal consistency of the scale was investigated by calculating Cronbach’s alphas and item-total correlations. Test–retest reliability was investigated through the inspection of intraclass correlation coefficients (ICC), with values ≥ 0.70 considered satisfactory. Robustness of construct validity was ascertained through the investigation of convergent and divergent validity and by testing a set of research hypotheses. Convergent-discriminant validity was investigated by calculating bivariate correlations (considering Pearson’s correlation coefficient) between each item of the PPOS and JSE, FATCOD-9IT, and PSS scores. It was expected that scales supposed to be conceptually related with PPOS items (i.e., JSE and, to a lesser degree, FATCOD-9IT, which is supposed to be conceptually related only with the Caring and not with the Sharing dimension) would be moderately positively correlated (i.e., Pearson r > 0.30). Conversely, it was expected that PSS, which is postulated to have little in common with PPOS items, would not be significantly correlated. Research hypotheses on between-group differences on PPOS score were developed and tested by specifying one-way multivariate analysis of variance (MANOVA) models with Scheffé post hoc test to address unequal group sizes, Cohen’s standardized effect size partial η 2 , and α set at 0.01. It was postulated that students who were females and had declared themselves more interested in humanistic and relational aspects of medical practice and prone to outpatient practice would score higher on the PPOS than those who demonstrated more interest toward technical and interventional medical procedures (e.g., lab test, surgery). Participants in this study were 1543 first-year medical students at the beginning of their medical training. All the students approached in the classroom agreed to participate in the study (response rate = 100%), accounting for the 93.5% of the total cohorts resulting from the sum of the students enrolled in three academic years . Of the participants, 596 (38.6%) were males, 943 were females (61.1%), and 4 were non-binary (0.3%). The mean age was 19.7 ± 1.8. VAS for psychological wellbeing was 6.8 ± 2.0 (cm). Most of the participants stated they were more interested in technical and interventional medical procedures rather than in humanistic and relational aspects of medical practice ( n = 776, % = 50.3 vs n = 601, % = 39.0) and they felt more prone to visit and care for outpatients rather than work in operating room or lab ( n = 799, % = 51.8 vs n = 578, % = 37.5). A relevant number of participants ( n = 166, % = 10.8) expressed no choice with respect to interest or preference for their future medical practice. In this study, floor and ceiling effects correspond to having answered to a PPOS item “1 = strongly agree” and “6 = strongly disagree,” respectively. Floor effect ranged from 21.3% (item 5) to 40.1% (item 18). Ceiling effect ranged from 17.9% (item 8) to 74.9% (item 6). Full details are given in Table 1 . Table 1 Floor/ceiling effect and factor structure of the Italian version of the Patient-Practitioner Orientation Scale (PPOS) PPOS item % of scores Factor loading Low High F1 F2 F3 h 2 ITC 7. If doctors are truly good at diagnosis and treatment, the way they relate to patients is not that important 0.9 41.6 0.577 0.060 0.061 0.340 0.428 16. It is not that important to know a patient’s culture and background in order to treat the person’s illness 0.3 52.4 0.476 0.065 0.033 0.232 0.356 2. Although healthcare is less personal these days, this is a small price to pay for medical advances 1.3 9.3 0.446 0.045 0.147 0.223 0.376 3. The most important part of the standard medical visit is the physical exam 6.1 1.8 0.425 0.141 0.057 0.203 0.405 11. If a doctors’ primary tools are being open and warm, the doctor will not have a lot of success 1.5 21.5 0.421 0.107 0.058 0.201 0.338 15. The patients must always be aware that the doctor is in charge 4.7 10.5 0.413 0.376 0.175 0.342 0.379 1. The doctor is the one who should decide what gets talked about during a visit 2.0 6.2 0.326 0.117 0.092 0.128 0.307 6. When doctors ask a lot of questions about a patient’s background, they are prying too much into personal matters 0.1 74.9 0.324 − 0.096 0.112 0.127 0.218 9. Patients should be treated as if they were partners with the doctor, equal in power and status 3.1 23.8 0.118 13. A treatment plan cannot succeed if it is in conflict with a patient’s lifestyle or values 4.4 13.6 0.031 18. When patients look up medical information on their own, this usually confuses more than it helps 40.1 0.9 − 0.051 0.650 0.099 0.435 0.416 5. Patients should rely on their doctors’ knowledge and not try to find out about their conditions on their own 21.3 4.0 0.084 0.636 0.082 0.418 0.458 12. When patients disagree with their doctors, this is the sign that the doctor not have the patient’s respect and trust 3.0 13.3 0.260 0.358 0.145 0.217 0.283 * 10. Patients generally want reassurance rather than information about their health 7.6 4.9 0.042 0.141 0.565 0.341 0.277 17. Humor is a major ingredient in the doctor’s treatment of the patient 11.8 3.6 0.134 0.035 − 0.394 0.174 0.180 # 4. It is often best for patients if they do not have a full explanation of their medical condition 1.5 30.8 0.093 0.083 0.358 0.144 0.254 8. Many patients continue asking questions even though they are not learning anything new 2.8 17.9 0.156 14. Most patients want to get in and out of the doctor’s office as quickly as possible 13.1 3.9 0.119 % variance explained 9.9 7.0 5.0 Cronbach’s 0.657 0.566 0.399 ICC 0.704 0.789 0.661 Low/high , % of lowest (1) and highest (6) possible scores; F1/F2 , factor 1 and factor 2 extracted; h 2 , communalities; ITC , item-total correlation; ICC , intraclass correlation coefficient (test–retest reliability); *Cronbach’s α = 0.608 if deleted; # Cronbach’s α =0.411 if deleted; Factor loadings >0.3 are reported in bold Assumptions of sampling adequacy (KMO measure was 0.748, i.e., middling) and data factorizability were met. In the first round, with the Kaiser-Guttman criterion applied, EFA identified six factors with eigenvalues > 1, explaining 52.2% of the variance. The inspection of the scree plot showed two points of inflection at factors 3 and 4, suggesting to retain two or three factors. The two-factor solution explained 26.6% of the variance, with five items (9, 13, 4, 8, 17) non-retainable due to low loading (< 0.30). The three-factor solution explained 34.4% of the variance and showed slightly higher communalities and higher loadings than the former, with four non-retainable items (9, 13, 8, 14). Considering these little improvements, the solution found with Kaiser’s criterion and the large sample size, three factors were retained in the final second round of EFA. Table 1 shows factor loadings and communalities of the three-factor solution. Internal consistency was questionable for factor 1 ( a = 0.657), poor for factor 2 ( a = 0.566), and unacceptable for factor 3 ( a = 0.399). Item-total correlations for factor 1 and factor 2 were > 0.3, except for item 6 (ITC = 0.218) and item 12 (ITC = 0.283). Item total-correlations for factor 3 were all < 0.3. Test–retest reliability was acceptable for factor 1 (ICC = 0.704) and factor 2 (ICC = 0.789) and questionable for factor 3 (ICC = 0.661). Details of reliability analysis are provided in Table 1 . PPOS items were not strongly correlated with JSE, FATCOD-9IT, and PSS total scores. As expected for convergent-discriminant analysis, significant medium-to-weak correlations were found between PPOS items and JSE total score (except for items 4, 10, 14, 18), significant small correlations were found between few PPOS items (i.e., 6, 12, 14, 15, 15, 17) and FATCOD-9IT total score, whereas non-significant correlations were observed between PPOS items and PSS total score. Details of correlation analysis are provided in Table 2 . Table 2 Bivariate correlations of the Patient-Practitioner Orientation Scale (PPOS) items and sums of scores of the Jefferson Scale of Empathy (JSE), the 9-item version of the Frommelt Attitude Toward the Care Of the Dying (FATCOD-9IT) Scale, and the Perceived Stress Scale (PSS) PPOS item JSE FATCOD-9IT PSS 1 0.188** 0.019 − 0.011 2 0.298** 0.032 0.028 3 0.245** 0.012 0.046 4 0.047 0.062 0.025 5 0.097** 0.030 0.027 6 0.191** 0.110** − 0.046 7 0.434** 0.048 − 0.084 8 0.143** 0.033 − 0.013 9 − 0.289** 0.015 0.078 10 0.023 0.071 − 0.019 11 0.324** − 0.055 0.070 12 0.122** 0.095* 0.009 13 − 0.143** − 0.044 − 0.040 14 0.032 0.120** − 0.020 15 0.194** 0.077* 0.068 16 0.373** 0.090* 0.097 17 − 0.329** 0.081* 0.101 18 0.049 0.044 0.011 Pearson r < 0.30 indicates weak correlation; * p < 0.05; ** p < 0.01 Research hypotheses developed about between-group differences on PPOS were confirmed. MANOVA showed significant effect of gender ( p < 0.001), preferential interest ( p < 0.001), and setting ( p < 0.001) in medical practice on PPOS score. Female students and those who had declared themselves more interested in humanistic and relational aspects of medical practice and prone to outpatient practice scored higher on the PPOS than males and those who demonstrated more interest toward technical and interventional medical procedures (see Table 3 ). MANOVAs showed significant effect also of age ( p = 0.007) and psychological wellbeing ( p = 0.044) on PPOS score, with follow-up univariate analyses mostly non-significant (except for psychological wellbeing on factor 1). Details of MANOVA are provided in Table 3 . Table 3 Between-group differences on Patient-Practitioner Orientation Scale (PPOS) score Variable Λ Part η 2 F1 p F2 p F3 p Gender 0.936** 0.033 < 0.001 0.001 0.060 Male 4.2±0.6 2.8±0.9 3.6±0.8 Female 4.5±5.6 2.9±0.9 3.7±0.7 Age group 0.987* 0.006 0.058 0.497 0.025 18–19 4.4±0.6 2.8±0.9 3.7±0.7 20–21 4.4±0.6 2.9±0.9 3.7±0.7 > 22 4.5±0.6 3.0±0.9 3.5±0.8 Interest in medical practice 0.915** 0.085 Doctor-patient relationship 4.6±0.6 < 0.001 2.9±0.9 0.139 3.7±0.7 0.023 Technical aspects and interventions 4.2±0.5 2.9±0.9 3.6±0.7 Setting in medical practice 0.970** 0.030 < 0.001 0.186 0.516 Examination room 4.5±0.6 2.9±0.9 3.7±0.7 Operating room/lab 4.3±0.6 2.9±0.9 3.6±0.7 Psychological wellbeing (quartiles) 0.991* 0.005 0.010 0.891 q 0.409 Lower 4.5±0.6 2.9±0.9 3.7±0.7 Middle 4.4±0.6 2.9±1.0 3.6±0.7 Upper 4.3±0.6 2.9±0.9 3.6±0.7 Λ , Wilks’ lambda; * p < 0.05; ** p < 0.01; Part η 2 , partial eta squared; F1/F2 , factor 1 and factor 2 extracted Although the PPOS has been widely used in healthcare and medical education research in the last two decades, its psychometric properties have not yet been fully ascertained and its factor structure is still discussed. Since it is the only tool available to date to assess patient-centeredness attitudes of healthcare students and professionals, further research was required, also to shed more light on factors affecting doctors’ patient-centered behavior. In detail, the aim of this study was to investigate the PPOS reliability, factor structure, and construct validity on a large sample of undergraduate medical students. Taken together, results of this study showed that the PPOS has psychometric limitations when used to measure patient-centeredness among first-year medical students (Q1), the assumption of two-dimensionality for the scale construct is not respected in this population (Q2), and the scale adequately differentiates the patient-centeredness based on gender, preferential interest, and setting toward medical practice (Q3). These findings suggest that the PPOS 18-item version might contain items with poor validity and the construct it intends to measure is quite weak and unstable, as it changes when assessed in different populations. These results corroborate previous observations and strengthen the need for scale revision . Findings from reliability and item analysis suggest that PPOS includes a cluster of items with poor validity, having little variance in common, that are not very homogeneous each other, consistently with previous studies . Findings about PPOS factor structure suggest multiple unclear not well separated solutions, as PAF pointed to possible six-, three-, and two-factor structures. The retained three-factor solution, which turned out to be the best in terms of percentage of variance explained and item numerosity observed in this study, nonetheless evidenced poor definition and functioning of many items, given low communality values and low factor loadings (items 9, 13, 8, 14 and were excluded from the final solution due to loadings < 0.30 and item 15 loaded on two factors), suggesting the weakness of scale construct. In the three-factor solution found in this study, F1 consisted of eight items (1, 2, 3, 6, 11, 15, 16) in partial overlap with the Caring dimension of the original PPOS, with the exception of items 1 and 15; F2 consisted of three items (8, 12, 15) all belonging to the Sharing dimension in the original PPOS; and F3 consisted of three items (4, 10, 17), of which the former two belonged to the Sharing dimension whereas item 17 belonged to Caring dimension in the original PPOS . According to the PAF performed in this study, F1 seems to refer to the doctor’s relational style and disposition to be welcoming to the patient; F2 seems to refer to the doctor’s disposition to share power with the patient; and F3 seems to refer to the doctor’s disposition to be protective of the patient. F1 is the better-defined factor, more numerous and reliable, that matches the Caring dimension of the original PPOS, and is generally preserved in most studies [ 15 , 17 – 21 ]. F2 and F3 were found to be less defined, numerous, and reliable. Items in these factors were observed to be mixed in previous studies , suggesting that the Sharing dimension may be weaker and questionable. The items excluded from the final factor solution seem to be part of a critical and uncorrelated cluster with low ITCs and inter-item correlations; items 9 and 13, two of the three statements phrased in patient-oriented style, were excluded also in previous studies , and items 8 and 14 were not retained in confirmatory factor analysis due to poor statistical indexes in PPOS shortening validation studies . Moreover, item 17, retained in this study even though its deletion would have increased F3 internal consistency, was deleted in multiple studies . In addition to the documented psychometric weakness of the PPOS, one of the possible reasons behind the inconsistency of the construct the scale intends to measure could concern the instrument was designed and used to assess attitudes in population highly different in training, experience, and role, including healthcare students at different stages of their curricula (preclinical vs clinical), physicians, nurses, and patients. As correctly argued by Jiang and colleagues, understanding the concept of patient-centeredness and acknowledging the way of behaving in accordance with it is not the same as acting the attitude in clinical work . In this study, first-year medical students asked to give an opinion on the statements of the PPOS, appeared to respond with confidence to the items of the Caring dimension, and seemed they were in trouble providing a personal evaluation on the items of the Sharing dimension. This should not be surprising, as while the Caring attitude reflects the purpose of the medical practice, in which the motivation to become a doctor of many young medical students is rooted, the Sharing attitude deals more with experience in clinical practice and the development of a personal clinical style. If so, the malfunctioning of F1 and F2 observed in this study might be due to its use with preclinical medical students. Findings from convergent-discriminant analysis showed the construct the PPOS measures correlates with empathy for patient care and attitude toward the care of the dying, while remaining distinct from them, and does not correlate with perceived stress, according with research hypotheses. Lastly, PPOS showed satisfactory measurement invariance across age and psychological wellbeing groups, according to research hypotheses. Findings in this study support the conclusion that the 18-item version of the PPOS has psychometric limitations as regard reliability and item validity. Moreover, the robustness of the underlying construct is not proven. This means that it should be used with caution when assessing first-year medical students’ patient-centeredness. Further studies are needed to address these limitations, confirm the multidimensionality of scale construct, and ascertain that previous attempts of scale shortenings have not caused loss of content and original construct validity. | Review | biomedical | en | 0.999996 |
PMC11699140 | Tumor boards, characterized by their convergence of diverse medical professionals and complex patient cases, represent an untapped educational resource for undergraduate medical students. The multidisciplinary nature of these boards has been shown to improve patient outcomes across a spectrum of cancers, in both adult and pediatric settings, and in diverse socio-economic contexts, from high-income to resource-limited settings [ 1 – 6 ]. This global and inclusive perspective is particularly relevant for medical students, who will be practicing in an increasingly interconnected and diverse world . This paper presents a roadmap of 12 strategies to achieve these educational goals. By engaging with the rich, real-world learning environment of tumor boards, students can gain a deep understanding of the collaborative, patient-centered, and evidence-based approach that underpins successful oncological care. These strategies serve as a guide to transform the tumor board experience into a dynamic, immersive learning journey for students, equipping them with the knowledge, skills, and attitudes necessary for effective contribution within a multidisciplinary team. The 12 strategies proposed in this paper are synthesized from a comprehensive review of existing literature and best practices. Encourage students to participate actively by assigning tasks that align with their level of expertise — summarizing patient histories or presenting relevant research findings. Active engagement stimulates critical thinking, promotes problem-solving skills, and instills a sense of responsibility towards their learning journey . Moreover, this participation fosters reflection on interprofessional interactions, encouraging students to consider how their contributions are influenced by the multidisciplinary team . Research suggests that such active engagement in multidisciplinary meetings significantly enhances learning experiences . Enhance the learning experience with structured, post-meeting case-based discussions. Select complex, multi-faceted cases from tumor boards for in-depth exploration in smaller student-led discussion groups. These discussions can delve into the nuances of each case, allowing students to critically analyze the patient history, evaluate diagnostic options, devise potential treatment strategies, and anticipate challenges in patient management. They also provide a platform for students to reflect on how multidisciplinary inputs shape the understanding and management of a case, thereby highlighting the value of diverse professional perspectives in patient care . Where possible, arrange for students to have direct interactions with patients whose cases are being discussed in the tumor board. These interactions could involve taking a patient history, conducting a physical examination, or discussing the patient’s experiences and perspectives about their illness. Such experiences can provide invaluable insights into the patient’s perspective, enhancing empathy and grounding students’ learning in real-world clinical practice . Supplement this learning with opportunities to interact with patients in a clinical setting under supervision, where students can see the real-life implications of the decisions made during tumor boards. These experiences will help students understand the critical role of patient-centered care in shaping clinical decisions and outcomes. Post-meeting reflective writing exercises can serve as a powerful tool for consolidating learning and fostering self-awareness . Encourage students to maintain a reflective journal where they can record their observations, thoughts, and questions about each tumor board meeting. The journal entries can focus on a variety of aspects such as the complexity of the cases, the interprofessional dynamics observed, the ethical dilemmas encountered, and the decision-making processes followed. Additionally, they should reflect on their own contributions and interactions within the team and how these experiences are shaping their understanding of oncology and their professional development. The journals can be reviewed periodically with a faculty mentor to provide feedback and guide further learning. Reflective writing has been recognized in medical education literature as an effective method for promoting critical thinking, self-awareness, and professional growth . Incorporate digital learning tools specifically designed to augment the tumor board learning experience . Utilize online platforms for curating a library of key cases discussed in the tumor board, complete with anonymized patient histories, diagnostic images, pathology reports, and summaries of the decisions made . This can serve as a valuable resource for self-directed learning and future reference. Further, create a protected online discussion forum dedicated to each tumor board meeting. Here, students can continue the discussion beyond the meeting, share additional research findings, pose unanswered questions, and engage in peer learning . Faculty members can participate in these forums, providing guidance and feedback and addressing student queries. These digital initiatives can offer a flexible, diverse, and multifaceted learning environment, catering to the varied learning preferences of today’s digital-native students . By integrating digital tools into the tumor board learning experience, we can enhance student engagement, foster active learning, and ensure continuous feedback and improvement . Transform the seemingly complex decision-making process of tumor boards into a structured learning experience . Begin by deconstructing previous decisions made during tumor boards, spotlighting how patient preferences, clinical guidelines, research evidence, and professional expertise are collectively considered . Also, highlight the role of effective communication, mutual respect, and consensus building within the team . Then, facilitate student participation in this process by creating mock decision-making scenarios, for example, through the implementation of tactical decision games, enabling them to apply these principles in a safe learning environment . Promote a culture of critical reflection on the nature and quality of interprofessional interactions within the tumor board [ 33 – 35 ]. Implement structured reflective debriefing sessions after each tumor board meeting, where students can share their observations about the dynamics of teamwork, the communication patterns they noticed, and the process of collaborative decision-making. These sessions can be facilitated by a faculty member who can provide insights, clarify misconceptions, and guide students in deriving learning from these reflections. Patient-centered care is a crucial aspect of modern healthcare and a key element in the proceedings of a tumor board . While patients themselves may not be physically present in these meetings, their experiences, preferences, and needs are central to the discussions . As such, it is essential that students understand how to place the patient at the center of their learning experience within the tumor board context . Encourage students to review each patient’s history, imaging, pathology, and the psychosocial aspects of their case critically. Highlight how the unique aspects of each patient’s case — their comorbidities, lifestyle, personal preferences, and social circumstances — influence the diagnostic and treatment decisions made during the tumor board. In oncology education, particularly within tumor board discussions, it is crucial to emphasize holistic ethical considerations, for example, by integrating the concept of “total pain” alongside traditional ethical dilemmas. Total pain, a term coined by Cicely Saunders, refers to the multifaceted nature of pain experienced by patients, encompassing physical, psychological, social, and spiritual dimensions . Use the cases to bring ethical considerations in oncological care such as informed consent, patient confidentiality, end-of-life decisions, and balancing treatment benefits and harms to the forefront . Encourage students to reflect on these aspects in dedicated ethics sessions, guiding them to understand the complexity of patient suffering and ethical decision-making in oncology. This approach, incorporating both conventional ethical dilemmas and the broader concept of total pain, aims to develop healthcare professionals who are not only ethically astute, able to reflect on these issues, debate differing viewpoints, and develop their own reasoned stance, but also deeply empathetic towards the multifaceted suffering of their patients . In conclusion, the integration of medical students into tumor board discussions presents a multifaceted opportunity to enhance their learning experience, equipping them with the skills and knowledge necessary for effective interdisciplinary collaboration in oncology care. This paper has outlined a comprehensive strategy, centered around active engagement, structured learning, patient interaction, reflective practice, and the use of digital tools, to maximize the educational potential of tumor boards. These strategies aim to foster a deep understanding of the complexities of cancer care, emphasizing the importance of evidence-based practice, patient-centered approaches, and ethical considerations. By engaging students in this dynamic, real-world context, we not only prepare them for their future roles in healthcare teams but also contribute to the cultivation of a more collaborative, informed, and compassionate oncology workforce. The emphasis on interprofessional education within tumor boards underscores the necessity for future physicians to navigate and contribute to multidisciplinary teams effectively. The skills developed through such exposure—critical thinking, effective communication, ethical reasoning, and empathetic patient care—are fundamental to the practice of medicine and transcend the specific context of oncology. As medical education evolves, the incorporation of such experiential learning opportunities as tumor boards will be crucial in preparing students for the complexities of modern healthcare delivery. Ultimately, this approach aligns with the broader objectives of medical education to produce healthcare professionals who are not only clinically competent but also capable of addressing the multifaceted challenges of patient care in a collaborative, ethical, and patient-centered manner. The strategies presented herein, therefore, not only enhance the educational value of tumor boards for medical students but also serve as a model for integrating experiential learning across the medical curriculum. As we continue to explore and refine these methodologies, it is imperative that we evaluate their impact on student learning outcomes and patient care, ensuring that our educational practices remain aligned with the evolving needs of our healthcare systems and the patients they serve. | Other | biomedical | en | 0.999997 |
PMC11699163 | Many medical schools in the past 20 years have integrated team-based learning (TBL) into their medical curriculum [ 1 – 5 ]. In fact, the AAMC reported that approximately 54% of medical schools utilized TBL in the 2019–2020 academic year . Historically, the TBL structure is as follows: pre-class preparation, individual readiness assurance test (IRAT), team readiness assurance test (TRAT), immediate feedback/clarification, and clinical problem-solving activities . The pre-class preparation typically involves the use of pre-reading or online lectures as prework for the TBL in the medical curriculum . The use of TBL in the preclinical years of medical education has been shown to provide many benefits including improved performance on exams and positive student feedback . A study from Carrasco et al. concluded “team-based learning scores and participation are better predictors of successful course performance than case-based learning performance” . The AAMC reported that approximately 97% of medical schools utilized case-based learning (CBL) in the 2019–2020 academic year . Though an extremely popular method of medical education, the use of CBL as preparation for TBL is less widely used and largely unstudied . Creighton University School of Medicine (CUSOM) has modified the typical structure to use CBL as prework and preparation for TBL instead of the traditional TBL pre-reading or online recordings . Students at CUSOM first work through the clinical aspects of a case with their small group in CBL. Then, students attend a TBL based on the material presented in the CBL. Students are tested individually and as part of their team/small group. In this approach, TBL assesses the material that students learn with their small group in CBL, leading us to question the relationships between aspects of learning in CBL and performance in TBL. Though students at CUSOM spend the majority of time learning from traditional lectures, students complete one to three TBL’s per week, making up a significant portion of their instruction. Student data from two medical campuses of CUSOM were collated to examine the relationship of the identified variables from 29 cases across 6 courses throughout the academic year 2022–2023 that met inclusion criteria as discussed below. Data from the Omaha campus included the entire M2 cohort that consisted of 128 students in the M2 year. Data from the Phoenix campus included the entire M2 cohort that consisted of 99 students in the M2 year. The following variables were collected: time spent in CBL (in minutes; extracted from the learning management system), IRAT and TRAT performance (extracted from Intedashbaord), and exam performance on related questions (extracted from ExamSoft). Of note, exam performance refers only to final exam questions that tested material directly related to CBL/TBL sessions, not to overall final exam performance in the course. CBL cases are available through the learning management system which requires students to click through the case in order to reach the next step of the case. Once the team has completed the case, a student moderator must hit submit in order to see the correct answers for questions that were completed throughout the case. With the submission of the case, completion time is recorded. Data was merged by student ID and then de-identified. Case times were calculated by subtracting the start time recorded from the completion time recorded. Outliers with respect to time (longer than 160 min for a scheduled 120-min time slot) were removed. Only cases with associated exam questions were included in analysis. A mean score for each variable across all cases was computed for each student, taking the final score from each relevant case and finding the mean average across all cases for IRAT, TRAT, and exam data. Data was transformed through centering and standardization, resulting in z -score data for each observation of each variable to allow for easier interpretation of comparisons. Z -scores were chosen for the variables due to the ease of interpretation with all variables being on the same scale, save for exam scores, which were kept on a 0 to 1 scale as provided in the data; Z -scores also allow for results of regression modeling to be interpreted in standard deviations. Computing variable values to z -scores also prevents additional weight from being assigned to variables on different scales. There was a significant relationship between IRAT and exam performance, controlling for time and TRAT scores, with exam performance as the dependent variable (adj. R 2 = 0.42, F (6, 73) = 10.3, p = 0.00). The coefficient from this model was β = 0.40, indicating that a 1 standard deviation increase in IRAT score would lead to an approximately 0.40 standard deviation increase in exam score . Predictions of these exam performances resulted in a distribution of residuals with a rightward skew, indicating a higher-than-observed estimation by the model for exam scores based on IRAT scores and controlling for time and TRAT scores . This data supports previous research showing a strong correlation between TBL performance and final exam performance when compared to other learning methods . Fig. 1 Distribution of estimated effect sizes from the chosen model Fig. 2 Approximate normal distribution of residuals for the chosen model, with the rightward skew indicated by the points above the trend line on the lower left and below the trend line on the upper right portion of the graph There was not a significant relationship between time and exam performance (adj. R 2 = 0.00, F (1, 78) = 1.02, p = 0.317). There was not a significant relationship between average time and average IRAT performance (adj. R 2 = 0.00, F (1, 78) = 1.07, p = 0.30). There was not a significant relationship between average time and average TRAT performance (adj. R 2 = 0.00, F (1, 78) = 13.12, p = 0.71). This indicates that exam performance, average IRAT performance, and average TRAT performance cannot be predicted by the average time spent on the case. There was a significant relationship between average IRAT and average exam performance, controlling for time and mean TRAT scores, with exam as the dependent variable (adj. R 2 = 0.33, F (3, 70) = 13.12, p < 0.01). The coefficient from the model was β = 0.18, indicating that a 1 standard deviation increase in mean IRAT score would lead to an approximately 0.18 standard deviation increase in mean exam score . Predictions of these exam performances resulted in a non-normal distribution of residuals, indicating that not all patterns within the data between mean IRAT scores and mean exam scores, when controlling for time and mean TRAT performance, were captured by the model. Figure 4 also indicates that there may exist outliers in the tail end of the residual distribution. Fig. 3 Distribution of estimated effects sizes from the chosen model Fig. 4 Non-normal distribution of residuals for the chosen model, shown by small systematic deviations near center of distribution. An outlier exists in the rightward tail of the distribution There was not a significant relationship between average time and exam performance (adj. R 2 = 0.00, F (1, 72) = 0.46, p = 0.50). There was not a significant relationship between IRAT average performance and average time (adj. R 2 = 0.00, F (1, 105) = 0.46, p = 0.78). There was a significant relationship between TRAT average performance and average time (adj. R 2 = 0.03, F (1, 105) = 4.25, p = 0.04). However, a low coefficient of determination indicates that very little variation in average TRAT scores can be explained by average time. These results indicate that exam performance and average IRAT and TRAT performance cannot be predicted by average time spent on the case. Overall, our findings suggest that there is a relationship between performance on TBL and exam performance. Our data supports previous literature indicating better course performance stemming from these active learning methods . We found no statistical relationship for time spent on CBL and related assessment performance, which may provide valuable feedback to medical educators on the use of CBL and TBL in preclinical medical education. | Review | biomedical | en | 0.999994 |
PMC11699177 | Patient cases are important and widely used in healthcare education . Within undergraduate medical education, cases are an integral aspect of case-based learning (CBL) and problem-based (PBL) modalities. CBL, an example of active learning, has been shown to have benefits over standard lecture and team-based learning [ 1 – 5 ]. Many medical schools have adopted some form of CBL in their curricula and face the challenge of developing, maintaining, and updating their own teaching cases. Resources have been published and can help guide the process of developing and evaluating individual cases . There is also guidance for improving diversity, inclusion, and equity in curricular materials in medical education [ 8 – 13 ]. Core attributes of effective individual cases have been identified as relevant, realistic, engaging, challenging, and instructional . When individual cases are combined to create a case catalog, desired attributes should be expanded to include being consistent, current, diverse and inclusive, patient-centered, and mission-centered . While the overall effectiveness of PBL and CBL as learning modalities has been studied, limited data are available on the effectiveness of the cases themselves, which can take a variety of forms and be used differently at each institution [ 1 – 5 ]. Similarly, most published student feedback on CBL appears to be focused on the overall CBL experience rather than the content or quality of the teaching cases . The University of Ottawa used medical student perspectives of the positives and negatives of their cases to develop a detailed tool for creating and reviewing CBL cases, focused primarily on case structure and inclusion of factual information on pathophysiology, differential diagnosis, and treatment . As many medical schools move to integrate more clinical aspects into pre-clerkship curricula, student perspectives on effectiveness of CBL cases that include communication skills, ethics, professionalism, and person-centered care are important. Given the centrality of the cases to our curriculum, a case oversight team was established in 2018 to design and implement a case catalog quality improvement process, a key component of which was the collection and use of student feedback . We initially prioritized humanizing case patients, increasing case patient diversity, and updating diagnosis and treatment details. We also made the cases more complex and realistic with increased coverage of social determinants of health, mental health concerns, ethical and professionalism topics, and modeling of communication and patient education. Additionally, we increased coverage of primary care scenarios, supporting the mission of our institution to produce more primary care doctors for our state. We report here the thematic analysis of qualitative case feedback obtained from multiple cohorts of medical students to identify themes for high-quality CBL cases to improve learning and guide systematic improvements in a pre-clerkship case catalog. Themes for suggested improvement from both years included requests for more media, clarifying/adjusting lab information, and more details to increase complexity and realism. Year 2 students also requested clarification of treatment rationale and details. Example quotes illustrating the themes are provided in Table 1 . Fig. 2 Case aspects for improvement Table 1 Sample quotes illustrating case themes Case aspects that facilitated learning Sample student quotes Inclusion of high-quality media “The imaging was very helpful for understanding the progression of the disease.” “This was our first pediatrics case… and the video demonstrating breathing retractions on physical exam was helpful.” Complexity with rich details “Having two patients with different pathologies helped facilitate the learning. There are various types of brachial plexus injuries, and it was interesting to compare and contrast them. The multigenerational aspect was a nice aspect. It was realistic that Mr. Gonzalez may have sought medical help after seeing similar symptoms in his grandson.” Realism “The case… was relevant to recent events with Covid-19, had a more realistic feel, incorporated complexity of the patient's conditions (i.e., progression of complications), and was relevant to our position as medical students.” Curricular alignment “Having the case correlated directly to a lecture was helpful because it allowed time to think and ask questions outside of group. Having a unique dynamic with a family member that has issues as well was useful and accurate.” Incorporation of psychosocial/contextual factors “We appreciated the inclusion of social determinants of health which facilitated important discussion on behalf of the healthcare provider.” Foster broad differential “Made you consider a large differential… [and] realistic in terms of loss of follow up, [and] ethical/ prognostic factors of chemotherapy.” Curricular reinforcement “Great broad case that covered a lot of material from this week… and some from previous weeks. Great revisiting shock and ARDS. Having to think about the DDx when considering her occupation (nurse) and the etiology of [pneumonia].” Case aspects for improvement Inclusion of more media “Would be nice to see CXR from Wednesday… also an EKG of atrial fibrillation. More pictures please i.e., radiographs, pictures of clinical manifestations.” Clarification/adjustment of lab information “Panel of lab result values to interpret would be useful.” “Would have been nice to have lipase for pancreatic function.” Inclusion of more details to increase complexity and realism “Could be made more complicated by having Sjogrens.” “Could be more realistic in terms of the logistics/difficulties of her initial hospitalization.” The importance of realism, complexity, and media in curricular cases were themes in our study and are consistent with existing literature that has identified authenticity, realism, being “engaging and challenging” (i.e., complexity), and use of media as important components of effective case-based learning cases . Although person-centered, diverse, and inclusive attributes have been described as important aspects of CBL cases and case catalogs, it is unclear how commonly these factors are included in standard CBL cases . For our students, inclusion of psychosocial and contextual factors was another positive theme, especially in year 2. In addition, this study documented changes to student priorities as they progressed through the pre-clerkship curriculum with increased interest in differentials, psychosocial factors, treatment, and curricular reinforcement. Careful crafting of cases can provide students with complex and diverse scenarios in which to practice contextualizing pathophysiology and clinical management in the setting of realistic biopsychosocial circumstances. A strength of this study was analysis of extensive data including multiple years of free-text feedback on over 100 individual cases from students at different stages of pre-clerkship training. Another strength was the consistency between coders working with a robust code set to generate themes. The findings reported in this study, as well as the process for obtaining and incorporating feedback, may be applied to other health professions’ case-based curricula at various levels of training. To improve learning outcomes, it is important to understand which aspects of curricular cases are highly valued by medical students. This study has demonstrated that increased realism, complexity, and media were strong themes for our pre-clerkship learners. Based on the results of this study, educators could consider a review of cases in their curriculum to ensure they contain valued attributes and are aligned with the curriculum. Medical educators may also consider implementing a process for collecting student feedback to guide systematic case improvements and development of future cases, with an overall goal of promoting the growth of well-rounded physicians who are better prepared to deliver patient-centered care. | Review | biomedical | en | 0.999995 |
PMC11699192 | As the artificial intelligence industry continues to gain momentum, there is a rising trend of integrating virtual reality (VR) technology into diverse sectors, including medical education in the form of high fidelity training simulation . Studies show that virtual simulation-based training is effective in teaching procedural medical skills [ 2 – 9 ]. However, during VR experiences, some users may suffer from troublesome symptoms that are similar to motion sickness, termed virtual reality sickness. Motion sickness can potentially affect learning ability. When an individual experiences virtual reality sickness, they may feel nauseous, dizzy, and uncomfortable, which can lead to a range of cognitive and physical effects that may impact the ability to learn . People rely on different sensory organs to understand orientation and movement. The vestibular, visual, and proprioceptive senses are particularly important in forming a unified perception of self-motion in three-dimensional surroundings. Harmonious processing of all these sensory inputs results in effortless and accurate recognition of position and movement in space. Motion sickness can arise when there is a disparity between visual information and dynamic vestibular input, leading to sensory conflicts between the incoming signals about the current state. When the primary sensory input triggering motion sickness is the visual stimuli, the term visually induced motion sickness (VIMS) is used. The VIMS, depending on the situation, may be called gaming sickness, simulator sickness, cinerama sickness, or virtual reality sickness (VRS) . The VRS is described in the literature by a variety of terms, including cybersickness, simulator sickness, VIMS, and VR-induced symptoms. Prevalence of VRS during or post VR experience has been reported to be as high as 80% . Various factors, including attributes of the intervention itself (such as content and hardware utilized) and individual subjective characteristics, contribute to the determination of VRS symptom severity. According to prior studies, the factors that affect VRS can be classified into VR hardware (the weight, the comfortability, the user interface), the content of the VR simulation (technicality, exposure duration, visual simulation, locomotion), and human-related aspects (prior experiences, eye-related condition, screen time, age, gender) . One of the significant factors influencing VRS is the user’s familiarity with the head-mounted display (HMD) within the virtual environment and the quality of the simulation training content. Furthermore, in the context of emergency simulation training, various locomotion challenges are encountered. The implementation of appropriate locomotion techniques in VR is imperative to ensure a high level of immersion and a sense of presence while navigating and engaging with the virtual environment . In emergency situations, healthcare professionals are required to possess knowledge and skill concerning machine assembly, equipment operations, vital signs monitoring, communication, and a range of medical competencies. In emergency setting, time is critical and delays in responding or making decisions can have significant consequences in emergency situations. These issues can add more burden to the locomotion challenge they already possessed with virtual reality training. Consequently, visual acuity, spatial awareness, effective communication, and seamless coordination play essential roles and are indispensable for a comprehensive and high-quality training in virtual reality simulation experience for emergency . This study is a part of VR-TBI (Virtual Reality-Traumatic Brain Injury) and VR-LAST (Virtual Reality-Local Anaesthetic Systemic Toxicity) project. The project is developed by the Department of Anaesthesiology and Intensive Care, Faculty of Medicine, Universitas Indonesia to evaluate the use of VR technology in emergency simulation training. The purpose of this study is to determine the incidence of VRS in an emergency setting simulation-based training and the related factors, hence providing information and mitigation plan of the virtual reality-related side effects to enhance and optimize learning outcomes. This study is a prospective observational study with cross-sectional design that took place in April–June 2023. The target population of our study comprises individuals who voluntarily participate in virtual reality-based simulation training. The study populations, during the designated period, were confined to doctors and doctors in training affiliated with the Faculty of Medicine at the Universitas Indonesia. The Simulation Sickness Questionnaire (SSQ) is used to measure the VRS as the study outcome. It was first derived from the 1983 Motion Sickness Questionnaire (MSQ) that was originally used to evaluate motion sickness in various forms of transportation. In 1993, Kennedy et al. reported the SSQ for the first time, proposing 16 items derived from MSQ that was divided into three categories: nausea, oculomotor, and disorientation (Table 1 ) . Table 1 Symptoms in SSQ SSQ items Nausea Oculomotor Disorientation General discomfort 1 1 Fatigue 1 Headache 1 Eyestrain 1 Difficulty focusing 1 1 Increased salivation 1 Sweating 1 Nausea 1 1 Difficulty concentrating 1 1 Fullness of head 1 Blurred vision 1 1 Dizzy (eyes open) 1 Dizzy (eyes closed) 1 Vertigo 1 Stomach awareness 1 Burping 1 Total (1) (2) (3) SSQ Simulation Sickness Questionnaire Formulas for scoring each component of the SSQ were proposed as Table 2 . It is possible to obtain four SSQ scores: nausea, oculomotor, disorientation, and total. The score of each category is defined as the sum of its symptom scores multiplied by a constant scaling factor. Based on a large sample of SSQ data gathered from military pilots, it is suggested that total scores can be associated with negligible (<5), minimal [ 5 – 10 ], significant [ 10 – 15 ], and concerning [ 15 – 20 ], and bad (>20) symptoms . Table 2 Computation of SSQ scores SSQ components Computation Nausea (1) × 9.54 Oculomotor (2) ×7.58 Disorientation (3) × 13.92 Total [(1) + (2) + (3)] × 3.74 SSQ Simulation Sickness Questionnaire There were two training scenarios provided within this study: the traumatic brain injury and the local anaesthetic systemic toxicity . Each participant can choose one scenario to enrol based on their preference. The traumatic brain injury (TBI) scenario consists of three settings; prehospital setting, ambulance setting, and emergency room setting. The player took role as doctor who treat the emergency patient. If the sequence is properly followed and player does not encounter problems, the TBI scenario can be completed in 20–30 min. The local anaesthetic systemic toxicity (LAST) scenario consists of 15–20 min single setting scene in the operating room. The players take role as resident doctors treating patient who experienced seizure after epidural administration. Each simulation is structured with checklists of the tasks players need to accomplish by the end of the simulation. Fig. 1 The scenario inside VR-TBI (left) and VR-LAST (right) The study involved 75 students who participated voluntarily. Table 3 shows the baseline characteristics of participants. The majority of the participants had no prior experience with VR. During the study, several factors were observed that could influence the VRS among the participants. These factors included a history of vertigo (1.3%), myopia (34.7%), astigmatism (14.7%), wearing glasses (29.3%), and screen time. The median screen time among participants is 10 h. Additionally, another factor associated with VRS was the type of VR mode used. Among the voluntary participants, roughly equal proportions of them used room scale (52%) and stationary (48%) VR mode. There were 57.3% participants participating in TBI scenario, and the rest 42.7% participated in LAST scenario. Table 3 Baseline characteristics Characteristics Frequency (percentage)/median (Min-Max) Gender Female 37 (49.3) Male 38 (50.7) Occupation Resident 32 (42.7) General practitioner 12 (16) Medical student 31 (41.3) VR experience Never 61 (81.3) Once 9 (12) More than once 5 (6.7) Age * 27 (20–32) *Data is presents as median (min-max) Out of 75 participants, the mean score for nausea, oculomotor, disorientation, and total score is 5.97 (SD 6.4), 6.26 (SD 6.5), 125.6 (SD 132), and 9.03 (SD 9.5) respectively. The SSQ score can be classified into 5 categories based on the severity, and it is shown in Fig. 5 . Fig. 5 Sickness severity on each SSQ category Of the observed factors, we tried to measure the contribution of the factors to the VRS with logistic regression. The OR has been calculated as adjusted OR while controlling for one or more additional variables that could confound the results. This adjustment is done to isolate the effect of the primary variable of interest. Adjusted OR of these factors, gender, astigmatism, and VR mode are associated with VRS, as can be seen in Table 4 . Table 4 Virtual reality sickness factors Factor VRS p value Adjusted OR * (CI95%) Negative Positive Gender Female 22 15 0.028 2.82 (1.1–7.2) Male 13 25 Vertigo history No 32 42 1 - # Yes 0 2 Myopia No 25 24 0.045 2.8 (1.1–7.9) Yes 7 19 Astigmatism No 32 32 0.002 - # Yes 0 11 Wearing glasses No 26 27 0.082 2.56 (0.87–7.57) Yes 6 16 VR mode Room scale 24 15 0.001 5.6 (2.02–15.47) * Stationary 8 28 Previous VR experience Never 28 33 0.782 0.848 (0.26–2.71) At least once 7 7 Screen time (hour) 10.38 10.16 0.8 0.21 (−1.06–1.48) + *Adjusted OR from logistic regression # OR cannot be calculated due to one cell with 0 number + Mean difference (standard deviation) In this study, we observed that virtual reality sickness (VRS) affected 57.3% of the 75 participants . This number aligns with findings from previous studies, which reported an incidence rate between 60 and 70% [ 13 , 18 – 21 ]. The mean SSQ score in our study was 9 with a range of 0 to 29, indicating lower score than previous study by Saredakis et al. which reported a pooled mean of 28 with a range of 14.30 to 35.27 . Among the SSQ components, disorientation scored the highest at 125.6, followed by oculomotor at 6.36, and nausea at 5.97 . These results are consistent with previous research reporting pooled mean SSQ scores of 16.72, 17.09, and 23.5 for nausea, oculomotor, and disorientation, respectively . According to Kenendy et al., total SSQ score of 9 indicates minimal symptoms . Although majority of the participants in our study experienced negligible to significant symptoms, there were 16% of them that experienced severe symptoms, and further mitigation plan should be prompted. In the context of emergency setting simulations, healthcare professionals are mandated to possess not just the knowledge base, but a comprehensive technical skill set. This proficiency encompasses the continuous monitoring of hemodynamic changes subsequent to specific medical procedures like looking at the monitor over and over after performing a medical procedure. This well involved repetitive rotational movement of the neck. Other technical skill healthcare professionals need to understand are the assembly and operation of medical equipment, effective communication, and a diverse array of medical competencies, such as airway management, resuscitation, and the precise administration of pharmaceuticals. In an emergency scenario, temporal considerations are of paramount importance, as any delays in response time or decision-making may yield profound and potentially life-altering consequences. Moreover, the demanding nature of emergency setting simulations, characterized by intricate and occasionally repetitive actions, can engender VRS. The disorientation component of the SSQ, encompassing symptoms such as difficulty focusing, nausea, fullness of the head, blurred vision, dizziness with eyes open or closed, and vertigo, exhibited a high occurrence of bad scores (>20) in 43 participants (57.3%) as seen in Fig. 5 . Our research findings have illuminated that VRS, particularly of a severe degree, predominantly falls within the disorientation category. This observation implies that the performance of users engaged in simulation exercises may be disrupted and hindered, preventing them from effectively showcasing their genuine competencies during training simulations. This can further impact learning process. Kelly et al. discovered that virtual reality users are particularly susceptible to disorientation, especially when using locomotion interfaces lacking self-motion cues. To address this issue, incorporating environmental cues, such as boundaries, into the design of locomotion interfaces is crucial to reduce disorientation-related effects . Our study found that hardware-related issues, specifically the VR mode, were associated with VRS. Participants using the stationary mode were five times more likely to experience VRS. This is in line with previous research indicating that nonstationary VR modes, especially physically walking mode, can help reduce the incidence of VR sickness . Chang et al. classified VRS factors into three domains: hardware, content, and human factors. Hardware factors encompassed various VR device manipulations, including display type, display mode, time delay, and device weight. Content factors involved altering VR scenes or scenarios, such as graphics, task-related features, duration, and controllability. Human factors considered individual differences, such as age, gender, BMI, postural sway, previous VR experience, and eye-related measures like interpupillary distance, refractive error, and eye-hand coordination . In Saredakis et al.’s study, the mean SSQ scores for nausea, oculomotor, and disorientation were 22.6, 22.4, and 28.5, respectively, in the stationary group, whereas in the walking group, they were 13.2, 15.3, and 18.5, respectively . Our findings revealed that male are more prone to VRS. This is the opposite of previous studies. Schmitz et al. found gender as an essential variable associated with motion sickness in VR systems . Males were 2.8 times more likely to experience VRS than females. However, a large-scale meta-analysis reported no significant gender difference in VRS . Former VR experience was believed to increase the likelihood of VRS . Surprisingly, our study did not find any significant effect of previous VR experience on VRS. Further research is needed to fully comprehend the underlying pathophysiology of this phenomenon . Young et al. discovered that ocular refraction disorders influenced motion sickness during head-mounted display experiences. Myopic and astigmatic participants showed significantly higher SSQ scores for nausea and disorientation. Our findings are aligned with these results, as we observed a higher incidence of VRS in participants with myopia and astigmatism. This study has several limitations. Firstly, its cross-sectional design makes it difficult to establish causal relationships between factors. It would be better if further research could address the dose-response relationship between the amount of time spent in VR-based learning and development of fatigue. Conducting further research using cohort or randomized trials would be more suitable for addressing this issue. Secondly, the consecutive sampling method used may introduce bias as it is a nonprobability sampling technique. Thirdly, the lack of pre-treatment SSQ scores before participants engaged in the VR experience could lead to additional biases in interpreting the results. Based on the current report, we recommend using the room-scale mode as the locomotion interface for VR to minimize VRS. Additionally, participants with refractive disorders should receive proper treatment by wearing glasses for refractive correction before engaging in VR activities. These strategies are essential to ensure all participants have the best possible environment to enhance learning outcomes in VR-based simulation training for traumatic brain injury management. The incidence of virtual reality sickness was associated with male gender, myopia, astigmatism, and the use of stationary VR mode. To prevent virtual reality sickness during VR simulation training, it is essential to engage in VR activities in room-scale or walking mode and ensure appropriate correction of any refractive disorders. Future research should address the dose-response relationship between the amount of time spent in VR-based learning and development of virtual reality sickness. | Study | biomedical | en | 0.999997 |
PMC11699197 | The integration of digital health tools into healthcare has transformed how individuals manage their health, providing opportunities for improved quality of life (QoL) and psychological adjustment. These tools encompass mobile health applications, wearable devices, and telemedicine platforms that enable users to monitor health metrics, access personalized feedback, and engage in health-promoting behaviors . While much of the focus has been on managing chronic conditions such as diabetes and hypertension , there is increasing recognition of their potential to address the unique challenges faced by specific populations, including long-term blood donors. Blood donation, a critical public health activity, relies heavily on the sustained contributions of long-term donors. Long-term blood donors, defined in this study as individuals who donate blood at least twice per year for a minimum of three years, play a crucial role in maintaining a stable and reliable blood supply. These donors are often highly committed to the practice, driven by altruism and a sense of social responsibility. However, these individuals often face both physical challenges, such as fatigue, iron depletion, and dizziness , and psychological concerns, including stress, anxiety, and societal perceptions about repeated donations . These challenges can disrupt social relationships, impact emotional well-being, and ultimately affect donors' long-term commitment . Within Saudi Arabia, maintaining a stable blood supply is essential, yet achieving this goal is complicated by a geographically dispersed population, varying health literacy levels, and cultural perceptions around blood donation . Digital health tools offer a promising avenue for addressing these issues by improving donor engagement, providing health monitoring, and supporting psychological well-being. While studies have highlighted the benefits of digital platforms in healthcare, there is a lack of localized research exploring their application among Saudi blood donors. This study aims to fill this gap by examining how digital health tools can improve both the QoL and psychological adjustment of long-term donors in Saudi Arabia, emphasizing the cultural and healthcare system context. Research demonstrates the effectiveness of digital health tools in managing chronic health conditions. For example, Debon et al. showed that mobile applications for individuals with hypertension enhanced their ability to monitor blood pressure and make informed decisions, contributing to improved QoL . Similarly, wearable devices tracking physical activity, heart rate, and sleep patterns have been linked to better disease management and psychological outcomes in individuals with chronic conditions . These tools enable real-time monitoring, personalized feedback, and tailored interventions, making healthcare more accessible and proactive. In contrast, research on the use of digital health tools specifically for blood donors remains limited. Although some parallels exist between the needs of long-term donors and individuals with chronic conditions such as the necessity for ongoing health monitoring and strategies for psychological support, the challenges faced by donors are unique . Psychological stressors, including anxiety about health risks and societal stigmas, can discourage continued participation in blood donation programs . Long-term donors must navigate these barriers to sustain their commitment, which makes targeted interventions essential . Studies have acknowledged the potential role of digital tools in supporting blood donors. For instance, gamified mobile applications have been used to educate donors, improve physical health outcomes, and foster engagement . Telemedicine platforms offering remote consultations have provided continuous support, enhancing adherence to donation schedules and overall well-being . However, these efforts predominantly focus on physical health and do not adequately address psychological adjustment or QoL, particularly within specific cultural contexts. This study aims to investigate the impact of digital health tools in enhancing the QoL and psychological adjustment of long-term blood donors in Saudi Arabia. The research will focus on the development and application of culturally relevant digital interventions, assessing their effectiveness in mitigating physical and emotional challenges and supporting sustained donor participation. An online cross-sectional survey design was adopted in this study conducted in Saudi Arabia, targeting long-term blood donors who have participated in at least 10 donation events over the past five years at blood banks and healthcare facilities. The study encompassed both urban and rural settings to capture the variability in access to healthcare and digital tools, as well as differing cultural attitudes toward blood donation. Participants were selected from key regions, including Riyadh, Jeddah, and the Eastern Province, representing diverse socioeconomic backgrounds, levels of health literacy, and technological adoption. This approach ensured the representation of donors with varying experiences in terms of healthcare access, physical and psychological challenges, and their use of digital health tools, thereby providing a comprehensive understanding of the long-term blood donor population in Saudi Arabia. The inclusion criteria for participants were: individuals aged 18-60 years, who have been regular blood donors (donating at least twice per year) for at least 3 years, and who voluntarily agreed to participate in the study. The age range of 18-60 years was selected to align with international blood donation guidelines, which typically recommend eligibility between these ages for ensuring donor safety and adequate physical capacity for donation . Additionally, this range encompasses the majority of active donors, as older populations often face age-related health issues that may limit donation frequency . Exclusion criteria included individuals with physical or mental health conditions that could interfere with their ability to complete the survey or participate in the digital health tools usage assessment. This exclusion was based on ethical considerations to avoid placing additional burdens on individuals whose health conditions might limit their ability to engage with the study interventions or skew results due to unrelated health factors. Similar studies on digital health tools emphasize the importance of excluding individuals with confounding health conditions to maintain study validity and participant safety . The sampling strategy employed in this study was purposive , aiming to recruit long-term blood donors with extensive blood donation experience, defined as individuals who have donated regularly for at least three years. Purposive sampling was chosen due to the need to focus on a specific subgroup of blood donors who could provide in-depth insights into the physical, psychological, and behavioral aspects of long-term blood donation. This approach allows researchers to ensure that the participants align closely with the study’s objectives, which require experience with repeated donation and potential interactions with digital health tools. While purposive sampling has the potential to introduce selection bias, it is particularly suitable for exploratory research where the goal is to generate insights from a targeted population rather than to achieve statistical generalizability . By selecting participants across diverse demographics, including age, gender, and geographic location (urban and rural regions within Saudi Arabia), the study aimed to mitigate this limitation and ensure the representation of a wide range of donor experiences. This approach is supported by previous studies in healthcare research, where purposive sampling has been successfully employed to explore the nuanced experiences of specific populations . Participants were selected from blood donation centers in key urban and rural areas, ensuring the sample's diversity and representativeness. Recruitment took place in collaboration with local hospitals, blood banks, and healthcare providers, who assisted in reaching potential participants. Additionally, healthcare professionals at the donation centers provided verbal invitations to eligible donors during their routine visits. Interested individuals were then directed to the study's online platform or to on-site recruitment staff for further details about participation. The target sample size is approximately 500 participants (based on an estimated sample of 383 calculated using Cochran’s formula ), distributed proportionally across the selected regions. This sample size is designed to provide sufficient statistical power for analyzing variations in public perceptions, behaviors, and attitudes toward air quality and respiratory health across different demographic and environmental contexts in Saudi Arabia. To measure the quality of life and psychological adjustment of the participants, two validated instruments were employed: the WHO Quality of Life-Brief Version (WHOQOL-BREF) and the Coping Orientation to Problems Experienced Inventory (Brief-COPE) inventory . WHOQOL-BREF is a widely recognized instrument developed by the WHO to assess the QoL across multiple dimensions. It consists of 26 items that measure four domains: physical health, psychological well-being, social relationships, and environmental factors. The WHOQOL-BREF is particularly suitable for this study as it provides a comprehensive evaluation of how digital health tools may impact various aspects of the participants’ lives, from their physical health and psychological state to their social interactions and environmental satisfaction. Questions are rated on a five-point Likert scale, with higher scores indicating better quality of life. This instrument's broad scope makes it ideal for capturing the multifaceted effects of digital health tools on long-term blood donors. The Brief COPE scale is a validated tool used to assess how individuals cope with stress and challenges in their lives. It is a shortened version of the COPE inventory and includes 28 items that measure 14 different coping strategies, such as active coping, planning, positive reframing, and seeking social support. The Brief COPE is particularly relevant for this study as it allows researchers to evaluate the coping mechanisms employed by long-term blood donors when managing the psychological demands associated with regular blood donation. By analyzing the use of digital health tools in relation to these coping strategies, the study aims to determine whether these tools facilitate more effective coping and contribute to better psychological adjustment. These tools were adapted to suit the cultural and linguistic context of Saudi Arabia. The adaptation process included translation into Arabic using a forward-translation and back-translation method to ensure semantic equivalence, as recommended for cross-cultural research . Additionally, a panel of bilingual experts in psychology and public health reviewed the scale for cultural relevance and clarity, making adjustments to items that may not align with local norms or experiences. For example, coping strategies involving social support were contextualized to reflect the importance of family and community in Saudi culture. A pilot test was conducted with a small sample of Saudi blood donors (n = 30) to assess the tool's reliability and validity in the target population. The data collected from this pilot study were analyzed, and Cronbach's alpha coefficient for all items was calculated. The coefficient exceeded 0.7, indicating robust internal consistency and reliability of the questionnaire . Feedback from the pilot study was used to refine the language and ensure the scale accurately captured the coping mechanisms relevant to this group. A total of 523 participants were included in the study; however, 25 participants did not complete the full survey, leading to a final sample of 498 participants. As shown in Table 1 , the demographic data revealed a balanced gender distribution among participants, with a slight predominance of females (50.8%) over males (49.2%). The age distribution highlighted a younger sample, with 34.7% of participants aged 18-30, followed by 22.5% in the 31-40 age group. Middle-aged groups (41-50 years and 51-60 years) represented a combined 34.2% of the sample, while only 8.6% of participants were over 60, suggesting that the findings might be more relevant for younger and middle-aged demographics. Education levels showed diverse representation: participants with a diploma were the largest group (27.3%), followed by those with primary or secondary education (20.7%). Bachelor's degree holders constituted 20.3%, while those with a Master’s degree or higher represented 17.1%, and uneducated participants made up 14.7%. This range across educational levels may have contributed to varied perspectives within the sample, supporting generalizability across different educational backgrounds. Table 2 provides an overview of participants' perceptions regarding the impact of digital health tools on their QoL and psychological adjustment. Across all factors, the mean scores generally hovered around 3.25-3.36, suggesting a moderate to positive perception of these tools. Self-distraction and denial received the highest mean scores ± SD (3.36 ± 0.97 and 3.33 ± 0.98, respectively), indicating that participants perceive these tools as particularly helpful in managing stress or emotional challenges. On the other hand, substance use and venting had the lowest mean scores (3.21 and 3.24, respectively), suggesting that while digital health tools were still perceived positively, they may have less of an impact on managing these specific behaviors. Active coping and positive reframing scored relatively high as well, suggesting that participants viewed digital tools as useful in fostering adaptive coping strategies. Regarding psychological adjustment, participants rated physical health and psychological well-being highly (3.30 ± 1.04 and 3.31 ± 0.95, respectively), signifying the positive role of digital health tools in improving overall health and mental well-being. Social relationships and environmental factors were also perceived positively, with mean scores of 3.27 ± 0.88 and 3.29 ± 0.80, respectively, indicating that participants felt these tools support not only individual well-being but also social and environmental contexts. Overall, the data suggests a broadly favorable perception of digital health tools in enhancing both quality of life and psychological adjustment, with some factors, such as denial and self-distraction, standing out as particularly beneficial. The one-way ANOVA of the data (Table 3 ) revealed meaningful insights into how long-term blood donors' perceptions of digital health tools vary across different age groups. The significant differences observed suggest that older participants tend to perceive digital health tools as more beneficial for their psychological adjustment and QoL compared to younger donors. This may be attributed to a variety of factors, such as the greater experience and possibly more pronounced health concerns among older individuals, which could make them more receptive to health interventions. Conversely, younger blood donors may have different health-related priorities or expectations, potentially explaining their lower ratings across most variables. The one-way ANOVA of the data (Table 4 ) highlighted notable trends regarding the impact of educational attainment on long-term blood donors' perceptions of digital health tools in enhancing their QoL and psychological adjustment. Statistically significant differences were observed across various educational groups, with participants possessing higher levels of education consistently reporting more favorable perceptions. For instance, those with a Bachelor’s degree or higher demonstrated higher mean scores in most factors, such as active coping, use of emotional support, self-blame, physical health, and psychological well-being, suggesting a stronger positive response to digital health tools compared to those with lower educational levels. In contrast, participants with lower levels of education, such as uneducated or with primary/secondary education, tended to report the lowest scores across the majority of measures. The t-test results (Table 5 ) revealed significant gender-based differences in the perceptions of long-term blood donors regarding the impact of digital health tools on their QoL and psychological adjustment. Across all factors, female individuals reported consistently higher mean scores compared to males, indicating that women perceive digital health tools as more beneficial in enhancing their QoL and psychological well-being. For example, women scored notably higher in factors like active coping, denial, substance use, use of emotional support, and self-blame, reflecting a more positive engagement with these tools. The results of this study reveal a generally positive perception among long-term blood donors in Saudi Arabia regarding the impact of digital health tools on their QoL and psychological adjustment. These findings align with previous research, which indicates that digital health tools such as mobile apps, wearable devices, and telemedicine can enhance users' well-being and provide vital support for chronic health management . Specifically, our findings show that participants value these tools for stress management, health monitoring, and emotional support, similar to what other studies have observed in chronic disease management contexts . The objective of this study, which was to investigate the impact of digital health tools in enhancing the QoL and psychological adjustment of long-term blood donors in Saudi Arabia, was assessed from different contexts. Age-based differences in perceptions emerged, with older participants perceiving digital health tools as more beneficial than younger donors. This result could be linked to a greater reliance on health support and a heightened awareness of health-related issues in older populations, as observed in the literature . Furthermore, younger blood donors may have different expectations from digital health tools, potentially finding them less useful for their specific needs. This finding suggests the need for age-tailored digital health interventions to maximize engagement and relevance across age groups, an approach recommended by similar studies addressing chronic health conditions . Tailoring these tools to better suit the needs and preferences of younger blood donors might help bridge the gap and increase the overall effectiveness of digital health interventions. Furthermore, the significant statistical differences underscored the importance of considering age as a key factor in designing and evaluating digital health solutions, as the response to such tools may not be uniform across different demographic groups. This could inform future strategies for improving the psychological well-being and coping mechanisms of long-term blood donors through targeted digital health initiatives. Gender differences in the perception of digital health tools were also observed, with female participants consistently reporting higher perceived benefits in terms of emotional and psychological support. This aligns with prior research showing that women may be more likely to engage with health management tools for emotional support and stress management than men . This highlights the importance of gender-sensitive design in digital health tools, potentially increasing their appeal and impact by addressing specific user needs more effectively. Although digital health tools are often perceived as universally beneficial, our findings suggest that factors such as age, gender, and educational background significantly influence user perceptions and experiences. This aligns with the study by Al-Hajri et al., which emphasized the need for culturally and demographically relevant adaptations of digital health tools for diverse populations . In the present study, the gender gap was particularly pronounced in self-distraction, behavioral disengagement, venting, and humor, with females again reporting higher mean scores, suggesting that women may experience greater emotional benefits or engagement with coping mechanisms facilitated by digital health interventions. On the other hand, male individuals tended to score lower on these factors, which could indicate differences in coping styles or willingness to use digital tools for emotional support. The results suggest that gender may influence how digital health tools are perceived and utilized, with women perhaps benefiting more in terms of emotional support and psychological adjustment. This could imply that digital health tools need to be adapted or tailored to account for these gender differences, potentially offering more targeted interventions for males to increase engagement and psychological benefit. Educational differences further underscore the need for tailored interventions. Participants with higher educational levels reported greater benefits from digital health tools, perhaps reflecting better digital literacy and health awareness, as has been suggested in previous studies on health tool usability . Participants with lower education levels might face barriers related to technology use or may be less aware of potential benefits, emphasizing the importance of user-friendly design and accessible health information for all educational levels. The findings from the present study suggest that education may play a significant role in how individuals perceive and utilize digital health interventions, possibly due to differences in digital literacy, health awareness, or the ability to engage with more complex health-related information. For example, those with a Master’s degree or higher consistently showed the highest mean scores in self-blame, substance use, and venting, which may reflect a better understanding of psychological health and coping mechanisms. The study also highlights the limitations of current digital health tools in addressing the specific psychological needs of long-term blood donors. While tools for general stress management were beneficial, aspects such as substance use and behavioral disengagement were perceived as less impacted. This finding reflects gaps identified in the reviews by López et al. and Lattie et al. , which noted that many existing digital tools inadequately address complex psychological needs or fail to provide personalized interventions for specific behavioral challenges. Addressing this gap could involve incorporating evidence-based psychological strategies within digital platforms, such as cognitive-behavioral components or motivational modules tailored to donor-specific stressors. This study has several limitations. First, the cross-sectional design restricts causal inferences regarding the impact of digital health tools on long-term blood donors' QoL and psychological adjustment. Additionally, reliance on self-reported data may introduce response bias, as participants' perceptions might not fully reflect the objective impact of these tools. The focus on Saudi Arabia limits the generalizability of the findings, as cultural factors, such as attitudes toward health technology and gender dynamics in healthcare usage, may have influenced participants' experiences. Moreover, the non-random purposive sampling limits generalizability. Future research should consider using a random sample and include diverse populations to enhance external validity. Longitudinal studies are needed to establish causal relationships, and further exploration of specific digital features and culturally tailored interventions could provide more targeted insights for improving donor retention and well-being. The findings of this study contribute to the theoretical understanding of digital health’s role in enhancing QoL and psychological adjustment, extending previous research primarily focused on chronic disease management to the unique context of long-term blood donors. The results underscore the need to consider demographic factors such as age, gender, and education level in digital health research, as these factors significantly influence user engagement and perception of benefits. Practically, these insights suggest that healthcare providers and developers should prioritize the customization of digital health tools to better meet the diverse needs of long-term donors. Tailoring these tools to address specific psychological stressors, coping mechanisms, and cultural considerations can potentially improve user experience, engagement, and donor retention, supporting a more resilient and sustainable blood donation system. This study underscores the positive role of digital health tools in enhancing the QoL and psychological adjustment of long-term blood donors in Saudi Arabia. Findings indicate that these tools are perceived as beneficial for managing physical and psychological challenges, especially among older and more educated donors, highlighting the influence of demographic factors on engagement. The findings of the study indicate that digital tools can play a significant role in facilitating coping mechanisms, promoting emotional well-being, and encouraging donor retention. However, it is important to exercise caution in making causal claims due to the cross-sectional design, which limits the ability to establish temporal relationships. While the data provide evidence for the effectiveness of digital health tools, areas such as the role of substance use and behavioral disengagement in coping strategies require further exploration. These factors may influence how donors engage with digital tools and should be studied in greater depth to understand their impact fully. Similarly, the influence of demographic variables such as age, gender, and socioeconomic status on the use and effectiveness of digital health tools was not exhaustively analyzed, leaving room for further investigation. Future research should focus on these dimensions to strengthen the link between the study’s results and its conclusions. Longitudinal studies examining the interplay between coping mechanisms, digital engagement, and demographic factors could provide more robust insights, allowing for tailored interventions to optimize the benefits of digital health tools for diverse donor populations. | Review | biomedical | en | 0.999997 |
PMC11699213 | Several studies have investigated the sociodemographic characteristics of women who are at higher risk of not participating in breast cancer screening (BCS) programmes. In the most recent international systematic review, Mottram et al. observed that migrant women, women with lower socioeconomic status, without home ownership, and those who experienced false positives had the lowest attendance rates . In a scoping review of the German context, Pedrós Barnils et al. identified native women, women with lower incomes, women living in rural areas, and those not cohabiting with their partners as those with the lowest lifetime BCS attendance rates. However, the author also highlighted considerable heterogeneity in methods and, therefore, results . Usually, inequalities in attendance are documented based on independent social dimensions. To explore correlations between social dimensions and BCS attendance, most studies incorporate variables deemed relevant (i.e. based on specific assumptions) into statistical models and then, in multivariate analyses, estimate the independent effect of each social dimension with the effects of other covariates held constant. However, as no individual can be defined by a single social dimension alone , it is unlikely that examining the independent effect of each social dimension will provide a comprehensive understanding of the inequalities in accessing cancer screenings. Over the past two decades, in the field of population health, quantitative intersectionality has given rise to new methodological approaches. The most commonly used methods for describing intersectional inequalities within a population range from simple cross-classification descriptions or regressions to methods that account for discriminatory accuracy (e.g. analysis of individual heterogeneity and discriminatory accuracy (AIHDA) and multilevel analysis of individual heterogeneity and discriminatory accuracy (MAIHDA)) or are data-driven (e.g. decision trees) . To build cross-classification regression and AIHDA or MAIHDA, the (potentially) relevant social dimensions are usually selected on the basis of the available evidence, and these dimensions are combined to identify intersectional subgroups. This is a deductive approach. In contrast, decision trees and analogous heuristic procedures employ an inductive methodology to identify which variables are most predictive of an outcome assuming non-linear relationships between the variables . This enables a data-driven determination of the social dimensions that will constitute intersectional subgroups, often previously unnoticed . Decision trees have been applied as statistical exploratory tools for classification in population health . To the author's knowledge, no explicit comparisons between these approaches to identify intersectional inequalities in breast cancer screening have been conducted; besides, no study has employed an intersectional approach for reporting inequalities in breast cancer screening in Germany. Consequently, the present study aims to identify intersectional groups of women aged 50–69 who are at higher risk of never attending BCS in Germany comparing two analytical strategies: a) evidence-informed regression and b) decision tree-based regression. The primary outcome of this study was self-reported breast cancer screening attendance via mammography at least once in a lifetime for women aged 50–69 in Germany. Responses were dichotomised, excluding those who indicated “unknown” or left the question unanswered to prevent uncertainty about whether the respondent reported never attending BCS (no = 0, yes = 1). Descriptive analytics, including frequencies and percentages, were calculated for all variables. A complete case analysis was conducted, i.e. cases with missing data were excluded listwise. The total sample was restricted to women aged 50–69 . Among these women, those who did not respond on whether they underwent mammography (n = 15), their place of residence (n = 384), the degree of urbanisation of their place of residence (n = 213), the household's income (n = 122), their level of education (n = 14), their social network dimensions (n = 11), their perceived social support (n = 46), the available help (n = 81), the type of household (n = 56), their marital status (n = 13), their partnership cohabitation status (n = 30), their working situation (n = 10), their country of origin (n = 11), their citizenship (n = 7), their GALI (n = 7) were excluded. Hence, the final total sample size of the study was 4761 participants. Sampling weights were not used in analyses, as the sampling weights provided in the German EHIS data were derived from variables included in the analyses (education, urbanisation and age), which could lead to multicollinearity and biased standard error estimation. We report sensitivity analyses applying the sampling weights in both analytical strategies in Appendix A and show the correlations between sampling weights and variables in the analyses in Appendix B . The central aim of this article was to compare the estimation of women at higher risk of never attending BCS using two different analytical strategies: (a) evidence-informed regression and (b) decision tree-based regression. For the purpose of comparison, univariate models were initially constructed for each of the four individual predictors and age. Next, a multivariable model that included all main effects was estimated. Following this, a multivariate logistic regression with the full cross-classification matrix as the main predictor was performed to estimate the odds ratio (OR) of never attending BCS adjusted by age. Discriminatory accuracy (DA) was estimated through the area under the receiver operating characteristics curve (AUC) with a 95% confidence interval (CI), indicating how well each model discriminates between women attending and women never attending BCS. DA is considered absent or very small when 0.5 ≤AUC≤ 0.6, moderate when 0.6< AUC ≤0.7, large when 0.7< AUC ≤0.8 and very large AUC>0.8 . These statistical procedures were carried out using Stata version 17.0. The second analytical strategy consisted of two steps. First, building an explorative decision tree with the total sample size to identify homogeneous subgroups of women at higher risk of never attending BCS in Germany. Second, performing a multivariate logistic regression using the outcome of the decision tree adjusted by age to estimate the OR of never attending BCS. There is no consensus on which decision tree better operates on binary outcomes. In this study, we trained three different algorithms: Classification and Regression Tree (CART), Conditional Inference Tree (CIT) and C5.0. The CART algorithm makes splitting decisions based on the lowest gini impurity (or entropy) coefficient among all potential splits (i.e. every category or step of every variable) . CART does not provide statistical significance measures and potentially overestimates the influence of variables with many categories. CIT addresses these limitations by utilising a formal statistical hypothesis in growing decision trees and mitigating variable selection bias by splitting the selection process into two steps . C5.0 uses the entropy coefficient of the imputed variables to generate splits plus adaptative boosting and winnowing . All three decision tree algorithms (CART, CIT, C5.0) were built using the entire dataset and the same subdivision of the data when performing cross-validation. Cost weights were applied to distribute the sums of weights equally for cases and non-cases, given the (relative) rareness of the outcome in the dataset (10.38% prevalence). Parameters were hypertuned and optimised by two performance measures: sensitivity (i.e. enhancing detection of positive cases) and the Area Under the Precision-Recall Curve (i.e. improving overall precision-recall performance for unbalanced datasets) . Decision trees were grown using the tune function from the “mlr3tuning” optimisation R packages in R version 4.4.0. This package integrates essential packages for building CART “rpart” , CIT “partykit” , and C5.0 “C50” . After inductively identifying the best-performing decision tree, the final nodes were deductively used as predictors for a multivariate logistic regression adjusted by age, where the ORs and DA of the model were estimated. This statistical procedure was performed using the Stata version 17.0. Estimations, performance and interpretability of both analytical strategies were compared and discussed. Summary descriptive statistics of the sample can be found in Table 2 . The total sample size is 4761. Of those, 4267 attended BCS at least once in their lifetime, and 494 did not. Relative frequencies for never attending BCS among the different PROGRESS-Plus characteristics were assessed. As expected, women aged 65–69 had the lowest prevalence (6.55%), and women aged 50–54, had the highest prevalence (18.58%). For SES, women in the lowest quintile attended BCS the least (14.07%), and those in the highest quintile attended BCS the most (9.79%). Almost contradicting, women with the highest education attainment, doctoral or equivalent, attended BCS the least (14.75%) and women with bachelor or equivalent educational attainment the most (9.10%). Based on the country of origin, women born in Germany (10.56%) had the lowest attendance rates compared to women born elsewhere. However, women of another European nationality attended the least (12.05%) and women of German nationality the most (10.31%). Regarding the place of residence, women living in cities (11.16%), women living in Berlin (13.69%) and Saarland (13.39%) had the lowest BCS attendance rate. Table 2 Descriptive PROGRESS-Plus characteristics on BCS attendance among targeted women in Germany. Relative frequencies per column and variable are displayed. Table 2 Attended BCS Never attended BCS (N = 494) Total Age 50-54 916 (21.5%) 209 (42.3%) 1125 (23.6%) 55-59 1149 (26.9%) 116 (23.5%) 1265 (26.6%) 60-64 1104 (25.9%) 92 (18.6%) 1196 (25.1%) 65-69 1098 (25.7%) 77 (15.6%) 1175 (24.7%) Income 1Q 458 (10.7%) 75 (15.2%) 533 (11.2%) 2Q 687 (16.1%) 76 (15.4%) 763 (16.0%) 3Q 795 (18.6%) 89 (18.0%) 884 (18.6%) 4Q 1010 (23.7%) 111 (22.5%) 1121 (23.5%) 5Q 1317 (30.9%) 143 (28.9%) 1460 (30.7%) Educational group Lower secondary or lower 219 (5.1%) 28 (5.7%) 247 (5.2%) Upper secondary 1515 (35.5%) 170 (34.4%) 1685 (35.4%) Post-secondary 609 (14.3%) 78 (15.8%) 687 (14.4%) Bachelor or equivalent 1120 (26.2%) 112 (22.7%) 1232 (25.9%) Master or higher a 804 (18.8%) 106 (21.4%) 900 (19.2%) Country of origin Germany 3948 (92.5%) 466 (94.3%) 4414 (92.7%) Outside of Germany a 319 (7.5%) (28) b (5.6%) 347 (7.3%) Citizenship German or other a 4267 (100%) 494 (100%) 4761 (100%) Degree of urbanisation City 1712 (40.1%) 215 (43.5%) 1927 (40.5%) Town or suburb 1803 (42.3%) 210 (42.5%) 2013 (42.3%) Rural area 752 (17.6%) 69 (14.0%) 821 (17.2%) Region Baden-Württemberg 459 (10.8%) (43) b (8.7%) 502 (10.5%) Bavaria 522 (12.2%) 71 (14.4%) 593 (12.5%) Berlin/Brandenburg a 503 (11.8%) 83 (14.7%) 576 (12.1%) Hesse 260 (6.1%) (31) b (6.3%) 291 (6.1%) Lower Saxony/Bremen a 368 (8.8%) (35) b (7.1%) 403 (8.5%) North Rhine-Westphalia/Rhineland-Palatinate a 991 (23.2%) 102 (20.6%) 1093 (22.9%) Saarland 427 (10.0%) 66 (13.4%) 493 (10.4%) Saxony/Saxony-Anhalt/Thuringia a 377 (8.8%) (37) b (7.4%) 414 (8.7%) Schleswig-Holstein/Hamburg/Mecklenburg-Vorpommern a 360 (8.4%) (36) b (7.2%) 397 (8.3%) Quality of social network 1–2 or less a 557 (13.1%) 82 (16.6%) 639 (13.5%) 3-5 2086 (48.9%) 252 (51.0%) 2338 (49.1%) >6 1624 (38.1%) 160 (32.4%) 1784 (37.5%) Perceived social support A lot 930 (21.8%) 116 (23.5%) 1046 (22.0%) Some 2529 (59.3%) 260 (52.6%) 2789 (58.6%) Uncertain 523 (12.3%) 77 (15.6%) 600 (12.6%) Little or none a 275 (6.7%) (41) b (8.3%) 326 (6.8%) Available help Very easy 1414 (33.1%) 165 (33.4%) 1579 (33.2%) Easy 1702 (39.9%) 176 (35.6%) 1878 (39.4%) Possible 726 (17.0%) 93 (18.8%) 819 (17.2%) Difficult 291 (6.8%) (34) b (6.9%) 325 (6.8%) Very difficult 134 (3.1%) (26) b (5.3%) 160 (3.4%) Marital status Single 481 (11.3%) 95 (19.2%) 576 (12.1%) Married 2706 (63.4%) 267 (54.0%) 2973 (62.4%) Widowed 449 (10.5%) (37) b (7.5%) 486 (10.2%) Divorced 631 (14.8%) 95 (19.2%) 726 (15.2%) Type of household Alone 1219 (28.6%) 168 (34.0%) 1387 (29.1%) With children 145 (3.4%) (21) (4.3%) 166 (3.5%) With a partner 2100 (49.2%) 172 (34.8%) 2272 (47.7%) With a partner and children 435 (10.2%) 86 (17.4%) 521 (10.9%) Other 368 (8.6%) (47) b (9.5%) 415 (8.7%) Working situation In paid employment 2531 (59.3%) 332 (67.2%) 2863 (60.1%) Unemployed/Others a 135 (3.1%) (20) b (4.0%) 155 (3.3%) Retired 1258 (29.5%) 90 (18.2%) 1348 (28.3%) Household work (unpaid) 181 (4.2%) (25) b (5.1%) 206 (4.3%) Unable 162 (3.8%) (27) b (5.5%) 189 (4.0%) Partner cohabitation Yes 2767 (64.8%) 280 (56.7%) 3047 (64.0%) No 1500 (35.2%) 214 (43.3%) 1714 (36.0%) Experienced limitation Severely limited 320 (7.5%) (44) (8.9%) 364 (7.6%) Mildly limited 1339 (31.4%) 137 (27.7%) 1476 (31.0%) Not limited 2608 (61.1%) 313 (63.4%) 2921 (61.4%) a Multiple categories were displayed collapsed when cell sizes <20 observations to avoid re-identifiability according to EHIS anonymisation rules. b Cells containing between 20 and 49 observations are individually flagged according to EHIS anonymisation rules. Univariate logistic regression analyses separately estimated the effects of these four variables ( Table 3 ). Only cohabitation significantly predicted BCS attendance, with women living alone having higher odds of never attending. Age also had a significant relationship with BCS attendance. Table 3 Univariate logistic regression on never attending BCS in Germany. Table 3 Sociodemographic variables OR 95% CI R 2 model AUC model Income High 1 Low 1.20 (0.98–1.47) 0.0010 0.5187 Country of origin Germany 1 Not Germany 0.74 (0.50–1.11) 0.0007 0.5090 Degree of urbanisation Urban 1 Rural 0.87 (0.72–1.05) 0.0007 0.5170 Partner cohabitation Yes 1 No 1.41∗∗∗ (1.17–1.70) 0.0039 0.5408 Age 50–54 1 55–59 0.44∗∗∗ (0.35–0.56) 60–64 0.37∗∗∗ (0.28–0.47) 65–69 0.31∗∗∗ (0.23–0.40) 0.0317 0.6225 Multivariate logistic regression was performed to capture the effects of each predictor when adjusting for covariates and age ( Table 4 ). Here, the only relationship that showed a statistically significant relationship with BCS attendance was partner cohabitation, with 1.45 higher odds (p < 0.001) for women not cohabitating with their partners. Table 4 Multivariate logistic regression on never attending BCS in Germany (main effects model). Table 4 Sociodemographic variables OR 95% CI Income High 1 Low 1.21 (0.98–1.49) Country of origin Germany 1 Not Germany 0.68 (0.46–1.02) Degree of urbanisation Urban 1 Rural 0.91 (0.75–1.11) Partner cohabitation Yes 1 No 1.45∗∗∗∗ (1.19–1.76) Age 50–54 1 55–59 0.43 ∗∗∗ (0.34–0.56) 60–64 0.36 ∗∗∗ (0.28–0.47) 65–69 0.29 ∗∗∗ (0.22–0.38) R 2 0.0394 AUC-ROC 0.6539 A complete case analysis only based on the variables would have resulted in 300 more participants, but the results do not change meaningfully – see Appendix C . Following this, an unweighted logistic regression was performed . As a reference group, we chose the one expected to have the highest attendance rate - based on the multivariate regression and Pedrós Barnils et al. - high-income women born outside Germany, living in urban areas with a partner (HOUY). Table 5 Full cross-classified multivariate logistic regression with evidence-informed intersectional groups. Table 5 OR 95% CI Intersectional groups HOUY 1 HGUY 2.49 (0.88–7.04) HGUN 2.97∗ (1.04–8.45) HGRY 1.92 (0.69–5.40) HGRN 2.84 (0.99–8.14) LGUY 1.96 (0.62–6.18) LGUN 3.71∗ (1.27–10.89) LGRY 2.81 (0.98–8.08) LGRN 3.24∗ (1.11–9.47) HOUN 3.11 (0.85–11.39) HORY 0.74 (0.16–3.46) HORN 3.15 (0.64–15.48) LOUY 0.66 (0.07–6.26) LOUN 4.00 (0.91–17.49) LORY 0.52 (0.05–4.82) LORN 9.48∗∗ (2.24–40.10) Age 50–54 1 55–59 0.43∗∗∗ (0.33–0.54) 60–64 0.35∗∗∗ (0.27–0.45) 65–69 0.29∗∗∗ (0.22–0.38) R 2 0.0445 AUC-ROC 0.6618 ∗p-value <0.05; ∗∗p-value <0.01; ∗∗∗ p-value <0.001. HGUY - high-income, born in Germany, urban, cohabitation. HGUN - high-income, born in Germany, urban, no cohabitation. HGRY - high-income, born in Germany, rural, cohabitation. HGRN - high-income, born in Germany, rural, no cohabitation. LGUY - low-income, born in Germany, urban, with cohabitation. LGUN - low-income, born in Germany, urban, no cohabitation. LGRY - low-income, born in Germany, rural, cohabitation. LGRN - low-income, born in Germany, rural, no cohabitation. HOUY - high-income, born outside Germany, urban, cohabitation. HOUN - high-income, born outside Germany, urban, no cohabitation. HORY - high-income, born outside Germany, rural, cohabitation. HORN - high-income, born outside Germany, rural, no cohabitation. LOUY - low-income, born outside Germany, urban, cohabitation. LOUN - low-income, born outside Germany, urban, no cohabitation. LORY - low-income, born outside Germany, rural, cohabitation. LORN - low-income, born outside Germany, rural, no cohabitation. Fig. 2 Odds Ratio (OR) with evidence-informed intersectional groups on never attending BCS in Germany. Fig. 2 Four intersectional groups were significantly associated with never attending BCS. Low income women not born in Germany and living in rural areas with no partner (LORN) showed the highest odds (OR = 9.48, p = 0.002). The confidence intervals for all these estimations were rather wide, increasing the uncertainty of the predicted estimations. The DA of the full cross-classification model was moderated and 0.0079 points higher than the main effects model. That indicates that the regression with intersectional groups discriminates slightly better between women attending or never attending BCS than the main effects model. Out of the three algorithms, CART showed the highest sensitivity and balanced accuracy performance. For more information on the hypertuned models, see Appendix D . The inner performance (i.e. evaluated on trained data) of CART was: 72.47% sensitivity, 51.35% specificity, 61.91% balanced accuracy, 14.71% positive predictive value and 94.15% negative predictive value. The moderate sensitivity suggests reasonable confidence in CART detecting women not attending BCS. However, the low specificity suggests small confidence in CART to identify negative cases (i.e. women attending BCS). The small positive predictive value indicates that many cases classified as positive (i.e. not attending BCS) are false positives. Nevertheless, the high negative predictive value indicates very few false negatives and, therefore, very high confidence that those cases classified as negative are negative (i.e. not assuming that a woman is attending BCS when she is not). Fig. 3 and Table 6 show the final decision tree and the emerged intersectional groups. Fig. 3 CART decision tree on never attending BCS in Germany. Fig. 3 Table 6 Intersectional groups on never attending BCS in Germany based on CART. Table 6 Group Intersectional groups Rank a Size, Prevalence H Women living with a partner, retired or doing unpaid household work 1 N = 882 Pr = 0.0454 E Widowed women living alone, with children, with a partner and children or other arrangements, residing in Baden-Württemberg, Berlin, Hesse, Mecklenburg-Vorpommern, Lower Saxony, North Rhine-Westphalia, Rhineland-Palatinate, Saxony, Saxony-Anhalt, and Schleswig-Holstein or Thuringia 2 N = 316 Pr = 0.0506 C Single, married or divorced women living in other living arrangements, with some or no perceived social support 3 N = 211 Pr = 0.0616 G Women living with a partner, who are either employed, unemployed, unable to work, or in other categories, and residing in Baden-Württemberg, Brandenburg, Hesse, Mecklenburg-Vorpommern, Lower Saxony, North Rhine-Westphalia, Rhineland-Palatinate, Saxony, Saxony-Anhalt, or Schleswig-Holstein 4 N = 918 Pr = 0.0730 B Single, married or divorced women living alone, with children, with a partner and children, with some or no perceived social support 5 N = 953 Pr = 0.1301 F Women living with a partner who are either employed, unemployed, unable to work, or in other working categories and residing in Bavaria, Berlin, Bremen, Hamburg, Saarland or Thuringia 6 N = 472 Pr = 0.1377 D Widowed women living alone, with children, with a partner and children or other arrangements, residing in Bavaria, Brandenburg, Bremen, Hamburg, or Saarland 7 N = 136 Pr = 0.1471 A Single, married or divorced women living alone, with children, with a partner and children or other arrangements, with little, uncertain or a lot of perceived social support 8 N = 873 Pr = 0.1707 An unweighted logistic regression with CART intersectional groups adjusted by age was carried out using the group with the lowest never-attended BCS prevalence (Group H) as the reference category . Table 7 Multivariate logistic regression with CART intersectional groups on never attending BCS in Germany. Table 7 OR 95% CI CART intersectional groups A 3.02∗∗∗ (2.02–4.50) B 2.18∗∗∗ (1.45–3.27) C 1.12 (0.58–2.18) D 3.43∗∗∗ (1.91–6.10) E 1.05 (0.57–1.90) F 2.54∗∗∗ (1.62–3.98) G 1.26 (0.81–1.96) H 1 Age 50–54 1 55–59 0.49∗∗∗ (0.37–0.62) 60–64 0.46∗∗∗ (0.35–0.60) 65–69 0.44∗∗∗ (0.32–0.60) R 2 0.0534 AUC-ROC 0.6726 Fig. 4 Odds Ratios (OR) from CART intersectional groups on never attending BCS in Germany. Fig. 4 After adjusting by age, four CART intersectional groups showed a statistically significant difference compared to group H. Group D showed the highest odds of never attending BCS (OR = 3.43; p < 0.001), and Group E the lowest odds (OR = 1.05; p = 0.88). This study aimed to identify intersectional groups of women aged 50–69 at higher risk of never attending BCS in Germany comparing two different analytical strategies: evidence-informed regression and decision tree-based regression. The decision tree-based approach also identified household type as a relevant variable, revealing that women living with a partner generally had a lower risk of never attending BCS than those in other living arrangements. Several authors have previously conveyed the importance of partnership cohabitation and breast cancer screening attendance . Furthermore, living arrangements seem to play a role for those with some or no social support. The risk of never attending BCS was found to be half that of women with some or no social support who were living in other arrangements, compared to those living alone, with children, or with a partner and children. Furthermore, the role of perceived social support in the intersectional group identified as relevant (i.e. single, married or divorced women living alone, with children, with a partner and children or in other arrangements) is unclear, as previously noted in the literature . The interpretability of the decision tree-based regression was slightly enhanced compared to the cross-classified regressions since it entailed fewer intersectional groups. Moreover, this reduction in dimensions did not entail a loss of information. On the contrary, the discriminatory accuracy of the model was slightly higher than the evidence-informed regression. Furthermore, the confidence intervals of the decision tree-based regression estimations are reduced (i.e. smaller variance), suggesting a more precise estimation of effect sizes. Conversely, the decision tree-based approach uses statistical algorithms to inductively identify patterns and relationships from the dataset . This approach is advantageous in revealing combinations of social dimensions not previously identified or explored, enabling more targeted interventions. Nevertheless, decision trees are susceptible to data quality, and their hierarchical structure might produce spurious results (i.e. the initial split has a significant impact on subsequent splits) . Moreover, given the uncommon application of decision trees in public health, no standardised procedures are yet defined, hence, many decisions are left to the discretion of the researcher (i.e. researcher bias). From a quantitative intersectionality perspective, the decision tree approach yields certain advantages for answering the question , “Who is at higher risk of never attending BCS?“ . The regression with a full cross-classification based on evidence-identified variables inevitably results in a loss of information due to the category simplification required to build the matrix . To maintain cells with sufficient size to preserve statistical power, variable categories are dichotomised, compromising the possibility of identifying non-linear patterns amid these categories . Decision trees allow for high dimensionality in the included variables (i.e. without risk of multicollinearity) and their categories (i.e. no need for dichotomisation). To the authors’ knowledge, this is the first study to compare regression- and decision tree-based approaches for identifying intersectional subgroups of women at higher risk of not attending BCS. However, the study is not without limitations, in particular the cross-sectional design of the survey, which impedes any causal inference from being drawn, and its self-report methodology, which may introduce response bias. The response rate for EHIS wave 3 in Germany was 21.6%, highlighting the necessity for caution when interpreting findings from studies utilising this dataset. Lastly, previous studies have indicated that EHIS may underestimate disparities in access to screening programmes . The combination of regression and decision tree-based approaches provides a comprehensive strategy for identifying intersectional groups at higher risk of an outcome. In this study, the evidence-informed regression identified that low-income women who were not born in Germany lived in rural areas, and did not cohabit with their partner as being at the highest risk of never attending BCS. Conversely, the decision tree-based approach identified widowed women living alone, with children, with a partner and children, or in other arrangements, and residing in specific federal states (i.e. Bavaria, Brandenburg, Bremen, Hamburg, or Saarland) as the highest risk group. The decision tree-based approach slightly outperformed the regression-based approach in its overall performance and interpretability and added a nuanced, data-driven layer of analysis, that enhances the overall understanding of the PROGRESS-Plus characteristics that determine BCS attendance in Germany. | Review | biomedical | en | 0.999996 |
PMC11699221 | Research is an integral component of evidence-based medicine. Relevant and timely information about novel disease management, pharmacological advancements, and public health interventions is critical in modern healthcare. In this context, research has become a considered method of skill development and literature interpretation for clinicians . In addition to this, medical research has a direct impact on the relationship between patients and the healthcare system, and their trust in ever-evolving medical practices . Engaging pre-qualification/registration (i.e. primary degree to obtain registration as a doctor) medical students in research from their training period could be a useful strategy to facilitate capacity building and a positive research culture right from the start. Although medical students view research opportunities as catalysts to obtaining entry into specialty training pathways, their participation in research remains limited . There is minimal research requirement as part of speciality training, and it depends on the interest of the individual along with the institution as to whether the individual is involved in research activities. Existing literature reveals several barriers that hinder medical student research participation including lack of opportunities, difficulty finding suitable supervisors and mentors, and a lack of time . Whilst the benefits of research involvement in medical school are favoured, student perception of research in this phase appears to be less favoured . The benefits of students participating in research do not appear to translate to higher levels of participation . A comprehensive synthesis on the current evidence related to enablers and barriers of medical student participation in research is required to inform policy and practice to mitigate these issues. Previous research has utilised the micro, meso, and macro frameworks to unpack enablers and barriers of an investigated topic . Macro factors involve policy considerations including legal, regulatory, and economic factors. Meso factors are related to the organisation and community, and micro factors are at the team and individual levels in the context of day-to-day practice. This review will utilise this framework to systematically unpack the enablers and barriers of medical student research participation at these levels. Doing so will not only enhance translation of review findings to policy and practice, but it will also strengthen the scholarship in this area. A preliminary mapping exercise of enablers and barriers to the macro, meso, and micro levels, based on information from the preliminary scoping searches, was completed during protocol development . The aim of this review was to synthesise the evidence on the enablers and barriers of research participation among students undertaking their pre-qualification medical studies, building on existing conceptual frameworks. Whilst using rapid review methodology considering available resources to undertake the review, systematic search methods were employed to ensure rigour. The review followed the World Health Organization (WHO) and the Cochrane guidelines for the conduct of rapid reviews . A protocol was developed and registered on the Open Science Framework . The WHO checklist for rapid reviews to demonstrate quality assurance of the review can be found in Supplementary Table 1 . The databases searched in this review included PubMed, EMBASE, and PsycINFO. This decision was made following a preliminary scoping search to identify sources with the most relevant citations of the review topic. Detailed PICo (Population, Investigated phenomena and Context; Table 1 ) domains were used to create inclusion and exclusion criteria. Essentially, quantitative, qualitative, and mixed-methods studies of medical students investigating the enablers and barriers to research participation during their medical school years, within university and healthcare settings, were included. Supplementary Table 2 contains search strategies for all three included databases. Searches were run in July 2022 and updated in October 2023. Table 1 Inclusion and exclusion criteria Inclusion criteria Exclusion criteria Population Medical students (both undergraduate and postgraduate entry) Research masters (i.e. M.Phil) or PhD students, non-medical students (i.e. nursing, allied health, etc.), provisional entry students (i.e. pre-medical degree) Investigated phenomena Barriers and/or enablers to participation in research during medical school Participation in activities other than research (e.g. other work experience, teaching, training) Context Research in medical school or health settings while on placement Research outside of these contexts Study design Primary research studies -Quantitative designs (including RCTs, cohort studies, pre-post, cross-sectional etc.) -Qualitative designs (including interviews, focus groups, case studies) -Mixed-methods designs Secondary research (systematic reviews, other reviews) Editorials, opinion pieces, commentaries Position papers Conference abstracts and posters Research protocols Other English-language literature Publications dated 2012 onwards Full texts Non-English literature Non-published studies (e.g. grey literature, thesis and dissertation manuscripts) All citations retrieved from the search were imported into Endnote X9™ and de-duplicated. Screening of titles and abstracts against the inclusion criteria was conducted using Covidence™ . For the title and abstract screening stage, 50 articles were screened by three reviewers (CM, HB, PM) together as a pilot exercise, with subsequent screening completed by two reviewers (CM, HB). Subsequently, at the full-text screening stage, three reviewers (CM, HB, PM) screened ten articles together to validate the process, with subsequent screening completed by two reviewers (CM, HB). During screening, conflict resolution was provided by a third reviewer (PM). Articles which met inclusion criteria were progressed to data extraction, while those which did not were excluded. The updated search did not yield any additional relevant articles. Methodological quality of included studies was assessed using the modified McMaster Quantitative Critical Appraisal tool and the McGill Mixed Methods Appraisal Tool (MMAT) . They were chosen as they are freely available and are widely used. Methodological quality was assessed by two reviewers (CM, HB) and verified by a third reviewer (PM). All discrepancies were resolved through mutual discussions by the review team. All studies, regardless of their methodological quality, underwent data extraction and synthesis. A directed content analysis approach of a deductive nature was used to analyse the extracted data. Codes were developed using the micro, meso, and macro frameworks for both enablers and barriers. The data were then synthesised and mapped against these codes, and categories were developed for reporting . This approach was chosen as it enables conceptual extension of theory to progress scholarship. Directed content analysis is a more structured process than traditional content analysis and can complement a structured review process . Data synthesis was performed by three reviewers (CM, HB, PM) through regular discussions until consensus was reached to enable researcher-triangulation. The fourth reviewer (DD) validated the findings. A total of 521 studies were extracted from the database search. Following removal of 73 duplicates, 448 articles were progressed to title and abstract screening. Subsequently, 50 studies were progressed for full-text screening. Of these, 23 studies were excluded based on wrong setting ( n = 3), wrong outcomes ( n = 3), wrong intervention ( n = 5), wrong study design ( n = 7), and wrong study population ( n = 5), leaving 27 studies in the final review. A flow diagram of included studies is provided in Fig. 1 . Further information on excluded studies with reasons is available in Supplementary Table 4 . Fig. 1 PRISMA flowchart of included studies Of the 27 included studies, eight originated from India, three each from New Zealand and Saudi Arabia, and four from Pakistan. One study each was from Australia, Colombia, Nigeria, Poland, South Africa, Sweden, and the United Arab Emirates. Lastly, one study was conducted across Egypt, Algeria, Sudan, Joran, Syria, and Palestine, while another was conducted across the UK, New Zealand, Malaysia, Canada, and France. The majority of studies ( n = 22) were conducted in a university setting. The remaining studies were conducted in a hospital ( n = 2), at a student conference ( n = 2), or in a research programme setting ( n = 1). Twenty-five studies surveyed only undergraduate students. One study surveyed undergraduate and intercalating medical students and one surveyed both undergraduate and post-graduate students. The sample size of the studies ranged from 48 to 2989 participants. The most widely used study design was a quantitative survey ( n = 26), while only one study employed a mixed-methods design. There were no qualitative studies. Common variables measured across most studies included student demographic information, level of research knowledge, research practices/experiences (including publication rates), general attitudes towards research, enablers/motivators for research involvement, barriers to research involvement, and future research goals/intentions. Further information about study characteristics is available in Table 2 . Table 2 Study characteristics Author Country Setting Participants Study design/measurement tool Outcome measures Alamri et al. 2019a New Zealand University Undergraduate students ( n = 48) Mixed-methods: qualitative, cross-sectional survey + retrospective analysis Publication rates, perceived barriers and motivators to pursuing research projects, supervisor satisfaction with research programme Alamri et al. 2019b New Zealand University Undergraduate students (years 2 and 3, n = 249) Quantitative, cross-sectional survey Research awareness and attitudes, motivators, and barriers to research Alamri et al. 2021 New Zealand University Junior medical students ( n = 253), intercalating medical students ( n = 10), senior medical students ( n = 323) Quantitative, cross-sectional survey Research engagement, motivation type (intrinsic vs extrinsic), effect of motivation type on other factors surrounding research engagement AlGhamdi et al. 2014 Saudi Arabia University Years 4 and 5 undergraduate students ( n = 172) Quantitative, cross-sectional survey Perceptions of research importance/impact; obstacles to research; research practice and experiences; sociodemographic information Assar et al. 2018 Egypt, Algeria, Sudan, Jordan, Syria, Palestine University Undergraduate medical students Multi-centre, quantitative, cross-sectional survey Demographics, knowledge, attitudes, practices, perceived barriers Awofeso et al. 2020 Nigeria University Undergraduate medical students ( n = 221) Quantitative, cross-sectional survey Knowledge of medical research, perceived barriers to research Baig et al. 2013 Pakistan University (private = 2, public = 2) Medical students ( n = 398) Quantitative, cross-sectional survey Year, institution, participation in research and intention, previous experience, satisfaction, incentives, suggestions for improvement Bonilla-Escobar et al. 2017 Colombia University Medical students ( n = 133) Quantitative, cross-sectional survey Sociodemographic information, research influences, affiliations, productivity, extracurricular activities, research motivations, career-research relations, research benefits and professional plans, personal attributes Chellaiyan et al. 2019 India University (private) Undergraduate students ( n = 344) Cross-sectional survey Demographic information, knowledge/perceptions of research, attitudes, practice, barriers Funston et al. 2016 UK ( n = 454) NZ ( n = 115) Canada ( n = 110) Malaysia ( n = 106) France ( n = 105) University Medical students Demographic information, research perceptions (experience, barriers, financial implications), future goals (incentives, barriers, career goals) Hada et al. 2021 India Medical college, hospital Undergraduate medical students ( n = 292) Cross-sectional survey Demographic information, student attitude towards research, attitude for sustaining research interest (if reported involvement) Jimmey et al. 2013 India University, medical conference Undergraduate (2nd–5th year MBBS, n = 114) Cross-sectional survey Research involvement, difficulties faced during research, reasons for research involvement Kharraz et al. 2016 Saudi Arabia University Undergraduate students (years 2 and 3, n = 221) Cross-sectional survey Perceived barriers towards research engagement Kini et al. 2017 India University Undergraduate students ( n = 220) Cross-sectional survey Attitude towards research and its career impacts, perceptions towards research, obstacles to research Kumar et al. 2019 Pakistan University Undergraduate (years 3–5, n = 687) Cross-sectional survey Barriers experienced by medical students in conducting research Mahmood-Shah et al. 2017 Pakistan University Undergraduate ( n = 294) Cross-sectional survey Attitudes, knowledge, and practices of research; perceived motivation, barriers, and interventions; intentions for future research Muhandiramge et al. 2021 Australia University Undergraduate and postgraduate ( n = 704) Cross-sectional survey Research experience; understanding of research terms; attitudes towards research (barriers, motivators) Nel et al. 2014 South Africa University Undergraduate ( n = 733) Cross-sectional survey Demographic information, interest in specialisation, extent of previous research involvement, general attitude towards research, factors influencing attitude towards research Noorelahi et al. 2015 Saudi Arabia University Undergraduate (years 3–5, n = 233) Cross-sectional survey Attitudes to and practice of research, barriers to conducting research Omprahash et al. 2019 India University Undergraduate (year 1, n = 205) Cross-sectional survey Knowledge of research, factors promoting positive attitude towards research and encouraging research involvement, perceived barriers to research Rani and Priya 2014 India University Undergraduate 2nd + 3rd years ( n = 308) Cross-sectional survey Demographic information, knowledge, and attitude of research methodology/statistics, research practices Sayedalamin et al. 2018 UAE Medical student conference Undergraduate ( n = 288) Cross-sectional survey Research attitudes and practices, barriers to research Shahab et al. 2013 Pakistan University Undergraduate (year 2–5, n = 160) Cross-sectional survey Student knowledge of research activities, perceived barriers, student attitudes Singh et al. 2021 India Teaching hospital Undergraduate (year 2, n = 217) Cross-sectional survey (pre and post 2-year workshop) Comparison of knowledge, attitude, and practice between pre and post survey Sobczuk et al. 2022 Poland University Undergraduate (2nd year n = 391, 5th year n = 695) Cross-sectional survey Scientific interest and participation, opinions on research issues, perception of potential barriers to research Stockfelt et al. 2016 Sweden University Undergraduate ( n = 471) Cross-sectional survey, mixed methods Medical student interest in research, disincentives for research, how students get involved, types of research Yerpude and Jogdand 2016 India University Undergraduate (final year, n = 282) Cross-sectional survey Demographic information; student knowledge, attitudes, and barriers A quality assessment was performed for each of the included studies, using the Modified McMaster’s Tool for quantitative studies ( n = 26) and MMAT ( n = 1) as outlined in Supplementary Tables 5a and b . All quantitative studies reported the study purpose and incorporated a relevant literature review. All studies were cross-sectional. All studies reported the sample size, but only 16 studies justified the sample size. Most outcome measures used were not reliable, and only under a quarter of studies used valid measures. A majority of studies ( n = 20) reported results in terms of statistical significance. All but one study used appropriate analysis methods and all studies reported on clinical importance of findings. All but two studies presented appropriate conclusions. The different components of the mixed-methods study did not adhere to the quality criteria of each tradition of the methods involved but met all other criteria on the MMAT tool. Utilising the macro, meso, and micro frameworks, enablers and barriers identified were mapped to the corresponding levels. Enablers of medical student participation in research were mapped to macro (career and financial incentives) and micro (skill acquisition and interest in research) levels, with no identified enablers at the meso level. Barriers were identified at all three levels. At the macro level, lack of training/information and financial constraints were noted. At the meso level, studies described difficulty finding supervisors, and at the micro level a lack of time, interest, and impact on studies/training were highlighted. Lack of training or information and financial constraints were mapped to this level. The absence of formal research training or a general lack of research awareness in universities was associated with decreased knowledge of available research opportunities and skills to participate in 18 studies [ 2 , 5 , 7 , 8 , 10 , 21 , 25 – 27 , 29 – 36 ]. Fourteen studies identified financial limitations as a barrier to research participation. Research was commonly completed on a volunteer basis or in conjunction with minimal financial aid through the enrolling university, which reduced student participation [ 1 , 2 , 5 , 7 , 21 , 27 – 29 , 31 – 36 ]. A lack of time, interest, and perceived impact on medical studies were barriers mapped to the micro level. Identified by 20 studies, increased workload and educational commitments related to medical studies limited time available to undertake research [ 1 , 2 , 5 , 7 , 10 , 21 – 30 , 32 – 36 ]. Ten studies identified a lack of interest in research as a barrier to participation during medical studies . Finally, participants in six studies noted that research participation in medical school might impact their medical education and prolong training . A systematic review and meta-analysis of medical student research in 2015 highlighted the association between medical student participation in research and improved short- and long-term scientific productivity, more informed career choices, and long-term success in academia . It further provided considerations for policy, decision makers, and researchers to progress this area. Despite these calls, engagement and participation of medical students in research and the resulting outputs and outcomes remain low . Our review explored enablers and barriers to medical student participation in research in order to further understand the prevailing gap. We subsequently mapped the enablers and barriers onto a conceptual framework to unpack different layers involved. Barriers to participation appear to outweigh enablers, thus substantiating the low participation rates in the literature . While identified enablers were mapped only at two levels of the framework, namely macro and micro, identified barriers were mapped to all three levels, indicating the wider extent of barriers across the continuum. The review by Amgad and colleagues exposed the lack of well-controlled high-quality prospective studies in this field . Eight years later, our review too echoes this finding. Studies included in our review were predominantly cross-sectional in design and utilising surveys to investigate participant perspectives. Apart from one mixed-methods study, all studies were quantitative, with an absence of qualitative studies to unpack the ‘how’ and ‘why’. Further, the surveys used in the included studies did not have established psychometric properties. The risk with in-house developed surveys without established psychometric properties is well-known. The field can only move forward when robust measures and tools are available . Given the diversity of approaches to research in different medical schools across the globe, availability and use of established surveys measures are essential. Furthermore, there is a need for qualitative studies so as to obtain in-depth experiences of students participating in research and their supervisors . Without these in-depth perspectives, available evidence will remain restricted. Medical students are a potential untapped resource that can be channelled to boost research outcomes. Medical students are generally interested and motivated to acquire new skills and education that enable them to progress their careers. However, some may find it hard to carry out research while juggling medical studies and associated workload. This could result in lower engagement and project completion rates. A recent Australian study by Fox and colleagues found that 33% of completed research projects medical students were involved in led to a peer-reviewed publication, while 51% led to outputs including conference presentations . This is slightly higher than the rates reported in the systematic review and meta-analysis by Amgad and colleagues in which only 30% of medical student projects resulted in peer-reviewed publications. Unless barriers at all levels of the system are tackled, these rates are unlikely to improve . Structured and targeted support that streamlines student participation and involvement in research from an early stage can make a difference. This can be enacted at several levels. At the macro level, institutions can provide medical students with a research framework, educational resources to enable research, mentorship, and supervision. Integrating research into the mandatory curriculum may be more facilitatory than undertaking research in an extracurricular capacity . Provision of incentives and/or a good support and supervision structure could also assist students in not only engaging with research but also completing it to a high standard. At the meso and micro levels, research supervisors can improve research culture by providing adequate and high-quality supervision, and promoting and educating medical students on the outcomes of research, thus boosting interest and motivation. Further, early adoption of research, training, education, supervision, and culture is expected to ultimately improve medical student research participation. There are more perceived barriers than enablers of medical students’ participation in research. These can be addressed at several levels including academic and healthcare institutions, research supervision and mentorship, financial incentives to students, and research and provision of a supportive and positive research culture. Academic and healthcare institutes can partner in several ways to provide more support, structure, and incentives for students to engage in research. Further studies are needed, especially using qualitative methods, to understand in-depth experiences of students and their supervisors engaging with research during the student’s medical training period. | Other | biomedical | en | 0.999996 |
PMC11699231 | Environmental pollutants include industrial and agricultural chemicals, heavy metals, pharmaceutical agents, as well as products with hormonal activity . They lead to interruptions in the reproductive endocrine circuits, cytotoxicity, mutagenicity , growth arrest, hemato-biochemical deteriorations, immunosuppression, and redox imbalance in fish. Still further, these contaminants accumulate in fish tissues, potentially harming human well-being through the food chain . Pyrogallol, alternatively termed 1,2,3-trihydroxybenzene or 1,2,3-benzenetriol, is a phenolic compound derived from plants . It has a historical background in hair dyeing and continues to have diverse applications in modern industry . Its uses range from being a corrosion inhibitor and developer in holography and photography to being present in insecticides, colloidal metal solutions, and various medical and scientific products . Pyrogallol is naturally found in aquatic plant and as a contaminant in tannins, anthocyanins, flavones and alkaloids and released into environment during its isolation, disposal and industrial use . Pyrogallol improved the immune response by increasing myeloperoxidase activity, leukocyte respiratory burst activity, and lysozyme activity in zebrafish ( zebra danio ) against Acinetobacter baumannii infection . Pretreating brine shrimp ( Artemia franciscana ) with pyrogallol succeeded in counteracting Vibrio harveyi infection by its prooxidant action involving generation of hydrogen peroxide against . Microalgae are promising ingredients for feed sources due to their cellular metabolites comprising a blend of triglycerides, pigments, vitamins, and vital amino acids. Besides being a major component in aquafeed, their diverse range of biologically active ingredients can enhance the survival rates of farmed species and elevate the pigmentation and quality of edible meat [ , , , , ]. Along with their role as a cornerstone in aquaculture nutrition, they are highly valuable in combating ecotoxicity . Spirulina platensis , Chlorella vulgaris , and Moringa oleifera serve as effective interventions in mitigating the harmful effects of environmental contaminants on fish, and thereby potentially improving their health and overall well-being. These supplements could ameliorate hematological, hepatic, renal, and histopathological alterations caused by different toxicants, such as chlorpyrifos, diazinon, and oxyfluorfen . Haematological outcomes provide straightforward and convincing methodological tools to comprehend the physiological processes and diagnose the somatic conditions of intoxicated fish . They identify anomalies within individuals even before observable negative effects on the ecosystem were noted . Despite various morphological, biochemical, and molecular analysis for evaluating the health status of fish, histopathologic evaluation remains a commonly employed technique in ecotoxicological investigations . C. gariepinus holds a prominent position as a widely favored freshwater fish globally, playing a crucial role as a significant nutritional source and a thriving species In terms of ecotoxicology, C. gariepinus is highly regarded as an outstanding model for evaluating the plausible harm of chemotoxicants, both within natural environments and controlled laboratory setups . As an indispensable source of human food, the toxicity in C. gariepinus could be transferred to human beings through the food chain. Therefore, the search for naturally occurring bioremediation strategies, with broad safety profiles, is of immense importance. These methods play a significant role not only in protecting the aquatic environment but also in ensuring human health. Upon thorough examination of scholarly articles, a notable gap exists in understanding the defensive properties of microalgae against toxicity triggered by pyrogallol in fish. Consequently, this study designs to address this gap by investigating hematological, biochemical, and histological markers as endpoints to explore this issue. The animal study conducted in this research underwent thorough review and received approval from Al-Azhar University, Assiut Branch, Egypt. The Ethical Committee of the Faculty of Science at Al-Azhar University, Assiut Branch, granted approval for the experimental design and fish handling protocols, referencing approval number . A total of 150 C. gariepinus , weighing approximately 200 ± 25 g and measuring 25 ± 5 cm in length, were sourced from the Aquaponic Unit of Assuit University, Assuit, Egypt. Before the experiments, the fish were housed in 120-liter tanks for 14 days, where they were provided with dechlorinated tap water at a temperature of 26 ± 2 °C, pH levels between 7.2 and 7.6, oxygen levels maintained above 80 %, and a natural light cycle of 12 h each of light and dark. They were fed twice daily using commercial SKRETTING food formulated in line with Guideline No. 203 for testing the acute toxicity of chemicals in fish , containing 30 % protein. To minimize the impact of fish waste and ensure consistency, daily, 40 % of the water was exchanged. After the adaptation period, fifty fish were separated in a random fashion into five groups (30 fish per group). Each group was represented by three tanks, 10 fish each. Pyrogallol concentration was selected based on a previous research . The experimental groups were as follows: Group (1); control, Group (2); 10 mg/L of pyrogallol, Group (3); 10 mg/L of pyrogallol + S. platensis (20 g/kg diet) , Group (4); 10 mg/L of pyrogallol + C. vulgar is (50 g/kg diet) , Group (5); 10 mg/L of pyrogallol + M. oleifera (5 g/kg diet) . After being exposed for 15 days, six fish were chosen randomly and then subjected to an ice bath to relieve stress . Blood samples were gathered in tubes containing heparin for hematological evaluations, while non-heparinized tubes were employed for assessing biochemical markers and ions. This was done after tail cutting to collect blood from the caudal blood vessels. The red blood cell count (RBC, 10^6/mm^3) was determined using a Thoma hemocytometer chamber and Dacie's diluting fluid. The hematocrit ratio (Hct, %) was measured with a capillary hematocrit tube. Hemoglobin concentration (Hb, g/dL) was assessed through spectrophotometry at 540 nm utilizing the cyanomethemoglobin method described by Blaxhall and Daisley . Mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were computed using formulas provided by Bain et al. . Peripheral blood smears stained with May-Grunwald-Giemsa were examined at × 100 oil immersion to analyze differential leukocytes. The count of white blood cells (WBC, 10 3 /mm 3 ) was determined following McKnight's protocol from 100 leukocytes on each slide to identify lymphocytes, neutrophils, and monocytes, as detailed by Kaya et al. . After the exposure phase, blood smears were attained by making an incision in the caudal area and placing the blood on clean microscopic slides. These smears were then fixed in absolute methanol for 10 min and allowed to dry at room temperature. Afterward, the slides underwent staining with hematoxylin and eosin, followed by examination using a 40X objective lens and 10X eyepiece to detect micronucleated and morphologically changed RBCs. The identification requirements for micronuclei (MN) were based on established guidelines from prior studies to guarantee precise evaluation. Blood samples were collected and then centrifuged at 4000 rpm for 10 min to separate the serum, following . The isolated serum was analyzed for different biochemical parameters using a T80+ UV/VIS spectrophotometer (Bioanalytic Diagnostic Industry, Co.). Plasma alanine aminotransferase (ALT) , aspartate aminotransferase (AST) , total protein , glucose , total cholesterol (TC) , and creatinine were assessed using commercial kits from the Egyptian Company for Biotechnology, Egypt, according to the manufacturer's instructions. Alkaline phosphatase (ALP) was measured using a colorimetric kit from Novus Biologicals, USA, and uric acid (UA) was measured using a kit from Abnova, Taiwan. All these biochemical parameters were analyzed according to Rifai . Malondialdehyde (MDA) was quantified in serum according to the thiobarbituric acid reaction . Superoxide dismutase (SOD) activity was measured by its capacity to inhibit the phenazine methosulfate-mediated reduction of nitroblue tetrazolium dye, which produces a red product . Total antioxidant capacity (TAC) was determined following the protocol outlined by Koracevic et al. . Levels of interleukin-6 (IL-6) were measured according to an established method . Samples of freshly sacrificed fish liver and kidney (N = 6) were immersed in 10 % neutral buffered formalin for preservation. The fixed samples underwent standard processing using paraffin embedding techniques. Subsequently, the samples were sliced into 5 μm-thick sections and stained with Hematoxylin and Eosin for comprehensive histological investigation , and Periodic Acid Schiff (PAS) staining was used to detect mucopolysaccharides . To assess each histopathological parameter, six randomly chosen sections from four fish in each treatment group were examined. These parameters were categorized as follows: absent (-), scored 0-2%; slight (+), <25%; moderate (++), (25-50%); and severe (+++), indicating (> 50%) of the sections involved. An Olympus microscope model BX50F4 (Olympus Optical Co., Ltd., Tokyo, Japan) was used for scrutiny of the sections. Six biological replicates for each parameter outline above were statistically processed using the SPSS software (V 25) at a designated significance level of p < 0.05. Data were tested for normality using the Shapiro-Wilk test and for homogeneity of variance to ensure that the data were normally distributed, and the variances were consistent. One-way ANOVA followed by Duncan post-test. Data are depicted as mean ± SE. For each parameter, values in the same row with distinct superscript letters (a, b, c …) indicate a difference among groups. Regarding hematological parameters, pyrogallol-exposed fish experienced a significant decrease in PCV and lymphocyte count, which was significantly ameliorated by microalgae interventions. C. vulgar is and M. oleifera were equally effective in normalizing PCV levels and significantly increasing lymphocyte counts. Neutrophil and eosinophil counts were significantly elevated in pyrogallol-exposed fish, and while S. platensis didn't significantly affect neutrophil levels, it significantly reduced eosinophil counts. C. vulgar is and M. oleifera supplementation significantly normalized these levels to those of the control group ( Table 1 ). Table 1 Effects of the studied microalgae on the hematological parameters in pyrogallol intoxicated- C. gariepinus. Table 1 Parameter Group Control Pyrogallol Pyrogallol + S. platensis Pyrogallol + C. vulgaris Pyrogallol + M. oleifera P value RBCs (million/mm 3 ) 3.17 ± 0.07 2.75 ± 0.05 3.17 ± 0.24 3.03 ± 0.07 3.19 ± 0.09 0.104 Hb (g/dL) 8.94 ± 0.49 7.85 ± 0.45 8.32 ± 0.21 8.23 ± 0.20 9.49 ± 0.50 0.070 PCV (%) 35.28 ± 0.31 32.33 ± 0.29 Φ 33.57 ± 0.35 Ω 34.21 ± 0.58 Ω 35.34 ± 0.33 Ω 0.000 MCV (μm 3 ) 109.48 ± 1.56 116.60 ± 2.50 106.63 ± 7.90 111.91 ± 2.43 109.99 ± 2.14 0.522 MCH (pg) 27.67 ± 0.98 28.24 ± 1.35 26.32 ± 1.64 26.92 ± 0.85 29.42 ± 0.81 0.417 MCHC (%) 25.00 ± 1.16 24.02 ± 1.31 24.54 ± 0.64 23.80 ± 0.27 26.55 ± 1.19 0.353 Platelet count ( × 10 3 /mm 3 ) 216.53 ± 5.40 205.92 ± 2.68 211.61 ± 4.87 214.48 ± 1.52 222.02 ± 5.48 0.160 WBCs count ( × 10 3 /mm 3 ) 11.67 ± 0.21 10.74 ± 0.20 11.24 ± 0.28 11.63 ± 0.25 11.50 ± 0.39 0.160 Large lymphocytes (%) 57.77 ± 0.23 54.45 ± 0.70 Φ 55.69 ± 0.62 56.89 ± 0.74 Ω 57.10 ± 0.51 Ω 0.010 Small lymphocytes (%) 24.20 ± 0.32 21.53 ± 0.25 Φ 23.02 ± 0.47 Ω 23.89 ± 0.29 Ω 24.75 ± 0.35 Ω 0.000 Neutrophils (%) 11.11 ± 0.26 13.86 ± 0.57 Φ 13.12 ± 0.47 12.06 ± 0.70 Ω 11.27 ± 0.29 Ω 0.005 Monocytes (%) 2.96 ± 0.01 1.98 ± 0.40 2.23 ± 0.25 2.47 ± 0.29 2.69 ± 0.24 0.144 Eosinophils (%) 2.72 ± 0.25 7.18 ± 0.47 Φ 4.70 ± 0.85 Ω 3.20 ± 0.24 Ω 2.20 ± 0.24 Ω 0.000 RBCs: red blood cells; Hb: hemoglobin; PCV: packed cell volume; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration; WBCs: white blood cells. Results are expressed as the mean ± SEM of 6 fish per group (One-way ANOVA followed by Duncan post-test). Φ Significant difference at p < 0.05 between control and pyrogallol groups; Ω Significant difference at p < 0.05 between pyrogallol and microalgae groups. In the control group, RBCs displayed a typical shape with a nucleus positioned centrally. However, smears from the pyrogallol group exhibited poikilocytosis in RBCs after the exposure periods. Various morphological changes were evident, including tear-drop cells, spinocytes, crenated cells, acanthocytes, eccentric nuclei, kidney-shaped cells, and schistocytes . The frequency of cell modifications and nuclear irregularities in RBCs notably rose in the intoxicated group in comparison to the control group . Fig. 1 The percentage of cell alterations and nuclear abnormalities in RBCs of pyrogallol-intoxicated C. gariepinus after 15 days of treatment with S. platensis (SP), C. vulgaris (CV), and M. oleifera (MO). Results are expressed as the mean ± SEM of 6 fish per group (One-way ANOVA followed by Duncan post-test). Φ Significant difference at p < 0.05 between control and pyrogallol groups; Ω Significant difference at p < 0.05 between pyrogallol and microalgae groups. Fig. 1 Fig. 2 Represented blood smears in C. gariepinus showing (a) the normal erythrocytes, (b and c) the deformed ones after exposure to 10 mg/L pyrogallol, (d) the deformed ones after exposure to 10 mg/L of pyrogallol + S. platensis (20 g/kg diet), (e) 10 mg/L of pyrogallol + C. vulgaris (50 g/kg diet) and (f) 10 mg/L of pyrogallol + M. oleifera (5 g/kg diet); Tr, tear-drop cell; Sp, spinocyte; Cr, crenated cell; Ac, acanthocyte; Eco, ecocentric nucleus; Bn, Bionucleus; Kn, kidney shape and Sh, schistocytic (H & E stain, scale bar: 100 μm). Fig. 2 Upon a 15-day exposure to 10 m/L of pyrogallol alone, the observed alteration percentages were 47.5 ± 3.59 % for cell alterations and 9 ± 1.41 % for nuclear abnormalities compared to the control group. Nevertheless, following the same exposure duration, the observed alteration percentages were 13.7 ± 0.82 %, 13.1 ± 0.73 %, and 14.2 ± 1.54 % for cell alterations, and 4 ± 0.81 %, 4.1 ± 0.8 %, and 4.5 ± 0.7 % for nuclear abnormalities when exposed to 10 mg/L pyrogallol + S. platensis , 10 mg/L of pyrogallol + C. vulgar is, and 10 mg/L of pyrogallol + M. oleifera , respectively. Thus, the supplementation of pyrogallol-exposed groups with S. platensis , C. vulgar is, and M. oleifera showed a significant reduction in cellular and nuclear abnormalities. The pyrogallol-intoxicated groups showed a significant increase in blood glucose levels compared to the control. However, treatments with C. vulgar is and M. oleifera significantly reduced these elevated glucose levels, while SP did not yield significant changes. Pyrogallol-exposed fish displayed increased serum creatinine and TC levels. Yet, all studied microalgae significantly decreased these elevated levels. M. oleifera exhibited superior efficacy compared to S. platensis in reducing serum creatinine and TC levels. Remarkably, there were no significant alterations in serum AST, ALT, and TP between the groups ( Table 2 ). Table 2 Effects of the studied microalgae on hepatic and renal damage biomarkers in pyrogallol intoxicated- C. gariepinus. Table 2 Parameter Group Control Pyrogallol Pyrogallol + S. platensis Pyrogallol + C. vulgaris Pyrogallol + M. oleifera P value AST activity (μ/l) 34.89 ± 1.06 35.33 ± 0.78 34.80 ± 0.89 34.41 ± 0.88 32.58 ± 0.73 0.253 ALT activity (μ/l) 17.23 ± 0.53 18.09 ± 0.54 17.48 ± 0.44 17.14 ± 0.51 16.57 ± 0.53 0.364 ALP activity (μ/l) 47.40 ± 1.70 43.31 ± 1.36 45.29 ± 1.48 46.07 ± 2.11 44.58 ± 1.74 0.532 Glucose level (mg/dl) 70.22 ± 1.61 78.61 ± 0.45 Φ 74.18 ± 2.15 72.91 ± 1.68 Ω 72.39 ± 1.01 Ω 0.016 TP level (mg/dl) 3.98 ± 0.16 4.56 ± 0.28 4.33 ± 0.20 4.21 ± 0.23 4.17 ± 0.17 0.441 TC level (mg/dl) 210.09 ± 0.63 223.25 ± 3.30 Φ 215.57 ± 1.42 Ω 212.75 ± 1.62 Ω 208.29 ± 1.89 Ω 0.000 Creatinine level (mg/dl) 0.36 ± 0.01 0.46 ± 0.02 Φ 0.39 ± 0.01 Ω 0.38 ± 0.01 Ω 0.35 ± 0.01 Ω 0.000 UA level (mg/dl) 22.84 ± 0.25 23.27 ± 0.37 22.67 ± 0.12 22.73 ± 0.23 22.03 ± 0.21 Ω 0.043 AST: aspartate aminotransferase; ALT: alanine transaminase; ALP: alkaline phosphatase; TP: total protein; TC: total cholesterol; UA: uric acid. Results are expressed as the mean ± SEM of 6 fish per group (One-way ANOVA followed by Duncan post-test). Φ Significant difference at p < 0.05 between control and pyrogallol groups; Ω Significant difference at p < 0.05 between pyrogallol and microalgae groups. Pyrogallol exposure resulted in a significant reduction in serum SOD activity and TAC, along with an elevation in serum MDA levels. Diets enriched with C. vulgaris and M. oleifera significantly normalized these parameters, while S. platensis did not produce any significant changes. All microalgae failed to improve TAC except for M. oleifera , which significantly increased it. MDA levels returned to the control level equally and significantly. IL-6 levels did not show significant changes between any of the groups ( Table 3 ). Table 3 Effects of the studied microalgae on serum oxidative stress parameters and pro-inflammatory interleukin-6 in pyrogallol intoxicated- C. gariepinus. Table 3 Parameter Group Control Pyrogallol Pyrogallol + S. platensis Pyrogallol + C. vulgaris Pyrogallol + M. oleifera P value SOD activity (U/ml) 2.68 ± 0.06 1.91 ± 0.10 Φ 2.23 ± 0.13 2.63 ± 0.19 Ω 2.67 ± 0.05 Ω 0.000 TAC (nmol/l) 56.86 ± 4.28 42.65 ± 1.16 Φ 43.76 ± 0.40 48.61 ± 3.10 50.40 ± 1.20 0.007 MDA level (nmol/ml) 14.94 ± 1.28 30.57 ± 2.60 Φ 17.97 ± 0.39 Ω 16.59 ± 0.95 Ω 16.87 ± 1.37 Ω 0.000 IL-6 level (pg/ml) 51.15 ± 0.34 53.42 ± 0.23 Φ 55.77 ± 0.47 58.04 ± 1.75 55.72 ± 2.91 0.060 SOD: superoxide dismutase; TAC: total antioxidant capacity; MDA: malondialdehyde; IL-6: interleukin-6. Results are expressed as the mean ± SEM of 6 fish per group (One-way ANOVA followed by Duncan post-test). Φ Significant difference at p < 0.05 between control and pyrogallol groups; Ω Significant difference at p < 0.05 between pyrogallol and microalgae groups. Liver sections stained with Hematoxylin and Eosin from the control group showed a normal structure. The hepatocytes appeared polygonal with unstained cytoplasm and centrally or eccentrically located rounded vesicular nuclei. Cords of hepatocytes radiated from central veins with blood sinusoids located between these cords. In the pyrogallol group, hepatic tissue exhibited numerous necrotic areas, pycnotic nuclei of hepatocytes, and congested dilated blood sinusoids . Inflammatory cell infiltration was observed. Mild improvement was detected in liver sections from the pyrogallol + S. platensis group . There was congestion of central veins and blood sinusoids, and pycnotic nuclei of some hepatocytes were noted. Necrotic areas were dispersed throughout the hepatic tissue. The liver of the pyrogallol + C. vulgaris group showed moderate improvement compared to the pyrogallol group, with some hepatocytes resembling those of the control group. Necrotic areas were still clearly observed. Marked improvement was observed in the hepatic tissue of the pyrogallol + M. oleifera group . The hepatocytes closely resembled those of the control group, although some blood sinusoids remained congested. Pigments were observed near the central vein. The histopathological scoring of lesions observed in the hepatic tissue is represented in Table 4 . Fig. 3 Photomicrographs of liver sections in C. gariepinus stained by H&E (bars = 50 μm) showing: (a) control group, (b) Pyrogallol group, (c) Pyrogallol + S. platensis , (d) Pyrogallol + C. vulgaris and (e) Pyrogallol + M. oleifera ; CV, central vein ; Δ , rounded vesicular nucleus of hepatocyte; ▲, congested blood sinusoid; ↑ , pyknotic nucleus; black asterisk , necrotic area; yellow arrow , inflammatory cells infiltration, red asterisk , congested central vein and red arrow , pigments. Fig. 3 Table 4 Scoring of histopathological lesions in the liver and kidney of the examined groups. Table 4 Lesions Groups Control Pyrogallol Pyrogallol + S. platensis Pyrogallol + C. vulgaris Pyrogallol + M. oleifera Liver Hepatocytes with pycnotic nuclei – +++ +++ + ++ Necrotic areas – +++ ++ ++ + Inflammatory cells infiltration – +++ ++ + + Congestion of blood vessels – ++ +++ + + Dilated blood sinusoids – ++ ++ + + Hemorrhage – + + – – Kidneys Bowman's capsule thickening – +++ +++ + – Glomeruli shrinkage – ++ + + – Glomerular tuft vacuolar degeneration + ++ + – + Complete degeneration of renal tubules – +++ ++ + – Separation of tubular cells from basement membrane + +++ ++ + + Pyknotic nuclei of renal tubular cells + +++ ++ + + Vacuolated renal tubular cells – +++ ++ + + Inflammatory cells infiltration + ++ ++ + + Melanomacrophage centers + +++ ++ ++ ++ Hematopoietic tissue + +++ +++ ++ + (−) Absent, (+) Slight (<25 %), (++) Moderate (from 25 to 50 %), and (+++) Severe lesion (>50 %). The liver sections stained with PAS staining revealed the glycogen content in the hepatic tissue . The magenta color observed in the liver sections from the control group indicated a normal amount of glycogen . A significant reduction in glycogen levels was observed in the pyrogallol-treated group , which was statistically significant compared to the control group . Glycogen content increased significantly in the pyrogallol + C. vulgaris and pyrogallol + M. oleifera groups compared to the pyrogallol group, but not significantly in the pyrogallol + S. platensis group. There were no significant differences in glycogen content among the three treated groups (pyrogallol + S. platensis , pyrogallol + C. vulgaris , and pyrogallol + M. oleifera ) and the control group . Fig. 4 Examination of liver glycogen in C. gariepinus . (a – e) : photomicrographs of liver sections stained by PAS stain (bars = 50 μm) showing: (a) control group with normal glycogen content as represented by magenta color, (b) Pyrogallol group, (c) Pyrogallol + S. platensis (SP), (d) Pyrogallol + C. vulgaris (CV), and (e) Pyrogallol + M. oleifera (MO). (f) liver glycogen in sections from all experimental groups. Bars represent means ± SE. Different letters indicate significant differences among treatments (p < 0.5). Fig. 4 The kidney of the control group showed Malpighian corpuscles with Bowman's space separating glomerular tufts and Bowman's capsule. Renal tubules were composed of cells with rounded vesicular nuclei. Hematopoietic tissue appeared normal in its content within the renal tissue, and melanomacrophages were also detected. In the Pyrogallol group, the kidney exhibited significant deterioration in renal tissue . There was thickening of Bowman's capsule around the Malpighian corpuscles, and the glomeruli showed vacuolar degeneration. Renal tubules had vacuolated cytoplasm and pyknotic nuclei, with necrotic areas also present. The hematopoietic tissue was excessively represented with densely packed components, and melanomacrophages were widespread throughout the renal tissue. Fig. 5 Photomicrographs of kidney sections in C. gariepinus stained by H&E (bars = 50 μm) showing: (a) control group, (b) Pyrogallol group, (c) Pyrogallol + S. platensis , (d) Pyrogallol + C. vulgaris and (e) Pyrogallol + M. oleifera ; Δ, Bowman's capsule; ▲, glomerulus; ↑ , renal tubular cell; yellow arrow , melanomacrophage; red asterisk , hematopoietic tissue; black asterisk , necrotic area; green arrow , thickening of Bowman's capsule; yellow arrowhead , glomerular vacuolar degeneration; blue arrow , vacuolated renal tubular cell with pyknotic nucleus; red arrow , shrunken glomerulus and orange arrow , separation of renal tubular cells from basement membrane. Fig. 5 In the kidney sections from the pyrogallol + S. platensis group , mild improvement was observed compared to the pyrogallol group. Shrunken glomeruli of Malpighian corpuscles were observed, and renal tubular cells showed separation from their underlying cell membranes. Hematopoietic tissue was excessively present, and necrotic areas and melanomacrophages were detected. In pyrogallol + C. vulgaris kidney sections , mild to moderate improvement in renal tissue was observed. Shrunken glomeruli of Malpighian corpuscles were observed, along with vacuolated renal tubular cells with pyknotic nuclei. Some necrotic areas and melanomacrophages were also present. Moderate improvement was detected in the renal tissue of the pyrogallol + M. oleifera group . Normal Malpighian corpuscles and vesicular rounded nuclei of renal tubular cells were observed. Separation of some renal tubular cells from their basement membranes was detected, along with the presence of hematopoietic tissue and melanomacrophages. Staining kidney sections with PAS staining revealed the polysaccharide content in the renal tissue . The magenta color observed in the glomeruli of Bowman's capsule, basement membranes, and brush borders of renal tubules from the control group indicated a normal amount . A marked depletion of polysaccharide content was detected in the pyrogallol group , and this decrease was statistically significant compared to the control group . The number of polysaccharides increased significantly in the three treated groups (pyrogallol + S. platensis , pyrogallol + C. vulgaris , and pyrogallol + M. oleifera ) compared to the pyrogallol group . There were no significant differences in polysaccharide content between the control group and the pyrogallol + C. vulgaris and pyrogallol + M. oleifera groups. Fig. 6 Examination of polysaccharides of kidney sections in C. gariepinus . (a – e) : photomicrographs of kidney sections stained by PAS stain (bars = 50 μm) showing: (a) control group with normal polysaccharides content as represented by magenta color in glomeruli, basement membranes and brush borders of renal tubules, (b) Pyrogallol group, (c) Pyrogallol + S. platensis (SP), (d) Pyrogallol + C. vulgaris (CV) and (e) Pyrogallol + M. oleifera (MO). (f): kidney polysaccharides of sections from all experimental groups. Bars represent means ± SE. Different letters indicate significant differences among treatments (p < 0.5). Fig. 6 As reported earlier , C. gariepinus encountered pyrogallol induced a marked drop in PCV which could be due to hemodilution, osmoregulatory issues, or kidney function disruption . The notable enhancement in PCV observed in our study, resulting from the dietary incorporation of microalgae, mirrors findings in O. niloticus supplemented with C. vulgaris after deltamethrin-induced hematotoxicity , as well as in C. gariepinus supplemented with S. platensis after chlorpyrifos exposure , and O. niloticus supplemented with M. oleifera after chlorpyrifos exposure . The marked reduction in lymphocytic populations following pyrogallol exposure indicates weakness of immuno-potency, as documented earlier . The decline in circulating lymphocytes could occur due to stress-triggered cell death in lymphocytes , heightened oxidative injury, and suppressed antioxidant shield . The expansion of lymphocytic clones is crucial for phagocytosis and immune responses against xenobiotic agents as seen in our microalgae-supplemented groups. The rise in lymphocytic count following feeding with microalgae-enriched diet confirms their immune-boosting impacts and stress-alleviating features of C. vulgar is . The immune-related impacts of dietary S. platensis could be linked to its diverse bioactive components like phycocyanin, phycobilins, xanthophylls, and allophycocyanin . The immune enhancement in C. gariepinus fed on the M. oleifera -enriched additive may be due to a variety of phytochemical ingredients . These include the rich content of polyphenolic compounds, volatile oils, and vitamins known for their immune-boosting properties . A previous study on the hematotoxic effects of pyrogallol in C. gariepinus triggered neutrophilia and eosinophilia . On the contrary, the studied microalgae succeeded in counteracting these abnormalities by enhancing the expression of anti-inflammatory mediators and suppressing the expression of pro-inflammatory mediators [ , , ]. The potential of pyrogallol to oxidize the sulfhydryl moiety located on fatty acid chains present within the cell membrane might be the underlying reason for the poikilocytosis observed in this study. In addition, excessive generation of reactive oxygen species heightens lipid peroxidation, hampers intracellular protein function, and causes DNA harm. This process can impact mitochondrial oxidative phosphorylation, prompting the discharge of numerous inflammatory substances and ultimately resulting in execution of cell suicide . The cytoprotective capability of microalgae aligns with findings observed in O. niloticus challenged with fipronil , largemouth bass ( Micropterus salmoides ) , and rats experiencing cobalt-induced apoptosis . S. platenesis mitigates the genotoxic effects by alleviating the chromosomal structural aberrations and DNA mutations due to its exceptional polysaccharides, which boost DNA repairing enzymes . Carotenoids, the active phytoconstituents in S. platenesis , encourage the activity of redox stabilizers and detoxifying enzymes, promoting cell proliferation . C. vulgar is demonstrates an anti-apoptotic impact by boosting the expression of anti-apoptotic genes, while reducing the expression of pro-apoptotic genes in M. salmoides . It increased the expression of peroxisome proliferator-activated receptor α, reduced activator protein 1 gene expression in the liver, and restored chromosomal stability and mitotic index of bone marrow cells in rats subjected to gibberellic acid-associated cytogenotoxicity . Quercetin found in M. oleifera lessens micronucleus occurrences and chromosomal abnormalities by directly interacting with xanthine oxidase and nitric oxide synthase, thereby decreasing the oxidative burden inside the cellular milieu . Kaempferol, a polyphenolic agent in M. oleifera , boosts b cell lymphoma-2 expression, consequently suppressing the presence of cell suicidal indicators like caspase-3 and poly (ADP-ribose) polymerase . Given the dose and duration employed in our study, the oxidative infliction and inflammatory damage in our model might not have been intense enough to disrupt the cellular membranes or lead to the discharge of hepatic enzymes into the circulating blood. Further research is therefore needed to explore the dose-dependent effects of pyrogallol on liver function. The elevated glucose levels noticed in pyrogallol-exposed fish might result from cortisol-induced hyperglycemia . This observation is supported by the depletion of hepatic and renal glycogen reserves, evident in histochemical analysis, primarily attributed to increased mRNA expression of genes encoding enzymes of glycogenolysis . The hypoglycemic activity of the studied microalgae is similar to that occurred in rainbow trout ( Oncorhynchus mykiss ) , crucian carp ( Carassius auratus ) , and O. niloticus . The improvement in insulin sensitivity , glycogenesis, glycolysis , inhibition of gluconeogenesis, and activation of insulin receptors may contribute to the antihyperglycemic effect of S. platensis . C. vulgar is exhibits analogous action by reducing insulin resistance and safeguarding beta-cell function from peroxidative insult , as well as inhibiting pancreatic alpha-amylase . Studies have demonstrated that M. oleifera can improve insulin resistance through multiple mechanisms. It activates the phosphoinositide 3-kinase/AKT and 5′ AMP-activated protein kinase pathways, enhances oxidative metabolism through the nicotinamide adenine dinucleotide-related deacetylase-peroxisome proliferator–activated receptor α pathway, and hinders fatty acid peroxidation . The hypercholesterolemia following pyrogallol intoxication is aligned with . The increase in TC levels might stem from alterations in liver cell permeability and disturbances in lipid metabolism, potentially associated with the buildup of the contaminants in the liver . Consistent with other research, microalgae exhibited beneficial effects on lipid balance [ , , ]. The hypolipidemic effects of S. platensis are thought to stem from its key components like linoleic acid, gamma linolenic acid, phycocyanin, phenolic compounds, and niacin . S. platensis demonstrates its hypolipidemic effects by activating lecithin cholesterol acyltransferase, a crucial element in the reverse cholesterol transport process, and by hindering 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, an enzyme vital for the production of cholesterol . C. vulgar is is an abundant provider of omega-3 polyunsaturated fatty acids which directly hinder acetyl-CoA carboxylases and reduce the mRNA expression of sterol regulatory element–binding proteins , thereby decreasing the production and function of lipogenic enzymes. Lipid-lowering properties of M. oleifera might be linked to its chemical profile, containing alkaloids, flavonoids, saponins, and cardiac glycosides . Saponins have the potential to lower cholesterol levels by either adhering to cholesterol in the intestines, thereby impeding its absorption, or by attaching to bile acids. This interaction may reduce the recycling of bile acids between the intestines and the liver, leading to their increased excretion in feces . Although the oxidant and antioxidant biomarkers were measured in the serum, they reflect redox disturbances that may adversely impact the hepatic and renal structures [ , , ]. The redox alterations induced by pyrogallol, marked by increased serum MDA levels and decreased serum TAC and SOD activity, align with the findings of Hamed et al. ). Like other chemotoxicants, this can be explained by the overproduction of reactive oxygen species , along with the downregulation of antioxidant gene expression , redox-linked transcription factors, and antioxidant responsive mediators . The oxidative stress caused by pyrogallol intoxication may be associated with the histological lesions and cytological modifications . The ability of microalgae to counteract the redox abnormalities induced by pyrogallol is comparable to the effects observed in O. niloticus exposed to imidacloprid , deltamethrin , and oxyfluorfen toxicity. The presence of bioactive components namely β-carotene, C-phycocyanin , γ-linolenic acid and α-tocopherol could underlie the marked antioxidant ability of S. platenesis against pyrogallol-induced oxidative injuries. These compounds, whether independently or synergistically, neutralize free radicals like, alkoxyl, peroxyl, and hydroxyl radicals . Additionally, C-phycocyanin can decrease nitrite generation and block lipid peroxidation . The defensive characteristics of C. vulgaris against the disruption in antioxidant shield may be related to its carotenoid profile, including β-carotene, lutein, canthaxanthin, and astaxanthin . They are effective in quenching the singlet oxygen and eliminating reactive oxidants . The phenolic constituents in M. oleifera , including kaempferol and quercetin glucosides , along with its total flavonoid content , are responsible for its antioxidant activity. Increased serum IL-6 levels, inflammatory cell infiltration in liver tissue, along with an elevated count of blood neutrophils and eosinophils, suggest that inflammation may play a significant role in pyrogallol toxicity. In a previous study , pro-inflammatory cytokines, including IL-1 beta and IL-6, were found to be elevated in the serum of C. gariepinus following pyrogallol contamination. This outcome could be attributed to the stimulation of the inflammatory cascade through the upregulation of the NF-κB signaling pathway . Pyrogallol depletes dissolved oxygen levels in water tanks . The depletion of oxygen in the ecosystem has been associated with increased levels of pro-inflammatory mediators in Chilean salmon ( Oncorhynchus kisutch ) . Similar to other reports , inflammatory cell accumulation was observed in the intestinal and muscular tissues of pyrogallol-intoxicated C. gariepinus. This finding might be due to the release of chemoattractants, which act as mediators for inflammatory cellular recruitment. IL-6 plays a role in neutrophil migration by inducing the production of chemokines, such as IL-8 and monocyte chemoattractant protein-1, and by triggering the expression of adhesion molecules on endothelial cells . Following the trafficking of neutrophils to the challenge site, they release chemokines to stimulate tissue-resident cells and recruit more leukocytes to the injured area, thereby intensifying inflammation . Inflammatory response can lead to a marked increase in eosinophil count . Eosinophils release inflammatory mediators through degranulation and function as antigen-presenting cells during inflammation . A previous study on the hematotoxic effects of pyrogallol in C. gariepinus reported neutrophilia and eosinophilia . Conversely, the studied microalgae succeeded in counteracting these abnormalities by enhancing the expression of anti-inflammatory mediators and suppressing the expression of pro-inflammatory mediators . Free radicals have the potential to harm the renal cells directly, activate intracellular signaling routes, or prompt systemic reactions that result in kidney damage . In a previous cited article, pyrogallol-treated C. gariepinus displayed extensive damage to the tubular structure, including complete splitting of the tubular epithelium with the loss of the basement membrane and various changes in cell nuclei. Additionally, the intertubular hematopoietic tissues exhibited higher content of RBCs alongside increased pigmented cell counts . In parallel with the histopathological disruptions in renal tissues and in accordance with previous research , serum creatinine levels markedly raised in the pyrogallol group. Estimation of creatinine can be applied to identify renal filtration rate and as an evaluation for renal inadequacy . The increased creatinine can be ascribed to the reducing effect of pyrogallol on glomerular filtration or increase in protein breakdown rates . Xenobiotics are recognized for causing liver damage through generating metabolic intermediates that can interact with cellular elements resulting in oxidative injury, antioxidants exhaustion, lipid peroxidation, and heightened membrane penetrability . The cellular alterations in the liver of pyrogallol-exposed C. gariepinus mirror those documented previously , and these changes are linked to the capacity of pyrogallol to stimulate pro-inflammatory cytokines which play important role in hepatic parenchymal cell damage . At the cellular level, cytoplasmic vacuoles are formed from elements of the endoplasmic reticulum or endosomal-lysosomal organelles due to cellular osmotic disturbance and exhaustion of ATP reserve . The histopathological deteriorations observed in the kidney of fish exposed to pyrogallol resemble those reported in a previous study . Extensive necrosis in hepatic tissue and the degeneration of renal tubules and glomerular tuft are commonly documented during exposure to various toxicants and may be mediated by the disruption in cytoskeletal elements . Pyknosis, the irreversible condensation of chromatin within a cell's nucleus, serves as an indicator of cellular apoptosis . An elevated occurrence of melanomacrophage aggregates in the kidney signifies notable tissue lesions and elevated immune response . The observed histological enhancements from the investigated microalgae are consistent with findings previously documented in studies by Refs. . The reduction in quantities of MMCs in microalgae-supplemented groups could be due to their immunostimulatory properties . The hematopoietic system in fish exhibits significant homeostatic potential, typically compensating for cell loss through the initiation of mitotic replication . The cytoprotective effect of S. platensis arises from its ability to up-regulate the expression of proliferating cell nuclear antigen which is responsible for DNA replication and regulation of cell cycle . In C. gariepinus , C. vulgar is counteracted the hepatorenal toxicity induced by microplastic particles by restoring the carbohydrate content in the microvilli and underlying membranes of kidney tubules and glomeruli, as well as within the cytoplasm of liver cells . C. vulgar is possesses chlorella growth mediators, promoting cellular multiplication and tissue regeneration. It also activates the immunity to assist in clearing away deceased cells . M. oleifera prompted the expression of PCNA and Ki-67 in the renal tissues of rats experiencing melamine-induced nephrotoxicity, indicating an improved capacity for cellular multiplication and renewal . Based on our results, exposure of C. gariepinus to pyrogallol led to alterations in blood granulocytes percent, erythrocytic cellular and nuclear features, and the redox defensive mechanism. Pyrogallol did not induce hepato-renal dysfunction and microalgae intervention did not enhance hepato-renal function. Based on the dose and duration used in our study, the oxidative burden and inflammatory effects in our experimental model may not have reached a threshold sufficient to disrupt the cellular membrane and cause the leakage of hepatic metabolizing enzymes into the bloodstream. Nevertheless, pyrogallol caused elevated serum glucose, TC, and creatinine levels, indicating metabolic deterioration confirmed by cytopathological lesions. Conversely, the investigated microalgae showed promise as potential remedies for mitigating these abnormalities. In this regard, M. oleifera was the most effective, followed by C. vulgaris , with S. platensis being the least effective. Consequently, incorporating these nutritional supplements into the diet could be pivotal in the aquaculture sector to alleviate the impact of environmental pollutants. However, dose-dependent studies are strongly recommended to thoroughly investigate the toxicological effects of pyrogallol on hepato-renal function and the potential benefits of microalgae. | Study | biomedical | en | 0.999998 |
