*2.2. NLC Preparation and Optimization*

NLC were prepared based on a previously described melt-emulsification technique [16,24]. Four different formulations were developed, combining solid and liquid lipids. The solid lipid Precirol ATO 5 ® (melting point: 56 ◦C) was used in all formulations; however, different liquid lipids in a different solid:lipid ratio were used, as shown Table 1. All nanoformulations shown in Table 1 were performed in triplicate.

**Table 1.** The composition of the different nanoparticles, using Precirol® ATO 5 and different Ω-3 fatty acids or Mygliol® as a liquid lipid with different solid:liquid lipid ratios.

DHA, its hydroxylated derivate in ethyl ester form (DHAH-EE) and Mygliol® (caprylic/capric glyceride) were liquid at room temperature; however, the hydroxylated derivate of DHA in triglyceride form (DHAH-TG) was slightly more viscous. We used two different variants of DHAH, its hydroxylated derivate in ethyl ester form (DHAH-EE) and triglyceride form (DHAH-TG) in order to select the most suitable one to develop our new nanocarrier.

The lipid phase, containing both the solid and the liquid lipid, was heated 5 ◦C above its melting point until a clear and homogeneous phase was obtained. The lipid phase was formed with a different solid:liquid lipid ratio, as described in Table 1. The aqueous solution was composed of Tween 80 (3% *w*/*v*) and poloxamer 188 (2% *w*/*v*) for a final volume of 4 ml. The aqueous phase was warmed and added to the melted oily phase and sonicated for 60 s at 50 W (Branson® Sonifier 250, Fisher Scientific, Madrid, Spain). The obtained nanoemulsion was maintained under magnetic stirring for 15 min at room temperature and stored at 4 ◦C overnight to allow the recrystallisation of the lipid for NLC formation. On the following day, the nanoparticle dispersion was centrifuged in an Amicon filter (Amicon, "Ultracel-100k", Millipore Sigma Life Sciences, Madrid, Spain) at 2500 rpm (MIXTASEL, JP Selecta, Barcelona, Spain) for 15 min and the nanoparticles were washed three times with milli Q water. Finally, 15% *w*/*w* trehalose was added as cryoprotectant, and the NLCs were lyophilized for 42 h (LyoBeta 15, Telstar, Spain).

Prior to the NLC coating process, TAT was covalently linked to CS by a surface activation method previously described by our research group [16] The CS: TAT employed ratio was 1:0.01 (*w*/*w*). TAT conjugation with CS was prepared through a carbodiimide-mediated coupling reaction [37]. The reaction was made though the EDC/NHS technique, in which the reactive sulfo-NHS ester reacted with the amine functionality of proteins and peptides via the formation of an amide bond [38,39]. Briefly, 250 µL EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) in solution (1 mg/mL) and 250 µL of sulfo-NHS (*N*-hydroxysulfosuccinimide) in 0.02 M phosphate buffered saline (PBS) were added dropwise to a 4 mL CS solution (0.5% *w*/*v* in PBS 0.02 M) under magnetic stirring (2 h at room temperature). For the coupling of TAT, a 250 µL TAT solution (1 mg/mL) in PBS (0.02 M; pH 7.4) was added dropwise to the activated CS under gentle agitation. The TAT-CS solution was maintained under agitation for another 4 h at room temperature and then incubated at 4 ◦C overnight. The next day, the NLC were coated with TAT-CS; for that purpose, an NLC dispersion previously prepared was added dropwise to the TAT-CS solution under continuous agitation for 20 min at room temperature. After the coating process, CS-NLC-TAT nanoformulation was centrifuged in Amicon filters (Amicon, "Ultracel-100k", MiIlipore, Millipore Sigma Life Sciences, Madrid, Spain) at 2500 rpm (MIXTASEL, JP Selecta, Barcelona, Spain) for 15 min and washed three times with Milli Q water. Finally, the nanoformulation was freeze-dried with the cryoprotectant trehalose at a final concentration of 15% (*w*/*w*) of the weighed lipid, and then it was lyophilized for 42 h (LyoBeta 15, Telstar, Terrassa, Spain). This final step, regarding the coating with CS and TAT, was only performed after the election of the lipid type and the solid:lipid ratio, resulting in the formulations named (CS-TAT) DHAH-NLC, or just DHAH-NLC, and (CS-TAT) Mygliol-NLC, or just Mygliol-NLC, used in the following described studies in primary cell cultures.


**Author details**

*Acupuncture - Resolving Old Controversies and Pointing New Pathways*

training and the length of experience of the practitioner.

explore everything that could be beneficial to the patient.

**3.7 A treatment for a few open-minded people**

largely from patient to patient.

**3.8 Rejection by a part of clinicians**

using a blunt device. All of them can produce false-positive results.

**3.6 Professional competencies are not universally standardized**

(with skin perforation), or a simulated needle insertion (without skin perforation)

Questions about the competency to practice acupuncture are discussed world-

In our consumerist society, most clients seek immediate solutions for their health problems. Compared to drug treatment, acupuncture may seem a boring, tardy, and expensive option to many people. The treatment with acupuncture typically involves several sessions and follow-up, which may be inconvenient for some patients. Many times, a private treatment has to be paid out-of-pocket, when it is not covered by health insurance policies. The cost of a course of sessions varies widely between providers, and the whole treatment can be expensive. Furthermore, it is unpredictable what will happen when the patient reduces the sessions' frequency or stops the treatment. Patients have to be adequately informed on what to expect, in order to be motivated by realistic prospects. They also must understand that the effectiveness of acupuncture cannot be generalized, as its effect varies

Some clinicians state they find no reason to indicate stranger therapeutic systems, since the value and success of conventional biomedical science are just enough. For clinicians who think so, having to learn the principles and indications of other practices would be an unnecessary task. Such colleagues would indicate acupuncture only when they have no further resources to offer (or even when they want to get rid of the patient, etc.). In some cases, disregarding acupuncture is actually an excuse (conscious or unconscious) for lack of time (or commitment) to

As the game goes on, acupuncture needs to prove on a daily basis that it has nothing to do with other picturesque therapies. A lot of energy must be spent to unstick acupuncture of weird East prescriptions such as the powder of rhinoceros' horn, a useless pinch of keratin that is leading this animal to extinction. However, this is a fact: acupuncture came to stay. The opposition to this therapeutic modality would never eliminate it from the canon of good complementary therapies. However, such opposition can clutter many advances for acupuncture use, from no

wide. Today, each country has its local legislation to assign who is allowed to perform acupuncture. Sometimes, anyone with some qualifications or experience is allowed to be called acupuncturist, even people without formal graduation in a healthcare course. In these cases, the practitioner receives education only focusing on acupuncture and/or TCM, and acupuncture will be offered outside a clinical environment. The risk of diagnosis or treatment delay, due to symptoms masking on a serious disease, is increased if clinical red flags are ignored [11]. The patient protection must be the priority when deciding on training standards and licensure requirements for practitioners. In acupuncture, clinical results vary with the level of

**6**

**4. In short**

Marcelo Saad1 \* and Roberta de Medeiros2

1 Spiritist Medical Association of S. Paulo, S. Paulo, SP, Brazil

2 Centro Universitário S. Camilo, S. Paulo, SP, Brazil

\*Address all correspondence to<EMAIL_ADDRESS>
© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


**1. Introduction**

376 Chronic Kidney Disease

Saydah, S., Eberhardt, M., Rios-Burrows, N., Williams, M., Geiss, L. (2007) "Prevalence of

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Shapiro, E., V. Hartanto, et al. (1992). "The response to alpha blockade in benign prostatic

Styles, R. A., D. E. Neal, et al. (1988). "Long-term monitoring of bladder pressure in chronic

Sutaria, P. M. and D. R. Staskin (2000). "Hydronephrosis and renal deterioration in the

Terris, M. K., N. Afzal, et al. (1998). "Correlation of transrectal ultrasound measurements of

Thomas, A. Z., A. A. Thomas, et al. (2009). "Benign prostatic hyperplasia presenting with

Thorpe, A. and D. Neal (2003). "Benign prostatic hyperplasia." *Lancet* 361(9366): 1359-1367. Tseng, T. Y. and M. L. Stoller (2009). "Obstructive uropathy." *Clin Geriatr Med* 25(3): 437-443. Wasson, J. H., D. J. Reda, et al. (1995). "A comparison of transurethral surgery with watchful

Wei, J. T., E. Calhoun, et al. (2008). "Urologic diseases in america project: benign prostatic

Wein, A. J., Kavoussi, L.R., Novick, A.C., Partin, A.W., Peters, C.A., Ed. (2007). *Campbell-*

Woo, H. H., P. T. Chin, et al. (2011). "Safety and feasibility of the prostatic urethral lift: a

Xia, Z., R. O. Roberts, et al. (1999). "Trends in prostatectomy for benign prostatic hyperplasia

Yamasaki, T., T. Naganuma, et al. (2011). "Association between chronic kidney disease and

Yamasaki, T., Naganuma, T., Iguchi, T., Kuroki, Y., Kuwabara, N., Takemoto, Y., Shoji, T.,

secondary to benign prostatic hyperplasia (BPH)." *BJU Int* 108(1): 82-88. Wu, S. L., N. C. Li, et al. (2006). "Natural history of benign prostate hyperplasia." *Chin Med J*

rate, and post-void residual volume." *Urology* 52(3): 462-466.

