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Abstract Wooden Panel Paintings (WPPs) stand as invaluable cultural artefacts from the past. These works present an intriguing challenge in understanding their complex mechanical behaviour and ensuring their long-term preservation. The present study assumes as founding paradigm the unicity of each WPP, in terms of its material composition, historical background, physical dimensions, and the specific environmental conditions it has been subjected to over time, and their complex behaviour, which requires the knowledge of both mechanical and materials specificity. These characteristics need to be considered, and studied in-depth for each individual WPP, particularly if the aim is to develop a comprehensive understanding of its individual mechanical behaviour. The study provides new computational models calibrated to reproduce the physical and mechanical behaviour of artworks and acting as their 'digital twins'. The models developed contribute significantly to the understanding of the mechanics of these artworks, including the impact of environmental thermo-hygrometric fluctuations and the role of structural elements such as crossbeams. The results, corroborated by experimental analyses, indicate that environmental variations, both short and long term, exert specific and quantifiable effects on the WPPs, and that the presence of crossbeams significantly influences the distribution of stresses within the panel, particularly affecting the pictorial layers that are often the most susceptible to damage. In this context the generated digital twins serve as an invaluable tool, offering the potential to simulate various risk scenarios or to evaluate the effectiveness of engineered conservation interventions on the WPPs.

The concept of participation as a learning process is essential to foster professional identity development. Faculties are expected to provide a curriculum that supports students' participation in the profession's context. Curriculum evaluation is needed to assess the extent to which curriculum implementation supports participation. In this regard, this study aims to develop instruments that measure students' perceptions of the medical education curriculum. The blueprint for the instrument's development was based on the concept of participation in communities of practice theory. Qualitative research, which involved 17 pre-clinical and clinical medical students as participants, was conducted to explore medical students' perception about formal learning activities that encourage participation. The results were used to generate the items. A series of review processes, item reduction, revisions, and analysis generated 20 items in four factors, namely: engagement support, imagination support, convergence, and feedback. This shows that the instrument is multidimensional. The instrument also has good discriminant validity and composite reliability.

Purpose - The purpose of this paper is to conduct systematic reviews on Indonesian papers, to examine the most recent evidence of the efficacy of the combination of cognitive training and physical exercise, and to make recommendations in order to improve prevention, care and treatment services in elderly patients with mild cognitive impairment (MCI). Design/methodology/approach - The databases of Cochrane, Medline, NIH (US National Library Medicine), ProQuest, EbscoHost, Clinical Key, EMBASE, Medical Librarian (TWE) in Ovid, Science Direct, Scopus, The Lancet Global Health, PubMed, Emerald, Indonesian National Library, Google Scholar, Google Indonesia, and Garuda Portal were systematically searched using Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines to obtain empirical papers published between June 1976 and January 2018. Findings - Out of the 3,293 articles collected, 10 were included in this analysis. The result of this combined meta-analysis compares the combination therapy group (cognitive therapy and physical exercise) with a control group. It shows that the control group was likely to experience MCI 1.65 times more often than the combination therapy group. According to the result acquired from the synthesized meta-analysis, the control group experienced MCI 1.65 times higher than the combination therapy. The finding is proven to be statistically significant (95% CI= 1.42–1.93). Research limitations/implications - The research considers only English and Indonesian articles. Practical implications - It is important to explore the most effective training characteristics in a special combined intervention differentiated by the duration, frequency, intervention, type and combination mode. There is a need for further investigation that focuses on the physiological mechanisms underlying the positive effects, by inserting a more comprehensive neuro-imaging measurement to assess specifically the domain that benefits in terms of cognitive functions and molecular markers. Finally, exploratory studies are definitely required, which will specifically examine maintenance and treatment effects as well as derive theoretical explanations related to the interventions and predictors. Social implications - A combination of cognitive training and physical exercise intervention may improve the global health or cognitive functions. Originality/value - A combination of cognitive training and physical exercise has been found to improve prevention, care and treatment services in elderly patients with MCI. There is an increase in value in comparison to the study of Karssemeijer, which considered five Indonesian articles.

The aim of this study was to investigate the impact of early leaf removal (ELR) and cluster thinning (CT) on the phenolic composition of cv. Teran berries and wines, and the sensory profile of wines at 6 and 24 months after fermentation. Although only ELR increased the concentration of total anthocyanins in the berries as compared to the untreated control (UC), both ELR and CT had higher concentration of total anthocyanins and greater colour intensity in the resultant wines than the UC. Similar effects were obtained with total phenolics in the berries and the wines; while CT obtained higher concentration of total phenolics in the berries than UC only in one season, same treatment obtained higher concentration of total phenolics in the wine in both seasons as compared to UC. Only minor sensory differences in wines were obtained at the age of 6 months, while treatments were highly discriminated 24 months after fermentation, when both ELR and CT wines had more enhanced fruitiness, aromatic intensity, complexity, body, balance and wine overall quality than UC. The obtained results indicate that adequate wine aging period is needed for a relevant assessment of the impact of vineyard management practices on wine sensory characteristics.