PMC11699243 | Table 2 displays essential descriptive data for the daily returns series of assets studied in this study. The results suggest that mean returns are lower and almost identical in most asset classes. The highest and lowest volatilities are observed in the case of clean energy (SPCE) and green bond (SPGRNB), respectively. However, regarding uncertainty, GPR exhibits greater volatility, followed by EPU shocks. Table 2 Descriptive analysis and unit root tests. Table 2 Variables Mean SD Skew Kurt Jarque-Bera ADF PP SPESG 0.03 0.69 −0.78 30.29 113628.9∗∗∗ −38.25∗∗∗ −55.55∗∗∗ MASCIESGL 0.03 0.68 −0.75 31.98 128037.4∗∗∗ −38.12∗∗∗ −55.79∗∗∗ SPGRNB 0.01 0.28 −0.15 15.76 24770.11∗∗∗ −39.10∗∗∗ −58.23∗∗∗ SPCE 0.02 1.09 −0.29 17.90 33816.58∗∗∗ −36.75∗∗∗ −60.61∗∗∗ DJSI 0.02 0.73 −0.83 26.44 83953.87∗∗∗ −38.22∗∗∗ −55.64∗∗∗ DJIM 0.03 0.74 −1.06 28.00 95740.04∗∗∗ −40.56∗∗∗ −60.61∗∗∗ SPG1200 0.03 0.69 −0.76 28.99 103098.8∗∗∗ −38.25∗∗∗ −55.35∗∗∗ GPR 0.00 53.13 0.15 4.92 573.00∗∗∗ −55.16∗∗∗ −256.36∗∗∗ EPU 0.00 51.81 −0.01 6.64 2012.11∗∗∗ −37.50∗∗∗ −237.88∗∗∗ VIX −0.02 5.98 0.78 11.39 11074.84∗∗∗ −59.01∗∗∗ −61.06∗∗∗ Notes: The table reports the summery statistics. SD stands for standard deviation, and Skew and Kurt are used to denote skewness and kurtosis. The ADF and PP are the Augmented Dickey-Fuller and Phillips-Perron test statistics for unit roots that take into account constants and trends. The ‘∗∗∗’ signify the significance level at 1 %. To validate the use of the quantile-based technique, we use the BDS (Broock, Dechert, and Scheinkman) test to assess the non-linearity of our series. Table 3 shows the BDS test results based on time series residuals using a vector autoregressive (VAR) technique in many dimensions (m = 2, 3, 4, …, 6). The null hypothesis of linearity is rejected for all the cases; thus, the non-linearity is further evidenced when implanting diverse dimensions in the BDS test. Consequently, all the diagnostic tests stress us choosing a non-linear estimate technique, which can capture the heterogeneous association between the time series. Table 3 BDS test results for nonlinearity from the VAR model-based residuals. Table 3 Variables m = 2 m = 3 m = 4 m = 5 m = 6 SPESG 0.03∗∗∗ 0.05∗∗∗ 0.06∗∗∗ 0.06∗∗∗ 0.06∗∗∗ MSCIESGL 0.03∗∗∗ 0.05∗∗∗ 0.06∗∗∗ 0.06∗∗∗ 0.06∗∗∗ SPGRNB 0.02∗∗∗ 0.03∗∗∗ 0.03∗∗∗ 0.02∗∗∗ 0.02∗∗∗ SPCE 0.03∗∗∗ 0.05∗∗∗ 0.05∗∗∗ 0.05∗∗∗ 0.04∗∗∗ DJSI 0.03∗∗∗ 0.04∗∗∗ 0.05∗∗∗ 0.05∗∗∗ 0.05∗∗∗ DJIM 0.01∗∗∗ 0.03∗∗∗ 0.04∗∗∗ 0.04∗∗∗ 0.05∗∗∗ SPG1200 0.03∗∗∗ 0.05∗∗∗ 0.06∗∗∗ 0.06∗∗∗ 0.06∗∗∗ GPR 0.02∗∗∗ 0.04∗∗∗ 0.04∗∗∗ 0.04∗∗∗ 0.04∗∗∗ EPU 0.02∗∗∗ 0.04∗∗∗ 0.04∗∗∗ 0.05∗∗∗ 0.04∗∗∗ VIX 0.03∗∗∗ 0.05∗∗∗ 0.05∗∗∗ 0.05∗∗∗ 0.04∗∗∗ Notes: The table presents the BDS test results, which assesses the null hypothesis of independence and identical distribution (i.i.d.) for the time series against an unspecified alternative hypothesis. ∗∗∗ represent the significance level at 1 %. Following conventions stated in extant literature [ , , ], this study sets two stringent stationary processes of factors ( R t ) t ϵ z , with mechanisms R t = ( R t , x , R t , y ) , where x and y are substitutions for a couple of variables. Hence, in the following way (Equation (1) ), the quantile coherency between these two procedures ( R x , y ) can be outlined as follows: (1) R x , y ( ω ; τ 1 , τ 2 ) ≔ f x , y ( ω ; τ 1 , τ 2 ) ( f x , x ( ω ; τ 1 , τ 1 ) f y , y ( ω ; τ 2 , τ 2 ) ) 1 / 2 , where − π < ω < π implies the quantile cross-spectral density and ( τ 1 , τ 2 ) ∊ . f x , y , f x , x , and f y , y state to the quantile spectral densities of procedures R t , x , and R t , y , which can be achieved using the Fourier transform of a matrix of quantile cross-covariance kernels Γ ( τ 1 , τ 2 ) ≔ ( f ( ω ; τ 1 , τ 2 ) ) x , y , where (Equation (2) ) (2) Υ k x , y ≔ C o v ( I { J t + k , x ≤ q x ( τ 1 ) } , I { J t , y ≤ q y ( τ 2 ) } ) , for x , y ∊ { 1 , … , d } , k ∊ z , τ 1 , τ 2 ∊ , and scenario A is specified by function I{A}. We obtain an urgent indication of the cross-section dependency and serial correlation, respectively, by selecting x ≠ y and diversifying k. The quantile cross-spectral density kernel matrix is produced by this frequency's domain as f ( ω ; τ 1 , τ 2 ) ≔ ( f ( ω ; τ 1 , τ 2 ) ) x , y , where (3) f x , y ( ω ; τ 1 , τ 2 ) ≔ ( 2 π ) − 1 ∑ k = − ∞ ∞ Υ k x , y ( τ 1 , τ 2 ) e − i k ω We estimate quantile coherency as follows (Equation (4) ), based on Baruník, Kley utilizing smoothed quantile cross-periodograms: (4) G ˆ n , R x , y ( ω ; τ 1 , τ 2 ) ≔ 2 π n ∑ s = 1 n − 1 W n ( ω − 2 π s n ) I n , R x , y ( 2 π s n , τ 1 , τ 2 ) , Where I n , R x , y and W n denote rank-based copula cross-periodograms matrix and order of weight functions respectively. Then, in the following way (Equation (5) ), the quantile coherency valuation can be expressed: (5) R ˆ n , R x , y ( ω ; τ 1 , τ 2 ) ≔ G ˆ n , R x , y ( ω ; τ 1 , τ 2 ) ( G ˆ n , R x , x ( ω ; τ 1 , τ 1 ) G ˆ n , R y , y ( ω ; τ 2 , τ 2 ) ) 1 / 2 This research examines the coherency matrices in connection with three quantiles labeling to upper (0.95), average (0.5), and lower (0.05) quantiles, with the amalgamation of three quantile levels—right (0.95|0.95), middle (0.5|0.5), and left (0.05|0.05)—tails of the joint distribution. Additionally, we separate the coherency into three time horizons: quarterly high value, half-yearly medium value, and annual low value corresponding to ω ∊ 2 π { 1 / 3 , 1/6, 1/12}. From Equation (3) , we can decompose the cross-spectral density of kernels { f x , y ( ω ; τ 1 , τ 2 ) } into real and imaginary parts . The actual portion denotes the co-spectrum of the processes ( I { R t , x ≤ q x ( τ 1 ) } ) t ϵ z and ( I { R t , y ≤ q y ( τ 2 ) } ) t ϵ z , 4 while the imaginary portion represents the quadrature spectrum, which removes a number of sources of noise coherence. Nonetheless, we only provide the actual portions of our quantile coherency calculations due to readability and improved display considerations. 5 As a robustness test, we further analyze our data using the wavelet coherence (WC) approach to explore the lead/lag association between asset classes and uncertainties. It decomposes data into time-frequency space, capturing short-term and long-term patterns. This helps understand market behaviors and interactions between asset classes. The lead-lag relationship is crucial for portfolio management and risk assessment. Wavelet coherency and phase relation techniques reveal correlation and directional intensity over time, providing insights into causality and connectedness. This wavelet coherence (WC) approach eventually proves that the findings of previous methodologies are quite robust and reliable. The WC method, developed by Whitcher, Craigmile , helps scrutinize the time-varying and intensity of co-dependence between pairs in time and frequency domains. Additionally, any possible non-stationarity, nonlinearities, structural breaks, or seasonal patterns in a pair's connection may be controlled with this method . We make use of the cross-wavelet transform (WT) in conjunction with the WC [ , , ]. Among the couple of series x ( t ) and y ( t ) , here is an illustration of the cross-wavelet using continuous wavelet transformations: (6) W x , y ( u , s ) = W x ( u , s ) W y ∗ ( u , s ) , where Equation (6) is the cross-wavelet transformation, and s and u are the scale and position index, respectively. The wavelet transforms of x ( t ) and ( t ) are signified by W x ( u , s ) and W y ( u , s ) , respectively, where ″ ∗ ″ denotes the complex conjugate. According to Torrence and Webster , the co-movement between the two series, x(t) and y(t) , across time and frequency may be evaluated by the WC and is expressed as follows (Equation (7) ): (7) R 2 ( u , s ) = | S ( s − 1 W x y ( u , s ) ) | 2 S ( s − 1 | W x ( u , s ) | 2 S | W y ( u , s ) | 2 ) , where, S states the smoothing operator between time and frequency, recognized by S ( W ) = s s c a l e ( s t i m e ( W n ( s ) ) ) . The coefficient of R 2 ( u , s ) justifies the conditions 0 ≤ R 2 ( u , s ) ≤ 1 in the time-frequency space. R 2 ( u , s ) values closer to zero indicate a poor correlation between the two-time series, which are shown as blue, while values closer to one indicate a high connection, which are shown as warmer (red) colors. There is no link between the series in the cold (blue) zones. Since the WC's theoretical distribution is unknown in this instance, the statistical significance of the coherence is investigated at the 5 % significance level using Monte Carlo simulations . Nevertheless, it is impossible to discern between the lead-lag connection and positive or negative coherency due to the squared value of WC. Torrence and Compo, present a wavelet phase-difference analysis to capture these insufficiencies . (8) Ø x y ( u , s ) = tan − 1 ( J ( S ( s − 1 W x y ( u , s ) ) ) R ( S ( s − 1 W x y ( u , s ) ) ) ) , where (Equation (8) ), the WC phase differences are stated by Ø x y ( u , s ) . The unreal and real parts of the smoothed power spectrum are denoted by J and R , respectively, and are shown with arrows inside the regions classified as having high coherence. The two directions shown by the black arrows are the lead/lag phase relationship and the negative or positive correlation between the couples. The WC estimations across asset classes and uncertainty indices are shown in Fig. 4 , Fig. 6 . The vertical axis' monthly frequencies, such as 4, 8, and 16, represent the short-, medium-, and long-term investment horizons, respectively. 6 This section looks at how our sample assets have performed against the ten largest shocks that stemmed from three uncertainty factors during the sample period. Table 4 illustrate the returns of SPG1200, DJIM, SPESG, DJSI, SPGRNB, SPCLEN and MSCIESGL in the ten largest daily shocks of uncertainty indicators. We notice that the COVID-19 pandemic was responsible for most of the largest shocks to uncertainty indices, as these shocks happened in 2020 when COVID-19 struck the world unprecedentedly. However, Panel A of Table 4 reports the returns of SPG1200, DJIM, SPESG, DJSI, SPGRNB, SPCLEN and MSCIESGL on the ten biggest shocks derived from the GPR index. The results show that SPG1200, SPESG, DJSI and MSCIESGL returns are positive for six of the ten days; DJIM shows positive returns throughout the whole 10 days, and both SPGRNB and SPCLEN exhibit positive returns for four out of ten days. Table 4 Ten major shocks from five uncertainty factors. Table 4 Panel A: Ten biggest shocks from GPR Date GPR SPG1200 DJIM SPESG DJSI SPGRNB SPCLEN MSCIESGL 07-01-2020 420.29 −0.090 0.013 −0.123 −0.195 −0.225 −0.144 −0.116 09-01-2020 402.58 0.646 0.863 0.579 0.411 −0.155 0.553 0.585 17-11-2015 361.02 −0.050 0.001 −0.061 −0.037 −0.126 −0.220 −0.055 18-11-2015 318.07 −0.050 0.001 −0.061 −0.037 −0.126 −0.221 −0.055 16-11-2015 297.27 0.390 0.258 0.480 0.409 −0.047 −0.928 0.439 12-04-2018 292.84 0.447 0.395 0.396 0.355 −0.365 −0.008 0.407 09-05-2018 289.47 0.636 0.639 0.663 0.447 0.022 −0.528 0.555 05-06-2017 274.77 −0.065 0.027 −0.062 −0.066 −0.035 −0.094 −0.063 06-08-2019 272.20 0.513 0.745 0.518 0.189 0.091 0.646 0.547 19-09-2016 266.86 0.148 0.289 0.141 0.279 0.038 0.482 0.150 Panel B: Ten biggest shocks from EPU 17-05-2020 861.10 1.006 0.004 0.911 0.753 0.053 1.538 0.945 26-04-2020 855.17 0.553 0.008 0.565 0.482 0.036 0.737 0.599 05-05-2020 807.66 0.959 1.244 1.012 1.208 −0.215 1.142 1.043 29-03-2020 743.13 0.702 0.001 0.774 0.863 −0.022 −0.098 0.959 05-04-2020 739.55 1.861 0.001 1.859 1.603 −0.020 1.864 1.934 02-04-2020 738.02 1.290 1.503 1.380 0.896 0.454 0.907 1.333 26-12-2020 699.78 0.076 0.267 0.071 0.033 0.097 −0.265 0.045 26-03-2020 670.30 4.612 4.683 4.725 4.768 2.013 3.274 4.934 22-03-2020 651.07 −1.020 −0.004 −1.018 −1.122 0.035 −2.038 −1.091 27-04-2020 642.66 0.550 1.427 0.562 0.480 0.036 0.732 0.595 Panel C: Ten biggest shocks from VIX 16-03-2020 82.69 −3.345 −9.632 −3.333 −2.986 −0.180 −3.575 −3.426 18-03-2020 76.45 −5.212 −4.415 −5.061 −4.482 −2.410 −10.558 −5.227 17-03-2020 75.91 3.993 3.871 3.992 3.666 −1.587 6.101 4.029 12-03-2020 75.47 −9.976 −9.303 −10.220 −10.61 −2.367 −12.497 −10.250 15-03-2020 74.40 −3.237 −0.003 −3.225 −2.90 −0.180 −3.452 −3.312 19-03-2020 72.00 −0.267 0.493 0.554 0.775 −1.298 1.371 0.751 14-03-2020 66.12 −3.135 −0.003 −3.125 −2.818 −0.180 −3.337 −3.206 20-03-2020 66.04 −2.327 −2.229 −2.365 −1.335 −0.375 1.372 −2.717 27-03-2020 65.54 −2.487 −2.396 −2.509 −2.346 0.763 −5.140 −2.487 21-03-2020 64.56 −1.010 −0.004 −1.008 −1.109 0.035 −1.998 −1.080 Note: The table shows the returns of all assets against the ten biggest uncertainty indices shocks. To further provide comprehension into the co-movement of variables under study in the time-frequency domain, the WC methodology is employed. Fig. 5 , Fig. 6 , Fig. 7 depict the relationships between three global risk and uncertainty factors and seven distinct asset classes in terms of their WC plots. WC metric evaluates the time-frequency associations as well as the causal relationships between the respective variable pairs. The time dimension is represented by the horizontal axis, while the frequency dimension is depicted by the vertical axis. The degree of association is shown by the color on the right side of the bar chart. From blue (near low coherence) to red (high coherence), the coherence color code is used. The black U-shaped curve represents the cone of influence (COL). The areas of significant coherence within COL are delineated by dark contours. The scale (number of days) shows the frequency domain, highlighting that the short, medium, and long-term frequency domains imply the short, medium, and long-run investment horizons, respectively. Fig. 5 Wavelet coherence between GPR and seven assets. Note: The figures exhibit the wavelet coherence (WC) pairwise plots between GPR and seven assets. The horizontal axis displays time frames, whereas the vertical axis represents frequency in days. The arrows show the phases that correlate to the variables. The two series are in phase (anti-phase), as shown by the arrows on the right (left) side. The first (second) variable is shown as leading by the arrows, which are pointing right-side down (up) and left-side up (down). Fig. 5 Fig. 6 Wavelet coherence plots between EPU and seven assets. Note: The figures exhibit the wavelet coherence (WC) pairwise plots between EPU and seven assets. The horizontal axis displays time frames, whereas the vertical axis represents frequency in days. The arrows show the phases that correlate to the variables. The two series are in phase (anti-phase), as shown by the arrows on the right (left) side. The first (second) variable is shown as leading by the arrows, which are pointing right-side down (up) and left-side up (down). Fig. 6 Fig. 7 Wavelet coherence plots between VIX and seven assets. Note: The figures exhibit the wavelet coherence (WC) pairwise plots between VIX and seven assets. The horizontal axis displays time frames, whereas the vertical axis represents frequency in days. The arrows show the phases that correlate to the variables. The two series are in phase (anti-phase), as shown by the arrows on the right (left) side. The first (second) variable is shown as leading by the arrows, which are pointing right-side down (up) and left-side up (down). Fig. 7 The present study employs two distinct methodologies, namely quantile coherence and wavelet coherence, to comprehend the time-frequency, time domain and lead-lag associations between risks and seven asset classes. The quantile coherence findings suggest that the variables exhibit co-integration with time-frequency, indicating the existence of three distinct markets: bearish, normal, and bullish. In contrast, the wavelet coherence analysis reveals the presence of co-movement among the variables across various time domains, including short, medium, and long-run periods, along with quantile coherence. | Study | other | en | 0.999994 |
PMC11699256 | Sometimes people remember information about the source of a prior event correctly (such as background, location, or other kinds of context) without fully recollecting the details of the event. 1 , 2 , 3 For instance: “this place seems familiar, but I don’t know why or what from” or “I know the general place where I read something (e.g., the bottom left hand part of a page of a book), but I can’t remember what it was (e.g., neither the page nor the specific information)”. 4 , 5 It is not clear how this phenomenon occurs in memory. Cognitive models have typically accounted for episodic memory through two processes (for Review, see 6 ): “familiarity” recognizes an item from the past but with varying levels of memory strength and lacking contextual details, whereas recollection retrieves the item bound within the contextual details it was experienced with in the past, such that researchers typically used context retrieval as a proxy for recollection. Familiar recognition of a context in the absence of recollection is thus a puzzle for memory theories because it is difficult to resolve the paradox with just the two processes of recollection and familiarity, but these memories happen nonetheless. 1 , 3 , 4 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 A prior study investigated this conundrum using event-related potentials (ERPs) to focus on the unique memory condition in which people reported low confidence recognition of an item but had accurate source memory for the item’s context. 1 It was found that familiar context can be retrieved without relying upon recollection, thereby demonstrating that these constructs are dissociable and not dependent .The ERPs associated with this condition were electrophysiologically distinct from those associated with the memory process of recollection. The condition was therefore named “context familiarity”, based upon the framework of the binding-in-context (BIC) model, 15 , 16 , 17 which proposes that items and contexts are processed differently in the parahippocampal gyrus of the medial temporal lobe, and that the hippocampus binds the item within the context it was experienced in, e.g., recollection. Figure 1 Memory test schematic and combinations of conditions analyzed A (left): For each of 216 items studied and 108 new item lures, participants responded to a memory test that first asked them to rate their recognition confidence on a scale from 1 to 5 for if they thought the word on the screen was an old item from the study phase or if they thought it was a new item. Immediately following the item recognition judgment, participants were asked to indicate their source memory, e.g., if they remembered which study task the item was encountered in during the study phase. If they could not remember, they were instructed to then press the ‘3’ key to indicate that the source was unknown. (B) (right): schematic identifying the response profile for the three main conditions of interest analyzed in the current study. Green: item recognition hits collapsed across confidence levels, and which received responses of having no source memory, were operationalized as “item familiarity” (iFam. 18 , 19 ). Blue: low confidence recognition hits (responses of ‘4’) that received correct source memory judgments were operationalized as ‘context familiarity’ (cFam. 1 ). Red: high confidence recognition hits (responses of ‘5’) that received correct source memory judgments were operationalized as “recollection” (Rec. 1 , 15 , 20 , 21 , 22 ). Right: Representative ERP results of context familiarity and recollection are depicted from the original findings 1 (reproduced with permission of the owner), shown as topographic maps of each condition plotted in comparison to correct rejections during the latency of 600–800 ms in the memory epoch, with color scale depicting difference amplitudes (from −2 to 2 μV) of each condition as compared to correct rejections. The ERP effects for this “context familiarity” manifested generally as a widespread fronto-central-parietal negative-going effect. This occurred at approximately 600 milliseconds (ms) after stimulus presentation and extended later in the epoch from 800 to 1200 ms when compared to both correct rejections as well as when compared to incorrect source judgments. 1 This relatively late, broad central negativity (BCN) was dissociated from the late positive component (LPC) effect that is well-associated with recollection and did not show signs of an FN400 effect often related to familiarity. However, that study did not directly address whether the effects of context familiarity were dissociable from those of item familiarity. Therefore, it has remained unknown whether there is one type of overarching familiarity process that includes representations of both item and contexts (e.g., the BIC model) or if there might be two separable forms of familiarity: one for items and one for context. As a brief primer, in human long-term memory, theoretical models of conscious episodic recognition have been largely governed by the dual cognitive processes of familiarity and recollection. 6 , 15 , 16 , 20 , 21 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 Recollection is operationalized as the declarative retrieval of episodic information (i.e., an event) specifically with both the item and context of the event bound together into a cohesive retrieval of the episode (for review see 16 ). Recollection is usually associated with the confident retrieval of contextual information surrounding the item of the event (for reviews see 6 , 28 , 36 ). The item in the event, however, may be retrieved without recollection, instead relying upon a process of general familiarity of the episodic information, typically conceptualized as declarative, conscious retrieval of an item from a prior episode but without the associated confidence, recollection, or contextual information in which it occurred. These two memory phenomena are dissociable cognitive processes 21 with differential neural substrates in the medial temporal lobes, 38 , 39 , 40 , 41 are neuropsychologically dissociable among patient impairments, 22 , 42 , 43 and demonstrate spatiotemporally distinct patterns of electrophysiology at the scalp (ERPs) 1 , 44 , 45 , 46 ; for review see. 47 , 48 Familiarity has been associated with positive ERP differences in memory trials during a negative-going ERP peak at the mid-frontal scalp sites that occurs at approximately 400–600 ms post stimulus, often called the mid-frontal old-new effect, or FN400 (for f rontal- N 400 effect). On the other hand, recollection has been associated with positive differences between memory conditions occurring at a peak in the ERP at the parietal region of the scalp from approximately 600 to 900 ms, often referred to as a late parietal component (LPC) or parietal old-new effect 1 , 49 ; for reviews see. 45 , 47 , 48 Among studies of episodic recollection, “source memory” has been one of the most reliable methods of assessing recollection for episodic events in both behavioral and cognitive neuroscience paradigms. That is, it has traditionally been assumed that if someone remembers the source of information, then they must have retrieved information about the context of that memory and hence “must” be recollecting that episode. As such, source memory has served as one of the most useful proxies for understanding memory and amnesia deficits, 22 despite researchers widely acknowledging that source memory is not process-pure as an exclusive measure of recollection. 3 , 35 , 50 , 51 , 52 , 53 , 54 , 55 , 56 Prior research has also identified the role that other processes can contribute to source memory, 57 including item-familiarity 52 , 58 , 59 , 60 and unitization of two items into a new association. 60 , 61 , 62 , 63 , 64 , 65 The findings of ERP dissociations for context familiarity and recollection as occurring at different times and different sites on the scalp 1 provided direct physiological evidence in support of the BIC model’s theory and related variants 6 , 15 , 16 , 17 , 27 , 48 ; however, there remained limitations. In particular, while those findings had demonstrated that context familiarity was reliably distinct from recollection, 1 it was unknown if context familiarity is also directly dissociable from item familiarity or whether it is operating merely as a component element of the same memory process of generic familiarity that governs both items and contextual representations. 16 The notion that context can be differentially familiar than item information has been suggested in prior studies, 4 , 5 , 66 but was viewed only as context familiarity being merely a variant of the same underlying process of familiarity, and lacked evidence of an independent process until the aforementioned ERP study. 1 Here, we put forth evidence for a fundamentally different view: that familiarity of context differs qualitatively from item familiarity. The focus of the present investigation was to assess if the ERP effects for context familiarity can be reliably dissociated from those for item familiarity, and to add novel insights from behavioral measures of response times. When prior investigations observed dissociable ERP activity in time and topography for conditions of recollection (LPC) and familiarity (FN400), it was considered compelling evidence of two independent memory processes, 44 , 46 , 47 that reflect dual-process models of episodic recognition, since a single process would not have (and has not) been able to account for such patterns of neurophysiology without substantial post-hoc modifications. 6 , 48 In turn, if the effects of context familiarity are found to be dissociable in ERPs and/or behavior, it could be taken as evidence that context familiarity is a distinct additional process of episodic memory that extends beyond the traditional dual processes of recollection and item-familiarity. Toward that end, we re-analyzed a published ERP dataset ( N = 54) 18 , 67 that utilized the same paradigm as prior studies 1 , 22 and tested a combination of item recognition confidence and source memory judgments. We directly assessed behavioral measures of response times and physiological measures of ERPs for the three conditions of “item-only hits with source unknown” (‘item familiarity’ 18 , 19 ), “low-confidence recognition hits that had correct source memory” (‘context familiarity’ 1 ), and “high-confidence hits that had correct source memory” (‘recollection’ 1 ) . To preface our findings, we found that context familiarity, item familiarity, and recollection were each dissociable in neural activity patterns occurring at different times and places on the scalp, as well as in behavioral measures of response times for both item and source memory judgments (Experiment 1), and that the electrophysiological findings replicated across several independent studies (Experiment 2). In the current study, when people had low-confidence recognition (item 4 judgments) they went on to provide accurate source discrimination judgments significantly above chance (considered as 0.50, see Method) (M = 0.572, SD = 0.18, SE = 0.03; t(47) = 2.85, one-tailed p = 0.003; Cohen’s d = 0.40, 95% CI [0.521, 0.623]) ( Table 1 ), which was (and replicated) the same level of source memory performance as was previously reported 1 (M = 0.576, SD = 0.15, SE = 0.03, 95% CI [0.517, 0.635]). This null difference was quantified by finding no significant differences between the present study and the preceding one 1 (t(71) = 0.081, p = 0.936, 95% CI [-0.080, 0.087], BF 01 = 3.95). Having established the reproducibility of the prior behavioral performance of memory responses, 1 we next explored the response speeds and electrophysiological correlates of this paradoxical combination of memory response patterns. Table 1 Proportions of overall response patterns for recognition hits and source judgment combinations for old items during the memory test Response Proportions Source Incorrect Unknown Source Correct Total Sure Unsure Unsure Sure Per total Old Items Item 4: Unsure ‘Old’ <0.01 0.05 0.02 0.06 0.02 0.16 Item 5: ‘Sure Old’ 0.12 0.07 0.04 0.11 0.30 0.64 Total 0.14 0.14 0.22 0.18 0.33 Per Confidence Level Item 4: Unsure ‘Old’ 0.05 0.32 0.14 0.38 0.10 1.00 Item 5: ‘Sure Old’ 0.19 0.11 0.07 0.17 0.47 1.00 Per Total Hits Item 4: Unsure ‘Old’ 0.01 0.07 0.03 0.08 0.02 0.20 Item 5: ‘Sure Old’ 0.15 0.09 0.05 0.13 0.37 0.80 Total 0.16 0.15 0.08 0.21 0.39 Each participant ( N = 54) had 216 trials of old items. Left hand column represents item recognition confidence responses, and successive columns denote source memory responses. Top subsection provides the overall proportion of responses of high- and low-confidence recognition hits for each level of source judgment, out of all old trials (214 trials for each of N = 54 participants). Middle subsection indicates the proportion of responses for each source judgment per high- and low-confidence recognition hits; e.g., when subjects did have a recognition hit, it indicated what proportion of times it ended up in each of the source memory categories of interest for the current investigation. Bottom subsection indicates proportion of responses for each source memory judgment out of total recognition hits (collapsing across high and low confidence levels). We analyzed only subjects who had provided pairwise responses in each of the six conditions of interest ( N = 38). In our paradigm, participants first made a recognition judgment immediately followed by a source memory judgment for each item ; we separately assessed the response times for the item and source judgments ( Table 2 ). Table 2 Descriptive statistics of response time (ms) for item and source memory judgements Item Memory Source Memory Condition M SD SE M SD SE Recollection 1810 422 068 2596 1126 182 Context Familiarity 2710 962 156 2075 0968 157 Item Familiarity 2361 684 111 2612 1577 255 Time is reported in milliseconds, and data represents the sample of participants with pairwise observations included in the reported results ( N = 38). First, in item recognition judgments, reaction times were assessed across the factor of the three memory conditions (recollection, context familiarity, and item familiarity), using the same low confidence source memory responses in recollection and context familiarity conditions as was done in prior studies, 1 since it held constant the variable of source memory confidence. Response times for the three memory conditions were subjected to a one-way repeated measures ANOVA with three levels, which revealed a significant main effect of condition (F(2,37) = 25.16, p < 0.001, n 2 p =0 .41). Post hoc pairwise comparisons using the Bonferroni correction revealed that responses significantly differed for item familiarity and context familiarity, such that context familiarity was responded to reliably slower than item familiarity (MD = 348.57 msec, p = 0.048, 95% CI [2.26, 694.88], Cohen’s d = 0.48, as well as recollection (MD = 900.15 msec, p < 0.001, 95% CI , Cohen’s d = 0.76) . Item familiarity responses were also slower than recollection (MD = 551.58 msec, p < 0.001, 95% CI [297.97, 805.19], Cohen’s d = 1.24). Figure 2 Reaction speeds for item recognition and source memory judgments Mean times in milliseconds are shown in black with standard error of the mean for responses of item recognition (left) and source memory judgments (right), for three different conditions of memory response patterns, N = 38. Green: item familiarity (iFam.), operationalized as retrieval of recognition hits that did not have source memory (received responses of “source unknown”). Blue: context familiarity (cFam.), operationalized as retrieval of low-confidence item recognition that had correct source memory responses. Red: recollection (Rec.), operationalized as retrieval of high-confidence item recognition that had correct source memory responses. The boxplots demonstrate the median value, notches (95% confidence interval), and interquartile range (IQR) with lower and upper whiskers (Q1-1.5∗IQR; Q3+1.5∗IQR). The violin plots show density curves centered around the median value. Plots were generated using the scripts created by. 68 Within source memory judgments, response times for the three memory conditions revealed a significant main effect of condition (F(1.51,55.81) = 8.28, p = 0.002, n 2 p = 0.18). Further investigation using post hoc tests with Bonferroni correction revealed that source memory judgments for context familiarity responses were faster than those for both recollection and item familiarity (MD = 521.06, p < 0.001, 95% CI [ 273.01, 769.10], Cohen’s d = 0.42; MD = 537.21, p = 0.012, 95% CI [100.20, 974.23], Cohen’s d = 0.43). . Response times during source memory judgments for recollection and item familiarity conditions did not differ (MD = 16.16, p = 1.00, 95% CI [−399.64, 431.96], BF 01 = 5.70). Because item- and context-familiarity conditions also differed in recognition confidence , it was possible that differences between item- and context-familiarity conditions could have been attributable to the high confidence (‘5’) recognition responses included in the item familiarity condition. To test this directional hypothesis, we conducted a targeted analysis of these conditions while using only “low confidence recognition (‘4’) with source unknown” (item familiarity Low, no source memory responses), and low confidence recognition with source correct (context familiarity Low , all correct source memory responses). (Authors thank an Anonymous Reviewer for suggesting this valuable control analysis). While this excluded high-strength item-familiarity responses (high confidence recognition responses of ‘5’), such that a null finding would not be able to falsify the hypothesis, it nevertheless represented a valuable challenge of the alternative hypothesis using only weaker-strength item familiarity matched for item-familiarity strength. 18 , 20 , 21 , 33 , 69 Since these control analyses’ conditions were specifically defined by removing response types and thereby inherently weakening its power to detect differences, they nevertheless still produced significant results, and they also went on to be mirrored by similar results in the physiological measurements. The reliable difference originally observed between item- and context-familiarity responses was still preserved (t(36) = 2.09, p = 0.022, one-tailed, Cohen’s d = 0.34 95% CI [-inf, −49.49]; cFam Low : M = 2703, SD = 1033, SE = 149; iFam Low : M = 2896, SD = 852, SE = 140), and maintained a comparable pattern for source memory responses as were seen in the main analysis above (t(36) = 1.94, p = 0.030, one-tailed, Cohen’s d = 0.32, 95% CI [-inf, 57.76]; iFam Low : M = 2418, SD = 1697, SE = 279). These findings ensured that differences between familiarity responses (context, item) were not confounded by inclusion of higher confidence levels of memory strength in the item familiarity condition. The EEG analyses began with an assessment of general memory ERPs (hits vs. correct rejections) to identify if, consistent with the behavioral findings, there were three different physiological patterns of activation supporting episodic memory retrieval. This adopted the same approach as originally taken to characterize the conventional dual processes of recollection and familiarity 46 , 70 but was applied here to three different locations and latencies selected based upon hypotheses from prior findings. 1 , 22 This was then followed by a series of more specific analyses of the particular memory condition contrasts. For the first analysis, we used a basic contrast of measuring ERPs for hits vs. correct rejections in every subject ( N = 54) using a priori predictions from the same sites as the previous reports 1 , 22 : mid-central site Cz from 400 to 600 ms for the FN400, left parietal site P3 from 600 to 900 ms for the LPC, and fronto-central site Fc1 from 800 to 1200 ms for the BCN. These ERPs were subjected to a 2 × 3 × 3 repeated measures ANOVA with factors being memory, time, and site. Results revealed main effects of site (F(1.20, 53) = 46.71, p < 0.001, n 2 p = 0.47) and time (F(1.6, 53) = 13.271, p < 0.001, n 2 p =0 .20) and, importantly, a significant 3-way interaction of memory x site x time (F(2.29, 53) = 20.018, p < 0.001, n 2 p =0 .27) [in addition to interactions of memory x site (F(1.57, 53) = 13.60, p < 0.001, n 2 p = 0.20), memory x time (F(1.61, 53) = 22.89, p < 0.001, n 2 p =0 .30) and site x time (F(1.75, 53) = 14.58, p < 0.001, n 2 p = 0.22) . Figure 3 Old-new effects of recognition memory Top panel: Topographic maps depicting memory effects of the subtraction of hits minus correct rejections, N = 54; color scale of topographic maps denotes amplitude differences from −2 to 2 mu-volts. Maps are plotted for each of the three time latencies of 400–600 ms (ms), 600 to 900 ms, and 800 to 1200 ms that have been associated with the frontal negative old-new effect (FN400), late parietal component (LPC), and later broad central negativity (BCN), respectively. White boxes denote the sites from the topographic maps that are shown for the corresponding ERP figures beneath the maps, as well as where statistical analyses were performed. Bottom panel: Event-related potential (ERP) amplitudes (μV) of the individual conditions of recognition hits (dashed line) and correct rejections (solid line) plotted through the 1200 ms epoch and beginning with a −200 ms baseline period. Shaded areas depict the standard error or the mean for each condition. Consistent with our a priori directional hypotheses, hits were more positive than correct rejections at Cz from 400 to 600 ms (i.e., an FN400 effect; t(53) = 3.86, p < 0.001, one-tailed, Cohen’s d = 0.53, 95% CI [0.47, inf]; Hit: M = −3.66, SD = 3.51, SE = 0.48; CR: M = −4.50, SD = 3.72, SE = 0.51) and at P3 from 600 to 900 ms (an LPC effect; t(53) = 3.10, p = 0.002, one = tailed, Cohen’s d = 0.42, 95% CI [0.38, inf], Hit: M = 3.46, SD = 2.54, SE = 0.346; CR: M = −0.430, SD = 2.28, SE = 0.310), but were more negative at fronto-central site Fc1 from 800 to 1200 ms (t(53) = 2.84, p = 003, one-tailed, Cohen’s d = 0.39, 95% CI -[inf, −0.348]; Hit: M = −3.86, SD = 3.31, SE = 0.451; CR: M = −3.01, SD = 3.28, SE = 0.45) with comparable results also evident from 800 to 1200 ms at adjacent site Cz, t(53) = 2.50, p = 0.007, one-tailed, Cohen’s d = 0.34, 95% CI [-inf, −0.242]; Hit: M = −2.27, SD = 3.08, SE = 0.42; CR: M = -1.54, SD = 3.11, SE = 0.423). These findings provide strong support for three dissociable neural signals underlying basic episodic recognition, operating in different times and places on the scalp. 46 , 47 , 59 , 71 , 72 , 73 , 74 A series of subsequent analyses were then carried out to systematically characterize each condition (context familiarity, recollection, and item-familiarity) as compared to correct rejections, 46 , 48 , 70 described below. Since the current project hypothesized that context familiarity is distinct from recollection, we first sought to establish that the dataset validly reproduced prior findings 1 before endeavoring to explore differences within familiarity of items and context. A sample of N = 27 subjects provided observations in both conditions of interest for this analysis and satisfied inclusion criteria for numbers of valid ERP trials (see Method). A 2 × 3 × 2 repeated measures ANOVA for factors of memory condition (recollection, context familiarity), time interval , and site (P3, Cz) was performed based upon a priori predictions derived from the preceding analyses and prior reports dissociating context familiarity from recollection-based processing. 1 Results revealed a significant main effect of memory condition (F(2,26) = 9.418, p = 0.005, n 2 p = 0.27), a main effect of time interval (F(1.138, 26) = 6.262, p = 0.015, n 2 p = 0.19), and a main effect of site (F(1,26) = 15.89, p < 0.001, n 2 p = 0.38), along with a significant time by site interaction (F(1.35, 26) = 18.81, p < 0.001, n 2 p =0 .42) and a memory x time × site interaction (F(1.5), 26 = 7.69, p = 0.002, n 2 p = 0.23) . Figure 4 Three physiological effects of episodic memory retrieval Left panels: Topography maps are shown for each memory condition as compared to correct rejections in Experiment 1: (top) high-confident item recognition judgments that also had correct source memory retrieval, N = 27; (middle) low-confident item recognition judgments of that also had correct source memory retrieval, N = 27; and (bottom) recognition hits (collapsed across both high and low confidence) that did not have source memory retrieved (received responses of “source unknown”), N = 19. Color scale of topographic maps denotes amplitude differences for each condition as compared to correct rejections, ranging from −2 to 2 mu-volts. Right panels: Event-related potential (ERP) amplitudes (μV) from representative sites of maximal activation for each of the respective effects during the memory retrieval epoch of −200 to 1200 ms (ms); shaded areas for each plot represent the standard error of the mean. ERPs for context familiarity compared to the conventional ERP control condition of correct rejections revealed reliable evidence of the same negative-going effect from 800 to 1200 ms that had been directionally hypothesized from previous reports 1 , 22 , while also reproducing the prior findings of there being no evidence for ERP correlates of recollection or familiarity (LPC, FN400). 1 ERPs for context familiarity were more negative-going than correct rejections (CR) from 800 to 1200 ms at mid central site Cz (t(26) = −2.53, p = 0.009, one-tailed; Cohen’s d = 0.49, 95% CI [-inf, −0.369]; cFam: M = −2.91, SD = 3.575, SE = 0.69; CR: M = −1.772, SD = 3.72, SE = 0.72). Alternatively, there was no LPC effect evident for context familiarity from 600 to 900 ms (t(26) = 0.161, p = 0.437, one-tailed; Cohen’s d = 0.03, 95% CI [-0.732, inf, BF 01 = 4.32]; cFam.: M = −0.857, SD = 3.00, SE = 0.58; CR: M = −0.932, SD = 2.15, SE = 0.413), nor any evidence for an FN400 effect for context familiarity from 400 to 600ms (t(26) = −0.056, p = 0.522, one-tailed, Cohen’s d = 0.01, 95% CI [-0.753, inf]), BF 01 = 5.12; cFam: M = −5.241, SD = 3.83, SE = 0.737; CR: M = −5.217, SD = 4.03, SE = 0.78). ERPs for the recollection condition were also found to be consistent with the prior findings 1 when compared to correct rejections : eliciting both an FN400 effect at Cz from 400 to 600ms, (t(26) = 2.82, p = 0.004, one-tailed, Cohen’s d = 0.54, 95% CI [0.487, inf]; rec: M = −3.939, SD = 3.54, SE = 0.68; CR: M = −5.22, SD = 4.03, SE = 0.78) and an LPC at left parietal site of P3 from 600 to 900ms (t(26) = 2.98, p = 0.003, one-tailed, Cohen’s d = 0.57, 95% CI [0.665, inf]; rec: M = 0.627, SD = 2.94, SD = 0.57; CR: M = −0.932, SD = 2.15, SE = 0.41) but no evident differences during the later time of 800–1200 ms (t(26) = 0.002, p = 0.499, one-tailed; Cohen’s d = 0.00, 95% CI = [−0.923, inf], BF 01 = 4.91). These results indicated that the neurophysiology for conditions of recollection and context familiarity were indeed distinct in time and place on the scalp. Overall, these results thus reproduced the prior findings that neural processing for the retrieval of familiar contexts was distinct from recollection, 1 and established the foundation for exploring if the context familiarity may also be directly dissociable from item familiarity. ERPs for the item familiarity condition also reliably exhibited their predicted effects of an early old-new effect from 400 to 600 ms, maximal at site Cp1 ( N = 19, t(18) = 2.02, p = 0.029, one-tailed, Cohen’s d = 0.46, 95% CI [0.160, ∞]; iFam: M = −1.32, SD = 3.47, SE = 0.797; CR: M = −2.47, SD = 2.57, SE = 0.59), providing concurrent validity with similar other findings of posterior distributions attributed to absolute familiarity. 75 , 76 , 77 The condition did not exhibit any evidence for an LPC from 600 to 900 ms at P3 (t(18) = 1.22, p = 0.119, one-tailed, Cohen’s d = 0.28, 95% CI [−0.238, ∞], BF 01 = 1.27; iFam: M = −5.45, SD = 3.49, SE = 0.802; CR: M = −1.11, SD = 2.37, SE = 0.543) nor any later negative effect from 800 to 1200 ms at Cz (t(18) = 0.910, p = 0.187, one-tailed, Cohen’s d = 0.21, 95% CI [∞, 0.576], BF 01 = 1.83; iFam: M = −1.93, SD = 3.60, SE = 0.83; CR: M = −1.29, SD = 2.92, SE = 0.67). Together, the consistent findings of distinct ERP effects of an FN400, LPC, and BCN among both the established general conditions (Hits vs. CR) and the more specific conditions of response combinations (iFam, cFam, Rec., each vs. Cr.) provided convergent validity of similar findings observed from different measures, while also demonstrating concurrent validity of the newer specific ERP measures of memory processing with the more established general ERP measures of memory processes. To test whether the context familiarity condition reflected the same or different kind of familiarity processing as traditional item-familiarity, we investigated if the two conditions varied during the conventional time window traditionally reported for familiarity-based processing of the FN400 (300–500 ms 46 , 47 ), performing a within-subjects ( N = 11) targeted analysis on mid-central site Cz where prior analysis identified peak activation for these conditions in both the present and prior study 1 that predicted a one-directional difference of context familiarity being more negative than item familiarity. ERPs for context familiarity were revealed to be significantly more negative than those for item familiarity (t(10) = −2.57, p = 0.015, one-tailed, Cohen’s d = 0.76, 95% CI [-inf, −0.622]; cFam: M = −4.62, SD = 3.32, SE = 0.70; iFam: M = −2.42, SD = 3.22, SE = 1.33; Figure 5 A), and topographies indicated a broad negative-going distribution of this effect across central scalp sites and throughout the epoch. To assess if these effects varied throughout the epoch a 2x3 ANOVA was performed using factors of memory condition (item familiarity, context familiarity) and time interval . Results revealed a marginal main effect of memory (F(1, 10) = 4.251, p = 0.066, n 2 p = 0.298, Cohen’s d = 0.65), a significant main effect of time (F(1.18, 10) = 5.13, p = 0.039, n 2 p = 0.339), and no memory × time interaction (F(1.34, 10) = 1.48, p = 0.255). Figure 5 Within-subjects ERP differences of context familiarity and item familiarity Results depict findings from Experiment 1. (A) within-subject effects for participants who shared pairwise response trials in both conditions of item familiarity and context familiarity ( N = 11; note: N = subjects, n = trials, see Methods section 2.3.2). Effects were evident at 300–500 ms and distributed broadly across the scalp while being maximal over central midline site Cz. Topographic maps of difference waves for effects results from the subtraction of item familiarity from context familiarity conditions, respectively, with color scale depicting difference amplitudes from −2 to 2 mu-volts; white boxes denote regions of peak activity and targeted analyses; time intervals for each topography map is noted beneath it in milliseconds (ms). Shaded areas of event-related potentials (ERPs) depict the standard error or the mean for each condition. (B) Results from a sample of participants ( N = 25) meeting a lower inclusion criterion for signal-to-noise ratio ( n = 5 artifact-free ERP trials pairwise). (C) Control analysis of the same conditions but while holding constant the variable of item recognition confidence (depicting results from only the low confidence ratings of ‘4’ for each condition, and excluding the high confidence ‘5’ responses from item familiarity, N = 3, n > 11). To test if these results could be attributable to the potential confound of high confidence recognition responses included in the item familiarity condition, we sought a similar control test as was described for the behavioral data of response times: by removing the high-confidence responses from the item-familiarity condition so that it was matched to the low-strength responses of the context familiarity condition (Authors thank an anonymous Reviewer for suggesting this valuable analysis). ERPs for this contrast were available from only a limited sub-sample of the primary dataset due to there being only N = 3 participants available meeting our strict a priori inclusion criteria of the “item4+source unknown” item familiarity condition 67 (see Methods section 2.3.1) (though we note that the present paradigm has repeatedly been shown in prior studies to be effective and sensitive to detecting reliable differences in similar small sample sizes of specialized clinical patients on ERP measures of familiarity, confidence, and implicit memory repetition effects, 18 , 22 , 78 in addition to conditions of metacognition, 67 and that subsequent analyses conducted in Experiment 2 obtained the same results using a larger sample size of N = 12 in an independent dataset). We used a directional t-test based upon the hypothesis that context familiarity exhibits more negative-going ERPs than item familiarity at the same mid-central size (Cz) and time (300–500 ms) that was found in the primary analysis . Despite the limited sample, ERPs for context familiarity Low remained more negative than those for item familiarity Low : while this difference was not initially significant from 300 to 500 ms (t(2) = 1.67, p = 0.119, one-tailed, Cohen’s d = 0.962, 95% CI [-∞, −1.37], BF 01 of 0.610; iFam: M = −1.67, SD = 3.99, SE = 2.30; cFam: M = −3.49, SD = 2.19, SE = 1.27) there was a significant effect maintained in the nearly-identical time interval of 400–600 ms that has also been commonly used for measuring the FN400 in prior studies using this specific dataset 18 , 67 : (t(2) = 5.57, p bonf = 0.015, one-tailed, corrected, Cohen’s d = 3.21, 95% CI [-∞, −1.5]; iFam: M = −1.36, SD = 2.38, SE = 1.38, values: −3.29, −2.03, 1.31; cFam: M = −4.51, SD = 1.54, SE = 0.89, values: −5.40, −5.404, −2.73) , indicating that the main effect was preserved in the more stringent control analysis. Since we used a relatively conservative criteria for inclusion of participants in the within-subjects analysis ( n = 12 artifact-free ERP trials pairwise, see Method), it provided a rigorous threshold to foster better signal-to-noise ratio of ERP effects but also resulted in a fairly small sample ( N = 11). Therefore, we also performed a parallel analysis with a more liberal inclusion criteria for subjects, (Authors thank an Anonymous Reviewer for suggesting this valuable control analysis) which could increase the sample size and challenge the findings via testing them with reduced signal-to-noise ratio. 79 , 80 The same results persisted when lowering the trial inclusion criteria to n = 5, which provided a larger sample of N = 25 subjects: ERPs for context familiarity still exhibited a negative difference from ERPs of item familiarity that was maximal at adjacent site Fc1 and extending broadly across bilateral mid-central regions . We used a similar 2x3 ANOVA as employed in the primary analysis, using factors of memory condition (item familiarity, context familiarity) and adjacent time interval , and like the primary results using the more conservative inclusion criteria with a smaller sample, there was a main effect of memory (F(1, 24) = 4.37, p = 0.047, n 2 p = 0.15, Cohen’s d = 0.42) with no significant evidence for effects of time nor a memory × time interaction (both F’s < 1), indicating that ERPs for context familiarity were again significantly different from item familiarity beginning early in the epoch and persisting throughout despite lower signal-to-noise ratio. 79 , 80 Overall, the convergence of findings indicated that ERPs for item familiarity and context familiarity differed reliably during the traditional time window of familiarity processing, and thus that these two memory conditions represented reliably different kinds of familiarity processing on a trial-wise level within subjects. 46 , 47 , 59 , 71 , 72 , 73 , 74 As the preceding analysis demonstrated moment to moment differences of modular cognitive processes within the same individuals over time, we next sought to determine if these patterns would persist as individual characteristics across individuals. 67 The within-subjects approach used above required paired observations in both conditions of infrequent memory response patterns; however, the two conditions of item-familiarity and context-familiarity tend not to co-occur often within subjects. That is, those who tend to retrieve source memory well (i.e., better performers) also rarely report lacking source information (lesser memory), and those who tend to fail retrieving source memory do not also frequently report retrieving it. We thus utilized a between-subjects approach that treated those with sufficient trials in one condition but not the other as separate groups, in a mutually independent way (total sample of N = 26; item familiarity group: N = 9, context familiarity group = 17). This resulted in the exclusion of those from the within-subject analysis, but nevertheless converged to provide the same findings with an entirely different subset of participants, despite the inherent challenges to the hypothesis from a smaller sample. Using a between-groups t-test, the same pattern of ERPs persisted between subjects from 300 to 500 ms such that ERPs for context familiarity were significantly more negative-going than from item familiarity (t(24) = 2.82, p = 0.005, one-tailed, Cohen’s d = 1.16, 95% CI [1.93, inf], Figure 6 A), (cFam: N = 17, M = −4.62, SD = 4.63, SE = 1.23; iFam: N = 9, M = 0.250, SD = 3.34, SE = 1.12), and was not statistically significant during the later time intervals (600–900 ms: t(24) = 1.21, p = 0.119, one-tailed; 95% CI [−0.698, inf], BF 01 = 1.57; 800–1200 ms: t(24) = 0.664, p = 0.257, 95% CI [−1.72, inf], BF 01 = 2.27). Overall, this mirrored the results from the preceding within-subjects analysis, independently demonstrating that these are distinct physiological patterns for familiar memory responses both at the single trial level (within-subjects) and at the level of individual variability (between-subjects), and that the context familiarity condition cannot be attributed to merely a form of the same kind of familiarity process supporting familiarity of item recognition. Figure 6 Between-subjects ERP differences of context familiarity and item familiarity (A) Top: Between-subject effects in independent sub-groups of participants who had response trials in one condition but not the other (i.e.,: mutually exclusive N = 26 total; item familiarity: N = 9, context familiarity: N = 17), from Experiment 1. 18 , 67 Event-related potentials (ERPs) are shown from representative midline central site Cz; Shaded areas of event-related potentials (ERPs) depict the standard error or the mean for each condition. Bottom: range-normalized topographic maps of individual conditions from 300 to 500 ms, with color scale depicting voltage on the numerical scales indicated. (B) Between-subjects effects from participants of Experiment 2, 1 , 19 ( N = 24 total; item familiarity: N = 13, context familiarity: N = 11). Control analyses of range-normalized values revealed that the group differences between the memory conditions were not attributable to scaling artifacts or cohort effects that might be operating independent of the memory conditions. Since it is possible that the results of the between-subjects analyses could be driven in part by group-level differences in overall magnitudes of the ERP amplitudes between the ‘cohorts’ (e.g., scaling artifacts, 81 , 82 , 83 , 84 ; we conducted follow-up analyses to control for this possibility. We range-normalized the ERP amplitudes of the individual conditions within each group for item-familiarity and context-familiarity, respectively . This approach serves to overcome potential scaling artifacts of differential amplitudes, as it normalizes the ERPs in the dimension of amplitude magnitude and allows for investigation of where they are spatially located 46 , 73 , 84 as has been done in prior work. 18 , 46 , 85 Results from this control analysis revealed that range-normalized values for item- and context-familiarity ERPs remained significantly different from 300 to 500 ms at mid-central site Cz (t(24) = 2.36, p = 0.027, Cohen’s d = 0.97, 95% CI [-1.24, −0.084]; iFam: N = 9, M = 0.75, SD = 0.64, SE = 0.21; cFam: N = 17, M = 0.096, SD = 0.69, SE = 0.17) , and thus that the differences between the memory conditions were not attributable to scaling artifacts or cohort effects of groups that might be operating independent of the memory conditions. Collectively, these findings indicated that the observed differences in familiarity response types reflected differential neural processing at the individual subject level of variability, in addition to the trial-wise nature of the memory processing that was identified in the preceding within-subject analyses. These results thus converge to reveal that the two conditions represent fundamentally different kinds of familiarity operating in service of episodic recognition. If there are differential neural correlates of context familiarity and item familiarity operating as distinct processes of episodic memory, then the differences should be evident in other, independent experiments. Therefore, we assessed questions of reproducibility and sought validity of the findings through replication. 86 , 87 , 88 We investigated if the differences between ERPs for context familiarity and item familiarity could be reproduced among independent datasets that were aggregated from three previously-published studies using a nearly-identical paradigm and which had previously reported both traditional ERP correlates of item familiarity as well as ERP effects of context familiarity being separately dissociated from those of recollection, but had not directly contrasted conditions of item-familiarity to context-familiarity. 1 , 19 , 22 The analyses of Experiment 2 were conducted using the same sites and latencies that had been reported as representative of effects in the present and previous studies (mid-central Cz; bilateral frontal sites F3, F4 1 , 22 ). The null hypothesis for these analyses is that ERPs for the two conditions of context- and item-familiarity would exhibit no differences, if in fact the two memory responses were being supported by the same kind of underlying memory processing, and/or if the main results of Experiment were representing a form of type I error. The alternate hypothesis was that context familiarity would exhibit more negative-going ERPs than item-familiarity, and that if there is any such difference in ERP activity for the two conditions then it would be indicative that the two memory response types could not be measuring the same kind of memory process, and thus reflecting different kinds of memory processing people were using to making these different memory judgments. 6 , 21 , 42 , 46 , 47 , 48 First, we assessed if the within-subjects results observed between item familiarity and context familiarity in Exp. 1 could be seen at the mid-central site of Cz, using a 2x3 ANOVA with factors of memory (context familiarity, item familiarity) and time . This revealed that there was a significant main effect of memory (F(1,19) = 4.55, p = 0.046, n 2 p = 0.193, Cohen’s d = 0.48), a main effect of time (F(1.26, 19) = 19.72, p < 0.001, n 2 p = 0.509), but no significant memory × time interaction (F(1.68, 19) = 0.437, p = 0.616, n 2 p = 0.022) . These findings indicated that the two conditions differed reliably at the mid central site throughout the epochs from 300 to 1200 ms, and represented a faithful reproduction of the differences originally observed between ERPs for context- and item-familiarity in the present study’s main findings . Figure 7 Independent replication of ERP differences for context- and item-familiarity Within-subject effects for participants sharing pairwise response trials in both conditions of item familiarity and context familiarity ( N = 20) in Experiment 2 data aggregated from previously published studies 1 , 19 , 22 (see Method). (A) Top row: topographic difference maps of the resulting within-subjects difference between context familiarity minus item familiarity, in time intervals shown in milliseconds (ms). Color scale depicts difference amplitudes from −2 to 2 mu-volts, white boxes denote regions of targeted analyses revealing statistically significant effects. ERPs from representative sites Cz (mid-central), F3 (left frontal), and F4 (right frontal); shaded areas depict the standard error or the mean for each condition. (B) Same contrast as depicted as in (A) but excluding high confidence recognition responses from the item familiarity conditions , to match conditions on item recognition confidence levels ( N = 12). Because the previous datasets 1 , 22 had reported the ERP effects for context familiarity also manifesting at bilateral fronto-central sites, we expanded the aforementioned analyses to also include the frontal sites F3 and F4, using a 2 × 3 × 3 ANOVA. This analysis continued to provide a faithful replication of the original findings of the present work, as there was a main effect of memory (F(1,19) = 6.69, p = 0.018, n 2 p = 0.261, Cohen’s d = 0.49), an effect of time (F(1.29, 19) = 9.69, p = 0.003, n 2 p = 0.338), and a site × time interaction (F(2.24, 19) = 15.20, p < 0.001, n 2 p = 0.444) as the previously-reported frontal sites became more pronounced later in the epoch . Importantly, these results provide an independent replication of the critical findings reported in the present study comprised of entirely different participants from a different laboratory (the UC Davis Dynamic Memory Lab vs. California State University – San Bernardino, using different EEG hardware. Thus, the results of Experiment 1 in the main text of the present study are bolstered as valid, reliable findings observed across diverse participants from four different independent experiments. 1 , 19 , 22 , 67 One possible alternative interpretation of the ERP differences we observed between the conditions of item familiarity and context familiarity is that they could have been confounded by inclusion of high confidence recognition trials in the item familiarity condition. That is, the conditions of item- and context familiarity also vary on the factor of item recognition confidence, since the item familiarity condition includes both high and low confidence responses of strong and weak item familiarity levels, whereas the context familiarity condition contains only the low confidence recognition judgments that had correct source memory. One approach to address this issue is to exclude the high confidence responses from the item familiarity condition, (Authors thank an Anonymous Reviewer for suggesting this valuable control analysis) even though, as noted in Experiment 1, the context familiarity condition could still differ from strong forms of item familiarity that were excluded from the analysis. 29 , 32 , 33 This control analysis was performed in the behavioral results reported in Experiment 1 and was found to successfully preserve the findings that the conditions for item- and context-familiarity were reliably distinct in response times. For the ERP version of this control analysis in Experiment 1, there was only a small sample ( N = 3) meeting our inclusion criteria for ERPs of pairwise comparisons of the item familiarity condition with context familiarity that still provided a significant effect, but would benefit from an independent replication in a larger sample. In Experiment 2 there were more participants available ( N = 12). We thus assessed ERPs for “item4+source unknown” (iFam Low .) vs. ‘item4+source correct’ (cFam.), using a directional t-test based upon the a priori hypothesis derived from the prior findings in Experiment 1 that context familiarity exhibits more negative-going ERPs than item familiarity at the same mid-central size (Cz) and time (300–500 ms) as was found in Experiment 1. This revealed that there were still significant effects (t(11) = 1.97, p = 0.037, one-tailed, Cohen’s d = 0.60, 95% CI [-∞, −0.101]; iFam: M = −0.849, SD = 2.74, SE = 0.79; cFam: M = −1.99, SD = 2.06, SE = 0.593), and thus the main finding was replicated again and with a larger sample than original finding in Experiment 1. Thus, the inclusion of the high confidence recognition responses in the condition of item familiarity is not contaminating nor confounding the results we observed of reliable differences between item- and context-familiarity . The between-subjects ERP effects from Exp. 1 were also reproduced when aggregating across participants from prior studies 1 , 19 . As was done in the main results, participants were included in conditions of either item familiarity or context familiarity if they met the trial inclusion criterion of sufficient trial responses ( n = 12) in exclusively one condition but not the other, and these were entirely different participants than those included in the within-subjects analysis described above. This resulted in a subgroup of N = 13 for the item familiarity condition, and N = 11 for context familiarity. As familiarity effects for these prior studies were reported for 400 to 600 ms, 1 , 22 a targeted analysis was conducted during this latency to assess if context familiarity ERPs exhibited the same negative-going difference from item-familiarity ERPS at the mid-frontal site characteristic of previous familiarity effects reported in these participants 1 , 19 (Fz). A between-group t-test revealed the ERPs to reliably differ (t(22) = 1.923, p = 0.034, one-tailed, Cohen’s d = 0.79, 95% CI [-∞, −0.426]), with ERPs for context familiarity again being more negative-going than those for item-familiarity ( N = 11, M = −2.69, SD = 4.98, SE = 1.50; N = 13, M = 1.25, SD = 5.00, SE = 1.39, respectively). Next, the results of each individual condition’s memory effect were compared to correct rejections, as was done in Figure 4 of the main findings of Exp. 1. ERP correlates of context familiarity again exhibited a reliable effect from 800 to 1200 ms ( N = 33) that was distributed broadly across the central-posterior scalp and maximal at site Cp1 (t(32) = −3.22, p = 0.003, Cohen’s d = 0.56, 95% CI [−3.17, 7.12]; cFam: M = 1.84, SD = 4.35, SE = 0.76; CR: M = 3.78, SD = 3.94, SE = 0.69), but for which there was no significant FN400 effects evident from 400 to 600 ms (t(32) = 0.163, p = 0.113, 95% CI [−0.195, 1.75], BF 01 = 1.25; cFam: M = −0.006, SD = 4.24, SE = 0.74; CR: M = −0.782, SD = 3.84, SE = 0.67), nor any evident LPC at P3 from 600 to 900 ms (t(32) = 1.07, p = 0.293, 95% CI [−1.66, 0.515], BF 01 = 3.17); cFam: M = 2.19, SD = 4.42, SE = 0.77; CR: M = 2.76, SD = 3.66, SE = 0.64) . This finding reproduced and confirmed the Exp. 1 finding that context familiarity was associated with a broad central negative effect, that it held no evidence of association with the FN400 and provided an evidence for absence of the LPC effect. Figure 8 Physiological effects of episodic memory retrieval conditions Left panels: Topography maps are shown for each memory condition as compared to correct rejections in Experiment 2, comprising data from three previously published studies utilizing the same paradigm as the present study. 1 , 19 , 22 (top) High-confident item recognition judgments that also had correct source memory retrieval, N = 54; (middle) low-confident item recognition judgments of that also had correct source memory retrieval, N = 33; and (bottom) recognition hits (collapsed across both high and low confidence) that did not have source memory retrieved (received responses of “‘source unknown”), N = 38. Color scale of topographic maps denotes amplitude differences for each condition as compared to correct rejections, ranging from −2 to 2 mu-volts. Right panels: Event-related potential (ERP) amplitudes (μV) from representative sites of maximal activation for each of the respective effects during the memory retrieval epoch of −200 to 1200 ms (ms); shaded areas for each plot represent the standard error of the mean. On the other hand, item familiarity was associated with significantly more positive ERPs than correct rejections at mid-central Cz from 400 to 600 ms ( N = 38) , reproducing the FN400 effect reported in Figure 4 of the main results section (t(37) = 3.77, p < 0.001, Cohen’s d = 0.61, 95% CI [−2.38, −0.715]; iFam: M = 0.362, SD = 3.78, SE = 0.61; CR: M = −1.18, SD = 3.79, SE = 0.62). During the later latencies of 600–900 ms and then 800 to 1200 ms associated with recollection and context familiarity, respectively, there were no reliable differences evident between the two conditions (t(37) = 0.95, p = 0.348, 95% CI [−1.44, 0.519], BF 01 = 3.76; iFam: M = 3.27, SD = 4.47, SE = 0.725; CR: M = 2.81, SD = 4.34, SE = 0.703); t(37) = 0.160, p = 0.877, 95% CI [-1.08, 0.923], BF 01 = 5.66; iFam: M = 3.49, SD = 4.39, SE = 0.71; CR: M = 3.41, SD = 4.19, SE = 0.68) , reproducing the same pattern of results reported in the present study’s main results . These findings indicated that the neurophysiological correlates of the item familiarity condition were linked with an FN400 effect but no comparable evidence for any LPC or BCN effects (and with Bayes Factor evidence of absence for each other effect, respectively), consistent with the well-established profile of item-familiarity ERPs observed within and across the two present Experiments and among the broader literature of other studies. 1 , 45 , 46 , 47 , 59 , 85 Furthermore, similar to the findings of the present study, the recollection response condition exhibited both a reliable FN400 and LPC effect when compared to correct rejections ( N = 54), (t(53) = 6.14, p < 0.001, Cohen’s d = 0.84, 95% CI [1.43, 2.81]; Rec.: M = 0.207, SD = 4.92, SE = 0.668; CR: M = −1.915, SD = 4.53, SE = 0.61; t(53) = 4.73, p < 0.001, Cohen’s d = 0.64, 95% CI [0.942, 2.33]; Rec. M = 3.92, SD = 3.96, SE = 0.54), but provided no evidence for any BCN (t(53) = 0.585, p = 0.561, 95% CI [-1.55, 0.848], BF 01 of 5.72) . Collectively, these analyses of previously published datasets using the same memory paradigm as the present study provide a replication of the results observed in the current investigation. These patterns of effects reported here across both behavior and neurophysiology, both within and between subjects, and their replications among several other independent studies cannot be accounted for by a priori predictions from existing models of only a single- or dual processes of episodic memory. Figure 9 Old-new effects for recognition memory in Experiment 2 Data for Experiment 2 was aggregated from three previously published studies 1 , 19 , 22 ( N = 56). Top row: topographic difference maps of the resulting difference between hits minus correct rejections, plotted for each of the three time latencies of 400–600 ms (ms), 600 to 900 ms, and 1000 to 1400 ms that have been associated with the FN400, late parietal component (LPC), and late broad central negativity (BCN), respectively. Color scale depicting difference amplitudes from −2 to 2 mu-volts, white boxes denote regions of targeted analyses revealing statistically significant effects. Bottom panel: Event-related potential (ERP) amplitudes (μV) of individual conditions of recognition hits (dashed line) and correct rejections (solid line) plotted through the 1500 ms epoch and beginning with a −200 ms baseline period. Shaded areas depict the standard error or the mean for each condition. Using the same aggregated data also reproduced the other findings reported in the present study of a triple dissociation of ERP effects for item-familiarity, recollection, and context familiarity among the general memory contrast of hits minus corrections , whereby a 3-way interaction was originally found among an FN400, LPC, and BCN. For this analysis, we focused upon the latencies where effects had been observed in the prior studies for the FN400, LPC, and BCN (400–600 ms, 600 to 900 ms, 1000 to 1400 ms, respectively (Similar results were also found when assessing the time windows used in the present study of 400–600 ms, 600 to 900 ms, and 800 to 1200 ms: with main effects of memory F(1, 55) = 5.144, p = 0.027, n 2 p =0 .086, site (F(1.68, 55) = 7.70, p = 0.002, n 2 p = 0.123), time (F1.45, 55) = 44.797, p < 0.001, n 2 p = 0.449), and significant interactions of memory x time (F(1.41, 55) = 26.34, p < 0.001, n 2 p =0 .324), site x time (F1.56, 55) = 25.52, p < 0.001, n 2 p = 0.317), and a 3-way interaction of memory x site x time (F2.78, 55) = 4.66, p = 0.004, n 2 p = 0.078))), and focusing on sites Cz, P3, and Fc1 reported for effects in the present study ( N = 56). Results from a 2 × 3 × 3 ANOVA revealed that, similar to the main findings from Experiment 1 of the present study, there was no initial main effect of memory (F(1,55) = 2.19, p = 0.145, n 2 p = 0.038), but there were significant main effects of site (F(1.68, 55) = 5.82, p = 0.007, n 2 p = 0.096), and significant interactions of memory x time (F(1.52, 55) = 32.77, p < 0.001, n 2 p = 0.373), site x time (F(1.57, 55) = 23.59, p < 0.001, n 2 p = 0.300), plus a significant memory x site × time interaction (F(3.20, 55) = 4.77, p = 0.003, n 2 p = 0.080) . Consistent with the a priori hypotheses, results from directional t-tests based upon our a priori hypotheses revealed that there were significant effects of the FN400 (t(55) = 6.16, p < 0.001, one-tailed, Cohen’s d = 0.82, 95% CI [1.27, inf]; Hit: M = −0.208, SD = 4.39, SE = 0.588; CR: M = −1.95, SD = 4.48, SE = 0.599), LPC (t(55) = 3.06, p = 0.002, one-tailed, Cohen’s d = 0.41, 95% CI [0.402, inf]; Hit: M = 3.13, SD = 3.63, SE = 0.49; CR: M = 2.24, SD = 3.99, SE = 0.534), and BCN (t(55) = 1.85, p = 0.035, one-tailed, Cohen’s d = 0.25, 95% CI [-∞, −0.083]; Hit: M = 0.916, SD = 3.68, SE = 0.49; CR: M = 1.80, SD = 4.60, SE = 0.62). The BCN effect identified at the a priori selected location of Fc1 was observed to have extended broadly and been maximal at site Pz: t(55) = 4.33, p < 0.001, one-tailed, Cohens d = 0.58, 95% CI [-inf, −1.14], Together, these results revealed the same finding of a significant memory x site × time interaction in an independent dataset, effectively reproducing the original findings reported in the present study . Specifically, these results confirm that the three different memory effects are present within the most general measures of memory historically used in the field, in addition to the specific conditions described herein. While the responses given for context familiarity were defined as being explicit declarative responses from people for item recognition hits and also had overtly declarative correct source memory judgments, it nevertheless remained theoretically possible that they reflected a form of non-conscious processing such as that related to guessing from 100 to 300ms 89 or fluency that has been known to occur characteristically in earlier latencies of 200-400ms 85 , 90 , 91 for Review see. 76 Thus, to rule this possibility out, we sought to verify if the memory processing associated with the context familiarity condition’s effects in these participants was different from the hallmark signs of non-conscious, implicit memory signals that have been extensively characterized in the prior literature (i.e.,: recognition misses, 18 , 46 , 76 , 85 old items endorsed as 1 or 2 responses during the memory retrieval test). The null hypothesis of this analysis was that if the context familiarity condition was actually representing an implicit memory process, then it should show the same, or similar, activity as other implicit memory processing, such as the misses in the time windows when each effect is known to operate, respectively , and thus predict no significant differences in ERPs. We assessed ERPs for misses and context familiarity at the centro-parietal site of peak activity identified earlier for context familiarity (Cp1), using a within-subjects two-factor ANOVA with repeated measures on both factors ( N = 27). This analysis revealed no significant main effect of condition (F(1,26) = 1.03, p = 0.321, n 2 p = 0.038), identified a significant main effect of latency (F(1.91, 26) = 19.95, p < 0.001, n 2 p = 0.434), and importantly revealed a significant interaction of memory condition and latency (F(2.16, 26) = 3.78, p = 0.026, n 2 p = 0.127), which was further investigated with follow-up analyses . This revealed that ERPs for context familiarity were significantly more negative-going than misses at centro-parietal sites (Cp1) from 800 to 1200ms (t(26) = 2.31, p holm = 0.029, corrected, Cohen’s d = 0.44, 95% CI -2.40, −0.140]; Context Familiarity M = −2.69, SD = 3.46, SE = 0.66; Miss M =.-1.43, SD = 2.34, SE = 0.45) though not in the earlier latency of 200-400ms (t(26) = 0.443, p = 0.660; BF 01 = 4.37 indicating moderate evidence for the null). This demonstrated that the context familiarity condition was exhibiting substantively different physiology from recognition misses at centro-parietal sites during 800–1200 ms, refuting the null hypothesis that they were both reflecting the same implicit process outside of conscious awareness (e.g., guessing driven by implicit factors). Figure 10 Physiological effects of context familiarity versus recognition misses (A) Top: topographic maps of the difference map for activity of context familiarity minus activity for recognition misses ( N = 27). Maps are plotted for each time interval of 200–400 ms (ms), 400 to 600, 600 to 900 ms, and 800 to 1200 ms; color scale of topographic maps denotes amplitude differences from −2 to 2 mu-volts. Event-related potential (ERP) amplitudes (μV) for each condition plotted for representative sites of maximal activation (left frontal, F3, and centro-parietal Cp1), through an epoch of −200 to 1200 ms of a memory retrieval test. Shaded areas depict the standard error or the mean for each condition. (B) Replication data from Experiment 2. Data from three previously published studies ( N = 27). ERPs for each condition plotted for representative sites from original analysis (Cp1) and of maximal activation (right parietal Cp5, 200–400 ms), through an epoch of −200 to 1200 ms of a memory retrieval test. Since topography maps of Experiment 1 also indicated an early frontal activity pattern for this contrast, we conducted an exploratory analysis of the evident frontal region at its maximal left frontal site of F3. Inspection of these findings revealed that ERPs for context familiarity were more positive-going than misses in the early latency of 200-400ms (F3: t(26) = 2.55, p = 0.034 holm , corrected, Cohen’s d = 0.49, 95% CI [-2.07, −0.221]; cFam: M = 1.57, SD = 2.89, SE = 0.55; Miss: M = 0.427, SD = 3.01, SE = 0.57), continuing marginally 400–600 ms (t(26) = 1.90, p holm = 0.068, corrected, Cohen’s d = 0.37, 95% CI [-1.85, 0.07]; cFam: M = −4.36, SD = 3.62, SE = 0.697; Miss: M = −5.26, SD = 4.11, SE = 0.792) . Overall, these analyses revealed reliable, significant differences between conditions of context familiarity and implicit memory misses in Experiment 1 but were also assessed for the replicability in Experiment 2. The same pattern of differences between context familiarity and misses was also observed as being independently reproduced by targeted analyses in Experiment 2’s datasets ( N = 27) based upon directional hypotheses derived from the preceding findings for the same contrasts in Exp. 1, during the 800 to 1200 ms time interval of context familiarity effects, occurring at the same site as the primary results (Cp1: (t(26) = 1.79, p = 0.043, one-tailed, Cohen’s d = 0.34, 95% CI [0.046, ∞], cFam: M = 1.68, SD = 3.68, SE = 0.707; Miss: M = 2.68, SD = 3.35, SE = 0.644; maximal activation site of P4 (t(26) = 2.35, p = 0.013, one-tailed, Cohen’s d = 0.45, 95% CI [0.396, ∞], cFam: M = 0.689, SD = 3.33, SE = 0.64; Miss: M = 2.14, SD = 3.34, SE = 0.681) . Similar results to the primary findings in the earlier latencies of 200-400ms and 400–600 ms were also observed in Experiment 2’s datasets, revealing more positive ERP activity for context familiarity, albeit in more central-parietal sites of these participants (maximal at Cp5) (t(26) = 2.62, p = 0.007, one-tailed, Cohen’s d = 0.50, 95% CI [∞, −0.400], cFam: M = 1.93, SD = 2.71, SE = 0.52; Miss: M = 0.786, SD = 2.84, SE = 0.55; t(26) = 1.81, p = 0.041, one-tailed, Cohen’s d = 0.35, 95% CI [∞, −0.062], cFam: M = 0.019, SD = 3.08, SE = 0.594; Miss: M = −1.08, SD = 3.48, SE = 0.67, respectively). Together, these results from both experiments 1 and 2 revealed that neural activity for explicitly declared context familiarity judgments was reliably differentiated from that of implicit misses at the human scalp, during early latencies (200–400 ms) and later latencies , and across several different independent studies. This line of converging evidence refuted the null hypothesis that both conditions of context familiarity and implicit misses had the same functional significance or were representing the same cognitive process. 46 , 70 , 71 , 72 , 73 , 74 , 92 More broadly, recent work in the same experimental paradigm has also identified that other, more pure, kinds of implicit recognition processes can operate distinctly from misses, 18 but that pattern of implicit processing was entirely opposite of the negative-going central site patterns of ERPs characterized here for context familiarity, and thus not possible as a viable account of the present findings either. In sum, explicit context familiarity judgments were supposed to be equivalent to misses if representing similar implicit processing, but instead were significantly more positive than implicit misses in early epochs at frontal sites and then reversed to have significantly more negative voltage than at centro-parietal sites at later times in the epoch, thus providing clear evidence of divergent validity from the measures of implicit memory processing. This pattern for context familiarity cannot easily be attributable to accounts such as guessing or signals outside of episodic awareness and lending further weight against the possibility that context familiarity judgments were driven by implicit processes. We began the investigation to understand how (by which process) people make accurate source judgments for items they recognize with low confidence. Emphasizing reproducibility in science 86 , 87 , 88 , 93 , 94 , 95 , 96 , 97 the current study first replicated the original behavioral performance and ERP effects of context familiarity as previously reported. 1 Extending those findings, the main finding here was that the effects of item familiarity, context familiarity, and recollection were reliably dissociable in multiple measures of both behavior and neural activity; these ERP effects were then reproduced in independent datasets. These patterns of effects cannot be accounted for by a priori predictions made by models of only a single- or dual processes of episodic memory without making post-hoc modifications. Item- and context-familiarity thus appear to be operating as fundamentally different kinds of memory processes, based upon established norms for identifying cognitive processes via neuroimaging and behavioral data, 42 , 46 , 71 , 72 , 73 , 74 , 98 , 99 , 100 , 101 , 102 such that dissociable physiological activity occurring at different times, at different locations, and for different conditions, cannot be taken as evidence that they are reflecting the same cognitive process and must therefore reflect at least partially non-overlapping cognitive operations. Together, this provides strong evidence that these three observations of ERP effects of memory are reflecting mutually exclusive, distinct episodic memory processes, and therefore advancing an updated model of the organization of episodic memory as discussed in further detail across each finding throughout the sections below. Item familiarity and context familiarity were found to be behaviorally distinct in measures of response time, both in item judgments and in source judgments. Control analyses that held memory strength constant among conditions still preserved these differences and demonstrated that they were not confounded by recognition confidence levels. Item familiarity responses were faster during item judgments, whereas context familiarity responses were faster during source memory. It is difficult to account for these patterns of dissociations if attributing the responses to the same underlying cognitive processes of a single familiarity signal. 20 , 21 , 25 , 33 Context familiarity responses were also responded to significantly different than were responses of recollection, with recollection being faster than both item- and context-familiarity conditions during recognition and slower during source memory . There are two different ways that one can interpret these patterns of behavioral results: (1) They are consistent with existing findings of the relative speeds of recollection and familiarity (recollection being slower and familiarity being faster 21 , 55 ; (2) they are contrary to traditionally-held patterns of recollection being slower than familiarity. Each view will be discussed in more detail below, but importantly, regardless of interpretation, the memory conditions were reliably distinct in behavioral measures, indicating that mnemonic processing for context familiarity is distinct from item familiarity and recollection. Traditionally, recollection has been conceptualized as a slower, deliberative process, whereas familiarity was modeled as a faster, automatic process. 21 As reviewed in the Introduction, source memory judgments are typically considered a general (though not exclusive) measure of recollection, which in the present study was reliably slower during source memory than the conditions of both context- and item-familiarity responses. Those results from source memory RTs are thus consistent with traditional characterizations of recollection as a slower, deliberative process. 21 Accordingly, item recognition tasks are usually considered to be a relatively better measure of familiarity-based responding, and faster response times observed here for item-familiarity than for context-familiarity in the present findings can thus be seen as it being the item (not the context) that is familiar during this early stage of the memory task. In this view, that recollection responses were also faster in item recognition would be seen merely that their inclusion of the highest confidence responses was driving a concurrent higher item familiarity and thus faster recognition time, consistent with traditional patterns. As the context familiarity responses then go on to become faster than item familiarity responses during the source judgments, it preserves the traditional notion that familiarity is also a faster automatic process-this time it is familiarity for context that is differentially faster (not items). That is, in a traditional view of RTs results are seen as showing that the two different types of familiarity (item, context) are faster than each other in each of the respective domains in which they are tested for representing differentially (item recognition, source memory), and ultimately consistent with the common findings that familiarity operates faster, while recollection is a slower process. Alternatively, the pattern of faster recollection during item recognition responses (and slower than context familiarity in the source judgments) could also be seen as departing from common findings that recollection is generally a slower process than item familiarity. While some studies have found that recollection can sometimes be faster than familiarity in some instances, 103 , 104 that discrepancy was suggested to likely be due to instructions to participants that they respond after both memory processes are complete, thereby delaying the responses times. 21 The current paradigm also requires subjects respond to a combination of both item and source memory confidence ratings on each trial, which could similarly impose complex demands on their memory judgements. Moreover, recent studies noted below have reported recollection to have faster responses than familiarity during recognition, suggesting that the traditionally assumed speeds of recollection and familiarity should not necessarily always be taken as simply one-directional (i.e., that familiarity must always be faster than recollection) and that is likely more complex in directionality. For instance, a review of pupillometry studies of recognition memory concluded that, contrary to common observation of behavioral studies, recollection is not always the characteristic “slow and effortful” process typically assumed, but can also be quite “fast and easy” in its manifestation as a memory process. 105 When more advanced modeling methods were used for measuring the speeds of memory processes across six different experiments, recollection was again found to operate as both fast and slow, 106 similar to findings in the present data. Such patterns are also consistent with proposals that recollection is a two-stage process that includes both an initial rapid assessment of memory, plus a later second stage that is slower and more deliberative, 107 and with behavioral studies suggesting a third recognition process of familiarity. 108 Together, the response time profiles observed in the present work are consistent with other studies, 109 while also adding insight from specific memory response conditions that have not previously measured response times, to reveal that regardless of chosen interpretation views, the conditions remain nevertheless as neurocognitively distinct. In physiological measures, first, a triple dissociation of activity patterns was evident in general ERP effects observed during the basic memory measure of hits vs. correct rejections, qualified by a significant condition x time × location interaction that revealed a mid-central FN400 effect from 400 to 600 ms, a left parietal LPC Effect from 600 to 900 ms, and a BCN effect from 800 to 1200 ms, and replicated in a second independent dataset. Second, when investigating individual ERP effects of the specific memory conditions of item-familiarity and context-familiarity, each was found to be reliably different within-subjects and across subjects and was dissociated across the different latencies and locations on the scalp as noted above, and each was also dissociated from recollection. The same results were observed for each independent condition when contrasted against the standard ERP baseline condition of correct rejections; all of these findings were also reproduced in archival data from independent studies. This suggests that differential neurocognitive processes underlie each of the memory judgments, respectively. 46 , 71 , 72 , 73 , 74 , 102 The neural correlates for context familiarity did not exhibit the LPC nor the FN400 effects (the putative correlates of recollection and item-familiarity, respectively) in any analysis. The absence of such standard ERP effects was further bolstered by the Bayes factor results, which provided moderate evidence that both the LPC and FN400 were not present for these memory judgments instead of being merely undetectable. This conclusion is further bolstered by the finding that context familiarity exhibited significant effects present elsewhere in time and scalp location, so the absence of LPC and FN400 correlates could not be merely a case of insufficient statistical power to detect such differences. In our main ERP contrasts of each memory condition , context familiarity manifested effects later than item familiarity , and behavioral response times for context familiarity judgments were similarly later than those for item familiarity [although we note that during the source memory judgment context familiarity responses were faster than item familiarity ]. To a certain extent, the present work is not designed to directly answer why the times differ, but importantly is reporting the original finding that they are, in fact, different at all . One speculative reason is that context information may be broader than item information: encompassing additional elements/features that require neural pattern completion 110 , 111 , 112 , 113 , 114 - and that this could take the extra few hundred milliseconds that we observe here. We note here that the later ERP time interval was not the only time that context familiarity was found to exert an effect: it also differed reliably from item familiarity ERPs during the early epoch of 300–500 ms , too. We underscore that this early finding was important to the present work, because it demonstrated that the two memory conditions are occurring through differential patterns of neural activity early in the epoch (300–500 ms)- not just later. There should also be caution in over-interpreting the response time patterns of ERP effects earlier than the behavioral effects. The timings of ERP effects are occurring substantially before the later behavioral memory judgments, which might seem odd to researchers unfamiliar with ERP research on memory. However, this pattern of faster ERP times than behavioral times for the same memory processes has been generally true for most studies throughout the literature. This is because ERP effects have generally never been thought to represent a direct 1:1 mapping of memory process (as discussed in 22 ) but instead are widely regarded as “the putative neural correlates” of the memory processes. 47 That is, the ERPs are covert measures of memory-related activity, 80 but the overt memory behavior is generally thought to occur later in time (when people indicate their memory response) after the initial neural processing has set the stage for integrating the ensuing cognition and then deploying the actual motor response. So, there should be caution exerted in assuming too much about the timing of ERPs-what matters most, in our view here, is that they differ at all, and thus must be representing at least partially non-overlapping cognitive processing. 46 , 47 , 72 , 73 , 74 , 102 One possibility to account for the present findings is that results for context familiarity could perhaps “simply” reflect a variant of generic familiarity-based processing for which item and context are merely differences in levels but not kinds, such as the distinction between absolute (pre-experimental) and relative familiarity (in-the-experiment) of the stimuli. 75 , 76 This potential account would propose that the ERP measures of each condition of item- and context-familiarity would vary incrementally rather than fundamentally, similar to how ERP measures have been found to vary incrementally to incremental gradations of item familiarity strength. 1 , 115 , 116 Yet, that pattern was not found in the present study, which instead revealed the two conditions to vary in fundamental, not incremental, ways. The present effects of context familiarity were significantly dissociable from the effects of item familiarity-in measures of both behavior and physiology, so cannot be the same. 46 , 47 , 71 , 72 , 73 , 74 Thus, a shared, single generic process of familiarity cannot account for this pattern of memory responses that have been observed here and in several independent datasets. The ERPs for conditions of item- and context-familiarity were found to significantly differ in N400 potentials from 300 to 500 ms, and the N400 component has also been known to be influenced by factors such as context. 92 , 117 , 118 , 119 , 120 , 121 , 122 This association with context is consistent with the present interpretations of the psychological constructs we describe as “context familiarity” and was differentiated from the opposing patterns observed for item-familiarity. The posterior distribution of the FN400 effect for the item-familiarity condition is consistent with other findings ascribing it to a sub-type of absolute familiarity (as opposed to relative familiarity, which is reported to have a more anterior scalp distribution. 76 , 92 , 123 , 124 The FN400 effect observed in the present study had the same mid-central topography as was reported originally for this paradigm, 1 which is between the frontal and posterior distributions observed in other studies that manipulated factors of relative and absolute familiarity, 75 , 76 , 119 , 120 , 121 , 123 , 124 respectively. This mid-central distribution may likely reflect a combination of both relative and absolute familiarity with the stimuli in this particular paradigm since the study was not designed to differentiate those factors. Finally, accurate source memory has also been shown to be sometimes achieved through reliance on cognitive processes such as item familiarity 52 , 58 and unitization, 60 , 61 , 62 , 64 , 65 , 125 , 126 and the Source Monitoring Framework 55 , 56 has directly proposed that familiarity can support source judgments in some contexts. However, weighing against such possibilities of the source judgments being contributed to by those forms of unitized item-familiarity is that prior ERP findings for item familiarity and unitization contributing to source judgments are found as positive-going ERP difference effects, 52 , 76 , 125 instead of the significant negative-going ERP effects observed in the current data. Another possible account for the effects of the context familiarity condition is that perhaps recollection is a bit delayed and happening later in the epoch (Authors thank a previous Reviewer for suggesting this possibility). There are several broad reasons why it is likely that the ERP effects of the context familiarity condition could not plausibly be reflecting a “delayed recollection” process, which we will follow with several additional specific reasons. For instance, first there is scant evidence to support a contribution of recollection to the condition we measured as context familiarity: there has been no ERP evidence of the putative neural correlates of recollection for this condition across several different studies, and this absence of evidence has been quantified by Bayes Factor analyses providing consistent evidence of absence. Alternatively, the evidence that has accrued across various studies and measures continues to unambiguously indicate that the condition of context familiarity differs from recollection trials in substantive ways that include behavioral response times for both recognition and for source memory judgements, and several physiological measures across several experiments. The context familiarity condition also tends to be low confidence source judgments, which argues against the premise that a later process of slow recollection might be driving their trials following a delayed memory search. This is because if recollection occurred later then it could be assumed to have more high confidence correct source judgments (and yet the response data doesn’t have that profile). That is, if recollection is “delayed”, then the common features of recollection should also be evident in at least a reasonable proportion of those source memory response conditions for when that response is “eventually” happening, and those features of recollection commonly include high confidence responses for source memory. 20 , 21 , 34 , 35 , 54 , 115 , 127 , 128 , 129 Since that, too, runs contrary to the pattern of responses evident in the present data, it weighs against the theory of “delayed recollection”. This alternative account of delayed recollection would also de facto hold that a delayed recollection arrives later in time, even though the source judgments for this particular condition are provided faster than the source judgments in the actual recollection condition. As such, it is difficult for that to be reconciled in light of the pattern of behavioral responses. The “delayed recollection” account also would have trouble explaining why the context familiarity ERPs differed reliably from the item familiarity ERPs during the early latency of 300–500 ms if it was only representing a delayed process happening later after 1200 ms. For these reasons, it is improbable (and unsupported by evidence) to speculate that the effects of context familiarity could plausibly be reflecting a delayed recollection; however, more specific reasons to reject this theory also exist and are detailed below. The term “delayed recollection” appears only sparsely in the memory literature. One of the few extant studies of “delayed recollection” using ERPs 130 found that “delayed recollection” occurred under conditions of “changed viewing conditions”, which were not a part of, nor directly applicable, to the present study. Notably, that study did not find any negative-going ERP effects to be associated with their condition of ‘delayed recollection’, which was instead associated with the traditional, positive LPC measures (the opposite of the present findings). Since recollection typically produces a specific hallmark pattern of positive ERP effects, 44 , 45 , 47 , 48 and we instead observed a distinct opposite polarity of negative-going ERP effects, the logic to support a “delayed recollection” account remains lacking. That is, there is not a reasonable explanation for why the polarity of the canonical recollection-related physiology would have been reversed among our other control analyses demonstrating the standard positive-polarity of recollection effects in all other standard conditions of memory. This provides an additional line of evidence against a “delayed recollection” account and it is thus difficult to support as a viable account of our present findings. Another related ERP study 131 claiming “delayed recollection” did report a later negative-going difference for hits vs. correct rejections that they attributed as late parietal negativity (LPN, see section 5.4.3 below), but in their paradigm their reported LPN effect actually became more reduced in their “delayed memory condition”, and was instead larger in their condition of immediate memory retrieval. This, too, is the opposite of what would be predicted from a ‘delayed recollection’ account of the present findings, and thus it, too, ends up providing evidence against the “delayed recollection” account. Could the BCN effects we attribute to context familiarity instead be reflecting processes of non-criterial recollection 132 ? If that were so, one could predict that then there would still be an LPC associated with recollecting the non-criterial information-a pattern of results that we did not see in the present series of studies, and for which there was Bayes factor evidence of absence. The alternative theory of “delayed recollection” is further complicated by other problematic aspects: it would predict no differences in ERPs between the condition we call context familiarity and that of correct rejections because if there was not memory/processing yet (i.e., it occurred via a ‘delayed recollection’) then there would be no reason for the condition to differ from a non-memory condition baseline of correct rejections. But that is not what was found in the data: there is in fact a significant difference between the condition of context familiarity and correct rejections, and we need to then know why or, at least, be able to offer a plausible account to explain why that difference exists. Our account provides at least one viable and parsimonious explanation (it is the dissociable memory process of context familiarity), whereas the delayed recollection account does not provide an explanation. The theory of delayed recollection is additionally difficult to sustain when one thinks about it more deeply to its natural consequent end. It is, in effect, saying that recollection might be present later in time if only we were to look (a potentially unfalsifiable hypothesis). One problem with that is that it is improperly assuming a direct 1:1 mapping of cognitive processes to ERP effects, de facto assuming that the timing of one effect (ERPs) must coincide with the timing of another effect (the cognitive process). However, it is widely understood that ERP effects of memory are considered to be covert measures that occur separate to the overt subjective experience of conscious memory reported by the subject. 18 , 47 , 72 , 73 , 80 , 133 Thus, it requires acknowledging that both item-familiarity and recollection are both normatively occurring in a delayed manner evident in their reported response times (in the present data: 2.4 s and 1.8 s, respectively) that is preceded in time by their respective ERP correlates (e.g., ∼400 msec and ∼800 msec, respectively). That is, all recollection is delayed, at least in the sense that the traditional recollection condition’s responses are normatively delayed from the earlier ERPs effect correlates that are well-established as the LPC. Why then would it now be something different and special for this one condition of accurately retrieving the source-especially when considering that the context-familiarity source memory responses are notably faster (e.g., happening less delayed) than both recollection and item-familiarity? The post-hoc modifications needed to support the delayed recollection account’s uniqueness to the context familiarity condition while leaving the canonical ERP effects of item-familiarity and recollection unaccounted for are thus deemed untenable. Another common suggestion about the broad central negative (BCN) ERP effects in the present study is that they could represent the Late Parietal Negativity (LPN), (Authors thank two Reviewers for suggesting this possibility) which is a heterogeneous effect sometimes seen in studies of source memory and other tasks (for Reviews see 134 , 135 ). The LPN has been linked with a controlled search process for episodic memory and is coarsely thought to reflect the mental processes of re-constructing the prior episode. As a brief primer, the LPN was proposed to reflect a reconstructive or evaluative process in memory search when memory features are not fully recoverable. 134 , 135 , 136 Prior work studying the LPN has identified early and late variants of the LPN, with the earlier variant occurring approximately 600–1200 ms post stimulus onset and being variously interpreted as (1) a mnemonic search for context, 137 (2) re-activating context-specifying information from an encoded episode, 136 , 138 or (3) during later epochs that we did not measure , associated with the evaluation of fluency, 77 , 137 , 139 or (4) reflective of the absence of information . 140 The results here may appear at first glance to be similar to the late parietal negativity (LPN), in that they are negative-going ERP differences that occur relatively late in the epoch, and extend to some parietal regions (though observed here and previously to emanate from foci in more central regions 1 ). As such, the present findings could potentially be seen to fit one/some of those four different accounts of the LPN’s functional significance. However, upon closer inspection there are several reasons, described fully below, of why the LPN interpretation does not fit the observed patterns in the present datasets. Thus, the LPN interpretation remains problematic and difficult to reconcile. First in determining if the present study’s BCN might instead be the same effect as the late parietal negativity (LPN) is a direct appeal to what the authors of the primary review of the LPN have said about the matter, 135 where it was specifically noted that the LPN was not the same effect: “ … in the studies by Addante et al. … these components are unlikely the same ERP component as the LPN.” (page 631), and “Addante et al. examined ERP correlates of source memory for items that were recognized low confidence … the rather broad scalp distribution and the early onset of the negativity do, however, not necessarily support an LPN interpretation of this effect ”. (page 634 135 ). The original report of the negative ERP effects of context familiarity 1 also similarly provided why the two were not the same effects, concluding that: “… these factors make it difficult to attribute the observed effects as an LPN ” (page 448). So, neither of the authors of either of the two effects thought they were measuring the same things, and this bears some weight toward a measured interpretation of the findings. Another possible account of the present findings as an LPN effect is that they may reflect a “controlled search process” that is one of the core (albeit vague) functional significances ascribed to the LPN. 135 There are several lines of reasoning weighing against this possibility. In theory, every trial of the study should be inducing participants to engage a “controlled memory search” simply by virtue of the memory question being asked of them. That is, the participants are expected to be searching their memory on every trial for if they remember the item and the source of its encoding condition, then reporting it. In that sense, the controlled memory search would be a variable that is held constant across all conditions, thus canceling out in the contrasts, and leaving open the question of why this particular memory condition would show the negative ERP effect when compared to other conditions that also involve the searching of memory do not show the effect. Furthermore, if the present effect was representing a controlled search process, then it would have also presumably been seen in the recollection condition when compared to correct rejections, if participants searched memory for controlled recollection of the information. However, the same negative ERP effects weren’t seen in the recollection condition (which had far more trials included for statistical power, too), nor were the same effects seen for the item-familiarity condition in which participants were searching for the unknown source information as well, as described further below. Expanding upon this line of reasoning, consider the item familiarity condition (i.e., successful recognition memory hits, but lacking source memory): participants report that they do not have source memory and spend significantly longer response times on the source memory judgments. If participants had made a correct item judgment on the basis of item familiarity, and could not recollect the source of the event, then presumably they would certainly be engaging in a controlled search process of their memory to see if they could find that source information. This provides a solid premise to infer that the condition would also include a “controlled search of memory” for the source information that still comes up empty for them, and makes a clear hypothesis that we should see a major negative-going ERP effect on these trials if it reflected the same thing as the LPN. 135 Yet, that is not what the present data shows, nor in several of the independent replications of the finding, and instead we see that the item familiarity condition differs reliably and consistently from the context familiarity condition, and that they each reliably differ from a baseline condition of correct rejections. Hence, the present differences must be attributable to another variable than just a controlled search of memory, and this creates a challenge for the LPN interpretation. An additional point of differentiation of the present results from the LPN is the different patterns of topographic and temporal activities. Differences in topographical and temporal profiles of neural correlates of behavioral responses has been widely taken to reflect dissociable cognitive processes. 18 , 46 , 47 , 48 , 70 , 71 , 72 , 73 , 115 The LPN is generally found by other studies to be maximal in posterior parietal areas, whereas the current effect was observed in superior central regions of the scalp and distributed broadly to both frontal and central-parietal areas, and had been previously differentiated from posterior sites. 1 In regard to timing, the LPN has been previously characterized as beginning during periods after the epoch ended in the present study . 135 Among those irreconcilable factors was sensitivity to memory conditions: the LPN has been reported to either be invariant to source accuracy (i.e.,: it was present for both correct and incorrect conditions 137 ) or even to be larger for incorrect source judgments. 141 However, the current conditions’ effects have been found to be specific for correct source memory when compared directly to incorrect source memory. 1 Furthermore, other lines of evidence weighing against the LPN account of our findings comes from testing a direct prediction made that the LPN should be present “ not necessarily tied to successful memory retrieval but also observed during misses … ” (pg. 635 135 ). We did not find that prediction supported in the present data, as the present findings directly dissociated ERP effects of misses from the ERP correlates of context familiarity, which was also replicated, and thus adds to the considerable line of evidence weighing against an interpretation of the present findings for context familiarity as an LPN. A related problematic issue for an LPN account of the present findings is that the LPN is well-researched but poorly defined for its functional significance. The LPN is operationalized broadly as either a “reconstructing of the past” or a “controlled search process”, which, colloquially, only means “trying to remember”, which should occur on every memory retrieval trial to one extent or another and is difficult to reconcile with the present pattern of results. Due to the poor operational definitions available to the LPN, it is possible that the LPN findings from at least some other studies could instead be reflecting context familiarity. 138 , 142 That is, there is also the other possibility that some instances of reported results interpreted as an LPN might have actually been misinterpreted and reflecting the effects of context familiarity, instead of an LPN. In such cases, misinterpretation of effects could be due to lacking paradigmatic sensitivity (i.e.,: being able to dissociate influence of item familiarity from context familiarity, or recollection) or having lacking a theoretical framework to understand results as reflecting context familiarity (i.e.,: if one was only considering the existence of two episodic memory processes of recollection and generic familiarity). For instance, another laboratory’s study of contextual memory retrieval confidence of source information, their findings of a broad, Cz-centric ERP negativity at approximately 1000 ms occurring for source memory retrieval were interpreted as an LPN in that study 138 but their results’ characteristics could also be interpreted as reflecting context familiarity instead, given the successful retrieval of the context of source memory attributes in that study. Leynes et al. 142 also report BCN effects occurring from 500 to 800 ms post-stimulus in their Experiments 2 and 3 for recognition hits being more negative than correct rejections for the conditions of testing context in a masked word priming study, which could be seen context familiarity influencing mnemonic processing, much as was reported in an ERP study of cued recall with semantic primes. 85 In sum, the main, non-specific feature shared among the present findings and the LPN is that each is a negative-going ERP effect, but that in itself is not a very helpful descriptor, just as many other positive-ERP effects are not assumed to reflect the same thing just by virtue of their being a positive difference. The present findings are clear that the BCN observed in the present studies is evident to the specific condition of context familiarity and not reasonably ascribed to other negative-going ERP effects such as the LPN, nor cognitive operations associated with the LPN such as a controlled search process or reconstruction of the past, concurring with the prior observations of their differentiation, 1 , 135 respectively. It is also possible that the current results might reflect a form of guessing. In this view, effects may be reflecting conceptual implicit memory contributions to source memory, as has been seen in the form of guessing observed in prior studies of item recognition. 89 However, ERP findings of guesses indicated that guess processing was occurring much earlier in time (200–400 ms and 600–700 ms) than what we found in the current study , 89 so the current findings do not bear those hallmarks of guessing. The fact that two explicit declarative responses (item, source) were observed for judgments in the condition is also inconsistent with guessing, too, as is the above-chance performance of the responses. While it is possible people could have been successfully guessing in these item + source judgments, this possibility becomes increasingly less likely when considering that participants were given the option of saying they “don’t know” the source in our task’s paradigm , and which are thus excluded from the condition. Furthermore, if results were influenced by guesses, then it is notable that the purportedly accurate “guesses” would actually be driven by an implicit memory process (i.e., “recognition without awareness”). We conducted several direct analyses to assess if context familiarity ERP activity resembled the known hallmark signs of other implicit processing (i.e., misses) that would have been presumably supporting the guess behavior, but instead found that it was significantly different in both time and scalp location from implicit misses. That finding is further bolstered by another, different implicit activity recently observed in the same dataset for a purer form of implicit memory (IMAP) 18 that was found instead to manifest in right parietal regions in positive-going ERP effects from approximately 400 to 1000 ms . Prior work discussing the requirements for a condition being ‘recognition without awareness has established the criteria that information about context should be absent from the memory 143 – which also rules out the condition of interest for context familiarity in the present study. Taken together, the evidence weighs against interpretations of context familiarity being primarily the result of processes associated with guessing from underlying implicit memory signals. It is probable that, much like many explicit memory processes, context familiarity is also supported by forms of implicit processes (i.e., repetition priming, perceptual priming, contextual fluency) that then give rise to the explicit memory judgments, as has been found in other studies of item familiarity and cued recall. 85 , 144 , 145 Familiarity of context may naturally represent various forms of fluency in processing information which has linked negative-going ERP effects for old items to repetition and perceptual fluency. 52 , 90 , 91 , 120 , 146 As such, it is possible that these explicit response trials for context familiarity could be supported by an underlying fluency heuristic that leads one to accurate recognition and source judgments. According to this explanation, the negative ERP effect likely reflects the fluency heuristic. This effect might purportedly be obscured during high-confidence trials by the predominance of positive LPC effects associated with recollection that could override the presumed baseline negative ERP activity postulated to occur in recollection conditions as well. While such an explanation is hypothetically conceivable in this familiar context, where various possibilities exist, it remains speculative and lacks parsimony with the data. This is because it fails to explain why conditions of item familiarity did not display similar negative ERPs in the apparent absence of recollection. Additionally, the effects of context familiarity manifest later in time than the very early latencies (200–400 ms) where fluency is often found exerting its effects. 76 , 91 Through the series of systematic analyses here we demonstrate that there are still core distinctions between the kinds of familiarity represented by items and context, both of which surely are multiply determined by earlier sensory and contributory processes. We believe that based upon the combination of converging data and the divergent findings for studies measuring fluency at much earlier latencies, locations, and conditions, the present findings cannot be explained by mere fluency. There are several next steps which can be envisioned for future research based upon the present findings. For instance, intracranial measures of subsections of the medial temporal lobe regions such as the parahippocampal cortex and perirhinal cortex 147 , 148 , 149 , 150 , 151 , 152 , 153 , 154 , 155 , 156 offer clear hypothesis-driven tests of model predictions from the present work . Such kinds of studies can include both invasive and non-invasive stimulation approaches to both enhance 157 , 158 , 159 , 160 , 161 , 162 , 163 , 164 , 165 , 166 , 167 and disrupt 157 memory operations of different episodic processes, and assessing their relevance in real-world environments/context. 168 , 169 , 170 , 171 Other potential future approaches include exploring for neuropsychological dissociations among various types of MTL patients 172 or those with specified focal lesions of MTL subregions, 22 , 78 , 173 or electrophysiological searching for differential oscillatory bandwidths that may possibly be selective for each process. 19 , 147 , 148 , 149 , 150 , 162 , 174 , 175 , 176 , 177 , 178 , 179 , 180 , 181 , 182 , 183 , 184 , 185 , 186 , 187 , 188 , 189 , 190 , 191 In pursuing future studies, note that the specialized condition used in the present study to measure context familiarity (low-confidence recognition hits accompanied with accurate source memory) does not necessarily mean that it is the only potential way to measure context familiarity (i.e., the present study also found the ERP effects using general ‘old-new' recogntion contrasts, and in general source memory contrasts used in prior studies 22 ). Rather, it is likely just one of many possible approaches to capturing an elusive cognitive process. The findings here suggest the importance of including multiple measures of memory into experimental protocols, as it permitted the leverage needed to identify the combined conditions needed to measure context familiarity in the present experiments. We would expect future studies to find context familiarity to accordingly be measurable in other ways pending properly sensitive experimental designs that do not conflate it with recollection or item-familiarity or guessing. For example, we surmise that context familiarity could be found in future studies using different paradigms that systematically vary repetition of context while holding items constant and vice versa, similar to repetition paradigms used in prior fMRI studies 2 , 38 , 40 , 69 , 192 that found results similar to the present data. Our measure here was a combination of multiple responses (memory confidence scales) across multiple measures (item and source memory judgments), and thus reflects the sophisticated paradigmatic sensitivity that will likely be needed for follow-up explorations. Toward this end, studies should use an unequal confidence scale for source memory judgments to permit a “source unknown” response in order to avoid the guesses that are inherently integrated in balanced confidence scales 89 , 193 , 194 , 195 ; so, researchers should be sure to move from a commonly used 6-point confidence scale 38 to a 5-point confidence scale. 1 , 18 , 19 , 67 That is, including a “source unknown” option into source memory tests should be essential for future researchers to avoid contaminating source memory responses with instances of guessing or the source unknown. Another direction for future studies could be to assess the possibility of delayed recollection supporting the condition identified here as context familiarity. One way to do that could be by studying response locked analyses of ERP or oscillatory analyses, 175 , 176 , 177 , 178 , 179 , 196 as opposed to the stimulus-locked events studied here for the purposes of consistency to the literature of known ERP effects of episodic memory. 44 , 45 , 47 , 48 , 59 , 71 , 85 But it is important to also note that such studies of response-locked activity in this paradigm risk being confounded or conflated in their baseline period due to the preceding activity of the item recognition prompt/decision in in the present paradigm, so future studies would need to be very carefully designed to ensure those and other factors are properly disambiguated similar to as has been done in fMRI studies. 197 The medial temporal lobe region of the parahippocampus has been modeled by several frameworks to be a neural substrate supporting contextual representations of episodic memory 6 , 16 , 17 , 198 , 199 .This is based upon neuroimaging studies identifying activations of that region for contextual processing, 38 , 40 , 41 while ascribing item familiarity to the adjacent perirhinal cortex. 40 , 69 , 192 , 200 This has been paralleled by findings from studies using intracranial recordings showing temporal dissociations of familiarity and recollection processing amid the parahippocampal gyrus and hippocampus, respectively, 109 , 152 , 153 , 201 , 202 which has been similarly observed using functional neuroimaging methods. 151 Figure 11 Physiologically informed model of long-term memory processes Top, (A) The tri-component model presents an updated representation of the canonical declarative memory framework that is inclusive of physiological findings from physiological data indicating that recollection and familiarity are distinct processes of episodic memory, 22 , 46 and that context- and item-familiarity are also dissociable processes in episodic memory . Model depicts the emerging views that differential forms of implicit fluency contribute to driving explicit familiarity. 76 A sample schematic of recollection shown models it as a threshold process based upon physiological findings in the current and former data indicating that is limited to high-confidence responses and does not extend to low-confidence responses even if source memory is retrieved. 1 , 35 , 53 Context familiarity and item familiarity are each depicted as continuous processes of signal detection, with representative distributions of old and new information each. Bottom, (B) Anatomical and conceptual representation of medial temporal lobe processing of mnemonic information, based upon the “binding-items-in-context (BIC)” model, 6 , 16 updated here to include differential familiarity processes for items and context in the perirhinal and parahippocampal cortices, respectively. Note that the model presents a simplified representation of the breadth of complex memory operations and neuroanatomical substrates. Neuropsychologically, converging reports of the perirhinal cortex role in item familiarity come from a rare clinical patient, NB, with an epileptic resection lesion specific to the perirhinal cortex that left the parahippocampal cortex intact. 203 This patient was deficient in familiarity for items (words) but showed intact abilities of familiarity for pictures and faces, 173 , 204 , 205 , 206 which we consider here as broadly representing kinds of context in episodic events and this could be seen as performance consistent with the patient’s intact parahippocampus service of context familiarity (though it is possible that faces and/or picture stimuli might have potentially instead be treated by that patient as an item in certain studies with other contexts, or alternatively the face/picture memory performance might have potentially been supported by the other hemisphere’s intact perirhinal cortex). Similar reports of familiarity for contexts in the absence of object familiarity or recollection have emerged from neuropsychological 207 and normative studies using visual scenes of mazes, whereby it is the spatial layout of the scene that is experienced as familiarity and not the items or objects. 9 , 10 , 208 Together with the present studies, these findings converge to suggest an updated framework for the organization of long-term episodic memory from the canonical declarative memory model used ubiquitously throughout textbooks, 209 , 210 , 211 , 212 , 213 , 214 , 215 , 216 , 217 , 218 , 219 , 220 , 221 , 222 , 223 following from the BIC model that had originally motivated the hypotheses tested in the present work. 16 We propose an updated model to conceptualize the organization of specific episodic memory processes among a broader framework of long term memory , which is primarily based upon being specifically physiologically informed, whereas most prior models of long term memory have instead been either informed primarily by cognitive psychology, neuropsychology, or neuroanatomical/neuroimaging studies 6 , 15 , 16 , 30 , 31 , 210 , 224 , 225 , 226 , 227 (though see 76 for an example of a physiologically informed model of just familiarity-specific memory processing). Our new tri-component model provides an updated representation of the canonical declarative memory framework and BIC model, that is now inclusive of findings from physiological data indicating that recollection and familiarity are distinct processes of episodic memory, 1 , 22 , 46 , 47 and that context- and item-familiarity are also dissociable processes in episodic memory . This physiologically-informed model also encompasses the emerging views that differential forms of implicit fluency contribute to explicitly aware familiarity 76 , 90 , 119 , 120 , 121 , 124 , 146 , 228 and that other forms of implicit memory depend upon the hippocampus. 18 , 78 , 229 , 230 , 231 , 232 , 233 , 234 , 235 In our proposed framework , the episodic process of recollection is modeled as a threshold process based upon physiological findings in the current and former data, since the LPC effects were found to be limited to high-confidence responses and did not extend to low-confidence responses even if source memory is retrieved 1 ; for other related cognitive frameworks see. 34 , 53 , 58 Context familiarity and item familiarity are each depicted as distinct continuous processes of signal detection for relatively old and new information distributions, 1 , 115 , 116 , 236 based upon the current and prior data, and as opposed to the traditional model of them both merged within a single continuous process of general familiarity. The model also updates the understanding of the parahippocampal and perirhinal cortices as processing differential familiarity signals , as opposed to previous models depicting them to operate amid a single familiarity signal along both regions of the larger parahippocampal gyrus. 6 , 15 , 16 , 25 , 26 , 199 How might the subjective experience of context familiarity occur during cognition? We draw upon our proposed additions to the BIC model/variants 6 , 15 , 16 to suggest a cognitive framework that can be built to understand how the condition of context familiarity may arise or manifest (note: this is a proposal of how it might occur in some instances but should not be taken as suggesting that it is how recognition works in every case, which has inherent variability). This account both echoes and extends models provided by prior work investigating the role of context in episodic memory, 1 , 17 , 27 , 192 while integrating fluency heuristics. 76 , 90 , 120 It has been proposed that people use a fluency heuristic to make recognition judgments, 76 , 90 and the current findings extend that by suggesting the possibility that a fluency heuristic can also be used to support accurate source memory judgments, too. This would be based upon an underlying fluency for context, as opposed to fluency for items or conceptual/perceptual dimensions. By our account, as a retrieval probe is first shown on a screen by itself, the item does not at first seem familiar (i.e., no FN400 for item familiarity from ∼300 to 600ms that we observed). The context, however, may seem familiar, even if they do not recollect the details of either the item or how the context was bound with other details from the prior episode. Akin to how item familiarity can be derived from underlying fluency of the stimuli, so too would context familiarity be facilitated by a separate underlying fluency for the context. If the context does not initially seem familiar from ∼300 to 600 ms, then in the ensuing time (600–900 ms, LPC) the participant would continuing to mentally look back in time for any context in which to place the item being shown on the screen (e.g., ‘where might I have seen that before?’), seeking the pattern completion process to autocomplete a recollection of the relevant details from the prior event. During that time (600–900 ms), that autocompletion process of binding any existing neural signals of context together with the presented item fails (i.e., our observations of an absence of evidence for the LPC correlate of recollection), consistent with the slower response times observed for these item judgments. As time continues the person continues to assess their memory for any connections of details, something about the stimuli remains diagnostic for prior exposure (the BCN effect emerges to reliably differentiate from correct rejections and item familiarity), which could be either the explicit familiarity of a contextual detail such as the encoding source or the implicit sense of its contextual fluency for associated information about the item. The successful retrieval of some familiar contextual information would be enough to logically deduce that the item must have been old. 4 , 5 That is, one can then draw upon that awareness that the context seems familiar to derive the logical inference that the item is likely old, akin to how other studies have found item familiarity can lead to false alarms in associative recognition by similar logical deductions. 12 , 197 , 237 , 238 , 239 Ultimately, this basis of a retrieved prior context is sufficient to support an accurate hit in item recognition, albeit with low confidence because the item itself is not remembered directly, while the correct source is identified in the ensuing judgment based upon that retrieved familiarity of the context of its prior exposure. 5 The item is thus accurately recognized due to the context seeming familiar: context familiarity. Importantly, our tri-component account provides an improved precision in characterizing human episodic memory, and as such could help to better understand disparate findings from memory studies and neuropsychological patient impairments that occasionally arise in the literature. That is, if prior studies had been conflating distinct familiarity processes for items and context, and conflating context familiarity with instances of recollection, when occurring in differential proportions and under differential experimental conditions, it could easily produce disparate clinical findings that have been reported about source memory, context, recollection, and neuropsychological impairments thereof. 6 , 15 , 20 , 22 , 29 , 31 , 172 , 173 , 205 , 206 , 227 , 240 , 241 , 242 , 243 If we are now able to delineate between these three distinct but overlapping processes of memory, it could help illuminate a better understanding of the nature of memory impairments and lead to better precision in types of amnesia diagnoses. Here, we put forth a fundamentally different view: that production of familiar context does differ qualitatively from the production of familiar item information and provide converging lines of support that the familiarity of items and familiarity of contexts are evidently operating as separable independent neurocognitive processes. In conclusion, the field of memory research is replete with complexities in memory behaviors in normative and clinical patient populations that are difficult for existing theories to fully account for. We show here that the common measure of source memory can be supported by a disparate process that is independent of both recollection and item-familiarity. The memory condition of context familiarity was found to be distinct from item familiarity, in addition to previously identified dissociations from recollection. This conclusion was evident from triple dissociations observed in both behavioral and physiological measures. Moreover, the differences in item-familiarity and context familiarity reflected differential neural processing at both the individual subject level as well as trial-wise differences in memory processing that was identified within-subjects and were replicated across several independent studies. These findings cannot be accounted for by relying upon using only two processes of episodic memory for interpreting the results. 36 , 54 Item familiarity and context familiarity are therefore seen as fundamentally different kinds of familiarity processes independently operating in service of episodic recognition 46 , 70 , 71 , 72 , 73 , 74 : one process for items and another for context, and each distinct from recollection. This extends traditional dual process models of recognition to reflect that context familiarity constitutes a third process of episodic memory. ERP studies are inherently limited in spatial resolution of their topographic distribution of effects and are generally unable to provide source localization of the subdural cortical and subcortical source generators of the effects recorded at the scalp. 80 , 196 , 244 Therefore, spatial distribution of the physiological effects reported here should be expected to have a reasonable amount of variability in future studies, and may vary upon different experimental factors, protocols, stimuli, and paradigms. While the physiological effects of the present study’s main Experiment 1 18 , 67 were found to be reproducible in independent datasets collected from different demographics in different universities (Experiment 2), 1 , 19 , 22 , 78 Experiment 2’s archival datasets were unable to collect meaningful response times to memory judgments due to a protocol that asked participants to withhold their response until after viewing the memory probe for 1.5 s, thus rendering the response time uninformative and unable to asses for reproducibility of the results from Experiment 1. Therefore, while the physiological effects that formed the backbone of the present study were independently replicated, the behavioral results remain to be assessed for the extent of their potential replication among future studies of larger samples. The current study (Experiment 1) consisted of a re-analysis of previously published ERP data, 18 , 67 so the sample size was determined by the number of participants’ data that was available in the archival dataset; the study was not pre-registered. The participants consisted of 61 right-handed students free from neurological disorder and memory problems. Data was not used for four participants due to noncompliance issues (i.e., pressed only one button throughout the task or ignored experimenter’s instructions), data lost due to experimenter error (N = 1) or EEG data deemed unusable (N = 2) due to excess motion artifacts/noise that resulted in an exclusion of the majority of EEG trials. This presented a working data set of N = 54 for the current study, which was more than double the size of the original study reporting physiological correlates of context familiarity (N = 25), 1 and triple the sample size standards of many of the foundational ERP studies of recognition memory. 42 , 46 , 102 , 115 , 245 , 246 , 247 , 248 , 249 As described by the data’s original publication 67 : Participants were recruited through a combination of methods including advertisements placed around CSUSB or through the schoolwide research pool SONA. Participants recruited through advertisements were paid $10 an hour for sessions that lasted approximately 2 h. The majority of participants were women ( N = 48); 57% were Hispanic, 23% Caucasian, 11% Asian, and 10% identified as more than one ethnicity. The average age was 23.5 years old ( SD = 4.82). Findings do not apply to only one particular sex, gender, or demographic; the study was a secondary analysis of archival data, 1 , 18 , 19 , 22 , 67 and thus was constructed to be neutral in consideration of any particular sex or gender or other demographic factor. Inclusion of participants was based upon the nature of the archival data that was available (we included all datasets available), which had included anyone who volunteered to participate for the research from broad advertisement efforts on the campus of California State University – San Bernardino, and determined based upon self-reporting. Since the pursuit of the present study was one that sought to assess the reproducibility of prior findings, analyses were performed agnostic to sex and gender or other demographic factor in order to faithfully reproduce prior findings of that had adopted the same approach. There are no known reasons for why an absence of demographic-specific analyses would limit the study’s generalization, and the findings were observed to be reproducible across several independent studies that comprised different demographic proportions and produced results in line with traditional findings of ERP effects of memory in the field across many labs, countries, and demographic distributions since the early 1990s. None of the participants reported any visual, medical, or physical issues that would interfere with the experiment. Most participants spoke English as their first language ( N = 47) and those who had indicated speaking a different first-language had been speaking English for an average of 16.73 years ( SD = 4.74, SE = 1.22, Median = 17.0, Minimum = 7, Maximum = 25). The participants included in Experiment 2 were aggregated across three previously published studies 1 , 19 , 22 , 78 that used nearly identical experimental protocols of item recognition confidence followed by source memory judgments as were used in the present Experiment 1, 18 for a total sample of N = 56. These studies had only directly contrasted conditions of recollection and context familiarity, but had never directly assessed if the ERPs for item- and context-familiarity differed. 66 Each study’s cohort of participants have been previously reported for having intact levels of familiarity-based recognition on both behavioral and electrophysiological measures that did not significantly differ as a function of younger/middle aged adults. 22 , 66 , 78 They have therefore been previously treated as equivalent cohorts of familiarity-based recognition, which was the subject of the present investigation, 22 , 78 and thus we also treated them accordingly here as well. The participants were reported in prior studies 1 as consisting of twenty-five healthy right-handed undergraduate students (Mean age = 20.4 years [SD = 2.9, SE = 0.60, Min = 18, Max = 30], mean education level = 14.4 years [SD = 1.8, SE = 0.4, Min = 12, Max = 19] demographics of 52% Caucasian, 20% Hispanic, 20% Asian, 8% more than once race; seventeen female) and twenty-two 1 , 19 right-handed undergraduate students (Mean age = 20.9 years [SD = 3.1, SE = 0.70, Min = 18, Max = 29], Mean education 14.5 years [SD = 1.4, SE = 0.30, Min = 13, Max = 17], demographics of 55% Caucasian, 9% Hispanic, 5% African American, 32% Asian; ten females), all recruited from the University of California–Davis Psychology Department subject pool. They received credit for participation and were free from neurological, visual, motor, or other medical disorders. An additional nine middle-aged participants (six female; Mean = 45.4 years, SD = 12.5, SE = 4.2, Min = 27, Max = 57) were included whom were tested on the same memory protocol (six male) 22 , 78 and had a comparable profile of education (M = 16.8 years, SD = 5.7, SE = 1.9, Min = 12, Max = 31) and majority demographics (78% Caucasian, 22% more than once race). The paradigm used was the same item- and source-memory confidence paradigm that has been routinely used in our prior reports to characterize various facets of memory, 1 , 18 , 19 , 22 , 67 , 78 , 166 with slight modifications as noted below. 67 This paradigm consisted of an encoding phase containing four sequential study sessions during which participants studied 54 words per session, followed by a retrieval phase that contained six test sessions in which the participant’s memory was tested for 54 words in each session. Item recognition was tested on a five-point confidence scale with ‘1’ indicating being sure the item was new, and ‘5’ indicating being sure the item was ‘old’ . During the encoding phase, participants were given instructions to make a simple decision about the word presented. The participants were either asked to judge if the item was man-made or if the item was alive. The instructions were presented in one of two counterbalanced orders: ABBA or BAAB. The participants viewed four lists of 54 words during the encoding phase. After the encoding phase was complete, the EEG cap was applied to the scalp of participants. During the memory test, participants viewed a total of 324 words, 216 of which were presented in the encoding phase and 108 of which were unstudied items (new items). All words presented during study and test were presented in white font on black background screen. Word stimuli were selected from the same source as stimuli used in the original study in 2012 and preserved the same characteristics. Word stimuli were selected from the Medical Research Council Psycholinguistics Database ( http://www.psych.rl.ac.uk/MRC Psych Db.html). Word stimuli were all nouns, had an average rating of concreteness of 589 (min = 400, max = 670), imageability of 580 (min = 424, max = 667), Kucera–Francis Frequency of 30 (min = 3, max = 198), and an average number of 4.9 letters in each word (min = 3, max = 8). During the memory retrieval phase, the participants were read instructions asking them to judge if the stimulus word presented was old (studied during the encoding phase) or new (not studied before in the encoding phase) . To begin a trial, a screen with a small white cross at the center was presented for one of three randomly chosen times: 1 s, 2.5 s, or 3 s. Then the participants were presented with a word in the middle of the screen, the numbers 1, 2, 3, 4, and 5 evenly spaced beneath the word, the word “New” on the left by the number 1, and the word “Old” on the right under the number 5. Participants pressed any number between 1 and 5 to indicate if they confidently believed the word was old (5), believe the word was old but was not confident (4), did not know if the word was old or new (3), believe the word was new but was not confident (2), or confidently believed the word was new (1). This prompt was subject-paced, and participants were told to choose the response that gave the most accurate reflection of their memory, and to respond as quickly and accurately as possible. Immediately after each decision on item memory confidence, participants were asked to answer a source memory question about if the word came from the animacy decision task or the man-made decision task. The word and numbers remained on the screen but this time, word “Alive” was presented on the left by the number 1, and the word “Manmade” was presented on the right under the number 5. Participants were told to choose the response that gave us the most accurate reflection of their memory and could respond that they confidently believed the word was from the animacy task (1), believe the word was from the animacy task but was not confident (2), did not know the source of the word or had replied in the question directly before that the word was new (3), believe the word was from the manmade task but was not confident (4), or confidently believed the word was from the manmade task (5). Trials were terminated by subject response. Each session consisted of a list of 54 words. Six lists of 54 words were presented during the retrieval phase. The retrieval phase of the paradigm also included a simple metacognitive question to participants for estimating their performance, which occurred once every ten trials and for which the data has been previously reported elsewhere for metacognition effects. 67 The current investigation focused instead upon the memory related responses, as the metacognitive response data has been reported previously. Each session consisted of a list of 54 words; overall, six lists of 54 words were presented during the retrieval phase. Following common convention in the field, we operationalized these processes based on the combination of responses given for item recognition confidence and source-memory judgments, consistent with the way prior research has defined these conditions. For recollection, we followed the convention of prior findings that it represented instances of retrieving high-confidence item recognition followed by correct source information: item responses of ‘5’ accompanied by source judgments that were correct ( Figure 1 . 1 , 22 , 67 ; For context familiarity, we followed the same approach used to originally identify this process 1 : instances of low-confidence item recognition accompanied by correct source judgments . Item familiarity was defined based upon previous work 18 , 19 as instances in which participants retrieved successful item recognition (hits, combining 4 and 5 responses) but reported having no source memory information (source unknown, 3 responses) . These conditions of item- and context-familiarity are ones which have never been directly compared against each other in the literature while using behavioral or ERP measures, and so it remains to be determined if and how they may differ, and the extent to which the process of familiarity may be organized among these variables as a continuous or distinct process(es). Response times for item recognition and source memory responses were measured as follows: item recognition times were measured from the onset of the memory probe (i.e., the word) to the button press of the recognition decision; source memory response time was measured from the onset of the source memory prompt (which happened immediately upon the end of the item recognition judgment) to the button press of the source memory decision. Each subject was tested individually inside a private chamber. Stimulus presentation and behavioral response monitoring were controlled using Presentation software on a Windows PC. EEG was recorded using the actiCHamp EEG Recording System with a 32-channel electrode cap conforming to the standard International 10–20 System of electrode locations and was acquired at a rate of 1024 Hz. The EEG cap was sized while the participant’s face was wiped free of skin oil and/or makeup in preparation for attaching ocular electrodes. Five ocular electrodes were applied to the face to record electrooculogram (EOG): two above and below the left eye in line with the pupil to record electrical activity from vertical eye movements, two on each temple to record electrical activity from horizontal eye movements, and one electrode in the middle of the forehead in line horizontally with the electrode above the left eye as the ground electrode. EOG was monitored in the horizontal and vertical directions, and this data was used to eliminate trials contaminated by blinks, eye-movements, or other related artifacts. The EEG cap was placed on the participant’s head and prepared for electrical recording. Gel was applied to each cap site and impedances were lowered below 15 KOhms via gentle abrasion to allow the electrodes to obtain a clear electrical signal. Subjects were instructed to minimize jaw and muscle tension, eye movements, and blinking. Physiological measurements of brain activity were recorded using EEG equipment from Brain Vision LLC. All EEG data was processed using the EEGLAB and ERPLAB toolboxes in MATLAB. 250 , 251 The EEG data was first re-referenced to the average of the mastoid electrodes, passed through a high-pass filter at 0.1 Hz as a linear de-trend of drift components, and then down sampled to 256 Hz (Hz). The EEG data was epoched from 200 ms prior to the onset of the item recognition stimulus to 1200 ms after the stimulus was presented. 67 Independent components analysis (ICA) was performed using Infomax techniques in EEGLab 252 for artifact correction and the resulting data was individually inspected for artifacts, rejecting trials for eye blinks and other aberrant electrode activity. During ERP averaging, trials exceeding ERP amplitudes of +/− 250 mV were excluded, as described in the previous report of this data. 18 , 67 A 30 Hz low pass filter was applied to each subject’s ERPs as a non-causal, infinite impulse response (IIR, Butterworth) filter, implemented through ERPLAB toolbox. 251 In order to maintain sufficient signal-to-noise ratio (SNR), all pairwise comparisons relied upon including only those subjects who met a pre-determined criterion of having a minimum number of 12 artifact-free ERP trials per condition being contrasted. 79 , 85 , 253 , 254 , 255 As was noted in the Introduction, the present work is not claiming any specifically process-pure ERP measurements of memory, but instead we adopt what others 44 , 47 , 48 , 59 as well as our own previously published positions 1 , 18 , 22 , 49 , 67 , 78 , 85 described as the ‘putative neural correlates’ of recognition memory processes. The current study’s interest focused specifically upon combinations of item + source memory responses described below. Like most studies of source memory, we wanted to know how accurate people were in discriminating between the two sources of information in the experiment, and so excluded the source unknown responses from the performance calculation of source discrimination. This procedure collapses high- and low-confidence source judgments into general ‘correct’ and ‘incorrect’ conditions. We thus calculated this source memory performance in the same way as had been done in prior studies 1 : the total number of source correct trials divided by the total number of all correct & incorrect source memory judgments (excluding the source unknown responses) [ # correct source trials/(sum of # of correct + # of incorrect source trials)]. The source memory calculation we used is akin to the standard recognition measure of memory performance (%Hits-%FA) but applied to source memory discrimination, which would require excluding the 'source unknown' responses, since a false alarm in source memory would be a source misattribution (not the lack of source memory). When source memory discriminations occur between available options of the two sources for participants to choose from, they have a 50% chance or being right/wrong (source correct or source incorrect responses), thus chance levels are at 50% in that analysis (as opposed to 33% if assessing it relative to the three response options of correct, incorrect and unknown; see discussion below for further considerations of the limitations of such alternative calculation methods (Authors acknowledge a previous Reviewer for suggesting consideration of this alternative approach)). In excluding the ‘source unknown’ responses from the calculation of performance on source memory discrimination, it excludes the trials of when people had an absence of source memory. 194 , 195 This approach was taken because we did not want to know how frequently people remembered correct source information at all (i.e.,: out of all of the possible times that they could have), because this number would be artificially low, but rather how well they discriminated sources that they did remember. For example, consider a scenario in which people were tested on if episodic memory for where we learned various items of information in our lives. Then consider if the performance measures for memory measured the few times that we did remember correctly amid all the times when we couldn’t remember any source at all (likely most of the time, for naturalistic settings). Most people would [falsely] appear to be dense amnesiacs unable to retrieve source information above chance-despite likely having reasonable source discrimination capabilities for the times that they could retrieve source memory information. The same is true for clinical applications of the same principle: consider cases studying reality monitoring of auditory hallucinations in schizophrenia patients, whereby the pertinent question is about understanding how well the source determination is made when a source of the information is available to the patient-not the other instances in which it isn’t. Our approach to measuring source memory performance is a common one that is sensibly used in other source memory studies. 60 For example, our standard approach used to measure source memory performance is not just the same as we have published with previously 1 but is also the same approach used by additional other studies measuring source memory performance, 256 where they note that: “ Conditional source measures control for overall differences in recognition by calculating the probability that the correct source is identified given that the item was correctly identified as old (p|source| = source hits/source hits + source misattributions)” . For these reasons noted above, we utilized the present method of calculation for source memory discrimination that excluded the ‘source unknown’ responses, as it provides the most appropriate treatment of participant’s memory while minimizing the extent of possible confounds. This approach also avoided the potential confounds of not knowing what response criterion a subject is adopting when making the ‘unknown’ response for source memory, as no two participants could be presumed to use the same such criterion. Importantly, it also ensured that the pursuit of replication could be achieved by using the same measures as were used in the preceding work we sought to assess for reproducibility. 87 , 88 Our hypotheses focused upon identifying three different processes of episodic memory: recollection, item familiarity, and context familiarity. We hypothesized that recollection would exhibit both an FN400 for item-familiarity and an LPC for recollection but would not exhibit a late broad central negativity (BCN) effect, whereas context familiarity would exhibit only an BCN and neither an FN400 or LPC, and that item-familiarity would be associated with only an FN400 effect but not an LPC nor BCN effect. As the current investigation was based upon clear a priori-defined hypotheses derived from prior findings, 1 analyses utilized planned paired t-tests to assess differences between the targeted conditions (two-tailed unless indicated otherwise) with an alpha level of 0.05. In cases where exploratory analyses were conducted to explore unplanned comparisons, ANOVA was used to qualify potential differences that may exist and were corrected with Geisser-Greenhouse corrections when necessary. Analyses conducted to assess the extent to which prior results could be found to replicate in independent datasets and/or were principally based upon a priori predictions that effects would differ in one way (i.e.,: context familiarity exhibiting more negative-going ERP amplitudes than correct rejections, item-familiarity 1 ); such analyses thus applied targeted directional hypotheses utilizing one-tailed t-tests where noted in order to maintain a properly principled statistical approach, though most were also still significant with non-directional two-tailed tests as well (i.e.,: multiply the p value by 2). In reporting results of behavioral response times, we followed convention in the field to report the mean values of reaction times. When outlier data points were removed from the mean values ( N = 2, using criteria of exceeding 3 standard deviations from the mean) the results from the analysis of mean response times remained the same. In this case, the two outlier subjects had longer mean RT values, merely indicating that some subjects sometimes responded a few seconds slower on uncertain memory conditions, which is common knowledge of reasonable variability in cognitive capabilities during conditions of uncertainty. Also, these inclusive data points are the aggregate of multiple responses for a person instead of a singular outlying value that might otherwise be considered as potential artifacts. Removing outliers based upon their post-hoc values would thus be inappropriately biasing the data based upon post hoc visual inspection of it. Therefore, we report mean reaction times with outlier data points included, since they represent the real average response times of subjects. Because a non-significant p -value in conventional frequentist t-test methods is unable to inform whether there is actually evidence for the null hypothesis or if there is merely not sufficient evidence for any conclusion at all, analyses that revealed a null finding were subsequently quantified using Bayes Factor (BF) analysis. Bayes Factor analysis is a tool that allows researchers to quantify the relative strength of evidence for the null hypothesis (invariance). 258 , 259 , 260 , 261 , 262 , 263 , 264 , 265 , 266 , 267 , 268 A resulting Bayes Factor value can generally be considered as the ratio of how likely a hypothesis is as compared to the likelihood to another hypothesis (for specific details see 262 , 265 ). For interpreting Bayes Factor result values, it is conventionally viewed that a Bayes Factor (BF 01 , evidence for the null hypothesis) of 0–1 typically represents there being no evidence available for the null hypothesis, 1–3 typically represents anecdotal evidence for the null hypothesis, a BF of 3–10 represents relatively moderate evidence for the null hypothesis, and a BF of 10–30 represents relatively strong evidence for the null hypothesis. 262 , 263 , 268 Thus, Bayes Factor results are reported as such for the pertinent analyses as representing the relative strength of evidence for the null finding as noted above (BF 01 ). The Bayes Factor analyses were computed for paired or one-sample t-test designs. Results were calculated using the standard scale of r = 0.707 and the resulting outputs are provided as values in favor of the null hypothesis using the recommended Jeffrey-Zellner-Siow Prior (JZS, Cauchy distribution on effect size). 265 The reproducibility of the effects of context familiarity was assessed in several ways through the present investigation. First, the present investigation (Experiment 1) was an assessment of the extent to which previously-reported effects 1 , 19 , 22 of context familiarity originally differentiated from the neural correlates of recollection in a sample of N = 25 could be found to be reproducible amid a larger sample ( N = 54) that had been previously published in the domain of memory metacognition. 18 , 67 Those original effects replicated in entirety in the present data. The sample of data of the present study thus demonstrated sensitivity of effect sizes and statistical power to detect the ERP effects of context familiarity originally discovered by the original study 1 by virtue of it having reproduced those findings of differences between context familiarity and recollection in a sample ( N = 54) 67 that was more than double the original sample size ( N = 25). 1 Second, when the present findings (Experiment 1) then discovered that the neural correlates of context familiarity were dissociable from those for item familiarity, we sought to assess the extent to which these findings would have been observed in the original study 1 if those authors had only thought to look (they hadn’t). These effects of the present investigation were also found to be replicated in entirety (Experiment 2), both within-subjects and between-subjects, and across the same control analyses. Third, we also sought the extent of reproducibility of the findings across a range of several control analyses conducted in both Experiments 1 and 2, which systematically varied different conditions to assess potential confounds and possible alternative accounts. The control analyses found that the results remained reproducible even when reducing signal to noise ratio of trials and when reducing statistical power limited to only low levels of familiarity strength (excluding trials of strong familiarity responses). Fourth, as the present study was a secondary analysis of existing independent datasets that had already been published with sufficient power and sample size to detect differences in previously published papers, 1 , 18 , 22 , 67 it was not possible nor necessary 269 , 270 , 271 , 272 , 273 to perform an a priori power analyses for the present investigation since the sample size was already immutably established before the present work’s secondary investigation of analyses began. Similarly, widespread agreement in the literature that post-hoc power analyses would be both inappropriate and uninformative 269 , 271 , 272 precluded their use here. The present results were fully replicated in independent data, and in twice the sample size of previously published results that the present investigation also replicated; thus, the sufficiency for statistical power in the study is self-evident. | Review | other | en | 0.999999 |
PMC11699258 | Dental implant therapy is a popular method for replacing missing teeth; however, it can lead to technical and biological complications known as peri-implant diseases. 1 These biofilm-induced inflammations affect soft and hard bone tissues around osseointegrated implants. There are two categories of peri-implant diseases: peri-implant mucositis and peri-implantitis. 2 Peri-implant mucositis is a reversible inflammation of the mucosa around the implant, 3 while peri-implantitis involves progressive bone loss. 4 The prevalence of peri-implant mucositis ranges from 23.9% to 88%, and peri-implantitis varies from 8.9% to 45%. 5 , 6 Although plaque accumulation is the main cause, 7 other risk factors include smoking, history of periodontitis, lack of regular periodontal maintenance, diabetes, implant design or surface characteristics, and excess cement. 8 , 9 The standard approach for treating peri-implant mucositis is non-surgical treatment, which involves reinforcing oral hygiene practices, including professional and patient-administered plaque control techniques to mechanically remove microbial plaques from the implant surfaces. Studies have investigated adjunctive or alternative methods and non-surgical mechanical debridement for treating peri-implant mucositis. 10 These methods include antimicrobial photodynamic therapy, antiseptics, topical or systemic antibiotics, abrasive devices, laser therapy, home care mouthwashes, and probiotics. 11 - 14 However, it should be emphasized that regardless of the treatment used, adequate plaque control is important for the complete resolution of the condition. 15 Statins, commonly prescribed for lower lipid levels to prevent cardiovascular events, have shown potential for treating periodontal diseases. Studies have demonstrated that statins can reduce tooth mobility, tooth loss, and bone resorption in patients with chronic periodontitis. In addition to their lipid-lowering effects, statins possess anti-inflammatory, immunomodulatory, antioxidant, antithrombotic, and endothelium-stabilizing properties. They can also promote angiogenesis and stimulate bone formation. 16 , 17 Recent studies have shown that patients receiving statin treatment for chronic periodontitis have fewer pathological periodontal pockets than those not receiving such medication. Atorvastatin (ATV), in particular, demonstrates inhibitory effects on inflammatory cells and matrix metalloproteinases that play a crucial role in degrading connective tissue in periodontal diseases. 16 Akram et al. 18 found that 1.2% ATV gel applied locally improved clinical and radiographic parameters significantly. ATV was more effective than other statins, such as simvastatin, pravastatin, lovastatin, and fluvastatin, in lowering low-density lipoprotein. According to this study, ATV may be more effective than simvastatin in promoting bone regeneration in periodontal defects, reducing probing depth (PD) and clinical attachment level (CAL), and exerting anti-inflammatory effects. 17 , 19 Although none of the recent studies demonstrated the effectiveness of ATV gel once it is used locally to treat peri-implant mucositis, the present study was undertaken to evaluate the effectivity of 1.2% ATV gel in addition to mechanical debridement for the treatment of peri-implant mucositis. This study was necessary as statins are very effective in anti-inflammatory effects, and a limited number of studies have been conducted in this field. In this double-blinded randomized controlled clinical trial, 49 patients (20 males and 29 females, aged 40–60 years) diagnosed with peri-implant mucositis were selected from patients referred to a private periodontal office and the Faculty of Dentistry, Tabriz University of Medical Sciences. The patients were blinded to the type of treatment they received randomly (ATV or placebo gel), and the examiner was unaware of the patients’ allocation to the test or control groups. The researcher was aware of the interventions administered. Forty-nine patients were selected based on the selection criteria, and after enrollment by an examiner, the patients were randomly allocated to either the test or control group. The randomization method was simple randomization and conducted using the RandomIZE Randomization tool app. The sample size was estimated to compare the average of the two groups from the respective formula with 95% confidence and 80% power, and an effect size equivalent to that of a similar study by Pradeep et al 21 equaled 21 participants in each group. Owing to the existence of three stages of follow-up and the possibility of dropping samples by 30%, the final sample size increased to 27 people in each group and 54 in total. Throughout all analyses of the research findings, the investigators were not part of or aware of the randomization process. After mechanical debridement in both groups, 1.2% ATV gel (1.2 mg/0.1 mL) was injected into the pockets around the implant in the test (ATV) group and placebo gel in the control (placebo) group. Mechanical debridement was performed using a plastic curette at the baseline for each patient. Prepared 1.2% ATV gel (1.2 mg/0.1 mL) or placebo gel was injected into the pockets around the implant using an insulin syringe in the test and placebo groups . Cyanoacrylate tissue adhesive was used to protect the area. After treatment completion, the patients were not prescribed antibiotics or anti-inflammatory drugs. They were given specific instructions for a week, including refraining from chewing hard or sticky food, brushing near the treated areas, and using any interdental aid. At one and three months after the intervention, all clinical parameters were measured again in both groups in the same area. The evaluation involved recording various clinical parameters such as BOP, PD, CAL, and pain on probing (POP) at different time points: baseline (before mechanical debridement), 1 month, and 3 months. A custom-made acrylic stent and a color-coded periodontal probe (UNC-15, Hu-Friedy, Chicago, IL, USA) were used to ensure uniformity in measuring these parameters. An examiner blinded to each individual’s treatment recorded all the pre- and post-treatment clinical parameters. ATV gel was prepared by a pharmacist using standard methods described in pharmacology texts. 22 - 24 After intensive in vitro investigations for optimization and stability to prepare a multiple-dose solution of isotone and sterile ATV, the gel base was first prepared with sodium carboxymethyl cellulose (2.6%) and mannitol (9%), and the pharmaceutical stock solution was prepared at a concentration of 2% in propylene glycol solvent. Subsequently, at a 4:1 ratio, the drug solution was slowly added to the gel base. If opaque, 0.5% polysorbate 80 was added to the solution to increase the solubility of the drug. Finally, 1% benzyl alcohol was added to the solution as a preservative for injectable products. All steps were performed under laminar hood and aseptic conditions. The obtained solution was stored in sterile 1.5 mL of polyethylene microtubes and was stable at 2-8 °C for up to 1 month . SPSS 22 was used for the data analysis. Descriptive statistics (frequency, frequency percentage, mean, and standard deviation) and inferential statistics (chi-squared test, Fisher’s exact test, and independent t test) were used for data analysis. An independent t test was used to compare the results between the two groups, and a paired t test was used to compare intragroup results. Statistical significance was set at P < 0.05. The clinical parameters (BOP, PD, CAL, and POP frequency distributions) at both the baseline and follow-up visits are shown in Tables 1 and 2 . After one month, the independent t test showed no significant difference ( P > 0.05) in the BOP index between the control and test groups. However, it significantly decreased ( P < 0.001) in the test group after three months. There was no significant difference ( P > 0.05) in PD between the two groups after three months, but it was significant one month after the intervention ( P < 0.001). The CAL and POP variables showed significant differences 1 and 3 months after the intervention ( P < 0.001). A comparison of intra-group results using a paired t-test showed a significant difference in PD one and three months after the intervention ( P < 0.001). No significant difference ( P > 0.05) was found in CAL in the control group at baseline and one and three months after the intervention. However, within the test group, the difference was statistically significant ( P < 0.001). A comparison of the BOP results showed a significant difference ( P < 0.001) one and three months after the intervention. The POP results were significantly different ( P ≤ 0.05) one month after the intervention, both within the control and test groups. There was no significant difference ( P > 0.05) in the control group three months after the intervention, but the difference was significant ( P < 0.05) in the test group ( Table 3 ). Statins have antimicrobial activity against periodontal pathogens and exhibit anti-inflammatory and immunomodulatory effects. 25 Animal and clinical studies have supported the idea that statins can be used as an adjunctive treatment to scaling and root planing (SRP) to manage periodontal disease, including chronic periodontitis. 18 , 26 Numerous studies have investigated the effectiveness of statins on various clinical parameters of periodontitis. 27 Due to the pleiotropic (cholesterol-independent) effects of statins, such as immunomodulatory and anti-inflammatory effects, they are expected to improve periodontal clinical outcomes. 28 Several studies have reported positive clinical effects, such as reduced PD, CAL, and BOP, with local administration of statins. Therefore, statins are considered a valuable adjunct to non-surgical and surgical treatments for periodontal disease. 18 , 26 , 27 The current study evaluated the clinical efficacy of 1.2% ATV gel as a supplement to mechanical debridement in treating peri-implant mucositis. Compared with the placebo gel, the results revealed a significant improvement in clinical parameters. To the best of our knowledge, no study has directly compared the use of 1.2% ATV gel in treating peri-implant mucositis. In contrast, Saxlin et al 29 investigated the dual effects of statins on the periodontium. Their study revealed that statin use was associated with a higher risk of deep periodontal pockets in individuals without BOP. However, Kumari et al 30 discovered that using a local 1.2% ATV gel significantly improved clinical and radiographic parameters compared with a placebo gel. Similar results were also reported by Lindy et al, 31 who found that ATV or simvastatin led to 37% fewer pathological periodontal pockets than in the control group. In addition, animal models have suggested that statins have a beneficial impact on ligature-induced alveolar bone loss. 32 In the current study, BOP significantly decreased from baseline to three months, indicating that ATV may have anti-inflammatory properties. In the current study, no significant difference ( P > 0.05) was observed in the BOP index between the control and test groups at the one-month follow-up. However, after three months, the difference was significant ( P < 0.001) in the test group. However, the results for the PD variable one month after the intervention were significant ( P < 0.001). There was a significant difference in CAL and POP between the control and test groups one and three months after the intervention ( P < 0.001). Studies using local statins have reported significant improvements in clinical periodontal outcomes compared with those using SRP. The subgingival release of statins allows for high concentrations and low doses of drugs in the periodontal tissues, leading to high patient acceptance and the ability to control the long-term release of therapeutic agents at the target sites without causing systemic side effects. 33 Compared with oral administration, which results in rapid absorption and entry of the drug into the circulation, local application of the drug is preferred. 34 , 35 Therefore, it is safer to administer the drug locally, and clinical results have demonstrated that it improves chronic periodontitis. 27 Bertl et al 26 found that the type of statin used was associated with periodontal outcomes. One study showed that rosuvastatin was the most effective, whereas another reported statistically significant effects of ATV. In two clinical trials evaluating the application of statins as an adjunct to SRP, rosuvastatin produced the best results regarding clinical and radiographic parameters such as PD reduction, CAL gain, and radiographic defect fill. 27 The superior clinical advantages of rosuvastatin over ATV may be attributed to its stronger anti-inflammatory effect, which results in a greater reduction in C-reactive protein levels. 36 , 37 Simvastatin is the most commonly used statin in clinical trials and is administered locally at a concentration of 1.2%. Numerous studies have shown significant improvements in clinical and radiographic results when using simvastatin. 27 Retrospective studies have also shown that patients with severe chronic periodontitis who were treated with simvastatin or ATV had lower PD indices than those who did not receive statins. 38 In addition, a recent study by Fajardo et al 39 indicated that ATV may reduce alveolar bone loss and tooth mobility in individuals with periodontal disease. Goes et al 40 reported that ATV could prevent alveolar bone loss in rats with ligature-induced periodontitis. Pradeep et al 21 evaluated the use of 1.2% ATV gel as a supplement to SRP for treating suprabony defects in patients with chronic periodontitis. The ATV group showed a significant reduction in clinical parameters such as BOP, PD, and CAL at the 3-, 6-, and 9-month follow-ups compared to the placebo group, indicating the anti-inflammatory effect of ATV. Overall, the clinical parameters of the peri-implant mucosa improved using 1.2% ATV gel as an adjunct to mechanical debridement. The results of this study support the additional application of ATV gel for the treatment of peri-implant mucositis. By injecting 1.2% ATV gel into the pockets around implants with peri-implant mucositis, this clinical trial showed that it significantly reduced BOP, PD, POP, and CAL gain when used with mechanical debridement, compared with placebo gel. This may provide a new approach for treating the inflammation caused by peri-implant mucositis. The results of this study must be confirmed in long-term, multicenter, randomized, controlled clinical trials. | Review | biomedical | en | 0.999997 |
