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Quaternary glaciations severely altered landscape/seascape at high latitudes and had major consequences on species geographical ranges, population sizes, genetic differentiation and speciation rates. The Magellan province in southern South America, constitutes an interesting area to evaluate the effect of glaciations over near-shore marine benthic organisms. Existing data have showed clear signatures of population genetic bottlenecks during glacial maxima followed by recent recolonizations and expansions during the interglacial. Here, we present new population-based analyses in species of the patellogastropod genus Nacella (N. deaurata, N. magellanica, and N. mytilina). The species N. magellanica and N. deaurata inhabit rocky ecosystems while N. mytilina lives closely associated with kelps. The analyzed species exhibit narrow bathymetric ranges and consequently should have been severely affected by recurrent glacial cycles. We performed phylogeographic and demographic analyses in Nacella species including different localities across their respective distributions in the Magellan province including the Falkland/Malvinas Islands (F/M). Genetic analyses showed that all Nacella species exhibited low levels of genetic diversity, the presence of single dominant broadly distributed haplotypes, lack of correlation between geographical and genetic distance, and recent demographic growths, which are evidence supporting rapid postglacial expansions. Such results may have been facilitated by larval and/or rafting-mediated dispersal following the Cape Horn Current System. The three species showed strong and significant differentiation between F/M and southern Pacific margin (SPM) populations including localities across the Strait of Magellan and Cape Horn. Haplotype genealogies and mismatch analyses recognized older and more complex demographic histories in the F/M than in South America. Different glaciological histories between SPM and F/M may be responsible of the marked phylogeographic structure in the analyzed species of Nacella. Alternatively, as previously proposed, the F/M represent a glacial refugium for Nacella species (sink area), as well as a secondary contact zone where endemic haplotypes are found together with recently arrived South American ones. Historical and contemporary processes, contrasting glacial histories between the analyzed areas, as well as life history traits of the analyzed organisms are main factors explaining current phylogeographic patterns of shallow Magellan marine benthic organisms.

Abstract Mammalian captive dietary specialists like folivores are prone to gastrointestinal distress and primate dietary specialists suffer the greatest gut microbiome diversity losses in captivity compared to the wild. Marmosets represent another group of dietary specialists, exudivores that eat plant exudates, but whose microbiome remains relatively less studied. The common occurrence of gastrointestinal distress in captive marmosets prompted us to study the Callithrix gut microbiome composition and predictive function through bacterial 16S ribosomal RNA V4 region sequencing. We sampled 59 wild and captive Callithrix across four species and their hybrids. Host environment had a stronger effect on the gut microbiome than host taxon. Wild Callithrix gut microbiomes were enriched for Bifidobacterium, which process host-indigestible carbohydrates. Captive marmoset guts were enriched for Enterobacteriaceae, a family containing pathogenic bacteria. While gut microbiome function was similar across marmosets, Enterobacteriaceae seem to carry out most functional activities in captive host guts. More diverse bacterial taxa seem to perform gut functions in wild marmosets, with Bifidobacterium being important for carbohydrate metabolism. Captive marmosets showed gut microbiome composition aspects seen in human gastrointestinal diseases. Thus, captivity may perturb the exudivore gut microbiome, which raises implications for captive exudivore welfare and calls for husbandry modifications.

Young children's eating behavior is crucial for any further development of healthy eating. Early eating behavior are often assessed through parental report. The Children's Eating Behaviour Questionnaire (CEBQ) is a widely used parental questionnaire that has been validated in families of different gender, age and cultural background. Research has shown that the 8-factor structure has some inconsistencies and sample characteristics such as age, gender, and culture can influence the results. To which extent such sample characteristics might influence results within a multi-lingual culture has not been investigated so far. Therefore, the aim of the study was to evaluate the factor structure of the CEBQ among 511 preschool children of the French and German parts of Switzerland, aged 2 to 6 years (Mean 3.85 years; SD 0.69). Confirmatory Factor Analysis showed a modified structure of the original questionnaire, with a 7-factor structure providing a reasonable fit to the data (TLI = 0.954, CFI = 0.952, RMSEA = 0.063 and SRMR = 0.067). The subscale 'Desire to drink' was removed, and a few items moved to other subscales as they loaded higher on a different subscale compared to the original model. Reliabilities based on the coefficient omega were acceptable to satisfying across the seven factors, ranging from 0.66 to 0.90. There were no significant gender or age differences, but French speaking children showed higher levels of 'Satiety responsiveness' and lower 'Enjoyment of food' than German speaking children. Yet, these effects were small. The German and French CEBQ are valid and reliable versions of the original CEBQ and can be used in a multicultural context.