Background: It is very important to develop professional identity among medical students, as it will affect how they work in the future. Factors affecting professional identity in medical education context need to be explored. This study aimed to develop a model of professional identity in medical students which examine the role of motivation, self-efficacy, personal reflection, social interaction, curriculum in action perceived by students and participation in determining professional identity. Methods: A cross-sectional survey was conducted among medical students in Mataram University. A total of 303 pre-clinical (year 1 to 4) and clinical students (year 5 to 6) filled out the questionnaires. Partial least square - structural equation model analysis was performed. Results: The modification model had a good model fit (GoF 0.404). The average path coefficient was 0.303 (p

Purpose - Community-based HIV testing and counselling (HTC) has been recommended for improving access to prevention, care, and treatment services in at-risk populations. Earlier systematic reviews and meta-analyses have been undertaken, but due to some methodological limitations, their findings do not yet provide a practical significance. The purpose of this paper is to re-examine the recent evidence of the efficacy of community-based HTC approaches on the uptake of HTC in at-risk populations. Design/methodology/approach - The database of PubMed online, Science Direct, the Lancet Global Health, the Cochrane Central Register of Controlled Trials, and Google Scholar were systematically searched using Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines to obtain empirical papers published between March 2013 and December 2015. Findings - Of 600 collected papers, there were 6 cluster randomized trials papers which met the inclusion criteria. Compared to the health facilities-based HTC, community-based HTC approaches have been shown to improve the uptake of HIV testing from 5.8 to 37 per cent, and improve HIV testing in men and their partners together from 6.8 to 34 per cent. The community approaches also detected lower HIV-positive cases (0.29 per cent as compared to 4 per cent), improved access to treatment services from 0.3 to 25 per cent, demonstrated higher cluster differentiation 4 count in newly diagnosed patients (median of 400-438 cells/µl), and increased the rate of first-time HIV testing from 9 to 11.8 per cent. With respect to social and behavioural outcomes, community-based HTC increased social norms for HIV testing by 6 per cent (95 per cent CI 3-9), decreased multiple sex partners by 55 per cent (95 per cent CI 42-73), lowered casual sex by 45 per cent (95 per cent CI 33-62), increased knowledge about HIV (83.2 vs 28.9 per cent), improved positive attitudes towards HIV patients (73.0 vs 34.3 per cent), and increased the use of condoms (28.0 vs 12.3 per cent). Originality/value - Community-based HTC combined with behavioural interventions have been found to be more effective in increasing the uptake of HIV testing as well as other outcomes as compared to the conventional health facilities-based testing and counselling approaches.

Background: Effective learning should be based on an understanding of how students learn. Students who are well adjusted will be actively involved in learning. The role of motivation will encourage individuals to have appropriate strategies to guide their learning, to depthly process the information which in turn produces better learning achievement. Environment creates a learning atmosphere that influences students learning motivation. This study was aimed to find out the correlation between students’ perception of clinical learning environment and learning motivation. Method: The study used quantitative method with cross sectional approach. 70 Ners students of Aisyiyah University of Yogyakarta academic year 2015/2016 answered the Clinical Learning Environment and Supervision (CLES) and Motivated Strategies for Learning Questionnaire (MSLQ) questionaires. Pearson test was used to analyze the data. Results: Most of the students (77.1%) showed positive perception about the clinical learning environment. They were more likely to be motivated extrinsically than intrinsically.The highest value of task value motivation subscales shows a mean value of 5.79 (SD = + 0.67) while the lowest value in the anxiety test sub-scale shows a mean of 3.52 (SD = + 1.23). The clinical learning environment of the head of the room leadership style sub-scale has a weak positive correlation with the motivation to learn (r = 0.297 and p = 0.012). Conclusion: There is a weak positive correlation between students’ perception of clinical learning environment and learning motivation. Clinical learning environment needs to be well prepared to meet the needs and improve students learning motivation.

The purpose of this study was to characterize gait initiation of persons with leg amputation and determine whether prosthetic alignment was a critical parameter in the initiation process. Gait initiation was chosen for study because of the difficult neuromuscular demands placed on the body in negotiating the transition from stance to ambulation. In this investigation, ground reaction force data were collected on seven persons with below-knee amputation. These subjects underwent a series of gait initiation trials while varying prosthetic alignment. An analysis of the data demonstrated key elements in the gait initiation process, including the motion of the center of gravity in preparation for steady-state walking. Significant asymmetries in the force profiles of the residual and nonamputated limbs were also found; gait initiation forces were consistently higher for the prosthetic limb and the timings of maxima and minima were indicative of an intact limb preference. Relatively small changes in prosthesis alignment proved not to have statistically significant effects on generalized force parameters. This result is consistent with the findings of other studies that gait initiation is a motor program with certain invariant characteristics. [ABSTRACT FROM AUTHOR]

Background: Several elements of PBL have been identified and prepared with a questionnaire to be reused. Variables of PBL elements that influence students' learning decisions are assessed using a questionnaire of PBL elements that affect students’ learning decisions. This study is one part of a study reported in a different journal of medical education. The objective of this study was to validate a questionnaire of PBL elements that influence learning decisions of students in FKIK UMY Methods: A total of 584 students from major A, B, C, and D in FKIK UMY on the introductory tutorial groups with Indonesian as discussion language were invited to fill out a questionnaire. Construct validity was assessed by testing the validity of the items, Crönbach alpha, factor analysis, convergent and discriminate validity. Results: A total of 582 students of the major A, B, C, and D FKIK UMY on the tutorial groups with Indonesian as the language of instruction participated in the study (response rate 99.6%). Data showed that the questionnaire on PBL elements that affect student learning decisions have a high construct validity. Conclusion: The questionnaire of PBL elements that influence students’ learning decisions in Indonesian version is valid and can be reused. A deeper, further qualitative research on the other PBL elements can be conducted to improve the questionnaire.