PMC11699263 | The anterior region of the maxilla undergoes many surgical interventions. Dental implant placement, surgeries of supernumerary impacted teeth and cysts, and orthognathic surgeries are some of these interventions in the anterior maxilla. 1 However, the most important of all these is the increasing demand for dental implants. Thus, a more precise anatomical investigation of this segment is essential due to the presence of canalis sinuosus (CS) in the anterior maxilla. 2 CS is a neurovascular canal about 2 mm in diameter, which carries a branch of nerves of the infraorbital canal, the anterior superior alveolar nerve (ASA), and related vessels. 3 , 4 The infraorbital nerve is a branch of the maxillary nerve, which is the second branch of the fifth cranial nerve, i.e., the trigeminal nerve. The skin distribution of the infraorbital nerve extends to the upper lip, cheeks, lower eyelids, outer nose, and nasal cavity. 5 On cone-beam computed tomography (CBCT) images, CS is a curved bone canal originating laterally from the infraorbital canal. It passes through an internal and anterior course to reach the maxillary anterior region, passes through the lateral wall of the nose, and is placed in the marginal part of the nasal cavity floor; then, lateral canals branch off, eventually opening next to the incisor canal in the palate. 6 CS detection is important because damage to such structures might cause sensory disorders. In addition, such structures may be mistaken for other anatomical structures or lesions, leading to unnecessary or incorrect procedures. 7 Therefore, accurately identifying the anatomy of the face, mouth, and jaws and using radiographic images, specifically CBCT in surgical procedures, are necessary to avoid the destruction of blood vessels and nerves in this area. 8 The anatomy of the CS 9 - 13 and its accessory canals 14 - 19 has been evaluated in numerous studies. A systematic review study conducted in 2023 considered CS and its accessory canals as anatomic structures due to their high prevalence. 20 However, a few studies have quantitatively evaluated the canal’s relationship with adjacent structures. 4 Therefore, this study investigated the exact position of CS relative to adjacent teeth and its distance from adjacent anatomical structures in CBCT images of an Iranian population. The CBCT images were captured using an 8-cm field of view of Planmeca Promax 3D mid (Helsinki, Finland) with the following conditions: voxel size: 200 μm; time: 12 s; mA: 10; kVp: 90. Two observers investigated all the images twice with a time interval of one month. The first observer was an experienced periodontist, and the second observer was an experienced oral and maxillofacial radiologist. Observations and measurements were carried out by Planmeca Romexis software version 3.8.1. The presence of CS was defined in axial and sagittal planes, and its clear extension was confirmed in the coronal sections. CS diameter was measured on the axial plane. Canals with a diameter of > 1 mm and a definite extension to the infraorbital canal were considered . Slice thickness and interval were 0.5 in all the sections. The axial plane in which the CS had the largest diameter was chosen to measure the distance of the CS to the adjacent teeth and the nasopalatine canal. The tooth with the closest distance to the CS was considered the main tooth; then, the mesial, mid, or distal position of the main tooth related to the CS was determined. The distances of CS from the nasal floor, ridge crest, buccal cortical plate, and the distance of canal extension from the main tooth apex were measured on the cross-sections perpendicular to the axial plane at the CS site . Intra- and inter-observer reliability was evaluated in 10% of the CBCT images after two weeks using the intraclass correlation coefficient (ICC). The data were analyzed with SPSS 22 using descriptive statistics, i.e., maximum, minimum, mean, and standard deviation. The ICC value was ˃0.80 for both intra- and inter-observer reliabilities. Pearson’s correlation coefficient was used to determine the relation between two quantitative variables. Fisher’s exact test was used to compare CS distribution in terms of sex and location. T-test was used to compare distance measurements between males and females or between the right and left sides. P ≥ 0.05 was considered statistically insignificant. CBCT images of 400 patients were investigated, with 185 males (46.3%) and 215 (53.7%) females. The mean age of the patients was 43.06 ± 13.60, with a range of 20–86 years. Forty-two patients (10.5%) had at least one CS with a clear extension towards the infraorbital canal, with a diameter of > 1 mm; 20 (47.6%) were female, and 22 (53.4%) were male. In 13 patients (30.95%), the canal was bilateral, and in 29 (69.05%), the canal was unilateral. In 25 (86.2%) patients, the canal was on the right side, and in 4 (13.80%), the canal was on the left side; generally, 55 canals were observed in images. Table 1 shows the mean diameters of CS in both sexes. There was no significant difference in the frequency of cases between the two genders ( P > 0.05). No significant correlation was observed between CS diameter and age ( P = 0.101) or sex ( P = 0.284). CS was more frequent on the right side than on the left side, but this difference was not significant ( P > 0.05) ( Table 2 ). The CS had the closest distance to the lateral incisor in most cases. The mid-position of the CS compared to the main tooth was significantly more common than other positions ( P = 0.04). The average distances of CS from the nasal floor, ridge crest, buccal cortical plate, main tooth apex, and nasopalatine canal were higher in males than in females; this difference was not statistically significant except for the buccal cortical plate distance and perpendicular distance to the nasopalatine canal ( Table 3 ). The distance of CS from neighboring structures on the right and left sides was not statistically significant ( P > 0.05). The prevalence of CS in this study was 10%, which was close to a study by de Oliveira-Santos et al. (15.7%) 21 ; this prevalence was about 35% in a study by Manhães Júnior et al, 4 66.5% in a study by Aoki et al, 13 88% in a study by Wanzeler et al, 5 and 98.5% in a study by Yeap et al. 22 However, the prevalence of CS in a study by Gurler et al 23 was 100%. The possible reasons for this discrepancy include differences in the slice thickness of the CBCT images, 23 the software used, sample size, and the content of studies. 11 In addition, in this study, cases traceable to the infraorbital canal with a diameter of > 1 mm were selected, which could be another reason for the difference. The canal was bilateral in 13 patients (30.95%) of the 42 patients with CS. The frequency percentage of bilateral cases of CS was various in other literature as follows: de Oliveira-Santos et al. 21 reported 21.4%, Manhães et al 4 reported 24.3%, Aoki et al 13 reported 54.1%, and Gurler et al 23 reported 100%. Gender distribution of CS was not statistically significant, consistent with studies by Gurler et al, 23 Von Arks et al, 6 Machado et al, 19 and Wanzeler et al. 5 In a study by Anatoly et al, 24 the prevalence of CS was significantly higher in females, while this prevalence was significantly higher in males in Aoki et al. study. 13 The mean (SD) diameter of CS was 1.06 (0.26) mm, which was significantly ( P = 0.284) higher in males [mean (SD) diameter in males = 1.10 (0.31) vs. females = 1.02 (0.28)]. In the study by Aoki et al, 13 this difference was not significant, either. Gurler et al. 23 reported a significantly higher mean diameter of CS in males ( P = 0.001). Because of the difficulty of some cases in precisely attributing the CS to the specific tooth, 16 Beyzade et al 25 modified the classification used by Oliveira-Santos et al. 21 For the same reason, in this study, CS’s distance to the neighboring teeth was determined in axial plane in which the CS had the most diameter, at first. The position of the CS with regard to the closest tooth was determined at the second. CS was most related to the mid-position of lateral incisors. CS was more commonly associated with this tooth in other populations. 4 , 11 , 22 , 24 In this study, CS-to-the-buccal cortical plate and CS-perpendicular-to-nasopalatine canal distances were significantly higher in males, so the CS location was more palato-lateral. In the Manhães Júnior et al 4 study, the nasal cavity distance was higher than in our study, and the ridge crest and the buccal cortical plate distances were lower than in our study, so the CS was located in a more buccal position and closer to the crest in Manhães Júnior and colleagues’ study. | Study | biomedical | en | 0.999996 |
PMC11699265 | Four factors should be addressed when evaluating an implant site in an esthetic zone: smile line, soft-tissue morphology, tooth morphology, and osseous architecture. 2 The placement of an implant in an appropriate connection to the intended restoration is critical for the best esthetic and functional outcomes, which would require sufficient alveolar bone volume and position. 3 - 5 Various surgical methods, such as distraction osteogenesis, 6 guided tissue regeneration, 7 and graft procedures, have been suggested to maintain or repair the alveolar ridge. 8 These techniques can be used to treat the ridge defect at the time of extraction or later. Orthodontic forced eruption (OFE) is another method of soft and hard tissue augmentation. According to Heithersay 9 and Ingber, 10 orthodontic treatment causes the periodontal ligament to be pulled during eruption, which increases bone volume and causes osteoblastic activity to occur where the periodontal attachment is located. 11 The gingiva and bone linked by the periodontal ligaments migrate in the same direction of the tooth’s coronal movement. 12 Additionally, this method may be used to move the root, creating room and anchoring for an implant. In 1993, Salama and Salama 13 suggested modifying the forced eruption method. By forced orthodontic extrusion of “hopeless” teeth and their periodontal structures, this novel technique, known as “orthodontic extrusive remodeling,” was employed to improve the soft and hard tissue profiles of possible implant sites. 13 Hence, the present study aimed to assess the effect of site preparation of dental implants using OFE in hopeless teeth. This systematic review was conducted according to the PRISMA (Preferred Reporting Items for Systemic Reviews and Meta-Analyses) guidelines statement. The following focused question was formulated to outline the search strategy: “What is the effect of orthodontic extrusion on site development for implant placement in the esthetic zone?” with “site development” referring to the optimization of the implant spatial positioning and minimizing the need for adjunctive regenerative treatments. A search of electronic databases, including Scopus, PubMed/MEDLINE, and Google Scholar, was performed from January 2000 to November 2023. The search was limited to English-language studies with available full texts. Furthermore, a hand search was conducted to assess the studies that were not electronically available. The search was performed based on the following keywords: [orthodontic AND extrusion] AND [dental AND implant], [orthodontic AND extrusion] AND [site AND development], [orthodontic AND extrusion] AND [dental AND implant] AND/OR [[soft OR hard] AND tissue AND [regeneration OR augmentation]]. All the clinical studies that used orthodontic extrusion for further implant placement in the esthetic zone were included. Abstracts, letters, and reviews were excluded. Studies were excluded if orthodontic extrusion was performed solely or adjacent to a dental implant. De-duplicating was done manually, and finally, eligible studies were included, and their full texts were obtained. Two independent reviewers assessed the full texts, and the following data were extracted and further classified in a table: type of study, number of patients, gender, age, number of tooth/teeth, type of jaw (maxilla/ mandible), orthodontic movement procedure, implant characteristics, follow-up, and outcome. Any disagreement between reviewers was resolved following discussion. Initially, 57 articles were identified via a search through the above-mentioned databases. After removing duplicate investigations, 37 studies were further considered. After evaluating titles, 7 studies, and after assessing the titles with their corresponding abstracts, 7 more studies were excluded. The full texts of the 23 remaining studies were evaluated based on the predetermined inclusion/exclusion criteria; consequently, 15 studies were included in the current systematic review. Figure 1 shows the strategy flowchart of the present investigation. All the included studies were case reports. Sixteen individuals have been documented in the literature, where orthodontic extrusion was used to establish implant sites in the esthetic zone. Patients’ age varied from 22 to 57 years and consisted of 11 women and 5 men. A total of 21 teeth underwent extrusion and further extraction and implant placement. All these teeth were maxillary anteriors, and central incisors were the most common. The orthodontic extrusion period varied noticeably between studies, from the shortest period of 3 months 14 to the longest one of 12 months. 15 In two studies, implant insertion was performed 6 15 and 7 16 months after tooth extraction, and in 8 studies, an additional bone grafting procedure was performed before or simultaneous with implant placement. 14 , 16 - 22 The most chosen implant diameter was 3.75 × 11 mm in two studies. 23 , 24 Dental implant placement has become the gold standard treatment for replacing missing teeth for a long time. 29 , 30 Bone and tissue loss following inflammatory disease in periodontium could lead to departure from normal alveolar morphology and further difficulties for implant placement where hopeless teeth exist. 24 , 31 OFE has been recommended as the only non-surgical adjunctive way to enhance hard and soft tissue conditions for implant placement. 31 , 32 Hence, the present review evaluated the effect of OFE for implant placement in the esthetic zone. Almost all the 15 studies included in our review reported positive outcomes of OFE from clinical, radiographic, and esthetic aspects. Continuous light force was the preferred approach in most of the studies. According to Kim and colleagues’ study, this light force could induce the stretch of gingival and periodontal fibers and further formation of new bone and gingiva in the coronal part. 24 In multiple studies, noticeable advantages of OFE for hopeless teeth before implant placement were mentioned: first, the hopeless teeth could aid in oral rehabilitation procedures. Second, the patient’s discomfort would decrease since the hopeless teeth solve esthetic issues, and third, by following this treatment, periodontal ligaments’ capacity as a distraction osteogenesis means would be used by stimulation of biological potential of periodontal ligament and modifying the morphology of an intrabony defect to a desirable one. 11 , 28 , 33 , 34 However, this treatment modality is not without complications; esthetic problems due to the presence of wires and brackets, phonetic discomfort, difficulty in orthodontic force control, undesirable external root resorption of adjacent teeth, gingival recession, and buccal bone dehiscence could be considered the drawbacks of the above-mentioned technique. 24 , 28 As was observed in Kim and colleagues’ study, the radiographic evaluation revealed apical root resorption in the lateral incisor adjacent to the targeted tooth. This phenomenon mainly contributed to a greater magnitude of force than intended. 24 To precisely perform case selection, good plaque control, resolution of periodontitis, presence of at least 1/3 to 1/4 of apical attachment, and feasibility of adequate stabilization period must be considered. 35 Magnitude, duration, and retention period varied in the studies. In Joo’s study, 1 mm/month of movement had similar results to 1 mm/week of coronal displacement. 27 However, Isola’s study emphasized using low and controlled ( < 100 g) forces to obtain < 1 mm/month movement. 33 The retention period was reported to be 6‒12 weeks, 27 while in Alsahhaf and Att’s study, a minimum of 3‒6 months was suggested. 30 This variation in OFE procedures necessitates careful examination and individualized treatment planning. The current review has its limitations. First, the number of available studies in the literature is limited, which does not allow for a comprehensive conclusion to be drawn. The lack of a long-term follow-up negatively affects the reliability of the outcomes. Moreover, all the studies included in this review are case reports or case series that will negatively affect the quality of the generated evidence, and the interpretation of the results should be carried out with caution. In conclusion, OFE of hopeless teeth seems an acceptable alternative to enhance soft and hard tissue conditions for future implant placement. Nevertheless, additional interventions such as submerged healing and guided bone regeneration should be considered in special cases to achieve the best outcome in the esthetic zone. | Study | biomedical | en | 0.999996 |
PMC11699266 | Despite the evidence showcasing the high success rate of dental implants in oral rehabilitation, healthcare providers must be aware of possible complications after implant placement. 1 , 2 Peri-implant mucositis affects the soft tissue surrounding implants and can progress into peri-implantitis if left untreated. Peri-implantitis is characterized by bleeding on probing (BoP) and/or suppuration, increased probing depth and/or marginal recession alongside progressive radiographic bone loss compared to previous visits . 3 The average prevalence rate of peri-implantitis is 22%, with a range of 1%‒47%. 4 The incidence of peri-implantitis is associated with the accumulation of bacterial plaque primarily consisting of the microorganisms involved in periodontitis. 3 , 5 However, Porphyromonas gingivalis , Tannerella forsythia , and Treponema denticola are found at higher concentrations in samples obtained from peri-implantitis patients. 6 Factors such as smoking, untreated periodontitis, irregular maintenance, and diabetes mellitus have been identified as risk factors for peri-implantitis. 7 - 10 Local factors such as excess cement, incorrect prosthesis seating, implant malpositioning, implant micro- and macro-design, abutment connection type, and excessive mechanical loads can all contribute to disease progression. 5 , 11 Peri-implantitis treatments primarily aim to eradicate tissue inflammation, stop disease progression and bone loss, regenerate lost supportive tissues, and restore osseointegration. 12 These treatments encompass diverse surgical and non-surgical approaches, such as mechanical debridement, application of antiseptics, antibiotic therapy, surgical flaps, and resective or regenerative surgeries. 13 - 19 Resective surgery is ideal for shallow defects, while deeper intrabony defects are better suited to regenerative strategies. 20 , 21 A regenerative approach would be preferred if the bony defect has a minimum depth of 3 mm, is enclosed by three or four walls, and sufficient keratinized mucosa is present. 22 In cases where a failing implant is predicted to have a poor prognosis or the aforementioned treatment strategies do not lead to success, explantation is the inevitable choice. 23 The regenerative approach involves surface decontamination and the use of bone grafts with or without a barrier membrane. 24 When selecting a treatment strategy, decisions must be made concerning the decontamination method, graft material, barrier membrane, and surgical technique. Decontamination can be achieved through mechanical or chemical methods, such as using acids, antiseptics, abrasives, and lasers. 25 - 28 Moreover, different graft materials can be used, including allografts, autografts, xenografts, etc. 28 , 29 If necessary, a range of resorbable or non-resorbable membranes can be used to cover the graft. 30 Given the multiple biomaterials and techniques reported in the literature, numerous protocols can be used to manage peri-implantitis. 24 , 31 However, the consensus is that no specific biomaterial or treatment protocol for peri-implantitis has proven superior to others. 32 Hence, this systematic review aimed to compare various surgical regenerative interventions for peri-implantitis management based on their clinical and radiographic enhancements. In January 2022, the initial search was performed in electronic databases, including PubMed, Embase, Web of Science, Scopus, Google Scholar, Cochrane CENTRAL, and ProQuest (for grey literature). An updated search was also conducted in May 2022. The search terms used in these electronic databases included the following: ((peri-implantitis) OR (peri-implant disease) OR (peri-implant disease)) AND ((regenerative medicine) OR (biomaterial) OR (regenerative surgery) OR (surgical regeneration) OR (bone graft) OR (bone substitute) OR (membrane) OR (growth factor)) AND ((treatment) OR (management) OR (therapy)). The results were imported into EndNote X20 software (Clarivate Company, Philadelphia, USA), and the duplicates were removed. The titles and abstracts of the remaining articles were independently screened by two reviewers who were unaware of each other’s decisions. After omitting irrelevant results, the full texts of the remaining articles were meticulously read and compared against the inclusion and exclusion criteria. In case of disagreements between the two reviewers, a third reviewer was consulted to reach an agreement. Two authors independently performed the risk of bias assessment concurrent with data extraction. The Cochrane risk of bias tool for randomized clinical trials was used. 34 The tool evaluates bias across five distinct domains: randomization process, deviations from intended interventions, missing outcome data, outcome measurement, and selection of reported results. A study was deemed to have a “low risk of bias” if all domains displayed a low risk. Conversely, the presence of high risk in even one domain classified the study as having a “high risk of bias.” If a study presented some concerns in at least one domain but did not manifest a high risk in any domain, it was categorized as having “some concerns.” The standardized mean difference (SMD) was computed for each outcome measure (BoP, PD, PI, BL, and BG) using Cohen’s d or Hedges’ g. Meta-analysis was performed using the random-effects restricted maximum likelihood (REML) method in Stata version 17 (Stata Corp., College Station, Texas, USA). Potential sources were examined through meta-regression analysis to assess the presence of heterogeneity. The initial search in electronic databases, hand-search, and update search yielded 5457 results, which were reduced to 4737 after deduplication. Further screening of titles and abstracts led to the exclusion of 4638 studies, leaving 99 potentially relevant studies. After a thorough examination of the full texts, 15 studies were chosen for data extraction . The remaining 84 studies were excluded for various reasons, such as not providing a disease definition or providing an unclear definition, a follow-up duration < 12 months, an undesired study design, not employing a regenerative strategy, and exhibiting a high risk of bias. The 15 remaining studies included 12 original randomized clinical trials and three long-term follow-up studies from these original trials. The total number of patients who received treatments for peri-implantitis was 455 (507 implants), with a mean age of 63.13 ± 10.72 years (ranging from 54.4 to 73.5 years). The examined implants were in function for 7.06 ± 3.08 years on average (ranging from 4.82 to 14 years). Ten studies included smoking patients, 29 , 35 - 43 one excluded smokers, 44 and one did not report smoking status. 45 Smokers comprised 31.85% of the participants in studies reporting smoking status (ranging from 15% to 69.6%). BoP was reported in 11 studies, 29 , 35 - 43 , 45 and the initial measurements showed a minimum of 15.4% and a maximum of 100%. The pre- and postoperative PDs were measured in 12 studies. 29 , 35 - 45 The baseline PD ranged from 4.9 mm to 7.6 mm. PI was reported in 10 studies, but two different indices were used. Seven studies used the O’Leary index, 29 , 35 , 38 - 40 , 42 , 43 and three used Silness & Löe. 36 , 37 , 45 The baseline measurements showed a minimum of 13% and a maximum of 45% through the former index and a minimum of 0.5 and a maximum of 1.21 through the latter. In three out of seven studies reporting BL, the implant shoulder was considered the coronal reference point, 38 , 40 , 44 and four studies did not clarify their reference points. 35 , 39 , 42 , 43 The baseline BL ranged from 3.91 mm to 5.3 mm in the first group and 3.6 mm to 5.6 mm in the second group. The reports of 11 studies evaluating BG showed a minimum value of 0.2 mm 29 , 42 and a maximum of 3.58 mm. 35 However, some interventions led to bone loss, with a maximum loss of 1.9 mm in a study by Andersen et al ( Tables 1 and 2 ). 41 Six studies included clinical and radiographic measurements for follow-up periods exceeding one year ( Table 3 ). Peri-implant bone defects were filled with various materials, including xenografts, autografts, alloplasts, growth factors, etc. ( Table 2 ). 29 , 35 - 46 Aghazadeh et al 29 reported a greater bone fill through xenograft insertion compared to autograft. In another study, Polymeri et al 38 found no significant difference between the two types of xenografts, namely EndoBon and Bio-Oss. In six studies, collagen or concentrated growth factor (CGF) membranes were used to cover the grafting materials. 29 , 36 , 37 , 40 , 43 , 45 Isler et al 37 compared collagen and CGF membranes for covering similar bone substitutes. The results demonstrated significant improvements with both modalities, but using collagen membranes resulted in superior outcomes. Among the seven studies mentioning the merging status following the treatment, five selected non-submerged healing, 29 , 35 , 38 , 40 , 45 and two opted for submerged healing. 37 , 41 The decontamination phase of the treatments involved a combination of mechanical and chemical techniques. Mechanical methods included plastic curettes, Ti curettes/brushes, ultrasonic devices, sonic devices, and implantoplasty. 35 - 37 , 39 - 41 , 44 , 45 Chemical agents such as saline, H 2 O 2 , NaCl, ozone, and EDTA were employed during chemical debridement. 29 , 35 - 45 A comparison of decontamination methods was conducted in the study by De Tapia et al, 40 which revealed that the additional use of a Ti brush resulted in a significant PD reduction. One study used an Er:YAG laser to decontaminate the peri-implant site. 45 However, laser application failed to obtain significantly superior outcomes compared to conventional decontamination via plastic curettes. In terms of postoperative care, the most frequently prescribed medications included ibuprofen, amoxicillin, azithromycin, metronidazole, and CHX. 29 , 35 - 45 Of the six studies reporting bone gain or loss, 35 , 39 , 41 - 44 one showed a deterioration of 1.9 mm, 41 while another showed a maximum BG of 3.63 mm during the first year. 35 The former study used porous Ti granules (PTG) as the bone substitute without membrane coverage, while the latter observed BG solely through curettage without graft or membrane materials. Due to the limited number of included studies and the diversity in the interventions they examined, only the six articles in the first category were sufficient to conduct a meta-analysis. Consequently, additional comparisons between different bone substitutes, membranes, healing status, and decontamination methods could not be established. The results of the meta-analyses for six parameters at baseline and one-year follow-up can be found in Figures 3 and 4 . Notably, the study by Jepsen et al 35 reported each parameter separately for the mesial and distal aspects. Thus, a separate meta-analysis was conducted for this study to obtain a single value for each parameter and avoid biased weighting compared to the other studies. At baseline, no statistically significant difference was observed between the studies regarding BoP, PD, PI, or BL . Regarding BoP at the one-year follow-up, the overall effect size for implementing bone grafts was 0.04 (95% CI: -0.26‒0.35) . However, this intervention did not result in a significantly lower BoP compared to not using bone grafts. The PD analysis at the one-year follow-up indicated that using bone substitutes in regenerative treatments did not show a significant advantage over approaches without these materials . In this regard, the overall effect size was -0.08 (95% CI: -0.42‒0.27). An overall effect size of 0.37 (95% CI: 0.08‒0.65) was obtained for bone substitutes regarding PI at one year . It was concluded that using bone grafts, regardless of their type, significantly boosted the decrease in PI values ( P = 0.01). When it came to BL comparison between the studies at one year , it was observed that using a bone graft during surgical regeneration had a significant positive impact on BL improvements, with an overall effect size of -0.44 (95% CI: -0.84 to -0.03). Similar to BoP and PD, bone grafts did not significantly influence the amount of BG following a one-year interval . The overall effect size equaled 0.16 (95% CI: -0.68‒1.01) for BG. The P values for the test of θ i = θ j were > 0.05 for all parameters at baseline, indicating that the studies were homogeneous at baseline. At the one-year follow-up, the BoP, PD, and PI analyses showed homogeneity, while the P values for BL and BG were ≤ 0.05 ( P = 0.05 and P = 0.00, respectively), indicating that the studies were heterogeneous in terms of these two parameters. The risk of bias assessment revealed that five studies had a low risk of bias, and seven raised some concerns ( Table 4 ). In addition, one study exhibited a high risk of bias and, as a consequence, was omitted due to exclusion criteria. This systematic review aimed to compare the clinical and radiographic outcomes of different regenerative protocols for peri-implantitis treatment. The findings revealed significant improvements in PI and BL one year after using bone grafts. However, using bone substitutes did not significantly affect the BoP, PD, and BG values. Various factors, including the decontamination method, postoperative care, and graft type, can also impact treatment outcomes alongside the surgical approach. Renvert et al. 43 recorded the lowest BoP (8.3%) one year after peri-implantitis treatment. This favorable outcome was achieved through decontamination with 3% H 2 O 2 and saline. Similarly, a significant decrease in BoP was recorded in the study by Leonhardt et al. 49 after applying H 2 O 2 for decontamination. In contrast, the highest BoP (83%) was observed in the study by Andersen et al. 41 after using a Ti curette accompanied by 24% EDTA gel. EDTA does not possess antimicrobial properties per se, and the additional usage of other chemicals, such as CHX, has been suggested for improved decontamination. 50 Ramanauskaite et al. 15 reported that regenerative interventions alongside conventional peri-implantitis treatment did not significantly enhance BoP changes. Supporting this finding, Daugela et al. 31 showed that a regenerative strategy could not improve BoP reduction significantly, whether a barrier membrane was used or not. One year after peri-implantitis treatment, decontamination with 3% H 2 O 2 and saline in conjunction with a xenograft as the bone substitute resulted in the most favorable PD (2.6 mm). 42 In line with this finding, Roccuzzo et al. 51 achieved significantly reduced PDs by using xenograft for bone substitution. However, their decontamination phase involved 24% EDTA and 1% CHX gels. On the contrary, Emanuel et al. 44 reported the least favorable PD (5.43 mm) following chemical decontamination with ultrasonic and saline. Luengo et al. 52 reported that ultrasonic decontamination yielded less favorable results than air-polishing or Ti brushes, particularly when cleaning the implant threads within the apical third. Based on our findings, the use of bone grafts did not significantly differ from treatment approaches without bone substitution in terms of PD changes. Consistently, Li et al. 53 concluded that the additional use of bone grafts did not significantly alter the changes in PD. When comparing regenerative, resective, and access flap procedures, it was found that PD reductions were relatively similar. 18 On the contrary, Ramanauskaite et al. 15 observed a greater PD reduction in studies using regenerative techniques along with conventional peri-implantitis treatment. At the one-year follow-up, the lowest PIs were observed in the studies by Isehed et al. 43 (0%) and Isler et al. 36 (0.22 via the Silness-Löe index). The former performed open flap debridement (OFD) using an ultrasonic device, Ti instruments, and saline irrigation. In the latter, decontamination was executed using Ti curette, saline, and ozone DTA, while the bone defect was filled with xenograft and CGF. In several studies, ozone therapy has been shown to improve PI and PD. 54 - 56 McKenna et al. 57 reported that ozone application significantly decreased PI in patients with peri-implant mucositis, which aligns with the results of Isler et al. 36 On the other hand, PI was the highest in the studies by Renvert et al. 42 (25%) and Schwarz et al. 45 (1.1 via the Silness-Löe index). The former executed chemomechanical decontamination using 3% H 2 O 2 , saline, and Ti curettes, while the latter used implantoplasty, saline, and plastic curettes for decontamination, as well as xenograft covered with collagen membrane for bone substitution. According to Monje et al., 58 incorporating implantoplasty into resective or reconstructive strategies of peri-implantitis treatment did not significantly improve clinical parameters, including PI. Furthermore, incorporating implantoplasty into regenerative treatment did not necessarily result in a marked amelioration in clinical measurements. 59 Also, regenerative treatments may not necessarily be superior to non-regenerative methods in PI reduction. However, it should be remembered that employing a barrier membrane in regenerative protocols would give rise to significant PI enhancement. 18 The highest one-year BL change (3.57 mm) was obtained in a study by Jepsen et al. 35 This change resulted from decontamination with Ti curette, 3% H 2 O 2 , and saline, as well as bone substitution with PTG. In another study, using PTGresulted in greater improvements in periodontal indices compared to a xenograft. 60 The most undesirable BL change (-0.33 mm) occurred in a study by Emanuel et al., 44 in which ultrasonic and saline were used for decontamination, and D-PLEX 500 was used to fill the bony defect. D-PLEX 500 is a biodegradable, prolonged-release antibiotic-formulated bone graft that contains β-tricalcium phosphate granules coated with doxycycline hyclate. In contrast to the mentioned finding, De Tapia et al. 40 concluded that implementing β-tricalcium phosphate as the bone substituting material would significantly enhance BL. Sanz-Martín et al. 18 compared regenerative, resective, and access flap treatment methods and concluded that a regenerative approach could lead to more significant BL gains. Jepsen et al. 35 reported the highest BG at one year, with 3.58 mm in the control group. This study utilized OFD using a Ti curette, a Ti brush, 3% H 2 O 2 , and saline. On the contrary, the least favorable outcome (1.9 mm bone loss) was found in a study by Andersen et al., 41 following OFD with a Ti curette and EDTA gel and bone substitution using PTG. Conversely, Jepsen et al. 35 concluded that employing PTG outperformed OFD regarding defect fill. Moreover, Guler et al. 60 reported significant superiority for PTG over xenograft placement. It can be assumed that the undesirable outcomes in the study of Andersen et al. 41 might be attributed to factors other than bone graft material. Based on our findings, bone grafts did not significantly affect the amount of BG. Another systematic review exploring various surgical regenerative treatments reported the greatest increase in marginal BL in three studies using enamel matrix derivative (EMD), platelet-derived growth factor (PDGF), bovine-derived xenograft, and PTG. Notably, xenografts and PTGs can appear radiopaque, making it difficult to distinguish them from regenerated bone. 31 Overall, combining regenerative measures with conventional surgical peri-implantitis treatments would achieve greater defect fill. 15 However, the complete resolution of a bony defect following guided bone regeneration (GBR) cannot be predicted with certainty. 61 Diverse materials, such as xenografts, autografts, etc., were used in the reviewed studies to fill bony defects. Some research outside this review combined bovine hydroxyapatite with nanocrystalline calcium sulfate, resulting in enhanced and stable outcomes. 62 Mandelaris and DeGroot 63 used a bone graft made of mineralized freeze-dried bone allograft (FDBA) and xenograft, paired with recombinant human platelet-derived growth factor (rhPDGF). Wen et al. 64 combined FDBA and mineralized bovine and autogenous bone, proving their efficacy in peri-implantitis reconstructive procedures. The application of platelet-rich fibrin has also demonstrated successful resolution of bony defects. 65 , 66 Kadkhodazadeh et al. 67 successfully managed extensive peri-implant defects by employing a Ti mesh, autogenous bone, FDBA, and acellular dermal matrix. Augmentation in bone height and attachment level can be achieved by impregnating bone grafts with tetracycline, vancomycin, or tobramycin during the GBR of peri-implantitis-affected sites. Local application of antibiotics would be advantageous concerning the absence of side effects associated with systemic administration. 68 , 69 After intrabony defect debridement, various types of cells can proliferate within the defect, including epithelial cells, connective tissue cells, bone cells, and periodontal ligament (PDL) cells. Barrier membranes can be employed to selectively allow bone cells to occupy the defect and provide physical stability for the bone substitute. 70 Among the six studies using barrier membranes, collagen membranes and CGF were the two options. 29 , 36 , 37 , 40 , 43 , 45 However, there is ongoing debate regarding the benefits of covering bone grafts with membranes. Isler et al. 37 compared two different barrier membranes, CGF and collagen, along with the same bone substitute. Collagen membranes were reported to yield more satisfactory results at the one-year follow-up. Monje et al. 71 depicted that adding a resorbable cross-linked barrier to allograft did not impact the results of defect filling. Chan et al. 72 suggested that applying barrier membranes in conjunction with graft materials may enhance outcomes compared to grafts alone. In contrast, Daugela et al. 31 showed that the additional use of barrier membranes did not significantly improve clinical outcomes. In essence, current knowledge does not necessarily support the superiority of using barrier membranes over not using them. Despite the previously mentioned materials for bone graft coverage, Dong et al. 73 reported encouraging outcomes after applying a nanofiber barrier membrane made up of magnesium oxide as the antibacterial agent alongside parathyroid hormone as the pro-osteogenic drug. In a case report, a non-resorbable Ti-reinforced polytetrafluoroethylene membrane was coupled with an absorbable collagen membrane to achieve immobility in the reconstructed region and enhanced wound healing. 63 Human amnion-chorion membranes tested for GBR showed promising results after peri-implantitis treatment. 74 Membrane exposure is a potential complication after GBR, reducing the success rate extensively. 75 Garcia et al. 76 noted that barrier exposure during the treatment of peri-implant defects would decrease healing chances by 27%. Alarmingly, human studies report exposure rates up to 87.6%. 72 Despite their potential benefits, barrier membranes can be costly, time-consuming, and technically sensitive, which might not justify their use in specific configurations such as three-wall defects. 72 In addition to conventional decontamination techniques, the Er:YAG laser was applied in one study. 45 At the 2- and 7-year follow-ups, laser-treated subjects did not exhibit significant differences in BoP or CAL reduction. 45 , 47 However, plastic curette debridement demonstrated a significantly greater reduction in both BoP and CAL after four years. 48 There is limited research regarding the advantages of using lasers to treat peri-implantitis. Chala et al. 77 found that the benefits of applying lasers are confined to a short-term follow-up of three months. Even the short-term clinical benefits of the Er:YAG laser for surface decontamination were refuted in another study. 78 Additional use of ozone alongside saline has yielded improved clinical and radiographic outcomes. 36 Ozone has also diminished bacterial adhesion to Ti and zirconia surfaces in vitro without inhibiting osteoblast proliferation. 79 In brief, the techniques employed for implant decontamination did not significantly impact the results following surgical regenerative procedures, and none exhibited superiority over others. 78 , 80 Additionally, the decontamination technique must be tailored to implant surface characteristics for optimal biofilm removal. 81 Antibiotics, namely amoxicillin and metronidazole, were the most common options. Although the prescription of these two antibiotics has been shown to be beneficial for peri-implantitis treatment, 78 the efficacy of local or systemic administration of metronidazole remains unclear. 82 CHX, an antiseptic, was widely used in almost all studies. The combination of azithromycin, ibuprofen, and CHX resulted in satisfactory periodontal improvements, 42 , 43 while amoxicillin in conjunction with metronidazole was another proper choice for postoperative care. 35 , 38 Overall, a personalized evaluation must be performed before the prescription of systemic antibiotics due to insufficient evidence supporting the integration of this drug delivery route into the standard treatment protocol. 83 Although some research favors non-submerged healing, a consensus report advocates for submerged healing, as it stimulates protected physiological wound closure. 32 Keeping the suprastructure in place during surgical treatment may negatively affect the efficacy of postoperative oral hygiene maintenance, intraoperative decontamination, flap design, and numerical measurements. 32 The non-submerged approach has also satisfied clinicians regarding clinical and radiographic improvements following peri-implantitis therapy. 84 The debate continues since any relationship between the success of peri-implantitis treatment and the postoperative merging status has been refuted. 85 Regardless of the materials and techniques implemented throughout peri-implantitis treatment, other factors such as implant location, defect morphology, and implant surface characteristics can be differential. 21 , 36 , 86 - 88 In addition to the higher prevalence of peri-implantitis within the upper jaw, 89 maxillary implants are more responsive to regenerative treatments. 36 Although Roccuzzo et al. 90 reported no significant association between defect configuration and defect resolution, Aghazadeh et al. 86 observed enhanced defect fill in four-wall and deeper defects. Also, Schwarz et al. 21 found a higher likelihood of resolution for circumferential defects than for dehiscence-type defects. A review of animal studies highlighted the crucial role of surface characteristics in peri-implantitis progression and treatment outcomes as opposed to the onset of the disease. In detail, treated surfaces represented the minimum BL and most desirable outcomes. 87 Furthermore, improvements were more pronounced around sandblasted and acid-etched implants than Ti plasma-sprayed implants after regenerative treatment. 88 Re-osseointegration has also been reported to occur more frequently around smooth-surface implants than around moderately rough implants. 91 Peri-implantitis risk factors can be divided into five categories, including factors associated with the patient, implant design, implant site, prosthesis, and clinician. 92 Achieving satisfactory long-term outcomes becomes possible when the primary cause is accurately identified and addressed. A 3% recurrence rate for peri-implantitis has been reported following surgical intervention, potentially resulting in a 36% implant loss in the long term. 93 Factors such as deep residual PD, recessed marginal BL, and implant surface modification during surgical peri-implantitis treatment were identified as contributors to disease recurrence at a surgically treated site. 94 Despite the limited number of histological examinations on re-osseointegration following regenerative treatment on previously contaminated implant surfaces, 95 re-osseointegration seems feasible given that an effective decontamination method and suitable regenerative strategy are employed. 91 , 96 Some argue that peri-implantitis is more of a foreign-body reaction than a bacterial-triggered disease such as periodontitis, suggesting that peri-implant bone loss can be traced to an osteolytic immune reaction. As in most cases, a physiologic balance is often established between osteoblast and osteoclast activity, making the long-term survival of implants feasible. However, when other factors, such as genetic variations, smoking, excessive cement, bacterial contamination, and technical issues, are added to the foreign-body reaction, the equilibrium gets disrupted, leading to bone loss. 97 , 98 Skeptics of this theory argue that there is insufficient evidence to solidify the pivotal role of foreign-body reactions in the pathogenesis of peri-implantitis. They assert that dental plaque biofilm is the principal causative agent of peri-implantitis, which should be the focus of both preventative and therapeutic measures. 99 The vast variability in peri-implantitis treatment components affected the reliability of inter-study comparisons and prevented the establishment of a standardized protocol. Differing disease definitions and outcomes may have also added heterogeneity. Soft tissue parameters and factors such as smoking or genetics were not addressed. Lastly, the full text of one study was unavailable, so data from its 7-year follow-up was used instead. 41 Following the comparison between various surgical regenerative protocols in peri-implantitis treatment, it was concluded that employing bone grafts did not significantly improve the parameters of BoP, PD, and BG, yet PI and BL showed significant enhancements. Decontamination predominantly relied on Ti instruments and chemicals such as H 2 O 2 . A variety of bone substitutes, including xenografts and CGF, were employed. Approximately half of the studies utilized collagen or CGF membranes, while others opted for none. Postoperative care involved a mix of antibiotics, CHX, and analgesics. Given the diverse materials and peri-implantitis definitions, more standardized trials are needed to establish a standardized protocol. | Review | biomedical | en | 0.999997 |
PMC11699267 | Periodontal regenerative treatment aims to provide suitable conditions for periodontal regeneration. Gingival recession can compromise esthetics and lead to root surface caries and tooth hypersensitivity. Several techniques have been suggested for root coverage, including pedicle flap, free gingival graft, guided tissue regeneration, and allografts. Autogenous grafts are procured from the palate or alveolar ridge and have limitations such as donor site morbidity and limited availability. Acellular dermal matrix (ADM) is an alternative to autogenous grafts. ADM has applications for root coverage, augmentation of keratinized tissue around teeth and implants, and treatment of gingival recession. 1 It eliminates the need for autogenous grafts and subsequent pain and discomfort. However, the absence of vasculature and cells in ADM slows down the unity and blending of the graft with the host tissue compared to autogenous grafts. Also, allograft requires cell attachment and anastomosis of the vasculature for maturity and reorganization. 2 Tissue engineering enables the fabrication of structures with the desired shape using biomaterials and progenitor cells and also allows cell proliferation and differentiation on suitable scaffolds. 3 ADM also serves as a temporary matrix for tissue regeneration, enhances the adhesion and proliferation of cells, and plays a key role in the transfer of MSCs to the defect site. 4 An ideal scaffold must be biocompatible, easy to use, and easily fixed at the site. Also, it should have interconnected porosities to allow the growth and proliferation of mesenchymal stem cells (MSCs) and angiogenesis. Moreover, it should have osteoconductive and osteoinductive properties. 5 The strength and stability of the scaffold also play an important role in the proliferation and differentiation of MSCs. 6 The size of porosities in the scaffold also affects the attachment, proliferation, and differentiation of MSCs. 7 Large pores provide less surface for the attachment of cells, and numerous pores increase the number of attachments. 8 MSCs are commonly used for cell therapy and tissue engineering due to their self-renewal property and differentiation ability. 9 These cells can be isolated from different human tissues. 10 The present in vitro study was conducted on two types of ADM scaffolds, namely scaffold type I (CenoDerm®, Tissue Regeneration Corporation, Tehran, Iran) and scaffold type II (Acellular Dermis, Iranian Tissue Product Co., Tehran, Iran). Of each scaffold, 26 samples were evaluated in this study. Also, 26 empty wells served as controls 11 (78 samples). One of the samples in each group was used to evaluate the morphologic characteristics of the cells. The scaffolds were coded to blind the operator to the group allocation of scaffolds. MSCs isolated from a sample of the buccal fat pad were seeded and cultured. The tissue specimens were immersed in sterile phosphate-buffered saline (PBS) (Sigma, USA) supplemented with 100-U/mL penicillin (Sigma, USA), 100-μg/mL streptomycin (Sigma, USA), and 2-mg/mL collagenase type IV (Sigma, USA) and incubated at 37°C for 90 minutes. After filtering the cell suspension using a 70-μm filter (SPL, Korea), they were cultured in a 75-cm 2 cell culture flask (SPL, Korea) containing alpha modification of Eagle’s medium (SPL, Korea) supplemented with 100-μg/mL streptomycin, 15% fetal bovine serum (SPL, Korea), 100-U/mL penicillin, 200-mM L-glutamine and 100-mM ascorbic acid 2-phosphate (Sigma, USA). The cells were incubated with 5% CO 2 and 95% air at 37 °C for 24 hours. After this period, unattached cells were rinsed off with PBS. The medium was refreshed every three days. Twenty-six rectangular pieces from each scaffold group, measuring 1.5 × 1 cm, with 0.2‒0.6 mm thickness, were rinsed with sterile saline solution (SPL, Korea) in 500-mL flasks for 10 minutes according to the manufacturer’s instructions. The samples were adapted to the bottom of 52 wells in six plates (SPL, Korea). Scaffolds I and II were placed in five wells on each of the five plates. Five empty wells were also considered as the control group in each plate. The sixth plate containing one sample of each scaffold and one empty well as control was used to assess cell morphology. The cell suspension with a density of 16,000 cells/mL was added to the scaffolds and control wells and incubated at 37 °C and 5% CO 2 for 12, 24, and 84 hours and 7 days. In total, two plates were used for cell attachment assessment using 6,4-diamidino-2-phenylindole (DAPI) staining and methyl thiazole tetrazolium (MTT) assay at 12 hours, and 3 plates were used to assess cell proliferation using the MTT assay at 24 and 84 hours and 7 days. 12 Five replicates were performed in every assessment at each time interval. One plate was used for cell morphology assessment at 24 hours. DAPI staining: The cell fixation was performed by 12 hours of incubation with 2.5% glutaraldehyde (Sigma, USA) and stained with 50 μg/mL of DAPI stain (Sigma, USA) for 30 minutes. The samples were washed with PBS to eliminate unattached cells. Then, the cells were observed under a fluorescence microscope at a 290 nm wavelength and counted in five points (four points at the corners and one at the center). 12 This was repeated for five samples in all the three groups. MTT assay: Optical density (OD) was measured 12 hours after culture to determine the primary attached cells in all the groups with five repetitions. 12 The cell viability and proliferation on the scaffolds and the control group were assessed 24 and 84 hours and 7 days after culture using the MTT assay. In this way, 200 μL of RPM1640 and 20 μL of fresh MTT solution (5 mg/mL) (Sigma, USA) were added to the cell culture wells, followed by incubation at 37 °C under 5% CO 2 for 4 hours. 12 Tetrazolium salt present in MTT was absorbed by biologically active cells, resulting in formation of purple formazan crystals, which were dissolved by adding isopropanol (Sigma, USA), including 0.1-N HCL (150 mL/well). The OD of the solution was read by a microplate spectrophotometer (SPL, Korea) by decreasing the wavelength from OD690 to OD570. 13 For assessment of cell proliferation and attachment, cell viabilityat each time interval was performed separately for five samples of each group, and determined based on a linear diagram representing the correlation between OD and cell number. To assess cell morphology, the cells were cultured on scaffolds and a control group and incubated for 24 hours. Then, they were washed twice with PBS, fixed with 2.5% glutaraldehyde for one hour at room temperature, and dehydrated with six graded concentrations of ethanol (from 50% to 100%), and hexamethyldisilazane (Sigma, USA). The samples were then gold-coated and evaluated under a scanning electron microscope (SEM; Nikon, Japan) at × 1000 magnification. One sample of each group was scanned under the SEM. Two parameters were assessed, including scaffold surface area covered with cells (in square micrometers) and roundness of the cells (smaller-to-larger diameter ratio of the cells). 14 Cell morphology assessment was performed on one sample of each group. Cell proliferation and attachment experiments were performed in five replicates. All the results were statistically analyzed using SPSS 25 (SPSS Inc., USA). Means ± standard deviations were used for adhesion and proliferation data analysis. The Mann-Whitney test was used to statistically analyze cell attachment, while ANOVA was applied to assess the proliferation of cells. Post hoc Tukey tests were applied for pairwise comparisons in cases of significant differences. A P value of < 0.05 with a 95% confidence interval was considered statistically significant. Cell attachment was assessed in 30 samples using the MTT assay and DAPI staining (five samples from each group for each test) at 12 hours. In the MTT assay, the scaffold II group had the highest cell attachment, followed by the control group. ANOVA showed no significant difference in cell attachment between the three groups ( P = 0.4). In DAPI staining , the highest attachment was noted in scaffold I, followed by the control group. ANOVA showed that the difference between the three groups was not significant ( P = 0.4) ( Table 1 ). In the assessment of cell proliferation, 45 samples were evaluated at 24 and 84 hours and 7 days in all the groups (5 samples in each group) with MTT assay ( Table 2 ). Over time, cell proliferation increased in all the three groups. At 24 hours, the highest proliferation rate was noted in the scaffold II group, followed by scaffold I. ANOVA showed that the proliferation rate was significantly higher in scaffolds I and II groups compared to the control group ( P < 0.001). Pairwise comparisons by Tukey’s test showed that the difference between the two scaffolds was not significant ( P = 0.8). At 84 hours, the highest proliferation rate was noted in the control group, followed by the scaffold I group. ANOVA showed no significant difference in this regard between the control and scaffold groups ( P = 0.2) or between the two scaffold groups ( P = 0.9). At seven days, the highest proliferation rate was noted in the control group, followed by the scaffold I group. The difference in this regard between the three groups was statistically significant ( P = 0.01). In all the three groups, the proliferation rate increased over time (shown by the MTT assay) such that in the control group, multiple comparisons revealed significant differences in the proliferation rate over time ( P < 0.001). In scaffold I, ANOVA showed that the difference in the proliferation rate was statistically significant over time ( P = 0.01). In scaffold II, ANOVA showed that the proliferation rate difference was not significant over time ( P = 0.2). In the three groups, the highest proliferation rate was noted at 84 hours and 7 days (the highest cell count was noted in the control group, with the lowest in the scaffold II group). Figure 2 shows the OD of MSCs of all the groups at all time intervals. Assessment of cell morphology under SEM at 24 hours revealed greater cell expansion with more appendages in the control group, followed by the scaffold I group compared to the scaffold II group . In this study, two commonly used scaffolds, CenoDerm and Acellular Dermis, were used. Attachment (at 12 hours) and proliferation (at 24 and 84 hours and 7 days) of MSCs cultured on these scaffolds were assessed using the MTT assay plus DAPI staining and MTT assay, respectively. Morphological properties of cells were also evaluated under SEM at 24 hours. The results showed no significant difference between these scaffolds concerning cell attachment at 12 hours. However, better results were achieved with CenoDerm at 24 hours and 7 days concerning cell morphological properties and cell proliferation, respectively. Cell attachment is the first response of the cell to scaffold. 15 Primary cell attachment to scaffold depends on the size and amount of porosities, water, and protein absorption 16 and plays an essential role in the proliferation of cells. According to Pabst et al, 17 an autologous scaffold enhancing proliferation of human gingival fibroblasts, endothelial cells, osteoblasts, and oral keratinocytes in vitro can also show higher angiogenic properties in vivo. Thus, scaffolds might show more favorable behaviors in vitro and have higher applicability in vivo. In the present study, the MTT assay showed no significant difference at 12 hours in cell attachment between the three groups. Similarly, Ma et al 18 used an MTT assay and showed proper attachment of fibroblasts to bilayer dermal equivalent. They indicated that using bilayer dermal equivalent also resulted in optimal regeneration in vivo. Thus, the results of MTT can be generalized to the clinical setting. Hussein et al 19 also assessed the attachment of fibroblasts to scaffolds with different sterilization methods using the MTT assay and DAPI staining, with both tests showing similar results. Thus, DAPI staining can confirm the results of the MTT assay in vitro. Also, the results of DAPI staining in vitro can be generalized to the clinical setting. In the present study, to confirm the results of the MTT assay, DAPI staining was also performed after 12 hours, which showed the same results, and both showed no significant difference between the three groups in cell attachment. Regarding the current study analysis, it might be concluded that the attachment of cells was the same in two types of scaffolds. Thus, they probably have the same efficacy for use in the clinical setting regarding attachment of MSCs. SEM showed morphological differences, demonstrating the superiority of scaffold I to scaffold II, which indicates the more biologically active cells. 20 Greater expansion of cells in the control group might be due to the smoother surface of wells in the control group compared to the porous surface of scaffolds. 21 The current study assessed the proliferation of MSCs in the three groups after 24 and 84 hours and 7 days using the MTT assay. It showed that the proliferation rate significantly increased over time, which was not significant in scaffold II between time intervals. At 24 or 84 hours, there were no significant differences between the scaffold groups in this respect. At 7 days, the significantly lowest cell population rate was noted in the scaffold II group. Overall, cell proliferation in the scaffold I group was higher than in the scaffold II group. The higher proliferation rate in the control group at each time interval might be attributed to the smooth surface of the plate compared to the porous surface of scaffolds. As previously confirmed, surface topography affects the attachment and differentiation of cells. 22 Osteoblasts have a greater attachment to rougher surfaces, while fibroblasts and MSCs better adhere to smoother surfaces. 23 - 25 We assessed MSC morphology, attachment, and proliferation, which are essential parameters in wound healing and repair. Using both DAPI and MTT assays simultaneously was a strength of our study. Also, no previous study has compared these two scaffolds concerning MSC behavior. The study was performed blindly, and each measurement was repeated five times. Hydrophilicity, pore size, biocompatibility, mechanical properties, composition, and solvent or toxic compounds in the scaffold all affect cell seeding. 13 The structure of biomaterials in the cellular matrix is also important and affects cell behaviors such as attachment, proliferation, and differentiation. 26 Attachment and proliferation of cells on scaffolds depend on the availability of nutrients, porosity, and interconnection between pores. 27 The Strength and density of the scaffold also affect cell morphology. 28 According to the above, further studies are recommended to compare these scaffold structural properties and the efficacy of these scaffolds in MSCs’ behavior in clinical situations. Both scaffolds showed similar efficacy in attachment of MSCs in vitro, but the proliferation of MSCs after 7 days was higher on scaffold I compared to scaffold II. Also, MSCs on scaffold I were more active, expanded more, and had more cellular appendages. Scaffold I was superior to scaffold II in terms of proliferation and morphology of MSCs in vitro. | Review | biomedical | en | 0.999995 |
PMC11699300 | Schizophrenia is a complex psychiatric disorder characterized by positive (e.g., delusions, hallucinations), negative (e.g., social withdrawal, lack of motivation), and cognitive (e.g., impairments in processing speed, working memory, and executive function) symptoms . Cognitive impairment associated with schizophrenia (CIAS) predicts poor functional outcomes , is likely a barrier to medication adherence and is associated with substantial burden on patients' daily lives . To relieve the burden on patients and reduce the financial and social burden of illness, CIAS must be adequately treated. However, there are currently no approved pharmacotherapies specifically targeting cognitive symptoms in schizophrenia . Positive, negative, and cognitive symptoms of schizophrenia have been linked with N -methyl- d -aspartate (NMDA) receptor hypofunction in the glutamatergic pathway, which is involved in synaptic plasticity and cognition . While studies of multiple compounds, including glutamatergic agents (i.e., D-cycloserine, cycloserine, glycine, and glycine transporter inhibitors) have suggested small beneficial effects on cognition, their clinical relevance is debatable . Computerized Cognitive Training (CCT) refers to software designed to improve cognition by promoting activity-dependent neuroplasticity practice by engaging cognitive functions . CCT may be combined with psychosocial programs in cognitive remediation therapy ; . Although only a few studies have demonstrated some beneficial effects for pharmacological interventions , evidence suggests CCT interventions may have beneficial effects on the glutamatergic system in patients with schizophrenia . It is possible that, alongside CCT, pharmacological interventions may act synergistically to improve cognition. There has been limited testing of glutamatergic agents administered in combination with CCT (referred to as Pharmacologically Augmented Cognitive Training [PACT]) as a potential facilitating strategy . Iclepertin is a novel, potent, and selective glycine transporter-type-1 (GlyT1) inhibitor in development for the treatment of CIAS that increases glycine levels in the synaptic cleft . Glycine acts as a co-agonist for NMDA receptors in excitatory glutamatergic neurotransmission . Increased synaptic glycine concentration by GlyT1 inhibition may normalize NMDA receptor hypofunction in patients with schizophrenia, strengthening glutamatergic signaling and synaptic plasticity, resulting in cognitive improvement . Phase I studies with iclepertin have demonstrated its safety and tolerability in healthy volunteers in single and multiple doses with a favorable pharmacokinetic profile . A Phase II proof-of-concept trial demonstrated improved cognition in adults with schizophrenia after 2—25 mg iclepertin treatment versus placebo at Week 12 . The current Phase II trial assessed the efficacy and safety of PACT using 10 mg iclepertin combined with self-administered at-home CCT. We hypothesized that the low-level cognitive stimulation provided by the at-home CCT would augment the effects of iclepertin in treating the symptoms of CIAS in patients with schizophrenia on stable antipsychotic treatment. A detailed description of the study design, including full eligibility and exclusion criteria, methodology, and a complete list of endpoints has been published previously . Briefly, in this Phase II, 12-week, multicenter, multinational, randomized, double-blind, placebo-controlled, parallel-group exploratory trial , patients with schizophrenia across approximately 40 centers in 6 countries (USA, UK, Canada, Australia, France, New Zealand) were randomized 1:1 to receive iclepertin 10 mg or placebo for 12 weeks . 10 mg was chosen as the dose level to be investigated in the present study based on the results from a proof of mechanism trial where central target engagement of iclepertin was demonstrated by a dose-dependent increase of glycine in rat and human cerebrospinal fluid (CSF). This study demonstrated a mean increase of 50 % in CSF glycine after multiple doses of 10 mg iclepertin in healthy volunteers . Fig. 1 Study design. aDuring screening, patients received CCT (HappyNeuron Pro) for a 2-week run-in period, and those with sufficient adherence to CCT (completion of at least 2 h per week for 2 weeks during the run-in period) were eligible for randomization. bApproximately 30 h over 12 weeks; target of approximately 2.5 h/week. CCT, computerized cognitive training; QD, once daily; R, randomization; S, screening. Fig. 1 During screening, patients received CCT (HappyNeuron Pro) for a 2-week run-in, and those with sufficient adherence to CCT (completion of at least 2 h per week for 2 weeks during the run-in period) were randomized in a blinded fashion via Interactive Response Technology at Visit 2. All randomized patients received access to an adjunctive self-administered at-home CCT for the full duration of the 12-week treatment period. Previous studies have demonstrated similar efficacy for cognitive performance with home-based self-administered training compared with office-based interventions ; at-home training was chosen to evaluate its feasibility in a patient population who may have difficulty accessing office-based programs . Target CCT compliance was 2–3 h per week, based on the results of a large meta-analysis . The minimum compliance threshold was at least 1 h per week and any less than this in a given week was recorded as a ‘CCT low compliance’ protocol deviation and triggered a follow-up from the CCT coach. Patients who completed no CCT training for three consecutive weeks or more despite follow-up from the CCT coach were classed as ‘CCT non-compliance’ protocol deviations. The trial was conducted in accordance with the Declaration of Helsinki, the International Council for Harmonization of Good Clinical Practice guidelines, applicable regulatory requirements, and BI standard operating procedures. The trial protocol and informed consent form were reviewed by the Independent Ethics Committees and/or Institutional Review Boards of the participating centers. The trial included patients with schizophrenia (18–50 years of age) who were clinically stable outpatients, with no hospitalizations for worsening of schizophrenia or symptom exacerbation within 3 months before randomization. Patients could have no more than moderate positive symptoms (per the Positive and Negative Syndrome Scale [PANSS]), had to be on stable antipsychotic treatment of 1–2 antipsychotics (except for clozapine) for ≥3 months and be on their current dose for ≥30 days before randomization. All patients had to demonstrate compliance with CCT during the run-in period and have a consistent study partner to provide reliable input into the Schizophrenia Cognition Rating Scale (SCoRS) and PANSS evaluation. The CCT program available through HappyNeuron Pro was adapted for this trial to include 22 different training tasks to train major cognitive functions often impaired in patients with schizophrenia, including processing speed, selective attention, working memory, visual memory, verbal memory, and executive functions. Task difficulty was rated from 1 to 10 for each exercise and was titrated so that progression to increasingly difficult tasks followed successful completion of previous exercises. The first time a patient completed an exercise, they began at level 1. Patients were required to successfully (≥80 % accuracy) complete this exercise twice to move up a difficulty level at their next attempt, while two consecutive failures (<60 % accuracy) would result in the patient moving down a level for their next attempt. Progression scores were generated by the HappyNeuron Pro algorithm: individual progression scores for each exercise were calculated as an average from the last two attempts of that exercise, with attempted level of difficulty adjusted for accuracy (missing attempts were scored zero). Overall progression scores were calculated as an average of all 22 individual exercise progression scores. Participants were allowed to train on their preferred tasks and training engagement was measured across the training protocol. The primary efficacy endpoint was the change from baseline in the Measurement and Treatment Research to Improve Cognition in Schizophrenia (MATRICS) Consensus Cognitive Battery (MCCB) neurocognitive composite T-score after 12 weeks. The MCCB comprises 10 tests assessing 7 cognitive domains, including speed of processing, attention/vigilance, working memory, verbal learning, visual learning, reasoning and problem solving, and social cognition . Social cognition is distinct from non-social cognition (i.e., neurocognition) , thus the MCCB neurocognitive composite T-score was calculated without the social cognition domain. Several studies have shown that the MCCB is sensitive to CCT effects in participants with schizophrenia . The key secondary endpoints were the change from baseline after 12 weeks in the SCoRS interviewer total score and the PANSS total score. The SCoRS is a 20-item interview-based assessment of cognitive deficits and the degree to which they affect day-to-day functioning that has shown preliminary evidence of sensitivity to treatment. The PANSS is a 30-item rating scale to assess the presence and severity of psychopathology . The change from baseline in the Virtual Reality Functional Capacity Assessment Tool (VRFCAT) adjusted total time T-score was an exploratory endpoint. The VRFCAT is a functional capacity measure in which participants are assessed for their ability to perform routine activities of daily living. The VRFCAT has been shown to improve with a social-cognition-focused CCT intervention in a study focused on social cognition . In a post hoc analysis, linear regressions of change from baseline at Week 12 in MCCB neurocognitive T-score, SCoRS interviewer total score, and VRFCAT adjusted total time T-score with CCT training time were conducted. Additionally, linear regressions of the change from baseline in these cognitive measures at Week 12 with the CCT progression score by quartiles of CCT overall training time (≤11.65, >11.65 ≤ 19.38, >19.38 ≤ 29.83, >29.83) were performed. Safety was assessed as the percentage of patients with adverse events (AEs), serious AEs (SAEs), clinically relevant abnormalities of physical examination, vital signs, electrocardiogram, and laboratory tests. Additional safety assessments were protocol-specified AEs of special interest (AESIs), worsening of disease state (assessed by PANSS) and assessment of suicidality through electronic Columbia Suicide Severity Rating Scale (eC-SSRS). A detailed description of the planned statistical analysis has been previously reported . In summary, a sample size of 200 patients was considered sufficient to achieve the aims of this exploratory trial, which was not powered for any formal hypothesis testing. Demographics, baseline characteristics, and safety analyses were performed on the treated set . For the primary, secondary, and VRFCAT (exploratory) endpoints, analysis was carried out on the full analysis set. The changes from baseline after 6 and 12 weeks in MCCB neurocognitive composite T-score were analyzed by a restricted maximum likelihood (REML) estimation of a mixed model for repeated measures (MMRM) adjusting for treatment by visit, baseline by visit, change from screening to baseline, and age strata. Within-patient correlation was modelled with an unstructured variance-covariance matrix. The same MMRM analysis was performed for each pre-defined subgroup with ≥25 patients. For SCoRS and VRFCAT endpoints, an analysis of covariance was performed, whereas REML estimation of a MMRM was performed for PANSS; these analyses were adjusted for age strata and the corresponding baseline value . Post hoc exploratory regression analysis of associations between the cognitive measures, CCT training time, and CCT progression score were only performed in patients receiving placebo. Of 406 screened patients, 200 were randomized (iclepertin group: iclepertin 10 mg + CCT [ n = 99]; placebo group: placebo + CCT [ n = 101]) and 154 (77.0 %) completed the trial medication. A total of 46 patients (23.0 %) prematurely discontinued treatment; the discontinuation rate was similar across groups . Most patients were randomized in the USA (148/200 [74.0 %]), 136 (68.0 %) were male, and 98 (49.0 %) were white. The VRFCAT scores were relatively high at baseline for both treatment groups ( Table 1 ). There was no substantial difference in alcohol and nicotine use between treatment groups. The mean exposure to trial medication and CCT was comparable for both treatments ( Supplementary Table 1 ). The adjusted mean (standard error [SE]) change from baseline in MCCB neurocognitive composite T-score after 12 weeks was 1.6 (0.6) points for iclepertin and 2.5 (0.6) points for placebo. The MMRM analysis did not show a treatment difference between iclepertin 10 mg + CCT and placebo + CCT groups . However, the adjusted mean [SE] change from baseline in the MCCB neurocognitive composite T-score at Week 12 indicated a slight improvement for the iclepertin 10 mg + CCT group, albeit not reaching statistical significance ( Table 2 ). Fig. 2 Patient disposition. a Of the 176 patients who did not meet the inclusion/exclusion criteria, 47 patients were unable to properly use the CCT device or were non-compliant with the requirements of the at-home CCT during the 2-week run-in period. AEs, adverse events; CCT, computerized cognitive training; COVID-19, coronavirus disease 2019; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. Fig. 2 Table 1 Patient demographics and baseline assessments. Table 1 Baseline Characteristic Placebo + CCT ( n = 101) Iclepertin 10 mg + CCT ( n = 99) Total ( N = 200) Demographics Male, n (%) 71 (70.3) 65 (65.7) 136 (68.0) Female, n (%) 30 (29.7) 34 (34.3) 64 (32.0) Race, n (%) White 51 (50.5) 47 (47.5) 98 (49.0) Black or African American 40 (39.6) 44 (44.4) 84 (42.0) Asian 5 (5.0) 4 (4.0) 9 (4.5) Other ⁎ 5 (5.0) 2 (2.0) 7 (3.5) Age (years), mean (SD) 38.5 (7.7) 37.9 (8.1) 38.2 (7.9) Time since diagnosis of schizophrenia (years), mean (SD) 13.9 (8.4) 13.1 (8.4) 13.5 (8.4) Baseline assessments MCCB neurocognitive composite T-score, mean (SD) 34.0 (11.3) 33.5 (12.0) 33.7 (11.6) SCoRS interviewer total score, mean (SD) † 34.8 (8.9) 36.3 (8.6) 35.5 (8.7) PANSS total score, mean (SD) 63.3 (14.5) 65.8 (14.5) 64.5 (14.5) VRFCAT summary score, mean (SD) 44.9 (15.2) 45.1 (13.9) 45.0 (14.6) Antipsychotic concomitant therapies Patients with 1 antipsychotic, n (%) 79 (78.2) 78 (78.8) 157 (78.5) Patients with 2 antipsychotics, n (%) 20 (19.8) 20 (20.2) 40 (20.0) Patients with oral antipsychotics, n (%) 78 (77.2) 75 (75.8) 153 (76.5) CCT, computerized cognitive training; MCCB, MATRICS Consensus Cognitive Battery; PANSS, Positive and Negative Syndrome Scale; SCoRS, Schizophrenia Cognition Rating Scale; SD, standard deviation; VRFCAT, Virtual Reality Functional Capacity Assessment Tool. ⁎ Includes American Indian or Alaska Native, Native Hawaiian or Other Pacific Islander, or those in multiple racial categories. † Baseline was available for 94 and 91 patients in the placebo + CCT arm and iclepertin + CCT arm, respectively. Table 2 Change from baseline in MCCB neurocognitive composite T-score by treatment groups at Weeks 6 and 12 (FAS). Table 2 Week 6 Week 12 (EoT) Placebo + CCT ( n = 86) Iclepertin 10 mg + CCT ( n = 88) Placebo + CCT ( n = 86) Iclepertin 10 mg + CCT ( n = 88) Change from baseline Adjusted mean (SE) 1.8 (0.5) 1.8 (0.5) 2.5 (0.6) 1.6 (0.6) 95 % CI (0.8, 2.7) (0.9, 2.8) (1.2, 3.7) (0.4, 2.8) Iclepertin vs placebo Adjusted mean difference (SE) 0.1 (0.7) −0.9 (0.9) 95 % CI (−1.3, 1.4) (−2.6, 0.8) p -value 0.9099 0.3101 Data was analyzed by a MMRM model using an unstructured within-subject variance-covariance matrix and included the following fixed effects: categorical factors of planned treatment, visit, planned treatment by visit interaction, and age group as well as continuous covariates of baseline value, baseline by visit interaction, and change from screening to baseline value. Increases in MCCB scores indicate improvements. CCT, computerized cognitive training; CI, confidence interval; EoT, end of treatment; FAS, full analysis set; MCCB, MATRICS Consensus Cognitive Battery; MMRM, mixed models for repeated measures; SE, standard error. While the subgroup analysis by CCT training adherence was generally consistent with the overall analysis, a slight trend towards a higher improvement in the placebo arm was observed for the subgroup of patients with cumulative CCT compliance ≥ median . Fig. 3 Treatment effects versus placebo at Week 12 for MCCB neurocognitive composite T-score overall, and by cumulative CCT compliance (FAS). The adjusted mean difference between iclepertin and placebo groups was obtained by a REML estimation of a MMRM model with unstructured variance-covariance matrix fitted to each subgroup level. CCT, computerized cognitive training; CI, confidence interval; FAS, full analysis set; MCCB, MATRICS Consensus Cognitive Battery; MMRM, mixed model for repeated measures; REML, restricted maximum likelihood. Fig. 3 There was no treatment difference between iclepertin and placebo groups in the SCoRS interviewer total score or PANSS total score at Week 12 ( Table 3 A and 3B ). Similar to the primary endpoint, the adjusted mean [SE] change from baseline in the SCoRS interviewer total score at Week 12 indicated a slight improvement (not statistically significant) for the iclepertin 10 mg + CCT group ( Table 3 A ). Table 3 Change from baseline in (A) SCoRS interviewer total score at Week 12 (FAS), (B) PANSS total score at Week 6 and Week 12 (FAS), and (C) VRFCAT total time T-score at Week 12 (FAS). Table 3 A. SCoRS Placebo + CCT ( n = 75) Iclepertin 10 mg + CCT ( n = 82) Change from baseline at Week 12 (EoT) Adjusted mean (SE) −3.1 (0.7) −2.5 (0.6) 95 % CI (−4.4, −1.8) (−3.7, −1.3) Iclepertin vs placebo Adjusted mean difference (SE) 0.6 (0.9) 95 % CI (−1.2, 2.4) p-value 0.5237 B. PANSS Week 6 Week 12 (EoT) Placebo + CCT (n = 85) Iclepertin 10 mg + CCT (n = 88) Placebo + CCT ( n = 85) Iclepertin 10 mg + CCT (n = 88) Change from baseline Adjusted mean (SE) −2.0 (1.0) −0.6 (0.9) −2.1 (1.0) −2.7 (1.0) 95 % CI (−3.9, −0.1) (−2.5, 1.2) (−4.1, −0.1) (−4.7, −0.8) Iclepertin vs placebo Adjusted mean difference (SE) 1.3 (1.3) −0.7 (1.4) 95 % CI (−1.3, 4.0) (−3.5, 2.2) p-value 0.3194 0.6420 C. VRFCAT Placebo + CCT ( n = 65) Iclepertin 10 mg + CCT ( n = 62) Change from baseline at Week 12 (EoT) Adjusted mean −2.6 −3.4 95 % CI (−5.0, −0.2) (−5.8, −1.0) Iclepertin vs placebo Adjusted mean difference (SE) −0.8 (1.7) 95 % CI (−4.2, 2.6) p-value 0.6542 Data was analyzed using the ANCOVA model which included the following fixed effects: categorical factors of planned treatment and age group as well as a continuous covariate of baseline value. Decreases in SCoRS rating indicates improvements. Data was analyzed by a MMRM model using an unstructured within-subject variance-covariance matrix and included the following fixed effects: categorical factors of planned treatment, visit, planned treatment by visit interaction, and age group as well as a continuous covariates of baseline value and baseline by visit interaction. Decreases in PANSS scores indicate improvements. Data was analyzed by an ANCOVA model which included the following fixed effects: categorical factors of planned treatment and age group as well as a continuous covariate of baseline value. Decreases in VRFCAT summary scores indicate worsening performance. ANCOVA, analysis of covariance; CCT, computerized cognitive training; CI, confidence interval; EoT, end of treatment; FAS, full analysis set; MMRM, mixed models for repeated measures; PANSS, Positive and Negative Syndrome Scale; SCoRS, Schizophrenia Cognition Rating Scale; SD, standard deviation; SE, standard error; VRFCAT, Virtual Reality Functional Capacity Assessment Tool. There was also no treatment difference between iclepertin and placebo groups in the VRFCAT adjusted total time T-score . However, the adjusted mean [SE] change from baseline in the VRFCAT adjusted total time T-score at Week 12 indicated a slight deterioration for the iclepertin 10 mg + CCT group ( Table 3 C ). In placebo patients, there was no correlation between CCT overall training time and the change from baseline at Week 12 in MCCB neurocognitive composite T-score (estimated slope, SE: 0.08 [0.05] per hour) or VRFCAT adjusted total time T-score (−0.17 [0.12] per hour), while the data suggest a negative correlation between CCT overall training time and changes from baseline at Week 12 in SCoRS interviewer total score . Fig. 4 CCT Training time versus change from baseline at Week 12 in (A) MCCB neurocognitive composite T-score, (B) SCoRS interviewer total score, and (C) VRFCAT adjusted total time T-score. CCT, computerized cognitive training; MCCB, MATRICS Consensus Cognitive Battery; PANSS, Positive and Negative Syndrome Scale; SCoRS, Schizophrenia Cognition Rating Scale; VRFCAT, Virtual Reality Functional Capacity Assessment Tool. The CCT progression score of an attempt is based on the level attempted adjusting for accuracy: level×10 for 80 % or higher accuracy, (level – 0.5) × 10 for 60 % to < 80 % accuracy, (level – 1.5) × 10 for < 60 % accuracy, and 0 for no attempts, for exercise levels that progress from 1 to 10. Fig. 4 The linear regression of change from baseline in cognitive measures at Week 12 on CCT progression score was also analyzed by subgroups of CCT overall training time quartiles. Generally, no pattern of correlation is suggested by the data, except a positive correlation between the CCT progression score and the MCCB neurocognitive composite T-score at Week 12 in patients with >19.38 to 29.83 h of overall CCT training, and a negative correlation between the CCT progression score and the SCoRS interviewer total score at Week 12 in patients with ≤11.65 h of overall CCT training. Overall, 39/99 (39 %) patients in the iclepertin group and 57/101 (56 %) patients in the placebo group reported ≥1 AE, and most AEs were mild to moderate ( Table 4 ). Between treatment groups, similar proportions of AEs led to discontinuation of trial medication. There were very few severe AEs and SAEs, and no AESIs or deaths. Table 4 Overall summary of adverse events (treated set). Table 4 Category of AE, N (%) Iclepertin 10 mg + CCT (n = 99) Placebo + CCT (n = 101) Any AE 39 (39.4) 57 (56.4) Severe AEs 1 (1.0) 3 (3.0) Investigator-defined drug-related AEs 17 (17.2) 21 (20.8) AEs leading to discontinuation of trial medication 3 (3.0) 4 (4.0) AESIs 0 0 SAEs a 1 (1.0) 2 (2.0) Fatal 0 0 Life-threatening 0 0 Persisting or significant disability/incapacity 0 0 Requiring or prolonging hospitalization 1 (1.0) 1 (1.0) Congenital anomaly or birth defect 0 0 Other 0 1 (1.0) An adverse event was defined as any untoward medical occurrence. An SAE was any AE which fulfilled one of the following criteria: resulted in death, was life-threatening, required inpatient hospitalization, required prolonging of existing hospitalization, resulted in a persistent/significant disability/incapacity, or deemed serious based on appropriate medical judgment. AE, adverse events; AESI, adverse events of special interest; CCT, computerized cognitive training; SAE, serious adverse events. a A patient could be counted in >1 seriousness criterion. Overall, no clinically relevant changes in laboratory parameters and vital signs were observed. A similar number of patients in both groups (iclepertin [ n = 8]; placebo [ n = 7]) had disease worsening indicated by the PANSS positive score ≥ 5 and 4 patients in each treatment group with PANSS total score ≥ 15. In the iclepertin and placebo groups, there were 7 and 8 patients, respectively, with ≥1 event of suicidal ideation during treatment. No events of C-SSRS type 4 or 5, suicidal behavior, or self-injury were reported. Results from this Phase II study to assess the efficacy and safety of iclepertin versus placebo for the treatment of CIAS demonstrated that iclepertin 10 mg had a favorable safety profile and was well tolerated. Consistent with previous Phase I and II trials, the iclepertin group had lower incidence of AEs versus placebo (39 % versus 56 %), and headache was the most common AE . However, the primary and secondary efficacy endpoints were not met in this trial. After 12 weeks of treatment, no difference was observed between the iclepertin and placebo groups in the MCCB neurocognitive composite T-score, SCoRS interviewer total score, or PANSS total score. Further, results from the exploratory VRFCAT endpoint also indicated no improvements in functional capacity. Although the present study failed to show positive efficacy results, 3 Phase III trials are ongoing to examine the efficacy, safety, and tolerability of iclepertin in improving cognition and functioning in a larger group of patients, over a longer treatment period (26 weeks); however, these trials do not include an adjunctive CCT: CONNEX-1, CONNEX-2, and CONNEX-3 . It is unclear whether the failure to meet efficacy endpoints in the current study truly indicates that iclepertin does not affect CIAS in its own right, or if there are methodological considerations which may have contributed to the lack of positive results. In the previous Phase II proof-of-concept trial that demonstrated improved cognition in adults with schizophrenia after 2—25 mg iclepertin the effect size seen was significant, but numerically small. It is possible that in the current study the sample size was too small and baseline VRFCAT scores too high to detect iclepertin monotherapy effects. Results from the larger, ongoing Phase III study are expected soon and should help answer this question. Similarly, it is difficult to say whether the results of the current study indicate iclepertin has no augmenting effect on CCT. Use of CCT to improve cognition is a relatively recent development, and there are minimal comparative data regarding efficacy of specific CCT programs. Although similar studies have demonstrated comparable efficacy of home-based and office-based interventions , it is possible that providing an office-based training environment and observation by healthcare professionals could have a slightly higher impact on training engagement and subsequent improvement in outcomes. Most previous studies using the HappyNeuron Pro CCT program deployed the training system as part of a comprehensive rehabilitation program, which includes training in areas such as strategic monitoring, bridging, and functional adaptation skills in order to achieve large and durable effects on functional competence and real-world functioning . In contrast, the intention of using CCT in the present study was not to effect cognitive change per se, but rather to provide a low level of cognitive stimulation to augment the effects of pharmacological treatment, in the most standardized possible way. Further to this, providing CCT in the home setting allowed us to evaluate the feasibility of at-home training in this patient population who may have had difficulty accessing traditional training programs . These differences mean a direct efficacy and mechanism of action comparison to the use in the current study is not possible . In two recent meta-analyses of cognitive training for the treatment of psychosis, at-home HappyNeuron Pro was not used in any of the cited articles . The lack of available efficacy data for HappyNeuron Pro alone makes the present results difficult to interpret, and it is difficult to speculate as to whether larger effects may have been seen from using a different CCT program. In the subgroup analysis of change from baseline in the MCCB neurocognitive composite score, a slight tendency towards greater improvement in the placebo + CCT group was observed for the subgroup of patients with total CCT compliance ≥ median 2.17 h/week. However, there was no correlation between CCT training time and cognitive outcomes. These mixed findings suggest no strong association between CCT and efficacy outcomes and add to the difficulty of differentiating treatment effects from CCT effects. Another factor that complicated the interpretation of the trial results was the absence of a non-CCT placebo arm. A recent publication reported results from a similar trial to the current study, wherein the impact of a GlyT1 inhibitor was investigated in combination with computer-based cognitive training (HappyNeuron Pro) to treat CIAS in a cross-over trial with no non-CCT placebo arm, and found no augmentation of the effects of cognitive training . One possible explanation offered by the authors is that it may be difficult to interpret the augmenting effects of two interventions in the context of one another, and an additional non-CCT placebo arm may help to produce clearer results , a consideration which may also be relevant in the present study. In contrast, a three-arm study comparing computerized cognitive training alone, in-person, group delivered functional adaptation skills training alone, or a combination of both (which met the criteria for being designated as “cognitive remediation”) and had no inactive placebo control arm, found statistically significant cognitive gains in both CCT and combined treatment arms, as well as gains in everyday functioning that were significant in the combined treatment arm . A potential limitation of this study is the high scores on VRFCAT assessment at baseline. The VRFCAT total time T-scores of ∼45 were observed in both treatment groups in this study; while average VRFCAT total time T-scores of 49.7 and 32.5 have previously been obtained from healthy volunteers without cognitive impairment, and patients with schizophrenia, respectively . While these may appear to be in contrast with the MCCB baseline scores of ∼34.0 (both treatment groups), which were in the expected, impaired range, it is important to remember that the MCCB is a paper and pencil test, while the VRFCAT is completed digitally. It is plausible that participants with advanced technological skills may have been more inclined to volunteer for a computer training study, meaning the technological performance of the population may have outstripped their general cognitive ability. This could partly explain the lack of efficacy observed in the VRFCAT endpoint and suggests a potential selection bias of patients with a high level of functional capacity. Another limitation of this study is that the trial site selection was restricted, given that the CCT program was only available in English and French languages. Finally, the study was exploratory in nature, which may affect its interpretation, and not powered to detect differences between the treatment groups beyond the primary measure. Despite the lack of positive efficacy results, the present study had several strengths. Foremost, this was the largest trial of its kind to combine CCT and pharmacotherapy for schizophrenia in 200 patients with CIAS. The recent publication by Surti TS et al. examined the effects of a GlyT1 inhibitor in combination with CCT in a smaller sample (71 patients randomized) . The successful implementation of the at-home CCT component represents an additional strength of the current study; on average, patients completed approximately 21.3 h of CCT in the iclepertin group and 19.4 h in the placebo group over a duration of 12 weeks. The adherence results presented here compare favorably to previous home-based training interventions . To our knowledge, this trial is the largest of its kind combining daily pharmacotherapy for CIAS with at-home CCT. Even though efficacy could not be demonstrated, the safety results of iclepertin 10 mg were consistent with previous clinical studies and no new risks were identified. Three multinational Phase III trials (CONNEX-1, −2, and − 3) are ongoing to further investigate the efficacy and safety of iclepertin in improving cognition and daily functioning (without utilizing an adjunctive CCT) in patients with schizophrenia. | Review | biomedical | en | 0.999995 |
PMC11699310 | The traditional methods of horse breeding have been based on simple selection and crossing of animals with desirable traits. Although such methods are still in use in horse selection programs, their sole usage limits the breeding progress. Modern practices imply the use of the data on genetic mechanisms underlying economically import traits such as productivity. Genomic and marker associated selection (MAS) became an important field of animal breeding as the increasing data on genetic polymorphism stimulate studies not only elucidating genetic properties of breeds but also endorsing them for use in selection practice. The study of breeds, using molecular techniques is very important and useful for their characterizing [ , , ]. Conservation of genetic diversity in animal species requires the proper performance of conservation superiorities and sustainable handling plans that should be based on universal information on population structures, including genetic diversity resources among and between breeds . Genetic diversity is an essential element for genetic improvement, preserving populations, evolution and adapting to variable environmental situations . On the other hands, determination of gene polymorphism is important in farm animals breeding in order to define genotypes of animals and their associations with productive, reproductive and economic traits [ , , ]. Considering meat productivity, the living mass and size of an animal are the primary traits of interest. Body mass is a multifactorial quantitative trait affected by both genetic and ambiance factors. For example, heritability of weight and body measures have been shown previously [ , , ]. Particularly, heritability of Zhabe body mass and measurements have been estimated . The more detailed studied on genetic factors affecting horse body parameters involve the advances of horse genomics. The availability of continuously updated reference assembly of horse genome (current version EquCab3.0 ) assists studies on genetic bases of various traits of interest involving thousands and millions of SNPs . Genome wide association studies (GWAS) have identified genes LCORL/NCAPG, HMGA2, ZFAT, and LASP1 as the major genetic factors associated with body size in horses [ , , , ]. Our previous study was the biggest to date and involved total 2020 horses of six traditional types and breeds of Kazakh horses analyzed using 80,000 SNP microarray . This study have revealed the surprising absence of genetic differentiation between studied breeds. The observed genetic structure was an evidence for existence of broadly defined landrace corresponding to Kazakh horses without notable genetic demarcation between rational types and breeds. It has been also hypothesized that the traditional nomadic ways of horse breeding on the territory of contemporary Kazakhstan could have lead to formation of Kazakh horse landrace without strong pressure of artificial selection . The present article is focused on horses of Zhabe type and uses previously published data combined with newly genotyped sample of Zhabe horses to provide more details on the genetic structure is type of Kazakh horses including GWAS for body weight and size, the traits associated with meat productivity. A special attention was paid to population genetic parameters potentially reflecting the way of horse selection. Genetic material (hairs from tails and/or manes) of Zhabe horses was collected at private farms “Alakol Asyl” (Zhetysu region) and “Akzhar” (Pavlodar region); additionally, samples of white horses “Zhetysu Asyly” were collected at A. Sophin's private farm (Zhetysu region) ( Table 1 ). All collected materials were stored at 4 °C until further use. DNA was isolated from hair follicles using the kit ‘‘DNK-Extran2” (Syntol, Russian Federation) following the manufacturer's protocol and quantified using Qubit 4 Fluoremeter with the Qubit dsDNA Broad Range reagent (Thermo Fisher Scientific, USA) for downstream analysis. Table 1 Summary of samples and data used in the study. Table 1 Zhabe horses, Kazakhstan Population code Husbandry Region Number of individuals with available data Final number of individuals used for analysis Data source Genotype Phenotype (weight, size) 37 Alakol Asyl Zhetysu a 421 427 384 Present study 39 b A. Sophin Zhetysu a 42 88 42 25 Akzhar Pavlodar 81 c 173 64 , present study 33 Agro-Damu Pavlodar 153 186 152 3 Kalka Zhambyl 143 163 141 28 Zhaksylyk Almaty 86 122 86 34 Anar Pavlodar 76 77 76 32 Sayakhat Almaty 48 0 47 29 Kyzylsok Almaty 46 46 46 Total 1096 1282 1038 Foreign breeds Breed code Breed name Breed origin Number of genotypes Sample origin Data source AKTK Akhat-Teke Turkmenistan 19 USA, Russia ARR Arabian Middle East 24 USA CSP Caspian Persia 19 USA MON Mongolian Mongolia 19 Mongolia TB_UK Thoroughbred UK 19 UK TB_US 17 USA a Former part of Almaty region. b Horses of the derivative “Zhetysu Asyly” breed. c Including 17 newly analyzed samples. SNP genotyping of collected samples was performed using Equine80k SNP array on the iScan system (Illumina, USA) according to the manufacturer's protocol. Genotype calling and primary data filtering were performed using GenomeStudio software (Illumina, USA). The primary filtering criteria included call rate ≥0.9, median GC score ≥0.8 for samples; call frequency ≥0.95 and GT score ≥0.7 for SNP; indel markers included into array were excluded. PLINK1.9 software was used for further data filtering to exclude SNPs with minor allele frequencies <0.05 and those deviating from Hardy-Weinberg equilibrium with a P-value threshold of 1 × 10 −5 . Additional sample filtering was performed based on data availability for particular individuals when needed. Newly obtained genotyping data were combined with previously reported data on Zhabe horses . Previously obtained unfiltered data were merged with current results and filtered (SNP call rate ≥0.9, minor allele frequency ≥0.05, Hardy-Weinberg equilibrium under p- value threshold 10 −5 ). The obtained dataset was used for genetic structure analysis using ADMIXTURE software and GWAS using PLINK1.9. ADMIXTURE run was set for K numbers of clusters from 2 to 20 with ten-fold cross-validation error estimate; results were vizualized using CLUMPAK web server . For comparative analyzis the dataset on Zhabe horses was further merged with data on foreign horse breeds and filtered anew with the same criteria. The following breeds were chosen: Akhal-Teke, Caspian, Arabian, Mongolian, and Thorougbred (two populations)( Table 1 ). The analysis included principal component analysis and evaluation of linkage disequilibrium (LD), expected and observed heterozygosity, and pairwise between population Wright's F ST using PLINK1.9 and R . Neighbor net for pairwise F ST between Zhabe populations and foreign breeds was calculated and plotted using R package phangorn . Additionally, ADMIXTURE analysis was performed for the combined dataset with the same running conditions. Genome wide association study was conducted using general linear model implemented in PLINK1.9. Sex and age of animals in years were used as covariates of the model. Animals without available phenotypic data (e.g. population 32 “Sayakhat”) or identified as outlying genotypes by population structure analysis were excluded from the analysis. The four size measurements (HW, OBL, CC, and CBC) were normalized by subtracting the mean and dividing by standard deviation and then used to perform principal component analysis using built-in R functions. The first principal component was further used in GWAS as a synthetic variable describing animal body size. GWAS was performed separately for animal body weight and size using --linear command of PLINK with age and sex as covariates (model Y = b 0 + b 1 X ADD + b 2 X AGE + b 3 X SEX + e, where Y – phenotype , X ADD – genotypes encoded as additive model (0, 2 for homozygote, 1 for heterozygote), X AGE – covariate of age, X SEX – covariate for sex, b n – regression coefficients, e – residual error term). P- values were estimated using 1000 Monte-Carlo permutation test with adaptive number of permutations (PLINK's--perm command) and used to construct Manhattan plot using ‘ggplot2’ R package. Variant Effect Predictor and DAVID tools were used to annotate significant SNPs. Results for SNPs associated with body weight and size were combined and filtered to include only variants with available annotations and exclude variants with duplicate annotations. As a final result of genotyping and data filtering, genotypes of 443 horses have been obtained including 401 samples of Zhabe horses and 42 samples of white horses ‘Zhetysu Asyly’, in addition to previously obtained data ( Table 1 ). The final combined dataset included 1038 individuals with 43,422 SNPs. The combined dataset including five foreign breeds consisted of 24,764 SNP after additional filtering. Calculated values of expected/observed heterozygosity and pairwise between population F ST are shown in Table 2 . Among Kazakh horses, the lowest hetezygosity level was in population 39 containing Zhetysu Asyly horses; expected value per chromosome 0.28 (SD 0.010), observed value 0.308 (SD 0.12). Other populations corresponding attributed Zhabe horses displayed expected values from 0.314 in population 34 to 0.334 in population 3, and observed values from 0.317 in populations 33 and 34 to 0.342 in population 32; the former population had the biggest deviation between expected and observed heterozygosity whereas other populations had only slightly differing values. All Kazakh horse populations had low F ST values not exceeding 0.010 between each other except for population 39 with F ST from 0.027 to 0.034. The foreign breeds displayed higher coefficients when compared to Zhabe horses: from 0.006 to 0.030 for Mongolian horses to 0.095–0.106 for the Thoroughbred horses. Thus, the Mongolian horses displayed more similarity to Zhabe horses, and the Thoroughbred horses were the most dissimilar. As it can be seen in the neighbor net plot of F ST , populations of Zhabe horses form close group except for popultaion 3 which demonstrated shift towards Thoroughbred horses. Population 39 (Zhetysu Asyly) formed independent branch distant from Zhabe populations. Table 2 Population genetic parameters calculated for combined dataset (24,764 SNP). Table 2 Population N H Echr H Ochr F ST 25 28 29 32 33 34 37 39 3 AKTK ARR CSP MON TB_UK TB_US 25 64 0.319 ± 0.009 0.325 ± 0.009 – 0.001 ± 0.001 0.003 ± 0.001 0.003 ± 0.001 0.003 ± 0.001 0.003 ± 0.001 0.003 ± 0.001 0.030 ± 0.007 0.008 ± 0.002 0.044 ± 0.006 0.057 ± 0.006 0.029 ± 0.004 0.008 ± 0.003 0.099 ± 0.017 0.096 ± 0.017 28 86 0.318 ± 0.008 0.318 ± 0.010 0.001 ± 0.001 – 0.004 ± 0.001 0.005 ± 0.001 0.002 ± 0.001 0.002 ± 0.001 0.003 ± 0.001 0.030 ± 0.007 0.008 ± 0.002 0.045 ± 0.007 0.058 ± 0.007 0.029 ± 0.004 0.007 ± 0.002 0.101 ± 0.018 0.098 ± 0.017 29 46 0.318 ± 0.009 0.326 ± 0.009 0.003 ± 0.001 0.004 ± 0.001 – 0.004 ± 0.001 0.003 ± 0.001 0.006 ± 0.001 0.004 ± 0.001 0.030 ± 0.007 0.007 ± 0.001 0.044 ± 0.006 0.057 ± 0.007 0.029 ± 0.005 0.008 ± 0.003 0.095 ± 0.015 0.092 ± 0.016 32 47 0.320 ± 0.009 0.342 ± 0.011 0.003 ± 0.001 0.005 ± 0.001 0.004 ± 0.001 – 0.006 ± 0.001 0.004 ± 0.001 0.006 ± 0.001 0.034 ± 0.007 0.011 ± 0.001 0.047 ± 0.006 0.059 ± 0.006 0.032 ± 0.004 0.012 ± 0.002 0.098 ± 0.017 0.095 ± 0.018 33 152 0.321 ± 0.009 0.317 ± 0.010 0.003 ± 0.001 0.002 ± 0.001 0.003 ± 0.001 0.006 ± 0.001 – 0.002 ± 0.001 0.002 ± 0.000 0.028 ± 0.007 0.006 ± 0.001 0.042 ± 0.006 0.055 ± 0.006 0.026 ± 0.004 0.006 ± 0.002 0.097 ± 0.016 0.094 ± 0.016 34 76 0.314 ± 0.009 0.317 ± 0.009 0.003 ± 0.001 0.002 ± 0.001 0.006 ± 0.001 0.004 ± 0.001 0.002 ± 0.001 – 0.004 ± 0.001 0.030 ± 0.006 0.010 ± 0.002 0.048 ± 0.006 0.061 ± 0.007 0.031 ± 0.005 0.007 ± 0.002 0.106 ± 0.018 0.104 ± 0.017 37 384 0.321 ± 0.008 0.319 ± 0.009 0.003 ± 0.001 0.003 ± 0.001 0.004 ± 0.001 0.006 ± 0.001 0.002 ± 0.000 0.004 ± 0.001 – 0.027 ± 0.007 0.008 ± 0.002 0.043 ± 0.006 0.057 ± 0.007 0.027 ± 0.004 0.005 ± 0.002 0.101 ± 0.017 0.099 ± 0.017 39 42 0.298 ± 0.010 0.308 ± 0.012 0.030 ± 0.007 0.030 ± 0.007 0.030 ± 0.007 0.034 ± 0.007 0.028 ± 0.007 0.030 ± 0.006 0.027 ± 0.007 – 0.032 ± 0.006 0.069 ± 0.009 0.079 ± 0.011 0.054 ± 0.007 0.034 ± 0.007 0.122 ± 0.017 0.120 ± 0.018 3 141 0.334 ± 0.008 0.324 ± 0.008 0.008 ± 0.002 0.008 ± 0.002 0.007 ± 0.001 0.011 ± 0.001 0.006 ± 0.001 0.010 ± 0.002 0.008 ± 0.002 0.032 ± 0.006 – 0.037 ± 0.006 0.050 ± 0.006 0.028 ± 0.004 0.015 ± 0.003 0.064 ± 0.012 0.060 ± 0.011 AKTK 19 0.300 ± 0.009 0.299 ± 0.012 0.044 ± 0.006 0.045 ± 0.007 0.044 ± 0.006 0.047 ± 0.006 0.042 ± 0.006 0.048 ± 0.006 0.043 ± 0.006 0.069 ± 0.009 0.037 ± 0.006 – 0.067 ± 0.009 0.058 ± 0.008 0.056 ± 0.009 0.097 ± 0.016 0.094 ± 0.017 ARR 24 0.299 ± 0.011 0.294 ± 0.016 0.057 ± 0.006 0.058 ± 0.007 0.057 ± 0.007 0.059 ± 0.006 0.055 ± 0.006 0.061 ± 0.007 0.057 ± 0.007 0.079 ± 0.011 0.050 ± 0.006 0.067 ± 0.009 – 0.059 ± 0.008 0.074 ± 0.01 0.109 ± 0.017 0.107 ± 0.018 CSP 18 0.297 ± 0.009 0.309 ± 0.013 0.029 ± 0.004 0.029 ± 0.004 0.029 ± 0.005 0.032 ± 0.004 0.026 ± 0.004 0.031 ± 0.005 0.027 ± 0.004 0.054 ± 0.007 0.028 ± 0.004 0.058 ± 0.008 0.059 ± 0.008 – 0.036 ± 0.004 0.110 ± 0.015 0.107 ± 0.015 MON 19 0.298 ± 0.009 0.306 ± 0.011 0.008 ± 0.003 0.007 ± 0.002 0.008 ± 0.003 0.012 ± 0.002 0.006 ± 0.002 0.007 ± 0.002 0.005 ± 0.002 0.034 ± 0.007 0.015 ± 0.003 0.056 ± 0.009 0.074 ± 0.01 0.036 ± 0.004 – 0.116 ± 0.020 0.113 ± 0.020 TB_UK 19 0.306 ± 0.010 0.321 ± 0.014 0.099 ± 0.017 0.101 ± 0.018 0.095 ± 0.015 0.098 ± 0.017 0.097 ± 0.016 0.106 ± 0.018 0.101 ± 0.017 0.122 ± 0.017 0.064 ± 0.012 0.097 ± 0.016 0.109 ± 0.017 0.110 ± 0.015 0.116 ± 0.02 – 0.003 ± 0.008 TB_US 17 0.310 ± 0.012 0.323 ± 0.016 0.096 ± 0.017 0.098 ± 0.017 0.092 ± 0.016 0.095 ± 0.018 0.094 ± 0.016 0.104 ± 0.017 0.099 ± 0.017 0.12 ± 0.018 0.060 ± 0.011 0.094 ± 0.017 0.107 ± 0.018 0.107 ± 0.015 0.113 ± 0.02 0.003 ± 0.008 – N – number of individual samples. H E chr – expected heterozygosity per chromosome, mean ± standard deviation. H Ochr – observed heterozygosity per chromosome, mean ± standard deviation. F ST – Wright's fixation index between populations. Fig. 1 Plot of linkage disequilibrium and pairwise F st of Zhabe horses; A) Linkage disequilibrium decay in nine studied populations of Zhabe horses; B) Linkage disequilibrium decay in Zhabe horses in comparison with Akhal-Teke, Caspian, Mongol, Arabian, and Thoroughbred horses; C) Neighbor net plot of pairwise FST between Zhabe horse populations and foreign breeds. Fig. 1 Linkage disequilibrium (LD) analysis displayed higher values of R 2 coefficient in population 39 comparing to other Zhabe populations. Foreign breeds except Mongolian horses have shown much higher values; the highest R 2 coefficients and slower LD decay was in two Thoroughbred populations . Mongolian horses had closer LD values to Zhabe horses than to other foreign breeds. Principal component analysis have shown a presence of outlying genotypes of Zhabe (populations 3 and 33) shifted towards Thoroughbred whereas most Zhabe samples have been combined into a single dense cluster. Principal component 1 (PC1) explaining 21.73 % of total variance contributes the most to discrimination between Zhabe horses and Thoroughbred, and PC2 with 8.27 % of explained variance separated Arabian horses. Whereas first two components place population 39 close to other Zhabe horses, PC3 has shown its significant deviation. Mongolian horses were placed close to Zhabe. Akhal-Teke and Caspian horses formed distinct cluster close to Zhabe. Fig. 2 Principal component analysis of Zhabe horses in comparison to five foreign breeds; A) Scatterplot of components 1 and 2; B) Scatterplot of components 2 and 3. Population codes according Table 1 . Fig. 2 ADMIXTURE analysis was performed for numbers of expected clusters K from 1 to 20. Cross-validation test have not revealed the best K value; although the error value was the lowest at K = 17, the absence of following inclining trend on the plot makes its selection doubtful . Fig. 3 A and B, demonstrates patterns for K= 2, 5, and 10 selected based on examination of diagrams as demonstrating most significant features of the obtained results. With K= 2 outlying genotypes persistent across all K configurations have been identified (shown orange in all diagrams). Comparative analysis with foreign breed allowed to attribute these individuals to inclusion of Thoroughbred into selection process. Population 39 was classified as a unique cluster dissimilar to either Zhabe or foreign breeds. Higher K patterns reveal within population heterogeneity of Zhabe breeds. Fig. 3 Results of ADMIXTURE analysis of Zhabe horses in comparison to five foreign breeds; A) Data on Zhabe horses, 43,422 SNP; B) Data on Zhabe horses combined with five foreign breeds (24,764 SNP); C) Plot of cross-validation error for data A (blue line) and B (red line). Population codes according Table 1 . Fig. 3 For genome wide association study, horse individuals identified as outlying genotypes by PCA and ADMIXTURE analysis were excluded from consideration. Total 823 animals were used for analysis, including 391 newly obtained genotypes and 432 genotypes from previously published data . The summary of phenotypic data used is shown in Table 3 . As a result, 126 SNPs had p- value adjusted by Monte-Carlo permutation test for association with body weight, and 99 SNPs for body size. All variants satisfying the selected significance threshold P < 0.001 were distributed occasionally, and no regions with high occurrence of significant SNPs were observed . After variant annotation and filtering of the results, total 57 SNPs have been retained, including 14 and 23 SNPs associated only with body size and weight, correspondingly, and 20 variants associated with both traits ( Table 4 ). Gene CFI (complement factor I) contained three revealed SNPs; for genes DLG2, KIT , and PRKG2 two variants have been identified; other genes contained single SNPs. Most annotated genes were have been involved into various regulatory processes and signal transmission. Table 3 Summary of phenotypic traits of Zhabe horse populations. Table 3 Population code Sex (percentage of mares in the sample) Age Weight HW OBL CC CBC mean sd mean sd mean sd mean sd mean sd mean sd 25 92.19 8.111 3.399 417.328 41.505 142.422 2.308 146.719 4.022 173.406 7.272 18.805 0.568 28 91.86 6.209 3.505 394.128 38.763 141.372 3.002 145.977 3.700 173.012 6.934 17.878 0.659 29 93.48 5.435 2.177 359.326 61.695 139.348 4.729 142.391 7.120 168.891 8.817 17.413 0.896 3 91.43 6.993 3.771 397.679 52.221 141.157 3.740 146.036 5.756 171.571 8.259 18.050 0.896 32 100.00 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 33 100.00 7.993 2.272 388.901 12.641 139.605 1.479 143.645 1.673 164.757 2.258 17.688 0.372 34 89.33 8.507 0.891 401.320 28.991 140.293 2.198 144.427 2.579 167.160 5.514 18.600 0.539 37 96.88 3.786 1.657 396.997 44.904 141.882 2.838 145.743 3.456 169.322 5.589 17.732 0.870 39 80.95 4.190 1.194 380.286 37.050 140.119 2.596 143.810 3.763 168.976 4.176 17.788 0.678 nd. - no data available. Fig. 4 Manhattan plots of P-values adjusted by the Monte-Carlo permutation test from genome-wide association analysis for body size (A) and weight (B) in Zhabe horses. Red line indicates significance threshold p= 0.001. Fig. 4 Table 4 Single nucleotide polymorhisms associated with horse weight and size annotated using VEP and DAVID tools. Table 4 Variant ID Location Gene P -value GO term – biological process Size Weight BIEC2_316765 15:68252917 ALK – 0.0008545 GO:0006468∼protein phosphorylation AX-103265166 2:55284182 BNIP3L 2.3 × 10 −5 1.80 × 10 −5 GO:0016239∼positive regulation of macroautophagy AX-104903715 4:93924679 BRAF 0.0007523 0.0004619 GO:0000165∼MAPK cascade BIEC2_946110 6:29507974 CACNA1C 0.0002397 – GO:0002520∼immune system development BIEC2_140284 11:13962473 CACNG4 – 3.181 × 10 −5 GO:0019226∼transmission of nerve impulse Affx-102683437 4:79003820 CADPS2 – 0.0001952 GO:0006887∼exocytosis AX-104609861 6:85773796 CCT2 – 0.0007669 GO:0006457∼protein folding BIEC2_508762 2:116459352 CFI – 0.0004362 GO:0006508∼proteolysis BIEC2_508766 2:116465026 CFI 0.0002012 0.0001068 GO:0006508∼proteolysis BIEC2_508769 2:116466442 CFI 0.0001972 0.0001277 GO:0006508∼proteolysis TBIEC2_331745 15:68417648 CLIP4 – 0.0002872 GO:0031122∼cytoplasmic microtubule organization Affx-101130275 4:60087882 CPVL 0.0009387 – GO:0006508∼proteolysis BIEC2_700308 27:2259858 CSGALNACT1 – 0.0004961 GO:0001958∼endochondral ossification BIEC2_725599 28:5564261 CSRP2 0.0003285 – GO:0045214∼sarcomere organization AX-103706694 1:120490380 CYP11A1 1 × 10 −6 1.00 × 10 −7 GO:0006700∼C21-steroid hormone biosynthetic process TBIEC2_498975 2:40275747 DISP3 0.0004661 – GO:0045665∼negative regulation of neuron differentiation AX-103721396 7:63690864 DLG2 0.0008281 1.00 × 10 −7 GO:0007268∼chemical synaptic transmission BIEC2_1004624 7:62981215 DLG2 0.0009895 – GO:0007268∼chemical synaptic transmission BIEC2_722382 27:39199656 DLGAP2 – 0.0002175 GO:0023052∼signaling AX-103583267 26:37333692 DSCAM – 0.0004288 GO:0007156∼homophilic cell adhesion via plasma membrane adhesion molecules Affx-103045508 2:27182213 EPB41 0.0008206 – GO:0007049∼cell cycle TBIEC2_889720 4:8502727 EPDR1 – 0.0004253 GO:0007160∼cell-matrix adhesion BIEC2_852365 3:115596394 EVC 0.0009599 – GO:0003416∼endochondral bone growth BIEC2_416905 18:63653165 FAM171B – 0.0009746 GO:0001525∼angiogenesis Affx-102845142 28:31765971 FBXO7 0.0003695 0.0001627 GO:0000422∼mitophagy BIEC2-472336 2:37787520 FHAD1 0.0003518 −0.0004809 – BIEC2_14481 1:32086433 FRAT1 5.366 × 10 −5 0.0006401 GO:0090263∼positive regulation of canonical Wnt signaling pathway Affx-102013817 17:62060245 GPC5 – 0.0004061 GO:0016477∼cell migration Affx-102676537 6:24907627 HDAC4 1.5 × 10 −5 3.00 × 10- 7 GO:0000122∼negative regulation of transcription from RNA polymerase II promoter BIEC2_14318 1:30611785 HPSE2 – 0.0002113 GO:0008283∼cell proliferation ITGA2B_19245752_GT 11:19245752 ITGA2B 1 × 10 −6 – GO:0001525∼angiogenesis KIT_79540741_W16 3:79540741 KIT 1 × 10 −6 1.00 × 10 −7 GO:0001541∼ovarian follicle development KIT_79580000_W7 3:79580000 KIT 1 × 10 −6 1.00 × 10 −7 GO:0001541∼ovarian follicle development BIEC2_320442 15:75799411 LDAH 0.0003885 – GO:0019915∼lipid storage Affx-101492057 10:19641430 NOSIP 6 × 10 −6 1.00 × 10 −7 – AX-104335949 5:40161837 NUP210L 1 × 10 −6 1.00 × 10 −7 – PAX3_11199140_SW4 6:11199140 PAX3 1 × 10 −6 1.00 × 10 −7 GO:0006355∼regulation of transcription AX-104963491 12:30951642 PITPNM1 2 × 10 −6 1 × 10 −7 GO:0015914∼phospholipid transport BIEC2_887173 5:2222786 PM20D1 0.0002478 – GO:0006520∼cellular amino acid metabolic process BIEC2_783022 3:57169047 PRKG2 – 0.0007432 GO:0006468∼protein phosphorylation BIEC2_783026 3:57171222 PRKG2 – 0.0003162 GO:0006468∼protein phosphorylation BIEC2_885537 4:107978404 PTPRN2 – 0.0009618 GO:0006470∼protein dephosphorylation BIEC2_591905 22:33100482 PTPRT 0.0006552 – GO:0006470∼protein dephosphorylation Affx-103079316 12:30867790 RAD9A – 3.127 × 10 −5 GO:0000076∼DNA replication checkpoint AX-104886193 25:31276468 RALGPS1 0.0001003 8.00 × 10 −7 GO:0007264∼small GTPase mediated signal transduction Affx-102836572 17:50354202 SLAIN1 0.0004986 1.00 × 10 −6 GO:0007020∼microtubule nucleation SLC36A1_25884457_Champagne 14:25884457 SLC36A1 1 × 10 −6 1.00 × 10 −7 GO:0015808∼L-alanine transport BIEC2_11418 1:24106986 SORCS1 0.0006665 – GO:0006892∼post-Golgi vesicle-mediated transport BIEC2_954544 6:50669754 SOX5 – 0.0009407 GO:0006357∼regulation of transcription from RNA polymerase II promoter BIEC2_1011868 7:87211576 SOX6 0.0006732 – GO:0006357∼regulation of transcription from RNA polymerase II promoter AX-104226721 9:36874369 SPIDR 1 × 10p 4.00 × 10 −7 GO:0000724∼double-strand break repair via homologous recombination AX-104855978 28:39009737 TEF 0.0007493 – GO:0006357∼regulation of transcription from RNA polymerase II promoter BIEC2_486399 2:69215803 TLL1 – 0.0006598 GO:0006508∼proteolysis BIEC2_1004550 7:62165511 TMEM126B – 0.0004348 GO:0032094∼response to food BIEC2_195234 12:24939247 TMEM138 – 6 × 10 −7 – AX-103728731 9:84365361 ZC3H3 0.0005481 2.00 × 10 −6 – Affx-101705964 10:25531136 ZNF787 0.0002468 – GO:0000122∼negative regulation of transcription from RNA polymerase II promoter The obtained results on genetic structure of Zhabe horses are in line with our previous findings . As we discussed in the mentioned paper, the specific conditions of traditional nomadic husbandry could affect genetic composition of Kazakh horses. Almost free grazing of horses on summer and winter pastures with low control of mating by the herders have been closer to the ways of existence of horses in the wild nature rather than horse breeds under strictly controllable stable conditions. Here we have examined 443 newly genotyped horse individuals in addition to the previously analyzed and reported horse samples (616) to have a deeper look on genetic structure of Zhabe horses in comparison with several foreign horse breeds, Akhal-Teke, Arabian, Caspian, Mongolian, and Thoroughbred (data by Ref. ). These particular breeds have been selected under specific considerations: Mongolian and Caspian horses have previously shown high genetic similarity to Kazakh horses ; Akhal-Teke breed was selected as another ancient breed with Central Asian origin; Arabian and Thoroughbred horses have been selected as reference well known breeds of stable maintenance and high breeding control; moreover, Thoroughbred horses are known to be involved to later breeding practice for improvement of Kazakh horses. Indeed, the presence of presumably hybrid genotypes between Zhabe and Thoroughbred horses was revealed by PCA and ADMIXTURE analysis. Population 3 (Kalka) which included the most of Thoroughbred hybrids was also shown closer to this breed by F ST analysis. In comparison, Zhabe horses display lower degree of LD comparing to foreign breeds. Such population genetics concepts as LD decay and ROH are considered as selection signatures in domestic animals . Comparing to foreign breeds, we have shown in our study that Zhabe horses indeed have these signatures less expressed. Lower LD parameters indicate higher individual variability within populations which is also illustrated by results of ADMIXTURE analysis. As Fig. 3 shows, besides individuals with identified admixture with Thoroughbred horses, higher K patterns demonstrate additional within populaion variability which is, however, is not associated with particluar populations. It was shown previously that such internal diversity within populations may be caused by the influence of groups of closely related horses, e.g. originated from the same productive sires . However, the lack of pedigree data on Kazakh horses due to low control of mating makes it impossible to evaluate such possible substructure in this study. Population 39 representing unique white horses ‘Zhetysu Asyly’ posed a special interest. This lineage is a result of amateur selection and have not been an object of scientific evaluation to date. Although it was stated by the breeder (personal communication) that this breed have been established based on Zhabe and other Kazakh horses, no documents on the selection history are available. Our results reveal surprisingly high level of differentiation of population 39 comparing to Zhabe horses. Patterns of LD and F ST of these horses is more similar to foreign breeds, however PCA, F ST and ADMIXTURE analysis place this population as unique cluster. Considering strict artificial selection towards color, the observed differences should be considered selective signatures. If the declared assumption about their origin from Kazakh horses is correct, their genetic composition could be an example of rapid transformation of animal's genome under pressure of targeted selection. However, lack of reliable descriptive and genealogical data makes possible assumption speculative. Thus, so called ‘Zhetysy Asyly’ horses are a potential object for further deeper investigation. Previously, we have attempted GWAS to identify variants associated with body weight and size in Kazakh horses . The results of that work have demonstrated the absence of genomic regions strongly associated with the considered body traits. The variants with significant associations were distributed occasionally and did not form notable islands corresponding to genomic regions to increased association. Moreover, no SNPs associated with known major horse body size factors, genes LCORL , NCARG , and ZFAT , have been revealed. In the present study, we obtained similar results for selection of Zhabe horses. Interestingly, the loci identified here showed no correspondence to previously reported ones. Such a distribution of occasional SNPs could be a sign of the absence of strong selection signatures for body size and weight in Kazakh horses and, particularly, Zhabe horse type. Thus, these traits of Zhabe horses, as well as other Kazakh horse varieties, could be determined by environment to higher extent than genetic factors. Along with the previous findings, our results illustrate specific genetic properties of Kazakh horses. Traditional nomadic ways of horse breeding have lead to formation of unique landrace with low presence of selection signatures. We could suggest that this lack of dominating genetic factors developed by selection is the reason of high versatility and adaption of Kazakh horses to a range of conditions of Central Asia, from steppes to mountains. On the other hand, the genetic identity of Kazakh horses is vulnerable before targeted breeding practices and hybridization. As we have shown here , relatively recent hybridization events involving Thoroughbred horses have left distinctive signature allowing to identify source of admixture. Horses “Zhetysu Asyly” (population 39) resulted from targeted selection of Kazakh horses for white color demonstrate significant changes in their genetic composition from Zhabe horses and, considering low variability between breeds , Kazakh horses in general. Thus, Kazakh horses and, particularly, Zhabe type should be considered a unique legacy landrace requiring special protection. The obtained results provide genetic data on the Kazakh horses of Zhabe type which are traditional but yet understudied horse breed. To conserve their genetic composition and properties the breeding programs should be focused on the internal genetic resources of Kazakh horses rather than hybridization with distant breeds. Still, Kazakh horses may be a valuable genetic resource for developing new horse breeds due to their unique properties and adaption to environmental conditions. The present work provides important insights into the genetic structure of Zhabe horses and other Kazakh horse populations. The analysis revealed that traditional nomadic breeding practices, characterized by minimal human interference, have shaped these horses into a unique landrace with low selection signatures and high genetic diversity. Population 39, the white horses known as ‘Zhetysu Asyly,’ displayed significant genetic differentiation, likely due to targeted breeding for coat color. The findings highlight the importance of conserving the genetic integrity of Kazakh horses, particularly Zhabe, to preserve their adaptability and historical significance, while recognizing their potential as a genetic resource for future breeding programs. | Study | biomedical | en | 0.999997 |
PMC11699314 | Soils are complex physicochemical matrices with a porous structure, a huge surface area, and an extremely variable supply of organic materials, nutrients, and water, providing a range of habitats for a multitude of organisms . They make up one of the most diverse habitats on Earth. Nowhere in nature are species so densely clustered as in soil communities . Microbial communities account for a large part of this biodiversity. Estimates of soil microbial diversity range from thousands to a million microbial species in just a few grams of soil . Soil microbial communities (Archaea, Bacteria, Fungi) play a crucial role in ecosystem functioning and resilience, particularly in biogeochemical cycles, organic matter mineralization, plant growth and productivity, soil structure maintenance, and soil pollutant reduction [ , , , , ]. Soil microbial communities are good indicators for monitoring soil quality and serve as early bioindicators of disturbances due to their essential role in soil functions and their high sensitivity to changes in soil conditions, combined with their short generation time . Recent advances in molecular biology have provided access to a large part of the soil microbial diversity, e.g., taxonomic richness or community composition . Moreover, several studies have reported that soil microorganisms serve as early and robust indicators of the impact of agricultural practices that threaten crop production sustainability, due to their small size, rapid generation time, and sensitivity to soil disturbances . Soils are fundamental resources for agricultural production and food security. As providers of more than 95 % of global food, they depend on intensive agricultural practices . Sustainable agriculture should satisfy global needs, i.e., not only food supply but also a range of other issues involving energy use, nutrient recycling, and the effects on adjacent ecosystems, including the impact on water bodies and climate change . Aiming for an agroecological transition, the biogas sector can represent a sustainable opportunity by developing (i) the production of renewable energies such as electricity, or biomethane, and (ii) the management of organic waste (OW) . Biogas is produced from the anaerobic digestion of organic matter. A wide variety of feedstock can be used (crop residues, animal manure, the organic fraction of municipal and industrial solid waste, or wastewater sludge) . This process converts biowaste into two economically useful products: renewable energy (biogas) and a fertilizer (digestate) . However, reports on the impacts of digestates on the soil microbiota are scarce, and rather contradictory, as reviewed by Karimi et al. . Most of the studies conducted to assess the impact of digestates on soil microbiota have been carried out under laboratory conditions often poorly linked to real-life agronomic conditions . For instance, some studies in short- ( ≤ 1 year) and mid- ( ≤ 5 years) term on field experiments reported positive or neutral effects after digestate application on soil organic carbon content (SOC) and some microbial parameters (e.g., microbial biomass and diversity) [ , , , , ]. Interestingly, Coelho et al. observed that microbial communities from different liquid digestates failed to establish in the soil after two years of repeated applications. However, studies conducted on long-term fertilization (≥5 years; organic and/or mineral fertilization) have reported more persistent impacts on soil characteristics , plant growth , and microbial diversity and activity [ , , ], suggesting that repeated digestate application over five years could have a lasting effect on soil microbial communities To our knowledge, no studies have assessed the long-term dynamics of soil microbial communities following repeated digestate applications. Therefore, no objective and robust conclusions can be drawn regarding the impact of digestate on soil microbial biodiversity, particularly in a real field context, with repeated applications over the long term . The field experiment “Effluents d'Elevage et Environnement” (EFELE; Rennes, France) was set up in 2012. It aims to comprehensively understand the long-term effects of repeated fertilization with different types of organic wastes (OW) on the soil quality and functioning. The experiment involved different fertilization practices, including mineral and organic inputs (cattle manure, pig slurry and pig slurry digestate). The present study aimed to assess the response of soil microbial communities (Archaea, Bacteria, Fungi) to 10 years of mineral and OW fertilization regimes by molecular DNA-based tools targeting the soil microbial biomass, soil microbial diversity, and soil microbial community structure. To our knowledge, no report had as yet employed an in-situ approach to monitor the dynamic effect of the repeated application of a digestate over a decade on soil microbial parameters. We hypothesized that (i) long-term historical and repeated digestate applications would strongly influence the edaphic soil properties, and in turn, affect the soil microbial parameters; and (ii) lasting modifications would only occur after several years of repeated applications. We assessed the long-term impact of historical and repeated fertilization practices on soil microbial communities, using digestate as an organic fertilizer. We also evaluated whether the digestate feedstock had a similar impact to that of the same undigested feedstock and other commonly used types of fertilizers. The experiment was based on a winter wheat ( Triticum aestivum L.) and maize ( Zea mays L.) crop rotation, with a catch crop of white mustard ( Sinapis alba ) . The crop residues (including aboveground residues) were not returned to the soil. Before the start of the experiment in 2010 and 2011, maize was grown as a homogenization crop. The soil management activities performed from 2012 to 2022 are presented in Fig. 1 . The fertilization treatments included a mineral N fertilizer (MIN) in the form of ammonium nitrate (comprising 50 % nitrate and 50 % ammonium), a control (ON) that did not receive any organic or mineral nitrogen input (but fertilized with P and K), and three different OW: cattle manure (CM), pig slurry (PS), and the digestate obtained after anaerobic digestion of the same pig slurry used in the study (PS-DIG). The design of the trial was a complete randomized block where each treatment had 4 replicates, and each plot had a surface area of 109 m 2 (12.5 m × 8.7 m). MIN, PS, and DIG-PS were applied once a year in early spring (late March to mid-April), and CM every two years. The mean characteristics of the OW applied from 2012 to 2022 are given in Table 1 . The index of residual organic carbon (I ROC ) represents the proportion of organic matter that may be incorporated into the soil organic matter following OW application . The mean rate of MIN applied from 2012 to 2021 represented 110.9 kg N ha −1 yr −1 , 52.5 kg P 2 O 5 ha −1 yr −1 , and 78 kg K 2 O ha −1 yr −1 . The detailed amounts of OW and mineral fertilizer applied per year are presented in Table S 1 , Figure S 2 , and Figure S 3 . The application rates for the liquids OW (PS-DIG and PS) were determined according to their N fertilizer value, aligning with the regulations of the EU Nitrates Directive (91/676/EEC ). The CM inputs (solid OW) were reasoned based on the first limiting element, phosphorus, aiming not to exceed the threshold of an annual input of 100 kg P 2 O 5 ha −1 yr −1 . The application rate of MIN was calculated using the mineral N balance method recommended in France . Except for the ON plots, all plots, regardless of the treatments, showed similar yields throughout the years (18.65 t DM/ha for maize silage; 8.23 t DM/ha for wheat straw and 82.06 quintal/ha for wheat grain). Fig. 1 Agricultural practices at the EFELE from 2012 to 2021 on all treatments, including crop succession, fertilization, and soil management activities. The experiment was based on a winter wheat ( Triticum aestivum L.) and maize ( Zea mays L.) crop rotation, with a cover crop of white mustard ( Sinapis alba ). Wheat and maize were harvested, whereas mustard was mulched. Tillage was classified depending on soil depth (superficial <10 cm; deep >10 cm). Fertilization treatments: MIN, mineral fertilizer; ON, control that received no organic or mineral N input; three different OW: CM, cattle manure; PS, pig slurry; PS-DIG, pig slurry digestate. “Others” management practices grouped: mechanical weeding, pesticide spreading, and liming. The liming rate was 370 kg ha −1 (granules containing 54.5 % CaO and 0.5 % MgO). Soil samples were collected in March every year from 2012 to 2022. Fig. 1 Table 1 Mean characteristics of the organic wastes (OW) applied in the EFELE plots from 2012 to 2021. Table 1 Treatment Applied quantity Dry matter Organic C Total N Organic N I ROC C/N pH (water) t FM ha −1 yr −1 % kg FM ha −1 yr −1 kg FM ha −1 yr −1 kg FM ha −1 yr −1 % Organic C CM 25a (0.0) 26.2a (3.6) 4692.5a (818.8) 313.3a (60.9) 248.1a (59.2) 58.8a (6.6) 15.2a (2.4) 9.4a (0.4) PS 31.4a (8.4) 7.2b (2.5) 844.5b (397.5) 160.6b (47.1) 41.6b (17.7) 49.3a (10.4) 5.2b (1.3) 8.5a (1.0) PS-DIG 32.9a (8.0) 5.8b (1.7) 645.3b (217.3) 159.5b (34.8) 36.8b (8.3) 54.7a (10) 4.0b (0.9) 9.0a (1.0) Fresh matter (FM). Values are means and letters denote the significant effect between treatments (p < 0.05) based on Tukey's HSD test p-value adjusted by the BH method. The standard error of the means is indicated in parentheses. CM, cattle manure; PS, pig slurry; PS-DIG, pig slurry digestate. Soil samples were collected in early March (about 11 months after the latest fertilization treatment) every year from 2012 to 2022. The last soil sampling was in March 2022. Each sample was composed of 8 soil cores taken from the 0–25 cm horizon at random locations in each plot, and mixed and homogenized by 4 mm mesh sieving to remove above-ground plant material, other plant debris, roots, and stones. The sieved soil was freeze-dried and stored at −40 °C at the GenoSol soil conservatory (INRAE, Dijon, France). 2 DNA extraction and molecular analyses were conducted in 2022 on soil samples collected at the beginning of the trial in 2012, as well as 5 and 10 years later . This led to a total of 60 soil samples (5 treatments × 4 replicates × 3 years). A portion of the same soils sampled in 2012, 2016, and 2021 was air-dried for physicochemical analysis to determine soil particle size distribution, pH, SOC, soil total nitrogen, the soil C/N ratio, total phosphorus, and the cation exchange capacity (CEC). These analyses were performed at the INRAE Soil Laboratory Analysis. 3 The soil physicochemical data are given in Table 2 . Table 2 Soil physicochemical parameters of the EFELE plots according to the fertilization treatment and the sampling time. Table 2 Treatment Organic carbon Total N P 2 O 5 (Olsen) C:N pH (water) CEC g kg −1 g kg −1 g kg −1 cmol + kg −1 2012 CM 10.77a (0.574) 1.13a (0.05) 0.18a (0.02) 9.53a (0.05) 6.14a (0.05) 6.17a (0.20) MIN 11.45a (0.532) 1.19a (0.06) 0.18a (0.03) 9.61 ab (0.08) 5.97a (0.24) 6.10a (0.65) ON 11.16a (0.935) 1.15a (0.09) 0.19a (0.02) 9.72b (0.11) 6.14a (0.27) 6.36a (0.55) PS 10.95a (0.443) 1.13a (0.04) 0.19a (0.02) 9.62 ab (0.08) 6.16a (0.21) 6.44a (0.58) PS-DIG 10.51a (0.878) 1.10a (0.09) 0.17a (0.01) 9.57 ab (0.05) 5.99a (0.25) 5.71a (0.41) 2016 CM 11.12a (0.36) 1.19a (0.02) 0.14a (0.02) 9.37a (0.09) 6.16a (0.11) 6.63a (0.47) MIN 10.8a (0.53) 1.16a (0.06) 0.13a (0.02) 9.28a (0.03) 5.91a (0.20) 6.01a (0.63) ON 10.42a (0.84) 1.12a (0.07) 0.14a (0.02) 9.26a (0.14) 6.13a (0.19) 6.29a (0.48) PS 10.95a (0.64) 1.17a (0.06) 0.15a (0.02) 9.38a (0.08) 6.26a (0.14) 6.62a (0.50) PS-DIG 10.45a (0.65) 1.12a (0.07) 0.14a (0.01) 9.29a (0.02) 6.07a (0.16) 5.92a (0.26) 2021 CM 11.27b (0.39) 1.25b (0.05) 0.14a (0.01) 9.03a (0.09) 6.36a (0.14) 6.55a (0.28) MIN 9.89a (0.23) 1.11a (0.04) 0.14a (0.02) 8.89a (0.17) 5.85b (0.16) 5.80a (0.45) ON 9.61a (0.39) 1.08a (0.05) 0.17a (0.03) 8.93a (0.09) 6.17 ab (0.31) 5.67a (1.21) PS 10.14a (0.32) 1.15 ab (0.04) 0.15a (0.01) 8.83a (0.07) 6.14 ab (0.13) 6.38a (0.46) PS-DIG 9.79a (0.53) 1.09a (0.06) 0.14a (0.01) 8.94a (0.04) 6.06 ab (0.22) 5.82a (0.28) Data pertains to air-dried soil. Values are means and letters denote the significant effect of treatment for each sampling time (p < 0.05) based on Tukey's HSD test p-value adjusted by the BH method. The standard error of the means is indicated in parentheses. CM, cattle manure; PS, pig slurry; MIN, mineral fertilizer; ON, no fertilization; PS-DIG, pig slurry digestate. Total DNA was extracted from 1 g (dry weight) of soil using a single procedure standardized by the GenoSol platform (INRAE, Dijon, France) 2 . This protocol is based on three main steps: (i) microbial cell lysis by physical and chemical action; (ii) deproteinization; and (iii) alcohol precipitation and washing of the extracted nucleic acids. The DNA concentrations of the crude extracts were determined by electrophoresis in 1 % agarose gel stained with ethidium bromide, using calf thymus DNA as a standard curve. Quantified crude DNA was used as an estimate of the soil molecular microbial biomass . In order to separate residual impurities – particularly humic substances – 100 μL of crude DNA was purified using a Nucleospin® Soil kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany). The concentrations of purified DNA were finally measured using a Quantifluor staining kit (Promega, Madison, Wisconsin, USA), according to the manufacturer's instructions. Prokaryotic (bacterial-archaeal) diversity was estimated from each DNA sample by metabarcoding of the 16S rRNA gene following the method described by . A 440-base fragment targeting the V3 to V4 regions was first amplified with the corresponding primers F479 (5′CAG CMG CYG CNG TAA NAC3′) and R888 (5′CCG YCA ATT CMT TTR AGT3′). Fungal diversity was estimated by metabarcoding of the 18S rRNA gene. A 350-base fragment targeting the V7 to V8 regions was first amplified with the corresponding primers FR1 (5′ANC CAT TCA ATC GGT ANT3′) and FF390 (5′CGA TAA CGA ACG AGA CCT3′) according to . For each sample, PCR amplifications were performed with 5 ng of DNA in a total reaction mixture volume of 25 μL. The thermal profile of the prokaryotic PCR was as follow: initial denaturation at 94 °C for 2 min, 35 cycles of denaturation at 94 °C for 30 s, annealing at 52 °C for 30 s, and extension at 72 °C for 1 min, followed by a final extension period at 72 °C for 7 min. The thermal profile of the fungal PCR was as follow: initial denaturation at 94 °C for 3 min, 35 cycles of denaturation at 94 °C for 30 s, annealing at 52 °C for 1 min, and extension at 72 °C for 1 min, followed by a final extension period at 72 °C for 5 min. All PCR products were purified with an Agencourt® AMPure XP kit (Beckman Coulter, Brea, California, USA) and quantified with a Quantifluor staining kit (Promega, Madison, Wisconsin, USA). A second PCR was run on the purified PCR products (7.5 ng of DNA for bacteria and archaea, 5 ng for fungi), using 10-base-pair multiplex identifiers (MIDs) added at the 5′ and 3’ ends of the primers for subsequent sample identification. The thermal profile of the second PCR for the preparation of bacterial and archaeal rDNA amplicon libraries was the same as previously described, but with only 7 cycles. The same procedure was followed for the preparation of fungal rDNA amplicon libraries (same thermal profile as in the first PCR, but only 7 cycles). Moreover, the denaturation step lasted 1 only min. All PCR products were purified using a MinElute PCR purification kit (Qiagen NV) and quantified with a Quantifluor staining kit (Promega, Madison, Wisconsin, USA). For all libraries, the samples were pooled at equimolar concentrations and purified using an Agencourt® AMPure XP kit (Beckman Coulter, Brea, California, USA) to remove excess nucleotides, salts, and enzymes. Finally, sequencing was carried out with a NovaSeq Illumina instrument (Illumina Inc. San Diego, California, USA) producing 250-bp paired reads. Bioinformatic analyses were performed using the BIOCOM-PIPE pipeline . First, all 16S and 18S raw reads were sorted according to the MID sequences. Data preprocessing included initial trimming of raw reads using PRINSEQ and merging of paired-end reads using FLASH. The low-quality reads were discarded based on their minimum length, number of ambiguities (Ns), and primer sequences. Then, the reads were dereplicated to save computing time in the subsequent steps of the pipeline (i.e., clustering of strictly identical sequences). The dereplicated reads were aligned using Infernal alignment tool , and clustered into operational taxonomic units (OTUs) with a similarity threshold of 95 %. A filtering step was carried out to remove chimeras based on the quality of their taxonomic alignments . Finally, the retained reads were homogenized by random selection of 10,000 reads to compare the datasets efficiently and avoid biased community comparisons. As observed in previous studies, if sequencing depth is sufficient, such a homogenization step will produce robust results when describing patterns in α- and β-diversity . The retained high-quality reads were used for (i) the re-clustering step using ReClustOR to improve OTU consistency based on a reference OTU database taken from the RMQS project (French Soil Quality Monitoring Network) ; (ii) taxonomy-independent analyses to calculate diversity indexes by Hill numbers (richness, Shannon, Inverse Simpson) using the OTU dataset; and (iii) taxonomy-based analyses by similarity approaches against curated reference databases from SILVA r132 . The raw datasets are available in the EBI database system under project accession number PRJEB71034. All statistical analyses were performed with R studio using the free statistics software R . OW characteristics, soil physicochemical parameters, soil molecular microbial biomass, alpha diversity index, and the relative abundance of soil prokaryotic and fungal phyla were processed by a two-way mixed analysis of variance (ANOVA). The model included the plot identity as a random factor to account for repeated measures within each plot, with ‘time’ as the within-subject factor and ‘treatment’ as the between-subject factor. A Tukey's HSD post hoc test with the Benjamini & Hochberg (BH) correction method was applied to adjust the p-values. Alpha diversity was analyzed using Hill numbers generated by the vegan package . Hill numbers allow interpreting alpha diversity linearly by progressively considering the abundances of rare and dominant OTUs using the scaling parameter q (order of diversity) . Therefore, q = 0 represents richness, q = 1 corresponds to the exponential of Shannon entropy, weighing OTUs by their frequency without disproportionate favoring either rare or abundant ones, and q = 2 represents the inverse of Simpson index, where the abundant OTUs are overweighed . Similarities in the compositions of the prokaryotic and fungal communities among different fertilization treatments over time were evaluated by an analysis of similarities (ANOSIM) and an analysis of multivariate homogeneity of group dispersions (beta-dispersion), using 999 permutations and a significance threshold p < 0.05, based on the robust Aitchison dissimilarity distance . To visualize the distribution patterns of microbial communities for each fertilization treatment over time, the non-metric multidimensional scaling (NMDS) approach was employed based on the robust Aitchison dissimilarity distance , using the metaMDS function from the vegan package . The significance of the differences between treatments and over time was assessed using a non-parametric permutational multivariate analysis of variance (PERMANOVA) based on the robust Aitchison dissimilarity distance . The relative effects of ‘time’, ‘treatment’, and ‘time × treatment’ were tested using the adonis2 function in the vegan package with 999 permutations, and a significance threshold p < 0.001; the “strata” option was used to constrain the permutations of samples within each time point. The abundance of soil prokaryotic and fungal phyla was subjected to a differential abundance analysis using the DESeq2 method to compare the cumulative effect of digestate to the other treatments . Finally, a variance partitioning approach was used to determine if the amounts of organic carbon and fertilizing elements (NPK) applied over 10 years could explain the variability observed in the dynamics of the soil molecular microbial biomass and the soil microbial community structure. For this purpose, the most parsimonious additive model was selected according to a forward selection procedure by minimizing the Akaike information criterion (AIC), using the rda and ordistep functions in the vegan package . Over a decade of repeated application of different organic wastes (OW), no statistically significant differences were observed between the physicochemical properties of undigested (PS) and digested (PS-DIG) organic matter ( p > 0.05, Table 1 ). In contrast, cattle manure (CM) exhibited significantly higher concentrations of carbon and nitrogen ( p < 0.05, Table 1 ). In 2012, before any treatment application, the physicochemical properties of the soil were similar in all plots ( p > 0.05, Table 2 ). Similarly, in 2016, no statistically significant differences were observed between treatments ( p > 0.05, Table 2 ). After a decade, in 2022, the soil chemical properties were lastingly modified, and the magnitude of the changes was treatment-dependent. Specifically, the soil organic carbon (SOC) content significantly decreased by an average of 7 % in the plots treated with PS and PS-DIG, and by 14 % in the MIN and ON plots ( p < 0.05, Table S 2). The only plots that maintained a stable SOC were those amended with CM ( p > 0.05, Table 2 and Table S 2). All treatments resulted in a significant decrease in the C/N ratio over time, by 7 % on average ( p < 0.05, Table S 2). Significant soil acidification was observed in the MIN plots ( p < 0.05, Table 2 and Table S 2), while the soil pH remained stable over time in the ON, PS, and PS-DIG plots ( p > 0.05, Table 2 and Table S 2). In contrast, the pH significantly increased in the CM plots ( p < 0.05, Table 2 and Table S 2). A significant ‘year’ effect was observed on soil molecular microbial biomass regardless of the treatment . Overall, across all plots, soil molecular microbial biomass decreased by 32 % in 2017 compared to the beginning of the trial . In 2012, before any fertilizer application, a heterogeneity in soil molecular microbial biomass was observed, particularly between plots selected for mineral fertilization treatment and those designated to receive digestate . After ten years , significant differences were observed between plots amended with cattle manure (CM) and those unfertilized control plots (ON) , where the highest soil molecular microbial biomass was recorded when CM was repeatedly applied over a decade and lowest in ON plots . The redundancy analysis (RDA) model identified the parameters that explained variations in soil molecular microbial biomass over a decade ( Table 3 ). The temporal factor (‘year’) strongly influenced the model, explaining 43.3 % of the variance. In contrast, cumulative C organic inputs explained 4.2 % of the variation in soil molecular microbial biomass. Fig. 2 Soil molecular microbial biomass dynamics by year at the EFELE (experimental field site in Rennes, France) from 2012 to 2022. Treatments: MIN, mineral fertilizer; ON, control that received no organic or mineral N input; three different OW: CM, cattle manure; PS, pig slurry; PS-DIG, pig slurry digestate. Capital letters, differences among sampling time; lower-case letters, effect of the treatment for each sampling time. Different letters indicate significant differences ( p < 0.05) based on Tukey's HSD test; p -value adjusted by the BH method. Fig. 2 Table 3 Effects of the cumulative inputs during the decade at the EFELE site on the soil molecular microbial biomass and microbial community structure. Table 3 Parameter Explanatory variables F P -value Variance explained (%) Soil molecular microbial biomass Year 23.09 0.001 43.3 C organic inputs 4.49 0.041 4.2 Residual – – 52.5 Structure of soil Prokaryotic community Year 8.14 0.001 21.3 C/N inputs 8.99 0.001 11.8 Residual – – 66.8 Structure of soil Fungal community Year 50.98 0.01 64.1 Residual – – 35.9 Variance partitioning approach, using Redundancy Analysis (RDA) to select the most parsimonious additive model, according to a forward selection procedure. F (Fisher's F-value), and p-value of Fisher's F from ANOVA test of RDA selected model. Scores from the NMDS ordination based on the robust Aitchison dissimilarity distance were employed for the variance partitioning approach of the soil microbial community structure. Following a decade of different fertilization regimes, significant differences in prokaryotic diversity indices were only observed in 2022 between digestate (PS-DIG) and pig slurry (PS) treated plots . Plots fertilized with digestate displayed higher values of richness (q = 0), OTUs frequency (q = 1), and effective number of dominant OTUs (q = 2) compared to PS plots . Regarding fungal community diversity indices, at the beginning of the experiment in 2012, significant differences in richness (q = 0) and OTUs frequency (q = 1) were observed, particularly among plots designated to receive pig slurry (PS) and digestate (PS-DIG) . Repeated digestate application over a decade induced a higher soil fungal effective number of dominant OTUs (q = 2) than in soils receiving slurry (PS) or cattle manure (CM) . Fig. 3 Soil prokaryotic community diversity dynamics by year at the EFELE (experimental field site in Rennes, France) based on Hill numbers. Hill numbers allow interpreting alpha diversity linearly by progressively considering the abundances (q-values) of rare and dominant OTUs. Therefore, ( A ) q0 represents richness, ( B ) q1 is the exponential of Shannon entropy, and ( C ) q2 is the inverse of Simpson index. Treatments: MIN, mineral fertilizer; ON, control that received no organic or mineral N input; three different OW: CM, cattle manure; PS, pig slurry; PS-DIG, pig slurry digestate. Lower-case letters: effect of the treatment for each sampling time. Different letters indicate significant differences ( p < 0.05) based on Tukey's HSD test; p -value adjusted by the BH method. Fig. 3 Fig. 4 Soil fungal community diversity dynamics by year at the EFELE (experimental field site in Rennes, France) based on Hill numbers. Hill numbers allow interpreting alpha diversity linearly by progressively considering the abundances (q-values) of rare and dominant OTUs. Therefore, ( A ) q0 represents richness, ( B ) q1 is the exponential of Shannon entropy, and ( C ) q2 is the inverse of Simpson index. Treatments: MIN, mineral fertilizer; ON, control that received no organic or mineral N input; three different OW: CM, cattle manure; PS, pig slurry; PS-DIG, pig slurry digestate. Lower-case letters: effect of the treatment for each sampling time. Different letters indicate significant differences ( p < 0.05) based on Tukey's HSD test; p -value adjusted by the BH method. Fig. 4 The analysis of similarities revealed significant differences in the microbial community structure (prokaryotic and fungal) among years and treatments (ANOSIM; p < 0.05, Table 4 ), although the ‘year’ effect seemed greater than the ‘treatment’ effect (ANOSIM; R statistic, Table 4 ). The analysis of multivariate homogeneity of group dispersions demonstrated that the dispersion of community structure differs significantly between years but not between treatments (beta-dispersion; Table 4 ). NMDS ordination confirmed these findings , which clearly highlighted distinct clustering by ‘year’ , indicating that the changes of soil microbial communities were more influenced by time than by fertilization type. PERMANOVA confirmed that both ‘year’ and ‘fertilization type’ had a significant effect on the prokaryotic and fungal community structure ( p < 0.05, Table 5 ). Pairwise comparisons revealed lasting modifications over time, with the largest differences between 2012 and 2022 . After 10 years, the microbial community structure in the plots receiving digestate (PS-DIG) differed significantly from those amended with cattle manure (both prokaryotic and fungal community structure) and pig slurry (fungal community structure) . In contrast, no significant differences were observed when comparing the microbial community structures between digestate fertilized plots and mineral fertilized (MIN) or unfertilized ones (ON) . Table 4 Analysis of similarities (ANOSIM), and analysis of multivariate homogeneity of group dispersions (beta-dispersion) depicting the differences in the prokaryotic and fungal community compositions between and within groups (‘treatment’ and ‘time’) based on the Robust-Aitchison distance matrices at the EFELE (experimental field site in Rennes, France), according to the treatment over time (999 permutations, significance threshold p < 0.05). Table 4 Prokaryotic community Fungal community ANOSIM Beta-dispersion ANOSIM Beta-dispersion R statistic P -value P -value R statistic P -value P -value Treatment 0.14 0.002 0.24 0.13 0.002 0.88 Year 0.29 0.001 0.04 0.60 0.001 0.01 R statistic: degree de separation between test groups ranging from −1 to 1; R = 0, not different; R = 1, completely different. Significance values were based on 999 permutations. Fig. 5 Non-metric multi-dimensional scaling (NMDS) ordination and PERMANOVA pairwise comparisons derived from robust Aitchison dissimilarity distances for ( A, B ) the prokaryotic community at the EFELE (experimental field site in Rennes, France) over 10 years of trial. Treatments: MIN, mineral fertilizer; ON, control that received no organic or mineral N input; three different OW: CM, cattle manure; PS, pig slurry; PS-DIG, pig slurry digestate. Big circles represent centroids, and little circles represent samples. (A) prokaryotic community structure colored by year, (B) prokaryotic community structure colored by treatment. Fig. 5 Fig. 6 Non-metric multi-dimensional scaling (NMDS) ordination and PERMANOVA pairwise comparisons derived from robust Aitchison dissimilarity distances for ( A, B ) the fungal community at the EFELE (experimental field site in Rennes, France) over 10 years of trial. Treatments: MIN, mineral fertilizer; ON, control that received no organic or mineral N input; three different OW: CM, cattle manure; PS, pig slurry; PS-DIG, pig slurry digestate. Big circles represent centroids, and little circles represent samples. (A) fungal community structure colored by year, and (B) fungal community structure colored by treatment. Fig. 6 Table 5 Permutational multivariate analysis of variance ( PERMANOVA) results depicting the differences in the prokaryotic and fungal community compositions based on the robust Aitchison distance matrices, at the EFELE (experimental field site in Rennes, France), according to the treatment over time (999 permutations, significance threshold p < 0.05). Table 5 Prokaryotic community Fungal community SS R 2 F P -value SS R 2 F P -value Treatment 0.44 0.11 1.78 0.001 0.69 0.07 2.88 0.001 Year 0.47 0.12 3.83 0.02 1.79 0.19 5.92 0.001 Treatment × Year 0.43 0.11 0.88 0.87 1.53 0.16 1.26 0.001 Residual 2.51 0.65 – – 5.44 0.57 – – Total 3.84 1 – – 9.46 1 – – SS (Sum of Squares), R 2 (Determination coefficient), F (Fisher's F-value), and p-value of Fisher's. The redundancy analysis (RDA) model identified the parameters that explain variations in the microbial community structure over a decade ( Table 3 ). The temporal factor (‘year’) had the strongest influence in both models. It represented 21.3 % and 64.1 % of the explained variance of the prokaryotic and fungal community structure, respectively ( Table 3 ). The C/N ratio of the applied product represented 11.8 % of the explained variance of the soil prokaryotic community structure. In contrast, none of the chemical properties of the treatments applied (cumulative organic carbon or NPK fertilizing elements) were able to explain the dynamics of variation in the structure of soil fungal communities ( Table 3 ). The temporal changes in the relative abundances of dominant soil microbial phyla over a decade following different fertilization regimes are illustrated in Fig. 7 , Fig. 8 . Overall, 13 prokaryotic and 6 fungal major phyla were identified across all treatments and years considered. The prokaryotic phyla were Acidobacteria, Actinobacteria-p, Bacteroidetes, Chloroflexi, Cyanobacteria, Firmicutes, Gemmatimonadetes-p, Nitrospirae, Planctomycetes, Proteobacteria, Rokubacteria , Thaumarchaeota , and Verrucomicrobia . The fungal phyla were Ascomycota, Basidiomycota, Chytridiomycota, Cryptomycota, Mucoromycota , and Zoopagomycota . When comparing digestate to other OW, the repeated digestate application over a decade induced lower relative abundance in Thaumarchaeota but higher in Actinobacteria-p, Gemmatimonadetes-p, and Rokubacteria . For the fungal phyla, digestate favored Basidiomycota and Cryptomycota . The differential abundance analysis conducted using the DESeq2 method provided insights into the changes in the abundance of various microbial phyla in response to different fertilization treatments. Notably, when comparing the cumulative effect of a decade of annual digestate application to other treatments, fewer significant differences at the phylum level were observed between PS-DIG and MIN-treated plots, specifically in the Archaeal phyla Thaumarchaeota and Euryarchaeota , and the fungal phylum Blastocladiomycota . Fig. 7 Relative abundances of the major prokaryotic phyla by year at the EFELE (experimental field site in Rennes, France) from 2012 to 2022. Treatments: MIN, mineral fertilizer; ON, control that received no organic or mineral N input; three different OW: CM, cattle manure; PS, pig slurry; PS-DIG, pig slurry digestate. Relative taxonomic abundances below 5 % were grouped in “Others”. Lower-case letters, effect of the treatment for each sampling time. Different letters indicate significant differences ( p < 0.05) based on Tukey's HSD test; p -value adjusted by the BH method. Fig. 7 Fig. 8 Relative abundances of the major fungal phyla by year at the EFELE (experimental field site in Rennes, France) from 2012 to 2022. Treatments: MIN, mineral fertilizer; ON, control that received no organic or mineral N input; three different OW: CM, cattle manure; PS, pig slurry; PS-DIG, pig slurry digestate. Lower-case letters, effect of the treatment for each sampling time. Different letters indicate significant differences ( p < 0.05) based on Tukey's HSD test; p -value adjusted by the BH method. Fig. 8 Comparing to laboratory approaches, employing an in-situ methodology in real agronomical and pedological conditions provides a more comprehensive understanding of short- ( ≤ 1 year), mid- ( ≤ 5 years), and long- ( ≥ 6 years) term impact of digestates on soil microbiota. To our knowledge, no prior study has employed an in-situ approach to monitor the effects of repeated digestate applications over a decade. The overall objective of this study was to assess the long-term effects of repeated digestate applications as an organic fertilizer on soil microbial parameters, including molecular microbial biomass, diversity, structure, and composition. Furthermore, we aimed to evaluate how these effects relate to changes in edaphic soil properties, hypothesizing that significant modifications would emerge only after several years of repeated applications. As previously observed with a liquid digestate by Ref. , the repeated application of digestate did not induce a lasting increase of SOC content as commonly observed with classical OW such as farmyard manure or compost . The stabilization or increase of the SOC content was observed depending on the OW type and quantity applied. In the present study, CM plots resulted in a stable SOC content throughout the 10 years of the experiment, primarily attributed to the large quantity of organic carbon applied (total organic C (TOC) in the CM treatment: 28,155 kg ha _1 ), significantly higher than the other treatments. In contrast, a lower quantity of organic carbon was applied via digestate and pig slurry , explaining why the SOC content could not be maintained throughout the years. These findings align with the literature, emphasizing that increasing total C input is the primary driver for maintaining or rising SOC stocks . Moreover, as previously reported for organic farming systems, rising SOC levels is a gradual process closely related to biological soil quality and the quality of inputs materials . The treatments induced distinct dynamics of the total soil nitrogen content. These variations were attributed to differences in the proportions of total N applied via each treatment; specifically, the plots fertilized with digestate and mineral fertilizer recorded lower total soil N content at the end of the experiment. Indeed, over a decade of repeated fertilization, the cumulative total nitrogen supplied was lower in plots fertilized with digestate or mineral fertilizer compared to plots amended with organic products (cattle manure and pig slurry) . In line with prior studies, differences in carbon and nitrogen contents in the OW led to high variability in the C/N ratios across treatments . The C/N ratio of the OW is an indicator of their nitrogen mineralization capacity. When OW with C/N ratios below 20 are added to soils, net mineralization dominates . The C/N ratios below 10 favor rapid organic matter mineralization, while values above 10 result in slower mineralization ; microbial N immobilization becomes the dominant process with higher C/N ratios . An optimal C/N ratio in the range of 5–6 has been proposed for OW . In the present study, the C/N ratios of digestate ranged between 2.9 and 5.8, and for the other OW (pig slurry and cattle manure), the values ranged between 3.5 and 8.3 (PS) and 12.8 and 19 (CM), respectively. The lower C/N ratios of digestate may also explain why these inputs did not contribute to increases in the SOC content, favoring rapid mineralization of the organic matter and potentially resulting in different fates for nitrogen in the amended soils . Regarding soil pH, our study found no significant changes after 10 years of repeated digestate application, consistent with previous in-situ studies that also reported stable soil pH levels following repeated digestate inputs [ 51 , , , ]. During the biogas production process, anaerobic microorganisms degrade most of the readily available carbon, leading to the subsequent production of methane and carbon dioxide . Consequently, a reduced amount of readily available carbon for soil microorganisms, and therefore a lower microbial biomass, can be expected when digested organic matter is used as an amendment compared to undigested organic matter . Contrary to these expectations, our findings highlighted that the soil molecular microbial biomass content was similar in plots treated with OW (digestate (PS-DIG), undigested organic matter (PS), and cattle manure (CM)), although their SOC content differed. As reported by Ref. , increases in SOC content were associated with improvements in soil biological activity and particularly in microbial biomass. Moreover, higher soil microbial biomass has been observed in plots amended with cattle manure . The results obtained in our study could be explained by a buffering effect of agricultural practices (e.g., crop rotation, the reasoning of the input doses, cover and catch crop) which exert a compensatory effect on soil molecular microbial biomass, not allowing for the observation of a more pronounced treatment-effect. Indeed, it is known that certain practices, such as the diversity of crop rotations or soil tillage , can impact soil microbial biomass. In addition, the variance partitioning approach identified the temporal parameter (‘year’) as the main factor explaining variations in soil molecular microbial biomass, with a significant and systematic decrease in biomass in all treatments in 2017. The winter of 2016/17 was drier and colder than usual . Typically, drainage seasons commenced between November and January, except for the winter of 2016/17, which started in February, lasted only one month, and had notably lower rainfall, resulting in reduced drainage volumes . This underscores the crucial role of soil temperature and moisture in driving seasonal changes in soil microbial communities. By influencing temperature and precipitation, climate can either mitigate or exacerbate the impact of land use on the soil microbial biomass . Moreover, the season of soil sampling may exert a greater influence on soil microbial communities than the application of digestate . Thus, in our study, land use and soil management practices probably buffered the effects of the treatments, whether organic or mineral, on the soil microbial biomass. These findings highlight the importance of fertilization and soil amendment, and the critical need for a long-term assessment of the potential impacts of any agricultural practice on soil microorganisms. As observed in previous studies, our results highlighted that repeated application of different fertilization treatments induced minor significant differences over time in alpha diversity indices (richness, the exponential of Shannon entropy, and the inverse of Simpson index) . Our findings highlighted that the long-term fertilization with digestate stimulated dominant OTUs more than other OW (pig slurry and cattle manure). Furthermore, our results revealed shifts and lasting modifications of the prokaryotic and fungal community structures in relation to time and the fertilization treatment, consistent with prior research, yearly organic and mineral fertilization treatments had a significant long-term impact on the soil microbial community structure . Field experiments have shown no significant short-term changes in the soil microbial community structure following digestate application . This indicates a different response of the microbial community structure to short- and long-term nutrient additions, whose effects take time before they become noticeable. Moreover, our results highlighted that (i) at least 5 years went by before lasting modifications of the soil microbial community structure were detected, and (ii) the distribution of the prokaryotic structure of the digestate fertilized plots was in between those of the mineral fertilizer and the other OW (pig slurry (PS) and cattle manure (CM)). The variance partitioning approach revealed that the temporal factor (‘year’) and, to a lesser extent, the cumulative C/N inputs best explained variations in the microbial community structure. As observed for the molecular microbial biomass, these findings underscored that the ‘year’ factor exerted a strong influence, likely reflecting environmental, climatic, and seasonal effects that could mask the impact of repeated applications of different fertilization treatments on soil microorganisms. Nevertheless, the nature of the inputs also plays a significant role in shaping these microbial communities. These findings align with previous studies which emphasize that both mineral and organic long-term fertilization can lead to changes in the soil microbial community composition , due to the stimulation of soil-borne microbes responding to the persistent modifications of the soil physicochemical properties resulting from the chemical composition of the applied OW . In our study, the variations of the soil microbial community composition were further reflected by the distinct proportions of the fungal and prokaryotic groups. The most abundant taxa were Proteobacteria (∼27 %) , Thaumarchaeota (∼25 %) , Bacteroidetes (∼19 %), and Actinobacteria-p (∼6.4 %). These are the major bacterial and archaeal taxa found in French soils . A decade of repeated digestate application resulted in a lower abundance of Thaumarchaeota and a higher abundance of Actinobacteria-p compared to the other OW (pig slurry and cattle manure). The phylum Thaumarchaeota comprises dominant archaea in the soil prokaryotic community, known for their pivotal role in soil ammonia oxidation by converting ammonia to nitrite and, subsequently, nitric oxide . Thus, a higher abundance of Thaumarchaeota could be expected after ammonia inputs via the digestate. However, contrary to these expectations, an antagonistic effect may be revealed as previously reported that oxygen availability might be suboptimal for soil-aerobic Thaumarchaeota ; therefore, our results could suggest a potential long-term consequence of the repeated application of an anaerobic digested product on the abundance of this phylum. However, to date, the impact of digestate application on the soil Thaumarchaeota phylum remains poorly documented. The phylum Actinobacteria is a Gram-positive, saprophytic, ubiquitous bacteria. They can produce a wide array of extracellular hydrolytic enzymes that degrade complex macromolecules that can break down animal and plant biomass. This makes them central organisms in carbon recycling . They are considered as oligotrophs because they dominate the decomposition of organic material when nutrients are limited . An increase in bacteria belonging to this phylum has already been observed after 42 days of digestate application . Furthermore, members of the Actinobacteria phylum have been reported as potassium-solubilizing and/or phosphate-solubilizing bacteria . The observed changes in the relative abundance of Actinobacteria-p may be linked to the cycling of phosphorus in the soil because the phosphorus content of the OW is one of the limiting elements to determine the application rates at the EFELE site. The most abundant fungal phyla in our in-situ approach were Ascomycota (∼43 %) , Basidiomycota (∼25 %) , Mucoromycota (∼16 %), and “Unknown Fungi” (∼8 %). These are worldwide dominant taxa in soil fungal communities . Ascomycota and Basidiomycota are major contributors to soil carbon cycling . Ascomycota members are generally defined as copiotrophic saprophytes. They are typically characterized as fast-growing, effective decomposers of labile carbon and are abundant dwellers of soils with high C availability . By contrast, Basidiomycota members are defined as oligotrophic saprophytes and symbiotrophs . Basidiomycota are assumed to possess improved metabolic capacities over Ascomycota for decomposing more recalcitrant soil C compounds . In 2022, the higher abundances of Ascomycota in plots amended with traditional organic products (PS and CM) may be attributed to organic carbon availability within these treatments. In contrast, the higher relative abundance of Basidiomycota in plots receiving digestate or mineral fertilizer may be explained by the reduced amount of readily available carbon for soil microorganisms when digested organic matter is used as an amendment, having a similar effect to a mineral fertilizer . This study provides the first field-based analysis of the dynamic effects of repeated digestate applications over a decade, assessing whether the impacts of digestate differ from those of its undigested feedstock and other commonly used fertilizers. Our findings provided new insights regarding the long-term effects of one type of digestate on soil microorganisms. After a decade of repeated applications of different OW (cattle manure, pig slurry and digestate of pig slurry) or mineral fertilizer, lasting changes were observed in the soil's physicochemical properties and microbial parameters across all treatments. These changes were primarily explained by the effect of time and to a lesser extent by the quality of the applied product. Our results highlight that the response of soil microbial communities to repeated digestate application is similar to that of mineral fertilizer but strongly influenced and buffered by other agricultural practices and environmental and climatic conditions. Hence, when assessing the impact of digestate on soil microbial communities, associated agricultural practices should be considered. Further research is needed to better understand the cumulative effects of repeated digestate applications on soil microbial communities, particularly across diverse agro-pedological contexts. This includes not only soil type but also interactions with agricultural practices, such as fertilization history, additive fertilization (additional nutrient inputs), tillage, and plant cover (e.g., crop type, cover crops, rotation, and plant diversity). | Study | biomedical | en | 0.999999 |