IntroductionHIV pre-exposure prophylaxis (PrEP), in which people take HIV medication to prevent HIV acquisition, underpins global HIV transmission elimination strategies. Effective prevention needs people to adhere to PrEP and remain in care during periods of risk, but this is difficult to achieve. We undertook a process evaluation of Scotland's PrEP programme to explore barriers and facilitators to PrEP adherence and retention in care and to systematically develop evidence-based, theoretically-informed recommendations to address them.MethodsWe conducted semi-structured interviews and focus groups (09/2018-07/2019) with patients who identified as gay or bisexual men and were either using PrEP, had declined the offer of PrEP, had stopped PrEP, or had been assessed as ineligible for PrEP (n = 39 of whom n = 5 (13%) identified as trans, median age 31 years and interquartile range 14 years), healthcare professionals involved in PrEP provision (n = 54 including specialist sexual health doctors and nurses of various grades, PrEP prescribing general practitioners, health promotion officers, midwifes, and a PrEP clinical secretary), and clients (n = 9) and staff (n = 15) of non-governmental organisations with an HIV prevention remit across Scotland. We used thematic analysis to map key barriers and facilitators to priority areas that could enhance adherence and retention in care. We used implementation science analytic tools (Theoretical Domains Framework, Intervention Functions, Behaviour Change Technique Taxonomy, APEASE criteria) and expert opinion to systematically generate recommendations.ResultsBarriers included perceived complexity of on-demand dosing, tendency for users to stop PrEP before seeking professional support, troublesome side-effects, limited flexibility in the settings/timings/nature of review appointments, PrEP-related stigma and emerging stigmas around not using PrEP. Facilitators included flexible appointment scheduling, reminders, and processes to follow up non-attenders. Examples of the 25 recommendations include: emphasising benefits of PrEP reviews and providing appointments flexibly within individualised PrEP care; using clinic systems to remind/recall PrEP users; supporting PrEP conversations among sexual partners; clear on-demand dosing guidance; encouraging good PrEP citizenship; detailed discussion on managing side-effects and care/coping planning activities.ConclusionsPrEP adherence and retention in care is challenging, reducing the effectiveness of PrEP at individual and population levels. We identify and provide solutions to where and how collaborative interventions across public health, clinical, and community practice could address these challenges.

Abstract The Brazilian buffy-tufted-ear marmoset (Callithrix aurita), one of the world’s most endangered primates, is threatened by anthropogenic hybridization with exotic, invasive marmoset species. As there are few genetic data available for C. aurita, we developed a PCR-free protocol with minimal technical requirements to rapidly generate genomic data with genomic skimming and portable nanopore sequencing. With this direct DNA sequencing approach, we successfully determined the complete mitogenome of a marmoset that we initially identified as C. aurita. The obtained nanopore-assembled sequence was highly concordant with a Sanger sequenced version of the same mitogenome. Phylogenetic analyses unexpectedly revealed that our specimen was a cryptic hybrid, with a C. aurita phenotype and C. penicillata mitogenome lineage. We also used publicly available mitogenome data to determine diversity estimates for C. aurita and three other marmoset species. Mitogenomics holds great potential to address deficiencies in genomic data for endangered, non-model species such as C. aurita. However, we discuss why mitogenomic approaches should be used in conjunction with other data for marmoset species identification. Finally, we discuss the utility and implications of our results and genomic skimming/nanopore approach for conservation and evolutionary studies of C. aurita and other marmosets.