*Data*(230): 1-16.

*Pharmacol* 31: 333-341.

*Prostate* 21(4): 297-307.

*Med J* 102(2): 43-44.

*Engl J Med* 332(2): 75-79.

*(Engl)* 119(24): 2085-2089.

*(Carlton)* 16(3): 5.

1159.

hyperplasia." *J Urol* 179(5 Suppl): S75-80.

*Walsh Urology*, Saunders Elsevier

*Nephrology (Carlton)* 16(3): 335-339.

dilatation." *J Urol* 140(2): 330-334.

*Int Urol Nephrol* 32(1): 119-126.

Chronic Kidney Disease and Associated Risk Factors --- United States, 1999--2004."

hyperplasia is related to the percent area density of prostate smooth muscle."

retention of urine: the relationship between detrusor activity and upper tract

elderly due to abnormalities of the lower urinary tract and ureterovesical junction."

prostate and transition zone size with symptom score, bother score, urinary flow

renal failure--what is the role for transurethral resection of the prostate (TURP)?" *Ir*

waiting for moderate symptoms of benign prostatic hyperplasia. The Veterans Affairs Cooperative Study Group on Transurethral Resection of the Prostate." *N*

novel, minimally invasive treatment for lower urinary tract symptoms (LUTS)

among black and white men in the United States: 1980 to 1994." *Urology* 53(6): 1154-

small residual urine volumes in patients with benign prostatic hyperplasia."

Nakatani, T. (2010). "Association between chronic kidney disease and small residual urine volumes in patients with benign prostatic hyperplasia." *Nephrology*  Chronic kidney disease is an increasing public health problem. In the United States, the prevalence is estimated to be approximately 11% of the adult population. Chronic kidney disease may progress to end-stage renal failure, a condition associated with high morbidity and mortality. Diabetes mellitus (DM) is one of the main causes of kidney disease and endstage renal failure. In the United States, DM is the primary diagnosis in 44% of all new cases of renal replacement therapy. Vascular complications are the most common cause of diabetic nephropathy, but it is possible that urinary tract infections (UTIs) also contribute to renal insufficiency in patients with DM.

The urinary tract is normally sterile. However, asymptomatic bacteriuria (ASB), which is defined as the presence of a positive urine culture with at least 10e5 cfu/ml collected from a patient without symptoms of a UTI, is a common phenomenon, especially in women. Different studies report a prevalence of approximately 1-5% among healthy young women, increasing to over 20% in the elderly and 12-26% in women with DM. A Swedish study among 1,462 adult women showed that women with bacteriuria at study entry had an increased risk of having bacteriuria six and twelve years later, compared to women without bacteriuria (Odds Ratio (OR) 6.9 and 3.1, after six and twelve years, respectively). Another Swedish study among 116 schoolgirls with ASB showed that at baseline renal parenchymal reduction was found in 10.3%, while reflux was found in 20.7%, but only 30% of the 116 patients had a history referable to an earlier UTI. A 3-year follow-up of these 116 schoolgirls with ASB (treated or untreated) showed that the risk of developing renal damage as a result of ASB in a schoolgirl with a roentgenographically normal urinary tract seemed to be small.

*Escherichia coli* is the most prevalent causative microorganism in both symptomatic and asymptomatic bacteriuria, accounting for more than 80% of uncomplicated UTIs. Previous studies have demonstrated that patients with renal scarring due to pyelonephritis are at increased risk for the development of hypertension and chronic kidney disease. Results from previous in vitro and in vivo studies indicate that a UTI with *E. coli* can lead to renal damage, either by the microorganism itself or by the following host response. For instance, it has been shown that type 1 fimbriae (the adhesive organelles at the outer surface of the bacterial

Asymptomatic Bacteriuria (ASB), Renal Function and Hypertension 379

distribution in stages of renal function was not different for women with bacteriuria

no (n=442) yes (n=48) **E.coli-bacteriuria**

Fig. 1. Differences in creatinine clearance between women WITHOUT DM with and without ASB. (Meiland R, Stolk RP, Geerlings SE, Peeters PH, Grobbee DE, Coenjaerts FE, Brouwer EC, Hoepelman AI. Association between *Escherichia coli* bacteriuria and renal function in

To obtain follow-up information on end-stage renal failure, we obtained data from the Renal Replacement Registry Netherlands (RENINE) that were available May 2002. RENINE is a foundation in which all Dutch nephrologists participate and where patients are registered who at one time have used kidney replacing therapy (hemodialysis or renal transplantation), with a coverage rate throughout the years of nearly 100%. Data from the baseline cohort and RENINE were matched on (maiden and married) name combined with date of birth to select the cases. A group consisting of four times the number of cases was randomly selected from the baseline cohort to form the control group. Four women participated in the follow-up cohort and were also selected as one of the cases who received kidney replacing therapy during follow-up; one woman underwent kidney transplantation before blood withdrawal (and was excluded for the cohort analysis), three women developed end-stage renal failure thereafter (and were included in both analyses). After excluding four individuals with a missing urine sample 49 cases and 206 controls were included. Among the cases, the mean duration until the date of kidney replacing therapy was 13.8 ± 7.4 years, with a minimum and maximum duration of 1.6 and 25.5 years, respectively. In the control group, the mean follow-up (i.e. the time from participation in the

women: long-term follow-up. Arch Intern Med. 2007 Feb 12;167(3):253-7.)

baseline cohort until study-endpoint in May 2002) was 27.0 ± 0.2 years.

compared to women without bacteriuria.

0

**2.3 Nested case-control study population**

30

60

90

**creatinine clearance (mL/min)**

120

150

membrane) can cause scarring in the renal parenchyma of rats, with large foci of inflammation. This might be due to the activation of polymorphonuclear leukocytes by type 1 fimbriatedstrains, which leads to the release of tissue destroying enzymes. Mice models have shown that although neutrophils are important in bacterial clearance, they can also cause renal damage.

In a clinical study, renal scarring was detected in 29 of 63 adult women ten to twenty years after hospitalization for pyelonephritis. In contrast, no study has convincingly shown that ASB can lead to a clinically relevant decline in renal function in otherwise healthy women. Several authors in the first half of the twentieth century have suggested a role of bacteriuria in the etiology of hypertension, but the pathogenesis is not understood.


**2. Methodological approach**

#### **2.1. Research design**

Constructivist grounded theory (conGT) was adopted in this project. This approach was inspired by Rodwell's [28] constructivism approach to research and Charmaz's [29] work on 'Constructing grounded theory: A practical guide through qualitative analysis'. Their writing provided a methodological map in clarifying the strategies and perspectives for understanding the phenomenon on decision-making in residential care homes for older people. In the research process, knowledge was co-created through the reciprocal relationship that was formed between the researcher and participants as they worked towards exploring an interpretative understanding of the participants' experiences and acknowledging they created multiple meanings of their worlds in which they live in [30].

#### **2.2. Sampling and setting**

**1.3. Defining the issues at play**

224 Gerontology

degree of involvement in making decisions.

**2. Methodological approach**

**2.1. Research design**

In HK, the maintenance of family harmony and filial piety is still very much the core value of the Confucius Chinese traditional practice, despite evidence suggesting that the practice of filial duties has weakened [22–23]. Policy reforms have continued to affirm the view of the 'warm, supportive and stable families are what counts in nurturing the healthy development of individuals' ([24] p. 28). Potentially, there is an expectation that Chinese families still have an important role to play as providers of care, even after older people are admitted into the homes. However, there is very scant information available to understand the respective roles of families and their influences on the care of older people following entry into residential care homes in HK. Despite efforts to involve older people and their significant others in making decisions that affect their care, participation and involvement remain generally minimal and decisions are mainly made by staff based on what they consider to be in the older persons best interests [25]. This is further supported by work undertaken in HK which found that residents became dependent much earlier than needed [26]. It was found that staff acted on behalf of all residents, even for those who did not need their assistance, and perceived the tasks to be within their capabilities. Indeed, there is a need to develop mutual understanding about how to provide care which can truly reflect the competence of older people and minimize unnecessary dependence [27].