PMC11699321 | An increased focus has been placed on the production of polymer composites reinforced with natural fibres over the past few decades. This is primarily due to concerns surrounding sustainability and degradability, as evidenced by various scholarly sources . The use of natural fibers like Fique fibers are being proposed for creating composites that are lightweight and less expensive than traditional synthetic fibers, including carbon fiber, Kevlar, and fiber glass [ , , , ]. Furthermore, reinforced polymer composites made from natural fibers may be an eco-friendly option. The mechanical and physical characteristics of pure cellulose fibers are influenced by their source and characteristics, including but not limited to diameter, length, and specific gravity. These properties play a pivotal role in identifying the potential applications of these fibers. The use of natural fibers, including sugar palm fiber, OPEFB fiber, kenaf fiber, flax fiber, pineapple leaf fiber, banana fiber, and jute in polymer matrix composites (PMCs) present numerous benefits, such as recyclability, reduced density, biodegradability, accessibility, enhanced mechanical and physical characteristics, and favorable thermal steadiness of the resultant materials [ , , , ]. The use of natural fibers, including but not limited to kenaf, jute, and hemp, as reinforcement for polymer composites has resulted in exceptional thermal properties that are comparable to those of synthetic fibers. This has led to an expansion of their potential applications in various fields, including aerospace and construction industries, as evidenced by studies [ , , , , ]. Before their implementation in structural applications, it is necessary to use testing methodologies to examine the composite structure and assess its performance under cyclic loading and thermal conditions. The categorization of natural fiber can be delineated into two primary classifications based on its source: inorganic and organic. Typically, inorganic natural fibers consist of mineral fibers, including wollastonite, fibrous brucite, and asbestos. Various plant species, including grass, bamboo, wheat, and bagasse, can produce fibers from their stalks. The constituents of plant fibers primarily comprise three fundamental elements, namely cellulose, hemicelluloses, and lignin. The fibers in question are commonly known as lignocellulosic materials, because cellulose constitutes the primary chemical, alongside varying quantities of lignin and hemicellulose . Mechanical characteristics of fibers are attributed to the hydrogen bonds present between macromolecules, as well as the orientation of crystalline cellulose fibrils within the cell wall. Lignin exhibits strong adherence to hemicellulose and forms cross-links with polysaccharides, thereby occupying the interstitial spaces within the cell wall matrix composed of pectin, cellulose, and hemicellulose constituents, thereby conferring mechanical stability to the cell wall . The primary constraints associated with natural fibers pertain to their hydrophilic characteristics, which can augment moisture absorption and undermine the bonding interplay between the fibers and polymer matrices [ , , , ]. The drawbacks associated with natural fibers, such as inadequate interfacial bonding and the manifestation of hydrophilic properties, can be overcome by using diverse chemical processing that modify the compatibility of the natural fibers with the matrix. The application of chemical processing results in the formation of natural fibers that are fully cured and cross-linked. These fibers serve to reinforce the polymeric matrix, thereby leading to enhanced mechanical strength and thermal stability. This has been documented in previous studies . Improved compatibility among the natural fibers and polymer binder might be achieved through the efficient removal of surface contaminations and hydroxyl group constituents . This phenomenon facilitates a robust chemical adhesion at the interfaces of two phases, resulting in a well-established network. This process has the potential to enhance the utility of natural fibers in various domains, including marine and automobile sectors, by augmenting their resistance to wear, tear, and creep [ , , , ]. The augmentation of these characteristics has the potential to enhance the longevity of the material, particularly when subjected to severe circumstances, such as elevated temperatures, higher levels of humidity, and acidic environment [ , , , ]. The investigation of thermal behavior under diverse conditions is a crucial aspect to be considered for the design and development of hybrid composites for multiple applications. The nonlinearity of thermal property fluctuations in hybrid composites presents a challenge in determining and forecasting thermal conductivity [ , , ]. The incorporation of filler particles of silicon carbide into the pristine epoxy polymer composites results in an enhancement of thermal conductivity, which is directly proportional to the percentage of volume fraction. The thermal conductivity of silicon carbide in isolation is notably higher than that of hybrid composites. However, when combined with epoxy resin, silicon carbide exhibits reduced thermal conductivity due to the relatively diminutive size of the filler particles, limiting their efficacy in producing conductive pathways is limited . Consequently, insufficient cooling may lead to the malfunction of electronic devices. The operational temperature range limit is a critical factor that determines the resilience and recital of electronic devices. Precise management of thermal conditions is a crucial factor in determining the high performance of semiconductors . The efficacy of using high-performance computing systems is depends on the selection of filler materials with superior thermal conductivity. The use of hybrid nano-fillers in the modification of polymer matrices has opened up extensive avenues for research, enabling the attainment of superior thermal and mechanical properties applicable across various fields. According to previous research, various nanoparticle fillers are frequently used to enhance the thermal conductivity and electrical resistivity of polymers . Owing to the inherently low thermal conductivity of polymers, various fillers including mica, zinc oxide, glass fiber, boron nitride, aluminum nitride, silicon carbides, and alumina are incorporated . The enhancement of thermal resistance can be observed in silica hybrid composites, which can be attributed to the robust interfacial bonding between silica particles and epoxy resin, as reported in Ref. . The incorporation of graphene nanoplates (GNPs) at a concentration of 1 % by weight resulted in a notable improvement in thermal conductivity. This effect can be attributed to the uniform distribution of GNPs within the epoxy polymer matrix as well as the establishment of internal bonding. Some studies involved a comparative analysis of the experimental thermal conductivity values obtained for polymer composites filled with particulate matter, with the mathematical methods, namely the rule of mixture thermal conductivity model, parallel thermal conductivity model, and Maxwell thermal conductivity model . To enhance the thermal conductivity and thermal stability of polymer composites, fillers including graphene, CNT, copper mesh and powder, can be incorporated into carbon fiber–reinforced polymer (CFRP) to create hybrid polymer composites . The use of hybrid fillers including boron nitride, SiC, and aluminum nitride in polymer composites has been observed to be a more efficacious approach in reducing the CTE . A few experiments were conducted by adding SiC nanoparticles into glass fiber-based epoxy composites to evaluate their thermal stability. The results of thermal experiments indicated that composites with 5 % and 20 % SiC contents exhibited relatively high thermal conductivities . Thermogravimetric analysis (TGA) was conducted on composites comprising polyester resin, glass fiber, and jute fiber. The results indicated that the composite demonstrated a lower weight loss with increasing temperature . In a TGA comparison study of hybrid polymer composites, carbon fiber composite, and glass fiber composite, it was found that the thermal stability of the hybrid composites was superior . The TGA of the composite material consisting of PMMA toughened glass and epoxy revealed a greater degree of mass loss, whereas the incorporation of SiC helped to maintain its temperature stability . The findings suggest that the incorporation of GNPs, graphene oxide (GO), reduced graphene oxide (rGO), and multi-walled carbon nanotubes (MWCNTs) results in an increase in thermal conductivity . The impact of filler materials on the glass transition temperature of polymers is significant. Certain composites undergo testing for structural stability at low temperatures, as indicated in Ref. . Hence, from all the above discussions it could be understood that that the incorporation of natural or inorganic nanoparticles exhibited better thermal characteristics when compared with the composites without nanoparticles. Accordingly, the focus of the current review article was to articulate the significance of nanoparticle addition to the natural fiber–based polymer composites in improving the thermal characteristics. Natural fiber polymer composites could be analyzed for their disintegration by temperature rise and weight loss in relation to temperature, including residue material beyond maximum heating, using TGA. The lignocellulosic fiber combined polymer matrix that makes up natural fiber composites. The rate at which these materials degrade in heat depends on the reinforcement and matrix used, with each component degrading at distinct temperatures. When something degrades at a certain temperature, it loses mass in accordance with that temperature. The process of reducing weight in fiber composites can be broken down into three distinct phases: first, because of the evaporation of water; second, owing to the breakdown of the fiber reinforcement; and third, because of de-polymerization in the binder itself. Recently, the hybridization process has enhanced the thermal resistance of fiber composites. Among the hybridization influences in the thermal resistance of sisal composites are the following. Researchers evaluated the thermal properties exhibited by the jute or sisal hybrid composites. As a result of the initial drying process, the composites lost approximately 5 wt%. The deterioration of binder and fiber caused 75 % of the composite's mass loss when the temperature began to rise. The composites completely degraded when subjected to high temperatures. Maximum thermal stability was observed in a composite made up of 50 % jute and 50 % sisal fiber. Additionally, alkaline treatment was found to be advantageous in improving the composites' thermal stability . Gupta obtained comparable findings from a different study that investigated identical jute or sisal epoxy composites with varying fiber weight fractions . Similarly, the temperature resistance of sisal material was improved by glass fiber incorporation. This increase can be attributed in large part to the fact that glass fiber has more thermal resistance than sisal fiber. The TGA revealed that the sisal or glass fiber composites generated significantly more charred residue than any of the other composites developed . There was some variation in thermal characteristics between sisal with glass hybrid composites made using an extrusion process and those made using an injection molding technique . The research findings point to the importance of fiber dispersion in the matrix for improving the thermal resistance of composites. The influence of particle reinforcement on the thermal characteristics of sisal polyester composite was reported by Venkatram et al. . Comparison research was conducted after adding 3 wt% nanoclay to an existing sisal polyester composite. The composite without nanoclays was less stable at high temperatures than those reinforced with nanoclay. At 290 °C, the sisal composites lost 82 % of their weight whereas the hybrid composite lost only 79 % of its weight . Charcoal units added to sisal-based composites exerted a positive influence on the deterioration and temperature resistance of the hybrid composite . High degradation temperatures are required to break down the charcoal particles. For this reason, the hybrid mixture with 8, 6, in addition, 4 wt% charcoal retained 30%–40 % of weight regardless of its decomposition at 450 °C. After being heated to 750 °C, the mixture of composite with 8 wt% charcoal retained nearly 38 % of the composite remnant. The thermal characteristics of sisal with SiC-blended composites were reported to be negatively impacted by Teja et al. . The thermal degradation of composites made with 30 wt% sisal fiber and either 5 or 10 wt% SiC particles increased, but the composites still exhibited high thermal stability compared to the pure 30 wt% sisal composites . Sisal, banana plus coir fiber were used to reinforce unique hybrid composites developed by Sumesh et al. . Nanoparticle incorporation improved the thermal resistance of the hybrid composites. Particle reinforcement increased the deterioration temperature of these hybrid composites from 50 % to 70 %. Though particle reinforcement was enhanced by the incorporation of 5 % nanoparticles, mechanical and thermal properties were degraded as an outcome of a greater aggregation of particles . Pappu et al. found that a hybrid composite of polylactic acid (PLA) and sisal and hemp fibers had the same thermal characteristics as pure PLA material. The TGA revealed no change in the thermal properties of the composite after natural fiber was added to the PLA matrix. The hybrid material lost weight in two distinct phases. At temperatures of 300 °C, weight loss began and at 410 °C, it accelerated significantly. At temperatures above 15 °C, the composite began to degrade rapidly and completely, as revealed by Samal et al. . In 2009, Jarukumjorn and Suppakarn explored the rise in heat distortion temperature (HDT) of sisal with glass hybrid PP composites as a function of fiber weight percentage. Adding more reinforcement in the form of sisal fibers raised the HDT. The stiffness of these composites was improved by the addition of the sisal fiber reinforcement, which might account for the observed increases in HDT. The thermal characteristics of a composite made of 10 % sisal fiber with 20 % glass fiber was found to be superior . Govindan and Srinivasan observed identical findings from their study of sisal with basalt hybrid PLA composite. At 250 °C, hybrid composites made of PP and sisal fiber degraded at a lower rate than composites made of sisal fiber alone. Nimanpure et al. reported on the thermal resistance of kenaf with sisal hybrid composites. Thermal degradation was different between composites of different weight percentages. High thermal stability was observed in the hybrid composite made up of 20 % sisal fiber and 20 % kenaf fiber. At 300 °C, this composite lost 10 % of its weight. By improving the fiber–matrix interface, chemical processing of fiber enhanced the thermal stability of the composites. Meenakshi and Krishnamoorthy found that the deterioration temperature of epoxy composites reinforced with sisal, flax, and glass fiber layers was between 306 and 315 °C. Desai et al. showed that carbon epoxy composites benefit from reinforcement with silane-treated sisal fiber. Further, it was reported that the enhanced bonding prevents the free radicals formed during degradation initiation from further spreading. Sisal fiber's degradation temperature is changed when it is reinforced in a hybrid composite because of the removal of organic material during treatment . Maleic anhydride-grafted polypropylene (MAPP) was found to improve the thermal stability of thermoplastic hybrid composites . MAPP was shown to improve the thermal stability of a hybrid polypropylene composite material with coir fiber and sisal fiber combined yarn reinforcement . Asaithambi et al. investigated the thermal characteristics of a banana with sisal fiber–reinforced PLA hybrid composite that had been chemically treated with benzoyl peroxide. The composites' first deterioration along with the final deterioration temperature were slowed by the chemically treated fiber reinforcement. Jute fibers are the common reinforcements in polymer matrices and are compatible with almost all other natural fibers and fillers when it comes to hybridization. Composites maintain the 40 % jute fabric weight incorporating the 3 wt% of into a polyester with jute composite significantly improve their tensile, flexural, and impact properties, as shown in Fig. 1 . Fourier-transform infrared spectroscopy (FTIR) spectroscopy confirmed the presence of OH connection among ATH, ZHO, with jute fabric, which are responsible for the enhanced mechanical characteristics. After performing a scanning electron microscopy (SEM) examination of the broken surfaces, it was determined that the synergy of the nanoparticles led to enhanced interfacial bonding. The TGA additionally demonstrated that the thermal resistance of the composites improved. Endothermic disintegration regarding ATH and ZHO fine particles as a char layer along with water molecules over combustion significantly improved the flame retardancy for the prepared composites, as evidenced by cone calorimetry along with horizontal igniting tests. By working together, ATH and ZHO particles improve mechanical, thermal, and fireproof qualities of polyester with jute composites . Fig. 1 Hydroxide-based nanoparticles addition in jute/polyester composites . Fig. 1 The environmentally beneficial natural fiber–reinforced polylactide composite (NFPC) has been the subject of some attempts to fortify it, but conventional methods reduce its ductility. To simultaneously increase the NFPC's strength and toughness, the authors of the present research synthesized rigid–soft core-shell nanoparticles. Nano-silica along with poly (butyl acrylate) rubber was chosen for their molecular properties, which made them ideal candidates for the core and shell roles, respectively. Crystalline, thermal, as well as mechanical characteristics of NFPC were studied in the presence of core-shell nanoparticles. The findings demonstrated that the core-shell nano-fillers can aid in the formation of a deeper crystalline grain within the PLA matrix and an increase in the material's thermal stability. A bonus is that the elongation at the break of NFPC was not compromised while the nanoparticle's rigid–soft core-shell construction increased the material's strength and stiffness. Furthermore, the resilience enhancement techniques and synergistic effect of core-shell nanoparticles have been demonstrated through field-emission SEM. Toughness is enhanced due to the core-shell filler inducing micro-cracks, shear bands, and matrix fibrillation . Natural fibers are increasingly used in the manufacture of polymer composites, even though these substances have become highly combustible. In this research, titanium dioxide, polydopamine, and diammonium phosphate were added to flax fiber composites made with a polyurethane-based shape memory polymer matrix (PUSMPM) to increase their fire resistance. Polydopamine, a shape memory polymer matrix reinforced using titanium dioxide, was applied to flax fibers that had been processed using diammonium phosphate. The defensive char layer of polydopamine can be seen in SEM images within the generated composites both before and following combustion. Owing to this coating, the composite material burned with much less of a loss of mass than it would have without it. PDA-coated TiO 2 -doped flax fiber composite showed substantial enhancement over uncoated flax fiber composite, with a limiting oxygen index value of 24 % compared to the noncoated flax fiber composite's 20 %. The flame propagation rate was reduced by 81 down to 21 mm/min during the horizontal burning experiment. Using TGA, along with differential thermal analysis, we looked at how polydopamine and titanium dioxide affected fibers and the PUSMPM. Titanium dioxide, polydopamine, and diammonium phosphate were shown in this investigation to significantly increase the combustibility of natural fiber composites made from a PUSMPM . Impact of nanoscale iron oxide (Fe 2 O 3 ) particles on medium-density fiberboard (MDF) physical and chemical characteristics was studied. In this investigation, experimented with untreated poplar fibers along with a variety of nano iron oxide loadings (0.5, 1.5, and 2.5 wt%). Before being embedded into natural fibers, Fe 2 O 3 nanoparticles were mixed into urea formaldehyde resin using a high-vacuum mechanical stirrer. Dry mat layers were created by winding untreated poplar fibers onto metal frames. Twenty distinct composite specimens were created. Thickness swelling, retention of water, moisture content, and density were measured for each composite specimen using industry standards EN-322, EN-317, EN-323, and ASTM D570. Results showed that using iron oxide nanoparticles that were uniformly dispersed greatly enhanced thickness swelling ( T s ). At the highest loading, 3 wt%, water absorption (WA) increased by as much as 49.2 % and 34.5 %, respectively. This study used SEM to examine the nanoparticles' microstructure and determine whether or not iron oxide nanoparticles demonstrate favorable interactions with urea formaldehyde along with poplar wood fibers. A differential scanning calorimetry (DSC) and a TGA were performed to look into the effects of Fe 2 O 3 nanoparticles on heat and mass transfer. Fe 2 O 3 improved the resin's curing temperature as well as thermal stability. To regulate the dissemination of Fe 2 O 3 a one-way analysis of variance was created. As a result, the incorporation of iron oxide nanoparticles into an epoxy polymer results in a more rigid matrix, which in turn increases nano-MDF's potential for exhibiting improved physical characteristics . Table 1 explains the types of nanoparticles reinforced with natural fiber-based composites. Table 1 Types of nanoparticles in natural fiber–reinforced composites. Table 1 Nanoparticle Concentration (wt.%) Natural fiber Reinforcement Matrix Ref. Nanoclay 0, 1, 3, 5 Sisal Polypropylene 0, 2, 4 Bagasse High-density polyethylene (HDPE) 1–4 Bagasse Polypropylene 0, 1, 3, 5 Sisal Epoxy 0, 2, 4 Bagasse Polyethylene 0, 1, 3, 5 Bamboo High-density polyethylene (HDPE) + malleated polyethylene (MAPE) Bacterial cellulose Surface treated Sisal Acrylated epoxidized soybean oil (AESO) Hemp Poly ( l -lactic acid) + cellulose acetate butyrate (CAB) Sisal Acrylated epoxidized soybean oil (AESO) MWCNTs 0.1, 0.5, 1 Palm oil Epoxy Oil palm CNTs 0, 0.5, 1-2 Flax Epoxy Graphene 0.01–0.05 Palm oil Epoxy Nanoclay 0, 5, 8, 10 Flax Epoxidized soybean oil (ESO) Titanium dioxide, TiO 2 0–4 Jute Epoxy Zinc oxide, ZnO 0–4 Jute Epoxy Silica, SiO 2 0, 1.5, 3 Flax Polypropylene (PP) 0, 5 Sisal Polyester Zirconium oxide, ZrO 2 Grafted Flax Poly (lactic acid) Graphene – Bagasse Polypropylene 0–5 Kenaf Alumina, Al 2 O 3 0–3 Flax/PLA Epoxy MWCNTs 0–0.6 Ramie Epoxy Titanium dioxide, TiO 2 0–7 Flax Epoxy Oil palm CNTs 0.15 Bamboo Epoxy Silica, SiO 2 0, 2, 5 Bagasse High-density polyethylene (HDPE) Nanoclay 0–1.5 Hemp Unsaturated polyester (UP) + epoxidized methyl soyate (EMS) Titanium dioxide, TiO 2 0, 2 Bagasse Ethylene co-vinyl acetate (EVA) Nanoclay 1, 2.5, 5, 10 Hemp Poly (lactic acid) Cenosphere – Bamboo Epoxy Bacterial cellulose Surface treated Sisal Poly ( l -lactic acid) + cellulose acetate butyrate (CAB) Oil palm CNTs – Kenaf Epoxy 0, 0.5, 1–4 Hemp Graphene 0.3, 1, 3 Jute Epoxy Alumina, Al 2 O 3 0, 5, 10, 15 (μm) Jute Epoxy 10 (μm) Coir Titanium dioxide, TiO 2 Grafted Flax Poly (lactic acid) Exfoliated graphite, xGnp 0, 1, 3, 5 Kenaf Poly (lactic acid) Graphite is composed of numerous layers of carbon atoms piled on top of each other. Graphene is a monolayer of graphite that can be mechanically exfoliated. The material exhibits notable characteristics such as exceptional strength, low mass, and excellent resistance to corrosion. In comparison to metals, it also has superior electrical and thermal conductivities. Additionally, its remarkable sensitivity renders it suitable for use in nanosensor technology. The advantageous characteristics of graphene, such as its lack of metallic contaminants and two-dimensional structure, make it a valuable material in the field of nanofabrication. Graphene can be synthesized using various techniques, such as mechanical exfoliation, intercalation, chemical synthesis, and burning of agricultural waste materials. This substance finds utility in a diverse range of applications. Graphene exhibits superior thermal conductivity compared to copper, enabling more effective heat dissipation from miniature electronic devices such as computer chips. Numerous industries anticipate the use of materials or nanocomposites that possess distinctive features. The integration of graphene into polymers has been found to considerably enhance the attributes of composites, including electrical conductivity, mechanical strength, thermal stability, and fire resistance . It effectively prevents the initiation and propagation of cracks when subjected to periodic loading. The results of the fatigue studies indicate that after 800 cycles, the hybrid composite material consisting of bamboo, glass fibers, and nanoparticles exhibits a load capacity that is 22.5 % greater than that of all other composite mixtures. The fatigue life of hybrid composites exhibits an upward trend when the filler dimension decreases. The thermal resistance of the hybrid bamboo/glass FRP composite with nanoparticles was enhanced, as indicated by the findings of TGA/DTG studies . The investigation examines the features of the micro- and nanoparticles of hydrophobic graphene oxide (HO) and HO Janus (HOJ) by the use of several analytical techniques, including field-emission scanning electron microscopy, TGA, energy dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy, and contact and centrifuge angle tests. Various micro- and nanocomposite samples of PS/PMMA were fabricated using solution mixing techniques and subsequently evaluated through thermal conduction experiments. Fig. 2 depicts the optical microscopic (OM) images of the PMMA/PS blends containing HOJ nanoparticles. The findings of the study indicate that the incorporation of HO micro- and nanoparticles in either phase of the PS/PMMA blend resulted in a slight increase in the conduction coefficient (K). However, it was observed that the combined presence of these particles in both phases had a more substantial effect on K. In contrast, while evaluating the outcomes of the blend specimens with and without the incorporation of HOJ micro- and nanoparticle, a significant enhancement in the heat conductivity of the polymer with polymer interface was seen as a result of the inclusion of Janus particles. Furthermore, it was observed that the thermal resistance of the samples exhibited a reduction as their thermal conductivity increased. This trend was particularly pronounced in the samples that contained both HO and HOJ micro- and nanoparticles . Fig. 2 OM images depicting the presence of HOJ nanoparticles in PMMA/PS polymer blends . Fig. 2 Nanoparticle-based polymer composites are potential candidates for various engineering applications apart from natural-fiber and hybrid composites. Polymer composites are normally reinforced with organic or inorganic nanoparticles and were assessed for their physical and thermal properties in most cases. Cellulose-based nanocomposites and carbon or graphene-based nanocomposites were analyzed by numerous researchers for their thermal and electrical properties [ , , , ]. Bioactive polymeric nanocomposites are materials that are considered essential and have gained significant interest due to their wide range of applications within the human organism. A complex of poly (hydroxyethyl methacrylate) (pHEMA) was fabricated using hydroxyethyl methacrylate (HEMA) as the primary material with the aid of a twin-screw extruder. The nanocomposites based on pHEMA were fabricated by incorporating titanium oxide nanoparticles (1 wt % TiO 2 ) and graphene oxide (0.1 wt% GO) and their thermomechanical properties were investigated. The PT and PTG nanocomposites exhibited notable alterations in microstructural behavior, resulting in enhanced thermomechanical characteristics as compared to pure pHEMA. This study investigated the suitability of pure pHEMA as well as PT and PTG nanocomposites for implantation in dental implications . A few experimental works used betel nut husk fiber (BNHF) derived nanocellulose reinforced in polyvinyl alcohol (PVA) matrix with various weight fractions and their thermal behaviour was evaluated using TGA and DSC techniques. It was understood from the results that the addition of nanocellulose to PVA matrix enhanced thermal stability of the nanocomposites due to the establishment of strong hydrogen bonding between the composite constituents . In some other studies, vinyl laurate micro fibrillated cellulose (VL-MFC) were reinforced in PLA matrix and their rheological and thermal properties were evaluated. It was observed from the results that the thermal stability of the PLA matrix was increased due to the addition of VL-MFC while the storage and loss modulus of the VL-MFC nanocomposites increased with the addition of the micro cellulose. These changes were supported by the better interfacial adhesion between the nanoparticles and the matrix and due to the liquid-to-solid transition in the composites when subjected to change in temperatures . From the above discussions, it could be understood that the thermal stability of the polymer composites were improved by the incorporation off the nanoparticles. The composite polymer electrolytes are specifically designed to be compatible with the 3D printing process known as fused deposition modelling. The composite materials used in this study were composed of altered poly (ethylene glycol) (PEG), lithium bis-(trifluoromethylsulfonyl) imide (LiTFSI), and nanofillers based on SiO 2 . The end group modification of PEG was effectively carried out, resulting in the production of telechelic PEG that incorporates either ureidopyrimidone (UPy) or barbiturate moieties. These modified PEG molecules possess the ability to form supramolecular networks through hydrogen bonding interactions, thereby providing self-healing properties to the electrolyte system. Silica NPs were used as a filler to modify the mechanical properties of the electrolyte to facilitate its 3D-printing capability. It is anticipated that the surface modification of the NPs using an ionic liquid (IL) or hydrophobic alkyl chains will result in an enhanced dispersion of the NPs within the polymer matrix. The rheological properties of composites containing varying concentrations of nanoparticles (in increment of 5 %, from 5 % to 15 %) and LiTFSI salt were examined to acquire an in-depth understanding of their suitability for 3D printing. Additionally, the ionic conductivity of these composites was assessed using broadband dielectric spectroscopy. The successful 3D printing of the composite electrolyte, PEG with other constituents in combination with 15 % NP-IL, demonstrates its potential as a viable option for printable composite electrolytes. TGA experiment was performed using the Netzsch TG 209 F3 instrument. Samples weighing between 5 and 10 mg were carefully positioned into alumina crucibles. These crucibles were then subjected to controlled heating in an environment free of reactive gases, with a heating rate of 10 K −1 . DSC measurements were conducted using a Netzsch DSC 204 F1 instrument. Before measurement, the samples were subjected to an air-drying process in a vacuum environment at 80 °C. Subsequently, the dried samples were carefully positioned in aluminum pans and measurements were carried out in the presence of a nitrogen atmosphere. The thermal history of the samples was eliminated with the application of heat at 100 °C. The cooling process was conducted until a temperature of −20 °C was reached, with a cooling rate. Subsequently, heating curves were recorded up to a temperature of 170 °C, using a heating rate. The samples underwent a drying process at 80 °C under vacuum conditions for 24 h. Subsequently, measurements of shear rate vs viscosity were conducted at various temperatures. The silica nanopowder, which incorporated alkyl groups (referred to as NP-alk), demonstrated the stretching vibration of the alkene of C–H within the range of approximately 3000 cm −1 . The findings from the thermogravimetric study are illustrated in Fig. 3 . The nanoparticles that underwent modification exhibited a reduction in weight when exposed to elevated temperatures, which can be attributed to the breakdown of the organic modifiers present on their surfaces. This outcome serves as evidence of the effective functionalization of the nanoparticles. The Ludox SM30 nanoparticles functionalized with ionic liquid groups (referred to as NP-IL) exhibited thermal stability up to 350 °C, consistent with previous findings reported in the literature for nanoparticles functionalized with ILs . In contrast, the thermal stability of NP-alk was found to be considerably lower, reaching only up to 250 °C. The estimation of the size of the nanoparticles (with a range of 10–120 nm) was conducted using dynamic light scattering and TEM measurements. Furthermore, proton nuclear magnetic resonance ( 1 H NMR) and solid-state cross-polarization/magic angle spinning silicon-29 nuclear magnetic resonance (CP/MAS 29Si-NMR) techniques were also used to characterize NPs . Fig. 3 Thermal degradation curves of nanocomposites with silica nanocomposites with surface modification done using ionic liquids (blue line) and Alkyl compounds (black line) . Fig. 3 Natural fiber composites have become the first-choice materials in various engineering applications due to their economic feasibility, technicality and environmental friendliness. Though many research works were undertaken for developing novel composite materials using natural fibers, various technical and economic constraints retarded the full-scale implementation of natural fiber composites in different commercial applications. A high level of priority must be given to the thermal ability of the natural fiber composites during their processing and the retention of their inherent properties even after exposure to thermal and other heat-related environments. In this context, the thermal analysis of the composite materials paved way for the development of newer and novel composite materials if the selection process of the materials is rightly optimized. Various approaches were analyzed in the previously published literature to improve the thermal properties of the natural fiber–reinforced polymer composites. Some of the commonly adopted techniques were tailoring the fiber and matrix type, addition of organic and inorganic fillers specifically in nanoscale, orientation, chemical modification, and loading of the natural fibers. In most of the studies, it was pointed out that the addition of nanoparticles resulted in a better enhancement of the thermal properties of the composites. Some of the commonly analyzed thermal behavior includes the mass loss of the nanoparticles reinforced composites when heated, the temperature of complete degradation of the composites, their glass transition temperature, and the viscoelastic behavior of the composites. The addition of nanoparticles facilitated the composites to attain better thermal properties. Specifically, the viscoelastic properties of the natural fiber–reinforced polymer composites could be readily improved by the addition of nanoparticles, which promote good interfacial adhesion between the matrix and the reinforcements and improve the energy dispersion. The addition of nanoparticles also governed the thermal degradation of the polymer composites by enhancing the molecular mass of the composites, increasing the cross-linkages, and increasing tacticity, which in turn affects the glass transition properties of the composites. Additionally, the hybridization of the natural fiber composites with the nanoparticles improved the crystallization kinetics, heat of fusion, heat capacity, melting behavior, and liquid crystal transition temperature, which enhance the thermal characteristics of the composites. From all the above studies, it could be concluded that the nanoparticles incorporated in natural fiber composites exhibit better thermal behavior and are highly suitable as renewable materials for various high-temperature applications. The trend of using nanoparticles for enhancing the thermal properties of the natural fiber–reinforced polymer nanocomposites was analyzed using a patent database due to its frequently updated nature. This analysis provides a path for researchers in this domain to take direction on decision making of their research work. This also paves a pathway to critically assess the application point-of-view of the material through the identification of the innovations using this material . It could be understood from many of the earlier research works that natural fiber composites with nanoparticles as hybrid reinforcements possess better thermal behaviour and thermal stability for various engineering applications. Since the thermal behaviour of the nanoparticle-reinforced natural fiber composites were found to be better, they can capture a significant market share in electrical and sports applications. To further enhance the potential of the nanoparticle-reinforced natural fiber composites, the weathering behaviour of the composites specifically in outdoor environments must be assessed in detail. Exposure of the composites to sunlight, moisture and UV light can be monitored and their behaviour can be evaluated under such conditions to enhance the applications spectrum of the composites . | Study | biomedical | en | 0.999997 |
PMC11699328 | The use of unconventional oil and gas production technologies is increasing due to the continuous increase in energy demand . Accordingly, the exploitation of unconventional reservoirs through hydraulic fracturing is an inevitable choice in the industry . Hydraulic fracturing (HF) operations inject large and highly pressurized volumes of fracturing fluid into tight shale formations to fracture the rock and allow the extraction of trapped oil and gas through the created flow pathways . The most widely used type of fracturing fluid is slick-water , which is a water-based fluid that contains a fraction of added chemicals (e.g., friction reducers) and proppants . Flowback and produced water returning to the surface during production consist of the fraction of the injected fracturing fluid that is recovered and the brine, respectively . Reported fracturing fluid flow-back recovery values vary significantly across different formations, reflecting that a substantial volume of fluid typically remains in the subsurface . For example, recovery rates for the Marcellus shale are reported at 7.8 % , for the Qusaiba formation at 9.6 % , for the Montney formation at 36 % , and for the Haynesville formation at 5 % . These examples highlight the generally low flow-back recovery rates across formations, which contributes to notable fluid retention in the subsurface. Meanwhile, environmentalists are constantly warning on the potential risks associated with the migration of the remaining fraction of this fluid (>70 %) which imbibed into the shale formation. Particularly, there are concerns on possible groundwater contamination because the additives present in the fracturing fluid might find their way to water aquifers . Toxicity information available about fracturing fluid chemicals show that being exposed to fracturing fluid poses serious health hazards on humans [ , , , ]. Public pressure concerning this issue is constantly intensifying because toxicity data is limited and disclosures of fracturing fluid are questionable. Previously, voluntary disclosures were minimal , and mandatory disclosure regulations used to have shortcomings concerning the chemicals' identification requirements, imposed penalties in case of insufficient reporting, and lenient timelines of reporting . A recent study by Bonetti et al. showed that revisiting and tightening disclosure mandates led to a decrease in the quantity of toxic chemicals used. Assessing the environmental impact of fracturing fluid, monitoring its fate, and tracking the diffusion of the fluid's constituents post injection are necessary to eliminate possible hazards. Additionally, although hydraulic fracturing is widely recognized for its use in the oil and gas industry, its application has expanded into sustainability-focused projects such as carbon capture and storage (CCS) and enhanced geothermal systems (EGS). In these areas, hydraulic fracturing is used to improve permeability, create heat reservoirs in EGS, and enhance CO₂ injectivity in storage reservoirs. Although the types and concentrations of chemicals used in fracturing fluids for CCS and EGS are typically simpler and lower than those in oil and gas applications, there remain critical environmental risks—such as induced seismicity, fluid migration, and CO₂ leakage—that are important to consider. These shared concerns highlight the broader relevance of hydraulic fracturing fate studies, as they illuminate fluid retention and migration behaviors that could affect subsurface and surface water resources across applications. By evaluating hydraulic fracturing's impacts in CCS and EGS alongside traditional oil and gas contexts, we gain a more comprehensive understanding of how this technology can support, rather than undermine, sustainability goals. Addressing these risks proactively is essential to ensuring that hydraulic fracturing contributes positively to energy transition efforts and environmental protection measures [ , , , , , , , ]. Peer reviewed articles from scientific journals, Environmental Protection Agency (EPA) reports, and conferences papers were analyzed and discussed in order to describe the fate, transport and recovery of fracturing fluids, in addition to their environmental and health impacts. The 100+ papers referenced in this review included both numerical simulations and case studies. Accordingly, the articles referenced are classified into three categories representing the main areas of the study, which are (1) environmental impact of HF fluid and associated risks; (2) fate of HF fluid; and (3) induced seismicity triggered by HF fluid diffusion and its correlation to flow back. First, HF fluid composition and toxicity are illustrated to emphasize the criticalness of surface and subsurface risks associated with the transport and migration of HF fluid. Next, the fate of HF fluid was examined due to the subsurface threats posed by its migration, particularly the retention of a significant fraction of HF fluid in the subsurface post-production. This analysis involved exploring the potential reasons behind the low flowback recovery values and establishing correlations between HF fluid recovery and the various factors affecting it. Finally, a correlation between induced seismicity and load recovery is demonstrated by highlighting the spatial and temporal link between seismic activity and hydraulic fracturing operations in which HF fluid migrates to critically stressed faults. Moreover, this research identifies the limitations and gaps in the literature, which are related to the scarcity of data. Disclosure data about the composition of the fracturing fluid is limited , and subsurface monitoring data utilized to characterize fluid migration are rare in comparison to the large number of hydraulically fractured wells . Such limitations impede groundwater contamination investigations and restrict the publishing of relevant studies, leading to shortcomings in irrefutable documentation of drinking water contamination by fracturing fluid . In studies dedicated to evaluating toxicity of additives present in hydraulic fracturing fluids, authors usually follow the same approach; compiling lists of disclosed chemicals, then scanning databases for toxicological data, and possible health effects. Key findings from the studies provide different pieces of information about the type of toxicity associated with the chemicals, toxicity ratings, hazard rankings, and associated health effects. Table 2 shows some of the interesting findings presented in the literature. Furthermore, Kahrilas et al. reviewed the toxicity of biocides used in fracturing fluids, highlighting their potential as serious irritants to sensory organs and their considerable acute toxicity to aquatic life. Data from their study are summarized and illustrated in Fig. 2 to show the types of chronic toxicity associated with certain biocides and their degradation products used in fracturing fluid formulations. Table 2 Summary of toxicity data availability for hydraulic fracturing fluid chemicals as reported in each study, including the types of data presented and the biological systems affected. Table 2 Number of chemicals studied Toxicity data availability Toxicity data available/most affected systems Reference 1076 10 % Chronic oral toxicity values 36 22 % Chronic oral toxicity values Note: Additives reported in fracturing fluids in at least 10 % of wells in the U.S. 600 58.8 % • >75 % affect sensory organs, respiratory and gastrointestinal systems • 52 % affect nervous system, immune and cardiovascular systems, and kidneys • 37 % affect endocrine system • 25 % cause cancer 81 69 % • 16 % low/moderate toxicity • 31 % lacked toxicity data • 53 % considered as non-toxic 168 N/A 14 % associated with reproductive and developmental toxicity 320 60 % Chronic and/or acute hazard screening values 924 21 % Reproductive and developmental toxicity reported 28 82 % Mammalian and aquatic acute toxicity measurements Note: Additives used in more than 10 % of fracturing treatments in California 20 45 % Mammalian and aquatic acute toxicity measurements Fig. 2 Toxicological data of biocides used in hydraulic fracturing operations . Fig. 2 Numerous studies have compiled and analyzed spill records [ 8 , 54 , 71 , 75 , 76 , 80 , , , ]. Scientists are able to categorize spills linked to hydraulic fracturing by type of material, pathways, sources, causes, and volume spilled . For example, the EPA report examined data of more than 36,000 spills recorded over a period of 6–7 years in 11 states in the U.S. and found that the most prevalent material types that are spilled are flow-back and produced water (49 % of the total material spilled), the source of these spills are mainly from storage (46 % of the total source type of spills), and the cause is mainly due to human error (33 % of the time) and equipment failures (27 % of the time). A detailed description is synthesized in Fig. 3 . Fig. 3 Percent distribution of material related to hydraulic fracturing that are spilled, their source and cause. These values were calculated using the number of spills per material, source, and cause types relative to the total number of spills with data synthesized from the U.S. EPA . Fig. 3 It must be highlighted that although the spill rates usually reported might be misinterpreted as inconsequential, the volume per spill ranges from small values like 1 Gal to critically high values reaching 1 MGal , thereby amplifying exposure risks. Estimating fracturing fluid spill rates serves as a basis for spill assessment. Spills of all material types, even if non-toxic, must be incorporated into spill probability assessments, since a spill during transport due to a wreck incident has the same likelihood whether the fluid is fracturing fluid or any other chemical; if the content is not safe it will pose a serious environmental hazard. Another important factor in assessment and mitigation practices is monitoring how changes in reporting requirements impact the spill rates recorded, where stricter mandates are leading to an improvement in accuracy, details, and number of reported spills. Digiulio & Jackson assessed the effect of fracturing operations on subsurface drinking water sources. The motivation behind this work was based on an EPA investigation done in 2008 in response to complaints in the Pavillion Field about changes in taste and odor of drinking water from domestic wells. To evaluate potential upward migration of fracturing fluid additives, the EPA drilled a couple of monitoring wells near the production wells (with a separating distance of 200–300 m) and sampled them. The two wells “MW01” and “MW02” were screened at intervals of 233–239 m and 296–302 m below ground surface, respectively. In the Pavillion Field, the maximum depth of groundwater use was 322 m (at the time the study was conducted) and some stimulation activities (acid stimulation/hydraulic fracturing) in the area occurred at depths as shallow as 213 and 322 m below ground surface. In the sampling results of the monitoring wells, several constituents of the fracturing fluid and/or their corresponding degradation products were detected, in considerable concentrations. Organic compounds that were detected include naphthalene, alkylbenzenes, diethylene glycol, octylphenol, and trimethylbenzene. These results provide extra credibility to the suggested scenario of the infiltration of fracturing fluid chemical additives into the drinking water sources. Based on facts and discussions analyzed in this section, it must be noted that even when the hydraulic fracturing fluid does not or takes several years to migrate towards the underground drinking water sources, it remains toxic posing a risk to human health, especially through the other existing exposure pathways. Human exposure to fracturing fluid chemicals primarily results from accidental spills and operational accidents. However, these chemicals are not exclusive to fracturing fluids; many are also components of other operational fluids or are, for example, hydrocarbon derivatives . This overlap complicates the attribution of contamination solely to hydraulic fracturing, especially since detected chemicals in environmental samples sometimes consist of complex mixtures of organic compounds, like petroleum distillates . Additionally, there exists a lack of robust pre-injection water quality checks in some regions, which complicates the ability to always demonstrate that the injection caused the contamination. So, to accurately pinpoint and limit contamination directly related to fracturing fluid injection, concentration of additives should not exceed the limits established by regulatory acts involved with monitoring the quality of drinking water quality . In addition, not all the operators disclose fracturing fluid constituents they use, and the toxicity-related information is somewhat limited . This obstructs investigations examining environmental hazards linked to the use of fracturing fluid. Discrepancy in flow-back rates and ambiguity surrounding the fate of the fracturing fluid have garnered research interest due to the uncertainty in the causes and the risk of aquifer contamination , which is strongly dictated by fluid migration and directly attributed to low flow-back ratios. The associated downhole risks have been previously addressed in sub-section 3.3.2 . Low recovery values are the consequence of hydraulic communication resulting from the intersection between induced hydraulic fractures and permeable pathways previously existing in the formation, like faults and abandoned wells. There is a large number of published studies that investigated the upward flow of HF fluid and that gave, indirectly, insights regarding the fate of HF fluid that is not produced back [ 6 , , , , , , , 97 , 98 ]. For instance, Birdsell et al. conducted a study where they simulated hydraulic fracturing fluid migration post injection into a Marcellus shale reservoir and the results showed that, over a period of 20 years, 68 % of the injected fracturing fluid was extracted by the production well, 0.1 % infiltrated into the overlying aquifer, and the remaining diffused elsewhere in the subsurface. The parameters that are positively correlated to, and control, the speed of fluid migration (presented from the most influential to least influential) are: 1) the proximity of a permeable pathway to HF, 2) permeable pathway permeability, 3) injection pressure, 4) permeable pathway width, 5) overburden heterogeneity, and 6) overburden permeability. As an illustration, the speed of fluid migration is higher in the case of higher proximity between HF region and the fault (i.e. shorter distance), when compared to cases of lower proximity . Correlations existing between load recovery and certain parameters (e.g. fracture network complexity, and shut-in period) have become a target of studies discussing fracturing fluid recovery, as illustrated in Table 4 which shows correlations derived specifically from numerical simulation studies. Negative correlation means that an increase in the ‘parameter studied’ yields a decrease in the load recovery, whereas a positive correlation means that an increase in the ‘parameter studied’ yields an increase in the load recovery. Table 4 Correlations between formation and operational parameters and load recovery. Negative correlation means that an increase in the ‘parameter studied’ yields a decrease in the load recovery, whereas a positive correlation means that an increase in the ‘parameter studied’ yields an increase in the load recovery. Table 4 Parameter studied Relation with load recovery References Shut-in period Negatively correlated Natural fracture density Negatively correlated Positively correlated Fracture closure Negatively correlated Positively correlated Rock wettability Hydrophobicity: positively correlated Hydraulic fracture permeability Positively correlated Reservoir pressure Positively correlated Geochemical interactions Negatively correlated Fracture network complexity Negatively correlated Complex fracture networks developed through multi-stage hydraulic fracturing in tight reservoirs can contribute to low flow-back percentages due to fluid retention in the fracture network and fluid leak-off from the fracture to the matrix . Fluid leakage phenomenon, known as “fluid loss”, is defined as the leakage of the fracturing fluid via the main fracture into the formation. Various numerical models dedicated to fluid leak-off simulation have been developed [ , , , , 105 ], and controlling properties were identified. Fluid leak-off is positively correlated to net fracture pressure, fracture length, natural fracture permeability and density, reservoir temperature and brine salinity. Conversely, reservoir pressure and fracturing fluid viscosity are negatively correlated with fluid leak-off . Using flow-back data from 18 wells in the Horn River Basin, Ghanbari & Dehghanpour categorized wells based on flow-back performance and gas production, linking higher fracture complexity to lower water recovery and higher gas production. To backup the basis of their categorization, Ghanbari & Dehghanpour developed a numerical model of a flow-back process. To account for complexity in the model, a fracture complexity index (FCI) controlled by the volume of natural and hydraulic fractures was introduced and showed that increased fracture complexity restricts load recovery while promoting gas production. Liao et al. employed the same FCI equation developed by Ghanbari & Dehghanpour and derived similar conclusions. As anticipated, their results revealed that as the fracture network complexity increases, fracturing fluid load recovery declines. To illustrate, the load recovery decreased from 90 % to 43.9 % as the fracture complexity index increased from 0 to 0.75. However, despite demonstrating the same trend in the relation between fracture complexity and fluid recovery, there is a substantial disparity in load recovery values, with an order of magnitude difference. This discrepancy can be attributed to variations in values of model parameters, such as matrix permeability (0.01 mD in Liao et al.’s study vs. 0.001 mD in Ghanbari & Dehghanpour's study) and hydraulic fracture permeability . Effect of fracture network complexity on fluids' flow and recovery can be explained as follows: During injection, the hydraulic fracturing fluid and proppants both fill the created hydraulic fracture while only the fluid flows into the natural fractures. Thus, the hydraulic fracture's aperture becomes greater than the natural fracture's aperture and the production of fluids from the hydraulic fracture becomes easier when compared to that from natural fractures. However, the higher the complexity of the fracture system created, the wider the contact surface area that is between the matrix and fractures. Hence, imbibition amplifies leading to an increase in the fraction of the water that gets imbibed into the shale matrix during shut-in, and displaces accumulated gas in fractures, which in-turn gets produced at higher ease by the production well. This leads to constrained load recovery and enhanced gas production during early production, and vice versa . Natural fracture density is one of the fracture network characteristics that determines the degree of complexity. The findings for the numerical simulations by Liu et al. contradict other models' that proposed that the increased contact area due to an increase in natural fracture density results in the retention of higher volumes of water in the subsurface. Liu et al.’s results may be considered as a demonstration of the counter-effect of an increased contact area, which could yield a boost in hydraulic communication and hence in water recovery. The apparent contradiction might be due to the different scales and input of these models. For example, natural fracture permeability in Liao et al.’s study is three orders of magnitude higher than the value used in Liu et al.’s study. Another noteworthy feature is the fracture closure. Fracture closure hinders the flow of a fraction of the fracturing fluid from the fractures back to the well, thereby increasing retained volume . While some studies support that accounting for fracture compressibility variations leads to lower load recovery , the study done by Seales et al. shows that fracture closure mechanisms might actually “squeeze” the water out of the saturated fractures and enhance early flow-back. This divergence in findings could stem from differing modeling assumptions. In the studies by Liu et al. and Liao et al. , fracture permeability varied with factors like fracture closure and pressure changes, whereas Seales et al.’s model assumed it to be constant. In conclusion, while fracture network complexity generally leads to lower fluid recovery and higher hydrocarbon production, the specific outcomes depend on a range of factors, including fracture closure properties and the interactions between natural and induced fractures. These complexities highlight the challenges in accurately predicting well performance and fluid flow-back. Additionally, in complex geological settings with local heterogeneities, in-situ stress fields, spatial inconsistency in rock mass properties, and pre-existing bedding planes and fault systems, hydraulic fracturing operations become more challenging. These factors, coupled with the complexities in fracture network characterization, highlight the difficulties in accurately predicting flow-back and hydrocarbon recovery . In studies examining seismicity referred to hydraulic fracturing activities, the primary focus is often on seismic events arising from wastewater injection . This process involves the disposal of flowback water, which consists of the original fracturing fluid (i.e. water and chemical additives) along with naturally present constituents from the formation, into designated wells . This emphasis is due to the fact that earthquakes triggered by wastewater injection generally exhibit higher magnitudes than those directly induced by hydraulic fracturing, with recorded events reaching magnitudes of 5.6 , 5.8 , and even 5.9 . These elevated magnitudes are typically ascribed to the substantial volumes of injected wastewater and the resultant pore pressure increases, which can stimulate seismic activity . Nonetheless, induced earthquakes recorded in areas near hydraulic fracturing sites can be attributed to hydraulic fracturing processes [ , , , , 44 , 45 , 87 , , , , , , , , ], especially under specific hydrogeological conditions . A spatial and temporal link has been developed in regions like the Western Canada Sedimentary Basin (WCSB) [ , , ] and the Sichuan Basin, China . This association is supported by data from well monitoring and the compilation of operational data and seismic activity into catalogs . An example of such data is the one compiled by Verdon & Rodriguez-Pradilla (see Table 6 ), showcasing seismic activity related to fracturing in 12 major shale plays in the Western Canada Sedimentary Basin (WCSB) and the United States using data from regulators (i.e., Alberta Energy Regulator and the British Columbia Oil and Gas Commission) and FracFocus ( www.fracfocus.org ), respectively. Table 6 Hydraulic fracturing-induced seismic activity in 12 major shale plays (adapted from ). Table 6 Play Number of wells Total HF volume (x 10 6 m 3 ) Number of associated earthquakes Largest event magnitude Number of associated wells Bakken 15,375 288 1 3.3 3 Barnett 6427 45 2 2.9 3 Duvernay 1946 44 569 4.1 145 Eagle Ford 15,825 407 389 3.7 731 Fayetteville 2863 28 421 3.9 336 Haynesville 3650 208 18 3.8 9 Horn River 344 14 30 3.6 25 Marcellus 11,530 456 2 2.8 2 Montney 8281 80 388 4.4 603 Niobrara 17,314 367 9 2.6 45 Utica 3146 157 37 3.8 24 Woodford 4424 198 794 3.6 584 One of the fundamental processes responsible for fluid induced seismicity is determined to be fluid diffusion, succeeding fluid injection. This diffusion is enabled due to the hydraulic communication established between hydraulic fractures and faults, allowing the fracturing fluid to reach existing faults. Hence, pre-existing faults are reactivated due to pore pressure increase during or after hydraulic fracturing that is enough to trigger earthquakes . Fluid pressurization and injection have become key research attractions due to their role in fault reactivation . He & Li used the data presented in Table 7 and conducted a simulation study that demonstrated that amplified fluid injection increases the probability of fault reactivation. As per the data presented by Wu et al. in their review paper (see Table 7 ), we can highlight that fluid injection in any unconventional energy project has the potential to induce seismicity, which broadens the scope of research concerned with the injection and fate of the fracturing fluid. Table 7 Data showing the link between flow back and HF and induced seismicity. Table 7 Reference Presented Data Treatment stage Flow-back after the stage Seismicity associated with the stage II No flow-back First seismic event III Aggressive flow-back Weak seismicity IV No flow-back Significant seismicity V Aggressive flow-back Weak seismicity Overall flow-back 20 % Location Reason Significant recorded magnitude Pohang, South Korea Geothermal reservoir stimulation MW = 5.5 Sichuan, China Shale gas production MW = 4.7 Oklahoma, United States Wastewater disposal MW > 4.0 Data Value Five earthquakes recorded during fracturing MW > 4.0 Magnitude of largest recorded event MW = 4.7 Distance separating the source of seismicity and injection point 2–3 km Magnitude of largest recorded event MW = 3.9 Time of occurrence of largest recorded event Two weeks after fracturing completion Distance separating clusters from fractured wells 2 km Flow-back 7 % Period between start of HF and first recorded seismic event 5 days Period between start of HF and largest recorded seismic event 3 months Magnitude of largest recorded event MW = 2.2 Flow-back 40 % According to Schultz et al. , the injected fluid volume is the dominating operational parameter that is directly positively correlated to both the number of seismic events recorded and their magnitude. Although this behavior is expected, a fluid injection over a long period also boosts fluid seepage into surrounding formation , which in turn affects flow-back recovery. In their report done to study induced seismicity in the Bowland Shale in the UK, De Pater & Baisch represented a plausible relation between fracturing flow-back and induced seismicity. Table 7 illustrates the degree of seismicity associated with each stage of hydraulic fracturing as a function of the well flow-back. They suggested that the pattern of seismicity shown is the result of flow-back routines, where strong seismicity is associated with low fluid flow-back volumes, and the fact that only about 20 % of the injected fluid was recovered in this operation supports their observations. Hence, they recommended aggressive fluid flow-back and reduced injected volumes as possible seismicity mitigation practices. Likewise, Bao & Eaton compiled data of a four-month period for six hydraulic fracturing well pads in the Duvernay formation, and the most interesting data are presented in Table 7 . Moreover, Tan et al. used available injection data from two well pads, named “N5” and “N7”, in Sichuan Basin, as a basis for their study. Data derived from pad N5 shows the time period separating the onset of fracturing activities and seismic events occurrence ( Table 7 ), and the flow-back ratio. Values synthesized in Table 7 prove the suggested link between hydraulic fracturing and induced seismicity as per the discussion above, and provide evidence that the earthquake distributions are spatially concentrated nearby hydraulic fracturing well pads and follow chronologically the timing of stimulation activities by few days or weeks. Although conclusions discussed in this section might be considered redundant, they act as support to the hypothesis that connects induced seismicity to low fracturing fluid flow-back recovery since authors present fluid diffusion as one of the major phenomena contributing to induced seismicity in the proximity of hydraulic fracturing locations. For instance, Tan et al. and Bao & Eaton reported load recovery values in cases where fluid diffusion was believed to be the main trigger behind seismic events (i.e., earthquakes), and these values show that more than 50 % and 90 %, respectively, of the injected fluid remained trapped in the subsurface in these cases. Nevertheless, it is worth highlighting that the magnitude of the events is a result of a combination of factors including the injection pressure, cumulative injected volumes, position of created fractures with respect to existing faults, and hydraulic communication between the fractures and these faults which is also discussed in Yehya et al. . To further improve environmental safety and performance, researchers are exploring the use of alternative additives in these fluids. These include biodegradable and green surfactants, nanocomposite friction reducers, hydrogel additives, and amino acid-based surfactants, each offering enhanced biodegradability and lower ecological impact . By incorporating such components, fracturing fluids are engineered to deliver higher lubricity, increased temperature resistance, and better proppant transport, thus supporting efficient hydraulic fracturing with reduced environmental risks . In this work, the main objective is not to discuss the risks related to all the stages of a hydraulic fracturing job, but to discuss the environmental impact of the fracturing fluid. First, we discuss the environmental and health risks associated with HF fluid and its implications on drinking water and highlight reported cases of contamination. Then, we inspect available field data on flow-back recovery rates and load recoveries of the HF fluid, and summarize the parameters and hydrogeological conditions controlling the values of recovery. Hence, we show how HF fluid recovery correlates with the fluid's upward migration through permeable pathways, retention in fracture networks, leak-off into the formation, and induced seismicity. Main conclusions derived from the literature can be summarized as follows: 1. The high number of toxic chemicals added to the HF fluid and the limited disclosure of toxicity data highlight the importance of addressing the surface and subsurface risks of HF fluid. 2. Load recovery is correlated to fracture properties (e.g. natural fracture density) and operational parameters such as shut-in period. Most evidently long shut-in period leads to low hydraulic fluid recovery values. 3. Fluid migration in the subsurface via a permeable pathway, which contributes to low recovery and aquifer contamination, is mostly sensitive to properties of the existing permeable pathway. For instance, a strong positive correlation exists between speed of fluid migration and permeability of the pathway. 4. Fluid leak-off from the fracture to the matrix is considered to be a strong contributor to the low load recovery values due to the retention of the HF fluid. 5. Higher fracture network complexity boosts oil/gas recovery and curbs water recovery. 6. Field evidence support the hypothesis suggesting a negative correlation between induced seismicity in the vicinity of fracturing operations and load recovery; which is explained by fluid diffusion into fractured regions. Further research is essential to specifically address the environmental impacts of fracturing fluids as complex chemical mixtures, independent of the hydraulic fracturing processes. Although existing studies provide insight into potential environmental risks, the majority concentrate on the operational aspects of hydraulic fracturing rather than the toxicity and long-term environmental effects of the fluid's chemical constituents. Comprehensive risk assessment requires more extensive chemical analysis of fracturing fluids, as well as toxicological and epidemiological data that quantify exposure risks to humans and animals. Also, detailed exposure assessments are necessary to understand the pathways through which these chemicals affect biological systems. This will assist in the development of appropriate safety precautions and disclosure mandates that reduce the harm. | Study | other | en | 0.999998 |
PMC11699343 | Flavor is a critical quality attribute in food and is a primary driver for consumer acceptance and food purchasing decisions. The development of flavor in food is a complex process, involving numerous chemical molecules, including volatiles, non-volatiles and high-molecular weight compounds . The sensation of flavor results from simultaneous stimulation of our chemical senses, mainly odor and taste, triggered by these chemicals originally present in raw food material or generated during food processing and storage. Traditional flavor research focused on identifying key odor-active and taste-active chemicals in food, which are less than a few hundred compounds . Advances in analytical techniques and artificial intelligence (AI), which can handle large datasets, have led recent flavor research toward a more systemic assessment of flavor-related chemicals. This includes the measurement of compounds that are tasteless and odorless but impact flavor perception (e.g., flavor enhancers) and compounds that interact with other molecules to modify flavor profile . Studies on flavor precursors and flavor synthetic pathways have also increasingly been introduced in fresh fruits and vegetables, as well as processed food products . With these trends, a new omics subdiscipline named ‘flavoromics’ was born in 2008 and has addressed various challenges in food flavor research . Flavoromics combines analytical chemistry, sensory evaluation, and data science to comprehensively understand the relationships between chemical compositions and flavor traits in food. To characterize a wide range of compounds, including unknown molecules that might affect flavor formation and regulation, flavoromics often employs untargeted chemical analysis using analytical techniques such as gas chromatography–ion mobility spectrometry (GC–IMS), gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–high-resolution mass spectrometry (LC–HRMS) . A similar term, sensomics, uses a combination of sensory evaluation and instrumental analysis, like flavoromics, while sensomics concentrates on the identification and quantification of aroma-active compounds at the molecular level, which is assisted by experiments such as aroma extract dilution analysis (AEDA) and aroma recombination and omission . Multi-omics, the integration of more than one omics approach (e.g., metabolomics with transcriptomics), is another emerging trend to understand in-depth biochemical mechanisms and regulatory processes behind the flavor phenotypes of food across different biological layers . Sugars and organic acids are significant chemical components in fresh fruits, contributing to their balanced sweetness and sourness. The total soluble solids to titratable acidity (TSS/TA) ratio is commonly used to assess the flavor quality and ripeness of fruits . However, efforts have been made to measure individual sugars and organic acids for a better understanding of fruit flavor, because each compound may have a different taste activity value (TAV), and their contents and ratios vary among fruits. Sugars, acids, and their proportions and distribution were examined in a cherry species ( Prunus pseudocerasus ) by high-performance liquid chromatography–ultraviolet/refractive index detection (HPLC–UV/RID) . With the support of sensory evaluation and the TSS/TA ratio, key indicators of cherry flavor, including glucose, fructose, and maltose were identified, and new cherry grading criteria were proposed. Liu, Song, et al. explored alterations in flavor-related compounds in yellow and white-fleshed loquats ( Eriobotrya japonica ) using HPLC–UV/RID and LC–MS-based metabolomics approach. Differentially accumulated metabolites, including sugars and organic acids (e.g., malic acid), were found in loquats of different colors and at different development stages. Interestingly, one collected article used a targeted metabolomics strategy, and reported the identification of ethyl vanillin, a ‘synthetic’ vanilla flavoring compound, in strawberry ( Fragaria × ananassa ) . It was the first time that ethyl vanillin had been observed in natural food. The presence of ethyl vanillin in strawberry was thoroughly confirmed by multiple analytical techniques including GC–MS/MS, LC–HRMS and LC–MS/MS with the use of isotope-labeled ethyl vanillin and a global metabolite library (NIST). The same research group published another article regarding the discovery of a novel bitter-masking compound in allspice ( Pimenta dioica ) using sensory-guided isolation . The structures of the isolated molecules were determined by nuclear magnetic resonance (NMR) and LC–HRMS, and one molecule exhibited promising bitter masking activity against quinine, as determined by sensory evaluation. Molecular docking analysis indicated that the compound could act as an antagonist of one of the bitter receptors, TAS2R14. In addition to the above topics, the flavor qualities of buckwheat ( Fagopyrum spp.) , pear ( Pyrus spp.) , wampee ( Clausena lansium ) , vanilla beans ( Vanilla planifolia ) and pepper seeds ( Capsicum annuum ) were assessed by characterizing flavor-related compounds using gas chromatography with flame ionization detection (GC–FID), GC–MS, GC–IMS, HPLC–UV/RID and/or LC–MS/MS, followed by multivariate statistical analysis. One study employed widely targeted metabolomics and annotated nearly 1000 metabolites in pear . The relationship between metabolites and sensory attributes was uncovered through statistical correlation network analysis, and key sensory-related metabolites in pear were identified based on gene significance and module memberships. Another study examined flavor precursors and downstream aroma volatiles in pepper seeds, demonstrating that certain fatty acids and amino acids were highly correlated with the formation of pepper aroma . For solid processed foods, key odorants associated with the warmed-over flavor of stewed beef after refrigeration and reheating were characterized using a sensomics approach, which combined sensory evaluation with GC–IMS/MS analysis . Shao et al. explored the effect of different starter cultures on the microbial communities and flavor compounds of fermented sausages using 16S rRNA gene sequencing and chemical analysis, demonstrating that a mixed starter enhanced the flavor of fermented sausages by generating a high level of umami taste-related compounds. With growing attention on plant-based foods, one study utilized a soybean by-product as an additive for the production of plant-based patties and assessed the quality of the patties via physicochemical measurements and GC–MS analysis . The developed patties showed increased water-holding capacity, improved texture profiles and lower levels of undesirable flavor volatiles (e.g., benzaldehyde, nonanal, 2-heptanone) compared to control patties without the soybean by-product. Luo et al. introduced a novel fermented food using a pineapple by-product and whey protein. The effect of fermentation on the product quality was clarified by measuring changes in carbohydrate and amino acid profiles, organoleptic properties, and the microbial community of the product. For liquid processed foods, the influence of roasting conditions on the physicochemical properties and flavor quality of coffee ( Coffea arabica ) was assessed by analyzing caffeine, chlorogenic acid, total flavonoid content, antioxidant capacity, flavor, and flavor compounds using various instruments such as HPLC–UV, electronic tongue, electronic nose, and GC–MS-olfactometry (GC–MS-O) . Ouyang et al. employed untargeted volatile analysis using two-dimensional gas chromatography-olfactometry–quadrupole-time-of-flight mass spectrometry (GC × GC-O–QTOF-MS) to exhaustively characterize the odorants of black tea from different provinces in China. A total of 190 volatiles were identified in the tea, and among them, 23 compounds were confirmed as key odorants contributing to the distinct aromas of black tea in different regions. In another study, the aroma profiles of Italian wines were determined by GC–IMS during spontaneous and inoculated fermentation processes, demonstrating that spontaneous fermentation effectively enhanced the aroma characteristics of wine, with increased levels of corresponding volatiles, showing its aging potential . Barba et al. examined the effect of sugar alcohols on the release of aroma compounds in soda beverages using GC–MS and NMR, which may help guide the use of sugar alcohols and aroma volatiles in the formulation of sugar-free or reduced-sugar beverages. Additionally, a new sample preparation technique, called dispersive liquid-liquid microextraction, was introduced and applied to the determination of toxic α-dicarbonyl compounds, such as glyoxal and methylglyoxal, in sesame oil, which has potential for use in the quality and safety control of oil . Several studies in this issue utilized multi-omics strategies to support and confirm their hypotheses across different biological layers in food products, such as tea , cheese and fermented foods . An integrated metabolomics-transcriptomics approach was employed in oolong tea research, revealing dynamic changes in metabolites and flavor formation during tea manufacturing process . Differentially expressed metabolites and genes were associated, and central metabolic pathways and network (sugar, amino acid and flavonoid metabolism) altered during different tea processing steps were identified. Another study examined variations in metabolite profiles and bacterial communities in Liupao tea during tea processing using a joint metabolomic and metagenomic analysis . The two omics datasets were correlated, and the results indicated that metabolic changes in Liupao tea were not primarily derived from bacterial activities, but rather from other processing conditions, such as high temperature and humidity. A combination of metabolomics and metagenomics was also used in yak cheese , and fermented dairy and vegetable products to elucidate the fundamental mechanisms behind the flavor formation of these products in different contexts. Chemicals in food are the end products of cellular signaling processes in raw foods or products generated during processing and storage in processed foods. These chemicals directly reflect food phenotypes, such as flavor, which is linked to food quality, safety and sensory properties. This special issue presents recent advances in the flavor chemistry of food and introduces the most up-to-date technologies and approaches for food flavor research. However, challenges remain in the field, as flavor is a complex trait influenced by numerous compounds, their levels, compositions, interactions and reactions. Future advancements in analytical techniques, along with the application of AI technologies such as machine learning and deep learning algorithms, are expected to enhance the measurement and assessment of food flavor from large and complex datasets across various research topics. Finally, the guest editors sincerely thank the authors, reviewers, editors, editorial office, and all those who contributed to this special issue. | Review | biomedical | en | 0.999994 |
PMC11699348 | Low back pain (LBP) presents a remarkably prevalent affliction and has been the leading cause of years lived with disability (YLDs) . In 2020, an estimated 619 million people worldwide were affected by low back pain, and the number is estimated to be 843 million (759–933) by 2050 . Various elements including vertebrae, sacroiliac joint, intervertebral disc, neurovascular structure, and soft tissue can alone or jointly result in low back pain . Intervertebral disc degeneration (IVDD), which contributes to spinal canal stenosis, spondylolisthesis, and deformity, has been identified as one of the major causes of LBP . Notably, annual economic costs related to IVDD exceed US$100 billion in the United States (see Table 1 , Table 2 , Table 3 , Table 4 , Table 5 ). Table 1 Applications of chondroitin sulfate-based biomaterial in IVDD regeneration and repair. Table 1 Material Biological factor, agent or cell Preparation method Type of study Function and advantage Ref Hyaluronic acid (HA), tetra poly (ethylene glycol) with maleimide end groups (Teran-PEG-MAL), and chondroitin sulfate (CS) Ex situ solution penetration In vitro, in vivo (rat) Injectable anti-inflammatory hydrogel, with Diels-Alder reaction-mediated sequential crosslinking enhanced mechanical property and pH-responsive release function Chondroitin sulfate, poly (ethylene glycol)- b -poly (trimethylene carbonate)-acrylate (PEG- b -PTMC-Ac) Bovine NPCs Ex situ solution penetration In vitro In situ forming, mechanically resilient hydrogel, with high NPCs viability and ECM production. poly (N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS), alginate microparticles Growth differentiation factor 6 (GDF-6) Solution mix Ex vivo, in vitro In situ forming hydrogel, enhanced initial injectability, bioadhesiveness, and mechanical performance. Decellularised extracellular matrix of bovine coccygeal discs functionalized with chondroitin sulfate Solution mix In vitro Injectable, self-assembled biomimetic hydrogel, boosting sGAG production and preserving rounded cell morphology of nasal chondrocytes Table 2 Applications of hyaluronic acid-based biomaterial in IVDD regeneration and repair. Table 2 Material Biological factor, agent or cell Preparation method Type of study Function and advantage Ref Hyaluronic acid-1,4-butanediol diglycidyl ether (HA-BDDE), HA-poly (N-isopropylacrylamide) (HA-pNIPAM) NP cells In situ penetrating In vitro, ex vivo (bovine IVD) Support high cell viability, facilitate ECM production, and withstand confined cyclic axial compression. Type Ⅰ collagen, tyramine-substituted hyaluronic acid BM-MSCs Solution mix In vitro Promote NP ECM biopolymers, increase hydration degree, and ensure cell adhesion and survival. Fibrin, collagen, and hyaluronic acid Solution mix In vitro Maintain cell viability, and increase the sGAG to collagen ratio. Hyaluronic acid, zirconium oxide Nanoparticles addition into solution In vivo (goat) and ex vivo (rabbit) Real-time visualization and monitoring, support disc height and promote anabolism of IVD Polyethylene glycol, hyaluronic acid, PPS Mesenchymal precursor cells Ex situ penetrating In vitro Injectable, enzymatically-crosslinked hydrogel, enhance MPCs differentiation to NP-like tissue. HA and poly (N-isopropylacrylamide) (pNIPAM) Stromal cell-derived factor 1 (SDF-1) Direct amidation reaction Ex vivo (bovine IVD) Increase both the number of recruited cells and their migration distance within the intervertebral disc (IVD) tissue. Collagen, HA, and gelatin microspheres Transforming growth factorβ3 (TGF-β3) Ex situ penetration In vitro and in vivo (mouse) possess superior viscoelastic properties and injectability, support MSC and NC differentiation HA, butanediol diglycidyl ether In situ crosslinking In vitro and in vivo (rabbit) Injectable, restore the disc hydration and function Aldehyde hyaluronic acid, poly (amidoamine) siRNA Solution mix In vitro and in vivo (rat) Transport siSTING and silence STING signaling pathway in NPC, alleviate IVD inflammation and degeneration Methacrylated hyaluronic acid microspheres circSTC2 silencing gene vectors Microfluidics In vitro and in vivo (rat) Silence circSTC2 expression in NPCs to maintain the ECM metabolism balance in the nutrient-restricted microenvironment and hinder IVDD Oxidized and methacrylated glycosaminoglycan, fibrin, and poly (ethylene glycol) diacrylate (PEGDA) In situ solution penetration In vitro and ex vivo (bovine) Injectable two-part hydrogel with high adhesion strength, low cytotoxicity, and mechanical compliance Thiolated HA and PEG vinylsulfone Ex situ penetration In vitro HA with lower molecular weight is advantageous for maximizing cell viability and sGAG synthesis. Hyaluronic acid, collagen In situ crosslinking Ex vivo (rat) Restore disc hydration and demonstrate superior instantaneous and equilibrium moduli Table 3 Applications of cellulose-based biomaterial in IVDD regeneration and repair. Table 3 Material Biological factor, agent or cell Preparation method Type of study Function and advantage Ref Carboxymethylcellulose Esterification of hydroxyl groups In vitro Injectable, photocrosslinked, and water-soluble hydrogel, with NP cells viable and mechanical integrity Carboxymethylcellulose (CMC) and methylcellulose (MC) Esterification of hydroxyl groups In vitro, ex vivo (bovine caudal IVDs), in vivo (rat) Thermoresponsive, tunable, biocompatible hydrogel with long-term structurally stable Carboxymethylcellulose Human mesenchymal stem cells (hMSCs) Esterification of hydroxyl groups In vitro Photocrosslinked and biocompatible hydrogel, NP ECM elaboration, improved mechanical properties [ , , ] Chitosan carboxymethyl cellulose Adipose-derived stem cells (ASCs) Ex situ solution penetration In vivo (ovine), in vitro Long-term stabilization of injectable composite hydrogel in degenerated IVDs in a large animal model Poly (ethylene glycol) dimethacrylate and nano-fibrillated cellulose Ex situ solution penetration Ex vivo (bovine IVDs) In situ injection and photopolymerization, restore IVD height and maintain after 0.5 million loading cycles Methacrylated gellan-gum reinforced with cellulose nanocrystals Solution mix In vitro Injectable composite hydrogel with improved scaffold stiffness that augments matrix entanglement Cellulose-alginate double network hydrogel-based annulus fibrosus and cellulose hydrogel-based nucleus pulposus Growth differentiation factor-5, SKP peptide (MSC homing peptide) and RGD peptide (cell adhesion peptide) In situ addition In vitro and in vivo (rats) Recruit and induce MSCs differentiation to NP-like phenotype, has high compressibility, shape memory property and comparable mechanical strength Cellulose nanofiber-based AF and type Ⅱ collagen-based NP Rat NPCs and AFCs Bioprinted bacterial cellulose by the fermentation of Acetobacter xylinum on a micropatterned template In vitro and in vivo (rats) Mimic native IVD and manifest distinguished structural and functional performance Table 4 Applications of alginate-based biomaterial in IVDD regeneration and repair. Table 4 Material Biological factor, agent or cell Preparation method Type of study Function and advantage Ref Alginate, collagen-cryogel, hyaluronic acid Froze and lyophilize alginate and collagen, crosslinked with EDC/NHS In vivo (rat) Ameliorate mechanical allodynia, preserve IVD hydration, and maintain the structure integrity of the disc. Alginate In situ crosslink by calcium carbonate and glucono-δ-lactone In vitro, ex vivo (bovine caudal IVDs) Reestablish function by reducing height loss during long-term cyclic loading and remains contained within the disc with no leakage at the injection site Ultra-purified alginate BMSCs, bone marrow aspirate concentrate (BMAC) In situ gelling In vitro, in vivo (rabbits) Hinder IVD degeneration in histological grade and promote the repair of IVD defects. Alginate beads Human NPCs, Wharton's jelly MSC-derived extracellular vesicles Solution mix In vitro Preserve cell viability, promote ECM deposition while inhibiting inflammation. Alginate, poly (ethylene glycol) diacrylate (PEGDA) microcryogels Mesenchymal stem cells (MSCs) Ex vivo solution penetration In vitro, in vivo (dog), ex vivo (canine IVD) Injectable hydrogel with enhanced elasticity, alleviate IVDD in an ex vivo organ culture model and an in vivo canine model. Alginate Syndecan-binding peptides (AG73), integrin-binding peptides (cyclic RGD) Strain Promoted Azide Alkyne Cycloaddition (SPAAC) click chemistry, maleimide-thiol click chemistry, solution mix. In vitro cRGD and AG73 loaded alginate hydrogel, contribute to higher cell viability, adhesion, biosynthetic property, and NPC phenotype expression. Alginate, poly (N-isopropylacrylamide) (PNIPAAm), silicate ceramics NPCs Solution mix, in situ crosslinking In vitro, in vivo (rat) Thermosensitive, immunoregulatory, promote matrix synthesis. Mesoporous bioactive glasses, sodium alginate melatonin Solution mix In vitro, in vivo (rat) Possess favorable physical and mechanical properties, and alleviate IL-1β triggered oxidative stress and inflammation. Ultra-purified alginate Human IVD cells Solution dilution In vitro, in vivo (sheep and rabbit) Reduced cytotoxicity, favorable biomechanical property, and anabolism enhanced phenotype. Ultra-purified alginate Rabbit BMSCs Solution dilution In vitro, in vivo (rabbit) Upregulate ECM and phenotypic marker, augment IVD regeneration. Ultra-purified alginate Rapidly expanding clones of human MSCs Solution mixture In vivo (rat) Suppress inflammatory cytokine production, reduce nociceptive behavior of rats. Sodium alginate, gelatin Antagomir-204–3p Solution mix In vitro, in vivo ( Improve IVD mechanical property, sustain hydration, and preserve structural integrity. Oxidized alginate microbeads, fibrin RGD peptides, AFCs Genipin-crosslinking, ex situ solution penetration In vitro, ex vivo (bovine IVD) Preserve high cell viability, inhibit AFCs apoptosis, reduce herniation risk in bovine caudal IVD organ for a long-term. Alginate IL-1 receptor antagonist (IL-1Ra), soluble tumor necrosis factor receptor-1 (sTNFR1) Cell encapsulation in solution In vitro, ex vivo (human cadaveric IVD) Inhibit IL-1β and TNF expression, promote proteoglycan and collagen production Alginate, poly (ε-caprolactone) microfibers AFCs and NPCs Ex situ fabrication In vitro, in vivo (rat) Enhance ECM deposition and mechanical properties. Table 5 Applications of chitosan-based biomaterial in IVDD regeneration and repair. Table 5 Material Biological factor, agent or cell Preparation method Type of study Function and advantage Ref Chitosan-glycerophophate NP cells Solution mix In vitro Thermosensitive and biocompatible hydrogel that retains proteoglycan of NP cells Chitosan-glycerophosphate Mesenchyme stem cells Solution mix In vitro Temperature-sensitive hydrogel, capable of inducing proteoglycans and collagens production of MSCs Chitosan-glycerophosphate (SHC0.075BGP0.1) NP cells Solution mix In vitro, ex vivo (human cadaveric IVDs) Similar mechanical properties to the human NP tissue, induction of highest GAG N -hexanoyl glycol chitosan N -hexanoylation reaction of glycol chitosan In vitro, in vivo (rat), ex vivo (pig) Thermos-sensitive injectable hydrogel with no cytotoxicity and no adverse effects in a rat model, maintaining the stability of defective porcine IVD for 28 days. Chitosan and poly-(γ-glutamic acid nanoparticles diclofenac Ex situ solution penetration In vitro, in vivo (rat) Ch/Df/γ-PGA NPs inhibits pro-inflammatory mediator production and reverse ECM synthesis. [ , , ] Methacrylate chitosan and aldehyde polyethylene glycol Solution mix, Schiff base reaction In vitro, in vivo (rat) Rapidly in situ forming injectable hydrogel with low cytotoxicity and enhanced mechanical strength. Chitosan, gelatine Link N Solution mix In vitro Improved mechanical properties in compression and pro-anabolism function Chitosan, poly (l-lactic acid) Human umbilical cord mesenchymal stem cells (hUCMSCs) Solution mix In vivo (rabbit) Beneficial for cell adhesion and proliferation, enhance bone connection between adjacent vertebrae and promote AF tissue regeneration Chitosan with dissolved perfluorotributylamine (core), polycaprolactone (shell) Annulus fibrosus stem cells (AFSCs) Coaxial electrospinning, solution mix In vitro, in vivo Persistent oxygen release, promoting proliferation, migration and ECM production of AFSCs. Chitosan, poly (butylene succinate-co-terephthalate) (PBST), poly (ether ether ketone) (PEEK) IVD cells In vitro fabrication In vitro, in vivo (pig) Support IVD cell growth, possess a good compressive stress and elastic moduli. Chitosan, poly (butylene succinate-co-terephthalate) copolyester AF and NP cells In vitro fabrication In vitro, in vivo (rabbit) Maintain the biological function of IVD cells and retain the height of intervertebral IVDD is a complex and multifactorial process that involves biochemical, structural, and mechanical changes in the disc tissue. The pathological process includes (a) progressive cell senescence and cell death , (b) cell dysfunction and declined anabolic production of extracellular matrix (ECM) , (c) inflammatory cells and cytokines, and increased catabolic activity . The loss of hydration and structural integrity accelerates the biomechanical property to fail and results in pain and disability. Furthermore, there exist complex interactions between the degenerated disc, adjacent vertebral endplates, and the paraspinal muscles. Changes in the endplates can alter nutrient transport to the disc, exacerbating degeneration, while the paraspinal musculature may undergo compensatory changes, further influencing spinal biomechanics and pain perception . Understanding these interconnected factors is critical for developing holistic therapeutic strategies. Although progress in understanding the molecular basis of IVDD has been made, the current treatments for IVDD, either conservative or operative options, are limited. Non-surgical treatments including immobilization, analgesic drugs, and other physical therapy, mainly aim to alleviate the pain . In severe cases, invasive surgical treatments like interbody fusion or arthroplasty are necessary to provide mechanical structural support and stability . Although pain control can be beneficial for patients, the long-term prognosis of anti-inflammatory drug use is not optimal. Additionally, the risk of adjacent segment degeneration (ASD) exists with invasive surgical procedures . Essentially, conservative treatments or surgical options can not address the underlying etiology or facilitate the regeneration of the degenerated disc tissue. In comparison, polysaccharide-based biomaterials possess the potential to alleviate the inflammatory microenvironment , halt cell senescence and apoptosis , improve biomechanical instability , and prevent IVD from degeneration. This is beneficial for addressing IVDD from the etiological source. Since traditional treatments could not reverse degenerative progress, restore tissue homeostasis, and reconstruct the mechanical stability of the spine. New strategies aimed to counteract intervertebral disc degeneration are urgent. Many attempts have been made in the following fields. (a) anti-inflammatory strategies to regulate the inflammatory microenvironment ; (b) vascularization and innervation growth inhibition ; (c) anti-catabolic strategies ; (d) pro-anabolic strategies ; (e) endogenous and exogenous cell supplementation . The main purposes of ingenious therapeutics are to alleviate inflammation in IVD which contributes to discogenic pain and degenerative symptoms and to remodel anabolic/catabolic balance in the nucleus pulposus (NP). Among these regenerative approaches, emerging materials such as synthetic hydrogels, decellularized tissue matrices, peptide-based scaffolds, and stimuli-responsive smart materials have shown promise in supporting disc cell viability, mimicking the disc's extracellular matrix, and enhancing mechanical resilience [ , , , ]. Advanced cellular approaches, including stem cell therapies, gene editing, and endogenous or exogenous biological factor recruitment are also under investigation to directly repair or regenerate damaged tissue [ 8 , , , ]. Against this backdrop, polysaccharide-based biomaterials stand out due to their biocompatibility, tunable mechanical properties, and ability to interact with native disc cells and matrix components. In situ IVD injection has become the paradigm for drug delivery in that systematical administration can barely reach the disc due to avascularity and immune privilege. However, puncture itself is an invasive procedure that may impair annulus fibrosus (AF) and therefore facilitate IVDD and the simply-delivered drugs are susceptible to transient clearance, limited absorption by nucleus pulposus cells (NPCs), and off-target side effects . In such a situation, biomaterial delivery systems based on tissue engineering hold promise in IVD repair and regeneration. The bioinspired delivery platform can not only bear the mechanical pressure of the IVD but also improve drug efficacy due to their excellent biocompatibility, biodegradability, accurate tissue targeting, and sustained release , of which polysaccharide-based materials are ideal candidates . The intervertebral disc, a fibrocartilaginous joint between adjacent vertebrae, consists of three main components: the gelatinous NP as the core, the annulus fibrosus surrounding the periphery, and the cartilage endplates (CEP) sandwiching aforesaid two structures . The NP contains two types of cells, NPCs and notochordal cells, the latter of which only exist in the embryonic and fetal stages. Its extracellular matrix (ECM) is defined by a network of irregularly organized type Ⅱ collagen embedded in amorphous proteoglycans . The AF is made up of 15–25 concentric layers of type I collagen fibers, with inner and outer regions, primarily responsible for resisting tensile forces . The upper and lower CEPs, through which the main source of nutrition is diffused and metabolites are excreted, interface the vertebral body and the disc . Fig. 1 (A) A schematic view of the human IVD (Adapted from Ref. ). (B and C) Representative H&E staining and representative T2-weighted MRI scans of disc samples from normal, moderately degenerated and severely degenerated groups. Fig. 1 Proteoglycans are proteins with glycosaminoglycan chains covalently attached. The glycosaminoglycan chains endow aggrecan osmotic properties to swell and withstand compressive pressure, to which the remarkable chondroitin sulfate (CS) and keratin sulfate (KS) chains are ascribed . Of note, as a key constituent of ECM (65 % in NP and 20 % in AF), aggrecan is essential in endowing IVD mechanical property to resist axial compression in that negatively charged glycosaminoglycans strongly attract water molecules and bind growth factors and cell adhesion molecules . Recent advances have demonstrated that aggrecan is critical for the preservation of appropriate stiffening of the ECM by providing biomechanical cues during IVD development . In addition, aggrecan restrains endothelial cell adhesion and cell migration, therefore helping maintain the avascular condition as well as inhibiting nerve growth . In particular, chondroitin sulfate (CS), as one kind of glycosaminoglycan, contributes to hydration, cell survival, and the non-innervated state of IVD . With growth and development, glycosaminoglycan chains undergo several changes including that the CS shortens while the KS elongates, accompanied by sulfation site transferring from the 4-carbon to the 6-carbon of N-acetyl galactosamine . IVDD defines the crucial pathological basis of degenerative disc disease (DDD), triggered by multiple factors including genetics, environmental factors, and mechanical factors . Cytokines, for example, IL-1β and TNF-α, aggravate the degenerative process and promote cellular senescence, autophagy, and apoptosis. The inflammatory cascade mediates catabolism through upregulating extracellular matrix enzymes such as MMP3 and ADAMTS production and restrains anabolism by inhibiting proteoglycan expression [ , , ]. The reduction of proteoglycans is the most prominent biochemical variation and is recognized as a hallmark of IVDD . With proteoglycan and type-Ⅱ collagen collapsing, the strength-bearing capacity of IVD and biomechanical integrity was impaired, leading to AF tear and subsequent NP exposure to the circulatory system and immune system . In the meantime, evoked inflammatory cytokines, IL-6, prostaglandin E2 (PGE2), and pro-innervating factors nerve growth factor (NGF) stimulate nerve ingrowth, leading to symptomatic pain . Upregulated vascular endothelium growth factor (VEGF) and chemokines, CCL1, 3 and 13, CXCL10 by NPCs provoke vascularization and immune cells infiltration, which accumulates the inflammatory cytokines including IL-1β and TNF-α, aggravating catabolism and ECM loss . In addition, CEP calcification evolves with aging, breaking the supply/demand balance. The nutrient diffused inclines while demand rises due to increased infiltrated immune cells, which accelerates the disorder of energy metabolism . This imbalance restrains the nutrients for NPCs and annulus fibrosus cells (AFCs) and further affects cell viability. Furthermore, a deteriorating extracellular environment characterized by significant acidification with metabolite cumulating induces further cell senescence and apoptosis and aggravates degradative enzyme production . Fig. 2 Schematic representation of biomechanical causes, biological factors and microenvironment changes in the process of IVDD. Multifactorial changes are involved in IVDD, including cellular changes, anabolism/catabolism imbalance, inflammatory response, mitochondrial dysfunction, calcification and endplate changes, and altered biomechanics. Loss of notochordal cell marks the beginning of the degeneration process, followed by nucleus pulposus cell decrease. Reduced matrix production and aggravated matrix degradation define phenotypic changes, leading to disc dehydration and loss of height. Pro-inflammatory cytokines upregulation and immune cell infiltration exacerbate disc microenvironment. Neovascularization and nerve infiltration are associated with discogenic pain. Mitochondrial dysfunction including accumulated ROS, impaired mtRNA, and altered mitochondrial dynamics contributes to and perpetuates disc degeneration. Endplate calcification and subchondral sclerosis limit nutrient supply to disc. IL-1β: Interleukin 1β, IL-6 and IL-17: Interleukin 6 and 17, TNF-α: Tumor necrosis factor α, PGE2: Prostaglandin E2, MMPs: Metalloproteinases, TIMPs: Tissue inhibitors of metalloproteinases, BMP: Bone morphogenetic protein, TGF-β: Transforming growth factor β, NGF: Nerve growth factor, ADAMTS: A disintegrin and metalloproteinase with thrombospondin motifs, VEGF: Vascular endothelium growth factor, ROS: Reactive oxygen species, mtRNA: mitochondrial RNA. Fig. 2 During IVDD, proteoglycan in NP undergoes a significant decrease and leads to the ratio of NP/AF proteoglycan dropping, which indicates the importance of high proteoglycan concentration in healthy IVD . Hyaluronic acid, as another major component of proteoglycans with aggrecan monomers linked, imbibes water and subsequently renders IVD mechanical strength. In addition, hyaluronan demonstrates an anti-inflammatory property by inhibiting tetratricopeptide repeats 3 (IFIT3) and insulin-like growth factor binding protein-3 (IGFBP3) and a matrix remodeling action by improving anabolism of extracellular matrix and reducing ADAMTS4 . Although the content of aggrecan declines in NP during aging, there is a compensating rise of aggrecan concentration in the inner AF . Furthermore, as MMPs and aggrecanases cleave aggrecan, whether fragmented aggrecan is harmful to disc needs to be elucidated. Mitochondrial dysfunction is associated with the development of IVDD through affecting a series of changes including mitochondrial fusion, autophagy, and cell apoptosis . Interestingly, hyaluronic acid can preserve mitochondrial function via activating mitophagy in NPCs, thus attenuating IVDD . Conversely, free hydroxyl radicals from hydrogen peroxide and superoxide are detrimental for HA to depolymerization . Polysaccharides are made up of monosaccharides and connected by O-glycosidic bonds. Widespread in nature, a variety of sources of polysaccharides includes algae (agarose), plants (starch, pectin), microorganisms (xanthan gum, gellan gum) and animals (chondroitin, heparin, hyaluronan) . With regard to their chemical structure, the various functional groups of different polysaccharides lay the foundation for physicochemical modifiability, so as to endow more characteristics and functionalities . In addition to the similarities between polysaccharides and the extracellular matrix (ECM), polysaccharides exert essential biological roles in molecular recognition, cell adhesion, and signal transduction . Therefore, considering their impressive merits including biocompatibility, biodegradability, hydrophilicity, nontoxicity, bioactivity, and water retention capacity, polysaccharides have garnered significant attention for diverse biomedical applications including drug delivery, tissue repair, as well as targeted therapy . Several common forms of polysaccharide-based biomaterials include hydrogels (e.g. hyaluronic acid, chondroitin sulfate, and alginate), scaffolds (e.g. chitosan and cellulose), nanoparticles (e.g. chitosan, hyaluronic acid, and alginate), microspheres (e.g. alginate and chitosan), nanofibers (chitosan, collagen, and silk fibroin), and nanocrystals (e.g. cellulose). These forms of polysaccharide-based materials provide a versatile toolkit for addressing different aspects of IVDD. As a polymeric carbohydrate, a polysaccharide is composed of more than 10 monosaccharides, among which the same monosaccharides constitute homopolysaccharides while different monosaccharides constitute heteropolysaccharides. According to the glycosidic linkage type and electrical charges, the polysaccharides can be classified into straight or branched chains and polyanion, electroneutral, or polycation polysaccharides respectively . Polysaccharides that derive from natural sources have their own drawbacks including microorganism pollution, batch variation, different hydroxide content, and unstable mechanical strength . However, the chemical modifications minimize these limitations and impart versatile functionalization to polysaccharides. There are diverse approaches including carboxymethylation, oxidation, sulfation, and polymer graft to achieve this transformation. Hydrated polysaccharides in an aqueous environment define the process of production of the hydrogel. As such, one of the most extensive usages of polysaccharides in tissue engineering and biomedical applications is hydrogel establishment. The mechanism whereby polysaccharides form network structure is mainly divided into physical crosslinking that is defined by ionic interaction, of which alginate represents the paradigm and chemical crosslinking that includes radical polymerization (e.g. dextran), aldehyde (e.g. amine contained polysaccharides), addition reaction, and condensation reaction. Mammalian polysaccharides including hyaluronic acid, keratan sulfate, and chondroitin sulfate naturally exist in vivo and constitute the ECM of the intervertebral disc. Glycosaminoglycan chains covalently attach to core protein and compose proteoglycans, contributing to structure integrity and microenvironmental homeostasis of intervertebral discs. In the following section, the fabrication and application of polysaccharide-based materials and biological properties in intervertebral disc repair and regeneration will be discussed. Widely distributed in vertebrates, invertebrates, and bacteria, chondroitin sulfate is characterized as a linear polysaccharide composed of repeated disaccharides of N-acetyl galactosamine and glucuronic acid. In regard to different sulfated residues at carbon positions, CS is classified into CS-A (chondroitin-4-sulfate), CS-C (chondroitin-6-sulfate), CS-D (chondroitin-2,6-sulfate) and CS-E (chondroitin-4,6-sulfate) . There has been reports that CS significantly inhibited NF-κB activation and nuclear translocation, along with the production of inflammation cytokines and inducible nitric oxide synthase . As a prominent constituent of the ECM, apart from anti-inflammatory, anti-oxidant, anti-coagulating, and immunomodulatory functions, CS serves as a signaling molecule that can influence cell signal transmission and promotes the formation of collagen and proteoglycans . Moreover, CS has been reported to promote ECM production via the integrin-WNT5A pathway and notochordal cells-derived CS has been observed to inhibit hyperinnervation and peripheral nociception . CS is now extensively utilized in biomedical applications because of its renowned compatibility with biological systems, biodegradability, and hydrophilicity . Based on the anti-inflammatory properties of CS, Luo et al. engineered a versatile hydrogel system (HA/CS). This hydrogel is injectable and remarkably self-crosslinked post-injection through boronate ester bonds. Further enhancement of mechanical strength through sequential coupling through the Diels-Alder reaction to simulate the human NP. CS can degrade under physiological stimuli such as hyaluronidase enzyme, reactive oxygen species, and pH change, thus enabling site-specific release. As such, this hydrogel system continuously protects NPCs from apoptosis and preserves extracellular matrix synthesis. In the rat model, the HA/CS hydrogel inhibits inflammatory cascade and remarkably alleviates IVDD . Fig. 3 Chondroitin sulfate-based biomaterials for intervertebral disc regeneration and repair. (A) Injectable self-antioxidant hydrogels grafted with inflammation inhibitory chondroitin sulfate formed via dynamic boronate ester bonding between furan/phenylboronic acid and furan/dopamine-modified hyaluronic acid and mechanically enhanced by Diels-Alder reaction-induced secondary crosslinking, and (i) CLSM images of double-crosslinked HA/CS0 and HA/CS2.0 hydrogels (Adapted from Ref. ). (B) Injectable Disc-Derived ECM Hydrogel functionalized with chondroitin sulfate via amine groups of collagen and (ii) Assessment of the structural stability of cell free ECM-derived hydrogels and SEM images (Adapted from Ref. ). Fig. 3 A prospective treatment attempt involves resecting the compromised NP tissue and restoring it with NPCs encapsulated in situ forming hydrogel. Riahinezhad et al. designed such a hydrogel using thiolated chondroitin sulfate crosslinked 4-arm [poly (ethylene glycol)-b-poly (trimethylene carbonate)-acrylate] via Michael type reaction. This hydrogel delivers NPCs with high viability and proliferating ability. These cells exhibit collagen type Ⅱ deposition instead of collagen type Ⅰ. The instantaneous moduli of this hydrogel are adjusted according to the native NP and resistant to cyclic compressive loading . An innovative graft copolymer, poly (N-isopropyl acrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS) combines the thermoresponsive behavior of the latter with the biocompatibility and bioactivity of CS to form a moisture-absorbing and fluid substance below the lower critical solution temperature and precipitated hydrogel above it . However, this combination has inadequate solution viscosity and adhesiveness. Christiani et al. embedded alginate microparticles into PNIPAAm-g-CS to enhance surface roughness and toughness. The structure provides an optimized platform for sustaining, multiplying, and differentiating adipose-derived stem cells (ADSCs) into native NP-mimicking phenotype due to incorporated GDF-6, a potent chondrogenic factor. The ex vivo implantation of this formation into degenerated porvine IVDs demonstrated promising results in restoring the compressive and neutral zone stiffness to levels seen in undamaged discs . Chondroitin sulfate and type Ⅱ collagen constitute the major ECM of NP. Borrelli et al. designed a self-assembled hydrogel of decellularized ECM functionalized with chondroitin sulfate. Of note, the inclusion facilitates a rounded morphology and higher sGAG synthesis of nasal chondrocytes which have been recognized as a promising cell source for NP regeneration with their robust matrix synthesis capacity . Basically, the effect of chondroitin sulfate on the chondrogenic phenotype of mesenchymal stem cells has been reported to be associated with Smad1/5/8 signaling inhibition and p38 signaling upregulation . Hyaluronic acid (HA) is a nonsulfated, linear polyanion mucopolysaccharide and the only glycosaminoglycan that is ubiquitous in almost all mammalian animals, with additional production capabilities in certain bacteria . This material is widely present in the extracellular matrix. It plays a vital part in diverse biological processes, including cell development, migration, as well as influencing the physiological activities of the human body . Specifically, CD44, the best-characterized HA receptor, mediates cell migration of MSCs through interaction with extracellular HA, and its expression on activated lymphocytes aids in the primary rolling to the inflammatory sites in a HA-dependent manner . Hyaluronic acid is a versatile and extensively used material in tissue engineering because of its biocompatibility, biodegradability, nonimmunogenicity, and viscoelastic properties . Its applications span from scaffold fabrication and drug delivery platforms to wound care, ophthalmology, and orthopedics. The extensive study and successful utilization of HA and its derivatives underscore their considerable potential in advancing regenerative therapies and improving patient outcomes in various medical fields. One ideal biomaterial as a novel treatment for IVD regeneration is able to support the expansion, differentiation, and function of encapsulated cells while providing sufficient mechanical stability. It has been shown that HA grafted with poly (N-isopropyl acrylamide) supports high cell viability and maintains differentiation potential . HA crosslinked with 1,4-butanediol diglycidyl ether (BDDE) has superior comprehensive mechanical testing performance . Therefore, Guo et al. combine HA-BDDE and HA-pNIPAM to form an interpenetrating polymer network as a cell carrier. The IPN hydrogel demonstrates rheological properties similar to native NP tissue. This network promotes GAG production and new matrix deposition of loaded NPCs after implanted ex vivo in organ cultured IVD . Using hyaluronic acid and type Ⅰ collagen to mimic the native nucleus pulposus tissue, encapsulated human BMSCs exhibit high gene expression of NP marker under a differentiation medium containing GDF5 and TGFβ1. Of note, cells tend to have a rounded morphology to NPCs . Similarly, Gansau et al. incorporated collagen and hyaluronic acid into fibrin-based hydrogel. Cell viability and proliferation are enhanced due to the addition of ECM components. Furthermore, incorporating HA elevates sGAG accumulation and inhibits collagen expression . Granular hydrogels have become favorable biomaterials with injectability and microscale porosity in tissue engineering. Muir et al. designed a hyaluronic acid granular hydrogel while encapsulating radiopaque zirconium oxide nano-powder. This radiopaque hydrogel can be monitored over time to track its location, degradation, and interaction with surrounding tissues, providing visualization of the placement of the hydrogel during injection. The large animal goat model of IVDD confirms that the granular hydrogel impedes disc height loss, increases proteoglycan production, and decreases collagen deposition . Simple biomaterials or scaffolds may lack the ability to enhance cellular functions and direct stem cells to specific lineages. Therefore, biological factor-loaded biomaterials harness the power of active molecules to improve the efficacy of medical treatments. Pentosan polysulfate (PPS), a semi-synthetic GAG-like polysaccharide, has been proven to boost viability and induce MSCs differentiating to chondrocytes, and induce NP-like ECM production . Frith et al. incorporated soluble PPS into polyethylene glycol and hyaluronic acid hydrogel system and encapsulated MPCs. The composites form NP-like tissue, particularly type Ⅱ collagen deposition. This hydrogel is immunologically tolerant using a rat subcutaneous implantation model . Afterward, the research group determined that a covalently bound PPS possesses a strong potential to mediate IVD regeneration . Pereira et al. attempted to add stromal cell-derived fator-1 (SDF-1) to an HA-poly (N-isopropylacrylamide) hydrogel to attract Hu-MSCs. The composite enhances the migration of MSCs into compromised discs in comparison to HAP or SDF-1 alone . Tsaryk et al. combined TGF-β3 delivery by gelatin microspheres with collagen-low molecular weight hyaluronic acid hydrogel. Of note, the material promotes nasal chondrocytes and MSCs to expand and differentiate without triggering inflammation or inducing cytotoxicity . Hyaluronic acid scaffold seeded with human Wharton's Jelly-derived MSC (WJ-MSC) expressing high levels of TGF-β receptor Ⅰ and TGF-β receptor Ⅱ significantly restore the disc hydration in a rabbit model compared to scaffold along or WJ-MSC-lowTR-seeded HA scaffold. The morphological and histological analyses are consistent with T2-weighted MRI analysis . Targeting RNA molecules is a promising therapeutic strategy for IVDD. This approach focuses on modulating the expression and function of various RNAs including long noncoding RNAs (lncRNAs), circular RNAs (cirRNAs), and micro RNAs (miRNAs) to influence cellular processes including apoptosis, proliferation, and extracellular matrix remodeling in NPCs . Recent studies demonstrate that stimulators of interferon genes (STING) are critical in diverse inflammatory and degenerative diseases. Chen et al. revealed that the STING signaling pathway is an intervention target for IVDD. A siSTING delivery hydrogel of aldehyde HA and poly (amidoamine)/siRNA complex is fabricated and the hydrogel system alleviates intervertebral inflammation and degeneration in the IVDD rat model . Likewise, circSTC2 is elevated in degenerative NP tissue, and the silence of circSTC2 can restore the extracellular matrix anabolism and catabolism balance. Chang et al. grated circSTC2 silencing genes-loaded 1,2-dioleoyl-3-trimethylammonium-propane/cholesterol/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine cationic liposomes on methacrylated hyaluronic acid microspheres. With the excellent merits including degradability, dispersibility, swellability, and injectability of HAMA microspheres, lipoplexes are persistently released over 27 days. This psh-circSTC2-lipo@MS enhances ECM anabolism and impedes catabolism in NPCs in vitro as well as in an in vivo rat IVDD model . Fig. 4 Hyaluronic acid-based biomaterials for intervertebral disc regeneration and repair. (A) Injectable self-healing hydrogel with continuous siRNA delivery property and (i) Representative H&E and Safranin O and Fast Green staining images demonstrate siSTING@HP gel slowing the IVD degeneration progress (Adapted from Ref. ). (B) A two-part biomaterial adhesive strategy involving dual-modified glycosaminoglycan and interpenetrating network hydrogel for annulus fibrosus repair and (ii) Cross-sectional view of HAMA Aldehyde treated AF and phase-contrast images of HAMA Aldehyde treated AF cells. (iii) Picrosirius red/alcian blue staining (Adapted from Ref. ). Fig. 4 HA can be combined with different biomaterials to ameliorate pain and inflammation due to its anti-inflammatory properties. Fragmentation of ECM accompanies IVDD. However, research indicates that hyaluronic acid fragments induce inflammatory and catabolic mediators such as interleukin-1β, IL-6, cyclooxygenase-2, MMP-1, and MMP-13. Through siRNA intervention or antibody neutralization, it is suggested that hyaluronic acid fragments result in IVDD via the Toll-like receptor 2 signaling pathway . Currently, there are some challenges and limitations associated with using hydrogel systems for sealing annulus fibrosus defects. For example, many hydrogels exhibit poor adhesion to wet tissue and insufficient mechanical strength that may lead to potential dislodgement and failure to seal the defect effectively. Tyler et al. designed a combined regeneration system by integrating oxidized and methacrylated glycosaminoglycan and fibronectin-linked fibrin and poly (ethylene glycol) diacrylate. The former absorbs the latter interpenetrating network and binds this hydrogel to ECM in the IVD covalently. The results show that the dual-modified hydrogel presents excellent adhesive strength without any observable cytotoxic effects and demonstrates mechanical compliance that aligns with the herniation risk of the current clinical standard . Interestingly, HA itself demonstrates an anti-inflammatory action in AF-defected and IFNα 2 β stimulated discs by downregulating IFIT3, ADATS4, and pro-apoptotic IGFBP3. Moreover, HA intervention increases collagen Ⅰ and aggrecan. Therefore, HA exhibits an anti-inflammatory property and matrix modulatory effect in annulus fibrosus . Hyaluronic acid (HA)-poly (ethylene glycol) (PEG) composite hydrogels are a prominent area of research due to their unique properties. Jeong et al. used artificial neural network analysis to screen HA-PEG formulation to match NP and AF cells. It turns out that lower-molecular weight HA leads to the highest NPC and AFC numbers and higher sGAG production . Based on the concept of IVD as integrity, Sloan et al. attempted to repair AF defect with riboflavin cross-linked high-density collagen gel and repair NP with modified HA hydrogel. Qualitative T2 MRI shows that combined therapies restore disc water content and preserve intact IVD morphology. Particularly, HA injection into NP recovers 35 % and 40 % of the effective instantaneous and equilibrium moduli . Similarly, Mohd et al. confirmed that HA hydrogel attenuated IL-6 and IL-1β in degenerated IVD validated by upstream regulations of c-Fos, phosphorylated p38 MAPK, and phosphorylated NF-κB in both AF and NP and regulated ECM deposition, such as higher CS, collagen Ⅱ, and fibronectin, via Smad3-TGF-β1 signaling . It has been implied that CS increases ECM production and correlates with peripheral sensory innervation and nociceptor. However, whether there exists direct action of chondroitin needs to be fully elucidated. Furthermore, HA hydrogel implantation inhibited injury-induced peripheral sensory hyperinnervation and pro-nociceptive TRPV1 and Trk-A expression in AF and NP tissues . These results suggest that HA hydrogel has a potential therapeutic application and could be translated clinically in patients with discogenic back pain due to IVDD. Cellulose is a linear polysaccharide in which D-glucose units are connected by β-(1,4) bonds . Due to its superior mechanical properties and biocompatibility, nanoscale cellulose has garnered significant attention in recent years . Cellulose nanomaterials can be classified into two types: cellulose nanostructure nanomaterials including cellulose microcrystals and microfibrils, and cellulose nano-objects including cellulose nanocrystals and cellulose nanofibrils . In comparison to the former nanocelluloses, cellulose nanocrystals and cellulose nanofibrils exhibit elevated specific surface areas and a more uniform distribution of particle sizes, therefore promoting the formation of mechanically robust and spontaneously organized structures such as hydrogel . Cellulose-based scaffolds have remarkable merits such as non-immunogenicity, nontoxicity, strong biomechanical properties, as well as injectability and in situ gelling that, therefore, hold a potential for NP replacement . Anna et al. first encapsulated NPCs in photocrosslinked carboxymethylcellulose (CMC) in which CMC was functionalized with methacrylate groups and the latter endow water solubility. Under normal pH conditions, the deprotonation of the carboxylic acid within the carboxymethyl groups leads to the negative charge of hydrogel that resembles the negatively charged glycosaminoglycans of ECM of NP. In addition, this polymer demonstrates support for NP cell viability with the occurrence of spherical cells and the composition of chondroitin sulfate proteoglycan . Varma et al. developed a dual-polymer network hydrogel composed of methacrylated carboxymethylcellulose and methylcellulose (MC). The thermogelling MC facilitates the gelation of CMC in situ consistently within intervertebral discs. After injection into IVDs following discectomy, the CMC-MC composite is able to recover IVD height and biomechanical properties . In a follow-up study to examine the sustaining stability of the structure, the CMC-MC hydrogel showed restoration of height and manifested similar herniation risk and fatigue resistance to those with standard nucleotomy. Additionally, the hydrogel injection only results in a limited foreign body reaction, evidenced by analyzing fibrous capsule formation and macrophage infiltration during a 12-week period . Similarly, Gupta et al. introduced human MSCs encapsulated carboxymethylcellulose hydrogel stimulated with transforming growth factor-beta 3 (TGF-β3). This hydrogel system confirmed significant glycosaminoglycan and type Ⅱ collagen deposition that fit in with the ECM of NP . Furthermore, the research group dug into the impact of the duration of TGF-β3 exposure on matrix metabolism and the best macromer concentration of CMC hydrogel. The results showed that a lower macromer concentration (1.5 % weight/volume) demonstrates stronger NP-like ECM elaboration and improved mechanical functionality and short-term TGF-β3 exposure augments sufficient ECM accumulation . Prior investigations have mainly focused on small animal models and short-term outcomes spanning just for a few weeks. Extensive long-term studies in relevant large animal models are imperative for translating into clinical application. An injectable chitosan CMC hydrogel system supplemented with adipose-derived stem cells (ASCs) was evaluated in an ovine model for 12 months. Of note, this scaffold proved a long-term stabilization that alleviated IVDD and sustained intervertebral height in comparison to injured and untreated IVDs . Incorporation of cellulose nano-objects with synthetic polymer (e.g. poly (ethylene glycol) (PEG)) matrix can produce hydrogels that have improved mechanical stability. Schmocker et al. developed a photopolymerized PEG dimethacrylate (MA) cellulose nanofibrils (CNFs) composite hydrogel. The material was finely tailored to fit the elastic modulus and water content of native NP. By utilizing a newly designed device to realize in situ injection and photopolymerization, the composite hydrogel is capable of rebuilding disc height within an ex vivo bovine organ model even after 0.5 million loading cycles and histological analyses indicate a superb tissue integration of this hydrogel implant . Celluloses are usually used as reinforcement by incorporating cellulose nanofibrils and nanocrystals into synthetic or natural polymers. Pereira et al. fabricated an injectable nanocellulose reinforced methacrylated gellan-gum (GGMA) composite hydrogel for AF substituting. The hydrogel system manifests compressive modulus consistent with native AF tissue and improved matrix entanglement with augmented scaffold stiffness. In vitro experiments indicated that nanocomposite hydrogel enhanced cell survival of encapsulated bovine AFCs and maintained AFCs physiological morphology . Individual NP and AF regeneration strategies have been broadly developed to alleviate IVDD. However, the integrity of IVD implies that individual therapy is insufficient and emphasizes the importance of combined AF and NP therapies. Hydrogels based on a cellulose-alginate dual network for the AF and a cellulose hydrogel for the NP are designed to replace IVD. This scaffold increased endogenous MSC homing and adhesion due to functionalized SKP peptide and RGD (Arg-Gly-Asp) peptide and induced MSCs differentiation to chondrocyte and NP-like phenotype with the delivery of growth differentiation factor 5 (GDF-5). Moreover, many other advantageous merits include enhanced compressibility, shape retention capability and comparable biomechanical strength. Of particular, the rat caudal IVDD model demonstrated that the implant impeded the degeneration progress and significantly reestablished both AF and NP . Similarly, Yang et al. created a more complex IVD implant that consists of type Ⅱ collagen-based NP and bioprinted bacterial cellulose-based AF. The former involves rat NPCs while the multilamellar bacterial cellulose layers with micropattern in ± 30° alternative direction contain AFCs. Animal studies confirmed that this tissue-engineered IVD can integrate with the adjacent vertebral and withstand tensile stresses for as long as 3 months . Fig. 5 Cellulose-based biomaterials for intervertebral disc regeneration and repair. (A) Peptide-functionalized (RGD and SKP peptides) cellulose/alginate double network hydrogel and (i) T2‐weighted MRI imaging of the caudal spine (marked with red boxes) (Adapted from Ref. ). (B) Reverse reconstruction and bioprinting of bacterial cellulose-based functional IVD consist of micropatterned bacterial cellulose aligned in ± 30° direction as AF and gelated type Ⅱ collagen as NP and (ii) a-d) Phase contrast microscopy imaging of microgrooves. e-h) SEM image for the BC membrane. n-q) Alexa Fluor 488 phalloidin/Hoechst staining. iii) a,b) Designed pattern of the AF template in opposing orientations ( ± 30°) within adjacent lamellae. c,d) SEM transection images for AF. e) Live/dead staining of APCs (Adapted from Ref. ). Fig. 5 Alginate, a naturally occurring polysaccharide extracted from brown seaweed and certain bacteria, features linear chains of repeating units of β-D-mannuronic acid and α-L-guluronic acid, which are connected together by 1,4-glycosidic bonds . Previous studies have shown that Alginate can reduce ROS levels in cells by chelating metal ions. Moreover, phosphorylation of IκB-p65 complex is downregulated by inhibiting nuclear translocation of P65. It may also reduce phosphorylation of P-Erk1/2 and P-JNK in a concentration-dependent manner . Its unique characteristics, determined by the ratio of mannuronic acid to guluronic acid, allow it to form hydrogels that can be tailored for specific uses, including cell encapsulation, delivering drugs, wound repair, and scaffold fabrication . The ability to customize its mechanical and biological properties makes alginate an essential material for advancing regenerative medicine and other therapeutic applications. Due to its excellent biocompatible property, alginate hydrogel is one of the most extensively employed biomaterials and has been shown to maintain a juvenile rounded morphology . Different gelling crosslinker concentrations may lead to distinct mechanical strength and degradability. Kalaf et al. determined that a ratio of 60 mM calcium carbonate to 120 mM glucono-δ-lactone is identified as having the most optimal properties compared to 2 % alginate crosslinked with calcium chloride. Injecting in situ gelling 2 % alginate into an enzyme-induced comprised IVD reestablishes functionality by reducing height reduction during prolonged cyclic loading and remains contained within the disc with no leakage at the injection area . Simple alginate hydrogel may present insufficient to rebuild compromised IVD. BMSCs or bone marrow aspirate concentrate (BMAC) loaded ultra-purified alginate (UPAL) gels have been delineated as effective in the repair of IVD defects . Similarly, Tilotta et al. trapped human NPCs in alginate spheres and then stimulated them with Wharton's Jelly MSC-derived extracellular vesicles. The intervention leads to cell content increase and cell death decrease. Nitrites, increasing when NPCs face oxidative stress, drop drastically after extracellular vesicle treatment. Moreover, proteoglycan content, indicated by Alcian blue staining, is upregulated. Therefore, extracellular vesicles ameliorate IVD degeneration and inflammation while enhancing ECM production . Traditional simple alginate hydrogels lack mechanical strength when implanted into animal models. Zeng et al. seeded MSCs in alginate and reinforced with macroporous poly (ethylene glycol) diacrylate (PEGDA) microcryogels (PM). The system creates 3D microscale niches and shows improved elasticity. Reinforced alginate hydrogel impedes cell leakage and enhances cell survival. In a canine animal model, PM-alginate hydrogel rescues IVDD after 6 months compared to other treatments . Preserving the juvenile phenotype of NPCs is crucial for IVD regeneration. Syndecan binding peptide (AG73) has been shown to promote NPCs adhesion and the RGD peptide motif has been shown to modulate the mechanotransduction in NPCs . Tan et al. designed alginate hydrogels combined with AG73 and cyclic RGD. The results show that peptides hydrogel demonstrate high cell viability, ECM synthetic property, and induce NPC phenotype, like N-Cadherin . With the aim of regulating local inflammatory microenvironment, Jiang et al. developed a hydrogel composite composed of sodium alginate, silicate ceramics, and poly (N-isopropylacrylamide). Poly (N-isopropylacrylamide) crosslinks in response to body temperature and forms an interpenetrating network with sodium alginate. Silicate ceramics (SC) release calcium ion that promotes alginate sodium crosslink. Magnesium ion from SC contributes to M2 macrophage polarization and regulates the immune microenvironment in a controlled manner, hence enhancing cell matrix production in which collagen Ⅱ and aggrecan are elevated . Similarly, Wu et al. add melatonin, recently shown to possess antioxidant and anti-inflammatory action, to mesoporous bioactive glasses (MBG) and sodium alginate (SA) composite hydrogel. MBG provides sufficient mechanical strength and acts as a melatonin reservoir to facilitate continuous release. The melatonin-MBG-SA hydrogel ameliorates IVD inflammation including TNF, ADAMTS5, MMP-3, and MMP-13 triggered by IL-1β by interacting with cell membrane melatonin receptors (MT1 and MT2). Additionally, melatonin enhances cell viability and facilitates continuous cell differentiation of MSCs by scavenging ROS and activating Nrf2 signaling. In vivo rat model shows that this composite hydrogel effectively inhibits inflammation and restores mechanical stability . Koo et al. used an alginate construct loaded with collagen and hyaluronic acid to form a shape-memory structure (SMS). The findings indicate that the SMS is effective in reducing pain, maintaining hydration, and preserving the structural integrity and function of the IVD matrix, together with supporting cell viability and the production of essential matrix components . Fig. 6 Alginate-based biomaterials for intervertebral disc regeneration and repair. (A) Injectable mesoporous bioactive glass/sodium alginate hydrogel loaded with inflammation suppressor melatonin and (i) Immunofluorescence analysis of COL-II expression in NPCs (Adapted from Ref. ). (B) Shape-memory collagen/alginate scaffold and in vivo procedures including nucleotomy, implantation of SMSs, and injection of HA, and (ii) Immunofluorescent analyses of collagen type II (Adapted from Ref. ). (C) Engineered high-strength biohydrogel consists of oxidized sodium alginate, gelatin, antagomir-204–3p, and Zn 2+ as a multifunctional platform to deliver nucleic acid. iii) SF staining images of IVD (Adapted from Ref. ). Fig. 6 Natural sodium alginate is usually gelated by calcium chloride in which endotoxins are relatively high with regard to in vivo implant. Ultra-purified alginate (UPAL) has been suggested to have a favorable lower concentration of endotoxins. Of note, UPAL hydrogel shows superior biomechanical characteristics and does not exhibit material extrusion following discectomy. Furthermore, UPAL gel enhances anabolism in NP, and maintains the moisture of IVDs in rabbits and sheep models. Surprisingly, the gel recruits endogenous NP progenitor cells in NP tissues . Subsequently, the same research group encapsulated BMSCs into UPAL gel. Co-culture of NPCs and BMSCs demonstrates elevated ECM synthesis, phenotypic markers, and bioactive factors. Combined BMSCs and UPAL hydrogel display augmented potential to rebuild degenerated IVDs . With the aim to administrate into the IVD by quick injection in situations involving persistent low back pain, a mixture of rapidly expanding clones of human MSCs and non-gelling UPAL solution is shown to impede inflammatory cytokine production and improve nociceptive behavior of rats . Of note, these UPAL gels are presently carried out in an initial human clinical trial in young patients, aged 20 to 40, after discectomy . Utilizing bioactive materials to deliver nucleic acids proposes a promising treatment for IVD regeneration. Antagomir-204–3p (AM) is capable of restraining NPCs apoptosis with lower immunogenicity. Chen et al. used zinc-oxidized sodium alginate-gelatin (ZOG) to distribute AM in which the former is able to crosslink with NP. In vivo results demonstrate that ZOG-AM rejuvenates the intervertebral disc height, sustains disc hydration, and upholds tissue integrity . Cell therapy holds potential for IVD repair, but the biomaterials used for cell delivery, which can seal annulus fibrosus defects and restore biomechanical function, often exhibit subpar biological performance. With the aim to keep the mechanical and biological performance balance, Panebianco et al. fabricated a composite hydrogel system utilizing AFCs seeded-oxidized alginate microbeads (MBs) loaded into fibrin (FibGen) hydrogels through genipin-crosslinking. AFCs encapsulated in microbeads demonstrate high cell survival, which is vital for long-term healing while high-modulus fibrin hydrogels provide immediate stabilization to degenerated IVDs. Subsequently, the FibGen-MB hydrogels are functionalized with RGD peptides and demonstrate significantly reduced AFC apoptosis and maintained phenotypic gene expression. The cell-encapsulated FibGen-MB-RGD hydrogels are assessed during a prolonged bovine caudal IVD organ model and exhibit a low risk of herniation . Inflammatory cytokines including interleukin-1 and tumor necrosis factor α are elevated in IVDD and participate in inflammation cascade and ECM degradation. Kakutani et al. determined that concurrent treatment inhibits IL-1β and TNF-α production of NPCs, AFCs, and explants encapsulated in alginate by using IL-1 receptor antagonist (IL-1Ra) and soluble tumor necrosis factor receptor-1 (sTNFR1) and promotes proteoglycan and collagen production in the NPCs and AFCs. Additionally, NPCs and AFCs produce these two cytokines in autocrine or paracrine manners via feedback regulation . Du et al. designed a biomimetic annulus fibrosus and nucleus pulposus composite consisting of circumferential poly (ε-caprolactone) microfibers laden with AFCs and an NPCs encapsulated alginate hydrogel core. The artificial IVD exhibits increased ECM deposition and organization, as well as strengthened mechanical strength . Chitosan is a biopolymer obtained from chitin, an abundant polysaccharide present in the exoskeletons of crustaceans and in fungal cell walls. Chitosan is formed by deacetylating chitin, a process that removes acetyl groups from the polymer chain . Structurally, chitosan is composed primarily of β-(1–4)-linked D-glucosamine units (deacetylated units) and N-acetyl-D-glucosamine units (acetylated units). The extent of deacetylation (DD) determines the ratio of glucosamine to N-acetylglucosamine units in the polymer chain . An acid hydrolysis product of chitosan, chitosan oligosaccharide (COS), has been shown to have significant anti-inflammatory properties. It can inhibit LPS-induced inflammatory cytokines such as TNF-α and IL-6 and nitric oxide secretion in a dose-dependent manner, thereby suppressing phosphorylation of JNK and translocation of p65, a subunit of NF-κB, into the nucleus . The unique combination of biocompatibility, biodegradability, antimicrobial properties, and ability to form films and gels of chitosan makes it a versatile biomaterial in diverse fields, for example, biomedicine, food technology, agriculture, and industrial applications . Based on the specific hypothesis that the cationic chitosan, due to its positive charge, creates an ideal environment for the entrapment of large quantities of newly synthesized anionic proteoglycans. Roughley et al. found that various formulations of cell-encapsulated chitosan hydrogels preserved a considerable portion of the proteoglycan generated by NPCs inside the hydrogel matrix, therefore suggesting the capability of chitosan as a platform for cell-loaded supplementation aimed at restoring disc function and structure . Furthermore, thermosensitive chitosan-glycerophosphate hydrogel has been proved to promote MSCs secreting proteoglycans and collagens in a proportion that is similar to that of nucleus pulposus cells . Among these different formulations, a physical crosslinked hydrogel that contains 2 % (w/v) chitosan, 0.075M sodium hydrogen carbonate, and 0.1M β-glycerophosphate proves to simulate the human nucleus pulposus tissue in mechanical properties and stimulates the highest production and retention of glycosaminoglycans by the loaded cells within the gel . Through N-hexanoylation of glycol chitosan, Li et al. developed a new hydrogel system with tunable thermos-sensitivity and enhanced stability. Biochemical serum profiles and histological examination of hexanoyl glycol chitosan-treated rats demonstrate excellent biocompatibility compared to saline-treated rats. The hydrogel maintains its stability for over a month in an ex vivo porcine system, supporting its potential use in IVDD . Inflammation is critical in the pathogenesis of IVDD, with macrophages and inflammatory pathways significantly contributing to disc degeneration . Chronic low-grade inflammation and cellular senescence further exacerbate the condition. Targeting inflammatory processes offers a promising strategy for the treatment and management of IVDD . Diclofenac, one of non-steroidal anti-inflammatory drugs (NSAIDs), has been reported to impede the pro-inflammatory polarization of human macrophages. Teixeira et al. evaluated the anti-inflammatory action of nanoparticles (NP) of chitosan and poly-(γ-glutamic acid) with diclofenac (Df). After internalization of IVD cells, nanoparticles inhibit IL-6, IL-8, PGE2, and MMPs expression and upregulate aggrecan and type Ⅱ collagen production . Subsequently, the same research group improved the nanocomplexes at pH 7.4 instead of 5.0 in that they found NPs under such an environment promote ECM anabolism including type Ⅱ collagen and sulfated glycosaminoglycan production . Furthermore, via systematic analysis of immune cell responses, the results show that pro-inflammatory intradiscal administration leads to aggravated IVD lesions, as indicated by increased CD4 + T cells (CD4 + CD8-TcR + CD161-) in the lymph nodes and blood and more pro-inflammatory cytokines, such as IL-1β, IL-6, and COX-2. In contrast, intradiscal anti-inflammatory administration slows the breakdown of the injured IVD and is associated with elevated systemic myeloid cell level (CD11b + MHCⅡ+) . Fig. 7 Chitosan-based biomaterials for intervertebral disc regeneration and repair. (A) Anti-oxidant and anti-inflammatory effect of mesenchymal stem cell-derived exosomes derived from C57BL/6 mice in an IVDD rabbit model and possible signal pathway. (i) Immunocytochemistry of NLRP3 and TXNIP. (ii) TEM to evaluate the microscopic structure of mitochondria (Adapted from Ref. ). (B) In situ forming an injectable Chitosan/PEG hydrogel shaped by Schiff base reaction with light exposure under 405 nm LED and (iii) Immunohistochemistry staining of Aggrecan and collagen II (Adapted from Ref. ). (C) Core-shell oxygen-releasing fibers via coaxial electrospinning, actions of PFTBA on the viability and functions of AFSCs, and implantation of the PFTBA core-shell scaffold in AF repair after discectomy and (iv) Fluorescence microscopy of the core-shell structure. (v) X-ray, MRI and Micro-CT (Adapted from Ref. ). Fig. 7 Traditionally, simple chitosan hydrogel possesses low mechanical strength, therefore limiting its use to biomedical applications. Huang et al. introduced a fast in situ crosslinking injectable chitosan/polyethylene glycol (PEG) hydrogel with improved machinal strength via incorporating a photo-crosslink of methacrylate and Schiff base reaction between these two components simultaneously. In vitro experiments have proved the biocompatibility and proliferation of NPCs. Micro-CT and MRI at 4 and 8 weeks claim promising effects on retarding the IVDD in rat tails . Similarly, Adoungotchodo et al. fabricated a chitosan-based hydrogel incorporating gelatine and Link N (LN), a peptide that naturally occurs in cartilage and the ECM of IVD. Rheological and compressive mechanical tests demonstrate that gelatine mildly affects hydrogel gelling but significantly strengthens mechanical properties in compression. Moreover, LN induces notably high glycosaminoglycan production in degenerative environments . Li et al. used poly (l-lactic acid) to enhance mechanical strength by distributing the former throughout chitosan scaffolds with human umbilical cord mesenchymal stem cells encapsulated. In vitro cell culture claimed that this hydrogel network is suitable for cell adhesion and proliferation. After being inserted for lumbar fusion, chitosan/PLLA enchance bone connectivity between adjacent vertebrae and, in the meantime, aids in the regeneration of annulus fibrosus tissue . Based on the distinctive biomechanical characteristics of native cellulose nanofibers (CNFs), a 3D printed, nature-inspired CNF-filled CHI hydrogel was proposed in which the former brings in essential mechanical reinforcement, whereas the latter acts as a biocompatible matrix that promotes cell growth. Because chitosan and cellulose are purely natural and are fabricated without any modification of components and the addition of a chemical crosslinker, this integrated hydrogel matrix supports 3D cell seeding with superior cell viability. The composite hydrogel opens up new possibilities for designing demanding engineered tissue of intervertebral discs . To date, there has been limited information available regarding strategies to address the challenge of hypoxia in annulus fibrosus (AF) defect sites. However, in a recent study, researchers have explored the use of perfluorotributylamine (PFTBA) core-shell fibers (10 % chitosan, chitosan: PCL, 1:6) fabricated via coaxial electrospinning. This approach aims to fabricate an oxygen-releasing platform designed to promote natural reconstruction processes in the annulus fibrosus following resecting disc. In vitro studies demonstrate that hydrogels facilitate proliferation, migration and ECM synthesis in annulus fibrosus stem cells under hypoxic conditions and in vivo studies show notable improvements in vertebral spacing and structural unity observed in animal models treated with these oxygen-releasing biomaterials . Tissue-engineered intervertebral discs (TE-IVDs) have been considered an effective therapeutic solution for treating IVDD. Yang et al. developed an artificial IVD employing chitosan hydrogel to mimic the central nucleus pulposus region, enclosed by a poly (butylene succinate-co-terephthalate) fiber layer to represent the inner annulus fibrosus and employing a poly (ether ether ketone) ring to simulate the outer annulus fibrosus with IVD cells seeded. The novel tissue-engineered IVD establishes a conducive setting for supporting the development of IVD cells. Overall morphology and biological functions of the scaffolds closely resemble those of natural porcine IVDs . Afterward, Yuan et al. constructed similar TEIVDs using poly (butylene succinate-co-terephthalate) copolyester as inner annulus fibrosus (IAF) while solid PBST as outer annulus fibrosus (OAF), and using chitosan as NP. AF and NP cells are loaded onto the corresponding scaffolds. Of note, the IVD cells express cell-specific extracellular matrix, therefore suggesting that TE-IVD is biologically functional. X-ray and MRI examinations indicated that the intervertebral disc space was maintained 4 weeks later after TE-IVD was engrafted into New Zealand white rabbits . Polysaccharide-based materials hold significant promise as therapeutic agents for IVDD, offering a versatile and biocompatible platform for tissue regeneration. Of these materials, hyaluronic acid and chondroitin sulfate, two key components of the NP's extracellular matrix, possess the strongest ability to stimulate the nucleus pulposus environment with the ability to retain water and resist compressive forces. Chitosan has superior adhesivity to alginate, which has moderate adhesivity, and hyaluronic acid. With regard to biomechanical strength, alginate stands out with its tunable mechanical strength and stability. Moreover, compared to other inorganic materials or synthetic polymers which may require harsher processing conditions and yield harmful byproducts, polysaccharides can be processed under mild conditions such as low temperature, neutral pH and degrade via enzymatic hydrolysis. In the meanwhile, polysaccharides possess excellent bioactivity and inherent hydrophilic properties to retain hydration similar to native NP tissue. Of note, while proteins like collagen and synthetic polymers like PLGA offer tunability, polysaccharides like HA or chitosan can reach a unique balance of bioactivity and mechanical properties. To conclude, these polysaccharide-based biomaterials, including hyaluronic acid, chitosan, and alginate have demonstrated the ability to mimic the extracellular matrix of the NP, support cell survival, and enhance matrix synthesis, making them ideal candidates for disc repair and regeneration. Their capacity to deliver bioactive molecules directly to the degenerative site as well as their cost-effectiveness further amplifies their therapeutic potential. Nevertheless, several challenges still remain despite of all these advances. The intricate biomechanics of the IVD necessitate the use of materials that are capable of providing support for cellular functions while simultaneously withstanding the mechanical loads and stresses that are inherent to the spinal environment. The AF and NP are anatomically and functionally interdependent and the integrity of NP and AF in IVD are needed to be considered. For instance, a weakened AF may lead to NP herniation, while a degenerated NP can lead to AF collapse. Herein, the combined therapy targeting both the NP and AF is critical for achieving comprehensive and durable outcomes. Furthermore, the transition of these materials from laboratory to clinical settings requires comprehensive investigations into their long-term biocompatibility, degradation profiles, and potential immunogenic responses. The development of injectable and minimally invasive polysaccharide-based systems is also of great importance in order to ensure clinical applicability and patient compliance. In the future, the integration of advanced technologies, including three-dimensional bioprinting, nanotechnology, and gene editing, may provide new avenues for enhancing the efficacy of polysaccharide-based therapies. Polysaccharide-based materials can be customized to suit the specific requirements of both AF and NP, for instance, customized stiffness and elasticity through chemical modifications, functionalized polymer grafts, or in combination with other materials to fabricate composites, which may pave the way for meeting combined AF and NP therapy. 3D bioprinting can facilitate the precise fabrication of complex disc structures, while nanotechnology may be employed to design nanoparticles for targeted drug delivery within the disc environment. Gene editing technologies, such as CRISPR, may facilitate the modification of cells encapsulated within these hydrogels to produce specific proteins or growth factors that enhance tissue regeneration. In conclusion, polysaccharide-based materials represent a promising and adaptable approach for the therapy of IVDD. Continued research and technological integration are essential to overcome current limitations and realize their full potential in clinical settings. Advancing these materials and strategies may facilitate the development of effective, long-lasting solutions for patients suffering from intervertebral disc degeneration. | Study | biomedical | en | 0.999997 |
PMC11699362 | The umbilical cord is a delicate, adaptable structure that associates the embryo to the mother through the placenta from the 6th week of pregnancy until birth. It contains one vein, which carries oxygenated, nutrient-rich blood to the fetus, and two arteries which carry deoxygenated, nutrient-depleted blood away from the fetus. The stump continuously dries, shrinks, and isolates from the body, typically somewhere in the range of 5 and 15 days after birth with shading change from a yellow-green to dark as it dries out. During this period, the umbilical cord ought to be kept perfect and dry to dodge contamination . Numerous further intercessions to forestall umbilical string contamination have been attempted. The two that have been studied most extensively are 70° alcohol and 4 % chlorhexidine, administered in different dosage forms (alcoholic or aqueous solution, gel or powder). Other antiseptics used for the purpose are triple dye (a combination of 3 disinfectant solutions frequently used in the United States), povidone, iodine, and salicylic acid, among others. Topical antibiotics such as silver sulfadiazine, tetracyclines, or neomycin have also been used. However, the application of any CHX to the umbilical cord of the newborn led to a 23 % reduction in all-cause neonatal mortality . WHO recommends dry cord care for nations with good obstetric care and low neonatal mortality rates. Recent studies suggest applying chlorhexidine on the umbilical cord stump during the first week of life for neonates delivered at home in settings with high NMR of 30/1000 live births or higher . The Ethiopian government introduced the Community Based Newborn Care (CBNC) program in 2013, to improve maternal and newborn health outcomes. Chlorhexidine was included as one of the components in CBNC after consensus building, and it was manufactured locally. So Chlorhexidine is a feasible and acceptable intervention in the Ethiopian setting . In addition to this and antiseptic mothers' awareness about cord care practices and maternal higher educational level were important factors in maintaining the cord in a healthy state . Even though Chlorhexidine application delays the time to cord separation, its application to the cord reduces the risk of neonatal mortality and omphalitis in infants born at home in high-NMR settings . A single intervention of 4 % CHX has been displayed by numerous studies to effectively reduce the incidence of omphalitis-related mortality by approximately 25 % . The sample size for this particular study was determined by using a single population proportion formula using a basic assumption of a 95 % confidence level and, a 5 % margin of error by considering the proportion of safe cord care was 68.3 % with a study conducted in Nekemte city Oromia region, Ethiopia . n i = z 2 P ( 1 − P ) d 2 , ( 1.96 ) 2 0.683 ( 1 − 0.683 ) ( 0.05 ) 2 = 333 Where, Multi-stage sampling technique was used to select the study participants. The study was conducted in eight Kebele's of hetosa district which is 30 % of the total kebeles. From twenty-five Kebele's found in the district, eight (seven rural and one urban) Kebele's were selected using simple random sampling method. The sample size was allocated to each Kebele proportional to the number of women who gave birth in the last six months from selected Kebele. Skip interval was determined for each kebele by dividing the estimated population by the respective sample size (i.e. Kth = N/n). The expected total number of women who gave birth in the last six months in selected kebeles was N = 1241. Hence, each participant was selected by systematic random sampling with a skip interval of 2 at each kebele. Using the lottery method, 1 was selected and used as a starting number based on the mother's registration book. Subsequently, every other mother was included until the desired sample size was achieved . K = N n f K = 1241 550 = 2.25 ≈ 2 Where, N = total Number of mothers who gave birth in the last six months in selected kebeles. Fig. 1 schematic representation of sampling technique to assess cord care practices among mothers who gave birth in the last six months in Hetosa district, Arsi zone, Ethiopia, 2021. Fig. 1 The purposive sampling technique was used for the qualitative study. The open-ended questionnaire was prepared to obtain an in-depth understanding of neonatal cord care practices and associated factors. A total of 12 in-depth interviews (IDIs) were conducted. The IDIs involved 4 mothers, 3 traditional birth attendants (TBAs), 3 Health Extension Workers (HEWs), and 2 grandmothers all of whom were selected purposively based on their close relation with mothers and neonates, involvement in the community and an assumption to be the richness of information on the topic of the study. The number of IDIs was determined based on the level of saturation of the required information. The pre-coded data were entered into Epi data version 3.1 software and then it was exported to SPSS version 25 for statistical analysis. A score of one was given for good cord care practice and zero for poor cord care practice. A binary logistic regression was used to identify the association of the independent variables with the dependent variable. Each variable that has p-value less than 0.25 was added to the final model to control the confounders. Variables that have a p-value <0.05 with a 95 % confidence interval in the final model were declared statistically significant. Knowledge of mothers on newborn cord care was assessed using a series of six close-ended questions and a composite variable was generated from these questions to categorize mothers as having “Good/poor” knowledge. A scoring system was used to analyze responses to closed-ended questions on Knowledge: 1 = coded as correct response and 0 = coded as incorrect response. Accordingly, mothers who correctly responded to at least 3 questions were categorized as having a good knowledge of newborn cord care. The descriptive data were presented using frequency, tables, figures, mean, and standard deviation. Hosmer and Lameshow goodness of fit test was conducted to test the model fitness and the model was adequate (p = 0.266). Multicollinearity was checked by using VIF and it was <10. Primarily, audio-recorded data were heard repeatedly until the principal investigator became intimately familiar with the contents. The audio-recorded qualitative data were transcribed into the English language. Then, codes or terms were identified and tallied to come up with some categories, which were later used to establish themes based on the objective of the study. Unique concepts were identified and trials were made to elaborate more and report on the final result. Finally, thematic analysis was done manually and the findings were triangulated with the quantitative one. A total of 541 mothers who gave birth in the last six months participated in this study giving a response rate of 98.36 %. The mean (mean ± SD) age of the respondents was 28.41 ± 5.72 years. Of all participants, 488 (90.2 %) mothers were married and 471 (87.1 %) were Oromo in ethnicity. Among the total participants, 263 (48.6 %) mothers attended primary school and about 286(52.9 %) women were orthodox in religion. About occupational status housewife 397 (73.4 %) take a larger proportion. The majority 405(74.9 %) mothers were rural residents and 224 (53.5 %) respondents used radio as a mass media source of information ( Table 1 ). Table 1 Socio-demographic characteristics of mothers who gave birth in the last six months in Arsi zone, Hetosa district, Ethiopia, 2021, (n = 541). Table 1 Variable Category Freq Variable Category Freq Age ≤24 years 135(25.0 %) Maternal occupation Housewife Farmer Government employer Other(merchant, daily labourer, student) 397(73.4 %) 21(3.9 %) 69(10 %) 25–32 years 281(51.9 %) ≥33years 125(23.1 %) Marital status Single 26(4.8 %) Do you have a source of Information/mass media Yes No 419(22.6 %) 122(77.4 %) Married 469(86.7 %) Other (windowed, divorced, separated) 46(8.5 %) Ethnicity Oromo 471(87.0 %) Type of information source you have Radio Television 224(53.5 %) 195(46.5 %) Amhara 42(7.8 %) Other(Tigree, Gurage, Woliyta) 28(5.2 %) Religion Orthodox 285(52.7 %) Monthly income ≤1999 2000–3999 ≥4000 282(52.1 %) 154(28.5 %) 105(19.4 %) Muslim 189(34.9 %) Protestant 56(10.4 %) Other (catholic) 11(2.0 %) Education level No formal education 81(15.0 %) Residence Rural Urban 405(74.9 %) 136(25.1 %) primary 263(48.6 %) secondary and above 197(36.4 %) From the total mothers who participated in this study 320(59.1 %) were multigravidas. The majority of the respondents delivered their babies at a health facility 418 (77.3 %) while 123 (22.7 %) had home deliveries. Almost all of the respondents delivered their babies at health facilities attended by health professionals. On the other hand majority of those who had their birth in the home were attended by families 46(37.7). Among the total participants, 216 (39.9 %) mothers had postnatal care after their deliveries of recent baby. Slightly more than half of the respondents 295(54.5) gave male babies ( Table 2 ) Table 2 Obstetric characteristics of mothers who gave birth in the last six months in Arsi zone, Hetosa district, Ethiopia, 2021, (n = 541). Table 2 Variable Category Frequency Percent % Number of ANC visit One 23 4.6 Two 80 15.8 Three 147 29.1 ≥four 255 50.5 Place of ANC Private clinic 72 14.3 Hospital 58 11.5 Health center 284 56.2 Other (health post) 91 18.0 Gravidity Primigravida 142 26.2 Multigravida 320 59.2 Grand multipara 79 14.6 Place of birth Home 123 22.7 Health facility 418 77.3 Birth attendant Health professional 418 77.3 TBA 45 8.3 Family 46 8.5 Friend 8 1.5 Other 24 4.4 Baby's sex Male 295 54.5 Female 246 45.5 ANC = antenatal care, TBA = traditional birth attendant. Slightly more than half 298(55.1 %) respondents received information regarding umbilical cord care and most of them 217(72.8 %) received the information from health professionals either before or after the delivery of their recent baby. Among the participants who had ANC visits only 82 (16.2 %) said they were informed about umbilical cord care during their ANC visit ( Table 3 ) Table 3 Source of information of mothers who gave birth in the last six months in Arsi zone, Hetosa district, Ethiopia, 2021, (n = 541). Table 3 Variable Category Frequency Percent % Received information regarding UCC before or after delivery Yes 298 55.1 No 243 44.9 A person who gave the information regarding UCC Health professional 217 72.8 Family 31 10.4 Neighbor 28 9.5 TBA 12 4.0 Mass media 7 2.3 Other (friend) 3 1.0 A place where the information received regarding UCC Health facility 194 65.1 Home 67 22.5 Neighbor 30 10.1 Other 7 2.3 Umbilical cord care education during ANC Yes 82 16.2 No 423 83.8 ANC = antenatal care, UCC = umbilical cord care. With regard to knowledge, 91.9 % of the respondents acknowledged that they always wash their hands with soap and water before umbilical cord care. However, 283 (52.3 %) of them knew that no substance was applied to the cord stump except chlorohexidine. Of the participants, 450(83.2 %) and 396(73.2 %) knew that cord-cutting and cord-tying material should be sterile respectively ( Table 4 ). Table 4 Knowledge of mothers who gave birth in the last six months in Arsi zone, Hetosa district, Ethiopia, 2021, (n = 541). Table 4 Variables Category Frequency Percent(%) Hand wash with soap and water before UCC Yes 497 91.9 No 44 8.1 No substance should be applied on the cord stump except chlorohexidine Yes 283 52.3 No 258 47.7 The diaper should not cover the cord Yes 185 34.2 No 356 65.8 Newborn bathing should be sponge bath till the cord is cut off Yes 152 28.1 No 389 71.9 Cord-cutting material should be sterile Yes 450 83.2 No 91 16.8 Cord tying material should be sterile Yes 396 73.2 No 145 26.8 Overall knowledge Good 380 70.2 Poor 161 29.8 UCC = umbilical cord care. Almost all participants applied those substances for different reasons. Among them, 123(22.7 %) applied substances to moisturize and prevent dryness of the cord stump whereas, 116(21.4 %) applied to facilitate cord separation. Others 84(15.5 %) applied ∗that substance to promote of the respondents who applied the substance, 129(23.8 %) mothers started to apply the substance on the second day, and 80(24.7 %) applied once daily. Regarding a person applying a substance on the cord, in 189(58.3 %) cases the substance was applied by the baby's mother and of those respondents who applied substance 210(64.8 %) always washed their hands before they applied the substance. Among those respondents who applied substance on the cord stump majority of them 222(68.5 %) had received information about the substance they applied and most of them 133(59.9 %) received the information from family members. Among study participants, the majority of mothers wash their child's body by immersion of whole body in water. Out of those interviewed 195 (36.0 %) correctly stated that the cord should be dry after washing their baby's body and majority of them dry their baby by loosely covered using a clean piece of cloth. Of the participants in this study, 480(88.7 %) didn't notice any complication on the cord. However, some of the mothers 61(11.3 %) notice complications such as reddening of the umbilicus 28(5.2 %), pus at the umbilicus 20(3.7 %), cord bleeding 10(1.8 %), swelling of the umbilicus 3(0.6 %). Most of the mothers 444(82.1 %) did allow the cord to separate on its own. But some of them 97(17.9 %) interfere with the cord separation process or try to separate the cord rather than allow the cord to separate on its own. Checked and found it to be satisfactory. Consequently, in bivariable logistic regression, the age of the mother, educational status of the mother, marital status, mother's occupation, having mass media, gravidity, place of birth, and knowledge status of the mother were included in multivariable analysis. However, after controlling the effect of confounding in multivariable analysis the final result confirmed that mothers' age, education, place of birth, and level of knowledge on umbilical cord care were found to be statistically significant at p-value <0.05. Hence, those who had primary, secondary, and above educational status had 5.26 times (AOR = 5.26, 95 % CI: 2.50, 11.04) and 3.63 times (AOR = 3.63, 95 % CI: 1.61, 8.18) more likely to practice good cord care than mothers who had no formal education, respectively. The odds of having a good cord care practice among age groups of ≤ 24years and 25–32years was 4.56 times (AOR = 4.56, 95 % CI; 2.08, 9.96) and 3.70 times (AOR = 3.70, 95 % CI; 1.98, 6.94) higher than mothers whose age ≥ 33 respectively. Hence younger mothers practice good cords as compared to older ones. The odds of delivering in a health facility was 5.09 times (AOR = 5.09, 95 % CI; 2.95, 8.78) was found to significantly associate good practice of cord care than among those who delivered outside the health facility and mothers who had good knowledge of newborn cord care practice were eight times more likely to practice good cord care than those who had poor knowledge (AOR = 8.58, 95 % CI; 5.09, 14.46) ( Table 5 ) Table 5 Bivariable and multivariable logistic regression model predicting the likelihood of good cord care practice among mothers who gave birth in the last six months in Arsi zone, Hetosa district, Ethiopia, 2021, (n = 541). Table 5 Variables Practice COR (95%CI) AOR (95%CI) P-value Good(%) poor (%) Age group (years) ≤24 92(31.8 %) 43(17.1 %) 7.08(4.08, 12.28) 4.56(2.08, 9.96) 0.000 a 0.000 a 25–32 168(58.1 %) 113(44.8 %) 4.92(3.04, 7.94) 3.07(1.98, 6.94) ≥33 29(23.2 %) 96(38.2 %) 1 1 Educational status No formal education 15(5.2 %) 66(26.2 %) 1 1 0.000 a 0.002 a Primary 148(51.2 %) 115(45.6 %) 5.66(3.07, 10.43) 5.26(2.50, 11.04) Secondary and above 126(43.6 %) 71(28.2 %) 7.80(4.15, 14.68) 3.63(1.61, 8.18) Mothers' occupation Housewife 192(66.4 %) 205(81.3 %) 0.33(0.18, 0.58) 0.46(0.20, 1.06) Farmer 10(3.5 %) 11(4.4 %) 0.32(0.11, 0.88) 1.03(0.24, 4.33) Government employer 36(12.5 %) 18(7.1 %) 0.70(0.32, 1.54) 0.81(0.28, 2.30) Other 51(17.6 %) 18(7.1 %) 1 1 Marital status Single 20(6.9 %) 6(2.4 %) 1 1 Married 240(83.1 %) 229(90.9 %) 0.31(0.12, 0.79) 0.59(0.13, 2.23) Other 29(10.0 %) 17(6.7 %) 0.51(0.17, 1.52) 1.20(0.26, 5.49) Access to mass media Yes 237(82.0 %) 182(72.2 %) 1.75(1.16, 2.63) 1.58(0.91, 2.73) No 52(18.0 %) 70(27.8 %) 1 1 Gravidity Primigravida 101(34.9 %) 41(16.3 %) 6.39(3.46, 11.76) 1.41(0.58, 3.41) Multigravida 166(57.4 %) 154(61.1 %) 2.79(1.63, 4.78) 0.83(0.39, 1.73) Grand multipara 22(7.6 %) 57(22.6 %) 1 1 Birthplace Health facility 260(90.0 %) 159(63.1 %) 5.24(3.30, 8.31) 5.09(2.95, 8.78) 0.000 a Home 29(10.0 %) 93(36.9 %) 1 1 Knowledge level Good 260(90.0 %) 120(47.6 %) 9.86(6.24, 15.56) 8.58(5.09, 14.46) 0.000 a Poor 29(10.0 %) 132(52.4 %) 1 1 a = p-value <0.05, COR = crude odds ratio and AOR = adjusted odds ratio. But this finding was higher than the study conducted in Hossana town, Hadiya zone, southern Ethiopia, 32.9 %, in Garissa county, Kenya which was 43 %, a population survey in eastern Uganda 38 % and in Cameroon 19.7 % [ 18 , , , ]. And this difference might be due to relatively a recent increase in antenatal care visits and institutional delivery services. The difference can also be a result of chlorhexidine provision among mothers. In addition, the current community-based interventions by health extension workers might have contributed to the reduction of harmful cord care practices among mothers in the study area. This is supplemented by qualitative findings in Pemba Tanzania which revealed that there was consensus among respondents that CHX liquid cord cleansing could be successfully implemented in the community with appropriate education and awareness . In this study, younger women were more likely to practice good cord care than those who are older women. The possible reason behind this can be those younger mothers were more likely educated than older ones. In addition, older mothers may not be equipped with the latest cord care guidelines which have been modified in recent times. Another explanation may be as the age of women increases the probability of having other previous pregnancies or children increases. This may lead women to apply more cultural practices received from their experience. This finding is opposed to studies conducted in Ibadan, Nigeria, in Benin City, Edo State, Nigeria, and the 2013 edition of the Nigeria Demographic and Health Survey (NDHS) which show that older women practice better cord care than younger mothers . This difference may be a result of the fact that older mothers tend to have delivered more times thann younger mothers and as such are more experienced in child care as they may have learned hard lessons as well following harmful cord care over the years. | Other | biomedical | en | 0.999997 |
PMC11699366 | In the absence of disease-modifying therapies for Parkinson's disease (PD), much research focuses on improving quality of life, health, and wellbeing. A new care model, Proactive and Integrated Management and Empowerment in Parkinson's (PRIME) , is a multicomponent intervention being evaluated within a randomized controlled trial (RCT) which is enrolling patients with parkinsonism, and therefore includes those with frailty, multimorbidity and/or cognitive impairment as well as informal caregivers . This intervention aims to improve the experience of living with PD and align care with ‘what matters most’ to individuals . We sought to assess benefit of this innovative care model using an outcome which is aligned to the project aims, captures the hypothesised impact and is meaningful to patients. Patient-reported outcome measures (PROMs), which collect outcomes directly from the people who experience them are increasingly being used alongside or instead of biomarkers and measures of morbidity, mortality and healthcare use . Many of these are disease-specific which has limitations, particularly in older people, as well as those with multiple long-term conditions, disability or short life expectancy in whom there are often trade-offs when considering multiple interventions/treatments . Given the complexity and heterogeneity of PD, PROMs focusing on a single domain, such as motor symptoms, are often not appropriate and risk overlooking the spectrum of experience. Conventional outcome measures may not be validated for proxy completion on behalf of someone lacking capacity and, whilst some outcomes have been endorsed for use in PD dementia , floor and ceiling effects may render them unsuitable for use in individuals with intact cognition, precluding their use in a trial recruiting across the disease spectrum. When considering a complex intervention like the PRIME-UK care model, there is a need for PROMs which capture the downstream effects of the multiple components, which are aiming to produce impact across physical, psychological and social domains. Given the limitations of traditional PROMs, novel outcomes that can more holistically capture personal experiences and wellbeing have been proposed. Cools et al . suggested happiness as an outcome in PD studies but this also has limitations since wellbeing is a multi-dimensional construct encompassing psychological wellbeing, not simply the pursuit of happiness . Traditional outcomes can be made more patient-centred by reframing them into goal-orientated outcomes: instead of using a pain inventory for arthritis, we may assess pain control sufficient to carry out an activity important to the patient . Instead of disease-free survival, the goal-orientated outcome would be survival until a personal milestone, or there may be no survival outcome if this were not a patient priority . Goal attainment scaling (GAS) requires individuals to set meaningful goals with progress subsequently rated on a standardised scale and a formula applied to calculate an aggregated score . In addition to clinical use as an intervention to motivate behavioural change, goal-orientated approaches have been used to evaluate interventions in research settings, including amongst people with multiple sclerosis , older adults living with frailty and PD patients [ , , , , ]. We aimed to select a suitable primary outcome measure and adapt it for use within the PRIME-UK RCT. The PRIME RCT, approved by the London-Harrow Research Ethics Committee , aimed to enrol a ‘real-world’ population to make the findings generalisable to clinical practice. The outcome measure therefore needed to be suitable for use in participants across the spectrum of disease stage and phenotype, including those with cognitive impairment and multimorbidity, as well as meaningful to patients and able to capture the effect of a complex intervention acting across multiple domains. Goal-orientated outcomes offered a way to elicit an individual's goals, and generate a numerical score to allow assessment of attainment over time and comparison between patients with multiple personalised goals. From a literature review, we identified four goal-based measures which have been used to evaluate interventions, rather than goal-setting as an intervention itself, each of which we deemed to have pros and cons based on the practicality of use within a trial, prior use in PD and psychometric properties ( Table 1 ). We selected the Bangor Goal-setting Interview (BGSI) as the PRIME RCT primary outcome measure because it was developed for research purposes, rather than for clinical settings with subsequent adaptation, and has advantages for this context ( Table 1 ), including a freely-available manual and the flexibility to adapt it to specific settings . It has also been shown to be feasible for use with people with PD dementia and Dementia with Lewy Bodies . Table 1 Interventional studies, including trials, which included a goal-orientated outcome measure. Table 1 Goal-orientated outcome measure Brief description of measure Examples of uses including study population Pros Cons Goal attainment scaling (GAS) Main problem area/domain is identified during an interview with the patient and goal(s) agreed jointly; the expected outcome defined as achievement level 0 is agreed and descriptors are pre-agreed for level −1/-2 and +1/+2, representing a worse or better outcome than expected respectively. Each goal is rated on the 5-point scale at follow up. - A single blind RCT of cognitive training, transfer training and psychomotor training in people with PD and MCI - A pilot, double-blind RCT of memantine versus placebo for PD dementia - A service evaluation of dance classes for PD - An RCT of comprehensive geriatric assessment as an adjunct to usual care in frail older patients - Goals can be weighted to account for the importance to the patient and/or anticipated difficulty - There is no requirement to record baseline scores (although these are usually rated as −1) - Performance is judged against predefined levels. - Potentially time-consuming to define pre-determined levels for each of the 5 outcome score levels (−2 to +2) - Zero and minus scores can be discouraging for patients - If baseline score is recorded as −1 (which allows for deterioration to −2), it is not possible to record if a goal is partially achieved (but does not warrant the expected outcome level of 0) . Patient-specific functional scale (PSFS) A self-reported measure in which patients list up to five activities they struggle to do because of their condition/problem. For each, they rate from 0 to 10 their ability to perform the activity at baseline and then subsequently at follow-up. - A prospective, observational pre-post study of goal-orientated rehabilitation in individuals with a neurological condition (including PD and MS) - RCTs in populations with COPD , low back pain and cervical radiculopathy . - May be less time-consuming and require fewer resources than a semi-structured interview as the process is patient-directed - Reported to have good reliability, validity and responsiveness in a range of musculoskeletal conditions including mechanical back pain . - Does not involve a semi-structured interview so the patient is not guided/supported through the process which may be challenging for some patient groups including those with cognitive impairment - Lack of evidence for use in neurological disease . Bangor Goal-setting interview (BGSI) The patient sets goals in a collaborative interview with the option for an ‘informant’ to provide input. Goal attainment descriptors are pre-defined to indicate what would constitute e.g. 0, 50, 100 % attainment. Current attainment is rated on a 1 to 10 scale, with option to rate importance and motivation to change. Attainment is rated from 1 to 10 at follow-up. - Primary outcome in a single blind pilot RCT of cognitive rehabilitation versus relaxation therapy for PD dementia/Lewy Body Dementia - Primary outcome in a single-blind RCT of goal-orientated cognitive rehabilitation in early-stage dementia, the’GREAT’ trial - Developed for research purposes and the manual includes specific guidance on use of BGSI in an RCT - The manual and recording sheet are freely available for researchers to use and include resources to guide the interview, written in an accessible format, suitable for individuals with cognitive impairment - Incorporates the option, not only for an informant to support the process, but also for a caregiver to independently rate attainment - The structure allows some flexibility to adapt the measure for use in different settings - Descriptors set at baseline can help with subsequent rating of attainment - Potentially time-consuming to define the descriptors at baseline - Limited information on the psychometric properties Canadian Occupational Performance Measure (COPM) Semi-structured interview in which the patient is guided to consider 3 areas: productivity, self-care and leisure. Patients choose up to 5 problems and rate current performance and satisfaction on a scale of 1–10, where a higher score represents greater performance or satisfaction. Performance and satisfaction are rated again at follow-up. - A case series (pre and post testing) of an 8-week treatment program to train for the use of compensatory external aids to achieve personalised goals - Evidence supports the reliability, construct validity and responsiveness . - Specification of the 3 areas (productivity, self-care and leisure) may not be relevant to all individuals or interventions - The measure and related resources must be purchased GAS: goal attainment scaling; RCT: randomized controlled trial; PD: Parkinson's Disease; PSFS: patient-specific functional scale; MS: Multiple Sclerosis; BGSI: Bangor Goal-setting Interview; COPM: Canadian Occupational Performance Measure. 3.1 Use of the Bangor Goal-setting Interview within the PRIME-RCT Agreement of goals before randomization avoids prior knowledge of intervention status biasing goal selection . During a collaborative interview the participant sets three to five goals with a trained researcher, with input from a family member/caregiver (an ‘informant’), particularly for participants with cognitive impairment . Unlike the original BGSI, we do not pre-specify domains within which participants must identify goals since these are expected to be highly personalised. After goal selection, percentage descriptors are specified to indicate what would constitute, for example, 50 % goal attainment. The participant is then asked to rate their current attainment, perceived importance and readiness to change numerically for each goal, with an option for an enrolled caregiver to independently rate attainment. The four steps are summarised in Fig. 1 , described in more detail below and illustrated in Table 2 with an example. Fig. 1 The procedure for using the Bangor Goal-Setting Interview within the PRIME randomized controlled trial prior to randomization. Fig. 1 Table 2 Applying the adapted Bangor Goal-setting interview to set and follow up a goal within the PRIME randomised controlled trial, where text in italics indicates how the researcher would guide the participant through the process. Table 2 STEP 1 Specify the area (domains not pre-specified a ) “Parkinson's can impact on daily life in various ways. There are different things that people may want to change to make their lives more enjoyable. We are asking everyone taking part in this trial to come up with several goals that they would like to achieve. A goal might be something that you currently find difficult to do and wish you could do more easily, or without getting frustrated. Or a goal might be something that you are currently not doing and would like to do more of. Or it might be something that you would like to learn how to do.” Going to the gym STEP 2 SMART goal statement (100 % attainment) I will go to the gym to exercise 3 times per week for 45–60 min Description of current attainment (0 % attainment) I have membership but do not go at all Goal attainment descriptor b (50 %) I will go 1–2 times per week or 3 times for a shorter session (e.g. 20 min s) STEP 3 Rating of attainment Image 1 1 Rating of importance b Image 2 7 Rating of readiness to change b Image 3 6 STEP 4 (e ach 3 monthly follow up conducted by blinded assessor b ) Free text record of current activity in relation to goal “Last time we spoke about going to the gym. Your goal was to go to the gym 3 times per week to exercise for 45–60 min. How are things going with that?” “Due to a recent small fracture of my right ankle I had to wear a boot and rest for 6 weeks, but I'm gradually starting to exercise again. In the last month, I have gone to the gym 4 times for 30–40 min” Participant raw attainment score (without reminder of prior rating in step 3 b ) “What score would you give yourself on a scale of 1-10 for how well you are doing now? 10 would be that you are doing it very successfully, 1 would be that you are not doing it at all or not successfully at all." 3 Calibrated attainments scores a “At your baseline visit, you said you had membership but were not going to the gym and you gave yourself an attainment score of 1. We decided at that time that achieving your goal by 50 % or making it half way to your goal would be going to the gym 1–2 times per week or going 3 times for shorter sessions ( e.g. 20 min). Based on what you have just told me, it sounds like you are more than 50 % of the way to achieving your goal. I wonder whether the new score should be somewhere around 6- would you agree with this?" Interviewer-decided attainment score calibrated from baseline attainment score using % descriptors6 Calibrated score as decided by participant following discussion5 SMART: Specific, Measurable, Achievable, Relevant, Time-bound. a Indicates an aspect of the process which is specific to the operationalisation of BGSI within PRIME-RCT. b Indicates an aspect of the process which was specified as an option within the BGSI manual and which was incorporated into the PRIME-RCT. The primary outcome is change in participant-rated attainment, calculated as shown in Fig. 2 . Whereas for classical measurement instruments a cut-off score may indicate a particular diagnosis and individual scores can be compared to normative data, intervention evaluation using a goal-based outcome relies on comparison of two group means . Follow-up at all 3-monthly timepoints will facilitate a secondary longitudinal analysis to determine whether any improved goal attainment is maintained. Secondary analyses will include change in caregiver-rated attainment and for the three goals rated as most important. Fig. 2 A worked example showing the process for calculating the change in attainment for one participant who had set three goals. Fig. 2 We have described how a goal-orientated outcome measure was adapted for use in a trial involving individuals with complexity, frailty and multimorbidity. As the trial progresses, we will gain crucial real-world experience of applying this in people with parkinsonism, including the time and resources required. We will also gather quantitative data on the numbers of goals set, typical domains chosen, agreement between patient and caregiver scores and the proportion unable to rate attainment (e.g. due to cognitive impairment). The lack of a defined MCID for the BGSI is a limitation; determining this in a parkinsonism population would add to its utility and help to inform whether BGSI is better able to detect a meaningful difference than some other outcomes, as suggested [ , , ]. Whilst the evidence for responsiveness is encouraging , evidence for the validity and reliability of goal-orientated outcome measures is limited and relates mainly to the COPM . Future work could establish whether an improvement in goal attainment is associated with downstream benefits such as reduced unplanned hospitalisation, which would demonstrate predictive validity. There are many potential benefits to wider use of goal-orientated outcome measures in clinical trials. They provide a holistic outcome not limited to specific conditions and with universal relevance, important for trials with broad inclusion criteria, enrolling heterogenous populations including adults with frailty and multimorbidity. Above all they aim to provide a meaningful measure of “what matters”. Knowledge gained from use of the adapted BGSI in the PRIME 10.13039/100014144 RCT will help to support its implementation in conditions besides neurodegenerative disease, the focus of its use so far. | Study | biomedical | en | 0.999999 |