Abstract Metagenomic and traditional paleolimnological approaches are suitable to infer past biological and environmental changes, however, they are often applied independently, especially in tropical regions. We combined both approaches to investigate Holocene Prokaryote and Eukaryote diversity and microbial metabolic pathways in ancient Lake Chalco, Mexico. Here, we report on diversity among a large number of lineages (36,722 OTUs) and functional diversity (27,636,243 non-clustered predicted proteins, and 6,144 annotated protein-family genes). The most abundant domain is Bacteria (81%), followed by Archaea (15%) and Eukarya (3%). We also determined the diversity of protein families and their relationship to metabolic pathways. The early Holocene (> 11,000 cal years BP) lake was characterized by cool, freshwater conditions, which later became warmer and hyposaline (11,000–6,000 cal years BP). We found high abundances of cyanobacteria, and fungi groups associated with mature forests in these sediments. Bacteria and Archaea include mainly anaerobes and extremophiles that are involved in the sulfur, nitrogen, and carbon cycles. We found evidence for early human impacts, including landscape modifications and lake eutrophication, which began ~ 6,000 cal years BP. Subsaline, temperate conditions were inferred for the past 5,000 years. Finally, we found nitrogen-fixing bacteria and protein-family genes that are linked to contaminated environments, as well as several fungal pathogens of crops in near-surface sediments.

Abstract Two species of scallop, Austrochlamys natans (“Ostión del Sur”) and Zygochlamys patagonica (“Ostión patagonico”) are presently exploited in the southern part of the Magallanes Province (MP). The lack of clarity in taxonomic identification and ecological aspects is generating both erroneous extraction statistics and an unperceived harvesting pressure on A. natans and Z. patagonica. We aim to discriminate these Magallanes scallops accurately, improve our understanding of their complex natural history and discuss possible implications for their management and conservation status, given the current fisheries statistics. To achieve these goals, we present a complete review of the historical identification of the Magallanes scallop and a multi-locus molecular phylogeny which allowed us to recover the phylogenetic position of A. natans. We sampled 54 individuals from five localities across the southern Pacific coast of the MP. We calculated the depth of the byssal notch (BND) and shell height (VH) ratio from morphological characters and conducted phylogenetic reconstructions with mitochondrial (12S and 16S) and nuclear markers (28S) using Bayesian and maximum likelihood analyses. Both morphology and molecular phylogeny identified two distinct entities, Z. patagonica and a distinct, highly divergent lineage that corresponds to A. natans. Our study provides integrative evidence to alert the current fishery management and the need for further conservation studies.

Abstract The aim was to compare short-term results of transvaginal hybrid-NOTES (NSR) with traditional laparoscopic technique in sigmoid resection (LSR) in cases of diverticulitis. Natural Orifice Transluminal Endoscopic Surgery has been evolved as a minimally invasive procedure to reduce the operative trauma due to the absence of specimen extraction through the abdominal wall causing less postoperative pain, and shorter hospital stay. Despite the increasing use and published case series of NSR for diverticulitis as a laparoscopic procedure with transvaginal stapling and specimen extraction, there are no studies comparing this procedure with LSR. Twenty NSR patients operated at the Cologne-Merheim Medical Center have been documented and compared with 20 female LSR patients matched for body mass index, American Society of Anesthesiologists-classification (ASA), Hansen/Stock classification, and age. To ensure comparability regarding peri- and postoperative care, only procedures performed by the same surgeon were included. Procedural time, intra- and postoperative complications, conversion rate, postoperative pain, the duration of an epidural catheter, analgesic consumption, and postoperative length of hospital stay were analyzed. There were no significant differences in the sum of pain levels (p = 0.930), length of procedure (p = 0.079), intra- and postoperative complications, as well as duration of an epidural catheter. On the contrary, there were significant positive effects for NSR on morphine requirement at day seven and eight (p = 0.019 and p = 0.035 respectively) as well as the postoperative length of hospital stay (p = 0.031). This retrospective study reveals significant positive effects for NSR compared to LSR regarding length of hospital stay as well as morphine consumption after removal of the epidural catheter, whereas there were no significant differences in complication rate and procedural time. In summary, NSR is an adequate alternative to traditional laparoscopic sigmoid resection considering the surgeons experience and the patient’s personal preferences.