Growing old and living in residential care homes need not mean that making decisions becomes a thing of the past. Rather, being able to make informed decisions about personal choices and preferences continues to be important and should be promoted regardless of mental and physical frailty. If the ethos of caring is about understanding what is wanted from older people, merely getting things done for them will not enhance their satisfaction or quality of life. Identifying older people's preferences and values on how care needs ought to be met and their capacity and capability to continue to make decisions about daily living will help to inform appropriate care decisions. Such information will enable staff to re-prioritize and re-organize their work patterns by responding to the older person's expectations of care, and thereby involve those older people who wish to participate in decisions that directly concern their welfare. It is with this intention that this study was undertaken to explore the decision-making experiences and processes taking place in residential care homes from older residents, their families and staff, particularly how residents' needs were met, and their

Constructivist grounded theory (conGT) was adopted in this project. This approach was inspired by Rodwell's [28] constructivism approach to research and Charmaz's [29] work on 'Constructing grounded theory: A practical guide through qualitative analysis'. Their writing provided a methodological map in clarifying the strategies and perspectives for understanding the phenomenon on decision-making in residential care homes for older people. In the research process, knowledge was co-created through the reciprocal relationship that **Table 1** depicts the participants and study settings. Purposive sampling was used to recruit the initial sample and to identify participants who were most likely to provide rich information about the experiences or phenomena of interest [31]. As data collection progressed I recruited people with diverse backgrounds to achieve maximum variation and ensure multiple perspectives [28]. Theoretical sampling followed to sample people, activities and events as guided by the emerging codes and categories. The selection criteria were:

Data collection used a 3 × 3 design with three datasets from residents, families and staff in the three distinct homes. Data collection in home one was completed before commencing concurrent data collection in the second and third homes. In home one, data were collected to provide an orientation and overview of the research problem from the three participant groups. These data informed the selection of another two homes and participants. Homes two and three moved from general discovery of issues to more targeted probing among the participants [28]. The emerging categories were refined to focus on the (co-) constructions that emerged in the data. Data collection terminated when in-depth information, with maximum

**Table 1.** Overview of participants in the three study homes.

variation in participants' views, and a high degree of consensus about the categories and constructions was achieved.

collapsed into broader categories [29]. Similar coded data were compared against the existing extensive data, and the incoming data from other transcripts. The identified sub-categories were compared with the verbatim data and codes to ensure that the 'emergent set of categories and their properties fit the data, work, and were relevant for integrating into a theory' ([35] p. 56). The researcher moved from specific incidents to abstraction, by comparing incidents to incidents and incidents to concepts to determine similarities and differences in an iterative process [33]. Once the codes were all collapsed to form categories, possible relationships among and between the categories were examined to establish a conceptual link between them [29] to theoretically explain decision-making experiences among the stakeholder groups.

Decision-Making Experiences and Patterns in Residential Care Homes for Older Residents…

http://dx.doi.org/10.5772/intechopen.74267

227

Ethical approval was obtained from the Survey and Behavioral Research Ethics Committee of the University to conduct this study. The superintendents of the homes gave me permission to conduct the study. In upholding the ethical principle to respect autonomy, participants were wellinformed about the inquiry, and were given time to ask questions before agreeing to consent to participate. The provision of informed information safeguarded the rights and respects the participant's choice to participate. All participants signed the consent form before being interviewed. They were informed of their rights to stop the interviews, refuse to answer questions and withdraw from the study at any time. They were advised that consenting to the inquiry was entirely voluntary and withdrawing from the study did not influence the care provided by the homes.

Based on the case analyses of the three homes, distinct patterns of decision-making and the influences on the residents' approach to decision-making could be delineated. The processes by which residents were facilitated or hindered from decision-making were strongly influenced by the pattern of decision-making that predominated in the homes. **Table 2** summarizes the three processes and six elements that were identified to capture the subtleties of the process of decision-making for residents that unfolded from entry into the homes to how they

Residents proceeded through a fairly logical approach and had varying degrees of involvement in decision-making. These processes demonstrated how residents settled into the home by becoming familiar with it and then becoming more involved in the decisions that influenced their lives. As residents moved through these decision-making processes that were practiced in the homes, the degree of negotiation that was possible determined the extent to which they were able to negotiate successful, less successful and unsuccessful decisions. As residents engaged in these processes the extent to which the family and staff ensured that decisions were successfully negotiated to meet needs or needs were not met were also highlighted. In comparing the similarities and differences in these processes across the three homes, three decision-making patterns were identified as negotiated, partially-flexible and constrained patterns of decision-making. Through the interactions between the participant groups, negotiations and/or compromises occurred that either facilitated or hindered residents' involvement

**3. Patterns of decision-making and influences on residents**

continued to make decisions to adjust to the living environment.

**2.5. Ethical considerations**

#### **2.3. Interview procedure**

Interviews were the main data collection method used. The main emphasis of interviewing pointed to what, why and how questions were asked, and being able to listen to the participants answering the questions in order to understand the experiences, and deriving interpretations from it [32]. Interviews therefore provided a tentative impetus to make decisions about 'where to go', 'what to look for', 'from whom', and 'how to ask questions' ([28], p. 21). The intention was not to impose a rigid order to the interviews by following each question in a particular sequence, but to allow the interview content to unfold by following the lead of the participants who were telling the story [33]. In fact, the researcher and participants were allowed to go 'beneath the surface of ordinary conversation and examine earlier events, views and feelings' ([29] p. 26–27). The participants were asked to reflect deeply on their experiences and encouraged to talk more. Clarifying and encouraging more information helped to articulate intentions and meanings. Therefore, only a few broad, open-ended semi-structured questions were needed to focus the interviews. A guiding principle in framing the questions was to 'direct questions to collective practices first and then attend to the individual's participation in and views of those practices' ([34] p. 679).

Family and staff members were interviewed once, and residents were interviewed twice (at 2 weeks and between 2 and 3 months after admission). As the study examined how residents made decisions with the support from staff, residents' first interviews and staff interviews were conducted almost simultaneously. Families were interviewed after the residents' second interview. This allowed me to hear the residents' experiences and how staff responded to them before comparing the accounts of residents and staff with the families' to gain fuller perspectives of those experiences.

#### **2.4. Analysis strategy**

Simultaneous data analysis and data collection occurred as far as possible. Constant comparative analysis was the method of data analysis used to generate concepts and to develop the theory through an inductive process of defining, categorizing, comparing data and, explaining and seeking relationships in the data [29]. Each interview was transcribed verbatim as soon as possible after each interview. Once the transcripts were checked for accuracy, the data were subjected to initial and focused coding. Initial coding enabled me to examine the fragments of data (words, lines, segments and incidents) and to give them a label that best summarized and described the data [29], using the participants' words to help me stay close to the data. A table of codes was compiled for each participant group, to which constant comparison of data was undertaken to check whether participants from each care home had identified similar concepts. The idea was to keep or reword existing codes, and only add new codes when new information was forthcoming [33]. The table was updated after changes were made to the coding scheme. The table at a glance helped to identify convergent and divergent opinions, and find gaps in the coded data to direct subsequent data collection [29]. Focused coding synthesized larger segments in the data by examining which significant initial codes could be collapsed into broader categories [29]. Similar coded data were compared against the existing extensive data, and the incoming data from other transcripts. The identified sub-categories were compared with the verbatim data and codes to ensure that the 'emergent set of categories and their properties fit the data, work, and were relevant for integrating into a theory' ([35] p. 56). The researcher moved from specific incidents to abstraction, by comparing incidents to incidents and incidents to concepts to determine similarities and differences in an iterative process [33]. Once the codes were all collapsed to form categories, possible relationships among and between the categories were examined to establish a conceptual link between them [29] to theoretically explain decision-making experiences among the stakeholder groups.

#### **2.5. Ethical considerations**

variation in participants' views, and a high degree of consensus about the categories and

Interviews were the main data collection method used. The main emphasis of interviewing pointed to what, why and how questions were asked, and being able to listen to the participants answering the questions in order to understand the experiences, and deriving interpretations from it [32]. Interviews therefore provided a tentative impetus to make decisions about 'where to go', 'what to look for', 'from whom', and 'how to ask questions' ([28], p. 21). The intention was not to impose a rigid order to the interviews by following each question in a particular sequence, but to allow the interview content to unfold by following the lead of the participants who were telling the story [33]. In fact, the researcher and participants were allowed to go 'beneath the surface of ordinary conversation and examine earlier events, views and feelings' ([29] p. 26–27). The participants were asked to reflect deeply on their experiences and encouraged to talk more. Clarifying and encouraging more information helped to articulate intentions and meanings. Therefore, only a few broad, open-ended semi-structured questions were needed to focus the interviews. A guiding principle in framing the questions was to 'direct questions to collective practices first and then attend to the individual's participation

Family and staff members were interviewed once, and residents were interviewed twice (at 2 weeks and between 2 and 3 months after admission). As the study examined how residents made decisions with the support from staff, residents' first interviews and staff interviews were conducted almost simultaneously. Families were interviewed after the residents' second interview. This allowed me to hear the residents' experiences and how staff responded to them before comparing the accounts of residents and staff with the families' to gain fuller

Simultaneous data analysis and data collection occurred as far as possible. Constant comparative analysis was the method of data analysis used to generate concepts and to develop the theory through an inductive process of defining, categorizing, comparing data and, explaining and seeking relationships in the data [29]. Each interview was transcribed verbatim as soon as possible after each interview. Once the transcripts were checked for accuracy, the data were subjected to initial and focused coding. Initial coding enabled me to examine the fragments of data (words, lines, segments and incidents) and to give them a label that best summarized and described the data [29], using the participants' words to help me stay close to the data. A table of codes was compiled for each participant group, to which constant comparison of data was undertaken to check whether participants from each care home had identified similar concepts. The idea was to keep or reword existing codes, and only add new codes when new information was forthcoming [33]. The table was updated after changes were made to the coding scheme. The table at a glance helped to identify convergent and divergent opinions, and find gaps in the coded data to direct subsequent data collection [29]. Focused coding synthesized larger segments in the data by examining which significant initial codes could be

constructions was achieved.

in and views of those practices' ([34] p. 679).

perspectives of those experiences.

