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Pure Basaloid Squamous Cell Carcinoma of the Uterine Cervix: A Case Report # Introduction Basaloid squamous carcinoma of the uterine cervix is a rare tumor type characterized by an ulcerated, infiltrating growth pattern; nests or cords of small basaloid cells; prominent peripheral palisading of cells in the tumor cell nests; and the absence of significant stromal reaction [bib_ref] Basaloid carcinoma of uterine cervix, Daroca [/bib_ref]. These tumors can arise from various anatomic sites, including the hypopharynx, base of the tongue, salivary glands, esophagus, anal canal, prostate, thymus, vulva, and urinary bladder [bib_ref] Basaloid-squamous carcinoma of the tongue, hypopharynx and larynx: report of 10 cases, Wain [/bib_ref] [bib_ref] Basaloid-squamous carcinoma of the upper aerodigestive tracts, Batsakis [/bib_ref] [bib_ref] Basaloid squamous cell carcinoma of floor of mouth, Coppola [/bib_ref] [bib_ref] Basaloid carcinomas of salivary glands, Gallimore [/bib_ref] [bib_ref] Basaloid-squamous carcinoma of the esophagus: a clinicopathologic, DNA ploidy, and immunohistochemical study..., Abe [/bib_ref] [bib_ref] Basaloid and warty carcinomas of the vulva: distinctive types of squamous cell..., Kurman [/bib_ref] [bib_ref] Carcinoma of the anal canal: a study of 79 cases, Dougherty [/bib_ref] [bib_ref] Thymomas and thymic carcinomas, Walker [/bib_ref] [bib_ref] Basaloid carcinoma of the prostate gland: histogenesis and review of the literature, Denholm [/bib_ref] [bib_ref] Basaloid squamous cell carcinoma occurring in the urinary bladder, Vakar-Lopez [/bib_ref] , but origin of uterine cervix is rare. Basaloid squamous carcinoma of the uterine cervix is neither recognized nor included as a specific histologic subtype in the current World Health Organization (WHO) classification of cervical tumors. Since basaloid squamous carcinomas are thought to behave aggressively [bib_ref] A reappraisal of ''basaloid carcinoma'' of the cervix, and the differential diagnosis..., Grayson [/bib_ref] but the evidence of supprting this behavior is not powerful, accurate diagnosis and accumulated data of this tumor are important for their clinical management and prognosis. ## Case report A 70-yr-old woman, gravida 8 para 3, was referred to a tertiary medical center from a local hospital for vaginal bleeding of 3 weeks' duration. She had undergone a punch biopsy at the local hospital, which resulted in a preliminary diagnosis of adenoid cystic carcinoma or carcinoid tumor. Her medical history included diabetes mellitus (DM) for 10 yr which had been controlled by medication; in addition, her mother died of uterine cervical cancer. Physical examination showed uterine cervical erosion. The biopsy specimen taken at the local hospital showed pathologic evidence of a high grade malignant epithelial tumor, with features unusual for a cervical tumor. She therefore underwent a loop electrosurgical excision procedure (LEEP) cone biopsy, which revealed a basaloid squamous cell carcinoma. A colonoscopy, intravenous pyelogram, and cystoscopy showed no evidence of metastatic disease. Magnetic resonance imaging showed a 2 cm sized cancerous mass confined to the cervix, with no evidence of invasion of the vagina or fornix and no evidence of pelvic lymphadenopathy . The tumor was classified as clinical stage Ib1. A radical hysterectomy was performed, along with bilateral salpingo-oophorectomy, pelvic lymph node dissection, and paraaortic lymph node sampling. The pathologic diagnosis was basaloid squamous cell carcinoma. The depth of invasion was 5/7 mm full thickness of the cervical wall. There was no evidence of tumor in sections taken from 26 lymph nodes. The resection margin of the vaginal cuff was Pure Basaloid Squamous Cell Carcinoma of the Uterine Cervix: ## A case report Basaloid squamous cell carcinoma of the uterine cervix is an extremely rare malignancy of the female genital tract with a poorer clinical outcome than squamous cell carcinoma of the uterine cervix. We report a case of pure basaloid squamous cell carcinoma of the uterine cervix. A 70-yr-old woman with vaginal bleeding was referred to our institute. A basaloid squamous cell carcinoma of the uterine cervix, of International Federation of Gynecology and Obstetrics (FIGO) stage Ib1, was diagnosed by a loop electrosurgical excision procedure cone biopsy. A radical hysterectomy was performed, along with bilateral salpingo-oophorectomy, pelvic lymph node dissection, and para-aortic lymph node sampling. Pathologic findings were consistent with a basaloid squamous cell carcinoma confined to the cervix without an extracervical tumor. No further treatment was administered and there was no clinical evidence of recurrence during the 12 months of follow-up. Follow-up for the patient is ongoing. Although basaloid squamous cell carcinoma of the uterine cervix is thought to behave aggressively, accumulation of data on these rare tumors is necessary to determine whether their behavior differs significantly from that of conventional cervical squamous cell carcinoma of similar clinical stage. These data would be useful for defining the best diagnosis and treatment for these rare tumors. clear. No adjuvant treatment was administered, and the patient was discharged. In the 12 months since discharge, she has shown no evidence of recurrent or metastatic disease. Follow-up is ongoing at Asan Medical Center. ## Pathologic findings A well-defined, fungating firm mass (1.5×1.0×0.6 cm) was present in the posterior wall of the cervix and invaded 5 mm into the cervical wall (full thickness, 7 mm). The parametria and vaginal cuff showed no tumor invasion. The cut surface of the mass was gray and granular. The tumor cells were immunopositive for p63 and immunonegative for S-100 pro-tein. The cells appeared basaloid with small hyperchromatic nuclei, distinct nucleoli and scanty cytoplasm. There was also peripheral palisading, supporting the above diagnosis . # Discussion Cervical basaloid carcinoma has recently been classified as a specific histologic subtype, with "pure" basaloid carcinomas being extremely rare [bib_ref] Malignant mullerian mixed tumors of the uterine cervix: a report of nine..., Clement [/bib_ref]. Often few incidence of this diagnosis can cause clinical and pathological misinterpretation. In our case, based on a punch biopsy specimen, our patient was initially diagnosed at the local hospital with adenoid cys- tic carcinoma or carcinoid tumor, which was not an accurate diagnosis. To obtain an accurate diagnosis, an LEEP cone biopsy, which yields a larger tissue sample, was performed and resulted in a diagnosis of basaloid carcinoma of the uterine cervix, indicating that diagnostic difficulties can be avoided by obtaining larger specimens for pathologic diagnosis [bib_ref] Adenoid basal epitheliomas of the uterine cervix: a reevaluation of distinctive cervical..., Brainard [/bib_ref]. The term "basaloid carcinoma of the uterine cervix" refers to any neoplastic lesion of the uterine cervix analogous to a cutaneous basal cell carcinoma (BCC), and may include some of the recognized morphologic variants of BCC. Among the morphologic characteristics of basaloid carcinomas of the uterine cervix are nests or islands of small basaloid cells, hyperchromatic epithelial cells with high nucleus-to-cytoplasm ratios, and a tendency to palisade at the periphery of the tumor islands. A basaloid carcinoma of the cervix may be associated with squamous dysplasia, in situ SCC, or invasive SCC [bib_ref] Basaloid squamous cell carcinoma of the esophagus: diagnosis and prognosis, Sarbia [/bib_ref] [bib_ref] Basaloid-squamous carcinoma of the upper aerodigestive tract and so-called adenoid cystic carcinoma..., Tsang [/bib_ref]. Immunocytochemistry can occasionally be helpful in identifying the epithelial origin of a basaloid carcinoma of the cervix. Some of these tumors have been shown to be strongly positive for high molecular weight cytokeratin [bib_ref] Basaloid squamous carcinoma in the liver, Bastiaan De Boer [/bib_ref] [bib_ref] Basaloid squamous cell carcinoma of the maxilla: a case report and immunohistochemical..., Tulunay [/bib_ref] , whereas others have shown little or no expression of cytokeratins [bib_ref] Basaloid-squamous carcinoma of the upper aerodigestive tract and so-called adenoid cystic carcinoma..., Tsang [/bib_ref] [bib_ref] Basal cell (basaloid) carcinoma of the lung: a new morphologic and phenotypic..., Brambilla [/bib_ref]. Most tumors are positive for epithelial membrane antigen (EMA), but show little or no expression of vimentin, smooth muscle actin, desmin or neuroendocrine markers [bib_ref] Basaloid squamous carcinoma in the liver, Bastiaan De Boer [/bib_ref] [bib_ref] Basaloid squamous cell carcinoma of the maxilla: a case report and immunohistochemical..., Tulunay [/bib_ref]. The major differential diagnosis of basaloid SCC includes the solid variant of adenoid cystic carcinoma (ACC), small cell carcinoma, and large cell neuroendocrine carcinoma (LC NEC) of the cervix. Solid ACC is distinguished from basaloid SCC by the focal presence of basement membrane material enveloped by basaloid neoplastic cells; in addition, the solid variant of ACC may show malignant squamous differentiation. Small cell carcinoma may be composed of variably sized nests of relatively small, hyperchromatic tumor cells, which may mimic adenoid basal carcinoma (ABC) and smaller ABC-like nests. Rare cases may present with large neoplastic islands having peripheral palisading of tumor cells, which may be confused with the solid variant of ACC [bib_ref] The solid variant of adenoid cystic carcinoma of the cervix, Albores-Saavedra [/bib_ref]. Small cell neuroendocrine carcinomas may be diagnosed by immunohistochemical or ultrastructural demonstration of neuroendocrine differentiation. Cervical LCNEC, another neoplasm omitted from the current WHO classification of uterine cervical neoplasms, is characterized by large cells with vesicular nuclei and prominent nucleoli, a mitotic index in excess of 10 per 10 high-power fields, geographic areas of tumor necrosis, and positive staining with appropriate immunohistochemical markers of neuroendocrine differentiation [bib_ref] Detection of human papillomavirus in large cell neuroendocrine carcinoma of the uterine..., Grayson [/bib_ref] [bib_ref] Terminology of endocrine tumors of the uterine cervix. Results of a workshop..., Albores-Saavedra [/bib_ref]. Cervical LCNEC composed of large basaloid islands may mimic basaloid SCC, and may also show a considerable degree of morphologic overlap with solid ACC, a lesion that may contain trabecular structures composed of basaloid cells that palisade at the periphery [bib_ref] Detection of human papillomavirus in large cell neuroendocrine carcinoma of the uterine..., Grayson [/bib_ref]. Accurate diagnosis is of prognostic importance because of the biologically aggressive na-ture of this uncommon type of cervical cancer. The lack of standard diagnostic criteria for pure basaloid squamous cell carcinoma of the uterine cervix has made it difficult to predict their precise biologic behavior and to design optimal management strategies. Accumulation of data on these rare tumors is therefore necessary to determine whether their behavior differs significantly from that of conventional cervical SCCs of similar clinical stage. Long-term follow-up of this patient and other such patients is therefore important. [fig] Figure 1, Figure 2: MRI findings in this patient. (A) T2 weighted image (B) T2 weighted image using an endorectal coil. The white arrow indicates a 2.0×1.6 cm cervical mass, comparable with cervical cancer. Histopathologic findings. The tumor is mainly composed of immature basaloid squamous cells with scanty cytoplasm and peripheral palisading (arrow). Keratinization foci are seen in the center of the nests (arrowhead). Both tumor components show p63 immunopositivity (A: H&E, ×200; B: p63, ×200). A B [/fig]
Understanding the mental health impact and needs of public healthcare professionals during COVID-19 in Pakistan : a qualitative study # Introduction Worldwide, health service providers are under insurmountable psychological pressures as they are expected to deal with a number of issues that arise due to the COVID-19 situation. [bib_ref] Prevalence of depression, anxiety and post-traumatic stress disorder in health care workers..., Li [/bib_ref] First, due to the increased clinical workload associated with COVID-19 cases, health service providers face increasingly long work hours, often with limited resources and weak infrastructure. [bib_ref] Public responses to the novel 2019 coronavirus (2019-nCoV) in Japan: mental health..., Shigemura [/bib_ref] Second, they face physical discomfort associated with donning and doffing of personal protective equipment (PPE), or the unavailability of it, which is essential to keep them safe from exposure to the virus.Third, health service providers feel unprepared to deal with a new and unfamiliar task since the evidence-based clinical treatments for COVID-19 continue to evolve.Finally, there is a very valid fear of autoinoculation and the risk of transmitting the virus to family members. Unsurprisingly, all these factors take a toll on the mental health of healthcare professionals (HCPs), and result in various psychiatric issues including fear, [bib_ref] Frontline nurses' burnout, anxiety, depression, and fear statuses and their associated factors..., Hu [/bib_ref] ## Strengths and limitations of this study ⇒ This study provides an in-depth exploration of experiences of healthcare professionals regarding the mental health impact of COVID-19, as well as their mental health needs and suggestions to address these needs in a pandemic crisis. ⇒ Unlike previous urban-focused studies, the participants in this study were invited across 22 urban and rural districts in the two most populous provinces of Pakistan. ⇒ One key limitation of the study was that the interviews were conducted by phone owing to restricted mobility during the COVID-19 pandemic, which may have introduced some risk of bias as the interviewers did not have the opportunity to build rapport as in face-to-face interviews. Open access anxiety, 1 depression, 1 burnout, 7 trauma 8 and insomnia. [bib_ref] Factors impeding health-care professionals to effectively treat coronavirus disease 2019 patients in..., Raza [/bib_ref] Evidence shows that mental health implications for health workers who are involved in the provision of healthcare during epidemics and pandemics are long-lasting. While quantitative studies have significantly outnumbered qualitative investigations in terms of assessing the magnitude of mental health problems among HCPs resulting from COVID-19, they are limited in providing an in-depth understanding of HCPs' perspectives and experiences regarding the impact of COVID-19 on their mental health. Moreover, most qualitative investigations explored the general experiences of HCPs during the COVID-19 pandemic, whereas very few have specifically focused on their mental health experiences and these are mainly from high-income countries. [bib_ref] The psychological impact of COVID-19 on front line nurses: a synthesis of..., Huerta-González [/bib_ref] A qualitative study from England concluded that patients with COVID-19 brought a significant emotional toll, and strained relationships between immediate frontline staff and their families. At the beginning of the pandemic, staff were driven by adrenalin and optimism; but over time this dissipated to be replaced by exhaustion, numbness and dreadful expectation of a 'second wave'. [bib_ref] COVID-19 Confessions: a qualitative exploration of healthcare workers experiences of working with..., Bennett [/bib_ref] Another qualitative study from Pakistan [bib_ref] Exploring stress coping strategies of frontline emergency health workers dealing Covid-19 in..., Munawar [/bib_ref] presented a thorough understanding of how the frontline emergency healthcare workers (HCWs) are dealing with the COVID-19 pandemic, their stress-coping strategies or protective factors, and challenges while dealing with patients with COVID-19. These studies are limited in providing evidence on health systems' preparedness to respond to the mental health needs of HCPs. Earlier evidence during the SARS epidemic showed that system-level interventions were effective in reducing health service providers' symptoms of anxiety and depression. By and large, there is not only a dearth of qualitative studies on mental health impact of COVID-19 on HCPs from low/middle-income countries but there are also gaps in existing literature. [bib_ref] Health care workers' experiences during the COVID-19 pandemic: a scoping review, Chemali [/bib_ref] For example, there is a lack of information about the psychosocial and mental health needs of HCPs and how well the health system has responded to those needs. [bib_ref] The psychological impact of COVID-19 on front line nurses: a synthesis of..., Huerta-González [/bib_ref] Furthermore, these qualitative studies had a predominant focus on hospitals situated in urban settlements or metropolitan cities, while their sample did not have adequate representation from rural settings. Also, the HCPs included in many quantitative studies were mainly doctors and nurses; few studies have gathered perspectives of other HCPs-such as health administrators. [bib_ref] The psychological impact of COVID-19 on front line nurses: a synthesis of..., Huerta-González [/bib_ref] To the best of our knowledge, there is a paucity of qualitative evidence to understand the mental health needs of HCPs and the health system response, which is essential to develop context-specific mental health interventions. This study is the first to explore the mental health impact of COVID-19 as well as the mental health needs of health service providers in public healthcare settings of Pakistan from the perspective of health managers and health service providers including doctors and nurses. Our study aims to answer the following research questions: ► What is the perceived mental health impact of the COVID-19 pandemic on the performance of health service providers in secondary and tertiary care settings in Pakistan? ► What are the health service providers' mental health needs working amid the COVID-19 pandemic, and how well the health system is responding to them? ► What are the recommendations by the health service providers to address their mental health needs? # Methods ## Country context Pakistan has a population of over 225 million inhabitants.The first COVID-19 case was identified in February 2020, and since then a total number of confirmed cases that exceed 1.5 million along with 30 574 deaths have been registered (until 29 August 2022). The country has experienced five waves-each with a different variant including omicron, more recently. With a focus on vulnerable groups of HCPs and elderly (65+ years) people, the country embarked on a nationwide vaccine campaign for COVID Healthcare in Pakistan is delivered through a threetiered health system. Secondary-level healthcare facilities concern with provision of technical, therapeutic and diagnostic services. Specialist consultation and hospital admissions fall into this category. The availability of specialised services vary from facility to facility across districts depending on the catchment population and their service needs and demands.It includes Tehsil Headquarters (THQs) and District Headquarters (DHQs) Hospitals. Tertiary healthcare hospitals are for more specialised inpatient care supported by availability of required resources. With few exceptions, these are also affiliated with the medical teaching institutions for graduates and postgraduates. [bib_ref] Healthcare system of Pakistan, Hassan [/bib_ref] The government of Pakistan has taken several measures to combat this deadly pneumonia virus 21 but it leaves a lot to be desired. [bib_ref] COVID-19 and its challenges for the healthcare system in Pakistan, Khalid [/bib_ref] The health system of Pakistan is weak,with critical shortage of health workforce which is not prepared to sustain a major surge in COVID-19 cases. [bib_ref] The health workforce crisis in Pakistan: a critical review and the way..., Abdullah [/bib_ref] Recent quantitative studies show the considerable impact of COVID-19 on the psychological health of HCPs. Generally in Pakistan, mental illnesses are stigmatised and widely perceived to have supernatural causes. The psychiatrist-to-population ratio is about 1 psychiatrist to 0.5-1 million people 27 ; this-combined with limited task-sharing interventions-results in a huge treatment gap in the country. The economic burden of mental illnesses in Pakistan is US$4.2 billion annually. [bib_ref] Economic burden of mental illnesses in Pakistan, Malik [/bib_ref] Design, setting and participants The Consolidated criteria for Reporting Qualitative research checklist has been used for designing this qualitative study [bib_ref] Consolidated criteria for reporting qualitative research (COREQ): a 32-item checklist for interviews..., Tong [/bib_ref] (see online supplemental file 1). This research employed an exploratory qualitative research design using semistructured interviews and a criterion purposive sampling approach. Our study was Open access conducted at secondary-level and tertiary-level public sector hospitals of the two most populous (Sindh and Punjab) provinces of Pakistan. A total of 8 teaching hospitals, 8 DHQs, and 25 THQs were randomly selected from Sindh and Punjab provinces. The study was conducted in close collaboration with provincial health departments. The data collection methods for this research included in-depth interviews (IDIs) with health service providers (doctors, nurses), head of department (HoD) and medical superintendent (MS). In terms of participants' involvement in the care of patients with COVID-19, health managers including MS and HoD were indirectly involved in the management of patients with COVID-19; however, health service providers including doctors and nurses were directly involved in the care of patients with COVID-19. For secondary-level health facilities, we contacted the MS who was invited to participate in the study. Moreover, he/she was further asked to nominate service providers from their respective health facilities who may or may be not involved in the provision of care to patients with COVID-19. Similarly, for teaching hospitals, we contacted the administrative head who identified a focal person on his/her behalf for further coordination. Similar request was made to the focal person for identification of department heads and service providers. Only nine participants refused to participate mainly due to their busy schedule and lack of interest. ## Eligibility criteria Health managers and health service providers employed in public sector hospitals of Sindh and Punjab provinces and were dealing directly or indirectly with patients with COVID-19 were eligible to participate in the study. Health service providers and hospital managers either belonging to private sector hospitals or from provinces other than Sindh and Punjab were excluded. Non-clinical staff such as janitors and technicians were also excluded from the study. ## Data collection Adapting to the COVID-19 situation, a total of 56 (Sindh: 34; Punjab: 22) semistructured telephonic IDIs were conducted individually in the local language (Urdu), by trained female data collectors who had a background in sociology, anthropology and psychology. The interviews are conducted at the Aga Khan University Hospital under close supervision of research investigators. Keynotes were also taken by data collectors during the interviews. It was ensured that the participant is comfortable during the interview and no one is sitting near to them whose presence could possibly influence his/her responses. To ensure data quality, a few initial interviews were conducted by WH (PhD scholar-health system), ASF (PhD scholarpublic health) and NA (BS psychology) in the presence of trained data collectors. The IDIs were conducted until data saturation was achieved. [bib_ref] Are we there yet? data Satur et? data saturation in qualitative research, Fusch [/bib_ref] Data collection and data analysis were conducted iteratively to determine the point of data saturation. All interviews were completed in the first interaction. Consistent with Creswell and Poth's 31 guidelines on interviewing, all IDIs lasted approximately between 45 and 60 min. The interviews were audiorecorded on tablets with consent from study participants, which were later transcribed in the English language by a transcriptionist. The data collection took place between August and October 2020. The transcripts were not returned to the participant due to their busy schedule in the hospitals. A semistructured interview guide was designed for IDIs through an extensive literature review and consultative approach seeking expert opinions. This guide was developed to interview all research participants. The guide includes five major themes: (a) mental health impact of COVID-19 on health service providers; (b) mental health needs of health service providers involved in the COVID-19 crisis; (c) support mechanisms available to address mental health needs of health service providers; (d) challenges in addressing mental health needs of health service providers; (e) recommendations to address mental health needs of health service providers. A free flow of information was encouraged, using the participants' verbatim account of the conversation to understand their perception of mental health needs during the COVID-19 pandemic. The interview guide was used flexibly to allow participants to construct their accounts on their own terms. The guide was pilot tested with a non-study sample which shares the same characteristics as the study sample.The pilot testing provided evidence-based direction to improve the interview guide questions 32 (see online supplemental file 2). # Data analysis All study data including notes, recordings and transcriptions were uploaded into the NVivo V.11.0 software for data analysis.The analysis looked at convergent and divergent lines of inquiry from the two main study groups, that is, health managers and health service providers (doctors and nurses). All qualitative data included were analysed using conventional content analysis. Two independent researchers (ASF and NA) read each transcript from beginning to end, and coded text that appeared to describe the mental health impact of COVID-19 as well as the mental health needs of health service providers in public healthcare settings of Pakistan. After open coding of initial few transcripts, independent researchers met to discuss and decide on the preliminary codes (codebook). Researchers then coded the remaining transcripts (and recoded the original ones) using the codebook and added new codes when they encountered data that did not fit into an existing code. Once all transcripts had been coded, researchers examined all data within a particular code. Some codes were combined during this process, whereas others were split into subcategories. The discrepancies in coding were resolved through Open access a discussion with a senior researcher. Throughout the analysis, the researcher made reflective notes (memos) to document important learning from the data. [bib_ref] Memoing in qualitative research: probing data and processes, Birks [/bib_ref] ASF and NA independently performed coding to ensure that interpretations are coherent, plausible and grounded in the study data.Please see online supplemental file 3 to see an example of content analysis. Trustworthiness of the study Of the five markers suggested by Tracy [bib_ref] Qualitative quality: eight "big-tent" criteria for excellent qualitative research, Tracy [/bib_ref] and Lincoln et alto ensure trustworthiness and methodological rigour, we used validity and credibility and reliability/dependability. To ensure credibility, the study triangulated data via two basic types of triangulation: data source triangulation (exploring insights of different groups-hospital managers and health service providers) and investigator triangulation (use of multiple researchers in analysis phase-ASF and NA).In addition, the primary researcher maintained a reflexive journal during all stages of the research to recognise and acknowledge biases during the research process. For example, reflexivity was used to develop the interview guides and modify it based on the inputs from other investigators and after the initial interviews, and analyse the data in different groups to tell a story about how the health system responded to deal with the mental health needs of health service professionals during the COVID-19 pandemic. ## Patient and public involvement We sought inputs from target audience through pretesting of interview guides to ensure comprehension of the questions. Furthermore, in consultation with provincial health departments, we selected districts where COVID-19 cases were high. The focal person in each selected district was oriented about the study who connected our team with those in charge in the health facility to avoid any miscommunication. Lastly, they were also advised on the dissemination of research findings-that is, they specifically emphasised sharing it through research briefs to reach a wider audience in the country. # Results A total of 56 semistructured IDIs were conducted to explore the mental health needs of health service providers amidst the COVID-19 pandemic in Pakistan. Of 56 IDIs, 16 IDIs were conducted with health service providers, and 40 IDIs were conducted with health managers. All the participants (n=56) who were approached by the study team agreed to participate. The demographic information for all the study participants is illustrated in table 1. Based on the directed content analysis, three overarching themes were identified: (1) mental health impact of COVID-19 on health service providers; (2) mental health needs of health service providers involved in the COVID-19 crisis and available support from the health system; and (3) suggestions to address mental health needs of health service providers. These themes and their categories are presented in table 2. ## Mental health impact of covid-19 on health service providers fear of infection, isolation and stigma The interviews with hospital managers and health service providers revealed that all health service providers felt fearful of acquiring the COVID-19 infection and transmitting the infection to their family members. A hospital manager mentioned that health service providers are particularly concerned about their parents and children who are vulnerable and might get infected. They (HCPs) were afraid for their families that our parents are old and if we carry coronavirus from here then we might not get them infected. Even if we don't show any symptoms, we would get them infected or we would get our children infected. So this fear was also prevailing. (Hospital Manager, Punjab) Health service providers were fearful that if they did acquire the infection, they would have to isolate themselves from their families. Many workers could not afford to isolate themselves in the event they tested positive for COVID-19. They used to say that if the report comes back positive, then how they will stay at home because their homes are small. (Hospital Manager, Sindh) Immediately after that, there was fear of isolation, like if you put yourself into isolation who will look Both groups of respondents highlighted that few health service providers resigned from their jobs since their families were getting infected because of them. In particular, female health service providers had higher pressure on them from their families to leave their jobs. While some providers refused to work in COVID-19 wards, others threatened health managers to commit suicide, if they were posted there. Health managers verbalised that a lot of the health service providers posted in COVID-19 wards revealed the fear of being isolated and stigmatised. Health workers were hospital-bound and were afraid of living alone and being cut off from their families. They also felt the stigma from the people around them. (Hospital Manager, Sindh) Stress due to poor availability of PPE A major concern among the health service providers was the lack of protective gear available to them. Thus, treating COVID-19-positive or suspected cases without PPE was escalating their fear of getting infected. They felt that they had been sent to fight a war without weapons. Firstly, the PPEs we were using, we had to use them twice or thrice… this was our main issue. We had to wear the mask for two to three days even one week continuously. (Health Service Provider, Punjab) On the other hand, health managers revealed that some health service providers labelled them as being insensitive and unaware of the problems faced by workers due to a lack of available PPE. However, managers themselves were helpless due to PPE's lack of availability in the market and ended up buying masks and other protective equipment for the staff themselves, but this protective gear was not up to the mark. That time if anyone didn't get PPE they (health service providers) would get hyper but we provided PPEs at our facility. We would provide requisite equipment soon so we didn't have to face so much trouble. (Hospital Manager, Punjab) ## Anxiety due to uncertainty of covid-19 Health service providers reported feeling extremely anxious working with patients with COVID-19. Owing to the novelty and unfamiliarity of the virus, along with workers being inadequately equipped with the required knowledge about the disease, they were uncertain about the consequences. Health service providers were also having trouble identifying cases of COVID-19 since there were both symptomatic and asymptomatic cases. They were not clear about the mode of transmission of the virus. One can call it anxiety, you can see it, and they were under a depressive state. All healthcare workers were. From doctors to nursing staff, paramedics, our sweepers, all our workers were going through depression. (Health Service Provider, Sindh) Some hospital managers had contrasting views about the difficulties faced by their staff while working during the pandemic. They claimed that health service providers were not stressed while working during the COVID-19 pandemic and that none of them came to the managers to express their concerns. I don't think their mental health was affected, I don't think so, and there was stress only in the beginning when they were not aware of the atmosphere. (Hospital Manager, Sindh) No there was no pressure on them, we were doing many meetings, we were explaining it to them, that there's no issue, this time has come we will solve it Insha'Allah, we will be successful, there wasn't an issue like this in our staff, they were definitely cooperating with us, that's why there was no stress or any problem like that. (Hospital Manager, Sindh) ## Open access Anxiety and nervousness due to media exaggeration Both groups of respondents reported that the media has a strong role in mounting anxiety and distress among HCWs. The media was seen to exaggerate the magnitude of the situation and there was a bombardment of information. News regarding deaths of workers due to COVID-19 was also creating panic among the majority of the HCWs. But this novel coronavirus made hype through digital media as no one knew what it was as initially people were stressed. (Hospital Manager, Sindh) False information was seen to circulate about the negative consequences for people testing positive for COVID-19, which was creating a lot of distress among HCWs currently working in COVID-19 wards. In addition, news related to the long-term complications of COVID-19 increased nervousness among HCWs. If you take my personal opinion, the hype created regarding this is too much, that has spread too much tension. That has bothered people a lot about what will happen, God forbid if somebody has corona then he will be taken away or this will happen or that will happen, etc or because of me, my children will suffer. (Health Service Provider, Punjab) ## Excessive workload Both groups of respondents mentioned that health service providers felt strained and exhausted because of the clinical workload. Due to the shortage of health workforce, the existing pool of health service providers had to bear the burden of extra clinical work. Their duty hours increased along with the number of shifts they had to do per week, which resulted in increased psychological distress among health service providers. So far, no one told me that their mental health has been affected… yes, some people have said that they are depressed… they are feeling tired and have motion sickness due to extra working hours. (Health Manager, Punjab) The COVID-19 outbreak among health service providers led to further staff shortages and increased mental pressure among health service providers. Both groups of respondents mentioned that even after returning from sick leave, health service providers were not performing at their best. Some of them seem to become weak and some of them would faint. In addition, some workers' tests were negative for the COVID-19 infection, but they still had cough symptoms which would make their coworkers anxious. Hence, they also had to be given leave. As the work load increased, so did the pressure on the HCWs and their efficiency began to decrease. (Health Manager, Sindh) Both groups of respondents mentioned that their commute time to the hospital also significantly increased due to the unavailability of public transport during the lockdown, which further increased the psychological burden among health service providers. Our doctors faced a lot of transport issues but we tried our best to support them. Few of our staff were punctual initially, but some of them were not coming at all due to fear of COVID-19. Their wages are less so we reduced the duration of their duties and counselled them a lot after which they started coming to work. They come from far off areas so they should be given space here at the hospital so that they can perform their duties easily. Psychological needs of the health workforce involved in the COVID-19 crisis and available support from the health system Need to provide counselling services and safe working conditions Health service providers and health managers highlighted the need for counselling services for health service providers working under pressure to provide care to patients with COVID-19. Health service providers mentioned the desire to receive one-to-one counselling sessions from mental health professionals to reduce the stress and anxiety they were experiencing because of working during the COVID-19 pandemic. All hospitals should at least provide one-to-one… counselling sessions… to reduce staff anxiety and stress. (Health Service Provider, Sindh, male) Since no proper counselling services from mental health professionals were available, informal counselling to health service providers was provided by hospital managers. Although the managers were trying to support their staff, they lacked formal training in providing counselling. They motivated them via reinforcing health providers' professional and moral obligations but it did not prove fruitful for most of the workers. Whatever knowledge I had regarding the disease and its spread, and I also told them that we must serve humanity, and death is inevitable and I did whatever I could, regarding the moral values, whatever I could make them understand, some of them understood and most of them didn't. (Hospital Manager, Sindh) I told them with a passion that the death Allah has written, if we die on this job, they will be martyred. (Hospital Manager, Punjab) Health managers from few facilities reported that the government had arranged webinars for health service providers to help them manage psychological stress. However, the effectiveness of this training was quite low because many health workers had duties due to which they were unable to attend or sit through training. In addition, health service providers highlighted the need of working in a safe and secure working environment. They further mentioned that the lack of safety was largely attributed to the inadequate supply of protective equipment. To make up for the lack of PPE, health service Open access providers resorted to using the same protective gear they had repeatedly used for multiple days. In many places, we have seen that doctors and paramedical staff were affected and all this is due to the lack of what was needed, for example, the lack of hand sanitizers and masks. (Hospital Manager, Sindh) Need for paid rest periods, timely payment of workers' salaries and incentives Health service providers also highlighted the need for extra rest periods so that they could get enough time to relax from the exhaustive routine and then come back to work. A common strategy employed by the health managers was to give short leave to the health service providers most affected by the COVID-19 stress. This leave helped the staff in regaining their mental and physical strength to work. They (health service providers) used to say we will do work for 15 days and then require 15 days for holidays, we will relax during that time, I said ok I will give that to you. (Hospital Manager, Sindh) In addition, the majority of health service providers and health managers reported that the lack of additional allowance for staff directly dealing with patients with COVID-19 was a demotivating factor especially for paramedical staff. The poor paramedical staff who are the first ones to interact with the patients get paid very less. The government did not do anything for them because the staff used to complain that we are getting the same salaries while having maximum exposure to COVID-19. (Health Service Provider, Punjab) Health service providers also wanted to be given monetary incentives because working during the pandemic meant that their lives were at stake. This would also help to increase their motivation to work during such difficult conditions. They should be given incentive because they are keeping their life on risk. (Health Service Provider, Sindh) Need for appreciation and motivation to work in the pandemic Hospital managers and health service providers reported that workers needed appreciation for all the hard work they were doing. Moreover, providing appreciation would, in turn, lead to the workers becoming motivated to continue their efforts. Health service providers were putting their lives at stake while working in such difficult conditions without adequate resources, hence their efforts should be commended. Then they (health managers) should arrange programs in which they appreciate and motivate their employees and provide them proper guidance then it will have major positive effects on mental health. (Health Service Provider, Punjab) It was also suggested that the staff should be given certificates to appreciate the work they were doing and to hold regular meetings with them to see work progress and understand their needs. Health service providers mentioned that motivating sessions are also needed to boost morale of the workers who are working in such difficult conditions. I told my hospital director that we need a psychologist. I also highlighted that our human resource team should be given appreciation certificates other than monetary incentives which might boost their motivation to work. Meetings should also be conducted with them to ask them about their work and what else they need since this is also lacking currently. (Hospital Manager, Sindh) ## Suggestions to address mental health needs of hcps Establish mental health services to address the mental health needs of health workers Both groups of respondents stressed that there needed to be an increase in the budget allocated to the health sector to establish mental health services for health service providers working during the COVID-19 pandemic. More funds and resources would lead to the betterment of HCWs. Hiring mental health professionals including psychologists and psychiatrists to support health service providers psychologically was strongly recommended and emphasised repeatedly by health managers. With regard to the roles of provincial governments in improving the mental health of HCPs, there was a need for the implementation of formal programmes addressing mental health. The government was also advised to arrange seminars on mental health for health service providers alongside launching a mobile application for providing relevant information on the topic. There should be seminars and sessions and the Punjab government should launch an app regarding this so that we get information from it regarding mental and physical health and keep on updating it. This will improve the situation. (Health Service Provider, Punjab) Hospital managers were also of the view that government officials should visit hospitals and have discussions with all levels of health service providers to understand their experiences and needs. This would enable the government to formulate effective strategies in dealing with problems faced by health service providers. Some health service providers complained that their health managers were not bothered if their staff were facing any issues. Nobody bothers to discuss any issue faced by workers. They only give orders to perform their duties. Our hospital managers, they don't have any concerns Sorting logistical issues such as providing transportation facilities for health service providers to and from quarantine centres was also stressed upon. ## Conduct formal training of health managers in managing mental health needs Since hospital managers were directly overseeing the health service providers and had the most interaction with them, their formal training in providing basic counselling to the staff was deemed necessary. Health service providers wanted managers to hold daily meetings with them where they could discuss the difficulties they were facing while caring for COVID-19-positive patients. The administrator of the hospital should talk about it from every doctor. He should do meeting with them to see if they face any mental pressure or not and if he has so the problem then he should sort out his problem. (Health Service Provider, Sindh) Some health service providers also remarked that hospital managers should be more approachable so that the staff feel comfortable in sharing their concerns with them. If employees are guided properly in monthly or weekly meetings, their issues are being heard whatever they are. There should be less difference of job between an employee and MS or head of the department so that he can go and discuss his problems with him, so many of problems can be solved. (Health Service Provider, Punjab) Assess and address mental health needs of the health workforce Another recommendation for the future was for the government and health managers to plan all the measures the health system would need to take in the event of such a crisis. They should have observed the situation in countries such as China where the outbreak first began and started thinking of strategies to get a better grip of the impending problems. We don't know if this pandemic will subside or not. God Forbid if this second waves comes and traffic of patients increases, then what will we do? This way we will get depressed, so proper training should definitely be given, mental health workers should come, go to the institution and guide the HCWs. (Health Manager, Sindh) Health service providers also suggested that keeping in mind the difficult conditions they were eventually going to have to work in, there should have been sessions that would mentally prepare them for these difficult conditions. When you send a doctor in the COVID-19 ward, there should have been a week-long training with a psychologist, to explain them the situation, what will be their duties, the government has selected you, for this task, they should have been prepared and there is nothing to worry about, the mortality rate is low, and most people recover. (Health Service Provider, Sindh) # Discussion The large-scale unprecedented public health catastrophe, in the form of COVID-19, placed the frontline HCPs, who are directly involved with all aspects of care of patients with COVID-19, at risk of developing psychological distress and other mental health symptoms. [bib_ref] The psychological impact of the SARS epidemic on hospital employees in China:..., Wu [/bib_ref] Our study provided an in-depth investigation into the mental health impact of COVID-19 on HCPs and mental health needs of different cadres (health service providers and administrators) in public health sector settings. We documented an adverse perceived mental healht impact experienced by health service providers while working during the pandemic as revealed in other studies from other countries. The health service providers expressed several explanations for psychological stress including the fear of acquiring the infection and transmitting it to their family members, fear of social isolation and stigma, anxiety related to the uncertainty of COVID-19, nervousness due to media exaggeration and stress associated with excessive workload. Evidence shows that exaggeration by the media tends to negatively affect the mental health of HCPs. Similarly, lack of knowledge about COVID-19 infection is likely to increase anxiety/ depression among physicians [bib_ref] COVID-19 pandemic-knowledge, perception, anxiety and depression among frontline doctors of Pakistan, Amin [/bib_ref] and inadequate precautionary measures in the workplace cause fear and anxiety among HCPs. [bib_ref] Frontline healthcare workers' experiences with personal protective equipment during the COVID-19 pandemic..., Hoernke [/bib_ref] Furthermore, overburdening due to high intensity of care and increased volume of clinical services has been associated with work-related stress, burnout and post-traumatic stress disorder, which in turn negatively affect job performance and quality of services. Perceptions of the two groups of respondents (service providers and health managers) on most issues were largely similar. However, at some instances, we noted divergent views among the two groups regarding the impact of COVID-19 on the mental health of health service providers. It is worrisome to note that while health service providers claimed to experience significant adverse psychological effects due to COVID-19, some health managers denied that their staff faced any stress or difficulty working during the pandemic. These contradictory views may be attributed to a disconnect or lack of communication between service providers and health managers. Another reason for this denial from health managers could be an attempt to express that things are managed well by them since they are generally responsible for the safety and well-being of service providers. Nonetheless, it is important to investigate the causes of such contrasting views about the same phenomenon by Open access different groups, especially when it affects the health system at large. The study revealed the mental health needs of health service providers including the need to provide counselling services and safe working conditions, the need for paid rest periods and timely payment of workers' salaries, and the need for appreciation and motivation to work in the pandemic. This finding is consistent with other studies where HCWs expressed similar needs. For example, previous experience from the Ebola outbreak in western Africa highlighted the need to provide risk allowance for motivating and retaining HCWs. [bib_ref] Burnout among healthcare workers during COVID-19 pandemic in India: results of a..., Khasne [/bib_ref] Our study also highlighted the need to provide risk allowance to health service providers. In addition, our study emphasised the need to provide timely salaries to health service providers and extra rest periods. Both groups of respondents revealed that the health system response in the given scenario has been weak given the lack of existing support mechanisms to address the mental health needs of the health workforce during the COVID-19 pandemic. Inefficient management of the pandemic from the healthcare system was also reported in other countries. [bib_ref] Caregivers' perception of the caring challenges in coronavirus crisis (COVID-19): a qualitative..., Mohammadi [/bib_ref] Finally, the health service providers and health managers provided three suggestions to address the mental health needs of health service providers in the future including the provision of mental health services to address the mental needs of health service providers, and conducting formal training of health managers in managing and anticipating the mental health needs of the health workforce to guide future initiatives. Experiences from similar outbreaks suggest that an early mental health intervention and the establishment of early support systems are particularly important to promote the emotional release and improve health service providers' well-being. [bib_ref] A qualitative study on the psychological experience of caregivers of COVID-19 patients, Sun [/bib_ref] Similar interventions have been used in the past to address mental health issues in HCWs during an infection disease outbreak. [bib_ref] Interventions to address mental health issues in healthcare workers during infectious disease..., Zaçe [/bib_ref] Capacity building of health facility staff on the provision of psychosocial support is also recommended in the WHO (2020) interim guidance document on COVID-19.Call centres have been established in many countries-including Pakistan 52 -to provide needs-based mental health support to HCPs. In Pakistan, the 'WeCare' Programme probably needs to expand its scale of services to effectively address the psychosocial and mental health needs of HCPs. Our study will use knowledge translation tools to share the key findings of this qualitative study with a diverse audience including policymakers, knowledge users, clinicians, researchers, health service providers and hospital managers. Concerning future research direction, insights from the qualitative study would help in streamlining health system response in addressing the mental needs of the health workforce working during the COVID-19 pandemic. # Strengths and limitations The study has several strengths and limitations. First, the study used multiple cadres of the health system (health service providers and health managers), which allowed researchers to look for converging and diverging lines of inquiry to identify common themes/concepts and incongruence between data sources. Second, our study sample provides adequate representation from rural settings in the country. Third, the study was conducted in close collaboration with the provincial governments. However, this study is also subject to limitations. First, the study was conducted with a focus on public sector hospitals in only two provinces of Pakistan (Sindh and Punjab), which would limit the transferability of the findings to other provinces. [bib_ref] Generalizability and the Single-Case Study, Donmoyer [/bib_ref] Guba and Lincoln argue for the concept of 'fittingness', which emphasises analysing the degree to which a situation studied matches other situations in which one is interested, and provides a more workable way of thinking about transferability. [bib_ref] Generalizability and the Single-Case Study, Donmoyer [/bib_ref] Thus, this study may provide insights for similar public hospitals across Pakistan that are interested in understanding the mental health impact and mental health needs of health service providers during the COVID-19 pandemic. Second, the study was unable to carry out member checking with study participants, which may have affected the validity of study findings. Lastly, the authors did not have the opportunity to build rapport and obtain non-verbal cues during interviews since the participants were interviewed telephonically due to the COVID-19 situation. In addition, the interviewees might have been distracted during interviews by the activities in their environment and might have experienced fatigue due to long telephonic interviews. # Conclusion Our findings show that the health system of Pakistan was not prepared to deal with the mental health needs of health service professionals during the COVID-19 pandemic. This information is crucial in anticipating and preparing for tackling possible future pandemics. Health managers and health service providers shared similar concerns about working during the outbreak. Multiple recommendations are made to the government stakeholders for initiating nationwide initiatives for catering to the mental health needs of HCPs. At the system, (a) communication strategies should be devised to ensure better communication between health managers and health service providers at all hierarchies so that the needs and concerns are timely heard and taken care of; and (b) there is a need to establish a specialised unit that should provide mental health services to mitigate the detrimental effects of COVID-19 on the mental health of HCPs. At the staff level, HCPs should be given recognition to boost their morale either through monetary incentives or through formal appreciation based on their performance. Open access time and made themselves available for the interviews. Last but not the least, we thank our data collectors and project management team: Zafar Dehraj, Imran Sheikh, Sajid Brohi and Ghani Muhammad who managed the implementation of this study in a proficient manner during the pandemic. Contributors WH and BA conceptualised the study with inputs from BK, ZF and SS. WH, BA, BK, ZF and ASF developed the interview guide. HJ and MAW facilitated the study conduct. ASF and NA performed data analysis, supported by WH. WH and ASF took the lead in writing the manuscript. All authors critically reviewed the manuscript and provided feedback in shaping the write-up, analysis and presentation of results. WH addressed comments from coauthors and finalised the manuscript. WH is the overall content guarantor. Funding This work was co-funded by the Aga Khan University, Pakistan (ID: 20051) and the WHO (ID: 202568710-1). Disclaimer The views expressed in this manuscript are those of the authors and contributors, and do not necessarily reflect those of the WHO or the organisation to which the authors are affiliated. Competing interests None declared. Patient and public involvement Patients and/or the public were involved in the design, or conduct, or reporting, or dissemination plans of this research. Refer to the Methods section for further details. ## Patient consent for publication obtained. Ethics approval Ethical approval for this study was obtained from the Aga Khan University Ethical Review Committee (AKU-ERC; 2020-5186-11602) and National Bioethics Committee of Pakistan (4-87/COVID-45/NBC/20/393). Provenance and peer review Not commissioned; externally peer reviewed. ## Data availability statement no data are available. Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise. Open access This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://creativecommons.org/ licenses/by/4.0/. [table] Table 1: Characteristics of study participants [/table] [table] Table 2: Themes and subthemes : mental health needs of the health workforce involved in the COVID-19 crisis and available support from the health system 2.1 Need to provide counselling services and safe working conditions 2.2 Need for paid rest periods, timely payment of workers' salaries and incentives 2.3 Need of appreciation and motivation to work in the pandemic Theme 3: suggestions to address the mental health needs of healthcare professionals 3.1 Establish mental health services to address the mental needs of health workers 3.2 Conduct formal training of health managers in managing mental health needs 3.3 Assess and address mental needs of the health workforce [/table]
Ezetimibe/Simvastatin 10/20 mg versus Rosuvastatin 10 mg in high-risk hypercholesterolemic patients stratified by prior statin treatment potency Objective: This post-hoc analysis compared the lipid-altering efficacy of Ezetimibe/Simvastatin 10/20 mg (EZ/Simva) versus Rosuvastatin 10 mg (Rosuva) in patients stratified by statin potency/dose prior to randomization.Methods: Patients with elevated low-density lipoprotein cholesterol (LDL-C) despite prior statin treatment (n = 618) were randomized 1:1 to EZ/Simva 10/20 mg or Rosuva 10 mg for 6 weeks. Percent change from baseline in lipids and attainment of lipid targets were assessed within each subgroup (low potency n = 369, high potency n = 249). Consistency of the treatment effect across subgroups was evaluated by testing for treatment-by-subgroup interaction. No multiplicity adjustments were made.Results: Significant treatment-by-subgroup interaction occurred for LDL-C (p = 0.013), total cholesterol (p = 0.025), non-HDL-C (p = 0.032), and apolipoprotein B (p = 0.016) with greater between-treatment differences in favor of EZ/Simva observed in patients from the high potency stratum vs low potency stratum. Individual and triple target attainment was higher for Eze/Simva compared with Rosuva in both strata.Conclusions: Compared with Rosuva, switching to EZ/Simva provided greater reductions in LDL-C, total cholesterol, non-HDL-C and apolipoprotein B and higher target attainment in patients on prior statin treatment, regardless of potency, although patients treated with higher potency statins prior to randomization experienced greater between treatment differences in favor of EZ/Simva. Trial Registration: Registered at ClinicalTrials.gov: NCT00479713. # Background Low-density lipoprotein cholesterol (LDL-C) is the primary target of therapy in patients with hypercholesterolemia [bib_ref] European guidelines on cardiovascular disease prevention in clinical practice: executive summary. Fourth..., Graham [/bib_ref]. If therapeutic lifestyle changes do not lower LDL-C to recommended levels, interventions such as HMG-CoA reductase inhibitors (statins) are indicated for further LDL-C reductions. Rosuvastatin has demonstrated more effective LDL-C reductions across its dose range than other statins across their dose ranges and greater lipid-lowering effects compared with the other marketed statins [bib_ref] Achieving LDL cholesterol, non-HDL cholesterol, and apolipoprotein B target levels in high-risk..., Ballantyne [/bib_ref] [bib_ref] Rosuvastatin in the management of hyperlipidemia, Cheng [/bib_ref]. However, despite the substantial lipid-lowering associated with the use of statins, LDL-C reductions beyond that achieved by currently used statin therapies-even those considered to be of highest potency-are recommended for a considerable number of patients [bib_ref] EUROASPIRE III: a survey on the lifestyle, risk factors and use of..., Kotseva [/bib_ref] [bib_ref] The lipid treatment assessment project (L-TAP): a multicenter survey to evaluate the..., Pearson [/bib_ref] [bib_ref] Incremental cholesterol reduction with ezetimibe/simvastatin, atorvastatin and rosuvastatin in UK General Practice..., Mccormack [/bib_ref]. Many patients at high cardiovascular risk and/or with severely elevated LDL-C do not achieve the rigorous treatment targets recommended by European, Canadian and US guidelines using established statin treatment regimens [bib_ref] European guidelines on cardiovascular disease prevention in clinical practice: executive summary. Fourth..., Graham [/bib_ref] [bib_ref] Perspectives on low-density lipoprotein cholesterol goal achievement, Catapano [/bib_ref] [bib_ref] Canadian Cardiovascular Society/Canadian guidelines for the diagnosis and treatment of dyslipidemia and..., Genest [/bib_ref]. Clinical trial results have demonstrated that combining ezetimibe with a statin more effectively lowers LDL-C than therapy with either of the individual components alone [bib_ref] Incremental cholesterol reduction with ezetimibe/simvastatin, atorvastatin and rosuvastatin in UK General Practice..., Mccormack [/bib_ref] [bib_ref] Ezetimibe: cholesterol lowering and beyond, Bays [/bib_ref]. In a study of ezetimibe combined with simvastatin vs rosuvastatin in treatment-naïve patients, at each dose comparison and across doses, ezetimibe/ simvastatin reduced LDL-C levels significantly more than rosuvastatin [bib_ref] Efficacy of ezetimibe coadministered with simvastatin versus atorvastatin in patients with hypercholesterolemia, Ballantyne [/bib_ref]. That trial utilized a placebo runin, which may not reflect usual clinical practice in which high cardiovascular risk patients are usually already being treated with a statin and require dose adjustments or treatment changes to achieve desired lipid levels. In the trial presented here patients were treated prior to randomization with statins of varying potency but had not achieved LDL-C <100 mg/dL [bib_ref] Lipid-altering efficacy of ezetimibe/simvastatin 10/20 mg compared with rosuvastatin 10 mg in..., Farnier [/bib_ref]. The results of the primary analysis of this trial demonstrated significantly greater reductions in LDL-C and other lipid levels and significantly greater achievement of prespecified LDL-C treatment targets with ezetimibe/simvastatin 10/20 mg (EZ/Simva) versus rosuvastatin 10 mg (Rosuva) in the overall population following 6 weeks of treatment [bib_ref] Lipid-altering efficacy of ezetimibe/simvastatin 10/20 mg compared with rosuvastatin 10 mg in..., Farnier [/bib_ref]. The objective of this post hoc analysis was to compare the lipid-altering efficacy of two treatment regimens recommended for clinical use. Analyses were done in patients grouped by statin potency/dose prior to randomization. # Methods The methods for this study were previously published [bib_ref] Lipid-altering efficacy of ezetimibe/simvastatin 10/20 mg compared with rosuvastatin 10 mg in..., Farnier [/bib_ref]. Briefly, this was a multicenter, randomized, double-blind, parallel-group study conducted at 85 sites in 10 countries in Europe between March 2007 and March 2008. The study protocol was approved by the appropriate ethics committees and institutional review boards at each site, and all patients provided written informed consent before any study procedures were administered. ## Patients Men and women 18 to 80 years of age with documented hypercholesterolemia [LDL-C ≥100 and ≤190 mg/dL at screening and ≥100 and ≤160 mg/dL at randomization] and high cardiovascular risk who were taking a stable daily dose of atorvastatin 10 or 20 mg; fluvastatin 80 mg; pravastatin 40 mg; rosuvastatin 5 mg; or simva 20 or 40 mg for at least 6 weeks prior to the study randomization were enrolled in the study. Patients were considered high cardiovascular risk if they had a history of CHD (i.e. stable and unstable angina, revascularization procedure, myocardial infarction, documented silent myocardial ischemia), or with established vascular atherosclerotic disease (i.e. peripheral vascular disease, ischemic stroke); type 2 diabetes without a history of vascular disease and with high cardiovascular risk (i.e. renal impairment [proteinuria >300 mg/24 h or creatinine clearance (standardized for body surface area) <1.002 ml/s] and/or at least two CHD risk factors per Framingham risk calculation); or CHD risk >20% over 10 years as determined by the Framingham risk calculation. All patients must have had fasting triglyceride levels <350 mg/dL, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels ≤1.5 × the upper limit of normal (ULN), and creatine kinase (CK) levels ≤ 3 × ULN at baseline. Patients were excluded if they had conditions or medications other than reported statin medications that could have affected lipid levels; active liver disease; congestive heart failure; poorly controlled diabetes [hemoglobin A1c (HbA1c) ≥8.5%]; uncontrolled hypertension (systolic >160 mmHg or diastolic >100 mmHg); or impaired renal function (creatinine ≥176.8 mmol/L). Patients taking prescription and/or over-the counterdrugs with the potential for significant lipid effects (other than study drug or above listed statins), or with potential drug interactions with the statins were also excluded from the study. ## Treatments At screening, patients were enrolled in the open-label run-in phase during which they continued to receive their current dose/brand of statin medication for 6 weeks. At randomization, eligible patients received either EZ/Simva 10/20 mg/day or rosuvastatin 10 mg/ day for 6 weeks. Patients were counseled regarding diet and exercise at each clinic visit. ## Efficacy measures In patients grouped by potency of statin at baseline (low potency stratum included simvastatin 20 mg, pravastatin 40 mg, fluvastatin 80 mg, atorvastatin 10 mg and high potency stratum included simvastatin 40 mg, atorvastatin 20 mg, rosuvastatin 5 mg), the percent change from baseline in LDL-C, total cholesterol, HDL-C, non-HDL-C, triglycerides, hs-CRP, LDL-C/HDL-C ratio, total cholesterol/HDL-C ratio and Apo B were assessed. In addition, the percentage of patients achieving LDL-C <100 mg/dL, <77 mg/dL, or <70 mg/dL; non-HDL-C <130 mg/dL or <100 mg/dL; Apo B <90 or <80 mg/dL; and achievement of the combination of LDL-C <100 mg/dL, non-HDL-C <130 mg/dL and Apo B <90 mg/dL was assessed. # Statistical methods The analyses were based on the Full-Analysis-Set (FAS) population which included all patients who received randomized treatment and had a baseline and at least one measurement performed after treatment start. The efficacy analyses for LDL-C, total cholesterol, HDL-C, non-HDL-C, LDL-C/HDL-C ratio, total cholesterol/ HDL-C ratio and Apo B were analyzed using a parametric ANOVA model. The consistency of the treatment effect across the two strata subgroups (low/high potency) was assessed through the significance of the treatment-by-subgroup interaction term in the ANOVA model with terms for treatment, baseline efficacy variable (categorized based on quartiles), study center, stratum subgroup and the treatment-by-stratum subgroup interaction. The least-square (LS) means and 95% confidence intervals (CIs) within each subgroup level were used to quantify the difference between treatment groups from the above ANOVA model (except for the last two terms involving subgroup). Consistency of the treatment effects on triglycerides and hs-CRP were assessed through the significance of the treatment-bysubgroup interaction term in the ANOVA model on ranks of these efficacy variables with terms for treatment, baseline triglycerides (or hs-CRP) categorized based on quartiles, study center, stratum potency status and the treatment-by-stratum subgroup interaction. The difference between treatment groups was quantified using the difference in medians and 95% CIs using Hodges-Lehmann estimates within each subgroup. The percentages of patients that achieved specified targets at endpoint were analyzed via a logistic regression model. The consistency of the treatment effect across the two strata subgroups was assessed through the significance of the treatment-by-subgroup interaction term in the logistic regression model with terms for treatment, baseline lipid variable as continuous variable, stratum status, and treatment-by-stratum subgroup interaction. Odds ratio estimates and 95% CIs were derived from the logistic regression model with similar terms except for the last two involving subgroups. Due to the exploratory nature of this analysis, no multiplicity adjustments nor power computation were employed. # Results ## Baseline The flow of patients through the study was previously published [bib_ref] Lipid-altering efficacy of ezetimibe/simvastatin 10/20 mg compared with rosuvastatin 10 mg in..., Farnier [/bib_ref]. In the EZ/Simva group (N = 314) there were 189 patients taking low-potency statins and 125 patients taking high-potency statins at baseline. In the rosuvastatin group (N = 304), there were 180 patients taking low-potency statins and 124 patients taking highpotency statins at baseline. Within each potency strata group, baseline demographic and clinical characteristics were generally well-balanced, although there were more patients with diabetes in the low-potency statin group compared with the high-potency statin group [fig_ref] Table 1: Baseline demographics and risk factors [/fig_ref] and [fig_ref] Table 2: Baseline clinical characteristics [/fig_ref]. ## Efficacy endpoints Both treatment regimens resulted in improvements in LDL-C, non-HDL-C, total cholesterol, triglyceride, Apo B, LDL-C/HDL-C ratio and total cholesterol/HDL-C ratio levels after 6 weeks of treatment, although the percent change from baseline was greater in patients treated with EZ/Simva 10/20 mg for all endpoints compared with patients treated with rosuvastatin 10 mg in both potency strata groups [fig_ref] Figure 1: Percent change in lipid and hs-CRP levels after 6 weeks of treatment... [/fig_ref] and . Significant treatment-by-stratum interactions were observed for LDL-C (p = 0.013), total cholesterol (p = 0.025), non-HDL-C (p = 0.032), and Apo B (p = 0.016), with greater between treatment differences in favor of EZ/Simva observed in patients from the high-potency stratum compared with the low-potency stratum [fig_ref] Figure 1: Percent change in lipid and hs-CRP levels after 6 weeks of treatment... [/fig_ref] and . Treatment effects were consistent across the two subgroups (low-potency/high-potency) with the overall population in HDL-C, LDL-C/HDL-C, total cholesterol/HDL-C ratio, hs-CRP and triglyceride levels. Higher percentages of patients achieved the specified treatment targets in the EZ/Simva group compared with patients treated with rosuvastatin in both potency groups [fig_ref] Figure 4: Percent of patients achieving lipid targets at 6 weeks [/fig_ref]. The magnitude of between-group differences was generally greater in the high-potency statin group compared with the lowpotency statin group. In the low-potency group, the predictive odds of attaining all individual specified lipid levels and the triple combination were greater in patients treated with EZ/Simva compared with rosuvastatin (see table under . In the high-potency group, the predictive odds of attaining all individual specified lipid levels and the triple combination were greater in patients treated with EZ/Simva compared with rosuvastatin (see table under [fig_ref] Figure 4: Percent of patients achieving lipid targets at 6 weeks [/fig_ref]. In the highpotency group, the predictive odds of attaining the lipid levels were higher compared with those in the lowpotency groups, although the treatment effects on attainment of specified levels for LDL-C, non-HDL-C, Apo B and the triple combination target were generally consistent across the two potency groups. # Discussion This post hoc analysis demonstrated that in patients with hypercholesterolemia at high risk for CHD who had not attained specified LDL-C treatment levels, switching to EZ/Simva 10/20 from high-or low-potency statins resulted in greater further reductions in most lipid levels compared with switching to rosuvastatin 10 mg for 6 weeks. In addition, switching to EZ/Simva 10/ 20 from high-or low-potency statins results in superior achievement of specified lipid targets vs rosuvastatin 10 mg treatment. The mean duration of hypercholesterolemia in patients enrolled in this study was 8 years, which suggests that patients were either candidates for or had received longterm treatment prior to study start but had not yet achieved suggested therapeutic targets. One would expect that with high-potency statin treatment, over time patients would achieve lower lipid levels than with low-potency Viigimaa et al. Lipids in Health and Disease 2010, 9:127 http://www.lipidworld.com/content/9/1/127 statin treatment. However, in this population of patients who had been treated with statins for a minimum of 6 weeks prior to study start, and quite possibly for much longer than 6 weeks, baseline lipid levels were similar between the two statin potency groups. Therefore, even with what may be considered more intensive statin monotherapy, a considerable number of patients did not achieve therapeutic targets on statin monotherapy. Of note the magnitude of response to the combination treatment appeared to be greater in terms of percent change from baseline in patients in the high-potency group vs patients in the low-potency group. Moreover, the odds of attaining most specified lipid levels (especially the LDL-C targets) in the high-potency group was double or triple the odds of attaining specified lipid levels in the low-potency group. These results suggest that some subjects in the high-potency group may be poor responders to statins. One reason for poor response to statin therapy and inadequate goal achievement may be inter-individual variability in LDL-C lowering, which has been reported with high-potency statins [bib_ref] Comparison of the effects of maximal dose atorvastatin and rosuvastatin therapy on..., Van Himbergen [/bib_ref]. Similar inter-individual variability has also been shown with ezetimibe [bib_ref] Effect of ezetimibe coadministered with statins in genotype-confirmed heterozygous FH patients, Pisciotta [/bib_ref] , but to date, even if no studies have been published demonstrating this same variability with the combination treatment, the complimentary mechanisms targeting the synthesis and absorption of cholesterol may explain the better efficacy in patients who are poor responders to statins. TC/HDL-C 3.9 (0.9) 3.9 (0.9) 3.9 (0.9) 3.9 (0.9 Although the mechanisms are poorly understood, research has begun to illuminate potential reasons why the response to statins and other drugs varies widely among individuals. Non-modifiable factors that may impact statin response include sex and age but these associations have not been fully elucidated. Variability associated with age may be attributed to poor compliance [bib_ref] Management of hyperlipidemia in older adults, Alexander [/bib_ref] or altered kidney function, which may be remedied through close monitoring and dose adjustment [bib_ref] Considerations in the treatment of dyslipidemia associated with chronic kidney failure and..., Davidson [/bib_ref] ; and hormone fluctuations may play a role in the variability observed in the elderly and between sexes [bib_ref] Endogenous steroids measured by high-specificity liquid chromatography-tandem mass spectrometry and prevalent cardiovascular..., Naessen [/bib_ref]. Some differences in statin response may be attributed to genetic variations in pharmacokinetic-and pharmacodynamic-related genes. Genetic variations in LDL receptor expression were shown to be associated with diminished statin response (i.e., diminished effects of statins on in vivo lipid reductions and on in vitro LDL receptor induction); and similarly, attenuated statin-mediated changes were observed in subjects with genetic mutations in HMG CoA reductase [bib_ref] Combined influence of LDLR and HMGCR sequence variation on lipid-lowering response to..., Mangravite [/bib_ref]. Without solid scientific evidence and pharmacogenetic tests to inform clinical decisions, individual patient response to statin monotherapy is likely the only way to assess the need to modify dosages or therapies to attain lipid goals. Although statins are typically considered the first line of therapy for lipid-lowering, this can prove to be a time-consuming and expensive endeavor for patients who respond suboptimally to statins. These patients are candidates for combination therapy with complimentary mechanisms of action, such as ezetimibe added to statin; and clinical trial results support an approach to lipid-lowering that targets the synthesis and the absorption of cholesterol in patients with suboptimal response to statin monotherapy [bib_ref] Lipid-altering efficacy of ezetimibe/simvastatin 10/20 mg compared with rosuvastatin 10 mg in..., Farnier [/bib_ref] [bib_ref] Tershakovec AM: Efficacy and safety of ezetimibe added on to atorvastatin (20..., Conard [/bib_ref] [bib_ref] Tershakovec AM: Efficacy and safety of ezetimibe added on to atorvastatin (40..., Leiter [/bib_ref]. Using a Markov model, the use of ezetimibe in combination with simvastatin was projected to be a cost-effective alternative to continuing or doubling the dose of a statin, [bib_ref] Projected Cost-Effectiveness of Ezetimibe/Simvastatin Compared with Doubling the Statin Dose in the..., Reckless [/bib_ref] and the results of the present analyses further support the use of EZ/Simva 10/20 mg vs rosuvastatin 10 mg, a high potency statin, for improving hypercholesterolemia in patients who had not achieved LDL-C targets on previous statin monotherapy, regardless of potency. Studies to demonstrate the efficacy of ezetimibe/statins vs statins on clinical outcomes are under way [bib_ref] An update on the IMProved reduction of outcomes: Vytorin Efficacy International Trial..., Califf [/bib_ref]. [fig] E: = ezetimibe; BMI = body mass index; CHD = coronary heart disease; n = number; R = rosuvastatin; S = simvastatin; SD = standard deviation; y = year *Low potency stratum included: simvastatin 20 mg, pravastatin 40 mg, fluvastatin 80 mg, atorvastatin 10 mg † High potency stratum included: simvastatin 40 mg, atorvastatin 20 mg, rosuvastatin 5 mg ‡ Missing data for 1 patient in the high potency Z/Simva group [/fig]
The Homeobox gene, HOXB13, Regulates a Mitotic Protein-Kinase Interaction Network in Metastatic Prostate Cancers Normal Prostate Prostate Tumor Cell line AR-ChIP seq Query DataSets for GSE56288 a. b Supplementary Figure 1. a. Flow chart: Identification of the HOXB13 direct targets in human prostate tumors. Integration of data sets to find overlaps of 536 genes with ~2677 DEGS following HOXB13 reduction in metastatic CRPC model. b. IGV analysis for AR and HOXB13 binding sites. HOXB13 or AR-ChIP sequencing in GSE56288 for binding in the proximity of the putative AR/HOXB13 target genes in normal prostate (1-7 lines, green peaks), human prostate tumors (8-20 lines, orange peaks) and FOXA1 ChIP-seq in tumor (orange peaks-line 21), HOXB13 ChIP-seq tumor (orange peaks-line 22), HOXB13 ChIP-seq in LNCaP (blue peaks-line 23), AR ChIP-seq in normal prostate cell line LHSAR (expressing HOXB13, FOXA1, HOXB13+FOXA1 (#1) HOXB13 +FOXA1 (#2)) ( blue peaks lines, 24-27). (a) VCaP cells were transfected control or HOXB13 siRNA and grown in charcoal-stripped androgendepleted media. Total RNA was isolated, followed by qRT-PCR with HOXB13, AR, AR-V7 and AR target genes (PSA, TMPRSS2 and NKX3.1) primers. (b) C4-2B cells were transfected with control or HOXB13 siRNAs (#1 is individual and #2 is pooled). Total RNA was isolated, followed by qRT-PCR with HOXB13, c-MYC, AR, and AR target genes (PSA and NKX3.1) primers. Data are represented as mean ± SEM and normalized to actin in (a-b). ****p<0.0001, ***p<0.0005, ns: not significant. + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ++ Overall survival (Months) ## Supplementary Survival probability + + + + + + + + + + + +++ + + + + ++ ++ + ++ + + + + + ++ + + ++ + + + + + + + + ++ ++ + + + ++ + + + +++ + + + + ++ ++ ++ + ++ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ++ + ++ + + ++ + ++ + +++ + + + + Overall survival (Months) + +++ + + ++ + ++ +++ + + ++ + +++ + + + +++ + + ++ + + + + ++ + ++ + + + + + + ++ ++ ++ ++ + + + + ++ + + + + + +++ + + + + + + + + ++ + + + + + + ++++ + + + + + + ++ + + ++ + ++ + + + + + + + + + + + + + ++ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ++ Overall survival (Months) [formula] + + + p = 0 [/formula] Hormone Therapy − Metastasis + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ++ + + + + + + + + + + + + + + + ++ + + + + + + + Margin Status − Primary Cancer + ++ + + + + + + + + + + + + + + + + + + + + ++ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Gleason score − Primary Cancer + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ++ + + + + + + + + + + ++ + + + + + (a-g) Kaplan-Meier overall survival curves based on categorized gene expression levels for HOTPAM9 genes in the Moffitt TCC data set. The following parameters were considered: age at diagnosis for primary and metastatic prostate cancer, hormone therapy for primary and metastatic prostate cancer, Gleason score, clinical tumor stage and margin status for primary disease. p values were generated by the Logrank test and univariate Cox proportional hazards regression models. b + + + + + + + ++ + + + + + + ++ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Age at Diagnosis − Metastasis Cancer c d + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ++ + ++ + + + + ++ + + ++ ++ + + + + + + + + + Hormone Therapy − Metastasis Cancer + + + + ++ + + + + + + + + + + + + + + + + + + + + + + + + ++ + ++ + + + + + + + + + + ++ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ++ + + ++++ + + + + + + Gleason score − Primary Cancer + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ++++ + ++ + + + ++ + + + + + ++ + + + + + ++ + + + + + + + + + + + + + + + + + + ++ + ++++++ + + + + Clinical Tumor Stage − Primary Cancer + ++ + + + + + + + + + + + + + + ++ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +++ +++ ++ + + + ++ + ++ + + + + (a-g) Kaplan-Meier RFS survival curves based on categorized gene expression levels for HOTPAM9 genes in the MSKCC data set. The following parameters were considered: age at diagnosis for primary and metastatic prostate cancer, hormone therapy for primary and metastatic prostate cancer, Gleason score, clinical tumor stage and margin status for primary disease. p values were generated by the log-rank test. Survival analysis: Moffitt TCC Data set − HOTPAM9 genes + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + p < 0.0001 Overall survival (Months) BUB1 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + p < 0.0001 Overall survival (Months) NEK2 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ++ p < 0.0001 Overall survival (Months) NUF2 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Overall survival (Months) Survival probability NCAPG + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Overall survival (Months) AURKA + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + HSPB8 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ++ + + + ++ + + + + + + ++ + + + ++ + + + + + + + + + + + + + + + + +++ + + + + + + + + + ++ + + + ++ + ++ + + + ++ + + ++ + + + + + + + + + + + + + + + + + + ++ + + + + + + + + + + + + + + + + + ++ + ++ + + + + p < 0.0001 Recurrence−free survival (Months) Probability of recurrence−free survival BUB1 + + + + + + + ++ + + + + + + + ++ ++ + + + + + + + + ++ + + + + + + + + + ++ + + + + + + + + + + ++ + + ++ + + + + + + + + + ++ + + +++ + ++ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ++ + + + ++ ++ ++ + + + + p < 0.0001 Recurrence−free survival (Months) Probability of recurrence−free survival NEK2 + + + + + + + + + + ++ + ++ + + + + ++ + + + + + + + ++ + + + + + + + + + + + + + + + + + + + + + + + + + +++ + + + + + + + ++ + +++ ++ + + + + + + + + + + ++ + + + + + + + + + + + + ++ + + + + + + + + + + + + + + + ++++++ + + + + + + + Probability of recurrence−free survival NUF2 + + + + + + + + + + + + + ++ + + + ++ ++ + + + + + + ++ + + ++ + + + + + + + + + + + + + + + + + + +++ + + + + + + ++ + + + ++ ++ + + ++ + + ++ + + + + + + + + + + + + + + + + + + ++ + + + + + + + + + + + + + + + ++ +++ + + + + + + + + + Recurrence−free survival (Months) Probability of recurrence−free survival NCAPG + + + + ++ + + + + + + + + + + + ++ + + + + + + + + ++ + + + + + + + + + + + + + + + + + + + + + + ++ + ++ + + + + + + + + + + ++ + + + + ++ + ++ +++ ++ + + + + + ++ + + + + + + + + + + + + + + + + + + + + + + + + ++ + ++ ++++ ++ + + Recurrence−free survival (Months) Probability of recurrence−free survival AURKA + + + + + + + + + + + + + + +++ + ++ + + + + + + + + + ++ + + + + + + + + + + + + + + + + +++ + ++ + + + + + + + + + + + + + + ++ ++ + + ++ + + + + + + + + + + + + + + + + + + + + + + + + + ++ + + + + + + + + + + + + + + + +++++ ++ + + + Recurrence−free survival (Months) Probability of recurrence−free survival MK1I677 MSKCC Survival Analysis + + + + + + + + + + + + + + + + + + + + + + + + ++ + + ++ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ++ + + +++ + + + + + + + + Recurrence−free survival (Months) Probability of recurrence−free survival HSPB8 + + + + + + + + + + + + + + + + + + + + + + + + + + ++ + + ++ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ++ + ++ + + + + + + + + + + + + + + + + +++++ + + + + + + + + + + + + + + + + + + + + + + ++ ++ ++ + + +
Cuprate-like Electronic Properties in Superlattices with AgIIF2 Square Sheet Using the generalized gradient approximation augmented with maximally localized Wannier functions analysis, we present the formation of cuprate-like electronic structures in Ag II F 2 -related superlattices resulted from the confinement together with structural chemical modification. The out-of-plane electronic reconstruction leads to electron doping of AgF 2 plane and gradually destablizes the antiferromagnetic state. Eventually a stable nonmagnetic metallic state emerges by applying in-plane tensile strain, in which the shape of effective Fermi surface of AgF 2 plane exhibits the key feature of high-temperature cuprate superconductor.T he discovery of high-temperature superconductivity in cuprates 1 initiated the quest for exploring superconductivity in other transition-metal compounds. Ag 21 is isoelectronic with Cu 21 (d 9 configuration). F 2 and O 22 are also isoelectronic ions, closed-shell species, moreover both F 2 and O 22 are weak-field ligands. Given the existence of the superconducting cuprates, one might be naturally interested in searching the superconductivity in Ag 21 -F 2 solids. Theoretical study 2-4 of Grochala and Hoffmann has also suggested that properly hole-or electron-doped Ag II fluorides might be good superconductors, due to similarity in structure and properties between the Ag II fluorides and the cuprate superconductors. Particularly, Jaron and Grochala have predicted that the high pressure s form of AgF 2 compound (.15 GPa) is a layered material with antiferromagnetic (AFM) ordering 4 , as seen for the parent compounds of high-temperature cuprate superconductors 5 .It is well known that [CuO 2 ]' plane with tetragonal tetra coordination of Cu (weak apical Cu-O bonds), is an essential structural element for superconductivity in cuprates. The basic band structure of the doped cuprates is a single 2D Cu À d x 2 {y 2 {like band deviating from half filled. In this situation, AFM fluctuations prevail in the undoped parent compounds and are often believed to mediate the superconductivity. The Fermi surface (FS) from this d x 2 2y 2 band has been observed in many overdoped cuprates and agrees with the density functional theory based band structure calculations 5,6 .As Grochala and Hoffmann pointed out in their review paper 2 , analogous [AgF 2 ]' plane with tetracoordinate Ag has not been reported experimentally. Noteworthy recent development of heterostructure interface technology, superlattices (SLs) containing Ag II F 2 square lattices can be prepared by using appropriate synthetic techniques to incorporate alternating layers of different transition metal compounds 7-14 , even technically a single atomic layer 11 . Here, interface can be used to modulate electronic structure for manipulating physical properties and generating novel phases which are not present in the bulk constituents. Whereby the quest for Ag II superconductors could be achieved by the aforementioned novel paradigm on designing and fabricating two dimensional materials 15-18 . In our paper, our research focuses on artificial superlattice materials design and their electronic properties and doping effects, different from research on real bulk Ag II fluorides materials 2-4 .To identify possible superconductivity in 2D Ag 21 -F 2 square sheet, we investigate electronic structures, magnetic states, model hamiltonian parameters and effective FSs for three proposed superlattices: SrTiO 3 / AgF 2 (STO/AgF 2 ), BaTiO 3 /AgF 2 (BTO/AgF 2 ) and SrTiO 3 /CsAgF 3 (STO/CsAgF 3 ), as illustrated in the top panels ofFig. 1, and compare these with corresponding properties of the cuprate superconductors. We find cuprate-like band structures and strong AFM fluctuations in all these SLs. More importantly, large cation size increases cation-anion polarization strength and corresponding apical Ag-O/F distance, and makes oxygen band edge shift above the Fermi level to exchange charge with Ag À d x 2 {y 2 band, leading to out-of-plane electronic reconstruction. Here, similar to charge transfer in cuprates and recently studied La 2 CuO 4 -related heterostructure 18 , the TiO 2 plane actually serves as a charge reservoir block and transfer electrons to the AgF 2 plane. As a result, AFM state presents an unstable trend. Finally, the applied in-plane tensile strain drives a novel phase transition from the AFM metallic state to a stable nonmagnetic (NM) metallic state in STO/CsAgF 3 SL. Model hamiltonian OPEN SUBJECT AREAS: ELECTRONIC STRUCTURE ELECTRONIC PROPERTIES AND MATERIALS DENSITY FUNCTIONAL THEORY parameters and FS character of STO/CsAgF 3 are extracted and compared to La 2 CuO 4 (LCO) and HgBa 2 CuO 4 (HBCO), indicating that STO/CsAgF 3 is a promising candidate for Ag II superconductivity. # Results For the in-plane lattice constant a of SLs, we first took that of STO (3.905 Å ), often used as the substrate. The lattice constant c and atomic z coordinates were fully relaxed. The main effect of the relaxation, is to make the negatively charged O/F and positively charged cations displaced relative to each other in SrO, BaO and CsF atomic layers, and thereby polarize the cation and anion planes. Cation size affects significantly polarization strength and corresponding apical Ag-O(F) bond length. AgF 2 layer acts as the mirror plane of whole unit cell. In STO/AgF 2 and BTO/AgF 2 SLs, oxygen atoms move symmetrically towards and against AgF 2 plane by 0.077 Å and 0.064 Å respectively. As a result, apical Ag-O bond length in the BTO/AgF 2 is slightly larger than that in the STO/AgF 2 by 0.143 Å , due to the larger size of the Ba 21 cation. The largest cation-anion polarization occurs in CsF plane in the STO/CsAgF 3 , and is 0.623 Å against AgF 2 plane. This polarization distortion produces a local ionic dipole moment, and together with in-plane strain it also leads to a larger increment in the apical Ag-F distance (d Local ionic dipole moment perturbs electrostatic potential and changes band positions around the Fermi Level. An evolution of Ag-e g states with structural chemical modification can be clearly observed in band structures. Spin-polarized GGA calculations give paramagnetic ground state for all these superlattices. shows energy bands of STO/AgF 2 , BTO/AgF 2 and STO/CsAgF 3 SLs in a 13 eV region around the Fermi level e F ; 0 and along the symmetry- [formula] lines C{X{M{C~0,0,0 ð Þ{ p a ,0,0 { p a , p a ,0 { 0,0,0 ð Þ. [/formula] The energy bands of bulk LCO and HBCO are also plotted in for comparison. For three SLs, electronic properties around e F are still mainly controlled by Ag-e g bands, which are above the filled O/F-2p and Ag-t 2g bands, and below the empty Ti-3d bands. We plot d x 2 {y 2 (dark cyan) and d 3z 2 {1 (orange) fatbands around e F to disclose their orbital contribution. Ag-e g antibonding bands have similar band width in superlattice configuration STO/AgF 2 and BTO/AgF 2 , and resemble that of LCO. Note that oxygen 2p bands become closer to Fermi Level in BTO/AgF 2 due to atomic polarization distortion in z direction. However, in STO/CsAgF 3 case, the antibonding band between Ag À d 3z 2 {1 and F-p states disappears due to the weak mixing of Ag-3d and F-p states in z direction. As a result, e g bands from 24 to 2 eV looks more like that of HBCO with a larger apical Cu-O distance of 2.784 Å . Most importantly, oxygen 2p band edge of TiO 2 plane upshifts eventually above the Fermi level and exchanges charge with AgÀd x 2 {y 2 band, as seen in layer-projected density of states in the left panel of [fig_ref] Figure 2 |: Layer-projected density of states [/fig_ref]. In order to investigate the microscopic orbital physics of polarization-induced electron doping, we plot the typical partial charge density of the unoccupied bands of STO/CsAgF 3 from Fermi level to 0.25 eV in the right panel of interface and AgÀd x 2 {y 2 orbital. Here, AgÀd x 2 {y 2 has considerable covalent hybridization with in-plane fluorine atoms' p x , p y orbitals, similar to Cu 21 -O 22 bonds. Interestingly, O-p x ,p y state around the Fermi level is mainly located at TiO 2 interface and far away from the AgF 2 plane, as illustrated in [fig_ref] Figure 2 |: Layer-projected density of states [/fig_ref] , which favors the realization of possible superconductivity in 2D Ag II F 2 plane. Next, we discuss the stability of magnetic states in three superlattices under GGA 1 U d scheme. AFM band structures with Ag-e g orbitals character (see of supplementary materials) indicate that STO/AgF 2 SL presents an AFM insulating ground state with a energy gap of 0.45 eV. While in BTO/AgF 2 electronic correlation drives a weak electron doping in AgÀd x 2 {y 2 state, which is absent in GGA bands, leading to an insulator-metal transition. For STO/ CsAgF 3 , an AFM metallic ground state is obtained. By analyzing layer-projected density of states of STO/CsAgF 3 from GGA 1 U d calculations and corresponding partial charge density isosurfaces for the bands around Fermi level (see [fig_ref] Figure 2 |: Layer-projected density of states [/fig_ref] [fig_ref] Table I |: The in-plane and apical bond length d AgðCuÞ{FðOÞ in Å , energy... [/fig_ref] , and finally to a much smaller value 4.105 meV/Ag in STO/CsAgF 3 . The process of AFM state instability is also companied by reduction of magnetic moment on Ag/Cu atoms. For superlattice structures, electron doping of AgF 2 plane emerges with the change of apical Ag-O/F distance, as we discussed above, which is an important derivation of AFM state instability, especially in STO/CsAgF 3 with much smaller E NM -E AFM value and magnetic moment of 0.184 m B /Ag. Furthermore, we investigate the effect of in-plane strain on magnetic state, because electronic properties are subject to electron-and orbital-lattice couplings in perovskite-like materials. Similar calculations are made for STO/CsAgF 3 with three additional in-plane lattice constants of 3.790, 4.005 and 4.105 Å . We find that compressive strain can effectively increase E NM -E AFM to 7.595 meV/Ag, but tensile strain decreases it to 0.335 meV/Ag for a 5 4.005 Å . Extraordinarily, STO/CsAgF 3 SL goes through phase transition to a stable NM metallic ground state under tensile strain a 5 4.105 Å , suggesting that the tuning of in-plane lattice constant can serve as an effective tool to modulate magnetic properties and even superconductivity. We know that effective low-energy hamiltonians containing the minimal set of bands are important tools for understanding chemical trends. Based on the aboved GGA simulations, we extract model hamiltonian parameters by MLWFs downfolding technique. In this work, we choose to downfold to a 6-band hamiltonian describing the in-plane d x 2 {y 2 , p x , p y orbitals, and out-of-plane d 3z 2 {1 , two p z orbitals (see. In particular, six parameters capture the essential physics: the e g crystal field splitting energy D 1~ex 2 {y 2 {e 3z 2 {1 , the in-plane charge-transfer energy D 2~ex 2 {y 2 {e p x y ð Þ , the direct in-plane and out-of-plane Ag-F (Cu-O) hopping t pd and t z pd , and the two shortest-ranged O-O hoppings t pp , and t pp . The extracted values are tabulated in [fig_ref] Table I |: The in-plane and apical bond length d AgðCuÞ{FðOÞ in Å , energy... [/fig_ref] , and corresponding interpolated band structure are shown inof supplementary materials. The hopping integrals t pd and t pp of LCO and HBCO are in good agreement with the 3-band model results by Weber et al. [bib_ref] Scaling of the transition temperature of hole-doped cuprate superconductors with the charge-transfer..., Weber [/bib_ref]. While D 2 and t pp' are further corrected in our model by including three additional out-of-plane orbitals. From [fig_ref] Table I |: The in-plane and apical bond length d AgðCuÞ{FðOÞ in Å , energy... [/fig_ref] :003, 2.053 Å (see bottom subtable), one can find that parameters evolve towards those in cuprates, except for t pp and t pp . In, we plot the localized Wannier functions of Ag À d x 2 {y 2 and {d 3z 2 {1 in STO/CsAgF 3 SL. Both look like those of cuprates, and have a strong p-d covalent hybridization characteristic. Ag À d 3z 2 {1 is more localized due to the big apical Ag-F distance (.3 Å ). The FSs centered at C point for LCO and HBCO are shown in the first row of. Compared to LCO (transition temperature T c 5 40 K), the FS of HBCO (T c 5 90 K) has the typical shape of high-T c cuprates superconductor with constant-energy surface obviously bulging toward C point. The FS shape of STO/AgF 2 and BTO/AgF 2 (see the second row ofis far away from that of HBCO or LCO. However, for STO/CsAgF 3 (the third row ofwith polarized electron-doping in AgF 2 plane, effective FSs from AgÀd x 2 {y 2 band presents the considerable similarity to that of HBCO. # Discussion We analyze the cuprate-like electronic structures and strong AFM fluctuations in the proposed superlattice with 2D Ag II F 2 square sheet. Atomic polarization induces out-of-plane electronic reconstruction occurring between O-p x ,p y orbitals in TiO 2 plane and covalent hybrid orbitals of AgÀd x 2 {y 2 and F-p x ,p y in AgF 2 plane, which is an important origin of AFM state instability. A stable NM metallic ground state emerges in STO/CsAgF 3 SL subjected to in-plane tensile strain, meanwhile corresponding Wannier functions of Ag À d x 2 {y 2 , Ag À d 3z 2 {1 , and FS shape present considerable similarity to those in cuprates with the high-T c . Therefore, d 9 Ag II F 2 -related superlattices are promising because their physics contains the main ingredients of high-temperature superconductivity. # Method We carried out the numerical calculations using the Vienna ab initio Simulation Package (VASP) [bib_ref] Ab initio molecular dynamics for liquid metals, Kresse [/bib_ref] [bib_ref] Ab initio molecular-dynamics simulation of the liquidmetal-amorphous-semiconductor transition in germanium, Kresse [/bib_ref] [bib_ref] Efficient iterative schemes for ab initio total-energy calculations using a plane-wave basis..., Kresse [/bib_ref] [bib_ref] Efficiency of ab-initio total energy calculations for metals and semiconductors using a..., Kresse [/bib_ref] within the framework of the generalized gradient approximation (GGA) (Perdew-Burke-Ernzerhof exchange correlation functional) [bib_ref] Generalized gradient approximation made simple, Perdew [/bib_ref] , and recently developed maximally localized Wannier functions (MLWFs) downfolding technique [bib_ref] Maximally localized generalized Wannier functions for composite energy bands, Marzari [/bib_ref] [bib_ref] Wannier90: A tool for obtaining maximally-localised wannier functions, Mostofi [/bib_ref]. The ion-electron interaction was modeled by the projector augmented wave (PAW) method 27,28 with a uniform energy cutoff of 500 eV. Spacing between k points was 0.02 Å [bib_ref] Efficient iterative schemes for ab initio total-energy calculations using a plane-wave basis..., Kresse [/bib_ref]. The structures of the SLs were optimized by employing the conjugate gradient technique, and in the final geometry, no force on the atoms exceeded 0.01 eV/Å . For magnetic states calculations, we used U d 5 7.5 eV and J d 5 0.98 eV for Ag-d and Cu-d states [bib_ref] Band theory and Mott insulators: Hubbard U instead of Stoner I, Anisimov [/bib_ref]. Table II | Tight-binding parameters of the six-band p-d model, containing the in-plane d x 2 {y 2 , p x , p y orbitals and out-of-plane d 3z 2 {1 , p z orbitals for LCO, HBCO, STO/AgF 2 , BTO/AgF 2 and STO/CsAgF 3 without (top subtable) and with (bottom subtable) in-plane strain. Parameters include e g crystal field splitting energies D 1~ex 2 {y 2 {e 3z 2 {1 , charge-transfer energies D 2~ex 2 {y 2 {e p xðyÞ , the three nearestneighbor (intra-cell) hoppings t pd , t z pd , t pp , and the inter-cell oxygen-oxygen hopping t pp . The in-plane and apical bond lengths d [fig] . 2, Figure 1 |: Obviously, electron doping originates from charge transfer between O-p x ,p y orbitals at Schematic geometrical structures and GGA bandstructures of bulk LCO, bulk HBCO, STO/AgF 2 , BTO/AgF 2 and STO/CsAgF 3 SLs from left to right. The Fermi level e F is set at zero. Dark cyan and orange fatbands represent contribution of d x 2 {y 2 and d 3z 2 {1 orbitals respectively. [/fig] [fig] Figure 2 |: Layer-projected density of states (left panel) of STO/CsAgF 3 SL, and corresponding partial charge density isosurfaces (right panel) for the unoccupied bands from Fermi level to 0.25 eV. [/fig] [fig] Figure 3 |: (a) Parameters of the six-band p-d model for the CuO and AgF(O) octahedral in cuprate superconductors and the proposed AgF 2 -related superlattices; (b) Localized Wannier functions of Ag{d x 2 {y 2 and {d 3z 2 {1 orbitals in STO/CsAgF 3 ; (c) Effective Fermi surface centered at C point in first Brillouin Zone from d x 2 {y 2 band for bulk LCO, bulk HBCO, STO/AgF 2 , BTO/AgF 2 and STO/CsAgF 3 SLs.www.nature.com/scientificreports SCIENTIFIC REPORTS | 4 : 5420 | DOI:10.1038/srep05420 [/fig] [table] Table I |: The in-plane and apical bond length d AgðCuÞ{FðOÞ in Å , energy differences E NM -E AFM in meV/Ag(Cu), and Ag/Cu atom's magnetic moment of AFM state in m B /Ag(Cu), for LCO, HBCO, STO/AgF 2 , BTO/AgF 2 and STO/CsAgF 3 without (top subtable) and with (bottom subtable) in-plane strain ð Þ , energy difference E NM -E AFM , and magnetic moment on Ag/Cu atom in AFM state. With the increasing apical Ag-O/F distance, E NM -E AFM value decreases gradually from 91.160 meV/Ag for STO/AgF 2 to 34.525 meV/Ag for BTO/AgF 2 , similar to the trend for cuprates (e.g. from 206.51 meV/Cu for LCO to 117.47 meV/Cu for HBCO in [/table] [bib_ref] Structure-property relation of SrTiO 3 /LaAlO 3 interfaces, Huijben [/bib_ref]
The Alpha Variant (B.1.1.7) of SARS-CoV-2 Failed to Become Dominant in Mexico [bib_ref] Assessing transmissibility of SARS-CoV-2 lineage B.1.1.7 in England, Volz [/bib_ref] [bib_ref] Deep mutational scanning of SARS-CoV-2 receptor binding domain reveals constraints on folding..., Starr [/bib_ref] [bib_ref] Detection of a SARS-CoV-2 variant of concern in South Africa, Tegally [/bib_ref] [bib_ref] Estimated transmissibility and impact of SARS-CoV-2 lineage B.1.1.7 in England, Davies [/bib_ref] [bib_ref] Spike mutation D614G alters SARS-CoV-2 fitness, Plante [/bib_ref] [bib_ref] Loss of furin cleavage site attenuates SARS-CoV-2 pathogenesis, Johnson [/bib_ref] [bib_ref] Estimated transmissibility and impact of SARS-CoV-2 lineage B.1.1.7 in England, Davies [/bib_ref] [bib_ref] Emergence and rapid transmission of SARS-CoV-2 B.1.1.7 in the United States, Washington [/bib_ref] [bib_ref] Danish COVID-19 Genome Consortium. 2021. Risk of hospitalisation associated with infection with..., Bager [/bib_ref] [bib_ref] Risk of mortality in patients infected with SARS-CoV-2 variant of concern 202012/1:..., Challen [/bib_ref] [bib_ref] Increased mortality in community-tested cases of SARS-CoV-2 lineage B.1.1.7, Davies [/bib_ref] [bib_ref] Genetic analysis of SARS-CoV-2 variants in Mexico during the first year of..., Taboada [/bib_ref] # Results Demographic and patient information of SARS-CoV-2. As of the first week of July 2021, the epidemic in Mexico had presented three main waves, and there had been 2,546,017 positive cases of SARS-CoV-2 in Mexico since the first case was detected on February 27, 2020 [fig_ref] FIG 1: Distribution of SARS-CoV-2 cases and B [/fig_ref]. The first peak was reached at the end of July 2020, having around ;50,000 new cases in epidemiological week 28 (W28; 40.01 cases per 100,000 inhabitants). The second peak was reached at the beginning of January of 2021, with ;109,000 new cases in the first epidemiological week (W1) of the year (86.95 cases per 100,000 inhabitants). The third wave peaked by mid-August 2021, with ;126,000 new cases in W32 (100.47 cases per 100,000 inhabitants). To determine the genetic diversity and the epidemiological characteristics of the B.1.1.7 lineage in Mexico, 1,620 sequences were considered in the analysis (Tables S1 and S2 in the supplemental material). As shown in [fig_ref] FIG 1: Distribution of SARS-CoV-2 cases and B [/fig_ref] , the B.1.1.7 lineage peaked from April to June 2021; therefore, the demographic characteristics of the patients were studied in this period [fig_ref] TABLE 1: Demographic and status information for Mexican patients during April to June 2021... [/fig_ref]. For comparison, the same period was analyzed for the B.1.1.519 lineage, which was the most prevalent cocirculating lineage in Mexico during this time [fig_ref] FIG 1: Distribution of SARS-CoV-2 cases and B [/fig_ref]. Briefly, the average age of the patients with the B.1.1.7 variant was 42 years old (range of 0 to 93), while it was 47 years old in those infected with B.1.1.519, which was significantly different (Wilcoxon test, P = 2.2 Â 10 212 ). Since the vaccination campaign for the population older than 60 years of age started in February 2021 and continued with younger groups in subsequent months, we observed an overall decrease in the median age of patients after May, in agreement with the age ranges of vaccination [fig_ref] FIG 1: Distribution of SARS-CoV-2 cases and B [/fig_ref]. The nation-wide longitudinal data on confirmed cases show that major drops in the prevalence of the corresponding age group occurred after the vaccination campaign started for each group. In contrast, the 0 to 17 age group, which remained unvaccinated during this time frame, saw a continuous and linear increase in prevalence [fig_ref] FIG 1: Distribution of SARS-CoV-2 cases and B [/fig_ref]. Although the difference between the median ages of B.1.1.7-and B.1.1.519-infected patients may be in part because of the variant itself, it is difficult to rule out factors such as vaccination, given that the B.1.1.519 prevalence started to decline in May when the older population completed their vaccination scheme, time during which the cases of B.1.1.7 started rising. The proportion of females and males showed no statistical differences by lineage, and comparing the proportion of ambulatory, hospitalized, and deceased patients between lineages B.1.1.519 and B.1.1.7 resulted in a significant difference (chi-square test, P = 0.017). An increase in hospitalized patients was observed for B.1.1.7 during W20 to W24 (16 May to 19 June), corresponding to the prevalence peak of this lineage. However, patient status data correspond to the collection date and do not necessarily reflect the final clinical outcome. Geographical and temporal distribution of B.1.1.7. B.1.1.7 was first identified in Mexico in a single sample collected on 31 December 2020, followed by a low frequency of 1% in January and February 2021 and a slight increase in March (2.5%) and April (8.9%) [fig_ref] FIG 1: Distribution of SARS-CoV-2 cases and B [/fig_ref]. The highest prevalence of B.1.1.7 was observed in May, reaching 18.8% at the national level but decreasing again in June (15.30%). This trend contrasts with the United Kingdom, the United States, and most of the European countries (Denmark, Finland, Italy, Spain, and Portugal among others), where, in general, after 3 to 5 months of community transmission, B.1.1.7 became the dominant variant, reaching more than 50% in the United States and 97.7% in the United Kingdom [fig_ref] FIG 1: Distribution of SARS-CoV-2 cases and B [/fig_ref]. Interestingly, as shown in [fig_ref] FIG 1: Distribution of SARS-CoV-2 cases and B [/fig_ref] , the low prevalence of B.1.1.7 in Mexico was also observed in South American countries, such as Brazil, Chile, and Peru. The median number of B.1.1.7 sequences per state was 199.5 (interquartile range [IQR] = 66 to 342.5), being identified in 31 of the 32 Mexican states [fig_ref] FIG 1: Distribution of SARS-CoV-2 cases and B [/fig_ref]. [fig_ref] FIG 2: Temporal distribution of the B [/fig_ref] shows B.1.1.7 variant dispersion and prevalence through time in the seven regions [fig_ref] FIG 1: Distribution of SARS-CoV-2 cases and B [/fig_ref] of Mexico defined in this study. Most of the low-prevalence states were in the Central South (CS), Central North (CN), and West (W) regions, while the Northwest (NW; [fig_ref] FIG 2: Temporal distribution of the B [/fig_ref] and the Northeast (NE; [fig_ref] FIG 2: Temporal distribution of the B [/fig_ref] regions, especially the states located in the Mexico-U.S. border (Baja California, Chihuahua, Coahuila, and Tamaulipas), had the [fig_ref] TABLE 2: Frequency of additional mutations for the B [/fig_ref]. This large number of genetic changes contrasts with the observation that the SARS-CoV-2 virus accumulates around two nonsynonymous substitutions per month. The lineage-defining amino acid changes and deletions were present in 98.6% (n = 1,224) of the Mexican viral genomes [fig_ref] TABLE 2: Frequency of additional mutations for the B [/fig_ref]. In addition to the lineage-defining changes, 20 nonsynonymous mutations were identified in at least 5% of the Mexican virus genomes [fig_ref] TABLE 2: Frequency of additional mutations for the B [/fig_ref] , with some of them being more prevalent in Mexican isolates than globally. For example, ORF1a:M2259I and ORF1b:P218L changes were found in more than 30% of Mexican sequences, while their worldwide frequencies were 5.3% and 13.7%, respectively. Also, in the spike protein, S:S98F, S:E1258D, and S:D138H mutations were identified in more than 15% of genomes, while globally, they were detected in less than 2%. Finally, 68.7% of the remaining amino acid substitutions were observed within one or two B. [fig_ref] FIG 4: Time-scaled maximum likelihood phylogenetic tree of the B [/fig_ref]. In this figure, multiple international and U.S. sequences can be observed in abundance by October 2020, while Mexican sequences appear later. Five large monophyletic groups are highlighted [fig_ref] FIG 4: Time-scaled maximum likelihood phylogenetic tree of the B [/fig_ref] , clades A to E), which are polytomies with an internal branch structure. Clades A, B, and C contain mostly Mexican sequences, while clade D is composed mainly of international viruses. In contrast, clade E, the largest one, has a subclade (E.1) with primarily Mexican viruses. The distribution of Mexican sequences throughout the phylogeny suggests that multiple introduction events occurred since they did not form a monophyletic group. Interestingly, many Mexican sequences were singletons (only one observed sequence) or formed small clades, suggesting that many introductions did not lead to community transmission. However, in some cases, these introductions resulted in large local community transmission chains, as observed in the internal subclades (C1 to C5) of clades A, B, C, and E. A haplotype network was constructed to discern the relationship between sequences at the tips of the tree and to document multiple introductions, local transmission, and spread patterns of B.1.1.7 lineage in Mexico [fig_ref] FIG 5: Haplotype network using genome-wide single-nucleotide variations of the B [/fig_ref]. The haplotype network shows that some clusters are unique for Mexican virus genomes, in agreement with the phylogeny [fig_ref] FIG 4: Time-scaled maximum likelihood phylogenetic tree of the B [/fig_ref] , with several of them containing sequences from a single or few Mexican states, sometimes directly deriving from other Mexican haplotype clusters and possibly representing local transmission chains. Moreover, the high-frequency substitutions identified in the B.1.1.7 Mexican sequences were associated with only one or a few clusters [fig_ref] TABLE 2: Frequency of additional mutations for the B [/fig_ref] , suggesting the existence of separate local transmission chains that circulated for several months. In the center of the network, a large cluster of Mexican and international sequences is located (central nodes in [fig_ref] FIG 5: Haplotype network using genome-wide single-nucleotide variations of the B [/fig_ref]. However, since the Mexican sequences did not form a single subgroup (the majority are singletons), these likely correspond to separate introduction events from the United States (black) or the rest of the world (gray), which did not result in sustained community transmission. Nevertheless, insufficient sampling cannot be ruled out. Three large clusters of Mexican sequences (multicolored vertices indicate different states) can be observed in the network and are marked as C1, C4, and C5, representing the largest transmission chains in Mexico. In C1, sequences mainly from Tamaulipas (yellow) can be identified. A subgroup of Tabasco sequences (blue) branches out from the initial group, including a smaller subgroup of sequences from Yucatán (brown). These data suggest a flow of viruses in C1 from the Northeast (Tamaulipas) toward the Southeast (Tabasco and Yucatán). In agreement with this observation, the spatiotemporal distribution of C1 sequences [fig_ref] FIG 3: Genomic changes of Mexican B [/fig_ref] shows that the earliest identification and higher prevalence was in the northern state of Tamaulipas, followed by dispersion to other states, especially in the south. Even though many sequences from Tamaulipas and Yucatán were present in C1, other haplotypes were in circulation in those states during the same period, for instance, the sequences in C3 from Tamaulipas (yellow) or in C2 from Yucatán (brown). C5 contains samples from Chihuahua (dark pink) and Sinaloa (light pink) states in the northwest of the country. A subgroup within C5 shows the presence of sequences from central Mexico (red and orange colors), including Mexico City and Hidalgo. This cluster also suggests a transfer of viruses from the northwest (Chihuahua and Sinaloa, among others) toward the country's center. [fig_ref] FIG 3: Genomic changes of Mexican B [/fig_ref] corroborates the introduction of C5 in Chihuahua at the Mexican-U.S. border and then its spread to central and western Mexico in parallel to the dispersion observed for C1. In contrast, C2 sequences show a possible introduction of B.1.1.7 in the southeastern state of Quintana Roo, in which the tourism destination of Cancun is located [fig_ref] FIG 3: Genomic changes of Mexican B [/fig_ref]. A limited dispersion to neighboring states can then be observed, although it remained at frequencies lower than 5%. However, C4 shows more diversity in geographical provenance location but with a common origin by a single introduction of an international haplotype (not from the United States). Small subclusters in the periphery of the central node indicate statespecific transmission, but, overall, the circulation pattern seems to be nationwide. On the other hand, dashed circles indicate other small subgroups, many originating in a single state and potentially representing other local minor transmission chains. The list of sequences comprised in clusters C1 to C5 is reported in . Finally, as mentioned before, the observation that the Mexico-U.S. border states showed the highest prevalence of the B.1.1.7 variant and the largest transmission chains (clusters C1 and C5) suggests dispersions of the virus from those states into the country's interior. To further explore this possibility, an additional phylogenetic analysis [fig_ref] FIG 4: Time-scaled maximum likelihood phylogenetic tree of the B [/fig_ref] was done to search for possible introduction events from the United States. The phylogeny shows that many Mexican sequences interspersed within sequences from the United States, forming many Mexican singletons, suggesting numerous introductions without local transmission, at least in northern Mexico. However, some Mexican clades, particularly C1 and C5, were grouped with a sister clade formed by U.S. sequence(s), supporting the idea that the largest national transmission chains entered from the United States. # Discussion In Mexico, the prevalence of B.1.1.7 remained at low frequency during the first trimester of 2021, ranging between 1% and 3% and rising to [bib_ref] Estimated transmissibility and impact of SARS-CoV-2 lineage B.1.1.7 in England, Davies [/bib_ref]. This period of rapid evolution led to the acquisition of 14 lineage-specific amino acid substitutions and three deletions in around 14 weeks, being more divergent from the root and its parental lineage than expected according to the estimated mean mutation rate of approximately 24 substitutions per year. Evolutionary jumps have been observed in other VOIs/VOCs, and, interestingly, B.1.1.519 also shows this pattern of discontinuous evolution, having obtained seven key mutations in a short period Although limited phenotypic information of lineage B.1.1.519 is available, it shares S:T478K with the Delta variant, which has been more thoroughly characterized. The T478K substitution confers to the S protein a less than 2-fold increase in affinity to ACE2, significantly lower than the 7-fold affinity increase reported for the S:N501Y change [bib_ref] Reduced neutralization of SARS-CoV-2 B.1.617 by vaccine and convalescent serum, Liu [/bib_ref]. T478K also resulted in a 1.5fold increase in cell entry, as measured in a pseudovirus assay. The presence of these substitutions might confer B.1.1.519 some advantage over the lineages circulating in Mexico at the end of 2020 but not necessarily against B.1.1.7, which was able to spread worldwide. Also, this competition took place when overall virus transmission was low, between the second and third waves, which may have given transmission superiority to the B.1.1.519 dominant virus despite the high transmissibility of B.1.1.7; also, as mentioned before, the introduction of other VOCs could have contributed to the B.1.1.7 low prevalence. Distinct Mexican geographical clusters of the B.1.1.7 variant were identified using phylogenetic and haplotype network approaches. The presence of clusters suggests different local transmission chains, alongside multiple repeated introduction events from other countries that failed to produce sustained community transmission events and do not cluster with other Mexican sequences. A large cluster (C1) with sequences from the northern state of Tamaulipas, with collection dates spanning May to June 2021, showed a possible migration event toward the southeast (Tabasco and Yucatán) of the country, where most sequences were collected in June. Another possible introduction from the northern region can be observed in C5, with sequences from Chihuahua (located at the Mexico-U.S. border) predating those from central Mexico. Together with their phylogenetic relatedness to U.S. sequences, these results support the idea that introduction events that resulted in continued local community transmission occurred in the northern states before spreading south. However, not all B.1.1.7 introductions occurred at the northern border. For instance, C2 sequences appeared first in Quintana Roo in the southeast, but its dispersion was limited, probably due to the circulation of other B.1.17 haplotypes and the dominant B.1.1.519 variant in this region and the introduction of other VOCs. However, given the limitations of sequencing efforts, the necessary subsampling of international data, and the limited diversity of the viruses, the elucidation of the exact origin of most Mexican clades is not feasible. Recently, some global variants of B.1.1.7 have been designated sublineages (Q.1 to Q.8). The circulation of these sublineages has been local, and none of them have been distributed beyond a few countries. Only three of these have been detected in Mexico, albeit at extremely low frequencies. Q.3, the most sampled sublineage, only reached 2.5% of all Alpha sequences, whereas for Q.1 and Q.8, only one sequence was detected. Some of nonsynonymous mutations, with a prevalence higher than 5% in Mexican sequences, have been reported to be associated with some of the sublineages of B.1.1.7. Interestingly, 10 of the additional prevalent amino acid changes identified abundantly in B.1.17 Mexican sequences were exclusive to one of the five clusters described in this work. Additionally, three mutations were common to two clusters, supporting the idea that most of the prevalent mutations may be associated with events of community transmission chains within Mexico. In conclusion, in this work, we have established that the circulation of B.1.1.7 in Mexico was temporally and geographically limited, contrary to reports from European countries and the United States. This finding highlights that lineage dynamics is a complex multifactorial phenomenon that is difficult to predict until a more thorough characterization of all variants and a comprehensive analysis of social dynamics is attained. Therefore, to better understand and cope with emerging variants on the global scale that carry mutations of potential biological significance, higher sequencing and surveillance across Mexico are necessary. Briefly, for RT-qPCR, 5 mL of RNA was used in a 25-mL reaction using the Superscript III one-step RT-PCR system (Invitrogen, Darmstadt, Germany). Reverse transcription was done for 10 min at 55°C, followed by PCR for 45 cycles, 95°C for 15 s, and 58°C for 30 s. # Materials and methods SARS-CoV-2 whole-genome sequencing and genome generation. The extracted RNA from the 6,585 samples that tested RT-qPCR positive for SARS-CoV-2 were subjected to amplification and next-generation sequencing. From the remanent RNA, total cDNA was synthesized by Superscript III reverse transcriptase (Thermo Fisher, USA) and random hexamers. Next, the POLAR nCoV-2019 amplicon sequencing protocol was used with the V3 primer set. Samples were then barcoded using the native barcode kits. Nextera XT sequencing libraries were prepared using the ligation sequencing kit, followed by sequencing on a midoutput kit in the NextSeq500 platform using 2 Â 150 cycles of paired-end runs with an insert size of 500 bp. FASTQ reads were generated by the Illumina pipeline at BaseSpace (https://basespace.illumina.com). Adapters, low-quality bases, dereplication, and off-target reads were removed for each sample using a customized pipeline described previously [bib_ref] Genomic analysis of early SARS-CoV-2 variants introduced in Mexico, Taboada [/bib_ref]. Then, unique and high-quality reads were mapped with Bowtie2 v2.3.4.3 [bib_ref] Ultrafast and memoryefficient alignment of short DNA sequences to the human genome, Langmead [/bib_ref] against the Wuhan-Hu-1 (MN908947) reference genome. Consensus calling was performed with iVar (v1.3.1) (28) using bases scored with a Q value of .20 and a minimum read coverage depth of 20Â, bases with lower depth were assigned as N. In total, 6,352 genome sequences comprised at least 90% coverage of the Wuhan-Hu-1 reference genome and were considered useful for genetic diversity and lineage composition analyses. Mexican B.1.1.7 genomes data set. SARS-CoV-2 genome sequences were initially assigned to viral lineages according to the nomenclature proposed by Rambaut et al. [bib_ref] A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist genomic epidemiology, Rambaut [/bib_ref] using the Pangolin v3.1.7 desktop application. In total, 473 virus genome sequences assigned as B.1.1.7 lineage were deposited in the Global Initiative on Sharing All Influenza Data (GISAID) platform (https://www.gisaid.org/) and GenBank (Table S1 in the supplemental material). To better characterize the genetic and distribution data of the B.1.1.7 lineage in Mexico, we obtained all other Mexican SARS-CoV-2 sequences from GISAID from the same dates as our genomes that were assigned to the B.1.1.7 lineage using the Pangolin database web interface (v3.1.7; https://github.com/cov-lineages/pangolin, accessed on 24 July 2021; [fig_ref] TABLE 1: Demographic and status information for Mexican patients during April to June 2021... [/fig_ref]. We obtained 1,141 additional sequences with their metadata, resulting in a total of 1,620 genomes of the B. Sequence alignment. From the 1,620 genome sequences of B.1.1.7 lineages from Mexico, only those with less than 1% of Ns (undetermined nucleotides) were selected (n = 1,241). International B.1.1.7 sequences available in GISAID were subsampled for temporal and spatial analysis using the following strategy: one random sequence per country and month (excluding Mexico) was selected from March to October 2020. From November 2020 to July 2021, no more than 20 sequences per month were selected from Europe, 20 from North America (10 from the United States), 20 from Asia, 10 from Africa, 5 from South America, and 5 from Oceania. In total, 709 international sequences were included in the alignment. The sequences were aligned against the reference sequence from Wuhan (NC_045512.2) using MAFFTv7 (30) (with the parameters -addfragments). The alignment was manually edited to remove untranslated (UTR) regions. Regional lineage distribution. To assess the differences in lineage distribution, we divided the country into seven regions as follows: Northeast (NE; Coahuila, Nuevo León, and Tamaulipas), Northwest (NW; Baja California, Baja California Sur, Chihuahua, Durango, Sonora, and Sinaloa), Central North (CN; Aguascalientes, Guanajuato, Querétaro, San Luis Potosí, and Zacatecas), Central South (CS; Mexico City, Estado de México, Morelos, Hidalgo, Puebla, and Tlaxcala), West (W; Colima, Jalisco, Michoacán, and Nayarit), Southeast (SE; Guerrero, Oaxaca, Chiapas, Veracruz, and Tabasco), and South (S; Campeche, Yucatán, and Quintana Roo). For each region, we built a stack density plot showing lineage circulation through time using the package ggplot2 in R. Haplotype network and amino acid changes analysis. Aligned sequences, considering 1,241 Mexican sequences of high quality along with the other 709 international genomes, were used to generate a haplotype network. The population analysis with reticulate trees (PopArt v1.7) software (31) was used to construct a statistical parsimony, at a 95% confidence level, TCS network, which is based on an agglomerative approach, where clusters are progressively combined with one or more connecting edges. Sites with more than 5% of undefined states were masked. Also, to estimate geographical relationships of haplotype groups, the network was colored using the respective state where samples were taken by using a trait in the nexus data. The Nextclade single nucleotide variant (SNV) calling system was used to identify synonymous/nonsynonymous substitutions in the Mexican sequences, enabling us to determine the association of nucleotide SNPs to a particular haplotype or geographical group. Also, Nexclade amino acid mutations annotation was used to compare between variants and to estimate evolutionary rates. For the most prevalent haplotypes, a map series was done showing their distribution through time in the country using the package mxmaps in R. Phylogenomic analysis. The multiple sequence alignment was used to reconstruct a maximum likelihood phylogeny using iqtree v.2.1.1 [bib_ref] Corrigendum to: IQ-TREE 2: new models and efficient methods for phylogenetic inference..., Minh [/bib_ref] with the GTR1F1R3 substitution model [bib_ref] ModelFinder: fast model selection for accurate phylogenetic estimates, Kalyaanamoorthy [/bib_ref] and the feature LSD2 to scale the resulting phylogeny based on collection date to ensure all child nodes had a later collection date than their parent node [bib_ref] Fast dating using least-squares criteria and algorithms, To [/bib_ref]. ggtree v.3.0.2 (37) and treeio v.1.16.1 [bib_ref] Treeio: an R package for phylogenetic tree input and output with richly..., Wang [/bib_ref] packages were used to plot the tree using R. Additionally, to explore further the relationship between B.1.1.7 viruses of Mexico and the United States, a phylogeny was built in Nextstrain using all high-quality sequences from Mexico's northern states plus 100 sequences per month from the United States and the global subsampling from Nextstrain. Statistical analysis. The statistical analyses and plots were generated with R using the ggplot2 and stats packages available from the CRAN repository. Medians, interquartile ranges (IQRs), and statistical tests to compare groups were calculated and performed in R. Statistically significant differences of the median patient age distribution grouped by lineage were assessed by Wilcoxon rank sum test. In contrast, differences between gender or patient status per lineage were evaluated using the chi-square test. Data availability. The generated sequences of SARS-CoV-2 used in this study have been publicly shared through the Global Initiative on Sharing All Influenza Data (GISAID) repository and have also been deposited in the GenBank NCBI database. Accession numbers are listed in [fig_ref] TABLE 1: Demographic and status information for Mexican patients during April to June 2021... [/fig_ref]. # Supplemental material Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 7.3 MB. [fig] FIG 1: Distribution of SARS-CoV-2 cases and B.1.1.7 variant (Alpha) viruses identified in Mexico between 1 March 2020 and 7 July 2021. (A) Confirmed cases, positivity rate, and case fatality rate (proportion of the number of deaths among confirmed cases) in the country based on confirmation date. (B) Relative frequency of B.1.1.7 and other variants. (C) Prevalence of B.1.1.7 in Mexico and other global places through time estimated from whole-genome sequencing. (D) Prevalence at the state level, considering only sequences from April to June 2021 (months of higher prevalence). highest prevalence, reaching its peak (29.5%) in May and June. Moreover, B.1.1.7 was identified as soon as January 2021 in the NE, suggesting that the north of Mexico may have been an entryway for this lineage. Interestingly, in the NW and NE, the B.1.1.519 lineage never achieved the dominant prevalence observed in other regions (Fig. 2C to G). Around June, B.1.1.7 and the other cocirculating lineages, including B.1.1.519, showed a decrease in their prevalence due to the entry of other VOCs in the country, initially Gamma and later Delta. Interestingly, the NE and NW regions exhibited the highest lineage diversity and the lowest prevalence of B.1.1.519 at the time of the introduction of Alpha (Fig. 2). These conditions may have contributed to this variant's relative success in northern Mexico compared to in the rest of the country. Mutations observed in the B.1.1.7 sequences. The B.1.1.7 lineage is characterized by 18 amino acid changes compared to the reference sequence Wuhan Hu-1 (four inherited from its parental B.1.1 lineage) and three in-frame deletions (open reading frame 1a [ORF1a]:del3676/3678, S:del69/70, and S:del144; [/fig] [fig] FIG 2: Temporal distribution of the B.1.1.7 variant and other lineages by region, including Northwest (A), Northeast (B), Central North (C), Central South (D), West (E), South (F), and Southeast (G [/fig] [fig] FIG 3: Genomic changes of Mexican B.1.1.7 and B.1.1.519 variants compared to the reference Wuhan-Hu-1 estimated by the Nextclade tool using sample collection date. (A) Nucleotide changes. (B) Amino acid changes. [/fig] [fig] FIG 4: Time-scaled maximum likelihood phylogenetic tree of the B.1.1.7 lineage of Mexican and international sequences. All Mexican isolates are in different colored circles according to sampling location; black circles are from the United States, and gray circles represent other countries. The correspondence of clades with the major Mexican haplotype clusters is indicated. [/fig] [fig] FIG 5: Haplotype network using genome-wide single-nucleotide variations of the B.1.1.7 lineage from Mexican and international sequences. All Mexican isolates are in different colored circles according to sampling location; black circles are from the United States, and gray circles represent other countries. The size of the circles corresponds to the number of samples within the same haplotype (scale is provided). Some Mexican clusters are highlighted with dashed rectangles and circles. InFig. S2in the supplemental material, a high-resolution haplotype network is provided, with mutations between sequences represented by the number of dashes in the connecting lines. [/fig] [table] TABLE 1: Demographic and status information for Mexican patients during April to June 2021 Wilcoxon sum of ranks test. b Chi-square test.compared to 1 for B.1.1.519 (Fig. 3A, red linear regression). This higher nucleotide evolution rate for the B.1.1.7 lineage was also observed in nonsynonymous mutations (Fig. 3B, yellow linear regression), having on average 0.83 amino acid changes per month for B.1.1.7 in contrast to 0.38 for the B.1.1.519 variant(Fig. 3B, red linear regression).Phylogenetic and haplotype analysis of B.1.1.7 Mexican sequences. A time-scaled maximum-likelihood phylogenetic tree was constructed to understand the temporal and spatial evolutionary relationships of Mexican B.1.1.7 sequences with international isolates [/table] [table] TABLE 2: Frequency of additional mutations for the B.1.1.7 lineage observed in sequences of Mexico as well as other abundant mutations identified Clusters of the haplotype network where the mutation was detected. An en dash indicates the mutation is not present in any analysed cluster. c Only some sequences have the amino acid change. [/table] [bib_ref] Spike mutation D614G alters SARS-CoV-2 fitness, Plante [/bib_ref]
Early prediction of postoperative liver dysfunction and clinical outcome using antithrombin III-activity Background and aimsAntithrombin III (ATIII) has been reported to be associated with liver pathologies and was shown to predict outcome in patients undergoing liver resection for hepatocellular carcinoma. We now aimed to assess whether perioperative ATIII-activity could predict postoperative outcome in patients without underlying liver disease, as well as in a routine clinical setting of patients undergoing hepatic resection.MethodsATIII-activity was evaluated preoperatively and on the first (POD1) and fifth day after liver resection in a retrospective evaluation cohort of 228 colorectal cancer patients with liver metastasis (mCRC). We further aimed to prospectively validate our results in a set of 177 consecutive patients undergoing hepatic resection.ResultsPatients developing postoperative liver dysfunction (LD) had a more pronounced postoperative decrease in ATIII-activity (P<0.001). ATIII-activity on POD1 significantly predicted postoperative LD (P<0.001, AUC = 84.4%) and remained independent upon multivariable analysis. A cut-off value of 61.5% ATIII-activity was determined using ROC analysis. This cut-off was vital to identify high-risk patients for postoperative LD, morbidity, severe morbidity and mortality (P<0.001, respectively) with a highly accurate negative predictive value of 97%, which could be confirmed for LD (P<0.001) and mortality (P = 0.014) in our independent validation cohort. Further, mCRC patients below our cut-off suffered from a significantly decreased overall survival (OS) at 1 and 3 years after surgery (P = 0.011, P = 0.025).The routine laboratory parameter ATIII-activity on POD1 independently predicted postoperative LD and was associated with clinical outcome. Patients with a postoperative ATIII-activity <61.5% might benefit from close monitoring and timely initiation of supportive therapy.Trial registrationClinicalTrials.gov NCT01700231Patients and methodsStudy cohortsWithin our retrospective evaluation cohort, patients suffering from mCRC, resected between March 2001 and December 2009, were included using our prospectively maintained institutional database. To confirm our findings in a routine clinical setting a prospective validation cohort was recruited from January 2010 to October 2014. The extent of resection was characterized following the IHPBA Brisbane 2000 nomenclature in minor (< 3 liver segments) and major (! 3 liver segments)[22]. Blood samples were collected routinely prior to surgery Antithrombin III-activity and clinical outcome after liver resection PLOS ONE | https://doi. [formula] a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 [/formula] # Introduction In a great number of patients suffering from liver malignancies surgical resection represents the treatment of choice and is frequently the only potentially curative treatment option [bib_ref] Hepatic resection associated with good survival for selected patients with intermediate and..., Zhong [/bib_ref] [bib_ref] Surgical resection of hepatic metastases from colorectal cancer: a systematic review of..., Simmonds [/bib_ref] [bib_ref] Towards a pan-European consensus on the treatment of patients with colorectal liver..., Van Cutsem [/bib_ref] [bib_ref] Improved survival in metastatic colorectal cancer is associated with adoption of hepatic..., Kopetz [/bib_ref]. It is important to preserve at least 20-25% of healthy liver volume, while resecting every radiological and macroscopically detectable tumour, to ensure sufficient postoperative liver function [bib_ref] Improving resectability of hepatic colorectal metastases: expert consensus statement, Abdalla [/bib_ref]. However, postoperative morbidity may compromise patients' outcome and can often be related to a delay in liver regeneration [bib_ref] Hepatic insufficiency and mortality in 1,059 noncirrhotic patients undergoing major hepatectomy, Mullen [/bib_ref]. As a member of the serpin family Antithrombin III (ATIII) is a main inhibitor of proteinases active in coagulation such as thrombin and factor Xa [bib_ref] Mechanism of heparin activation of antithrombin: evidence for an induced-fit model of..., Desai [/bib_ref]. ATIII is primarily synthesized by hepatocytes [bib_ref] Measurement of Blood Coagulation Factor Synthesis in Cultures of Human Hepatocytes, Heinz [/bib_ref]. Furthermore, levels of ATIII have been shown to be altered in patients with liver disease [bib_ref] Procoagulant imbalance aggravated with falling liver function reserve, but not associated with..., Tang [/bib_ref]. Recently, ATIII was reported to be a valuable marker for clinical outcome in hepatocellular carcinoma (HCC) patients undergoing curative intended liver resection [bib_ref] Prognostic significance of antithrombin III levels for outcomes in patients with hepatocellular..., Iwako [/bib_ref] [bib_ref] Serum antithrombin III level is well correlated with multiple indicators for assessment..., Mizuguchi [/bib_ref]. In particular, ATIII has been proposed as a marker for overall and progression free survival in HCC patients [bib_ref] Prognostic significance of antithrombin III levels for outcomes in patients with hepatocellular..., Iwako [/bib_ref]. Moreover, Mizugichi et al. showed a predictive potential of ATIII for postoperative liver dysfunction (LD) in a cohort of 158 HCC patients [bib_ref] Serum antithrombin III level is well correlated with multiple indicators for assessment..., Mizuguchi [/bib_ref]. It is well known, that HCC is often associated with cirrhosis [bib_ref] Epidemiology of Viral Hepatitis and Hepatocellular Carcinoma, El-Serag [/bib_ref] or fatty liver disease [bib_ref] Clinical gastroenterology and hepatology: the official clinical practice journal of the, White [/bib_ref]. However, the value of ATIII as a predictive marker in colorectal cancer liver metastases (mCRC) patients undergoing liver resection, who commonly do not suffer of primary liver pathologies [bib_ref] Characteristics of common solid liver lesions and recommendations for diagnostic workup, Assy [/bib_ref] , has not been investigated so far. Of note, while systemic levels of other factors involved in the coagulation cascade are largely affected by systemic treatments, postoperative substitution, deficiencies and diseases, ATIII seems to be comparably less affected and seems to be primarily altered by mutations, consumption or hepatocellular dysfunction. Within this study, we now aimed to explore the role of perioperative dynamics of ATIII as a predictive marker in a retrospective cohort of our prospectively maintained institutional database including a homogenous cohort of mCRC patients undergoing liver resection. Importantly, we aimed to further validate our results in a prospective validation cohort, consecutively including patients undergoing liver resection within a routine clinical setting. (PRE-OP) as well as on the first (POD1) and fifth (POD5) day after liver resection. The study was approved by the institutional ethics committee ((#424/2010; #2032/2013) All patients gave written informed consent. Furthermore the study has been registered at a clinical trials registry (ClinicalTrials.gov Identifier: NCT01700231). ## Definition and classification of postoperative ld and morbidity Postoperative LD was classified according to the ISGLS criteria [bib_ref] Posthepatectomy liver failure: a definition and grading by the International Study Group..., Rahbari [/bib_ref]. Briefly, the criteria were met if serum bilirubin (SB) concentration and prothrombin time (PT) did not reach normal levels on POD5 after liver resection or thereafter. If SB and PT differed from standard values prior to the operation the criteria were fulfilled when both parameters were elevated for two succeeding days on or after POD5. Additionally, patients who reached normal SB or PT values before POD5 and thus had no further routine blood collection were considered as "no LD". Morbidity after surgery was assessed using the classification described by Dindo et al. [bib_ref] Classification of surgical complications: a new proposal with evaluation in a cohort..., Dindo [/bib_ref]. The severity of postoperative complications was sub-classified in grade I-V. In case of multiple complications per patient, the most serious one was used for analysis. Further, patients with morbidity grade III-V were classified as patients with severe morbidity (SM). Postoperative mortality was defined as death within 90 days after surgery. [bib_ref] Thirty-day mortality leads to underestimation of postoperative death after liver resection: A..., Schiergens [/bib_ref] Quantification of blood parameters All perioperative parameters of liver function (SB, PT, aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), alkaline phosphatase (AP), and albumin) as well as C-reactive protein (CRP), fibrinogen and platelet counts were measured in appropriate samples by routine laboratory blood tests. Moreover, ATIII-activity was assessed in routine laboratory blood tests and used as a surrogate parameter for absolute levels of ATIII. # Statistical analysis All analyses are performed using data from subjects with valid marker values only. Statistical analyses were carried out using SPSS software (SPSS, Inc., Chicago, IL, USA, Version 23) and GNU R version 3.2.3. Exploratory analysis of the evaluation samples. We compared ATIII-activity distributions between subjects with and subjects without LD, morbidity, SM and mortality using boxplots as well as Wilcoxon-Mann-Whitney U-Tests. Boxplot illustrations are given without outliers and extreme values to improve resolution of the interquartile ranges. Given the exploratory character of this analysis corresponding p-values are reported unadjusted. Furthermore, we fit receiver operating characteristic (ROC) curves for postoperative ATIII-activity for the prognosis of LD and mortality within 90 days after surgery. To further elucidate clinical relevance of ATIII-activity in the prediction of liver dysfunction following partial hepatectomy, we aimed to compare it to postoperative parameters that are already in clinical usage [bib_ref] Prognostic utility of postoperative C-reactive protein for posthepatectomy liver failure, Rahman [/bib_ref]. Therefore we performed a ROC analysis for postoperative ATIII-activity, fibrinogen, CRP, PT and SB. Furthermore, we included postoperative markers of liver injury, such as GGT, AST and ALT To define a suitable cut-off value for the classification of patients with high risk for postoperative LD we computed the threshold value maximising the Youden-Index (i.e. the sum of sensitivity and specificity). We estimated corresponding values for sensitivity and specificity and computed bootstrap 95% confidence intervals. Proportions of subjects with LD, morbidity, SM and 90 day mortality are compared using relative proportions and the chi-square test (p-values are reported unadjusted). Validation of the predictive potential of ATIII-activity. Prognostic potential of ATIIIactivity on POD1 for the prognosis of LD, morbidity, SM and 90 day Mortality was validated using only data from the validation sample. We estimate sensitivity and specificity of ATIIIactivity on POD1 using the cut-off chosen based on the evaluation sample. We tested whether subjects with high and low ATIII-activity (according to the cut-off chosen in the evaluation sample) show significantly different incidence of LD, morbidity, SM and postoperative mortality using chi-square tests. Considering this as the primary confirmatory analysis of this article, p-values of the four hypothesis tests are adjusted for multiplicity using the Bonferoni-Holm procedure. Test results are deemed statistically significant if the adjusted two-sided p-value is below 0.05. Uni-and multivariable analysis. To further elucidate the prognostic value of ATIII-activity in combination with other potential markers we fit univariable and multivariable logistic regression models and perform a model selection using baseline variables and all available POD1 markers as candidate variables. Given the small number of LD cases in the validation sample, we combined the evaluation and validation sample for this analysis to achieve a better model fit. In a first step, separate univariable logistic regression models were performed for baseline characteristics and postoperative blood parameters. Second, we fit a multiple logistic regression model with stepwise forward selection to assess the effect of the respective parameters on LD status and postoperative mortality. The initial model included all parameters with a p-value below 0.05 in the univariable analysis. The p-value limit for a parameter to stay in the model was 0.1. Classification tree. Finally to investigate potential interactions and nonlinear relationships between predictive markers and outcome variables we fitted binary classification treefor the prediction of postoperative LD using postoperative markers and baseline characteristics. Tree depth was chosen to minimise the relative cross-validation error. Similar to the multivariable model we used the combined sample for this analysis due to the small number of cases with LD. # Results ## Patients and cohorts In total 405 patients were included into this study. The evaluation cohort consisted of 228 mCRC patients from our prospectively maintained institutional database and was analysed retrospectively. Succeeding, a prospective validation cohort consisting of 177 patients was recruited to confirm the results obtained within our evaluation set. The prospective validation cohort set out to reflect a routine clinical setting and accordingly consisted of mCRC (N = 84), HCC (N = 33), cholangiocellular carcinoma (CCC, N = 26), benign neoplastic entities (N = 16) and other liver pathologies requiring resection (N = 18). Baseline characteristics of both cohorts are given in [fig_ref] Table 1: Patients demographics [/fig_ref]. Median follow up for the evaluation and validation cohort were 67 and 38 months, respectively. Missing data is visualized in S1 Values for ATIII-activity could be obtained from the majority of patients prior to the operation as well as on POD1. Due to early postoperative dismissal and immediate postoperative death missing vales increase on POD5 (53% in evaluation set, 49% in validation set). Postoperative follow up concerning the outcome parameters LD (patients dismissed prior to POD5 with already normal PT and SB were classified as noLD-see methods section), morbidity, postoperative mortality, as well as OS, could be recorded from every patient. Postoperative levels of ATIII-activity decrease after liver resection and correlate with extent of resection First, we investigated the perioperative dynamics of ATIII-activity after liver surgery in our evaluation cohort [fig_ref] Fig 1: Perioperative ATIII-activity dynamics [/fig_ref]. We observed a significant decrease in ATIII-activity on POD1 (median ATIII PRE OP = 107%, median ATIII POD1 = 73.5%, P<0.001), which persisted upon POD5 (median ATIII POD1 = 73.5%, median ATIII POD5 = 72%, P = 0.347). Moreover, we found that ATIII-activity followed the extent of hepatic resection [fig_ref] Fig 1: Perioperative ATIII-activity dynamics [/fig_ref]. While no preoperative difference in ATIII-activity was observed (median ATIII before minor resection = 107%, median ATIII before major resection = 107%, P = 0.634), we found that patients undergoing major resection had a more pronounced decrease in ATIII-activity compared to patients undergoing minor resection on POD1 (median ATIII after minor resection = 76%, median ATIII after major resection = 64%, P<0.001) as well as on POD5 (median ATIII after minor resection = 78%, median ATIII after major resection = 57.5%, P<0.001). Interestingly, patients receiving minor resections were able to partially restore levels of ATIII-activity from POD1 to POD5 (P = 0.004), while patients with major resection further decreased significantly (P = 0.047). Postoperative ATIII-activity is associated with LD after liver resection We further aimed to evaluate ATIII-activity in patients with and without postoperative LD [fig_ref] Fig 1: Perioperative ATIII-activity dynamics [/fig_ref]. In our evaluation cohort a total of 26 patients suffered from LD after surgery. Preoperative ATIII-activity did not differ between both groups (median ATIII no LD = 107%, median ATIII LD = 107%, P = 0.517), whereas patients developing LD after surgery had extensively reduced levels of ATIII on POD1 (median ATIII no LD = 75.5%, median ATIII LD = 52.5%, P<0.001) and POD5 (median ATIII no LD = 77.5%, median ATIII LD = 46.5%, P<0.001). Of note, regarding the postoperative time course, ATIII-activity increased in patients who did not suffer from LD (P = 0.018), whereas it decreased significantly in patients who developed postoperative LD (P = 0.009). Postoperative ATIII-activity correlates with poor clinical outcome and predicts mortality within 90 days We further evaluated ATIII-activity concerning postoperative morbidity according to Dindo et al. [bib_ref] Classification of surgical complications: a new proposal with evaluation in a cohort..., Dindo [/bib_ref] [fig_ref] Fig 1: Perioperative ATIII-activity dynamics [/fig_ref]. In total 81 patients in the evaluation cohort suffered from postoperative morbidity (grad I-V according to Dindo et al. [bib_ref] Classification of surgical complications: a new proposal with evaluation in a cohort..., Dindo [/bib_ref] during their postoperative course. These patients were found to have significantly lower levels of ATIII-activity on POD1 (median ATIII no morbidity = 76%, median ATIII morbidity = 68%, P = 0.001) and on POD5 (median ATIII no morbidity = 78%, median ATIII morbidity = 60%, P<0.001), without showing significant differences in their preoperative ATIII-activity (median ATIII no morbidity = 106%, median ATIII morbidity = 107.5%, P = 0.766). Patients without postoperative morbidity were able to partially restore ATIII (P = 0.023), while patients with postoperative morbidity did not recover but tended to further decrease (P = 0.238) during the postoperative period. We further sub-classified postoperative complications in severe morbidity, namely patients developing postoperative morbidity grade III-V according to Dindo et al. [bib_ref] Classification of surgical complications: a new proposal with evaluation in a cohort..., Dindo [/bib_ref] [fig_ref] Fig 1: Perioperative ATIII-activity dynamics [/fig_ref]. As for morbidity, patients developing severe morbidity had significantly decreased levels of ATIII-activity on POD1 (median ATIII no severe morbidity = 75%, median ATIII severe morbidity = 56%, P<0.001) and on POD5 (median ATIII no severe morbidity = 76%, median ATIII severe morbidity = 51%, P = 0.001). Of note, preoperative ATIII-activity in patients suffering from severe morbidity in the postoperative time course did not differ from patients who recovered well after surgery (median ATIII no severe morbidity = 107%, median ATIII severe morbidity = 108%, P = 0.732). Seven patients died within 90 days after surgery. There were no preoperative differences in ATIII-activity levels (P = 0.459, [fig_ref] Fig 1: Perioperative ATIII-activity dynamics [/fig_ref]. However, patients that suffered from postoperative mortality had substantially lower ATIII-activity on POD 1 (median ATIII no mortality = 74%, median ATIII mortality = 54%, P<0.001), as well as on POD5 (median ATIII no mortality = 74%, median ATIII mortality = 42%, P = 0.025). Postoperative ATIII-activity challenges established postoperative markers of LD Next, we performed a ROC analysis for postoperative ATIII-activity, fibrinogen, CRP, PT, SB, as well as GGT, AST and ALT in a subgroup of patients that do not miss any of those markers (N = 187). Strikingly, our results showed that ATIII-activity on POD1 was the strongest predictor of postoperative LD in our study cohort (AUC = 87.4%, P<0.001, 95%-CI: 0.783-0.964, [fig_ref] Fig 2: ATIII-activity on POD1 competes other markers in the prediction of postoperative LD [/fig_ref]. Values of ATIII-activity on POD1 below 61.5% specifically identify patients with postoperative LD and poor clinical outcome To further assess the value of ATIII-activity to predict postoperative LD after liver resection we performed a ROC analysis in our evaluation cohort. Accordingly, we found that ATIIIactivity on POD1 was able to significantly predict postoperative LD (AUC = 84.4%, P<0.001, 95%-CI: 0.753-0.936, [fig_ref] Fig 3: ATIII-activity on POD1 predicts postoperative LD [/fig_ref]. To identify high-risk patients that will most likely develop postoperative LD, we defined a cut-off value at 61.5% ATIII-activity on POD1 with a high specificity of 84.9% (95%-CI: 0.797-0.896) and an equally good sensitivity of 80.8% (95%-CI: 0.654-0.962). This leads to a positive predictive value (PPV) of 42% and a negative predictive value (NPV) of 97%. According to this cut-off, a high-risk group (ATIII low ) consisting of 50 patients was identified, while the remaining 168 patients were classified as a low-risk group (ATIII high ). Patients within our high-risk group suffered more frequently from postoperative LD (P<0.001, 21 of 50 [42%] in ATIII low vs. 5 of 168 [3%] in ATIII high ) as shown in [fig_ref] Fig 4: Patients with low postoperative ATIII-activity suffer from poor clinical outcome [/fig_ref] Furthermore, we aimed to evaluate if patients within our high-risk group suffered from an increased incidence of postoperative morbidity and mortality. Indeed, patients in our high-risk group developed postoperative morbidity (P = 0.001, [bib_ref] Inherited antithrombin deficiency: a review, Patnaik [/bib_ref] Prospective validation cohort confirms predictive potential of ATIIIactivity and cut-off value After having observed a predictive potential of postoperative ATIII-activity for postoperative LD and clinical outcome in our evaluation cohort of 228 patients, we aimed to validate our findings in a prospective validation cohort of 177 consecutive patients, reflecting a routine clinical setting. To validate the predictive potential of postoperative ATIII-activity for LD after liver resection, again ROC analysis was performed. Interestingly, results showed an equally good predictive potential for ATIII-activity with an AUC of 84.2% (P<0.001, 95%-CI: 0.752-0.932) and using 61.5% as a cut-off yields a specificity of 85% (95%-CI: 0.790-0.902) and a sensitivity of 62.5% (95%-CI: 0.375-0.813 95%). Moreover, comparison of ATIII-activity to established markers of postoperative LD as performed in the evaluation cohort via ROC analysis was performed and strikingly, even in the validation cohort ATIII-activity provided the highest AUC of 83.8% (P<0.001, 95%-CI: 0.741-0.935, S1 . Consequently, the validation cohort was divided in risk groups using the same cut-off value as determined in the evaluation cohort. In line with the findings in the evaluation cohort, patients in the validation cohort within the high-risk group had a significantly higher in ATIII high ), as visualized in [fig_ref] Fig 4: Patients with low postoperative ATIII-activity suffer from poor clinical outcome [/fig_ref] Incidences for postoperative morbidity and severe morbidity did not significantly differ between risk groups in our validation cohort. However, a similar tendency as in the evaluation cohort could be observed for both morbidity (P = 0.096, Antithrombin III-activity and clinical outcome after liver resection Using a cut-off of 61.5% ATIII-activity, patients were divided in a high-risk group (ATIII low ) and a low-risk group (ATIII high ). Incidence of postoperative LD, morbidity, severe morbidity (SM) and mortality are illustrated according to risk groups in our evaluation cohort (A) and were confirmed in our validation cohort (B). Overall survival (OS in %) for mCRC patients in the entire collective was assessed for both risk groups after 1, 3, 5 and 10 years (C). * P < 0.05; ** P < 0.005. ATIII-activity remains an independent marker for postoperative LD and postoperative mortality upon multivariable analysis and is associated with overall survival As we had observed a significant predictive potential of ATIII-activity for LD after hepatic resection, we aimed to explore if ATIII-activity on POD1 can independently predict postoperative LD. Therefore, multivariable analysis (MVA) was performed including the entire cohort of 405 patients (evaluation and validation cohort) to achieve sufficient statistical power. Extent of resection, ATIII-activity, SB, PT, GGT, AST, ALT, and albumin gave p-values below 0.05 in the univariable models for LD and were consequently included in the initial mulitivariable model. After step-wise forward selection extent of resection, ATIII, SB and ALT remained. The results from the final model fit are shown in [fig_ref] Table 2: Multivariable analysis for liver dysfunction and 90 days mortality [/fig_ref]. ## Of 33 [64%] in atiii Further, we aimed to explore whether postoperative ATIII-activity can independently predict death within 90 days after liver resection. Again, we performed a MVA, including all significant parameters for postoperative mortality from univariate analysis, which were age, ATIII-activity, SB, PT, GGT, AST. Interestingly, after step-wise forward selection exclusively ATIII-activity remained highly significant with an odds ratio of 0.863 (95% Confidence interval 0.809-0.920, p-value <0.001; [fig_ref] Table 2: Multivariable analysis for liver dysfunction and 90 days mortality [/fig_ref]. Moreover, we aimed to evaluate the association between postoperative ATIII-activity and long term overall survival (OS). To allow for a homogenous cohort, exclusively patients treated for mCRC (N = 312) were included in OS analyses. Hence we found, that postoperative ATIIIactivity was associated with long term overall survival. In particular, ATIII-activity was significantly reduced for patients that died within the first (median ATIII survivors = 74%, median ATIII dead = 62%, P = 0.002), third (median ATIII survivors = 75%, median ATIII dead = 68%, P = 0.001), fifth (median ATIII survivors = 76%, median ATIII dead = 71%, P = 0.005) and up to tenth year after surgery (median ATIII survivors = 80%, median ATIII dead = 72%, P = 0.009). Furthermore, in regards to our proposed cut off, the low-risk group had a significantly higher overall survival rate after one (P = 0. Antithrombin III-activity and clinical outcome after liver resection Combination of ATIII-activity with SB on POD1 determined using a classification tree represents a useful tool for risk stratification in our cohort To investigate whether any interactions between postoperative markers or nonlinear relationships influence the predictive potential of ATIII-activity, we fited a binary classification tree to predict LD using all baseline characteristics and postoperative markers as candidate variables, identifying ATIII-acitivity as the variable with the best discriminatory capacity. After correction for overfitting the resulting classification tree first classified subjects with ATIII-activity levels above 60.5 (entire cohort) as low risk for LD and among the remaining subjects only those with SB values above 2.845 mg/dl as high risk patients. This provided a sensitivity of 42% and a specificity of 98% that corresponds to a PPV of 72% and an NPV of 93%. The resulting decision tree diagram is shown in [fig_ref] Fig 5: Decision tree for postoperative risk stratification [/fig_ref] # Discussion Despite substantial improvements in liver surgery, postoperative LD, morbidity and mortality still represent a major clinical concern [bib_ref] Hepatic insufficiency and mortality in 1,059 noncirrhotic patients undergoing major hepatectomy, Mullen [/bib_ref] [bib_ref] Thirty-day mortality leads to underestimation of postoperative death after liver resection: A..., Schiergens [/bib_ref]. In this study, we were able to demonstrate that low levels of ATIII-activity as early as on the first day after surgery could predict postoperative outcome in a retrospective evaluation cohort of 228 patients suffering from mCRC. To our Antithrombin III-activity and clinical outcome after liver resection knowledge, this represents the first clinical evidence for a close association of postoperative clinical outcome with ATIII-activity in patients without primary liver diseases. Importantly, we were further able to validate our results within a prospective validation set of 177 consecutive patients undergoing liver surgery in a routine clinical setting. Ultimately, we were able to document the independent association of ATIII-activity with postoperative LD and mortality upon multivariable analysis. ATIII is an α 2 -globulin and mainly synthesized by the liver. Via inhibition of thrombin, factor Xa, but also of factors IXa, XIa, XIIa, tissue plasminogen activator (tPA), trypsin, plasmin, urokinase and kallikrein, ATIII acts as a physiologic anticoagulant [bib_ref] Inherited antithrombin deficiency: a review, Patnaik [/bib_ref]. It is well known that PT is prolonged by a series of conditions, including direct acting anticoagulants (among them argatroban, dabigatran, rivaroxaban, apixaban, edoxaban) [bib_ref] Poor comparability of coagulation screening test with specific measurement in patients receiving..., Testa [/bib_ref] and intake of vitamin K antagonists, vitamin K deficiency, coagulation factor deficiency, lupus anticoagulans, polycitemia vera. Similarly, it was shown that factor VII deficiency is frequent in patients with infections, neoplasia, trauma, nephrotic syndrome, left heart failure, penicillin intake and clearly in patients under vitamin K dependent anticoagulation [bib_ref] Isolated acquired factor VII deficiency: review of the literature, Mulliez [/bib_ref]. However, ATIII should only be altered due to mutations [bib_ref] Causative genetic mutations for antithrombin deficiency and their clinical background among Japanese..., Sekiya [/bib_ref] , consumption [bib_ref] Observational study to compare antithrombin and thrombomodulin for disseminated intravascular coagulation, Murata [/bib_ref] or hepatocellular dysfunction [bib_ref] Coagulation inhibitors in alcoholic liver cirrhosis, Raya-Sanchez [/bib_ref] [bib_ref] Coagulation and fibrinolysis in primary biliary cirrhosis compared with other liver disease..., Segal [/bib_ref]. Our data support the hypothesis that ATIII-and subsequently ATIII-activity as a surrogate parameter for absolute amount of circulating ATIII-represents a synthesizing parameter of the liver as we observed a decrease after liver resection and a more pronounced reduction in patients undergoing major liver resection. As hemodynamic management after surgery is highly standardized at our institution and variability in postoperative fluid management is limited, we do not believe that hemodilution is responsible for the observed dynamics of ATIIIactivity. Additionally, intraoperative blood loss is kept to a minimum. In fact, we could not observe significant differences in the distribution of postoperative ATIII-activity between patients that did or did not receive intraoperative blood transfusion (data not shown). Still, consumption of ATIII after liver resection is highly increased. This drastically shortens halflife of ATIII, which might suggest that hepatocellular synthesis may be the major determinant for postoperative levels of ATIII that are reflected by ATIII-activity values. Besides its association with hepatocellular function, experimental studies suggest a potential pathophysiologic role of ATIII during liver regeneration. Preclinical data report on the regulatory effect of ATIII on intrahepatic inflammation and apoptosis [bib_ref] Antithrombin prevents apoptosis by regulating inflammation in the liver in a model..., Isik [/bib_ref]. In particular, ATIII has been shown to reduce liver apoptosis via induction of IGF-1 in a rodent model [bib_ref] Antithrombin prevents reperfusion-induced hepatic apoptosis by enhancing insulin-like growth factor-I production in..., Harada [/bib_ref]. As IGF-I is known to be centrally involved during liver regeneration [bib_ref] Impaired liver regeneration in Nrf2 knockout mice: role of ROS-mediated insulin/IGF-1 resistance, Beyer [/bib_ref] ATIII might potentially act as an inducer of hepatic regeneration. In this context, we would like to point out that recently clinical data on the beneficial effect of postoperative ATIII administration was reported by Kuroda et al.. Patients that received ATIII substitution after curative resection for HCC had a significantly lower risk of postoperative LD, suggesting a potential role of ATIII during liver regeneration. However, the authors hypothesized that ATIII might reduce incidences of LD through a suppression of coagulopathies. Previously it was shown that ATIII reduces ischemia/reperfusion injury in a rodent model [bib_ref] Antithrombin prevents apoptosis by regulating inflammation in the liver in a model..., Isik [/bib_ref]. This might be due to reduced thrombosis. However, while thrombosis is a major effector in ischemia/reperfusion injury, its relevance in LD after partial hepatectomy remains poorly understood. In our entire cohort 7 patients developed coagulopathies (details given in [fig_ref] Table 3: Detail on postoperative thromboembolic incidences [/fig_ref]. Yet, patients in our study cohort suffering from thromboembolic incidences did not significantly differ in terms of their ATIII-activity on POD1 (median ATIII no thromboembolisms = 73%, median ATIII thromboembolisms = 69%, P = 0.238). Further, we did not observe a correlation between thromboembolic occurrence and postoperative LD (r = 0.077, P = 0.120). This might suggest that additional effects of ATIII could support postoperative liver regeneration in patients without underlying liver disease. Still, due to the limited number of thromboembolic events, we are ultimately unable to sort out weather patients with low levels of ATIII-activity are prone to develop microthrombosis after liver resection. Besides possible therapeutic benefits of ATIII administration, the major finding of our investigation was the striking predictive potential of ATIII-activity for postoperative LD and clinical outcome in mCRC patients without underlying liver disease, as well as in a routine clinical setting of hepatic resections. Accordingly, ATIII-activity allowed to identify high-risk patients timely after liver resection. Interestingly, the severity of postoperative LD, scored from A to C following the ISGLS criteria [bib_ref] Posthepatectomy liver failure: a definition and grading by the International Study Group..., Rahbari [/bib_ref] showed highly significant differences within our risk groups (S2A Further, incidence of grades of postoperative morbidity classified according to Dindo et al. [bib_ref] Classification of surgical complications: a new proposal with evaluation in a cohort..., Dindo [/bib_ref] tended to be increased in our high-risk group (S2B Of note, only patients belonging to our high-risk group suffered from life threatening or lethal complications, graded with IVb and V according to . This striking association might allow us to tailor postoperative treatment for patients at risk shortly after liver surgery. Accordingly, growing evidence suggest that liver support devices such as the molecular adsorbent recirculating system (MARS) are primarily effective, if they are introduced early [bib_ref] Meta-analysis of survival with the molecular adsorbent recirculating system for liver failure, He [/bib_ref]. In this context ATIII-activity could be helpful to guide treatment decisions. Furthermore, high-risk patients might benefit from a timely radiologic assessment or precise monitoring of liver function parameters for early detection of postoperative LD. As patients suffering from postoperative LD are prone to develop infectious complications, decreased postoperative ATIII-activity levels could trigger rapid postoperative antibiotic therapy, if indicated. Further, we want to point out that postoperative ATIII-activity was able to compete with established postoperative markers of LD. Importantly, ATIII-activity showed a superior potential to predict postoperative LD as compared to postoperative PT, which represents an internationally acknowledged marker for postoperative liver function. Of note, despite the fact that the ISGLS criteria use PT and SB on POD5 to define patients with postoperative LD, ATIIIactivity showed a comparable predictive potential for postoperative LD on POD1 as SB in an independent manner as shown by MVA. These findings further underline the clinical importance of ATIII-activity in risk-assessment after liver resection. However, ultimately the combination of ATIII-activity with SB on POD1 within our classification tree model showed the highest predictive accuracy for postoperative LD and might represent the clinically most useful tool of our investigation (see also . As the fitting algorithm identified the best cut-off for ATIII-activity in the pooled cohort (60.5%), the cut-off was close to, yet not exactly the same as our proposed cut-off of 61.5% identified in the homogenous evaluation cohort. Ultimately, an ideal cut-off for ATIII-activity will range between 60.5 to 61.5%. However, the fitted classification tree nicely illustrated, that after risk stratification via ATIII-activity, further subclassification using SB (cut-off of 2.845 mg/dl) may further improve predictive accuracy. Of note, ATIII-activity had highest discriminatory capacity in this model, which again points towards the use of this routine laboratory marker in patients undergoing liver resection. An additional major finding of this study is the correlation of low levels of ATIII-activity and postoperative mortality. In our entire study collective 10 patients died in the postoperative period. Interestingly, death of all those patients could be predicted using our cut off for ATIII Antithrombin III-activity and clinical outcome after liver resection on POD1. In particular, 15.9% of our high-risk patients died within the first 90 postoperative days, whereas no postoperative mortality was observed in our low risk group (based on our entire collective). ROC analysis of ATIII-activity on POD1 showed a remarkable predictive potential with an AUC of 95.5% for postoperative 90 days mortality (P<0.001, 95%-CI: 0.916-0.994). Moreover, we showed that ATIII-activity on POD1 is significantly reduced in mCRC patients that died within 1, 3, 5 and 10 years after liver surgery. In line with these findings, again our ATIII-activity cut-off was able to predict survival within one and three postoperative years. Accordingly, this finding further underlines the predictive relevance of postoperative ATIII-activity for postoperative overall outcome. Taken together, to our knowledge this study presents the first clinical evidence for a fundamental impact of ATIII-activity on postoperative risk stratification in a routine clinical setting of patients undergoing curative liver resection, while given clinical evidence primarily relays on HCC patients [bib_ref] Prognostic significance of antithrombin III levels for outcomes in patients with hepatocellular..., Iwako [/bib_ref] [bib_ref] Serum antithrombin III level is well correlated with multiple indicators for assessment..., Mizuguchi [/bib_ref]. Postoperative ATIII-activity was found to be a valuable and independent predictor for postoperative LD and poor clinical outcome after hepatic resection. Additionally, preoperative ATIII-activity showed a predictive potential for postoperative mortality. Because ATIII-activity is easily assessable and included in most clinical routine laboratory reports, this parameter might reliably be used to identify high-risk patients timely after surgery that will benefit from close monitoring and early induction of supportive treatment. Although we implied ATIII as a synthesizing parameter of the liver, the protein might have an additional physiologic involvement during liver regeneration. However, this hypothesis needs further investigation. This is of specific clinical interest, as therapeutic efficiency of postoperative ATIII substitution might not be limited to HCC patients as reported previously, but be presumably beneficial in all patients undergoing liver resection independent of underlying liver disease. Patients were characterized in risk groups regarding their ATIII-activity levels, leading to a high-risk group (ATIII low ) and a low-risk group (ATIII high ). Incidences of LD graded following the ISGLS score system were compared for patients in our high-and lowrisk group (A ## Supporting information [fig] Fig 1: Perioperative ATIII-activity dynamics. ATIII-activity was evaluated prior to surgery (PRE OP), as well as on the first (POD1) and fifth (POD5) postoperative day. Perioperative dynamics are shown in A for all patients of the evaluation cohort. Patients were divided in groups according to their resection extent (B). Further, dynamics of ATIII-activity in patients with and without postoperative liver dysfunction (LD) were assessed and are illustrated in C. Moreover, patients were grouped depending on postoperative outcome. ATIIIactivity is shown for patients with or without postoperative morbidity (D), postoperative severe morbidity (SM) (E) and mortality within 90 days after surgery (F). * P < 0.05; ** P < 0.005. [/fig] [fig] Fig 2: ATIII-activity on POD1 competes other markers in the prediction of postoperative LD. Predictive value of ATIII-activity, CRP, fibrinogen, SB, PT, AST, ALT and GGT on POD1 to predict postoperative LD using receiver operating characteristics (ROC) analysis was compared. [/fig] [fig] Fig 3: ATIII-activity on POD1 predicts postoperative LD. Receiver operating characteristic (ROC) curve for ATIII-activity on POD1 in the evaluation sample. Point with confidence bars gives sensitivity and specificity for the cut-off maximizing the sum of sensitivity and specificity.https://doi.org/10.1371/journal.pone.0175359.g003 [/fig] [fig] Fig 4: Patients with low postoperative ATIII-activity suffer from poor clinical outcome. [/fig] [fig] Fig 5: Decision tree for postoperative risk stratification. Combination of ATIII-activity and SB values on POD1 allows highly specific discrimination of patients risk for postoperative LD. https://doi.org/10.1371/journal.pone.0175359.g005 [/fig] [fig] S1: Fig. ATIII-activity on POD1 competes other markers in the prediction of postoperative LD in the validation cohort. Predictive value of ATIII-activity, CRP, fibrinogen, SB, PT, AST, ALT and GGT on POD1 to predict postoperative LD using receiver operating characteristics (ROC) analysis was compared. (TIF) S2 Fig. ATIII-activity risk groups correlate with ISGLS grades for LD and Dindo et al. grades for morbidity. [/fig] [table] Table 1: Patients demographics. [/table] [table] Table 2: Multivariable analysis for liver dysfunction and 90 days mortality.A. Multivariable analysis for liver dysfunction in the entire collective. [/table] [table] Table 3: Detail on postoperative thromboembolic incidences. https://doi.org/10.1371/journal.pone.0175359.t003 [/table]
Evaluation of teaching effect of first-aid comprehensive simulation-based education in clinical medical students [bib_ref] A qualitative analysis of statements on motivation of applicants for medical school, Wouters [/bib_ref] [bib_ref] Basic science right, not basic science lite: medical education at a crossroad, Fincher [/bib_ref] [bib_ref] Standardized patients: a promising tool for health education and health promotion, Weaver [/bib_ref] ## Figure The general flowchart of the whole research. SBE, simulation-based education. # Methods ## An ## Participants The full-time medical students in Grade 2, 3, and 4 of clinical medicine in Shantou University Medical College were eligible for admission to the study. The total number of grade 2 students in Shenzhen class is 35. Excluding those who have received first aid (such as cardiopulmonary resuscitation, endotracheal intubation, and electric defibrillation), 30 students are enrolled. There are a total of 34 students in Grade 3 Shenzhen class, 4 students who have received first aid training were excluded. There are 35 students in Shenzhen class of grade 4. Excluding those who have received first aid training, 30 students are enrolled. The study was undertaken from September 1 to December 31 in 2021. The written consent was obtained prior to the commencement of the scenario. The Ethics Approval was obtained from the Ethics Committee of the third Affiliated Hospital of Shenzhen University. ## Study procedure The medical students in Grade 2, 3, and 4 received different situational SBE, respectively. The design and implementation of the first aid SBE for each grade were carried out by the teachers from the Department of Critical Care Medicine of the Third Affiliated Hospital of Shenzhen University. For the 2nd-year medical students, the SBE is a single skill module which included cardiopulmonary resuscitation, endotracheal intubation, and electric defibrillation training. For the 3rd-year medical students, the SBE is a single subject module that includes cardiovascular and respiratory system training. For the 4th-year medical students, the SBE is the integrated multidisciplinary module that combined first-aid skills, clinical thinking, and teamwork training. The study procedure was listed in . The mannequins used in SBE are realistic, anatomically correct, and computer-driven, which can simulate the physiological reactions of real patients. Each SBE was carried out within 4 h, with 30 students in each grade. The student-teacher ratio is 5:1. ## Outcome measures The primary outcome measured in this research project was expert evaluation and peer evaluation. The expert evaluation was performed through the expert evaluation table. Peer evaluation was performed through the peer evaluation form. The students' satisfaction with SBE was the secondary outcome of this project and was measured with training feedback forms. The questionnaire was measured on a 5-point Likert scale ranging from 0 (Not at all) to 4 (Totally). In the first 5 min of each class, all students who participated in these classes were required to take a pre-training test, and the expert evaluation form and peer evaluation form were required to complete. All of these forms were collected before the beginning of each training. At the end of the training courses, the students were required to take a post-training test and the same two forms, together with the students' own learning feedback forms. All questionnaires were issued in paper forms. # Statistical analysis All analyses were performed by using SPSS version 22 (IBM Inc., New York, United States). All statistical tests were 2sided, and p-value < 0.05 indicated statistical significance. An independent t test was performed to compare the continuous variables of the normal distribution, and a Mann-Whitney U test was performed to compare the continuous variables of skew distribution. The scores were expressed as means plus or minus standard deviations (SD). # Results For the cohort of 90 medical students enrolled in the study, none was excluded or declined to participate. Questionnaire response rates for each of the three SBE scenarios and learning reactionnaire are 100%. In all of the single skill SBE cardiopulmonary resuscitation, endotracheal intubation, electric defibrillation, the single subject SBE (cardiovascular, respiratory), or the comprehensive disciplines SBE, the scores in the expert evaluation and peer assessment after the training were significantly higher than before the training, and the differences were statistically significant (p < 0.05, see . The highest scores pre-and post-test were in the single-skill SBE, and the lowest scores preand post-test were in the comprehensive subject SBE. There was a 100% response rate to the learning reactionnaire. The results indicated that 93.3% of the students scored ≥ 3 (A lot), and 100, 96.7, 96.7, 96.7, 100, and 100%, of the students scored ≥ 2 (Moderately) (see , indicating that the medical students had a positive feedback on the training effect of SBE. A total of 90 students from the SBE group provided their complete feedback on the SBE . For this course, 95.5% of the students were satisfied with our SBE and more than 80% have a good evaluation (evaluation of the necessity, 95.5%; # Discussion Medical education is rapidly changing, influenced by many factors including the changing healthcare environment, the changing role of the physician, altered societal expectations, rapidly changing medical science, and the diversity of pedagogical techniques [bib_ref] Using technology to meet the challenges of medical education, Guze [/bib_ref]. The traditional teaching method is faced with many problems such as fewer clinical patients and uncooperative patients; compared with traditional teaching methods, the advantages of simulation-based education are step-by-step learning, a "hands-on learning experience and the opportunities for repetition until proficiency is reached" [bib_ref] Current concepts in simulation-based trauma education, Cherry [/bib_ref] [bib_ref] Simulation in healthcare education: a best evidence practical guide. AMEE Guide No..., Mcgaghie [/bib_ref]. At present, the first-aid training for clinical 2-4 grade students in medical colleges in China is only in the traditional mode of theoretical teaching by teachers and skills training on models. This teaching method makes medical students lack of clinical thinking, team cooperation, and humanistic concepts. Without the training of various clinical situations, medical students, even after such training, often do not know what to do when they meet real patients in clinical practice, especially in critically ill patients (14). In real situations, with the development of society, the enhancement of the legal awareness of the whole people and the clear right to know, consent and privacy of patients, as the subject of traditional teaching, patients have the right to refuse to be the object of teaching, especially the examination of some private parts. Most patients refuse to cooperate with the teaching (15). This makes clinical teaching often into an awkward situation. Moreover, the use of real patients in clinical teaching may lead to medical disputes, so that clinical teaching hospitals often sacrifice teaching, medical students should master the various clinical skills which cannot be properly trained (14). According to China's "Medical Practitioners Law", medical students can only be legally engaged in medical practice after obtaining a medical qualification and at the side of patients and their families, especially critically ill patients, they do not cooperate with clinical teaching and refuse to be managed by interns [bib_ref] Current concepts in simulation-based trauma education, Cherry [/bib_ref]. At the side of clinical teachers, they do not arrange interns to deal with critically ill patients due to concerns about medical disputes. In the present course of medical students' skill training, the attention is only paid to the cultivation of composition skills, without systematic logical reasoning, multidisciplinary cooperation, and development of emergency management ability to deal with critically ill patients. In order to solve these problems, it is very urgent to construct first-aid simulation scenarios and conduct comprehensive SBE on this basis, The prevailing view is that SBE is superior to traditional teaching methods [bib_ref] Debriefing 101: training faculty to promote learning in simulation-based training, Paige [/bib_ref] [bib_ref] The Importance of debriefing in simulation-based learning: comparison between debriefing and no..., Ryoo [/bib_ref]. In medical education, the traditional education model is generally that students listen to their teacher, and then learn knowledge through their own understanding or observation. The traditional education model fails to provide simulated learning opportunities that affect their transition from theory to practice (19). SBE is also an increasingly important educational strategy that plays an important role in improving patient safety. SBE trainees can improve professionally by learning from their mistakes, which will help them avoid the same mistakes in real life. The main motivation for this study was to evaluate the effectiveness of SBE in training undergraduate students. A study with a sample size of 1,178 students suggested that SBE could have a positive impact on college education. Students in the SBE group had more positive communication with classmates and teachers, which could explain their great improvement in humanistic care and doctor-patient communication [bib_ref] Exploration of simulation-based medical education for undergraduate students, Wang [/bib_ref]. The purpose of the simulation is to imitate real patients, clinical tasks, and the real environment provided by medical services. It can provide feedback after simulation, with repetitive practice habits, course integration, and appropriate difficulty level. The researchers point it out that in order to achieve proficiency, students firstly need to acquire the component skills, then practice to integrating them, and proceed to know when to apply the skills they have learned (21). The significance of simulation teaching lies not only in the application of simulated mannequins, but also in the integration of clinical situations. It is very important to integrate the clinical context into the teaching scenarios. First of all, the single skill training teaches only component skill, resulting in students' deficiency of clinical thinking. Secondly, without the experience of simulated scenarios, medical students often do not know how to deal with real clinical situations. Thirdly, the integration of clinical situation is helpful to train the medical students' teamwork, humanistic spirit, and to lead students to systematically integrate the knowledge and skills they have learned, thus helping them to reach the level of proficiency [bib_ref] Does simulation-based medical education with deliberate practice yield better results than traditional..., Seam [/bib_ref]. Therefore, we set up three levels of situation simulation for medical students in grades 2, 3, and 4, and evaluate the teaching effect of first-aid comprehensive simulation-based education in these clinical medical students. Our study showed that whether in the single skill SBE [cardiopulmonary resuscitation (CPR), endotracheal intubation, electric defibrillation], the single subject SBE (cardiovascular, respiratory, or the comprehensive disciplines SBE, the scores in the expert evaluation and peer assessment after training were significantly higher than scores before training, and the differences were statistically significant (see . Our study is consistent with the reports of Issenberg SB et al. Our results also show that while the integrated subject SBE scored lowest in pre-and post-school tests, there was a significant increase in scores. Our integrated clinical situations are modified according to real cases by clinicians, which can simulate the clinical situations to the greatest extent. Not only can we provide medical students with very realistic clinical situations, but also the training is very safe and effective. Medical students often lack confidence in handling common clinical emergencies. Morris MC et al. (25) designed a new sub-module to incorporate high-fidelity simulation into the undergraduate medical curriculum and found that students showed a very positive attitude toward this new teaching method, especially in integrating previously learned knowledge and skills, indicating that simulation-based teaching is feasible and effective in the undergraduate setting. Among the 2nd-year medical students' single skill training, the skill of electrical defibrillation increased the most after training, and the score of endotracheal intubation increased the least after training. This may be due to the relatively few training steps for electric defibrillation and the complexity steps for endotracheal intubation. Therefore, we need to increase theoretical learning of endotracheal intubation and increase the number of trainings to better master this clinical skill. Simulation-based medical education is a complex service intervention that needs to be planned and practiced with attention to organizational contexts. In our study, we arranged an adequate teaching staff for intensive training and timely feedback (the student-teacher ratio of 5:1), adequate time for repetitive practice (each SBE was carried out within 4 h), curriculum design and integration from real cases by clinicians, realistic computer-driven mannequins to ensure simulation fidelity, providing different difficult level of SBE to different grades of students, and pre-and post-tests for outcome measurement. These factors are consistent with the features and best practices of SBE that is convinced to be used in medical simulation technology to maximum educational benefits (27). Other studies have also shown that medical simulation teaching can provide opportunities and conditions for medical students to contact the clinical practice in the early stage of school, which can improve medical students' clinical skills, operation ability, bedside comprehensive diagnostic thinking ability, and is beneficial to the cultivation of students' professional ethics and code of conduct [bib_ref] The standardized live patient and mechanical patient models -their roles in trauma..., Ali [/bib_ref]. Simulation teaching advocates medical teaching in a way that is as close to the real clinical environment . /fpubh. . as possible and more in line with medical ethics, and uses all simulated hand segments to create simulated patients, simulated scenes, simulated classrooms for various skills training and assessment, simulated wards, simulated operating rooms, and other software and hardware conditions (13). As an effective supplement to theoretical teaching and clinical practice, simulation teaching has changed the traditional teaching model, provided a safe teaching environment, cultivated agile and correct clinical thinking, and reduced the occurrence of medical accidents and disputes in clinical practices (30). Our study has limitations. First, only 30 medical students of each grade were included. More participants are needed to further confirm the effectiveness of the first-aid-integrated comprehensive SBE. Second, although improvements were observed in our study post-training when compared with pre-training; there is no comparison to standard educational methods. Thirdly, this research was only based on a single center. Only grade 2-4 medical students were included, so the conclusion cannot be generalized to other populations. Finally, the participants are all from the Shantou University Medical College and may not represent students from other medical colleges. # Conclusion The first-aid comprehensive simulation-based education in grade 2-4 clinical medical students, basing on timely feedback, repetitive practice, curriculum integration, simulation fidelity, and outcome measurement are effective in improving the students' proficiency in managing the real emergencies. # Data availability statement The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. # Ethics statement The studies involving human participants were reviewed and approved by Shenzhen Luohu People's Hospital. The patients/participants provided their written informed consent to participate in this study. # Author contributions MP and NS prepared the tables and drafted the manuscript. RH, HG, FC, WZho, WZha, JZ, and ZY reviewed the manuscript for its intellectual content. WC were responsible for revising the manuscript. All authors have read and approved the final manuscript. # Funding This study was supported by the clinical teaching reform research project from the Education Office of Guangdong Province in 2019 (2019 JD115). This article is a summary of the research project. . /fpubh. . [table] TABLE The feedback: of students in the SBE group. [/table]
Septum Pellucidum Chronic Encapsulated Hematoma With Osseous Metaplasia Mimicking Recurrent Astrocytoma and Shunt-Related Foreign Body Granuloma # Introduction Chronic encapsulated intracerebral hematoma (CEIH) is a well-known complication of spontaneous intraparenchymal hemorrhage (ICH) characterized by the slow enlargement of the hematoma due to recurrent micro-hemorrhages within its capsule. The frequency of CEIH after spontaneous ICH was found to be 4.9% in a recent study where the presence of a radiological "layer sign" surrounding the hematoma (as a sign of recurrent bleeding) was found to be predictive of progressive hematoma expansion. In patients with no clinical history of symptomatic spontaneous hemorrhage, vascular malformations, previous radiation therapy, or radiosurgery, or when other intracranial conditions are present (as it was in our patient), the diagnosis may be challenging. Midline structures, such as the corpus callosum and the septum pellucidum, are uncommon sites for spontaneous ICH and therefore not the expected anatomical location for the formation of an CEIH. We present a very rare case of chronic encapsulated hematoma involving the septum pellucidum and the foramen of Monro that by location, radiological appearance, and clinical history was mimicking a recurrent astrocytoma or a shunt-related foreign body granuloma. To the best of our knowledge, no other cases of CEIH involving the septum pellucidum and the foramen of Monro have been previously reported in the literature. ## Case presentation A young adult presented to our evaluation after an accidental fall with head injury and brief loss of consciousness. He had a history of cognitive disability with short-term memory deficit since early childhood when he was operated for a resection of a hemispheric frontal juvenile pilocytic astrocytoma (JPA), as per family report. As a child, he also sustained a severe head injury in a motor vehicle accident (MVA) that led to a posttraumatic hydrocephalus (HCP) that was treated with the placement of a right frontal ventriculoperitoneal (VP) shunt. After a few years, the patient underwent a shunt revision for recurrent HCP where the extracranial portion of the old shunt was tied and disconnected and a new right parietal shunt was placed. The patient had no further follow-ups with the neurosurgery service, and his cognitive deficits remained stable, with stable mild gait ataxia/instability and occasional urinary urgency. At ER evaluation, a CT of the head and MRI of the brain revealed the presence of a heterogeneous solid mass involving the region of the right foramen of Monro and septum pellucidum with a large right frontal cystic component. The solid and deep mass had only minimal faint enhancement after gadolinium injection, and there were no peripheral enhancement or HCP. There was no restricted diffusion on DWI (diffusion-weighted imaging) and unenhanced T1weighted images, but was some increased signal intensity consistent with small areas of hemorrhage. The mass was encasing the tip of a shunt catheter, whereas another ventricular catheter was ending within the right lateral ventricle. These findings were consistent with the patient's history of VP shunt placement and revision. The patient had no history of recurrent headaches, loss of consciousness, and no progressive symptoms as per family members' report. Aside from the MVA, no other recurrent head traumas were reported. In light of the clinical and radiological findings, a differential diagnosis comprehensive of foreign body granuloma and recurrent JPA was raised. The patient (with the support of his family) elected to undergo a right frontal craniotomy for resection of the mass, decompression, and diagnosis, which was uneventful. He recovered well, his gait improved, and he was able to resume some work activities a few weeks after the surgery. At the time of the surgery and after decompressing the frontal cyst, a solid mass consistent with an organizing hematoma was found and removed from the surrounding gliotic parenchyma. There were no feeders to the mass, and an old frontal flanged, spring reinforced, Portnoy ventricular catheter was found at the center of the organizing hematoma. Pathology examination revealed a mass tightly adherent to the Portnoy shunt catheter. The lesion wall had multiple calcified foci with central hemorrhagic necrotic friable material. Histological sections reveal a dense fibrous hyalinized capsule with osseous metaplasia. Various stages of organizing hematoma were present within the mass. There were no foreign body giant cells or granulomas to suggest a foreign body granuloma. Surrounding this encapsulated hematoma, there was non-neoplastic brain tissue with reactive gliosis and hemosiderin. No evidence of residual or additional tumor was seen in the entirely sampled tissue. The findings were consistent with the final diagnosis of CEIH with osseous metaplasia. # Discussion CEIH is a rare type of intracerebral hematoma first described by Hirsh et al. and characterized by the presence of a fibrotic capsule surrounding an organizing hematoma. CEIH is a wellknown complication of spontaneous ICH and its formation is thought to be secondary to recurrent episodes of micro-hemorrhages within its capsule, which lead to hematoma growth and the possible onset of slowly progressing neurological symptoms. Radiological diagnosis with CT and MRI scans is not always straightforward, as multiple hemorrhages in different stages of evolution can render every case peculiar; therefore the use of thallium-201 singlephoton emission CT has been suggested during the preoperative diagnosis. CEIH formation has been described as a late sequela after stereotactic radiosurgery for arteriovenous malformations (AVMs), with activation of vascular endothelial growth factor pathways that leads to hematoma capsule neovascularization, subsequent recurrent mural bleedings, and progressive hematoma enlargement. CEIHs have been reported to involve the hemispheric convexity, the basal ganglia, and the cerebellum. Although they are more commonly seen in adults, pediatric cases have also been described. These hematomas can be clinically indolent and paucisymptomatic or present with slowly progressing neurological symptoms due to recurrent bleeding within the hematoma capsule. Some patients may also present with a history of epilepsy or intractable seizures. On pathology examination, the hematomas appear well capsulated with the capsule made of an outer fibro-collagenous layer and an inner granulation layer with high concentrations of vascular endothelial growth factor. Chronic encapsulated hematomas have been reported in association with vascular lesions, such as cavernous malformations and ruptured AVMs, and have been recognized as a late complication of radiosurgery treatment when the AVMs were not completely obliterated. They may also mimic hemorrhagic primary and secondary brain tumors, and, in the presented case, the hematoma surrounding an old ventricular flanged catheter raised the suspicion for the presence of a shunt-related foreign body granuloma. The case presented is original in that the chronic encapsulated hematoma developed years after a resection of a low-grade glioma (frontal JPA), was strictly associated with a first-generation retained nonfunctioning ventricular catheter, and demonstrated osseous metaplasia of the capsule. The anatomical location of the pathology was also very uncommon and not previously reported. The presence of immature bone formation (osseous metaplasia) within the hematoma capsule has been previously described in subdural, and rarely interdural, hematomasand was a distinctive and rare feature on the specimen reported here. The differential clinical and radiological diagnosis included recurrent low-grade tumor and foreign body granuloma. Pilocytic astrocytomas are low-grade gliomas most commonly seen in childhood and can be cured with complete surgical resection. In adults, they can present as adult pilocytic astrocytomas or reflect a recurrent tumor after an incomplete surgical resection at an early age. Pilocytic astrocytoma demonstrates a myxoid background and numerous monomorphous bipolar cells, whose processes often radiate from prominent blood vessels. The brain tissue in this specimen did not demonstrate myxoid change or enough increased cellularity and vascularity for a diagnosis of recurrent pilocytic astrocytoma to be considered. Foreign body granulomas have been reported as a complication of CSF shunting procedures, both intracranially and in the spine, with the granuloma formation elicited as a reaction to the presence of the catheter material. The old Portnoy flanged ventricular catheter was spring reinforced, therefore adding to the possibility of an allergic chronic reaction to the shunt placement. Granulomas around a foreign body consist of inflammatory cells infiltrate, with giant cells, lymphocytes, plasma cells, epithelioid cells, and perivascular cuffing. All these findings were absent in the presented case. In our case, it is unclear if the initial ICH that led to the formation of the CEIH was related to an intraoperative bleeding in the region of the foramen of Monro/choroid plexus at the time of the shunt revision, was secondary to a minor trauma, or was possibly related to a rare hemorrhage within a residual JPA. # Conclusions CEIHs involving the septum pellucidum and the foramen of Monro are very rare, and, when associated with the presence of a ventricular shunt, may mimic the appearance of a shuntrelated foreign body granuloma. Although radiological preoperative examinations can facilitate narrowing the differential diagnosis, surgical resection of the encapsulated hematoma is both diagnostic and curative.