Background: In the process of becoming a doctor, a medical student experiencing the development of professional identity. In this process, students need a broad picture of medical profession. They need to orient themselves in the medical profession to determine the future professional identity as a doctor. Source of the picture of medical profession needs to be explored further. This study aims to explore the ways students get picture of the medical profession. Method: Qualitative research by conducting in-depth interviews to 17 preclinical and clinical medical students. Determinants and emergent coding performed to analyze the results of the interview. Results: Source of imagination can be derived from the curriculum and outside the curriculum. Students get two important things, first was overview of the work environment and how the physician work and second was physician professional values. Characters of role models identified primarily from student interaction with faculties. The characters include faculty’s role as teacher, as doctor, their personal atributes and how they interact with students. Conclusion: Formal curriculum plays important roles in providing and facilitating students to get overview about the work environment and how the doctors work in the health service. Although the transfer of professional value to students is determined by the hidden curriculum, but the formal curriculum can facilitate by providing activities that allows the interaction between generations or conduct review on fiction and non-fiction medical stories and histories that is obtained from media.

Background: Motivation is the force that drives a person to do something. Motivation can affect student learning achievement. The purpose of this study was to examine the relationship between motivation and student learning achievement. Method: This research method was a cross sectional survey. The study was conducted at the Medical Faculty of Lampung University. The study population was students that taking Medical Basic Science (MBS) blocks 3. Samples were from all population. The instrument used to assess motivation was Motivated Strategies of Learning Questionnaire (MSLQ). The questionnaire comprises 6 dimensions divided into 31 questions that intrinsic, extrinsic, task value, control of learning beliefs, self-efficacy and anxiety. Learning achievement were measured by the MCQ which. Questionnaire data were taken at the end of the block MBS3, which analyzed using Pearson Product Moment correlation. Results: The correlation between intrinsic motivation and student’s achievement obtained -0.020 with 0.805 significance (p> 0.05). Relationships intrinsic motivation and student’s achievement showed an inverse relationship. Extrinsic motivation and student’s achievement obtained 0.670 with 0.397 significance (P> 0.05). The correlation task value and student’s achievement 0.066 with 0.403 significance (p> 0.05). The correlation control of learning beliefs and student’s achievement of 0.054 with 0.339 significance (p> 0.05). The correlation of self-efficacy and student’s achievement of 0.054 with 0.496 significance (p> 0.05). The correlation test anxiety and student’s achievement -0.060 with 0.447 significance (p> 0.05). The correlation of total score of motivation and student’s achievement 0.034 with 0.670 significance (p> 0.05). All correlation dimension obtained motivation was weak and not significance. Conclusion: The results showed very weak positive correlation between the total score of motivation to learning achievement.

Background: Evaluation and faculty development program for dental faculty at Muhammadiyah University of Yogyakarta are necessary to improve clinical teacher abilities in skills laboratory. The objective of this study is to conduct pilot study of training program on clinical skills laboratory instructors’ teaching abilities; to measure instructors’ teaching ability before and after the training; and to understand instructors’ perception on the given training. Method: This study was a pretest-posttest single group design accompanied with qualitative method. The subjects are 12 clinical teachers and 91 students of Dental School, Muhammadiyah University of Yogyakarta. Quantitative data analysis by measuring effect size. Focus group discussion conducted after quantitative data were collected Result: (1) There were moderate effect of clinical skills laboratory teacher’s training program to communication and clinical teaching abilities (d> 0,50). (2) The qualitative analysis showed the good perception of participants and feasibility of teacher training program, the lack of teacher of teacher training program, and the advice to improve the quality of teacher training program. Conclusion: Training of skills-lab instructors was needed to improve communication and teaching ability among skills-lab instructors.

Background: Peer teaching is a teaching method that widely applied in many medical institutions. This research was designed to evaluate group discussion effectiveness, intrinsic motivation, and student achievement differences between peer tutor−facilitated students and faculty tutor−facilitated students. Method: This research is an experimental research using post−test only control group design during the implementation of third and fourth module of the Cardiovascular Block (consist of five modules). Fifty-nine third year students were randomly assigned into two groups. Three experimental groups (n= 30) was facilitated by peer tutors (PT) and three control groups (n= 29) was facilitated by faculty tutors (FT). Two students were randomly elected from each PT group as peer tutor. A six hour training was given to the peer tutors. In the end of the third and fourth module, group discussion effectiveness, intrinsic motivation, and student achievement in those modules were evaluated using Tutorial Group Effectivenes Instrument (TGEI) questionnaire, Intrinsic Motivation Inventory (IMI) questionnaire, and 20 multiple choice questions, respectively. Mean differences between groups were measured by Cohen's d with the practical significance at Cohen's d > 1. Results: There was no significant differences of group discussion effectiveness (Cohen’s d= -0.41) and intrinsic motivation (Cohen’s d= -0.83) between two groups. Significant difference in student achievement is found between those groups, peer tutor−facilitated students score higher than faculty tutor−facilitated students (Cohen’s d= 2.96). Peer tutors showed higher achievement than discussants (Cohen’s d= 1.86). Conclusion: The results showed that peer tutor can facilitated group discussion as well as faculty tutor in group discussion effectiveness, intrinsic motivation and student achievement paramaters. There was advantage for students who given role as peer tutor.