**2.4. Analysis strategy**

**2.3. Interview procedure**

226 Gerontology

Ethical approval was obtained from the Survey and Behavioral Research Ethics Committee of the University to conduct this study. The superintendents of the homes gave me permission to conduct the study. In upholding the ethical principle to respect autonomy, participants were wellinformed about the inquiry, and were given time to ask questions before agreeing to consent to participate. The provision of informed information safeguarded the rights and respects the participant's choice to participate. All participants signed the consent form before being interviewed. They were informed of their rights to stop the interviews, refuse to answer questions and withdraw from the study at any time. They were advised that consenting to the inquiry was entirely voluntary and withdrawing from the study did not influence the care provided by the homes.


*2.8. An Epoxy Ring-Opening Reaction*

Next, 2,2-dimethylpropanoyl chloride (0.5 mL) was added to an aqueous solution of **6** (12.0 mg) at room temperature, and the solution was stirred for 12 h. After the evaporation of the solvent, the product was dissolved in MeOH and purified by reversed-phase HPLC (Phenomenex Hydro-RP column) eluted with CH3OH-H2O (50:50) to yield compound **4** (3.7 mg) and compound **5** (6.4 mg).

#### *2.9. Cell Lines and Culture Conditions*

The human normal cell lines HEK 293T and MRC-9 cell lines were purchased from ECACC (Salisbury, UK). Human normal cell lines PNT2 and RWPE-1 as well as human cancer cell lines PC-3, DU145, 22Rv1, VCaP and LNCaP, as well as a human normal prostate line were purchased from ATCC (Manassas, VA, USA). Human normal cell line HUVEC (passage 11) was kindly donated by Prof. Sonja Loges (University Medical Center Hamburg-Eppendorf, Hamburg, Germany). The human keratinocytes cell line HaCaT was kindly provided by Prof. N. Fusenig (Cancer Research Centre, Heidelberg, Germany). All the cells had a passage number ≤ 30.

Cells were incubated in humidified 5% CO2 at 37 ◦C. The cells were continuously kept in a culture for 3 months maximum and regularly checked for mycoplasma infection using MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, Karlsruhe, Germany) and stable phenotype using light microscopy [17].

The following culture media were used: RPMI medium supplemented with GlutamaxTM-I (Invitrogen, Paisley, UK) with 10% fetal bovine serum (FBS, Invitrogen) and 1% penicillin/streptomycin (Invitrogen) for PC-3, DU145, LNCaP, 22Rv1 and PNT2 cells. DMEM medium supplemented with GlutamaxTM-I (Invitrogen) containing 10% FBS and 1% penicillin/streptomycin (Invitrogen) for MRC-9, HEK 293 and VCaP cells. Clonetics® EGMTM-2 SingleQuots® medium (Lonza, Walkersville, MD, USA) containing 10% FBS for RWPE-1 cells. Clonetics® EGMTM-2 SingleQuots® medium (Lonza, Walkersville, MD, USA) containing 10% FBS for HUVEC cells. DMEM medium (BioloT, St. Petersburg, Russia) containing 10% FBS and 1% penicillin/streptomycin (Invitrogen) for HaCaT cells.

#### *2.10. MTT Assay*

Cytotoxicity of the isolated compounds to mammalian cells was evaluated using an MTT assay as previously reported with minor modification [18]. In brief, 6000 cells/well were seeded in 96-well planes in 100 µL/well and were incubated overnight. Then the media were exchanged with fresh media containing tested compounds in different concentrations. Following 72 h of incubation, 10 µL of MTT solution (5 mg/mL, Sigma-Aldrich, Munich, Germany) was added to each well, the cells were incubated for 2–4 h. Then the media was carefully aspirated and the plates were dried for 2 h. Then 50 µL/well of DMSO was added to each well to dissolve formazan crystals and the absorbance was measured using plate reader according to the manufacture's protocol. The data were analyzed and the IC50s values were calculated using GraphPad Prism software v.9.1.1 (GraphPad Software, San Diego, CA, USA).

#### *2.11. Sortase Activity Inhibition Assay*

The enzymatic activity of sortase A from *Staphylococcus aureus* was determined using SensoLyte 520 Sortase A Activity Assay Kit \* Fluorimetric \* (AnaSpec AS-72229, AnaSpec, San Jose, CA, USA) in accordance with the manufacturer's instructions. Substances **1**–**6** were dissolved in DMSO and diluted with reaction buffer to obtain a final concentration of 0.8% DMSO, which did not affect enzyme activity. DMSO at a concentration of 0.8% was used as a control. PCMB (4-(hydroxymercuri)benzoic acid) was used as sortase A enzyme activity inhibitor. Fluorescence was measured with a plate reader PHERAStar FS (BMG Labtech, Offenburg, Germany) for 60 min, with a time interval of 5 min. The data were processed with MARS Data Analysis v. 3.01R2 (BMG Labtech, Offenburg, Germany). The results were presented as relative fluorescent units (RFUs) and percentage of the control data, calculated using STATISTICA 10.0 software [14].

#### *2.12. Antimicrobial Activity*

The antibacterial activity of compounds **1**–**6** was evaluated as described previously [19]. The bacterial culture of *Staphylococcus aureus* ATCC 21027 (Collection of Marine Microorganisms PIBOC FEBRAS) was cultured in a Petri dish at 37 ◦C for 24 h on solid medium Mueller Hinton broth with agar—16.0 g/L.

The assays were performed in 96-well microplates in appropriate Mueller Hinton broth. Each well contained 90 µL of bacterial suspension (109 CFU/mL). Then, 10 µL diluted at concentrations from 1.5 µM to 100.0 µM using two-fold dilution was added to compounds **1**–**6** (DMSO concentration < 1%). Culture plates were incubated overnight at 37 ◦C, and the OD620 was measured using a Multiskan Spectrum spectrophotometer (Thermo Labsystems Inc., Beverly, MA, USA). Gentamicin was used as a positive control in concentration 1 mg/mL; 1% DMSO solution in PBS as a negative.

#### *2.13. Biofilm Formation*

The inhibition of the reducing biofilm formation and growth was assessed using the crystal violet biofilm assay as described [20]. Mueller Hinton broth was inoculated with 109 CFU/mL of *S. aureus* overnight cultures. A total of 90 µL of this cell suspension was then dispensed into 96-well microtiter plates containing 10 µL of different concentrations of compounds **1**–**6**. After 24 h growth at 37 ◦C the plates were washed with PBS to remove unbound cells. Next, the wells were stained with 0.1% crystal violet solution for 10 min at 37 ◦C. At the completion of the incubation, plates were washed 3 times with PBS and dried. Then, the crystal violet dye from the biofilm was solubilized with 100 µL of ethanol. A total of 100 µL of this solution was then moved to a new microtiter plate for absorbance measurement at λ = 570 nm. The results were reported as percent inhibition normalized to the wild-type control.

#### *2.14. Co-Cultivation of HaCaT Cells with S. aureus*

Co-cultivation of HaCaT cells with *S. aureus* was carried out as described [21]. HaCaT cells at a concentration of 1.5×104 cells per well were seeded in 96-well plates for 24 h. Then, culture medium in each well was changed with *S. aureus* suspension (102 CFU/mL) in full DMEM medium. Fresh DMEM medium without *S. aureus* suspension was added in other wells as needed. Compounds **1**–**6** at a concentration of 10 µM were added in wells after 1 h. HaCaT cells and *S. aureus* were cultured at 37 ◦C in a humidified atmosphere with 5% (*v*/*v*) CO2 for 48 h.

After incubation, the plate was centrifuged at 250× *g* for 10 min and 50 µL of supernatant from each well was transferred into the corresponding wells of an optically clear 96-well plate. An equal volume of the reaction mixture (50 µL) from LDH Cytotoxicity Assay Kit (Abcam, Cambridge, UK) was added to each well and incubated for up to 30 min at room temperature. The absorbance of all samples was measured at λ = 450 nm using a Multiskan FC microplate photometer (Thermo Scientific, Waltham, MA, USA) and expressed in optical units (o.u.).

#### *2.15. Statistical Analysis*

All the experiments were performed in biological triplicates. Statistical analyses were performed using GraphPad Prism v.9.1.1 (GraphPad Software, San Diego, CA, USA) or STATISTICA 10.0 software. The data are reported as mean ± SD (standard deviation). For the analysis of statistical differences between the control and drug-exposed group, a one-way ANOVA test followed by Dunnett's post-hoc test was used. Asterisk (\*) indicates statistically significant difference between the treated group and control group if *p* < 0.05.

#### **3. Results and Discussion**

#### *3.1. Isolated Compounds from Asteromyces cruciatus*

The fungus *Asteromyces cruciatus* was cultivated on a solid rice medium for 21 days. The ethyl acetate extract of the mycelium was fractionated on silica gel, followed by C18- SiO2-column chromatography, and reversed-phase HPLC to produce compounds **1**–**6** (Figure 1).