ObjectiveTo evaluate if previously found associations between low serum bilirubin concentration and kidney function decline is independent of hemoglobin and other key confounders.Research design and methodsClinical trial data from the SAVOR-TIMI 53 trial as well as the UK primary care electronic healthcare records, Clinical Practice Research Datalink (CPRD), were used to construct three cohorts of patients at risk of chronic kidney disease (CKD). The randomized clinical trial (RCT) cohort from the subset of SAVOR-TIMI 53 trial consisted of 10,555 type-2 diabetic patients with increased risk of cardiovascular disease. The two observational data cohorts from CPRD consisted of 71,104 newly diagnosed type-2 diabetes (CPRD-DM2) and 82,065 newly diagnosed hypertensive (CPRD-HT) patients without diabetes. Cohorts were stratified according to baseline circulating total bilirubin levels to determine association on the primary end point of a 30% reduction from baseline in estimated glomerular filtration rate (eGFR) and the secondary end point of albuminuria.ResultsThe confounder adjusted hazard ratios of the subpopulation with lower than median bilirubin levels compared to above median bilirubin levels for the primary end point were 1.18 (1.02-1.37), 1.12 (1.05-1.19) and 1.09 (1.01-1.17), for the secondary end point were 1.26 (1.06-1.52), 1.11 (1.01-1.21) and 1.18 (1.01-1.39) for SAVOR-TIMI 53, CPRD-DM2, CPRD-HT, respectively.ConclusionOur findings are consistent across all cohorts and endpoints: lower serum bilirubin levels are associated with a greater kidney function decline independent of hemoglobin and other key confounders. This suggests that increased monitoring of kidney health in patients with lower bilirubin levels may be considered, especially for diabetic patients.

Background COVID-19 has so far affected more than 250 million individuals worldwide, causing more than 5 million deaths. Several risk factors for severe disease have been identified, most of which coincide with advanced age. In younger individuals, severe COVID-19 often occurs in the absence of obvious comorbidities. Guided by the finding of cytomegalovirus (CMV)-specific T cells with some cross-reactivity to SARS-CoV-2 in a COVID-19 intensive care unit (ICU) patient, we decided to investigate whether CMV seropositivity is associated with severe or critical COVID-19. Herpes simplex virus (HSV) serostatus was investigated as control. Methods National German COVID-19 bio-sample and data banks were used to retrospectively analyze the CMV and HSV serostatus of patients who experienced mild (n = 101), moderate (n = 130) or severe to critical (n = 80) disease by IgG serology. We then investigated the relationship between disease severity and herpesvirus serostatus via statistical models. Results Non-geriatric patients (< 60 years) with severe COVID-19 were found to have a very high prevalence of CMV-seropositivity, while CMV status distribution in individuals with mild disease was similar to the prevalence in the German population; interestingly, this was not detectable in older patients. Prediction models support the hypothesis that the CMV serostatus, unlike HSV, might be a strong biomarker in identifying younger individuals with a higher risk of developing severe COVID-19, in particular in absence of other co-morbidities. Conclusions We identified ‘CMV-seropositivity’ as a potential novel risk factor for severe COVID-19 in non-geriatric individuals in the studied cohorts. More mechanistic analyses as well as confirmation of similar findings in cohorts representing the currently most relevant SARS-CoV-2 variants should be performed shortly.