Background: Short version instruments are being developed to meet the needs of social sciences researchers who simulataneously use a number of instruments. The Approaches to Learning and Studying Inventory (ALSI) is a short version instrument to measure learning approaches used by students. This analysis is part of a study that is published in different medical education periodicals. The objective of this assessment was to validate the ALSI in a population of second year medical students of the Gadjah Mada University Faculty of Medicine (GMU FM), Yogyakarta. Method: 225 second year students in the regular program at the GMU FM were invited to complete the ALSI questionnaire. To suport construct validities of ALSI, an item’ validity, Crönbach alpha, factor analysis, convergent validity, and discriminant validity were measured. Results: A total of 160 students participated in this study (71,11% response rate). The data collected did not support the construct validities of ALSI. Conclusion: ALSI requires improvement, in particular the items of the surface approach subscale. Instead of the initial five subscales, it is probably more appropriate if the instrument is divided in only three subscales - surface approach, effort management-organized studying, and deep approach-monitoring studying.

Abstract Background Patients with Adult Respiratory Distress Syndrome (ARDS) and Acute Lung Injury (ALI) have low concentrations of disaturated-phosphatidylcholine and surfactant protein-B in bronchoalveolar lavage fluid. No information is available on their turnover. Objectives To analyze disaturated-phosphatidylcholine and surfactant protein-B turnover in patients with ARDS/ALI and in human adults with normal lungs (controls). Methods 2H2O as precursor of disaturated-phosphatidylcholine-palmitate and 113C-Leucine as precursor of surfactant protein-B were administered intravenously to 12 patients with ARDS/ALI and to 8 controls. Disaturated-phosphatidylcholine and surfactant protein-B were isolated from serial tracheal aspirates, and their fractional synthetic rate was derived from the 2H and 13C enrichment curves, obtained by gas chromatography mass spectrometry. Disaturated-phosphatidylcholine, surfactant protein-B, and protein concentrations in tracheal aspirates were also measured. Results 1) Surfactant protein-B turned over at faster rate than disaturated-phosphatidylcholine both in ARDS/ALI patients and in controls. 2) In patients with ARDS/ALI the fractional synthesis rate of disaturated-phosphatidylcholine was 3.1 times higher than in controls (p < 0.01), while the fractional synthesis rate of surfactant protein-B was not different. 3) In ARDS/ALI patients the concentrations of disaturated-phosphatidylcholine and surfactant protein-B in tracheal aspirates were markedly and significantly reduced (17% and 40% of the control values respectively). Conclusions 1) Disaturated-phosphatidylcholine and surfactant protein-B have a different turnover both in healthy and diseased lungs. 2) In ARDS/ALI the synthesis of these two surfactant components may be differently regulated.

Extracellular vesicles (EVs) are membrane-bound particles released by cells that play vital roles in intercellular communication by transporting diverse biologically active molecules, including RNA molecules, including mRNA, miRNA, lncRNA, and other regulatory RNAs. These RNA types are protected within the lipid bilayer of EVs, ensuring their stability and enabling long-distance cellular interactions. Notably, EVs play roles in infection, where pathogens and host cells use EV-mediated RNA transfer to influence immune responses and disease outcomes. For example, bacterial EVs play a crucial role in infection by modulating host immune responses and facilitating pathogen invasion. This review explores the complex interactions between EV-associated RNA and host-pathogen dynamics in bacteria, parasites, and fungi, aiming to uncover molecular mechanisms in infectious diseases and potential therapeutic targets.

Objectives: To assess long-term sexual outcome and quality of life after perineal reconstruction by pedicled anterolateral thigh (ALT) flaps after Fournier's gangrene. Postoperative surgical outcomes were assessed; quality of life and sexual function were assessed at long term follow-up (>12 months) with 2 scientifically validated questionnaires., Methods: We conducted a retrospective analysis of a prospectively maintained database. Long-term sexual function and quality of life were assessed by standardized questionnaires. Descriptive statistics were conducted., Results: 8 patients were included in our study, 5 patients responded to quality of life and sexual function analysis. Surgical outcomes were in line with literature: one minor complication (minor dehiscence requiring a skin graft), one major complication (flap loss, requiring a second, contralateral flap) occurred. No reconstructive failure occurred. Average time to complete wound healing was 17 days (SD ±5). Quality of life scores over 70/100 in four out of five categories; social function was rated lowest: patients reported very few residual complaints. Sexual outcome analysis emphasizes the positive impact of the reconstruction. As expected, Fournier's Gangrene heavily affected patient's sexual health., Conclusions: Perineal reconstruction with ALT shows excellent quality of life, and good sexual health outcomes. Currently the lack of reliable and relatable data leads to the impossibility to compare different reconstructive procedures. We emphasize that assessing sexual function and quality of life after perineal reconstruction is paramount to weigh reconstructive success., Competing Interests: 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., (© 2022 Rossi, de Schoulepnikoff, Guillier, Raffoul and Di Summa.)