#### *3.2. Structural Characterization of New Compounds*

The molecular formula of **1** was determined as C11H15ClO3, based on the analysis of HRESIMS (*m/z* 229.0625 [M – H]– calcd for C11H14ClO3, 229.0637), showing the characteristic isotope pattern with one chlorine atom, and confirmed by NMR data. A close inspection of the 1H and 13C NMR data (Tables 1 and 2; Figures S13–S19) of **1** by DEPT and HSQC revealed the presence of one methyl (δH 1.80, δC 23.5), two methylenes (δH 1.65, 1.92, δC 33.1; δH 5.14, 5.18, δC 120.7), and five methines (δH 2.96, δC 28.5; δH 4.00, δC 65.7; δH 3.92, δC 68.2; δH 3.57, δC 70.2 and δH 3.81, δC 71.9), including three oxygen-bearing methines, one *sp2* quaternary carbon and one triple bond (δC 81.7 and 91.1). The 1H–1H COSY correlations of H-1(OH)/H-2(OH)/H-3/H-4(OH)/H2-5/H-6/H-1 together with the 1H-13C HMBC correlations (Figure 2) OH-1 (δH 4.89)/C-6 (δC 28.5); OH-2 (δH 4.92)/C-1 (δC 71.9), C-2 (δC 70.2), C-3 (δC 65.7) and OH-4 (δH 5.06)/C-4 (δC 68.2), C-5 (δC 33.1) indicated the presence of a penta-substituted cyclohexane ring and the location of the hydroxy groups at C-1, C-2 and C-4 in **1**. These data, as well as the chemical shifts of CH-3 (δH 4.00, δC 65.7), indicated the location of the chlorine atom at C-3.

**Figure 1.** Chemical structures of **1**–**6**.

**Figure 2.** Key 1H–1H COSY, 1H–13C HMBC (**a**) and ROESY (**b**) correlations of **1**.

The HMBC correlations from H-40a (δH 5.14) to C-20 (δC 81.7) and C-30 (δC 126.8), from H3-50 (δH 1.80) to C-20 , C-30 and C-40 (δC 120.7) and cross 1H–1H COSY correlations between H2-40 and H3-50 revealed the presence of a 3-methyl-3-buten-1-ynyl side chain in **1**. The correlations H-6 (δH 3.19)/C-10 (δC 87.5) and C-20 (δC 84.1) observed in the HMBC spectrum, recorded in CDCl3 (Figures S20–S22), established the position of the side chain at C-6.

The relative configurations of **1** were assigned based on ROESY correlations (Figure 2) H-6 (δH 2.96)/H-2 (δH 3.57); OH-4 (δH 5.06)/H-5β (δH 1.65) and H-2; H-3 (δH 4.00)/H-2β (δH 1.92), OH-1 (δH 4.89) and 1H-1H coupling constants (Table 1). For further investigation, we analyzed the stereoconfigurations of diol at C-1 and C-2 and for protection of these groups before MTPA-esters obtaining the acetonide derivative (**1a**) of compound **1** (Figure 3) was prepared. The small coupling constant (*J*1,2 = 4.3 Hz) and dissimilar magnetic environment of acetonide methyls (∆ = 0.13 ppm) (Figure S57) indicate an *erythro* configuration of the diol group at C-1 and C-2 [22]. The absolute configuration of **1** was established by the modified Mosher's method [23]. Esterification of **1a** with (*S*)- and (*R*)- MTPA chloride occurred at the C-4 hydroxy group to yield the (*R*)-and (*S*) MTPA esters **1a-1** and **1a-2**, respectively. The observed chemical shift differences ∆δ(δ*S*-δ*R*) (Figure 3) indicated the 4*R* configuration and, therefore, the absolute configurations of **1** were established as 1*R*,2*R*,3*R*,4*R*,6*S*. Compound **1** was named acrucipentyn A.

**Figure 3.** Chemical structure of **1a** (**a**) and ∆δ(δS-δR) values (in ppm) for MTPA esters of **1a** (**b**).

The HRESIMS of **2** and **3** showed the peaks of [M–H]– at *m/z* 229.0634 and *m/z* 229.0631, respectively. These data, coupled with 13C NMR spectral data (DEPT), established the molecular formulas of **2** and **3** as C11H15ClO3 for both. A close inspection of the 1H and 13C NMR data (Tables 1 and 2 and Figures S23–S35) of **2** and **3** by DEPT and HSQC revealed the presence of a penta-substituted cyclohexane ring with three hydroxy groups and a 3-methyl-3-buten-1-ynyl side chain.

The main 1H–1H COSY and HMBC correlations (Figures S26 and S28) indicated that compound **2** has the same planar structure as **1**. The relative configuration of **2** was assigned based on 1H-1H vicinal coupling constants (Table 1) and ROESY (Figure S29) correlations H-6 (δH 2.87)/H-4 (δH 4.22) and H-5β (δH 1.91)/H-1 (δH 3.92). Due to the small amount of compound **2,** the absolute configuration establishing by Mosher's method was impossible. Compound **2** was named acrucipentyn B.

The 1H–1H COSY correlations of H-1(OH)/H-2/H-3(OH)/H-4(OH)/H2-5/H-6 together with the 1H-13C HMBC correlations (Figure S34) OH-1 (δH 4.38)/C-1 (δC 74.2), C-2 (δC 68.9) and C-6 (δC 35.0); OH-3 (δH 4.50)/C-2, C-3 (δC 79.5) and C-4 (δC 71.1), and OH-4 (δH 4.04)/C-3, C-4 and C-5 (δC 34.2) indicated the location of the hydroxy groups at C-1, C-3, C-4 and a chlorine atom at C-2 in a penta-substituted cyclohexane ring of **3**. The structure of the 3-methyl-3-buten-1-ynyl side chain and its position at C-6 in **3** were determined by HMBC correlations (Figure S34), as for acrucipentyn A (**1**).

The relative configurations of the chiral centers in **3** were determined based on 1H-1H coupling constants (Table 1). Using the Mosher's method to determine absolute configurations of compound **3** was unsuccessful, due to lability in this compound. Compound **3** was named acrucipentyn C.

The HRESIMS of **4** showed the peak of [M – H]– at *m/z* 227.0471. These data, coupled with 13C NMR spectral data (DEPT), established the molecular formula of **4** as C11H13ClO3. The 1H and 13C NMR (Tables 1 and 2 and Figures S36–S42), DEPT and HSQC spectra showed the presence of three hydroxy protons (δH 5.25, 5.17, 5.04), one methyl group (δH 1.86, δC 23.0), one olefinic methylene (δH 5.32, 5.27, δC 122.2) and five methines (δH 4.25, δC 63.3; δH 4.37, δC 65.1; δH 4.06, δC 67.9; δH 3.87, δC 68.6 and δH 5.92, δC 136.0), including three oxygen-bearing methines and one olefinic methine, two *sp2* quaternary carbons and one triple bond (δC 88.2 and 89.9).

The HMBC correlations (Figure 4) from H-3 (δH 4.25) to C-1 (δC 67.9), C-2 (δC 68.6), C-4 (δC 65.1) and C-5 (δC 136.0); from H-5 (δH 5.92) to C-1, C-4, C-6 (δC 123.5) and C-10 (δC 88.2), and from OH-4 (δH 5.25) to C-4, C-5, together with 1H–1H COSY correlations of H-1(OH)/H-2(OH)/H-3/H-4(OH)/H-5, indicated the presence of a penta-substituted cyclohexene ring with ∆ 5,6 double bond, the location of the hydroxy groups at C-1, C-2, C-4 and a chlorine atom at C-3 in **4**. The structure of the 3-methyl-3-buten-1-ynyl side chain and its position at C-6 in **4** were determined by HMBC correlations (Figure S41), as for acrucipentyn A (**1**).

**Figure 4.** Key 1H–1H COSY, 1H–13C HMBC correlations of **4** (**a**) and chemical structure of acetonide derivatives **4a** (**b**).

The relative configuration of **4** was assigned based on 1H-1H coupling constants (Table 1) and ROESY correlation (Figure S42) H-2 (δH 3.87)/OH-4 (δH 5.25). The acetonide derivative (**4a**) of compound **4** (Figure 4) was prepared for further investigation of the stereochemistry at the diol position. The small coupling constant (*J*1,2 = 5.7 Hz) and dissimilar magnetic environment of acetonide methyls (∆ = 0.01 ppm) (Figure S63) indicate an *erythro* configuration of the diol group at C-1 and C-2 [21]. Esterification of **4a** with (*S*)- and (*R*)-MTPA-Cl led to destruction of the compound. Etherification of compound **4** with (*S*)- and (*R*)-MTPA chloride resulted in the formation of esters at three hydroxyl groups, which made it impossible to establish the absolute configuration using the modified Mosher's method. Compound **4** was named acrucipentyn D.

The HRESIMS of **5** showed the peak of [M – H]– at *m/z* 227.0468. These data, coupled with 13C NMR spectral data (DEPT), suggested the molecular formula of **5** as C11H13ClO3. The 1H and 13C NMR data (Tables 1 and 2 and Figures S43–S49) of **5** revealed the presence of a penta-substituted cyclohexene ring with three hydroxy groups and a 3-methyl-3-buten-1-ynyl side chain, the same as in **4**. The location of the hydroxy groups at C-1, C-3, C-4, a chlorine atom at C-2 and a 3-methyl-3-buten-1-ynyl side chain at C-6 in **5** were determined by 1H–1H COSY and HMBC correlations (Figures S46 and S48), as for acrucipentyn C (**3**).