PMC11699391 | As with many other diabetic complications, the poor bone quality and increased fracture risk in diabetes has already aroused great concerns. 1 , 2 It was well recognized that hyperglycemia exposure inhibits proliferation and differentiation of osteoblasts, 3 but its specific mechanism is still not clear, and no definitive pharmaceutical remedies for diabetic osteoporosis are available except for those traditional medications to treat primary osteoporosis. Energy metabolism is an important physiological basis for maintaining cell function. The function of mitochondria, as the hub of energy metabolism, is the key to maintaining normal life activities. Mitochondrial dysfunction-related diseases have expanded from rare monogenic disorders to common polygenic diseases, including metabolic, cardiovascular, neurodegenerative, and neuromuscular diseases in nowadays. 4 , 5 Lots of effort was made to explore signaling pathways to reverse mitochondrial dysfunction. 6 Recent studies have suggested a mutual cross-talk between bone remodeling and glucose homeostasis. 7 , 8 So far, the best characterized metabolic pattern for osteoblast maturation is the glucose metabolism reprogramming from oxidative phosphorylation (OXPHOS) toward aerobic glycolysis, coupling with diminished mitochondrial respiration. 9 , 10 Suppression of glucose metabolism restricts osteoblast differentiation. 11 Thus, it is interesting to investigate whether and how the disturbed metabolic reprogramming causes impaired osteoblastogenic differentiation and poor bone quality in diabetes, and how to reverse it. Mitophagy is a mechanism that eliminates damaged mitochondria and controls mitochondrial quality and quantity. Simultaneously, mitophagy mediates metabolic reprogramming, either into an OXPHOS phenotype or into a glycolytic phenotype, to satisfy the metabolic requirements of cells. 12 , 13 Mitophagy dictates stem cell fate by facilitating glycolytic phenotype swift. 14 The deficiency of mitophagy induces reactive oxygen species (ROS) stress, and leads to the development of neurodegenerative disorder, 15 cardiomyopathy, 16 and acute kidney injury. 17 In type 2 diabetes patients, mitophagy pathway gene expression was downregulated. 18 In β-cells, decreased glucose-responsive insulin secretion was associated with impaired mitophagy. 19 Mitophagy is mediated by PTEN-induced kinase 1 (PINK1)/Parkin pathway. PINK1 is a mitochondrial serine/threonine-protein kinase. It can sense mitochondrial stress, accumulate on defective mitochondria, recruit Parkin, and finally trigger mitophagy. 20 Loss of PINK1/Parkin may contribute to several diseases, such as Parkinson’s disease. 21 As to bone metabolism, PINK1-mediated mitophagy is involved in cartilage degeneration seen in osteoarthritis. 22 , 23 , 24 Therefore, our research interest is what role mitophagy plays in hyperglycemia-induced osteoblastogenic defects and whether the intervention of related mechanisms can restore diabetic osteoporosis. To investigate the effect of high glucose level on the osteoblastogenesis of MC3T3-E1 cells in vitro , alkaline phosphatase (ALP) and Alizarin red staining were performed. Alizarin red staining in H-Glu group showed significantly fewer mineralization nodules compared with L-Glu group in the late stage (14 days, 21 days) of osteogenic differentiation. However, there was no significant difference in ALP staining between the two groups in the early stage (1 day, 7 days) of osteogenic differentiation . Furthermore, the mRNA expression levels of Runx2, Osx, Col1a1, and ALP were reduced in H-Glu group at day 14, while there was no significant difference between H-Glu and L-Glu group at day 1, and only Osx expression decreased significantly in the H-Glu group at day 7 . Consistently, the western blot (WB) results showed that the protein levels of Runx2 and Osx were decreased in H-Glu group at day 14 . These results suggest that high glucose inhibits the late stage of osteogenic differentiation in MC3T3-E1 cells. Figure 1 High glucose inhibited osteoblastogenic differentiation and respiration rate of MC3T3-E1 cells The concentration of glucose in the medium of the L-Glu group was 5.5 mmol/L, and that in the medium of the H-Glu group was 25 mmol/L. (A) ALP staining was performed in L-Glu and H-Glu cultured MC3T3-E1 cells after 1 and 7 days of osteogenic induction. Alizarin red S staining was performed in L-Glu and H-Glu cultured MC3T3-E1 cells after 14 and 21 days of osteogenic induction. Scale bars, 500 μm. (B–D) MC3T3-E1 cells were cultured in an osteogenic medium for 1, 7, and 14 days. Expressions of osteogenic markers at different time points were determined by RT-qPCR. (E) MC3T3-E1 cells were cultured in osteogenic medium for 14 days. Expressions of Runx2 and Osx were determined by WB. (F–Q) MC3T3-E1 cells were cultured in osteogenic medium for 1, 7, and 14 days. Mitochondrial oxidative capacity and glycolytic capacity were measured in real time. Basal respiration, ATP-linked respiration, maximal respiration, spare respiratory capacity, basal, proton leak, glycolysis, glycolytic capacity, and glycolytic reverse were calculated by WAVE software. Data presented as mean ± SD. n = 3 biological replicates. One-way ANOVA was used for comparison among multiple groups. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. Ns, no significance. Since the disruption of energy metabolism in osteoblasts may lead to impaired osteoblast function, 25 , 26 we then detected two key energy metabolic pathways, aerobic respiration and glycolysis in MC3T3-E1 cells using the Seahorse extracellular flux analyzer. On day 1 of osteogenic differentiation, no noteworthy distinctions of basal respiration, maximal respiration, spare respiration capacity, and ATP-linked respiration, which are pivotal parameters that underscore mitochondrial functionality, were observed between H-Glu group and L-Glu group . As to cellular glycolytic function, H-Glu group exhibited elevated glycolysis and glycolytic capacity compared to the L-Glu group, with no statistically significant variance in glycolytic reversal between the two groups . Subsequent to 7 days of osteogenic induction, exclusive reductions in maximal respiration and glycolysis were discerned in H-Glu group relative to L-Glu group . Following a 14-day osteogenic differentiation period, the mitochondrial stress test revealed a substantial attenuation in basal, maximal, and ATP-linked respiration in H-Glu cells as contrasted with those in L-Glu group . Simultaneously, outcomes from the glycolytic rate examination exhibited notable diminutions across glycolysis, glycolytic capacity, and glycolytic reversal in H-Glu cells in comparison to L-Glu counterparts . Collectively, these findings provide evidence that the late stage of osteogenic differentiation is notably affected by a discernible inhibition of osteoblastic mitochondrial function induced by the high glucose level. Mitochondrial proliferation may indicate the engagement of feedback loops to compensate for compromised mitochondrial function. Alterations in gene expression profile involved in mitochondrial biogenesis, and changes of concentrations and activities of candidate respiratory complexes in cells may lead to energetic dysfunction. 27 Hence, the peroxisome proliferator-activated receptor gamma coactivator 1a (PGC-1α), nuclear respiratory factors (NRF-1 and NRF-2), and mitochondrial transcription factor A (TFAM) that are the key regulators of mitochondrial biogenesis were determined in osteoblasts. The results showed that there was no difference in the expression of the aforementioned genes between H-Glu and L-Glu groups . Figure 2 High glucose did not affect mitochondrial biogenesis but elevated ROS production and decreased mitochondrial membrane potential (MMP) in MC3T3-E1 cells (A) MC3T3-E1 cells were cultured in osteogenic medium for 14 days. The total DNA of the cells was extracted. Using HK2 as reference gene, expression ND1 and 16s were determined by RT-qPCR. (B) MC3T3-E1 cells were cultured in osteogenic medium for 14 days. Expressions of mitochondrial biogenesis markers were determined by RT-qPCR. (C) MC3T3-E1 cells were cultured in osteogenic medium for 14 days. Expressions of respiratory chain complexes were determined by WB. (D–F) MC3T3-E1 cells were cultured in osteogenic medium for 14 days. ROS generation was observed by using flow cytometry following staining with DCFH-DA. (G and H) MC3T3-E1 cells were cultured in osteogenic medium for 14 days. MMP of living cells was assessed by the Image-iT TMRM Reagent. Scale bars, 50 μm. Data presented as mean ± SD. n = 3 biological replicates. A t test was used for comparison between two groups. One-way ANOVA was used for comparison among multiple groups. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. Ns, no significance. Mitochondrial biogenesis involves the integration of multiple transcriptional pathways controlling both nuclear and mitochondrial gene expression. For example, mitochondrial DNA (mtDNA) transcription is activated by the family of PGC-1 proteins (PGC-1α and PGC-1β), from which PGC-1α is considered the master regulator of mitochondrial biogenesis. The pathway is initiated by PGC-1α activation, followed by stimulation of a series of nuclear transcription factors, that is the NRF-1 and NRF-2 and by the increase in expression of TFAM, the final effector of mtDNA transcription and replication. 28 To further measure mitochondrial biogenesis, we quantitated relative mtDNA copy number by qPCR in MC3T3-E1 cells. As shown in Figure 2 B, mtDNA copy number in osteoblasts of H-Glu group remained similar to L-Glu group. Additionally, we examined the relative levels of the 5 OXPHOS complexes in mitochondria by WB. The result was in accordance with the mitochondrial biogenesis gene expression. Mitochondrial respiratory chain complex protein levels were not significantly different between the two groups . These results demonstrate that high glucose level does not affect the mitochondrial biogenesis of MC3T3-E1 cells and suggest that high glucose level may affect mitochondrial function through other mechanisms. Mitochondria is one of the main sources of ROS, as it utilizes oxygen for energy production. Mitochondrial dysfunction may lead to increased radical production and consequent “oxidative stress.” Therefore, cell ROS level is one of the recognized indicators to evaluate mitochondrial function. 29 The flow cytometry results in this study showed that the number of positively stained cells in H-Glu group was significantly higher than that in L-Glu group . Fluorescence intensity quantitative analysis revealed that the ROS level in H-Glu group was significantly higher than that in L-Glu group . Mitochondrial function can also be assessed through monitoring changes in MMP. A decrease in the MMP indicates mitochondrial dysfunction. 30 We examined the MMP using a cell-permeant dye tetramethylrhodamine (TMRM), which can accumulate in active mitochondria with intact membrane potentials. The TMRM signals in living cells were observed by confocal laser microscopy, quantitative analysis of TMRM fluorescence intensity in images showed that the average fluorescence intensity of cells in H-Glu group was 36% lower than that in L-Glu group. The MMP of H-Glu group cells were reduced . These results suggest that high glucose level impairs mitochondrial function in MC3T3-E1 cells. The aforementioned results demonstrated that high glucose level inhibited the late stage of osteogenic differentiation and impaired mitochondrial function of these cells. Then, what is its molecular mechanism? To address this question, MC3T3-E1 cells cultured in low glucose or high glucose osteoblastogenic induction media for 14 days were examined by RNA sequencing (RNA-seq) analysis . Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that the major pathways involved in the differential genes between the two groups were mitophagy, autophagy, and cell cycle . Gene ontology (GO) functional enrichment showed that the major pathways involved in the differentially expressed genes were ubiquitin proteasome system and mitophagy . As shown in Figure 3 E, further analysis of mitophagy genes revealed that transcription levels of PINK1 and dynamic-associated protein 1 (Drp1) were significantly reduced in H-Glu group compared to L-Glu group. We further verified the expression of Drp1 and PINK1 by qPCR . It is worth mentioning that we also detected mitophagy marker genes of non-ubiquitination-dependent pathways by qPCR and there were no significantly difference between the two groups . These findings suggest that marker gene PINK1 of mitophagy signaling pathway and Drp1 may be important factors involved in the mechanism by which high glucose inhibits osteogenic differentiation. Figure 3 RNA-seq data analysis of differentially expressed genes in MC3T3-E1 cells cultured in L-Glu and H-Glu MC3T3-E1 cells were cultured in osteogenic medium for 14 days, then eukaryotic transcriptome sequencing was performed. (A) The volcano map showed significant differences in gene expression between the two groups. (B) The enrichment analysis of KEGG pathway based on the significantly different genes in the volcano map showed that the main pathways involved in the damage of osteoblasts by high sugar were mitochondrial autophagy, cell autophagy and cell cycle. (C) GO ontology functional enrichment showed that the main pathways involved in the damage of osteoblasts by high glucose were ubiquitin-proteasome system, ubiquitin-proteasome system, and mitochondrial autophagy. (D) The KEGG pathway loop map showed the main mechanisms of hyperglycemic damage: mitochondrial autophagy, autophagy, and cell cycle. (E) Genes of interest with significant differences. n = 4 biological replicates. (F) MC3T3-E1 cells were cultured in osteogenic medium for 14 days. Expressions of PINK1 and Drp1 were determined by RT-qPCR. (G–I) MC3T3-E1 cells were cultured in osteogenic medium for 1, 3, 7, and 14 days. Expressions of marker genes involved in ubiquitin-dependent mitophagy pathway at different time points were determined by RT-qPCR. Data presented as mean ± SD. n = 3 biological replicates. One-way ANOVA was used for comparison among multiple groups. A t test was used for comparison between two groups. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. Ns, no significance. To further validate the functionality of PINK1 and Drp1 in osteogenic differentiation, we inhibited the expression of PINK1 and Drp1 in MC3T3-E1 cells by siRNA. Figures 4 A and 4B demonstrated that the expression of PINK1 and Drp1 genes was significantly knocked down. We substantiated the involvement of PINK1/Drp1 signaling in the process of osteogenic differentiation through gene knockdown under normal glucose culture. Evidently, the targeted suppression of PINK1 or Drp1 significantly inhibited the expression levels of Runx2 and Osx . We also observed that inhibiting the expression of PINK1 resulted in the downregulation of Drp1 protein. Conversely, inhibiting Drp1 did not affect the level of PINK1 protein, implying that PINK1 acts as the upstream signaling molecule of Drp1 . Consistently, the quantification of mineralized nodules within osteoblasts, following the downregulation of PINK1 and Drp1, exhibited a substantial reduction compared to the control group . Figure 4 High glucose inhibited PINK1/Drp1 signaling, leading to impaired osteoblastogenic differentiation (A and B) Expressions of PINK1 and Drp1 genes were determined by RT-qPCR after siRNA transfection. (C) MC3T3-E1 cells were cultured in osteogenic medium (L-Glu) for 14 days after siRNA transfection. Expressions of PINK1, Drp1, Runx2, and Osx were determined by WB. (D) MC3T3-E1 cells were cultured in osteogenic medium (L-Glu) for 21 days after siRNA transfection. Alizarin red S staining was performed. Scale bars, 500 μm. (E) Expression of PINK1 gene was determined by RT-qPCR after PINK1 plasmid transfection. (F) MC3T3-E1 cells were cultured in osteogenic medium (H-Glu) for 14 days after plasmid transfection. Expressions of PINK1, Drp1, Runx2, and Osx were determined by WB. (G) MC3T3-E1 cells were cultured in osteogenic medium (H-Glu) for 21 days after plasmid transfection. Alizarin red S staining was performed. Scale bars, 500 μm. (H) MC3T3-E1 cells were cultured in osteogenic medium (L-Glu) for 14 days after siRNA transfection. MMP of living cells was assessed by the Image-iT TMRM Reagent. Scale bars, 20 μm. (I) MC3T3-E1 cells were cultured in osteogenic medium (L-Glu) for 14 days after siRNA transfection. Mitochondrial reactive oxygen species of living cells were assessed by the MitoSox Reagent. Scale bars, 50 μm. (J) MC3T3E1 cells were transfected with the mito-Keima plasmid and cultured in osteogenic medium (L-Glu) for 14 days. The fluorescent dots of mito-Keima were observed by confocal microscopy. Scale bars, 20 μm. (K) MC3T3-E1 cells were cultured in osteogenic medium (H-Glu) for 14 days after plasmid transfection and siRNA transfection. Expression of PINK1, Drp1, Runx2, and Osx were determined by WB. (L) MC3T3-E1 cells were cultured in osteogenic medium (H-Glu) for 14 days after plasmid transfection and siRNA transfection. Alizarin red S staining was performed. Scale bars, 500 μm. (M) MC3T3-E1 cells were cultured in osteogenic medium (H-Glu) for 14 days after plasmid transfection and siRNA transfection. MMP of living cells was assessed by the Image-iT TMRM Reagent. Scale bars, 50 μm. (N) MC3T3-E1 cells were cultured in osteogenic medium (H-Glu) for 14 days after plasmid transfection and siRNA transfection. Mitochondrial reactive oxygen species of living cells were assessed by the MitoSox Reagent. Scale bars, 50 μm. (O) MC3T3-E1 cells were cultured in osteogenic medium (H-Glu) for 14 days after plasmid transfection and siRNA transfection. The fluorescent dots of mito-Keima were observed by confocal microscopy. Scale bars, 50 μm. Data presented as mean ± SD. A t test was used for comparison between two groups. One-way ANOVA was used for comparison among multiple groups. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. Ns, no significance. We further constructed an overexpressed PINK1 plasmid and transfected osteoblasts. Then, we induced osteogenic differentiation of MC3T3-E1 cells in high glucose medium. Figures 4 E and 4F showed that the expression of PINK1 gene and protein were significantly increased after transfection. Notably, this augmentation in PINK1 expression coincided with noteworthy enhancements in the expressions of Runx2 and Osx . Alizarin red staining showed that the number of mineralized nodules of osteoblasts overexpressing PINK1 increased significantly under high glucose medium culture . We also examined the mitochondrial function of osteoblasts that had knocked down PINK1 and Drp1, respectively. In Figure 4 H, TMRM staining was performed on living cells in each group to detect MMP. The results showed that inhibiting the expression of either Drp1 or PINK1 could decrease the MMP of cells. In Figure 4 I, MitoSox was used to detect mitochondrial reactive oxygen species, and the results showed that knockdown of Drp1 or PINK1 could increase the superoxide level of cellular mitochondria. In Figure 4 J, MCT3E1 cells were transfected with pMito-Keima to detect mitophagy, and the results showed that mitophagy was weakened in cells with the Drp1 or PINK1 knocked down. To verify Drp1 is a downstream target of PINK1, we further knocked down Drp1 in the presence of overexpression of PINK1 in MCT3E1 cells and examined the osteogenic phenotype and mitochondrial function of osteoblasts in high glucose medium. Figures 4 K and 4L showed that the protein levels of Runx2 and Osterix of cells in high glucose medium were significantly increased when PINK1 overexpressed, and the formation of mineralized nodules was increased; while Drp1 depletion significantly weakened this phenotype. Correspondingly, the MMP decreased and the level of mitochondrial superoxide of PINK1 overexpressed cells in high glucose medium increased significantly after Drp1 knockdown . Furthermore, the level of mitophagy of PINK1-overexpressed cells in high glucose medium was also significantly decreased after Drp1 was knocked down . Collectively, these findings indicate that the PINK1/Drp1 signaling exerts positive influence on mitochondrial function of osteoblasts and osteogenic differentiation. Overexpression of PINK1 can rescue the impaired osteogenic differentiation under high glucose medium. The aforementioned results show that high glucose level inhibits osteogenic differentiation and impairs mitochondrial function of MC3T3-E1 cells, so we are interested in whether there are molecules that can protect the mitochondrial function and promote osteogenic differentiation of MC3T3-E1 cells exposed to high glucose level. Our previous mice studies have demonstrated that bone morphogenetic protein 9 (BMP9) is effective in improving both ovariectomized (OVX) and senile osteoporosis. 31 , 32 In this study, an obvious less mineralized nodule formation was observed in H-Glu group compared to L-Glu group. However, the impaired osteogenic differentiation induced by high glucose treatment was partially reversed by BMP9 treatment: mineralized nodule formation was increased after BMP9 treatment relative to L-Glu group . Moreover, expression levels of ALP, Runx2, Osx, Ocn, and Col1a1 were significantly increased after BMP9 treatment . Consistently, the WB results showed that the protein levels of Osx and Runx2 were increased after BMP9 treatment . Figure 5 BMP9 reversed both the osteoblastogenic differentiation impairment and mitochondrial dysfunction induced by high glucose in MC3T3-E1 cells The concentration of glucose in the medium of the L-Glu group was 5.5 mmol/L, and that in the medium of the H-Glu and H-Glu+BMP9 group was 25 mmol/L. The final concentration of BMP9 was 100 ng/ml. (A) Alizarin red S staining was performed in MC3T3-E1 cells after 21 days of osteogenic induction. Scale bars, 500 μm. (B) MC3T3-E1 cells were cultured in osteogenic medium for 14 days. Expressions of osteogenic markers were determined by RT-qPCR. (C) MC3T3-E1 cells were cultured in osteogenic medium for 14 days. Expressions of Runx2 and Osx were determined by WB. (D and E) MC3T3-E1 cells were cultured in osteogenic medium for 14 days. Mitochondrial oxidative capacity was measured in real time. Basal respiration, ATP-linked respiration, maximal respiration, and spare respiratory capacity were calculated by WAVE software. (F and G) MC3T3-E1 cells were cultured in osteogenic medium for 14 days. MMP of living cells was assessed by the Image-iT TMRM Reagent. Scale bars, 50 μm. (H and I) MC3T3-E1 cells were cultured in osteogenic medium for 14 days. Mitochondrial reactive oxygen species of living cells were assessed by the MitoSox Reagent. Scale bars, 50 μm. Data presented as mean ± SD. n = 3 biological replicates. One-way ANOVA was used for comparison among multiple groups. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. Ns, no significance. We used extracellular flux analysis to real-time monitor mitochondrial respiratory capacity in vitro . BMP9 treatment dramatically increased basal, ATP production-coupled respiration, maximal respiration, and the spare respiratory capacity . Furthermore, the result of TMRM signal showed that the MMP was significantly increased after BMP9 treatment . We also examined superoxide in the mitochondria of live MC3T3-E1 cells using mitochondrial superoxide indicator MitoSOX Red. In the MC3T3-E1 cells cultured in high glucose, superoxide production was higher than that in control cells, and BMP9 significantly reduced superoxide production . Collectively, these data indicate that BMP9 could improve mitochondrial function of MC3T3-E1 cells under high glucose medium culture. To explore the regulatory mechanism of BMP9 on mitophagy, we inhibited the expression of PINK1 and Drp1 in cells by siRNA. After Drp1 or PINK1 depletion, the protein levels of Runx2 and Osterix of cells in H-Glu+BMP9 group were significantly decreased , and the formation of mineralized nodules was inhibited, as compared with only treated with BMP9 . Correspondingly, after PINK1 or Drp1 knockdown, the MMP decreased, and the level of mitochondrial superoxide of cells in H-Glu+BMP9 group increased significantly . Furthermore, the level of mitochondrial autophagy of cells in H-Glu+BMP9 group was also significantly decreased after Drp1 or PINK1 was knocked down . Figure 6 BMP9 activated PINK1/Drp1-mediated mitophagy, leading to the reversal of high glucose-induced osteoblast differentiation impairment in MC3T3-E1 cells The concentration of glucose in the medium of the L-Glu group was 5.5 mmol/L, and that in the medium of the H-Glu, H-Glu+BMP9, Drp1 siRNA and PINK1 siRNA groups was 25 mmol/L. The final concentration of BMP9 in H-Glu+BMP9, Drp1 siRNA and PINK1 siRNA groups was 100 ng/ml. (A) MC3T3-E1 cells were cultured in osteogenic medium for 14 days after siRNA transfection. Expressions of PINK1, Drp1, Runx2, and Osx were determined by WB. (B) MC3T3-E1 cells were cultured in osteogenic medium for 21 days after siRNA transfection. Alizarin red S staining was performed. Scale bars, 500 μm. (C) MC3T3-E1 cells were cultured in osteogenic medium for 14 days after siRNA transfection. MMP of living cells was assessed by the Image-iT TMRM Reagent. Scale bars, 20 μm. (D) MC3T3-E1 cells were cultured in osteogenic medium for 14 days after siRNA transfection. Mitochondrial reactive oxygen species of living cells were assessed by the MitoSox Reagent. Scale bars, 20 μm. (E) MC3T3E1 cells were transfected with mito-Keima plasmid and siRNA and cultured in osteogenic medium for 14 days. The fluorescent dots of mito-Keima were observed by confocal microscopy. Scale bars, 20 μm. (F) Representative electron micrographs show mitochondria and mitophagosomes (arrowhead) in MC3T3-E1 cells of different groups. Scale bars, 500 nm. Data presented as mean ± SD. A t test was used for comparison between two groups. One-way ANOVA was used for comparison among multiple groups. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. Ns, no significance. We further observe the ultrastructure of mitochondria of MC3T3-E1 cells by transmission electron microscope. Figure 6 F showed mitochondria of L-Glu cells showed a bilayer-membrane rod-like structure with clear mitochondrial ridge. Mitochondria of cells in H-Glu group were swollen, and mitochondrial ridge structure was destroyed and disappeared. The ridge structure of some mitochondria in H-Glu+BMP9 group was blurred, and the damaged mitochondria could be seen to phagocytosed and degraded by double-layer membrane structure . While mitochondrial membrane structure was damaged but mitochondrial autophagy was not observed in PINK1 or Drp1 knockdown groups. Our results indicate that BMP9 effectively ameliorates osteogenic differentiation of cells under high glucose condition by activating PINK1/Drp1 signaling axis. We conducted experiments to explore whether high glucose inhibits osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) through the mitophagy-associated PINK1/Drp1 signaling pathway and whether BMP9 has a reversal effect. After Drp1 or PINK1 depletion, the protein levels of Runx2 and Osterix of cells in H-Glu+BMP9 group were significantly decreased , and the formation of mineralized nodules was inhibited, as compared with only treated with BMP9 . Correspondingly, the MMP of cells in H-Glu+BMP9 group significantly decreased after PINK1 or Drp1 knockdown . Furthermore, the level of mitophagy of cells in H-Glu+BMP9 group was also significantly decreased after Drp1 or PINK1 was knocked down . The results indicate that BMP9 can also effectively ameliorate osteogenic differentiation of BMSCs under high glucose by augmenting the activity of the PINK1/Drp1 signaling axis. Figure 7 BMP9 activated PINK1/Drp1-mediated mitophagy, leading to the reversal of high glucose-induced osteoblast differentiation impairment in BMSCs The concentration of glucose in the medium of the L-Glu group was 5.5 mmol/L, and that in the medium of the H-Glu, H-Glu+BMP9, Drp1 siRNA and PINK1 siRNA groups was 25 mmol/L. The final concentration of BMP9 in H-Glu+BMP9, Drp1 siRNA and PINK1 siRNA groups was 100 ng/ml. (A) BMSCs cells were cultured in osteogenic medium for 14 days after siRNA transfection. Expressions of PINK1, Drp1, Runx2, and Osx were determined by WB. (B) BMSCs cells were cultured in osteogenic medium for 21 days after siRNA transfection. Alizarin red S staining was performed. Scale bars, 1 mm. (C) BMSCs cells were cultured in osteogenic medium for 14 days after siRNA transfection. MMP of living cells was assessed by the Image-iT TMRM Reagent. Scale bars, 20 μm. (D) BMSCs cells were transfected with mito-Keima plasmid and siRNA and cultured in osteogenic medium for 14 days. The fluorescent dots of mito-Keima were observed by confocal microscopy. Scale bars, 20 μm. To further verify the effects of BMP9 on bone mineral density (BMD) and bone quality in vivo , we constructed an streptozotocin (STZ) induced diabetes mice model. The design of the animal experiment is shown in Figure 8 A. All diabetic mice remained hyperglycemic until the end of the study. Although serum BMP9 concentration was higher in STZ+BMP9 group, blood glucose was similar between STZ and STZ+BMP9 group. . Figure 8 Diabetic mice models induced by STZ were treated with BMP9 (A) Flow charts for the establishment of STZ mice model. (B) Fasting blood glucose concentrations were measured and compared every two weeks from week 2 to week 18. (C) Fasting blood glucose concentrations at week 18 were compared between groups. (D) Serum BMP9 levels were detected at week 18. Data presented as mean ± SD. n = 8 biological replicates. One-way ANOVA was used for comparison among multiple groups. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. Ns, no significance. As shown in Figure 9 A, a 3D reconstruction of the cortical bone and trabecular bone at the distal femurs clearly showed thinner cortical bone and the breakage of cancellous bone in STZ diabetic mice, and the administration of BMP9 improved the cortical bone thickness and microarchitecture of the distal femur trabecular bone. Moreover, micro-CT analysis of the distal femur showed decreased cortical BMD in the STZ mice and improved BMD in the STZ+BMP9 group ( p < 0.05) . Other cortical bone parameters, including BV/TV and Ct.Th, were significantly decreased in STZ mice as compared with controls ( p < 0.05). The Ct.Th value of STZ mice treated with BMP9 increased compared to that in the STZ group ( p < 0.05) , while the difference in BV/TV value between the two groups was not statistically significant . Micro-CT analysis also showed that compared with mice in the control group, STZ mice exhibited obvious decrease in BV/TV, Tb.N, and BMD and increased Tb.Sp of the distal femur trabecular bone, while the BV/TV, Tb.N, and BMD of mice treated with BMP9 increased by 43% ( p < 0.05), 30% ( p < 0.05), and 29% ( p < 0.01), respectively . Consistent with the micro-CT analysis results, H&E staining of the distal femur sections confirmed a reduction in trabecular number and trabecular thickness in STZ mice compared with that of the control, and these reductions were attenuated by BMP9 treatment . Figure 9 BMP9 promoted mitophagy markers to the control levels in diabetic mice in vivo (A) Representative images derived from micro-CT analysis, including 2D image construction of distal femur, 3D images reconstruction of trabecular bone of distal femur, and 3D image reconstruction of the femoral midshaft corticoid bone. (B–D) Quantitative analysis of the vBMD, BV/TV and Ct.Th of corticoid bone by micro-CT. (E–I) Quantitative analysis of the BV/TV, Tb.N, Tb.Sp, Tb.Th, and vBMD of trabecular by micro-CT. (J) Representative images of HE-stained decalcified femur sections. Scale bars, 200 μm. (K–M) The right femur was isolated and subjected to biomechanical properties analysis. The maximum load, elastic modulus and SMI were evaluated for each group. (N) Representative images derived from micro-CT analysis, including 2D image construction and 3D image reconstruction of L3 lumbar vertebra. (O–S) Quantitative analysis of the vBMD, BV/TV, Tb.N, Tb.Th, and Tb.Sp of L3 by micro-CT. (T and U) The levels of serum bone turnover parameters PINP and CTX-I were detected by ELISA. (V) WB analysis of protein levels of mitophagy marker in skull. (W) Expressions of ALP, Osx, Drp1, and PINK1 in skull were determined by RT-qPCR. (X) Immunofluorescence analysis of Drp1 and PINK1 expression in femur sections. Scale bars, 10 μm. (Y) Immunofluorescence analysis of PINK1 expression in femur sections. Scale bars, 20 μm. (Z) Immunofluorescence results of double labeling of osteoblasts with PINK1 and Drp1 in femur sections. Scale bars, 20 μm. Data presented as mean ± SD. n = 8 biological replicates. One-way ANOVA was used for comparison among multiple groups. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. Ns, no significance. Furthermore, compared with the control group, the maximum load and Young’s modulus of femur in STZ group were significantly decreased ( p < 0.05), the maximum load and elastic modulus of femur in STZ+BMP9 group were increased by 27.1% ( p < 0.01), and the value of elastic modulus was increased by 21.6% compared with STZ mice, but there was no statistical difference. The structural simulation index was not significantly different from that of STZ group . Additionally, we examined the parameters of lumbar vertebrae in mice. The bone density and 3D reconstruction images of the third lumbar vertebra in mice were shown in Figure 9 M. Compared with the normal control group, the lumbar-spine BMD in the STZ group decreased by 16.9% ( p < 0.01) and increased by 14.1% ( p < 0.01) in STZ+BMP9 group. The BV/TV, Tb.N, and Tb.Th values of lumbar cancellous bone in the STZ group were significantly decreased compared with the control group, and the Tb.Sp value was significantly increased in the STZ mice. Compared with the STZ group, the BV/TV and Tb.N values of lumbar cancellous bone in the STZ+BMP9 group were increased by 10.7% and 7.5%, respectively, and there was no significant difference compared with the control group. The Tb.Sp and Tb.Th values of lumbar vertebrae in STZ+BMP9 group were not significantly different from those in STZ group . We then examined bone turnover markers in mice. Compared with the normal control group, the serum procollagen I N-terminal propeptide (PINP) level of mice in STZ group was significantly decreased ( p < 0.01), but was significantly increased in STZ+BMP9 group compared with the STZ mice group ( p < 0.01) . There was no significant difference in serum C-terminal telopeptide of type 1 collagen (CTX-I) concentration between three groups . These results suggest that BMP9 has the ability to ameliorate diabetic osteoporosis. The favorable bone effect of BMP9 in diabetic STZ mice was investigated mechanistically. The protein from parietal bone in mice was quantified by WB. The results demonstrated that the expressions of mitophagy related proteins PINK1 and Drp1 were significantly increased in mice treated with BMP9, and the ratio of autophagy marker proteins LC3A/BⅡ and LC3A/BⅠ were significantly increased in STZ+BMP9 group . Moreover, qPCR results showed expression levels of ALP, Osx, Drp1, and PINK1 were significantly increased in BMP9 treatment group . We further stained the bone sections of the femur in mice by immunofluorescence. As shown in Figure 9 X, the expression of Drp1 protein in osteoblasts located on the bone trabecular surface of mice in STZ group was significantly decreased compared with that of control mice, and was significantly increased after BMP9 administration, which was consistent with the variation trend of PINK1 in Figure 9 Y. In order to further confirm the aforementioned results, double-label immunofluorescence staining was performed on the femur section. Osx was used to label osteoblasts and the expression of PINK1 in osteoblasts was detected. Compared with the control group, the expression of PINK1 in osteoblasts in the STZ group was significantly decreased. The expression of PINK1 in osteoblasts in STZ+BMP9 group was significantly higher than that in STZ group . These results suggested that BMP9 can promote the expression of mitophagy protein in bone tissue of diabetic mice, promoting mitophagy may be an effective means to improve the bone mass and quality in diabetic mice. In this study, we revealed that the suppressed PINK1/Drp1-mediated mitophagy is a molecular pathway responsible for impaired osteogenic differentiation in diabetes. Activation of mitophagy by BMP9 can mitigate osteoporosis induced by hyperglycemia. Osteoblast-mediated bone formation requires enough ATP, which is generated through OXPHOS and glycolysis. 33 Our research found that high glucose condition lasting to the late stage of differentiation could induce the decrease in basal and maximal respiration, ATP synthesis, and glycolytic rate of osteoblast. We also found mitochondrial dysfunctions, including the increased ROS, and decreased MMP in osteoblast cultured under high glucose level. Our RNA sequencing analysis suggested that impaired mitophagy might be the molecular mechanism for high glucose-induced inhibition of osteogenic differentiation. Upon mitochondrial stress, mitophagy is activated and can selectively eliminate the damaged mitochondria, and thus stabilizing cellular homeostasis. 34 Autophagy and mitophagy play a vital role in regulating osteoblasts’ proliferation and differentiation. Thus, we further explored the role of mitophagy in high glucose-induced inhibition of osteogenic differentiation and its related signaling pathways. PINK1 is an important promotor of mitochondrial autophagy. When MMP declines, the Ser228 and Ser402 groups of PINK1 will be phosphorylated, 35 upon activation of PINK1 phosphorylation, ubiquitin on the mitochondrial outer membrane can be phosphorylated, then initialing mitophagy. We found that siRNA knockdown of PINK1 inhibited the ameliorative effect of BMP9 to impaired mitophagy and osteogenic differentiation in high glucose conditions, while overexpression of PINK1 can reverse the inhibitory effect of high glucose level on osteogenic differentiation. Mitophagy and mitochondrial dynamics are close related. Drp1, a GTPase of the dynein superfamily, is the most important molecule in promoting mitochondrial fission. After Drp1 is recruited into the outer membrane of mitochondria, it will form helical oligomers, thereby inducing contraction and severing of the mitochondrial membrane. 36 Our RNA sequencing analysis showed the decreased expression level of Drp1 in osteoblasts cultured in high glucose medium, suggesting the potential interplay between Drp1 and PINK1-induced mitophagy in impaired osteogenic differentiation caused by hyperglycemia. Then, we conducted a series of experiments to address the regulatory effect between Drp1 and PINK1. We found that the expression of Drp1 was downregulated after the knockdown of PINK1. In contrast, the expression of PINK1 did not change following the inhibition of Drp1, suggesting Drp1 as a downstream signaling molecule of PINK1. Our results also demonstrated that Drp1 knockdown can inhibit osteogenic differentiation and mitophagy and impair mitochondrial function. PINK1 was shown to directly phosphorylate S616 of Drp1 to regulate mitochondrial fission, 37 which was consistent with our results. Taken together, we hypothesized that PINK1/Drp1-mediated mitophagy plays a critical role in osteogenic differentiation, and its suppression contributes to hyperglycemia-induced impaired osteoblastogenesis. To further verify this hypothesis, we then tried to find a molecule to activate this signaling pathway to maintain mitophagy of osteoblast in hyperglycemia and treat diabetic osteoporosis in vivo . BMP9, a member of the transforming growth factor beta (TGF-β) superfamily, regulates various physiological processes such as angiogenesis, glucose metabolism, neurogenesis, and tumorigenesis. Our previous research found that BMP9 promotes osteoblastogenesis by upregulating LGR6 and activating the Wnt/β-catenin pathway and suppresses osteoclast differentiation by inhibiting the nuclear factor-κB (NF-κB) pathway. 31 Our team’s other research also found that BMP9 reduces expression of senescent genes and alleviates senescence-associated secretory phenotype in bone microenvironment, simultaneously increasing bone mass and bone biomechanical properties in aged mice. 32 As a continuation of work, we investigated the regulatory role of BMP9 on mitophagy in osteoblast and its protective effect on diabetic osteoporosis in this study. In vitro , we observed that BMP9 improved the osteogenic differentiation of MC3T3-E1 cells under high glucose conditions. This protective effect of BMP9 on osteoblasts could be mediated by the regulation of mitophagy through its capacity to upregulate PINK1/Drp1 expression. Whereas knockdowns of PINK1 and Drp1 can abolish the bone protective function of BMP9 under high glucose conditions. Mitophagy dysfunction participates in pathogenesis diabetes and diabetes-related complications that include β-cell damage, 38 glucose intolerance, 39 cardiomyopathy, 40 insulin resistance, 41 and diabetes-associated cognitive dysfunction. 42 Previous study has found that non-imprinted protein 2 in the Prader-Willi/Angelman syndrome region (NIPA2), a mitophagy-related molecule, was decreased in the bone tissue of diabetic mice. 43 Our research found that PINK1/Drp1-mediated mitophagy was inhibited in the bone of diabetic mice and its activation by BMP9 ameliorated diabetic osteoporosis, indicating that PINK1/Drp1-mediated mitophagy may be a therapeutic target for diabetic osteoporosis. BMP9 is known to be a powerful promoter of osteogenic differentiation. Our research found that BMP9 rescued diabetic osteoporosis by addressing various suboptimal bone phenotypes, including promoting bone formation, improving bone density, enhancing the microstructure of cancellous and cortical bones, and ameliorating femur biomechanics. Notably, all these favorable bone effects of BMP9 in diabetes are not related to changes in blood glucose. These results suggest that BMP9 may act directly and specifically on bone tissue in diabetic mice, and this advantage makes BMP9 a potential therapeutic drug for diabetic osteoporosis. In conclusion, our findings suggest the suppressed PINK1/Drp1-mediated mitophagy is responsible for the impairment of osteogenic differentiation in diabetes. BMP9 facilitates osteogenic differentiation and maturation under diabetic conditions through activating PINK1/Drp1-mediated mitophagy, ultimately exerting a bone-protective effect in diabetic mice. Further research on PINK1/Drp1-mitophagy pathway and its agonists, such as BMP9, is a promising way to explore the potential pathogenesis and therapeutic strategy for osteoporosis in diabetes. This study had several limitations. Firstly, the precise mechanism through which BMP9 activates PINK1/Drp1 signaling pathway requires further elucidation. Secondly, the late onset of skeletal phenotype in diabetic mice led to a limited number of extracted skull primary cells or mesenchymal stem cells with diminished activity. Thirdly, we did not use osteoblast conditional gene knockout mouse models to verify the impaired PINK1/Drp1-mediated mitophagy as a pathogenesis of diabetic osteoporosis. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse Anti-Hsp90 antibody Santa Cruz Cat# sc-13119 Rabbit Anti-PINK1 antibody Novus Cat# BC100-494S Rabbit Anti-Drp1 antibody Abcam Cat# ab184247 Rabbit Anti-Runx2 antibody CST Cat# 8486 Rabbit Anti-Osx antibody Abcam Cat# ab209484 Anti-VDAC1/Porin antibody Abcam Cat# ab306581 LC3A/B antibody CST Cat# 4108 HRP-conjugated Alpha Tubulin antibody Proteintech Cat# HRP-66031 HRP-linked Anti-Mouse antibody CST Cat# 7076 HRP-linked Anti-Rabbit antibody CST Cat# 7074 CF 488 tyramide Runnerbio China Cat# Bry-880488 AF 647 tyramide Runnerbio China Cat# Bry-880647 Chemicals, peptides, and recombinant proteins α-modified minimal essential medium Gibco Cat# 32571101 Fetal bovine serum Gibco Cat# 10091148 Penicillin/streptomycin Beyotime Cat# C0222 β-glycerophosphate disodium salt hydrate Sigma-Aldrich Cat# 50020 L-ascorbic acid Sigma-Aldrich Cat# A4544 Lipofectamine 2000 Invitrogen Cat# 11668027 Streptozotocin Sigma-Aldrich Cat# S0130 MitoSOX™ Red mitochondrial superoxide indicator Invitrogen Cat# M36008 Image-iT tetramethylrhodamine (TMRM) reagent Invitrogen Cat# I34361 Hoechst 33342 Invitrogen Cat# H3570 Seahorse XF DMEM medium Agilent Cat# 103575–100 Hematoxylin Staining Solution Beyotime Cat# C0107 Super sensitive ECL luminescence reagent Meilunbio Cat# MA0186 Tri reagent Sigma-Aldrich Cat# T9424 Recombinant Mouse BMP-9 Biolegend Cat# 553204 Critical commercial assays BCIP/NBT Alkaline Phosphatase Color Development Kit Beyotime Cat# C3206 Alizarin Red S Solution Solarbio Cat# G1450 Mitochondrial isolation kit Thermo Scientific Cat# 89874 Bicinchoninic acid (BCA) protein assay kit Thermo Scientific Cat# 23227 ROS assay kit Beyotime Cat# S0033S ELISA Kit for Procollagen I N-Terminal Propeptide (PINP) USCN Life Science Cat# SEA957Hu ELISA Kit for Cross Linked C-Telopeptide Of Type I Collagen (CTXI) USCN Life Science Cat# CEA665Hu Mouse BMP-9 ELISA Kit RayBiotech Cat# Q9WV56 Seahorse XF Cell Mito Stress Test Kit Agilent Cat# 103015–100 PrimeScript reverse transcript master mix TaKaRa Cat# RR036Q ChamQ Universal SYBR qPCR Master Mix Vazyme Cat# Q711-02 Total OXPHOS Rodent WB antibody cocktail Abcam Cat# ab110413 Bicinchoninic acid (BCA) protein assay kit Thermo Scientific Cat# 23227 Deposited data RNA sequencing data This paper Zenodo: https://doi.org/10.5281/zenodo.14174646 Experimental models: Cell lines MC3T3-E1 cells ATCC Cat# CRL-2593 Experimental models: Organisms/strains C57BL/6 mice Charles River Cat# 213 Oligonucleotides PINK1 siRNA GenePharma N/A Drp1 siRNA GenePharma N/A siRNA oligonucleotide sequences, see Table S1 This paper N/A Primer sequence of target genes in RT-qPCR, see Table S2 This paper N/A Recombinant DNA Mitochondria-targeted mKeima-Red expression plasmid MBL Cat# AM-V0251 PINK1 expression plasmid GenePharma N/A Drp1 expression plasmid Shanghai Xitubio Biotechnology N/A AAV-BMP9 Hanbio N/A Software and algorithms Zen software Zeiss N/A R (version 4.3.2) R Core Team https://www.r-project.org GraphPad Prism 9 GraphPad https://www.graphpad.com/features Other Instron 5569 Instron https://www.instron.com/en/products/testing-systems/out-of-production-systems/electromechanical SkyScan 1176 Bruker https://www.accela.eu/bruker-biospin/skyscan-1176 Seahorse Extracellular Flux Analyzer XFe96 Agilent https://www.agilent.com/cs/library/usermanuals/public/usermanual-xfe96-xf96-cell-characterization-cell-analysis-5994-0368en-agilent.pdf QuantStudio Dx Real-Time PCR Instrument Applied Biosystems https://www.thermofisher.com/hk/en/home/clinical/diagnostic-testing/instruments-automation/genetic-analysis-instruments/quantstudio-dx-systems.html NanoDrop ND-2000 spectrophotometer Thermo Scientific Cat# ND-2000C eBlot Touch Imager eBlot https://www.e-blot.com/ JEM-1400Flash electron microscope JEOL https://www.jeol.com/products/scientific/tem/JEM-1400Flash.php TissueFAXS system TissueGnostics, Austria https://tissuegnostics.com/products/scanning-and-viewing-software/tissuefaxs-imaging-software The preosteoblast line MC3T3-E1 cells (American Type Culture Collection) were cultured in α-modified minimal essential medium (α-MEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Beyotime) at 37°C with 5% CO2. The culture medium was changed every two days. BMSCs were isolated from the bone marrow of mice. First, mice were euthanized, followed by the dissection of its tibia, femur, and humerus. The ends of the bones were cut off with sharp scissors. The bone marrow was then flushed out from the bone cavities using α-MEM containing 10% FBS. The aspiration of bone marrow was filtered through a 70-micron mesh, then centrifuged, washed, and resuspended in α-MEM containing 10% FBS. The culture dishes were placed at 37°C in a 5% CO2 incubator. Non-adherent cells were removed after 24–72 h by changing the culture medium. Once the culture reached 70–90% confluence, the cells were subcultured at a 1:3 split ratio. Osteoblastogenic induction medium consisted of α-MEM (Gibco) supplemented with 10% FBS (Gibco), 1% penicillin/streptomycin (Beyotime), 10mM β-glycerophosphate disodium salt hydrate (Sigma-Aldrich), and 50μM L-ascorbic acid (Sigma-Aldrich). The osteoblastogenic induction media were formulated with two concentrations of glucose: low glucose (5.5 mmol/L) and high glucose (25 mmol/L). BMP9 intervention was achieved through the supplementation of 100 ng/mL BMP9 (Biolegend) in the osteoblastogenic induction media. MC3T3-E1 cells or BMSCs were cultured in the osteoblastogenic induction media to induce osteogenic differentiation. Based on the glucose concentrations of the media and BMP9 intervention during osteogenic differentiation, the MC3T3-E1 cells or BMSCs were assigned to three groups: the low-glucose group (L-Glu), the high-glucose group (H-Glu), and the high-glucose group supplemented with BMP9 (H-Glu + BMP9). We purchased commercial and customized siRNAs and plasmids: a PINK1 siRNA (GenePharma), a Drp1 siRNA (GenePharma), a mitochondria-targeted mKeima-Red expression plasmid (pMT-mKeima-Red, MBL), and a PINK1 expression plasmid (GenePharma). MC3T3-E1 cells were transiently transfected with siRNA or plasmids using Lipofectamine 2000 (Invitrogen), following the transfection protocol provided by the Lipofectamine 2000 reagent. The siRNA oligonucleotide sequences were listed in Table S1 . Alp staining of differentiated MC3T3-E1 cells was performed using a 5-bromo-4-chloro-3-indolyl phosphate/tetranitro blue tetrazolium chloride (BCIP/NBT) alkaline phosphatase color development kit (Beyotime) according to the manufacturer’s instructions. In brief, MC3T3-E1 cells were washed and fixed with 4% paraformaldehyde following the 7-day differentiation induction, and the staining solution was applied for 15 min at room temperature. Level of differentiation was quantified by visual examination of cells stained purple. Alizarin red S staining assay (Solarbio) was performed after 14 and 21 days of osteoblastic induction according to the manufacturer’s instructions. In brief, MC3T3-E1 cells were fixed with paraformaldehyde for 10 min. The mineralized nodules were stained with 0.2% Alizarin red and washed three times with PBS. Total protein was extracted by radioimmunoprecipitation (RIPA) lysis buffer (Biocolors, Shanghai, China) supplemented with Protease Inhibitor Cocktail (APExBIO, Houston, USA). Mitochondrial protein fraction was extracted using a mitochondrial isolation kit (Thermo Scientific) for cultured cells according to the manufacturer’s instruction. The concentration of protein was measured using a Bicinchoninic acid protein assay kit (Thermo Scientific), then the degenerated protein lysates were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) for electrophoresis and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). Membranes were blocked in 5% fat-free milk (Sangon Biotech) for 1.5 h at room temperature and then incubated overnight at 4°C with the primary antibodies: mouse anti-Hsp90, rabbit anti-PINK1, rabbit anti-Drp1, rabbit anti-Runx2, rabbit anti-Osx, anti-VDAC1/Porin antibody, LC3A/B antibody and antibodies of OXPHOS complexes I–V from total OXPHOS Rodent WB antibody cocktail (Abcam). HRP-conjugated secondary antibody incubation was performed at room temperature for 1 h. Immunoblotting signals were detected by super sensitive ECL luminescence reagent (Meilunbio, China) and visualized by the eBlot Touch Imager (eBlot, China). For RNA extraction, total RNA was extracted from tissues or cultured cells using Tri reagent (Sigma-Aldrich). The absorbance ratio at 260/280 nm and the RNA concentration of all samples were detected using a NanoDrop ND-2000 spectrophotometer (Thermo Scientific). Reverse transcription was performed using the PrimeScript RT Master Mix (TaKaRa), and RT-qPCR was conducted using the ChamQ Universal SYBR qPCR Master Mix (Vazym). The measurements were taken using the QuantStudio Dx Real-Time PCR Instrument (Applied Biosystems, USA). The ΔΔCT method was used to evaluate the relative mRNA expression that was normalized to 36b4. Primer sequences were listed in Table S2 . Mitochondrial function was determined by measuring the oxygen consumption rate (OCR) in real time using a Seahorse Extracellular Flux Analyzer XFe96 (Agilent). Cells were seeded in an XF96 microplate at 3 × 10³ cells/well. The culture medium was changed to Seahorse XF DMEM buffer (Agilent) containing 10 mM glucose, 2 mM glutamine, and 1 mM pyruvate, and the plate was incubated at 37°C in a non-CO₂ incubator for 1 h. The compounds from the Seahorse XF Cell Mito Stress Test Kit (Agilent) were loaded into the injection port to reach the final concentrations of 1 μM oligomycin, 2 μM carbonyl cyanide- p -trifluoromethoxyphenylhydrazone (FCCP), 0.5 μM antimycin A, and 0.5 μM rotenone to detect respiratory parameters, including basal respiration, ATP production-coupled respiration, maximal respiration, and spare respiratory capacity. The OCR in each microplate well was normalized to the total protein of each well. The glycolytic function was evaluated by measuring the extracellular acidification rate (ECAR) in real time using a Seahorse Extracellular Flux Analyzer XFe96 (Agilent). Briefly, cells were seeded in an XF96 microplate at 3×10 3 cells/well. The culture medium was changed to Seahorse XF DMEM buffer (Agilent) and the plate was incubated at 37°C in a non-CO2 incubator for 1 h. The compounds were loaded into the injection port in sequence to reach the final concentration of 10mM glucose, 2μM oligomycin, and 0.1M 2-Deoxy-D-glucose (2-DG). The final ECAR results were normalized to total protein. The intracellular levels of ROS in cells were assessed by ROS assay kit (Beyotime) that contains DCFH-DA fluorescent probe. Briefly, cells were seeded in 12-well plates and induced into osteoblastogenic differentiation for 14 days. Subsequently, washed cells were incubated with 10 μM DCFH-DA for 20 min in dark and then harvested and washed with PBS. Finally, the intensity of DCFH-DA fluorescence was determined by flow cytometry (BD FACSLyric) at 480 nm (excitation) and 530 nm (emission). MMP was assessed using Image-iT tetramethylrhodamine (TMRM) reagent (Invitrogen) following the manufacturer’s instructions. In brief, cells were seeded in 35 mm glass-bottom microwell dishes (MatTek) and induced to undergo osteoblastogenic differentiation for 14 days. Subsequently, the washed cells were incubated with 100 nM TMRM reagent and 0.1 mg/mL of the nuclear stain Hoechst 33342 (Thermo Scientific) at 37°C in an incubator for 30 min, followed by washing with PBS. Finally, the microscope was configured to image TMRM (Ex 548/Em 574) and Hoechst 33342 (Ex 350/Em 460), and images were acquired from multiple sites per well at 20× magnification for fluorescence expression using a laser scanning confocal microscope (LSM880, ZEISS). Mitochondrial superoxide in cells was detected by MitoSOX Red mitochondrial superoxide indicator (Invitrogen). Cells were seeded in 35 mm glass bottom microwell dishes (MatTek) and induced to undergo osteoblastogenic differentiation for 14 days. Subsequently, the washed cells were incubated with 5 μM MitoSOX and 0.1 mg/mL of the nuclear stain Hoechst 33342 (Thermo Scientific) at 37°C in an incubator for 10 min, followed by washing with PBS. Finally, the microscope was configured to image TMRM (Ex 510/Em 580) and Hoechst 33342 (Ex 350/Em 460), and images were acquired from multiple sites per well at 20× magnification using a laser confocal microscope (LSM880, ZEISS). To visualize mitophagy using a mitophagy-specific probe, MCT3E1 cells were transfected with pMT-mKeima-Red (MBL). Keima in neutral conditions was observed using excitation of 430 ± 20 nm and emission of 624 ± 20 nm. Keima in acidic conditions was observed using excitation of 562 ± 20 nm and emission of 624 ± 20 nm. Images were obtained by an LSM880 confocal microscope (Zeiss). For primary fixation, MC3T3-E1 cells were enriched by centrifugation, then immersed in 2.5% glutaraldehyde and stored at 4°C for 24 h. The samples were washed with 0.1 M phosphate buffer for 15 min, three times, and then immersed in 1% osmium tetroxide for 2 h for post-fixation. For dehydration, the samples were washed with 0.1 M phosphate buffer for 15 min, three times, and then step-by-step immersed in dehydrating agents (50% ethanol, 75% ethanol, 80% ethanol, 95% ethanol, 100% ethanol, and 100% acetone). The samples were immersed in infiltration agent A (a 2:1 solution of acetone and 812 embedding agent) for 2 h, then in infiltration agent B (a 1:2 solution of acetone and 812 embedding agent) overnight. For embedding, the samples were placed into a mold with embedding medium and then baked at 37°C and 60°C for 12 h, respectively. The samples were cut into sections (60–80 nm thick). For lead-uranium double staining, the sample sections on the copper grid were immersed in a 2% uranyl acetate staining solution for 30 min in the dark, and then in lead citrate staining solution for 15 min. Image Acquisition: The organelle morphology was captured using the JEM-1400Flash electron microscope (JEOL). Mice assigned to the STZ group were administered low doses of STZ at 40 mg/kg for five consecutive days to induce diabetic osteoporosis. Mice in the control group were injected with a sodium citrate solution. Mice assigned to the STZ+BMP9 group were administered STZ for five consecutive days, then were administered AAV-BMP9 (Hanbio) at week 5. Specimens were collected at week 18, and elevated BMP9 levels were maintained until the mice were euthanized. All procedures involving micro-CT were performed according to the recommendation of the American Society for Bone and Mineral Research. 44 Briefly, the right femurs of mice were isolated, fixed in 4% paraformaldehyde for 48 h, and then maintained in 75% ethanol. Quantitative analysis of distal femoral metaphysis and midshaft femoral diaphysis were performed using a high-resolution ex vivo micro-CT scanner, Skyscan 1176 (Bruker). Using 2D data from scanned slices, 3D analysis was performed to calculate morphometric parameters by a microCT Software (Bruker). The following trabecular morphometric indexes were analyzed: vBMD, percentage of bone volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) and structure model index (SMI). The cortical thickness (Ct.Th) was assessed at the midshaft of femurs. For biomechanical testing, the left femurs were cleaned of adherent tissue, wrapped in saline-soaked gauze, and tested immediately. A three-point bending test was carried out at the midshaft of the femurs using a mechanical testing machine, the Instron 5569 (USA). The elastic modulus, bending stiffness, maximum bending load, and fracture energy were evaluated. Mice were fasted for at least 12 h before being euthanized. Blood was collected and allowed to clot at room temperature for 1 h, then centrifuged at 4,000 rpm for 15 min at 4°C. Serum samples were stored at −80°C. Serum levels of bone turnover markers were determined using enzyme-linked immunosorbent assay (ELISA) kits for mouse PINP (USCN, Wuhan, China), CTX-I (USCN, Wuhan, China), and BMP9 (RayBiotech, USA). The fixed lumbar spines of mice were decalcified in 23% ethylenediaminetetraacetic acid at 4°C for 5–7 days and embedded in paraffin. Bone sections (4 μm) were deparaffinized and washed with water for 3 min. The bone section was stained with hematoxylin staining solution (Beyotime) for 10 min, then washed with water and hydrochloric acid ethanol for 30 s, counterstained with eosin solution, and dehydrated. The expression of Drp1 and PINK1 in bone was investigated using multiplex immunohistochemistry staining enhanced by tyramide signal amplification. Bone tissue was prepared into paraffin sections. The bone sections were blocked with 5% BSA and then washed three times with PBS. The sections were incubated overnight in a dilution of anti-Drp1 antibody. After washing three times with PBS, the sections were incubated in a dilution of HRP-linked anti-mouse secondary antibody. Following another round of washing with PBS, the sections were incubated in a dilution of AF 647 tyramide for 10 min. Consistent with the above process, the bone sections were stepwise incubated in a dilution of anti-PINK1 antibody, HRP-linked anti-mouse secondary antibody, and CF 488 tyramide. Finally, the bone sections were incubated in antifade mounting medium with DAPI. The bone sections were scanned using the TissueFAXS system (TissueGnostics, Austria). Flow cytometry data were analyzed with FlowJo 10, and fluorescence intensity was quantified by ImageJ software. | Review | biomedical | en | 0.999995 |
PMC11699392 | Radical prostatectomy (RP) is the primary treatment for prostate cancer. However, a considerable percentage of patients (approximately 15–45 %) experience biochemical recurrence (BCR) after the procedure . Accurately predicting the timing of postoperative BCR is crucial in determining subsequent treatment approaches. In 1999, a predictive model was established incorporating various factors such as preoperative prostate-specific antigen (PSA) levels, pathological Gleason scores (GS), extracapsular extension (ECE), seminal vesicle invasion (SVI), lymph node invasion, and positive surgical margins (PSM). This model remains a valuable tool for determining the appropriate treatment strategies. Kattan developed a nomogram capable of predicting the BCR in patients who underwent RP for prostate cancer. This nomogram was subsequently validated at multiple centers in the United States [ , , , ]. Several prediction models inspired by the Kattan postoperative nomogram were subsequently introduced [ , , ]. The primary focus of these models is to predict the probability of long-term BCR. However, it is noteworthy that approximately two-thirds of BCR cases manifest within the first 3 years after undergoing RP . Consequently, a model that can accurately forecast the BCR probability within this critical timeframe holds greater significance than previous models. Contrastingly, previous models have predominantly relied on clinical or pathological data obtained during the perioperative period, neglecting the inclusion of short-term postoperative PSA levels acquired during follow-up. Nonetheless, PSA is a serum marker requiring regular monitoring after RP. Previous studies have demonstrated the potential importance of short-term postoperative PSA levels in predicting BCR. [ , , ] In theory, prostatectomy completely eliminates prostate tumors, leading to a decline in PSA levels to an undetectable range within 6 weeks. However, persistent elevated PSA levels after prostatectomy suggest the presence of residual microtumor foci that require further management. Hence, this study aimed to develop a novel nomogram prediction model based on postoperative elevated PSA level recurrence. This model sought to enhance the accuracy of predicting the probability of BCR after robot-assited prostatectomy, simplifying existing clinical follow-up strategies and aiding in the early prediction of patients at high risk of BCR. A total of 1700 patients from University-based hospital who underwent robot-assisted radical prostatectomies between August 2009 and December 2022 were included in this study. Data were collected prospectively and entered into an electronic database by trained personnel. Regular quality control checks were performed to ensure the accuracy of the data. With a median follow-up of 61 months (range, 12–162 months). Consider excluding cases with observation periods less than one year from the analysis. The primary focus of this study was to investigate BCR as a primary outcome. In this study, BCR was defined as the occurrence of two consecutive elevations in PSA levels after undergoing RP, with both measurements surpassing 0.2 ng/mL. However, it is important to note that there is currently no universally accepted international standard for the PSA threshold that indicates BCR following RP. Since the publication of the consensus by European urological experts in 2004, two distinct criteria have been widely used, and more researchers have recognized consecutive PSA levels above 0.2 ng/mL . Due to the improved lymph node yield, better staging, and theoretical improvement in the control of micro-metastatic disease, guidelines have supported the use of pelvic lymph node dissection (PLND) in patients deemed to be at intermediate or high risk of lymph node involvement and cases with lymph node involvement were excluded. Neoadjuvant conventional androgen deprivation therapy agents (luteinizing hormone–releasing hormone (LHRH) analogues with or without first generation antiandrogens) in the setting of neoadjuvant therapy before radical prostatectomy for patients with clinically localized high risk-prostate cancer [goserelin monotherapy, cyproterone monotherapy and combination therapy with either leuprolide plus flutamide or goserelin plus flutamide]. We used Cox regression analysis to identify the risk factors associated with BCR. The univariate regression analysis identified important variables, which were subsequently incorporated into the multivariate analysis. The multivariate analysis employed a backward stepwise regression approach, leading to the exclusion of non-significant variables. The remaining significant variables were utilized in constructing the nomogram model, employing the regression modeling strategy package in R software (Windows version 4.3.1; http://www.r-project.org/ ). The consistency index (C-index) of the nomogram model was computed, and a calibration curve was generated to compare predicted values from the nomogram with actual values. Internal validation was performed via 500 iterations of repeated sampling using the bootstrap method, while external validation utilized data from the validation group. The validation cohort was selected from a separate institution using the same inclusion criteria, ensuring that the group represents a comparable patient population. The clinical and pathological characteristics of patients are shown in Table 1 . Table 1 Clinical and pathological Features of patients. Table 1 Total Total 1700 Age, years Median (IQR) 67 (63,71) Age, number <65 561 (33.0) ≥65 1137 (66.9) Unknown 2 (0.1) PSA(ng/mL) Median (IQR) 7.5 (5.5–10.7) PSA (ng/mL) <10 1194 (70.2) 10–19.00 391 (23.0) ≥20 112 (6.6) Unknown 3 (0.2) BMI (Kg/m 2 ) <18.5 38 (2.2) 18.5–22.9 635 (37.4) 23–24.9 491 (28.9) 25–29.9 492 (28.9) ≥30 34 (2.0) Unknown 10 (0.6) Clinical T stage 1–2b 1287 (75.7) 2c 289 (17.0) 3–4 120 (7.1) Unknown 4 (0.2) Clinical Gleason score 6 393 (23.1) 7 872 (51.3) 8–10 431 (25.4) Unknown 4 (0.2) Preoperative potency No erection 660 (38.8) Erection 548 (32.2) Penetrate 462 (27.2) Unknown 30 (1.8) D'Amico risk classification High 698 (41.1) Intermediate 769 (45.2) Low 229 (13.5) Unknown 4 (0.2) Neoadjuvant treatment None 1090 (64.1) ADT 166 (9.7) Anti-A 178 (10.5) NACHT 246 (14.5) Unknown 20 (1.2) Nerve-sparing technique None 497 (29.2) Bilateral 207 (12.2) Unilateral 996 (58.6) Pathologic T stage 1–2b 393 (23.1) 2c 1026 (60.4) 3–4 281 (16.5) Pathologic Gleason score 6 167 (9.8) 7 1191 (70.1) 8–10 342 (20.1) Surgical margin Negative 1354 (79.6) Positive 346 (20.4) Extraprostatic extension Negative 1,469 (86.4) Positive 231 (13.6) Seminal vesicle involvement Negative 1606 (94.5) Positive 94 (5.5) Independent risk factors associated with BCR were identified By applying Cox regression to the clinical and pathological information of the modeling group ( Table 2 ). Univariate analysis revealed that PSA, clinical T stage, biopsy Gleason score, D'Amico risk classification, pathological T stage, pathologic Gleason score, extraprostatic extension, SVI, and positive surgical margins were independent risk factors for BCR. Multivariate analysis showed a significant association with BCR. Multivariate analysis showed that high PSA ≥20 ng/mL (HR: 1.93; p = 0.034), pathological T stage 3–4 (HR: 1.89; p < 0.001), pathological Gleason score 8–10 (HR: 5.43; p < 0.001), extraprostatic extension (HR: 1.41; p < 0.001), SVI (HR: 1.92; p = 0.018), and positive surgical margin (HR: 2.73; p < 0.001) were independent predictors of BCR considered the independent risk factors and established in the final model. Table 2 Independent predictors of BCR with Cox proportional hazards model. Table 2 Variables Univariate analysis Multivariate analysis Hazard ratio (95 % CI) P value Hazard ratio (95 % CI) P-value Age: 0.83 (0.59–1.17) 0.289 ≥65 vs < 65 PSA (ng/mL) <10 Ref. 10–19.99 1.51 (1.03,2.19) 0.033 1.18 (0.79–1.76) 0.424 ≥20 2.75 (1.65,4.59) <0.001 1.93 (1.05–3.53) 0.034 Clinical Gleason score 6 Ref. Ref. 7 2.77 (1.58,4.86) <0.001 1.84 (0.87–3.9) 0.11 8–10 4.2 (2.35,7.53) <0.001 1.81 (0.74–4.45) 0.195 Clinical T stage 1–2b Ref. 2c 1.86 (1.25–2.76) 0.003 1.24 (0.66–2.3) 0.504 3–4 2.67 (1.61–4.43) <0.001 1.71 (1.02–2.89) 0.043 D'Amico risk classification Low Ref. Ref. Intermediate 2.79 (1.26–6.2) 0.66 1.75 (0.75–4.07) 0.084 High 4.82 (2.2–10.56) <0.001 2.16 (0.9–5.2) 0.196 Pathologic T stage 1–2b Ref. Ref. 2c 0.74 (0.48–1.13) 0.216 0.66 (0.42–1.03) 0.173 3–4 2.87 (1.82–4.51) <0.001 1.89 (0.82–4.38) <0.001 Pathologic Gleason score 6 Ref. Ref. 7 3.61 (1.31–9.95) 0.011 2.74 (0.98–7.65) 0.054 8–10 8.67 (3.09–24.29) <0.001 5.43 (1.89–15.57) <0.001 Extraprostatic extension 2.72 (1.86–3.97) <0.001 1.41 (1.02–2.18) <0.001 Yes vs. No Seminal vesicle involvement 3.92 (2.4–6.39) <0.001 1.92 (1.12–3.29) 0.018 Yes vs. No Surgical margin 3.42 (2.44–4.79) <0.001 2.73 (1.87–3.98) <0.001 Yes vs. No The nomogram model serves as a graphical representation of the underlying regression equation. To establish a scoring system, we assigned scores based on the magnitude of the regression coefficients for each independent variable. These scores were then allocated to different levels of each independent variable. Consequently, for every patient, a comprehensive score was computed, enabling the determination of the probability associated with the outcome time for that specific patient. This calculation was facilitated by employing a conversion function that links the score to the corresponding probability of the outcome . Fig. 1 Nomogram predicting BCR after robot-assisted radical prostatectomy. A vertical line perpendicular to the horizontal axis was made according to the corresponding state of each predictor, and the intersection point between the vertical and upper score lines was the score of the predictor. The score of the five predictors was added to obtain the final score of the patient. Fig. 1 Internal validation of the prediction model showed that the C-index's accuracy was 0.743 (95 % confidence interval [CI]: 0.741–0.745). Calibration of the new model in the validation group also demonstrated good consistency between the predicted and actual values , indicating that the prediction model performed reasonably well in distinguishing individuals with varying outcome probabilities. Moreover, the area under the receiver operating characteristic (ROC) curve (AUC) was 0.744. This indicates that the model has a significant accuracy in predicting BCR in this context . Fig. 2 Calibration curve of the new model. Fig. 2 Fig. 3 The ROC curve of the prediction model. Fig. 3 The BCR-free survival rate for the entire cohort at the median follow-up time was 25 %, and the 1-, 3-, and 5-year estimates were 90 %, 37.5 %, and 22.5 %, respectively. Fig. 4 shows the BCR-free survival curve after RARP. Fig. 4 Kaplan-Meier survival curve of biochemical recurrence-free survival. Fig. 4 BCR is a crucial factor in determining whether a change in treatment strategy is necessary in patients who have undergone RP. Approximately 34 % of patients may progress to distant metastasis without additional radiotherapy or endocrine therapy after experiencing BCR . Currently, a sole clinical approach is available for identifying the incidence of BCR. Nevertheless, research findings indicate that the rate of BCR within a decade after undergoing RP is 34.3 % . Throughout the years, researchers have dedicated their efforts to developing a statistical model that can precisely forecast the likelihood of BCR by utilizing clinical and pathological data from patients. The postoperative nomogram introduced by Kattan et al., in 1999 is widely recognized as the most prominent model in this field . Utilizing preoperative PSA levels and postoperative factors such as the Gleason score, ECE, SVI, and PSM, this nomogram demonstrated a high degree of effectiveness in estimating patients' probability of BCR. Subsequently, several scholars proposed a range of prediction models based on the foundation of the Kattan postoperative nomogram. These models incorporate specific variables that emphasize the robotic approach to enhance the overall prediction accuracy of the model [ , , , 17 ]. With efforts to enhance the model's prediction accuracy, the C-index was 0.743 (95 % CI: 0.741–0.745) indicating that the predictive model for BCR after robotic-assited prostatectomy performed well in discriminating between patients at different risk levels. These findings suggest that the model exhibits reasonably accurate ability to predict the occurrence of BCR within this population. Prediction models that amalgamate multiple indicators to anticipate disease occurrence or progression have been extensively used in various medical research domains. The “Transparent Reporting of a Multivariable Prediction Model for Individual Prognosis or Diagnosis” statement published in 2015 aimed to standardize the process of establishing prediction models . Furthermore, Alba et al. specified standard statistical methods for constructing a prediction model in 2017 . A reliable prediction model typically comprises two essential components: discrimination and calibration. Discrimination pertains to using an indicator to differentiate the risk of a specific event within a group, often assessed using the AUC or C-index. Calibration, another crucial evaluation index, reflects the agreement between predicted and actual values. Integrated Discrimination Improvement, proposed by Pencina et al., in 2008, is a statistical method used to compare two models . However, as reported by Wessler et al., only 63 % of prediction model studies reported discrimination, and 36 % reported calibration . The ROC curve and corresponding AUC offer valuable insights into the overall performance of the BCR prediction model, enabling clinicians and researchers to evaluate its predictive capabilities. These results indicate that the new model enhances the prediction performance, particularly in RARP. Moreover, about the sentinel node technique and its relevance to pelvic lymph node dissection (PLND) in prostate cancer [ , , ]. Unfortunately, data on variant histologies and aberrant growth patterns were not available for our cohort.According to the European Association of Urology guidelines, PSA levels are typically assessed every 6 months for the initial 3 years post-prostatectomy and yearly thereafter . A limitation of this study is that the long time span of the study may introduce variation due to changing surgical techniques and case volumes. External validation using independent data is crucial and we have alsosentien performed external validation. The predictive nomogram model provides a user-friendly and precise means to estimate the likelihood of achieving a BCR-free status within a three-year timeframe for each individual patient. This empowers healthcare providers to tailor follow-up strategies specifically to each patient, effectively minimizing the unnecessary allocation of medical resources. This model holds significant clinical utility, offering a valuable tool for optimizing patient care. Its potential benefits warrant further dissemination and widespread adoption in clinical practice. However, as the data were obtained from a single center, data from other centers is needed for further validation. | Review | biomedical | en | 0.999997 |
PMC11699394 | Rhythm is ubiquitous in human behavior: music and dance are inherently rhythmic, and most daily activities depend on finely timed motor control to coordinate our movement with the environment. Cognition also partly relies on rhythm, as evidenced by temporal predictability in language, 1 and even memory and attention seem to be sustained by rhythmic brain processes. 2 , 3 , 4 Reciprocally, rhythm processing is cognitively demanding; for example, stabilizing synchronization with increasingly complex rhythmic patterns requires additional attentional load. 5 , 6 , 7 , 8 , 9 One prominent theory in psychology, dynamical systems theory, has sought to explain how the brain rhythmically deploys attention to the right moments in time. Dynamical systems theories assume that self-organized neural and behavioral systems coordinate perceptual and cognitive processes. 10 Oscillator models are central mathematical and conceptual tools in dynamical system theories, and are particularly suited to study the anticipatory processes sustaining attentional and rhythmic mechanisms in human behavior. 11 , 12 , 13 In these models, internal and behavioral oscillatory systems (e.g., electrical activity in populations of neurons, rhythmic movements like gait) synchronize to external events for more fluent processing. 14 , 15 , 16 , 17 , 18 , 19 These oscillations appear spontaneously (i.e., in the absence of any external cues) at different rates for each individual. 20 These spontaneous motor rhythms can be measured behaviorally by asking participants to tap at their own comfortable rate. Spontaneous rates have been shown to influence participants’ abilities to synchronize both to other people and music. 21 , 22 Specifically, participants more optimally synchronize to rates that are closer to their own spontaneous rates. These findings may arise because smaller phase adjustments require fewer attentional resources or because oscillatory attention processes respond more efficiently via resonance-like properties. However, neither of these possible attentional mechanisms has yet been directly explored. One simple yet reliable way to measure such attentional processes is via pupillary activity due to its tight coupling to noradrenergic activity from the locus coeruleus. 23 , 24 , 25 Greater task-evoked pupil dilations and pupil size have consistently been linked to increased attention allocation since the 1960s 26 , 27 , 28 , 29 and, more recently, oscillatory pupil activity (particularly in the delta range between ∼0.5 and 2 Hz) has been demonstrated to entrain to rhythms and appears to be related to movement. 30 , 31 , 32 Unlike electroencephalography, the event-related pupil dilation response is so sluggish (typically peaking between ∼500 and 1,200 ms post-event onset 33 , 34 ) that phase-aligned pupil dilations can be interpreted as entrained rather evoked. This is particularly relevant for testing theories like dynamical systems where the difference between evoked and entrained responses is crucial to the underlying neural mechanism. 35 , 36 , 37 , 38 , 39 Thus, pupillometry offers a robust and convenient way to investigate (1) whether synchronizing to rates beyond one’s spontaneous rate demands more attention and (2) whether attention is better entrained (and therefore more efficiently processed according to dynamical systems theory) to one’s own spontaneous rate relative to faster and slower tempi. To this end, we first recorded the spontaneous rates of 25 healthy adults in an unpaced tapping tapping (spontaneous motor tempo, SMT) and created three metronomes consisting of 60 woodblock sounds for each participant, one corresponding to their average SMT, one 20% faster, and one 20% slower than this rate. Participants were then tasked with either passively listening or synchronizing their finger taps to the metronomes across three trials per condition while their pupil size was recorded with an eye-tracker . We hypothesized that average pupil sizes would be larger while synchronizing relative to listening, particularly at the faster and slower tempi, reflecting increased attentional demands. Furthermore, we hypothesized that pupil activity would be better entrained (as assessed with phase coherence) at participants’ SMT where, according to dynamical systems theory, their internal oscillators are already primed to resonate for more efficient attentional processing. Figure 1 Experimental design The study design. Participants either tapped or listened to metronomes that matched their SMT or were 20% faster or slower. Half of the participants tapped to the metronomes first followed by listening to the metronomes (synchronization then perception) while the other half of the participants listened to the metronomes first followed by tapping to them (perception then synchronization). As in previous studies, 21 , 22 , 40 , 41 participants’ SMTs spanned a relatively broad range from 462 to 1,192 ms (ITI, M = 692.34, SD = 190.34) . Despite this considerable variability, all participants nevertheless managed to synchronize their taps to each of their three custom metronomes with equal success as indicated by equivalent vector lengths (tapping consistency, F (1,44) = 1.37, p = 0.26, η2G = 0.11) and directions (tapping precision, F (1,44) = 2.42, p = 0.10, η2G = 0.13) in all tempo conditions. Thus, the following pupillometry results reflect the attentional processes employed to achieve this performance rather than attentional differences related to errors or their corrections. Figure 2 Distribution of participants’ SMTs Mean individual spontaneous motor tempi, ordered from fastest to slowest. Error bars indicate standard errors of the mean. Pupil size by task and tempo condition are presented in Figure 3 . The repeated measures analysis of variance (ANOVA) on average task-evoked pupil sizes over the entire trial revealed a modest effect of task ( F (1,22) = 12.530, p = 0.002, η2G = 0.142), indicating that synchronizing one’s taps to the metronome was more cognitively demanding than passively listening to the same metronome. Crucially, there was also an interaction with tempo ( F (2,44) = 4.533, p = 0.016, η2G = 0.030) where post-hoc t tests demonstrated that the main effect of task was driven exclusively by the faster ( t (22) = 3.301, p = 0.004, d = 0.69) and slower ( t (22) = 4.434, p < 0.001, d = 0.92) metronomes evoking greater pupil sizes while synchronizing relative to listening whereas pupil sizes did not differ at their preferred rates ( t (22) = 1.165, p = 0.257). These results show that synchronizing to the metronome only required more attention than listening to it when its tempo was outside of one’s comfortable rate. Figure 3 Evoked pupil size by task and tempo Raincloud plots for average pupil size by task and tempo condition. Black diamonds denote the mean while error bars represent one standard error of the mean. Each point depicts a single subject’s average pupil size and gray lines connect these averages between Task conditions. ∗∗ p < 0.01, ∗∗∗ p < 0.001 (post-hoc t tests). An additional participant was missing too much data from the SMT condition, unbalancing the design, and thus had to be excluded. Nonetheless, the repeated measures ANOVA on entrained pupillary activity revealed a main effect of task ( F (1,21) = 5.000, p = 0.036, η2G = 0.052) with no effect of tempo ( F (2,42) = 1.131, p = 0.332) or interaction ( F (2,42) = 0.932, p = 0.402). This demonstrates that attention was more efficiently deployed to the frequency of the metronome while participants were synchronizing their taps relative to passively listening . Figure 4 Pupillary entrainment by task Raincloud plots depicting the main effect of task on entrained pupil activity. Black diamonds denote the mean while error bars represent one standard error of the mean. Each point depicts a single subject’s average phase coherence and gray lines connect these averages between task conditions. ∗ p < 0.05 (ANOVA). One could argue that we might have observed the same pattern of results had we instructed participants to move without the presence of the metronome cues. That is to say that these effects could be mere motor effects rather than sensorimotor synchronization effects per se. As an additional control analysis, we analyzed participants’ average pupil dilations and entrained pupillary activity during their SMT recordings and compared this to their respective signals while listening or synchronizing to metronomes at this tempo. Two participants’ pupillary recordings during the SMT recordings were corrupted or missing and so were omitted from these control analyses. There was no effect of task on either the average evoked pupil size ( F (2,40) = 2.514, p = 0.094) or pupillary phase coherence ( F (2,40) = 1.823, p = 0.175). The results are plotted in Figure 5 in the following. Figure 5 Control analyses Raincloud plots for the control analyses only on the SMT condition while listening, synchronizing, and tapping without a metronome. Black diamonds denote the mean while error bars represent one standard error of the mean. Each point depicts a single subject’s average pupil size (A) or phase coherence (B) and gray lines connect these averages between Task conditions. There were no significant differences observed. Here we tested whether synchronizing to metronomes at one’s preferred motor rate demands fewer cognitive resources (assessed by evoked pupil size) and whether it is associated with more efficient allocation of those resources (assessed by pupillary entrainment) compared to faster and slower rates. We found that synchronizing, relative to listening, evoked greater average pupil sizes at rates outside of participants’ preferred tempo, indicating that more attention was deployed to achieve the same level of synchronization to the faster and slower metronomes. Despite this overall increase in attention while synchronizing to faster and slower tempi, pupillary phase coherence was modestly greater while synchronizing compared to listening but in a similar manner across all tempi conditions. Our average evoked pupil size results demonstrate that more attentional resources are allocated to synchronize to tempi outside of one’s comfortable rate, confirming our first hypothesis. This is consistent with previous results showing that more attentional resources are necessary for synchronizing with more complex stimuli, as reflected in behavioral and neurophysiological processes. 5 , 6 , 7 , 8 , 9 Interestingly, these differences in attention allocation were not symmetrical; the effect was larger for slower metronomes ( d = 0.90) than faster metronomes ( d = 0.66). This asymmetry fits with previous studies where synchronization tends to be worse for slower tempi than faster tempi. 40 , 41 , 42 This may be an effect of expertise, since the asymmetry seems to arise in nonmusicians more than in musicians who are more flexible in their ability to synchronize, including to slower tempi. 41 Why this asymmetry exists, however, is still unclear. The precision of timing processing decreases with larger intervals (Weber’s law), 43 leading to increased tapping variability and asynchronies at slower rates. 44 , 45 The increased pupil size when tapping at slow rates may therefore reveal cognitive and attentional resources needed to overcome the natural tendency to tap with less precision and accuracy at slow tempi, as predicted by Weber’s law. The oscillatory pupil activity results demonstrate that synchronous movements result in greater attentional entrainment. While most participants generally displayed a modest degree of entrainment even while listening, this entrainment was greater while synchronizing their taps to the metronomes. This is consistent with theories of active sensing and embodied cognition where action plays a central role in cognitive processes like attention. 46 , 47 , 48 , 49 Contrary to our second hypothesis, there was no interaction with tempo, indicating that this motor effect is persistent across the range of tempi. Given participants’ successful behavioral synchronization at all tempi, this is rather unsurprising in hindsight, especially since all of our participants’ metronome rates fell well within the ecological range that bounds human rhythm perception and production. 50 , 51 Thus, it seems that attentional deployment is more entrained for successful behavioral synchronization relative to passive listening. Taken together, these results paint a more nuanced picture of dynamical systems theories of attention. As dynamical systems theories predict, synchronizing beyond one’s preferred tempo does seem to require more attention as evidenced by our evoked pupil dilation effects. However, our oscillatory pupillary activity results seem to contradict dynamical systems accounts based on resonance properties that would predict worse pupillary entrainment at tempi faster and slower than one’s SMT. It is possible that a reduction of pupillary entrainment would be observed at larger deviations from one’s SMT than the ones used in this study; this should be addressed in future experiments to test whether pupil oscillations are flexible or relatively rigid resonators that only entrain to a narrow set of frequencies. Entrained pupil dilations may also represent a form of active sensing, i.e., a top-down sampling routine that sharpens sensory representations of the environment. 47 , 52 , 53 , 54 Under this view, the locus coeruleus may proactively release norepinephrine, dilating the pupils, to maximize the encoding of sensory information at attended moments in time, specifically those coinciding with our own movements, regardless of the motor rate. Our study demonstrates the usefulness of considering participants’ comfortable rates in experimental settings where tempo is manipulated because there could be latent yet systematic differences in the attentional demands among subjects. This would be particularly important to consider in experiments where the complexity or difficulty of tracking the rhythm is of interest. For instance, there are well-known interactions between rhythmic complexity and tempo in both perception and synchronization 55 , 56 ; these interactions may partially reflect the distance from participants’ SMT in addition to differences stemming from the physical stimulus rates. These considerations may be even more important outside of the lab, particularly in clinical settings for movement disorders. For instance, if existing rhythmic- and movement-based interventions for Parkinson’s disease are titrated to each individual’s preferred rate, these treatments could be less attentionally demanding to adhere to which could in turn boost retention. Our pupillary entrainment results may similarly have clinical implications for attention and developmental disorders. For example, both children and adults with attention deficit hyperactivity disorder (ADHD) display more variable tapping performance so pupillometry could provide a cheap, non-invasive biomarker for diagnosis alongside tapping behavior. 57 , 58 , 59 Moreover, children with dyslexia have been found to have faster SMTs and more variable sensorimotor synchronization, indicating that pupillary biomarkers may exist for them as well. 60 , 61 , 62 Additionally, because attention was better entrained while synchronizing, rhythmic interventions may offer a promising avenue for treating attention disorders. We provide evidence that the pupil signal can capture variations in attentional demands when synchronizing at different tempi. Consistent with modern theories of rhythmic attention such as dynamical systems and active sensing, we observed pupil oscillations aligned to the frequency of the perceived metronomes, with stronger alignment when movement coordination was required. These results thus demonstrate that pupillary activity is a useful measure of both attentional demands and attention allocation over time, providing opportunities for scientists and clinicians alike. One limitation of this work is that our analysis to control for purely motor (rather than synchronization) effects was limited to participants’ SMT tempo. It’s possible that moving without metronome cues at rates faster or slower than their comfortable rate could elicit results similar to our synchronization condition. However, were we to implement a movement only condition at the manipulated tempi, we would introduce a secondary task demand of moving at a faster or slower rate in addition to simply moving at a comfortable rate. This increased cognitive demand would induce larger pupil sizes, and thus confound a pure motor effect. Future work could investigate this possibility more directly using a synchronization-continuation paradigm where participants synchronize to a metronome cue and then continue tapping at that tempo after the metronome stops. That said, the continuation window would have to be long enough (e.g., longer than ∼4–10 s) for the pupil size to restabilize after the orienting response dilation from the metronome stopping, but not so long that the participants’ tapping rate drifts to a new tempo. Another limitation is that the influence of sex and gender was not tested in this study. The sample size was not sufficient to address this question. However, we do not expect that gender or sex would influence the results, as no studies on SMT and synchronization reported any effects from these factors. 63 Tapping data were collected using a Roland V-drums snare. The auditory stimuli consisted of metronome sequences composed of 60 cues (using a woodblock sample) created with MATLAB 2019a. They were played at a comfortable volume from two Genelec speakers using custom scripts in MATLAB. The auditory signal and the Midi signal were recorded by a Roland V-drums Sound Module TD17 connected to a Windows computer using Reaper v. 6.45 (Cockos Inc., NY, USA). Pupil size was continuously recorded at 500 Hz with an EyeLink Portable Duo and a chin rest 70 cm from the eye-tracker in a well-lit room. The experiment started with participants’ spontaneous motor tempi being recorded. To familiarize them with the procedure, the participant completed a short practice trial where they produced a sequence of ten taps at an uncued rate. Once they understood the task, they completed three experimental trials consisting of 60 taps. Each participant’s SMT was then calculated as the mean intertap interval (ITI, in ms) across the three SMT trials after the first ten taps were discarded from each trial. This value was used to create the metronomes; one metronome matched their SMT (SMT condition), one was 20% faster (Faster condition), and one was 20% slower (Slower condition). At the start of the main experiment, participants completed a five-point (cross-shaped) calibration procedure for the eye-tracker. Subsequently, one of the three metronome sequences were played, selected pseudo-randomly such that all three tempi were played before being randomly shuffled again. Participants were instructed to fixate within a black ring presented in the center of a gray background while either passively listening to the metronome or synchronizing their taps to the metronome with their dominant index finger. Participants completed nine trials of either listening or tapping before completing nine more trials of the other for a total of 18 trials with the task order counterbalanced across subjects. Thus, the experiment followed a within-subject design with Tempo (SMT, Faster, Slower) and Task (Listen, Tap) as factors with three trials per condition. The study design is schematized in Figure 5 below. Each finger tap was matched with the nearest metronome cue. Signed asynchrony values were calculated as the participant’s tap onset time minus the auditory cued onset time (in ms). A negative asynchrony value indicates that the tap occurred before the cue while a positive value indicates that the tap occurred after the cue time. The first four taps of each sequence were also discarded to allow for tempo stabilization as is standard procedure. Some taps (i.e., when participants tapped too softly) were not captured by the midi touchpad. Therefore, a minimum of forty taps was necessary to analyze a trial; ten trials (out of 225, 4.4%) were discarded because there were fewer than 40 recorded taps. Finally, outliers, defined as taps with signed asynchronies of more than three standard deviations from the mean signed asynchrony in a trial, were discarded (fewer than 0.5% of the taps in total). There was at least one trial per condition for each participant after these data cleaning procedure. In order to compare the magnitude and variability of the asynchronies across tempi conditions, circular statistics 64 , 65 were conducted on the synchronization data using the MATLAB Circular Statistics Toolbox. 66 The interval between metronome cues was represented on a 360° circular scale, and individual taps were represented as unit vectors with a given angle. A mean resultant vector R was calculated based on each individual tap in a trial. The length of the resultant vector, between zero and one, indicates synchronization consistency (i.e., the reciprocal of variability; the closer to one, the less variable the performance). The angle of the vector (in degrees) represents the relative phase, an index of synchronization accuracy. Negative angles indicate that participants tapped before metronome cue. Pupil data were exported offline from Eyelink DataViewer 67 for preprocessing with custom R scripts using functions from the “pupillometry” and “gazeR” packages in R. 68 , 69 First, the timeseries for each participant’s right pupil was read in and locked to the stimuli onsets. Blinks were removed along with their preceding and succeeding 100 ms to exclude artifacts arising from partial occlusions of the pupil by the eyelid during a blink. Each trial was then smoothed using a 4 ms moving average and gaps smaller than 1000 ms were interpolated using cubic splines. Afterward, each trial was baseline-corrected by subtracting the median pupil value recorded during the 1000 ms of silence preceding stimuli onsets to remove random fluctuations in pupil size between trials. 24 At this point, surviving artifacts were removed in a tripartite fashion: trials with more than 33% of missing data were discarded, samples containing rapid pupil size changes were detected and eliminated using the median absolute deviation of the dilation speed time series and a constant of 16 as suggested by Kret & Sjak-Shie, 70 and visual inspection of a histogram of all remaining pupil sizes as recommended by Mathôt and colleagues. 71 Each subject’s cleaned pupil size was averaged over the entire trial. To investigate pupillary entrainment, additional processing was needed. First, the data from the first four taps needed to be discarded because of the orienting responses elicited by the metronome onset. Next, each trial for each subject was high-pass filtered with a third order Butterworth filter at 0.05 Hz to remove the signal drift, and then the data were z-scored according to the preprocessing steps employed and validated in past research. 30 , 31 The time series for the remaining 56 taps were grouped into 14 four-tap phrases and timelocked to the start of each phrase for greater statistical power. Fourier series at the frequency of the metronome in each condition were extracted from each phrase in each trial using a fast Fourier transform. 72 Three neighboring frequency bins were kept as well to control for spectral leakage as done in past work. 73 As in previous studies, 74 , 75 pupillary phase coherence was calculated by dividing the Fourier coefficients by their absolute values to get the phase angles, summing the resulting phase angles, and then dividing them by the total number of phrases per trial, and then taking the absolute value of the complex mean. The complex mean was then normalized, so that it can be compared across tempi (between zero and one, the closer to one, the less variable the performance). Synchronization (Vector Length, Vector Direction) and pupillometry (Pupil Size, Phase Coherence) indices were used as the dependent variables in a three (Faster, SMT, Slower) by two (Listen, Tap) repeated measures ANOVA. Following a significant interaction, Welch’s paired sample t-tests were conducted and corrected for multiple comparisons using the false discovery rate method. 76 In all figures, significance is denoted with a single asterisk for p < 0.05, a double asterisk for p < 0.01, and a triple asterisk for p < 0.001. | Review | biomedical | en | 0.999996 |
PMC11699398 | In particular, A. Bonaca et al. demonstrated that the perturber must be surprisingly dense to account for the spur and gap features in the GD-1 stream. Assuming a Hernquist density profile, the perturber's mass is estimated to be in the range of 10 5.5 –10 8 M ⊙ , with a scale radius of ≲20 pc; recent encounters within the last 1 Gyr are favored. The perturber is significantly denser than the subhalos predicted in the standard cold dark matter (CDM) model, at the ~3 σ level. Thus, even if the perturber were a known satellite galaxy of the Milky Way, its unusually high density would remain puzzling. Furthermore, none of the known globular clusters can match the orbit of the inferred perturber . In this work, we assume that the GD-1 perturber is a dark matter subhalo and explore its formation in within the framework of self-interacting dark matter (SIDM); see S. Tulin & H.-B. Yu and S. Adhikari et al. for reviews and references therein. The gravothermal evolution of an SIDM halo occurs in two sequential phases. In the core-forming phase, dark matter self-interactions transport heat inward, resulting in a shallow density core, while in the core-collapsing phase, heat transfer reverses, leading to a higher central density than in the CDM counterpart . Notably, SIDM models with large cross sections could explain the high density of the strong lensing perturber for SDSSJ0946+1006 and the low density of the Crater II satellite galaxy , both challenging CDM. It is intriguing to explore the SIDM scenario to account for the high density of the GD-1 perturber. We will analyze progenitors of CDM subhalos from a zoom-in cosmological simulation of a Milky Way analog from D. Yang et al. and E. O. Nadler et al. and explicitly show that their inner densities are systematically lower than those inferred for the GD-1 perturber. We then take one of the progenitor halos, with a mass of ~10 8 M ⊙ , and evolve it in the tidal field of the Milky Way, including both halo and stellar components. For a self-interacting cross section in the range σ / m = 30–100 cm 2 g −1 at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} ${V}_{{\mathrm{\max }}}\unicode{x0007E}10\,{\mathrm{km}}\,{{\mathrm{s}}}^{-1}$\end{document} V max ~ 10 km s − 1 , the SIDM halo enters the collapse phase within 3–6 Gyr while evolving in the tidal field. By the final snapshot, its enclosed mass within the inner 10 pc is increased by more than 1 order of magnitude compared to its CDM counterpart, making it consistent with the high density of the GD-1 perturber. Additionally, we will discuss future investigations aimed at further improvement. We first present progenitor halos from a cosmological zoom-in CDM-only simulation of a Milky Way analog , with initial conditions drawn from the suite in Y.-Y. Mao et al. . This simulated system includes a main halo with a mass of 1.14 ×10 12 M ⊙ h −1 ≈ 1.6 × 10 12 M ⊙ ( h = 0.7) and a Large Magellanic Cloud analog. The simulation has a particle mass of 4 ×10 4 M ⊙ h −1 , a Plummer-equivalent softening length of ϵ = 0.08 kpc h −1 , and a spline length of ℓ = 2.8 ϵ =0.22 kpc h −1 , the characteristic length scale of the smoothing kernel used to calculate gravitational forces between particles . We select subhalos of the main halo with the virial mass larger than 10 8 M ⊙ h −1 at z = 0 and then identify their progenitors at infall. With the mass cut, there will be at least 2500 simulation particles for each progenitor halo so that we can accurately reconstruct its density profile. The radial resolution of the cosmological simulation ℓ ≈ 0.3 kpc is more than 1 order of magnitude larger than the radial scale relevant for the GD-1 perturber. To overcome this resolution limit, we fit each progenitor halo with a truncated Navarro–Frenk–White (NFW) profile for the region r > 0.3 kpc: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{eqnarray*}\rho (r)=\frac{{\rho }_{s}}{\left(r/{r}_{s}\right){\left(1+r/{r}_{s}\right)}^{2}}\times \frac{\exp (-r/{r}_{{\mathrm{cut}}})}{{(1+{r}_{s}/{r}_{{\mathrm{cut}}})}^{0.3}},\end{eqnarray*}\end{document} ρ ( r ) = ρ s r / r s 1 + r / r s 2 × exp ( − r / r cut ) ( 1 + r s / r cut ) 0.3 , where ρ s and r s are the scale density and radius, respectively, and r cut is the truncation radius due to tidal stripping. We determine the three parameters for each progenitor at infall, achieving excellent overall fit quality. The truncated NFW profile is then extrapolated inward to compute the total enclosed mass within r = 10 pc. Additionally, we confirm that many progenitor halos can be well-fitted with the standard NFW profile, while some exhibit density profiles slightly steeper than r −3 in the outer regions. For these cases, the standard NFW fit may introduce bias and overestimate the central density. However, the truncated NFW profile provides a significantly better fit. Figure 1 (left) shows the density profiles for the 125 progenitor halos at infall (blue). We also present the fit to one of the progenitors (black), which will be used as the initial condition for our SIDM simulations; see the detailed comparison in the inset panel. For this halo, the standard NFW profile provides a good fit. The simulated density profile is flattened for r ≲ 0.3 kpc due to the resolution limit. However, we expect that the NFW profile provides a good approximation for extrapolating the density inward before the halo undergoes significant tidal stripping. Figure 1 (middle) shows the enclosed mass within 10 pc versus virial mass of the progenitor halos at infall. For comparison, we include a reference case from the viable parameter region of the GD-1 perturber in A. Bonaca et al. 2019 : a Hernquist scale radius of r H = 15 pc and a total mass of M = 4.6 × 10 5 M ⊙ , which approximately corresponds to a substructure with the minimum density required to explain the spur and gap features of the GD-1 stream. For this reference case, the enclosed mass within 10 pc is ≈7.4 × 10 4 M ⊙ , as denoted by the horizontal line in the middle panel. We see that none of the CDM progenitor halos are sufficiently dense to be the perturber, and this conclusion holds when comparing the enclosed mass within r = 15 pc. The inner density of these CDM halos would further decrease as they evolve within the Milky Way's tidal field. The progenitor CDM halos shown in Figure 1 correspond to subhalos with masses >10 8 M ⊙ h −1 at z = 0. We plan to relax this mass threshold and examine halos with lower masses. Based on the resolution limit in the cosmological CDM simulation , we expect to reconstruct the density profiles of progenitors for subhalos with masses a few times 10 7 M ⊙ h −1 using the truncated NFW profile in Equation ( 1 ). A more detailed investigation will be deferred to future work. For the CDM progenitor halos, we estimate the timescale of gravothermal collapse in SIDM : \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{eqnarray*}{t}_{c}=\frac{150}{C}\frac{1}{{r}_{s}{\rho }_{s}\left({\sigma }_{{\mathrm{eff}}}/m\right)}\frac{1}{\sqrt{4\pi G{\rho }_{s}}},\end{eqnarray*}\end{document} t c = 150 C 1 r s ρ s σ eff / m 1 4 π G ρ s , where C = 0.75 is a numerical factor and σ eff is the effective cross section . For simplicity, we assume a constant cross section in this work. Figure 1 (right) shows the collapse time versus virial mass for the progenitors, where we have taken σ / m = 50 cm 2 g −1 . About one-third of the halos are expected to collapse within 10 Gyr. Since tidal stripping could speed up the onset of the collapse , we expect that more halos would be in the collapse phase after they evolve in the tidal field, and their overall mass would be reduced as well. We choose the CDM progenitor with the earliest infall time among the five halos that have t c < 10 Gyr, and its density profile is shown in Figure 1 (left, black). For this halo, the fitted NFW parameters are ρ s = 7.5 × 10 7 M ⊙ kpc −3 and r s = 0.50 kpc. The maximum circular velocity and the associated radius are \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} ${V}_{{\mathrm{\max }}}=14.8\,{\mathrm{km}}\,{{\mathrm{s}}}^{-1}$\end{document} V max = 14.8 km s − 1 and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} ${r}_{{\mathrm{\max }}}=1.1\,{\mathrm{kpc}}$\end{document} r max = 1.1 kpc , respectively. We use the public code SpherIC to generate the initial condition, and the total halo mass is M = 3.25 × 10 8 M ⊙ . The simulation has a particle mass of 32.5 M ⊙ , a total number of 10 7 particles, and a softening length of ϵ = 2 pc. We use the public N -body code GADGET-2 implemented with an SIDM module from D. Yang et al. , which follows the algorithm in A. Robertson et al. with small modifications. As indicated in Figure 1 (right), the halo would collapse within 10 Gyr for σ / m = 50 cm 2 g −1 even if it is isolated. In our N -body simulations, we consider three values, σ / m = 30 cm 2 g −1 (SIDM30), 50 cm 2 g −1 (SIDM50), and 100 cm 2 g −1 (SIDM100), to explore a wide range of cross sections. A viable SIDM model should exhibit a velocity-dependent cross section that is large at low velocities while decreasing toward high velocities to evade constraints on massive halos around cluster scales ≲0.1 cm 2 g −1 at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} ${V}_{{\mathrm{\max }}}\unicode{x0007E}1000\,{\mathrm{km}}\,{{\mathrm{s}}}^{-1}$\end{document} V max ~ 1000 km s − 1 . Nevertheless, for a specific halo, we can use a constant effective cross section to characterize its gravothermal evolution . In our case, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} ${V}_{{\mathrm{\max }}}$\end{document} V max decreases from ~15 to 7 km s −1 due to tidal mass loss. Thus, the σ / m values we consider can be regard as effective cross sections for \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} ${V}_{{\mathrm{\max }}}\unicode{x0007E}10\,{\mathrm{km}}\,{{\mathrm{s}}}^{-1}$\end{document} V max ~ 10 km s − 1 on average, which overall align with SIDM models proposed to explain diverse dark matter distributions in galaxies . The Milky Way is modeled as a static potential that contains three main components. 1. A spherical NFW halo: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{eqnarray*}{{\mathrm{\Phi }}}_{\,\mathrm{DM}\,}(r)=-4\pi G{\rho }_{s}{r}_{s}^{3}\frac{\mathrm{ln}\left(1+r/{r}_{s}\right)}{r},\end{eqnarray*}\end{document} Φ DM ( r ) = − 4 π G ρ s r s 3 ln 1 + r / r s r , with ρ s = 8.54 × 10 6 M ⊙ kpc −3 and r s = 19.6 kpc. G is the Newton constant. 2. A spherical stellar bulge with a Hernquist profile : \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{eqnarray*}{{\mathrm{\Phi }}}_{{\mathrm{b}}}(r)=-\frac{G{M}_{{\mathrm{b}}}}{{r}_{{\mathrm{H}}}+r},\end{eqnarray*}\end{document} Φ b ( r ) = − G M b r H + r , with M b = 9.23 × 10 9 M ⊙ and r H = 1.3 kpc. 3. Two stellar disks and two gas disks with an axisymmetric Miyamoto–Nagai profile : \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{eqnarray*}{{\mathrm{\Phi }}}_{{\mathrm{}}}(R,z)=-\frac{G{M}_{{\mathrm{d}}}}{{\left[{R}^{2}+{\left({a}_{{\mathrm{d}}}+\sqrt{{z}^{2}+{b}_{{\mathrm{d}}}^{2}}\right)}^{2}\right]}^{1/2}}.\end{eqnarray*}\end{document} Φ ( R , z ) = − G M d R 2 + a d + z 2 + b d 2 2 1 / 2 . The parameters for each disk are as follows. Thin stellar disk: M d = 3.52 × 10 10 M ⊙ , a d = 2.50 kpc, and b d = 0.3 kpc; thick stellar disk: M d = 1.05 × 10 10 M ⊙ , a d = 3.02 kpc, and b d = 0.9 kpc; thin gas disk: M d =1.2 × 10 9 M ⊙ , a d = 1.5 kpc, and b d = 0.045 kpc; and thick gas disk: M d = 1.1 × 10 10 M ⊙ , a d = 7.0 kpc, and b d = 0.085 kpc. These parameters are motivated by the Milky Way mass model in P. J. McMillan . Note that the stellar and disk density profiles in P. J. McMillan use exponential functions, which are challenging to implement in controlled N -body simulations due to the lack of analytical expressions for their corresponding potentials. Nevertheless, we have verified that the difference in the total potential remains within 2% in the regions with \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $\sqrt{{R}^{2}+{z}^{2}}> 10\,{\mathrm{kpc}}$\end{document} R 2 + z 2 > 10 kpc , which are most relevant for our simulated subhalo. Since the host halo is treated as a static potential, we neglect dark matter particle scatterings between the host halo and the subhalo. This approximation is well justified for velocity-dependent SIDM models with σ / m ≲ 1 cm 2 g −1 at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} ${V}_{{\mathrm{\max }}}\unicode{x0007E}200\,{\mathrm{km}}\,{{\mathrm{s}}}^{-1}$\end{document} V max ~ 200 km s − 1 . A. Bonaca et al. found that the best-fit orbit of GD-1 has a pericenter of r peri = 13.8 kpc and an apocenter of r apo = 22.3 kpc, while the orbit of its perturber remains highly uncertain. For our simulation, we adopt an orbit with r peri = 17 kpc and r apo = 142 kpc, with the simulated subhalo undergoing five pericenter passages over 10 Gyr. Although we do not aim to explicitly model the encounter event, at t ≈ 10 Gyr, the simulated subhalo's coordinates are R.A. = 21 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $\mathop{.}\limits^{\unicode{x000b0}}$\end{document} . ° 5 and decl. = −7 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $\mathop{.}\limits^{\unicode{x000b0}}$\end{document} . ° 9, consistent with the inferred position range of the present-day GD-1 perturber . While this orbit differs from that of the progenitor halo selected from the cosmological merger tree , it remains typical for many subhalos in the simulation. We emphasize that gravothermal collapse is intrinsic to SIDM halos, and the overall properties of our simulated perturber are robust regardless of the specific orbit chosen. Figure 2 (left) shows the evolution of the enclosed mass within the inner r = 10 pc for CDM (blue), SIDM30 (amber), SIDM50 (orange), and SIDM100 (pink) subhalos. For CDM, the inner mass decreases monotonically due to tidal stripping. In contrast, for SIDM, the mass initially decreases sharply due to core expansion, followed by an increase as core collapse occurs. By t ≈ 10 Gyr, the inner mass of the SIDM subhalos is 1 order of magnitude higher than that of the CDM subhalo, aligning well with the reference Hernquist profile (horizontal line). Additionally, the collapse times are t c ~ 6, 4, and 2 Gyr for SIDM30, SIDM50, and SIDM100, respectively, about a factor of 2 shorter than those estimated using Equation 2 , which is calibrated for isolated halos. In a subhalo, tidal stripping reduces the velocity dispersion of dark matter particles from the intermediate to outer regions as a result of mass loss. Consequently, a negative “temperature” gradient—a necessary condition for the onset of core collapse—is more easily established compared to an isolated halo . In Figure 2 (middle), we show the evolution of the total bound mass for the simulated CDM and SIDM subhalos. Initially, the halo mass is 3.25 × 10 8 M ⊙ and is reduced by 1 order of magnitude by t ≈ 10 Gyr due to tidal stripping. As expected, the total mass loss is more significant as the cross section increases. For SIDM, the final halo mass ranges from 4 × 10 6 to 10 7 M ⊙ , which falls well within the favored mass range of the GD-1 perturber 3 × 10 5 –10 8 M ⊙ . Figure 2 (right) shows the corresponding density profiles at t = 10 Gyr for the CDM and SIDM subhalos, along with the initial NFW profile. For comparison, the viable region for the GD-1 perturber (shaded gray), converted from Figure 6 of A. Bonaca et al. , and the reference Hernquist profile (dashed gray) are also shown. Compared to CDM, the density profiles of the SIDM subhalos are significantly steeper and overall consistent with the favored Hernquist profiles from A. Bonaca et al. . This indicates that dark matter self-interactions can both increase central density and accelerate tidal mass loss in the outer regions. Consequently, an SIDM subhalo can become more compact and dense than its CDM counterpart. We note that the CDM subhalo has a small density core near the center. This is due to the resolution limit as ℓ = 2.8 ϵ ≈ 5.6 pc, although the simulated subhalo contains more than 6 × 10 5 simulation particles at t = 10 Gyr. We fit the density profile using the analytical function \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $\rho (r)={\rho }_{{\mathrm{cut}}}\exp (-r/{r}_{{\mathrm{cut}}}){r}_{{\mathrm{cut}}}/r$\end{document} ρ ( r ) = ρ cut exp ( − r / r cut ) r cut / r from R. Errani & J. F. Navarro , which is proposed to model a tidally stripped CDM halo. With ρ cut ≈ 1.2 × 10 8 M ⊙ kpc −3 and r cut ≈ 0.22 kpc, we find a good fit for the region r ≳ 10 pc; see Figure 2 (right, dotted blue). The fitted function has a cusp ρ ( r ) ∝ r −1 near the center, and it provides a correction to the core due to the resolution limit. For the fitted profile, the enclosed mass within 10 pc is 1.6 × 10 4 M ⊙ . However, even with this correction, the CDM subhalo remains insufficiently dense to explain the high density of the GD-1 perturber. In Figure 2 (right), we also present the density profile of the globular cluster NGC 2419 (cyan), modeled using the King profile from H. Baumgardt et al. . Interestingly, this profile closely resembles the density profile of the SIDM30 subhalo within 30 pc. This similarity is not coincidental, as the formation of globular clusters follows the same mechanism as the collapse of SIDM halos. This suggests that distinguishing between SIDM and globular cluster scenarios in explaining the GD-1 perturbation could be challenging. However, NGC 2419 itself cannot be the GD-1 perturber, as its orbit does not align with the perturbation . If the perturbation is caused by an undetected globular cluster that emits light, it could be identified in future astronomical surveys. Furthermore, narrowing down the favored parameter space in the mass–size plane for the perturber would help us distinguish the two scenarios. For instance, if the perturber's mass is further constrained to the range 10 7 –10 8 M ⊙ , the SIDM scenario would be favored, as globular clusters typically have masses below a few times 10 6 M ⊙ . Another intriguing possibility is that the perturber is an SIDM substructure hosting stars, as we will discuss later. It may have undergone significant tidal stripping, resulting in an ultrafaint dwarf with mass and structural properties similar to those of a massive globular cluster . Confirming this scenario would require detecting a stellar counterpart at the inferred location of the perturber. Distinguishing between these possibilities will require dedicated observational campaigns and detailed modeling efforts, making this an exciting avenue for future research. The inner density profiles of our simulated SIDM subhalos ( r ≲ 10 pc) could be underestimated due to numerical issues in N -body simulations when the halo is deeply collapsed . Specially, numerical artifacts introduce additional “energy” that heats the simulated halo, slowing down or even preventing further increases in inner density; see M. S. Fischer et al. for discussions about potential causes. As shown in Figure 2 (left), for the SIDM subhalos, the enclosed mass within inner 10 pc stalls after t ≈ 4.5–6.5 Gyr, suggesting that they may suffer from the artificial heating effect. To further test this, we conducted an isolated simulation without the tidal field for the same initial NFW profile and σ / m = 50 cm 2 g −1 . Since the isolated halo experiences no tidal mass loss or heating, its total energy can be computed straightforwardly. See Appendix A for details on the isolated simulation and comparison with the subhalos. Indeed, we find that the energy increases when the isolated halo enters the deep collapse phase, corresponding to a Knudsen number of Kn ≈ 0.4 within 10 pc, i.e., the ratio of the mean free path to the gravitational height . For the SIDM subhalos, the stalling behavior occurs when their Kn values reach 0.3–0.6. In comparison, the total energy of the simulated SIDM halo in M. S. Fischer et al. starts to increase when Kn reaches 0.1. Even at Kn = 0.01 energy conservation violation is at the 1.5% level, better than our simulation. This is likely because M. S. Fischer et al. adopted a more accurate criterion for the gravity computations while at a higher computational cost. Since the artificial heating effect leads to an underestimation of the inner density profile for a collapsed SIDM halo, our results are conservative in this regard. Nevertheless, it will be important to further improve the SIDM prediction as future measurements of the GD-1 stream could narrow down the viable parameter space of the perturber . When modeling the Milky Way, we used static potentials for both halo and stars, calibrated with present-day measurements. Simulations show that Milky Way–like systems could grow significantly over the last ~6 Gyr due to mergers and accretion . If these effects were incorporated, our simulated subhalo would experience weaker tidal stripping in the early stages. However, we note that this is degenerate with the orbital parameters; similar results can be achieved by lowering the pericenter if a weaker potential is adopted at early times. In Appendix A , we will see that even for an isolated halo, the SIDM50 case can still collapse to the viable parameter region. Additionally, encounters between the GD-1 stream and the perturber are likely to have occurred within the last 1 Gyr , and hence the growth history of the Milky Way may not directly impact the inference of the perturber's properties. The subhalo we used to demonstrate the SIDM scenario for the GD-1 perturber has an infall mass of ≈3 × 10 8 M ⊙ . Interestingly, this is near the upper limit on the peak mass of subhalos that host currently observed satellite galaxies in the Milky Way . Thus, it remains an open question whether the perturber is a truly dark substructure, devoid of a galaxy. To further investigate detectability, we conducted additional simulations for the CDM and SIDM50 cases with live stellar particles, assuming a Plummer stellar profile with a scale radius of 0.3 kpc and a total mass of 3.2 × 10 4 M ⊙ , motivated by hydrodynamical simulations of the Local Group . At t = 10 Gyr, the bound stellar masses are 1.8 × 10 4 and 1.3 × 10 4 M ⊙ for the CDM and SIDM50 cases, respectively, with the latter also exhibiting a steeper stellar density profile toward the central regions. These substructures fall into the category of ultrafaint dwarf galaxies and could potentially be detected in the near future through observations, e.g., with the Rubin Observatory . More work is needed along these lines. For instance, the stellar–halo mass relation becomes increasingly steep in the ultrafaint regime and exhibits significant scatter , which must be taken into account. Additionally, since the Rubin Observatory can only detect objects in the southern hemisphere, it would be crucial to assess Rubin's sky coverage in conjunction with the orbital information of the GD-1 perturber from A. Bonaca et al. . We leave these investigations for future work. Furthermore, our scenario should also apply to smaller infall masses below ~10 8 M ⊙ . Indeed, for SIDM models with large velocity-dependent cross sections, the population of core-collapsing (sub)halos increases as the mass decreases . Thus, stellar streams like GD-1 can probe both population and density profile of core-collapsing subhalos even below the mass threshold for galaxy formation. We used the Hernquist profile for the GD-1 perturber from A. Bonaca et al. as a reference to assess the simulated subhalos. It would be intriguing to take the SIDM subhalo and directly model its encounter with GD-1, incorporating the influence of the Large Magellanic Cloud . We could use the parametric model to generate a population of collapsed SIDM subhalos in Milky Way analogs. To overcome the numerical issues in N -body simulations of core-collapsing halos, we may complement them with the semianalytical fluid model to better capture the dynamics in the the central regions . In summary, we have conducted controlled N -body simulations and shown that a core-collapsed SIDM halo could explain the high density of the GD-1 stellar stream perturber. For progenitor halos from the cosmological simulation of a Milky Way analog, the required self-interacting cross section σ / m ≳ 30 cm 2 g −1 for ~10 8 M ⊙ halos with \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} ${V}_{{\mathrm{\max }}}\unicode{x0007E}10\,{\mathrm{km}}\,{{\mathrm{s}}}^{-1}$\end{document} V max ~ 10 km s − 1 . Dark matter self-interactions can both increase inner density and accelerate tidal mass loss in the outer regions, producing a compact and dense perturber to explain the spur and gap features of the GD-1 stream. Our findings demonstrate that stellar streams provide a novel probe into the self-interacting nature of dark matter. We have also outlined future investigations to further improve this promising approach. | Study | other | en | 0.999996 |
PMC11699422 | Medical imaging is an essential tool, crucial in diagnosis and treatment planning in the medical field. Various approaches, such as Magnetic resonance imaging (MRI), computed tomography (CT), digital mammography, X-ray, ultrasound, positron emission tomography (PET), and digital pathology, are used to generate images that aid in a variety of tasks such as pathology localization, the study of anatomical structure, treatment planning, and even computer-integrated surgery, among others [ , , ]. Humans have traditionally performed medical image analysis and interpretation for several years. However, with the rapid advancement of artificial intelligence (AI), the medical field increasingly embraces computer-assisted tools as a resource to solving diagnostic problems with precision and efficiency. This allows for real-time prediction of various diseases and in-depth examination of different treatment options , avoiding limitations such as inter-observer and intra-observer variability and error resulting from significant variations in pathologies and the possible exhaustion of human experts . The bibliometric analysis allows for uncovering emerging trends and patterns in the scientific knowledge of a specific domain in a quantitative approach , by combining disciplines such as information science, mathematics, and statistics . Deep learning application in medical image analysis has been significantly exploited in recent years due to its current relevance. Many bibliometric analysis works have focused on various fields of research including the usage of artificial intelligence in healthcare , medical image segmentation , the application of deep learning in the medical field , medical data mining research , and machine learning-based disease diagnosis . To the best of our knowledge, bibliometric and qualitative analysis research has not been published on applying object detection algorithms in the medical field. In this work, we propose to conduct a comprehensive quantitative and qualitative analysis of the literature related to applying deep learning object detectors in medical imaging and to reveal potential trends in this field. Our contributions include. ⁃ A bibliometric analysis of the application of deep learning object detectors was conducted using two of the most widely used databases: Scopus and the Web of Science (WoS). ⁃ The reporting of several relevant quantitative indicators, such as annual publication analysis, research area analysis, journal publication analysis, publications by country and author, and keyword analysis. By conducting and employing bibliometric analysis, we aim to evaluate the overall status of the application of object detection in the medical field and identify possible divergences among distinct screening methods and anatomical areas. Furthermore, our work also reflects on. • The publications trends on this topic, and concretely the growth trajectory of object detection applications in medical imaging. • The most influential authors, i.e., the most prolific and leading contributors concerning object detection in medical field research. • The international collaborations existing in this field. • The most highly cited papers and the topics they cover. • The emerging research themes in this topic, how those themes evolved over time, and the ones that gained the most attention in recent years. • The academic journals representing the main outlets for the research on object detection in the medical field. • The algorithms, screening methods, and anatomical areas under study that experienced the most significant growth in terms of publications. • The data sets used, their availability, the sample size of these data sets, whether external or internal data sets are used in the different studies, and the nature of the study (retrospective or prospective). By assessing the frequency at which different algorithms are applied, we aim to understand the diversity of approaches used in object detection for medical imaging, giving insights into the popularity and effectiveness of various algorithms. The occurrence analysis of the algorithms allows us to identify which approaches are commonly employed, which may lead to better performance, and which areas might require further research. The structure of this work is as follows: in the first section, a brief introduction, background to the problem, and purpose of this work are provided. The following section outlines the research methodology, data collection procedures, bibliometric tools, and screening methods. The third section presents a quantitative and descriptive view supported by a bibliometric analysis of the filtered information. In the fourth section, a qualitative analysis of the most cited papers is conducted, and an exploratory analysis is performed on the incidence of different object detectors applied and the medical fields where object detection is more prevalent. In the final section, the conclusions of this study and the overall findings are presented. The framework for gathering and filtering data was based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The query search, conducted on January 20th, 2023, was centered around three main topics that align with our purpose, excluding the concept of segmentation on keywords or titles, and limiting the results to articles written in English, as depicted in the Supplementary File 1. Those results comprise papers up until the end of 2022. Removing duplicated articles resulted in a database of 951 reports that underwent a screening process for the title and abstract for enhanced filtering. Of those, 390 were excluded, 197 were included in the final analysis, and 364 were subjected to a comprehensive report screening as their titles and abstracts were inconclusive. Of the latter, 250 articles were rejected as they were unrelated to object detection; the remaining were added to the final inclusion list. This resulted in a total of 311 articles pertaining to the topic of interest. Those 311 papers are the object of quantitative analysis. From those, papers with an annual citation rate lower than five (n = 231) were removed, and the remaining (n = 80) were further analyzed following a qualitative approach. The citations of these 311 publications were analyzed as a whole, as shown in Table 1 . In total, those articles were cited 5052 times, and only 5.79 % (n = 18) of the publications have more than 50 citations. On the other hand, 77.81 % (n = 242) of the total publications have at least one citation. Table 1 Article papers and citations per year of object detection in medical imaging field. a Table 1 Year TP TC AC Total publications with citations ≥50 ≥20 ≥10 ≥5 ≥1 2018 13 1062 81.69 6 9 12 13 13 2019 28 799 28.54 6 14 22 23 27 2020 57 2358 41.37 6 18 36 47 57 2021 78 593 7.60 0 7 19 42 73 2022 135 240 1.78 0 0 2 14 72 % 100.00 % 5,79 % 15.43 % 29.26 % 44.69 % 77.81 % a TP stands for total papers, TC for total citations, and AC for average article citations. An analysis of the number of publications according to their quantity of citations (higher or equal to 50,20,10,5 and 1) is performed for each year. Endoscopy allows the visual inspection of an internal organ or tissue in detail . One possible application of endoscopic procedures is to identify colorectal lesions, where techniques such as high-resolution microendoscopy, fluorescence imaging, and enhanced endoscopy are available to improve the detection rate of tumors using endoscopy . Pathology Imaging uses static images, live streaming of images, and whole slide imaging (WSI) to make pathology diagnoses. The latter allows the digitalization of entire glass slides ,enabling quantitative analyses for the entire landscape of tissue morphology without the need for microscopy . Digital photography plays an important role in various specific medical specialties, enabling the identification and diagnosis of potential lesions and diseases in a cost-effective, noninvasive, readily accessible, and real-time manner. The usage of this approach encompasses a wide range of tasks, including the detection of skin cancer, dermoscopy imaging, assessment of oral and dental health, and the diagnosis of hair and nail conditions . Microscopy images can support quantitative analysis for diagnosing and characterization of several diseases, playing an essential role in computer-aided diagnosis and prognosis . Associated with Histopathology, the field of study of human tissue using a microscope, it allows, through staining, the visualization of specific parts of tissues that enables the identification of different diseases such as kidney cancer, lung cancer, and breast cancer, among others, through nuclei and cell detection . Conventional angiography and contrast-enhanced magnetic resonance angiography is regarded as the reference examination for investigation of atherosclerotic lesions of the supra-aortic extracranial vessels, and especially for detecting stenosis of the carotid artery, being the latter one the less invasive and with similar results to the former . As shown in Table 5 , on abdominal organs, the research is focused on the colon, gallbladder, liver, stomach, cervix, and uterus. Table 5 Publications in which object detection algorithms are applied to abdominal organ images. a Table 5 Reference C Modality Organ Algorithm-based Task 98 E Colon YOLO Polyp detection 52 E Colon SSD Polyp detection 16 E Colon YOLO Polyp detection 7 E Colon SSD Polyp detection 14 E Colon RefineNet Polyp detection 21 E Gallbladder YOLOv3 Detection of bile duct injury 41 CT Gallbladder YOLOv3 Detection of gall stones 17 US Liver Faster R-CNN Abnormality detection 25 E Stomach Faster R-CNN Detection of gastric lesions 9 E Stomach YOLOv3 Detection of gastric lesions and neoplasm prediction 13 PH Cervix R-CNN Cervical cancer detection 27 PH Cervix Faster R-CNN Cervical cancer detection 18 PH Cervix Faster R-CNN Cervical cancer detection 30 US Uterus YOLOV2 Detection of cardiac structural abnormalities 27 US Uterus Custom Detection of cardiac structural abnormalities a Overview of papers using object detection techniques for detecting abnormalities in abdominal organs, where E stands for endoscopy, CT for computed tomography, PI for pathology image, PH for photography, and US for ultrasound. In the header of the table, C stands for the number of citations. Five of the selected papers are dedicated to colon polyps detection, using images obtained through endoscopy. Among those publications, YOLO and Faster R-CNN are applied twice, while RefineNet is applied once. In the first study in which YOLO is implemented, an improved framework is developed to detect polyps in a dataset consisting of more than 17,000 frames, yielding a precision of 88.6 % and a recall of 71.6 % at a prediction speed of 6.5 frames per second . In the second study, YOLOv3 and YOLOv4 architectures are applied to two datasets, achieving a precision of 90.6 % and a recall of 91.0 % at a processing speed of 100 frames per second .This last publication combines the YOLO structure with CSPNet to obtain a higher-performance model. In the SSD Implementations, one of the studies is validated in 7077 images, where the model was able to identify correctly 1073 colon polyps out of the existent 1172, with a sensitivity of 90 %, and a processing time of 20 frames per second . In the second implementation, a multiscale pyramidal fusion single-shot multibox detector network is applied, resulting in an mAP of 93.4 % , with a testing speed of 62.5 FPS. RefineNet is also employed for polyp detection and applied to a combination of four public data sets. In this study , the authors compare RefineNet with different algorithms such as YOLO and Faster R-CNN, achieving the best performance among all the tested approaches with an mAP of 73.5 %, while YOLOv3 and SSD achieve the fastest inference with 60 FPS. Two studies have been conducted using YOLOv3, with distinct objectives concerning gallbladder organs. In the first one, endoscopic images were used to detect four landmarks to prevent bile duct injury in 23 short videos. The authors of this study recognize the system's suboptimal performance . In contrast, the second study uses 223,846 CT images to detect gall stones, achieving an mAP of 89.5 % for granular and muddy stones . Regarding the liver, one study has been conducted in which cysts and primary hepatic carcinoma detection in ultrasound imaging were accomplished using Faster R-CNN with a ResNet backbone. In this publication , the authors achieved an mAP of 60 %, representing an improvement over the results obtained with YOLOv2, which had an mAP of 51 %. In two different studies, the detection of gastric lesions on endoscopic imaging was approached. In the Faster R-CNN implementation , more than one million images were employed to train and test the system. The authors of this study enhance the significant difference in detection time between the automatic approach and clinicians’ performance. The results are reported by sensitivity values, ranging from 87.2 % to 99.3 %. In the second publication, where YOLOv3 was applied, the data was obtained from over 100,00 patients, and the final model sensitivity ranged between 91.7 % and 96.9 % . Three of the fifteen papers on abdominal organs are focused on cervical cancer detection, obtained through applying two-stage object detection approaches on photographic images. In a study published in 2022 , a generative adversarial network was developed using an R-CNN to accurately detect small cervical cells and classify them into normal, precancerous, or cancerous. The remaining two publications employed Faster R-CNN. The first study created a system based on 2853 images labeled as high-risk or low-risk, achieving an AUC value of 0.87 . In the second study, an improved version of Faster R-CNN was developed and tested on 2528 images with 5294 lesion regions, achieving an mAP of 80.37 %. This represented better results than those obtained with SSD (mAP = 74.27 %), YOLOv3 (mAP = 77.11 %), and Faster R-CNN (mAP = 75.89 %). Nine of the eighty studies selected based on the annual citation rate are employed in bone imaging, as exposed in Table 6 . Table 6 Publications in which object detection algorithms are applied to bone images. a Table 6 Reference C Modality Algorithm Task 73 CT Faster R-CNN Detection of distal radius fractures 66 CR DetectNet Detection of mandible fractures 28 CR DetectNet Detection of maxillary sinus lesions 7 US YOLOv3 Detection of vertebras in spine 7 CT Ensemble Detection of wrist fractures 13 CR YOLOv3 Detection of hip dislocation 12 MRI YOLOv3 Detection of Lumbar Disc Herniation 22 CR Faster R-CNN Skeletal bone age assessment 10 CT YOLOv3 Detection of vertebral fractures a Overview of papers using object detection techniques on bones, where CT stands for computed tomography, CR for computed radiography, US for ultrasound and MRI for magnetic resonance imaging. In the table's header, C stands for the number of citations. On CT imaging, three studies use object detection to locate fractures, namely on the distal radius , on the wrist , and on the vertebras . In the first one, a comparison is made between the automatic approach using a Faster R-CNN, and professional assessment. The authors conclude that the network achieved performances similar to those obtained from orthopedists and superior to radiologists. In a second study, wrist fractures are located with an Ensemble technique based on WBF. Ten base models are tried out, and the highest AP Score was obtained with the ensemble version obtained from the base learners. The authors enhance the potential of ensemble techniques in this field. In the third publication, vertebral fractures are detected using a YOLOv3 architecture, and one remark appointed by the authors is the highest interobserver reliability of the automatic system compared to human observers. CR imaging is used in object detection studies with different purposes. Two publications, in particular, apply DetectNet , a network developed in DIGITS. The first study employs a model built on 210 training images to locate mandible fractures, achieving a sensitivity of 88 % in the testing data . The second study, also utilizing DIGITS, identifies maxillary sinus lesions, with a sensitivity of 100 % on healthy and inflamed maxillary sinuses and 98 % and 89 % on distinct data sets for cyst maxillary sinus region . Another study trains YOLOv3 to assess the risk of hip dislocation on 1490 radiographs from dislocated cases and 91,094 from non-dislocated cases, achieving a sensitivity of 89 % . A Faster R-CNN is employed to assess skeletal bone age by detecting the ossification centers of the epiphysis and carpal bones , with a performance measured by MAE, achieving values of 0.48 and 0.51 in two tested data sets. On ultrasound imaging, various object detectors, such as SSD, YOLOv3, and YOLOv4, were trained to detect the vertebrae in the spine . While SSD achieved the highest mAP with a value of 90.84 %, YOLOv3 was chosen as the final model due to its short inference time of around 7 FPS, around ten times faster than SSD's. An MRI is a powerful tool for producing detailed images of the brain, which can be used to detect a wide range of conditions, such as tumors, injuries, strokes, and blood vessel problems. In recent years, some object detection algorithms have been applied to this field to automate the diagnostic process and increase the accuracy of results. Table 7 exposes those studies. Table 7 Publications in which object detection algorithms are applied to brain images. a Table 7 Reference C Modality Algorithm Task 9 MRI YOLOv5 Brain tumor diagnosis 20 MRI YOLOv4 Brain tumor diagnosis 11 MRI Faster R-CNN Stroke lesion detection a Overview of publications that employ object detection techniques on the brain, where MRI stands for magnetic resonance imaging. In the header of the table, C stands for the number of citations. In the publications considered for qualitative analysis, two studies applied the YOLO architecture to brain tumor diagnosis, while another study applied Faster R-CNN for stroke lesion detection. In the study where YOLOv5 is applied , the authors compared the performance of different algorithm variants on 800 images to brain tumor detection. Unsurprisingly, the larger version of the model achieved the best mAP at 91.2 % using YOLOv5x, while the smaller versions of YOLOv5 (YOLOv5n and YOLOv5s) achieved the worst performances with an mAP of 85.2 % and 87 %. However, the trade-off is that larger versions take more time to train. In a second study for brain tumor diagnosis and detection, a transfer learning approach and fine-tuning techniques were applied to YOLOv4 to build a model on a data set of 3064 MRI scans, which was able to detect three distinct tumors, namely glioma, meningioma, and pituitary. This improved version of YOLO achieved a final mAP of 93.14 % . A third study on Brain MRI scans is focused on stroke lesion detection . In this study, the authors compared the performance of Faster R-CNN, YOLOv3 and SSD, and concluded that SSD performed the best, with the highest mAP (89.77 %). When comparing the inference time, YOLO was the fastest approach, but this advantage was reflected in a significant decrease in the mAP, with a value of 74.9 %. Breast cancer is the most common cancer diagnosed worldwide, with 685,000 deaths associated in 2020, being estimated that in 2040 this value will rise to 1 million . With eight publications associated, as expressed in Table 8 , object detectors in breast imaging focus on detecting lesions. Table 8 Publications in which object detection algorithms are applied in breast images. a Table 8 Reference C Modality Algorithm Task 364 CR Faster R-CNN Breast lesion detection 90 CR YOLO 9000 Breast lesion detection 78 US SSD Breast lesion detection 43 CR Custom Breast lesion detection 61 CR RetinaNet Breast lesion detection 21 MRI RetinaNet Breast lesion detection 6 CR Mask R-CNN Breast lesion detection 10 CR YOLOv3 Breast lesion detection a Overview of publications using object detection techniques for breast lesion detection, where CR stands for computed radiography, US for ultrasound and MRI for magnetic resonance imaging. In the header of the table, C stands for the number of citations. Regarding CR imaging, six studies were conducted using different algorithms as a basis. One of the publications applied Faster R-CNN to detect and classify malignant or benign lesions on a mammogram in a data set with 2620 screening exams. The system achieved an AUC of 0.85, with 10 % of the malignant lesions being missed. RetinaNet was tested in seven different data sets in the same year and, according to the authors, outperformed the conventional mass detection models . In 2020, two publications addressed this task. One of these applied YOLO 9000 on two distinct data sets, comprising 600 and 360 images, respectively. 99.17 % of the total cases were predicted correctly on these data sets, with 0.83 % being false detections . In the second case, a custom network was built to address class imbalance for small lesion detection on breast . Another publication used a YOLO-based model on 235 publicly available mammograms and 487 privately collected mammograms, achieving a detection accuracy rate of more than 95 % for the distinct datasets used and an inference time of around 0.55 s per image . In 2022, a Mask R-CNN-based framework was developed and implemented on public and in-house datasets. The model's performance was analyzed only using recall, achieving values of 0.82 for the first data set and 0.87 for the private data set, with an IoU threshold of 0.5 . In 2019, ultrasound images were applied on distinct object detectors, including Fast R-CNN, Faster R-CNN, YOLO, YOLOv3 and SSD. YOLO, and SSD performed significantly better than the other methods, and SSD was chosen as the most suitable model for this task, achieving an mAP of 96.89 % in a total of 1041 cases . Regarding MRI imaging, only one publication was included in this qualitative analysis , using RetinaNet. The authors compared the sensitivity, specificity, and AUC between the AI system and human readers, concluding that the former had a better diagnostic performance. In thoracic image analysis of both computed radiography and tomography, the detection of pulmonary tuberculosis and COVID-19 were the most commonly addressed applications, as seen in Table 9 . Table 9 Publications in which object detection algorithms are applied in chest images. a Table 9 Reference C Modality Algorithm Task 1306 CR YOLO 9000 COVID-19 detection 9 CT CenterNet Pulmonary tuberculosis detection 7 CR SSD COVID-19 detection 19 CR Faster R-CNN Pulmonary tuberculosis detection a Overview of publications that apply object detection techniques on chest imaging for COVID and pulmonary tuberculosis detection, where CT stands for computer tomography and CR for computed radiography. In the table's header, C stands for the number of citations. SSD and YOLO 9000 are applied to COVID detection on CR imaging. Several backbones have been tested with SSD, including VGG, Residual Network, DarkNet, and DenseNet, and their performance has been compared. DenseNet achieves the highest performance, with a precision of 0.93 and a recall of 0.94 . The most cited publication in our qualitative research focuses on implementing YOLO for COVID detection. The proposed model is developed to provide accurate diagnostics for binary classification (COVID vs No-findings) and multi-class classification, where Pneumonia is added to the former classes. The authors report an accuracy of 98.08 % on the binary problem and 87.02 % for the multi-class problem . In pulmonary tuberculosis detection, a Faster R-CNN adapted model has been applied to a multi-class problem on CT images, which encompasses co-existing cases of exudation, calcification, nodules, miliary tuberculosis, and other related conditions. This model achieves an mAP of 53.74 %, outperforming the original Faster R-CNN model, which reached 22.66 % mAP and surpassing the results obtained with FPN, which achieved 50.96 % mAP . Additionally, CenterNet has also been used on 892 CT scans for pulmonary tuberculosis detection, yielding an mAP of 68 % . Concerning the digestive system, object detection techniques have been applied to various forms of medical imaging, including esophagus imaging obtained through endoscopy, tongue images obtained through photography, and teeth images obtained through CR imaging, as seen in Table 10 . Table 10 Publications in which object detection algorithms are applied to images of digestive system organs. a Table 10 Reference C Modality Organ Algorithm Task 92 E Esophagus YOLOV2 Early esophageal neoplasia detection 44 E Esophagus SSD Early esophageal adenocarcinoma detection 29 PH Tongue R-CNN Tooth-marked tongue detection 117 CR Teeth Faster R-CNN Tooth detection 58 CR Teeth DetectNet Root fracture detection 24 CR Teeth DetectNet Tooth detection 26 CR Teeth Faster R-CNN Periodontal compromised teeth detection 8 CR Teeth Faster R-CNN Permanent teeth detection 11 CR Teeth Faster R-CNN Dental disease detection 5 CR Teeth Faster R-CNN Marginal bone loss around implants detection a Overview of publications that apply object detection techniques on digestive system organs, where CR stands for computed radiography, E for endoscopy and PH for photography. In the header of the table, C stands for the number of citations. In particular, YOLOv2 and SSD have been applied to endoscopy imaging, with the former being used to detect early stages of esophageal neoplasia and the latter being used to detect early stages of esophageal adenocarcinoma . In the first study, 916 images were used to train the model, and 458 images were used to test it, achieving an mAP of 75.33 % for narrow bone imaging and 80.19 % for near-focus images at a speed of 45 FPS. In the second study, the performance of SSD and Faster R-CNN were compared in terms of average recall rate (ARR), average precision rate (APR), sensitivity (SE), specificity (SP), and F-measure (FM) using a 5-fold cross-validation method. SSD achieved the highest values with 0.7 on APR, 0.90 in SE, 0.88 in SP, and 0.88 in FM, being surpassed by Faster R-CNN only in ARR with a difference of 0.04. In a tongue photography study, the goal was to identify tooth-marked areas on the tongue using a data set containing 641 images, 297 of which were tooth-marked. An adapted version of R-CNN was applied, and the authors reported an average accuracy of 72.7 %, a TPR of 69.1 %, and a TNR of 76.2 % . Tooth detection appears to be a prevalent subject in the realm of object detection frameworks in medical imaging. Seven of the 80 studies analyzed were dedicated to tooth detection and related tasks, and the Faster R-CNN algorithm was applied in five of these studies. In the first study , the model achieved a precision and recall rate of over 90 %, with a mean average precision (mAP) of 91 %. These values were only attainable after implementing several postprocessing procedures. In another study, Faster R-CNN was employed for Periodontal compromised teeth detection , resulting in an accuracy rate of 80 %, a positive predictive rate of 81 %, a sensitivity of 84 %, a specificity of 88 %, and a F-measure of 81 %. The authors of this study concluded that the system exhibited satisfactory detection abilities. Faster R-CNN was also applied to permanent teeth detection in a study where the authors reported results of 0.99 for recall and precision. Additionally, the algorithm was used in another work to detect dental diseases such as decay, periapical periodontitis, and periodontitis at varying levels of severity. The results showed that decay and periapical periodontitis lesions were detected with precision, recall, and average precision values of less than 0.25 for mild levels, while moderate and severe levels had values ranging from 0.2 to 0.3 and 0.5–0.6, respectively. In the more recent study where Faster R-CNN was applied, the goal was to detect Marginal bone loss around dental implants . The system was evaluated using a dataset of 1670 images and was assessed in terms of sensitivity, specificity, diagnostic error rate, omission diagnostic rate, and positive predictive value. Kappa statistics were also compared between the system and dental clinicians, and the authors concluded that there was a high level of agreement between the automatic system and the clinicians. On DetectNet, two studies are implemented, both applied to CR imaging. In one of these studies, the primary objective was the detection of root fractures . The study employed 300 images, containing a total of 330 root fractures. Out of these, 267 were successfully detected by the automatic approach, while twenty were falsely detected, resulting in a recall of 0.75, a precision of 0.93, and an F measure of 0.83. In a second study , DetectNet was applied to tooth detection. However, the authors acknowledged several limitations in their study, such as the small data set used, which was composed of only 75 training cases and 25 test cases. The growing availability of large-scale gigapixel whole-slide images of tissue specimens has made digital pathology and microscopy a highly popular application area for deep learning techniques . These imaging modalities have been applied to various purposes, such as cell detection and malaria detection, among others, as seen in Table 11 . Table 11 Publications in which object detection algorithms are applied to digital pathology and microscopy images. a Table 11 Reference C Modality Organ Algorithm Task 93 PI Blood Faster R-CNN White blood cells detection 37 PI Tissue Faster R-CNN Glomerular detection in multistained human renal tissue 27 M Others Faster R-CNN Human intestinal organoid detection 16 M DNA Faster R-CNN Detection of DNA damage 29 PI Cervical cells YOLOv3 Cervical cell recognition 31 M Blood YOLO Malaria detection 73 PI Blood SSD Peripheral leukocyte recognition 53 PI Blood YOLO Blood cells detection 18 PI Blood YOLOv4 Blast cell detection 15 PI Blood RetinaNet Malaria detection 9 PI Brain tissue YOLOv3 Alzheimer's diagnose 22 PI Blood R-CNN Cells detection 15 M Others Faster R-CNN Detection of cellular organelles 12 PI Liver and Kidney tissue RetinaNet Diatom detection 24 M Others RetinaNet Pulmonary Hemosiderophages detection 8 M Others YOLOv4 Sperm detection a Overview of publications that apply object detection techniques on digital pathology and microscopy imaging, where PI stands for pathological imaging and M for microscopy imaging. In the header of the table, C stands for the number of citations. The YOLO algorithm is regarded as the most prevalent among these studies, with six out of sixteen publications devoted to its application. In particular, three studies have explored the use of YOLO in the analysis of blood samples, specifically for detecting blood cells , blast cells , and malaria . In the first study, YOLO was trained and tested using 364 images with red blood cells, white blood cells, and platelets. The results showed an mAP of 82.58 % using the VGG16 backbone. Another study compared YOLOv3 with other popular models, including R-CNN, Fast R-CNN, Faster R-CNN, and SSD. Ultimately, the Faster R-CNN model arose as the winner with an mAP of 71 % and an inference time of 609 ms per image with VGG16. A third study on blood smears applied 14,700 images of peripheral leukocytes with eleven distinct categories, resulting in an mAP of 93.10 % for SSD and an inference time of 53 ms per image. Although YOLOv3 also performed well, it achieved only 92.10 % mAP. The blast cell detection study applied YOLOv4 on blood smears to aid in early leukaemia diagnosis and achieved an mAP of 95.57 % with an inference value of 50 FPS. In the malaria detection study with microscopic images, YOLOv3 and YOLOv4 were compared, with the last being the superior model, achieving an mAP of 96.32 % and an inference rate of 29.60 frames per second. Another study applied RetinaNet to detect malaria in 169 microscopic images for train and 130 for validation, with results reported in terms of sensitivity (0.92), specificity (0.90), accuracy (0.91), positive predicted value (0.92) and negative predicted value (0.90). The YOLO object detector has also been used for cervical cell recognition and sperm detection . In the first study, YOLOv3 was applied to cervical cytology screening and achieved an mAP of 63.4 %, with 95.7 % of sensitivity and 67.8 % of specificity, on a data set of 12,909 images split into ten distinct categories. The last study on YOLO was on Alzheimer's diagnosis . The YOLOv3 was trained to detect five tau lesion types, including neuronal inclusions, neuritic plaques, tufted astrocytes, astrocytic plaques, and coiled bodies, on 2522 immunostained slides images of the motor cortex, achieving a maximum mAP of 74.4 %. The Faster R-CNN algorithm was the second most used among the studies, appearing in five of the sixteen works available. In the study of glomerular detection in multistained human renal tissue , Faster R-CNN was used to automate the task and was trained on 33,000 images. The results were reported regarding recall, precision, and F measure for four different classes. Detection of human intestinal organoids was performed using Faster R-CNN on a data set of 1750 image patches with 14,242 ground truths, resulting in an mAP of 80 %. The authors argue that the quality of the automatic detection was similar to human capability but substantially faster. Another study, examining DNA damage detection , reported an mAP of 74 % with Faster R-CNN, while SSD and YOLOv3 achieved mAPs of 22 % and 30 %, respectively. The detection of cellular organelles was also considered using Faster R-CNN, with results reported solely for classification purposes. The RetinaNet algorithm was used in three studies. Excluding the malaria study also reported in this subsection, this algorithm was used on liver and kidney tissue for diatom detection , and to detect Pulmonary Hemosiderophages . The first study obtained an average precision of 0.82 and an average recall of 0.88. In the second study, a RetinaNet was trained on seventeen completely annotated cytology whole slide images (WSI) containing 78,047 hemosiderophages and achieved an mAP of 66 % for the five classes approached, exceeding human expert concordance. The qualitative analysis of the eighty studies revealed that only two of them have been applied to eye imaging, as expressed in Table 12 . Table 12 Publications in which object detection algorithms are applied to eye images. a Table 12 Reference C Modality Algorithm Task 29 FP Faster R-CNN Diabetes-Based Eye Disease Detection 12 FP YOLOv3 Lesion detection a Overview of publications that apply object detection techniques on eye imaging, where FP stands for fundus photography. In the header of the table, C stands for the number of citations. The first study used FRCNN, a Keras implementation of Faster R-CNN, to identify diabetes-based eye diseases, including diabetic retinopathy, diabetic macular edema, and glaucoma . The authors achieved an mAP of 94 % with the proposed model, outperforming two other approaches, SSPnet and R-CNN, which achieved mAPs of 85 % and 89 %, respectively. In the second study , the authors used YOLOv3 to identify lesions in eye images. The results were reported in terms of average precision (0.08 and 0.52), average recall (0.86 and 0.91), and F1 Score (0.16 and 0.66) for both the number of lesions and the number of images analyzed. This final subsection lists papers that address multiple applications, where the organ is not unique, and diverse applications that were not included in the previous subsections, as seen in Table 13 . Table 13 Publications in which object detection algorithms are applied to various topics. a Table 13 Reference C Modality Organ Algorithm Task 38 PH Foot Faster R-CNN Diabetic foot ulcers detection 37 PH Skin Mask R-CNN Melanoma detection 19 PH Skin Faster R-CNN Scalp health diagnosis 98 US Thyroid Faster R-CNN Thyroid papillary cancer detection 27 A Vessels Custom Intracranial aneurysm detection 10 CT Lymph nodes DetectNet Cervical lymph nodes detection 228 CT Several Faster R-CNN Lesion detection 54 CT Several Faster R-CNN Organ localization 20 US Several RFCN Region detection in Ultrasounds 25 E Several Fast R-CNN Wireless capsule endoscopy abnormal pattern detection 12 CT Several Mask R-CNN Lesion detection 22 E Several YOLO Upper gastrointestinal disease 26 MRI Several SSD Tuberculous and pyogenic spondylitis diagnosis a Overview of publications that apply object detection techniques with various purposes, where PH stands for photography, US for ultrasound, A for angiography, CT for computed tomography, E for endoscopy and MRI for magnetic resonance imaging. In the header of the table, C stands for the number of citations. Seven of the thirteen studies analyzed in this subsection focus on object detection without specifying a particular anatomical area. For the lesion detection task, two studies have been carried out. The first study uses an algorithm based on Faster R-CNN and a dataset that was compiled with 23,735 lesions from 32,120 CT scans. The results show a sensitivity rate of 81.1 %, with five false positives per image. The second study proposes a universal lesion detection in CT imaging. The data set was created by merging images from data sets related to the liver, lymph nodes, and the entire body, resulting in a total of over 30,000 lesions. The average sensitivity of the proposed model was reported to be 47.6 %. Lesions in various gastrointestinal regions, including the esophagus, stomach, pylorus, duodenum, and cardia, can be detected in gastroscopic images using YOLOV3 . The authors reported an mAP of 58.10 %. Additionally, this performance was compared against the of SSD and RetinaNet performances. Organ localization is the focus of a study where Faster R-CNN is used in CT imaging. In this work, the authors combined two datasets, one containing images of eleven body organs and the other containing images of twelve head organs, achieving an mAP of 73.01 % for the former and 84.78 % for the latter. A study on region detection in ultrasounds employs Faster R-CNN, and its performance is compared against SSD and RFCN. This study diagnoses various diseases, including gallbladder stones and polyps, hydronephrosis, kidney stones, renal cysts, hemangiomas, and fatty liver. RFCN emerged as the top performer of the three models with an mAP of 78.7 %, followed closely by SSD with 76.7 % and Faster R-CNN with 75.4 %. In medical imaging, tuberculous and pyogenic spondylitis diagnosis is carried out using an SSD approach on MRI scans . The results of this study show that the model achieved an AUC of 0.80, with a sensitivity of 85 % and a specificity of 67.9 %. Additionally, the skin is also the subject of research in automatic detection systems. A study was conducted on melanoma detection using Mask R-CNN , to identify potential skin lesion areas and crop those areas for further analysis by a classifier algorithm. Another study analyzed scalp health and analyzed four distinct scalp hair symptoms . The results showed that Faster R-CNN achieved an mAP of 91.75 %, while SSD achieved an mAP of 87.16 %. The detection of diabetic foot ulcers is assessed using Faster R-CNN on a photographic imaging dataset consisting of 2000 training images, 200 validation images, and 2000 testing images . In this study, the authors compare the performance of different algorithms in terms of mAP and other metrics, with Faster R-CNN achieving an mAP of 69.40 %, YOLOv3 an mAP of 65.60 %, Cascade DetNet an mAP of 63.94 %, YOLOv5 an mAP of 62.94 %, and EfficientDet an mAP of 56.94 %. In CT imaging for patients with oral cancers, a DetectNet is utilized for cervical lymph node detection . The sensitivity for metastatic lymph nodes was identified as 73 %, and for non-metastatic lymph nodes, a sensitivity of 52.5 % was observed. For intracranial aneurysm detection on digital subtraction angiography, a custom object detection network is employed on vessel imaging . The proposed architecture attained an accuracy of 93 % and an AUC of 0.942. In this study, the proposed network is also compared to YOLOv3 and RetinaNet, both with lower performance results. This study conducted a thorough bibliometric analysis of the relevant studies in the rapidly growing field of the application of object detection in medical image analysis. A PRISMA methodology was applied to identify 311 relevant publications in this field. Our research findings led us to understand that this is a recent area of study but one in which research is steadily increasing and has the potential to grow. Most of the publications were included in the research areas of Medicine, Computer Science, and Engineering and were published in top journals belonging to the first quartile. China, the United States, and Japan were the most productive countries, with the United States being the country with the highest level of international cooperation among these three countries. The majority of the authors with the highest number of citations were associated with only one published paper, and only six authors had five or more publications associated. In the authors’ keyword analysis, it was clear that concepts such as object detection, deep learning, artificial intelligence, and convolutional neural networks were prevalent, while in index keyword analysis, medical concepts were introduced with higher relevance. Despite the emergence of deep learning object detection methods in 2014, medical imaging only adopted this approach in 2018, when the algorithms had evolved and demonstrated improved performance capabilities, which is a high demand in the medical field. Regardless of its relatively late arrival in the field, the introduction of object detectors in medical imaging has been modest, and only thirteen studies were published in 2018. Nonetheless, our research findings indicate this is a rapidly developing study area. Four years later, in 2022, the number of publications has increased by more than ten times, demonstrating the significant growth of the field. Additionally, it can be understood that while the establishment of object detectors in the field was initially cautious, it quickly expanded into various medical imaging techniques and a diversity of anatomical application domains. Taking into account the results obtained in the quantitative approach, several insights can be obtained concerning the application of object detection algorithms in medical imaging, namely. • This is a promising field of future research, weakly explored due to the late arrival of these techniques in the medical field. With the expansion and development of new and optimized frameworks of object detection, it is possible nowadays to obtain models with high performance, and with the demand of real-time detection needed for some specific fields of medicine, such as in endoscopies. • The publications in this area have a bi-discipline nature, being almost equally distributed between Medicine-related and Computer Science fields. This dual can bring some advantages, such as comprehensive solutions and interdisciplinary insights if research is done in cross-domain collaboration. When this collaboration is not applied, there is frequently a lack of consensus regarding the terminology used in both approaches, particularly on how to evaluate the performance of those models in a manner that facilitates comparison across studies and enables the identification of best approaches and procedures for specific cases and data sets. • China, the United States, and Japan stand as the most productive countries in this area, with the United States having the highest level of international cooperation among these countries. • When comparing the authors' keywords analysis with the index keyword analysis, the former has a prevalence of keywords related to the algorithms themselves, such as deep learning, object detection, and medical field. In the latter analysis, medical concepts were found to be of higher relevance, suggesting the growing interest in applying advanced computational techniques to solve medical challenges, with a high number of those articles published on journals medicine- oriented. Of the 311 papers that were analyzed, 80 of them underwent a rigorous qualitative analysis from three distinct perspectives: the algorithms utilized, the imaging modalities employed, and the anatomical areas of application. The analysis results revealed that most of the publications employed Faster R-CNN and YOLO (regardless of their specific variant) in their research, indicating that both one-stage and two-stage object detectors play a significant role, depending on the trade-off between performance and training speed. For instance, in applications where real-time predictions are a demand, such as endoscopic exams, the authors prefer one-stage algorithms because they can make faster inferences on new images. It is clear, however, that there is no consensus on the best algorithm to use since its selection depends on several factors, such as the quantity of data available, the high need for precision versus the increased demand for inference speed, and even the particular characteristics of the objects of interest in the problem at hand. With regards to medical imaging modalities, object detection algorithms have been applied to a wide variety of imaging techniques, including CR scans, pathology images, and endoscopic imaging, which are among the most commonly used, and this diversity is also reflected in the broad scope of anatomical areas to which these automatic systems have been applied. Nevertheless, object detection is also applied to CT scans, photography, MRI, ultrasound, fundus photo, and angiography imaging. Regarding the anatomical applications of object detection algorithms, digital pathology and microscopy imaging of tissues and blood smears are the most commonly explored. However, other areas are also frequently studied in this field, such as abdominal organs focusing on colon polyp detection, digestive system organs emphasizing tooth-related tasks, bone imaging for fracture identification, and breast CT scans for breast lesion detection. In the studies evaluated, we also examined the proportion of samples used for each partition. As good practice, datasets should be divided into three non-overlapping partitions: train (to train the model), validation (to tune the hyperparameters of the model) and testing (to evaluate the model's performance on unseen data and assess generalization) . Several studies follow this best practice, often splitting the dataset into two or three partitions or using external datasets (an approach that is also effective in preventing overfitting and ensuring that the test set remains independent). Although this important detail is considered in several studies, it is not consistently applied, and the proportions of each partition vary significantly, indicating a lack of standardization. Based on these findings, it becomes evident that the research field could significantly benefit from increased public availability of data or making data accessible upon request. Such an initiative would consequently lead to improved performance in the models utilized within these studies. Furthermore, an evident gap exists in the lack of prospective studies in the conducted research. Encouraging prospective studies is crucial for comprehending the long-term effectiveness of the developed approaches and for collecting data with the specific purpose of implementing these techniques. This approach allows researchers to exercise control over the data collection methods, ensuring data quality, reliability, and minimizing bias. Moreover, the collected data can be customized to address specific research questions in development. Such an emphasis on customization can foster further improvements and innovations in object detection techniques applied to medical imaging. It is also important to note that some studies highlight the high capability of these automatic systems in detecting the objects of interest, sometimes with equal or higher performance than human specialists. • The choice of an object detection algorithm is, in most cases, independent of the task itself and the screening method. As an example, Faster R-CNN is applied to the detection of gastric lesions , abnormality detection on the liver , detection of distal radius fractures , stroke lesion detection , pulmonary tuberculosis detection , among others. Exceptions are made when those tasks demand predictions at a faster rate or in a real-time manner, such as in endoscopies, where one-stage algorithms are preferred, such as YOLO , SSD and RefineNet , in colon polyp detection. • There is no consensus on the best algorithm to use, as it depends on various factors, including available data, precision needs, and inference speed requirements. • Due to the bi-discipline nature of the publications mentioned above, the lack of agreement on the metrics used leads to a difficult task, if not impossible, to compare different approaches even when the same data set is used. This gap reveals the need in the research field to define ways of evaluating object detection results in an agreed manner, independently of the academic nature of the authors. Taking as an example the chest organ studies approached in this work, where pulmonary conditions such as COVID and tuberculosis are detected, some authors use precision and recall to evaluate their results on the object detection framework , while others focused on accuracy , and others on mAP , only in four distinct articles. In other tasks, other metrics are used, such as specificity , AUC ,F-measure , among others. • YOLO framework has gained importance during time, with prior versions of YOLO being associated with lower performance; however, latter versions achieve similar if not superior performance to two-stage algorithms, indicating that one-stage approaches tend to be preferred in the future. In 2018, only one study applied YOLO , and four studies implement Faster R-CNN . In 2022, YOLO studies were in the number of 6 , while the Faster R-CNN algorithm was only applied in two studies . This continuous advancement in algorithms reflects the evolving nature of the field and, consequently, the necessity of ongoing research and ad-hoc optimization for specific tasks. • Independently of the screening methods, object detection procedures can show satisfactory results, if not better equal or even higher than human specialists, as defended in Refs. , showing the versatility and potential impact of these systems in several medical fields. Based on two widely used academic databases, the quantitative and qualitative analysis performed in this study has employed the PRISMA methodology to screen for relevant articles. However, due to the bi-disciplinary nature of the field of study, combining both medicine and computer science, it is apparent that each discipline utilizes distinct concepts and terminologies to address similar issues. One of the primary challenges in this study was to evaluate the performance of various models across different tasks. There is a lack of consensus among researchers on which metrics should be used to make such comparisons, with some advocating for the use of commonly accepted metrics such as Average Precision (AP) and mean Average Precision (mAP) , while others prefer to utilize alternative metrics such as sensitivity, specificity, and accuracy. The search strategy used in this study was focused on the title, abstract, and keywords of the publications. However, it became evident that many medical-oriented studies did not include AI concepts as a primary topic, while computer science-oriented works did not focus on medical concepts. To capture a more comprehensive set of relevant publications, future research should consider adjusting the search queries to account for the different terminology used in both disciplines. Furthermore, a more extensive comparison between the developed work should be addressed, including important details such as the data sets used, the metrics used to evaluate the models and their corresponding performance, and the remarks of the most relevant publications. Additionally, the search was limited to articles and focused on English-language publications, which may have resulted in a biased selection. This filtering may have missed relevant studies conducted in different formats and languages. Furthermore, the quantitative analysis can be further explored, including topics such as subgroup analysis for different countries. Considering these limitations, future research should aim to broaden the search scope, exploring a wider range of relevant studies in various formats and languages. Concerning the models and the data exposed in the publications under analysis, we acknowledge the significance of addressing safety and privacy concerns. While assuming that the journals publishing the analyzed studies adhere to strict procedures that take into account those concerns, our study did not comprehensively explore the potential safety and privacy implications associated with each individual study. This concern is of utmost importance and should be highlighted, taking into account the possible implications that these models can lead when applied in real-world situations. The guidelines and future work that this particular study can induce should always be accompanied by a thorough understanding of the safety and privacy characteristics of the baseline publications. The tools and techniques developed and investigated in these studies should be viewed as supplementary procedures able to reinforce the medical expertise rather than substitutes for it. | Other | biomedical | en | 0.999997 |