BackgroundIncreased physical activity levels and their determinations are essential issues worldwide. The Longitudinal Study of Physical Activity Determinants (ELDAF) aims to understand the roles of psychosocial and environmental factors in workers' physical activity levels.MethodsA prospective cohort study of non-faculty civil servants from a public university (approximately 1,200 individuals) will start in 2022 (baseline). The primary measurements will be accelerometer- and questionnaire-based physical activity, social support, social network, socioeconomic status, bereavement, job stress, body image, common mental disorders, depression, and neighborhood satisfaction. Additional measurements will include necessary sociodemographic, physical morbidity, lifestyle and anthropometric information. Participants' places of residence will be geocoded using complete addresses. All participants will furnish written, informed consent before the beginning of the study. Pilot studies were performed to identify and correct potential problems in the data collection instruments and procedures. ELDAF will be the first cohort study conducted in Latin America to investigate physical activity and its determinants.

[This corrects the article DOI: 10.1371/journal.pone.0263385.].

Abstract To better understand the dispersion strategies of Triatoma infestans (Klug) (Hemiptera: Reduviidae, Triatominae), we evaluated the spatial effect of infested peridomicile and density vegetation cover in a historically endemic area for Chagas disease. The study was conducted in rural houses of the northwest of Córdoba province, Argentine, during 2012-2013. Active search of triatomines were made in domicile and peridomicile habitats. To characterize vegetation coverage, a thematic map was obtained considering five types of vegetation cover (closed/open forest, closed/open shrubland and cultural land). From each house we extracted the area of vegetation coverage, housing density and infested peridomiciles density. We used generalized linear models to evaluate the effect of these variables on the occurrence of infested peridomicile. According to our results, the probability of a peridomicile to be infested increases by 1.34 (95%CI [0.98; 1.90]) times more when peridomicile structures are in environments with higher housing density and by 1.25 (95%CI [0.84; 1.88]) more times when houses are surrounded by open shrublands. Among the multiple ecological determinants of peridomestic infestation, the influence of vegetation cover has been poorly studied. In this study we discussed the effect of the vegetation as a potential modulator of the dispersion strategies of T. infestans.

Plants deploy numerous receptor-like kinases (RLKs) to respond to pathogens. Here the authors show that NIK1, an RLK that positively regulates antiviral immunity, negatively regulates the response to bacteria by modulating FLS2/BAK1 complex formation, suggesting crosstalk between bacterial and viral immunity.

Abstract Membranolytic anticancer peptides represent a potential strategy in the fight against cancer. However, our understanding of the underlying structure-activity relationships and the mechanisms driving their cell selectivity is still limited. We developed a computational approach as a step towards the rational design of potent and selective anticancer peptides. This machine learning model distinguishes between peptides with and without anticancer activity. This classifier was experimentally validated by synthesizing and testing a selection of 12 computationally generated peptides. In total, 83% of these predictions were correct. We then utilized an evolutionary molecular design algorithm to improve the peptide selectivity for cancer cells. This simulated molecular evolution process led to a five-fold selectivity increase with regard to human dermal microvascular endothelial cells and more than ten-fold improvement towards human erythrocytes. The results of the present study advocate for the applicability of machine learning models and evolutionary algorithms to design and optimize novel synthetic anticancer peptides with reduced hemolytic liability and increased cell-type selectivity.

Abstract The enzyme acetylcholinesterase participates in the end of cholinergic transmission and it has been shown that its activity is increased in some diseases that affect the brain, including Alzheimer disease. The objective of this study was to investigate the effect of purple grape juice consumption with or without high-fat diet in the gestational and lactation period on acetylcholinesterase activity and oxidative stress parameters in the hippocampus of female descendants. During pregnancy and lactation, 40 female Wistar rats received a control diet or a high-fat diet, with half of them receiving grape juice. After lactation, the female descendants received water and control diet in ad libitum until euthanasia on the 120 postnatal day. Hippocampus from were removed for analysis of AChE activity, protein oxidation and lipid peroxidation. It was observed that high-fat diet consumption during the pregnancy increased the AChE activity and the grape juice reduced this activity in descendants. The same was observed in protein oxidation, the descendants from high-fat diet had significantly highest values, and grape juice decreased the levels. We conclude that dietary choices during pregnancy can alter the acetylcholinesterase levels and grape juice is an important alternative to improve this function in adulthood.