The relative configuration of **5** was assigned based on ROESY correlations (Figure S49) H-2 (δH 3.69)/H-4 (δH 3.97), OH-1 (δH 5.73), OH-3 (δH 5.48); H-3 (δH 3.31)/H-1 (δH 4.05), OH-4 (δH 5.31) and 1H-1H coupling constants (Table 1). The attempts to obtain an acetonide of compound **5** were unsuccessful. Etherification of compound **5** with (*S*)- and (*R*)-MTPA-Cl resulted in the formation of esters at three hydroxyl groups, which made it impossible to establish the absolute configuration by the modified Mosher's method. Compound **5** was named acrucipentyn E.

The molecular formula of compound **6** was determined as C11H12O3, based on the analysis of HRESIMS (*m/z* 191.0705 [M – H]−, calcd for C11H11O3 191.0714) and NMR data (Tables 1 and 2; Figures S50–S56). The 1H and 13C NMR data for this compound were similar to those obtained for (+)-asperpentyn [24,25], with the exception of the CH-2 and CH-3 proton and carbon signals. These data, as well as the biogenetic relationship of compound **6** with acrucipentyns D (**4**) and E (**5**), led us to suggest a configuration of asymmetric centers for it, different from the known asperpentyns.

The ROESY spectrum data and 1H-1H coupling constants were useless to establish the relative stereochemistry of **6** unambiguously. Therefore, an epoxy ring-opening reaction was carried out in **6**. The reaction of compound **6** with 2,2-dimethylpropanoyl chloride in an aqueous medium (Figure 5) yielded two products, the spectra of which (1H, 13C NMR, HRESIMS and CD) were identical to acrucipentyns D (**4**) and E (**5**). The presence of only two reaction products confirmed the SN2 mechanism of the epoxy ring opening, which corresponded to the literature data [26]. The orientation of the hydroxyl groups in the reaction products corresponds with the configuration of the epoxy ring in the initial compound. These data made it possible to establish the relative configuration of **6**. Compound **6** was named acrucipentyn F.

**Figure 5.** Scheme of an epoxy ring reaction in compound **6**.

It should be noted that acrucipentyn F is a stereoisomer of well-known fungal metabolites (−)-asperpentyn [27,28] and (+)-asperpentyn [24]. To the best of our knowledge, acrucipentyns are the first chlorine-containing asperpentyn-like compounds. However, it should be noted that several other related groups of 3-methylbutenynyl cyclohexanols, e.g., truncateols from the marine-derived fungi *Truncatella angustata* [29,30] and oxirapentyns from the marine-derived fungi *Beauveria felina* KMM 4639 [31], also have chloro-containing members.

#### *3.3. Biological Activity*

We evaluated the safety and toxicity of compounds **1**–**6** in various human cells.  Indeed, none of the investigated compounds exhibited any significant cytotoxicity at concentrations up to 100 µM, following 72 h of treatment (IC50 > 100 µM, Figure S65). Additionally, no morphological changes of the cells exposed to the isolated compounds (100 µM for 72 h) could be detected (Figure S66). Therefore, the isolated acrucipentyns A–F were assumed to be nontoxic to mammalian (human) cells.

The inhibitory effect of compounds **1**–**6** on sortase A enzyme from *Staphylococcus aureus* activity was investigated to detect their antibacterial potential (Figure 6).

**Figure 6.** The effect of compounds **1**–**6** on sortase A enzymatic activity. (**a**) The effect of compounds **1**–**6** on sortase A enzymatic activity measured after 10 min of incubation with the substrate. (**b**) The time-dependent effect of compound **3** (50 µM) on sortase A enzymatic activity. Data presented as relative fluorescent units (RFU). DMSO (0.8%) did not show any inhibition activity in comparison with sortase A assay buffer and was used as a control. The sortase inhibitor—4-(hydroxymercuri)benzoic acid (PCMB) in DMSO 0.8% was used as a positive control. All experiments were performed in three independent replicates and the data presented as a mean ± standard error mean (SEM). \* indicates the significant differences with *p* ≤ 0.05.

Compounds **1**, **3**, and **5,** at a concentration of 50 µM, significantly decreased sortase A activity by 18%, 30%, and 21%, respectively (Figure 6a). Compounds **4** and **6** showed less significant effects on sortase A enzymatic activity and compound **2** was inactive in this test. It was observed that an increase in concentrations of **1**–**6** up to 80 µM resulted in some decrease in their sortase A inhibitory activity, which was sometimes detected [32]. The inhibitory effect of compound **3** on sortase A activity was detected throughout the entire period of data acquisition (Figure 6b) and the effects of other studied compounds were similar.

To detect the antibacterial activity of isolated acrucipentyns A–E (**1**–**6**), their inhibitory effects on bacterial growth and biofilm formation of *Staphylococcus aureus* were investigated (Figure 7).

**Figure 7.** Effect of acrucipentyns A–F (**1**–**6**) on growth and biofilm formation of *Staphylococcus aureus*. All experiments were performed in three independent replicates and data were presented as a mean ± SEM.

Compounds **1** and **6** have shown the most pronounced antimicrobial effects against Gram-positive bacterium *S. aureus*. Compound **6** almost completely inhibited the growth of *S. aureus* at a concentration of 100 µM; a two-fold decrease in the concentration of the substance also halved the antimicrobial activity. Compound **1** at a concentration of 100 µM reduced the bacterial growth by 60%. A decrease in concentration to 12.5 µM reduced antimicrobial activity up to 50%. Compound **3** at 100 µM inhibited *S. aureus* growth by 50%. A decrease in the concentration of compound **3** led to a two-fold decrease in activity. The antimicrobial effects of **2**, **4,** and **5**, even at the highest used concentration of 100 µM, do not exceed 50% inhibition of bacterial growth.

When studying the effect of compounds **1**–**6** on the ability to inhibit the biofilm formation by Gram-positive bacteria *S. aureus*, it was noted that compounds **3**, **4**, and **6** have the most pronounced inhibitory activity at a concentration of 100 µM, in the case of which the formation of biofilms is almost absent. A high level of inhibition of biofilm formation in the case of substances **3** and **6** is kept up to concentrations of 12.5 and 25 µM, respectively. When substance **4** is diluted twice, its effect on biofilm formation is halved. Compound **1** inhibits the biofilm formation at concentrations of 12.5–100 µM by 50–70%, respectively. Compounds **2** and **5** at a concentration of 100 µM inhibited biofilm formation by 30%.

Sortase A is an essential component of *S. aureus* virulence because it is responsible for the covalent anchoring of many virulent factors of Gram-positive bacteria onto the cell wall (Appendix A) and, as a result, sortase A plays a key role in the pathogenic processes of *S. aureus* infection [33]. The decrease in sortase activity leads to the abolition of bacterial adhesion to mammalian cells and, thus, is one of the mechanisms preventing the formation of biofilms, which are the predominant form of bacterial existence [34].

The inhibitory effect of investigated compounds **1**, **3**, **4**, and **6** on the biofilm formation correlated with an ability to affect the activity of sortase A. Compound **2**, which did not show a significant effect on biofilm formation, also did not have any effect on the sortase A activity. Opposite to this, compound **5**, in the same experiments, had a significant effect on the activity of sortase A, but had a weaker inhibitory effect on the biofilm formation, in comparison with compounds **1**, **3**, **4**, and **6**. Thus, substances **1**–**6** can be assumed as anti-Staphylococcal agents.

To further confirm their antibacterial properties in a model of infectious damage to human cells, we investigated their effects on lactate dehydrogenase (LDH) release from human keratinocytes HaCaT, co-cultivated with *S. aureus* (Figure 8).

**Figure 8.** Effect of acrucipentyns A–F (**1**–**6**) on LDH release from human keratinocytes HaCaT cocultivated with *Staphylococcus aureus* (Sa) for 48 h. All compounds were tested at a concentration of 10 µM. All experiments were performed in triplicates and data are presented as a mean ± SEM. The difference between control (without Sa) and HaCaT/Sa co-cultivation was statistically significant with *p* < 0.05 (one-way ANOVA test). Asterisk (\*) indicates significant differences (*p* < 0.05) between HaCaT/Sa without compounds and HaCaT/Sa with compounds variants.

In normal conditions, LDH weakly releases from cells to culture media. *S. aureus* caused a significant increase in the LDH release from keratinocytes during co-cultivation. The addition of compounds **1**–**6** at a concentration of 10 µM reduced the LDH release by 30–50%. The greatest effect was registered for compounds **1**, **2**, **4,** and **5**.

Interestingly, compounds **2** and **4**, which showed significant activity in co-cultivation HaCaT cells with *S. aureus*, did not show high antibacterial and anti-biofilm-forming activity, in contrast to substances **1** and **5**. We assume that the cytoprotective effect of substances **1**–**6** in the in vitro infectious skin lesions could be due not only to their anti-*S. aureus* effects, but also to their anti-inflammatory and other cytoprotective effects. Recently, we observed similar dual effects during an investigation on flavuside B, an inhibitor of sortase A enzymatic activity derived from fungi. Flavuside B was able to inhibit *S. aureus* growth and biofilm formation, as well as protect HaCaT keratinocytes against *S. aureus* infection in a co-culture model via an anti-inflammatory pathway [14].

Our work is the very first detailed investigation of anti-Staphylococcal activity of isoprenylated cyclohexanols. To the best of our knowledge, earlier, only oxirapentyns A and D were found as antimicrobial agents among related compounds [35]. Thus, this group of secondary metabolites of marine fungi is interesting for future study, including their structure–activity relationships.