Determinants of relapse periodicity in Plasmodium vivax malaria Plasmodium vivax is a major cause of febrile illness in endemic areas of Asia, Central and South America, and the horn of Africa. Plasmodium vivax infections are characterized by relapses of malaria arising from persistent liver stages of the parasite (hypnozoites) which can be prevented only by 8-aminoquinoline anti-malarials. Tropical P. vivax relapses at three week intervals if rapidly eliminated anti-malarials are given for treatment, whereas in temperate regions and parts of the sub-tropics P. vivax infections are characterized either by a long incubation or a long-latency period between illness and relapse -in both cases approximating 8-10 months. The epidemiology of the different relapse phenotypes has not been defined adequately despite obvious relevance to malaria control and elimination. The number of sporozoites inoculated by the anopheline mosquito is an important determinant of both the timing and the number of relapses. The intervals between relapses display a remarkable periodicity which has not been explained. Evidence is presented that the proportion of patients who have successive relapses is relatively constant and that the factor which activates hypnozoites and leads to regular interval relapse in vivax malaria is the systemic febrile illness itself. It is proposed that in endemic areas a large proportion of the population harbours latent hypnozoites which can be activated by a systemic illness such as vivax or falciparum malaria. This explains the high rates of vivax following falciparum malaria, the high proportion of heterologous genotypes in relapses, the higher rates of relapse in people living in endemic areas compared with artificial infection studies, and, by facilitating recombination between different genotypes, contributes to P. vivax genetic diversity particularly in low transmission settings. Long-latency P. vivax phenotypes may be more widespread and more prevalent than currently thought. These observations have important implications for the assessment of radical treatment efficacy and for malaria control and elimination. # Introduction In endemic areas of Asia, Oceania, Central and South America, and in the horn of Africa Plasmodium vivax malaria is a major cause of morbidity. It is an important contributor to early pregnancy loss and reduced birth weight which increases mortality in infancy . Plasmodium vivax is a sophisticated and resilient malaria parasite which was once prevalent over much of the inhabited world. It has receded from North America, Europe and Russia, but in the tropics vivax malaria remains a major cause of childhood illness. In most endemic areas, P. vivax cohabits with Plasmodium falciparum. Mixed infections with the two species are common. P. vivax is more difficult to control and eliminate than P. falciparum because of its tendency to relapse after resolution of the primary infection. In endemic areas relapse of vivax malaria is a major cause of malaria in young children, and an important source of malaria transmission. Relapse also occurs in Plasmodium ovale infections and in several of the simian malarias, notably Plasmodium cynomolgi, which has often been used as an animal model of vivax malaria. The factors which control relapse and determine their remarkable periodicity are not known. ## History The history of clinical research on vivax malaria contains a wealth of valuable information that is not widely known or recognised. Most studies were conducted over fifty years ago and are not readily accessible to the modern reader. Indeed more may have been forgotten than has been learned about vivax malaria in recent years. The tendency of malaria infections to recur was well known since Roman times. From the 1630s onwards a specific treatment (Cinchona bark) was available for agues (although for most of those infected it was unaffordable). This treatment was often followed by frequent relapses of the periodic fever, and so opinions differed widely on its efficacy. Following Laveran's discovery of the blood parasite that caused malaria in 1880 [bib_ref] Nouveau parasite du sang, Laveran [/bib_ref] , understanding of malaria epidemiology, pathology, and treatment was placed on a rational basis. In 1885, Golgi in Pavia distinguished the parasites responsible for tertian and quartan fevers [bib_ref] Sulle febbri malariche estivo-autumnali di Roma, Golgi [/bib_ref]. In 1890, P. vivax was identified as a separate species by Grassi and Feletti , although debate continued into the early 1920s as to whether there were indeed separate human malaria species, or just one single polymorphic species . Studies of the time often tended to consider the responses of the benign (vivax) and malignant (falciparum) tertian malarias together. The Dutch physician Pel is generally credited with the first postulation of an exoerythrocytic stage of malaria in 1886 . In 1897, the American physician WS Thayer gave a very clear description of a long-latency relapse of malaria 21 months after the initial attack in a physician who had probably not been re-exposed between the two events . Thayer and Bignami (in Italy) both surmised that relapses of malaria resulted from a "spore" deposited in the internal viscera which remained inert "only to be set free as a result of some insult, the nature of which is still not appreciable to us" . Self-experimentation then provided remarkably accurate descriptions of long latencies in vivax malaria. In 1900 in London Sir Patrick Manson was investigating the mosquito transmission theory proposed by his protégé Ronald Ross. Upon request Bignami and Bastianelli kindly provided him with P. vivax infected anopheline mosquitoes that had been fed on malaria patients in the Ospedale Spirito Santo in Rome. The mosquitoes were taken to the British Embassy in Rome, and thereafter by the "Indian mail", and duly arrived in London 48 hours later. His son Patrick Thorburn Manson, and George Warren (a laboratory technician) both volunteered to be bitten. Acute attacks of malaria followed after a two-week incubation period, in September 1900. These were treated successfully with quinine [bib_ref] Experimental proof of the mosquito-malaria theory, Manson [/bib_ref]. On June 1st the following year, after a latent period of nine months, the younger Manson experienced a sudden onset of rigors. Relapse of his vivax malaria was confirmed by microscopy [bib_ref] Experimental malaria: recurrence after nine months, Manson [/bib_ref]. Meanwhile in India, at the end of 1900, Major CF Fearnside infected himself with mosquitoes which had fed on vivax malaria patients in the Madras jail. His primary attack of malaria began on January 14th, he suffered a relapse on , and a second relapse followed on November 11th apparently without the possibility of interim reinfection [bib_ref] Experimental inoculation of malaria with a relapse after eight months, Fearnside [/bib_ref]. These precise observations describing latencies of some eight to nine months were complemented by Korteweg's detailed and painstaking prospective epidemiological observations over more than two decades in the village of Wormeveer in The Netherlands. Celli had long surmised that the spring wave of benign tertian (P. vivax) malaria in Northern Italy resulted from relapsebut it was Korteweg who provided convincing evidence that the vivax malaria which emerged in the early summer had been acquired in the autumn of the previous year [bib_ref] Long-latency in Plasmodium vivax infections in a temperate zone, Winckel [/bib_ref] [bib_ref] The seasonal periodicity of malaria and the mechanism of the epidemic wave, Gill [/bib_ref]. The considerable vivax malaria experience of the First World War (in the Balkans, Mesopotamia, and the Jordan valley), the secondary cases in England which followed the year after the return of infected soldiers, studies in American soldiers serving in Panama and the Philippines, studies in British soldiers serving in India, studies in Japanese soldiers who had invaded China, and further epidemiological observations and individual case reports in Europe and the United States all pointed to a disease which could relapse within two months of stopping quinine treatment, but also tended to relapse some eight to ten months later . Until the 1920s it was common to recommend an eight-week course of quinine treatment for malaria. Despite this protracted treatment relapses were common, but whether these resulted from persistence of the blood stage infection (i.e. recrudescence), or derived from a latent tissue stage was unresolved. ## Early observations on relapse In 1913, Bignami proposed that relapses of malaria all derived from persistence of small numbers of parasites in the blood [bib_ref] Concerning the pathogenesis of relapses in malarial fevers, Bignami [/bib_ref]. Although this theory explained the late recurrences of Plasmodium malariae infections in man, it did not explain several features of P. vivax and P. ovale infections. A much clearer understanding of relapse in P. vivax malaria was to come from Julius Wagner-Jauregg's discovery that malaria could cure neurosyphilis. Between the 1920s and the 1950s thousands of patients confined in mental hospitals with neurosyphilis were treated with malaria (malaria therapy). General paralysis of the insane was then a uniformly lethal condition, and at least half the malaria therapy treated patents were improved and over one fifth were cured. It was a remarkable period, unique in the history of medicine, when, as Henry Dale put it, "Man was the experimental animal". The majority of the malaria therapy experience was with a relatively small number of parasite "strains" transmitted either by blood passage, or more usually by the bites of several infected anopheline mosquitoes. On the 8th September 1922 Professor Warrington Yorke and JWS MacFie inoculated blood from a patient with simple tertian malaria (P. vivax), which had been acquired in India, into a patient with neurosyphilis at the Whittingham Mental Hospital near Liverpool [bib_ref] Observations on malaria made during the treatment of general paralysis, Yorke [/bib_ref] [bib_ref] Further observations on malaria made during the treatment of general paralysis, Yorke [/bib_ref]. This "strain" was then used to infect multiple patients initially by blood passage, and later by mosquito transmitted infections. It soon became apparent that recurrences of P. vivax (and P.ovale) malaria followed a different temporal pattern to those of P. falciparum [bib_ref] Some general results of a study of induced malaria in England, James [/bib_ref] [bib_ref] Clinical and parasitological observations on induced malaria, James [/bib_ref] [bib_ref] Report on the first results of laboratory work on malaria in England, James [/bib_ref]. Recurrences of vivax malaria commonly occurred many months after apparently successful treatment of the primary infection. Furthermore whereas recurrence in blood transmitted vivax malaria could be prevented by curing the blood stage infection, recurrence in mosquito transmitted vivax malaria could not be [bib_ref] Observations on malaria made during the treatment of general paralysis, Yorke [/bib_ref] [bib_ref] Further observations on malaria made during the treatment of general paralysis, Yorke [/bib_ref] [bib_ref] Some general results of a study of induced malaria in England, James [/bib_ref]. This pointed to an exoerythrocytic stage of malaria, but its anatomical location remained elusive. The Dutch malariologists were able to show that their indigenous P. vivax could have a short incubation period if patients were bitten by a large number of infected mosquitoes, but, in a further example of courageous self-experimentation, they proved that bites by one or two infected mosquito were followed by vivax malaria 8 to 9 months later [bib_ref] Report on the first results of laboratory work on malaria in England, James [/bib_ref] [bib_ref] Pathology -Experimental malaria with protracted incubation, Schuffner [/bib_ref]. The Northern European and Russian "strains" of P. vivax therefore proved unsatisfactory for malaria therapy because blood passage was required to ensure an acute illness within weeks [bib_ref] Observations on malaria made during the treatment of general paralysis, Yorke [/bib_ref] [bib_ref] Further observations on malaria made during the treatment of general paralysis, Yorke [/bib_ref] [bib_ref] Some general results of a study of induced malaria in England, James [/bib_ref] [bib_ref] Clinical and parasitological observations on induced malaria, James [/bib_ref] [bib_ref] Report on the first results of laboratory work on malaria in England, James [/bib_ref] [bib_ref] Pathology -Experimental malaria with protracted incubation, Schuffner [/bib_ref] [bib_ref] Dates of onset of relapses and the duration of infection in induced..., Tiburskaja [/bib_ref] whereas mosquito infection, even with multiple infected anophelines, often failed to produce an early febrile illness. Importantly it was also noted in The Netherlands that relapse rates in naturally acquired infections were higher than with mosquito-transmitted malaria therapy with local "strains". The most widely used parasite used for malaria therapy in Europe A.atroparvus was a P. vivax "strain" isolated from an Indian merchant seaman whose last port of call had been Madagascar [bib_ref] Concerning the pathogenesis of relapses in malarial fevers, Bignami [/bib_ref]. The "Madagascar" strain of P. vivax was extensively investigated in England by James and later in several European centres . It reliably produced an acute infection with high fevers, sometimes relapsed again within a few weeks, and then relapsed again approximately eight months later. With serial passage through man and mosquito the Madagascar strain apparently became even more virulent [bib_ref] The Madagascar strain of Plasmodium vivax, Shute [/bib_ref]. James' terminology, which was adopted by several others, was different to that used today. A recurrence of malaria (any species) before eight weeks was termed a recrudescence, recurrence from 8-24 weeks was termed relapse, and return of malaria after 24 weeks was called a recurrence. The pattern observed by James in England, Swellengrebel in The Netherlands, and Ciuca in Romania with the Madagascar strain of P. vivax was very similar to patterns of the St Elizabeth and McCoy strains, of indigenous origin, used for malaria therapy in the United States [bib_ref] Pathology -Experimental malaria with protracted incubation, Schuffner [/bib_ref] [bib_ref] Dates of onset of relapses and the duration of infection in induced..., Tiburskaja [/bib_ref] [bib_ref] The Madagascar strain of Plasmodium vivax, Shute [/bib_ref] [bib_ref] On strains or races of the malaria parasites, Boyd [/bib_ref] [bib_ref] Studies on malaria with special reference to treatment. XV. Does the strain..., Sinton [/bib_ref] [bib_ref] A controlled technique for the employment of naturally induced malaria in the..., Boyd [/bib_ref] [bib_ref] Studies in human malaria. XVI. Results of massive sub-inoculation during latency from..., Cooper [/bib_ref] [bib_ref] On the duration of homologous immunity to Plasmodium vivax, Boyd [/bib_ref] [bib_ref] The influence of sporozoite dosage in vivax malaria, Boyd [/bib_ref]. These strains were all chosen because of their suitability for malaria therapy, so they probably represented the upper end of the spectrum of abilities to produce early infection and relapse. The European studies, investigations in India by Sinton and colleagues, and in Florida 28 weeks 7 weeks The temporal pattern of illness recurrence in patients with neurosyphilis artificially infected for malaria therapy with Plasmodium falciparum (87 patients) and the "Madagascar" strain of P. vivax (105 patients) studied by SP James and colleagues at the Horton Hospital, Epsom, England [bib_ref] Some general results of a study of induced malaria in England, James [/bib_ref] [bib_ref] Clinical and parasitological observations on induced malaria, James [/bib_ref] [bib_ref] Report on the first results of laboratory work on malaria in England, James [/bib_ref] between 1925 and 1930. The vivax relapses had a bimodal pattern with the majority having a long latent period (mode 28 weeks) before the relapse. by Boyd suggested that some "strains" relapsed after a few weeks while others had only a primary infection, and then exhibited the long-latency seen with the Madagascar and St Elizabeth strains [bib_ref] Some general results of a study of induced malaria in England, James [/bib_ref] [bib_ref] Clinical and parasitological observations on induced malaria, James [/bib_ref] [bib_ref] Report on the first results of laboratory work on malaria in England, James [/bib_ref] [bib_ref] Pathology -Experimental malaria with protracted incubation, Schuffner [/bib_ref] [bib_ref] Dates of onset of relapses and the duration of infection in induced..., Tiburskaja [/bib_ref] [bib_ref] The Madagascar strain of Plasmodium vivax, Shute [/bib_ref]. The distinction was often unclear as characterization of latency required reliable long-term follow-up, and the development of immunity with a febrile illness of many weeks duration became a significant confounder. As the object of malaria therapy was a prolonged high fever, the consequent long illness obscured early relapses. These single isolate (presumably single or highly related genotype) infections eventually resulted in solid immunity such that after several episodes of protracted fever reinfection with the same isolate was not possible [bib_ref] Clinical and parasitological observations on induced malaria, James [/bib_ref] [bib_ref] Report on the first results of laboratory work on malaria in England, James [/bib_ref] [bib_ref] Pathology -Experimental malaria with protracted incubation, Schuffner [/bib_ref] [bib_ref] Dates of onset of relapses and the duration of infection in induced..., Tiburskaja [/bib_ref] [bib_ref] The Madagascar strain of Plasmodium vivax, Shute [/bib_ref] [bib_ref] On strains or races of the malaria parasites, Boyd [/bib_ref] [bib_ref] On the duration of homologous immunity to Plasmodium vivax, Boyd [/bib_ref]. This acquired immunity suppressed later homologous relapses. Importantly for our current understanding of P. vivax epidemiology asymptomatic parasitaemia (and gametocytaemia) tended to persist for weeks as disease controlling immunity was acquired. Boyd and Kitchen, who studied the McCoy strain in Florida extensively, noted that relapse did not follow infections which had terminated spontaneously (indicating an effective immune response) or infections in which the primary illness lasted for ≥ 48 days [bib_ref] On strains or races of the malaria parasites, Boyd [/bib_ref] [bib_ref] On the duration of homologous immunity to Plasmodium vivax, Boyd [/bib_ref]. By contrast all agreed that if prompt anti-malarial treatment was Combined results of the preliminary mosquito infection studies of James and Shute in the Horton Hospital, Epsom, England and the self experimentation of Shüffner, Korteweg, Swellengrebel-de Graaf, Swellengrebel, de Buck and de Moor in The Netherlands as illustrated by Shüffner et al [bib_ref] Pathology -Experimental malaria with protracted incubation, Schuffner [/bib_ref]. This confirmed the 8 to 9 month interval from being bitten by one or two infected anopheline mosquitoes and developing vivax malaria White Malaria Journal 2011, 10:297 http://www.malariajournal.com/content/10/1/297 The family history between May 1925 and May 1933 of the "Madagascar strain" of Plasmodium vivax as used in malaria therapy at the Horton hospital [bib_ref] The Madagascar strain of Plasmodium vivax, Shute [/bib_ref]. The number within the circles refers to the number of patients infected with the "Madagascar strain" at each time and the number over the lines refers to the batch number of the infected mosquitoes. Overall 24,361 mosquitoes and 1739 patients were infected. given to terminate the infection then symptomatic relapses of vivax malaria were common. During the malaria therapy experience it became clear that both the incubation period and the number of relapses were determined by the numbers of sporozoites inoculated [bib_ref] Clinical and parasitological observations on induced malaria, James [/bib_ref] [bib_ref] Report on the first results of laboratory work on malaria in England, James [/bib_ref] [bib_ref] Pathology -Experimental malaria with protracted incubation, Schuffner [/bib_ref] [bib_ref] Dates of onset of relapses and the duration of infection in induced..., Tiburskaja [/bib_ref] [bib_ref] The influence of sporozoite dosage in vivax malaria, Boyd [/bib_ref]. The Dutch had shown first [bib_ref] Pathology -Experimental malaria with protracted incubation, Schuffner [/bib_ref] , and others later confirmed, that low sporozoite inocula often resulted in an extended incubation period of 7-10 months. The more sporozoites that were inoculated, the more likely was an early infection (incubation period two weeks), and the more relapses that followed -provided that prompt anti-malarial treatment (quinine) was given each time. Later clinical and experimental studies, reported after the Second World War, were to confirm these observations. In order to ensure that there was a short incubation period preceding the malaria illness malaria therapy infections were produced typically by the bites of 5-10 infected mosquitoes, and either no treatment or partial suppressive treatment was given. Previous theories that seasonal influences were important determinants of relapse were largely rejected as the intervals from primary illness to relapse in neurosyphilis patients were generally similar whichever month the infection started [bib_ref] Observations on malaria made during the treatment of general paralysis, Yorke [/bib_ref] [bib_ref] Further observations on malaria made during the treatment of general paralysis, Yorke [/bib_ref] [bib_ref] Some general results of a study of induced malaria in England, James [/bib_ref] [bib_ref] Clinical and parasitological observations on induced malaria, James [/bib_ref] [bib_ref] Report on the first results of laboratory work on malaria in England, James [/bib_ref]. Between the 1920s to the 1940s the long-latency infection was regarded as the "usual" P. vivax phenotype. Throughout the endemic areas of Europe vivax malaria peaked in the late spring and early summer (largely from inoculations the previous year) [bib_ref] Clinical and parasitological observations on induced malaria, James [/bib_ref]. In southern Europe there was often a bimodal pattern with a late summer peak of falciparum malaria [bib_ref] The seasonal periodicity of malaria and the mechanism of the epidemic wave, Gill [/bib_ref]. The epidemiological studies of malaria epidemics in Sind (now Pakistan) and Ceylon (Sri Lanka) followed a similar pattern [bib_ref] The seasonal periodicity of malaria and the mechanism of the epidemic wave, Gill [/bib_ref] [bib_ref] Clinical and parasitological observations on induced malaria, James [/bib_ref] [bib_ref] The study of a regional epidemic of malaria in Northern Sind, Covell [/bib_ref]. This suggested that long-latency P. vivax was also responsible for the peak of vivax malaria cases which occurred the year after the falciparum malaria epidemics in these two tropical areas (although other interpretations are also possible). During the Second World War observations on Allied soldiers fighting in North Africa, Italy, the Caucasus, and Greece, and further observations in Japanese occupation forces in China, all pointed clearly to long-latency P. vivax with similar illness patterns to the Madagascar and St Elizabeth strains [bib_ref] Induced and war malaria, Höring [/bib_ref] [bib_ref] Studies on imported malarias the parasitological pattern of relapsing Plasmodium vivax in..., Eyles [/bib_ref] [bib_ref] Office of the Surgeon General, Department of the Army, Mowrey [/bib_ref]. In contrast the soldiers on both Boyd's 10 year experience with mosquito transmitted P. vivax malaria therapy in 375 patients studied at the Florida State Hospital [bib_ref] Renewed clinical activity in vivax malaria, Boyd [/bib_ref]. Most infections were with the McCoy strain, and some were with other local "strains" which he considered to behave similarly. Infections which were allowed to continue until self termination did not relapse subsequently [bib_ref] On strains or races of the malaria parasites, Boyd [/bib_ref]. The median interval to relapse was approximately 9 months. Inset is the study of Coatney et al [bib_ref] Studies in human malaria. XVIII. The life pattern of sporozoite-induced St. Elizabeth..., Coatney [/bib_ref] describing 403 mosquito transmitted infections with the St Elizabeth strain of P. vivax. ## Onset of secondary attacks in weeks from inoculation sides fighting in the Indo-Burman and South Pacific campaigns encountered vivax malaria with a very different relapse pattern. Relapses were frequent and the relapse rate was very high -in some companies all soldiers were infected and all relapsed. Infections occurred at three week intervals if quinine was given, and seven week intervals following mepacrine treatment [bib_ref] Studies on imported malarias the parasitological pattern of relapsing Plasmodium vivax in..., Eyles [/bib_ref] [bib_ref] Office of the Surgeon General, Department of the Army, Mowrey [/bib_ref] [bib_ref] Clinical trials of anti-malarial drugs, Most [/bib_ref] [bib_ref] Natural course of Southwest Pacific malaria, Noe [/bib_ref] [bib_ref] Long term observation of Plasmodium vivax malaria, Bianco [/bib_ref]. Multiple relapses were very common and there was no evidence of long latency. The "type strain" for this tropical frequent relapse P. vivax phenotype was the "Chesson strain" isolated from a soldier of that name who had acquired the infection in New Guinea [bib_ref] The Chesson strain of Plasmodium vivax malaria; clinical aspects, Whorton [/bib_ref] [bib_ref] The Chesson strain of Plasmodium vivax malaria. I. Relationship between prepatent period,..., Craige [/bib_ref] [bib_ref] Relapses with Chesson strain Plasmodium vivax following treatment with chloroquine, Jeffery [/bib_ref]. In volunteers infected with the Chesson strain 80% of relapses occurred within 30 days of initial treatment with quinine. ## Discovery of the liver stages In 1902, three years before his death, the eminent protozoologist Fritz Schaudinn reported that he had observed direct infection of erythrocytes by sporozoites. There was therefore no need to postulate a tissue stage of malaria. By the 1930s this theory was largely discredited as others had tried and failed to reproduce the observations, and by then tissue stages of bird malarias had been demonstrated unequivocally. The existence of a tissue stage of the malaria life cycle in humans was considered sufficiently likely that the Malaria Commission of the League of Nations in 1933 suggested that the sporozoite in human malaria went on to divide in cells of the endothelial system as did Haemoproteus in birds. In 1937 James and Tate showed that the exoerythrocytic development of Plasmodium.gallinaceum took place in the brain capillaries of chickens [bib_ref] New knowledge of the life cycle of malaria parasites, James [/bib_ref]. After the Second World War, Fairley's brilliant work at the Cairns experimental station showed that sporozoites were cleared from the blood within one hour of mosquito feeding on volunteers, and in the case of P. vivax, parasites only returned to the blood one week later [bib_ref] Chemotherapeutic suppression and prophylaxis in malaria, Fairley [/bib_ref] [bib_ref] Sidelights on malaria in man obtained by subinoculation experiments, Fairley [/bib_ref]. Several researchers had noted earlier that in the latent or interrelapse period even transfusion of as much as 500 mL blood could not transmit P. vivax to a volunteer recipient, whereas if P. falciparum recurred -it could always be transmitted beforehand by blood transfusion. It was clear then that there was a pre-erythrocytic tissue stage which preceded the blood stage infection, and also that subsequent relapses originated from an exoerythrocytic stage -but where was it? SP James, the eminent British malariologist, was so convinced that there must be an exoerythrocytic stage in the primate malarias that he told the young PCC Garnham to stay in East Africa until he had found it -and so he did! In 1947, Garnham identified the pre-erythrocytic development of Hepatocystis (then Plasmodium) kochi in the hepatocytes of African monkeys [bib_ref] Exoerythrocytic schizogony in Plasmodium kochi Laveran. A preliminary note, Garnham [/bib_ref]. Shortly afterwards in England definitive studies by Shortt and Garnham identified the site of pre-erythrocytic development in primate malarias as the liver, first in P.cynomolgi infected Rhesus monkeys [bib_ref] Pre-erythrocytic stage in mammalian malaria parasites, Shortt [/bib_ref] [bib_ref] Demonstration of a persisting exo-erythrocytic cycle in Plasmodium cynomolgi and its bearing..., Shortt [/bib_ref] , and then in a heroic experiment with P. vivax in a very heavily infected volunteer who underwent open liver biopsy [bib_ref] The pre-erythrocytic stage of human malaria, Plasmodium vivax, Shortt [/bib_ref] [bib_ref] The pre-erythrocytic stage of mammalian malaria, Shortt [/bib_ref] [bib_ref] Relapses and latency in malaria, Garnham [/bib_ref]. This classic work still did not identify the persistent stage, although later primate work suggested that relapses might arise from arrested development of hepatic pre-erythrocytic schizonts [bib_ref] Relapses and latency in malaria, Garnham [/bib_ref] [bib_ref] The pre-erythrocytic development of Plasmodium cynomolgi and Plasmodium vivax, Shortt [/bib_ref]. Forty years later Krotoski, working with Garnham and colleagues at Imperial College, finally identified the dormant stages or "hypnozoites" of P.cynomolgi and P. vivax responsible for relapses in the liver [bib_ref] Relapses in primate malaria: Discovery of two populations of exoerythrocytic stages. Preliminary..., Krotoski [/bib_ref] [bib_ref] Demonstration of hypnozoites in sporozoite-transmitted Plasmodium vivax infection, Krotoski [/bib_ref] [bib_ref] Swellengrebel lecture. Hypnozoites and 'relapses' in Plasmodium vivax and in vivax-like malaria, Garnham [/bib_ref] [bib_ref] The life-cycle of primate malaria parasites, Bray [/bib_ref]. Although parasite bodies, which are probably hypnozoites, have since been demonstrated in liver cell cultures [bib_ref] Towards an in vitro model of Plasmodium hypnozoites suitable for drug discovery, Dembele [/bib_ref] remarkably little is known of their biology. The term relapse is now used specifically to describe recurrences of malaria derived from persistent liver stages of the parasite (hypnozoites) whereas recrudescence refers to a recurrence of malaria derived from persistence of the blood stage infection. The relapse arises after the "awakening" of these hypnozoites and the subsequent intrahepatic schizogony followed by blood stage multiplication. The question remained unanswered as to how the hypnozoites were woken, and what determined their remarkable periodicity. ## Phenotypic variation in p. vivax Today there is a tendency to regard all P. vivax together as a single homogenous species, but the human malaria therapy and volunteer studies showed that there was substantial phenotypic variation between Plasmodium vivax "strains". There has been corresponding taxonomic debate over how these "sub-species" should be defined [fig_ref] Figure 6: Diagram of the different P [/fig_ref]. Studies conducted over fifty years ago indicated that incubation periods, numbers of merozoites per blood schizont, antigenic relationships, intrinsic drug susceptibility, virulence, and relapse intervals all differed between "strains". At that time the Plasmodium vivax with infection phenotypes similar to those of the "Madagascar" and "St Elizabeth" strains which were prevalent in the United States and Southern Europe were considered the "typical" P. vivax infections [bib_ref] Some general results of a study of induced malaria in England, James [/bib_ref] [bib_ref] Clinical and parasitological observations on induced malaria, James [/bib_ref] [bib_ref] On strains or races of the malaria parasites, Boyd [/bib_ref] [bib_ref] Studies in human malaria. XVIII. The life pattern of sporozoite-induced St. Elizabeth..., Coatney [/bib_ref]. A primary illness followed approximately two weeks after mosquito inoculation, and, although relapse could follow some three weeks later, there was often a 7 to 10 month interval before a subsequent relapse. Sometimes the latency could be as long as one year, and there were well documented, but apparently unusual, cases reported of latencies greater than two years. Further north in the Netherlands, Northern Germany, Scandinavia, Finland and Central Russia the long incubation phenotypes were prevalent (P. vivax hibernans) in which the primary infection occurred 8 to 10 months after inoculation [bib_ref] Long-latency in Plasmodium vivax infections in a temperate zone, Winckel [/bib_ref] [bib_ref] The seasonal periodicity of malaria and the mechanism of the epidemic wave, Gill [/bib_ref] [bib_ref] Further observations on malaria made during the treatment of general paralysis, Yorke [/bib_ref] [bib_ref] Some general results of a study of induced malaria in England, James [/bib_ref] [bib_ref] Pathology -Experimental malaria with protracted incubation, Schuffner [/bib_ref] [bib_ref] Dates of onset of relapses and the duration of infection in induced..., Tiburskaja [/bib_ref] [bib_ref] Features specific to the Moscow strain of P. vivax, Tiburskaya [/bib_ref] [bib_ref] Explanation for the difference of incubation type and features of alteration of..., Moshkovsky [/bib_ref] [bib_ref] Endemic malaria: an 'indoor' disease in northern Europe. Historical data analysed, Huldén [/bib_ref]. It seemed that the proportion of infections which had a short incubation period (circa two weeks) declined steadily with increasing latitude (and shorter summer mosquito breeding seasons). In artificial infection studies latency was independent of season [bib_ref] Observations on malaria made during the treatment of general paralysis, Yorke [/bib_ref] [bib_ref] Further observations on malaria made during the treatment of general paralysis, Yorke [/bib_ref] [bib_ref] Some general results of a study of induced malaria in England, James [/bib_ref] [bib_ref] Clinical and parasitological observations on induced malaria, James [/bib_ref] [bib_ref] Report on the first results of laboratory work on malaria in England, James [/bib_ref] [bib_ref] Studies in human malaria. XVIII. The life pattern of sporozoite-induced St. Elizabeth..., Coatney [/bib_ref]. In the past twenty years infections with intermediate relapse characteristics have been reported but not well described from sub-tropical areas, although as will be explained later, there may be other explanations for relapses which emerge two to eight months after sporozoite inoculation. There was one very important and puzzling feature of the long-latency P. vivax infections which still requires explanation in any theory which seeks to explain relapse periodicities; once the relapse had occurred (after a latency of 7-10 months) subsequent relapses would then usually occur with intervals of approximately 3 to 4 weeks following quinine -or 6 to 8 weeks if chloroquine was given for treatment [bib_ref] The seasonal periodicity of malaria and the mechanism of the epidemic wave, Gill [/bib_ref] [bib_ref] Further observations on malaria made during the treatment of general paralysis, Yorke [/bib_ref] [bib_ref] Studies in human malaria. XVIII. The life pattern of sporozoite-induced St. Elizabeth..., Coatney [/bib_ref] [bib_ref] Features specific to the Moscow strain of P. vivax, Tiburskaya [/bib_ref] [bib_ref] Explanation for the difference of incubation type and features of alteration of..., Moshkovsky [/bib_ref] [fig_ref] Figure 7: Schematic diagram by Hankey et al[79]of relapse patterns following Korean vivax malaria [/fig_ref]. Thus after the long latent period the subsequent intervals were similar to the incubation period in temperate infections with early relapses, and the inter-relapse intervals in the tropical frequent relapse "Chesson" phenotype vivax malaria [bib_ref] Studies in human malaria. XXX. A summary of 204 sporozoite-induced infections with..., Coatney [/bib_ref]. In contrast infections from some parts of Russia were documented which had a second long-latency after the first relapse(s). Following the Second World War artificial infection studies were conducted in several locations to study different anti-malarial treatment regimens. The administration of an effective schizontocidal drug allowed better definition of relapse periodicity than the malaria therapy studies (where the objective had been to produce a sustained high fever). In the more prevalent tropical strains of P. vivax treated in soldiers fighting in the Indo-Burmese and South-West Pacific campaigns in the Second World War, the relapses were documented to occur at intervals of 3 to 4 weeks (as in the Chesson strain) [bib_ref] Studies on imported malarias the parasitological pattern of relapsing Plasmodium vivax in..., Eyles [/bib_ref] [bib_ref] Office of the Surgeon General, Department of the Army, Mowrey [/bib_ref] [bib_ref] Clinical trials of anti-malarial drugs, Most [/bib_ref] [bib_ref] Natural course of Southwest Pacific malaria, Noe [/bib_ref] [bib_ref] Long term observation of Plasmodium vivax malaria, Bianco [/bib_ref] [bib_ref] The Chesson strain of Plasmodium vivax malaria; clinical aspects, Whorton [/bib_ref] [bib_ref] The Chesson strain of Plasmodium vivax malaria. I. Relationship between prepatent period,..., Craige [/bib_ref] [bib_ref] Relapses with Chesson strain Plasmodium vivax following treatment with chloroquine, Jeffery [/bib_ref] [bib_ref] Southwest Pacific vivax malaria; clinical features and observations concerning duration of clinical..., Hill [/bib_ref]. These observations were similar to those in the seminal chemotherapy studies of Sinton and colleagues in Kasauli, Himachal Pradesh, India in the 1920s and 1930s [bib_ref] Studies on malaria with special reference to treatment. XV. Does the strain..., Sinton [/bib_ref] [bib_ref] Some lacunae in our knowledge of the malaria parasite, Sinton [/bib_ref] [bib_ref] Studies in malaria, with special reference to treatment. Part II. The effects..., Sinton [/bib_ref]. Interest in the long-latency phenotype revived during the early 1950s as P. vivax malaria which had a long-latency was an important problem in soldiers fighting in the Korean war [bib_ref] Korean vivax malaria. I. Natural history and response to chloroquine, Hankey [/bib_ref]. During this period patients who had received very heavy inoculations, typically soldiers from the Second World War, would return to clinics for many years complaining of recurrences of malaria. The long latencies and the long relapse intervals in some infections and the multiple relapses in others despite anti-malarial treatment gave rise to the old saw that "you never got rid of malaria". ## Relapse intervals effects of sporozoite inoculum For long-latency vivax malaria from Northern climes the sporozoite dose determined the clinical phenotype [bib_ref] Pathology -Experimental malaria with protracted incubation, Schuffner [/bib_ref] [bib_ref] Dates of onset of relapses and the duration of infection in induced..., Tiburskaja [/bib_ref] [bib_ref] The influence of sporozoite dosage in vivax malaria, Boyd [/bib_ref] [bib_ref] Studies in human malaria. XVIII. The life pattern of sporozoite-induced St. Elizabeth..., Coatney [/bib_ref] [bib_ref] Features specific to the Moscow strain of P. vivax, Tiburskaya [/bib_ref] [bib_ref] Explanation for the difference of incubation type and features of alteration of..., Moshkovsky [/bib_ref]. In long term observations of infections with the St Elizabeth strain there was a clear bimodal pattern in which a long pre-patent period (~300 days) only occurred following smaller sporozoite inocula (more reflective of natural infection) [bib_ref] Studies in human malaria. XVIII. The life pattern of sporozoite-induced St. Elizabeth..., Coatney [/bib_ref]. Similar observations were made with a North Korean strain used for malaria therapy in a Moscow hospital from 1953 to 1968 [bib_ref] Dates of onset of relapses and the duration of infection in induced..., Tiburskaja [/bib_ref]. When the inoculum of sporozoites was small (10-100 sporozoites) the initial parasitaemic illness did not occur for nine or 10 months, or longer. If ≥ 1000 sporozoites were inoculated illness occurred after a "normal" incubation period of two weeks. By contrast when increasing sporozoite doses of the tropical "Chesson" strain were inoculated the incubation period shortened and there was no evidence for a long prepatent period [bib_ref] Studies in human malaria. XXX. A summary of 204 sporozoite-induced infections with..., Coatney [/bib_ref] [bib_ref] Prepatent periods of a tropical strain of Plasmodium vivax after inoculations of..., Ungureanu [/bib_ref]. This, and a series of experimental investigations in chimpanzees [bib_ref] Prepatent periods of a tropical strain of Plasmodium vivax after inoculations of..., Ungureanu [/bib_ref] [bib_ref] Observations on early and late post-sporozoite tissue stages in primate malaria. IV...., Krotoski [/bib_ref] , led Garnham to propose that the ratio of hypnozoites to immediately developing forms in the Korean P. vivax strain was 999:1 compared with the Chesson strain where he estimated the ratio as 50:50 [bib_ref] Swellengrebel lecture. Hypnozoites and 'relapses' in Plasmodium vivax and in vivax-like malaria, Garnham [/bib_ref] [bib_ref] The life-cycle of primate malaria parasites, Bray [/bib_ref]. [fig_ref] Figure 6: Diagram of the different P [/fig_ref] Several other important observations were made in the artificial infection studies conducted in humans and experimental primates. It was noted that even with a single infected mosquito bite some Chesson strain P. vivax infections relapsed after intervals which were as long as a year after a series of regular "short interval" relapses [bib_ref] Studies in human malaria. XXX. A summary of 204 sporozoite-induced infections with..., Coatney [/bib_ref]. These terminal long intervals did not appear to have a fixed periodicity . In contrast the initial inter-relapse intervals of Chesson strain P. vivax in volunteers, and also P.cynomolgi in Rhesus monkeys, were remarkably regular although they gradually lengthened with each successive relapse [bib_ref] The Chesson strain of Plasmodium vivax malaria. I. Relationship between prepatent period,..., Craige [/bib_ref] [bib_ref] Relapses with Chesson strain Plasmodium vivax following treatment with chloroquine, Jeffery [/bib_ref] [bib_ref] Studies in human malaria. XXX. A summary of 204 sporozoite-induced infections with..., Coatney [/bib_ref] [bib_ref] Compatibility of relapse patterns of Plasmodium cynomolgi infections in Rhesus monkeys with..., Schmidt [/bib_ref] [bib_ref] Studies on the chemotherapy of the human malarias; the anti-malarial activity of..., Berliner [/bib_ref] [fig_ref] Figure 10: Lengthening intervals between sequential relapses in individual volunteers who were infected with... [/fig_ref]. Two factors are likely to have explained these gradually lengthening intervals -numerical probabilities and immunity. The simultaneous activation of several hypnozoites will shorten the relapse interval because the interval is measured until the progeny of the most rapidly growing parasites cause patent infection. For example if ten genetically identical hypnozoites are activated the relapse interval will in 91% of occasions be shorter than if one hypnozoite is activated. This is because there is natural phenotypic variation even amongst genetically identical organisms and it is the progeny of the earliest activated and most rapidly multiplying parasite that become patent first. This is best illustrated in the studies of Coatney et al with the long-latency St Elizabeth strain [bib_ref] Studies in human malaria. XVIII. The life pattern of sporozoite-induced St. Elizabeth..., Coatney [/bib_ref]. The interval from inoculation to relapse (~9 months later) shortened with increasing inocula. Thus the more hypnozoites that are activated the shorter is the average interval between relapses [fig_ref] Figure 11: In infections of volunteers with the St Elizabeth strain of P [/fig_ref]. This relationship is also clearly seen in Schmidt's studies of P.cynomolgi in monkeys [bib_ref] Compatibility of relapse patterns of Plasmodium cynomolgi infections in Rhesus monkeys with..., Schmidt [/bib_ref]. This is an important consideration for concomitant activation of hypnozoites with different genotypes in endemic areas as will be discussed later. In natural settings multiple genotypically distinct hypnozoites may be activated but, on many occasions, only one or two genotypes will be 397 days Relapse patterns in volunteers in the USA who were infected by a single mosquito bite with the Chesson strain of P. vivax studied by Coatney et al [bib_ref] Studies in human malaria. XXX. A summary of 204 sporozoite-induced infections with..., Coatney [/bib_ref]. Some were rechallenged as indicated. detected subsequently at clinical relapse. The other hypnozoites' progeny may reach patency later, or asexual growth may be suppressed by fever, illness, immune response, and treatment such that they never reach patency. ## Effects of immunity The second factor accounting for lengthening interrelapse intervals in artificial infections was the acquisition of blood stage immunity against the single infecting genotype. In the studies of the St Elizabeth strain there was clear evidence that late relapses were attenuated if there was an early infection, but this did not affect the interval to latency [fig_ref] Figure 11: In infections of volunteers with the St Elizabeth strain of P [/fig_ref] , whereas with increasing size of sporozoite inoculum there was a corresponding shortening of the interval. These observations suggest that inoculum size is more important than immunity in determining the duration of latency or the inter relapse interval -at least for the first relapses with genetically homologous parasites [bib_ref] Studies in human malaria. XVIII. The life pattern of sporozoite-induced St. Elizabeth..., Coatney [/bib_ref]. In Schmidt's primate studies the lengthening of inter-relapse intervals was Relapse patterns of P.cynomolgi infections in Rhesus monkeys studied by Schmidt [bib_ref] Compatibility of relapse patterns of Plasmodium cynomolgi infections in Rhesus monkeys with..., Schmidt [/bib_ref]. The infections were induced with different numbers of injected sporozoites as indicated and treated repetitively with chloroquine. Monkeys in groups A, B, C, and D were inoculated, respectively, with 5 × 10 6 , 5 × 10 4 , 5 × 10 2 and 5 sporozoites. Some monkeys were rechallenged and some were finally given radical treatment with primaquine in addition. less prominent than in the human investigations [bib_ref] The Chesson strain of Plasmodium vivax malaria. I. Relationship between prepatent period,..., Craige [/bib_ref] [bib_ref] Relapses with Chesson strain Plasmodium vivax following treatment with chloroquine, Jeffery [/bib_ref] [bib_ref] Studies in human malaria. XVIII. The life pattern of sporozoite-induced St. Elizabeth..., Coatney [/bib_ref] [bib_ref] Compatibility of relapse patterns of Plasmodium cynomolgi infections in Rhesus monkeys with..., Schmidt [/bib_ref] [bib_ref] Studies on the chemotherapy of the human malarias; the anti-malarial activity of..., Berliner [/bib_ref] and increasing inter-relapse intervals were seen only after 12 or so relapses [bib_ref] Compatibility of relapse patterns of Plasmodium cynomolgi infections in Rhesus monkeys with..., Schmidt [/bib_ref] , although in these experiments the inocula were very large, and all animals received chloroquine treatment which was given early (on the second day of patency). Schmidt also observed in the Rhesus monkeys infected with P. cynomolgi [bib_ref] Compatibility of relapse patterns of Plasmodium cynomolgi infections in Rhesus monkeys with..., Schmidt [/bib_ref] , as Coatney had done earlier in volunteers infected with the Chesson strain of P. vivax [bib_ref] Studies in human malaria. XXX. A summary of 204 sporozoite-induced infections with..., Coatney [/bib_ref] , that occasionally a very long interval would follow a series of short intervals. Thus the steadily increasing intervals between the homologous strain relapses result from both the "running out of hypnozoites" with successive relapses, which results in reversion to the mean intervals associated with single hypnozoite activation, together with slower asexual growth rates resulting from the acquisition of asexual stage immunity. Blood stage immunity against homologous strains of P. vivax, which persists for many months, was a consistent observation in malaria therapy and challenge studies [bib_ref] A retrospective examination of reinfection of humans with Plasmodium vivax, Collins [/bib_ref]. Boyd noted that if the initial infection was allowed to run its natural course, then relapse did not occur and reinfection with the homologous strain was not possible . ## Effects of drugs Since the introduction of mepacrine (atebrine, quinacrine) in 1932, a drug which has a terminal elimination half-life of over one month, it was observed that early relapses were delayed by approximately thirty days compared with quinine treatment (i.e. from three to seven weeks) [bib_ref] Clinical trials of anti-malarial drugs, Most [/bib_ref]. Later chloroquine, which also has a terminal half-life of over one month, but a different elimination profile to mepacrine, was found to delay early relapse appearance by two to six weeks [bib_ref] Clinical trials of anti-malarial drugs, Most [/bib_ref]. Larger doses of the anti-malarials resulted in longer intervals to relapse consistent with a concentration-dependent slowing of asexual growth rates. Slowly eliminated anti-malarials delayed the onset of P. vivax relapse, and consequently reduced their frequency, but importantly these drugs did not appear to reduce the overall number of relapses experienced [bib_ref] Clinical trials of anti-malarial drugs, Most [/bib_ref] [fig_ref] Figure 12: Cumulative proportions of relapses in soldiers with vivax malaria acquired in the... [/fig_ref]. Only the 8-aminoquinolines reduced or prevented relapses. The relapse preventing properties of these drugs, and their synergistic action with quinine, were demonstrated within years of their introduction in the mid 1920s [bib_ref] Studies in malaria, with special reference to treatment. XII. Further researches into..., Sinton [/bib_ref] [bib_ref] The use of plasmoquine in the prevention of malarial infections, James [/bib_ref]. Two other key observations were made during this early era of malaria therapy and drug evaluation which were not satisfactorily explained until decades later. It was noted that haemolytic reactions occurred sporadically with plasmoquine in patients of Asian, African or South European descent, but were uncommon in Caucasians originating further north [bib_ref] Studies on the chemotherapy of the human malarias. IX. Effect of pamaquine..., Earle [/bib_ref]. This was explained later by the epidemiology of glucose-6-phosphate dehydrogenase deficiency. In the Southern United States it proved difficult of impossible to infect patents or volunteers of West African descent with P. vivax . This was later shown to reflect the absence of the Duffy blood group receptor for P. vivax invasion of erythrocytes in this population. ## Contrasting artificial with natural infections Artificial infections provided invaluable information but they differed from natural infections in several important respects [bib_ref] Are the experimental data of therapeutic malaria applicable to conditions obtaining in..., Winckel [/bib_ref]. The infections were in non-immune adults whereas the burden of vivax disease in endemic areas was in children. Adults in the malaria endemic areas have usually developed significant immunity to a broad range of local parasites which controls symptoms and reduces parasite densities. The artificial infections in the majority of volunteer studies and in malaria therapy followed the bites of 5-10 infected anopheline mosquitoes selected for maximal infectivity based on salivary gland sporozoite loads in sibling mosquitoes. The timing of inoculation in malaria therapy and experimental studies to coincide with maximum infectivity contrasts with the natural setting where anopheline mosquitoes display a wide range of infectivities depending on sporozoite age and other factors. The inocula in artificial infections were therefore usually "supranormal". This resulted in reliable infections but did not bring out the important stochastic component of P. vivax epidemiology resulting from low sporozoite inocula in areas of low seasonal transmission. Median sporozoite inocula in natural infections are estimated to be less than 10 sporozoites [bib_ref] Feeding behaviour and sporozoite ejection by infected Anopheles stephens. i, Ponnudurai [/bib_ref] [bib_ref] An estimation of the number of sporozoites ejected by a feeding mosquito, Rosenburg [/bib_ref] [bib_ref] Quantitation of Plasmodium falciparum sporozoites transmitted in vitro by experimentally infected Anopheles..., Beier [/bib_ref]. If Garnham's estimates (50:50 ratio of immediately developing parasites to hypnozoites) are correct this corresponds to a median of 5 hypnozoites in tropical P. vivax infections. The "strains" of P. vivax used in malaria therapy were likely to have been of a single (albeit evolving) genotype or very closely related interbreeding genotypes which were passaged through a very large number of patients over many years. Even if these infections originated as a mixed genotype infections in the donor patient it is likely that with multiple passage in malaria therapy the "strains" became purified through successive interbreeding to a very closely related group of genotypes. In contrast multiple unrelated genotype infections are common in natural infections. ## Day of late relapse onset Days of patent parasitaemia The malaria therapy patients usually had neurosyphilis and were often very frail. Overall the mortality associated with P. vivax malaria therapy was approximately 7% (and was 10% with P.malariae infections -generally regarded as the mildest human malaria). This high mortality reflects the underlying condition, although it was undoubtedly contributed to by the infection as well. The objective of treatment in natural infections is cure, but in malaria therapy quinine was used to "damp down" the more severe infections, not to eliminate them. Reinfection with a different genotype was usual in endemic areas but in malaria therapy this was undertaken only occasionally if the first infection was insufficient, and in volunteer studies was performed to demonstrate the "strain specificity" of the immune response. ## The proportion of infections which relapse The proportion of P. vivax infections which relapse is often thought of as an intrinsic property of the malaria parasites which varies considerably by geographic region. Tropical "strains" relapsed more than temperate "strains". But the relapse proportion is also clearly a function of the sporozoite inoculum and immunity (if any). As described earlier if artificial sporozoite induced infections were allowed to continue for weeks until selftermination the relapse did not usually occur and reinoculation was usually unsuccessful [bib_ref] A retrospective examination of reinfection of humans with Plasmodium vivax, Collins [/bib_ref] [bib_ref] Renewed clinical activity in vivax malaria, Boyd [/bib_ref]. In this setting the probability of relapse with a presumed single genotype depended on the duration of preceding illness. In total, the treatment responses in over 87,000 patients with acute vivax malaria have been reported in the available medical literature in English (> 300 publications) of whom~17,000 relapsed [fig_ref] Figure 13 P: intermediate group, if it exists, has not been well characterized [/fig_ref] and. This experience of a wide variety of antimalarial treatment regimens, is heavily biased to adults and military reports. Two-thirds of the reports are over fifty years old. The reported relapse rates varied from 0 to 100%! Many relapse rates are likely to have been underestimated as follow-up periods, particularly in recent studies, were often two months or less, and in the majority of studies radical treatments were being evaluated. On the other hand studies conducted in endemic areas could not exclude reinfection. The relapse rate was very high in soldiers who were generally non-immune and presumably experienced intense exposure. In the Second World War 70 to 80% of soldiers fighting in the South Pacific had relapses (and in many units all soldiers had relapses). In Hill and Amatuzio's series of 222 patients, 9% had 10 or more relapses [bib_ref] Southwest Pacific vivax malaria; clinical features and observations concerning duration of clinical..., Hill [/bib_ref] [fig_ref] Figure 15: Proportions of P [/fig_ref]. Relapses were estimated to account for about half of all vivax malaria hospitalizations in the Second World War. Once chemoprophylaxis was enforced in soldiers it became clear that almost all malaria could be prevented, but that several weeks after the mepacrine (quinacrine, atebrine) was stopped P. vivax relapses started to occur [bib_ref] Clinical trials of anti-malarial drugs, Most [/bib_ref]. Without radical treatment the proportion of patients who experience one relapse, the proportion of these patients who have a second relapse, and the proportion of these who have a third and so on, is constant [fig_ref] Table 1: Relationship between the proportion of patients relapsing with vivax malaria and total... [/fig_ref]. This appears to obtain in each different epidemiological or experimental setting [bib_ref] Further observations on malaria made during the treatment of general paralysis, Yorke [/bib_ref] [bib_ref] Some general results of a study of induced malaria in England, James [/bib_ref] [bib_ref] Dates of onset of relapses and the duration of infection in induced..., Tiburskaja [/bib_ref] [bib_ref] Induced and war malaria, Höring [/bib_ref] [bib_ref] Studies on imported malarias the parasitological pattern of relapsing Plasmodium vivax in..., Eyles [/bib_ref] [bib_ref] Clinical trials of anti-malarial drugs, Most [/bib_ref] [bib_ref] Natural course of Southwest Pacific malaria, Noe [/bib_ref] [bib_ref] Long term observation of Plasmodium vivax malaria, Bianco [/bib_ref] [bib_ref] The Chesson strain of Plasmodium vivax malaria. I. Relationship between prepatent period,..., Craige [/bib_ref] [bib_ref] Relapses with Chesson strain Plasmodium vivax following treatment with chloroquine, Jeffery [/bib_ref] [bib_ref] Features specific to the Moscow strain of P. vivax, Tiburskaya [/bib_ref] [bib_ref] Southwest Pacific vivax malaria; clinical features and observations concerning duration of clinical..., Hill [/bib_ref] [bib_ref] Korean vivax malaria. I. Natural history and response to chloroquine, Hankey [/bib_ref]. In other words the relationship between the numbers of patients who experience one or more, two or more, three or more etc relapses is exponential. Thus if "x" is the fraction of patients experiencing one or more relapses, then the fraction experiencing "n" or more relapses is approximately x n A plot of the logarithm of the proportions versus number of relapses is, therefore, linear with slopes varying depending on the geographical and epidemiological setting [fig_ref] Figure 6: Diagram of the different P [/fig_ref]. Thus, for areas with 50% relapse rates approximately 6.25% of patients have four or more relapses per incident symptomatic infection. This is important when considering radical curative drug efficacy as it provides an indication of the minimum burden of hypnozoites (because each relapse must derive from ≥ 1 hypnozoite) (text box on pharmacodynamics). It follows that once symptomatic relapse rates exceed 50%, then relapse becomes the predominant cause of vivax malaria illness. ## Geographic distribution Overall there was good evidence for the presence of the long-latency "Madagascar" relapse phenotypes in Europe, Central Asia, Southern Russia, North Africa, the horn of Africa, Madagascar, the Middle East, Afghanistan, Pakistan and Northern India, Central parts of China, Korea, North and Central America [fig_ref] Figure 7: Schematic diagram by Hankey et al[79]of relapse patterns following Korean vivax malaria [/fig_ref]. Long incubation period P. vivax ("hibernans") was prevalent in Northern Europe and more Northern parts of Russia. Frequent relapse "strains" were reported in parts of South America, India, South East Asia and Oceania. It is highly likely that in those areas where malaria has not been eradicated this geographic distribution of P. vivax phenotypes pertains today, although there is very little contemporary information apart from reports from South East Asia and the island of New Guinea (which has much higher levels of malaria transmission than most other P. vivax endemic areas of the world). It is also evident that both phenotypes overlap in geographic distributions [fig_ref] Figure 7: Schematic diagram by Hankey et al[79]of relapse patterns following Korean vivax malaria [/fig_ref]. Where both are present together it would be very easy for the long-latency infections to go unrecognized, although the presence of a spring vivax malaria peak while vector densities are still low would be an epidemiological pointer [bib_ref] The seasonal periodicity of malaria and the mechanism of the epidemic wave, Gill [/bib_ref]. There has been very little recent interest in this question despite its obvious importance. It is generally thought, but is by no means certain, that the majority of the global burden of vivax malaria today is the tropical frequent relapse type (although interestingly the first P. vivax to be sequenced fully was a long-latency phenotype from El Salvador-Sal 1) [bib_ref] Studies on the characterization of Plasmodium vivax strains from Central America, Contacos [/bib_ref] [bib_ref] Comparative genomics of the neglected human malaria parasite Plasmodium vivax, Carlton [/bib_ref]. The greatest uncertainty is the epidemiology of relapse phenotypes in the Indian sub-continent, because India harbours the majority of P. vivax in the world and clearly has both phenotypes. This remarkable gap in our understanding of the global epidemiology of vivax malaria seems to have gone unrecognized outside the sub-continent. Prospective studies from India over the past 25 years have recorded relapse rates following chloroquine treatment of between 8.6% (Orissa) and 8.9% (Madhya Pradesh) and 40.1% (Delhi) [bib_ref] Plasmodium vivax polymorphism in a clinical drug trial, Adak [/bib_ref] [bib_ref] Relapse pattern of Plasmodium vivax in Mumbai: a study of 283 cases..., Gogtay [/bib_ref]. In Mumbai 19 out of 150 patients with vivax malaria and treated with chloroquine only and who were followed for one year had a relapse (17 within six months) [bib_ref] Relapse pattern of Plasmodium vivax in Mumbai: a study of 283 cases..., Gogtay [/bib_ref]. Reinfection was considered unlikely. Higher rates were reported from the Delhi areawhere the authors concluded "Based on the foregoing epidemiologic features, three distinct relapse patterns were observed in the present study, and it can be concluded that the P vivax population in northern India is polymorphic. Group I is the tropical or Chesson strain type of frequent relapsing P. vivax with a short period of latency between the primary attack and the first relapse, which is similar to other Southeast Asian strains such as those from Thailand and Vietnam. Group III is the temperate or St. Elizabeth-type strain that has a long period of latency between the primary attack and the first relapse. Group II is intermediate between these two types". This White Malaria Journal 2011, 10:297 http://www.malariajournal.com/content/10/1/297 [bib_ref] Malaria: patterns of relapse and resistance, Saifi [/bib_ref]. Overall relapse rates from India have been relatively low by comparison with South-East Asia [bib_ref] Plasmodium vivax polymorphism in a clinical drug trial, Adak [/bib_ref] [bib_ref] Relapse pattern of Plasmodium vivax in Mumbai: a study of 283 cases..., Gogtay [/bib_ref] [bib_ref] Malaria: patterns of relapse and resistance, Saifi [/bib_ref] [bib_ref] Efficacies of 5-and 14-day primaquine regimens in the prevention of relapses in..., Gogtay [/bib_ref] [bib_ref] Radical treatment of vivax malaria in Madhya Pradesh, Singh [/bib_ref] [bib_ref] Relapse/reinfection patterns of Plasmodium vivax infection: a four year study, Prasad [/bib_ref] [bib_ref] Malaria relapses and chloroquine resistance at the BHEL industrial complex, Sinha [/bib_ref] [bib_ref] Treatment of vivax malaria in an endemic area on the western border..., Luxemburger [/bib_ref] [bib_ref] Clinical trial of oral artesunate with or without highdose primaquine for the..., Silachamroon [/bib_ref]. Data from travellers returning from the Indian sub-continent have suggested that long incubation-period P. vivax may be common in the Punjab, and this is consistent with evidence in the past from Northern India, Pakistan and Afghanistan [bib_ref] Prolonged incubation period of imported P. vivax malaria in London, Warwick [/bib_ref] [bib_ref] The seasonal pattern of Plasmodium vivax malaria in Glasgow, Walker [/bib_ref] and also the early observations of Fearnside, Yorke, and MacFie with artificial infections of Indian origin [bib_ref] Experimental inoculation of malaria with a relapse after eight months, Fearnside [/bib_ref] [bib_ref] Observations on malaria made during the treatment of general paralysis, Yorke [/bib_ref] [bib_ref] Further observations on malaria made during the treatment of general paralysis, Yorke [/bib_ref]. There are few data on temporal patterns of relapse in travellers in recent years, although studies in Eritrean immigrants to Israel, Turkish immigrants to Germany, and US soldiers returning from Somalia suggest the presence of longlatency phenotypes in the countries of origin [bib_ref] Relapsing vivax malaria cluster in Eritrean refugees, Israel, Kopel [/bib_ref] [bib_ref] Malaria tertiana bei Kindern und Erwachsenen aus dem Epidemiegebiet der südlichen Türkei, Eichenlaub [/bib_ref] [bib_ref] Plasmodium vivax infections in U.S. Army troops: failure of primaquine to prevent..., Smoak [/bib_ref]. Most travellers receive radical treatment for vivax malaria in temperate countries, which may be more effective against the long incubation or long-latency infections than against the tropical frequent relapse phenotypes. The tropical frequent relapse phenotype was documented by Sinton and colleagues in British soldiers stationed in India in the 1920s and 1930s, and was observed in soldiers fighting in North-East India and Burma, and in prisoners of war in Thailand. More recently Luxemburger et al studied 342 children with acute vivax malaria treated with chloroquine on the north-western border of Thailand in 1995 and 1996 [bib_ref] Treatment of vivax malaria in an endemic area on the western border..., Luxemburger [/bib_ref]. Reappearance of P. vivax occurred in one patient on day [bib_ref] Concerning the pathogenesis of relapses in malarial fevers, Bignami [/bib_ref] Otherwise as for [fig_ref] Figure 13 P: intermediate group, if it exists, has not been well characterized [/fig_ref] Thailand, or Cambodia) with acute vivax malaria who were treated with either 5 days (N = 157) or 7 days (N = 159) of artesunate monotherapy [bib_ref] Clinical trial of oral artesunate with or without highdose primaquine for the..., Silachamroon [/bib_ref]. Relapse rates within 28 days were 52.2% and 47.8% respectively. The timing of the relapses suggested that very few if any relapses emerged from the liver before the eighth day after starting anti-malarial treatment. In Papua Indonesia the relapse rate estimated at six weeks following artemether-lumefantrine treatment was 38% (the total number may well have been higher because of later emerging relapses) [bib_ref] Two fixed-dose artemisinin combinations for drug-resistant falciparum and vivax malaria in Papua,..., Ratcliff [/bib_ref]. In French Guiana the relapse rate in children was 70% (nearly all relapses occurred within three months) but both recrudescence and reinfection could not be excluded [bib_ref] Determination of the Plasmodium vivax relapse pattern in Camopi, French Guiana, Hanf [/bib_ref]. Although South American P. vivax is generally regarded as "tropical frequent relapsing" in phenotype, a recent report from Rio de Janeiro that six of 80 travellers presenting with vivax malaria (who had returned from the Amazon region and not received chemoprophylaxis) had an incubation period of between three to 12 months, and another from Brasilia describing long-latency in three patients, suggest that long-latency forms may coexist with frequent relapse phenotypes in Brazil [bib_ref] Unexpectedly long incubation period of Plasmodium vivax malaria, in the absence of..., Brasil [/bib_ref] [bib_ref] Vivax malaria with long incubation period, detected in the Federal District: three..., Tauil [/bib_ref]. ## The effects of age In malaria endemic areas, such as the north-western border of Thailand, the age profile of P. vivax malaria suggests much more rapid acquisition of immunity than for P. falciparum [bib_ref] The epidemiology of malaria in a Karen population on the western border..., Luxemburger [/bib_ref]. Entomological studies suggested similar transmission rates (at least in terms of measured entomological inoculation rates) so it is likely that relapse contributes to much of this difference. It also suggests that relapses are probably partially suppressed in older patients. Thus both the proportion of infections which relapse and the number of symptomatic relapses per mosquito sporozoite inoculation decline with age. It is likely that immunity and therefore age is a significant confounder in epidemiological assessments based on passive case reporting in many P. vivax endemic areas. In many areas adults are more likely to present to malaria clinics than children. In India the peak age of malaria presentation is often in young adults (and often with a predominance of young males). Yet in the indigenous population living in transmission areas a [bib_ref] Long term observation of Plasmodium vivax malaria, Bianco [/bib_ref] [bib_ref] Southwest Pacific vivax malaria; clinical features and observations concerning duration of clinical..., Hill [/bib_ref] , German soldiers who acquired vivax malaria in Greece [bib_ref] Induced and war malaria, Höring [/bib_ref] , US soldiers with vivax malaria acquired in the Mediterranean area (two series) [bib_ref] Office of the Surgeon General, Department of the Army, Mowrey [/bib_ref] [bib_ref] Clinical trials of anti-malarial drugs, Most [/bib_ref] and Italy, patients receiving malaria therapy with a local "strain" in Moscow [bib_ref] Dates of onset of relapses and the duration of infection in induced..., Tiburskaja [/bib_ref] , British patients receiving malaria therapy with the Madagascar strain [bib_ref] Some general results of a study of induced malaria in England, James [/bib_ref] [bib_ref] Clinical and parasitological observations on induced malaria, James [/bib_ref] , patients receiving malaria therapy with the McCoy strain in the United States [bib_ref] Renewed clinical activity in vivax malaria, Boyd [/bib_ref] , volunteers infected with the Chesson strain in the United States [bib_ref] Studies in human malaria. XXX. A summary of 204 sporozoite-induced infections with..., Coatney [/bib_ref] and children followed prospectively in an evaluation of an ineffective malaria vaccine (SPf66) in northwestern Thailand [bib_ref] Randomised double-blind placebo-controlled efficacy trial of the SPf66 vaccine against falciparum malaria..., Nosten [/bib_ref]. Inset shows proportions on a log scale and numbers of patients studied. White Malaria Journal 2011, 10:297 http://www.malariajournal.com/content/10/1/297 significant degree of immunity should have been gained (by both sexes) by early adulthood which reduces the number of relapses. Usually in endemic areas it is children who bear the brunt of malaria. This applies to both falciparum and vivax malaria when malaria is uncontrolled, but with increasing control falciparum declines more rapidly than vivax, and their epidemiology separates. The paucity of data from children may contribute to the low apparent relapse rates reported from the Indian sub-continent. It seems likely then that malaria clinic data are not necessarily representative of disease epidemiology in some endemic areas, and that studies of children living in the endemic areas of the Indian subcontinent might reveal higher relapse rates. ## Drug effects on relapse Before the Second World War most malaria infections were treated with quinine. Early relapses of P. vivax were observed to occur approximately three to four weeks after starting quinine treatment. The 8-aminoquinoline plasmoquine (plasmochin, pamaquine) was evaluated clinically shortly after its discovery in 1924 in Germany. Sinton and colleagues in India soon provided evidence that plasmoquine synergized with quinine in the treatment of acute vivax malaria and also reduced the rate of recurrence (mainly relapse) [bib_ref] Studies in malaria, with special reference to treatment. XII. Further researches into..., Sinton [/bib_ref]. "Sinton's regimen" of one week's quinine plus plasmoquine was endorsed by the League of Nations and generally replaced the two-month regimens previously in vogue Long latency P. vivax ## Frequent relapse p. vivax Frequent relapse + long latency P. vivax? Approximate historical distribution of P. vivax latency phenotypes. Areas where tropical "frequent relapse phenotypes" are prevalent are shown in pink. Areas where both frequent relapse and long-latency phenotypes have been reported are shown in purple, and areas where long-latency phenotypes were prevalent are shown in grey. Although both South America and India are generally considered to harbour frequent relapse phenotypes predominantly, there is evidence that long-latency phenotypes are present in both areas (particularly across the North of India), and without genotyping it may be difficult or impossible to distinguish the two phenotypes within an endemic area [fig_ref] Figure 22: Potential similarity of relapse patterns with the long-latency and frequent relapse P [/fig_ref]. The epidemiology of vivax and falciparum malaria in an area of low seasonal transmission on the Western border of Thailand; age incidence profiles [bib_ref] Treatment of vivax malaria in an endemic area on the western border..., Luxemburger [/bib_ref].. In the 1930s the newly introduced sulphonamides were shown to have activity against malaria, but were more effective against P. falciparum than P. vivax [bib_ref] Clinical trials of anti-malarial drugs, Most [/bib_ref]. The discovery of mepacrine (quinacrine, atebrine) in 1932 and its subsequent introduction provided a simpler, somewhat better tolerated, treatment although there was a pharmacokinetic interaction with plasmoquine which resulted in increased plasmoquine concentrations and oxidative toxicity, and precluded using the drugs simultaneously. Mepacrine was very slowly eliminated and extensively distributed [bib_ref] The pharmacological basis for the rational use of atabrine in the treatment..., Shannon [/bib_ref]. It was an effective treatment of vivax malaria. Mepacrine treatment markedly delayed the early relapse of tropical P. vivax which, usually then presented six to eight weeks after the primary infection rather than three weeks later, as was usual following quinine. When mepacrine was used as a prophylactic it suppressed infections for at least one month after stopping the drug [bib_ref] Clinical trials of anti-malarial drugs, Most [/bib_ref] [bib_ref] The pharmacological basis for the rational use of atabrine in the treatment..., Shannon [/bib_ref] [bib_ref] Atabrine studies in the field; the relation of serum atabrine level to..., Schaffer [/bib_ref]. Thereafter relapses followed soon afterwards in tropical areas where frequent relapse "strains" were prevalent, and many months later where long-latency "strains" were prevalent. Extended follow-up studies suggested that although the relapses were delayed, they were not prevented [bib_ref] Clinical trials of anti-malarial drugs, Most [/bib_ref] [fig_ref] Figure 12: Cumulative proportions of relapses in soldiers with vivax malaria acquired in the... [/fig_ref]. It was assumed that the slowly eliminated treatment had suppressed the expansion of the blood stage infection (post treatment prophylaxis). It was unclear whether the relapse seen later was the first relapse which had been suppressed for three or more weeks, or whether the first relapse had been prevented altogether and it was in fact the second relapse (but the first clinical evidence of relapse) that became patent six to eight weeks after starting initial treatment. Mepacrine (atebrine, quinacrine) was the main drug used by all sides in the Second World War. The British were more willing to use plasmoquine than the US forces who discontinued its use during the war. They considered the risk exceeded the benefit [bib_ref] The present status of anti-malarial drugs, Napier [/bib_ref] , possibly because reactions were common in African-American soldiers (presumably G6PD deficient individuals who haemolysed). Relapse of vivax malaria was a major problem in soldiers, and was well documented in all the tropical theatres of war. Immediately after the Second World War the enormous research effort to find new anti-malarial drugs gave chloroquine (discovered in 1934 and initially overlooked), proguanil, and new more effective and less toxic 8-aminoquinolines. Chloroquine was clearly the best anti-malarial yet to be discovered, but like quinine and mepacrine, it acted only on the blood stage parasites. Proguanil, and later pyrimethamine, were both shown to have pre-erythrocytic activity but not radical curative activity. Furthermore resistance arose readily to the antifol anti-malarials in P. vivax. Although plasmoquine was moderately effective it was relatively poorly tolerated in the dose regimens necessary to prevent relapse (radical cure) [bib_ref] Studies on the chemotherapy of the human malarias; the anti-malarial activity of..., Berliner [/bib_ref] [bib_ref] A retrospective examination of reinfection of humans with Plasmodium vivax, Collins [/bib_ref] [bib_ref] Studies in malaria, with special reference to treatment. XII. Further researches into..., Sinton [/bib_ref] [bib_ref] The use of plasmoquine in the prevention of malarial infections, James [/bib_ref] [bib_ref] Studies on the chemotherapy of the human malarias. IX. Effect of pamaquine..., Earle [/bib_ref]. The more effective and better tolerated 8-aminoquinolines pentaquine and then primaquine were developed and evaluated between 1944 and 1955 [bib_ref] Pentaquine (SN-13,276) a therapeutic agent effective in reducing the relapse rate in..., Alving [/bib_ref] [bib_ref] Primaquine, SN 13272, a new curative agent in vivax malaria; a preliminary..., Edgcomb [/bib_ref] [bib_ref] Studies in human malaria. XXXI.: Comparison of primaquine, isopentaquine, SN-3883, and pamaquine..., Cooper [/bib_ref] [bib_ref] Korean vivax malaria. V. Cure of the infection by primaquine administered during..., Coatney [/bib_ref] [bib_ref] Korean vivax malaria. II. Curative treatment with pamaquine and primaquine, Robinson [/bib_ref]. Primaquine took over as the standard radical treatment of vivax malaria (except in the USSR where the positional isomer quinocide was preferred). The 8-aminoquinolines also have significant blood stage activity against P. vivax (and Plasmodium knowlesi) although resistance in the blood stage infection can be induced experimentally [bib_ref] Induced primaquine resistance in vivax malaria, Arnold [/bib_ref]. The widely used chloroquine-primaquine regimen should therefore be considered a combination treatment. The 8-aminoquinoline development programme also uncovered the reason why some patients of African or Asian origin developed severe haemolysis, whereas Caucasian patients originating from Northern Europe did not haemolyse significantly. X-linked Glucose-6-phosphate dehydrogenase (G6PD) deficiency was discovered as the most common genetic defect of humans [bib_ref] Alving AS: The hemolytic effect of primaquine. VII. Biochemical studies of drug-sensitive..., Beutler [/bib_ref] [bib_ref] Enzymatic deficiency in primaquine-sensitive erythrocytes, Alving [/bib_ref]. The dose of primaquine recommended globally was chosen largely as a result of studies on the relatively drug-sensitive Korean P. vivax [bib_ref] Korean vivax malaria. V. Cure of the infection by primaquine administered during..., Coatney [/bib_ref] [bib_ref] Korean vivax malaria. II. Curative treatment with pamaquine and primaquine, Robinson [/bib_ref] [bib_ref] Korean vivax malaria. IV. Curative effect of 15 milligrams of primaquine daily..., Lorenzo [/bib_ref]. After a very high rate of relapses was observed in US soldiers returning in 1950 from the Korean war , all soldiers were given a radical curative regimen of 15 mg base/day for two weeks during their return by sea [bib_ref] Korean vivax malaria. V. Cure of the infection by primaquine administered during..., Coatney [/bib_ref]. This proved very effective. The Chesson strain had been shown to be more "resistant" to 8-aminoquinolines [bib_ref] Studies in human malaria. XXXI.: Comparison of primaquine, isopentaquine, SN-3883, and pamaquine..., Cooper [/bib_ref] , but recommendations for a higher dose of primaquine (adult dose: ## Number of p. vivax relapses Exposure Numbers of US servicemen admitted to hospital in the USA each week with vivax malaria following their return from the Korean war. Exposure was predominantly in 1950. ## Mg base/day) were applied initially only in oceania. In hindsight there was no very good reason for this, and it might have been better to recommend higher doses for all frequent relapse parasites (i.e. in South-East Asia). It remains possible that all temperate "strains" are equally sensitive to primaquine, and all tropical strains are equivalent but have higher burdens of hypnozoites which could be activated immediately and so require a higher dose (i.e. 0.5 mg/base/kg for 14 days) [fig_ref] Table 2: Radical treatment pharmacodynamics [/fig_ref]. This highlights the need for better discrimination of the tropical and temperate phenotypes and a better understanding of the epidemiology of relapse, and the pharmacokinetic-pharmacodynamic basis of radical treatment. One particular area of therapeutic relevance is the question of synergy. Sinton's work in India had suggested that quinine and plasmoquine were synergistic in the prevention of relapse in vivax malaria [bib_ref] Studies in malaria, with special reference to treatment. XII. Further researches into..., Sinton [/bib_ref] and studies during and after the Second World War [bib_ref] Clinical trials of anti-malarial drugs, Most [/bib_ref] provided further support for this notion. Ruhe et al showed that concurrent quinine and pamaquine (plasmoquine) was more effective in prevention of relapse with the St Elizabeth strain than sequential administration [bib_ref] Studies in human malaria. XV. The therapeutic action of pamaquine against St...., Ruhe [/bib_ref]. In the 1950s, Alving and colleagues conducted a formal interaction study which provided evidence of marked synergy between both quinine and chloroquine and primaquine [bib_ref] Potentiation of the curative action of primaquine in vivax malaria by quinine..., Alving [/bib_ref]. The reason for this synergy (i.e. was it pharmacokinetic or pharmacodynamic?), and whether it extends to other quinolines or related compounds has not been explored further. The mid 1950s saw a decline in clinical research on vivax malaria. Meanwhile most countries adopted the 15 mg base/day primaquine regimen usually given for 14 days. Five day regimens of primaquine (total adult dose 75 mg base) were recommended in the Indian subcontinent, although there was no evidence they were effective [bib_ref] Plasmodium vivax polymorphism in a clinical drug trial, Adak [/bib_ref] [bib_ref] Relapse pattern of Plasmodium vivax in Mumbai: a study of 283 cases..., Gogtay [/bib_ref] [bib_ref] Malaria: patterns of relapse and resistance, Saifi [/bib_ref] [bib_ref] Efficacies of 5-and 14-day primaquine regimens in the prevention of relapses in..., Gogtay [/bib_ref] [bib_ref] Radical treatment of vivax malaria in Madhya Pradesh, Singh [/bib_ref] [bib_ref] Relapse/reinfection patterns of Plasmodium vivax infection: a four year study, Prasad [/bib_ref] [bib_ref] Malaria relapses and chloroquine resistance at the BHEL industrial complex, Sinha [/bib_ref] [bib_ref] Regional differences in the response of Plasmodium vivax malaria to primaquine as..., Goller [/bib_ref] ]. Baird has recently pointed out that the much improved tolerability of primaquine when taken with food was not emphasized sufficiently at this time and thus later dosage recommendations may have been limited by perceived or observed poor tolerability [bib_ref] Resistance to therapies for infection by Plasmodium vivax, Baird [/bib_ref]. In the recent reawakening of interest in malaria it has been suggested that resistance to the radical curative activity of primaquine has emerged [bib_ref] Primaquine resistance in Plasmodium vivax, Collins [/bib_ref] -but it is not at all clear if there has been any significant change in susceptibility. As noted earlier, because the tropical phenotype is usually associated with a greater number of relapses than the temperate phenotype, then it requires a greater proportional reduction in activatable hypnozoites to prevent all relapses. More clinical pharmacology evidence on these important points is needed. ## Vivax malaria following falciparum malaria In July, 1921 Major JA Sinton VC was put in charge of the Indian quinine and malaria inquiry under the newly formed Central Malaria Bureau. Between March 1924 and July 1925 Sinton compared two different quinine regimens in the treatment of falciparum malaria in British soldiers. These soldiers were followed for eight weeks at Kasauli (above the level of malaria transmission at an altitude of 6,000 feet in Himachal Pradesh). Of 76 soldiers completing follow-up 30 (39%) had subsequent vivax malaria [bib_ref] Some lacunae in our knowledge of the malaria parasite, Sinton [/bib_ref]. In East Asia and Oceania a similar pattern pertains today. In East Asia a remarkably high proportion (20-50%) of symptomatic infections with P. falciparum are followed by an infection with P. vivax [bib_ref] High rate of Plasmodium vivax relapse following treatment of falciparum malaria in..., Looareesuwan [/bib_ref] [bib_ref] Mixed-species malaria infections in humans, Mayxay [/bib_ref] [bib_ref] Plasmodium vivax recurrence following falciparum and mixed species malaria: risk factors and..., Douglas [/bib_ref]. In Ethiopia where early relapse rates in vivax malaria are much lower, this proportion is also lower; 7% [bib_ref] In vivo efficacy of artemetherlumefantrine against uncomplicated Plasmodium falciparum malaria in Central..., Hwang [/bib_ref]. The intervals between the onset of treatment for the acute P. falciparum infection and the subsequent P. vivax infection are very similar to the intervals between acute vivax malaria and the first relapse . As the treatments given for falciparum malaria are highly effective, and therefore should be curative against the blood stage infections of P. vivax, these recurrent malaria infections are highly likely to be relapses. Furthermore, as with relapses after vivax malaria, the probability of vivax recurrence after falciparum malaria is also age related [bib_ref] Plasmodium vivax recurrence following falciparum and mixed species malaria: risk factors and..., Douglas [/bib_ref]. Several lines of evidence Thus we would expect 13 to 20 times more relapses in group A compared to group B. This hypothetical example simply points out that the apparent differences in primaquine "resistance" may reflect differences in the biology of the parasite rather than drug susceptibility per se. White Malaria Journal 2011, 10:297 http://www.malariajournal.com/content/10/1/297 detailed below point to these vivax episodes being relapses of latent hypnozoites acquired before the P. falciparum inoculation. ## The periodicity of relapse Various theories to explain the remarkable periodicity of Plasmodium vivax infections have been proposed [bib_ref] Population studies of Plasmodium vivax. 1. The theory of polymorphism of sporozoites..., Lysenko [/bib_ref] including reinfection of liver cells from released merozoites, intrinsic differences in latency periods of the inoculated sporozoites (tachyzoites, bradyzoites), and activation of dormant parasites by external stresses or seasonal stimuli [bib_ref] Latency and long-term relapses in benign tertian malaria, Shute [/bib_ref] [bib_ref] The hypnozoite and relapse in primate malaria, Cogswell [/bib_ref]. In bird malarias there is reinfection of tissues from blood stage parasites. Following their seminal discovery of the pre-erythrocytic developmental stage in the liver, Shortt and Garnham initially suggested that this might also occur in the primate malarias [bib_ref] Pre-erythrocytic stage in mammalian malaria parasites, Shortt [/bib_ref] [bib_ref] Demonstration of a persisting exo-erythrocytic cycle in Plasmodium cynomolgi and its bearing..., Shortt [/bib_ref] [bib_ref] The pre-erythrocytic stage of human malaria, Plasmodium vivax, Shortt [/bib_ref]. However there is no convincing evidence to support this theory, and most are now agreed that reinfection from the blood stage back to the liver to produce secondary tissue stages does not take place in the primate malarias. The temperate zone P. vivax usually had an incubation period of 8-9 months so emergence of an infection acquired in late summer or autumn could coincide with vector emergence in the following late spring or summer. Further south in temperate climes P. vivax infections had a primary illness two to three weeks after mosquito inoculation but the first relapse occurred 8-9 months later. Although the interval ("latency") between the primary infection and first relapse for this "Madagascar phenotype" was long (8-10 months), the subsequent inter-relapse intervals were short [fig_ref] Figure 7: Schematic diagram by Hankey et al[79]of relapse patterns following Korean vivax malaria [/fig_ref]. In fact they were similar to the intervals from primary infection to early relapse which occurred in some Madagascar/St Elizabeth phenotypes and all Chesson phenotypes. This observation sits uncomfortably with a simple preprogrammed biological clock conjecture in which each sporozoite has a programmed latency. Lysenko et al pointed out that if the inoculated sporozoites were indeed a mixture of short and longlatency "zoites" then long latencies would only be evident if the sporozoite inocula were very small (otherwise there would almost invariably be one or more tachyzoites in the inoculum, and its emergence and subsequent treatment would obscure any later emergence of bradyzoites) [bib_ref] Population studies of Plasmodium vivax. 1. The theory of polymorphism of sporozoites..., Lysenko [/bib_ref]. Under this conjecture then perhaps failure to relapse early simply implies that long-latency (bradyzoites) only were inoculated. Much of the theorizing preceded discovery in 1982 of the dormant or persistent liver stage-now called the hypnozoite [bib_ref] Relapses in primate malaria: Discovery of two populations of exoerythrocytic stages. Preliminary..., Krotoski [/bib_ref] [bib_ref] Demonstration of hypnozoites in sporozoite-transmitted Plasmodium vivax infection, Krotoski [/bib_ref] [bib_ref] Swellengrebel lecture. Hypnozoites and 'relapses' in Plasmodium vivax and in vivax-like malaria, Garnham [/bib_ref] [bib_ref] The life-cycle of primate malaria parasites, Bray [/bib_ref]. After the discovery of the hypnozoite the biological clock model was refined. The generally accepted theory (as described, and disputed, by has been that "sporozoite populations of all strains of P. vivax, P. ovale, and relapsing simian malarias such as P. cynomolgi, include a subpopulation that completes development promptly and is responsible for the early primary attack, and a group of subpopulations that undergo partial development to the resting hypnozoite stage. Subsets of these dormant pre-erythrocytic stages are preprogrammed to resume development at different times and, via this built-in time clock, evoke the sequence of relapses that characterize sporozoite-induced infections with the aforementioned plasmodia" [bib_ref] Compatibility of relapse patterns of Plasmodium cynomolgi infections in Rhesus monkeys with..., Schmidt [/bib_ref]. As discussed previously it is difficult to understand how multiple relapses could occur at regular intervals with generally small inocula (median~five hypnozoites) and a simple clock mechanism. This remarkable efficiency suggests that some activation or feedback mechanism must operate in addition. A recent suggestion is that mosquito bites themselves might provide the trigger -perhaps by parasite sensing of a mosquito protein [bib_ref] Activation of the hypnozoite: a part of Plasmodium vivax life cycle and..., Hulden [/bib_ref]. This is difficult to reconcile with the similarity of latency periods in indigenous peoples living in malaria endemic areas and the latency periods observed in malaria therapy patients and returned soldiers (i.e. away from seasonal mosquitoes and independent of season). There are relatively few recent data on relapse patterns in frequent relapse "tropical" vivax malaria, but the available evidence confirms the remarkable periodicity documented in the volunteer studies with the "Chesson strain". Most informative are studies in which anti-malarials such as quinine or the artemisinin derivatives have been used as these drugs are eliminated rapidly and do not suppress or delay the emergence of subsequent relapse [bib_ref] Clinical trial of oral artesunate with or without highdose primaquine for the..., Silachamroon [/bib_ref] [bib_ref] Therapeutic responses to different antimalarial drugs in vivax malaria, Pukrittayakamee [/bib_ref] [bib_ref] A comparison of two short-course primaquine regimens for the treatment and radical..., Pukrittayakamee [/bib_ref]. Much of the early volunteer data with tropical "strains" was confounded partly by slowly eliminated drug effects and inoculations of unnaturally large numbers of sporozoites. Coatney realized this and so, in his classic series of 204 sporozoite-induced infections with the Chesson strain [bib_ref] Studies in human malaria. XXX. A summary of 204 sporozoite-induced infections with..., Coatney [/bib_ref] , he made detailed longitudinal observations following a single infected mosquito bite . The number of relapses varied considerably. In the seven volunteers who were not reinfected the median (range) number of relapses following a single bite was five (one to nine) and 11 of the 39 relapses in this group (28%) occurred more than six months after the initial infection. The interval from one relapse to the next was remarkably similar but overall the inter-relapse intervals gradually lengthened. Importantly there could then be very long intervals between relapses (four relapses occurred with preceding latencies exceeding six months. The maximum documented interval was 397 days). This proves that long-latency does occur with the tropical frequent relapse phenotype. These patterns of relapse are also illustrated well in the primate model ; the Rhesus monkey infected with P.cynomolgi (the primate malaria "equivalent" of P. vivax) [bib_ref] The life-cycle of primate malaria parasites, Bray [/bib_ref]. In Schmidt's detailed longitudinal series [bib_ref] Compatibility of relapse patterns of Plasmodium cynomolgi infections in Rhesus monkeys with..., Schmidt [/bib_ref] he noted that "The mean days separating each of the first four attacks (primary and first three relapses) were essentially identical in infections treated with chloroquine in combination with potentially curative agents, varying from 18.8 to 21.8 days from onset of patency in infections induced with the smaller inoculum and from 18.4 to 22.1 days in infections larger inoculums". In these monkey experiments where different sporozoite inocula were evaluated, it was noted that, although there was no clear difference in the number of relapses between monkeys inoculated with 5 × 10 2 sporozoites up to 5 × 10 6 sporozoites, "the intervals between relapses were related to size of inoculum, being distinctly shorter in monkeys inoculated with 5 × 10 6 sporozoites than in those challenged with 5 × 10 2 sporozoites, with recipients of 5 × 10 4 sporozoites occupying an intermediary position". Taken together with the absence of any relapses following an inoculum of only 5 sporozoites in three monkeys this argues for activation of a proportion of hypnozoites per relapse, and is consistent with the earlier observations in soldiers and malaria therapy patients of a fixed fraction of relapses following each illness episode in vivax malaria [fig_ref] Figure 6: Diagram of the different P [/fig_ref]. It is unfortunate that inocula between 5 and 500 sporozoites were not studied in the primate model. ## The biology of relapse Any theory seeking to explain the remarkable biology of Plasmodium vivax relapse must accommodate the following 1. Relapses show remarkable periodicity. 2. Early relapses reach patency around three weeks after starting treatment which suggests emergence from the liver at least one week earlier. [bib_ref] Nouveau parasite du sang, Laveran [/bib_ref]. Not all P. vivax primary infections are followed by a relapse. In Thailand approximately 50% of infections are followed by a subsequent relapse within 28 days if a rapidly eliminated anti-malarial drug (artesunate) is given for treatment of the primary infection and primaquine is not given [bib_ref] Clinical trial of oral artesunate with or without highdose primaquine for the..., Silachamroon [/bib_ref]. Elsewhere the probability of relapse generally varies between 20% and 80%. Animal experiments, the malaria therapy experience, and volunteer studies all suggest this proportion is a function of sporozoite inoculum. 4. Multiple relapses are common, particularly in young children, even though sporozoite inocula are thought to be relatively small (median 6-10 sporozoites). It is not uncommon in tropical areas for children to have four to six relapses at 4-6 week intervals and sometimes more following an incident infection. Even larger numbers of relapses were observed in soldiers following intense exposure and in Rhesus monkeys receiving very large sporozoite inocula. Importantly the fraction of people experiencing a relapse after each illness episode in a particular location appears constant 5. In long-latency phenotypes there is commonly a period of 8-9 months either before the first symptomatic infection, or between the first symptomatic infection and the first relapse. This long-latency interval appears to be normally distributed (mode 28 weeks for the Madagascar strain [bib_ref] Some general results of a study of induced malaria in England, James [/bib_ref] [bib_ref] Clinical and parasitological observations on induced malaria, James [/bib_ref]. Sometimes there are several short interval relapses followed by a long interval. Conversely long latencies may also occur after multiple relapses in the tropical frequent relapse phenotype [bib_ref] Studies in human malaria. XXX. A summary of 204 sporozoite-induced infections with..., Coatney [/bib_ref]. . if there are further relapses after the long latent period then they occur frequently with short intervals which are very similar to those observed in the tropical "strains". 7. The relapses in clinical studies conducted in endemic areas are commonly with a genotype which is different to that identified in the primary infection (48% in Columbian isolates, 55% in Indian isolates, 61% in Thai and Burmese isolates, and 71% in East Timor isolates) [bib_ref] Relapses of Plasmodium vivax infection usually result from activation of heterologous hypnozoites, Imwong [/bib_ref] [bib_ref] Relapses of Plasmodium vivax infection result from clonal hypnozoites activated at predetermined..., Chen [/bib_ref] [bib_ref] High genetic polymorphism of relapsing P. vivax isolates in northwest Colombia, Restrepo [/bib_ref]. 8. A remarkably high proportion of acute infections with Plasmodium falciparum are followed by an episode of P. vivax infection. The proportion is currently 30% in Thailand [bib_ref] High rate of Plasmodium vivax relapse following treatment of falciparum malaria in..., Looareesuwan [/bib_ref] [bib_ref] Mixed-species malaria infections in humans, Mayxay [/bib_ref] [bib_ref] Plasmodium vivax recurrence following falciparum and mixed species malaria: risk factors and..., Douglas [/bib_ref] and 50% in Myanmar [bib_ref] Effectiveness of five artemisinin combination regimens with or without primaquine in uncomplicated..., Smithuis [/bib_ref]. The intervals between the acute P. falciparum malaria illness and the subsequent P. vivax malaria are similar to those between acute P. vivax malaria and the subsequent P. vivax relapse. The epidemiological characteristics suggest that these are all relapses . It is also interesting and perhaps relevant that in endemic areas, despite often low seasonal transmission, P. vivax maintains a high degree of genetic diversity ## Relapses of vivax malaria arise from activation of latent hypnozoites (alh) Swellengrebel and de Bucknoted that relapse rates were higher in naturally acquired P. vivax infections (up to 50%) than in mosquito-borne infections with the local strains in neurosyphilis patients (despite larger inocula in the latter). They ascribed this to immunity, and this was undoubtedly a contributor, but another explanation is possible. That is that in endemic areas a high proportion of the population have latent P. vivax hypnozoites which can be activated by a sufficient stimulus. Four lines of evidence support this "Activation of latent hypnozoites" (ALH) hypothesis. ## Mixed species infections Mixed blood stage infections with P. falciparum and P. vivax are underestimated by microscopy, but, even with sensitive PCR techniques, this proportion is insufficient to explain the 30% to 50% of patients in SE Asia who experience vivax malaria following falciparum malaria [bib_ref] Plasmodium vivax recurrence following falciparum and mixed species malaria: risk factors and..., Douglas [/bib_ref]. The interval between the primary P. falciparum infection and the subsequent P. vivax malaria strongly suggests this is a relapse . Persuasive supporting evidence that these are P. vivax relapses and not simultaneously acquired infections comes from entomology studies in which single anopheline mosquitoes have been examined for both species [bib_ref] A review of mixed malaria species infections in anopheline mosquitoes, Imwong [/bib_ref]. If these mixed species infections resulted from simultaneous inoculation then 30 to 50% of anopheline vectors carrying one species should also carry the other. In fact finding vectors with both P. falciparum and P. vivax sporozoites is relatively uncommon (overall in Asia 6.6% of P. falciparum sporozoite positive mosquitoes (17 of 258) also contained P. vivax). In the published literature not one of the 45 individually examined malaria positive wild anopheline vectors trapped in Thailand contained both malaria species [bib_ref] A review of mixed malaria species infections in anopheline mosquitoes, Imwong [/bib_ref]. This is also supported by the rarity of finding patients or healthy subjects with gametocytes of both species in the blood at the same time. Development rates in the mosquito are also slightly different (P. vivax being more rapid). Although space-time clustering of infections may occur in low transmission areas it is implausible that over 20% of P. falciparum inoculations would be associated with a separate P. vivax inoculation within one or two days, particularly when individuals receive on average less than one infectious bite per year. There is other supporting anecdotal evidence from travellers and from soldiers with brief periods of exposure in SE Asia. These groups have much lower rates of P. vivax following P. falciparum. The most plausible explanation for these findings is that the majority of these P. vivax episodes arise from hypnozoites which were latent in the liver of the patient at the time of acquiring P. falciparum (ALH). The remarkable similarity of both the timing of the P. vivax recurrences and their variance strongly suggest that latent P. vivax hypnozoites are activated by acute falciparum malaria. The median (IQR, range) Intervals between acute P. falciparum malaria (in green) and acute P. vivax malaria (in red) and the subsequent vivax malaria episode in patients studied in Thailand following different anti-malarial treatments. The figures in brackets are the number of patients studied [bib_ref] High rate of Plasmodium vivax relapse following treatment of falciparum malaria in..., Looareesuwan [/bib_ref] [bib_ref] Mixed-species malaria infections in humans, Mayxay [/bib_ref] [bib_ref] Plasmodium vivax recurrence following falciparum and mixed species malaria: risk factors and..., Douglas [/bib_ref]. * reinfection excluded. ## Heterologous genotypes If P. falciparum malaria activates latent P. vivax hypnozoites then there is no reason why P. vivax malaria should not do the same. This would explain satisfactorily the finding of heterologous genotypes in one half to two thirds of P. vivax relapses [bib_ref] Relapses of Plasmodium vivax infection usually result from activation of heterologous hypnozoites, Imwong [/bib_ref] [bib_ref] Relapses of Plasmodium vivax infection result from clonal hypnozoites activated at predetermined..., Chen [/bib_ref] [bib_ref] High genetic polymorphism of relapsing P. vivax isolates in northwest Colombia, Restrepo [/bib_ref]. In these cases where the original genotype was not detected in the malaria recurrence then either the inoculated infection did not relapse, or its hypnozoite(s) were activated but their progeny were outcompeted by the earlier activation or more rapid growth of the progeny of the activated latent hypnozoite(s). In approximately one third of P. vivax relapses studied in South East Asia, the malaria parasites isolated are either identical or closely genetically related to the primary infection. Relatedness could occur if there is little genetic diversity in the area where P. vivax malaria was acquired, or could result from recombination in the anopheline vector with the production of genetically related sibling sporozoites (i.e. simultaneous inoculation). If the "cross" took place several cycles of infection previously then subsequent infections may contain highly related parasites through successive interbreeding between related siblings. More work on this important area is needed. However in the majority of relapses the parasites are clearly genetically unrelated. In the one third of patients in whom the relapse is homologous or highly related with the primary infection, either there were no latent hypnozoites (as in volunteer studies), or the homologous infection's hypnozoites' progeny won the race to patency against the heterologous hypnozoites' progeny. It is evident then that close "races" between different genotypes to reach patency commonly result in gametocyte genotype mixtures in relapses (which may then recombine in the mosquito). Further support for the ALH hypothesis comes from observations in mothers and their infants living in a malaria endemic area on the north-west border of Thailand. The relapses of vivax malaria in the babies' mothers were usually genetically different to those which caused the primary infection, as in other patients studied in this area, whereas the relapses which followed the first P. vivax infection of life in their babies were usually of the same genotypes as those which caused the initial infection [bib_ref] The first Plasmodium vivax relapses of life are usually genetically homologous, Imwong [/bib_ref]. Obviously the infants could not have any previously acquired latent hypnozoites in their livers to be activated by the illness. ## Natural versus artifical infection relapse rates Higher rates of vivax relapse in indigenous compared with artificial infection would also be explained by the ALH hypothesis. The incidence and number of relapses depends on the number of sporozoites inoculated. If all relapses derived from the inoculated infection then artificial infections which follow inoculations with 5-10 times more sporozoites should have higher, not lower rates of relapse. Relapse rates were particularly high in soldiers who were immunologically naïve and underwent intense exposure. If all relapses derived from the most recent inoculum then there should be no relationship between intensity of exposure and number of relapses. 4. Long-latency also occurs in the tropical frequent relapse "Chesson" P. vivax phenotypes Four of seven volunteers receiving a single infected mosquito bite in Coatney's series had relapses of the Chesson strain of P. vivax with variable but long latency periods -all exceeding six months after their preceding relapse [bib_ref] Studies in human malaria. XXX. A summary of 204 sporozoite-induced infections with..., Coatney [/bib_ref]. It is not uncommon to encounter patients returning from malaria endemic areas where tropical phenotypes are prevalent with relapses more than 3 months after either a primary infection or return to the non-endemic area (of course these might also be longlatency phenotypes particularly if the interval is eight -10 months). This proves that some hypnozoites of frequent relapse phenotypes can remain dormant for long periods. ## Mechanisms of hypnozoite activation If acute malaria activates latent hypnozoites and thereby causes vivax malaria relapses then it seems that a significant systemic illness is necessary for this activation. In the first half of the twentieth century, it was widely believed that a variety of external stresses could bring on a relapse of malaria [bib_ref] Population studies of Plasmodium vivax. 1. The theory of polymorphism of sporozoites..., Lysenko [/bib_ref]. By analogy, relapses in the bird haemosporidian parasite Leukocytozoon are provoked by the stresses of egg laying and the exhaustion of long migratory flights. It was even taught in some textbooks of tropical medicine that Cinchona alkaloids should be given before surgery to pre-empt a relapse of malaria. However formal studies to provoke relapses of vivax malaria, which included forced route marches, simulated altitude, and induced hypoxia, did not yield convincing results [bib_ref] An interim report on the treatment of malaria, Ross [/bib_ref] [bib_ref] Effect of altitude anoxia in provoking relapse in malaria, Howe [/bib_ref]. Nevertheless it is interesting that soldiers in field hospitals in the Mediterranean region between 1940 and 1945 (who had acquired P. vivax in North Africa or Italy) were apparently considerably more likely to experience vivax malaria (presumably a relapse) if they had pneumonia or hepatitis compared with trench foot [bib_ref] Clinical aspects of malaria, Levine [/bib_ref]. In our own series of children seen every day for over 18 months on the Thai-Burmese border during a study of the SPf66 malaria vaccine [bib_ref] Randomised double-blind placebo-controlled efficacy trial of the SPf66 vaccine against falciparum malaria..., Nosten [/bib_ref] P. vivax episodes were associated with malaria, but not with minor illnesses (S Lee; personal communication), so it seems that a substantial systemic stimulus is required. Periodicity can be generated in several ways in biological systems. Illness generally (and the associated cytokine responses) or malaria specifically could provide the activating or inhibitory necessary stimuli to generate periodic relapses in vivax malaria. Overall it seems most likely that the illness associated with the infection itself is the activator in P. vivax relapses , and that each symptomatic episode provides an activation stimulus which may give rise to the next relapse. A simple clock mechanism with a single unimodal distribution of latencies alone is inadequate to explain the observed phenomena. Equally, as explained previously, a multiplicity of different latencies with a single harmonic is very complex and difficult to reconcile with the efficient use of relatively small sporozoite inocula (median~1 0 sporozoites). For efficient yet periodic activation of small numbers of hypnozoites (i.e. so that they do not all activate together) it is necessary either to hypothesise a negative feedback mechanism, which may occur with illness being the negative feedback, or propose simply a relatively broad temporal distribution of latencies (analogous to seeds germinating or eggs hatching), and a low individual susceptibility to activation. Either results in several relapses at regular intervals, each activated by the preceding illness. Importantly the proportion of Proposed mechanism and sequence of Plasmodium vivax relapse activation in a malaria endemic area. In an illustrated example, at the time of infection (sporozoite inoculation) the individual already has hypnozoites of two different genotypes acquired from two previous inoculations which are latent in the liver. The different genotypes are denoted by different colours (red and white). Half the newly acquired infection sporozoites (blue) develop into preerythrocytic schizonts and half become dormant as hypnozoites (the estimated proportions for tropical "strains") [bib_ref] Pre-erythrocytic stage in mammalian malaria parasites, Shortt [/bib_ref] [bib_ref] The life-cycle of primate malaria parasites, Bray [/bib_ref]. Illness associated with the blood stage infection activates a small fraction of the hypnozoites (inset shows a "classic" P. vivax fever pattern in relation to asexual parasite development). In this example there is one hypnozoite of each genotype and each is activated by the illness and each develops into a pre-erythrocytic schizonts. By chance the progeny of one of the preexisting latent hypnozoites reach pyrogenic densities before the progeny of the recently inoculated hypnozoite (inset shows the logarithmic growth of the three genotypically different infections-vertical axis shows number of parasites in the body). The consequent febrile illness then suppresses further multiplication of the blood stage infection so that the progeny of the other two prerythrocytic schizonts may not reach transmissible densities. The ensuing illness activates some of the remaining hypnozoites (the same fraction as were activated initially) and relapses continue until either the number of hypnozoites is exhausted or some fail to be activated. If there are some hypnozoites which fail to be activated these may be activated at a later date by a subsequent malaria infection. susceptible hypnozoites activated would be fractional, at least initially, which fits with the observed fixed proportions of patients who experience multiple relapses [fig_ref] Figure 6: Diagram of the different P [/fig_ref]. It also may leave a significant proportion of unactivated (but activatable) hypnozoites which remain latent until either they die (for example when the host hepatocyte dies) or they are activated by a systemic illness such as malaria. This would explain the high rate of vivax malaria (presumably relapses) following falciparum malaria. It follows that the number of immediate relapses per mosquito inoculation will decrease with increasing age in endemic areas, as the stimulus to hypnozoite activation declines with increasing disease controlling immunity, and immunity increases the probability that the relapse is asymptomatic. In this respect it is interesting that in Thailand the incidence of symptomatic P. vivax peaks in childhood [bib_ref] Treatment of vivax malaria in an endemic area on the western border..., Luxemburger [/bib_ref] , but P. vivax malaria following P. falciparum malaria shows a weaker age relationship. P. falciparum may contribute significantly to P.vivax transmission, particularly in young adults, through this mechanism. It also follows that with small inocula it is quite possible for all sporozoites to develop immediately (early infection, no relapses) or for all to result in hypnozoites, and all the hypnozoites to become latent (no early infection, first infection follows activation stimulus such as a malaria illness). For temperate strains of P. vivax, as suggested by several investigators previously, there are clearly at least two populations of hypnozoites, one becoming activatable early (as for tropical P. vivax) and another remaining latent and not immediately activatable. The study of Cooper et al, in which blood induced homologous (St Elizabeth strain) infections were induced 120 days after infectious mosquito bites, is informative in this respect. The blood inoculations reliably gave rise to symptomatic malaria but critically did not affect the timing of the subsequent relapse. This suggests that the hypnozoites (half way through their sleep) were absolutely refractory at this time. It is likely that once hypnozoites do become activatable, that there is a background relatively low rate of spontaneous activation in order to account for the distribution of latencies. Thus for the long-latency P. vivax the first relapse after the long latent period is usually spontaneous, but the illness then activates further hypnozoites, accounting for the subsequent short inter-relapse intervals. If this is correct it follows that first relapses which occur with a longlatency interval (i.e. 8-10 months after the primary infection) should usually be of a similar genotype to the initial infection, whereas relapses at other times could be heterologous. If there are subsequent relapses i.e. which follow the first relapse after the latent period (i.e. with a short periodicity) then these could be genetically heterologous. Plasmodium vivax in temperate zones has clearly evolved to adapt to the long winters across the Northern hemisphere [bib_ref] The seasonal periodicity of malaria and the mechanism of the epidemic wave, Gill [/bib_ref] [bib_ref] Pathology -Experimental malaria with protracted incubation, Schuffner [/bib_ref] [bib_ref] Dates of onset of relapses and the duration of infection in induced..., Tiburskaja [/bib_ref] [bib_ref] Korean vivax malaria. I. Natural history and response to chloroquine, Hankey [/bib_ref] [bib_ref] Long incubation of Plasmodium vivax multinucleatum as demonstrated in three experimental human..., Jiang [/bib_ref]. It is interesting to speculate that the large proportion of sporozoites dedicated to latency, and the relatively low number of relapses compared with the tropical strains may reflect the high natural wastage of hypnozoites in hepatocytes which die before the hypnozoites become activatable. In experimental P. cynomolgi bastianelli infection in the Rhesus monkey Garnham observed a ten-fold reduction in the number of hypnozoites in serial liver biopsies over a nine month period, but it is not clear how much of this reduction resulted from activation and how much was from cell death [bib_ref] Swellengrebel lecture. Hypnozoites and 'relapses' in Plasmodium vivax and in vivax-like malaria, Garnham [/bib_ref]. The hypnozoite can be considered as an unactivated sporozoite. If the duration of pre-erythrocytic development of the liver stage is similar for sporozoites and hypnozoites, as seems likely, this puts activation at the time or shortly after presentation with acute malaria (of any species). If the ALH hypothesis is correct then the key biological difference between frequent relapse and long-latency P. vivax phenotypes lies in the temporal distribution of susceptibility to activation among the sporozoites. This can then be subdivided into the proportion of sporozoites which activate immediately and the subsequent temporal distribution of susceptibility to activation among the remaining hypnozoites. The genetic basis for this regulation may be difficult to find, as it could well reflect subtle quantitative rather qualitative differences. Recent studies in murine malaria indicate a central role for iron sequestration in controlling pre-erythrocytic development through malaria illness induction of the iron regulatory hormone hepcidin [bib_ref] Host-mediated regulation of superinfection in malaria, Portugal [/bib_ref] [bib_ref] Superinfection in malaria: Plasmodium shows its iron will, Portugal [/bib_ref]. It is possible that iron availability also plays an important regulatory role in controlling hypnozoite activation and pre-erythrocytic development. One possible mechanism contributing to regular short interval (three weeks) relapses is a malaria illness related temporary halt to liver-stage development (mediated by reduced iron availability), which is then lifted with clinical recovery. Some of these hypotheses may be testable in the ex-vivo hepatocyte culture systems [bib_ref] Towards an in vitro model of Plasmodium hypnozoites suitable for drug discovery, Dembele [/bib_ref]. In summary this ALH hypothesis proposes that there is indeed a biological clock (which is most evident in temperate strains) determining latency in Plasmodium vivax. This clock (which could be an intrinsic parasite clock, or could reflect a host-parasite interaction) determines the length of the interval before the hypnozoite becomes susceptible to activation, but that there is a separate sensing mechanism which determines whether or not activation does occur. The trigger could be either a positive activation stimulus or removal of inhibition. This mechanism may activate spontaneously once the hypnozoite has become susceptible, and spontaneous activation presumably usually explains the first relapse after a long latent period, but once susceptible activation is much more likely with an external systemic illness such as malaria. Activation must involve signaling via the infected hepatocyte (which is very sensitive to systemic inflammatory responses). Importantly the individual probability of activation for each hypnozoite is low, allowing accumulation of latent but "activatable" hypnozoites after each sporozoite inoculum. This implies that people living in vivax endemic areas commonly harbour latent but "activatable" hypnozoites. In endemic areas of South-East Asia, if patients who acquire falciparum malaria are representative of the population, this figure is approximately one quarter to one third. The periodicity of P. vivax relapses is derived from the sequential activation of hypnozoites by illness. Mathematical modeling will provide valuable insights into which activation-inhibition model best fits the observed malaria therapy, volunteer, and epidemiological information. ## Implications for epidemiological assessment If the ALH theory is correct it also explains why relapse phenotypes in tropical malaria endemic areas may be difficult to characterize, and why in areas with longlatency P. vivax frequent relapse patterns may still be observed [fig_ref] Figure 22: Potential similarity of relapse patterns with the long-latency and frequent relapse P [/fig_ref]. This is because a primary illness with long-latency P. vivax of the "Madagascar" or "St Elizabeth" phenotype may activate previously acquired latent hypnozoites (of similar phenotype) -giving an early relapse. The illness caused by the relapse could then activate further latent homologous or heterologous hypnozoites. Thus several relapses may follow at short intervals even though all the parasites are of the longlatency type. The net result would be indistinguishable from the epidemiological pattern of frequent relapse vivax malaria [fig_ref] Figure 22: Potential similarity of relapse patterns with the long-latency and frequent relapse P [/fig_ref]. The converse -long-latency with the tropical frequent relapse phenotypes was demonstrated clearly in the studies of Robert Coatney and his colleagues [bib_ref] Studies in human malaria. XXX. A summary of 204 sporozoite-induced infections with..., Coatney [/bib_ref]. If both types are prevalent in the same area then identifying phenotypes from illness patterns becomes even more difficult, and it may be impossible to discern the long-latency phenotypes amongst the frequent relapses. In temperate areas, such as Southern Europe, the early spring-summer wave of vivax malaria which preceded the appearance of anopheline vectors was an obvious clue, but in tropical regions with longer transmission seasons this may be difficult to discern [bib_ref] The seasonal periodicity of malaria and the mechanism of the epidemic wave, Gill [/bib_ref]. The presence of long-latency phenotypes may only be evident in travellers and soldiers who spend a brief period in the endemic area, do not receive primaquine, and then return to a non-endemic area and relapse many months later without re-exposure [bib_ref] Prolonged incubation period of imported P. vivax malaria in London, Warwick [/bib_ref] [bib_ref] The seasonal pattern of Plasmodium vivax malaria in Glasgow, Walker [/bib_ref] [bib_ref] Relapsing vivax malaria cluster in Eritrean refugees, Israel, Kopel [/bib_ref] [bib_ref] Malaria tertiana bei Kindern und Erwachsenen aus dem Epidemiegebiet der südlichen Türkei, Eichenlaub [/bib_ref] [bib_ref] Plasmodium vivax infections in U.S. Army troops: failure of primaquine to prevent..., Smoak [/bib_ref]. These cases provide an important epidemiological signal. There is uncertainty over the true epidemiology of relapse patterns over a large proportion of the P. vivax endemic world. Despite a resurgence of interest in malaria, and the belated recognition of the importance of Plasmodium vivax, this large knowledge gap seems to have gone unnoticed. It is quite possible that long-latency phenotypes are present in many tropical areas [fig_ref] Figure 7: Schematic diagram by Hankey et al[79]of relapse patterns following Korean vivax malaria [/fig_ref]. Defining these patterns is an essential prerequisite for therapeutic assessments, control and elimination planning, and the evaluation of novel interventions such as vaccines. ## Implications for the emergence of anti-malarial drug resistance The factors determining de-novo selection of anti-malarial drug resistance are likely to be similar across all malaria parasites (pattern of drug exposure, parasite biomass, frequency and stability of the genetic or epigenetic mechanism, fitness cost) [bib_ref] Anti-malarial drug resistance, White [/bib_ref]. The much lower parasite biomass in P. vivax malaria makes de-novo selection less likely than in P. falciparum infections, some of which may comprise very large numbers of parasites (> 10 12 ). In malaria, recrudescence is necessary for onward transmission of a de-novo resistant mutant parasites' progeny [bib_ref] Anti-malarial drug resistance, White [/bib_ref]. In P. vivax malaria the de-novo recrudescent infection is effectively in a race with the relapse-either of heterologous or drug sensitive homologous (sibling) parasites. Resistant parasites will only be transmitted if their progeny reach transmissible parasite densities before the relapse does. If the de-novo resistance is high grade then the resistant parasites may expand in numbers rapidly and may not be impeded by the relapse. However if the first step in resistance is a small reduction in susceptibility, as is more usual in anti-malarial drug resistance, then resistance cannot be transmitted onward in infections in which the relapse becomes patent before the recrudescence would have . This is because parasite multiplication falls rapidly once symptoms have developed, and treatment usually follows soon afterwards. Of course the resistant parasites are less drug-sensitive so they will have a "second chance" to recrudesce later if the same drug is used for treatment of relapse, but treatment may change, primaquine may be added, and immune responses are strengthening -all of which reduce the chance of subsequent recrudescence. The relapse therefore pre-empts the expanding intra-host population of de-novo resistant parasites. Together with the lower biomass, and the use of primaquine in "combination therapies", this may explain the slower emergence of low-grade anti-malarial drug resistance in P. vivax compared with P. falciparum. Slowly eliminated drugs interefere with the resistance protection provided by relapse. Chloroquine has a slow multiphasic elimination phase and suppresses the relapse of sensitive parasites for approximately two weeks (from two to six weeks). Mefloquine has a different profile of elimination, and provides longer suppression of vivax relapse. The residual drug levels reduce potential interference with resistance emergence by relapse providing greater suppression of the sensitive compared with the resistant parasites and they therefore reduce the delay on resistance emergence. In summary early relapse provides a brake on the emergence and spread of low-grade resistance in Plasmodium vivax by pre-empting recrudescence. ## Implications for genetic diversity Activation of hypnozoites from different preceding inoculations will commonly result in two or more genotypes reaching patent parasitaemia at similar times. As gametocytogenesis in P. vivax occurs simultaneously with asexual stage development this provides an effective method of increasing the likelihood that a mosquito will ingest gametocytes of different genotypes, thereby facilitating meiotic recombination between genetically unrelated P. vivax parasites. This must be an important contributor to the relatively high degree of genetic diversity in P. vivax often found in areas with very low seasonal transmission. ## Practical implications If the ALH theory is correct then the epidemiology of malaria relapse and the biology of interaction between the species need reconsideration. If long-latency Plasmodium vivax still contributes a significant proportion of vivax malaria in the Indian sub-continent and further west then current methods of assessing drugs and vaccines may also need reconsideration . . Three relapses occur with similar periodicities, and a later randomly activated relapse occurs 8 months later. In the lower panel the long-latency phenotypes are present. Activation occurs as above and the long-latency relapse emerges nine months later. The initial relapse patterns associated with the two different phenotypes are identical. This illustrates the difficulty in excluding the presence of long-latency phenotypes in malaria endemic areas. ## New infection relapses ## Radical treatment therapeutics In therapeutic assessments a follow-up period of six months or less may miss a significant proportion of the relapses. If the majority of relapses in endemic areas derive from heterologous latent hypnozoites then malaria control interventions which are effective will not prevent relapses emerging for months or years, although their number will reduce as the reduced transmission will result in less illness and therefore less hypnozoite activation. Mass drug administration with radical curative regimens (currently primaquine is the only option) would be the only way to eliminate this reservoir of infection quickly. Epidemiological assessments in older children and adults in endemic areas may underestimate the burden of vivax malaria as partial immunity (and premunition) will ameliorate disease severity and may lead to reduced activation of relapses. This would result in relatively low relapse rates. The proportion of acute falciparum malaria infections, which are followed by P. vivax may be a better indicator of the prevalence of latent hypnozoite carriage. It is possible that much of the variance in responses to primaquine is explained by differences in rates and burdens of latent hypnozoite carriage and degree of immunity and not variation in drug susceptibility. The same factors which affect therapeutic responses in the blood stage infection appear to affect the responses to hypnozoitocidal treatment i.e. organism load and immunity. We have tended to consider 8-aminoquinoline efficacy only from the perspective of the drug, and we do not take into account either organism load, organism phenotype, or host immunity. Taking a quantitative approach to assessing 8-aminoquinoline radical curative activity based on hypnozoite burdens may be a valuable approach. Patients with very large liver burdens of hypnozoites from either a very heavy inoculation or multiple inoculations and little or no immunity (such as soldiers) would be expected to have a larger number of relapses than travellers who have a brief period of exposure. Studies of radical curative activity in soldiers may not be comparable to studies in travelers. Studies in adults may not be comparable to studies in children. The apparent radical curative activity of primaquine would be expected to improve as malaria transmission falls. Lower dose regimens may have useful efficacy in this context. Long term follow up (minimum one year) Relapse Relapse pre-empts the emergence of de-novo antimalarial drug resistance. De-novo drug resistance is a rare event and usually there is only one mutant resistant parasite which multiplies while the sibling drug sensitive parasite population declines (green) [bib_ref] Superinfection in malaria: Plasmodium shows its iron will, Portugal [/bib_ref]. Only a highly resistant parasites' progeny (red line) can reach transmissible densities in blood (total numbers circa 100,000,000 in the body) before the relapse (comprising drug sensitive parasites) becomes patent and the consequent illness (and any treatment effect) suppresses multiplication. Slowly eliminated anti-malarial drugs reduce this protective effect by reducing multiplication of the relapse parasites more than multiplication of the de-novo resistant parasites. Epidemiological implications of well characterized patients with parasite genotyping in low transmission settings should help to dissect the contributions of pre-existing versus recently inoculated hypnozoites to relapse. These should be accompanied by entomology and genotyping studies in wild-trapped anopheline vectors to determine mixed genotype and genetic recombination rates. ## Immunity It is interesting to speculate on the role of relapse in enhancing the opportunities for recombination and how, in the pre-anti-malarial era, relapse would have contributed to persistence of the untreated infection. Rechallenge experiments showed that a "strain specific" immunity developed after protracted symptomatic infection with that P. vivax strain. But this did not always prevent subsequent parasitaemia, particularly if rechallenge was many years later [bib_ref] Further observations on the duration of immunity to the homologous strain of..., Boyd [/bib_ref]. Despite considerable investment in a P. vivax vaccine there is relatively little information how immunity to vivax malaria is acquired and maintained in the context of frequent infection and relapse with different genotypes [bib_ref] Differential patterns of infection and disease with P. falciparum and P. vivax..., Lin [/bib_ref]. It is generally recognized that asymptomatic P. vivax infections are common in endemic areas but their overall contribution to P. vivax transmission in malaria-endemic areas, and the importance of relapse in maintaining these asymptomatic infections has not been characterized adequately. However the malaria therapy experience suggested that asymptomatic infections were a very important source of infection [bib_ref] Further observations on the duration of immunity to the homologous strain of..., Boyd [/bib_ref]. An effective vaccine which did not confer longlasting immunity and required frequent boosting might provide a selective pressure towards longer latency. There are major questions about the basic biology and the epidemiology of Plasmodium vivax relapse which first need recognizing, and then need answering, if we are to address seriously controlling and eliminating this important malaria parasite [bib_ref] The neglected burden of Plasmodium vivax malaria, Mendis [/bib_ref]. [fig] Figure 6: Diagram of the different P. vivax phenotypes and the usual patterns of primary illness and relapse (after Bray and Garnham[69]). The thickness of the lines gives a rough approximation of proportions. [/fig] [fig] Figure 7: Schematic diagram by Hankey et al[79]of relapse patterns following Korean vivax malaria (upper panel) and tropical frequent relapse P. vivax (lower panel). Note the frequent relapse pattern after a long interval with Korean vivax malaria. [/fig] [fig] Figure 10: Lengthening intervals between sequential relapses in individual volunteers who were infected with the Chesson strain of P. vivax, each of whom was treated with quinine for 16 days[83]. Diamonds represent median values. [/fig] [fig] Figure 11: In infections of volunteers with the St Elizabeth strain of P. vivax[71], there was no evident relationship between the occurrence or duration of the primary illness and the long-latency interval before illness (left panel), whereas there was an inverse relationship between sporozoite inocula (assessed semi-quantitatively from sporozoite numbers in the salivary glands and number of infectious bites) and the latency interval. [/fig] [fig] Figure 12: Cumulative proportions of relapses in soldiers with vivax malaria acquired in the Pacific and treated subsequently with different anti-malarial drug regimens in Chicago. Regimens were quinine; 2 g/day for 14 days (N = 75), mepacrine 0.4 g/day for 7 days (N = 69), chloroquine 1.5 -2 g/day for 4-7 days (N = 82), and quinine 2 g/day and plasmoquine 60 mg/day for 14 days (N = 72)[47]. [/fig] [fig] Figure 13 P: intermediate group, if it exists, has not been well characterized. The ratio of short to long-latency relapse phenotypes in Aligarh, Uttar Pradesh was estimated as 4. vivax relapse rates without radical treatment in published studies conducted between 1920 and 2010. Each circle represents one study arm. The horizontal scale represents the study location from West (United States) to East (Pacific). A very wide range of treatments were used in a very diverse range of patient groups. [/fig] [fig] Figure 14 P: vivax relapse rates following treatments which included 8-aminoquinolines (mainly plasmoquine or primaquine). [/fig] [fig] Figure 15: Proportions of P. vivax relapses in 222 US servicemen who had fought in the South Pacific in the Second World War[76]. [/fig] [fig] Figure 16: The proportions of patients experiencing successive P. vivax relapses taken from eight different clinical series: These were US soldiers with vivax malaria acquired in the South Pacific (2 series) [/fig] [fig] Figure 22: Potential similarity of relapse patterns with the long-latency and frequent relapse P. vivax phenotypes. The different colours and symbols represent different genotypes in two hypothetical infections. The upper panel shows the relapse pattern where the frequent relapse phenotypes are prevalent. The patient has hypnozoites from three preceding inoculations present at the time of illness from the newly acquired (red) infection. These are sequentially activated as proposed in [/fig] [table] Table 1: Relationship between the proportion of patients relapsing with vivax malaria and total number of relapses experienced [/table] [table] Table 2: Radical treatment pharmacodynamics; Quantitative considerationsConsider two groups of patients who represent two extremes Without radical treatment group A has an 80% relapse rate (eg some soldiers who fought in the Pacific in the Second World War) Without radical treatment group B has a 20% relapse rate (eg some soldiers who fought in the Korean War) Assuming a fixed fractional proportion of relapses and no acquisition of immunity, then the total number of relapses/100 patients is These numbers represent the minimum number of viable activatable hypnozoites (VAH) i.e. there are 16 times more in group A compared to group B. It is likely that the distribution of VAH is random among the patients and therefore conforms to a Poisson distribution. [/table]
Distinct gene expression profiles associated with the susceptibility of pathogen-specific CD4+ T cells to HIV-1 infection # Background HIV infection causes the progressive depletion of CD4+ T cells. Contrary to the early loss of CD4 response to opportunistic pathogens like Candida albicans, cytomegalovirus (CMV)-specific CD4 response is persistent when total CD4+ T cell number is low. The mechanism is less clear. Despite considerable knowledge for the impact of HIV infection on total CD4+ T cells and their subsets, little is known about HIV infection of CD4+ T cells of different pathogen/antigen (Ag) specificity. # Methods PBMC from HIV-negative donors were CFSE-labeled and stimulated ex vivo with pathogen-specific antigens including viral (CMV), bacterial (Tetanus Toxoid: TT) and fungal (Candida albicans) antigens. HIV infection of Ag-specific CD4+ T cells was determined by intracellular p24 production in CFSE-low population. # Results While TT-and Candida-specific CD4+ T cells were permissive, CMV-specific CD4+ T cells are highly resistant to both X4 and R5 HIV independent of coreceptor useage. Quantification of HIV DNA in sorted, antigenspecific CD4+ T cells demonstrated a reduction of both strong-stop and full-length HIV DNA in CMV-specific CD4+ T cells. β-chemokine neutralization enhanced HIV entry and viral replication in TT-and Candidaspecific CD4+ T cells, whereas HIV infection in CMVspecific CD4+ T cells remained low despite increased HIV entry by β-chemokine neutralization, suggesting both entry and post-entry HIV restriction in CMVspecific cells. Microarray analysis revealed distinct gene expression profiles that involved selective upregulation of a broad array of antiviral genes in CMV-specific CD4+ T cells, whereas TT-and Candida-specific CD4+ T cells mainly upregulated a Th17 inflammatory response. # Conclusion Our data suggest a mechanism for the persistence of CMV-specific CD4 response and the earlier loss of mucosal Th17-associated TT-and Candida-specific CD4 response in AIDS patients. The model described is useful in HIV vaccine studies by evaluating the susceptibility of vaccine-specific CD4 responses to HIV infection. © 2012 Hu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Correlation Analysis of Serum Pepsinogen, Interleukin, and TNF-α with Hp Infection in Patients with Gastric Cancer: A Randomized Parallel Controlled Clinical Study Background. Gastric cancer pathological biopsy and visual examination have been the gold standard for gastric cancer diagnosis, but their operation is costly, demanding, and risky, so it is especially important to find an effective examination method in clinical practice. Aims. To investigate the correlation between serum pepsinogen I (PGI), pepsinogen II (PGII), pepsinogen I and II ratio (PGR), IL-6, and TNF-α and Helicobacter pylori (Hp) infection in patients with gastric cancer. Materials and Methods. Fifty patients with Hp-infected gastric cancer admitted to the Department of Gastroenterology of our hospital from January 2019 to December 2021 were selected for the study as the observation group, and another 50 patients without Hp-infected gastric cancer were selected as the comparison group to compare the correlation analysis of PGI, PGII, PGR, IL-6, and TNF-α with Hp infection between the two groups after admission and treatment. Results. After measurement, PGI and PGII in the observation group were significantly lower than those in the comparison group, and TNF-α, IL-18, and IL-6 in the observation group were significantly higher than those in the comparison group, and the comparative differences were all statistically significant (P < 0:05). The results of multivariate logistic regression model analysis of independent risk factors for gastric cancer showed that IL-18, hs-CRP, and tumor necrosis factor-(TNF-) α were risk factors for Hp infection in gastric cancer. Conclusion. The expression of IL-18, hs-CRP, and TNF-α factors in Hp-infected gastric cancer patients is correlated. IL-6, IL-18, and TNF-α are involved in the entire process from the onset to the development of Hp-positive gastric mucosal inflammation in patients, which is of great value in the diagnosis of gastric cancer and helps to assess the degree of progression and prognosis of gastric cancer. # Introduction Epidemiological surveys in recent years have shown that the global morbidity and mortality of gastric cancer have been decreasing year by year, but it is still a common clinical malignant tumor [bib_ref] Atypical diametric polyps and the incidence of diametric cancer: a retrospective cohort..., Jacobs [/bib_ref]. The incidence of gastric cancer in my country ranks first among various malignant tumors, and it also accounts for 35% of the global incidence [bib_ref] Proliferation in postmenopausal endometrial polyps-a potential for malignant transformation, Adomaitienė [/bib_ref]. Epidemiological surveys have shown that with age, the incidence of H. pylori infection will continue to increase [bib_ref] Endometrial stromal polyps in rodents: biology, etiology, and relevance to disease in..., Davis [/bib_ref]. In recent years, studies have confirmed that H. pylori is closely related to the occurrence of gastric cancer, because H. pylori can cause damage to the gastrointestinal mucosa and promote the regeneration of gastrointestinal cells, thereby increasing the risk of injury [bib_ref] Risk factors associated with endometrial pathology in premenopausal breast cancer patients treated..., Lee [/bib_ref]. H. pylori infection can increase the generation of oxygen free radicals, which leads to the peroxidation of gastric mucosal epithelium, which induces symptoms such as acid reflux, nausea, upper abdominal pain, and belching and is closely related to the incidence of gastric cancer [bib_ref] Atypical endometrial polyps and concurrent endometrial cancer, De Rijk [/bib_ref]. Interleukin-18 (IL-18), high-sensitivity C-reactive protein (hs-CRP), and tumor necrosis factor-α (TNF-α) are highly sensitive markers of inflammatory factors [bib_ref] Uterine polyps, adenomyosis, leiomyomas, and endometrial receptivity, Munro [/bib_ref]. Studies have confirmed that these inflammatory factors can induce and aggravate inflammatory responses, and IL-18 is involved in the occurrence and development of various malignant tumors including gastric cancer through various mechanisms . After the inflammatory reaction, due to the continuous increase of free radicals and the generation of superoxide, the cells undergo peroxidative damage, resulting in longterm cellular tumors and gastric cancer. H. pylori is a Gram-negative bacterium with multiple genotypes and secretes a variety of exogenous toxins [bib_ref] Newly Edited Practical Obstetrical and Gynecological Ultrasonography, Wu [/bib_ref]. It can damage the gastric mucosal epithelium, and the damage mechanisms include cell DNA damage, mitochondrial base expression affecting gastric mucosal epithelial cells, and cell proliferation disorders . Studies have shown that H. pylori is closely related to gastritis, peptic ulcer, and gastric cancer. H. pylori infection is an important factor in the incidence of gastric cancer and is listed as the first carcinogen by the World Health Organization [bib_ref] Diagnosis and management of endometrial polyps: a critical review of the literature, Salim [/bib_ref]. The relationship between H. pylori infection and cell proliferation and apotheosis is a current research hotpot [bib_ref] Endometrial polyps and abnormal uterine bleeding (AUB-P): what is the relationship, how..., Clark [/bib_ref]. The stability of the gastric environment requires a balance between proliferation and apotheosis of gastric colossal cells. Although the rate of cell loss caused by apotheosis is comparable to the rate of new cell formation, H. pylori infection will affect this balance and lead to the occurrence of many gastric diseases. It has been suggested that Hp infection affects PG changes, and Hp infection plays an important role in gastric carcinogenesis, but its exact mechanism of action is not clear. Hp is a trigger for a number of diseases, and Hp activates disease cells, causing them to accumulate in the gastric mucosal tissues, creating a mediated inflammatory response, and the levels of cytokines in chronic gastritis vary significantly. The higher the level of Hp in the patient, the more severe the degree of infection of the patient's gastric mucosa, and the degree of erosion of the Hp-positive gastric mucosa by disease cells has an important relationship with the density of Hp. Peptic ulcer is a common gastrointestinal disease with a complex etiology, and Hp infection is one of its important causes. We believe that Hp infection affects PG changes and Hp infection plays an important role in gastric carcinogenesis, but its specific mechanism of action is not clear. The reason why Hp affects PG changes and causes cancer may be related to chronic inflammatory stimulation of Hp leading to atrophic intestinalization of gastric tissues and damage to PG genes, and some scholars believe that it is related to the host's own immune disorder caused by Hp. In this study, 100 patients admitted to our hospital from January 2016 to January 10, 2020, were selected for exploration and analysis and the correlation analysis of serum pepsin, IL-6, and TNFα with Hp infection in patients with gastric cancer. The report is as follows. # Material and methods ## Research Object. Fifty cases of Hp-infected gastric cancer patients admitted to the Department of Gastroenterology of our hospital were selected for the study as the observation group, and another 50 cases of patients without Hp infection were selected as the comparison group. Inclusion criteria [bib_ref] Vaginoscopy for office hysteroscopy: a systematic review & metaanalysis, Silva [/bib_ref] : (i) after gastroscopic biopsy histopathological examination, pathology and imaging consistent with the diagnosis of gastric cancer; (ii) age 18-65 years, no history of special medication (gastric mucosal protective agents, nonsteroidal antiinflammatory drugs, acid suppressants, and antibacterial drugs) in the previous 2 months, all selected patients accepted and voluntarily joined the experiment; (iii) no history of relevant vaccinations, approved by the hospital ethics committee and those who signed the informed consent. Exclusion criteria: (i) with infectious diseases, (ii) with severe liver and kidney impairment, and (iii) with mental illness (interfering with our study or affecting the results of the trial). ## 2.2. Methods. Serum-related factor assay: 2 ml of fasting elbow venous blood was collected from both groups in the early morning, and the serum was separated after centralization at 3000 r/min for l0min, and the level of CRP was measured by enzyme-linked immunodeficient assay kit (Bioengineering Shanghai Co., Ltd.); IL-18 and TNF-α were measured by IMMU-NITE1000 luminescence analyzer (Thermo Fisher) (kit: Institute of Radiology and Immunology, PLA General Hospital). The 2000 luminescence instrument from Abbott and the accompanying PGI and PGII reagents were used to determine PGI and PGII levels and calculate the PGI/PGII (PGR) ratio, which was determined by the luminescence method; patients were kept in a fasting state before undergoing gastropod, 3 ml of venous blood was collected, serum was separated to obtain serum and tested, and the degree of gastric colossal inflammation was measured. The degree of inflammation of the gastric mucous was observed with a low-mounted microscope, and the values of IL-6, IL-18, and TNF-α were recorded and analyzed. Hp assay: the anti-Hp antibody characterization kit (Shanghai Changchun Technology Co., Ltd., enzymelinked immunodeficient assay) was used to detect the level of Hp-IgG antibody in the serum of the two groups of patients. # Statistical analysis. All statistical data in this study were entered into Excel software by the first author and the corresponding author, respectively, and the statistical processing software was SPSS 25.0 for calculation. Repeated measures analysis of variance between groups was used to measure the measurement expressed as mean ± standard deviation (X ± S). Count data expressed as a percentage (%) were tested by χ 2 . Univariate and logistic multivariate regression analysis was used to compare the influencing factors, and the risk factors with significant differences were screened. Correlation test used logistic regression linear correlation analysis. Included data that did not conform to a normal distribution were described by M (QR), using the Mann-Whitney test. All statistical tests were two-sided probability tests. The statistical significance was P < 0:05. # Results ## 3.1. Comparison of Baseline Data. The differences in mean age, gender, lesion diameter, tumor classification, and body mass index between the two groups were not statistically significant (P > 0:05). See [fig_ref] Table 1: Comparison of baseline information between the two groups of patients [/fig_ref]. ## Serum index comparison. After measuring, PGI (95:76 ± 3:14) and PGII (16:36 ± 4:90) in the observation group were significantly lower than those in the comparison group, and TNF-α (82:28 ± 4:24), IL-18 (19:76 ± 3:14), and IL-6 (9:13 ± 1:01) in the observation group were significantly higher than those in the comparison group, and the differences were statistically significant (P < 0:05). See . ## Comparison of gastric cancer stage and related Indicators. The higher clinical stage of gastric cancer led to higher serum gastrin-17, CEA, and CA199 levels and lower pepsinogen I levels, and the comparative differences were statistically significant (P < 0:05). However, the differences of pepsinogen I/pepsinogen II and H. pylori positivity rates in patients with different gastric cancer stages were not statistically significant (P > 0:05). See 3.4. Multiword Regression Analysis. The variables with significant differences were assigned, namely, IL-18, CA199, CEA, hs-CRP, and TNF-α (normal = 1, abnormal = 0). The results of multivariate logistic regression model analysis of independent risk factors for gastric cancer showed that IL- : Comparison of serum indicators. In this study, the statistics of the pain VAS scores of the two groups of patients were entered into Excel software by the first and corresponding author, respectively, and the included data were tested using the Shapiro-Wilk method of mean ± standard deviation of the measured data conforming to a normal distribution. And independent sample or paired sample t-tests were implemented between or within groups. PGI and PGII in the observation group were significantly lower than those in the comparison group, and TNF-α, IL-18, and IL-6 in the observation group were significantly higher than those in the comparison group, and the comparative differences were all statistically significant (P < 0:05). 18, hs-CRP, and TNF-α were risk factors for Hp infection in gastric cancer, and the comparative differences were all statistically significant (P < 0:05). See [fig_ref] Table 2: Multiword logistic regression analysis [/fig_ref]. # Discussion Hp infection is an important trigger of chronic gastritis and peptic ulcer, and research data show that some immune mechanisms and inflammatory responses play an important role in the pathogenesis of gastric mucosa, and nowadays, diseases due to cytokine triggers are getting more and more attention [bib_ref] Cysto-vaginoscopy of a 3D-printed cloaca model: a step toward personalized noninvasive preoperative..., Krois [/bib_ref]. Hp-positive chronic gastritis and peptic ulcer make some cells in patients to form an immune response, in which endocrine secretion of serum IL-6, IL-8, and TNF-α and other cytokines is endocrine raised [bib_ref] Use of hysteroscope for vaginoscopy or hysteroscopy in adolescents for the diagnosis..., Johary [/bib_ref]. Hp is the trigger of some diseases, Hp activates disease cells and causes them to accu-mulate in the gastric mucosal tissue, forming a mediated inflammatory response, and the levels of cytokines in chronic gastritis differ significantly [bib_ref] Management of mid-urethral slings erosions by vaginoscopy using the Gel-POINT® system (with..., Dubuisson [/bib_ref]. The higher the level of Hp in the patient, the more severe the degree of infection of the patient's gastric mucosa, and the degree of erosion of the Hp-positive gastric mucosa by the disease cells has an important relationship with the density of Hp [bib_ref] Colonoscope-mediated vaginoscopy for diagnostic evaluation of colovaginal fistulas, Latchana [/bib_ref]. Peptic ulcer is a common gastrointestinal disease with a complex etiology, and Hp infection is one of its important causes [bib_ref] Vaginoscopy compared to traditional hysteroscopy for hysteroscopic sterilization. A randomized trial, Chapa [/bib_ref]. We found that Hp can secrete a large number of pathogenic factors via regulation of relevant signaling pathways, and long-term persistent Hp infection induces immune and inflammatory responses and produces carcinogenic substances, thus playing a role in the progression of precancerous diseases [bib_ref] The role of examination under anesthesia (EUA) and vaginoscopy in pediatric and..., Nakhal [/bib_ref]. In addition, persistent Hp infection leads to an increased rate of DNA damage, and inflammation can lead : Comparison of gastric cancer staging and related indicators. In this study, statistics of pain VAS scores for both groups were entered into Excel software by the first and corresponding authors, respectively, and measures of gastrin-17, CEA, CA199, PGI, and PGI/ PGII were tested for inclusion by the Shapiro-Wilk method of mean ± standard deviation. And independent sample or paired sample t -tests were implemented between or within groups, and HP-positive rate count data were expressed as whole numbers and found by chisquare test. Higher clinical stage of gastric cancer resulted in higher serum gastrin-17, CEA, and CA199 levels and lower PGI levels, all with statistically significant differences in comparison (P < 0:05). However, the differences in PGI/PGII and HP-positive rates were not statistically significant in patients with different gastric cancer stages (P > 0:05). to the production of large amounts of superoxide and free radicals, reducing the concentration of vitamin C in the gastric juice [bib_ref] Vaginal foreign body: successful management with vaginoscopy, Yıldız [/bib_ref]. This makes the cells less tolerant to oxidative damage and prone to peroxidative damage [bib_ref] Evaluation of vaginoscopy for the diagnosis of clinical endometritis in dairy cows, Leutert [/bib_ref]. The possible mechanisms linking Hp infection to the development of gastric cancer are considered to include the following: oxidative damage, type of Hp strain, genetic variants and differences in their expression, and abnormal kinetics related to gastric mucosal epithelial cells [bib_ref] Laparoscopylike operative vaginoscopy: a new approach to manage mesh erosions, Billone [/bib_ref] [bib_ref] Randomized study of vaginoscopy and H Pipelle vs traditional hysteroscopy and 6..., Ngu [/bib_ref] [bib_ref] An earring incidentally diagnosed and removed through two-step vaginoscopy in a pubertal..., Di Spiezio [/bib_ref] [bib_ref] Resection of vaginal recurrence of granulosa cell tumor by pneumovaginal endoscopic surgery, Kita [/bib_ref]. Our study showed showing that serum IL-6 and 1L-18 has an important factor in gastrointestinal diseases and also provides a diagnostic basis for patients with early gastric cancer [bib_ref] Extended lactation in high-yielding dairy cows. I. Effects on reproductive measurements, Niozas [/bib_ref]. Elevated serum IL-6 levels have been reported to be positively correlated with the development of bone tumors, and higher levels of IL-6 have been found in tumor cells and tumor-associated macrophages, and it has been found that tumor cells may produce higher levels of IL-6 during proliferation, invasion, or metastasis [bib_ref] Tumoral and pseusotumoral processes of the vagina in the pediatric population: a..., Thibault [/bib_ref]. Elevated serum IL-6 levels were positively correlated with tumor load and disease progression, and IL-6 levels were significantly elevated in patients with gastric cancer with lymph node metastasis [bib_ref] Utilizing colpocleisis to repair a vesicovaginal fistula in a cervical cancer patient..., Dahman [/bib_ref]. Serum pepsinogen, an endoproteinase with digestive function, is divided into two subgroups, PGI and PGII, according to immunology and biochemistry [bib_ref] A challenging diagnosis and management of Herlyn-Werner-Wunderlich syndrome in low-resource settings: a..., Jomaa [/bib_ref]. Among them, PGI is mainly expressed in cervical mucus cells and principal cells of the gastric fundus and is especially highly expressed in the gastric mucosa of embryos [bib_ref] Vaginoscopic incision of oblique vaginal septum in adolescents with OHVIRA syndrome, Cheng [/bib_ref]. Among them, PGII is mainly expressed with all duodenal Brunner's gland and gastric glands and to a lesser extent in prostate and pancreas [bib_ref] The invisible external cervical os. Tips and tricks to overcome this challenge..., Di Spiezio [/bib_ref]. Most of the synthesized pepsinogen is secreted into the gastric lumen and activated into pepsin by the action of acidic gastric juice, and the pepsinogen that enters the circulation is very stable [bib_ref] Management of cervix atresia with hematometra by "genitoscopic ultrasound-guided cervix fenestration and..., Jan [/bib_ref]. The measurement of serum pepsinogen changes can reflect the degree of gastric mucosal lesions and differentiation, which is beneficial for the early diagnosis of gastric cancer and is important for the prevention of gastric cancer, and it is also considered to be the best serological indicator for the histology of gastric mucosa [bib_ref] In-office hysteroscopic treatment of Herlyn-Werner-Wunderlich syndrome: a case series, Fascilla [/bib_ref]. Therefore, it is considered that serum pepsinogen can be used for the initial screening of gastric cancer and as an ideal tumor marker for the diagnosis of gastric cancer [bib_ref] Does cervical preparation before outpatient hysteroscopy reduce women's pain experience? A systematic..., Cooper [/bib_ref]. In our study, pepsinogen I and pepsinogen II in the observation group were significantly lower than those in the comparison group, and TNF-α and IL-6 in the observation group were significantly higher than those in the comparison group after measurement. The findings may suggest that elevated IL-6 levels may be an indicator of further deterioration of gastric cancer patients or suggest that the tumor may metastasize, suggesting appropriate therapeutic measures to control the development of the disease, and the experimental results have some clinical guidance [bib_ref] Overcoming the challenging cervix: identification and techniques to access the uterine cavity, Wood [/bib_ref]. TNF-α is one of the cytokines with the strongest antitumor effect, and both in vivo and ex vivo experiments have shown that TNF-α has very significant antitumor effect. Patients with higher tumor TNF-α values are more prone to metastasis and recurrence after treatment [bib_ref] Cloacal malformations: technical aspects of the reconstruction and factors which predict surgical..., Wood [/bib_ref]. Therefore, TNF-α can be used as an important marker for tumor recurrence and metastasis, as well as an indicator for identifying pretumor lesions, and the experimental results are consistent with the literature [bib_ref] Ulcerative colitis of the neovagina in a toddler with cloaca and chronic..., Erculiani [/bib_ref]. The mean value of serum TNF-α in patients with lymph node metastasis from gastric cancer was nearly 6-fold higher than normal, and the mean value of serum TNF-α in patients with postoperative recurrence was nearly 3-fold higher than normal, which may be due to increased tumor load and excessive release of TNF-α from activated lymphocytes in vivo, resulting in increased serum TNF-α levels [bib_ref] Disk battery as a vaginal foreign body in a five-yearold preadolescent child, Al-Oufi [/bib_ref]. Our study also suggests that the determination of TNF-α activity in serum of gastric cancer patients, like the determination of IL-6, can be used as an adjunct to observe the progress of patients' disease and judge the deterioration or tumor metastasis, which has some reference value for clinicians [bib_ref] Robot-assisted repair of complex vesicovaginal fistulae: feasibility and outcomes, Chandna [/bib_ref]. Since the level of serum pepsinogen directly reflects the function of gastric mucosa, the significant decrease in serum pepsinogen I level in gastric cancer patients suggests that the secretion capacity of gastric mucosa of gastric cancer patients is reduced, and the significant decrease in serum pepsinogen I level in gastric cancer patients is related to atrophy, intestinalization, and reduced secretion of gastric mucosa in gastric cancer patients . There was no statistically significant change in serum PC II levels in gastric cancer patients, which may be related to the wide distribution of 11 cells secreting pepsinogen . A significant decrease in serum pepsinogen I is clinically important for the monitoring of early gastric cancer, and pepsinogen I and pepsinogen II are elevated in patients with gastric ulcer, whereas serum PGI and pepsinogen II are significantly lower in patients with cancer . Multifactorial logistic regression analysis in our study showed that serum pepsinogen I, TNF-α, and IL-6 in gastric cancer were independent risk factors for Hp infection as a complication of gastric cancer. It indicates that serum pepsinogen and IL-6 tests can predict Hp infection in gastric cancer . We believe that serum pepsinogen and IL-6 predicting Hp infection in gastric cancer also has some limitations to some extent, but the combination of separate tests can be used to improve the sensitivity of detection, early detection of Hp infection in gastric cancer, early intervention, and improve the survival rate of patients . Existing clinical studies have shown that peripheral blood of gastric cancer patients showed a significant increase in serum pepsinogen, IL-6, which was correlated with tumor diameter and highly pelvic lymph node metastasis in gastric cancer patients, which is considered a factor associated with gastric cancer development and prognosis. IL-6 is a functional protein, mainly expressed by macrophages and epidermal cells, and is a common inflammatory factor with immunomodulatory and inflammation-mediated response. It has an important role in tissue injury and repair stages. Studies have shown phantom that the expression capacity of IL-6 is significantly lower in healthy humans than in patients with gastrointestinal diseases. IL-8 is mainly secreted by macrophages and epidermal cells and is also a common inflammatory factor with inflammation-mediating and endothelial cell proliferation effects, accelerating angiogenesis role. IL-8 has an important role at the beginning of the pathogenesis of diseases such as insulin, inflammation, and 5 Computational and Mathematical Methods in Medicine xanthogranuloma. TNF-α is a class of cytokines with multiple activities, which can overactivate leukocytes. This increases the adhesion of leukocytes to endothelial cells and makes leukocytes more easily phagocytized. The current study showed that when patients were infected with Hp, the concentration of TNF-α in the organism was significantly increased. In conclusion, the expression of IL-18, hs-CRP, and TNF-α factors in Hp-infected gastric cancer patients is correlated, and IL-6, IL-18, and TNF-α are involved in the entire process from the onset to the development of inflammation in the Hp-positive gastric mucosa of patients, which is of great value in the diagnosis of gastric cancer and helps to assess the degree of progression and prognosis. ## Data availability No data were used to support this study. ## Conflicts of interest There are no conflicts of interest. # Authors' contributions Shunxin Hao and Minyue Shou contributed equally to this work. [table] Table 1: Comparison of baseline information between the two groups of patients. [/table] [table] Table 2: Multiword logistic regression analysis. [/table]
Loci That Control Nonlinear, Interdependent Responses to Combinations of Drought and Nitrogen Limitation Crop improvement must accelerate to feed an increasing human population in the face of environmental changes. Including anticipated climatic changes with genetic architecture in breeding programs could better optimize improvement strategies. Combinations of drought and nitrogen limitation already occur world-wide. We therefore analyzed the genetic architecture underlying the response of Zea mays to combinations of water and nitrogen stresses. Recombinant inbreds were subjected to nine combinations of the two stresses using an optimized response surface design, and their growth was measured. Three-dimensional response surfaces were fit globally and to each polymorphic allele to determine which genetic markers were associated with different response surfaces. Three quantitative trait loci that produced nonlinear surfaces were mapped. To better understand the physiology of the response, we developed a model that reproduced the shapes of the surfaces, their most characteristic feature. The model contains two components that each combine the nitrogen and water inputs. The relative weighting of the two components and the inputs is governed by five parameters, and each QTL affects all five parameters. We estimated the model's parameter values for the experimental surfaces using a mesh of points that covered the surfaces' most distinctive regions. Surfaces computed using these values reproduced the experimental surfaces well, as judged by three different criteria at the mesh points. The modeling and shape comparison techniques used here can be extended to other complex, high-dimensional, nonlinear phenotypes. We encourage the application of our findings and methods to experiments that mix crop protection measures, stresses, or both, on elite and landrace germplasm.KEYWORDS Crop improvement will need to accelerate in the coming decade, as the human population increases and the abiotic environment changes [bib_ref] Climate change impacts on global food security, Wheeler [/bib_ref]. Improving cultivars to be more re-silient to stress is key. While breeding programs can increase the rate of genetic gain by using genotype prediction to shorten the breeding cycle time [bib_ref] Does genomic selection have a future in plant breeding?, Jonas [/bib_ref] , crop improvement depends on the immediate agricultural context, complicating selection schemes [bib_ref] Predicting the future of plant breeding: complementing empirical evaluation with genetic prediction, Cooper [/bib_ref]. So far, cultivar improvement in maize has not significantly increased stress tolerance on a large scale [bib_ref] Greater sensitivity to drought accompanies maize yield increase in the U, Lobell [/bib_ref]. But there is ample potential for improvement: in yield competitions, the maximum is typically one-third higher than the average [bib_ref] Yield potential, yield stability and stress tolerance in maize, Tollenaar [/bib_ref]. Many breeding programs select germplasm in multiple environments, and model weather and soil inputs using multivariate methods or crop models. A crop model is an agronomic model of a crop's productivity that incorporates genotypic, environmental, and management information into a system of equations [bib_ref] Use of crop growth models with whole-genome prediction: application to a maize..., Cooper [/bib_ref]. Models used in predicting crop productivity via genomic selection and crop modeling make two sets of assumptions. The first set includes additivity and the choice of testing environments. To fit growth and to predict genotype-environment interactions, the current models typically assume an additive relationship among individual loci, the environmental and managerial contexts, and the phenotypes of interest. Using single genetic coefficients and a one-SNP-one-input relationship, the models partition the phenotypes' variances into additive components that are each determined by a single QTL [bib_ref] Use of crop growth models with whole-genome prediction: application to a maize..., Cooper [/bib_ref]. Thus, these models imply that any QTL candidate can be exchanged for another that exhibits an effect. Epistasis among the QTL is not considered, except as a last resort in interpreting the data [bib_ref] The genetic architecture of quantitative traits cannot be inferred from variance component..., Huang [/bib_ref]. To predict the phenotypes of a line in a novel environment, current approaches attempt to infer performance from the same or genotypically related lines in a set of testing environments. These environments do not necessarily include the most predictive ones under climate change [bib_ref] Identification of drought, heat, and combined drought and heat tolerant donors in..., Cairns [/bib_ref]. Further, the equations used to characterize environments can be nonlinear in the crop models, but the genotypes' interactions with the environments are usually modeled linearly. These complications decrease prediction accuracy in many circumstances (for example, see [bib_ref] Use of crop growth models with whole-genome prediction: application to a maize..., Cooper [/bib_ref] [bib_ref] Contribution of crop models to adaptation in wheat, Chenu [/bib_ref]. To understand the second set of assumptions, it helps to clarify several ideas about phenotypes. We define a complex phenotype as a function of at least three dimensions, of which at least one is an input, or independent, variable and at least one is an output, or dependent, observed variable. The set of dimensions can include measurements of different features of the phenotype, genotypic information, and environmental and cultivation conditions. The set of dimensions forms a point in an n-dimensional space, and each point corresponds to an experimental unit such as a plant, row, or plot. Each phenotypic point represents a particular instantiation of the phenotype. Together, the set of points that have the same definitions for each dimension comprise a phenotype. The points lie in a phenotypic space, and the surface they delimit is a phenotypic surface. (Throughout, we use the more general term "surface" to denote surfaces in any number of dimensions, including the "curves" traced by the successive positions of a single point in an n-dimensional space.) Because the points are high-dimensional, the phenotypes they denote are also surfaces, not scalars. We will refer to the phenotype defined by a group of phenotypic points as the phenotypic collection when needed to distinguish that from the common genetic usage of "phenotype". The second set of assumptions is that the phenotypic surface is determined by a mathematically linear function of the genotypes and the cultivation contexts. Each of the terms in an equation relating a phenotypic response to genotypes, contexts, and noises has a coefficient and a single term whose exponent is either 0 or 1. The phenotypic surface is planar when viewed in n-dimensional space: no terms bend the surface. The mathematical mappings among the phenotypic responses, the markers, and the contexts are one-to-one and onto. But the multiple stresses of field environments fundamentally change the mathematics of the analysis in its dimensionality, independence, linearity, and mappings. Each measured stress and observed trait are dimensions of the complex phenotype of crop performance and the equations that describe it. When the response of a single trait to a single stress is a twodimensional function lying in a plane, many of the two-dimensional techniques familiar to geneticists are valid. Combinations of two or more stresses, or changes in more than one phenotypic dimension, push the phenotypic response function from the plane to surfaces in three or more dimensions, making the phenotype complex. Here, two-dimensional regression is uninformative, since infinitely many two-dimensional functions lie equally well on a three-or higherdimensional surface. Moreover, the mappings can now be few-to-many or even many-to-many. Many statistical methods assume that the input dimensions are independent of each other and have the same distribution (independent, identically distributed variates). In practice, both assumptions are often violated by real data. When two or more dimensions are related to linked mechanistic causes, their behaviors can jointly change in unexpected ways that are not apparent from covariance analysis. This interdependence of dimensions can occur both among the inputs and between inputs and outputs. Moreover, the distributions of values for each of the dimensions can be very different, even after rescaling, recentering, and other modest transformations. Finally, regression and many dimension reduction techniques assume that linear relationships exist between independent input dimensions and independent output dimensions. (Throughout, we use the term "linear" in its strict mathematical sense when referring to models. The statistical "linear" models we fitted included quadratic terms (see Equations 1 and 3), as statisticians use the term.) In at least some situations, these input-output relationships are not linear. Plotted in two or more dimensions, curvy or bumpy responses signal nonlinear relationships among the dimensions, and scatter beyond measurement and physiological noise can signal important missing dimensions or many-to-many mappings. To understand performance in the out-ofrange field environments of the future, we will need a mechanistic view that incorporates climate prediction, an understanding of the genetic architecture and physiology of complex phenotypes, and analytical treatments that are appropriate to the data [bib_ref] Predictable 'meta-mechanisms' emerge from feedbacks between transpiration and plant growth and cannot..., Tardieu [/bib_ref] [bib_ref] Characterizing the crop environment 2 nature, significance and applications, Chenu [/bib_ref] [bib_ref] Predicting the future of plant breeding: complementing empirical evaluation with genetic prediction, Cooper [/bib_ref] [bib_ref] Integrating environmental covariates and crop modeling into the genomic selection framework to..., Heslot [/bib_ref]. Genetic analysis of combined stress environments is a first step toward this mechanistic view. There are examples of genetic dissection of abiotic stress combinations for heat and drought [bib_ref] Identification of drought, heat, and combined drought and heat tolerant donors in..., Cairns [/bib_ref]. In the [bib_ref] Identification of drought, heat, and combined drought and heat tolerant donors in..., Cairns [/bib_ref] study, genotypes with good performance in drought or heat did not perform well when the stresses were combined. The poor predictive ability of single stresses for stress combinations illustrates the interdependence of single stress inputs in conferring the response. A controlled greenhouse-scale analysis of high UV stress combined with moderate drought also indicated that loci important for one stress did not have an important effect in the combined stress treatment; and that the combined stress effect level was less than additive, indicating a nonlinear protective interaction between the two stresses [bib_ref] Genotype to phenotype maps: multiple input abiotic signals combine to produce growth..., Makumburage [/bib_ref]. This suggests that experiments should incorporate multiple levels of each input abiotic stress in order to detect loci that are important for the interactions between stresses. Though analysis of the genetic control of the response to combined stresses is rare, we have more information about physiological responses for combinations within one or a few genotypes. For example, plant protection chemical mixtures show interaction effects [bib_ref] Abscisic acid and the herbicide safener cyprosulfamide cooperatively enhance abiotic stress tolerance..., Dashevskaya [/bib_ref] , as do biotic/abiotic combinations [bib_ref] Signaling events in plants: stress factors in combination change the picture, Prasch [/bib_ref] [bib_ref] Enhancing crop resilience to combined abiotic and biotic stress through the dissection..., Kissoudis [/bib_ref] [bib_ref] Abiotic and biotic stress combinations, Suzuki [/bib_ref]. A common theme across all the types of combinations examined is that the effect of the stress interaction is not easily derived from single-effect responses. It is also clear that a single, often severe stress treatment does not predict the response at lower stress levels [bib_ref] Abiotic stress, the field environment and stress combination, Mittler [/bib_ref] [bib_ref] Any trait or trait-related allele can confer drought tolerance: just design the..., Tardieu [/bib_ref] [bib_ref] Plant adaptations to the combination of drought and high temperatures, Zandalinas [/bib_ref]. Work on mixtures of toxins illustrates the classes of phenotypes one might see in response to combined stresses. Organismal responses to mixtures of drugs and chemical toxins are grouped into modes of action such as concentration addition, independent action, synergy, antagonism, dose-level, and dose-ratio [bib_ref] Significance testing of synergistic/antagonistic, dose level-dependent, or dose ratio-dependent effects in mixture..., Jonker [/bib_ref]. The shape of the responses to the mixtures defines these different modes of interaction. In favorable cases, mechanistic inferences can be drawn from an analysis of the phenotypic responses to increasing levels of two abiotic stresses. Models of high-dimensional response surfaces can then be translated into network models [bib_ref] Plant systems biology: network matters, Lucas [/bib_ref] [bib_ref] Predictive modelling of complex agronomic and biological systems, Keurentjes [/bib_ref] [bib_ref] Combining quantitative trait loci analysis and an ecophysiological model to analyze the..., Reymond [/bib_ref]. World-wide, one of the most important stress combinations in maize is co-occurring drought and nitrogen deficiency. Increased growth and yield in maize under drought and low nitrogen are genetically correlated; selection for one stress results in enhanced performance in the other stress environment [bib_ref] Efficiency of managed-stress screening of elite maize hybrids under drought and low..., Weber [/bib_ref]. However, the correlation can vary by trait and by the specifics of the stress level and genotype used [bib_ref] Interactive effects of nitrogen and water stresses on biomass accumulation, nitrogen uptake,..., Bennett [/bib_ref] [bib_ref] Improvement of crop yield in dry environments: benchmarks, levels of organisation and..., Sadras [/bib_ref]. Typically, only a few levels of nitrogen and drought are applied; and factorial analyses are used instead of surfacefitting approaches that could compare equivalent stress intensities. The maize inbred lines B73 and Mo17 have different responses to drought and nitrogen. B73 exhibits top-fire and Mo17 barrenness under drought , and their response to nitrogen differs by % 25% [bib_ref] Response of maize inbred lines to N fertilizer, Balko [/bib_ref]. These two inbreds are known to combine well as a hybrid, with the hybrid having very good performance under drought stress (A. . We infer that there are interactions between alleles of one or more genes in these parents that confer increased stress tolerance in the hybrid. If true, a population of offspring from these parents would generate a range of allele combinations from those inherited alleles, and would exhibit different responses to varying combinations of stresses. We also infer from stress combination experiments and toxicological data that sets of specific alleles should be able to control the sensitivity of growth to combinations of stresses, and thus shift the stress-tolerance system in maize among more-responsive and less-responsive states. Those states delimit parts of the phenotypic space that defines the set of high-dimensional, nonlinear stress response surfaces. The difficulties in predicting the effects of stress combinations from experiments using single stresses indicate physiological interactions among these dimensions. Mapping the alleles that control these shifts in phenotypic space identifies the portions of the system controlling these responses. To better understand the mechanisms of field-relevant stress responses, the interaction between limited nitrogen and limited water in maize should be examined over a large range of levels of the stresses and in multiple genotypes with appropriate comparisons of the surfaces, rather than scalar summary statistics. In this paper, we map several alleles controlling overall responses to combined stresses, and identify the most parsimonious nonlinear producing function that describes their underlying mechanism. Identifying alleles, response surfaces, and models will be helpful in optimizing crop improvement strategies. # Materials and methods ## Seed stocks The Zea mays intermated recombinant line population (IBM94) derived from inbreds B73 and Mo17 was provided by the Maize Co-op (http://maizecoop.cropsci.uiuc.edu/). Seed stocks were increased using standard nursery conditions at the North Carolina Central Agricultural Station, Clayton, NC. Seed lots were genotyped using eight simple sequence repeat markers; lines Mo066 and Mo062 failed genotyping quality control, and were thus removed from the data analysis. The B73 parent inbred was used for random checks across factor levels within the experiment. ## Experimental design and plant growth A face-centered cubic experimental design [bib_ref] Optimal Design of Experiments, Pukelsheim [/bib_ref] with five levels of drought and five levels of nitrogen was used to examine dose response surfaces for mixtures of the two stresses. The statistical program JMP v.6's (SAS, Inc., Cary, NC, USA) experimental design module was used to compare design matrices and to generate the face-centered cubic sample points (see . This experimental design has more biological replicates in the center portions of the response surface to enable better fit of nonlinear functions. We used an unbalanced design to increase the power for detecting the genotype-stress combination QTL, as QTL analysis was the primary goal of this experiment. In our face-centered cubic experimental design, the maximum sample size was either n ¼ 4 or n ¼ 8: The seeds of each of the 89 IBM RILS and parental Mo17 and B73 inbreds were randomly assigned within the cubic centered face design stress levels; replication existed within these levels of the experimental design. Pots in water levels were grouped to reduce human error when administering the water to the plants. Therefore, the only variation that would confound the experiment would be spatial variation within the greenhouse, i.e., if there was fluctuation or variation between parts of the greenhouse table within a water level that affected the estimation of the genotype-environment interaction. We considered adding spatial modeling of water blocks to our analysis but the additional complexity could not be justified, since differences in temperature or light across the greenhouse tables were not detected. The experiment was conducted in the Cape Fear Community College horticulture greenhouse (GPS coordinates Lat: N 34°199 24 99 (34.324°) Lon: W 77°529 45 99 (-77.879°), weather station KNCCASTL2) from May-July 2011. Greenhouse maximum temperature was set to 38°C. Slow-release fertilizer was custom-mixed by Coor Farm Supply, Smithfield, NC, with clay pellets containing standard trace minerals, 15% potassium, 15% phosphate, and nitrogen levels of 0, 2.5, 7.5, 12.5, and 15% fertilizer treatment level. 6.36 kg of fertilizer pellets were mixed with a 0.08 m 3 bag of MetroMix360 potting mix (SunGro, Vancouver, BC, CA). Deep plant pots (MT38, 0.9 l, Stuewe and Sons, Tangent, OR, USA) were filled with soil-fertilizer mix. Random soil-filled pots were weighed, with an average weight of 350 g per pot. Seeds were planted 1 cm below the soil surface. A random number was generated for each plant pot within each water level using SAS v9.2 (SAS Inc. Cary, NC, USA). The plant pots were sorted by random number within each water level, so that neighboring plants were of randomly chosen genotypes and nitrogen levels. Water evaporation in plant pots containing B73 checks in the greenhouse was examined May 20-24; the average difference in weight between fully wet and dry pots over 24 hr was 170 g. Drought was applied to experimental groups using this average, with drought levels of 8, 20, 50, 80, and 92% of full weight (13 ml water, 34 ml water, 85 ml water, 136 ml water, and 156 ml water applied per day per pot). Selective watering in different amounts was applied from 20-30 days after planting, beginning when the check plants were at the four-leaf growth stage. Soil water potential was measured with a conductivity meter (EC-5, Decagon Devices, Pullman, WA, USA); the selective watering was stopped when the B73 check 8%-weight plant pots had an average water potential of 2%. All pots were watered fully for five days after drought treatment. ## Trait data collection Each plant pot was photographed against a 1 cm grid background 14 days after planting, before selective watering. Plants were re-photographed using the same setup and focal length 35 days after planting, after recovery from selective watering. Plant photographs were measured using ImageJ [bib_ref] NIH image to ImageJ: 25 years of image analysis, Schneider [/bib_ref] , with the internal centimeter ruler in each image used to calibrate the pixel lengths for each measurement session. Each person analyzing the images practiced on a calibration image set until his or her accuracy was greater than 95%. All plant images are available upon request. The complete trait data file is included as Supplemental Data Files 2 and 3. # Parental inbred analysis The initial plant height was subtracted from the final height to generate Z ijk ; the growth variable difference_in_height. Measured initial, final, and difference in plant heights for each parental inbred for each stress treatment were fit with a full factorial model (see Equation 1) using JMPv11 (SAS Inc., Cary, NC). ## Mixture surface parameter fits For the check B73 inbred with adequate data points, the height difference data were analyzed by the procedure of [bib_ref] Significance testing of synergistic/antagonistic, dose level-dependent, or dose ratio-dependent effects in mixture..., Jonker [/bib_ref] using Excel Mixtox analysis tools provided by C. Svendsen. The Mo17 data had too few points to generate a fit. The first step in the Mixtox analysis procedure was to fit a single dose response relationship to the height difference data using the log-logistic two-dimensional surface as a dose response model to determine the separate, single parameter effects of water and nitrogen. To fit the surface to the water level, the data were filtered to only include points with the lowest level of nitrogen that had a variation in the water levelin this case, the nitrogen level was held constant at 2.5% in order to analyze the single parameter effect of water. Similarly, the surface was fit for single-parameter nitrogen by holding the water level constant at 20%. These fitted surfaces are the empirical analogs of the discrete partial differentials of the response surface with respect to water or nitrogen, constrained to lie on the planes where nitrogen ¼ 2:5% and water ¼ 20%; respectively. The fits of the loglogistic surfaces were further refined using the Solver add-in in Excel to minimize the sum of squares (SS) between the actual data points and the predicted model values. The second step in the analysis was to fit the Mixtox mixture dose-ratio and dose-level reference models and the deviation models to the data. In order to optimize use of the solver, which was set up to use one measurement rather than replicates, and was not optimized for a face-centered cubic design, an average was taken for each of the treatment combinations in the larger data set. # Qtl analysis To determine which markers are responsible for creating significantly different dose response surfaces, we fit the response data for each marker, then compared these to the surface fit to all the markers from all lines using an F-test (see Supplemental Materials 6). Since our experimental design was optimized to detect interactions among markers and stresses, we fit the data to a quadratic function; and we focused on smoothed surfaces to incorporate all the information across levels efficiently. As seeds germinate at different times, all the recombinant inbred analyses were conducted on trait measurements adjusted for initial plant size. This approach fits the data for Z ijk ; the difference in height, using Equation 1: [formula] Z ijk ¼ m þ a i þ b 1 x w;j þ b 2 x n;k þ b 3 x 2 w;j þ b 4 x 2 n;k þ b 5 x w;j x n;k þ e ijk ;(1) [/formula] where Z ijk is the difference in height for line i before and after the combined water x w ; and nitrogen, x n ; stresses; m is the mean of the height differences before and after stresses; a i is a random effect due to line i; x w;j is a covariate for the j th amount of water; x n;k is a covariate for the k th amount of nitrogen; and x w;j x n;k is the interaction term between j ¼ 1; 2; . . . ; J for J amounts of water and k ¼ 1; 2; . . . ; K for K amounts of nitrogen. b Á are the regression coefficients, and e is the residual error in the fit to the data. To fit the data to this equation and detect QTL, we first fit an allinclusive model that included all the markers from all the lines. Then for each marker, data from all the lines and all combinations of water and nitrogen were divided into two groups according to whether the genotype of the marker was B73 or Mo17. The SAS procedure PROC MIXED was used to model each surface. Due to non-random relatedness between recombinant inbred lines, we incorporated kinship matrix information into the analysis, as recommended by [bib_ref] Gene and QTL detection in a three-way barley cross under selection by..., Malosetti [/bib_ref]. Kinship matrices were calculated using the SPAGEDI method [bib_ref] SPAGEDI: a versatile computer program to analyse spatial genetic structure at the..., Hardy [/bib_ref] within the TASSEL v3 program [bib_ref] TASSEL: software for association mapping of complex traits in diverse samples, Bradbury [/bib_ref]. The sums of squares for the individual marker models were compared to the sums of squares of the all-inclusive model via an F-statistic. The resulting raw P-values from the analysis were adjusted using the approach described by [bib_ref] Phenotype uniformity in combined-stress environments has a different genetic architecture than in..., Makumburage [/bib_ref] , by grouping correlated adjacent marker P values with the Simes function in SAS (PSMOOTH). SAS data steps were used to scan the Simes-adjusted P values for groups of significant markers adjacent along the chromosome. A false discovery rate of 0.05 and a Sidak adjustment of 0.05 were each separately used to adjust for multiple testing. Raw and adjusted P-values, marker data for each mapping line, and SAS code for surface fits and P-value adjustment are provided in the Supplemental Data and Methods files (1, 4, and 5). ## Response surfaces of the markers We generated the phenotypic response surfaces of the lines using the parameters obtained by linear regression and Equation 1. These surfaces were plotted in three-dimensional Cartesian space using the R package rgl, recentering the intervals for water and nitrogen . Plotting details and code are provided in Supplemental Methods File 11. For each marker, two response surfaces were plotted for the B73 and Mo17 alleles in the QTL region. ## Annotation of qtl loci QTeller (http://www.qteller.com/) was used to assemble a list of maize genes in the three chromosomal regions containing QTL that changed the difference in height Z ijk ; and the gene IDs were placed into AGRIGO [bib_ref] agriGO: a GO analysis toolkit for the agricultural community, Du [/bib_ref] for annotation analysis. All GO annotations within each QTL were used to create scaled semantic-similarity plots through the Revigo interface [bib_ref] REVIGO summarizes and visualizes long lists of gene ontology terms, Supek [/bib_ref]. ## Mathematical model of the phenotype The most distinctive and robust feature of the plant height phenotype is the shapes of the response surfaces. We sought a function that was simpler than Equation 1, that would reproduce the shapes of the experimental phenotypes, and that would not assume the hypothesized interactions. The simplest such producing function is the sum of two components, an elliptical paraboloid and a plane, shown in Equation 2. [formula] z ¼ c À ax 2 w þ bx 2 n Á |fflfflfflfflfflfflfflfflffl ffl{zfflfflfflfflfflfflfflfflffl ffl} elliptical paraboloid þ dx w þ ex n |fflfflfflfflfflffl{zfflfflfflfflfflffl} plane ; [/formula] (2) z is the difference in height; x w ; water; x n ; nitrogen. a; b; and c are parameters governing the paraboloid's shape and orientation (c) and its weighting of input water (a) and nitrogen (b). d and e tilt the plane along the water and nitrogen axes, respectively. To confirm the model's correctness, we tested many different candidate components by analysis and substitution, the importance of each term by deletion, and the effects of the parameters by simulation. Using R, the function was evaluated over the recentered water and nitrogen intervals ½242; 42 and ½27:5; 7:5 with a step size of 0.5, and surfaces were plotted in a standard orientation (Supplemental Files 12 and 16). Package viridis was used to color these and subsequent plots, since it produces color maps that are less problematic for those with color blindness . ## Estimation of model parameters We estimated the parameters of the producing function (Equation 2) for each allele's surface using standard linear regression, analogous to the fit of Equation 1 but omitting the constant. We set the value of c to f21; 1g for the non-Mo17 and Mo17 shaped experimental surfaces, respectively, and estimated the values of ða; b; d; eÞ for [formula] Z ¼ c À aX 2 w þ bX 2 n Á þ dX w þ eX n ;(3) [/formula] where Z is the vector of empirical heights at a mesh of points at coordinates ðX w ; X n Þ: The mesh points were chosen to emphasize surface regions that had the most empirical data and were most comparable among the surfaces. The points lie at the intersections of a set of contours at fixed z values and a set of rays defined with respect to a local axis. For the non-Mo17 surfaces, the local axis was defined as the apparent major axis of the distorted ellipsoid. For Mo17, the local axis was defined as midway between the asymptotes of the trough. The rays extended from the surfaces' peaks at fixed angles relative to the local axes. We call these the "absolute mesh points" to distinguish them from the relative mesh points of the next section. We used the linear solvers Solve and lsei in the R package limSolve [bib_ref] An R function for sampling linear inverse problems, Van Den Meersche [/bib_ref]. Both gave identical parameter values. We report those obtained by lsei in , since that algorithm also computes a scalar error measure cumulated over the surface, the square root of the least squares error fit. We repeated these computations using the relative mesh points described in the next section. Those fits were considerably worse except for Mo17, as judged by the scalar error. Code for these computations is in File S15. The resulting surfaces were projected into the ðx w ; x n Þ plane and plotted using the image function of package graphics (R Development Core . ## Comparison of surfaces' shapes We assessed how well the surfaces generated by the model using the fitted parameters reproduced the intrinsic shapes of the experimental surfaces. For all possible pairs of experimental and simulated surfaces, we scored differences in signed Euclidean distance, r; relative rotation of the surfaces projected into the ðx w ; x n Þ evaluation plane, u; and relative gradients in z along a set of rays, dz r : r estimates the displacement of the simulated surface in ðx w ; x n ; zÞ space relative to the experimental, due to either or both components of the model. u accounts for different amounts of rotation over the surfaces, due to tilting of the planar component of the producing function. dz r captures differences in the "bending" of the surfaces, due to either or both components of the model. We discretized the shapes using 10 mesh points placed at the same relative distances from the peak along each of six ray segments of fixed slopes (the "relative mesh points"). The segments are bounded by the peak and the edges of the evaluation plane. Non-Mo17 surfaces had ray segments at slopes f0; 0:05; 0:099; 0:175; 0:32; 0:75g relative to the origin. For Mo17, the slopes were f0; 2 0:05; 2 0:099; 2 0:175; 2 0:32; 2 0:75g This discretization divides the surface into nine adjacent bands whose tilts and twists reflect shape changes in that region. R code to generate mesh points, compute comparisons, and plot heatmaps is in File S14. We used the R packages superheat and viridis for the heatmaps . ## Data and code availability All supplemental files (input data, SAS analysis code, outputs, supplemental methods, supplemental results, and outputs are available from FigShare at https://figshare.com/s/3ef69b44d24d0953d625. Code for modeling, fits, and simulations is on GitHub at https://github.com/ tonikazic/univariate_dose_response.git in a public repository. shows the parameter values obtained by fitting Equation 1 to the experimental data for the parental lines (first and last rows). The response surfaces generated using these parameters are plotted in [fig_ref] Figure 1: Parental Inbred Response Surfaces to Combined Drought and Nitrogen Deprivation [/fig_ref] for the parental lines. The parental inbreds exhibited different responses to combinations of water and nitrogen deprivation, with B73 showing more variation in its response surface than Mo17. The plots are rotated to show the most severe stresses in the front center corner, placing the normal full-water and full-nitrogen combination in the back. The intersections of the surface with the nitrogen-height (ðx n ; zÞ) and water-height (ðx w ; zÞ) planes define the phenotypic response at constant amounts of water and nitrogen, respectively, corresponding to the discrete partial differentials dz=dx n and dz=dx w : B73 exhibited a domed surface, with the peak at moderate amounts of water and nitrogen [fig_ref] Figure 1: Parental Inbred Response Surfaces to Combined Drought and Nitrogen Deprivation [/fig_ref]. This surface is convex upward in the sense that it opens downward toward negative values of z; and has a relatively high, and highly curved, peak (large z max and small discrete curvature), that lies in the region of relatively high nitrogen and water [bib_ref] Curvature measures for discrete surfaces, Sullivan [/bib_ref]. While B73 declined under the most severe conditions (front center corner), it showed modest growth under both moderate drought and very low nitrogen (e.g., the maximum of dz=dx w at the surface's right edge) and high drought and middle nitrogen (e.g., the maximum of dz=dx n at the surface's left edge). The worst condition was minimum water and maximum nitrogen (rear left corner). # Results ## Effect of stress combinations on parental inbreds n Regression Coefficients and Constant Terms for the Experimental Data Regressed to Equation 1. The regression coefficients, b Á ; are ordered by the degree of the applied stresses. Thus, b 1 ; x w ; b 2 ; x n ; b 3 ; x w 2 ; b 4 ; x n 2 ; and b 5 ; x w;n : The columns are ordered to highlight the elliptical paraboloid (b 3 x 2 w;j þ b 4 x 2 n;k ), hyperbolic paraboloid (b 5 x w;j x n;k ), and plane (b 1 x w;j þ b 2 x n;k ), components of Equation 1 in that order. The lumped constant ℓ incorporates all the constant terms in the equation, ℓ ¼ m þ a i;0 þ e i;w;n : In contrast, Mo17 had little growth change under any stress [fig_ref] Figure 1: Parental Inbred Response Surfaces to Combined Drought and Nitrogen Deprivation [/fig_ref]. Its surface is a very shallow trough, or is concave upward; the nadir, z min ; is small; its discrete curvature is larger; and the peak lies on a corner. For Mo17, reducing nitrogen affected growth slightly more severely than reducing water (compare the slopes along the left front and right front faces of [fig_ref] Figure 1: Parental Inbred Response Surfaces to Combined Drought and Nitrogen Deprivation [/fig_ref]. We investigated whether this small difference in Mo17's change of heights could be due to its decreased overall growth. We compared the initial plant heights of B73 and Mo17 and found no significant differences between them (P ¼ 0:18). When we compared the inbred plant heights after deprivation, we found that the B73 plants had more growth and greater differences between treatments than Mo17 (comparing the factors inbred, nitrogen level, water level, and inbred by nitrogen level, at P , 0:05; the numerous sample sizes and confidence intervals are reported in File S9). But when we scaled the differences in height to adjust for the smaller Mo17 plants at the beginning of the experiment, plant growth during the experiment was more pronounced for Mo17 in mid-level nitrogen and low water-level treatment combinations. In contrast, B73 growth was typically greater when more water was available. This indicates that the Mo17 inbred line is less sensitive to drought provided at least some nitrogen was present. Comparisons at very low nitrogen levels did not exhibit any trend toward differences between parental inbreds . For both B73 and Mo17, the slopes of the four lines intersecting each pair of the surfaces' corners are different, indicating the plants' responses vary with extremal stress combinations. Mixture toxicity models with two shape parameters, , a and b . for water and nitrogen, were used to analyze the shape differences in the parental B73 inbred. (These two parameters are not the same as the a and b of Equation 2, and we have typeset them in a different font than is normally used in mixture toxicity papers to emphasize this distinction.) B73 had the best fit to a dose-ratio surface. , a and b . indicating that the antagonistic effect of combined stresses is caused mainly by nitrogen deprivation. This is consistent with the curvature of the discrete partial differentials at the edges of the B73 response surface in [fig_ref] Figure 1: Parental Inbred Response Surfaces to Combined Drought and Nitrogen Deprivation [/fig_ref] : dz=dx w is more sharply curved and has a higher local maximum than dz=dx n : [formula] marker b 3 b 4 b 5 b 1 b 2 ℓB73 [/formula] ## Qtl that change phenotypic response surfaces Chromosomal loci with significant interaction P values for the response surface were fit to nitrogen deprivation and drought. The interaction effect is visualized as the intersections between the surfaces in the illustrative plots in . The interaction between marker allele and each stress combination covariate was fit with a standard linear model, and the parameters derived from the model were those used in mapping the QTL. We found three highly significant QTL that changed the response surface fitted from the data on all lines and an additional 50 QTL when false discovery rate-adjusted P-values less than 0.05 were considered. The three QTL with P values below the experiment-wise Sidak-adjusted significance threshold of 0.05 are shown in . shows the parameter values obtained by fitting Equation 1 to the data for the smoothed QTL response surfaces. For all three QTL, the response surface for the Mo17 allele is upwardly convex, a shape resembling that of the B73 allele and parent, and very different from the upwardly concave shape of the Mo17 parental surface. The three QTL's alleles differ from each other and from parental inbreds in many details, including the magnitudes of z over their surfaces, the relative magnitudes of the B73 and Mo17 surfaces, and the value and position of z max : The Mo17 allele's surface for QTL1, shown in , is pushed upward along the z axis, far above the range of the Mo17 parent's response in [fig_ref] Figure 1: Parental Inbred Response Surfaces to Combined Drought and Nitrogen Deprivation [/fig_ref]. For extremal combinations of water and nitrogen, changes in the growth of the Mo17 allele exceed those for the B73 allele. QTL2's and QTL3's Mo17 surfaces lie mostly above those of their B73 alleles. For these loci, the B73 alleles exhibit better performance under extremal conditions (see . Thus, the phenotypic response surface can differ in shape and magnitude within the population, and surface shape can be quite different than the parental response surface in offspring carrying some QTL allele combinations. Because the experimental design was optimized to detect marker-stress treatment interactions using Equation 1, these QTL display a crossover interaction between the allele surfaces. Like their parents, none of these alleles have surface corners that lie on parallel lines. The differences in surface shape between allele fits in all three QTL, with highly-domed surfaces that have increased combined stress peaks in the middle of the response surface, indicate that nitrogen and water stress have nonlinear effects on plant growth. These B73-like surface shapes indicate that the better-performing allele at mid-range combined stress is typically not the allele that provides best performance under extreme conditions. The highest water and nitrogen input conditions, which might naïvely be assumed to support the most growth, exhibit less growth and could be favored by a different allele than the mid-range combinations. ## The shapes of the alleles' response surfaces The most distinctive and robust feature of the phenotypes is the shapes of the surfaces, rather than their absolute placement in ðx w ; x n ; zÞ space. The shapes show the patterns of the alleles' responses to the stresses, and are less sensitive to the effects of errors due to small sample sizes. The smoothed experimental surfaces fall into four nondisjoint categories: domed more sharply domed, highest amplitude surfaces with peaks in the high nitrogen, high water region (B73 and QTL1-B73); hybrid higher amplitude domed surfaces with the peaks displaced from the high nitrogen, high water corner toward the center of the water-nitrogen plane (QTL2-Mo17 and QTL3-Mo17); shoulder lower amplitude surfaces, with lower peaks on the high water edge that slope more gradually downward as nitrogen decreases (QTL1-Mo17, QTL2-B73, and QTL3-B73); and trough a very low amplitude trough with a peak at the lowest water and highest nitrogen corner (Mo17). These categories are illustrated by examining the positions of the peaks in ðx w ; x n ; zÞ [fig_ref] Table 2: Comparison of Numerical Values of the Experimental Peaks' Positions and the Nondisjoint... [/fig_ref] , and by projecting the surfaces into the water-nitrogen plane (first and third columns of . We emphasize that the membership of the middle categories depends on the classification criteria. If one considers just the position of the peak in the ðx w ; x n Þ plane, then B73 and QTL1-B73 would form the domed class, and QTL2-Mo17 and QTL3-Mo17 would form the hybrid class. Weighting the peak's position in z more than in ðx w ; x n Þ would classify only B73 as domed and shift QTL1-B73 into the hybrid class with QTL2-Mo17 and QTL3-Mo17. Binning the z max more coarsely would eliminate the hybrid category altogether. Adding other criteria, singly or in combination, might further change the classification. ## Annotations of gene function in qtl regions Gene annotations under QTL provide a qualitative new data type that can provide additional context to the mapping of chromosomal loci. Annotations such as "response to abiotic stress" in the two QTL on Chromosome 1 [fig_ref] Figure 3: Gene Ontology Annotations for QTL [/fig_ref] and 3B) are consistent with our identification of these QTL as important for response to drought and nitrogen fertilizer. QTL3 in bin 9.03 does not have unique annotations in stress response [fig_ref] Figure 3: Gene Ontology Annotations for QTL [/fig_ref] ; this may indicate that a novel gene type is responsible for the causal allele difference at this locus. The marker with the smallest P value within the QTL1 region was IDP168, which tags gene GRMZM5G828396. This gene is annotated as a basic Helix-Loop-Helix (BHLH) transcription factor. The marker with the lowest P value in the second QTL interval was umc1446, which tags gene GRMZM2G162508; this gene is annotated as a polyketide-synthase-like protein. The marker with the smallest P value in the third QTL in bin 9.03 was mmp17b, which is between the genes GRMZM2G538859 and GRMZM2G093187; neither gene model has assigned annotations. ## Modeling the response surfaces with a producing function What is the simplest physiological model that produces the phenotypes? If one assumes the phenotypes are produced by a single network in the plant, then the corresponding producing function is the most parsimonious network. Tuning the function's parameters to reproduce the observed phenotypic points is equivalent to tuning the network's parameters, rather than its connectivity. The phenotypic points form an n-dimensional space, with each phenotype a point in this space. The greater the range of observed phenotypes, the more the phenotypic space is sampled, and the more constraints an hypothesized function must satisfy. Such a function can also predict novel phenotypic points that lie in the space. Equation 1 is not the best choice for the producing function. Its large constant, lumped together as ℓ in , shifts the surfaces along the z axis without modifying their shapes. It assumes the hypothesisan interaction between water and nitrogenvia the hyperbolic paraboloid term. Finally, it has a fairly large number of terms. Loci with Significant Effects on the Phenotypic Response Surface. Three QTL with a Sidak-adjusted significant interaction surface for differences in height were identified. At each QTL, the trait data were fit with a quadratic response surface separately for each allele. The B73 allele is shown in orange and the Mo17 allele in blue. Each panel shows the QTL's location (chromosome bin and marker range indicated above) and surface fits for the allele differences at the locus. (A) Surfaces for QTL1, gpm906a -IDP2465; (B) surfaces for QTL2, umc1838a -mmp22; and (C) surfaces for QTL3, ufg71 -IDP1681. n We therefore searched for a simpler function that would reproduce the surfaces' shapes without explicitly assuming an interaction between the inputs. The single, sharp global maxima of the B73-like surfaces immediately suggested an elliptical paraboloid. This is the simplest function that generates a single peak; changing the sign of one parameter flips the peak to produce Mo17's trough. An orthogonal projection of the paraboloid into the ðx w ; x n Þ evaluation plane gives an ellipse, whose ratio of major and minor axes reflect the relative weighting of the water and nitrogen inputs for that phenotypic point. We considered other functions that generate peaks, but they are structurally more complicated, harder to flip, and need more assumptions that are more difficult to justify. For example, a two-dimensional Gaussian function is structurally more elaborate, and flipping requires the reciprocal of the Gaussian. Periodic functions, such as transcendental or Bessel functions, would have forced us to assume either that their other peaks lie outside the evaluation interval or that the functions are severely damped. However, three asymmetries in the phenotypes indicate the producing function is not just an elliptical paraboloid. First, the maxima are not where they would be for an elliptical paraboloid, at ð0; 0; zÞ: Second, the surfaces are tilted: intersecting each surface with the four planes perpendicular to the evaluation plane yields a set of different surfaces. Third, the surfaces are not symmetric around the peaks, but distorted. While the peak can be shifted by adding a constant along x w ; x n ; or both, tilting and distorting the surfaces requires the addition of another component to the producing function. We experimented with many possibilities for this second component, including exponential and transcendental functions and operators to combine the first and second components, and found the simplest approach was to add a plane. Thus, the simplest producing function is that shown in Equation 2, reproduced here: [formula] z ¼ c À ax 2 w þ bx 2 n Á |fflfflfflfflfflfflfflfflffl ffl{zfflfflfflfflfflfflfflfflffl ffl} elliptical paraboloid þ dx w þ ex n |fflfflfflfflfflffl{zfflfflfflfflfflffl} plane ; [/formula] where z is the difference in height; x w is water; and x n is nitrogen. a and b modify the paraboloid's size, shape, and weighting of input water (a) and nitrogen (b). c modifies the paraboloid's size and shape and flips it: c , 0 makes the surface convex upward like the B73-like surfaces, while c . 0 produces the concave upward surface of Mo17. d and e tilt the plane along the water and nitrogen axes, respectively. This shifts, stretches, rotates, and tilts the paraboloid to change its shape with a set of affine transformations. The relative weighting of the paraboloid and plane components is controlled by all five parameters: cða þ bÞ . d þ e emphasizes the paraboloid features of the surface. The relative weightings of water and nitrogen within each component are different (a and b; d and e), and independent of the weightings of the two components. We tested the model by deleting terms and by varying the values of the parameters. Both components are essential to reproducing the experimentally observed surfaces. Omitting the elliptical paraboloid makes it impossible to reproduce any experimental surface, since all have domes or troughs; and omitting the plane makes all of the surfaces symmetric about the major and minor axes of the paraboloid, placing the maxima and minima at ð0; 0; zÞ: Each term in the equation corresponds to a node in the network, drawn in [fig_ref] Figure 4: Modeling of Response Surfaces with the Producing Function [/fig_ref]. The simulated surfaces in [fig_ref] Figure 4: Modeling of Response Surfaces with the Producing Function [/fig_ref] show good qualitative agreement with the experimentally observed surfaces of the B73 and Mo17 parental inbreds in [fig_ref] Figure 1: Parental Inbred Response Surfaces to Combined Drought and Nitrogen Deprivation [/fig_ref]. Thus, the phenotypes we observe are products of the entire network. ## Parameter estimation for the producing function How well does Equation 2 reproduce the shapes of the alleles' response surfaces? To answer this question one must first estimate the model's parameters for each allele. However, the surfaces' nonlinearity entails several trade-offs that affect the accuracy of the estimates. The edges of the surfaces were under-sampled to optimize the experiment for peak detection. By omitting a hyperbolic paraboloid, the model cannot reproduce the apparent nonplanar, twisted quadrilateral of the surfaces' corners. Circumventing those issues by fitting with data near the peaks makes the estimates more sensitive to the smoothing errors inherent in flatter peaks, and inadvertant affine transformations of the surfaces due to slight changes in a; b; d; or e: Linear regression is simpler, faster, and more likely to converge than nonlinear methods, but will exaggerate the peak regions since their z values are so much greater. With these caveats, we estimated the model's parameters for each line by linear regression to Equation 3. We used a set of absolute mesh points that sampled the central, best-determined parts of the smoothed experimental surfaces. We preset c to f21; 1g to simplify estimation, and estimated parameters using and omitting the peaks. summarizes the values of a; b; d; and e; and the square root of the solution norm scalar errors, s, for each experimental surface. The residual norms for all the parameters were 0. In general, the estimated values distribute the numerical weight more evenly among the four parameters than the fits to the regression model of Equation 1, shown in . The exception is QTL3-Mo17, where nearly all of the numerical weight is concentrated in e. The parameters shown were estimated by including the experimental peaks. Judged by s, omitting the peaks slightly degraded the quality of the estimates for all lines except Mo17 [fig_ref] Figure 1: Parental Inbred Response Surfaces to Combined Drought and Nitrogen Deprivation [/fig_ref]. The right side of summarizes the patterns of parameter signs, and compares these to the independently derived classification of the surfaces' shapes shown in [fig_ref] Table 2: Comparison of Numerical Values of the Experimental Peaks' Positions and the Nondisjoint... [/fig_ref]. Grouping by signs makes it more visually obvious how the surfaces in connect to the parameters of the producing function, and emphasizes the overlap among the categories. The sign patterns match the shape classification shown in [fig_ref] Table 2: Comparison of Numerical Values of the Experimental Peaks' Positions and the Nondisjoint... [/fig_ref] except for QTL1-B73 and QTL3-Mo17. QTL1-B73's classification strongly depends on how the classification criteria are weighted, and its estimated values for b and d are much closer to those for the hybrid shape of QTL2-Mo17 than B73's. QTL3-Mo17 is a hybrid surface based on the peak position in ðx w ; x n ; zÞ [fig_ref] Table 2: Comparison of Numerical Values of the Experimental Peaks' Positions and the Nondisjoint... [/fig_ref] , the projection into the evaluation plane , and its estimate for e, but has a distinct sign pattern that reflects the small estimated value of b. Both the sign pattern and the estimates of a; b; d; and e for the shouldered lines are much more internally consistent. ## Comparison of experimental and simulated surfaces To see how well the model reproduces the shapes of the phenotypes, we compared the smoothed experimental response surfaces to those generated by the model. The experimental surfaces were generated by Equation 1 and [fig_ref] Table 2: Comparison of Numerical Values of the Experimental Peaks' Positions and the Nondisjoint... [/fig_ref]. shows these surfaces projected into the ðx w ; x n Þ evaluation plane. They are plotted to show each surface's amplitude by using its minimum and maximum to set the scales. The producing function reproduces the major shape features of the phenotypes. All four shape categories are generated with the correct membership and ambiguities. The surfaces illustrate the caveats of using linear regression for nonlinear phenomena for the values in . Relative to the generated experimental surfaces, all the model surfaces are rotated counter-clockwise and their amplitudes exaggerated. Model surfaces are translated relative to the evaluation plane:domed and hybrid surfaces toward the low-nitrogen edge; the shouldered surfaces toward the high water edge; and Mo17 toward the low nitrogen, high water corner. We assessed the intrinsic shapes of the surfaces using three types of similarities, computed on a mesh of points that extend from the peaks leftwards and downward. Unlike the absolute mesh points used in the linear regression that are very sensitive to affine transformations of the surfaces, these relative mesh points are placed at constant relative distances along a set of ray segments that cover the lower left quadrant of the surfaces, and are adequately robust to translation of the surfaces relative to the evaluation plane. Three similarity scores were computed. r, the signed Euclidean distance, evaluates the displacement of the model surface in ðx w ; x n ; zÞ space relative to the experimental due to either or both components of Equation 2. u, the rotation angle between the surfaces projected into the ðx w ; x n Þ evaluation plane, accounts for different amounts of relative rotation due to tilting the planar component. dz r ; the gradients of relative discrete differences along the rays as one moves away from the peaks, captures differences in the "bending" of the surfaces due to either or both components. We compared the shapes' similarities for each pair of generated experimental (e) and model (s) surfaces at each corresponding pair of relative mesh points. The QTL3-Mo17 and Mo17 parameters produced surfaces that were displaced too far in the evaluation plane to be included in the comparison. Values for r; u; and dz r for each pair of comparisons are shown as heatmaps in . In the heatmaps, each experimentally-derived surface (e) is compared to all available model surfaces (s), beginning with its model sibling. Each comparison is a row, and the columns are ordered to form concentric rings around the peaks. The first ray segment in each ring is parallel to x w ; the water axis, and the last nearly parallel to x n ; the nitrogen axis. Bluer shades indicate higher similarity for r; for u and dz r ; the greener shades indicate higher similarities. For dz r ; bluer shades indicate the simulation's gradients are steeper compared to the experimental; yellower shades signal the simulation's gradients are shallower. Shape reproduction by the model is good, with different pairs of surfaces showing reasonable accuracy over their entirety for the different similarity scores. Blocks of similar blue and green colors dominate all three scoring matrices, and correspond to the nondisjoint shape categories. The same experimental surface was often fit equally well over most of its surface by multiple simulations, as judged by two or more criteria. For example, generated experimental shouldered surfaces QTL1-Mo17, QTL2-B73, and QTL3-B73 were best emulated by non-Mo17 surfaces; B73 was poorly emulated; and Mo17 was never well emulated by simulations of the non-trough surfaces. Occasionally the self comparisons were not the best fit, suggesting we did not overfit the model's parameters. For example, the shouldered surfaces were less well emulated by their simulated selves, compared to B73. Changes in gradients along the shapes were usually least for the experimental surfaces compared to their simulated siblings. All simulated non-Mo17 surfaces tended to descend more sharply toward the tails of the surfaces in several comparisons. Mo17 descended more shallowly when compared to any other simulations, as one would expect. Some nuances deserve highlighting. As expected, the accuracy of shape reproduction is not uniform over the surfaces for any similarity score, and the three scores often differ for any given comparison. Reproduction is better nearer the peaks and for the more central ray segments. The regression to Equation 3 was based on those regions, but the performance of the model outside them is often almost as good for many comparisons. The scores are still somewhat sensitive to affine transformations. For example, the repeating yellow stripes in u for ray segment r1 in are an artifact of the way u is calculated. As the peaks from which they are drawn shift in the ðx w ; x n Þ plane, the absolute lengths of the ray segments compared increases, increasing the apparent rotation. Thus, the maximum rotation we observe along r1 reflects greater length differences in the r1 ray segments relative to the others. # Discussion We observed that the parental B73 and Mo17 inbreds show different responses to combined stresses [fig_ref] Figure 1: Parental Inbred Response Surfaces to Combined Drought and Nitrogen Deprivation [/fig_ref]. The position of maximum response and the surfaces' shapes differ markedly among the parental and recombinant lines and [fig_ref] Table 2: Comparison of Numerical Values of the Experimental Peaks' Positions and the Nondisjoint... [/fig_ref]. All three of the QTL we identified have better performance for one allele in the mid-range combined stress, at moderate levels of water and nitrogen. Mo17 is the least responsive of all the lines, with a flat trough responding to all water-nitrogen combinations. In constrast, lines with Mo17 QTL in mixed RIL backgrounds have responses that show much greater, more B73-like amplitudes and convexities. All Mo17 QTL exhibit B73-like convexity, but their other effects on the baseline B73 response vary by QTL: QTL2-Mo17 and QTL3-Mo17 lift the response above their B73 alleles. The responses of the B73 alleles are damped, and shifted by the addition of Mo17 germplasm in the background across all environments [fig_ref] Table 2: Comparison of Numerical Values of the Experimental Peaks' Positions and the Nondisjoint... [/fig_ref]. These differences in the response surfaces illustrate the effects of multiple segregating genes for this population in combinations of water and nitrogen. The QTL we identified have contrasting and intersecting response surfaces. To estimate an effect size for these QTL, we would need a way to express the shape differences in multiple dimensions for fitting. Simply calculating the slope along a slice of the surface, or at z max ; would not capture the full effect of the QTL and would mis-estimate the effect size. One interpretation of the nonlinear response surfaces is that they arise from epistatic interactions between the QTL and other modifier loci in the background. Antagonistic, damped responses to combinations of stresses were previously observed in this IBM population in an experiment measuring changes in height under combinations of UV and drought, which identified different QTL than we see here for drought and low nitrogen [bib_ref] Genotype to phenotype maps: multiple input abiotic signals combine to produce growth..., Makumburage [/bib_ref]. Our results are consistent with the importance of nitrogen for growth for modern corn linesand the ability of lower levels of nitrogen fertilization to ameliorate the effect of severe drought under certain conditions [bib_ref] Improvement of crop yield in dry environments: benchmarks, levels of organisation and..., Sadras [/bib_ref]. For example, the B73 MixTox analysis indicates that low nitrogen "over-shadows" drought; in low nitrogen, having additional water available does not improve growth. Since combinations of other stresses may have different genetic control, dose response analyses for more stress combinations, and perhaps more complex surface fits such as dose-ratio [bib_ref] Significance testing of synergistic/antagonistic, dose level-dependent, or dose ratio-dependent effects in mixture..., Jonker [/bib_ref] , would be needed. While epistatic interactions are common, they are difficult to detect in small mapping populations using combinatorial or variance component methods [bib_ref] Influence of gene interaction on complex trait variation with multilocus models, Mäki-Tanila [/bib_ref] [bib_ref] The genetic architecture of quantitative traits cannot be inferred from variance component..., Huang [/bib_ref]. Although we weighted our statistical model (Equation 1) for overall polygenic similarity, we did not have sufficient data to test for specific epistatic interactions. However, it is likelier that any epistasis one might deduce from a linear model is instead the result of attempting to fit a linear model to a nonlinear response such as those seen here, or n Parameter Values, Errors, Sign Patterns, and Shape Types for Experimental Surfaces Fit to Equation 3. The absolute experimental mesh points used in these estimates included the peak of each surface. The value of c was preset to produce the appropriate convexity. s is the square root of the solution norm. The residual norms were all 0. mis-assigning the meaning of the various variance components [bib_ref] Detecting high-order epistasis in nonlinear genotype-phenotype maps, Sailer [/bib_ref] [bib_ref] The genetic architecture of quantitative traits cannot be inferred from variance component..., Huang [/bib_ref]. Instead, one would solve for the nonlinear function and its matrix of coefficients. The latter captures both the interactions among genes and their entailed products and reactions; and the magnitudes of those interactions (the effect size). Such a function and its matrix define a physiological model of the network that, as an entirety, produces the phenotypic collection. Paths through the network select subsets of terms from the full nonlinear model, fragmenting the phenotypic collection into multiple approximations that can appear as epistasis. Epistatic interaction terms can also arise from linear transformations of nonlinear responses, which decouple the network into a matrix of connecting interactions and a matrix of the interactions' magnitudes. Better nonlinear models would fold these apparent epistases into the inherent nonlinearity of the response. Nonlinear changes in growth in response to combined stresses form a complex phenotype. Just from the surfaces fit to the experimental data, one can rule out an unbranched network sensitive to both water and nitrogen, because the response's peak does not scale with the sum or product of the inputs. Similarly, one can exclude two completely independent nodes, one for each input. Instead, the two inputs interact so that the response varies as a function of both: the phenotypic collection is produced by the action of the entire network, rather than just paths through it [fig_ref] Figure 4: Modeling of Response Surfaces with the Producing Function [/fig_ref]. The nonlinear producing function of Equation 2 defines this interaction as the sum of two components, each of which is sensitive to both water and nitrogen. It successfully accounts for all the important qualitative features of the experimentally observed phenotypic collection without assuming the large constants that dominate the solution in the QTL analysis (Equation 1 and . Surfaces generated using parameter values obtained by linear regression approximated the shapes of the experimental surfaces well, but were shifted downward in three-dimensional space . The heatmaps illustrate how much the surfaces can change with the parameter values. Slightly tilting the plane component of Equation 2 can strongly shift and rotate the position of the peak in ðx w ; x n ; zÞ: This suggests the model is quite sensitive to variation in d and e, and is consistent with our simulations (data not shown). Unlike crop models that frame the organism's physiology as a system of equations that involve large numbers of parameters that may not be directly measured [bib_ref] Use of crop growth models with whole-genome prediction: application to a maize..., Cooper [/bib_ref] [bib_ref] Integrating crop growth models with whole genome prediction through approximate bayesian computation, Technow [/bib_ref] , the producing function is a simpler, more coarsely-grained model that approximates the behavior of the system. It explicitly treats the parameters as fundamental model components that adjust the organism's physiological response to input water and nitrogen. Environmental perturbations, such as varying the available water and nitrogen, change the intervals over which the producing function is evaluated by the plant. Genetic perturbations, such as the alleles of the QTL identified in this work, delimit different regions in the phenotypic space in which possible responses lie. Of course, considering additional phenotypes or phenotypic dimensions might necessitate changing the function. The usual objective of QTL experiments is to isolate loci that exert a change on a single dimension of a phenotype, such as the mean. The assumptions are that the mappings between QTL and the parameter space, and between the parameter space and the phenotypic space, are one-to-one and onto; that the association function is linear and additive; and that one QTL can be freely exchanged for another with an effect of similar magnitude. However, our data clearly show none of these assumptions hold. The phenotypic collection we observe falls into four overlapping categories, and the close similarities of the phenotypic points within each category, and the overlap between the domed and hybrid categories depending on the classification criterion, suggest that the parameter values governing them can fall into rather broad ranges. This is a hallmark of sloppiness in model systems that breeders commonly call equifinality, and statisticians call "parameter nonidentifiability" [bib_ref] Sloppiness and emergent theories in physics, biology, and beyond, Transtrum [/bib_ref] [bib_ref] Parameter identifiability, constraint, and equifinality in data assimilation with ecosystem models, Luo [/bib_ref] [bib_ref] Parameter non-identifiability of the Gyllenberg-Webb ODE model, Hartung [/bib_ref] [bib_ref] Determination of parameter identifiability in nonlinear biophysical models: A Bayesian approach, Hines [/bib_ref]. This interpretation is supported by extensive simulation experiments: so far, we have been unable to identify unique combinations of parameter values that determine each phenotypic point. The parameter ranges for the observed phenotypic points are not disjoint, another characteristic of sloppy systems (data not shown) [bib_ref] Sloppiness and emergent theories in physics, biology, and beyond, Transtrum [/bib_ref]. Thus, our data divide both the parameter and the phenotypic spaces into nondisjoint subspaces, and confirm the mapping is many-to-many. Our current approach detects single QTL that control shifts among points in the phenotypic space, changing entire response surfaces. How Heatmaps of Surface Similarity Scores r, u, and dz r : The pairwise comparison of all 60 relative mesh points for each pair of experimental (e) and simulated surfaces (s) are the rows; the values for the pair of mesh points are the columns. The columns are ordered by concentric rings around the experimental peaks, so that the leftmost six columns are closest to the peak (:1) and the rightmost six furthest away (:10). Within each set of six columns, the ray segments (r0; r1; . . . ; r5) are arranged in order of increasing slope (non-Mo17) or decreasing slope (Mo17). For dz r ; bluer colors correspond to steeper gradients for the simulated surfaces, relative to the experimental ones, and yellower colors the opposite. The scales are heatmap-specific. could one detect portions of the network that affect different aspects of the phenotypic collection? For example, are there QTL that influence just the elliptical paraboloid or that restrict which shape categories can be reached from another? One would need to look for sets of QTL that jointly influence phenotypic features: these phenotypic subspaces, identified by specific loci, correspond to a higher resolution view of portions of the system. The many-to-many mappings among markers, the parameter space, and the phenotypic space would also have to be explicitly considered. We can think of two ways to approach this problem. The first way is to ask for markers that jointly affect subsets of parameters, shifting the phenotypic points from one phenotypic subspace to another. The overlapping categories of shapes we observe may reflect genuine nondisjointness in phenotypic space or missing dimensions that would separate the categories in a higher dimensional space. The second approach is to ask how many distinct groups of phenotypic points are in the collection by unsupervised clustering of all the experimental units. The membership of the resulting clusters would not necessarily be coincident with the genotypic or stress labels, nor would they necessarily be disjoint. One would then ask for intersecting sets of loci within and across all clusters, which allows many-to-many mappings. The clusters in phenotypic space correspond to allowable states of the underlying system and its producing function, joined by appropriate transitions among the clusters. The sets of loci may not be mutually disjoint, and subspaces of the parameters will differ in their sloppiness. These approaches could be tested first by simulations that randomly assign markers to different simulated surfaces. The success of this endeavor hinges first on the ability to recognize similarities among high-dimensional surfaces. The technique we used of scoring similarities by multiple criteria over a set of mesh points can be readily extended to higher dimensional surfaces, and used for clustering phenotypic points, optimizing parameter searches, and genetic mapping. For example, looking across all three scoring matrices in would produce a vector in 179-dimensional space. The second condition is the ability to fit parameters to nonlinear phenomena. Estimating parameters by fitting is more challenging with nonlinear phenomena. Two questions arise: what should be fitted? and how should it be fit? The first question is the fit criterion. We tested a wide variety of objective functions, singly and in combination, to describe the surfaces through a set of proxies that are faster to compute than the scores over the mesh points. None effectively discriminated among somewhat similar surfaces. This is not surprising in retrospect, given the many-to-many mapping between parameter and phenotypic spaces. The second question is one of fitting method. The most common approach approximates the higher degree polynomial with a linear statistical model, as we have done here. Our results illustrate the limitations of that approach. A more sophisticated, but compute-intensive, approach would be to fit a set of hyperplanes, approximating the surface as a set of n 2 1-dimensional splines. Another approach is to optimize the fit of the model to the data using one of many nonlinear optimization techniques (Bartholomew-Biggs 2008). Nonlinear optimizations require a good initial guess for the parameter values. We found the values generated by the linear fits were not good guesses, evidently placing the initial point outside the feasible region of the optimization. Further experimentation will be needed to improve the fits using this approach. Finally, one can search systematically for parameter combinations that generate surfaces that match the experimental ones. This is one way of asking how sensitive the phenotypic surfaces are to variations in the parameters' values. For nonlinear polynomial functions, the relationship between sets of parameter values and generated surfaces will not be regular or easily anticipated: stepwise changes in parameter values will produce "clumps" of generated phenotypes, consistent with the nonlinearity and sloppiness of the system. We encourage application of our response surface and shape modeling approaches to experiments mixing crop protections or stresses; developing methods to discover producing functions and the various mappings; and detecting and characterizing subspaces that subsume expressed phenotypic points. Methods to more efficiently traverse phenotypic spaces have the potential to accelerate breeding gains. [fig] Figure 1: Parental Inbred Response Surfaces to Combined Drought and Nitrogen Deprivation. A quadratic surface was fit to the measured trait of differences in plant height (z) and is shown for each parental inbred on the same scales. (A) The B73 inbred response surface; (B) the Mo17 response surface. [/fig] [fig] Figure 3: Gene Ontology Annotations for QTL. All known genes in each QTL region were scanned for significant annotations. GO process annotations are shown. Annotations were ordered by semantic similarity(Supek et al. 2011), with single genes under the QTL having higher uniqueness (redder shades). (A) QTL1, bin 1.06, bounded by markers gpm906a to IDP2465; (B) QTL2, bin 1.08, bounded by umc1838a to mmp22; and (C) QTL3, bin 9.03, bounded by ufg71 to IDP1681. [/fig] [fig] Figure 4: Modeling of Response Surfaces with the Producing Function (Equation 2). (A) The producing function drawn as a network. The elliptical paraboloid component is on the left, and the planar component on the right. Each term in the function is a node; the operators are edges. The parameters of the producing function (Equation 2) are in magenta; results of mathematical operations on the input nodes are shown in orange. The final equation is in yellow. (B) Sample simulated response surfaces and their projections into the ðx w ; x n Þ plane. (left) B73-like response surface, ða; b; c; d; eÞ ¼ ð0:260; 0:305; 2 0:175; 2:575; 0:500Þ; (right) Mo17-like response surface, ða; b; c; d; eÞ ¼ ð0:0330; 0:4250; 0:0030; 2 0:0025; 0:5500Þ:The color scale for each is the same for its surface plot and projection. [/fig] [table] Table 2: Comparison of Numerical Values of the Experimental Peaks' Positions and the Nondisjoint Shape Classification. These values were obtained from the surfaces generated using Equation 1 and the fitted parameter values ofTable 1. [/table]
App-based symptoms screening with Xpert MTB/RIF Ultra assay used for active tuberculosis detection in migrants at point of arrivals in Italy: The E-DETECT TB intervention analysis BackgroundFrom 2014 to 2017, the number of migrants who came to Italy via the Mediterranean route has reached an unprecedented level. The majority of refugees and migrants were rescued in the Central Mediterranean and disembarked at ports in the Sicily region. Rapid on-spot active TB screening intervention at the point of arrival will cover most migrants arriving in EU and by detecting TB prevalent cases will limit further transmission of the disease.Material and methodsBetween November 2016 and December 2017 newly arrived migrants at point of arrivals in Sicily, were screened for active Tuberculosis using a smartphone application, followed in symptomatic individuals by fast molecular test, Xpert MTB/RIF Ultra, on collected sputum samples.ResultsIn the study period 3787 migrants received a medical evaluation. Eight hundred and ninetyone (23.5%) reported at least one protocol-defined Tuberculosis symptom. Fifteen (2.7%) were positive to at least one microbiological test revealing a post-entry screening prevalence rate of 396 per 100.000 individuals screened (95% CI: 2.22-6.53). In logistic regression analysis, those with cough and at least one other symptom had an increased probability of testing positive compared to persons with symptoms other than cough. Whole-genomesequencing demonstrate two separate cases of transmission.DiscussionTo our knowledge this study reports first-time results of an active TB case finding strategy based on on-spot symptom screening using a smartphone application, followed by fast molecular test on collected sputum samples. Our preliminary findings reveal a post-entry screening prevalence rate of 396 per 100.000 individuals screened (95% CI: 2.22-6.53).Integrated on-the-spot screening strategy for active tuberculosis detection in migrants PLOS ONE | https://doi. [formula] a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 [/formula] Introduction From 2014 to 2017, the number of migrants who came to Italy via the Mediterranean route has reached an unprecedented level, with a peak of more than 181,400 new arrivals in 2016. In the same period, 29,595 and 21,653 migrants entered by sea Greece (eastern Mediterranean route) and Spain (west Mediterranean route), making Italy the main point of entrance in Europe for refugees and migrants. The migrant population, departing mainly from Libya, includes newly arrived individuals, who subsequently either claim asylum or passing through Italy move to other EU countries. Since the last two decades migration from high tuberculosis (TB) burden countries have contributed to slowing down the decreasing trend in TB incidence in European countries. In low TB incidence EU/EEA, these trends are mainly affected by migration dynamics with TB incidence rates among foreign-born individuals usually several times higher than among nonforeign-born individuals. Italy is a low TB incidence country, with a notification rate of 6.6 cases per 100,000 population and a prevalence of 4032 TB cases in 2016. Since 2009, more than 50% of TB cases notified each year (and most of MDR TB cases) have occurred in the foreign-born population. Hence, screening for TB among newly arrived migrants and asylum seekers represents a cornerstone of tuberculosis prevention and control strategies. In 2015 to face the disproportionate migratory pressures, Italian National Authorities agreed to implement the so-called "hotspot-hubs" approach. The "hotspot-hubs" approach aims to identify, register and properly process (including relocation and enforced return decisions for irregular migrants) new arrivals in few designated centers at key arrival points. In 2017 four "hotspots" (Lampedusa, Pozzallo and Trapani in Sicilian region and Taranto in Apulia) were operational, with a total official capacity of approximately 1,600 places. However, as the number of new arrivals has exceeded their capacity, mobile "hotspot" units were organized in Sicily and South Italy for identification and assistance purpose. After completing the identification procedures in place, migrants applying for refugee status are usually transferred within two or three days to regional first-line reception centers (so-called "hubs") where they wait for the decision on their application. While the national guideline for screening refugees has recently been reviewed, the use of a national protocol on TB screening procedures is difficult to implement and highlydemanding. Nationwide data for the number of asylum seekers screened, and the active TB cases found in newly arrived refugees are scarce in Italy, mainly due to a fragmented reception and public health system. The national law requires notification of tuberculosis cases only. Different approaches are adopted by both the Italian regions and by the different health-care providers in the reception centers. In hotspots active case-finding is based on TB symptoms screening, while in first aid reception centres interventions are often limited to passive case finding, performed in symptomatic migrants who report themselves to health centres or who visit outpatient clinics for unrelated medical conditions. Microbiological or radiological investigations of suspected cases among new arrivals at the hotspot required authorised transport to local hospital, limiting the number of people successfully screened at arrival centres. An Early DETECTion of tuberculosis consortium (E-DETECT TB) has recently been formed with the support of the Health program of the European Union bringing together national public-health agencies, academic centres and TB experts, with the aim to face the disproportionately high disease-burden of TB in vulnerable groups in European countries by using trans-national evidence-based intervention. The aim of the study is to report the data on an innovative active TB case finding strategy to screen newly arrived migrants at point of arrivals in Sicily, based on symptom screening through a smartphone application, the E-detect App and on-spot sputum analysis relying on fast, sensitive and specific molecular assay such as the Xpert Ultra. (Cepheid, Sunnyvale, CA, USA). # Material and methods ## E-detect tb app One of the main challenges in screening activities in non-healthcare settings is the need for fast data recording and prompt sharing of clinical records with the referral hospital as well as with relocation sites. Digital technologies are changing healthcare delivery globally. In TB field there is increasing recognition that digital technologies can support TB treatment adherence in diverse settings. An electronic-health (eHealth) system called E-DETECT TB has been developed to address the need for a fast and user-friendly mobile tool to log screening results directly at the point of arrival and at relocation sites. E-Detect TB App is a m-Health systemthat helps health-care staff to perform active and latent TB screening practice according to the World Health Organization (WHO) recommendations. The system includes three components. A touch-screen icon-based application for Android smartphone, structured in five modules (Questionnaire, Smear, Diagnostics, Latent TB infection, Treatment and follow-up). To record information, the user can click on the relevant icons or take photos. The application additionally allows the user to verbally record and save any further comments if necessary. At the end of the questionnaire all data collected is automatically transmitted to a Web database. The Web database is a java-based software system with secure access to the medical staff of the reference hospital. The system receives all the information registered by the phone application during the visit and allows the clinical staff of the hospital to monitor the data collected. The data collected can be subsequently exported into an electronic database for scientific and epidemiological purposes. At the end of the equestionnaire all data collected is automatically transmitted to the Medical Unit within the referral hospital. In the absence of cellular network coverage, it is still possible to use the application and store the data locally until an area with network coverage is reached. ## Microbiological procedures OMNIgene-sputum. OMNIgene-sputum (DNA Genotek-Ontario)is a highly stable non-toxic reagent that liquefies and decontaminates sputum samples at the point of collection, preserving MTB viability at ambient temperatures and minimizing contamination and crosscontamination. All samples were decontaminated onspot or within a day from the sputum collection using OMNIgene-sputum, according to the manufacture instruction. Briefly, the specimens were mixed with an equal volume of OMNIgene-sputum and stored at ambient temperature. Samples in OMNIgene-sputum were shipped at ambient temperature to the Emerging Bacterial Pathogen Unit at san Raffaele Scientific Institute within a week. Upon arrival OMNIgene-sputum was removed by centrifugation at 2800 g for 20 min, and then the sediment was resuspended in 2 ml of phosphate buffer pH 6.7 for further processing. Xpert MTB/RIF Ultra. TB screening approaches based on symptoms screening usually include a second step referral for further radiological/microbiological investigation. E-detect strategy uses fast molecular tests such as the Xpert MTB/RIF Ultra to confirm the diagnosis, thus resulting in a single step approach, feasible in outreach settings. Xpert MTB/RIF assay is an automated diagnostic test that can identify Mycobacterium tuberculosis DNA and resistance to rifampicin in less than 2 hours, compared to standard cultures that can take up to 6 weeks. Xpert Ultra incorporates two different multicopy amplification targets (IS6110 and IS1081) and uses improved assay chemistry and cartridge design, resulting in an approximately 1-log improvement in the lower limit of detection compared to the previous version of the test. The results of a first diagnostic accuracy study show that the sensitivity of Xpert Ultra is superior to that of the standard Xpert for tuberculosis case detection, especially in sputum smear-negative pulmonary tuberculosis, thus facilitating tuberculosis diagnosis at the earlier stages of disease. Sputum samples were tested by smear microscopy, solid and liquid culture, Xpert MTB/ RIF (Xpert G4) and the new XpertMTB/RIF-Ultra (Xpert Ultra). Xpert G4 and Xpert Ultra assays were performed by adding decontaminant reagent to the collected sputum specimens, as per the manufacturers instruction. Xpert G4 and Xpert Ultra present different grading system according to the number of cycles of amplification necessary to detect M. tuberculosis genome in the sample. Precisely Xpert Ultra has a new category "Trace" besides the four standard categories of "very low, low, medium and high" already present in the older Xpert G4. Smear microscopy was carried out using Ziehl-Neelsen (ZN staining) staining while the decontaminated sediment (0.5 ml) was inoculated in BBL MGIT tubes and incubated BAC-TEC MGIT960 instrument (BD Microbiology Systems, Sparks, MD, USA) and again 0.2 ml of decontaminated sediment was inoculated on Löwenstein-Jensen medium. Positive cultures were tested for ZN staining for the presence of Acid fast bacilli and subsequently tested by SD Bioline TB Ag MPT64 (Standard Diagnostics Inc.-South Korea) for identification of M. tuberculosis complex (Mtb) isolates. Positive Mtb cultures were tested for drug susceptibility testing for first line drugs. Whole Genome Sequencing (WGS). DNA was extracted for genomic analysis. Genomic DNA was extracted and purified by automated Maxwell system (Promega, Madison, WI, USA) Illumina (San Diego, CA, USA) technology was used for paired-end WGS applying the Nextera XT DNA sample preparation kit and the "benchtop" MiniSeq platform (300 cycles). Similarly, amplicons retrieved directly from amplification external chamber of the Xpert Ultra cartridges were sequenced for the detection of IS6110, IS1081 and rpoB gene to confirm the presence of Mtb DNA in samples categorized as "Trace". The WGS transmission analysis was based on a multi locus sequence type (MLST) based on the analysis of allelic variants in the core-genome by use of Ridom SeqSphere+ (Ridom GmbH). Both this approach and formal contact investigation were carried out in order to confirm inter-human transmissions[21]. We defined two isolates in cluster when the maximum number of allelic variants were less or equal to 5 units. ## E-detect tb intervention Between November 2016 and December 2017 individuals hosted in first line reception centres were screened for active TB, with the use of a e-questionnaire followed by sputum sample collection in symptomatic individuals. The screening was performed in 3 reception centers: the first aid reception centers of Mineo in Catania (whole period), the hotspot of Lampedusa and the temporary hotspot in Agrigento (from May 2017 to December 2017). The study population included asylum seekers defined as individuals seeking safety from persecution or serious harm in a country other than his or her own and awaits a decision on the application for refugee status. All those arriving or already hosted in the centres were offered the opportunity to participate in a voluntary screening which was performed through a standardised E-questionnaire carried out by medical staff. The e-questionnaire assessed sociodemographic data including age, sex, country of origin, travel time as well as TB risk factors (including HIV status if known), previous TB disease and TB contact. Symptoms suggestive of TB were investigated: cough for more than 2 weeks duration, fever for more than 1-week duration, night sweats, weight loss and haemoptysis. On the spot sputum sample collection were requested if at least one symptom was present. Whenever possible collection of sputum sample were performed during the visit. On the spot decontamination was also performed using OMNIgene-sputum (DNA Genotek-Ontario)on the collected samples. Participants were asked to provide only one sputum specimen. The aims and the methods of the study were explained to the participants verbally and through a printed leaflet in 4 different languages. Cultural mediators were presents in the centres when e-questionnaire were administered. Besides, regardless the participation to the study, information was provided on the main symptoms of TB disease and individuals were encouraged to visit the local health centres whenever these symptoms develop in the future. The screening intervention took place in agreement with local Authorities (Prefettura of Catania and Prefettura of Agrigento) and in collaboration with local health care providers and NGOs responsible for first support in the centres. The study was approved by the San Raffaele Institute Ethic Commission (protocol number 709624) and informed written consent was obtained from each subject before data collection. Microbiologically confirmed patients were address to the local Hospital for treatment and follow-up. # Statistical analysis Descriptive statistics are reported as proportions. For individuals reporting at least protocoldefined tuberculosis symptom, we investigated the association with lack of referral or refusal/ non-attendance to sputum collection of relevant characteristics using multinomial logistic regression analysis through Relative Risk Ratios (RRR, sometimes interpreted as conditional odd ratios) and their 95% confidence intervals (95% CI; significance was set at p <0.05). Gender, age, TB incidence in the country of origin, presence of comorbidities, symptoms and time since arrival in Italy were included in the multivariable model which was also adjusted for center and screening period. A univariate logistic regression was used to assess, among symptomatic individuals providing at least one sputum sample, variables associated with positivity of at least one microbiological test, calculating odds ratios (OR) and their 95% confidence intervals (95% CI). Sensitivity and specificity for each of the molecular diagnostic tests were estimated as compared to culture (as a gold standard) and reported with 95% confidence intervals. Cohen's kappa is used for assessing agreement between two molecular diagnostic tests (GeneXpert MTB/RIF vs GeneXpertMTB/RIF-Ultra). Data management and statistical analysis was performed using IBM SPSS Statistics version 25 (IBM Corp., Armonk, N.Y., USA) and STATA version 13 (Stata Corp LP, College Station, TX, USA). # Results ## Characteristics of the study population Between November 2016 and December 2017, 3787 migrants, hosted at the participating reception centres, received a first medical evaluation, which included the TB symptom screening, at the participating reception centers. Characteristics of these individuals are summarized in. The median age was 22 years (IQR:, the vast majority were male, reflecting data on refugees arrivals to Italy which shows a prevalence of males (79%). Almost half of the study population arrived in Italy since less than one month (48.2%). Overall, 39 The flow of enrolled individuals through the screening process is depicted inEight hundred and ninety-one (23.5%) reported at least one protocol-defined tuberculosis symptom. Among them, 489 (54.9%) reported cough, 468 (52.5%) weight loss, 261 fever (29.3%), 218 (24.5%) night sweats, and 126 (14.1%) haemoptysis; 268 individuals (30.1%) reported 2 symptoms, and 176 (19.8%) 3 or more. Among the 891 symptomatic individuals, 592 underwent sputum collection, 140 refused or not attended sputum collection. When an alternative diagnosis was already present a sample was not collected (159 individuals). ## Sputum yield and risk factors Among the 563 persons with symptoms, for whom a valid sputum sample was available, 15 (2.7%) were positive for at least one microbiologic test. This yield represents 0.40% of migrants included in the study; a point prevalence of 400 per 100,000. In logistic regression analysis compared to persons with symptoms other than cough, those with cough and at least one other symptom had an increased probability of testing positive (OR = 4.41, p<0.10;but this estimate is imprecise (95% CI: 0.97-20.11) and the association is not statistically significant at the conventional p-value<0.05. ## Yield among those with no symptoms Among the 2896 individuals not reporting protocol defined TB symptoms, 28 non-symptomatic persons (0,9%) underwent sputum collection based on personal will and physician judgement: 5 of them reported previous active disease and 4 of them were recent contact of active TB patients and want to be tested. Out of 28 individuals, 3 had at least one microbiologic test positive. One of them was a HIV positive woman in antiretroviral therapy, who wanted to be tested as her daughter was currently affected by pulmonary TB. The second patient had active TB disease in 2016 but the length of the treatment was unknown. No information is available on the third positive individual. ## Head to head comparison between xpert and xpert ultra Among the 591 migrants with a sample collected, with or without symptoms, 18 were positive for at least one microbiologic test: 11 individuals had culture-positive sputum (two of them, were positive only to culture), 9 had Xpert G4 positive results and 16 samples had Xpert Ultra positive results. None was rifampicin-resistant based on phenotypic drug susceptibility testing and Xpert results (both Xpert G4 and Xpert Ultra) Results of the comparison between Xpert and Xpert Ultra sensitivity and specificity are shown in. Sensitivities of Xpert Ultra and Xpert G4 were 81.8% and 54.6%, respectively, having culture as reference comparator. Specificities of Xpert Ultra and Xpert G4 for case detection were 98.7% and 99.5%. The overall agreement between the two tests was 0.71 (0.64-0.79 95% CI). Integrated on-the-spot screening strategy for active tuberculosis detection in migrants The two tests gave concordant results for 9 subjects, while discordant results were found in 7 subjects. All discordant results scored negative to the Xpert G4 and positive to the Xpert Ultra assay. Most of the discordant results showed positivity scoring Trace (6 Trace, 1 Very Low). The characteristics of subjects with discordant results are shown in . Sequencing of amplicons was performed on specimens for which Xpert Ultra gave "Trace and Rifampicin indeterminate" results, Sequencing of the amplicons obtained from the 6 samples with apparent false-positive results showed the presence of insertion sequences (IS6110 and IS1081) and rpoB gene in all tested samples (for 1 sample the amplicon was not available) Whole Genome Sequencing (WGS) analysis has been performed for all the culture positive samples. Genomes comparison demonstrated two separate cases of transmission probably occurred at the first aid reception centre of Catania. # Discussion The migrant population accounts for an increasing large proportion of TB cases in EU/EEA and challenges TB controls strategies. Currently two main TB control strategies has been applied among migrants: active TB cases finding using symptom screening followed by referral of symptomatic patients to hospital for further investigations, and CXR-based screening. However, both strategies are resource-demanding and difficult to implement at point of arrival due to logistic as well as security issues. In the last few years, the Central Mediterranean route from North Africa to Italy has experienced an unprecedented increase in population movement. The majority of refugees and migrants were rescued in the Central Mediterranean and disembarked at ports in the Sicily region. Rapid on-spot active TB screening intervention at the point of arrival will cover most migrant arriving in EU and by detecting TB prevalent cases will limit further transmission of the disease. The present study report data of post-entry active TB screening of asylum seekers performed in Italian hotspots and primary reception centers located in Sicily using a smartphone application for data collection, followed by on-spot collection of sputum sample and and molecular rapid test using Xpert Ultra assay. To our knowledge the study first report the active TB prevalence among newly arrived migrant in Sicily. Out of 3787 migrants receiving first medical evaluation, 891 reported at least one symptom of TB and were referred to sputum collection that was actually done for approximately 60% of them. Adherence to tuberculosis case finding interventions based on symptom screening in migrants and refugees/asylum seekers has been evaluated in previous studies conducted in primary care centers and mobile clinics in Italy. In these studies, diagnostic evaluation provided in TB clinics was completed by 30% of symptomatic individuals. This suggest that on site provision of diagnostic test increase the adherence to screening procedure. Our results reveal a post-entry screening prevalence rate of 3.96 per 1000 individuals screened (95% CI: 2.22-6.53). Several systematic reviews assessed the yield of active TB case among migrants. Screening yield was highly heterogenous across the studies reflecting the heterogeneity of post-arrival programs (type of migrant screened, timing and setting of the screening intervention, TB incidence in the country of origin and TB screening procedures). The overall yield of CXR to detect active TB in post-arrival settings was 350 cases/100.000. Vanino et al. published data of CXR-based TB screening intervention among asylum seekers arriving in the "hub" of Bologna. The study showed a post-entry screening prevalence rate of 535 (95% confidence interval, 317-844) per 100000 individuals screened. Thus, the yield of our intervention appears to be of the same order of magnitude of other interventions using different approaches. CXR screening has been reported to have higher sensitivity of (98%)compared to symptoms screening (78%). On the other hand, CXR has a poor specificity for TB, and radiographic findings need to be confirmed by laboratory tools. Our screening approach, by using symptoms screening, may miss some prevalent asymptomatic TB cases. However, it will reduce the use of local resource and therefore is more suitable to be applied in emergency settings, where radiographic facilities are not available, and transport is limited. The 23.5% of the population screened reported at least one symptom, cough was the most frequently reported (54.9%). 15 cases of TB were microbiologically detected. In the logistics regression analysis individuals who reported cough associated to other symptoms seem to have a greater probability of TB compared to migrants reporting one of the symptoms other than cough (even if the estimate is imprecise and the association does not reach statistical significance). This finding is consistent with the fact that our approach allows diagnosis of highly symptomatic patients while may be less effective for sub-clinical and pauci-symptomatic TB forms. Two TB cases were found among asymptomatic persons demonstrating the limits of symptom-based screening. One of them was a HIV positive woman in antiretroviral therapy, who wanted to be tested as active pulmonary TB was confirmed in the daughter. The second had active TB disease in 2016. He was previously hospitalized and treated but duration of antituberculosis treatment was uncertain. A third patient reported an Xpert Ultra positive (trace), Xpert G4 and culture negative sputum. He was a young asymptomatic male, who did report neither past disease nor previous TB contact but willing to be screened. As per WHO recommendations in absence of symptoms as well as predisposing factors to TB development, Ultra were repeated on a second and third sputum specimen. Trace positivity was not confirmed. The main difference between the strategy here described and the majority of TB screening approaches based on symptoms screening is the use of fast molecular tests, as the Xpert MTB/ RIF Ultra (Xpert Ultra), to confirm the diagnosis. Xpert MTB/RIF assay (Xpert G4 and Xpert Ultra) is an automated diagnostic test that can identify Mycobacterium tuberculosis DNA and resistance to rifampicin in less than 2 hours. Results of first diagnostic accuracy study show that the sensitivity of Xpert Ultra is superior to that of the Xpert G4 for tuberculosis case detection especially in sputum smear-negative pulmonary tuberculosis. In clinical practice, the high sensitivity of Xpert Ultra could facilitate diagnosis of tuberculosis at earlier stages of disease. In our study Xpert Ultra sensitivity was higher than that of the standard Xpert for TB case detection in participants with culture positive sputa (81.8% vs 54.6%, respectively). Specificities of Xpert Ultra and Xpert for case detection were 98.7% and 99.5% respectively. The two tests show an overall good agreement. Discordant results were found in 7 subjects: they all scored negative to the Xpert G4 and positive to the Xpert Ultra assay. In most of the discordant samples, the semiquantitative Xpert Ultra results correspond to the lowest bacillary burden (trace). Three of them were culture positive-Xpert Ultra positive samples, which will be missed out by Xpert G4 (one of them was a HIV patient). Four samples were culture negative-Xpert G4 negative but Xpert Ultra positive samples. One of them was an asymptomatic individual, whose positivity were not confirmed by further microbiological analysis. None of them reported past TB disease. False-negative cultures from over-decontamination has been also propose as possible explanations for a positive nucleic-acid amplification test result and negative sputum cultures. In this regard the use of OMNI-gene sputum to perform on spot decontamination may have caused an over-decontamination as indicated by the low rate of contaminated culture found (1.8% in liquid medium) WGS analysis has been performed for all the culture positive samples. The sequencing results demonstrate two separate case of potential transmission most probably occurred in the Catania Hubs. The first transmission identified (1 allelic variant), confirmed by WGS and epidemiological links, was between two relatives (a mother and the daughter) sharing the same room. In the second case (no allelic variant found) the two patients involved were two young men from Guinea and Liberia, they are not home-contact and apparently, they do not know each other. At the time of the diagnosis they have been hosted in the centre for 6 and 10 months respectively. No transmission were found in the hotspot of Lampedusa or Agrigento or between people hosted in different reception centers. Notably, the reception Centre of Catania, is the only hub included in the study. While accommodation in hotspot is strictly limited to the time need to carry out operations to defined the legal position, hubs are structures where migrants formalised their asylum request and wait for the approval. Social mixing, poor housing condition and overcrowding could facilitate the transmission of TB, therefore active TB case finding and systematic contact investigation should be implemented. The study has several limitations. The foremost limitation is that the study design did not allow to measure to what extent our approach may have increase the detection rate of active TB and/or reduced diagnostic delay. We could not find data on the number of TB cases notified before or after our intervention during a similar period of time at the enrolment centers. The small number of active TB cases identified limits our ability to investigate risk factors for a positive test. Similarly, we are unable to formally compare this strategy with CXR screening as this was not performed. To our knowledge this study reports for the first-time results of an innovative active TB case finding strategy based on-spot symptoms screening using a smartphone application followed by fast molecular test on sputum samples collected. The use of a phone application standardised data collection, allowed screening in the "field" and transfer of patients information directly to the referral Hospital. Fast molecular assays facilitated rapid identification of TB cases and prompt referral to the hospital in case of TB detection. It has been reported that TB screening at point of arrival is more acceptable in migrant patients. Moreover, single step for screening test, outreach settings and quick turnover of results increase the effectiveness of screening approach among migrantsOur screening approach offering TB symptoms screening and immediate on spot microbiological analysis may reduce the loss in the cascade of care. In our setting Xpert Ultra is more sensitive compared with the standard version of the test. Major advantages of the Xpert MTB/RIF assay in this setting are the rapid results, minimal technical training required to run the test, and the future possibility to perform the cartridge base molecular test directly on spot by using platform such as the portable Xpert OMNI. Supporting information S1 The questionnaire includes individuals' personal data, date of arrival in the centre, past medical history, past TB history, risk factors for TB and protocol-defined symptoms of TB (cough, fever, haemoptysis, night sweats and weight loss) Questionnaire is available in three languages (English, French, and Arabic). (DOCX)
Co-existing Gastrointestinal Hemorrhage and Deep Vein Thrombosis in a Patient with Hereditary Hemorrhagic Telangiectasia: Management Dilemma # Introduction The incidence of an autosomal dominant disorder known as hereditary hemorrhagic telangiectasia (HHT) or Osler-Weber-Rendu disease is one in 8000 people. The complex pathophysiology of HHT includes molecular abnormalities that disrupt the cellular pathways, leading to abnormal vascular development. Patients with HHT have various defective genes, but the three most common are endoglin (HHT1), activin receptor-like kinase (ALK1/HHT2), and SMAD4 (HHT associated with juvenile polyposis) [bib_ref] Hereditary haemorrhagic telangiectasia: a clinical and scientific review, Govani [/bib_ref]. Endoglin and ALK1 upregulate transforming growth factor beta (TGF-B) on endothelial cells, which promotes angiogenesis and helps vascular homeostasis [bib_ref] Transforming growth factor-beta signal transduction in angiogenesis and vascular disorders, Bertolino [/bib_ref]. A mutation of endoglin or ALK1 makes vessels vulnerable [bib_ref] Hereditary haemorrhagic telangiectasia: a clinical and scientific review, Govani [/bib_ref] 2 3 4 5 Open Access Case Report DOI: [bib_ref] Efficacy and safety of thalidomide for the treatment of severe recurrent epistaxis..., Invernizzi [/bib_ref] to damage. Atypical vessel formation causes recurrent epistaxis and GI bleeding, which leads to anemia. Dilated mucocutaneous blood vessels are often evident on physical examination. A review of the patient's history, physical examination, and laboratory workup is necessary. The diagnosis of HHT is made by using the Curacao criteria [bib_ref] Diagnostic criteria for hereditary hemorrhagic telangiectasia (Rendu-Osler-Weber syndrome), Shovlin [/bib_ref]. ## Case presentation A 69-year-old Caucasian male presented with a complaint of constant fatigue and weakness for multiple years. The patient had a history of epistaxis since childhood. According to the patient, tilting his head backward exacerbated the nosebleed and sitting upright alleviated the bleeding. The patient also had multiple first-and second-degree relatives with arteriovenous malformations and epistaxis. The patient also complained of a productive cough with clear sputum for the past six months. Additionally, the patient reported having exertional dyspnea and intermittent paroxysmal nocturnal dyspnea. His past medical history consisted of anemia, GI bleeding, gastric ulcer, melena, diabetes mellitus type 2, bilateral DVT, hypertension, arteriovenous malformation of the small bowel, occasional orthostatic lightheadedness, and scarlet fever. Past surgical history included multiple esophagogastroduodenoscopy (EGD) procedures. Upon physical examination, the patient was not in acute distress. His vitals were as follows: a blood pressure of 119/70 mmHg, a pulse of 68 beats per minute (bpm), a temperature of 103 F, and a respiratory rate of 16 breaths per minute (bpm). At presentation, the patient had multiple vascular malformations on the fingers, upper palate, tongue, lower lips, ears, and the face (as shown in [fig_ref] FIGURE 6: Telangiectasias of the face [/fig_ref] , respectively). The S1 and S2 sounds were audible with a regular rate and rhythm. The tenderness was present on deep palpation in the left lower quadrant. There was no leg swelling, warmth, or redness. Peripheral pulses were palpable. The deep tendon reflex was normal and the cranial nerves were intact. ## Hospital course Considering the consistently low hemoglobin (Hb), fatigue, and occult positive stool results, the patient was admitted to the hospital and was given two units of blood. The epistaxis episode resolved spontaneously. On day one, the patient's Hb level was 4.9, and he was treated with four units of packed red blood cells (PRBC), and the Hb increased to 8.0. On day two, the patient's Hb dropped to 7.7, and he was infused with another unit of PRBC. The colonoscopy result showed bleeding in the ascending colon. The patient also underwent esophagogastroduodenoscopy (EGD), showing multiple 4-millimeter angiodysplasias with active bleeding at the cardia and fundus. There were also non-bleeding angiodysplasias in the duodenum and the jejunum, which were treated with argon beam coagulation and photodynamic therapy. # Discussion The Curacao criteria are established by the scientific advisory board of the HHT Foundation International. The criteria are characterized by recurrent epistaxis, visceral bleed, mucocutaneous telangiectasia, and a family history of HHT. The presentation of three or more symptoms is required to establish a definitive diagnosis. Patients presenting with two symptoms are considered at risk of developing the disease [bib_ref] Transforming growth factor-beta signal transduction in angiogenesis and vascular disorders, Bertolino [/bib_ref]. Epistaxis and GI bleeding are the most common causes of anemia in HHT. The patient is often born without symptoms. Epistaxis is more common in younger patients while GI bleeding presents in the fifth decade of life. Frequent rupture of angiodysplasias and blood loss worsens in adults. In one of the studies, gastrointestinal bleeding was reported in 33% of the patients with HHT. The same study also reported that 25% of HHT patients greater than 60 years of age suffered from severe gastrointestinal bleeding [bib_ref] Gastrointestinal bleeding in patients with hereditary hemorrhagic telangiectasia, Kjeldsen [/bib_ref]. Additionally, patients with HHT not only have a higher bleeding tendency but they also have an increased risk of thromboembolism. A study conducted by Livesey et al. reported that the risk of thromboembolism increased by almost two-fold in a patient with HHT compared to the general population. Recurrent bleeding in HHT decreases serum iron levels. The low iron has an inverse relationship with factor VIII and contributes to these patients having a 2.5-fold increased risk of venous thromboembolism [bib_ref] Low serum iron levels are associated with elevated plasma levels of coagulation..., Livesey [/bib_ref]. The management of HHT is a complicated and frustrating process for both patient and physician. Multiple bleeding events lead to iron deficiency anemia. The patient is often given a dietary plan, which includes an iron-rich diet and an iron supplement. Even with a proper diet, the patient often requires a regular blood transfusion. Therapeutic decisions are based on the bleeding symptoms, but the challenge arises in a patient with DVT. To our knowledge, in the current literature, there is no definitive treatment for GI bleeding in an HHT patient with a history of recurrent DVT. Hormonal treatment has been used to decrease recurrent epistaxis episodes and GI bleeding. Tamoxifen or raloxifene work at a molecular level by upregulating multiple genes. Their effect is on the transcription factor at the promoter region of endoglin and ALK1 through an increased expression of their receptors on the cell surface. The upregulation of these proteins has a pro-angiogenic effect on vessels. It may decrease the bleeding complication, but it is a contraindication in our patient due to its adverse effect of potentially exacerbating the DVT. Thalidomide and bevacizumab are antiangiogenic drugs that have shown to improve recurrent nose bleeds and incidences of telangiectasia. Bevacizumab affects the vascular endothelial growth factor (VEGF) receptors and prevents abnormal angiogenesis. Long-term treatment with intravenous bevacizumab has shown an improvement in the cessation of bleeding and helps improve the hemoglobulin level. Similarly, thalidomide not only affects VEGF but also downregulates TGF-B. Along with angiogenesis, thalidomide also has an anti-inflammatory property that provides additional benefits in a patient with HHT. Thalidomide also decreases the epistaxis and improves the telangiectasia spots. Despite the improvement in bleeding problems, the severe adverse effect has been seen in patients who are taking bevacizumab and thalidomide. The study conducted by Cohen et al. described several adverse effects of bevacizumab, such as an increased risk of arterial thromboembolic events, delayed wound healing, hemorrhage, and gastrointestinal perforation. Thalidomide also causes thromboembolic complications [bib_ref] FDA drug approval summary: bevacizumab (Avastin) plus carboplatin and paclitaxel as first-line..., Cohen [/bib_ref] [bib_ref] Long-term therapy with bevacizumab in hereditary hemorrhagic telangiectasia, Brinkerhoff [/bib_ref] [bib_ref] Thalidomide effects in patients with hereditary hemorrhagic telangiectasia during therapeutic treatment and..., Peng [/bib_ref] [bib_ref] Efficacy and safety of thalidomide for the treatment of severe recurrent epistaxis..., Invernizzi [/bib_ref]. Argon plasma coagulation (APC) is a medical endoscopic procedure used primarily to control bleeding from certain lesions in the gastrointestinal tract by a high-frequency current distributed on the tissue through ionizing argon gas. Kwan et al. conducted a prospective study, which showed an improvement in hemoglobin levels and a decrease in the recurrence of blood transfusions in a patient with APC treatment [bib_ref] Argon plasma coagulation in the management of symptomatic gastrointestinal vascular lesions: experience..., Kwan [/bib_ref]. Immediate hemostasis rates range from 85 to 100 percent in various reports and treatment can result in the long-term control of bleeding [bib_ref] Argon plasma coagulation in the management of symptomatic gastrointestinal vascular lesions: experience..., Kwan [/bib_ref] [bib_ref] Argon plasma coagulation (APC) in gastroenterology: experimental and clinical experiences, Johanns [/bib_ref] [bib_ref] Argon beam coagulation for treatment of symptomatic radiation-induced proctitis, Fantin [/bib_ref]. In hospitals, a patient with current DVT and a history of GI bleeding (with HHT) is often treated with short-term anticoagulant or antiplatelet therapy. The extended use of anticoagulant therapy is a contraindication due to severe hemorrhagic complications. # Conclusions Chronic GI bleeding causes low iron, which increases the risk of DVT. Once the disease progresses, the treatment to control recurrent bleeding and DVT becomes more laborious. The use of currently available hormonal medications for HHT becomes a contraindication due to the risk of DVT. Regular monitoring for anemia and ultrasounds to assess for DVT formations in these patients are often required. Early preventative treatment for patients who have firstdegree relatives with HHT can prevent delayed complications. We hope our case report highlights the management dilemma in treating co-existing GI bleeding and DVT in a patient with HHT. Furthermore, we also hope that it possibly helps dictate the further research needed for the early diagnosis and management of these patients. [fig] FIGURE 6: Telangiectasias of the face (orange circle) [/fig]
Platelet-to-lymphocyte ratio as a predictive index for delirium in critically ill patients Delirium is a neuropsychiatric syndrome commonly encountered in critically ill patients, and systemic inflammation has been strongly implicated to underlie its pathophysiology. This study aimed to investigate the predictive value of the platelet-to-lymphocyte ratio (PLR) for delirium in the intensive care unit (ICU).In this retrospective observational study, we analyzed the clinical and laboratory data of 319 ICU patients from October 2016 to December 2017. Using the Locally Weighted Scatterplot Smoothing technique, a PLR knot was detected at a value of approximately 100. Logistic regression was used to investigate the association between the PLR and delirium.Of the 319 patients included in this study, 29 (9.1%) were diagnosed with delirium. In the delirium group, the duration of mechanical ventilation was significantly longer than that in the no-delirium group (40.2 ± 65.5 vs. 19.9 ± 26.5 hours, respectively; P < .001). A multiple logistic regression analysis showed that PLR > 100 (odds ratio [OR]: 1.003, 95% confidence interval [CI]: 1.001-1.005), age (OR: 2.76, 95% CI: 1.110-6.861), and the ratio of arterial oxygen partial pressure to the inspired oxygen fraction (OR: 0.996, 95% CI: 0.992-0.999) were independent predictors of delirium.In our study, a high PLR value on ICU admission was associated with a higher incidence of delirium. Owing to easy calculability, the PLR could be a useful delirium predictive index in ICUs, thereby enabling early interventions to be implemented.Abbreviations: APACHE II = acute physiology and chronic health evaluation II, CI = confidence interval, ICU = intensive care unit, IL = interleukin, LOS = length of stay, OR = odds ratio, PaO 2 /FiO 2 = ratio of arterial oxygen partial pressure to the inspired oxygen fraction. # Introduction Delirium is a common neuropsychiatric syndrome in the intensive care unit (ICU) that presents as impaired consciousness, cognition, and attention,with a reported incidence that varies from 11% to 87%.Previous studies indicated that delirium could contribute to prolonged mechanical ventilation duration and ICU length of stay (LOS), as well as a higher mortality rate.However, effective therapies for delirium, including pharmacologic and nonpharmacologic, remain limited. Thus, identifying patients who are at high risk of delirium is critical. According to recent findings, neuroinflammationis a potential underlying mechanism for delirium. Nonetheless, its pathophysiology remains unclear. Studies have confirmed that inflammatory markers, such as interleukin (IL)-1, IL-8, cortisol, and tumor necrosis factor a, were significantly elevated in patients with delirium.Recently, the platelet-to-lymphocyte ratio (PLR) was reported as a marker for the inflammatory response in various diseases, including coronary artery disease,acute kidney injury,and various cancers.However, whether a similar association exists between PLR and delirium in critically ill patients remains unclear. Therefore, we performed a retrospective analysis to explore the predictive effect of PLR in delirium. # Methods ## Study design and patients This was a retrospective, observational study performed from October 2016 to December 2017 in a tertiary teaching hospital that has a 20-bed general ICU. The study was approved by the Ethical Committee of Dongyang People's Hospital. Informed consent was waived because of the study's observational nature. All patients in the ICU received the standard treatment to prevent delirium-the 'ABCDE' bundle,including Awake, Breathing coordination, Choice of sedation, Delirium monitoring and treatment, and Early mobility and exercise. All adult patients admitted to the ICU were screened. The exclusion criteria were as follows: (1) inability to communicate (coma, profound dementia); (2) a history of schizophrenia or Parkinson's disease; (3) brain injury; and (4) stay in the ICU of less than 24 hours. ## Data collection We collected the data from the electronic medical records system within the first 24 hours after ICU admission. We obtained the patients' demographic data, including sex, age, education level, Acute Physiology and Chronic Health Evaluation II (APACHE II) score, surgical types, comorbidities, tracheal intubation, and drinking and smoking status. Laboratory measurements included the ratio of arterial oxygen partial pressure to the inspired oxygen fraction (PaO 2 /FiO 2 ), hemoglobin, platelet, lymphocyte, white blood cell, neutrophil, hematocrit, serum potassium, sodium, total protein, albumin, glucose, creatinine, and C-reactive protein levels. The PLR was calculated by dividing the platelet count by the lymphocyte count on ICU admission. The primary outcome was delirium diagnosed after ICU admission. The secondary outcomes included the hospital LOS, ICU LOS, and mechanical ventilation duration. ## Definition of delirium Diagnosis of delirium was made according to the Confusion Assessment Method for ICU by nursing staff who had undergone specialist training to make diagnoses of delirium, which included acute onset or fluctuating course of inattention, disorganized thinking, and an altered level of consciousness. All patients were evaluated for delirium twice daily until ICU discharge. ## Grouping methods for plr in the logistic models The locally weighted scatterplot smoothing technique was used to detect the cutoff values of the PLR that predicted delirium. Thus, in the multivariate logistic regression, PLR was divided into 2 groups (100 and >100). ## Statistical analyses Continuous data were presented as means ± standard deviation or medians (interquartile range) and count data were presented as proportions. The Student's t-test was applied for normally distributed continuous variables and the x2 test for categorical variables. For non-normally distributed continuous variables, the Mann-Whitney U-test was used. A univariate 1-way analysis of variance was performed to assess significant differences in the demographic data and laboratory measurements. Predictive variables from the univariate analysis with a P < .1 were included in the multivariate models, which were constructed using the stepwise backwards method. Finally, the age, APACHE II score, serum glucose level, surgery, serum albumin level, respiratory disease, PaO2/FiO2, and PLR were included in the logistic models. The STATA 11.2 software (College Station, TX) was used for all statistical analyses. A 2tailed test was used and P < .05 was considered statistically significant. # Results In total, 319 patients were included in this study after screening. The baseline comparisons are shown in. The mean age was 62.6 ± 16.8 years and 49.5% were males. Twenty-nine patients (9.1%) were diagnosed with delirium. In total, 278 patients (87.1%) were admitted to the surgical services and the most common diagnoses were cardiac disease (31.7%) and orthopedic diseases (17.2%). There were 84 patients (26.3%) with a history of cigarette smoking and 63 (19.7%) with a history of alcohol drinking. Significant differences were found between the delirium and no-delirium groups in age, APACHE II score, history of respiratory diseases, history of surgery, PaO2/FiO2, serum glucose level, albumin level, platelet count, and PLR. Regarding the clinical outcomes, a longer duration of mechanical ventilation was observed in the delirium group compared to the no-delirium group (40.2 ± 65.5 vs 19.9 ± 26.5 hours, respectively; P < .001). The differences in the ICU LOS (7.9 ± 7.8 vs 5.3 ± 9.4 days, respectively; P = .11) and hospital LOS (25.7 ± 13.2 vs 29.3 ± 30.3 days, respectively; P = .50) were not significant. In the locally weighted scatterplot smoothing curve between the PLR and delirium, a knot was detected at a PLR value of approximately 100, on ICU admission. Thus, in the multivariable logistic regression model ( # Discussion The results of this study show that the PLR was significantly higher in the ICU patients with delirium. A high PLR value (>100) on ICU admission was associated with a higher incidence rate of delirium. In addition, age and PaO2/FiO2 were also determined to be independent risk factors for delirium. In our study, 9.3% of the patients developed delirium, which is less than the reported incidences in previous studies.This discrepancy could be caused by the fact that we applied a quality control circle to reduce the occurrence of delirium. Additionally, the delirium incidence rate was significantly lower among surgical patients than among medical patients. We found that the proportion of patients with diabetes mellitus was 13.2%; however, it is likely that there were undiagnosed cases. There were significant differences between those with delirium, nodelirium, and serum glucose level, but not with diabetes. Our results are similar to those reported by van et al.The neuroinflammatory hypothesis is a popular hypothesized mechanism underlying the development of delirium.It is theorized that acute peripheral inflammatory stimulation, brain parenchymal cell activation, as well as proinflammatory cytokine expression could lead to neuronal cell apoptosis and synaptic dysfunction,promoting the development of delirium. It is well known that delirium is common in end organ dysfunction after sepsis. Martin et al found that sepsis-induced delirium is the most common cause of delirium in the ICU and that sepsis and delirium are closely related. Furthermore, systemic inflammation could be an important trigger,there is increasing evidence indicating that proinflammatory factors, such as procalcitonin,IL-8,IL-6, and S100 beta,have important roles in the development of delirium. In addition, C-reactive protein APACHE-II = acute physiology and chronic health evaluation, ICU = intensive Care Unit, NLR = neutrophil to lymphocyte ratio, PaO2/FiO2 = ratio of arterial oxygen partial pressure to fraction of inspired oxygen, PLR = platelet to lymphocyte ratio. Jiang et al. Medicine (2020) 99:43 www.md-journal.com levels were foundto predict severity and duration of postoperative delirium. Overall, inflammation is a potential underlying mechanism of delirium. In previous studies, it was reported that platelets may exhibit an important effect on inflammatory modulationby promoting the release of inflammatory cytokines that initiate the inflammatory process.In addition, evidence has also indicated that lymphocytes are also an important inflammatory factor in different diseases, such as cardiovascular diseasesand type 2 diabetes. Therefore, the PLR was proposed as a new marker for inflammation in various disorders. For example, an elevated PLR was associated with adverse survival probabilities in patients with colon,breast,and small cell lung cancer,which may be caused by an intensified systemic inflammatory reaction. Kurtul et alexamined 1,016 patients with acute coronary syndrome and reported that an increased PLR (>150) was significantly associated with severity and complexity of coronary atherosclerosis. Similarly, a possible explanation for this may be an increased inflammatory response. In another study, the PLR was found to be lower in the early stages of diabetes, but significantly higher in the later stages.Our results showed that a high PLR value (>100) was an independent risk factor for delirium, even after adjusting for potential confounders. As it is easily calculated, the PLR could be a useful predictor of delirium in critically ill patients, thereby enabling early interventions to be implemented. # Limitations First, this was a pilot observational study; therefore, selection bias could have occurred. In clinical practice it is difficult to evaluate for delirium during invasive mechanical ventilation in critically ill patients, so a proportion of patients with delirium may not have been diagnosed, and so these results should be interpreted with caution. Second, the number of patients with delirium in this study was small and we were not able to perform stratified analyses for subtypes of delirium. Finally, the study population was heterogeneous, including medical and surgical patients, and among the latter, patients who had undergone different types of surgery. However, our results can be generalized to all critically ill patients. # Conclusion Delirium is a neurobehavioral syndrome; it is unlikely that a single cascade will explain the phenomena of delirium. Our study found that a high PLR value on ICU admission was associated with a higher incidence of delirium. The PLR is simple to calculate, inexpensive, and could be used to predict delirium in critically ill patients. APACHE-II = acute physiology and chronic health evaluation, CI = confidence interval, OR = odds ratio, PaO2/FiO2 = Ratio of arterial oxygen partial pressure to fraction of inspired oxygen, PLR1 = platelet to lymphocyte ratio<100, PLR2 = platelet to lymphocyte ratio>100.