We previously reported xanthohumol (XN), and its synthetic derivative tetrahydro-XN (TXN), attenuates high-fat diet (HFD)-induced obesity and metabolic syndrome in C57Bl/6J mice. The objective of the current study was to determine the effect of XN and TXN on lipid accumulation in the liver. Non-supplemented mice were unable to adapt their caloric intake to 60% HFD, resulting in obesity and hepatic steatosis; however, TXN reduced weight gain and decreased hepatic steatosis. Liver transcriptomics indicated that TXN might antagonize lipogenic PPARγ actions in vivo. XN and TXN inhibited rosiglitazone-induced 3T3-L1 cell differentiation concomitant with decreased expression of lipogenesis-related genes. A peroxisome proliferator activated receptor gamma (PPARγ) competitive binding assay showed that XN and TXN bind to PPARγ with an IC50 similar to pioglitazone and 8–10 times stronger than oleate. Molecular docking simulations demonstrated that XN and TXN bind in the PPARγ ligand-binding domain pocket. Our findings are consistent with XN and TXN acting as antagonists of PPARγ.

Cardiac pumping depends on the morphological structure of the heart, but also on its subcellular (ultrastructural) architecture, which enables cardiac contraction. In cases of congenital heart defects, localized ultrastructural disruptions that increase the risk of heart failure are only starting to be discovered. This is in part due to a lack of technologies that can image the three-dimensional (3D) heart structure, to assess malformations; and its ultrastructure, to assess organelle disruptions. We present here a multiscale, correlative imaging procedure that achieves high-resolution images of the whole heart, using 3D micro-computed tomography (micro-CT); and its ultrastructure, using 3D scanning electron microscopy (SEM). In a small animal model (chicken embryo), we achieved uniform fixation and staining of the whole heart, without losing ultrastructural preservation on the same sample, enabling correlative multiscale imaging. Our approach enables multiscale studies in models of congenital heart disease and beyond.

Dependent peptide searching is a method for discovering covalently-modified peptides-and therefore proteins-in mass-spectrometry-based proteomics experiments. Being more permissive than standard search methods, it has the potential to discover novel modifications (e.g., post-translational modifications occurring in vivo, or modifications introduced in vitro). However, few studies have explored dependent peptide search results in an untargeted way. In the present study, we sought to evaluate dependent peptide searching as a means of characterising proteins that have been modified in vitro. We generated a model data set by analysing N-ethylmaleimide-treated bovine serum albumin, and performed dependent peptide searches using the popular MaxQuant software. To facilitate interpretation of the search results (hundreds of dependent peptides), we developed a series of visualisation tools (R scripts). We used the tools to assess the diversity of putative modifications in the albumin, and to pinpoint hypothesised modifications. We went on to explore the tools' generality via analyses of public data from studies of rat and human proteomes. Of 19 expected sites of modification (one in rat cofilin-1 and 18 across six different human plasma proteins), eight were found and correctly localised. Apparently, some sites went undetected because chemical enrichment had depleted necessary analytes (potential 'base' peptides). Our results demonstrate (i) the ability of the tools to provide accurate and informative visualisations, and (ii) the usefulness of dependent peptide searching for characterising in vitro protein modifications. Our model data are available via PRIDE/ProteomeXchange (accession number PXD013040).