#### **4. Conclusions**

Six new isoprenylated cyclohexanols acrucipentyns A–F (**1**–**6**) were isolated from the alga-derived fungus *Asteromyces cruciatus* KMM 4696. The absolute configuration of acrucipentyn A was assigned by the modified Mosher's method and ROESY data. Acrucipentyns A–E are the very first members of chlorine-contained monocyclic cyclohexanols containing a 3-methylbutenynyl unit. The compounds have shown inhibitory activity against sortase A from *Staphylococcus aureus,* as well as inhibition of *S. aureus* growth and biofilm formation, while no cytotoxicty to mammalian cells was observed. Moreover, acrucipentyns A–F (**1**–**6**) protected human keratinocytes HaCaT from *S. aureus* toxicity in skin infection in an in vitro model. Thus, the isolated compounds hold a good potential as antimicrobal agents and should be further investigated.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/jof8050454/s1, Figures S1–S12: CD and UV spectra of compound **1**–**6**, Figures S13–S22, S23–S28, S29–S35, S36–S43, S44–S49 and S50–S56: NMR spectra of compounds **1**–**6**, Figures S57–S58, S59–S60, S61–S62 and S63–S64: NMR spectra of compounds **1a**, **1a-1**, **1a-2**, **4a**, Figure S65: Viability of the various cells exposed to the tested compounds, Figure S66: Microphotographs of human cells exposed to the tested compounds.

**Author Contributions:** Conceptualization, O.I.Z.; Investigation, G.K.O., A.S.A., N.N.K., D.N.P., A.B.R., A.S.M., R.S.P., N.Y.K., A.R.C., E.A.C., O.O.V. and S.A.D.; Methodology, E.A.Y., D.N.P. and A.N.Y.; Project administration, O.I.Z.; Resources, O.I.Z., I.V.G., G.v.A. and A.N.Y.; Supervision, G.v.A. and A.N.Y.; Validation, G.v.A. and A.N.Y.; Visualization, E.A.C.; Writing—Original draft, O.I.Z.; Writing—Review and editing, G.v.A., S.A.D. and A.N.Y. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Russian Science Foundation, grant number 19-74-10014.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are contained within the article or Supplementary Material.

**Acknowledgments:** The study was carried out on equipment at the Collective Facilities Center "The Far Eastern Center for Structural Molecular Research (NMR/MS) PIBOC FEB RAS". The study was carried out using the Collective Facilities Center "Collection of Marine Microorganisms PIBOC FEB RAS".

**Conflicts of Interest:** The authors declare no conflict of interest.


*4.2. Dust Nebulae*

With the SPITZER MIPSGal survey more than 400 small (∼ 1) single bubbles were detected in 24 μm emission [66,67]. An extended sample was even derived using citizen-science and machine-learning methods [68]. For most of these bubbles no optical counterpart is known, making them heavy obscured gas and/or pure dust bubbles. Some of the bubbles contain central, NIR bright stars (some even faintly visible in the optical), while others do not show central sources at all, not even in SPITZER IRAC images 3.6 & 4.5 μm from the GLIMPSE surveys. The nature of these small bubbles were an enigma, until first classification spectra of some the bright central sources were taken [69,70]. Several of those turned out to be massive evolved stars, like blue supergiants, LBV candidates and Wolf-Rayet stars. Others were red supergiants and AGB stars. The nature and origin of the emission of the bubbles however remained uncertain. It could be hot dust, or MIR lines of ionized gas, or both. Taking SPITZER IRS spectra of several of the bubble revealed that all cases exist [71,72]: bubbles for which no central stars are detected seems to be dominated by line emission (mostly the high ionization [OIV] *λ*25.9 μm line), and are therefore most likely planetary nebulae. Bubbles with NIR visible central stars that show dust dominated IRS spectra are even less frequent. Stellar NIR spectroscopic classification again prove that the central stars are dominated by evolved massive stars [73] of various types, with several Wolf-Rayet stars, e.g., [74,75], and a number of LBV candidates or related stars [76–78]. Two of these candidates can now be seen as established LBVs (see Table 2, WS1 [79] and MN48 [80]. Also, several previously known LBVs show MIR nebulae, e.g., MWC 930 [81] or HR Car [82].

Still there are some problems with interpreting the small MIR bubbles and especially the LBV candidate interpretation. Most of these bubbles are round/spherical, e.g., [69], and bipolar structures are rare among the 24 μm bubbles. While this is consistent with the morphology of circumstellar gas nebulae of Wolf-Rayet stars, it seems to contradict the results found for LBV nebulae [52] which have as reported above a preference for bipolar morphologies. From the 24 μm images alone it is not clear, whether the nebulae are (a) dust only (b) partly dust, partly gas, or (c) dominated by ionized gas. The distribution and kinematics of the dust and gas are often different for circumstellar nebulae of massive stars, as e.g., shown for the nebula of the classical LBV AG Carinae [61]. Gail et al. [83] were among the first to investiage the problem of dust formation in an CNO precessed material like LBV envelops. Later hydrodynamical simulations of gas and dust nebulae, e.g., [84], show the small dust grains follow the gas quite well, but the larger grains show their own unique distribution. The morphologies problem may therefore be dominantly a wavelength bias. Last but not least one can speculate about the bubbles being signposts of a more general, previously overlooked short high mass-loss phase in the evolution of many massive stars. We are currently running a program with the LBT infrared spectrograph LUCI to classify more of the central stars using HK band spectroscopy.


**5.1 Other applications**

When used simultaneously with multiple modalities, this combined information can create a better estimate of the robot's environment. Since the panoramic attention map encompasses the robot's entire sensory range of motion beyond the immediate visual field, entities not immediately in camera range can still be tracked. The ability for the robot to obtain a greater sensory awareness of its environment is utilized in the following applications:


4.2.2. CNN-Based Handwritten Arabic Digit Recognition on the Local Data

We separated the local data into 80% training and 20% test sets. The local dataset is balanced in terms of digit types. Then, we applied the CNN architecture shown in Figure 8 to train a model for the local data and tested on the remaining separate test set.

**Figure 8.** The CNN architecture is shown. The layers in the green rectangle are used for feature extraction in DTL. In order to train a model for local data, all layers are used. Conv2D stands for 2D

Convolutional Layer.


*4.3. Resolution of the Localization Problem in a Compact Model*

Image retrieval is an inefficient process due to the fact that the maps are usually composed by a huge number of images and the descriptors have a high dimensionality. Therefore, the computational cost could be a problem. In this case, clustering is used to compact the map. Additionally, indoor environments may present visual aliasing. As explained in Section 3, after clustering, the map M will be formed only by a set of clusters *C* = {*C*1, ..., *Cnc*}, where *nc* is the number of clusters. For each cluster, a representative descriptor was calculated as the average of the descriptors in it and the coordinates of those representatives as the average coordinates of the descriptors that compose that cluster. Thus, a set of cluster representatives {#»*r*1, ..., # »*rnc*} and the coordinates of each representative {(*x*, *y*)*r*1 , ...,(*x*, *y*)*rnc* } are known (ground truth).

The localization in this hierarchical map is carried out as follows. (1) The robot captures a new image *imt* from an unknown position (*xt*, *yt*), which must be estimated, and (2) the descriptor corresponding to the new captured image is obtained (#»*dt*) by using any of the description algorithms explained in Section 2 (FS, HOG, or *gist*). (3) The distance vector is obtained #»*lt* = {*lt*1, ..., *ltnc*} where *ltj* = *dist*{ #»*dt*, #»*rj* } is the distance (one of the three types explained in Section 4.1) between the descriptor #»*dt* and each representative #»*rj* . Finally, (4) the estimated position of the robot (*xe*, *ye*) is the position associated with the nearest neighbour *dnn t* |*t* = *arg* min*j ltj*.

The coordinates of the representatives are not used in the localization step. However, to measure the goodness of the estimation, the geometric distance between (*xt*, *yt*) and the centre of the corresponding cluster (obtained as the average position among the positions of the images that belong to that cluster) is calculated: *error* = (*xe* − *xt*)2 + (*ye* − *yt*)2. Furthermore, the required computational cost to estimate the localization is calculated.


**4.2. Social impacts**

Ecotourism had helped in increasing mutual help and cooperation, controlling antisocial activities and conserving religious and cultural heritage in Ghandruk. It had also played a great role in maintaining peace and prosperity in the society. There is increase in cooperation of people with religious beliefs and religious tolerance [5].

The local people felt that the presence of visitors in remote area enhanced their pride in Barpak. Increased excitement of children was the positive outcomes of ecotourism. Success of ecotour‐ ism is due to coordination of local community members, line agencies and various levels of government, transparency in the development of the homestay progras, clear guidelines and standards, hospitality training and monitoring of environment and bottom up approach of women taking local level ownership and control. Also, ecotourism entrepreneurs sit in a meeting every month reviews the activities and takes feedback to improve their services. Ecotourism addresses gender inequities by the active involvement of women in economic activities associated with tourism [38].