Co-application of ACC-deaminase producing PGPR and timber-waste biochar improves pigments formation, growth and yield of wheat under drought stress Besides other deleterious effects, drought elevates ethylene level too in plants. Increased ethylene concentration reduces root elongation and development that consequently retard plant growth andyield. There are certain PGPR which produce ACC-deaminase. The ACC-deaminase converts ACC (an immediate precursor of ethylene biosynthesis in methionine pathway in higher plants) into ammonia and α-ketobutyrate instead of ethylene. Regularization of ethylene level in plants mitigate the effects of drought. On the other hand, biochar has been reported to be rich in nutrients and exhibiting higher water holding capacity. So, a pot study was conducted with the hypothesis that the combined application of ACC-deaminase producing PGPR and biochar would minimize the drought effects on wheat growth. The ACC-deaminase producing PGPR were applied on wheat seeds in combination with two biochar doses. Three moisture levels were maintained throughout the trial. The data obtained revealed that B. amyloliquefaciens + 2BC improved the chlorophyll a, chlorophyll b, photosynthetic rate, transpiration rate, 100-grain weight, and grain N, P and K up to 114%, 123%, 118%, 73%, 59%, 58%, 18% and 23%, respectively, under drought conditions. It is concluded that co-application of PGPR and biochar is an effective technique to mitigate the drought effects.www.nature.com/scientificreports www.nature.com/scientificreports/ conductance. However, stomatal conductance was maximum as compared to the control through 2BC (56%), B. amyloliquefaciens + 2BC (146%) and B. amyloliquefaciens + 2BC (5.62-fold) at normal moisture (NM), MD and SD levels , respectively. Shoot length and electrolyte leakage. Both main and interactive effects of T and D were significantly different for shoot length and electrolyte leakage in wheat leaves. At SD level, the B. amyloliquefaciens + 2BC and P. aeruginosa + 2BC remained significantly better as compared to rest of the treatments for shoot length(Fig. 1). The 1BC and 2BC remained statistically alike but significantly different as compared to the control at SD for shoot length. However, B. amyloliquefaciens + 2BC also remained significantly better among other treatments for shoot length at MD level. Maximum increase, 153% and 73% in shoot length was noted at SD and MD levels respectively, as compared to the control where B. amyloliquefaciens + 2BC was applied(Fig. 1a). In case of electrolyte leakage at SD, A. fabrum + 2BC and B. amyloliquefaciens + 2BC were found to be significantly better as compared to the control. The 1BC and 2BC remained statistically alike but significantly different as compared to the control at SD for electrolyte leakage(Fig. 1b). The L. adecarboxylata, P. aeruginosa, B. amyloliquefaciens without BC also decreased the electrolyte leakage as compared to the control at SD. Maximum reduction (50%) in electrolyte leakage was noted as compared to the control where A. fabrum + 2BC and B. amyloliquefaciens + 2BC were applied at SD. Carotenoids and proline. Both main and interactive effects of T and D were significantly different for carot-Scientific RepoRts | (2019) 9:5999 | https://doi. Various biotic (pests, pathogens) and abiotic (soil compaction, drought, salinity, waterlogging, heavy metals, poor nutrition etc.) stresses are a big cause of low crops productivity around the globe 1 . Drought stress is very common in worldwide arid and semi-arid areas. Moreover, climate change is going to create the worst situation in this regard [bib_ref] Fulvic Acid Application Improves the Maize Performance under Well-watered and Drought Conditions, Anjum [/bib_ref] [bib_ref] Climate Change-Induced Water Stress Suppresses the Regeneration of the Critically Endangered Forest..., Zhang [/bib_ref] [bib_ref] Evaluating Climate Change Induced Water Stress: A Case Study of the Lower..., Griffin [/bib_ref] [bib_ref] Compounding Impacts of Human-Induced Water Stress and Climate Change on, Mehran [/bib_ref] [bib_ref] Alleviation of drought stress in pulse crops with ACC deaminase producing rhizobacteria..., Saikia [/bib_ref]. The demand for irrigation water is expected to increase by 10% up to 2050 [bib_ref] Multimodel Projections and Uncertainties of Irrigation Water Demand Under Climate Change, Wada [/bib_ref]. Under drought stress, growth and yield of crops are usually decreased due to less intake of nutrients, poor photosynthesis [bib_ref] Crop Production under Drought and Heat. Stress: Plant Responses and Management Options, Fahad [/bib_ref] and limited supply of water [bib_ref] A Critical Evaluation of Traits for Improving Crop Yields in Water-Limited Environments, Ludlow [/bib_ref]. In addition, drought accelerates the biosynthesis of ethylene [bib_ref] The ethylene gas signal transduction pathway: a molecular perspective, Johnson [/bib_ref] [bib_ref] A Model For the Lowering of Plant Ethylene Concentrations by Plant Growth-promoting..., Glick [/bib_ref] which retards the roots elongation and development [bib_ref] Plant growth-promoting bacteria that confer resistance to water stress in tomatoes and..., Mayak [/bib_ref] [bib_ref] Ethylene and plant responses to stress, Morgan [/bib_ref] [bib_ref] Rhizobacteria with ACC-Deaminase Activity Improve Nutrient Uptake, Chlorophyll Contents and Early Seedling..., Danish [/bib_ref]. Although, traditional breeding, water management and genetic engineering are thought to be useful tools to alleviate drought stress but high technicalities are involved to adopt and implement these approaches [bib_ref] Drought-tolerant plant growth-promoting rhizobacteria associated with foxtail millet in a semiarid and..., Niu [/bib_ref]. However, the use of plant growth promoting rhizobacteria (PGPR) is an alternative technique for mitigation of drought effects [bib_ref] Drought-tolerant plant growth-promoting rhizobacteria associated with foxtail millet in a semiarid and..., Niu [/bib_ref]. A large number of rhizospheric bacteria are well documented that show growth promotion in plants under stressful conditions [bib_ref] Rhizobacteria with ACC-Deaminase Activity Improve Nutrient Uptake, Chlorophyll Contents and Early Seedling..., Danish [/bib_ref]. As far as regularization of ethylene biosynthesis under drought stress is concerned, using 1-aminocyclopropane-1-carboxylate deaminase (ACC-deaminase) producing PGPR is found to be quite effective [bib_ref] The Role of ACC Deaminase Producing PGPR in Sustainable Agriculture, Saraf [/bib_ref] [bib_ref] Bacterial-mediated drought tolerance: Current and future prospects, Ngumbi [/bib_ref] [bib_ref] Perspective of plant growth promoting rhizobacteria (PGPR) containing ACC deaminase in stress..., Saleem [/bib_ref] [bib_ref] Enhancement of drought stress tolerance in crops by plant growth promoting rhizobacteria, Vurukonda [/bib_ref] [bib_ref] Metabolism of 1-aminocyclopropane-1-carboxylic acid, Honma [/bib_ref]. The ACC-deaminase cleaves the ACC (1-aminocyclopropane-1-carboxylic acid, an immediate precursor of ethylene biosynthesis through methionine pathway in higher plants) into ammonia and α-ketobutyrate instead of ethylene [bib_ref] A Model For the Lowering of Plant Ethylene Concentrations by Plant Growth-promoting..., Glick [/bib_ref] [bib_ref] Bacterial ACC deaminase and the alleviation of plant stress, Glick [/bib_ref] [bib_ref] ACC deaminase is central to the functioning of plant growth promoting rhizobacteria, Glick [/bib_ref]. Besides ethylene regularization, the PGPR also help in better root development [bib_ref] Characterization of plant growth promoting rhizobacteria isolated from polluted soils and containing..., Belimov [/bib_ref] , secretion of growth hormones (auxins or cytokinins) [bib_ref] ACC deaminase is central to the functioning of plant growth promoting rhizobacteria, Glick [/bib_ref] and solubilization of immobile nutrients (phosphorus, potassium etc.) [bib_ref] Phosphate solubilizing rhizoplane bacteria on growth and yield of transplant aman rice, Alam [/bib_ref] [bib_ref] Co-inoculation of potassium solubilizing and nitrogen fixing bacteria on solubilization of waste..., Basak [/bib_ref]. On the other hand, the imperative role of organic amendments in mitigation of drought stress by improving soil water holding capacity and availability of nutrients cannot be denied [bib_ref] Influence of biochar on growth and photosynthetic attributes of Triticum aestivum L...., Danish [/bib_ref] [bib_ref] Effects of various biochars on seed germination and carbon mineralization in an..., Qayyum [/bib_ref]. Biochar (BC), is a black carbon compound which is a good source of nutrients. It is produced through pyrolysis at high temperature under low or no supply of oxygen [bib_ref] Effects of various biochars on seed germination and carbon mineralization in an..., Qayyum [/bib_ref] [bib_ref] Bio-energy in the black, Lehmann [/bib_ref] [bib_ref] Characterisation and evaluation of biochars for their application as a soil amendment, Singh [/bib_ref]. The physio-chemical properties of BC depend on the nature of waste material used and temperature of the pyrolysis [bib_ref] Closing the loop on organic waste management: biochar for agricultural land application..., Navia [/bib_ref] [bib_ref] Ameliorating physical and chemical properties of highly weathered soils in the tropics..., Glaser [/bib_ref]. High surface area and pore spaces of BC-structure improve soil water and nutrients holding capacity [bib_ref] Charcoal effects on soil solution chemistry and growth of Koeleria macrantha in..., Gundale [/bib_ref] [bib_ref] Characteristics of Biochar -Micro-chemical Properties, Amonette [/bib_ref] [bib_ref] Ranking the magnitude of crop and farming system effects on soil microbial..., Hartmann [/bib_ref]. Wheat (Triticum aestivum L.) is an important cereal crop and staple food in most parts of the world. It contains 55% carbohydrates and 8-12% proteins [bib_ref] Postprandial metabolic utilization of wheat protein in humans, Bos [/bib_ref]. It is an important crop due to its worldwide trade too. Cultivation of wheat under a limited supply of water significantly decreases the yield 38 while its demand is increasing at the rate of 1.6%/annum [bib_ref] Plant growth promotion by Bacillus megaterium involves cytokinin signaling, Ortíz-Castro [/bib_ref]. The need of time is to enhance wheat yield even in the areas under drought stress. In recent past, the researchers focused on the application of either ACC-deaminase containing PGPR or BC in separate to mitigate the drought stress. The novelty and aim of the present study are to examine the combined effect of ACC-deaminase producing PGPR and timber-waste BC for the alleviation of drought effects. Keeping in mind the importance of wheat, the current study was conducted with the hypothesis that the co-application of drought tolerant ACC deaminase producing PGPR and timber waste BC could be very effective to alleviate drought effects. # Results Gas exchange attributes. Main effects of treatments (T) and various levels of drought (D) were significantly different, while their interaction (T × D) remained similar for the rate of photosynthesis and transpiration. For stomatal conductance, both main and interactive effects of T and D were significantly different. The photosynthetic rate was significantly improved as compared to the control where P. aeruginosa and B. amyloliquefaciens were applied . The BC application without PGPR significantly enhanced photosynthetic rate as compared to the control but the application rate 2BC was more effective than 1BC for the improvement in photosynthetic rate. However, among all the treatments P. aeruginosa + 2BC and B. amyloliquefaciens + 2BC remained the best to significantly increase the photosynthetic rate. For transpiration rate, a significant improvement was noted as compared to the control in all the treatments. Application of 1BC and 2BC without PGPR gave statistically similar results regarding transpiration rate. However, P. aeruginosa + 2BC and B. amyloliquefaciens + 2BC remained significantly better for transpiration rate as compared to L. adecarboxylata, A. fabrum, P. aeruginosa, B. amyloliquefaciens, 1BC and the control. Maximum increase in photosynthetic rate (118%) and transpiration (73%) rate was noted as compared to control where B. amyloliquefaciens + 2BC was applied. Application of 1BC and 2BC remained significantly better at severe drought (SD) level as compared to the control for stomatal conductance. However, at mild drought (MD) level, application of 2BC remained significantly better than 1BC and control regarding stomatal conductance. The B. amyloliquefaciens + 2BC at SD level while 2BC, P. aeruginosa + 2BC and B. amyloliquefaciens + 2BC at MD level performed significantly better among other treatments for stomatal enoids and proline contents. In case of carotenoids, the treatments, L. adecarboxylata + 2BC, A. fabrum + 2BC, P. aeruginosa + 2BC and B. amyloliquefaciens + 2BC were statistically alike but differed significantly as compared to the control for carotenoids at SD [fig_ref] Figure 2: Effect of drought tolerant ACC deaminase containing PGPR and various levels of... [/fig_ref]. The 2BC proved significantly better as compared to 1BC and the control for carotenoids at SD. The 1BC, L. adecarboxylata + 1BC, A. fabrum + 1BC, P. aeruginosa + 1BC, B. amyloliquefaciens + 1BC, 2BC, L. adecarboxylata + 2BC, A. fabrum + 2BC, P. aeruginosa + 2BC and B. amyloliquefaciens + 2BC were statistically alike but significantly different as compared to the control for carotenoids at MD [fig_ref] Figure 2: Effect of drought tolerant ACC deaminase containing PGPR and various levels of... [/fig_ref]. Maximum increase i.e., 42%, 48% and 220% in carotenoids was noted at NM, MD and SD respectively as compared to the control where B. amyloliquefaciens + 2BC was applied. For proline reduction, A. fabrum, P. aeruginosa and B. amyloliquefaciens differed significantly as compared to the control at SD. The 1BC significantly decreased proline too as compared to control, but the 2BC was statistically far better for proline reduction as compared to 1BC at SD. The PGPR strains remained significantly better with 2BC for proline reduction as compared www.nature.com/scientificreports www.nature.com/scientificreports/ to with 1BC at SD [fig_ref] Figure 2: Effect of drought tolerant ACC deaminase containing PGPR and various levels of... [/fig_ref]. However, the B. amyloliquefaciens + 2BC showed maximum decrease in proline i.e. 34% at SD as compared to the control. Photosynthetic pigments. Main effects of T and D were significantly different but their interactions remained similar for chlorophyll a, chlorophyll b and total chlorophyll contents in wheat leaves. The treatments L. adecarboxylata, A. fabrum, P. aeruginosa and B. amyloliquefaciens were statistically alike but differed significantly as compared to the control for chlorophyll a content. With and without PGPR, the application of 1BC and 2BC gave statistically similar results but remained significantly different as compared to the control for chlorophyll a . For chlorophyll b contents, with and without 1BC, all the PGPR treatments (except B. amyloliquefaciens + 1BC) were statistically alike but differed significantly as compared to the control. Without PGPR, the 2BC was significantly better than 1BC as compared to the control for chlorophyll b contents. However, A. fabrum + 2BC and B. amyloliquefaciens + 2BC proved significantly better as compared to other treatments regarding chlorophyll b contents. In case of total chlorophyll contents, the treatments L. adecarboxylata, A. fabrum, P. aeruginosa and B. amyloliquefaciens differed significantly as compared to the control. Statistically, 2BC was significantly better than 1BC for total chlorophyll. However, the 1BC and 2BC significantly increased total chlorophyll as compared to the control. The 2BC, L. adecarboxylata + 2BC, A. fabrum + 2BC, P. aeruginosa + 2BC and B. amyloliquefaciens + 2BC were statistically alike and proved better among the other treatments for total chlorophyll contents. Maximum increase in chlorophyll a (114%), chlorophyll b (123%) and total chlorophyll (115%) was noted through B. amyloliquefaciens + 2BC as compared to the control. N, P and K in shoot. Main effects of T and D were significantly different but their interaction remained similar for nitrogen (N) and phosphorus (P) concentration in shoot. For potassium (K) concentration in shoot, both main and interactive effects of T and D were significantly different. The treatments, L. adecarboxylata, A. fabrum, P. aeruginosa and B. amyloliquefaciens differed significantly as compared to the control for shoot N concentration. The 2BC remained significantly better as compared to 1BC for N concentration in shoot . The PGPR (L. adecarboxylata, A. fabrum, P. aeruginosa and B. amyloliquefaciens) with 2BC performed significantly better as compared to 1BC (except B. amyloliquefaciens + 1BC) for N concentration in shoot. For phosphorus concentration in shoot, 1BC and 2BC significantly differed as compared to the control. In case of P concentration in shoot, L. adecarboxylata, A. fabrum and B. amyloliquefaciens were statistically alike but significantly different as compared to the control. The 2BC significantly increased (i.e., by 28%) P concentration in shoot as compared to 1BC. The treatments A. fabrum + 2BC, P. aeruginosa + 2BC and B. amyloliquefaciens + 2BC proved significantly better for P concentration in shoot. Maximum increase in N (73%) and P (150%) concentration in shoot was noted as www.nature.com/scientificreports www.nature.com/scientificreports/ compared to the control where B. amyloliquefaciens + 2BC was applied. In case of K concentration in shoot, all the treatments remained significantly better as compared to control at SD. The treatments 1BC and 2BC remained statistically alike without PGPR for K concentration in shoot at MD and SD. The B. amyloliquefaciens + 1BC, A. fabrum + 2BC, P. aeruginosa + 2BC and B. amyloliquefaciens + 2BC were significantly better for K concentration Treatments Chlorophyll a (mg g −1 ) Chlorophyll b (mg g −1 ) Total Chlorophyll (mg g −1 ) Various levels of drought (D) www.nature.com/scientificreports www.nature.com/scientificreports/ in shoot at MD. Maximum increase of 38%, 85% and 138% in K concentration in shoot was noted where B. amyloliquefaciens + 2BC was applied as compared to the control at NM, MD and SD, respectively. Yield attributes. Both main and interactive effects of T and D were significantly different for grain yield pot −1 and straw yield pot −1 . In case of 100-grain weight, main effects of T and D were significantly different but their interaction remained statistically similar. The PGPR with and without 1BC, as well as, 1BC and 2BC were statistically at par with the control at SD for grain yield pot −1 . However, the L. adecarboxylata + 2BC, A. fabrum + 2BC, P. aeruginosa + 2BC and B. amyloliquefaciens + 2BC differed significantly as compared to the control for grain yield pot −1 at MD and SD . The treatments B. amyloliquefaciens + 1BC also remained significantly better as compared to the control and other PGPR with 1BC at MD for grain yield pot −1 . At NM, the B. amyloliquefaciens + 1BC, 2BC, L. adecarboxylata + 2BC, remained significantly different as compared to the control for grain yield pot −1 . Maximum increase, 40%, 155% and 215% in grain yield pot −1 was noted at NM, MD and SD as compared to the control where L. adecarboxylata + 2BC, P. aeruginosa + 2BC and B. amyloliquefaciens + 2BC were applied, respectively. For 100-grain weight, all the treatments (except A. fabrum) remained significantly better as compared to the control. The 1BC and 2BC remained statistically alike for 100-grain weight. The 1BC, A. fabrum + 1BC, B. amyloliquefaciens + 1BC, 2BC, L. adecarboxylata + 2BC, A. fabrum + 2BC, P. aeruginosa + 2BC and B. amyloliquefaciens + 2BC remained significantly better for 100-grain weight. Maximum increase (59%) in 100-grain weight was noted as compared to the control where B. amyloliquefaciens + 2BC was applied . In case of straw yield pot −1 , the PGPR with and without BC remained significantly better as compared to the control at NM, MD and SD. However, 1BC and 2BC remained statistically alike for straw yield pot −1 . The L. adecarboxylata + 1BC, A. fabrum + 1BC, P. aeruginosa + 1BC, B. amyloliquefaciens + 1BC, 2BC, L. adecarboxylata + 2BC, A. fabrum + 2BC, P. aeruginosa + 2BC and B. amyloliquefaciens + 2BC remained significantly better for straw yield pot −1 . Maximum increase i.e., 181% and 178% in straw yield pot −1 was noted as compared to the control where B. amyloliquefaciens + 2BC and A. fabrum + 2BC were applied at SD and MD respectively. N, P and K in grains. Both main and interactive effects of T and D were significantly different for N, P and K concentration in grain. At SD, the L. adecarboxylata, A. fabrum, P. aeruginosa and B. amyloliquefaciens with and without 1BC and 2BC differed significantly as compared to the control for grain nitrogen [fig_ref] Table 5: Effect of ACC deaminase containing PGPR in combination with various rates of... [/fig_ref]. The B. amyloliquefaciens, 1BC, L. adecarboxylata + 1BC, A. fabrum + 1BC, P. aeruginosa + 1BC and B. amyloliquefaciens + 1BC, 2BC, L. adecarboxylata + 2BC, A. fabrum + 2BC, P. aeruginosa + 2BC and B. amyloliquefaciens + 2BC remained significantly better as compared to the control for grain nitrogen at MD. Application of A. fabrum + 2BC proved significantly better as compared to the control for grain nitrogen at NM. For grain phosphorus, all the treatments (except P. aeruginosa) were significantly different as compared to the control at SD. However, at MD and NM all the treatments were statistically similar to the control for grain P. In case of grain K, all the treatments were significantly different as compared to the control at SD. The A. fabrum and P. aeruginosa at 2BC remained significantly better than at 1BC for grain K at SD. At MD, inoculation of PGPR with 1BC and 2BC www.nature.com/scientificreports www.nature.com/scientificreports/ proved significantly better than the control for grain K. The performance of A. fabrum was significantly better at 2BC than at1BC for grain K at MD. Among all the treatments A. fabrum + 1BC, B. amyloliquefaciens + 1BC, 2BC, L. adecarboxylata + 2BC, A. fabrum + 2BC, P. aeruginosa + 2BC, B. amyloliquefaciens + 2BC remained significantly better at NM. The 2BC proved significantly better than 1BC for grain K at NM, MD and SD. Maximum increase in grain N (58%), P (18%) and K (23%) was noted through B. amyloliquefaciens + 2BC as compared to the control at SD. ## Ie (t × d) (means of 3 replicates) me (t) ie (t × d) (means of 3 replicates) me (t) ie (t × d) (means of 3 replicates) me (t) nm Discussion. In the current study, reduction in shoot length of wheat at MD and SD in the control might be due to competition for water and nutrients between root and shoot. Gargallo-Garriga et al. [bib_ref] Opposite metabolic responses of shoots and roots to drought, Gargallo-Garriga [/bib_ref] stated that drought stress deactivated shoot metabolic activity to conserve water and food which would have facilitated roots elongation. The reduction in water and nutrients movement in shoot, enhanced the up-regulation of ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) 41 while significant amount of ACC generated ethylene restricted the root elongation 12 . Glick et al. [bib_ref] A Model For the Lowering of Plant Ethylene Concentrations by Plant Growth-promoting..., Glick [/bib_ref] suggested the mechanism for reduction in stress ethylene through the activity of ACC-deaminase [bib_ref] A Model For the Lowering of Plant Ethylene Concentrations by Plant Growth-promoting..., Glick [/bib_ref]. According to Glick et al. [bib_ref] A Model For the Lowering of Plant Ethylene Concentrations by Plant Growth-promoting..., Glick [/bib_ref] , the synthesis of indole acetic acid (IAA) by PGPR stimulates the elongation of plants cells and activates ACC synthase which converts S-adenosyl methionine to ACC. A significant amount of ACC is exuded by plants roots and seeds in rhizosphere which is hydrolyzed by ACC deaminase into NH 3 and α-ketobutyrate, resulting in better roots elongation. Secretion of roots exudates (phytosiderophores, sugars, organic acids, amino acids, vitamins, nucleosides and mucilage) also attracts PGPR that colonize roots and facilitates better uptake of water and nutrients [bib_ref] Nature and role of root exudates: Efficacy in bioremediation, Shukla [/bib_ref] [bib_ref] Control of the Cooperation Between Plant Growth-Promoting Rhizobacteria and Crops by Rhizosphere..., Drogue [/bib_ref]. Besides imperative role of PGPR, high surface area, ion exchange capacity, nutrients and water holding capacity of BC make it an effective amendment for better intake of nutrients and water in plants [bib_ref] Production and characterization of biochar from different biological wastes, Shenbagavalli [/bib_ref] [bib_ref] Effect of in-situ aged and fresh biochar on soil hydraulic conditions and..., Paetsch [/bib_ref]. A significant improvement in photosynthetic rate, transpiration rate and stomatal conductance under MD and SD, signified the effectiveness of co-application of L. adecarboxylata, A. fabrum, P. aeruginosa, B. amyloliquefaciens with 2BC, comparative to their sole application and the control. The increase in gas exchange attributes might be due to the better uptake of water and nutrients, improvement in soil water holding capacity (WHC), PGPR colonization and reduction in ethylene through the co-application of 2BC [bib_ref] Characterization of designer biochar produced at different temperatures and their effects on..., Novak [/bib_ref] [bib_ref] Biochar effects on soil biota -A review, Lehmann [/bib_ref] and ACC deaminase containing PGPR [bib_ref] Application of ACC-deaminase containing rhizobacteria with fertilizer improves maize production under drought..., Zafar-Ul-Hye [/bib_ref] [bib_ref] Interactive effect of biochar and plant growth-promoting bacterial endophytes on ameliorating salinity..., Akhtar [/bib_ref]. According to Zheng et al. [bib_ref] Influence of soil texture on fertilizer and soil phosphorus transformations in Gleysolic..., Zheng [/bib_ref] and Borch et al. [bib_ref] Ethylene: A regulator of root architectural responses to soil phosphorus availability, Borch [/bib_ref] the deficiency of nitrogen and phosphorus significantly decrease the growth of crops. Siddique et al. [bib_ref] & Islam Drought stress effects on photosynthetic rate and leaf gas exchange..., Siddique [/bib_ref] suggested the less stomatal conductance as main cause of reduction in rate of transpiration under drought. Tholen et al. [bib_ref] The role of ethylene perception in the control of photosynthesis, Tholen [/bib_ref] argued that drought stress decreased the intake of CO 2 due to less stomatal conductance which restricted the carboxylation. Hence reduced the rate of photosynthesis [bib_ref] Photosynthesis under drought and salt stress: Regulation mechanisms from whole plant to..., Chaves [/bib_ref]. However, A significant improvement in chlorophyll a, chlorophyll b, total chlorophyll, shoot and grain nutrients concentration validated the efficacious functioning of co-application of ACC deaminase producing PGPR and 2BC that also significantly increased the yield attributes (100-grains weight, straw yield and grains yield pot −1 ) of wheat plants at MD and SD. Richardson et al. [bib_ref] Plant mechanisms to optimise access to soil phosphorus, Richardson [/bib_ref] and Zahir et al. [bib_ref] Comparative effectiveness of Pseudomonas and Serratia sp. containing ACCdeaminase for coinoculation with..., Zahir [/bib_ref] , suggested better roots elongation and secretion of organic acids by PGPR for P and K solubilization as key factors, responsible for better nutrients uptake, improvement in dry weight and yield of crops. Moreover, the biochar ability to sorb nutrients also reduced the losses of N and improved its uptake in plants [bib_ref] Nutrient shifts modeling in Spinacea oleracea L. and Trigonella corniculata L. in..., Younis [/bib_ref] [bib_ref] Application of biochar in dryland soil decreasing loss of nitrogen and improving..., Decai [/bib_ref]. Chan et al. [bib_ref] Using poultry litter biochars as soil amendments, Chan [/bib_ref] suggested www.nature.com/scientificreports www.nature.com/scientificreports/ that the high surface area of biochar is a basic reason for improved cation exchange sites in soil which resulted in better bioavailability of nutrients. Results of the current study also showed that electrolyte leakage in the wheat plants leaves was decreased where L. adecarboxylata, A. fabrum, P. aeruginosa, B. amyloliquefaciens with 2BC were applied comparative to the control under MD and SD. The reduction in electrolyte leakage might be due to the activity of ACC deaminase, better availability of water and nutrients by co-application of PGPR and 2BC. Senaratna and McKersie 61 observed a significant increase in electrolyte leakage because of cell membrane damage by drought stress which made it more permeable. According to Matile et al. [bib_ref] Planta Localization of chlorophyllase in the chloroplast envelope, Matile [/bib_ref] , cell usually lost its membrane integrity as a result of lipid degradation by ethylene. When the lipids in cell membrane become degraded, the ethylene comes in contact with the chloroplast and activates the chlorophyllase (chlase) gene which causes severe damage to the chlorophyll 62 . However, the addition of 2BC and PGPR in combination significantly decreased the electrolyte leakage which might be due to activity of ACC deaminase, better availability of water and nutrients uptake. # Conclusion. It is concluded that the co-application of drought tolerant ACC deaminase producing PGPR and 2BC is comparatively better approach than their sole application to mitigate drought stress in wheat. Though Leclercia adecarboxylata and Pseudomonas aeruginosa were also effective enough but Agrobacterium fabrum and Bacillus amyloliquefaciens with 2BC gave maximum increase in the gas exchange attributes, nutrients concentration in shoot and grain, photosynthetic pigments and yield of wheat. More investigations are needed at field level to introduce A. fabrum and B. amyloliquefaciens with 2BC to improve growth and yield of wheat under drought stress. # Materials and methods Out of 23 initially screened rhizobacteria [fig_ref] Table 7: Characteristics of PGPR [/fig_ref] isolated from wheat rhizospheric soil, collected from Old Shujabad Road (30.11°N and 71.43°E) and Akramabad (30.16°N and 71.29°E), four most efficient drought-tolerant ACC-deaminase producing PGPR were screened out after a laboratory hydroponic trial in the Department of Soil Science, Bahauddin Zakariya University Multan, Pakistan. For screening of most effective drought tolerant PGPR strains, polyethylene glycol 6000 (PEG-6000) was used (0, 10 and 20%) to maintain osmotic potential (0.05, −0.23 and −0.78 MPa) to introduce drought stress [bib_ref] Influence of PEG generated osmotic stress on shoot regeneration and some biochemical..., Piwowarczyk [/bib_ref] [bib_ref] Characterization of osmotolerant rhizobacteria for plant growth promoting activities in vitro and..., Paul [/bib_ref]. Molecular identification of the most efficient drought tolerant ACC deaminase producing PGPR was done by 16S rRNA gene sequencing using PCR primers 1492R 5′ (TAC GGY TAC CTT GTT ACG ACT T) 3′ and 27F 5′ (AGA GTT TGA TCM TGG CTC AG) 3′. The gene sequencing primers were 907R 5′ (CCG TCA ATT CMT TTR AGT TT) 3′ and 785F 5′ (GGA TTA GAT ACC CTG GTA) 3′. Finally, 16S rRNA gene sequences were aligned and relationships were deduced using BLAST analysis [bib_ref] Isolation, characterization, and use for plant growth promotion under salt stress, of..., Siddikee [/bib_ref]. Most efficient drought-tolerant ACC-deaminase producing PGPR were identified as AbW 1 = Leclercia adecarboxylata (NR_104933.1), CbW 2 = Agrobacterium fabrum (NR_074266.1), CbW 3 = Bacillus amyloliquefaciens (FN_597644.1) and AbW 5 = Pseudomonas aeruginosa (CP012001.1). These PGPR strains were able to grow at the osmotic potential −0.78 MPa generated through 20% polyethylene glycol 6000 (PEG). The DF minimal salt medium (4.0 g KH 2 PO 4 , 6.0 g Na 2 HPO 4 , 0. To confirm the presence of AcdS gene that plays key role in synthesis of ACC deaminase NCBI gene bank was consulted. From NCBI gene bank it was confirmed that B. amyloliquefaciens (https://www.ncbi.nlm.nih. gov/nuccore/KX709841.1/), A. fabrum (https://www.ncbi.nlm.nih.gov/protein/PZP48640.1/) and P. aeruginosa (https://www.ncbi.nlm.nih.gov/nuccore/CP014948.1/) have AcdS gene while work is yet continued on L. adecarboxylata. For assessing the ACC deaminase produced by PGPR methodology of El-Tarabily 68 and Honma and Shimomura 20 was used. Pikovskaya's medium was used to examine the phosphorus solubilizing activity of PGPR as described by Vazquez et al. [bib_ref] Phosphate-solubilizing microorganisms associated with the rhizosphere of mangroves in a semiarid coastal..., Vazquez [/bib_ref]. Potassium solubilizing activity of PGPR was assessed according to the methodology of Candra Setiawati and Mutmainnah [bib_ref] Solubilization of Potassium Containing Mineral by Microorganisms From Sugarcane Rhizosphere, Setiawati [/bib_ref]. Characterization of the PGPR isolates is provided in [fig_ref] Table 7: Characteristics of PGPR [/fig_ref]. . Characteristics of soil, timber waste biochar (BC). www.nature.com/scientificreports www.nature.com/scientificreports/ For the production of biochar, timber waste was collected from local timber market. The timber waste was initially sun-dried and then pyrolyzed at 389 °C for 80 min in an especially designed pyrolyzer as described by Qayyum et al. [bib_ref] Effects of various biochars on seed germination and carbon mineralization in an..., Qayyum [/bib_ref]. All the pyrolyzed material (biochar) was then crushed in a grinder and passed through 2 mm sieve. Finally, the fine powder of timber waste biochar (BC) was stored in air tight plastic jars [bib_ref] Effects of various biochars on seed germination and carbon mineralization in an..., Qayyum [/bib_ref]. The pH and ECe of BC were determined by mixing the BC and water with the ration, 1:20 (w/v) as described by . Di-acid (HNO 3 : HClO 4 ) digestion 71 of biochar was done for the analysis of total phosphorus following yellow color method on spectrophotometer, and those of potassium and sodium on flame photometer [bib_ref] Qualitative and Chemical Analysis of Rice Kernel To Time of Application of..., Nadeem [/bib_ref]. For the determination of nitrogen, H 2 SO 4 digestion 72 was done followed by distillation on Kjeldahl's distillation apparatus. The volatile matter and ash content of biochar were analyzed according to Qayyum et al. [bib_ref] Kinetics of Carbon Mineralization of Biochars Compared with Wheat Straw in Three..., Qayyum [/bib_ref] by heating the biochar in muffle furnace at 450 °C and 550 °C respectively. The fixed carbon in biochar was assessed The plastic bag (30 cm deep × 20 cm in diameter) was used as a pot, having capacity to carry 8 kg soil. The soil was collected from the plough layer of bank of the Chenab River, Multan, Punjab, Pakistan. The soil of selected area was previously characterized as dark yellowish brown, moderately calcareous, weakly structured and well drained with Cambic subsurface horizon and an Ochric epipedon [bib_ref] Biochar increased photosynthetic and accessory pigments in tomato (Solanum lycopersicum L.) plants..., Abid [/bib_ref]. The soil texture was determined by hydrometer methodwhich was sandy loam (USDA triangle) with mixed hyperthermic Haplocambids. The organic matter in soil was determined by Walkley [bib_ref] An Examination of Methods for Determining Organic Carbon and Nitrogen in Soils, Walkley [/bib_ref]. The organic nitrogen in soil was determined using the equation: = Organic N (%) Soil Organic Matter/20 [bib_ref] Fulvic Acid Application Improves the Maize Performance under Well-watered and Drought Conditions, Anjum [/bib_ref] For extractable soil P determination, Olsen and Sommers 80 method was used. Similarly, the extractable K in soil was determined according to the method described by Nadeem et al. [bib_ref] Qualitative and Chemical Analysis of Rice Kernel To Time of Application of..., Nadeem [/bib_ref]. In each plastic pot, 8 kg soil was filled. To fulfil macro nutrients requirement nitrogen (N), phosphorus (P) and potassium (K) fertilizers were added at the rate of 120: 90 and 60 kg ha −1 respectively, as recommended dose keeping in mind the nutrients concentration of biochar where it was applied [bib_ref] Integrated effects of rhizobial inoculum and inorganic fertilizers on wheat yield and..., Adnan [/bib_ref]. The urea was added in three split doses. As far as diammonium phosphate (DAP) and muriate of potash (MOP) fertilizers are concerned, the recommended rates of fertilizers were applied in a single dose at the time of sowing. Timber waster biochar was added at three different rates including: control i.e., no biochar (0BC), 0.75% of soil (60 g biochar per 8 Kg soil) biochar (1BC) and 1.50% of soil (120 g biochar per 8 Kg soil) biochar (2BC). The seeds of wheat (Galaxy-2013) were obtained from the certified seed dealer of the Government of Punjab, Pakistan. Healthy seeds were separated from broken and weak seeds. The seeds were surface-sterilized with www.nature.com/scientificreports www.nature.com/scientificreports/ sodium hypochlorite (5%) followed by 3 washes with ethanol (95%). Finally, all the seeds were washed three times with sterilized deionized water [bib_ref] Cadmium-tolerant bacteria induce metal stress tolerance in cereals, Ahmad [/bib_ref]. For PGPR inoculation, 10 ml of inoculum (0.5 optical density at 535 nm wavelength) [bib_ref] Rhizobacteria containing ACC-deaminase confer salt tolerance in maize grown on saltaffected fields, Nadeem [/bib_ref] was added along 10% sugar (glucose) in 100 g sterilized seeds. After proper mixing of seeds, inoculum and sugar solution, top dressing of seeds was done with a mixture of peat and clay (3:1 ratio) as described by Ahmad et al. [bib_ref] Synergistic Effect of Rhizobia and Biochar on Growth and Physiology of Maize, Ahmad [/bib_ref]. Before inoculation of seeds, the peat and clay mixture was sterilized at 121 °C for 20 min in an autoclave [bib_ref] Rhizobacteria containing ACC-deaminase confer salt tolerance in maize grown on saltaffected fields, Nadeem [/bib_ref]. All the control treatment seeds were also top dressed with peat and clay mixture along with 10% sugar solution without inoculum [bib_ref] Effect of plant growth promoting rhizobacteria containing ACC-deaminase on maize (Zea mays..., Shaharoona [/bib_ref]. In each pot, 10 seeds of wheat were initially sown. In control, the soil normal moisture (NM) was maintained at the level of 70% of field capacity (FC 70 ) throughout the experiment on weight basis. However, to introduce mild drought (MD) and severe drought (SD) stress as per treatment plan, the soil moisture was maintained at the level of 50% and 30% of field capacity (FC 50 and FC 30 ), respectively, throughout the trial as suggested by Boutraa et al. [bib_ref] Effect of water stress on growth and water use efficiency (WUE) of..., Boutraa [/bib_ref]. After germination of seeds, five healthy seedlings were kept in each pot by thinning. The pot experiment was conducted in the research area of the Department of Soil Science, Bahauddin Zakariya University Multan, Pakistan under drought stress on wheat. There were 15 treatments with 3 replications, following factorial completely randomized design (CRD). The treatments included: Control (No PGPR + No BC), L. adecarboxylata, A. fabrum, P. aeruginosa, B. amyloliquefaciens, 1BC, L. adecarboxylata + 1BC, A. fabrum + 1BC, P. aeruginosa + 1BC, B. amyloliquefaciens + 1BC, 2BC, L. adecarboxylata + 2BC, A. fabrum + 2BC, P. aeruginosa + 2BC and B. amyloliquefaciens + 2BC. Leaf gas exchange parameters (net photosynthetic rate, net transpiration rate and stomatal conductance) were determined with the help of Infra-Red Gas Analyzer (CI-340 Photosynthesis system, CID, Inc. USA) by joining 4 leaves of wheat. On a sunny day, the readings were taken between 10:30 and 11:30 AM at saturating intensity of light [bib_ref] Involvement of ethylene in reversal of salt-inhibited photosynthesis by sulfur in mustard, Nazar [/bib_ref]. After 50 days of sowing, the seedlings were harvested from each pot for the measurement of shoot length and determination of electrolyte leakage, proline contents, photosynthetic pigments level and nutrients concentration in the shoot. The electrolyte leakage (EL) was determined following the procedure given by Lutts et al. [bib_ref] NaCl-induced Senescence in Leaves of Rice (Oryza sativa L.) Cultivars Differing in..., Lutts [/bib_ref]. The leaves were washed with deionized water and then cut using a steel cylinder having diameter 1 cm. One gram of uniform sized leaf pieces were immersed in a test tube containing deionized water (20 ml) and incubated at 25 °C for 24 h. The electrical conductivity (EC1) was determined using pre-calibrated EC meter. The second EC (EC2) was noted heating the test tubes in a water bath at 120 °C for 20 min. The final value of EL was calculated using the equation as follows; = × Electrolyte Leakage (EL) EC1/EC2 100 [bib_ref] Climate Change-Induced Water Stress Suppresses the Regeneration of the Critically Endangered Forest..., Zhang [/bib_ref] For proline assessment in wheat leaves, methodology stated by Bates et al. [bib_ref] Rapid determination of free proline for water-stress studies, Bates [/bib_ref] was followed. The proline was extracted from fresh (0.1 g) leaves in 2 ml of 40% methanol. After extraction, the 1 ml mixture of glacial acetic acid and 6 M orthophosphoric acid (3:2 v/v) was mixed in 1 ml extract along with 25 mg ninhydrin. Then the solution was incubated at 100 °C for 60 min. After cooling down, 5 ml Toluene was added. For the estimation of proline contents, absorbance was noted on spectrophotometer at 520 nm wavelength. The chlorophyll a, chlorophyll b and total chlorophyll contents were determined in the fresh leaves of wheat according to the protocol given by Arnon [bib_ref] Copper Enzymes in Isolated Chloroplasts. Polyphenoloxidase in Beta vulgaris, Arnon [/bib_ref]. The extract was taken from the leaves using acetone (80%) solution. For the estimation of chlorophyll a and chlorophyll b, the absorbance was taken at 663 and 645 nm wavelength, respectively on spectrophotometer. Final calculations were made using the following relations; = . . = + Total Chlorophyll (mg/g) Chlorophyll a Chlorophyll b [bib_ref] Alleviation of drought stress in pulse crops with ACC deaminase producing rhizobacteria..., Saikia [/bib_ref] where, OD = Optical density (wavelength). V = Final volume made. W = Fresh leaf made (g). The samples were digested with sulfuric acid 72 followed by distillation on Kjeldahl's distillation apparatus. The yellow colour method was used for the determination of phosphorus concentration noting absorbance at 420 nm on spectrophotometer. As far as the K concentration in wheat shoot and grain is concerned, the samples were digested and then run on flame photometer as described by Nadeem et al. [bib_ref] Qualitative and Chemical Analysis of Rice Kernel To Time of Application of..., Nadeem [/bib_ref]. The wheat plants were harvested after 125 days of sowing for the determination of grains yield pot −1 , straw yield pot −1 and 100-grain weight. Weight of 100 grains, straw and grains yield pot −1 were assessed on top weight balance. For straw yield, plants were harvested at 4 inches above the ground surface. Sun dried 100 grains of wheat were counted randomly and manually and then weighed on top weight balance. Total wheat grains collected from a single pot were weighed and considered as grain yield pot −1 . Statistical analyses of the data were carried out using standard statistical procedures. All the treatments were compared using Tukey's test at p ≤ 0.05. ## Data availability No datasets were generated or analyzed during the current study. All the analyzed data can be accessed after publication by requesting the corresponding author. [fig] Figure 1: Effect of drought tolerant ACC deaminase containing PGPR and various levels of timber waste biochar on shoot length (a) and electrolyte leakage (b) in wheat leaves under various levels of drought (D). Means sharing the same letter are statistically similar. Error bars represent ± standard error. NM = Normal Moisture; MD = Mild Drought; SD = Severe Drought. [/fig] [fig] Figure 2: Effect of drought tolerant ACC deaminase containing PGPR and various levels of timber waste biochar on carotenoids (a) proline (b) in wheat leaves under various levels of drought (D). Means sharing the same letter are statistically similar. Error bars represent ± standard deviations. NM = Normal Moisture; MD = Mild Drought; SD = Severe Drought. [/fig] [fig] 2: Effect of ACC deaminase containing PGPR in combination with various rates of timber waste biochar (0BC, 1BC and 2BC) on photosynthetic pigments under various levels of drought (D). Means sharing different letters are significantly different (p ≤ 0.05). Non-significant interactive effect (T × D) did not have any letter. ME indicates main effect; IE indicates interactive effect; NM = Normal Moisture; MD = Mild Drought; SD = Severe Drought. [/fig] [table] Table 5: Effect of ACC deaminase containing PGPR in combination with various rates of timber waste biochar (0BC, 1BC and 2BC) on grains N, P and K concentration under various levels of drought (D). Means sharing different letters are significantly different (p ≤ 0.05). Non-significant interactive effect (T × D) did not have any letter. ME indicates main effect; IE indicates interactive effect; NM = Normal Moisture; MD = Mild Drought; SD = Severe Drought. [/table] [table] Table 7: Characteristics of PGPR. (2019) 9:5999 | https://doi.org/10.1038/s41598-019-42374-9 [/table]
Capillary leak syndrome induced by neoadjuvant cisplatin and gemcitabine in a patient with bladder cancer Capillary leak syndrome (CLS) is a rare disorder associated with an increased capillar permeability due to an endothelial damage, causing leakage of plasma and proteins into the interstitial compartment. CLS is characterized by rapidly developing edema, hypotension and hypoproteinemia. We observed CLS in a 54-year-old man affected by muscle-invasive bladder cancer who received neoadjuvant treatment with Cisplatin and Gemcitabine. Treatment with infusion of albumin and increasing corticosteroid doses and diuretics led to a complete regression of all signs and symptoms related to the disorder. Of note, the patient showed an objective complete response to chemotherapy and underwent radical surgery on schedule. # Introduction Capillary leak syndrome (CLS) is a rare clinical condition characterized by rapid and reversible increase in endothelial permeability, with consequent leakage of plasma proteins into the interstitial space, with peripheral edema. [bib_ref] Capillary leak syndrome: etiologies, pathophysiology, and management, Siddall [/bib_ref] A similar phenomenon is observed during sepsis and less frequently in some manifestations of autoimmune diseases. This syndrome can be suspected in patients with hypotension, hypoalbuminemia without organ failure, partial or generalized edema, hemoconcentration and possible presence of paraproteins in the blood. It can be idiopathic, called Clarkson's syndrome, or induced by other factors. In all cases, it can be a life-threatening condition. Among the causes that trigger this syndrome are counted some drugs, the engraftment syndrome, promyelocytic leukemia which undergoes induction treatment with either all-trans retinoic acid or arsenic trioxide, ovarian hyperstimulation syndrome, Hhemophagocytic lymphohistiocytosis, hemorrhagic fever and snakebite. Acute renal failure, hypovolemic shock and coagulopathies are often found in the conditions mentioned above. Among the drugs that have been reported to lead to CLS in the literature, there is gemcitabine. This is a chemotherapic drug used in several types of malignancies, including bladder cancer. So far, few cases, less that ten, of CLS induced by gemcitabine are found in literature. Herein we report a case of CLS developed during last cycle of neoadjuvant treatment with cisplatin and gemcitabine in a patient with muscle-invasive bladder cancer. ## Case A 54-year-old male, affected by locally advanced, muscle-invasive high grade papillary urothelial cancer of bladder, without any other significant comorbidities, was proposed for neoadjuvant chemotherapy with cisplatin 80 mg/mq and gemcitabine 1000 mg/mq 1,8 q21 for four cycles, as for guidelines. [bib_ref] Bladder cancer, version 3.2020, NCCN clinical practice guidelines in oncology, Flaig [/bib_ref] During first three cycles, the patient did not receive the gemcitabine on day 8 for grade 3 neutropenia. The fourth cycle was completed. Five days after from receiving last dose of gemcitabine, the patient developed fever and petechiae on legs and trunk and he was hospitalized. The blood count showed anemia (hemoglobin 9,6 g/dL), leukopenia (1900 mm 3 ) and thrombocytopenia (21,000 mm 3 ). The renal function, hepatic function and coagulation parameters were within normal ranges. Albuminemia was decreased (3.07 g/dL). He was infused with platelets concentrate and started granulocyte colony-stimulating factor (G-CSF), 30 million units/die. He also started antibiotic therapy. On day 6th, the patient showed contraction of diuresis, hypotension and edema of the trunk and arms, bilaterally. Eco-color-doppler of the arms excluded venous thrombosis. Cardiac ecocolordoppler and Angio-CT excluded thrombo-embolic events of arterial vessels. Edema progressively extended to lower limbs [fig_ref] Figure 1: Edema of lower limbs on the day of lower level of albuminemia [/fig_ref] and the persistence of fever, neutropenia, hypotension posed the suspect of sepsis, excluded by serial hemocoltures at feverish peak, urine cultures and by normal blood procalcitonin levels. On day 8th, total protein value and level of albuminemia further decreased (4,4 g/dL and 2,45 g/dL, respectively) [fig_ref] Figure 2: Fig [/fig_ref]. There was no proteinuria in the urine test and we started treatment with albumin 20% 50 ml once daily. He also received intravenous furosemide (20 mg, once daily) and prednisone 25 mg, once daily and received transfusions of platelets and red blood cells concentrates. The clinical manifestations persisted, without improvement, until day 12th, when we increased prednisone dose to 50 mg daily. On day 14th, we observed a reduction of edema of the trunk and arms, an increasing of blood cells count and normalization of diuresis and blood pressure. Renal function was within normal range. The complete normalization of clinical condition was observed on day 17th. No other cycles of chemotherapy were administered. Interestingly, a Chest-Abdomen CT was performed and showed an objective complete response to neoadjuvant chemotherapy . The patient successfully underwent radical cystectomy and bilateral pelvic lymph node dissection, with construction of cutaneous ureteroileostomy ad modum Bricker. At histopatological examination, there was no evidence of infiltrating tumor on bladder wall, but the presence of carcinoma in situ, mild lymphocytes infiltration and focal granulomatous inflammatory reaction with macrophages and multinucleated giant cells. Nonspecific reactive lymphadenitis in 10 lymph nodes examined was detected. # Discussion In this clinical experience, we have witnessed a rare adverse event of gemcitabine, for the first time reported in a patient with bladder cancer receiving neoadjuvant treatment. We cannot exclude that cisplatin, associated with gemcitabine, might also have played a role in the pathogenesis of the disorder. Nevertheless, in view of the absence of the syndrome during the first cycles in which the patient had not sustained the eighth day of treatment with gemcitabine alone, a dose-dependent effect and a particular susceptibility of the patient in metabolizing the drug may be assumed. Increasing in corticosteroid dosage led to a rapid improvement in symptoms, demonstrating an almost total ineffectiveness of prednisone at lower doses of 0.5 mg/kg, despite simultaneous diuretic therapy. Combination of steroids and diuretics at an appropriate dosage has allowed the complete reversibility of symptoms 8-10 days after the start of treatment. Similar experiences have already been published, [bib_ref] Gemcitabine-induced systemic capillary leak syndrome, De Pas [/bib_ref] underlying the need for prompt recognition of CLS and treatment, that need to be continued for some weeks before being able to see concrete clinical benefits. The effectiveness of the therapy, with the complete clinical response to the chemotherapy treatment, has largely justified the risk of toxicity. The graphic shows decreasing of total proteins and albumin levels and blood pressure until day 8th from last administration of gemcitabine and subsequent gradual normalization of this values, more evident after improving therapy with prednisone on day 12th. The patient was subsequently operated and he returned to his previous daily routine. Cases of CLS have recently been reported to be associated with the administration of immune checkpoint inhibitors (ICIs), including Nivolumab 4 and during infection with SARS-CoV-2. [bib_ref] SARS-CoV-2 Induces Acute and Refractory Relapse of Systemic Capillary Leak Syndrome (Clarkson's..., De Chambrun [/bib_ref] These experiences suggest a probable pathogenesis due to a dysregulation of the immune response with cytokine alterations, in the absence of a cellular inflammatory response, impacting on endothelial permeability. # Conclusion CLS remains a clinical challenge, not just as a side effect of chemotherapy. The main goal is a prompt recognition of the symptoms, regardless of the underlying cause, because even if CLS is a rare syndrome, it needs to be treated properly to avoid serious and irreversible damages. Focusing on the pathophysiological mechanism that underlies this syndrome, a predictive role of response to chemotherapy could be assumed, for example by inducing an increased activity of the inflammatory response. This hypothesis could be investigated by other clinical experiences. ## Declaration of competing interest The authors report no conflicts of interest in this work. ## Fig. 3. Comparative CT with contrast images of the bladder neoformation, before and after neoadjuvant chemotherapy treatment; A. Image before neoadjuvant treatment; C. Image after neoadjuvant treatment: we observed a total disappearance of the parietal lesion; B. Image before neoadjuvant treatment; D. Image after neoadjuvant treatment: the filling defect is no more detectable. [fig] Figure 1: Edema of lower limbs on the day of lower level of albuminemia (2,45 g/ dL on day 8th). [/fig] [fig] Figure 2: Fig. 2. The graphic shows decreasing of total proteins and albumin levels and blood pressure until day 8th from last administration of gemcitabine and subsequent gradual normalization of this values, more evident after improving therapy with prednisone on day 12th. [/fig]
Connected, Respected and Contributing to Their World: The Case of Sexual Minority and Non-Minority Young People in Ireland Outcome 5 of the Irish Better Outcomes, Brighter Futures national youth policy framework ("Connected, respected, and contributing to their world") offers a suitable way to study psychosocial determinants of adolescent health. The present study (1) provides nationally representative data on how 15-to 17-year-olds score on these indicators; (2) compares sexual minority (same-and bothgender attracted youth) with their non-minority peers. We analyzed data from 3354 young people (aged 15.78 ± 0.78 years) participating in the Health Behaviour in School-aged Children (HBSC) study in Ireland. Age and social class were associated with the indicators only to a small extent, but girls were more likely than boys to report discrimination based on gender and age. Frequency of positive answers ranged from 67% (feeling comfortable with friends) to 12% (being involved in volunteer work). Sexual minority youth were more likely to feel discriminated based on sexual orientation, age, and gender. Both-gender attracted youth were less likely than the other groups to report positive outcomes. Same-gender attracted youth were twice as likely as non-minority youth to volunteer. The results indicate the importance of a comprehensive approach to psycho-social factors in youth health, and the need for inclusivity of sexual minority (especially bisexual) youth. # Introduction ## The human ecology of child and adolescent health Health is situated within a complex, interactive network of determinants [bib_ref] Determinants of health, Keleher [/bib_ref]. These determinants can be understood as the personal, social, economic, and political conditions in which people grow, live, work, and age, and the structural drivers behind those conditions [bib_ref] Changes not for the fainthearted: Reorienting health care systems toward health equity..., Baum [/bib_ref]. Research, as well as practice, often concentrates on intra-individual factors at the expense of social determinants, despite the need to also address these social factors if we want to achieve structural equality in health [bib_ref] Pushing the international health research agenda towards equity and effectiveness, Mccoy [/bib_ref]. Bronfenbrenner's ecological systems theory, originally developed to better understand human development and children's health, provides a useful framework to understand how these complex factors shape health outcomes across individual, micro-, meso-exo-, and macro-levels over the life-course of an individual [bib_ref] Interacting systems in human development, Bronfenbrenner [/bib_ref]. Currently, we are seeing a shift in focus from negative to positive determinants and outcomes in child and adolescent health research [bib_ref] Adolescence and the social determinants of health, Viner [/bib_ref]. These include concepts such as resilience, self-esteem, sense of connectedness, and identity development. Such factors are embedded within the microsystems, including young persons' family and peer networks In Ireland, the health and well-being of youth are prioritized in line with the State's commitments under the United Nations Convention on the Rights of the Child. This was the basis of the development of Better Outcomes, Brighter Futures (BOBF), the national policy framework that sets out the Government's agenda and priorities in relation to children and young people up to the age of 24 years. The BOBF indicator setwas developed to track progress for children and young people across five national outcomes for the purposes of identifying trends, contributing towards priority setting, and to inform policy and service provision. The five dimensions ("outcomes") of BOBF state that children and young people will (1) be active and healthy; [bib_ref] Changes not for the fainthearted: Reorienting health care systems toward health equity..., Baum [/bib_ref] achieve in all areas of learning and development; (3) be safe and protected from harm; (4) experience economic security and opportunity; and (5) be connected, respected, and contribute to their world. The indicators reflect a broad picture of children and young people's lives and their experiences. These outcomes are examined against an indicator set that was completed following a comprehensive development and consultation process. The BOBF indicator set includes more than 100 indicators across 70 indicator areas. A national research priority is that country-level population health studies provide data to these indicators. One of the main data providers of BOBF is the Health Behaviour in School-aged Children (HBSC) Ireland study. HBSC is a World Health Organization collaborative crossnational study initiated in 1983 and thence conducted in four-year cycles, in a constantly growing number of countries. Currently, 51 countries in North America, Europe, and the former Soviet region participate in the study [bib_ref] Spotlight on Adolescent Health and Well-Being, Inchley [/bib_ref]. The Irish HBSC asks children and young people aged 10-17 about their health and well-being and health behaviors within their social contexts-home, school, and with family and friends. It is a school-based survey, with data collected through self-completion questionnaires administered by teachers in the classroom. There is a strong conceptual and methodological connection between the HBSC study and the BOBF framework. This is illustrated by the fact that in the 2018 survey round, the HBSC Ireland study was the data source for more than one third [bib_ref] Stigma and minority stress as social determinants of health among lesbian, gay,..., Hatzenbuehler [/bib_ref] of the BOBF indicators. The fifth outcome of BOBF-"Connected, respected, and contributing to their world"contains four aims [fig_ref] Table 1: Better Outcomes, Brighter Futures [/fig_ref] : Young people (1) develop a sense of identity and are free from discrimination; [bib_ref] Changes not for the fainthearted: Reorienting health care systems toward health equity..., Baum [/bib_ref] are part of positive network of friends, family, and community; (3) are civically engaged, socially and environmentally conscious; and (4) are aware of their rights and are responsible and respectful of the law. These aims are assessed with thirteen indicator areas, and HBSC Ireland provides data on ten of these [fig_ref] Table 1: Better Outcomes, Brighter Futures [/fig_ref]. The aims, indicator areas, and the actual indicators collected by HBSC are outlined in the Method section. There are some areas within BOBF Outcome 5 where other studies provide data to the indicator set. The Quarterly National Household Survey, Special Module on Equality, conducted by the Central Statistics Office of Ireland provides data on perceived discrimination in 18-to 24-year-olds. The Growing Up in Ireland longitudinal study provides data on peer support and having at least one caring and consistent adult to confide in among 9-year-olds. The Programme for International Student Assessment (PISA) provides data on positive parent and family relationships [bib_ref] Learning for the Future: The Performance of 15-Year-Olds in Ireland on Reading..., Mckeown [/bib_ref]. However, the HBSC indicators relevant to BOBF Outcome 5 cover the majority of the indicator areas and therefore provide an extensive picture of adolescents' connectedness, sense of being respected, and contribution to their world. The first aim of the present article is to demonstrate how population-level descriptive data on BOBF Outcome 5 ("Connected, respected, and contributing to their world") can inform the implementation of a national level policy to improve adolescents' lives. We hypothesize that age, gender, and socio-economic status will impact some of the BOBF indicators among 15-to 17-year-old youth in Ireland. ## Connectedness, self-esteem, and societal engagement in sexual minority youth An increasing number of scholars studying sexual (and gender) minority youth health acknowledge that the dominant narratives describing lesbian, gay, and bisexual (LGB) youth as universally vulnerable should be shifted to focus on the positive aspects of queer identities and lives [bib_ref] Speaking back to dominant constructions of LGBT lives: Complexifying 'at riskness' for..., Bryan [/bib_ref]. Such a positive, "after-queer" [bib_ref] After-queer' tendencies in queer research, Talburt [/bib_ref] approach does not mean neglecting or downplaying that many sexual and gender minority youth face challenges and adversity, including experiences of stigma and discrimination [bib_ref] Stigma and minority stress as social determinants of health among lesbian, gay,..., Hatzenbuehler [/bib_ref]. It rather emphasizes that a disproportionate focus on negative aspects of sexual and gender minority lives may overshadow or impede the benefits and positive aspects of LGBT identities. Riggle and Rostoskydiscuss many positive LGB (and transgender) experiences uncovered by their research, including living an authentic life, having empathy and compassion for others, belonging to a community, and experiencing strong emotional connections with others. These latter themes of belonging and connectedness were also highlighted by a recent systematic review of qualitative research on sexual and gender minority youth [bib_ref] LGBTQI+ youth and mental health: A systematic review of qualitative research, Wilson [/bib_ref]. The authors found that connectedness with others, whether as part of small groups like Gay-Straight or Gender-Sexuality Alliances, wider networks of sexual and gender minority youth, or online friendships, can be a source of empowerment as well as a vital protective factor. However, evidence on such positive factors which promote resilience are scarce, the findings are inconsistent, and-just like with many other facets of LGB research-the majority of studies were conducted in North America [bib_ref] Research on adolescent sexual orientation: Development, health disparities, stigma and resilience, Saewyc [/bib_ref] [bib_ref] Sexual orientation differences in the self-esteem of men and women: A systematic..., Bridge [/bib_ref]. While there have been some studies with sexual minority youth in Ireland [bib_ref] School Climate Survey Report: The Experience of Lesbian, Gay, Bisexual and Trans..., Pizmony-Levy [/bib_ref] [bib_ref] My World Survey 2: The National Study of Youth Mental Health in..., Dooley [/bib_ref] [bib_ref] The school-based lives of LGBT youth in the Republic of Ireland, Reygan [/bib_ref] , the research landscape is rather bleak, and the existing studies concentrate on the negative aspects of belonging to sexual minorities. The LGBTI+ National Youth Strategy 2018-2020prioritizes conducting more comprehensive and balanced research to better understand the specific needs, and developmental assets, of sexual and gender minority youth. In recent years, Ireland has advanced LGBTI+ equality at a structural level, with the passing of marriage equality legislation and the Gender Recognition Act in 2015. However, there is still much work to do in achieving more inclusive environments for LGBTI+ people. For instance, LGBTI+ young people still report a lack of understanding and acceptance, as well as barriers to accessing inclusive and supportive services [bib_ref] Tackling social and health inequalities: Vulnerability among the young lesbian, gay and..., Mannix-Mcnamara [/bib_ref]. Stigmatization and discrimination are contingent on social and cultural context and occur across different levels of the socio-ecological spectrum [bib_ref] The Health Stigma and Discrimination Framework: A global, crosscutting framework to inform..., Stangl [/bib_ref]. Sexual minority youth who experience higher levels of stigma may be at increased risk of adverse health outcomes, which can vary depending on the mechanism of stigma experienced [bib_ref] Stigma and minority stress as social determinants of health among lesbian, gay,..., Hatzenbuehler [/bib_ref] [bib_ref] How does sexual minority stigma "get under the skin"? A psychological mediation..., Hatzenbuehler [/bib_ref]. LGB young people can also experience compound or intersecting stigmas, related to other aspects of their identity, including chronic conditions and disability status, race, ethnicity, faith, sex, and gender identity. For instance, a meta-analysis found a stronger correlation between minority stress and negative mental health outcomes in lesbian and bisexual females than in gay and bisexual males [bib_ref] Minority stress and mental health in lesbian, gay male, and bisexual youths:..., Dürrbaum [/bib_ref]. These findings highlight the need to adopt a more nuanced approach that considers the heterogeneity of young people's lives. Such an approach should account for the wide variety of context-specific, environmental, social, and structural factors involved in shaping LGBT youth health and well-being and balance the focus to involve positive determinants of health. One of the potential ways to utilize the BOBF indicator set is to explore whether perceived discrimination affects the sense of being respected and valued and community engagement. Comparing sexual minority and non-minority youth on these dimensions speaks to the LGBTI+ National Youth Strategy 2018-2020 of Ireland, the first governmental strategy in the world specifically aimed to improve the health and well-being of sexual and gender minority young people. This strategy is directly aligned with the BOBF framework but sets specific objectives to improve the health and lives of sexual and gender minority youth in Ireland. An additional benefit of applying the BOBF framework in the study of sexual minority young people is that the findings may add to the thin evidence basis on connectedness and self-esteem in LGB youth. Therefore, the second aim of our paper is to compare sexual minority and non-minority youth in Ireland across the indicators included in BOBF Outcome 5. We hypothesize that compared to their non-minority peers, sexual minority youth will be more likely to feel discriminated based on their sexual orientation and other grounds; that they will be less likely to report high levels of social support (from families and peers); and that they will be more likely to be involved in volunteer work. # Method ## Procedure HBSC is a cross-sectional epidemiological study conducted every four years with nationally representative samples of children and adolescents. In line with the international HBSC protocol, a two-stage cluster sampling was conducted to ensure national representativity. First, a proportional sample of schools was randomly selected from the eight geographical regions of Ireland; subsequently, class groups within the participating schools were also randomly selected. School principals were contacted by post; 63% of the invited schools agreed to participate. Data collection took place between April and September 2018. In the present study, responses from an average of 11 young people from 297 classes were analyzed. The study instrument was a paper-based questionnaire that participating youth filled in during school hours. The study was carried out in adherence to the international HBSC study protocoland was approved by the Research Ethics Committee of the National University of Ireland Galway under Decision Ref. REC17-Nov-13. Informed consent was obtained from all participating youth as well as their parents/guardians and school principals. It was at the discretion of the school principals and boards whether active or passive consent from parents was required. Evidence shows that reported health outcomes in young people are independent from the type of parental consent [bib_ref] Does the requirement of getting active consent from parents in school-based research..., Jelsma [/bib_ref] , therefore, we have not used it as a control variable. No reimbursement was offered or provided for participation. Young people were told that they were free to not answer any questions in the survey and to withdraw their participation at any time. ## Measures ## Sociodemographic variables Gender was assessed by a single item: "Are you a boy or a girl?", with response options "A boy"/"A girl". Age was computed from the year and month of birth reported by the respondents, in combination with the time of data collection. Social class was manually coded based on the parents' occupation and employment reported by young people, into the categories used by the Central Statistics Office. The highest social classes included children of professional and managerial or technical workers. Medium social class groups comprised children of non-manual and skilled manual workers. The lowest social class groups included children of semi-skilled and unskilled workers. ## Romantic attraction A single item was used to assess romantic attraction in young people, developed by members of the sexual health working group of the international HBSC Network [bib_ref] Love and dating patterns for same-and both-gender attracted adolescents across Europe, Költő [/bib_ref] : "Are you attracted to . . . ", with response options "Girls"/"Boys"/Both girls and boys/"I am not attracted yet to anyone". Boys attracted to girls and girls attracted to boys were coded as attracted to opposite-gender partners. Boys attracted to boys and girls attracted to girls were classified as attracted to same-gender partners. Those indicating being attracted to both girls and boys were classified as attracted to both gender partners. Those reporting not being attracted to anyone were classified as not attracted. ## Bobf outcome indicators Perceived discrimination was assessed by a grid of items [fig_ref] Table 1: Better Outcomes, Brighter Futures [/fig_ref] : "How often are you treated unfairly or negatively . . . ", based on the eight grounds listed by the Equal Status Act 2000 . The items included unfair or negative treatment because of where the respondent, their parents, or grandparents were born; because of the respondents' gender; their age; their disability; their race; their sexual orientation; their religion; or their membership of the Traveller community. The frequency of these were rated by the respondents on a five-point Likert scale, with response options "Never" = 1, "Hardly ever" = 2, "Sometimes" = 3, "Often" = 4, and "Very often" = 5. The numeric responses were used as continuous outcome variables. A ninth item, discrimination based on other reason(s), was also administered. Alongside the frequency, respondents could provide reason(s) in a text box. Perceived discrimination based on other ground(s) is not included in the present analysis. Single items were used to cover the following indicator areas [fig_ref] Table 1: Better Outcomes, Brighter Futures [/fig_ref]. Experience of sense of freedom: "In general, do you feel you have freedom in your life?" Having at least one caring and consistent adult to confide in: "In general, do you have a caring adult you can tell anything to?" Perceptions of being valued and respected: "In general, do you feel valued and respected?" Belief in being able to make a positive contribution to the world: "In general, do you feel that you make a positive contribution to the world?" Volunteering and altruism: "In general, do you take part in volunteer work?" Children and young people's awareness of their rights: "In general, do you know your rights as a young person?" For these seven areas, the response options were: "1 (Not at all)"/"2"/"3"/"4 (Very much)". The response options were dichotomized so that 1 reflects very much, while 0 reflects responses less than very much. The eight discrimination items described above and six single items, developed by the HBSC Ireland team specifically to cover BOBF Outcome 5, were tested in a sample of 237 young people [bib_ref] New Questions for the Health Behaviour in School-Aged Children, Költő [/bib_ref]. This pilot phase included asking young people to mark on their questionnaire words or phrases within the questions or response options they did not understand. Following completion of the questionnaire, a classroom discussion was facilitated to ensure that the items were understandable and acceptable. Apart from one respondent who asked what "race" is, and another asking what "sexual orientation" is, young people reported no problems with the content or wording of any item [bib_ref] New Questions for the Health Behaviour in School-Aged Children, Költő [/bib_ref]. For Peer acceptance and respect, we used an item developed by children in Ireland as part of a youth consultation process: "Do you feel comfortable being yourself while with your friends?" Response options were: "Always"/"Often"/"Sometimes"/"Never". These were dichotomized into 1 (always) and 0 (less often than always) [fig_ref] Table 1: Better Outcomes, Brighter Futures [/fig_ref]. Positive parent and family relationships were operationalized by the Family subscale of the Multidimensional Scale of Perceived Social Support (MSPSS) [bib_ref] The Multidimensional Scale of Perceived Social Support, Zimet [/bib_ref]. This subscale contains four items which capture helpfulness and emotional availability of the family. Respondents rated all items on a seven-point Likert scale where 1 indicates very strong disagreement and 7 very strong agreement. In line with the international HBSC protocol, scores for the four items are summed and divided by four, and the overall score is dichotomized in a way that a score 5.5 or above indicates high family support, while a score lower than 5.5 indicates low family support. In our sample, the scale had high internal consistency (Cronbach's alpha = 0.956). Positive relationships with peers were assessed by the Friends subscale of the MSPSS [bib_ref] The Multidimensional Scale of Perceived Social Support, Zimet [/bib_ref]. Similar to the Family subscale, it contains four items on friends' helpfulness and emotional availability. Response options and scoring are identical to that of the Family subscale, thus, based on the cut-off score of 5.5 or above, we coded high friend support, while a score of less than 5.5 was coded as low friend support. The items showed high internal consistency (Cronbach's alpha = 0.959). [fig_ref] Table 1: Better Outcomes, Brighter Futures [/fig_ref] shows three BOBF indicator areas where HBSC is not providing data. The area 18-24-year-olds who vote in local, regional, national, or European elections and referenda concerns young people outside the age range of the HBSC Ireland study. Respect for laws and the judicial process and Perception of fairness of the law fall outside the scope of HBSC. Therefore, these three indicator areas are not featured in the present analysis. ## Sample The present study includes 15-to 17-year-old young people attending post-primary schools in Ireland. The selection of the sample is displayed in [fig_ref] Figure 1: Selection flowchart. [/fig_ref]. From the full sample, we eliminated those who were younger than 15 years, or did not provide information on their gender, age, social class, or romantic attraction. At this stage, the sample comprised data from 3354 young people, with a mean age of 15.78 (SD = 0.78), with 55.1% girls; 56% belonging to the highest social classes, 34.3% to the medium social classes, and 9.7% to the lowest social classes. Attraction-wise, 88.4% reported being exclusively attracted to opposite-gender partners, 3.0% exclusively to same-gender partners, 6.3% to both gender partners, and 2.4% reported no attraction. scores for the four items are summed and divided by four, and the overall score is dichotomized in a way that a score 5.5 or above indicates high family support, while a score lower than 5.5 indicates low family support. In our sample, the scale had high internal consistency (Cronbach's alpha = 0.956). Positive relationships with peers were assessed by the Friends subscale of the MSPSS [bib_ref] The Multidimensional Scale of Perceived Social Support, Zimet [/bib_ref]. Similar to the Family subscale, it contains four items on friends' helpfulness and emotional availability. Response options and scoring are identical to that of the Family subscale, thus, based on the cut-off score of 5.5 or above, we coded high friend support, while a score of less than 5.5 was coded as low friend support. The items showed high internal consistency (Cronbach's alpha = 0.959). [fig_ref] Table 1: Better Outcomes, Brighter Futures [/fig_ref] shows three BOBF indicator areas where HBSC is not providing data. The area 18-24-year-olds who vote in local, regional, national, or European elections and referenda concerns young people outside the age range of the HBSC Ireland study. Respect for laws and the judicial process and Perception of fairness of the law fall outside the scope of HBSC. Therefore, these three indicator areas are not featured in the present analysis. ## Sample The present study includes 15-to 17-year-old young people attending post-primary schools in Ireland. The selection of the sample is displayed in [fig_ref] Figure 1: Selection flowchart. [/fig_ref]. From the full sample, we eliminated those who were younger than 15 years, or did not provide information on their gender, age, social class, or romantic attraction. At this stage, the sample comprised data from 3354 young people, with a mean age of 15.78 (SD = 0.78), with 55.1% girls; 56% belonging to the highest social classes, 34.3% to the medium social classes, and 9.7% to the lowest social classes. Attraction-wise, 88.4% reported being exclusively attracted to opposite-gender partners, 3.0% exclusively to same-gender partners, 6.3% to both gender partners, and 2.4% reported no attraction. Subsequently, all those who provided answers to the BOBF Outcome 5 indicators were retained for the analyses (3190 ≤ n ≤ 3342). Since the responses on discrimination items were analyzed together, those who did not answer one or more items on discrimination had to be excluded, resulting in n = 3213 for the discrimination analyses. Subsequently, all those who provided answers to the BOBF Outcome 5 indicators were retained for the analyses (3190 ≤ n ≤ 3342). Since the responses on discrimination items were analyzed together, those who did not answer one or more items on discrimination had to be excluded, resulting in n = 3213 for the discrimination analyses. ## Analytic approach Analyses were carried out in SPSS 24.0 (IBM Corp., Armonk, NY, USA). Since the variables on perceived discrimination were continuous, while all other items were dichotomized, we used two arms of statistical analysis for each study aim. For Aim 1 (presenting population-level descriptive statistics), first the means and standard deviations are provided by gender and social class groups. Discrimination perceived by boys and girls was compared by Student's t-tests. Perceived discrimination across social groups was compared by one-way variance of analysis (ANOVA). Effect size r for t-tests and omega-squared effect sizes for ANOVA were calculated. For all other BOBF Outcome 5 variables, the percentages of positive responses are provided. The association of the variables with gender and social class were examined by chi-square tests. Cramér's V effect sizes are provided for these associations. For Aim 2 (analysis of the outcomes across romantic attraction groups), first means and standard errors of perceived discrimination are provided for the four attraction groups. Data on discrimination were heavily skewed (the majority of young people reported never or hardly ever being discriminated on any grounds). According to Kolmogorov-Smirnov tests, all of these variables deviated from normal distribution (p < 0.001). Therefore, medians for each grounds of discrimination across romantic attraction groups were obtained. To the same end, the means were compared using non-parametric (Kruskal-Wallis test) as well as parametric methods (ANOVA). For the latter, indices of omega-squared effect size and statistical power are also provided. The magnitudes of the effect sizes were interpreted following Cohen's (1988) guidelines. We compared means pairwise across romantic attraction groups, adjusted for Šidák criteria. To make interpretation easier, mean values of perceived discrimination across romantic attraction groups are displayed in a vertical profile chart [fig_ref] Figure 2: Discrimination [/fig_ref] , where the y axis represents grounds of discrimination, and the x axis indicates mean frequency of perceived discrimination. The vertical lines in the chart represent the pattern of discrimination experienced by the different groups. A similar profiling approach is used in studies of personality [bib_ref] NEO Personality Inventory-Revised, Costa [/bib_ref] : an example of such vertical profile charts is provided by Carver and Scheier, p. [bib_ref] The school-based lives of LGBT youth in the Republic of Ireland, Reygan [/bib_ref]. For all other BOBF Outcome 5 variables, binary logistic regression models were computed, with opposite-gender attracted young people as the reference group. Significances of the models were tested by Wald chi-square tests. Odds ratios were obtained to assess whether same-gender attracted, both-gender attracted, and not attracted young people had different outcomes than their opposite-gender attracted peers. For all odds ratios, 95% confidence intervals (CIs) were calculated. Statistical significance for all analyses was set at p < 0.05. At different stages of the analysis, we conducted statistical tests adjusted for gender, social class and other variables, including area of residence, parental immigrant status, young people's disability, or chronic conditions, and Traveller ethnicity. Ultimately, we decided not to adjust the analyses, for two reasons. First, their effect was usually not significant, or even if significant, the effect sizes were marginal and/or lacking adequate statistical power. Second, including these as control variables or covariates poses methodological and theoretical problems, which will be outlined in the Discussion. # Results ## Descriptive findings: discrimination Mean scores for frequency of perceived discrimination, along with standard deviations, are displayed in . Means are given for boys and girls, and the three social class groups separately. Boys and girls reported similar levels of discrimination based on their disability, race, sexual orientation, and religion. Boys were significantly more likely to report being discriminated based on their, their parents' or grandparents' place of birth, or belonging to the Traveller community, but the effect sizes were low. Girls were significantly more likely to be discriminated based on their age and gender. The effects were medium sized. Across social classes, no significant difference was found in perceived discrimination based on young people's gender, age, race, or religion. Young people from lower social classes were more likely to report discrimination based on their (or their parents') birthplace or their Traveller status, but the effect sizes were marginal. Young people from medium social classes reported somewhat more frequent discrimination based on their sexual orientation than their peers in high or low social classes, but this effect was also marginal. The means rarely exceeded 2, which indicates that the majority of young people never or hardly ever experienced discrimination based on any of the eight grounds. ## Descriptive findings: positive bobf outcome 5 variables Percentages of young people giving a positive answer to the positive BOBF Outcome 5 indicators (e.g., reporting that they feel freedom in their lives, always feeling comfortable being themselves while they are with their friends, very much agreeing with having a caring adult, etc.), are presented in the upper part of [fig_ref] Table 3: Descriptive statistics for positive BOBF Outcome 5 indicators in the overall sample,... [/fig_ref]. These showed a large variation: more than two thirds of young people reported always feeling comfortable being themselves while they are with their friends and almost 60% reported high level of peer support, while only around a fifth indicated feeling that they made a positive contribution, and around one in ten reported that they took part in volunteer work. Perceived freedom, feeling comfortable while being with friends, high family support, feeling valued and respected, and being aware of one's rights as a young person were not associated with gender. Girls were significantly more likely to report having a caring adult and taking part in volunteer work, and boys were more likely to feel that they made a positive contribution than girls, but the effects were small. Girls were significantly more likely to report high peer support; this effect was medium in size. In general, the BOBF Outcome 5 indicators were not associated with social class. However, young people in medium social class groups were significantly more likely to feel comfortable with their friends, and those in high social classes more likely to report high family support compared to their peers in other social class groups (p ≤ 0.004); the effects were small to medium in size. ## Comparing sexual minority and non-minority youth: discrimination Numerical data and comparisons of perceived discrimination across romantic attraction groups is presented in [fig_ref] Table 4: Descriptive statistics and univariate analysis of variance of discrimination, across romantic attraction... [/fig_ref]. The mean values for discrimination are also displayed in a vertical profile chart [fig_ref] Figure 2: Discrimination [/fig_ref] , where the romantic attraction groups are marked with separate lines. The means and the medians indicate that most young people, regardless of their romantic attraction, never or only rarely experienced discrimination based on any grounds. Both parametric ANOVA and non-parametric Kruskal-Wallis tests rendered significant results in almost all cases, indicating that there are significant differences in perceived discrimination across romantic attraction groups. Nevertheless, the models had marginal effect size for discrimination based on birthplace, disability, race, and religion. Discrimination across romantic attraction based on belonging to the Traveller community was not significant when non-parametric comparison was used, which may reflect the small proportion of Traveller youth in our sample, and that only a fraction of them reported being attracted to same-gender or both gender partners. This is supported by the model being on the border of acceptable statistical power (0.827). There were some grounds, though, on which same-and especially both-gender attracted young people were more likely to feel discriminated than their opposite-gender attracted and not attracted peers. Significant differences were detected across romantic attraction groups for gender and age, with small effect sizes. Pairwise comparisons revealed that both-gender attracted youth were significantly more likely than the other three groups to feel that they were discriminated based on their gender and age. In other cases, apart from belonging to the Traveller community, both-gender attracted young people also experienced more discrimination than the other groups. Discrimination based on sexual orientation was significantly more likely to be experienced by same-and both-gender attracted young people than their opposite-gender attracted and not attracted peers. The effect size was large. ## Comparing sexual minority and non-minority youth: positive bobf outcome 5 variables The lower part of [fig_ref] Table 3: Descriptive statistics for positive BOBF Outcome 5 indicators in the overall sample,... [/fig_ref] presents the proportion of positive responses to BOBF indicators other than discrimination. Perceived freedom, feeling that one made a positive contribution, and knowing their rights as a young person were not significantly associated with romantic attraction. Feeling comfortable while being with friends, having a caring adult, high family and peer support, feeling valued and respected, and taking part in volunteering work were significantly associated with romantic attraction, but the effect sizes were small. This pattern was reflected in the binary logistic regression models [fig_ref] Table 5: Relative odds for same-gender attracted, both-gender attracted, and not attracted young people,... [/fig_ref] : romantic attraction, overall, did not have a significant effect on perceived freedom, feeling that they made a positive contribution, and knowing their rights as a young person. In other words, same-and both-gender attracted and not attracted young people had odds similar to their opposite-gender attracted peers of reporting these positive outcomes. It should be noted, though, that both-gender attracted youth were 0.7 times less likely, compared to their opposite-gender attracted peers, to feel that they made a positive contribution; while same-gender attracted youth were 1.3 times more likely to report that they knew their rights as a young person. Note: a The overall model was not statistically significant. Odds ratios highlighted in boldface indicate that there is a statistically significant difference in the odds of adolescents in the given romantic attraction group compared to their opposite-gender attracted peers. Both-gender attracted young people were, significantly, 0.8 times less likely to report having a caring adult compared to their opposite-gender attracted peers; similarly, they were less likely to report high family support and feel that they made a positive contribution. Same-gender attracted and not attracted youths' odds were not significantly different from that of their opposite-gender attracted peers. On the other hand, both-gender attracted and not attracted youth had significantly lower odds of reporting feeling comfortable while being with their friends compared to those who reported opposite-gender attraction. No significant difference was found for the odds of same-gender attracted youth. Not attracted young people were also less likely than the other three groups to report high peer support. Same-gender attracted young people were almost twice as likely as their oppositegender peers to report taking part in volunteer work. Both-gender attracted youth were also somewhat more likely to report volunteering, but this was not statistically significant. # Discussion In this study, we analyzed the prevalence of psycho-social phenomena under the Better Outcomes, Brighter Futures (BOBF) national youth policy framework's fifth outcome ("Connected, respected, and contributing to their world") in a nationally representative sample of 15-to 17-year-old young people in Ireland, demonstrating the utility of population-based data in informing policy makers. Data for the majority of the BOBF Outcome 5 indicators are provided by the Irish Health Behaviour in School-aged Children (HBSC) study, a World Health Organization collaborative cross-cultural study, therefore, we examined a subsample of the HBSC Ireland study, conducted in 2018. The first aim of the present study was to provide population-level data on these outcomes. The second aim was to showcase the utility of the BOBF framework by comparing how sexual minority (same-and bothgender attracted) young people fare on these indicators compared to their non-minority (opposite-gender attracted and not attracted) peers. ## Descriptive findings: gender and social class have a moderate role In relation to Aim 1, a positive finding of the study is that a large majority of young people were not likely to report that they experienced discrimination based on any of the eight grounds listed by the Equal Status Act of : these include where the respondent, their parents, or grandparents were born; the respondents' gender; their age; their disability status; their race; their sexual orientation; their religion; or their membership of the Traveller community. In general, perceived discrimination was not associated with gender or social class. Even where associations were statistically significant, the sizes of the effect were small. In that sense, our hypothesis outlined in Section 1.2 was not confirmed by the data. Remarkable exceptions were that girls were more likely to feel that they had been discriminated based on their gender or their age, with medium effect sizes. While there is little consensus in how to characterize and analyze intersectional stigma [bib_ref] Challenges and opportunities in examining and addressing intersectional stigma and health, Turan [/bib_ref] , this result suggests that one component that contributes to intersecting discrimination is being female. Ageism-prejudices based on somebody's biological age-is often experienced by young people; this is often combined with sexism at the expense of girls or young women, especially if they are involved in activism or act in a non-traditional manner [bib_ref] An inconvenienced youth? Ageism and its potential intergenerational roots, North [/bib_ref]. On the other hand, girls were significantly more likely to report high levels of peer support than boys. This is in line with earlier findings, i.e., that girls spend more time with their friends and perceive their friendships as more cohesive and emotionally close than boys [bib_ref] Gender, grade, and relationship differences in emotional closeness within adolescent friendships, Johnson [/bib_ref]. These results indirectly suggest that interpersonal solidarity among minority girls may, to some extent, counterbalance the negative effects (e.g., stress and anxiety) stemming from discrimination [bib_ref] A snapchat story: How black girls develop strategies for critical resistance in..., Kelly [/bib_ref]. This may be an important asset in interventions that aim to improve the lives of sexual minority girls. Only 11.5% of the young people agreed very much with that they are participating in volunteer work. In a seven-country study, adolescents reporting civic commitments, regardless of their gender or country, were more likely to consider public interest an important life goal [bib_ref] Ties that bind: Correlates of adolescents' civic commitments in seven countries, Flanagan [/bib_ref]. Percentages of young people reporting ever being engaged in volunteering work ranged from 16% (boys in Russia) to 68% (girls in Hungary), but they indicated their volunteering activity on a binary "yes-or-no" question, while we have used more stringent dichotomization. Had we included all youth who reported doing any amount of volunteer work, the percentages would have been 60.2% in boys and 63.9% in girls. Existing evidence unequivocally supports that young people who volunteer show more favorable outcomes in a large range of health, well-being, and psycho-social indicators than their peers who are not engaged in volunteering [bib_ref] The effects of volunteering on the young volunteer, Moore [/bib_ref]. However, research on prosocial behaviors such as volunteering in adolescents is scarce, and the causal links between positive health, psycho-social characteristics, and prosocial behaviors are not fully understood [bib_ref] Young people and volunteerism: A model of sustained volunteerism during the transition..., Marta [/bib_ref]. A potential explanation is that engaging in volunteering can increase a person's social capital, which is known to have benefits for health outcomes [bib_ref] Social capital and health: A decade of progress and beyond, Kawachi [/bib_ref]. Another mechanism may be that being connected to communities can improve confidence and self-esteem in young people [bib_ref] Strengthening social ties to increase confidence and self-esteem among sexual and gender..., Romijnders [/bib_ref]. A key element of psychotherapeutic interventions for gay and bisexual men is to support them in considering how local LGBTI+ communities may help them in reducing stress and anxiety [bib_ref] Psychotherapy for the spectrum of sexual minority stress: Application and technique of..., Burton [/bib_ref]. Facilitating joining such communities and/or being engaged in volunteering may be particularly beneficial for bisexual or both-gender attracted young people; however, it should be noted that a barrier for them belonging to LGBTI+ communities may be the anticipation that they will be discriminated against based on their bisexuality [bib_ref] Bisexual women's discriminatory experiences and psychological distress: Exploring the roles of coping..., Craney [/bib_ref]. More explorative studies are required to better understand the specific needs and experiences of bisexual youth in communities and with voluntary work. ## Better outcomes, brighter futures in sexual minority adolescents: discrimination, resilience, and social agency Our study's second aim was to showcase the utility of the BOBF Outcome 5 indicator set by comparing young people belonging to sexual minorities (operationalized by being attracted to same-and both gender partners) to their non-minority peers (being attracted exclusively to opposite-gender partners or not reporting being attracted). Sexual minority groups were more likely to perceive discrimination based on their sexual orientation, which is in line with our second hypothesis. This finding indirectly supports that assessing romantic attraction may correctly identify young people whose self-defined sexual orientation would have been lesbian, gay, or bisexual [bib_ref] Sexual identity, attraction and behaviour in Britain: The implications of using different..., Geary [/bib_ref]. Elsewhere, our team have argued that classifying sexual minority young people based on the gender of partners they are attracted to may result in a larger group than assessing self-defined sexual orientation, since there may be young people who have not recognized and/or disclosed their non-heterosexual orientation [bib_ref] Love and dating patterns for same-and both-gender attracted adolescents across Europe, Költő [/bib_ref]. Sexual minority young people, especially those who reported being attracted to both boys and girls, were more likely than their peers (even more than exclusively same-gender attracted youth) to feel discriminated against based on their age and gender. This result is in line with findings from the Hungarian LGBT Survey conducted in 2007, where researchers observed that members of the lesbian, gay, bisexual, or transgender community sample, compared to a nationally representative sample, were more likely to experience discrimina-tion based on not only on the grounds of their sexual orientation or gender identity, but on other grounds as well [bib_ref] Social Exclusion of Lesbian, Gay, Bisexual and Transgender (LGBT) People in Hungary;..., Takács [/bib_ref]. The most cited other grounds for discrimination, similar to our study, were gender and age. We hypothesize that this additional discrimination may be attributed to a "negative halo effect", sometimes termed "reverse halo effect" or "horns effect". This phenomenon denotes that one characteristic that is considered as negative sheds a negative light on the whole person. Thus, it is antithetical to the "halo effect", described 100 years ago by E. L. Thorndike, when a (positive) characteristic of a person favorably biases how others evaluate their (otherwise unrelated) attributes [bib_ref] A constant error in psychological ratings, Thorndike [/bib_ref]. Perceived freedom, the feeling that one makes a positive contribution to the world and knowing their rights as a young person were not significantly associated with romantic attraction. However, the pattern of the data suggests that had the subsample sizes in sexual minority and not attracted youth been larger, we would probably have seen significant differences. Not attracted young people were less likely than their opposite-gender attracted peers to feel comfortable with their friends or report high peer support. A probable explanation is that young people who have never been involved in romantic relationships in general seem to have problems with establishing and building friendships with their peers [bib_ref] Adolescents' anxiety in dating situations: The potential role of friends and romantic..., Greca [/bib_ref] , although from this association we cannot infer causality. Both-gender attracted young people were less likely than their non-minority peers to report high family support, feeling comfortable while being with friends, or to feel valued and respected. Remarkably, no such difference was found for exclusively samegender attracted youth. This result echoes earlier findings that adolescents who identify as bisexual face disproportionate health risks across a wide range of determinants and health outcomes compared to their peers who identify as heterosexual, or even lesbian or gay [bib_ref] Sexual orientation disparities in adolescent cigarette smoking: Intersections with race/ethnicity, gender, and..., Corliss [/bib_ref] [bib_ref] Racial/ethnic differences in mental health, substance use, and bullying victimization among self-identified..., Feinstein [/bib_ref]. Our international HBSC team found that among adolescents from eight European countries or regions, both-gender attracted youth were the most likely to report substance use [bib_ref] Romantic attraction and substance use in 15-year-old adolescents from eight European countries, Költő [/bib_ref] or rate their health as poor and report multiple health symptoms [bib_ref] Self-reported health and patterns of romantic love in adolescents from eight European..., Költő [/bib_ref]. This pattern of unfavorable outcomes for both-gender attracted or bisexual youth may be because bisexuality is often "invisible" or denied by members of the individuals' social network. Bisexual people may be mis-classified by others as either heterosexual or lesbian/gay. Sometimes bisexual individuals experience rejection and biphobic harassment or monosexism (the belief that hetero-and homosexuality are superior to bisexuality) not only from their heterosexual peers but even by those who identify as gay or lesbian. Some findings indicate that bisexual identity and gender interact with each other in their impact on health outcomes [bib_ref] Global health/LGBTQ health, Poteat [/bib_ref]. While from our data we cannot infer the reason for these disparities, it is worth noting that similar to many other Western countries, there is a growing acceptance towards LGBT individuals and issues in Ireland. However, it seems that bisexual people do not benefit from this shift as much as their lesbian and gay peers. While being lesbian or gay can be perceived by others as a stable identity, bisexuality is often seen as "just a phase" and bisexual individuals report frequent discrimination, identity invalidation, and erasure [bib_ref] Perceived discrimination, coping mechanisms, and effects on health in bisexual and other..., Doan Van [/bib_ref]. Therefore, not only individuals need support and empowerment in being more bi-inclusive, but LGBTI+ communities as well. Youth exclusively attracted to same-gender partners were almost twice as likely as their opposite-gender attracted peers to report that they are involved in community work. A similar, albeit not significant, tendency was observed among both-gender attracted youth. The work of Riggle and Rostoskydemonstrates that sexual minority people, due to their numerous experiences of discrimination and rejection, may become more aware of social injustice and empathetic with marginalized groups-not only based on sexual orientation or gender identity, but on other grounds too. This may motivate them to develop compassion towards others who are oppressed and stigmatized, and combat injustice and marginalization. In a recent landscape and knowledge gap analysis to explore and synthesize research done with LGBTI+ young people in Europe, we have found some studies tangentially dealing with community work, volunteering, and activism. These imply that many sexual and gender minority young people may want to do volunteer work for their local LGBTI+ communities, which provides them with a sense of belonging, or gives an opportunity to help others [bib_ref] Young Life and Times Survey, Schubotz [/bib_ref]. Mayock [bib_ref] Supporting LGBT Lives in Ireland: A Study of the Mental Health and..., Mayock [/bib_ref] found that LGBT young people in Ireland saw their community as a source of resilience and support, with themes of connectedness, safety, and solidarity frequently mentioned in respondents' accounts of belonging to the LGBT community. Our findings, however, suggest that this may not be the case for all subgroups within sexual minority youth. Findings elsewhere indicate that LGBT young people report that dealing with adversity can result in a sense of empowerment [bib_ref] Factors enhancing adaptive coping and mental health in lesbian youth: A review..., Kulkin [/bib_ref] , sometimes conceptualized as "coming out growth" [bib_ref] Coming out growth: Conceptualizing and measuring stress-related growth associated with coming out..., Vaughan [/bib_ref]. Such intra-individual processes, coupled with LGBT community connectedness at the micro-and meso-level [bib_ref] Systematic mapping of relationship-level protective factors and sexual health outcomes among sexual..., Johns [/bib_ref] and societal acceptance and tolerance at the macro-level, may act to boost self-esteem, increase resilience, and buffer the negative effects of stigma and discrimination. # Limitations and strengths Both gender and romantic attraction questions provided only binary (boy-girl) response options which exclude gender diverse (transgender, non-conforming, or non-binary) young people, or those living with an intersex variation. Romantic attraction is only one dimension of sexual orientation beside sexual identity, behavior, and fantasies, and there may be young people who would have described their gender in other terms than "boy" or "girl". While dimensions of sexual orientation are generally overlapping [bib_ref] Sexual identity, attraction and behaviour in Britain: The implications of using different..., Geary [/bib_ref] , an inclusive assessment of sexual orientation needs to capture them. The BOBF is a comprehensive framework to improve the health and well-being of all young people containing a large suite of indicators, but it inevitably misses some key aspects of sexual and gender minority youths' lives (e.g., coming out or disclosing someone's sexual orientation or gender identity) to different persons. We have not assessed adolescents' romantic relationship status either. Other key aspects to provide a balanced, non-victimizing account of sexual minority youths' life and well-being would require that we assess resilience in the face of sexual orientation-and gender identity-based harassment, exclusion, and bullying, as well as belonging to LGBT communities. Finally, it should be noted that since HBSC collects data in classrooms, young people who were absent on the day of the data collection or attend youth centers or out of school services will inevitably be excluded from the sample. Given that sexual minority youth tend to miss school due to health problems [bib_ref] Self-reported health and patterns of romantic love in adolescents from eight European..., Költő [/bib_ref] or due to fear of harassment and bullying [bib_ref] School Climate Survey Report: The Experience of Lesbian, Gay, Bisexual and Trans..., Pizmony-Levy [/bib_ref] , they are probably underrepresented in the present study. Further work is needed to include a broader sample of young people both within and outside the traditional school setting (e.g., by using community sampling). As noted in the Method section, adjusting for "background" variables caused a dilemma. In earlier stages of the analysis, we controlled the comparisons of romantic attraction groups for gender and social class, but either these were not significant predictors of the outcomes, or-due to the very low subsample sizes-they resulted in inadequate statistical power. However, there is the broader problem of intersectionality [bib_ref] Mapping the margins: Intersectionality, identity politics, and violence against women of color, Crenshaw [/bib_ref]. It has to be noted that the results, especially those on perceived discrimination, are confounded with sexual minority and non-minority young people potentially belonging to other minority or marginalized groups, for instance, based on their ethnicity, place of origin, belonging to the Traveller community, being a young carer, or living with a disability or chronic condition. Excluding them would have rendered much smaller subsamples, inevitably reducing statistical power. Potential solutions for these problems are case-control matching and identifying young people with multiple or intersecting minority statuses. Our team has previously used such analytic techniques, but these are not without methodological challenges. On the other hand, we believe that our study has some strengths. It is based on a nationally representative sample of 15-to 17-year-olds from an international study using rigorous methodology. Micro-and meso-level psycho-social determinants of young people's health were analyzed using an established, comprehensive indicator set. Some areas explored in this study (e.g., perceived discrimination, feeling valued and respected, and volunteering) are relatively less studied, especially among sexual minority adolescents. ## Implications for practice, policy, and research In recent years, Ireland has seen substantial change in acceptance and tolerance towards LGBTI+ individuals, with the Marriage Equality Bill in 2015 allowing same-sex couples equal rights in marriage passed in a referendum by a popular vote of 62%. This demonstrated a remarkable shift in the attitudes towards the LGBTI+ community in a country where the constitution and societal values are historically grounded in the Catholic faith. However, this change in itself does not necessarily mean that sexual (and gender) minorities are now free from stigma and discrimination. Our findings, in line with earlier studies conducted in Ireland, found that sexual minority youth, especially both-gender attracted or bisexual young people, are more likely than their non-minority peers to experience discrimination and less likely to score favorably on psycho-social variables. It is therefore imperative that evidence-based interventions that increase awareness of the LGBTI+ community and inclusivity towards them are developed and implemented in society. Such interventions should be initially aimed at young people themselves, adults working with young people, and healthcare professionals. This will support youth to flourish and live a full and balanced life, irrespective of their sexual orientation or gender identity. It is also important to increase opportunities for young people to contribute to society through volunteering, thus increasing the sense of citizenship and social agency in young people, as well as their sense of cohesiveness. Ireland is one of the leading countries with regard to having a clear focus on children's well-being, and its commitment to children and youth is demonstrated by the appointment of a Minister for Child and Youth Affairs, as well as the development of the Better Outcomes, Brighter Futures national youth policy framework and indicator set. Ireland was the first country in the world to develop a dedicated national strategy for LGBTI+ youth. The European Union's LGBTIQ Equality Strategy also highlights the importance of combating inequalities in educational settings. Nevertheless, there is still a need for national and local strategies and policies that are based on the current needs of young people. These should include policies on gender neutral spaces, improving awareness of non-binary gender identities, encouraging inclusion and diversity, and, more specifically, prohibiting discrimination based on gender identity and sexual orientation, in schools and in the workplace. Such policies, and their appropriate implementation, will allow for children of all backgrounds, genders, and sexual orientations to grow and thrive safely in society. While this study focuses on sexual minority young people, it should be noted that one of the groups reporting perceived discrimination is girls. Discrimination, social exclusion, and inequalities based on gender, as well as the intersectional effects of being a girl and belonging to a minority group [bib_ref] Mapping the margins: Intersectionality, identity politics, and violence against women of color, Crenshaw [/bib_ref] are important, especially given the effort that societies make to close the gender gaps in various dimensions and venues. That girls still feel discriminated against is a sign that we must not assume that we have overcome gender discrimination, even if there has been progress at a policy level. Multiple interventions are necessary, most importantly, "gender-proofing" policies and practice developments. Just as practice and policy need to adapt to accommodate the needs of marginalized young people, so does research. Research often lags behind societal changes, resulting in matters that are deemed important to participants but not to researchers not being included study designs and questionnaires. This highlights the need for child participation in informing research, but also emphasizes the need to include underrepresented populationswhen setting the research agenda. For example, sexual orientation and gender identity are rarely included in youth population studies outside North America, limiting our ability to understand the health disparities in sexual and gender minority young people [bib_ref] Re-doing research: Best practices for asking about gender and sexuality in education..., Jones [/bib_ref]. Our study clearly demonstrates the difficulties in examining intersectionality, even in a nationally representative youth population study. To allow for better understanding of the impact of intersectionality, researchers should consider oversampling minority groups where possible, or to conduct more targeted research using similar research instruments to allow for comparison with the general population. Temporal changes in sexual and gender minority youths' health (the effect of the chronosystem) should be investigated with studies using longitudinal designs or repeated measurement at subsequent points. has pointed out that data on depression, mental ill-health, and suicide are "too often taken as a point of departure when contemplating the lives of LGBT identified young people". According to , discourses that reproduce sexual minority youth as "always-already victims" have been critiqued as "essentialist presumptions" that socially construct LGB lives as inevitably problematic, disempowered, and traumatized [bib_ref] Popular culture, the 'victim' trope and queer youth analytics, Marshall [/bib_ref]. For instance, mental ill-health and suicidal distress in sexual and gender minority youth can be motivated by a complex web of factors and experiences of which LGB identity is but one small piece of the puzzle [bib_ref] Speaking back to dominant constructions of LGBT lives: Complexifying 'at riskness' for..., Bryan [/bib_ref] [bib_ref] Supporting LGBT Lives in Ireland: A Study of the Mental Health and..., Mayock [/bib_ref]. We believe that shifting research from the predominant at-riskness and victimizing narrativeto a resource-and resilience-oriented agenda may provide clearer direction to those interested in improving sexual and gender minority youths' lives and well-being. Such a shift is also coherent with the "after-queer" approach in LGBTI+ research [bib_ref] After-queer' tendencies in queer research, Talburt [/bib_ref]. # Conclusions Adolescents in Ireland, in general, very rarely experience discrimination on any grounds, and perceived discrimination is not strongly affected by gender or social class. A notable exception is that girls were more likely than boys to feel they have been discriminated based on their age and gender. Sexual minority (same-and both-gender attracted) youth perceive more discrimination based on their sexual orientation than their nonminority peers (those who were exclusively attracted to opposite-gender partners or did not report romantic attraction). In addition, those who are attracted to both gender partners reported that they experience discrimination based on their gender and age more often than the other three attraction groups. These findings imply that girls and sexual minority, especially both-gender attracted or bisexual, youth are more vulnerable to stigmatization on multiple grounds, which may be attributed to a negative halo effect. Additionally, of note is the finding that only around 60 percent of young people report high peer and family support, and positive responses on other indicators of the Better Outcomes, Brighter Futures Outcome 5 ("Connected, respected, and contributing to their world") are even less frequent. Only around a fifth of young people agreed very much that they made a positive contribution or had freedom in their lives, and around one in ten reported very much taking part in volunteer work. Given that these features contribute to health and well-being, it would be desirable that practice and policy support initiatives that facilitate social skills, connectedness, and civic engagement. In general, these indicators were not strongly associated with gender or social class. Girls were, however, substantially more likely than boys to report high peer support. Both-gender attracted and not attracted young people were less likely than their opposite-or same-gender attracted peers to report that they feel comfortable being themselves while being with their friends, and not attracted youth were also less likely than the other three groups to report high peer support. Both-gender attracted adolescents were around half as likely to feel valued and respected compared to their non-minority peers. These findings suggest that even within sexual minority young people, psycho-social determinants of health may work differently. Our results are in line with other findings in the literature that young people attracted to both genders or identifying as bisexual are disproportionately affected by stigma, discrimination, social exclusion, and their negative health consequences than their exclusively same-or opposite-gender attracted (lesbian, gay, or heterosexual) peers. Interventions and policies should tackle not only heterosexism and homophobia but monosexism and biphobia as well. Same-gender attracted young people were almost twice as likely as their oppositegender attracted peers to report that they take part in volunteer work. Although we do not have data on what type of volunteer work they were engaged in, this finding may indicate that sexual minority youth are more perceptive of social injustice than their nonminority peers and are more motivated to partake in activism and community work. The next iterations of the LGBTI + National Youth Strategy of Ireland and similar governmental or state-level initiatives should facilitate civic engagement and volunteering in sexual minority youth as well as an awareness of the specific needs of both-gender attracted or bisexual youth. We believe that further studies in this area have the potential to balance the predominant victimizing narrative on sexual minority young people into a more positive, "after-queer" discourse. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. # Data availability statement: The datasets analyzed in this study can be accessed via the webpage of the HBSC Ireland research team, in accordance with the HBSC data access policy: http://www. nuigalway.ie/hbsc/dataaccess/. [fig] Figure 1: Selection flowchart. [/fig] [fig] Figure 2: Discrimination [/fig] [fig] Table 2: Descriptive statistics for discrimination, by gender and social class ( [/fig] [fig] Author: Contributions: Conceptualization, A.K. and S.N.G.; Formal analysis, A.K. and A.G.; Funding acquisition, S.N.G.; Investigation, A.K., A.G., E.V., C.K., M.M. and S.N.G.; Methodology, A.K., A.G., E.V., C.K., M.M. and S.N.G.; Project administration, A.K. and S.N.G.; Resources, S.N.G.; Supervision, S.N.G.; Visualization, A.K.; Writing-original draft, A.K., A.G. and E.V.; Writing-review and editing, C.K., M.M. and S.N.G. All authors have read and agreed to the published version of the manuscript. [/fig] [fig] Funding: This research was funded by the Department of Health, Republic of Ireland. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Research Ethics Committee of the National University of Ireland Galway under Decision Ref. REC17-Nov-13 (13 November 2017). [/fig] [table] Table 1: Better Outcomes, Brighter Futures (BOBF) framework Outcome 5: "Connected, respected, and contributing to their own world"-Aims and indicator areas within the framework and indicators/response options within the Health Behaviour in School-aged Children (HBSC) Ireland study. [/table] [table] Table 3: Descriptive statistics for positive BOBF Outcome 5 indicators in the overall sample, by gender, social class, and romantic attraction.Note: a Cramér's V effect size. Cells highlighted in boldface indicate a statistically significant association with gender, social class, or romantic attraction. [/table] [table] Table 4: Descriptive statistics and univariate analysis of variance of discrimination, across romantic attraction groups: opposite-gender attracted (Note: a Discrimination based on the birthplace of the respondent, their parents, or their grandparents. [/table] [table] Table 5: Relative odds for same-gender attracted, both-gender attracted, and not attracted young people, compared to their opposite-gender attracted peers, to show positive outcomes on positive BOBF Outcome 5 indicators. [/table]
Tobacco use and sinonasal cancer: a case-control study. The risk for sinonasal cancer associated with tobacco use was examined in a case-control study in males diagnosed between 1978 and 1981 in the Netherlands. Of the 116 cases of sinonasal cancer and 259 controls identified, interviews were completed for 92 (79%) of the cases and 195 (75%) of the controls. Everusers of cigarettes had a moderately elevated risk for sinonasal cancer. The association was strongest for squamous cell carcinoma among recent users of tobacco (RR=3.1, P<0.05, one-sided). For recent tobacco users, there was also a trend in risk associated with the amount of cigarette use (P<0.05, one-sided). Associations between tobacco use and adenocarcinoma were inconsistent, and no positive associations were found for the other histologic types, largely undifferentiated tumours. The study findings indicate that tobacco use, and in particular recent tobacco use, is associated with the development of squamous cell sinonasal cancer. Over the last 50 years, several occupational risk factors for sinonasal cancer have been identified, including exposure to nickel, chromium, wood and leather dust [bib_ref] Towards control of nasal cancer. Lancet i, 856, Anon [/bib_ref] [bib_ref] Nasal cavity and paranasal sinuses, Redmond [/bib_ref]. Although this body of literature provides an important basis for primary prevention in the workplace, only a small proportion of all nasal cancers can be attributed to these factors. Few non-occupational factors, however, have been associated with nasal cancer. Among these, tobacco smoking would appear to be a prime suspect. Yet, until recently there was little epidemiologic evidence to support this association . Several large cohort studies on cigarette smoking had failed to report an elevated risk for nasal cancer, although this may have been the result of small numbers for this rare tumour site or of the policy of combining cancer sites for statistical analysis [bib_ref] Mortality in relation to smoking: Ten year observations of British doctors, Doll [/bib_ref] [bib_ref] Smoking in relation to death rates of one million men and women, Hammond [/bib_ref]. Two recent casecontrol studies from Canada [bib_ref] Wood exposure and smoking: Association with cancer of the nasal cavity and..., Elwood [/bib_ref] and the US [bib_ref] A case control study of cancers of the nasal cavity and paranasal..., Brinton [/bib_ref] have shown elevated risks for nasal cancer associated with tobacco use. We have examined this relationship in a case-control study in the Netherlands. # Methods In early 1982 the records from the six major institutions in the Netherlands for the surgical and radiation treatment of tumours of the head and neck were reviewed to identify cases for this study. One hundred and sixteen males resident in the Netherlands, aged 35 to 79, who were newly diagnosed between 1978 and 1981 with a histologically confirmed primary epithelial cancer of the nasal cavities (ICD9: 160.0) or of the accessory sinuses (ICD9: 160.2-160.5) were identified for study. In reviewing the medical records for all potential cases, cases diagnosed as having nasal cancer but for whom the site of origin was unspecified were excluded from study. At the time of study implementation, 74 cases were alive and 42 (36%) were deceased. Of the 116 cases, 67 (58%) were squamous cell carcinomas; 28 (24%) were adenocarcinomas; and 21 (18%) were tumours of other types, mostly (18 cases) undifferentiated tumours. Based upon the available clinical data, 50% of the cases with adenocarcinoma originated in the accessory sinuses; while for the squamous cell and other tumour types 25% and 38%, respectively, were so described. The control group consisted of age-stratified random samples of living male residents of the Netherlands in 1982 (in the ratio of 2: 1 for all cases, living and deceased) and of deceased (all causes) males in the Netherlands in 1980 (in the approximate ratio of 1:1 for deceased cases). Two-hundred and twenty-three living controls were selected from the municipal resident registries and 36 deceased from the records of the Central Bureau of Genealogy, yielding 259 controls eligible for study. Study group cases or, for the deceased, their nearest relative were requested by the treating physician to participate in this study. Control group members or, for the deceased, the nearest relative were approached by letter followed by a telephone call or, if necessary, a house visit to request participation. The interviews of study subjects or their next of kin were carried out by experienced interviewers who had special training for this study. Interviews included information on the time period of beginning and stopping use and the extent of use of manufactured cigarettes, hand-rolled cigarettes, cigars, pipe tobacco, chewing tobacco, and snuff. For the purposes of this analysis, 50 g of hand rolling tobacco is considered the equivalent of 40 manufactured cigarettes, and the data for amount is presented as combined cigarette equivalent use. Ever-use of cigarettes was defined as lifetime use of 100 cigarettes or more; while for cigars and pipe, everuse was defined as regular use for 6 months or more. Chewing tobacco use was reported by 4 squamous cell cancer cases, 2 adenocarcinoma cases and 10 controls. Snuff use was reported for only one subject, with an adenocarcinoma. Due to the small numbers, chewing tobacco and snuff use will not be further considered in this analysis. The study participation rate was 79% (92/116) for the cases and 75% (195/259) for the controls. Among cases, participation was highest in the older ages, but the reverse was true among the controls. Among those alive, 86% of the cases and 77% of the controls participated in the study. For those deceased, interviews were obtained from respondents for 64% of the cases and 64% of the controls. For the cases all interviews took place in the home. Among the controls 20 (10%) of the interviews were carried out by telephone. The measure of statistical association used in this study is the exposure odds ratio. This measure, as well as confidence limits (90%) were derived by the maximum likelihood method [bib_ref] Exact and asymptomatic methods for the combination of 2 x 2 tables, Thomas [/bib_ref]. Statistical tests of an excess risk (P < 0.05, one-sided) for nasal cancer were derived as equivalent to the lower 90% confidence limit. The Chisquare test for trend with stratification for age is used to examine whether disease risk increases with increasing levels of exposure. To assess the possible confounding effects of wood dust exposure and of possibly inter-related tobacco use variables, logistic regression analysis was carried out. # Results Examining selected demographic features of the study respondents in, 18% (17/92) of the cases and 10% (20/195) of the controls were not married (P<0.05, one sided), while there were no statistical differences for level of education. Although the control series was selected to be of similar age distribution as the cases, the control respondents were on average somewhat younger than the respondent cases. After adjustment for age, the finding for marital status was unchanged. Inthe association between ever use of tobacco and nasal cancer is presented. For all histological types combined, there is a non-significant increase in risk associated with cigarette use, while for cigar and pipe use the risk estimates are not elevated. By histologic type, cigarette use is associated with elevated risks for squamous cell carcinoma and adenocarcinoma, but not with the tumours of other types. Cigar and pipe use is associated with elevated risks for adenocarcinoma only. None of the associations are, however, statistically significantly elevated. Statistical control for occupational wood dust exposure did not modify these findings. The confidence intervals, particularly for cigarette smoking, are wide largely due to the scarcity of non-smokers in this study series. As shown in, 48 of the 50 squamous cell cases and 23 of the 24 adenocarcinoma cases had smoked cigarettes. All of the cases of these two histologic types had reported use of some form of tobacco. Thus virtually all of the pipe and cigar smokers had also smoked cigarettes. The risk for tumours of other types, largely undifferentiated tumours, appears if anything to be negatively associated with ever use of tobacco. Estimating daily dose, the reported usual amounts of cigarettes, cigars or pipe tobacco consumed were not associated with any of the histologic types of nasal cancer. When examined by total duration of use, statistically significant trends were found for the number of years of use of cigarettes (P< 0.05, one-sided) and for the number of years of use of all tobacco (P< 0.05, one-sided) with the risk for squamous cell carcinoma. When analyses were restricted to living respondents, the associations found for duration were somewhat stronger. Inthe use of tobacco, as cigarettes, cigars or pipe, is presented for the study groups by the recency of use. Those who previously smoked are categorized as long-term aAdjusted for age (30-59, 60-69, and 70-79 years); n is the number exposed. (>10 years) and recent (0-9 years) quitters. To examine whether recent tobacco use is associated with an excess risk for nasal cancer, never users of cigarettes, cigars, or pipe, and those who had quit 10 or more years ago were considered as non-exposed and compared to those who reported still smoking 9 or fewer years before diagnosis. The resultant age adjusted relative risks were 3.1 (P <0.05, onesided) for squamous cell carcinoma, 1.4 for adenocarcinoma, and 0.3 for the other tumour group. Further control in logistic regression analyses for wood dust exposure, age begun cigarette use, and for usual cigarette use did not substantially change these findings. Among living respondents the risk for squamous cell carcinoma in this comparison was 2.2 (NS). When the risk among recent users was compared to that of long-term quitters, excluding never smokers, the risk for squamous cell carcinoma was 2.3 and no longer statistically significant. An analysis was carried out with respect to cigarette smoking only. For cigarette smoking the age adjusted relative risks for recent smoking were 2.3 (90% CI: 1.2-4.8) for squamous cell carcinoma, 1.2 (90% CI: 0.5-2.8) for adenocarcinoma, and 0.6 (90% CI: 0.2-1.5) for the other tumour group. Again considering only the recent smokers as exposed as above, Table IV presents the associated risks by the extent of cigarette use. There is an increase in risk for squamous cell carcinoma associated with an increase in level of usual cigarette consumption (P <0.05, one-sided). This finding could not be attributed to age at start of smoking, vital status, or occupational exposure to wood dust. Removing subjects who had never smoked from these analyses resulted in similar associations although the associated relative risks were no longer as strong. No such association is noted for the adenocarcinoma or the other cell type groups. However, with the small numbers involved it is clearly not possible to rule out a similar association, particularly for the adenocarcinomas which show some elevation in risk. For living respondents the associated risks were similar, although somewhat higher than for all respondents combined. Similar analyses were carried out for usual cigar and pipe use. No positive associations were found for pipe or cigar use with any of the histologic cell types. The association between duration of cigarette use and risk of squamous cell cancer was also examined, excluding the recent (0-9 years) smoking history. When this recent experience was not included in the calculation of duration, no association was found between duration of use and risk of disease. [bib_ref] Wood exposure and smoking: Association with cancer of the nasal cavity and..., Elwood [/bib_ref] was the first to report an association between tobacco use and nasal cancer, particularly for cigarette smoking. The risk increased with the amount of cigarettes used. The author reported that smoking most frequently occurred in patients with squamous cell and transitional cell carcinomas, but that an association with all histologic types could not be ruled out. Information on smoking recorded in existing medical records was used and a control group was chosen from patients with other forms of cancer. No smoking data was available for more than one-third of the study subjects who were grouped in the analysis with the non-smokers. These aspects of the study design may have introduced important biases. [bib_ref] A case control study of cancers of the nasal cavity and paranasal..., Brinton [/bib_ref] also reported an association between nasal cancer and tobacco use. Cigarette smoking was most strongly related to squamous cell tumours , and there was a significant linear relationship of risk with years of cigarette smoking. The association of squamous cell tumours with tobacco use prevailed for both males and females. Associations with pipe and tobacco smoking and snuff usage were also predominantly for squamous cell tumours. # Discussion The current study further indicates that tobacco use is associated with an elevated risk for nasal cancer, particularly of the squamous cell type. We found a statistically nonsignificant elevation in risk for squamous cell tumours among ever users of cigarettes of RR=3.0, as compared to the finding of [bib_ref] A case control study of cancers of the nasal cavity and paranasal..., Brinton [/bib_ref] of RR = 1.8. As they did, we also found a statistically significant trend in risk among ever smokers for duration of cigarette use. However when we considered time period of smoking, we found that the excess risk for squamous cell cancer was most evident among those who were recent smokers (9 years or less before diagnosis). In fact when the recent smoking experience was excluded in calculating the duration of tobacco use, the association of duration with risk disappeared. Among recent users of cigarettes, there was a statistically significant trend for risk of squamous cell cancer by the level of cigarette use. The findings could not be attributed to the vital status of the respondents. Clearly, these analyses in which histologic type and time period of exposure are considered are based on small numbers and the findings need further support. If the association with recent smoking were to be established in further studies, it would be consistent with the findings that quitting cigarette smoking reduces the subsequent risk for developing lung and bladder cancer, compared to the risk for those who continue to smoke . For ever-users of cigarettes, cigars and pipes we found elevated risks of 2 to 3-fold for adenocarcinoma. However, no statistically significant associations were found when. examined by duration, level, or recency of use. In fact, the assessment of risk associated with pipe or cigar use in the absence of cigarette smoking was not possible. We previously reported [bib_ref] Wood related occupations, wood dust exposure and sinonasal cancer, Hayes [/bib_ref] that adenocarcinoma was strongly associated with occupational wood dust exposure (RR=26.3) in this group. Eighteen of the 23 adenocarcinoma cases had occupational exposure to wood dust. Statistical control for wood dust exposure did not alter the results for tobacco use, although it is extremely difficult to assess the independent influence, if any, of tobacco use in this small group. For the other histologic types, largely undifferentiated tumours, no excess risk associated with tobacco use was identified. Any association present would appear to be a negative one. Twenty-five percent of the cases in this group reported no use of tobacco, compared to 5% in aAdjusted for age (30-59, 60-69, and 70-79 years); bNone includes never smokers of cigarettes and those who had quit use of cigarettes, cigars, and pipes 10 or more years before study. the two other case groups. Other than as a statistical artefact due to small numbers, no explanation for this finding is evident. In summary, the study findings indicate that tobacco use, and in particular recent tobacco use, is associated with the development of squamous cell sinonasal tumours. [table] Table II: The relative riska and confidence intervals (90%) for nasal cancer by ever use of tobacco and by tumour histologic type, [/table] [table] Table IHI: The number and percent (%) of study group members and relative risks by recency of [/table] [table] Table IV: The usual amount of cigarettes smoked among recent tobacco users and the relative riska of nasal cancer by histologic type,Netherlands, 1978-81 Level of recent use (cigarette equivalents per day) [/table]
High temperature environment reduces olive oil yield and quality Global warming is predicted to have a negative effect on plant growth due to the damaging effect of high temperatures. In order to address the effect of high temperature environments on olive oil yield and quality, we compared its effect on the fruit development of five olive cultivars placed in a region noted for its high summer temperatures, with trees of the same cultivars placed in a region of relatively mild summers. We found that the effects of a high temperature environment are genotype dependent and in general, high temperatures during fruit development affected three important traits: fruit weight, oil concentration and oil quality. None of the tested cultivars exhibited complete heat stress tolerance. Final dry fruit weight at harvest of the 'Barnea' cultivar was not affected by the high temperature environment, whereas the 'Koroneiki', 'Coratina', 'Souri' and 'Picholine' cultivars exhibited decreased dry fruit weight at harvest in response to higher temperatures by 0.2, 1, 0.4 and 0.2 g respectively. The pattern of final oil concentration was also cultivar dependent, 'Barnea', 'Coratina' and 'Picholine' not being affected by the high temperature environment, whereas the 'Koroneiki' and 'Souri' cultivars showed a decreased dry fruit oil concentration at harvest under the same conditions by 15 and 8% respectively. Regarding the quality of oil produced, the 'Souri' cultivar proved more tolerant to a high temperature environment than any other of the cultivars analyzed in this study. These results suggest that different olive cultivars have developed a variety of mechanisms in dealing with high temperatures. Elucidation of the mechanism of each of these responses may open the way to development of a variety of olives broadly adapted to conditions of high temperatures. OPEN ACCESS Citation: Nissim Y, Shloberg M, Biton I, Many Y, Doron-Faigenboim A, Zemach H, et al. (2020) High temperature environment reduces olive oil yield and quality. PLoS ONE 15(4): e0231956. https:// # Introduction Fluctuations in temperature occur naturally during plant growth and reproduction. However, extreme hot summers can damage the intermolecular interactions needed for proper growth, thus impairing plant development and fruit set. The increasing threat of climate change is already having a substantial impact on agricultural production. High temperatures may cause visual symptoms of sunburn, leaf abscission and growth inhibition of plantswith two treatments at differing temperatures. They found that fruit dry weight was not affected by average temperatures within a range of 16-25˚C, but was reduced with further increases in temperature. Oil concentration decreased linearly at 1.1%˚C −1 across the entire range (16-32˚C) of average seasonal temperatures explored, while oleic acid concentration decreased 0.7%˚C −1 over the same range. In the one month long experiment, an additional 7˚C above the control had a permanent negative effect on oil concentration at final harvest, particularly when the exposure to high temperature was at the beginning of oil accumulation. Oleic acid concentration was also negatively affected by the high temperature treatment. However, oleic acid concentration recovered upon removal of the chamber, with the exception being that just as with total oil accumulation, oleic acid accumulation was retarded when the heat treatment was applied at the beginning of fruit development. In general, they concluded that high temperatures during oil accumulation negatively affected olive oil yield and quality in warm regions, particularly if the high-temperature event occurs early. Garcia-Inza et al.used chambers which may distort the effects of a natural environment. Other studies tested the effect of high temperatures comparing fruits of various cultivars in different locations during different years. When comparing the yields of trees from different orchards, which were subjected to different methods of agriculture, many factors can influence the parameters being tested. In order to avoid these problems, we compared adult plants in pots, all of the same age, and whose fertilization, pruning and other treatments were equal, the only difference between the two groups being their location during the period of fruit development. Olives are a major crop in Israel and are planted all over the country including semi-desert regions such as the area surrounding Tirat Zvi, where summer temperatures often rise above 40˚C. The objective of this study was to characterize the effect of a high temperature environment during olive oil accumulation, on oil yield and quality. In order to address this we used five year old potted olive trees of five selected cultivars and placed one group in Tirat Zvi (high temperatures during the summer) during the period of fruit development, and a second group in Tzuba, a village with relatively mild summers, for two consecutive years. Characterizing fruit development, oil accumulation and oil quality in both groups revealed that unsurprisingly, high temperatures during oil accumulation can decrease fruit size, oil yield and oil quality. We demonstrated however, that sensitivity to high summer temperatures is genotype dependent. # Materials and methods ## Experimental design The olive cultivars 'Barnea', 'Coratina', 'Koroneiki', 'Souri' and 'Picholine' were used in this study. Twelve olive trees from each of the five cultivars brought from the Boxer nursery (Bnei Darom, Israel) and planted in pots in 2010 at the Volcani Center, Israel (31˚59'N 34˚49'E; 42 m above sea level). During the summer of 2015, the trees were replanted in 50 liter pots. Right after fruit set, at the beginning of May 2016, six saplings of each cultivar were placed in an extremely hot climate zone at Tirat Zvi village (32˚25'N 35˚31'E; 225 m below sea level). The remaining six trees of each cultivar were placed in a more temperate environment at Tzuba village (31˚47'06.0"N 35˚06'33.5"E; 705 m above sea level). Both locations are private land and the owners of the land gave permission to conduct the study on these sites. Temperature measurements were determined using a HOBO USB Micro battery-powered station, which is a weatherproof data logger for monitoring temperature. The HOBO devices were stationed in the shade at both locations and measured the temperature every two hours throughout the entire period of the experiment. Data regarding humidity, rainfall and wind speed was taken from the Israel Meteorological Service database (https://ims.data.gov.il), measured at meteorological stations located 5 km from Tirat Zvi and 4.5 km from Tzuba. The plants were watered using a drip irrigation system which delivered 2L of water per hour spread over three 25 minute cycles per day (07.00, 12.00 and 17.00 h) at Tzuba, the moderate temperature (MT) site, and four such cycles (06.00,.00,.00 and 18.00 h) at Tirat Zvi, the high temperature (HT) site. Each plant was irrigated with four drippers. An additional fifteen minutes of irrigation was added at each location in mid-summer to ensure an adequate supply of water. Irrigation was delivered in excess, in order to prevent dry soil. The pots had perforated bottoms in order to allow the escape of excess water. Taking into account the differences in evaporation between the two locations, the trees in the HT site were watered one third more than the trees at the MT site. In total, each tree in the HT site was watered with 13.3 liters per day and the trees in the MT site with 10 liters per day per tree. During July and August 2 liters per day per tree were added in both locations. Weeds were controlled by manual removal. Olive fruit fly control was done only in Tzuba site using Vertimec EC, Vertigo EC and Score EC as necessary, as well as Rogor L-40 EC which was applied once a month throughout the experiment. Plants were fertilized at the beginning of May with Osmocote, smartrelease plant food; one application contains 11 essential nutrients and is effective for 6 months. In order to address the effect of a high temperature environment on olive fruit development as well as oil accumulation and quality, we characterized various physiological parameters of the five olive cultivars 'Barnea', 'Picholine Languedoc', 'Koroneiki', 'Souri' and 'Coratina' during the fruit development period of two consecutive years. Trees of each cultivar were placed at two different locations, two weeks after full bloom: The HT site (Tirat Zvi), was characterized by very warm summers and the MT site, Tzuba, with a mild summer. Temperatures were measured at both locations every two hours during the entire season by portable data loggers. Olives from each of the five cultivars on both locations were sampled every month throughout the experiment for physiological and histological analysis as well as for evaluation of olive oil content. At the end of the season, all fruits remaining on the trees were harvested, the oil extracted and analyzed for quality. Trees were returned to the Volcani center for the winter and spring, and in May 2017, two weeks after full bloom, the trees were transported once more to Tirat Zvi (HT site) and Tzuba (MT site). Transportation in both years was carried out very carefully and no loss of fruit was experienced as a result of the move. In total, the experiment lasted from the beginning of May till the end of October 2016 and from the beginning of May till Mid December 2017. At the end of the 2016 season, we harvested only a limited number of 'Souri' and 'Picholine' fruits. Therefore, the 'Souri' fruits were only partially analyzed and the 'Picholine' fruits were not analyzed at all. In 2017, all five cultivars were fully analyzed. Fruit load was not measured, even though it may have had an effect on fruit weight and oil concentration. However, plants were randomly chosen to be placed in either of the two locations and therefore we believe that the average fruit load was similar at the HT and the MT sites. ## Evaluating olive oil concentration Whole fruit fresh weight was recorded, and the fruit was then dissected to mesocarp and seed for further analysis. The fresh weight of each dissected section was recorded. Olive mesocarp and endocarp was oven dried at 90˚C for 48 h and the dry weight was recorded. Oil content (dry weight basis) was determined using chemical oil extraction with petroleum ether as a solvent in quintuple (approximately 5 gr each). # Cell size-histological analysis A section of the fruit was analyzed for the number of cell layers (S1 cell area using differential staining. Fresh fruit sections from each sampling date were preserved throughout the experiment using FAA (Formaldehyde 10%, Ethanol 50%, Acetic Acid 5% and water 35%) as a fixative. At the end of the experiment, we analyzed only samples representing different developmental stages. The beginning of June, 50 days post anthesis (DPA), represented the developmental stage preceding pit hardening. The beginning of July, 83 DPA, represented the developmental stage just after pit hardening, and September, 146 DPA, represented the period before ripening and the end of fruit growth. The last time-point for this analysis was at harvest. Analysis was done as describedwith safranin/fast green staining. # Oil drops-histological analysis A fresh section of the fruits was analyzed for oil drop size and density using Sudan IV staining protocol. The specimens were stained for 6 min with Sudan IV (0.5% w/v in 90% ethanol) and then transferred to a slide, differentiated rapidly in ethanol 50% to remove excess stain and the images observed and photographed under a light microscope DMLB (Leica, Germany) with a DS-Fi1 camera attached (Nikon, USA). Measurement of oil drop size and density was done using NIS elements software (Nikon, USA). ## Cold-press-olive oil extraction At the end of the experiment, olives of each cultivar and location were harvested after developing a semi-black skin color, and a maturity index (MI (of approximately three, which occurred around November. The MI was calculated from three repeats of 100 fruits, as a subjective evaluation of the skin color and flesh as developed in the Research Station of Venta del Llano (Jaen, Spain) and proposed by Uceda and Frias. Oil was extracted from healthy fruits using a laboratory-scale Abencor system (Comercial Abengoa, S.A., Seville, Spain) equipped with a hammer crusher, malaxer and centrifuge that simulates the industrial process of EVOO production. ## Determination of phenolic compounds In 2016, Phenolic compounds were isolated from a solution of oil in hexane by double-extraction with methanol/water (60:40, v/v). Total phenols, expressed as tyrosol equivalents (ppm), were determined with a UV-visible spectrophotometer (Beckman Coulter, Fullerton, CA, USA) at 735 nm using Folin-Ciocalteu reagent. In 2017, the phenolic compounds were isolated from olive oil to evaluate Ortho-diphenols by solid-phase extraction as developed by Mateos et al.,. Determination of biophenols by HPLC was done according to International Olive Council (COI/T.20/Doc No 29/Rev.1). The calculation of Biophenol content, expressed in mg/kg, was calculated by measuring the sum of the areas of the related chromatographic peaks. ## Determination of oil fatty acid composition Fatty acids were transformed into fatty acid methyl esters (FAMEs (using trans-esterification with cold methanolic solution of potassium hydroxide, according to International Olive Council (COI/T.20/Doc. 24) and European Union (EU Regulation-EN 1991R2568) protocols. Data analyzed by Chemstation software. The calculation of the percentage of each fatty acid identified by gas chromatograph was done according to the formula: %fatty acid = (area of fatty acid x 100) / (total area) # Statistical analysis The parameters tested were fresh and dry fruit weight, dry fruit oil concentration, wet oil concentration, amount of oil per fruit, cell size and number of cell layers and oil droplet size and density. These were subjected to three-way analysis of variance (ANOVA) including full factorial analysis for each year, for their dependence on the three independent variables of sampling date, tree location and cultivar type, including the various interactions between them. Fruit was sampled during the entire period from fruit set to ripening. Obviously, fruit growth and oil accumulation continued throughout this period. Therefore, we chose two sampling days and performed a full factorial two-way ANOVA analysis for the independent variables of tree location and cultivar type for each. The first sampling date chosen (146 days postanthesis), was that on which the differences between the two locations were most evident. Harvesting time was chosen as the obvious summation date of the experiment. When we encountered significant interaction between factors, a Tukey-Kramer test was performed in order to rank the various levels of interaction. All statistical analyses were performed using JMP software. # Results ## Different locations typify different climate conditions Summer temperatures in Tirat Zvi (the HT site) were higher than in Tzuba (the MT site) by almost 10˚C in daytime and 5˚C at night. For example, in 2016 the average daily maximum temperature at the HT site was above 40˚C during June, July August and September, whereas at the MT site it was 32.8, 32.8, 32.6 and 30.8˚C respectively during these months. During this period, there was only one day with a temperature above 40˚C in the MT site (40.6˚C). The average daily minimum temperature at the HT site was above 21˚C from June to September, whereas in the MT site it never exceeded 20˚C. During 2017, the maximum monthly temperature was above 40˚C in the HT site from May till October. During November and December, temperatures decreased dramatically at the HT site. During July, the maximum temperature at the HT site was 45.8˚C whereas at the MT site it was 37.8˚C. In 2017, the average daily minimum temperature in the HT site was above 20.5˚C from June to September, and below 20.5˚C in the MT site. The average difference in maximum daily temperature between the HT site and the MT site was 9.2˚C and 7.5˚C during 2016 and 2017 respectively and a difference of 4˚C and 4.15˚C in the average minimum daily temperatures during 2016 and 2017 respectively . During both seasons, there were only few rainy days. The humidity at the MT site was slightly higher compared to the HT site and wind speed in both locations differed in an average speed of 0.47 m/s (S2 . Since all trees were kept together at the volcani center until fruit set, differences in flowering time of the various cultivars were not recorded. In both years, fruits were harvested at a maturity index of approximately 3. In 2016, the time of ripening of the fruits was uniform for all cultivars at both locations and the harvest was carried out at the beginning of November with no significant differences between maturity index at the time of harvest for all cultivars at both locations. In 2017, fruits at the MT site ripened earlier than at the HT site. In contrast to the MT site, differences in ripening time between cultivars were observed at the HT site. Therefore, in the MT site, all cultivars were harvested at 168 days post anthesis (October 23 rd ). In the HT site, the cultivars 'Koroneiki', 'Picholine' and 'Souri' were harvested 198 days post anthesis (November 22 nd ) and 'Barnea' and 'Coratina' were harvested 226 days post anthesis (December 20 th ). The maturity index for the various cultivars is presented in S3A high temperature environment affects fruit weight and oil accumulation The difference between summer temperatures at the HT site and the MT site was greater in 2016 compared to 2017. Therefore, differences between the two locations in all parameters measured were greater in 2016. Interaction between sampling date, cultivar type and tree location was found to be significant for dry fruit weight as well as dry fruit oil concentration (Three-ways ANOVA-P<0.001 and P<0.005 in 2016 and P<0.001 and P<0.001 in 2017 respectively). We tested the effects of cultivar type and tree location and the interaction between them on dry fruit weight and dry fruit oil concentration, by two-ways ANOVA at 164 DPA and at harvest in 2016 and at 146 DPA and harvest time in 2017 (S4A and S4F . These independent variables and the interaction between them were found to have significant effects on both parameters at both sampling dates for both years. 'Barnea' dry fruit weight in August 2016 was significantly lower in the HT site trees than that measured at the MT site. In 2017, dry fruit weight of 'Barnea' in the HT site was significantly lower from August till the end of October (harvest time at the MT site). However, when temperatures decreased in November-December, 'Barnea' dry fruit weight at the HT site rose to the same level as at the MT site. 'Koroneiki' dry fruit weight during 2016 was significantly higher at the MT site compared to the HT site, since its dry fruit weight at the HT remained constant from July till harvest. In 2017, dry fruit weight was equal at both locations from fruit set till the end of July. However, during August and September 'Koroneiki' fruit weight increased dramatically at the MT site, while at the HT site fruit weight was roughly constant during this period. At harvest, 'Koroneiki' fruit weight at the MT site was significantly higher than at the HT site (405.3 and 254 mg respectively). 'Coratina' dry fruit weight was equal at the HT and MT sites until July, but after July of both years, there was a significant rise in dry weight at the MT compare to the HT site. At the 2016 harvest, dry fruit weight was double at the MT site compared to the HT site. In 2017, at the MT harvest time, 'Coratina' dry fruit weight at the MT site was 1.38 g, and at the HT site, 0.871. However, when temperatures decreased in November-December, fruit weight at the HT site rose and at HT harvest time reached 1.12 g, still significantly lower compared to the MT 'Coratina' dry fruit weight at harvest. 'Souri' cultivar dry fruit weight was significantly higher at the MT site from the end of July till harvest during both years. 'Picholine' dry fruit weight was significantly higher at the MT site than at the HT site, from August until harvest. During harvest, 'Picholine' dry fruit weight was 1.66 g at the MT site, significantly higher than 1.28 g at the HT site. Fresh fruit weight, commercial oil percentage and oil per fruit data are presented in S5The 'Barnea' dry fruit oil content was not significantly different between the two locations in 2016 except in August when dry fruit oil percentage was 30.6 at the MT site and only 16.3 at the HT site. In 2017, beginning in September, dry fruit oil content was higher in the MT trees compared to the HT site (34% and 23.1% respectively). However, since the HT 'Barnea' was harvested much later than in the MT, the final dry fruit oil content at harvest was similar at both locations. The dry fruit oil content in the 'Koroneiki' cultivar was significantly higher at the MT compared to the HT site during both years from August until harvest. The oil percentage of dry fruit of the 'Coratina' cultivar was significantly higher at the MT site compared to the HT site during most of the season. However, in both years, at harvest time, the dry fruit oil percentage was similar at the MT and HT sites (43.6 and 40% in 2016, 46.3 and 44.8% in 2017 respectively). From August till harvest time, the dry fruit oil percentage of the 'Souri' cultivar was significantly higher at MT compared to the HT site during both years. The dry fruit oil percentage of the 'Picholine' cultivar was significantly higher at the MT compared to the HT site during August and September. However, at harvest, the dry fruit oil percentage in the MT and HT sites were similar (32.7% and 30.6% respectively). We analyzed the variation in the gain of dry fruit weight and dry fruit oil concentration in all the cultivars tested, during each month from June to September at the two locations in 2016 and 2017. We found that the main disparity in gain of fruit weight between the HT and MT sites occurred during August of both years. Similarly, the main difference in oil concentration between the HT and MT sites was measured during July and August of 2016 and in August of 2017. Since the interaction between cultivar type and tree location was significant for both traits, during August (164 DPA in 2016 and 146 DPA in 2017) and at harvest time, we ranked the cultivars by their performance at the MT site compared to that at the HT site (S4B-S4E and S4G-S4J . In 2016, at harvest, the, 'Koroneiki' and 'Coratina' cultivars showed the most significant difference in dry fruit weight, whereas the 'Barnea' cultivar exhibited the least difference between the MT and HT sites. A similar analysis for oil content of dry fruit demonstrated that the 'Koroneiki' cultivar showed a highly significant difference between the two locations, whereas variation for this trait in the 'Coratina' and 'Barnea' cultivars was significantly lower. In 2017, at harvest, the 'Souri' cultivar showed the most significant difference in dry fruit weight between the MT and HT sites. The cultivars 'Koroneiki', 'Picholine' and 'Coratina' exhibited lesser differences between sites, whereas the 'Barnea' cultivar exhibited the least difference between the MT and HT sites. A similar analysis for oil content of dry fruit demonstrated that the 'Koroneiki' and 'Souri' cultivars showed a highly significant difference between the two locations, while the cultivars 'Picholine', 'Coratina' and 'Barnea' had significantly lower variation between locations. We also analyzed the association between the gain of dry fruit weight and dry fruit oil concentration in all analyzed cultivars during each month in relation to the monthly average maximum daily temperature (Tmax), minimum daily temperature (Tmin) and average daily temperature (Tmean). We found that dry fruit weight in all cultivars was correlated (negatively and significantly) to all the above temperature variables. In contrast however, dry fruit oil concentration was not correlated to any of those temperature variables monitored. When we applied these analyses to those cultivars in which dry fruit weight was sensitive to high temperatures ('Koroneiki', 'Coratina', 'Souri' and 'Picholine'), we found that gain of dry fruit weight exhibited a significantly negative correlation with all temperature variables. Similarly, when we applied our analysis to those cultivars in which oil concentration had proved itself to be sensitive to high temperatures ('Koroneiki' and 'Souri'), we found a significant negative correlation between dry fruit oil concentration and Tmax (S6 . ## High temperature environment affects mesocarp growth In order to address the effect of a high temperature environment on fruit growth, we measured mesocarp cell size and the number of cell layers in the mesocarp at various developmental stages. The effect of cultivar type and tree location on mesocarp cell size and the number of cell layers in the mesocarp was found to be significant at both 146 DPA and harvest time. The interaction between cultivar type and tree location was found to have a significant effect on cell size at both sampling dates, but affected the number of cell layers at harvest time alone (S4F . The 'Barnea' fruit weight curve at the MT site exhibits the expected double sigmoid pattern, in which fruit weight increases linearly after fruit set, stays steady during pit hardening and increases again after pit hardening until September, just before ripening. At the HT site, the 'Barnea' fruit weight curve increases in approximately linear fashion from fruit set to harvest. The number of cell layers in the 'Barnea' fruits at the MT site did not change statistically during fruit growth from the beginning of June until harvest-time at the end of October. However, cell size increased dramatically from 1181 μm 2 at the beginning of June to 6604 μm 2 at the end of October. The number of cell layers in 'Barnea' fruits at the HT site was constant from June till September. However, it increased from 27 to 37 layers from September until harvest time in December. The cell size curve of the 'Barnea' fruits at the HT site showed the opposite tendency and increased from June till September but remained steady from September until December. The 'Koroneiki' fruit weight curve at the MT and HT sites is similar to that of 'Barnea'. At the MT site it exhibits double sigmoid growth and at the HT site growth is linear. However, the slope of the 'Koroneiki' curve at the HT site is flatter than in the 'Barnea'. The number of mesocarp cell layers as well as cell size in the 'Koroneiki' fruits in the MT site increased during the entire period of fruit development. In June, the 'Koroneiki' mesocarp in the MT site consisted of 20 layers of 1596 μm 2 cells, whereas in October (at harvest) it consisted of 34 layers of 6210 μm 2 cells. At the HT site, mesocarp cell size and the number of cell layers increased dramatically from June to July, before pit hardening, and then increased at a slower pace till harvest. During this period cell size as well as the number of cell layers was significantly lower in the mesocarp of 'Koroneiki' fruits from the HT site compared to those which grew at the MT siteThe 'Coratina' mesocarp showed a tendency similar to that of 'Koroneiki', however the number of cell layers, unlike in the 'Koroneiki' mesocarp, was higher at the MT site all season. The number of cell layers in the 'Souri' and 'Picholine' mesocarp was similar at both sites, whereas cell size in both cultivars was significantly higher at the MT site compared to the HT site in three of the four dates sampled. September fruit weight of all five cultivars at the MT site was significantly higher than at the HT site. However, at that time, the number of mesocarp cell layers in the fruit of all cultivars at the MT site was not significantly higher than at HT. In contrast to 'Barnea', the mesocarp of the other four cultivars consisted of significantly larger cells in the MT site trees compared to the same cultivars grown at the HT site ## A high temperature environment affects oil drop size and density In order to address the effect of a high temperature environment on oil accumulation, we measured oil drop size and density at various stages of development: 50, 83 and 146 DPA. At 146 DPA, the effects of cultivar type and tree location and their interaction, on oil drop size, was found to be significant. In contrast, the effect of tree location on oil drop density was not found to be significant. However, the effects of cultivar type and the interaction between cultivar type and tree location were significant (S4F . 'Barnea' fruit at the HT site, at 50 DPA, showed significantly larger, denser oil drops compared to those at the MT site. Average oil drop size was 75 and 54 μm 2 and average oil drop densities were 476 and 352 drops per cm 2 , in the HT and the MT sites respectively. However, later in the season, at 83 as well as 146 DPA, the oil content and oil drop size were higher at the MT compared to the HT site, whereas oil drop density was similar at both locations. Oil drop sizes at 83 DPA were 286 and 220 μm 2 at the MT and the HT sites respectively and at 146 DPA, 860 and 658 μm 2 . The differences between the two locations on both dates are significant. Oil drop densities at 83 DPA were 747 and 650 drops per cm 2 and at 146 DPA, 677 and 645 drops per cm 2 at the MT and the HT sites respectively. These differences on both dates are not statistically significant. 'Koroneiki' olives showed the same trend as the 'Barnea' with bigger oil drops at the HT site at 50 DPA and bigger oil drops at the MT site at 83 DPA and at 146 DPA. However, at 146 DPA, in 'Barnea', the ratio of oil drop size between the MT and the HT sites is 1.3 and in 'Koroneiki', the ratio reaches 2.6. In addition, at 146 DPA, oil drop density of 'Koroneiki' olives is higher in trees at the HT compared to the MT site. 'Coratina' oil drop density was higher at the MT site compared to the HT site. However, the differences between the two sites was not significant at all three sample dates. Surprisingly, 'Coratina' oil drops were significantly larger at the HT site compared to the MT site at 83 DPA. However, later in the season, at 146 DPA, oil drops were significantly larger at the MT site compared to the HT site. The oil drop density in 'Souri' and 'Picholine' was similar at both sites at all sampling dates. However, oil drops were significantly larger at the MT sites compared to the HT sites in both cultivars. By September, the oil content of all five cultivars was significantly higher in olives at the MT site than in those grown in the HT site. At that time, oil drop size was also significantly greater in the MT site olives than in those from the HT site. However, oil drop density in 'Koroneiki' olives was higher at the HT than at the MT site ## High temperature environment affects oil composition In order to characterize the effect of high temperature environments on oil composition and quality, we measured the polyphenol content in the oil extracted from the various cultivars at both locations. During the 2016 season, polyphenol levels varied between cultivars. In general, levels were almost double in oil extracted from fruits from the MT site compared to those grown at the HT site. In 2017, polyphenol levels in all cultivars at the HT site were lower than at the MT site. The lowest polyphenol level, 156 mg/g oil, was measured in the oil extracted from 'Barnea' from the HT site. The polyphenol level in oil extracted from 'Barnea' grown at the MT site was more than double that from the HT fruits-404 mg/g oil. Oil extracted from 'Coratina' showed the same pattern as that of 'Barnea'. Polyphenol levels in oil extracted from 'Picholine' grown at the MT site were more than three times higher than in oil extracted from 'Picholine' from the HT site (893 and 291 mg/g oil respectively). Polyphenol levels in oil extracted from 'Koroneiki' at the MT site were 150% higher than the levels at the HT site. The smallest difference between polyphenol levels in oil extracted from the two locations was found in oil extracted from the 'Souri' cultivar. This was mainly due to the unusually high level of polyphenols found in oil extracted from 'Souri' grown at the HT site, 772 mg/g oil. The main fatty acid found in olive oil is oleic acid. We found that oil extracted from all five cultivars grown at the HT site contained lower levels of oleic acid than oil extracted from olives grown in a milder environment. During 2016, the differences between sites in the percentage of oleic acid in oil extracted from olives grown at the MT site compared to olives from the HT site was 8, 4 and 7% in 'Barnea', 'Koroneiki' and 'Coratina' respectively. During 2017, oleic acid levels were higher than in 2016 for all cultivars in both climate regions. Oleic acid levels in oil extracted from olives grown at the MT site reached 74% in 'Koroneiki' and 76% in 'Coratina', whereas the levels in oil extracted from olives grown at the HT site was 67 and 69% respectively. Oleic acid level in oil extracted from 'Picholine' olives grown at the HT site was 51.8%, whereas at the MT site it was 60.4%. We also characterized all other fatty acids in the oil of the various cultivars in both climate regions (S7 . The decrease in oleic acid content in the oil extracted from olives grown at the HT site coincides with an increase in the level of palmitic acid (C16:0) and linoleic acid (C18:2) in oil extracted from olives grown there. The palmitic acid content was about 2% more in oil from the HT site compared to the MT olives in all cultivars during both years. Excessively high levels of palmitic and linoleic acid were detected in 'Barnea' oil extracted in 2016, and in 'Picholine' oil extracted in 2017. 'Picholine' oil extracted from olives grown in 2017 in the HT site contained 21.6% palmitic acid, and 'Barnea' oil extracted from olives grown in 2016 at the HT site contained 23.3% linoleic acid. # Discussion The most critical stage of development of olive fruit, when fruit growth as well as oil accumulation rate are maximized, is between 60 and 120 days after flowering, between pit hardening and fruit ripening. In the northern hemisphere, this period falls in July and August. In both 2016 and 2017, this period was particularly hot at the HT site. Temperatures reached 46˚C in 2016 and 45˚C in 2017, whereas the mean daily maximum temperatures during July and August were 42.5˚C in 2016 and 40.4˚C in 2017. In general, the summer of 2016 at the HT site was hotter than the summer of 2017. This may be the reason for the early ripening in 2016 at the HT site compared to 2017. However, it was shown that several parameters such as irrigation regime or fruit size can affect ripening time. Olive oil yield is the function of the number of fruits, average fruit weight and oil concentration at maturity. In this study, we addressed the effects of an environment of consistently high temperatures on fruit development, oil accumulation and oil composition in five olive cultivars. We found that different olive cultivars respond to high temperatures of the environment in a genotypic specific manner. A high temperature environment repressed fruit development and oil accumulation and also affected oil composition. We found that the 'Koroneiki' and 'Souri' cultivars are highly temperatures sensitive, and were affected to an extreme degree by the high environmental temperatures. We demonstrated that fruit weight and oil accumulation in 'Koroneiki' and 'Souri' olives harvested in the HT site were much lower compared to these parameters at the MT site. In contrast to 'Koroneiki' and 'Souri', the 'Barnea' cultivar exhibited greater tolerance to high temperatures, and although fruit growth as well as oil accumulation were at some stages significantly lower at the HT site, the final fruit weight as well as oil percentage at harvest was virtually the same at both the HT and MT sites (S4 . These results were repeated in 2016 and 2017. Oil quality, defined by oleic acid content as well as by polyphenol levels in the oil after harvest, was found to be affected by high temperatures in all five cultivars. Whether the effects of high temperatures can be attributed mainly to average, maximum or minimum daily temperatures or to other temperature parameters, is still not clear. We found significant association between dry fruit weight and Tmax, Tmin and Tmean. However, when we analyzed sensitive cultivars only, we found a significant association between Tmax and dry fruit oil concentration (S6 . Daily maximum temperature differences between the HT and the MT site were higher in 2016 compared to 2017. On the other hand, differences between the HT and MT site in the daily minimum temperatures were higher in 2017. Differences in dry fruit weight and dry fruit oil concentration were also higher in 2016. It would seem therefore, that the most relevant parameter for predicting the effects of high temperatures, is the daily maximum temperature. A recent studyfound that olive fruit dry weight showed a tendency to decrease with increasing mean temperature, while the proportion of oil in the fruit exhibited a significant correlation with mean daily thermal amplitude, and weaker correlation with mean daily maximum and minimum temperatures. The proportion of oleic acid in the oil showed a negative correlation with mean daily minimum temperature and with mean daily temperature, and a linear relationship with mean daily thermal amplitude. This study used lower maximum temperatures to cause heat stress compared to the ambient temperatures at our HT site (S1 . We can therefore surmise that olive plants suffered from high temperatures as a result of the maximum daily temperatures encountered at the HT site. ## A high temperature environment affects fruit weight We found that final dry fruit weight at harvest of 'Barnea' cultivar was not affected by the location of the trees nor by the resulting difference in temperatures. In comparison to 'Barnea', the 'Koroneiki', 'Coratina', 'Souri' and 'Picholine' cultivars showed a decreased dry fruit weight at harvest in response to the higher temperatures at the HT site. The influence of temperature during early stages of fruit development has been identified in several fruit tree species. Dry fruit weight in the olive cultivar 'Arauco' was not affected by temperatures in the range of 16-25˚C. However, at higher temperatures it showed a decrease of 0.08 grams in dry fruit weight for each additional 1˚C. We found a cultivar dependent decrease of between 0.026 to 0.044 grams in 2016, and 0.015 to 0.078 grams in 2017, in dry fruit weight per 1˚C increase, measured in September, before harvest. It has been suggested that temperature during fruit development may have a greater effect on cell division than its effect on cell expansion in tomato fruits. Another study found that continuous heating of tomato fruit reduced cell expansion. We found that in 'Barnea' olives grown at the MT site, cell expansion alone contributed to mesocarp growth beginning 7 weeks after full bloom. At the HT site, 'Barnea' fruits followed this pattern from 50 to 146 days (7 to 21 weeks) after full bloom. However, during the period of 146 to 247 days after full bloom, cell expansion ceased and cell division alone contributed to mesocarp growth. In the 'Koroneiki' cultivar we found a similar pattern to 'Barnea', the exception being, that at the MT site, cell division also contributed to mesocarp growth in the late stages of fruit development. 'Coratina' showed the same trend as 'Koroneiki' whereas in 'Souri' and 'Picholine', as in 'Barnea', it was cell expansion alone that was responsible for the differences between the two sites. Rallo and Rapoportfound that in olives, as in other drupes, both cell division and cell expansion contribute to mesocarp growth at early stages of fruit development. Six weeks after full bloom, cell division ceased, and cell expansion alone contributed to further mesocarp growth. Hammami et al.found that fruit size differences among six olive cultivars are due to cell division throughout fruit growth, which occurs mainly in the first six weeks after bloom. However, they were surprised to find that a substantial number of cells formed after these six weeks, and continued during the following 6 months. The six cultivars they tested did not include the five cultivars tested in this study. In our study, it is possible that in reaction to the unusually high temperatures at the HT site during the early stages of fruit development, the "Barnea" cultivar delayed mesocarp cell division until the arrival of milder weather (October-December). The 'Koroneiki' cultivar, which has been shown to be relatively sensitive to high temperatures, may have been stressed by the temperatures at the MT site during the spring and as a result, exhibited delayed cell division. In September 2017, when fruit weight differences between olives grown at the HT site and those of the MT site were at their greatest, the number of cell layers was identical, but cell area was significantly greater at the MT site in all cultivars but the 'Barnea'. This suggests that unlike satsuma mandarins, the main effect of high temperatures on olive fruit weight is through repression of cell expansion and not cell division ## High temperature environment affects oil accumulation We found that the effect of a high temperatures environment on oil concentration is genotype dependent. In 2016, the average difference in maximum daily temperature between the HT and the MT site was 9.2˚C, and that of minimum daily temperature was 4˚C. In 2016 these temperature differences did not affect 'Barnea' and 'Coratina's' final oil concentration. However, the 'Koroneiki' cultivar of the same year had a final dry fruit oil concentration at harvest of 45.6% at the MT site and 29.1% at the HT site, which is an average of about a 1.79% decrease per degree of increased maximum daily temperature and 4.13% decrease per degree of increased minimum daily temperature. The 'Souri' cultivar had a 2.08% decrease per degree of increased maximum daily temperature and a 4.78% decrease per degree of increased minimum daily temperature. In 2017, decrease in oil concentration as a function of the severity of high temperatures, was more moderate than in 2016. However, just as in 2016, dry fruit oil concentration of 'Barnea' and 'Coratina' cultivars was not affected by the location of the trees and the resulting difference in temperatures, whereas the 'Koroneiki' and 'Souri' cultivars showed a decreased oil concentration at harvest in response to the higher temperatures at the HT location. 'Picholine' dry fruit oil concentration, like that of 'Barnea' and 'Coratina', was similar at both locations. Other studies have found that heat stress reduced oil concentration in sunflower hybrids by 6% [50]. However, corn which had undergone heat stress during grain fill, was found to have the same oil content as control plants . Olive oil concentration as a percentage of dry weight in 'Arauco' cultivars was found to decrease linearly at 1.1% per degree of increased temperature.. Our finding is also consistent with the results of Rondanini et al.which measured the oil accumulation of six olive cultivars, at three locations over two years and found a negative association between oil concentration and average temperature. The response to heat stress treatment of potted 'Coratina' and 'Arbequina' trees was found to reduce dry fruit weight by 0.34 and 0.22 g, respectively. In this study, fruit oil concentration (%) was 4.6 and 6.2% less on a dry-weight basis in fruit exposed to elevated temperatures. Higher temperature was found to promote vegetative growth but negatively affected oil concentration. Trentacoste et al.found that fruit oil concentration in 10 olive varieties decreased with increasing maximum daily temperature. It is accepted that oil accumulation begins only after pit hardening. However, while pit hardening occurs about 10 weeks after flowering, Matteucci et al.has shown that oil bodies appear in the mesocarp cells 7 weeks after flowering, and large oil droplets, derived from their fusion, are present in each cell 10 weeks after flowering. In accord with Matteucci et al., we also observed very clear oil bodies 50 days after flowering. In September, 146 days after flowering, oil drops were significantly larger at the MT site compared to the HT site in all five cultivars. At this stage, oil drops comprise between 3% to 17% of the cell volume. However, these values do not represent the true oil concentration in the mesocarp, because each cell may include many small oil drops that had not yet fused at the time the picture was taken. ## Differences between years of the experiment Although development patterns of the olive fruits were similar during both years of the study, the differences between those grown at the MT site and those at the HT site, were significant. Humidity, wind speed and rainfall were similar in 2016 and 2017 (S2 . However, the yearly average temperature difference between the HT and the MT site was higher in 2016 compared to 2017. Another result of our study indicates that during the course of the growing season, August is the month in which temperature has the greatest effect on fruit weight and oil accumulation, since during this month we found the greatest divergence between the two locations in 2016 and 2017 (S6 . ## High temperatures environment affects oil quality Among the effects of high temperatures on chemical parameters characterized in the current study, were total polyphenols and the fatty acid profile of olive oil. Polyphenol levels are known to decrease during fruit development . These levels are often positively correlated with water stress but in some cases show a divergent trend . Our analysis shows that a high temperature environment caused a decrease in the total polyphenol content of all analyzed cultivars. In 2016, total polyphenol level of oil from the HT site was approximately 55% of the level in oil from the MT site in all 3 analyzed cultivars. In 2017, total polyphenol level oil from the HT site was approximately 35% the level found in oil from the MT site in 'Barnea', 'Picholine' and 'Coratina'. However, in the 'Koroneiki' cultivar, polyphenol level at the HT site was 65% the level in the MT site, and in 'Souri', polyphenol level was very high in the HT site olives, reaching 772 mg/kg oil, compared to 905 mg/kg oil in the MT site. The decrease in total polyphenols in the HT site may also be explained by the differences in the irrigation regime. It is recognized that excess irrigation during fruit development results in lower phenolic concentrations in the olive oil due to changes in the biosynthetic and catabolic polyphenol pathways in the olive fruit [56-60]. The olive trees in the HT site got one third more water than those in the MT site; In our opinion, the difference between polyphenol levels at the two locations are too dramatic to be explained only by the level of irrigation . The total polyphenol levels in 2017 were higher than in 2016 and were very high compared to levels found in previous studies . This can be explained as an effect of climate change. Total polyphenol levels were dramatically lower in oil from the HT compared to the MT site in all cultivars except for the 'Souri', which as mentioned above, had a very high total polyphenol level at the MT site as well as at the HT. This might suggest that, at least in terms of oil quality, the 'Souri' cultivar is relatively tolerant to high temperatures. One of the major criteria of oil quality is its fatty acid composition. In accord with other studies on olive oil, in our study all cultivars showed a reduction in their oleic acid content in both years in response to the high temperature environment at the HT site. In sunflower oil, heat stress caused an increase in oleic acid content and a reduction of linoleic acid [50]. In contrast, it has been demonstrated that in the olive, high temperatures caused a decreased level of oleic acid and an increase of linoleic and palmitic acids. This may be partly explained by the gene expression level of genes involved in the oil biosynthesis pathway [61]. The cultivars 'Arbequina', 'Barnea', 'Koroneiki', 'Manzanillo' and 'Picual', showed lower level of oleic acid and higher level of linoleic acid in regions with high temperatures compared to the levels found in areas of mild temperatures. This study compared olive oil composition in Northern New South Wales and Southern Queensland, which is a warm area to Tasmania, a milder region. The 'Koroneiki' fatty acid composition was not analysed in Tasmania. However, the 'Barnea' and 'Coratina' showed a decrease in oleic acid content of 16.6 and 7.8% respectively in the warm region compared to the mild region. Our results showed smaller differences between oil extracted from the HT compare to the MT sites in oleic acid content. The temperatures during the 2 season analysed by Mailer et al.were not mentioned. However, the larger decrease in oleic acid content compared to our findings can be explained by larger differences in temperatures or in other agronomic parameters, since the sites in their experiment differed in many variables: they were cultivated by different farmers, possibly with different soil, water quality and many other parameters. The influence of temperatures during the growing season on the fatty acids composition of 188 Italian cultivars was studied between 2001 and 2005. Significantly lower oleic acid and higher palmitic and linoleic acids levels were found in the warmest year of the study (2003) compared to the coolest year (2005). The cultivars used in our study all exhibited, in both years of the trial, elevated levels of palmitic as well as linoleic acids as a result of exposure to the high temperatures of the HT site. According to the International Olive Council (IOC; http://www.internationaloliveoil.org), olive oil must contain 55-83% oleic acid and 3.5-21% linoleic acid. The oil of the 'Barnea' grown at the HT site in 2016 contained 23.34% linoleic acid and 'Picholine' oil from the HT site in 2017 contained 51.8% oleic acid and 21.62% linoleic acid. Thus, neither meets the standards of the IOC. Analysis of the oils extracted from the five cultivars in 2017, showed 'Souri' to have the smallest disparity in its oil composition between olives grown at the MT site compared to those from the HT site. Oleic acid in 'Souri' decreased by less than 4% and linoleic acid increased by only 1.5% at the HT compared to the MT site. These results, taken with those of total polyphenol levels, suggest that in regard to oil quality, the 'Souri' cultivar is more tolerant to high temperature environments than any of the other cultivars analyzed in this study. # Conclusions Our study demonstrates the negative effect of a high temperature environment on several key characteristics critical to the quality of olive oil. High temperature environments are shown to negatively influence fruit development as well as oil accumulation and thereby reduce yield. These high temperatures diminish oil quality by modifying fatty acid composition and causing a reduction of polyphenols and oleic acid, the most important components of olive oil. However, high temperature effects are genotype dependent and each cultivar responds differently to this stress. We found that each of the tested cultivars responded differently to high temperatures environment; none were completely heat tolerant. The 'Koroneiki' cultivar was negatively affected by high temperatures in all analyzed parameters. In the 'Picholine' and 'Coratina' cultivars, fruit development and oil quality were negatively affected by the high temperatures of the HT site, but oil concentration remained unaffected. The 'Souri' cultivar responded negatively to the high temperature environment with regard to fruit development and oil concentration, but was relatively tolerant to high temperatures in terms of oil quality. In contrast, in the 'Barnea' cultivar, exposure to high temperatures reduced oil quality, but did not impair final fruit weight or oil concentration. Although our results should be treated cautiously since the experiment was carried out for only two years, we found that the tested cultivars were tolerant regarding the effect of a high temperature environment on some of the traits examined, while exhibiting sensitivity to others. This suggests that the response to a high temperature environment is different for each of these traits and indicates induction of three different signal transduction mechanisms, resulting in a reduction of fruit development, oil accumulation and oil quality. Different olive cultivars have developed a variety of mechanisms to deal with different aspects of high temperature damage. Elucidation of the mechanism of each of these three responses is a vital step in the process of developing a variety of olives tolerant to high temperature damage. ## Supporting information