The new generation of organoids derived from human pluripotent stem cells holds a promising strategy for modeling host-bacteria interaction studies. Organoids recapitulate the composition, diversity of cell types, and, to some extent, the functional features of the native organ. We generated lung bud organoids derived from human embryonic stem cells to study the interaction of Streptococcus pneumoniae (pneumococcus) with the alveolar epithelium. Invasive pneumococcal disease is an important health problem that may occur as a result of the spread of pneumococcus from the lower respiratory tract to sterile sites. We show here an efficient experimental approach to model the main events of the pneumococcal infection that occur in the human lung, exploring bacterial adherence to the epithelium and internalization and triggering an innate response that includes the interaction with surfactant and the expression of representative cytokines and chemokines. Thus, this model, based on human minilungs, can be used to study pneumococcal virulence factors and the pathogenesis of different serotypes, and it will allow therapeutic interventions in a reliable human context. IMPORTANCE Streptococcus pneumoniae is responsible for high morbidity and mortalities rates worldwide, affecting mainly children and adults older than 65 years. Pneumococcus is also the most common etiologic agent of bacterial pneumonia and nonepidemic meningitis, and it is a frequent cause of bacterial sepsis. Although the introduction of pneumococcal vaccines has decreased the burden of pneumococcal disease, the rise of antibiotic-resistant strains and nonvaccine types by serotype replacement is worrisome. To study the biology of pneumococcus and to establish a reliable human model for pneumococcal pathogenesis, we generated human minilungs from embryonic stem cells. The results show that these organoids can be used to model some events occurring during the interaction of pneumococcus with the lung, such as adherence, internalization, and the initial alveolar innate response. This model also represents a great alternative for studying virulence factors involved in pneumonia, drug screening, and other therapeutic interventions.

Buttock augmentation is a commonly performed aesthetic surgery. Several methods have been described, but only the use of implants or autologous fat is consensually deemed safe and effective. Synthetic fillers in gluteal augmentation have been described despite potential severe long-term complications, both medical and aesthetic. The aim of this study is to report a series of 2 consecutive cases who underwent buttock and hip augmentation with large volumes of permanent copolyamide filler requiring surgical removal due to significant complications. Based on these cases and a review of recent literature, a management algorithm is proposed. The authors conducted a retrospective chart review of 2 consecutive cases of failed copolyamide filler augmentations in the gluteal and inguinal regions. The authors conducted a literature overview using PubMed (National Institutes of Health, Bethesda, MD) and Google Scholar (Google, Mountain View, CA) to include all articles concerning the removal of large quantities of permanent copolyamide fillers. Based on the physical properties of copolyamide, resection of the filler was performed by percutaneous aspiration with liposuction cannulas, with varying infiltration protocols. Both cases showed successful removal of major parts of the filler; however, residual material tended toward migration, requiring a secondary intervention. Hydrated low-pressure aspiration can manage nonintegrated gluteal copolyamide filler but will achieve only partial resection. Literature shows that radical excision is possible, however, with major drawbacks in function and aesthetics. Moreover, in acute inflammation and infection, an open approach should be preferred., (© 2022 The Aesthetic Society.)

As there are more than 6 million human deaths due to mycoses each year, there is an urgent need to develop fungal vaccines. Moreover, given the similarities among pathogenic fungi, it may be possible to create a multi-fungi vaccine. In this study, we combined immunoproteomic and immunopeptidomic methods, for which we have adapted a technique based on co-immunoprecipitation (Co-IP) that made it possible to map Histoplasma capsulatum epitopes for the first time in a natural context using murine dendritic cells (DCs) and macrophages (Mφ). Although polysaccharide epitopes exist, this research focused on mapping protein epitopes as these are more immunogenic. We used different algorithms to screen proteins and peptides identified by two-dimensional electrophoresis (2-D) and Co-IP. Seventeen proteins were revealed by 2-D gels, and 45 and 24 peptides from distinct proteins were presented by DCs and Mφ, respectively. We then determined which epitopes were restricted to MHC-I and II from humans and mice and showed high promiscuity, but lacked identity with human proteins. The 4 most promising peptides were synthesized, and the peptides with and without incorporation into glucan particles induced CD4+ and CD8+ T cell proliferation and produced a Th1 and Th17 response marked by the secretion of high levels of IFN-γ, IL-17 and IL-2. These epitopes were from heat shock protein 60, enolase, and the ATP-dependent molecular chaperone HSC82, and they each have a high degree of identity with proteins expressed by other medically important pathogenic fungi. Thus, the epitopes described in this study have the potential for use in the development of vaccines that could result in cross-protection among fungal species., Competing Interests: 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., (Copyright © 2021 Kischkel, Boniche-Alfaro, Menezes, Rossi, Angeli, de Almeida, Palmisano, Lopes-Bezerra, Nosanchuk and Taborda.)