**3.1.2 Rigid objects**

56 Haptics Rendering and Applications

physical one. The former describes the geometrical representation of the shape of the proxy and the objects touched, while the latter comprises the effects of deformation and material properties like friction. In terms of temporal scale it is possible to organize contact distinguishing between first impact and then continuous contact, having two different time

At beginning of haptic rendering research, contact has been modeled by representing the proxy as a single point like in the reference god-object algorithm (Zilles & J. Salisbury, 1995) or as a rigid sphere (Ruspini et al., 1997). In both cases the interaction between the proxy and the objects is based on geometrical considerations with the objective of avoiding penetration. The proxy is mass-less, and it produces a force on the handle proportional to the material's stiffness without being affected by such force. This model is effective for rendering tool proxies that are rigid, with the advantage of high performance, requiring only point or sphere contact with object geometry. This model contains several simplifications that allow discussing the later improvements. First, both the proxy and the object are considered rigid in geometrical terms, while for realistic contact it will be necessary to represent soft fingers and deformable bodies. Second, the contact has no friction, an aspect that can be integrated with or without a complete physic simulation of the proxy. Third, the feedback has only a force component, while contact for grasping requires a torsion component. Fourth, contact is quasi static because there is low frequency contact

There is anyway a general result that has been applied by later approaches with different geometry models and physical properties: the proxy is constrained to move over the surface of the object without penetration while the haptic handle pulls it around. The other general result is the importance of a collision detection method that allows to identify or to predict the intersection of the proxy with the object. For a review on the topic see (Teschner et al., 2005). As in this case it is not necessary that both proxy and object have the same geometrical representation, it is instead more usual the case of adopting an asymmetric

Contacts between objects can be represented with few entities that do not depend on the geometrical representation and the collision detection algorithm. For two contacting objects A and B we identify the two points P and Q that are computed as the innermost points of collision respectively on the two objects. The normal of contacting surfaces can be a general n vector not necessarily directed along QP, and assumed to be toward the inner part of A. In addition, the velocities of the two points are provided, all in world coordinates. The Signorini law of contact expresses the contact of two generic objects by means of two functions: the first is the stress exerted on an object at a given point, and the second is the gap, or penetration depth, that is the projection of the QP vector on the normal. For the assumptions before a positive gap means that the two objects are penetrating. The Signorini model states that, when the contact is resolved, the gap is zero and the object B exerts a pressure toward the object A at point P, or the gap is negative and there is no pressure. When pressure is exerted it can be represented by a force directed along the normal n.

scheme, knowing that the proxy object is under the control of the user.

scales and physical modeling.

transient.

**3.1.1 Contact model**

Objects for haptic interaction can be represented in several ways like implicit functions describing the surface (K Salisbury & Tar, 1997), volumetric objects based on voxels, or distance fields, but the most common are triangulated meshes that allow to rely on proven techniques from the fields of simulation and computer graphics. Due to the timing constraints of haptic rendering these representations can take advantage of boundary representation for collision detection or hierarchical representation of the object for reducing the computational effort. An interesting example is the technique of sensation preserving simplification by Otaduy (Otaduy & Lin, 2003) in which an object is represented by a hierarchy of variations of the object, each more detailed than the parent. Every level is represented by an aggregation of convex parts. In this approach the proxy is also an aggregate of convex parts, while collision detection is performed at a given level by comparing pairs of convex elements using the effective GJK algorithm (Gilbert, Johnson, & Keerthi, 1988). The sensation preservation is taken into account when the algorithm has to decide if it is necessary to descend into the hierarchy or to compute the force feedback at the current level. Surface properties of the pair are used to evaluate if the additional details can provide more sensation information or they are not influent.


**1. Introduction**

The World Health Organization foodborne disease burden epidemiology reference group (WHO-FERG) in 2016, estimated and identified the global burden of 31 microorganisms as a leading cause of deaths from foodborne diseases amounting to 2.8 million disability-adjusted life-years (DALY). Because of this, they must be given due consideration as a possible differential diagnosis in areas of high prevalence of cysticercosis, which is termed to be acquired only from the fecal-oral route (ingestion of infected eggs) and ingestion of the cysticerci in undercooked pork that may lead to intestinal taeniasis. The *Taenia solium* tapeworm infections can lead to cysticercosis, which is a disease that can cause seizures, so it is important to seek treatment. In recent times, researchers reported that cysticercosis is prevalent in most West African countries where favorable conditions for parasitic transmission in both humans and pigs occur widely within the region and as such, defecation in the open field, illicit slaughtering of pigs, and unhygienic way of handling meat with unqualified meat inspectors involved in the process [1–3]. The aim of this is to provide updated knowledge as regards cysticercosis diagnostic tools challenges outlined by WHO report in endemic resource-limiting settings and specify needs for adoption to local context considering practical implementation to advance the desire goal for test determinants.


**2.6. Genome clustering pipeline**

type is designed to ignore differences in protein lengths and tuned to measure dissimilarity in internal parts of the sequences. The subsequent genomic distance averages over the majority

Consider proteins i and j, having the best aggregated BLAST alignment of length *L ij* with

, *sii* =*Sii* / *L i*

( ) 1 min 1, min , *ij*

Distance (1) is an identity-like characteristic calculated from the aggregated BLAST [17] scores (using positives based on BLOSUM62 matrix [22]). For full-length alignment, it can be reduced

= - ç ÷

. Assume that the proteins have lengths *L i* and *L j* and self-scores *Sii*

*ii jj*

) . However, when lengths are different; distance (1) avoids penalizing nona‐

. Then robust genomic distance is defined by the formula:

( ) ( )

*d l*

*l*

( )

1 ,

are corresponding alignment length. The marker-protein distances are weighted by

in order to provide where possible results similar to the original method

*h* −2Δ*ij*

*s s*

*s*

æ ö

ç ÷ è ø

, *sjj* =*S jj* / *L j*

.

*a* types of markers found in both genomes, with *Nij*

*h* ). Order marker distances in the ascending

and

(1)

*h* of

(2)

allows filtering out

distances in the middle. For each

of marker-distances, ignoring the outliers.

234 Next Generation Sequencing - Advances, Applications and Challenges

. Define normalized scores: *sij* =*Sij* / *L ij*

*ij*

ligned ends of the proteins, taking into account only mutation events.

*h*

*D*

= å

in [8] based on concatenation of proteins. However, the use of offset Δ*ij*

4 , 1 + *Nij*

*a* − *Nij*

1

phylum level group, an agglomerative hierarchical clustering tree is built using the complete

*p N p p ij ij p ij p N p ij p*

*h ij ij ij h ij ij ij*

å

= -D - =D = -D - =D

*d*

*2.5.1. Protein distances*

aggregated score *Sij*

Then define protein distances:

*S jj*

to 1− *Sij* min(*Sii* , *S jj*

order: *dij*

where *l ij* ( *p*)

(0) ≤*dij* (1) ≤...≤*dij* (*Nij h* −1)

alignment lengths *l*

*2.5.2. Genomic distances*

Suppose that genomes *i* and *j* have *Nij*

*ij* ( *p*)

linkage clustering algorithm [19, 20].

outliers since the averaging in (2) is performed over *Nij*

them having acceptable BLAST hits.

Define the offset Δ*ij* =max(3, *Nij*

The pipeline for calculating genome clades consists of three major components (see Figure 1). The first is the collection of the input data from NCBI main sequence repositories. The genomic data are dynamic: hundreds of new genomes and assembly updates are submitted to NCBI each day. We create a snapshot of all live genome assemblies and their nucleotide sequence components (chromosomes, scaffolds, and contigs) and store them in an internal relational database: UniCol. The genome data set is organized into large groups (phyla and superphyla defined by NCBI Taxonomy). The assemblies are then filtered by quality and passed to the processing script. Ribosomal protein markers are predicted in every genome to overcome problems with the genome annotations (missing and/or incorrect annotations) and to normal‐ ize markers' data set. Marker predictions are performed by aligning reference protein markers against full genome assemblies. Assemblies with at least 17 markers are passed to the next step. Genome distance is calculated as an average of pairwise protein distances of markers shared in a pair of genomes. Finally, agglomerative hierarchical clustering trees are built within phylum-level groups. Clades at the species level are calculated using species-aware algorithm. Superclade trees are constructed by sectioning the trees at the distance of 0.25.

**Figure 1.** Dataflow of ribosomal-marker-based clade (genome group) processing. Ribosomal markers (in green) are maintained outside of the main pipeline (in blue). Clades and markers are available on NCBI FTP site: ftp:// ftp.ncbi.nlm.nih.gov/genomes/GENOME\_REPORTS/CLADES/ ftp://ftp.ncbi.nlm.nih.gov/genomes/ GENOME\_REPORTS/MARKERS/


**Abstract**

Computer games and, of course, the development associated with them have been in the spotlight for many years; recently, also with technologies, such as virtual reality or extended reality. The main part of this chapter presents the classification of collaborative XR systems, the concept of the major application architectures and the consistency models of shared virtual game environments. The next subchapter briefly deals with the sharing of property ownership. The mentioned concepts and examples used in the chapter are implemented in many works and projects using a collaborative environment (also in gamified form) developed in the laboratory LIRKIS, the home laboratory of the authors. The knowledge presented in this chapter may provide tips and inspiration for some other game projects, and practical and useful notes on the advantages or disadvantages of some systems will be interesting and useful.

**Keywords:** extended reality, virtual reality, mixed reality, augmented reality, web-based collaborative environments