Antidiabetic Actions of Endogenous and Exogenous GLP-1 in Type 1 Diabetic Patients With and Without Residual β-Cell Function OBJECTIVE-To investigate the effect of exogenous as well as endogenous glucagon-like peptide 1 (GLP-1) on postprandial glucose excursions and to characterize the secretion of incretin hormones in type 1 diabetic patients with and without residual b-cell function.RESEARCH DESIGN AND METHODS-Eight type 1 diabetic patients with (T1D+), eight without (T1D2) residual b-cell function, and eight healthy matched control subjects were studied during a mixed meal with concomitant infusion of GLP-1 (1.2 pmol/kg/min), saline, or exendin 9-39 (300 pmol/kg/min). Before the meal, half dose of usual fast-acting insulin was injected. Plasma glucose (PG), glucagon, C-peptide, total GLP-1, intact glucose-dependent insulinotropic polypeptide (GIP), free fatty acids, triglycerides, and gastric emptying rate (GE) by plasma acetaminophen were measured.From the RESULTS-Incretin responses did not differ between patients and control subjects. Infusion of GLP-1 decreased peak PG by 45% in both groups of type 1 diabetic patients. In T1D+ patients, postprandial PG decreased below fasting levels and was indistinguishable from control subjects infused with saline. In T1D2 patients, postprandial PG remained at fasting levels. GLP-1 infusion reduced GE and glucagon levels in all groups and increased fasting C-peptide in T1D+ patients and control subjects. Blocking endogenous GLP-1 receptor action increased endogenous GLP-1 secretion in all groups and increased postprandial glucose, glucagon, and GE in T1D+ and T1D2 patients. The insulinogenic index (the ratio of insulin to glucose) decreased in T1D+ patients during blockade of endogenous GLP-1 receptor action. CONCLUSIONS-Type 1 diabetic patients have normal incretin responses to meals. In type 1 diabetic patients, exogenous GLP-1 decreases peak postprandial glucose by 45% regardless of residual b-cell function. Endogenous GLP-1 regulates postprandial glucose excursions by modulating glucagon levels, GE, and b-cell responsiveness to glucose. Long-term effects of GLP-1 in type 1 diabetic patients should be investigated in future clinical trials. Diabetes 60:1599-1607, 2011 A t time of diagnosis and during the first year, prevalence of residual b-cell function in patients with type 1 diabetes is nearly 100% [bib_ref] Prevalence of residual B cell function and its metabolic consequences in type..., Madsbad [/bib_ref]. After alleviation of initial hyperglycemia with exogenous insulin, patients enter a remission period with improved b-cell function, where insulin treatment can be paused in up to 20-30% of the patients without loss of target glycemic control [bib_ref] Natural course of remission in IDDM during 1st yr after diagnosis, Martin [/bib_ref]. Persistence of residual insulin secretion is associated with reduced risk of ketosis (4), lower HbA 1c levels [bib_ref] Residual insulin production, glycaemic control and prevalence of microvascular lesions and polyneuropathy..., Sjöberg [/bib_ref] , lower insulin doses, less risk of hypoglycemia, and reduced long-term complications [bib_ref] Correlation between minimal secretory capacity of pancreatic b-cells and stability of diabetic..., Fukuda [/bib_ref]. However, after disease duration of 5-10 years, the prevalence of residual b-cell function has declined to about 15% [bib_ref] Prevalence of residual B cell function and its metabolic consequences in type..., Madsbad [/bib_ref]. Even though lack of insulin is considered to be the most important factor for the hyperglycemia in type 1 diabetic patients, other metabolic disturbances may also play a role: the glucagon response to carbohydrate and protein ingestion has been shown to be abnormal [bib_ref] Abnormal alpha-cell function in diabetes. Response to carbohydrate and protein ingestion, Müller [/bib_ref] and there is evidence that postprandial hyperglycemia is because of lack of insulin as well as inappropriately elevated glucagon levels [bib_ref] The essential role of glucagon in the pathogenesis of diabetes mellitus, Unger [/bib_ref]. The gut hormone, glucagon-like peptide 1 (GLP-1), reduces glucagon levels, increases insulin secretion [bib_ref] The influence of GLP-1 on glucose-stimulated insulin secretion: effects on b-cell sensitivity..., Kjems [/bib_ref] , and inhibits gastric emptying rate (GE), thereby reducing postprandial glucose excursions [bib_ref] The physiology of glucagon-like peptide 1, Holst [/bib_ref]. The insulinotropic and the glucagonostatic properties of GLP-1 are glucose dependent [bib_ref] Effects of glucagon-like peptide 1 on counterregulatory hormone responses, cognitive functions, and..., Nauck [/bib_ref] , and exogenous GLP-1, therefore, does not produce hypoglycemia. Several studies have found lowering of fasting and postprandial glucose by GLP-1 or GLP-1 agonists in type 1 diabetic patients with [bib_ref] Glucagon-like peptide I reduces postprandial glycemic excursions in IDDM, Dupre [/bib_ref] [bib_ref] Subcutaneous glucagonlike peptide I combined with insulin normalizes postcibal glycemic excursions in..., Dupré [/bib_ref] [bib_ref] Effects of exenatide alone and in combination with daclizumab on b-cell function..., Rother [/bib_ref] as well as without [bib_ref] Glucagon-like peptide 1 improved glycemic control in type 1 diabetes, Behme [/bib_ref] [bib_ref] Glucagonostatic actions and reduction of fasting hyperglycemia by exogenous glucagon-like peptide I(7-36)..., Creutzfeldt [/bib_ref] [bib_ref] Exendin-4 normalized postcibal glycemic excursions in type 1 diabetes, Dupré [/bib_ref] [bib_ref] Antidiabetogenic effect of glucagon-like peptide-1 (7-36)amide in normal subjects and patients with..., Gutniak [/bib_ref] residual b-cell function. Some studies suggested that the glucose lowering effect was because of the enhancement of insulin sensitivity [bib_ref] Antidiabetogenic effect of glucagon-like peptide-1 (7-36)amide in normal subjects and patients with..., Gutniak [/bib_ref] , whereas others concluded that delay of gastric emptying [bib_ref] Glucagon-like peptide I reduces postprandial glycemic excursions in IDDM, Dupre [/bib_ref] [bib_ref] Subcutaneous glucagonlike peptide I combined with insulin normalizes postcibal glycemic excursions in..., Dupré [/bib_ref] or reduction of glucagon levels [bib_ref] Glucagonostatic actions and reduction of fasting hyperglycemia by exogenous glucagon-like peptide I(7-36)..., Creutzfeldt [/bib_ref] was the most important mechanism. In animal studies, treatment with GLP-1 or GLP-1 agonists has been shown to delay diabetes development or reverse recent onset diabetes in NOD mice [bib_ref] Treatment of type 1 diabetic patients with glucagon-like peptide-1 (GLP-1) and GLP-1R..., Kielgast [/bib_ref] , ascribed to an improved function of existing b-cells rather than through increments in b-cell mass. However, there is also evidence that GLP-1, in combination with gastrin, increases b-cell mass and restores normoglycemia in recent onset diabetic NOD mice [bib_ref] Combination therapy with glucagon-like peptide-1 and gastrin restores normoglycemia in diabetic NOD..., Suarez-Pinzon [/bib_ref] and that GLP-1 combined with gastrin is able to expand b-cell mass of human islets implanted under the renal capsule of immunodeficient diabetic NOD mice [bib_ref] Combination therapy with glucagon-like peptide-1 and gastrin induces beta-cell neogenesis from pancreatic..., Suarez-Pinzon [/bib_ref]. In freshly isolated human islets, GLP-1 has been reported to inhibit b-cell apoptosis [bib_ref] Glucagon-like peptide 1 inhibits cell apoptosis and improves glucose responsiveness of freshly..., Farilla [/bib_ref]. However, in C-peptide-positive subjects with longstanding type 1 diabetes treated with exenatide for 6 to 9 months with or without daclizumab, insulin dose was significantly reduced, primarily because of the reduction of prandial insulin, but b-cell function was not improved [bib_ref] Effects of exenatide alone and in combination with daclizumab on b-cell function..., Rother [/bib_ref]. Four weeks of treatment with vildagliptin (a DPP-4 inhibitor that increases endogenous GLP-1 levels) in 11 well-controlled type 1 diabetic patients with longstanding disease decreased postmeal glucagon and glucose levels [bib_ref] Effect of vildagliptin on glucagon concentration during meals in patients with type..., Foley [/bib_ref] , and in adolescents with minimal or no endogenous insulin secretion treated with exenatide, postprandial glucose excursions were reduced despite 20% reduction of insulin dose [bib_ref] The role of adjunctive exenatide therapy in pediatric type 1 diabetes, Raman [/bib_ref]. Therefore, GLP-1-based therapies have potential for treatment of type 1 diabetes alone or-more likely-in combination with insulin. Because of controversies regarding secretion of incretin hormones in type 1 diabetes [bib_ref] Effect of nutrient ingestion on glucagon-like peptide 1 (7-36 amide) secretion in..., Lugari [/bib_ref] [bib_ref] Incretin secretion in relation to meal size and body weight in healthy..., Vilsbøll [/bib_ref] , assessment of the meal-related GLP-1 secretory responses in patients with diabetes is of interest [bib_ref] Secretion of glucagonlike peptide-1 (GLP-1) in type 2 diabetes: what is up,..., Nauck [/bib_ref]. We therefore studied incretin secretion as well as the antidiabetic actions of both endogenously secreted and exogenously infused GLP-1 during a mixed meal in type 1 diabetic patients with and without residual b-cell function. ## Research design and methods Subjects. Sixteen type 1 diabetic Caucasian patients and eight healthy control subjects were studied. Eight patients had residual b-cell function (T1D+), and eight were without (T1D2). All diabetic subjects were diagnosed between 8 and 40 years old; all were normal or underweight at diagnosis and were treated with multiple daily insulin injections from time of diagnosis. None had other diseases affecting glucose metabolism, overt diabetes complications, or symptoms of autonomic nerve dysfunction. Control subjects were healthy, had a normal oral glucose tolerance test, and no family history of diabetes. Patients and control subjects were matched with respect to age, sex, and BMI. T1D+ and T1D2 patients had similar HbA 1c , age, and BMI, but T1D2 patients had a longer duration of diabetes (10.6 6 2.2 vs. 3.2 6 0.7 years [mean 6 SEM; P , 0.05]) and required more insulin (47.4 6 3.5 vs. 31.9 6 3.6 IE/day [mean 6 SEM, P , 0.05]). Subject characteristics are presented in [fig_ref] TABLE 1: Data are mean 6 SEM [/fig_ref]. The protocol was approved by the Ethical Committee for Region Hovedstaden and the Danish Data Protection Agency and conducted according to the principles of the Helsinki Declaration 2. Glucagon test. To assess residual b-cell function, a glucagon stimulation test (GST) was performed at blood glucose levels above 7 mmol/L. Fasting plasma C-peptide concentrations were measured before and 6 min after intravenous bolus-injection of 1 mg glucagon. Patients were classified as T1D+ when stimulated plasma C-peptide concentration was $200 pmol/L and T1D2 if below the detection limit of 0.1 ng/mL (;33 pmol/L) (See [fig_ref] TABLE 1: Data are mean 6 SEM [/fig_ref]. Experimental protocol. Patients were investigated after an overnight fast (10 h). The night before, they injected normal dose of long acting insulin. An individual difference in fasting plasma glucose of no more than 3 mmol/L between study days was allowed. Cannulas were inserted in forearms: one for infusions and one for blood sampling with arterialization of the venous blood using the heated hand technique. After basal blood sampling, GLP-1 (1.2 pmol/kg/min), saline, or the GLP-1 receptor antagonist exendin 9-39 (Ex9-39; 300 pmol/kg/min) was infused for 3 h. This dose of Ex9-39 blocks 95% of the insulinotropic and completely antagonizes the glucagonostatic effects of physiological levels of GLP-1 [bib_ref] Exendin(9-39)amide is an antagonist of glucagon-like peptide-1(7-36)amide in humans, Schirra [/bib_ref]. Infusions were randomized and single blinded. Thirty minutes after infusion start (time zero), participants consumed a mixed meal of 2.036 KJ (;485 kcal: 23.6% protein, 15.2% fat, and 45.9% carbohydrates). One gram of acetaminophen was added to the yogurt, which was part of the meal. The whole meal was eaten within 20 min, and the yogurt was eaten within the first 10 min. Before the meal, patients injected half-dose of their usual fast acting insulin (Novorapid was injected at time zero, and Actrapid at time 215 min). For each subject, insulin dose, injection site, as well as exact time until meal completion were identical on the three study days. Blood samples for analysis of plasma glucose, C-peptide, insulin, free fatty acids (FFA), triglycerides (TG), glucagon, intact GIP, total GLP-1, and Ex9-39 were drawn at 245, 240, 235, 230 (infusion start), 220, 210, 0 (meal served), 10, 20 (meal finished), 30, 40, 50, 60, 75, 90, 105, 120, 150, and 180 min. Analytical methods. Plasma glucose was analyzed bedside with an ABL800 blood gas analyzer (Radiometer, Copenhagen, Denmark). C-peptide responses to the GST were analyzed with solid-phase two-site chemiluminiscent immunometric assay (Immulite 2000), with a detection limit of 0.1 ng/mL (;33 pmol/L). Plasma C-peptide during the meal test was measured by autoDELPHIA automatic flouroimmunoassay (Wallac, Turku, Finland), with a detection limit of 17 pmol/L. Blood was collected in chilled heparinized tubes and centrifuged immediately, and plasma was held on ice and stored at 280°C until analyzed. Blood samples for glucagon, total GLP-1, and intact GIP were sampled in tubes containing EDTA and DPP-4 inhibitor (valpyr). The antiserum of glucagon assay (code no. 4305) is directed against the C-terminus of glucagon and reacts specifically with pancreatic glucagon [bib_ref] Proglucagon products in plasma of noninsulin-dependent diabetics and nondiabetic controls in the..., Orskov [/bib_ref]. Unexpectedly, we observed weak cross-reaction with Ex9-39 in concentrations above 10 27 mol/L. All glucagon samples were therefore also measured with the LINCO assay (Milipore, Billerica, MA), which did not cross-react with Ex9-39 at all (own results) but had a poorer sensitivity. Total GLP-1 concentrations were measured using antiserum no. 89390, reacting equally with intact GLP-1 (7-36) amide and its primary N-terminally truncated metabolite GLP-1 (9-36) amide [bib_ref] Tissue and plasma concentrations of amidated and glycine-extended glucagon-like peptide I in..., Orskov [/bib_ref]. Intact GIP was measured using antiserum no. 98171, reacting with the N-terminus of GIP, but not with the metabolite, GIP 3-42 [bib_ref] Degradation of endogenous and exogenous gastric inhibitory polypeptide in healthy and in..., Deacon [/bib_ref]. Detection limits and intra-assay coefficients were 1 and 2 pmol/L and less than 6% for GLP-1 and GIP, respectively. Ex9-39 was measured in EDTA plasma using antibody 3145 obtained from rabbits immunized with exendin 4. It shows 100% cross-reactivity with Ex9-39 but ,0.01% cross-reactivity with GIP, glucagon, or GLP-1. Intraassay coefficient of variation is ,6%, with a detection limit of 0.5 pmol/L. Serum non-esterified FFA and TG were measured by oxidase colorimetric methods (Wako Chemicals, Neuss, Germany; and products VITROS Chemistry, Ortho-Clinical Diagnostics, Buckinghamshire, U.K.). Acetaminophen was analyzed by fluorescence polarization immunoassay technology (Abbott Laboratories, Abbot Park, IL). Insulin secretion rates. For T1D+ patients and control subjects, prehepatic insulin secretion rates (ISR) were calculated through deconvolution of peripheral C-peptide concentrations using two-compartment model of C-peptide kinetics [bib_ref] ISEC: a program to calculate insulin secretion, Hovorka [/bib_ref] [bib_ref] Measuring pre-hepatic insulin secretion using a population model of C-peptide kinetics: accuracy..., Hovorka [/bib_ref] and population-based C-peptide kinetics with adjustment for metabolic parameters such as age, sex, and BMI [bib_ref] Estimation of insulin secretion rates from C-peptide levels. Comparison of individual and..., Van Cauter [/bib_ref]. ISR is expressed in units of picomoles 3 kilograms 21 3 minutes 21 . Insulinogenic index. Insulinogenic index was calculated as the ratio of incremental integral ISR and plasma glucose from start of infusion (230 min) until termination of the 3-h meal test (180 min), (ISR/PG210) during saline and Ex9-39 infusion, respectively. (2) and with (+) residual b-cell function and control subjects. GAD-65, glutamic acid decarboxylase-65; ICA, islet cell antibody. *P , 0.05 vs. T1D+. calculating the incremental values. Normally distributed data were evaluated using Student t test; paired within groups and unpaired between groups and nonnormally distributed data using nonparametric tests. Three or more datasets were evaluated using one-way ANOVA between groups and repeatedmeasurements ANOVA within groups. Differences resulting in P values # 0.05 were considered statistically significant. # Results GIP. Data are presented in -C and [fig_ref] TABLE 2: Postprandial incretin responses related to infusion type [/fig_ref]. With saline, baseline intact GIP (iGIP) concentrations were 14-18 pmol/L and did not differ within or between the three groups on either study day, and peak plasma concentrations were 82 6 8, 67 6 7, and 85 6 8 pmol/L in the T1D2, T1D+, and control subjects, respectively (P 5 0.2). When compared with saline, plasma concentration of iGIP was significantly decreased in all three groups during GLP-1 infusion but did not change under Ex9-39 infusion [fig_ref] TABLE 2: Postprandial incretin responses related to infusion type [/fig_ref]. Total AUC 0-180 min of iGIP during saline was 9. [fig_ref] TABLE 2: Postprandial incretin responses related to infusion type [/fig_ref]. When compared with saline, Ex9-39 significantly increased secretion of GLP-1, but the GLP-1 response achieved during Ex9-39 did not differ between groups (P = 0.6) [fig_ref] TABLE 2: Postprandial incretin responses related to infusion type [/fig_ref]. Exendin 9-39. Data are presented in . On Ex9-39 days, plasma levels of Ex9-39 increased to 60 nmol/L in all three groups before the meal was served, corresponding to levels where insulinotropic effects of physiological GLP-1 levels was completely abolished [bib_ref] Glucagon-like peptide 1 has a physiological role in the control of postprandial..., Edwards [/bib_ref]. Ex9-39 levels continued to increase reaching plateau levels of 100-120 nmol/L about 120 min after the meal was served. Total AUC 0-180 min of Ex9-39 did not differ between groups; 16.8 6 1.0, 17.3 6 2.5, and 19.7 6 1.3 nmol/L 3 min in T1D2, T1D+, and control subjects, respectively (P = 0.5). There was no correlation between Ex9-39 and glucagon concentrations. Glucose. Data are presented in -C and . Within each group, baseline plasma glucose (PG) did not differ between the 3 study days, but were higher in both diabetic groups compared with control subjects. During saline, peak PG were 6.9 6 0.2 mmol/L in control subjects and 14.2 6 0.9 and 16.2 6 1.5 mmol/L in T1D+ and T1D2 patients, respectively, but with GLP-1 infusion, peak PG decreased to 8. . Ex9-39 significantly increased total and incremental AUC 230 to 180 min in T1D+ patients, but not in T1D2 patients or control subjects . However, compared with saline, Ex9-39 significantly increased total AUC 02180 min from 2.53 6 0.23 to 2.90 6 0.21 (P , 0.05) and from 2.19 6 0.14 to 2.46 6 0.14 (P , 0.01) mol/L 3 min in T1D2 and T1D+ patients, respectively, whereas no differences were found in control subjects. Glucagon , and described below. Glucagon data measured by the LINCO assay (no cross-reaction with Ex9-39) are presented in [fig_ref] TABLE 1: Data are mean 6 SEM [/fig_ref] With saline, neither incremental nor total AUC 230 to 180 min differed between groups (P = 0.9 and P = 0.2, respectively; . When compared with saline, GLP-1 infusion decreased total and incremental AUC 230 to 180 min of glucagon in all three groups and Supplementary Data). The increase was mainly due to increments in postprandial levels; total AUC 0-180 min increased to 3.38 6 0.26 (P , 0.001), 4.40 6 0.69 (P , 0.001), and 2.74 6 0.21 (P = 0.018) (vs. saline) in T1D2, T1D+, and control subjects. The supplementary glucagon data (without cross-reaction with Ex9-39) shows that Ex9-39 significantly increased total and incremental AUC of glucagon in both groups of type 1 diabetic patients and increased incremental AUC 230 to 180 min in control subjects. C-peptide. C-peptide data are presented in -I and . None of the T1D2 patients displayed C-peptide levels above 33 pmol/L. During GLP-1 infusion, total and incremental AUC 230 to 180 min of C-peptide decreased significantly in T1D+ patients In control subjects, C-peptide showed the same pattern as in T1D+ patients; GLP-1 decreased total AUC 230 to 180 min , but increased fasting incremental AUC 230 to 0 min of C-peptide from 20.61 6 1.13 (saline) to 7.68 6 2.7 (GLP-1) nM 3 min (P , 0.01). There were no significant differences in fasting or postprandial C-peptide responses between Ex9-39 and saline, but C-peptide tended to be lower during Ex9-39 . ISR. In T1D+ patients, Ex9-39 tended to decrease incremental AUC 230 to 180 min of ISR from 0.28 6 0.9 to 0.22 6 0.1 nmol 3 kg 21 (P = 0.06; vs. saline) but no difference was found in the control subjects (P = 0.5). However, in T1D+ patients, the insulinogenic index, calculated as the ratio of incremental integral ISR and PG from 230 to 180 min (ISR/PG210), decreased with Ex9-39: 2.09 6 0.48, and 4.61 6 1.22 nM 3 min (P , 0.01, P , 0.01, and P , 0.05 vs. saline) in T1D2, T1D+, and control subjects, respectively. Time-to-peak increased to 159 6 14, 140 6 17, and 150 6 15 min (P , 0.05 compared with saline). Ex9-39 decreased time-to-peak by about 20 min in all three groups, but the difference reached significance only in the T1D2 group. However, when the two diabetic groups were considered together, Ex9-39 shifted AUC 0-180 min from 7.9 6 0.3 to 8.6 6 0.8 nmol/L 3 min (P = 0.046) and time-to-peak from 61 6 7 to 42 6 5 min (P = 0.02 compared with saline). Effect of endogenous and exogenous GLP-1 on plasma lipids. Data are presented in [fig_ref] FIG. 4: A-C [/fig_ref] -F. Fasting TG concentrations tended to be higher in the healthy subjects (1.1 6 0.2 mmol/L) than in T1D+ (0.7 6 0.09 mmol/L) and T1D2 (0.6 6 0.08 mM) patients, on the saline day (P = 0.08). Neither GLP-1 nor Ex9-39 infusions significantly affected TG concentrations. Fasting FFA did not differ within or between groups (fasting FFA ;0.4-0.5 mmol/L) except for T1D+ subjects on the day assigned to Ex9-39 where fasting FFA levels were almost twice as high (;0.7 mmol/L). FFA concentrations decreased in all groups after ingestion of the meal regardless of infusion type. # Discussion We conclude that incretin responses to a mixed meal test are similar between type 1 diabetic patients with as well as without residual b-cell function and healthy subjects. This is in contrast with results from Lugari et al. [bib_ref] Effect of nutrient ingestion on glucagon-like peptide 1 (7-36 amide) secretion in..., Lugari [/bib_ref] , who reported absence of postprandial GLP-1 responses in type 1 diabetes, but in agreement with data from Vilsbøll et al. [bib_ref] Incretin secretion in relation to meal size and body weight in healthy..., Vilsbøll [/bib_ref] , who found incretin responses in type 1 diabetic patients equal to that of lean healthy controls. We have no clear explanation for this discrepancy other than differences in methodology or subject characteristics [bib_ref] Secretion of glucagonlike peptide-1 (GLP-1) in type 2 diabetes: what is up,..., Nauck [/bib_ref]. Our study confirms that chronic hyperglycemia in lean type 1 diabetic patients in otherwise good metabolic control does not decrease L-cell secretory response to a mixed meal. Blocking the effect of endogenous GLP-1 with Ex9-39 increased postprandial GLP-1 consistent with earlier findings in healthy subjects [bib_ref] Endogenous glucagon-like peptide 1 controls endocrine pancreatic secretion and antro-pyloro-duodenal motility in..., Schirra [/bib_ref] and in type 2 diabetic patients [bib_ref] Effect of endogenous GLP-1 on insulin secretion in type 2 diabetes, Salehi [/bib_ref]. This could theoretically be the result of 1) increases in GE (with more nutrients reaching the intestine and the L-cells [bib_ref] Emptying of the gastric substitute, glucagon-like peptide-1 (GLP-1), and reactive hypoglycemia after..., Miholic [/bib_ref] , 2) blockade of a negative feedback system between the GLP-1 and the L-cell [bib_ref] Somatostatin restrains the secretion of glucagon-like peptide-1 and -2 from isolated perfused..., Hansen [/bib_ref] , 3) changes in plasma clearance of GLP-1 (because the GLP-1 receptor [GLP-1R] is expressed in tubular cells of the kidneys and might be involved in the very high renal extraction of GLP-1 [bib_ref] Glucagon-like peptide 1 undergoes differential tissue-specific metabolism in the anesthetized pig, Deacon [/bib_ref] , and 4) cross-reactions with Ex9-39 in the GLP-1 assay as previously described [bib_ref] Glucagon-like peptide 1 has a physiological role in the control of postprandial..., Edwards [/bib_ref] , but this was excluded in the current study. During infusion of saline, GIP increased to the same extent in the three groups but was clearly decreased during GLP-1 infusion, presumably because of delayed GE. GLP-1 infusion, despite a 50% dose-reduction of usual fast-acting prandial insulin, effectively reduced postprandial glucose-excursions in T1D+ and T1D2 patients. In T1D+ patients postprandial glucose decreased and became indistinguishable from those of healthy subjects receiving saline, and in T1D2 patients, it remained at fasting values. This clearly antidiabetic effect is in accordance with a previous study [bib_ref] Antidiabetogenic effect of glucagon-like peptide-1 (7-36)amide in normal subjects and patients with..., Gutniak [/bib_ref] , where GLP-1 infusion decreased the isoglycemic meal-related insulin requirement by 50%, which was thought to be because of increased glucose utilization as well as a decrease in glucagon release. However, another study did not find evidence of an Hormone responses [bib_ref] Effect of glucagon-like peptide 1 (7-36 amide) on insulin-mediated glucose uptake in..., Meneilly [/bib_ref]. In yet another study of type 1 diabetic patients with residual b-cell function who omitted their prandial insulin, postprandial glucose elevations were completely prevented without stimulation of endogenous insulin, whereas glucagon and pancreatic polypeptide (PP) were suppressed [bib_ref] Glucagon-like peptide I reduces postprandial glycemic excursions in IDDM, Dupre [/bib_ref]. Six to nine months of exenatide treatment in patients with longstanding type 1 diabetes and measurable C-peptide levels decreased preprandial insulin by 30% but did not affect b-cell function or glucagon levels [bib_ref] Effects of exenatide alone and in combination with daclizumab on b-cell function..., Rother [/bib_ref]. The patients' C-peptide level in that study was relatively small (mean stimulated of about 270 pmol/L). Our T1D+ patients had stimulated C-peptide levels about twice as high, which could explain the difference in effect of GLP-1 treatment on glucose metabolism. However, we also found a strong GLP-1-induced glucose lowering effect in our T1D2 patients. The remaining b-cells in our T1D+ patients responded well to GLP-1 stimulation, as clearly shown in the premeal period (where PG levels were still above 6 mmol/L), and improved insulin secretion may have contributed to the reduction of postprandial glucose excursions. It is possible that the glucose lowering effect observed here-regardless of residual insulin secretionis mainly caused by the combination of reduced glucagon levels and inhibition of GE. The observation that DPP-4 inhibition reduces postprandial glucose excursions in type 1 diabetic patients through b-cell independent suppression of glucagon (24) but has no or little effect on GE (44), supports a particular role for glucagon suppression. Blocking the effect of endogenous GLP-1 decreased postprandial (0-180 min) C-peptide level in the T1D+ patients despite slightly elevated PG values, suggesting that endogenously secreted GLP-1 might affect b-cell responsiveness to glucose. Accordingly, Ex9-39 significantly decreased the insulinogenic index (PG/ISR210) in the T1D+ patients and increased postprandial glucagon levels most pronounced in the patients with diabetes. Thus endogenous GLP-1 seems to participate in the regulation of postprandial glucagon secretion in type 1 diabetic patients and enhances the b-cell responsiveness to glucose during a mixed meal. The absence of an effect of GLP-1R blockade on glucose excursions and b-cell function in control subjects may be because of the low glycemic index of the meal resulting in modest glucose excursions and therefore reduced insulinotropic effects of incretin hormones. Furthermore, because insulinotropic effect of GIP is severely impaired in type 1 diabetes (45), but explains about half of the incretin effect in healthy humans [bib_ref] Both GLP-1 and GIP are insulinotropic at basal and postprandial glucose levels..., Vilsbøll [/bib_ref] , Ex9-39 (which only antagonizes GLP-1, but does not affect insulinotropic action of GIP) might abolish the incretin effect relatively more in the patients as observed. As expected, GLP-1 strongly decreased GE in all groups accompanied by a reduction of GIP secretion. Ex9-39 accelerated GE in type 1 diabetic patients but did not significantly affect GE rate in control subjects. Using the same antagonist but scintigraphy for detection of emptying rates, Deane et al. [bib_ref] Endogenous glucagon-like peptide-1 slows gastric emptying in healthy subjects, attenuating postprandial glycemia, Deane [/bib_ref] found accelerated GE in healthy humans, influencing glucose absorption and postprandial glycemia. Therefore, insufficient statistical power may be partly responsible for the insignificant effect observed here. Furthermore, in healthy subjects and in type 1 diabetic patients, hyperglycemia decelerates GE, whereas hypoglycemia increases it [bib_ref] Insulin-induced hypoglycemia accelerates gastric emptying of solids and liquids in long-standing type..., Russo [/bib_ref] [bib_ref] Hypoglycaemia increases the gastric emptying rate in patients with type 1 diabetes..., Schvarcz [/bib_ref] [bib_ref] Hypoglycemia increases the gastric emptying rate in healthy subjects, Schvarcz [/bib_ref] [bib_ref] Physiological hyperglycemia slows gastric emptying in normal subjects and patients with insulin-dependent..., Schvarcz [/bib_ref] , and differences in glucose levels by infusion type may therefore indirectly have affected GE in our patients. FFA decreased during the meal in all three groups regardless of infusion type, but we have no explanation for the elevated fasting FFA in the T1D+ patients on the Ex9-39 day other than natural variation. It has been shown that treatment with GLP-1 and DPP-4 inhibitors reduces intestinal lipid production and absorption, preventing postprandial rise in TG [bib_ref] One-year treatment with exenatide improves b-cell function, compared with insulin glargine, in..., Bunck [/bib_ref] [bib_ref] Vildagliptin therapy reduces postprandial intestinal triglyceride-rich lipoprotein particles in patients with type..., Matikainen [/bib_ref]. In our study, TG was unaffected by the meal as well as by infusion type, except for a weak (nonsignificant) trend of lower postprandial values during GLP-1 infusion in all groups. The tendency of lower TG levels in the patients compared with control subjects is probably the result of exogenous insulin [bib_ref] One-year treatment with exenatide improves b-cell function, compared with insulin glargine, in..., Bunck [/bib_ref]. In a recent study, GLP-1R signaling was found to be essential for the control of postprandial lipoprotein biosynthesis and secretion at least in rodents (54), but our results suggest that this may not apply to humans. Conclusions. We conclude that type 1 diabetic patients have normal incretin responses to a mixed meal and that infusion of GLP-1 at the rate we used decreases peak postprandial glucose by approximately 45% in type 1 diabetic patients regardless of b-cell function. Endogenously secreted GLP-1 plays a role in the regulation of postprandial glucose excursions in type 1 diabetes by modulating glucagon levels, GE rate, and b-cell responsiveness to glucose. We suggest that long term effects of GLP-1-based therapies should be investigated in future clinical trials of type 1 diabetic patients with as well as without residual b-cell function. [fig] 6: 6 1.3 and 7.5 6 0.7 mmol/L in T1D2 and T1D+ patients and to 5.8 6 0.2 mmol/L in the control subjects (P , 0.01 vs. saline for all three groups). GLP-1 infusion decreased peak PG by 45.4 6 17.7% in the T1D2 patients and 45.1 6 16.2% in the T1D+ patients compared with saline. Peak PGs in both diabetic groups were similar to control subjects receiving saline (P = 0.3). GLP-1 also significantly decreased total and incremental AUC 230 to 180 min of glucose in all three groups, and in both groups of patients, total AUC 230 to 180 min did not significantly differ from control subjects receiving saline [/fig] [fig] .: Glucagon data measured by the sensitive RIA (weak cross-reaction with Ex9-39) are presented in FIG 1 A-C: Intact GIP (pM) D-F: Total GLP-1 (pM) during infusion with GLP-1 (red symbols), saline (gray symbols), and Ex9-39 (blue symbols) in T1D2 (■), T1D+ (•), and control subjects (▲) during a mixed meal test Solid arrow: start of infusion; open arrow: meal start T1D2: type 1 diabetic patients without residual b-cell function; T1D+: type 1 diabetic patients with residual b-cell function [/fig] [fig] Figure 3D -: F, [/fig] [table] TABLE 1: Data are mean 6 SEM. Characteristics of type 1 diabetic patients without [/table] [table] TABLE 2: Postprandial incretin responses related to infusion type [/table]
Mobile laminar air flow screen for additional operating room ventilation: reduction of intraoperative bacterial contamination during total knee arthroplasty Background Surgical site infections are important complications in orthopedic surgery. A mobile laminar air flow (LAF) screen could represent a useful addition to an operating room (OR) with conventional turbulent air ventilation (12.5 air changes/h), as it could decrease the bacterial count near the operating field. The purpose of this study was to evaluate LAF efficacy at reducing bacterial contamination in the surgical area during 34 total knee arthroplasties (TKAs). Materials and methods The additional unit was used in 17 operations; the LAF was positioned beside the operating table between two of the surgeons, with the air flow directed towards the surgical area (wound). The whole team wore conventional OR clothing and the correct hygiene procedures and rituals were used. Bacterial air contamination (CFU/m 3 ) was evaluated in the wound area in 17 operations with the LAF unit and 17 without the LAF unit. Results The LAF unit reduced the mean bacterial count in the wound area from 23.5 CFU/m 3 without the LAF to 3.5 CFU/m 3 with the LAF (P \ 0.0001), which is below the suggested limit for an OR with ultraclean laminar ventilation. There were no significant differences in the mean bacterial count in the instrument table area: 28.6 CFU/m 3 were recorded with the LAF (N = 6) unit and 30.8 CFU/m 3 (N = 6) without the LAF unit (P = 0.631). During six operations with LAF and six without LAF, particle counts were performed and the number of 0.5 lm particles was analyzed. The particle counts decreased significantly when the LAF unit was used (P = 0.003). Conclusion When a mobile LAF unit was added to the standard OR ventilation, bacterial contamination of the wound area significantly decreased to below the accepted level for an ultraclean OR, preventing SSI infections. # Introduction Surgical site infections (SSI) represent one of the most common complications in surgery. In particular, deep periprosthetic infections in orthopedic surgery constitute a disaster for both patient and surgeon. Conservative estimates of infection rates average 1-2% for hip implants and 2-4% for knee implants [bib_ref] Evaluation and treatment of infection at the site of a total hip..., Hanssen [/bib_ref] [bib_ref] The outcome of perioperative wound infection after total hip and knee arthroplasty, Abudu [/bib_ref]. The number of joint replacements is expected to double in the next 20 years, and if the infection rate is not reduced, the incidence of infection will also double, yielding increased morbidity, hospital stays, and costs for the healthcare system [bib_ref] Evaluation of measures to decrease intra-operative bacterial contamination in orthopaedic implant surgery, Knobben [/bib_ref] [bib_ref] Health and economic impact of surgical site infections diagnosed after hospital discharge, Perencevich [/bib_ref]. Periprosthetic infection rates have been shown to correlate with the number of airborne bacteria within 30 cm of the wound [bib_ref] Airborne contamination of wounds in joint replacement operations: the relationship to sepsis..., Lidwell [/bib_ref]. This is influenced by several factors relating to either the surgical environment (number of operating theater personnel, their clothing, type of ventilation system used) or the surgical procedure employed (approach, duration of exposure, use of a tourniquet). The source of pathogens can be the patient (endogenous infection), the bacteria present in the OR air, instruments used, or the surgeon's hands (exogenous infection). However, it is generally accepted that the main cause of surgical site infections (SSIs) after clean operations is bacterial contamination of the OR air, predominantly from contaminated skin scales shed by the surgical team, instruments used, or the surgeon's hands [bib_ref] The importance of airborne bacterial contamination of wounds, Whyte [/bib_ref] [bib_ref] The addition of a mobile ultra-clean exponential laminar airflow screen to conventional..., Friberg [/bib_ref] [bib_ref] A mobile laminar air flow unit to reduce air bacterial contamination at..., Pasquarella [/bib_ref]. Small numbers of organisms, including those of low pathogenicity, can cause orthopedic implant infections, and give rise to a considerable degree of morbidity and also mortality. It has been estimated that as few as ten colony forming units (CFU) are sufficient to cause deep infection in a prosthetic replacement arthroplasty. Bacteria that cause infection in the joint after total hip or knee replacement are inoculated into the wound at the time of insertion of the prosthesis [bib_ref] The importance of airborne bacterial contamination of wounds, Whyte [/bib_ref] [bib_ref] The effect of ultraclean air in operating rooms on deep sepsis in..., Lidwell [/bib_ref] [bib_ref] Predicting bacterial populations based on airborne particulates: a study performed in nonlaminar..., Stocks [/bib_ref]. The number of airborne bacteria in the OR is also dependent on the number of people present as well as their activities and behavior. Use of a ventilation system and appropriate personnel dress and discipline are therefore ways to reduce air contamination in the OR [bib_ref] Evaluation and treatment of infection at the site of a total hip..., Hanssen [/bib_ref] [bib_ref] The outcome of perioperative wound infection after total hip and knee arthroplasty, Abudu [/bib_ref] [bib_ref] Health and economic impact of surgical site infections diagnosed after hospital discharge, Perencevich [/bib_ref] [bib_ref] The addition of a mobile ultra-clean exponential laminar airflow screen to conventional..., Friberg [/bib_ref] [bib_ref] A mobile laminar air flow unit to reduce air bacterial contamination at..., Pasquarella [/bib_ref] [bib_ref] The effect of ultraclean air in operating rooms on deep sepsis in..., Lidwell [/bib_ref] [bib_ref] Mobile zoned/exponential AF screen: a new concept in ultra-clean air technology for..., Friberg [/bib_ref] [bib_ref] Importance of air quality in and related factors in the prevention of..., Gosden [/bib_ref] [bib_ref] Environmental controls in operating theatres, Dharan [/bib_ref] [bib_ref] A review of air distribution patterns in surgery rooms under infection control..., Pereira [/bib_ref] [bib_ref] Behaviours and rituals in the operating theatre. A report from the Hospital..., Woodhead [/bib_ref] [bib_ref] The index of microbial air contamination, Pasquarella [/bib_ref] [bib_ref] Bacterial contamination of air and surgical wounds during joint replacement operations. Comparison..., Scheibel [/bib_ref] [bib_ref] Ultraclean air and antibiotics for prevention of postoperative infection. A multicenter study..., Lidwell [/bib_ref] [bib_ref] Molecular epidemiology of microbial contamination in the operating room environment: is there..., Edmiston [/bib_ref] [bib_ref] Prosthetic osteomyelitis with special reference to the knee: risks, treatment and costs, Bengtson [/bib_ref] [bib_ref] The economic impact of infected joint arthroplasty, Sculco [/bib_ref] [bib_ref] The impact of surgical-site infections following orthopedic surgery at Community Hospital and..., Whitehouse [/bib_ref] [bib_ref] Current concept for clean air anf total joint arthroplasty: laminar airflow and..., Evans [/bib_ref]. A laminar air flow ventilation system is recommended for an OR where orthopedic implant surgery is performed [bib_ref] The effect of ultraclean air in operating rooms on deep sepsis in..., Lidwell [/bib_ref]. Unfortunately, LAF systems are very expensive and complicated to install in a pre-existing OR. The introduction of a mobile LAF screen to complement the use of a standard ventilation system could be an effective but inexpensive way to decrease bacterial air contamination, as noted by Friberg et al. [bib_ref] The addition of a mobile ultra-clean exponential laminar airflow screen to conventional..., Friberg [/bib_ref] [bib_ref] Mobile zoned/exponential AF screen: a new concept in ultra-clean air technology for..., Friberg [/bib_ref] and Pasquarella et al. [bib_ref] A mobile laminar air flow unit to reduce air bacterial contamination at..., Pasquarella [/bib_ref]. The aim of this study was to evaluate the efficacy of LAF at reducing bacterial contamination in the surgical area during orthopedic implant surgery in an OR with conventional turbulent air ventilation. # Materials and methods ## Surgery This study focused on 34 total knee replacements carried out over a period of two months. All operations were performed in the same OR, early in the morning, and by the same surgeon; all cases received spinal anesthesia, tourniquet, and standard short-term antibiotic prophylaxis. The additional LAF screen was used in 17 operations, while the remaining 17 operations were performed under ordinary conditions without the additional LAF screen. ## Operating room The experiments were performed within a standard OR (&120 m 3 ) equipped with a conventional turbulent ventilation system (with 12.5 air changes/h). Mean thermohygrometric parameters were: temperature, 20.6°C (0.1); relative humidity, 44.6% (3.1) [fig_ref] Table 1: OR data [/fig_ref] ; . ## Additional laf screen The additional LAF screen used in the study (Toul-400, Toul Meditech, Vasteras, Sweden) is a mobile unit that produces ultraclean exponential laminar air flow in a predefined area (the wound in our case). The additional mobile unit is a box with a fan and a HEPA filter (CAMFIL type, 99.997% particles [0.3 lm). The screen produces a laminar air flow of 0.5-0.7 m/s onto the wound and 0.4 m/s at the periphery. This exponential air flow prevents the entrainment of OR air outside the LAF [bib_ref] The addition of a mobile ultra-clean exponential laminar airflow screen to conventional..., Friberg [/bib_ref] [bib_ref] A mobile laminar air flow unit to reduce air bacterial contamination at..., Pasquarella [/bib_ref]. This air flow is turbulence-free and not impeded by the movements of the surgical team in the defined air flow area. The existing regular ventilation system does not affect the functioning of the unit. A camera assists in determining the direction of air flow, and an integrated sensor determines the correct distance from the surgical site for maximum effect. An integrated display allows the user to easily check and verify the setup (Toul Meditech data). The LAF screen was positioned beside the operating table and between the two surgeons, with the air flow directed towards the surgical area, as shown in . ## Surgical team The surgical team consisted of a chief surgeon, an assistant surgeon, and two residents; there were also one chief anesthetist, one resident, one scrub nurse, one room nurse, and a technician. The team numbered between six and eight during all 34 operations. Each member of the team wore conventional OR clothing during all operations: the surgeons, scrub nurse, and technician wore sterile nonwoven gowns, facemasks, surgical caps, and sterile gloves; the anesthetist and room nurse wore a woven OR uniform (shirt and trousers), facemask, surgical cap, and nonsterile gloves. # Sampling methods Air contamination (in CFU/m 3 ) was studied during all operations using a Biotest (Rockaway, NJ, USA) RCS Plus sampler (50 l/min) using Biotest HYCON agar strips TC-c (c-irradiated Total Count Tryptic Soy agar). The sampler was located 30 cm from the wound. Air counts were performed during 17 operations with the LAF screen and 17 operations without the LAF. Air quality on the instrument table was also investigated in six operations with and without the LAF unit. Sampling periods were always 20 min, and 1 m 3 of air was sampled, as suggested by ISPESL guidelines ;. The agar strips were incubated for 48 h at 37°C before counting the CFU, and the results were expressed in CFU/m 3 [fig_ref] Table 3: Bacterial air contamination [/fig_ref]. Particle counts were performed in the wound area during 12 operations (six with the LAF screen and six without) at the same time as the bacteria count using the Biotest (Dreieich, Germany) APC Plus (2.8 l/min). The sampling periods were again 20 min ;. Results were expressed in particles/m 3 , and ISO values for 0.5 lm particles were considered when interpretating the results . Statistical analysis SPSS (Statistical Package for Social Sciences) was used for statistical evaluations. The Mann-Whitney U test was used to establish significant differences between means. P B 0.02 was regarded as significant. # Results Mean bacterial air contamination in the wound area was 23.5 CFU/m 3 under standard ventilation conditions; when the LAF unit was added to the standard ventilation, the mean bacterial count in the wound area decreased to 3.5 CFU/m 3 (P \ 0.0001), a reduction of about 85%, which is below the accepted limit (\10 CFU/m 3 ) for ultraclean laminar ventilation [bib_ref] The importance of airborne bacterial contamination of wounds, Whyte [/bib_ref] [bib_ref] The effect of ultraclean air in operating rooms on deep sepsis in..., Lidwell [/bib_ref] [bib_ref] Importance of air quality in and related factors in the prevention of..., Gosden [/bib_ref]. In the instrument table area, the mean bacterial air contamination was almost the same whether or not the LAF unit was used (P = 0.631 = ns): 28.6 CFU/m 3 with the LAF unit and 30.8 CFU/m 3 without the LAF [fig_ref] Table 3: Bacterial air contamination [/fig_ref]. When the LAF unit was used, there was a significant correlation between bacterial air contamination on the wound and instrument table (P = 0.0004); without the screen, no significant correlation was found (P = 0.361). The mean numbers of 0.5 lm particles in the wound area and the instrument table area are shown in . Without the LAF screen, the mean value in the wound area was 970,533 particles/m 3 ; upon adding the LAF screen, this mean was reduced to 17,361 particles/m 3 (P = 0.003). In the instrumental table area, the mean particles/m 3 value ranged from 1,224,367 with the LAF unit to 1,380,181 without the LAF unit (P = 0.521 = ns). No significant correlation was found between the particles/m 3 values in the wound area and the instrument table area when the LAF unit was not used (P = 0.262); but when the LAF unit was added, a significant correlation was observed (P = 0.004). # Discussion Many studies have demonstrated a correlation between airborne bacterial contamination and postoperative joint sepsis in arthroplasty surgery [bib_ref] Airborne contamination of wounds in joint replacement operations: the relationship to sepsis..., Lidwell [/bib_ref] [bib_ref] The importance of airborne bacterial contamination of wounds, Whyte [/bib_ref] [bib_ref] The effect of ultraclean air in operating rooms on deep sepsis in..., Lidwell [/bib_ref] [bib_ref] Importance of air quality in and related factors in the prevention of..., Gosden [/bib_ref] [bib_ref] Molecular epidemiology of microbial contamination in the operating room environment: is there..., Edmiston [/bib_ref]. Correct rituals, surgical clothing, and the use of ultraclean laminar ventilation are strongly recommended in orthopedic implant surgery in order to reduce postoperative SSIs and respect the accepted limit of 10 CFU/m 3 in the wound area for an ultraclean OR . We tested the efficacy of using an LAF unit in addition to a conventional air ventilation system in an implant surgery OR (12.5 air changes/h). We studied the effect of the screen on bacterial OR contamination during 34 total knee replacement operations. During the experiments, the surgical team respected the OR rituals and hygiene procedures and wore proper surgerical clothing. In our study, we used an active sampling method, as suggested in the ISPESL guidelines. Bacterial sampling was performed in the wound area and the instrument table area to get an indication of bacterial contamination levels present under standard ventilation conditions and when the LAF screen is also used. We also decided to perform particle sampling in the same places and at the same time as the bacterial sampling during 12 operations (six with LAF and six without LAF), as an additional indicator of LAF unit efficacy. The results suggested that the LAF screen is very effective at reducing bacterial contamination; the CFU/m 3 value in the wound area was below the accepted limit for an ultraclean OR: the contamination in the wound area dropped from 23.5 CFU/m 3 under standard ventilation conditions to 3.5 UFC/m 3 when LAF was used, which is well below the limit of 10 CFU/m 3 accepted for ultraclean laminar ventilation [bib_ref] The importance of airborne bacterial contamination of wounds, Whyte [/bib_ref] [bib_ref] The effect of ultraclean air in operating rooms on deep sepsis in..., Lidwell [/bib_ref] [bib_ref] Importance of air quality in and related factors in the prevention of..., Gosden [/bib_ref] and that of the UK (NHS) standard HTM 2025, which states that a limit of 20 CFU/m 3 should not be exceeded in an OR with ultraclean laminar ventilation during surgical operations . This reduction in the CFU/m 3 value in the wound area is statistically significant. Bacterial contamination of the instrument table area did not change upon adding the LAF screen: it was 28.6 CFU/m 3 with the LAF unit and 30.8 CFU/m 3 without the LAF unit; no significant correlation between the level of contamination and whether LAF was functioning was observed, meaning that the influence of the LAF unit was limited to the focal area (in this case the wound area). The count of 0.5 lm particles in the wound area dropped from 970,533 particles/m 3 when LAF was added to 17,361 particles/m 3 when LAF was not working. This means that, for the wound area, the OR complied with ISO Class 8 standard conditions, and with ISO Class 6 standard conditions when the LAF unit was used. In the instrument table area, the particles count complied with ISO Class 8 conditions. In conclusion, the prevention of infection is preferable to treatment in terms of both patient outcome and cost of treatment [bib_ref] Health and economic impact of surgical site infections diagnosed after hospital discharge, Perencevich [/bib_ref] [bib_ref] Prosthetic osteomyelitis with special reference to the knee: risks, treatment and costs, Bengtson [/bib_ref] [bib_ref] The economic impact of infected joint arthroplasty, Sculco [/bib_ref] [bib_ref] The impact of surgical-site infections following orthopedic surgery at Community Hospital and..., Whitehouse [/bib_ref]. Employing an additional ultraclean LAF unit reduced bacterial contamination and bacteriacarrying airborne particles in the surgical area (the wound) during total knee replacement operations. The CFU/m 3 value in the wound area was reduced to below the limit suggested for implant surgery performed in an OR with ultraclean laminar ventilation. A complete ultraclean LAF ventilation system is very expensive, and can sometimes be impossible to install in pre-existing premises without extensive rebuilding [bib_ref] Evaluation of measures to decrease intra-operative bacterial contamination in orthopaedic implant surgery, Knobben [/bib_ref] [bib_ref] The addition of a mobile ultra-clean exponential laminar airflow screen to conventional..., Friberg [/bib_ref]. Employing an additional LAF screen could be an interesting, effective, and inexpensive complement to OR standard ventilation when laminar air flow is required; in other words, for high-risk surgery (implant surgery, neurosurgery, transplant surgery), and in situations with insufficient ventilation or clothing facilities to reduce bacterial contamination and prevent SSI infections [bib_ref] The addition of a mobile ultra-clean exponential laminar airflow screen to conventional..., Friberg [/bib_ref]. ## Conflict of interest none. Ethical standards The authors state that the study conforms to the Declaration of Helsinky as revised in 2008. All the patients provided informed consent to be involved in the study. No ethical commitee evaluation was requested since the research focussed on the environment of the Operating Room, not on the patients, who received a standard-of-care treatment. Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credited. [table] Table 1: OR data: relative humidity and temperature are expressed as mean (standard deviation) [/table] [table] Table 3: Bacterial air contamination (CFU/m 3 ) in the wound area and the instrument area with and without an additional LAF screen; mean (standard deviation) values and significant differences are indicated in boldTable 4Particle counts (0.5 lm) in the wound area and instrument area with and without the additional LAF screen; mean (standard deviation) values and significant differences are shown in bold Particle count (0.5 lm/m 3 ) of the: [/table]
A multimodal approach to improve asthmatic adolescents’ self-efficacy in Taiwan Background:An efficient asthma self-management for adolescents must be based on adolescents' needs, increase self-efficacy and adherence to treatment. The effects of such program are likely be dose dependent.Aim:To examine the impact of the dose-effect of multiple components on an asthma self-management program for adolescents aged 12-18 years in Taiwan.Methods: A scoring system was developed to classify intervention groups into high-(19-23), medium-(11-18) and low (< 11) dose according to the number of components completed by participants. The impacts of the dose level on outcomes of asthma self-efficacy, prevention behaviors, asthma medication adherence, and asthma symptoms were examined.Results/Conclusion:Our results suggest that a high dose of the intervention can improve adolescents' self-efficacy, asthma prevention behavior, and medication adherence. # Introduction Adolescents aged 10-14 y with asthma have a lower adherence to prescribed medications compared with other age groups and this can lead to poorer health outcomes and more emergency department (ED) visits. [bib_ref] Treatment adherence in adolescents with asthma, Kaplan [/bib_ref] National Taiwan statistics show an ED visit rate of 14.1% in adolescents, with only 9.7% of the young people using bronchodilators when having an asthma attack compared with a 1.6% ED visit rate and 60% of adults, respectively. Effective asthma self-management for adolescents must be based on adolescents' needs, increase self-efficacy and adherence to treatment, reduce exacerbations and improve overall quality of life. The effects of such programs are likely be dose (the quantity and strength of the program delivered) dependent. 4 # Methods A secondary data analysis of a randomized controlled trial 5,6 dataset was undertaken to evaluate the effects of intervention dose. Recruited adolescents aged 12-18 y were randomly allo-cated to either the control or the intervention group. The endpoints of asthma management self-efficacy, preventive behaviors and asthmatic symptoms were measured by the Self-efficacy and Prevention Behavior Index 3 and Asthma Control Test questionnaires. The outcome of asthma medication adherence was examined via patients' self-report diaries. A scoring system was developed and used to categorize participants (n=83) into three groups (high-, medium-and low-dose) according to completing different components of the intervention. Patients who completed all the components had a total dose score of 23 (12+2+8+1) comprising: 4×3 times face-toface meetings; 2×1 telephone consultation; 1×8 text messages; and/or 1×1 booklet. The intervention dose was categorized into high (19-23), medium (11-18) and low (<11) according to the number of components completed. An ANOVA was used to determine differences in continuous variables between the low-, medium-and high-dose groups. For data that were not normally distributed (i.e. medication adherence), the Kruskal-Wallis test was used. Categorical variables were compared with a χ 2 test. # Results and discussion A total of 83 patients completed the study. There were no significant differences in demographic data among the three groups. Average time to asthma diagnosis was 6.09 y. The majority of adolescents were male (n=47, 56%, p=0.18), with a median age (IQR=3) of 14 y (p=0.37). The median time to diagnosis with asthma was 6.0 (IQR=5), 6.5 (IQR=7.5) and 4.5 (IQR=7) y for participants in the low-, medium-and high-dose Asthma Self-Management Program (ASMP), respectively (p=0.60). The results examined by ANOVA revealed that participants who completed all components (high dose) had significantly greater self-efficacy, prevention behaviors and medication adherence than the low-dose group (see for the mean differences). This is an improvement over previously implemented approaches that failed to improve asthma self-management in adolescents. The high-dose group demonstrated adolescents' improved self-confidence (F (2, 80)=3.7, p=0.03) in carrying out asthma self-management behaviors (F (2, 80)=7.04, p=0.002). It is likely that varied channels used in ASMP enhanced patients' learning interests and subsequently enabled behavioral changes. The results of adherence to asthma medication from the Kruskal-Wallis test showed that there was a statistically significant effect of ASMP dose on medication adherence (χ 2 =10.6, p<0.01), with a mean rank of asthma adherence of 38.4 for the low dose of ASMP, 36.2 for the medium dose of ASMP and 50.7 for the high dose of ASMP. Pairwise comparisons were made to determine which pairs of groups differed significant. The only significant difference in medication adherence among the groups was between patients who had received a high dose of ASMP and those who had received a low dose (U=333.5, p<0.01). The scores of the asthma control test measuring asthmatic symptoms among the three dose groups after completion of the intervention (χ 2 =5.07, p=0.08) suggested that the ASMP did not produce any significant improvement . The results suggest that multiple delivery modes (face-toface, telephone follow-ups and text messages) can contribute to effective preventive behaviors. The face-to-face meeting encourages interactions between facilitators and participants; therefore, it is more likely to have an influence on promoting commitment to behavior change. 4 An additional point of concern noted was the length of time to asthma diagnosis (median 5 y). This highlights a need for further research and resources for young people to be diagnosed and learn effective self-management techniques in a timely manner. # Limitations Some limitations must be noted. First, there were the potential accumulative effects of the intervention. Our study reported the dose effect of a multicomponent ASMP. However, comparisons of outcomes from individual components (face-to-face, telephone or text messages) were not examined. Thus, there is the possibility of overestimating the effect of the intervention. Second, participants who did not receive all components of ASMP may have reported fewer outcomes. Third, age-stratified analysis was not conducted, and the outcome of adherence may vary between a 12-and an 18-y-old participant. Another limitation acknowledged is the dependence on self-reported diaries of medication adherence. Patients in the low or medium group may be adhering to their medication regimen, but not reporting adherence in their diaries. T.-J. Tseng and C.-J. Wu ## Practical implications High dose levels through multiple components resulted in an increase in self-efficacy, preventive behaviours and treatment adherence. It is important to note that as long as the medium dose was reached, asthma preventive behaviors were positively impacted. Hence, it is recommended to aim for high dose levels of intervention in the planning phase of delivering asthma selfmanagement programs for adolescents, in order to reach at least a medium dose level for the majority of participants in the implementation phase. # Conclusion Our results suggest that a high dose of ASMP can improve adolescents' self-efficacy, asthma prevention behavior and medication adherence. [table] International Health Table 1: Mean differences in self-efficacy, preventive behavior, symptoms of asthma and asthma medication adherence [/table]
Ecology of blood stream infection and antibiotic resistance in intensive care unit at a tertiary care hospital in North India a b s t r a c tObjective: To analyse the prevalent microorganisms and their antimicrobial resistance among intensive care unit patients in a tertiary care centre in New Delhi.Methods: A retrospective study of all consecutive blood cultures from various intensive care unit patients in the hospital during four years (January 2008 to December 2011). Antibiotic consumption data in the intensive care units were also analysed during the same period.Results: Out of the total 22,491 blood cultures processed, 2846 samples were positive and 3771 microorganisms were isolated. The blood culture positivity was estimated as 12.7% of which 67.5% were monomicrobial and 32.5% polymicrobial infections. Gram negative bacilli, Gram positive cocci, and fungi were isolated in 49%, 33%, and 18% cases, respectively.Coagulase negative staphylococcus was the commonest single isolate followed by Candida spp. A drastic shift in the distribution of Candida spp. towards nonalbicans along with high resistance to azole group of antifungals suggest echinocandins for the empiric therapy of candidemia. High penicillin resistance in Gram positive isolates suggest vancomycin, linezolid and tigecycline as the options for empiric therapy, whereas tigecycline and colistin are the only options remaining for highly resistant Gram negative isolates. Aminoglycosides were observed to have better sensitivity and reduced usage when compared with cephalosporins and ␤-lactam + ␤-lactam inhibitor combinations.Conclusions: High frequencies of multidrug resistant organisms were observed in intensive care units which is a warning as to use the only few effective antimicrobials wisely to reduce selective pressure on sensitive strains. # Introduction Infections caused by multidrug resistant bacteria constitute a serious problem for intensive care patients throughout appropriate antimicrobials, which in turn can reduce the length of stay as well as the morbidity and mortality in ICU patients. [bib_ref] Blood stream infection in the ICU, Valles [/bib_ref] The paradox remains where the antibiotics are needed the most (ICU), there the highest resistance, is observed due to multiple reasons. Random use of high-end antimicrobials has the risk of eventually selecting out mutants that are multidrug resistant. Every hospital must recognize the epidemiology of its microorganisms to recommend initial appropriate presumptive antimicrobial therapy. The application of hospital-wide antibiograms to guide clinicians in the initial choice of antimicrobials is the usual approach adopted. If more resistant organisms are isolated from ICU patients, this important information would be masked by the use of hospital-wide antibiogram. Here we have made an attempt to analyse the prevalent microorganisms causing blood stream infection (BSI) and their antimicrobial resistance as seen among the ICU patients in a tertiary care centre in New Delhi. # Materials and methods ## Patients This retrospective study was conducted for a period of four years (January 2008 to December 2011) in a 650-bed tertiary care centre in New Delhi. A total of 22,491 blood samples were analysed from the patients admitted in various ICUs in the hospital, which include general adult ICU, coronary care unit (CCU), surgical ICU, and the liver, kidney and bone marrow transplant ICUs. ## Sample collection and processing Five to 10 mL of blood sample was collected and were inoculated immediately into BacT/ALERT culture bottles (bioMerieux, Durham, North Carolina, USA) under complete aseptic conditions and were processed as per the manufacturer's specifications. ## Identification and susceptibility testing of isolates Positive blood culture isolates were identified using VITEK 2 automated system (bioMerieux, Durham, North Carolina). MIC was confirmed using E-test (bioMerieux, France) in case of VRE. In case of any discrepancy E-test result was taken as final. Susceptibility testing was performed by Kirby Bauer method for tigecycline (15 g, Oxoid Ltd., Basingstoke, Hampshire, England), and colistin (10 g, HiMedia Laboratories, Mumbai) and results interpreted as per Clinical Laboratory Standards Institute (CLSI) guidelines 2 and British society for Antimicrobial Chemotherapy (BSAC) guidelines where ever CLSI guidelines were not available.Daptomycin MIC was determined for Staphylococcus aureus and Enterococcus spp. using E-test during the period of 2010 and 2011. Antibiotic resistance data were extracted from the hospital information system (Intersystems, Cambridge, MA, USA) using a software Speedminer (Petaling Jaya, Malaysia) and analysed using a customized software, Wattal-Protech, Delhi, India. ## Screening for carbapenem resistance Because of the observation of high carbapenem resistance among Enterobacteriaceae at our facility, we tried to assess the magnitude of different mechanisms of carbapenem resistance. All consecutive isolates of Enterobacteriaceae were screened for carbapenem resistance by modified Hodge test (MHT) during the period of December 2010 to April 2011. All MHT positive isolates were further tested for metallo-␤lactamase (MBL) by MBL E-test strips (bioMerieux, France). Further confirmation of all MBL positive isolates for New Delhi Metallo-beta-lactamases (NDM-1) was done using a real time polymerase chain reaction (PCR) assay for the detection of the bla NDM-1 using Taq Man probes. 4 ## Antifungal susceptibility testing Antifungal susceptibility testing was performed against amphotericin B, 5-flucytosine, fluconazole, itraconazole, and voriconazole by broth microdilution using API system (ATB FUNGUS 3, bioMerieux, France), during 2008. For isolates wherein API system was not recommended by the manufacturer, sensitivity was done using E-test for amphotericin B, fluconazole, and voriconazole. During 2009-2011, antifungal susceptibility testing was done using VITEK 2 automated system except for caspofungin, which was done using E-test. Results were interpreted as per the CLSI guidelines. 5 ## Antimicrobial consumption data Four years antimicrobial consumption data were evaluated from the antibiotic dispensing data from the hospital information system. Customized software Speedminer was used to extract the data from the hospital information system. The amount of antimicrobial drug in grams was converted into the number of defined daily doses (DDDs)/100 bed-days using ABC calc. 6 # Ethical considerations The study was approved by the hospital ethics committee (reference number EC/09/12/414 and approval letter dated 31/01/2013). Consent was waived since this was an anonymised study with retrospective evaluation. # Results A total of 2846 samples were positive from 22,491 blood cultures received from the study group. The blood culture positivity rate in our ICUs was estimated to be 12.7%. A total of 3771 microorganisms were isolated from 2846 episodes of BSIs. 67.5% (1921/2846) of infections were monomicrobial, while 32.5% (925/2846) were polymicrobial [fig_ref] Figure 1 -: Distribution of the various microorganisms from blood culture from ICU patients [/fig_ref]. Analysis of these isolates showed that the commonest single isolate among our ICU patients was coagulase negative staphylococcus (CoNS) (20.3%) followed by Candida spp. (17.5%). Gram negative bacilli (GNB), Gram positive cocci (GPC), and fungi were isolated in 49%, 33%, and 18% cases, respectively. Among GNB, Klebsiella spp. was the commonest ## Antimicrobial susceptibility testing Patterns of antimicrobial susceptibility for the major pathogens are shown in [fig_ref] Table 1 -: Percentage resistance of antimicrobial agents in the Gram negative bacilli [/fig_ref]. The evidence generated here indicates that antibiotics like penicillins and most cephalosporins would not be effective against Gram positive and negative bacteria in the ICU. For highly resistant bacteria, especially Klebsiella spp. and Acinetobacter spp., which were also the commonest gram negative isolates in our ICUs even ␤-lactam + ␤-lactam inhibitor (BL + BLI) combinations and carbapenems were not effective. Amikacin was slightly better and the only remaining effective antibiotics were tigecycline and colistin. ## Carbapenem resistant enterobacteriaceae A total of 34 Enterobacteriaceae isolates (7 Escherichia coli and 27 Klebsiella pneumoniae) were subjected for carbapenemase screening. The overall prevalence of carbapenemase production (MHT positive isolates), MBLs (MBL E-test positive isolates) and KPC (MHT positive and MBL negative) was 52.9%, 41.2% and 11.8%, respectively. Interestingly all the phenotypically MBL positive isolates, tested for NDM-1 by PCR were positive, implying 100% concordance between the phenotypic presence of MBLs and presence of bla NDM-1 . Fourteen K. pneumoniae (51.9%) and no E. coli isolates were observed to be NDM-1 producers. The amount of antimicrobials used during the same time period is given in [fig_ref] Table 3 -: Antimicrobial use during 2008-2011 [/fig_ref]. The distribution of various candida species and their antifungal susceptibility pattern are given in . # Discussion Keeping in view the source of patients to our ICU being from all over the city and other parts of the country including tertiary care centres, this ecology could be representative of most of the ICUs in the country. In our study, coagulase negative staphylococci were the most common blood culture isolate (20.3%) [fig_ref] Figure 1 -: Distribution of the various microorganisms from blood culture from ICU patients [/fig_ref]. Some other studies in our country (Mukherjee et al., 61%) as well as from other countries (Japoni et al., 67.7%, Karlowsky et al., 42%) also observed CoNS as the commonest blood stream isolate. [bib_ref] Nosocomial infections in geriatric patients admitted in ICU, Mukherjee [/bib_ref] [bib_ref] Multidrug-resistant bacteria isolated from intensive-care-unit patient samples, Japoni [/bib_ref] [bib_ref] Prevalence and antimicrobial susceptibilities of bacteria isolated from blood cultures of hospitalized..., Karlowsky [/bib_ref] In a review by Valles et al. who reviewed more than five recent publications from different parts of the world also observed CoNS as the commonest microorganism (20-30%) causing BSIs among ICU patients. [bib_ref] Blood stream infection in the ICU, Valles [/bib_ref] Appropriate antimicrobial therapy and control measures could be adopted to prevent cross infection of multidrug-resistant CoNS bacteria from patient-to-patient and ICU environment. Moreover, CoNS isolates are often skin colonizers and appear in blood culture as common contaminants at the time of sample collection. [bib_ref] The clinical significance of positive blood cultures in the 1990s: a prospective..., Weistein [/bib_ref] Meticulous skin disinfection at the time of venepuncture can limit this potential contaminant species to a large extent. Clinical correlation of the blood culture isolate and determination of time to positivity of CoNS isolates can help to differentiate between potential contaminants and true pathogens. 11,12 -Percentage resistance of common candida isolates to antifungals. Considering the fact that CoNS isolated from blood cultures are often contaminants, [bib_ref] The clinical significance of positive blood cultures in the 1990s: a prospective..., Weistein [/bib_ref] Candida spp. (17.5%) was the commonest single isolate in the current study [fig_ref] Figure 1 -: Distribution of the various microorganisms from blood culture from ICU patients [/fig_ref]. Studies have proven the various risk factors associated with candida infection in ICU patients which include exposure to multiple antibiotics, indwelling catheters, parenteral nutrition, previous surgery and presence of a solid organ malignancy. [bib_ref] Nosocomial candidemia risk factors, Diaz [/bib_ref] Candida remains a major cause of BSIs in ICU patients worldwide. [bib_ref] Secular trends in candidemia-related hospitalizations in the US, Zilberberg [/bib_ref] [bib_ref] Candidemia in Norway (1991 to 2003): results from a nationwide study, Sandven [/bib_ref] In fact a major increase in candida BSI started during the 1980s. [bib_ref] Secular trends in nosocomial primary blood-stream infections in the United States, 1980-1989..., Banerjee [/bib_ref] According to the surveillance data from the US Centers for Disease Control and Prevention (CDC), candida accounts for 12% of all hospital-acquired BSIs. [bib_ref] Antimicrobial-resistant pathogens associated with healthcare-associated infections: annual summary of data reported to..., Hidron [/bib_ref] Another interesting observation is the shift in the distribution of Candida spp. responsible for BSI to non-albicans species. In our study Candida albicans accounted for only 15.3% of candida BSIs. This epidemiologic trend has also been observed in many studies in different parts of the world. [bib_ref] Epidemiologic trends in nosocomial candidemia in intensive care, Bassetti [/bib_ref] [bib_ref] Epidemiology of candidemia in intensive care units, Bouza [/bib_ref] This could be attributed to the increased use of fluconazole as observed in another study in our hospital. [bib_ref] Evolution of non-albicans Candida species in blood stream infections in a tertiary..., Oberoi [/bib_ref] Even though C. tropicalis, the commonest candida isolate in our ICUs still remains susceptible to most of the antifungals, C. haemulonii, the 2nd commonest isolate, has shown very high resistance to fluconazole and even to amphotericin B. In other studies also, C. haemulonii has shown increased MICs and frank resistance to both amphotericin B and fluconazole and has resulted in clinical failure. [bib_ref] Infections due to Candida haemulonii: species identification, antifungal susceptibility and outcomes, Ruan [/bib_ref] Our susceptibility data suggests that reduced susceptibility to fluconazole is common in Candida glabrata and Candida parapsilosis, and it may prove simpler in clinical practice to use an alternative agent to treat candidaemia due to these species. Reduced susceptibility to fluconazole in C. glabrata was consistent with previously reported data. [bib_ref] One year prospective survey of Candida bloodstream infections in Scotland, Odds [/bib_ref] [bib_ref] A retrospective analysis of antifungal susceptibilities of Candida bloodstream isolates from Singapore..., Tan [/bib_ref] In contrast, C. parapsilosis has usually been reported to be sensitive to azoles. Enterococcus spp. also was observed as an important pathogen in our study (8.4%). Enterococci form part of the normal flora of the gastrointestinal tract and female genitourinary tract. Over the past decade, vancomycin resistant enterococci (VRE) have emerged as a leading cause of nosocomial infections due to its spread by direct patient-to-patient contact and by indirect transmission via hospital personnel, environmental surfaces and hospital equipment. In our study, 23% of enterococci were VRE, while Japoni et al. reported 10.5% VRE among blood stream isolates in ICU. [bib_ref] Multidrug-resistant bacteria isolated from intensive-care-unit patient samples, Japoni [/bib_ref] A study by the CDC also reported high prevalence of VRE (29%), similar to our study, among the enterococcal infections in ICU settings.Still vancomycin can serve as an important antibiotic in our ICU to control CoNS, S. aureus and streptococcal infections. In the case of S. aureus and CoNS, 47% and 94% of isolates were resistant to methicillin respectively. Our findings are similar to other studies that report an MRSA rate in ICU varying between 47 and 65%. [bib_ref] Prevalence and antimicrobial susceptibilities of bacteria isolated from blood cultures of hospitalized..., Karlowsky [/bib_ref] [bib_ref] Risk factors for ICU-acquired methicillin-resistant Staphylococcus aureus infections, Oztoprak [/bib_ref] [bib_ref] Abd-Elsayed AA. Nosocomial blood stream infection in intensive care units at Assiut..., Ahmed [/bib_ref] All the MRSA and VRE isolates in our study were sensitive to vancomycin/teicoplanin, linezolid, tigecycline as well as daptomycin. Penicillin resistance was observed in 16% (five isolates) of S. pneumoniae isolates, all of which were having an MIC in the moderately sensitive range (MIC 0.25-1 g/mL). The rate of resistance was similar to some other studies in India quoting 5-18% penicillin resistance. [bib_ref] Prospective multicenter hospital surveillance of Streptococcus pneumonia disease in India. Invasive Bacterial..., Lalitha [/bib_ref] [bib_ref] Antimicrobial resistance in invasive and colonizing Streptococcus pneumoniae in North India, Goyal [/bib_ref] [bib_ref] Reporting emerging resistance of Streptococcus pneumoniae from India, Chawla [/bib_ref] There was no frank penicillin resistance or multidrug resistance, unlike some other studies from India. [bib_ref] Antimicrobial resistance in invasive and colonizing Streptococcus pneumoniae in North India, Goyal [/bib_ref] [bib_ref] Reporting emerging resistance of Streptococcus pneumoniae from India, Chawla [/bib_ref] All the isolates were sensitive to ceftriaxone and quinolones. Gram negative bacteria caused more than half of the BSIs in the current study of ICUs. This observation in our study is in agreement with many other studies from India as well as from different parts of the world, even though the distribution of various Gram negative pathogens tend to vary among the different ICUs. [bib_ref] Microbial isolates from patients in an intensive care unit, and associated risk..., Tennant [/bib_ref] [bib_ref] Prospective surveillance of nosocomial device-associated becteremia in three adult intensive units in..., Gopal Katherason [/bib_ref] This study documents the extent of drug resistance among the frequently isolated Gram negative isolates from intensive care patients. An interesting observation of this analysis is the comparatively better sensitivity of aminoglycoside against the majority of GNB. Similar observations of better aminoglycoside sensitivity (50-60%) among respiratory isolates was observed by other authors as well. [bib_ref] Changing trend of antimicrobial resistance among Gram-negative bacilli isolated from lower respiratory..., Gagneja [/bib_ref] [bib_ref] The study of prevalence and antimicrobial susceptibility of tracheal bacterial strains isolated..., Jafari [/bib_ref] High resistance to cephalosporins and BL + BLI combinations observed in our study might be due to the selective influence of extensive usage of these antimicrobials as compared to aminoglycoside (see [fig_ref] Table 3 -: Antimicrobial use during 2008-2011 [/fig_ref]. Our results show a high prevalence of carbapenem resistance among non-fermenters, higher in Acinetobacter spp. (83%) followed by Pseudomonas aeruginosa (67%). Carbapenem resistance observed in Acinetobacter isolates was much higher compared to some recent studies in ICUs where the resistance is reported to be 16-17%. [bib_ref] Multidrug-resistant bacteria isolated from intensive-care-unit patient samples, Japoni [/bib_ref] [bib_ref] Microbial isolates from patients in an intensive care unit, and associated risk..., Tennant [/bib_ref] [bib_ref] Multidrug-resistant bacteria isolated from patients hospitalized in intensive care units, Wroblewska [/bib_ref] The trend of carbapenem resistance in the past decade indicates significant increase in resistance in Acinetobacter spp. with no significant change in P. aeruginosa. [bib_ref] Trend analysis of antimicrobial consumption and development of resistance in non-fermenters in..., Goel [/bib_ref] [bib_ref] Nonsusceptibility trends among Pseudomonas aeruginosa and other nonfermentative Gram negative bacteria from..., Livermore [/bib_ref] Our study shows that, among Enterobacteriaceae, K. pneumoniae has emerged as a problematic species in our health care settings as compared to E. coli due to high prevalence of carbapenemases, which are predominantly NDM-1. Though KPC was previously thought to be the main mechanism of resistance among Enterobacteriaceae, the data in our study shows that MBL has replaced other mechanism of carbapenem resistance. In-fact all isolates positive for MBL phenotypically were also positive for NDM-1 by PCR. The rapidly emerging NDM-1 is of concern from the perspective of infection control not only because of their broad ␤-lactam resistance coverage but also due to their transmissibility. Additionally we tested few strains of non-fermenters (20 P. aeruginosa and seven Acinetobacter spp.) for the presence of bla NDM-1 . Surprisingly we detected the presence of bla NDM-1 in one isolate of Acinetobacter baumannii. Sporadic cases of NDM-1 producing acinetobacter strains have also been reported around the world 37 highlighting its spread to non-fermenters also. Tigecycline, introduced in 2006 in our hospital for MDR bacteria, has lost its effectiveness in 47% of Acinetobacter spp. and 28% of Klebsiella spp. among our blood stream isolates in ICU. Currently, colistin remains the only therapeutic option for the majority of ICU patients, although high-quality pharmacokinetic data and clinical outcome studies are lacking for polymyxin therapy. Among Enterobacteriaceae, Klebsiella spp. with 77% carbapenem resistance appears to be at par with the Acinetobacter spp. (83%). The rapidly increasing prevalence of Enterobacteriaceae harbouring carbapenemases is alarming. Some other recent studies have also reported high carbapenem resistance (77.8%) in K. pneumonia from ICU. [bib_ref] Risk factors for bloodstream infection with Klebsiella pneumoniae producing VIM-1 metallo-␤-lactamase, Daikos [/bib_ref] On studying the pattern of consumption of various antibiotics in the ICUs, an increase in the usage of second-line antibiotics with broad spectrum of activity and reserved for multidrug resistant organisms was observed in our ICUs. The selection pressure associated with the use of high-end antimicrobials might have led to the predominance of multidrug resistant isolates in our ICUs. Even though a direct relationship between the quantity of antimicrobials used and the development of resistance is not easy to establish, studies have documented that the indiscriminate use of antimicrobials has led to the selection of drug-resistant organisms. [bib_ref] Trend analysis of antimicrobial consumption and development of resistance in non-fermenters in..., Goel [/bib_ref] [bib_ref] Increasing resistance to antibiotics: a public health crisis?, Percival [/bib_ref] An increase in the prevalence of non-fermenters might have led to an increase in the usage of second-line antibiotics such as ␤-lactam/inhibitor combinations and carbapenems. In conclusion, BSI caused by multidrug resistant pathogens including, MRSA, VRE, carbapenem resistant Enterobacteriaceae, non-fermenters and non-albicans Candida spp. comprise the current infection profile of the ICUs. Linezolid, tigecycline and vancomycin are the most reliable treatment options for GPC and colistin alone or in combination with carbapenems or aminoglycosides appear to be the remaining treatment options for such Gram negative infections. Deescalation of the high-end antimicrobials once the sensitivity pattern of the isolate is known is suggested to reduce the antimicrobial pressure. More aggressive measures such as routine screening cultures to identify and isolate carriers and the various environmental sources are also recommended, as well as periodical antibacterial sensitivity assessment in ICUs because of the continuous changes in the antibacterial susceptibility patterns. ## Conflicts of interest The authors declare no conflicts of interest. [fig] Figure 1 -: Distribution of the various microorganisms from blood culture from ICU patients.followed by Acinetobacter spp. Candida tropicalis and Candida haemulonii were the most frequently isolated Candida spp. [/fig] [table] Table 1 -: Percentage resistance of antimicrobial agents in the Gram negative bacilli. ICU, Intensive care unit; NT, not tested. [/table] [table] Table 2 -: Percentage resistance of Gram positive bacteria to antimicrobial agents. [/table] [table] Table 3 -: Antimicrobial use during 2008-2011. DDD, defined daily doses; BL + BLI = ␤-lactam + ␤-lactam inhibit. [/table]
Epigenetic Signatures Discriminate Patients With Primary Sclerosing Cholangitis and Ulcerative Colitis From Patients With Ulcerative Colitis # Introduction Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease, characterized by inflammation and fibrosis of the intraand extrahepatic bile ducts. The etiology of PSC is largely unknown and there is still no medical treatment with a proven benefit on disease progression [bib_ref] New Therapeutic Strategies for Primary Sclerosing Cholangitis, Williamson [/bib_ref]. The male to female ratio is 2:1, and interestingly, up to 80% of PSC patients has concomitant inflammatory bowel disease (IBD), of which the majority has ulcerative colitis (UC) [bib_ref] Inflammatory Bowel Disease With Primary Sclerosing Cholangitis: A Danish Population-Based Cohort Study, Sorensen [/bib_ref] [bib_ref] Patient Age, Sex, and Inflammatory Bowel Disease Phenotype Associate With Course of..., Weismuller [/bib_ref]. Comparing patients with UC and patients with PSC-UC could give more insights in pathophysiological processes underlying PSC. The co-occurrence of PSC and IBD has led to various hypotheses linking these two disease entities. One of these hypotheses is the aberrant gut-homing lymphocyte paradigm, which hypothesizes that circulating T-lymphocytes that are primed in the gut and express gut-homing molecules integrin a4b7 and C-Chemokine Receptor 9 (CCR9), can infiltrate in the liver via aberrantly expressed adhesion molecules in PSC-affected liver [bib_ref] Return to Sender: Lymphocyte Trafficking Mechanisms as Contributors to Primary Sclerosing Cholangitis, De Krijger [/bib_ref]. This mechanism may be due to genetic predisposition of the host and/or epigenetic changes in circulating lymphocytes. Over the past few years, several genome-wide association studies (GWAS) in PSC have identified multiple susceptibility loci for PSC-IBD, with none of the loci being associated with this gut-lymphocyte homing paradigm. Interestingly, PSC-IBD patients and IBD patients share only a limited number of risk loci, suggesting that the combination of PSC-IBD comprises a distinct disease manifestation [bib_ref] Genome-Wide Association Study of Primary Sclerosing Cholangitis Identifies New Risk Loci and..., Ji [/bib_ref] [bib_ref] Analysis of Five Chronic Inflammatory Diseases Identifies 27 New Associations and Highlights..., Ellinghaus [/bib_ref]. Nevertheless, genetic variation alone cannot account for total disease liability in PSC-IBD, emphasizing the role of internal and external exposures (the "exposome") including epigenetic factors that may contribute to the disease [bib_ref] Epigenetics in the Primary Biliary Cholangitis and Primary Sclerosing Cholangitis, Cheung [/bib_ref]. Epigenetics comprises heritable processes that involve transcriptional regulation without changing the nucleotide sequence. One of the most studied epigenetic marks is DNA methylation, which represents the addition of a methyl to a base, typically a cytosine that is directly followed by a guanine (CpG). DNA methylation in the promotor region of a gene has been inversely associated with gene expression and is thought to prevent binding of transcription factors, thereby silencing gene expression [bib_ref] DNA Methylation and Gene Expression, Razin [/bib_ref] [bib_ref] Cpg Islands and the Regulation of Transcription, Deaton [/bib_ref]. Differences in DNA methylation of blood cells has been described previously in the context of IBD, where patients with Crohn's disease (CD) differed from patients with UC [bib_ref] DNA Methylation Profiling in Inflammatory Bowel Disease Provides New Insights Into Disease..., Mcdermott [/bib_ref] and healthy controls [bib_ref] Peripheral Blood Methylation Profiling of Female Crohn's Disease Patients, Yim [/bib_ref]. Similar observations have been made in colonic tissue of IBD patients [bib_ref] DNA Methylation Profiling in Inflammatory Bowel Disease Provides New Insights Into Disease..., Mcdermott [/bib_ref]. Furthermore, patients with PSC with and without IBD share common methylation differences compared to controls [bib_ref] Genome-Wide Resolution Peripheral Blood Methylome Profiling Reveals Signatures for Cholestatic Liver Disease, Moore [/bib_ref]. Recently, it was reported that in patients with PSC the DNA methylation age, as estimated using the so-called Horvath clock, was higher than the chronological age [bib_ref] Methylation Signatures in Peripheral Blood Are Associated With Marked Age Acceleration and..., Trauner [/bib_ref] [bib_ref] DNA Methylation Age of Human Tissues and Cell Types, Horvath [/bib_ref]. This age acceleration has proven to have predictive properties regarding disease activity in different diseases, both based on peripheral blood as well as, amongst others, in liver tissue [bib_ref] Methylation Signatures in Peripheral Blood Are Associated With Marked Age Acceleration and..., Trauner [/bib_ref] [bib_ref] DNA Methylation Age of Human Tissues and Cell Types, Horvath [/bib_ref] [bib_ref] DNA Methylation-Based Measures of Biological Age: Meta-Analysis Predicting Time to Death, Chen [/bib_ref]. In this study, we hypothesized that the peripheral blood DNA methylome of patients with concomitant PSC-UC is distinct from that of patients with UC without PSC and healthy controls (HC). Accordingly, we investigated the DNA methylome and performed supervised classification analyses to see whether we could accurately discern PSC-UC from UC and identify CpG loci that contributed to this classification. To ascertain that discriminating differences in DNA methylation between PSC-UC and UC were not solely the result of underlying differences in cellular composition in these different disease states, we also performed mass cytometry to characterize the cellular heterogeneity. # Materials and methods ## Patients Patients included in the current study were selected from a wellcharacterized cohort of the 'Epi PSC PBC project', a large population-based cohort to study patients with cholestatic liver diseases [PSC and primary biliary cholangitis (PBC)] as well as IBD patients in the Netherlands [bib_ref] Population-Based Epidemiology, Malignancy Risk, and Outcome of Primary Sclerosing Cholangitis, Boonstra [/bib_ref]. Patients with prior liver transplantation, colorectal carcinoma, cholangiocarcinoma or prior bowel surgeries were excluded. Out of 1183 cases, 18 patients with PSC-UC, 17 patients with UC, as well as 12 healthy controls (HC) were selected for DNA methylation analysis. Only male patients were included, and groups were matched for age, UC and medication use [fig_ref] TABLE 1 |: Baseline characteristics [/fig_ref]. For mass cytometry analysis, peripheral blood was collected from 10 patients with PSC-UC, 10 patients with UC and 9 healthy volunteers [fig_ref] TABLE 1 |: Baseline characteristics [/fig_ref]. Again, only male patients were included, and biological use was an exclusion criterion to minimize influences on immunological cell distribution. Both protocols were approved by the Medical Ethics Committee at the Amsterdam UMC, University of Amsterdam (METC 06-267/E and METC 2018-050). All samples were collected with written informed consent. manufacturer protocol) and stored at 4°C. Genomic DNA was sent to GenomeScan (Leiden) for bisulfite conversion and DNA methylation profiling. In short, bisulfite converts unmethylated cytosines into uracil, whereas the methylated cytosines remain unchanged. DNA methylation profiling was performed using the Illumina HumanMethylation Infinium EPIC BeadChip (850K) array, yielding the methylation status of approximately 850.000 CpG sites. Samples were randomized across the different chip slides to reduce possible batch effects. ## In silico dna methylation analysis Raw microarray data was imported in R statistical programming environment (v4.0.2) using the Bioconductor (v3.11) package minfi (v1.32.0). Quality control was performed using MethylAid (v1.22.0)and shinyMethyl (v1.24.0), suggesting no apparent technical issues, such as slide-related batch effects. The raw data was then normalized either through functional normalization for differential methylation analyses using limma (v3.44.3) (23), or through noob for classification analyses [bib_ref] Low-Level Processing of Illumina Infinium DNA Methylation Beadarrays, Tjjr [/bib_ref]. Preprocessing of the data consisted of removing probes that were associated to known genetic variants and cross-reactive probes [bib_ref] Identification of Polymorphic and Off-Target Probe Binding Sites on the Illumina Infinium..., Mccartney [/bib_ref]. Allosome-associated probes were not specifically filtered out in the preprocessing steps as the cohort consisted of only males. Repetitive element-binding probes were not excluded as their effect on the percentage methylation was found to be minimal [bib_ref] Comprehensive Characterization, Annotation and Innovative Use of Infinium DNA Methylation Beadchip Probes, Zhou [/bib_ref]. Quality control based on principal component analysis resulted in the removal of one sample with PSC-UC due to its outlier status. Differential methylation analyses using limma were performed using the following design matrix: [formula] methylation ∼ age + steroids + UC : PSC, [/formula] where we compared PSC-UC with UC and PSC-UC with HC. The resultant p-values were adjusted for multiple testing using the Benjamini-Hochberg method. For the hypothesis-driven approach we extracted all probes of length N gene associated to a particular gene of interest and calculated the aggregated p-value using the Fisher method. Next, we constructed a null distribution of p-values by aggregating p-values calculated from 5000 randomly selected stretches of N gene consecutive of probes thereby capturing the correlated nature of DNA methylation that occurs in a particular region. By comparing the observed aggregated p-value with that of the null distribution, we obtained the final p-value. Visualizations were generated using ggplot2 (v3.3.2) (27) and ggbio (v1.36.0) [bib_ref] Ggbio: An R Package for Extending the Grammar of Graphics for Genomic..., Yin [/bib_ref]. ## Dna methylation acceleration analysis The DNA methylation age in years was calculated using the Horvath clock as implemented in wateRmelon (v2.0.0) [bib_ref] DNA Methylation Age of Human Tissues and Cell Types, Horvath [/bib_ref] [bib_ref] A Data-Driven Approach to Preprocessing Illumina 450K Methylation Array Data, Pidsley [/bib_ref]. The difference between the DNA methylation age and the chronological age called the age acceleration. ## Dna methylation blood cell estimation The blood cell distribution as estimated from the DNA methylation data using the estimateCellCounts2 function as implemented in the FlowSorted.Blood.EPIC (v1.12.1) package. In short, this package estimates the cellular composition per sample using a quadratic programming approach. Resultant estimates were subsequently compared between groups using a two-way ANOVA test as implemented in R. # Gradient boosting analysis Gradient boosting analysis was used to classify patients with PSC-UC from patients with UC. To identify the CpGs that contributed the most to the predictive performance, covered information disentanglement (CID) was implemented [bib_ref] Differential DNA Methylation in Familial Hypercholesterolemia, Reeskamp [/bib_ref] [bib_ref] Faecal Microbiota Transplantation Halts Progression of Human New-Onset Type 1 Diabetes in..., De Groot [/bib_ref] [bib_ref] Effects of Fecal Microbiota Transplant on DNA Methylation in Subjects With Metabolic..., Van Der Vossen [/bib_ref]. In short, data was split up in a train (2/3) and test (1/3) set, whereupon the classifier was trained through repeated crossvalidation on the training set. The performance of the resulting model was subsequently evaluated on the withheld test set. The area under the receiver operating characteristic (AUROC) scores were computed within each repetition of cross-validation and averaged for the final test AUROC. We reported the CID-derived feature importance scores for the CpG sites that contributed the most to the prediction model [bib_ref] Differential DNA Methylation in Familial Hypercholesterolemia, Reeskamp [/bib_ref]. ## Dna sequencing ninj2 DNA sequencing of NINJ2-associated loci was performed through Sanger sequencing using the BigDye Terminator v1.1 Cycle Sequencing kit. In short, primers were designed against the region encompassing the CpG loci of interest (Supplementary [fig_ref] FIGURE 5 |: High dimensional mass cytometry [/fig_ref] and . Genomic DNA of all patients included in the DNA methylation data was used as input for specific PCR amplification of the region of interest. A comprehensive overview of the PCR amplification protocol can be found in the supplementary methods. The resultant PCR products were subsequently sequenced at the Core Facility Genomics, Amsterdam UMC. Reads were then aligned using BioEdit and the CpGs of interest were analyzed for variants that might have introduced bases other than cytosine. Seven samples (n=3 PSC-UC and n=4 UC) were excluded after quality control. # Mqtl database analysis The mQTL database was interrogated for the NINJ2-associated predictor CpGs to identify the potential relationship with catalogued genetic variants (mQTL; http://www.mqtldb.org/). ## Bisulfite conversion, pcr and illumina miseq sequencing Technical validation of the NINJ2-associated loci of interest (cg26654770 and cg14911689) annotated to the NINJ2 gene was performed through targeted amplicon sequence analysis using the Illumina MiSeq platform. Primers were designed in MethPrimer (35) (Supplementary . DNA samples from five PSC-UC, two UC and two HC patients (cohort 1, [fig_ref] TABLE 1 |: Baseline characteristics [/fig_ref] were bisulfite converted according to standard protocol using the EZ DNA methylation kit (Zymo Research) [bib_ref] Performance evaluation of kits for bisulfite-conversion of DNA from tissues, cell lines, Holmes [/bib_ref]. Amplicons were made from bisulfite converted DNA PCR and further purified with the Agencourt AMPure PCR purification kit (Beckman Coulter). During a second PCR, amplicons were elongated using TruSEQ indices and Illumina sequence adapters, whereupon they were purified and pooled in stoichiometric amounts. Quality control of the amplicon length within the pools was performed using Agilent 2100 BioAnalyzer. DNA concentrations were measured using Qubit 2.0 Fluorometer (ThermoFisher) and equalized to equimolar concentrations for all subject pools. MiSeq amplicon sequencing was then performed according to the standard protocol. Raw sequence data was mapped, aligned, and analyzed using Bismark [bib_ref] Bismark: A Flexible Aligner and Methylation Caller for Bisulfite-Seq Applications, Krueger [/bib_ref] and visualized using Integrative Genomics viewer (v 2.3.57) against the bisulfite-converted human genome hg19. A minimum of 120 reads per samplederived amplicon was deemed successful. ## Quantitative real-time polymerase chain reaction Messenger RNA was extracted from frozen PBMCs using the Bioline ISOLATE II RNA mini kit (GC biotech B.V. Alphen a/d Rijn, the Netherlands) according to manufacturer's instructions. RNA concentration was measured using the Nanodrop 1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). cDNA was synthesized using the Revertaid first strand cDNA synthesis kit (Fermentas, St. Leon-Rot, Germany). A quantitative polymerase chain reaction (qPCR) was performed using SensiFAST SYBR No-ROX (GC Biotech B.V.) on a BioRad (CFX96 real-time qPCR thermocycler). Resultant gene expression levels were calculated using LinRegPCR [bib_ref] Amplification Efficiency: Linking Baseline and Bias in the Analysis of Quantitative PCR..., Ruijter [/bib_ref]. After stability analysis in geNorm, two human reference genes were selected for normalization; Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) and Hypoxanthine-guanine-fosforibosyl-transferase (HPRT) [bib_ref] Accurate Normalization of Real-Time Quantitative RT-PCR Data by Geometric Averaging of Multiple..., Vandesompele [/bib_ref]. Primers were either obtained by Qiagen or synthesized by Sigma (Supplementary . ## Mass cytometry Cryopreserved PBMCs were thawed and washed with medium (RPMI+20% fetal bovine serum (FBS)) whereupon they were resuspended in PBS. For cellular viability assessment, single-cell suspensions were incubated with Cisplatin (5 µM, Fluidigm) for 5 minutes and washed with cell staining buffer (CSB, Fluidigm). Cells were incubated with Human TruStain FcX Fc receptor blocking solution (Biolegend), after which cells were stained with a mix of metal-conjugated antibodies against cell surface markers (Supplementary Normalized.fcs files were uploaded in to Cytobank [bib_ref] Web-Based Analysis and Publication of Flow Cytometry Experiments, Kotecha [/bib_ref] for analysis and quality control. Viable CD45 + singlets were selected according to gating strategy previously described [bib_ref] Automated Data Cleanup for Mass Cytometry, Bagwell [/bib_ref]. Both batches of samples included a technical replicate. Potential batch effects were investigated through manual inspection of marker distributions and overlap in clusterings. Different lineages (B-cells, CD4 + T-cells, CD8 + T-cells, myeloid cells and NK-cells) were clustered and color-coded using FlowSOM and subsequent manual annotation [bib_ref] Using Self-Organizing Maps for Visualization and Interpretation of Cytometry Data, Van Gassen [/bib_ref]. Data is visualized using viISNE, a visualization tool for high-dimensional single-cell data based on the t-dDistributed Stochastic Neighbor Embedding (t-SNE) algorithm [bib_ref] Visne Enables Visualization of High Dimensional Single-Cell Data and Reveals Phenotypic Heterogeneity..., Amir El [/bib_ref]. ## Patient characteristic statistical analyses Patient characteristics are presented as median and interquartile range (IQR; 25th-75th percentile). Dichotomous variables are presented as percentage (%) of the cohort. Differences were calculated with the chi-square test or Fisher's exact test for categorical variables. Numerical data were compared using a Mann-Whitney U test or One-way ANOVA, or a Kruskal-Wallis test with Dunn's correction for multiple testing. Statistical analysis was performed in SPSS statistical software for Windows version 26.0 (SPSS, Chicago, USA) or GraphPad Prism 8. A p-value <0.05 was considered statistically significant. # Results ## The genome-wide methylome of patients with psc-uc is comparable to that from patients with uc and healthy controls DNA methylation was investigated in peripheral blood samples from 17 patients with PSC-UC, 17 patients with UC and 12 HCs. The characteristics of the included patients are shown in [fig_ref] TABLE 1 |: Baseline characteristics [/fig_ref] (cohort 1). To limit the number of variables, we included only male patients, with all groups being matched for age (median age 41, 34 and 39 years for PSC-UC, UC and HC, respectively), UC duration (16 and 11 years for PSC-UC and UC, respectively) and medication use (all patients with PSC-UC and UC used mesalazine, 50% used thiopurins and none used biologicals). Median PSC duration at time of inclusion was 5 years. None of the patients had undergone liver transplantation at the time of sampling. Global methylation analysis through principal component (PC) analysis did not present a clear separation of the samples according to disease status [fig_ref] FIGURE 1 |: The genome-wide methylome of patients with PSC-UC is comparable to that from... [/fig_ref]. Notably, PC1 presented a larger separation between HC and UC (p-value = 0.021), than HC and PSC-UC (p-value = 0.347) or PSC-UC and UC (p-value = 0.292) [fig_ref] FIGURE 1 |: The genome-wide methylome of patients with PSC-UC is comparable to that from... [/fig_ref]. Next, we specifically investigated whether any of the probes were differentially methylated when comparing PSC-UC with UC. While we were able to observe differentially methylated positions (DMPs) that were visibly differentially methylated [fig_ref] FIGURE 1 |: The genome-wide methylome of patients with PSC-UC is comparable to that from... [/fig_ref] , they were not statistically significant after correction for multiple testing [fig_ref] TABLE 2 |: Differentially methylated positions [/fig_ref]. The most differentially methylated probe was cg02169981 (annotated to WNT11), which displayed hypermethylation among patients with UC compared to patients with PSC-UC (Supplementary [fig_ref] FIGURE 1 |: The genome-wide methylome of patients with PSC-UC is comparable to that from... [/fig_ref]. Expanding our search to regions of contiguous differential methylation (DMRs) when comparing either PSC-UC with UC, or PSC-UC with HC yielded no statistically significant differences (Supplementary [fig_ref] TABLE 1 |: Baseline characteristics [/fig_ref] Having identified no clear differences in methylation at a genome-wide level, we adopted a hypothesis-driven approach where we interrogated the methylation status of previously reported loci/genes of interest, obtained from previous genome-(GWAS) or transcriptome-wide association studies (TWAS) [bib_ref] Genome-Wide Association Study of Primary Sclerosing Cholangitis Identifies New Risk Loci and..., Ji [/bib_ref] [bib_ref] Analysis of Five Chronic Inflammatory Diseases Identifies 27 New Associations and Highlights..., Ellinghaus [/bib_ref] [bib_ref] Gene Expression by PBMC in Primary Sclerosing Cholangitis: Evidence for Dysregulation of..., Aoki [/bib_ref] [bib_ref] Genetic Association Analysis Identifies Variants Associated With Disease Progression in Primary Sclerosing..., Alberts [/bib_ref] [bib_ref] Fine Mapping and Replication of Genetic Risk Loci in Primary Sclerosing Cholangitis, Srivastava [/bib_ref] [bib_ref] Extended Analysis of a Genome-Wide Association Study in Primary Sclerosing Cholangitis Detects..., Folseraas [/bib_ref] [bib_ref] Genome-Wide Association Analysis in Primary Sclerosing Cholangitis Identifies Two Non-HLA Susceptibility Loci, Melum [/bib_ref]. When comparing PSC-UC with UC, only two genes were significantly enriched for nominally differentially methylated probes: BACH2 and ASAP2 (Supplementary [fig_ref] TABLE 2 |: Differentially methylated positions [/fig_ref] and [fig_ref] FIGURE 2 |: Gradient boosting analysis distinguishes PSC-UC from UC [/fig_ref]. Comparing PSC-UC with HC indicated that probes associated with eight genes showed a statistically significant difference (Supplementary [fig_ref] TABLE 2 |: Differentially methylated positions [/fig_ref]. UBASH3A, the most significant SNP in a large GWAS study and associated with a lower risk of PSC (8), showed differential methylation centering around the promotor region when comparing PSC-UC with HC (Supplementary [fig_ref] FIGURE 2 |: Gradient boosting analysis distinguishes PSC-UC from UC [/fig_ref]. In a recent study, it was reported that patients with PSC presented an increased DNA methylation age relative to their chronological age, which was especially apparent in samples obtained from patients at an advanced disease state [bib_ref] Methylation Signatures in Peripheral Blood Are Associated With Marked Age Acceleration and..., Trauner [/bib_ref]. Using the same method we estimated the DNA methylation age of all samples and compared the estimated and chronological age [bib_ref] DNA Methylation Age of Human Tissues and Cell Types, Horvath [/bib_ref]. Overall, we found that the DNA methylation age correlated well with the chronological age [fig_ref] FIGURE 1 |: The genome-wide methylome of patients with PSC-UC is comparable to that from... [/fig_ref] ; Spearman's Rho 0.942, p-value < 0.001). Calculating the difference between the DNA methylation age and the chronical age revealed a median difference of 2.1 years in patients with PSC-UC (range -5.6-8.6), which did not significantly differ from either patients with UC or HCs (median 2.3 (range -3.3-9.7) and 1.5 (range -3.4-10.7), respectively (p-value = 0.874, [fig_ref] FIGURE 1 |: The genome-wide methylome of patients with PSC-UC is comparable to that from... [/fig_ref]. We note however that the use of different normalization methods resulted in larger or smaller differences between the predicted age and the chronological age, with a more notable deviation when using quantile normalization. However, even with the larger differences from the quantile normalization, we did not identify any significant differences between phenotypes (Supplementary [fig_ref] FIGURE 3 |: Predictive CpGs annotated to NINJ2 were all hypermethylated in PSC-UC compared to... [/fig_ref]. Taken together, we observed no big differences between the three groups at a DNA methylome-wide level, nor did we find any statistically significant differences between DMPs or DMRs of patients with PSC-UC and either patients with UC or healthy controls. ## Classification analysis distinguishes psc-uc from uc Next, we investigated whether DNA methylation could be of use for distinguishing patients with PSC-UC from patients with UC without PSC. To this end, we performed a classification analysis using repeated cross-validation with gradient boosting, where we sought to capitalize on potential non-linear relationships between the CpG loci of interest and the presence of PSC-UC [bib_ref] Effects of Fecal Microbiota Transplant on DNA Methylation in Subjects With Metabolic..., Van Der Vossen [/bib_ref]. Altogether, the classification analysis yielded a predictive model with an area under the receiver-operator characteristic curve (AUROC) of 0.80. Subsequent permutation analyses indicated that 18 CpGs contributed significantly to the model [fig_ref] TABLE 3 |: Predictor CpG loci capable of distinguishing PSC-UC from UC [/fig_ref] and [fig_ref] FIGURE 2 |: Gradient boosting analysis distinguishes PSC-UC from UC [/fig_ref]. Of these 18 CpGs, PSC-UC-associated hypermethylation was observed for NINJ2, cg12219587, SERPINB9, DNAJC17, cg19079513, OR51A7, cg12313868 and SOX6 whereas hypomethylation was found for cg00980980, TTC15, THUMPD1, TRAPPC12 and CYP4F22 [fig_ref] FIGURE 2 |: Gradient boosting analysis distinguishes PSC-UC from UC [/fig_ref]. While most predictive CpGs were embedded in a gene as single CpG, four were found to be all localized within the gene NINJ2. All four of the NINJ2-associated predictor CpGs were adjacent to one another and were located in an intronic region [fig_ref] FIGURE 3 |: Predictive CpGs annotated to NINJ2 were all hypermethylated in PSC-UC compared to... [/fig_ref] and Supplementary [fig_ref] FIGURE 5 |: High dimensional mass cytometry [/fig_ref]. Technical validation through bisulfite sequencing of two of the four predictor CpGs (cg26654770 and cg14911689) confirmed the methylation pattern [fig_ref] FIGURE 3 |: Predictive CpGs annotated to NINJ2 were all hypermethylated in PSC-UC compared to... [/fig_ref]. Notably, the observed methylation signal of the NINJ2-associated DMPs presented itself as a clustered pattern around 0%, 50% and 100% methylation [fig_ref] FIGURE 3 |: Predictive CpGs annotated to NINJ2 were all hypermethylated in PSC-UC compared to... [/fig_ref] , a pattern characteristic of underlying genetic variants [bib_ref] Eureas_Gplex-a New Snapshot Assay for Continental Population Discrimination and Gender Identification, Daca-Roszak [/bib_ref]. However, sequencing the underlying region of interest revealed no single nucleotide polymorphism (SNP) or other local genetic variants at the loci of interest [fig_ref] FIGURE 3 |: Predictive CpGs annotated to NINJ2 were all hypermethylated in PSC-UC compared to... [/fig_ref]. As more distal genetic variants might affect DNA methylation, which have been termed methylation quantitative trait loci (mQTL), we interrogated the mQTLs database (http://www.mqtldb.org) (51) to identify potential catalogued genetic variants that have been associated with the DNA methylation of our sites of interest. Altogether, we identified 1330, 1374, 1329 and 1329 mQTLs where genetic variation was strongly associated with the loci cg01201512, cg26371957, cg14911689 and cg26654770, respectively (Additional File_mQTLs). Next, we sought to investigate whether the relation between CpG methylation that we found associated with PSC-UC was reflected in differential gene expression of this particular gene, through measuring NINJ2 transcripts using quantitative PCR. However, we found no differential expression of NINJ2 between PSC-UC and UC patients [fig_ref] FIGURE 3 |: Predictive CpGs annotated to NINJ2 were all hypermethylated in PSC-UC compared to... [/fig_ref] , suggesting that the observed difference in methylation does not affect the expression of NINJ2. Additional classification analysis on PSC-UC and HC samples yielded a predictive model with an area under the receiver-operator characteristic curve (AUROC) of 0.83. The CpG positions observed in this classification did not overlap with the CpGs as compared to the predictive model on PSC-UC and UC (Supplementary [fig_ref] TABLE 3 |: Predictor CpG loci capable of distinguishing PSC-UC from UC [/fig_ref]. ## Peripheral blood cell distribution is comparable between patients with psc-uc and uc Due to the epigenetic and hence cell-specific nature of DNA methylation, observed differences could be the result of differences in the cellular composition of peripheral blood between PSC-UC and UC patients [bib_ref] Epigenetics in Health and Disease: Heralding the EWAS Era, Murphy [/bib_ref] [bib_ref] Accounting for Cellular Heterogeneity is Critical in Epigenome-Wide Association Studies, Jaffe [/bib_ref]. We therefore estimated the cellular composition using the algorithm described in Houseman et al. [fig_ref] FIGURE 4 |: High-dimensional mass cytometry [/fig_ref] [bib_ref] DNA Methylation Arrays as Surrogate Measures of Cell Mixture Distribution, Houseman [/bib_ref]. We found no evidence that any of the estimated lineages (CD4 + T-cells, CD8 + T-cells, B-cells, natural killer (NK)-cells, monocytes and neutrophils) were different when comparing PSC-UC with UC. By contrast, we observed a significantly lower abundance of NK cells in the PSC-UC and UC samples as compared to HC (pvalue = 5.33E-04). ## A b As cellular estimates based on DNA methylation are currently limited in their resolution, we explored the peripheral blood mononuclear cellular distribution in more detail using mass cytometry. Blood samples were collected from a new cohort of PSC-UC (n = 10), UC (n = 10) and HC (n = 9). The median age at inclusion was 40, 58 and 30 years (p=0.022) [fig_ref] TABLE 1 |: Baseline characteristics [/fig_ref] , cohort 2), for PSC-UC, UC and HC respectively. UC duration was significantly longer in patients with UC without PSC (median 22 years (IQR 14-34) compared to 9 years (IQR 5-19) in patients with PSC-UC (p=0.019). The majority of PSC-UC and UC patients used Mesalazine (90% in both groups) with only one patient with UC (without PSC) using a thiopurine. Different lineages were identified based on cellular phenotype and visualized in tSNE plots [fig_ref] FIGURE 5 |: High dimensional mass cytometry [/fig_ref] , [fig_ref] FIGURE 4 |: High-dimensional mass cytometry [/fig_ref]. Akin to our [fig_ref] FIGURE 4 |: High-dimensional mass cytometry [/fig_ref] and Supplementary [fig_ref] FIGURE 4 |: High-dimensional mass cytometry [/fig_ref]. Notably, we observed a similar trend in NK cell distribution as seen in the Houseman algorithm estimation. The myeloid lineage was significantly more abundant in patients with UC compared to patients with PSC-UC (p-value = 0.013, [fig_ref] FIGURE 4 |: High-dimensional mass cytometry [/fig_ref]. Disentangling the myeloid lineage into the constituent monocyte subtypes revealed that this difference was predominantly observable in the classical monocyte (CD14 ++ CD16 -) population [fig_ref] FIGURE 4 |: High-dimensional mass cytometry [/fig_ref]. Within the classical monocyte populations, we particularly saw a decrease in CD45RA + CCR7 + classical monocytes and CD45RO + CD2 DIM CD69 DIM classical monocytes among PSC patients compared to UC patients. Although the overall # Discussion Distinguishing patients with UC and concomitant PSC from patients with UC could give more insights in disease pathophysiology of PSC and would be of clinical value for early diagnosis. In this explorative study, we performed genome-wide DNA methylation and mass cytometry analysis on whole peripheral blood from patients with PSC-UC, UC and HCs. We show that minor differences exist in the peripheral blood methylome when comparing male patients with PSC-UC to male patients with solely UC. Notwithstanding, classification analysis yielded a predictive model capable of distinguishing patients with PSC-UC and UC. The overall lack of large-scale differences in methylation between the various groups corroborates the observations made by Moore et al., where the authors did not observe large global methylation changes in the peripheral blood of patients with PSC compared to healthy controls and between patients with PSC with and without IBD [bib_ref] Genome-Wide Resolution Peripheral Blood Methylome Profiling Reveals Signatures for Cholestatic Liver Disease, Moore [/bib_ref]. While peripheral blood is a practical tissue for biomarker use due to its ease of access, it contains a heterogeneous population of cells, which might be less representative for disease features, such as the PSC-UCassociated phenotypes that manifest primarily in the liver and gut tissue. Subtle differences between PSC-UC, UC and HC may therefore remain hidden in the analysis performed. Indeed mass cytometry analyses revealed subtle differences in blood cell composition across patient groups. While we demonstrated that the peripheral blood cell distribution was largely comparable between patients with PSC-UC and UC, the myeloid lineage, the monocyte CD14/CD16 subsets in particular, were more abundant in patients with UC compared to PSC-UC and HCs. As DNA methylation signal can vary between different cell types, observed differences might be reflective of changes in the underlying population [bib_ref] Accounting for Cellular Heterogeneity is Critical in Epigenome-Wide Association Studies, Jaffe [/bib_ref]. Importantly, we could not confirm an increased DNA methylation age in PSC patients as was reported by Trauner et al. [bib_ref] Methylation Signatures in Peripheral Blood Are Associated With Marked Age Acceleration and..., Trauner [/bib_ref]. The discrepancy between our observations might be related to their phenotype of interest, namely progression of fibrosis, for which we have no information of at the time of sampling. The fact that the median PSC duration was low in our cohort (5 years), might have influenced our accelerated age differences. Notwithstanding, we believe that the observed differences were likely influenced by the choice of normalization as Trauner et al. utilized quantile normalization, a method that presented one of the largest offset as compared to other normalization methods [bib_ref] Systematic Evaluation of DNA Methylation Age Estimation With Common Preprocessing Methods and..., Mcewen [/bib_ref]. Classification analysis yielded a predictive model, which enables a distinction between peripheral blood of patients with PSC-UC and UC with an AUROC of 0.80, indicating that these two disease entities do have a distinct epigenetic architecture. The discrepancy between our DMP analysis and the classification analysis may be due to the non-linear relationships identified through gradient boosting. Whereas none of the predictive CpG-associated genes had been described within the context of PSC previously, two genes are associated to hepatocellular carcinoma, namely CUL2 and SOX6 (58-60). Moreover, both CUL2 and SOX6 are associated with colitis as well, while SERPINB9 and NINJ2 have been associated with colorectal cancer [bib_ref] Deep Resequencing of GWAS Loci Identifies Independent Rare Variants Associated With Inflammatory..., Rivas [/bib_ref] [bib_ref] Structural Remodeling of the Human Colonic Mesenchyme in Inflammatory Bowel Disease, Kinchen [/bib_ref] [bib_ref] Expression of Serpin B9 as a Prognostic Factor of Colorectal Cancer, Vycital [/bib_ref] [bib_ref] Ninjurin 2 Overexpression Promotes Human Colorectal Cancer Cell Growth In Vitro and..., Li [/bib_ref]. The four predictive CpG associated to NINJ2 were found to be hypermethylated in PSC-UC compared to UC patients, which we validated through bisulfite sequencing. While the four CpGs presented a methylation pattern reminiscent of an underlying genetic variant (65), we did not identify any genetic variants through Sanger sequencing at the sites of interest. This does not eliminate the possibility that other genetic variants might have conferred the observed methylation signal, such as copy number variations (CNV), indels and more distal SNPs associated with differences in DNA methylation [bib_ref] Systematic Identification of Genetic Influences on Methylation Across the Human Life Course, Gaunt [/bib_ref] [bib_ref] Genetic Control of Individual Differences in Gene-Specific Methylation in Human Brain, Zhang [/bib_ref] [bib_ref] Association of Cnvs With Methylation Variation, Shi [/bib_ref] , which we were not able to pick up with the techniques utilized in the current study. With over a thousand catalogued mQTLs associated to the four NINJ2-associated predictive CpGs, the possibility is present that distal genetic variants may influence the methylation status of NINJ2 [bib_ref] Systematic Identification of Genetic Influences on Methylation Across the Human Life Course, Gaunt [/bib_ref]. The biological consequence of the observed difference in methylation of NINJ2 remains to be established. The encoded Ninjurin 2 or nerve injury induced protein 2 (NINJ2) is an adhesion molecule expressed in neurons and glial cells and is involved in nerve regeneration [bib_ref] Ninjurin2, a Novel Homophilic Adhesion Molecule, is Expressed in Mature Sensory and..., Araki [/bib_ref]. Notably, Ninjurin 1, a homologue of Ninjurin 2, as well as NINJ2 are highly expressed in myeloid cells and peripheral leukocytes, suggesting a role in immune-mediated diseases as well [bib_ref] Gene Expression by PBMC in Primary Sclerosing Cholangitis: Evidence for Dysregulation of..., Aoki [/bib_ref] [bib_ref] Ninjurin1 is Expressed in Myeloid Cells and Mediates Endothelium Adhesion in the..., Ahn [/bib_ref] [bib_ref] NINJ2-a Novel Regulator of Endothelial Inflammation and Activation, Wang [/bib_ref]. In vascular endothelial cells it was found that NINJ2 regulates monocyte-adhesion as well as endothelial inflammation through the expression of pro-inflammatory cytokines such as IL-1b, TNF-a, IL-8, IL-6, ICAM-1 and Eselectin [bib_ref] NINJ2-a Novel Regulator of Endothelial Inflammation and Activation, Wang [/bib_ref]. Through mass cytometry we observed a diminished abundance of the myeloid population among the PSC-UC patients relative to the UC patients. The observed differences in myeloid cell population abundance may therefore be related to the observed difference in NINJ2 methylation. However, while NINJ2 expression was reportedly associated with DNA methylation in CD4 + T-cells (71), we observed no difference in NINJ2 gene expression in the current cohort. This might be attributable to the differences in methylation being observed in the first intron rather than the promotor region. In summary, the role of NINJ2 in relation to PSC-UC remains unclear. Our study is not without its limitations: we only included male subjects to reduce variables and limit confounding in DNA methylation analysis. While having a same-sex cohort decreases variance, it makes the observations less translatable to the general PSC-IBD population. While previous EWAS have indicated that DNA methylation profiles differ between males and females [bib_ref] Sex Differences in DNA Methylation Assessed by 450 K Beadchip in Newborns, Yousefi [/bib_ref] , limited to no sex-associated difference were reported in PSC-associated EWAS [bib_ref] Genome-Wide Resolution Peripheral Blood Methylome Profiling Reveals Signatures for Cholestatic Liver Disease, Moore [/bib_ref] [bib_ref] Methylation Signatures in Peripheral Blood Are Associated With Marked Age Acceleration and..., Trauner [/bib_ref]. One study did show an enrichment of differentially methylated CpG sites located on chromosome X in patients with PSC-IBD and PSC without IBD compared to controls but did not make a comparison with the methylomes of patients with PSC alone [bib_ref] Genome-Wide Resolution Peripheral Blood Methylome Profiling Reveals Signatures for Cholestatic Liver Disease, Moore [/bib_ref]. While the majority of the patients with PSC are male, we acknowledge that further validation experiments in a larger and more heterogeneous population with both male and female subjects are necessary to properly ascertain the robustness of the predictive CpGs and the potential utility in the clinic in defining PSC-UC. # Conclusions Our study provides a novel approach of exploring DNA methylation analysis to differentiate patients with PSC-UC from patients with UC. Further validation in a different cohort has to confirm the biomarker potential of these methylation differences for early detection of PSC. # Data availability statement The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://ega-archive. org/, EGAS00001005832. # Ethics statement The studies involving human participants were reviewed and approved by Medical Ethics Committee at the Amsterdam UMC, University of Amsterdam, Ref. nrs: METC 06-267/E and METC 2018-050. The patients/participants provided their written informed consent to participate in this study. # Author contributions Concept and design of the study: MK, CP, and WJ. MK assembled the patient cohorts, prepared samples for the DNA methylation array and performed the mass cytometry. PvH performed RNA isolation. JV, JGV, and MW analyzed the mass cytometry data. IH and TH performed Sanger sequencing and next generation sequencing. ALY and EL analyzed and visualized the results acquired from the HumanMethylation EPIC BeadChip array. EL performed machine learning. MK, IH, and ALY drafted the manuscript. PH, TH, CP, and WJ supervised the study. All authors read and approved the final manuscript. ACKNOWLEDGMENTS M.S. Tulek, K. Boonstra: collection peripheral blood samples of PSC-UC patients, UC patients and healthy controls. We thank the Microscopy and Cytometry Core facility at the VU Medical Center Amsterdam for use of the Helios and support in sample preparation and acquisition. We thank the company Genome Diagnostics B.V (GENDX) for use of the Illumina Miseq machine to perform next generation sequencing [fig] FIGURE 1 |: The genome-wide methylome of patients with PSC-UC is comparable to that from patients with UC and healthy controls.(A) Principal component (PC) analysis of the methylome of patients with PSC-UC (light grey), UC (grey) and HC (dark grey). (B) Association of PC1 with all groups. (C) Heatmap of top 50 differentially methylated positions between PSC-UC (blue) and UC (green). (D) Correlation between chronological age (years) and estimated DNA methylation age (years) in patients with PSC-UC, UC and HC. (E) Comparison of accelerated age (difference between chorological age and estimated age) between patients with PSC-UC, UC and HC. A p-value <0.05 was considered statistically significant (*p < 0.05). de Krijger et al. Epigenetic Signatures in PSC-UC [/fig] [fig] FIGURE 2 |: Gradient boosting analysis distinguishes PSC-UC from UC. (A) Heatmap of 18 differentially methylated positions contributing to the predictive model distinguishing PSC-UC from UC. (B) Radar plot depicting the 15 most predictive CpG loci that are capable of distinguishing patients with PSC-UC from UC. The axes represent the mean scaled changes for the top 15 most discriminative CpG sites. [/fig] [fig] FIGURE 3 |: Predictive CpGs annotated to NINJ2 were all hypermethylated in PSC-UC compared to UC patients. (A) Visualization of NINJ2 by plotting the actual percentage methylation for PSC-UC, UC and HC ("Methylation") as well as the difference between PSC-UC and UC in percentage methylation ("Methylation difference") relative to the position on the genome. (B) Visual correlation of the percentage methylation observed for cg26654770 and cg14911689 as calculated using the 850k DNA methylation array and through MiSeq sequencing for 5 PSC-UC, 2 UC and 2 HC patients. (C) Differences in percentage methylation (beta value) in patients with UC (n = 17) and PSC-UC (n = 17). (D) Representative images of Sanger sequencing traces surrounding the CpG loci of interest (marked in grey) that are annotated to NINJ2 in PSC-UC (n = 14), UC (n = 13) and HCs (n = 12). (E) Relative mRNA expression of NINJ2 normalized to the household genes GAPDH and HPRT in peripheral blood mononuclear cells of PSC-UC (n = 10), UC (n = 10) and HCs (at the level of DNA methylation, the abundance of CD4 + T-cells, CD8 + T-cells and B-cells was not statistically different between PSC-UC, UC and HCs ( [/fig] [fig] FIGURE 4 |: High-dimensional mass cytometry (cyTOF) analysis reveals that peripheral blood cell distribution is comparable between patients with PSC-UC, UC and HC. (A) Estimated cell proportions as derived from Houseman algorithm cell mixture deconvolution from DNA methylation data of PSC-UC (blue), UC (green) and HC (red). (B) The lineages CD4 + T-cells, CD8 + T-cells, B-cells, myeloid cells and NK-cells were clustered for PSC-UC, UC and HC and color-coded visualized using vISNE. (C) Differences in frequencies of CD4 + T-cells, CD8 + T-cells, B-cells, Myeloid lineage and NK-cells as percentage of total CD45 + cells in HC (n = 9), UC (n = 10) and PSC-UC (n = 10). (D) Differences in frequencies of classical monocytes, intermediate monocytes and non-classical monocytes percentage of total CD45 + cells in HC (n = 9), UC (n = 10) and PSC-UC (n = 10). Statistical testing was performed using Kruskal Wallis with Dunn's correction for multiple testing. A p-value < 0.05 was considered statistically significant (cell population did not differ between PSC-UC and UC, we observed distinct proportions of memory T-cells expressing CD161, a c-type lectin like-receptor expressed by NK cells, T helper 17 cells and mucosal invariant T (MAIT) cells (55) between patients with PSC-UC and UC (SupplementaryFigure 4). [/fig] [fig] FIGURE 5 |: High dimensional mass cytometry (cyTOF) analysIs reveals that peripheral blood cell distribution is comparable between patients with PSC-UC, UC and HC. The lineages CD4 + T-cells, CD8 + T-cells, B-cells, myeloid cells and NK cells were clustered for PSC-UC, UC and HC and color coded visualized using vISNE, a visualization tool for high dimensional single cell data based on the t Distributed Stochastic Neighbor Embedding (t-SNE) algorithm. [/fig] [fig] FUNDING: ALY was funded by the European Union's Horizon 2020 research and innovation program under Grant Agreement No. ITN-2014-EID-641665. ALY and IH were funded by the Helmsley charitable trust. [/fig] [table] TABLE 1 |: Baseline characteristics. [/table] [table] Table 5 ,: 'pre-fixation'), washed with CSB and fixed with 1.6% PFA. Cells were permeabilized by Maxpar Barcode Perm Buffer (Fluidigm), incubated with mass tag barcodes and stained with the remaining metal-conjugated antibodies (SupplementaryTable 5, Fluidigm). For intracellular staining, cells were washed with Perm-S buffer (Fluidigm) and incubated with antibodies against CTLA-4 and CES-1 (SupplementaryTable 5, 'nuclear staining'), washed and incubated with the corresponding secondary antibodies. Antibodies were fixated with 1.6% PFA/PBS, washed and incubated overnight with 191/193 Ir DNA intercalator (1:4000) diluted in Fix-and-Perm Buffer (Fluidigm). Acquisition was performed on the Cytometry by time of flight (CyTOF)3-Helios. Sample was diluted in H2O and supplemented with 10% v/v of EQ Four Element Calibration beads (Fluidigm). After acquisition data was normalized and individual files were deconvoluted using the CyTOF software v6.7 functions. [/table] [table] TABLE 2 |: Differentially methylated positions (DMPs) when comparing PSC-UC with UC.The 10 most differentially methylated positions. *Neighbour gene. P-values were calculated through linear regression using limma and adjusted for multiple testing using the Benjamini-Hochberg method. [/table] [table] TABLE 3 |: Predictor CpG loci capable of distinguishing PSC-UC from UC.The 18 CpG loci returned by gradient boosting that were capable of distinguishing PSC-UC from UC. [/table]
Alcohol-Related Brain Damage in Humans Chronic excessive alcohol intoxications evoke cumulative damage to tissues and organs. We examined prefrontal cortex (Brodmann's area (BA) 9) from 20 human alcoholics and 20 age, gender, and postmortem delay matched control subjects. H & E staining and light microscopy of prefrontal cortex tissue revealed a reduction in the levels of cytoskeleton surrounding the nuclei of cortical and subcortical neurons, and a disruption of subcortical neuron patterning in alcoholic subjects. BA 9 tissue homogenisation and one dimensional polyacrylamide gel electrophoresis (PAGE) proteomics of cytosolic proteins identified dramatic reductions in the protein levels of spectrin b II, and aand b-tubulins in alcoholics, and these were validated and quantitated by Western blotting. We detected a significant increase in a-tubulin acetylation in alcoholics, a non-significant increase in isoaspartate protein damage, but a significant increase in protein isoaspartyl methyltransferase protein levels, the enzyme that triggers isoaspartate damage repair in vivo. There was also a significant reduction in proteasome activity in alcoholics. One dimensional PAGE of membrane-enriched fractions detected a reduction in b-spectrin protein levels, and a significant increase in transmembranous a3 (catalytic) subunit of the Na + ,K + -ATPase in alcoholic subjects. However, control subjects retained stable oligomeric forms of a-subunit that were diminished in alcoholics. In alcoholics, significant loss of cytosolic aand b-tubulins were also seen in caudate nucleus, hippocampus and cerebellum, but to different levels, indicative of brain regional susceptibility to alcohol-related damage. Collectively, these protein changes provide a molecular basis for some of the neuronal and behavioural abnormalities attributed to alcoholics. # Introduction Chronic excessive alcohol consumption is a global healthcare problem of epidemic proportion [bib_ref] Global burden of disease and injury and economic cost attributable to alcohol..., Rehm [/bib_ref]. Alcoholics experience a number of cognitive deficiencies such as learning and memory deficits, impairment of decision making, and problems with motor skills, as well as suffering behavioural changes that include anxiety and depression [bib_ref] The neurotoxicity of alcohol, Harper [/bib_ref] [bib_ref] Ethanol and cognition: indirect effects, neurotoxicity and neuroprotection: a review, Brust [/bib_ref]. These debilitating morbidities arise from the cumulative effects of intoxication and alcohol withdrawal, and may be further exacerbated by nutritional deficiencies such as Wernicke-Korsakoff disorders [bib_ref] The neurotoxicity of alcohol, Harper [/bib_ref] [bib_ref] Ethanol and cognition: indirect effects, neurotoxicity and neuroprotection: a review, Brust [/bib_ref] [bib_ref] The neuropathology of alcohol-related brain damage, Harper [/bib_ref] [bib_ref] Clinical and pathological features of alcohol-related brain damage, Zahr [/bib_ref] [bib_ref] Alcoholrelated brain damage: report from a Medical Council on Alcohol Symposium, Thomson [/bib_ref]. Excessive alcohol consumption can result in a reduction of brain weight, with regional brain atrophy [bib_ref] The neurotoxicity of alcohol, Harper [/bib_ref] [bib_ref] The neuropathology of alcohol-related brain damage, Harper [/bib_ref] [bib_ref] Clinical and pathological features of alcohol-related brain damage, Zahr [/bib_ref]. The production of toxic ethanol metabolites, and their post-translational modification (PTM) and damage of cellular proteins is one of the proposed mechanisms that contribute to neuronal damage [bib_ref] Clinical and pathological features of alcohol-related brain damage, Zahr [/bib_ref] [bib_ref] Evidence of acetaldehyde-protein adduct formation in rat brain after lifelong consumption of..., Rintala [/bib_ref] [bib_ref] Acetaldehyde adducts in the brain of alcoholics, Nakamura [/bib_ref] [bib_ref] Detection and localization of proteinacetaldehyde adducts in rat brain after chronic ethanol..., Upadhya [/bib_ref]. The first toxic metabolite of ethanol, acetaldehyde, can form adducts with the e-amino group of lysine residues, and it has been suggested that these covalent modifications can disrupt the function of proteins resulting in cellular injury [bib_ref] Acetaldehyde and microtubules, Tuma [/bib_ref]. One of the major targets of acetaldehyde-mediated adduction is a-tubulin [bib_ref] Acetaldehyde substoichiometrically inhibits bovine neurotubulin polymerization, Smith [/bib_ref] , and site specific a-tubulin acetylation can influence microtubule polymerisation [bib_ref] Acetaldehyde and microtubules, Tuma [/bib_ref] [bib_ref] Acetaldehyde substoichiometrically inhibits bovine neurotubulin polymerization, Smith [/bib_ref]. Acetaldehyde-protein adducts have been detected within the white matter, frontal cortex, midbrain, dentate gyrus, and cerebellum in ethanol-fed rats, and in the frontal cortex and midbrain of an alcoholic [bib_ref] Evidence of acetaldehyde-protein adduct formation in rat brain after lifelong consumption of..., Rintala [/bib_ref] [bib_ref] Acetaldehyde adducts in the brain of alcoholics, Nakamura [/bib_ref] [bib_ref] Detection and localization of proteinacetaldehyde adducts in rat brain after chronic ethanol..., Upadhya [/bib_ref]. Furthermore, alcohol metabolism may also trigger the production of detrimental protein adducts from products of lipid peroxidation, including 4-hydroxy-2-nonenal (4-HNE), that can bind tubulins and trigger loss of microtubular structures via an increase in their insolubility [bib_ref] The lipid peroxidation product 4-hydroxynonenal inhibits neurite outgrowth, disrupts neuronal microtubules, and..., Neely [/bib_ref] [bib_ref] Residue-specific adduction of tubulin by 4-hydroxynonenal and 4-oxononenal causes cross-linking and inhibits..., Stewart [/bib_ref] [bib_ref] Mechanism of destruction of microtubule structures by 4-hydroxy-2-nonenal, Kokubo [/bib_ref]. In liver, ethanol consumption also increases protein damage as isoaspartate via the impact of ethanol on the methionine metabolic pathway [bib_ref] Accumulation of proteins bearing atypical isoaspartyl residues in livers of alcohol-fed rats..., Kharbanda [/bib_ref] [bib_ref] Proteomics reveal a concerted upregulation of methionine metabolic pathway enzymes, and downregulation..., Kharbanda [/bib_ref]. Ethanol consumption triggers a reduction in the levels of the methyl donor S-adenosylmethionine (SAM), and an increase in the levels of the metabolite S-adenosylhomocysteine (SAH). This reduced SAM:SAH ratio inhibits the activity of many methyltransferases including protein isoaspartyl methyltransferase (PIMT), an enzyme that functions to trigger the repair of isoaspartate damaged proteins [bib_ref] Proteomic identification of novel substrates of a protein isoaspartyl methyltransferase repair enzyme, Vigneswara [/bib_ref] [bib_ref] Formation, localization, and repair of Lisoaspartyl sites in histones H2A and H2B..., Carter [/bib_ref] [bib_ref] The role of protein L-isoaspartyl/D-aspartyl O-methyltransferase (PIMT) in intracellular signal transduction, Furuchi [/bib_ref]. However, it has yet to be determined whether a similar mechanism of ethanol-induced inhibition of PIMT and elevation of isoaspartate damage exists in brain tissue. Regional brain alcohol-induced pathology may impact upon motor-neuron function, and also influence cognitive behaviour. In an attempt to assess protein damage within a brain region involved in cognitive and social behaviour, we first examined postmortem brain tissue from the prefrontal cortex (region BA 9) from 20 human alcoholics and 20 age, gender, and postmortem delay matched control subjects. We compared control and alcoholic neuronal tissue histology, and then employed protein profiling to identify prominent neuronal tissue protein changes. An identification of the major brain protein changes provided an insight into structural damage in alcoholic's brains, for which functional deficits were extrapolated. # Materials and methods ## Human brain samples and ethics statement The human postmortem samples used in this study belong to the brain collection of the Neuropsychopharmacology Research Group at the Department of Pharmacology of the University of the Basque Country (UPV/EHU). (http://www.farmacologia.ehu.es/s0026home/en/contenidos/informacion/s0026_presentacion/en_farm/ presentacion.html). Brain collection is registered in the National Biobank Register of the Spanish Health Department with the number C.0000035 (https://biobancos.isciii.es/ListadoColecciones.aspx). Human brains were obtained at autopsy from 20 alcoholic and 20 control subjects in the Basque Institute of Legal Medicine, Bilbao, Spain. Brain collection was developed in compliance with policies of research and ethical review boards for postmortem brain studies (Basque Institute of Legal Medicine, Bilbao). Spanish legislation at the time of sample collection did not require written informed consent from the next of kin for use of these postmortem samples in research. Furthermore, the analysis of postmortem brain specimens is not defined as human research by the United States Department of Health and Health Services (DHHS) and Food and Drug Administration (FDA) regulations. The diagnosis of alcoholism was carried out according to the Diagnostic and Statistical Manual of Mental Disorders (DSM-III-R, DSM-IV or DSM-IV-TR; American Psychiatric Association) or International Classification of Diseases criteria (ICD-10; World Health Organization). All diagnoses were established by clinicians in charge of the patients prior to death. This group included 20 alcoholic subjects with no other diagnosed psychiatric disease. Each alcoholic case was carefully matched for gender, age and postmortem delay with a control subject who died by sudden and violent cause with no antemortem history of any neurologic or psychiatric disorder. Blood toxicological screening for alcohol and psychotropic drugs was undertaken for each study participant. Tables 1 and 2 summarise the demographic characteristics and the drug history of the subjects included in the microscopy and biochemical studies respectively. Samples from the BA 9 region were macroscopically dissected at the time of autopsy and processed according to the type of study. Brain samples from 10 case-control matched pairs were processed for microscopy, and brain samples from another 10 matched case-control pairs immediately frozen and stored at 280uC until required for biochemical studies. For 5 pairs of BA9 samples used for biochemical studies, additional participant brain tissue from the caudate nucleus, hippocampus, and cerebellum were also obtained. ## Brain tissue sectioning and microscopy Brain tissues were cryosectioned to 8 mm thicknesses using a Microm HM 550 cryostat, and then mounted onto glass slides and stained with H & E. Brain tissue sections were imaged using a Leica DM4000B light microscope fitted with a 100x/1.25NA, 50x/0.9NA, 20x/0.4NA or 10x/0.25NA N Plan objective lens. Images were captured by a MicroPublisher 3.3RTV camera (QImaging) controlled by OpenLab software (Improvision/ PerkinElmer). Regional image capture and analysis were undertaken for all control and alcoholic samples, for which representative images from a control and alcoholic matched pair are included as [fig_ref] Figure 1: Histological examination of prefrontal cortex neuronal cells from controls and alcoholics [/fig_ref]. ## Brain tissue homogenisation Brain tissue samples (,100 mg) were homogenised at 4uC in 20 volumes of phosphate buffer (200 mM Na 2 HPO 4 , 40 mM NaH 2 PO 4 , 1 mM EDTA, pH 7.4) containing protease inhibitor cocktail (Sigma), using an Ultra-Turrax T8 homogeniser (IKA Labortechnik, Germany). Homogenates were centrifuged at 5006g for 10 min at 4uC in a benchtop centrifuge to pellet the nuclear fraction. The supernatant was then centrifuged at 21,3006g for 40 min at 4uC to produce a crude cytosolic preparation. The pellet from this centrifugation, corresponding to the plasma membrane enriched fraction, was resuspended in 10 volumes of the homogenisation buffer. Nuclear, cytosolic, and membrane enriched fractions were flash-frozen and stored at 280uC until required. Protein concentration in cytosolic and membrane fractions was determined by Lowry protein assay (DC, BioRad, UK) using bovine serum albumin (BSA) as a protein standard. ## One dimensional polyacrylamide gel electrophoresis (1d-page) Protein homogenate was separated on 10% Bis-Tris NuPAGE Novex pre-cast gels (20 mg/gel lane) run with 2-(N-morpholino)ethanesulfonic acid (MES) running buffer as described previously [bib_ref] Proteomic identification of novel substrates of a protein isoaspartyl methyltransferase repair enzyme, Vigneswara [/bib_ref]. Resolved proteins were either stained with colloidal Coomassie blue and then densitometry performed using an Odyssey laser scanner (LI-COR Biosciences, Lincoln, NE), or proteins electroblotted at 80 V for 2 hours to a polyvinylidene difluoride (PVDF) (Millipore) membrane for Western blotting. All protein homogenates were resolved 2-3 times by gel electrophoresis, and differentially expressed proteins excised from gels and identified by mass spectrometry. Similarly, protein extracts were all resolved 2-3 times for Western blotting analysis, with representative figures from protein staining and blotting experiments included as Figures. ## Matrix assisted laser-desorption ionisation-time of flight (maldi-tof) mass spectrometry Differentially stained proteins were excised from gels and analysed by MALDI-TOF mass spectrometry using a Micromass MALDI (Waters, UK) as described previously [bib_ref] Analytical approaches to investigate protein-pesticide adducts, Carter [/bib_ref]. ## Liquid chromatography and tandem mass spectrometry (lc ms/ms) Tryptic peptides from excised protein bands were run on a Waters QTOF2 hybrid quadrupole mass spectrometer incorporating an integrated capillary LC system as described previously [bib_ref] Proteomic identification of novel substrates of a protein isoaspartyl methyltransferase repair enzyme, Vigneswara [/bib_ref]. Further details detailing peptides identified by MALDI-TOF mass spectrometry and LC MS/MS are included as Data S1. Western (immuno) blotting: PVDF membranes were stained with Coomassie blue (Safestain, Invitrogen), and then destained with 50% methanol (v/v), 10% acetic acid (v/v) to visualise proteins, and ensure even protein transfer across the blot. Membranes were then washed in phosphate buffered saline (PBS) containing 0.05% (v/v) Tween 20 (PBS-T), blocked for 1 h at room temperature with 5% (w/v) milk protein in wash buffer, and then incubated overnight at 4uC with the primary antibody of interest in blocking buffer. The antibodies used in this study were directed against the following proteins, and at the dilutions specified: goat polyclonal to spectrin b II (Santa Cruz, sc-7468) at 1:250; rabbit polyclonal to btubulin (Santa Cruz, sc-9104) at 1:250; mouse monoclonal to atubulin (Santa Cruz, sc-8035) at 1:250; mouse monoclonal to acetylated a-tubulin (Santa Cruz, sc-23950) at 1:250; mouse monoclonal to protein-L-isoaspartate O-methyltransferase (Santa Cruz, sc-100977) at 1:500; rabbit polyclonal to actin (Santa Cruz, sc-1616-R) at 1:1000; mouse monoclonal to the a1 catalytic subunit of the Na + ,K + -ATPase (Abcam, ab7671) at 1:500. A mouse monoclonal antibody to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Abcam, ab8245) was used at 1:3000 as a gel loading control for cytosols, and rabbit polyclonal to actin (Santa Cruz, sc-1616-R) at 1:1000 as a gel loading control for membrane fractions. Blots were washed in PBS-T and then incubated for 1 hour at room temperature with their corresponding horseradish peroxidase conjugated anti-species secondary antibody (polyclonal goat anti-rabbit, goat anti-mouse or rabbit anti-goat immunoglobulins, all purchased from Dako, products P0448, P0447, P0449 respectively) at 1:1000 dilution in PBS-T. Antibody immunoreactivity was visualised using SuperSignal West Pico Chemiluminescent substrate (Pierce) with the light generated captured on CL-Xposure X-ray film (Pierce), or using a Chemi Doc device (Bio-Rad). Films were densitometrically scanned using a CanoScan LiDE 700F flatbed scanner (Canon), and the levels of proteins quantified using the public domain image processing program ImageJ, developed at the National Institutes of Health. Alternatively, blots were quantified using QuantityOne software intrinsic to the Chemi Doc charge-coupled device. The results are expressed as GAPDH or actin normalised values. The mean signal density for quantitation of control samples was set at 100% of protein signal. Blots probed for multiple antibody analyses were intermittently antibody stripped with stripping buffer (Restore, Thermo Scientific) by shaking at 125 rpm at 50uC for 30 minutes. ## Quantification of isoaspartate levels The level of isoaspartate in control and alcoholic cytosolic extracts was quantified based upon the method of Aswad and Deight [bib_ref] Endogenous substrates for protein carboxyl methyltransferase in cytosolic fractions of bovine brain, Aswad [/bib_ref]. Protein extracts (100-200 mg of protein) were incubated in a final reaction volume of 100 ml, containing 40 mM K-MES, pH 6.2, 20 mM S-Adenosyl-L-[ 3 H-methyl]methionine ( 3 H-SAM) (8250 dpm/pmol, final specific activity), and 2 mM recombinant PIMT (Promega). Methylation reactions were initiated by the addition of PIMT and incubation for 30 minutes at 30uC. Methylation was terminated by the addition of 1 ml of ice-cold 7% (w/v) trichloroacetic acid (TCA), and protein precipitation on ice for 10 minutes. Protein precipitate was retained by centrifugation for 5 minutes in a refrigerated centrifuge (Eppendorf 5415R) at maximum speed (16 1006g). The supernatant containing unincorporated 3 H-SAM was removed to waste, and then the precipitate washed with 100 ml of . Demographic characteristics, postmortem delay (PMD), cause of death, and blood toxicological screening of the control (C) and alcoholic (A) subjects analysed by light microscopy. . Demographic characteristics, postmortem delay (PMD), cause of death, and blood toxicological screening of the control (C) and alcoholic (A) subjects used for biochemical studies. The * indicates the subjects for which caudate nucleus, hippocampus, and cerebellum samples were obtained in addition to the prefrontal cortex. doi:10.1371/journal.pone.0093586.t002 88% (v/v) formic acid. One ml of 7% TCA was added and protein precipitation performed as before. Precipitate was again collected by centrifugation and extraneous unincorporated 3 H-SAM removed. The pellet was washed as previous with 100 ml of 88% (v/v) formic acid, and then precipitation with 1 ml of 7% TCA repeated. The protein precipitate was then solubilised in 100 ml of 0.5 M NaOH, 1% (v/v) methanol, 5% (v/v) Triton X-100, and transferred to 10 ml of scintillant and counted for radioactivity incorporation using a Packard Tri-Carb counter. Replicate assays were performed for all assay points from which an average value of radiolabel incorporation determined. Radiolabel incorporation into brain extracts in the absence of PIMT addition, due to endogenous methylation was subtracted from assay values. Isoaspartate quantitation was performed on the 10 matched pairs of control and alcoholic brains. ## Proteasome activity assay Control or alcoholic brain tissue was homogenised in a buffer of 20 mM Tris-HCl pH 7.5, 2 mM ATP, 5 mM MgCl 2 , 1 mM DTT at 4uC. The homogenate was centrifuged at 10, 0006g to pellet cellular debris, and the supernatant transferred to a fresh eppendorf. Fifty mg of homogenate was assayed for proteolytic Alcohol-Related Brain Damage in Humans PLOS ONE | www.plosone.org activity against N-succinnyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin by fluorimetry (excitation wavelength 360 nm, emission wavelength 460 nm). Fluorescent assays were performed in duplicate from which an average value was obtained. ## Statistics Statistical comparison between data means was performed using a Student's t-test using GraphPad Prism 4 software, with results expressed as means 6 SEM. A p value of ,0.05 was regarded as statistically significant. # Results ## Histological examination of prefrontal cortex neuronal cells from controls and alcoholics To assess the presence of structural changes within BA 9 tissue that may arise from cumulative ethanol intoxications, we examined cells within brain tissue slices from 10 alcoholics and 10 control subjects matched for age, gender and postmortem delayrefer to . Light microscopy of cells within the cortical pyramidal cell layers revealed that cells from alcoholics exhibited disrupted cytoplasm surrounding their nuclei. These differences were particularly evident with a 100x lens -refer to arrows in [fig_ref] Figure 1: Histological examination of prefrontal cortex neuronal cells from controls and alcoholics [/fig_ref] , upper two panels. An examination of subcortical neurons within the white matter of the alcoholic brain tissue slices also highlighted a reduction in the level of organised cytoplasm surrounding the nuclei. Additionally, subcortical neurons from alcoholics lacked the ordered parallel arrangement of cells seen with control subjects. These differences were well distinguished with a 50x lens -refer to arrows in [fig_ref] Figure 1: Histological examination of prefrontal cortex neuronal cells from controls and alcoholics [/fig_ref] , lower two panels. ## Profiling of cytosolic proteins from the prefrontal cortex of control and alcoholic subjects and western blotting To investigate further the reduced and disorganised cytoplasm or cytoskeleton observed for alcoholic brain tissue, we homogenised BA 9 tissue from control and alcoholics and examined protein profiles by 1D PAGE. Due to limited sample availability, and to further validate this observation with more subjects, 1D PAGE was performed with an additional population of 10 matched control and alcoholic subjects, the details of which are listed in . Visual inspection of 1D PAGE protein profiling of cytosolic proteins revealed only two major recurrent protein level differences between control and alcoholic groups. There was a prominent reduction in the level of an ,270 kDa protein and an ,50 kDa protein, and these changes were visible in all alcoholics when compared to their control matched counterparts -refer to [fig_ref] Figure 2: Profiling of cytosolic proteins from the prefrontal cortex of control and alcoholic... [/fig_ref] , upper section. Protein bands at these molecular weights from six individuals were removed and proteins identified by mass spectrometry. The ,270 kDa protein band was identified as spectrin b II, and the ,50 kDa protein band identified as a mixture of peptides from different forms of a-tubulin and b-tubulin -refer to [fig_ref] Table 3: Mass spectrometry identification of differentially expressed proteins from the prefrontal cortex of... [/fig_ref] and Data S1. To validate and quantify this dramatic loss of the cytoskeletal proteins b-spectrin, and aand btype tubulins specifically within alcoholic brain tissue, we assessed their protein levels by Western blotting - [fig_ref] Figure 2: Profiling of cytosolic proteins from the prefrontal cortex of control and alcoholic... [/fig_ref] lower section. Western blot analyses confirmed a significant 36% decrease in spectrin b II (p = 0.0002), significant 56% decrease in a-tubulin (p = 0.0017) and significant 83% decrease in b-tubulin levels (p,0.0001) for the 10 alcoholic subjects - [fig_ref] Figure 3: Quantification of protein level and activity differences between control and alcoholic subjects [/fig_ref]. The alcoholic subjects for which the level of either aor b-tubulins were below the Western blotting detection threshold have been expressed as 100% protein loss. Cytosolic GAPDH levels were stable throughout all control and alcoholic subjects investigated and were used for protein normalisation. The cellular cytoskeletal network is composed of microtubules, intermediate filaments, and microfilaments. The major protein constituent of microfilaments is actin, hence we also quantified actin protein levels to determine if cytoskeletal loss was extended to actin in microfilaments. Actin levels increased by 16% in alcoholic subjects, but this was not significant -Figures 2 & 3. ## Examination of protein ptm and damage in control and alcoholic brain tissue We investigated possible PTMs of tubulins that may have contributed to an alteration of their cytosolic level, or that may influence tubulin stability or turnover. We quantified the level of acetylated a-tubulin in all cytosolic protein samples by Western blotting with an antibody that specifically recognizes acetylated Lysine-40- [fig_ref] Figure 2: Profiling of cytosolic proteins from the prefrontal cortex of control and alcoholic... [/fig_ref]. The relative proportion of acetylated atubulin to total a-tubulin was quantified as a significant 56% increase (p = 0.0094) for alcoholic subjects when compared to their matched controls - [fig_ref] Figure 3: Quantification of protein level and activity differences between control and alcoholic subjects [/fig_ref]. Secondly, aand b-tubulins can accumulate isoaspartate damage in vivo if PIMT activity is inhibited or removed [bib_ref] Proteomic identification of novel substrates of a protein isoaspartyl methyltransferase repair enzyme, Vigneswara [/bib_ref] [bib_ref] Molecular aging of tubulin: accumulation of isoaspartyl sites in vitro and in..., Najbauer [/bib_ref] [bib_ref] Protein repair in the brain, proteomic analysis of endogenous substrates for protein..., Zhu [/bib_ref]. We quantified the levels of total isoaspartate protein damage in the cytosolic proteins from control and alcoholic brains, and also the levels of cytosolic PIMT enzyme by Western blotting - [fig_ref] Figure 2: Profiling of cytosolic proteins from the prefrontal cortex of control and alcoholic... [/fig_ref] There was a 9% increase in total cytosolic isoaspartate levels within the alcoholic brains but this did not reach significance. In contrast there was a significant 28% increase (p = 0.0438) in cytosolic PIMT protein levels in alcoholics. Due to the relatively low total levels of isoaspartate within control or alcoholic brains, an examination of the specific levels of isoaspartate protein damage within aand b-tubulins was not undertaken. The proteasome complex is responsible for the turnover of the majority of cytosolic proteins including tubulins [bib_ref] Parkin binds to alpha/beta tubulin and increases their ubiquitination and degradation, Ren [/bib_ref] [bib_ref] Assembly, structure, and function of the 26S proteasome, Bedford [/bib_ref]. Hence we also quantified proteasomal activity of brain tissue from the 10 control and 10 alcoholic subjects. There was a significant 25% reduction in proteasomal activity in the alcoholic subjects (p = 0.0426) -refer to [fig_ref] Figure 3: Quantification of protein level and activity differences between control and alcoholic subjects [/fig_ref]. We also examined the levels of aand b-tubulin within nuclear and membrane fractions to assess whether the reduced cytosolic protein levels seen in alcoholics arose from protein translocation to these other cellular compartments. After 1D PAGE and Western blotting there were no significant changes in the levels of aor btubulins in the nuclear or membrane fractions of alcoholic brain tissue when compared to control subjects - [fig_ref] Figure 4: Western blot of aand b-tubulins in nuclear and membrane fractions from the... [/fig_ref]. This suggested that a and b-tubulin protein loss in the prefrontal cortex of alcoholics was limited to the cytosol. ## Profiling of membrane-fraction proteins from the prefrontal cortex of control and alcoholic subjects One dimensional PAGE profiling of proteins from the membrane fractions of control and alcoholic subjects resulted in the detection of two visually prominent protein changes. There was a reduction in the protein levels of an ,270 kDa protein in alcoholic subjects, and an increase in the levels of a protein doublet at ,112 kDa protein - [fig_ref] Figure 5: Profiling of membrane-fraction proteins from the prefrontal cortex of control and alcoholic... [/fig_ref] upper panel. Protein bands at these molecular weights from 5 control and 5 alcoholic subjects were excised from gels and analysed by mass spectrometry. Similar to the cytosolic protein analysis, the reduced ,270 kDa protein was identified as spectrin b II. Both protein doublet bands at ,112 kDa protein were identified as the a3 subunit of the Na + ,K + -ATPase -refer to [fig_ref] Table 3: Mass spectrometry identification of differentially expressed proteins from the prefrontal cortex of... [/fig_ref] , and Data S1. In concurrence with the protein staining data for the Na + ,K + -ATPase a-subunit, Western blotting confirmed a broader immunoreactivity of monomeric, membrane-associated a-subunit in alcoholic subjects; quantified as a combined 31% significant increase (p = 0.0002). Western blotting also showed that oligomeric forms of a-subunit present in control subjects were virtually absent in alcoholics, suggesting a functional deficit in a-a oligomerisation - [fig_ref] Figure 5: Profiling of membrane-fraction proteins from the prefrontal cortex of control and alcoholic... [/fig_ref] middle and lower panels. ## Profiling of cytosolic proteins from the caudate nucleus, hippocampus and cerebellum of control and alcoholic subjects We wanted to ascertain if loss of aand b-tubulins in alcoholic brains was a regional phenomenon restricted to the prefrontal cortex. Thus for those brain regions for which we also had access to additional tissues, we examined the levels of cytosolic aand btubulins within caudate nucleus, hippocampus, and cerebellum from 5 matched pairs of control and alcoholic subjects (subject details included in . Cytosolic proteins from each of the brain regions were resolved by 1D PAGE and Western blotted with the specific anti-a-and anti-b-tubulin antibodies - [fig_ref] Figure 6: Profiling of cytosolic proteins from the caudate nucleus, hippocampus and cerebellum of... [/fig_ref] upper and middle panels. Quantitation of Western blots showed a significant reduction in the total levels of aand b-tubulins in all brain regions, but the level of reductions were region specific. There was a significant 30% decrease (p = 0.0198) in a-tubulin and a significant 31% decrease (p = 0.0081) in b-tubulin in the caudate nucleus. There was a significant 50% (p = 0.0004) and 47% (p = 0.0054) decrease in aand b-tubulin respectively in the hippocampus, and a significant 54% (p = 0.0044) and 36% (p = 0.0486) decrease in aand b-tubulin in the cerebellum. However, the prefrontal cortex displayed the greatest reductions in a-tubulin (56% decrease, p = 0.0002) and b-tubulin levels (84%, p,0.0001) - [fig_ref] Figure 6: Profiling of cytosolic proteins from the caudate nucleus, hippocampus and cerebellum of... [/fig_ref] lower panels. # Discussion An examination of the molecular abnormalities that arise as a consequence of cumulative ethanol intoxications will assist with an understanding of the development of tissue pathology. In addition, a characterisation of ethanol-induced molecular changes may also provide an insight into the molecular adaptations associated with tolerance, dependence, and an alcoholic's behavioural abnormalities. Collectively, research in this field will provide a basis for targeted therapeutics that counter debilitating morbidity, and lower the incidence of mortality. We assessed alcohol-related neuronal tissue damage of the prefrontal cortex using light microscopy, and were intrigued to see clear histological distinction between cells of alcoholics and their matched controls. This led us to undertake one dimensional protein profiling of cytosolic proteins from alcoholics and controls and we identified the major protein losses as that from aand btubulins, and spectrin b-chain. Alpha and b-tubulins localise as heterodimers, and are major components of microtubules. Microtubules are non-covalent cytoskeletal polymers that provide a cellular protein network. Microtubules influence cell shape, motility, and stability, and are central to the functioning of countless cellular processes including mitosis, and vesicular transport [bib_ref] Microtubule assembly, organization and dynamics in axons and dendrites, Conde [/bib_ref] [bib_ref] Post-translational regulation of the microtubule cytoskeleton: mechanisms and functions, Janke [/bib_ref]. Microtubules exist as both dynamic and stable polymers, with transitions between these states in sub-populations of microtubules influenced by PTMs and microtubule-associated proteins (MAPs) [bib_ref] Microtubule assembly, organization and dynamics in axons and dendrites, Conde [/bib_ref] [bib_ref] Post-translational regulation of the microtubule cytoskeleton: mechanisms and functions, Janke [/bib_ref]. Hence the loss of functional tubulins seen in the BA 9 tissue of alcoholics would be expected to result in a disruption of the cytoskeletal network, and impinge on the many cellular trafficking processes that are tethered to the cytoskeletal architecture. Similar to tubulins, spectrins are a family of proteins that are major structural components of the cytoskeleton. Spectrins (as a and b heterodimers) act as molecular scaffolds that associate with the cytoplasmic surface of the plasma membrane, and form a molecular lattice that links the plasma membrane to actin and the microtubule cytoskeleton [bib_ref] The neural cell spectrin skeleton: a review, Goodman [/bib_ref] [bib_ref] Association of brain spectrin isoforms with microtubules, Riederer [/bib_ref]. Spectrin functions include maintenance of cell shape, and arrangement of transmembranous proteins. Additionally, brain spectrins function in the association of vesicles to the microtubular network, and influence synaptic vesicle stabilization and release [bib_ref] The neural cell spectrin skeleton: a review, Goodman [/bib_ref] [bib_ref] Presynaptic spectrin is essential for synapse stabilization, Pielage [/bib_ref]. Hence the reduction in spectrin levels seen with alcoholic subjects would also likely disrupt cellular trafficking processes, and the triggering of synaptic neurotransmission events. Thus collectively, we propose that a reduction of the cytoskeletal architecture provides a rationale for the profound differences in the prefrontal cortex neuronal histology of alcoholics, and likely contributes to the cognitive and learning impairments experienced by alcoholics. We next considered molecular mechanisms that could explain the selective damage and loss of these cytoskeletal proteins in alcoholics. Ethanol can be converted in the cytosol to acetaldehyde via the action of alcohol dehydrogenase, and in brain this conversion may also be undertaken by catalase and cytochrome P450 enzymes [bib_ref] Brain metabolism of ethanol and alcoholism: an update, Hipolito [/bib_ref]. Acetaldehyde is a reactive compound and readily adducts a number of cytosolic proteins including tubulins [bib_ref] Acetaldehyde and microtubules, Tuma [/bib_ref] [bib_ref] Acetaldehyde substoichiometrically inhibits bovine neurotubulin polymerization, Smith [/bib_ref]. We detected an ,1.5-fold increase in the ratio of acetylated a-tubulin to total a-tubulin in the alcoholics. Similarly, an increase in the relative proportion of acetylated a-tubulin (to total a-tubulin) within liver or liver cells as a consequence of ethanol consumption or exposure has been reported [bib_ref] Alcohol-induced alterations of the hepatocyte cytoskeleton, Shepard [/bib_ref]. In liver, this increased a-tubulin acetylation influences microtubule hyperstabilisation and inertness, with an associated impairment of protein trafficking [bib_ref] Alcohol-induced alterations of the hepatocyte cytoskeleton, Shepard [/bib_ref] [bib_ref] Alcohol-induced protein hyperacetylation: mechanisms and consequences, Shepard [/bib_ref] , but at present the influence of ethanol consumption on brain tubulin acetyltransferases or deacetylases, and the functional consequences of increased tubulin acetylation have not been determined. Another potential source of ethanol-induced damage to aand b-tubulin could arise through an increase in their protein damage as isoaspartate. We detected a 9% increase in total cytosolic isoaspartate levels within the alcoholic brains, although this did not reach significance. It is still feasible that isoaspartate levels may increase significantly specifically within aand b-tubulins, but since the total levels of cellular isoaspartate were low, quantitation of individual protein isoaspartate levels was not attempted. An increase in isoaspartate protein damage in alcoholic subjects could be countered by upregulation of cellular PIMT levels to trigger isoaspartate repair. We detected a significant 28% increase in cytosolic PIMT protein levels in alcoholic tissue. Similarly, a proteomic study of synaptic proteome changes in the superior frontal gyrus (SFG) and occipital cortex (OC) of control and alcoholic postmortem tissue reported a significant 30% increase in PIMT protein levels in the SFG and a 50% increase in PIMT protein levels in the OC of alcoholic subjects [bib_ref] Synaptic proteome changes in the superior frontal gyrus and occipital cortex of..., Etheridge [/bib_ref]. The molecular mechanism by which PIMT protein levels are elevated has yet to be determined, and it will be of interest to establish if it reflects a compensatory means to counter an increase in cellular stress and isoaspartate protein damage. The 26S proteasome complex is the predominant cellular protease that target proteins for degradation after their prior ubiquitination [bib_ref] Assembly, structure, and function of the 26S proteasome, Bedford [/bib_ref]. Additionally, oxidised, modified and/or damaged proteins may also be degraded by 20S proteasomes directly [bib_ref] Protein degradation and protection against misfolded or damaged proteins, Goldberg [/bib_ref] [bib_ref] The proteasome and its role in the degradation of oxidized proteins, Jung [/bib_ref]. Hence one possible mechanism to explain the loss of these cytoskeletal proteins is that ethanol induces PTMs and protein damage, and this triggers protein elimination by the proteasome. However, when quantified there was a significant 25% fall in the proteasome activity of alcoholic tissue. Thus although the proteasome may still target clearance of these proteins, its global cellular activity was compromised in the brains of alcoholics. The reduced levels of cytosolic proteins did not reflect translocation to either the nuclear or membrane fractions. Protein profiling of the membrane fraction also demonstrated that spectrin b II was present at a reduced level in the membrane-enriched fraction from alcoholics. The membrane-associated a-subunit of the Na + ,K + -ATPase was visualised and evidenced as two protein bands of similar molecular weight. Western blotting enabled us to quantify a significant increase in the transmembranous level of the a-subunit of the Na + ,K + -ATPase, which presumably reflected immunoreactivity of both bands of the protein doublet. The Na + ,K + -ATPase catalyses the hydrolysis of ATP coupled to the asymmetric exchange of intracellular Na + for extracellular K + across the plasma membrane, and participates in generation and maintenance of a membrane potential [bib_ref] Isozymes of the Na-K-ATPase: heterogeneity in structure, diversity in function, Blanco [/bib_ref]. Native Na + ,K + -ATPase is heterotrimeric, composed of three subunits: a, b, and c. The a-subunit is the catalytically active ion transporting subunit. The a1 isoform is ubiquitously distributed, whereas the a3 isoform is expressed in neurons [bib_ref] Isozymes of the Na-K-ATPase: heterogeneity in structure, diversity in function, Blanco [/bib_ref] [bib_ref] Na,K-ATPase and the role of alpha isoforms in behavior, Lingrel [/bib_ref]. Western blotting with the Na + ,K + -ATPase a-subunit antibody revealed that stable higher molecular weight (oligomeric) forms of the a-subunit present in controls were absent (or just visible with long Western blot exposures) in alcoholic subjects. The existence of a-a oligomers that are resistant to SDS-PAGE conditions have been documented by independent groups [bib_ref] The alpha subunit of the Na,K-ATPase specifically and stably associates into oligomers, Blanco [/bib_ref] [bib_ref] Thermal denaturation of the Na,K-ATPase provides evidence for alpha-alpha oligomeric interaction and..., Donnet [/bib_ref]. Ethanol exposure can partially inhibit Na + ,K + -ATPase pump activity in the cerebellum of rats, although the precise mechanism for pump inhibition has yet to be defined [bib_ref] Alcohol excites cerebellar Golgi cells by inhibiting the Na+/K+ ATPase, Botta [/bib_ref]. Defective Na + ,K + -ATPase a-subunits result in a rapid-onset dystonia Parkinsonism, and other behavioural and cognitive deficits [bib_ref] Na,K-ATPase and the role of alpha isoforms in behavior, Lingrel [/bib_ref] [bib_ref] Mutations in the Na+/K+ -ATPase alpha3 gene ATP1A3 are associated with rapid-onset..., De Carvalho Aguiar [/bib_ref] [bib_ref] Deficiency in Na,K-ATPase alpha isoform genes alters spatial learning, motor activity, and..., Moseley [/bib_ref] [bib_ref] Mania-like behavior induced by genetic dysfunction of the neuron-specific Na+, K+-ATPase alpha3..., Kirshenbaum [/bib_ref]. Hence it is provocative to postulate that alterations in a-subunit levels or function and/or oligomerisation could also contribute to some of the behavioural symptoms exhibited by alcoholics. We also examined tissue from additional brain regions and detected a significant reduction in aand b-tubulin protein in the caudate nucleus, hippocampus, and cerebellum, but not to the levels of protein loss recorded for the prefrontal cortex. The differential vulnerability of brain regions could reflect varied penetrance across the blood brain barrier of the toxic metabolites of ethanol, such as acetaldehyde and/or their specific regional production. # Study limitations Although we have undertaken a quantitative evaluation of protein levels using Western blotting we appreciate this is not without methodological limitations. Antibody binding to a target protein may be masked or sterically hindered due to the presence of PTMs. This provides a rationale to explain why the antibody directed against acetylated a-tubulin exhibited a higher immunoreactivity signal than the antibody directed against the full-length a-tubulin protein -refer to [fig_ref] Figure 2: Profiling of cytosolic proteins from the prefrontal cortex of control and alcoholic... [/fig_ref]. Hence the total levels of cytoskeletal protein degradation in alcoholic brain tissue are clearly significant (and visibly prominent after 1D-PAGE and protein staining), but levels of protein degradation may possibly be overestimated using immunological reactivity. Direct comparison with other proteomic studies of alcoholic tissue from the prefrontal cortex is problematic due to differences in alcoholic subject age, cumulative alcohol intake, as well as variability of regional sampling, protein preparation and methodology etc [bib_ref] The application of proteomics to the human alcoholic brain, Lewohl [/bib_ref] [bib_ref] Differential protein expression in the prefrontal white matter of human alcoholics: a..., Alexander-Kaufman [/bib_ref] [bib_ref] Proteomics approach in the study of the pathophysiology of alcohol-related brain damage, Matsumoto [/bib_ref]. Nevertheless, other independent two-dimensional (2D) proteomic studies have also reported reductions in alcoholic's brains of the protein levels of aand b-tubulins [bib_ref] The application of proteomics to the human alcoholic brain, Lewohl [/bib_ref]. Within our study patients differ in many parameters such as blood toxicology at death, but by matching each pair of controls and alcoholics for age, gender, and postmortem delay, robust and universal changes in protein levels and modifications were revealed. A more substantive proteomic analysis by 2D separation techniques may reveal additional protein level changes, however, we were primarily concerned with prominent visual differences for which a related function could be postulated; hence a comprehensive 2D-PAGE analysis was considered beyond the scope of this primary publication. Furthermore, 2D proteomic studies utilise protein fractionation and protein precipitation methods to enrich components of the proteome for analyses [bib_ref] Synaptic proteome changes in the superior frontal gyrus and occipital cortex of..., Etheridge [/bib_ref] [bib_ref] The application of proteomics to the human alcoholic brain, Lewohl [/bib_ref] [bib_ref] Differential protein expression in the prefrontal white matter of human alcoholics: a..., Alexander-Kaufman [/bib_ref] [bib_ref] Proteomics approach in the study of the pathophysiology of alcohol-related brain damage, Matsumoto [/bib_ref] , and this could mask protein differences visible by primary 1D PAGE screening. An insight into changes within postmortem tissue of the frontal cortex of alcoholic subjects has also been provided using gene arrays [bib_ref] Gene expression in human alcoholism: microarray analysis of frontal cortex, Lewohl [/bib_ref] [bib_ref] Patterns of gene expression in the frontal cortex discriminate alcoholic from nonalcoholic..., Liu [/bib_ref] [bib_ref] Altered gene expression profiles in the frontal cortex of cirrhotic alcoholics, Liu [/bib_ref]. These studies document numerous changes in gene expression levels that include reduced expression of elements of the ubiquitin-proteasome system in alcoholics [bib_ref] Gene expression in human alcoholism: microarray analysis of frontal cortex, Lewohl [/bib_ref] [bib_ref] Patterns of gene expression in the frontal cortex discriminate alcoholic from nonalcoholic..., Liu [/bib_ref] [bib_ref] Altered gene expression profiles in the frontal cortex of cirrhotic alcoholics, Liu [/bib_ref] , and reduced expression of a beta III spectrin in cirrhotic alcoholics [bib_ref] Altered gene expression profiles in the frontal cortex of cirrhotic alcoholics, Liu [/bib_ref]. Thus it remains likely that significant protein level changes evidenced in the prefrontal cortex of alcoholic subjects arise from alteration of transcriptional and translational mechanisms. In summary, we report profound reductions in the levels of the cytoskeletal proteins aand b-tubulin, and spectrin b II in alcoholic subjects that correlate with altered neuronal cell organisation and cell patterning visible by light microscopy. The known susceptibility of these proteins to protein damage or regulatory PTMs provides a putative mechanism to explain their targeted loss of protein level and function. Altered protein level and PTMs of the cytoskeletal architecture, and also disruption of the functional activity of the Na + ,K + -ATPase provides a molecular basis for some of the altered neuronal and behavioural manifestations encountered by alcoholics, and may also contribute to the atrophic brain macrostructure that is phenotypic of an alcoholic. ## Supporting information Data S1 MALDI-TOF and LC-MS/MS data for atubulin, b-tubulin, spectrin b-chain, and the a3-subunit of Na + ,K + -ATPase. (DOCX) [fig] Figure 1: Histological examination of prefrontal cortex neuronal cells from controls and alcoholics. Cortical pyramidal cells (upper two panels), and subcortical neurons (lower two panels) of control and alcoholic tissue sections were visualised by light microscopy. White bar represents 50 mm for 10x, 10 mm for 50x, and 5 mm for 100x lenses. Examples of the differences in the cytoplasm surrounding the nuclei of cells from alcoholic or control tissue sections have been marked with white arrows. doi:10.1371/journal.pone.0093586.g001 [/fig] [fig] Figure 2: Profiling of cytosolic proteins from the prefrontal cortex of control and alcoholic subjects. Control (C) or alcoholic (A) prefrontal cortex cytosolic proteins were resolved by 1D PAGE and proteins stained with colloidal Coomassie. Alcoholic subjects displayed a prominent reduction in the level of protein staining at protein bands of ,270 kDa and ,50 kDa (upper panel). Protein levels were visualized by Western blotting (lower panel). Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PIMT, protein-L-isoaspartate O-methyltransferase. doi:10.1371/journal.pone.0093586.g002 [/fig] [fig] Figure 3: Quantification of protein level and activity differences between control and alcoholic subjects. Protein levels were quantified by Western blotting, and the levels of isoaspartate protein damage and proteasome activity determined. For quantitation of the ratio of acetylated atubulin to total a-tubulin, the figure is representative of those subjects that displayed visible total a-tubulin signal (6 of the 10 subjects assayed). For marked significance: * = p,0.05, ** = p,0.01, *** = p,0.001. Abbreviations: PIMT, protein-L-isoaspartate O-methyltransferase. doi:10.1371/journal.pone.0093586.g003 [/fig] [fig] Figure 4: Western blot of aand b-tubulins in nuclear and membrane fractions from the prefrontal cortex of control and alcoholic subjects. Control (C) or alcoholic (A) prefrontal cortex nuclear and membrane fractions were resolved by 1D PAGE and proteins Western blotted for aand b-tubulins. doi:10.1371/journal.pone.0093586.g004 [/fig] [fig] Figure 5: Profiling of membrane-fraction proteins from the prefrontal cortex of control and alcoholic subjects. Control (C) or alcoholic (A) prefrontal cortex membrane fraction proteins were resolved by 1D PAGE and proteins stained with colloidal Coomassie. Alcoholic subjects displayed a prominent reduction in the level of protein staining at a protein band of ,270 kDa and upregulation at ,112 kDa (upper panel). Protein levels of transmembranous (,112 kDa) Na + ,K + -ATPase a-subunit, and oligomeric forms of the a-subunit were visualized by Western blotting (middle panel). The level of transmembranous a-subunit was quantified using actin for normalisation (lower panel). For marked significance: *** = p,0.001. doi:10.1371/journal.pone.0093586.g005 [/fig] [fig] Figure 6: Profiling of cytosolic proteins from the caudate nucleus, hippocampus and cerebellum of control and alcoholic subjects. Control (C) or alcoholic (A) cytosolic proteins from caudate nucleus, hippocampus, and cerebellum were resolved by 1D PAGE and proteins stained with colloidal Coomassie (upper panel). The positions of the ,50 kDa aand b-tubulin protein bands are marked with arrows. Cytosolic proteins were Western blotted for aand b-tubulin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (middle panel), and protein level changes quantified using GAPDH for normalization (lower panel). For marked significance: * = p,0.05, ** = p,0.01, *** = p,0.001. doi:10.1371/journal.pone.0093586.g006 [/fig] [table] Table 3: Mass spectrometry identification of differentially expressed proteins from the prefrontal cortex of control and alcoholic subjects. [/table]
Determinants of an autism spectrum disorder diagnosis in childhood and adolescence: Evidence from the UK Millennium Cohort Study Characteristics of the MCS sample by the timing of diagnosis for autism spectrum disorder Characteristics of the MCS sample by the timing of diagnosis for autism spectrum disorder Supplementary Table S1 Total MCS sample at age 5 interview (N = 15,431) a ASD (n = 581) No diagnosis of ASD by age 14 (n = 14,850) Note. Adjusted Relative risk ratios (95% confidence intervals) are reported. a The Before school group is taken as reference. b Significant difference between During primary school group and During secondary school group. All analyses adjusted for multiple birth indicator.