Due to its location, the fungal cell wall is the compartment that allows the interaction with the environment and/or the host, playing an important role during infection as well as in different biological functions such as cell morphology, cell permeability and protection against stress. All these processes involve the activation of signaling pathways within the cell. The cell wall integrity (CWI) pathway is the main route responsible for maintaining the functionality and proper structure of the cell wall. This pathway is highly conserved in the fungal kingdom and has been extensively characterized in Saccharomyces cerevisiae. However, there are still many unknown aspects of this pathway in the pathogenic fungi, such as Cryptococcus neoformans . This yeast is of particular interest because it is found in the environment, but can also behave as pathogen in multiple organisms, including vertebrates and invertebrates, so it has to adapt to multiple factors to survive in multiple niches. In this review, we summarize the components of the CWI pathway in C. neoformans as well as its involvement in different aspects such as virulence factors, morphological changes, and its role as target for antifungal therapies among others.

Treatment modalities for systemic mycoses are still limited. Currently, the main antifungal therapeutics include polyenes, azoles, and echinocandins. However, even in the setting of appropriate administration of antifungals, mortality rates remain unacceptably high. Moreover, antifungal therapy is expensive, treatment periods can range from weeks to years, and toxicity is also a serious concern. In recent years, the increased number of immunocompromised individuals has contributed to the high global incidence of systemic fungal infections. Given the high morbidity and mortality rates, the complexity of treatment strategies, drug toxicity, and the worldwide burden of disease, there is a need for new and efficient therapeutic means to combat invasive mycoses. One promising avenue that is actively being pursued is nanotechnology, to develop new antifungal therapies and efficient vaccines, since it allows for a targeted delivery of drugs and antigens, which can reduce toxicity and treatment costs. The goal of this review is to discuss studies using nanoparticles to develop new therapeutic options, including vaccination methods, to combat systemic mycoses caused by Candida sp., Cryptococcus sp., Paracoccidioides sp., Histoplasma sp., Coccidioides sp., and Aspergillus sp., in addition to providing important information on the use of different types of nanoparticles, nanocarriers and their corresponding mechanisms of action., (Copyright © 2020 Kischkel, Rossi, Santos Junior, Nosanchuk, Travassos and Taborda.)

Amphotericin B (AmB) is the antifungal with the strongest fungicidal activity, but its use has several limitations, mainly associated with its toxicity. Although some lipidic and liposomal formulations that present reduced toxicity are available, their price limits their application in developing countries. Flucytosine (5FC) has shown synergistic effect with AmB for treatment of some fungal infections, such as cryptococcosis, but again, its price is a limitation for its use in many regions. In the present work, we aimed to identify new drugs that have a minor effect on Cryptococcus neoformans , reducing its growth in the presence of subinhibitory concentrations of AmB. In the initial screening, we found fourteen drugs that had this pattern. Later, checkerboard assays of selected compounds, such as erythromycin, riluzole, nortriptyline, chenodiol, nisoldipine, promazine, chlorcyclizine, cloperastine, and glimepiride, were performed and all of them confirmed for their synergistic effect (fractional inhibitory concentration index [FICI] < 0.5). Additionally, toxicity of these drugs in combination with AmB was tested in mammalian cells and in zebrafish embryos. Harmless compounds, such as the antibiotic erythromycin, were found to have synergic activity with AmB, not only against C. neoformans but also against some Candida spp., in particular against Candida albicans In parallel, we identified drugs that had antifungal activity against C. neoformans and found 43 drugs that completely inhibited the growth of this fungus, such as ciclopirox and auranofin. Our results expand our knowledge about antifungal compounds and open new perspectives in the treatment of invasive mycosis based on repurposing off-patent drugs., (Copyright © 2020 American Society for Microbiology.)

: The increasing incidence in systemic fungal infections in humans has increased focus for the development of fungal vaccines and use of monoclonal antibodies. Invasive mycoses are generally difficult to treat, as most occur in vulnerable individuals, with compromised innate and adaptive immune responses. Mortality rates in the setting of our current antifungal drugs remain excessively high. Moreover, systemic mycoses require prolonged durations of antifungal treatment and side effects frequently occur, particularly drug-induced liver and/or kidney injury. The use of monoclonal antibodies with or without concomitant administration of antifungal drugs emerges as a potentially efficient treatment modality to improve outcomes and reduce chemotherapy toxicities. In this review, we focus on the use of monoclonal antibodies with experimental evidence on the reduction of fungal burden and prolongation of survival in in vivo disease models. Presently, there are no licensed monoclonal antibodies for use in the treatment of systemic mycoses, although the potential of such a vaccine is very high as indicated by the substantial promising results from several experimental models., Competing Interests: The authors declare no conflict of interest.