PMC11699423 | Medicinal plants have been an essential component of conventional health care around the world for ages since Ayurveda . According to these natural resources provide a wide range of chemicals that are bioactive with a variety of therapeutic potential. Tinospora cordifolia , often called Amrita or Guduchi, holds a great significance in Ayurveda owing to the presence of various secondary metabolites . Giloy leaves have historically been utilized to cure a variety of ailments, such as diabetes, rheumatism, jaundice, and skin diseases . Because of its historical use and the increasing popularity of natural therapies, scientists are looking into the possible therapeutic uses of giloy leaves and the underlying mechanisms behind their medicinal action . This review aims to reduce this gap by thoroughly analyzing immunomodulatory potential of giloy leaves. The research findings about its capacity to regulate the immune system, with particular attention to important pathways such as NF-κB signalling, inflammatory regulation, induction of apoptosis, phagocytosis stimulation, and interleukin modulation. Giloy leaves have been used in the creation of functional food products by comprehending these processes. Through the integration of contemporary scientific findings with traditional wisdom, our goal is to provide insight into the immunomodulatory mechanisms of Giloy leaves and their potential to enhance overall health and well-being. Then, by utilizing this knowledge, innovative giloy leaves-based functional food products can be made, bridging the gap between conventional wisdom and current medical developments. A systematic search of significant electronic databases such as PubMed, Scopus, and Google Scholar has been done to thoroughly examine the immunomodulatory activities of Giloy ( Tinospora cordifolia ) leaves. The search strategy included various terms that addressed multiple aspects of immune system regulation and prospective uses. Terms such as “ Tinospora cordifolia ” or “Giloy leaves” were used here, along with terms on general immunomodulatory effects, cellular mechanisms of the immune response, and product development, such as “cytokines,” “macrophages,” and “natural killer cells,” as well as terms related to immune system function and anti-inflammatory properties. This comprehensive strategy intended to collect all relevant data on how Tinospora cordifolia affects the immune system and to investigate the potential use of giloy leaves in the development of future functional food products. The review process involves a two-tiered selection procedure with a primary focus on research that clarifies the mechanisms of action by which giloy ( Tinospora cordifolia ) leaves affect the immune system. The featured research examines how Tinospora cordifolia affects immune cells or function in animal models or in vitro (lab-based), is published in scholarly journals that undergo peer review, and provides clear evidence of Tinospora cordifolia's action mechanisms on immunological function, including the involvement of specific bioactive chemicals extracted from its leaves. Tinospora cordifolia is a member of the Menispermaceae family, which is part of the Ranunculales order in the Magnoliopsida class (commonly known as dicots) of the plant world. This classification includes it amid a wide range of blooming plants, while the Menispermaceae family is best recognized for its climbing vines and bushes. Tinospora cordifolia , often known as Guduchi or Gurjo, is an ancient medicinal plant from the Menispermaceae family of moonseeds . The Menispermaceae family is abundant in tropical lowland environments, with 70 genera and 450 species. Tinospora is one of the most common genera in the Menispermaceae family, with around 15 different species . Tinospora cordifolia is extensively widespread in Tropical India and may reach elevations of 1000 feet in South Asia, Indonesia, Philippines, Thailand, Myanmar, China, and Sri Lanka . Ayurvedic and folk medicine systems frequently employ this compound, which may be found in a wide range of soil types (from acidic to alkaline) and only needs a small amount of soil moisture . Giloy leaves are a potent source of nutrients and other essential elements. It was found that dehydrated giloy leaves contained high levels of calcium, protein, iron, crude fiber and ash as depicted in Table 1 . All parts of Tinospora cordifolia , including leaves, stems, fruits, and roots, are used as functional foods. Tinospora cordifolia leaves are rich sources of nutrients, essential macronutrients, and micronutrients as shown in Table 1 . Table 1 Nutrient content of Tinospora cordifolia leaves per 100g . Table 1 Nutrients Fresh Dehydrated References Moisture % 31.36 9.64 [ , , ] Ash % 2.3 5.880 Carbohydrates (g) 3.34 7.53 Protein (g) 2.30 5.23 Fat (g) 0.36 1.05 Fibre (g) 11.321 52.295 Iron (g) 5.87 22.55 Calcium (g) 85.247 210 Vitamin C (mg) 56 16 Beta Carotene (μg) 303.7 428.5 Energy (Kcal) 88.64 240 The significant immunomodulatory qualities of Tinospora cordifolia leaves are attributed to a plethora of bioactive substances . These active ingredients include alkaloids (found primarily in the stem) and steroids (beta-sitosterol, d-sitosterol, and g-sitosterol), as well as glycosides (18-norclerodane glycosides, furanoid diterpene glycosides, and tinocordiside) and palmatine D, choline D, tinosporine, magnoflorine, tetrahydropalmatine, and isocolumbin) . Additional components found in its aerial sections include diterpenoid lactones (furanolactone, tinosporon, and columbin) as well as many other substances such cordifolioside A, 11-hydroxymuskatone, N-methyl-2-pyrrolidone, octacosanol, heptacosanol, and nonacosan-15-one . Interestingly, the alkaloid palmatine is also present in the root. RR1, a polysaccharide extracted from the leaves, has been found to possess immune stimulatory properties and has the potential to be used as an adjuvant to boost immunity . Additionally, the various bioactive ingredients present in giloy leaves affect the immune system by increasing cytokine synthesis, enhancing immune cell mitogenicity, and activating specialized immune cells like B cells and macrophages, which can trigger targeted immune responses , . Further research into the exact mechanisms of these active ingredients will help to clarify the immunomodulatory potential of giloy leaves. The various mechanisms through which bioactive compounds of leaves help in boosting the immunomodulatory properties of the body have been illustrated in Fig. 2 . Fig. .2 Immunomodulatory effects of giloy leaves. Fig. .2 Giloy leaf extract has a high concentration of bioactive components such as alkaloids, glycosides, and terpenoids, contributing to its well-known immunomodulatory activities. The leaf extract constitutes bioactive components that stimulate a variety of immune cells, including natural killer (NK) cells, B cells, and T cells, resulting in the generation of favourable immuno-stimulating cytokines [ , , ]. In addition, among HIV-positive patients, giloy leaves lower total leucocyte, neutrophil, and eosinophil counts . These immuno-stimulating effects are linked to several bioactive chemical compounds found in the leaf, including 11-hydroxymuskatone, N-methyl-2-pyrrolidone, cordifolioside A, magnoflorine, tinocordioside, and Synringin . These compounds specifically target immune cells present inside the human body such as B lymphocytes and macrophages, implying a regulated activation of the immune response . This increases haemoglobin levels and polymorphonuclear leukocytes, indicating an overall increase in immune response . These compounds are well known for their ability to promote a greater immune response by enhancing the proliferation and differentiation of lymphocytes. Thus, it increases total WBC, bone marrow cellularity and alpha esterase-positive cells in the marrow of bone which leads to an increase in humoral immune response. Some of the bioactive components appear to considerably raise IgG antibody levels, which are important components of the immunological response for complimentary inhibition of the pathway . Inflammation is an immune system response that is necessary for healing, but it can be harmful if it is severe or chronic. Herbal medicines such as giloy leaves have long been utilized to treat inflammation, however, worries about potential side effects have been highlighted . It's important to note that while giloy ( Tinospora cordifolia ) has been used in Ayurvedic medicine for a long time, it should be used with caution due to potential negative effects, especially when taken in large amounts. Research indicates that exceeding the recommended dosage can lead to gastrointestinal issues such as nausea, upset stomach, and constipation . Some studies conducted by researchers exhibits potential hepatotoxic effects due to Giloy's influence on liver enzymes and metabolism . While the blood-sugar-lowering properties of giloy can be beneficial for diabetics, excessive use or concurrent use with blood-sugar-lowering medications can increase the risk of hypoglycemia (low blood sugar) . Finally, allergic reactions are a possibility, particularly for individuals sensitive to plants of the Menispermaceae family . These reactions may include skin rashes and swelling. Giloy leaf extract intake has been associated with the activation of phagocytic cells that aid in wound healing, as well as the acceleration of skin regeneration when applied topically to wounds and bruises. Giloy leaves' immunomodulatory properties, which have been explained by their bioactive chemical constituents such as alkaloids, glycosides, terpenoids, and polysaccharides, which contribute to their capacity to modulate immune responses and decrease inflammation [ , , ]. Tinospora cordifolia leaf extract encompasses bioactive chemical compounds that help to reduce inflammation and decrease immunological responses. The leaf extract contains a variety of metabolites, some of which have anti-inflammatory and antioxidant activities . A molecule known as 7,9-Di-tert-butyl-1-oxaspiro (4,5) deca-6,9-diene-2,8dione was discovered as a potential dual inhibitor of COX enzymes, which is critical for treating inflammation. This chemical compound has been demonstrated high binding energies against COX 1 and COX 2, showing its potential as a drug like molecule for inflammatory therapy . In addition, T. cordifolia leaf extract has been studied for in-vitro anti-inflammatory action and shown to decrease protein denaturation, a fundamental process in inflammation. These findings indicate that the chemicals in Tinospora cordifolia leaf extract produce anti-inflammatory actions and modify immunological responses by inhibiting COX and suppressing protein denaturation ,. Compounds in giloy leaves decrease inflammatory mediators, inhibit enzyme activity such as COX and alter immune cell function. Giloy leaves have the potential anti-inflammatory properties through a variety of pathways as shown in Fig. 3 about Inflammation and its action mechanism. More research will be required to determine their clinical benefits in controlling inflammatory diseases. Fig. 3 Lipopolysaccharide-induced inflammation and its attenuation by giloy leaf components through NF-kB pathway. Fig. 3 Giloy leaves anti-inflammatory mechanisms that have been studied. According to studies giloy leaf extract prevented lipopolysaccharide (LPS)-stimulated macrophages from releasing prostaglandin E2 (PGE2) and nitric oxide (NO) . Another study reported that giloy leaf extract reduced the expression of tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), two pro inflammatory cytokines in LPS-stimulated macrophages , . Studies demonstrated by that giloy leaf extract suppressed the activation of the transcription factor-kappa B (NF-κB) signalling pathway, which is crucial for the inflammatory response . Wilkinson showed that giloy leaf extract inhibited the activity of an enzyme produced by the human body cyclooxygenase-2 (COX-2), which plays a role in PGE2 synthesis . These studies suggest that giloy leaves have multiple anti-inflammatory mechanisms, including the inhibition of NO and PGE2 production, the reduction of pro-inflammatory cytokine expression, and the suppression of NF-κB and COX-2 activity. These mechanisms make giloy leaves a promising natural anti-inflammatory agent with potential uses in the treatment of a variety of inflammatory conditions, which has been illustrated in Fig. 3 . Apoptosis is a process which helps in regulating the population of immune cells. For instance, after an immune response, excess immune cells may undergo apoptosis to prevent overactivity of the immune system. Apoptosis is used as a mechanism to eliminate infected cells . When a cell is infected with a virus or bacteria and gets damaged or destroyed beyond repair, it can trigger apoptosis to prevent the spread of the infection , . Some of the bioactive compounds of giloy leaves such as aporphine, magnoflorine, palmatine, tinocordiside, and cordifolioside -A, have demonstrated potential as anti-cancer agents . They induce apoptosis and inhibit the growth of cancer cells, suggesting their significance in cancer therapy. Cancer cells undergo apoptosis, a process of planned cell death, when exposed to giloy leaf extract. It triggers nuclear condensation, the production of apoptotic bodies, and the activation of the essential apoptotic enzyme caspase-3 . It also causes apoptosis in cancer cells by suppressing anti-apoptotic genes like Bcl-2 and upregulating pro-apoptotic genes like Bax (Bcl-2 associated X protein). Giloy leaf extract effectively inhibits cancer cell development by interfering with the G1 phase of the cell cycle , . Cell division and DNA replication depend on this stage. Giloy leaf extract stops the cell cycle at this point, which stops cancer cells from proliferating and growing. Nitric oxide (NO) generation is enhanced by the stimulation of immune cells, specifically macrophages, by giloy leaf extract. NO causes cancer cells to become cytotoxic, leading to their destruction , . Giloy leaves contain several polyphenolic chemicals, including tannins, magnoflorine, jatrorrhizine, tembetarine, tinosporine, isocolumbin, palmatine, and tetrahydropalmatine. These substances are good candidates for antioxidant therapy because of their demonstrated potent antioxidant properties . The giloy leaves extract's antioxidant capacity employs a range of in vitro tests. The extract exhibited noteworthy efficacy against DPPH, hydroxyl, and nitric oxide radicals, as demonstrated by the obtained results. The existence of energy-producing polyphenolic compounds that fight off free radicals and stop oxidative damage is the cause of this waste activity , . The antioxidant activity of giloy leaf extracts in human lymphocytes against oxidative stress - driven through DNA damage. Studies have indicated that these extracts can mitigate DNA damage resulting from exposure to hydrogen peroxide, which is a strong inducer of oxidative stress . This protection is due to the antioxidant capabilities of giloy leaves polyphenols, which remove free radicals and prevent DNA oxidation . The effect of giloy leaf extract on oxidative stress and antioxidant state in rats with experimental liver injury . Studies indicate that these extracts can enhance Antioxidant enzymes such as glutathione peroxidase, catalase, and superoxide dismutase as well as lower oxidative stress markers like malondialdehyde . According to these results, giloy leaf extract may shield the liver from oxidative stress and liver damage . The majority of the research indicates that the polyphenols present in giloy leaves have significant antioxidant properties and can be used to treat a variety of oxidative disorders associated with stress using antioxidant-based therapy. The immune-modulatory function of Tinospora cordifolia is associated with its bioactive compounds such as terpenes, glycosides, alkaloids, steroids, flavonoids, and polysaccharides . IL-17, Th17 Cells, and the JAK-STAT Pathway interplay is crucial for understanding the potential immunomodulatory activity of Giloy leaf extract ( Tinospora Cordiofolia ). Proinflammatory cytokines like interleukin-17 (IL-17) are released by Th17 cells among other immune cells . It is essential for protecting the body from infections, especially those caused by fungus and extracellular bacteria. Studies have shown that Autoimmune diseases and chronic inflammatory illnesses have been associated with elevated or dysregulated IL-17 generation . T helper 17 (Th17) cells are a subset of CD4 + T lymphocytes that primarily secrete IL-17. Although they may contribute to autoimmune disorders, they are necessary for immune-mediated defence from external infections . The differentiation and activation of diverse immune cells, including Th17 cells, are facilitated by the JAK-STAT pathway, a crucial signalling cascade . It involves the phosphorylation of Janus kinases (JAKs) by cytokines attaching to particular cell surface receptors. Following JAK activation, STAT proteins (Signal Transducers and Activators of Transcription) relocate to the nucleus and regulate the expression of genes required for Th17 development and synthesis of IL17 . Studies have shown that Tinospora cordifolia extract can reduce the amount of IL-17 generating cells in CD4 + T cells developed under Th17-polarizing circumstances suggesting an immunomodulatory effect . Another study discovered that the water-soluble extract of Tinospora cordifolia improves macrophage phagocytic capabilities and significantly increases nitric oxide generation by stimulating splenocytes and macrophages at a dose of 1 mg/kg Research found that 100 μg/ml of Tinospora cordifolia extract caused 90 % cytotoxicity in B16F10 murine melanoma cells after 72 h of treatment . Another research identified a polysaccharide from Tinospora cordifolia, RR1, which has been demonstrated to have innate immune stimulatory capabilities and can offer adjuvant-like action in the formation of a Th1-type immune response to an antigen , . To put it in simple terms, Tinospora cordifolia activates the immune system by influencing cytokine synthesis, mitogenicity, and immune-effector cell activation, as well as enhancing phagocytic power and nitric oxide generation in macrophages . The specific mechanism of interleukin modulation by which Tinospora cordifolia extract produces these benefits is unknown and will require more investigation and study. Research on the uses of giloy ( Tinospora cordifolia) leaf extract in various value-added products has revealed its significant immune-boosting properties and health benefits. Giloy extracts have been incorporated in different forms such as powder, fresh leaves, and stem juice to enhance the nutritional value and taste of the products which has been shown in detail in Table 2 , Table 3 . For instance, a combination of giloy leaf powder, besan, wheat flour, and adusa leaves was utilized to produce biscuits and sev, resulting in nutrient-rich products confirmed through tests like DPPH and Folin-Ciocalteu . The phytochemicals present in giloy have been found to have a positive impact on the immune system, leading to increased consumption of homemade kadha (a herbal concoction) containing fresh giloy leaves and other ingredients during the COVID-19 pandemic . Moreover, products like value-added cookies made with giloy and tulsi powders, herbal squash made with giloy leaf extract and pineapple juice, and herbal lassi made with giloy stem juice have displayed enhanced nutritional properties . Table 2 Immunomodulatory giloy leaves-based value-added products. Table 2 S. No. Objective of study Value added Product Overview of Study Reference 1. Evaluation of Herbal Leaves for the Development of Value-Added Food Product Biscuit and Sev In this study, the giloy leaves were used to make value-added products such as biscuits and sev. The greatest method to add medicinal plants' nutritional advantages to humans' everyday diets to help fight degenerative illnesses is to provide value to food items by including them. It shows that the nutrients in these products can help in the fight against diseases and improve immunity. 2. Consumption of natural products and Ayurvedic decoctions “Kadha” as immunity-boosting measures during the spread of COVID-19 in Delhi Ayurvedic Decoction “Kadha” This study aimed to explore the prevalence of consumption of natural products and Ayurvedic decoctions “kadha” as immunity-boosting measures during the initial phase of the COVID-19 pandemic. Around 540 responses were taken through an online survey which shows the usage of immunity-boosting measures and Ayurvedic decoctions “kadha” among the adult residents of Delhi belonging to different age groups was effective. 3. Development of Giloy leaves-based Herbal Squash incorporated with Pineapple Giloy leaves based Herbal Pineapple Squash This research work was an effort in the direction of producing value-added instant beverages. Due to the functional and nutritional goodness of both plants, it can be further exploited in the development of healthy beverage products to develop giloy leaves-based herbal squash incorporated with pineapple and to determine its physical and biochemical properties. 4. Development of value-added cookies supplemented with giloy leaves and Tulsi powder Cookies with giloy leaves and tulsi powder In this study, giloy leaves powder was utilized in place of whole wheat flour to create herbal biscuits. When preparing herbal cookies, whole wheat flour partially replaces the powdered tulsi leaves and giloy leaves stems. Table 3 Traditional Giloy leaves Based Value-added Products. Table 3 S. No. Objective of study Value-added Product Overview of Study Reference 1. Development of herbal lassi using giloy leaves stem juice Herbal lassi Based on the current study, giloy leaves may be utilized to successfully make herbal lassi. Based on microbiological characteristics, the produced lassi was deemed safe for consumption. Natural antioxidants, such as giloy leaves, effectively lower the risk of heart disease, cancer, and many inflammatory processes, and have a positive impact on cardiovascular illnesses. It also strengthens immunity. 2. Development of Antioxidant-Rich Herbal Tea Bags Herbal Tea Bags with Giloy leaves This study was conducted to develop herbal tea bags using giloy leaves and different herbs which can provide antioxidant properties, boost immunity and also enhance the efficacy of white blood cells which helps in fighting against infections and bacteria-causing diseases. 3. Value Addition and Fortification in NonCentrifugal Sugar (Jaggery): A Potential Source of Functional and Nutraceutical Foods Jaggery fortified with giloy leaves. Jaggery is classified as a nutraceutical since it contains a range of vital amino acids, antioxidants, phenolics, minerals (calcium, phosphorus, iron), and vitamins. Jaggery is a more natural source of nutrients for health benefits and might be utilized as a healthier nutritional option for white sugar. Giloy leaves are incorporated as a health-supporting herb to generate even superior antioxidant, detoxifier, digestive, and immune booster products with potential functional and nutraceutical value. 4. Formulation of Herbal Candies Containing Giloy Leaves Satva: A Nutritious and Palatable Herbal Confectionery Option Herbal Candies Containing Giloy leaves The addition of giloy leaves satva in confections shows potential for improving immunity as it fights free radicals and boosts the body's mechanisms for defence against infections. It offers an intriguing discipline of study and innovation, providing a natural and pleasant method for immunomodulation 5. Sensory Evaluation of Laddu Enriched with Giloy leaves ( Tinospora Cordifolia) - Iron-Rich Powder Laddu Enriched with Giloy leaves The purpose of this study is to formulate and establish a standard giloy leaves laddu for people with anaemia and to determine its level of acceptability. The goal is to make a healthy laddu and put giloy leaves powder into it. 6. A study on incorporation of giloy leaves for the development of shelf-stable goat milk-based functional beverage Goat milk incorporated with giloy leaves By combining debittered giloy leaf juice with goat milk, a study was carried out to develop a shelf-stable giloy leaves goat milk beverage. The product profile was examined in depth, including its proximate composition, bioactive qualities, sensory, rheological, and structural characteristics. The addition of giloy leaves in this creates a functional beverage with a longer shelf life that targets health issues and boosts immunity. Different Varieties of modern value-added products are available nowadays, while traditional value-added products are very limited as shown in Table 3 , Table 4 . Table 3 , Table 4 illustrate the contrast between traditional and current applications of giloy leaves. Table 4 displays the Giloy-based products available in the market, while Table 3 outlines traditional uses. There are various value-added products in today’s modern era like capsules, juice, giloy leaves powder, giloy leaves extract and many more while traditional value-added products were limited to most consumers where churan, and kadha (juice blend) are common among all. Modern products are fortified with different vitamins and minerals to enhance the nutritional value of the product while traditional products are not fortified and produced as it is consumed. Modern giloy leaves products are widely available in pharmacies, local stores and online platforms, and are greatly accessible to consumers worldwide, which is not the same in the case of traditional value-added products. Modern giloy leaves products offer a convenient form of consumption, making it easier for individuals to consume and incorporate into their daily routines plus they are less time-consuming. Modern giloy leaves products have a high shelf life as they are preservatives with chemicals like sodium benzoate and potassium sorbate etc. or with various modern preserving methods. Traditional giloy leaves products typically use natural and herbal ingredients plus no chemicals were used resulting in less shelf life of the product. Traditional value-added products are made by methods that are passed down from generation to generation. Which involves manual labour, the use of natural ingredients and traditional equipment, which helps in preparing the perfect desired product. In Modern value-added products, advanced technologies and machines are used for production which works on standardized methods. Automatic machines, standard measurements, and quality control techniques are very common for preparing required products. Table 4 Commercially giloy leaves based on value-added products. Table 4 Product Name Brand Name Health Claims Ingredients Reference Capsules/Tablets rowhead 1. Guduchi Immunity Wellness Himalaya ● Strengthens Immunity ● Fights infections ● Supports detoxification ● Helps in increasing the performance of white blood cells Giloy leaves Stem Extract https://www. amazon. in/HimalayaWellness-Herbs-Guduchi-Immunity/dp/B00B8ROM1M 2. Giloy leaves Immunity Booster Zandu ● Protection Against Infections & Immunity Booster. ● Promotes Liver Health ● Stress Relief ● Anti-oxidant Compounds for Good Skin Health. Giloy leaves Stem Extract https://www. amazon. in/ZanduGuduchi-herbs-immunitywellness/dp/B07V6TPRQ8 3. Guduchi (Giloy leaves) Ghanbati Baidyanath ● Boost Immunity ● Reduces anxiety and improves mental strength. ● Rich in antioxidants and has anti-allergic, antifungal & anti-bacterial benefits. ● Maintain strength and vitality ● Aids pitta disorders & improves digestion. Giloy leaves Stem Extract. Ashwagandha Amla Gorakhmundi https://www. amazon. in/Baidyanath-Guduchi-Giloy leaves-GhanBati/dp/B08CY425YB 4. Divya Giloy leaves Ghanvati Patanjali ● Immune boosting ● Antioxidant properties and anti-inflammatory effects. ● Provide liver support ● Promote respiratory health. Giloy leaves Stem Extract https://www. amazon. com/Patanjali-Giloy leaves-Ghan-Vati-Tablets/dp/B07HWTJ2G3 5. Jiva Giloy leaves Jiva ● Boosts Immunity. ● Helps fight respiratory problems. ● Helps reduce joint pain. ● Improve digestion. ● Helps in detoxification. Giloy leaves Stem Extract https://www. amazon. in/Jiva-Giloy leaves-Capsule-Respiatory-Detoxification/dp/B093KTSXX8 Powder rowhead 1. Organic Giloy leaves/Guduchi Stem Powder CARMEL ORGANICS ● Boosting the immune system. ● Improving digestion. ● Reducing stress and anxiety. ● Controls blood sugar level. Giloy leaves stem powder https://www. amazon. in/CertifiedAmruthavalli-Tinospora-cordifolia-Preservative/dp/B08CHGD429 2. Natural Organic Guduchi Powder/Giloy leaves Powder MY HERB ●Aids in detoxification, increases antibodies, and stimulates vitality. ●Relieves stress and anxiety while also replenishing the body. ●Combats illnesses related to respiration. Demonstrates anti-inflammatory and antiarthritic activities Giloy leaves stem powder https://www. amazon. in/GuduchiPowder-Tinospora-cordifoliaMetabolism/dp/B08MTZPL1C 3. Giloy leaves Satva Baidyanath ●Useful in treating burning sensation in hands & feet, headache, metallic taste in mouth & excessive thirst. ●Enhances immunity of the body. Giloy leaves extract powder https://www. amazon. in/Baidyanath-Giloy leaves-Satwa-Pack-140/dp/B08KT8VB8P Teas Moringa & Giloy leaves Green Tea with Lemongrass Care ●Support to build an Immune system. ●Make bones and joints healthy. ●Help in losing weight. ●Enhance metabolism. Moringa Giloy leaves Licorice Ginger Lemongrass https://www. amazon. in/Moringa-Lemongrass-Immunity-Management-Ayurvedic/dp/B0836MJH7G 2. Giloy leaves Immunity Tea Jiva ●Effective immunity booster. ●Improve skin condition and reduce acne and pimples. ●Helps with seasonal colds and coughs. ●Helps to improve overall health & well-being. Giloy leaves stem powder https://www. amazon. in/Jiva-Giloy leaves-150gm-Pack-Immune-Tea/dp/B085S4ZFZQ 3. Giloy leaves Tea One Herb ●Immunity booster. ●Improves digestion. ●Helps reduce skin infections. ●Relieves chronic fever. ●Help regulate blood sugar levels. ●Boosts metabolism and aids weight management. Giloy leaves stem powder https://www. amazon. in/One-Herb-Ultimate-Immunity-Infections/dp/B08VNLYKTW 4. Ayurvedic Herbs Giloy leaves Green Herbal Tea Rishtpusht ●Immunity booster. ●Helps maintain a healthy heart and nervous system. ●Fight allergies and common infections. ●It improves blood circulation and eliminates harmful substances from the body. Giloy leaves Satva, Dry ginger, Clove, Cardamom, Lemon Basil, Green tea, Cumin seed, Sugar. https://www.amazon.in/Rishtpusht-Ayurvedic-Immunity-Booster-Natural/dp/B091YTDSK8 Juice rowhead 1. Giloy leaves juice Dabur ●Natural immunity booster ●Antioxidant properties ●Good for liver and skin health ●Natural detoxifier Giloy leaves extract https://www. amazon. in/DABUR-Giloy leaves-Neem-JuiceTulsi/dp/B087DJ9L3K 2. Giloy leaves tulsi juice. Vansaar ●Cleans and detox the gut ●Boost immunity & fight against common cough cold ●Antioxidant ●Anti-microbial ●Anti-bacterial ●Anti-inflammatory Giloy leaves stem Tulsi leaves https://www. amazon. in/Enhancing benefits-Handpicked-all-round-Immunity/dp/B07CYYPFFR 3. Giloy leaves juice Baidyanath ●Boost immunity ●Rich source of antioxidant ●Helps maintain a healthy heart and nervous system Giloy leaves extract https://www. amazon. in/Baidyanath-Boost-Immunity-Natural-Giloy leaves/dp/B087LTSC5M 4. Wild tulsi and giloy leaves juice Krishna’s herbal & ayurveda ●Helps regulate blood sugar levels. ●Boost metabolism & improve digestion ●Stimulates insulin secretion ●Helps manage weight Giloy leaves, Jamun, Bel Patra, Amla, Methi, Karela, Kutki, Vijaysar, Tulsi, Gudmaar Neem https://www. amazon. in/Krishnas-Herbal-Ayurveda-GeloyTulsi/dp/B07PLYBMMB 5. Giloy leaves vital AVG health organics ●Immunity booster ●Anti-pyretic ●Reduces inflammation & pain in the body ●Glucose metabolism Giloy leaves Ashwagandha Basil Amla berry https://www. amazon. in/AVGHealth-Organics-Ashwagandha-Premium/dp/B08CYCPTV6 Tinospora cordifolia , also known as Giloy, is a revered medicinal plant in the Indian Ayurvedic tradition, renowned for its diverse therapeutic applications . However, recent studies have raised concerns about the potential hepatotoxic effects associated with the consumption of giloy leaves powder. The traditional use of giloy in Ayurveda has been well-established, with the plant being recognized for its anti-pyretic, anti-inflammatory, and hepatoprotective properties. However, a growing body of evidence suggests that prolonged or excessive consumption of Giloy leaves powder may lead to liver damage, a phenomenon known as hepatotoxicity. Several in vitro and animal studies have investigated the potential hepatotoxic effects of giloy. Researchers have observed that exposure to giloy extracts can result in increased lipid peroxidation, lactate dehydrogenase release, and a decline in glutathione-S-transferase activity, all of which are indicative of cellular stress and potential liver injury . Alkaloids (berberine, palmatine, and jatrorrhizine) and sinapic acid are believed to contribute to its hepatoprotective effects. Berberine, for instance, has been shown to reduce inflammation by inhibiting TNF-α-mediated proinflammatory pathways and nitrosative stress by suppressing iNOS activity . Beyond its hepatoprotective properties, Tinospora cordifolia exhibits a broad spectrum of biological activities, including anticancer, anti-inflammatory, antimicrobial, and antioxidant effects. It is generally considered safe at doses up to 2000 mg/kg. The pharmacological actions of Tinospora cordifolia are attributed to a diverse array of phytochemicals, including polyphenols, alkaloids, steroids, terpenoids, and glycosides . Hepatotoxicity, or liver damage, can be caused by a variety of factors, including the ingestion of toxic substances, overdose of medications, and even certain herbal preparations . Although information on the immunomodulatory qualities of giloy leaves is growing, there is still a significant lack of understanding about the precise processes behind these effects. The precise molecular pathways and cellular interactions underlying the observed antioxidant activity, inflammation modulation, Th17 cell regulation, inhibition of the NF-κB pathway, interleukin modulation, and immunosuppressive and white blood cell-enhancing properties remain unclear. To understand the full therapeutic potential of giloy leaves, it is imperative to close this gap between data and intricate processes. Therefore, future research on several areas should be done including important signalling pathways like NF-κB, which have previously been connected to giloy leaves effects, should be the focus of research. These processes have to be clearly understood. Targeted therapies need a clearer comprehension of the molecular interactions that components of giloy leaves have with these pathways. Extensive research is required to determine the particular pathways through which giloy leaves constituents interact with other types of immune modulatory cells, such as Th17 cells, macrophages, and natural killer cells. Having a thorough understanding of how giloy leaves affect these cells' activation, differentiation, and function would help develop therapeutic applications. Investigate the possible benefits that giloy leaves may have when combined with other widely consumed foods that are high in vitamins, minerals, or antioxidants. This can help with dietary suggestions for enhancing immune function and for nutraceutical development. Examine the impacts of food matrices and determine which food ingredients can improve the bioavailability of the bioactive chemicals found in giloy leaves. This information can help formulate products in a way that maximizes health benefits. Contrast the immunomodulatory properties of giloy leaves with known immunomodulatory medications or natural compounds. This analysis can shed light on the distinct processes of giloy leaves and discover potential synergies in combination therapy. Through establishing a connection between current data and comprehensive research, scientists can fully realize the potential of giloy leaves as a medicinal agent. This all-encompassing strategy will open the door for focused therapies and enhanced formulations for particular immune-related ailments. In Future researchers should prioritize the development of standardized protocols for giloy leaves to ensure consistent quality and efficacy. Further research is necessary for negotiating the regulatory environment for giloy-based applications in order to commercialize the product. In conclusion, the study we did on the immunomodulatory properties of giloy leaves states that they have many health benefits such as boosting immunity, regulating apoptosis in immune cells and acting as antioxidants. They have active compounds such as alkaloids, polysaccharides, flavonoids, and terpenes present in them that help in immune cell activation which stops cell damage and immune cells like macrophages and B cells which help in inflammation reduction and stop cancer cells from increasing. Giloy leaves modulate interleukins and Th17 cells which helps in protection against infections and increasing immunity. | Other | biomedical | en | 0.999995 |
PMC11699432 | Malaria is a parasitic disease transmitted by Anopheles mosquitoes. In 2022, malaria caused 249 million cases and 608,000 deaths, with a high burden in Africa particularly for children under 5 years and pregnant women . The World Health Organization has put in place a global technical strategy targeting the reduction of malaria incidences and mortality rates by 90 % by 2030 with vector control as a cornerstone approach to reaching said target . Common malaria vector control strategies include the use of long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) . However, malaria-transmitting mosquitoes, have developed resistance to public health insecticides , and selective pressure has shifted their biting behaviors to spaces, times, and alternative blood sources not protected by LLINs or IRS including feeding outdoors, early in the evening, and upon peri-domestic cattle . Ivermectin is an antiparasitic drug used in livestock and humans that works by blocking signal transmission in nervous systems, opening chloride channels causing hyperpolarization that results in flaccid paralysis and death in insects . In mammals, the drug has an excellent safety profile . Since ivermectin is effective against mosquitoes (when ingested via blood meal), but safe for mammals, it is under evaluation for mass drug administration (MDA) in humans and livestock as a potential complementary strategy to reduce malaria transmission [ , , , ]. About 80 to 90 % of administered ivermectin and its metabolites in cattle are excreted in feces . Dung fauna such as flies, dung beetles, termites, and others feed on excrement and help decompose pats from livestock and return nutrients to soils . Previous studies in temperate settings have demonstrated that ivermectin and its metabolites affect dung fauna, causing delayed dung degradation , which could cause decreased pasture productivity and economic losses . In contrast to temperate settings, the environmental effects of ivermectin and its metabolites in tropical settings are understudied . It is unclear if differences in conditions such as higher sun light exposure and different climate pattern at lower latitudes could mitigate the negative impact of ivermectin on dung fauna. If ivermectin MDA in cattle is determined to be effective for malaria control, it will be mostly applied in the tropical countries, making it important to understand how ivermectin treatments in cattle will affect dung fauna and degradation in tropical settings where malaria is prevalent. Here we studied the impact of ivermectin on dung fauna and degradation in a tropical dry savanna setting in southeastern Tanzania. Specifically, we conducted qualitative observations of dung fauna colonization, and quantified termite colonization, wet weight, water content, dry weight, organic matter, and larval abundance in dung. These data will support the assessment of the environmental impacts of the drug when used for MDA in cattle for malaria vector control. We conducted the study in Sagamaganga village (8°3′50.352″ S, 36°47′46.254″ E), located in Kilombero District within Kilombero Valley, Morogoro Region, southeastern Tanzania. This valley has a wet season from February to June and a dry season from July to January with average annual temperatures between 20 °C and 32 °C . The inhabitants in this valley are involved in human land use activities including livestock husbandry (e.g. cattle). However, the fieldwork was conducted during the dry season from July 2022 to October 2022 but monitored until March 2023. We selected 22 Tanzanian short horn zebu cattle from a herd that fed and grazed in the same area. These were allocated 1:1 to the control and ivermectin treatment groups using a random computer-generated sequence in R. Cattle ranged in age from 1 to 4 years, weighed between 90 and 351 kg, and had not been exposed to antiparasitic drugs in the preceding three months. A veterinarian checked the cattle before the study, qualifying them as physiologically mature. Similar studies have used cattle of different age ranges . We had 11 cattle (8 males and 3 females) in the control group and 11 ivermectin treated cattle (7 males and 4 females). The control group had weight of (186 ± 93.5) kg and age (2.0 ± 1.1) years while the ivermectin group weighed (148 ± 45.7) kg and aged (1.5 ± 0.5) years. We employed a randomized controlled experimental design to evaluate ivermectin's effect on dung degradation and fauna. On days 15, 30 and 45 in the field, we first measured wet weight of each entire pat, and then randomly scooped 10 g from its surface for further analysis of organic matter content. These 10 g sub-samples were sent to the Ifakara Health Institute laboratory, where they were refrigerated for 5 to 7 days at 4 °C until further analysis. 110 treatment and 110 control samples were collected on those specific days. Termite infestation observations were also done on day 15 and 30 for each pat. For processing, 10 g sub-samples were homogenized by grinding them with a mortar and pestle. We put the 10 g sub-samples in a crucible which we previously weighed to the nearest 0.01 g. We then dried the sub-samples in the oven for 6 h at 110 °C to remove all moisture content . After drying, the sub-samples in the crucibles were cooled in a desiccator for 30 min before weighing to prevent moisture absorption. We calculated the percentage dry matter as the remaining weight of a sub-sample after drying . We determined organic matter in the dung pat using the loss on ignition method also known as ashing which helps separate mineral content (ash) from organic constituents that are susceptible to decomposition by: heating the oven dried samples from the previous step (sub samples heated at 110 °C that were cooled and weighed) in a pre-heated furnace at 500 °C overnight (12h) and then cooled them for 30 min before weighing. We then calculated organic matter content by determining percentages from the differences in weight of subsamples at 500 °C and at 110 °C . We used standard methods to assess dung insect larval abundance [ , ]. In this case fresh dung for this experiment was collected on the same dates from the control and treated animals and counts performed on larvae. For the larval study, on day 3, 10 and 29 after ivermectin administration, we haphazardly collected dung from ivermectin and untreated cattle as they were deposited in the field. We then mixed the individual pats thoroughly. From the collected pats, we made 1 kg standardized treated and control composite pats ( N = 10 each) and placed them on a wire mesh in the field to aid recovery. Following a 1 day field exposure period, we collected pats and placed them in an emergence cage to recover insects . We recorded visitations of arthropods to the dung and documented beetle larval counts for the larval study and termite presence in the two treatments for the degradation study. Termites were only observed and recorded in the pats that were used in the dung degradation study. We used R version 4.2.1 for all statistical analyses. To account for the longitudinal nature of our data with repeated measures of individual pats, we used mixed models to study the effect of ivermectin treatment on wet weight, water content, dry weight, organic matter, termite infestation and larval abundance. In these models with the three degradation outcomes as response variables, we added random intercepts for individual dung pats, and we added treatment × time interactions as fixed effects . Similarly, we used Poisson mixed models to study effects the ivermectin treatment on larval abundance. Here, we used larval counts as our response variable and accounted for the source of the data (sets 1 and 2, see above) with random intercepts and treatment, scaled days and scaled days squared as fixed effects. The latter was done to account for the initial increase and then decrease of new larvae emerging. All mixed models were implemented using the lme4 package . We used the emmeans package to extract estimated marginal means and compute standard errors and confidence intervals . Ivermectin treated pats were significantly more likely to be infested by termites on day 15 (odds ratio: 23.2 (3.9 to 136.2); df = ∞; z = −3.48; p < 0.001), but not on day 30 (odds ratio: 4.7 (0.9 to 23.3); df = ∞; z = −1.89; p = 0.059; see Fig. 1 ). The point estimate for the probability of termite infestation in ivermectin pats decreased from 15 to 30 days ( p = 0.484), while the reverse occurred in control pats, though neither was significant ( p = 0.142). Fig. 1 Probability of termite infestation and 95 % confidence intervals after 15 and 30 days. Termites significantly preferred ivermectin treated pats compared to control pats on day 15, but not on day 30. ( n = 220). Fig. 1 Both treatment and control wet weights decreased from Day 0 to Day 30. By Day 15, there was a significant effect of treatment, as control pats were 13.4 % lighter than treatment pats (control pats = 291.9 g (275.7 to 308.1) versus treatment pats = 337.2 g (321.0 to 353.3), mean difference: −45.3 g (68.2 to −22.4); df = 435.1; t = −3.89; p < 0.001, Fig. 2 ). The difference was 12.4 % by Day 30 (control pats = 277.8 g (261.6 to 294.1) versus treatment pats = 317.0 g (300.7 to 333.2), mean difference: −39.2 g (−62.1 to −16.2); df = 437.8; t = −3.35; p < 0.001). The difference was also maintained to a smaller, non-statistically significant, extent (9.3 %) on Day 45 (control pats = 238.5 g (222.2 to 254.7) versus treatment pats = 262.9 g (246.7 to 279.2), mean difference: −24.5 g (−47.4 to −1.5); df = 437.8; t = −2.09; p = 0.037); see Fig. 2 ). Fig. 2 Mean wet weight and 95 % confidence intervals of whole pats over time. Overall, ivermectin treated pats displayed significantly slower wet weight decrease in comparison to control, with the largest effect occurring from day 0 to 15. ( n = 220). Fig. 2 We tested whether termites affected the wet weight decrease, but we did not find a relationship between termite presence and wet weight decrease on Day 15 (controls: 11.1 g (−7.8 to 30.0) ivermectin: 20.3 g (2.8 to 37.9); mean difference: −9.2 g (−35.0 to 16.6); df = 214; t = −0.7; p = 0.482). Water content decreased over the three time points for both treatment and control dung pats. The treatments did not differ with respect to water content on day 15 (control pats = 16.4 % (14.5 to 18.2) versus treatment pats = 15.7 % (13.9 to 17.6), mean difference: 0.6 % (−2.0 to 3.3); df = 643.9; t = 0.47; p = 0.639, day 30 (control pats = 13.6 % (11.7 to 15.5) versus treatment pats = 12.4 % (10.6 to 14.3), mean difference: 1.2 % (−1.5 to 3.8); df = 644.1; t = 0.88; p = 0.381) nor day 45 (control pats = 6.5 % (4.6 to 8.3) versus treatment pats = 6.0 % (4.1 to 7.9), mean difference: 0.5 % (−2.2 to 3.1); df = 643.9; t = 0.35; p = 0.724); see Fig. 3 A). Fig. 3 Change of percent water, dry weight and organic matter content of subsamples. A Water content (%) and 95 % confidence interval over time. Ivermectin did not affect water content on day 15, 30 and 45 and the ivermectin and control pats lose water content on similar trajectories.( n = 220) B Dry matter content (%) and 95 % confidence intervals over time. Ivermectin did not affect dry weight content on day 15, 30 and 45. These results show that ivermectin and control pats dry weight content increase similarly.(n = 220) C Organic matter content and 95 % confidence intervals over time. Ivermectin did not affect organic matter content, on day 15, 30 and 45. These results show that organic matter in sub-samples decreased similarly in ivermectin and control pats.(n = 220). Fig. 3 Dry weight content of sub-samples increased over the three time points for both treatment and control dung pats. The control and ivermectin treated pats did not differ on day 15 control 83.6 % (81.8 to 85.5); ivermectin: 84.3 % (82.4 to 86.1); mean difference: −0.6 % (−3.3 to 2.0); df = 643.9; t = −0.47; p = 0.639), day 30 control: 86.4 % (84.5 to 88.3); ivermectin: 87.6 % (85.7 to 89.4); mean difference: −1.2 % (−3.8 to 1.5); df = 644.1; t = −0.88; p = 0.381), or day 45 control: 93.5 % (91.7 to 95.4); ivermectin: 94.0 % (92.1 to 95.9); mean difference: −0.5 % (−3.1 to 2.2); df = 643.9; t = −0.35; p = 0.724; see Fig. 3 B). There was no effect of treatment on organic matter content. Ivermectin treated pats and control pats did not differ at day 15 (control: 59.9 % (57.2 to 62.6); ivermectin: 59.8 % (57.1 to 62.5); mean difference: 0.0 % (−3.8 to 3.8); df = 631.8; t = 0.02; p = 0.983), day 30 (control: 46.5 % (43.7 to 49.2); ivermectin: 47.1 % (44.4 to 49.8); mean difference: −0.6 (−4.5 to 3.2); df = 632.8; t = −0.31; p = 0.758), or day 45 (control: 45.6 % (42.9 to 48.3); ivermectin: 44.8 % (42.1 to 47.5); mean difference: 0.8 % (−3.0 to 4.6); df = 632.1; t = 0.41; p = 0.682; see Fig. 3 C). Both treatment and control total organic matter/pat, as interpolated from subsamples to whole pats, decreased from Day 0 to Day 30. There was a significant effect of treatment on total organic matter remaining in the pats, with control pats having approx. 13.2 % lower mass, or an estimated mass at 144.1 g (135.5 to 152.7) versus treatment pats at 166.1 g (157.5 to 174.7). This is a mean absolute difference of −22.0 g (−34.1 to −9.8; df = 563.5; t = −3.55; p < 0.001). By day 30 control pats had approx. 14.7 % less mass, or 106.8 g (98.1 to 115.5) remaining of controls, compared to 125.2 g (116.5 to 133.8) of ivermectin treated pats for a mean absolute difference of −18.4 g (−30.6 to −6.1; df = 568.1; t = −2.93; p = 0.003). On day 45, this was no longer significant (control pats approx. 6.2 % lower mass; control pats: 100.5 g (91.9 to 109.2); ivermectin pats: 107.1 g (98.5 to 115.7); mean difference: −6.6 g (−18.8 to 5.6); df = 564.9; t = −1.06; p = 0.291, Fig. 4 A). Fig. 4 A Mean mass of total organic matter/pat and 95 % confidence interval over time. Ivermectin affects the average mass of total organic matter on day 15, 30 and 45 in the dung pats.( n = 220) B Mean larvae count and 95 % confidence interval over time. There were fewer larvae in ivermectin treated pats in comparison to control. These results are significant and show that ivermectin negatively impacts larvae abundance. ( n = 40). Fig. 4 We found a significant effect of ivermectin treatment on larvae counts, with the difference between treatment and control increasing over time . There was a significant effect of treatment on larvae population from day 1 to day 124, (mean counts on day 124 (95 %): control pats = 82.1 (78.1 to 86.2), treatment pats = 11.9 (11.3 to12.6), corresponding to an increase by 689 % (666 to 712); df = ∞; z = 112.92; p < 0.001). We determined the impact of ivermectin MDA in cattle on dung degradation and dung fauna in a tropical dry savanna setting in Tanzania, focusing on dung degradation in terms of wet weight, dry weight, loss of organic matter and larval abundance. In our study, ivermectin treated pats degraded more slowly in terms of wet weight and the total organic matter/pat . Additionally, we found that more beetle larvae lived in control than ivermectin treated pats. Originally, we aimed to count adults that would have emerged from dung in the two groups. Intense hot weather in the field led to a modification of the study to a semi-field setting for further observation. During the single day in the field, adult insects visited the pats. By the end of the day, the pats had started drying out, particularly on the surface, which had become crusty. Once moved to the semi-field setting, we periodically counted larvae in the pats. Interestingly, we observed only beetle larvae and no fly larvae. The intense weather conditions and the drying dung could likely be unsuitable for fly larvae survival. We continued counting larvae until the pats completely dried out. However, no adult insects emerged during the experiment. Wet weight of whole pats decreased in the treatments by a difference of 14 % in 15 days, 12 % by day 30 and 9 % by day 45. Dry weight of sub-samples increased and water content decreased, presumably because of moisture loss associated with dry weather for our study area . There was no statistically significant treatment difference in the organic matter content among the sub-samples. However, extrapolating the organic matter to whole pats, we found significant differences in the first month of degradation, but not the period after that. This extrapolation has uncertainties as insects moved the pats and some left hollow spaces in the dung pats. In these cases, less dung or soil remained in the samples. Still, a sensitivity analysis restricted to pats without termite infestation upheld the main result. In summary, we find these data to be compelling evidence that ivermectin given to cattle decreases dung degradation in the field of a tropical environment . A number of studies have found similar results in the temperate settings . The similarity is somewhat surprising, as ivermectin degrades quickly in sunlight and metabolites like 3′′- O -demethylivermectin (3DI) go below the threshold for detection on the upper dung layer in a week's time post dung placement . Despite the higher solar irradiation at the lower latitudes, ivermectin and its metabolites must have had an effect on dung before their degradation in our study. We observed termites throughout the dry season, corroborating previous observations that point to their ability to tolerate harsh dry conditions . Termites specifically add soil contents in dung during mound making facilitating microbial activity. Termites also help organic matter break into nutrients taken up by plants . Despite this important role of termites in dung degradation, there was no association between termite infestation and dung degradation, measured in terms of wet weight, in this study. Maybe such an effect would become evident after the 45 days that we allotted to our experiment. Surprisingly, termites preferentially associated with ivermectin-treated pats. This was a novel observation that echoes a similar report on dung beetles [ , , ]. Wardhough and Manon suggested that some volatile metabolites of ivermectin in the dung as well as changes in the gut flora of cattle caused by ivermectin may be involved in the bias to ivermectin-treated dung . However, a recent study found no evidence that dung undergoes direct changes in the volatile compounds releases or if there is a change in gut microbiota in cattle . In our study, given the overall lower rates of colonization by insects, perhaps the termites face less competition in the ivermectin- treated pats. Further research on the phenomenon is needed. We did not observe adult helminths in any dung pats. While less obvious in our study, colonization of pats with other dung organisms may also be crucial. For example, beetles remove dung , and adult beetles consume the liquid portion of dung, but their survival seems to be unaffected by ivermectin with further evidence of past exposures not impacting the future ecosystem at the area of exposure . Although as dung organisms make their movements, they facilitate drying and decomposition . Less larval activity in ivermectin pats is problematic since larvae contribute to dung degradation by direct decomposition of organic matter, mixing, aerating, and breaking dung into smaller particles enabling microbial decomposition . The number of larvae in dung increases with time; however, in the field, their numbers quickly decreased as dung lost its moisture in the tropical dry season tropical setting , explaining their inability to survive . In our study, adult Diptera were only observed on the surface of dung pats for one day, when they were still moist on the top layer. Afterwards, conditions for Diptera development were not favorable . Adult beetles, on the other hand, seemed less affected, but the lower larvae counts in ivermectin treated pats in the semi-field experiment points to a reduction in the ability to produce viable offspring under these conditions [ , , ]. We conducted our study during the dry season and loss of dung diversity due to dry conditions may exacerbate the effects of ivermectin during that time . On the other hand, increased ivermectin photodegradation may have lessened the impact of ivermectin treatments on dung degradation . There is a critical need to conduct similar studies during wet season in the tropics to rule out the dry condition effect on microbial and insect activity and investigate further the ivermectin effect during this period crucial to malaria spread. Furthermore, it is unknown what level of difference in dung degradation rate is environmentally relevant and may lead to downstream effects like pasture fouling due to prolonged presence of dung pats, particularly in the extensive livestock management systems found in tropical climates. Despite these limitations, our study represents an important first step if we want to evaluate the environmental impact of mass drug administration in cattle to curb the spread of malaria in humans. In summary, results from tropical Tanzania add to the previous findings from the temperate region studies which demonstrate that ivermectin negatively affects dung degradation and larval development. Therefore, if ivermectin MDA in cattle proves to be an effective method for malaria control, its non-target environmental consequences should be further quantified and evaluated using a One Health approach to better account for the full costs and benefits of such a control strategy. | Review | biomedical | en | 0.999997 |
PMC11699457 | The eyebrows are critical facial structures serving a multitude of functions. They play a significant role in facial recognition, emotional expression (sadness, surprise, anger), gender identification, and aesthetics. Additionally, they serve a protective role, shielding the eyes from sweat and minor injuries. 1 Eyebrow abnormalities can significantly impact a patient's confidence and quality of life. 2 Reconstructing an eyebrow defect presents a unique challenge in plastic surgery due to the location and characteristics of eyebrow hair. Existing techniques for eyebrow reconstruction range from simple closure to more complex procedures, including local flaps, scalp flaps, hair grafts, and composite grafts. 3 While pedicle superficial temporal artery flaps have been used successfully for large eyebrow defects, there is a lack of documented cases utilizing free superficial temporal artery flaps for this purpose. This report presents the first documented case of total eyebrow reconstruction using a free superficial temporal artery flap. This novel approach offers a potential solution for patients with large eyebrow defects, potentially surpassing the limitations of existing techniques. An 18-year-old female patient presented with a giant congenital melanocytic naevus involving her left eyebrow and temporal region . The naevus was excised in 2015 and reconstructed with a supercharged superficial cervical artery perforator flap harvested from the scapular region. The flap's blood supply was enhanced by anastomosing the circumflex scapular artery to the superficial temporal artery . Six months later, she desired eyebrow reconstruction. Figure 1 Pre-operative views: giant congenital melanocytic naevus. Figure 1 Figure 2 Post-tumour removal view: absence of left eyebrow. Figure 2 For eyebrow reconstruction, a free flap based on the right superficial temporal artery was chosen. Doppler ultrasound was used to preoperatively map the right and left superficial temporal arteries for the flap pedicle and recipient vessels. The flap design considered the size and hair direction of the contralateral eyebrow. The surgery was performed under local anaesthesia with 3.5 times magnification. Lidocaine 2 % was injected subcutaneously and intradermally to both the donor and recipient sites prior to incision and was repeated every 45–60 min as needed for patient comfort. No general anaesthesia was required. The patient was placed in a supine position. The recipient site was prepared by exposing the left superficial temporal vessels. The planned anastomosis site and the required vascular pedicle length were determined. A skin and subcutaneous tissue flap was harvested from the right temporal region based on the frontal branch of the superficial temporal artery. The dissection aimed to preserve the underlying hair follicles. The lateral branches of the artery were ligated and divided. The vascular pedicle was approximately 10 cm in length. The flap was detached and transferred to the recipient site. The superficial temporal vessels of the flap were anastomosed in an end-to-end fashion to the left superficial temporal vessels using 9–0 Prolene sutures. The flap margins were meticulously sutured to the recipient eyebrow defect using 6–0 Prolene sutures . Haemostasis was confirmed, and the donor and recipient sites were closed in layers. A drainage tube was placed at the reconstruction site. The total surgical time was 2 h, and a total of 400 mg of lidocaine (20 ml) was used. Figure 3 Intraoperative views. Figure 3 This case report describes the successful reconstruction of a total eyebrow defect in an 18-year-old female patient following the removal of a giant congenital melanocytic naevus. While naevi are typically managed with excision, significant post-operative defects can negatively impact a patient's appearance and quality of life, particularly when facial structures like the eyebrow are involved. Traditionally, eyebrow reconstruction employs a variety of techniques depending on the size and location of the defect. These techniques range from simple closure to more complex procedures like local flaps, scalp grafts, and follicular unit transplantation. 3 Selecting the most suitable approach involves careful consideration of factors like the defect size and colour, eyebrow orientation, and the patient's natural features. Balancing functional and aesthetic outcomes while minimizing donor site morbidity is also crucial. 3 For partial eyebrow defects, local flaps harvested from the uninjured contralateral eyebrow remain the gold standard due to their colour and hair growth characteristics matching the recipient site. 4 However, recent decades have seen the rise of superficial temporal artery island flaps for eyebrow reconstruction. These flaps offer advantages like hair-bearing skin with appropriate texture and thickness. 5 , 6 Other authors recommended using the advancement V-Y flap with an orbicularis oculi-based pedicle for defects less than half of the eyebrow length and the superficial temporal artery island flap for defects exceeding half of the eyebrow length. 7 While effective, island flaps become unsuitable when the ipsilateral temporal region or blood vessels are compromised. 8 Free flaps offer a solution in such cases. While they can be technically demanding and require longer operative times compared to island flaps, they provide unmatched versatility and can be used even when the donor site has been damaged. 9 Notably, free superficial temporal artery flaps share the same structural and aesthetic properties as the recipient eyebrow, including hair growth characteristics. Additionally, the rich vascular supply of the superficial temporal artery allows for successful microsurgical anastomosis, enabling reconstruction under local anaesthesia with shorter hospitalization and recovery times compared to traditional free flaps. 9 While local anaesthesia is typically sufficient, the type of anaesthesia should be discussed with patients preoperatively. The amount of lidocaine administered should be carefully calculated to prevent potential toxicities. For patients with significant surgical anxiety, sedation or general anaesthesia may be considered. This case report presents the first documented instance of total eyebrow reconstruction using a free superficial temporal artery flap. The successful outcome, with complete flap survival and hair growth, highlights the potential of this technique as a viable option for reconstructing large or complex eyebrow defects, especially when traditional methods are not feasible. Further studies with larger patient populations are warranted to validate the long-term efficacy and refine the surgical approach. | Clinical case | biomedical | en | 0.999997 |
PMC11699482 | Chronic orofacial neuropathic pain can arise from damage to peripheral branches of the trigeminal nerve. Such injuries often result in striking changes in sensitivity to both tactile and noxious stimuli applied to the head and orofacial regions, as well as intense episodic or persistent pain . Trigeminal neuropathic pain is described as particularly distressing and intensely disabling, and most current treatments are largely ineffective over the long term . The complexity of neural changes triggered by damage to peripheral nerves underlies the significant failures in the long-term management of most neuropathic pain conditions. Preclinical models of neuropathic pain play an important role in simplifying this complexity and are contributing significantly to defining the neural and non-neural changes triggered by nerve damage. Transection, compression, or constriction of either maxillary ( infraorbital n. ) or mandibular ( inferior alveolar n. or mental n. ) branches of the trigeminal nerve or damage to the trigeminal ganglion in the rat have each provided clinically relevant models of trigeminal neuropathy . Injury of a peripheral nerve triggers immediate changes at the site of damage, an immediate inflammatory response is mounted. The damaged axons and their associated Schwann cells release inflammatory mediators such as NGF and TNF-⍺, and immune cells, usually circulating macrophages migrate to the site, attracted by monocyte chemoattractant protein-1 (MCP-1), followed by invasion of the disrupted structures by these cells. These macrophages are usually a mixture of both pro- and anti-inflammatory phenotypes . This process often results in an increased firing of the nerve both spontaneously and in response to activation of its sensory terminals . Once established, these processes trigger changes along both damaged and undamaged nerve fibres as they project towards their central nervous system targets, i.e., the chief sensory nucleus of the trigeminal, as well as along the trigeminal tract to the spinal nucleus (SpV), and the dorsal horn of the upper cervical spinal cord (UCC). In response to injury to a branch of the trigeminal nerve, the ganglion also shows injury related changes, both resident and infiltrating macrophages are reported to accumulate within the division of the ganglion through which the damaged nerves pass, and where their damaged cell bodies reside . This macrophage invasion and proliferation is driven both by increased cytokines, for example chemokine C-C motif ligand 2 (CCL2) released from damaged ganglion cells, as well as by the barrages of spontaneous activity triggered by the damaged fibres . Satellite glial cells are reported to show activated states in some reports and begin to express gap junctions, specifically connexin-43, which facilitates glial-glial communication, the result of which drives increased activity in ganglionic cells . This increase in neural activity is also enhanced by the release of TNF-⍺ from the infiltrating macrophages . The ganglionic changes then drive further change in brainstem recipient regions specifically in the SpV and the UCC, a large portion of the SpV shares a similar laminar architecture to the UCC and is the region that receives most of the trigeminal nociceptive input. This region sends projections to higher brainstem and thalamic pain processing regions, including the parabrachial region, the midbrain periaqueductal gray matter (PAG), hypothalamus, amygdala, medial thalamus as well as the medial part of the ventroposterior thalamus . Neuronal activity in these regions has been shown to change in preclinical models of trigeminal neuropathic pain . Microglia are the “resident macrophages” of the central nervous system, although occasionally, circulating macrophages can enter the parenchyma, including the brainstem. Trigeminal nerve injuries are reported to trigger the activation of microglia within the SpV, and they may also contribute significantly to the pain sensations in the immediate injury phase . Microglia share similar roles to macrophages, one of which is the production of cytokines that have pro-inflammatory effects (for example TNF-⍺, BDNF, IL-1b, IL-6). These cytokines act via several different mechanisms to increase neuronal excitation . This sustained neural activity is believed to contribute significantly to the pain of trigeminal nerve injury. Microglial activity can be sustained over time and in principle could contribute to the chronicity of pain, however inhibition of microglial activity has not shown consistent or substantial successes in attenuating ongoing pain in preclinical models . Microglial activation drives activity in co-located astrocytes, thus trigeminal nerve injury is also associated with significant changes in astrocyte activity in the same anatomical regions; importantly modulation of astrocyte activity appears to have a stronger attenuation on injury evoked pain . It is clear that recruitment and activation of macrophages, microglia, and astrocytes, at the site of injury, in the sensory ganglion and the brainstem nuclei, plays a critical role in the establishment of neuropathic pain by increasing neural excitability . These findings of non-neuronal contributions to the development of neuropathic pain are derived almost exclusively from post-mortem histological/immunohistochemical analyses. In this study we used translocator protein 18 kDa (TSPO) PET imaging with [ 18 F]PBR06 to visualise in-vivo , the activity of non-neuronal cells (macrophages and glia) following trigeminal nerve injury in the rat. TSPO ligands have been used successfully for brain imaging in several human pain conditions, despite suggestions of low specificity . This success highlights the great potential for use in preclinical models, thus enhancing translational opportunities, which includes identifying and understanding specific cellular contributions to the PET signal . Using chronic constriction injury of the infraorbital nerve (ION-CCI) our aim was to describe temporal changes in TSPO binding in male rats, prior to, and up to 28 days after ION-CCI compared to sham-injured and naïve counterparts. Sites of significant TSPO signal change were evaluated qualitatively for evidence of macrophage accumulation and glial reactivity in the infraorbital nerve, trigeminal ganglion, and the brainstem trigeminal complex. Male Sprague-Dawley rats (n = 79) were sourced from the Monash Animal Research Platform and kept for the duration of the experiment on a 12:12hr light:dark cycle in a temperature controlled environment. Rats at the transition from adolescence (6 weeks) to adulthood were used in these studies to ensure the optimal range of head size for the scanners over the duration of the study. Rats arrive at the facility at 6 weeks of age, and habituate for at least 1 week. Animals were housed 2–3 per cage with ad libitum access to standard laboratory chow and water. At seven weeks of age, rats were arbitrarily allocated to one of three groups: (i) infraorbital nerve chronic constriction injury (ION-CCI) (n = 35), (ii) sham surgical procedures (sham) (n = 23), or (iii) uninjured controls (naïve) (n = 21). The ION-CCI and sham surgical rats were then assigned to one of four imaging time points post ION-CCI or sham surgery: 2 days ( D2 ) (sham n = 6; ION-CCI n = 8), 7 days ( D7 ) (sham n = 5; ION-CCI n = 9), 14 days ( D14 ) (sham n = 7, ION-CCI n = 10), or 28 days ( D28 ) (sham, n = 5; ION-CCI n = 8). The naïve rats (n = 21) were placed into a single group for comparison with the ION-CCI and sham groups at each timepoint. On their allocated imaging day, each rat was scanned once for approximately 60 min in a 9.4 Tesla Bruker MRI scanner and then transferred to a PET/CT scanner for further imaging. On completion of the PET/CT scan, rats were permitted to recover from anaesthesia. Two days later, the naïve rats and the ION-CCI and sham rats at each time point ( D2-D28 ) were culled and their tissues were collected. Rats were anaesthetised by induction with isoflurane (4 % in 100 % oxygen), delivered via an airtight induction chamber. Surgical level anaesthesia was maintained with 1.5–2 % isoflurane in 100 % oxygen administered via a custom-made facemask. Body temperature was maintained by a thermal blanket. ION-CCI was performed as described by Vos and colleagues . The following procedures were performed under x3.5 magnification using Galilean Loupes . The midline above the nasal bones and between the eyes was shaved and sterilised with povidone-iodine. Once the blink reflex and pedal reflex were abolished, an incision was made approximately 12 mm long, and 2 mm medial to the supraorbital ridge of the right orbit, following the curvature of the frontal bone. The fascia and muscle were gently teased from the bone using blunt dissection until the contents of the orbit could be gently retracted laterally. Following retraction of the orbital contents, the ION was visualised approximately 8 mm deep within the orbit, lying within the infraorbital canal of the maxillary bone. Once visualised, 5 mm of the ION was gently freed from the surrounding connective tissue via blunt dissection. Two chromic gut ligatures (Chromic Gut 5–0 sutures, #687G, Ethicon, Inc.) were loosely tied around the ION using a square knot, tight enough to slightly depress the nerve, but not enough to occlude epineural circulation. The ligatures were spaced approximately 2 mm apart, and the ION was repositioned in the infraorbital canal of the maxillary bone. The incision was sutured closed with nylon sutures . The incision site was cleaned, and antibiotic powder was dusted over the suture site. The rat was moved to a heated recovery cage and monitored closely until ambulatory and eating and drinking. Sham surgical procedures were conducted identically, the nerve was isolated and exposed for the same duration as the application of the ligatures in the nerve-injured group, but no ligatures were applied to the ION. All procedures were performed at the Alfred Research Alliance-Monash Biomedical Imaging (ARA-MBI) facility (Alfred Centre, Melbourne, Australia). PET/CT imaging was performed using a Mediso NanoScan PET/CT (Mediso Ltd., Hungary). The [ 18 F]PBR06 was synthesized at the Peter MacCallum Cancer Centre with the radiochemical purity of [ 18 F]PBR06 always exceeding 95 %, and the molar activity ∼ 18,500 MBq/µmol. A maximum of six animals were imaged per imaging session at the different endpoints, D2 to D28 as described above. Rats were anaesthetised with isoflurane for the duration of all scanning, they were induced in an air-tight box (3–4 %), then transferred to a custom-built facemask, where isoflurane level was maintained at 1–3 %. Each rat then underwent a series of MRI scanning sequences for 1 h and 15 min prior to be transferred to the PET/CT (animal kept under anaesthesia for the whole process). Animals were placed into the PET/CT in a supine position, headfirst, with the brain and heart in the field of view (FoV). At the start of the dynamic PET acquisition, animals were injected via the dorsal penile vein with a bolus of 0.1987 mCi to 0.6343 mCi of [ 18 F]PBR06 in volumes ranging from 0.04 to 0.45mls. PET scans were acquired continuously for a total of 60 min with 1–3 coincidence mode, a coincidence time-window of 5 ns and an energy window of 400–600 keV. Following the PET scan, two CT scans were acquired over a period of 10 min. The first CT image was acquired using the same field of view as the PET and was used for attenuation correction (70 kVp at 700 µA, 480 projections per bed position, helical mode, scan length of 98.2 mm, 300 ms exposure time and 1:4 binning). The second CT image covered the head only and was used for co-registration (70 kVp at 700 µA, 720 projections per bed position, semi-circular single FoV mode, with medium zoom, scan length of 37.1 mm, 300 ms exposure time and 1:1 binning). The raw PET list-mode data was reconstructed using the Mediso software, Tera-Tomo 3D reconstruction method, 2 iterations of a 3-dimensional ordered subsets expectation maximization (3D OSEM) algorithm (6 subsets) and with a matrix size of 142x142x159, an isotropic voxel size of 0.6 mm and with the following corrections, attenuation, scatter, random and 18 F decay. The length of the dynamic scan frames were 5x1min, 5x2min, 3x5min, 3x10min. Time-activity-curves were obtained to assess the kinetics of the [ 18 F]PBR06 radiotracer in the brain. The last 30 min, corresponding to the relatively stable transient equilibrium, were used to calculate the Standard Uptake Values (SUVs 30-60min ), which is SUV 30-60min body weight [g/ml] = (Tissue activity/Decay corrected injected dose)*Weight*1000. To account for the amount of radiotracer that crossed the blood–brain barrier, these SUV values were divided by the whole brain SUV for each rat to obtain the SUV ratio (SUVr 30-60min ) . PET images were then pre-processed using Amide’s A Medical Image Data Examiner , Statistical Parametric Mapping 12 (SPM12) and in-house Matlab scripts . CT and SUV and SUVr images were co-registered to the Waxholm Space atlas of the Sprague Dawley rat brain . These co-registered brain maps were then resliced at 0.3x0.3x0.3 mm voxel size and smoothed using a 0.9 mm full-width-at-half-maximum (FWHM) Gaussian kernel. To determine significant differences between groups, using SPM12, voxel-by-voxel comparisons were made using the co-registered, resliced, and smoothed SUV and SUVr brain images. Our primary goal was to determine the effects of ION-CCI over time on TSPO binding, we compared ION-CCI groups at each timepoint (D2, D7, D14, D28) with naïve and sham-injured rats. There were no significant differences between ION-CCI rats and sham-injured rats, therefore we analysed each group compared to naïves. Significant differences in TSPO binding (SUVr brain maps) between groups were determined using second level, two sample, random effects analyses (p < 0.05, family wise error [FWE] corrected for multiple comparisons). Because there were no differences between ION-CCI and sham groups, for each significant cluster derived from the ION-CCI versus naïve analysis at a particular time point, we extracted the SUVr values from the naïve group and the ION-CCI and sham groups at each timepoint and the mean (±SEM) was calculated and plotted. Significant differences between groups then were determined between groups using two-sample t-tests (p < 0.05, Bonferroni corrected for multiple comparisons). Comparisons between ION-CCI and sham groups did not reveal significant differences between nerve-injured and sham-injured groups. Since we hypothesised that changes in glial reactivity would occur along the ascending trigeminal pain pathway, we restricted this initial analysis to the right (ipsilateral to injury) spinal trigeminal nucleus (SpV), left ventroposterior medial thalamus (VPM) and left primary somatosensory cortex (S1). These masks were created using the volume-of-interest (VOIs) masks within the Waxholm Space atlas of the Sprague Dawley rat brain. To further explore changes in SpV, we also created effect size maps for each animal and then created a mean effect size map for each ION-CCI group and for the naïve group. We created effect size difference maps by subtracting each ION-CCI group from the naïve group, masked the right (ipsilateral to injury) SpV, and overlaid the resultant SpV maps onto a rat brain template for each of the four timepoints. In addition, we conducted a non-masked voxel-by-voxel analysis of the brain to determine significant increases or decreases in TSPO binding between ION-CCI groups at each time point and the naïve group. Since we found no significant differences at FEW corrected levels, we used a more liberal threshold of p < 0.001, uncorrected to assess more subtle group differences. To limit the potential for Type 1 errors we used a minimum cluster size of 20 contiguous voxels. In addition to exploring TSPO binding changes in the brain, we hypothesized that in the ION-CCI groups, the injured trigeminal nerve/ganglion would also display significantly increased TSPO binding. For each timepoint, significant differences in TSPO binding (SUV maps) between the ION-CCI groups and naïve group were determined using second level, two sample, random effects analyses. We restricted this initial analysis to the right trigeminal nerve/ganglion using a custom mask. Since we found no significant differences at FWE corrected levels, we used a liberal threshold of p < 0.05, uncorrected to assess more subtle group differences. To limit the potential for Type 1 errors we used a minimum cluster size of 20 contiguous voxels. For each significant cluster at a particular time point, SUV values were extracted for all ION-CCI, sham and naïve groups and the mean (±SEM) calculated and plotted. SUV was used for analysis of peripheral structures because of the relatively small volumes of the structures analysed, whereas for the much larger brain structures SUVr was used for analyses. Significant differences were determined between groups using two-sample t -test (p < 0.05, Bonferroni corrected for multiple comparisons). Comparison between ION-CCI and sham groups did not reveal significant differences between nerve-injured and sham-injured groups. Forty-eight hours following the imaging session, animals were briefly anaesthetised with 5 % isoflurane mixed in oxygen 1.5 L/minute and a lethal dose of pentobarbitone administered (130 mg/kg, intraperitoneally). Each rat was then perfused trans -cardially with 400 ml of ice cold heparinised 0.9 % (w/v) saline followed by 400 ml of 4 % paraformaldehyde in sodium acetate-borate buffer (pH 9.6, 4 °C) (PFA). The left and right infraorbital nerves (ION), their corresponding trigeminal ganglion (TG) and the brain and cervical spinal cord were dissected free and post-fixed in PFA for two hours at room temperature, then cryoprotected in 10 % (w/v) sucrose in 0.1 M phosphate buffered saline, pH 7.4 (PBS) and stored at 4 °C until processing. All tissue was sectioned using a cryostat , a 1 in 6 series of transverse sections of the infraorbital nerve (14 µm); and the trigeminal ganglia (14 µm) were cut onto 2 % (w/v) gelatinised slides and stored at -20 °C until processing. The medulla was blocked caudally with a coronal cut at approximately the C3 level of the cervical spinal cord, and rostrally at the level of the subnucleus interpolaris of the SpV (−12.00 mm bregma . Coronal sections of the medulla were cut at 40 µm, as a 1 in 10 series and stored in antifreeze at −20 °C until processed. ION, TGs, and sections of medulla were immunoreacted to determine the co-localisation of CD68-immunoreactive (CD68-IR) macrophages, Peripheral-Type Benzodiazepine Receptor (PBR) immunoreactivity (PBR-IR), blood vessels (tomato lectin immunoreactivity [TL + ]), ionized calcium binding adaptor molecular 1 (IBA1) immunoreactive microglia, or Glial Fibrillary Acidic Protein (GFAP) immunoreactive (GFAP-IR) astrocytes. ION and TG mounted slides were washed in 0.1 M phosphate buffered saline, pH7.4 (PBS) and blocked in PBS containing 5 % (v/v) normal horse serum (NHS) and 0.05 % (v/v) Tween-20 (Sigma-Aldrich, Australia). Sections were incubated for 16-hours at room temperature with mouse anti-CD68 [ED-1] and rabbit anti-PBR [EPR5384] , in PBS containing 5 % (v/v) NHS and 0.05 % (v/v) Tween-20. Sections were then washed in PBS and incubated for 2-hours in Alexa-488 conjugated donkey anti-mouse and Cy3 conjugated Donkey anti-rabbit . Sections were washed in PBS and then incubated for 3-hours with DyLight™ 594 conjugated Tomato [Lycopersicon Esculentum] Lectin (TL) in PBS . Slides were washed in PBS and then incubated for 20-mins with DAPI in PBS. Slides were washed in PBS before being cover slipped with Prolong Gold Antifade mounting medium and stored at 4 °C until microscopy. Medulla sections were washed in PBS and then blocked in 5 % (v/v) NHS in PBS for 30-minutes at room temperature. PBR/GFAP/TL immunofluorescence: Sections were incubated for 16-hours at 4 °C with rabbit anti-PBR [EPR5384] and mouse anti-GFAP . Sections were then washed in PBS and incubated for 2-hours in Alexa-488 conjugated donkey anti-mouse and Cy3 conjugated donkey anti-rabbit . PBR/IBA1/TL immunofluorescence: Sections were incubated for 16-hours at 4 °C with rabbit anti-PBR [EPR5384] , sections were then washed in PBS and incubated for 2-hours in Cy3 conjugated donkey anti-rabbit . Sections were washed and then incubated for 16-hours at 4 °C with rabbit anti-IBA1 , washed in PBS and incubated for 2-hours in Alexa-488 conjugated donkey anti-rabbit . Following antibody incubations all sections were then washed in PBS and incubated for 3-hours with DyLight™ 594 conjugated Tomato [Lycopersicon Esculentum] Lectin (TL) in PBS . Slides were washed in PBS and then incubated for 20-mins with DAPI in PBS. Slides were washed in PBS before being mounted and cover slipped with Prolong Gold Antifade mounting medium and stored at 4 °C until analysed under the microscope. Slides were imaged using a Nikon C2 confocal microscope. The large image function was used to ‘stitch’ multiple images (100x magnification) to allow the visualisation of the entire infraorbital nerve including ligated areas, trigeminal ganglia, and coronal medulla sections. Representative z-stack images were captured at 600x magnification to visualise co-localisation of CD68-IR macrophages, PBR-IR, TL + blood vessels, IBA1-IR microglia, or GFAP-IR astrocytes. Analysis of the trigeminal ganglion and nerve revealed that relative to the naïve group, there was a significant increase in binding in the trigeminal ganglion ipsilateral to the nerve constriction in the ION-CCI rats at day 14, and no differences at days 2, 7 or 28 . A similar increase was observed also in the sham-injured rats at this time point , although the TSPO binding was somewhat higher in sham-injured rats, at days 2 and 7 post-ION-CCI, this was not significantly different. Qualitative histological observations of the ganglion of ION-CCI rats revealed PBR-IR in the maxillary division of the ganglion co-localized with CD68-IR macrophages, and TL + endothelial cells of the ganglionic vasculature . In ganglia from sham-injured rats, PBR-IR was also observed . Naïve rats showed little PBR-IR. At the site of injury, PBR-IR was frequently colocalized with CD68-IR macrophages and TL + endoneurial vasculature . Fig. 1 TSPO binding assessed in infra-orbital nerve/ganglion in chronic constriction injury (ION-CCI), sham-injured and naïve groups. Significant binding increases (hot colour scale) at day 14 (D14) in the ION-CCI compared with naive groups occurred in the region encompassing the right (ipsilateral to nerve injury) trigeminal (V) ganglion. Clusters are overlaid onto a T2-weighted rat brain template anatomical image set. To the top right of each image is the approximate location relative to bregma derived from Paxinos and Watson . To the right are plots of mean ± SEM standard uptake value (SUV) for all three groups at day 2 ( D2 ), day 7 ( D7 ), day 14 ( D14 ) and day 28 ( D28 ). The horizontal grey line and grey shading indicates the mean and SEM of the naïve group. *p < 0.05 derived from voxel-by-voxel analysis, # p < 0.05 post hoc two-sample t-tests sham versus naïve (values extracted from significant clusters derived from ION-CCI vs naïve voxel-by-voxel analysis). Table 1 Significant cluster properties including SUVr values in the ascending trigeminal pathway and SUV values for the trigeminal ganglion. * p < 0.05 voxel-by-voxel analysis. # p < 0.05, two sample t -test. Day 2: day 2; D7: day 7; D14: day 14; D28: day 28; ION-CCI: infra-orbital nerve chronic constriction injury; SEM: standard error mean; SpV: spinal trigeminal nucleus; SUV: standard uptake values; SUVr: standard uptake values ratio. Ascending trigeminal pathway: ION-CCI > naive cluster size t- value Day SUVr mean ± SEM naive sham-injured ION-CCI D2: SpV cluster 8 3.27 D2 1.77 ± 0.03 # 1.80 ± 0.08* D7 1.59 ± 0.03 1.65 ± 0.07 1.80 ± 0.07 # D14 1.54 ± 0.09 1.70 ± 0.05 # D28 1.47 ± 0.08 1.53 ± 0.03 D7: SpV cluster 53 3.91 D2 1.79 ± 0.03 # 1.79 ± 0.07 # D7 1.61 ± 0.03 1.69 ± 0.06 1.84 ± 0.06* D14 1.57 ± 0.09 1.72 ± 0.05 # D28 1.48 ± 0.07 1.52 ± 0.04 Trigeminal ganglion/nerve: ION-CCI > naive SUV mean ± SEM D14: trigeminal ganglion cluster 116 2.31 D2 4.38 ± 0.37 3.90 ± 0.30 D7 3.96 ± 0.19 4.25 ± 0.35 3.81 ± 0.19 D14 4.75 ± 0.17 # 4.64 ± 0.23* D28 3.90 ± 0.34 3.84 ± 0.27 Fig. 2 Representative photomicrographs of PBR-IR co-localization. A: trigeminal (V) ganglion TSPO binding cluster overlaid on a T2-weighted rat brain template anatomical image. B: stitched 40X magnification photomicrograph of the trigeminal ganglion and corresponding ophthalmic (V1), maxillary (V2) and mandibular (V3) divisions with PBR-IR (orange), CD68-IR (green) and TL (red) co-localization with immunofluorescence. The PBR-IR in the V2 of (C) naïve, (D) sham-injured and (E) ION-CCI rats are shown at 600X magnification. Example of co-localization of (F) CD68-IR and PBR-IR, (G) TL + and PBR-IR and (H) CD68-IR, PBR-IR and TL + . White arrows show co-localized immunofluorescent signals. Scale bars represent 20 µm or 500 µm in panel B. Analysis of TSPO binding in the brainstem trigeminal complex, revealed significant increases in the SpV/paratrigeminal region (Pa5) of the ION-CCI group compared to the naïve group at day 2 and day 7 . Increased binding was restricted to the right Pa5. Extraction of SUVr values from the significant Pa5 cluster at day 2, revealed that compared with the naïve group, ION-CCI groups displayed greater TSPO binding at days 2, 7 and 14, but not at day 28 ( Table 1 ). Sham-injured rats showed a significant increase in TSPO binding compared to the naïve group at day 2, but not at days 7, 14 and 28. Once again, extraction of SUVr values from the significant Pa5 cluster at day 7, revealed that compared with the naïve group, ION-CCI rats had greater TSPO binding at days 2, 7 and 14, but not day 28. Sham-injured rats displayed a significant increase in TSPO binding compared with naïve group at day 2, but not at days 7, 14 and 28. Qualitative observations of the Pa5 region confirmed PBR-IR localised to this region which was usually co-localized with IBA1-IR microglia, and TL + endothelial cells in the vasculature, but never in GFAP-IR astrocytes in the sections observed (not illustrated). Fig. 3 TSPO binding assessed in the ascending trigeminal pathway in infra-orbital nerve chronic constriction injury (ION-CCI), sham-injured and naïve groups. A : Significant binding increases (hot colour scale) at day 2 ( D2 ) and day 7 ( D7 ) in the ION-CCI compared with naive groups. Binding increases occurred in the right (ipsilateral to nerve injury) brainstem trigeminal complex. Clusters are overlaid onto a T2-weighted rat brain template anatomical image set. To the top right of each image is the approximate location relative to bregma derived from a rat atlas Paxinos and Watson . Below the overlays are plots of mean ± SEM standard uptake value ratios (SUVr) for all three groups. The horizontal grey line and grey shading indicates the mean and SEM of the naïve group. *p < 0.05 derived from voxel-by-voxel analysis, # p < 0.05 post hoc two-sample t-tests sham versus naïve (values extracted from significant clusters derived from ION-CCI vs naïve voxel-by-voxel analysis). Inset to the right shows the volumes of interest (light-blue) used for analysis. B: Representative photomicrographs of PBR-IR co-localization within the Brainstem Trigeminal Complex. (i) TSPO binding cluster overlaid on a T2-weighted rat brain template anatomical image of the brainstem trigeminal complex at approximately −13.44 mm bregma (ii) stitched 40x magnification photomicrograph of the brainstem trigeminal complex, with the paratrigeminal nucleus (Pa5) outlined in white and PBR-IR (orange), Iba1-IR (green) and TL (red) co-localization with immunofluorescence. Example of co-localization of (iii) PBR-IR, Iba1-IR and TL+; (iv) PBR-IR and TL+; (v) Iba1-IR and TL+; and (vi) PBR-IR and Iba1-IR. White arrows depict co-localized immunofluorescent signals. Scalebars represent 20 µm or 500 µm in panels ii-vi. Fig. 4 shows overlays of the mean effect size differences within the right SpV and shows increased TSPO binding across the rostro-caudal extent of the SpV at days 2–14 and by day 28, these increases remain only in the Pa5 region, at approximately −12.8 mm to 13.5 mm from bregma. Histological analysis of the SpV region confirmed PBR-IR specific to this region and was co-localized with IBA1-IR microglia, and TL + endothelial cells in the vasculature, and GFAP-IR astrocytes . Fig. 4 Effect size increases (hot colour scale) of TSPO binding in infra-orbital nerve chronic constriction injury (ION-CCI) compared with naïve groups within the right brainstem trigeminal complex volumes of interest at day 2, day 7, day 14 and day 28 overlaid onto coronal slices of a T2-weighted rat brain anatomical image set. On the left are shown the terminal projections of the infraorbital nerve traced using WGA- conjugated horse-radish peroxidase, the data are adapted from Fig. 4 of . To the top right of each image is the approximate location relative to bregma derived from a rat atlas. Fig. 5 A: Top row shows significantly greater TSPO binding in the infra-orbital nerve chronic constriction injury (ION-CCI) compared with naïve groups at day 7 overlaid onto a series of coronal T2-weighted anatomical images between −13.68 mm and −14.76 mm relative to bregma. Below are photomicrographs of coronal sections of the brainstem trigeminal complex at equivalent levels stained for PBR-IR (orange), GFAP-IR (green) and TL (red) co-localization with immunofluorescence. The upper row of photomicrographs shows sections from naïve control rats and the lower row of rats following ION-CCI. All data are from rats at day 7 post-injury. Scale bar 500 µm. B: Photomicrographs of coronal sections of the brainstem trigeminal complex at day 7 post-injury. Top (i) large photomicrograph, stitched 40x magnification of the brainstem trigeminal complex, with high magnification insets (to right) taken from the region of the SpV outlined in white. PBR-IR (orange), GFAP-IR (green) and TL+ (red) co-localization with immunofluorescence. To the right, insets show examples of co-localization of: (ii) PBR-IR, & GFAP; (iii) PBR-IR & TL+; (iv) GFAP-IR & TL+; and (v) PBR-IR, GFAP-IR & TL + . White arrows depict co-localized immunofluorescent signals. Scalebars represent 20 µm or 500 µm. Bottom Panel: Left: large photomicrograph, stitched 40x magnification of the brainstem trigeminal complex, with high magnification insets (to right) taken from the region of the SpV outlined in white. PBR-IR (orange), Iba1-IR (green) and TL+ (red) co-localization with immunofluorescence. To the right, insets show examples of co-localization of: (a) PBR-IR, & Iba1-IR; (b) PBR-IR & TL+; (c) Iba1-IR −IR & TL+; and (d) PBR-IR, Iba1-IR −IR & TL + . White arrows depict co-localized immunofluorescent signals. Scalebars represent 20 µm or 500 µm. Analysis of supra-medullary brain regions revealed a number, of TSPO binding differences between ION-CCI and naïve rats within the 28-day period. On day 2, TSPO binding was significantly increased in the area encompassing both the ipsilateral primary motor cortex (M1) and primary somatosensory cortex (S1) . Extraction of SUVr values from the cluster located in this combined M1/S1 cortical region, revealed that compared with the naïve group, the ION-CCI rats also displayed greater TSPO binding at day 28 whereas the sham-injury group did not display a significant TSPO binding change at any time point. On day 7, there was a significant decrease in TSPO binding in the ION-CCI group relative to the naïve group within the midline cerebellar cortex. No change occurred in this region at any other timepoint in either the ION-CCI or sham-injury groups. On day 14, there were multiple regions where TSPO binding was significantly reduced in the ION-CCI compared with naïve groups. Binding decreases occurred in the region of the right septal nucleus, right hippocampus and in two clusters in the right caudate/putamen. These decreases did not occur at any other timepoint and in the sham-injury group only occurred at day 14 in the septal nucleus and one of the caudate/putamen clusters. Finally, on day 28, there were two regions that displayed increased binding in the ION-CCI compared with naïve groups. One cluster in the region of the right M1 that also increased at day 2, and another at day 28 only in the region of the right S1. The sham-injury group also displayed increased binding in these two regions only at day 28. Fig. 6 TSPO binding assessed over the entire brain in infra-orbital nerve chronic constriction injury (ION-CCI), sham-injury and naïve groups. Significant binding increases (hot colour scale) or decreases in ION-CCI compared with naïve groups at day 2 ( D2 ), day 7 ( D7 ), day 14 ( D14 ) and day 28 ( D28 ) are displayed. Clusters are overlaid onto a T2-weighted rat brain template anatomical image set. To the top right of each image is the approximate location relative to bregma derived from Paxinos and Watson . To the right are plots of mean ± SEM standard uptake value ratios (SUVr) for all three groups. The horizontal grey line and grey shading indicates the mean and SEM of the naïve group. *p < 0.05 derived from voxel-by-voxel analysis, # p < 0.05 post hoc two-sample t-tests sham-injury versus naïve (values extracted from significant clusters derived from ION-CCI vs naïve voxel-by-voxel analysis). CuP: caudate/putamen; M1: primary motor cortex; S1: primary somatosensory cortex; SpV: spinal trigeminal nucleus; SUVr: standard uptake values ratio. Table 2 Significant cluster properties including SUVr values in the whole brain analysis. * p < 0.05 voxel-by-voxel analysis. # p < 0.05, two sample t -test. Day 2: day 2; D7: day 7; D14: day 14; D28: day 28; ION-CCI: infra-orbital nerve chronic constriction injury; M1: primary motor cortex; S1: primary somatosensory cortex; SEM: standard error mean; SpV: spinal trigeminal nucleus; SUVr: standard uptake values ratio. cluster size t-value day SUVr mean ± SEM naive sham ION-CCI D2: ION-CCI > naive right M1/SI 273 6.00 D2 1.30 ± 0.05 1.42 ± 0.03* D7 1.16 ± 0.03 1.33 ± 0.06 1.23 ± 0.03 D14 1.14 ± 0.04 1.16 ± 0.04 D28 1.37 ± 0.09 1.36 ± 0.05 # D7: ION-CCI < naive Right SpV 50 4.04 D2 1.63 ± 0.02 # 1.64 ± 0.06 # D7 1.49 ± 0.03 1.53 ± 0.05 1.68 ± 0.05* D14 1.42 ± 0.08 1.55 ± 0.04 D28 1.40 ± 0.08 1.42 ± 0.02 cerebellar cortex 51 3.85 D2 1.60 ± 0.03 1.71 ± 0.09 D7 1.63 ± 0.04 1.50 ± 0.04 1.41 ± 0.04* D14 1.56 ± 0.06 1.66 ± 0.07 D28 1.75 ± 0.10 1.76 ± 0.05 D14: ION-CCI < naive right septal nucleus 38 3.59 D2 0.79 ± 0.01 0.78 ± 0.01 D7 0.83 ± 0.02 0.77 ± 0.03 0.82 ± 0.02 D14 0.75 ± 0.03 # 0.73 ± 0.02* D28 0.93 ± 0.08 0.86 ± 0.04 right hippocampus 27 3.69 D2 1.07 ± 0.02 0.97 ± 0.02 D7 1.03 ± 0.01 0.96 ± 0.02 # 0.99 ± 0.02 D14 0.99 ± 0.03 0.94 ± 0.01* D28 0.97 ± 0.03 1.00 ± 0.02 right caudate/putamen 1 26 3.71 D2 0.90 ± 0.02 0.85 ± 0.02 D7 0.88 ± 0.01 0.85 ± 0.02 0.86 ± 0.02 D14 0.79 ± 0.02 # 0.78 ± 0.01* D28 0.93 ± 0.05 0.90 ± 0.02 right caudate/putamen 2 22 3.50 D2 0.75 ± 0.03 0.74 ± 0.02 D7 0.77 ± 0.02 0.73 ± 0.02 0.75 ± 0.02 D14 0.69 ± 0.03 0.66 ± 0.02* D28 0.84 ± 0.05 0.80 ± 0.02 D28: ION-CCI > naive right M1 55 4.12 D2 1.11 ± 0.06 1.24 ± 0.03 # D7 1.02 ± 0.03 1.09 ± 0.03 1.06 ± 0.03 D14 0.97 ± 0.03 0.99 ± 0.03 D28 1.25 ± 0.11 # 1.23 ± 0.04* right S1 373 4.12 D2 1.16 ± 0.02 1.18 ± 0.04 D7 1.07 ± 0.03 1.15 ± 0.05 1.09 ± 0.03 D14 1.06 ± 0.02 1.03 ± 0.03 D28 1.21 ± 0.04 # 1.27 ± 0.03* In this study our specific aim was to use PET imaging to visualise in-vivo, the activity of macrophages and microglia during the development of trigeminal neuropathic pain in male rats. Using chronic constriction injury of the infraorbital nerve (ION-CCI) we aimed to describe temporal changes in TSPO binding 2, 7, 14, and 28 days after ION-CCI, and to compared this to the binding in both sham-injured and naïve control rats. Unexpectedly, we identified almost identical changes in TSPO binding in rats that had undergone the sham-injury procedures, ie., the surgical isolation of the ION without ligation, to those seen in nerve injured rats. It is clear from these data that within this 28 day window, the surgical approach taken for nerve isolation, which involves skin incision and retraction of orbital contents is sufficient to evoke TSPO binding changes akin to those that are suggested to underly the development of the neuropathic pain state. The sham-injury effect that we have detected may share similarities to the model of persistent post-surgical pain triggered by skin-muscle incision and retraction (SMIR model) described in the hindlimb . This procedure tiggers microglial activity in lumbar spinal segments attributed to the duration of exposure of the surgical site . We note however, that similar levels of microglial activity are not reported for sham-injury conditions in hindlimb neuropathic models, suggesting that sham-injury procedures in these instances are brief and perhaps more discrete, and likely that in preclinical trigeminal neuropathy models, the surgery for sham-injury is potentially more disruptive. We reflect that the sham condition is often considered a condition closer to the uninjured state, which is not always the case as surgical procedures still trigger significant tissue disruption with associated sensory hypersensitivity, as capitalised on in models of post-surgical pain. Further, we have recently described significant neuroimmune changes following identical sham-injury procedures on the infraorbital nerve and so the fact that we detected TSPO binding changes in the sham-injured rats is perhaps not quite so surprising. We evaluated the main PET findings with immunohistochemistry using a combination of antibodies to identify TSPO binding sites (anti-PBR), combined with markers for glial cells and macrophages. Our immunofluorescence results indicated that in both infra-orbital nerve injured, and sham-injured rats, TSPO was likely binding to microglia, macrophages, astrocytes, vascular endothelial cells, as well as other non-identified classes of cell. Therefore, changes in TSPO signal are unlikely to reflect only macrophage accumulation or changes in glial cells. Secondly, we found that TSPO binds to specific sites along the trigeminal pain pathway at distinct timepoints. In addition, we found changes in TSPO binding in additional central sites including the cerebellum, the motor and somatosensory cortices, septum, hippocampus, and the striatum. To reduce the testing burden for both ethical and practical reasons, in this study we did not evaluate the rat’s behavioural responses to facial stimulation. We are however confident of the overall trajectories of both nerve injury-evoked, and sham-injury evoked changes in behaviour, based on data from a large-scale study that was conducted concurrently, and recently published . We consider the timing and location of the TSPO binding changes with regards to the changes described in that study and current literature. There is broad agreement that there are three distinct phases in the response to injury of the infraorbital nerve, these are characterized by both behavioural and neuroimmune markers. The first of the phases, described as the “early” period, lasts for up to one week, it correlates with the period of Wallerian degeneration and is characterized by hypo-responsiveness . The second phase appears to be an “intermediate”, or transition period (12–21 days post-injury) where behavioural reactivity increases and animals show aversive or defensive responses to tactile stimulation . The final or “late” phase appears after 28 days and was described by Vos and colleagues , to last at least 130 days post ION-CCI and is characterised by well-established hyper-responsiveness and clear reductions in facial grooming. We will consider our PET findings using this temporal framework of ION-CCI evoked changes, from the site of injury and then at each level of the trigeminal pain pathway. As highlighted above, TSPO was selected so that we could identify changes in microglia and macrophages, however it is important to note however that we need to broaden this consideration to include reactive astrocytes, vascular endothelial cells, and possibly smooth muscle cells and neurons . At the site of nerve injury, our histological analysis showed a significant accumulation of macrophages with “frothy” and vacuolated appearances, some of these cells colocalized with PBR-IR, indicating a likely site for TSPO binding. Following sham-surgery, there were small numbers of macrophages at the site of nerve exposure on each of the days of testing. PBR-IR was also colocalized with the endoneurial vasculature, we expect that this would be largely attributed to staining of the vascular endothelial cells. PET scans did not detect significant TSPO binding at the nerve-injury site at any timepoint. TSPO binding in the trigeminal ganglion was increased at day 14 in both the ION-CCI and sham-injury groups when compared to naïve rats. We also noted an increase in PBR-IR at this time point, however the numbers of CD68-IR macrophages in the ganglion were quite low and few of these contained PBR-IR. This raises the likelihood that the TSPO binding is not an indicator of macrophage accumulation. We noted that PBR-IR was present in the lumen of TL + vessels raising the possibility that TSPO was binding to endothelial cells, as well as other non-identified classes of cells. Binding of TSPO to endothelial cells has previously been reported in a mouse model of CRPS triggered by tibial fractures . It has been reported that nerve injury can lead to increased vascularization of ganglia associated with the damaged nerve . These vessels can play a critical role in the development of neuropathic pain, through local release of inflammatory mediators such as cytokines and chemokines that directly, and indirectly alter the function of the resident satellite glial cells and sensory neurons . The day 14 timepoint at which these observations were made is during the transitional phase, during which we have previously observed decreased numbers of macrophages relative to the acute injury phase, i.e., 2–7 days post ION-CCI, and prior to an apparent second wave of macrophage accumulation seen during the late phase, which includes 28 days post-injury and beyond . This transitional phase may well reflect the change from a phase of clearance of injury related cell damage, to a later phase of nerve repair and attempted functional recovery. Our observations in sham-injured rats suggest that the surgical procedures for nerve injury might also contribute significantly to these changes. Similar to the trigeminal ganglia, in both nerve-injured and sham-injured rats, PBR-IR was co-localized with the neurovasculature along the rostro-caudal extent of the brainstem trigeminal complex ( pars interpolaris , pars caudalis , and Pa5) and the NTS, as well as in astrocytes and microglia. As such, the changes in TSPO binding that we observed along the brainstem do not appear to correspond to a single cell type, rather it emphasizes the close relationship between glial activity and the neurovasculature. Astrocytes are well known to regulate neuronal function and directly interact with the vasculature as well as other glial cells . Increased TSPO binding was observed in a discrete region of the brainstem trigeminal complex ipsilateral to the injury, and surgical site, this region included the Pa5, the dorsal portion of SpV ( pars interpolaris and pars caudalis ), and the trigeminal portion of the NTS. These areas have all been described to receive direct inputs from fibres of the infraorbital nerve specifically and more generally from the maxiliary division of the trigeminal nerve. The paratrigeminal nucleus is a small interstitial system of the spinal trigeminal tract, which consists of small diffuse nuclei located in the dorsal lateral medulla . It receives sensory inputs not only from the trigeminal nerve, but also from the glossopharyngeal and vagal nerves and is involved in the integration of both cardiovascular, and respiratory responses, in addition to pain mechanisms . Its cells send efferent projections to both the parabrachial nucleus, the nucleus of the solitary tract, lamina 1 of the pars caudalis of the SpV, and the ventroposterior medial nucleus of the thalamus (VPM) . Similarly, the NTS receives direct sensory input from the trigeminal nerve, the glossopharyngeal and vagal nerves as well as inputs from the superficial laminae of the SpV, pars caudalis . The pars interpolaris and pars caudalis of SpV also receive direct inputs from the infraorbital nerve, via both the maxillary and ophthalmic divisions of the trigeminal nerve . These anatomical distributions of sensory inputs correspond well with the effect size analysis that we report here, which revealed increased TSPO binding ipsilateral to the nerve-injury, and the associated surgical site in the dorsolateral aspect of the laminar part of the SpV, as well as the Pa5 region and the dorsal NTS (trigeminal portion). Our data suggest that the surgical procedures for ION-CCI contributes significantly to these changes based on our observations of significant binding in sham-injured rats. The dynamic changes observed in TSPO binding in the brain over the experimental period were similar to those seen at the site of nerve injury, and the trigeminal ganglion and suggest a common phasic response profile to both nerve injury and sham surgery. The largest increases in TSPO binding were noted 2 days after nerve injury and sham surgery, within the early response phase. In rats, an initial and rapid microglial response in SpV pars caudalis has been observed after inferior alveolar nerve transection and peaks between 1–3 days this response profile is also seen following ION-CCI . We note, as discussed above, that such a microglial response is not reported in sham-injured counterparts in other studies of ION-CCI, although it is reported for the SMIR model of post-surgical pain, which our sham-injury procedure may more closely resemble. This microglial response is not related to an immediate (1–3 days) increase in neuronal activity, however 7 days after ION-CCI, phosphorylated ERK (pERK) expression, a marker of neuronal excitation, provides evidence of increased neuronal excitation in SpV pars caudalis . Similarly, cFos expression increases at 9–12 days after ION-CCI, again indicating an injury evoked increase in neuronal activity sometime after the microglial peak response . Furthermore, at 7 days post injury the mean background discharge and after-discharge rates of wide dynamic neurons were significantly larger in ION-CCI rats in the superficial laminae of the pars caudalis . The delay between the peak microglial response and the increased neuronal activation may be a result of microglial modulation of the extracellular matrix, whereby the microglia degrade the perineuronal nets forming the matrix, which facilitates neuronal excitation in a similar fashion to that described following sciatic nerve injury and dorsal horn neurons . Glia-glial interactions can also result in the initiation and maintenance of chronic neuropathic pain. At 7 days post injury, although microglia are still present, their numbers are decreased, and the presence of GFAP-IR hypertrophic astrocytes are increased following trigeminal nerve transection . Microglial derived factors such as C1q are known to increase astrocyte reactivity and subsequently nociceptive signaling 7 days after ION-CCI . TSPO binding at 14 days post injury appears to show a more elongated distribution along the rostro-caudal axis of the SpV, and extends into its deeper laminae, the binding in the dorsal NTS and Pa5 is maintained at this timepoint also. This timepoint corresponds to the transitional phase where it is suggested that the primary glial activity is that of astrocytes. GFAP-IR is sustained in astrocytes 14- and 21-days post trigeminal transection injury as well as ION-CCI . It is important to note that GFAP-IR astrocytes during this transitional phase are present across both superficial and deep laminae of pars caudalis . We note that in the literature, there are fewer studies of glial expression following trigeminal nerve injuries at these extended timepoints, with most studies focusing on 7–14 days post injury. In our rats at 28 days post injury or sham-injury, the TSPO binding contracts along the rostrocaudal axis, and becomes most concentrated in the dorsolateral aspect of the SpV/Pa5 region and the dorsal NTS. At this late phase of injury, microglia are reported to be still present following trigeminal nerve transection, albeit in much smaller numbers, and GFAP-IR astrocytes are sustained but also in smaller numbers following trigeminal nerve transection and ION-CCI . The contraction of TSPO signal around the Pa5 region is intriguing as this subregion has been shown to be more sensitive to nociceptive-specific C-fibre inputs than the SpV, and is thought to drive the affective-emotional elements of pain via its outputs to midbrain structures including the parabrachial nucleus and the PAG . Our analyses of TSPO using binding density versus effect size analysis revealed differences in peak signals within the trigeminal brainstem complex. While these signals all fall well within the boundaries of termination of the infraorbital nerve derived from anatomical tracing studies as illustrated in Fig. 4 , the shift deserves comment. These differences may reflect limitations of the resolution of the PET technique, or the TSPO ligand in particular, or they may in fact reflect salient neurobiological features of ION-CCI and sham surgical procedures. Whether the locations with the greatest effect sizes contribute to the sensory and behavioural consequences of ION-CCI and sham-injury , when compared with the sites showing the statistically significant binding increases, warrants future investigation. Primary motor cortex (M1) and S1): In the current study, we also observed increased TSPO binding in a variety of forebrain structures including S1 and M1, ipsilateral to the nerve injury and surgical site, in both ION-CCI and sham-injured rats. This was evident during both the early phase ( D2 ) and late phase ( D28 ) after both ION-CCI and sham-injury. While contrary to the classical view that ascending nociceptive pathways terminate exclusively contralaterally, there is substantial evidence for an ipsilateral nociceptive pathway transmitting trigeminal pain . Our observations could suggest surgery and nerve injury-evoked cortical plasticity; this proposal is based on observations that sciatic nerve ligations trigger astrocyte activation in the S1 within the first week of injury, and that this correlates with changes in extracellular glutamate concentrations and dendritic spine turnover indicative of neuroplasticity . Of note are data from Loggia and colleagues, who have shown in humans with chronic back pain, that significant increases in TSPO binding are present in both the S1 and M1 which provides strong evidence for prolonged glial activity that persists long after the initial injury . TSPO binding was also noted in a region of the midline cerebellar cortex, at approximately lobules 4 and 5 of the anterior lobe. This region receives direct sensory projections from the trigeminal nerve via the superior cerebellar peduncles , providing a route for injury and surgery mediated, direct modulation of glial-neurovascular contributions to cerebellar function. Sub-cortical Forebrain: During the transitional phase (D14) the septal nuclei, hippocampus, and dorsal striatum showed decreased TSPO binding on the side ipsilateral to the nerve injury and sham-injury , consistent with the cortical changes described above. TSPO binding decreases are difficult to interpret, they may indicate a transient decrease in glial-neurovascular interactions. Altered metabolism, measured by [ 18 F]fluorodeoxyglucose in awake rats has been shown at this timepoint in a range of subcortical forebrain structures following the spinal nerve ligation model of neuropathic pain and striatal monoamine levels are significantly disrupted 13 days after a peripheral sciatic nerve CCI . A second possibility is nerve injury and surgery triggered mitochondrial dysfunction at specific central sites. TSPO is mitochondrial protein, to which [ 18 F]PBR06 binds, decreased mitochondrial function/expression may lead to decreased [ 18 F]PBR06 binding identified by regional reductions of the PET signal. It is of note that mitochondrial dysfunction is often associated with neuropathic pain-like symptoms . Both of these suggested changes likely correlate with disruptions to both cognitive and affective behaviours, resulting for example, in impaired decision making. We provide in vivo evidence of transient increases in TSPO binding in the trigeminal ganglion and trigeminal brainstem complex following both infra-orbital nerve constriction injury and sham-injury procedures that isolate the ION in identical fashion, but do not ligate it. The use of in vivo imaging of glial cells in combination with other imaging techniques such as structural and functional magnetic resonance imaging, can provide a novel view of the complex interactions between neural and non-neural cells in preclinical models of neuropathic pain, and as suggested by observations in sham-injured rats in this study, in models of persistent post-operative pain. Given that these in vivo techniques can be used to acquire similar information in humans, our results provide the platform to begin to translate findings from preclinical models into human patients with chronic pain or the risk of persistent post-surgical pain and offer an opportunity to begin to explore the effects of treatments on neural and non-neural cells in both preclinical models and humans. | Review | biomedical | en | 0.999998 |
PMC11699484 | In this report we present new aDNA-based studies of the Khvalynsk population in combination with traditional archaeological and anthropological studies. The formal presentation of the ancient DNA data along with a comprehensive set of genetic analyses will be made in a separate publication; here, we summarize results where they are relevant to understanding the Khvalynsk cemetery population. In addition, we present specific analytical summaries of radiocarbon dates and stable isotopes, copper artifacts, animal sacrifices, and polished stone maces. We argue that Khvalynsk exhibits remarkable diversity in its population and equally remarkable segmentation between groups of individuals in its grave offerings . We discuss how the Khvalynsk cemetery was related to other sites and archaeological cultures during the steppe Eneolithic. We also discuss cranio-facial types, skeletal pathologies, and new data from aDNA on family relationships within the Khvalynsk cemetery, with broader comments on the evolution of “steppe ancestry” that later characterized the Yamnaya populations. Anthony refers to groups defined by similarities in their aDNA as mating networks . A mating network is a population that shared a distinctive cluster of autosomal genetic traits such that individuals from that chronological period and region can be assigned to a space in a principal components analysis (PCA) plot that does not overlap significantly with the spaces occupied by other contemporary mating networks. Mating networks were maintained by the long-term, multi-generational exchange of daughters and/or sons as mates, creating significant gene flow between groups within the network. It cannot be assumed that mating networks were culturally relevant or even known to ancient populations, although borders between mating networks probably were recognized. Khvalynsk is the largest excavated Eneolithic cemetery in the Don-Volga-Ural steppes (201 recorded graves). It has the largest copper assemblage of the late fifth millennium BC in the steppes (373 objects) and the largest assemblage of sacrificed domesticated animals (at least 106 sheep-goat, 29 cattle, and 16 horses). The human skeletons have been sampled extensively for ancient DNA, but genome-wide data from only three individuals has been published to date . Here we discuss relevant results from 32 analyzed individuals. The whole genomes of three additional Eneolithic individuals from graves in the North Caucasus steppes dated 4400–4100 BC at the Progress-2 and Vonyuchka-1 3 cemeteries, broadly contemporary with Khvalynsk, were previously recognized as similar in ancestry and in PCA space to the published three from Khvalynsk . This discovery expanded the range of the Khvalynsk mating network 1000 km to the south, from the Middle Volga steppes to the North Caucasus steppes. Unpublished samples from Volga Eneolithic cemeteries do not significantly alter the relationships or PCA space observed in the initial published samples, but rather form a cline between Khvalynsk and Progress-2. The Khvalynsk/Progress-2 ancestry cline represents a distant genetic ancestor, although not the exclusive or proximate ancestor, for the typical pattern of genetic ancestry exhibited in Yamnaya individuals. Yamnaya individuals cluster near Khvalynsk/Progress-2 in PCA space, but their distributions overlap only marginally . Yamnaya genomes had additional Anatolian Farmer ancestry (typical for agricultural populations in southeastern Europe and the North Caucasus) not present in Khvalynsk/Progress-2 . Khvalynsk is important because of its size, its unique concentration of copper artifacts and domesticated animal sacrifices, and its genetic and ritual connections with the Yamnaya culture. For Khvalynsk I, the ten dates previously published were from the Kiiv, UPI, and Groningen laboratories ( Table 1 ). Three dates on shell beads are clearly subject to a variable freshwater reservoir effect; they are usually ignored in discussions of Khvalynsk chronology. Four new dates from the Pennsylvania State University Accelerator Mass Spectrometry laboratory (PSUAMS) are presented here, making 14 dates from Khvalynsk I. For Khvalynsk II, eight dates were published previously by the Oxford, Groningen, and Arizona laboratories, and two dates by PSUAMS . To these ten previously published dates we now add 24 new PSUAMS dates from Khvalynsk II, making 34 dates from Khvalynsk II ( Table 1 ). Table 1 presents 48 dates for the Khvalynsk cemetery, 34 from Khvalynsk II and 14 from Khvalynsk I, including 28 new dates. In addition, Table 1 presents data on dietary stable isotopes (δ 13 C and δ 15 N) from 30 individuals, not all dated by radiocarbon. We have direct evidence of such skewing from two graves published by Shishlina et al. , one at Khvalynsk I and the other at Khvalynsk II, with radiocarbon dates from domesticated cattle and sheep bones, not subject to reservoir effects . Calibrated, the two samples produced statistically the same age: 4450–4350 BCE. A date of 4450–4355 BCE was obtained on a ring made of sheep bone from grave 147 at Khvalynsk I . The human female buried with this bone ring was dated 4789–4618 BCE , about 300 years older ( Table 1 ). The second date, 4448–4362 BCE , was obtained on a cow bone from grave 10 at Khvalynsk II. The human female in this grave was dated by Oxford to 4730–4530 BC , about 300 years older; but recently has been re-dated to 5210–5017 BC , about 600 years older than the cow bone in the same grave ( Table 1 ). It seems possible that a mistake was made in labeling one of these two human samples, but in any case, we can be confident that both came from Khvalynsk II. 5 The offsets between faunal and human dates from the same grave indicate the presence of an FRE, in which consumption of aquatic resources (fish, shellfish, aquatic birds) leads to the incorporation of ‘old carbon’ into human tissues . Therefore, the faunal dates of 4450–4355 calBCE from Khvalynsk I and 4448–4362 calBCE from Khvalynsk II provide the best estimate currently available for the true age of the two cemeteries. If we compare the midpoint of these dates, ca. 4400 calBCE, to the midpoints of the calibrated age ranges for the humans, the resulting offsets range between 43 yr (essentially no offset given the inbuilt uncertainty in radiocarbon dating) and 860 yr, with a mean FRE offset of 401 ± 288 yr ( Table 1 ). The details of the genetically-determined family trees at Khvalynsk are examined below . Here our narrow purpose is to use family relationships as a chronological check on the FRE connected with radiocarbon dates. In three of the five families at Khvalynsk II (Grey, Purple, and Orange), individuals who were nearly contemporary (1st to 3rd degree relatives) have 14 C dates more than 100 years apart, and the older 14 C dates are associated with lower δ 13 C values. In the extreme case (Orange), a father and son are dated minimally 247 years apart (between the 95% confidence intervals of the two dates), and the older date is linked to a lower δ 13 C value. The dates for related individuals confirm that the 14 C dates at Khvalynsk II do not identify contemporary graves, so they are not reliable relative to each other. However, in some related pairs of individuals the older 14 C date (indicating depleted 14 C) is from the individual with lower δ 13 C values (indicating depleted δ 13 C). This is reflected in a moderate negative correlation between δ 13 C values and calibrated age, accounting for nearly half the variation in the latter ( r 2 = 0.474, p < 0.001, n = 29) . There is a clear outlier (K-I, grave 17) with a predicted offset removed by nearly three standardized residuals from the assumed date of 4400 calBCE. Its removal improves the regression considerably ( r 2 = 0.663, p < 0.001, n = 28; the slope of the regression line remains similar). Extending the slope to the y-intercept suggests that a diet with no 14 C offset would result in a δ 13 C value of ca. −20.3‰ (or ca. −20.0‰ if the outlier is excluded). In contrast, there is no relationship between δ 15 N values and calibrated age ( r 2 < 0.001, p = 0.995, n = 29) . This is unexpected, since aquatic foods are typically significantly 15 N-enriched compared to terrestrial flora and fauna , and therefore a positive relationship with radiocarbon offsets is often observed . Since most of the analyses were made on the petrous bone, the core of which forms in infancy and does not remodel , it is possible that some samples retain a partial nursing signal , which could obscure the relationship between δ 13 C and δ 15 N values. However, the relationship between radiocarbon offsets and both δ 13 C and δ 15 N values is complex . Aquatic systems are often 13 C-depleted, as seems to be the case on the Volga, but they may also be elevated relative to C 3 terrestrial ecosystems . And fish from adjacent watersheds, or even different parts of the same river, can exhibit variable 14 C offsets, leading to different relationships with both stable isotopes . In the Upper Lena river system north of Lake Baikal, Siberia, a program of paired human–fauna dating from the same graves identified a comparable relationship in which 14 C offsets were better predicted by δ 13 C values than by δ 15 N . This differed from Lake Baikal itself, where both isotopes were significant predictors, but δ 15 N accounted for the larger amount of the variability in 14 C offsets. While this is not the case for Y-chromosome haplogroups, there is some indication of such a relationship between the estimated FRE offset and mitochondrial haplogroups. Limiting the comparison to haplogroups with more than five samples, the estimated mean FRE offsets relative to the faunal date of 4400 cal BC differ significantly for mt-haplogroups U2, U4 and U5 (ANOVA, F = 4.268, p = 0.031, n = 20). Bonferroni post-hoc tests show that the significant difference is between U2 and U4 ( p = 0.029), with mean 14 C offsets of 295 ± 256 yr and 624 ± 114 yr, respectively . Note that the same result would obtain if the means of the calibrated dates were used directly, since the same offset is applied to all the individuals. The Volga River appears to have been depleted in both δ 13 C and 14 C in some of its catchments, creating a mild correlation between older ages and lower δ 13 C in the bones of people who regularly ate Volga fish from those parts of the river. The maternal mtDNA haplogroup U2 (represented in two lineages, U2e1b and U2e2a) differed significantly from U4 in its smaller average FRE offsets (with U5 being intermediate), perhaps suggesting that U2 females came from a riverine catchment with less depleted δ 13 C and 14 C. Females with U2 maternal ancestry occur in both Khvalynsk I and II ( Table 6 ). The richest grave at Khvalynsk II contained an older brother (Khvalynsk II:24) and a younger sister (II:25) who carried U2 mtDNA ancestry. The mean faunal date of 4400 calBC probably is the most accurate estimate of the midpoint date for the Khvalynsk cemetery. A relatively short span of time is suggested by the fact that 70% of the individuals analyzed from Khvalynsk II were related to other individuals in ways that could fit within a 5-or-6 generation span, or about 140–170 years . Stable isotopes indicate a diet in which riverine fish played a large role, causing a strong FRE in radiocarbon dates on human bones and teeth. Variation in δ 13 C seems to identify Volga riverine catchments that were depleted in carbon. δ 13 C also correlated with mtDNA haplogroups, suggesting that females at Khvalynsk came from different riverine catchments, while the men’s Y-haplogroups did not display such patterning. Balkan ores probably were the source of the copper imported to Khvalynsk, although most of the imported metal was worked into rings and beads by local artisans. A Balkan source is surprising given the distance between Khvalynsk and the lower Danube valley. But ‘clean’ Balkan ores, specifically copper ores of groups B1-B2 and B3-B6 from Ai Bunar in Bulgaria, match the trace elements in Khvalynsk copper better than Caucasus ores do . Courcier argued that relatively ‘clean’ copper ores also were found in the Caucasus in some Chalcolithic artifacts, as at Menteshtepe . But the Menteshtepe ‘clean’ copper had trace amounts of arsenic measured in the high tenths of one percent (range 0.6–0.9% arsenic). E.N. Chernykh analyzed 41 copper objects from Khvalynsk with methods capable of detecting arsenic, and only ten (12.2%) had any arsenic trace elements; more than 80% had no detectable arsenic. Of the ten exhibiting some arsenic, seven were in the range 0.0034–.1% , like the copper from Cucuteni-Tripolye sites, which ranged 0.007–0.1% . The trace elements in 70% of the tested Khvalynsk copper objects with arsenic fell into the range of the trace elements in Balkan copper rather than Caucasian copper. Three of the ten tested objects had arsenic outside the range of the tested Balkan copper objects, but not by very much: 0.2, 0.3, and 0.42. These three rings all were worn by adult females. Their slightly elevated arsenic might have resulted from a mixture with copper from Caucasian ores, so might indicate trade with the south. According to our interpretation of these sources, the animal bones recovered from Khvalynsk I and II represented the funeral sacrifices of at least 151 mammals. Three mammalian taxa were sacrificed: at least 106 domesticated sheep-goat (70%), 29 domesticated cattle (19%), and 16 horses (11%) whose domesticated status is debated. No obviously wild mammals were included in the funeral sacrifices, although wild species were represented in bone tools, ornaments, and flutes or whistles; and moose ( Alces alces ), red deer, horses, beavers, and fish were important in the diet at regional Eneolithic settlements . At the Eneolithic Ivanovska settlement on the upper Samara River, dated 4360–4220 BCE (68%) , with pottery of the ‘Samara’ type, distinct from Khvalynsk pottery, horses contributed 40.2% of the 6068 animal bones, domesticated cattle 11.4%, domesticated sheep-goat 7%, moose 17%, and beaver 22.5% , not counting fish or birds. Sheep-goat were ten times more frequent in the funeral deposits at Khvalynsk than at the Ivanovska settlement. However, in seasonal (winter?) camps containing Khvalynsk pottery on the lower Volga, as at Kair-Shak VI, dated 4400 BCE, sheep-goat were 60–70% of bones, and wild saiga antelope and onagers were 15% . The sacrifices at Khvalynsk did not include the wild game animals that were prominent in the diet at both settlements. Instead, domesticated mammals were exclusively used to communicate with the spirit world. The inclusion of horses in graves with humans and domesticated animals, and the equally interesting exclusion of obviously wild animals such as moose, suggests that at Khvalynsk the symbolic status of horses had started to move toward the domesticated pole on the wild-domesticated continuum by 4500 BC. Horses were treated like domesticated animals in three ways: they were buried with humans and domesticated animals in graves that excluded obviously wild animals; at S’yezzhe they were arranged in head-and-hoof deposits like the cattle and sheep-goats at Khvalynsk; and horse images were new symbolic artifacts. Decorative bone plaques shaped like horses were found at S’yezzhe and zoomorphic mace-heads that might represent horse heads were found at Khlopkov Bugor, 130 km south of Khvalynsk; and at Lebyazhinka IV, an Eneolithic settlement near Samara . The evidence for a significant change in the human treatment of horses during the fifth millennium BC is symbolic rather than zoological, but it should not be ignored. In addition, recent studies of ancient horse DNA indicate that the horses in the Don-Volga steppes in this era were the genetic ancestors of the modern domesticated horses that first appeared in fully modern form about 2200–2100 BCE in the Don-Volga region. The symbolic changes in the human treatment of horses seen at Khvalynsk, S’yezzhe, and other Volga sites signal the earliest phase in an experimental selection process between humans and horses in this region that produced a gradually improving partnership over the next two millennia, culminating in horses genetically and behaviorally suited for warfare, like modern horses. Perhaps the Khvalynsk horses could be trained to ride in quiet settings such as herding. The male aged 20–25 (II:24) was buried with a female aged 8–9 (II: 25), his sister, with the partial remains of three other individuals at their feet: a male 16–19 years old (a 3 rd -degree relative), and two infants (not analyzed for aDNA). Whole genome analysis revealed that 24 & 25 were brother and sister, although born at least 10 years apart. She wore many decorative belts of riverine Unio shell beads. Copper rings and beads were found only on the male, and beside him was an ‘eared’ mace like the broken one in Grave 108, and 14 bones from a sheep and a goat . He was buried wearing multiple mid-body belts of Unio shell beads, multiple mid-body belts made of 194 beaver incisors (or a shirt covered with beaver incisors?); a boar’s tusk chest pendant; fossil Glycemeris shell pendants (a marine shell also used at Varna for ornaments); a tubular bird bone; and 14 copper ornaments consisting of beads, rings, a spiral ornament or coil of wire , and bands that might have been wrapped around wooden shafts . He also had two lumps of melted copper, perhaps signs of metal craft working, or perhaps copper trade ingots. The male’s Y-chromosome haplogroup was Q1a1b, a Siberian, northern haplogroup; for example, almost all the males at Murzikha, a contemporary cemetery in the forest zone, were Q1a . His and his sister’s MtDNA haplogroup was U2e1b, also found in Mesolithic individuals in Latvia and Siberia, so again a northern lineage. His paternal ancestry contrasted with the paternal ancestry of most of the males at Khvalynsk II. The radiocarbon dates on human bone are skewed too old by FRE, but we also have dates on animal bones or teeth unaffected by reservoir effects. In Table 4 we present five dates from Ekaterinovka Mys from terrestrial animals, including a goat, a sheep, and a beaver. Like beavers at other sites , the beaver incisor dated here has stable isotopes indicating a terrestrial diet (δ13C −20.7, δ15N 6.7). The human from grave 45, buried with the dated goat kid, yielded a FRE-skewed radiocarbon date 800 years older . An organic residue from a potsherd gave a date like three animal bone/tooth dates; one date on a sheep tooth was somewhat older. The average midline for the five dates was 4618 BCE, about 200 years older than the average midline from terrestrial animals at Khvalynsk, 4400 BCE. The sheep and goat dates are among the oldest dates for domesticated sheep and goats in the Volga steppes (excluding anomalous dates on organic residues) . But the male in grave 45 was buried with much more than a domesticated goat. He had three maces. These oval, saucer-shaped depressions in the parietal bone are called “ritual trepanations” by Khokhlov, who counted 10 cases at Khvalynsk II, all adults , and nine cases at Ekaterinovka Mys, including the male in grave 45. The gouged-but-not-hit skulls at Khvalynsk II were noticed also by Murphy in her internal report for the Samara Valley Project, but she did not describe them in print . In the cases at Khvalynsk and Ekaterinovka Mys, the outer layer of skull bone was scraped away, making small ovoid depressions that did not penetrate through the inner layer of bone. Gresky et al. documented a similar but more extreme skull modification ritual in the North Caucasus steppes at Progress-2 and Vonyuchka-1 (also known as Konstantinovskii-1), in which full trepanations were conducted, with penetration through both the inner and outer layers of bone, on people who showed no sign of skull trauma or injury. These features did not exhibit the radiating cracks or crushed edges that accompany a violent blow, but were created for unknown reasons, perhaps (by analogy with actual trepanations) to relieve other sources of head pain. They represent a confusing factor in attempting to evaluate the level of inter-personal violence in the Eneolithic, because they look very much like head trauma and might disguise trauma, but they are not themselves the result of violence. Gresky et al. counted these features on fully 10% of the Eneolithic skulls they examined from graves between the North Caucasus steppes and the lower Don. They document a ritual that was shared across the Volga-Don-Caucasus steppes among Eneolithic people who also exhibited similar genetic ancestries and similar styles of polished stone maces. Of the 26, 18 (70%) were related to at least one other individual and 17 of the 18 (95%) were males . The only female related to another individual (II: 25) was a 9-year-old girl buried with her older brother, the mace chief (II: 24). The other five females at Khvalynsk II that passed screening were unrelated to the males or to each other, within three degrees. Assuming that they were wives of the men, cross-cousin marriage, a type ascribed to Proto-Indo-European speakers by Benveniste , is disproved for the Khvalynsk II population, since no adult female was first cousin to any man. Moreover, no mother-son or father-daughter relationships were detected at Khvalynsk II; all mothers, sisters (with one exception), and daughters were buried elsewhere. We can identify one such female relative: a grandmother or great-aunt of the Yellow-family male in II: 4 was buried 130 km downstream at Khlopkov Bugor (KB7). The absence of female relatives at Khvalynsk II was unlike the older cemetery at Ekaterinovka Mys, where three mother-son relationships were recognized in the cemetery population of about 100. In contrast, Khvalynsk II contained three father-son pairs, and brother’s sons (II:22,27) were buried near their paternal uncles (II:12, 13). Patrilineal family relationships connected 70% of the individuals analyzed at Khvalynsk II, which might have been reserved for a paternally related male sodality, with some unrelated females and immatures. In this hypothetical sodality males died at a younger average age than the males in Khvalynsk I . As noted above, 70–80% of the individuals in Yamnaya kurgans were adult males, as at Khvalynsk II, suggesting that Yamnaya funeral customs could have evolved from the conventions of an Eneolithic male sodality rather than a “culture”. The colors in Figure 18 designate sex-linked haplogroups, not families. The mtDNA haplogroup is the main color and the Y haplogroup group is the corner color. The corner colors for Y-haplogroups in Figure 18 are used again in Table 6 , which shows all sex-linked haplogroups detected at Khvalynsk I and II. Nine mtDNA haplogroups and four Y haplogroups are listed. R1b-L754 was the most common Y-haplogroup and U5a was the most common mitochondrial group. Five individuals from Khvalynsk I had four mitochondrial haplogroups (T2a, U2e, U4a, U5a), all of which were shared at Khvalynsk II; and two paternal Y-haplogroups, one of which (I2a-L699) was unique, while the other (R1b-L754) was shared at Khvalynsk II. In Table 6 , Y-haplogroups are listed with both their alpha-numeric code out to three digits (as in R1b) and their Y-full tree ( https://www.yfull.com/tree/ ) designation (as in R1b-L754). Evolutionary lineages within a Y-haplogroup branch are shown as in R1b-L754 > L389 > V1636, where L754 is basal, L389 is derived from L754, V1636 is derived from L389, and all the SNPs out to V1636 are preserved. The difference between R1b-L754 and R1b-V1636 can be caused by differential preservation of SNPs, so does not indicate different paternal ancestry. Figure 18:A presents a best-fitting family tree for the Yellow family individuals, limited by making II:4 among the oldest Yellow individuals, and by Y-chromosome and mtDNA haplogroups ( Table 6 ) as well as permissible degrees of relationship ( Table 5 ). Six maternal mtDNA haplogroups were present in the Yellow family , and one paternal Y-haplogroup (R1b). The oldest modeled Yellow male, II:4, was a 3 rd -degree relative, probably a paternal great-uncle, of the Yellow brothers II:12&13. If this sequence is correct, then after the brothers II:12&13 were buried, three more Yellow family males (22 and 27, then finally 31) were buried on either side of II:12&13. The Yellow family is modeled as present at Khvalynsk over five generations, although we lack graves from generation two, between II:4 and the II:12&13 brothers. The R-V1636 form of R1b seen in the Yellow family occurred also at Ekaterinovka Mys, where it was abundant among males, at Progress-2, and at the Eneolithic cemetery at Berezhnovka II on the lower Volga, so seems to have been widespread in the Volga-North Caucasus steppe mating network in the final centuries of the 5 th millennium BCE. This is a separate side branch of the R1b that led to the typical Yamnaya form of R1b. Figure 18:B presents a similar analysis for the Grey family, including the mace chief in II:24 and his sister in II:25, in this case representing one possible family tree among several equally plausible trees. The Grey family had Y-haplogroup Q1a2b , a patriline with northern forest-zone and Siberian connections, and three maternal mtDNA haplogroups, including U2e, linked above with an isotopically distinct riverine catchment. None of their haplogroups, maternal or paternal, were shared with the Yellow family. Like the Yellows, the Grey patriline buried at Khvalynsk was divided into two primary avuncular branches. But the Greys also included a male related only through his mother. Grey males II:34 and II:1 were 3 rd -degree relatives, probably cousins whose mothers were sisters (U5a1i), sharing only maternal relatives in a cemetery dominated by paternal relations ( Table 6 ). Also, II:1’s mother had married an R1a (R-M459) husband, making II:1 the only R1a (R-M459) male at Khvalynsk II. Male II:1 was included in the Grey family through his relationship with his mother’s sister’s son, hinting at the continuing importance of maternal marriage links in this paternally dominated society. Additionally, a male unrelated to anyone was included in grave II:26, with Y-haplogroup J1 ( Table 6 ). Some males at Khvalynsk II were associated with but not genetically related to the others. The two dominant patrilines at Khvalynsk II had distinct histories and fates. The Q1a Y-haplogroup is also found at the cemetery of Murzikha II, located 400km north of Khvalynsk in the forests of the Volga-Kama region, and chronologically contemporary with Khvalynsk or slightly later . Most men at Murzikha II were Q1a, but from a different lineage (Q1a1) than the Grey family at Khvalynsk (Q1a2). A migrant from the steppes buried in Hungary at Csongrad-Kettëshalom Bastanya, contemporary with Khvalynsk, also had Y-haplogroup Q1a2, like the Grey family, and autosomal DNA similar to Khvalynsk. This steppe male was part of a diaspora of steppe males into the Danube valley that occurred about 4400–4200 BCE. The Q1a patriline was then mobile and wide-ranging, and at Khvalynsk II was accorded the richest grave at the cemetery. However, most of the men at Khvalynsk II, and all the Yellow family, were R1b of the R-L754 > R-L389 > R-V1636 lineage. A millennium later, when the Yamnaya culture appeared, the Q1a Y-haplogroup would be eliminated from steppe patrilines and a different branch of the R1b family, R-Z2103, would become dominant. The cranio-facial types of Khvalynsk and neighboring Eneolithic sites were studied by A. A. Khokhlov in Samara as part of a quantitative metric analysis of 549 skulls from the Volga-Ural region . Cranio-facial metrics showed that the Khvalynsk population was an admixture of two major components, one (robust, broad-faced) derived from the northern forest zone and the other (more gracile, narrow-faced) from the southern steppes, a conclusion borne out by aDNA data that came to the same conclusion (see below). Khokhlov further divided each major regional type into two sub-types, so two northern sub-types (Lapp-like and Uralic) and two southern (perhaps lower Don and Caucasus steppe). Khokhlov was uncertain about the exact metric source of the southern component at Khvalynsk, which is also true of the geneticists’ uncertainty about the exact source of the southern genetic component (CHG). His metrics also identified the cranio-facial similarities between most of the first-order relatives discussed above: 12 & 13 brothers, noted as very similar by Khokhlov ; 29 & 30 father-son ; 24 & 25 brother-sister ; and 18 & 33 brothers, noted as similar in Khokhlov . Also Khokhlov felt that the Khvalynsk II burial plot was designated for the burial of some special group of males who died young, compared to the males in Khvalynsk I. In many ways, cranio-facial metrics, traditional demographic research, and aDNA findings confirmed each other at Khvalynsk. The Khvalynsk population was genetically admixed between northern and southern ancestry types, in general agreement with Khokhlov’s interpretation based on cranio-facial data. The northern type, Eastern Hunter-Gatherers (EHG), evolved in northern Eurasia; and the southern type, designated Caucasus Hunter-Gatherers (CHG), was defined initially by Mesolithic and Early Neolithic inhabitants of Georgia and western Iran . Both the EHG and CHG labels were first applied to hunter-gatherers, but afterwards were extended to genetically similar individuals regardless of economy. Wang et al. recognized that EHG & CHG ancestry like Khvalynsk was shared by Eneolithic individuals at Progress-2 and Vonyuchka-1 (also known as Konstantinovskii-1) in the North Caucasus steppes. They are dated 4336–4173 calBCE ; and 4233–4047 calBCE . In the aDNA literature, “steppe ancestry” is a phrase used since Allentoft et al. and Haak et al. to refer to the typical Yamnaya pattern of genetic ancestry. The principal components of steppe ancestry were EHG & CHG, each in robust proportions, like Khvalynsk, although often with more CHG than in the Khvalynsk/Progress-2 population, with an added component of Anatolian Farmer (AF) ancestry (5–15%) that was absent from the Khvalynsk/Progress-2 populations . Also, the Khvalynsk/Progress-2 mating network has not yet yielded the Y-haplogroup mutations that were directly ancestral to the typical Yamnaya form of R1b . The R-V1636 form of R1b, found in males at Khvalynsk, Ekaterinovka Mys, Berezhnovka II, and Progress-2, identifies a branch that split from the Yamnaya branch defined by R-P297 > R-M269 > R-L23 > R-Z2103 ( yfull.com ). This entire branch is absent from the sampled Eneolithic males from the steppes, appearing for the first time in Yamnaya males. The evolution of Yamnaya Y-haplogroup ancestry occurred in a still-unsampled Eneolithic population. The Eneolithic populations around the Dnieper Rapids were even more different from Yamnaya. All those sampled were admixtures of EHG (primarily) and Western European Hunter-Gatherers (WHG) similar to the Iron Gates Mesolithic populations . Among 30 published individuals from three Neolithic and Eneolithic cemeteries (Dereivka-1, Vil’nyaka, and Vovnigi) in the Dnieper River valley dated 5200–4400 BC, assigned to the Dnieper-Donets culture, there were a few individuals with minor (<10%) CHG ancestry, but most had none . The Khvalynsk/Progress-2 populations had substantial CHG ancestry but no WHG ancestry, ubiquitous in Dnieper-valley populations. This indicates that the Dnieper-Donets mating network did not extend eastward to the Volga, nor westward to the Criş and early Tripol’ye farmers, whose ancestry was typical of European farmers (AF or EEF) . The Dnieper-Donets people seem to have been an endogamous population focused on the rich resources of the Dnieper Rapids. Their substantial WHG ancestry, nearly absent in Yamnaya individuals, rules them out from being a major source for the Yamnaya. The Sredni Stog culture succeeded and replaced the Dnieper-Donets culture in the strategic Dnieper Rapids and throughout the steppes of Ukraine beginning around 4500–4300 BCE and ending in the late fourth millennium BCE with the appearance of Yamnaya. Unpublished Sredni Stog male genomes exhibit admixture ‘cocktails’ with the same basic elements as Yamnaya (EHG & CHG & AF). The CHG & EHG component was like Khvalynsk/Progress-2, suggesting an eastern origin for at least part of the Sredni Stog population, and the AF component could have come from either the early Maikop or Tripol’ye populations. Sredni Stog introduced into the Ukrainian steppes new funeral customs (the Khvalynsk or ‘Yamnaya’ position), ceramic types (shell-tempered like Khvalynsk), and economies (large numbers of horse bones) that had appeared earlier on the Volga. Sredni Stog has for decades been recognized as an Eneolithic ancestor of Yamnaya influenced by late Khvalynsk, early Maikop, and the Tripol’ye and Varna cultures. But neither R1b Z-2108 nor its immediate ancestral forms are found among sampled Sredni Stog males, most of whom belonged to the R1a or I2a haplogroups, unlike Volga males. The sampled Sredni Stog populations included individuals who autosomally resembled Yamnaya a millennium before the Yamnaya culture appeared. But within that population the Yamnaya Y-haplogroup patriline evolved in a region that has not been sampled. | Study | biomedical | en | 0.999997 |
PMC11699511 | All data on the 2181 identified specimens currently in the IMBE collection were input in a table format. Latitude and longitude coordinates of each capture location were obtained either directly from the label or inferred from the location description. In the second case, coordinates were retrieved using the geoportail.gouv.fr website. Coordinates originally in Lambert93 format and in degrees minutes seconds were transformed to standard GPS format (latitude and longitude in decimal degrees). The locations were then verified using the https://www.geoportail.gouv.fr/ website. Coordinates uncertainty (‘coordinateUncertaintyInMeters’ column) was set to at least 100 m, depending on the level of detail provided on the capture location on the specimen label. All formats follow GBIF Darwin Core specification, to ensure interoperability with other international databases. Six species in the IMBE collection are classified as “Near Threatened” by the IUCN at the European level : Andrena ovatula , Colletes albomaculatus , Lasioglossum prasinum , Lasioglossum pygmaeum , Lasioglossum sexnotatum and Dasypoda argentata . In addition, D. argentata is also generally recognised as a rare species. The identification of these species is crucial for conservation efforts, as it guides habitat management and protection initiatives. Understanding the status of near-threatened species helps preserve biodiversity and maintain essential ecosystem services provided by pollinators, while raising public awareness and support for bee conservation initiatives. Finally, a total of 68 species recorded in the IMBE collection are currently classified as 'Data Deficient'. This status underscores significant gaps in our understanding of these species' biology, distribution and conservation needs. It highlights the critical importance of maintaining and enhancing monitoring efforts to gather the data necessary for informed conservation decisions. Ensuring the availability of updated and comprehensive information on these species is essential to address potential threats and support their long-term survival. | Study | biomedical | en | 0.999996 |
PMC11699512 | The Diptera family Chironomidae (also known as non-biting midges) comprise the most diverse and frequently the most abundant insect group found in freshwaters. Although larvae of some species can be semi-aquatic or terrestrial, the majority are strictly aquatic . The immature stages of chironomids can inhabit a wide range of water types, from thin water film on glaciers to plant-held waters, streams, rivers, lakes, ponds, reservoirs, brackish waters and shoreline habitats . Besides their widespread distribution, the species richness of Chironomidae is usually amongst the highest of aquatic insect families, occasionally exceeding 100 species per site in many aquatic habitats . In case of larvae and pupae, all individuals were identified from samples containing less than 300 individuals, while subsamples of ca. 300 individuals were taken from larger samples. We used multi-level identification methods that included: separating specimens based on macroscopic characteristics; conducting detailed investigations without preparation and using light microscope; and mounting specimens on microscope slides for examination at higher magnification. Exuviae were mounted on microscope slides in every instance. For morphological identification of larvae and pupae/exuviae, multiple keys were used. For further details, see Suppl. material 1 . Over 140,000 chironomid individuals were collected from 456 samples. A total of 207 taxa identified to at least genus level were documented, belonging to five subfamilies (32 Tanypodinae , 8 Diamesinae , 3 Prodiamesinae , 103 Orthocladiinae , 61 Chironominae ) and 74 genera, providing 5,481 occurrence records. A total of 170 taxa were identified to species level (82% of all taxa), accounting for 3,613 occurrence data (66% of all records). We recorded 117 taxa (93 species) from Croatia, 127 taxa (102 species) from Hungary and 128 taxa (104 species) from Czechia. Across the three countries, Micropsectra sp. (representing different species) provided the highest number of occurrence records amongst taxa, while Parametriocnemus stylatus accounted for the highest number of species-level occurrences, ranking first in both Croatia and Hungary. However, in Czechia, Polypedilum convictum was the most frequent species. DNA was extracted from a total of 31 specimens (14 from Hungary, 10 from Croatia and 7 from Czechia) that were uploaded to BOLD. Sequences had a median length of 658 bp, ranging from 569 to 659 bp, meaning all the sequences were high-quality barcodes (≥ 500 bp) being assigned to a Barcode Index Number . The 31 COI-sequences were assigned to 23 BINs, including eight unique BINs that are new to BOLD. Half of the specimens were identified to species level; however, in the case of 16 specimens, there were no matches passing the 1.6% threshold, preventing conclusive species-level identification. Clinotanypus – In most parts of Europe, C. nervosus can be found , but from Russia and Romania, larvae identified as C. pinguis have also been recorded . The difference between the two species is that C. nervosus has a 5-tooth ligula, while C. pinguis has ligula with 6–7 teeth . However, C. nervosus is regarded as the only western Palaearctic representative of the genus, while C. pinguis is widely distributed in North America and, to our best knowledge, has never been found in the Palaearctic as adult. In our study, Clinotanypus larvae were found in Hungary and all of them had 6-toothed ligula. DNA barcoding revealed that this morphotype belongs to C. nervosus , proving that two morphotypes of this species exist and the new morphotype can be dominant in some populations. Furthermore, our results raise the possibility that the occurrence of C. pinguis in Europe might be based on misidentification. Zavrelimyia – According to the broadened generic conception of Zavrelimyia , the genus contains more former genera as subgenera . In our study subgenera Zavrelimyia s. str. and Zavrelimyia ( Paramerina ) occurred, with larvae identifiable only to subgenera by morphological characters. Some pupae/exuviae were identified as Z. ( Z. ) barbatipes and as Z. ( P. ) cingulata . The pupal form Paramerina spec-Griechenland was found in Croatia and sequences from DNA barcoding matched (100% and 98.48% similarity) with one sequence under the name Zavrelimyia divisa in BOLD Systems. However, these sequences differed more than 9% from other sequences of Z. divisa , which questions the correctness of species identification. The clear morphological differences between Z. ( P. ) divisa and P. spec-Griechenland and the inconsistencies in molecular results suggest that the pupal form represents a different species. Diamesa – There are many difficulties in separating Diamesa species as larvae (e.g. Janecek , Rossaro and Lencioni ) and as pupae by morphological characters. In our study, identification was possible only to the species group level in many cases ( D. cinerella -Gr., D. zernyi -Gr.). Amongst D. insignipes / cinerella / tonsa , only pupae with very typical characters (see Langton and Visser ) were identified as D. insignipes or as D. tonsa . In the case of larvae, D. tonsa was relatively easily recognisable, based on the colouration of the head , but D. insignipes was only accepted when DNA barcoding confirmed the species identity (one specimen from Croatia and two specimens from Czechia). Epoicocladius – The Palaearctic E. ephemerae and Nearctic E. flavens are currently recognised as separate species (e.g. Ashe and O’Connor , Andersen et al. ); however, some authors suggest they are synonyms (e.g. Evenhuis and Pape ). Accordingly, we used the name E. ephemerae for our records. Larvae collected in Butižnica River catchment were somewhat different from those in other populations, as the setae on body segments were remarkably less numerous and paler. DNA barcoding resulted in no match with any Epoicocladius sequences, raising the possibility that these larvae represent a different species within the genus. Eukiefferiella – In the case of E. devonica , larvae were identified to species only as pupae and to species group as larvae. Eukiefferiella minor and E. fittkaui were separated neither as larvae nor as pupae due to very little morphological differences . A strange larval morphotype ( Eukiefferiella sp. BUT) was found in the Butižnica River catchment: its morphology fits the generic description of Eukiefferiella , but the thorax is covered with dense, relatively long (approx. half the width of the thoracic segment) setae , which is unique in the genus. In the Velička River catchment, a peculiar pupa ( Eukiefferiella sp. VEL) was collected. In the key of Langton and Visser , the specimen could be identified as either E. claripennis or E. brevicalcar due to the swollen base of the thoracic horn, but the length of its apical filament separates this morphotype from both species (longer than in E. claripennis , but shorter than in E. brevicalcar ). Although the pattern of the hooks on abdominal segments resembles E. claripennis , it is not clear whether it is a new pupal form or a non-typical representative of one or the other species. Orthocladius – Larvae and pupae belonging to the subgenus Orthocladius s. str. were identified to species level only occasionally due to unclear taxonomy and little morphological differences between the species . Nearly all larvae and pupae belonging to the subgenus Euorthocladius were separated to species using multiple keys . One larva was morphologically different from the identifiable ones and either the DNA barcoding resulted in no match in BOLD Systems. Accordingly, this larva represents a new morphotype and a species that, at least as larvae, has not yet been described. In the subgenus Symposiocladius , larvae were identified as O. ( S. ) lignicola and as O. ( S. ) holsatus based on the L4 hair tuft and morphology of the mentum . The occurrence of O. ( S. ) lignicola in Bükkösdi-víz catchment was also confirmed by pupae . However, both larval types might represent other species as well, for example, the larva of O. ( S. ) ruffoi is not known . Larvae from the Butižnica River catchment closely resembled O. ( S. ) holsatus , but the L4 hair tuft was much shorter than it is depicted in Cuppen and Tempelman ; accordingly, they only were identified to subgenus level. Parametriocnemus – Larvae from all catchments were identified as P. stylatus , based on their morphology (e.g. Janecek ) and co-occurrence of conspecific pupae . However, DNA barcoding of a larva from the Bükkösdi-víz catchment resulted in no match with P. stylatus , suggesting that it is rather a species complex (see Orendt and Bendt ). Some pupae collected in the Velička River catchment proved to be Parametriocnemus Pe1; DNA barcoding also resulted in no match in BOLD systems in this case. Genus unknown – The morphology of some larvae from the Velička River catchment clearly matches with the taxon Orthocladiinae sp. “Berka vor dem Hainich, Thüringen” in Orendt and Bendt . Our results proved that the distribution of this taxon is not limited to Germany. Orthocladiinae larvae and a pharate larvae with unique morphological characteristics were found in Bükkösdi-víz catchment ( Orthocladiinae Gen. sp. III.). Neither larval nor pupal characteristics resemble any species or genera included in the keys used in our work. Some larval characters suggest that this morphotype belongs to Parakiefferiella , but the thoracic horn is completely different from that characteristic for the genus. DNA barcoding also resulted in no match in BOLD Systems. Chironomus – Based on DNA barcoding, a larva from the Bükkösdi-víz catchment identified earlier as C. entis by morphological characters, proved to be C. plumosus . As sequences can also be found for C. entis in the BOLD Systems, we accept that this result is correct. In our larva, the upper surface of the antennal pedestal was completely light brown, which is a distinctive character for C. entis according to Vallenduuk . Moreover, the dimensions of the parts of the head fell in the range of this species, but it must be noted that these characters can vary and overlap between the species. According to Orendt and Spies , C. plumosus can be recognised by a distinct dark streak between the antennal pedestal and the upper eyespot. Our specimen proved that this pigmentation could spread further on the upper surface of the antennal pedestal. Accordingly, all characters used to separate the two species may overlap and should be regarded with caution. DNA barcoding performed on a larva identified as C. bernensis resulted in the closest match with a sequence under the name C. annularius . However, in this BIN, there are also specimens under the name C. bernensis MOTU7, which was analysed by Pfenninger et al. , with the result that the nominal species C. bernensis might be a cryptic species complex. However, the taxonomy of C. annularius is also not clear with more biological species mentioned under this name by various authors . Nevertheless, the two larval morphotypes that represent these names can clearly be separated by the presence of lateral gills on abdominal segment VII in C. annularius , while these gills lack in C. bernensis (e.g. Orendt and Spies , Vallenduuk ). As the larvae we found had no lateral gills on segment VII, accordingly we listed them as C. bernensis . Phaenopsectra – Larvae from the Bükkösdi-víz and Velička River catchments were identified as P. flavipes , which was confirmed by DNA barcoding of an associated pupa. Another larval form was collected in the Butižnica River catchment, with the mentum and mandible being identical with those depicted as Phaenopsectra sp. in Andersen et al. . Micropsectra – We had difficulties with the identification of a larval Tanytarsini morphotype (cf. Micropsectra sp.) from Bükkösdi-víz catchment. The bifid premandible suggested Micropsectra , but the remarkably small body size, the lack of spur on antennal pedestal and the small number of claws on posterior parapods rather resemble Tanytarsus . The separation of the two genera is very difficult and probably the best character is the shape of the premandible . Based on the bifid premandible, our larvae most probably belong to Micropsectra . In addition, co-occurring small Micropsectra pupae were also collected. These pupae resemble those of M. notescens , but are much smaller and less pigmented. The similar size of the larvae and pupae raises the possibility that they might represent the same species. The pupal morphotype Micropsectra sp. BUK1 was collected at one site in the Bükkösdi-víz catchment. This morphotype is most similar to M. auvergnensis due to the shagreen on the pleura of segments II-V, but, in the case of the latter species, pleura of segments II-VII have shagreen. Furthermore, male hypopygium was observable in one pupa and it clearly differed from that of M. auvergnensis . Further studies are in progress to clarify the taxonomic status of this species. Using DNA barcoding is an effective method for species identification; however, its success largely depends on the availability of appropriate reference databases . With the increasing effort in barcoding campaigns, more data are becoming available for Chironomidae . However, reference databases of the Central European Chironomidae are incomplete, as the number of public barcodes from our study area demonstrates: from Czechia, 21 COI-sequences are available, while the number of public barcodes is five from both Hungary and Croatia. In addition, we found some inconsistencies in molecular results (e.g. Zavrelimyia species, see Notes on selected taxa) and these did not fit with the results based on morphological investigation in some cases, highlighting the conspicuous gaps in the databases. It is widely recognised that combining traditional taxonomy with molecular tools can contribute to the better documentation of a region’s Chironomidae fauna and, accordingly, DNA barcoding has contributed to our taxa list in a few cases. | Other | biomedical | en | 0.999994 |
PMC11699514 | The class Dothideomycetes is megadiverse and the largest class of fungi in the phylum Ascomycota . Currently, the class is divided into 54 orders and 226 families and contains about 20,000 described species . The best-known and studied orders are Capnodiales s. lato, Dothideales , Botryosphaeriales , and Pleosporales . Other orders of Dothideomycetes are less known, primarily because their members are less common in the environment, although some of them are important for human life and the economy . The high species diversity observed in Dothideomycetes is reflected by the wide geographical range and diversity of lifestyles of its members. They are known from all areas of the world and habitats, even capable of colonizing extreme habitats such as cold deserts , deep-sea sediments, including methane sediments , saline waters , acidic environments , and resin exudates . Species of Dothideomycetes are plant pathogens, saprobes, rock-inhabiting fungi, lichens, endophytes, and epiphytes. Hongsanan et al. defined fungal epiphytes as species occurring on the living plant surfaces, especially leaves, which belong to the following orders of Dothideomycetes : Asterinales , Capnodiales , Microthyriales , Zeloasperisporiales , and Meliolales in Sordariomycetes . However, epiphytes are also known in other, less obvious lineages . The special kind of epiphytes are sooty moulds, which live on leaves/needles covered with exudates of phloem-feeding insects, especially honeydew secreted by aphids . Recently, we isolated an enigmatic fungus from a sooty mould colony on Symphoricarpos albus in southern Poland, which showed affinities to members of the poorly known order Neodactylariales . In this study, a novel species accommodated in a new genus is described for this fungus. The phylogenetic placement of this new genus and its most closely related genera are analysed, and emended descriptions of the order Neodactylariales and family Neodactylariaceae are provided. The strain was isolated from the sooty mould community on Symphoricarpos albus leaves planted in municipal greenery in southern Poland . Macroscopic features of cultures were observed and photographed using 4-week-old colonies grown on MEA and PDA at 6 °C, 15 °C, and 25 °C, as well as 15-week-old colonies grown on MEA and PDA at 15 °C. Description of culture characteristics is based on 4-week-old colonies. The morphology of colonies observed after 15 weeks is briefly mentioned in the subsection “notes”. Growth at different temperatures was assessed by measuring the colony diameter after 4 weeks. Microscopic features were analysed using colonies older than 8 weeks grown on MEA at 15 °C. Hyphae and conidia taken from the edge of the colony were mounted in lactic acid (80%) on microscope slides and examined under a Nikon Eclipse 80i light microscope. Microscopic structures were measured and photographed using NIS‐Elements BR 3.0 imaging software. The holotype is a dried specimen obtained from culture and deposited in the fungal collection of the W. Szafer Institute of Botany, Polish Academy of Sciences, Kraków ( KRAM F ). Culture is preserved in the culture collection of the Westerdijk Fungal Biodiversity Institute ( CBS ) and in the W. Szafer Institute of Botany, Polish Academy of Sciences, Kraków. The morphological characteristics of Beaucarneamyces and Pseudoarthrographis used for the emendation of Neodactylariales and Neodactylariaceae are taken from the literature . After growth of cultures for about one month, genomic DNA was extracted from a portion of mycelium using the DNeasy® Plant Mini Kit (Qiagen, Germany). To conduct molecular studies, four genetic loci were amplified, namely ITS1-5.8S-ITS2 rDNA (= ITS), 28S D1–D2 rDNA (= LSU), a small subunit rDNA (= SSU ), and a protein-coding gene—a partial DNA-directed RNA polymerase II second largest subunit ( rpb2 ). To amplify the loci mentioned above, four different primer pairs were used: ITS1 and LR5 for a fragment containing ITS and LSU , NS1 and NS4 for SSU , and fRPB2-5F and fRPB2-7cR for rpb2 . Polymerase chain reactions ( PCR ) for all loci were performed in a reaction mixture prepared as described in Piątek et al. . Amplification conditions for PCR reactions of the fragment containing ITS and LSU were performed as described by Piątek et al. ; in turn, amplification conditions for SSU and rpb2 were described by Piątek et al. . The PCR products were checked on 1% agarose gels and enzymatically purified using Exo-BAP Mix (EURx, Gdańsk, Poland). DNA sequencing was carried out in both directions by Macrogen Europe B.V. (Amsterdam, the Netherlands). ITS was sequenced using primers ITS1 and ITS4; LSU was sequenced using primers LSU1Fd and LR5; and SSU and rpb2 were sequenced with the same pairs of primers that were used for amplification. The affinities of obtained ITS, LSU, SSU , and rpb2 sequences of the isolated fungus were performed in the NCBI’s GenBank nucleotide database using the megablast search tool . To resolve its phylogenetic position, the multilocus dataset containing LSU, ITS, SSU , and rpb2 sequences of representatives of Dothideomycetes used by Maharachchikumbura et al. was obtained. The dataset was, among others, augmented by sequences of representatives of the orders Neodactylariales ( Beaucarneamyces , Neodactylaria , Pseudoarthrographis ) and Oncopodiellales ( Diplocladiella , Oncopodiella ) (Suppl. material 1 ). Sequences were separately aligned for each single-gene dataset using the MAFFT algorithm in Geneious 11.1.5 and concatenated. Phylogenetic relationships were inferred using the concatenated LSU-ITS- SSU - rpb2 alignment by the maximum likelihood ( ML ) analysis using RAxML-NG v. 1.1.1 , with 1000 bootstrap replicates. The best-fit substitution models were selected with ModelTest-NG v. 0.2.0 using the Bayesian Information Criterion ( BIC ) . The final phylogenetic tree was visualized using FigTree v1.4.3. The alignment was deposited at figshare.com . The concatenated multilocus dataset (LSU, ITS, SSU , and rpb2 ) included sequences of 154 representatives of most of the orders of the class Dothideomycetes , including all representatives of Neodactylariales , and a member of the class Arthoniomycetes ( Schismatomma decolorans ) used as an outgroup. The concatenated alignment contained 4483 characters . The best-fit substitution models selected for single-gene alignments were as follows: GTR+I+G4 for both ITS and LSU, TrNef+I+G4 for SSU , and TPM3uf+I+G4, TPM2uf+I+G4, and TIM2+I+G4 for rpb2 (three codons). The phylogenetic tree resulting from maximum likelihood analysis is shown in Fig. 1 . Representatives of Dothideomycetes formed lineages that correspond to the orders that were well supported, but relationships between orders were mostly not resolved. The strain of a new genus and species, Szaferohypha enigmatica , clustered within the lineage assigned to the order Neodactylariales , but its relationship to the remaining genera of this order ( Beaucarneamyces , Neodactylaria , and Pseudoarthrographis ) was not resolved. The clustering of genera assigned to Neodactylariales was well supported (MLB = 85%), and this order formed a strongly supported (MLB = 98%) sister group to the lineage assigned to the order Oncopodiellales that was fully supported (MLB = 100%). Asexual morph from human-associated organs or saprobic on plant debris or epiphytic on living leaves . Conidiophores acroauxic, macronematous or micronematous , mononematous, branched or unbranched, or reduced to conidiogenous cells . Conidiogenous cells mono- and polyblastic, sympodially extended or not . Conidia solitary or in branched or unbranched chains , hyaline or pale pigmented, smooth, verrucous, or echinulate. Chlamydospores sometimes present. Sexual morph not observed. Mycelium superficial or immersed, composed of branched, septate, hyaline to subhyaline hyphae. Conidiophores macronematous or micronematous , mononematous, straight or flexuous, septate, unbranched, or reduced to conidiogenous cells . Conidiogenous cells terminal or intercalary, monoblastic or polyblastic, sympodial or not , with short-cylindrical denticles or without denticles . Conidial secession schizolytic. Conidia solitary or in branched or unbranched chains , smooth or finely echinulate. Chlamydospores sometimes present. Sexual morph not observed. Colonies erumpent, spreading, umbonate, grayish-brown, with a velvety surface caused by abundant aerial mycelium, margin undulate. Mycelium composed of branched, septate, hyaline, subhyaline, pale brown, or brown, smooth or verrucose, usually thick-walled hyphae. Conidiophores micronematous, reduced to conidiogenous cells, rarely macronematous. Conidiogenous cells terminal, rarely lateral, monoblastic, hyaline, subhyaline, pale brown, or brown. Conidia globose, subglobose, rarely broadly ellipsoid, hyaline, subhyaline, or brown, aseptate, rarely with 1–2 septa or muriformly septate, smooth or finely verrucose, thick-walled, sometimes produced intercalary. Mycelium composed of branched, septate, hyaline, subhyaline, pale brown, or brown, smooth or verrucose, usually thick-walled hyphae, 2–4 µm, sometimes with oil guttules; wall ca. 0.5 µm thick. Conidiophores micronematous, reduced to conidiogenous cells, rarely macronematous. Conidiogeneous cells terminal, rarely lateral, monoblastic, hyaline, subhyaline, pale brown, or brown, 3.5–13.5 × 2.5–4.5 µm. Conidia globose, subglobose, rarely broadly ellipsoid, hyaline, subhyaline, or brown, aseptate, rarely with 1–2 septa or muriformly septate, smooth or finely verrucose, thick-walled, 6.5–15 × 6–13.5 µm, sometimes germinating into hypha or produced intercalary, wall ca. 0.5–1.5 µm thick. Based on a megablast search of NCBI’s GenBank nucleotide database, the closest hits of the named species using the ITS sequence are Pseudoarthrographis phlogis (strain CPC 32759, GenBank MH327796 ; identities = 370/414 (89%), 11 gaps (2%)), Oncopodiella trigonella (strain FMR 10788, GenBank KY853455 ; identities = 356/408 (87%), 11 gaps (2%)), and Xylographa parallela (voucher Z. Palice 22099 (PRM), GenBank MK778618 ; identities = 344/396 (87%), seven gaps (1%)). The closest hits of the named species using the LSU sequence are Beaucarneamyces stellenboschensis (strain CPC 45687, GenBank PP791445 ; identities = 826/873 (95%), four gaps (0%)), Pseudoarthrographis phlogis (strain CPC 32759, GenBank NG_064540 ; identities = 835/883 (95%), no gaps), and Umbilicaria hypococcinea (strain A12, GenBank JQ739991 ; identities = 876/927 (94%), eight gaps (1%)). The closest hits using the SSU sequence are Cophinforma atrovirens (strain CSM_72, GenBank MF436134 ; identities = 857/870 (99%), no gaps), Gloeopycnis protuberans (specimen DAOM 745762, GenBank NG_067652 ; identities = 857/870 (99%), no gaps), and Botryosphaeria mamane (strain CBS 117444, GenBank KF531821 ; identities = 857/870 (99%), no gaps). The closest hits using the rpb2 sequence are Shiraia bambusicola (voucher SICAUCC 23-0005, GenBank OR424351 ; identities = 237/283 (84%), six gaps (2%)), Lindra obtusa (strain AFTOL-ID 5012, GenBank FJ238382 ; identities = 207/252 (82%), three gaps (1%)), and Natonodosa speciosa (strain CLM-RV86, GenBank MH745150 ; identities = 217/266 (82%), six gaps (2%)). In the case of SSU and rpb2 , the sequences of these two regions are not available for representatives of the most closely related genera, namely Beaucarneamyces and Pseudoarthrographis . In this study, morphological and phylogenetic analyses were conducted to identify the enigmatic fungal strain isolated from sooty mould biofilm on the surface of Symphoricarpos albus leaves. In consequence, the isolated fungus is described as a new genus and species, Szaferohypha enigmatica , and assigned to the order Neodactylariales . Three genera were previously included in this order, namely Beaucarneamyces , Neodactylaria , and Pseudoarthrographis , and Szaferohypha is morphologically different from all of them. Beaucarneamyces , with type species B. stellenboschensis , was described from dead leaves of Beaucarnea stricta in South Africa and is characterized by hyaline conidiophores reduced to conidiogenous cells that are polyblastic with several apical denticles. Conidia in this genus are solitary, fusoid-ellipsoid, hooked, 3-septate, hyaline but with pale brown central cells . Neodactylaria is typified with N. obpyriformis described from human bronchoalveolar lavage in the USA . The second species in this genus is N. simaoensis , described from submerged unidentified dicotyledonous leaves in China . Neodactylaria is characterized by having straight or flexuous, brown conidiophores with polyblastic, sympodial, and denticle-like conidiogenous loci, which form solitary, obpyriform or rostrate, 0-1-septate, pale brown conidia . Pseudoarthrographis , with a type species Pseudoarthrographis phlogis described from Phlox subulata in New Zealand, has smooth hyphae and smooth, 0–1-septate cylindrical arthroconidia with truncate ends produced in branched or unbranched chains. In culture it also forms smooth, globose chlamydospores occurring in chains . The order Neodactylariales , with one family Neodactylariaceae , was originally described to accommodate only one genus, Neodactylaria , containing two species in China and the USA . Recently, two monotypic genera, Beaucarneamyces and Pseudoarthrographis , containing species known in South Africa and New Zealand, respectively, were assigned to this order by Crous et al. . Here, yet another monotypic genus, Szaferohypha , known from Poland, is added to Neodactylariales . Thus, currently this order includes fungi living in opposite corners of the world. The order Neodactylariales and family Neodactylariaceae were originally circumscribed based on characters known in the genus Neodactylaria . They therefore need emendation by the features of the genera Beaucarneamyces , Pseudoarthrographis , and Szaferohypha. In our phylogenetic analysis, Neodactylariales is related to the order Oncopodiellales that is going to be described . Two genera, Diplocladiella and Oncopodiella , assigned to two families, Diplocladiellaceae and Oncopodiellaceae , are included in this order . The relationships of Neodactylariales and Oncopodiellales are revealed here for the first time. Sooty mould communities are an underexplored source of rare and undescribed species, of which many are probably extremophilic since they live in extreme environments . They were previously studied using classical morphological methods that are, however, inadequate to reveal their true diversity, and only recently started to study in modern ways using morphological and molecular methods, though mainly in tropics and much more rarely in temperate regions . In the course of our ongoing studies of sooty mould communities covering the surface of leaves/needles of ornamental plants in Poland, i.e., in the temperate climate, about 190 species were isolated from these communities (M. Piątek et al. unpublished data), being either as constant or accidental colonizers, of which many are undescribed. These include four new Rachicladosporium species , a new Lapidomyces species , a new Xenoramularia species , a new Pseudopezicula species , and many more that are waiting for description. | Other | biomedical | en | 0.999998 |