Leveraging community health worker system to map a mountainous rural district in low resource setting: a low-cost approach to expand use of geographic information systems for public health Background: Geographic Information Systems (GIS) have become an important tool in monitoring and improving health services, particularly at local levels. However, GIS data are often unavailable in rural settings and village-level mapping is resource-intensive. This study describes the use of community health workers' (CHW) supervisors to map villages in a mountainous rural district of Northern Rwanda and subsequent use of these data to map village-level variability in safe water availability.Methods: We developed a low literacy and skills-focused training in the local language (Kinyarwanda) to train 86 CHW Supervisors and 25 nurses in charge of community health at the health center (HC) and health post (HP) levels to collect the geographic coordinates of the villages using Global Positioning Systems (GPS). Data were validated through meetings with key stakeholders at the sub-district and district levels and joined using ArcMap 10 Geo-processing tools. Costs were calculated using program budgets and activities' records, and compared with the estimated costs of mapping using a separate, trained GIS team. To demonstrate the usefulness of this work, we mapped drinking water sources (DWS) from data collected by CHW supervisors from the chief of the village. DWSs were categorized as safe versus unsafe using World Health Organization definitions. Result: Following training, each CHW Supervisor spent five days collecting data on the villages in their coverage area. Over 12 months, the CHW supervisors mapped the district's 573 villages using 12 shared GPS devices. Sector maps were produced and distributed to local officials. The cost of mapping using CHW supervisors was $29,692, about two times less than the estimated cost of mapping using a trained and dedicated GIS team ($60,112). The availability of local mapping was able to rapidly identify village-level disparities in DWS, with lower access in populations living near to lakes and wetlands (p < .001). Conclusion: Existing national CHW system can be leveraged to inexpensively and rapidly map villages even in mountainous rural areas. These data are important to provide managers and decision makers with local-level GIS data to rapidly identify variability in health and other related services to better target and evaluate interventions. # Background The spread of Geographic Information Systems (GIS)a set of tools to capture, store, transform, analyse, and display spatial data -has improved spatial analysis of health related services and population health [bib_ref] The application of geographical information systems to important public health problems in..., Tanser [/bib_ref]. Health data combined with geographic information allows us to analyse the spatial variation of diseases burden, mortality, morbidity, physical access to health care and social or environmental determinants of health outcomes [bib_ref] Geographical Information System (GIS) as a tool for monitoring and analysing pesticide..., Kaminska [/bib_ref] [bib_ref] The Atlas of human African trypanosomiasis: a contribution to global mapping of..., Simarro [/bib_ref] [bib_ref] Geographic information systems and public health, Ricketts [/bib_ref] [bib_ref] Using a GIS-based floating catchment method to assess areas with shortage of..., Luo [/bib_ref] [bib_ref] Spatial mapping of temporal risk characteristics to improve environmental health risk identification:..., Wen [/bib_ref]. The transformation of detailed data into maps can facilitate communication of geographic distribution of health challenges in different communities and identify areas for intervention [bib_ref] The application of geographical information systems to important public health problems in..., Tanser [/bib_ref] [bib_ref] Geographical Information System (GIS) as a tool for monitoring and analysing pesticide..., Kaminska [/bib_ref] [bib_ref] Health and GIS: toward spatial statistical analyses, Chung [/bib_ref]. The use of GIS in low resource settings has been hampered by a number of factors including data availability, software, expertise, and economic resources needed [bib_ref] The application of geographical information systems to important public health problems in..., Tanser [/bib_ref] [bib_ref] The Atlas of human African trypanosomiasis: a contribution to global mapping of..., Simarro [/bib_ref] [bib_ref] Fuzzy expert systems and GIS for cholera health risk prediction in southern..., Fleming [/bib_ref]. Participatory mapping is a map production process by local communities with the support of governmental institution, non-governmental organizations (NGOs) or academic institutions. The United Nations (UN) Millennium Development Goals (MDGs) and the World Summit on the Information Society suggested utilization of a participatory approach to promote equal access to information and knowledge sharing. This approach could offer a relatively low resource method to map communities in rural settings. Partners In Health/Inshuti Mu Buzima (PIH/IMB) has partnered with the Rwandan Ministry of Health (MoH) since 2005 to provide support to three districts (Kayonza, Kirehe and Burera) in rural Rwanda, including supporting the community health workers (CHW) system to provide community-based care. Since 2009, PIH/IMB has used GIS to supplement existing monitoring and evaluation efforts in supported areas to better target district-wide health system strengthening interventions [bib_ref] Local use of geographic information systems to improve data utilisation and health..., Sudhof [/bib_ref]. Village location mapping was done using a trained GIS team of PIH/IMB in Kayonza (Rwinkwavu District Hospital catchment area only) and Kirehe districts and was relatively resource intensive. In the third district, Burera, a mountainous rural district with poor road access, we adapted a community participatory approach to conduct villagelevel mapping. We describe this approach of leveraging Rwanda's national CHW program to map village locations, and demonstrate the utility of this process through identification of geographic disparities in critical services through mapping of village-level access to safe water. # Methods Study area: Burera district, one of the 30 districts of Rwanda, is located in the Northern Province, neighboring Uganda. Burera is a mountainous rural district with a topography ranging between 1728 m to 4098 m of altitude above sea level with an area of 646 km 2 [fig_ref] Figure 1: Location of the study area as well as its topography, sectors, cells,... [/fig_ref]. The district is subdivided by Rwandan administrative boundaries into 17 sectors, with 69 sub-sector divisions (cells) and 573 villages, with each cell containing between five and 16 villages [fig_ref] Figure 1: Location of the study area as well as its topography, sectors, cells,... [/fig_ref]. Rwanda National CHW network: Each village has four CHWs in charge of community health and each cell has a CHW supervisor. CHW supervisors live in the community, and have completed a minimum of primary school education. Each health center (HC) or health post (HP) is staffed with a nurse who supervises the community health activities in the catchment area. Community mapping: Four meetings were held for authorities from the district, district hospital (DH), HCs and some CHW supervisors in order to encourage participation and ownership in the mapping process. During these meetings we reviewed the approach and goals of GIS mapping and potential to help efforts to improve health care in Burera district. Training process: We developed a three day training program and training manual in English and Kinyarwanda to build knowledge around the use of Global Positioning System (GPS) device and the skills needed to collect the data. The first day of the training included the introduction and explanation of the purpose of the activity, the value of map analysis, and an introduction to GPS functionalities. The second day focused on the use of the GPS device; emphasizing taking GPS coordinates points. The last day was field-based practice, where trainees collected GPS coordinates that were then validated, and also discussed challenges and solutions. CHW supervisors who had more difficulty using GPS were identified by trainers and given coaching on their first day of data collection to ensure data quality. In total 111 people were trained, 69 cell-level CHW supervisors, 17 sector-level CHW supervisors, and 25 nurses in charge of community health at HCs and HPs. A full-time Burera-based District GIS project assistant was hired by PIH/IMB who provided to training, validation and support for the CHW supervisors field work and data entry. CHW staff: All CHW supervisors and nurses in charge of community health at HC and HP level were included in the training. Different roles were attributed to each cadre: cell-level CHW supervisors collected the village GPS coordinates; sector-level CHW supervisors and community health nurses provided supportive supervision and organization of the field data collection along with the GIS project assistant. GPS data collection: Cell-level CHW supervisors collected GPS coordinates of all villages in their cell over five days. The GPS device was used to collect longitude and latitude information of a specific location, and the collected data was stored as point features in the device. Every data collector had a GPS with two extra fully charged batteries. We started data collection in the central part of the district, and then continued north to the mountainous part of the district in the dry season (July, September, and October) due to transportation-related challenges presented by the rainy season. Village location was mapped based on where population gathered for meeting places like village office or village chief 's house Drinking Water Sources (DWS) and population data: Information on DWS and population was provided by the elected chief of the village in 2013 through a brief survey administered by the CHW supervisor. DWSs were classified as safe water (water from Improved Drinking Water Source (IDWS)) and unsafe water (water from Unimproved Drinking Water Source (UDWS)) using the World Health Organization (WHO) definitions. IDWSs were defined as adequately protected from outside contaminations, including piped household water connection, public standpipe borehole, protected dug well, protected spring and rainwater collection. UDWSs were those inadequately protected from outside contamination, including unprotected dug well and spring, surface water, vendor-provided water (cart with small tank/drum, tanker truck), and bottled water (bottled water is considered improved only when the household use another improved source for cooking and personal hygiene). Since there may be multiple drinking water sources in a village, we classified the most frequently used drinking water source as primary and others were classified as secondary. Other information on the DWS and population data and combined with district lakes, and wetlands. Map validation and distribution: CHW supervisorcollected GPS coordinates were first validated by the GIS assistant. He randomly recollected at least two villages GPS coordinates for each cell, and compared them to those collected by the CHW supervisor. Secondly, we met with local authorities from cell, sector, district, HC and DH to validate villages GPS locations mapped. Seventeen sector-level validation meetings were held with 124 authorities' participants. The district map was then validated by district-level authorities during a validation meeting, including 27 participants (vice mayors and district office authorities in charge of health, land and environment, district hospital administrators and other district officials). Once validated, the maps were printed out, laminated and distributed to the district, sector, cell and health facilities for posting and administrative use. Cost: Costs of the mapping process were estimated from a health system perspective, using the data from program budgets and financial activity records. Before starting the mapping in Burera district, the GIS team at PIH/IMB had just concluded a similar mapping exercise in Kirehe district using a trained full-time PIH/IMB GIS team. We compared the costs of mapping by the CHW supervisors in Burera, with modelled estimates that would have been incurred if staffing resources similar to those of the Kirehe mapping were utilized. To get the cost of personnel and equipment (transport, computers and other devices), we first estimated their capacity rates in hours available for mapping activities during the entire mapping period (weekday hours minus holidays, time-offs and weekends). The concept of useful-life was used to estimate the depreciation and present value of equipment such as vehicles, computers, and GPSs that last for more than a year. Indirect and overhead costs such as PIH/ IMB's organizational and administrative spending related to this mapping were very minimal in both mapping methods, and were therefore ignored. The main cost categories were training, data collection and mapping, validation, and dissemination [fig_ref] Table 1: Cost of the intervention using CHW supervisors compared to GIS team of... [/fig_ref]. Unit and total costs were calculated in Rwandan Francs (RWF) and converted into USD using the median exchange rates of April 2011 to March 2012, actual spending period. Mapping village level DWS: DWS was overlaid onto the maps to create a village-level district-wide map of access to safe water. We categorized villages as proximal to lakes and wetlands if they were located within one kilometer Euclidean distance and then used chi squared test for association between lake and wetland proximity and DWS type. # Ethical consideration This project was reviewed by the institutional review board of University of Rwanda, College of Medicine and Health Sciences, School of Public Health, and was given exempt status. # Results ## Mapping and validation Between April 2011 and March 2012, CHW supervisors successfully mapped the 573 villages in Burera district [fig_ref] Figure 2: Villages mapped by quarter [/fig_ref]. During the 18 validation meetings, we received 73 comments (64 comments in sector validation meetings and nine in the district validation meeting). Based on the feedback, locations of 32 villages were recollected and corrected. Following validation, we produced the 17 sector maps and the overall district map, with sector-level maps distributed in hard and soft copy to the 69 cells-level offices, 17 sector-level offices and the 18 HCs and 7 HPs. The overall district map was distributed to the district and DH offices. ## Costs The total cost of mapping using CHW supervisors was $29,692, compared to the $60,112 cost which would have been needed for mapping using the existing GIS team of PIH/IMB. For the participatory mapping method, data collection and mapping accounted for most of the costs (68%), followed by the training (15%), map validation (11%) and dissemination (6%) [fig_ref] Table 1: Cost of the intervention using CHW supervisors compared to GIS team of... [/fig_ref]. ## Use of chw-system derived maps for rapid assessment of dws Three-quarters of Burera's population had two DWS, with 75.1% of primary DWS categorized as safe (IDWS), and 44.6% for secondary DWS. We found that 94.1% of the population had access to safe water for at least one DWS, with 76.2% having access to IDWS through their primary water source. Over two-thirds (69.1%) of the population had their primary DWS located inside their village. Overlay of the DWS information onto the maps allowed rapid assessment of geographic differences in access to IDWS. For example, access to safe water at the sector level ranged from 32.0% to 100%, with the northwest and central part of the district that had better access to safe water than the southeast part [fig_ref] Figure 3: Drinking water distributions by village and sector [/fig_ref]. GIS data also allowed analysis of geographic factors associated with better or worse access to IDWS. For example, villages near to lakes and wetlands were more likely to identify an unsafe source as a primary DWS (29.4% versus 19.5% respectively, p < .001). # Discussion We successfully leveraged an existing national CHW network to map a mountainous district in Rwanda at lower cost and with effective engagement of the community and other key stakeholders. Following a three-day practical training and supported by a district-based staff member to provide support, CHW supervisors mapped this mountainous rural district in less than one year despite poor roads and no vehicle access to parts during the rainy season. There were several advantages to using CHW supervisors to collect the geographic data. They were already travelling by foot to the villages as part of their CHW supervisory activities, so no additional transport was needed and they already knew the physical location of the villages. The approach overcame the transportation challenges facing mapping in rural areas with limited road infrastructure. The participation of CHW supervisors in mapping villages and safe water access was also highly valued by government authorities as part of local capacity building and strengthening integration and participation of the local community in decision making. Not surprisingly, using CHW supervisors resulted in lower cost compared to using a trained and dedicated GIS team. Using program costing data, we estimated that using PIH/IMB's GIS team would have been about two times more costly compared to the mapping using CHW supervisors (saving about $30,420). The main difference in cost using the CHW supervisor network approach was in the data collection and mapping largely due to salary and transportation, ($20,166 versus $54,988) [fig_ref] Table 1: Cost of the intervention using CHW supervisors compared to GIS team of... [/fig_ref]. The process could have been accelerated if the CHW supervisors had more time to do the mapping, but we worked to integrate into their usual activities so that no additional salaries were needed, increasing the feasibility for replication by groups with limited funds. Community engagement was another important benefit of our approach. Involvement of local community and authority for decision making is recognized as important in addressing gaps in population health and service. Integrating the community into mapping work has also been successfully used in others settings such as the participatory mapping in Tanzania for malaria project [bib_ref] Participatory mapping of target areas to enable operational larval source management to..., Dongus [/bib_ref]. We found similar engagement in both the planning and validation processes increased the acceptability and value of the maps by district and sub-district authorities. Village level maps were the first ever produced in the district and were given to local authorities to use them for their daily activities and decision making. The work also resulted in a database that can be used for future geographic analysis at this local level. The assessment of the spatial distribution of safe water by the villages was evidence of this value. For example, we were able to rapidly map and illustrate that safe water was unevenly distributed across villages of the district. The access to safe water that we found (76.2%) was similar to the NISR findings in 2011 (76.7%) for the northern province where Burera district was located, providing evidence that the approach was able to provide results consistent with other data sources. These maps also had value to help to inform decision makers. When the maps showing village and sector-level variability in IDWS access were shared with the district authorities, they were able to intervene for sectors with low access to safe water by increasing supply of safe water through the WASH (Water, Sanitation and Hygiene) project on-going in the Burera district of Rwanda. We learned a number of lessons which can inform similar approaches to feasibly integrate GIS into monitoring, evaluation and program planning at the local level. Leveraging existing community-level work has the advantage of both reducing cost as well as increasing the local ownership of the data. In addition, ensuring a rapid feedback loop of resulting maps is important to ensure engagement of local authorities and the community in decision-making. Focusing on building capacity and local ownership throughout the mapping process, supported by practical training and skills-based supervision resulted in engaged CHW supervisors motivated to learn new skills and perform the mapping and participate in data-driven decision making without additional reimbursement. For this new mapping activity, we also found that a locallybased GIS assistant was important to supervise initial data collection and address challenges during the process. While we used a proprietary software, the costs could also be lowered through use of free open source software and open e-learning for geo-analysis tools. There were some limitations to our approach. There were not readily recognized village centers, as village location was mapped based on where population gathered for meeting places like village office or village chief 's house. We did not test the accuracy of maps produced using CHW supervisors versus maps produced using GIS team of PIH/IMB. It was also more difficult to validate the location of villages far from known features such as roads, rivers, lakes, and wetland. In addition, the safe water assessment by village was based on the assumption that the entire village's population used the primary DWS available within or outside the village. This may have resulted in under or over estimates of access to safe drinking water. Modeling the cost of transport from Kirehe to Burera mapping using PIH/IMB's GIS team, assumed similar driving conditions like road, topography, etc. which might not be the same case. # Conclusion The existing CHW system can be leveraged to inexpensively map rural areas. Involvement of local authorities from health and political sectors ensures community buy-in and ownership of the results for future decisionmaking. The creation of a village-level GIS database is an important to help local officials identify geographic disparities in access to important resources such as safe water, communicate these challenges through maps, and better target interventions to improve population health and reduce inequity. Our approach of leveraging an existing community health network supported through skills-based training and supervision and focused on local engagement and ownership could be replicated in other resource limited countries with community health worker programs similar to the Rwandan model . [fig] Figure 1: Location of the study area as well as its topography, sectors, cells, and villages. A: location of Rwanda in Africa, B: location of Burera district as one of 30 district of Rwanda, C: Burera district subdivisions; 17 sectors, 69 cells and 573 villages, and the Digital Elevation Modal (DEM) showing elevation and terrain of Burera district. [/fig] [fig] Figure 2: Villages mapped by quarter. A: 125 villages (three sectors) in quarter two of 2011 (April -June). B: 220 villages (six sectors) in quarter three of 2011 (July -September). C: 116 villages (four sectors) in quarter four 2011 (October -December). D: 112 villages (four sectors) in quarter one of 2012 (January -March). E: Total of 573 villages of 17 sectors in four quarters. [/fig] [fig] Figure 3: Drinking water distributions by village and sector. A: The dot represented the location of villages while the size of dot was proportion to the number of population in the village. The dots in green color represented safe water while dots in red color represented unsafe water. Area in yellow was a one kilometer Euclidian distance from lake in blue and wetland in green and while lines. B: Represented the percentage of population using safe drinking water by sector which decreased from green to yellow and red, from 100% (the highest) to 32% (the lowest). [/fig] [table] Table 1: Cost of the intervention using CHW supervisors compared to GIS team of PIH/IMB [/table]
Novel Analysis of Immune Cells from Nasal Microbiopsy Demonstrates Reliable, Reproducible Data for Immune Populations, and Superior Cytokine Detection Compared to Nasal Wash The morbidity and mortality related to respiratory tract diseases is enormous, with hundreds of millions of individuals afflicted and four million people dying each year. Understanding the immunological processes in the mucosa that govern outcome following pathogenic encounter could lead to novel therapies. There is a need to study responses at mucosal surfaces in humans for two reasons: (i) Immunological findings in mice, or other animals, often fail to translate to humans. (ii) Compartmentalization of the immune system dictates a need to study sites where pathogens reside. In this manuscript, we describe two novel non-invasive nasal mucosal microsampling techniques and their use for measuring immunological parameters: 1) using nasal curettes to collect cells from the inferior turbinate and; 2) absorptive matrices to collect nasal lining fluid. Both techniques were well tolerated and yielded reproducible and robust data. We demonstrated differences in immune populations and activation state in nasal mucosa compared to blood as well as compared to nasopharyngeal lumen in healthy adults. We also found superior cytokine detection with absorptive matrices compared to nasal wash. These techniques are promising new tools that will facilitate studies of the immunological signatures underlying susceptibility and resistance to respiratory infections. Fig 7. Tolerability of novel nasal sampling methods. The percentage of volunteers rating (A) nasal curettage and (B) nasosorption on discomfort, pain and lacrimation. Conceptualization: DMF SPJ J. Rylance KP SBG. Data curation: AS KP SPJ J. Reiné. Formal analysis: SPJ KP J. Rylance. Funding acquisition: DMF SBG. Investigation: SPJ KP J. Rylance HA BFC AC JFG CH HH J. Reiné AS CS AT ADW SZ DMF. Methodology: DF SPJ SBG. [formula] a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 [/formula] # Introduction Respiratory tract disease is an important cause of morbidity and mortality worldwide [bib_ref] Global and regional mortality from 235 causes of death for 20 age..., Lozano [/bib_ref]. In addition, lower respiratory tract infections and chronic respiratory disease, such as asthma, have both been highlighted as leading causes of disability [bib_ref] Disability-adjusted life years (DALYs) for 291 diseases and injuries in 21 regions,..., Murray [/bib_ref] [bib_ref] Global, regional, and national incidence, prevalence, and years lived with disability for..., Gbod [/bib_ref]. The nasal mucosa is the key niche in the pathogenesis of respiratory disease. For example, carriage of Streptococcus pneumoniae (the pneumococcus) in the nasopharynx has been identified as an essential step in developing both localised and systemic infection [bib_ref] The fundamental link between pneumococcal carriage and disease, Simell [/bib_ref]. Alteration of the nasal mucosa, for example by viruses, has also been shown to increase susceptibility to pneumococcal infection [bib_ref] Modulation of nasopharyngeal innate defenses by viral coinfection predisposes individuals to experimental..., Glennie [/bib_ref]. Moreover, viral infection of the upper respiratory tract is associated with exacerbation of asthma and excessive inflammation in the upper respiratory tract is a risk factor for asthma [bib_ref] Viral-associated exacerbations of asthma and COPD, Traves [/bib_ref] [bib_ref] Upper airway inflammatory diseases and bronchial hyperresponsiveness, Eggleston [/bib_ref]. A greater understanding of the immunological responses to pathogens in the nasal mucosa and the pathogenesis of respiratory diseases may provide targets for new treatments or vaccinations against disease. There is increasing evidence that mucosal immune responses vary significantly at different sites within the body [bib_ref] Phenotypic distribution of T cells in human nasal mucosa differs from that..., Jahnsen [/bib_ref] [bib_ref] Distribution and compartmentalization of human circulating and tissue-resident memory T cell subsets, Sathaliyawala [/bib_ref]. This compartmentalisation necessitates specific study of the nasal mucosa as it is a key component of host-pathogen interaction. Developing non-invasive techniques to study the nasal mucosa in humans offers clear advantages over animal models, which frequently lack translational applicability [bib_ref] Genomic responses in mouse models poorly mimic human inflammatory diseases, Seok [/bib_ref]. Currently, the most used method for collecting cells from within the nasopharynx is a nasal wash (NW) procedure [bib_ref] Experimental human pneumococcal carriage, Gritzfeld [/bib_ref]. The NW procedure is generally well tolerated but not suitable for all groups of patients especially those who are particularly young or unwell. In addition, luminal cell populations can vary significantly from intra-mucosal cell populations [bib_ref] Phenotypic distribution of T cells in human nasal mucosa differs from that..., Jahnsen [/bib_ref] [bib_ref] The role of neutrophils during intestinal inflammation, Fournier [/bib_ref]. An improved method of sampling the nasal mucosa would lead to a greater understanding of the cellular components of the immune response in the nasopharynx. Cell collection using nasal curettes has previously been used to collect epithelial cells for culture, as well as for gene expression analysis [bib_ref] Culturing of human nasal epithelial cells at the air liquid interface, Muller [/bib_ref] [bib_ref] Gene expression profiles during in vivo human rhinovirus infection: insights into the..., Proud [/bib_ref]. Nasal brushes have also been used to collect samples to investigate epithelial cell phenotype [bib_ref] CFTR expression analysis in human nasal epithelial cells by flow cytometry, Van Meegen [/bib_ref]. Here we use for the first time cell collection with nasal curettes to analyse the composition and activation state of immune cells using flow cytometry. A NW is also typically performed to measure cytokine and other soluble immune mediators. An absorptive matrix to collect nasal fluid (nasosorption) has been used in neonates, and has the potential to be better tolerated and more widely applicable [bib_ref] Neonatal cytokine profile in the airway mucosal lining fluid is skewed by..., Folsgaard [/bib_ref]. This technique has recently been used to investigate nasal responses to grass pollen, LPS and rhinovirus [bib_ref] The nasal mucosal late allergic reaction to grass pollen involves type 2..., Leaker [/bib_ref] [bib_ref] Nasal Lipopolysaccharide Challenge and Cytokine Measurement Reflects Innate Mucosal Immune Responsiveness, Dhariwal [/bib_ref] [bib_ref] IL-33-dependent type 2 inflammation during rhinovirus-induced asthma exacerbations in vivo, Jackson [/bib_ref]. Here, we aimed to compare cytokine detection between nasal wash and nasosorption techniques. Here we describe the novel use of two non-invasive nasal microsampling techniques. We present data on the reproducibility, utility and tolerability of these techniques in measuring immunological responses within the nasal mucosa. # Methods ## Recruitment of volunteers and ethical statements We recruited healthy non-smoking adults aged between 18-60 years of age. Volunteers gave written informed consent. Inclusion criteria were: capacity to give informed consent, aged 18-50 years and speak fluent English. Exclusion criteria were: current involvement in another study unless observational or in follow-up phase (non-interventional), influenza vaccination in the last 2 years, clinically diagnosed with influenza in the last 2 years, egg allergy, previous significant adverse reaction to any vaccination/immunisation, close contact with at risk individuals (children under 5 years, immunosuppressed adults, elderly, chronic ill health), current regular smoker, >10 pack years smoking history, asthma or respiratory disease, pregnant, women of child-bearing potential without effective birth control in place, allergic to penicillin/ amoxicillin/ gentamicin, on medication that may affect the immune system in any way e.g. steroids, steroid nasal spray, regularly taking acetylsalicylic acid (aspirin), been involved in a clinical trial involving experimental pneumococcal carriage over the last 3 years, current acute severe febrile illness, taking long term antibiotics. Volunteers were screened for S. pneumoniae carriage and only carriage-negative volunteers were analysed. In addition, volunteers with nasal and general symptoms were excluded from the analysis. Ethical approval was given by NHS Research and Ethics Committee (REC)/Liverpool School of Tropical Medicine (LSTM) REC, reference numbers: 15/NW/0146 and 14/NW/1460 and Human Tissue Authority licensing number 12548. The individuals in this manuscript have given written informed consent (as outlined in PLOS consent form) to publish these case details Nasal sampling procedures All participants underwent nasal wash procedures as described previously using 20mL of sterile saline [bib_ref] Experimental human pneumococcal carriage, Gritzfeld [/bib_ref]. A selection of participants underwent nasal curettage and nasosorption. Nasal curettage uses a small probe to collect cells from the nasal mucosa (S1 . The nasal inferior turbinates were visualised with a light with the participant being seated with the head tilted posteriorly. The curette (ASL Rhino-Pro © , Arlington Scientific) was used to scrape a small collection of cells from the nasal mucosa (S1 Video). Two scrapes per nostril were taken and placed in a 15mL Falcon tube placed on ice containing phosphate-buffered saline (PBS) + 0.5% heat-inactivated fetal bovine serum (FBS) and 2.5 mM ethylenediaminetetraacetic acid (EDTA) (PBS++, all ThermoFisher). For nasosorption collection, an adsorptive matrix strip (Nasosorption™, Hunt Developments) was inserted into the nostril and placed against the nasal lining for 2 minutes and then placed in its transport tube (S2 . # Flow cytometry analysis Cells were dislodged from the curette by repeated pipetting with PBS+. Cells were spun down (440 x g for 5 minutes) and resuspended in PBS++ containing LIVE/DEAD1 Fixable Aqua Dead Cell Stain (ThermoFisher). After 15 minute incubation on ice, an antibody cocktail containing Epcam-PE, HLADR-PECy7, CD16-APC, CD66b-FITC (all Biolegend), CD3-APCCy7, CD14-PercpCy5.5 (BD Biosciences) and CD45-PACOrange (ThermoFisher) was added to the cells. Following a further 15 minute incubation on ice, cells were filtered over a 70 μm filter (ThermoFisher). Cells were spun down (440 x g for 5 minutes), resuspended in PBS++ and acquired on a flow cytometer (LSRII, BD). Samples with less than five hundred events (15% of all measured) were excluded from further analysis. Nasal washes were similarly processed excluding the dislodging step. To 100 μL heparinized blood, the viability dye and antibodies were directly added sequentially. After the final incubation step, BD FACS lysing solution was used to remove red blood cells according to manufacturer's instruction. Flow cytometry data was analysed using Flowjo V. 10 (Treestar). ## Cytokine detection Nasal lining fluid was extracted from nasosorption strips by centrifugation (1880 x g for 10 minutes) and frozen until use at -80C. Supernatant from nasal wash was collected by centrifugation at 3000 rpm for 3 minutes and was stored at -80C until use. Interferon gamma-induced protein 10 (IP10), interleukin 8 (IL-8) and monocyte-chemoattractant protein 1 (MCP1) were measured by enzyme-linked immunosorbent assay (ELISA) (BD OPTEIA) according to manufacturer's instructions. The human magnetic 30-plex cytokine kit (ThermoFisher) was used to detect thirty cytokines simultaneously on a LX200 with xPonent3.1 software (Luminex) following manufacturer's instructions. ## Scoring tolerability of nasal sampling procedures Following nasal wash, nasal curettage and nasosorption, participants rated by 5-point modified Likert scale how 'painful' and how 'uncomfortable' each procedure was, and how much it made their 'eyes water'. Thirty-nine participants also completed a symptoms log for 7 days documenting both local and general symptoms with severity ratings from 1-7. ## Multi-dimensional scaling and heat map generation Multi-dimensional scaling and heat map representations were generated using R. Flow cytometry data (epithelial cell yield, immune composition and activation) was log-transformed and a distance-matrix was calculated. The Kruskal stress was calculated using the 'MASS' package. Heat maps of log-transformed cytokine data were generated using the 'gplots' package # Statistical analysis Non-parametric two-tailed tests were used throughout using Prism 5 (Graphpad). If two groups were compared, a Mann-Whitney test was used. If multiple groups were compared, a Kruskal-Wallis test was used, followed by a Dunn's post-test. A Spearman test was used to measure correlations between two continuous variables. Analysis of similarity (ANOSIM) testing was performed using the 'vegan' package in R. # Results ## Nasal curettage yields robust and reproducible data We collected nasal cells from the inferior turbinate using curettes (Supplementary Video 1). In total 240 samples were collected from 139 healthy individuals to investigate their use for studying immune responses at the mucosal level. To verify the repeatability of nasal curettage, we initially collected samples from the left and right nostril of three healthy volunteers and performed flow cytometry to identify cellular composition [fig_ref] Fig 1: Repeatability of nasal curettage sampling [/fig_ref]. Samples from both nostrils were processed independently and frequencies of granulocytes, monocytes and T cells were compared for each of the three volunteers. Cellular samples collected from the two nostrils were similar for each of the three volunteers, compared to samples collected from the other two volunteers. This demonstrates the repeatability of nasal curettage as well as the presence of inter-individual variation in immune cells in the nose. To verify that nasal curette sampling yields stable data, cells were collected from healthy volunteers (n = 117) over a five month period . The percentage of granulocytes and T cells among immune cells was stable during this period . Moreover, for a subset of volunteers, up to four nasal samples were collected during a thirty-three day period. The levels of both granulocytes and T cells correlated on an intra-individual level between repeated sampling . This demonstrates that despite variation between individuals, the immunological profile in the nose is stable in the absence of disease or immune intervention such as vaccination. ## Nasal curettage and nasal wash yield different cell populations We then compared the yield and composition of nasal cells collected using curettes to those collected using a NW, the currently most used method for collecting nasopharyngeal cells . Nasal curettage yielded a median of 4367 (interquartile range (IQR): 1511-10348) immune cells and 1407 (IQR: 570-3194) epithelial cells, respectively. The number of immune cells obtained was similar between NW and nasal curette. In contrast, there were 22.7 fold increased numbers of epithelial cells collected by nasal curette p < 0.05). the composition of the collected immune cell. NW immune cells consisted almost exclusively of granulocytes (median 96%, IQR: 93-97%). In contrast, nasal curette samples contained predominantly granulocytes (median 64%, IQR: 39-79%, p < 0.0001 compared to NW), but also consisted of a larger fraction of T cells than NW (median 16%, IQR: 9-38%, p < 0.0001 compared to NW). A median of 2591 (IQR: 691-7666) granulocytes and 633 (IQR: 210-1740) T cells were acquired per sample. Nasal curette samples also contained more lineagehuman ## Differences in proportions and activation state of immune cells in blood and nasal cells Next, we compared immune populations from nasal samples with those found in blood [fig_ref] Fig 4: Comparison of samples from nasal mucosa and blood [/fig_ref]. Levels of neutrophils, T cells and Lineage -HLA-DR + were similar between nasal curette samples and blood. In contrast, levels of monocytes/macrophages were decreased in nasal curette samples compared to blood (0.3% and 5.4%, respectively, p < 0.0001). Immune cells from nasal mucosa differed from blood cells in their activation state [fig_ref] Fig 4: Comparison of samples from nasal mucosa and blood [/fig_ref]. Levels of HLA-DR + T cells were significantly increased in nasal curette samples compared to blood (p < 0.0001). Similarly, levels of HLA-DR were significantly increased on granulocytes in nasal curette samples and NW samples compared to blood (p < 0.001). Granulocytes from nasal curette samples and NW samples also displayed high CD66b expression relative to blood (p < 0.001), a marker for degranulation. Granulocyte activation and degranulation was even further increased in NW compared to nasal curette samples (p < 0.05 and p < 0.001, respectively). When taking epithelial cell yield, immune cell composition and activation state into account, nasal curette samples clustered separately from blood and NW [fig_ref] Fig 4: Comparison of samples from nasal mucosa and blood [/fig_ref]. ## Macroscopic red blood cell contamination does not affect immune cells recovered using nasal curettage Red blood cells were visible macroscopically in 44% of collected nasal curette samples (52 of 117 assessed). Blood leukocytes did not contaminate the recovered immune cells in these samples, as levels of monocytes among immune cells were not increased in the presence of red blood cells [fig_ref] Fig 5: Comparison of nasal curettes with visible red blood cell contamination [/fig_ref]. Moreover, T cell activation and neutrophil activation and degranulation were not decreased when red blood cells were present [fig_ref] Fig 5: Comparison of nasal curettes with visible red blood cell contamination [/fig_ref]. ## Cytokine detection from nasal lining fluid using nasosorption devices To investigate cytokines in the nose, we used nasosorption devices to collect nasal lining fluid. The median volume of nasal lining fluid returned using this technique was 42.5μL (IQR: 29.25-71.25 μL, n = 41). We measured the levels of thirty cytokines in these samples by multiplex ELISA. To investigate whether concentrations of cytokines collected locally by nasosorption can be used to detect intra-individual differences, we measured levels of three cytokines by ELISA in paired NW samples as a control [fig_ref] Fig 6: Comparison of cytokine levels in samples collected by nasal wash and using... [/fig_ref]. Levels of IL-8 (r 2 = 0.37, p < 0.0001), IP10 (r 2 = 0.48, p < 0.0001) and MCP1; r 2 = 0.08, p = 0.04) correlated across the two sample types and detection techniques. We then compared cytokine levels in nasal lining fluid and NW supernatant for thirty cytokines by Luminex [fig_ref] Fig 6: Comparison of cytokine levels in samples collected by nasal wash and using... [/fig_ref]. Levels of different cytokines varied considerably, with median levels of IL1 receptor antagonist (IL-1RA) at 212,000 pg/mL and granulocyte macrophage colony-stimulating factor (GM-CSF) at 2 pg/mL in nasal lining fluid. Relative cytokine abundancy correlated well between nasosorption and NW such that cytokines that were abundant in nasosorption were also highly present in NW. Of interest, T cell cytokines (IL-10, IL-17, Interferon gamma (IFNγ), Tumour necrosis factor alpha (TNFα), IL-4, IL-5, IL-2) were only present at low levels. This correlates well with the absence of T cells in the lumen . Growth factors as epidermal growth factor (EGF), hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) were expressed at moderately high levels, reflecting the homeostatic nature of mucosal surfaces. Levels of cytokines were higher in nasosorption compared to NW (median 4.7x, IQR: 3.1-8.0x; [fig_ref] Fig 6: Comparison of cytokine levels in samples collected by nasal wash and using... [/fig_ref]. However, some cytokines had a ratio between nasosorption and NW that differed substantially from this: IL-1RA and IL-5 were respectively 51.5x and 45.2x higher in nasosorption than in NW. In contrast, Monokine induced by IFNγ (MIG), Regulated on activation, normal T-cell expressed and secreted (Rantes) and IP10 were found at similar levels in nasosorption and in NW (ratio of 1.5x, 1.0x and 0.9x, respectively). While all cytokines were within the limit of detection of the assay (0.5 pg/mL) in the nasal lining fluid, several cytokines could not be detected in one or more NW samples. Five cytokines (GM-CSF, TNFα, Rantes, IL-5 and Macrophage inflammatory protein (MIP)-1a) were not detectable in the NW of any of the six volunteers. One cytokine (IL-17) could not be detected in 3/6 samples. Two cytokines, IL-2 Receptor (IL-2R) and G-CSF, were below the detection limit for 2/6 samples and an additional 9 cytokines were undetectable in 1/6 NW samples. ## Methods of nasal microsampling are well tolerated by volunteers and do not lead to symptoms Using the 5-point modified Likert scale, twenty participants gave ratings for nasal curettage (eighty-eight ratings) and nasosorption (sixty ratings) with regards to pain, discomfort and lacrimation. For nasal curettage and nasosorption, the proportion of responses that reported any degree of pain (any score > 1) were 73% and 10%, respectively . For nasal curettage and nasosorption, the proportion of responses that reported any degree of discomfort (score > 1) were 86% and 47%, respectively . For nasal curettage and nasosorption, the proportion of responses that reported any degree of lacrimation (score > 1) were, 84% and 28%, respectively . For nasosorption the maximum rating was moderately for levels of discomfort, pain or causing lacrimation. A small proportion of responses rated nasal curettage as very painful (2%), uncomfortable (8%) or causing lacrimation (6%). Finally, we assessed whether these sampling techniques led to increased general and nasal symptoms over a longer period in thirty-nine healthy volunteers (S1 . All participants had nasal wash procedures and twenty of those participants had nasal curettage and nasosorption to investigate whether these additional sampling methods affect nasal symptoms. Daily symptom logs for nasal and general symptoms were completed by all volunteers. Age and sex distribution was similar in each group. The median ratings for overall nasal symptoms were 1 (range 1-4) and 1 (range 1-5) in the group with and without additional nasal sampling, respectively. In the symptoms log, a score of 1 represented 'none to occasional symptoms' and 5 represented 'moderately bothersome'. Comparison of area under the curve between the two groups showed no significant difference in either overall nasal symptoms or general symptoms. In addition, we compared nasal and general symptoms between participants with and without additional nasal sampling on day 1 (before additional sampling) and day 3 (after additional sampling). There were no significant differences between nasal or general symptoms with or without additional nasal sampling at these time points. # Discussion We have shown that non-invasive sampling of the nasal mucosa is well tolerated and reproducible, yielding immune cells that are stable over time. In particular, our results demonstrate the reliability of nasal curettage as a method for studying the nasal immune response. The ability to measure cellular phenotype during infection could shed light on the mechanisms that are involved in pathogen clearance. This technique could also be used in vaccine testing to define cellular correlates of protection at a mucosal level [bib_ref] Low Thymic Activity and Dendritic Cell Numbers Are Associated with the Immune..., Schulz [/bib_ref]. Moreover, the use of these sampling techniques will facilitate the investigation of cellular responses in asthma, which includes an important immunological component of the upper respiratory tract [bib_ref] Innate and adaptive T cells in asthmatic patients: Relationship to severity and..., Hinks [/bib_ref]. Immune cells from the nose differed from those in the blood in composition and activation state. Both T cells and granulocytes collected from the nose displayed an increased activation profile compared to blood leukocytes, consistent with earlier reports [bib_ref] Distribution and compartmentalization of human circulating and tissue-resident memory T cell subsets, Sathaliyawala [/bib_ref]. Moreover, luminal granulocytes had an increased activation profile compared to those found using nasal curettes. As neutrophil migration through the epithelial layer depends on interaction with molecules as Intercellular adhesion molecule 1 (ICAM-1) [bib_ref] Transmigrated neutrophils in the intestinal lumen engage ICAM-1 to regulate the epithelial..., Sumagin [/bib_ref] , it is possible that these granulocytes become activated by the transmigration event itself. The only difference in proportion of immune cells found between blood and nasal mucosa was the decreased presence of monocytes in nasal samples. It has been demonstrated that intestinal macrophages lack CD-14 expression [bib_ref] Intestinal macrophages lack CD14 and CD89 and consequently are down-regulated for LPS-and..., Smith [/bib_ref]. Staining with additional macrophage markers (CD163 and CD206) confirmed the low levels of monocytes/macrophages in nasal mucosa (S3 Similar to our findings, monocytes were described to be present only at low levels in nasal aspirates of healthy children [bib_ref] Differential recruitment of dendritic cells and monocytes to respiratory mucosal sites in..., Gill [/bib_ref]. In the presence of influenza A infection however, these cells were recruited to the nose [bib_ref] Differential recruitment of dendritic cells and monocytes to respiratory mucosal sites in..., Gill [/bib_ref]. It will be useful to compare nasal cell profiles from healthy and diseased individuals using this new sampling technique. Differences in immune cells were also present between NW and nasal curette samples as NW yielded almost exclusively granulocytes. The lack of T cells in NW reflects earlier findings in other body compartments: neutrophils readily enter the lumen in the gut, while T cells are mostly associated with the epithelial layer [bib_ref] Phenotypic distribution of T cells in human nasal mucosa differs from that..., Jahnsen [/bib_ref] [bib_ref] The role of neutrophils during intestinal inflammation, Fournier [/bib_ref]. The nasal curette sampling method allows the collection of such sub-epithelial and intra-epithelial cell populations, while NW is limited to sampling luminal populations. However, Natural killer (NK) cells and epithelial cells were found at high levels in nasal washes in a study by Horvath et al [bib_ref] Nasal lavage natural killer cell function is suppressed in smokers after live..., Horvath [/bib_ref]. As this study collected NW through 40 sprays of 100uL of saline followed by forceful expulsion rather than the more gentle NW approach we employed [bib_ref] Experimental human pneumococcal carriage, Gritzfeld [/bib_ref] , it is possible that different collection NW techniques yield different cell populations. Our flow cytometry panel did not include markers that could assess NK cell frequency in samples collected using NW or curettes. Increased collection of epithelial cells with nasal curettage compared to NW is likely due to the technique itself (S1 Video) rather than a difference in sample sites. Although blood and nasal immune cells displayed different profiles, a visible presence of red blood cells in nasal samples did not change the phenotype of the collected nasal cells. As the level of leukocytes in blood is relatively low it is not surprising that the presence of red blood cells at macroscopically visible levels does not indicate a substantial blood leukocyte contamination [bib_ref] Distinguishing cerebrospinal fluid abnormalities in children with bacterial meningitis and traumatic lumbar..., Bonadio [/bib_ref]. Of all immune cells, eighty percent could be identified in both blood and nasal curette samples. The remaining twenty percent like consists of various types of cells that could not be analyzed using this flow cytometry panel, such as innate lymphocytes and basophils. Increasing the number of markers will allow the further study of this fraction, which could potentially identify other differences in immune composition between blood and nose. As cell yields are low, the capacity to study rare cell populations or perform functional assays using such collected cells is limited however. The second nasal sampling technique that we assessed was the use of nasosorption devices to collect nasal lining fluid. Importantly, the nasal lining fluid contained cytokines in concentrations that were increased compared to NW, allowing for the investigation of several cytokines that were undetectable in NW supernatant. A correlation was seen between cytokine levels that were detected in both nasal lining fluid and those from NW. However, the ratio between cytokine concentrations in nasal lining fluid and NW was not similar for all cytokines assessed. One potential explanation is differences in levels could exist for some cytokines in different nasopharyngeal compartments. As nasal washes sample the entire nasopharynx in contrast to a localized sample coming from the nasosorption strip this might lead to different returned cytokine levels. Another possible explanation is that differences exist between cytokines in their propensity to bind to the nasosorption paper. NW is currently the most commonly used method to obtain samples from the nasopharynx. Despite this, it has limited application in multiple clinical scenarios. Here we have demonstrated that nasosorption has greater sensitivity than the traditional wash and is extremely well tolerated . A potential problem with sample collection using nasosorption devices can be poor return volume, in particular if volunteers are dehydrated. In conclusion, non-invasive mucosal sampling yields nasal cells and nasal lining fluid that can be used to study both cellular and soluble immune responses at the mucosal surface. Such sampling is well-tolerated and does not lead to a change in nasal symptoms. These techniques can be easily implemented and provide researchers with an effective tool to study immunological parameters in the upper respiratory tract. ## Supporting information [fig] Fig 1: Repeatability of nasal curettage sampling. Nasal cells were collected from the left (L) and right (R) nostril of three volunteers, processed independently and their composition was assessed by flow cytometry. After excluding debris and doublets, epithelial cells were identified by Epcam expression. A viability dye and CD45 were used to identify live immune cells. Among those cells, side scatter, CD66b, CD14 and CD3 were used to identify granulocytes, monocytes and T cells respectively.doi:10.1371/journal.pone.0169805.g001 Superior Cytokine Detection and Reproducible Data from Nasal Microbiopsy Fig 2. Nasal curettage yields reproducible and consistent results over time. (A) The percentage of granulocytes (closed circles) and T cells (open circles) in 218 nasal cell samples collected over a five month period (n = 117 volunteers, sampled up to five times). Individual samples and loess curves are depicted for both populations. (B, C) The correlation for individuals in four repeated measurements over a 33-day period for (B) granulocytes and (C) T cells. doi:10.1371/journal.pone.0169805.g002 Superior Cytokine Detection and Reproducible Data from Nasal Microbiopsy PLOS ONE | DOI:10.1371/journal.pone.0169805 January 20, 2017leukocyte antigen-antigen D related (HLA-DR) + cells, which are likely to consist of B cells and dendritic cells (median 1.7%, IQR 0.9-3.2%, p < 0.001 compared to NW). Of all immune cells collected by curettage and NW, 81.7% and 96.4% could be characterized, respectively. [/fig] [fig] Fig 4: Comparison of samples from nasal mucosa and blood. (A) Median proportions of granulocytes, T cells, monocytes, lineage -HLA-DR + and uncharacterized cells among immune cells in blood (n = 10) and nasal curette (n = 139). **** p < 0.0001 Mann-Whitney test. (B) The percentage of HLA-DR + T cells in blood and nasal curette samples and mean fluorescent intensity (MFI) of HLA-DR and CD66b on granulocytes was measured for blood, nasal curette and nasal wash (n = 8) samples. Median and interquartile range are shown. *p < 0.05, ***p < 0.001 Kruskal-Wallis, followed by Dunn's Multiple Comparison Test. (C) Multi-dimensional scaling analysis shows the clustering of samples from blood (grey circles), nasal curette (open squares, 11 randomly selected) and nasal wash (black triangles). The epithelial cell yield, activation state of granulocytes and composition of the immune cells were taken into account. Kruskal stress = 5.8% and Analysis of Similarity ANOSIM p-value = 0.001. doi:10.1371/journal.pone.0169805.g004 Superior Cytokine Detection and Reproducible Data from Nasal Microbiopsy PLOS ONE | DOI:10.1371/journal.pone.0169805 January 20, 2017 [/fig] [fig] Fig 5: Comparison of nasal curettes with visible red blood cell contamination (n = 55) or not (n = 62). (A) Median proportions of granulocytes, T cells, monocytes, lineage -HLA-DR + and uncharacterized cells among immune cells. (B) The percentage of HLA-DR + T cells (left axis) and mean fluorescent intensity (MFI) of HLA-DR and CD66b on granulocytes (right axis) of nasal curette samples visibly contaminated with erythrocytes (open squares) or not (open circles). Individuals samples and median and interquartile range are shown. doi:10.1371/journal.pone.0169805.g005 Superior Cytokine Detection and Reproducible Data from Nasal Microbiopsy PLOS ONE | DOI:10.1371/journal.pone.0169805 January 20, 2017 [/fig] [fig] Fig 6: Comparison of cytokine levels in samples collected by nasal wash and using nasosorption devices. (A) Levels of IL-8, IP-10 and MCP1 were measured by Luminex in nasosorption devices and by ELISA from paired nasal washes (n = 41). The r 2 and p indicate goodness-of-fit and p-value calculated by linear regression analysis (B) A heat map depicts log-transformed cytokine concentrations, with white and black indicating low and high levels, respectively. Each of the columns corresponds to one sample (n = 6 nasal wash and nasosorption) and each of the rows to one cytokine. A legend assigning color gradient to log-transformed cytokine levels is displayed. (C) The ratio of cytokine concentrations measured in paired nasosorption and nasal wash (median and IQR are shown, n = 6).doi:10.1371/journal.pone.0169805.g006 [/fig] [fig] S1: Fig. Image of nasal curette (ASL Rhino-Pro © , Arlington Scientific) before insertion into nostril. (TIF) S2 Fig. Image of adsorptive matrix strip (Nasosorption™, Hunt Developments) before insertion into nostril. (TIF) S3 Fig. Flow cytometry data for one volunteer showing with absence of CD163 and CD206 staining in the non-granulocyte immune gate. Some CD14 positive events can be seen for this volunteer indicating the presence of monocytes in the sample. (TIF) Table. Demographics and symptoms of the groups with and without additional nasal sampling. (DOCX) Video. Supplementary video demonstrating correct nasal curette sampling technique. The curette is placed on the inferior turbinate and then dragged forward to collect cells from the nasal lining. (MP4) [/fig]
The Impact of Purifying and Background Selection on the Inference of Population History: Problems and Prospects SuppFigure 15: Performance of demographic inference by MSMC (red lines) and fastsimcoal2 (blue lines) under different scenarios of neutrality, when the true model is equilibrium: (a) there is variation in recombination and mutation rates, (b) there is variation in recombination and mutation rates and the centromeric region is masked, (c) there is variation in recombination and mutation rates, and short regions resembling repeats (comprising 10% of each chromosome) are randomly masked across the genome, and (d) there is variation in recombination and mutation rates, and the centromere as well as small-sized repeats are randomly masked across the genome. The maximum and minimum fold change detected in every scenario is indicated on the upper right corner. Detailed methods including command lines can be found here: https://github.com/paruljohri/demographic_inference_with_selection/blob/main/CommandLines/ SuppFigure15-20.txt. ## Supplementary information Supp : Performance of fastsimcoal2 under neutrality and demographic equilibrium with varying chromosome sizes, when the correct model was specified. Inference was performed using 50 diploid individuals and all SNPs, and a comparison is given between simulations performed in SLiM (3.1) as well as msprime (0.7.3). The number of replicates for each chromosome size was set to 100, except for 1Gb chromosomes simulated in SLiM for which we report 10 replicates. Detailed methods including command lines can be found here: https://github.com/paruljohri/demographic_inference_with_selection/blob/main/CommandLines/ SuppTable1.txt.SuppSupp , and DFE0 refers to neutrality. Results are shown when all SNPs were used for inference, when 5%, 10% or 20% of the genomes were exonic (with exonic sites masked), and the standard AIC penalty was used. Detailed methods including command lines can be found here: https://github.com/paruljohri/demographic_inference_with_selection/blob/main/CommandLines/ SuppFigure5.txt. Supp : Model selection by fastsimcoal2 in the presence of BGS, when chosen from four possible models: equilibrium, instantaneous size change, exponential size change, and instantaneous bottleneck. The DFEs are specified in , and DFE0 refers to neutrality. Results are shown when SNPs used for inference were separated at a distance of 100 kb, 20% of the genomes were exonic, and the AIC penalty was 2× (standard) or 25× the number of parameters. Detailed methods including command lines can be found here: https://github.com/paruljohri/demographic_inference_with_selection/blob/main/CommandLines/ SuppFigure6.txt. : Effects of BGS on inference of growth or decline by fastsimcoal2. The inferred model was classified as growth if < and as decline if > . The DFEs are specified in . Results are shown for all SNPs, when 5%, 10% and 20% of the genomes were exonic (with exonic sites masked), and a standard AIC penalty was used. Detailed methods including command lines can be found here: https://github.com/paruljohri/demographic_inference_with_selection/blob/main/CommandLines/ SuppFigure7.txt. : Inferred demography from MSMC (red lines) and fastsimcoal2 (blue lines) in the presence of background selection, with the true DFE shown to the left of the panel, for 2-fold instantaneous decline (right column) and 2-fold exponential growth (left column). In this case, 20% of the genome was exonic (and exonic sites were masked). The true demographic models are shown as black lines. Detailed methods including command lines can be found here: https://github.com/paruljohri/demographic_inference_with_selection/blob/main/CommandLines/ SuppFigure8.txt. : Inference of demography by MSMC (red lines; 10 replicates) and fastsimcoal2 (blue lines; 10 replicates) under demographic equilibrium (left column), 30-fold exponential growth (middle column), and ~6-fold instantaneous decline (right column) in the presence of direct purifying selection (i.e., directly selected sites are not masked). The true demographic model is depicted in black lines. Exonic sites experience purifying selection specified by the following DFEs (defined in : (a) DFE1, (b) DFE2, (c) DFE3, (d) DFE4, (e) DFE5, (f) DFE6. In this case, 20% of the genome was exonic and all SNPs were used for inference, including exonic sites. Detailed methods including command lines can be found here: https://github.com/paruljohri/demographic_inference_with_selection/blob/main/CommandLines/ SuppFigure9.txt. : Inference of the timing of 30-fold exponential growth by fastsimcoal2 in the presence of BGS compared to neutrality (DFE0). The DFEs are specified in . Results are shown for the case where (a) 5%, (b) 10%, and (c) 20% of the genome is exonic (with exonic sites masked), and all non-exonic SNPs are used for inference. The black horizontal line represents the true timing. : Inference of the timing of 2-fold exponential growth by fastsimcoal2 in the presence of BGS compared to neutrality (DFE0). Results are shown for the case where (a) 5%, (b) 10%, and (c) 20% of the genome is exonic (with exonic sites masked), and all non-exonic SNPs are used for inference. The black horizontal line represents the true timing. presence of BGS compared to neutrality (DFE0). The DFEs are specified in . Results are shown for the case in which (a) 5%, (b) 10%, and (c) 20% of the genome is exonic (with exonic sites masked), and all non-exonic SNPs are used for inference. The black horizontal line represents the true timing. Boxplots are presented in green if decline was inferred, in yellow if growth was inferred, and in blue if a bottleneck was inferred. : Inference of the timing of 2-fold instantaneous decline by fastsimcoal2 in the presence of BGS compared to neutrality (DFE0). The DFEs are specified in . Results are shown for the case where (a) 5%, (b) 10%, and (c) 20% of the genome is exonic (with exonic sites masked), and all non-exonic SNPs are used for inference. The black horizontal line represents the true timing. Boxplots are presented in green if decline was inferred, in yellow if growth was inferred, and in blue if a bottleneck was inferred. : Distribution of lengths of repeat regions in the human genomes (hg19). Shown above is the distribution of lengths up to 1000 bp, although lengths of repeat regions range between 6 to 160602 bp. Detailed methods including command lines can be found here: https://github.com/paruljohri/demographic_inference_with_selection/blob/main/CommandLines/ SuppFigure14.txt. : Performance of demographic inference by MSMC (red lines) and fastsimcoal2 (blue lines) under different scenarios of neutrality, when the true model is 30-fold exponential growth: (a) there is variation in recombination and mutation rates across the genome, (b) there is variation in recombination and mutation rates, and the centromeric region is masked, (c) there is variation in recombination and mutation rates, and short regions resembling repeats are randomly masked across the genome (comprising of 10% of each chromosome), and (d) there is variation in recombination and mutation rates, and the centromere as well as small-sized repeats are randomly masked across the genome. Detailed methods including command can be found here: https://github.com/paruljohri/demographic_inference_with_selection/blob/main/CommandLines/ SuppFigure15-20.txt. : Performance of demographic inference by MSMC (red lines) and fastsimcoal2 (blue lines) under different scenarios of neutrality, when the true model is 6-fold instantaneous decline: (a) there is variation in recombination and mutation rates across the genome, (b) there is variation in recombination and mutation rates, and the centromeric region is masked, (c) there is variation in recombination sand mutation rates, and short regions resembling repeats are randomly masked across the genome (comprising of 10% of each chromosome), and (d) there is variation in recombination and mutation rates, and the centromere as well as small-sized repeats are randomly masked across the genome. Detailed methods including command lines can be found here: https://github.com/paruljohri/demographic_inference_with_selection/blob/main/CommandLines/ SuppFigure15-20.txt. : Performance of demographic inference by MSMC (red lines) and fastsimcoal2 (blue lines) in the presence of background selection, under different scenarios when the true model is 30-fold exponential growth: (a) there is variation in recombination and mutation rates, (b) there is variation in recombination and mutation rates and the centromeric region is masked, (c) there is variation in recombination and mutation rates, and short regions resembling repeats (comprising 10% of each chromosome) are randomly masked across the genome, and (d) there is variation in recombination and mutation rates, and the centromere as well as small-sized repeats are randomly masked across the genome. Exons comprise of 20% of the genome, experience purifying selection given by DFE4 (f0 = f1 = f2 = f3 = 0.25), and are masked when performing inference. Detailed methods including command lines can be found here: https://github.com/paruljohri/demographic_inference_with_selection/blob/main/CommandLines/ SuppFigure15-20.txt. : Performance of demographic inference by MSMC (red lines) and fastsimcoal2 (blue lines) in the presence of background selection, under different scenarios when the true model is a 6-fold instantaneous decline: (a) there is variation in recombination and mutation rates, (b) there is variation in recombination and mutation rates and the centromeric region is masked, (c) there is variation in recombination and mutation rates, and short regions resembling repeats (comprising 10% of each chromosome) are randomly masked across the genome, and (d) there is variation in recombination and mutation rates, and the centromere as well as small-sized repeats are randomly masked across the genome. Exons comprise of 20% of the genome, experience purifying selection given by DFE4 (f0 = f1 = f2 = f3 = 0.25), and are masked when performing inference. Detailed methods including command lines can be found here: https://github.com/paruljohri/demographic_inference_with_selection/blob/main/CommandLines/ SuppFigure15-20.txt. : Performance of the ABC method when recombination rate is mis-specified. (a) The true recombination rate is 2-fold higher than that assumed, and (b) the true recombination rate is 2-fold lower than that assumed. Boxplots in green show the posterior estimates when the recombination rate is higher or lower than assumed. For comparison, boxplots in red show the posterior inferred when the corresponding recombination rate is correctly specified. The black line displays the true ancestral population size ( ) and the gray line represents the true current population size ( ). Detailed methods including command lines can be found here: https://github.com/paruljohri/demographic_inference_with_selection/blob/main/CommandLines/ SuppFigure21.txt. ## Supp ## Supp ## Supp ## Supp ## Supp ## Supp ## Supp ## Supp ## Supp ## Supp ## Supp ## Supp ## Supp
Comparative immune profiling of acute respiratory distress syndrome patients with or without SARS-CoV-2 infection ## In brief Roussel et al. characterize the immune profile of COVID-19 + and COVID-19 À patients, both presenting an acute respiratory distress syndrome (ARDS) and COVID-19 + without ARDS. They identify a COVID-19 signature associating CD169 + S100A9 + monocytes, plasmablasts, and Th1 cells. CD14 + HLA-DR lo and CD14 lo CD16 + monocytes increase during the ICU stay, correlating with an unfavorable clinical course. # Introduction The severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) virus has rapidly affected >30 million people worldwide, requiring admission to intensive care units (ICUs) for >2 million patients. [bib_ref] Factors associated with COVID-19-related death using OpenSAFELY, Williamson [/bib_ref] Whereas most patients exhibit mild-to-moderate symptoms, acute respiratory distress syndrome (ARDS) is the major complication of coronavirus disease 2019 (COVID-19), [bib_ref] Clinical Characteristics of Coronavirus Disease 2019 in China, Guan [/bib_ref] [bib_ref] Clinical features of patients infected with 2019 novel coronavirus in Wuhan, Huang [/bib_ref] leading to prolonged ICU stays and a high frequency of secondary complications, notably cardiovascular events, thrombosis, pulmonary embolisms, and strokes. [bib_ref] Factors associated with COVID-19-related death using OpenSAFELY, Williamson [/bib_ref] [bib_ref] High risk of thrombosis in patients with severe SARS-CoV-2 infection: a multicenter..., Helms [/bib_ref] The immune system plays a dual role in COVID-19, contributing to both virus elimination and ARDS development. [bib_ref] COVID-19 and the human innate immune system, Schultze [/bib_ref] Excessive inflammatory response has been proposed as the leading cause of COVID-19-related clinical complications, thus supporting intensive efforts to better understand the specificities and mechanisms of SARS-CoV-2-induced immune dysfunction. [bib_ref] Clinical and immunological features of severe and moderate coronavirus disease 2019, Chen [/bib_ref] Moreover, even if therapies such as those provided by convalescent plasma or neutralizing antibodies at an early stage of the disease can lower the viral burden, this was demonstrated only in specific populations such as patients older than age 75, [bib_ref] Early High-Titer Plasma Therapy to Prevent Severe Covid-19 in Older Adults, Libster [/bib_ref] and no antiviral treatment has yet been able to definitively prevent the evolution of some patients toward deregulated inflammation and critical respiratory complications. The benefit of corticosteroids in severe COVID-19 for lowering overall mortality is now widely acknowledged. 9,10 Conversely, steroid therapy was shown to be harmful in other ARDS etiologies, such as in influenza-associated ARDS, [bib_ref] The effect of corticosteroids on mortality of patients with influenza pneumonia: a..., Ni [/bib_ref] suggesting specific biological features of COVID-19-related ARDS. A detailed understanding of the COVID-19-specific immune dysfunctions underlying ARDS development and severity is thus a high priority and will, it is hoped, help us to adopt a specific therapeutic strategy. A number of high-resolution studies have recently concentrated on the determination of circulating markers that can distinguish severe from mild forms of COVID-19, providing a tremendous amount of data describing phenotypic and functional alterations in T cell, B cell, and myeloid cell subsets. [bib_ref] Expansion of myeloid-derived suppressor cells in patients with severe coronavirus disease (COVID-19), Agrati [/bib_ref] [bib_ref] Systems biological assessment of immunity to mild versus severe COVID-19 infection in..., Arunachalam [/bib_ref] [bib_ref] T cell responses in patients with COVID-19, Chen [/bib_ref] [bib_ref] A distinct innate immune signature marks progression from mild to severe COVID-19, Chevrier [/bib_ref] [bib_ref] Impaired type I interferon activity and inflammatory responses in severe COVID-19 patients, Hadjadj [/bib_ref] [bib_ref] Longitudinal analyses reveal immunological misfiring in severe COVID-19, Lucas [/bib_ref] [bib_ref] Longitudinal immune profiling reveals key myeloid signatures associated with COVID-19, Mann [/bib_ref] [bib_ref] Deep immune profiling of COVID-19 patients reveals distinct immunotypes with therapeutic implications, Mathew [/bib_ref] In particular, CD14 + HLA-DR low , CD14 + CD16 + , and immature monocytes were demonstrated to be increased among peripheral blood mononuclear cells (PBMCs) from critically ill COVID-19 patients. [bib_ref] A distinct innate immune signature marks progression from mild to severe COVID-19, Chevrier [/bib_ref] Interestingly, the monocyte number is reduced in COVID-19 patients compared to influenza patients, suggesting specific myeloid dysregulation. Various COVID-19-related alterations of lymphoid cells have also been described, including a T cell lymphopenia, predictive of patient outcome; a broad T cell activation, including T helper cell 1 (Th1), Th2, and Th17; an alteration of B cell and T cell repertoires; and a strong increase in plasmablasts, most prominently in COVID-19 ARDS patients. [bib_ref] T cell responses in patients with COVID-19, Chen [/bib_ref] [bib_ref] Longitudinal analyses reveal immunological misfiring in severe COVID-19, Lucas [/bib_ref] Importantly, COVID-19 ARDS immune profiling was performed using healthy donors as a control, thus precluding any conclusions on whether reported immune alterations could be related to COVID-19 and/ or ARDS status. Answering this question has the potential to decipher whether ARDS induced by SARS-CoV-2 is mechanistically different from other ARDS etiologies. To fill this gap, we performed a high-throughput mass cytometry approach on PBMCs obtained from 3 complementary series of 18 COVID-19 À ARDS + , 18 COVID-19 + ARDS + , and 20 COVID-19 + ARDS À patients, including exploratory and validation cohorts. We report common myeloid cell alterations in all COVID-19 patients, which are absent from non-COVID-19 ARDS patients. This includes in particular a strong increase in an unusual population of activated monocytes showing the upregulated expression of CD169, associated with major COVID-19specific alterations of T and B cell compartments. # Results ## Study population Analyses were performed on cohort 1 of 63 cryopreserved PBMC samples isolated from 42 patients included in ICUs (n = 36) or infectious standard wards (n = 6). The demographic characteristics of patients included are provided in [fig_ref] Table 1: Patients' characteristics for cohort 1 [/fig_ref]. All patients but 1 were classified as severe at admission, requiring oxygen at a flow rate >2 L/min. ARDS was defined in accordance with international guidelines. 34 Patients were classified in 3 groups: COVID-19 À ARDS + (n = 12, ARDS stages: 1 mild, 4 moderate, 7 severe), COVID-19 + ARDS + (n = 13, ARDS stages: 8 moderate, 5 severe), and COVID-19 + ARDS À (n = 17, including 11 from ICUs and 6 from infectious standard wards). In the COVID-19 + ARDS À , no statistical differences were noticed for immune cell abundance or phenotype between ICU and standard ward patients. Within the COVID-19 À ARDS + group, ARDS etiologies were bacterial pneumonia (n = 9), anti-synthetase syndrome (n = 1), and unknown (n = 2) [fig_ref] Table 1: Patients' characteristics for cohort 1 [/fig_ref]. For 21 patients, a second blood sample obtained on day 7 (D7) after enrollment was studied (n = 7 for COVID-19 À ARDS + , n = 8 for COVID-19 + ARDS + , and n = 6 for COVID-19 + ARDS À ). In addition, a validation cohort (cohort 2) was set up with 16 patients, with demographic data detailed in Tables S1 and S2. Patients were classified in 3 groups: COVID-19 À ARDS + (n = 6), COVID-19 + ARDS + (n = 5), and COVID-19 + ARDS À (n = 3); additionally, COVID-19 À ARDS À (n = 2) samples were included. None of our patients received corticosteroids at the time of the study nor immunomodulators. The presence of SARS-CoV-2 in respiratory specimens (nasal and pharyngeal swabs or sputum) was detected by real-time reverse transcription-polymerase chain reaction (RT-PCR) methods. To rule out undetected infections, negative RT-PCR samples were confirmed when possible by the absence of neutralizing antibodies. Neutralizing antibodies were undetectable for the 11 samples of the 18 COVID-19 À patients for which material was available. In contrast, neutralizing antibodies were detected in 29 of 30 COVID-19 + tested. The timeline of the sample collection is shown in [fig_ref] Figure 1: SARS-CoV-2 induces specific phenotype of circulating immune cells CellCnn analysis performed on... [/fig_ref]. ## Sars-cov-2 induces phenotypic changes in circulating immune cells To decipher the impact of SARS-CoV-2 on circulating immune cells, we characterized PBMCs from COVID-19 + versus COVID-19 À patients at admission using two separate mass cytometry panels exploring myeloid and lymphoid subsets, respectively . The full pipeline of analysis is depicted in [fig_ref] Figure 1: SARS-CoV-2 induces specific phenotype of circulating immune cells CellCnn analysis performed on... [/fig_ref]. We performed an unbiased discovery approach with CellCnn, a neural network-based artificial intelligence algorithm allowing the analysis of single-cell data and detection of cells associated with clinical status. 35-37 During training, CellCnn learns combinations of weights for each marker in a given panel that best discriminate between groups of patients. These weight combinations, called filters, can be used to highlight the specific profiles of cells associated with patient status. We identified the best-performing CellCnn filters for both the myeloid and the lymphoid panels, highlighting a population of cells significantly enriched in COVID-19 + patients as compared to COVID-19 À patients (p < 0.0001 for both panels) [fig_ref] Figure 1: SARS-CoV-2 induces specific phenotype of circulating immune cells CellCnn analysis performed on... [/fig_ref]. Projecting these cells on t-distributed stochastic neighbor embedding (t-SNE) maps generated with either the myeloid or the lymphoid panels revealed that they fell into several distinct areas [fig_ref] Figure 1: SARS-CoV-2 induces specific phenotype of circulating immune cells CellCnn analysis performed on... [/fig_ref]. The cells selected by the CellCnn filter on the myeloid panel showed high expression for CD169, CD64, S100A9, CD11b, CD33, CD14, and CD36 compared to background, while the cells selected by the CellCnn filter on the lymphoid panel showed high expression for CD38 and CXCR3 [fig_ref] Figure 1: SARS-CoV-2 induces specific phenotype of circulating immune cells CellCnn analysis performed on... [/fig_ref]. These results were replicated in cohort 2 [fig_ref] Figure 3: Monocyte metaclusters enriched in COVID-19 are correlated with effector memory T cells... [/fig_ref] and confirmed on a public set of data by using the CellCnn analysis, showing high expression of CD14, CD36, CD64, and CD169 cells on COVID-19 + patients [fig_ref] Figure 4: Evolution of immune cell subsets between D0 and D7, defines high-risk clinical... [/fig_ref]. [bib_ref] A distinct innate immune signature marks progression from mild to severe COVID-19, Chevrier [/bib_ref] As a whole, this broad and unbiased approach reproducibly showed that immune markers, in particular related to monocytes, segregated COVID-19 À and COVID-19 + patients. ## Sars-cov-2 induces cd169-expressing monocyte subsets To investigate circulating monocyte heterogeneity and define consistent phenotypes, we used the FlowSOM algorithm. This approach led to the identification of 15 monocyte metaclusters from the myeloid panel [fig_ref] Figure 2: CD169 monocytes are enriched in SARS-CoV-2-infected patients [/fig_ref]. In particular, Mo30, Mo11, and Mo28 metaclusters were defined by higher expression of CD16 and lower expression of CD14, CD36, and CD64, corresponding to a non-classical monocyte phenotype. Mo21 and Mo22 were defined by the high expression of S100A9 and the low expression of CD36. Finally, Mo243 and Mo180 strongly expressed S100A9, CD169, and CD36. To assess the phenotypic changes in monocytes during SARS-CoV-2 infection, we determined the frequencies of these metaclusters in each patient at admission and performed hierarchical clustering on these values [fig_ref] Figure 2: CD169 monocytes are enriched in SARS-CoV-2-infected patients [/fig_ref]. The upper branch of the hierarchical clustering included 20 COVID + (10 ARDS À and 10 ARDS + ) patients and 1 COVID À ARDS + patient, whereas the lower branch included 10 COVID + (7 ARDS À and 3 ARDS + ) and 11 COVID À ARDS + (chisquare test = 0.001) [fig_ref] Figure 2: CD169 monocytes are enriched in SARS-CoV-2-infected patients [/fig_ref]. We then analyzed the abundance of individual metaclusters and identified only 4 of 15 metaclusters as differentially represented between the 3 groups of patients [fig_ref] Figure 2: CD169 monocytes are enriched in SARS-CoV-2-infected patients [/fig_ref]. In particular, within ARDS + patients, Mo11 and M181 were less abundant in COVID-19 + patients (p < 0.01 and p < 0.05, respectively), while Mo243 and Mo180 were more abundant (p < 0.05 and p < 0.001) [fig_ref] Figure 2: CD169 monocytes are enriched in SARS-CoV-2-infected patients [/fig_ref]. No differences were detected within COVID-19 + groups (ARDS + versus ARDS À ) [fig_ref] Figure 2: CD169 monocytes are enriched in SARS-CoV-2-infected patients [/fig_ref]. Interestingly, Mo243 and Mo180 were both enriched in cells highly expressing CD169, CD64, CD36, and CD14 [fig_ref] Figure 2: CD169 monocytes are enriched in SARS-CoV-2-infected patients [/fig_ref]. In addition, Mo22 was present only in some COVID + patients and also expressed CD169 [fig_ref] Figure 2: CD169 monocytes are enriched in SARS-CoV-2-infected patients [/fig_ref]. Taken together, Mo243, Mo180, and Mo22 metaclusters were highly enriched in COVID-19 + patients when compared to COVID-19 À patients (p < 0.0001), with no difference regarding the ARDS status [fig_ref] Figure 2: CD169 monocytes are enriched in SARS-CoV-2-infected patients [/fig_ref]. Accordingly, CD169 was differentially expressed in COVID-19 + versus COVID-19 À patients (p < 0.001) [fig_ref] Figure 2: CD169 monocytes are enriched in SARS-CoV-2-infected patients [/fig_ref]. Our study including COVID-19 and non-COVID-19 critically ill patients suggests a , these metaclusters also presenting a trend for high CD169 expression [fig_ref] Figure 3: Monocyte metaclusters enriched in COVID-19 are correlated with effector memory T cells... [/fig_ref]. [formula] Chronic respiratory disease, n (%) 1 (8.3) 0 (0) 0 (0) Chronic kidney disease, n (%) 0 (0) 2 (15.4) 0 (0) Cancer, n (%) 3 (25) 0 (0) 0 (0) [/formula] ## Monocyte metacluster enrichment in covid-19 is correlated with a specific increase in effector memory t cells and plasma cells To define a more global immune pattern and the relationship between immune cells in the context of the SARS-CoV-2 infection, we sought correlation between the frequencies of clusters of T, natural killer (NK), B, and plasma cells (n = 136 clusters from the lymphoid panel; [fig_ref] Figure 1: SARS-CoV-2 induces specific phenotype of circulating immune cells CellCnn analysis performed on... [/fig_ref] and the 4 monocyte metaclusters (Mo11, Mo181, Mo243, and Mo180) previously described. This analysis identified 70 clusters with significantly correlated variations (p < 0.05) [fig_ref] Figure 2: CD169 monocytes are enriched in SARS-CoV-2-infected patients [/fig_ref]. To strengthen the relevance of these correlations, we restrained further analysis to the 29 strongest relationships (R > 0.5 or < ÀÀ0.5 and p < 0.01) between Mo180 or Mo243 (the 2 metaclusters enriched in COVID-19 patients) and other immune cell subsets [fig_ref] Figure 3: Monocyte metaclusters enriched in COVID-19 are correlated with effector memory T cells... [/fig_ref] ; . As expected, Mo180 and Mo243 metaclusters were correlated (R = 0.93). Moreover, they were positively correlated with 18 clusters of T (n = 6), NK (n = 10), and plasma cells (n = 2), and inversely correlated with 11 clusters of T (n = 9) and NK cells (n = 2) [fig_ref] Figure 3: Monocyte metaclusters enriched in COVID-19 are correlated with effector memory T cells... [/fig_ref]. Among positively correlated clusters, plasmo_183 and plasmo_198 similarly expressed CD38, CD44, and CD27, whereas plasmo_183 was high for Ki-67 and human leukocyte antigen-DR isotype (HLA-DR), corresponding to an early plasma cell phenotype [fig_ref] Figure 3: Monocyte metaclusters enriched in COVID-19 are correlated with effector memory T cells... [/fig_ref]. NK cells were marked by CD7 and T-bet expression, NK_209 being CD8 high , and NK_241 and NK_197 displaying a Ki-67 high proliferating phenotype. The related T8_147 and T8_161 clusters exhibited a CD45RA high CD45RO low CCD7 low CD27 low Tbet high CD38 high effector phenotype. Few T4 clusters were positively correlated with Mo180 and Mo243; among them, T4_106 displayed an effector memory proliferating phenotype (Ki-67 high CD45RA low CCR7 low CD45RO high CD27 high and CTLA4 high PD1 high ). T4_25 was also marked by an effector memory phenotype (CD45RA low CCR7 low CD45RO + ) and displayed a CD27 low CD127 + CCR6 + CxCR3 À CD161 + Th17 profile [fig_ref] Figure 3: Monocyte metaclusters enriched in COVID-19 are correlated with effector memory T cells... [/fig_ref]. Conversely, some T4 clusters were inversely correlated with Mo_180 and Mo_243-in particular clusters T4_6, T4_20, and T4_34-all three corresponding to naive cells (CD45RA high CD45RO low CCR7 high ), and T4_59 expressing a Th2 phenotype (CCR4 high ). We then compared the abundance of these 29 lymphoid clusters correlated with Mo180 and Mo243 and highlighted the 22 differentially represented lymphoid clusters between the 3 groups of patients (p < 0.05) [fig_ref] Figure 2: CD169 monocytes are enriched in SARS-CoV-2-infected patients [/fig_ref]. Only 7 clusters of CD4 T cells and 2 clusters of CD8 T cells were at lower abundance in COVID-19 + ARDS + patients compared to COVID-19 À ARDS + patients. As previously discussed, T4_6, T4_20, and T4_34 corresponded to naive cells, whereas within the effector memory cells, T4_7 and T4_45 were CD127 low , T4_24, T8_99, and T8_113 were CD127 high and T4_59 was CCR4 high . Conversely, 13 clusters were enriched in COVID-19 + ARDS + compared to COVID-19 À ARDS + , including: (1) CTLA4 high PD1 high effector memory activated CD4 T cells (T4_106); (2) Tbet high Th1-like CD8 effector phenotype (T8_146, T8_147, and T8_161); (3) cytotoxic mature CD16 + CD56 low CD7 + Tbet + CD127 À NK cells (NK_209, NK_241, NK_242, and NK_244), with proliferating Ki-67 high NK cells (NK_241); and (4) proliferating plasmablasts (plasmo_183) and mature plasma cells (plasmo_198) [fig_ref] Figure 3: Monocyte metaclusters enriched in COVID-19 are correlated with effector memory T cells... [/fig_ref]. Of note, no cluster was differentially expressed between COVID-19 + ARDS + and COVID-19 + ARDS À groups [fig_ref] Figure 2: CD169 monocytes are enriched in SARS-CoV-2-infected patients [/fig_ref]. Then, to explore the whole immune profile and define relationships with groups of patients, we performed a correspondence analysis (CA) using, as a variable, the abundance of the myeloid (n = 4) and the lymphoid (n = 22) clusters differentially expressed between groups of patients [fig_ref] Figure 3: Monocyte metaclusters enriched in COVID-19 are correlated with effector memory T cells... [/fig_ref]. CA was developed to analyze frequency tables and visualize similarities between patients and cooccurrence of cell subsets. [bib_ref] An Immune Atlas of Clear Cell Renal Cell Carcinoma, Chevrier [/bib_ref] The first and second dimensions of the CA explained 80.5% and 13.5% of the difference, respectively [fig_ref] Figure 3: Monocyte metaclusters enriched in COVID-19 are correlated with effector memory T cells... [/fig_ref]. The top 10 cell populations accounting for the difference between COVID + and COVID À patients were Mo243, Mo180, T8_146, NK_244, and T8_161 being increased and Mo181, T4_6, Mo11, T8_99, and T4_45 being decreased in COVID + . These subsets corresponded to an increase in inflammatory monocytes (CD169 high CD64 high ), Tbet high Th1-like CD8 T cells, and mature NK cells and a decrease in naive T4 cells and effector memory T4 and T8 cells. Interestingly, only the first dimension of the CA segregated COVID-19 + ARDS + from COVID-19 À ARDS + (p < 0.001), and no statistical differences was found between COVID-19 + ARDS + and COVID-19 + ARDS À [fig_ref] Figure 3: Monocyte metaclusters enriched in COVID-19 are correlated with effector memory T cells... [/fig_ref]. [fig_ref] Figure 4: Evolution of immune cell subsets between D0 and D7, defines high-risk clinical... [/fig_ref]. The first and second dimensions of the CA explained 85.1% and 9% of the differences acquired between D0 and D7. The first dimension captured the difference between D0 and D7 only for COVID-19 + ARDS + (p < 0.01) [fig_ref] Figure 4: Evolution of immune cell subsets between D0 and D7, defines high-risk clinical... [/fig_ref]. Because of the limited number of samples, only a trend was observed for COVID + ARDS À (p = 0.062). The top 5 enriched populations accounting for the differences between D0 and D7 for COVID-19 + ARDS + patients were Mo11, Mo181, T8_113, T4_34, and NK_197, corresponding to an enrichment in non-classical monocytes (CD14 low CD16 high CD64 low CD36 low S100A9 high ), in monocytic myeloid-derived suppressor cell (M-MDSC)-like (HLA-DR low S100A9 high ), in effector memory CD127 high T8 cells, in T4 naive cells, and in Ki-67 high proliferating NK cells. These 5 cell subsets were integrated in an immune score combining their fold change between D0 and D7. To define the relevance of this immune score in discriminating COVID-19 patients with unfavorable prognosis, we built a clinical score as the sum of events occurring during ICU stay (thromboembolic, ICU-acquired infection, septic shock, renal failure, and death) [fig_ref] Table 1: Patients' characteristics for cohort 1 [/fig_ref]. Interestingly, both the clinical and the immune scores were found to be correlated in severe COVID-19 patients, irrespectively of their ARDS status (Spearman R = 0.71; p = 0.006) [fig_ref] Figure 4: Evolution of immune cell subsets between D0 and D7, defines high-risk clinical... [/fig_ref]. Finally, we analyzed changes between D0 and D7 of genes involved in the interferon (IFN) pathway. We found an upregulation of IFNAR1 (interferon alpha and beta receptor subunit 1 gene) and IFNAR2 during time in COVID + ARDS + . Conversely, the evolution of IFN type I target genes (ISG15, IFI27, IFI44L, RSAD2, and IFIT1) revealed a specific downregulation in COVID + ARDS + samples. Interestingly, both IFNAR score and type I IFN score, obtained by combining the expression of IFN receptors and targets, respectively, presented a trend of correlation with the immune score , and the type I IFN score was significantly correlated with the CD169 expression . # Discussion Immune response to COVID-19 infection has been recently intensively studied at both transcriptomic and proteomic levels. However, most studies focused on either the lymphoid [bib_ref] Deep immune profiling of COVID-19 patients reveals distinct immunotypes with therapeutic implications, Mathew [/bib_ref] or the myeloid compartments, [bib_ref] Expansion of myeloid-derived suppressor cells in patients with severe coronavirus disease (COVID-19), Agrati [/bib_ref] and only a few performed a wide analysis of the circulating immune landscape, [bib_ref] Systems biological assessment of immunity to mild versus severe COVID-19 infection in..., Arunachalam [/bib_ref] [bib_ref] Impaired type I interferon activity and inflammatory responses in severe COVID-19 patients, Hadjadj [/bib_ref] [bib_ref] Comprehensive mapping of immune perturbations associated with severe COVID-19, Kuri-Cervantes [/bib_ref] [bib_ref] A dynamic COVID-19 immune signature includes associations with poor prognosis, Laing [/bib_ref] thus precluding the definition of complex patterns of immune parameter alterations associated with COVID-19 severity or physiopathology. Moreover, these studies were designed to identify differences in immune cell subset frequencies between COVID-19 patients and healthy donors, and eventually correlated with the severity of the disease, but did not include severe non-COVID-19 patients as controls, although critically ill patients were previously largely demonstrated to display immune reprogramming. [bib_ref] The immune system's role in sepsis progression, resolution, and long-term outcome, Delano [/bib_ref] ARDS is a major adverse event occurring during ICU stay, leading to an overall mortality rate of 40% to 60%. Whether COVID-19-associated ARDS is clinically and biologically similar to other causes of ARDS remains controversial. [bib_ref] Clinical features, ventilatory management, and outcome of ARDS caused by COVID-19 are..., Ferrando [/bib_ref] [bib_ref] COVID-19 Does Not Lead to a ''Typical'' Acute Respiratory Distress Syndrome, Gattinoni [/bib_ref] To address this point, we characterized for the first time, by mass cytometry, the immune landscape in COVID-19associated ARDS compared to other causes of ARDS. We demonstrated that an increase in CD169 pos monocytes, correlated with specific changes of T, plasma, and NK cell subsets, defines COVID-19-associated ARDS and is not found in bacteria-associated ARDS, suggesting a COVID-19-specific immune reprogramming. The amplification of CD169 + circulating monocytes has already been highlighted in the context of COVID-19, [bib_ref] A distinct innate immune signature marks progression from mild to severe COVID-19, Chevrier [/bib_ref] [bib_ref] Monocyte CD169 Expression as a Biomarker in the Early Diagnosis of Coronavirus..., Bedin [/bib_ref] [bib_ref] Whole blood immunophenotyping uncovers immature neutrophil-to-VD2 T-cell ratio as an early marker..., Carissimo [/bib_ref] and is reminiscent of other inflammatory conditions found in viral infections, such as with human immunodeficiency virus or Epstein-Barr virus, in which the CD169 sialoadhesin is induced in an IFN-dependent manner on the surface of circulating monocytes. [bib_ref] Epstein-Barr virus lytic infection promotes activation of Toll-like receptor 8 innate immune..., Farina [/bib_ref] [bib_ref] Sialoadhesin expressed on IFN-induced monocytes binds HIV-1 and enhances infectivity, Rempel [/bib_ref] Consistent with the inflammatory response, we showed that the accumulation of CD169 pos monocytes in COVID-19 + patients is positively correlated with an increase in plasmablasts and mature plasma cells, Th1-like CD8 effector T cells, cytotoxic mature NK cells, and activated CD4 effector memory T cells displaying a CTLA4 high PD1 high phenotype. CD169 + activated monocytes were detected in mild disease 23 and were proposed to rise rapidly and transiently in patients with COVID-19, in association with a high expression of IFN-g and CCL8. [bib_ref] A distinct innate immune signature marks progression from mild to severe COVID-19, Chevrier [/bib_ref] This could be due to the transient nature of this monocytic population, either losing CD169, being short-lived, or being recruited into tissues as CD169 + macrophages, as suggested by the high expression of CCR2 on Mo243 and Mo180, the 2 monocyte subsets identified here in COVID-19 patients, and the local inflammation and lung tissue destruction mediated by monocyte-derived macrophages in severe cases of SARS-CoV-2 infections. [bib_ref] Explore COVID-19 IPH group; Explore COVID-19 Marseille Immunopole group (2020), Carvelli [/bib_ref] [bib_ref] Single-cell landscape of bronchoalveolar immune cells in patients with COVID-19, Liao [/bib_ref] Interestingly, we also found an upregulation of cytoplasmic S100A9 in monocyte subsets specifically amplified in COVID-19 patients irrespective of their ARDS status. These data suggest that, in the early stage of the disease, monocytes could contribute to the burst of circulating calprotectin (S100A8/ S100A9), recently proposed to contribute to the secondary cytokine release syndrome described in severe COVID-19 and attributed to neutrophils.Despite phenotypic alterations, our data revealed a specific alteration of the response to type I IFN in COVID-19 + versus COVD-19 À ARDS patients after a short stay in the ICU, with an upregulation of IFN receptors without induction of IFN target genes. These results are reminiscent of the demonstration that deficiency of the type I IFN pathway is associated with poor outcomes in COVID-19 patients. [bib_ref] COVID-STORM Clinicians; COVID Clinicians; Imagine COVID Group, Zhang [/bib_ref] Whereas a seroconversion score was recently associated with huge modifications in immune parameters reflecting B, T, and NK cell function in non-ICU COVID patients,our ICU patients clearly stand at a later stage of the disease, with 22 of 29 already carrying neutralizing antibodies at D0. It is thus highly unlikely that the differential evolution of monocytic markers identified between D0 and D7 in our study could be attributable to seroconversion. Within severe COVID-19 patients, we detected no significant differences between ARDS + and ARDS À immune profiles, indicating a specificity of the phenotype induced by SARS-CoV-2 infection, irrespective of the respiratory complications. While most published studies showed differences between mild and severe COVID-19 diseases, some of their conclusions may be obscured by the fact that ARDS by itself, mechanical ventilation, and/or nonspecific treatments may affect immune parameters. [bib_ref] Norepinephrine Dysregulates the Immune Response and Compromises Host Defense during Sepsis, Stolk [/bib_ref] A strength of our study comparing 2 groups of severe COVID-19 patients with or without ARDS is to highlight features directly related to the viral infection rather than to its respiratory complications or their treatment. Importantly, our cohort was homogeneous regarding treatment, with, in particular, no immunosuppressive therapy at the time of sampling. The small size of our cohort did not allow us to pinpoint a mortality prognostic factor based on our phenotypic data. However, we identified a specific immune pattern associated with the [fig_ref] Figure 2: CD169 monocytes are enriched in SARS-CoV-2-infected patients [/fig_ref] from all patients at D0 (COVID-19 À ARDS + [n = 12], COVID-19 + ARDS + [n = 13], and COVID-19 + ARDS À [n = 17]). Only strong correlations (Spearman R > 0.5 or R < À0.5 and p < 0.01) are shown (see all significant correlations [p < 0.05] in [fig_ref] Figure 2: CD169 monocytes are enriched in SARS-CoV-2-infected patients [/fig_ref] and . (B) Heatmap showing marker expression for the lymphoid clusters (Spearman R > 0.5 or R < À0.5 and p < 0.001) strongly correlated with Mo180 and Mo243 (see heatmap for all clusters and markers in [fig_ref] Figure 2: CD169 monocytes are enriched in SARS-CoV-2-infected patients [/fig_ref]. (C) Abundance of lymphoid clusters differentially expressed between groups, among singlet cells analyzed. Kruskal-Wallis test with Dunn's multiple comparison correction, *p < 0.05, **p < 0.01, ***p < 0.001 [see all clusters in [fig_ref] Figure 2: CD169 monocytes are enriched in SARS-CoV-2-infected patients [/fig_ref] Article ll OPEN ACCESS occurrence of the major adverse clinical events (thrombosis, nosocomial infection, septic shock, acute renal failure, and death) described in COVID-19 and combined as a clinical score. In particular, an increase in non-classical CD14 low CD16 + monocytes (Mo11), and CD14 + HLA-DR low M-MDSC-like (Mo181), both not expressing CD169, are markers of adverse events. This suggests that besides the early increase in CD169 + monocytes in all COVID-19 patients associated with T cell dysfunctions, the immunological response to SARS-CoV-2 infection features multiple alterations of monocytic subsets reflecting the severity of the disease. Consistent with these data, it was shown that CD14 + HLA-DR low cells were increased in critical COVID-19 patients,,26,56-58 while CD14 low CD16 + monocytes, able to migrate to the lung, were correlated with the length of stay in the ICU. [bib_ref] A distinct innate immune signature marks progression from mild to severe COVID-19, Chevrier [/bib_ref] [bib_ref] COVID-19 severity associates with pulmonary redistribution of CD1c+ DCs and inflammatory transitional..., Sá Nchez-Cerrillo [/bib_ref] Our study correlates the accumulation of nonclassical monocytes and M-MDSCs occurring during the first days of ICU to adverse events. # Limitations of study Besides the low number of included patients, our study has other limitations. By focusing on severe patients with and without ARDS, we cannot reach conclusions about phenotypic changes in mild and moderate diseases. The analysis would also benefit from comparison with other virus-associated ARDS. We thus analyzed a published dataset of flu-like illness and COVID patients, analyzed by mass cytometry.Interestingly, by using CellCnn, we were able to define a filter that accurately discriminates flu-like illness from COVID samples, suggesting immune differences between both diseases [fig_ref] Figure 4: Evolution of immune cell subsets between D0 and D7, defines high-risk clinical... [/fig_ref]. Moreover, since the mass cytometry was conducted on PBMCs, we lack information on the neutrophil lineage, which appears affected in COVID-19 disease.It would also be interesting to link these data with in situ data from lung tissue samples and bronchoalveolar lavages. Unfortunately, at the time of the study, bronchoalveolar fluid collection was not allowed in our institution for patients who were positive for SARS-CoV-2. However, our detailed analysis of circulating immune cells shows that immune monitoring of severe COVID-19 patients brings interesting prognostic biomarkers independent of their clinical classification in ARDS + versus ARDS À . Moreover, we demonstrated that at the biological level, COVID-19-associated ARDS is different from other causes of ARDS, and may benefit from personalized therapy in addition to standard ARDS management. 23,60 # Star+methods Detailed methods are provided in the online version of this paper and include the following: [formula] d KEY RESOURCES [/formula] ## Detection of sars-cov-2 neutralizing antibodies The viral strain (RoBo strain), which was cultured on Vero-E6 cells (ATCC CRL-1586), used for the nAb assay was a clinical isolate obtained from a nasopharyngeal aspirate of a patient HOS at the University Hospital of Saint-Etienne for severe COVID-19. The strain was diluted in Dulbecco's modified Eagle's medium-2% fetal calf serum in aliquots containing 100-500 tissue culture infectious doses 50% (TCID50) per ml. Each serum specimen was diluted 1:10 and serial twofold dilutions were mixed with an equal volume (100 mL each) of virus. After gentle shaking for 30 min at room temperature, 150 mL of the mixture was transferred to 96-well microplates covered with Vero-E6 cells. The plates were then placed at 37 C in a 5% CO2 incubator. Measurements were obtained microscopically 5-6 days later when the cytopathic effect of the virus control reached $100 TCID50/150 mL. The serum was considered to have protected the cells if > 50% of the cell layer was preserved. The neutralizing titer is expressed as the inverse of the higher serum dilution that protected the cells. Thresholds were set on the filter response scores to select Covid-19-associated cells by calculating the relative frequencies of selected cells in each sample at 100 different thresholds for each filter, then performing a logistic regression to predict sample labels. For each threshold, the data was first split in a stratified manner into a training set, comprising 60% of samples, and a test set, comprising 40% of samples. The logistic regression was performed on the training set, and the accuracy of resulting predictions was calculated on the test set. This procedure was performed 10 times, with randomly chosen training/test splits, and the mean of the resulting accuracies for each threshold was calculated. For the lymphoid panel, one threshold (9.63) achieved the highest accuracy and was set as the final threshold. For the myeloid panel, multiple thresholds achieved the same level of accuracy; the lowest of these (4.96) was set as the final threshold. The relative frequencies of cells in each sample with filter response scores greater than or equal to the respective thresholds were calculated and compared using a Wilcoxon rank-sum test. viSNE, FlowSOM, and hierarchical clustering We first performed a dimension reduction for both panels (i.e., myeloid and lymphoid) and all cleaned-up 63 files were first analyzed using viSNE, based upon the Barnes-Hut implementation of t-SNE. Equal downsampling was performed, based on the lowest event count in all files (lymphoid panel) or on the maximum total events allowed by Cytobank (myeloid panel). For the myeloid panel, the following parameters were used: perplexity = 45; iterations = 5000; theta = 0.5; all 37 channels selected. For the lymphoid panel the parameters were as follows: perplexity = 45; iterations = 7500; theta = 0.5; all 36 channels selected. Then we applied a clustering method using the FlowSOM clustering algorithm. FlowSOM uses Self-Organizing Maps (SOMs) to partition cells into clusters based on their phenotype, and then builds a Minimal Spanning Tree (MST) to connect the nodes of the SOM, allowing the identification of metaclusters (i.e., group of clusters). We performed the FlowSOM algorithm on the previous viSNE results, using all events and panel channels, and the following parameters: clustering method = hierarchical consensus, iterations = 10, number of clusters = 256, number of metaclusters = 30. For both panels, each metacluster (containing a given number of clusters) was manually annotated based on his marker expression phenotype, his projection on the viSNE and his localization in the FlowSOM MST. We first analyzed the myeloid panel. Out of 30 metaclusters defined by the FlowSOM approach, we identified 13 metaclusters with monocyte markers, other metaclusters contained other cell types, low count of cells or remaining doublets or dead cells. We visually identified 2 (Mo18 and Mo26) out of the 13 metaclusters that were heterogeneous. These 2 metaclusters were manually split into 2 new metaclusters (identified respectively as Mo180, Mo181 and Mo214, Mo243) [fig_ref] Figure 1: SARS-CoV-2 induces specific phenotype of circulating immune cells CellCnn analysis performed on... [/fig_ref]. Thus, altogether we analyzed 15 metaclusters of myeloid cells. Regarding the lymphoid compartment, we noticed that FlowSOM defined metaclusters at the lineage level, thus we retain all the 136 clusters included in 10 metaclusters of interest (i.e., containing lymphoid lineage markers) [fig_ref] Figure 1: SARS-CoV-2 induces specific phenotype of circulating immune cells CellCnn analysis performed on... [/fig_ref]. All metaclusters and clusters phenotypes including their abundances and mean marker intensity were then exported from Cytobank for [fig] Figure 1: SARS-CoV-2 induces specific phenotype of circulating immune cells CellCnn analysis performed on single cells from myeloid (top) and lymphoid (bottom) panels on 39 samples at admission (day 0) (COVID-19 À [n = 9] and COVID-19 + [n = 30]) (A) Frequencies of cells discovered by the best-performing CellCnn filter in COVID-19 À (blue) and COVID-19 + (orange) patients for each panel. Mann-Whitney tests, ****p < 0.0001. (B) Cells defined by the best-performing CellCnn filters enrichment shown on tSNE and representative markers for each panel (CD14 and CD38 [see additional markers in Figure S2]). [/fig] [fig] Figure 2: CD169 monocytes are enriched in SARS-CoV-2-infected patients (A) Heatmap of the 15 monocyte metaclusters defined after FlowSOM analysis. (B) Relative abundance of metaclusters among monocytes for each patient and hierarchical clustering of COVID-19 À ARDS + (n = 12, green), COVID-19 + ARDS + (n = 13, blue), and COVID-19 + ARDS À (n = 17, red). (C) Abundance of metaclusters differentially expressed between groups, among singlet cells analyzed. (D) Expression of the corresponding markers (mean metal intensity) for background (gray), Mo11 and Mo181 (orange), and Mo243 and Mo180 (blue) metaclusters. (E) Abundance of Mo22, Mo180, and Mo243 and expression of CD169 (box and whiskers with 10th and 90th percentiles). (F) Uniform manifold approximation and projection (UMAP) from scRNA-seq of COVID-19 patients (COVID-19) and healthy donors (healthy) highlighting CD14 and CD169 expression (data adapted from Wilk et al. 25 ). Kruskal-Wallis test with Dunn's multiple comparison correction, *p < 0.05, **p < 0.01, [/fig] [fig] Figure 3: Monocyte metaclusters enriched in COVID-19 are correlated with effector memory T cells and plasma cells (A) Correlation between Mo180 and Mo243 and lymphoid clusters (see heatmap for all lymphoid clusters and markers in [/fig] [fig] Figure 4: Evolution of immune cell subsets between D0 and D7, defines high-risk clinical grade COVID-19 patients (A) Two first dimensions of correspondence analysis accounting for 94.1% of the association between immune clusters differentially expressed between groups (n = 4 monocyte and n = 22 lymphoid clusters) and patients for which a follow-up of 7 days was available (COVID-19 À ARDS + [n = 7], COVID-19 + ARDS + [n = 8], and COVID-19 + ARDS À [n = 6]). For clarity, patients and immune cells are shown on 2 different plots. Dimensions 1 and 2 coordinates were compared between D0 and D7 for each group of patients. Wilcoxon matched-pairs signed rank tests, **p < 0.01.(B) Spearman correlation between immune and clinical score for COVID-19 + patients (ARDS + [n = 8] and ARDS-[n = 6]). 24. Song, J.-W., Zhang, C., Fan, X., Meng, F.-P., Xu, Z., Xia, P., Cao, W.J., Yang, T., Dai, X.P., Wang, S.Y., et al. (2020). Immunological and inflammatory profiles in mild and severe cases of COVID-19. Nat. Commun. 11, 3410. 25. Wilk, A.J., Rustagi, A., Zhao, N.Q., Roque, J., Martínez-Coló n, G.J., McKechnie, J.L., Ivison, G.T., Ranganath, T., Vergara, R., Hollis, T., et al. (2020). A single-cell atlas of the peripheral immune response in patients with severe COVID-19. Nat. Med. 26, 1070-1076. 26. Giamarellos-Bourboulis, E.J., Netea, M.G., Rovina, N., Akinosoglou, K., Antoniadou, A., Antonakos, N., Damoraki, G., Gkavogianni, T., Adami, M.E., Katsaounou, P., et al. (2020). Complex Immune Dysregulation in COVID-19 Patients with Severe Respiratory Failure. Cell Host Microbe 27, 992-1000.e3. 27. Merad, M., and Martin, J.C. (2020). Pathological inflammation in patients with COVID-19: a key role for monocytes and macrophages. Nat. Rev. Immunol. 20, 355-362. 28. Ong, E.Z., Chan, Y.F.Z., Leong, W.Y., Lee, N.M.Y., Kalimuddin, S., Haja Mohideen, S.M., Chan, K.S., Tan, A.T., Bertoletti, A., Ooi, E.E., and Low, J.G.H. (2020). A Dynamic Immune Response Shapes COVID-19 Progression. Cell Host Microbe 27, 879-882.e2. 29. Reizine, F., Lesouhaitier, M., Gregoire, M., Pinceaux, K., Gacouin, A., Maamar, A., Painvin, B., Camus, C., Le Tulzo, Y., Tattevin, P., et al. (2021). SARS-CoV-2-Induced ARDS Associates with MDSC Expansion, Lymphocyte Dysfunction, and Arginine Shortage. J. Clin. Immunol. 41, 515-525. 30. Mudd, P.A., Crawford, J.C., Turner, J.S., Souquette, A., Reynolds, D., Bender, D., Bosanquet, J.P., Anand, N.J., Striker, D.A., Martin, R.S., et al. (2020). Distinct inflammatory profiles distinguish COVID-19 from influenza with limited contributions from cytokine storm. Sci. Adv. 6, eabe3024. 31. De Biasi, S., Meschiari, M., Gibellini, L., Bellinazzi, C., Borella, R., Fidanza, L., Gozzi, L., Iannone, A., Lo Tartaro, D., Mattioli, M., et al. (2020). Marked T cell activation, senescence, exhaustion and skewing towards TH17 in patients with COVID-19 pneumonia. Nat. Commun. 11, 3434. 32. De Biasi, S., Lo Tartaro, D., Meschiari, M., Gibellini, L., Bellinazzi, C., Borella, R., Fidanza, L., Mattioli, M., Paolini, A., Gozzi, L., et al. (2020). Expansion of plasmablasts and loss of memory B cells in peripheral blood from COVID-19 patients with pneumonia. Eur. J. Immunol. 50, 1283-1294. 33. Vabret, N., Britton, G.J., Gruber, C., Hegde, S., Kim, J., Kuksin, M., Levantovsky, R., Malle, L., Moreira, A., Park, M.D., et al.; Sinai Immunology Review Project (2020). Immunology of COVID-19: Current State of the Science. Immunity 52, 910-941. 34. Ranieri, V.M., Rubenfeld, G.D., Thompson, B.T., Ferguson, N.D., Caldwell, E., Fan, E., Camporota, L., and Slutsky, A.S.; ARDS Definition Task Force (2012). Acute respiratory distress syndrome: the Berlin Definition. JAMA 307, 2526-2533. 35. Arvaniti, E., and Claassen, M. (2017). Sensitive detection of rare diseaseassociated cell subsets via representation learning. Nat. Commun. 8, 14825. [/fig] [table] Table 1: Patients' characteristics for cohort 1 [/table]
Kinetochores Generate Microtubules with Distal Plus Ends: Their Roles and Limited Lifetime in Mitosis Figure S1, (associated withFigure 1).(A) Schematic drawing of a CEN reactivation assay Using this assay, we can displace a centromere further away from a spindle pole. See detail in main text and inTanaka et al., 2005.(B) Microtubules extend from kinetochores prior to their interaction with spindle-pole microtubules (i) Short microtubules often extend from kinetochores in most of the cells in the absence of mild osmotic stress. PGAL-CEN3-tetOs GFP-TUB1 PMET3-CDC20 cells (T7673) were treated as inFig 1B (note that an array of tetOs was not visualized in this strain). GFP (tubulin) images were acquired every 1.5 sec for 10 min. Representative time-lapse images (top) with schematic diagrams (bottom) show extension of short (about 500 nm) microtubules from the CEN3-associated punctate GFP Tub1 signal. Results: We often observed a punctate GFP signal apart from the spindle and the majority of such GFP signal subsequently interacted with spindle-pole microtubules. We interpreted that the GFP signal was associated with CEN3.(ii) Distribution of the maximum extension of kinetochore (KT)-derived microtubules (MTs) in the absence (blue bars) and presence (red bars) of osmotic shock. PGAL-CEN3-tetOs TetR-4mCherry GFP-TUB1 PMET3-CDC20 cells (T7715) were treated as inFig 1B and Fig 1C,respectively. GFP (tubulin) images were acquired every 2 sec. At the start of imaging, mCherry images were also acquired in order to confirm that GFP signals were associated with mCherry signals (i.e. CEN3). In each condition, 7 cells were analyzed for 5 min. Each bar shows the average number of microtubules (per cell per min) with the categorized length at maximum extension. (C) Dynamics of kinetochore-derived microtubules: comparison with spindle-pole microtubules (i) Growth and shrinkage rate. T3828 cells (seeFig 1B)were treated as inFig 1B (with no osmotic stress) and inFig 1C (with mild osmotic stress: striped), and images were acquired every 30 sec as inFig 1C.Graphs show the growth rate (red) and shrinkage rate (blue) of spindle-pole microtubules with no CEN3 attached, and of kinetochore-derived microtubules. n: number of observed events. Error bars show SEM. n.s.: not significant.(ii) Kinetochore-derived microtubules did not show microtubule rescue, in contrast to spindle-pole microtubules that were associated with CEN3. T6718 cells (seeFig 5B)were treated as inFig 1C.mCherry (Bik1; green), YFP (tubulin; red), and CFP (CEN3; blue) images were acquired every 20 sec for 25 min. The frequency of microtubule rescue (per min) was categorized as shown in the graph. For spindle-pole microtubules, only those that were associated with CEN3 were taken into consideration. n: number of observed microtubules.(D) Quantifying the number of tubulin molecules localizing at kinetochoresIn normal S phase and in the centromere reactivation system, the intensity of punctate GFP-Tub1 signals, associated with KTs, was compared with that of Cse4-GFP. Cse4 is a histone H3 variant at a centromere and 32 molecules (2 molecules per centromere; Furuyama and Biggins, 2007) should be present close to a single spindle pole where 16 centromeres are clustered in telophase. From this comparison, we estimated how many GFP-Tub1 molecules were present at each kinetochore. (E) Short microtubules, not connected to an SPB, are found during S phase using electron tomography Wild-type cells (K699, W303 background) were treated with -factor and subsequently released to fresh media. After 20-30 min, cells were collected and frozen under high pressure. The samples were then freeze-substituted, embedded, cut into sections and post-stained. Tilt series of electron micrographs were acquired and reconstituted to 3D tomograms. (i) A representative electron micrograph (left) showing an SPB, spindle-pole microtubules and the nuclear envelope (coloured in right). (ii) 3D reconstitution of image series of the example shown in (i). A presumptive short microtubule (white arrow) was not connected to an SPB as its both ends were within the 280 nm section (see images at left and right). (iii) A series of electron micrographs of the presumptive short microtubule, indicated by a white arrow in (ii). Its edges are shown in green lines at bottom. Note that its top end seems to have a sheet-like structure as its right-hand edge extends longer than its left. Results: Electron tomography analysis identified structures that seemed to be short microtubules, which were not connected to a spindle pole, during early S phase when kinetochores, not associated with spindle-pole microtubules were present (seeFig 1A; Fig 6A, B); these microtubules may originate from kinetochores. However the positions of kinetochores could not be determined with this method, as the electron beam does not produce sufficient contrast to visualise yeast kinetochores(O'Toole et al., 1999). , (associated with . After CEN3 has nucleated microtubules, it occasionally leaves their minus ends and moves along their lateral surface PGAL-CEN3-tetOs TetR-3CFP YFP-TUB1 BIK1-4mCherry PMET3-CDC20 cells (T6718) were treated as in mCherry (Bik1; green), YFP (tubulin; red), and CFP (CEN3; blue) images were acquired every 30 sec for 25 min. (A) Percentages of cells in which CEN3 migrated to the lateral side of the microtubule that had been originally extended from CEN3. Cells were classified according to the maximum distance from the minus end, which CEN3 had reached. (B) A time-lapse image sequence, in which CEN3 had reached the vicinity of the plus-end of a CEN3-derived microtubule (rare event: see A). Zero time is set arbitrarily for the first panel, in which a cell shape is outlined in white. (A) The appearance of tubulin signals at CEN3 and time course of CEN3 interaction with spindle-pole microtubules in mutants (associated with ii) (i) Wild-type (T3828), ndc10-1 (T4230), okp1-5 (T7983), dsn1-7 (T4293), spc24-1 (T4231), dam1-1 (T4232), ipl1-321 (T4414), tub4-1 (T4409), spc98-1 (T4410), stu2-10 (T4296) and spc72-7 (T8075) cells with PGAL-CEN3-tetOs TetR-3CFP YFP-TUB1 PMET3-CDC20 were treated as in YFP (tubulin) and CFP (CEN3) images were acquired at time points indicated. Time 0: transfer to glucose-containing medium. The results are presented as in . "Tubulin localization on KT" was scored "+" in ii when tubulin signals were observed (red bars) on the majority of CEN3, which was not yet associated with spindle-pole microtubules (magenta rectangles), at least one time point (5 min or later). (ii) Wild-type (T3828), bim1 (T4315) and bik1 (T4318) cells with PGAL-CEN3-tetOs TetR-3CFP YFP-TUB1 PMET3-CDC20 were treated as in Images were acquired and the results are presented as in A. (B) Frequency of microtubule extension from CEN3 in mutants (associated with iii) Top: Wild-type (T3531), ndc10-1 (T2832), okp1-5 (T3375), dsn1-7 (T3528), spc24-1 (T2902), dam1-1 (T2897), ipl1-321 (T2863), tub4-1 (T3996), spc98-1 (T3999) and stu2-10 (T2862) cells with PGAL-CEN3-tetOs TetR-GFP YFP-TUB1 PMET3-CDC20 were treated as in except that temperature was raised to 35 C, 30 min before cells were suspended in synthetic complete medium containing glucose and methionine. YFP (tubulin) and GFP (CEN3) signals were acquired together every 20 sec for 25 min at 35 C. Kinetochore (KT)-derived microtubules were scored when they were observed for 2 or more time points and their maximum length was > 0.5 m. The scored number of microtubules during the total observation time (the sum of observation time in multiple cells) is shown on the right. Bottom: Wild-type (T3531), bim1 (T2839) and bik1 (T2840) cells with PGAL-CEN3-tetOs TetR-GFP YFP-TUB1 PMET3-CDC20 were treated as in The number of microtubules extended from CEN3 was scored as in the top. (C) Components of the -tubulin complex localize at spindle poles, but not at CEN3 TUB4-3GFP (T4045) and SPC97-3GFP (T3906) cells with PGAL-CEN3-tetOs TetR-3CFP CFP-TUB1 PMET3-CDC20 were treated as in YFP (CEN3, Tub1: red) and GFP (Tub4 or Spc97: green) images were acquired. Representative images are shown. Results: Tub4 and Spc97 signals were found with high intensity at spindle poles, but no signals were observed at CEN3. (D) Localization of Bik1 at the plus ends of kinetochore-derived microtubuless, but not at kinetochores T6718 cells (see were treated as in mCherry (Bik1; green), YFP (tubulin; red), and CFP (CEN3; blue) images were acquired every 30 sec (time 0; arbitrary). See also Movie S3. (E) Stu2 localization at CEN3 in mutants (associated with iv) Wild-type (T3680), ndc10-1 (T4480), okp1-5 (T4486), dsn1-7 (T4487), spc24-1 (T4482), dam1-1 (T4503), ipl1-321 (T4011), tub4-1 (T5407), spc98-1 (T5409), spc72-7 (T8123) and bik1 (T5541) cells with PGAL-CEN3-tetOs TetR-3CFP CFP-TUB1 STU2-3GFP PMET3-CDC20 were treated as in 35 C) and . GFP (Stu2) and CFP (CEN3, tubulin) images were acquired. (i) Representative images. (ii) Percentages of cells in which Stu2 signals were detected at CEN3 that was not associated with spindle-pole microtubules. When Stu2 signals were found in <15, 15-30, >60% of cells, the results were summarized as Note. Spc72, which shows a limited homology to TACC proteins, is not involved in the generation of microtubules at kinetochores. At centrosomes in metazoan cells, Stu2 orthologues are associated with TACC-family proteins: they cooperate to facilitate microtubule nucleation [bib_ref] The TACC proteins: TACC-ling microtubule dynamics and centrosome function, Peset [/bib_ref]. Similarly Spc72, which shows a limited homology to TACC proteins [bib_ref] Identification of the substrates and interaction proteins of aurora kinases from a..., Tien [/bib_ref] , binds Stu2 and cooperates for microtubule nucleation at SPBs in budding yeast [bib_ref] The yeast spindle pole body component Spc72p interacts with Stu2p and is..., Chen [/bib_ref] [bib_ref] The XMAP215 homologue Stu2 at yeast spindle pole bodies regulates microtubule dynamics..., Usui [/bib_ref]. We therefore tested a possible role of Spc72 in generating microtubules at kinetochores, using the centromere reactivation assay. In spc72-7 mutants [bib_ref] Receptors determine the cellular localization of a gamma-tubulin complex and thereby the..., Knop [/bib_ref] , microtubule/tubulin signals were not reduced at kinetochores, microtubules extended similarly to a wild-type control (data not shown) and Stu2 localized at kinetcohores similarly to a wild-type control. Moreover Spc72 protein was not detected at kinetochores while its bright signals were detected at spindle poles (data not shown). Thus, it is unlikely that Spc72 is involved in microtubule generation at kinetochores. After 10 min incubation with medium containing glucose and methionine, cells were fixed with paraformaldehyde. Meanwhile PGALS-STU2-GFP-LacI REC102::lacOs CFP-TUB1 PMET3-CDC20 (T4837) cells were treated as in After 60 min incubation with medium containing galactose and methionine, cells were fixed with paraformaldehyde. The two fixed samples were mixed, and then GFP (Stu2; green) and CFP (CEN3, tubulin; red) images were acquired. (i) Images of representative T5477 and T4837 cells (left and right, respectively). Two kinds of cells were distinguished by the presence CFP signals for CEN3 or the spindle. Scale bar represents 1 µm. (ii) The intensity of Stu2-GFP signals at the subsets of CEN3 (T5477) and REC102 (T4837), on which tubulin signals were detected. To obtain these results six microscopy fields were analyzed, and there were no significant differences in the Stu2-GFP signal intensity of each strain between microscopy fields. Thick lines indicate mean values. Results: The average accumulation level of Stu2 at its tethered site was higher than that at centromeres. Nonetheless several cells showed similar Stu2 signal intensity at the tethered site to that of centromeres, but still were associated with tubulin signals. (B) Tubulin signals at the Stu2-tethered site are found in an ndc10-1 mutant, as in wild-type control NDC10 + (wild type NDC10 + ; T4689) and ndc10-1 (T5464) cells with REC102::lacOs PGAL-STU2-GFP-LacI CFP-TUB1 PMET3-CDC20 were treated as in except that the temperature was raised to 35 C, 30 min before galactose was added to the medium. Previous data suggested that incubation at 35 C for 30 min was sufficient to impair the kinetochore assembly in ndc10-1 mutant [bib_ref] Molecular mechanisms of kinetochore capture by spindle microtubules, Tanaka [/bib_ref]. GFP (Stu2 fused with LacI) and CFP (tubulin) images were acquired. Percentages of cells for each category are shown in coloured lines. Results: The percentage of cells, in which microtubule/ tubulin signals were found at the Stu2tethered site, increased similarly between the two strains, suggesting that microtubule nucleation at the Stu2-tethered site does not require a kinetochore component Ndc10. (C) Stu2-tethered sites interacted with spindle-pole microtubules and were transported towards a spindle pole in some cells T4837 cells (see A) were treated as in GFP and CFP images were acquired every 10 sec. Results: In some cells (10%, 6/63), Stu2-tethered sites interacted with the lateral sides of spindlepole microtubules. Similar results were obtained in the presence (this figure) and absence of a mild osmotic stress (data not shown). This behaviour of Stu2-tethered sites was reminiscent to the behaviour of authentic kinetochores, which initially interact with the lateral sides of spindle-pole microtubules, subsequently slide along them towards a spindle pole, then attach to the plus ends of spindle-pole microtubules (end-on attachment) and move further poleward as they shrink [bib_ref] Molecular mechanisms of kinetochore capture by spindle microtubules, Tanaka [/bib_ref]. However, in contrast to kinetochores, Stu2-tethered sites did not preferentially show sliding towards a spindle pole. Instead, Stu2-tethered sites were associated with the plus ends of spindle-pole microtubules (end-on attachment) and transported poleward (as shown in this figure), which was similar to the later stage of kinetochore transport towards a spindle pole . However, while the end-on attachment of kinetochores to spindle-pole microtubules were stable and detachment was very rare , Stu2-tethered sites often detached from the end of these microtubules (data not shown). (A) In some cases, kinetochores are loaded onto the lateral surface of spindle-pole microtubules, which is followed by interaction between kinetochore-derived and spindle-pole microtubules Experiments were carried out as in In some cases, CEN3s interacted with spindle-pole microtubules first, followed by the association between kinetochore-derived and spindle-pole microtubules in a parallel (i) or antiparallel manner (ii). Scale bar: 1 µm. (B) New growth of microtubules from a spindle pole is discerned even if they overlapped with microtubules that have extended earlier from the same spindle pole T6718 (see cells were treated as in To discern overlapping spindle-pole microtubules, Bik1 was visualized with mCherry at the plus ends of growing microtubules. Results: New growth of a microtubule from a spindle pole was recognized even if they overlapped with a microtubule that extended earlier from the same spindle pole, as: 1) Bik1 moved along the microtubule that extended earlier, and 2) tubulin signals showed higher intensity along the region where the microtubules overlapped. (ii) Percentage of time intervals, during which microtubules were newly extended from CEN3 (that were not yet associated with spindle-pole microtubules). Results: The increase of this percentage at later time points suggests that microtubules are generated more frequently when kinetochore interaction with spindle-pole microtubule is delayed: the same conclusion has been also obtained in a normal S phase (see . (D) The amount of Stu2 at CEN3 decreases soon after CEN3 is loaded onto the surface of spindle-pole microtubules (supplemental to Experiments were carried out and data were analyzed as in (i) The quantified intensity of Stu2 signals at CEN3 in 8 individual cells, where CEN3 interacted with spindle-pole microtubules and no rescue occurred for these microtubules. Time is shown relative to the CEN3 encounter with spindle-pole microtubules. (ii) As a control (shown in the same colour as [i]) for each cell in (i), Stu2 intensity was measured at the same time points in a neighbouring cell in the same microscope field, in which CEN3 did not interact with spindle-pole microtubules. By collecting controls in this way, we could avoid possible bias due to photo-bleaching and possible variance in the Stu2 signal intensity between different microscopy fields. (A) Kinetochore-derived microtubules have their plus ends distal to kinetochores in a physiological condition We investigated the polarity of microtubules generated at kinetochores during normal S phase (which overlaps with mitosis; [bib_ref] Kinetochore microtubule interaction during S phase in Saccharomyces cerevisiae, Kitamura [/bib_ref]. MTW1-4mCherry NDC80-4mCherry GFP-TUB1 (T8179) and BIK1-4mCherry GFP-TUB1 (T7972) cells were treated as in mCherry (kinetochores or Bik1; green) and GFP (tubulin; red) images were acquired as in Top: Representative images. Rectangles (magenta; magnified at right) show microtubules generated at kinetochores that were not yet associated with spindle-pole microtubules. Bottom: Scored localization patterns of Mtw1/Ndc80 (authentic kinetochore components) and Bik1 (a +TIP protein that localizes at the plus ends of growing microtubules). Results: Tubulin often showed globular signals co-localizing at kinetochores (as also found in , from which rod-like tubulin signal extended distal to kinetochores. The globular signals may correspond to unpolymerized tubulins or very short additional microtubules at kinetochores. Crucially, in most cases, Bik1 localized distal to the globular tubulin signals (at the end of rod-like signals); i.e. Bik1 localized at the microtubules ends distal to kinetochores. The results suggest that kinetochore-derived microtubules have their plus ends distal to kinetochores in normal S phase. (B) stu2-10 mutant cells, but not tub4-1 or spc98-1 mutants, show reduction in microtubule/ tubulin signals at kinetochores in a physiological condition Wild-type control (T7199), stu2-10 (T7057), tub4-1 (T8327) and spc98-1 (T8331) cells with MTW1-4mCherry NDC80-4mCherry YFP-TUB1 were treated as in except that temperature for cell culture was shifted to 35 C when  factor was washed out. Images were acquired at 35 C from 20 min after washout of  factor. mCherry (kinetochores) and YFP (tubulin) images were collected every 10 sec for 10 min. Percentage of time points (out of all time points observed in 8 cells), during which kinetochores were detected but not yet associated with spindlepole microtubules, with positive, marginal or negative tubulin signals at kinetochores. See more detail in supplemental experimental procedures. (C) Microtubule/tubulin signals are found more frequently at kinetochores with brighter signals Using the data set shown in the numbers of time points, at which tubulin signals (cyan box in were present or absent at kinetochores, are shown by blue and orange bars, respectively, in correlation with the presence and absence of brighter kinetochore signals (orange dots in . ## Supplemental experimental procedures ## Yeast genetics and molecular biology The background of yeast strains (W303) and methods for yeast culture and -factor treatment were as described previously. Cells were cultured at 25 ºC in YP medium containing glucose, unless otherwise stated. Constructs of PGAL-CEN3-tetOs [bib_ref] Molecular mechanisms of kinetochore capture by spindle microtubules, Tanaka [/bib_ref] , TetR-3CFP [bib_ref] Mating type-dependent constraints on the mobility of the left arm of yeast..., Bressan [/bib_ref] , PMET3-CDC20 [bib_ref] Cleavage of cohesin by the CD clan protease separin triggers anaphase in..., Uhlmann [/bib_ref] , TetR-GFP [bib_ref] Cohesins: chromosomal proteins that prevent premature separation of sister chromatids, Michaelis [/bib_ref] and REC102-lacOs [bib_ref] GFP tagging of budding yeast chromosomes reveals that protein-protein interactions can mediate..., Straight [/bib_ref] [bib_ref] Cdc14 phosphatase induces rDNA condensation and resolves cohesin-independent cohesion during budding yeast..., Sullivan [/bib_ref] were as described previously. Temperature-sensitive mutants ndc10-1 [bib_ref] NDC10: a gene involved in chromosome segregation in Saccharomyces cerevisiae, Goh [/bib_ref] , okp1-5 [bib_ref] A putative protein complex consisting of Ctf19, Mcm21, and Okp1 represents a..., Ortiz [/bib_ref] , dsn1-7 [bib_ref] Interactions between centromere complexes in Saccharomyces cerevisiae, Nekrasov [/bib_ref] , spc24-1 [bib_ref] The Ndc80p complex from Saccharomyces cerevisiae contains conserved centromere components and has..., Wigge [/bib_ref] , dam1-1 [bib_ref] Mitotic spindle integrity and kinetochore function linked by the Duo1p/Dam1p complex, Cheeseman [/bib_ref] , ipl1-321 [bib_ref] The conserved protein kinase Ipl1 regulates microtubule binding to kinetochores in budding..., Biggins [/bib_ref] , tub4-1 , spc98-1 , stu2-10 [bib_ref] Stu2 promotes mitotic spindle elongation in anaphase, Severin [/bib_ref] were as described previously. To make bim1, bik1 and kar3, whole open reading frames of the relevant genes were replaced with antibiotic-resistance genes, using a one-step PCR method. Yeast genes were tagged at their C-termini at their original gene loci by a one-step PCR method [bib_ref] Molecular mechanisms of kinetochore capture by spindle microtubules, Tanaka [/bib_ref] , using 3GFP-KanMX6 (pSM1023; [bib_ref] Yeast Cdk1 translocates to the plus end of cytoplasmic microtubules to regulate..., Maekawa [/bib_ref] and 4mCherry-NatMX6 (pT909) cassettes as PCR templates, unless otherwise stated. pT909 was constructed by multiplying the mCherry gene in pKS391 [bib_ref] Multistep and multimode cortical anchoring of tea1p at cell tips in fission..., Snaith [/bib_ref]. CFP-TUB1 [bib_ref] Four new subunits of the Dam1-Duo1 complex reveal novel functions in sister..., Janke [/bib_ref] and YFP-TUB1 (pDH20, obtained from Yeast Resource Centre, Seattle, USA) plasmids were integrated at auxotroph marker loci. Strains with the tagged genes grew normally at temperatures used in this study. To construct TEL15R-tetOs, 1198-bp genomic DNA fragment between YOR387C and FDH1 (close to the right telomere of chromosome XV) was cloned into a plasmid carrying a hygromycin-resistance gene and an array of tet operators (112 copies); this plasmid (pT548) was cut with KpnI within the genomic DNA fragment and integrated at this locus. To construct TEL15L-tetOs, 1369-bp genomic DNA fragment between YOL159C-A and YOL159C (close to the left telomere of chromosome XV) was cloned into a plasmid carrying a clonNAT-resistance gene and an array of tet operators (112 copies); this plasmid (pT543) was cut with NheI within the genomic DNA fragment and integrated at this locus. To construct PGALS-GFP-LacI and PGALS-STU2-GFP-LacI alleles, the GALS promoter [bib_ref] Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use..., Mumberg [/bib_ref] was subcloned into pAFS135 (pRS303-GFP12-LacI; [bib_ref] Time-lapse microscopy reveals unique roles for kinesins during anaphase in budding yeast, Straight [/bib_ref] at XhoI site (pT776). Subsequently STU2 coding sequence was subcloned into pT776 at XhoI site (pT780). pT776 and pT780 were then cut with NheI and integrated at HIS3 locus. ## Live-cell imaging The procedures for time-lapse fluorescence microscopy were described previously [bib_ref] Molecular mechanisms of microtubuledependent kinetochore transport toward spindle poles, Tanaka [/bib_ref]. Time-lapse images were collected at 25 ºC (ambient temperature) unless otherwise stated. For image acquisition, we used a DeltaVision RT microscope (Applied Precision), UPlanSApo 100 objective lens (Olympus; NA 1.40), a CoolSnap HQ CCD camera (Photometrics) and SoftWoRx software (Applied Precision). We acquired 5-9 (0.7 mm apart) zsections, which were subsequently deconvoluted and analyzed with SoftWoRx and Volocity (Improvision) software. For figures, z stacks were projected to two-dimensional images. GFP signals were discriminated from YFP, using the JP3 filter set (Chroma). CFP, YFP (or GFP) and mCherry signals were discriminated with the 89006 ET filter set (Chroma). GFP and YFP signals were acquired together, using the YFP channel of the 89006 ET filter set. ## Electron tomography Samples were processed and analyzed as described previously [bib_ref] Organization of interphase microtubules in fission yeast analyzed by electron tomography, Hoog [/bib_ref] with minor modifications. From 10 ml culture (OD=0.6), yeast cells were collected on polycarbonate filters (Millipore) using vacuum filtration, transferred to gold-plated flat specimen carriers and frozen by using the EM PACT2 high-pressure freezing machine (Leica Microsystems). The samples were then freeze-substituted at -90°C for 2 days in 0.1% OsO4, 0.1% glutaraldehyde and 0.2% uranyl acetate in acetone using the EM-AFS1 device (Leica Microsystems). Then the samples were gradually warmed up to -45 ºC and embedded in Lowicryl resin (HM20), which was polymerized under a UV lamp. Serial thin sections (280-300 nm thick) were cut using a Reichert Ultracut-E microtome (Leica Microsystems) and collected on Formvar-coated nickel grids. Sections were poststained with 4% uranyl acetate in 70% methanol for 4 min and with lead citrate for 1 min. Digital images were taken at 300kV from -60º to +60º tilt with 1º increment on a Tecnai TF30 electron microscope equipped with a Eagle 4K CCD camera (FEI: pixel size 1.18 nm at 20,000 magnification). Tomograms were then generated by R-weighted back projection, modelled and analyzed using IMOD software [bib_ref] Computer visualization of three-dimensional image data using IMOD, Kremer [/bib_ref]. In the diameter of spindle-pole microtubules was 18 nm. The smaller diameter of these microtubules than the standard 25 nm is probably due to sample processing for electron microscopy. The diameter of the presumable short MT in was very similar to that of spindle-pole MTs observed in the same cell. ## Analyzing dynamics of kinetochores, microtubules and associated proteins To evaluate the length of MTs and position of centromeres, we took account of the distance along the z-axis as well as distance on each z plane. The rate of microtubule growth and shrinkage was evaluated only for approximate linear changes (R 2 >0.85 in linear regression analyses) of microtubule length of more than 2 m. Statistical analyses were carried out with the Fisher's exact test [fig_ref] Figure S6: [/fig_ref] , a paired t-test by comparing with an individual control), a chisquare test for trend ii, S5C ii, S6B) or a log-rank test for survival curves , an unpaired t-test , chi-square test [fig_ref] Figure S6: [/fig_ref] or a Wilcoxon matched pairs test [fig_ref] Figure S6: [/fig_ref] , using the Prism (Graph pad) software. All p values are twotailed. All error bars in figures represent SEM. To visualize tubulins and MTs in other figures, the YFP-TUB1 construct was integrated in the genome (e.g. at trp1 locus) of yeast cells that have the original TUB1 (non-tagged) at its original locus. The integration of YFP-TUB1 may have increased the total amount of Tub1 in cells and kinetochore-associated microtubule/tubulin signals might have been due to overexpression of Tub1. To address this possibility, cells with and without YFP-TUB1 were analyzed using Western blotting with an anti-tubulin antibody (data not shown). The ratio of YFP-Tub1 and Tub1 amount was approximately 1: 5 and the total Tub1 expression level (YFP-Tub1 plus Tub1) was similar in the presence and absence of the extra YFP-TUB1 copy, which is consistent with previous reports [bib_ref] Dominant effects of tubulin overexpression in Saccharomyces cerevisiae, Burke [/bib_ref] [bib_ref] Cell cycle-dependent changes in microtubule dynamics in living cells expressing green fluorescent..., Rusan [/bib_ref]. Therefore the appearance of kinetochoreassociated microtubule/tubulin signals was not due to tubulin overexpression. other figures, microtubules extended for greater lengths from reactivated CEN3 when cells were exposed to a mild osmotic stress. This was originally found when we fortuitously added 1/10 volume of concentrated glucose (20%) to culture medium after removing galactose to reactivate CEN3, instead of replacing galactose-containing culture medium with one containing 2% pre-diluted glucose as we usually do. This seemed to be due to osmotic stress, as it also happened when 1/10 volume of 1 M sorbitol was added immediately after replacement of galactose-containing medium with 2%, pre-diluted glucose-containing medium. In these conditions, spindle-pole microtubules did not change their dynamics (growth and shrinkage rates) significantly . If a higher-concentration of sorbitol (1/10 volume of 2 M) was added, polymerization and depolymerization of spindle-pole microtubules, as well as the extension of microtubules from CEN3, were halted for up to 12 min. This is consistent with a recent report that severe osmotic stress (addition of 1/2 volume of 2.4 M sorbitol) causes a cessation of microtubule dynamics [bib_ref] Stress-regulated kinase pathways in the recovery of tip growth and microtubule dynamics..., Robertson [/bib_ref]. It is still unclear how a 'mild' osmotic stress facilitated generation of longer microtubules from CEN3. Nonetheless, treatment of cells with a mild osmotic stress gave us a useful means to make long CEN3derived microtubules and observe their behaviour. ## As shown in In PGAL-CEN3-tetOs TetR-3CFP GFP-TUB1 PMET3-CDC20 cells (T6721) were treated as in . Subsequently GFP (tubulin, Cse4) and CFP (CEN3) images were acquired. Separately, GFP-TUB1 MTW1-4mCherry NDC80-4mCherry cells (T6298) were mixed with CSE4-GFP cells (T6799). Then GFP (tubulin, Cse4) and mCherry (Mtw1 Ndc80) images were acquired. In T6721 cells, we focused on CEN3s that had been reactivated but not yet associated with spindle-pole microtubules. In T6298 cells, we found and focused on kinetochores, which had been reassembled away from a spindle pole during S phase but not yet associated with spindle-pole microtubules. At these CEN3s and kinetochores, the numbers of associated GFP-Tub1 and total Tub1 molecules, as well as the total length of associated microtubules were estimated as follows. We first quantified GFP signals (Tub1) at CEN3/kinetochores in T6721 and T6298 cells and compared them with GFP signals (Cse4) at a spindle pole in telophase of T6799 cells within the same microscopy field. Cse4 is a histone H3 variant at a centromere and 32 molecules (2 per centromere; [bib_ref] Centromere identity is specified by a single centromeric nucleosome in budding yeast, Furuyama [/bib_ref] should be present close to a single spindle pole where 16 centromeres are clustered). From this comparison, we estimated how many GFP-Tub1 molecules were present at each kinetochore (as in [bib_ref] Molecular architecture of a kinetochore-microtubule attachment site, Joglekar [/bib_ref]. Next we quantified the intensity of GFP-Tub1 signals along a single spindle-pole microtubule (which was found and confirmed as described in [bib_ref] Molecular mechanisms of kinetochore capture by spindle microtubules, Tanaka [/bib_ref] and estimated how many GFP-Tub1 molecules are contained along 1 µm of a single nuclear microtubule. We calculated how many Tub1 molecules should be present along 1 µm of the microtubule, and estimated that 6.9 % of the total Tub1 molecules are GFP-tagged. Finally, assuming that all CEN3/kinetochore-associated Tub1 molecules belong to either single or multiple short microtubules, and that the ratio of GFP-tagged Tub1 molecules to the total Tub1 molecules is the same at CEN3/kinetochores as along a spindle-pole microtubule, we estimated the total length of microtubules associated with each CEN3/kinetochore. We predicted that, if microtubules were longer than approximately 300 nm, their signals would show extended shapes (e.g. . Therefore, when the total length of CEN3-associated microtubules was longer than 300 nm but GFP-Tub1 signals did not show extension matching this length, CEN3/kinetochores were probably associated with multiple short microtubules. In , the extension of spindle-pole microtubules in the nucleus was scored as follows. Wild-type (T3270), ndc10-1 (T3446), okp1-5 (T3429), dsn1-7 (T3392), spc24-1 (T3274), dam1-1 (T2897), ipl1-321 (T2863), tub4-1 (T5426), spc98-1 (T5428), stu2-10 (T3390), bim1 (T3410), and bik1 (T3514) cells with PGAL-CEN3-tetOs TetR-GFP YFP-TUB1 NIC96-YFP KIP2-3GFP PMET3-CDC20 (except for T2897 and T2863, where NIC96-YFP and KIP2-3GFP were not present, and for T3514 where KIP2-3GFP was not present) were treated as in but cultures were shifted to 35˚C, 30 min before transfer to glucosecontaining medium (except for bim1 and bik1 cells and their control wild-type, where temperature was maintained at 25˚C). Subsequently, YFP (Tubulin and Nic96) and GFP (CEN3 and Kip2) time-lapse images were acquired every 10 sec for 30 min at 35˚C (25˚C for bim1 and bik1). Using Nic96 signals (visualizing the nuclear envelope) and Kip2 signals (visualizing the plus ends of cytoplasmic microtubules), nuclear and cytoplasmic microtubules were distinguished [bib_ref] Molecular mechanisms of kinetochore capture by spindle microtubules, Tanaka [/bib_ref]. When NIC96-YFP and KIP2-3GFP were not present, nuclear microtubules were discerned by enhancing TetR-GFP signals that are present in the nucleus (TetR-GFP had a nuclear localization signal) but unbound to tetOs. These two methods gave almost identical results in 'wild-type' cells (data not shown). Because the extension of individual microtubules from spindle poles is generally observed more frequently with a monopolar spindle (for example, in stu1-5 mutant: see supplementary note 5 in [bib_ref] Molecular mechanisms of kinetochore capture by spindle microtubules, Tanaka [/bib_ref] than with a bipolar spindle (in which, many spindle-pole microtubules inter-digitate in the middle of the spindle and not recognized as individual microtubules), the results of tub4-1 and spc98-1 cells, largely showing monopolar spindles, were not included for comparison in the table. Also note that a part of the results in , have been already published in [bib_ref] Molecular mechanisms of kinetochore capture by spindle microtubules, Tanaka [/bib_ref]. In tubulin YFP signals at CFP-labeled CEN3 were scored negative when their integral signal intensity (from Voxel with the default setting of Volocity), colocalizing with the CEN3-CFP signal, was ≤ 95 and positive for > 95. In the intensity of integral Mtw1/Ndc80 GFP signals, which colocalize with the CEN3-CFP signal, was scored as negative for ≤ 130 and positive for > 130. In , the extension of MTs were counted only when they were observed for two or more consecutive time points and their maximum length was > 0.5 m. In kinetochore assembly was scored when its integral signal intensity was > 80 and the signal moved towards a spindle pole at later time points; brighter kinetochores were scored when its integral signal intensity was > 132. Tubulin signals at kinetochores were scored positive when their intensity was > 107. From the regression curve obtained in we reasoned that some events of kinetochore reassembly and subsequent interaction with spindlepole MTs were not recognized in the time interval between the two was very short. The addition of hypothetical 18 and 15 events of kinetochore assembly, followed by kinetochore interaction with spindle MTs at 10 and 20 sec, respectively, gave the best fit to the regression curve in and they were therefore taken into consideration when black dots were plotted in to plot the number of free kinetochores at time 0 in Because in these hypothetical events the assembled kinetochores rapidly interacted with spindle-pole MTs, we assumed that "brighter kinetochore signal" and "tubulin signal at kinetcohore" were not positive. In and ii, we evaluated the effect of kinetochore-associated microtubules /tubulins on efficient kinetochore interaction with spindle-pole microtubules. However this effect is probably underestimated by the method used there, because of the following reason. To estimate this effect, we plotted how quickly the percentage of free kinetochores decreased after tubulin signals had first appeared at kinetochores (blue and magenta curves in i, ii). We then compared these curves with the control curve (black curves) showing the decrease of free kinetochores since their reassembly had been detected, including all 75 samples shown in One might think that, a more precise estimation of the above effect could be obtained by excluding the samples in where tubulin signals appeared at kinetochores, from the control curve. Unfortunately this did not prove a good option because this exclusion would lead to a bias towards samples that showed rapid kinetochore interaction with spindle-pole microtubules, because tubulin signals preferentially appeared at kinetochores when this interaction was delayed (see . It was therefore inevitable to use all samples in order to avoid such a bias in making a control curve. Because the control included both samples where tubulin signals did and did not appear at kinetochores, we probably underestimated the effect of kinetochore-associated microtubules/ tubulins in facilitating kinetochore interaction with spindle-pole microtubules. In both Fig 6 E i and ii, "time for 50%" was shorter, relative to the control curve. However, in a log-rank test for survival curves, the effect was significant (see p values) only when tubulin signals appeared at kinetochores at 2 or more consecutive time points. Nonetheless, because of this probable under-estimation, the tubulin-signal appearance may indeed have the effect even if they did not appear at consecutive time points. In [fig_ref] Figure S6: [/fig_ref] kinetochore assembly was scored positive when its integral signal intensity (from Voxel with the default setting of Volocity) was >80. We noticed that it took longer time for kinetochore signals (since their appearance) to interact with spindle-pole microtubules in stu2-10, tub4-1, spc98-1 mutant cells (actually, in the majority of stu2-10 cells, the interaction did not happen during observation) than in wild-type control, due to the reduced number of spindle-pole microtubules (data not shown). On the other hand, tubulin signals appeared at kinetochores more frequently when kinetochore interaction with spindle microtubules was delayed (see . Therefore, if we had considered for scoring tubulin signals all the time points during which kinetochores were not associated with spindle pole microtubules in the mutant cells, this might have given a bias towards higher tubulin signals due to a delay in kinetochore interaction with spindle microtubules in these cells. We therefore scored tubulin signals at kinetochores by making 8 pairs between each mutant and wild-type cells in the order of the time length required for kinetochore interaction with spindle-pole microtubules (or, in stu2-10 cells, the time length, during which kinetochores were observed without this interaction, until the end of the imaging): for example, a mutant cell, in which kinetochores were observed for the shortest time length without this interaction, was matched with the wild-type cell with the quickest interaction, second with second, so on. In all pairs, mutant cells gave the same or a larger number of time points, in which kinetochores were not associated with spindle microtubules, than wild-type cells. We then scored tubulin signals only at the time points (since the appearance of kinetochore signals; relative to this appearance) in each mutant cell, at which kinetochores were not yet associated in the paired wild-type cell. The time points were also paired in this way between paired mutant and wild-type cells, in order to score tubulin signals between the paired time points using a Wilcoxon matched pairs test (used for comparison of paired non-parametric samples). In addition to the above experiment, we also compared timing of kinetochore assembly in stu2-10, tub4-1 and spc98-1 mutant cells and wild-type control, after CEN detachment from microtubules upon CEN DNA replication. For this, we used cells with similar genotype to [fig_ref] Figure S6: [/fig_ref] but with CFP-labelled CEN5, treated cells as in [fig_ref] Figure S6: [/fig_ref] collected mCherry (kinetochores Mtw1/Ndc80), YFP (tubulin) and CFP (CEN5) images. Time from CEN5 detachment (from spindle-pole microtubules) until Mtw1/Ndc80 reappearance at CEN5 was similar between the 4 strains (data not shown). Thus, timing of kinetochore assembly in stu2-10, tub4-1 and spc98-1 mutants was similar to that in wild-type cells. [fig] Figure S3: (associated with Figure 3). [/fig] [fig] Figure S4 ,: (associated withFigure 4).(A) Comparison of the intensity of Stu2-GFP signals at CEN3 and at the Stu2 tethered site PGAL-CEN3-tetOs TetR-3CFP STU2-GFP PMET3-CDC20 cells (T5477) were treated as inFig 1B. [/fig] [fig] Figure S5 ,: (associated withFigure 5). [/fig] [fig] Figure S6: (associated with Figure 6). [/fig]
## S2 text. predicting choices using eye-fixations Several recent studies have shown that the more one look at an alternative, the more likely this alternative will be chosen . Here, we examined whether this pattern holds in our data, by predicting the probability of choosing alternative A(x 1 , p 1 ) over alternative B(x 2 , p 2 ) using logistic regressions based on the: i) EU and CPT subjective utilities functions, and ii) relative number of fixations (or dwell-times) on each alternative. The EU based models which we examined were: : ( 1 , 1 ; 2 , 2 ) = ( 1 - 1 − 2 - 2 ) : ( 1 , 1 ; 2 , 2 ) = ( 1 - 1 - 1 − 2 - 2 - 2 ) [formula] : ( 1 , 1 ; 2 , 2 ) = ( 1 - 1 - 1 − 2 - 2 - 2 ) [/formula] where is the risk-parameter of EU, t 1 and t 2 correspond to the relative looking time on the two alternatives (i.e., normalized by the total looking time), f 1 and f 2 correspond to the relative number of fixations (i.e., normalized by the total number of fixations) on the two alternatives, is a saturation parameter for fixations, and ( ) is the logistic function, which depends on a slope-parameter, θ: [formula] ( ) = 1 1+ − - . [/formula] The Traditional EU model (Eq. 1) corresponds to a probabilistic specification of the EU, using an exponential version of Luce's choice rule , which takes into account the EU differences between the lotteries (see S3 Text for detailed description of the EU model). The EU Dwell time model (Eq. 2) includes the dwell-times on the two alternatives, so that the EU value of each alternative increases with its dwell time. The EU Fixations model (Eq. 3) is similar to the EU Dwell time model, except that instead of using dwell-time, we use the number of fixations to each alternative. Note that in both the latter models, we included the parameter τ, which represents the marginally decreasing effect of the dwell-time/number of fixations (lower values of this parameter indicate higher degrees of saturation; for example, if τ=0.5, an increase of the fixation number by a factor of four will only increase the utility by a factor of two). The CPT based models were analogues to the EU ones, except for using subjective probabilities (decision weights) rather than objective probabilities (as in EU): where ( ) = ( +(1− ) ) 1 is the decision weight function, and is a free parameter capturing the curvature of this function (see S3 Text for further details). The quantitative fits of the models were evaluated using two selection criteria: prediction-accuracy and AIC (see S1 Methods for detailed description of each measure). The results indicate that: i) using eye-movements improve the prediction accuracy and AIC compared with the traditional models , and ii) the fixations based models showed equal (for EU) or better (for CPT) performance than the dwell-time based models . ## Impact of overall fixations in eu and cpt type models ## Model aic prediction-accuracy
Unequal excess mortality during the Spanish Flu pandemic in the Netherlands A century after the Spanish Flu, the COVID-19 pandemic has brought renewed attention to socioeconomic and occupational differences in mortality in the earlier pandemic. The magnitude of these differences and the pathways between occupation and increased mortality remain unclear, however. In this paper, we explore the relation between occupational characteristics and excess mortality among men during the Spanish Flu pandemic in the Netherlands. By creating a new occupational coding for exposure to disease at work, we separate social status and occupational conditions for viral transmission. We use a new data set based on men's death certificates to calculate excess mortality rates by region, age group, and occupational group. Using OLS regression models, we estimate whether social position, regular interaction in the workplace, and working in an enclosed space affected excess mortality among men in the Netherlands in the autumn of 1918. We find some evidence that men with occupations that featured high levels of social contact had higher mortality in this period. Above all, however, we find a strong socioeconomic gradient to excess mortality among men during the Spanish Flu pandemic, even after accounting for exposure in the workplace. # Introduction In 1918-1919 the Spanish Flu took the lives of an estimated 50-100 million people worldwide [bib_ref] Updating the accounts: Global mortality of the 1918-1920 ''Spanish'' Influenza pandemic, Johnson [/bib_ref]. In the Netherlands, estimates of contemporaries suggest that over 41.000 persons died, of which about 30.000 perished during the autumn wave of 1918 . At the time, it was thought that social differences in mortality to the disease were relatively small, both in the Netherlands and abroad. For example, the Dutch Health Council reported that there were notable differences in mortality between age groups and regions, but found no reason to conclude that occupation or wealth affected mortality. In recent decades, however, evidence has grown that there existed a socioeconomic gradient in Spanish Flu mortality [bib_ref] A socially neutral disease? Individual social class, household wealth and mortality from..., Mamelund [/bib_ref] [bib_ref] Social inequalities in infectious diseases, Mamelund [/bib_ref] [bib_ref] What explains cross-city variation in mortality during the 1918 influenza pandemic? Evidence..., Clay [/bib_ref]. Yet, uncertainty remains, a large part of the literature is based on aggregate data, and the mechanisms that linked together socioeconomic factors and mortality risk to the Spanish Flu remain unclear. In this paper, we use new micro-level data containing occupations and dates of death for five provinces in the Netherlands to further establish whether and how mortality risk during the Spanish Flu was linked to socioeconomic and occupational characteristics. have contributed to risk of infection with and risk of mortality during the Spanish flu. Particularly, we are interested in the social gradient in mortality while taking into account occupation-related risk of infection. Such a social gradient may point to the relevance of resources and existing health differences in shaping mortality during the pandemic, whereas a relationship with occupational characteristics may point towards the role of infection risk in the workplace. Our case is the Spanish Flu in the Netherlands, the history of which has largely remained unwritten (for exceptions, see [bib_ref] De huisarts en 'de Spaansche griep' 1918-1920, De Melker [/bib_ref] [bib_ref] Retracing hotbeds of the 1918-19 influenza pandemic. Spatial differences in seasonal excess..., Mourits [/bib_ref]. We focus on the second wave of the Spanish Flu, in autumn 1918, which was by far the deadliest phase of the pandemic. We use occupations registered on death certificates to measure socioeconomic status of the deceased and create a new occupational classification whether work led to frequent social contact and whether it took place in enclosed spaces. We use this to isolate socioeconomic status from occupational exposure to viral transmission. We find evidence for a social gradient in mortality among men both in strongly and more mildly affected places. Our findings suggest that part of the socioeconomic patterns in Spanish flu mortality identified by previous studies exist independently from occupational differences in the frequency of social contact with others and conditions for viral transmission at work. Other factors related to socioeconomic status are thus likely to have been important and may have included the opportunity to isolate and viral transmission away from work, and existing health differences in the population. ## Theory and literature # Historical background The 1918 influenza pandemic has been described as ''the mother of all pandemics'' [bib_ref] Influenza: the mother of all pandemics, Taubenberger [/bib_ref]. After a mild summer wave in 1918, a deadly second wave took hold during the final months of the First World War. Between September and December 1918, the pandemic took more lives than the preceding four years of warfare. The flu was notorious for its lethality, but especially feared as it heavily affected young adults between ages 20-40, who are normally least affected by infectious diseases. Especially young men and pregnant women were affected. The infected initially showed common symptoms of the flu, which could quickly develop into pneumonia. Death often followed within days. The horror is perhaps best described in the memoirs of the surgeon general of the US army Vaughan: ''I see hundreds of young, stalwart men in the uniform of their country [. . . ] placed on the cots until every bed is full and yet others crowded in. The faces soon wear a bluish cast; a cough brings up the blood stained sputum. In the morning the dead bodies are stacked about the morgue''. In the 20th century, the Spanish Flu was mostly perceived as a socially neutral disease that affected both the poor and the rich. Immediately after the outbreak, many doctors and scientists maintained that there was no socioeconomic gradient in who was affected [bib_ref] Social class and excess mortality in Sweden during the 1918 influenza pandemic, Bengtsson [/bib_ref]. For the Netherlands, the Dutch Health Councilwrote that for influenza-related mortality: ''One cannot conclude that there is any distinction, neither by occupation, nor between the more and less affluent''. The idea of the Spanish Flu as a socially neutral disease persisted in the remainder of the 20th century. Doctors and scholars thought that the influenza virus infected and killed all classes equally because the disease was so highly transmissible . ## Social differences in early 20th-century mortality The image of Spanish Flu as a socially neutral disease has changed in the past decades. Statistical evidence has since then shown that mortality to the Spanish Flu was higher in poorer countries [bib_ref] Updating the accounts: Global mortality of the 1918-1920 ''Spanish'' Influenza pandemic, Johnson [/bib_ref] [bib_ref] Estimation of potential global pandemic influenza mortality on the basis of vital..., Murray [/bib_ref] , even though there are recent studies arguing that Spanish Flu was unrelated to pre-pandemic economic characteristics. Furthermore, in more developed countries mortality among the poor was higher, even though there was no perfect social gradient in mortality [bib_ref] Social class and excess mortality in Sweden during the 1918 influenza pandemic, Bengtsson [/bib_ref] [bib_ref] A socially neutral disease? Individual social class, household wealth and mortality from..., Mamelund [/bib_ref]. Using micro-level data from the Swedish census, [bib_ref] Social class and excess mortality in Sweden during the 1918 influenza pandemic, Bengtsson [/bib_ref] show that this disadvantage of the lower classes was especially notable among men and less pronounced among women The discussion about the role of socioeconomic inequalities during the Spanish flu pandemic links up to a larger debate on the origins of socioeconomic differences in mortality. Across Europe, the social gradient in health was growing in the first half of the 20th century [bib_ref] Socioeconomic inequalities in death from past to present: An introduction, Bengtsson [/bib_ref]. In industrialised and urbanised England, survival differences by socioeconomic group were already present around 1900 [bib_ref] Social class, life expectancy and overall mortality, Antonovsky [/bib_ref] [bib_ref] The hazards of wealth: Adult mortality in pretwentieth-century England, Razzell [/bib_ref]. In some regions, such as Southern Sweden, a health gradient by social class emerged relatively late. In the Netherlands in 1918, mortality differences after age 50 were only to a limited extent affected by social class [bib_ref] Exceptional lives, extraordinary families, Mourits [/bib_ref] , but in younger age groups, which were especially affected by the Spanish Flu, there existed a social gradient in health from the end of the 19th century. [bib_ref] Time trends in social class mortality differentials in the Netherlands, 1820-1920: An..., Van Poppel [/bib_ref] found that between ages c. 35-55, the elite in the Netherlands had a survival advantage over farmers and the middle class, whereas the working class had a survival disadvantage. Both existing differences in health as well as other social class characteristics related to knowledge and skills, income, and occupational characteristics may have played a role in excess mortality during the Spanish Flu pandemic. According to the fundamental cause theory [bib_ref] Social conditions as fundamental causes of disease, Link [/bib_ref] , a socioeconomic gradient in health persists even when the disease environment changes, as socioeconomic resources provide health advantages regardless of what the dominant diseases and causes of death are. However, the specific advantages linked to socioeconomic resources are dependent on the local context [bib_ref] A social history of disease: Contextualizing the rise and fall of social..., Clouston [/bib_ref]. For diseases that are not well understood or cannot be treated effectively, such as Spanish Flu, socioeconomic differences in disease burden or mortality are still expected. For example, resources can help people isolate from sick relatives or co-workers, improve access to information and understanding of the appropriate behaviour to avoid exposure, and ease access to health care. The topic of the Spanish Flu has seen renewed interest by historians and social scientists in recent years [bib_ref] Disease and fertility: Evidence from the 1918-19 influenza pandemic in Sweden, Boberg-Fazlic [/bib_ref] [bib_ref] A bitter epidemic: The impact of the 1918 influenza on sugar production..., Gallardo-Albarrán [/bib_ref] , though few studies provide in-depth analyses of socioeconomic differences in mortality. Evidence for socioeconomic contrasts in Spanish Flu mortality comes from a number of studies, predominantly at the aggregate level and showing correlations between local conditions and excess mortality. [bib_ref] The north-south divide: Social inequality and mortality from the 1918 influenza pandemic..., Herring [/bib_ref] show that the poorer neighbourhoods of Hamilton, Ontario had significantly higher influenza mortality rates than the richer neighbourhoods. [bib_ref] The diffusion of the influenza pandemic of 1918 in hartford, Tuckel [/bib_ref] andfound that immigrant groups from Italy and Eastern Europe in Hartford, Connecticut and Norwood, Massachusetts, often of low socioeconomic status, were harder hit by the Spanish Flu pandemic than non-immigrants. Similarly, [bib_ref] Pollution, infectious disease, and mortality: Evidence from the 1918 Spanish influenza pandemic, Clay [/bib_ref] find that the local share of foreign-born immigrants and prepandemic typhoid rates predict higher all-cause mortality during the pandemic. [bib_ref] What explains cross-city variation in mortality during the 1918 influenza pandemic? Evidence..., Clay [/bib_ref] also find that high illiteracy rates and prepandemic infant mortality predict higher mortality during the Spanish Flu. Studies on socioeconomic gradient in Spanish Flu mortality based on individual-level data remain scarce, however. Among the factors that may have contributed to higher risk among lower socioeconomic status groups, higher exposure to disease figures prominently. 2 Socioeconomic status is and was closely related to the degree and intensity of social contact, and households living in crowded conditions may have had a larger chance of exposure to the Spanish flu, especially in urban slums. Specific factors for socioeconomic differences in Spanish Flu mortality include hygiene and crowded housing, implying that the sick could not be cared for in a separate room. Such living conditions were also related to existing health before the arrival of the pandemic. Tuberculosis in particular may have been a risk factor, as it may have contributed to the chance of a secondary infection with bacterial pneumonia as well as reduced access to health care [bib_ref] Disparities in influenza mortality and transmission related to sociodemographic factors within Chicago..., Grantz [/bib_ref] [bib_ref] A socially neutral disease? Individual social class, household wealth and mortality from..., Mamelund [/bib_ref]. Not surprisingly, then, working class neighbourhoods and areas with small housing had higher death rates during the Spanish Flu in Oslo, Norway [bib_ref] A socially neutral disease? Individual social class, household wealth and mortality from..., Mamelund [/bib_ref]. ## Occupational exposure to the spanish flu Exposure to disease does not only occur at home but also at the workplace. Evidence for a relation between occupational characteristics and Spanish flu mortality largely stems from macro-level studies. For Chicago it was found that local unemployment rates were related to decreased mortality rates and transmission, indicating that social contact and not poverty itself could have been a key factor in mortality to the Spanish flu [bib_ref] Disparities in influenza mortality and transmission related to sociodemographic factors within Chicago..., Grantz [/bib_ref]. From the early 20th century, working conditions in factories were recognised as a general risk factor for transmission of infectious diseases. In 1898, Dutch socialist politician Domelawrote about the poor hygiene, and exposure to toxins, dust, and small particles among the working poor. By 1917, critique of labour conditions had become more widely accepted. Statistics Netherlands reported that working men aged 35-54 working outdoors or near furnaces had lower all-cause and tuberculosis-related mortality than those who were subjected to organic dust from tobacco, flower, and textiles, worked with chemicals and toxins, or -even more detrimental -were exposed to grinding dust from glass, metal, porcelain, or stone . For the same period in Sweden, reports by healthcare professionals addressed how poor air quality and unhygienic conditions in factories made workers more susceptible to tuberculosis. In a study of the Swedish case using individual level data, [bib_ref] Social class and excess mortality in Sweden during the 1918 influenza pandemic, Bengtsson [/bib_ref] provide evidence that the ability to maintain distance from others (now commonly known as ''social distancing'') may have affected mortality rates favourably. In southern Sweden, farmers were least affected by the Spanish flu, and low-skilled and unskilled workers the most. Although this points towards the importance of solitary work, the protective effect of farming was only found for men, and white collar workers had no mortality advantage compared to the working class. To understand whether social contact affected Spanish Flu mortality, social contact at the workplace needs to be measured in more detail. Differences in mortality by social class may have been directly affected by job characteristics and working conditions that are not fully captured by social class schemes. Recent studies have highlighted substantial heterogeneity in mortality rates within social classes but between occupational groups. The decades 2 There are also studies that focus on vulnerability to infection. One way through which vulnerability could lead to higher mortality during the Spanish Flu was through air pollution, to which the poor were more exposed. [bib_ref] Pollution, infectious disease, and mortality: Evidence from the 1918 Spanish influenza pandemic, Clay [/bib_ref] show that pollution due to coal-fired electricity generation worsened the impact of Spanish Flu in US cities, as the air pollution probably made lungs more susceptible to infection. Similarly, [bib_ref] Deaths from bacterial pneumonia during 1918-19 influenza pandemic, Brundage [/bib_ref] argue that secondary infections, rather than the virulence of Spanish flu itself, played a key role in the high death rates and age profile of the deceased as well as differences between occupational groups. before 1918 were dominated by infectious disease mortality, and occupational differences in mortality may have been, at least partly, related to exposure to infectious disease. For example, among white collar men in Sweden, especially among health professionals, there was a mortality disadvantage, but not among religious professionals. It is contested whether the clergy may have had a better lifestyle than the general population, as Bavarian monks had no survival advantage over non-cloistered men before the 1950s [bib_ref] Causes of male excess mortality: Insights from cloistered populations, Luy [/bib_ref]. More likely, the difference in mortality rates between doctors and religious professionals can be explained by exposure to infectious disease due to the nature of their occupation. In this paper, we take an in-depth look at men's occupational characteristics and mortality during the Spanish Flu pandemic. We overcome some of the shortcomings of earlier studies by studying individual-level data for the entire population of part of the Netherlands, including social and occupational characteristics. Our primary variable of interest is socio-economic status. However, instead of analysing specific occupations, as has been done in earlier studies to allcause and infectious disease mortality, we also measure occupational risk factors for broad categories of occupations. We supplement existing coding systems for occupational skill and status with a coding for occupational risk of exposure to infectious disease. Specifically, we look into the degree of social contact at work and whether workers worked in indoor environments, at the time often in cramped and poorly ventilated conditions with workers working closely together, conditions under which viral transmission tends to be higher than outdoors. # Methods and data ## Death certificates and the civil registry We use death certificates from the Dutch civil registry to calculate excess mortality in 1918 in comparison to deaths in the period 1910-1917. The Dutch civil registry was introduced in 1811 and provided legal proof of birth, marriage, and death. By the turn of the twentieth century the procedure was well-established. For everyone who died in the Netherlands a death certificate was issued, in duplicate so that safekeeping was ensured. Digitisation of these certificates has been ongoing since the 1990s by local and provincial archives. Death records list the date of death, full name, age, sex, place of residence, and the occupation of the deceased. These individual-level records allow us to analyse mortality during the Spanish Influenza pandemic in more detail than before. Other historical sources, such as municipal reports or national health reports, give only aggregated deaths per year and do not split out observations by age, sex, or occupation (see for example the Historical Database Dutch Municipalities [bib_ref] Historische database nederlandse gemeenten, v5, Mourits [/bib_ref]. The aggregated number of deaths per municipality is available by age group, but without information on occupation or other indicators of socioeconomic status. Some records of individual-level causes of death data survive in the Netherlands, but they are only available for isolated periods and municipalities. Death certificates are thus the only remaining source that combines individual-level characteristics with sufficient nationwide coverage to generate meaningful insights into mortality patterns along socioeconomic lines. Dutch death certificates contained no cause-specific mortality information. However, total numbers of deaths from death registration are sometimes deemed the most accurate way to address the impact of an epidemic. Shifts in overall mortality rates do not depend on potentially wrong or missing diagnoses of the cause of death, includes individuals who had multiple diseases at the time of death, and includes indirect deaths from the Spanish Flu . Death certificates have been digitised in all Dutch provinces, but not for all municipalities ( Some certificates were entered by more than one archive, or were digitised multiple times by the same archive, resulting in duplicates. These have been removed from the data. Certificates were digitised on a per-archive basis, and therefore there is variation in what information from the certificates was digitised [fig_ref] Figure 1 ,: top left panel [/fig_ref] , and we have to drop further municipalities for this reason. We can see clear bimodal distributions of missing data, with municipalities clustering at low or high percentages. We therefore exclude municipalities where the share of reported ages, sex, and full dates is below 50%. 5 In the case of occupations, many of the deceased would not be expected to have an occupation, especially in the case of children, the elderly, and women. Because nearly half the deaths are among women, and the very young and the elderly dominate among those who died in the years 1910-1918, the local share of recorded occupations can be low. We exclude municipalities where the share of recorded occupations is below 10%. We explore the impact of using stricter thresholds in Appendix A. The changes in the sample due to excluding further municipalities are shown in [fig_ref] Table 1: Summary statistics and selection steps [/fig_ref] , showing that it leaves us with 220 795 certificates for 215 municipalities, concentrated in the provinces of Gelderland, Overijssel, Zeeland, Drenthe, and Zuid-Holland. The most important bias is that three of the four largest cities are missing from the data: Amsterdam, Rotterdam, and Utrecht. However, our data does contain large and medium-sized cities (The Hague, Arnhem, Nijmegen, Leiden, and Apeldoorn), including a number of textile and industrial centres. Because of this and because very small municipalities are underrepresented, the average size of a municipality in our final dataset is 7850 inhabitants, compared to 6015 for the country as a whole. Comparing the average excess mortality rate (EMR) of municipalities that were dropped from our sample due to missing variables (excluding Amsterdam as there is no data available), we find that the difference is negligible: 1.38 outside our sample, and 1.40 in our sample. [fig_ref] Table 1: Summary statistics and selection steps [/fig_ref] also shows the results of further selection steps, and how each step affects the composition of the included sample. As explained below, we calculate excess mortality based on the months September-December, and exclude the year 1914. We only keep certificates where the deceased is aged 16-78. We only include men in the analyses. Female occupations were not always recorded due to norms about female labour force participation (especially in marriage records [bib_ref] De vermelding des beroeps: eene ijdele formaliteit? 'Twee eeuwen vrouwelijke beroepsarbeid in..., Walhout [/bib_ref]. Consequently, for every 11 men with a recorded occupation, we only have information about the occupation of one woman. Moreover, recorded occupations for women fall in a small number of categories, such as maids, seamstresses, and nurses. The high percentage of maids in particular makes the women in our data less skilled than the male population. In part, these are real effects, as labour market opportunities for women were limited, especially in more skilled and high-status occupations. At the same time, we believe the data contain insufficient detail about women's occupations to include them in the current work. We therefore omit women from our analyses. As we discuss below, our results are robust to including women. ## Occupational coding Standardised occupations of the deceased were matched against standardised occupational titles from the Historical Sample of the Netherlands (HSN) . 6 This dataset contains over 280 000 Dutch occupational titles, with numerous spelling variations, coded in HISCO (Historical Classification of Occupations) and HISCLASS (Historical International Social Class Scheme). Almost 98 per cent of occupational titles on the death certificates could be coded this way. 7 To infer the skill level of each occupation, we used the skill category of its corresponding HISCLASS, ranging from high (HISCLASS 1-2), medium (HISCLASS 3-4, 6-8), low , to unskilled (HISCLASS 11-13). We decided to aggregate to these skill level groupings to avoid ending up with very small totals in some of the HISCLASS categories, the higher skilled occupations in particular. As an alternative to the HISCLASS system, we also use HISCAM occupational classification scores [bib_ref] The construction of HISCAM: A stratification scale based on social interactions for..., Lambert [/bib_ref]. This is a continuous measure that infers distance between occupations in an occupational hierarchy from the social interaction of the occupations incumbents, specifically historical marriage behaviour. The advantage of HISCAM is that it is based on actual behaviour, indirectly accounting for many implicit social factors that affect social stratification, making it a worthwhile addition to the HISCLASS skill dimension. HISCAM also provides a useful alternative coding of farmers. In the HISCLASS skill scheme, farmers are coded as medium skilled workers, while in HISCAM they are viewed as a relatively low status profession. Both arguments are valid insofar farmers had to run a complicated business, but the certificates we rely on do not always distinguish between the operators of large farms, smallholders and farm labourers. To analyse exposure to infectious disease in the workplace, we used the HISCO classification to create an occupational indicator for exposure. Comparing occupations with higher and lower excess mortality could lead to ad-hoc explanations, whereas creating this new indicator allows us to systematically account for characteristics in occupations that lead to risk of exposure. Moreover, like the skill levels derived from HISCLASS above, this scheme avoids very low regional counts for certain occupations. Exposure to infectious disease due to working conditions was coded on two dimensions: (1) working in an indoor environment (yes/no), and (2) regular social interaction (yes/no). Coding was done at the three-digit HISCO level. For example, an office clerk was considered to be working indoors, but had infrequent social contact, especially with strangers. A tailor also worked indoors but would have had frequent social contact through dealings with customers. Conversely, a farm worker worked neither indoors, nor in close contact with others. Occupations were independently coded at least two researchers and then compared. Approximately 75 per cent of occupations were coded identically in the first round, and the remaining occupational groups were discussed by the coders until an unanimous decision was reached. The remaining differences were often due to differing interpretation of the underlying occupations in the HISCO unit or the actual tasks performed in an historic occupation, which could often be solved by consulting the documentation of HISCO. 9 Although our scoring omits regional and within-occupation variance in occupational characteristics, it captures perceived conditions for exposure to and transmission of the virus on the work floor. Moreover, the scores measure something different from the skill levels we also use in our analyses. Correlations between these dummy variables range from −0.27 to +0.32 (see [fig_ref] Table 1: Summary statistics and selection steps [/fig_ref] in Appendix B). ## Excess mortality As death certificates in the Netherlands do not include a cause of death, we work with all-cause mortality. We use excess mortality as our outcome variable: the number of deaths in excess of what would be expected based on previous years. 10 This approach is used because The Dutch term landbouwer which is frequently used on the certificates is not specific enough to make this distinction. Appendix B provides our coding of the most frequent occupations in the dataset. The full coding scheme is described in [bib_ref] HSNDB index for exposure to infectious disease, Schalk [/bib_ref] , and the data can be found at https://hdl.handle.net/10622/TFG1DQ. Unless stated otherwise, excess mortality in the results and conclusion section is used to refer to excess all-cause mortality during the second Spanish Flu wave. the 1918 municipal population at risk (by age, sex, and occupation) is not available. We calculate excess mortality rates (EMRs) for 1918, comparing the number of deaths between September 1st to December 31st in 1918 by age group, municipality, and occupational cluster to mortality in the preceding years. 11 Baseline mortality was established by calculating the average mortality of the same occupational groups (by age group) in autumn 1910-1917 (September through December). The Netherlands remained neutral during the First World War, so dramatic changes to the population at risk and mortality rates that characterised many other countries at the time are not expected . The exception was the year 1914, when the Netherlands briefly took in 1 million Belgian refugees, who largely returned before the end of the calendar year. We excluded 1914 from the baseline to make sure that our estimates of excess mortality are not biased by the events of the First World War in the Netherlands. Our approach relies on the assumption that the population at risk is relatively stable in the period leading up to the Spanish Flu. [fig_ref] Figure 2: Weekly number of deaths by age and sex in the Netherlands from... [/fig_ref] shows that relative to the huge mortality spike of the Spanish Flu, weekly death number were stable in the 2.5 years preceding the pandemic, especially in the age categories that we are interested in. Moreover, [fig_ref] Figure 3: Line graph of annual deaths in the months September-December, 1910-1918, broken down... [/fig_ref] shows that the annual number of deaths for the main subgroups of interest, in the selection of the data described above, were also stable over the 1910-17 period. We also use external demographic data to investigate the possibility of strong demographic shifts in the Netherlands in the 1910-17 period. The Historical Database Dutch Municipalities shows that across all municipalities, the coefficient of variation in population was on average only 0.04 [bib_ref] Historische database nederlandse gemeenten, v5, Mourits [/bib_ref]. Total Dutch population figures from the Human Mortality Database show that the coefficient of variation in population by 10-year age groups averaged to, again, 0.04 (Human Mortality Database, 0000, July 2021 version). While the total Dutch population in this period grew 1.5% per year, implying growth in the subgroups, year-to-year shifts in mortality are small compared to the events of 1918. In [fig_ref] Table 2: Excess mortality rates in September-December 1918 by occupational skill group and their... [/fig_ref] , we show excess mortality rates by occupational skill level. Less skilled people had higher excess mortality. We now turn to analysing these differences further. # Analysis Excess mortality is modelled using OLS-regressions with the ratio of deaths in September-December 1918 over the average number deaths A. Rijpma et al. in these months for the period 1910-17 (excluding 1914) as the dependent variable. The distribution of these ratios is highly skewed, so we take the logarithm of the dependent variable. 12 However, as we calculate excess mortality per municipality by age, and social class, for 59% of our observations for 1918 there are no deaths, resulting in a ratio of zero. We do not discard these data because the fact that these groups had zero mortality during the Spanish Flu is important. To include these observations, we add one to the dependent variable. This means we estimate excess mortality in region , month , occupational group , and age group as follows: [formula] log ⎛ ⎜ ⎜ ⎝ ℎ =1918 ℎ + 1 ⎞ ⎟ ⎟ ⎠ = 0 + 4 ∑ 1, + 3 ∑ 2, + 3 + 3 ∑ 4, + + +(1) [/formula] Standard errors are clustered by region [bib_ref] Various versatile variances: An object-oriented implementation of clustered covariances in R, Zeileis [/bib_ref]. For regions we use Economic Geographic Regions (EGG): clusters of municipalities defined by Statistics Netherlands below the NUTS-3 level. We prefer to work at this relatively low level of aggregation to be able to control for the spatial patterns of the pandemic and differences between urban and rural regions which the EGG regions capture, but NUTS-3 region in the Netherlands do not. To verify the validity of our outcomes, we conduct a number of robustness checks. First, we explore whether the level of geographic aggregation affects our results. Larger geographic areas mean there are fewer regions without deaths in 1918, and, more generally, will increase precision in our data, albeit at the expense of a lower number of observations. In addition to the EGG level, we also estimate our models for municipalities, COROP 14 (NUTS-3) regions, and provinces (NUTS-2). For similar reasons we also provide separate estimates for high and low mortality regions. This allows us to focus on the regions most affected by the Spanish Flu pandemic, and again, exclude many cases of zero deaths. Additionally, this provides a check for the importance of high-mortality outliers. The cutoff was an overall excess mortality 2.5 times the mortality we would usually expect in September-December. In addition, we also look at a variety of alternative types of models to deal with the zeros in our data. We also check whether population density affect our estimates due to easier transmissibility in cities compared to the countryside. We also check whether the 1914-1918 mobilisation of troops affects our results by controlling for the presence of army bases. We also include alternative specifications for our occupational exposure measures and estimate the models including women. Finally, we estimate our models again but using stricter thresholds in the data selection process. [fig_ref] Table 3: Regression models of log excess mortality rate [/fig_ref] shows the results of this model, with progressively more controls to come to our preferred model in the rightmost column. For each estimate we report the coefficient, standard error, and significance level. # Results In the most basic model, it can be seen that there is a skill gradient in the excess mortality rates of 1918. Compared to higher-skilled workers, medium skilled men had 55 percent higher mortality than they would experience in a normal year (exp(0.44) -1). For unskilled workers this was higher still: their excess mortality was 68 percent higher than that of higher skilled workers. Lower skilled workers also had substantially higher excess mortality than higher-skilled workers, but not as high as unskilled workers and medium-skilled workers. An important driver of this break in the skill gradient are farmers. They are a large group in our dataset with relatively high mortality who are classified as medium-skilled in the HISCLASS scheme. We return to this point in more detail below. Here we observe that if we separately control for farmers (model 2 onwards), excess mortality is still not strictly increasing with occupational skill, but closer to a full social gradient. Next, we introduce our occupational exposure variables (model 3). Here, we find that contact occupations had nearly 20 percent higher excess mortality compared with occupations that had neither of the exposure characteristics. In this specification with limited controls, the other occupational exposure variable, indoor work, is not associated with higher excess mortality. Once further controls are added it also shows a positive association, but it is not estimated with sufficient precision to draw firm conclusions. However, an F-test does show that the exposure variables are jointly significant (model 7 has the lowest F-statistic at 4.05, p = 0.006). Of particular interest is what happens to the skill gradient when we introduce these exposure variables (model 4). We find that when controlling for exposure, the skill gradient in excess mortality remains largely unchanged, although the mortality estimate for farmers increases. Overall, we find that working indoors does not predict higher excess mortality. However, occupational exposure to infectious disease We also note that the increase in standard errors is limited compared to model 2 and 3, suggesting the moderate correlations between these two sets of variables discussed in Section 3.2 are not an issue in the model. due to frequent contact predicts somewhat higher excess mortality, though skill remains a more important predictor. Our next model adds demographic controls. Adding age controls reduces the social gradient in excess mortality describe above somewhat, but it is still clearly present. This is important because we know that Spanish influenza affected younger men in particular, and we can expect them to have lower-skilled occupations due to career effects. Indeed, keeping all else constant, we find the highest excess mortality for age groups 16-30 (reference category), with progressively lower mortality predicted for older age groups. Adding month dummies to capture the phase of the epidemic also leaves the estimates for skill level largely unchanged. We finally add region fixed effects to adjust for the fact that some regions were struck harder than others (the rural, poorer North-East in particular, see [bib_ref] Retracing hotbeds of the 1918-19 influenza pandemic. Spatial differences in seasonal excess..., Mourits [/bib_ref]. This increases the skill gradient again. In our preferred model, including the full set of demographic controls and region and time fixed effects medium skilled workers have 46 percent higher excess mortality compared to high skilled workers; lower skilled workers 23 percent and unskilled workers have 70 percent higher excess mortality. The predicted excess mortality due to the exposure characteristics of the occupations of the deceased is 19 percent for contact occupations compared to the baseline of occupations characterised by neither exposure risk. To further analyse the socioeconomic gradient gradient in excess mortality during the Spanish Flu, we turn to the role of farmers in our results and an alternative measure of social status -HISCAM. These alternative models are tested in . In column 2, the preferred model from [fig_ref] Table 3: Regression models of log excess mortality rate [/fig_ref] is estimated again, but for a dataset where excess mortality was calculated without farmers. In the next column, as another alternative, we recode farmers as lower skilled, rather than medium skilled as in the HISCLASS scheme. Omitting farmers from our dataset makes only a limited impact on the social gradient we find. Recoding farmers as lower skilled does make a difference. The N-shaped gradient, where, compared to higher A. Rijpma et al. ## Table 4 Regression models of log excess mortality rate, with different treatments of farmers, and with HISCAM scores rather than HISCLASS-based skill levels. Region-clustered standard errors between parentheses. skilled workers, lower skilled workers had lower excess mortality than medium skilled workers, disappears. Medium and lower skilled workers now have similar excess mortality. In this model, our occupational exposure variables show the counter-intuitive result, where working indoors now predicts lower excess mortality. We surmise that this is because the composition of the reference group is changed strongly by farmers group who were classified as having neither an indoors nor a contact occupation. We also estimate our preferred model with HISCAM scores instead of HISCLASS. Again, we find a socioeconomic gradient gradient in excess mortality. The model using HISCAM shows that, keeping all else constant, higher occupational status predicts lower excess mortality during the Spanish Flu. The HISCAM scores in our data range from 0.40-0.99, so going from the lowest to the highest status occupation predicts a decrease in excess mortality of 23%. To assess if high-mortality regions drive our results, compares how our preferred model performs in regions with relatively low and high overall mortality. Making this distinction is a more direct assessment of the impact of regional Spanish Flu severity versus occupational or demographic characteristics. The cutoff was an overall excess mortality rate of 2.5, that is 2.5 times the mortality we would usually expect in September-December. As expected, we find that most estimates in the high mortality areas are higher compared to low mortality areas, and we lose some precision on the estimates due to the lower number of observations. Otherwise, the overall results are similar, suggesting that the basic occupational patterns in excess mortality during the Spanish Flu were present regardless of the presence of high-mortality clusters. Regression models of log excess mortality rate for low (EMR below 2.5) and high (above 2.5) excess mortality regions. Region-clustered standard errors between parentheses. ## Robustness checks To verify the robustness of our results, we included a number of alternative models in the Appendix. In we estimate the preferred model from [fig_ref] Table 3: Regression models of log excess mortality rate [/fig_ref] at different levels of geographic aggregation: municipalities, EGG regions, Corop (NUTS-3) regions, and provinces (NUTS-2). While we prefer to work at a relatively low level of aggregation, a concern could be that this results in cells in our dataset with few observations, either for our 1910-17 baseline, or for the Spanish Flu pandemic deaths. Overall, the results are similar to the preferred EGG aggregation level, though overall effect sizes tend to be larger at the higher aggregation COROP (NUTS-3) level, and we lose some precision at higher levels of aggregation. Important factors in the spread of any infectious disease are population density and geographic mobility, as these make it easier for the disease to move from one person to the next. While the inclusion of region fixed effects in our models should capture any time invariant aspects of these factors, we explore this possibility further in [fig_ref] Table 7: Regression models of log excess mortality rate including controls for presence of... [/fig_ref] , Appendix A, where we show the effect of including population density and the presence of army bases or hospitals in a municipality. Because these variables are measured at the municipality level, we aggregate our data to this level as well for these checks. As expected, the overall socioeconomic gradient is the same as these effects would also be captured by the location fixed effects included in the models in . The effect of the population variable itself is positive. However, population density does not have a significant effect on excess mortality, even if we exclude population size from our model. In the same table, we also include a control for municipal military bases in part of our models for more reliable estimates of excess mortality. Troop movements and crowded conditions at army bases could are suspected to be important contributors to the spread of the Spanish Flu [bib_ref] Plagues & wars: the 'Spanish Flu' pandemic as a lesson from history, Flecknoe [/bib_ref]. Although the Netherlands remained neutral during World War I, the Dutch army was mobilised from 1914 to the end of 1918. Living conditions in the barracks and other army camps were poor [bib_ref] Ziekte en dood binnen de gemobiliseerde Nederlandse krijgsmacht 1914 -1918, Koten [/bib_ref] , and young adult men were affected by the Spanish Flu in large numbers. As many soldiers were conscripts, their occupation listed at death would not be 'soldier', but their occupation before the War. Only 0.2% of the death certificates have occupational titles that can be linked to the military between 1910 and 1918. Given that over 200.000 men out of 6.5 million inhabitants were conscripted, this is surely an underestimation of deaths among conscripts. To control for this potentially strong increase in local excess mortality around military bases, we add a set of dummy variables to indicate whether divisions of the Dutch army were present in 1918 at the places of death recorded in the certificates. These locations were obtained from a list of military bases in 1918 compiled by [bib_ref] Vredesgarnizoenen van 1715 tot 1795 en 1815 tot 1940, Ringoir [/bib_ref] and from the locations where Dutch soldiers were admitted to military hospitals in 1913, listed in annual reports [bib_ref] Statistisch Overzicht der Behandelde Zieken van Het Leger Hier te Lande in..., Landmacht [/bib_ref]. We used military hospitals for 1913 because no military hospital reports are available between 1914 and 1918, and the recording procedure changed after the War. The presence of army bases shows a negative, though statistically insignificant association with excess mortality, which is surprising given positive results found by other studies [bib_ref] What explains cross-city variation in mortality during the 1918 influenza pandemic? Evidence..., Clay [/bib_ref]. It should be mentioned, however, that one of the most important military bases in the Netherlands, the main navy base in Den Helder, had some of the highest excess mortality in the country (see [fig_ref] Figure 1 ,: top left panel [/fig_ref] , but had to be excluded from our dataset because the occupations for these certificates were not digitised. There are a number of ways in which the occupational exposure variables could have been used in the models, and in , Appendix A we explore a number of these specifications. We first look at a model that omits the interaction between indoors and contact occupations. This reduces the size of the effects we find and they are no longer statistically significant. While we think the combination of the two kinds of exposure is important to include in the model, we also note that this further shows that the occupational exposure effect is not consistent in our data. We next look at an additive measure of occupational exposure, by adding up the two dummy variables so that no risk equals zero, one of both indoors or contact risk results in a score of one, and both being present gets a score of two. We find that when we enter this variable as a linear predictor it does not have a statistically significant association with excess mortality. We prefer the specifications where occupational exposure enters as separate dummies because it does not require us to make assumptions about the size of the two variable's effect sizes as adding them up would. In [fig_ref] Table 9 continued.: Pref [/fig_ref] in Appendix A we check whether the thresholds we used in our data selection procedure impact our results. Specifically, we check whether using higher coverage thresholds (at least 80% of age, sex, and date observations in a municipality have to be present rather than 50%). For presence of occupations, we use a threshold of 20% rather than 10%. The results are very similar, the main differences being that the estimates on our exposure variables have become stronger. We also check whether including women alters our results [fig_ref] Table 1: Summary statistics and selection steps [/fig_ref] , applying the same model as in [fig_ref] Table 3: Regression models of log excess mortality rate [/fig_ref] , with the addition of a dummy variable for sex. We find that men in this data indeed had higher excess mortality during Spanish Flu. Beyond that, our main findings are similar across these two models. As a final check of our results, we investigate whether the way we handled the cases of zero deaths during the Spanish Flu in our model affects our results. The following alternative approaches are explored: dropping cases of zero deaths (column 2), not taking the logarithm of the dependent variable (column 3), a quasiPoisson model with the ratio of 1918 to baseline deaths as the dependent variable , a quasiPoisson model of the number of deaths during the Spanish Flu pandemic with the log baseline deaths as the offset (column 5), and taking the inverse hyperbolic sine of the ratio (Bellemare and Wichman, 2020; Burbidge et al., 1988, column 6). Appendix A, [fig_ref] Table 1: Summary statistics and selection steps [/fig_ref] shows that the socioeconomic gradient in excess mortality and the higher excess mortality for contact occupations we find in our preferred specification is found in the alternative models as well. The main exception here is the model where we exclude groups with zero deaths during the Spanish Flu pandemic, in which the patterns we find elsewhere are reversed. As mentioned earlier, we think the absence of deaths during the Spanish Flu pandemic is a relevant outcome that should be included in the model. The precision of our estimates is also lower in the two quasiPoisson models. Beyond that, what stands out is that the alternative models provide higher estimates of the same pattern. # Conclusion This paper explored whether there was a socioeconomic gradient in excess mortality among male workers during the Spanish Flu in the Netherlands. Using individual level occupational information and all-cause mortality data, we calculated excess mortality and found it to be highest among farmers and unskilled labourers and lowest among higher-skilled labourers. Excess mortality for medium-skilled and lower-skilled workers was in between these groups. These findings are in line with evidence from a recent study on Sweden [bib_ref] Social class and excess mortality in Sweden during the 1918 influenza pandemic, Bengtsson [/bib_ref] and earlier regional evidence, e.g. for Kristiania (Oslo, Norway) [bib_ref] A socially neutral disease? Individual social class, household wealth and mortality from..., Mamelund [/bib_ref] , and strongly contrast with the perceptions of contemporaries that the Spanish flu had a relatively egalitarian impact across the society [bib_ref] A socially neutral disease? Individual social class, household wealth and mortality from..., Mamelund [/bib_ref]. The national death registers we relied on included individual information on the age, occupation, and date of death as well as sex and the municipality of residence of the deceased. This information allowed us to estimate the association between social class and excess mortality, while also taking occupational characteristics, age, and regional variation in excess mortality into account [bib_ref] Geography may explain adult mortality from the 1918-20 influenza pandemic, Mamelund [/bib_ref]. Moreover, controlling for municipality size and presence of local army bases, or analysis of specific regions with high excess mortality resulted in similar estimates of the magnitude of socioeconomic differences in excess mortality during the Spanish flu. In other words, there was a clear socioeconomic gradient in excess mortality during the autumn wave of the 1918-19 influenza pandemic. As we study excess mortality, these estimates come on top of the existing social gradient in mortality among individuals age 35-55 in the Netherlands [bib_ref] Time trends in social class mortality differentials in the Netherlands, 1820-1920: An..., Van Poppel [/bib_ref] , indicating that the Spanish Flu increased existing social inequalities in mortality. Our analyses take into account whether the social differences in excess mortality during the Spanish Flu could have been driven by occupational characteristics, including social interaction at the workplace and indoor work. For COVID-19, it has been shown that occupational conditions are related to workplace infections [bib_ref] What explains the socioeconomic status-health gradient? Evidence from workplace COVID-19 infections. SSM..., Godefroy [/bib_ref]. Similarly, working together with others in an enclosed space or regular social interaction at the workplace could have been related to increased chances of infection and death with the Spanish Flu in autumn 1918. To test this hypothesis, a contact index was constructed. We found some evidence that social contact at the workplace predicted higher excess mortality during the Spanish Flu pandemic. However, contrary to our expectations, it did not seem to matter whether individuals worked together indoors or not. While the estimates of our contact index were generally robust for controlling for demographics, municipality size, local army bases, regional fixed effects, or specifically looked at regions with high excess mortality, it should be noted that the effect we find for social contact depends on how these exposure variables are included in the model. Our findings indicates that the higher social strata were, to some degree, able to protect themselves against the Spanish flu. It is tempting to think that such differences result from differences in income, as educational divides in mortality were uncommon before the second half of the 20th century. Earlier studies show inequalities in Spanish flu at the macro level. Neighbourhoods with larger housing had lower Spanish flu mortality [bib_ref] A socially neutral disease? Individual social class, household wealth and mortality from..., Mamelund [/bib_ref] and, across the globe, poverty was related to higher Spanish flu mortality [bib_ref] Estimation of potential global pandemic influenza mortality on the basis of vital..., Murray [/bib_ref]. In the Netherlands in 1918, lower-skilled and medium-skilled labourers were by no means rich, but they could afford significantly better housing and food than unskilled labourers. However, material resources are unlikely to fully account for the advantages that social status provides, as non-material resources -such as existing health conditions, occupational hazards, knowledge about infectious diseases, and access to health care -have been at least equally important in explaining survival differentials by social status in other social contexts [bib_ref] Social conditions as fundamental causes of disease, Link [/bib_ref] [bib_ref] A social history of disease: Contextualizing the rise and fall of social..., Clouston [/bib_ref] [bib_ref] Social class differentials in health and mortality: Patterns and explanations in comparative..., Elo [/bib_ref] [bib_ref] Old age, health, and social inequality: Exploring the social patterns of mortality..., Edvinsson [/bib_ref] including the ongoing COVID-19 pandemic [bib_ref] A population-based cohort study of socio-demographic risk factors for COVID-19 deaths in..., Drefahl [/bib_ref]. We found that farmers had higher excess mortality during the Spanish Flu, especially after we control for occupational skill levels, occupational social contact and age composition. This contradicts earlier work on mortality during the Spanish Flu in Sweden [bib_ref] Social class and excess mortality in Sweden during the 1918 influenza pandemic, Bengtsson [/bib_ref]. More generally, at the turn of the 20th century, farmers were known to outlive their peers in many populations [bib_ref] Once were farmers: Occupation, social mobility, and mortality during industrialization in Saguenay, Gagnon [/bib_ref] [bib_ref] Exceptional lives, extraordinary families, Mourits [/bib_ref] [bib_ref] Social class, social mobility and mortality in the Netherlands, Schenk [/bib_ref] [bib_ref] Effects of childhood and middle-adulthood family conditions on later-life mortality: Evidence from..., Smith [/bib_ref]. In the Netherlands, excess mortality rates during the Spanish Flu were especially high in Drenthe and parts of Groningen and Overijssel -rural and poor regions where many people were involved in farming [bib_ref] Retracing hotbeds of the 1918-19 influenza pandemic. Spatial differences in seasonal excess..., Mourits [/bib_ref]. Locals noted that the poverty, bad living conditions, and lack of medical care might have elevated mortality in this region. In normal circumstances, farmers were able to outlive their peers even if they were poor, as they continued to be physically active at advanced ages and had access to fresh food in a time when refrigeration was not commonplace [bib_ref] Exceptional lives, extraordinary families, Mourits [/bib_ref] [bib_ref] Lifestyle and nutrition related to male longevity in Sardinia: An ecological study, Pes [/bib_ref]. However, there is no evidence that farmers in the Netherlands lived longer than the middle class or elite between ages 35 and 55 [bib_ref] Time trends in social class mortality differentials in the Netherlands, 1820-1920: An..., Van Poppel [/bib_ref] , which was part of the age group hit hardest by the Spanish flu. It could be that poverty protected farmers from lifestyle diseases, enabling them to live to old age, but made them vulnerable to outbreaks of infectious disease due to poor housing, malnutrition, and lack of medical care. Some drawbacks of our approach and the data used should be mentioned. We analyse all-cause excess mortality, while ideally we would use sufficiently detailed and reliable cause-specific mortality. High excess mortality during the Spanish Flu means we should mostly pick up Spanish Flu related deaths, but uncertainty remains. Second, incomplete digitisation of death certificates as well as our data selection steps mean we lose some of the large cities in the Netherlands, most notably Amsterdam. While our sample showed no large biases on the basis of observable characteristics, differences in the spread of the virus in high population density settings are possible, and could impact our results. Third, the specific age structure within occupational groups could affect results, if relatively more young men -who were affected hardest by the Spanish Flu -were unskilled labourers, for example. Finally, in this work we have only included men. Occupational characteristics of women were hard to reconstruct from the death certificates we use. Since both mortality and occupational structure are known to have differed between men and women, this is an important point for future research. The Spanish flu did not hit the Dutch population evenly. Higher social classes experienced lower excess mortality rates. These differences between socioeconomic groups were not explained by occupationspecific risk factors included in the analyses in this work. Frequent social contact at the workplace was associated with increased excess mortality during the Spanish Flu pandemic, but cannot account for the socioeconomic gradient in excess mortality. Overall, the social factors that put populations at higher risk during the pandemic may also have been factors that determined existing health differences across the population, exacerbating existing social gradients in mortality. ## Credit authorship contribution statement Auke Rijpma: Conception and design of study, Acquisition of data, Analysis and/or interpretation of data, Drafting the manuscript, Revising the manuscript critically for important intellectual content. Ingrid K. van Dijk: Conception and design of study, Analysis and/or interpretation of data, Drafting the manuscript, Revising the manuscript critically for important intellectual content. Ruben Schalk: Conception and design of study, Acquisition of data, Analysis and/or interpretation of data, Drafting the manuscript, Revising the manuscript critically for important intellectual content. Richard L. Zijdeman: Acquisition of data, Analysis and/or interpretation of data, Revising the manuscript critically for important intellectual content. Rick J. Mourits: Conception and design of study, Acquisition of data, Analysis and/or interpretation of data, Drafting the manuscript, Revising the manuscript critically for important intellectual content. # Funding and acknowledgements We would like to thank Svenn-Erik Mamelund, Mar Rodríguez-Girondo, Niels van den Berg, participants to OsloMet's PANSOC webinar and two anonymous referees for their comments on earlier drafts of this paper. Any remaining mistakes are our own. Auke Rijpma, Ruben Schalk, Richard Zijdeman and Rick Mourits acknowledge financial support from the Netherlands Organization for Scientific Research (NWO) for the . Ingrid van Dijk was funded through the research programme ''Landskrona Population Study'' and project ''An Age Old Advantage?'', Swedish Foundation for the Humanities and Social Sciences (Riksbankens Jubileumsfond, RJ). All authors approved the final version of the manuscript. A. ## Appendix a. robustness checks See Tables 6-9. Regression models of log excess mortality rate at different levels of aggregation: EGG (Economic Geographic Regions), COROP: NUTS-3 regions, Provinces: NUTS-2 regions. Standard errors between parentheses, clustered at the relevant regional level. R 2 0.14 0.14 0.14 0.14 Adj. R 2 0.14 0.14 0.14 0.14 Num. obs. 4650 4650 4650 4650 * * * < 0.001; * * < 0.01; * < 0.05. ## Municipalities ## Table 8 Regression models of log excess mortality rate, with alternative specifications for the exposure variables. Region-clustered standard errors between parentheses. ## Table 9 Robustness checks for regressions of log excess mortality rate, using stricter data selection steps: municipalities with higher occupation coverage (20 percent), and higher ( 3114 3551 * * * < 0.001; * * < 0.01; * < 0.05. ## Table 11 Regressions of log excess mortality rate, using alternative model forms to deal with observations of 0 excess mortality. Region-clustered standard errors between parentheses. [fig] Figure 1 ,: top left panel): the digitised part covers 85 per cent of the 1118 municipalities existing in 1918. A large part of the missing death certificates are from the city of Amsterdam, where digitisation has not yet begun. [/fig] [fig] Figure 1: Maps of geographic coverage of certificates of death (panel a), certificate dates (panel b), coverage of individuals' age (panel c), coverage of sex (panel d), and occupations (panel e) on death certificates. Panel f shows municipal excess mortality rates during the Spanish Flu pandemic (September-December 1918). Source: Openarch Death Certificates.The maps represent the completeness of digitisation of death certificates (panel a) and specific information from certificates (panel b-e). Differences between municipalities exist due to differences in digitisation procedures.A.Rijpma et al. [/fig] [fig] Figure 2: Weekly number of deaths by age and sex in the Netherlands from death certificates, 1916-1919. Source: Openarch Death Certificates. [/fig] [fig] Figure 3: Line graph of annual deaths in the months September-December, 1910-1918, broken down by occupational skill level (left panel) and exposure (right panel). Number of deaths is on a semilogarithmic scale. Source: Openarch Death Certificates. [/fig] [table] Table 1: Summary statistics and selection steps. Source: Openarch Death Certificates. The table shows the stepwise selection of cases for analysis. The database contains a total of 741 758 death certificates. We select only death certificates from municipalities fully available in the database (715 703 cases) and where there is sufficient coverage for our variables (220 795 cases). We also drop certain periods, age groups, and women (16 786), and finally select complete cases (11 469). [/table] [table] Table 2: Excess mortality rates in September-December 1918 by occupational skill group and their differences, with bootstrapped standard errors between parentheses. Difference refers to the difference between the EMR in that row and the previous. Source: Openarch Death Certificates. [/table] [table] Table 3: Regression models of log excess mortality rate. Region-clustered standard errors between parentheses. [/table] [table] Table 7: Regression models of log excess mortality rate including controls for presence of army base and military hospitals as well as population density controls. Data is aggregated to the municipal level. Region-clustered standard errors between parentheses. [/table] [table] 80: percent) coverage of sex, age, and date variables. Region-clustered standard errors between parentheses. [/table] [table] 59: * * * < 0.001; * * < 0.01; * < 0.05. [/table] [bib_ref] Retracing hotbeds of the 1918-19 influenza pandemic. Spatial differences in seasonal excess..., Mourits [/bib_ref] [bib_ref] Retracing hotbeds of the 1918-19 influenza pandemic. Spatial differences in seasonal excess..., Mourits [/bib_ref]