Candida auris is an emerging fungal pathogen of great concern among the scientific community because it is causing an increasing number of hospital outbreaks of difficult management worldwide. In addition, isolates from this species frequently present reduced susceptibility to azole and echinocandin drugs. For this reason, it is necessary to develop new antifungal strategies to better control the disease caused by this yeast. In this work, we screened drugs from the Prestwick chemical library, which contains 1,280 off-patent compounds that are already approved by the Food and Drug Administration, with the aim of identifying molecules with antifungal activity against C. auris . In an initial screening, we looked for drugs that inhibited the growth of three different C. auris strains and found 27 of them which it did so. Ten active compounds were selected to test the susceptibility profile by using the EUCAST protocol. Antifungal activity was confirmed for seven drugs with MICs ranging from 0.5 to 64 mg/L. Some of these drugs were also tested in combination with voriconazole and anidulafungin at sub-inhibitory concentrations. Our results suggest synergistic interactions between suloctidil and voriconazole with fractional inhibitory concentration index (FICI) values of 0.11 to 0.5 and between ebselen and anidulafungin (FICI, 0.12 to 0.44). Our findings indicate that drug repurposing could be a viable alternative to managing infections by C. auris .

Cryptococcus neoformans is an encapsulated pathogenic yeast that can change the size of the cells during infection. In particular, this process can occur by enlarging the size of the capsule without modifying the size of the cell body, or by increasing the diameter of the cell body, which is normally accompanied by an increase of the capsule too. This last process leads to the formation of cells of an abnormal enlarged size denominated titan cells. Previous works characterized titan cell formation during pulmonary infection but research on this topic has been hampered due to the difficulty to obtain them in vitro. In this work, we describe in vitro conditions (low nutrient, serum supplemented medium at neutral pH) that promote the transition from regular to titan-like cells. Moreover, addition of azide and static incubation of the cultures in a CO2 enriched atmosphere favored cellular enlargement. This transition occurred at low cell densities, suggesting that the process was regulated by quorum sensing molecules and it was independent of the cryptococcal serotype/species. Transition to titan-like cell was impaired by pharmacological inhibition of PKC signaling pathway. Analysis of the gene expression profile during the transition to titan-like cells showed overexpression of enzymes involved in carbohydrate metabolism, as well as proteins from the coatomer complex, and related to iron metabolism. Indeed, we observed that iron limitation also induced the formation of titan cells. Our gene expression analysis also revealed other elements involved in titan cell formation, such as calnexin, whose absence resulted in appearance of abnormal large cells even in regular rich media. In summary, our work provides a new alternative method to investigate titan cell formation devoid the bioethical problems that involve animal experimentation., Competing Interests: The authors have declared that no competing interests exist.

Cryptococcus neoformans is an environmental encapsulated yeast that behaves as an opportunistic pathogen in immunocompromised individuals. The capsule is the main virulence factor of this pathogen. This structure is highly dynamic, and it can change its size and structure according to the environmental conditions. During infection, C. neoformans significantly enlarges the size of the capsule by the addition of new polysaccharide. It is believed that capsule growth is an energy-cost process, but this aspect has never been addressed. In this work, we have evaluated the role of mitochondrial activity on capsule growth using specific inhibitors of the electron respiratory chain. We observed that capsule growth was impaired in the presence of inhibitors of the respiratory chain as salicylhydroxamic acid or antimycin A. Furthermore, capsule growth correlated with an increase of the mitochondrial membrane potential and higher production of reactive oxygen species. Our results confirm that capsule growth depends on mitochondrial activity, and open new insights to understand the regulation of this process.

Pathogenic fungi have developed many strategies to evade the host immune system. Multiple escape mechanisms appear to function together to inhibit attack by the various stages of both the adaptive and the innate immune response. Thus, after entering the host, such pathogens fight to overcome the immune system to allow their survival, colonization and spread to different sites of infection. Consequently, the establishment of a successful infectious process is closely related to the ability of the pathogen to modulate attack by the immune system. Most strategies employed to subvert or exploit the immune system are shared among different species of fungi. In this review, we summarize the main strategies employed for immune evasion by some of the major pathogenic fungi.

Cryptococcus neoformans and C. gattii are fungal pathogens that are most commonly found in infections of the central nervous system, which cause life-threatening meningoencephalitis and can grow as a biofilm. Biofilms are structures conferring protection and resistance of microorganism to the antifungal drugs. This study compared the virulence of planktonic and biofilm cells of C. neoformans and C. gattii in Galleria mellonella model, as well as, the quantification of gene transcripts LAC1, URE1, and CAP59 by real time PCR. All three of the genes showed significantly increased expressions in the biofilm conditions for two species of Cryptococcus, when compared to planktonic cells. C. neoformans and C. gattii cells in the biofilm forms were more virulent than the planktonic cells in G. mellonella. This suggests that the biofilm conditions may contribute to the virulence profile. Our results contribute to a better understanding of the agents of cryptococcosis in the host-yeast aspects of the interaction.