PMC11699517 | The urbanisation process represents one of the main threats to biodiversity , causing fragmentation, modification and loss of habitats and consequently resulting in richness and abundance loss . However, studies quantifying the effects of urbanisation on biodiversity seem to provide conflicting results. Recent works have shown that the urban environment, particularly urban green areas, can even have positive effects on the conservation of biodiversity compared to other ecosystems strongly impacted by anthropogenic activities, such as areas subject to intensive agriculture . Not all species are adversely affected by urbanisation and urban areas can provide unique opportunities for conservation . For example, the positive effects on pollinator biodiversity may be the result of biotic factors, such as a greater diversity of plant species in urbanised areas than in rural areas due to the presence of exotic and ornamental plants that make trophic resources available for a longer period during the year and increase the diversity of habitats and microclimates . Another possible factor can be the degradation in the quality of habitats in rural areas . Abiotic factors can also have important effects. The 'heat island effect' produces higher air temperatures and more stable climatic conditions in cities than in surrounding areas , affecting insect phenology and survival . For this reason, urban green spaces can represent important locations for the conservation of several animal groups of vertebrates and invertebrates , particularly for insect pollinators such as bees. Despite the considerable efforts made in recent years through studies and investments, there is still a lack of knowledge regarding insect urban diversity and ecology. In particular, few studies have focused on pollinators in cities. Italy is a biodiversity hotspot for wild bees . Focusing on the most important and complete papers published on Apoidea biodiversity in Italy in recent decades, 1173 species were reported in Comba . Lhomme et al. reduced the number to 1028 species using data provided by Ascher and Pickering and showing that Italy has the eleventh bee richness in the Mediterranean Basin. Wood et al. , while revising Italian Andrena , subsequently added nine more species to those previously listed, confirming that Italy hosts one of the largest Andrena faunas in the world. This addition resulted from the revision of eight species and the description of a new one. Cornalba et al. recently provided eight new faunistic records for the Italian fauna, taking the number of species to 1045. The diversity of Hymenoptera Apoidea inhabiting Rome was previously discussed in Comba and Comba , in which 450 species were reported for the Latium Region and, in Comba and Comba , 378 wild bee species were recorded within the Grande Raccordo Anulare (GRA; see Materials and Methods). Despite the large number of species listed in these works, unpublished data and specimens housed in the Maurizio Mei collection (Museum of the Institute of Zoology, Sapienza University, Rome, Italy) suggest that the number of species present in the urban area of Rome might exceed 400. One of the most recent papers on this topic is that of Geppert et al. , who studied the functional traits and ecology of wild bees in Rome and provided a list of bee species. The objective of this study is to provide precise and original georeferenced faunistic data on the Apoidea Anthophila inhabiting the urban natural reserves of Rome, making them immediately available in the standard Darwin-core format to facilitate their use in future scientific work focused on the biodiversity of pollinating insects in urban areas. In addition, these data constitute an integration and update of the previously available information, thus setting the new faunistic reference to be used as a starting base. Producing this type of data for a city such as Rome, which is amongst the largest and most urbanised in Europe, is an important starting point for understanding how urbanisation defines and might affect animal biodiversity. Providing precise information on Apoidea distributions and insects in general in large urban green areas is essential for implementing plans to protect and enhance the positive effects on the diversity and abundance of pollinator communities. This type of data and information can also be used to provide guidelines for urban green management actions aimed at encouraging the presence of pollinating insects in the urban environment. Sampling was carried out along 250-metre-long fixed transects (the transects remained the same in length and position throughout the study) and was repeated once a month, from April to September, in all eight nature reserves selected for the study. The number of transects performed in each site depended on the size of each nature reserve. In total, we performed 15 fixed transects, one each 250 hectares of extension, for a maximum of three transects per nature reserve. The transect locations were chosen where the vegetation was representative of the area and at least 100 metres apart. In three out of the eight sites, this ratio was not mathematically respected due to some specific characteristics of the area and limits to accessibility. The total extension of TDM is 774 hectares, but we performed two linear transects, due to the ban on access to a large archaeological private area. In VAN, we performed two transects because the park has elongated and narrow shapes that extend to the sides of a part of the Aniene River. It increased greatly the ratio between the perimeter and the surface of the area and meant that a large part of the park is occupied by the watercourse. In addition, a relevant percentage of the remaining walkable surface is made up of roads to move inside the park reducing the areas where a transect could be performed. APP is a huge urban green area. It extends from the centre of the city to the outskirts and presents, within it, a mosaic of inhabited, grazing, recreational and archaeological areas, but also integral reserves, accessible to citizens. Here, we carried out the transects in three areas, homogeneously distributed along the park. The investigations were conducted under sunny, low-wind weather conditions between 10:00 am and 3:00 pm. Wild bees were collected via an entomological net within 2 m on both sides of each sample line . Each transect was carried out by walking at a constant speed for 45 minutes. All the observations were conducted by the same person (LF) walking along the sample lines. All the samples were collected while foraging on flowers along the transects. All collected samples were stored in Falcon tubes, exposed to ethyl acetate and dry-pinned. Morphological identification was performed using reference literature on the group and taxonomic keys such as those provided by Michez et al. for genera identification, Amiet et al. for Apidae , Smith for Nomada , Amiet et al. and Pauly for Halictidae , Amiet et al. for Megachilidae , Dathe for Hylaeus , Amiet et al. for Colletes , Wood for Andrena and taking as reference the collection from the Museum of Zoology of La Sapienza University. The identifications have been validated by an expert taxonomist author (MM). The final list of species collected during the study is reported below. The checklist respects the alphabetical order and the latitude/longitude given for each specimen are those of the mid-point of the transect where it was collected. The taxonomic arrangement followed that of Ghisbain et al. , Wood and Orr et al. . The survey allowed the collection of 2139 specimens (spc.) of Apoidea belonging to 208 species (spp.) in 36 genera and six different families. Amongst the areas we investigated, APP presented both the greatest number of specimens and the greatest number of species collected (448 spc., 110 spp.), followed by INS (329 spc., 93 spp.), VDC (278 spc., 84 spp.), VAN (313 spc., 83 spp.), TDM (269 spc., 73 spp.), LAU (145 spc., 68 spp.), MMA (186 spc., 58 spp.) and ACQ (171 spc., 53 spp.). LAU presented the smallest number of specimens, whereas ACQ presented the lowest number of species (Table 1 ). The most abundant species were Halictus scabiosae (148 spc.), Lasioglossum malachurum (110 spc.) and Bombus pascuorum (63 spc.). The species we recorded in the nature reserves selected for the study represent approximately 74% of the known bee species for the area within the GRA of Rome since 1997, 46% of the Latium Region and approximately 20% of Italy. In April, the first month of sampling, we collected the greatest number of bees compared to the other months, while we observed a clear decrease in abundance in August and September compared with the previous months. We found the highest number of species in May (114 spp.), while we found the lowest number of species in September (22 spp.) . Halictidae is the most common family, with 788 spc. representing 36% of the samples belonging to 40 species and three genera (19% of the total species) . This bee family is the most abundant in six out of the eight nature reserves, except for INS and LAU . Apidae counts 505 spc., representing 24% of the samples and belonging to 48 species and 11 genera (23% of the total species) . Apidae is the most abundant family in INS and LAU . Andrenidae represents 17% of the samples , belonging to 48 species and two genera (23% of the total species) . Megachilidae represents 15% of the samples , belonging to 52 species and 14 genera (25% of the total species) . Colletidae counts 148 spc., representing 7% of the samples and belonging to 19 species and two genera (10% of the total species) . Mellittidae is represented just by Dasypoda hirtipes . It represents 1% of the total samples . We did not collect Mellittidae from APP, MMA or VDC, whereas, in ACQ, it represented 11% of all the samples . We found eight species included in the European Wild Bees Red List. One species, Trachusa interrupta , is classified in the category Endangered (EN) and seven species are in the category Near Threatened (NT) (Table 2 ). Twenty-four species amongst those collected are new records for the urban area of Rome (Table 3 ). Although the entire experiment and sampling took place over a single year, carried out in eight green areas, using only one collection method (entomological net along fixed transects) and performed by only one operator (LF), we collected 208 species, representing 74% of the wild bee species reported within the urban area of Rome , 46% of the Latium Region and 20% of the species reported for Italy . The percentage of species detected in this experiment, relative to all those cumulative and historically reported for the study area and the addition of twenty-four new taxa represents, in our opinion, a good achievement, showing that an intensive sampling effort may yield new results regarding the distribution of wild bees in southern Europe. To highlight the effectiveness of the work and show with as little approximation as possible the state of the diversity of bees, it is possible to compare the methodological strategy and faunal results reported with the recent article by Geppert et al. , which represents a landmark study on the topic of wild bees in Rome from an ecological point of view. The two sampling strategies are not comparable in terms of collection effort, but they provide an idea of what may be the best approach to use if the goal is to study the alpha diversity of an area. The work of Geppert et al. resulted in the collection of 3280 wild bees belonging to 96 species, representing 34.5% of the species reported in the urban area of Rome. Geppert's data were collected during four months of sampling, which was carried out at thirty-six sites of different sizes within the GRA and using yellow pan traps. Compared with passive sampling methods such as yellow pan traps, active sampling using entomological netting more exhaustively captures alpha diversity, but results in a bias in the abundances of the groups collected because it is highly dependent on sampler capabilities . In this study, we collected data over two more months (April and May), but in only eight large green areas and we collected more than twice the number of species recorded by Geppert et al. . In this way, we provide a recent snapshot of the presence and distribution of wild bee species in the nature reserves of Rome, representing a starting point for monitoring them in the future. In addition, not all the species collected were reported for the area; in fact, twenty-four species (11% of the spp. collected) are new reports for the urban area of Rome. In terms of the abundance data, April was the month in which the greatest number of bees were collected overall . As we expected, from April to July, wild bees were more abundant than they were in August and September, the months in which a significant decrease in abundance was evident. The peak in the species diversity was detected during the May session (114 spp.) and declined steadily until September when only 22 species were detected. In April, the first month of sampling, the number of species detected already amounted to 82. Flower plants provide sustenance to bees and almost all herbaceous plants observed during sampling had completed their flowering period by August. The sampling period was selected according to the guidelines given in the literature for sampling pollinating insects in the Mediterranean environment , but in April, the peak of abundance for wild bees may have already been reached and a relatively good number of species is already active. The “heat island effect” produced by large urban centres and the average warmer and drier climate cause pollinators to be active early. For this reason, it would probably be correct to start sampling for pollinator insect studies in early March or even mid-February to obtain more complete information on the presence and abundance of species with early phenology. Amongst those species we collected, eight are included in the European Red List of bees . The presence of Red-listed species within the city shows that large urban green areas can be a refuge not only for wild bee species with a generalist ecology, but also for species with a more restricted ecology and that the nature reserves of Rome allow the survival of rarer species. The work of Fusco et al. , for example, studied ground spider diversity in APP itself and showed that it is true for other groups of arthropods. Focusing on APP, it is the largest urban nature reserve in Europe. It represents a unique case, being a single continuous green area that extends from the city centre to the area outside the city. In this way, APP offers ecological continuity and hosts an exceptionally high biodiversity of botanical species . The diversity of plants in the area is due to the presence of spontaneous, non-native and ornamental herbaceous and shrubby species in villas and home gardens distributed throughout the Reserve. Many plant species can have many different flowering periods that overlap. It can ensure continuity in the supply of resources for pollinators and spontaneous plants, promoting more functionally diverse bee communities . This study revealed that, given its unique combination of natural and semi-natural elements embedded in the urban matrix, Rome hosts and offers shelter to a remarkable variety of Apoidea Anthophila , including multiple threatened species. Notably, the bee community was probably not subject to any particular erosion in terms of richness between 1997 and 2022, indicating that the current complexity and repartition of green areas within or at the margin of Rome possibly buffered some of the drivers causing pollinators to decline. However, from a conservation perspective, the protection of urban fauna has to pass through the adoption of correct green management and planning strategies. Furthermore, faunistic surveys aimed at investigating bee diversity in urban ecosystems should consider the ‘heat island effect’ that occurs in large urban centres, a phenomenon that affects bee phenology and enhances flowering in plants in an urban context. In this regard, an earlier sampling period in February and March would be desirable to intercept the more precocious species, which we probably did not encounter in the present survey. | Study | biomedical | en | 0.999997 |
PMC11699519 | Interventional radiology (IR) plays a pivotal role in the management of complex urological conditions, particularly in cases where traditional approaches prove challenging . This technical note introduces an IR technique that addresses recurrent urinary tract infections (UTIs) and discomfort experienced by patients with nephroureteral stents with an ileal conduit urostomy. This technique demonstrates a practical solution to internalizing nephroureteral stents to ureteral stents in a patient with an ileal conduit. Our patient also highlights the clinical utility of this technique to avoid complications with external nephroureteral stents. A 61-year-old female patient was presented with a challenging medical history of a hysterectomy, lymph node dissection, and adjuvant radiotherapy in 2009 for curative treatment of stage IB squamous cell carcinoma of the cervix. Unfortunately, this resulted in a right ureteral stricture. Complications further ensued with the need for nephrostomy placement, ureteral stent insertion, and an ileal conduit urinary diversion in August 2018. The patient had long-term bilateral nephroureteral stents, which over prolonged time, were associated with multiple urinary tract infections . The patient also developed skin irritation from urinary leaks at the skin entrance site and significant discomfort from the nephroureteral stents. We hypothesized that if the nephroureteral stents were long enough, they could easily be changed retrogradely every three months, allowing the patient to live without the external nephrostomy component of the stents. An alternative approach used at our institution involves retrogradely cannulating the ureters from the ileal conduit and placing pigtail stents, similar to the study by Maher et al. . However, retrograde placement without endoscopic guidance is technically more challenging and time-consuming than antegrade stent placement, particularly in the setting of distal ureteric strictures. Antibiotic prophylaxis was administered and pre-procedure imaging, blood tests, and medications were reviewed. The patient was positioned prone on the fluoroscopic imaging table in the interventional radiology suite. The bilateral nephroureterostomies were prepped and draped with Betadine. Periprocedural sedation was given by the nursing staff using fentanyl and midazolam. The right nephroureterostomy was approached first, followed by the left. Injection of contrast into the stent under fluoroscopic guidance was performed. The position of the nephroureteral stent was confirmed. A 0.35", 180 cm stiff Terumo wire (Somerset, NJ: Terumo Medical) was inserted into the nephroureteral stent and into the ileal conduit . The nephroureteral stent was removed over the wire and exchanged for a 65 cm Berenstein catheter (Bloomington, IN: Cook Medical). The Terumo wire was removed and contrast injection via the Berenstein catheter outlined the ileal conduit . A 0.35" super stiff Amplatz wire (Marlborough, MA: Boston Scientific) was placed through the catheter and into the ileal conduit stoma bag . An 8 Fr angiographic percutaneous sheath was placed into the renal pelvis to secure the wire access to the kidney. Bilateral Amplatz wires were secured for change of position with tape. The patient turned into the supine position and the ileal conduit stoma bag was removed and prepped with povidone-iodine (Betadine). Lidocaine hydrochloride gel (Instillagel; Melsungen, Germany: CliniMed) was administered to the stoma. The super-stiff Amplatz wires were secured at the ileal conduit site with a 10 Fr angiographic sheath. New 8 Fr × 26 cm regular double-J ureteral stents (Boston Scientific) were sequentially placed over the Amplatz wires under fluoroscopic guidance and the pigtails formed in each renal pelvis . Contrast injection via the angiographic sheath within the renal pelvis was used to confirm the position of the proximal left ureteral stent . The wire was removed and ureteral stents were confirmed to be in a satisfactory position. The proximal and distal sheaths were then removed . The distal pigtails were formed external to the ileal conduit. This technique of internalizing nephroureteral stents to internalized ureteral stents within an ileal conduit represents a helpful advancement in the management of complex urological cases . The successful application and safety of this technique depend on the patient (e.g., type of ileal conduit, anatomy, and BMI) and technical, environmental, and device factors . Technical proficiency plays a paramount role in the execution of the internalization procedure. Interventional radiologists must possess the requisite skills and experience to navigate the intricate steps involved, which include wire insertion, stent removal, and precise stent placement. A commitment to maintaining meticulous sterile technique and fostering effective communication among the procedural team, particularly when switching from prone to supine, is equally indispensable. The choice of devices and equipment employed during the procedure significantly influences its outcome. Employing high-quality wires, catheters, and stents is crucial for facilitating smooth wire passage, stent exchange, and secure placement. Ongoing advancements in device technology, with a focus on tailoring equipment to the internalization technique, can enhance procedural efficiency and safety . Bander stents are an alternative specialized ureteral diversion stent used in ureteroileal conduit construction which can be 6 or 7 Fr and are polytetrafluoroethylene (PTFE)-covered stents . Similar to other devices, these facilitate drainage from the kidney to an external stoma, and they include color-coded radiopaque markers to assist in accurate positioning. The design ensures the stents are secure and can be easily replaced. Another option is to use biliary stents depicted in Figure 8 , which are held in place by a gastrostomy disc. Close collaboration between interventional radiologists and device manufacturers holds promise for driving innovations that can further enhance patient outcomes and reduce procedural complexity . The type of ileal conduit employed also influences the procedure's application and success. The standard ileal conduit (Bricker procedure) is the most common, but variations include the Studer orthotopic neobladder and cutaneous ureteroileostomy . Patients with different conduit types may require tailored approaches for effective stent management. While the internalization technique may not be directly applicable in some cases, the consideration of how to manage stents within these various conduit types remains a crucial aspect of patient care. Anatomical or physiological complexities pose challenges to the procedure's execution. Factors such as ureteral strictures, anatomical variations, peri-conduit adhesions, active infection, inflammation, and obesity can complicate wire passage, stent placement, and overall procedural success . A meticulous pre-operative assessment and procedural planning are essential for addressing challenging anatomical factors, minimizing risks, and ensuring patient safety. In conclusion, the technique of internalizing nephroureteral stents to ureteral stents within an ileal conduit is useful in the management of complex urological cases. Success depends on a nuanced understanding of patient, technical, environmental, and device factors. Additionally, an awareness of the types of ileal conduit and anticipation of challenging anatomical factors is crucial in tailoring the procedure to individual patient needs. As interventional radiology continues to evolve, ongoing collaboration between clinicians and device manufacturers holds promise for further enhancing the technique's safety and efficacy. This technique holds promise for similar cases and highlights the importance of patient-centered approaches in interventional radiology. | Other | biomedical | en | 0.999997 |
PMC11699541 | Bone defects can be caused by numerous reasons; the most significant ones in dental medicine include periodontal disease, trauma, peri-implantitis, and atrophy. In these cases, bone loss not only affects the esthetic appearance but also diminishes the structural integrity and function of the dental and facial framework. In order to overcome these issues, the demand for advanced treatment is increasing, and the search for innovative materials and techniques to facilitate bone regeneration has become extremely important . Guided bone regeneration (GBR) and guided tissue regeneration (GTR) have been recognized as gold standards among the numerous techniques employed. These methods use barrier membranes in a strategic manner to create a protective environment that prevents the infiltration of unwanted cell populations such as fibroblasts and epithelial cells while also supporting osteogenesis . However, traditional membranes, including polytetrafluoroethylene (PTFE) and collagen-based alternatives, are often limited by insufficient mechanical strength, a high risk of soft tissue invasion, and in some cases, the need for surgical removal on non-resorbable variants . The introduction of the cortical laminar bone membrane (CLBM), derived from xenogeneic bone tissue has revolutionized regenerative dentistry. CLBM's unique properties, including high mechanical stability, controlled degradation, and osteoconductivity, enable it to overcome the limitations of traditional membranes. Particularly, in the context of large or complex defects, CLBM has demonstrated remarkable clinical outcomes. The present review probes into the structural characteristics, biological mechanism, clinical applications, and challenges associated with CLBM supported by evidence from recent studies. CLBM is characterized by its rigidity, which significantly surpasses the mechanical strength of traditional collagen membranes. This rigidity ensures that the membrane effectively maintains space within bone defects, preventing collapse under soft tissue pressure. This property is critical when regenerating extensive or irregular defects, where maintaining structural integrity is essential for predictable outcomes . The integrity of the membrane is maintained by adapting it onto the site and fixing it in place using stainless steel mini screws . A study by Rossi et al. highlights the use of CLBM in cases of severe bone atrophy . The ability of the membrane to stabilize graft materials and maintain defect morphology was instrumental in achieving successful regeneration. Such mechanical stability is particularly important in demanding clinical scenarios, such as lateral ridge augmentation or large vertical defects, where traditional membranes often fail to provide adequate support . Since CLBM is derived from xenogeneic bone, it undergoes stringent processing to eliminate antigenic proteins while preserving its osteoconductive properties. This ensures compatibility with human tissues and creates an optimal surface for osteoblast attachment and mesenchymal stem cell differentiation . Tumedei et al. emphasized that CLBM not only supports the adhesion and proliferation of osteogenic cells but also facilitates their integration into the host bone tissue. This compatibility with surrounding tissues is a crucial factor in achieving long-term stability and functionality in regenerated bone . Unlike rapidly degrading collagen membranes, CLBM has a much slower and more controlled resorption profile. This property ensures that the membrane provides prolonged protection against soft tissue invasion while supporting the healing process over an extended period . Schuh et al. investigated the use of CLBM in multilayer regenerative techniques and found that its longevity significantly improved outcomes, particularly in esthetically demanding areas. The controlled degradation rate allows for sufficient time for bone remodeling, thus reducing the chances of complications such as soft tissue intrusion or incomplete regeneration . CLBM functions as a scaffold that mimics the natural extracellular environment of bone. Its surface promotes the adhesion of osteogenic cells, facilitating the migration and differentiation of mesenchymal stem cells into osteoblasts. This process is essential for the formation of new bone and the eventual remodeling of the defect site . Furthermore, the membrane supports osteoclastic activity, which is vital for remodeling and integration with the host bone. This dual support for osteogenesis and remodeling distinguishes CLBM from purely barrier-focused materials, such as PTFE membranes. CLBM acts as a reservoir for bioactive molecules, including bone morphogenetic proteins (BMPs) and vascular endothelial growth factor (VEGF). BMPs are critical for osteogenesis, while VEGF stimulates angiogenesis, ensuring the delivery of oxygen and nutrients to the regenerating site . Studies have shown that the synergistic effects of BMPs and VEGF enhance both bone and vascular tissue regeneration, making CLBM particularly effective in complex defect management. For example, Lee and Kim demonstrated how CLBM's ability to modulate these growth factors contributed to accelerated healing and improved clinical outcomes . The dense structure of CLBM prevents the infiltration of epithelial and fibroblastic cells into the defect site. This barrier function is crucial in maintaining an undisturbed environment for bone regeneration, particularly in GTR procedures where the regeneration of periodontal ligament and connective tissue is prioritized . CLBM has proven effective in treating both vertical and horizontal periodontal bone defects. Recent studies have shown its potential to augment the ridge laterally . When combined with bone grafts, the membrane enhances clinical attachment levels and reduces probing depths, making it a valuable tool in managing periodontitis . In cases of peri-implantitis, CLBM has demonstrated its ability to regenerate bone around failing implants. Clinical studies have reported substantial bone gain and improved implant stability when CLBM is paired with xenografts, highlighting its utility in restoring compromised implant sites . For ridge augmentation procedures, CLBM provides the necessary mechanical strength to stabilize graft materials and create adequate bone volume for implant placement. Deepika-Penmetsa et al. reported successful outcomes in lateral ridge augmentation, with the membrane ensuring graft stability and promoting long-term bone regeneration . Also, a recent review highlighted the superior vertical bone gains achieved with cortical lamina compared to traditional membranes . In sinus lift procedures, CLBM prevents graft displacement while promoting new bone formation. High success rates for implant placement have been documented in sites treated with CLBM, making it a reliable choice for sinus augmentation procedures . CLBM holds several advantages over conventional membranes, which are listed in Table 1 . The rigidity of CLBM is advantageous for maintaining space, but at the same time, it poses a challenge to adapt the membrane to irregular or complex defects such as morphologies . Unlike more flexible collagen-based membranes, CLBM requires precision in trimming and shaping to ensure optimal coverage of the defect. This inflexibility may increase surgical time and the potential for errors during placement. The use of cortical lamina requires precise handling and customization, making it technique-sensitive . Deepika-Penmetsa et al. emphasized that achieving proper adaptation demands advanced surgical expertise, which may limit its use among less experienced clinicians or in high-stress clinical scenarios . Due to its slower degradation rate, CLBM is prone to exposure if not adequately covered by soft tissue. Proper flap management is critical to minimizing this risk . Membrane exposure not only compromises the regenerative outcome by increasing the risk of infection but can also necessitate additional surgical interventions to address complications . Rossi et al. noted that poor flap management or insufficient soft tissue coverage could lead to exposure, particularly in cases involving thin gingival biotypes or significant defect dimensions . This highlights the need for careful case selection and advanced soft tissue management techniques. The production of CLBM involves stringent processing to remove antigenic proteins while preserving its osteoconductive properties. This complex manufacturing process contributes to the high cost of the material, limiting its availability and adoption, particularly in resource-constrained settings . Tumedei et al. pointed out that these costs could present a barrier to widespread clinical use, particularly when compared to more economical alternatives such as resorbable collagen membranes . As cost efficiency becomes an increasingly important consideration in healthcare, this limitation highlights the need for continued innovation to make CLBM more accessible. Although numerous studies have demonstrated the short- to mid-term success of CLBM in regenerative procedures, there is a relative lack of long-term clinical trials comparing its outcomes with traditional membranes or other advanced biomaterials. As Schuh et al. noted, long-term follow-up studies are essential to assess the durability and predictability of CLBM in diverse clinical contexts . The absence of such data makes it challenging for clinicians to fully evaluate its advantages and risks over extended periods. To expand the use of CLBM, ongoing research aims to enhance its handling properties, reduce production costs, and enhance antimicrobial properties. Additionally, incorporating bioactive molecules or growth factors could further enhance its regenerative potential. Long-term clinical trials and comparative studies are essential to validate its superiority over other advanced biomaterials. CLBM represents a significant step forward in regenerative dentistry. With their unique combination of mechanical stability, biocompatibility, and osteoconductive properties, CLBM addresses many of the limitations associated with traditional membranes. While challenges such as handling difficulties and costs remain, the transformative potential of CLBM positions it as a cornerstone in modern regenerative techniques. Ongoing innovations and research will continue to refine its applications, ensuring its role in achieving predictable and successful clinical outcomes. | Other | biomedical | en | 0.999997 |
PMC11699556 | Endometrial cancer (EC) is a gynecological tumor occurring in the endometrium . In China, the incidence of EC and its mortality are on the rise every year, posing a serious threat to society . Several factors, including tumor stage, histological types, and age, determine the prognosis of EC . Based on statistics, the 5-year survival rate of EC patients that are diagnosed and treated early exhibits a range of 70–90%, whereas those diagnosed late typically experience 20–60% due to tumor metastasis . Recent advancements in gene-targeted therapy for EC have expanded treatment options, offering patients more precise and efficacious interventions, with the potential to enhance future survival rates and quality of life [ 6 – 8 ]. Microfibril-associated protein 5 (MFAP5), an important extracellular matrix (ECM) protein, regulates the structure and function of ECM by mainly binding to microfibrils . In cancer, MFAP5 was found to function in tumor growth, metastasis, and angiogenesis . For example, MFAP5 may activate the epithelial–mesenchymal transition (EMT) program to promote basal-like breast cancer growth and aggressiveness . MFAP5 modulated EMT-related pathways, which led to a reduction in cervical cancer cell migration and invasion . MFAP5 facilitated the proliferation and metastasis of breast cancer cell, as reflected by the elevated levels of matrix metalloproteinase 2 (MMP2) and MMP9 after MFAP5 overexpression . However, there is no clear understanding of MFAP5’s role in EC. Herein, down-regulated MFAP5 was found by retrieving the TCGA database ( https:// tcga -data.nci.nih.gov/ tcga ). Subsequently, a series of in vitro experiments was conducted to investigate the effect of MFAP5 expression on the malignant behavior of EC cells. Normal human cervical epithelial cells (HcerEpic) and EC cell lines (JEC, KLE, HEC-1A, and Ishikawa) were purchased from ATCC (Rockville, MD, USA). Cells were grown in DMEM medium with 10% FBS at 37°C with 5% CO 2 . Adenovirus vector overexpressing MFAP5 (ad-MFAP5) and adenovirus-negative control (ad-NC) were prepared by Genepharma (Shanghai, China). Then, transfection of ad-NC or ad-MFAP5 was conducted with 50 multiplicity of infection for 48 h. The blank group (Control) was taken as a control for the experiment. Cells (1,000 cell/well) were planted into 96-well plate for 48 h. Whereafter, each well was injected with 10 µL of CCK8 reagent (Dojindo, Shanghai, China). A 2 h incubation was followed by the measurement of absorbance at OD 450 nm and the determination of cell viability. Cells (2,000 cell/well) were inoculated into six-well plates and grown at 37°C for about 2 weeks. Afterward, a 30 min fixation with methanol was followed by staining the cells with 0.2% crystal violet. Photographs of the colonies were captured with a camera. After collecting the cells, they were washed with buffer containing PBS. Following that, according to Annexin V-FITC detection kit (Dojindo), cells were stained with FITC-conjugated Annexin V and PI. A flow cytometry (FC500, BECKMAN COULTER, USA) was employed to analyze cell apoptosis within 1 h. Cells were suspended in serum-free medium and added to the upper compartment of a transwell. The lower compartment contained medium with 10% FBS. After cultivating for 24 h at 37°C, the upper chamber was removed, fixed in methanol, and stained with Giemsa. After cleaning, unmigrated cells were removed using a cotton swab. Next, the cells were visualized under the microscope (Leica Microsystems, Wetzlar, Germany) and counted. For cell invasion assay, a layer of Matrigel matrix was applied to the bottom of the transwell chamber. Cell lysates were prepared using RIPA buffer, then centrifuged and denaturated. Protein samples were then run on a 10% SDS-PAGE and transferred to PVDF membranes. After blocking, incubation of primary antibodies on the membranes at 4°C overnight was followed by secondary antibody incubation for 1 h. Table 1 lists the primary antibodies used. An ECL luminescent solution was utilized to render the color, and the protein bands were analyzed and quantified using Image J. Based on TCGA analysis, the results revealed that MFAP5 expression was lower in EC tissues compared to normal tissues . Subsequent results from western blot showed that MFAP5 protein expression was observably decreased in EC cell lines . These data suggest that MFAP5 is expressed at low levels in EC. An overexpression of MFAP5 in HEC-1-A and Ishikawa cells was conducted to study the function of the protein in EC. Western blot results showed a significantly elevated protein level of MFAP5 in cells after MFAP5 overexpression . Furthermore, CCK8 assay and colony formation assay indicated significantly reduced cell viability and clonogenic activity in cells overexpressing MFAP5 compared to the control cells . Moreover, results of flow cytometry showed that the MFAP5 overexpression group displayed a significant increase in apoptosis rates . These data demonstrate that overexpression of MFAP5 suppressed proliferation and facilitated apoptosis of EC cells. Further, MFAP5 was examined for its impact on cell migration and invasion by transwell assays. As shown in Figure 3a , comparing the overexpressing MFAP5 group with the control and ad-NC groups, the number of migrating and invading cells was both significantly reduced. In addition, western blot results showed that MMP2 and MMP9 protein levels were observably decreased in the MFAP5 overexpression group . These data indicate that overexpression of MFAP5 in EC cells inhibited their migration and invasion capabilities. Using western blot analysis, we further quantified the levels of EMT markers expressed in cells overexpressing MFAP5. As shown in Figure 4 , MFAP5 overexpression resulted in an increase in E-cadherin and a decrease in N-cadherin and α-SMA. These findings suggest that EMT in EC cells may be inhibited by overexpressing MFAP5. Next, the phosphorylation of AKT and mTOR was monitored to explore whether overexpressing MFAP5 in EC cells mediated the activity of the AKT/mTOR pathway. Western blot analysis revealed that phosphorylations of AKT and mTOR were reduced when MFAP5 was overexpressed in EC cells . These data suggest that MFAP5 overexpression decreased AKT/mTOR activity. Poor prognosis of EC is largely determined by tumor metastasis . Patients with cancer cells spreading to other organs make treatment more challenging, and the prognosis worsens . Therefore, investigating the mechanisms of tumor metastasis in EC may help to develop effective treatment strategies to improve patient outcomes. In this study, reduced level of MFAP5 was tested in EC cells, and cell proliferation was suppressed while cell apoptosis was promoted by overexpressing MFAP5. Importantly, our results demonstrate that MFAP5 overexpression inhibited migration, invasion, and EMT of EC cells. These data show a vital role of MFAP5 in EC cell malignant behaviors. MFAP5, a matrix glycoprotein consisting of microfibers, serves as a multifunctional protein implicated in endothelial cell behavior and mediated cell adhesion . Recently, most research studies indicated that MFAP5 expression is high in various tumor types, leading to enhanced tumor proliferation, endothelial cell motility, and angiogenesis [ 11 – 13 , 20 , 21 ]. For example, MFAP5 has been reported to enhance the stem cell features of non-small cell lung cancer cells, while the knockdown of MFAP5 has been shown to exert the opposite effect . Another study has found that MFAP5 silencing resulted in apoptosis and growth inhibition in cervical cancer cells . Herein, our TCGA database analysis revealed contrasting findings, showing underexpression of MFAP5 in EC tumor tissues, which differs from previous studies. Later, we detected diminished MFAP5 expression in EC cells. The invasion and metastasis of tumor cells are intricately regulated by the ECM, with MMPs, particularly MMP2 and MMP9, playing a crucial role in facilitating tumor formation and metastasis by degrading matrix proteins . Dysregulation of MMPs can compromise the integrity of tissue barriers, facilitating tumor invasion . This process promotes the degradation of the ECM and basement membrane, thereby enhancing tumor penetration, invasion of adjacent tissues, and metastasis to distant sites . Our findings demonstrate that MFAP5 overexpression led to a decrease in MMP2 and MMP9 levels, thereby confirming the inhibitory effect of MFAP5 on EC cell migration and invasion. However, how MFAP5 affects MMPs expression should be investigated further. EMT is a biological phenomenon in which cancer cells undergo a phenotypic change from epithelial to mesenchymal cells, resulting in enhanced abilities for proliferation, migration, and resistance to apoptosis [ 26 – 28 ]. MFAP5 has been shown to play a role in EMT process in cancer progression . Similar to previous results, our further data found an increased level of E-cadherin and a decreased N-cadherin and α-SMA in cells overexpressing MFAP5, indicating that MFAP5 overexpression inhibited EMT in EC cells. AKT, a protein kinase, can be activated by various growth factors, subsequently activating mTOR, thus promoting cellular proliferation and growth . In addition, EMT can be accelerated by activating AKT . Research indicates that dysregulated activation of the AKT/mTOR pathway in EC tissues can enhance cancer proliferation, invasion, and metastasis, while suppressing apoptosis and accelerating tumor malignancy [ 34 – 36 ]. Consequently, the modulation of the AKT/mTOR pathway has emerged as a prominent focus in the realm of EC therapy. In this study, the activity of the AKT/mTOR pathway was found to be reduced in EC cells overexpressing MFAP5, manifested by the inhibition of AKT and mTOR phosphorylation levels. However, further experiments will be required to determine whether MFAP5 exerts its biological role in EC by mediating the AKT/mTOR pathway. In addition, whether MFAP5 has the same biological function in vivo and the further regulatory mechanisms need in-depth investigation. In conclusion, our findings confirmed that MFAP5 regulates the EMT and malignancy in EC cells, and inactivation of the AKT/mTOR pathway may be one of the mechanisms , suggesting that targeting MFAP5 may represent an attractive strategy for EC treatment. | Review | biomedical | en | 0.999997 |
PMC11699559 | Retinal diseases such as age-related macular degeneration (AMD), diabetic retinopathy (DR), and uveitis are major causes of vision impairment and blindness worldwide . These conditions involve complex processes such as chronic inflammation, oxidative stress, and abnormal angiogenesis. Inflammation, in particular, plays a crucial role in the progression of these diseases by disrupting the delicate balance of pro- and anti-inflammatory cytokines, thus leading to tissue damage and abnormal blood vessel growth . Cytokines are small signaling proteins that regulate various aspects of the immune response, including inflammation, cell proliferation, and cell death. Among these, the interleukin-6 (IL-6) family of cytokines, including Oncostatin M (OSM), has received significant attention due to their dual role in both promoting and resolving inflammation. These cytokines are implicated in the development of various inflammatory and autoimmune diseases, including those affecting the retina . This review aims to explore the potential therapeutic effects of OSM and the broader IL-6 family of cytokines in retinal diseases. By examining the molecular mechanisms through which these cytokines influence the health and disease of the retina, as well as by reviewing preclinical and clinical studies, this article aims to provide a comprehensive understanding of their potential as targets for novel therapies. Additionally, this review will discuss the challenges and future directions for translating these findings into clinical practice. OSM is a significant member of the IL-6 cytokine family, characterized by the presence of the gp130 signal receptor subunit in their receptor complexes. This family includes various cytokines such as IL-6, IL-11, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), cardiotrophin-1 (CT-1), cardiotrophin-like cytokine factor 1, and IL-27 . OSM was first discovered by Zarling et al. in the U-937 human lymphoma cell line . The gene encoding human OSM is located on chromosome 22q12.2. The OSM gene encodes a polypeptide that initially consists of 252 amino acid residues. This polypeptide includes an N-terminal sequence of 25 amino acids and a C-terminal sequence of 32 amino acids. After proteolytic processing, the mature OSM protein consists of 195 amino acid residues. The mature human OSM protein has a molecular weight of approximately 28 kDa. Structurally, OSM is characterized by four α-helices arranged in an “up–up–down–down” topology [ 6 – 8 ]. This structural arrangement is typical of cytokines in the IL-6 family and is critical for their biological activity. OSM is widely expressed in vivo and plays a crucial role in various physiological and pathological processes. Several types of immune cells are known to produce and secrete OSM [ 9 – 12 ]. The receptor complexes for OSM are heterodimers, and their composition defines the type of signaling they mediate. Two main types of OSM receptor (OSMR) complexes exist: type I and type II . The type I OSMR complex consists of the α subunit gp130 and the β subunit leukemia inhibitory factor receptor (LIFR) β subunit and the type II OSMR complex is composed of the α subunit gp130 and OSMR β subunit . The receptor complex diversity mediates the different biological functions of OSM through the activation of multiple signaling pathways. The OSMR complex operates in a dynamic and species-specific manner, with its activation dependent on the presence of its subunits and ligand (OSM) . In the absence of OSM, the two subunits of the OSMR complex, gp130 and either OSMR or LIFR, exist separately in a non-associated state. This separation prevents spontaneous activation of the downstream signaling pathways. When OSM is present, it first forms a low-affinity heterodimer with gp130. This initial interaction is crucial for the subsequent recruitment of the second subunit. After forming a heterodimer with gp130, the complex then recruits either the OSMR or LIFR subunit, depending on the specific receptor complex involved. The recruitment leads to the formation of a high-affinity receptor complex that is now capable of signaling . Computational techniques including homology modeling, protein–protein docking, and molecular dynamics simulations were used by Du et al. to identify several critical amino acid residues involved in the binding process between OSM and OSMR . These residues are crucial for the stability and specificity of the interaction, ensuring that OSM can effectively bind and activate its receptor, revealing potential “hot spots” that could serve as targets for inhibitor design to block this pathway. Moreover, it has been showed the OSM/OSMR signaling module exhibits a unique microenvironment-restricted expression pattern, suggesting that targeting this pathway could mitigate the side effects associated with anti-IL-6 therapies, strengthening the demonstrated safety and tolerability of humanized anti-OSM antibodies as a potential therapeutic strategy for inflammatory diseases and cancer treatment . Also, it has been revealed that OSM and LIF share structural similarities, which explains why both cytokines can bind to the LIFR receptor . Despite these similarities, OSM and LIF have distinct biological functions, attributed to differences in their overall structure and the specific receptor subunits they recruit. In mice, OSM predominantly binds to type II OSMR complexes, which include gp130 and OSMR. Mouse OSM generally does not activate LIFR, except at very high concentrations, where it can weakly stimulate LIFR. There is a single report that mouse-derived OSM can activate LIFR specifically in mouse osteoblasts, suggesting a degree of context-dependent flexibility . OSM from rats and humans can bind to both type I (gp130 and LIFR) and type II (gp130 and OSMR) receptor complexes within their respective species. This dual-binding capacity allows OSM from these species to participate in a broader range of biological functions and signaling pathways compared to mouse OSM . The interaction between OSM and its receptor complexes is a finely tuned process that depends on the presence of specific receptor subunits and the structural characteristics of OSM. The formation of the OSMR complex, particularly the role of OSMR in type II complexes, enables OSM to activate unique signaling pathways that differentiate its functions from other cytokines in the IL-6 family. The species-specific nature of OSM binding further underscores the complexity and specificity of its biological activities, making OSM a critical cytokine in both normal physiology and disease states Abnormal levels of OSM are indeed linked to various inflammatory diseases. Compared to healthy controls, OSM levels are elevated in patients with pulmonary fibrosis . It has been demonstrated that increased the expression of OSM and OSMR in many inflammatory bowel disease lesions . Elevated levels of Oncostatin M receptor beta subunit (OSMRβ) in fibroblasts and dermal endothelial cells have been observed in patients with scleroderma, a chronic autoimmune disease characterized by fibrosis, vascular abnormalities, and immune system dysregulation . Studies have indicated that cytokines such as OSM and interleukin-31 (IL-31) play a critical role in promoting itch and inflammation in this condition, particularly through their interaction with dermal cells expressing IL-31RA (IL-31 receptor A) and OSMRβ . Elevated levels of OSM in gingival crevicular fluid have been observed in patients with periodontal disease, and research indicates that OSM concentrations increase in correlation with the severity of the disease, progressing from early-stage periodontal disease to chronic periodontitis [ 26 – 28 ]. In patients with coronavirus disease 2019 (COVID-19), OSM levels have been found to be elevated in peripheral blood plasma compared to healthy controls. These elevated levels of OSM are associated with the severity of the disease, suggesting that OSM plays a significant role in the immune response and inflammation characteristic of severe COVID-19 cases . The role of OSM in promoting angiogenesis, particularly in the context of cancer, is supported by accumulating evidence . OSM, a cytokine in the IL-6 family, can significantly influence angiogenesis through its effects on capillary endothelial cells and its activation of various signaling pathways. The recombinant human OSM has been shown to activate the STAT3 signaling pathway in endothelial cells. This activation leads to increased angiogenesis, particularly in tumor environments where new blood vessels are required to supply nutrients and oxygen to the growing tumor mass . In osteosarcoma, a highly aggressive bone cancer, the activation of the OSM/JAK2/STAT3 axis has been linked to both increased angiogenesis and the invasive behavior of cancer cells. The upregulation of MMP2 and vascular endothelial growth factor (VEGF) by this pathway not only promotes the formation of new blood vessels but also enhances the cancer cell’s ability to invade surrounding tissues, contributing to the aggressive nature of osteosarcoma . Neutrophils, which are often recruited to the tumor microenvironment, can produce OSM. Neutrophil-derived OSM interacts with the type II OSMR (composed of gp130 and OSMRβ) on breast cancer cells, leading to the induction of VEGF expression . Indeed, while OSM and other IL-6 family cytokines share the ability to activate the STAT3 signaling pathway, their effects on angiogenesis can be inconsistent. This variability is influenced by several factors, including the specific cytokine involved, the cellular context, and the microenvironment. CNTF exhibits anti-angiogenic effects in vascular endothelial cells and the retina, suggesting that CNTF-induced STAT3 signaling can inhibit rather than promote angiogenesis in certain contexts. This contrasts with the generally pro-angiogenic effects of other IL-6 family cytokines and highlights the complexity of cytokine signaling pathways. This study sheds light on the nuanced role of STAT3 in angiogenesis and reveals the complex and context-dependent role of STAT3 signaling in angiogenesis, particularly in response to different IL-6 family cytokines like CNTF and OSM . The findings underscore that the effects of STAT3 on angiogenesis are not uniform but are influenced by the specific cytokine involved, the concurrent activation of other signaling pathways, and the overall cellular environment. IL-6, a key cytokine in the IL-6 family, has been shown to induce the expression of VEGF mRNA in various cancer cell lines, including A431 cells (a human epidermoid carcinoma cell line) and C6 cells (a rat glioma cell line). This induction of VEGF expression highlights IL-6’s role in promoting angiogenesis, particularly in the context of tumor growth . The study by Wei et al. using nude mice demonstrated that IL-6 plays a significant role in promoting tumor growth in human cervical cancer, specifically in the C33A cell line, through a mechanism that is dependent on VEGF-mediated angiogenesis . In several cancers, including hepatocellular carcinoma, renal cell carcinoma, colorectal cancer, and glioblastoma, increased levels of circulating IL-6 have been linked to a poor response to therapies that target the VEGF/VEGFR pathway. These therapies include sunitinib, a tyrosine kinase inhibitor, and bevacizumab, an anti-VEGF antibody . Animal models have played an essential role in advancing our knowledge of retinal diseases and assessing potential treatments like OSM and the IL-6 family of cytokines. Among these models, rodents, particularly mice and rats, are favored due to their genetic adaptability and close resemblance to human retinal structures. One notable model is the oxygen-induced retinopathy (OIR) model in mice, which replicates the abnormal blood vessel growth seen in DR and ROP . By subjecting mice to high oxygen levels followed by a return to normal levels, this model induces retinal ischemia and new blood vessel formation, mirroring the processes observed in human diseases. This model has been used to investigate the roles of various cytokines, including OSM and IL-6, in retinal inflammation and abnormal blood vessel growth and researchers have been trying to unveil the intricate and multifaceted roles of OSM and the IL-6 family in the context of retinal diseases. The IL-6 cytokine family is a diverse group of inflammatory and pleiotrophic cytokines, encompassing IL-27, IL-11, IL-6, LIF, CT-1, CLC, CNTF, and OSM. These cytokines utilize similar receptor complexes and commonly activate the STAT3 signaling pathway within cells. The angiomodulatory effect of CNTF-driven STAT3 signaling in different mouse models of vasoproliferative eye diseases involves both a direct anti-angiogenic impact on vascular endothelial cells and an indirect effect mediated by Müller cells . Also, intravitreal injection of LIF can increase the vascular density in contrast to the OSM injection through changes in the cathepsin B and L expression . OSM, in particular, exhibits a duality in its effects, displaying both protective and detrimental impacts contingent on the disease stage and the specific retinal microenvironment. Other preliminary studies showed that OSM treatment promotes the proliferation of choroidal and retinal endothelial cells, in contrast to its effect on aortic endothelial cells . Rapp et al. observed a positive angiomodulatory effect of OSM following its intravitreal injection in the OIR model. The treatment activated STAT3 signaling in various retinal cell types, including Müller and vascular endothelial cells. In vitro studies indicate that OSM influenced the secretory profile of Müller cells. OSM promotes the regeneration of rods and cone photoreceptors in the early stage of degeneration via interacting with muller cells through activating STAT3. Also, it has been the regenerative role of OSM reported in the muscle, bone , and heart . Additionally, RNA sequencing of isolated retinal vascular endothelial cells from OIR P17 mice revealed a transcriptomic shift resulting from OSM treatment at P12. These findings highlight the critical role of Müller cells in vasoproliferative eye diseases and suggest that the effects of angiogenic agents may vary significantly between in vitro and in vivo environments. Also, it suggests that angiomodulatory agents like OSM might produce unanticipated and occasionally opposing effects in vivo versus in vitro , depending on the cell types involved within the intricate retinal environment. OSM shows a positive angiomodulatory influence on retinal angiogenesis, with Müller cells being key contributors to vascular homeostasis. Another study highlights the intricate role of STAT3 signaling in vascular endothelial cells in response to cytokine treatment, leading to diverse angiogenic outcomes . The angiomodulatory effects of STAT3 appear to be influenced by the activity of other intracellular signaling pathways and its specific localization within the cytosol, mitochondria, or nucleus. OSM and CNTF differ in their intracellular signaling mechanisms and STAT3 specificity, resulting in distinct transcriptomic profiles and metabolic activities . Another significant model is the streptozotocin (STZ)-induced diabetic mouse model, which allows for the study of the long-term impacts of high blood sugar on the retina, particularly in the context of DR. Through STZ administration, the destruction of pancreatic β cells occurs, leading to elevated blood sugar levels and consequent damage to retinal blood vessels . Robinson et al. demonstrated that inhibiting IL-6 trans-signaling significantly reduces diabetes-induced oxidative damage systemically and in the retina in an STZ-induced diabetic mouse model . Another study discovered that high levels of IL-6, a proinflammatory cytokine in the eye, were significantly linked to the advancement of proliferative diabetic retinopathy (PDR) and poor outcomes after eye surgery. This indicates that IL-6 within the eye may play a crucial role in the development of abnormal blood vessel growth associated with PDR, possibly by increasing VEGF expression . Mason et al. systematically review and analyze the changes in cytokine levels in the eyes of patients with nonproliferative DR. The meta-analysis highlights significant alterations in inflammatory cytokines, which could potentially contribute to the pathophysiology of the disease and may inform future therapeutic strategies . Moreover, other constituents of the IL-6 family, such as LIF and CNTF, have exhibited potential as neuroprotective agents in diverse models of retinal degeneration. For instance, LIF has demonstrated the ability to shield and protect the choriocapillaris, and yet, the photoreceptors, in a mouse model of dry-AMD, induced with sodium iodate . Yang et al. showed that a single intravitreal injection of CNTF-NP or OSM-NP provided protection for retinal ganglion cells in an acute glaucoma model and safeguarded photoreceptors and vision for over 70 days in a rat model of retinitis pigmentosa . The investigation of OSM and the IL-6 family as potential therapeutic targets in retinal diseases has progressed from animal experiments to clinical trials, summarized in Table 1 . Clinical studies have mainly focused on the role of IL-6 in retinal diseases. One important clinical trial examined the use of tocilizumab, a monoclonal antibody targeting IL-6, in patients with macular edema related to non-infectious uveitis. The trial demonstrated that tocilizumab (at both 4 and 8 mg/kg doses) effectively enhances visual acuity, reducing vitreous haze and central macular thickness in eyes with noninfectious uveitis. Tocilizumab is generally well-tolerated but requires careful patient monitoring due to risks such as neutropenia and gastrointestinal perforations. It offers a new avenue for managing challenging cases of retinal inflammatory diseases . Sarilumab is a fully human anti-IL-6Rα mAb that blocks the IL-6 signaling pathways by binding to membrane and soluble forms of IL-6Rα. After receiving approval for rheumatoid arthritis, it is undergoing clinical trials for use in the management of posterior segment non-infectious uveitis. In the SATURN study, patients with non-infectious uveitis showed reduced vitreous haze and lower steroid dosing with 200 mg subcutaneous dose every 2 weeks. The treatment improved visual acuity and central macular thickness with fewer side effects than other therapies, but adverse events like neutropenia and elevated alanine aminotransferase levels were reported . Designed specifically for intravitreal delivery, EBI-031 blocks both free IL-6 and the IL-6 receptor complex. Clinical trials have been conducted to assess the effectiveness and safety of EBI-031 in patients with diabetic macular edema . The current Phase I clinical trial involving KSI-501 marks a significant leap forward in the medical field. This trial aims to combat neovascular AMD and Diabetic Macular Edema (DME) by targeting both IL-6 and VEGF. KSI-501 is an innovative bispecific inhibitor specifically engineered to concurrently address IL-6 and VEGF, with the goal of overcoming the limitations encountered in prior studies by combining anti-inflammatory and anti-angiogenic effects. Initial findings from this trial have displayed encouraging results in terms of safety and preliminary effectiveness, and more comprehensive data are anticipated to unveil the therapeutic potential of this dual-target approach . Many trials are currently underway to evaluate the effectiveness of blocking the activation of OSM pathways, but none are focused on the retinal field. For instance, a humanized anti-OSM monoclonal antibody developed by GSK for rheumatoid arthritis showed limited efficacy in clinical trials due to poor binding affinity and was eventually discontinued . Another GSK anti-OSM monoclonal antibody demonstrated safety in early trials for systemic scleroderma but showed no significant efficacy, and all patients in the highest dose group experienced adverse effects . Researchers have developed a specific binding protein targeting OSM to inhibit its interaction with gp130 or LIFR , potentially applicable to inflammatory skin diseases and cancer . Boise State University developed small molecule inhibitors targeting Site III of OSM , particularly SMI-10B, to reduce tumor cell detachment and metastasis, confirmed through molecular dynamics simulation . Upcoming clinical trials will explore the combination of cytokine modulation with established treatments, such as anti-VEGF therapies, to enhance therapeutic results. Moreover, there is growing interest in personalized medicine strategies, where patient-specific cytokine profiles could guide the selection and combination of therapies to optimize treatment effectiveness and minimize adverse effects. The potential for therapies targeting OSM in retinal diseases also continues to be an area of intense study, with preclinical data suggesting the potential neuroprotective benefits of OSM modulation in certain retinal conditions. Modulating the activity of OSM and the IL-6 family offers a promising approach to treating retinal diseases. The cytokine network in the retina is complex, and strategies to modulate this network must be carefully designed to achieve therapeutic benefits without unintended adverse effects. One approach to cytokine modulation involves using inhibitors targeting the IL-6 receptor (e.g., tocilizumab) are being assessed for their ability to halt disease progression by blocking IL-6 signaling pathways . These therapies aim to reduce the inflammatory and angiogenic processes that contribute to retinal degeneration. Another potential strategy involves using agonists that activate protective cytokine pathways. For instance, recombinant forms of neuroprotective cytokines like CNTF have been investigated for their ability to slow down the degeneration of light-sensitive cells in conditions like retinitis pigmentosa . CNTF works by activating survival pathways that counteract signals that lead to cell death in the retina, thereby preserving vision. Despite promising preclinical results, challenges exist in ensuring the effective and targeted delivery of such therapies to the retina. One of the key challenges in cytokine modulation is achieving precise control over cytokine activity. Over-inhibition of cytokines like IL-6 could impair the retina’s natural defense mechanisms, leading to unintended consequences. Conversely, excessive activation of protective cytokines could potentially trigger unintended effects or lead to immune system imbalances. Therefore, the development of cytokine-based therapies must take into account the balance between efficacy and safety . Combination therapies that target multiple pathways simultaneously represent a promising approach to enhancing treatment efficacy in retinal diseases. For example, an ongoing Phase I clinical trial is investigating a bispecific inhibitor that targets both IL-6 and VEGF . By simultaneously addressing the inflammatory and angiogenic components of retinal diseases, this dual-target approach has the potential to provide superior therapeutic outcomes compared to monotherapy. Additionally, combining cytokine modulation with existing treatments, such as anti-VEGF therapies, may offer synergistic effects. For instance, in AMD and DME, where VEGF plays a central role in pathological angiogenesis, adding cytokine-targeting agents could further suppress disease progression by modulating the inflammatory environment that supports neovascularization. Personalized medicine also holds great potential in the context of cytokine modulation for retinal diseases. By tailoring treatments based on individual patient profiles, including specific cytokine expression patterns and genetic predispositions, clinicians could optimize therapeutic efficacy while minimizing side effects. For example, patients with elevated levels of IL-6 or OSM may benefit more from therapies targeting these cytokines, while others may require different approaches. Future research will likely focus on identifying biomarkers that can guide personalized treatment strategies and on developing advanced delivery systems that ensure precise targeting of cytokine modulators to the retina. As our understanding of the cytokine network in retinal diseases deepens, the potential for personalized and combination therapies to transform the management of these conditions will continue to grow. In this review, we have examined the various roles of OSM and the IL-6 family of cytokines in relation to retinal diseases. The evidence emphasizes the significant influence of these cytokines on inflammation, neovascularization, and cell survival within the retina. Preclinical studies show the potential effectiveness of targeting these pathways while emerging clinical data indicate encouraging avenues for therapeutic intervention. The increased expression of IL-6 and OSM in retinal diseases, such as DR and AMD, may reflect a failure of anti-inflammatory pathways to properly regulate chronic low-grade inflammation, which is a key feature in the progression of these diseases. This disruption could lead to a compensatory yet maladaptive overproduction of pro-inflammatory cytokines, contributing to ongoing retinal damage. More studies need to determine whether this cytokine upregulation is a primary mechanism or a secondary response to deficiencies in anti-inflammatory signaling, which could offer new insights into therapeutic approaches targeting inflammation regulation in retinal diseases. To address both inflammation and vascular remodeling, targeting the OSM pathways may offer a promising alternative to current therapies for retinal diseases. While potential challenges like antibody formation exist, strategies to minimize immunogenicity and combine these treatments with anti-VEGF agents may enhance therapeutic efficacy. The optimal treatment regimen will depend on patient-specific responses, and further studies are needed to refine these approaches and ensure long-term effectiveness. Moreover, there are challenges, such as the requirement for specific and safe delivery methods and the management of potential side effects. Future research should concentrate on refining these therapeutic approaches, including exploring combination therapies and personalized medicine strategies tailored to individual patient profiles. Continued clinical investigation is essential to fully realize the potential of OSM and IL-6 family cytokine modulation, which could revolutionize therapeutic approaches for retinal diseases and enhance patient outcomes . Table 1 summarizes various anti-IL-6, anti-OSM, and therapeutic agents under investigation or clinical use, highlighting their molecular structure, targeted pathways, current FDA-approved indications, and potential future applications. | Review | biomedical | en | 0.999995 |
PMC11699560 | Gestational diabetes mellitus (GDM) is a prevalent metabolic disorder that occurs during pregnancy, characterized by glucose intolerance that typically manifests in the second and third trimesters . This condition is associated with several adverse outcomes, including abnormal embryonic development, insulin resistance, hyperinsulinemia, and hyperglycemia . The incidence of GDM has been rising, primarily due to increasing maternal obesity and advanced maternal age . GDM significantly increases the risk of severe complications for both mother and infant, such as macrosomia, dystocia, and neonatal hypoglycemia . Despite ongoing research, the pathogenesis of GDM remains incompletely understood. Known etiological factors include oxidative stress, insulin resistance, and inflammation, which are triggered by placental hormones and strongly linked to the progression of GDM . Therefore, the identification of novel and effective therapeutic agents is critical for managing GDM. Baicalein is a flavonoid compound extracted from the roots of Scutellaria baicalensis Georgi, known for its diverse pharmacological effects . Recent studies have revealed various biological functions of Baicalein, including its role in regulating different diseases. For instance, Baicalein can alleviate oxidative stress and promote autophagy, and was shown to improve cardiac hypertrophy in mice . Additionally, Baicalein targets the GPX4/ACSL4/ACSL3 axis to inhibit ferroptosis, thereby reducing cerebral ischemia-reperfusion injury . In hyperlipidemic pancreatitis, Baicalein modulates the miR-192-5p/TXNIP axis to inhibit the NLRP3/Caspase-1 pathway, thereby mitigating pyroptosis and inflammation . Moreover, Baicalein regulates the AhR/IL-22 pathway to enhance the intestinal epithelial barrier, which alleviates ulcerative colitis . Notably, Baicalein has been shown to target the miR-17-5p-Mfn1/2-NF-κB pathway in trophoblast cells, reducing high glucose-induced inflammation and apoptosis . Despite these findings, the regulatory effects and mechanisms of Baicalein in GDM progression in vivo remain unexplored. In this study, we demonstrate that Baicalein inhibits the NLRP3 inflammasome and alleviates placental inflammation and oxidative stress in a GDM mouse model, suggesting that Baicalein holds potential as a therapeutic agent for managing GDM. C57BL/KsJ +/+ (wild-type) and C57BL/KsJ db/+ (db/+) mice (10 weeks old, n = 24 per sex) were obtained from the Nanjing Model Animal Center (Nanjing, China). All animal procedures were conducted in accordance with the guidelines of the Ethics Committee of Zhejiang Provincial People’s Hospital. Female mice were individually paired with male mice of the same genotype. The presence of copulatory plugs the following morning was used to confirm mating and designate gestation day (GD) 0. Pregnant female mice were then divided into four groups: Control (normal pregnancy, C57BL/KsJ +/+ ), GDM (C57BL/KsJ db+ (db/+) mice treated with DMSO), GDM + 20 mg/kg Baicalein (C57BL/KsJ db+ (db/+) mice treated with 20 mg/kg Baicalein), and GDM + 40 mg/kg Baicalein (C57BL/KsJ db+ (db/+) mice treated with 40 mg/kg Baicalein). Each group consisted of six mice. Baicalein was dissolved in DMSO and administered orally at doses of 20 or 40 mg/kg daily for 20 days starting from pregnancy. On GD 10, glucose and insulin tolerance were assessed using intraperitoneal glucose tolerance tests (IPGTT) and intraperitoneal insulin tolerance tests (IPITT). On GD 20, fasting blood glucose levels were measured with a glucometer (Roche Diagnostics, Risch-Rotkreuz, Switzerland), and insulin levels were quantified using an Ultra-Sensitive Mouse Insulin ELISA kit (ALPCO Diagnostics, Salem, NH). Cesarean sections were performed on GD 20 to collect placentas and fetuses for further analysis. Litter size and birth weight were recorded. For the IPGTT, after a 6 h fast, the mice received an intraperitoneal injection of glucose (2.0 g/kg). Blood glucose levels were measured at 0, 30, 60, 90, and 120 min using an ACCU-CHEK Advantage glucometer (Roche Diagnostics, Risch-Rotkreuz, Switzerland). For the IPITT, after a 6 h fast, insulin (0.75 U/kg) was administered intraperitoneally. Blood glucose levels were then measured at 0, 30, 60, 90, and 120 min. The levels of TNF-α , IL-1β , and IL-6 were quantified using commercial ELISA kits according to the manufacturer’s protocols. Oxidative stress markers, including malondialdehyde , glutathione , myeloperoxidase , and superoxide dismutase , were measured using commercial kits in placental tissue samples. Proteins from mice placental tissues were extracted using RIPA lysis buffer (Thermo Fisher Scientific, Inc.), separated using 10% SDS-PAGE, and transferred to PVDF membranes (Beyotime, Shanghai, China). After blocking, membranes were incubated with primary antibodies at 4°C for 12 h. Secondary antibodies were then applied. Protein detection was performed using a chemiluminescence detection kit (Thermo Fisher Scientific, Inc.). Our results showed that the glucose tolerance in GDM mice was impaired, but this impairment could be reversed using Baicalein (40 mg/kg) . Similarly, insulin tolerance was reduced in GDM mice, and similarly, this reduction could be mitigated using Baicalein (40 mg/kg) . Additionally, fasting blood glucose and plasma insulin levels were elevated in GDM mice, and these elevations could be normalized following Baicalein (40 mg/kg) treatment . Overall, our findings indicate that Baicalein could improve metabolic symptoms in GDM mice. In the GDM group, the number of offspring per litter was reduced, but this reduction was alleviated by Baicalein (40 mg/kg) treatment . Furthermore, the birth weight of the offspring was increased in the GDM group; this effect was attenuated with Baicalein (40 mg/kg) administration . Thus, Baicalein positively influenced reproductive outcomes in GDM mice. ELISA results showed that levels of IL-6, IL-1β, and TNF-α were elevated in GDM mice, but these increases were significantly reduced following Baicalein (40 mg/kg) treatment . Additionally, markers of oxidative stress, including MDA and MPO, were elevated, while SOD and GSH levels were decreased in GDM mice. Baicalein (40 mg/kg) treatment normalized these oxidative stress markers . Taken together, these findings indicate that Baicalein could effectively alleviate inflammation and oxidative stress in GDM mice. NLRP3 protein expression was elevated in the placentae of GDM mice, but this elevation was reduced following Baicalein (40 mg/kg) treatment . Similarly, levels of IL-1β and IL-18 were increased in GDM mice, but these increases were diminished after Baicalein (40 mg/kg) administration . Collectively, Baicalein decreased NLRP3 inflammasome activation in the placentae of GDM mice. Numerous extracts from Chinese herbs have been shown to influence the progression of gestational diabetes mellitus (GDM). For instance, Polyphyllin I modulates the AMPK pathway to reduce inflammatory damage in GDM . Similarly, Astragaloside IV alleviates placental inflammation and oxidative stress in GDM mice , and Resveratrol activates the AMPK pathway to improve GDM progression in mice . Baicalein, a flavonoid with diverse biological functions, has been implicated in the management of various diseases . However, its regulatory roles and mechanisms in the context of GDM have not been fully elucidated. This study demonstrates that Baicalein improves metabolic symptoms in GDM mice by enhancing glucose and insulin tolerance, and normalizing fasting blood glucose and plasma insulin levels. Additionally, Baicalein positively affected reproductive outcomes, including the number of offspring per litter and birth weight. Inflammation and oxidative stress play pivotal roles in the progression of GDM . Consequently, many researchers have concentrated on modulating these factors to slow the progression of GDM. For instance, cryptotanshinone has been shown to alleviate placental inflammation and oxidative stress in GDM mice . Similarly, N-acetyl-L-cysteine helps reduce both inflammation and oxidative stress associated with GDM . Additionally, miR-875-5p targets TXNRD1, thereby influencing oxidative stress and inflammation in GDM rats . Myrtenol has also been demonstrated to modulate the TLR4/MyD88/NF-κB pathway, thereby mitigating inflammation and oxidative stress in GDM rats . In alignment with these findings, our study observed that inflammation and oxidative stress were elevated in GDM mice. However, these effects were significantly mitigated following treatment with Baicalein (40 mg/kg), suggesting that Baicalein can also effectively alleviate inflammation and oxidative stress in the context of GDM. Inflammatory factors such as TNF-α, IL-6, and C-reactive protein are known to contribute to insulin resistance in GDM . The nucleotide-binding oligomerization domain-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome, a macromolecular complex consisting of NLRP3, caspase-1, and ASC, plays a critical role in this process . The interaction between caspase-1 and pathogen-associated molecular patterns or damage-associated molecular patterns activates the NLRP3 inflammasome, leading to the secretion of IL-1β and IL-18 from immune cells and promoting inflammation . Previous studies have shown that Baicalein can target the miR-17-5p-Mfn1/2-NF-κB pathway in trophoblast cells, reducing high glucose-induced inflammation and apoptosis . Despite these findings, the effects of Baicalein on the NLRP3 inflammasome in the context of GDM progression in vivo have not been well characterized. In this study, we observed that protein levels of NLRP3, IL-1β, and IL-18 were increased in GDM mice. However, these effects were attenuated following Baicalein treatment, indicating that Baicalein inhibits the NLRP3 inflammasome. In conclusion, Baicalein effectively inhibited the NLRP3 inflammasome and mitigated placental inflammation and oxidative stress associated with GDM in mice. Nevertheless, this study has certain limitations, including the need for further investigation using human samples, additional cell models, and a broader range of phenotypes. Future research should focus on these areas to provide a more comprehensive understanding of Baicalein’s effects and potential therapeutic applications. | Review | biomedical | en | 0.999995 |
PMC11699562 | TAU is a major microtubule-associated protein (MAP) in the human brain, where it contributes to microtubule (MT) stabilization and regulates MT dynamics. Normally, TAU is sorted and restricted mainly to the axon, where it is associated with MTs. In pathological conditions, it can dissociate from MTs, mislocalize into the somatodendritic compartment, and form insoluble aggregates, known as neurofibrillary tangles (NFTs). These NFTs are the hallmark lesions of tauopathies, which include Alzheimer’s disease (AD), frontotemporal dementia, atypical Parkinson’s syndromes like corticobasal degeneration, progressive supranuclear palsy, and other tauopathies . Missorted TAU leads to or coincides with the breakdown of the MT system, compromised axonal transport, synaptic dysfunction, and neurodegeneration . Much is known about the downstream effects of missorted TAU, but the upstream events that lead to TAU missorting or the mechanisms enabling physiological sorting under healthy conditions are poorly understood. In the case of AD, we know that the loss of TAU’s MT-stabilizing function and its downstream effects constitute a distinct cause leading to neurodegeneration, aside from the toxic gain-of-function of TAU aggregates . Besides AD, many other neurodegenerative diseases display the effects of a disrupted MT network – for example, intracellular protein aggregates disturb axonal transport in synucleinopathies, ALS or Huntington’s disease , and defects of motor proteins lead to intellectual disabilities . Moreover, mutations in MT-related genes cause defective cell migration, as seen in lissencephaly, or hinder MT-cytoskeletal rearrangements during neuronal development, like in autism spectrum disorders . Importantly, MTs in neurons have a particular organization that differs significantly from other cell types and also within the compartments of the neuron itself . The minus end is anchored in the MTOC, which is classically the centrosome in most dividing eukaryotic cells. In spherical somatic cells, e.g., fibroblasts or unpolarized neuronal progenitors, MTs would classically grow out centrifugally from a central centrosome. In postmitotic neurons, the centrosome loses its function as an MTOC during development, and instead, the MTs grow out from Golgi outposts in dendrites or by non-centrosomal MTOC proteins, e.g., on behalf of augmin from pre-existing MTs or from the MAP CAMSAP2 at the proximal axon . Aside from atypical growth origins, MTs have particular features differing between axons and dendrites, including their orientation, stability, bundling, and chemical tubulin modifications . MTs in the axon are uniformly orientated with their plus end outwards, thus growing anterogradely from the soma, while MTs in the dendrite show a mixed polarity, with a varying fraction of anterograde and retrograde growing MTs depending on the species and neuronal cell type . In the dendrite, anterograde MTs make up a portion of approximately 65% in mice in vivo , 11% in Drosophila , 55% in hippocampal rat neurons , and 85% in human . The different orientation in axons and dendrites seems to be important for two mechanisms: (a) the uniform orientation of MTs in the axon is important for the axonal outgrowth and specification and (b) the different polarity of MTs directly drives polarized cargo transport. Kinesin-driven cargo is directed towards the plus end and targeted primarily to the axon, whereas dynein-driven vesicles drive toward the minus end, preferentially on dendritic MTs . This is important because the cargos themselves contain proteins that determine the axonal or dendritic fate, e.g., synaptic receptors for the axonal synapse. Neuronal MT dynamics are being studied i.a. in primary neurons derived from rodents, the standard model for mammalian neurobiology in vitro . Unfortunately, human disease is only partially recapitulated in rodents and rodent-based human disease models, and there are notable differences in gene and protein sequences and isoform composition of disease-associated players, which is in particular true for TAU . This makes it difficult to draw conclusions from rodent models, but primary human neurons are very rarely available (e.g., from abort material or from neurosurgically removed peripheral ganglia in case of injury) and unsuitable for research purposes. With biotechnological progress over the past two decades, inducible pluripotent stem cells (iPSCs) can now be differentiated into various types of human cells, including neurons and their numerous subtypes . Somatic cells are genetically reprogrammed into pluripotent iPSCs, which can be transformed into neuronal precursor cells and finally neurons, by exposing them to external morphogens – a method called “directed differentiation” – or via genetic fating usually achieved by induced expression of a neurotransmitter driving differentiation into specific neuronal subtypes . An alternative human neuronal cell system, besides induced pluripotent stem cell (iPSC)-derived neurons (iN), are secondary neuronal cell lines derived from neuronal tumors. An extensively used type is the human neuroblastoma cell line SH-SY5Y. Treatment of this cell line with several chemicals, including retinoic acid (RA), brain-derived neurotrophic factor (BDNF), neuronal growth factor, or others, results in the generation of excitatory neuronal cells . SH-SY5Y cells can be differentiated within 2–3 weeks in cultures showing neuronal morphology and neuronal markers . Undifferentiated SH-SY5Y neuroblastoma cells show catecholaminergic characteristics but during differentiation, they develop into neurons inheriting cholinergic, noradrenergic, and dopaminergic properties, of which all three neuronal subtypes are present in AD-affected loci . As pre-mitotic cells, SH-SY5Y cells undergo cell division and can provide a vast quantity of neuronal cells for experimental arrays. Their differentiation is less time- and cost-consuming than those of iN and yields cultures of higher homogeneity and robustness. Here, we set out to test MT dynamics in iN and SH-SY5Y-derived neurons (SHN) and in different neuronal subcompartments. We first tested proper neuronal compartmentalization, i.e., axodendritic development, axon-initial-segment (AIS) establishment, and polarized sorting of TAU and MAP2. We compared the efficiency of axonal sorting of TAU and MAP2, and expression of the AIS-marker proteins AnkG and TRIM46 in iN to SHN and the standard model of neurobiology, mouse primary neurons (mPN). We found that iN and SHN showed remarkably similar TAU sorting properties, which were also comparable to mPN, but SHN lacked expression of AnkG and showed reduced expression of TRIM46. We then investigated MT dynamics via EB3-live-imaging and found that i.a. MTs are orientated anterogradely in axons of both iN and SHN, but more retrogradely in iN dendrites. MT polymerization speed was much alike in the three subcompartments of SHN and iN, whereas in SHN, we observed generally more growth events. Conclusively, overall cell polarization and neuronal compartmentalization do take place in SHN and iN, but only iN express and enrich AnkG and TRIM46 at the AIS and their MT-dynamics appear more reminiscent of mature neurons. Both SH-SY5Y cells and iPSCs followed a differentiation protocol into neuronal cell cultures with a duration of 2–3 weeks. Both cell models were cultivated in 6-well, 12-, and 24-well plates (VWR) or in imaging dishes (ibidi). Well plates have partly been equipped with coverslips (VWR) for microscopy experiments. If not stated otherwise, the amount of solutions and media was 2 ml for a well of a six-well plate, 1 ml for a well of a 12-well plate and 0.5 ml for a well of a 24-well plate. Media reagents were prewarmed at 37°C and cells were kept in a humidified incubator at 37°C with 5% CO 2 . Undifferentiated SH-SY5Y cells were cultivated in DMEM/F-12 GlutaMAX™ supplemented with 10% fetal bovine serum (FBS, Biochrom AG), and Antibiotic–Antimycotic (referred to as SHM-10 medium) in uncoated T75 flasks (VWR). SHM-10 medium was changed twice per week and SH-SY5Y cells were passaged when reaching 80% confluence. For passaging cells were washed with phosphate-buffered saline (PBS, TFS), trypsinized (0.05% trypsin/EDTA, TFS) for 3–5 min, and centrifuged for 3 min at 1,000 × g . The cell pellet was re-suspended in 10 ml SHM-10 medium, diluted to 1:10, and seeded for further cultivation. For differentiation, SH-SY5Y was seeded onto plates or imaging dishes, previously coated with 20 µg/ml poly- d -lysine in PBS. The PDL coating solution was added to the well plates and incubated for at least 3 h at 37°C. The coating solution was then discarded and wells were washed three times with Dulbecco’s phosphate-buffered saline (DPBS). For imaging experiments, glass coverslips (VWR) were placed into the wells (25 mm for a 6-well plate, 12 mm for a 24-well plate) before adding the coating solution. Seeding density was 8,000 or 5,500 cells/cm 2 when seeding one, respectively, 2 days prior to the start of differentiation. SH-SY5Y cells were differentiated into SHN within 2 weeks performing sequential medium exchanges with the supplements RA and BDNF. SHM-10 medium was exchanged to SHM-10 with 10 μM of RA on days 0, 3, and 5. On day 7 of differentiation, SHM-10 + RA (Sigma-Aldrich) medium was removed, cells were washed once with DPBS and SHM-10 medium without FBS (referred to as “SHM-0”), supplemented with 10 ng/ml BDNF (Peprotech) was added. On day 10, another medium exchange with SHM-0 + BDNF was carried out. On day 14, cell medium was again changed to SHM-10 without supplements. Differentiation was finished at this point with a differentiation efficiency of about 75% and SHN could be used for further experiments. Cultivation continued in SHM-10 medium with medium exchange every 3 days. hiPSCs were transformed into cultures of cortical glutamatergic neurons following a differentiation protocol, adapted from . The used iPSC cell line WTC11 carries a Neurogenin 2 (Ngn2) transgene, inducible by doxycycline, a transcription factor that rapidly converts iPSCs into neurons (for detailed protocol including troubleshooting guide, see ). For routine cultivation, undifferentiated iPSC colonies were grown in cBrew with medium exchanges every 2–3 days on Geltrex-coated (TFS) 6-well plates. Plates were coated on the day of use with 200 μg/ml Geltrex for 30 min at 37°C. Habitually, certain iPSCs differentiated spontaneously before starting induced differentiation and were hence removed manually with a P200 pipette under a sterile hood. iPSCs were passaged when they reached a confluence of about 80%. For passaging, iPSCs were washed with PBS and dissociated with Versene (TFS) for 3–5 min. Versene was aspirated and 1 ml of cBrew was added. Then, the colonies were carefully detached with a cell scraper and the cell suspension was transferred to a 15 ml tube (Falcon). Cell clumps within the suspension were dissolved by trituration with a 5 ml Pasteur pipette. The cell suspension was diluted in cBrew to a routine passaging ratio of about 1:10. Additionally, Thiazovivin (Axon Med Chem) was added in a concentration of 1:5,000 to the cell suspension, which was then distributed to the culture wells. On the next day, the medium was changed to fresh cBrew without Thiazovivin. Cells would typically reach 80% confluency again after 3–4 days. iPSCs were transformed into cortical glutamatergic neuronal cultures following a 3-day pre-differentiation (d3 to d0), followed by a multiple-week-long differentiation (d0 to d21 or further) protocol. Three days before starting differentiation (d3), iPSCs were passaged onto Geltrex-coated plates. Cells were washed once with DPBS and then dissociated with Accutase (Merck). Cells were incubated in Accutase for 5–8 min at 37°C. Dissociation was stopped by adding 3 ml of DPBS, then cells were collected in a 15 ml tube (Falcon) and centrifuged for 5 min at 400 × g . The supernatant was discarded and the cell pellet was re-suspended in 1 ml of cBrew. iPSCs were diluted in a pre-differentiation medium, supplemented with Thiazovivin (1:5,000), and seeded in a density of 1.5–2 × 10 5 cells/cm 2 . The medium was changed on days 2 and 1 to a fresh pre-differentiation medium without Thiazovivin. For differentiation (d0), pre-differentiating iPSCs were passaged onto PDL/Cultrex-coated plates. Plates were first coated with 20 μg/ml PDL in DBPS 1 day before the start of differentiation (d–1), incubated overnight at 37°C, washed once with DBPS, and then coated with 20 μg/ml Cultrex in DPBS for another hour (d0). Plates were used within 24 h and washed twice with DPBS before seeding the iPSCs. For seeding, dissociation with Accutase, collection, and centrifugation were performed as described during the pre-differentiation, except for re-suspending the cell pellet in 1 ml of neuronal maturation medium (NMM), instead of using cBrew. Cells were diluted in NMM containing freshly added Geltrex (1:100) to reach a density of 0.5–0.8 × 10 6 cells/well and 2.5–4 × 10 6 cells/well in a 24-well plate and 6-well plate or imaging dish, respectively. Half of the amount of NMM was exchanged every weeks (d7, d14, d21) while iPSCs differentiated progressively into iN. Pre-differentiation medium: KO DMEM/F-12 1× neuropan-2 supplement 1× non-essential amino acids 10 ng/ml BDNF 10 ng/ml NT-3 1.5 μg/ml laminin 2 μg/ml doxycycline 1× Antibiotic–antimycotic solution NMM: 50% neurobasal medium 50% DMEM/F-12 0.5× neuropan-2 supplement 1× non-essential amino acids 0.5× B27 supplement 10 ng/ml BDNF 10 ng/ml NT-3 1.5 μg/ml laminin 2 μg/ml doxycycline 1× antibiotic–antimycotic solution SHN were transfected using the polymer-based PolyJet DNA transfection reagent (SignaGen) at d7 of differentiation. Transfection was performed according to the manufacturer’s protocol with the following modifications: prior to transfection, 1/2 of the conditioned medium was collected from each well and stored at 37°C with 5% CO 2 . For 1 well of a 24-well plate, 0.33 μg of plasmid DNA and 1 μl PolyJet reagent were separately mixed with 25 ml DMEM each, then joined, incubated for 10–15 min, and added dropwise to the culture medium. After 3 h, cells were washed once with DMEM and then cultivated in a previously collected medium filled up with fresh SHM-10. The transfection efficiency ranged widely depending on the used plasmid between 5 and 20%. iN were transfected using the LipoStem transfection reagent (TFS) 2–4 days before experimental use, according to the manufacturer’s protocol. Prior to transfection, 1/2 of the conditioned medium was collected from each well and stored at 37°C with 5% CO 2 . For 1 well of a 24-well plate, 0.5 μg plasmid DNA was mixed with 25 μl of Opti-MEM (mix A) and 0.5 μl LipoStem was mixed with 25 µl DMEM (mix B). Mixes A and B were joined, incubated for 10 min, and added dropwise to the cell culture. After 24 h, cells were washed with warm KO-DMEM and cultivated in a previously collected medium filled up with fresh NMM. Cells were fixed with 3.7% formaldehyde (FA) in PBS. After incubation for 18 min, cells were stored in 55% (v/v) glycerol in PBS (storage solution) at −20°C for later analysis or washed three times with PBS for direct staining. For immunofluorescence staining, FA or storage solution was aspired and cells were washed three times in PBS. Cells were permeabilized and blocked in 5% BSA (Carl Roth) and 0.2% Triton X-100 (Carl Roth) in PBS for 10 min. Afterward, cells were washed once in PBS and then stained with primary antibodies at 4°C overnight. On the following day, coverslips were washed three times in PBS and incubated with the secondary antibody for 3 h. Finally, coverslips were washed three times with PBS and stained with Hoechst 33342 (NucBlue™, TFS) in PBS for another 25 min at room temperature. Cells were washed twice with deionized water and then mounted on objective slides with a Poly-Mount mounting medium (Polysciences). Mounted slides were dried for at least 24 h before imaging and kept at 4°C for long-term storage. Microscopy images were taken with a widefield fluorescence microscope (Zeiss), equipped with an LED lamp (Colibri 7, Zeiss) and a fluorescence camera (Axiocam 503 mono, Zeiss). Images were acquired using the Zen imaging software (Blue pro, Zeiss). Images were taken with magnifications of 200×, 400×, or 630×, using 10×, 20×, and 40× objectives air based and the 63× objective with immersion oil (Immersol 518F, Zeiss). Exposure time and laser power were adjusted so that the fluorescence signal was not oversaturated. Within the same experiment, all images were taken with identical LED intensity and exposure time to ensure statistical comparability of the protein expression levels. For investigation of MT dynamics by live imaging, SHN and iN were transfected with a plasmid expressing tdTomato-tagged EB3 (ptdTomato-EB3) following the transfection protocol described above and before . SY5Y-cells were transfected on day 7 while iN were transfected on days 12–14 post-differentiation. Generally, the neurons expressed the EB3-tdTomato 2–3 days after transfection. The movement of fluorescently tagged EB3 particles was recorded with a Leica DMi8 S Platform microscope equipped with a Leica-DFC9000 fluorescence camera and the image acquisition software Las X Life Sciences (Leica Microsystems). The cells were continuously covered with a CO 2 incubation unit and a microscope chamber to protect the cells from external light radiation and were left to accommodate 15 min before imaging. The time-lapse video was composed of images taken at a rate of 1 image per 2 s over a period of 2 min (for in-depth protocols, see also ). A quantitative RT-qPCR was performed to assess the expression of TRIM46 and AnkG in different cell types. Cells were harvested and lysed and total RNA was isolated using PureLink RNA Mini Kit (TFS). Reverse transcription into cDNA was carried out with ProtoScript II First Strand cDNA Synthesis Kit (NEB). Quantitative RT-qPCR was performed with SYBR green I (TFS) on the following cycling protocol: denaturation at 95°C for 10 s, annealing at 58.5°C for 30 s, and elongation at 68°C for 60 s, 30 cycles. The HPRT gene was used as a reference gene for relative quantification. Used primers were the following: TRIM46 Primer 1 (GCAGCTGCACAACAGGATTG) and Primer 2 (ATCATAGGCAAAGGTGCGCT); AnkG Primer 1 (GTCTGAGCAAAAGCAGGGAGA) and Primer 2 (ACCGTTCGCTGTTACGAGTG). The acquired movie files were registered and exported with LasX Image acquisition software. The movie file was then analyzed with ImageJ/Fiji software (OpenSource) as follows: a 30 μm line region of interest (ROI) was drawn along the proximal axon and the dendrite, respectively, and a 50–200 μm line ROI along the distal axon (at least 400 μm away from soma) with the segmented line tool. The ImageJ plugin Kymograph Reslice Wide generated kymographs from the EB3 comets moving along this linear ROI, which were then read out for (comet number/30 μm/min), comet direction (anterograde/retrograde), and comet speed (μm/min). The comet speed was calculated from the gradient of the comet track in the kymograph. All experiments were conducted in 3 experimental replicates of 20–60 cells each. To compare EB3 dynamics in the different neuronal compartments, it was essential to distinguish axons from dendrites. Axons were distinguished from dendrites using established morphological characteristics (Bell et al., ; Tjiang and Zempel, ; Zempel et al., ), e.g., a constant small diameter, longer outgrowth than dendrites (>300 μm), and branching pattern of 90°. In case of uncertain morphological discrimination, we fixed the neurons after live imaging, re-identified the imaged cells on the gridded imaging chamber, and stained for TAU, MAP2, and AnkG/TRIM46 to ascertain that we analyzed/imaged the appropriate neuronal subcompartments . To quantify the enrichment of TAU and MAP2 in the axon, relative to the soma, we calculated an AEF (AEF Tau , AEF MAP2 ). For both proteins, we measured the mean fluorescent intensity (MFI) in ROIs drawn in the soma (MFI S ), the axon (MFI A ) (>100 µm distal from the AIS), as well as in the empty space next to the soma (MFI bgS ) and axon (MFI bgA ), to normalize for background noise. The ROI in the soma was drawn, so it would not overlap with the nuclear signal. We subtracted MFI S – MFI bgS (=MFI Soma ) and MFI A –FI bgA (=MFI Axon ) to exclude background fluorescent noise. Next, an axon-to-soma ratio was calculated, for TAU and MAP2 each (MFI Axon /MFI Soma ). Finally, to account for volume bias in the fluorescent intensities, we normalized the TAU/MAP2 axon-to-soma ratios to the axon-to-soma ratio of the randomly distributed volume markers tdTomato or GFP e.g., AEF Tau = ( MFI Axon / MFI Soma ) Tau ( MFI Axon / MFI Soma ) tdTomato \documentclass[10pt]{article}\usepackage{wasysym}\usepackage[substack]{amsmath}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{amsbsy}\usepackage[mathscr]{eucal}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\[\left(\phantom{\rule[-0.75em]{}{0ex}},\text{e}\text{.g}\text{.,}\hspace{.25em}{\text{AEF}}_{\text{Tau}}=\frac{{({\text{MFI}}_{\text{Axon}}/{\text{MFI}}_{\text{Soma}})}_{\text{Tau}}}{{({\text{MFI}}_{\text{Axon}}/{\text{MFI}}_{\text{Soma}})}_{\text{tdTomato}}}\right)]\]\end{document} . ROIs for tdTomato, GFP, MAP2, and TAU were identical. Quantitative data for AEF of SHN from Figure 1b (fourth bar of quantification) was obtained from Michael Bell-Simons from Bell et al. and replotted for comparison. To quantify the localized enrichment of TRIM6 and AnkG (AIS-EF TRIM46 , AIS-EF AnkG ) at the beginning of the axon, a ratio between the peak fluorescence intensity (all values >70% of maximum value) and the baseline fluorescence intensities along the AIS (first 80 μm) was calculated. The peak intensity ( y ) was determined as the mean value of all values greater than 70% of the maximum fluorescence signal, along the peak zone of a certain length ( x ). The baseline intensity was determined as the mean value of all fluorescence intensities over the same distance of ( x ) μm at the end of the 80 μm ROI. AIS enrichment factor (AIS EF) was calculated from three experimental replicates for all cell types. Quantitative data for AIS plot profile and AIS enrichment factor of SHN and mPN were obtained from Michael Bell-Simons from Bell et al. and replotted for comparison. In AD and related neurodegenerative tauopathies (neurodegenerative diseases [NDD]/tauopathy), cell polarity is impaired, reflected by TAU mislocalization into the somatodendritic compartment . To have a better understanding of the cell culture systems, we first tested which human neuronal cell model is able to recapitulate neuronal cell polarity in terms of successful axonal targeting of TAU and somatodendritic retention of MAP2 similar to primary rodent neurons. We differentiated SH-SY5Y cells to neurons (SHN) using our established protocol, which resulted in a relatively high yield of differentiated cells (∼75%), successful axodendritic polarization, and convincing TAU enrichment in the axon after 14 days of differentiation using RA and BDNF, as detailed previously . We also differentiated our iN as established previously using doxycycline-dependent expression of Ngn2, resulting in a differentiation efficiency of >90%, axodendritic polarization, and successful TAU sorting . Finally, we used the established standard model of mammalian neurobiology, mouse primary forebrain neurons, which shows excellent axonal targeting of endogenous TAU and even allows to discriminate subtle differences of axodendritic targeting of, e.g., different isoforms of pseudophosphorylated TAU . We found that all neuron types investigated, after sufficient differentiation as appropriate and necessary (i.e., 14 days for human neurons, >7 days for murine neurons, for details, see ), developed axons and dendrites. When stained for the axonal marker TAU and the somatodendritic marker MAP2 and subsequently imaged via immunofluorescence microscopy (IF), we found that TAU was strongly enriched in the axons, but not MAP2, which remained largely in the soma and the dendrites . Morphologically, dendrites were larger, both in diameter and length, in iN and primary neurons compared to SHN . Quantification of the AEF, a measure of enrichment of a protein in the axon compared to the soma (see Section 2 for details) revealed that in all cell types, MAP2 is only slightly enriched in the axon compared to the soma, while TAU is distinctly enriched in the axon. Axonal TAU enrichment is strongest in iN and increases during maturation from ∼5-fold at day 7 after differentiation (d7) to ∼18-fold at d21. Axonal TAU enrichment is equally strong in mPN (∼15-fold) and moderately strong in SHN (∼9-fold). Nonetheless, this means that both SHN and iN show strong axonal enrichment of TAU, but not of MAP2, comparable to murine primary neurons, indicative of successful axodendritic development and establishment of neuronal cell polarity. Next, we compared the AIS development in three neuronal cell systems: primary mouse neurons, iN, and SHN. We conducted AnkG and TRIM46 immunostainings and measured the fluorescent intensities, localization, and local enrichment using IF and calculation of the AIS enrichment factor (AIS-EF), as well as their mRNA amounts via qPCR. Both iN and primary neurons showed pronounced expression of AnkG as well as TRIM46 along the proximal axon and displayed the typical morphology of an AIS in IF . The length and location of their AIS, situated approximately 5–40 μm distal to the soma in the proximal axon, were identical: The proximal enrichment of AnkG (mPN: 9.7-fold, iPSC: 7.3-fold) and TRIM46 (mPN: 9.1-fold, iN: 7.8-fold) was pronounced in both mPN of age DIV9 and in iN at day 17 of differentiation. Moreover, the maximal AnkG and TRIM46 fluorescence were situated ±10 μm distally from the soma and had the same spatial distance of ±2 μm from each other in both cell models . In contrast, IF showed no immuno-labeling of AnkG in SHN. Nevertheless, TRIM46 was expressed in this cell line after differentiation, however, relatively moderate in comparison to iN. Moreover, the local enrichment of TRIM46 (1.6-fold) was much less pronounced than in iN (7.8-fold) and primary neurons (9.1-fold) . Secondary antibody control showed no unspecific binding . To confirm the lower expression level of the AIS-related proteins in SHN, we conducted qPCR to assess the mRNA levels of TRIM46 and AnkG in iN, differentiated SHN, undifferentiated naive SH-SY5Y cells, and HEK cells. The HEK cells served as a negative control for qPCR since no expression of AIS proteins is expected in these cells derived from human embryonic kidney cells. In line with the findings from the IF, there was no expression of AnkG in undifferentiated, naive SH-SY5Y cells (<1% compared to iN). But notably, also AnkG mRNA levels in differentiated SHN were negligible (<1% compared to iN). However, we detected expression of TRIM46 in SHN, which augmented from 10.7% (naive) to 39.9% (differentiated neurons, both values compared to iN) during their differentiation, which is consistent with the moderate signal of TRIM46 in the immunostainings. As expected, we found no expression (<1% of iN) of neither AnkG nor TRIM46 in HEK cells ( n = 1–2 experiments). Taken together, these data demonstrate that iN develop an AIS, morphologically alike of that in primary mouse neurons. On the other hand, SHN only express TRIM46 in the axon upon neuronal differentiation but lack AnkG, which likely results in a differently organized AIS structure. After observing the absence of a classic AIS, in terms of no detected AnkG expression and low TRIM46 levels, in SHN compared to iN, we investigated if this influences the MT dynamics, which are of great importance for neuronal polarization as well as the pathophysiology of AD and NDD in humans. To investigate the dynamics and orientation of MTs in different compartments and cell types, we transfected both our human SHN and human iN with tdTomato-tagged EB3 on day 7, respectively, days 12–14 (Seq. S1). The protein EB3 binds to the plus tips of growing MTs and, when conjugated with a fluorescent protein like tdTomato, enables live-tracing of MT growth events on MT plus ends . These moving EB3 accumulations appear as comet-like structures in the time-lapse recordings, whose direction, speed, and quantity can be read out from kymographs, generated from the time-lapse videos (Movie S1). Kymographs are imaging tools that visualize dynamic processes in biological systems by capturing time-lapse images along a specified axis. Each pixel represents a position and time, creating a two-dimensional graph where the x -axis denotes space and the y -axis indicates time. This technique is particularly effective for studying the movement of cellular structures along cytoskeletal elements, revealing insights into transport mechanisms and motility. We recorded and analyzed EB3 movements in the proximal axon, distal axon, and dendrites of the neurons on days 14–18 of differentiation when cell polarity is well-established. We used morphological characteristics and/or post-imaging immunostaining to distinguish axons from dendrites (see Section 2.9.1 ). While in both cell types identification of the axon is relatively simple (as the axon is smaller and constant in diameter, there are different branching patterns of axons and dendrites, and very different lengths of axons and dendrites as described before , we nonetheless fixed the cells, re-identified the imaged cells, and stained for TAU, MAP2, and ANKG/TRIM46 in initial experiments to ascertain that we analyzed/imaged the appropriate neuronal subcompartments . Counting the EB3 comets, we found that in all cell compartments and in both cell types, the majority of MTs was orientated anterogradely (thus with the growing plus end directed to the periphery), represented by EB3 moving outwards from the soma; however, there were notable differences: In SHN, the proportion of anterograde comets was equally high in the proximal axon (94.9% ± 2.0) and distal axon (95.8% ± 0.8), whereas the proportion of anterograde comets was lower in the dendrites (79.8% ± 3.6). Consequently, the percentage of retrograde comets was ∼4–5 times higher in the dendrites (20.2% ± 3.6) than in the axonal compartments (proximal: 5.1% ± 2.0, distal: 4.2 ± 0.8%). A similar distribution was observed in iN, where anterograde comets accounted for 89.0% ± 4.1 in the proximal and 94.3% ± 1.3 in the distal axon of all moving comets. However, the proportion of anterograde comets in the dendrites (72.4 ± 3.4%) was even lower than in the dendrites of SHN, resulting in a significantly higher portion of retrograde comets (27.6% ± 4.7) compared to the other compartments and cell types . We also quantified the number of moving comets (comet number/30 μm/min), representing the amount of dynamic MTs and thus the overall growth dynamics . Generally, in both cell models, the highest amount of total moving comets was observed in the dendrites (SHN: 6.0 ± 0.4, iN: 6.1 ± 2.6), followed by those in the proximal axon (SHN: 4.8 ± 1.0, iN: 4.3 ± 1.5) whereas the number of moving comets in the distal axon was significantly lower (SHN: 3.1 ± 0.7, iN: 1.9 ± 0.6). Strikingly, the number of moving comets in the SHN exceeded those in the iN in all three compartments for anterogradely moving comets, but not for retrogradely moving comets. Finally, we analyzed the speed of the moving comets from the gradient of the comet traces within the kymographs . The average speed of all moving comets was 12.9 ± 1.1 μm/min in SHN and 12.1 ± 1.1 μm/min in iN. Broadly, the average speed was similar between the two cell types and also between the different compartments, with differences of only 1–2 μm/min. In the SHN, comets moved fastest in the distal axon (14.1 ± 2.1 μm/min), whereas comets of iN had the highest speed (13.9 ± 1.1 μm/min) in the dendrites. Interestingly, the speed of anterograde comets was always ∼1–2 µm/min higher than the retrograde ones in both cell types with the exception of the dendritic compartment in iN, where retrograde comets moved faster than the anterograde ones. However, the differences appeared overall marginal and not statistically significant. In summary, MTs in SHN and iN behaved remarkably similarly regarding orientation, dynamics, and speed of growth, but with a higher proportion of retrogradely moving comets particularly in iN dendrites. In this study, we set out to evaluate whether and which type of polarized human neurons would be a suitable cell model to investigate compartment-specific MT dynamics, in order to test human model systems also for future studies in the context of axodendritic TAU (mis-)sorting, AD, and associated tauopathies/neurodegeneration (NDD). We used the SH-SY5Y cell line and neurons derived thereof (SHN), which are cost-efficient and which we had judged to be potentially suitable for TAU-based research , and iPSC that can be differentiated via doxycycline-induced expression of Ngn2 into forebrain neurons (iN), which are also valuable for studying, e.g., neuronal activity in disease settings, all of which we have described previously . We used as a reference the standard model of cellular neurobiology, mouse primary neurons (mPN) . We assessed if the three neuronal models acquired neuronal polarity, which includes the structural separation of axons and dendrites with compartment-specific enrichment of MAP2 in dendrites and TAU in axons . Our findings indicate that both SHN and iN models show strong axonal enrichment of TAU, comparable to murine primary neurons. Specifically, axonal TAU enrichment was strongest in iN, increasing from approximately 5-fold at day 7 to 18-fold at day 21 post-differentiation. This enrichment was equally strong in mPN (∼15-fold) and moderately strong in SHN (∼9-fold). These results suggest that both SHN and iN are suitable models for studying TAU sorting and neuronal polarity, with iN showing the highest degree of TAU enrichment. Strikingly, architectural properties of axons and dendrites were obtained and axonal outgrowth occurred extensively not only in the AIS-positive iN and primary neurons but also in the AnkG-deficient SHN. Additionally, immunostainings for MAP2 and TAU revealed a clearly polarized distribution of the two proteins in all cell types. We also compared AIS development in iN, primary mouse neurons, and SHN, whereas the AIS composition was much alike in primary neurons and iN, we found that SHN did not express AnkG, but only TRIM46 in moderate amounts . Importantly, TRIM46 expression augmented during differentiation, which is in line with its reported role in initial neuronal polarization . AnkG was reported to maintain neuronal polarity and gate the axonal identity , but SHN showed both axon formation as well as polarized protein sorting despite AnkG deficiency. This is in line with AnkG as the sole actor governing the axonal identity being questioned in various studies . So what is the interaction and the role of TRIM46 and AnkG? We found that while AnkG and TRIM46 are both involved in establishing neuronal polarity, they play distinct roles. TRIM46 appears to act earlier to initiate axon formation and polarized protein sorting, while AnkG is important for maintaining the axon-dendrite separation later in development . Several lines of evidence suggest that TRIM46 can localize and function independently of AnkG at the axon initial segment (AIS): TRIM46 clusters at the proximal axon earlier than AnkG during development, with 70% of neurites in stage 2 neurons being TRIM46+/AnkG− . In dorsal root ganglion (DRG) neurons that lack AnkG, TRIM46 is still confined to the proximal axon. AnkG knockdown in young hippocampal neurons caused only moderate mislocalization of TRIM46 from the proximal axon . Treating mature hippocampal neurons with AnkG shRNA for over a week reduced TRIM46 levels by only 30% . This suggests that TRIM46 may be recruited to the AIS by binding partners other than known AIS components like ankyrin, spectrins, or sodium channels. The data indicate that TRIM46 can autonomously localize to establish the initial MT organization and polarity at the nascent AIS, independent of AnkG. A key finding is that axonal TAU enrichment correlates with the uniform MT orientation established by TRIM46, even in AnkG-deficient SHN. This could suggest that TAU axonal sorting depends on the MT lattice organization rather than a physical diffusion barrier at the AIS, as proposed previously . Yet, TRIM46 may be sufficient to generate this polarized MT array conducive for axonal TAU sorting, independent of AnkG. MT dynamics, crucial for neuronal polarization and function, were investigated using EB3-comet tracking. Both SHN and iN showed an axodendritic polarity in MT orientation: while MTs were orientated anterogradely in 90–95% in axons of both SHN and iN, dendrites showed MTs of mixed polarity with only 70% (iN) to 80% (SHN) of MT plus-ends directed anterogradely. Importantly, this polarized organization occurred independently of AnkG, which is absent in SHN, supporting the hypothesis that TRIM46 is sufficient to establish axonal MT polarity . The extent of MT reorientation from mixed/multipolar in stage 2 neurons to uniform in stage 3 axons matched previous reports in rodent neurons . Recent investigations in iN displayed a similar trend: in unpolarized stage 2 neurons, 80% of MTs had anterograde orientation which increased to 90% in axons of stage 3 neurons. The fraction of plus-end-out MTs in the dendrites decreased from 80% to 60% in stage 3 neurons . The higher proportion of retrograde dendritic MTs in iN in comparison to SHN could thus hint towards a higher grade of maturity, consistent with structural differences between the two neuronal cultures. Nevertheless, both SH-SY5Y and iN show the MT cytoskeleton of a polarized neuron. Can we now hypothesize that axonal TAU sorting depends on uniform MT orientation in the axon? Indeed, polarized protein sorting correlated with MT directionality in iN and SHN. In both neurons, MAP2 accumulated in regions with mixed MT polarity, whereas TAU was enriched in axons with unidirectional MTs. Consistently, TAU was distributed evenly throughout unpolarized stage 2 iN (correlating well to our d7 iNs), which have multipolar MTs in their developing neurites . Hence, axonal MT orientation may drive anterograde TAU mobility, e.g., as suggested previously by kinesin motors . Additionally, MT properties – orientation, PTM, spacing – in the AIS may enforce TAU binding and thereby a diffusional gradient of soluble TAU from the soma to the axon . Contrarily, MAP2 might be unable to couple to axonal MT motors, as described for other somatodendritic cargo , and therefore be hindered to drive along axonal MTs. It is thus possible that differential MT orientation in axons and dendrites drives polarized protein sorting by selective acquisition of motors. These results would be compatible both with TAU being simply an MT binder, but also with a functional role of TAU modifying MT liability as proposed previously . Interestingly, SHN exhibited more growth events in all compartments compared to iN, indicating higher overall MT dynamics. This might be due to a higher immaturity of SHN. The speed of EB3 comets was consistent among the compartments as well as among the two cell models, with an average speed of 12.1 μm/min in SHN and 12.9 μm/s in iN. Anterograde growth events were slightly faster than retrograde, except for dendritic MTs in iN. Brown-Handerson et al. reviewed MAPs that regulate MT growth, i.e., catastrophe frequency, and rescue frequency and also growth speed. The differential composition of accelerating MAPs on plus and minus tips may result in a faster growth rate of anterograde MTs. Indeed, Jakobs et al. propose that faster polymerization of anterograde MTs contributes to uniform MT orientation in the axon . Nevertheless, as the polymerization rate is not significantly faster in axons than in dendrites (here and elsewhere ), the extensive outgrowth and specification of the axon likely results from differential MT orientation, reduced catastrophe frequency, or increased rescue frequency – as mediated by for TRIM46 – and not by faster growth rates. Consistently, silencing of TRIM46 does not decrease the MT polymerization rate but the portion of anterograde MTs and thereby axonal outgrowth . Interestingly, growth speed in human neurons was substantially faster than in numerous other neurons observed in former studies, e.g., 0.12 μm/s (7 μm/min) in Drosophila dendrites , 5 μm/min in axons of Drosophila , 0.1 μm/s (6 μm/min) in Caenorhabditis elegans , 0.1 μm/s (6 μm/min) in cortical mouse neurons , or 0.08 μm/s (4.8 μm/min) , respectively, and 0.2–0.3 μm/s (13–19 μm/min) in hippocampal rat cultures . Importantly, the growth rate appears not related to the fluorescent + TIP used. Thus, these differences could hint toward species-dependent regulation of MT growth: possibly, MT polymerization is regulated faster in humans, since MTs have to accomplish a much longer neurite growth than in neurons of rodents or non-vertebrates. Additionally, we investigated the absolute number of MT growth events within a defined distance of 30 µm along the neurites. Both in SHN and iN, we observed most MT growth events in the dendrites, followed by the proximal axon, and least in the distal axon . This can be explained either by a higher density of MT in the dendrites or alternatively by a greater number of dynamic MTs compared to stable MTs in dendrites, as reported before . How neurons establish a polarized MT network is a broadly debated question in neurobiology. Likely a combination of parallel bundling by TRIM46, local MT nucleation sites, translocation of anterograde and retrograde MTs, de novo MT generation from pre-existing MTs, differential growth behaviors of anterograde and retrograde MTs controlled by associated MAPs, is responsible for unipolar MTs in axons. We can now hypothesize that besides axonal outgrowth and neuronal transport, polarized MT orientation is essential for axonal TAU sorting, but further studies are needed to elucidate the regulators of MT polymerization and its impact on TAU sorting in humans. In sum, in this study, we show that while SHN compared to iN lack a proper AIS as defined by the presence of AnkG, axonal targeting of TAU and axodendritic MT polarization is comparable, making both human neuron cell culture types suitable for studying neuronal cell polarity and further understanding of MT-related features. This holds true in particular for SHN, if the focus is not the AIS. Finally, to assess both the influence of the AIS and MT-associated factors on TAU sorting, iN are the more appropriate model to gain a holistic understanding of pathomechanisms in AD/NDD and associated dysfunction of neuronal cell polarity. | Review | biomedical | en | 0.999998 |
PMC11699588 | Several collaborative efforts involving physicians, dentists, and pharmacists have been implemented to encourage dental consultations; however, reports on the involvement of community pharmacists in these initiatives remain limited. Takaki et al. examined the impact of a periodontal disease awareness campaign conducted at pharmacies on the dental consultation rate among patients with diabetes and reported that 8.2% of patients sought dental care after promotion. However, > 50% of the patients did not consult a dentist, highlighting the need for continuous guidance. Additionally, despite the short three-month duration of the study, approximately 20% of pharmacists reported it as a burden, and 35% stated that the three-month period was too long, raising concerns regarding the continuity of such efforts at pharmacies. Thus, we developed follow-up content on periodontal disease for patients with diabetes using the "FollowNavi ® " platform (Unike Software Research Co., Ltd., Minato, Japan), a LINE ® -based medication follow-up support tool. We aimed to investigate whether follow-up by community pharmacists could improve dental consultation rates, enhance the understanding of dental care, and raise awareness of oral hygiene among patients with diabetes. The primary outcomes were changes in dental consultation and checkup rates from baseline to six months. Data were collected at baseline and six months later from all participants. HbA1c levels of the patients were determined from the blood test results in the most recently recorded medical history during the six-month follow-up. Information regarding age, sex, and medication use was obtained from the medical history records. Furthermore, information regarding the duration of diabetes, dental consultations (for therapeutic purposes), participation in dental checkups (for cavity and periodontal disease screening, plaque removal, and brushing instruction), oral care status, willingness to prevent periodontal disease, and understanding of periodontal disease were evaluated using questionnaire surveys administered to patients. This study included 113 patients (55 men and 56 women; 2 unspecified). The median age at baseline was 60 years (interquartile range [IQR]: 52-68), and the median HbA1c level was 6.7% (IQR: 6.3-7.0%). Overall, 26 patients (23.0%) had been diagnosed in less than 5 years, 85 patients (75.2%) had been diagnosed more than five years, and two patients (1.8%) did not provide an answer. The median follow-up period was 182 days (IQR: 125-266), and all patients underwent four follow-ups. The median number of responses to these follow-ups was 3.0 (IQR: 1.0-4.0). The median number of diabetes medications used was 3.0 (IQR: 2.0-4.0). The usage rates of diabetes medications were as follows: biguanides, 62.8%; sodium-glucose cotransporter 2 inhibitors, 51.3%; dipeptidyl peptidase IV inhibitors, 44.2%; glucagon-like peptide 1 receptor agonists, 31.0%; sulfonylureas, 16.8%; thiazolidinediones, 15.9%; insulin, 15.0%; α-glucosidase inhibitors, 13.3%; meglitinides, 12.4%; and imeglimin, 3.5% (multiple responses allowed) (Table 1 ). The dental consultation and checkup rates at baseline and after six months are shown in Table 2 . A total of 98 patients were included in the analysis, after excluding 15 patients for whom data at either baseline or six months were unavailable. The dental consultation rate increased from 40.8% to 41.8%, and the dental checkup rate increased from 57.1% to 59.2%; however, the changes were not statistically significant (P = 1.000, 0.824). HbA1c levels after six months were 6.8% (IQR: 6.4-7.2), with no significant change (P = 0.062). In the questionnaire, the question "Do you know that having periodontal disease can worsen blood sugar levels?," the number of patients who answered "Yes" increased from 64 (65.3%) at baseline to 85 (86.7%) (P < 0.001). In response to the question "Do you know that diabetes is a risk for periodontal disease?," 72 patients (73.5%) answered "Yes" at baseline, but 93 (94.9%) after six months (P < 0.001) (Table 3 ). In response to the question, "Do you want to prevent periodontal disease (by consultation, the dentist, undergoing checkups, and brushing your teeth properly)?," the number of patients who answered "Strongly agree" or "Agree" increased significantly from 84 (86.6%) at baseline to 92 (94.8%) after six months (P = 0.039). In response to the question "Do you try to keep your teeth healthy?," 62 people (63.3%) answered "Yes" at baseline, and 64 people (65.3%) answered "Yes" after six months (P = 0.845) (Table 4 ). Munenaga et al. reported that in patients with T2DM moderate to severe periodontal disease, HbA1c levels improved significantly three months after periodontal treatment. Engebretson et al. showed that in a meta-analysis of nine RCTs, periodontal treatment significantly reduced HbA1c levels by 0.36%. These studies were conducted on periodontal patients and on patients who had undergone dental treatment. In our study, the rate of dental consultation did not improve significantly, and not all patients had periodontal disease, so it seems that there was no improvement in HbA1c levels. Regarding the questionnaire survey, the patients' understanding of the relationship between periodontal disease and glycemic control increased significantly, as well as their awareness that diabetes is a risk factor for periodontal disease. At baseline, 65.3% and 73.5% of the patients had this knowledge, and these rates increased significantly to 86.7% and 94.9%, respectively, after six months. This suggests that the follow-up of periodontal content by community pharmacists effectively enhances patient understanding. In terms of motivation for oral care, the proportion of patients who answered "Strongly agree" or "Agree" to the question, "Do you want to prevent periodontal disease (by visiting the dentist, undergoing checkups, and brushing your teeth properly)?" significantly increased from 86.6% at registration to 94.8% after six months (P = 0.039). Additionally, in response to the question after follow-up "Did you consider visiting a dentist by message?", 46 patients (40.7%) answered "Strongly agree" and 39 (34.5%) answered "Agree." These results confirm that follow-ups improved patients' awareness and motivation for oral care. Despite these changes in awareness, the actual consultation rate did not improve, possibly because behavioral changes take time. According to the transtheoretical model of health behavior change , there are five stages of behavior change: pre-contemplation (not yet acknowledging that there is a problem behavior that needs to be changed), contemplation (acknowledging that there is a problem but not yet ready, sure of wanting, or lacks confidence to make a change), preparation (getting ready to change), action (changing behavior), and maintenance (maintaining the behavior change). The survey results suggested that the willingness to visit dentists improved, indicating a transition from the pre-contemplation stage to the contemplation stage. However, for patients to move to actual behavioral changes, a shift from the contemplation stage to the preparation and action stages is necessary, and building a trusting relationship is considered crucial for this transition . In the pharmacist's questionnaire, some respondents mentioned the challenge of "being unable to obtain non-verbal information, such as the patient's feelings and thoughts," highlighting the limitations of mechanical follow-ups. To build trust and promote behavioral change among patients, combining mechanical follow-ups with in-person consultations and guidance is necessary. In the patient questionnaire survey regarding usability, 52 (46.0%) answered that the messages were "Easy," while 12 (10.6%) answered that they were "Slightly easy.” These values were lower than those obtained in our study using diabetes follow-up data . This may be due to the high number of non-answers to this question (20.4 %). Given that a combined total of 7.1% of patients answered that it was "Slightly difficult" or "Difficult," the contents were adequately understood. Regarding the number of questions, 70.8% answered that it was "Appropriate,” and regarding the frequency of questions, 71.7% answered that it was "Appropriate,” indicating that the number was suitable. In this study, follow-ups were conducted only once every two months. In our previous report , follow-ups were planned for each consultation and conducted approximately once a month, with > 90% responding that this was appropriate. Although it is difficult to generalize the results because the participants and contents of follow-ups varied, the frequency of follow-up may not be an issue, depending on the patient. Therefore, pharmacists should consider medication and patient information and conduct frequent follow-ups. This study has two limitations. First, this study had a high rate of incomplete answers in the questionnaires. In the usability questionnaire, approximately 20% of the questions remained unanswered. Thus, improving the questionnaire method and questions is necessary for future surveys. Second, data, such as duration of diabetes, dental consultations, and participation in dental checkups, were obtained via questionnaire surveys administered to patients. Therefore, there is a possibility of recall bias and lack of accuracy. Further studies are needed to obtain accurate medical data and evaluate them. In the six-month follow-up using the periodontal contents of the "FollowNavi ® " platform, which leverages LINE ® for the follow-up system, there was no significant improvement in the dental consultation, checkup rates, or HbA1C levels in patients with diabetes. However, the awareness and understanding of oral care improved, and there was an increase in dental consultations at pharmacies. The results of this study, which confirmed the usefulness of dental follow-ups for patients with diabetes regardless of the prescribed medication, are useful when considering the expansion of follow-ups in the future and ways of involvement in the dental field. | Study | biomedical | en | 0.999998 |
PMC11699589 | Joint degeneration characterized by cartilage deterioration and bone wear is the hallmark of osteoarthritis (OA), a condition that worsens over time . This debilitating disorder often manifests as persistent discomfort, potentially leading to impaired mobility and function. The impact of OA extends beyond the physical changes in joint structures, as the chronic pain associated with this condition can significantly compromise an individual's ability to perform daily activities . Epidemiological data indicate that the Americas bear the highest overall OA burden globally, while Asia demonstrates a disproportionately high prevalence of knee-specific OA. To mitigate this substantial health challenge , it is needed to implement comprehensive prevention strategies and targeted interventions aimed at addressing modifiable risk factors associated with OA in order to reduce the increase in cases annually . One of the main reasons for the increase in the number of patients suffering from OA is the increase in the number of aged populations with other risk factors, such as obesity and not performing physical activities . Total knee arthroplasty (TKA), typically performed in advanced osteoarthritis cases, offers modest improvements in proprioception with higher safety than other interventions . However, some sensory deficits may persist post-surgery . Additionally, patients often experience significant quadriceps weakness on the operated side, with strength potentially reduced by up to 30% compared to the unaffected limb . These strength and balance impairments can lead to various functional issues, including uneven weight distribution between legs, compromised balance, modified movement patterns, and overall reduced physical functionality . TKA can be performed using cemented, cementless, or hybrid fixation methods. Previous meta-analyses have indicated no significant differences in clinical outcomes among these fixation techniques, with the availability of these techniques being performed in a conventional way or robotic-assisted . Most of the studies use low-intensity training as a therapeutic procedure, although recent research indicates that higher-intensity training protocols may yield superior outcomes for knee rehabilitation . These more vigorous exercise regimens are associated with improved functional strengthening of the knee and enhanced overall knee function . Importantly, these intensive training approaches have been found to maintain safety standards, suggesting they can be effectively implemented without increasing risk to patients . This study's objective is to evaluate and contrast the safety and efficacy of intensive therapy training (ITT) versus conventional therapy training (CTT) programs implemented preoperatively for TKA patients. We seek to provide clear guidance for clinical professionals on optimizing preoperative physical therapy to enhance recovery, improve functional outcomes, and ensure patient safety. We performed our meta-analysis according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist and the guidelines that were mentioned in the Cochrane Handbook for Systematic Reviews and Meta-Analysis . The PICOS (Population, Intervention, Comparison, Outcome, and Study Design) framework was used to guide the development of search terms, strategy, and inclusion criteria for the study . The framework components were defined as follows: the population included individuals who recently underwent knee arthroplasty and performed preoperative exercise; the intervention was pre-operative intensive physical therapy; the comparison was standard pre-operative physical therapy; the outcomes included clinical measures and questionnaire results; and the study designs considered were randomized and non-randomized controlled trials. Only English articles with full texts were included To maintain data integrity, two independent researchers extracted information from the selected trials. In cases of disagreement, additional authors provided oversight and facilitated consensus. We used two different tools according to the study type: for randomized controlled trials (RCTs), the Cochrane Risk of Bias (RoB) 2 (Cochrane Methods, London, UK) tool was utilized, evaluating five critical domains . Non-randomized studies were assessed using the Risk of Bias in Non-randomized Studies of Interventions (ROBINS-I) tool (Cochrane Methods, London, UK) . Data extraction was performed independently by two authors using Excel spreadsheets (Microsoft Corporation, Redmond, USA), encompassing three main categories of information. The first category, summary data, included study timing and locations, design, protocol number, total patient count, ITT and CTT details, follow-up duration, and primary outcome. The second category covered baseline patient characteristics such as study groups, age, gender, BMI, height, and knee arthroplasty laterality and location. The third category focused on clinical outcomes, further divided into physical measures (including 6 or 10-minute walk test, quadriceps strength, range of motion for extension and flexion, isometric knee extension and flexion stair test, and Timed Up and Go test) and questionnaires (comprising the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and Visual Analog Scale (VAS) . The analytical component of the study utilized Review Manager version 5.4 software (Cochrane Methods, London, UK). For continuous outcomes, results were presented as mean differences (MD) with 95% confidence intervals . Statistical significance was established at a p-value threshold of 0.05. Heterogeneity assessment employed χ2 and I-square (I2) tests, with heterogeneity considered present when the χ2 p-value was below 0.1 or the I2 value exceeded 50%. In cases where heterogeneity was detected, a random-effects model was applied; otherwise, a fixed-effects model was used. When heterogeneity persisted despite the use of a random-effects model, a leave-one-out sensitivity analysis was conducted. After applying our search strategy, we found a total of 917 articles, and after the removal of the duplicates, a total of 605 studies proceeded for the title and abstract screening. We performed a title and abstract screening that led to the elimination of 561 articles. Finally, after full-text screening, a total of seven studies were included. The full, detailed PRISMA flowchart (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) is shown in Figure 1 . Details of excluded studies after full-text screening are in the Appendices. Seven studies were included, with a total of 490 participants. The locations of the studies were Japan, China, Switzerland, and Greece. Various rehabilitation programs were used, whether for intensive therapy or the conventional ways. There are some differences regarding the type of exercise, duration, and number of sessions. The follow-up duration ranged from three months to 13 months. Full details about the summary characteristics are shown in Table 1 . The majority of the included participants were females and elderly people more than 60 years of age. The BMI of the included population ranged from 29 to 35. The majority of the patients had unilateral knee arthroplasty. Full details about the baseline characteristics are shown in Table 2 The risk of bias assessment using the RoB 2 tool revealed varying levels of potential bias across the included studies. No studies demonstrated a high risk of bias. However, three studies were associated with some concerns due to randomization domain or others as deviation from the intended intervention or missing outcome domain. The remaining two RCTs were associated with a low risk of bias. A comprehensive visual representation of these assessments is provided in Figure 2 . On the other hand, regarding the ROBINS-I, one study showed a low risk of bias, and the other showed a serious risk due to bias in the measurement of the outcomes domain . Six- or 10-minute walk test (meters): In the follow-up of one to two months, two studies were included, and the result showed a significant increase in the distance that patients were able to walk in the ITT when compared to CTT with results of (MD = 47.71, 95% CI). Moreover, in the follow-up period of three to six months, there were significant differences between the two comparators (MD = 42.62, 95% CI (23.15 to 62.10), p-value < 0.0001). The overall results demonstrated a significant difference favoring ITT, and pooled results were (MD = 45.07, 95% CI (31.04 to 59.1), P-value < 0.000001). The overall results of the study were homogenous as p-value = 0.34 and I2 =11% . Isometric knee extension (Kg): In the follow-up period ranging from one to two months, there was no significant difference in the isometric knee extension (MD = 1.06, 95% CI (-2.09 to 4.20), P-value = 0.51). Additionally, the follow-up period ranging from three to six months showed a significance favoring CTT over ITT (MD = 7.83, 95% CI (4.11 to 11.55), p-value < 0.0001). Regarding the long-term follow-up (12 months), only one study was included, with no significant difference between the comparators. The overall results favored the CTT over ITT (MD = 3.45, 95% CI (0.49 to 6.41), p-value = 0.02) . Isometric knee flexion (Kg): In the follow-up period ranging from one to two months, there was no significant difference in the isometric knee flexion. Additionally, the follow-up period ranging from three to six months showed no significant difference between CTT and ITT (MD = 2.97, 95% CI (-0.21 to 6.16), p-value =0.07). Regarding the long-term follow-up (12 months), only one study was included with no significant difference between the comparators. The overall results favored the ITT significantly over CTT (MD = 2.32, 95% CI (0.07 to 4.56), p-value = 0.04). The results were heterogeneous and I2=90% . Quadriceps strength (Kg): In the follow-up period ranging from one to two months, there was a significant difference between ITT and CTT regarding the strength (MD = 0.06, 95% CI (0.02 to 0.10), P-value = 0.0003). The follow-up period ranging from three to six months showed another significant difference favoring ITT (MD = 0.08, 95% CI (0.02 to 0.13), p-value =0.005). The overall results favored the ITT over CTT (MD = 0.07, 95% CI (0.07 to 0.10), p-value <0.0001). The results were heterogeneous and I2=90% . ROM extension: In the follow-up period ranging from one to two months, there was no significant difference between ITT and CTT regarding the extension. The follow-up period ranging from three to six months showed another insignificant difference between the two comparator groups. The results were heterogeneous and I2=83% . ROM flexion: In the follow-up period ranging from one to two months, there was no significant difference between ITT and CTT regarding the flexion. The follow-up period ranging from three to six months showed another insignificant difference between the two comparator groups. Finally, the overall result showed that ITT was significantly improved in comparison to CTT (MD = 4.29, 95% CI (0.35 to 8.22), p-value = 0.03). The results were homogenous (p-value= 0.77) and I2=0% . SF-36 physical component: In the follow-up period ranging from one to two months, two studies were included showing a significant difference in the SF-36 (MD = 0.96, 95% CI (0.43 to 1.48), p-value = 0.0003). Additionally, the follow-up period ranging from three to six months showed a significant difference between CTT and ITT (MD = 1.00, 95% CI (0.09 to 1.91), p-value =0.03). Regarding the long-term follow-up (12 months), only one study was included with a significant difference between the comparators. The overall results favored the ITT over CTT (MD = 2.32, 95% CI (0.07 to 4.56), p-value = 0.04). The results were heterogeneous and I2=90% . Stair test (seconds): In the follow-up period ranging from one to two months, two studies were included showing a significant difference in the stair test (MD = -3.21, 95% CI (-4.76 to -1.67), p-value < 0.0001). Additionally, the follow-up period ranging from three to six months showed a non-significant difference between CTT and ITT (MD = -2.30, 95% CI (-5.54 to 0.93), p-value =0.16). Regarding the long-term follow-up (12 months), only one study was included with a non-significant difference between the comparators. The overall results favored the ITT over CTT (MD = -2.01, 95% CI (-3.61 to -0.41), p-value = 0.01). The results were heterogeneous and I2=89% . Timed up and go: In the follow-up period ranging from one to two months, two studies were included showing a non-significant difference in the time up and go (MD = -0.21, 95% CI (-4.76 to -4.35), p-value =0.93). Additionally, the follow-up period ranging from three to six months showed a significant reduction in time favoring ITT (MD = -1.26, 95% CI (-2.43 to -0.09), p-value =0.04). Regarding the long-term follow-up (12 months), only one study was included with a non-significant difference between the comparators. The overall results favored the ITT over CTT (MD = -1.12, 95% CI (-2.04 to -0.20), p-value = 0.02). The results were heterogeneous (p-value= 0.003) and I2=75% . VAS score: No results were found significant according to the included studies at the VAS score results. In the follow-up period, which ranged from one month to two months, three studies were included, and the result showed a non-significant decrease in pain in the ITT on VAS when compared to CTT. Additionally, in the follow-up ranging from three to six months, there were no significant differences between the two comparators with three included papers. The long-term follow-up of 12 months was only mentioned in one study without any significant difference. The results of the outcome were heterogeneous as p-value <0.0001 and I2 = 85% . WOMAC score: Results showed a significant difference according to the included studies on WOMAC score. In the follow-up period that ranged from one to two months, two studies were included, and the result showed a significant difference in the ITT on WOMAC score when compared to CTT (MD = -10.87, 95% CI (-19.23 to -2.51), p-value = 0.01). Additionally, in the follow-up ranging from three to six months, there was a significant difference between the two comparators with two included papers (MD = -6.22, 95% CI (-8.70 to -3.74), p-value <0.00001). The overall results showed a significant difference in favoring ITT over CTT (MD = -8.43, 95% CI (-13.63 to -3.22), p-value = 0.28). The results of the outcome were heterogeneous as P-value < 0.0001 and I2 = 87% . This systematic review and meta-analysis analyzed seven studies comparing ITT to CTT for knee arthroplasty patients. The study populations were predominantly female and elderly, with no weight specification. The results demonstrated that ITT generally outperformed CTT across several outcomes. Significant improvements were observed in walking tests, quadriceps strength, range of motion flexion, physical component scores, stair tests, timed up and go tests, and WOMAC scores. These benefits were noted across various follow-up periods, ranging from short-term to medium-term and long-term post-intervention. However, the results showed CTT showed better outcomes in isometric knee extension, particularly in the medium-term follow-up period The selection of appropriate training intensity and duration is critical for optimizing muscle strength gains. While many previous studies focused on low to moderate-intensity regimens , recent research has highlighted the potential benefits of higher-intensity approaches . Notably, a randomized controlled trial demonstrated that preoperative high-intensity strength training can yield significant improvements. This intensive training was found to reduce pain levels, significantly increase lower limb muscle strength, and enhance performance in functional tasks, suggesting that higher-intensity protocols may offer superior outcomes for patients preparing for knee surgery . Rooks et al. found that preoperative progressive resistance training improved postoperative functional performance and muscle strength but did not significantly affect patient-reported outcomes . This suggests a potential disconnect between objective physical improvements and patients' subjective experiences. A comprehensive approach to TKA rehabilitation should integrate both preoperative and postoperative physiotherapy programs . Preoperative training for patients with end-stage osteoarthritis has been shown to enhance initial outcomes and improve patient satisfaction following TKA . Conversely, postoperative exercise regimens primarily focus on pain management, edema reduction, and improvement of knee range of motion. This dual-phase approach aims to optimize overall patient outcomes by addressing both preoperative conditioning and postoperative recovery needs . Our study has several strengths. By including only RCTs and non-randomized controlled trials, we ensured the inclusion of high-quality evidence, excluding observational studies to minimize potential biases. Furthermore, our study is the first meta-analysis to focus specifically on the preoperative differentiation between ITT and CTT, addressing a critical gap in the literature. The key guidance from our findings highlights that preoperative physical therapy tailored to intensity levels has significant implications for optimizing TKA outcomes. Specifically, the results suggest that ITT protocols may be more effective in improving functional recovery and postoperative strength, while CTT approaches might still hold value in specific patient populations or settings where ITT is less feasible. Our study also had some limitations, as long-term follow-up (12 months) was limited, with only two studies reporting outcomes at this time point . Moreover, various training programs were introduced with different intervention settings which could add possible heterogeneity and make the results affected. The included studies exhibited considerable variation in exercise training protocols which were observed in the duration of training periods, frequency of sessions, and intensity of exercises. This diversity in intervention approaches may have contributed to heterogeneity among the study populations, potentially influencing the comparability and generalizability of results across studies. It's important to note that although ITT generally appears more effective, individual responses to therapy may vary as there is a high heterogenicity among the included studies. Additionally, taking into consideration that some outcomes had no significant difference in the specified follow-up durations and gave overall significance according to the analysis. Previous meta-analyses have suggested that robotic-assisted TKA may offer certain advantages over conventional surgical techniques, particularly in terms of knee alignment and functional outcomes which may add another factor to be considered for investigation and increase the requirement of having a homogenous population . We recommend more long-term follow-up studies beyond 12 months, which are crucial to assess the sustained benefits of intensive therapy compared to conventional approaches. Given the observed heterogeneity in outcomes, future studies should aim to identify patient subgroups that benefit most from intensive therapy, enabling more personalized rehabilitation approaches. Additionally, economic analyses should evaluate the cost-effectiveness of intensive therapy, considering both short-term resource utilization and long-term functional outcomes. Clinical guidelines for post-operative care in TKA should be reviewed and potentially updated to reflect these benefits. Finally, patient education materials should be developed to inform individuals about the potential advantages of intensive therapy and encourage adherence to more rigorous rehabilitation protocols. Special training programs should be made to fit every participant, considering their health and condition. We conclude that ITT generally demonstrates overall superior outcomes compared to CTT for patients undergoing knee arthroplasty when done preoperatively. ITT showed significant improvements across a range of functional and patient-reported outcomes, including walking capacity, quadriceps strength, range of motion, and quality of life measures. On the other hand, the isometric knee extension showed superiority for the CTT group. We recommend that healthcare providers should consider implementing preoperative intensive therapy protocols for TKA patients due to their superior outcomes across multiple recovery domains. However, further research is needed to optimize these protocols by focusing on specific exercises, frequency, and duration for the best results. | Review | biomedical | en | 0.999998 |
PMC11699598 | Out-of-hospital-cardiac arrest (OHCA) is a significant health concern with approximately 275,000 annual cases in Europe and 420,000 in the United States. 1 Immediate recognition of cardiac arrest, initiation of cardiopulmonary resuscitation (CPR) and early defibrillation are key factors to improve survival. 2 However, bystander CPR are only reported on average in 58 % (range 13 % to 83 %) of episodes. 2 In addition, there is considerable variation in the quality of bystander CPR, with only a minority demonstrating high quality CPR. 3 Simulation studies have shown that video assisted CPR (V-CPR) may improve cardiac arrest recognition, improve the quality of basic CPR and that it can be performed in a real-life setting. 4 , 5 In OHCA, clinical studies show promising results of V-CPR on CPR quality, return-of-spontaneous-circulation (ROSC) rates and survival to hospital discharge. 6 , 7 , 8 , 9 However, there are considerable knowledge gaps regarding possible benefits of applying this technology in patients with cardiac arrest. 10 We present a case with witnessed OHCA, where dispatcher V-CPR was initiated early and considered essential to provide a tailored approach to a complex OHCA. A 58-year-old male with known paroxysmal atrial fibrillation (AF) sustained an OHCA at 9:47 a.m. while changing tires on his jacked-up car together with his son. An emergency call was received at 9:48 a.m. The patient was reported unconscious with agonal respiration and no CPR was performed. Guided by the Norwegian Index for Medical Emergency Assistance, a stepwise tool for decision-making and to guide callers in emergency medical situations by the Emergency Medical Coordination Centre (EMCC), the patient was identified as unresponsive and not breathing normally. 11 The medical dispatcher immediately instructed the two laypersons on-scene to perform CPR, which they did without hesitation. None had previous CPR experience. Emergency medical services (EMS) and the on-call general practitioner were also immediately dispatched. No other emergency resources (e.g. volunteer first responders) were available during the incident. Since there were two persons on-scene, the medical dispatcher decided to initiate a video call (VC) to better guide the ongoing layperson CPR. The video transmission was initiated two minutes after the emergency call and showed high-quality chest compressions, and a patient with apparent spontaneous respiration and limb movements. After three minutes, they paused compressions according to Norwegian CPR guidelines. 12 The medical dispatcher then observed that the patient‘s breathing pattern changed into agonal respiration. When compressions were continued, the respiration was again considered normal. Because the breathing pattern was deemed normal due to well performed chest compressions, the medical dispatcher decided not to instruct in mouth-to-mouth ventilations according to standard CPR guidelines. The patient therefore received compression-only CPR until arrival of EMS at 10.21 am, 33 min after start of bystander CPR, at which point V-CPR was discontinued. The two laypersons alternated in performing CPR during the whole incident prior to EMS arrival. On EMS arrival, the patient was still in cardiac arrest and presented with ventricular fibrillation (VF). A direct current (DC) defibrillation with 200 Joule was delivered by EMS personnel before they continued CPR. The patient obtained ROSC at 10:23 am. However, the ROSC only lasted for 30–40 s, and the patient resumed to VF. Another two DC 200 J defibrillations were delivered, but the patient remained with VF. A physician-staffed air ambulance helicopter arrived on-scene at 10:30 am. With ongoing CPR with the patient in VF, the patient showed signs of life with head and limb movements, verbal sounds and a normal respiration pattern with a respiration rate of 12 breaths per minute during chest-compressions. A fourth DC 200 Joule defibrillation was delivered which resulted in sustained ROSC with AF rhythm at 10:33, 46 min after the time of cardiac arrest. Before transportation a rapid sequence induction with intravenous (iv) analgesia (fentanyl 0.2 mg iv), sedation (ketamine 75 mg iv), muscle relaxation (rocuronium 50 mg iv) and endotracheal intubation was performed without any complications. An arterial line was inserted and an intravenous ketamine infusion (1.5 mg/kg/hour) was started for maintenance of sedation during transport to hospital. The patient was hemodynamically stable in-flight with a blood pressure of 150/80 and a pulse rate of 70–90/min, except a brief period of ventricular tachycardia during take-off from the scene. A dose of intravenous amiodarone 100 mg was administered, and the patient converted to AF. The measured oxygen saturation level was 98 % and the patient was normothermic. Transport to the nearest university hospital was uneventful and he was admitted to hospital at 11:59 a.m. The first arterial blood gas showed a metabolic acidosis with pH 7.20, pCO2 5.7 kPa, pO2 12.2 kPa, base excess −11.2 mmol/L, lactate 5.5 mmol/L, bicarbonate 17 mmol/L and glucose 13.6 mmol/L. Other values were normal. A percutaneous coronary intervention was performed with a successful revascularisation and stent implementation of partly occluded left anterior descending and circumflex arteries. A one-chamber cardioverter-defibrillator was implanted and he was discharged to a local hospital for follow-up on day 11, with full neurological recovery. Access to timely EMS response in time-critical situations (e.g. cardiac arrest) is a vital factor to improve patient outcome. 13 The EMS response time in this case was 33 min, which is often the case in sparsely populated areas, such as rural Norway. 14 However, the massive development of smartphone technology during the last two decades has opened new telemedicine possibilities in modern healthcare. 15 The use of VC to assist callers and EMS has the potential to compensate for longer response and travel times. However, despite considerable interest in this technological development, the evidence of effect on patient survival and outcome in real-life OHCA is limited as the specific effect of V-CPR has been difficult to isolate. Other promising technological developments, such as the use of drones carrying defibrillators in OHCA, have shown the potential for increased access to advanced devices and additional decision-making, especially in rural areas. 16 , 17 However, like V-CPR, there is a need for further testing in real-life situations to evaluate efficiency and how these interventions impact final outcome. 16 , 17 A recent meta -analysis found that V-CPR can improve bystander CPR during simulated cases, but that the process is substantially affected by poor video signals and lack of guidance procedures. 18 Several studies have found improvements in specific items of the CPR process, with better compression rates and hand placement. 10 , 5 , 6 , 7 In our opinion, the main effect of V-CPR in this case was a more tailored approach and thorough guidance of laypersons without previous CPR experience in a complex setting with long-lasting CPR without back-up of present EMS, resulting in high-quality CPR, CPR induced consciousness (CPRIC) and a good neurological outcome. VCs were introduced in 2020 in Norwegian EMCCs for the use at the medical dispatcher’s discretion, in order to increase dispatcher situational awareness and improve the quality of care given by lay persons. 19 Currently, no specific guidelines exist on how VC can be used in the most appropriate way. The solution used in this case was developed by the Norwegian Air Ambulance Foundation and implemented in EMCC nationally in collaboration with the Norwegian Directorate of Health in 2020. 20 The EMCC sends a link to the caller’s mobile phone after approval by the caller, and the video link allows the medical dispatcher to use the available mobile phone camera. 20 , 21 Preliminary evaluations pertaining to general use have described high user-friendliness and that the medical dispatcher’s perception of patient’s acuity was affected in about half of the cases. 19 We strongly believe that video assistance by trained medical personnel has future unsolved potential. However, it is important to recognize the need for 1) clear guidance protocols 2) training in video-assisted dispatch and 3) training in the ability to instruct lay rescuers in psychological distress. It is also important to keep in mind, that the availability of live video transmission from scenes may expose the EMCC dispatchers to unpleasant visual impressions. 22 . Apparent signs of life (i.e. breathing and head/limb movements) were observed in this case during compressions by the laypersons and after arrival of EMS personnel. This situation added extra complexity, which potentially could have affected the decision to continue or stop ongoing CPR. In case reports and a systematic review describing the phenomena, the observation of breathing efforts has been inconsistently reported. 23 , 24 Agonal gasps or breathing efforts also may be present in patients with cardiac arrest not receiving CPR, so this sign may be less sensitive on CPRIC than movements. Increasing number of CPRIC events are reported, most likely due to a combined effect of community CPR responder programs, improved CPR quality and increased focus on the chain of survival. 25 A noteworthy point is that in a prospective study focusing on cognitive experiences in cardiac arrest survivors, only 2 % of patients retrieved visual or auditory awareness following their incident and no patients remembered any experience of pain. 26 In the described case, the patient could not recall any memory of the incident. | Clinical case | clinical | en | 0.999997 |