RE: “Socioeconomic status and overweight/obesity in an adult Chinese population in Singapore” [bib_ref] Prevalence of obesity in Taiwan, Chu [/bib_ref] [bib_ref] Association between simple anthropometric indices and cardiovascular risk factors, Ho [/bib_ref] [bib_ref] Prevalence of obesity in Korea, Kim [/bib_ref] [bib_ref] Sex differences in the association of socioeconomic status with obesity, Wardle [/bib_ref] [bib_ref] Socioeconomic inequality of obesity in the United States: do gender, age, and..., Zhang [/bib_ref] [bib_ref] Socioeconomic status in relation to obesity and abdominal obesity in Korean adults:..., Yoon [/bib_ref] [bib_ref] The relation of gender, race and socioeconomic status to obesity and obesity..., Paeratakul [/bib_ref] [bib_ref] Socioeconomic status and obesity: a review of the literature, Sobal [/bib_ref] [bib_ref] The influence of socioeconomic status, ethnicity and lifestyle on body mass index..., Sundquist [/bib_ref] [bib_ref] Obesity and overweight in young adults: the CARDIA study, Burke [/bib_ref] [bib_ref] Socioeconomic inequality of obesity in the United States: do gender, age, and..., Zhang [/bib_ref] [bib_ref] Central and total obesity in middle aged men and women in relation..., Langenberg [/bib_ref] [bib_ref] Social status and health risks in Canadian adults: 1985 and 1991, Millar [/bib_ref] [bib_ref] Socioeconomic status and obesity: a review of the literature, Sobal [/bib_ref] [bib_ref] Body mass index and its association with socioeconomic and behavioral variables among..., Reddy [/bib_ref] [bib_ref] Socio-economic status and risk factors for cardiovascular disease: a multicentre collaborative study..., Anonymous [/bib_ref] ## Study population The Tanjong Pagar survey was a population-based cross-sectional survey of adult Chinese aged 40-81 years residing in the Tanjong Pagar district in Singapore. The survey was conducted between October 1997 and August 1998. Detailed population selection and methodology have been previously reported. [bib_ref] Prevalence and risk factors for refractive errors in adult Chinese in Singapore, Wong [/bib_ref] [bib_ref] Education, socioeconomic status, and ocular dimensions in Chinese adults: the Tanjong Pagar..., Wong [/bib_ref] [bib_ref] The prevalence of glaucoma in Chinese residents of Singapore: a cross-sectional population..., Foster [/bib_ref] In brief, the 1996 Singapore electoral register in the district of Tanjong Pagar was used as the sampling frame in this study. The electoral register listed 15,082 Chinese names aged between 40 and 79 years residing in the district. Two thousand (13.3%) names were selected using a disproportionate (with more weights given to the older age groups), stratified, clustered, random sampling method. This involved selecting residents randomly, 500 from each of 4 age strata: 40-49, 50-59, 60-69 and 70-79 years, residing in 50 area clusters defined by street name (out of a total of 84), located within specified boundaries of the pre-designated study clinic. The area clusters selected were those with the largest concentration of persons in the district (82% of the population). Among the 2,000 names selected, 46 had died and 235 had moved to addresses outside the district before the study period, and 2 people were excluded on grounds of ill health, leaving 1,717 subjects considered eligible to participate in this study. Among the 1717 subjects eligible for the survey, 1232 (71.8% of eligible subjects) participated, and 1,090 (63.4%) attended the clinic examination. The number of persons with data on body mass index (BMI) was 1,082. The final sample included for analysis, after eliminating persons with missing information on the SES variables considered, was 942. Compared to those who were excluded in the final analysis, those who were included were younger, taller, heavier, # Ethical issues Informed written consent was obtained from all participants, and ethics approval was obtained from the Singapore National Eye Centre. Demographic and behavioral characteristics of the study population and the distribution of SES indicators by gender are shown in . The study involved predominantly older individuals (average age=58.0 years). Men were significantly more likely to be current smokers, to have higher educational achievement, and a higher income. Spearman correlation for men and women between income and education was 0.51 and 0.57, between income and housing type was 0.39 and 0.24, and between education and housing was 0.39 and 0.32. In the whole cohort, 33% were overweight/obese, with a mean BMI of 23.6kg/m 2 (standard deviation of 3.8kg/m 2 ). The mean CIPROP programme) by each SES indicator was calculated. The association of education, income, and housing type with overweight/obesity was assessed using logistic regression models after controlling for significant confounding factors (age and smoking status); variables also considered as potential confounding factors, but not included in the final model included diabetes (absent, present), hypertension (absent, present) and systolic and diastolic blood pressures (mm Hg). Tests for trend were performed using each SES indicator as ordinal variable. Odds ratios (ORs) and 95% CIs of overweight/obesity were estimated by using the highest SES category as the reference for each SES indicator. Statistical interaction between gender and each SES indicator was examined in the corresponding logistic regression model by including cross-product interaction terms. All reported p values were based on two sided tests and compared to a significance level of 5%. Statistical interaction was deemed significant if pinteraction was <0.10. All analyses were performed using SAS ® version 9. : Income was based on individual monthly income in Singapore dollars (SGD). : Housing: (1) small size public apartments (1-2 room), (2) medium size public apartments (3 room), and (3) large public apartments (4-5 room) or private housing. ciated with overweight/obesity (p<0.05). The prevalence of overweight/obesity was highest (37%) among low income earners among women. Among the 3 housing types we examined, majority (54%) of the study population lived in medium housing (3 room public apartments) in both genders. In women the lowest prevalence of overweight/obesity (25%) was observed among those with large housing (>3 room or private housing). These associations with education, income, and housing type were not observed in men. [fig_ref] Table 3: Odds ratios [/fig_ref] shows the OR of overweight/obesity in relation to SES by gender. Among women, in separate analyses, lower categories of educational level, income, and housing were positively associated with overweight/obesity; similar association was not observed in men. To formally evaluate the observed effect modification of SES and overweight/obesity by gender, we included cross product interaction terms in the corresponding multivariable BMI decreased with increasing levels of education, (p-trend <0.0001). Current smokers had lower BMI. Increasing age categories had a lower prevalence of overweight/obesity and lower BMI, (p-trend <0.05). In a linear regression analysis, BMI was found to be inversely related to increasing age categories (indicator variable), after adjusted for education ( = -0.041, SE=0.012, p=0.0007) The prevalence of overweight/obesity by SES indicators and by gender is shown in . An inverse relationship was observed between the level of education and the prevalence of overweight/obesity in women (p<0.01). Prevalence of overweight/obesity was highest (38%) among the primary or lower educated women and lowest (12%) among women with post-secondary education . In men, the prevalence of overweight/obesity was also lowest (23%) among those with post-secondary education. Among women, income was significantly asso- [fig_ref] Table 3: Odds ratios [/fig_ref]. Finally, in a supplementary analysis that incorporated sampling weights in the logistic regression models, the results were similar. In this study, data from a representative sample of Chinese population aged 40-81 years in Singapore suggests that low SES, defined by categories of education, income, and housing, was associated with a higher prevalence of overweight/obesity in women. In men, no significant relations between these SES indicators and overweight/obesity were found. The overall prevalence of overweight/obesity in the current study was 33%, similar to previous reports from national surveys in Singapore conducted by the Ministry of Health (33% in 2004 and 30% in 1998).In the present study, current smoking was inversely associated with overweight/obesity, a finding similar to previous studies. [bib_ref] Associations between smoking and body weight in the US population: analysis of..., Albanes [/bib_ref] [bib_ref] The relationship between body mass and cigarette smoking using a biochemical index..., Klesges [/bib_ref] [bib_ref] Relationship of smoking status, energy balance, and body weight: analysis of the..., Klesges [/bib_ref] These consistent findings with previous studies indirectly suggest that our results have reasonable internal validity. In the current study, SES was found to be inversely associated with overweight/obesity among adult Singaporean women. In our study, we used commonly used measures of SES such as education, income, and housing to assess the prevalence of overweight/obesity. These findings from Singapore, a newly industrialized Asian country, are similar to the previously reported models. There was a significant gender educational status interaction (p-interaction=0.03), gender income interaction (p-interaction=0.003), and gender housing type interaction (p-interaction=0.03). We performed several sets of supplementary analyses. First, we examined the association between SES variables and overweight/ obesity after excluding men and women in the oldest age group (70-81 years). Among women, the multivariable OR (95% CI) of overweight/ obesity was 2. : Adjusted for age and smoking status : Income was based on individual monthly income in Singapore dollars (SGD) : Housing: small size public apartments (1/2 room), medium size public apartments (3 room), and large size public apartments (> 3 room) or private housing : P-interaction for sex and income=0.003, sex and education=0.03, and sex and housing=0.03 BMI 35-37 may contribute to inconsistent associations between socioeconomic position and BMI among men 2 particularly given our crude measure of smoking. Our findings are also similar to studies involving adolescents, which showed a strong inverse relationship of SES to overweight prevalence in white adolescent females. [bib_ref] Obesity and overweight in young adults: the CARDIA study, Burke [/bib_ref] [bib_ref] The relationship of ethnicity, socioeconomic factors, and overweight in US adolescents, Gordon-Larsen [/bib_ref] Similar analogous findings are noted in cardiovascular studies where low SES exerts a stronger adverse influence on cardiovascular risk factors of women than it does on those of men [bib_ref] Social gradients in cardiovascular risk factors and symptoms of Swedish men and..., Manhem [/bib_ref] [bib_ref] A multilevel analysis of income inequality and cardiovascular disease risk factors, Diez-Rouxa [/bib_ref] and in diabetic studies where a negative association between SES and prevalence of diabetes was found only among women. [bib_ref] Sex differences in the associations of socioeconomic status with undiagnosed diabetes mellitus..., Rathmann [/bib_ref] [bib_ref] Gender-related differences in the association between socioeconomic status and self-reported diabetes, Tang [/bib_ref] Not all studies have consistently shown these gender patterns. In the 1996 Health Survey in England, higher educational attainment was associated with a lower risk of obesity/overweight in both men and women, although higher occupational status was associated with a lower risk only among women. [bib_ref] Sex differences in the association of socioeconomic status with obesity, Wardle [/bib_ref] An inverse association between obesity and education was found only in men in Finland. [bib_ref] Determinants of weight gain and overweight in adult Finns, Rissanen [/bib_ref] Because of the cross-sectional nature of the survey, it is difficult to determine whether there is a temporal relationship between SES and overweight/obesity. We do not have information on physical activity or other relevant lifestyle factors which could confound the observed association. Also the lack of information on household income, marital status, or the size of the family would be a potential limitation, because SES of women would more likely to be influenced by these factors. Further, it is possible that the associations seen in our Chinese population in Singapore may differ from other ethnic groups, with dissimilar genetic and environmental exposures, different distributions of height and weight and higher rates of obesity. The strengths of our study includes the use of population based sampling strategy, the use of measured rather than self-reported heights and weights to calculate BMI, the availability of several indicators of SES, and the inclusion of potentially confounding variables in the multivariate analysis. We believe that our study sample is fairly representative of the adult Chinese population in Singapore for the following reasons: (1) Tanjong Pagar district is centrally located in Singapore and includes a representative range of social and economic backgrounds and housing types, and (2) we selected subjects using a variation of the stratified, random sampling strategy from the population-based electoral register which is regularly updated by Elections department under the Prime Minister's office and almost 100% complete.In conclusion, lower SES as defined by education, income and housing type was associated with a greater risk of overweight/obesity only in women. In men no significant associations between SES indicators and overweight/obesity were found. The observation of gender differences and SES differences in overweight/obesity warrants further research to investigate the underlying reasons and to plan appropriate public health intervention strategies. inverse association between SES variables and overweight/obesity in developed Western societies. [bib_ref] Socioeconomic status and obesity: a review of the literature, Sobal [/bib_ref] [bib_ref] The influence of socioeconomic status, ethnicity and lifestyle on body mass index..., Sundquist [/bib_ref] [bib_ref] Socioeconomic inequality of obesity in the United States: do gender, age, and..., Zhang [/bib_ref] [bib_ref] Central and total obesity in middle aged men and women in relation..., Langenberg [/bib_ref] [bib_ref] Social status and health risks in Canadian adults: 1985 and 1991, Millar [/bib_ref] Also, as expected, these results are dissimilar to reports of a direct, positive association between SES and overweight/obesity in developing countries from Asia. [bib_ref] Body mass index and its association with socioeconomic and behavioral variables among..., Reddy [/bib_ref] [bib_ref] Socio-economic status and risk factors for cardiovascular disease: a multicentre collaborative study..., Anonymous [/bib_ref] Previous studies in adults show that SES is one of the most consistent correlates of body weight. 2,7,38-40 SES factors such as education, income, and closely related occupation are related to variations in behaviors which change energy consumption, energy expenditure and metabolism. [bib_ref] Obesity and socioeconomic status: a framework for examining relationships between physical and..., Sobal [/bib_ref] Education enables people to integrate healthy behaviors (e.g., specific dietary patterns, lack of exercise), into a coherent lifestyle, gives them a sense of control over their health [bib_ref] Education, personal control, lifestyle and health -A human capital hypothesis, Mirowsky [/bib_ref] and limits exposure to negative influences associated with the social and physical environment in which one lives and works. [bib_ref] Social class disparities in risk factors for disease: eight-year prevalence patterns by..., Winkleby [/bib_ref] Income may reflect access to medical care resources, good housing and working conditions, and provides opportunities for healthy lifestyles.Also higher SES is positively associated with weight control behaviors such as physical activity, access to healthy foods, and less time spent watching television. 2,45-47 Based on our findings, a corollary observation is that the previously reported association between low SES and mortality 48 may be explained, at least in part, by the association between low SES and overweight/ obesity. In the current study, we found that an inverse association between SES factors and overweight/obesity was present only in women, but not in men. These are consistent with previous studies on gender differences in the risk of overweight/obesity and SES classes. [bib_ref] Sex differences in the association of socioeconomic status with obesity, Wardle [/bib_ref] [bib_ref] Socioeconomic inequality of obesity in the United States: do gender, age, and..., Zhang [/bib_ref] [bib_ref] Socioeconomic status in relation to obesity and abdominal obesity in Korean adults:..., Yoon [/bib_ref] [bib_ref] Central and total obesity in middle aged men and women in relation..., Langenberg [/bib_ref] [bib_ref] Gender and race differences in the correlation between body mass and education..., Leigh [/bib_ref] [bib_ref] Sex differences in the associations of socioeconomic status with undiagnosed diabetes mellitus..., Rathmann [/bib_ref] In a prospective birth cohort study, adult social classes were inversely related to overweight/obesity among women, but not among men. [bib_ref] Central and total obesity in middle aged men and women in relation..., Langenberg [/bib_ref] In the US Third National Health and Nutrition Examination Survey (NHANES III), 1988-1994, a stronger, inverse association between SES and obesity was observed in women compared with men. [bib_ref] Socioeconomic inequality of obesity in the United States: do gender, age, and..., Zhang [/bib_ref] The KORA survey 2000 conducted in Germany, found a stronger inverse association between BMI and SES indicators including education, income, and occupational status in women. In men, these associations were weaker or absent. [bib_ref] Sex differences in the associations of socioeconomic status with undiagnosed diabetes mellitus..., Rathmann [/bib_ref] In the 1998 Korean National Health and Nutrition Examination Survey, education was inversely associated with obesity in women, but not in men. [bib_ref] Socioeconomic status in relation to obesity and abdominal obesity in Korean adults:..., Yoon [/bib_ref] The observed gender differences in the relation between SES and overweight/obesity may have a number of plausible explanations. In developed/industrialized societies, men and women may have different attitudes towards body weight and have different practices for controlling body weight. [bib_ref] Socioeconomic status and weight control practices among 20-to 45-year-old women, Jeffery [/bib_ref] [bib_ref] Socioeconomic status and weight control practices in British adults, Wardle [/bib_ref] [bib_ref] Gender differences in food choice: the contribution of health beliefs and dieting, Wardle [/bib_ref] In most Western societies, women hold a more negative attitude towards overweight/obesity than men. [bib_ref] Socioeconomic status and weight control practices in British adults, Wardle [/bib_ref] Social pressure for slimness is stronger among women than men, particularly among high SES women, 2 with associated dieting behavior [bib_ref] Socioeconomic status and weight control practices among 20-to 45-year-old women, Jeffery [/bib_ref] and with more physical activity. [bib_ref] Physical activity behaviors in lower and higher socioeconomic status populations, Ford [/bib_ref] Furthermore, the absence of association between low SES and overweight/obesity in men could be explained by the fact that low SES men were more likely to have physically demanding occupations, which reduced the risk of obesity, than high SES men. Also, the association of smoking with a lower [table] Table 3: Odds ratios (ORs) and their 95% confidence intervals (Cis) for the prevalence of overweight/obesity in relation to socioeconomic status (SES) in men and women. [/table]
Hybrid approach to fabrication of hollow internally weighted mandibular denture: A case report Preservation of ridge dimensions is critical for denture success. For long the concept of an internally weighted denture, which suggested that gravity and the additional weight to the mandibular complete denture aids in prosthetic retention is widely accepted. However, excessive weight and pressure can accelerate bone resorption. Here, we describe a unique modification of internally weighted metal denture base for the resorbed mandibular ridge with an incorporated additional hollow section over the anterior knife-edge ridge. The weight provided retention and stability while the hollow portion prevented further resorption of the bone. Figure 4: Weighted metal denture base with hollow anterior segment # Introduction The stability and retention of mandibular denture are difficult due to extreme resorption and reduced denture bearing area. Although weighted denture contributes to retention and stability, [bib_ref] Gold base lower dentures, Grunewald [/bib_ref] decreasing the lower denture weight prevents leverages, resorption and overtaxing of the remaining supporting structures. [bib_ref] Responses of jawbone to pressure, Carlsson [/bib_ref] [bib_ref] Some clinical factors related to rate of resorptionof residual ridges, Atwood [/bib_ref] [bib_ref] An experimental study on histopathological changes in the tissue covered with denture..., Nakashima [/bib_ref] Here we described an innovative technique to fabricate a hybrid prosthesis for resorbed mandibular ridge with the anterior knife edge section. ## Review of literature The weighted denture should have the same weight as the missing tissue for optimal clinical outcomes, closest possible adaptation, resistance to lateral deformation, less breakage, and minimal tissue changes. Moreover, a metal base helps prevents bacterial fermentation due to its less porosity and better thermal conduction. Most lower dentures weigh less than half as much as the teeth and supporting tissues lost to promote prosthetic retention. [bib_ref] Gold base lower dentures, Grunewald [/bib_ref] However several studies [ [fig_ref] Table 1: Studies concluding that weight and pressure on the ridge causes RRR [/fig_ref] ] recommended addition of extra weight and added pressure for residual ridge resorption (RRR). [bib_ref] An experimental study on histopathological changes in the tissue covered with denture..., Nakashima [/bib_ref] [bib_ref] The continuing reduction of the residual alveolar ridges in complete denture wearers:..., Tallgren [/bib_ref] [bib_ref] Internal and external changes in the edentulous mandible, Yung [/bib_ref] [bib_ref] Oral status and prosthetic factors related to residual ridge resorption in elderly..., Xie [/bib_ref] [bib_ref] Post-extraction remodeling of the adult mandible, Kingsmill [/bib_ref] [bib_ref] Effect of weight change of mandibular complete dentures on chewing and stability:..., Ohkubo [/bib_ref] [bib_ref] Factors affecting mandibular residual ridge resorption in edentulous patients: A preliminary report, Zmyslowska [/bib_ref] [bib_ref] Clinical evaluation of mandibular ridge height in relation to aging and length..., Jagadeesh [/bib_ref] We described here a case wherein we adopted a blended approach to achieve the optimal prosthodontics outcome. ## Case report A male patient, 66 years of age, visited the Department of Prosthodontics, with the chief complaint of difficulty in mastication. On intraoral examination, the patient presented with a mandibular anteriorly high knife-edge ridge and a resorbed posterior section [ [fig_ref] Figure 1: Intra oral view of anterior knife-edge ridge with posterior resorbed section S144... [/fig_ref] ]. No history of any systemic illness and medications was reported. Following radiographic and clinical investigations a modification in the internally weighted denture was planned to relieve the ridge crest of pressure. Further the region above the Case Report anterior knife edge was planned to be made segmentally hollow to relieve it of the pressure and hence prevent the resorption while increasing the retention of the prosthesis. The ethical clearance committee of the institute approved the study, and a written consent was obtained from the patient after explaining the entire treatment plan. Primary impressions were made in impression compound (Rolex impression composition, Ashoo Sons, Delhi, India). Border molding was performed with low fusing impression compound (Dental products of India [DPI], The Bombay Burmah Trading Corporation Ltd, Mumbai, India) and final impression was made with zinc oxide eugenol impression paste (DPI, The Bombay Burmah Trading Corporation Ltd, Mumbai, India) and the impression was poured in type III dental stone (Gyprock, Rajkot, Gujarat, India). The final cast was duplicated. On the duplicated cast two sheets of modeling wax were adopted on the residual alveolar ridge (Rolex, Ashoo Sons, New Delhi, India). One sheet was then cut and removed from the area, which the metal casting would occupy, leaving the anterior part of the ridge so that the thin knife-edge ridge was relieved. This assembly was then duplicated, and a refractory cast was obtained. On the refractory cast, a spacer wax was adopted in the prepared slot for casting and retentive finger-like extensions for acrylic were incorporated. Once the casted metal framework was prepared, it was readapted over the master cast [ ]. Next a permanent record base incorporating the metal framework was fabricated (Trevalon-HI, Dentsply, Germany).The occlusal vertical dimension and the centric relation of the patient were determined with the help of tissue conditioners (GC, Coe-Comfort Professional, GC America Inc.,) neutral zone was recorded [ [fig_ref] Figure 3: Neutral zone recording using tissue conditioner Figure 2 [/fig_ref] ]. Teeth arrangement was done in balanced occlusion and try in was done. The mandibular trial denture was duplicated in type three gypsum product and a thermoplastic sheet was adapted over the external surface. This thermoplastic template was letter used to verify the extent of the hollow segment. In order to make the hollow section over the anterior knife edge area, a roll of modeling wax was adapted over the sharp ridge after dewaxing. Autopolymerizing acrylic resin Tallgren [bib_ref] The continuing reduction of the residual alveolar ridges in complete denture wearers:..., Tallgren [/bib_ref] 1972 Alveolar bone loss was 4 times more in the lower arch. The lower ridge is more likely to respond to the various functional forces transmitted through the dentures. One of the contributing factors with regard to the anterior mandibular resorption is long-term complete denture wearing with impaired retention and stability Yung [bib_ref] Internal and external changes in the edentulous mandible, Yung [/bib_ref] 1975 A localized high incidence of pressure causes circumscribed complete osteolysis and replacement of bone by fibrous tissue and greatest resorption taking place on the external surface of the knife edge residual ridge Nakashima et al. [bib_ref] An experimental study on histopathological changes in the tissue covered with denture..., Nakashima [/bib_ref] 1994 In a study using experimental dentures to load continuous pressure on the palate of the molar region of rats it was demonstrated that the low pressure (1.5 kPa) did not cause bone resorption, but higher pressure (3.4 and 4.9 kPa) caused RRR. It was concluded that osteoclastic bone resorption was a pressure thresholdregulated phenomenon with a lower threshold for continuous than for intermittent pressure Xie et al. [bib_ref] Oral status and prosthetic factors related to residual ridge resorption in elderly..., Xie [/bib_ref] 1997 The rate of resorption in the anterior alveolar ridge is most rapid during the 1 st year of denture-wearing Kingsmill [bib_ref] Post-extraction remodeling of the adult mandible, Kingsmill [/bib_ref] 1999 Prolonged pressure occludes the fine periosteal plexus of vessels, stimulating osteoclastic resorption by altering the local oxygen tension and reducing the pH Ohkubo and Hosoi [bib_ref] Effect of weight change of mandibular complete dentures on chewing and stability:..., Ohkubo [/bib_ref] 1999 Concluded that weighted dentures did not affect retention or stability. Patients were often dissatisfied due to compression of gingival tissues by the weighted mandibular denture Zmyslowska et al. [bib_ref] Factors affecting mandibular residual ridge resorption in edentulous patients: A preliminary report, Zmyslowska [/bib_ref] 2007 Continuous pressure is more harmful than intermittent pressure Jagadeesh and Patil [bib_ref] Clinical evaluation of mandibular ridge height in relation to aging and length..., Jagadeesh [/bib_ref] 2013 In RRR, there is an osteoclastic activity, especially on the external surface of the crest of residual ridges RRR: Residual ridge resorption How to cite this article: Hazari P, Mishra SK. Hybrid approach to fabrication of hollow internally weighted mandibular denture: A case report. J Nat Sc Biol Med 2015;6:S143-5. (DPI-RR Cold Cure, Mumbai, India) in dough consistency was adapted over the wax roll. Once the acrylic resin was cured, the wax roll was then removed by flushing hot water into the tunnel created by autopolymerizing resin [ ]. The thermoplastic template was used to verify the extent of the hollow segment. The hollow segment was within the confines of the template with sufficient space for heatcure acrylic resin. The two open ends of the tunnel were closed using autopolymerizing acrylic resin. Dentures were processed with high impact heat-polymerized acrylic resin (Trevalon-HI, Dentsply, Germany). The prosthesis was retrieved and polished for the denture insertion. ## Source of # Discussion Several studies have reported that the lower ridge is more likely to respond to the various functional forces transmitted through the dentures. [bib_ref] The continuing reduction of the residual alveolar ridges in complete denture wearers:..., Tallgren [/bib_ref] Constant pressure is one of the major cause for RRR. [bib_ref] Post-extraction remodeling of the adult mandible, Kingsmill [/bib_ref] Longterm complete denture wearing with impaired retention and stability further exacerbates anterior mandibular ridge resorption. [bib_ref] The continuing reduction of the residual alveolar ridges in complete denture wearers:..., Tallgren [/bib_ref] The functional parameters of force such as frequency, intensity, duration, and direction are transformed into cell activity with stresses which deviate from the normal particularly excessive compressive forces bringing about RRR, which may be because of gravitational reasons. This stress causes an area of complete osteolysis and formation of fibrous tissue on the external surface of the knife edge residual ridge. [bib_ref] Internal and external changes in the edentulous mandible, Yung [/bib_ref] [bib_ref] Factors affecting mandibular residual ridge resorption in edentulous patients: A preliminary report, Zmyslowska [/bib_ref] [bib_ref] A comparative study of the resorption of the alveolar ridges in denture-wearers..., Campbell [/bib_ref] Hence for this case we preferred a modification of the lower denture. The metal insert was given only in the posterior region of the residual ridges, which had provided the necessary retention by the action of gravity and also reduce the weight of the denture. The hollow section given over the anterior knife-edge ridge prevented overloading of the mucosa and bone, thus preventing the bone resorption in the anterior region. # Conclusion For simultaneously aiding retention preventing the important residual ridge from resorption, modified prosthesis presented in this case report was found to be an optimal alternative. In cases such as anterior knife-edge ridges, diabetic patients, were alveolar ridge is more prone to resorption, these dentures may provide good results. These dentures can also be successfully adopted in cases where processing and lateral deformations are anticipated and in patients with neuromuscular disorders since the metal incorporated provides the needed rigidity and strength. This hybrid denture successfully combined the advantages of two concepts eliminating the disadvantages of both. [fig] Figure 1: Intra oral view of anterior knife-edge ridge with posterior resorbed section S144 Journal of Natural Science, Biology and Medicine | August 2015 | Vol 6 | Supplement 1 [/fig] [fig] Figure 3: Neutral zone recording using tissue conditioner Figure 2: Modified metal casting to relieve anterior ridge of pressure S145 Journal of Natural Science, Biology and Medicine | August 2015 | Vol 6 | Supplement 1 [/fig] [table] Table 1: Studies concluding that weight and pressure on the ridge causes RRR [/table]
Microbial Experimental Evolution – a proving ground for evolutionary theory and a tool for discovery Microbial experimental evolution uses controlled laboratory populations to study the mechanisms of evolution. The molecular analysis of evolved populations enables empirical tests that can confirm the predictions of evolutionary theory, but can also lead to surprising discoveries. As with other fields in the life sciences, microbial experimental evolution has become a tool, deployed as part of the suite of techniques available to the molecular biologist. Here, I provide a review of the general findings of microbial experimental evolution, especially those relevant to molecular microbiologists that are new to the field. I also relate these results to design considerations for an evolution experiment and suggest future directions for those working at the intersection of experimental evolution and molecular biology. # Introduction Experimental studies of evolving populations now constitute one of the foundations of the theory of evolution. In particular, studies of microbial populations in the laboratory bring greater power and precision to experimental evolution studies, providing a means to carry out elaborate tests of theory and explore new ideas in evolutionary biology . A typical microbial evolution experiment starts with a culture, just like any other in a microbiology laboratory. Cells are inoculated into media and left to grow until the culture reaches a high population density. Instead of throwing out or using all of the resultant population, the experimental evolutionist transfers or dilutes the culture to allow continued growth and division. This cycle can be continued indefinitely, and as the generations accumulate, natural selection will drive the population to adapt to the laboratory environment. This simple process can be carried out in the laboratory using a range of experimental systems, summarised inLong-and short-term experimental approaches to study evolution Perhaps the most striking advantage of experiments with microbes is the access to long evolutionary time scales. The short generation times of microbes allow for up to tens of generations of evolution to pass every day. In theory, an evolution experiment is limited only by how long the experimentalist can maintain regular transfers. A microbial population is easily stored in the freezer, for an indefinite period, so populations can be saved as a frozen snapshot of evolution or used to restart the experiment when inevitable accidents happen. The longest running, and probably most famous, microbial evolution experiment is the long-term evolution experiment (LTEE). This experiment is comprised of 12 replicate populations of E. coli, started in 1987 [3] and still passaged daily over 68,000 generations later (see here for a recent review of this experiment . What can be learned from running an evolution experiment for so long? Twenty years ago, an evolutionary biologist might have predicted that these populations of E. coli would have reached optimal fitness after a few thousand generations. However, we now know that each population continues to adapt after 61,500 generations . A key discovery has been the evolution of the utilisation of citrate (cit+ phenotype), a carbon source used as a buffer in the growth media. The evolution of this phenotype is especially significant because a species-defining characteristic of E. coli is that citrate is unable to be utilised under oxidising conditions . The effect of mutations that explicitly cause the cit+ phenotype is dependent on other "potentiating" mutations that do not seem to directly influence citrate utilisation and occurred within the first 20,000 generations of the experiment . In other words, this particular trait is unlikely to have evolved in a short-term experiment. However, there are quicker routes to study many generations of evolution. An alternative to propagating a few experimental replicates for the long term is to evolve many replicate populations for a shorter period of time. As long as selection is strong, populations can adapt rapidly. Adapting E. coli to high temperatures, Tenaillon et al propagated 115 experimental populations for 2,000 generations . Increasing the number of replicates by another magnitude, Lang et al evolved 1,000 replicate populations of Saccharomyces cerevisiae for 1,000 generations . The massive replication of these studies confers the statistical power to detect evolutionary change, which may be more difficult to detect after only hundreds of generations of evolution. The discovery of the cit+ phenotype shows that there are some questions these highly replicated shortterm studies cannot address; however, there are trends emerging that are consistent across both long-and short-term experiments , reviewed below. Repeatability, diminishing returns and rapid diversification: predictable trends in experimental evolution Parallel evolution is the evolution of the same phenotypes, and sometimes the same genetic mutations, in independently evolving populations . Parallelism is often driven by natural selection and has been observed in both short-and long-term experiments across a range of species . Repeatability in evolution experiments is interesting because it suggests that the phenotypic outcomes of evolution could be predictable. To anticipate the evolutionary response to environmental changes is a major goal of evolutionary biology , and the capacity to make accurate predictions of the outcomes of evolution would be desirable. However, it is unclear whether predictions about evolution could ever be precise enough to be useful, and this is subject to ongoing studies using microbial models . At the onset of an evolution experiment, adaptation tends to be rapid and then slows down over time . In the E. coli LTEE, the rate of fitness increase follows a power law, which suggests that there is no optimal fitness that can be attained by the evolving population . The slowed rate of adaptation over time can be explained by epistatic interactions that cause the fitness effects of beneficial mutations to be lower in a better adapted population . Experiments show that beneficial mutations engineered into a lowfitness genetic background have a larger effect than if they are engineered into a high-fitness background. This "diminishing returns" epistasis has been observed using beneficial mutations from the experimental evolution of M. extorquens and S. cerevisiae [25], as well as from the E. coli LTEE . While diminishing return epistasis makes no predictions about specific phenotypic outcomes of evolution, it does allow for robust predictions to be made about the ongoing rate of adaptation in a population, although this might not be true in populations experiencing fluctuating or complex environments. Most evolution experiments use unicellular organisms adapting to defined-nutrient environments. One of the more surprising findings in evolution experiments has been the capacity for these simple experimental systems to evolve diverse, co-existing subpopulations adapted to different niches, evident in both short-and long-term studies of evolution [6,27,28]. Diverse subpopulations can evolve in response to environmental heterogeneity introduced by the experimenter, or due to a process called eco-evolutionary feedback . As evolution happens in microbial populations, the altered production of waste products or rates of consumption can cause modifications to the environment. This change in ecology alters the selective pressures experienced by individuals and can drive further evolution . The observation of eco-evolutionary feedback in evolution experiments emphasises its importance in real microbial communities and suggests one mechanism that could drive the continuous evolution observed in long-term evolution experiments. Experiments with microbes facilitate precise control over fundamental parameters of evolution: environment, population size and mutation Understanding, and manipulating, evolution in natural populations is difficult due to the large number of factors that can influence the outcomes of evolution. A major benefit of working with laboratory populations of microbes is the control that can be exerted over the key parameters of evolution: the environment, population size, mutation rate and founding genotype [2]. The environment determines the selective pressures experienced by an evolving population and therefore drives the genetic and phenotypic outcomes of evolution. The capacity to maintain many controlled experimental replicates while manipulating a single variable makes possible the observation of potentially subtle effects. The fundamental importance of the environment for interpreting and setting up evolution experiments is discussed below. Population size (N) determines the strength of the selective forces experienced by the population. The minimum effect size of a mutation that can be detected by natural selection, expressed as a selection coefficient (s), is 1/N, where "N" is the population size, so that selection is ineffective when Ns < 1 . A small population is more likely to experience fluctuations in allele frequency due to genetic drift, the random sampling of allele frequencies across generations. This can lead to the chance fixation of deleterious mutations or loss of beneficial mutations. As a consequence of Glossary clonal interference slowed rates of fixation in an asexual population due to competition between lineages that each carry a beneficial mutation coverage the length of concatenated DNA-sequence read data divided by genome length de novo mutation a mutation that occurs spontaneously during a period of evolution fixed the state at which an allele for a given genetic locus is at a frequency of 1 in a population genetic barcode a short DNA sequence that is used to identify an individual or lineage haplotype the set of genetic variants physically linked on a single chromosome HGT horizontal gene transfer lineage a set of individuals that share a common ancestor within a given time period LN natural log LTEE long-term evolution experiment N population size parallel evolution the evolution of similar phenotypes and genotypes in independently evolving populations selection coefficient(s) a quantitative representation of relative fitness or reproductive success standing genetic variation genetic variation that is present in a natural or laboratory population before the period of evolution considered by the observer genetic drift, small populations can expect slower rates of adaptation and, in extreme cases, population extinction . Some experiments are designed to explore the consequences of variation in population size , and may deliberately bottleneck the population to 1-10 individuals. If the goal of the experiment is to avoid genetic drift, a dilution rate that does not bottleneck the population to < 10 3 -10 4 individuals is recommended. Variation in the mutation rate allows the experimenter to vary how much genetic variation, the "fuel" of evolution, is supplied to the population [37]. The rate of evolution is proportional to the amount of genetic variation in the population ## D emulsion ## Fast but inefficient strain ## After growth ## Slow but efficient strain In emulsion after inoculation Experimental evolution of antimicrobial resistance The evolution of antibiotic resistance is a global health challenge that, like experimental evolution, sits at the intersection of the disciplines of evolutionary biology, microbiology, molecular biology and genomics . Evolution experiments can be used to measure the fitness costs of the mutations that underlie antibiotic resistance, and the rate and probability of the evolution of resistance. Mutations that cause antibiotic resistance often occur in genes for important biological functions and are therefore expected to cause a reduction in growth rate or viability. Fitness assays (see Box 1: How to measure fitness) have shown that the effects of the mutations that confer antibiotic resistance are actually highly variable, and do not always come at a cost. When resistance mutations are costly, a resistant microbe can adapt by secondary mutations that compensate for the effects of primary resistance mutations. Since antibiotic resistance is a consequence of evolutionary processes, strategies for the amelioration of antibiotic resistance, especially resistance to multiple drugs, should take evolution into account. One promising line of research is to characterise the fitness effects and antibiotic susceptibilities of multidrug-resistant strains. In order to attain resistance to multiple drugs, multidrugresistant strains are likely to have evolved multiple primary resistance mutations as well as several compensatory mutations. It is possible that some multidrug-resistant strains will be less able to evolve resistance to an additional antibiotic. Knowledge of the susceptibilities that evolve with multidrug resistance could facilitate the targeted use of drug combinations based on the genotype of clinical pathogenic strains. ## Experimental evolution of bacteriophages Bacteriophages are another source of antimicrobial therapies, and experiments with bacteriophage provided some of the first insights into the genetics of adaptation in evolving laboratory populations. Bacteriophage genomes are small, and whole-genome sequencing of experimental phage populations was possible before the rise of next-generation sequencing technologies. This head start was exploited to identify the mutations that evolved in phage evolution experiments and to measure the fitness effects and epistatic interactions of these mutations . The relative ease of propagating bacteriophage and bacteria in co-culture has also led to insights into coevolutionary dynamics. For instance, bacteria that are propagated with an infecting phage experience more rapid molecular evolution than bacteria evolved in isolation. If the bacteria are propagated with diverse bacteriophage, the rate of evolution increases with the number of phage types present in the culture. Bacteriophages typically bind to a membrane protein to gain entry to the cell. Experimental evolution has facilitated the detailed molecular analysis of the mechanism by which phage k can evolve to bind a new site on the E. coli membrane. Conversely, bacterial resistance to a bacteriophage can evolve by modifying or deleting the gene that encodes that protein. Since antibiotic resistance is sometimes conferred by multidrug efflux pumps, it has been hypothesised that a phage that targets such a pump could be given in tandem with the antibiotic and thus comprise an "evolution proof" treatment strategy. This principle was demonstrated with a phage selection experiment, which drove the selective loss of the MEX efflux pump, thereby restoring antibiotic sensitivity to a multidrug-resistant strain of P. aeruginosa. ## Experimental evolution as a tool Microbial evolution experiments are often designed with the goal of providing insights into evolutionary theory, or the evolution of a particular trait. However, experimental evolution is also a tool that can be used to evolve organisms for specific applications. The introduction of genetic modifications designed to confer useful properties often results in slowed growth or other reductions in performance. In yeast, engineered strains can be crossed with "wild-type" strains and then passaged to promote recombinants that possess the engineered features as well as the productivity of fast-growing strains. Continuous passaging has been used widely to restore growth rates in S. cerevisiae engineered for the production of ethanol and the consumption of xylose. In a recent example, the engineered E. coli strain C321 has been modified to replace all UAG codons with UAA. This strain provides the ideal genetic background for a range of biotechnological applications, such as the incorporation of codons for non-standard amino acids into the genetic code. However, the engineering of this strain caused slow growth. A 1,000-generation evolution experiment propagating this strain in the laboratory resulted in the evolution of restored high growth rates. Moreover, whole-genome re-sequencing of the evolved populations revealed the mutational targets of selection and therefore the causes of reduced growth rate in the founder strain. ◀. Mechanisms of propagation for experimental evolution. (A) Batch culture requires the regular dilution of culture into fresh media. These experiments are relatively easy to establish, since a range of vessels commonly used in a microbiology laboratory can be used for batch culture. These experiments can be scaled to a large number of replicates, for example when using 96-well plates. (B) Chemostat culture systems include mechanisms for the constant supply of fresh medium. This provides for the continuous cultures of populations and constant growth without large fluctuations in populations size or growth phase. (C) Microfluidics provides the most precise control over the supply of media and supplements to cell cultures. Microfluidics may need to be custom designed, and the number of replicates will be limited. (D) Emulsion cultures take advantage of small cellcontaining vesicles that form when mixing an oil, surfactant and cells. The number of cells in each vesicle is determined by the ratio cell, surfactant and oil. The cells can be mixed back into a single population by vortexing and centrifuging the solution. One advantage of evolving cells in a large number of small populations is that this can select for yield per-vesicle rather than rapid growth. (E) Mutation accumulation introduces a regular, single-cell bottleneck into each replicate population. This achieved by streaking out cells on a petri dish and then choosing a single colony (founded by a single cell) to streak out the next plate. (F) Microbial cultures can be introduced into a model organism, often a plant or a mouse, and left to propagate for a number of generations before it is recovered from the organism. The recovered cells can be analysed or subjected to further propagation in the organism. This mode of experimental evolution allows for the testing of unanticipated organism-specific features of the environment that are difficult to replicate in the laboratory. Experimental evolution can be used to adapt microbes to novel hosts, as well as novel laboratory conditions. Some species of Wolbachia bacteria spread quickly among their hosts by conferring a reproductive advantage to infected females. In addition, some Wolbachia strains are able to induce resistance to insect pathogens. A strategy was devised to spread resistance to the Dengue virus amongst mosquitos using a strain of Wolbachia originally discovered in D. melanogaster. However, this strain was not suited to rapid growth and dispersal in mosquito populations. In order to adapt this Wolbachia strain to grow in the A. aegypti mosquito's intracellular environment, it was passaged in a mosquito cell line for 2 years. After this period, the newly evolved Wolbachia strain was able to establish a stable infection in mosquitosand thereafter facilitate the eventual public dispersal of dengue resistance mosquitos in Australia. Next, I introduce important considerations for the design of evolution experiments by describing some key results in experimental evolution. This review does not provide a full historical treatment of experimental evolution. I recommend these books and these reviewsfor exhaustive treatments of earlier periods and non-microbial experimental evolution, and these reviewsfor different aspects of experimental evolution. You get what you select for: environment and the outcomes of experimental evolution One of the most important choices when starting a laboratory evolution experiment is the environment. Setting conditions beyond what is normally experienced by an organism will drive adaptation. Adaptation to a range of conditions has been described, including elevated temperatures [9,82], antibiotic gradientsand even high levels of ionising radiation. The environmental parameters that can be used to drive selection are limited only by the imagination. As long as the chosen environmental parameter provides a selection pressure that drives the differential survival of individuals in the population, adaptation will happen. While experimental populations can be relied upon to adapt regardless of the selective pressure, the types of adaptations that evolve can be difficult to predict. Wildenberg et alused a fluorescence-activated cell sorter to select for the brightest fluorescent S. cerevisiae cells every 24 h. One anticipated outcome was that selection would drive the evolution of gene expression in order to modulate fluorescence. Instead, the population evolved to periodically form multicellular clusters that increased brightness and thereby conferred a selective advantage. This unpredicted outcome did not diminish the elegance of this experiment. However, it serves to demonstrate how unpredictability can thwart experiments that are designed to have specific outcomes. In general, the more complicated the selection regime, or subtle the strength of selection, the more unpredictable the outcomes of evolution. It should be noted that a complicated, but well-designed, experiment can still elicit the anticipated response to selection. One experiment sought to evolve multicellular traits by selecting for or against germ progenitor cells in cooperative mats of the bacteria P. fluorescens. Although the genetic mechanisms of evolution were unexpected, the experiment successfully applied selection pressures that led to the evolution of multicellular traits. Simple environments can drive the evolution of loss of function Natural environments expose microorganisms to a range of nutrients and stresses that vary across spatial and temporal scales. The complexity of natural environments is reflected in the large numbers of genes that organisms have evolved to utilise diverse nutrients and respond to stress. Laboratory experimental populations experience environments that are typically much less complex, and will adapt by mutations that inactivate genes that have become superfluous in the conditions of the experiment . Many evolution experiments are carried out in growth media that contain a single carbon source, usually glucose. In the LTEE, glucose is supplied as the sole carbon source in a concentration that limits population growth . In an evolution experiment, the regular supply of glucose every 24 h can lead to the evolution of a reduction in the "lag time", the time required for the population to enter the log growth phase. In the LTEE, this is achieved via mutations in pykF, which became fixed in every population within the first 2,000 generations of the experiment . Adaptation for specialisation on a single carbon source can come at the cost of growth on other carbon sources. Studies of the LTEE after 2,000 generations of evolution showed that the rbs operon, which encodes proteins required for the utilisation of ribose, has been disrupted or deleted in all 12 replicate populations. Measurements of the selective benefit of rbs loss using competitive fitness assays determined the fitness gain to be~1%. Since then, whole-genome sequencing has revealed the disruption of other genes, including genes for the utilisation of carbon sources, such as maltose, that may be superfluous in the minimal media environment. Other evolution experiments have propagated the yeast S. cerevisiae in media containing high concentrations of glucose, in a range of culture conditions. Genes that evolve beneficial mutations in multiple replicate populations during adaptation to high glucose concentrations in batch culture have been shown to be targets for selection across different experiments . Whole-genome sequencing of evolved populations has revealed that over half of the replicate populations adapt by mutations that disrupt genes that encode negative regulators of the RAS/PKA pathway . These mutations increase RAS/PKA pathway activity and result in the rapid utilisation of glucose, even in experimental cultures carried out in a range of glucose concentrations. Mutations in RAS/PKA pathway genes have also been discovered in chemostat experiments with glucose-limiting concentrations. Interestingly, in these experiments the most frequently recovered mutations increase the amount of glucose transport. Since glucose is the limiting nutrient in these experiments, increasing transport of Box 1: How to measure fitness Fitness is a quantitative measure of the capacity of an organism to contribute offspring to the next generation. Fitness assays are carried out to determine the degree of adaptation of a population after experimental evolution and to validate the fitness effect of specific mutations. Fitness can be experimentally determined by a wide range of assays. Growth rates, total carrying capacity, biomassand speed of colony boundary expansionhave all been used as measures of fitness in evolution experiments. The gold standard for fitness measurement in the laboratory is competitive fitness assays. The starting point for a fitness assay is to obtain or construct a marked reference strain. This is typically the ancestor of the evolution experiment, modified to be readily distinguished from the evolved strain. The nature of the genetic marker can influence the accuracy of the experiment. For instance, if a fluorescent marker is used to differentiate the ancestor from an evolved strain, the proportions of each genotype can be measured using flow cytometry [10] and 10s of thousands of cells can be counted in order to measure ratios. Alternatively, the mixture can be spread onto agar plates containing supplements that provide for the distinction of genotypes, and this allows for the counting of hundreds of cells. Initially, each strain to be measured should be mixed with the marked reference strain in a 1:1 ratio. Even if care has been taken to mix the competitors in a 1:1 ratio, it is very important to measure the initial starting frequency, since small difference in this ratio can have a large effect on the calculations for fitness. Once a portion of the mixture has been taken aside to measure the starting ratio, the mixture of competing cells is diluted and incubated for a set period of time, allowing for the two genotypes to compete. After this period of competition, the proportions of the genotypes are measured again. The selection coefficient can be calculated from these two measurements by counting number of evolved individuals and dividing by the number of reference individuals. This is done for the initial time point and the final time point. The final ratio is divided by initial ratio, and the natural log (LN) of the quotient gives a measure of the performance of the evolved strain, compared with the reference strain. This value is divided by the number of generations that passed between the final and initial time points, yielding a per-generation selection coefficient (s). The period of competition between these measurements must be chosen carefully. If left too long, then one genotype will drive the other to extinction, thus reducing the precision of the calculation of s. If the competition is too short, then the genotype frequencies will not have changed enough to allow the detection of differences between the genotypes. glucose into the cell may be a rapid path to adaptation. However, the frequent recovery of mutations in genes that increase activity of the RAS/PKA pathway in experiments with high and low glucose, and both batch and chemostat culture, suggests that glucose may be the selective force driving the recurrent evolution of these mutations. Evolution experiments have shown that genes required for functions beyond metabolism are also targets for loss-of-function mutations. After 1,500 generations, replicate populations of M. extorquens were found to have sustained large deletions of up to 10% of their genome, covering a wide range of gene functions. S. cerevisiae evolution experiments often employ strains that have been genetically altered to reduce the probability that they can mate. Propagation without sexual reproduction can cause selection for mutations that interrupt the genes that encode components of the mating pathway . Careful measurements have shown the fitness gain derived from eliminating the expression of an unnecessary gene. The precise cost of expression of one gene in the mating pathway was determined to be approximately 1%, demonstrating that the expression of un-needed genes is a costly trait that can be targeted by selection in evolution experiments. The selective benefit of loss-of-function mutations drives their fixation. However, genetic target size for this class of mutations is another factor that makes these genes more likely to contribute towards adaptation. Any frameshift or change in a key amino acid can result in a non-functional protein. Gain-of-function mutations require modification of certain amino acids that will increase the activity or function of that protein . Any given mutation that occurs in a gene is therefore more likely to cause a loss than a gain in function. This trend of evolution by loss of function has been borne out in bacterial evolution studies , and studies of haploid S. cerevisiae [11,. However, the few studies that have studied the evolution of haploid and diploid S. cerevisiae in similar environments have found different molecular patterns of adaptation. This may be because the inactivation of one of two gene copies in a diploid is less likely to cause a phenotypic change. It should be noted that relatively few mutations that occur in evolution experiments have been characterised and that mutations that occur across multiple experiments are more likely to be studied. The striking difference between molecular adaptation in diploid and haploid S. cerevisiae suggests that small differences in experimental conditions can lead to large differences in the outcomes of evolution and that the lessons learned in one experiment should only be tentatively applied to other experimental systems. ## Spatial structure selects for diversification Many microbiology protocols specify that cultures are well-shaken and aerated. In experimental populations, this generates a homogenous distribution of nutrients and oxygen and promotes a uniform selection pressure through the microcosm. When cultures are incubated statically, without shaking, new environmental niches become available on surfaces and across nutrient gradients, and the outcomes of evolution can be quite different. In 1,000 experimental populations of S. cerevisiae, 10% of replicates were found to have evolved stable, co-existing subpopulations, one that can attach to the wall of the growth chamber, and another that grows at the bottom [10]. Whole-genome sequencing and genetic reconstructions revealed that this wall-attachment adaptation was repeatedly conferred by mutations that disrupted ergosterol biosynthesis . The most comprehensive exploration of evolution in static microcosms has been carried out using P. fluorescens . In an adaptive radiation that reliably unfolds over 7 days, a planktonic ancestor diversifies into genetically distinct lineages. The best studied of these is the "wrinkly spreader", which adapts by forming a mat of stuck-together cells that float on the broth surface and attach to the glass walls of the microcosm. This adaptive strategy provides access to oxygen, a limiting nutrient in a non-shaken broth, and to nutrients in the liquid phase. The mutations that cause the wrinkly spreader phenotype modify expression of a secondary messenger molecule, c-di-GMP, causing the constitutive expression of cellulose. Even though there are over 25 c-di-GMP-producing enzymes in the P. fluorescens genome, only three of these are ever mutated during wrinkly spreader evolution . If all three of the operons encoding these enzymes are removed from the genome, then this triple deletion mutant can evolve the wrinkly spreader phenotype by mutations in some of the other genes that encode cdi-GMP proteins . Parallel evolution is commonly observed in natural and experimental populations, and it has long been hypothesised to be due to the organisation and content of the genome as well as natural selection . The molecular genetic analysis of the trait combined with a delete-and-evolve strategy provided an early demonstration of the genetic constraints on evolutionary outcomes. The technique of static incubation in the presence of a surface has been used to explore adaptation to surface attachment in other species, such as the pathogens B. cenocepacia [18], P. aeruginosa, S. typhimuriumand V. cholerae. A clever study of attachment and biofilm evolution was carried out by the Cooper laboratory . A plastic bead coated with a B. cenocepacia biofilm was incubated in media containing another bead. After 24 h of incubation, this second bead was removed and used to found a new culture. This was continued for 143 days (~1,500 generations)and drove the evolution of the rapid and robust biofilm colonisation of the second bead by the inoculating bead. The authors found that three types would reliably evolve, forming a biofilm community of increased productivity relative to a biofilm formed by any one type. Interestingly, the variants that evolved in this experiment also carried causal mutations in c-di-GMP-regulating enzymes, similar to the genetic causes of adaptation in the P. fluorescens "wrinkly spreader" experiments described above. This experiment highlights the potential for experimental evolution to advance the understanding of bacterial attachment and biofilm formation, which is associated with pathogenesis and antibiotic resistance in a range of species. Microfluidics provides a system for the growth and continuous propagation of experimental populations. Since the flow rate of media can be carefully controlled, cells can attach to surfaces, while allowing for some population turnover and the constant provision of nutrients. Microfluidic systems have been used to demonstrate the increased productivity of a simple engineered community of P. putida and Acinetobacter sp.. Despite interspecies interactions frequently occurring in naturally occurring microbial communities, establishing a stable, long-term co-culture can be difficult in batch or chemostat experimental systems. The capacity to engineer and control the space where microbes interact can facilitate co-culture experiments and has been used to systematically screen the potential for multiple strains of P. aeruginosa to coestablish biofilms. Microfluidic devices have been underutilised in experimental evolution. One reason may be the perception that specialist knowledge is required to design and construct microfluidic systems and to propagate many replicate populations in parallel. Currently, the design of scalable experimental systems is making microfluidics more accessible. Attachment can also occur on biological surfaces. The gut-on-chip systems developed for culture of mammalian cells can support the propagation of bacteriophage. Similar systems could be exploited to study the evolution of microbial colonisation on biological surfaces. ## High-throughput methods for identifying and tracking beneficial mutations in evolving populations Whole-genome re-sequencing of replicate evolved populations is now a routine part of experimental evolution. The gold standard for assembling and analysing whole genome short-read data from microbial evolution experiments is breseq, a set of tools developed in the Barrick Lab. An important consideration before undertaking a genome sequencing experiment is the method of sampling the population. One approach is to sequence individual clones sampled from evolved populations. In experiments where multiple clones from a single population have been sequenced, it has been found that, as well as mutations that have fixed in the population, the clone will contain mutations that are unique to that clone, also called private mutations. The conclusions that one may draw from the sequencing of a single clone from an evolved population are quite limited since it will be impossible to tell which mutations are rare, and which are high-frequency mutations that are more likely to have contributed to adaptation. One way to obtain detailed information about the frequency of each mutation within the population is to carry out whole-population sequencing . The lower bound of allele frequencies that can be detected using whole-population sequencing is determined by the average sequencing depth or coverage. The theoretical minimum frequency that can be detected is the inverse of coverage (C) of the genome. For example, an experiment that obtains whole-population whole-genome coverage of 100-fold depth will not be able to detect mutations with a frequency of < 1%. In reality, due to variations in coverage and the risk of false positives, a conservative approach in this case would be to reject as spurious mutations that do not exceed a frequency of 10% in the sequence data . The whole-genome whole-population sequencing approach has several weaknesses. The first is the inability to determine which mutations are physically linked on chromosomes (haplotypes). This can be resolved by supplementing whole-population sequencing with the sequencing of clones. It can be also difficult to detect structural rearrangements, large indels and changes in ploidy, although variations in read depth can provide some information for high coverage data. The only way to unambiguously resolve these types of mutations is to incorporate longread sequencing data into the genome assembly. There are now tools available for combining long-and short-read data to assemble closed genomes. ## Identifying and validating beneficial mutations Before next-generation sequencing, it was difficult to discover mutational changes in experimentally evolved populations. Now, the challenge is to determine which of the many mutations revealed by sequencing are actually the cause of adaptation, a problem that has also emerged with the sequencing of tumour genomes. If multiple populations have been sequenced, the repeated observation of mutations in the same gene (parallel evolution), across independent replicate populations, can indicate the action of natural selection . Statistical tests comparing the observed number of parallel mutations to a null model can be used to determine a conservative cut-off. This null model should take into account gene size, since large genes are more likely to be mutated during an experiment. It should be noted that the absence of parallel evolution is not evidence for the absence of natural selection. These strategies can identify candidate beneficial mutations; however, validation requires that the mutation is either engineered into the ancestral genome or replaced by the ancestral sequence in the evolved strain. While CRISPR-cas9-based technologies are making genetic reconstruction realistic for a growing number of experimental models, it is difficult to engineer and measure more than 10s of individual mutations in a single experiment. Experimental systems, such as S. cerevisiae, that allow mating or recombination, can overcome this limitation. Haploid evolved clones that carry several mutations can be crossed with the haploid ancestor strain of the opposite mating type. The resulting diploid can be sporulated, generating millions of recombinant haploid offspring, with every different combination of the mutations. This pool of mutants is purged of one mating type, to prevent mating of the recombinants and then propagated for a period of 100 generations. The population is then sequenced at multiple time points. This technique is dependent on the capacity for recombination, and so can only be adapted to other sexual eukaryotes or potentially, naturally competent bacterial systems. ## Clonal interference and recombination impact evolutionary outcomes Whole-population (metagenome) sequencing across different time points of an evolution experiment makes it possible to track the dynamics of individual mutations that arise and segregate in an evolving population . Studies that employ this technique provide a direct view of the trajectories of mutations and how they interact during adaptation. A phenomenon that has been explicated by these studies is clonal interference. Clonal subpopulations arise and compete in experimental populations that contain multiple beneficial mutations. If beneficial mutations arise on different genetic backgrounds, and recombination cannot bring them together, these beneficial mutations will compete. Since the result of clonal interference is one beneficial lineage outcompeting another, some beneficial mutations are driven extinct in the population . Clonal interference thus slows population adaptation. The tracking of mutations through time provides an opportunity to study sex and recombination. Sexual recombination or horizontal gene exchange is common in natural populations of microbes. However, the direct observation of the recombination of mutations during adaptation in laboratory populations of microbes is difficult. One of the main reasons for this is that many model laboratory organisms, such as E. coli, S. cerevisiae or Pseudomonas species, do not undergo recombination or HGT under commonly used experimental conditions. These challenges can be overcome by engineering strains of S. cerevisiae and E. colithat are capable of repeated bouts of sex, or conjugative gene exchange, in an experimental evolution setting. Sex can accelerate adaptation in laboratory experimental populations Saccharomyces cerevisiae, with two mating types and the capacity to reproduce both sexually and asexually, provides an ideal experimental system for the study of sexual recombination. This has long been recognised, and the effects of recombination on the rate of adaptation have been investigated in some depth . A recent study has added to this by providing details of the genetics of adaptation in recombining populations of S. cerevisiae. In control populations that did not undergo recombination, significantly deleterious mutations were able to fix. This is possible because, as long as the cumulative effect of the mutations in any given genetic background has an overall beneficial effect, a strongly beneficial mutation can mask the effect of a deleterious mutation. In populations that were able to recombine the genomes of individuals, these deleterious mutations were decoupled from the beneficial mutations and purged from the population. Another potential benefit of recombination is the resolution of clonal interference . In populations that do not have recombination, different beneficial mutations were unable to be brought together onto the same genetic background. However, sexual populations were able to fix all of the beneficial mutations that were measured. Experimental evolution of HGT in bacteria has been more difficult to study; however, recombination has been incorporated into E. coli experiments using conjugation systems. One of these studies confirmed results in S. cerevisiae that incorporating recombination into an experimental system will speed up adaptation. The benefits of recombination depend on the presence of multiple mutations concurrently segregating in the population. Interestingly, an experiment showed that combining high mutation rates with recombination could further increase the rate of adaptation. Amplicon sequencing facilitates the tracking of a multitude of lineages Whole-population sequencing provides for the detection of any mutations that have occurred across the genome; however, this comes at the cost of sequencing depth. If a population is sequenced to 100-fold coverage, in theory, only mutations that are present in at least 1% of individuals can be detected. Practically, wholepopulation sequencing projects can reliably track mutations that exceed a frequency of 5-10% at multiple time points. This means that within a population of 10 7 individuals, mutations that attain a sub-population size of < 100,000 individuals will not be discovered. One way to circumvent this limit is to sequence a much smaller part of the genome that has been modified to be highly variable (bar-coded). By sequencing only this portion of the genome to ultrahigh depth (10 5 -10 6 coverage), many lineages can be tracked. An experiment pioneering this approach has been carried out in a S. cerevisiae. Amplicon sequencing at several time points in large populations in batch culture facilitated the tracking of 500,000 individual lineages for 160 generations of evolution. Estimations of fitness effects of the mutations that defined each of these lineages revealed the large amounts of beneficial genetic variation that lie hidden in large populations, and provided the most comprehensive picture of the distribution of the fitness effects of new mutationsand their dynamics. One limit of this technique is that mutations can only be tracked in the short term. As soon as one mutation has fixed in the population, then one version of the barcode also fixes, sweeping out all other barcode variants. Transposon mutagenesis is a classic technique in bacterial genetics for the functional characterisation of a genome. Lineage tracking by amplicon sequencing can be used in combination with transposon mutagenesis to discover novel gene functions. Selection is applied to the library by continuously passaging the population in defined growth conditions. By sequencing at different time points, the proportions of all mutations both before and after selection can be compared, and precise fitness coefficients calculated. Alternatively if the selection is applied over longer terms, the presence and absence of certain mutants after selection can be recorded. This technique has been used effectively to systematically identify the genes important for antibiotic resistance in a range of bacteria. ## Future outlook Experimental evolution of model microbiomes Experimental evolution of microbes has provided a detailed picture of the molecular details of adaptation. The most comprehensive understanding has come from the simplest evolution models, typically haploid populations of non-recombining microbes, evolving in isolation from other species. On the other hand, the culture-free, sequence-based characterisation of microbiomes has revealed that natural and clinical populations are more likely to evolve as part of Time (generation) a complex community of microbes and to engage in horizontal gene transfer. A new challenge for experimental studies is to understand how evolution happens in these communities and whether the "rules" of evolution discovered so far hold true in these systems. So far, experimental co-evolution has mainly been explored using bacteria and phage as models of predator-prey interactionsor using naturally interacting sets of uncharacterised bacteria. There is potential to undertake co-evolution experiments with well-characterised prokaryotic and eukaryotic microbes to study the genetic basis of a wider range of co-evolutionary interactions (see Box 2: In need of answers). Recent studies have shown that recombination can increase the power of natural selection. This insight has been applied in the field of directed evolution, which use recombination to generate diverse combinations of protein domains. Incorporating recombination into evolving populations also has the capacity to improve the directed evolution of whole organisms and genomes, not just single proteins. Currently, microbial experimental systems are limited by an inability to readily exchange DNA without cycles of genetic manipulation and induced transformation. However, there are model systems, such as H. pylori, where recombination and genetic exchange are constant within the evolving population. Such model systems could speed adaptation in directed evolution experiments. Experimental evolution as a genetic screen for functional annotation of hypothetical genes Despite being studied intensely for most of the last century, E. coli and S. cerevisiae still have many genes that have only been assigned hypothetical functions. Deletion collections for E. coli, S. cerevisiae and now other species have revealed the epistatic interactions of many of these genes and provide resources for the connection of gene function with environmental conditions. For instance, the systematic plating out of every viable gene mutant on media containing a drug quickly reveals which genes are essential for detoxification or toxicity of the drug. However, some discoveries require the passage of multiple evolutionary generations. One relatively unexplored path towards understanding the importance of these genes could lie in propagating these mutants, or libraries of mutants, in complex conditions. Natural populations of microbes rarely experience a constant environment, yet the default for any experiment with microbes is monocultures growing in constant experimental conditions. Moreover, in natural systems almost no species evolves in isolation from other species. The last 10 years of microbiome metagenomics has revealed that natural microbial ecosystems are complex. Could it be that the genes of unknown function are involved in interspecies interactions? One way to address this is experiments with libraries of deletion mutants in co-culture with another species. Controlled experiments that compare the performance of deletion mutants in co-culture and mono-culture may reveal new gene functions. Microbes, microbiomes, and the drugs and molecules that they produce emerged from evolutionary processes. Previous progress in molecular biology has relied on converting discoveries such as restriction enzymes, transposons and DNA polymerases into tools to generate new insights. The creative application of experimental evolution to problems in molecular biology has the potential yield the next generation of discoveries into basic and applied biology.
Multifaceted roles of GRAS transcription factors in growth and stress responses in plants The GAI-RGA-and -SCR (GRAS) proteins regulate a myriad of biological functions in plants. The C-terminus of GRAS proteins is highly conserved, whereas the N-terminus is hypervariable. So far, GRAS proteins have been reported in more than 50 plant species. However, not many GRAS proteins are characterized, thus limiting the revelation of their many functions. This review provides a recent update on GRAS proteins, including their structural features, evolutionary gene family expansion/diversification, and interacting protein partners. Also, a mechanistic insight on GRAS protein-mediated plant growth and abiotic stress response is provided. For this, we assessed the transcriptional dynamics of GRAS genes in rice (monocot) and Arabidopsis (dicot) at different developmental stages and under several abiotic stresses. Lastly, the usage of genome-editing tools such as the CRISPR/Cas9 system to understand GRAS molecular functions is highlighted, with the ultimate goal of developing improved agronomic and climate-resilient traits in plants. # Introduction The GAI-RGA-and -SCR (GRAS) transcription factors (TFs) are an important plant-specific TF family that derives the name from three members, GIBBERELLIN-ACID INSENSITIVE (GAI), REPRESSOR of GA1 (RGA), and SCARECROW (SCR). This TF is characterized by the presence of a conserved C-terminus GRAS domain consisting of five distinct motifs, including LHRI (Leucine Heptad Repeat I), VHIID, LHR II (Leucine Heptad Repeat II), PYRE, and SAW, and a highly variable N-terminus region containing homopolymeric stretches of specific amino acids, intrinsically disordered proteins (IDD) and conserved DELLA domain [bib_ref] Structural basis of the specific interactions of GRAS family proteins, Hakoshima [/bib_ref] [bib_ref] A functionally required unfoldome from the plant kingdom: intrinsically disordered N-terminal domains..., Sun [/bib_ref]. These structural features of GRAS proteins suggest that GRAS can potentially function as TFs in plants. Moreover, GRAS proteins contain nuclear localization sequences (NLSs), suggesting their site of action is the nucleus [bib_ref] Genome-Wide analysis of the GRAS gene family in rice and Arabidopsis, Tian [/bib_ref]. The regulatory role of GRAS has been shown in various plant developmental and stress responses. For instance, GRAS proteins act as a key regulator of root development in Arabidopsis [bib_ref] Surfing along the root ground tissue gene network, Pauluzzi [/bib_ref]. SHR, also known as a moving transcriptional regulator, moves from the stele into the adjacent layer. Further, the heterodimerization of SHR-SCR leads to the sequestration of SHR into the nucleus that blocks SHR movement in the endodermis. The formation of the SHR-SCR complex results in the upregulation of several TF genes, including zinc finger (ZF) of the BIRD/IDD family [bib_ref] Arabidopsis JACKDAW and MAGPIE zinc finger proteins delimit asymmetric cell division and..., Welch [/bib_ref]. This regulatory network comprising BIRD/IDD and SCR with SHR dictates tissue specificity and developmental plasticity [bib_ref] Transcriptional control of tissue formation throughout root development, Moreno-Risueno [/bib_ref]. Comparable homologs of AtSCR are found in maize (ZmSCR) and rice (OsSCR), which showed almost similar expression patterns with slight variability in OsSCR expression, suggesting regulatory activity during radial root patterning in dicots and monocots [bib_ref] Conservation and diversification of SCARECROW in maize, Lim [/bib_ref]. Also, NSP1 and NSP2 can form homo and heterodimers to direct the nodulation process [bib_ref] GRAS proteins form a DNA binding complex to induce gene expression during..., Hirsch [/bib_ref]. Also, NSP2 can interact with RAM1 protein [bib_ref] A GRAS-type transcription factor with a specific function in mycorrhizal signaling, Gobbato [/bib_ref]. Moreover, RAM1 also interacts with RAD1 for transcriptional regulation of several downstream genes [bib_ref] Hyphal branching during arbuscule development requires reduced arbuscular mycorrhiza, Park [/bib_ref]. Evidence that shows direct DNA-binding of GRAS proteins with promoters is not available, however, in vivo chromatin immunoprecipitation (ChIP) assays show association with several putative DELLA target genes [bib_ref] Global analysis of DELLA direct targets in early gibberellin signaling in Arabidopsis, Zentella [/bib_ref]. Likewise, in vivo study showed that SHR binds to its own promoter and the promoters of other SHR targets [bib_ref] An evolutionarily conserved mechanism delimiting SHR movement defines a single layer of..., Cui [/bib_ref]. The GRAS protein isolated from lily activated the transcription in yeast and plant systems, suggesting GRAS acts as a transcriptional activator. The confirmatory evidence on the role of GRAS as classic TFs was shown in Medicago truncatula where in vivo and in vitro studies revealed that NSP1 binds directly to the promoter of a nodulation gene [bib_ref] GRAS proteins form a DNA binding complex to induce gene expression during..., Hirsch [/bib_ref]. Being a plant-specific TF, the GRAS family has been reported in more than 50 species, including Arabidopsis [bib_ref] Genome-Wide analysis of the GRAS gene family in rice and Arabidopsis, Tian [/bib_ref] , Oryza sativa [bib_ref] Genome-Wide analysis of the GRAS gene family in rice and Arabidopsis, Tian [/bib_ref] , Glycine max, Manihot esculenta, Hordeum vulgare , Brassica rapa, Solanum lycopersicum [bib_ref] Genome-wide identification, phylogeny and expression analysis of GRAS gene family in tomato, Huang [/bib_ref] , Nicotiana tobacum, Camellia sinensis [bib_ref] Genomewide identification and expression analysis of GRAS family transcription factors in tea..., Wang [/bib_ref] , and Rosa chinensis [bib_ref] Genome-wide identification of GRAS transcription factors and their potential roles in growth..., Kumari [/bib_ref]. Based on conserved domains and functions, the GRAS family was initially classified into eight different subfamilies, including SCR, SCARECROW-LIKE3 (SCL3), SHORT-ROOT (SHR), DELLA, LATERAL SUPPRESSOR (LS), LIGHT SIGNALING through INTERACTIONS LIGHT-RESPONSIVE TRANSCRIPTION FACTOR PIFs (LISCL), HAIRY MERISTEM (HAM) and PHYTOCHROME A SIGNAL TRANS-DUCTION1 (PAT1) [bib_ref] Genome-wide identification of GRAS transcription factors and their potential roles in growth..., Kumari [/bib_ref] [bib_ref] Large-scale analysis of the GRAS gene family in Arabidopsis thaliana, Lee [/bib_ref] [bib_ref] Genome-Wide analysis of the GRAS gene family in rice and Arabidopsis, Tian [/bib_ref] ; however, in some plant species number of subfamilies reported the higher number of subfamilies (for details, see the later section). These TFs are reported to regulate multiple biological processes and molecular functions, including gibberellic acid (GA) signaling [bib_ref] The Arabidopsis GAI gene defines a signaling pathway that negatively regulates gibberellin..., Peng [/bib_ref] , brassinosteroid (BR) signaling [bib_ref] DWARF and LOW-TILLERING, a new member of the GRAS family, plays positive..., Tong [/bib_ref] , shoot and axillary meristem maintenance [bib_ref] Molecular analysis of the LATERAL SUPPRESSOR gene in Arabidopsis reveals a conserved..., Greb [/bib_ref] [bib_ref] Shoot meristem maintenance is controlled by a GRAS-gene mediated signal from differentiating..., Stuurman [/bib_ref] , radial organization of the root , phytochrome signal transduction [bib_ref] PAT1, a new member of the GRAS family, is involved in phytochrome..., Bolle [/bib_ref] , nodulation and arbuscular mycorrhiza (AM) symbiosis [bib_ref] A GRAS-type transcription factor with a specific function in mycorrhizal signaling, Gobbato [/bib_ref] [bib_ref] Lotus japonicus Nodulation requires two GRAS domain regulators, one of which is..., Heckmann [/bib_ref] [bib_ref] Nodulation signaling in legumes requires NSP2, a member of the GRAS family..., Kaló [/bib_ref] [bib_ref] A CCaMK-CYCLOPS-DELLA complex activates transcription of RAM1 to regulate arbuscule branching, Pimprikar [/bib_ref] [bib_ref] Role of the GRAS transcription factor ATA/RAM1 in the transcriptional reprogramming of..., Rich [/bib_ref] and abiotic stress responses [bib_ref] The salt-and drought-inducible poplar GRAS protein SCL7 confers salt and drought tolerance..., Ma [/bib_ref]. The green revolution in the 1960s also exploited the GRAS genes as mutants of Reduced height1 (Rht1) and Rht2 alleles encode DELLA proteins, which have reduced sensitivity for the GA [bib_ref] The genes of the green revolution, Hedden [/bib_ref]. These alleles result in the development of semi-dwarf plant stature with improved yield and lodging resistance. Further, SHR and SCR form a complex to regulate asymmetric cell division in roots [bib_ref] An evolutionarily conserved mechanism delimiting SHR movement defines a single layer of..., Cui [/bib_ref]. A GRAS protein, AtSCL14, interacts with TGACG motif-binding factor (TGA) TFs and regulates stressresponse in Arabidopsis . Another GRAS gene, PeSCL7 provides tolerance to high salinity, osmotic, and drought stresses [bib_ref] The salt-and drought-inducible poplar GRAS protein SCL7 confers salt and drought tolerance..., Ma [/bib_ref]. These reports demonstrate the diversified functions of GRAS TFs, which made them interesting candidates for further studies to delineate their precise roles in plant growth, development, and stress response. Genome-scale studies in different plant species have identified several GRAS TFs candidates that are not functionally characterized to date. Implementing functional genomics approaches coupled with overexpression and genome editing tools will enable the development of overexpression and mutant lines, which can further be studied to understand the involvement of GRAS TFs in regulating agronomic as well as stressresilient traits. Thus, the functional characterization of GRAS TFs possesses excellent application potential. Given this, the present review provides comprehensive information on the GRAS protein structure, GRASinteracting proteins, and their involvement in various biological processes, such as growth and development, and phytochrome-mediated signaling, AM association, and response to biotic and abiotic stresses. Importantly, RNA sequencing data mining was performed to elucidate the expression dynamics of GRAS TFs in different tissues across the developmental scale and under abiotic stresses. In the end, we summarize genetic manipulation studies on GRAS by using genome editing approaches, and future perspectives on the usage of GRAS to improve agronomic traits and develop climate-resilient crops are discussed. The GRAS genes have been reported in more than 50 plant species, and the number of genes ranged from 33 (Arabidopsis) to 188 (wheat) [fig_ref] Table 1: Details of GRAS genes reported in different plant species [/fig_ref]. However, their classification into different subfamilies is still not standardized. Initially, the GRAS genes were clustered into eight subfamilies, including-(i) DELLA, (ii) HAM, (iii) LISCL, (iv) PAT1, (v) LS, (vi) SCR, (vii) SHR, and (viii) SCL9 in case of rice and Arabidopsis [bib_ref] Large-scale analysis of the GRAS gene family in Arabidopsis thaliana, Lee [/bib_ref] [bib_ref] Genome-Wide analysis of the GRAS gene family in rice and Arabidopsis, Tian [/bib_ref]. Similar to Arabidopsis and rice, eight subfamilies of GRAS genes were also reported in apple . The conserved C-terminus GRAS domain comprises five distinct motifs, LHRI, VHIID, LHRII, PYRE, and SAW. The LHRI, VHIID, LHRII, PFYRE motifs are part of the a-helical cap, whereas LHRII, PFYRE, and SAW are the components of the a/b core subdomain. The N-terminus comprises variable motifs and regions. (A) DELLA subfamily consists of two motifs DELLA and VHYNP and a poly-S/T/V region, (B) Short-root (SHR) subfamily comprises of three poly-Q, poly-T, and poly-S/H regions, and (C) SCARECROW (SCR) subfamily comprises of three poly-S/P, poly-S, and poly-Q/P regions on their N-terminus. (D) Ribbon diagram of the GRAS protein of Os-SCL7. The conserved motifs and regions are shown in different colors: LHRI in cyan, VHIID in yellow, LHRII in blue, PFYRE in red, SAW in orange; disordered regions in green. iScience Review , walnut [bib_ref] Genome-wide identification, classification, expression and duplication analysis of GRAS family genes in..., Quan [/bib_ref] , and barrel clover. However, in other plant species, the number of subfamilies is higher; for instance, 13 subfamilies were identified in the case of populus [bib_ref] Genome-wide comparative analysis of the GRAS gene family in populus, Arabidopsis and..., Liu [/bib_ref] , tea [bib_ref] Genomewide identification and expression analysis of GRAS family transcription factors in tea..., Wang [/bib_ref] , and castor bean [bib_ref] Genome-Wide identification, evolutionary analysis, and stress responses of the GRAS gene family..., Xu [/bib_ref] and 16 subfamilies were identified in bottle gourd [bib_ref] Genome-wide identification and analysis of GRAS transcription factors in the bottle gourd..., Sidhu [/bib_ref]. Whereas combined phylogenetic analysis using eight different angiosperm species, regroup GRAS TF into 17 subfamilies [bib_ref] Evolutionary analyses of GRAS transcription factors in angiosperms, Cenci [/bib_ref]. To further understand the evolutionary relationship, we also performed the phylogenetic analysis using 581 GRAS proteins from 12 plant species and found that GRAS proteins were grouped into 17 subfamilies such as RAM1, NSP1, NSP2, etc. (Figures 2 and S1-S3). The studies mentioned above showed considerable divergence among the GRAS family genes in flowering plants. To understand the functional divergence of GRAS subfamilies in different species, we constructed an evolutionary Tree of Life for 42 plant species belonging to bryophyte, lycophyte, gymnosperm, basal angiosperm (Amborella), and angiosperms (monocot and dicot). The phylogenetic tree clearly shows the evolutionary emergence of NSP1, NSP2, and RAM1 in the higher plant species, suggesting the attainment of specialized functions such as nodulation or arbuscular mycorrhizal interactions [fig_ref] Figure 3: Phylogenetic tree of 42 species belonging to bryophyte, lycophyte, gymnosperm, basal angiosperm,... [/fig_ref]. In the future, more studies on GRAS genes in the plant kingdom may identify more subfamilies that can help trace the evolutionary history of addition/deletion or duplication of subfamilies. GRAS proteins are not present in algae but are identified in bryophytes (Marchantia polymorpha) . GRAS protein sequences across different plant species, including lower plants and higher plant species, showed 12 subclades in a phylogenetic tree. In flowering plants, the GRAS family showed maximum divergence. There is enormous variability in the number of subfamilies of GRAS TFs among different plant species [fig_ref] Table 1: Details of GRAS genes reported in different plant species [/fig_ref] , suggesting some species or lineage-specific retention of GRAS subfamilies during evolution. Further, the less conservation of the GRAS domains across different subfamilies, but more sequence similarities (up to 98%) within subfamilies, highlights similar functions of GRAS genes . For example, most of the genes characterized yet from the DELLA subfamily are involved in the GA signaling pathway. It has also been confirmed with GRAS protein functional characterization in different plants, which has indicated a conserved function/pattern among putative orthologues of each subfamily and/or subclade in different plants. Another good example is the NSP1 gene, conserved in leguminous and non-leguminous crops and associated with nodule formation [bib_ref] Lotus japonicus Nodulation requires two GRAS domain regulators, one of which is..., Heckmann [/bib_ref]. In bryophytes, ESTs similar to GRAS gene sequences were found, suggesting that this protein family arose before the evolution of land plants. The GRAS genes are mostly intronless in several plant species, including grapevine (88.46%), barley (88.2%), tomato (77.4%), capsicum (84%), mustard (83.3%), apricot (82.2%), Arabidopsis (67.6%), rice (55%), and populus (54.7%) [bib_ref] Structural and functional analysis of the GRAS gene family in grapevine indicates..., Grimplet [/bib_ref] [bib_ref] Genome-wide identification, phylogeny and expression analysis of GRAS gene family in tomato, Huang [/bib_ref] [bib_ref] Genome-wide comparative analysis of the GRAS gene family in populus, Arabidopsis and..., Liu [/bib_ref] [bib_ref] Genome-Wide analysis of the GRAS gene family in barley (Hordeum vulgare L.), To [/bib_ref]. The probable causes for the intronless genes in eukaryotic genomes are duplication events and retroposition of intron-containing genes [bib_ref] The roles and evolutionary patterns of intronless genes in deuterostomes, Zou [/bib_ref]. Intronless genes are a typical prokaryote feature since GRASlike proteins were initially reported in bacteria and involved in methylase activity. ## Intrinsically disordered proteins (idps) and functional versatility in gras In eukaryotes, 25-30% of proteins are intrinsically disordered proteins (IDPs) and play a significant role in various molecular and cellular functions. More than 70% of signaling proteins and 82-94% of TFs have IDRs . IDR, present within IDP, enables proteins to undergo disorder-to-order transitions to recognize and bind at specific binding interfaces among different partners . Computational and bioinformatics analysis revealed that the GRAS proteins also fall under the IDP category . The GRAS protein's N-terminus contains molecular recognition features (MoRFs), short interaction-prone sites within IDR that, upon interaction and recognition of partner's proteins, allow specific disorder-to-order transitions to form functional complexes . The functional diversification of GRAS proteins is attributed to the intrinsically disordered N-domain, which is reflected by (a) its [bib_ref] Genome-Wide analysis of the GRAS gene family in rice and Arabidopsis, Tian [/bib_ref] , Phoenix dactylifera (Al-Mssallem et al., , Theobroma cacao [bib_ref] The genome of Theobroma cacao, Argout [/bib_ref] , and Vitis vinifera [bib_ref] Structural and functional analysis of the GRAS gene family in grapevine indicates..., Grimplet [/bib_ref]. GRAS protein sequences are aligned using MUSCLE package available in MEGA X software [bib_ref] Molecular evolutionary genetics analysis (MEGA) for macOS, Stecher [/bib_ref]. The phylogenetic tree was created using the neighbor-joining (NJ) method with the Poisson model, pairwise deletion, and 1,000 bootstraps values. The phylogenetic tree was visualized using the iTOL online tool [bib_ref] Interactive Tree of Life (iTOL) v5: an online tool for phylogenetic tree..., Letunic [/bib_ref]. The GRAS proteins are clustered into 17 subfamilies, marked by various colors. expansion into different subfamilies, (b) role in transcriptional regulation along with signaling pathways, and (c) some proteins can show cross-talk in different signal pathways forming homo-or heterodimers. For example, BdSLR1 and BdSLRL1 can form homodimers but cannot form heterodimers . Intrinsic disorder characteristic in the GRAS proteins has provided them binding plasticity, which is directly correlated with the functional polymorphism of these proteins . In the DELLA subfamily, the N-terminus has been characterized as intrinsically disordered, whereas the C-terminus possesses only basic structural protein folding with a series of highly conserved motifs . This highly variable N-terminus domain with intrinsically disordered characteristics enables this gene family with multiple functions. The intrinsically disordered N-domain allows GRAS proteins to act as the key regulators in several signaling pathways. Moreover, phosphorylation and dephosphorylation have widely been observed for the functionality of GRAS proteins. Phosphorylation plays an essential role in adjusting disordered protein interactions and ultimately signal transduction events [bib_ref] Protein dynamics and conformational disorder in molecular recognition, Mittag [/bib_ref]. For instance, in rice, GID2 interacts specifically with phosphorylated SLR1 protein (a DELLA protein), not with unphosphorylated SLR1 protein; and strongly suggests that only the phosphorylated state of SLR1 protein can undergo GA-dependent degradation. Moreover, a rice kinase may also directly phosphorylate SLR1 proteins [bib_ref] Rice early flowering1, a CKI, phosphorylates DELLA protein SLR1 to negatively regulate..., Dai [/bib_ref]. In SLR1 proteins, Ser residues present within DELLA/TVHYNP and polyS/T/V domain (at N-terminus domain) undergo phosphorylation [bib_ref] Dissection of the phosphorylation of rice DELLA protein, SLENDER RICE1, Itoh [/bib_ref]. The experimental demonstrations have confirmed that rice SLR1 and Brachypodium BdSLR1 show that the DELLA domain and TVHYNP motif are the key regulators for transcriptional activation activity and GID1 interaction [bib_ref] Genome-wide identification of GRAS genes in Brachypodium distachyon and functional characterization of..., Niu [/bib_ref]. Also, CCaMK (calcium-and calmodulin-dependent protein kinase) is required to facilitate nodulation signaling that requires the GRAS family transcriptional NSP1 and NSP2 [bib_ref] Nodulation independent of rhizobia induced by a calcium-activated kinase lacking autoinhibition, Gleason [/bib_ref]. The reversible phosphorylation also plays a significant role in regulating the stress-induced response of some GRAS proteins. For instance, NtGRAS1 in tobacco [bib_ref] NtGRAS1, a novel stress-induced member of the GRAS family in tobacco, localizes..., Czikkel [/bib_ref] , and CIGR1 (chitin-inducible gibberellin-responsive) and CIGR2 in the case of rice [bib_ref] Two rice GRAS family genes responsive to N-acetylchitooligosaccharide elicitor are induced by..., Day [/bib_ref] are good examples. Therefore, the phosphorylation and dephosphorylation states of DELLA proteins are critical to governing the stability or degradation of proteins and regulating intracellular signal transduction under different stress conditions [bib_ref] NtGRAS1, a novel stress-induced member of the GRAS family in tobacco, localizes..., Czikkel [/bib_ref] [bib_ref] Two rice GRAS family genes responsive to N-acetylchitooligosaccharide elicitor are induced by..., Day [/bib_ref]. Although the knowledge about GRAS proteins' phosphorylation and dephosphorylation is still elusive, disorder-assisted phosphorylation may provide a different dimension to study GRAS proteins and their recognition patterns during signaling mechanisms. The IDPs also provided a functional advantage over structurally ordered proteins for molecular recognition via protein-protein interactions. To facilitate specific recognition, they undergo binding-induced conformational changes and display different binding sites to recognize different partners during protein interactions, giving binding plasticity to these proteins. The MoRFs are responsible for this disorder-to-order transition to recognize their interacting partners. The N-domain is highly variable between subfamilies, but it is highly conserved within the subfamily . For example, in the DELLA subfamily, repeated hydrophobic/aromatic residues are the restricted motifs in the N domain and are crucial for GID1 interaction, GA signaling, and ultimately plant growth [bib_ref] Gibberellin-induced DELLA recognition by the gibberellin receptor GID1, Murase [/bib_ref]. The GRAS TFs exhibit a higher level of divergence owing to MoRFs at their N-terminus region, which allows them to function as an activator in different signaling and molecular pathways. Whereas the C-terminus region is highly conserved in the GRAS family and interacts with downstream proteins [bib_ref] The role of GRAS proteins in plant signal transduction and development, Bolle [/bib_ref] [bib_ref] The GRAS gene family in Arabidopsis: sequence characterization and basic expression analysis..., Pysh [/bib_ref]. For instance, Leu heptad repeats in the conserved LHRI-VHIID-LHRII domain involved in protein-protein interactions, which has also been proved experimentally by following interacting pairs, where these interactions are conferred by either an individual motif or the entire region: DELLA-PIFs, DELLA-JAZ1, DELLA-GID2, SHR-SCR, NSP1-NSP2, SCL14-TGA, and SCL1-HDA19 [bib_ref] An evolutionarily conserved mechanism delimiting SHR movement defines a single layer of..., Cui [/bib_ref] [bib_ref] An auxin-responsive SCARECROW-like transcriptional activator interacts with histone deacetylase, Gao [/bib_ref] [bib_ref] GRAS proteins form a DNA binding complex to induce gene expression during..., Hirsch [/bib_ref] [bib_ref] DELLAs modulate jasmonate signaling via competitive binding to JAZs, Hou [/bib_ref]. It has been observed that the GRAS proteins are mostly transcriptional coactivators, which may block or enhance the activity of interacting partners . Another conserved C-terminus, the VHIID domain is involved in protein-DNA interactions [bib_ref] The GRAS gene family in Arabidopsis: sequence characterization and basic expression analysis..., Pysh [/bib_ref]. Therefore, this transition in protein structure for molecular recognition based upon intrinsic disorder-to-order transition owing to the presence of MoRFs provides a theoretical framework that can help explore more probable functioning of GRAS TFs in signaling and regulation pathways that can further be confirmed by experimental elucidations. ## Gras-interacting protein complexes Generally, transcriptional regulation of a particular gene is regulated by the action of TFs that directly binds promoter cis-elements of target genes. However, transcriptional regulation is also mediated by regulatory complexes, such as TFs interact with other TFs or transcriptional regulators (TRs) [bib_ref] Transcriptome diversity among rice root types during asymbiosis and interaction with arbuscular..., Gutjahr [/bib_ref]. GRAS proteins carry out their functions via forming homo-or heterodimers and/or interacting with other proteins. GRAS proteins primarily function via interaction with several protein partners such as TFs (e.g., IDD, TGA, MYB, SPL) and chromatin remodeling complexes (Pickle; PKL). In Arabidopsis, Indeterminate Domain (AtIDD) proteins regulate the expression of SCR by interacting with SHR and SCR, which are further involved in root tissue formation by accommodating their ZF motifs into the groove of the SHR GRAS domain [bib_ref] Gene regulation via the combination of transcription factors in the INDETERMINATE DOMAIN..., Aoyanagi [/bib_ref] [bib_ref] Structural basis of the specific interactions of GRAS family proteins, Hakoshima [/bib_ref]. Further, IDD proteins also act as transcriptional coactivators with DELLA proteins. The DELLA/IDD protein complex induces the expression of the SCL3 gene and regulates the GA signaling pathway in plants [bib_ref] DELLA protein functions as a transcriptional activator through the DNA binding of..., Yoshida [/bib_ref]. The TGA TFs belong to group D of bZIP TFs and are found ubiquitously in eukaryotes. TGA plays a vital role in Arabidopsis to recruit LlSCL/ SCL9 branch member SCL14 (SCARECROW-LIKE 14) to its target promoter region. The TGA/SCL14 protein complex activates the detoxification mechanism in plants against xenobiotic chemicals such as isonicotinic acid 2,4,6-triiodobenzoic acid . In eukaryotes, chromatin remodeling is essential for gene expression, and chromatin-remodeling factor PKL/ Enhanced Photomorphogenic1(EPP1) suppresses phytochrome signaling in plants . PKL interacts with regulators of hypocotyl growth such as PIF3 and BZR1 to inhibit the histone methylation of genes involved in cell elongation (i.e., IAA19, PRE1, TCH4) . Also, PKL interacts with DELLA proteins to negatively regulate its activity by constricting its binding ability. Therefore, the PIF-BZR1-PKL-DELLA module regulates H3K27me3 modification status and ensures plant seed etiolation [fig_ref] Figure 4: Role of GRAS TFs in different biological processes in plants [/fig_ref]. Several GRAS TFs are involved in AM symbiotic association and regulation of plant developmental transitions. RAM1 is a central regulator of arbuscular development and is involved in hyphopodia formation in AM fungi, whose transcription is regulated explicitly by CYCLOPS and DELLA [bib_ref] A CCaMK-CYCLOPS-DELLA complex activates transcription of RAM1 to regulate arbuscule branching, Pimprikar [/bib_ref]. The calcium-and calmodulin-dependent kinase (CCaMK) interacts with CYCLOPS and directly binds to the cis-element (GGCGCC box/AM-CYC box) of the RAM1 promoter [bib_ref] A CCaMK-CYCLOPS-DELLA complex activates transcription of RAM1 to regulate arbuscule branching, Pimprikar [/bib_ref]. CYCLOPS also interacts with DELLA, which further boosts its capability to transactivate the RAM1 promoter along with the promoters of other nodulation genes such as NIN (NODULE INCEPTION) and ERN1(ERF REQUIRED FOR NODULATION1) [bib_ref] DELLA proteins are common components of symbiotic rhizobial and mycorrhizal signalling pathways, Jin [/bib_ref] [bib_ref] A CCaMK-CYCLOPS-DELLA complex activates transcription of RAM1 to regulate arbuscule branching, Pimprikar [/bib_ref]. These studies suggest that the complex formed by GRAS proteins is crucial for establishing arbuscular mycorrhiza symbiosis between plants and fungi. BOTRYTIS SUSCEPTIBLE1 INTERACTOR (BOI) is an Arabidopsis RING protein that interacts with DELLA and suppresses the expression of GA-responsive genes and eventually affects seed germination, juvenile-to-adult phase transition, and flowering . ## Microrna-based regulation of gras MicroRNAs are 21-24 nucleotide long, highly conserved noncoding RNA sequences that regulate plant growth and developmental processes by directing cleavage of mRNA sequences or translational inhibition. The microRNAs regulate several developmental processes and environmental interactions in plants [bib_ref] Biochemical evidence for translational repression by Arabidopsis MicroRNAs, Lanet [/bib_ref] [bib_ref] MicroRNA control of PHABULOSA in leaf development: importance of pairing to the..., Mallory [/bib_ref]. The predicted target of miR171 is the AtSCL6 gene [bib_ref] Cleavage of scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA, Llave [/bib_ref]. The three SCL6 transcripts targeted by miR171 are critical for differentiating axillary meristem and promoting shoot elongation [bib_ref] MicroRNA171c-Targeted SCL6-II, SCL6-III, and SCL6-IV genes regulate shoot branching in Arabidopsis, Wang [/bib_ref]. Moreover, miR171 target transcripts SCL6, SCL22, and SCL27 and control chlorophyll biosynthesis . Similarly, in rice and barley, miR171 functions in phase transitions and floral meristem determinacy, thereby highlighting the role of GRAS genes in regulating developmental processes in monocots and dicots [bib_ref] Over-expression of microRNA171 affects phase transitions and floral meristem determinacy in barley, Curaba [/bib_ref]. The miR171 targets NSP2 transcripts in legumes such as barrel clover and lotus, thus regulating nodule formation and mycorrhiza association [bib_ref] Two MicroRNAs linked to nodule infection and nitrogen-fixing ability in the legume..., De Luis [/bib_ref] [bib_ref] MiR171h restricts root symbioses and shows like its target NSP2 a complex..., Hofferek [/bib_ref] [bib_ref] The microRNA miR171h modulates arbuscular mycorrhizal colonization of Medicago truncatula by targeting..., Lauressergues [/bib_ref]. Likewise, in soybeans, the GmSCL-6 and GmNSP2 mRNAs are cleaved by miRNAo and miR171q to regulate the nodulation process [bib_ref] Characterization of the spatial and temporal expression of two soybean miRNAs identifies..., Hossain [/bib_ref]. In tomatoes, the functional genomic study of the miR171-SlGRAS24 module demonstrated that miR171 regulates various agronomical traits in tomatoes via gibberellin and auxin homeostasis [bib_ref] Overexpression of a tomato miR171 target gene SlGRAS24 impacts multiple agronomical traits..., Huang [/bib_ref]. These studies emphasized the crucial role of miRNAs in regulating GRAS genes during different developmental stages of plants. ## Biological functions regulated by gras The GRAS TF family members play an essential role in regulating various biological processes, such as plant growth and development (GA signal transduction, shoot/root formation, male gametogenesis, phytochrome signaling, and symbiotic association) [fig_ref] Figure 4: Role of GRAS TFs in different biological processes in plants [/fig_ref]. Also, GRAS plays a crucial role in plant stress responses. The transcriptional reprogramming of GRAS genes occurs in different plant development stages and abiotic stress responses (cold, drought, heat, and salt) in Arabidopsis and rice. These diverse roles of GRAS TFs make them potential candidates for improving plants. iScience Review (LAS), a GRAS gene, was involved in axillary meristem initiation and lateral shoot formation through negative regulation of GA signal transduction. Similarly, in Arabidopsis, an AtLAS (ortholog of LeLs) gene was involved in axillary shoot formation [bib_ref] Molecular analysis of the LATERAL SUPPRESSOR gene in Arabidopsis reveals a conserved..., Greb [/bib_ref] , thus signifying a conserved mechanism for lateral shoot formation in tomato and Arabidopsis. The higher expression of AtLAS in leaf axile can be considered as a marker of meristem formation, and AtLAS along with STM gene switched on the expression of REVOLUTA (Rev) gene that is a potential regulator of shoot branching [bib_ref] Molecular analysis of the LATERAL SUPPRESSOR gene in Arabidopsis reveals a conserved..., Greb [/bib_ref] However, the ortholog in rice, i.e., MOC1, regulates tiller number, signifying the attainment of functional differences between monocotyledonous and dicotyledonous. A GRAS gene, Hairy Meristem (HAM), was identified in Petunia involved in lateral organogenesis and meristem maintenance. The ham mutants of Petunia showed non-maintenance of the meristem. Further, HAM and WUSCHEL (WUS) work in parallel during the specification and maintenance of meristem, and HAM is essential for prolonged response to TERMINATOR and SHOOTMERISTEMLESS (PhSTM) in Petunia [bib_ref] Shoot meristem maintenance is controlled by a GRAS-gene mediated signal from differentiating..., Stuurman [/bib_ref]. Recently, the SCL28 gene was reported to regulate the mitotic cell cycle via MYB3R TFs and modulates the meristematic cell development in Arabidopsis [bib_ref] The Arabidopsis GRAS-type SCL28 transcription factor controls the mitotic cell cycle and..., Goldy [/bib_ref]. It suggests that this pathway is implicated in meristem cell differentiation and maintenance. ## Shoot meristem and plant tillering MONOCULM 1 (MOC1) encodes a GRAS protein in rice, expressed in the axillary buds, and regulates axillary bud initiation, thus executing the tiller formation in rice. The moc1 null mutants exhibited only single culm (without tillering), whereas overexpression lines showed enhanced tillering [bib_ref] Rice APC/CTE controls tillering by mediating the degradation of MONOCULM 1, Lin [/bib_ref]. TILLER AND DWARF 1 (TAD1) recruit the MOC1 to ANAPHASE-PROMOTING COMPLEX (APC/C), and APC/C degrades the MOC1 owing to E3 ubiquitin ligase activity. In tad mutants, TAD1 gene is disrupted and thus A B C iScience Review could not recognize the MOC1, thus tad mutants show enhanced tillering. These studies suggested the MOC1 involved in downstream of TAD1 and control tillering in a positive manner, although the molecular mechanism behind it is still unilluminated. Gibberellin (GA) has long been known to inhibit tillering in plants [bib_ref] Control of tillering in rice, Li [/bib_ref]. Another gene, dwarf, and low-tillering (DLT), which encodes a GRAS protein, regulate plant morphology via BR signaling in rice. The mutant of the DLT gene showed a dwarf phenotype and low-tillering in rice suggested the involvement of the DLT gene in tillering, although the molecular mechanism is not known [bib_ref] DWARF and LOW-TILLERING, a new member of the GRAS family, plays positive..., Tong [/bib_ref]. ## Microsporogenesis, fruit ripening, and seed germination The GRAS proteins regulate anther development and fruit ripening. LiSCL is a nuclear-localized microsporocytes gene expressed explicitly during premeiotic anther developmental in Lilium longiflorum anthers. As shown by the transient expression assay, it is involved in meiosis and activates the pollen mother cell. The Lels mutants in tomatoes showed the blocked axillary meristem initiation iScience Review. Tomato GRAS1 is also differentially expressed in breaker and mature fruits emphasizing its role in fruit development [bib_ref] Genome-wide identification, phylogeny and expression analysis of GRAS gene family in tomato, Huang [/bib_ref]. Recently, it has been demonstrated that a GRAS TF, SlGRAS4, was involved in tomato ripening by regulating ethylene biosynthesis genes and MADS TFs . SlGRAS4 binds to the promoter sequence of the crucial ethylene biosynthesis genes (like SlACO1 and SlACO3), activates their expression, and enhances the fruit ripening. Further, SlGRAS4 also represses the SlMADS1 (negative regulator of fruit ripening) expression and accelerates the ripening . Seeds germination is influenced by external (temperature, moisture, and light) and internal (phytohormones such as ABA and GA) cues. ABA regulates seed dormancy via DELLA protein that belongs to the GRAS family [bib_ref] Seed dormancy and germination, Koornneef [/bib_ref]. The DELLA protein makes a complex with ABSCISIC ACID INSENSITIVE 3 (ABI3) and ABI5 and binds to the promoter of SOMNUS (SOM), which is involved in the negative regulation of the seed germination process [fig_ref] Figure 4: Role of GRAS TFs in different biological processes in plants [/fig_ref] [bib_ref] ABA-INSENSITIVE3, ABA-INSENSITIVE5, and DELLAs interact to activate the expression of SOMNUS and..., Lim [/bib_ref]. During unfavorable situations (heat stress), the levels of ABA increase, whereas GA decreases, which leads to the binding of the DELLA/ABI3/ABI5 protein complex on the SOM promoter. It causes transcription activation of SOM genes and inhibits seed germination during unfavorable environmental conditions [bib_ref] ABA-INSENSITIVE3, ABA-INSENSITIVE5, and DELLAs interact to activate the expression of SOMNUS and..., Lim [/bib_ref]. ## Radial patterning of root and shoot The asymmetric cell divisions pave the way for plant organ development and the formation of patterns. Almost 1.5 decades ago, AtSCR, a GRAS TF was identified to express in the cortex and endodermis (Di , shoot apical meristem, bundle sheath , and quiescent center [bib_ref] SCARECROW is involved in positioning the stem cell niche in the Arabidopsis..., Sabatini [/bib_ref]. The scr mutant in Arabidopsis showed aberrant plant growth with defective roots, hypocotyl stem, and inflorescence. This suggests that AtSCR is essential in root and shoot formation radial patterning. SHORT-ROOT (SHR) is another GRAS TF required for quiescent center identity and its role in radial root patterning [bib_ref] SCARECROW is involved in positioning the stem cell niche in the Arabidopsis..., Sabatini [/bib_ref]. AtSHR gene is exclusively expressed in provascular tissue [bib_ref] Intercellular movement of the putative transcription factor SHR in root patterning, Nakajima [/bib_ref] , and shr mutants did not possess one layer of cortex, unlike in scr mutants . Presence of SHR and SCR is critical for periclinal division and endodermis formation [bib_ref] A bistable circuit involving SCARECROW-RETINOBLASTOMA integrates cues to inform asymmetric stem cell..., Cruz-Ramírez [/bib_ref] [bib_ref] An evolutionarily conserved mechanism delimiting SHR movement defines a single layer of..., Cui [/bib_ref] [bib_ref] Mechanisms regulating SHORT-ROOT intercellular movement, Gallagher [/bib_ref]. In shr mutants, SCR expression levels are significantly low, suggesting that transcription of SCR is directly or indirectly controlled by the SHR gene [bib_ref] Intercellular movement of the putative transcription factor SHR in root patterning, Nakajima [/bib_ref]. The SHR gene transcript is initially present in the stele, from where it moves to the adjacent cell layers and interacts with its target SCR. SHR functions downstream to SCR and is primarily responsible for asymmetric cell division in Arabidopsis [bib_ref] Mosaic analyses using marked activation and deletion clones dissect Arabidopsis SCARECROW action..., Heidstra [/bib_ref]. However, SCR restricts SHR movement to endodermis [bib_ref] An evolutionarily conserved mechanism delimiting SHR movement defines a single layer of..., Cui [/bib_ref]. The SCL23 is another GRAS TF, which is operative in the same pathway. No alterations in the root pattern were observed in the single mutant under normal growth conditions, which suggests that it functions redundantly. However, the double mutant scrscl23 mimicked the shr phenotypes and resulted in smaller roots and plants. Therefore, SCL23, SCR, and SHR genes play a significant role in endodermis formation in the root meristem ) [fig_ref] Figure 4: Role of GRAS TFs in different biological processes in plants [/fig_ref]. Another GRAS gene SCL3 was found to be involved in the GA pathway and helps develop root endodermis [bib_ref] DELLA protein functions as a transcriptional activator through the DNA binding of..., Yoshida [/bib_ref]. The scl3 null mutant showed decreased GA responses and enhanced GA biosynthesis gene expression specifying that SCL3 functions as a positive regulator of the GA signaling pathway [bib_ref] SCARECROW-LIKE 3 promotes gibberellin signaling by antagonizing master growth repressor DELLA in..., Zhang [/bib_ref]. However, the tissue-specific maintenance of the GA pathway in endodermis is regulated by the joint action of SCL3 and SHR/SCR genes, highlighting a cross-regulatory network of the GRAS TFs in the root endodermis. In Zea mays, SCARECROW (ZmSCR), an ortholog of AtSCR, was found to be localized to the endodermal cell layer. Despite variation in the size and configuration of the quiescent center in the two species, it suggests that conserved pathways are involved in radial patterning [bib_ref] Molecular analysis of the SCARECROW gene in maize reveals a common basis..., Lim [/bib_ref]. An ortholog of Arabidopsis SCR is OsSCR found in rice. OsSCR was upregulated in the endodermis, whereas it is downregulated in the daughter cortex. OsSCR transcripts were found in leaf primordium at the P3 stage during stomatal development and in guard mother cells (GMCs). Also, a higher transcript level of OsSCR was found in the ligule initiation tissues. It suggests that OsSCR has multiple functions, including cortex/endodermis, stomata, and ligule development . Two orthologs of Arabidopsis SHR have been identified in rice, namely OsSHR1 and OsSCR. The OsSHR1 is expressed in leaves and roots, stomatal development, and specifically in P3 leaf primordia at stage 1. The OsSCR is predominately expressed in stomata, whereas the OsSHR1 transcript was almost ubiquitously found in the L1-layer cells and not restricted in the stomatal row. The co-expression of OsSHR1 iScience Review during different developmental tissues suggests that their co-operative networking is essential for forming root endodermis, stomata, and subsidiary cells. SHR acts as a key player that activates SCL23 and SCR genes in the endodermis. SHR TF enters into endodermis and forms protein complexes of SHR-SCR, SHR-SCL23, or SHR-SCR-SCL23 to regulate the SCR and SCL23, respectively, and a negative feedback loop functions to control each other levels to maintain cell specificity in both roots and shoots [bib_ref] Conservation and diversification of the SHR-SCR-SCL23 regulatory network in the development of..., Yoon [/bib_ref]. ## Stress responses GRAS genes play a significant role in providing tolerance against different abiotic and biotic stresses. For instance, the functional characterization of the PeSCL7 gene of populus emphasizes its involvement in salinity, osmotic and water stresses [bib_ref] The salt-and drought-inducible poplar GRAS protein SCL7 confers salt and drought tolerance..., Ma [/bib_ref]. Further, overexpression of Brassica napus gene BnLAS in Arabidopsis showed an increase in chlorophyll content, stomatal density, and enhanced tolerance to drought and salt stress [bib_ref] Overexpression of the Brassica napus BnLAS gene in Arabidopsis affects plant development..., Yang [/bib_ref]. Also, the enhanced epidermal wax deposition was observed in the leaves of transgenic plants, together with increased expression of wax biosynthesis and regulatory genes like CER1, CER2, KCS1, and KCS2. OsGRAS23 is a nuclear-localized stress-responsive GRAS TF, which belongs to the LISCL subfamily of GRAS proteins in rice. Overexpression of OsGRAS23 in rice led to enhanced tolerance against drought and oxidative stresses owing to decreased H 2 O 2 accumulation [bib_ref] OsGRAS23, a rice GRAS transcription factor gene, is involved in drought stress..., Xu [/bib_ref]. The OsGRAS23 positively regulates drought tolerance through binding to the promoters of several stress-related genes (anti-oxidants, protein kinases, proteinase inhibitors, and enzymes related to metabolism). Moreover, under drought stress, the expression of several GRAS genes is modulated in castor beans [bib_ref] Genome-Wide identification, evolutionary analysis, and stress responses of the GRAS gene family..., Xu [/bib_ref]. For instance, the expression of 16 RcGRAS members has been induced up to 5-fold and the expression of four RcGRAS genes has downregulated during drought stress. The PAT1 proteins implicated in phytochrome signaling are also involved in regulating abiotic stress responses. Overexpression lines of the VaPAT1 gene showed increased tolerance against multiple abiotic stresses (i.e., cold, drought, and salt stresses). The VaPAT1 gene positively regulates several stress-related genes (SIZ, CBF1, MYB34, MYC2, COR15A, RD29A) and leads to enhanced concentration of proline and soluble sugar contents in plant cells and provides abiotic stress tolerance [bib_ref] Overexpression of VaPAT1, a GRAS transcription factor from Vitis amurensis, confers abiotic..., Yuan [/bib_ref]. In wheat, TaSCL14 GRAS gene showed enhanced expression under high-light stress. TaSCL14 acts as a multifunctional regulator as the silencing of TaSCL14 produces various defects, including (i) lowered tolerance to photooxidative stress, (ii) increased injury to biological membranes, (iii) increased rate of leaf senescence, (iv) decreased photosynthetic capacity, and (v) inhibited plant growth [bib_ref] TaSCL14, a Novel Wheat (Triticum aestivum L.) GRAS gene, regulates plant growth,..., Chen [/bib_ref]. The DELLA genes regulate GA signals and the JA pathway, both of which play distinct roles in plant disease resistance [bib_ref] DELLAs modulate jasmonate signaling via competitive binding to JAZs, Hou [/bib_ref]. Furthermore, DELLA also involved in signal transduction during bacteria infection in Arabidopsis, and provides resistance to such bacteria infection [bib_ref] The Arabidopsis DELLA RGA-LIKE3 is a direct target of MYC2 and modulates..., Wild [/bib_ref]. In cassava, four MeDELLA proteins have been identified. The overexpression of these genes in Nicotiana benthamiana showed lesser bacterial growth, whereas the silencing of genes decreased resistance against bacteria, highlighting their role in bacterial blight resistance . Likewise, ecotypic expression of SCL14 (LlSCL/SCL9 subfamily GRAS protein) in Arabidopsis showed enhanced tolerance to toxins chemicals (isonicotinic acid and 2,4,6-triiodobenzoic acid, 2,4-D, p-chlorophenoxyisobutyric acid), whereas the mutants were highly susceptible. These results showed that SCL14 is essential for imparting tolerance against xenobiotic stress . SCL28 is a nuclear-localized transcriptional activator found in the root meristem zone in Arabidopsis. SCL28 orchestrates cell division and elongation patterns of root growth during stress conditions [bib_ref] Characterization of the GRAS transcription factor SCARECROW-LIKE 28's role in Arabidopsis root..., Choe [/bib_ref]. Also, GRAS homologs found in tobaco are induced by stress conditions such as antimycin A, H 2 O 2 , salicylic acid, and L-cysteine, which suggests their role under stress conditions [bib_ref] NtGRAS1, a novel stress-induced member of the GRAS family in tobacco, localizes..., Czikkel [/bib_ref]. Collectively, GRAS proteins are also involved in managing various biotic and abiotic stresses in plants along with their role in growth and development. However, there are still gaps in knowledge of how GRASs function in many plants in response to stresses, and further functional study is required. ## Symbiosis The plant forms mutualistic partnerships with mycorrhizal fungi and with rhizobial bacteria to exchange nutrients [bib_ref] Multiple control levels of root system remodeling in arbuscular mycorrhizal symbiosis, Gutjahr [/bib_ref] [bib_ref] Transcriptional regulation of arbuscular mycorrhiza development, Pimprikar [/bib_ref]. Plants recognize the Nod factor from rhizobial bacteria and Myc factor from mycorrhizal fungus, resulting in these interactions [bib_ref] Expression and roles of GRAS gene family in plant growth, signal transduction,..., Khan [/bib_ref] [bib_ref] Fungal lipochitooligosaccharide symbiotic signals in arbuscular mycorrhiza, Maillet [/bib_ref] [bib_ref] Symbiosis and the social network of higher plants, Venkateshwaran [/bib_ref]. The GRAS genes are actively involved in nodulation and the formation of fungal hyphae during the symbiotic associations [bib_ref] A GRAS-type transcription factor with a specific function in mycorrhizal signaling, Gobbato [/bib_ref] [bib_ref] Expression and roles of GRAS gene family in plant growth, signal transduction,..., Khan [/bib_ref]. A conserved signaling route is involved in both Nod and Myc factor signaling cascade, and this signaling pathway divides into two branches, both of which contain GRAS TFs, enabling targeted activation of nodulation or mycorrhizal-associated responses [bib_ref] A GRAS-type transcription factor with a specific function in mycorrhizal signaling, Gobbato [/bib_ref]. In this highly conserved signaling cascade (symbiosis-specific association), the GRAS TF has come into the picture after the induction of calcium signaling induced by CCaMK [bib_ref] A CCaMK-CYCLOPS-DELLA complex activates transcription of RAM1 to regulate arbuscule branching, Pimprikar [/bib_ref] [bib_ref] CYCLOPS, A DNA-binding transcriptional activator, orchestrates symbiotic root nodule development, Singh [/bib_ref]. The two GRAS genes, NSP1 and NSP2, are well characterized in M. truncatula and are involved Nod-factor signaling and nodule formation during rhizobial symbiotic association [bib_ref] GRAS proteins form a DNA binding complex to induce gene expression during..., Hirsch [/bib_ref] [bib_ref] Nodulation signaling in legumes requires NSP2, a member of the GRAS family..., Kaló [/bib_ref]. Further, LjNSP1 and LjNSP2 function as the Nod factor-activated transcription regulators in the lotus [bib_ref] Lotus japonicus Nodulation requires two GRAS domain regulators, one of which is..., Heckmann [/bib_ref]. NSP1 and NSP2 can form a heterodimer, which binds to the promoter of the Early Nodulin 11 (ENOD11) gene, and a single mutation in the LHR I domain of NSP2 affects its binding ability that causes nodule formation and nitrogen fixation [bib_ref] GRAS proteins form a DNA binding complex to induce gene expression during..., Hirsch [/bib_ref]. Other GRAS proteins also form homo-or heteropolymers, such as RAM1-NSP2 [bib_ref] A GRAS-type transcription factor with a specific function in mycorrhizal signaling, Gobbato [/bib_ref] and RAD1-LjNSP2, RAM1-RAD1 [bib_ref] Hyphal branching during arbuscule development requires reduced arbuscular mycorrhiza, Park [/bib_ref] , and DIP1-RAM1 and regulates the nodulation process in plants. A GRAS gene called Reduced Arbuscular Mycorrhization 1 (RAM1) acts as a major gene in the transcriptional activation of the late arbuscule-related genes that are essential during the nutrient exchange by targeting carbohydrate and lipid metabolism genes [bib_ref] Hyphal branching during arbuscule development requires reduced arbuscular mycorrhiza, Park [/bib_ref]. Moreover, in Petunia hybrida, the genes involved in the early stages are not affected by the ram1/ata mutation. However, the genes involved in late arbuscule functioning, for example, PT4 and STR, are activated explicitly by RAM1 [bib_ref] Role of the GRAS transcription factor ATA/RAM1 in the transcriptional reprogramming of..., Rich [/bib_ref]. In M. truncatula, RAM1 and NSP2 are required to form cutin monomers during fungal entry [bib_ref] Signaling at the root surface: the role of cutin monomers in mycorrhization, Murray [/bib_ref]. RAM1 from M. truncatula has also been reported to be involved in arbuscular development [bib_ref] A GRAS-type transcription factor with a specific function in mycorrhizal signaling, Gobbato [/bib_ref]. In rice, DELLA INTERACTING PRO-TEIN (DIP1) is a crucial gene for mycorrhizal colonization. Mutations in DIP1, RAM1, and DELLA (SLENDER RICE1) showed a substantial reduction in AM formation, although the exact mechanism of arbuscular branching has not been investigated yet. Further, the expression of RAM1 is regulated by CCaMK/ CYCLOPS and DELLA [bib_ref] A CCaMK-CYCLOPS-DELLA complex activates transcription of RAM1 to regulate arbuscule branching, Pimprikar [/bib_ref]. CCaMK interacts with CYCLOPS to activate RAM1 by directly binding at a conserved palindromic region (GGCGCC box) of the RAM1 promoter [bib_ref] A CCaMK-CYCLOPS-DELLA complex activates transcription of RAM1 to regulate arbuscule branching, Pimprikar [/bib_ref]. DIP1 interacts with mycorrhizal GRAS protein RAM1 to regulate mycorrhizal-associated gene expression. Other GRAS proteins are also involved in AM degeneration regulation, which involves the interaction of MYB1 with DELLA and NSP1. This interaction regulates the Cysteine protease 3 (CP3) gene, a wellknown player involved in AM degeneration . MYCORRHIZA-INDUCED GRAS (MIG1), a GRAS-domain TF found in M. truncatula, expresses in arbuscule-containing cells, suggesting a specific function during fungal association. It was also reported that MIG1 also interacts with DELLA, and this complex regulates radial cell expansion in roots to adjust fungal infection structures during AM symbiosis [bib_ref] Symbiotic fungi control plant root cortex development through the novel GRAS transcription..., Heck [/bib_ref] [fig_ref] Figure 4: Role of GRAS TFs in different biological processes in plants [/fig_ref]. Overall, these studies suggest that nodulation and arbuscule formation involves calcium and hormone signaling. The RAM1 promoter act as a central integration node of this calcium (CCaMK/CYCLOPS) and hormonal (DELLA/GA) signaling [fig_ref] Figure 4: Role of GRAS TFs in different biological processes in plants [/fig_ref] , which plays a vital role in the regulation of nodulation and AM development in plants. However, the function of GRAS proteins in nodulation and AM symbiosis remains largely unknown, and much more study is required to understand the details. ## Phytohormones and growth regulation Phytohormones are the master regulators of various biological processes in plants and play a central role in various growth and stress signaling pathways. For instance, GAs are involved in plant developmental processes, including leaf expansion, stem elongation, hypocotyl elongation, seed germination, pollen maturation, trichome development, and flowering. Most of the GA signaling pathway components have been identified in plants. The DELLA (a major GRAS TFs subfamily) acts as a central component of growth inhibition [bib_ref] Releasing the brakes of plant growth: how GAs shutdown DELLA proteins, Achard [/bib_ref]. In earlier reports, it has been shown that the GAI and RGA (DELLA proteins) degradation occurs via the E3 ligase components. A conformational change occurs by the interaction between GA-GID1-DELLA complex in the presence of GA, which is subsequently recognized by SCFSLY1/GID2 and causes the degradation of DELLA protein via the E3 ubiquitin ligase complex. The degradation of DELLA proteins subsequently activates the downstream genes (Dill and . DELLA gain-of-function mutants showed dwarf phenotype and insensitivity against GA; however, loss-of-function mutants showed enhanced GA sensitivity resulting in slender or tall phenotypes . Degradation of RGA proteins is delayed by Xanthomonas effector protein, which helps plants provide tolerance against different stress ll OPEN ACCESS iScience 25, 105026, September 16, 2022 15 iScience Review conditions. The ERF-associated amphiphilic repression (EAR) motif of Xanthomonas effector protein interacts with the DELLA motif of RGA proteins and regulates the GA-induced binding of GID1 [bib_ref] The Xanthomonas campestris effector protein XopDXcc8004 triggers plant disease tolerance by targeting..., Tan [/bib_ref]. In contrast to Arabidopsis, only one highly conserved DELLA was found in tomatoes, grapevines, and cereals. In tomatoes, a DELLA gene called PROCERA (PRO) has been characterized by a missense mutation in the GRAS domain region [bib_ref] Procera is a putative DELLA mutant in tomato (Solanum lycopersicum): effects on..., Bassel [/bib_ref] [bib_ref] morphology, cell division and expansion, and the auxin-signaling pathway during fruit-set and..., Carrera [/bib_ref]. This mutant showed partial responsiveness to GA, whereas an enhanced GA responsive mutant suggested the retention of its partial activity [bib_ref] Characterisation of the procera mutant of tomato and the interaction of gibberellins..., Van Tuinen [/bib_ref] , which confers that tomatoes have both DELLA-independent and DELLA-dependent pathways for GA-signaling. During the ''green revolution,'' dwarf cultivars (RHT-B1 and RHT-D1, an ortholog of GAI) have been selected from wheat, which lacks GA response. These dwarf mutants (Rht-B1 and Rht-D1) have reduced GA response because of truncation near the DELLA domain [bib_ref] Green revolution' genes encode mutant gibberellin response modulators, Peng [/bib_ref]. Similar truncation has been found in the D8 genes of maize. Maize contains two duplicated DELLA genes, DWARF PLANT 8 (D8) and DWARF PLANT 9 (D9), found on different chromosomes. Both D8 and D9 genes are gibberellin-insensitive and produce dwarfing phenotypes. The Arabidopsis transgenics carrying the dominant maize D8 and D9 alleles also showed a reduced length of rosette leaves, siliques, inflorescence, stems, root structures, shortened filaments, and produced dwarf floral structures with a few seeds [bib_ref] Maize DELLA proteins dwarf plant8 and dwarf plant9 as modulators of plant..., Lawit [/bib_ref] [bib_ref] Physiological genetics of the dominant gibberellin-nonresponsive maize dwarfs, Dwarf8 and Dwarf9, Winkler [/bib_ref]. In maize, D8 and D9 mutants also exhibited male sterility [bib_ref] Physiological genetics of the dominant gibberellin-nonresponsive maize dwarfs, Dwarf8 and Dwarf9, Winkler [/bib_ref]. The dwarf plant 8 (d8) is a homolog of Reduced height (Rht1d) from wheat, Slender1 from barley, and Slender rice1 from rice [bib_ref] Maize DELLA proteins dwarf plant8 and dwarf plant9 as modulators of plant..., Lawit [/bib_ref] [bib_ref] Physiological genetics of the dominant gibberellin-nonresponsive maize dwarfs, Dwarf8 and Dwarf9, Winkler [/bib_ref]. The Arabidopsis transgenic carrying the grapevine Gibberellin Insensitive1 (VvGAI1) mutant alleles with three different promoters of wheat (Rht-B1b), barley (Sln1d), and Brassica (Brrga1-d) [bib_ref] Mutants at the Slender1 locus of barley cv himalaya. Molecular and physiological..., Chandler [/bib_ref] [bib_ref] A novel dwarfing mutation in a green revolution gene from Brassica rapa, Muangprom [/bib_ref] showed very short internodes and dwarf phenotype regardless of which promoter was used [bib_ref] Characterization of grape Gibberellin Insensitive1 mutant alleles in transgenic Arabidopsis, Zhong [/bib_ref]. Two DELLA genes in Brachypodium distachyon (BdSLR1 and BdSLRL1) have been characterized in Arabidopsis and found to be involved in GA-mediated signaling and plant development. A similar function was also observed for Arabidopsis, rice, maize, and wheat orthologs . Single base deletion leading to frameshift mutation was observed in rice slender mutant, resulting in mutants exhibiting constitutive GA response phenotypeand reduced JA sensitivity , along with enhanced disease susceptibility. Unlike rice, in Arabidopsis, DELLA proteins are also found to promote susceptibility and resistance to the virulent biotrophs and necrotrophs, respectively. This differential function of DELLA proteins in Arabidopsis and rice might result from JA and SA signaling. It also throws light on the diverged functions of DELLA in rice and Arabidopsis, which might be owing to the evolutionary or signaling divergence between monocots and dicots. In barley, the SLN (for Slender, rice SLR1 ortholog) gene has been identified [bib_ref] Mutants at the Slender1 locus of barley cv himalaya. Molecular and physiological..., Chandler [/bib_ref] and found to be degraded in response to GA. Brassinosteroids (BRs) are steroidal phytohormones that regulate various biological processes, including agronomic traits in crop plants. OsDLT (DWARF AND LOW-TILLERING), a GRAS TF, was involved in BR signaling in rice, and dlt mutants showed phenotypic deformities including the reduced height, less tillering, and late-flowering, similar to that observed in BR-mutants [bib_ref] DWARF and LOW-TILLERING, a new member of the GRAS family, plays positive..., Tong [/bib_ref]. Gene OsDLT was down-regulated in the presence of BR; OsBZR1 can bind to its promoter to repress its expression, as experimentally proved by in vitro assays, which suggests that OsDLT and OsBZR1 regulate each other in opposite ways. In rice, upregulation of DWF4 (BR biosynthesis gene) enhances the tillering number, indicating a positive role of BR in tillering [bib_ref] Brassinosteroids regulate grain filling in rice, Wu [/bib_ref]. In contrast, the BR negatively controls the OsDLT resulting in a reduction in tiller number and grain size in the mutants. Therefore, phenotypes of growth and tiller number are controlled by the expression of DLT in rice. GRAS family members are also found to be involved in auxin signaling. An ortholog of AtSCL15 [bib_ref] The GRAS gene family in Arabidopsis: sequence characterization and basic expression analysis..., Pysh [/bib_ref] from Arabidopsis and BnSCL1 from B. napus are highly expressed in root tissue, and their expression is influenced by auxin [bib_ref] An auxin-responsive SCARECROW-like transcriptional activator interacts with histone deacetylase, Gao [/bib_ref]. Under the influence of auxin, BnSCL1 expression is upregulated, and BnSCL1 interacts with histone deacetylase 19 (HDA19) to regulate downstream genes involved in the radial patterning of root. SCL1 genes in Pinus radiate (PrSCL1) and Castanea sativa (CsSCL1) are the two GRAS genes predominately expressed in roots under the influence of exogenous auxin. ## Phytochrome signaling and growth regulation In addition, to regulate various developmental pathways, GRAS proteins also play a crucial role in light signaling. For instance, SCL21, SCL5, and PAT1 have been involved in phytochrome A (PhyA) signal transduction. Proteins encoded by these three genes contain conserve EAISRRDL motif, which is absent in other family ll OPEN ACCESS iScience 25, 105026, September 16, 2022 iScience Review members, and the mutants of these three genes showed common Phy-A related deformities [bib_ref] PAT1, a new member of the GRAS family, is involved in phytochrome..., Bolle [/bib_ref] [bib_ref] The GRAS protein SCL13 is a positive regulator of phytochrome-dependent red light..., Torres-Galea [/bib_ref]. Further, SCL13 (Scarecrow-like 13), another member of the PAT1 subfamily is involved in the phytochrome B (PhyB) pathway. It is mainly involved in red light signaling and is also known to modulate phyA responses in a phyB-independent manner. The SCL3 antisense lines showed lower sensitivity toward red light and executed their role in hypocotyl elongation in plants [bib_ref] The GRAS protein SCL13 is a positive regulator of phytochrome-dependent red light..., Torres-Galea [/bib_ref]. Two more cytoplasm and nuclear-localized proteins belonging to the PAT1 subfamily, i.e., AtPAT1 and AtSCL21, have also been characterized as positive regulators of phyA signal transduction in Arabidopsis. Moreover, the biochemical evidence showed that AtPAT1 and AtSCL21 interact physically. The double mutant showed an elongated hypocotyl under FR light, suggesting that SCL21 and PAT1 are essential for FR light-mediated signaling. These results suggested that the heterodimeric complex of SCL21 and PAT1 acts as a positive regulator of the phyA signaling pathway [bib_ref] PAT1, a new member of the GRAS family, is involved in phytochrome..., Bolle [/bib_ref] [bib_ref] Two GRAS proteins, SCARECROW-LIKE21 and phytochrome A signal TRANSDUCTION1, function cooperatively in..., Torres-Galea [/bib_ref]. GRAS proteins (especially DELLA) are also involved in regulation shade avoidance responses via phytochrome signaling in plants. In Arabidopsis, low red/far-red light ratio sense by the phyB photoreceptor and the phyB inactivates the PHYTOCHROME INTERACTING FACTOR 4 (PIF4) TFs through phosphorylation. PIF4 TFs are also a target by the GA signaling pathway, phyB promotes GA biosynthesis through regulating the expression of these two genes GA20ox and GA3ox. This leads to the degradation of DELLA proteins that otherwise prevent PIF4 function [bib_ref] The involvement of gibberellin 20-oxidase genes in phytochromeregulated petiole elongation of Arabidopsis, Hisamatsu [/bib_ref] [fig_ref] Figure 4: Role of GRAS TFs in different biological processes in plants [/fig_ref]. Overall, these studies suggest that GRAS forms the core functional module of light signaling involving phytochrome. ## Genome editing of gras genes for plant improvement With recent advances in genome-editing technologies [i.e., zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), RNA-guided CRISPR-Cas nuclease system], several avenues are open for improving plant traits. These genome-editing technologies can precisely target any gene of interest. Here, a summary of recent studies utilizing CRISPR-Cas9 based genome editing to manipulate GRAS genes is provided. CRISPR-Cas9 technology has been implemented to target the GRAS gene in plants . The DELLA domain in GAI locus was targeted in Arabidopsis, and one amino acid was deleted. This resulted in DELLA proteins becoming insensitive to GA-induced degradation, causing repressed growth and a dwarf phenotype . Similarly, in tomatoes, the mutant generated by CRISPR, where one amino acid was deleted from DELLA protein, resulted in a dwarf phenotype [bib_ref] Using CRISPR/Cas9 genome editing in tomato to create a gibberellin-responsive dominant dwarf..., Tomlinson [/bib_ref]. In rice, SLR genes (which encode DELLA) were edited by targeting the DELLA domain, and 16 lines were developed involving six types of mutation in SLR genes [bib_ref] Generation and transcriptome profiling of Slr1-d7 and Slr1-d8 mutant lines with a..., Jung [/bib_ref]. Out of these, two mutants were insensitive toward GA and were dwarf with shrunken leaf and short internode. The SHR subfamily (involved in root radial patterning) has also been edited through the CRISPR-Cas system. In G. max, mutants were developed with anomalous root radial patterning by editing SHR genes [bib_ref] CRISPR/Cas9-mediated genome editing in soybean hairy roots, Cai [/bib_ref]. GRAS genes have also been edited in polyploid plant species where multiple paralogous genes are present. For instance, in B. napus (allotetraploid), four genes (BnaA9.RGA, BnaC9.RGA, BnaA6.RGA, and BnaC7.RGA) belonging to the RGA subfamily of GRAS proteins were edited through CRISPR-Cas . The quadruple mutant obtained was significantly taller than the wild type, implying that GRAS genes can be edited precisely in diploid and polyploid plant species. These studies indicated that the CRISPR-Cas technology has significant potential for crop improvement and extensive usage in agriculture. Till now only a few GRAS genes (DELLAs, SHRs and RGAs) have been targeted that mainly regulate plant height. As GRAS genes were also involved in various biological processes, we suggested that the CRISPR-Cas system should be used to target potential GRAS genes for crop improvement against grain yield, abiotic and biotic stress enhancement. ## Conclusions and future outlook GRAS proteins play a plethora of biological functions, including regulation of growth and development, phytohormones and phytochrome signaling, symbiosis, biotic and abiotic stress regulation. For instance, DELLA acts as a master regulator of the GA gibberellin signaling pathway and is involved in several biological events of plant development, stress regulation, phytochrome signaling, and symbiotic associations. The SCL subfamily is also reported to regulate endodermis development, seed germination, root elongation, and multiple abiotic stress regulations. Similarly, RAM1 act as a central regulator of arbuscular development and is involved in hyphopodia formation in AM fungi, whose transcription is regulated explicitly by CYCLOPS and DELLA [bib_ref] A CCaMK-CYCLOPS-DELLA complex activates transcription of RAM1 to regulate arbuscule branching, Pimprikar [/bib_ref]. However, predominant studies are limited to genome-scale identification and characterization using in silico tools, followed by expression profiling in different tissues, contain the information of GRAS proteins in all the sequenced plant genomes and provide a base for functional studies. Also, efforts need to be directed toward identifying their upstream regulators and downstream target genes. This comprehensive information will help to delineate the cascade of molecular events involving GRAS proteins during the growth, development, and stress responses. Importantly, crystal structure elucidation and in vivo studies are needed to reveal the mode of action of GRAS proteins with their interacting partners and substantiate that GRAS acts as TFs. The genome sequencing data of various lower and higher plant species are available now, which provides an opportunity to explore the evolution and diversification of GRAS genes on an evolutionary timescale. The conserved functional mechanisms of the GRAS family genes among different species suggest its conservation among plant species. For instance, LS, LAS, and MOC1 from tomatoes, Arabidopsis, and rice, respectively, share only about a 50% sequence similarity and show a conserved function in promoting shoot branching or tillering. LAS genes in Arabidopsis and tomato are functionally similar, and a conserved control mechanism exists between two distantly related species. However, the differences in the mutant phenotypes in both species may be attributed to redundant gene activities during flower development in Arabidopsis, leading to variability in phenotypes [bib_ref] Molecular analysis of the LATERAL SUPPRESSOR gene in Arabidopsis reveals a conserved..., Greb [/bib_ref]. The expression of many GRAS TFs is triggered upon stress exposure, suggesting their potential for plant genetic engineering and genome editing (ZFNs, TALENs, CRISPR/Cas9) approaches to improve agronomic traits that can lead to the development of higher-yielding crop varieties. ## Supplemental information Supplemental information can be found online at https://doi.org/10.1016/j.isci.2022.105026. # Acknowledgments # Author contributions Conceptualization, V.G.; writing -original draft, V.J., M.K., P.K., V.G., and G.Z.; writing -review and editing, V.J., V.G., G.Z., and S.K.; data curation, V.J., M.K., and P.K.; visualization, V.J., M.K., P.K., and V.G.; supervision, V.G., G.Z., and S.K.; project administration, V.G. and S.K.; funding acquisition, V.G., G.Z., and S.K. # Declaration of interests The authors declare no conflict of interest. The coffee genome provides insight into the convergent evolution of caffeine biosynthesis. Science 345, 1181-1184. https://doi.org/10.1126/ science.1255274. Di Laurenzio, L., Wysocka-Diller, J., Malamy, J.E., Pysh, L., . The SCARECROW gene regulates an asymmetric cell division that is essential for generating the radial organization of the Arabidopsis root. [fig] Figure 1: Schematic depicting domains of the GRAS proteins (A-C) [/fig] [fig] Figure 2: Phylogenetic analysis of GRASs in plant speciesPhylogenetic tree of GRAS proteins in Arabidopsis thaliana(Lee et al., 2008), Amborella trichopoda(Albert et al., 2013), Capsicum annum, Coffea canephora(Denoeud et al., 2014), Fragaria vesca(Chen et al., 2019a), Gossypium hirsutum(Zhang et al., 2018), Lagenaria siceraria(Sidhu et al., 2020), Musa acuminate(D'Hont et al., 2012), O. sativa [/fig] [fig] Figure 3: Phylogenetic tree of 42 species belonging to bryophyte, lycophyte, gymnosperm, basal angiosperm, and angiosperms (monocot and dicot) visualized as a species tree The divergence times at different nodes are depicted in different geological time scales. The number of genes present/absent of NSP1, NSP2, RAM1, LISCL, DELLA, and PAT1 in different species are shown in columns. Dated phylogeny trees for 31 plant species were retrieved from TimeTree(Kumar et al., 2017). [/fig] [fig] Figure 4: Role of GRAS TFs in different biological processes in plants (A-C). Regulatory circuits mediated by GRAS transcription factors (TFs) during plant growth and development (A), nodulation and arbuscular mycorrhizal development (B), and phytochrome regulation (C). Black arrows indicate transcriptional controls; protein-protein interaction is denoted by red arrows; green denoted the movement of proteins; bars denote negative regulation; and the cross represents the confined movement. BR, brassinosteroid; PKL, pickle; ABI, abscisic acid insensitive; SOM, SOMNUS; AM, arbuscular mycorrhizal; Ca 2+ , calcium; FR, far-red; R, red; PIF4, Phytochrome Interacting Factor 4. ll OPEN ACCESS iScience 25, 105026, September 16, 2022 [/fig] [fig] Figure 5, Figure 6: Expression of GRAS genes at different developmental stages (A and B) Heat maps representing GRAS gene expression patterns in (A) Arabidopsis and (B) rice during various developmental stages. The expression levels of GRAS genes in different developmental stages were analyzed using the Genevestigator tool (Hruz et al., 2008). Heatmap color represents the % of expression potential (log 2 scale). Expression of GRAS genes under different abiotic stresses (A and B). Heat maps representing GRAS gene expression patterns in (A) Arabidopsis and (B) rice under abiotic stresses (cold, drought, heat, and salt). The expression levels of GRAS genes in response to different abiotic stresses were analyzed using the Genevestigator tool (Hruzet al. 2008) (red = upregulation and green = downregulation of genes). ll OPEN ACCESS 12 iScience 25, 105026, September 16, 2022 [/fig] [fig] Fan: S., Zhang, D.,Gao, C., Zhao, M., Wu, H., Li, Y., Shen, Y., and Han, M. (2017). Identification, classification, and expression analysis of GRAS gene family in Malus domestica. Front. Physiol. 8, 253. https://doi.org/10.3389/fphys.2017.00253., T., Li, X., Yang, W., Xia, K., Ouyang, J., andZhang, M. (2015). Rice osa-miR171c mediates phase change from vegetative to reproductive development and shoot apical meristem maintenance by repressing four OsHAM transcription factors. PLoS One 10, e0125833. https://doi.org/10.1371/journal.pone.0125833., Y., Wei, X., Lai, D., Yang, H., Feng, L., Li, L., Niu, K., Chen, L., Xiang, D., Ruan, J., et al. (2021a). Genome-wide investigation of the GRAS transcription factor family in foxtail millet (Setaria italica L.). BMC Plant Biol. 21, 1-19. https://doi. org/10.1186/s12870-021-03277-y. , Y., Yan, J., Lai, D., Yang, H., Xue, G., He, A., Guo, T., Chen, L., Cheng, X.B., Xiang, D.B., et al. (2021b). Genome-wide identification, expression analysis, and functional study of the GRAS transcription factor family and its response to abiotic stress in sorghum [Sorghum bicolor (L.) Moench]. BMC Genom. 22, 1-21. https://doi.org/ 10.1186/s12864-021-07848-z.Feng, Z.,Zhang, B., Ding, W., Liu, X., Yang, D.-L., Wei, P., Cao, F., Zhu, S., Zhang, F., Mao, Y., et al. (2013). Efficient genome editing in plants using a CRISPR/Cas system. Cell Res. 23, 1229-1232.https://doi.org/10.1038/cr.2013.114. Floss, D.S., Gomez, S.K., Park, H.-J., MacLean, A.M., Mü ller, L.M., Bhattarai, K.K., Lé vesque-Tremblay, V., Maldonado-Mendoza, I.E., and Harrison, M.J. (2017 [/fig] [table] Table 1: Details of GRAS genes reported in different plant species [/table]
Bone Microarchitecture in Obese Postmenopausal Chinese Women: The Chinese Vertebral Osteoporosis Study (ChiVOS) Background: Obesity is associated with improved bone mass and microarchitecture in Caucasian individuals, but evidence in obese Asian individuals is lacking.Objective: To analyze the areal bone mineral density (aBMD) and bone microarchitecture in normal-weight, overweight, and obese postmenopausal Chinese women.Methods: A total of 243 postmenopausal women from the Chinese Vertebral Osteoporosis Study (ChiVOS) were included and were divided into three groups (OB, obese group; OW, overweight group; NW, normal weight group) by BMI level. aBMD, trabecular bone score (TBS), and appendicular lean mass (ALM) were measured by dualenergy X-ray absorptiometry (DXA). Bone microarchitecture was measured by HR-pQCT at the distal radius and tibia. X-ray was performed to confirm vertebral fractures (VFs). Multiple linear regression was used to evaluate the correlations between bone parameters and ALM after adjusting for confounding variables.Results: The prevalence of VFs and clinical fractures were similar among the groups. Participants in the OB group showed a lower level of osteocalcin with comparable levels of other bone turnover markers (BTMs). The aBMD at several skeletal sites was higher in the OB group than in the NW group after adjusting for age (p<0.01 for all comparisons). At the radius, the OB group had a higher Ct.Ar, Tb.vBMD, Tb.BV/TV, Tb.N, Tb.Th, and Ct.Th than the NW group after adjusting for covariates (p<0.05 for all). Differences of a similar magnitude were found at the distal tibia. There was a trend of decreasing trend in Tb.Sp, Tb.1/N/SD, and Ct.Po among groups at both sites. However, the bone microarchitecture did not differ between participants with severe obesity (BMI≥35.0kg/m 2 ) and those with 30.0≤BMI<35 kg/m 2 . Multiple linear regression revealed that the associations between ALM and most of the bone microarchitecture parameters at both sites were much stronger than the association between body weight and bone parameters.Conclusion:We have observed significant improvements in aBMD, bone geometry, and bone microarchitecture in obese postmenopausal Chinese women. Except for a lower level of osteocalcin in the OB group, no significant differences in BTMs were found among the groups. Compared with body weight, ALM may explain greater variance in the improvement of bone microarchitecture parameters.Keywords: obesity, BMI -body mass index, postmenopausal women, HR-pQCT (high-resolution peripheral quantitative computed tomography), bone microarchitectureClinical Data CollectionEach participant completed an investigator-administered questionnaire, which contained questions regarding demographic information, personal habits and living environment, physical condition, bone status and fracture history, medications, and bone health knowledge. Alcohol intake (never/former/current drinking and average unit/week) and smoking history (never/former/current Qi et al. Bone Microarchitecture in Obese Women Frontiers in Endocrinology | www.frontiersin.org # Introduction Osteoporosis is characterized by lower bone mass and the deterioration of bone microarchitecture with an increased risk of bone fracture, which has already become a worldwide public health problem due to the high morbidity, mortality, and heavy socioeconomic burden. According to the most recent population-based study in China, the prevalence of osteoporosis in men aged 40 years or older was 5.0% (95% CI 4.2%-5.8%), and that in women was 20.6% (95% CI 19.3%-22.0%) [bib_ref] Prevalence of Osteoporosis and Fracture in China: The China Osteoporosis Prevalence Study, Wang [/bib_ref]. For women aged 80 years or older, the prevalence of osteoporosis in China is even higher than that in most American and European countries, with a prevalence of 67.5% (95% CI 56.5%-78.4%) [bib_ref] Treatment and Costs of Osteoporosis in Germany-the Boneeva Study, Haussler [/bib_ref]. Obesity is an important public health concern worldwide and has a great impact on both mortality and morbidity. Despite being a risk factor for cardiovascular disease and diabetes, the potential protective effect of obesity against osteoporosis is becoming a focus of medical care. Obesity is traditionally considered a protective factor for fractures due to increased loading through bodyweight increasing BMD and the protective effect of soft tissue padding [bib_ref] Obesity and Bone: A Complex Relationship, Rinonapoli [/bib_ref]. However, the relationship between obesity and the risk of fracture is controversial and appears to vary depending on sex and skeletal sites, especially for vertebral fractures (VFs) (5-7), despite improved areal bone mineral density (aBMD) [bib_ref] Bone Density, Microstructure and Strength in Obese and Normal Weight Men and..., Evans [/bib_ref] [bib_ref] In Obese Postmenopausal Women, Bone Microarchitecture and Strength Are Not Commensurate to..., Sornay-Rendu [/bib_ref]. Since dual-energy X-ray absorptiometry (DXA) may overestimate aBMD values at axial sites due to excess fat tissue [bib_ref] A New Perspective on the Causal Influence of Soft Tissue Composition on..., Bolotin [/bib_ref] , and other factors including bone geometry and microarchitecture might also be important determinants of bone strength in addition to BMD [bib_ref] Important Determinants of Bone Strength: Beyond Bone Mineral Density, Friedman [/bib_ref] , researchers have performed highresolution peripheral quantitative computed tomography (HR-pQCT) to measure volumetric densities and the cortical and trabecular compartments in obese individuals, which is less affected by soft tissue padding. Several studies have demonstrated the bone microarchitecture characteristics in obesity, with favorable cortical and trabecular parameters [bib_ref] In Obese Postmenopausal Women, Bone Microarchitecture and Strength Are Not Commensurate to..., Sornay-Rendu [/bib_ref] [bib_ref] Bone Density, Microarchitecture and Strength Estimates in White Versus African American Youth..., Reyes [/bib_ref] [bib_ref] Adiposity and Bone Microarchitecture in the Glow Study, Litwic [/bib_ref]. However, data on the bone microarchitecture across the BMI spectrum are lacking in postmenopausal Chinese women. Owing to the different diagnostic criteria and prevalence of obesity from Caucasian individuals, the characteristics of bone microarchitecture in the Chinese population remain unclear. Regarding this research gap, we conducted a cross-sectional study to evaluate bone microarchitecture in obese postmenopausal Chinese women compared with overweight and normal-weight women from the Chinese Vertebral Osteoporosis Study . The findings will increase our understanding of obesity and bone health. # Methods ## Study design and participants We conducted a subgroup study within a nationwide, observational, population-based study to investigate the prevalence of VFs among postmenopausal women in China (ChiVOS). Briefly, the ChiVOS randomly recruited 2,664 community-dwelling postmenopausal women aged 50 or over from 5 regions throughout China. All participants recruited in Beijing (n=274) were enrolled at Peking Union Medical College Hospital (PUMCH, Beijing, China), and underwent an additional HR-pQCT examination. The weight and height of each participant were measured by standard methods, and body mass index (BMI) was calculated as the weight in kilograms divided by height in meters squared. Since low-weight conditions have typically been regarded as an increased risk for fractures [bib_ref] Long-Term Fracture Risk Among Women With Anorexia Nervosa: A Population-Based Cohort Study, Lucas [/bib_ref] , we excluded participants with BMI<18.5kg/m 2 (n=6). After excluding 25 participants who lacked HR-pQCT measurements, 243 participants were finally included in this study. According to the cutoff points of the Chinese criteria [bib_ref] Department of Disease Control Ministry of Health PRC. The Guidelines for Prevention..., Chen [/bib_ref] , women with BMI≥28.0kg/ m 2 were grouped into the obese group (OB, n=73), women with BMI ranging from 24.0 to 27.9 kg/m 2 were grouped into the overweight group (OW, n=98), and women who had normal BMI, corresponding to a BMI of 18.5-23.9 kg/m 2 , were grouped into the normal-weight group (NW, n=72). Among the obese participants, we further categorized those with BMI≥35kg/m 2 into the severely obese group (n=9). We also performed a subgroup analysis to investigate whether abdominal obesity (AO) would affect bone microarchitecture, defined as waist circumference (WC) ≥85cm, according to the Health Industry Standards of the People's Republic of China classifications [bib_ref] Prevalence of Abdominal Obesity in China: Results From a Cross-Sectional Study of..., Zhang [/bib_ref]. Participants with a history of anti-osteoporosis drug use (n=5) were not excluded due to the small sample size. This study was approved by the ethics committee of PUMCH, and all participants provided written informed consent prior to engagement in any study activities. smoking and average cigarettes/day) were also obtained. Current smoking was defined as smoking at least 1 cigarette/day for more than 6 months, and participants who had quit smoking for more than 6 months when filling out the questionnaire were classified as former smokers. Current drinking was defined as 1 unit of alcohol per week for more than 6 months, and participants who had stopped drinking for more than 6 months when filling out the questionnaire were classified as former drinkers. Information about nutrition and dietary intake included weekly calcium, vitamin D, and milk supplementation status. Information about physical activity (hours and intensity/day) was also collected. A detailed history of self-reported fractures occurring after age 50 was collected. Prior and concomitant medications, history of corticosteroid use, hormone replacement therapy (HRT), and any antiresorptive drugs or teriparatide were recorded (medication name, daily dose, and treatment duration). ## Biochemical measurements The samples were collected from each participant in the morning (7-9 am) after fasting for at least 8 hours. The blood samples were centrifuged at 3,000 r/min for 4 minutes to separate the serum for analysis. Measurements of serum calcium (Ca), phosphorus (P), and alkaline phosphatase (ALP) were performed in each participant with a Beckman Automatic Biochemical Analyzer (AU5800, Beckman Coulter, Indianapolis, IN, USA). Bone turnover markers (BTMs) including beta-C-terminal telopeptide (b-CTX), N-terminal propeptide of type I procollagen (P1NP) and osteocalcin were measured by electrochemiluminescence immunoassay (Roche Cobas e601, Mannheim, Germany). Serum total 25-hydroxyvitamin D (total 25OHD) and parathyroid hormone (PTH) were measured by electrochemiluminescence immunoassay (Roche Cobas e601, Mannheim, Germany) and chemiluminescence (Siemens ADVIA Centaur, Munich, Germany) respectively at the end of enrollment using stored serum samples. The intraassay coefficients of variability of the main measurements are 2.2% -2.6% for PTH, 1.1% -3.1% for 25OHD, 2.7% -3.1% for b-CTX, and 0.9% -1.1% for P1NP; The inter-assay coefficients of variability of the main measurements are 2.8% -5.8% for PTH, 2.2% -4.3% for 25OHD, 2.9% -3.4% for b-CTX, and 1.4% -1.6% for P1NP. All these measurements were made in the central laboratory of PUMCH. ## Abmd, tbs, and appendicular lean mass measurements Certified technicians performed DXA on each participant to measure aBMD at the lumbar spine (LS, L1-L4), femur neck (FN), and total hip (TH), using GE-Lunar scanners (GE Healthcare, Madison, WI, USA). Absolute aBMD values were recorded. The trabecular bone score (TBS) was obtained retrospectively using DXA scans at the same region of interest (L1-L4) and TBS iNsight v2.1 software (Medimaps). Quality control and calibration of DXA machines were performed per routine practice at each site. DXA results were segmented into the trunk and limbs automatically by the software to determine the appendicular lean mass (ALM). The skeletal muscle index (SMI) was calculated as the ALM divided by the square of height (m 2 ) to indicate body-size normalized data and minimize the correlation between lean mass and height. The coefficient of variance (CV%) of DXA was approximately 0.9% to 1.5% for LS, 0.7% to 1.2% for TH, and 1.6% to 2.1% for FN respectively. ## Ascertainment of vfs Morphometric VFs were assessed by the lateral radiographs of the thoracolumbar spine in all women at the visit to our center. Two experienced radiologists independently evaluated the radiographs to diagnose VFs according to the semiquantitative (SQ) technique of Genant [bib_ref] Vertebral Fracture Assessment Using a Semiquantitative Technique, Genant [/bib_ref] in a blinded fashion. Radiographic VFs with SQ≥1 were categorized as clinical VFs. Discrepancies in the diagnosis between the radiologists were resolved by reassessment and ultimate consensus. ## Bone microarchitectural measurements All subjects underwent HR-pQCT of the nondominant distal radius and tibia (Xtreme CT II; Scanco Medical AG, Bassersdorf, Switzerland) to measure bone microarchitecture using a standard protocol [bib_ref] In Vivo Assessment of Trabecular Bone Microarchitecture by High-Resolution Peripheral Quantitative Computed..., Boutroy [/bib_ref]. The isotropic resolution of the images was 61 mm. The reference line was placed at the distal endplate of the nondominant radius and tibia. The first slice of the region of interest (ROI) was 9.0 mm and 22.0 mm proximal to the reference lines of the radius and tibia respectively, with a total of 168 parallel CT slices scanned for the 3D image reconstruction of the nondominant radius and tibia. The parameters consisted of bone geometry indices (Tt.Ar, Tb.Ar and Ct.Ar; Tb.Th and Ct.Th), indices of volumetric BMD (Tt.vBMD, Ct.vBMD and Tb.vBMD), trabecular bone parameters (Tb.BV/TV, Tb.Sp and Tb.N), and cortical porosity (Ct.Po). The CV% of HR-pQCT was 0.7% to 1.5% for Tt.vBMD and Tb.vBMD, 2.5% to 4.4% for trabecular architecture, and 0.9% to 1.5% for the Tt.Ar, Ct.Ar, Ct.Po, Ct.Th, and Ct.vBMD. # Statistical analysis Variables were tested for normality of distribution using the Shapiro-Wilk test. Continuous variables with normal distributions are presented as the means ± SDs, categorical variables are expressed as percentages, and continuous nonnormally distributed data are expressed as medians (interquartile range). Bone parameters were compared among groups by ANOVA and the Kruskal-Wallis H-test for normally and nonnormally distributed data respectively, followed by pairwise comparisons if the p-value for the overall was <0.05. Bonferroni post hoc analysis was used for multiple comparisons among groups. Chi-square or Fisher's exact test was performed for categorical variables. Multiple linear regression analysis was performed to evaluate the correlations between the bone parameters and weight or ALM with adjustment for confounding variables. For subgroup analyses of abdominal obesity (AO), Student's t tests or the chi-square tests were performed to compare parameters between groups. All statistical analyses were performed using IBM SPSS 26.0 software (IBM Corp., Armonk, NY, USA). A p-value<0.05 in a two-tailed test was considered statistically significant for all comparisons. # Results ## Clinical characteristics and the prevalence of vfs A total of 243 postmenopausal women were recruited for this study. [fig_ref] TABLE 1 |: Clinical characteristics in normal-weight [/fig_ref] showed the clinical characteristics of the three groups. The women in the OB group were younger than those in the OW group, with medians of 64.0 and 69.0 years old respectively (p=0.007). The groups did not differ in terms of height or age of menopause, but the OB and OW groups had markedly elevated height loss compared with NW group (p=0.047 and p=0.015, respectively). Obviously, weight, waistline, and BMI were highest in the OB group and lowest in the NW group, with the highest BMI being 37.2 kg/m 2 . Women in the OB group also showed higher ALM and SMI than those in the other two groups (p<0.001 for all comparisons). No significant differences were noted among groups in terms of fall risk regardless of whether the falls occurred in the last year or after age 50. Regarding fracture prevalence, the proportion of individuals with VFs was similar among the groups (16.44% vs. 18.37% vs. 13.89% in the OB, OW and NW groups respectively). Previous fractures (traumatic or nontraumatic) were reported in 5.48% of the OB group vs. 3.06% of the OW and 4.17% of the NW group, with no significant difference among groups. Smoking habits, alcohol intake, physical activity, and calcium, vitamin D, and milk supplementation status were comparable among groups. There was no significant difference in medication use among the groups. [fig_ref] TABLE 2 |: parameters Tb [/fig_ref] showed aBMD, TBS, and BTM measurements for all participants. When stratifying the population by BMI status, the aBMD at several skeletal sites showed a trend of being highest in the OB group, followed by the OW group, and lowest in the NW group, but there was no significant difference between the OW and NW groups. After adjusting for age, differences in aBMD between the OB and NW groups persisted. In contrast, the TBS values were comparable among the groups. The median (interquartile range) values in the OB group for total 25OHD were lower than those in the NW group For other BTMs, a lower level of osteocalcin was found in the OB group than in the OW and NW groups (p<0.05 for all), but no significant differences in serum levels of ALP, b-CTX or P1NP were found among the groups. . ## Comparison of abmd, tbs, and btms To evaluate the effect of severe obesity on bone microarchitecture, we further compared the HR-pQCT parameters of severely obese women (n=9), with those of age-and height-matched moderately obese women (n=9), who had BMIs from 30.0 to 35 kg/m 2 . [fig_ref] TABLE 4 |: Comparisons of bone microarchitecture in severely obese women and age-and height-matched moderately... [/fig_ref] shows the comparisons between the groups. The two groups had similar ages (69.67 ± 10.34 vs. 69.56 ± 9.68, p=0.982) and comparable FIGURE 1 | Comparisons of bone microarchitecture at the distal radius and tibia after adjusting for confound variables in normal-weight, overweight, and obese women. Ct.Ar=cortical area; Tt.vBMD=total volume bone mineral density; Tb.vBMD=trabecular volume bone mineral density; Tb.BV/TV=trabecular bone volume fraction; Tb.N=trabecular number; Tb.Th=trabecular thickness; Ct.Th=cortical thickness. The p-value was adjusted for age, current smoking, alcohol intake, supplementations of calcium or vitamin D, milk drinking, and physical activity. NS, no significant. ## Associations of hr-pqct parameters with body weight and alm To evaluate the role of lean mass in the increase in absolute values of bone microarchitecture parameters, we investigated the relationship between HR-pQCT parameters and ALM or body weight in postmenopausal women as shown in . There were universal associations between most of the bone microarchitecture parameters and body weight after adjusting for confounding variables. Bodyweight was positively associated with bone geometry indices, Tb.vBMD, trabecular bone parameters Tb.BV/ TV, Tb.N, and cortical thickness at both sites (p<0.05 for all comparisons), and negatively correlated with Tb.Sp and Tb.1/ N.SD at the radius and tibia (p<0.01 for all). However, no significant association was detected between body weight and Ct.vBMD or Ct.Po. Interestingly, the association was much stronger between HR-pQCT parameters and ALM than that between HR-pQCT parameters and body weight, indicating the much larger impact of ALM on bone microarchitecture. At the distal tibia site, the effect of ALM on bone geometry was even more pronounced than at the non-weight-bearing radius. Trabecular bone parameters were positively associated with lean mass with the same magnitude at both sites (p<0.05 for all comparisons). ## Subgroup analysis in abdominal obesity classified by waist circumference Since a few studies have suggested a relationship between abdominal obesity and bone health [bib_ref] Abdominal Fat Is Associated With Lower Bone Formation and Inferior Bone Quality..., Cohen [/bib_ref] [bib_ref] Association Between Abdominal Obesity and Fracture Risk: A Prospective Study, Yang [/bib_ref] , we conducted a subgroup study to examine the effect of abdominal obesity (AO) on bone microarchitecture. The women with AO showed higher aBMD at the lumbar spine and total hip with increased TBS (Supplemental [fig_ref] TABLE 2 |: parameters Tb [/fig_ref] , but no difference in VFs prevalence was found between groups (Supplemental [fig_ref] TABLE 1 |: Clinical characteristics in normal-weight [/fig_ref] [fig_ref] TABLE 2 |: parameters Tb [/fig_ref]. We also compared the bone microarchitecture parameters of AO and non-AO groups, but only individual parameters showed differences between groups (Supplemental [fig_ref] Table 3: showed the characteristics of geometric features, vBMD, and microarchitecture measurements at the... [/fig_ref]. # Discussion In this cross-sectional study, we found that obese postmenopausal Chinese women had favorable aBMD and vBMD, and higher values of cortical and trabecular parameters at both the radius and tibia than normal-weight postmenopausal women. However, the bone parameters were not further improved as BMI gradually increased to severe obesity. To the best of our knowledge, this is the first analysis of bone microarchitecture in normal-weight, overweight, and obese Asian women. Obesity has traditionally been regarded as having a beneficial effect on bone density and fracture risks. We also found increased aBMD at several skeletal sites in the OB group, which indicated the adaptations to the excess body weight. Despite the higher aBMD at the LS, postmenopausal women with obesity did not show a decreased prevalence of clinical fractures, suggesting the importance of evaluating the bone microarchitecture in individuals with obesity. Sornay-Rendu et al. [bib_ref] In Obese Postmenopausal Women, Bone Microarchitecture and Strength Are Not Commensurate to..., Sornay-Rendu [/bib_ref] reported greater volumetric density, greater trabecular number with lower trabecular TABLE 5 | Correlation of bone microarchitecture with weight and ALM in postmenopausal women (n=243). ## Radius Tibia separation, and higher Ct.Ar at the distal radius and tibia in obese postmenopausal France women previously. These results were further confirmed by other studies [bib_ref] Suboptimal Bone Microarchitecure in Adolescent Girls With Obesity Compared to Normal-Weight Controls..., Singhal [/bib_ref]. However, the evidence on bone microarchitecture in obese Asian women is insufficient. Our study demonstrated that OB postmenopausal Chinese women had favorable bone microarchitecture parameters compared with NW women, which was in agreement with the results of previous studies in Caucasian individuals [bib_ref] In Obese Postmenopausal Women, Bone Microarchitecture and Strength Are Not Commensurate to..., Sornay-Rendu [/bib_ref] [bib_ref] Suboptimal Bone Microarchitecure in Adolescent Girls With Obesity Compared to Normal-Weight Controls..., Singhal [/bib_ref]. Consistent with these results, we also found greater Tb.vBMD as a result of higher Tb.BV/ TV and Tb.N values. Evans and colleagues [bib_ref] Bone Density, Microstructure and Strength in Obese and Normal Weight Men and..., Evans [/bib_ref] found similar results, reporting a greater amount of trabecular bone and Tb.BV/TV at both the radius and tibia, but similar trabecular thickness, in favor of OB women compared with NW women (55-75 years old). We also noticed a decreasing trend in Tb.Sp at both sites as BMI category increased, as previously described (9), although this finding was not statistically significant. With a significant trend for increasing Ct.Ar and Ct.Th from NW to OB status, our results are generally in agreement with existing literature indicating that OB women had higher Ct.Ar and Ct.Th values. However, we did not find differences in Ct.Po or Ct.vBMD as previously reported [bib_ref] In Obese Postmenopausal Women, Bone Microarchitecture and Strength Are Not Commensurate to..., Sornay-Rendu [/bib_ref]. Racial differences may partly account for the discrepancy since Chinese women had thicker and denser cortices than white women, and the thicker cortices and more plate-like trabecular bone tended to persist with aging [bib_ref] Differences in Bone Microarchitecture Between Postmenopausal Chinese-American and White Women, Walker [/bib_ref]. These advantages may help to narrow the bone microarchitecture gap between OB and NW Chinese women. [formula] Weight ALM Weight ALM b (95%CI) p b (95%CI) p b (95%CI) p b (95%CI) p [/formula] In addition, OB women in our study had a lower BMI than those in previous studies .75] vs. 33.4 ± 3.5, 35.9 ± 5.0, 44.8 ± 0.9 kg/m 2 , respectively) [bib_ref] In Obese Postmenopausal Women, Bone Microarchitecture and Strength Are Not Commensurate to..., Sornay-Rendu [/bib_ref] [bib_ref] Suboptimal Bone Microarchitecure in Adolescent Girls With Obesity Compared to Normal-Weight Controls..., Singhal [/bib_ref] , owing to the different prevalence of obesity between Chinese and Caucasian individuals. The positive association between body weight and Tb.vBMD, Tb.N, Ct.Th and negative association between body weight and Tb.Sp further confirmed the improved bone mass in OB women to be a result of the adaptive effect of increased body weight, which was similar to results in several other studies [bib_ref] Bone Density, Microarchitecture and Strength Estimates in White Versus African American Youth..., Reyes [/bib_ref] [bib_ref] Suboptimal Bone Microarchitecure in Adolescent Girls With Obesity Compared to Normal-Weight Controls..., Singhal [/bib_ref] [bib_ref] Obesity Alters Cortical and Trabecular Bone Density and Geometry in Women, Sukumar [/bib_ref]. Although obesity is believed to be beneficial to bone health, recent studies have shown that there might be a suboptimal adaptation of BMD to greater body weight [bib_ref] Suboptimal Bone Microarchitecure in Adolescent Girls With Obesity Compared to Normal-Weight Controls..., Singhal [/bib_ref] , and morbid obesity could even reverse the improvement of some tibia parameters [bib_ref] Adiposity and Bone Microarchitecture in the Glow Study, Litwic [/bib_ref]. These studies provide insights into the complex relationship between obesity and bone health. Data from the UK arm of the GLOW cohort demonstrated that the improved bone microarchitecture was reversed in morbid obesity (BMI≥35kg/m 2 with hypertension or diabetes) with a decrease in several bone parameters especially the trabecular compartment Tb.vBMD, indicating the limited protection of obesity on bone strength [bib_ref] Adiposity and Bone Microarchitecture in the Glow Study, Litwic [/bib_ref]. We selected participants with BMI≥35kg/m 2 as the severely obese group, and the bone parameters were not further improved as BMI gradually increased to severe obesity. Similarly, Dytfeld and colleagues [bib_ref] Influence of Lean and Fat Mass on Bone Mineral Density (Bmd) in..., Dytfeld [/bib_ref] demonstrated that there was a parabolic relationship between BMI and aBMD in postmenopausal women. LS-BMD was highest in OW (BMI from 25 to 30 kg/m 2 ) women compared with NW or OB women. The GLOW cohort indicated that increased pelvic fracture risk could be seen at both extremes of BMI within a nonlinear model. The log-hazard for pelvic fracture dropped rapidly from the minimum BMI value to the minimum log-hazard at approximately BMI=30 kg/m 2 and then rose gradually [bib_ref] Relationship of Weight, Height, and Body Mass Index With Fracture Risk at..., Compston [/bib_ref]. Owing to the lack of enough participants with BMI over 35 kg/m 2 , expanded samples are required in future studies to investigate the possibility that bone parameters would reach a plateau and even drop gradually at a certain BMI value. The effect of body weight on bone strength is attributable to both lean mass and fat mass. The positive associations between fat mass and aBMD [bib_ref] The Relative Contributions of Lean Tissue Mass and Fat Mass to Bone..., Wang [/bib_ref] or bone microarchitecture parameters at the weight-bearing tibia (10) indicated the direct mechanical effect of fat on bone. However, the positive correlation between fat mass and bone mass was reversed when removing the mechanical loading effect of body weight as previously reported [bib_ref] Relationship of Obesity With Osteoporosis, Zhao [/bib_ref] , suggesting a detrimental effect of fat mass on bone. Conversely, the effect of lean mass on bone strength was much more pronounced than that of fat mass. In a longitudinal study, the absolute value of bone strength at the tibia seemed to be higher in OW children, but bone strength was adapted to the greater muscle instead of the excess of body fat [bib_ref] Bone Structure and Volumetric Bmd in Overweight Children: A Longitudinal Study, Wetzsteon [/bib_ref]. Sukumar and colleagues [bib_ref] Obesity Alters Cortical and Trabecular Bone Density and Geometry in Women, Sukumar [/bib_ref] revealed that lean mass explained greater variance than fat mass in geometric and strength indices of the distal tibia. Consistent with previous studies, we observed a universal positive association between bone parameters and appendicular lean mass not only at the tibia but also at the radius, which was much stronger than those with body weight. Altogether, these findings suggested that the positive effect of weight on bone appears to be mainly due to the relatively increased muscle mass. However, because obesity is associated with a lower increase in lean mass compared with fat mass, the improvements in bone strength are not adapted to the excess fat mass. The relatively lower level of lean mass in OB participants could partly explain why the bone strength changes in OB women are inappropriate to the excess body weight. Although conflicting data have been reported, there is considerable evidence supporting that BMI may not be directly related to the risk of VFs. Similar to the previous study conducted by in women from the UK, we found that OB women and women with AO had a similar risk of VFs compared with NW women. We should not forget that women with VFs tend to have a higher level of height loss, which may overestimate the BMI level. Conversely, BMI does not reflect the changes in body composition in older people, including the loss of muscle and increase in fat, even if the person does have too much body fat. Therefore, BMI might not be an appropriate parameter to evaluate the relationship between obesity and VF risk, and further studies are needed to investigate the influence of other measurements of obesity on VF risk. Some studies have revealed that body composition helps to determine the difference in bone status between VF and non-VF participants [bib_ref] Body Composition and Osteoporosis in Elderly Women, Gillette-Guyonnet [/bib_ref] [bib_ref] Associations Between Body Composition and Vertebral Fracture Risk in Postmenopausal Women, Kuo [/bib_ref]. found the prevalence of VFs is higher in postmenopausal women with increased BMI and fat percentage and in those with loss of lean mass. It's reported that the risk of having osteoporosis was double in individuals with sarcopenia than in normal individuals [bib_ref] Age-and Sex-Related Differences in Body Composition in Healthy Subjects Aged 18 to..., He [/bib_ref]. Together with the significantly positive associations between ALM and bone parameters in our study, these results emphasized the importance of body composition in the evaluation of VF risk. Unfortunately, there is a lack of sufficient data about body composition in our present study, except for ALM, to further investigate the relationship between obesity and VFs. It is commonly agreed that bone quality and bone strength are not directly related to the risk of fractures, which are also influenced by several other factors. Obesity is associated with several endocrine changes that could directly or indirectly affect bone metabolism. In addition to the lower bone turnover rate (10), obesity has been associated with increased levels of leptin and decreased IGF-1 [bib_ref] Skeletal Muscle Insulin Resistance in Morbid Obesity: The Role of Interleukin-6 and..., Mitrou [/bib_ref] [bib_ref] Vertebral Bone Marrow Fat Is Positively Associated With Visceral Fat and Inversely..., Bredella [/bib_ref] , which have a complex relationship with BMD and bone metabolism. The increased levels of pro-inflammatory cytokines in OB individuals are inversely associated with BMD and positively associated with bone resorption [bib_ref] Effects of Inflammatory and Anti-Inflammatory Cytokines on the Bone, Schett [/bib_ref]. Unfortunately, biochemical measurements of inflammatory cytokines, IGF-1, and leptin were not available in our study, and these may help to better clarify the relationship between obesity and bone health. Owing to the relatively small sample size, no significant difference in falling among groups was found in our study. Various studies have reported that the risk of falling in OB people is significantly increased [bib_ref] Sotiriadi-Vlachou S. Obesity and Its Relationship With Falls, Fracture Site and Bone..., Himes [/bib_ref] , due to postural instability (37), poorer balance capacity, and loss of muscle mass and strength [bib_ref] Associations of Sarcopenic Obesity and Dynapenic Obesity With Bone Mineral Density and..., Scott [/bib_ref]. In addition, obesity is associated with various diseases, which can be associated with general weakness and peripheral neuropathy, predisposing individuals to falling [bib_ref] Obesity and Bone: A Complex Relationship, Rinonapoli [/bib_ref]. There are several limitations in our present study. Given the crosssectional design, we can determine that there is an association between obesity and bone microarchitecture but not a causal relationship. Second, morbid obesity may have an influence on the association between obesity and bone microarchitecture. The sample size of women with extremely elevated BMI is very small, making it difficult to investigate whether the favorable microarchitecture would reach a plateau or even be reversed in morbidly obese individuals. Third, we did not measure the body composition or fat distribution, which may provide more information about the relationship between obesity and bone health. Further analyses of the relationship between body composition and HR-pQCT parameters are needed. Finally, we did not perform microstructural finite-element analysis (mFEA) to estimate bone stiffness and strength in this study. Future studies are needed to fill this research gap. # Conclusion We have observed significant improvements in bone geometry and bone microarchitecture at the radius and tibia in obese postmenopausal Chinese women, synchronously with the increased aBMD at several skeletal sites. Except for a lower level of osteocalcin in the OB group, no significant differences in BTMs were found among the groups. Compared with body weight, ALM may explain greater variance in the improvement of bone microarchitecture parameters. # Data availability statement The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author. # Ethics statement The studies involving human participants were reviewed and approved by The ethics committee of Peking Union Medical College Hospital. The patients/participants provided their written informed consent to participate in this study. # Author contributions WX designed the study and revised the manuscript. WQ analyzed all data and drafted the manuscript. WQ, YJ, WL, YC, RJ, QP, OW, ML, XX, WY, and WX collected all the data used in this study. WQ and WX are responsible for the integrity of the data analysis. All authors read and approved the final manuscript. [table] TABLE 1 |: Clinical characteristics in normal-weight (NW), overweight (OW), and obese (OB) postmenopausal women. 1Data presented as mean±SD or median (interquartile range) or percentages. 2Significant values are shown in bold. p1: NW vs. OW, p2: NW vs. OB, p3: OW vs. OB. 3BMI, body mass index; HRT, hormone-replacement therapy; ALM, appendicular lean mass; SMI, skeletal muscle index [SMI = ALM (kg) / height 2 (m 2 )]. [/table] [table] Table 3: showed the characteristics of geometric features, vBMD, and microarchitecture measurements at the distal radius and tibia obtained by HR-pQCT. After adjusting for confounding variables, the OB group showed favorable parameters in both the cortical and trabecular bone. At the distal radius, OB women had 14.79% higher Tt.vBMD and 24.06% higher Tb.vBMD than NW women, which were less improved in OW women. No statistically significant difference was found in Ct.vBMD among the groups. Both Tt.Ar and Ct.Ar were highest in the OB groups, followed by the OW group, and they were lowest in NW women. Notably, Ct.Ar was 14.95% higher in the OB group than in the NW group (p<0.001). The trabecular bone [/table] [table] TABLE 2 |: parameters Tb.BV/TV and Tb.N increased progressively from NW to OW to OB (p < 0.05 for all), and Tb.Sp tended to decrease from NW to OB. The same trend was observed for Tb.1/N.SD from OB to NW. For cortical bone, Ct.Th was greater in the OB group than in the OW group (p = 0.039). Similar results were found at the distal tibia for Ct.Ar, Tb.vBMD, Tb.BV/TV, Tb.Th, and Ct.Th, which were notably increased in the OB group (p<0.05 for all comparisons). Tt.vBMD was also significantly increased as BMI category increased. Tb.N, Tb.Sp, and Tb.1/ N.SD were comparable among groups at the distal tibia. No significant differences were detected in Tb.Ar and Ct.Po among the groups at either sites ( [/table] [table] TABLE 4 |: Comparisons of bone microarchitecture in severely obese women and age-and height-matched moderately obese women. Ar, total area; Ct.Ar, cortical area; Tb.Ar, trabecular area; Tt.vBMD, total volume bone mineral density; Tb.vBMD, trabecular volume bone mineral density; Ct.vBMD, cortical volume bone mineral density; Tb.BV/TV, trabecular bone volume fraction; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp= trabecular separation; Tb.1/N.SD, trabecular inhomogeneity of network; Ct.Th, cortical thickness; Ct.Po, cortical porosity. Ct.Ar, Tb.vBMD, Tb.BV/TV, Tb.Th, and Ct.Th with moderately obese women. [/table]