Cryptococcus sp. are responsible for around 1 million cases of meningitis every year. Fluconazole (FLU) is commonly used in the treatment of cryptococcosis, mainly in immunocompromised patients and the resistance is usually reported after long periods of treatment. In this study, the morphological characterization and virulence profile of FLU-susceptible and FLU-resistant clinical and environmental isolates of C. neoformans and C. gattii were performed both in vitro and in vivo using the Galleria mellonella model. FLU-susceptible isolates from C. neoformans were significantly more virulent than the FLU-resistant isolates. FLU-susceptible C. gattii isolates showed a different virulence profile from C. neoformans isolates where only the environmental isolate, CL, was more virulent compared with the resistant isolates. Cell morphology and capsule size were analyzed and the FLU-resistant isolates did not change significantly compared with the most sensitive isolates. Growth at 37°C was also evaluated and in both species, the resistant isolates showed a reduced growth at this temperature, indicating that FLU resistance can affect their growth. Based on the results obtained is possible suggest that FLU resistance can influence the morphology of the isolates and consequently changed the virulence profiles. The most evident results were observed for C. neoformans showing that the adaptation of isolates to antifungal selective pressure influenced the loss of virulence.

Members of the Paracoccidioides genus are the etiologic agents of paracoccidioidomycosis (PCM). This genus is composed of two species: Paracoccidioides brasiliensis and Paracoccidioides lutzii. The correct molecular taxonomic classification of these fungi has created new opportunities for studying and understanding their relationships with their hosts. Paracoccidioides spp. have features that permit their growth under adverse conditions, enable them to adhere to and invade host tissues and may contribute to disease development. Cell wall proteins called adhesins facilitate adhesion and are capable of mediating fungi-host interactions during infection. This study aimed to evaluate the adhesion profile of two species of the genus Paracoccidioides, to analyze the expression of adhesin-encoding genes by real-time PCR and to relate these results to the virulence of the species, as assessed using a survival curve in mice and in Galleria mellonella after blocking the adhesins. A high level of heterogeneity was observed in adhesion and adhesin expression, showing that the 14-3-3 and enolase molecules are the most highly expressed adhesins during pathogen-host interaction. Additionally, a survival curve revealed a correlation between the adhesion rate and survival, with P. brasiliensis showing higher adhesion and adhesin expression levels and greater virulence when compared with P. lutzii. After blocking 14-3-3 and enolase adhesins, we observed modifications in the virulence of these two species, revealing the importance of these molecules during the pathogenesis of members of the Paracoccidioides genus. These results revealed new insights into the host-pathogen interaction of this genus and may enhance our understanding of different isolates that could be useful for the treatment of this mycosis.

Cryptococcus neoformans is an encapsulated opportunistic fungal pathogen that is found in multiple niches in the environment and that can cause fatal meningoencephalitis in susceptible patients, mainly HIV+ individuals. Cryptococcus also infects environmental hosts such as nematodes, insects and plants. In particular, C. neoformans can kill the lepidopteran Galleria mellonella, which offers a useful tool to study microbial virulence and drug efficacy. Galleria mellonella immunity relies on innate responses based on melanization, accumulation of antimicrobial peptides, and cellular responses as phagocytosis or multicellular encapsulation. In this work we have investigated the immune response of G. mellonella during cryptococcal infection. We found that G. mellonella infected with C. neoformans had a high lytic activity in their hemolymph. This response was temperature- and capsule-dependent. During interaction with phagocytic cells, C. neoformans behaved as an intracellular pathogen since it could replicate within hemocytes. Non-lytic events were also observed. In contrast to Candida species, C. neoformans did not induce melanization of G. mellonella after infection. Finally, passage of C. neoformans through G. mellonella resulted in changes in capsule structure as it has been also reported during infection in mammals. Our results highlight that G. mellonella is an optimal model to investigate innate immune responses against C. neoformans.

This work aims to demonstrate that the gallic acid structure modification to the decyl gallate (G14) compound contributed to increase the antifungal activity against several species of pathogenic fungi, mainly, Candida spp., Cryptococcus spp., Paracoccidioides spp., and Histoplasma capsulatum, according to standardized microdilution method described by Clinical Laboratory Standard Institute (CLSI) documents. Moreover this compound has a particularly good selectivity index value, which makes it an excellent candidate for broad-spectrum antifungal prototype and encourages the continuation of subsequent studies for the discovery of its mechanism of action.

Fungal infections in humans have increased alarmingly in recent years, particularly in immunocompromised individuals. Among the infections systemic candidiasis, aspergillosis, cryptococcosis, paracoccidioidomycosis, and histoplasmosis mortality are more prevalent and more severe in humans. The current high incidence of dermatophytosis is in humans, especially as the main etiologic agents Trichophyton rubrum and Trichophyton mentagrophytes. Molecules pristimerin and maytenin obtained from the plant Maytenus ilicifolia (Celastraceae) are known to show various pharmacological activities. This study aimed to evaluate the spectrum of antifungal activity of maytenin and pristimerin and their cytotoxicity in human keratinocytes (NOK cells of the oral mucosa). It was concluded that the best spectrum of antifungal activity has been shown to maytenin with MIC varying from 0.12 to 125 mg/L, although it is also active with pristimerin MIC ranging between 0.12 and 250 mg/L. Regarding the toxicity, both showed to have high IC(50). The SI showed high pristimerin against some species of fungi, but SI maytenin was above 1.0 for all fungi tested, showing a selective action of fungi. However, when comparing the two substances, maytenin also showed better results. The two molecules can be a possible prototype with a broad spectrum of action for the development of new antifungal agents.