PMC11699613 | According to the International Agency for Research on Cancer, female breast cancer (BC) had 2.3 million new cases in 2020, making it the most prevalent cancer worldwide . About 7 % of patients die, mainly due to metastasis. Optimizing diagnostics and discovering new prognostic factors are crucial, alongside well-accepted clinico-pathological factors like biomarker status, patient age, comorbidity, tumor size, grade, and lymph node involvement . Recently, the histological features of the acellular tumor microenvironment (TME) have gained attention. High breast tissue density, due to increased collagen, is a significant risk factor for BC. Tumor-associated collagen signatures (TACS) may serve as prognostic markers , correlating with tumor grade and prognosis [ , , ]. Notably, TACS5 (aligned collagen fibers) and TACS6 (randomly aligned fibers) at the tumor's invasive front strongly correlate with poor prognosis . These collagen signatures might act as “migration highways” , facilitating cancer cell dissemination. Cancer cells respond to biomechanical cues from the TME, which influence migration and gene expression [ , , ]. For instance, cells prefer migrating parallel to topographical features , impacting cell morphology and migration efficiency . Matrix alignment, more than stiffness, enhances migration for BC cells by promoting directional persistence . Topography-based contact guidance depends on cell-cell interactions. Epithelial clusters respond less to topographical alignment than single cells . This underscores the role of EMT, where cancer cells gain mesenchymal traits, enhancing interaction with TME cues. EMT is linked to increased BC malignancy [ , , ], suggesting a synergistic relationship with TACS in metastasis. Bioengineering applications aim to understand the relationship between tissue-relevant topographies and EMT, which could advance BC diagnostics and treatment targets. Electrospinning, a technique producing fiber mats mimicking native ECM, is valuable in biomedicine . Electrospun scaffolds serve as drug delivery systems, cancer biosensors, and 3D in vitro models . This study established an electrospun 3D in vitro BC model using poly (ε-caprolactone) (PCL) fibers to mimic TACS. By culturing BC cell lines on this model, the study addressed how TACS-mediated contact guidance and EMT phenotype interrelate, how EMT/MET-like changes influence contact guidance, and how phenotypic changes affect cellular mechanics. This interdisciplinary approach aimed to elucidate the reciprocal dependence of cancer cells and the TME in tumor progression. MCF7 (and MCF7 miR200c_KO) cells, a Luminal A breast cancer cell line, were cultured in EMEM supplemented with 10 % FBS, 1× Pen/Strep, 1× MEM Non-Essential Amino Acids Solution and 2 mM L-glutamine. The HER2-positive breast cancer cell line HCC1954 was grown in RPMI-1640 Medium supplemented with 10 % FBS and 1× Pen/Strep. MDA-MB-231 (and MDA-MB-231 i-miR200c) cells, a triple negative breast cancer (TNBC) cell line, were cultured in high glucose DMEM, and 10 % FBS, 1× Pen/Strep and 2 mM glutamine were added to the medium. For miRNA induction in MDA-MB-231 i-miR200c cells, the medium was equipped with 5 μg/mL doxycycline hydrochloride which was replenished every 48 h. The latter three cell lines were cultured in a humidified atmosphere with 5 % CO 2 at 37 °C. The second TNBC cell line MDA-MB-468 (DSMZ, Braunschweig, Germany) was grown in L-15 Medium supplemented with 20 % FBS and 1× Pen/Strep. These cells were grown in a humidified incubator with 0 % CO 2 at 37 °C. The used electrospinning system was custom made by the Laboratory for Tissue Engineering and Cardiovascular Medical Technology of the Clinic of Cardiac Surgery at the Ludwig-Maximilian-University in Munich. Briefly, the test chamber included air conditioning for organic solvents to evaporate and contained molecular sieves to reduce humidity within the chamber. Either a static aluminum plate or a dynamic rotating cylinder was used as collectors to produce unaligned or aligned fibers, respectively. The cylinder (Ø 150 mm) itself was assembled out of 3D-printed constructs using the Keyence Agilista 3200W printer. The use of non-conductive AR – M2 (Keyence Corp, Osaka JP) acrylic polymer necessitated an additional wrapping step with aluminum foil to enable electrostatic attraction. A NEMA 17 motor (Nanotec Electronic GmbH, Feldkirchen, Germany) connected to a computer via an associated control card was used to drive the cylinder. The speed and direction of rotation were set using the “Plug & Drive Studio” (Nanotec) software. A high-voltage power supply from iseg GmbH (Radeberg, Germany) with a voltage range of 0–30 kV (positive and negative) was used to generate the electric field. The voltage supply was coupled to the needle and the collector by means of a high-voltage cable. The flow rate of the polymeric solution was controlled by a laboratory syringe pump (Fusion 100, Chemyx Inc., Stafford, TX, USA). PCL (20 g) was dissolved in 100 mL of DCM/DMF (40/60) 24 h prior to the electrospinning process. The solution was stirred over night at 400 rpm covered with aluminum foil to avoid evaporation of the organic solvents and degradation of PCL. To increase adhesion of fibers to glass coverslips, coverslips were incubated for 5 min in 4 % ( v/v ) of 3-(Trimethoxysilyl)propyl methacrylate in absolute ethanol. The solution was removed and rinsed 3 times with pure ethanol. Coverslips were subsequently baked at 100 °C for 40 min. To produce cell culture inserts, optimized (methacrylated) coverslips were mounted either on the plate collector or rotating cylinder using double-sided adhesive tape as can be seen in Fig. S1a . The rotation speed was set to 1800 rpm. A 20 mL syringe was loaded with the polymer (PCL) solution and connected to the syringe pump. The flow rate was set to 1.0 mL/h. The high-voltage supply (+7–10 kV) was connected to a needle (Ø 0.7 × 30 mm) placed 21–23 cm away from the collector. Opposite, constant charge was applied to the collector (-1 kV). In case of the rotating cylinder, a sliding contact was used. To assure constant fiber deposition during the 10 min of the electrospinning process, Taylor cone formation was monitored at the tip of the nozzle. As shown in Fig. 1 , fiber-coated coverslips (FCCs) were consequently either subjected to SEM analysis or further processed to fulfill cell culture requirements. To remove excess amounts of organic solvents, FCCs were placed in a chemical hood for 48 h. Afterwards FCCs were sterilized using UV-light for 1 h and thereafter placed into a 24-well plate under sterile conditions where they were exposed to UV-light for another hour. Subsequently, for cell culture work, FCCs were coated with collagen (I/IV) as described elsewhere . Methacrylated glass coverslips coated with collagen were used as reference samples (referred to as blank or conventional) for all methods, if not stated otherwise. For AFM and live-cell imaging purposes, collagen-coated FCCs were fixed onto the surfaces of culture dishes. Therefore, 3 μL of silicon-based glue were placed in the center of the bottom side of the FCCs and let dry under sterile conditions for 1 h prior to cell seeding. For the seeding process, 25.000–50.000 cells were suspended in 200 μL of the respective medium and then slowly pipetted on the scaffold starting at the center and spirally approaching the margin of the FCC with a blob emerging and covering the entire FCC. After 15 min of incubation, allowing cells to sediment onto the scaffold, each well was filled up with pre-warmed medium to 1 mL and subjected to various analyses. Fig. 1 (a, b) SEM image analysis: Upper panel shows representative SEM images of (a) TACS6- and (b) TACS5-like structures (10.000× magnification); mid panel highlights single fiber orientations with same colors representing same fiber orientation; lower panel depicts distribution intensity of fiber orientation (−90°–90°). (c) Fiber diameters and (d) inter-fiber distances of TACS5- and TACS6-like structures analyzed with Fiji software. (e) Residual amount of organic solvents (DCM and DMF) in ng/mg fiber were analyzed with HS-GC-MS. Errors indicate standard error of mean (SEM). Fig. 1 The topography of TACS5- and TACS6-like scaffolds (FCCs) were examined by scanning electron microscopy (SEM) using a JSM-6510LVLGS, 25 kV (JEOL, Peabody, MA, USA). For imaging, scaffolds were sprinkled on a stub covered with double-sided carbon tape and sputter-coated with gold (Ernest Fullan) under vacuum for 60 s. Topographic features including fiber orientation, diameter and density were analyzed from the SEM images by processing with the Fiji imaging software (Version 2.30/1.53q) . Fiber orientation and distribution of orientation were analyzed from representative samples of each collector type using the OrientationJ plug-in (written by Daniel Sage). Fibers represented with the same color indicate same directionality. Fiber diameters and inter-fiber distances were manually calculated from 10.000× images taken from samples of 2 independent electrospinning runs per collector type. Residual DCM and DMF content was analyzed by static headspace-gas chromatography-mass spectrometry (HS-GC-MS). An Agilent Technologies 7890B gas chromatograph (Waldbronn, Germany), equipped with an Agilent J&W DB-624 UI ultra-inert capillary column (6 % cyanopropyl phenyl and 94 % polydimethylsiloxane) 30 m × 0.25 mm × 1.4 μm and an Agilent Technologies 7010B triple quadrupole detector with high efficiency source (HES) was used for analysis. Helium (99.999 %) was used as mobile phase. DCM (HPLC grade), 1,2-dichloroethane (DCE, purity 97 %), DMF (purity HPLC grade), DMF-d7 (purity 99.5 %), and DMSO (purity HPLC grade) were purchased from Sigma-Aldrich (Schnelldorf, Germany). As samples, 15 mg fiber mesh (obtained from three independent electrospinning runs, respectively) were filled into a 20 mL headspace vial, 10 μL of DMF-d7 and DCE (each 10 μg/mL in DMSO) as internal standards were added, and the vial was closed tightly. After sealing, the sample was analyzed by HS-GC-MS (see Table S1 ). The MS was operated in single ion monitoring mode (SIM; EI 70 eV). The retention times and characteristic ions of DCM (5.6 min, m / z 84.1), DCE (internal standard; 8.2 min, m / z 62.1), DMF (9.8 min, m / z 73.2), and DMF-d7 (internal standard; 9.9 min, m / z 80.2) were used for identification and quantification. For detailed information on HS-GC-MS conditions, see Table S1 in the supplements. The cell viability of cells growing on TACS5- and TACS6-like (data not shown) structures was assessed by applying the LIVE/DEAD™ Cell Vitality Assay Kit (Invitrogen™) and subsequent fluorescence microscopy. For each sample, 35.000 MDA-MB-231 cells were seeded on collagen I – coated tissue plates, uncoated FCCs and coated FCCs. All samples were cultivated for 72 h and then stained according the manufacturer's protocol. Untreated cells on conventional culture dishes served as positive (live/red) control, whereas negative (dead/green) control cells were incubated for 30 min with ethanol 70 % ( v/v ) prior to the analysis. Fluorescence images were recorded with the Keyence BZ81000 Fluorescence microscope (Keyence, Osaka, Japan). Images of transmitted light, green and red channel were taken simultaneously with a 20× objective and further analyzed with Fiji software. Percentages of living/dead cells was calculated in duplicates (with each sample >100 cells), counting red/green fluorescence signals (cells) of the respective channels. Confocal laser scanning microscopy was used to assess morphological changes across different cell lines growing on glass coverslips, TACS5- and TACS6-like structures. As performed with the FCCs, coverslips were coated with collagen I and IV. Thereinafter, 35.000–50.000 cells of the respective cell lines were seeded in 24-well plates following the protocol described above. In case of growth factor (GF) treatment, cells were allowed to attach within the first 4 h. Medium was then removed and replenished with GF-containing medium. Samples were either supplied with 10 ng/mL of TGF-β1 or 25 ng/mL of EGF (MDA-MB-468). DMEM medium used for miR200c induction in modified MDA-MB-231 cells contained 5 μg/mL of doxycycline hydrochloride (DOX). After 72 h of incubation, wells were washed 3 times with PBS before cells were fixed for 15 min with a 4 % formaldehyde solution. To stain the actin cytoskeleton, cells were incubated with 8.25 μM rhodamine-phalloidin solution for 40 min. Hereinafter, cells were washed another 3 times with PBS. Nuclei were stained with a 0.5 μg/mL DAPI solution for 10 min. Finally, after an additional washing step, samples were mounted on glass slides using FluorSave and stored at 4 °C until the next day. Fluorescence images were acquired using a confocal laser scanning microscope (Leica SP8 inverted, Software: LAS X, Leica microsystems GmbH, Wetzlar, Germany) equipped with a HC PL APO CS2 63×/1.40 oil immersion objective. Diode lasers (405 nm) and a semiconductor laser OPSL (552 nm) were chosen for excitation. Emission was detected in blue (PMT1: 410–520 nm) and yellow (PMT2: 560 nm–760 nm), respectively. Images were further processed with Fiji image analysis software. Nuclear circularity was calculated as C N = 4 π A P 2 , (n > 30; A: area, P: perimeter), and the cellular aspect ratio as A R = d min d max , (n > 15). The axes (d min and d max ) were drawn manually. Results are given as Whiskers Plots with error bars indicating minimal and maximal values. One-way ANOVA with Bonferroni multiple comparison test was performed in GraphPad Prism software version 5.00 (Graph Pad Software, La Jolla, CA, USA) to calculate P-values at a 95 % confidence interval. Live cell imaging was performed using the Eclipse Ti inverted microscope (Nikon, Düsseldorf, Germany) with a 4/10 phase contrast objective and a charge-coupled device camera (DS-Qi1Mc; Nikon). After 48–72 h of incubation, nuclei of cancer cells were stained with Hoechst 33342 dye (Invitrogen™) according to the manufacturer's protocol. Briefly, cells were washed once with PBS and subsequently incubated for 20 min in the cell culture incubator in 300 μL of 0.5 μg/mL dye in PBS. Afterwards cells were washed another 2 times, and 1 mL of the respective medium was replenished. The 24-well plates were inserted into a 37 °C heating and incubation system that was flushed with actively mixed 5 % CO2 (0 % in case of MDA-MB-468 cells) at a rate of 10 L/h, and the humidity was kept at 80 % to prevent dehydration of cells. The cells were imaged in bright-field, and the nuclei were detected at 405 nm using the integrated fluorescence LED. Time-lapse videos were taken with a time interval of 5 min between images over 24 h. For single-cell trajectories, the position of nuclei was analyzed using the TrackMate plugin within the Fiji software . To extract nuclear circularity trajectory, the image series of the 405 nm channel was manually processed ( Enhance contrast (5.0 %) ≫ 2× Smooth ≫ Convert to mask ) and further analyzed with “Mask detector” function of the TrackMate plugin (see Table 1 ). Table 1 Description of variables calculated with the “Track analyzer” function of the TrackMate plugin (Fiji software) , which was used to evaluate differences in contact guidance amongst cell lines and treatments. Table 1 Track displacement Confinement Ratio Mean directional change Measure of the distance [μm] between first (0 h) and last spot (24 h) of a track. Indicative for how far a cell has migrated away from its starting point but not total distance it has traveled. = Linearity of forward progression = t r a c k d i s p l a c e m e n t [ μ m ] t o t a l d i s t a n c e [ μ m ] The confinement ratio indicates how efficient a cell displaced from its origin. Measures the angle between two succeeding links, which is then averaged over all links of a track. Low values indicate high consistency of directed migration/migratory persistence and vice versa . In 24-well plates, 40.000 cells of the respective cell line were seeded in triplicates. Importantly, to allow for the best possible comparison, all cells were seeded on collagen I coated coverslips or coated FCCs. GF and DOX supply was performed as described above. After 72 h incubation, cells were washed 3-times with PBS. Subsequently, 300 μL of ethanol-containing lysis buffer was added for 15 min. In order to maximize the amount of RNA for analysis, triplicates were pooled together prior to the RNA isolation step. Total RNA was isolated using the PureLink RNA mini kit according to the manufacturer's protocol with additional DNAse I digestion. Subsequently, 500 ng of RNA was used to synthesize cDNA using the High capacity cDNA synthesis kit. In the following, E-cadherin (CDH1)-, Snail (SNAI1)-, Vimentin (VIM)-, and XBP1-specific primers were used to amplify and quantify RNA using Power SYBR™ Green PCR Master Mix and the qTOWER real-time PCR thermal cycler (Analytik Jena, Jena, Germany). C t values were normalized to GAPDH RNA expression, and delta C t values were calculated for the comparison. One-way ANOVA with Bonferroni multiple comparison test was performed in GraphPad Prism software (Graph Pad Software, La Jolla, CA, USA) to calculate P-values at a 95 % confidence interval (n = 4). In 24-well plates, 40.000 cells of the respective cell line were seeded in triplicates. Importantly, to have the best comparison possible, cells were seeded on collagen I coated coverslips or coated FCCs (here: TACS5). DOX was supplied as described above. After 72 h incubation, cells were washed 3 times with PBS prior to cell lyses. Subsequently, 60 μL of proteinase- and phosphatase-inhibitor containing RIPA buffer was added to one well (of a triplicate) and cells were kept on ice for 20 min. Thereinafter, wells were thoroughly scraped and the extract was transferred to the next well of a triplicate. This step was repeated again for the last well of a triplicate (including 20 min of incubation). This step was necessary to maximize the protein yield. Finally, pooled triplicates were transferred into 1.5 mL microcentrifuge tubes. After a 10 min centrifugation step at 4 °C, total protein concentration was assessed according to the manufacturer's protocol (Pierce™ BCA Protein Assay Kit). Gels were loaded with 30 μg protein per sample, and electrophoresis was run for 90 min at 120 mV. Subsequent to 1 h of protein transfer at 100 mV, blots were washed, blocked and incubated overnight using CD44s-, CDH1-, VIM- and GAPDH-specific antibodies. HRP-bound secondary antibody was then added for 1 h under exclusion of light before blots were developed. All stiffness measurements were performed on a JPK Nano Wizard 4 in cell culture media using the AFM's contact mode (force spectroscopy mode). Tipless MLCT-D cantilevers (silicon nitride, resonance frequency 15 kHz, spring constant 0.03 N/m) glued to a glass bead (diameter microscopically determined) were used to indent the cells. These were calibrated with the contact-free method in liquid. The following parameters were used: set point 1 nN, z-length 10 μm and velocity 5 μm/s. The Petri dish was mounted onto the AFM stage and the cells were kept in cell culture media. A digital petri dish heater attached to the AFM stage was used to maintain the physiological temperature of 37 °C during the indentation. For every sample, 3–5 cells were indented overall, and force curves were collected. For measuring nuclear stiffness, the area of the nucleus was selected for the acquisition of force curves. One-way ANOVA with Bonferroni multiple comparison test was performed to calculate P-values at a 95 % confidence interval . For imaging the enhanced imaging QI mode of the AFM and pyramidal tip MLCT-D silicon nitride cantilevers were used (spring constant 0.03 N/m and resonance frequency 15 kHz), and the following parameters were taken: set point 1 nN, z-length 2500 nm, pixel time 40 ms. The acquiring area of the grid was set according to the cell size, and the pixel ratio was 256 ✕ 256. The data were analyzed using JPK data processing software. The stiffness/elastic modulus of the cells (Young's modulus) was calculated with the Hertzian contact model according to Ref. : E = 3 4 ∙ F ∙ ( 1 − υ 2 ) δ 3 ∙ R During electrospinning, the polymer jet from the nozzle experiences physico-chemical instabilities , causing random fiber deposition on the collector ( e.g. plate collector). These instabilities can be mitigated by using a rotating cylinder instead of a static collector, resulting in straight, parallel fibers. For cell culture inserts, glass coverslips were mounted on the collectors and coated with fibers during spinning . Scanning electron microscopy (SEM) images of fiber-coated coverslips (FCCs) revealed strong fiber alignment and straightening (“TACS5-like structures”) for samples spun with the rotating cylinder compared to random deposited fibers (“TACS6-like structures”) obtained from the plate collector . Image analysis confirmed efficient alignment (equally colored fibers) of TACS5-like structures with a narrow peak angle distribution of 30° and an intensity of 80,000, versus the chaotic deposition of TACS6-like structures ( Fig. 1 a, (baseline: 7,000; maximum: 15,000). Both setups had similar fiber diameters and inter-fiber distances , with unaligned fibers slightly larger at 0.760 μm and 2.405 μm compared to 0.664 μm and 2.022 μm for aligned fibers, respectively. Thus, fiber orientation was adjusted without significantly affecting individual fiber characteristics. The possible toxic effects of residual solvents or incompatibilities on cell growth were evaluated. GC analysis showed complete evaporation of DCM (below 10 ng) and negligible DMF residues (1.65 ng/mg), making it unlikely for residual solvents to affect cell viability . However, PCL is highly hydrophobic and unsuitable for cell attachment. To improve tissue mimicry and enhance cellular attachment, collagen coatings were applied. Confocal microscopy confirmed effective collagen deposition on fibers . Biological compatibility of the in vitro model was assessed using a fluorescence-based cell viability assay. Fig. S1c shows that cell viability on coated fibers remained high (4.6 % dead cells), with collagen coating enabling widespread cellular infiltration. Without coating, toxicity tripled to 15.1 %, with cells clustering and indicating poor matrix infiltration. Thus, the hydrophobic nature of the PCL fiber surface, not solvent residues, threatened cell viability. Due to the improved compatibility and bio-similarity of coated fibers, collagen coatings were used in subsequent evaluations. In a previous in vitro breast cancer study, our group established an EMT phenotyping system based on EMT-marker expression, morphological changes, and cellular motility. MCF7 and HCC1954 cell lines were designated EMT-negative, MDA-MB-468 as E/M-hybrid, and MDA-MB-231 as EMT-positive. Significant changes in cellular and nuclear morphologies, such as aspect ratio ( A R ) and nuclear circularity ( C N ), predicted EMT-like changes on the protein level during growth factor-dependent EMT . Morphologically, the three studied cell lines remained unaffected when grown on TACS6-like structures as revealed by confocal imaging . Similarly, growth on TACS5-mimicking scaffold did not induce prominent changes in A R and C N , neither in MCF7 nor in HCC1954 cells . However, TACS5-like fibers caused a significant decrease of A R and C N in MDA-MB-468 cells, indicating pronounced cellular alignment on the scaffold. Cancer cells dispersed within the scaffold in contrast to conventional 2D-cell culture and TACS6-like scaffolds where cells formed sheet-like aggregates. On TACS5-like scaffolds, however, cells lost their cobblestone-like morphology and stretched into spindle-like shape . Similarly, EMT-positive MDA-MB-231 cells aligned with fiber orientation, implying a correlation between EMT-status and cellular alignment . Significant C N alterations were only observed in MDA-MB-468 and MDA-MB-231 cells. Fig. 2 Confocal images (63×) and morphological analysis of (a) MCF7, (b) HCC1954 and (c) MDA-MB-468 cells growing on conventional cell culture dishes and TACS-like scaffolds. The blue fluorescence (DAPI) represents the nucleus and the red fluorescence (Rhodamine-Phalloidin) depicts the actin cytoskeleton. Yellow arrows indicated mesenchymal-like morphologies. The graphs below demonstrate the morphological features, nuclear circularity C N (left) and aspect ratio A R (right), which were analyzed and calculated with the Fiji image software and plotted as Whiskers plot in GraphPad Prism 5. (d) Schematic representation of relative mRNA-expression of EMT-relevant markers (CDH1, VIM, SNAI1, XBP1) in MCF7, HCC1954 and MDA-MB-468 cells after 72 h incubation with growth factors or growth on TACS5-mimics. Colors define changes in relative mRNA expression as shown in the right box (green: down; grey: unchanged; orange: up). (e) Mean migration speed of the latter three cell lines was recorded either on conventional coverslips or on TACS5-mimics. Means were calculated based on three individual videos with n (cells) > 50. Errors indicate standard deviation (SD). (f) Western blot of MDA-MB-468 cells stained for CDH1 and VIM after 72 h incubation on conventional coverslips or TACS5-mimics. Stars indicate statistical significance (∗∗∗P < 0.001; ∗∗P < 0.01; ∗P < 0.05). Stars in brackets (∗) indicate statistical significance after exclusion of 1 out of 4 biological replicates. Fig. 2 To correlate morphological changes on TACS5-like fibers with EMT-related gene expression, qPCR analysis was performed, also after growth factor treatments known to induce EMT-like changes . Expression levels of E-cadherin ( CDH1 ) and Vimentin ( VIM ) and mRNA levels of the EMT transcription factors SNAI1 and XBP1 (X-box binding protein 1) were monitored. Snail is known to downregulate CDH1 and upregulate VIM , while XBP1 has been recently linked to EMT and metastasis, impacting cancer progression and therapy outcomes . Fig. 2 d summarizes relative mRNA expression with green indicating downregulation and orange representing upregulation of gene expression. TACS5-like topographies decisively influenced gene expression depending on the cell line, with accessibility to contact guidance cues increasing with EMT-status . Overall, CDH1 expression was equally downregulated across cell line and stimulatory cue. Fiber topography induced significant upregulation of XBP1 (P < 0.001) in MCF7, VIM (≈1.7 fold) in HCC1954, and XBP1 (≈2.3 fold, P < 0.01), SNAI1 (≈2.1 fold) and VIM (≈1.9 fold) expression in MDA-MB-468. Western blotting confirmed topography-induced downregulation of CDH1 protein levels in MDA-MB-468, while VIM protein levels remained unaffected . Although similar EMT-related genes were affected by contact guidance, growth factor-mediated mRNA expression changes were 2–3 magnitudes higher . Another feature comprised in EMT-like changes is accelerated cellular motility. Live cell imaging estimated mean migration speed on conventional coverslips and TACS5-mimicking scaffolds. Fig. 2 e demonstrates that TACS5-like topographies strongly influenced random cell migration. Mean speeds of MCF7 and HCC1954 cells decreased by 28 % and 38 %, respectively, while velocity of MDA-MB-468 significantly increased by 22 % on the fibrous matrix. Differences in motility and contact guidance among all four cell lines on the TACS5 model were measured. Besides mean migration speed, three trajectory features indicative of directional persistence were analyzed (see Table 1 ). Directional persistence increases with higher track displacement and confinement ratio but decreases with mean directional change. MDA-MB-468 and MDA-MB-231 displaced significantly further (46 % and 144 %) than MCF7 and HCC1954 over 24 h . Fig. S2e shows that MDA-MB-468 cells moved fastest (4.76 nm/s), followed by MDA-MB-231 (3.32 nm/s), MCF7 (2.67 nm/s) and HCC1954 (2.12 nm/s). MDA-MB-231 cells migrated most efficiently along the fibers, with the highest confinement ratio and the lowest mean directional change . Cancer cells gain phenotypic plasticity during EMT-like changes, enhancing cell-substrate interactions within the TME . Efficient migration through a porous matrix requires nuclear deformation, as the nucleus is the greatest cell organelle and dictates cellular migration and invasion in cancer . Since MCF7 and HCC1954 cell migration slowed down on TACS5-mimicking scaffolds, without elongation of cellular and nuclear morphologies , EMT was induced using growth factors (EGF, TGF-β1) and ECM components (collagen I/IV), promoting cancer cell spreading and alignment within the scaffolds . Growth factor treatment reduced cell-cell contacts (green arrows) and increased membrane protrusion (yellow arrows) prominently, fostering a mesenchymal-like morphology (yellow arrows) on TACS5-like mimics, especially in TGF-β1-treated HCC1954 cells. This led to alignment with fiber orientation on the TACS5-mimicking scaffolds and significantly decreased C N values on TACS5-like but not TACS6-like structures indicating the importance of unidirectional fiber orientation and growth factor treatment. Similar results were observed in the MCF7 cell line , where growth factors enhanced cell-fiber interaction and promoted mesenchymal-like morphologies (yellow arrows) in comparison to untreated cells clustered in sheet-like aggregates (green circle). Fig. 3 Morphological alignment of nuclei of HCC1954 cells on (a) TACS5- and (b) TACS6-like scaffolds depending on growth factor stimulation (EGF, TGF-β1) and growth surfaces (collagen I/IV). C N -values were plotted as Whiskers plot. (a, b) The dotted line represents the mean C N -value of control cells growing on conventional culture dishes. (c) Relative mRNA expression of CDH1 and VIM in HCC1954 cells. ΔΔC t -values were calculated after 72 h of either growth on TACS5-mimics and/or treatment with 10 ng/mL TGF- β1 (n = 2). Error bars depict SD. (d-g) Comparison of trajectory analysis of untreated (control) HCC1954 cells and cells treated with TGF-β1 using Fiji software (TrackMate). Displacement, mean migration speed, confinement ration and mean directional change were plotted as whiskers plot (5–95 percentiles). Statistical significance was assessed with a t -test based on > 200 cells/condition. Stars indicate statistical significance (∗∗∗P < 0.001; ∗∗P < 0.01; ∗P < 0.05, ns = not significant). Fig. 3 To evaluate combinatorial cue effects, mRNA expression of CDH1 and VIM in HCC1954 was analyzed. Fig. 3 c displays equally downregulated CDH1 mRNA levels (18–20 %) among the three conditions. However, the combined treatment significantly increased VIM expression, showing a 15.2-fold increase compared to a 11.6-fold increase upon TGF-β1 treatment alone. These results demonstrated that TACS5-mimicking scaffolds alone can induce EMT-like changes in HCC1954 cells which is fostered by combinatorial action with GFs. Trajectory analysis revealed that TGF-β1-induced EMT significantly affected HCC1954 migration on TACS5-scaffolds. Cells migrated 70.5 % further and 39.3 % faster than control cells. With a 23 % increase in confinement ration and 12 % decrease in mean directional change, cells migrated more efficiently on the fibrous matrix, indicating reinforced contact guidance. The data indicated a reciprocal dependence between topographical cue and cancer phenotype. Growth factor-induced EMT-like changes enhanced cancer cells’ ability to use topographical input for cellular migration. To further confirm this observation and explore if MET-like changes reverse these effects, two genetically modified breast cancer cell lines were studied: miR200c knock-out MCF7 cells (EMT cell line), and miR200c-inducible expression in MDA-MB-231 cells (MET cell line) under doxycycline (DOX) exposure . First, CDH1 and VIM mRNA levels were measured in unmodified MCF7 cells (MCF7_WT), miR200c knock-out MCF7 (MCF7_KO), and inducible MDA-MB-231 (MB231_i-miR200c) cells with and without DOX stimulation. MiR200c knock-out in MCF7 decreased CDH1 expression by 50 % and increased VIM mRNA levels 3.2-fold. DOX-induced miR200c expression in MDA-MB-231 cells increased CDH1 mRNA 3.0-fold and decreased VIM expression by 40 % . CDH1 and VIM protein levels changed similarly, and CD44s protein levels, overexpressed in cancer stem cells and during EMT , were partially depleted upon DOX stimulation indicating MET . Fig. 4 (a) Relative expression of CDH1 and VIM mRNA in MCF7_miR200c_KO and DOX-treated MDA-MB-231_i-miR200c (MB231_i-miR200c) cells normalized to wild type (unmodified MCF7 or untreated MB231_i-miR220c, respectively). (b) Western blot of MB231_i-miR200c cells with and without doxycycline-induction stained for EMT-relevant marker CDH1, VIM and CD44s. (c) Whiskers plot showing alterations in C N -values (from 40× images) in MB231_i-miR200c cells with and without doxycycline-induction for growth on TACS5- and TACS6-like structures. One-way ANOVA with Bonferroni multiple comparison test was performed to calculate P-values at a 95 % confidence interval (n (cells) > 50). (d) Corresponding confocal images of (c) at 10× magnification. Cells were stained for nuclei (blue) and the cytoskeleton (red). Comparison of trajectory analysis of (e) WT cells with MCF7_KO or (f) MB231_i-miR200c, respectively, performed with Fiji software (TrackMate). Displacement, mean migration speed, confinement ration and mean directional change were plotted as whiskers plot (5–95 percentiles). Statistical significance was assessed with a t -test based on > 150 cells/condition. Stars indicate statistical significance (∗∗∗P < 0.001; ∗∗P < 0.01; ∗P < 0.05, ns = not significant). Fig. 4 Second, nuclear circularity and cell growth of MB231_i-miR200c cells in TACS5- and TACS6-mimicking scaffolds were monitored. After 48 h, untreated cells aligned with fiber orientation , while DOX-induced miR200c expression impeded morphological polarization on aligned fibers . C N analysis confirmed this with elevated values for DOX-treated cells and no significant differences between TACS5 and TACS6-like scaffolds . Third, live cell imaging-based trajectory analysis evaluated the effect of EMT-like (MCF7_KO) or MET-like (MB231_i-miR200c) changes on cellular motility on TACS5 mimics. miR200c KO increased migration speed significantly but did not improve contact guidance; track displacement remained unchanged, confinement ratio decreased (P < 0.001), and mean directional change increased (P < 0.001) . Conversely, miR200c induction in MDA-MB-231 cells reduced mean speed (WT: 3.625 ± 0.068 nm/s vs. i_miR200c: 3.151 ± 0.064 nm/s) and impaired contact guidance, affecting track displacement, confinement ratio and mean directional change . Overall, MET-like changes restricted contact guidance by TACS-like structures. Total cellular stiffness and nuclear stiffness of single cells were estimated using atomic force microscopy (AFM) on either the whole cell or the nucleus. The Elastic Young's modulus [Pa], a good approximation of cellular stiffness , was quantified. Cell mechanics varied significantly among the four cell lines. As demonstrated in Fig. 5 a, total cellular stiffness decreased with EMT-status, from 769.5 ± 54.5 Pa (MCF7) to 372.1 ± 20.8 Pa (MDA-MB-231). This trend was not observed for nuclear stiffness. While MCF7 cells had the highest Young's modulus (590.0 ± 280.0 Pa), the nuclear stiffness of the other three cell lines did not differ significantly . However, in Fig. 5 c, nuclear stiffness of the most invasive MDA-MB-231 cells contributed to the overall stiffness (92 %), whereas this contribution was less important for the other three cell lines (55–68 %). This indicates that the nucleus of this mesenchymal cell line is the main physical restriction for invasion. Lamins (A/C, B), nuclear envelope proteins, are thought to determine nuclear physical properties. Contrary to the literature , high levels of Lamin B1, instead of Lamin A/C, correlated with increased nuclear stiffness in the studied breast cancer cell lines . These findings imply that EMT induction would decrease total cellular stiffness and nuclear stiffness. To challenge this hypothesis, MDA-MB-468 cells were stimulated with EGF for 24 h or 48 h to undergo EMT, and their biomechanical properties were analyzed . After 24 h, epithelial cancer cells adopted a spindle-like morphology . Contrary to expectations, total cellular stiffness significantly increased upon EGF-treatment, with Young's moduli rising by 39.6 % and 28.9 %, respectively . Nuclear stiffness also increased by 17.6 % within the first 24 h. However, after 48 h, the Young's modulus decreased by 21.4 % below the values of control cells, indicating nuclear softening . To validate these findings, EMT was also induced in HCC1954 cells. Confocal scanning microscopy was used to monitor changes in the actin cytoskeleton, which is key to cell elasticity. Total cellular stiffness significantly increased upon TGF-β1 stimulation , with the Young's modulus rising by 117.9 % , surpassing the EGF-induced increase in MDA-MB-468 cells. However, nuclear stiffness did not change significantly upon TGF-β1 treatment. Image analysis illustrated cytoskeletal rearrangements explaining the dramatic cell stiffening. Control cells had a crosslinked, randomly orientated actin network , while TGF-β1-induced EMT elongated HCC1954 cells and restructured actin bundles into stress fibers . Actin fibers became significantly more parallel compared to the network-like structure in control cells, indicated by increased anisotropy . Next, the effect of cell growth on TACS5-topographies on biomechanical changes was assessed to correlate EMT-marker expression changes with cell mechanics. Cellular stiffness was compared between cells growing on collagen I-coated coverslip and coated fibers. The Young's modulus remained unaffected in MCF7 cells and slightly increased in MDA-MB-231 cells on TACS5-mimicking scaffold. Interestingly, a significant increase in cellular stiffness occurred in HCC1954 and MDA-MB-468 cells growing on the fibrous matrix, with Young's moduli increasing by 72.9 % and 95.2 %, respectively, similar to GF-mediated EMT induction. The goal of this in vitro study was to create a reproducible 3D electrospun matrix that mimics the topography and dimension of tumor-associated collagen signatures found in diseased mammary gland tissue. Two electrospinning collector types, namely a rotating mandrel and a plate collector, were used to mimic TACS5 or TACS6 structures, respectively. SEM images revealed that optimized PCL fibers on coverslips had either a parallel (mandrel) or a random (plate) orientation. Fiber diameters ranged from 400 to 1700 nm with comparable mean inter-fiber distances. Naturally occurring collagen structures have widths from a few hundred nanometers to one micron and inter-fiber distances in breast cancer tissues range from 2 μm to 3 μm . The optimized electrospinning setup imitated the in vivo architecture of collagen fibers by producing scaffolds with TACS5 and TACS6 orientations and appropriate submicron scale and fiber density. With negligible cytotoxicity on collagen-coated fibers, the in vitro model was successfully established. Several earlier studies demonstrated that breast cancer cells acquired a more malignant phenotype with EMT-like changes on fibrous 3D scaffolds compared to conventional 2D cell culture [ , , , , ]. This response to topographical cues, known as contact guidance, includes cellular polarization, alignment, altered cell motility, and gene expression. The transition magnitude depends on the fiber matrix’ physical characteristics and biological aspects of the cells, including scale (nanometric vs. micrometric), topography orientation (unidirectional vs. multidirectional), cell size and type, and the relation between cell size and substrate dimension [ , , , , , , ]. Additionally, the epithelial/mesenchymal-state of a cell influences the degree of contact guidance . This study found that cells with low EMT-status had weak contact guidance on the TACS5-mimicking model, while high EMT status related to strong guidance. TACS6-like structures did not provoke significant morphological changes. Cells were also described to align and migrate directionally on TACS5-like substrates but orientate randomly and migrate on multidirectionally on TACS6-like patterns . In case of MDA-MB-468 cells on TACS5-mimics , cytoskeletal and nuclear morphology changes coincided with EMT-phenotypic gene expression pattern , highlighting the predictive importance of (sub-)cellular morphologies. In contrast, MCF7 cells, which did not significantly change morphology, retained their epithelial expression pattern . Migration on TACS5-like scaffolds was slower in case of MCF7 and HCC1954 cells, while mesenchymal MDA-MB-468 cells demonstrated increased migration speed . The EMT-status was crucial in determining cellular responses, with cells having a partial mesenchymal phenotype showing polarization, pro-EMT transcription, and increased motility. TGF-β1-mediated downregulation of CDH1 and upregulation of VIM in HCC1954 supported alignment and migration within the TACS5-like fiber matrix . This increased scaffold interaction fostered mesenchymal transcription . As shown in Fig. 2 and Fig. S3 epithelial phenotypes in the TACS5-micking matrix tend to establish cell-cell contacts, while mesenchymal phenotypes interact more with the matrix. Focal adhesion-mediated recognition of topographical patterns likely induced cell polarization and actomyosin contractility, modulating EMT-related gene transcription by affecting chromatin arrangement [ , , ]. Ravikrishnan et al. demonstrated that HGF-dependent EMT-induction was necessary for epithelial cells to disintegrate, interact with, and migrate along nanofibers. E-Cadherin-based adherens junctions may have restrained contact guidance by counteracting anisotropic cell-substrate interaction . Additionally, the absence of vimentin in fully epithelial cells may explain impaired contact guidance, as vimentin aligns with grooved patterns in meningeal cells and affects focal adhesion maturation . Since vimentin templates microtubule-mediated directional migration , its presence and upregulation enhances contact guidance. These findings align with observations that miR200c-dependent MET induction in MDA-MB-231 cells downregulates vimentin, significantly reducing contact guidance on TACS5-like topographies . MET induction restricted cellular alignment and directed migration. Conversely, miR200c KO in MCF7 (EMT-induction) only marginally elevated contact guidance, likely because CDH1 mRNA expression still dominated over VIM expression. Biochemically induced EMT resulted in stronger overall EMT-like changes than topographical triggers within the 72 h , corroborating findings by Saha et al. regarding EMT-relevant genes affected by contact guidance. Moreover, to the best of our knowledge, the present study is the first report to link XBP1 to contact guidance, with significant upregulation in two of three cell lines after 72 h of cell growth on the fibers . XBP1, known to upregulate SNAI1 expression and induce EMT-like changes , was upregulated alongside SNAI1 in the MDA-MB-468 cell line. Future studies should clarify XPB1's role in contact guidance, potentially involving ER stress and unfolded protein response (UPR) due to spatial confinements, which could provide survival advantage in cancer cells . Fig. 5 (a) Total cellular stiffness of the 4 breast cancer cell lines shown as Whiskers plot (5–95 percentiles). (b) Nuclear stiffness of the 4 breast cancer cell lines shown as Whiskers plot (5–95 percentiles). (c) Nuclear stiffness stacked over total cellular stiffness. Numbers indicate contribution (in percent) of nuclear stiffness on total cellular stiffness. Impact of nuclear stiffness on total nuclear stiffness increases with EMT-status. Stars indicate statistical significance (∗∗∗P < 0.001; ∗P < 0.05; ns = not significant). Fig. 5 Fig. 6 (a) Transmitted light photography images of MDA-MB-468 cells measured in AFM contact mode. Black arrows indicate analyzed cell. (b) Total cellular stiffness of MDA-MB-468 cells treated with EGF for 24 and 48 h. (c) Nuclear stiffness of MDA-MB-468 cells treated with EGF for 24 and 48 h (c). Total cellular stiffness of HCC1954 cells treated with TGF-β1 for 48 h. (d) Nuclear stiffness of MDA-MB-468 cells treated with TGF-β1 for 48 h. (e) Anisotropy of actin fibers of control HCC1954 cells compared with TGF-β1 stimulated cells ( t -test; ∗∗P < 0.01). Confocal images of (f) untreated and (g) TGF-β1-treated HCC1954 cells showing nuclei (blue) and actin cytoskeleton (red) at 63× magnification. Right panel (zoom-in) highlights actin cytoskeleton architecture to either show a cross-linked network (f) or parallel stress fibers (g). Stars indicate statistical significance (∗∗∗P < 0.001; ns = not significant). Fig. 6 Fig. 7 AFM analysis of (a) MCF7, (b) HCC1954, (c) MDA-MB-468 and (d) MDA-MB-231 cell growth on TACS5-like scaffolds. Left panel shows surface topography. Right panel depicts total cellular stiffness of the respective cell lines growing either on conventional culture dishes or TACS5-mimics. Stars indicate statistical significance ( t -test: ∗∗∗P < 0.001; ∗P < 0.05; ns = not significant). Fig. 7 Cell mechanical properties are defined by the nucleus, the actin cytoskeleton, microtubules, intermediate filaments and cell membrane, and are altered by interactions with adjacent cells and the ECM . EMT-like changes reorganize the cytoskeleton, affecting cell mechanics. AFM indentation revealed that Young's moduli of total cellular stiffness decreased with increasing EMT-status , aligning with cancer cell malignancy [ , , , , , ]. However, probing nuclear stiffness revealed some inconsistencies. In line with earlier studies nuclei were less compliant in highly invasive MDA-MB-468 and MDA-MB-231 cells compared to poorly invasive MCF7 cells . Contrary to literature , nuclei were not stiffer than the surrounding cytoskeleton. Changes in cell mechanics during EMT were also investigated. While some studies report cell stiffening during GF-mediated EMT , others observed softening [ , , ]. This study supports cell stiffening within 24–48 h of GF induction in two cell lines . Phalloidin staining of HCC1954 cells demonstrated strongly aligned actin bundles under TGF-β1 stimulation , correlating stress fibers with increased stiffness [ , , , ]. Moreover, Tavares et al. revealed pre-metastatic cell stiffening before converting into a malignant but softer phenotype. Nuclear stiffness changes in MDA-MB-468 cells also supported initial stiffening followed by softening, highlighting the importance of transition time in biomechanical EMT evaluations. Vimentin, proposed to maintain mechanical integrity during invasion, correlated with cell stiffening in lung and breast cancer cells . However, vimentin expression did not directly correlate with cell stiffness in the studied breast cancer cell lines . MDA-MB-231 cells, with the highest vimentin levels , exhibited the lowest Young's modulus. Further studies on vimentin network assembly and cellular stiffness are needed to better understand context-dependent cell mechanics Presumably, as proposed elsewhere, geometry and organization of the vimentin network, which changes upon EMT, as well as interactions with other cells dictate vimentin's contribution to overall cell mechanics, which seems to be ultimately context-dependent . Cellular mechanics on TACS5 scaffolds conformed to prior findings. MCF7 cells showed no stiffness change in comparison to 2D cell culture , while already polarized MDA-MB-231 cells slightly stiffened. HCC1954 and MDA-MB-468 cells, undergoing EMT-like changes on the TACS5-like scaffold, significantly increased stiffness , similar to biochemically induced EMT. This suggests transient stiffening of breast cancer cells as a new marker for EMT-like changes. In future experiments, longer time intervals of the biomechanical evaluation need to be assessed to further study the kinetics of cellular mechanics of the EMT process. Ideally, understanding and interrupting the mechanical adaption may prevent tumor cell dissemination . Discrepancies with existing literature may stem from different biophysical techniques, culture dishes or probing parameters . Examining single-cell versus epithelial layer mechanics yields distinct outcomes due to cytoskeleton reorganization. AFM parameters likely caused observed deviations, as the cytoskeleton contribution to nuclear stiffness was negligible. This in vitro study provided strong evidence that TACS5-like structures support and promote EMT-like changes in breast cancer cells. Contact guidance highly depended on the EMT-status, not necessarily requiring a full transition to a mesenchymal phenotype. Epithelial depletions or mesenchymal acquisitions creating E/M-hybrids improved single-cell contact guidance of single cells. In vivo, EMT-phenotypic changes and restructuring of the stromal compartment's acellular fraction coincide in breast cancer. This co-action likely creates a positive feedback loop that is sustains tumor progression, ultimately leading to metastasis. A plausible strategy to inhibit this loop is to target initial transition into a (partial) mesenchymal phenotype or disturb biomechanical adaption of cancer cells. While the diagnostic value of TACS is yet to be confirmed, early detection may allow therapeutic prevention of disease progression. | Review | biomedical | en | 0.999997 |
PMC11699620 | Polylactic acid (PLA) stands one of the most studied and applied biodegradable aliphatic polyesters in the biomedical field. Produced from fermentation of sustainable sources like corn syrup, potatoes, and sugar cane, PLA is then synthesized by polymerization of lactic acid. 1 At the end of product life, PLA is degraded hydrolytically into lactic acid, a common metabolic product, and thus considered as biocompatible and environmentally friendly. 2 , 3 , 4 PLA has demonstrated the significant potential, either as a substitute for traditional petrochemical-based polymers in industry or as a prominent biomaterial for various medical applications. 5 Lactic acid is the monomer of PLA, and due to its asymmetric carbon atoms, there are two spatial isomers: the dextrorotatory form called r or d -lactic acid and the levorotatory form called s or l -lactic acid. 6 Since only l -lactic acid exits in mammalian metabolism, d -lactic acid or a mixture of d - and l -lactic acid is generally not advisable in the food, drink, and pharmaceutical industries to avoid metabolic issues. 7 , 8 , 9 The manufacturing process of PLA mainly consists of two steps: the preparation of high-purity lactic acid monomers and the synthesis of high molecular weight PLA. 10 There are two approaches for lactic acid production: chemical synthesis and microbial fermentation. 11 The primary method for chemical synthesis is through the addition reaction of acetic aldehyde and hydrogen cyanide, followed by hydrolysis to obtain lactic acid. The chemical synthesis approach has problems such as limited production capacity, high costs, and production of only racemic lactic acid. 12 Therefore, most of lactic acid worldwide is produced through microbial fermentation. 13 , 14 , 15 Lactic acid bacterial fermentation can be divided into homofermentative fermentation, where glucose is mainly converted by lactic acid bacteria into lactic acid, counting for more than 90% of the products, and heterofermentative fermentation, where glucose is converted by lactic acid bacteria into various products including lactic acid, ethanol, acetic acid, etc. 16 Currently, industrial companies commonly use engineered strains such as recombinant Escherichia coli to efficiently produce lactic acid monomers by adding neutralizing agents like calcium hydroxide during fermentation. 17 This method yields over 140 g per liter, which achieves high fermentation intensity and produces only l -lactic acid or d -lactic acid. 18 Additionally, acid-resistant yeast or Lactobacillus can produce lactic acid in non-neutralizing fermentation systems, though the lactic acid yield is relatively low. 19 , 20 The downstream purification process includes the removal of bacterial cells, separation of by-products, and the refining and purification of lactic acid monomers. 21 Currently, the widely used approach for the separation and purification of lactic acid monomers is the calcium lactate crystallization-acid hydrolysis process, which provides high yield and purity. Due to the technical maturity, operational simplicity, and high efficiency, traditional neutralization precipitation methods are still used in industrial production. Meanwhile, the development of emulsion liquid membrane extraction, reactive distillation, and advanced membrane separation techniques have shown great potential for purifying lactic acid in an energy-efficient manner. 22 , 23 After extraction and purification, the lactic acid monomers are polymerized through a polycondensation reaction to form PLA. There are three primary methods for massive PLA production: direct lactic acid polycondensation, lactic acid azeotropic dehydrative condensation, and ring-opening polymerization. 24 , 25 The direct polycondensation involves the continuous dehydration of lactic acid to form oligomers and then PLA. 26 However, water is slowly diffused in the viscous polymer melt during processing. The residual water in the PLA melt affects the efficiency of the polycondensation reaction and limits the molecular weight and performance of PLA. Therefore, azeotropic dehydrative condensation is developed for high molecular weight PLA, using a high-boiling-point organic solvent to achieve dehydration. The ring-opening polymerization involves the dehydration and polycondensation of lactic acid to form oligomers under high-temperature vacuum conditions, eliminating water. 27 Subsequently, these oligomers undergo depolymerization to produce lactide (cyclic dimer of lactic acid) under reduced pressure. Finally, purified lactide, separated from residual water and lactic acid, undergoes ring-opening polymerization reaction to form PLA. By using various catalysts, the ring-opening polymerization method can achieve both high molecular weight and high optical purity and has been the most used method for large-scale production of PLA. 28 , 29 Furthermore, the strategies for synthesizing PLA are constantly evolving. 30 , 31 , 32 , 33 , 34 In addition to optimizing the process for ring-opening polymerization and developing new catalysts, reports have indicated the successful construction of PLA metabolic pathways in chassis cells such as E. coli , 35 Yarrowia lipolytica , 36 and Saccharomyces cerevisiae , 37 using glucose as a substrate for the biosynthesis of PLA homopolymers. 38 In the biological polymerization process, the key enzyme propionyl-CoA transferase is responsible for converting lactic acid monomers into lactyl-CoA, which is further polymerized into PLA under the catalytic action of polyhydroxyalkanoate synthase. Compared to chemical processes, the biological polymerization of PLA faces challenges such as low polymer content within cells and low molecular weight of the polymers, directly impacting the production costs and material properties of PLA. 35 Currently at the forefront, synthetic biology has also proposed the utilization of cyanobacteria as host cells for synthesizing PLA directly from carbon dioxide. By employing a combination of metabolic engineering and high-density cultivation on a light-driven cyanobacteria platform, one-step biosynthesis of PLA using carbon dioxide achieved a PLA concentration as high as 108.0 mg/L. 39 This “negative carbon” production technology can not only address plastic pollution but also achieve carbon capture, offering multiple benefits on society, economy, and the environment. 40 Properties of PLA depend on its component isomers, molecular weight, annealing time, and processing temperature. 41 Component isomers can affect the crystallinity, melting temperature, and glass transition temperature. 42 PLA with poly l -lactic acid content higher than 90% tends to be crystalline, while a lower proportion often leads to amorphous PLA with reduced melting temperature and glass transition temperature. 43 Molecular weight primarily affects the degradation, mechanical strength, and solubility of polymers. PLA materials with a high molecular weight take approximately 2–8 years for complete resorption. 44 The PLA used in implants, such as orthopedic fixation screws, has a higher molecular weight, providing mechanical strength for a period, allowing the bone fracture to heal properly. In contrast, PLA materials used in drug delivery systems often have lower molecular weights, allowing for relatively rapid degradation and reduced drug accumulation in tissues. 44 Copolymerization of lactic acid and different monomers together may control the degradation time of PLA materials or enhance their hydrophilicity, flexibility, and ductility. 45 , 46 For example, copolymerization of lactic acid and glycolic acid forms poly(lactic-co-glycolic) acid (PLGA), which can shorten the degradation time of PLA from 6 months to 2 months. 47 Since the degradation time is negatively correlated with the content of polyglycolide, the biocompatible PLGA can be used in drug delivery systems with controlled medicine release. 46 , 48 , 49 , 50 , 51 In addition, hydrogels formed by copolymerization of lactide and polyethylene glycol (PEG) exhibit good biocompatibility, adjustable degradation rates, and enhanced flexibility. 48 , 52 These hydrogels can absorb large amounts of water, forming soft gel networks with excellent plasticity, and then be used in drug delivery systems and as cell carrier scaffolds for tissue regeneration engineering. 53 , 54 , 55 Moreover, the PLA-polycaprolactone copolymer is a semi-crystalline, biodegradable thermoplastic with good flexibility and a low melting point (∼60°C). 56 , 57 Compared with PLA, this copolymer demonstrates enhanced flexibility and ductility, reduced brittleness, and improved mechanical properties, making it an ideal material for biodegradable implants and three-dimensional (3D) printing. 58 , 59 Blending PLA with other polymers through melting or solution mixing can significantly change its properties, 60 , 61 offering a cost advantage over copolymerization. 61 , 62 Blending with heat-resistant polymers can be used for heat-resistant flame-retardant materials, while blending with flexible polymers can improve PLA’s toughness and elasticity for 3D printing applications. 63 However, the application of PLA blended materials in the medical field is relatively limited. 64 PLA materials can also be modified by adding fillers or plasticizers. 65 Since PLA is hydrophobic and lacks inherent bioactivity, appropriate fillers can promote cell migration, extracellular matrix deposition, and vascularization. 50 , 55 Calcium-based inorganic fillers, such as calcium phosphate and calcium silicate, have been shown to promote the migration and mineral deposition of mesenchymal stem cells, making them widely applied in PLA modification for bone tissue regeneration. 66 , 67 Various processing techniques have been developed to meet the thermal and mechanical demands of PLA materials, which include but not limited to drying and extrusion, 68 injection molding, 69 injection stretch blow molding, 3 and casting (film and sheet). 70 PLA can also be transformed into films, fibers, particulates, and porous structures of various shapes and sizes through techniques like solvent blending or melt blending. 62 These processes have largely broadened the applications of PLA materials. 70 , 71 PLA can be engineered into nanomaterials, unlocking exciting possibilities for various applications. 72 Advanced techniques like nanoparticle fabrication, electrospinning, and nanocomposite synthesis are employed in producing PLA nanomaterials. 73 These methods enable precise control over the size, shape, and composition of the nanoparticles or nanofibers. For example, PLA nanoparticles have been employed in drug delivery systems to enhance drug solubility and release profiles. 74 PLA nanofibers are utilized in applications such as tissue engineering scaffolds, wound dressings, and filtration membranes due to their high surface area-to-volume ratio and favorable mechanical properties. 75 3D printing is an innovative and forward-looking technology that enables the fabrication of multiscale, biomimetic structures according to specific patient needs and clinical requirements. 76 In clinical applications, 3D printing technology can be utilized to manufacture personalized medical devices, orthopedic scaffolds, artificial tissues, and biomedical models. In the treatment of bone defects, 3D printing, guided by imaging technologies like magnetic resonance and CT scans, can easily reconstruct the required 3D models. This enables the precise manufacturing of bone grafts that match the defect area accurately. 77 Compared with other bio-polymers, PLA is characterized for its biocompatibility, biodegradability, and plasticity in medical applications. Firstly, PLA is not toxic or carcinogenic and it undergoes degradation through non-enzymatic hydrolysis, where its monomer lactic acid is a common metabolic product and eliminated through the tricarboxylic acid cycle. 78 , 79 Secondly, the degradation rate of PLA can be controlled by adjusting factors such as molecular weight, d/l isomer purity, and environment temperature. The degradation time can vary from several hours in drug delivery systems to several months in orthopedic fixation implants, which allows PLA to achieve controlled degradation suitable for various medical treatments and repairs. 44 Thirdly, PLA can be processed into various forms, including films, fibers, particles, and porous structures. 62 New manufacturing techniques such as 3D printing technology and nanomaterials further expand its applications in the medical field. Therefore, PLA materials find a wide range of applications in the clinical treatments. 80 PLA and PLA blends have been investigated for various drug delivery approaches, encompassing nanosystems, hydrogels, films, and fibrous matrices. 81 In orthopedic and dental applications, PLA-based materials have found widespread use as fixation devices such as pins and screws in reconstructive surgeries for various fractures. 82 In tissue regenerative engineering, PLA-based biomaterials bring new developments for bone, ligament and cartilage regeneration. 75 PLA can also be used in wound management, such as surgical sutures. 83 The introduction of controlled release drug delivery has brought a significant transformation in the pharmaceutical sector, which provides the advantages of low side effects and specific targeting abilities. 84 , 85 PLA and its copolymers have been extensively investigated in this field because of their remarkable biocompatibility, biodegradability, low immunogenicity, and desirable mechanical properties. 86 For instance, PLA and PLGA have been approved by FDA for human use in formulations for controlled release drug delivery and as carriers for vaccine antigens. 87 Carried by PLA nanoparticles, macromolecules such as nucleic acids, peptides, proteins, etc., now can be used in therapeutics, by overcoming the challenges of rapid clearance through kidney or liver, enzyme degradation, cell membrane permeability, barriers between blood and other organs. 88 , 89 Additionally, nanoparticle drug delivery system can enhance the performance of various medications beyond conventional formulations, by reducing the size of compounds and modifying the surface characters. 90 For instance, nanoparticle drug delivery system can be utilized to encapsulate drugs, leading to (1) improved solubility especially for hydrophobic drugs, (2) protection from degradation, (3) increased absorption through epithelial barriers and extended circulation in the bloodstream, (4) precise targeting of drugs to specific cells, tissues, or organs, and (5) enhanced cellular uptake. A few methods have been employed to produce PLA nanoparticles, such as single or multiple emulsion, 91 , 92 nano precipitation or salting out, 93 melting based direct compositing method, supercritical fluids technique, 94 and template/mold based technique. Due to the presence of blood-tumor barrier, conventional drugs generally struggle to reach sufficient concentrations at tumor sites, especially for central nervous system tumors that are further blocked by blood-brain barrier. 88 , 95 , 96 In oncology, loading usually hydrophobic chemotherapy drugs onto PLA nanoparticles is a promising approach, which can enhance the targeting effect, increase drug concentration at the tumor site, reduce systemic toxicity, and particularly, facilitate penetration through barriers such as the blood-brain barrier. 97 Additionally, modifications can be made to PLA nanoparticles to change surface chemistry, such as surface charge, influencing the pathways of cellular uptake; or add macromolecules such as antibodies, proteins, nucleic acids, etc., enhancing targeted actions on tumors; or manipulate the chemical composition of nanoparticle themselves, controlling the degradation rate and drug release rate, enhancing drug metabolism kinetics. 98 , 99 , 100 For example, a study employed PEG-PLA nanoparticles to deliver peptides for effective tumor targeting and internalization. The F3 peptide, which specifically bound to the overexpressed nucleolin marker on glioma cells, was used to modify the PEG-PLA nanoparticles, resulting in greater accumulation in the glioma regions. 101 Gene therapy operates on the fundamental idea that polynucleotides delivered into cells can change the expression of a specific protein, leading to therapeutic advantages. This process encompasses the delivery of polynucleotides like DNA, RNA, anti-sense oligonucleotides, and small interfering RNA, either at a specific location or throughout the entire system. 102 Compared to viral vectors, non-viral vectors are mostly non-immunogenic, cost-effective, safer, and capable of carrying more genetic material. Among them, PLA nanoparticles can serve as non-viral carriers to deliver functional nucleic acids. 103 , 104 It has been reported that biodegradable polymeric nanoparticles through the combination of PLA, PLGA, and polyethylenimine exhibited DNA binding capabilities and performed well in vitro. 105 The therapeutic proteins and peptides have been approved by FDA to cure many diseases, including Alzheimer’s disease, diabetes and melanoma. 106 , 107 , 108 The nanoparticle-based systems have significantly enhanced the delivery of antigens for vaccine therapies, which can be custom-designed to resemble cellular components so that they can enter cells through endocytosis and pinocytosis. 109 Tetanus toxoid, serving as a representative antigen, was integrated into either PLA or PEG-PLA nanoparticles to facilitate nasal vaccine administration, and the properties of these formulations were examined both in laboratory settings and in living organisms. 110 PLA-based materials have found extensive use as fixation devices in orthopedic and dental applications. 82 Current orthopedic surgical procedures primarily involve the use of autografts, allografts, and metal and plastic implants. 111 However, metal and plastic implants face various challenges, including low fatigue strength, creep, poor adhesion, and biocompatibility issues with native tissue. Compared to traditional metal fixation materials such as steel or titanium alloys, PLA polymers with modifications to improve mechanical properties and corrosion and creep resistance, can also withstand the loads inside the body. 112 , 113 Moreover, PLA materials have advantages in biodegradability, and the degradation rate can be adjusted by modifications to match the patient’s tissue healing needs. Biodegradability also allows PLA materials to release stress to the affected area over time, facilitating tissue healing. Another significant benefit is the avoidance of a secondary surgical procedure to remove unnecessary hardware. This not only lowers medical expenses but also enables the gradual restoration of tissue function. 82 , 114 PLA polymers have been utilized in orthopedics for the fabrication of biodegradable screws, fixation pins, plates, and suture anchors, 115 which have been increasingly used in fractures in knee, shoulder, ankle, and foot. 116 , 117 The ligaments in knee and ankle joints are susceptible to tears or injuries because they bear weight and are exposed to high stress. During the surgical for reconstructions, orthopedic screws made by biodegradable polymeric are employed either independently or in conjunction with grafts, depending on the nature of the ligament injury. 118 Synthetic bioabsorbable screws have been integrated with bioceramic osteoconductive materials such as calcium phosphates and other composites to create biocomposite-based interference screws. 119 Osteoarthritis is characterized by progressive cartilage degeneration and inflammation, leading to severe joint pain. 120 Traditional intra-articular drug injection therapies often provide only short-term benefits due to the rapid clearance of drugs from the joint space. The transport of drugs into cartilage to reach cellular targets is also hindered by the dense and negatively charged extracellular matrix of cartilage. 121 , 122 Therefore, developing drug delivery systems capable of crossing the cartilage barrier and achieving long-lasting therapeutic responses has become a research focus. Among these, PLA nanoparticles have garnered significant attention due to their controllable degradation properties and modifiable surfaces, which enable prolonged retention of drugs within the joint and penetration of the extracellular matrix to deliver drugs specifically to diseased chondrocytes. Current research directions include the following aspects. (1) Development of synovial joint-targeted hybrid systems: this involves using a combination of hydrogels, liposomes, and nanoparticle carriers to target the synovial joint and alleviate pain and inflammation. 123 , 124 , 125 (2) Drug delivery systems targeting intra-cartilage components: these systems aim to penetrate cartilage by targeting components such as aggrecan, collagen II, and chondrocytes through surface protein or charge modifications, facilitating drug transport through the cartilage. 126 , 127 (3) Steroid-encapsulated polymer microparticles: these microparticles, which have been approved for clinical use, provide prolonged release of steroids to alleviate pain more effectively. 128 , 129 A novel multi-arm cationic nanostructure based on PLA has recently been developed, which possesses 28 covalent drug conjugation sites, allowing it to penetrate the entire thickness of the cartilage at high concentrations. When conjugated with dexamethasone, it achieves prolonged intra-cartilage retention and sustained drug release for up to two weeks. 130 PLA nanoparticles can also be loaded with various regenerative drugs and combined with structures such as hydrogels and cartilage cell membranes to serve as stem cell expansion vectors. 122 , 131 , 132 It has been reported that a novel porous microsphere was made by PLGA and loaded with kartogenin, serving as a stem cell expansion vector. Its advantages include a high cell-carrying capacity (up to 1 × 10 4 cells/mm 3 ) and the ability to effectively protect stem cells, promoting their controlled release in the osteoarthritis microenvironment, and inducing the differentiation of mesenchymal stem cells into chondrocytes . 133 Figure 1 Advantages and strategies of PLA-based targeted drug in osteoarthritis PLA-based targeted drugs in osteoarthritis can persist within the joint cavity for extended periods and effectively translocate across the cartilage barrier due to their controlled degradation properties and modifiable surfaces. 134 This figure illustrates two common strategies for PLA-based drug delivery: hydrogels and microspheres. Upon injection into the joint cavity, both hydrogels and microspheres form repair zones at cartilage defect sites. Stem cells encapsulated within these delivery systems can differentiate into new chondrocytes under the influence of the drugs, facilitating cartilage repair in osteoarthritis. 133 Both delivery systems utilize PLA-based nanofibers as fundamental units, capable of loading therapeutic agents and being modified to target synovium or cartilage. Common drugs in these systems include dexamethasone 135 and kartogenin. 133 The bone formation process, known as osteogenesis, can occur through either endochondral ossification or intramembranous ossification pathways. 136 , 137 In addition to being used as fixation devices, PLA is also utilized as a repair and filler material in orthopedics, dentistry, and neurosurgery. 138 In these fields, PLA can be used for bone defect repairs, providing temporary support and promoting the formation and regeneration of new bone. 139 Common applications include cranial bone plates in cranioplasty, 140 bio-substitute materials for mandibular fractures, bioabsorbable bone plates for orbital floor fracture 141 and filler materials for large bone defects. 142 Repair and filler materials made by PLA can also carry drugs, controlling the release of drugs during slow degradation to maintain the local concentration. The common drugs loaded are antibiotics that inhibit infection and various growth factors that promote tissue regeneration . 48 Moreover, researchers have explored more complex scaffold designs and material modifications to better support bone formation and the healing process. The characteristics required for bone tissue regeneration scaffolds are summarized in Figure 2 . In orthopedics, the latest technologies involve the use of 3D printing and electrospinning to fabricate composite scaffolds comprising organic and inorganic phases. Polyhydroxyapatite/PLA 3D composite scaffolds have been investigated in bone repair and exhibited improved compatibility, bioactivity, and osteoinductivity, with a reduced likelihood of inflammation. 143 Furthermore, the incorporation of antibacterial silver nanoparticles, graphene oxide, and selenium nanoparticles made from PLA materials, on the surface or within bone regeneration scaffolds, has been proven to be highly effective for addressing the issues of infection and accelerating bone regeneration after bone tumor treatment. 144 , 145 Also, coordinated growth factors, such as bone morphogenetic proteins, vascular endothelial growth factors, and fibroblast growth factors can be added to the scaffolds for stable and controlled release within the core region of bone regeneration. 146 , 147 A recent study reported a porous bio-composite PLA scaffolds integrated with nuciferine-loaded chitosan hydrogel through 3D printing, which demonstrated uniform pore distribution, sustained nuciferine release, and favorable cytocompatibility with mouse mesenchymal stem cells, leading to enhanced new bone formation. 148 Figure 2 Bone structure and the characteristics required for biomaterial-based bone regeneration scaffolds Bones are essential components of the human skeletal system with a complex anatomical structure. Osteon is the fundamental structural unit of bone, comprising a central canal, concentric bone matrix layers, and osteocytes embedded within. 136 , 137 Osteocytes are responsible for the production of new bone tissue. The central canal contains blood vessels and nerves, ensuring nutrient supply to the bone. The ordered arrangement of osteons imparts strength and stability to the bone. Biomaterial-based scaffolds for bone regeneration are expected possess several beneficial characteristics. 48 , 139 During ligament injury, the anterior cruciate ligament that lacks vascular tissue is typically difficult to heal and thus biomaterial-based regeneration has been employed. 149 Multiphasic scaffolds, designed to mimic the process of ligament-cartilage-bone regeneration, have been investigated for osteochondral regeneration. 150 In a recent report, braided PLGA is used to build multiphasic scaffolds, where the upper phase includes PLGA microspheres to encourage the development of non-calcified cartilage, the middle phase includes a small amount of bioglass added to PLGA microspheres to aid in cartilage tissue calcification, and the lower phase comprises PLGA microspheres with a higher bioglass concentration, promoting bone regeneration. 151 In the mid-to-late stages of osteoarthritis, when irreversible cartilage defects occur, drug treatments become very limited. At this point, additional surgical interventions are required. The main clinical treatments currently include microfracture, osteochondral transplantation, and autologous chondrocyte implantation. A recent study reported a novel 3D nanocomposite scaffold made from poly-ε-caprolactone and PLA, incorporating transforming growth factor β 1 (TGF-β1)-loaded chitosan-dextran nanoparticles, and fabricated using the electrospinning method to create a bead-free, semi-aligned nanofiber structure. The scaffold’s advantages include its biomimetic properties, high hydrophilicity, high porosity, and the sustained release of TGF-β1, which collectively enhance the expression of aggrecan and collagen type Ι genes, crucial for cartilage tissue engineering . 152 Figure 3 From repair fillers to composite and multiphase tissue regenerative scaffolds (A) In orthopedics, PLA was initially used for manufacturing repair fillers for bone defects. 139 These fillers are dense in structure and possess high strength, gradually degrading over time within the bone defect area while releasing antibiotics and bone regeneration-promoting growth factors. 48 As the filler degrades, new bone tissue grows into the defect site. With advancements in stem cell technology and 3D printing, repair fillers have evolved into composite tissue regenerative scaffolds. These scaffolds feature larger pores and are composed of both inorganic and organic phases. 153 The inorganic phase includes fillers such as hydroxyapatite, which promotes bone mineral deposition. 154 The organic phase is loaded with stem cells, antibiotics, and growth factors. (B) According to clinical practice, the latest multiphase scaffolds are frequently used for complex defects spanning bone, ligament, and joint tissues. 50 These scaffolds provide more comprehensive tissue repair and regeneration by replicating the natural transitions between different types of tissues. A multiphase bone-ligament scaffold for treating anterior cruciate ligament injuries. The scaffold includes three regions: the ligament region directly connecting with the ligament repair material, the bone region containing anchor points for fixation and a porous scaffold to promote bone regeneration, and the bone-ligament interface region replicating the gradual transition from fibrocartilage to bone, inducing a mechanical fixation to long-term biological fixation between the implant and the host bone. 151 , 155 The concept of tissue regenerative engineering has arisen from the fusion of tissue engineering, advanced materials science, stem cell research, and developmental biology with the aim of regenerating complex tissues that have been damaged. 156 Advanced materials science offers scaffolds featuring precise geometry and architecture, ensuring sufficient mechanical support and potentially regulating cellular activities by delivering chemical and biochemical materials in both spatial and temporal control. 156 The PLA-based nanofibers in tissue engineering possess unique characteristics, such as a high surface area-to-volume ratio, high porosity, and excellent mechanical strength. 105 Additionally, PLA nanoparticles containing growth factors or antimicrobial agents can also be embedded in regenerative scaffolds. 157 PLA nanoparticles can also be coated on the surface of regenerative scaffolds to improve the compatibility of the scaffold’s surface with tissue materials. 158 Additionally, the polymer surface can be modified by adding chemically charged end groups (e.g., –OH – , –COO – , and –NH 3 + ) to increase protein adsorption, or incorporating peptide segments or synthetically engineered proteins to enhance cell receptor binding and guide cell migration. 159 The effective treatment of severe peripheral nerve injuries remains an unmet clinical challenge, and biomaterials provide a hopeful avenue for stimulating nerve regeneration. 160 , 161 , 162 Scaffolds used in neural tissue engineering are typically manufactured in hollow tube-like structures, characterized for biocompatibility and biodegradability. They are designed to be neuro-compatible to support cell growth and the release of neurotrophic factors, thereby promoting regeneration. 163 , 164 Recent study reported that a multi-channel scaffolds made from electrospun poly- l -lactic acid and poly-ε-caprolactone have been developed, in which a suspension of autologous adipose-derived stromal and stem cells was injected during implantation in order to bridge a 10 mm gap in the rat sciatic nerve. 143 However, while the scaffold was observed to support nerve regeneration during the 4 weeks recovery period, the implanted cells also induced an inflammatory response. 165 Further research has reported the enhancement of neural repair in rats following spinal cord injury, by integrating PLA nano scaffolds fabricated using electrospinning and hydrogel coatings on the scaffold surfaces. Additionally, regenerative factors like brain-derived neurotrophic factor and stromal cell-derived factor-1α were incorporated into both the scaffolds and coatings, which released over time and promoted neural repair . 164 Figure 4 Hollow tubular PLA scaffolds in tissue regeneration engineering In tissue engineering, PLA scaffolds with a hollow tubular scaffold play a significant role in nerve tissue regeneration and cardiovascular tissue regeneration. This scaffold can mimic the morphology of natural tissues, such as nerve conduits and blood vessels, providing an appropriate environment for cell growth, tissue repair, and drug delivery. (A) Nerve regenerative scaffolds are commonly used to repair and regenerate damaged nerves, particularly in spinal cord 164 and peripheral nerve injuries. 166 A typical structure consists of a hollow tubular form, composed of PLA nanofibers coated with a hydrogel layer on the surface as the basic unit. This scaffold can carry neurotrophic factors and tissue growth factors that are gradually released over time to promote nerve cell growth and repair. (B) Vascular regenerative scaffolds are primarily used for small-diameter blood vessels and myocardial repair, divided into two common structures. The first is a multilayer tubular electrospun scaffold, featuring a porous outer layer that supports cell growth and tissue repair, facilitating the adhesion of endothelial cells and fibroblasts, and promoting capillary infiltration and endothelial regeneration. 167 The inner layer is drug-loaded, incorporating stem cells, antibiotics, and tissue regeneration factors. 168 The second structure is a multilayer rolled structure made from layered PLA composites, loaded with vascular-related cells. After rolling, it forms an arrangement of three types of vascular-related cells (endothelial cells, smooth muscle cells, and fibroblasts) from the inner to outer layers, mimicking the structure of artificial blood vessels. The inner layer degrades slowly to maintain vascular stability, while the outer layer degrades to promote cell growth and angiogenesis. 169 , 170 Cardiovascular disease is the most common cause of death globally, prompting a massive need of cardiovascular tissue engineering. 171 PLA nanofibrous have been used in vascular tissue engineering, especially in small-diameter blood vessels and cardiac muscle repairing. 172 , 173 The model of PLA fibrous vascular scaffolds, like nerve conduit scaffolds, is a tube with a hollow lumen. 73 Researchers have built a double-layered electrospun scaffold composed of a micro-scale PLA fibrous outer layer and an inner layer made by silk fibroin-gelatin nanofibrous. The outer layer was found to promote the growth and multiplication of mouse fibroblasts, while the inner layer facilitated the adhesion and growth of human umbilical vein endothelial cells. During treatment, there was less inflammatory response and more coinciding between a vascular network forming and the implant denigrating. 174 Additionally, a self-regulating multilayered polymeric rolled structure composed of polycaprolactone and PLGA has been reported for the fabrication of artificial blood vessels. This tubular structure is formed by the stress-induced rolling of composite polymer membranes and is loaded with three types of vascular cells (endothelial cells, smooth muscle cells, and fibroblasts). After rolling, the structure features an inner layer of polycaprolactone and an outer layer of PLGA, with the three cell types arranged in a manner similar to natural blood vessels . During degradation, the inner polycaprolactone layer expands outward while the outer PLGA layer contracts inward, effectively maintaining the structural stability of the artificial vessel and facilitating the growth of the loaded cells. 169 Skin is the body’s primary defense against external factors, and a compromised skin barrier can lead to various problems such as infection, hydration issues, and temperature dysregulation. 175 Nanofibrous mats made by PLA are exceptionally appropriate for promoting skin healing. The mesh structures replicate the natural structure of the dermal bed, aiding in wound protection, moisture retention, protein preservation, and exudate removal. 176 The large surface area-to-volume ratio of nanofiber mesh structures further promotes cell adhesion and growth, thereby facilitating the healing of large-area wounds. 177 Also, PLA nanofibrous mats can incorporate drug delivery systems of antibiotics, 178 silver nanoparticles, 179 or growth factors 180 to prevent infection, facilitate wound healing and promote the maturation of skin tissues. In dental, oral, and craniofacial tissue regenerative engineering, including teeth, dental pulp, periodontal tissues, hard and soft tissues of the craniofacial complex, polymeric materials have a broad range of applications as tissue engineering scaffolds, carriers for cell-based therapies, and delivery devices for drugs and biologics. 54 In oral medicine, PLA materials have been widely applied in the reconstruction and repair of bone tissues, especially in the alveolar bone, sinuses, and temporomandibular joint. The utilization of 3D printing technology to manufacture personalized bi-phasic bone and cartilage scaffolds, followed by seeding the scaffolds with isolated cells and bio-active molecules to promote bone formation, has been demonstrated to achieve favorable clinical outcomes. 181 Research has shown that in periodontal surgery for the treatment of periodontal intrabony defects, the use of nanohydroxyapatite powder in combination with PLA/PLGA as bone replacement grafts has improved postoperative periodontal parameters in patients. 182 Additionally, PLGA is efficacious in promoting chondrogenesis by facilitating the colonization and proliferation of mesenchymal stem cells, and it interacts with chondrocytes and other cells in the temporomandibular joint disc. 183 In periodontal and peri-implant tissue repair and dental pulp regeneration, regenerative scaffolds are primarily used post-periodontal surgery to prevent oral epithelial and soft tissue infiltration into bone defects, and promote the regeneration of periodontal soft and hard tissues. 184 For more complex dental pulp tissue regeneration, PLA materials combined with stem cell therapy hold significant potential. Recently a study presented an innovative approach for dental pulp regeneration by injecting simvastatin-functionalized GelMA cryogel microspheres loaded with stem cells from human exfoliated deciduous teeth. The system enhances stem cell functions, promotes vascularized pulp-like tissue formation, and demonstrates significant potential for clinical use in endodontic regenerative dentistry. 185 PLA and its copolymers have found extensive applications in surgical sutures and wound management. Because of the biocompatibility and biodegradability of PLA, PLA based suture is well-tolerated by the human body and cause less inflammation or foreign body reactions. Also, PLA breaks down naturally in the body, which is particularly beneficial in surgical sutures, as it eliminates the requirement for a second surgery to remove non-absorbable sutures. 54 PLA-based sutures can be engineered to have the necessary strength and flexibility for various surgical applications, so that surgeons can choose sutures with specific properties to suit the type of tissue being sutured. 186 Recent research has reported a new type of antibacterial PLA suture, which uses polyglycolide and polycaprolactone coatings as carriers to load the antibacterial drug ciprofloxacin onto PLA sutures. This drug-loaded antibacterial PLA suture has the dual advantages of being antibacterial and biodegradable. 187 The PLA materials can also serve as novel bandages and dressings for wound healing. Compared to traditional gauze and other biological dressings such as polyvinylpyrrolidone and sodium alginate fibers, PLA-based biomaterials exhibit more collagen deposition, angiogenesis, and cellular activity, resulting in accelerated wound healing. 188 , 189 This is attributed not only to PLA’s good biocompatibility and biodegradability but also to its moisture management properties, which mimic the extracellular matrix by absorbing and transferring moisture in the wound environment. 190 Additionally, when PLA materials are combined with antimicrobial agents such as antibiotics, silver, etc., they demonstrate enhanced ability to prevent wound infections. 191 , 192 Recent research reported a novel sandwich-structured PLA-based dressing for hard-to-heal diabetic wounds. 193 This dressing is composed of an electrospun three-layer structure of PLA-polyvinyl alcohol-PLA. The hydrophilic polyvinyl alcohol inner layer is loaded with metformin hydrochloride, which is slowly released during the wound healing process, promoting the healing of diabetic wounds. The hydrophobic PLA outer layers on both sides are loaded with erythromycin and puerarin, which possess antibacterial properties. This laminate film dressing demonstrated excellent mechanical properties, high water vapor permeability, and promoted wound healing in a diabetic animal model. 194 , 195 Surgical meshes are primarily used for tissue repair and reconstruction surgeries, such as hernia repair and soft tissue repair. The biocompatibility and biodegradability of PLA material make it an ideal choice for surgical meshes. 196 Abdominal wall hernias are conditions where visceral organs protrude due to weakened or lost continuity of the fascia or muscle, typically requiring surgical intervention. 197 In most hernia surgeries, mesh implants are commonly used for reinforcement. PLA materials are often blended with other polymers to enhance performance, improve thermal stability, and increase cell adhesion, as well as to add antimicrobial coatings. 198 , 199 PLA meshes provide a temporary support structure that helps repair abdominal wall defects while allowing new tissue to grow. During the healing process, the PLA mesh gradually degrades and is eventually replaced by newly formed tissue. 200 , 201 According to research, a novel 3D-printed PLA mesh combined with acellular dermal matrix composite material was used to repair abdominal wall defects in rats. This composite scaffold can effectively reduce surrounding inflammation and significantly promote the repair of abdominal wall defects. 202 Inserting cardiovascular stents is a crucial therapeutic approach for addressing coronary artery diseases. Compared with bare-metal stents, the development of bio-resorbable stents provides new potentials. 203 The degradation of bio-resorbable stents left no foreign objects in the blood vessel for an extended period, and thus the risks of late and very late stent thrombosis may be reduced or even eliminated. 204 The PLA polymer-based stents can effectively intermediate revascularization of coronary artery lesions and demonstrate relatively low rates of major adverse cardiac events in early follow-up. 173 , 205 The Igaki-Tamai stent (Kyoto Medical Planning Co., Ltd., Kyoto, Japan) made of PLA is the pioneering bio-resorbable stent employed in human patients, which undergoes self-expansion at body temperature until it reaches equilibrium with vessel wall dilation and resistance. 206 Biodegradable stents present an appealing alternative to self-expanding stents for managing biliary, coronary, and various duct-related ailments, 207 where other chemical components can be incorporated into PLA to tailor the characteristics, e.g., ranging from soft and elastic materials to rigid and high-strength materials. 208 There are also reports about the implantable, biodegradable and inflatable balloons made by polymers of PLA, 209 where the balloons were developed as a sub-acromial spacer in the treatment of extensive irreparable rotator cuff tears. 210 Antimicrobial materials are crucial in the medical field, particularly in surgical environments, to prevent infections and promote patient recovery. 196 Several additives, including natural compounds, peptides, enzymes, metals, chelating agents, and antibiotics, have been incorporated into PLA polymer matrix to impart antimicrobial activity. 211 , 212 , 213 , 214 , 215 PLA silver ion nanoparticles are synthesized by introducing silver ions into the PLA matrix. These nanoparticles combine the biocompatibility of PLA with the broad-spectrum antimicrobial properties of silver ions, resulting in significant antimicrobial efficacy. 216 Silver ions can disrupt bacterial cell membranes, interfere with bacterial DNA replication, and inhibit protein synthesis, effectively hindering bacterial growth. As a carrier, PLA allows for the sustained release of silver ions, extending the antimicrobial action, and degrades gradually within the body, reducing the side effects in long term. 217 , 218 Recent studies have reported a new antimicrobial surgical retractor manufactured using 3D printing technology with PLA materials, and a thin layer of silver ion PLA nanoparticles is uniformly deposited and fixed on the surface via sonochemical thin-film deposition technology. This retractor exhibits excellent antimicrobial properties and costs only one-tenth of the stainless-steel retractor. 216 Overall, surgical tools such as scalpels, sutures, and needles can be coated or embedded with PLA-metal nanoparticles to create a durable antimicrobial layer that protects wounds from bacterial invasion. 219 Additionally, these nanoparticles can be used to produce antimicrobial dressings, catheters, and implants, providing extensive antimicrobial protection. 220 Controlled biodegradability is considered the core value of PLA materials, but matching the degradation rate precisely with the clinical treatment cycle is still a challenge. 221 The degradation of PLA scaffolds needs to establish a give-and-take relationship with the regeneration of human tissues, and the drugs released from PLA nanoparticles also need to meet the pharmacokinetic demand of targeted tissue. Therefore, research efforts have been made to investigate the degradation rate of PLA materials in different microenvironments of the human body, e.g., tumor microenvironments, skeletal muscle tissues, blood vessels, etc. 222 Additionally, it is worth considering the local inflammatory effects caused by the lactic acid produced during PLA degradation. PLA is generally considered nontoxic and biocompatible, but when it degrades rapidly and the local tissue circulation is restricted, the accumulation of lactic acid may cause a decrease in pH, possibly leading to inflammation and tissue irritation, although the syndrome is usually limited and manageable. 223 , 224 Therefore, PLA material design needs to consider controlling the degradation rate to prevent excessive lactic acid buildup. The tensile strength of PLA is moderate among bioplastics, sufficient for static load applications, and PLA has a high compressive strength, allowing it to withstand substantial compression loads. It also possesses relatively high stiffness and modulus, providing good hardness and stability. 10 , 225 However, PLA has limitations in terms of brittleness and ductility, making it more prone to fracture under impact or tensile stress. 226 Additionally, its heat resistance is insufficient, with a glass transition temperature typically ranging from 60°C to 65°C, which may lead to softening and deformation in vivo. 227 When applied in the human body, the mechanical strength of PLA can become unstable under prolonged exposure to body temperature and humidity. 228 In tissue regeneration scaffolds, the porous and loose structure of PLA is beneficial for cell adhesion and growth, but it also reduces mechanical strength, compromising the long-term stability of the scaffold. 229 Therefore, new modification strategies are needed to expand the application range of PLA materials. Enhancing the mechanical performance of PLA can be achieved through copolymerization, blending, the incorporation of additives or fillers, and optimization of processing techniques. 10 , 225 , 230 , 231 The hydrophobicity of PLA material is a disadvantage for medical applications by impairing cell adhesion. 232 Surface modification methods such as plasma treatment, 233 chemical coating 234 or UV light-induced free radical reactions can be used to increase the hydrophilicity of PLA, 235 and then enhance cell adhesion and extracellular mineralization. 236 Additionally, blending PLA with other hydrophilic materials can increase the hydrophilicity of the composite. 237 However, uneven dispersion of the hydrophilic materials within the PLA matrix may happen during blending, leading to phase separation. 238 For example, when blending natural fibers with PLA, insufficient interfacial adhesion between the fibers and the PLA matrix can lead to uneven fiber distribution and reduced mechanical properties. 167 Using chemical coupling agents or compatibilizers, along with optimizing the blending process, can improve the interface bonding between PLA and hydrophilic materials, reduce uneven dispersion, and thereby enhance the overall mechanical properties and uniformity of the composite. 239 Enhanced hydrophilicity of PLA materials has been shown to improve cell adhesion and proliferation, thereby promoting tissue regeneration and extracellular mineralization in bone repair, and also accelerating wound healing. 236 Currently, 3D printing technology has been widely utilized in the processing of PLA materials, including bone defect implants based on the precise 3D modeling of imaging data, and porous, biologically labeled, multi-phase tissue regenerative scaffolds. 240 , 241 , 242 The newly proposed concept 4D printing refers to the additional feature that products printed in 3D can undergo shape and structural changes when stimulated externally. 243 The combination of PLA materials with 4D printing may find applications in bioresorbable coronary stents. 232 When placed inside blood vessels, a bioresorbable coronary stent changes its shape in response to temperature variations after coronary revascularization and degrades over time to reduce the occurrence of blood clots. Another concept to be mentioned is 3D bioprinting, which produces more complex structures to meet the requirements of multi-level and multi-space regeneration engineering. 244 Currently, PLA has been used in 3D printing to make a cartilage-bone biphasic scaffold for knee joint cartilage repair or a bone-ligament-muscle triphasic scaffold for ligament regeneration. 243 In the field of skin regeneration engineering, multiphase PLA scaffolds have been proposed to include epidermis, dermis, blood vessels, and nerves, along with PLA hydrogels or nanoparticles loaded with antimicrobial drugs and growth factors. Furthermore, 3D bioprinting may integrate stem cell materials into printing inks, which facilitates complete organ regeneration on PLA scaffolds. PLA has established itself as a remarkable and versatile material in the realm of medical applications. Its biocompatibility, biodegradability, controlled degradation, and versatile processing have made it indispensable in various medical fields, ranging from drug delivery systems, fixation devices and implants, tissue regenerative engineering to wound management and beyond. Nevertheless, the PLA material itself still has some drawbacks, which can be further improved in the future through material modification and advanced manufacturing. In summary, the PLA materials have shown a promising future in medical innovation and will contribute significantly to healthcare advancement and patient well-being. | Review | biomedical | en | 0.999995 |
PMC11699633 | Developed and first introduced in France in 2017, the Nutri-Score has now been adopted as the official FoPL in seven European countries, including Germany in 2020. A proposal for a harmonised mandatory FoPL at EU level, as part of the Farm to Fork strategy, is currently pending, with the Nutri-Score being one of the potential labels discussed . Although the use of the Nutri-Score is so far voluntary, food manufacturers or retailers that choose to use the Nutri-Score are obliged in general to display the FoPL on all their products within 2 years . The Nutri-Score undergoes regular revisions, including the most recent one in 2023, with the consistent goal to improve the discrimination between products with high and low contents of unfavourable nutrients. Smaller increments in the point allocation scale for instance for sugar allow for a stricter and therefore more adequate Nutri-Score rating. As the discrimination of sugary products was considered a key focus in this context, breakfast cereals were included as a priority product group . In Germany, the 2023 algorithm was introduced in January 2024, with a 24-month transition period . Data on breakfast cereals were gathered from August to December in 2019 and 2022, respectively, as part of the German monitoring of packaged food. Data collection comprised the energy and nutrient contents as stated in the mandatory nutrition declaration (fat, saturated fat, carbohydrate, sugar, protein, and salt) . Data on fibre was recorded when provided. Where available, additional labelling information such as ingredient lists, label on organic production or Global Trade Item Number (GTIN) was collected. In order to cover the market as broadly as possible, data was predominantly collected online via the manufacturers’ websites. To fill data gaps, the online research was complemented by enquiries with manufacturers and visits to grocery stores. The data were managed with a customised branded food module within the FoodCASE software, version 7.9.1 (Premotec GmbH, Winterthur, Switzerland). In the case of implausible values or missing data concerning energy or nutrient content, manufacturers were contacted to verify, correct, or complete the respective information. In case of no answer, these products were excluded from analysis. Detailed information on the design and methods of the German monitoring of packaged food is published elsewhere . In order to assess whether the nutrient composition of a given breakfast cereal changed over time, a subsample of breakfast cereals present on the market in both survey years was analysed. Such pairing of products over time is not performed in the German monitoring of packaged food but was carried out as part of the EU Joint Action Best-ReMaP, in which Germany took part . Pairing was done manually as follows: (1) GTIN for each product of 2022 was used to identify a matching GTIN in the 2019 data. (2) if there was no result of matching GTINs or no GTIN available, product information, including brand name, product name, legal name, flavour, and net weight were used to search for a match. The ingredient list and the nutritional values could be different, but product or legal name had to be the same or close. If two products of 2022 could be assigned to one product of 2019, the product nearest in net weight was counted as a match. Breakfast cereals that had changed the design over the years, resulting in a different classification of a product as child-targeting or not in 2019 and 2022, were classified according to the 2022 design for the subsample of paired products. The 2023 algorithm of the Nutri-Score for general foods was used to compute the overall nutritional value of breakfast cereals . In brief, ‘unfavourable’ points were allocated for energy (0 to 10 points), saturated fats (0 to 10 points), sugar (0 to 15 points), and salt (0 to 20 points), whereas ‘favourable’ points were allocated to proteins (0 to 7 points) as proxy for iron and calcium, fibres (0 to 5 points), and the percentage of fruits, vegetables, and legumes (henceforth described as F&V component) (0 to 5 points). The ‘unfavourable’ (0 to 55 points in total) and ‘favourable’ points (0 to 17 points in total) are balanced out in the computation of the final nutritional score (FNS). However, ‘favourable’ points for protein are disregarded, if a product scores more than 11 ‘unfavourable’ points. In consideration of this rule, the FNS may range from − 17 (best rating) to 45 points (worst rating). The FNS is attributed a Nutri-Score ranging from A to E (Table 3 ). In our study, products classified as C, D, or E (FNS ≥ 3) are defined as ‘less favourable’. The computation of the Nutri-Score is based on the declared nutritional values per 100 g for energy, saturated fats, sugar, protein, salt, and fibre. Based on the ingredient list, the percentage of F&V was estimated by an experienced nutritionist using the Nutri-Score FAQ document . If the ingredient list was not available on the manufacturer’s website or fibre content was not declared, the product was excluded. All statistical analyses were performed using the software R, version 4.3.2. The normality of the data distribution was tested using the Shapiro-Wilk test and rejected. Consequently, the non-parametric Mann-Whitney test for two independent samples was used to compare energy and nutrient contents as well as the FNS of children’s breakfast cereals respectively non-children’s breakfast cereals between survey years 2019 and 2022. For all results, p-values < 0.05 were considered as statistically significant. Furthermore, shares of pairs that showed a change in FNS were calculated. This subsample is further analysed as the individual scores of the FNS in 2019 and 2022 were plotted against each other and the effects on the Nutri-Score classification described. The comparison between the subsamples revealed significantly lower sugar and significantly higher fat contents in 2022 than in 2019. Children’s breakfast cereals demonstrated a greater percentage change, with a 25.5% reduction in sugar ( p < 0.001) and a 32.0% increase in fat ( p < 0.001) than non-children’s breakfast cereals with − 8.7% ( p < 0.001) and + 7.0% ( p = 0.036), respectively. For energy, no significant change was found ( p = 0.913; p = 0.844). Overall, mean FNS was only significantly reduced in children’s breakfast cereals, by 3 points ( p < 0.001) (see Table 4 ). The share of products with a ‘green’ (A or B) classification as well as products classified as Nutri-Score C was higher in 2022 than in 2019. At the same time, the share of Nutri-score D products was lower. The share of breakfast cereals classified as less favourable (Nutri-Score C-E) was higher for children’s breakfast cereals . At category level of children’s breakfast cereals, a reduction of sugar content was found in other cereal products (12.4%, p = 0.003) and an increase of fat content in muesli (39.0%, p = 0.016) and flakes (149.3%, p = 0.016). For non-children’s breakfast cereals, a reduction of sugar content was shown in muesli (9.2%, p < 0.001) and flakes (25.9%, p = 0.032). The latter also showed an increase in fat content (99.5%, p = 0.031). For details on changes at category level, see Supplement File 1. Table 4 Mean energy and nutrient contents of breakfast cereals surveyed in Germany in 2019 ( n = 888) and corresponding changes in 2022 Breakfast cereals Non-children‘s Children‘s mean in 2019 ( n = 761) mean change in 2022 mean in 2019 ( n = 127) mean change in 2022 ( n = 225) abs. rel. (%) sig. 1 abs. rel. (%) sig. 1 Energy [kcal/100 g] 400.7 + 0.8 + 0.2 390.3 + 1.0 + 0.3 Fat [g/100 g] 10.7 + 0.7 + 7.0 * 5.5 + 1.8 +32.0 *** Saturated Fat [g/100 g] 3.1 0.0 + 0.5 1.6 + 0.5 +33.7 ** Carbohydrate [g/100 g] 60.9 -2.5 -4.1 *** 73.9 -5.6 -7.6 *** Sugar [g/100 g] 15.7 -1.4 -8.7 *** 22.9 -5.8 -25.5 *** Protein [g/100 g] 10.9 + 1.0 + 8.9 *** 8.4 + 1.2 +14.0 *** Salt [g/100 g] 0.25 -0.03 14.1 ** 0.43 -0.20 -45.5 *** Fibre [g/100 g] 2 8.8 + 0.4 + 4.1 * 6.1 + 1.4 +22.8 *** Final nutritional score 3 (FNS) 4.7 -0.3 -5.5 8.7 -3.1 -35.9 *** 1 Statistically significant change in mean content in 2022 compared to 2019 (* p < 0.05; ** p < 0.01; *** p < 0.001) 2 Sample size differs, as the fibre content was not available for 123 resp. 154 non-children’s breakfast cereals and 29 resp. 22 children’s breakfast cereals 3 Sample size differs, as the fibre content and/or ingredient list was not available to calculate the FNS for 123 resp. 156 non-children’s breakfast cereals and 29 resp. 22 children’s breakfast cereals Changes in the FNS that led to a change in the Nutri-Score classification were in the range of -11 to -1 (improvement) and in the range of + 1 to + 9 (deterioration) points, respectively. The most often observed changes in FNS were in the range of -1 to -4 points. Most products achieved an improvement in FNS by reducing sugar (-0.1 to -10 g/100 g), often accompanied by FNS-affecting changes in fibre (+ 0.1 to + 3.0 g/100 g), protein (+ 0.1 to + 3.6 g/100 g), saturated fat (-0.1 to -5.2 g/100 g), or salt (-0.09 to -1.3 g/100 g), or a combination of these. The largest FNS improvements were primarily driven by reducing saturated fat, often by substituting palm oil with sunflower oil, or by reducing salt content. For most products with a reduction of at least 5 FNS points, the number of ‘unfavourable‘ points was below the limit of 11 points, resulting in considering the ‘favourable‘ points for protein content in the product of 2022, which were not counted in the product of 2019. As Fig. 4 illustrates, not only the magnitude of the change in the FNS score was decisive for a change in Nutri-Score classification, but also the initial position of a product (whether it was close to a class threshold or not) as well as the initial class itself. Since many of the products in category D were close to the threshold to category C, relatively small changes of 1–2 FNS points were already sufficient to change the Nutri-Score classification to C. Our sample indicated that, between 2019 and 2022, the number of breakfast cereal products on the German market grew substantially, with concomitant changes in the average nutrient content. Whereas the average sugar content decreased significantly, especially in the group of children’s breakfast cereals, the average fat content increased significantly. Our results are in line with findings from other countries that showed declining sugar contents in this product group in the period between 2004 and 2020 [ 27 – 29 ]. A concomitant significant increase in average fat content as in our study was only found for the Australian market in different subcategories (granola and clusters, hot flavoured and plain cereal (porridge), and muesli) as well as overall in a sample of paired products . Our results on subcategory level showed these concomitant changes only for flakes. This diverse subcategory ranges from plain, unsweetened flakes to sugar- or chocolate-coated flakes high in sugar and fat, respectively. Whether this interaction (fat up, sugar down) shown for the overall non-paired sample is also evident on the single product level was outside the scope of this study but changes in Nutri-Score could help answer this question. A first look at the subsample of paired breakfast cereals with changes in Nutri-Score due to a decrease of negative points by a sugar reduction (e.g. -8.0 g/100 g) indicates that this interaction exists, although the increase in fat was rather small (e.g. 0.5 g/100 g). Pairing the products from different time points revealed a fast-changing breakfast cereal market in Germany: out of 888 products in 2019, only 424 (47.7%) were still on the market in 2022. The product monitoring in France specifically analyses market changes and also shows that the overall market is mainly influenced by the introduction of new products and products that are withdrawn from the market. Between 2011 and 2018, 63% of the sample were newly introduced products . Therefore, the observed changes in average nutrient content are explainable by shifts in the portfolio of products over the years rather than the reformulation of existing products. Compared to 2019, the 2022 subsample contained substantially more mueslis (especially porridges), and these typically have lower sugar content but higher fat content than other categories . On the one hand, these market developments do appear beneficial for public health in terms of improving the food offer, as many mueslis in particular had come onto the market, many of which have a nutritionally favourable composition. Increasing demand for alternatives to classic cereals such as froot loops, honey pops or chocolate crispies (other cereals category), as well as more opportunities for product variation in the muesli category, could be the reason for the growing muesli market, which has had a major impact among children’s cereals in particular. This is supported by a pronounced shift in consumption shares away from other cereals towards muesli, but also rolled oats and cornflakes, in 2021 . Moreover, if a product had been reformulated, the product often fell just below the threshold of the next better Nutri-Score class, which is particularly noticeable in children’s products. One might argue that this again could be an artefact from using the stricter 2023 algorithm of the Nutri-Score, when the 2015 algorithm was the point of reference in 2019 and 2022. However, our observation is paralleled by recent results for the French market and based on the 2015 algorithm: even though the introduction of the Nutri-Score brought breakfast cereals with a more favourable nutritional composition to the market, an accumulation of products just below the threshold of the next better Nutri-Score class was recognised . Hence, products are not necessarily reformulated in a way that would make their being nutritionally favourable visible through a better Nutri-Score. Where improvements in Nutri-Score emerged, this was explained largely by decreases in sugar and salt content and increases in fibre and protein content . Our study extends these findings by exploring how changes in the individual nutrients interrelate to improve the Nutri-Score classification in the end. We found that the improvement was primarily due to a combination of sugar reduction and increase in protein or fibre content. Our analyses also revealed that the number of negative points can be considerably reduced, particularly by lowering saturated fat, as the range between two thresholds (1 g/100 g per level) are comparatively lower than for sugar (4.5 g/100 g per level). In this regard, substituting palm oil through e.g. sunflower oil, seems to be an easy leverage point for mueslis in particular to improve their overall rating, as we could determine for some crunchy mueslis. Further modelling studies at the individual food level are necessary to provide information as to what compositional changes would be the most feasible in order to achieve a better overall Nutri-Score. Although we were able to demonstrate that reformulation within breakfast cereals could be visualised via the Nutri-Score and thus can be an opportunity for food producers to positively highlight their own products in comparison to competing products receiving a less favourable rating, the visibility for consumers appears to remain limited so far. As per current EU regulation , usage of the Nutri-Score is and can only be voluntary. A first survey, conducted just after the implementation of the Nutri-Score in Germany, showed a labelling of less than 10% of the included cereals . A snapshot taken about a year later noted a coverage of 28% . Despite this increase, the data suggest that the vast majority of breakfast cereals on the German market do not carry a Nutri-Score. Unless the Nutri-Score is applied on all products, the empowerment of consumers to quickly identify nutritionally favourable products within a given food category remains impaired. Food manufacturers could send a strong signal of consumer support by adopting the Nutri-Score as the government-endorsed FoPL in Germany on all their products. This is supported by comprehensive evidence reviews concluding that summary indicators with or without colour-coding help consumers best in identifying and choosing the nutritionally favourable option . It must be borne in mind though that the Nutri-Score as a FoPL should not be used as an overall guidance on ‘healthy’ products. Food reformulation is often viewed as one of several means to reduce the prevalence of overweight and obesity. However, as observed in our data and already discussed, measures targeting only individual nutrients (e.g. sugar), can lead in some product groups to an increase in another nutrient with the same or even higher energy content , and may thus not achieve the overall goal of reducing energy density. Gressier et al. showed that reformulation strategies generally resulted in products with the same energy content and are hence unlikely to contribute to a lower energy intake in the population . Since simultaneous reduction in energy content is not always easy to achieve due to technological challenges, it is even more important that reformulation strategies focus on nutritionally favourable products overall, e.g. by increasing the proportion of wholegrain in breakfast cereals. Our study sheds light on several nutrition policy issues. We were able not only to show differences between single nutrients over time in breakfast cereals on the German market but also to compare the Nutri-Score classification, including possible shifts therein due to product reformulation. A strength of the study is the combined investigation of the overall market and the subgroup of paired products. This allows for concrete insights regarding nutritional changes on the overall market as well as for reformulated products . However, no clear conclusion could be drawn regarding the public health impact of the observed change in nutritional composition of the breakfast cereal supply, as the data could not be linked to sales and/or consumption data. Also, thorough as our approach for pairing products from different survey years was, we may have missed products where a change in name or presentation obscured the link. This study showed a reduction in sugar of over 25% in children’s breakfast cereals on the German market between 2019 and 2022, along with a significant improvement of the FNS. Since there were hardly any specific product reformulations, particularly ones that led to an improved Nutri-Score, it can be inferred that changes in the overall sample were mostly driven by shifts in the product portfolio. Unless improvements in nutrient content and FNS are reflected in widely purchased and consumed breakfast cereals, benefit for consumer health will remain limited. Reformulation guided by holistic product monitoring approaches and FoPLs like the Nutri-Score can be seen as synergistic public health tools to improve the nutritional quality of the packaged food supply and enable consumers to make nutritionally more favourable food choices. Using these tools to their fullest potential appears a worthy ambition towards the goal of reducing the prevalence of overweight and obesity and with it the burden on health care systems and people’s health. | Other | biomedical | en | 0.999997 |
PMC11699685 | Sarcoglycanopathies (SGPs) are autosomal recessive limb-girdle muscular dystrophies (LGMDs) caused by mutations in four genes: SGCA , SGCB , SGCG , and SGCD , which lead to α-SGP (LGMDR3), β-SGP (LGMDR4), γ-SGP (LGMDR5), and δ-SGP (LGMDR6), respectively . Since the protein products of SGCA , SGCB , SGCG , and SGCD form a sarcoglycan complex and work together, the symptoms among SGPs are almost similar. However, there are slight differences. For instance, LGMDR4 are more likely to be associated with cardiomyopathy, and LGMDR3 may have a milder symptom and slightly later onset and slower progression . Genetic analyses of patients with SGP from Algeria, China, and Europe have been reported [ 2 – 4 ], demonstrating that many share the same variants, and that alleles harboring variants are carried on specific haplotypes, indicating founder effects underlying SGPs in various countries [ 5 – 8 ]. In contrast, SGPs are relatively rare in Japan, although there have been a few reports of patients with LGMDR3, LGMDR4, or LGMDR5 [ 9 – 11 ]. Previous genetic research into SGPs has focused on variants within exons and their flanking intronic regions; however, technological advances in analysis techniques, including multiplex ligation-dependent probe amplification (MLPA), RNA-seq, and long-read sequencing, have enabled identification of genetic variations in intronic regions, as well as structural genomic changes . Further, the development of artificial intelligence-based prediction tools has contributed to more accurate and cost-effective identification of splice changes . The aims of this study were to profile the genetic variation in Japanese patients with SGP and identify variant founder effects. This retrospective cohort study was performed using samples and datasets sent to the National Center of Neurology and Psychiatry between January 1979 and June 2023. Cases with absent or reduced expression of sarcoglycan, detected by immunohistochemistry, and biallelic variants in SGCA , SGCB , SGCG , or SGCD , were selected for inclusion in these analyses. This study cohort included 39 patients with negative Western blotting result for SGCA . Muscle samples for histological examination were frozen in isopentane cooled with liquid nitrogen. A series of histochemical and immunohistochemical analyses, including assessment of sarcoglycan, were performed for diagnosis. Routine histochemical and immunohistochemical stainings were conducted using standard procedures . Genomic DNA was extracted from peripheral blood or muscle samples from patients, using standard procedures . All exons and flanking intron regions of SGCA , SGCB , SGCG , and SGCD were amplified; some cases were also analyzed by whole genome sequencing. All variants were confirmed by Sanger sequencing. MLPA was conducted to analyze suspected large deletions, as previously described , using SALSA MLPA Probemix P116 SGC (MRC Holland, Amsterdam, The Netherlands), according to the manufacturer’s instructions. We applied RNA-seq for 2 patients. As previously reported , total RNA was extracted from frozen muscle using the RNAeasy mini kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions. Libraries were prepared from total RNA using Ribo-Zero Gold (Illumina), according to the manufacturer’s instructions, and 100 bp paired-end sequencing was performed on the NovaSeq 6000 platform (Macrogen, Seoul, Korea). RNA-seq reads were mapped using Spliced Transcripts Alignments to Reference software ( https://github.com/alexdobin/STAR ). We confirmed the splicing abnormality by RT-PCR for 5 patients. Total RNA was extracted from frozen muscle samples using an RNAeasy mini kit. Complementary DNA was generated by reverse transcription of RNA using SuperScript IV VILO Master Mix (ThermoFisher Scientific, Waltham, MA, USA). Primers are listed in Table S1 (see Additional file). The sequences of the products were confirmed by Sanger sequencing. The pathogenicity of missense variant is predicted to be pathogenic by more than one in silico tool (PolyPhen-2 ( http://genetics.bwh.harvard.edu/pph2/ ), MutationTaster ( http://www.mutationtaster.org/ ), or CADD ( http://cadd.gs.washington.edu/ )). The criteria for pathogenicity by prediction tool include ‘Probably Damaging’ for PolyPhen-2, ‘Disease-causing’ for MutationTaster, and a CADD score of ≥ 20. SpliceAI and MaxEntScan were used to predict the effects of identified single nucleotide variants (SNVs) on aberrant splicing. SpliceAI calculates the changes in activities of splicing sites (AG, gain of acceptor site; AL, loss of acceptor site; DG, gain of donor site; DL, loss of donor site) as delta scores, where higher scores indicate that SNVs are more likely to alter splicing. MaxEntScan assigns a log odds ratio (MaxEnt score) to the sequences of 5' and 3' splice sites, where higher scores indicate higher probabilities that splice sites are used. PDIVAS was used to predict pathogenicity of deep-intronic variants . To verify the protein 3D structure, we applied the software AlphaFold ( https://alphafold.ebi.ac.uk/ ). We also applied the prediction software iPSORT ( https://ipsort.hgc.jp/ ) and WoLF PSORT ( https://wolfpsort.hgc.jp/ ) to predict the subcellular localization of protein based on amino acid sequencing. Long-read sequencing was performed using genomic DNA extracted from patient blood. Library preparation was performed using a Ligation Sequencing Kit (SQK-LSK109; Oxford Nanopore Technologies Inc., Oxford, UK) with CRISPR-Cas9. Guide RNA (crRNA) sequences are listed in Table S2 (see Additional file). Prepared libraries were applied to MinION flow cells and sequenced on an MK1C instrument (Oxford Nanopore Technologies Inc., Oxford, UK). Real-time base calling was performed using MinKnow integrated in Guppy. Sequences were mapped to the human genome reference sequence (GRCh38) using Minimap2 ( https://github.com/lh3/minimap2 ). Single nucleotide polymorphisms (SNPs) around the variants, c.229C > T in SGCA , c.325C > T in SGCB , and exon 6 deletion in SGCG , were selected as neutral markers for haplotype reconstruction, based on their genomic position and allele frequency (< 0.2). Primers are shown in Table S3 (see Additional file). We identified 53 Japanese families and 55 patients who harbored biallelic variants in any of the SGC genes analyzed . Variants in SGCA , SGCB , SGCG , and SGCD were identified in 30 (56%), 9 (17%), 11 (21%), and 3 (6%) families, respectively . Among 32 identified variants, 14 had previously been reported and 18 were novel. In previous reports, limited types of variants have been identified in the different SGC genes, regardless of the causative gene involved and in the current study, we identified 18, 6, 4, and 4 types of variants in SGCA , SGCB , SGCG and SGCD , respectively. Some recurrent variants were identified in multiple patients . Notably, allele frequencies of the most common variants in SGCA , SGCB , and SGCG in our study population reached 32%–64%. Fig. 1 STRAD flow diagram showing the genetic analysis carried out. We started with sequencing analyses, such as Sanger sequencing, panel analysis, whole-exome sequencing, or whole-genome sequencing. Aberrant splicing was detected using RNA-seq or RT-PCR in patients with exon–intron boundary or intronic variants. MLPA was applied to patients who were not diagnosed by sequencing analysis, and the breakpoints of a large SGCG deletion were identified using long-read sequencing Table 1 Variants found in Japanese patients with sarcoglycanopathy Family Age at onset Age at biopsy Sex Serum CK (UI/L) Symptoms Causative gene Variants F1 2y 4y F 5393 High CK SGCA c.37 + 6 T > C c.725 T > C, p.V242A F2 5 y 5 y M 4538 High CK SGCA c.38-2A > C c.320C > T, p.A107V F3 4 y 37 y F 206 Arm and leg weakness, respiratory failure SGCA c.158-2_167del c.626dupG, p.C209Wfs*10 F4 58 y 59 y M 14620 Muscle pain SGCA c.190G > A, p.A64T (homozygous) F5 50 y 72 y F 1141 Arm and leg weakness, calf hypertrophy SGCA c.190G > A, p.A64T (homozygous) F6 3 y 56 y M No data Leg weakness, respiratory failure, heart failure SGCA c.190G > A, p.A64T (homozygous) F7 9 y 42 y M 2630 Arm and leg weakness SGCA c.190G > A, p.A64T c.626dupG, p.C209Wfs*10 F8 6 y 18 y F 706 Leg weakness, calf hypertrophy SGCA c.220C > T, p.R74W (homozygous) F9 3 y 12 y M 7244 Leg weakness, calf hypertrophy SGCA c.229C > T, p.R77C (homozygous) F10 3 y 7 y M 13299 Leg weakness, calf hypertrophy SGCA c.229C > T, p.R77C (homozygous) F11 6 y 15 y M 5159 Leg weakness, calf hypertrophy SGCA c.229C > T, p.R77C (homozygous) F12 10 m 11 y F 7581 Leg weakness, muscle pain, calf hypertrophy SGCA c.229C > T, p.R77C (homozygous) F13 6 y 8 y M 9000 Leg weakness, calf hypertrophy SGCA c.229C > T, p.R77C (homozygous) F14 6 y 17 y F 3746 Leg weakness SGCA c.229C > T, p.R77C (homozygous) F15 1 y 9 y M 8415 Arm and leg weakness SGCA c.229C > T, p.R77C (homozygous) F16 2 y 6 y F 2599 Muscle pain SGCA c.229C > T, p.R77C c.320C > T, p.A107V F17 5 m 13 y M 1775 Arm and leg weakness muscle pain, calf hypertrophy SGCA c.229C > T, p.R77C c.320C > T, p.A107V F18 28 y 35 y M 1357 Muscle pain SGCA c.229C > T, p.R77C c.320C > T, p.A107V F19 2 y 2 y F 6280 Equinus foot SGCA c.229C > T, p.R77C c.409_410delinsCCTGGTGCGCAGCCAGG, p.E137delinsPGAQPG F20 2 y 2 y F 13766 Equinus foot SGCA c.229C > T, p.R77C c.626dupG, p.C209Wfs*10 F21-1* 3 y 9 y M 14418 Leg weakness, muscle pain, calf hypertrophy, equinus foot SGCA c.266 T > C, p.L89P c.749 T > G, p.V250G F21-2* 2 y 7 y M 7455 Leg weakness, muscle pain F22 1 y 6 y M 516 Muscle pain, equinus foot SGCA c.271G > A, p.G91S c.320C > T, p.A107V F23 6 y 48 y M 1328 Muscle pain, trunk and leg weakness SGCA c.320C > T, p.A107V c.409_410delinsCCTGGTGCGCAGCCAGG, p.E137delinsPGAQPG F24 62 y 70 y F 1115 Leg weakness SGCA c.320C > T, p.A107V c.662G > C, p.R221P F25 No data 71 y F No data No data SGCA c.584 + 1G > A c.662G > A, p.R221H F26 6 y 54 y M 887 Arm and leg weakness, calf hypertrophy SGCA c.584 + 5G > T (homozygous) F27 3 y 29 y M 465 Arm and leg weakness, respiratory failure, heart failure SGCA c.626dupG, p.C209Wfs*10 (homozygous) F28 6 y 80 y F 342 Arm and leg weakness SGCA c.634A > G, p.M212V (homozygous) F29 5 m 3 y M 555 Calf hypertrophy SGCA c.662G > A, p.R221H (homozygous) F30 59 y 67 y M 333 Arm and leg weakness SGCA c.662G > A, p.R221H (homozygous) F31 1 y 2 y M 42630 Leg weakness, calf hypertrophy SGCB c.214_221del, p.L72Pfs*24 (homozygous) F32 4 y 11 y M 8022 Leg weakness, calf hypertrophy, equinus foot SGCB c.214_221del, p.L72Pfs*24 c.325C > T, p.R109* F33-1* 3 y 3 y M 3994 Leg weakness, equinus foot, intellectual disability SGCB c.214_221del, p.L72Pfs*24 c.325C > T, p.R109* F33-2* 4 m 4 m M 2420 High CK F34 1 y 7 y F 9920 Arm and leg weakness, intellectual disability, respiratory failure SGCB c.325C > T, p.R109* (homozygous) F35 9 m 10 m F 32400 Muscle weakness, calf hypertrophy, equinus foot SGCB c.325C > T, p.R109* (homozygous) F36 8 y 9 y F 21944 Leg weakness, calf hypertrophy, equinus foot SGCB c.325C > T, p.R109* c.607G > C, p.A203P F37 4 y 6 y M 24880 Leg weakness, calf hypertrophy, equinus foot SGCB c.325C > T, p.R109* c.753 + 5G > A F38 3 y 4 y M 3254 Calf hypertrophy, equinus foot SGCB c.390_429dup, p.I144Afs*3 (homozygous) F39 3 y 7 y M 3370 Leg weakness, intellectual disability SGCB c.499G > A, p.G167S c.753 + 5G > A F40 1 y 8 y F 15772 Leg weakness, calf hypertrophy SGCG c.320C > T, p.S107L Exon 1–6 deletion F41 0 m 2 y F 17770 Leg weakness, calf hypertrophy, equinus foot SGCG c.320C > T, p.S107L (homozygous) F42 1 y 3 y M 8557 Leg weakness, calf hypertrophy SGCG c.320C > T, p.S107L Exon 6 deletion F43 2 y 6 y M 28300 Leg weakness, calf hypertrophy, equinus foot SGCG Exon 6 deletion (homozygous) F44 4 y 4 y M 10176 Leg weakness, calf hypertrophy SGCG Exon 6 deletion (homozygous) F45 2 y 5 y M 5524 Leg weakness, calf hypertrophy, equinus foot SGCG Exon 6 deletion (homozygous) F46 6 y 34 y F 1240 Leg weakness, calf hypertrophy, equinus foot SGCG Exon 6 deletion (homozygous) F47 7 y 8 y M 15336 Leg weakness SGCG Exon 6 deletion (homozygous) F48 6 y 25 y M 515 Leg weakness, calf hypertrophy, respiratory failure SGCG Exon 6 deletion Whole gene deletion F49 1 y 3 y M 9760 Leg weakness, calf hypertrophy SGCG Exon 6 deletion Whole gene deletion F50 1 y 1 y F 18630 Calf hypertrophy SGCG Exon 6 deletion Whole gene deletion F51 2 y 2 y F 17391 Calf hypertrophy, respiratory failure SGCD c.94A > G, p.K32E c.246_247del, p.S83* F52 4 y 7 y F 3290 Leg weakness, calf hypertrophy, equinus foot SGCD c.102delC, p.L35Cfs*9 c.246_247del, p.S83* F53 6 y 44 y F 1235 Leg weakness, intellectual disability SGCD c.502 + 24695G > T (homozygous) Bold characters indicate novel variants. y, years; m, months; CK, creatinine kinase *F21-1 and F21-2, and F33-1 and F33-2 are two pairs of siblings Fig. 2 Locations of amino-acid changes caused by identified genetic variants in predicted protein structures. Predicted three-dimensional structures of α-sarcoglycan (Q16586 (SGCA_HUMAN)), β-sarcoglycan (Q16585 (SGCB_HUMAN)), γ-sarcoglycan (Q13326 (SGCG_HUMAN)), and δ-sarcoglycan (Q92629 (SGCD_HUMAN)), obtained from Alphafold Protein Structure Database ( https://alphafold.ebi.ac.uk/ ). Red circles indicate the positions of missense variants. Red squares indicate in-frame deletions or insertions. Gray circles denote the positions of truncation of normal sequences in truncation variants. Yellow and white arrowheads show the 4 amino-acid deletion (13–16) caused by c.38-2A > C in SGCA and p.K32E encoded by c.94A > G in SGCD , respectively Fig. 3 Frequencies of variants and variant types in patients with sarcoglycanopathies. The frequency of ( a ) causative genes in Japanese patients with sarcoglycanopathies, and ( b )–( e ) variants in each causative gene: b SGCA , c SGCB , d SGCG , and e SGCD . Gray sections in pie charts indicate variants detected one family. f Effects of gene variants on protein products Two patients with a homozygous c.220C > T variant, the most common variant in the SGCA , tended to manifest symptoms in childhood and lost ambulation. However, 5 patients carrying a c.220C > T variant in combination with another variant exhibited milder symptoms, such as myalgia or elevated CK levels, compared to the homozygous patients. Patients with the c.190G > A, c. 320C > T and c.662G > A variants in the SGCA exhibited relatively mild symptoms like only high CK levels, maintaining ambulation into advanced age. All patients with the variants in the SGCB and SGCG showed childhood-onset. Adult patients with whole deletion of SGCG gene had lost ambulation and required respiratory support. A patient carrying the c.246_247del variant in the SGCD required respiratory support from an early age, while a patient with c.502 + 24695G > T variants preserved ambulation but presented with comorbidities such as intellectual disabilities. We identified three kinds of large deletions of the SGCG gene in nine families by MLPA, including deletions of exon 6, exon 1 to 6 and the entire gene (Table 1 ). To more accurately delineate the deleted regions, we applied Nanopore CRISPR/Cas9-targeted long-read resequencing. Genomic DNA samples from individuals 42–46 (F42-46) had deletions of precisely the same chromosome 13 region , which included exon 6 of SGCG . The 5'-breakpoint was within a short interspersed nuclear element (SINE) and the 3' breakpoint was within a long terminal repeat (LTR). In F40, 1.4-Mb sequence from downstream of LINC00424 to intron 6 of SGCG was deleted. Both 5’- and 3’- breakpoints were within SINE . In F49, a 1.6 Mb sequence from downstream of LINC00621 to upstream of PPAR4 , including the whole SGCG gene, was deleted. Similar sequences (approximately 7.3 kb) containing repeat elements (SINE, LTR, and long interspersed nuclear elements) were present in sequences flanking both ends of the 1.6 Mb deletion; therefore, we could not determine the exact breakpoints . In other cases, who had entire SGCG deletion, long genomic DNAs, which were required for Nanopore CRISPR/Cas9-targeted long-read resequencing, were unavailable. Fig. 4 Genome structures in patients with deletion of SGCG exon 6, SGCG exon 1 to 6 and the whole SGCG gene. a SGCG exon 6 deletion. The breakpoints were the same in all patients. b SGCG exon 1 to 6 deletion. c Whole gene deletion of SGCG ; similar sequences (identity: 94%) were present in the 5' and 3' breakpoints. Repeat sequence schema were retrieved from Ensembl ( https://asia.ensembl.org/index.html ). SINE, short interspersed nuclear element; LINE, long interspersed nuclear element; LTR, long terminal repeat Intronic single nucleotide variants were identified in five, two, and one families with LGMDR3, LGMDR4, and LGMDR6, respectively (Table 1 ) and we analyzed skeletal muscle transcripts from these patients to search for splicing abnormalities. We found a 59 bp extension to the 3’ end of exon 1 by RNA-seq in F1, since we could not obtain abnormal product by RT-PCR . One additional band from SGCA transcript was detected with some additional non-specific bands denoted by * in each of F2 and F3 by RT-PCR with primer pairs targeting SGCA exons 1–2 and 1–5, respectively; both products were amplified at comparable levels to those of the normal transcript. In The abnormal PCR product detected in F2 was truncated by 12 bp at the 5' end of exon 2, while that detected in F2 skipped exon 3 . RT-PCR analysis of samples from F25 and F26 with primers targeting exon 5–7 of SGCA generated the same longer product, which contained an 85 bp extension to the 3' end of exon 5, with an additional non-specific band denoted by *, in addition to the normal amplicon . In F37, which carried an SGCB variant, a smaller PCR product lacking exon 5, amplified at a comparable level to that of the normal product in control samples, was obtained by amplification of exon 4–6 . A deep intronic variant in SGCD was identified in F53 and found to result in transcription of a pseudoexon containing 91 bp of intron 6 . Fig. 5 Splicing abnormalities and MaxEnt scores generated by analysis of intronic variants. a F1 SGCA intron 1: c.37 + 6 T > C. Sashimi plots of RNA-seq data using control and patient muscles are shown. b – e RT-PCR products obtained from control and patient muscle samples separated on agarose gels and schema showing aberrant splicing events with MaxEnt scores. White squares are normal-sized exons and gray squares are altered-sized exons. In the right schema, genetic variants are shown in red and new splicing sites are shown in light blue. * indicate the non-specific products which were not related to SGC genes. b F2 SGCA intron 1: c.38-2A > C. c F3 SGCA intron 2/exon 3: c.158-2_167del. d F25 SGCA intron 5: c.584 + 1G > A and F26 SGCA intron 5: c.584 + 5G > T. e F37 SGCB intron 5: c.753 + 5G > A. f F53 SGCD intron 6: c.502 + 24695G > T. RT-PCR and Sashimi plots of RNA-seq data are shown; sequences 5' (upstream) of the variants included a possible branch point site, a polypyrimidine tract, and a splice acceptor site. ctrl, control sample; Ref, reference sequence To evaluate the pathogenicity of the identified intronic variants as determinants of aberrant splicing, we calculated the scores of the splicing acceptor and donor sites of affected exons using SpliceAI and MaxEntScan in silico . In F1, F2, F3, F25, F26, and F37, losses of splice sites near the genetic variants were predicted by SpliceAI. Similarly, the scores for those sites were greatly reduced in MaxEntScan analysis. Both software packages predicted the creation of alternative splice sites, including a splice acceptor site 12-bp downstream of the normal site in F2 and a splice donor sites, 59-bp downstream in F1, and 85-bp downstream in F25 and F26, with high delta scores for AG and DG, respectively. SpliceAI and MaxEntScan also successfully predicted the pseudoexon-creation observed in F53, with high scores for donor and acceptor site creation (DG = 1.00 and AG = 0.95) in SpliceAI, and a high score of 9.31 at the new donor site using MaxEntScan against 1.67 of the reference sequence. In addition, the pathogenicity of the pseudo-exon creation was also predicted by PDIVAS with a high score of 0.971 . Interestingly, this pseudoexon preserved the functional upstream sequence in the flanking intron; a branch-point-like structure at –29 nucleotides (nt), a polypyrimidine track at –22 to –5 nt, and a pseudoexon acceptor site. Together with the results of RT-PCR, these in silico predictions indicated that all variants identified in introns were pathogenic and the causes of aberrant splicing. Table 2 Summary of splicing abnormalities and SpliceAI predictions Family Gene Variant Effect on transcript Acceptor loss Donor loss Acceptor gain Donor gain ORF F1 SGCA Intron 1: c.37 + 6 T > C Exon 1 extension 0 0.51 0 0.25 Out of frame F2 SGCA Intron 1: c.38-2A > C Exon 2 shortening 0.96 0 0.68 0.09 In frame F3 SGCA Intron 2/exon 3: c.158-2_167del Exon 3 skipping 0.99 0 0.49 0.01 Out of frame F25 SGCA Intron 5: c.584 + 1G > A Exon 5 extension 0 0.99 0.01 0.54 Nonsense codon F26 SGCA Intron 5: c.584 + 5G > T Exon 5 extension 0 0.58 0.01 0.48 Nonsense codon F37 SGCB Intron 5: c.753 + 5G > A Exon 4 skipping 0 0.86 0.02 0 In frame F53 SGCD Intron 6: c.502 + 24695G > T Pseudoexon creation 0 0 0.95 1.00 Nonsense codon ORF, open reading frame Founder effects in patients with SGPs worldwide have been suggested in several previous reports [ 8 , 19 – 21 ]. Therefore, we analyzed the haplotypes containing the following variants: SGCA : c.229C > T, SGCB : c.325C > T, and SGCG : exon 6 deletion . We identified 5–10 SNPs in a homozygous state in individuals homozygous for the pathogenic variants, F11 and F12 with SGCA c.229C > T, F34 with SGCB c.325C > T, and F47 with SGCG exon 6 deletion. Among families with the SGCA c.229C > T variant, F11, F12, and F15 had the same haplotype, while F9, F13, and F14 had a distinct haplotype , indicating the presence of at least two haplotypes. Heterozygous individuals had compatible haplotypes, but were not informative, because they were hybrid for both genotypes. Families homozygous for SGCB c.325C > T (F34 and F35) shared the same haplotype, and those heterozygous for this variant (F32, F36, and F37) also possessed the same haplotype, indicating a single haplotype carrying SGCB c.325C > T. In families with SGCG exon 6 deletion, the homozygous individuals, F44, F45, and F47, and those hemizygous for the deletion, F48–F50, had the same haplotype; further, one heterozygous individual, F42, also showed compatible results, while the homozygotes, F43 and F46, had haplotypes with evidence of genetic recombination. Fig. 6 Haplotype analyses of alleles containing common single nucleotide polymorphism (SNP) variants. a Patients with SGCA c.229C > T. b Patients with SGCB c.325C > T. c Patients with SGCG exon 6 deletion. Bases in blue font indicate minor SNP alleles and bases in red font indicate variants. Orange shaded cells indicate homozygosity for minor SNP alleles. Yellow shaded cells indicate heterozygosity for minor SNP alleles. Blue shaded cells indicate homozygosity for major SNP alleles Notably, the frequencies of missense and truncation variants differed among causative genes of SGPs, as reported previously [ 2 – 4 ]. Missense variants in SGCA were frequent at 78.3%, while they were relatively rare in SGCB , SGCG , and SGCD , at 11.1%, 18.2%, and 16.7% respectively. By contrast, truncation variants were common in SGCB , SGCG , and SGCD. In this study, we conducted genetic profiling of 53 Japanese families with SGP. In Japan, SGCA was the most common causative gene, followed by SGCG , SGCB , and SGCD . The proportion of causative genes varies by region, with SGCA being the most common in Europe, the USA and Brazil , SGCB in Iran and SGCG in Algeria and India . No patients with LGMDR6 have previously been reported in Japan; here, we report the first identification of Japanese patients with LGMDR6, with biallelic variants in SGCD . Although the reason for the low prevalence of LGMDR6 is unclear, it has been suggested that SGCD is expressed in the heart, and that individuals with variants in SGCD may die in utero ; however, no cardiac disease was observed in our patients with LGMDR6, although genetically undiagnosed siblings of F53 are recorded to have died of cardiac disease. A few types of variants accounted for more than half of alleles in all causative genes. In particular, c.229C > T (R77C) in SGCA , which is also reported to be common in other countries, and has been suggested as a founder variant [ 3 , 25 – 27 ]. Analysis of the genotypes of Japanese patients with c.229C > T indicated the presence of two distinct haplotypes, suggesting that SGCA : c.229C > T can be considered a recurrent variant. The alleles harboring c.325C > T in SGCB and exon 6 deletion in SGCG , shared common haplotypes and were likely each inherited from a single common ancestor. This concept is significant, as genomic screening that prioritizes founder and recurrent variants enhances diagnosis efficacy and provide a foundation for the development of gene therapies. Interestingly, large deletions of SGCG are common in Japan, and SGCG contains numerous repetitive sequences in its introns, which may be prone to genetic recombination. The breakpoints of the Exon 6 deletion and Exon 1 to 6 deletion were within an LTR and SINE respectively, and the breakpoints flanking whole SGCG deletions contained similar repetitive elements, suggesting non-allelic homologous recombination as the likely mechanism underlying both types of deletion . Additionally, individuals with deletion of the entire SGCG gene also lacked five other genes, among which SACS and MIPEP are reported to cause spastic ataxia and combined oxidative phosphorylation deficiency 31, respectively . Thus far, no patient with SGP and homozygous deletion of the entire SGCG gene has been identified, as combined oxidative phosphorylation deficiency is a very severe condition. MLPA combined with long-read sequencing proved useful for the diagnosis of these variants. We also predicted the pathogenicity of detected intronic variants and altered transcript structures in silico, as well as successfully identifying their splicing abnormalities in all cases by RT-PCR and RNA-seq. The shifted PCR products detected in F2 and F37 had in-frame changes and were not subject to nonsense-mediated mRNA decay; therefore, they appeared to be expressed in comparable amounts to normal transcripts in control samples. In SGCD , creation of a pseudoexon deep in intron 6 is pathogenic. Exon-skipping therapy is an approved treatment for Duchenne muscular dystrophy and recently, we developed a therapeutic strategy using branchpoint-targeted antisense oligonucleotides for patients with Fukuyama congenital muscular dystrophy caused by an FKTN transcript involving a pseudoexon . Accurate diagnosis of SGP may also provide therapeutic opportunities. We found that many of the SGCA variants detected were predicted to be missense and to cause amino-acid substitutions in the protein, while most SGCB , SGCG , and SGCD variants were predicted to cause truncation of their protein products; this trend has been also reported in other cohort studies . Interestingly, the rates of missense/truncated variants in SGCA-SGCD may be related to the roles of each protein subunit in the formation and function of the sarcoglycan complex. Recent advances in the prediction of protein structures using Alphafold have allowed us to consider the pathogenicity of identified variants in the context of whole protein structures . α-Sarcoglycan has a globular domain in its central extracellular region and missense variants in SGCA are concentrated in this globular domain. Additionally, the central extracellular region of α-sarcoglycan has been reported to exhibit ecto-ATPase activity . By contrast, the extracellular regions of β-, γ-, and δ-sarcoglycans are predicted to have simple structures, without globular domains. The majority of variants in SGCB , SGCG , and SGCD cause protein truncation. Our previous model of sarcoglycan complex formation suggested that β-, γ-, and δ-sarcoglycans make up the core of the complex, while α-sarcoglycan binds later and is primarily on the outside of the complex . On the other hand, other reports demonstrated core structure of sarcoglycan complex consists of β- and δ-sarcoglycans and later incorporates α- and γ-sarcoglycan . Thus, amino-acid substitutions caused by missense variants in SGCA most probably affect the ecto-ATPase activity of α-sarcoglycan and also cause instability of the sarcoglycan complex, while truncations of β-, γ-, and δ-sarcoglycans might affect the de novo formation of the sarcoglycan complex. Additionally, we identified two novel variants which may prevent the resulting mutated proteins from undergoing normal intracellular processing. One of these novel variants is c.38-2A > C in SGCA , detected in F2, which causes aberrant splicing resulting in in-frame deletion of four hydrophobic amino acids in the signal peptide of α-sarcoglycan ; this deletion is predicted by iPSORT ( https://ipsort.hgc.jp/ ) to render the signal peptide non-functional . The other novel variant is c.94A > G in SGCD in F51, which is predicted to lead to a p.K32E substitution . This substitution decreases the positive charges on the cytosolic side of the transmembrane domain by replacing a basic amino acid, which was predicted to influence the membrane topology of δ-sarcoglycan using WoLF PSORT ( https://wolfpsort.hgc.jp/ ) . Further structural and functional studies on mutated proteins are required to clarify the exact roles and pathogenicity of the identified variants. In conclusion, both muscle biopsy and genetic analysis are essential for accurate diagnosis of SGPs; LGMDR3 is the most common type in Japan, while LGMDR6 is very rare. Patients with SGPs share relatively few variants. Missense variants are the most common type of change in SGCA , while truncation mutations account for a major proportion of SGCB , SGCG , and SGCD variants. Tools for prediction of splicing changes are useful for the identification of pathogenic intronic variants. | Review | biomedical | en | 0.999996 |
PMC11699709 | A stroke is caused by an arterial blockage or hemorrhage in one part of the brain tissue, which leads to focal neurological damage in the adjacent area. Ischemic stroke is induced by ischemia and hypoxia in the brain, which leads to limited ischemic necrosis or softening of the brain tissue, and it will cause severe neurological deficits, which can result in cell death if reperfusion is not possible in a short period, this type of stroke accounts for about 70% of all strokes . The incidence and prevalence of stroke have been reported to increase year by year, with high rates of disability and mortality, and is a major cause of death and disability in the world’s population, seriously jeopardizing human life and health . Currently, thrombolysis applied to achieve early reperfusion is the most valid method of treating acute cerebral ischemia. Still, this therapy is limited by the time window of thrombolysis, and successful thrombolysis causes substantial damage to brain tissue with cerebral ischemia (ischemia/reperfusion injury (I/R)), which may be related to inflammatory responses, mitochondrial dysfunction, increased production of ROS, oxidative stress, and activation of cell death pathways [ 3 – 8 ]. For many years, despite many studies in this field, we did not significantly improve the prognosis and treatment of ischemic stroke patients. Therefore, exploring new targets and therapies to reduce re-injury in cerebral ischemia remains a pivotal challenge for treating cerebral ischemia. Ferroptosis was a new mechanism of programmed cell death, a non-apoptotic programmed death pathway reliant on iron ions and ROS . Ferroptosis is characterized by a significant elevation of iron content and accumulation of lipid peroxides, and lead to cell death and neurological damage after stroke. During ischemia/reperfusion injury, the abnormal increase of cerebral microvascular endothelial cell permeability severely damages the blood–brain barrier (BBB), which allows a large amount of iron to enter the brain parenchyma and trigger iron overload. The increased iron content results in the disturbance of cerebral iron metabolism and eventually contributes to the occurrence of cellular ferroptosis . During cerebral ischemia, local cerebral tissue blood supply is insufficient, mitochondrial dysfunction occurs, and ATP can not be produced. After reperfusion of cerebral blood flow, oxidative stress increases mitochondrial damage further and generates excessive ROS . The ROS accumulation-mediated oxidative stress and lipid peroxidation are the key driving forces triggering cellular ferroptosis . In addition, overaccumulation accumulation of extracellular glutamate is also a major cause of neuronal cell death, which exerts oxidative toxicity by inhibiting cystine absorption through restraining the activity of System Xc-. As the light chain of the Xc system, SLC7A11 translocates a molecule of glutamate out of the cell and exchanges a molecule of cystine into the cell in a 1:1 ratio. Cystine is necessary for the synthesis of glutathione (GSH) . Intracellular GSH depletion reduces the activity of GPX4 and further leads to the dysfunction of lipid peroxides metabolism . Excess Fe 2+ oxidizes lipids employing the Fenton reaction, generating massive amounts of ROS and eventually leading to ferroptosis [ 19 – 21 ]. As shown in many studies, inhibition of ferroptosis is pivotal in rescuing neuronal damage after brain I/R injury. Electroacupuncture (EA), integrates traditional acupuncture with modern electric stimulation and has been widely used in treating stroke and poststroke with significant clinical effects, and less undesirable effects. EA therapy has been demonstrated to be neuroprotective against ischemic stroke by effectively attenuating a wide range of pathologic processes. In addition, the co-application of EA at the “Baihui” point and the “Zusanli” point had a synergistic protective effect on mitigating neuronal damage in MCAO rats by curbing endoplasmic reticulum stress and ameliorating the defective mitochondrial function . Therefore, EA can effectively restrain the ischemic-hypoxic cascade response and neuronal injury . Recent studies have found that EA intervention in MCAO rats can also repress the formation of ferroptosis, which protects damaged neuronal cells in MCAO rats . However, the mechanism of the way by which EA inhibits ferroptosis is still obscure. Investigating the specific mechanism of EA for ischemic stroke is anticipated to yield a new scientific basis for targeted therapy of relevant neurological diseases in the future. The etiopathogenesis of ferroptosis is sophisticated and involves multiple transcription factors including p53, NFE2L2, and Nrf2 . Among them, Nrf2, a crucial ferroptosis regulator, is known to transcribe several antioxidant response element (ARE)-containing genes to maintain redox homeostasis . Therefore, deficiency of Nrf2 causes increased susceptibility to brain injury [ 29 – 31 ]. Under normal conditions, Nrf2 binds to Kelch-like ECH-associated protein1 (Keap1), an interface protein for Cul3 E3 ubiquitin ligases, which is accountable for the ubiquitination and degradation of Nrf2 ; under oxidative stress, Nrf2 dissociates from Keap1, translocates to the nucleus and activates the transcription of the ARE. The ARE transcribes and drives a variety of antioxidant genes, including heme oxygenase-1 (HO-1), to exert antioxidant effects [ 34 – 38 ]. As a transcription factor, Nrf2 can directly regulate the expression of several important genes in the process of ferroptosis in the nucleus, including SLC7A11, GPX4, FTH1, etc. , which in turn regulates intracellular iron metabolism, GSH levels, GPX4 synthesis, lipid oxidation, etc. . Therefore, activation of Nrf2 is a valuable target for treating cerebral ischemia . The purpose of this research was to explore the molecular mechanisms regarding the beneficial efficacy of EA in the rat model of MCAO/R-induced brain injury, and it was found that EA could prevent neuronal ferroptosis after ischemic stroke by facilitating the Nrf2 nuclear translocation and activating the Nrf2/SLC7A11/GPX4 pathway, which in turn achieves a neuroprotective effect on the nervous system. Male SPF Sprague–Dawley (SD) rats (200 ± 20 g) were obtained from Hunan Slack (Certificate No.: SCXK (Xiang) 2019–0004) and reared in the Laboratory Animal Center under a 12 h light/dark cycle at 21 ± 2 °C and 60–70% humidity. These rats had unlimited access to food and water. All animal experiments were granted by the Animal Ethics Committee of Yunnan University of Traditional Chinese Medicine . The middle cerebral artery occlusion/reperfusion (MCAO/R) model was constructed with the Longa method . In brief, the rats were sterilized by intraperitoneal injection of 3% sodium pentobarbital (40 mg/kg) anesthesia and fastened to an operating platform, and the neck hair was removed, and the muscles and connective tissues were separated. Then the left common carotid artery, external carotid artery, and internal carotid artery. The external carotid artery was tied with a thin line and a minor incision was cut in the external carotid artery. A nylon monofilament (Beijing Sinon Technology Co., Ltd., Beijing, China) with a silicone coating at the tip was then plunged into the internal carotid artery through a tiny incision to a depth of approximately 18–20 mm at the bifurcation. During ischemia, these rats were placed on an insulating pad at 37 °C for 60 min. 60 min later, the nylon monofilament was withdrawn to establish reperfusion and the wound was sutured. Sham rats underwent all steps apart from insertion and extraction of the nylon monofilament. After 24 h of surgery, the success of MCAO/R was appraised with a neurobehavioral score. 72 rats with successful MCAO/R were randomly numbered and categorized into four groups ( n = 18): the MCAO/R group, the EA + MCAO/R group, the EA + MCAO/R + DMSO group, and the EA + MCAO/R + Brusatol group, the Sham group was used for control ( n = 18). EA intervention on Quchi (LI11), Baihui (GV20), Dazhui (GV14), and Neiguan (P6) was performed in the MCAO/R + EA group, the EA + MCAO/R + DMSO group, and the EA + MCAO/R + Brusatol group at 24 h after modeling . The Baihui point is located at the top of the head, where the Mai qi converges. Stimulating the Baihui point can directly or indirectly connect with the meridian system of the whole body, thus achieving the effect of nourishing the blood. The Dazhui point belongs to the Governor's Chakra, which has the effect of relieving spasms, relaxing contracture, calming the mind, and strengthening the body; Quchi point applies to all clinical disciplines and is particularly good in Chinese medicine internal diseases, among which paralysis in limb meridian diseases is the most specialized; Neiguan point has the effect of enlightening the mind and tranquilizing the mood, so Neiguan point can treat the diseases of the mind. Therefore, these four acupoints we have chosen are indicated for ischemic stroke treatment. The specific parameters of acupoint were as follows: Baihui point: located in the middle of the parietal bone, and obliquely stabbed forward for 2 mm; Dazhui point: located in the posterior midline and depression below the spinous process of the seventh cervical vertebrae, and stabbed straightly for 5 mm; the right Quchi point: located in the depression in the proximal end of the radius just anterior to the lateral side of the elbow joint, and stabbed straightly for 4 mm; and the right Neiguan point: the medial side of the forelimb, the About 3 mm from the wrist joint, between the ulnar-radial suture, straight stabbing 1 mm. The disposable acupuncture needle (Beijing Zhongyan Taihe Medical Instrument Co., Ltd., Beijing, China) was used for stabbing and then connected to the EA instrument (Changzhou Indy Electronic Medical Instrument Co., Ltd., Changzhou, China), with a current of 2 mA, the frequency of 2 Hz, sparse and dense waves, 30 min/time, 1 time/day for consecutive 7 days . Rats in the EA + MCAO/R + Brusatol group were injected intraperitoneally with the Nrf2 inhibitor Brusatol ((2 mg/kg; Shanghai Yuanye Bio-Technology, CAS number: 14907–98-3, (Shanghai, China)) at 30 min before EA treatment on the first day after modeling, and every other day until the seventh day. On the seventh day after MCAO/R, all rats were scored for neurologic severity. Neurological scoring was performed by the same investigator in a blinded manner, and the rats were rated for neurological deficits using a 7-point NSS scale. Higher NSS indicated poorer neurological function . All rats were blinded by the same researcher, and the Garcia score was based on six main characteristics: voluntary locomotion, body symmetry, forelimb extension, grasp, and climb ability, tactile reflexes on both sides of the body, and whisker touch response on both sides of the body. The total score is 18, with higher scores meaning less nerve impairment . Motor coordination in rats was evaluated by the foot fault test. The test was performed three times consecutively at 5-min intervals. The experiment was scored by the state of the rat's right forefoot grasping the horizontal ladder each time , and higher scores represented the better walking ability of the rats. The Rotarod test can assess the recovery of motor function and tolerance level of rats, the operation is as follows: the rats were placed on the rotating rod, the velocity of which was raised from 0 to 10 rpm in 10 s for 5 min. The test was performed three times, recording the time of the rats running on the rotating rod, and average values were taken for statistical analysis . On postoperative day 7, rats were dosed intraperitoneally with 3% sodium pentobarbital, and the brains were immediately removed and refrigerated for 5 min at −20 °C. The brains were then sliced into 2 mm thick sections. There were 6 slices in total, which were put into a Petri dish containing 2% TTC staining solution (Solarbio Company, Beijing, China), and then covered with tin foil for light protection, and then put into a 37 ℃ incubator to incubate for 15 min, and then brain slices were taken out and arranged in an anterior–posterior order, photographs were taken of these sections. The pale areas were characterized as infarcted. Infarct volume was counted using Image J software (Media Cybernetics, Rockville, MD, USA). Percentage of infarct volume (%) = (infarct volume/total volume) × 100%. Rats were perfused transcardially with 0.9% NaCl and 4% paraformaldehyde solution. The brains were taken out and fixated with paraformaldehyde solution for 24 h before being immersed in 30% sucrose solution at 4 °C for 24 h. After removal, the brains were embedded in paraffin and cut into 4 μm thick slices on a paraffin slicer in preparation for Nissl staining. Briefly, slices of each group were degreased in graded alcohol (70%, 95%, and 100% alcohol) for 3 min, followed by hydration in graded alcohol (95%, 70%, and 50% alcohol) for 3 min. Next, the slices were immersed in a toluidine blue solution at 50–60 °C for 40 min. Following washing with distilled water, the slices were dehydrated once in 70%, 80%, and 95% ethanol for a total of 3 min each, and then twice in 100% ethanol solution. Finally, the slices were immersed in 100% dimethylbenzene solution for 5 min and then sealed with a drop of neutral resin. Fe 2+ levels in brain tissue were measured by use of an iron content assay kit . Measure the absorbance at a wavelength of 593 nm with a spectrophotometer based on the manufacturer's instructions. ROS levels were assayed by a ROS kit (E004-1-1, Nanjing Jianjian Biotechnology Co., Ltd.) according to the manufacturer's instructions. Fluorescence intensity was measured with a luciferase marker (Spectra Max Gemini EM, Molecular Devices, USA) with 485 nm excitation wavelength and 530 nm emission wavelength. The MDA assay kit and SOD assay kit were performed to detect MDA and SOD levels. Nucleoprotein and cytoplasmic proteins were isolated using the Nucleoprotein Extraction Kit following the manufacturer's instructions. After obtaining nuclear and cytoplasmic proteins, Nrf2 was quantified in the nucleus and cytoplasm by Western blotting and Lamin-B1 was applied as an up-loading control for nuclear proteins. Data were resolved by Image J. Rat brain samples were cleaved with RIPA lysis solution (Solarbio, Beijing, China) containing PMSF and phosphatase inhibitors. Total proteins were collected and quantified by BCA protein assay kit (Biosharp, Beijing, China). Total proteins were detached in SDS-PAGE gels and shifted to PVDF (Merck, USA) membranes. The membranes were occluded with 5% skimmed milk (Biofroxx) for 2 h at room temperature and then incubated with primary antibodies at 4 °C overnight. Primary antibodies included Anti-Nrf2 , Anti-pNrf2 , Anti-GPX4 , Anti-HO-1 , Anti-FTH1 , Anti-SLC7A11 , Anti-β-actin , and Anti-Lamin-B1 . The next day, the membranes were cleaned with TBST three times, and then incubated with HRP-coupled goat anti-rabbit IgG secondary antibody at room temperature for 1 h. After three washings, the membranes were visualized on a chemiluminescence detection system (Clink Science Instruments Co., Ltd., Shanghai, China) with ECL chemiluminescence reagent (Biosharp Life Science Co., Ltd.) and analyzed with Image J software. Rats were anesthetized intensively and perfused transcardially with 0.9% NaCl and 4% paraformaldehyde. Whole brains were removed and immobilized in 4% paraformaldehyde through the night, and then dehydrated with a sucrose gradient at room temperature. The brains were encapsulated in OCT and sectioned into 10 μm thick slices using a cryosectioner. Sections were incubated with 5% bovine serum albumin (BSA) blocked at room temperature for 1 h and then hatched overnight at 4 °C with Anti-GPX4 , Anti-Nrf2 , and Anti-NeuN . Subsequently, sections were rinsed three times with PBS and incubated with a fluorescent secondary antibody (Alexa Fluor 488/594 AffiniPure Goat Anti-Rabbit IgG (H + L); Jackson ImmunoResearch) and then in a 37 °C incubator. The sections were then incubated with DAPI for 5 min at room temperature. Positive signals in the ischemic penumbra were visualized and photographed using a Zeiss LSM-710 confocal microscope. Fluorescence intensity was analyzed using ImageJ software. Brain tissues of 1 × 1 × 2 mm size from the ischemic penumbra were stabilized in 3% glutaraldehyde solution for 1 h and then in 1% osmium tetroxide solution for 2 h. The sections were then dehydrated in acetone and finally imbedded in Epon812. Ultrathin sections of 60–90 nm were fabricated using an ultrathin sectioning machine. The sections were then colored with uranyl acetate for 10–15 min at room temperature, then with lead citrate for 1–2 min, and lastly, the morphology of mitochondria was investigated by transmission electron microscopy . Data were statistically analyzed using SPSS 26.0 software. All experimental data were reported as (mean ± SD). One-way ANOVA with a 95% confidence interval was used to assess the differences between three or more experimental groups. Tukey’s multiple comparison post-hoc test was then performed, p < 0.05 denoted a statistically significant difference. Graphs were plotted using GraphPad Prism 9.5 software. The neurofunctional recovery was measured by NSS and Garcia score on days 7 after MCAO/R. The results indicate that the neurological function of rats in the MCAO/R group was markedly corrupted. EA markedly improved the neurological deficits. The NSS in the MCAO/R group was significantly higher than that in the Sham group, EA treatment significantly improved the neurological deficits compared with the MCAO/R groups . Similarly, the Garcia score was significantly lower in the MCAO/R group than in the Sham group, and the EA treatment improved the Garcia score . The results of the Foot-fault Test and the Rotarod Test revealed that exercise capacity was enhanced in the MCAO/R + EA group as compared to the MCAO/R group. Major cerebral infarcts were noticed in MCAO/R operated rats by TTC staining compared to the Sham group. Yet, the volume of cerebral infarction was diminished in the EA + MCAO/R group compared with the MCAO/R group. . Meanwhile, Nissl staining indicated that compared with the Sham group, the neuronal cells in the MCAO/R group were disorganized or missing, and the Nissl body staining was incomplete or even dissolved, suggesting that neuronal damage was induced by MCAO/R modeling. The infarct volume was decreased and the neuronal cell recovery in the ischemic rats with EA treatment compared with that of the ischemic rats without treatment . Fig. 1 Neuroprotective effect of EA against cerebral MCAO/R induced injuries. A – D Neurological deficits of each group were scored with the NSS, Garcia score, Foot-fault test, and Rotarod test at 7 days after surgery ( n = 12). E , F Representative images of TTC stained section and quantitation ( n = 6). G Representative photomicrographs of Nissl stained sections (Scale bar: 1 mM, 200 μM, and 50 μM, n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001 MDA and Fe 2+ levels were elevated distinctly in the MCAO/R and EA + MCAO/R groups relative to the Sham group. Yet, the levels of MDA and Fe 2+ in the EA + MCAO/R group were decreased compared with those in the MCAO/R group. Similarly, the expression of ferroptosis-related proteins, GPX4 , SLC7A11 , and FTH1 was reduced substantially in the MCAO/R group despite the Sham group; nonetheless, the EA intervention counteracted this trend in the EA + MCAO/R group. Fig. 2 EA inhibits ferroptosis in rats following MCAO/R. A , B The concentration of Fe 2+ , and MDA in brain tissue ( n = 6). C The representative Western blotting results of GPX4, SLC7A11, and FTH1 expression. D – F Quantification of GPX4, SLC7A11, and FTH1 expressions ( n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001 Ferroptosis damages the structure and functional impairment in mitochondria. We observed the mitochondrial ultrastructure by TEM. The samples exhibited irregular mitochondrial morphology, proliferation of vacuoles, and invagination of the mitochondrial membrane after MCAO/R, whereas the mitochondrial morphology in the MCAO/R + EA group was standardized with fewer vacuoles. . Fig. 3 EA inhibits ferroptosis in rats following MCAO/R. The bottom panels display the magnified images of regions indicated by yellow rectangles in the top panels. Arrows labeled: representative images of mitochondria. (Scale bar: 5 μM, 2 μM, and 1 μM, n = 3) To probe the molecular mechanism by which EA anti-ferroptosis potential, a crucial factor of the Nrf2 pathway was determined by the researchers. The outcomes revealed that the expression and phosphorylation values of Nrf2 and HO-1 were pronouncedly under-regulated in the MCAO/R group. However, the decrease of total Nrf2 , p-Nrf2 and HO-1 was rescued by EA treatment. Intriguingly, nuclear Nrf2 was slightly elevated in the MCAO/R group and markedly elevated in the EA + MCAO/R group, whereas cytoplasmic Nrf2 was strikingly diminished in both the MCAO/R group and the EA + MCAO/R group. Compared to the MCAO/R group, the cytoplasmic Nrf2 was less while the nuclear Nrf2 was more. We further observed the localization of Nrf2 by confocal. Our findings confirmed that Nrf2 mainly accumulates in the cytoplasm in the MCAO/R group, and EA efficiently promotes the nuclear translocation of Nrf2 . These results implied that EA promoted the phosphorylation of Nrf2 and nuclear translocation. Fig. 4 EA increases the expression of Nrf2 and promotes Nrf2 phosphorylation and nuclear translocation. A The representative Western blotting bands of Total Nrf2, p-Nrf2, HO-1, Nuclear Nrf2, and Cytoplasmic Nrf2 expression. B – F Quantification of Total Nrf2, p-Nrf2, HO-1, Nuclear Nrf2 and Cytoplasmic Nrf2 expressions ( n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001. G The confocal images of Nrf2. Scale bar: 5 μm. Nrf2 and DAPI were excited at 488 and 405 nm, respectively. Exemplary XY plane of confocal z-stack acquisition of Nrf2 (green signal) and treated with DAPI for nuclear staining (blue signal). Orthogonal views (XZ and YZ planes) extracted from z-stack are reported To further verify the effects of Nrf2 in MCAO/R-induced ferroptosis, we inhibited the Nrf2 by brusatol. The expression of Total Nrf2 , p-Nrf2 , HO-1 , Nuclear Nrf2 , and Cytoplasmic Nrf2 were examined by Western blotting, and the results indicated that brusatol reversed the effects of increased expression and promoted nuclear translocation induced by EA treatment. We further observed the location of Nrf2 by immunofluorescence . The results also revealed that EA treatment enhanced the content of Nrf2 and attracted the nuclear translocation of Nrf2 in the MCAO/R + EA group, while Brusatol suppressed the nuclear translocation of Nrf2 prominently. These observations confirm that Brusatol prominently suppressed the total content of Nrf2 and HO-1, and suppressed the phosphorylation and nuclear translocation of Nrf2 upon EA treatment. Fig. 5 Brusatol reduces the content of Nrf2 and inhibits Nrf2 phosphorylation and nuclear translocation. A The representative Western blotting results of Total Nrf2, p-Nrf2, HO-1, Nuclear Nrf2, and Cytoplasmic Nrf2 expression. B – F Quantification of Total Nrf2, p-Nrf2, HO-1, Nuclear Nrf2 and Cytoplasmic Nrf2 expressions ( n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001. G The confocal images were performed to determine the protein expressions of Nrf2. Scale bar: 5 μm. Nrf2 and DAPI were excited at 488 and 405 nm, respectively. Exemplary XY plane of confocal z-stack acquisition of Nrf2 (green signal) and treated with DAPI for nuclear staining (blue signal). Orthogonal views (XZ and YZ planes) extracted from z-stack are reported Compared with the MCAO/R + EA + DMSO group, the Fe 2+ content, ROS and MDA levels in MCAO/R + EA + Brusatol group were significantly higher, while, the SOD level in was significantly lower. Similarly, the results by Western blotting showed that Brusatol significantly down-regulated the expression of GPX4 , SLC7A11 and FTH1 proteins . The GPX4 expression level was further assessed by Double immunofluorescence staining. The findings displayed that GPX4 was predominantly co-localized with neurons (F i g. 6 I). Consistent with GPX4 protein levels, MCAO/R prominently degraded GPX4 expression. EA augmented GPX4 expression, while the Brusatol reversed the up-regulation of GPX4 induced by EA . These findings demonstrate that brusatol counteracts the protectiveness of EA through the Nrf2 pathway. Fig. 6 Brusatol diminishes the protective effect of EA that attenuates ferroptosis after MCAO/R. A – D The concentration of Fe 2+ , ROS, MDA, and SOD in each group's brain tissue ( n = 6). E The representative Western blotting results of GPX4, SLC7A11, and FTH1 expression. (F–H) Quantification of GPX4, SLC7A11, and FTH1 expressions ( n = 6). I , J Typical double immunofluorescence images (GPX4 (green), NeuN (red), DAPI (blue). Scale bar: 25 µm. n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001 Next, TEM was used to observe the morphology of mitochondria. As shown in the images, mitochondria in the Sham group had an intact outer membrane, abundant cristae, and normal morphology, MCAO/R led to the production of fragmented mitochondria with decreased cristae, even appeared to be vacuolated, which is a characteristic of ferroptosis. However, EA dampened MCAO/R-induced morphological changes in mitochondria . In contrast, Brusatol inverted the advantageous effects of EA on mitochondria. Fig. 7 Brusatol diminishes the protective effect of EA that attenuates ferroptosis after MCAO/R. Mitochondrial morphology associated with ferroptosis was determined by TEM. The bottom panels display the magnified images of regions indicated by yellow rectangles in the top panels. Arrows labeled: representative images of mitochondria. (Scale bar: 5 μM, 2 μM, and 1 μM, n = 3) Behavioral tests and TTC staining were utilized to appraise the effects of brusatlo on the neuroprotection provided by EA. The results presented that the score of NSS was significantly greater and the Garcia score was below in the MCAO/R + EA + Brusatol group than in MCAO/R + EA + DMSO group. Similarly, The results of the Foot-fault Test and the Rotarod Test suggested that functional motor restoration was inferior in the MCAO/R + EA + Brusatol group compared to the MCAO/R + EA + DMSO group. TTC staining revealed that the volume of cerebral infarction in rats in the MCAO/R + EA + Brusatol group was noticeably larger than that in rats in the MCAO/R + EA + DMSO group. . Meanwhile, Nissl staining results showed that compared with the MCAO/R + EA + DMSO group, the MCAO/R + EA + Brusatol group had an increased volume of cerebral infarction, disorganized neuronal cell arrangement, and more severe brain damage . These results suggested that EA provided a neuroprotection against stroke, while the neuroprotective effect was diminished by the inhibition of Nrf2. Fig. 8 Brusatol reverses the neuroprotective effect of EA. A – D Neurological deficits of each group were scored with the NSS, Garcia score, Rotarod Test, and Foot-fault Test at 7 days after surgery ( n = 12). E , F Evaluation of cerebral infarction volume of rats by TTC staining and analysis ( n = 6). G Representative photomicrographs of Nissl stained sections (Scale bar: 1 mM, 200 μM, and 50 μM, n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001 In this study, we established the MCAO/R model in rats and treated them with EA for 7 consecutive days starting from one day after modeling, which ultimately diminished infarct volume and improved neurological function and motor behavior scores. The possible neuroprotective mechanism was that EA alleviated MCAO/R-induced ferroptosis by promoting Nrf2 nuclear transposition and activating the Nrf2/SLC7A11/GPX4 pathway. Ischemic stroke is the prevalent type of stroke with high mortality and restricted functional recovery in survivors . Ischemic stroke occurs with ischemia and hypoxia, which drastically declines the provision of oxygen, glucose, and other nutrients, and blood supply to the brain, consequently leading to disruption of cellular energy metabolism and destruction of neuronal function . The region of infarction formed by a sharp diminution of blood flow to the brain in ischemic stroke is referred to as the ischemic core. Once the local blood flow supply is curtailed to below 20%, ischemia and hypoxia induce severe reactions such as ATP exhaustion, invalidation of Na + /K + pumps, proliferation of intracellular Ca 2+ , and discharge of neurotoxic substances in the ischemic core region within a short period, which results in speedy cell death. It is surrounded by the peri-infarct region or penumbra, which encloses brain tissue that is functionally damaged but potentially salvageable. Cells in the penumbra region are not electrically active but retain energy in the form of ATP, and thereby the cells are typically enabled to die in a modulated programmed manner, which prevents deleterious inflammation from occurring by rendering cellular contents from being released into the extracellular milieu . Hence, salvaging ischemic penumbral regions and facilitating cell survival are the primary goals of exploiting neuroprotective strategies to mitigate the severity of cerebral ischemic injury through immediate intervention . Recent research has revealed that ferroptosis is an influential factor in brain I/R injury, and its development is closely linked to various biological courses like iron, amino acid, polyunsaturated fatty acid metabolism, and GSH biosynthesis . Morphologically, ferroptosis occurs mainly within the cell, as evidenced by a reduction in mitochondrial volume, density of the bilayer membrane, and reduction or disappearance of the mitochondrial cristae, but with intact membranes and normal nuclear size . Biochemically, ferroptosis is manifested by intracellular GSH depletion, the reduction of GPX4 activity, and the inability of GPX4 to metabolize lipid peroxides to catalyze the reduction reaction, which generates a large amount of ROS and facilitates ferroptosis . Previous studies have indicated that high levels of GPX4 can safeguard neurons and mitochondria from oxidative damage . Meanwhile, reduced levels of GPX4 dramatically diminished the amount of NeuN-positive cells in the hippocampus and elicited neuronal ferroptosis , implying that GPX4 is a crucial factor in the modulation of neuronal ferroptosis. Similarly, diminished levels of SLC7A11 can generate a drop in intracellular cystine levels, leading to GSH depletion and inhibition of GPX4 activity, eventually activating ferroptosis [ 65 – 67 ]. Therefore, the expression level of SLC7A11 can directly mediate the susceptibility of cells to ferroptosis. In addition, FTH1 is known to carry out high levels of iron storage, thus ensuring normal biochemical reactions in vivo , and the genesis of ferroptosis is often accompanied by reduced levels of FTH1 . Our results showed that the levels of GPX4, SLC7A11, and FTH1 were decreased after MCAO/R, while EA treatment significantly augmented the expression of GPX4, SLC7A11, and FTH1. Iron is characterized by a diversity of metabolic roles and performs valuable physiological functions in vivo. Extracellular Fe 3+ combines with transferrin (TF) and is internalized to the nucleus through transferrin receptor 1 (TFR1) on the cell membrane, where Fe 3+ is reduced to Fe 2+ . Eventually, divalent metal transporter 1 (DMT1) discharges Fe 2+ from the nuclear endosome into the intracytoplasmic unstable iron pool in the cytoplasm. Furthermore, ferroportin 1 (FPN1) on the cell membrane frees surplus Fe 2+ to the exterior of the cell to ensure the maintenance of iron homeostasis . While the body's tissues or organs suffer damage, the cells are under stress and the intracellular redox balance is in jeopardy, with a resultant reduction of Fe 3+ to Fe 2+ . Overloaded Fe 2+ can catalyze the conversion of H 2 O 2 , a product of oxidative respiration in mitochondria, into hydroxyl radicals, which undergo nonenzymatic lipid peroxidation, eliciting membrane lipid peroxidation and mitochondrial damage, resulting in ferroptosis, which then exacerbates pathological damage . MDA as the terminal product of membrane lipid peroxidation reaction is a biomarker of lipid peroxidation and oxidized protein damage. SOD is among the prominent enzymes capable of scavenging ROS efficiently , and is liable for the conversion of superoxide anion, a generated ROS, into hydrogen peroxide, which is then converted to H 2 O by CAT (catalase), thus it is a crucial endogenous antioxidant factor for preserving cellular redox homeostasis [ 74 – 76 ]. Levels of MDA, SOD, and Fe 2+ have been recognized as signatures and pivotal indicators of ferroptosis; as yet, the definitive mechanisms governing ferroptosis downstream of lipid peroxidation remain elusive. Therefore, while the levels of MDA, SOD, and Fe 2+ cannot be regarded as the “gold standard” for ferroptosis, for the time being, these indicators can be one of the bases for determining if ferroptosis occurs in cells, and in the future, we will also further seek for more applicable indicators to refine the experiments. We showed that high accumulation of Fe 2+ in the brain of I/R-injured rats resulted in an imbalance of the antioxidant system, whereas EA treatment remarkably declined MDA levels, elevated SOD activity, and lessened ROS content, pointing to a striking amelioration of intracerebral lipid peroxidation and curtailing of ferroptosis, and that suppressing the origin and progression of cellular ferroptosis might be an equally valuable target for the exploration of novel therapeutic approaches for ischemic stroke in the future. The brain I/R injury belongs to the category of "stroke disease" in Chinese medicine, its cause is mainly due to the deficiency of healthy energy in the body, which leads to the inability to transport blood, so it stagnates in the body and produces stagnation. Although the vital signs have been stabilized during the recovery period, the blood stasis and phlegm turbidity have not been effectively eradicated , thus the treatment is centered on the principle of activating blood circulation, removing blood stasis, and regulating the meridians . Growing evidence indicates that EA is a potential strategy to promote neurological recovery in patients with ischemic stroke. As a safe and effective treatment, EA can be used to alleviate the symptoms of ischemic stroke and promote neurological recovery. Clinical trials and meta-analyses have revealed the effectiveness of EA in alleviating spasticity, reinforcing muscle strength, and achieving better overall health post-stroke, whilst improving patients' quality of life . It has been reported that EA not only protects neurovascular units by modulating cellular autophagy but also significantly reduces the level of oxidative stress and inhibits cellular ferroptosis . Compared to acupuncture alone, EA can produce a synergy effect through combining acupuncture and electrical stimulation . Four acupoints were chosen for this study: Baihui, Dazhui, Neiguan, and Quchi. Baihui and Dazhui have special therapeutic effects on brain disorders, and they are commonly used acupoints for emergencies as recorded in ancient Chinese medical literature, in the preventive period, acute period, relief period, and sequelae period of cerebral stroke, especially in the acute period of cerebral stroke in the state of coma is the preferred acupoints, which are worthy of in-depth study . Some research has found that acupuncture at Baihui and Dazhui points can reduce neuronal apoptosis in the ischemic area, inhibit oxidative stress, attenuate ischemic cerebral edema [ 83 – 85 ], and other mechanisms, that can promote the recovery of neurological function. In addition, acupuncture at the Quchi and Neiguan points has the property of replenishing vital energy and can cure hemiplegia, which are crucial points about health care. When we applied EA stimulation to these four acupoints, it significantly improved neurobehavioral scores and motor-behavioral outcomes after ischemic stroke and reduced cerebral infarct volume. We further confirmed that EA exerts this neuroprotective effect by inhibiting ferroptosis. Interestingly, we discovered that EA facilitated Nrf2 translocation to the nucleus, and thus we pursued further the correspondence between Nrf2 nuclear translocation and the preventive role of EA against stroke. Nrf2 is ubiquitously present in a broad range of cells and is redox-sensitive . At equilibrium, Nrf2 is bound to Keap1, continuously ubiquitinated by Cul3 E3 ubiquitin ligase, and subtracted by the proteasome, which maintains a high Nrf2 turnover rate. Under stress, Keap1 is oxidized and inactivated, resulting in the stability and translocation of Nrf2 to the nucleus, where it forms a heterodimer with the small Maf proteins, combines with the ARE, and triggers transcription of its target genes . Nrf2 regulates several downstream pathways, including apoptosis, inflammation, oxidative stress, calcium overload load, etc., assisting the body in upholding redox responses. Therefore, stabilizing Nrf2 activity is pivotal to maintaining redox balance and homeostasis in the brain. After a stroke, excess ROS activate Nrf2 , and incremental Nrf2 facilitates the expression of antioxidant genes and dampens ROS expression, which ultimately strengthens the mitochondrial antioxidant response and attenuates the destruction of the blood–brain barrier and neurological damage . HO-1, an ıncredibly essential downstream factor of Nrf2, which degrades heme to CO and biliverdin, products that are usually anti-inflammatory and antioxidant , and is a major mediator of the salutary effects of Nrf2 . By decomposing free radicals in the body into water and molecular oxygen, the Nrf2/HO-1 signaling pathway serves as a vital mechanism for the body's defense against oxidative stress, mitigating oxidative stress damage and curtailing the production of oxidative products, thereby rendering anti-inflammatory and antioxidant effects . As such, Nrf2 is located at the center of a sophisticated modulatory network that exerts protective effects against cerebral ischemia through multiple mechanisms . Our study found that EA promoted the translocation of Nrf2 from the cytoplasm to the nucleus and resulted in elevated HO-1 levels. To elucidate the protective mechanism of EA on MCAO/R, we applied Brusatol, an inhibitor of Nrf2, to block this pathway. The results showed that Brusatol decreased the content of Total-Nrf2, Nuclear-Nrf2, and HO-1 and decreased the locomotor ability, increased the infarct volume, increased the levels of lipid peroxides and Fe 2+ , inhibited the expression of GPX4, SLC7A11, and FTH1, and reversed the inhibitory effect of EA on ferroptosis. This finding suggests that EA therapy inhibited the occurrence of ferroptosis in cells by facilitating the nuclear translocation of Nrf2 and exerting its antioxidant effects. In the last decades, research on ischemic encephalopathy models using rodents has yielded very rewarding results, and many neuroprotective measures have been deemed as prospective therapies. Unfortunately, many drugs have mostly failed to show effectiveness in ischemic stroke patients in randomized clinical trials, however. Therefore, translating research findings into clinical practice is quite a challenging task, and we expect EA to become a more efficacious treatment modality in the clinical management of ischemic stroke. Additionally, modulation of iron metabolism and antioxidant pathways to inhibit ferroptosis is an encouraging goal for the future treatment of ischemic stroke. However, more studies are required to elucidate the functional alterations and molecular mechanisms of ferroptosis. In recent years, Nrf2 has become one of the hotspots in the research field due to its potent value in inhibitory inflammatory response and anti-oxidative stress, therefore, the detailed mechanism of EA on the Nrf2/SLC7A11/GPX4 axis needs to be further explored, such as how EA specifically regulates Nrf2 expression and entry into the nucleus to exert a neuroprotective effect. In addition, in vitro experiments are necessary to investigate the effect of activating Nrf2 in ferroptosis, which could help to further elucidate and verify this process. In this experiment, EA inhibited cellular ferroptosis by stimulating Nrf2 nuclear translocation, thus displaying a neuroprotective effect versus acute brain injury, which provides an emerging target for EA to remedy specific mechanisms of ischemic stroke. As our understanding of the mechanisms and pathology of stroke increases, neuroprotective strategies emerge as hopeful treatments. With multi-center, large-sample, well-quality laboratory, and clinical trials, further studies on the molecular mechanisms underlying the neuroprotective effects of electroacupuncture may provide a scientific basis for the selection of acupoints and therapeutic parameters for clinical treatment, as well as a more optimized strategy. The present study highlights the efficacy of EA at Baihui, Dazhui, Quchi, and Neiguan on MCAO/R rats, and simultaneously proposes a mechanism involving the promotion of Nrf2 nuclear translocation and activation of the Nrf2/SLC7A11/GPX4 pathway to suppress ferroptosis following ischemic stroke in rat, these suggesting that EA treatment could be applied as an effective therapeutic intervention to improve neurological function. These findings afford prospective therapeutic mechanisms for EA in the treatment of ischemic stroke at Baishui, Dashui, Quchi, and Neiguan, and render valuable doctrinal support for clinical application. | Other | biomedical | en | 0.999998 |
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