Physiological correlates of cognitive load in laparoscopic surgery Laparoscopic surgery can be exhausting and frustrating, and the cognitive load experienced by surgeons may have a major impact on patient safety as well as healthcare economics. As cognitive load decreases with increasing proficiency, its robust assessment through physiological data can help to develop more effective training and certification procedures in this area. We measured data from 31 novices during laparoscopic exercises to extract features based on cardiac and ocular variables. these were compared with traditional behavioural and subjective measures in a dual-task setting. We found significant correlations between the features and the traditional measures. The subjective task difficulty, reaction time, and completion time were well predicted by the physiology features. Reaction times to randomly timed auditory stimuli were correlated with the mean of the heart rate ( r = −0.29 ) and heart rate variability ( r = 0.4 ). completion times were correlated with the physiologically predicted values with a correlation coefficient of 0.84. We found that the multi-modal set of physiology features was a better predictor than any individual feature and artificial neural networks performed better than linear regression. The physiological correlates studied in this paper, translated into technological products, could help develop standardised and more easily regulated frameworks for training and certification.Laparoscopic surgery (LS) offers substantial clinical and economic benefits over open surgery, including decreased postoperative pain and better utilisation of hospital beds and antibiotics 1-3 . Consequently, LS is becoming increasingly routine in many surgical conditions. In the United States in 2013, surgeons performed cholecystectomy laparoscopically in 96% of cases 4 . The share of laparoscopy procedures within the total number of appendectomy and cholecystectomies in the United Kingdom in 2017 were respectively 74% and 92%, up from 41 and 84% in 2010 5 .During LS, surgeons coordinate their hands, eyes, and long-shaft instruments in trocars inserted at narrow incisions, while mentally translating two-dimensional real-time video into the three-dimensional intracorporal setting. They continuously translate hand movements into the inverted movements of tool-tips and receive limited haptic feedback. The additional difficulties of performing LS relative to open surgery are widely acknowledged 6 . These highlight further unmet needs in the context of training and assessment, which would be greatly helped by tools that can provide new insights into skill development 7,8 . www.nature.com/scientificreports/ next steps or detecting a potential problem before it becomes an emergency. Focussed on completing a suture, a surgeon may miss a crucial alarm; an instance of inattentional blindness/deafness [bib_ref] Failure to detect critical auditory alerts in the cockpit: evidence for inattentional..., Dehais [/bib_ref] [bib_ref] Inattention blindness in surgery, Hughes-Hallett [/bib_ref]. Or a trainee may reach a performance plateau where additional training does not improve overt patterns but continues to reduce the perceptual-motor and cognitive demands of the task. These considerations suggest that performance based metrics alone may not suffice to reveal the trainees' actual state of readiness. Although not as directly observable or well-defined as performance itself, cognitive load has nonetheless been operationalised in multiple ways. An established technique involves introducing a secondary task to be done concurrently, and observing the extent to which it affects the performance of the primary task 18 . Secondary task methods have been used to measure the cognitive load associated with LS [bib_ref] Single incision laparoscopic surgery (SILS) is associated with poorer performance and increased..., Montero [/bib_ref] [bib_ref] Differences in mental workload between traditional and single-incision laparoscopic procedures measured with..., Scerbo [/bib_ref]. A common technique is to employ a secondary task involving the operator's ability to respond to an environmental stimulus such as a visual or auditory change. In this case the reaction time (the duration between stimulus onset and the subject's response to it) and detection rate (e.g. the fraction of times the stimulus is responded to) are taken as the relevant performance measures [bib_ref] Assessment of cognitive load in multimedia learning with dual-task methodology: Auditory load..., Brünken [/bib_ref]. There are, in addition, numerous survey based approaches that obtain information from the self-reporting of the subjects' experience. One of the most widely used is the questionnaire known as NASA-TLX [bib_ref] NASA-task load index (NASA-TLX); 20 years later, Hart [/bib_ref] [bib_ref] Physical and mental workload in single-incision laparoscopic surgery and conventional laparoscopy, Koca [/bib_ref] [bib_ref] Higher mental workload is associated with poorer laparoscopic performance as measured by..., Yurko [/bib_ref]. The amount of effort thus measured can be assumed to indicate the operator's residual capacity available for dealing with unexpected events and for planning and strategy [bib_ref] On the economy of the human-processing system, Navon [/bib_ref] [bib_ref] Multiple resources and performance prediction, Wickens [/bib_ref]. Secondary task and subjective ratings were concurrently used in measuring the cognitive load of novices in the early phases of LS skills training, with subjects monitoring a patient's vital signs (secondary task) while tying surgical knots [bib_ref] Measuring cognitive load during simulation-based psychomotor skills training: Sensitivity of secondary-task performance..., Haji [/bib_ref]. These traditional measures face some limitations, as they may influence or interrupt what they are trying to monitor. As alternatives to secondary task and subjective methods, physiological measures are available for tracking operator states, often in the form of wearable devices. They are gaining in popularity as progress in sensor technology makes this approach less intrusive and capable of delivering continuous, multi-modal information. Physiological responses are tightly coupled to the experienced load during task performance, and cardiac and ocular measures are among the most informative physiological indicators [bib_ref] The influence of stress responses on surgical performance and outcomes: Literature review..., Chrouser [/bib_ref] [bib_ref] Measuring mental workload using physiological measures: A systematic review, Charles [/bib_ref] [bib_ref] Relationships between features of autonomic cardiovascular control and cognitive performance, Duschek [/bib_ref]. The heart rate (HR) and heart rate variability (HRV) are autonomic response markers which can quantify stress and cognitive load in the surgical context [bib_ref] Heart rate and heart rate variability as indirect markers of surgeons' intraoperative..., Rieger [/bib_ref]. The changes in intraoperative HR tend to be larger than can ascribed to physical and musculoskeletal demand [bib_ref] Stress impairs psychomotor performance in novice laparoscopic surgeons, Arora [/bib_ref]. Accordingly, HR has been used as an indicator of acute stress in surgeons [bib_ref] Increased cardiovascular and anxiety outcomes but not endocrine biomarkers of stress during..., Alobid [/bib_ref] [bib_ref] Heart rate as an indicator of stress in suegeons and anaesthetists, Payne [/bib_ref]. Under resting conditions HR typically oscillates, increasing/decreasing during inspiration/expiration, in what is known as the respiratory sinus arrhythmia (RSA) [bib_ref] Respiratory sinus arrhythmia in humans: How breathing pattern modulates heart rate, Hirsch [/bib_ref]. The abatement of RSA by the sympathetic system correlates with forms of arousal and cognitive load [bib_ref] Respiratory sinus arrhythmia: autonomic origins, physiological mechanisms, and psychophysiological implications, Berntson [/bib_ref]. HRV is used to describe such changes in RSA that are readily derivable from the electrocardiogram. HRV may detect autonomic changes even when measures of HR remain constant. It was found to decrease during the surgical decision making steps [bib_ref] The surgeon's mental load during decision making at various stages of operations, Czyżewska [/bib_ref] , could detect the cognitive load associated with LS [bib_ref] A prospective randomized trial on heart rate variability of the surgical team..., Böhm [/bib_ref] , and helped identify the segments of coronary bypass surgery that were differentially stressful for attending and resident surgeons [bib_ref] Intraoperative heart rate variability of a cardiac surgeon himself in coronary artery..., Song [/bib_ref]. Healthy, resting subjects spontaneously blink their eyes approximately 14 times per minute. These endogenous eye blinks occur more frequently than needed for maintaining the corneal tear film, which may be their primary function [bib_ref] Tear film stability: A review, Sweeney [/bib_ref]. Endogenous blinks (in contrast with the protective reflex and voluntary ones) are driven in part by dopaminergic processes originating from basal ganglia, and their frequency and duration reflect mental states [bib_ref] Blinks as an index of cognitive activity during reading, Orchard [/bib_ref] [bib_ref] What does eye-blink rate variability dynamics tell us about cognitive performance, Paprocki [/bib_ref] [bib_ref] On the act of blinking, Ponder [/bib_ref]. Since blinks momentarily block vision, it is plausible that they would be suppressed when expecting important visual input. Evidence supports this view [bib_ref] The timing and temporal patterns of eye blinking are dynamically modulated by..., Oh [/bib_ref] , but also suggests that the blink rate (BR) decreases with increasing attention and task engagement regardless of stimulus modality [bib_ref] Prediction of subjective states from psychophysiology: A multivariate approach, Fairclough [/bib_ref] [bib_ref] Predicting work performance in nuclear power plants, Hwang [/bib_ref] [bib_ref] Eyeblink activity as an index of cognitive processing: Temporal distribution of eyeblinks..., Ichikawa [/bib_ref] [bib_ref] Eye-blinks in choice response tasks uncover hidden aspects of information processing, Wascher [/bib_ref] [bib_ref] An analysis of mental workload in pilots during flight using multiple psychophysiological..., Wilson [/bib_ref]. The goal of the present study was to investigate the ability of physiological variables to track the level of effort associated with LS training tasks. Our study focussed on the major non-cerebral responses described by cardiac and ocular variables, HR, HRV and BR. These were derived from concurrent near-infrared spectroscopy (fNIRS) and electroencephalography (EEG) recorded from novice subjects. As they performed tasks on a LS trainer box, the subjects responded to randomly timed auditory stimuli by pressing a pedal. They reported their experience by filling out a questionnaire after each session. We postulated that the physiological metrics would track the overall effort as indexed by the traditional secondary task and subjective methods, and sought to quantify their ability to do so through statistical and machine learning techniques. At present, there is limited research on the extent of the agreement among these distinct measures of effort and no studies to our knowledge in the context of LS training. In our study, varying levels of effort were generated by differences in subject aptitude and task difficulty, as well as by learning and time on task effects. We believe physiological responses which track their effects are more direct than behavioural and verbal ones, and are more easily measured. Thus, a better understanding of the physiological expression of skill is of potential value for improving training and assessment in LS. # Results In Figs. 1 and 2 we present the descriptive statistics of the data collected. That is followed by the prediction of cognitive load from physiological variables. [fig_ref] Figure 3: Values of the individual predictor features and targets [/fig_ref] drill down into the details of the some of the associations revealed in [fig_ref] Table 1: Prediction performance for the task episodes [/fig_ref]. Further results elicited by analysis of HR and HRV are displayed in Figs. 5 and 6. The experience related and behavioural variables are given in [fig_ref] Figure 1: Experience and performance metrics grouped by type of experimental episode [/fig_ref]. The first row is the subjects' experience as reported in their NASA-TLX scores. The NASA-TLX questionnaire asked subjects to score the task along the dimensions Mental, Physical, Temporal, Performance, Effort and Frustration, on a scale from 0 to 100 in increments of 5. The average of these dimensions is shown in [fig_ref] Figure 1: Experience and performance metrics grouped by type of experimental episode [/fig_ref]. The overall shifts in scores were evident and consistent with expectations. Similar shifts in the average score were present in all the individual dimensions; we show only Effort scores in [fig_ref] Figure 1: Experience and performance metrics grouped by type of experimental episode [/fig_ref] www.nature.com/scientificreports/ repeated. In [fig_ref] Figure 1: Experience and performance metrics grouped by type of experimental episode [/fig_ref] the Effort scores are selected for display because most of the repetition effects were statistically significant. Performance scores (not shown) also contained changes that were statistically significant. There did not appear to be a significant difference between the experiences of the Ring Transfer and Threading tasks. The secondary-task based measures had a greater number of statistically significant between-group shifts as shown in the second row of the figure. The reaction time [fig_ref] Figure 1: Experience and performance metrics grouped by type of experimental episode [/fig_ref] was shortest during the Baseline, as expected, since the pedal press task was then being performed by itself. The reaction time tended to increase (both its median and range) during task performance, and visibly dropped when the first task was repeated. The nonresponse rate [fig_ref] Figure 1: Experience and performance metrics grouped by type of experimental episode [/fig_ref] showed a similar pattern of increase followed by a decrease related to repetition. The non-response rate was a significantly greater for Ring Transfer than for the Threading task. The non-response rate was the rate at which a subject failed to respond to the stimulus before the next stimulus onset. The primary task's completion time [fig_ref] Figure 1: Experience and performance metrics grouped by type of experimental episode [/fig_ref] also showed a strong repetition effect. The completion of Threading appeared to be significantly quicker than that of the Ring Transfer. The rates of error did not display strong trends [fig_ref] Figure 1: Experience and performance metrics grouped by type of experimental episode [/fig_ref] although the increase in the median of type 10 Error in going from the first to the second task appeared to mirror the experience and secondary task variables. Error type 10 was dropping a ring on the ring stack board; type 20 was dropping it outside the board; and type 30 was dropping it outside the trainer box. The physiological variables are shown in [fig_ref] Figure 2: Physiological metrics, heart rate [/fig_ref]. The left column of the figure shows robust differences between the Rest and Task episodes for HR, HRV and BR. In fact, each Rest episode was statistically significantly different from each Task episode; therefore, the usual asterisks were omitted from the left column to avoid cluttering. In addition, the Baseline values for HR and HRV were situated about halfway between Rest and Task, consistent with the expectation that performing only one task is easier than performing two at the same time. In the case of HR, the difference between Task 1 and its repetition was significant, while this was not the case for HRV or BR. [fig_ref] Figure 2: Physiological metrics, heart rate [/fig_ref] shows a significant difference in HR between the first and repeat performances of Threading, while [fig_ref] Figure 2: Physiological metrics, heart rate [/fig_ref] indicates that BR was far lower in Threading than in Ring Transfer. Outlier data are shown as the ' + ' signs. A large number of outliers (e.g. [fig_ref] Figure 2: Physiological metrics, heart rate [/fig_ref] suggests a non-normal distribution. Outliers were defined as points greater than q 3 + w(q 3 − q 1 ) or less than q 1 + w(q 3 − q 1 ) , where q k is the k th quartile and w = 2. Having found task-dependent patterns in the subjective, behavioural and physiological data with different degrees of inter-subject variability, we next proceeded to the central concern of this study, namely the extent to which the physiological variables could predict experience and behaviour that are traditionally used for measuring effort. [fig_ref] Table 1: Prediction performance for the task episodes [/fig_ref] shows how HR, HRV and BR derived variables (predictors) related to the traditional variables (targets), individually as well as collectively via machine learning approaches. The the Pearson correlation ( r ) for individual features. It also shows the adjusted-R and cross-validated-R, explained in the "Methods" and Supplementary Information sections, for assessing the performance of the linear regression (LR) and artificial neural network (ANN). Regarding the individual features in [fig_ref] Table 1: Prediction performance for the task episodes [/fig_ref] , the Pearson correlations show that HR's mean and standard deviation tended to increase with the experienced difficulty as measured by the NASA-TLX score. Without reaching statistical significance both were individually positively correlated with it. Mean HR was negatively correlated with the Reaction Time and Non-response Rate. Higher HR generally implied quicker and more accurate response in the secondary task and fewer errors in the primary task. HRV appeared to be an even better predictor. The minimum value of HRV was significantly negatively correlated with the NASA-TLX score. Many HRV features were significantly positively correlated with the Reaction Time, Completion Time, and Error Rate. This indicated that higher HRV (which is associated with respiratory modulation of HR and relaxation) degraded performance. The BR, although not as predictive as the others, was significantly negatively correlated with the Reaction Time. Note that many of the correlation values in [fig_ref] Table 1: Prediction performance for the task episodes [/fig_ref] may be considered as low or negligible [bib_ref] A guide to appropriate use of correlation coefficient in medical research, Mukaka [/bib_ref]. Those predictors do not have high degree of linear dependence on their target. Note, however, that the value the Pearson coefficient is distinct from its statistical significance. (The latter is indicated by an asterisk in the table.) We used linear correlation only as a baseline to compare with the performance of groups of physiological metrics that had predictive utility via machine learning (the right column of the table). www.nature.com/scientificreports/ With regard to the LR and ANN which used all available features (the last two columns on the right of [fig_ref] Table 1: Prediction performance for the task episodes [/fig_ref] , the results indicated that the full set of features collectively outperformed any individual feature in predicting subject experience and performance. In almost all rows, the table shows that R adj and R CV were greater than the absolute values of the individual Pearson correlations. Furthermore, ANN outperformed LR in almost all cases, except in predicting the Error Rate which was the unique case with R CV < R adj . Having shown how physiological variables were related to experience and behaviour, we looked more closely at the relationships that were quantified in [fig_ref] Table 1: Prediction performance for the task episodes [/fig_ref]. We examined the scatter plot of the predictor and target data points in [fig_ref] Figure 3: Values of the individual predictor features and targets [/fig_ref]. The figure shows in subplots a-f the cases shown in bold for individual features of [fig_ref] Table 1: Prediction performance for the task episodes [/fig_ref]. In subplots a-c, the dependences of the Reaction Time, Non-response Rate and Completion Time on HR are described. In subplots d-f, the same targets are shown plotted against HRV. Finally, since the NASA-TLX score was traditionally used as a measure of stress (as HR and HRV), we also show in subplots g-i the same targets plotted against the NASA-TLX scores. In addition to the linear regression shown as the thin black line in each subplot, we also computed a best fit by including the square of the predictor. Only if the fit was statistically significant, we included this quadratic regression as a thick grey curve in the plot. When the quadratic regression was significant (subplots b, c and i) the best fits were U-shaped curves, suggesting that there was a performance optimum. Continuing with the scrutiny of the results in [fig_ref] Table 1: Prediction performance for the task episodes [/fig_ref] , we show a scatter plot of the actual and predicted values of two variables in [fig_ref] Figure 4: Predictions of the artificial neural network plotted against the actual values [/fig_ref]. These correspond to the predictions by ANN shown in bold in the last column on the right of [fig_ref] Table 1: Prediction performance for the task episodes [/fig_ref]. Completion Time [fig_ref] Figure 4: Predictions of the artificial neural network plotted against the actual values [/fig_ref] was the most accurately predicted target, with R CV = 0.84 , while the NASA-TLX score (A) was the second best, with R CV = 0.60. www.nature.com/scientificreports/ We described in [fig_ref] Figure 2: Physiological metrics, heart rate [/fig_ref] the episode averages of the HR and HRV. However the heart beats approximately once per second, which is sufficiently frequent to allow the temporal variations of HR and HRV within episodes to be observed. These variations are illustrated in [fig_ref] Figure 5: Heart rate [/fig_ref] that shows the time courses of the subject mean (black curves) and inter-subject variability (shaded regions). For the task episodes only the beginning and end segments are shown since the task duration was different for each subject. The figure suggests that during task performance the HR and HRV reach values that are anti-correlated and, as such, potentially contain comparable amounts of information about effort. However, the intra-episode time courses reveals a significant difference between these two measures. While the HR climbs gradually up to its maximum level within the first minute or so, the HRV attains its task-related minimum without any visible delay. [fig_ref] Figure 5: Heart rate [/fig_ref] showed differences in the time-courses of HR and HRV. However, there are additional differences in how the change in these metrics depend on their starting point. This is partly revealed in [fig_ref] Figure 6: Relationships among the heart rate [/fig_ref] , a scatter plot of HR v HRV during the rest (black dots) and task (red dots) periods. The linear regression lines to the data are shown in the corresponding colours. The distribution of HR and HRV are also shown at the bottom and right. Task performance causes the HR distribution shift to the right as a whole, while the distribution of HRV changes shape and becomes more concentrated at small values. The reasons for these differences become evident when we consider how the change in HR (in [fig_ref] Figure 6: Relationships among the heart rate [/fig_ref] and HRV (in [fig_ref] Figure 6: Relationships among the heart rate [/fig_ref] depended on the subjects resting values. The best fit in [fig_ref] Figure 6: Relationships among the heart rate [/fig_ref] is almost flat suggesting that the shift in HR does not depend on its starting point, while it is sloping down in in [fig_ref] Figure 6: Relationships among the heart rate [/fig_ref] suggesting that the shifts in HRV are strongly negatively correlated to their starting points. This indicates that only the HRV of those subjects that have high resting HRV are affected by task performance. # Discussion This paper showed that the cognitive load on novice trainees during LS training has physiological correlates. We showed that physiological indicators of cognitive load predict experience and performance. As physiological metrics, we calculated from N = 31 subjects the heart rate, heart rate variability and blink rate. We measured their subjective experience by the NASA-TLX reports and their performance in a dual-task setting. We used regression and artificial neural networks to discover relationships among the variables. Our hypothesis was that the residual capacity revealed traditionally by reports and other measures such as reaction time would be reflected in their physiology. The motivation was to develop new metrics to guide training so that surgeons train not only to become good at their primary technical tasks, but to have sufficient capacity for planning and for unexpected events. Although some combination of HR, HRV and BR as well as NASA-TLX and dual-task performance have been used in the past to investigate workload in surgery, we are not aware of other studies which brought all of these variables and machine learning together, within the experimental setting of laparoscopy training. We found that changes in physiology, experience and performance depended on whether subjects were resting, performing the secondary task only (baseline) or both tasks [fig_ref] Figure 1: Experience and performance metrics grouped by type of experimental episode [/fig_ref]. Many individual physiological features had some predictive power [fig_ref] Table 1: Prediction performance for the task episodes [/fig_ref]. From least to most effective were BR, HR and HRV features. The superiority of HRV came at the cost of the additional computations that were required. ANN were more effective than LR in predicting experience and performance from physiology, suggesting that the relationships were nonlinear. However, the accuracy advantage of ANN also came at the cost of additional CPU time (1.8 s per round of cross-validation, as compared to 0.07 s for LR). In addition, the full set of features was collectively more predictive than any individual feature. Such synergistic operation of physiological indicators was noticed previously in measuring the mental workload of nuclear power plant operators [bib_ref] Predicting work performance in nuclear power plants, Hwang [/bib_ref]. HRV had the additional advantage of responding to task difficulty within the first few seconds, much faster than HR [fig_ref] Figure 5: Heart rate [/fig_ref]. The regression-to-the-mean which we observed in HRV [fig_ref] Figure 6: Relationships among the heart rate [/fig_ref] has been shown in a mental arithmetic task [bib_ref] Heart rate variability in mental stress: The data reveal regression to the..., Dimitriev [/bib_ref]. To interpret this in another way, note that HRV has a lower bound near zero and, and HRV that is already low at baseline consequently has little room to further decrease under task conditions. Here we have explored potential uses of HRV as a biomarker; the underlying mechanisms of HRV have been extensively probed in other studies [bib_ref] Heart rate variability: origins, methods, and interpretive caveats, Berntson [/bib_ref]. www.nature.com/scientificreports/ In [fig_ref] Table 1: Prediction performance for the task episodes [/fig_ref] both the predictor and target values were selected from the task episodes. We have repeated the calculations in two different ways. First, we repeated them by taking the predictor variables not from the task episodes but from the first rest episode. This was done in order to assess the amount of information carried by the subject's resting physiology about their experience and performance during task. Results showed that in this case all values in the table (except Reaction Time) became smaller and statistically insignificant. The predictability of the Reaction Time from the HR mean, HRV minimum, and LR, remained approximately the same. Second, we took the predictor variables as the change from rest to task. In this case, the predictability measures improved compared to the resting predictors. For example, many values in the last two columns on the right of the table remained significant. Nonetheless, all the values were lower, suggesting that the task physiology by itself was a better predictor than the physiology of task relative to rest, or rest alone. This is consistent with previous finding on the physiological correlates of cognitive performance and attention in non-surgery contexts [bib_ref] Relationships between features of autonomic cardiovascular control and cognitive performance, Duschek [/bib_ref] [bib_ref] Physiological correlates of cognitive functioning in an elderly population, Wright [/bib_ref]. Our results suggested that stress helps improve primary task performance, indexed by Completion Time, up to a point, after which it degrades performance [fig_ref] Figure 3: Values of the individual predictor features and targets [/fig_ref]. Here, stress was quantified by HR and by NASA-TLX score [bib_ref] The influence of stress responses on surgical performance and outcomes: Literature review..., Chrouser [/bib_ref]. This may be the first evidence (to our knowledge) for the Yerkes-Dodson law in the context of surgery training. The law postulates an inverted U-shaped relationship between unspecific arousal and cognitive performance [bib_ref] Yerkes-Dodson: A law for all seasons, Teigen [/bib_ref]. Given that the stress-performance curves may differ among surgeons, objectively evaluating them may improve training as well as optimise intra-operative conditions for performance. The physiological metrics we have found correlated well with subjective and dual-task measures, but not well enough to replace them. We believe physiological correlates may contribute to surgery training not necessarily by eliminating other metrics but by providing additional, sometimes more practical, tools; especially if they are encapsulated in wearable systems. We have only considered cardiac and blink variables, but the number of relevant physiological metrics could be vast, given the complexity of the physiological processes, which underlie skill. Although insight into such underlying mechanisms is still in progress, the metrics can still be used for prediction with the help of machine learning. Furthermore, our results do not depend on the specific modalities (fNIRS and EEG) which were used to extract this information. Their detection could be based on different principles, for example, blinks can be extracted unobtrusively from video. This study was motivated by the view that surgical skill is an important factor that impacts patient outcomes. It has been shown that this impact can be clearly distinguished from that of other factors that interact with it, such as the technology used, patient risk, and the overall quality of care. (An example of such interactions is that difficult cases may be taken up by more highly skilled providers, thereby selectively lowering their success rate.) Recent studies that controlled for these confounds have revealed that surgeons in the top quartile of skill level had significantly better outcomes than those in the bottom quartile [bib_ref] Surgical skill and complication rates after bariatric surgery, Birkmeyer [/bib_ref] [bib_ref] Surgeon specialization and operative mortality in United States: Retrospective analysis, Sahni [/bib_ref]. We were also driven by the view that wearable sensors stand to play an important role in conveniently tracking the physiological correlates of skill. However future research needs to focus more on the effects of the sensors themselves on stress as well as on the normal range of physiological responses. The types of physiological metrics investigated in this study may benefit skills training in robot-assisted surgery [bib_ref] Relationship between surgeon's brain functional network reconfiguration and performance level during robot-assisted..., Shafiei [/bib_ref] [bib_ref] Comparative assessment of physical and cognitive ergonomics associated with robotic and traditional..., Lee [/bib_ref] , in the wider field of human-robot interaction [bib_ref] Physiological and subjective evaluation of a human-robot object hand-over task, Dehais [/bib_ref] and in sports [bib_ref] Inside the brain of an elite athlete: the neural processes that support..., Yarrow [/bib_ref]. We believe that the need for training human surgeons is not likely to diminish in the near future, as surgery is not one of the occupations at risk of being automated [bib_ref] The future of employment: How susceptible are jobs to computerisation?, Frey [/bib_ref]. Our study had some limitations. These included: (i) Using ANNs without optimisation of parameters; (ii) Not implementing subject-specific calibration; (iii) Not controlling for levels of motivation or psychomotor aptitude among the novice subjects, although these variables may have accounted for the distribution in our results; (iv) Recruiting only novice subjects, while different known levels of expertise among the subjects would have provided additional insights; (v) Lack of training interventions accompanied by longitudinal tracking. We hope and expect that these limitations will provide opportunities for further study. Laparoscopy has been spreading, owing to its clear advantages in safety and cost, short hospital stays and rapid return to work. These advantages can translate into significant economic benefits, reducing national 62 and global [bib_ref] Practice, training and safety of laparoscopic surgery in low and middle-income countries, Alfa-Wali [/bib_ref] [bib_ref] Systematic review of laparoscopic surgery in low-and middle-income countries: Benefits, challenges, and..., Chao [/bib_ref] disparities in surgical care. The physiological correlates of surgical performance studied in this paper, translated into technological products, could shrink the learning curves and help develop standardised and more easily regulated frameworks for training and certification [bib_ref] A decade of imaging surgeons' brain function (part I): Terminology, techniques, and..., Modi [/bib_ref] [bib_ref] A decade of imaging surgeons' brain function (part II): A systematic review..., Modi [/bib_ref]. # Methods ## Participants. Thirty-eight healthy adult volunteers without any prior experience in laparoscopic surgery participated in one main trial to perform pre-determined basic laparoscopic tasks on a laparoscopic simulator. We excluded seven participants; one could not enrol in the experiment due to participant's hairstyle, that made it impossible to fit the cap and record EEG; for four participants, technical problems prohibited recording; two were excluded from the main analysis due to a problem with the time stamps routine. Therefore, complete data sets from 31 participants were used in this study (17 females and 21 males, age: 21.61 ± 2.12 years). All participants provided their written informed consent prior to the study commencing and received gift vouchers for participating after the experiment was completed. Participants had normal or corrected to-normal vision. The Ethical Committee of the College of Science and Technology at the Nottingham Trent University approved the study and all research was performed in accordance with relevant guidelines. www.nature.com/scientificreports/ tion with the surgical equipment (Maryland Grasper and Needle Holder, Inovus Medical, St Helens, UK) until they became competent before commencing on the training. The LS trainer box was equipped with a centrally mounted camera and a light source with entry ports of the instruments separated by 13.5 cm. The training bases (14 cm × 10 cm) were placed in the LS trainer box and centred in the camera's field of view. Laparoscopic video was projected onto the monitor that was in the direct view of the participant. The experiment started by performing the secondary task alone for two minutes, which was considered as a baseline [fig_ref] Figure 1: Experience and performance metrics grouped by type of experimental episode [/fig_ref]. For the rest of the experiment, participants performed primary and secondary tasks simultaneously. The primary tasks included two Fundamentals of Laparoscopic Surgery tasks, Ring Transfer and Threading, in alternating sequence (Task1 and Task 2) followed by repetition of the first task (Task 1 Repeated). Fingertip blood samples were taken at baseline and immediately after completion of all three LS tasks to determine the serum cortisol and brain-derived neurotropic factor (BDNF) concentrations. After each blood sample, participants filled in the NASA-TLX questionnaire for each LS task. A rest period of 2 min was taken after the initial secondary task performed alone and after each blood sample, prior to performing the next task. Blood sample analysis is not included in this paper. Participants were instructed to stand in front of the LS trainer box during the performance of Laparoscopic and secondary tasks. The laparoscopic tasks completion time was recorded, with a maximum time on task of 15 min after which the participants were told to stop. The Ring Transfer task involved grasping, lifting and relocating rings from one rod to another using both surgical instruments and was performed on a ring stack base (Inovus Medical, St Helens, UK) [fig_ref] Figure 1: Experience and performance metrics grouped by type of experimental episode [/fig_ref]. Four rods were selected and labelled A, B, C and D, at the left-hand bottom, top left-hand, top right-hand and bottom right-hand corners on the ring stack base respectively. Four rings were initially put over rod A at the beginning of the trial. The procedure includes picking up a ring from rod A and placing it onto rod B with the left-hand only. After transferring all four rings to rod B, participants used their left-hand only to grasp and lift up each ring, pass it to the right-hand and place it on rod C. The procedure was completed by moving the rings individually from rod C to D using the right-hand only. If any rings were dropped during the task completion, they were placed back on the rod they were taken from by an experimenter, and participants were allowed to continue the trial. The Threading task consisted of passing a piece of string through the holes in a pre-determined order. The holes were labelled 1-7 in a zigzag pattern on the Threading base [fig_ref] Figure 2: Physiological metrics, heart rate [/fig_ref]. Participants could use both surgical tools, however, no restriction was made on the use of right, left or both hands. Timing began upon first grasp of the string that was initially placed on the right-hand side of the Treading base. In order to simulate the potential distractions or disruptions (e.g. auditory alarms) that may arise in a realistic setting, an auditory task was added to the experiment. The secondary task, as a disruptive stimulus, provided an additional measure of the cognitive load on the participants in terms of reaction time and non-response rate. Participants had to respond to a series of beeps as quickly as possible by pressing down on a foot pedal. The beeps were generated with random frequencies (ranged from 1,000 to 2000 Hz), intervals (ranged from 3,000 to 10,000 Hz) and durations (ranged from 500 to 1,000 ms). ## Data collection. Participants' brain electrical activities were collected using a wireless data acquisition system by TMSi Mobita 67 with potentially up to 32 gel-based EEG channels, following the international 10-20 system of electrode placement. The EEG signal was collected at 2000 Hz using 19 electrodes out of 32 with average reference, and a ground electrode integrated in a wristband that was attached to the participant's wrist. The fNIRS data were acquired at 10 Hz using Artinis Octamon continuous wave system. The eight fNIRS channels (transmitter-receiver pairs with separations in the range 20-30 mm) covered a region approximately between FP1-F3-F7 on the left and its symmetric counterpart on the right. Hence they sampled parts of ventro-and dorsomedial prefrontal and orbitofrontal cortices [bib_ref] Shining new light on mammalian diving physiology using wearable near-infrared spectroscopy, Mcknight [/bib_ref]. These were combined with EEG electrodes into a single head-cap. EEG and fNIRS signals were synchronised using PortaSync from Artinis 70 , and Lab Streaming Layer (LSL) software 71 . The raw EEG and fNIRS data were analysed offline separately. In this study, EEG was used only for determining the eye blink event times and fNIRS was used only for extracting cardiac information. Analysis of brain activity is not included in this paper due to space limitations. pre-processing. The first step after recording the raw EEG data was to pre-process it to segregate the nonbrain signals, and preserve as much of the brain signal as possible for further analysis. We pre-processed the EEG data using ICA-based method to separate and remove artefacts from background EEG. As suggested in previous studies, additional steps have to be made prior to ICA decomposition. The continuous EEG data was initially epoched into segments based upon the baseline, each Laparoscopic surgery tasks and resting episode, and baseline-corrected using the whole epoch as the baseline [bib_ref] Identifying reliable independent components via split-half comparisons, Groppe [/bib_ref] [bib_ref] Influence of signal preprocessing on ICA-based EEG decomposition, Zakeri [/bib_ref]. The fragments of data that contain substantial noise with high frequency and high amplitude waves, which could have originated from the gross movements of the participants' body during EEG recording were deleted. This was done by using a sliding window of 1,000 ms length and removing the fragments in which at least one of the channels exceeded the identified threshold ± 200 µV. Since the EEG data typically have near zero kurtosis values, the electrodes at which the resulting potential values exceed a pre-defined value (e.g. kurtosis value more than 5) were considered as bad channels and removed from the data. The next pre-processing step was band-pass filtering the data using a zerophase Hamming windowed-sinc FIR filter with passband frequency of 0.16 Hz to 40 Hz to reduce the slow drifts and high frequency artefacts. Afterward, the EEG datasets were down-sampled to 200 Hz to reduce computational and storage cost. In order to segregate components such as eye blinks and eye movements automatically, ADJUST method 76 implemented in EEGLAB software package 77 , was used. Therefore, the filtered EEG data underwent ICA decomposition using Extended-Infomax algorithm to decompose EEG data into independent Scientific RepoRtS | (2020) 10:12927 | https://doi.org/10.1038/s41598-020-69553-3 www.nature.com/scientificreports/ components. Then the ADJUST method was applied to detect the independent components associated with eye blink, horizontal and vertical eye movements, and generic discontinuities automatically. The power spectrum, time series and topographic maps of the independent components were also visually inspected by the experimenter. After removing the components associated with artefacts, the pre-processed data was reconstructed for further analysis. In order to estimate the blink rate, we determined the blink times by detecting the sharp peaks in the associated independent components. In order to capture the cardiac features of interest while minimising other physiological patterns (e.g. Mayer waves) the fNIRS signals were first band-pass filtered in the frequency band 0.5-2 Hz. The signals were converted to hemoglobin concentration changes using the modified Beer-Lambert Law Analysis. The channel-averaged oxy-hemoglobin concentration changes contained a strong oscillatory component related to the cardiac pulsation. Accordingly, we assumed that the period of this signal (the interval between every other zero-crossing) was the period of the heartbeat. Next, from the heart rate (reciprocal of the period) the heart rate variability was found. To prepare for the Fourier transform of the HR, we interpolated its values to the regular temporal grid of the original fNIRS signal. We separately considered components in the low-frequency range 0.01-0.15 Hz and the high-frequency range 0.15-0.8 Hz. The latter envelops the frequencies of normal respiration. For every fixed time segment of the HR, the frequency band powers, LF and HF , in the low-and high-frequency ranges were computed. The heart rate variability was then calculated as HRV = HF (HF + LF) , thus providing a measure of the extent to which the heart rate was modulated by the respiratory rhythms. For every participant and experimental episode we calculated the mean, standard deviation, and the maximum and minimum of the HR and HRV. In the case of BR, only the episode mean was used since blinks were insufficiently frequent to allow reliable averaging within shorter time segments. These were used as features for predicting the cognitive load indexed by subjective experience or by performance. Thus, our predictors were the features derived from physiology while the targets of prediction were derived from NASA-TLX reports and behavioural quantities. Note that, following common usage in machine learning, we are using the term prediction to describe a statistical relationship, without implying that the predictor temporally precedes the target. One of our prediction methods was linear regression (LR). In the simplest case, this involved calculating the correlation between a feature and a target. The result was r , the Pearson correlation coefficient, which ranges in the interval −1 ≤ r ≤ 1 . Using r , rather than its square, allowed us to estimate the extent and type (positive or negative) of the association. More generally, multi-variate LR was employed to determine the association between a set of predictors and a target. In such cases the coefficient of determination, R 2 , could be used to indicate the goodness of fit. However R 2 tends to grow spuriously with the number of predictors. It was therefore preferable to use the adjusted coefficient of determination, R 2 adj , which remains commensurate across different numbers of predictors. We used R adj = R 2 adj in order to make it approximately comparable with r . The absolute value was taken because R 2 adj is not always positive. For further generalizability, we deployed an additional prediction method with the same set of predictor and targets. Cascade-forward (CF) artificial neural networks (ANN) with two hidden layers were trained using the Levenberg-Marquard backpropagation algorithm. CF networks are similar to feed-forward (FF) ones but include additional connections from each layer directly to all successive layers. We selected this configuration (hidden layer sizes 10 and 8) after exploring the performance of various FF and CF networks. The performance of the CF-ANN was determined by means of fivefold cross-validation (CV), which trained the network on part of the data (training set) and used it to predict the values of the remaining test set. At each round of CV, 80% of the full data set was selected as the training set. The process was repeated 5 times so that all values had been predicted. The Pearson correlation between the actual and predicted targets was then computed. The fivefold CV itself was repeated a large number of times (we chose 1,000 iterations which appeared more than sufficient for convergence) and the resulting Pearson correlations were averaged. We refer to the resulting average as the cross-validated correlation coefficient, denoted R CV . The CF-ANN was intended to capture non-linear dependencies which may be missed by LR and, in addition, help determine the effects of the prediction method on the accuracy. No effort was made to rigorously optimize the network parameters since this was not the main topic of our study. Further validation of R adj and R CV is described in Supplementary Information. In assessing group differences we used the Wilcoxon signed-rank test when the groups contained paired subjects (e.g. subjects that performed the first task v second task) and the Kolmogorov-Smirnov test when they did not (e.g. subjects that performed Ring Transfer v Threading as the first task). These tests were deemed suitable for our study, as they were relatively conservative and did not presuppose the normal distribution. We did not utilise a null hypothesis whose rejection would have required multiple comparisons. The Matlab functions regress, fitlm, cascadeforwardnet, autofindpeaks, cvpartition, corr, kstest2 and signrank were utilized for implementing some of the calculations described above (Matlab R2017b, Math-Works, Inc., Natick, Massachusetts, United States). The data that support the findings of this study are available on request from the corresponding author. [fig] Figure 1: Experience and performance metrics grouped by type of experimental episode (three boxes on the left of each subplot) and by task type, namely Ring Transfer, Ring Transfer Repeated, Threading and Threading Repeated (four boxes on the right). (a) NASA-TLX Average score. (b) NASA-TLX Effort score. (c) Reaction time. (d) Non-response rate. (e) Task completion time. (f) The rates of errors during the Ring Transfer task. In the boxes in Figs. 1 and 2, the central mark indicates the median, and the bottom and top edges of the box are the 25th and 75th percentiles. The whiskers extend to the most extreme data points not considered outliers. [/fig] [fig] Figure 2: Physiological metrics, heart rate (HR) and heart rate variability (HRV) extracted from fNIRS signals and blink rate (BR) from EEG. (a) HR grouped by experimental episode. (b) HR grouped by task type and taskspecific repetition. (c) HRV grouped by experimental episode. (d) HRV grouped by task type and task-specific repetition. (e) BR grouped by experimental episode. (f) BR grouped by task type and task-specific repetition. Significant differences were not marked in (a), (c) and (e) in order to avoid clutter. (*p < 0.05).Scientific RepoRtS | (2020) 10:12927 | https://doi.org/10.1038/s41598-020-69553-3 [/fig] [fig] Figure 3: Values of the individual predictor features and targets. Linear fits to the data are shown. The Pearson correlation, r , and the p-value of linear regression (black line) are shown. The quadratic fit (thick grey curve) and its p-value p 2 are shown only if p 2 < 0.05. [/fig] [fig] Figure 4: Predictions of the artificial neural network plotted against the actual values (x-axis) for selected cases. (a) NASA-TLX Average score with R CV = 0.60 . (b) Completion Time with R CV = 0.84 . The 45-degree diagonal line corresponds to perfect prediction. [/fig] [fig] Figure 5: Heart rate (a) and heart rate variability (b) extracted from NIRS signals for the experimental episodes, and smoothed by a 10 s moving window. The black curves represent subject average and the shaded regions are the standard deviation of subject variability. For the task episodes, 40 s segments at the beginning and end are shown.Scientific RepoRtS | (2020) 10:12927 | https://doi.org/10.1038/s41598-020-69553-3 [/fig] [fig] Figure 6: Relationships among the heart rate (HR) and heart rate variability (HRV) for Rest and Task. (a) HR and HRV in resting (black) and task performance (red). Black and red dots respectively represent individual subjects in the resting and task blocks. The linear fits to the data are shown as the black and red straight lines. The Pearson correlations are r = −0.32(resting) and r = −0.23 (task). The insets at the bottom and on the right illustrate the marginal probability distributions, with x-axes matched to the mainfigure.(b) Resting HR and the task related change in HR relative to resting ( r = 0.11 ). (c) Resting HRV and the task related change in HRV relative to resting ( r = −0.96).Scientific RepoRtS | (2020) 10:12927 | https://doi.org/10.1038/s41598-020-69553-3 [/fig] [table] Table 1: Prediction performance for the task episodes. All data from the tasks (T1, T2, T1R) were pooled. The last two columns on the right show the values of R adj and R CV for the performance of regression and artificial neural network, respectively, using the full set of features. Individual features of heart rate, heart rate variability and blink rate are shown in separate columns. For each feature the table shows the Pearson correlation ( r ) between the feature and target. The values in bold were chosen as examples to be examined further. (*p < 0.05). [/table]
A mild form of adenylosuccinate lyase deficiency in absence of typical brain MRI features diagnosed by whole exome sequencing Background: Adenylosuccinate lyase (ADSL) deficiency is a defect of purine metabolism affecting purinosome assembly and reducing metabolite fluxes through purine de novo synthesis and purine nucleotide recycling pathways. The disorder shows a wide spectrum of symptoms from slowly to rapidly progressing forms. The most severe form is characterized by neonatal encephalopathy, absence of spontaneous movement, respiratory failure, intractable seizures, and early death within the first weeks of life. More commonly, ADSL presents purely neurologic clinical picture characterized by severe psychomotor retardation, microcephaly, early onset of seizures, and autistic features (type I) or a more slowly progressing form with later onset, and major features including slight to moderate psychomotor retardation, and transient contact disturbances (type II). Diagnostic markers are the presence of succinylaminoimidazole carboxamide riboside (SAICAr) and succinyladenosine (SAdo) in extracellular fluids. ADSL is a rare disorder, although its prevalence remains unknown. Of note, the wide range of essentially nonspecific manifestations and lack of awareness of the condition often prevent diagnosis. Case presentation: We present here the case of particularly mild, late onset ADSL that has been unsuccessfully investigated until whole exome sequencing (WES) was performed. Conclusions: Besides emphasizing the valuable diagnostic value of WES, this report provides new data further documenting the relatively wide clinical manifestation of ADSL. # Background Adenylosuccinate lyase deficiency (ADSL, OMIM #103050) is an autosomal recessive defect of purine metabolism, resulting from biallelic inactivating mutations in the ADSL gene, and associated with a wide range of clinical manifestations. The disease was first described more than 30 years ago by Jaeken and van den Berghe, in three patients with severe psychomotor delay, autistic features, and succinylpurines in the cerebrospinal fluid (CSF), plasma and urine [bib_ref] Misleading behavioural phenotype with adenylosuccinate lyase deficiency, Gitiaux [/bib_ref]. The ADSL enzyme is involved in two pathways of purine nucleotide metabolism, i.e., the conversion of succinylaminoimidazole carboxamide riboside (SAICAr) into aminoimidazole carboxamide ribotide (AICAr) along the de novo pathway, and formation of adenosine monophosphate (AMP) from adenylosuccinate (S-AMP). ADSL deficiency results in marked elevation of succinyladenosine and SAI-CAr in various bodily fluids, particularly in CSF and urine. ADSL deficiency is characterized by marked clinical variability, ranging from a fatal neonatal form to milder conditions with infancy onset. The fatal form is characterized by neonatal encephalopathy, absence of spontaneous movement, respiratory failure, and intractable seizures, resulting in early death within the first weeks of life [bib_ref] Lethal fetal and early neonatal presentation of adenylosuccinate lyase deficiency. Observation of..., Mouchegh [/bib_ref]. A relatively milder but still severe phenotype includes severe psychomotor retardation, microcephaly, early onset of seizures, and autistic features (type I). A more slowly progressing form has also been described (type II), as having later onset, usually within the first years of life, slight to moderate psychomotor retardation, and transient contact disturbances, epilepsy, and visual impairment [bib_ref] Magnetic resonance imaging of the brain in adenylosuccinate lyase deficiency: a report..., Jurecka [/bib_ref]. The disease manifests symptoms along a continuum and, despite the utility of communicating with the above mentioned three descriptive categories, there are no fixed parameters to ascribe a particular patient to a single category. In principle, diagnosis could be possible by determining succinyladenosine and SAICAr in urine; the wide range of essentially nonspecific manifestations and lack of awareness of the condition, however, generally prevent a prompt and correct diagnosis, with direct impact on patient management and counseling. Here, we present the case of a mild, late onset ADSL with no obvious signs of disease progression and degradation, without "more classical signs" like visual impairment, hypomyelination or microcephaly that has been largely and unsuccessfully investigated until diagnosis was reached by whole exome sequencing (WES). ## Case presentation A girl of 9 years presented with a history of global development delay and epilepsy. First of two siblings of unrelated healthy parents, the patient was born at 41 weeks of gestational age after an uneventful pregnancy. The neonatal period was unremarkable with growth parameters in the normal range. Developmental milestones were globally delayed: she was sitting independently at the age of 9 months, started to walk independently at 30 months with an ataxic and uncertain gate, but she walked and ran normally at the age of 4 years. She showed severe language delay and, at the age of 9, pronounced around 20 words. She presented with seizures at 3 years and 9 months that ranged from partial seizures, myoclonus to generalized seizures only partially controlled by drugs. At 4 years, brain magnetic resonance imaging (MRI) revealed very mild abnormalities of the white matter and a mega cisterna magna. The corpus callosum appeared normal, and a mild increased spacing of the cerebellar folia was noted [fig_ref] Figure 1: MRI of the patient at age 4 years [/fig_ref]. The child had been evaluated at many different third level centers and a number of metabolic and genetic tests had been performed, including standard karyotype, array-CGH, SHANK3 mutation scan, and very long chain fatty acid, transferrin isoform, plasmatic amino acids and urinary organic acid analyses. All laboratory tests provided negative results. At the age of 9 years, the girl presented with a global developmental delay (her mental age at9, was 4 years), slight autistic feature (stereotypies such as hand movements, repetitive manipulation of toys, clapping hands, . Sagittal T1 weighted image shows a normal corpus callosum and a mild increased spacing of the cerebellar folia (arrow) (c). Coronal and axial T2 weighted images document peculiar fine stripes in T2 weighted image (b, e, f) rubbing feet, and stereotyped sounds), language delay, myoclonic seizure only partially controlled by drugs (sodium valproate in association with clobazam). In the main clinical and imaging features of ADSL compared with patient are reported. After a prior workup no additional targeted test were considered useful for the etiological evaluation, so the need for WES was considered. # Methods ## Whole exome sequencing Informed and written consent from all family was obtained. Targeted enrichment and massively parallel sequencing were performed on genomic DNA extracted from circulating leukocytes of the proband and her parents. Exome capture was carried out using SureSelect Human All Exon V.4 (Agilent). Sequencing data analysis was performed using an in-house implemented pipeline which mainly takes advantage of the Genome Analysis Toolkit (GATK V.3.4) framework [bib_ref] The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing..., Mckenna [/bib_ref] , as previously reported in detail [bib_ref] Mutations impairing GSK3-mediated MAF Phosphorylation cause cataract, deafness, intellectual disability, seizures, and..., Niceta [/bib_ref] [bib_ref] Biallelic mutations in TBCD, encoding the Tubulin folding cofactor D, perturb microtubule..., Flex [/bib_ref]. WES statistics and data analysis are provided in Additional file 1: . # Genetic analysis Sequence validation and family segregation analyses were performed by Sanger sequencing. ## Assay of adsl enzymatic activity and hplc analyses Packed erythrocytes were treated to obtain a 20% haemolisate, and 5 ml were incubated for 20 min at 37°C in presence of ADS 250 mM (Sigma-Aldrich), in 400 ml of a proper incubation buffer. ADSL activity was determined using a time-course assay, and calculated on the basis of the total amount of AMP produced in the reaction mixture during incubation, with respect to the value of this compound determined in control erythrocytes. Samples obtained at different times, were deproteinized as previously reported [bib_ref] Ion-pairing high-performance liquid chromatographic method for the detection of N-acetylaspartate and N-acetylglutamate..., Tavazzi [/bib_ref] , and 100 ml of supernatant were utilized for the AMP analysis, performed by an ion-pairing HPLC method, allowing the synchronous separation of several compounds for the chemical diagnosis and screening of inborn errors of metabolism [bib_ref] Simultaneous high performance liquid chromatographic separation of purines, pyrimidines, N-acetylated amino acids,..., Tavazzi [/bib_ref]. The same HPLC method was utilized for the S-Ado and SAICAr determination in urine sample of the patient. # Results # Wes analysis Among a total 57,433 called variants, data annotation predicted 12,363 high-quality variants having functional impact (i.e., non-synonymous, indels and splice site changes) (Supporting information, Additional file 1: . Among them, 310 private and rare changes were retained for further analyses. Only changes predicted to be deleterious by CADD v.1.3 (score > 15.0) and/or dbNSFP SVM v.2.8 (radial score > 0.0) algorithms were retained. Variants were prioritized on the basis of the functional relevance of genes, taking into account X-linked, autosomal dominant and autosomal recessive inheritance models (Additional file 1: . Variant filtering and prioritization allowed to identify two compound heterozygous variants in the ADSL gene as the only excellent candidates as causative event underlying the trait [fig_ref] Figure 2: Molecular characterization of ADSL mutations [/fig_ref]. Both variants were validated by Sanger sequencing and confirmed to be inherited in trans [fig_ref] Figure 2: Molecular characterization of ADSL mutations [/fig_ref]. One variant was missense, p.Arg309His (c.926 G > A), and affected a highly conserved arginine residue [fig_ref] Figure 2: Molecular characterization of ADSL mutations [/fig_ref] , while the other was a splice site change c.1191 + 5G > C inherited from the father and present also in the healthy brother. To verify the impact of the splice site change on transcript processing, RNA sequencing was performed, which confirmed aberrant processing of the ADSL transcript. Specifically, the mutated mRNA was 11-nucleotide shorter than the wildtype, and the missing nucleotide stretch corresponded to the distal sequence of exon 11 [fig_ref] Figure 2: Molecular characterization of ADSL mutations [/fig_ref]. Exon 12 was found to be spliced on to the remaining exon 11, at position c.1180, due to the use of a cryptic splice donor site. Translation of this aberrant transcript predictsan ADSL protein with a premature stop codon [fig_ref] Figure 2: Molecular characterization of ADSL mutations [/fig_ref]. ## Structural analyses To explore the impact of the p.Arg309His change on ADSL function, inspection of the structural consequences of the amino acid substitution on the holoenzyme organization was obtained. Arg 309 is located at the interface between subunits in the functional ADSL homotetramer contributing to holoenzyme stabilization by forming a salt-bridge with Asp 332 in a region close to the active sites of the enzyme [fig_ref] Figure 3: Three-dimensional mapping of the Arg309His substitution in the ADSL holoenzyme [/fig_ref]. Due to symmetry reasons, four identical interactions occur in the homotetramer contributing to its four equivalent catalytic regions. Of note, this substitution occurs in a region of densely packed residues [fig_ref] Figure 3: Three-dimensional mapping of the Arg309His substitution in the ADSL holoenzyme [/fig_ref]. Through the salt-bridge, with residue Asp 332 , Arg 309 also contributes to stabilize the conformation of residues flanking the Asp 332 side chain with the bulk and rigid imidazole ring, with possible impact of the conformation of the substrate binding region. Overall, these analyses predicted an effect of the amino acid substitution on the overall structure of the holoenzyme and its catalytic function. ## Biochemical analyses To test our in silico structural prediction, the ADSL catalytic activity was analyzed in vitro, and found to be significantly reduced in erythrocytes (80.95 IU/I), corresponding to approx. 25% of the lower value of normal range of healthy control subjects (320-450 IU/l erythrocytes). Contextually, concentration of urinary purine metabolites revealed a high elevation of S-Ado (115.45 mmol/mmol # Discussion and conclusion ADSL deficiency is a rare autosomal recessive progressive disorder with central nervous system involvement. The disease manifests symptoms along a continuum ranging from a neonatal lethal condition to a milder slowing progressing form generally manifesting in the first years of life. Clinical heterogeneity is especially prominent in the less severe end of the spectrum, and some patients with an "attenuated phenotype" may present no obvious signs of disease progression. Incidence of ADSL deficiency remains unknown. The wide clinical spectrum of the disease and absence of pathognomonic features account for difficulties in ADSL diagnosis and differential diagnosis. We report about an atypical case of ADSL deficiency, which has largely and unsuccessfully been investigated (three University Centers and a pediatric multispecialistic Hospital) until WES was performed. The patient presented with a quite unspecific clinical phenotype, in which the relatively mild form of the condition was characterized by the absence of some major characteristic features of ADSL deficiency, including visual impairment, microcephaly and hypomyelination [bib_ref] Attenuated adenylosuccinate lyase deficiency: a report of one case and a review..., Jurecka [/bib_ref]. When the disease is suspected, and the clinical picture is suggestive, clinical diagnosis of ADSL deficiency can be easily confirmed by urinary/plasma screen and/or ADSL mutation analysis. However, purine urinary screen is not widely available and, moreover, with ADSL being a rare and clinically variable disease, diagnosis based on clinical assessment can be challenging. In the case described, the diagnosis was reached through a reverse pathway (from molecular to biochemical confirmation), after a long period of diagnostic uncertainty. This report enlightens the power of WES as first-line tool, to obtain diagnosis of complex tracts or "orphan" disorder. In the absence of any gestaltic approach, the genetic heterogeneity of many Mendelian neurologic/metabolic disorders and variable clinical impact of mutations in individual genes often represent obstacles to the use of "phenotype"-driven genetic testing. Even with the availability of chromosomal microarray testing and more disease-specific genetic panels, prior to WES, the diagnostic rate in pediatric neurodevelopmental disorders has remained around 25%. The introduction of WES in the diagnostic workflow has significantly improved the diagnostic rate in pediatric neurology patients, up to 48% in some specific patient cohort. Moreover, stepwise diagnostic testing based on the use of single gene or gene panels in the evaluation of pediatric neurology patients is time consuming and costly, as in the specific case reported, placing the additional burden of prolonged diagnostic uncertainty on families. Besides the high diagnostic yield, WES also is emerging as an economically convenient diagnostic tool if performed early in the diagnostic evaluation. A recent paper estimated that with the use of WES there is the potential for an estimated average savings of $2465.62 and $502.52 (the average charge estimate on secondary genetic and metabolic testing, respectively) and up to $13,305.00 and $2340.00 (the maximum charge estimate on secondary genetic and metabolic testing, respectively). It is suggested that WES charge could be lower than the composite required for more conventional secondary genetic and metabolic laboratory testing [bib_ref] Whole exome sequencing in pediatric neurology patients: clinical implications and estimated cost..., Nolan [/bib_ref]. Limiting the molecular screening to the Mendeliome, which comprehends the genes that are known to cause a Mendelian disorder, WES appears a highly informative and convenient first-tier tool in the diagnostic setting. Of note, in unsolved cases, WES reanalysis can be expected to provide diagnosis since the constant improvements in WES data analysis and unceasing identification of new disease genes. Moreover, by providing a full profile of the extent of variation contained in the protein-coding portion of the genome, WES data reanalysis in a research-oriented environment is expected to allow the identification of potential candidate genes in a significant proportion of cases. Investigating causality of the sequence variants in human disease becomes an important part in NGS for the research and clinical applications. Recently, important guidelines on them have been published and will keep on updating. [bib_ref] Novel mutations in ADSL for Adenylosuccinate Lyase deficiency identified by the combination..., Mao [/bib_ref]. Implementing this approach in the clinical setting, however, requires tight collaboration between clinicians, geneticists and molecular biologists since the concerted effort required for a correct interpretation and validation of the functional impact and clinical relevance of the identified sequence variants. Increasing the diagnostic rate has high clinical relevance even if prospects for treatment or disease-altering management are not prominent, like in ADSL. Diagnosis helps the patient and family members gain access to appropriate patient management as well as to organizations that provide emotional support as well as practical advice. Furthermore, accurate diagnosis is vital so that families can receive genetic counseling about risk recurrence. WES can significantly improves the diagnostic rate in pediatric neurology patients and likely will become a valid first tier evaluation for a wide variety of neurologic disorders with nonspecific or complex phenotypes, contributing to the identification of new mutations and a delineation of the clinical variability characterizing disorder. Despite the increasing number of ADSL-deficient patients reported, there are only a few communications of therapeutic considerations and efforts. Two papers showed some beneficial effects (D-ribose and uridine administration) [bib_ref] Effect of D-ribose administration to a patient with inherited deficit of adenylosuccinase, Salerno [/bib_ref] [bib_ref] Effect of uridine administration to a patient with adenylosuccinate lyase deficiency, Salerno [/bib_ref]. D-ribose administration, which increases the provision of phosporibosylpyrophosphate (PRPP) and stimulates de novo purine synthesis, has been applied in a few ADSL patients. Although promising results in motor coordination and seizure control in a 13-year-old female after several months of D-ribose therapy [bib_ref] Novel features in the evolution of adenylosuccinate lyase deficiency, Pérez-Dueñas [/bib_ref] they have not been confirmed in further studies. In 2013 Werkhoven et al. evaluated S-adenosyl-l-methionine (SAMe) as a potential treatment for ADSL deficiency [bib_ref] Early diagnosis of adenylosuccinate lyase deficiency using a high-throughput screening method and..., Van Werkhoven [/bib_ref] but there was no clear response evidenced in urine metabolite levels or clinical. Reports about positive use of a ketogenic diet for treatment of refractory epilepsy have also been reported [bib_ref] Adenylosuccinate lyase deficiency, Jurecka [/bib_ref]. None of the mention treatments has been used for the patient. ## Additional file Additional file 1: . Whole exome sequencing data output. Availability of data and materials All data here presented are collected in the medical record of the patient, which can be consulted with the appropriate authorizations. Authors' contributions MM and AB: responsible for patient care and drafting the article SB, FC GZ, MN, EB, GL, AMA, EB, SR were involved in different stages of laboratory/ molecular analysis. EB, MT,FC revising the intellectual content of the article. All the authors have read and approved the final manuscript. Ethics approval and consent to participate No ethics approval was required for the type of report. ## Consent for publication Informed consent for publication was required and obtained from the parents. ## Competing interests The authors declare that they have no competing interests. [fig] Figure 1: MRI of the patient at age 4 years. The images show very mild abnormalities of the white matter that are barely visible in FLAIR (a, d) (arrow head) [/fig] [fig] Figure 2: Molecular characterization of ADSL mutations. a Schematic representation of the ADSL transcript (ENST0000062306) with positions of the two mutations identified by WES. b Electropherograms showing the missense c.926G > A, p.R309H, and splice site c.1191 + 5G > C mutations (indicated with red arrows) occurring in trans in the proband. On the right side of the panel the family tree and segregation are shown.c Multiple sequence alignment of the human ADSL sequence of the region encompassing the disease-associated amino acid change (red arrow) with its orthologues. Arg309 is evolutionarily conserved among vertebrates. d Schematic representation of proper and aberrant splicing of ADSL exons 11 (black box) and 12 (red box). In the presence of the c.1191 + 5G > C change, the normal splice donor site is no longer recognized and an alternative cryptic splice donor site is used instead, resulting in loss of the distal sequence of exon 11, and in a frameshift causing a premature stop of translation creatinine) and SAICAr (57.59 mmol/mmol creatinine) which are not detectable in control samples. [/fig] [fig] Figure 3: Three-dimensional mapping of the Arg309His substitution in the ADSL holoenzyme. On the left side of the panel, localization of Arg 309 in the ADSL protein homotetramer (the four subunits are in different colors) complexed with adenylosuccinic acid (S-AMP) substrate, and its enzymatic products adenosine monophosphate (AMP) and fumaric acid (FA) (PDB accession number 2VD6). On the right side of the panel, the inset shows a slab view of Arg 309 in one ADSL subunit forming a salt-bridge with Asp 332 of the neighboring subunit, and the nearby Leu 331 and Ser 334 that bind to the substrate (interactions are represented by dots between atoms) [/fig]
Risk and outcome in metastatic malignant melanoma patients receiving DTIC, cisplatin, BCNU and tamoxifen followed by immunotherapy with interleukin 2 and interferon alpha2a. Combined chemo-/immunotherapy has shown high objective response rates and a significant though small proportion of longterm complete responders in metastatic malignant melanoma. The purpose of this study was to determine response rates, freedom from treatment failure (FFTF) and overall survival in patients with advanced metastatic malignant melanoma treated with combined chemo-/ immunotherapy, and to determine the value of a prognostic model for prediction of treatment outcome, FFTF and survival. Sixty-nine patients with metastatic malignant melanoma received combined chemo-/immunotherapy consisting of up to four cycles of DTIC (220 mg m-2 i.v. days 1-3), cisplatin (35 mg m-2 i.v. days 1-3), BCNU (150 mg m-2 i.v. day 1. cycles 1 and 3 only) and tamoxifen (20 mg orally. daily). Two cycles of chemotherapy were followed by 6 weeks of outpatient immunotherapy with combined interleukin 2 (20 x 1 06 U m-2 days 3-5. weeks 1 and 4: 5 x 106 IU m-2 days 1. 3. 5. weeks 2, 3, 5. 6) and interferon-a (6 x 106 IU m-2 s.c. day 1. weeks 1 and 4: days 1. 3. 5. weeks 2, 3. 5. 6). All patients were evaluated on an intention-to-treat basis. Of 69 patients entered in the study, seven achieved complete remissions and 20 reached partial remissions with an objective response rate of 39% (95% confidence interval 28-52%). Median survival was 11 months. median FFTF was 5 months. Seven patients achieved ongoing long-term remissions, with maximum survival of 58 + months, and maximum FFTF of 58 + months. By Kaplan-Meier survival analysis and two-proportional Cox regression analysis, pretreatment performance status and serum lactic dehydrogenase were statistically significant and independent predictors of survival; risk groups could be defined as (a) the absence of both or (b) the presence of either one or both of these risk factors. Whereas survival and response were significantly influenced by patient risk. no infuence could be demonstrated for FFTF. This combined outpatient chemo-/immunotherapy is feasible and results in objective response rates and survival similar to earlier trials. Pretreatment risk, as defined by serum lactate dehydrogenase (LDH) and performance status, has a significant impact on treatment outcome and patient survival. responses may be achieved by introducing cx tokines into the therapeutic regimens. with interleukin 2 (IL-2) and interferon alpha (INF-a) beinc the most widely acclaimed. Monotherapx with IL-2 results in up to 29%c objective responses IRosenberr et al. 1994): combination regimens s ith cvtotoxic drugs reach up to 66%/c objective response rates depending on the treatment schedule [bib_ref] Durable complete responses in metastatic malignant melanoma treated with interleukin-2 in combination..., Legha [/bib_ref]. In a small proportion of patients. long-lasting remissions can be achieved [bib_ref] Durable complete responses in metastatic malignant melanoma treated with interleukin-2 in combination..., Legha [/bib_ref]. The present study shows results of a combined chemo-/ immunotherapx regimen. consisting of three cyvtyotoxic agents (DTIC. BCNU. cisplatin). tamoxifen and subcutaneous interleukin 2 and interferon alpha. We report objective response rates as sell as survival. freedom from treatment failure (FF-FT). sites of recurrence and prognostic factors for FFTF and survix al. ## Patients and methods patient characteristics Betwseen January 1991 and May 1996. 69 patients were entered into this combined chemo-/immunotherapy protocol. All patients had histologicall1 confirmed metastatic malignant melanoma. an ## Treatment and evaluation Treatment consisted of up to four cycles of DTIC (220 mg m-' iV. days 1-3). cisplatin (35 mg m-' i.v. days 1-3.) BCNIJT (150mg mi.v. day 1. cycles 1 and 3 only) and tamoxifen (20 mg orallyv daily). Two cvcles of chemotherapy were followed by 6 weeks of outpatient immunotherapy with combined IL-2 (20 x 106 IU m-' s.c. days 3-5. weeks 1 and 4: 5 x 106 IU m-s.c. days 1. 3. 5. weeks 2 3. 5. 6) and INF-ax2a (6 x 106 lIT m-' s.c. dav 1. weeks 1 and 4: davs 1. 3. 5. weeks 2. 3. 5. 6). Therapy was administered until progression of disease. grade III or IV toxicitv (WHO), or scheduled end of administration. Re-evaluation of patients was performed according to WHO criteria. Survival and FF-TF were measured from the initiation of therapy: response duration was measured from documentation of response to documentation of progression. Patients were evaluated on an intention-to-treat basis. # Statistical analysis The probabilitv of overall sun ival w as plotted over time according to the method of Kaplan and Meier. Differences between groups in overall survival were tested with log, rank statistics: variables demonstrating significant impact on survival in this univariate analysis were tested for statistical independence in a multivariate analysis using the Cox proportional hazards model with forward selection of variables. # Results ## Risk model The ability of vanious pretherapeutic clinical factors to predict clinical outcome as measured by overall survival was assessed by using univariate Kaplan-Meier sun-ival analysis and log rank statistics . The following factors demonstrated significant impact on survival: ECOG performance status, serum lactic acid dehydrogenase (LDH) level, brain metastases. liver metastases. lymphatic metastases and erythrocyte sedimentation rate. Using two-dimensional Cox regression analysis. statistical independence could be shown for serum LDH and performance status only. This allowed for definition of two risk groups: (a) low risk in patients without any of these factors (n = 38): (b) high risk for patients with either one or both of these factors (n = 31). Clinical variables tested before therapy and rendered statistically insignificant included: age, sex. time from first diagnosis to first appearance of metastases. haemoglobin level. C-reactive protein level. neutrophil count. bone metastases. pulmonary metastases. cutaneous or subcutaneous metastases. and visceral metastases. Patient nsk and treatment response [fig_ref] Table 3: Sites of disease and response to therapy [/fig_ref] shows sites of disease and response to therapy for the 69 patients entered into the study. Seven patients had a complete remission. 20 experienced a partial remission: the objective response rate was 39% (95%c confidence interval. 28-52%7). Seven objective remissions are ongoing [fig_ref] Table 4: Characteristcs of patents with ongoing partial or complete tumour regression [/fig_ref]. Stabilization of disease occurred in 14 patients. 28 patients continued to progress despite therapy. Overall objective response rates were 47.3%7 for low-risk patients (95% confidence interval. 31-64%) and 29%c for high-risk patients (95% confidence interval. 14-48%). Of patients achieving British Joumal of Cancer (1998) 78(8) into the study complete remissions. five were at low risk and two were at high risk. In contrast. progressive patients showed a predominance for the high-risk group. with 19 patients belonging to the high-risk group and only nine patients being at low risk. Median response duration was 7+ months (range. 1-55 months) with 27+ months for complete and 6 months for partial responders. there were no significant differences between low-and high-risk patients. In all patients. most common sites of relapse included lymph nodes (37%). liver (35%) and lun, (22%). Compared with metastatic sites before therapy (7%). the proportion of patients with CNS metastases was increased (19%). In contrast. proportions of patients exhibiting progressive lymphatic (minus 20%). pulmonary (minus 21%) and visceral metastases (minus 32%). respectively, were reduced after treatment when compared with initiation of therapy. Of patients with progressix e cerebral. hepatic and bone disease. 67'k. 59% and 63%. respectively, showed high-risk features. whereas patients with lymphatic. pulmonarv and cutaneous/subcutaneous metastases belonged predominantly to the low-risk group (60%. 64% and 67% respectively). Median overall sunrival was 11 months. with a range from 1 to 58+ months [fig_ref] Figure 1: Kaplan-eier plot of overall survival for the 69 patients entered [/fig_ref]. Median sunrival for the high-risk patients was 6 months, as opposed to 15 months in the low-risk group (P < 0.0005. [fig_ref] Figure 2: Overall survival of 69 patients entered into the study was stratified for... [/fig_ref]. For complete responders. median survival has not been reached at 39 months. In comparison (P < 0.007). partial responders and patients in disease stabilization had a median survival of 13 and 15 months. respectively. as opposed to a median survival of 6 months in progressive disease patients (P < 0.0001). Twelve patients remain alive. five with complete remissions. three with partial remissions, and four patients with transient disease stabilization. Of those patients alive. three showed high-risk and nine showed low-risk features at the beginning of therapy. Median FFTF was 5 months. with a range from 0 to 58+ months [fig_ref] Figure 3: Kaplan-Meier plot of overall FFTF for the 69 patients entered into the... [/fig_ref]. Median FFTF for complete and partial responders was 32+ and 8 months respectively (P < 0.003). Seven patients continue to be progression-free. four of whom showed low-risk features at initiation of therapy [fig_ref] Table 4: Characteristcs of patents with ongoing partial or complete tumour regression [/fig_ref]. Median FFTF for lowand high-risk patients showed no statistically significant difference. # Discussion Treatment of metastatic maligrnant melanoma has always been a field of particular disappointment. Although the most common combination chemotherapy regimen yielded response rates of 55%7c . these responses were usually short-lived with a median FFT.F of 5 months . This led to the introduction of cvtokines into the chemotherapeutic regimens. resulting in a significant proportion (approximatelv 10%lc) of long-term responders [bib_ref] Durable complete responses in metastatic malignant melanoma treated with interleukin-2 in combination..., Legha [/bib_ref]. The present sequential chemoimmunotherapy regimen showed an objective response rate of 39% that is comparable to earlier chemotherapy-cvtokine combination trials yielding response rates between 33% and 66%7. with 95% confidence inter-vals from 19% to 81 %e [bib_ref] Subcutaneous recombinant interleukin-2 plus chemotherapy with cisplatin and dacarbazine in metastatic melanoma, Guida [/bib_ref] [bib_ref] Durable complete responses in metastatic malignant melanoma treated with interleukin-2 in combination..., Legha [/bib_ref]. Whereas in most other trials IL-9 has been administered as continuous i.v. infusion in the hospital setting and with substantial toxicity. we used both IL-' and INF-a on an outpatient basis and could significantly reduce overall side-effects. In previous trials. the sequential administration of chemo-/immunotherapy has demonstrated therapeutic superiority over other treatment rerimens including those using concomitant chemo-/immunotherapy . The present study is among the first to employ a simple pretherapeutic risk model comprisinc elevated serum LDH and impaired performance. This has shown substantial influence on patient's response to therapy: significantly more complete and partial remissions were seen in the low-risk group when compared with high-risk patients. Also. analysis of sites of disease progressing after or during therapy demonstrated the predictiv-e value of our risk model: high-risk patients were more likely to progress at clinically serious or life-threatening sites. Most importantly. overall survival was highly risk-dependent in the present cohort of patients. Analysis of the correlation between remission and survival showed highly increased survival in complete responders. although no significant survival difference between partial responders and patients in stabilization could be seen: still. survival in these groups was substantially increased compared with progressive patients. Whereas our risk model showed substantial influence on a patient s response and survival. it did not predict FFTF. Except for 10% of patients achieving ongoing tumour remission and lonaterm survival. in the remaining patients survival appeared to be influenced by a patient s tumour biology rather than by treatment efficacy. Analysina the overall survival times. it is clear that only a small subset of patients was likely to benefit. which was comparable to previous reports [bib_ref] Durable complete responses in metastatic malignant melanoma treated with interleukin-2 in combination..., Legha [/bib_ref]. Recently published studies have introduced a risk model based on serum LDH and cross-sectional surface area of all measurable metastases : in this risk model. survival difference betw-een risk groups was only marginal. whereas the proportion of long-term responders appeared similar to our results. In the present study. the therapeutic relevance of additional outpatient interleukin 2 and interferon alpha remained unclear. To clearly demonstrate the usefulness of immunotherapy. a prospective randomized trial is necessarv and has been initiated by this multicentric study group. The current risk model suggests the possibility of preselecting patients that are likely to be long-term responders: on this basis. in future clinical trials. risk-adapted therapeutic strategies will lead to more aggressive and potentially curativ-e therapies for patients who are likely to be long-term responders. For malignant melanoma patients at high risk. therapeutic strategies must achieve effective tumour palliation. [fig] Figure 1: Kaplan-eier plot of overall survival for the 69 patients entered [/fig] [fig] Figure 2: Overall survival of 69 patients entered into the study was stratified for risk group as defined by absence of elevated pretreatnent serum Lactic dehydrogenase or impaired performance status or presence of either one or both of these risk factors. Kaplan-Meier plot Patient risk and survival [/fig] [fig] Figure 3: Kaplan-Meier plot of overall FFTF for the 69 patients entered into the study [/fig] [table] Table 1: Patient characteristics [/table] [table] Table 3: Sites of disease and response to therapy [/table] [table] Table 4: Characteristcs of patents with ongoing partial or complete tumour regression [/table]
Drug Susceptibility Testing of 31 Antimicrobial Agents on Rapidly Growing Mycobacteria Isolates from China Objectives. Several species of rapidly growing mycobacteria (RGM) are now recognized as human pathogens. However, limited data on effective drug treatments against these organisms exists. Here, we describe the species distribution and drug susceptibility profiles of RGM clinical isolates collected from four southern Chinese provinces from January 2005 to December 2012. Methods. Clinical isolates (73) were subjected to in vitro testing with 31 antimicrobial agents using the cation-adjusted Mueller-Hinton broth microdilution method. The isolates included 55 M. abscessus, 11 M. fortuitum, 3 M. chelonae, 2 M. neoaurum, and 2 M. septicum isolates. Results. M. abscessus (75.34%) and M. fortuitum (15.07%), the most common species, exhibited greater antibiotic resistance than the other three species. The isolates had low resistance to amikacin, linezolid, and tigecycline, and high resistance to first-line antituberculous agents, amoxicillin-clavulanic acid, rifapentine, dapsone, thioacetazone, and pasiniazid. M. abscessus and M. fortuitum were highly resistant to ofloxacin and rifabutin, respectively. The isolates showed moderate resistance to the other antimicrobial agents. Conclusions. Our results suggest that tigecycline, linezolid, clofazimine, and cefmetazole are appropriate choices for M. abscessus infections. Capreomycin, sulfamethoxazole, tigecycline, clofazimine, and cefmetazole are potentially good choices for M. fortuitum infections. Our drug susceptibility data should be useful to clinicians. # Introduction Nontuberculous mycobacteria (NTM) form a large class within the Mycobacteriaceae family. More than 100 NTM species are found in soil, potable water, food, and animals [bib_ref] Pedicure-associated rapidly growing mycobacterial infection: an endemic disease, Stout [/bib_ref]. In China, the proportion of NTM among all mycobacterial isolates has increased from 11.1% to 22.9% according to National surveys conducted in 1990 and 2010 [bib_ref] Tuberculosis prevalence in China, 1990-2010; a longitudinal analysis of national survey data, Wang [/bib_ref]. Thus, the rising percentage of NTM in China is now an important public health concern [bib_ref] Pedicure-associated rapidly growing mycobacterial infection: an endemic disease, Stout [/bib_ref] [bib_ref] Tuberculosis prevalence in China, 1990-2010; a longitudinal analysis of national survey data, Wang [/bib_ref]. NTM can be classified into rapidly growing mycobacteria (RGM) and slowly growing mycobacteria (SGM). More than 50 RGM species are able to produce mature colonies on agar plates within 7 days [bib_ref] Rapidly growing mycobacterial bloodstream infections, Helou [/bib_ref]. Many of these are important human pathogens that cause pulmonary and soft tissue infections and various other infections [bib_ref] Differential macrophage response to slow-and fastgrowing pathogenic mycobacteria, Helguera-Repetto [/bib_ref] [bib_ref] Nontuberculous mycobacterial ocular infections: a systematic review of the literature, Kheir [/bib_ref]. RGM comprise a diverse group of species, including M. abscessus, M. fortuitum, M. chelonae, and various rare species. Most studies have shown that M. abscessus accounts for 80% of the lung disease caused by RGM, and after M. fortuitum, M abscessus is the second most common RGM to cause extrapulmonary disease [bib_ref] Rapidly growing mycobacterial bloodstream infections, Helou [/bib_ref] [bib_ref] Diagnosis and treatment of infections caused by rapidly growing mycobacteria, Colombo [/bib_ref]. Diagnosing and treating RGM diseases is challenging for clinicians [bib_ref] Diagnosis and treatment of infections caused by rapidly growing mycobacteria, Colombo [/bib_ref] [bib_ref] Nontuberculous mycobacteria isolated from pulmonary specimens between 2004 and 2009: causative agent..., Bicmen [/bib_ref]. Over the past few decades, RGM infections have been diagnosed based on a patient's clinical characteristics, risk factors, and the results of antimicrobial susceptibility testing [bib_ref] Rapidly growing mycobacterial bloodstream infections, Helou [/bib_ref]. However, drug susceptibility patterns vary greatly between RGM species and optimally therapeutic regimens have not been established [bib_ref] Rapidly growing mycobacterial bloodstream infections, Helou [/bib_ref] [bib_ref] Diagnosis and treatment of infections caused by rapidly growing mycobacteria, Colombo [/bib_ref]. In this study, the antimicrobial susceptibility of 73 clinical RGM isolates and their corresponding standard strains were tested with 31 antibiotics. The tests were based on the recommendations of the Clinical and Laboratory Standards 2 BioMed Research International Institute (CLSI)for determining the clinical criteria for therapeutic treatment of RGM infections. # Materials and methods ## Clinical isolates and reference ## Species identification. Species identification of the isolates was conducted by sequence analysis of the hsp65 gene. When an hsp65 sequence match was less than 97%, the rpoB gene and the 16S-23S internal transcribed spacer region were also sequenced [bib_ref] Multiplex real-time PCR assay and melting curve analysis for identifying Mycobacterium tuberculosis..., Kim [/bib_ref] [bib_ref] Rapid identification of mycobacteria and drug-resistant Mycobacterium tuberculosis by use of a..., Pérez-Osorio [/bib_ref]. PCR products were sequenced by the Beijing Tsingke Bio Tech Co. Ltd. (Beijing, China). The sequences obtained were compared with those in the Gen-Bank (National Center for Biotechnology Information: http:// www.ncbi.nlm.nih.gov/) DNA sequence database; species identification was confirmed if a 97% match was achieved [bib_ref] Multiplex real-time PCR assay and melting curve analysis for identifying Mycobacterium tuberculosis..., Kim [/bib_ref] [bib_ref] Rapid identification of mycobacteria and drug-resistant Mycobacterium tuberculosis by use of a..., Pérez-Osorio [/bib_ref]. ## Rgm growth Medium. The strains tested were cultured using Difco Middlebrook 7H10 Agar (BD company). The medium was prepared as follows. First, 19 g of 7H10 (powder) was suspended in 900 mL of purified water containing 5 mL of glycerol and then mixed thoroughly. Next, the powder was completely dissolved by heating with frequent agitation for 1 min and then sterilized at 121 ∘ C for 10 min. Last, 100 mL of Middlebrook OADC enrichment solution (BD, Franklin Lakes, NJ, USA) was added aseptically to the medium after cooling to 50-55 ∘ C. ## Medium for drug susceptibility testing of rgm. The medium used for antimicrobial susceptibility testing of RGM was BBL cation-adjusted Mueller-Hinton (CAMH) Broth (BD, Franklin Lakes, NJ, USA). The medium was prepared by suspending 22 g of powder in 1 L of purified water and autoclaving the bottle at 121 ∘ C for 10 min, followed by supplementation with 20-25 mg/L of calcium and 10-12.5 mg/L of magnesium. ## Drug susceptibility tests. The strains were grown on 7H10 agar and incubated at 37 ∘ C in ambient air. Drug susceptibility tests were performed using the broth microdilution method according to CLSI recommendations [bib_ref] Diagnosis and treatment of infections caused by rapidly growing mycobacteria, Colombo [/bib_ref]. Tests on the strains were repeated at least twice using 96-well microplates. The final minimum inhibitory concentration (MIC) of each drug used for each strain was the average value of the two tests. Bacterial inocula were adjusted with normal saline to a density of a 0.5 McFarland standard with an organism density of approximately 1 × 10 7 colony forming units (CFU)/mL. Fifty microliters of the suspension, added to 10 mL of CAMH broth, was vortexed thoroughly to make a 1 : 200 bacterial dilution. First, 100 L of CAMH medium was added to each well of a 96-well microplate, except for the first well of the each row. CAMH medium (180 L) was added to the first well of every row, followed by a 20 L aliquot of a drug solution. The thoroughly mixed solution in the first well was serially diluted into the next well, and so on up to the 11th well. The 12th well in every row was a blank control. Second, 100 L of the bacterial dilution was added to the wells of the 96-well microplate. The final volume in each row was 200 L. Finally, the 96-well microplate was sealed in a plastic bag and incubated at 37 ∘ C. The concentration ranges for rifampicin, isoniazid, ethambutol, streptomycin, tobramycin, sulfamethoxazole, dapsone, amoxicillinclavulanic acid, cefoxitin, cefmetazole, thioacetazone, pasiniazid, minocycline, doxycycline, tigecycline, and meropenem were all 0.25-256 g/mL, those of amikacin, kanamycin, capreomycin, ofloxacin, ciprofloxacin, levofloxacin, moxifloxacin, sparfloxacin, clarithromycin, azithromycin, roxithromycin, clofazimine, rifapentine, and rifabutin were 0.03-32 g/mL, and the concentration of linezolid was 0.06-64 g/mL. All the drugs were purchased from Sigma-Aldrich (St. Louis, MO). The following two negative controls were used: CAMH broth plus inoculum (drug-free control), which was used to decide the optimal time to add Alamar blue to the assay; the other was only CAMH broth, which was used to decide the interference level of CAMH to Alamar blue. The plates were checked after 72 h. If the drug-free growth control showed sufficient bacterial growth, the indicator (20 L of Alamar Blue and 50 L of sterile 5% Tween-80) turned pink. Generally, the minimal inhibitory concentration (MIC) value was read on day 3 or 4 after addition of the inoculum. If bacterial growth in the drug-free control was insufficient on day 5, the test was repeated. The MIC values for clarithromycin were evaluated 3 to 5 days after inoculation and were incubated for a further 14 days at 37 ∘ C for the final reading. MIC values were defined as the lowest concentration of drug that inhibited the visible growth of the isolates tested. MIC 50 and MIC 90 values were defined as the drug concentrations at which 50% and 90% of the isolates tested showed no visible growth, respectively. The MIC breakpoints of antibiotics displaying susceptibility, intermediate susceptibility, and resistance were interpreted by the World Health Organization (WHO)and CLSI guidelines, except for sparfloxacin [bib_ref] Experimental and theoretical studies on the molecular properties of ciprofloxacin, norfloxacin, pefloxacin,..., Kłosińska-Szmurlo [/bib_ref] , clofazimine [bib_ref] Systematic review of clofazimine for the treatment of drugresistant tuberculosis, Gopal [/bib_ref] , azithromycin [bib_ref] Susceptibility of Ureaplasma urealyticum to tetracycline, doxycycline, erythromycin, roxithromycin, clarithromycin, azithromycin, levofloxacin..., Samra [/bib_ref] , roxithromycin [bib_ref] Susceptibility of Ureaplasma urealyticum to tetracycline, doxycycline, erythromycin, roxithromycin, clarithromycin, azithromycin, levofloxacin..., Samra [/bib_ref] , amoxicillin-clavulanic acid [bib_ref] Susceptibility of 186 Nocardia sp. isolates to 20 antimicrobial agents, Larruskain [/bib_ref] , cefmetazole, rifapentine [bib_ref] Antibacterial activity of rifamycins for M. smegmatis with comparison of oxidation and..., Staudinger [/bib_ref] , rifabutin [bib_ref] Antibacterial activity of rifamycins for M. smegmatis with comparison of oxidation and..., Staudinger [/bib_ref] , dapsone [bib_ref] High efficacy of clofazimine and its synergistic effect with amikacin against rapidly..., Shen [/bib_ref] , and thioacetazone [bib_ref] Synthesis, antitubercular activity and mechanism of resistance of highly effective thiacetazone analogues, Coxon [/bib_ref] [fig_ref] Table 1: MIC [/fig_ref]. ## Statistical [formula] Rifampicin - - ⩾1 [8] Isoniazid - - ⩾1 [8] Ethambutol - - ⩾4 [8] Streptomycin - - ⩾5 [8] Amikacin ⩽16 32 ⩾64 [8] Kanamycin - - ⩾4 [11] Capreomycin - - ⩾2.5 [11] Tobramycin ⩽2 4 ⩾8 [8] Ofloxacin - - ⩾2 [11] Ciprofloxacin ⩽1 2 ⩾4 [8] Levofloxacin ⩽2 4 ⩾8 [8] Sparfloxacin ⩽1 2 ⩾4 [12] Moxifloxacin ⩽1 2 ⩾4 [8] Linezolid ⩽8 16 ⩾32 [8] Clofazimine - - ⩾1 [13] Sulfamethoxazole ⩽38 - ⩾76 [8] Minocycline ⩽1 2-4 ⩾8 [8] Doxycycline ⩽1 2-4 ⩾8 [8] Tigecycline ⩽1 2-4 ⩾8 [8] Clarithromycin ⩽2 4 ⩾8 [8] Azithromycin ⩽2 4 ⩾8 [14] Roxithromycin ⩽2 4 ⩾8 [14] [/formula] Amoxicillinclavulanic acid [formula] ⩽8/4 16/8 ⩾32/16 [15] Cefoxitin ⩽16 32-64 ⩾128 [8] Cefmetazole ⩽16 32 ⩾64 [16] Meropenem ⩽4 8-16 ⩾32 [8] Rifapentine - - ⩾1 [17] Rifabutin - - ⩾1 [17] Dapsone - - ⩾4 [18] Thioacetazone - - ⩾8 [19] Pasiniazid - - ⩾2 [11] [/formula] # Results Among [fig_ref] Table 2: MIC [/fig_ref]. The strains were highly resistant to the four first-line antituberculous agents tested on them, especially M. chelonae, M. abscessus, and M. fortuitum. We found that aminoglycoside antibiotics including amikacin, kanamycin, capreomycin, and tobramycin, were effective antimicrobials for the RGM species. However, M. abscessus was resistant to tobramycin and M. chelonae was resistant to kanamycin and capreomycin. Fluoroquinolones (including ofloxacin, ciprofloxacin, levofloxacin, sparfloxacin, and moxifloxacin) also exhibited favorable in vitro activities against the standard RGM strains. However, M. chelonae was resistant to all four of the fluoroquinolones we tested. M. chelonae and M. abscessus were not susceptible to minocycline and doxycycline, but were susceptible to tigecycline. Clarithromycin, azithromycin, and roxithromycin all exhibited favorable in vitro activities (except for azithromycin) against M. septicum. The reference species were not susceptible to amoxicillin-clavulanic acid but were susceptible or moderately susceptible to cefoxitin and cefmetazole, apart from M. chelonae and M. septicum, which were not susceptible to cefoxitin. Unlike M. chelonae, the RGM species were susceptible to meropenem. Rifapentine had a more favorable MIC than rifabutin against M. chelonae, M. abscessus, and M. fortuitum. Linezolid, clofazimine, and sulfamethoxazole were highly active against the standard RGM organisms. However, dapsone, thioacetazone, and pasiniazid displayed poor activities against the reference species. The percentage of in vitro drug susceptibility values of the 31 antibacterial agents against the 73 clinical RGM isolates is shown in [fig_ref] Table 3: In vitro drug susceptibility percentage per species for rapidly growing mycobacteria isolates [/fig_ref]. Among the four first-line antituberculous drugs, no (0/73) strains were susceptible to isoniazid and 3 (4.11%), 2 (2.74%), and 4 (5.48%) strains were susceptible to rifampicin, ethambutol, and streptomycin, respectively. However, the M. chelonae isolates were less resistant to rifampicin than M. abscessus and M. fortuitum. Aminoglycosides and fluoroquinolones displayed a range of activities against the RGM isolates. Amikacin displayed the highest activity (72/73, 98.63%), while moxifloxacin displayed a range of activities (57/73, 78.08%). Tigecycline (70/73, 95.89%) had much higher activity against the isolates than minocycline # Discussion With the development of improved microbiological and laboratory techniques, more RGM have been identified [bib_ref] The therapeutic approach to non-tuberculous mycobacterial infection of the lung, Mcgrath [/bib_ref]. Effective treatment of RGM-related diseases is challenging to physicians because it is not obvious which drugs should be selected. In this study, the susceptibilities of 73 clinical RGM isolates and their corresponding reference RGM strains were examined for 31 antimicrobial agents using CAMH broth microdilution methodology. Some studies have shown that M. abscessus, M. fortuitum, and M. chelonae are important human pathogens among RGM isolates [bib_ref] The he epidemiology and geographic distribution of nontuberculous mycobacteria clinical isolates from..., Shao [/bib_ref] [bib_ref] Molecular epidemiology of nontuberculous mycobacteria isolates from clinical and environmental sources of..., Velayati [/bib_ref] [bib_ref] Epidemiology of nontuberculous mycobacteria among patients with cystic fibrosis in Scandinavia, Qvist [/bib_ref] [bib_ref] The epidemiology of pulmonary nontuberculous mycobacteria, Panagiotou [/bib_ref]. Here, we showed that M. abscessus (75.34%) is the predominant RGM species in the 73 clinical isolates, followed by M. fortuitum (15.07%). Both organisms were susceptible to amikacin, linezolid, tigecycline, cefmetazole, capreomycin, moxifloxacin, macrolides, and carbapenems, but were highly resistant to the firstline antituberculous drugs, dapsone, thioacetazone, and pasiniazid. The percentage of resistance to numerous drugs was higher in M. abscessus than in M. fortuitum, except for moxifloxacin, minocycline, doxycycline, roxithromycin, cefmetazole, and rifabutin. In a recent report, amikacin and clarithromycin were the optimal choices against infection with M. abscessus [bib_ref] Rapidly growing mycobacteria in Singapore, Tang [/bib_ref]. Additionally, quinolones and trimethoprim-sulfamethoxazole were effective against M. fortuitum [bib_ref] Rapidly growing mycobacteria in Singapore, Tang [/bib_ref]. In the present study, Amikacin was the most active drug against M. abscessus. Furthermore, we found that amikacin, capreomycin, and linezolid had the highest antibacterial activities against M. fortuitum. Previous studies have reported that numerous RGM strains were highly resistant to the first-line antituberculous agents [bib_ref] An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases, Griffith [/bib_ref] [bib_ref] Multidrug-resistant tuberculosis' may be nontuberculous mycobacteria, Shahraki [/bib_ref]. Our data confirms this finding. Elsewhere, researchers have shown that dapsone had little activity against RGM isolates [bib_ref] High efficacy of clofazimine and its synergistic effect with amikacin against rapidly..., Shen [/bib_ref]. Thioacetazone is used mainly as an antituberculous agent but has variable activity, and the drug was formerly used in conjunction with isoniazid [bib_ref] Synthesis, antitubercular activity and mechanism of resistance of highly effective thiacetazone analogues, Coxon [/bib_ref]. RGM strains have been shown to be highly resistant to pasiniazid [bib_ref] Antibiotic susceptibility pattern of rapidly growing mycobacteria, Gayathri [/bib_ref]. Our data shows that dapsone had little activity against M. abscessus and M. fortuitum isolates, while thioacetazone and pasiniazid had no activity against any of the RGM organisms. Aminoglycosides and quinolones, which are second-line antituberculous drugs, have good activities against RGM strains [bib_ref] Antibiotic susceptibility pattern of rapidly growing mycobacteria, Gayathri [/bib_ref] [bib_ref] Antimicrobial susceptibility of standard strains of nontuberculous mycobacteria by microplate Alamar Blue..., Li [/bib_ref] [bib_ref] In vitro antimicrobial susceptibility of Mycobacterium abscessus in Korea, Park [/bib_ref]. In our study, amikacin was found to have potential to be effective for treatment of RGM diseases and showed higher activity than the other aminoglycoside antibiotics we tested. However, a higher percentage of M. chelonae isolates were sensitive to tobramycin than M. abscessus, although the sample size of the latter was smaller. The third generation fluoroquinolone drugs levofloxacin and sparfloxacin displayed higher activities than ofloxacin. Moxifloxacin, a fourth generation fluoroquinolone, displayed higher activity than the third generation ones [bib_ref] In vitro antimicrobial susceptibility of Mycobacterium abscessus in Korea, Park [/bib_ref]. Quinolones exhibited better activity against M. fortuitum than M. abscessus, especially levofloxacin, against which M. fortuitum was more susceptible than M. abscessus. Minocycline, doxycycline, and tigecycline represent the newest tetracycline derivatives [bib_ref] Tetracycline antibiotics and resistance mechanisms, Nguyen [/bib_ref]. A recent study [bib_ref] An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases, Griffith [/bib_ref] showed that NTM displayed ∼50% susceptibility to doxycycline and minocycline, but in our research susceptibility to these two drugs was more than 20% [fig_ref] Table 3: In vitro drug susceptibility percentage per species for rapidly growing mycobacteria isolates [/fig_ref]. This finding may reflect the small sample number of M. fortuitum in this study. Tigecycline displayed activity against RGM organisms [bib_ref] An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases, Griffith [/bib_ref]. We found that tigecycline had more activity than minocycline and doxycycline, with lower MIC 50 and MIC 90 values. [formula] M. fortuitum ( = 11) (%) M. chelonae ( = 3) (%) M. septicum ( = 2) (%) M. neoaurum ( = 2) (%) INH 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) RFP 3 (5.46) 0 (0) 0 (0) 0 (0) 0 (0) 3 (4.11) EMB 2 (3.64) 0 (0) 0 (0) 0 (0) 0 (0) 2 (2.74) SM 3 (5.46) 0 (0) 0 (0) 0 (0) 10 (0) 0 (0) 0 (0) 5 (6.85) THI 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) PASI 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) [/formula] Note: : number of strains tested. RGM strains have been shown to be susceptible to the newer generation of macrolide antibiotics (i.e., clarithromycin, azithromycin, and roxithromycin) [bib_ref] Rapidly growing mycobacteria in Singapore, Tang [/bib_ref] [bib_ref] An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases, Griffith [/bib_ref] [bib_ref] In vitro antimicrobial susceptibility of Mycobacterium abscessus in Korea, Park [/bib_ref]. This drug class is a good alternative for treating RGM species because of its high activity and oral formulations. Clinical experience shows that azithromycin toxicity is dose dependent and most adult patients with M. avium complex (MAC) lung disease do not tolerate azithromycin doses greater than 300 mg/day because of frequent of adverse events, including gastrointestinal symptoms (primarily diarrhea) and reversible hearing impairment [bib_ref] An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases, Griffith [/bib_ref]. In our research, the isolates were less susceptible to clarithromycin than to azithromycin, making the latter more applicable in the future. Some reports suggest that clarithromycin resistance can be induced in M. abscessus and M. fortuitum, and the resistance is associated with erm (41) and erm (39) genes, respectively [bib_ref] An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases, Griffith [/bib_ref] [bib_ref] Carbapenemnonsusceptible strains of Klebsiella pneumoniae producing SHV-5 and/or DHA-1 -lactamases in a..., Chudáčková [/bib_ref]. The M. abscessus complex is subclassified into three closely related subspecies (M. abscessus, M. massiliense, and M. bolletii) [bib_ref] Recent progress towards understanding genetic variation in the Mycobacterium abscessus complex, Howard [/bib_ref]. Historically, the M. fortuitum group has included three species: M. fortuitum, M. peregrinum, and an unnamed third biovariant complex [bib_ref] Clinical experience in 52 patients with tigecycline-containing regimens for salvage treatment of..., Wallace [/bib_ref]. The different subspecies potentially exhibit different drug susceptibilities. Macrolide resistance in M. abscessus and M. fortuitum will be the subject of our future research. Cefoxitin and cefmetazole are second and third generation cephalosporin antibiotics and in previous studies cefoxitin was frequently found to have good mycobacterial activity [bib_ref] Rapidly growing mycobacteria in Singapore, Tang [/bib_ref] [bib_ref] An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases, Griffith [/bib_ref] [bib_ref] Antibiotic susceptibility pattern of rapidly growing mycobacteria, Gayathri [/bib_ref] [bib_ref] In vitro antimicrobial susceptibility of Mycobacterium abscessus in Korea, Park [/bib_ref]. The isolates were more susceptible to cefmetazole than cefoxitin, so cefmetazole can be used where cefoxitin is ineffective. In addition, the antibacterial mechanism for meropenem, a carbapenem antibiotic, occurs via inhibition of bacterial cell wall synthesis [bib_ref] Single or in combination antimicrobial resistance mechanisms of Klebsiella pneumoniae contribute to..., Tsai [/bib_ref]. Carbapenems include imipenem, meropenem, and ertapenem, among others. In previous studies, imipenem was widely used in experiments [bib_ref] Rapidly growing mycobacteria in Singapore, Tang [/bib_ref] [bib_ref] An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases, Griffith [/bib_ref] [bib_ref] In vitro antimicrobial susceptibility of Mycobacterium abscessus in Korea, Park [/bib_ref]. Here, among the RGM strains, meropenem was found to have good activity and a lower MIC value than cefoxitin. In clinical work, rifabutin has been used mainly to target SGM, and toxicity to this drug was dose related. Clarithromycin has been shown to increase rifabutin serum levels and this effect was likely to be related to the hepatic metabolism of rifabutin [bib_ref] An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases, Griffith [/bib_ref]. Our data indicates that rifabutin and rifapentine can be used to treat rifampicinresistant strains; the RGM isolates have better susceptibility to rifabutin than rifapentine, but rifapentine was more active against M. chelonae, M. abscessus, and M. fortuitum. Otherwise, the rifabutin dose should be reduced when used in combination with clarithromycin to treat infections with RGM strains. Several studies have shown that linezolid and clofazimine have potent activities against NTM [bib_ref] Rapidly growing mycobacteria in Singapore, Tang [/bib_ref] [bib_ref] An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases, Griffith [/bib_ref] [bib_ref] Antimicrobial susceptibility of standard strains of nontuberculous mycobacteria by microplate Alamar Blue..., Li [/bib_ref] [bib_ref] In vitro antimicrobial susceptibility of Mycobacterium abscessus in Korea, Park [/bib_ref] [bib_ref] In vitro synergy between clofazimine and amikacin in treatment of nontuberculous mycobacterial..., Van Ingen [/bib_ref]. In our experiments, linezolid had >95% activity against the strains tested and clofazimine > 60% susceptibility for the RGM isolates (with the exception of M. chelonae). The small M. chelonae sample size probably affected this result. In the future, a larger sample size should be used to determine the MICs of the 31 drugs used in this study. The RGM isolates used in our research were from four different Chinese provinces. Most of them (86.30%) were from Fujian Province, making it important to obtain more samples from different geographical areas in the future. The data presented here suggest that tigecycline or linezolid combined with clofazimine or cefmetazole should be the most efficacious drug combination for treating M. abscessus infections. For M. fortuitum, capreomycin, sulfamethoxazole, tigecycline, clofazimine, and cefmetazole in combination may be a good choice. # Conclusions In summary, our data provide useful information on antibiotics that are effective against RGM and this information may help to identify suitable therapy for patients infected with such organisms. Future studies should address whether combining two or more antimicrobial agents for treatment of RGM infections is better than treatment with a single drug alone. [table] Table 1: MIC ( g/mL) breakpoints of 31 antimicrobial agents. [/table] [table] Table 2: MIC ( g/mL) results of the antimicrobial susceptibility tests for five rapidly growing reference mycobacteria. Bold, italic values indicate drug susceptibility. Values shown in bold indicate moderate drug susceptibility.The MIC 50 and MIC 90 values of doxycycline were 16 g/mL and 32 g/mL, respectively, for M. abscessus, and 64 g/mL and 256 g/mL, respectively, for M. fortuitum. Azithromycin had better activity against M. abscessus than M. fortuitum, with MIC 50 values of 0.5 g/mL and 2 g/mL, respectively. [/table] [table] Table 3: In vitro drug susceptibility percentage per species for rapidly growing mycobacteria isolates. [/table] [table] Table 4: The MIC range, MIC 50 , and MIC 90 ( g/mL) per species of rapidly growing mycobacterial isolates for all the antibacterial agents tested.Note: : number of strains tested. [/table]
Haploidy in Tobacco Induced by PsASGR-BBML Transgenes via Parthenogenesis Background: Engineering apomixis in sexually reproducing plants has been long desired because of the potential to fix hybrid vigor. Validating the functionality of genes originated from apomictic species that contribute to apomixis upon transfer to sexually reproducing species is an important step. The PsASGR-BABYBOOM-like (PsASGR-BBML) gene from Pennisetum squamulatum confers parthenogenesis in this apomict, and its functionality was demonstrated in several sexually reproducing monocots but not in any dicots. Methods: We introduced the PsASGR-BBML gene regulated by egg cell-specific promoters, either AtDD45 or AtRKD2, into tobacco, and analyzed progeny of the transgenic lines resulting from self-pollination and crossing by flow cytometry. Results: We identified haploid progeny at a frequency lower than 1% in the AtDD45 pro lines, while at a frequency of 9.3% for an octoploid (2n = 8x) AtRKD2 pro line. Haploid production in the T 2 generation, derived from the tetraploid T 1 offspring of this original octoploid AtRKD2 pro line, was also observed. Pollinated by homozygous transgenic tobacco carrying a DsRed marker gene, 4x progeny of the AtRKD2 pro line yielded parthenogenetic embryos identified as DsRed negative. We verified that the DsRed negative seedlings recovered were haploid (2x). Conclusion: The PsASGR-BBML gene regulated by egg cell-specific promoters could enable parthenogenesis in tobacco, a dicotyledon species. # Introduction Flowering plants (angiosperms) can reproduce both sexually and asexually. In female sexual reproduction, a nucellar cell goes through meiosis to produce four megaspores, one of which divides by mitosis to develop into the female gametophyte (embryo sac) containing an egg cell with two flanking synergid cells at the micropylar end, a central cell with two polar nuclei, and antipodal cells at the chalazal end. Microspore mother cells in anthers go through meiotic division and develop into the male gametophytes (pollen grains). The haploid gametophytes complete the life cycle by developing gametes which fuse to initiate the diploid sporophyte. Angiosperms require the process of double fertilization; the egg cell (1n) merges with a sperm cell (1n) to form a zygote (2n) that develops into an embryo encapsulated within a seed, while the central cell (2n) fuses with the second sperm cell (1n) from the same pollen grain to initiate endosperm (3n) development. In dicots, the endosperm typically is absorbed during seed maturation, while in monocots it provides nutrition for germinating seeds. In contrast, as an asexual reproduction pathway, apomixis produces seeds without double fertilization. The mechanisms of apomixis can be complex and diverse across species and have been reviewed intensively. In general, various forms of gametophytic apomixis share three common components: (1) apomeiosis by which unreduced female gametophytes form; (2) parthenogenesis by which female gametes develop into embryos without fertilization; (3) formation of endosperm autonomously or following fertilization of the central cell by a sperm cell (pseudogamy). If parthenogenesis occurs # Materials and methods ## Vector construction Two cassettes of the genomic PsASGR-BBML sequence (EU559280) fused with egg cell-specific promoters were generated. The cassette AtDD45 pro :gPsASGR-BBML contained a 1008 bp promoter of Arabidopsis DD45/EC1.2 (At2g21740), the 3540 bp of the PsASGR-BBML gene from BAC p208, including 8 exons and 7 introns, and 609 bp 3 of the stop codon plus the predicted poly(A) signal. The cassette was subsequently ligated into pCambia1300 (CAMBIA, Canberra, Australia) for hygromycin selection in transgenic plants. The recombinant vector pCambia1300-AtDD45 pro :gPsASGR-BBML (ddBR1) was introduced into Agrobacterium strains AGL1 and LBA4404 for plant transformation. The 521 bp promoter region of AtRKD2 (At1g74480), previously verified as egg-cell expressing in Arabidopsis,was amplified from genomic DNA of Arabidopsis Columbia using the primer combination p3905/3906. The sequence of the AtRDK2 promoter was verified. The AtDD45 promoter in the cassette AtDD45 pro :gPsASGR-BBML was replaced with the AtRKD2 promoter and the new cassette AtRKD2 pro :gPsASGR-BBML was ligated into pCambia2300 for kanamycin selection in transgenic plants. Kanamycin selection offered a simpler scheme to separate transformant T1 progeny from non-transformant T1, compared with the use of hygromycin selection (see Section 2.5). The new recombinant vector pCambia2300-AtRKD2 pro :gPsASGR-BBML (RKD2gBB) was verified by additional restriction mapping, and introduced into Agrobacterium strain EHA105, as AGL1 led to relatively severe tissue browning during the transformation of ddBR1. ## Plant transformation Two vectors were introduced into tobacco by Agrobacterium-mediated transformation adapted from Clemente. Leaves from 1-month-old tobacco plants (PI 552484, N. tabacum cv. Xanthi NN, seeds purchased from Lehle Seeds, Round Rock, TX, USA) grown in the greenhouse were collected, immersed in reverse osmosis deionized (RODI) water, and then surface sterilized with 10% commercial bleach (Clorox, Oakland, CA, USA, (6% sodium hypochlorite)) for 10 min, followed by 5× rinse with sterile RODI water. After removal of the midribs, the leaves were cut into 1-2 cm 2 explants and cultured on a shoot induction medium (SIM) consisting of Murashige and Skoog salts and vitamins (M519, PhytoTechnology Laboratories, Lenexa, KS, USA), 3% (w/v) sucrose (Research Products International, Mt Prospect, IL, USA), 1 mg/L 6-benzylaminopurine (BA), 0.1 mg/L 1-naphthaleneacetic acid (NAA), and 8 g/l agar with pH adjusted to 5.7. After 2 days of culture, the leaf disc explants were inoculated with Agrobacterium by immersing the explants in Agrobacterium suspension (OD 600 = 0.5~0.6, in liquid SIM containing 200 µM acetosyringone and 0.02% (v/v) Silwet-77 (Lehle Seeds, Round Rock, TX, USA)) for 15~20 min. After blotting dry with sterile filter paper, explants were placed on fresh SIM for co-culture. After 2 days of co-culture, explants were transferred to SIM containing 30 mg/L meropenem (ABBLIS Chemicals, Houston, TX, USA), plus 30 mg/L hygromycin or 200 mg/L kanamycin for ddBR1 and RKD2gBB, respectively. Each plate contained 5-7 explants which were transferred to fresh SIM with antibiotic selection every 2 weeks. When shoots reached 2-3 cm, they were excised and transferred to a root induction medium (RIM) containing MS salts and vitamins, 3% (w/v) sucrose, 0.1 mg/L NAA, and 8 g/L agar with pH adjusted to 5.7, plus 30 mg/L meropenem and the same selective agent used in SIM. Rooted plantlets were transferred to the Magenta™ GA7 vessels (Sigma-Aldrich, St. Louis, MO, USA), containing 0MS medium (SIM without plant growth regulators) with 30 mg/L meropenem and the same selective agent used in SIM, for further development. Plantlets derived from different leaf disc explants were considered independent transformation events while those from the same explants were considered potentially the same lines. Plantlets of 5-8 cm height were transferred to soil for acclimation. After genotyping, at least two plants from each independent line with a full-length PsASGR-BBML transgene were grown in the greenhouse to set seeds. All tissue cultures were maintained at 26 - C with a 16/8 h light cycle. Unless otherwise noted, all chemicals were from Sigma-Aldrich (St. Louis, MO, USA). ## Genotyping Genomic DNA was isolated from T0 transgenic plants and their progeny by using the CTAB method. The presence of the full-length gPsASGR-BBML transgene in the T0 transgenic lines and the haploid T1 progeny was verified by polymerase chain reaction (PCR) using the primer combination p1792/1801and 50-100 ng genomic DNA in a 25 µL reaction followed by 35 cycles of amplification with a cycling condition of 15 s at 98 - C, 15 s at 60 - C, and 4 min at 68 - C. In order to confirm the presence of the transgenes in the haploid T2 progeny of line RKD2gBB, progeny were genotyped by PCR using primer combinations p3767/4127 and p4303/4304. PCR reactions consisted of 1× GoTaq ® Master Mix (M7123, Promega, Madison, WI) and 50-100 ng genomic DNA in a 20 µL reaction followed by 32 cycles of amplification with a cycling condition of 15 s at 95 - C, 15 s at 60 - C, and 30 s at 72 - C. PCR products were visualized under UV light after electrophoresis in 1% agarose gels and staining with ethidium bromide. ## Estimation of transgene copy numbers by quantitative pcr In order to estimate the copy number of the transgenes in T0 and T1 plants, genomic DNA from transgenic tobacco was digested overnight with EcoRI-HF ® (NEB, Ipswich, MA, USA), followed by purification using the DNA Clean and Concentrator Kit (D4033, Zymo Research, Irvine, CA, USA). The purified DNA, after dilution 20-fold, was used as a template for quantitative PCR (qPCR) using a LightCycler ® 480 system (Roche, Basel, Switzerland). PCR reactions were set up following the manufacturer's instruction of the LightCycler ® 480 SYBR Green I Master mix V13 (Roche) and conducted using the SYBR green I/HRM dye program with the primer combination p4303/4304 for the PsASGR-BBML transgene and the primer combination p4133/4134 for the tubulin gene (NCBI Reference Sequence: XM_016623993) as a reference gene. The amplification efficiency of each assay was estimated based on the qPCR data of a 5-log serial dilution (0.0016, 0.008, 0.04, 0.2, 1×) of a DNA mixture that contained an equal amount of DNA from each tested sample by using the absolute quantification method in the software of the Lightcycler ® 480 system. Each reaction had two technical replicates. The qPCR data were analyzed by using the advanced relative quantification method in the software of the Lightcycler ® 480 system to estimate the copy numbers of the PsASGR-BBML transgene in transgenic tobacco plants. ## Seed germination assay for transgenic progeny Seeds were surfaced sterilized using a 16-h chlorine gas treatment (2 mL 12 M HCl into 200 mL commercial bleach in a 10 L desiccator) or immersing in 10% commercial bleach (Clorox) for 10 min followed by 5× rinse with sterile RODI water. Since cotyledons could turn green despite the lack of transgene when seeds were germinated on hygromycin-containing medium and unreduced transgenic progeny potentially could outcompete the haploid plants with prolonged culture on hygromycin-containing medium, a selection scheme was developed to quickly separate unreduced transgenic progeny from their haploid siblings and non-transgenic with a short culture period on hygromycin-containing medium, based on a method developed for Arabidopsis. Seeds of the ddBR1 lines were sown on 0MS medium containing 60 mg/L hygromycin and incubated in the dark for 7-10 days. Seedlings displaying elongated hypocotyls (>0.5 cm, mostly 0.8~1 cm) were considered to be transgenic while seedlings that did not elongate (<0.5 cm, mostly 0.1~0.3 cm) were considered to be either non-transgenic or potentially haploid transgenic. After transfer to 0MS for further growth with a 16/8 h light cycle, short seedlings that turned green were considered potential transgenic haploids while seedlings that failed to turn green and grow further were considered to be non-transgenic. The ploidy levels of those potentially haploid transgenic plants were examined by flow cytometry within a month. Adapted from, seeds of the RKD2gBB lines were sown on 0MS containing 600 mg/L kanamycin which was intended to prevent non-transgenic escape. After a 2-week culture period, green seedlings were considered to be transgenic while seedlings showing chlorosis (i.e., fully or partially whitening) at cotyledons or the shoot apex were considered to be non-transgenic. Those resistant transgenic seedlings were transferred to 0MS for further growth and their ploidy levels were examined by flow cytometry within a month. In order to estimate the baseline of autonomous haploid production of the PI 552484, non-transgenic seeds were sown on 0MS and the seedlings that did not elongate after 7 days in the dark were considered to be potentially haploid. Those small seedlings were transferred to fresh 0MS for further growth with a 16/8 h light cycle, and their ploidy levels were examined by flow cytometry within a month. ## Flow cytometry Possible parthenogenesis in T0 transgenic plants was first determined by flow cytometric seed screen (FCSS). Preliminary microscopy study found most of the endosperm was absorbed by the embryo when capsules started to turn brown but were not completely desiccated (21-28 days after pollination). Capsules at this developmental stage were selected for FCSS as seeds were not too hard to chop while the flow cytometric signal would be mostly from the developing embryos rather than the endosperm. Bulked seed samples from two individual capsules were processed for each transgenic plant adapted from Conner. In brief, 50-100 developing seeds together with young leaf tissue of sorghum (Sorghum bicolor) or cowpea (Vigna unguiculata) as a genome-size standard were chopped in 200 µL LB01 lysis buffer consisting of 15 mM Tris, 2 mM Na 2 EDTA, 0.5 mM spermine tetrahydrochloride, 80 mM KCl, 20 mM NaCl, 0.1% (v/v) Triton X−100, pH 7.5 and 16 mM 2-mercaptoethanol, and filtered with a 30 µm CellTrics disposable filter (Sysmex Partec, Görlitz, Germany). After propidium iodide solution containing RNase (Cat# 550825, BD Biosciences, San Jose, CA, USA) was added at half volume, the filtered samples were incubated on ice for at least 15 min, followed by analysis using a BD Accuri C6 flow cytometer (BD Biosciences) with gating set by the selection of objects with a strong correlation between FL2 and FL3 signals using a flow rate of 35 µL sample per min. Events were collected within the gated region for each sample. In order to identify haploid seedlings, selected tobacco seedlings (see Section 2.5) were analyzed by flow cytometry when they reached at least the 4-leaf stage. Leaf tissues (1 mm 2 or less) from five seedlings were pooled together and processed as described previously by flow cytometry. If a bulk sample showed a haploid signal, leaf tissue from each individual comprising the bulk was collected and processed by flow cytometry to identify the haploid individuals. The efficiency of haploid production was determined based on the percentage of haploid seedlings among the total seedlings germinated. ## Transcription analysis of the psasgr-bbml transgene in tobacco ovules Ovules were isolated from 6-7 flowers at stage 11 of tobacco flower developmentand stored in the RNAlater™ Storage Solution (Invitrogen, Carlsbad, CA, USA). Total RNA was extracted using the RNeasy ® Plant Mini Kit (QIAGEN, Hilden, Germany), followed by a DNase (Cat# 18068015, ThermoFisher Scientific, Waltham, MA, USA) treatment according to the manufacturer's recommendation to remove genomic DNA. Single strand cDNA was synthesized with the SuperScript III First-strand Synthesis System (Invitrogen) by reverse transcription PCR (RT-PCR). The presence of the PsASGR-BBML transcript including 8 exons was verified by PCR using the primer combination p1792/1793. PCR reactions consisted of 2 µL of the first-strand cDNA synthesis reaction, 1× PrimeSTAR GXL Buffer, 200 µM dNTP, 0.2 µM primers, 0.625 U PrimeSTAR GXL DNA Polymerase in a 25 µL reaction (Takara Bio USA, Inc.) followed by 35 cycles of amplification with a cycling condition of 15 s at 98 - C, 15 s at 60 - C, and 2 min at 68 - C. PCR products were visualized under UV light after electrophoresis in 1% agarose gels and staining with ethidium bromide.were used to amplify the transcript from the start of the ORF through to the 3 UTR. Amplified PCR products from each sample were directly cloned into PCR4-TOPO (Invitrogen) vector with cloned inserts sequenced at Psomagen (Rockville, MD, USA). Sequencing data were analyzed using Geneious prime software (Biomatters Limited, Auckland, New Zealand). ## Tobacco crossing Flowers were emasculated at stage 10in the evening before anthesis. The emasculated flowers were pollinated the following morning, tagged and capped with tailored pollination envelopes. The 4x T1 plants of line RKD2gBB_6.1 (an octoploid, 2n = 8x) were pollinated by non-transgenic tobacco. The 4x T2 plants of line RKD2gBB_6.1 were pollinated by homozygous T3 plants of a transgenic tobacco line (2n = 4x) carrying a cassette of a DsRed gene regulated by the promoter of soybean (Glycine max) elongation factor 1a (GmEF1a, Glyma.17G186600, Phytozome v12) and the nopaline synthase (NOS) terminator that segregated as a single locus (Zhang unpublished). The embryos and seedlings without DsRed fluorescence were considered to be parthenogenetic, since these progenies did not inherit the DsRed transgene from the male parent. ## Embryo isolation and observation Capsules were harvested 9 through 17 days after pollination. Ovules were isolated and macerated in an enzymatic solutioncomposed of 12% (w/v) mannitol, 3 mM MES, 1% (w/v) cellulose R-10 (Research Products International), and 0.8% (w/v) Rhizopus sp. pectinase, with pH adjusted to 5.7, for 30 min with agitation at 60 rpm. The ovules were rinsed in a washing buffer composed of 12% (w/v) mannitol and 3 mM MES (pH 5.7) at least 3 times and then gently ground with a small pestle to release the developing embryos. Without removing integument debris, isolated embryos were mounted on a glass slide in the washing buffer and observed under a microscope (Zeiss, Thornwood, NY, USA) equipped with a PhotoFluor LM-75 illuminator (89 North, Williston, VT, USA) and a Ds-RED filter (excitation: 545/25 nm, emission: 605/70 nm, Chroma Technology, Bellows Falls, VT, USA). Images were taken using an AxioCam camera (Carl Zeiss, Oberkochen, Germany) and the AxioVision LE64 software. Embryos without visible damage were counted. Unless otherwise noted, all chemicals were obtained from Sigma-Aldrich. # Results ## Recovery of transgenic tobacco Thirty and seventeen T0 lines that contained all eight exons, seven introns and 3 UTR were recovered, respectively, for the cassettes AtDD45 pro :gPsASGR-BBML (ddBR1) and AtRKD2 pro :gPsASGR-BBML (RKD2gBB). In general, all transgenic lines displayed wild type vegetative growth, except for one ddBR1 line that was severely stunted. Upon entering the reproductive stage, most T0 lines showed normal flower development and produced seeds by self-pollination. In some T0 lines, short stamens and poor pollen production were observed. By hand-pollination with their own pollen when available, all lines with abnormal phenotypes produced viable seeds except for the severely stunted ddBR1 line. The PsASGR-BBML transcript including eight exons was detected in ovules of ten transgenic lines that were randomly selected for each construct at stage 11 of tobacco flower development. ## Haploid production of t0 transgenic tobacco As tobacco is an allotetraploid (2n = 4x), progeny were considered "haploid" when their genome size was calculated at half of their maternal parent. Five independent ddBR1 lines (5/29, 17.2%) and three independent RKD2gBB lines (3/17, 17.6%) displayed a haploid (2x since tobacco is tetraploid) signal when immature seeds were analyzed by FCSS. All T0 transgenic lines remained tetraploid (2n = 4x) except for three RKD2gBB lines (RKD2gBB_4.1, RKD2gBB_6.1 and RKD2gBB_7.1) which became octoploid (2n = 8x), probably due to genome duplication during tissue culture. According to FCSS, two of the haploid-producing RKD2gBB T0 lines (RKD2gBB_6.1 and RKD2gBB_7.1) were octoploid (2n = 8x,. The ploidy levels of the haploid-producing lines were confirmed by flow cytometry analysis using leaf tissue of the T0 plants, and these haploid-producing lines were analyzed in further detail. In a preliminary study, T1 seedlings of a haploid-producing ddBR1 line that germinated on 0MS containing 60 mg/L hygromycin and elongated after seven days of culture in the dark were all tetraploid (4x) as their T0 parent. Haploid (2x) seedlings were only identified among seedlings that failed to elongate during the first seven days on 0MS containing hygromycin. Thus, we decided to focus on the T1 seedlings of the ddBR1 lines that failed to elongate on 0MS containing 60 mg/L hygromycin within 10 days where haploid plants would be more likely to be identified. Haploid (2x) seedlings were identified among T1 seedlings of three haploid-producing ddBR1 lines (2n = 4x), ddBR1_204.2, ddBR1_205.1 and ddBR1_240.2, at a frequency lower than 1%, while no haploid T1 seedlings were recovered from the other two haploid-producing ddBR1 lines, ddBR1_13.1 and ddBR1_204.1, showing FCSS haploid peaks (Table 1,. This could be due to the frequency of haploid seedling production against the number of seedlings screened. In contrast, among the three haploid-producing RKD2gBB lines, haploid (4x) T1 seedlings were identified from one octoploid (2n = 8x) line (RKD2gBB_6.1) at a frequency of 9.3% while no haploid plant was identified in the other two lines, RKD2gBB_3.2 and RKD2gBB_7.1, showing FCSS haploid peaks (Table 1,. In comparison, 397 seedlings that remained stunted within the first two weeks of growth on 0MS from 3700 non-transgenic seedlings were analyzed by flow cytometry, and no haploid seedlings were identified. In a preliminary study, T1 seedlings of a haploid-producing ddBR1 line that germinated on 0MS containing 60 mg/L hygromycin and elongated after seven days of culture in the dark were all tetraploid (4x) as their T0 parent. Haploid (2x) seedlings were only identified among seedlings that failed to elongate during the first seven days on 0MS containing hygromycin. Thus, we decided to focus on the T1 seedlings of the ddBR1 lines that failed to elongate on 0MS containing 60 mg/L hygromycin within 10 days where haploid plants would be more likely to be identified. Haploid (2x) seedlings were identified among T1 seedlings of three haploid-producing ddBR1 lines (2n = 4x), ddBR1_204.2, ddBR1_205.1 and ddBR1_240.2, at a frequency lower than 1%, while no haploid T1 seedlings were recovered from the other two haploid-producing ddBR1 lines, ddBR1_13.1 and ddBR1_204.1, showing FCSS haploid peaks (Table 1,. This could be due to the frequency of haploid seedling production against the number of seedlings screened. In contrast, among the three haploid-producing RKD2gBB lines, haploid (4x) T1 seedlings were identified from one octoploid (2n = 8x) line (RKD2gBB_6.1) at a frequency of 9.3% while no haploid plant was identified in the other two lines, RKD2gBB_3.2 and RKD2gBB_7.1, showing FCSS haploid peaks (Table 1,. In comparison, 397 seedlings that remained stunted within the first two weeks of growth on 0MS from 3700 non-transgenic seedlings were analyzed by flow cytometry, and no haploid seedlings were identified. (f) a 2x T2 progeny of a 4x T1 plant K6.1_20. S2 G1 and S4 G2 designate 2n/2x/2c and 2n/2x/4c peaks of sorghum or cowpea, N2 G1 , N4 G1 , N4 G2 , N4 G1|G2 , N8 G2 , N8 G1|G2 , and N16 G2 designate 2n/2x/2c, 2n/4x/4c, 2n/2x/4c, 2n/4x/4c|2n/2x/4c, 2n/4x/8c, 2n/8x/8c|2n/4x/8c, and 2n/8x/16c peaks of tobacco, respectively. (f) a 2x T2 progeny of a 4x T1 plant K6.1_20. S2 G1 and S4 G2 designate 2n/2x/2c and 2n/2x/4c peaks of sorghum or cowpea, N2 G1 , N4 G1 , N4 G2 , N4 G1|G2 , N8 G2 , N8 G1|G2 , and N16 G2 designate 2n/2x/2c, 2n/4x/4c, 2n/2x/4c, 2n/4x/4c|2n/2x/4c, 2n/4x/8c, 2n/8x/8c|2n/4x/8c, and 2n/8x/16c peaks of tobacco, respectively.For the ddBR1 lines, the seedlings screened were from a sub-population of seedlings which did not elongate when germinated on 0MS medium containing 60 mg/L hygromycin and remained shorter than 0.5 cm within 10 days. All haploid-producing lines had a relatively simple integration of the transgene. Based on the elongation of T1 seedlings germinated in the dark, indicating resistance to hygromycin, the transgene likely segregated as a single locus in all five haploid-producing T0 ddBR1 lines except for line ddBR1_204.1 showing segregation distortion of the transgene and having an inflated number of T1 seedlings characterized as "susceptible". Among the haploid-producing RKD2gBB lines, the transgene segregated as a single locus in lines RKD2gBB_3.2 and RKD2gBB_7.1, and as two loci in the line RKD2gBB_6.1 which showed the highest efficiency in haploid production. When genotyped by PCR, all haploid T1 plants identified carried the full-length sequence of PsASGR-BBML transgene. Quantitative PCR further suggested that the haploid-producing T0 lines should have one or two copies of the transgene integrated except for one line. ## Haploid production of the progeny derived from line rkd2gbb_6.1 As tobacco is an allotetraploid, haploid T1 plants derived from the ddBR1 lines could not form normal embryo sacs due to the lack of homologous chromosomes for pairing at meiosis. As a result, they were sterile and produced no seeds. Since line RKD2gBB_6.1 was 8x, its haploid (4x) T1 were fertile and capable of producing seeds, providing an opportunity to verify the functionality of the PsASGR-BBML transgene among progeny lines even when the ploidy level reduced by half in the second generation. Similar to the T0 generation, T1 plants of line RKD2gBB_6.1 tended to have short stamens and many empty anthers. Nevertheless, seeds could still be produced by hand-pollination with their own pollen as long as some pollen grains were available. Among nine 4x T1 plants derived from line RKD2gBB_6.1 (2n = 8x), three were able to produce T2 seeds after hand-pollination. The other six plants produced no T2 seeds after attempted hand-pollination as functional pollen grains were not produced from these plants. Haploid (2x and 4x) T2 seedlings were identified from the three 4x T1 plants (K6.1_11, K6.1_20 and K6.1_23) and four 8x T1 plants (K6.1_1, K6.1_12, K6.1_17, and K6.1_22) analyzed, respectively (Table 2,. The efficiency of haploid production ranged from 2.7% to 27.3%, showing no clear correlation to the numbers of loci inherited or the copy numbers of the transgene. The T1 plants that inherited one locus or both were able to produce haploids. In general, the efficiency of haploid production tended to be higher in the 8x T1 than the 4x T1. The 8x T1 plants tended to set seeds more poorly with higher frequencies of haploid progeny associated with poorer seed setting and where fewer seeds were available for analysis. Haploid (2x) progeny were obtained at a frequency of 5.6% when a 4x T1 plant of line RKD2gBB_6.1 was pollinated by non-transgenic tobacco. The number of seedlings susceptible to kanamycin inflated in the F1 to about three times as many as the resistant seedlings . Segregation distortion was also observed in progeny from the 4x T1 plants of line RKD2gBB_6.1 that inherited one or both transgene loci. The number of resistant seedlings was much lower than expected when these T1 plants were self-pollinated and a relatively large proportion of seeds failed to germinate. When genotyped, all haploid T2 progeny identified carried the PsASGR-BBML transgene and the nptII marker gene. Attempts to recover homozygous T1 plants of any haploid-producing ddBR1 lines were unsuccessful. A homozygous T1 plant of line RKD2gBB_6.1 was likely recovered (K6.1_7,according to the copy number estimated by qPCR, but it only produced distorted flowers and no seeds. ## Transcription of the psasgr-bbml transgene in the 4x t1 of line rkd2gbb_6.1 The presence of the PsASGR-BBML transcript including eight exons was verified in all four 4x T1 plants of line RKD2gBB_6.1along with four T1 plants not displaying parthenogenesis. Sequencing data showed that the PsASGR-BBML transcript from two 4x T1 plants (K6.1_20 and K6.1_23) of line RKD2gBB_6.1 was identical to the original transcript, showing no alternative splicing or sequence alteration to explain the higher rate of parthenogenesis in this line. ## Observation of parthenogenetic embryos Attempts to observe parthenogenetic embryos via ovule clearing from emasculated flowers of the haploid-producing lines were unsuccessful. This method had been used to successfully identify parthenogenesis in pearl millet transgenic lines. Tobacco flowers abscised approximately six days after emasculation with no defined embryos observed in cleared ovules. Pollination was needed to prevent flower abscission, and to ensure that haploid embryos could further develop and be identified. In preliminary studies to enzymatically isolate embryos from the several hundred ovules of a capsule, only a small number of four-celled embryos were observed seven days after pollination. This suggested that either tobacco embryos at the four-cell stage were too fragile to isolate or that the majority of embryos might be at an earlier developmental stage that could be easier to damage during isolation or harder to identify after isolation. We decided to examine capsules at least nine days after pollination when the majority of embryos entered the eight-cell stage or later. After crossing with pollen from tobacco plants homozygous for a DsRed fluorescence marker gene driven by a strong constitutive promoter GmEF1apro, 4x T2 progeny derived from three 8x T1 plants of line RKD2gBB_6.1 that inherited one or both loci of the transgene were able to produce embryos or seeds without DsRed fluorescence,. The DsRed negative embryos were considered to be parthenogenetic due to the lack of the DsRed transgene transmitted by the paternal parent. Developing parthenogenetic embryos without DsRed fluorescence were observed at a frequency from 3.2% to 8.0% among different 4x T2 plants of line RKD2gBB_6.1which was comparable to the frequency of haploid production observed when a 4x T1 plant was pollinated by wildtype pollen. Those DsRed negative parthenogenetic embryos tended to develop as normally as the DsRed positive embryos from the same capsule, though abnormality (e.g., slow-growth, deformity, etc.) was occasionally observed in some of the DsRed negative embryos. Seedlings without DsRed fluorescence were also identified when the mature seeds from capsules obtained by crossing were germinated, and they were verified as haploid (2x) by flow cytometry . According to PCR genotyping, these 2x haploid seedlings carried the PsASGR-BBML transgene but not the DsRed gene. RKD2gBB_6.1 that inherited one or both loci of the transgene were able to produce embryos or seeds without DsRed fluorescence,. The DsRed negative embryos were considered to be parthenogenetic due to the lack of the DsRed transgene transmitted by the paternal parent. Developing parthenogenetic embryos without DsRed fluorescence were observed at a frequency from 3.2% to 8.0% among different 4x T2 plants of line RKD2gBB_6.1which was comparable to the frequency of haploid production observed when a 4x T1 plant was pollinated by wildtype pollen. Those DsRed negative parthenogenetic embryos tended to develop as normally as the DsRed positive embryos from the same capsule, though abnormality (e.g., slow-growth, deformity, etc.) was occasionally observed in some of the DsRed negative embryos. Seedlings without DsRed fluorescence were also identified when the mature seeds from capsules obtained by crossing were germinated, and they were verified as haploid (2x) by flow cytometry . According to PCR genotyping, these 2x haploid seedlings carried the PsASGR-BBML transgene but not the DsRed gene. # Discussion This report demonstrates that the PsASGR-BBML transgene, regulated by egg cell-specific promoters, enabled tobacco to produce haploid progeny via parthenogenesis as previously observed in sexual pearl millet, rice and maize. While haploid production by self-pollination has been reported in some tobacco varieties at a frequency of approximately 0.01%, the haploid frequency observed in the current study with transgenic tobacco was 20 to 900 times higher. In addition, haploid progeny was not identified among seeds resulting from self-pollination of the non-transgenic PI line used to produce the transgenic lines. This indicates that haploid production was a gain of function attributable to the transgene rather than a propensity caused by the genetic composition of the PI. Expression of PsASGR-BBML in egg cells presumably transforms the egg cells into a zygote-like state, inducing embryogenesis from unfertilized eggs. Use of an egg cell-specific promoter AtDD45 to regulate the gene PsASGR-BBML or OsBBM1 whose protein sequence clustered with the PsASGR-BBML protein in a phylogenetic subcladewas able to induce parthenogenetic embryos in rice. Compared with the monocot species tested, the efficiency of haploid production in transgenic tobacco where the PsASGR-BBML transgene was regulated by the AtDD45 promoter was low. Given that embryogenesis in monocots significantly diverges from dicots, the PsASGR-BBML transgene by itself may be inefficient, if sufficient, to transform the egg cell into a zygote-like cell in tobacco, which was also implied in the study of Arabidopsis where haploid progeny were not recovered from any transgenic lines expressing the PsASGR-BBML transgenes. The reasons why the PsASGR-BBML transgene functioned more efficiently in tobacco than Arabidopsis remains unknown. The higher efficiency of haploid production in the RKD2gBB line than the ddBR1 lines was likely attributable to temporal or spatial differences in the promoter activity. When haploid progeny were identified, the RKD2gBB line (RKD2gBB_6.1) yielded haploid progeny at a frequency at least 10 times higher than the ddBR1 lines. While AtDD45 pro and AtRKD2 pro were both considered to be egg cell-specific, gene expression regulated by the AtRKD2 pro was tightly restricted to the egg cellwhile the expression regulated by the AtDD45 pro was observed in the egg cell and synergids (the egg apparatus), as well as the early developing embryos from the zygote stage to the eight-cell stage. If the activity of these two promoters in tobacco was consistent with what was observed in Arabidopsis, such temporal and spatial differences in the PsASGR-BBML expression likely resulted in more haploid progeny recovered in the RKD2gBB line than the ddBR1 lines, which was comparable to the observation where egg ablation by using the barstar gene happened more frequently when the gene was regulated by the AtRKD2 pro than the AtDD45 pro. While RKD transcription factors play an essential role in maintaining the quiescent state of egg cells, expression of the PsASGR-BBML transgene in a temporal and spatial manner similar to the endogenous RKD genes appeared to relax the quiescent state of egg cells and promote parthenogenesis (inducing embryogenesis from unfertilized egg cells). In addition to the differences of the promoter activity, it could be argued that the genome duplication in line RKD2gBB_6.1 might contribute to the higher efficiency of haploid production. Since the 4x T1 progeny of line RKD2gBB_6.1 still produced more haploid progeny than the ddBR1 linesand two other octoploid (8x) RKD2gBB lines, line RKD2gBB_4.1 and line RKD2gBB_7.1, recovered in this study did not produce any haploid progeny or at a very low frequency, respectively, the increase in ploidy would not be the only cause of more haploid production. We are unable to exclude the possibility that the particular integrations of the transgene in the genome could magnify the transgene effect in line RKD2gBB_6.1 which would require further investigation. Regardless of a similar frequency of haploid-producing lines identified, none of the ddBR1 lines reached the same level of haploid production as line RKD2gBB_6.1. The efficiency of haploid production varied among lines, though a full-length of the PsASGR-BBML transgene was present in all these lines. Such variation was also observed in the previous study of rice and maize, probably due to positional differences in integration of the transgene. The transgenes seemed to segregate as a single locus in all haploid-producing lines except for line RKD2gBB_6.1 that had the transgenes integrated at two independent loci. Based on the haploid production in the limited number of RKD2gBB_6.1 T1 progeny tested, the transgene at one locus was sufficient to induce haploid production. A segregating population would be needed to determine whether both loci were functional and whether an additive effect of the two loci might lead to a higher frequency of haploid production. The segregating population could also help to unravel potentially unknown changes in the genome if any occurred that contributed to haploid production. Characterizing the integration sites of the transgene would be helpful to examine whether the transgene integration interrupted any genes associated with reproduction which might have enhanced the transgene effect on parthenogenesis. Homozygous progeny have not been recovered for any haploid-producing lines, except for one T1 plant (K6.1_7) of line RKD2gBB_6.1 that appeared to be a homozygote based on the qPCR data but was unable to set seeds due to the severe malformation of flowers. Previous study in rice and maize also had difficulty in recovering plants homozygous for the PsASGR-BBML transgenes. Homozygosity potentially brought about embryo lethality given the segregation ratio of the T1 progeny derived from some haploid-producing lines (i.e., a 2:1 ratio in. The lack of fertile homozygous plants made it difficult to address the question of why not all egg cells that inherited the transgene showed the parthenogenesis phenotype (penetrance). The inflated numbers of non-transgenic progeny and a large proportion of seeds that failed to germinate in some T0 and T1 plants (Tables S3 and S4) suggested that the transgene or its integration site might affect embryo development and survival, which was observed in rice carrying the PsASGR-BBML transgenes. Unlike sexual pearl millet carrying the PsASGR-BBML transgene where parthenogenetic embryos were observed in ovules of emasculated flowers without pollination, developing embryos were not observed in ovules of the haploid-producing transgenic tobacco lines unless emasculated flowers were hand-pollinated. Pseudogamous parthenogenesis observed in the PsASGR-BBML transgenic tobacco might be similar to what was observed in Boechera apomicts where self-pollination is still required to initiate the development of apomictic embryos as in sexual reproduction but the egg cell does not fuse with either sperm cell. Since parthenogenetic progeny were obtained as long as the 4x progeny of line RKD2gBB_6.1 were pollinated regardless of whether the pollen carried the PsASGR-BBML transgene or not, pollination could simply lead to fertilization of the central cell and endosperm formation to support development of parthenogenetic embryos into viable seeds, which was also observed and required in monocot species. Fertilization of the central cells could generate certain critical signals to regulate early embryo development as observed in Arabidopsisthat would affect embryo survival in tobacco. Pollination also could deliver some paternal signalsthat potentially influence parthenogenetic egg or zygote development. If parthenogenetic embryos began to develop without pollination, the slowness of embryo development in tobacco made it difficult to observe them, as most embryos only completed the first division in ovules seven days after pollination whereas flowers without pollination usually abscised six days after emasculation, as previously reported. Taking advantage of a homozygous tobacco line carrying a GmEF1a:DsRed:NOS cassette as a pollen donor enabled us to identify parthenogenetic embryos that could be distinguished from the sexually reproducing embryos due to their lack of the paternal trait DsRed fluorescence. The haploidy characteristic of the DsRed negative seedlings was verified, indicating that the DsRed negative embryos did result from parthenogenesis. # Conclusions Despite its origin from a monocot species, the PsASGR-BBML gene was shown to be functional in a dicot species, inducing haploid production in tobacco. Tuning the PsASGR-BBML transgene by altering the promoter seemed to improve the efficiency of haploid production. Compared with the monocot species previously studied, the lower penetrance of the gene in the dicot species tobacco probably stems from the divergence in the reproductive process and embryogenesis between monocots and dicots. Further investigation on the genetic network and protein interactions with the PsASGR-BBML transcription factor in egg cells may reveal the absence of monocot-specific co-factors or the presence of suppressors in dicots that hinder the full potential of the PsASGR-BBML gene to promote parthenogenesis. Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4425/11/9/1072/s1,: Flow cytometric seed screen (FCSS) analysis identified T0 plants that produced haploid progeny;: Expression of the PsASGR-BBML transgene in the ovules from the haploid-producing 4x T1 of RKD2gBB_6.1 compared with non-haploid-producing RKD2gBB T0 lines;: Sequences of primers used for PCR;: T0 lines randomly selected to verify expression of the PsASGR-BBML transgene in ovules of flowers at developmental stage 11 by RT-PCR;. Genetic segregation and copy number estimate of the transgenes in the haploid-producing T0 lines;. Genetic segregation and copy number estimate of the transgenes in T1 progeny of line RKD2gBB_6.1; . Genetic segregation in progeny of a cross between a 4x RKD2gBB_6.1 T1 and non-transgenic tobacco.
Finding multiple target optimal intervention in disease-related molecular network Drugs against multiple targets may overcome the many limitations of single targets and achieve a more effective and safer control of the disease. Numerous high-throughput experiments have been performed in this emerging field. However, systematic identification of multiple drug targets and their best intervention requires knowledge of the underlying disease network and calls for innovative computational methods that exploit the network structure and dynamics. Here, we develop a robust computational algorithm for finding multiple target optimal intervention (MTOI) solutions in a disease network. MTOI identifies potential drug targets and suggests optimal combinations of the target intervention that best restore the network to a normal state, which can be customer designed. We applied MTOI to an inflammation-related network. The well-known side effects of the traditional non-steriodal anti-inflammatory drugs and the recently recalled Vioxx were correctly accounted for in our network model. A number of promising MTOI solutions were found to be both effective and safer. # Introduction The one-target one-drug paradigm has been the dominating drug discovery approach in the past decades. The paradigm focuses on identifying a single chemical entity that binds to a single target. This single-target-based method was believed to be more efficient than the traditional in vivo approach because of its greater screening capacity and the associated rational drug discovery programs. However, the number of successful drugs did not increase as expected [bib_ref] Target-based drug discovery: is something wrong?, Sams-Dodd [/bib_ref]. Problems occur when the target itself is uncertain and when multiple targets have to be involved in disease control. It was long realized that the behavior of drug molecules in a disease network can be complex. Drugs with efficacy predicted only from their specific target-binding experiment may not have the same effect in clinical treatment due to interactions between pathways in the disease network [bib_ref] The efficiency of multi-target drugs: the network approach might help drug design, Csermely [/bib_ref]. Side effects often occur because of the unexpected effects of the drug. For example, in the case of inflammation, two pathways, the COX (cyclooxygenase) pathway and the 5-LOX (5-lipoxygenase) pathway, produce inflammatory mediators. Molecules important for normal physiology, such as vasoactive substance prostacyclin (PGI2) and thromboxane A2 (TXA2), are metabolized in the same system as well. Selective COX-2 inhibitors can effectively prohibit the production of certain inflammatory mediators, such as prostaglandins . But this leads to a concomitant increase of leukotrienes (LTs), another kind of inflammatory mediator [bib_ref] Leukotrienes in the pathogenesis of NSAIDinduced gastric and intestinal mucosal damage, Rainsford [/bib_ref] [bib_ref] Use and benefits of nonsteroidal anti-inflammatory drugs, Brooks [/bib_ref]. Even worse, it causes a cardiovascular side effect due to the induced unbalance between PGI2 and TXA2 [bib_ref] Cardiovascular hazard and non-steroidal anti-inflammatory drugs, Wang [/bib_ref]. To overcome the limitations of the single-target-based drugs, growing attention has been paid to drug discoveries involving multiple targets and at the level of disease networks [bib_ref] The efficiency of multi-target drugs: the network approach might help drug design, Csermely [/bib_ref] [bib_ref] Drug discovery: playing dirty, Frantz [/bib_ref] [bib_ref] A robustness-based approach to systems-oriented drug design, Kitano [/bib_ref] [bib_ref] Multi-target therapeutics: when the whole is greater than the sum of the..., Zimmermann [/bib_ref]. Some clinical data support the benefits of multi-target drugs. Examples of such strategy can be found in the combinatorial therapy of AIDS, atherosclerosis, cancer, and depression [bib_ref] How to design multi-target drugstarget search options in cellular networks, Korcsmaros [/bib_ref]. The discovery that some traditional remedies and empirically selected drugs act on multiple targets also suggests that multi-target drugs can be beneficial [bib_ref] From large networks to small molecules, Sharom [/bib_ref]. Several marketing successes of multicomponent therapies have already been reported: salmeterol/fluticasone (Advair; GlaxoSmithKline) [bib_ref] Advair: combination treatment with fluticasone propionate/salmeterol in the treatment of asthma, Nelson [/bib_ref] , nicotinic acid/lovastatin (Advicor; Kos Pharmaceuticals) [bib_ref] Lovastatin and extended-release niacin combination product: the first drug combination for the..., Gupta [/bib_ref] [bib_ref] Comparison of once-daily, niacin extended-release/lovastatin with standard doses of atorvastatin and simvastatin..., Bays [/bib_ref] and AZT-3TC (Combivir; GlaxoSmithKline) [bib_ref] Potential mechanism for sustained antiretroviral efficacy of AZT-3TC combination therapy, Larder [/bib_ref]. A diverse range of work has been carried out in the emerging field of multi-target drug design. Cell-based phenotypic assays were employed to construct better models of disease systems [bib_ref] Systematic discovery of multicomponent therapeutics, Borisy [/bib_ref] [bib_ref] Multi-target therapeutics: when the whole is greater than the sum of the..., Zimmermann [/bib_ref]. Ligand promiscuity was studied to design inhibitors against multiple targets [bib_ref] Can we rationally design promiscuous drugs?, Hopkins [/bib_ref]. Disease-related networks were reconstructed to gain insights at a global and systems level [bib_ref] A physical and functional map of the human TNF-alpha/NF-kappa B signal transduction..., Bouwmeester [/bib_ref] [bib_ref] Global reconstruction of the human metabolic network based on genomic and bibliomic..., Duarte [/bib_ref]. Various databases have been developed for systematic analysis [bib_ref] Development of human protein reference database as an initial platform for approaching..., Peri [/bib_ref] [bib_ref] Development of a large-scale chemogenomics database to improve drug candidate selection and..., Ganter [/bib_ref]. Theoretical and computational studies in this and related areas are also picking up pace. It was proposed that system-oriented drug design should take into account the intrinsic properties of biological systems, for example, robustness [bib_ref] A robustness-based approach to systems-oriented drug design, Kitano [/bib_ref]. Many mathematical models of disease-relevant pathways have been constructed with the potential to elucidate underlying mechanisms of diseases and to identify treatment strategies [bib_ref] Computational modeling of the dynamics of the MAP kinase cascade activated by..., Schoeberl [/bib_ref] [bib_ref] Systems modeling: a pathway to drug discovery, Rajasethupathy [/bib_ref]. Network properties were analyzed to find potential drug targets and to understand connectivity between them [bib_ref] In silico simulation of inhibitor drug effects on nuclear factor-kappaB pathway dynamics, Sung [/bib_ref] [bib_ref] Identification of information flow-modulating drug targets: a novel bridging paradigm for drug..., Hwang [/bib_ref]. However, practical and systematic computational strategies and methods are in need for multi-target drug design based on disease network modeling. In the present study, we develop a computational method for finding multiple target optimal intervention (MTOI) solutions in a disease network. For a given disease network, the method tries to identify effective points of intervention and the combination of interventions that can best restore the disease network to a desired normal state. Instead of focusing on a single drug target, the MTOI method analyzes the relevant network as a system to extract information about the interconversion of the disease and the normal state of the network. The method has two end products: (1) it generates a list of potential drug targets in a given disease network and their corresponding regulation (inhibition or activation) needed for treatment; (2) it suggests a list of optimal combinatorial multitarget intervention solutions for any user-defined therapeutic requirements. As a concrete example, we have applied MTOI to an inflammation-related network-the arachidonic acid (AA) metabolic network (AAnetwork). The long history of the antiinflammatory struggle has not yet succeeded in safe and effective drugs, but has accumulated abundant experimental and clinical data. This allowed us to construct an AAnetwork model in human polymorphonuclear leukocyte (PMN), presented in our earlier study [bib_ref] Dynamic simulations on the arachidonic acid metabolic network, Yang [/bib_ref]. Here, we further extended this model to other cell types to construct a more accurate model with improved toxicity prediction. With this new AAnetwork, we simulated the drug effects of popular antiinflammatory medicines, such as aspirin, Vioxx and so on. The known bleeding or cardiovascular side effects of these drugs were correctly reproduced. We applied MTOI to the new AAnetwork and identified five drug targets in the network: phospholipase A2 (PLA2), PGE synthetase (PGES), COX-2, 5-LOX and LTA4 hydrolase (LTA4H). A number of optimal combinations of target intervention (MTOI solutions) were found that are both effective in controlling the inflammation mediators and are safe with minimal side effects. # Results ## Mtoi for disease network Feedback loops, cross-talk and other network-intrinsic properties can make the effects of drug molecules much more complicated than what a linear one-drug one-target approach would predict [bib_ref] Proteins, drug targets and the mechanisms they control: the simple truth about..., Araujo [/bib_ref] [bib_ref] A robustness-based approach to systems-oriented drug design, Kitano [/bib_ref] [bib_ref] Chemical combination effects predict connectivity in biological systems, Lehar [/bib_ref]. It is therefore necessary to systematically analyze the disease network as a whole to find points of intervention that are the most effective and with the least side effects. We have developed a computational method for finding multiple target intervention solutions with user-defined therapeutic effects. This method contains two stages. The first stage is the identification of the potential drug targets. This stage can be skipped if the drug targets have been identified or are given. The second stage is the search and optimization of multiple target intervention solutions, which are the core functions of the method. Two network states are defined: the disease state and the normal (or desired) state. The disease state is a network state in which the production of disease-related molecules is abnormal. The normal (or desired) state is the network state one would like to achieve after taking medicine, which can be customer-defined. The main procedure of MTOI is to perturb the network and optimize it toward the desired state. The procedure can be carried out by various optimization algorithms; here, we used Monte Carlo simulated annealing (MCSA). By comparing the perturbed network that has achieved the desired state with the network that generated the disease state, we are able to find potential drug targets and the MTOI solutions that best restore the disease network state to the desired one. ## Drug target identification Before searching for the MTOI solutions, we need to identify the potential drug targets in the disease network. These targets are selected according to a criterion that measures their robust 'influence' in restoring the network state when being perturbed. Specifically, we define an objective function to be the difference between the current network state and the userdefined desired state. The function is then minimized with an MCSA algorithm , by changing the activity of drug target candidates, until the network state is sufficiently close to the desired one. We record the activities of drug target candidates in the final state, and this is called an acceptable MCSA run. After many acceptable runs of MCSA, many sets of activities of drug target candidates that achieved the desired state are obtained. Two parameters are then calculated to analyze the robust influence of a drug target candidate on the network state (see details in Materials and methods). One is the standard deviation (s.d.) of the activities of the drug target candidate in all acceptable runs. This parameter describes the consistency of the activity of the candidate in restoring the desired state. The other is the median deviation (m.d.) of the activities of the drug target candidate in the desired state from that of the disease state. Median deviation describes how far on average, does the activity of drug target candidate in the desired state deviate from that in the disease state. Drug target candidates with larger m.d. are more responsive to perturbations that restore the network to the desired state. The absolute value of the ratio of m.d. to s.d. is used to rank drug target candidates. The larger the |m.d./s.d.| is, the more likely that perturbing the target will have a significant effect in steering the network toward the desired state. Median deviation also provides information about the manner of regulation that should be adopted in the treatment. When the m.d. value is negative, the corresponding drug target should be inhibited in the treatment; otherwise, the target should be activated. With all the information above, a set of drug targets is selected, which will be the subject of the multiple targets control solution search in stage 2 of MTOI described below. ## Optimal multiple targets intervention solutions With the potential drug targets in the disease network identified, we search for the best sets of combinatorial control on these targets that can restore the network to the desired state. Similar to stage 1, MCSA is utilized to search for solutions that can turn the network state into the desired one . The difference with stage 1 is that (1) the optimization is performed only on a pre-specified set of drug targets that are selected in stage 1 and (2) the perturbation on the targets is modeled in detail with corresponding equations of inhibition or activation (see details in Materials and methods). Therefore, at the end of an acceptable MCSA run, which resulted in a network state sufficiently close to the desired one, we obtain a set of inhibition and/or activation intensities on the drug targets. We call this set an optimal solution of the multi-target control for the specified targets. After many runs of MCSA, we can, in principle, find all possible optimal solutions for the combinatorial multi- The flow chart of MTOI. MTOI functions in two stages: drug target search and optimal multi-target control solution identification. MCSA is employed as the optimization method. Differences in stages 1 and 2 are highlighted by light and dark gray, respectively. target control with desired therapeutic effects for the disease network. ## Application of mtoi to inflammation network The AAnetwork model in human PMN, endothelial and platelet cells Inflammation is a basic way in which the body reacts to infection, irritation or other injuries. Standard drug targets such as COX-2 and 5-LOX are among the key enzymes involved in the network responsible for generating inflammation mediators. However, essentially all single-target drugs so far have side effects in anti-inflammatory treatment. To find safer multi-target intervention solutions, we employed MTOI in an inflammation-related network, the AA metabolic network (AAnetwork) with a multi-cellular ensemble of human PMN, endothelial (EC) and platelet (PLT) cells , the separate AAnetworks in PMN, EC and PLT can be found in Supplementary . The model was developed partly based on our previous AAnetwork model for human PMN [bib_ref] Dynamic simulations on the arachidonic acid metabolic network, Yang [/bib_ref]. However, the metabolism of AA in different cell types is different. LTs are the major inflammatory mediators produced in PMN [bib_ref] The release of leukotriene B4 during experimental inflammation, Simmons [/bib_ref] [bib_ref] Cyclooxygenase and lipoxygenase metabolite synthesis by polymorphonuclear neutrophils: in vitro effect of..., Abbate [/bib_ref]. PGI2, PGF2a and PGE2 are the main eicosanoids detected in EC [bib_ref] Rate of vasoconstrictor prostanoids released by endothelial cells depends on cyclooxygenase-2 expression..., Camacho [/bib_ref]. PGE2 is an important inflammatory mediator causing fever and pain [bib_ref] Prostaglandin E2 as a mediator of fever: synthesis and catabolism, Ivanov [/bib_ref] [bib_ref] Membrane prostaglandin E synthase-1: a novel therapeutic target, Samuelsson [/bib_ref] , whereas PGI2 inhibits PLT aggregation and relaxes smooth muscle [bib_ref] Prostacyclin: a potent antimetastatic agent, Honn [/bib_ref]. PLT cells produce abundant TXA2, which is a potent inducer of PLT aggregation and vasoconstriction [bib_ref] Thromboxane A2, prostacyclin and aspirin: effects on vascular tone and platelet aggregation, Smith [/bib_ref]. The new AAnetwork model produced a better simulation of inflammation course in human blood vessel. In addition, the ratio of PGI2/TXA2 can be calculated to evaluate cardiovascular or bleeding side effects of the drugs [bib_ref] Prostacyclin and thromboxane in acute hemorrhagic pancreatitis in dogs, Kiviniemi [/bib_ref] [bib_ref] Thromboxane A2 and prostacyclin generation in the microvasculature of patients with atherosclerosis-effect..., Kyrle [/bib_ref] [bib_ref] Dose-related effects of low dose aspirin on hemostasis parameters and prostacyclin/thromboxane ratios..., Martin [/bib_ref] [bib_ref] Roles of thromboxane A(2) and prostacyclin in the development of atherosclerosis in..., Kobayashi [/bib_ref]. Ordinary differential equations (ODEs) were constructed to simulate the dynamic course of AA metabolism, where 24, 29 and 11 equations were used for PMN, EC and PLT, respectively (see details in Supplementary information). The concentrations of PMN, EC and PLT in the model were adopted according to their ratios in the human vessel (see details in Materials and methods). As signaling interactions among cells are complex in real circumstances and there is not enough experimental data, we did not include them in the current model. Among the 117 parameters in the ODE model, 46 were taken directly from experiments, whereas the others were determined by parameter fitting to experimental curves (see details in Supplementary information), including the production of LTB4 and o-LTB4 in PMN [bib_ref] Omega-oxidation is the major pathway for the catabolism of leukotriene B4 in..., Shak [/bib_ref] , the production of PGF2a, PGE2 and 6-keto-PGF1a in EC [bib_ref] Rate of vasoconstrictor prostanoids released by endothelial cells depends on cyclooxygenase-2 expression..., Camacho [/bib_ref] , and the production of TXA2 and TXB2 in PLT [bib_ref] Kinetic studies on the conversion of prostaglandin endoperoxide PGH2 by thromboxane synthase, Anderson [/bib_ref]. It is to be noted that in addition to these parameters, the concentrations of the enzymes and the initial concentrations of metabolites are also variables. Multiple parameter sets were obtained, which fit the curves equally well [fig_ref] Table I: Results from drug targets search by MTOI [/fig_ref]. ## Drug targets search in the aanetwork We performed MTOI to identify drug targets in the AAnetwork. The disease state of the AAnetwork is defined as a state where the output of PGs and LTs is markedly above the normal level. As the experimental curves used in parameter fitting corresponded to the abnormal metabolism of PGs and LTs, we used the parameter sets derived there to describe the disease state (see details in 'The AAnetwork model in human PMN, The metabolic network of arachidonic acid in human PMN, EC and PLT. Two pathways are responsible for the production of inflammatory mediators: COX-2 pathway and 5-LOX pathway. LTB4 and PGE2 are major inflammatory mediators produced in the AAnetwork. PGI2 and TXA2 are vasoactive molecules, the abnormal production of which causes bleeding and cardiovascular disease. The separate AAnetworks in PMN, EC and PLT can be found in Supplementary Figure S1. endothelial and platelet cells'). The desired state was defined as low output of inflammatory mediators, that is, the cumulative output of LTB4 and PGE2 should be smaller than 10% of that in the disease state, respectively. The threshold of 10% was selected here arbitrarily as it is impossible to give a definite cutoff to divide normal from disease in this case. The situation would depend on conditions such as the type of inflammation, the development level of disease and so on. We have tested the effect of cutoff on the results by changing the threshold to 20%. The same drug targets were selected by the new threshold (Supplementary [fig_ref] Table I: Results from drug targets search by MTOI [/fig_ref]. The entire 17 enzymes in the AAnetwork were selected as drug target candidates and their activities ( [formula] K cat [E]/K m , where [E] [/formula] is the concentration of an enzyme) were perturbed during optimization. The absolute value of m.d./s.d. was calculated to determine the importance of the enzyme, and the corresponding manner of regulation was determined by the sign of m.d. (see details in Materials and methods). To get reliable results, many runs of MCSA were performed until the s.d. and m.d. for all candidates converged. As multiple parameter sets can fit the experimental data equally well, the drug targets identified can, in principle, depend on which parameter set we use. However, we note that the top drug targets (scored according to |m.d./s.d.|) are always the same, independent of the parameter set used, although their rank order may vary. This implies that the results of MTOI are rather robust, which depend more on the network structure than on the value of the individual parameters. [fig_ref] Table I: Results from drug targets search by MTOI [/fig_ref] shows the result of the search for drug targets of one particular parameter set derived from the parameter fitting. Results for other parameter sets are summarized in Supplementary Table SII. In the five parameter sets that we studied, the enzymes identified as top the five in at least four parameter sets were selected as drug target candidates. They are PLA2, LTA4H, COX-2, 5-LOX and PGES. Interestingly, all the five drug targets have negative m.d. values, indicating that the corresponding drugs should be inhibitors. Some other enzymes also have close |m.d./s.d.| values to the top five targets, such as PHGPx in [fig_ref] Table I: Results from drug targets search by MTOI [/fig_ref]. However, they were not selected as drug targets for our further studies in stage 2, because their m.d. values are positive. Their corresponding drugs would be enzyme activators, which is normally difficult to realize in drug development. ## Mtoi solutions for the aanetwork We performed MTOI to search for optimal multi-target antiinflammatory intervention solutions. The disease state at this stage is the same as in the previous section, modeled by the parameter sets derived from fitting experimental curves. The desired state, on the other hand, is slightly modified. In addition to reducing the inflammatory mediators, we have added requirements that minimize possible side effects. Specifically, we require that the desired state should satisfy the following conditions: (1) the cumulative production of LTB4 must be below 10% of that in the disease state; (2) the cumulative production of PGE2 must be below 10% of that in the disease state and (3) the change in the PGI2/TXA2 ratio between the desired and disease states must be smaller than a preset small value (here, we use 20%). This condition ensures that the drugs will not break the balance between PGI2 and TXA2 to cause cardiovascular or bleeding side effects. We took the top five drug targets identified in stage 1 (in the previous section) for further optimization of multi-target control in this stage. Other enzymes have not been selected for their low |m.d./s.d.| value and positive m.d. Considering that most COX-2 inhibitors will inhibit COX-1 at the same time to a certain extent due to the high homology of the two enzymes, we included COX-1 in the drug target list and but constrained because the inhibition on COX-2 and COX-1 should coexist. Thus, six drug targets were selected at stage 2, in which five of them (excluding COX-1) were predicted to be subjected to inhibition at stage 1. We assumed that the drugs against these enzymes were all competitive inhibitors and defined their inhibition intensity as the ratio of inhibitor concentration to inhibition constant ([I]/K i ). The values of [I]/K i for the five drugs were perturbed in MCSA and the difference between the present network and the desired states was employed as the objective function for minimization (see details in Materials and methods). To obtain reliable results, many runs of MCSA were performed. The set of [I]/K i for inhibiting the six enzymes at the end of each acceptable run would correspond to an optimal multi-target intervention solution. These solutions from multiple runs were then clustered into distinct groups. We note that whereas at stage 1 all parameter sets studied gave the same top five drug targets, the results of stage 2 depended somewhat on parameter sets. We have used five different parameter sets in the study of stage 2. Although there is a large overlap in the solutions from different parameter sets, some solutions were found only in individual parameter sets [fig_ref] Table I: Results from drug targets search by MTOI [/fig_ref]. Perhaps this is not very surprising given the great uncertainty about the parameters. What is interesting is that there are common features in solutions among all the parameter sets. Thus, we can still make some concrete conclusions about the MTOI solutions even with partly uncertain parameters. First, none of the solutions in any parameter sets is single target, implying the necessity for multi-target control in this network. Second, the solutions involving the larger number of targets are more robust. The six-target solution and certain five-and four-target The parameter set used is the same as in [fig_ref] Figure 3: The distribution of [I]/K i of MTOI solutions [/fig_ref]. The top five enzymes were selected as drug targets and their corresponding regulations were all predicted to be inhibition. solutions appear in the solutions of all parameter sets. This result suggests a strategy when facing uncertainties in parameters: simultaneous intervention of many targets that are 'influential' for the network state. If one views genetic and other variations in the system as a source of parameter uncertainty, this strategy may also be used to maximize the 'bet' in the case of limited information. Ideally, the efficacy and safety of a robust multi-target intervention solution should not be sensitive to small changes in the inhibition intensities of the solution. We defined this sensitivity as the ratio of the percentage change of the objective function over that of the inhibition intensities (see details in Materials and methods). As can be seen from [fig_ref] Table I: Results from drug targets search by MTOI [/fig_ref] , this sensitivity is extremely small for the MTOI solutions we found. Furthermore, we analyzed the effective and safe range of the drug intensity ([I]/K i ) of the multi-target solutions ('therapeutic window'). These distributions of [I]/K i are obtained by sampling the intensity [I]/K i of the inhibitors to the multitargets specified by the solution, and recording the combination of [I]/K i that can change the disease state into the desired state. As the intensity space [I]/K i is highly dimensional (with the dimensionality being the number of targets), we project the distribution onto every pair of two dimensions. In [fig_ref] Figure 3: The distribution of [I]/K i of MTOI solutions [/fig_ref] , we show the [I]/K i distribution of the inhibitors for two different MTOI solutions, with three targets [fig_ref] Figure 3: The distribution of [I]/K i of MTOI solutions [/fig_ref] and four targets [fig_ref] Figure 3: The distribution of [I]/K i of MTOI solutions [/fig_ref] , respectively. Results of other MTOI solutions are listed in the Supplementary information. In general, inhibitors against more drug targets have larger therapeutic windows. It is to be noted that when all six targets are inhibited, the [I]/K i distributions not only have very wide ranges but are also essentially uncorrelated with each other. This makes the multitarget solutions much more practical, both in terms of the drug design and of the clinical use. The inhibition of PLA2 and/or COX-1/2 has strong effect on the ratio of PGI2/TXA2. When PLA2 is not inhibited, the ratio of COX-1 and COX-2 inhibition has to be fixed within a narrow range to maintain the ratio of PGI2/TXA2 [fig_ref] Figure 3: The distribution of [I]/K i of MTOI solutions [/fig_ref]. Balanced inhibition of these two COX isoenzymes would be important to avoid side effects. Selective COX-1/2 inhibitors were known to conduct either gastrointestinal or cardiovascular damage [bib_ref] Anti-inflammatory and side effects of cyclooxygenase inhibitors, Suleyman [/bib_ref]. With balanced inhibition, the therapeutic window of COX inhibition can be very large. As COX-1 and COX-2 are isoenzymes, designing an inhibitor with a fixed ratio of affinities should not be difficult. This fixed ratio of COX-1/2 inhibition is no longer necessary when PLA2 is inhibited simultaneously [fig_ref] Figure 3: The distribution of [I]/K i of MTOI solutions [/fig_ref]. ## Verification of the aanetwork model by simulating the effects of known non-steriodal anti-inflammatory drugs If the constructed AAnetwork is reasonably reliable, we should be able to simulate the behavior of anti-inflammatory drugs to assess their efficacy and potential side effects within the network model. We have made so far a long list of antiinflammatory drugs covering most currently used nonsteriodal anti-inflammatory drugs (NSAIDs). They include non-selective NSAIDs, selective COX-2 inhibitors, and dual functional COX-2/5-LOX inhibitor. The relative IC 50 values of these medicines were taken from the published experimental data [bib_ref] Cyclooxygenaseselective inhibition of prostanoid formation: transducing biochemical selectivity into clinical read-outs, Patrono [/bib_ref] [bib_ref] Licofelone, a balanced inhibitor of cyclooxygenase and 5-lipoxygenase, reduces inflammation in a..., Vidal [/bib_ref]. The ratio of PGI2/ TXA2 was calculated as the indicator for side effects-a large increase in this ratio after taking the medicine would imply bleeding risks, whereas a significant decrease of the ratio would raise cardiovascular risks [bib_ref] Dose-related effects of low dose aspirin on hemostasis parameters and prostacyclin/thromboxane ratios..., Martin [/bib_ref] [bib_ref] Roles of thromboxane A(2) and prostacyclin in the development of atherosclerosis in..., Kobayashi [/bib_ref]. The simulation results are shown in [fig_ref] Table I: Results from drug targets search by MTOI [/fig_ref]. Aspirin is a strong COX-1 inhibitor and a mild COX-2 inhibitor. COX-1 is the main COX isozyme in PLT, which is responsible for the major production of TXA2 in the model. A low concentration of aspirin is reported to have an antithrombotic effect [bib_ref] Cyclooxygenaseselective inhibition of prostanoid formation: transducing biochemical selectivity into clinical read-outs, Patrono [/bib_ref]. In our simulation, as aspirin strongly inhibited the production of TXA2, the value of PGI2/ TXA2 was much larger than before the administration of medicine. Thus, aspirin can prevent myocardial infarction and ischemic stroke but raise the risk of bleeding. When the relative IC 50 (COX-1:COX-2) increases, the inhibition effect of the medicine on TXA2 production and PLT aggregation decreases when compared with their inhibition effect on PGI2 production [bib_ref] NSAID induced gastric mucosal damage and its prevention in rats, Sun [/bib_ref] [bib_ref] Comparative inhibitory activity of rofecoxib, meloxicam, diclofenac, ibuprofen, and naproxen on COX-2..., Van Hecken [/bib_ref]. The bleeding risk of NSAIDs lessens following the reduced inhibition of COX-1. In our simulation, the value of PGI2/TXA2 was observed to decrease. When the relative IC 50 of COX inhibitors increased above 10, we observed a strong inhibition on the production of PGI2, whereas the production of TXA2 was hardly affected. The value of PGI2/TXA2 became significantly smaller than that before the administration of medicine. Cardiovascular side effects have been found in selective COX-2 inhibitors [bib_ref] Is nimesulide safe in a cardiovascularcompromised patient, Rahman [/bib_ref] [bib_ref] Merck withdraws arthritis drug worldwide, Singh [/bib_ref] [bib_ref] Cardiovascular risk associated with celecoxib in a clinical trial for colorectal adenoma..., Solomon [/bib_ref] , presumably due to the decrease in PGI2/TXA2. Indeed, in our simulation, Vioxx was correctly predicted to be a high cardiovascular risk. As COX inhibitors fail to control inflammation safely, licofelone, a dual functional inhibitor of COX-1/2 and 5-LOX, has been developed. This compound was reported to be effective in inflammation [formula] 1 O O - - - O 1 0.0011 2 O O O - - O 1 0.0011 3 - O O O O O 5 0.002 4 - O O - O O 5 0.0021 5 - - O O O - 2 0.0026 6 O O O O O O 5 0.0028 7 - O O O - O 5 0.0033 8 O O O - O O 5 0.0034 9 - O - O O O 5 0.0036 10 O O - O O O 5 0.0037 11 O O O O - O 5 0.0041 12 O O - - O O 5 0.0042 13 - O - - O O 5 0.0048 14 O - O O O - 1 0.0057 15 - - O O - - 2 0.0061 16 - - O - O - 3 0.0072 17 O O - O - O 5 0.0075 18 - O - O - O 5 0.01 19 O - O - O - 1 0.0123 20 O - O O - - 1 0.0241 21 O - - O O - 1 0.0431 22 O - - - O - 1 0.0569 23 O - - O - - 1 0.0624 [/formula] The frequency of MTOI solutions appearing in five parameter sets was also shown. The sensitivity /SS is averaged over the parameter sets in which the solution appeared. '-' denotes no regulation and 'O' denotes that the corresponding enzyme is inhibited. Controlling multiple targets in disease network K Yang et al control and in cardiovascular derangement prevention [bib_ref] The gastrointestinal tolerability of the LOX/COX inhibitor, licofelone, is similar to placebo..., Bias [/bib_ref] [bib_ref] The lipoxygenase-cyclooxygenase inhibitor licofelone prevents thromboxane A2-mediated cardiovascular derangement triggered by the..., Rotondo [/bib_ref]. Our simulation showed that licofelone can maintain the balance between PGI2 and TXA2, and at the same time effectively reduce PGE2 and LTB4 production. ## Drug efficacy prediction of mtoi solutions Several promising MTOI solutions were selected for further investigation. The ratio of PGI2/TXA2 was calculated as the indicator for side effects. The relative [I]/K i values of solutions were taken from MTOI analysis. Simulation results are summarized in [fig_ref] Table I: Results from drug targets search by MTOI [/fig_ref]. Unlike the currently used drugs that inhibit only COX-1/2 (with the exception of licofelone, which also inhibits 5-LOX), MTOI solutions achieved a balanced control on the entire network. The ratio of PGI2/TXA2 was almost unchanged after taking MTOI solutions, although the production of LTB4 and PGE2 was inhibited effectively at the same time. Recently, much attention has been drawn to the two downstream drug targets: PGES and LTA4H, as their inhibitors may produce fewer side effects when compared the with upstream enzyme blockers [bib_ref] mPGES-1 as a novel target for arthritis, Fahmi [/bib_ref] [bib_ref] Leukotriene A4 hydrolase/aminopeptidase, the gatekeeper of chemotactic leukotriene B4 biosynthesis, Haeggstrom [/bib_ref]. MTOI found that optimal therapeutic effects are achieved only when the two targets or one of them in combination with other targets are inhibited simultaneously; for example, the combination of PGES and LTA4H and the combination of COX-2/1 and LTA4H. Inhibiting more than one drug target is important in anti-inflammatory treatment. Another feature in MTOI solutions is that when multiple targets are inhibited simultaneously, a mild inhibition of each target is sufficient to achieve effective control of LTB4 and PGE2 production and there is a much larger therapeutic window. For example, simultaneously inhibiting COX-2/1 and LTA4H, the [I]/K i of COX-2 should be 400 to achieve effective treatment. In the case of simultaneously inhibiting PLA2, COX-2/1 and LTA4H, the value of [I]/K i of COX-2 needed is reduced to 80. This value is further reduced to 20 when all the six targets are inhibited. The reduced inhibition intensity may decrease the toxicity risk of the drugs. Furthermore, regulation on multiple enzymes can maintain the PGI2/TXA2 ratio more easily than single-target drugs. As discussed in the section of 'MTOI solutions for the AAnetwork', we found that the intervention of more targets gave more robust solutions. This may explain the possible benefits of traditional Chinese medicines (TCMs), in which mild control is directed to multiple drug targets. # Discussion We have developed a systematic method (MTOI) to search for optimal interventions in a disease network. The basic concept is simple: the goals are to identify effective points of intervention and the optimal combinations of interventions that restore the network to a normal state. Quantitatively, optimization is carried out with an objective function that specifies the customer-defined requirements of what the normal state should be, including safety conditions. An MCSA algorithm is employed here to optimize the network state with perturbations on network components and/or interactions. The end results include promising drug targets and optimal solutions of their combinatorial intervention. We have applied the method to a network responsible for inflammation-the AAnetwork. We have demonstrated the utility and power of the method with the robust identification of the drug targets and the multi-target intervention solutions that are both effective and safe. ## Discovering drug targets MTOI systematically analyzes the conversion between the disease and desired states to identify potential drug targets and to find multi-target intervention solutions with user-defined efficacy and safety thresholds. Compared with the traditional drug target search methods, such as parameter sensitivity analysis [bib_ref] Model complexity has a significant effect on the numerical value and interpretation..., Palsson [/bib_ref] [bib_ref] Automated oncogene detection in complex protein networks with applications to the MAPK..., Pant [/bib_ref] , MTOI emphasizes the influence of the drug target candidate's activity on the entire network state. Thus, it can be regarded as a network-state-based drug target sensitivity analysis method. Instead of relying on individual parameters for target identification, MTOI dynamically perturbs and searches the various combinations of the entire parameters of drug target candidates to accurately and robustly identify the relationship between target candidates and the network state. As a comparison, we also performed single parameter sensitivity analysis on the AAnetwork , see details in Supplementary information). Although the top list was consistent with the result of MTOI, the ways of invention (upregulate or downregulate) cannot be predicted from the analysis. When applied to the AAnetwork, MTOI has succeeded in identifying some well-known anti-inflammatory drug targets, including PLA2, COX-2, 5-LOX, PGES and LTA4H. PLA2 regulates the metabolism of AA upstream in the network. Inhibition of PLA2 can effectively control both PGE2 and LTB4, but will also downregulate all the downstream AA metabolites including vasoactive eicosanoids, which has important physiological functions. COX-2 and 5-LOX have been studied intensively and became regular anti-inflammatory drug targets. Medicines against these two enzymes, respectively, such as ibuprofen and zileuton, were developed for general clinical treatment. PGES and LTA4H are recently discovered downstream enzymes. Inhibitors against PGES or LTA4H attracted much attention due to the anticipated reduction of toxicity. MTOI has also identified several important enzymes, for example, 15-LOX, CYP4F3, for which increased activity is desired for anti-inflammatory treatment. Although it is The same parameter set used is the same as in [fig_ref] Figure 3: The distribution of [I]/K i of MTOI solutions [/fig_ref]. In the same way, the ratio of PGI2/TXA2 reflects possible side effects of the drug. After taking the medicine, if PGI2/TXA2 becomes much greater than the normal level, bleeding side effects may occur. If PGI2/TXA2 decreases significantly, cardiovascular risks will be raised. difficult to devise drugs that increase enzyme activity, this result may be useful to understand elevated disease susceptibilities or variations in drug response among individuals due to single nucleotide polymorphism [bib_ref] Genetic diversity and new therapeutic concepts, Shastry [/bib_ref]. ## Finding multiple target intervention solutions Identification of optimal multi-target intervention solutions is the core function of MTOI. The complexity of a network, due to connectedness, feedback, cross talk and so on, is exploited here to provide the researchers multiple choices for intervention solutions. Moreover, the desired network state is customer-designed by the user. This allows MTOI to be applicable in the regulation and intervention of other kinds of networks, for example, in studies of pesticides. The efficacy of currently used NSAIDs has been correctly predicted by the AAnetwork model. Except for licofelone, these compounds were found to have side effects both clinically and in our simulation. As MTOI can correctly reproduce the known side effects of NSAIDs, one may apply it in predicting the possible side effects for new antiinflammation drugs targeting the AAnetwork. Furthermore, MTOI study also gave several novel multi-target intervention solutions for the AAnetwork control with high efficacy and low toxicity. Although, theoretically it is better to inhibit all the six enzymes at the same time, practically, it might be difficult to design one medicine that can inhibit all the six enzymes simultaneously or to use a cocktail containing six different inhibitors. To simplify the case, we can start from the solutions with fewer enzymes. For example, we believe that there are several such solutions that are rather promising for developing anti-inflammation drugs: (1) the combination of PGES and LTA4H and the combination of COX-2/1 and LTA4H. These two solutions basically involve only two targets and should be relatively straightforward to achieve. However, care should be taken to make sure that the inhibitors should be used only in the proper dosing range. The valid [I]/K i range is quite narrow for LTA4H in the PGES and LTA4H solution, and the ratio of inhibition for COX-1/COX-2 should be maintained in a narrow range for the COX-2/1 and LTA4H solution. (2) The combination of PLA2, 5-LOX and COX-2/1 or the combination of PLA2, COX-2/1 and LTA4H. With more targets that are inhibited, the dosing ranges for these two solutions are much larger, making them very attractive candidates for multi-target drug design. Our MTOI study suggested that optimal solutions often involve mild but simultaneous interventions of multiple targets. This is reminiscent of TCM in which multiple targets are intervened with mild or low intensity. It would be interesting to explore the molecular mechanisms of TCM along this line. Of course, the targets of the most TCM are unknown, which presents a significant challenge. ## Multi-target drug design at systems level Given sufficient knowledge about the disease-related network, our results suggest that computational methods can play an important and unique role in the new era of network-based drug design. Construction of a network that is reasonably complete is the first key step. However, as almost surely the network is incomplete and with many parameters being unknown, a major challenge is to devise a robust computational algorithm that is able to extract desirable information based more on the overall structure of the network than on individual parameters. In principle, it is difficult to know when the knowledge about a network is sufficient to apply a network-based drug discovery method. One way to test this is to perturb the network and see how the solutions change. We have constructed a simplified AAnetwork model [fig_ref] Figure 4: The simplified network of arachidonic acid metabolism in human PMN, EC and... [/fig_ref] , parameters are summarized in [fig_ref] Table I: Results from drug targets search by MTOI [/fig_ref] using partial information of the network and applied MTOI to this model. In the simplified model, only the disease-related metabolites and enzymes were included and unknown parameters were evaluated by parameter fitting (see details in Supplementary information). MTOI has identified 14 multitarget solutions in the simplified AAnetwork model (Supple- [fig_ref] Table I: Results from drug targets search by MTOI [/fig_ref]. All of them are among the MTOI solutions of the full model [fig_ref] Table I: Results from drug targets search by MTOI [/fig_ref]. With the fast accumulation of experimental data and more disease networks revealed, we expect an increasingly critical role for computational approaches in systems drug design. # Materials and methods Finding multi-target intervention solutions in disease network MTOI contains two stages. MCSA is employed as the main optimization tool in both stages. The procedure of stage 1 includes the following steps: Step 1. Define the disease and desired states. A state in the MTOI program is defined as a steady or temporal state of the network, which can be a collection of concentrations of proteins and/or metabolites, metabolic fluxes or any other relevant information, for example, the temporal behavior of the network within some time window. Generally, the disease state can be obtained by experimental data from patients or cells in abnormal conditions. The desired state is specified by researchers, which can be the normal physiological state of the network. Step 2. Select reactions that can be controlled by drugs. Define a quantity that controls the corresponding reaction rate as the activity of the drug target candidate. For example, if the target is an enzyme, the activity is defined as K cat [E]/K m . Step 3. Starting from a randomly selected set of the activities of drug target candidates, perform MCSA to drive the system toward the desired state. The activities of drug target candidates are perturbed in MCSA, whereas the other parameters are maintained at the same values of the disease state. The difference between the present network and desired states is used as the objective function. Step 4. Record as 'acceptable' the set of the drug target candidates' activities, when the desired state is reached by MCSA. Calculate the standard deviation of the activities of each drug target candidate over all acceptable sets: [formula] s:d: i ¼ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi P n j¼1 ðA i;j À A i Þ 2 n À 1 v u u u tð1Þ [/formula] where A i,j is the activity of the ith drug target candidate in the jth set and A i ¼ 1 n P n j¼1 A i;j is the average activity of the candidate over all 'acceptable' sets. Step 5. Calculate the m.d. of the activity of drug target candidates between the desired and disease states: [formula] m:d: i ¼ medfDA i;1 ; Á Á Á DA i;j ; Á Á Á DA i;n g ð 2aÞ DA i;j ¼ A i;j À A disease; ið2bÞ [/formula] where A disease,i is the activity of the ith candidate in the disease state. Use |m.d. i /s.d. i | to determine the importance of the drug target candidate and the sign of m.d. i to decide whether inhibition or activation should be performed in the treatment. Step 6. Repeat steps 3-5 until s.d. and m.d. converge to stable values for all drug candidates (see details in Supplementary information). Stage 1 can be skipped if the drug targets have been identified by experiments or other methods. After identifying drug targets in the network, one starts stage 2, which includes the following steps: Step 1. Add reactants of drugs against the selected drug targets in the network. The reactants influence the fluxes and rates of the target reactions in different ways depending on specific reaction kinetics. For example, if the target is an enzyme and the reaction has the Michaelis-Menten kinetics: [formula] d½S dt ¼ K cat ½E t ½S K m þ ½Sð3Þd½S dt ¼ K cat ½E t ½S K m ð1 þ ½I=K i Þ þ ½Sð4Þ [/formula] where [I] is the concentration of the inhibitor and K i the inhibition constant: K i ¼ ½E½I=½EI. [I]/K i is defined as the intensity of the drug influence against the drug target. Step 2. Starting from the disease state and selecting a random set of intensities of drug influence against all drug targets, perform MCSA to drive the system toward the desired state. Only the intensities of drug influence are perturbed, whereas other parameters in the network are maintained at the values of the disease state. The difference between the present network and desired states is used as the objective function. Step 3. Record as 'acceptable' the set of the intensities of the drugs against all targets, when the desired state is reached by MCSA. This 'acceptable' set is one multi-target intervention solution for the disease network. Step 4. Repeat steps 2 and 3 until many solutions are found and the results are clustered in the space of intensities to find distinct solutions. ## Construction of the aanetwork model in human pmn, ec and plt cells The AAnetwork in human PMN, EC and PLT cells was set up based on our previous study and experimental data was published (see details in Supplementary information). As the ratio of PMN, EC and PLT depends on the size of the blood vessel, we simulated the metabolism of AA in a 1-cm long and 50-mm diameter vessel. The concentration of PMN and PLT in the model is set to the same value as in human blood, that is, 4500 and 2.5 Â10 5 cells/ml, respectively, so that the number of PMN and PLT in the considered region was 88 and 4908, respectively. The diameter of PMN and PLT was set to 10 and 2 mm, respectively. The number of EC depends on the size of the vessel. Only the inner layer of EC was considered and the diameter of EC was set to 50 mm, thus in the considered region the number of EC in the model was 628. On the basis of the AAnetwork of , a set of ODEs were constructed to describe cell behavior in inflammation. The ode15 s routine of Matlab 6.5 (The Mathworks Inc., http:// www.mathworks.com) was used to integrate the ODEs. Michaelis-Menten equations (equation (3)) were used to describe enzyme catalytic reactions in the network. Equation (4) was employed if competitive reversible inhibitors were involved in the catalysis. If the inhibition is irreversible, we assumed that the enzyme would decay according to [formula] d½E dt ¼ ÀK½E½I ð 5Þ [/formula] When activators were involved in the catalysis, we used: [formula] d½S dt ¼ K cat ð1 þ ð½U=KIÞ þ ð½U 0 =KI 0 ÞÞ½E t ½S K m þ ½Sð6Þ [/formula] To describe the reaction kinetics, where [U] is the concentration of the activator and KI is a constant. When upregulation occurred through transcription, we described its effect with the following equation: [formula] d½E dt ¼ k½g 2 ½g 2 þ K 2ð7Þ [/formula] where [g] is the concentration of the metabolite upregulating the transcription of the enzyme, K and k are constants. ## Mtoi application in the aanetwork model The MTOI procedure described earlier was applied in the AAnetwork model to find multi-target anti-inflammatory control solutions. Stage 1 of the procedure includes: Step 1. Define the disease and desired states. The disease state in AAnetwork was described by parameter sets derived from parameter fitting (see details in 'Construction of the AAnetwork model in human PMN, EC and PLT cells'). The desired state was a state where the 1 h cumulative production of LTB4 and PGE2 is less than 10% of that in the disease state. The fluxes of other metabolites are not monitored. Step 2. All the enzymes in the AAnetwork were selected as potential drug target candidates. The activity of an enzyme was defined as [formula] A ¼ K cat ½E K mð8Þ [/formula] Step 3. Perform MCSA as described earlier to find the desired state. In the process, the initial temperature was set to be 501C, the maximum number of Monte Carlo attempts under the same temperature was 500, the constant in the exponential cooling scheme was 0.7, and the final temperature in MCSA was 5 Â10 À6 . The perturbed range of enzyme activity was [0.01A disease , 100A disease ]. The objective function was [formula] F obj ¼ C T;net C T;disease LTB4 þ C T;net C T;disease PGE2ð9Þ [/formula] where C T,net and C T,disease were the 1 h cumulative production of the metabolite in the present network and disease states, respectively. Equation (9) optimized the production of LTB4 and PGE2 toward zero. In the calculation, when the production of LTB4 and PGE2 was below 10% of that in the disease state, respectively, the corresponding set of enzyme activities was accepted as the desired state. Step 4. Record as 'acceptable' the set of activities of the drug target candidates, when the desired state is reached by MCSA. Standard deviation of enzyme activity over all accepted sets was calculated as [formula] s:d: i ¼ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi X n j¼1 log 10 A desired;i; j A disease; i À log 10 A desired; i A disease; i 2 =n À 1 v u u tð10Þ [/formula] where A desired,i,j is the activity of the ith drug target candidate in the jth desired set. Because of the requirement of normalization, here we used log 10 ðA desired;i;j =A disease;i Þ for s.d. calculation instead of A i,j as in equation (1). This change does not influence the result on drug targets selection. Step 5. Mead deviation of enzyme activity between the desired and disease states was calculated as m:d: i ¼ medfDA i;1 ; Á Á Á DA i;j ; Á Á Á DA i;n g ð 11aÞ DA i;j ¼ log 10 A desired;i;j À log 10 A disease;i Again, we used equation (11) instead of (2) due to normalization. Step 6. Repeat steps 3-5 until s.d. and m.d. converge. The procedure of stage 2 was: Step 1. Add reactants of drugs against the selected drug targets in the network. Drugs in AAnetwork were assumed to be competitive reversible inhibitors, thus [I]/K i was defined as inhibition intensity. Step 2. Perform MCSA to find the desired state. The high temperature, the maximum number of Monte Carlo attempts under the same temperature, the constant in the exponential cooling scheme and the final temperature in MCSA adopted the same value as those in stage 1. [I]/K i was perturbed in a range of [0 100000]. The objective function was This objective function was set according to the following considerations: in the desired state, the production of LTB4 and PGE2 was below 10% of that of the disease state and the change of PGI2/TXA2 was less than 20%. Step 3. Record as 'acceptable' the set of the intensities of drug influence, when the desired state is reached by MCSA. Step 4. Repeat the above steps until no more new solutions are found. In the end, we also calculated the sensitivity of drugs' safety and effectiveness to [I]/K i as [formula] S ¼ DF 0 obj =F 0 obj P 5 j¼1 Dð½I=K i Þ j =ð½I=K i Þð13Þ [/formula] where F obj was calculated with equation (12). S measures how sensitive the drug effects are to small changes in the inhibition intensities. ## Supplementary information Supplementary information is available at the Molecular Systems Biology website (www.nature.com/msb). [fig] Figure 3: The distribution of [I]/K i of MTOI solutions. (A) Inhibition against LTA4H, COX-1 and COX-2. (B) Inhibition against PLA2, LTA4H, COX-1 and COX-2. [/fig] [fig] Figure 4: The simplified network of arachidonic acid metabolism in human PMN, EC and PLT. The separate AAnetworks in PMN, EC and PLT can be found in SupplementaryFigure S2. [/fig] [table] Table I: Results from drug targets search by MTOI [/table]
ASSOCIATION BETWEEN SMOKING, CRACK COCAINE ABUSE AND THE DISCONTINUATION OF COMBINATION ANTIRETROVIRAL THERAPY IN RECIFE, PERNAMBUCO, BRAZIL Despite the effectiveness of combination antiretroviral therapy in the treatment of people living with HIV/AIDS (PLWHA), nonadherence to medication has become a major threat to its effectiveness. This study aimed to estimate the prevalence of self-reported irregular use of antiretroviral therapy and the factors associated with such an irregularity in PLWHA. A cross-sectional study of PLWHA who attended two referral centers in the city of Recife, in Northeastern Brazil, between June 2007 and October 2009 was carried out. The study analyzed socioeconomic factors, social service support and personal habits associated with nonadherence to antiretroviral therapy, adjusted by multivariable logistic regression analysis. The prevalence of PLWHA who reported irregular use of combination antiretroviral therapy (cART) was 25.7%. In the final multivariate model, the irregular use of cART was associated with the following variables: being aged less than 40 years (OR = 1.66, 95%-CI: 1.29-2.13), current smokers (OR = 1.76, 95%-CI: 1.31-2.37) or former smokers (OR = 1.43, 95%-CI: 1.05-1.95), and crack cocaine users (OR = 2.79, 95%-CI: 1.24-6.32). Special measures should be directed towards each of the following groups: individuals aged less than 40 years, smokers, former smokers and crack cocaine users. Measures for giving up smoking and crack cocaine should be incorporated into HIV-control programs in order to promote greater adherence to antiretroviral drugs and thus improve the quality of life and prolong life expectancy. # Introduction Although the number of new HIV infections has declined globally by 19% over the past decade and the access to antiretroviral therapy in low-and middle-income countries has increased, HIV infection rates are increasing in several countries in Eastern Europe and central Asia, notably among injecting drug users and their sexual networks. Access to antiretroviral treatment (ART) has been primarily responsible for prolonging and improving the lives of people living with HIV/AIDS (PLWHA), with Brazil, being the first developing country to implement the universal distribution of these drugs [bib_ref] Adesão à terapia antiretroviral para HIV, Colombrini [/bib_ref] [bib_ref] Detrimental effects of continued illicit drug use on the treatment of HIV-1..., Lucas [/bib_ref]. Consequently, nonadherence to the use of antiretroviral drugs is the greatest risk for a non-effective response to treatment and the possibility of spreading resistant viruses [bib_ref] Risk-factors for non-adherence to antiretroviral therapy, Silva [/bib_ref] [bib_ref] Predictors of nonadherence to highly active antiretroviral therapy among HIV-infected South Indians..., Venkatesh [/bib_ref]. Moreover, poor adherence is one of the factors that may lead to a lower CD4 cell count, higher plasma viral RNA levels and delayed immune recovery, with progression of the disease that can lead to death [bib_ref] Psychosocial factors affecting medication adherence among HIV-1 infected adults receiving combination antiretroviral..., Do [/bib_ref]. In the treatment of chronic disease, the degree of adherence is always influenced by a number of different factors: characteristics of the individual, characteristics of the disease, social support network (family and friends) and factors related to the health service, such as difficulties in accessing it, and the relationship of the health team/individual [bib_ref] Adesão à terapêutica anti-retroviral por indivíduos com HIV/AIDS assistidos em uma instituição..., Gir [/bib_ref]. Amongst the characteristics of the individual, habits such as alcoholism, drug abuse and irregular use of antiretrovirals [bib_ref] Coping strategies and patterns of alcohol and drug use among HIV-infected patients..., Pence [/bib_ref] place the individual at further risk of secondary infections. Furthermore, studies have shown that the quantity of pills is also an important factor that influences the rate of adherence [bib_ref] Adesão à terapia antiretroviral para HIV, Colombrini [/bib_ref] [bib_ref] Psychosocial factors affecting medication adherence among HIV-1 infected adults receiving combination antiretroviral..., Do [/bib_ref]. For the treatment of HIV infection to be effective, several authors recommend an adherence level of at least 95% of the prescribed drugs to decrease the chance of viral resistance 6 . However, recent studies indicate that for boosted protease inhibitors an adherence level greater than 80% would be equally effective [bib_ref] Levels of adherence required for virologic suppression among newer antiretroviral medications, Kobin [/bib_ref]. Different methods have been employed in order to measure adherence to antiretrovirals, without a consensus gold standard. Due to their simplicity and the low costs involved, methods that use self-reports are widely used, and studies have demonstrated an association between the measure of self-reported adherence to antiretrovirals and HIV plasma viral load (with odds ratios and hazard ratios on the order of 2.0), making it possible to use this strategy in the conduct of PLWHA [bib_ref] Self-report measures of antiretroviral therapy adherence: a review with recommendations for HIV..., Simoni [/bib_ref] [bib_ref] Improving the self-report of HIV antiretroviral medication adherence: is the glass half..., Wilson [/bib_ref]. The aim of this study was to identify the prevalence of the factors 128 associated with the irregular use of antiretroviral drugs in people living with HIV, with special emphasis on socioeconomic factors and life habits, intending thereby to identify the groups at greatest risk so as to develop new strategies that set out to minimize the problem. # Methods The study was conducted in Recife, a city in northeastern Brazil, with an estimated population of 1,561,659 inhabitants. This cross-sectional study was carried out in June 2007 and October 2009, with people living with HIV/AIDS aged 18 years and over, who attended two HIV referral centers in the state of Pernambuco (Hospital Correia Picanço and Hospital Universitário Oswaldo Cruz), which attend around 60% of the state's patients. Individuals were invited to participate in the study either during routine consultations and/or hospitalization. Those who agreed to take part gave informed consent and were then interviewed by trained professionals who used a questionnaire developed specifically for this study. Hospitalized patients who could not answer the questionnaire due to their clinical condition were excluded from the study. The dependent variable was the self-reported irregular use of combined antiretroviral therapy (cART), categorized in 'yes' -when patients reported a discontinuation of treatment at some point on their own -and 'no'. The independent variables were classified as: demographic (age and race), socioeconomics (marital status, social service support, head of household's income), how long the patient has been aware of being HIV positive, life habits (alcoholism and drug abuse). Information regarding the use of cART was collected from medical records. Users of illicit drugs (crack cocaine and cocaine) fell into the following categories: non-users; intermittent (intermittent users who had abstained during the past year) and current users (who had been using during the past year). The criteria adopted for alcohol consumption was based on the number of drinks per day, according to the definition of alcohol consumption patterns approved by the Centers for Disease Control and Prevention (CDC). Patients were questioned about their alcohol consumption in the previous three months. Patients were classified as abstainers or light drinkers (non-drinkers or less than two drinks per day for men and one drink for women), and heavy drinkers (more than two drinks per day for men and more than one drink per day for women). The Student's t-test was used to compare the mean scores of the independent samples and variance was tested with Levene's test. All variables associated with the irregular use of cART in the univariate analysis with a p-value of less than 0.20 were included in the multivariate analysis, using logistic regression and Odds Ratio (OR) with a confidence interval of 95%. The variables whose association with the event was statistically significant (p < 0.05) remained in the final multivariate model. The software used was Stata 11.2 (Stata-Corp LP, College Station, TX). The study is part of a cohort, which has been carried out in two research centers, and was approved by the Ethics and Research Committee of the Centro de Ciências da Saúde da Universidade Federal de Pernambuco. # Results In the period from June 2007 to October 2009, 1815 PLWHA were interviewed, of which 1432 (78.9%) were taking cART. Of these, 52 (3.6%) did not answer the question referring to the irregular use of treatment, and were therefore excluded from the study. The study sample was made up of 1380 patients with a mean age of 40.6 years (18-80) and a median age of 40.0 years; 64.1% were male and 79.1% had a monthly family income of less than two minimum wages. The majority (84.7%) lived in the metropolitan region of Recife. The prevalence of people who reported irregular use of cART was 25.7%. Among people who reported irregular use of cART, 42% had stopped taking the pills in the two weeks preceding the study interview. A comparison of the mean ages, CD4 cell count and HIV viral load at the time of the interview revealed an association between self-reported irregular use of cART, being under 40 years old and a lower CD4 cell count [fig_ref] Table 1: Frequencies and univariate analysis of the association between characteristics of people living... [/fig_ref]. [fig_ref] Table 1: Frequencies and univariate analysis of the association between characteristics of people living... [/fig_ref] shows the frequency distribution of the studied factors and the Regarding the habits of the study population [fig_ref] Table 1: Frequencies and univariate analysis of the association between characteristics of people living... [/fig_ref] , 69.1% were considered abstainers or light drinkers of alcoholic beverages; most participants had never used cocaine or crack cocaine (90.1% and 93.3% respectively); in relation to smoking, most individuals (54.7%) were current or former smokers, the majority (84.2%) having consumed cigarettes for 10 years or more. In the multivariate analysis [fig_ref] Table 2: Multivariate analysis of the association between characteristics of people living with HIV/AIDS... [/fig_ref] , associations with the irregular use of cART in a statistically significant manner were, an age lower than or equal to 39 (p < 0.001), a former smoker (p = 0.023), currently smoking (p < 0.001) and currently using crack cocaine (p = 0.013). # Discussion After adjusting the socioeconomic and social service support variables, the discontinuation of combination antiretroviral therapy was associated with the following variables: being aged less than 40 years, a former smoker or a current smoker and a crack cocaine user. The prevalence of people who reported irregular use of cART was 25.7%. This prevalence was similar to the prevalence of non-adherence to treatment encountered in a number of studies in Brazil [bib_ref] Vulnerability and non-adherence to antiretroviral therapy among HIV patients, Minas Gerais State,..., Bonolo [/bib_ref] [bib_ref] Determinantes da adesão ao tratamento anti-retroviral em Brasília, DF: um estudo de..., Carvalho [/bib_ref] [bib_ref] Pessoas vivendo com HIV/AIDS: variáveis associadas à adesão ao tratamento anti-retroviral, Seidl [/bib_ref] [bib_ref] Risk-factors for non-adherence to antiretroviral therapy, Silva [/bib_ref]. Most studies conclude that taking less than 90-95% of prescription drugs is an indication of non-adherence, unlike the present study, which evaluated individual patient responses to questions regarding the discontinuation of treatment at some point in time. Studies also show the importance of including additional resources to self-reporting, such as testing tablets, to provide a better evaluation of adherence [bib_ref] Monitoramento e avaliação da adesão ao tratamento antirretroviral para HIV/aids: desafios e..., Polejack [/bib_ref]. Even without a consensus concerning the method of measuring this adherence, studies have indicated similar results of prevalence, demonstrating that the most appropriate method of certifying adherence to treatment was to question the patient directly. The strategy which uses, direct patient questioning, was chosen because it is easy to employ in everyday health care. We are aware that adherence may be overestimated due to a fear of displeasing the interviewer, the health professional or physician (false negative response); however those who actually report poor adherence are probably expressing the truth (decrease in false positive response) and are an important group to intervene with. The option for the question on abandonment of treatment at some point, without the restriction to a length time before and after the interview, aimed to expand the knowledge of the problem. We recognize that in the systematic care to the patient, the definition of a time span would be crucial in the evaluation of the patient's response to treatment and in the selection of the best intervention to be adopted to change the patient's behavior. It would also minimize recall bias. Most of the patients from this study were male and had a low monthly family income. These factors were related to poor adherence to antiretroviral treatment in another study [bib_ref] Adesão à terapêutica anti-retroviral por indivíduos com HIV/AIDS assistidos em uma instituição..., Gir [/bib_ref]. Low socioeconomic status may be a proxy of several factors associated to nonadherence, such as difficulties in accessing the health system, difficulties involved in understanding the individual regarding his/her health situation, etc. [bib_ref] Self-report measures of antiretroviral therapy adherence: a review with recommendations for HIV..., Simoni [/bib_ref]. Since the research involved a very low socioeconomic population, the study of the association of characteristics such as lifestyle, with the irregular use of cART, may be more specific to define the individual at risk. Research has indicated that older patients have a better adherence to antiretroviral treatment 15 , which also reinforces the findings of this study. It is possible that, due to a better perception of their own health, older people tend to better assimilate the importance of treatment and the consequences of interrupting it. This data was also encountered in another study undertaken in Brazil, where being younger was related to low adherence to the treatment 5 . Being a former smoker or a current smoker was associated with the discontinuation of combination antiretroviral therapy. Cigarette smoking has been associated with sexual behavior and drug abuse [bib_ref] Health risk behaviors and associated risk and protective factors among Brazilian adolescents..., Anteghini [/bib_ref] [bib_ref] Nicotine dependence and HIV risk behaviors among illicit drug users, Hershberger [/bib_ref] and with a lower inclination to develop healthy behaviors related to 131 diet [bib_ref] Prevalence of multiple chronic disease risk factors: 2001 National Health Interview Survey, Fine [/bib_ref] [bib_ref] Effects of smoking and smoking cessation on dietary habits of a Swiss..., Morabia [/bib_ref] and physical activity [bib_ref] Prevalence of multiple chronic disease risk factors: 2001 National Health Interview Survey, Fine [/bib_ref]. The same mechanism that predisposes all these behaviors may also lie behind, associated or not with these factors, a behavior less prone to adhere to medication. A study carried out by our group, in a cohort of people living with HIV/AIDS and under tuberculosis treatment, showed an association between smoking and the evasion of tuberculosis treatment [bib_ref] Risk factors for default from tuberculosis treatment in HIV-infected individuals in the..., Maruza [/bib_ref]. The present study corroborates the findings of other authors who found an association between smoking and lower adherence to antiretrovirals 4,24 , either smoking alone or as part of a risky behavior profile. Depression has been thought of as a possible mediator between smoking and adherence to antiretrovirals 28 but this is not a finding common to all authors 4 . People who use drugs tend to be more socially vulnerable and have a chaotic, unstable lifestyle that influences the adherence to any type of chronic treatment [bib_ref] Adesão à terapia antiretroviral para HIV, Colombrini [/bib_ref]. The use of illicit drugs has been associated with both nonadherence and a decrease in the viral and immunologic responses to antiretrovirals 18 , the former also being a finding in the present study. Research has demonstrated that cocaine (including the crack variation) has an influence over the pathogenesis of AIDS, accelerating its progression and the risk of mortality, morbidity and an increased viral load [bib_ref] Associations between use of crack cocaine and HIV-1 disease progression: research findings..., Cook [/bib_ref] [bib_ref] Crack cocaine, disease progression, and mortality in a multicenter cohort of HIV-1..., Cook [/bib_ref]. For this group, an increase in the viral load may be related to nonadherence to the treatment, which may lead to viral resistance and subsequently to an immunological decline, thus favoring the HIV virus and its replication. Studies show that direct administered antiretroviral therapy (DAART) in people living with HIV/AIDS who use drugs may improve adherence and thus improve the immune status of the individual 2,3 . Beyond DAART, other interventions such as counseling by motivational interviewing or video information, which is feasible in health care centers, improve adherence to antiretroviral treatment and maintain their efficacy three months later [bib_ref] A pilot randomized clinical trial of two medication adherence and drug use..., Ingersoll [/bib_ref]. Once adherence to treatment has been promoted through individual approaches and discussions by health professionals in the majority of the specialized services for treating PLWHA, differentiated attention towards smokers and crack users, as well as interaction with specific control programs, are central to good adherence to antiretroviral treatment and the consequent reduction of viral resistance. This study has some constraints. We are aware that the strategy used to measure the use of illicit substances may not be very accurate as a participant who has experienced crack cocaine once in the previous 12 months, an occasional user and a drug dependent (who used crack cocaine in the same time span), are all classified as current users of crack cocaine, although usage patterns and possibly behavior differ. The constraint posed to the interpretation of our findings by the discontinuation of combination antiretroviral therapy being measured has already been mentioned. Finally, a major limitation of this study, because of its cross-sectional design, is that it is not possible to establish the temporal sequence between exposure and outcome and differentiate cause and effect. Nevertheless the results pointed to groups which should be monitored differentially because of their association with discontinuation of combination antiretroviral therapy. Further studies, using a longitudinal design and more restricted definitions, could complement our findings. ## Resumo Associação entre tabagismo e o uso de crack com a descontinuidade da terapia antirretroviral combinada em Recife, Pernambuco, Brasil Apesar da eficácia da terapêutica antirretroviral combinada para o tratamento de pessoas vivendo com HIV/Aids, a não adesão aos medicamentos tem se tornado uma das maiores ameaças à efetividade dessa terapêutica. O objetivo desse estudo foi estimar a prevalência de uso irregular autorreferido da terapia antirretroviral e os fatores associados com essa irregularidade em pessoas vivendo com HIV. Foi realizado um estudo seccional de pessoas vivendo com HIV/Aids atendidas em dois centros de referência no Recife, Nordeste do Brasil, entre junho [table] Table 1: Frequencies and univariate analysis of the association between characteristics of people living with HIV/AIDS and the irregular use of antiretroviral treatment, Recife, Pernambuco, Brazil, 2009 [/table] [table] Table 2: Multivariate analysis of the association between characteristics of people living with HIV/AIDS and the irregular use of antiretroviral treatment, Recife, Pernambuco, [/table]
Reliable resolution of ambiguous hepatitis C virus genotype 1 results with the Abbott HCV Genotype Plus RUO assay Accurate subtyping of hepatitis C virus genotype 1 (HCV-1) remains clinically and epidemiologically relevant. the Abbott HCV Genotype Plus RUO (GT Plus) assay, targeting the core region, was evaluated as a reflex test to resolve ambiguous HCV-1 results in a challenging sample collection. 198 HCV-1 specimens were analysed with Gt Plus (38 specimens with and 160 without subtype assigned by the Abbott Realtime Genotype II (GT II) assay targeting the 5'NC and NS5B regions). Sanger sequencing of the core and/or NS5B regions were performed in 127 specimens without subtype assignment by GT II, with "not detected" results by GT Plus, or with mixed genotypes/subtypes. The remaining GT Plus results were compared to LiPA 2.0 (n = 45) or just to GT II results if concordant (n = 26). GT Plus successfully assigned the subtype in 142/160 (88.8%) samples. "Not detected" results indicated other HCV-1 subtypes/genotypes or mismatches in the core region in subtype 1b. The subtyping concordance between Gt Plus and either sequencing or LiPA was 98.6% (140/142). Therefore, combined use of GT II and Gt Plus assays represents a reliable and simple approach which considerably reduced the number of ambiguous HCV-1 results and enabled a successful subtyping of 98.9% of all HCV-1 samples.Chronic infection with hepatitis C virus (HCV) can progress to liver cirrhosis, hepatocellular cancer and death 1 . In 2015, 71 million people worldwide were estimated to be chronically infected 2 . Being highly genetically diverse, HCV has been classified into 8 major genotypes and 86 confirmed subtypes 3,4 . Genotypes 1, 2 and 3 are distributed worldwide, genotype 1 being the predominant one, specifically subtypes 1a and 1b 5 . In Spain, a 1.5% HCV seroprevalence has been estimated in the general population 6 , with genotype 1 being the most prevalent one (66.9%), with subtype 1b predominating over 1a 7 .Since the efficacy and barrier to resistance of non-pangenotypic direct-acting antiviral agents (DAAs) depend on the HCV genotype and subtype (especially for subtype 1a and 1b), HCV genotyping must be performed prior to treatment initiation and will determine the choice of therapy 8 . Additionally, genotyping upon treatment failure may differentiate between relapse and reinfection 9 . Furthermore, knowledge of circulating genotypes and subtypes is necessary for epidemiological purposes 10 .While commercial genotyping assays use sub-genomic regions such as core or NS5B in addition to the more conserved 5′ untranslated (5′NC) region, the high genetic variability and small differences between genotypes and subtypes still remain a challenge for both real-time PCR and line probe-based HCV genotyping assays. This also applies to HCV-1 subtyping 11-21 . In case of ambiguous results, the use of a second genotyping method may help guiding treatment selection. Nucleotide sequencing and phylogenetic analysis of the NS5B or core/E1 regions has been recommended as the reference genotyping method in consensus proposals 22 . However, even this method is not able to resolve every individual sample, e.g. due to failure of amplification11,13,17,23. Furthermore, the procedure is considered impractical for most clinical laboratories because it is time-consuming, less sensitive 21 , may be technically challenging, and does not readily allow for detecting mixed-type infections.The Abbott RealTime HCV Genotype II assay (GT II, Abbott Molecular Inc.) is a real-time PCR assay which includes specific probes for the identification of genotypes 1 to 6 (5′NC region), and subtypes 1a and 1b (NS5B region). Using this assay, about 5.4% of all genotypes 1 were not classified at the subtype level in our centre 14 . The novel HCV Genotype Plus RUO assay (GT Plus, Abbott Molecular Inc., Des Plaines, USA) has been designed to complement GT II or other assays as a reflex test in order to resolve ambiguous HCV-1 results. By targeting the core region, it identifies subtypes 1a, 1b and genotype 6.The aims of this study were: (i) to evaluate the performance of the GT Plus assay in a challenging collection of genotype 1 clinical specimens not subtyped by the GT II assay and obtained from three geographical regions; and (ii) to assess its accuracy at identifying 1a and 1b subtypes in comparison with the reference method. ## Material and methods study design. A flowchart diagram of the study design and the methods used is shown in [fig_ref] Figure 1: Flowchart diagram of the study design and methods used [/fig_ref]. A total of 198 samples were included in this study and classified into two groups. Group 1 consisted of 160 genotype 1 samples for which no subtype had been assigned by the GT II assay upon routine testing (including eight samples with mixed genotypes). These samples were retrospectively collected in three different geographic areas in order to assess the performance of the GT Plus assay in subtyping these challenging samples. Group 2 consisted of 38 genotype 1 samples with subtypes assigned by the GT II assay (16 identified as 1a, 16 as 1b including three mixed genotypes, and six mixed 1a + 1b subtypes). The Group 2 samples were included in order to assess the concordance between both Abbott assays (GT II and GT Plus). Overall, the two groups included 17 mixed infections (11 mixed genotypes and six mixed subtypes). All samples from Groups 1 and 2 underwent testing with the GT Plus assay. Subsequently, samples in Group 1 were subjected to the reference method for HCV genotyping based on Sanger sequencing and phylogenetic analysis of the targets of the two assays (NS5B region for GT II, and core region for GT Plus), except for 44 samples from Israel; GT Plus results for these 44 samples and another sample that failed Sanger sequencing were compared to the results of the line probe-based VERSANT HCV Genotype 2.0 Assay (LiPA, Siemens Healthcare GmbH, Erlangen, Germany) according to the manufacturer's procedure. Out of Group 2, only samples with discordant results between the two Abbott assays, including partially discordant results in the case of mixed genotypes or subtypes, were subjected to the reference sequencing method. The result of one discordant sample from Israel was compared to LiPA. Finally, all samples with suspected mixed genotypes or subtypes or discordant results between core and NS5B sequences were further characterised by next-generation sequencing (NGS). The HCV genomic targets of the molecular techniques used in this study are listed in . This study was approved by the Ethics Committee (Comitè d'Ètica de la Investigació Clínica -CEIC-, Germans Trias i Pujol University Hospital, coordinator centre), and was conducted in agreement with the Declaration of Clinical specimens. Left-over plasma or RNA samples archived at −80 °C were retrospectively selected from three laboratories in different geographic locations: (i) Germans Trias i Pujol University Hospital (Badalona, North-eastern Spain) (n = 74); (ii) Virgen de la Victoria University Hospital (Málaga, Southern Spain) (n = 64); and (iii) Maccabi Mega-Lab (Rehovot, Israel) (n = 60). These specimens were selected according to previous GT II assay results as follows: genotype 1 with no subtype assigned (Group 1, n = 160), or genotype 1 with subtype assigned (Group 2, n = 38) including 1a (n = 16), 1b (n = 16) and 1a + 1b (n = 6). www.nature.com/scientificreports www.nature.com/scientificreports/ Genotyping by Abbott GT II and GT Plus assays. Both genotyping assays are based on real-time PCR technology. Plasma specimens were tested with the fully automated Abbott m2000 system, including RNA extraction, PCR plate set-up, amplification and detection. When only previously extracted RNA was available, the amplification mix and PCR plate were prepared manually. The GT II assay identifies genotypes 1, 2, 3, 4, 5, and 6 with fluorescence-labelled oligonucleotide probes targeting the 5′NC region, as well as subtypes 1a and 1b targeting the NS5B region. When a fluorescent signal is only obtained from the HCV-1 specific probe (5′NC region) but 1a and 1b specific probes fail to provide a valid signal, HCV-1 subtype cannot be assigned. The GT Plus assay detects genotypes 1a, 1b, and 6 in a single reaction with probes targeting the core region. Results are interpreted by Abbott software as 1a, 1b, 6, or "not detected". All analyses were performed following the manufacturer's recommendations. Genotyping by NS5B and core sequencing and phylogenetic analysis. This method served as gold standard to assess (i) the accuracy of both Abbott genotyping assays in identifying 1a and 1b subtypes, and (ii) the presence of mismatches within the target regions for primers and probes of these assays. Starting from RNA extracted with the Abbott m2000sp system, retrotranscription reactions were performed in a 30 μl volume containing 7.5 μl eluted RNA, 0.25 mM dNTPs, 150 U Moloney murine leukaemia virus reverse transcriptase and 6 μl 5× RT buffer (Promega), 30 U RNasin RNase inhibitor (Promega), and 0.75 μg random hexadeoxynucleotides (Roche) in order to retrotranscribe the whole HCV genome preventing any bias during the reaction. The reactions were incubated at 42 °C for 2 min, 20 °C for 5 min, 25 °C for 5 min, 30 °C for 5 min, 35 °C for 5 min, 37 °C for 5 min, 42 °C for 45 min, and 2 min at 94 °C. NS5B region was partially amplified and sequenced using a pangenotypic strategy as previously described [bib_ref] Evaluation of a new assay in comparison with reverse hybridization and sequencing..., Martró [/bib_ref]. To amplify the whole core region of HCV genotype 1 (573 bp, H77 positions 342-914), three different strategies were used. lists primers and thermal cycler profiles used in each PCR strategy. The first strategy was performed as previously published [bib_ref] Baseline prediction of combination therapy outcome in hepatitis C virus 1b infected..., Saludes [/bib_ref]. The second strategy consisted of two PCR rounds performed each in a 50 μl reaction volume with 5 µl cDNA, 5 μl Pfu DNA polymerase 10× buffer, 1.5 U Pfu DNA polymerase (Promega, Mannheim, Germany), 0.25 mM dNTPs, and 0.3 µM primers. The third amplification alternative consisted of two PCR rounds performed each with the amplification mix described above. Amplification products were size selected and purified with Agencourt AMPure XP system (Beckman Coulter) following manufacturer's instructions, and subjected to bidirectional Sanger sequencing with second round amplification primers. Sequence readings were assembled and edited with the STADEN package v1.6 [bib_ref] The Staden package, Staden [/bib_ref]. Study sequences were aligned by ClustalW implemented in MEGA 6 27 together with reference sequences. Then, jModeltest 28 was used to obtain the evolutionary model that best fitted the data according to the Akaike information criterion. This model was used to reconstruct a maximum likelihood phylogenetic tree with PHYML [bib_ref] A simple, fast, and accurate algorithm to estimate large phylogenies by maximum..., Guindon [/bib_ref]. The robustness of the tree topologies was assessed by bootstrap analysis implemented in PHYML (500 replicates). Information regarding the presence of mutations in the primer/probe-binding regions of the commercial assays was kindly provided by Abbott as these sequences are proprietary. Genotyping with the VERSANT HCV Genotype 2.0 Assay (LiPA). The LiPA assay is based on the reverse hybridisation of biotinylated amplified products from the 5′NC and core regions. The genotypes/subtypes 1, 1a, 1b, 1a/1b, 2a/2c, 2b, 3, 4, 5a, 6 and 6c-6l can be identified. Probes in the core region allow for a more reliable identification of subtypes 1a, 1b and 6c-l than the previous version of this assay. RNA extraction was performed using the Abbott m2000sp instrument and reagents. Hybridisation and detection steps were performed manually, following the manufacturer's instructions. ## Assessment of mixed infections by ngs. NS5B amplicons, as well as 5′NC-core ones if necessary (spanning the first 130 bp of the core region), were subjected to NGS and subtype calling using the methodology and primers previously described [bib_ref] High-resolution hepatitis C virus subtyping using NS5B deep sequencing and phylogeny, an..., Quer [/bib_ref]. Briefly, a RT-PCR-nested amplification was performed, and purified PCR products were quantified using the PicoGreen assay (Invitrogen, Carlsbad, CA, USA), and quality analysed using the BioAnalyzer DNA 1000 LabChip (Agilent, Santa Clara, CA, USA) prior to sequencing. PCR products from multiple patients individually identified by a genetic barcode were mixed at equal molecular proportion, and www.nature.com/scientificreports www.nature.com/scientificreports/ sequenced using MiSeq sequencing platform with MiSeq reagent kit v3 (Illumina, San Diego, CA). The raw data was filtered and processed to obtain a fasta file per sample which then was subjected to a phylogenetic analysis together with reference sequences. This multiple alignment provided a matrix of genetic distances with the Kimura-80 model with a gamma shape parameter of 0.42. The query haplotype was classified as the subtype with the nearest reference sequence. For subtypes with multiple reference sequences, two other distances to the query haplotype were computed: the average and the farthest in each subtype. These distances were used as clustering quality scores. A high-quality clustering result was produced when the subtyping according to the shortest, the average, and the farthest distances coincide. A confidence score was produced by 200 cycles of bootstrap analysis on the multiple alignment obtained for each query haplotype and subtyping according to the shortest distance. Query haplotypes classified with discordant shortest-distance and average-distance subtyping or with bootstrap scores below 70 were tagged as doubtful. A minimum of 5 reads and of 1% abundance was imposed to infer a mixed infection by a minority subtype. The data analysis system consisted of four R modules, implementing the demultiplexing module, the quality filter and haplotype selection module, the phylogenetic analysis module, and the expert system module. # Results All HCV genotype and subtype results according to the different genotyping methods used are summarised in [fig_ref] Table 2: HCV genotype and subtype results according to the genotyping method used [/fig_ref]. Ability of the Gt Plus assay for subtyping genotype 1 samples with no subtype assigned by the GT II assay. Among the 160 samples in Group 1, the GT Plus assay resolved the subtype in 142 cases (88.75%; 1a in 20 cases and 1b in 122 cases), while in the remaining 18 cases (11.25%) a "not detected" result was obtained. For the latter, the reference method based on the NS5B and core sequencing and phylogenetic analysis was used for HCV classification. Five out of 18 "not detected" cases correctly showed a "not detected" result as per assay design since sequencing identified genotype 1 subtypes other than 1a or 1b in four cases (subtypes 1d, 1e, 1g, and 1h) and a genotype 4m in the one case which previously was genotyped as HCV genotype 1 + 4 by the GT II assay. Another 12 cases were classified as 1b; evaluation of the sequences revealed the presence of mismatches in the core region in which the 1b-specific probes or primers of the GT Plus assay anneal. No specific cluster was observed in the phylogenetic tree of these "not detected" core sequences , which include the region targeted by the GT Plus assay. In the remaining "not detected" case, sequencing failed in both regions due to low viral load (3.2 Log (IU/mL)) and LiPA was used for comparison to the GT Plus assay, which detected a subtype 1b. In samples with a "not detected" result, viral loads ranged from 3.2 to 6.9 Log (IU/mL) and, thus, were above the lower limit of detection of the GT Plus assay. Overall, among the samples with genotype 1 results without subtype assigned by GT II, the proportion of HCV subtype 1b isolates identified by sequencing or LiPA that could not be subtyped by the GT Plus assay was 9.6% (13/135) but varied geographically: 22.5% (9/40) in North-eastern Spain, 4.1% (2/49) in Southern Spain, and 4.3% (2/46) in Israel. Similarly, the proportion of non-1a non-1b subtypes, which are not covered by the GT Plus assay design, also varied geographically (1.8% in Spain and 4.0% in Israel), albeit the overall percentage was low. Concordance at the subtype level of the Gt Plus assay with the reference method or LiPA in genotype 1 samples with no subtype assigned by the GT II assay. Among the 160 samples of Group 1 with no subtype assigned by the GT II assay, 116 samples underwent sequencing using the reference method, while remaining samples without sequencing results were compared to LiPA. NS5B amplification and sequencing was successful in 115/116 samples (99.1%), and core sequencing in 111/113 samples (98.2%). Furthermore, HCV classification at subtype level based on the phylogenetic analysis of NS5B and core sequences agreed between the two target regions in all samples [fig_ref] Table 2: HCV genotype and subtype results according to the genotyping method used [/fig_ref] except for two, which were subjected to NGS (see below). In those cases in which the GT Plus assay assigned a genotype 1 subtype (n = 98), the concordance between this subtype call and the reference method was 18/19 for subtype 1a (94.7%; one sample was classified as subtype 1b by sequencing), and 78/79 for subtype 1b (98.7%; one sample was classified as subtype 1g by sequencing). Thus, the overall agreement between the GT Plus assay and the reference method was 98.0% (96/98). For the remaining 44 of 160 samples, the GT Plus assay assigned the subtype in all cases, and the LiPA assay was used as comparator to the GT Plus assay (Israel). The concordance between the genotype 1 subtypes identified by the GT Plus assay and by the LiPA assay, respectively, was 100% for both subtype 1a (1/1) and subtype 1b (43/43), respectively. Overall, the accuracy of subtype 1a or 1b assignment by the GT Plus assay in comparison to either sequencing or LiPA was 140/142 (98.6%); i.e. 19/20 (95%) for subtype 1a and 121/122 (99.2%) for subtype 1b. Concordance at the subtype level between the Gt Plus assay and the GT II assay in genotype 1a and 1b samples. When testing the 38 genotype 1a and 1b samples of Group 2 using the GT Plus assay, a genotype 1 subtype was assigned in 33 cases (86.8%). The agreement between both assays at subtype level was 15/16 for subtype 1a (93.8%; one sample was classified as mixed 1a + 1b subtypes by GT Plus), 11/11 for subtype 1b (100%), and 1/6 for those detected as mixed 1a + 1b subtypes (16.7%; two samples were classified as 1a by GT Plus and three as 1b). The GT Plus result was "not detected" in the remaining samples (n = 5): four of them were 1b by sequencing and the other one, initially detected as 1b + 2 by the GT II assay, was genotype 2 by LiPA. The overall concordance for single infections with either subtype 1a or 1b between both Abbott assays was 96.3% www.nature.com/scientificreports www.nature.com/scientificreports/ Assessment of mixed infections. Seven 1a + 1b mixed subtypes were detected either with the GT Plus assay (n = 2) and/or the GT II assay (n = 6). These mixed subtypes were assessed by Sanger (core and NS5B regions) and NGS (NS5B region) but none of these methods confirmed a presence of mixed subtypes . Evaluation of the generated sequences revealed perfect matches in the probe regions of the GT II or GT Plus assay for the subtypes that were confirmed by sequencing while mismatches were observed for the second subtypes. Eleven mixed genotypes were detected by the GT II assay, eight in Group 1 and three in Group 2. The GT Plus assay results in these samples were as follows [fig_ref] Table 4: Sequencing results in samples with mixed genotypes detected by the GT II... [/fig_ref] : (i) the genotype 1 subtype was resolved in seven specimens and results were confirmed either by the reference method in the targeted core region (n = 6; two 1a and four 1b) or by LiPA (n = 1; 1b); (ii) the GT Plus correctly showed a "not detected" result in one sample since sequencing revealed the presence of genotype 4m; (iii) similarly, a "not detected" result was obtained in one sample from Israel (I35) showing a result pattern across the assays that suggested a recombinant St. Petersburg variant consisting of genotype 2k in the 5'NC and core regions and 1b in NS5B 31 (GT II: genotype 2 in the 5′NC and 1b in the NS5B regions; LiPA: 2a/2c in 5'NC and core regions; and GT Plus: "not detected" in the core region since genotype 2 is not detected per assay design) but, unfortunately, there was no sample material left to perform sequencing for confirmation; and (iv) two samples with a "not detected" result of GT Plus turned out to be genotype 1b by sequencing of the targeted core region. Subsequently, all these samples with mixed genotypes were subjected to NGS, except for the three cases from Israel due to lack of residual sample material [fig_ref] Table 4: Sequencing results in samples with mixed genotypes detected by the GT II... [/fig_ref]. In one case (M49), NGS detected mixed 1a + 1b subtypes that had not been identified by any of the previous assays. The last two specimens in [fig_ref] Table 4: Sequencing results in samples with mixed genotypes detected by the GT II... [/fig_ref] (M51 and M54) showed discordant results at the genotype level between core and NS5B Sanger sequencing and, thus, NGS was performed targeting the 5′NC-core region in addition the NS5B to identify the cause of these discordant results. However, NGS did not detect any mixed genotypes but identified genotype 2i for specimen M51 and genotype 3a for specimen M54 in both target regions, respectively. Thus, NGS ruled out recombination between different genotypes as the cause of these rare discordant results. Nevertheless, since genotype 1 primers had been used to amplify the core region during Sanger sequencing and pangenotypic ones during NGS, it could be possible that the former ones had been able to amplify a minority population of 1b genomes that were below the NGS cut-off, i.e. Sanger sequencing of the core region could have revealed the Group GT II (5′NC, NS5B) GT Plus (core) NS5B sequencing Core sequencing LiPA (5′NC, core) (17) 1b (12) 1b (12) - www.nature.com/scientificreports www.nature.com/scientificreports/ presence of a 1b subpopulation besides genotype 2i for patient M51 and 3a for M54 being the dominant population. Therefore, taking all data into account for specimens M51 and M54, we consider mixed genotypes, with subtype 1b below the NGS cut-off, as the most probable result. Overall, NS5B NGS results confirmed those obtained by Sanger sequencing in 14/15 cases (93.3%) with suspected mixed subtypes or genotypes by Abbott assays. [formula] Group 1 1 (152) 1a (18) 1a (16) 1a (16) - - - 1a (1) 1b (1) 1b (1) - 1b (117) 1b (72) 1b (72) - 1b (1) failed (1) - 1b (1) - - - - 1b (42) 1 g (1) 1 g (1) - Not detected1d (1) - - 1e (1) 1e (1) - 1 g (1) 1 g (1) - 1 h (1) 1 h (1) - failed (1) failed (1) 1b (1) 1, 2 (1) 1b (1) 2i (1) 1b (1) - 1, 3 (2) 1b (2) 1b (1) 1b (1) - - - 1b (1) 1, 4 (5) 1a (2) 1a (2) 1a (2) - 1b (2) 1b (2) 1b (2) - Not detected (1) 4m (1) - - Group 2 1a (16) 1a (15) - - - 1a, 1b (1) 1a (1) 1a (1) - 1b (13) 1b (11) - - - Not detected (2) 1b (2) 1b (2) - 1b, 2 (1) Not detected (1) - - 2a/2c (1) 1b, 3 (1) Not detected (1) 3a (1) 1b (1) - 1b, 3, 4 (1) Not detected (1) 1b (1) 1b (1) - 1a, 1b (6) 1a, 1b (1) 1b (1) 1b (1) - 1a (2) 1a (2) 1a (2) - 1b (3) 1b (3) 1b (3) - [/formula] # Discussion In the era of direct-acting antivirals against HCV infection, an accurate genotyping as well as a reliable discrimination between subtype 1a and 1b remains clinically and epidemiologically relevant 9,10,32 . However, commercial genotyping assays may result in a low percentage of HCV-1 results with no subtype assigned [bib_ref] Characterization of Samples Identified as Hepatitis C Virus Genotype 1 without Subtype..., Mokhtari [/bib_ref] [bib_ref] Hepatitis C virus gene sequencing as a tool for precise genotyping in..., Ceccherini Silberstein [/bib_ref] [bib_ref] Evaluation of the Abbott realtime HCV genotype II RUO (GT II) assay..., Mallory [/bib_ref] [bib_ref] Accuracy of a commercially available assay for HCV genotyping and subtyping in..., González [/bib_ref] [bib_ref] NS3 genomic sequencing and phylogenetic analysis as alternative to a commercially available..., Neukam [/bib_ref] [bib_ref] Assessment of a Novel Automatic Real-Time PCR Assay on the Cobas 4800..., Nieto-Aponte [/bib_ref] [bib_ref] Evaluation of the new cobas ® HCV genotyping test based on real-time..., Stelzl [/bib_ref] [bib_ref] Cobas(R) HCV Gt for Use on the Cobas(R) 4800 System: A Highly..., Herman [/bib_ref]. The GT II assay, which targets the NS5B region for identifying subtypes 1a and 1b, may fail to resolve the subtype in about 5-12% of HCV-1 specimens [bib_ref] Characterization of Samples Identified as Hepatitis C Virus Genotype 1 without Subtype..., Mokhtari [/bib_ref] [bib_ref] Evaluation of the Abbott realtime HCV genotype II RUO (GT II) assay..., Mallory [/bib_ref] [bib_ref] Accuracy of a commercially available assay for HCV genotyping and subtyping in..., González [/bib_ref] [bib_ref] Utility of the Abbott RealTime HCV Genotype Plus RUO assay used in..., He [/bib_ref]. However, a recent GT II software update has reduced the number of genotype samples without subtype assignment (4% in Herman et al. [bib_ref] Cobas(R) HCV Gt for Use on the Cobas(R) 4800 System: A Highly..., Herman [/bib_ref]. An inability of the GT II assay to identify the genotype 1 subtype may be due to (i) the presence of genetic variability in the NS5B region in which the probes . Maximum likelihood phylogenetic subtree of the genotype 1 core region (which includes the region targeted by the GT Plus assay) for samples belonging to Group 1 with a "not detected" result by the GT Plus assay. Among the 18 "not detected" samples, sequencing of the core region failed for sample I51 and could not be performed for samples I23 and I45 (not available). Sequences available in the Los Alamos National Library HCV sequence database (http://hcv.lanl.gov/content/index) and International Committee on Taxonomy of Viruses (ICTV) databasewww.nature.com/scientificreports www.nature.com/scientificreports/ specific for subtype 1a or 1b detection anneal (majority in the present study), (ii) the presence of rare HCV-1 subtypes (four cases observed), or (iii) the presence of certain genotype 6 subtypes in which the 5′NC region targeted by the genotype 1-specific probe is identical to some 1b subtypes [bib_ref] Characterization of Samples Identified as Hepatitis C Virus Genotype 1 without Subtype..., Mokhtari [/bib_ref] [bib_ref] Evaluation of the Abbott realtime HCV genotype II RUO (GT II) assay..., Mallory [/bib_ref] [bib_ref] Utility of the Abbott RealTime HCV Genotype Plus RUO assay used in..., He [/bib_ref] (none observed in our study). We evaluated the performance and accuracy of the novel GT Plus assay in resolving HCV-1 specimens not subtyped by the GT II assay (Group 1) in comparison with the reference method or with LiPA. While three other evaluations of the GT Plus assay have previously been published [bib_ref] Characterization of Samples Identified as Hepatitis C Virus Genotype 1 without Subtype..., Mokhtari [/bib_ref] [bib_ref] Utility of the Abbott RealTime HCV Genotype Plus RUO assay used in..., He [/bib_ref] [bib_ref] Evaluation of the Abbott RealTime HCV genotype II plus RUO (PLUS) assay..., Mallory [/bib_ref] , the present study investigated HCV-1 specimens including the largest collection of very challenging clinical specimens with ambiguous HCV-1 results by GT II from three different geographical regions that was ever tested with the GT Plus assay and subsequently evaluated by a reference method for confirmation. Additionally, it was the first study in generating full core region sequences that shed some light into the reasons why this assay may not assign an HCV-1 subtype in rare cases. This HCV sequencing information will also be useful for other manufacturers of core-based genotyping assays. Herein, the GT Plus assay resolved the subtype in 88.8% (142/160) of Group 1 cases, while a "not detected" result was obtained for the remaining 18 specimens. Since the GT Plus assay is designed to identify HCV subtypes 1a and 1b as well as genotype 6, three main reasons may be pivotal if the GT Plus assay provides a "not detected" result for genotype 1 samples without subtype by the GT II assay. Firstly, HCV-1 subtypes other than 1a and 1b may be present which are not detected by the GT Plus assay due to its genotype coverage (i.e. 1a, 1b, 6) per assay design. In the current study, 4/18 (22%) "not detected" samples were classified as 1d, 1e, 1g, and 1h by sequencing, while the proportion of non-1a non-1b subtypes was higher in the study by in France (25/32, 78.1%) [bib_ref] Characterization of Samples Identified as Hepatitis C Virus Genotype 1 without Subtype..., Mokhtari [/bib_ref]. Secondly, another potential reason is the presence of mismatches in the core region targeted by HCV 1a-, 1b-and 6-specific primers and probes used by the GT Plus assay. In the present study, evaluation of the . Sequencing results in samples with mixed HCV 1a + 1b subtypes as detected by either the GT II and/or the GT Plus assay. GT II, Abbott RealTime HCV Genotype II assay (Abbott Molecular Inc.); GT Plus, HCV Genotype Plus RUO assay (Abbott Molecular Inc.); NGS, next-generation sequencing; B, Badalona; M, Málaga. *In case of mixed subtype results, perfect match in the probe target region for the subtype that was also identified by sequencing while the other subtype showed mismatches. **100% was not achieved because residual sequences appeared which were filtered and discarded as they were below 1%. www.nature.com/scientificreports www.nature.com/scientificreports/ core sequences confirmed this in 12/18 (66.7%) samples that were "not detected" by the GT Plus but classified as subtype 1b by sequencing (for another sample sequencing failed). This proportion was higher than in the studies by Mokhtari et al. 4%, 3 out of 32 cases, which were also 1b) [bib_ref] Characterization of Samples Identified as Hepatitis C Virus Genotype 1 without Subtype..., Mokhtari [/bib_ref] and Mallory et al. 1%, 1 out of 14 samples, being 1a) [bib_ref] Evaluation of the Abbott RealTime HCV genotype II plus RUO (PLUS) assay..., Mallory [/bib_ref]. Thirdly, in rare cases, a sample can be incorrectly genotyped as HCV-1 by GT II, and consequently, the GT Plus returns a "not detected" result. Indeed, we observed a "not detected" result in a sample originally identified as mixed 1 + 4 genotypes by the GT II assay that turned out to be subtype 4m by sequencing. This was also observed by Mallory et al., who reported a "not detected" result in a sample previously identified as HCV-1 with no subtype assigned by the GT II assay and classified as genotype 4 by sequencing [bib_ref] Evaluation of the Abbott RealTime HCV genotype II plus RUO (PLUS) assay..., Mallory [/bib_ref]. The polymorphisms present in specific HCV isolates circulating in different geographical areas (genetic diversity both among HCV-1 subtypes and within subtype 1b) influenced the proportion of "not detected" results by GT Plus in genotype 1 samples without subtype by GT II: it increased from 3.5% (2/57) in Southern Spain to 10% (5/50; including the genotype 4 sample) in Israel, and up to 20.8% (11/53) in North-eastern Spain (overall 18/160, 11.3%). However, it was lower than previously reported: 32/98 (32.7%) in France 11 , and 7/14 (50.0%) in the USA [bib_ref] Evaluation of the Abbott RealTime HCV genotype II plus RUO (PLUS) assay..., Mallory [/bib_ref]. The accurate recognition of subtypes 1a and 1b represents a common and difficult challenge to commercial assays. In the present study, the GT Plus assay demonstrated to be highly reliable in assigning 1a and 1b subtypes in specimens initially not subtyped by the GT II assay (Group 1), as the proportion of calls by the GT Plus assay that were confirmed by sequencing was very high (98.0%; 96 out of 98 samples). This proportion was higher than that observed in Mokhtari's study (84.3%; 43 out of 51 samples) [bib_ref] Characterization of Samples Identified as Hepatitis C Virus Genotype 1 without Subtype..., Mokhtari [/bib_ref] , but similar to that reported by ; 168 out of 173 samples) [bib_ref] Evaluation of the Abbott RealTime HCV genotype II plus RUO (PLUS) assay..., Mallory [/bib_ref]. In agreement with the latter study, we suggest a non-specific reactivity as the most probable cause for the rare discordant results observed in the present study; one subtype 1b misclassified by the GT Plus as subtype 1a, and one subtype 1g misclassified as subtype 1b. When we included LiPA results for those samples in which sequencing was not done, overall agreement at the subtype level (with either LiPA or sequencing) even slightly increased to 98.6% (140/142), as the agreement at HCV-1 subtype calling between the GT Plus and the LiPA assays was 100% (all being 1b except one 1a case). The LiPA assay also relies on the core region for the identification of subtypes 1a and 1b, and genotype 6. However, it may fail to correctly identify 1a and 1b subtypes in a variable percentage of cases (5-14%) [bib_ref] Using NS5B Sequencing for Hepatitis C Virus Genotyping Reveals Discordances with Commercial..., Chueca [/bib_ref] [bib_ref] Comparison of Abbott RealTime HCV Genotype II with Versant line probe assay..., Liu [/bib_ref] [bib_ref] Reassessment of genotype 1 hepatitis C virus subtype misclassification by LiPA 2.0:..., Guelfo [/bib_ref] [bib_ref] NS3 genomic sequencing and phylogenetic analysis as alternative to a commercially available..., Neukam [/bib_ref] , and tends to misclassify genotype 6 as genotype 1 [bib_ref] Comparison of Abbott RealTime HCV Genotype II with Versant line probe assay..., Liu [/bib_ref] [bib_ref] Comparison of three different HCV genotyping methods: core, NS5B sequence analysis and..., Cai [/bib_ref]. While the latter is unlikely in Spain and Israel, we have relied on the LiPA assay for assessment of GT Plus HCV-1 subtype calls in 31% of the cases. As this fact may be a limitation of our study, we have analysed the concordance between the GT Plus and LiPA assays separately from the concordance between the GT Plus assay and sequencing. The identification of mixed infections represents another challenge for current commercially available assays based on real-time PCR or line-probe hybridisation [bib_ref] Evaluation of the Abbott realtime HCV genotype II RUO (GT II) assay..., Mallory [/bib_ref] [bib_ref] Comparison of Abbott RealTime HCV Genotype II with Versant line probe assay..., Liu [/bib_ref] [bib_ref] Assessment of a Novel Automatic Real-Time PCR Assay on the Cobas 4800..., Nieto-Aponte [/bib_ref] [bib_ref] Cobas(R) HCV Gt for Use on the Cobas(R) 4800 System: A Highly..., Herman [/bib_ref] [bib_ref] Detection of Mixed Hepatitis C Virus (HCV) Infection Using Abbott RealTime HCV..., Lampinen [/bib_ref] [bib_ref] Ultra-Deep Sequencing Characterization of HCV Samples with Equivocal Typing Results Determined with..., Minosse [/bib_ref]. Mixed infections have been reported particularly in those patients exposed to multiple HCV infections such as people who inject drugs [bib_ref] Mixed HCV infection and reinfection in people who inject drugs-impact on therapy, Cunningham [/bib_ref]. It has been suggested that NGS may improve the detection of minor genotypes/subtypes in mixed infections; however, NGS assays including the one used herein 38 usually require comparably high viral loads, while real-time subtype-specific nested reverse transcriptase PCR can detect virus at low concentrations [bib_ref] Mixed HCV infection and reinfection in people who inject drugs-impact on therapy, Cunningham [/bib_ref]. It has previously been suggested that the GT II assay is able to reliably detect mixed infections if the minor genotype/subtype represents at least 20-30% of the circulating variants (similarly to Sanger sequencing) [bib_ref] Evaluation of the Abbott realtime HCV genotype II RUO (GT II) assay..., Mallory [/bib_ref] [bib_ref] Detection of Mixed Hepatitis C Virus (HCV) Infection Using Abbott RealTime HCV..., Lampinen [/bib_ref]. Furthermore, it is able to genotype at viral loads of 500 IU/mL and below depending on the genotype [bib_ref] Evaluation of Abbott RealTime Hepatitis C Virus (HCV) Genotype II Assay, Leckie [/bib_ref]. In our study, NGS of the NS5B region did not detect mixed 1a + 1b subtypes as previously identified by either the GT Plus (n = 1), the GT II (n = 5) or both (n = 1) assays. Alternatively, the detection of the two mixed 1a + 1b subtypes by the GT Plus assay could have been due to cross-reactivity between both subtypes. The latter is supported by our evaluation of the sequences that revealed no mismatches in the probe target region for the subtypes confirmed by sequencing while there were mismatches observed for the other subtypes, respectively. On the other hand, in a sample classified as mixed 1 + 4 genotypes without genotype 1 subtype assignment by GT II, only NS5B NGS identified mixed 1a + 1b subtypes while GT Plus and Sanger sequencing of both NS5B and core regions concordantly classified it as 1b only. Lastly, in order to further characterise two specimens with discordant results between core and NS5B Sanger sequencing (1b/2i and 1b/3a), two distant genomic regions of the virus (5′NC-core and NS5B) were subjected to NGS. While the NGS assay did not confirm the presence of mixed genotypes in these specimens, the evidence gathered from Sanger sequencing and real-time PCR assays nevertheless points to the presence of both genotypes, respectively. These few examples show that even the application of gold standard methods may lead to discordant results and cannot unambiguously resolve the HCV genotype of some specifically challenging samples. Hence, more data is required to establish the lowest viral load that can be reliably detected by NGS for minor types [bib_ref] Mixed HCV infection and reinfection in people who inject drugs-impact on therapy, Cunningham [/bib_ref]. Finally, when focusing on the results from North-eastern Spain (Germans Trias i Pujol University Hospital), 5.7% of all genotypes 1 from 2009 to 2016 (76 out of 1322) were not classified at the subtype level by the GT II assay. Among them, 53/76 left-over plasma samples were available and analysed with the GT Plus assay in this study. In 42/53 (79.2%) the subtype was successfully resolved. Thus, we can estimate that the use of the GT Plus assay as a reflex test after the GT II assay would have decreased the overall ratio of HCV-1 samples in need of subtyping by the reference method from 5.7% down to 1.1%, which translates into a successful resolution of 98.9% of all HCV-1 samples when the two Abbott genotyping assays are combined. This corresponds well to the recently reported significant reduction of genotype 1 cases without subtype assignment down to 0.4% 33 and to the increase of the successful genotyping rate to >99% 11 . In conclusion, in the largest study focused on HCV-1 specimens not subtyped by the GT II assay, the GT Plus assay was able to assign the subtypes 1a and 1b in the majority of cases (88.8%), and subtype calling was highly reliable (98.6%) in comparison with sequencing or LiPA. The GT Plus assay is fully automated and can easily and rapidly be performed in the laboratory. Consequently, combined application of the GT II assay followed by the GT Plus assay using the Abbott m2000 platform represented an adequate approach which substantially reduced the number of ambiguous genotype 1 samples requiring subtyping by alternative methods down to 1.1%. www.nature.com/scientificreports www.nature.com/scientificreports/ Data Availability Protocols used are specified or referenced in the manuscript. NS5B and core sequences obtained by Sanger sequencing were deposited in the EMBL sequence database (http://www.ebi.ac.uk/embl). All commercial assays were used following the instructions of the manufacturers. The Abbott HCV GT Plus RUO assay is not commercially available. Data other than proprietary information can be provided on request. [fig] Figure 1: Flowchart diagram of the study design and methods used. GT, genotype; ST, subtype; GT II, Abbott RealTime HCV Genotype II assay; GT Plus, Abbott HCV Genotype Plus RUO assay; LiPA, VERSANT HCV Genotype 2.0 assay; mGT, mixed genotypes; mST, mixed 1a and 1b subtypes; NGS, next-generation sequencing. [/fig] [table] Table 2: HCV genotype and subtype results according to the genotyping method used (n). GT II, Abbott RealTime HCV Genotype II assay; GT Plus, Abbott HCV Genotype Plus RUO assay; LiPA, VERSANT HCV Genotype 2.0 assay; NGS, next-generation sequencing. [/table] [table] Table 4: Sequencing results in samples with mixed genotypes detected by the GT II assay. Málaga. *Combination of results of all 3 assays is in agreement with a recombinant 2k/1b (St. Petersburg) variant, however, no material was left for sequencing to confirm. **100% was not achieved because residual sequences appeared which were filtered and discarded, being each of them below 1%. [/table]
Lettuce (Lactuca sativa) productivity influenced by microbial inocula under nitrogen-limited conditions in aquaponics The demand for food will outpace productivity of conventional agriculture due to projected growth of the human population, concomitant with shrinkage of arable land, increasing scarcity of freshwater, and a rapidly changing climate. While aquaponics has potential to sustainably supplement food production with minimal environmental impact, there is a need to better characterize the complex interplay between the various components (fish, plant, microbiome) of these systems to optimize scale up and productivity. Here, we investigated how the commonly-implemented practice of continued microbial community transfer from pre-existing systems might promote or impede productivity of aquaponics. Specifically, we monitored plant growth phenotypes, water chemistry, and microbiome composition of rhizospheres, biofilters, and fish feces over 61-days of lettuce (Lactuca sativa var. crispa) growth in nitrogen-limited aquaponic systems inoculated with bacteria that were either commercially sourced or originating from a pre-existing aquaponic system. Lettuce above-and below-ground growth were significantly reduced across replicates treated with a pre-existing aquaponic system inoculum when compared to replicates treated with a commercial inoculum. Reduced productivity was associated with enrichment in specific bacterial genera in plant roots, including Pseudomonas, following inoculum transfer from pre-existing systems. Increased productivity was associated with enrichment of nitrogen-fixing Rahnella in roots of plants treated with the commercial inoculum. Thus, we show that inoculation from a PLOS ONE PLOS ONE | https://doi. [formula] a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 [/formula] # Introduction Sustainable food production has been on the rise in recent decades as traditional agricultural practices, which contribute to large-scale environmental degradation and enormous resource consumption, fall short of fulfilling the demands of our growing human population. Aquaponics offers a sustainable alternative to traditional food production methods by combining hydroponic plant cultivation with aquaculture in a semi closed-loop systemthat minimizes water and fertilizer use, increases agricultural efficiency, and does not require arable land. Central to the health of fish and plants in these systems are microorganisms, which drive many critical functions such as nitrogen cycling, plant growth promotion, disease resistance, and nutrient uptake; however, a deeper understanding of microbial community composition and function in aquatic agricultural systems is central to engineering and scaling-up efficient, sustainable food systems with low natural resource dependence. While some work has been conducted on microbial communities in hydroponicsand aquaponics, our knowledge of the microbial ecology of aquaponics is mainly grounded in soil-based agricultural research. Interest among researchers and growers in aquaponic microbes has been focused on initiating nitrogen cycling and promoting plant growth via inoculation with plant growth promoting microbes (PGPMs). For this reason, one of two inoculation strategies are traditionally used to initiate cycling: 1) addition of commercially-derived nitrifying bacteria (Nitrosomonas, Nitrobacter, and Nitrospira) or 2) transfer of established bacteria from existing, healthy aquaponic systems. Despite the inclusion of PGPMs, a 2018 international survey found that 84.4% of aquaponic growers observed disease in their systems, of which 78.1% could not identify the causal agent. Therefore, understanding the effect that microbial transfer has on production in aquaponics is crucial not only to establish best practices and increase commercial profitability by way of improving efficiency, but also to decrease loss due to disease. Of the growers who observed disease, a mere 6.2% used pesticides or biopesticides against plant pathogens and relied, instead, on non-curative actions, likely due to a lack of knowledge among aquaponic growers regarding disease control measures. Knowledge of key associations between microbial genera and productivity throughout early stages of system establishment could enable the development of diagnostic tools for monitoring microbiome composition, potentially aiding in early detection and prevention of system collapse. Institute for Systems Biology (ISB) established Project Feed 1010 (PF1010) to promote education and research around sustainable food systems, such as aquaponics, to help combat global food insecurity. Through ISB high school internships supported by the Seattle Youth Employment Program, small-scale aquaponic experiments were designed and carried out in collaboration with researchers at the National Aeronautics and Space Administration (NASA) Ames Research Center to test how two inoculation strategies impacted productivity. The Institutional Animal Care and Use Committee (IACUC) protocol limited the number of fish per system to prioritize animal welfare, which meant that our aquaponic systems were nitrogenlimited compared to commercial systems. We examined how microbiome transfer from either pre-existing systems or commercial inoculum promoted or impeded plant productivity. We compared lettuce (Lactuca sativa var. crispa) production in these two distinct systems-those inoculated with the biofilter media from an established, fully cycled aquaponic system ("established inoculum treatment" or "EIT") and those inoculated with a commercially-available microbial consortium ("commercial inoculum treatment" or "CIT"). ## Plos one # Results and discussion An overview of the aquaponics systems and experimental design is shown inOver the 61-day study period, lettuce growth (height, number of leaves, and root length) was significantly reduced in all EIT replicates compared to growth in CIT replicates. Physicochemical properties did not significantly differ across aquaponic systems over the study period (S1 individual Welch t-test p>0.07), making it unlikely that the observed growth disparity can be explained by system-wide biogeochemical parameters. Therefore, we explored potential associations between microbial community composition and plant growth parameters. We first investigated whether microbiome transfer affected the nitrogen transformation process. As anticipated, all systems showed low levels of nitrogen due to the limited number of fish allowed per system to maintain compliance with IACUC. One CIT system (tank 3) showed a transient nitrogen spike associated with the death of one fish. However, areas under the nitrate curves (AUNC) were not significantly different between EIT and CIT tanks (Welch t-test p = 0.3) and AUNC variance was explained slightly better by the number of dead fish in each tank (R 2 of 0.37 vs 0.23, see Materials and methods). Similarly, there was https://doi.org/10.1371/journal.pone.0247534.g001 no significant difference in the number of fish that died between CIT and EIT systems. Previous studies of established aquaponic systems found a ubiquitous presence of nitrifiers such as Nitrosomonas, Nitrobacter, and Nitrospira, albeit in low abundances, and these organisms are described as major drivers of plant growth. We only detected nitrifying taxa in the established inoculum itself, as well as in two EIT samples from days 21 and 61, respectively. Instead, biofilter samples were dominated by Proteo-and Cyanobacteria (S2. Even though our protocol was validated to detect nitrifiers (S3 we also found no nitrifiers in the commercial inoculum, which was dominated by Rhodanobacter, a genus containing known denitrifiers. This suggests that the improved plant growth in CIT systems was likely independent of nitrifying bacteria. We also observed low nitrogen levels in our endpoint plant nutrient analyses (S2 and water chemistry (S1 suggesting that nitrogen was limiting. This finding could explain the increased abundance of nitrogen fixers, such as Rahnella, and reduced abundance of nitrifying species. We hypothesize that in nitrogen-limited aquaponic systems, nitrogen fixing bacteria may play an important growth-promoting role in supplementing the limited ammonia produced by fish by fixing atmospheric nitrogen and producing additional ammonia, which is a well-known nitrogen source for L. sativa. In examining whether microbiome transfer affected establishment of microbial communities in new systems, we found alpha-diversity increased with time in all compartments and achieved similar values for CIT and EIT tanks in biofilters and roots at day 61. This temporal development of alpha-diversity was not an artefact of a bias due to sequencing depth. A total of 44% of variation in beta-diversity was explained by a combination of compartment (25%), inoculum (11%), and an interaction term of the two (8%; all PERMANOVA p values < 0.02). Conversely, prior studies in nitrogen-replete systems found that the microbial composition of different compartments in established systems, with the exception of fish feces, were quite similar. Given the differences in microbial composition between EIT and CIT systems, we hypothesized there may be a negative effect of microbial transfer from a prior system on plant growth, which acts independent of nitrogen concentration and cycling timeline. Thus, we investigated whether the difference in plant growth could be explained by an enrichment in potentially growth-inhibiting bacteria from the established system inoculum. This microbiome-inhibition hypothesis is similar to negative plant-soil feedback (NPSF), which has long been studied in both field and agricultural soils. NPSF is thought to be characterized by enrichment of species-specific plant pathogens or the accumulation of allelochemicals that limit plant productivity in successive generations grown in the same soil. This self-inhibitory process promotes plant community diversity by allowing sub-dominant species to thrive, and has served as a justification for crop rotations as a practice in soil-based agriculture. Thus, we hypothesize that a similar negative feedback phenomenon may be responsible for the reduced aquaponics productivity. In order to explore putative biotic mechanisms for the growth-promotion and negativefeedback hypotheses outlined above, we examined whether there were distinct bacterial genera in the plant roots that were associated with inoculum source and plant growth phenotypes. To that end we conducted negative binomial likelihood-ratio tests (LRT) between plant phenotypes and microbial abundances with DESeq2. Association tests between bacterial genuslevel abundances and plant growth measures revealed that three highly abundant genera in the roots, namely Pseudomonas, Rahnella and Serratia, were significantly associated (LRT FDR corrected p<0.05) with plant growthsee Materials and methods). Pseudomonas and Serratia relative abundances were higher in the EIT systems and were thus associated with diminished plant growth, whereas Rahnella was more abundant in CIT systems and was therefore associated with improved plant growth. Some isolated incidents of fish death were observed across all systems, though it is unclear if this led to altered nitrate abundances (area under the nitrate curve, Spearman rho = 0.73, p = 0.1). However, no bacterial abundances in the roots were correlated with fish mortality, ammonia, or nitrate levels (all FDR corrected p>0.1), suggesting that establishment of the rhizosphere microbiota was independent of fish mortality or system-wide nitrogen cycling. Although Pseudomonas, Rahnella and Serratia were all associated with plant growth metrics, only members of the genus Pseudomonas were 1) previously described as plant pathogensand 2) found in both initial inoculum types. Across time points, we observed a relatively uniform accumulation of Pseudomonas in biofilters, with all biofilter microbiomes consisting of 5-30% Pseudomonas. However, detection of Pseudomonas in the rhizosphere was highly dependent on inoculum type. EIT rhizosphere microbiomes were dominated by Pseudomonas (with 50-80% Pseudomonas, LRT FDR-corrected p<0.05), whereas CIT rhizosphere microbiomes contained less than 20% Pseudomonas. Instead, CIT rhizospheres were enriched for Aeromonas and nitrogen-fixing Rahnella, which were associated with improved plant growth. In summary, we saw two divergent but not necessarily mutually exclusive signals associated with inoculum type and aquaponic productivity. First, the genus Pseudomonas was enriched within biofilters across all systems, but only dominated the L. sativa rhizosphere in systems inoculated with microbes from an established aquaponic system, suggesting that EIC-specific growth-inhibitory pseudomonads may be responsible for a reduced yield in EIC-treated systems. However, our MinION sequencing data cannot resolve taxonomy beyond the genus level, so we are unable to verify whether or not specific bacterial pathogens were present in our systems. Furthermore, it is possible that viral, fungal, or other eukaryotic plant pathogens could be enriched in the established inoculum, as we did not collect data on non-bacterial microbiota in this study. Second, we saw enrichment of nitrogen-fixing Rahnella in CIT rhizospheres. Rahnella species are known to form associations with plant roots, promote plant growth, and have been found with other diazotrophs in nitrogen-fixing root nodules. Given the low levels of nitrate and ammonia in our aquaponic systems, Rahnella strains may have promoted L. sativa growth. Overall, further work is necessary to distinguish whether associations between aquaponics productivity and inoculum treatment are due to bacterial plant pathogens, growth-promoting microbes, or factors including other plant pathogens (e.g., viral, fungal, protist) or archaeal species (such as ammonia-oxidizing archaea), which were not quantified in our study. Additional studies with larger sample sizes, multi-omic data collection, and bacterial isolate characterization will be necessary to identify pathogenic or growth-promoting strains, establish causality, and test our mechanistic hypotheses. However, our findings indicate that CIT aquaponics systems showed greater yields when compared to EIT systems under nitrogen-limiting conditions and therefore that established inocula are not optimal for nitrogen-limited aquaponic systems. The decreased yield (an approximate 36-fold decrease in plant biomass; S1 observed in this study could be financially devastating to aquaponic farmers. While most commercial aquaponic systems are run under higher nitrogen levels than in our study, it is likely that other research institutions will follow similar animal welfare standards in future aquaponic studies and therefore nitrogen will be limited. Therefore, our work is an important step towards characterizing aquaponic systems operated for research purposes, where more controlled experiments can be performed compared to commercial systems. Moving forward, metagenomic analyses, metabolomics, strain isolation, and whole-genome sequencing will provide deeper insights into the specific strains, functional genes, and small molecules involved in both positive and negative plant-inoculum feedbacksand could lead to targeted strategies for engineering the microbial ecology of aquaponics systems to improve yield and resource use efficiency. # Materials and methods ## Aquaponic system design Six independent aquaponics systems were constructed using a 4-compartment design, with three replicate systems treated with a commercial inoculum and three with an established inoculum, in an open-air greenhouse in Seattle, Washington. These treatments will henceforth be labeled CIT (commercial inoculum treatment) and EIT (established inoculum treatment). Each system housed one aquaculture tank connected to one hydroponic unit utilizing the deep water culture method. These units were separated by a biofilter, which housed approximately 60 L of K3 filter media, as well as an 18 L solids filter. The experiment commenced when two adult Red Nile Tilapia (Oreochromis niloticus) were added to each system. On the same day, the 6 systems were divided into two sets of triplicate systems and inoculated with either Microbe-Lift commercial bacteria (tanks 1-3; "CIT") or established microbes from an existing aquaponic system (tanks 4-6; "EIT"). Four weeks later, 13 lettuce seedlings, which had been sprouted in rockwool cubes outside the systems 3 weeks earlier, were added to the floating trays (Beaver Plastics 28-hole 2ft x 4ft Lettuce Raft) in each system and allowed to grow 35 days. ## Animal care Prior on-site research conducted at the Institute for Systems Biology under IACUC protocol number NB.01a.16 ("Educational Aquaponics"; OLAW #A4355-01 and AAALAC #001363) provided guidelines that were closely followed in this off-site study. Water chemistry, space, and fish food were all determined and maintained to optimize living conditions for all Nile Tilapia (Oreochromis niloticus) used in this study, and fish health and behavior were observed and recorded regularly. Fish were fed Stage 3 Intermediate AquaNourish Omnivorous Fish Feed pellets (Star Milling Co., Perris, California, USA; 37% crude protein, 10% crude fat, 2.2% crude fiber, 11% ash) every other day in an increasing amount (between 10-40 pellets per fish) during nitrogen cycling, so as not to produce excess, toxic ammonia at initial stages of cycling. Each approximately 203 L system housed two fish with a total length of 44 inches. To maintain compliance with our IACUC protocol, which prioritizes animal welfare, only two fish per system were used (the sum of their lengths being approximately 44cm). Because this stocking density is lower than that of most commercial aquaponic systems, this experiment was run under relatively nitrogen-limited conditions. Throughout this study, 5 fish died unexpectedly due to unknown causes in tanks 1, 2, 3, and 6. Water chemistry was ruled out as a cause, as there were no associations between water chemistry fluctuations and subsequent fish deaths. One possible cause may be stress-induced infection by a fish pathogen such as Saprolegnia or Aphanomyces. Although 16S rRNA analysis is not capable of detecting these fungal pathogens, Pseudomonas and Aeromonas were increasingly enriched throughout the study. Both of these genera are known to include species which can mitigate oomycete diseases in aquaculture by acting as an antipathogen agent. Fish deaths were included as factors in our statistical analyses, to ensure that our reported results were independent of these adverse events. ## Inoculation of systems Systems in the CIT were inoculated with Microbe-lift (Cape Coral, Florida, USA) commercial bacteria, marketed as containing Nitrosomonas, Nitrobacter, and Nitrospira, whereas EITs were inoculated with biofilter media from a previously established, highly-efficient L. sativaproducing aquaponic system where nitrogen had been fully cycled for approximately 2 years. To inoculate these systems, 10 bioballs (approximately 105.8 ng/mL bacteria DNA) from the existing aquaponic system were collected and transferred to the biofilter of each new EIT system. On the same day, 30 mL of commercial bacteria (company recommendation) was added to the CIT biofilters. ## Water chemistry maintenance Throughout the study period, water chemistry (pH, temperature, ammonia, nitrite, nitrate, chlorine, hardness, and alkalinity) and plant growth metrics (number of leaves, plant height, root length, and presence of disease or discoloration) were collected 3-4 times per week (S1 Water chemistry data was collected using test strips (Tetra EasyStrips 6-in-1 Aquarium Test Strips and Tetra EasyStrips Ammonia Aquarium Test Strips) and a multi-parameter probe (Hanna Instruments 1 GroLine Waterproof Portable pH/EC/TDS Meter). To account for evaporation and plant use, an average of 126.5 L of aerated, dechlorinated tap water was added to each tank throughout the experimental period. Approximately 45g of Instant Ocean 1 Sea Salt (Blacksburg, VA) was added to each tank 5 days into the experiment to reach a conductivity of approximately 900 uS/cm. ## Lettuce phenotypes Height, number of leaves, discoloration, and pest pressure, were recorded for each plant every other day at a consistent time. Height was measured from the base of each stem to the natural crest of the tallest leaf. Leaf count included cotyledons and emerging buds. Root length was recorded at the midpoint and end of the experiment and was measured from the base of each rockwool cube to the longest root of each plant. ## Sample collection Microbiome samples were strategically collected throughout the 61 day study period and analyzed from 3 compartments of each system: 1) plant roots in the grow bed, where root-associated bacteria are located, 2) biofilter media in the biofilter where bacterial nitrifiers typically carry out nitrification, and 3) fish feces from the solids filter, where gut-associated bacteria can be found. Sampling time points were reflective of key chemical species transformations occurring during nitrification, and therefore supposed microbial community shifts, across the study period (pre-cycling, during cycling, and post-cycling;. The experiment ended 61 days after microbial inoculation and plants were harvested. At the end of the experiment, wet and dry mass (biomass) of plants were collected (S1 . Nutrient analysis of dried leaf samples was performed by the University of Missouri Soil and Plant Testing Lab (S2 . ## Sample processing Roots. Time point 0 for the roots was taken from rockwool cubes that seedlings were growing in prior to transfer to the aquaponic systems. Nine rockwool cubes were collected from the grow tray and all liquid was squeezed from the rockwool into a 50 mL falcon tube. Tubes were centrifuged at 4,000 xg for 10 min, supernatant was discarded, and pellets were flash frozen in liquid nitrogen and stored at -80C until DNA extraction. The final time point for the roots was taken at the time of system disassembly (and plant harvest). Roots were clipped at the base of all plants in each system and collected in 50 mL falcon tubes. PBS was added to the tube and samples were sonicated at low frequency (intensity 1) for 5 minutes total (five 30 second bursts, each followed by a 30 second rest period) in order to remove cells from roots. Following sonication, roots were removed from the tube and samples were centrifuged at 4,000 xg for 10 min, supernatant was removed, and pellets were flash frozen in liquid nitrogen and stored at -80C until DNA extraction. Fish feces. Fish feces were collected from the bottom of the aquaponic solids filter using a 25 mL serological pipette. Samples were centrifuged at 4,000 xg for 10 minutes to pellet the feces samples, supernatant was discarded, and pellets were flash frozen in liquid nitrogen and stored at -80C until DNA extraction. Biofilter. Ten bioballs were collected from the established aquaponics biofilter using sterile forceps and stored in PBS. A single bioball was vortexed at maximum speed for 2 minutes in a 50 mL falcon tube and then sonicated at low frequency (intensity 1) for 5 minutes total (five 30 second bursts, each followed by a 30 second rest period). Following sonication, the bioball was removed from the tube and another bioball was added in, vortexed, and sonicated. This process was repeated until cells from 10 bioballs were collected for each sample. Cells from the 10 bioballs were then collected as a single pellet through centrifugation at 4,000 xg for 10 minutes, supernatant was discarded, and pellets were flash frozen in liquid nitrogen and stored at -80C until DNA extraction. ## Analysis of nitrogen cycling Continuous nitrogen measures for each tank were reduced to areas under the curve (AUCs) using the trapezoidal method. To identify the dominant covariates associated with nitrogen cycling, nitrate AUCs were regressed against a binary inoculum covariate and the number of dead fish with a linear model in R (formula "nitrate~inoculum + dead_fish"). Explained variance of each term was obtained from an ANOVA analysis on the fitted model. ## Dna extraction, 16s rrna sequence analysis Microbial genomic DNA was isolated from samples using two similar extraction kits marketed specifically for bacterial DNA isolation from agricultural environments (Samples T0F1, T0F2, T0F3, T0F4, T0F6, and T0B with Qiagen PowerSoil kit and all others with PowerBiofilm kit). 16S rRNA genes were amplified using universal primers suggested by MinION (27F 5'-AGAGTTTGATCMTGGCTCAG and 1492R 5'-CGGTTACCTTGTTACGACTT). A MinION Nanopore Sequencer was used for sequence analysis. ## Sequencing Amplicons were aliquoted to a starting concentration of 1 ug and were further processed and sequenced according to ONT's 1D Native barcoding genomic DNA (with EXP-NBD103 and SQK-LSK108) protocol (v. NBE_9006_v103_revN_21Dec2016). Processing started with an AMPure XP bead purification step and proceeded to end-repair/dA-tailing, barcoding ligation, and adapter ligation steps. # Analysis Basecalling for the raw MinION files was performed by Albacore (v2.0.2) to yield the corresponding FASTQ files. Reads were processed using "filterAndTrim" methods from DADA2. The first 10 bp of the 5' end were trimmed from all reads as they generally showed lower qualities. Raw reads were also trimmed to a maximum length of 1.5 kbp (the expected length of the 16S gene). Reads with more than 200 expected errors under Illumina error model (based onor more than 2 ambiguous base calls ("N" bases) were removed from the analysis (~70% of all reads passed these filters). PCR chimeras were removed using yacrd version 0.5.1. However, yacrd removed very few sequences (<1%) because the prior length cutoff likely removed the majority of PCR chimeras. The filtered reads were then aligned to the complete SILVA 16S database using minimap2 with the Oxford Nanopore preset allowing up to 100 alternative alignments per read. ## Validation and mapping improvement for high error rates We observed acceptable read qualities with a median of 15 corresponding to an average error rate of 3% under the Illumina model. However, quality measures are based on a predictive model and the nominal error rate is usually above 10%. In order to evaluate the accuracy of nanopore sequencing on the full length 16S gene we used a small validation data set consisting of two biological replicates of a long-running established aquaponic system sequenced with the same nanopore protocol as well as V4-V5 Illumina amplicon sequencing. V4-V5 sequencing data (515F 5'-GTGYCAGCMGCCGCGGTAA, 926R 5'-CCGYCAATTYMTTTRAG TTT;were analyzed using the DADA2 pipeline and used as the ground truth. Due to the high error rate of nanopore sequencing we expected a lot of spurious assignments when mapping to a 16S reference database such as SILVA. In order to limit those spurious matches, we employed an Expectation-Maximization (EM) algorithm similar to what is used in kallisto but without correction for gene length due to the fixed length of the 16S gene(implementation available at https://github.com/Gibbons-Lab/mbtools). Applying the EM algorithm led to a reduction of unique mappings for low abundance cutoffs compared to a "naive" counting algorithm that just uses the highest scoring match (S3A We observed good agreement of estimated abundances between Illumina and Nanopore sequencing down to the genus level if the taxon was observed in both sequencing technologies (S3Bbetween 0.5-0.8, Spearman rho between 0.73-0.85, all ANOVA p<10 −6 ). However, we also observed many spurious mappings only present in the nanopore data, with generally low abundancesWe found that using an abundance cutoff of read 300 counts removed >96% of all spurious mappings across all taxa ranks and >97% of all spurious mappings on the genus rank. This cutoff did not depend on library size, which itself varied between~9.8K to~125K reads per sample. Thus we employed this abundance cutoff throughout the manuscript. The final abundances as estimated by the EM algorithm were then annotated by the SILVA taxonomy down to the strain level where available and collapsed on the genus level. We also verified whether the nanopore 16S primers were capable of identifying the same nitrifiers that were amplified by the V4-V5 primers used in the Illumina run. We found that all known nitrifying taxa identified by Illumina sequencing were also found in the nanopore data (S3D ## Diversity metrics Diversity estimates often depend on the library size (sequencing depth) of the sample. Library sizes ranged from~9.8K reads to >125K reads across samples. In order to rule out bias based on library size, we applied rarefaction to 9000 reads to all samples. We also used rarefaction curves to judge whether sampling depth was sufficient to saturate the alpha diversity measure, which we found to be the case for all samples (see plateau in S4 . ## Association testing Read abundances across samples were normalized using the DESeq2 "poscounts" normalization strategy (similar to a centered log ratio transformation). Association tests were performed using DESeq2 with a prior filtering step to remove bacterial genera with either very low average abundances or low prevalence across samples (mean abundance >10 across all samples and present in at least 2 samples). This more permissive filter was used to allow for effective shrinkage for DESeq2. Significance was judged by a negative binomial log ratio test (LRT) comparing the full model to a model lacking the association term. Finally, we only considered significant tests for those genera which showed abundances larger than the default cutoff of 300 reads in at least two samples. Association tests between bacterial genus-level abundances and response variables were performed for each of the 3 environments (biofilters, roots, fish feces). The tested response variables were 3 plant growth measures (height, root length, number of leaves), inoculum type (CIT, EIT), and putative confounders (number of dead fish, area under ammonia and nitrate curves). False discovery rate was controlled by the Benjamini-Hochberg method. No associations in the fish feces passed an FDR cutoff of 0.1. ## Supporting information
Primary Care Networks and Starfield’s 4Cs: A Case for Enhanced Chronic Disease Management # Introduction As the world ages at a rapid pace, the number of patients with chronic conditions is set to increase in tandem. Chronic diseases are commonly characterised by their requirement for long term follow-up with healthcare providers, association with functional impairment or disability and need for holistic management of the patient. Inevitably, the uptick in chronic disease load had led to an overwhelming burden on healthcare infrastructure and national health expenditures. This affliction is accrued from the systematic stress precipitated by higher bed occupancies, hospital readmission numbers and emergency medicine interventions. This perpetuating strain has created the catalyst to provide chronic disease management services for stable patients at the community level in order to free up health care resources at the tertiary care interface. Therefore, the defining features of primary care which encompass comprehensiveness, continuity and coordination make this setting well equipped for community management of patients with chronic diseases . Importantly, shifting stable chronic cases to the primary care space for long term management is timely and cogent. Multiple studies had elucidated that health service expenditure reduction and overall more equitable health outcomes are derived when patients with chronic conditions are firmly anchored with their primary care providers. Essentially, prescient health policy manoeuvres that focus on enhancing primary care capacities for chronic disease management will generate multi-fold benefits for the healthcare system and should be explored in detail. ## Singapore's primary care landscape For many countries, primary healthcare is heralded as the bedrock of their healthcare systems, and Singapore is no exception. Although, in 2014, Singapore was ranked first for its efficiency in healthcare delivery, the strength of its primary care did not fare well when healthcare experts applied Barbara Starfield's seminal primary care framework. Therefore, being ranked as a country with one of the highest life expectancies in the world, with a greying population forecasted to swell to one in four by 2030 and one in two by 2050, Singapore needs to upend and strengthen its primary healthcare system so as to contain tertiary healthcare utilisation, improve continuity of care and overall health outcomes. At present, Singapore's primary care sector is divided between private general practitioners (GPs) and polyclinics which are government-subvented primary care entities. Polyclinics are multi-doctor (usually more than 10) clinics that provide a comprehensive range of services for the family, functioning as a one-stop care centre for acute and chronic conditions. In contrast, private GPs are solo practices with limited services. Currently, 1700 private GP clinics and 20 polyclinics operate island-wide. In terms of primary care utilisation, private GPs account for 80% of all primary care attendances, out of which only 20% are for chronic disease management. On the other hand, polyclinics fulfil 52% of chronic disease attendances, while government tertiary hospitals manage the remaining. With the imbalance in chronic disease attendances afflicting the polyclinics and tertiary sectors coupled with an increasing chronic disease burden, the primary care networks (PCNs) were commissioned by the Ministry of Health (MOH) as an enhanced mainstream primary care model in 2018 to mobilise more private primary care sector resources. This was envisaged through the voluntary enrolment of GP practices into a PCN of their choice. As of November 2020, a total of 561 private GP practices have been enrolled. In the PCN architecture, private GP practices are given the autonomy to allocate financial and manpower resources provided by MOH and dictate the internal workings of the network with two GP leaders at the helm. Such a ground-up approach to the PCN model enabled operational variegation to suit the specific characteristics of the different networks of GPs and their patients. Currently, there are ten PCNs; each a cluster of hitherto independent and separate GP practices working more closely together to offer extended care services for patients with complex chronic conditions. ## Starfield's "4cs" of primary care There is a general agreement in primary care that the four core pillars enshrined in Starfield's "4Cs" primary care framework, or more commonly referred to as the "4Cs", are associated with patient-centred services, cost containment, reduction in unequal access to medical care and an overall improvement in population health. Starfield explicated these four attributes as: C1: "Comprehensive care", which expands on the availability of a wide range of services in primary care to cater to a spectrum of health conditions. Offering a comprehensive scope of services permits a practice to provide health promotion, prevention, diagnosis and treatment services for a range of health conditions throughout a patient's life course. This can be enabled by building teams of professionals including GPs, registered nurses and allied health professionals that are based in the primary care space. Primary care teams can reduce the need for specialist referrals and services particularly when specialty services are made accessible at the primary care level. If the services are unavailable, the team can refer patients to community-based options to augment the existing primary care suite of services to provide sufficient breadth and depth of service coverage for holistic patient-centred care in the community. C2: "First Contact of care" which refers to care first sought from the primary care provider (i.e., services must be accessible and used by the population each time new health or medical need arises). Broadly, first contact access is widely associated with primary care providers being the main point of entry and subsequently, the preferred point of contact with the health system. First contact opens the doors to the rest of the health system and coordination with other health entities permits that, especially when a gatekeeper role is mandated. If prompt management of complications is required, GPs will consequently refer patients to higher levels of care. This has been shown to reduce overuse of specialist hospital resources and in turn, containment of ambulatory care expenditure. C3: "Coordination of care", which discusses the linking of health care visits and services so that patients receive appropriate care for all their health problems. Chronic diseases often requisites a wide range of services, some of which fall outside the ambit of primary care. This makes the transition of patients across care venues a requirement that is buttressed on seamless coordination functions. Studies have highlighted that facilitating inter-organisational and interprofessional care planning for patients is capacitated by mechanisms such as a shared electronic medical record platform and joint clinical case conferences between providers at different care interfaces. Harmonisation of cross level service delivery will enable both patients and providers to efficiently navigate within and beyond the province of primary care. C4: "Continuity of care", which describes the longitudinal use of a regular care provider, fostering a provider-patient relationship over time. Studies have discerned that continuity of care embraces three main forms, namely relational, informational and managerial which commonly results in longitudinal and personal continuity between GPs and patients. A strong patient-GP relationship is instrumental in improving the uptake of preventive care and enhance adherence to treatment. On the contrary, a lack of connected and coherent care had been shown to be associated with a higher risk of mortality, driving the need to build strengthen therapeutic relationships between patients and their GPs. This is particularly so for patients with chronic conditions like diabetes, where constant monitoring and follow-up with a care provider can potentially circumvent further complications. Since Starfield first articulated them in 1992, the "4Cs" have been used to design and plan primary care systems and develop novel ways of measuring primary care models such as the Patient-Centered Medical Home in the United States and Canada. After the first "4Cs" were well-established, other similar paradigms started emerging, using the original "4Cs" as cornerstones. The less well-known "10Cs", which include competence, cost-effectiveness, communication, collaboration, compliance and competing interests, were, to an extent, an offshoot from their parent framework. As primary care took centre stage in most health systems, an expanded version of the "4Cs" framework was developed, pivoted to evaluate the Patient-Centered Medical Homes. This became coined as the "9Cs" of accountable primary care, which moved beyond the medical home model. Again, additional attributes such as physician creditability, collaborative learning, cost-effectiveness, capacity expansion and career satisfaction were added to Starfield's original pillars of exemplary primary care. Notably, other primary care frameworks were also employed to assess the performance of similar primary care structures. For example, in Bodenheimer's ten building blocks of high performing primary care, a practical conceptual model was employed to evaluate PCMH. The model's foundation was built on Starfield's "4Cs", with other building blocks such as engaged leadership, data-driven improvements and patient-team partnerships that support the four essential functions of primary care. On the ground evaluation of a primary care system necessitates a practical evaluation tool, in this case, the Primary Care Assessment Tool (PCAT), a publicly owned instrument implemented by the World Health Organization that empirically measures essential primary care attributes, which are organised around the fields embraced by Starfield's "4Cs" while capturing other salient domains, such as family orientation, community associations and cultural relevance. Fundamentally, Bodenheimer's ten building blocks and PCAT, both of which are widely accepted and adopted, have the "4Cs" at their nexus, making Starfield's framework a suitable and validated one to be deployed for our research. With the advent of an increasing chronic disease burden, pathways to health system gains through cost containment, better health outcomes and more equitable access are widely accepted to be attainable through a primary care system which enshrines Starfield's "4Cs". To our knowledge, there are only two published quantitative studies conducted to date that evaluates the effectiveness of PCN in Singapore, both exclusively for diabetes management, and no prior study with an emphasis on mapping the characteristics of the PCN to the "4Cs" framework had been conducted despite the recent promulgation of the PCN. To fill the gap in research, this secondary qualitative analysis study aims to map the operational characteristics of PCN to Starfield's "4Cs" of high performing primary care and, in doing so, provide deep understanding of how PCN empowers GPs to manage patients with chronic conditions through the attainment of the "4Cs". # Methods ## Study design A secondary qualitative analysis was performed using qualitative data obtained from an earlier study exploring the perceived barriers and facilitators experienced by private GPs enrolled in PCNs. Consequently, it was considered valid to re-examine the collected data and explore how PCN manifests the "4Cs" when managing patients with chronic conditions. We used supplementary analysis, a specific type of secondary qualitative analysis for this study, which helps address aspects of data which were either not addressed or only partially addressed in the original study. In turn, enabling the research team to thematically analyse the data set from a different angle. ## Sample As the focus of this study was on mapping the operational characteristics of the PCN, we purposefully selected the transcripts of all the GPs (n = 30) who were involved in the original study. Within our sample, three were female and 27 were male. Our participants' age ranged from 31 to 68 years, with an average of 49 years and years of experience practising in primary care ranged from 3 to 35 years, with an average of 18 years. In addition, participants were recruited from 8 out of 10 PCNs in Singapore. ## Recruitment and data collection of the originalsstudy Participants from the original study were recruited using purposive and snowball sampling techniques. The inclusion criteria of sample necessitated that participants had to be private GPs enrolled in a PCN and practising at the time of interview. Prospective participants were contacted using their contact details such as email addresses and clinic telephone numbers which were made publicly available on public domain websites. For the original study, the interviews which were audio-recorded were conducted at a conducive location after signed informed consent was obtained from the participants. The interview guide used was piloted tested with two GPs before implementation. The feedback obtained from the pilot tests were used to iteratively modify the interview guide in terms of the questions' objectivity, brevity and tone, such that they could adequately draw relevant and in-depth responses from the main pool of participants when formally implemented. Overall, the interview guide comprised of questions on the present status of Singapore's primary care landscape and the PCN (Supplementary Material: Topic guide). No reimbursement was provided for participation in the study. Thirty private GPs participated in our interviews lasting 40 to 90 min and detailed field notes were also collected during the interviews. All interviews (which were conducted in English) were transcribed verbatim by a professional transcriptionist and assessed for accuracy by the study team. After the interviews, the audio recordings and subsequently, audio transcripts were anonymised by removing potentially identifying information such as names of clinics and GPs and replacing them with pseudonyms to ensure that the data could not be traced to the participants. Member checking, whereby interview transcripts were sent back to participants to be reviewed, was not performed. Consolidated criteria for reporting of qualitative research (COREQ) checklist was used throughout this study (Supplementary Material: . ## Ethics approval Ethics approval was obtained from the National University of Singapore, Institutional Review Board (NUS-IRB) before starting the study. The NUS-IRB reference code is S-19-005. # Data analysis For this study, using Starfield's "4Cs" conceptual framework, we performed a framework analysis of the transcripts to derive themes salient to the "4Cs" of primary care. This procedure allowed us to map the operational features of the PCN to the criteria set forth by Starfield's primary care components. To ensure rigor, research team members from the original study (n = 4), as well as new research team members (n = 2) with fresh perspectives uninfluenced by the findings of the first study, were involved in the secondary qualitative data analysis using uncoded transcripts from the original study. Transcripts were analysed inductively using thematic analysis, and deductively using Starfield's '4C' framework with NVivo version 12 for Windows software (QSR International Pty Ltd. Version 12, Ottawa, ON, Canada). We followed a five-step framework analysis approach proposed by Ritchie and Spencer to allow themes that were relevant to the "4Cs" to surface. In brief, we first familiarised ourselves with the transcripts, coded aspects salient to our research question and organised codes into themes, namely the "4Cs", so as to map the codes and themes to domains featured in Starfield's framework. In order to increase trustworthiness, the research team would raise and discuss any disagreements regarding the themes and codes until a consensus was reached. To further improve interrater reliability, the same five-step approach was employed by all coders. Thereafter, final themes and codes were agreed among all the authors after multiple rounds of iterative feedback and thematic saturation was reached. In addition, the research team drew reference from the field notes collected in the original study to guide the reflexive process and to give context during the coding process. # Results ## Main findings Our results showed that the PCNs did fulfil most of the criteria set forth by Starfield's "4Cs" and have summarised them inbelow. The various aspects of the "4Cs" and suggested enhancements will be elaborated in our discussion. Our results showed that the PCNs did fulfil most of the criteria set forth by Starfield's "4Cs" and have summarised them inbelow. The various aspects of the "4Cs" and suggested enhancements will be elaborated in our discussion. C1: Comprehensive care. For every PCN, ancillary services were provided around the premises of the GP clinics, which were traditionally not made available to them due to the lack of economies of scale to provide services at low cost to patients. In addition, PCNs were free to choose a model of service delivery that suited their operational needs, and our data showed that the most common mode of service provision was through a roving team of nurses. Each practice was equipped with the capacity to offer such services, transforming these practices into a "one-stop-shop" for consultation, health screening and self-management education. The mandated ancillary services for each PCN were limited to diabetes mellitus care such as diabetic retinal photography (DRP), diabetic foot screening (DFS) and nurse counselling (NC). DRP and DFS detected early signs of diabetes progression while NC offered education to patients on disease-modifying behaviours and self-management strategies, thus offering timely interventions for patients at these clinics and in turn preventing the deterioration of patients' conditions. C1: Comprehensive care. For every PCN, ancillary services were provided around the premises of the GP clinics, which were traditionally not made available to them due to the lack of economies of scale to provide services at low cost to patients. In addition, PCNs were free to choose a model of service delivery that suited their operational needs, and our data showed that the most common mode of service provision was through a roving team of nurses. Each practice was equipped with the capacity to offer such services, transforming these practices into a "one-stop-shop" for consultation, health screening and self-management education. The mandated ancillary services for each PCN were limited to diabetes mellitus care such as diabetic retinal photography (DRP), diabetic foot screening (DFS) and nurse counselling (NC). DRP and DFS detected early signs of diabetes progression while NC offered education to patients on disease-modifying behaviours and self-management strategies, thus offering timely interventions for patients at these clinics and in turn preventing the deterioration of patients' conditions. "I am able to offer a one-stop service for my diabetic patients. So, when they come here [PCN GP Clinic], they will have their DRP, DFS done and they come for consultation with the doctor and even for nurse counselling. It is all done at that one stop [PCN GP Clinic]. So, the patients actually like it and there is very little reason for them to want to go elsewhere." (R48) The PCNs also operated on an amorphous model which enabled the provision of additional services, such as spirometry and Holter monitor tests, if proposed by the PCN and subsequently authorised based on MOH's discretion. The costs of these ancillary services are decided by each PCN; however, the prices that were charged were lower than market rate (what is charged at the polyclinics) and some PCNs even went as low as $1 to make these services more attractive to their price-sensitive patients. "We charge a very nominal pricing [for ancillary services] for PCN [ . . . ] a PG [Pioneer Generation, elderly] patient will pay $10 flat for any of these three services whether it's eye, foot or nurse counselling. It is just $10." (R36) "Our nurse counselling is $1. So, my patients cannot say no, right? In fact, for example, eye check [DRP], foot check , are all priced at maybe half to one third of the polyclinic fees. In fact, that is the truth. My patient looked at the polyclinics and said that polyclinics are more expensive than us." (R17) C2: First contact of care. The provision of ancillary services is linked to another essential tenet of good primary care (i.e., first contact), as patients have increased access to ancillary services at their neighbourhood GP clinics without having to travel further to other care venues. With the availability of ancillary services within clinic premises, GPs could also perform opportunistic screening for patients when they present for their acute consultation visits. "[ . . . ] some of them could be seeing us for common illnesses and we will ask them have you had your eye screened before? Then if they say never, I say why don't you have it done here? So that is where it gives us a chance to have a conversation starter with them." (R48) Furthermore, most GPs operate long clinic hours which extended into the night on weekdays and on weekends unlike their polyclinic counterparts, creating increased accessibility, especially for the working population. "Some of them [patients] are agreeable [to seek treatment from private GPs] because they can come after office hours." (R30) The PCNs would have nurses from the mobile ancillary services team available on the clinic premises to present the benefits of receiving ancillary services from a PCN clinic. This would include going through the cost benefits of using the PCN's ancillary services. Having a lower price point as mentioned above will also posture PCN GP clinics as a first choice when coming to receiving these ancillary services. C3: Coordination of care. The PCNs received funding from MOH to hire primary care coordinators (PCCs) to coordinate with GPs across the network to arrange for the ancillary services. PCCs also liaise with individual GP practices to coordinate with their patients and remind patients to attend ancillary services and return for follow-up consultations. Thus, the additional manpower provided to the PCNs could facilitate the reduction in patients' non-attendance for appointments. "The PCCs will follow up with the patients on their appointments, they will book their appointments and then bring the provider [ancillary service provider] to provide their service in our clinic." (R46) The GP clinics use varied types of electronic medical record (EMR) systems. Hence, the lack of an interoperable EMR system with other GPs within their network impeded patient information sharing across the system, which might lead to fragmented care and miscommunication among providers. Furthermore, some GPs were still using hardcopy records to keep track of their patient information which added to the difficulty in seamless record keeping for the entire network. "About five of them, initially out of the 30 [GP practices] were on paper and pen, but they are now in flight to convert to some form of electronic medical system." (R36) C4: Continuity of care. Every PCN must maintain a chronic disease registry (CDR) that promotes care continuity by tracking process and clinical outcome indicators, such as the number of follow-up consultations, types of ancillary services attended and clinical parameters such as glycated haemoglobin, blood pressure and cholesterol levels. The CDR served as a reminder mechanism for GPs to ensure that necessary tests, ancillary services and follow-ups are performed which would otherwise have been difficult to monitor amid a busy clinical day as a solo GP. This judicious monitoring of patients in turn ensures that the clinical parameters remain within the optimal range for their patients thus preventing potential deterioration and subsequent need for specialist care. The CDR further promoted continuity of care as GPs would receive Care Plus Fee (CPF) (monetary reimbursement of SGD 100 per patient per year) when specific CDR indicators, such as a minimum number of follow-up appointments, were fulfilled within a stipulated timeframe (usually within one calendar year). This quantum was geared to offset the extended consultation time required to manage a chronic patient. This is because the revenue generated by private GP practices is volume-based, making it more profitable for GPs to see more acute cases. However, complex chronic patients require a lengthened consultation. Hence, the CPF was introduced as a way to reimburse clinics for that trade-off in revenue generation. "[ . . . ] CDR reminds especially the private doctors when your clinic so busy, a lot of times we will overlook, or we will you know forget certain things. So, this, in a way, is a constant reminder to make sure that this has been done for the patient." (R26) "Everyone, every clinic, every single clinic in the PCN should be eligible for the Care Plus Fee, if there are patients who satisfy the criteria required. I think three visits per year or two or three visits per year with the necessary blood investigations [amongst other requirements] being done for them to be eligible for the Care Plus Fee." (R18) "It [Care Plus Fee] is basically a process indicator and regularity of follow-up [ . . . ] that was a carrot [financial incentive] to tell the GPs don't be afraid to see complex patients [ . . . ] to recognise the increased time that you [GPs] are spending with complex patients." (R36) In terms of healthcare financing, in the private primary care sector, the Community Health Assist Scheme (CHAS) was implemented as a means-tested finite portable medical subsidy to enable patients to receive medical subsidies at private GPs, thus reducing out-of-pocket payments. However, patients who suffer from multiple chronic conditions require more medications, placing a financial burden on them despite having CHAS subsidies. These patients tend to return to the polyclinics due to its heavily subsidised medications as compared to the full unsubsidised prices they pay at private GPs. Therefore, the adverse financial gradient between private GPs and polyclinics due to the discrepancy in medication pricing severed patients' continuity of care with the GPs. "The CHAS subsidies help, but it is for simple chronic illness. For simple cases, it may be comparable to the polyclinic. But when it comes to more medications, it makes it very difficult, even with the CHAS subsidy." (GP R48) "If you are talking about chronic patients here, then the distribution between private and public chronic patients are highly steered towards the government side [polyclinics] because polyclinics are offering such a high subsidy that it makes no sense for patients to follow up with private . Most of the patients that follow up with private [GPs] are simple chronic patients, meaning that they are probably on one or two chronic medications [ . . . ] So that means out-ofpocket [costs] will not be so high per year. But what happens when the disease starts to deteriorate or what happens when people start to age, and they need more and more medications to control their chronic diseases? Their out-of-pocket [costs] in primary care in private sector is going to get higher and higher until a point where all these patients are driven back to the polyclinics." (R26) # Discussion Our results showed that the features of the PCN had potential to achieve Starfield's "4Cs" of primary care, for the management of patients with chronic conditions to an extent. The provision of ancillary services, chronic disease registry and financial incentives capacitate the PCNs to enshrine similitudes of comprehensiveness, first contact, coordination and continuity of care. Unfortunately, the lack of an integrated EMR, insufficient CHAS subsidy quantum and adverse financial gradient favouring polyclinics (all of which fall out of the province of the PCN policy) had made it challenging for PCNs to fully manifest the "4Cs" of primary care. Taking a broad view, our findings also pointed to the delicate interactions of the "4Cs" within and beyond each other in terms of optimising GPs' delivery of high-value chronic disease care within the sphere of primary care. Hereafter we will elaborate in detail on each "C", while concurrently arguing the dynamic interplay between them to paint a fuller picture of how the "4Cs" manifest themselves in PCN. The "4Cs" may either enhance or inhibit one another. For example, providing comprehensive ancillary services at a considerably lower prices at PCN GP clinics, as compared to polyclinics, had enticed patients to view PCN GP clinics as a first option to receive these services, and return for not only their consultations but regular ancillary check-ups as well. These findings corroborate with a study by Ann et al., which highlighted that comprehensive care is associated with improved continuity of care. However, in Singapore, only, DRP, DFS and NC were the mandated ancillary services offered by the PCNs, due to its heightened need as the prevalence of diabetes and its related complications in Singapore were projected to double from 7.3% in 1990 to 15% in 2050 and from 12.2% in 2007 to 24.3% in 2035, respectively. Additionally, the total economic burden of managing diabetes is estimated to climb from one billion USD in 2010 to 2.5 billion USD in 2050. Hence, the Singapore government launched a "war on diabetes" to re-orientate the healthcare system to have a stronger focus on diabetes management. However, the present iteration of the PCN has limited the range of ancillary services to diabetes care, posing a shortcoming to attaining full comprehensiveness, as comprehensive care encapsulates services addressing both breadth and depth of coverage by the primary care team beyond diabetes care. Although diabetes poses a threat to health security and has received rapid prioritisation and funding, other non-communicable diseases should also be covered. Hence, a wider array of services such as physiotherapy and social prescribing is merited for the next iteration of PCN to provide a heightened level of comprehensiveness. Another example is, before enrolling into PCNs, GPs used to coordinate care with ancillary service providers by arranging appointments, referrals, and following up of test results. However, being able to offer comprehensive ancillary services within the clinic premises enable less care coordination events by GPs and concurrently enhances patients' convenience. Additionally, providing a comprehensive range of services is found to nurture relationship continuity. These findings are supported by a study conducted by Freeman et al., which showed that providing a range of ancillary services, phone consultations, advance appointments and quality of consultation were crucial to securing relationship continuity. However, it is also worth noting that a highly comprehensive clinic may involve multiple providers, which in turn could diminish the patient's sense of continuity. Care coordination for PCNs in Singapore is facilitated by PCCs with a more limited role than individuals (usually called care navigators) in other contexts. Rather than managing patients' follow-up services across different levels of care (i.e., between specialist units and primary care clinics), within the purview of the PCN, PCCs manage patients exclusively within the ambit of the primary care interface. However, small steps pave way to big changes. The provision of PCCs has fostered integration of care within the private GP sector, which had otherwise been fragmented, where GPs tend to practice in siloes. Additionally, an interoperable EMR system, teleconsultations and email consultation should be made available at all facets of care to nurture care coordination such that patient information can be shared seamlessly across different provider settings. Having an electronic presence can make PCN GPs more palatable to patients to use them as their first point of care and continual care for non-emergent and follow-up consultations. First contact increases patients' possibility to see the same healthcare provider and in turn enhance care continuity. Studies have shown that patients rank care continuity as the highest among the other attributes of primary care because a long-standing relationship helps to build trust. Having built a trusting relationship through care continuity with their GPs, patients will be more willing to adhere to physician recommendations. One way to enforce care continuity is via a gatekeeping mechanism. This concept primarily holds true for some western countries. However, it is not the case for Singapore. The country's small land area, availability of numerous healthcare facilities island-wide, a highly efficient inter-connected transportation system and historical preference of patients seeking primary care services with a provider of their choice could be reasons for not having a mandated gatekeeping role for GPs. On the other hand, studies have shown that patients develop relational discontinuity with their GPs when care continuity is enforced via gatekeeping. This is because a mandated gatekeeping mechanism potentially limits patients' healthcare choice to a single primary care provider and might result in delayed diagnosis and treatment. Our study findings illustrated that patients developed relational discontinuity with their GPs due to the adverse financial gradient between the public and private primary care sector. Inevitably, first contact and care continuity at the private primary care interface are disrupted because of this. Patients with complex chronic conditions requiring multiple medications were consequently prompted to return to the heavily subsidised polyclinics to enjoy lower out-of-pocket expenses. More importantly, without cultivating a long-standing trusting relationship with the GPs, patients will want to seek care from tertiary hospitals or polyclinics even for health problems within the realms of the GP sector. While it is crucial to strengthen continuity of care, instead of enforcing a gatekeeping mechanism, other approaches can be explored. For example, the adverse financial gradient between the public and private primary care sector can be partially surmounted by developing community pharmacies whereby patients obtain governmentsubsidised medications after consulting a PCN GP. Moreover, educating patients on the value and advantages of seeking the same primary healthcare provider can be promoted by the PCNs and also made more cognizant at the policy level. # Conclusions The implementation of the PCN is highly pragmatic and timely against the looming backdrop of an ageing population. Therefore, our study findings provide evidence on the capacity of the PCN to confer enhanced chronic disease management capacity to GPs by sufficiently meeting the criteria of Starfield's "4Cs" through the provision of a suite of ancillary services, heightened financial and physical accessibility to services at GP clinics, manpower to coordinate between practices and liaise with patients for follow-up and a registry to ensure care processes are diligently fulfilled. Even though certain aspects such as the lack of a common EMR system, insufficient government subsidies at private GPs and adverse financial gradient favouring public primary care require refinement, the empowering features mentioned above outweigh the shortfalls in all important aspects of providing optimal chronic disease management in a primary care setting. While this analysis was limited to data collected from the previous qualitative study, the data were suitably rich to allow us to gain meaningful new insights. Moreover, this study's research question fitted well with that of the original study, as both studies were concerned with chronic disease management in PCNs. However, as data were limited to the providers' perspective, we propose a future study to understand how features of the PCN fit well with Starfield's "4Cs" framework from the patients' angle. Integrating patients' perspective to tweak health delivery models unlocks opportunities to improve patient satisfaction, patient experience and overall health outcomes. Therefore, though still in its early stage of development, PCN has proven to be well positioned to drive private GPs towards enhanced chronic disease management. The funding organisations had no role in the study design, data collection and analysis, interpretation of the data, writing the paper and the decision to submit the paper for publication. Institutional Review Board Statement: Ethics approval was obtained from the National University of Singapore, Institutional Review Board (NUS-IRB) before starting the study. The NUS-IRB reference code is S-19-005. Informed Consent Statement: Written informed consent was obtained from all participants involved in the study. # Data availability statement: The data presented in this study are available on reasonable request from the corresponding author.
Cannabidiol regulates apoptosis and autophagy in inflammation and cancer: A review Cannabidiol (CBD) is a terpenoid naturally found in plants. The purified compound is used in the treatment of mental disorders because of its antidepressive, anxiolytic, and antiepileptic effects. CBD can affect the regulation of several pathophysiologic processes, including autophagy, cytokine secretion, apoptosis, and innate and adaptive immune responses. However, several authors have reported contradictory findings concerning the magnitude and direction of CBD-mediated effects. For example, CBD treatment can increase, decrease, or have no significant effect on autophagy and apoptosis. These variable results can be attributed to the differences in the biological models, cell types, and CBD concentration used in these studies. This review focuses on the mechanism of regulation of autophagy and apoptosis in inflammatory response and cancer by CBD. Further, we broadly elaborated on the prospects of using CBD as an anti-inflammatory agent and in cancer therapy in the future. # Introduction Cell death plays an important role in physiologic and pathophysiologic processes, implying that the pathogenesis of human diseases is closely related to this process. Cell death can be broadly divided into two categories, namely programmed cell death (PCD) and simple necrosis. PCD refers to the ontogenetic, preprogrammed, and tightly controlled death of certain cells in a multicellular organism. PCD includes apoptosis, programmed necrosis, and pyroptosis. Apoptosis is usually caused by some proapoptotic stimuli, such as endoplasmic reticulum stress and accumulation of reactive oxygen species. These stimuli activate several caspases and eventually cause a series of irreversible changes in cells. Morphologically, apoptosis is characterized by cell shrinkage, chromatin condensation, blebbing, and the formation of apoptotic bodies [bib_ref] ICP6 prevents RIP1 activation to hinder necroptosis signaling, Hu [/bib_ref]. Despite this, the cell membrane remains intact and no inflammation occurs . The process of apoptosis avoids the occurrence of inflammatory reactions and tissue damage to achieve a steady state while maintaining an internal environment and protecting the host. Autophagy is a phylogenetic degradation process that depends on the formation of specialized membrane structures including phagosomes, autophagosomes, and autolysosomes. Autophagy plays a complex role in maintaining health and developing diseases [bib_ref] Biological functions of autophagy genes: A disease perspective, Levine [/bib_ref]. Eukaryotic cells maintain homeostasis and renew themselves through autophagy, which is an evolutionarily conserved mechanism [bib_ref] Autophagy induction promoted by m6A reader YTHDF3 through translation upregulation of FOXO3..., Hao [/bib_ref]. Autophagy can be divided into macroautophagy, microautophagy, and chaperone-mediated autophagy according to the different routes of cellular material transported to lysosomes; macroautophagy is the most common type among them. Autophagy is thought to be a major factor in determining the fate of the cells and can trigger apoptosis. This implies that autophagy can also represent a form of programmed cell death known as autophagic cell death or type II programmed cell death. Three sequential steps are involved in intact autophagy: induction, autophagosome formation, and autophagosomelysosome fusion and degradation. Bulk and selective are the two modes of autophagy [bib_ref] Cannabinoid signaling in auditory function and development, Gatica [/bib_ref] , contributing to metabolic adaptation and cellular homeostasis, respectively [bib_ref] Autophagy at the crossroads of catabolism and anabolism, Kaur [/bib_ref]. Autophagy is closely associated with several human diseases, such as cancer, neurodegenerative diseases, and infectious diseases [bib_ref] The critical role played by endotoxin-induced liver autophagy in the maintenance of..., Chung [/bib_ref] [bib_ref] LC3/GABARAPs drive ubiquitin-independent recruitment of Optineurin and NDP52 to amplify mitophagy, Padman [/bib_ref]. The endocannabinoid system is a naturally occurring lipid signaling system intricately related to a range of physiologic and disease processes. The endocannabinoid system comprises endocannabinoid receptors, endocannabinoids, and metabolic enzymes responsible for regulating the synthesis and decomposition of ligands. The endocannabinoids mainly exist in two forms in the body, arachidonoylethanolamine (AEA) and 2arachidonoylglycerol (2-AG). Endocannabinoid receptors mainly exist in mammalian tissues with two types of G protein-coupled receptors, CB1 and CB2. CB1 is mostly distributed in the central nervous system [bib_ref] Cannabinoid signaling in auditory function and development, Gatica [/bib_ref] [bib_ref] Epigenetic regulation of adipogenesis in development of metabolic syndrome, Pant [/bib_ref] , including the amygdala, cortex, hypothalamus, hippocampus, and cerebellum; the proportion of CB1 in these locations is higher than in other locations. CB2 is mainly expressed in peripheral immune cells (lymphocytes, macrophages, and splenocytes). The endocannabinoid system is important in the regulation of the autonomic nervous system, immune system, and peripheral microcirculation. The theory of clinical endocannabinoid deficiency syndrome (CEDS), proposed by Russo, suggests that some chronic diseases may occur because of a lack of endocannabinoid signaling [bib_ref] The endocannabinoid system of animals, Silver [/bib_ref]. Therefore, whether the supplementation of exogenous cannabinoids can maintain the homeostasis of the body and improve the prognosis of some diseases is worth examining. Tetrahydrocannabinol and CBD are the most common cannabinoids used in medical cannabis preparations. However, the use of THC, which has psychoactive features, has adverse effects on the nervous system. THC-treated mice showed obvious but transient neurological changes, slow movement and breathing, hyperactivity, secondary immune dysfunction, and impaired ability to eliminate infection [bib_ref] Changes in body temperature and oxygen consumption rate of conscious mice produced..., Fitton [/bib_ref] ]. In contrast, CBD is a nonpsychoactive terpenoid and has a wide range of biological effects, including anti-inflammatory, anticancer, and neuroprotective effects. Therefore, CBD has been the main focus of research on bioactive compounds for treating psychological disorders. This review aims to describe the critical role of CBD in the regulation of apoptosis and autophagy. This information may be useful in analyzing the prospects of using CBD as an anti-inflammatory agent and in innovative cancer therapy for clinical use . ## Effect of apoptosis on the pathophysiology of inflammatory diseases Apoptosis is regarded as a non-inflammatory process, but the lytic nature of necrosis triggers the release of intracellular damageassociated molecular patterns and eventually leads to inflammation. Apoptotic signaling pathways are of two types: mitochondrialmediated (intrinsic) and death receptor-mediated (extra) pathways. In the death-receptor-mediated pathway, FasL binds to Fas, leading to the recruitment of various proteins and activation of downstream caspase-8, caspase-7, and caspase-3. The intrinsic pathway of apoptosis is regulated by the B-cell lymphoma-2 (Bcl-1) family of antiapoptotic (such as Bcl-2 and Bcl-xL) and proapoptotic proteins (such as Bad, Bid, Bax, and Bim). The caspase-8-mediated cleavage of Bid into its active form (tBid) leads to mitochondrial dysfunction and cytochrome c release, which activates downstream caspase-9 and caspase-3, thereby mediating cell death. Crosstalk between extrinsic and intrinsic pathways can occur because of the similar mechanism of eliciting apoptosis . These pathways are initiated by effector caspases and result in DNA fragmentation, cytoskeletal reorganization, cytoplasmic condensation, and the formation of apoptotic bodies . Several types of cells are affected in the course of inflammation. One of the important mechanisms of inflammatory response involves the imbalance of apoptosis regulation that leads to abnormal distribution or quantity of cell types. Consequently, it is vital to clarify the relationship between apoptosis and inflammation, especially in the context of the various cell types involved. ## Figure 2 The mechanism of cell apoptosis. The extrinsic pathway involves the recruitment and activation of procaspase-8, and activated caspase-8 then directly activates the effector caspases such as caspase-3 to initiate the execution process. The intrinsic apoptotic pathway is mediated by the cleavage of BID (BH3 interacting domain death agonist), a BCL-2 homology 3 (BH3)-only protein. Truncated BID (tBID) subsequently translocates to the mitochondria and activates the BCL-2 family members BAX and BAK. Upon activation, BAX and BAK induce mitochondrial outer membrane permeabilization and the release of proapoptotic mitochondrial contents into the cytoplasm, such as cytochrome c. Released cytochrome c binds APAF1, and APAF-1 recruits procaspase-9 through the CARD-CARD interaction and forms the apoptosome, leading to proximity-induced activation of caspase-9, which in turn cleaves and activates effector caspases (Members of the IAP family including XIAP negatively regulate caspase activation and they can be inactivated by SMAC). APAF1, apoptotic protease activating factor-1; Bax/Bak, a proapoptotic member of Bcl-2 family; BID, BID protein; FADD, Fas associated death domain; MCLl, myeloid cell leukemia-1; MOMP, mitochondrial outer membrane permeabilization; SMAC, second mitochondria-derived activator of caspases; X1AP, X-linked inhibitor of apoptosis protein. Apoptosis or programmed cell death is essential for cell homeostasis, injury repair, immune tolerance, and inflammation resolution in multicellular organisms [bib_ref] Short treatment of peripheral blood cells product with Fas ligand using closed..., Rodionov [/bib_ref]. When apoptosis occurs, the apoptotic cells are cleared by specialized phagocytes (macrophages and immature dendritic cells) or other cells (such as endothelial and mesenchymal cells). Apoptosis inhibits the transcription of pro-inflammatory cytokine genes, downregulates the expression of CD40L and CD40, silences CD4 + and CD8 + T cells, and promotes the secretion of anti-inflammatory cytokines . In the acute phase of sepsis or acute organ injury, excessive inflammation induces and aggravates apoptosis . Further, apoptosis continues to aggravate the inflammatory injury effect, eventually causing irreversible organ function damage. A report on acute liver injury suggested that excessive generation of reactive oxygen species not only directly damages cells but also recruits inflammatory cells and activates pro-inflammatory cytokines, both of which induce mitochondrial apoptosis . In a study on microvascular endothelial cell injury, oxidative stress markedly activated the cAMP response element binding protein, which enhanced the transcription and expression of IP3R and VDAC, resulting in increased Ca2+ content and the release of cytochrome c into the cytoplasm, thereby activating the mitochondria-dependent death pathway. Excessive oxidative stress, increase in inflammatory responses, and cellular calcium overload are upstream activators of mitochondria-dependent apoptosis. Improper clearance of apoptotic cells results in the occurrence and progression of several human chronic inflammatory diseases such as autoimmune diseases, including rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), neurological disorders, obesity, type 2 diabetes, and atherosclerosis. Correspondingly, disruption of inflammatory reaction also disturbs cell apoptosis. While treating autoimmune disease, the use of certain drugs can promote caspasedependent cell apoptosis and reduce IL-10 and IL-12 secretion. The use of non-steroidal anti-inflammatory drugs for treating inflammatory diseases may be associated with the activation of peroxigenic proliferator-activated receptor (PPAR)γ, which ultimately induces apoptosis. In patients with SLE, the accumulation of myeloid-derived suppressor cells (MDSCs) is positively correlated to disease activity. Hydroxychloroquine may relieve the symptoms of SLE inflammation by regulating the expression of CD81 in MDSCs and inducing their apoptosis [bib_ref] Hydroxychloroquine induces apoptosis of myeloid-derived suppressor cells via up-regulation of CD81 contributing..., Ni [/bib_ref]. Taken together, promoting the apoptosis of key cells in inflammatory diseases and reducing the secretion of inflammatory cytokines are crucial in alleviating inflammatory responses. The efficient execution of apoptotic cell death followed by the clearance of the debris by phagocytes is a key mechanism in maintaining tissue homeostasis. ## Autophagy is connected to the process of inflammatory disease Autophagy is a metabolic, cytoplasmic quality control, and homeostasis-maintaining process having cytoprotective, tissueprotective, and anti-inflammatory effects. Autophagy contributes to the removal of cell-damaging irritants or invading pathogens in adaptive and innate immune cellular processes, asepsis, and infection-related inflammation. The close association between inflammatory diseases (dysregulation of inflammatory responses in multiple diseases leading to histopathologic changes in several human organs) and alterations in autophagy was first identified from the studies on the association between genetic polymorphisms and disease susceptibility in humans . Genetic susceptibility to various diseases, such as asthma, RA, SLE, multiple sclerosis, and other neurological disorders, has been linked to mutations in autophagy genes [bib_ref] Study of the common genetic background for rheumatoid arthritis and systemic lupus..., Orozco [/bib_ref] [bib_ref] Functional variant in the autophagy-related 5 gene promotor is associated with childhood..., Martin [/bib_ref] [bib_ref] The autoimmunity-associated gene CLEC16A modulates thymic epithelial cell autophagy and alters T..., Schuster [/bib_ref]. Inflammasomes are cytoplasmic high-molecular-weight protein complexes that activate caspase-1 in response to inflammation triggered by microbial infection or injury. They comprise nucleotide-binding oligomerization domain-like receptor (NLR) family protein, adaptor apoptosis-related spot-like protein containing caspaserecruitment domain, and effector protease caspase-1. Activated caspase-1 converts pro-inflammatory cytokines IL-1β and IL-18 precursors into their biologically active forms and cleaves GSDMD to induce pyroptosis. Autophagy can eliminate agonists (such as damaged mitochondria) and components of NLRP3 inflammasomes and regulate inflammation. Autophagy removes damaged mitochondria and a complete autophagy process is necessary to prevent the release of endogenous inflammasome agonists (reactive oxygen species and oxidized mitochondrial DNA) [bib_ref] A role for mitochondria in NLRP3 inflammasome activationin: Nature, Zhou [/bib_ref]. Currently, several authors have linked the promotion of autophagy with the prevention of inflammasome activation. MEFV/TRIM20, a member of the TRIM protein family, recognizes the pro-CASP1, NLRP1, and NLRP3 through its SPRY domain to recruit major regulators of autophagy, namely ULK1, BECN1/Beclin 1, and ATG16L1. This process, termed precision autophagy, involves direct recognition of the degradative targets by the core autophagic machinery. Although the autophagic components have a general inhibitory effect on the inflammasome, they induce the unconventional secretion of proinflammatory cytokine IL1β into the extracellular environment. Therefore, autophagy (in its involvement with inflammasomes and their substrates) appears to play a balancing role in supporting productive inflammatory responses while preventing excessive inflammatory responses and tissue damage. However, the relationship between autophagy and apoptosis is difficult to define. Autophagy may coincide with apoptosis and promote cell death [bib_ref] Selfconsumption: The interplay of autophagy and apoptosis, Mariño [/bib_ref] [bib_ref] Aldehyde dehydrogenase 2 protects against lipopolysaccharide-induced myocardial injury by suppressing mitophagy, Ji [/bib_ref] or antagonize apoptosis to promote cell survival [bib_ref] PIM-2 is an independent regulator of chondrocyte survival and autophagy in the..., Bohensky [/bib_ref] [bib_ref] The AMPK/p27Kip1 pathway as a novel target to promote autophagy and resilience..., Mckay [/bib_ref]. A close correlation between inflammation and autophagy has also been reported, indicating that enhancing autophagy may alleviate inflammation. Autophagy dysfunction in neurons or glial cells is associated with neurodegenerative diseases [bib_ref] PIM-2 is an independent regulator of chondrocyte survival and autophagy in the..., Bohensky [/bib_ref]. In a study of lipopolysaccharide (LPS)-induced neuroinflammation in N9 microglial cells, LPS inhibited the expression of autophagy flux and Vps34 through the activation of PI3KI/AKT/mTOR pathway, whereas rapamycin injection enhanced microglia autophagy and downregulated LPS-induced neuroinflammation . The authors suggested that promoting the early stages of autophagy could be a potential therapeutic approach for neuroinflammatory diseases. Atherosclerosis is a complex chronic disease caused by the formation of atherosclerotic plaques. The disease is characterized by the upregulation of endothelial dysfunction, leading to an inflammatory response [bib_ref] Genistein protects against ox-LDL-induced inflammation through MicroRNA-155/SOCS1-mediated repression of NF-ĸB signaling pathway..., Zhang [/bib_ref]. In a study on atherosclerosis, lncRNA-FA2H-2 alleviated the inflammatory responses induced by oxidized-low-density Frontiers in Pharmacology frontiersin.org 04 lipoprotein through autophagy flux induction [bib_ref] The role of the LncRNA-FA2H-2-MLKL pathway in atherosclerosis by regulation of autophagy..., Guo [/bib_ref]. In addition, activation of autophagy in mesenchymal stem cells (MSCs) decreased the concentration of inflammatory cytokines when cocultured MSCs regulated the recruitment and polarization of CD4 + T cells in vitro. In an in vivo study of whether microvesicles of MSCs can act on acute lung injury, investigators demonstrated that MSC microvesicles enhanced autophagy and partially alleviated acute lung injury by delivering miR-100 . Therefore, autophagy is hypothesized to be a key regulator of MSC-mediated immune regulation, which may be a potential new strategy to improve the efficacy of MSC-mediated therapy [bib_ref] Autophagy enhances mesenchymal stem cell-mediated CD4+ T cell migration and differentiation through..., Cen [/bib_ref]. In an animal inflammatory model of non-alcoholic steatohepatitis (NASH), ezetimibe ameliorated hepatic steatosis, inflammation, and fibrosis by inducing AMPK-mediated autophagy activation. Human nonalcoholic fatty liver or NASH liver shows decreased autophagic vesicle formation, impaired autophagy pathway, and decreased nuclear TFEB expression. Ezetimibe-induced autophagy markedly blocked NLRP3-inflammasome activation and subsequent IL1β release in macrophages . Overall, autophagy is closely related to the occurrence of inflammatory reactions, and autophagy dysfunction may induce inflammation. Enhancing autophagy can alleviate inflammation and reduce the level of inflammatory cytokines. While autophagy and inflammation are interdependent processes, macroautophagy is associated with most of the reported suppressive effects on inflammation, and the implications of other types of autophagy in inflammatory reactions are unclear. ## Cannabinoid ameliorates inflammatory diseases by regulating apoptosis Cannabis contains various complex cannabinoids, such as tetrahydrocannabinol (THC) and CBD, which affect inflammatory and lymphocyte activation pathways in the lymphoid tissue and brain. Therefore, it is increasingly being used in clinical settings. The use of cannabinoids in treating several diseases has been extensively studied in Western countries. Several studies on viral infections have revealed that cannabis inhibits pro-inflammatory pathways in HIV-infected adults and non-human primates infected with SIV [bib_ref] HIV-infected cannabis users have lower circulating CD16+ monocytes and IFN-γ-inducible protein 10..., Rizzo [/bib_ref] [bib_ref] Cannabinoids and inflammation: Implications for people living with HIV, Costiniuk [/bib_ref] [bib_ref] Cannabis exposure is associated with a lower likelihood of neurocognitive impairment in..., Watson [/bib_ref]. Apoptosis caused the depletion of CD4 + and CD8 + T cells in HIV-1 infection, leading to an increased risk of infection and increased mortality. In a cohort study of patients with HIV, increased CD4 + and CD8 + T cell counts were associated with the use of cannabinoids [bib_ref] Confirmed marijuana use and lymphocyte count in black people living with HIV, Keen [/bib_ref]. In an animal model of SIV-infected rhesus monkeys, the apoptosis of intestinal lymphocytes was reduced after THC treatment, suggesting that chronic THC administration can regulate duodenal T-cell populations, promote Th1/Th2 cytokine balance, and reduce intestinal cell apoptosis [bib_ref] Modulation of gut-specific mechanisms by chronic δ(9)-tetrahydrocannabinol administration in male rhesus macaques..., Molina [/bib_ref]. Overall, cannabinoids exert their classical anti-inflammatory effects on the cells of monocyte lineage in HIV infection while protecting T lymphocytes from apoptosis. Ulcerative colitis (UC) is a chronic immune-mediated inflammatory bowel disease that involves the dysfunctional immune response to the normal microbiota and dietary contents in the gastrointestinal tract. The inflammatory cascade activates T cells, which secrete excessive amounts of pro-inflammatory cytokines, including IL-1, IL-6, and TNF-α, thereby damaging healthy tissues. Endoplasmic reticulum stress and unfolded protein reaction are closely related to cell apoptosis, autophagy, and inflammatory reaction in the pathogenesis of UC [bib_ref] The unfolded protein response: Controlling cell fate decisions under ER stress and..., Hetz [/bib_ref] [bib_ref] Epithelial ER stress in crohn's disease and ulcerative colitis, Cao [/bib_ref]. Severe or persistent endoplasmic reticulum stress eventually triggers the internal apoptosis pathway, leading to cell death and aggravating UC [bib_ref] The endoplasmic reticulum stress sensor IRE1α in intestinal epithelial cells is essential..., Zhang [/bib_ref]. In an in vitro study on inflammatory bowel disease, CBD reduced the production of reactive oxygen species and lipid peroxidation in cells, thereby reducing the occurrence of apoptosis. Meanwhile, some authors suggest that CBD may indirectly activate the CB1 receptor in the intestine, thereby reducing intestinal motility and relieving UC symptoms [. Taken together, although there is definite evidence that CBD has a therapeutic effect in autoimmune diseases, its action mechanisms need to be explored further. Several authors have confirmed that A2A, A2B, and A3 receptors participate in the antiapoptotic effect of ATP through MEK/ERK1/2, PKA, and NOS pathways. The selective antagonists of A2A, A2B, and A3 receptors limit the antiapoptotic effect of ATP against hypoxia. In the LPS-induced acute lung injury animal model, CBD reduced the migration of leukocytes to the lung, thereby decreasing the concentration of albumin and pro-inflammatory cytokines in bronchoalveolar lavage fluid. The use of an A2A antagonist restored the inflammatory response, suggesting that CBD may play a role by binding with the A2A receptor [bib_ref] Cannabidiol, a non-psychotropic plant-derived cannabinoid, decreases inflammation in a murine model of..., Ribeiro [/bib_ref]. In addition, in the inflammatory model of demyelinating disease induced by encephalomyelitis virus, the application of CBD reduced leukocyte infiltration and activated microglia in the cerebral cortex, suggesting that CBD can limit inflammatory response by increasing adenosine signal transduction [bib_ref] Neurological benefits, clinical challenges, and neuropathologic promise of medical marijuana: A systematic..., Longoria [/bib_ref]. Overall, we suggest that CBD can inhibit inflammation; however, whether it plays an anti-inflammatory role by binding to adenosine receptors to inhibit apoptosis needs further investigation. Liver fibrosis is the basic pathologic change in the liver caused by chronic liver diseases. The activation of hepatic stellate cells (HSCs) leads to the accumulation of scar matrix and fibrotic liver. A basic study on HSCs revealed that CBD induced downstream activation of the IRE1/ASK1/c-Jun N-terminal kinase pathway that promoted apoptosis, leading to HSC death. The authors suggested that CBD can be used as a potential therapeutic agent for chronic hepatitis that leads to liver fibrosis by selectively inducing apoptosis of activated HSCs [bib_ref] Cannabidiol causes activated hepatic stellate cell death through a mechanism of endoplasmic..., Lim [/bib_ref]. ## Cannabinoids promote autophagy and ameliorate chronic inflammatory diseases Autophagy, a lysosomal catabolic process, is critical to cell homeostasis and is crucial to the neuroprotection of the central nervous system. Autophagy defects are observed in many neurodegenerative diseases [bib_ref] Fat cadherins in mouse models of degenerative ataxias, Baron [/bib_ref]. However, abnormal autophagy activation may aggravate the extensive ischemic and inflammatory processes caused by nerve traumatic diseases [bib_ref] Fibroblast growth factor 21 facilitates peripheral nerve regeneration through suppressing oxidative damage..., Lu [/bib_ref]. CBD, a non-psychoactive cannabinoid, is becoming a promising therapeutic agent for mental disorders and inflammatory diseases [bib_ref] Cannabidiol, neuroprotection and neuropsychiatric disorders, Campos [/bib_ref]. CBD has antidepressant , antiemetic [bib_ref] Synergy between cannabidiol, cannabidiolic acid, and Δ⁹-tetrahydrocannabinol in the regulation of emesis..., Rock [/bib_ref] , neuroprotective [bib_ref] Protective effects of cannabidiol against hippocampal cell death and cognitive impairment induced..., Schiavon [/bib_ref] [bib_ref] Protective effects of cannabidiol on lesion-induced intervertebral disc degeneration, Silveira [/bib_ref] , anticonvulsant, Frontiers in Pharmacology frontiersin.org antianxiety [bib_ref] Neural basis of anxiolytic effects of cannabidiol (CBD) in generalized social anxiety..., Crippa [/bib_ref] , and antipsychotic effects [bib_ref] Cannabidiol as a potential new type of an antipsychotic. A critical review..., Rohleder [/bib_ref]. CBD has pharmacological properties in the treatment of neurological diseases. Therefore, the mechanism of CBD-mediated potential autophagy activation needs to be explored further. The neuroprotective effects of autophagy occur because it reduces the effects of inflammation. Neuronal cell death caused by chronic inflammation, which is induced by dysfunctional mitochondria, is considered the main cause of neurodegenerative diseases [bib_ref] Mitochondrial dysfunction in the development and progression of neurodegenerative diseases, Johnson [/bib_ref]. When mitochondrial dysfunction occurs in cells, accurate autophagy can prevent the occurrence of chronic inflammation and delay degenerative diseases. PPAR agonists restore autophagy, enhance mitochondrial ß-oxidation, and stimulate mitochondrial biosynthesis; therefore, they can be potentially used to treat patients with steatohepatitis (caused by glycogen storage disease type Ia) [bib_ref] A liver-specific thyromimetic, VK2809, decreases hepatosteatosis in glycogen storage disease type Ia, Zhou [/bib_ref]. CBD mediates the functions regulated by PPARα and ? receptors and plays neuroprotective, antiinflammatory, and metabolic roles. PPARγ agonists have been shown to reduce inflammatory processes associated with chronic and acute nerve injury [bib_ref] Mechanisms of anti-inflammatory and neuroprotective actions of PPAR-gamma agonists, Kapadia [/bib_ref]. CBD can bind and activate PPARγ in vitro [bib_ref] Critical role of mast cells and peroxisome proliferator-activated receptor γ in the..., Hegde [/bib_ref] ; it can also protect rats from neurological and inflammatory damage caused by ß-amyloidosis [bib_ref] Cannabidiol reduces Aβ-induced neuroinflammation and promotes hippocampal neurogenesis through PPARγ involvement, Esposito [/bib_ref]. In addition to its role in inhibiting inflammation, some studies have evaluated whether CBD treatment is feasible for chronic inflammatory pain. In another study on neurodegenerative diseases, CBD played an important role in autophagy activation by regulating ERK1/2 and AKT kinase phosphorylation and participating in ULK1 [bib_ref] Cannabidiol induces autophagy via ERK1/2 activation in neural cells, Vrechi [/bib_ref]. In this study, low doses of CBD promoted autophagy flux without affecting cell viability, and CB1, CB2, and TRPV1 receptors mediated CBD-induced autophagy, which is crucial to maintain neuronal activity and function. CBD plays a role in the treatment of neurodegenerative diseases by promoting autophagy, participating in homeostasis, and regulating the circulation and degradation of cellular proteins through the lysosome degradation pathway; however, it exerts the opposite effect in autoimmune diseases. RA is characterized by an anoxic environment in the joints accompanied by mitochondrial dysfunction. The compounds that inhibit autophagy (chloroquine or hydroxychloroquine) are being clinically used to treat RA. In an animal model of RA chronic inflammation, CBD showed antiinflammatory and analgesic effects. In some studies, immune cells and synovial fibroblasts were reduced following CBD treatment to alleviate inflammation [bib_ref] Transdermal cannabidiol reduces inflammation and pain-related behaviours in a rat model of..., Hammell [/bib_ref]. In addition, CBD may also interact with antirheumatic drugs, such as methotrexate or JAK inhibitors to increase the absorption of cytotoxic chemotherapy drugs [bib_ref] CBD loaded microparticles as a potential formulation to improve paclitaxel and doxorubicin-based..., Fraguas-Sánchez [/bib_ref]. CBD is feasible as an adjuvant treatment for RA and can be used in combination with other antirheumatic drugs. The underlying mechanism may involve the binding of CBD to β2 adrenergic receptors and then activating downstream ß-arrestin2 to inhibit autophagy [bib_ref] Joints for joints: Cannabinoids in the treatment of rheumatoid arthritis, Lowin [/bib_ref] ]. ## Effect of apoptosis on the progression of cancer The occurrence and development of cancer represent the imbalance between cell proliferation and cell death, indicating that the rate of proliferation and mutation of tumor cells exceeds that of dead cells. The early understanding of cancer mainly revolved around cell oncogenes, cell proliferation, and cell transformation. Apoptosis can be induced as a part of cancer treatment because of the detection of DNA fragmentation and changes after chemotherapy [bib_ref] Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation, Wyllie [/bib_ref]. Cell apoptosis is a common tumor inhibition mechanism. The molecular mechanism of apoptosis has been elucidated in detail. The key is to activate caspases, which can activate the internal pathway through mitochondrial outer membrane permeabilization or activate the death receptors, such as Fas, DR4, and DR5, on the cell surface by their death-inducing ligands (FasL and TRAIL). In recent decades, research on cancer treatment has mainly focused on the development of drugs and radiotherapy to increase tumor cell death, reduce tumor volume, and block invasion. Although many survivalpromoting pathways have become established drug targets in oncology, it is significant to recognize that drugs ultimately induce apoptosis through the core apoptosis pathway [bib_ref] Apoptosis and molecular targeting therapy in cancer, Hassan [/bib_ref]. The discovery of the BCL-2 gene in patients with follicular lymphoma can be instrumental in controlling cancer growth by promoting cell apoptosis [bib_ref] BCL2 and miR-15/16: From gene discovery to treatment, Pekarsky [/bib_ref]. Moreover, FDA has approved a BCL-2 targeted drug that promotes cell apoptosis and controls the growth of cancerous cells. Selective Bcl-2 inhibitors were used in a clinical study of leukemia or non-solid tumors [bib_ref] Targeting Bcl-2/Bcl-XL induces antitumor activity in uveal melanoma patient-derived xenografts, Némati [/bib_ref] [bib_ref] Found in translation: How preclinical research is guiding the clinical development of..., Leverson [/bib_ref] [bib_ref] S55746 is a novel orally active BCL-2 selective and potent inhibitor that..., Casara [/bib_ref]. Bcl-xL inhibitors were used in combination with traditional chemotherapeutic drugs for treating some solid tumors (US National Library of Medicine, 2019). The inhibitor of apoptosis (IAP) proteins is overexpressed in several malignancies and negatively affect prognosis by preventing caspase activation, thereby promoting tumor cell survival [bib_ref] Targeting IAP proteins for therapeutic intervention in cancer, Fulda [/bib_ref]. Bcl-2-targeting and IAP-targeting inhibitors target the intrinsic pathways of apoptosis. The exogenous pathways are activated by extracellular signals that activate apoptosis-promoting death receptor (DR)-and TNF-receptor superfamily (TNFR) transmembrane proteins [bib_ref] Cell death signaling, Green [/bib_ref]. Death receptors include TNFR1, Fas (CD95 and APO-1), DR3, DR4 (TRAILR1), DR5 (TRAILR1), and DR6. Mapatumumab, a DR4receptor agonist, in combination with chemotherapy has shown limited clinical benefit in phase I clinical trials in patients with non-small cell lung cancer, colorectal cancer, and other solid tumors [bib_ref] A phase 1 study of mapatumumab (fully human monoclonal antibody to TRAIL-R1]..., Hotte [/bib_ref] [bib_ref] Phase II trial of mapatumumab, a fully human agonist monoclonal antibody to..., Von Pawel [/bib_ref]. Lexatumumab, a DR5 agonist, in combination with chemotherapy stabilized the growth of advanced solid tumors but some adverse reactions were also recorded. Therefore, targeting the apoptotic pathway in tumor cells is an effective anticancer strategy. Further, promoting the death of tumor cells helps to improve the clinical status of patients and reduce the chance of tumor recurrence. In addition to cancer cells, tumor lesions also contain other cell types, such as endothelial cells and cancer-related fibroblasts, which inhibit the development of cancer [bib_ref] Hallmarks of cancer: The next generation, Hanahan [/bib_ref]. In addition, innate and adaptive immune cells, which grow and infiltrate tumor tissues to fight tumors, combine with these cells to form the tumor microenvironment (TME). Although voluntary apoptosis constitutes a common tumor suppressor mechanism, apoptosis may promote tumor cell survival and resistance to therapy by modulating the TME. The complex interrelationship between cellular and non-cellular components of TME coordinates tumor formation, progression, invasion, and response to therapy [bib_ref] Metabolites and the tumour microenvironment: From cellular mechanisms to systemic metabolism, Elia [/bib_ref] , which may determine its different effects in the face of Frontiers in Pharmacology frontiersin.org apoptotic regulation. This phenomenon may be related to the subsequent phagocytosis of apoptotic cells. Apoptotic cells can be phagocytosed by cells in neighboring tissues, especially macrophages. Macrophages can recognize the "find me" (such as phosphatidylcholine, 1-phosphosphingosine, and chemokine CX3CL-1) and "eat me" (phosphatidylserine) signals on the surface of apoptotic cells (Boada-Romero et al., 2020). Tumor-associated macrophages (TAMs) occupy the majority of TME in several malignant tumors; TAM accumulation is closely related to tumor growth and angiogenesis. Apoptosis-driven tissue repair and regeneration responses may be involved in generating and supporting TME [bib_ref] Limited mitochondrial permeabilization causes DNA damage and genomic instability in the absence..., Ichim [/bib_ref] [bib_ref] Microenvironmental effects of cell death in malignant disease, Gregory [/bib_ref]. Apoptotic cells in the TME generate an oncoregenerative niche, which refers to cellular and tissue programs that are activated when these cells promote tumorigenesis and malignant disease progression through apoptosis-driven regeneration and tissue repair mechanisms, thereby promoting tumor expansion and invasion while inhibiting antitumor immunity [bib_ref] An apoptosis-driven 'onco-regenerative niche': Roles of tumour-associated macrophages and extracellular vesicles, Gregory [/bib_ref]. The main function of TAMs in cancer is to support tumor growth through various mechanisms, including activation of angiogenesis, production of growth and survival factors, support of invasion and metastasis, and inhibition of antitumor immunity [bib_ref] Cancer-related inflammation: Common themes and therapeutic opportunities, Balkwill [/bib_ref]. Apoptotic cells induce M2 -like activation of macrophages. Moreover, they are easily engulfed by M1 macrophages and can inhibit their antitumor activity [bib_ref] Modulation of macrophage antitumor potential by apoptotic lymphoma cells, Voss [/bib_ref]. However, more studies are needed to understand how apoptosis drives the M2like phenotype of TAMs in the oncoregenerative niche. Lactic acid produced by TAMs may be necessary to promote tumor activation by polarizing to an M2-like phenotype. Overall, apoptosis may be a "double-edged sword" for tumor tissue. On the one hand, it inhibits tumors by removing malignant or precancerous cells, and on the other hand, it promotes tumor progression by stimulating the repair and regeneration response in the TME. ## Role of autophagy in the regulation of cancer Autophagy maintains a steady state in the single cell, and damaged organelles, cytoplasmic substances, and misfolded proteins are subjected to lysosome degradation. Apoptosis occurs in multicellular organisms. The occurrence of autophagy-induced cell death represents the failure of the cell to resist a lethal factor. This form of cell death is often referred to as cell death associated with autophagy-independent caspases [bib_ref] The end of autophagic cell death?, Shen [/bib_ref]. Autophagy pathway relies on the formation of autophagosomes, which depends on the recruitment of various autophagy-associated (ATG) proteins. This activation process begins with the formation of a preinitiation complex composed of kinases UNC-51-like kinase 1 (ULK1), FIP200, and ATG13, which activates downstream initiation complexes composed of Beclin 1, Class III PI3K (Vps34), and protein kinase Vps15. This initiation complex leads to the production of the lipid phosphatidylinositol 3-phosphate (PI3P), which activates ATG5-12 and LC3-PE conjugation systems [bib_ref] The Beclin 1 interactome, He [/bib_ref]. Finally, LC3 is conjugated with phosphatidyl ethanolamine (PE) to form the LC3-PE conjugate (also known as LC3-II) to aid the formation of autophagosomes. Pre-initiation complex formation is regulated by two major metabolic checkpoints, mammalian target protein complex 1 (mTORC1) and AMP-activated protein kinase (AMPK) [bib_ref] Organismal carbohydrate and lipid homeostasis, Hardie [/bib_ref]. Initiation complex activity is negatively regulated by several independent signaling pathways (including the growth factor and PI3K-AKT signaling pathways) [bib_ref] Akt-mediated regulation of autophagy and tumorigenesis through Beclin 1 phosphorylation, Wang [/bib_ref] , antiapoptotic proteins Bcl-2 and Bcl-xL [bib_ref] Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy, Pattingre [/bib_ref] , death-related protein kinases, and JNK family kinases [bib_ref] JNK1-mediated phosphorylation of Bcl-2 regulates starvation-induced autophagy, Wei [/bib_ref]. Autophagy is a multistep lysosomal degradation pathway that supports nutrient cycling and metabolic adaptation. In the early stages of cancer, autophagy may play a role in limiting tumorigenesis. However, autophagy can help tumor tissue respond to intracellular and environmental stresses, such as hypoxia, nutrient limitations, and anticancer treatments during cancer progression and metastasis, thereby facilitating tumor progression. In the early stages of tumorigenesis, autophagy acts as a control mechanism to protect tissues from damage, prevent tumorigenesis and genetic accumulation and inhibit cell apoptosis by removing damaged organelles and defective proteins [bib_ref] The double-edge sword of autophagy in cancer: From tumor suppression to pro-tumor..., Chavez-Dominguez [/bib_ref]. When the pressure of the living environment of tumor cells is too severe, autophagy can repair damaged DNA and organelles, thus maintaining tumor expansion and survival [bib_ref] The dual role of autophagy in cancer, Eskelinen [/bib_ref]. Therefore, autophagy may also play a dual role in cancer by either enhancing tumor tolerance or regulating self-sacrifice to preserve tumor tissue integrity [bib_ref] Autophagy and cancer: Modulation of cell death pathways and cancer cell adaptations, Towers [/bib_ref]. The role of autophagy in cancer treatment depends on specific stimuli, tumor cell types, and the severity of the injury and is not completely determined by the progression of the tumor . Although autophagy plays a paradoxical role in the treatment of tumors, understanding the pathophysiology and functional correlation of autophagy in tumors is still crucial to avoid drug resistance and enhance the anticancer therapeutic efficacy. For example, although there are many examples suggesting that autophagy has a cell-protective function in lung cancer [bib_ref] The tumor suppressor, p53, contributes to radiosensitivity of lung cancer cells by..., Cheng [/bib_ref] , the cytotoxic function of autophagy has also been reported. Pemetrexed is a folate antimetabolite approved for the treatment of non-small cell lung cancer and has been shown to regulate autophagy. This drug in combination with a multikinase inhibitor sorafenib, increased the levels of AKT, p70 S6K, and/or phosphorylated mTOR, thereby enhancing tumor killing by promoting toxic forms of autophagy [bib_ref] Sorafenib enhances pemetrexed cytotoxicity through an autophagy-dependent mechanism in cancer cells, Bareford [/bib_ref]. Cisplatin is also a commonly used chemotherapeutic drug in the treatment of lung cancer, but the long-term treatment causes tumor resistance; the potential mechanism involved a reduction in autophagy [bib_ref] Long-term cisplatin exposure impairs autophagy and causes cisplatin resistance in human lung..., Sirichanchuen [/bib_ref]. Mechanically, long-term cisplatin treatment causes overexpression of Bcl-2, and the results of in vitro cell line studies have suggested that cisplatin resistance can be reversed by using triflurazine to induce autophagy. In some studies on radiation therapy for cancer, the autophagy inhibitor, 3-methyladenine, induced radiation resistance in liver cancer cells [bib_ref] Enhancement of autophagy is a potential modality for tumors refractory to radiotherapy, Kuwahara [/bib_ref] ]. Rapamycin, as an autophagy inducer, enhanced the sensitivity of glioma-initiating cells to radiation [bib_ref] Induction of autophagy promotes differentiation of glioma-initiating cells and their radiosensitivity, Zhuang [/bib_ref]. In addition to the interaction of Beclin-1 with class III phosphatidylinositol-3-kinase Vps34 to initiate the classical pathway of autophagy, non-classical pathways also exist. This implies that autophagy can be regulated from multiple directions. Some authors used resveratrol to inhibit the proliferation of human breast cancer MCF-7 cell line and promote cell death and found that typical autophagy biomarkers such as LC3-I and LC3-II accumulated in the presence of lysosome inhibitor E64 and gastric enzymostatin A Frontiers in Pharmacology frontiersin.org [bib_ref] Role of noncanonical Beclin 1-independent autophagy in cell death induced by resveratrol..., Scarlatti [/bib_ref]. Moreover, knockout of neither Beclin-1 nor hVPS34 gene eliminated resveratrol-induced autophagosome formation, suggesting the existence of a non-classical pathway independent of tumor suppressor Beclin-1 [fig_ref] FIGURE 3: Primary mechanism of autophagy [/fig_ref]. Autophagy and apoptosis lead to cell death in a parallel manner. However, in some cases, autophagy can also be used as a part of the apoptosis program and a backup cell death mechanism after the inhibition of caspases [bib_ref] Cell death by autophagy: Facts and apparent artefacts, Denton [/bib_ref]. When stress-induced apoptosis is blocked, autophagy can be another mechanism of cell death. Both these processes share key metabolic regulators, indicating that these pathways are used under similar cell death or survival pressures. The role of apoptosis in cancer tissues and TME is contradictory due to their complexity. Several new forms of autophagy, such as secretory autophagy, regulate cancer progression, which realizes intercellular communication through the release of tumorpromoting substances in TME [ [bib_ref] Autophagy-dependent secretion: Mechanism, factors secreted, and disease implications, New [/bib_ref]. Simultaneously, complex crosstalk happens between autophagy and epithelial-to-mesenchymal transformation (EMT), which enables cancer cells to develop invasive phenotypes and metastatic potential [bib_ref] Autophagy as a potential therapeutic target during epithelial to mesenchymal transition in..., Singla [/bib_ref]. Several components of autophagy pathways also mediate many non-autophagic functions. This shows that autophagy plays a role in many ways, including building complex signal networks with other cell elements, integrating various signals in the TME, and regulating the fate of cancerous and other cells in the microenvironment. Therefore, understanding the mechanism of autophagy inhibition in complex systems is crucial for developing strategies to use autophagy pathways as targets for controlling tumor growth. ## Potential application of the endogenous cannabinoid system (ecs) in cancer In the past decade, ECS has become the focus of anticancer therapies. ECS refers to the complex network of cannabinoid Primary mechanism of autophagy. The autophagic signaling pathway. Under metabolic stress, AMPK activation and/or mTORC1 inhibition lead to the activation of the preinitiation complex (ULK1, FIP200, and ATG13). The latter activates the initiation complex (beclin 1, VPS15, and VPS34) that generates PI3P and recruits ATG7 to the phagophore. ATG7 initiates two conjugation pathways necessary for membrane elongation and closure of the autophagosome. In the ATG5-ATG12 conjugation pathway, ATG12 is sequentially transferred to ATG7, ATG10, and ATG12. The ATG5-12 conjugate recruits ATG16L and forms a complex necessary to stabilize the phagophore and to complete the second conjugation pathway. In the LC3-PE pathway, LC3 is cleaved by ATG4 and sequentially conjugated to ATG7 and ATG3. The ATG5-ATG12-ATG16 L complex carries out the final step by transferring LC3 to PE to form an LC3-PE conjugate (also called LC3-II). LC3-II binds to the autophagosomal membrane, and form autolysosome. AMPK, AMP-activated protein kinase; Atg, autophagy related gene; Bcl2, antiapoptotic protein; Beclin1, myosin-like BCL2 interacting protein; LC3 (MAP1LC3), microtubule-associated proteins light chain three; mTORC1, mammalian target of rapamycin complex one; p62, p62 protein; PIP, polyphosphoinositides; Rag, Rag proteins (RagA-D); TFEB, transcription factor EB; ULK, Unc-51-like kinase; vATPases, vacuolar-type adenosine triphosphatases. Frontiers in Pharmacology frontiersin.org receptors, endogenous cannabinoid ligands, enzymes that drive its biosynthesis, degradation, and transportation, and all cells and neural pathways involved in endogenous cannabinoid signal transduction. Presently, ECS is thought to be related to the important processes of controlling the body, assisting and maintaining the homeostasis of the body, which explains its uncertain role in tumorigenesis and tumor inhibition. CB1 and CB2 are the main receptors in the ECS. They belong to the extensive class A rhodopsin-like G protein-coupled receptor family, and their main functions include completing the transduction of signal molecules from extracellular space to various cells [bib_ref] International union of basic and clinical Pharmacology. LXXIX. Cannabinoid receptors and their..., Pertwee [/bib_ref]. The signal transduction of endocannabinoids is different from that of neural signal transduction. The role of endogenous cannabinoids is mainly limited to their biosynthesis and storage in synaptic vesicles in advance and released to the receptors when required [bib_ref] An introduction to the endogenous cannabinoid system, Lu [/bib_ref]. Endogenous and exogenous cannabinoids have other in the central nervous system and tumor tissues [bib_ref] Cannabinoids modulate neuronal activity and cancer by CB1 and CB2 receptor-independent mechanisms, Soderstrom [/bib_ref] , including transient receptor potential channels, ligands, voltage-gated ion channels, and other orphan G protein-coupled receptors such as GPR55, GPR18, and GPR119. The exogenous cannabinoids may cause the activation, antagonism, or reverse activation of cannabinoid and noncannabinoid receptors because of the differences in the abundance of receptors and levels and activities of endogenous ligands. This may also be one of the reasons for the inconsistent effects of exogenous cannabinoids observed in many empirical studies. CB1 and CB2 and other members of the endogenous cannabinoid-like system have become novel targets to treat various cancer subtypes because of their dual roles in tumorigenesis and inhibition of tumor growth and metastasis. Although the clinical use of cannabinoids has been widely recorded in palliative treatment, clinical trials on their use as an anticancer drug are still in progress. Cannabinoid receptors and endocannabinoids are usually upregulated in tumors, and their expression levels may be related to tumor invasiveness [bib_ref] Update on the endocannabinoid system as an anticancer target, Malfitano [/bib_ref]. Some authors have revealed that the loss of CB1 accelerates tumor growth, and higher levels of endogenous cannabinoid in the body lead to the reduction of precancerous lesions [bib_ref] Increased endocannabinoid levels reduce the development of precancerous lesions in the mouse..., Izzo [/bib_ref] , which confirms the theory of CEDS (occurrence of some diseases is related to the disorder of ECS signal transduction). The relationship between the distribution and expression of CB1 and CB2 on tumor cells and the tumor itself is not clear. Some authors believe that the loss of CB1 and/or CB2 expression accelerates tumor growth; however, it is likely to be related to the type of tumor cells. More clinical trials are needed to confirm the accuracy of results to better understand the relationship between ECS and neoplasmic diseases. ## Cannabinoid compounds improve cancer treatment by regulating apoptosis and autophagy The endocannabinoids can bind to very rich channel sites in the body, which coincide with the receptors that simultaneously mediate the pathological process of cancer, such as PPAR. Targeted activation of PPARγ can inhibit cell proliferation and induce programmed cell death (apoptosis and autophagy) [bib_ref] A role for the PPARgamma in cancer therapy, Campbell [/bib_ref]. In an in vitro study, CBD could activate the apoptosis of lung cancer cells after binding to the PPARγ receptor [bib_ref] COX-2 and PPAR-γ confer cannabidiol-induced apoptosis of human lung cancer cells, Ramer [/bib_ref]. In another study on human cervical cancer cells (treated with met-AEA in advance), the occurrence of apoptosis depended on the production of PGD 2 and PGJ 2 and the activation of the PPARγ receptor [bib_ref] R(+)-methanandamide-induced apoptosis of human cervical carcinoma cells involves a cyclooxygenase-2-dependent pathway, Eichele [/bib_ref]. In addition to exogenous intake, regulating enzymes are also an important link in regulating endogenous cannabinoid levels. Fatty acid amidohydrolase (FAAH) and monoacylglycerol lipase are the two most important endocannabinoid hydrolases. In non-melanoma skin cancer cells, the use of FAAH inhibitor URB597 increased the apoptosis-promoting effect of AEA [bib_ref] Arachidonoyl ethanolamide (AEA)-induced apoptosis is mediated by J-series prostaglandins and is enhanced..., Kuc [/bib_ref]. Moreover, URB597 can increase the autophagic toxicity of AEA on neuroblastoma cells by inhibiting the hydrolysis of AEA by FAAH [bib_ref] Increasing antiproliferative properties of endocannabinoids in N1E-115 neuroblastoma cells through inhibition of..., Hamtiaux [/bib_ref]. These studies suggest that URB597 can increase cellular endocannabinoid levels and enhance the interaction between endocannabinoid and its molecular target by reducing the activity of hydrolases. In addition, several studies have suggested that endocannabinoids can play an anticancer role through the endoplasmic reticulum stress pathway. In hepatocellular carcinoma cells (HEPG1), the use of plant cannabinoids increased the phosphorylation of eIF2α and expression of CHOP10 transcript TRB3 [bib_ref] Involvement of PPARγ in the antitumoral action of cannabinoids on hepatocellular carcinoma, Vara [/bib_ref]. TRB3 is necessary to inhibit Akt/mTORC1 signal transduction and induce autophagy and apoptosis [bib_ref] Cannabinoid action induces autophagy-mediated cell death through stimulation of ER stress in..., Salazar [/bib_ref] , indicating that exogenous cannabinoid contributes to tumor autophagy and apoptosis. Synthetic cannabinoids have also been used in several studies. In human glioblastoma cell lines, the ability of THC to inhibit cell growth and induce apoptosis was associated with the activation of CB1 and CB2 receptors, which was validated by the use of selective receptor antagonists or by the silencing of receptor expression using specific small interfering RNA . Neuroblastoma is one of the most common childhood cancers, CBD induces apoptosis of neuroblastoma cells by activating serotonin and vanillin receptors. In this process, miRNA hsa-mir-1972 is upregulated, resulting in decreased expression of apoptosis-related genes BCL2L1 and SIRT2 and regulating the apoptosis of neuroblastoma cells [bib_ref] Role of miRNA in the regulation of cannabidiol-mediated apoptosis in neuroblastoma cells, Alharris [/bib_ref]. Synthetic cannabinoid receptor agonists are more widely used in research than naturally occurring cannabinoids. Synthetic cannabinoid receptor antagonists AM251, SR144528, and AM630 inhibit osteoclast formation and bone resorption in vitro. Cannabinoid receptor blockade caused osteoclast inhibition mostly through apoptosis of osteoclasts because AM251 and SR144528 markedly enhanced apoptosis in mature rabbit osteoclasts and RANKL-induced [bib_ref] Regulation of bone mass, bone loss and osteoclast activity by cannabinoid receptors, Idris [/bib_ref] ]. The effect of the CB receptors on cell apoptosis is gradually being determined. In a study on liver cancer, researchers found that Δ9-THC and JWH-015 (a CB2 cannabinoid receptor-selective agonist) reduced the viability of the human hepatocellular carcinoma cell lines HepG2 and HuH-7, an effect that relied on the stimulation of CB2 receptor [bib_ref] Erratum: Anti-tumoral action of cannabinoids on hepatocellular carcinoma: Role of AMPK-dependent activation..., Vara [/bib_ref]. JWH-015 also reduced pain in patients with bone cancer by improving impaired autophagy flux [bib_ref] Cannabinoid receptor 2-selective agonist JWH015 attenuates bone cancer pain through the amelioration..., Mao [/bib_ref]. Arvanil (N-arachidonoylvanillamine), a compound similar to AEA (Di [bib_ref] A structure/activity relationship study on arvanil, an endocannabinoid and vanilloid hybrid, Marzo [/bib_ref] , induced apoptosis of Jurkat cells, which was essentially mediated through mechanisms typical of type II apoptotic Frontiers in Pharmacology frontiersin.org cells and involved activation of the death-inducing signaling complex and caspase-8. This arvanil-induced apoptotic activity is independent of CB1 and has important implications in the development of antitumor drugs [bib_ref] The CB1/VR1 agonist arvanil induces apoptosis through an FADD/caspase-8-dependent pathway, Sancho [/bib_ref]. Some authors have reported that a synthetic cannabinoid WIN55212-2 can induce autophagy and apoptosis in human colorectal cancer and mantle cell lymphoma cells; this activity is related to the induction of cytotoxic endoplasmic reticulum stress [bib_ref] WIN55, 212-2 induces cytoplasmic vacuolation in apoptosis-resistant MCL cells, Wasik [/bib_ref] [bib_ref] WIN induces apoptotic cell death in human colon cancer cells through a..., Pellerito [/bib_ref]. Cannabinoids bind to CB1 and CB2 receptors to inhibit energy metabolism in pancreatic cancer cells and induce AMPK-dependent autophagy [bib_ref] Cannabinoids inhibit energetic metabolism and induce AMPK-dependent autophagy in pancreatic cancer cells, Dando [/bib_ref]. A new synthetic cannabinoid (CB83) has been used on human HT-29 colorectal adenocarcinoma cells; it induced the activation of endoplasmic reticulum stress-related signaling pathways by increasing ceramide production, ultimately leading to the activation of the intrinsic apoptotic pathway and inhibiting the proliferation of cancer cells [bib_ref] Cytotoxic effects of cannabinoids on human HT-29 colorectal adenocarcinoma cells: Different mechanisms..., Cerretani [/bib_ref]. Taken together, cannabinoids have great prospects in cancer treatment. We have reviewed the available information on natural or synthetic cannabinoid compounds that target cancer cell apoptosis and autophagy. However, it can bind to receptors other than CBR1 and CBR, or directly act on some pathways to exert its antitumor effects. CBD, a plant-derived cannabinoid, can combine with a variety of receptors not involved in the pathology of mental disorders; therefore, it has attracted much attention as an anticancer drug. # Conclusion and outlook PCD has been increasingly implicated as the gatekeeper of cell fate, with decisive roles in various diseases including inflammatory diseases, autoimmune diseases, degenerative diseases, and cancer. The role of cell death in the pathophysiology of inflammation and cancer needs to be explored further. Several in vitro/in vivo studies on exogenous cannabinoids have indicated that cannabinoids can affect the key functions of cells in inflammatory or cancer diseases, such as proliferation, migration, cytokine formation, differentiation, autophagy, and apoptosis. The diversity of cellular receptors of cannabinoid ligands may explain the wide range of biological effects of cannabinoid drugs in several diseases. More information on cannabinoid CB1/CB2-independent targets is needed to determine their in vitro effects and tolerance/resistance mechanisms and to properly interpret the results of available studies. Previous studies have shown that cannabinoids have an immunosuppressive effect on the immune system. Cannabinoid ligands inhibit phagocytosis, cell proliferation, antigen Schematic diagram of different mechanisms/signaling pathways of cannabinoids affecting autophagy and apoptosis. The mechanism of cannabinoidinduced apoptosis and autophagy in cell is depicted. AKT, protein kinase B; AMPK, AMP-activated protein kinase; ATF-4, activating transcription factor 4; Bax, pro-apoptotic protein; Bcl2, antiapoptotic protein; CB1/2, cannabinoid receptor 1/2; CHOP, C/EBP homologous protein; elF2α, eukaryotic translation initiation factor 2α; ERK, extracellular regulated kinase; GPR55, G protein-coupled receptor 55; H 2 O 2 , hydrogen peroxide; mTORC2, mammalian target of rapamycin complex two; p53, p53 protein; p21 ras, p21 ras protein; p38MAPK, p38 mitogen activated protein kinase; PI3K, phosphatidyl inositol three kinase; PKA, protein kinase A; TFEB, transcription factor EB; TRIB3, tribbles-homologue three; TRPV1, transient receptor potential cation channel V1. Frontiers in Pharmacology frontiersin.org presentation, and other properties of immune cells, and cannabinoid may be a safe and effective antitumor drug. Nevertheless, clinical studies to evaluate the effect of cannabinoids on the human body are limited. Only animal models or in vitro cell lines have been used to evaluate the feasibility of cannabinoid treatment and large-scale clinical trials have not been conducted. Therefore, the results may vary in the clinical setup. The prospects of cannabinoid treatment in inflammatory and cancer diseases are worth exploring. The influence of cannabinoids on cell fate should be explored further. A detailed understanding of the regulation of autophagy and apoptosis by cannabinoids will not only improve the understanding of the biology of the disease but will also be crucial in finding better therapeutic targets, thereby generating new combined therapies. Author contributions ZF, P-YZ, and HL codrafted the manuscript; X-HD, Y-XX, and ZF conceptualized; P-YZ, S-DH, and X-PY undertook and refined the searches. All authors have read and agreed to the published version of the manuscript. # Funding This work was supported by grants from Military Medical Innovation Project (18CXZ025) and Department of General Surgery, First Medical Center of the Chinese PLA General Hospital, Beijing, China. ## Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. ## Publisher's note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. [fig] FIGURE 3: Primary mechanism of autophagy. The autophagic signaling pathway. Under metabolic stress, AMPK activation and/or mTORC1 inhibition lead to the activation of the preinitiation complex (ULK1, FIP200, and ATG13). The latter activates the initiation complex (beclin 1, VPS15, and VPS34) that generates PI3P and recruits ATG7 to the phagophore. ATG7 initiates two conjugation pathways necessary for membrane elongation and closure of the autophagosome. In the ATG5-ATG12 conjugation pathway, ATG12 is sequentially transferred to ATG7, ATG10, and ATG12. The ATG5-12 conjugate recruits ATG16L and forms a complex necessary to stabilize the phagophore and to complete the second conjugation pathway. In the LC3-PE pathway, LC3 is cleaved by ATG4 and sequentially conjugated to ATG7 and ATG3. The ATG5-ATG12-ATG16 L complex carries out the final step by transferring LC3 to PE to form an LC3-PE conjugate (also called LC3-II). LC3-II binds to the autophagosomal membrane, and form autolysosome. AMPK, AMP-activated protein kinase; Atg, autophagy related gene; Bcl2, antiapoptotic protein; Beclin1, myosin-like BCL2 interacting protein; LC3 (MAP1LC3), microtubule-associated proteins light chain three; mTORC1, mammalian target of rapamycin complex one; p62, p62 protein; PIP, polyphosphoinositides; Rag, Rag proteins (RagA-D); TFEB, transcription factor EB; ULK, Unc-51-like kinase; vATPases, vacuolar-type adenosine triphosphatases. [/fig]
The parasite Trichomonas vaginalis expresses thousands of pseudogenes and long non-coding RNAs independently from functional neighbouring genes Background: The human pathogen Trichomonas vaginalis is a parabasalian flagellate that is estimated to infect 3% of the world's population annually. With a 160 megabase genome and up to 60,000 genes residing in six chromosomes, the parasite has the largest genome among sequenced protists. Although it is thought that the genome size and unusual large coding capacity is owed to genome duplication events, the exact reason and its consequences are less well studied. Results: Among transcriptome data we found thousands of instances, in which reads mapped onto genomic loci not annotated as genes, some reaching up to several kilobases in length. At first sight these appear to represent long non-coding RNAs (lncRNAs), however, about half of these lncRNAs have significant sequence similarities to genomic loci annotated as protein-coding genes. This provides evidence for the transcription of hundreds of pseudogenes in the parasite. Conventional lncRNAs and pseudogenes are expressed in Trichomonas through their own transcription start sites and independently from flanking genes in Trichomonas. Expression of several representative lncRNAs was verified through reverse-transcriptase PCR in different T. vaginalis strains and case studies exclude the use of alternative start codons or stop codon suppression for the genes analysed. Conclusion: Our results demonstrate that T. vaginalis expresses thousands of intergenic loci, including numerous transcribed pseudogenes. In contrast to yeast these are expressed independently from neighbouring genes. Our results furthermore illustrate the effect genome duplication events can have on the transcriptome of a protist. The parasite's genome is in a steady state of changing and we hypothesize that the numerous lncRNAs could offer a large pool for potential innovation from which novel proteins or regulatory RNA units could evolve. # Background The parabasalian flagellate Trichomonas vaginalis is a unique human parasite causing trichomoniasis, the most common sexually transmitted disease (STD) [bib_ref] Clinical and microbiological aspects of Trichomonas vaginalis, Petrin [/bib_ref]. The anaerobic protist possesses the ability to rapidly shift between an amoeboid and flagellated phenotype [bib_ref] Dramatic reorganisation of Trichomonas endomembranes during amoebal transformation: A possible role for..., Lal [/bib_ref] [bib_ref] The actin-based machinery of Trichomonas vaginalis mediates flagellate-amoeboid transition and migration across..., Kusdian [/bib_ref] , and was once considered to represent an early-branching eukaryotic lineage [bib_ref] Early branching eukaryotes?, Embley [/bib_ref]. At least 46,000 genes, and potentially up to 60,000, are encoded on six chromosomes, representing one of the highest coding capacities known [bib_ref] Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis, Carlton [/bib_ref] [bib_ref] Gene expression in the unicellular eukaryote Trichomonas vaginalis, Smith [/bib_ref]. Exhaustive coding capacity analyses in Trichomonas are generally hampered through the extensive presence of repeats and transposable elements that are thought to make up 45% of the genome [bib_ref] GiardiaDB and TrichDB: integrated genomic resources for the eukaryotic protist pathogens Giardia..., Aurrecoechea [/bib_ref]. The expansion of the genome appears recent [bib_ref] Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis, Carlton [/bib_ref] and might coincide with the colonization of new host habitats. The genome enlargement of this eukaryote was further fueled by a high amount of lateral gene transfer events [bib_ref] Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis, Carlton [/bib_ref] [bib_ref] Horizontal gene transfer in eukaryotic parasites: a case study of Entamoeba histolytica..., Alsmark [/bib_ref] and the massive expansion of some gene families [bib_ref] Trichomonas vaginalis vast BspA-like gene family: evidence for functional diversity from structural..., Noël [/bib_ref] [bib_ref] Deep sequencing of Trichomonas vaginalis during the early infection of vaginal epithelial..., Gould [/bib_ref]. It has been suggested that the frequency of pseudogenes in T. vaginalis is at least 5% and that unstable gene families that underwent many gene duplication events, thereby producing pseudogenes on the way, further contributed to the large genome of T. vaginalis [bib_ref] Trichomonas transmembrane cyclases result from massive gene duplication and concomitant development of..., Cui [/bib_ref]. The transcriptome of T. vaginalis and its many known strains is not well characterized, but some classes of non-coding RNAs (ncRNA) have been described. Genome annotations of T. vaginalis include 668 ribosomal RNAs (rRNA) genes of three types and 468 transfer RNAs (tRNA) genes of 48 types [bib_ref] Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis, Carlton [/bib_ref] [bib_ref] GiardiaDB and TrichDB: integrated genomic resources for the eukaryotic protist pathogens Giardia..., Aurrecoechea [/bib_ref]. RNA subunits of the ribonucleoproteins RNase P and MRP were also identified [bib_ref] Characterization of RNase MRP RNA and novel snoRNAs from Giardia intestinalis and..., Chen [/bib_ref] [bib_ref] Identification and analysis of ribonuclease P and MRP RNA in a broad..., Piccinelli [/bib_ref]. Furthermore, small regulatory RNAs (sRNA) have been discovered including potential microRNAs (miRNA) [bib_ref] High throughput genome-wide survey of small RNAs from the parasitic protists Giardia..., Chen [/bib_ref] [bib_ref] Malate dehydrogenase is negatively regulated by miR-1 in Trichomonas vaginalis, Lin [/bib_ref] [bib_ref] Identification of microRNA in the protist Trichomonas vaginalis, Lin [/bib_ref] [bib_ref] Identification of putative miRNAs from the deep-branching unicellular flagellates, Huang [/bib_ref] , small nuclear RNAs (snRNA) [bib_ref] Spliceosomal snRNAs in the unicellular eukaryote Trichomonas vaginalis are structurally conserved but..., Simoes-Barbosa [/bib_ref] and small nucleolar RNAs (snoRNAs) [bib_ref] Characterization of RNase MRP RNA and novel snoRNAs from Giardia intestinalis and..., Chen [/bib_ref] [bib_ref] High throughput genome-wide survey of small RNAs from the parasitic protists Giardia..., Chen [/bib_ref]. Genes of the Argonaute (AGO) and Dicer-like family are encoded by Trichomonas and hence suggest the existence of functional RNA interference mechanisms [bib_ref] Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis, Carlton [/bib_ref] [bib_ref] High throughput genome-wide survey of small RNAs from the parasitic protists Giardia..., Chen [/bib_ref] , although other studies question the functionality of identified miRNAs in this parasite [bib_ref] Do miRNAs have a deep evolutionary history?, Tarver [/bib_ref]. Regulatory RNAs are mostly small (<200 nucleotides), but recent reports of longer regulatory RNAs are accumulating [bib_ref] Noncoding RNA in development, Amaral [/bib_ref] [bib_ref] Regulation of mammalian cell differentiation by long non-coding RNAs, Hu [/bib_ref] [bib_ref] RNA maps reveal new RNA classes and a possible function for pervasive..., Kapranov [/bib_ref] [bib_ref] Long non-coding RNAs: insights into functions, Mercer [/bib_ref] [bib_ref] Specific expression of long noncoding RNAs in the mouse brain, Mercer [/bib_ref] [bib_ref] Long noncoding RNAs in C. elegans, Nam [/bib_ref] [bib_ref] Functionality or transcriptional noise? Evidence for selection within long noncoding RNAs, Ponjavic [/bib_ref] [bib_ref] Transcription of two long noncoding RNAs mediates mating-type control of gametogenesis in..., Van Werven [/bib_ref]. Recent deep-sequencing of the parasite's transcriptome has shed light on the expression potential of the genome and provided evidence for the expression of about 30,000 genes and a correlated co-expression of gene families induced by different stimuli [bib_ref] Deep sequencing of Trichomonas vaginalis during the early infection of vaginal epithelial..., Gould [/bib_ref] [bib_ref] Adaptive responses to glucose restriction enhance cell survival, antioxidant capability, and autophagy..., Huang [/bib_ref]. Long non-coding RNAs (lncRNAs) are often defined as transcribed but not translated RNA segments larger than sRNAs (>200 nucleotides) [bib_ref] Characterizing ncRNAs in human pathogenic protists using high-throughput sequencing technology, Collins [/bib_ref]. lncRNAs affect chromosomal dynamics, the telomeres and structural organization [bib_ref] Noncoding RNA in development, Amaral [/bib_ref] [bib_ref] Regulation of mammalian cell differentiation by long non-coding RNAs, Hu [/bib_ref] [bib_ref] Long non-coding RNAs: insights into functions, Mercer [/bib_ref]. Their expression can be regulated and restricted to certain developmental stages and tissues [bib_ref] Noncoding RNA in development, Amaral [/bib_ref] [bib_ref] RNA maps reveal new RNA classes and a possible function for pervasive..., Kapranov [/bib_ref] [bib_ref] Specific expression of long noncoding RNAs in the mouse brain, Mercer [/bib_ref]. Some are recognized by canonical transcription factors [bib_ref] Unbiased mapping of transcription factor binding sites along human chromosomes 21 and..., Cawley [/bib_ref] and their promoters can show evidence of purifying selection [bib_ref] Functionality or transcriptional noise? Evidence for selection within long noncoding RNAs, Ponjavic [/bib_ref]. However, the functionality of the majority of lncRNAs is unknown, and many are thought to represent "junk" RNA or transcriptional noise attributable to the promiscuity of RNA polymerase II [bib_ref] Transcriptional noise and the fidelity of initiation by RNA polymerase II, Struhl [/bib_ref]. It has been proposed that every euchromatic nucleotide in the human genome could be transcribed, albeit this does obviously not necessarily translate into every expressed nucleotide having a biological function [bib_ref] On the immortality of television sets: "function" in the human genome according..., Graur [/bib_ref]. Most lncRNA studies focus on metazoan organisms with yeasts representing a rare exception [bib_ref] Long noncoding RNAs in C. elegans, Nam [/bib_ref] [bib_ref] Transcription of two long noncoding RNAs mediates mating-type control of gametogenesis in..., Van Werven [/bib_ref] [bib_ref] Dynamic transcriptome of Schizosaccharomyces pombe shown by RNA-DNA hybrid mapping, Dutrow [/bib_ref] [bib_ref] The transcriptional landscape of the yeast genome defined by RNA sequencing, Nagalakshmi [/bib_ref] [bib_ref] Dynamic repertoire of a eukaryotic transcriptome surveyed at single-nucleotide resolution, Wilhelm [/bib_ref]. Although several thousand lncRNAs have been predicted to be functional [bib_ref] RNA maps reveal new RNA classes and a possible function for pervasive..., Kapranov [/bib_ref] [bib_ref] Long noncoding RNAs in C. elegans, Nam [/bib_ref] [bib_ref] Distinguishing protein-coding from non-coding RNAs through support vector machines, Liu [/bib_ref] , the number of experimentally validated functional lncRNA (about 200) remains low [bib_ref] lncRNAdb: a reference database for long noncoding RNAs, Amaral [/bib_ref] [bib_ref] Computational analysis of functional long noncoding RNAs reveals lack of peptide-coding capacity..., Niazi [/bib_ref]. Most lncRNAs contain only short open reading frames [bib_ref] Computational analysis of functional long noncoding RNAs reveals lack of peptide-coding capacity..., Niazi [/bib_ref] , but for yeast it has been demonstrated that more than a thousand short open reading frames are translated [bib_ref] Proto-genes and de novo gene birth, Carvunis [/bib_ref]. They were shown to be conserved between organisms and to fulfil biological functions [bib_ref] On the extent and role of the small proteome in the parasitic..., Ericson [/bib_ref] [bib_ref] Functional genomics of genes with small open reading frames (sORFs) in S...., Kastenmayer [/bib_ref] [bib_ref] Small open reading frames associated with morphogenesis are hidden in plant genomes, Hanada [/bib_ref]. Pseudogenes, like lncRNAs, do not encode functional proteins but can be identified through their sequence similarity to protein-coding genes from which they evolved. Some are expressed and translated, but most resemble non-processed genetic remnants [bib_ref] Pseudogenes in the ENCODE regions: consensus annotation, analysis of transcription, and evolution, Zheng [/bib_ref] [bib_ref] Pseudogenes: newly discovered players in human cancer, Poliseno [/bib_ref]. There are 1354 annotated pseudogenes in T. vaginalis (or~2% of predicted protein-coding genes), but based on gene family analysis it was estimated that a minimum of 5% of the proteincoding genes may represent pseudogenes and half of the Trichomonas transmembrane cyclase family appears to represent pseudogenes [bib_ref] Trichomonas transmembrane cyclases result from massive gene duplication and concomitant development of..., Cui [/bib_ref]. Expressed pseudogenes are essentially a sub-group of lncRNA, and for some a biological function has been identified [bib_ref] Pseudogenes: newly discovered players in human cancer, Poliseno [/bib_ref]. Antisense pseudogene transcripts can be processed into small regulatory RNAs [bib_ref] Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes, Tam [/bib_ref] [bib_ref] Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes, Watanabe [/bib_ref] or to complementarily bind to their functional counterparts and influence their expression [bib_ref] Transcriptional regulation of Oct4 by a long noncoding RNA antisense to Oct4-pseudogene..., Hawkins [/bib_ref] [bib_ref] Neuronal expression of neural nitric oxide synthase (nNOS) protein is suppressed by..., Korneev [/bib_ref]. One of the best-studied functional lncRNAs that participates in X chromosome inactivation in mammals is the Xist RNA. It is a lncRNA that originates from the pseudogenization of a protein-coding gene [bib_ref] The Xist RNA gene evolved in eutherians by pseudogenization of a protein-coding..., Duret [/bib_ref]. Here we identified and characterized lncRNAs of the parabasalian parasite T. vaginalis by screening available transcriptional data and 271 million novel RNA-Seq reads we generated. We found that almost one fifth of the transcripts originate from intergenic regions of the parasite. We have characterized these transcripts in terms of their potential coding capacity, flanking genomic regions and similarity to annotated genes, in order to elucidate their origin and determine what drives their expression. # Results and discussion ## General transcript mapping and homology We used 91,601 expressed sequence tags (ESTs) downloaded from TrichDB [bib_ref] GiardiaDB and TrichDB: integrated genomic resources for the eukaryotic protist pathogens Giardia..., Aurrecoechea [/bib_ref] and combined those with 271.3 million raw reads from our own RNA-Seq data. After assembling and merging the two data sets, we mapped in total 27,385 unique transcript contigs onto the genome of Trichomonas vaginalis in total. From those, 22,609 (83%) mapped onto regions encoding annotated genes and 4,606 (17%) did not. We refer to these datasets as CDS P and CDS N , respectively . The CDS P set overlapped with 24,950 protein-coding genes, representing only 42% of annotated genes and less than half of what was found for other protists [bib_ref] The Transcriptome of the Human Pathogen Trypanosoma brucei at Single-Nucleotide Resolution, Kolev [/bib_ref] [bib_ref] A comprehensive comparison of RNA-Seq-based transcriptome analysis from reads to differential gene..., Nookaew [/bib_ref] [bib_ref] Transcriptome analysis of the model protozoan, Tetrahymena thermophila, using deep RNA sequencing, Xiong [/bib_ref] [bib_ref] The transcriptome and proteome of the diatom Thalassiosira pseudonana reveal a diverse..., Dyhrman [/bib_ref]. Yet, these transcripts represent 93% of the gene families identified in Trichomonas [bib_ref] OrthoMCL-DB: querying a comprehensive multi-species collection of ortholog groups, Chen [/bib_ref] , indicating that (a) sequencing depth appears to be sufficient and that the numbers are not likely to change much with more sequencing data becoming available, and that (b) most of the functional proteome the genome encodes is expressed, but not all members of a gene family. The homology of CDS N transcripts to annotated genes was examined next. About half (2175; 47%) had no significant similarity to any annotated genes, hence representing lncRNAs of non-recognizable origin. The remainders of the CDS N transcripts (2431; 53%) were found to be significantly similar to annotated genes and were thus classified as expressed pseudogenes with functional homologous genes. These were additionally filtered to exclude contigs that mapped to the very proximal regions of genomic scaffolds and those with bad sequencing resolution, that is stretches of 'N'. 455 such contigs were identified. We termed the remaining identified set PSEUDO, and those loci without significant homologies LNCRNA . The repetitive nature of this parasite's genome is extensive. Using REPEATMASKERwe screened the genome for repetitive elements and subsequently for overlaps with associated genomic regions. About 30% of the PSEUDO and CDS P loci (31.5% and 28.9%, respectively) were associated with repeat regions, while for the LNCRNA loci this was the case for only 17.3%. Comparable to PSEUDO and CDS P , a dataset consisting of all T. vaginalis gene annotations showed an association with repeat elements for 29.5%. Therefore, these loci seem to be preferably embedded into the repeat structure of the genome, but do not show any specific links. LNCRNA loci varied more and this might be connected to specific sequence selection to form functional RNA structures. Data for the human genome suggests that half of the transcriptome consists of lncRNAs [bib_ref] RNA maps reveal new RNA classes and a possible function for pervasive..., Kapranov [/bib_ref] and in mouse 28,000 ncRNAs were identified [bib_ref] Distinguishing protein-coding from non-coding RNAs through support vector machines, Liu [/bib_ref]. For T. vaginalis only 17% of the transcripts did not map to any annotated genes. With more data for the parasite becoming available one will be able to determine whether this difference is due to sequencing depth or biological differences. Considering studies on other protists, which were able to cover most of the annotated genes with less sequencing depth, the former seems unlikely [bib_ref] The Transcriptome of the Human Pathogen Trypanosoma brucei at Single-Nucleotide Resolution, Kolev [/bib_ref] [bib_ref] A comprehensive comparison of RNA-Seq-based transcriptome analysis from reads to differential gene..., Nookaew [/bib_ref] [bib_ref] Transcriptome analysis of the model protozoan, Tetrahymena thermophila, using deep RNA sequencing, Xiong [/bib_ref] [bib_ref] The transcriptome and proteome of the diatom Thalassiosira pseudonana reveal a diverse..., Dyhrman [/bib_ref]. In any case, most will resemble transcriptional noise [bib_ref] Transcriptional noise and the fidelity of initiation by RNA polymerase II, Struhl [/bib_ref] and random expression caused for instance by sequences mimicking transcriptional promoters (see below), with only a few representing expressed and functional lncRNAs. We experimentally validated the expression of a random set of lncRNAs in the most frequently used laboratory strain T1, and the virulent T016 and highly virulent FMV1 strains. For all six cases we could verify expression in all the three T. vaginalis strains tested [fig_ref] Figure 2: Expression of lncRNAs is conserved among different T [/fig_ref] , which demonstrates lncRNA expression to generally be conserved across the different strains tested. ## Characterization of transcribed pseudogenes The PSEUDO set includes 7% of all transcripts analysed. It represents a lower bound on the pseudogene content of T. vaginalis, as this set does not include non-expressed pseudogenes, unitary pseudogenes, or pseudogenes erroneously annotated as functional genes. It has previously been estimated that at least 5% of the annotated genes of T. vaginalis could represent mis-annotated pseudogenes, and for one large gene family it has been shown that about half of its members could qualify as pseudogenes [bib_ref] Gene expansion in Trichomonas vaginalis: a case study on transmembrane cyclases, Cui [/bib_ref]. For the human genome it is estimated that 8 to 20% of all pseudogenes are expressed [bib_ref] Pseudogenes in the ENCODE regions: consensus annotation, analysis of transcription, and evolution, Zheng [/bib_ref]. If that is also true Schematic workflow of the data management. Sequenced reads and expressed sequence tags (ESTs) of Trichomonas vaginalis were mapped onto the genome as shown and sorted into the categories presented according to their best BLAST hits. for Trichomonas, the parasite could potentially harbour between 10,000 and 25,000 pseudogenes. In order to estimate the number of non-expressed pseudogenes in T. vaginalis we performed BLASTN searches (e value cutoff 10 −10 ) with annotated proteins to intergenic regions lacking expression evidence. This revealed approximately 50,000 intergenic loci, for which no expression evidence exists, but with a significant homology to annotated (and likely functional) genes. Although the absolute number is much higher, the value is comparable to that from human, where the amount of pseudogenes (up to 20,000) almost reaches that for the coding genes. High abundances of pseudogenes are generally known for mammals, but their number in less complex organisms is usually smaller [bib_ref] Large-scale analysis of pseudogenes in the human genome, Zhang [/bib_ref]. This would support a recent hypothesis that the Trichomonas genome (and maybe even proteome) faces constantly emerging and disappearing paralogs, and is in a steady state of changing [bib_ref] Trichomonas transmembrane cyclases result from massive gene duplication and concomitant development of..., Cui [/bib_ref]. Large gene families contain high a number of genes, where each one can pseudogenize or duplicate. We examined our transcribed and non-transcribed intergenic pseudogenes for a correlation between the number of pseudogenes and sizes of corresponding gene families. Although we observed a moderate Pearson correlation for non-transcribed pseudogenes (r = 0.54, P value <0.05), the correlation for transcribed pseudogenes (PSEUDO) was rather low (r = 0.19, P value <0.05), indicating a potential connection. But at least for the transcription of pseudogenes this factor seems less important. Functional categories of pseudogene datasets were analysed using EuKaryotic Orthologous Groups (KOGs; [bib_ref] A comprehensive evolutionary classification of proteins encoded in complete eukaryotic genomes, Koonin [/bib_ref] and it revealed similar distributions of categories for non-transcribed pseudogenes, transcribed pseudogenes (PSEUDO) and annotated transcripts (CDS P ). A clear difference occurred according to the frequency of genes, which were associated with KOG categories. While for CDS P 64% of loci remained unclassified, for the untranscribed pseudogenes and PSEUDO loci they accounted for 83% and 92%, respectively. 4% of unclassified loci in PSEUDO, which is low compared to 37% for non-transcribed pseudogenes, represented repetitive gene models described in Carlton et al. [bib_ref] Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis, Carlton [/bib_ref]. These findings indicate that these pseudogenes, which are still transcribed, predominantly are based on recent Trichomonasspecific functions. In order to compare homologies of PSEUDO, CDS P and intergenic regions (INTG; randomly picked intergenic loci, but with the same length distribution as the CDS N ) we examined the distributions of the best BLASTN hit e values [fig_ref] Figure 3: Comparison of potential coding capacities for the different sets of transcripts identified [/fig_ref]. All compared sets differed significantly (Kolmogorov-Smirnov test; P value <0.05; Additional file 1: [fig_ref] Table 1: Protein coding sequence features of the various sets analysed [/fig_ref] , with the INTG behaving similarly to the CDS N set. The BLASTN hits of the PSEUDO set revealed higher e values compared to those of the CDS P set, suggesting these homologies are less conserved and to only partially map onto the annotated gene sequences. The several cases of pseudogenes that retrieved hits with small e valuesindicating full sequence hitsmost likely represent novel pseudogenes that represent more recent gene duplications events and not falsely annotated genes. ## Transcript coding capacity of cds n The PSEUDO, LNCRNA and CDS P sets were compared in regard to their potential protein-coding capacities. Three control sets were used: the first represents the intergenic loci (INTG) mentioned above, the second was based on randomized CDS N sequences (RND N ) and the third simply comprised all annotated T. vaginalis genes that included also those lacking expression evidence (TVAG; [fig_ref] Table 1: Protein coding sequence features of the various sets analysed [/fig_ref] and [fig_ref] Figure 3: Comparison of potential coding capacities for the different sets of transcripts identified [/fig_ref]. We found that the PSEUDO and LNCRNA sets behaved similarly and were placed in between the protein-coding CDS P and the randomized CDS N sets. Differences between all datasets, except PSEUDO and LNCRNA in [fig_ref] Figure 3: Comparison of potential coding capacities for the different sets of transcripts identified [/fig_ref] , were statistically supported (Kolmogorov-Smirnov test; P value <0.05; Additional file 1: Table S1), where the P values suggested that CDS P differs the most. As expected for CDS P , this set's GC-content was found to be very similar to the GC-content described for annotated genes (34.6% versus 35%, respectively), while the GC-content of CDS N (30.5%) was more similar to that of the non-expressed intergenic sequences (28.8%). PSEUDO and LNCRNA subsets of CDS N alone differ only slightly from the total CDS N set, with the PSEUDO set showing a marginal tendency towards protein-coding gene sequences [fig_ref] Table 1: Protein coding sequence features of the various sets analysed [/fig_ref]. This suggests that the PSEUDO set does not contain many, if any, genes that are not yet annotated. The relatively high amount of lncRNAs with longer open reading frames (ORFs; 55-65% ≥50 amino acids) is noteworthy. Similarities of lncRNAs to protein-coding genes have been described before and a high density of ORFs among lncRNA noticed [bib_ref] Functionality or transcriptional noise? Evidence for selection within long noncoding RNAs, Ponjavic [/bib_ref] [bib_ref] Computational analysis of functional long noncoding RNAs reveals lack of peptide-coding capacity..., Niazi [/bib_ref]. We found a median ORF length of 177 nucleotides among the CDS N set, which is lower than the median of 250 nucleotides reported for mammalian lncRNAs [bib_ref] Computational analysis of functional long noncoding RNAs reveals lack of peptide-coding capacity..., Niazi [/bib_ref]. As expected the PSEUDO and LNCRNA sets showed a significantly lower coding capacity when compared to the CDS P set. It demonstrates that CDS N does not just represent erroneous protein-coding gene annotations, but largely non-coding transcripts similar to the non-expressed intergenic regions. Cui and colleagues [bib_ref] Gene expansion in Trichomonas vaginalis: a case study on transmembrane cyclases, Cui [/bib_ref] suggested stop codon readthrough could explain the high number of pseudogenes in T. vaginalis, and which are nearly identical to their evolutionary predecessors and functional counterparts. In consequence, a massive number of genes could have been missed during genome annotation. For a single candidate of the ABC transporter family, tentative evidence exists for stop codon suppression to occur in Trichomonas [bib_ref] The ATP-binding cassette proteins of the deep-branching protozoan parasite Trichomonas vaginalis, Kay [/bib_ref]. However, Western blot evidence for the translation of the full-length protein including its hemagglutinin (HA)-tag was not shown and the authors concluded: "…further experimental work would be required to substantiate this". In the current T. vaginalis genome annotation we found 2,293 cases, in which two annotated genes on the same strand are separated by a maximum of up to 33 codons [fig_ref] Figure 4: No evidence for stop codon suppression in Trichomonas vaginalis [/fig_ref] ; promoter and terminator sequences in the parasite are generally short, hence 99 nucleotides were chosen as an arbitrary cut-off value). For 219 of the 2,293 cases we found expression evidence existing across their combined length. These could represent misannotations, expressed pseudogenes, or cases of stop codon suppression leading to non-interrupted translation. We selected four candidate loci [fig_ref] Figure 4: No evidence for stop codon suppression in Trichomonas vaginalis [/fig_ref] and fused the two adjacent genes to a C-terminal HA-tag and checked for the transcription and translation of the fusion constructs in transfected cells. For one case (TVAG_354100 and TVAG_354110; together encoding the full-length elongation factor 1α) the mRNA reads we obtained and mapped, and our PCR amplification product, suggested an error in the genome assembly and an incorrect annotation (or a strain-specific difference), as the stop codon annotated between the two genes could not be verified. This construct served as an additional control next to the expression of TVAG_386160::HA. In all cases tested we found evidence for the expression of the full-length constructs, but not for their translation [fig_ref] Figure 4: No evidence for stop codon suppression in Trichomonas vaginalis [/fig_ref]. Only the control and the TVAG_354100:: TVAG_354110 construct were translated and detectable through the C-terminal HA-tag. Alternative start codons do not appear to be used by the parasite either (Additional file 2: and although the TAA stop codon is the most frequently encoded (64%), the other two, as expected, are functional (Additional file 2: . Hence, in summary, our results confirm a conservative codon usage by the parasite and that should stop codon suppression exist, it must be very rare and has yet to be experimentally verified. ## Distribution of cds n relative to flanking genes For yeast it has been reported that the expression of lncRNAs is associated with the expression of functional genes encoded in flanking regions [bib_ref] Transcriptional coupling of neighboring genes and gene expression noise: evidence that gene..., Wang [/bib_ref] [bib_ref] Ripples from neighbouring transcription, Ebisuya [/bib_ref]. We analysed the expression of the PSEUDO and LNCRNA sets of Trichomonas vaginalis depending on the four possible orientations to neighbouring genes: divergent (←CDS N →), convergent (→CDS N ←), co-oriented (→CDS N →) and antioriented (←CDS N ←). Distances and distributions of the orientations between PSEUDO and LNCRNA did show differences (see [fig_ref] Table 2: PSEUDO and LNCRNA sets are expressed with no statistic significance in correspondence... [/fig_ref]. The distance between PSEUDO loci and flanking genes was found to be larger compared to the LNCRNA set, while the LNCRNA loci were found in divergent orientations more frequently than a convergent one. Expression of PSEUDO and LNCRNA together with flanking genes in close proximity could indicate coexpression or even the expression as one RNA molecule. To statistically test the association of co-expression with upstream or downstream gens, we performed Yates' chisquared tests (Additional file 3: [fig_ref] Table 2: PSEUDO and LNCRNA sets are expressed with no statistic significance in correspondence... [/fig_ref]. All of the orientations tested, both for PSEUDO and LNCRNA, did not pass the false discovery rate (FDR; P value <0.05; [fig_ref] Table 2: PSEUDO and LNCRNA sets are expressed with no statistic significance in correspondence... [/fig_ref] , demonstrating that no statistically significant correlation regarding the expression of these sets together with their flanking genes. The mean intergenic distance between annotated genes in T. vaginalis was found to be 1165.4 nucleotides [bib_ref] Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis, Carlton [/bib_ref]. The mean distances to neighbouring genes for PSEUDO and LNCRNA range between 1100 and 1700 nucleotides [fig_ref] Table 2: PSEUDO and LNCRNA sets are expressed with no statistic significance in correspondence... [/fig_ref] , being quite similar to that of the annotated genes. Overall the CDS N , PSEUDO and LNCRNA sets behaved "autonomously" and appear independently scattered when compared to flanking, annotated gene orientation Annotated protein-coding genes. [bib_ref] Dramatic reorganisation of Trichomonas endomembranes during amoebal transformation: A possible role for..., Lal [/bib_ref] Intergenic regions without expression evidence randomly selected in size of CDS N . (3) Order of nucleotides randomized per sequence. [bib_ref] Early branching eukaryotes?, Embley [/bib_ref] In reading frame with lowest number of stop codons. and distance. Taken together this indicates that these transcripts are expressed independently from their neighbouring functional genes. PSEUDO and LNCRNA are transcribed, but lack obvious translation start motifs Several promoter motifs including the DNA initiator motif (Inr) have been identified in T. vaginalis [bib_ref] Novel core promoter elements and a cognate transcription factor in the divergent..., Smith [/bib_ref] , and some are linked to the expression of gene subsets induced through changing environmental conditions [bib_ref] Deep sequencing of Trichomonas vaginalis during the early infection of vaginal epithelial..., Gould [/bib_ref]. In order to identify known, as well as new, promoter sequences, the upstream regions of the expressed intergenic loci were screened for overrepresented motifs . A motif similar to the Inr motif of the CDS P (that is annotated and expressed protein-encoding genes) was well represented among upstream sequences of all expressed loci (PSEUDO, LNCRNA). With 16.8% for LNCRNA and 15.5% for PSEUDO, the frequency of the most prominent Inr motif was comparable to the 19.9% of the CDS P set (Additional file 4: [fig_ref] Figure 2: Expression of lncRNAs is conserved among different T [/fig_ref]. Among all loci we identified one non-functional pattern recently described as the M2 motif (AAAGTGAC) [bib_ref] Novel core promoter elements and a cognate transcription factor in the divergent..., Smith [/bib_ref] , but only among the CDS P set the translation-associated M4 motif (AAAAT[T/G]) was identified together with other translation start motifs containing ATG start codons . PSEUDO and LNCRNA display approximately the same amount of known transcription-associated motifs, while lacking any evidence for translation-associated motifs. INTG sequences, for which we found no expression evidence, do not encode any of the previously described motifs, except M2, but with very low frequency. Taken together this demonstrates that lncRNAs and pseudogenes in the parabasalian parasite are not expressed as by-products and in dependence to neighbouring genes Illustration of four selected candidates, in which two adjacent genes share the same reading frame and in combination match to a single BLAST hit. (C) RT-PCR demonstrates the full-length transcription of the gene pairs including the C-terminal HA-tag. RNA was isolated from transfected trichomonads, transcribed into complementary DNA and served as template for the PCR using specific forward and HA-reverse primers (+). RNA served as a negative control (−). (D) Multiplex western blot analysis of the same candidates demonstrates only candidate #1 is translated. 50 μg of protein extract loaded, anti-HA in blue, anti-SCS (succinyl-coenzyme A synthetase subunit alpha; TVAG_047890; 33 kDa) in pink as a loading control, and TVAG_337240::HA served as a positive control. For SCS the double bands are routinely observed [bib_ref] The Nterminal sequences of four major hydrogenosomal proteins are not essential for..., Zimorski [/bib_ref] , and the two additional bands migrating below the 44 kDa TVAG_337240::HA fusion protein likely represent degradation products. as found for other model organisms [bib_ref] Ripples from neighbouring transcription, Ebisuya [/bib_ref] , but because of their own transcriptional initiator motifs. As suggested by Carvunis and colleagues [bib_ref] Proto-genes and de novo gene birth, Carvunis [/bib_ref] , and supported by our data, it is possible that the LNCRNA loci only represent an intermediate and transient form of genetic elements with characteristics from both functional proteins and intergenic regions. In either case, they would not simply represent transcriptional noise, but could serve as a sequence pool for the development of novel functional genes. This would further explain the high number of ORFs identified among the loci and the presence of fully functional promoter motifs. However, it is too early to tell whether any of these fulfil an actual biological function. # Conclusion The vast majority of information available on lncRNA stems from mammals [bib_ref] lncRNAdb: a reference database for long noncoding RNAs, Amaral [/bib_ref]. No analysis dedicated to the characterization of lncRNA or pseudogene expression in protists apart from yeast [bib_ref] Transcription of two long noncoding RNAs mediates mating-type control of gametogenesis in..., Van Werven [/bib_ref] [bib_ref] The transcriptional landscape of the yeast genome defined by RNA sequencing, Nagalakshmi [/bib_ref] is currently available. Our results provide insight into the expression of lncRNAs of a representative of the not well-studied eukaryotic kingdom of excavates. The expression of lncRNAs and pseudogenes in the parabasalian parasite Trichomonas vaginalis is extensive. Almost one-fifth of the transcripts mapped onto non-coding genomic loci, and of which half showed no sequence similarity to annotated genes of the protist. These loci do not encode for canonical proteins, but are clearly distinct from the random sequences that were simultaneously analysed as controls. Intriguingly, and in contrast to yeast [bib_ref] Transcriptional coupling of neighboring genes and gene expression noise: evidence that gene..., Wang [/bib_ref] , the expression of intergenic DNA is not associated with annotated neighbouring genes, but driven by transcription start signals mimicking those of coding genes. The fact that half of the lncRNAs expressed are pseudogenes reflects the dynamic nature of the Trichomonas genome that is characterized by an unknown amount of duplications of at least parts of the genome and large gene families that are unusually frequent. # Methods ## Culture, rna isolation and cdna synthesis Trichomonas vaginalis strains T1, T016 and FMV1 were cultivated in tryptone-yeast extract maltose-medium (2.22% (w/v) tryptose, 1.11% (w/v) yeast extract, 15 mM maltose, 9.16 mM L-cysteine, 1.25 mM L(+)ascorbic acid, 0.77 mM RNA was additionally digested with DNase (DNase I, RNase-free, Therma Scientific). 1 μg of DNase digested RNA was transcribed into cDNA using the "SuperScript III First-Strand Synthesis System for RT-PCR Kit" (Invitrogen) with specific primers as stated below or the iScript Select cDNA Synthesis Kit (Bio-Rad) using its random primer mix according to manufacturer's protocol. The synthesized cDNA was used as template for test-PCRs using specific primers (Additional file 5: . Amplification products were sequenced for verification. ## Sequencing, mapping and assembly RNA-Seq reads were produced by Illumina sequencing of Trichomonas vaginalis under different conditions (Infection and/or oxygen stress at several time points). T. vaginalis was cultured and RNA isolated as described in [bib_ref] Deep sequencing of Trichomonas vaginalis during the early infection of vaginal epithelial..., Gould [/bib_ref] and deep-sequencing was performed by Eurofins MWG (Ebersberg, Germany). Two sequencing approaches had been used: 100 basepairs paired-end reads. The filtered and trimmed reads used here are deposited in Sequence Read Archive (SRA) [bib_ref] International Nucleotide Sequence Database Collaboration: The Sequence Read Archive: explosive growth of..., Kodama [/bib_ref] under Accession SRA059159 (3′-library) and SRA129698 (paired-end reads). Genomic scaffolds of Trichomonas vaginalis, sequences of annotated genes, genomic features (General Feature Format), orthologous gene clusters and additional EST sequences were downloaded from TrichDB V1.3 [bib_ref] GiardiaDB and TrichDB: integrated genomic resources for the eukaryotic protist pathogens Giardia..., Aurrecoechea [/bib_ref] [bib_ref] OrthoMCL-DB: querying a comprehensive multi-species collection of ortholog groups, Chen [/bib_ref]. KOG classifications were adopted from a previous study [bib_ref] Deep sequencing of Trichomonas vaginalis during the early infection of vaginal epithelial..., Gould [/bib_ref]. In order to determine repetitive elements in the genomic scaffolds REAPEATMASKER was used using default parameters, Trichomonas vaginalis as species definition and RMBLAST as the search engine. The reads of both RNA-Seq sequencings were mapped separately to the draft genome and the corresponding genome annotations of Trichomonas vaginalis using TOPHAT2 [bib_ref] TopHat: discovering splice junctions with RNA-Seq, Trapnell [/bib_ref]. Assembly of overlapping reads was performed by CUFFLINKS [bib_ref] Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching..., Trapnell [/bib_ref] and the results of the two samples were merged by CUFFMERGE [bib_ref] Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching..., Trapnell [/bib_ref]. We supplemented the RNA-Seq with additional ESTs from TrichDB. ESTs were matched to the T. vaginalis scaffolds using BLASTN [bib_ref] Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Altschul [/bib_ref] with disabled filtering. Best BLASTN hits with an identity of at least 95% and query coverage of at least 90% were extracted, and overlapping hits were merged to unique loci and combined with overlapping loci from the RNA-Seq experiments using BEDTOOLS [bib_ref] BEDTools: a flexible suite of utilities for comparing genomic features, Quinlan [/bib_ref]. Transcribed loci on smaller scaffolds (<1000 nucleotides) were discarded due to missing gene annotations [bib_ref] Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis, Carlton [/bib_ref]. ## Classification of transcribed loci Gene entries downloaded from TrichDB were used to search for overlap between our transcribed loci and the gene annotations. Overlapping regions were classified as CDS P , while those remaining were referred to as CDS N . Additionally we created two datasets to serve as controls. For the intergenic dataset (INTG) we extracted all sequences longer than 1000 basepairs from the T. vaginalis scaffolds that were not annotated as genes (with a designated TVAG number), not identified through mapped transcripts (CDS N and CDS P ) and were not found in close proximity to the ends of scaffolds. From these we randomly sampled sequences of the same lengths as those in CDS N , thus ensuring an identical length distribution. As a second control set we subjected CDS N sequences to a random permutation of nucleotide order (RND N ). Homologies to annotated T. vaginalis genes were inferred by BLASTN searches of CDS N , CDS P and INTG against the annotated gene sequences, with an e value cutoff of 10 −10 . CDS N loci without hits were classified as LNCRNA. CDS N loci with hits were removed, if either the hit or the query sequence included undetermined nucleotides ("N") or was prematurely terminated due to scaffold termination. Remaining CDS N loci were classified as PSEUDO. Estimates for non-expressed pseudogenes were produced by taking all BLAST hits of annotated genes to intergenic regions with an e value cutoff of 10 −10 and merging those with overlapping locations into single entries. Resulting pseudogene loci were assigned to gene families and KOG categories based on their best BLAST hit to annotated genes with the mentioned e value cutoff. Information on which strand transcribed loci are encoded was inferred by counting TOPHAT hits of the 3′-libraries that are overlapping with the corresponding gene locations. An orientation was assigned, if at least 90% of the matching hits lead to the same orientation. A control with CDS P and the corresponding genes, for which orientations are known, revealed that for 86% of them a unique orientation was identified and 95.4% of them were congruent with overlapping annotations. For CDS N we were able to assign orientations for 79% of the loci. Protein-coding capacities were examined by two different methods. The length of the longest ORFs was defined as the longest peptide sequence in any reading frame beginning with the start of the sequence or a methionine and ending at the next stop codon or the end of the sequence. We defined the frequency of stop codons as the minimum count found inspecting all six reading frames separately. ## Flanking regions and stop codon read-through For motif search upstream regions of transcribed loci were extracted −60 to 40 basepairs relative to the start position. Resulting sequences were clustered using CDHIT [bib_ref] Cd-hit: a fast program for clustering and comparing large sets of protein..., Li [/bib_ref] with a cutoff of 90%. A search for the most overrepresented motifs was conducted using the MEME software V4.7 [bib_ref] Fitting a mixture model by expectation maximization to discover motifs in biopolymers, Bailey [/bib_ref] with window size of 6-8 and zero or one occurrences per sequence. Orientations and distances of transcribed loci to surrounding annotated genes were extracted from genome annotations of scaffolds using their locations. Candidates for stop codon read-through were determined by examining locations of genome features. We searched for gene pairs on the same strand with a distance from 0 to 33 full codons. Transcription of connected genes was determined by using CUFFLINKS results for the pairedend libraries only. Assembled transcripts had to span at least from the stop codon of the one gene to the start codon of the other. ## Cloning and transfection All fragments were cloned into expression vector pTagvag2; for primer sequences refer to Additional file 5: . For lncRNA_ATG the artificial SCS promoter of pTagvag2 [bib_ref] Trichomonas hydrogenosomes contain the NADH dehydrogenase module of mitochondrial complex I, Hrdy [/bib_ref] was replaced by the putative, endogenous promoter region of the candidate (309 bp upstream of open reading frame). To check if all three classical stop codons are valid in T. vaginalis, we altered the stop codon of the HA-tag (TAA) into TGA and TAG and checked the length of the translation of the actin derivative TVAG_054030 (Additional file 2: . To identify potential stop codon suppression, pairs of adjacent genes, for which combined expression evidence was found based on our RNA-Seq data, fragments were amplified with the 5′ oligonucleotide binding to the start codon of first gene and the 3′ oligonucleotide replacing the stop codon of the adjacent gene with an HA-tag (Additional file 5: . All gene sequences were amplified using a proof-reading polymerase and verified through sequencing. 30 μg of the plasmid DNA was used for transfection of roughly 2.5×10 8 T. vaginalis cells using standard electroporation [bib_ref] Transient and selectable transformation of the parasitic protist Trichomonas vaginalis, Delgadillo [/bib_ref]. After four hours of incubation neomycine (G418) was added to a final concentration of 100 μg/ml for selection. Protein samples were separated through standard SDS-PAGE and blotted onto nitrocellulose membrane. Membranes were blocked in 5% milk powder in Tris-buffered saline pH7 (blocking buffer) for 30 min. Blots were incubated with the primary antibodies at a dilution of 1:5,000 in blocking buffer either overnight (ON) at 4°C or for 1 h at room temperature (RT) and then washed 3× with TBS-T (TBS +0.1% Tween 20), followed by the incubation with the secondary, fluorescent antibodies (1:10,000) and identical subsequent washes in the dark. Fluorescence signal was detected using a ChemiDoc™ MP System (Bio-Rad). Antibodies used: monoclonal HA-antibody (Sigma H9658), antibody against succinyl CoA synthetase alpha subunit SCSα [bib_ref] The Nterminal sequences of four major hydrogenosomal proteins are not essential for..., Zimorski [/bib_ref] , Alexa fluor 488 donkey anti-rabbit and Alexa fluor 594 donkey anti-mouse antibodies (Invitrogen). [fig] Figure 2: Expression of lncRNAs is conserved among different T. vaginalis strains. Reverse transcriptase (RT-)PCR was performed on complementary DNA (cDNA) generated from RNA of T. vaginalis strains T1, T016 and FMV1 in the presence (+) or, as control, absence of the reverse transcriptase enzyme (−). All six randomly chosen lncRNAs candidates were found expressed in the three different strains analysed. [/fig] [fig] Figure 3: Comparison of potential coding capacities for the different sets of transcripts identified. (A) shows proportions of BLASTN hits with a given e value to annotated genes of Trichomonas vaginalis. Relative frequencies of CDS P were calculated excluding those e values lower 10 −180 (the dashed bar illustrates the relation compared to all CDS P hits). (B) Distribution of the GC-contents in per cent, showing that CDS P behaves nearly identical to TVAG (TVAG representing annotated genes of the parasite). (C) Distribution of the sequence lengths of the longest ORFs relative to the corresponding full-length sequence. The ORFs of CDS P distribute very differently in comparison to the remaining datasets, while the intergenic regions behave similar to the PSEUDO and LNCRNA sets. (D) Distribution of stop codons over the relative positions in the full sequence of the reading frame showing the lowest number of stop codons. Counts were normalized according to total codons per bin. [/fig] [fig] Figure 4: No evidence for stop codon suppression in Trichomonas vaginalis. (A) Bar diagram of the frequency of annotated gene pairs and their distances in base triplets (light grey). Dark grey bars indicate the gene pairs, for which expression evidence exists. Note that the most abundant distances originate from highly conserved and large gene families. (B) [/fig] [table] Table 1: Protein coding sequence features of the various sets analysed [/table] [table] Table 2: PSEUDO and LNCRNA sets are expressed with no statistic significance in correspondence to flanking genes [/table]