The Quick Environmental Exposure and Sensitivity Inventory (QEESI) is a validated questionnaire used worldwide to assess intolerances to chemicals, foods, and drugs, and has emerged as the gold standard for assessing chemical intolerance (CI). Despite a reported prevalence of 8-33%, epidemiological studies and routine primary care clinics rarely assess CI. To help address this gap, we developed the Brief Environmental Exposure and Sensitivity Inventory (BREESI)-a 3-item CI screening tool. We tested the BREESI's potential to predict whether an individual is likely to be classified as chemically intolerant if administered the 50-item QEESI. We recruited 293 participants from a university-based primary care clinic and through online participation. The statistical sensitivity, specificity, and positive and negative predictive values of the BREESI were calculated against the validated QEESI. Ninety percent (90%) of participants answering "yes" to all three items on the BREESI fit the QEESI criteria for being very suggestive of CI based upon their chemical intolerance and symptom scores (positive predictive value = 90%). For participants endorsing two items, 93% were classified as either very suggestive (39%) or suggestive (54%) of CI (positive predictive value = 87%). Of those endorsing only one item, 13% were classified as very suggestive of CI, and 70% as suggestive. Of those answering "No" to all of the BREESI items, 95% were classified as not suggestive of CI (i.e., negative predictive value = 95%). The BREESI is a versatile screening tool for assessing potential CI useful for clinical and epidemiological applications, based upon individuals' past adverse responses in a variety of settings. Just as health care professionals routinely inquire about latex allergy to prevent adverse reactions, the BREESI provides an essential screen for CI. Together, the BREESI and QEESI provide new diagnostic tools that may help predict and prevent future adverse reactions to chemicals, foods, and drugs.

The RNA for ALS- and frontotemporal dementia-associated C9ORF72 gene is exported from nucleus via an unknown mechanism. This study shows that reduction of nuclear export adaptor SRSF1 can alleviate neuronal cell death and nuclear export of C9ORF72 inDrosophilaand patient-derived induced motor neurons.

PURPOSE:We investigated the stability, correlations and bidirectional relationship of light physical activities (PA), moderate and vigorous PA (MVPA), television viewing (TV) and video game/computer use (VG) in Brazilian adolescents. METHODS:Adolescent Nutritional Assessment Longitudinal Study-ELANA is a middle school cohort study conducted in Rio de Janeiro-Brazil in 2010-2012. Self-reported data on PA (International Physical Activity Questionnaire- IPAQ) and screen activities were obtained from 810 adolescents (mean ages of 10.9 years old (SD 0.78) for girls; 11 years old (SD 0.85) for boys) to perform autoregressive cross-lagged structural equation models in two time points for PA and three time points for screen activities. RESULTS:There was no significant stability of light PA and MVPA for boys and girls. Moderate stability of screen activities were found for both genders, with a significant coefficient of TV for boys (T1-T2:0.29; T2-T3:0.27 p

Drosophila neural stem cells (NSCs) are quiescent at early larval stages but how this is regulated is unclear. Here, Ding et al. show that quiescence of NSCs is mediated by cell-contact inhibition via the Hippo pathway transmembrane proteins Crumbs and Echinoid, which in turn are regulated by nutrient levels.

The maintenance of visual function is supported by the proper functioning of the retinal pigment epithelium (RPE), representing a mosaic of polarized cuboidal postmitotic cells. Damage factors such as inflammation, aging, or injury can initiate the migration and proliferation of RPE cells, whereas they undergo a pseudo-metastatic transformation or an epithelial to mesenchymal transition (EMT) from cuboidal epithelioid into fibroblast-like or macrophage-like cells. This process is recognized as a key feature in several severe ocular pathologies, and is mimicked by placing RPE cells in culture, which provides a reasonable and well-characterized in vitro model for a type 2 EMT. The most obvious characteristic of EMT is the cell phenotype switching, accompanied by the cytoskeletal reorganization with changes in size, shape, and geometry. Atomic force microscopy (AFM) has the salient ability to label-free explore these characteristics. Based on our AFM results supported by the genetic analysis of specific RPE differentiation markers, we elucidate a scheme for gradual transformation from the cobblestone to fibroblast-like phenotype. Structural changes in the actin cytoskeletal reorganization at the early stages of EMT lead to the development of characteristic geodomes, a finding that may reflect an increased propensity of RPE cells to undergo further EMT and thus become of diagnostic significance.

INTRODUCTION:Community-dwelling older persons often experience hindering health complaints that disturb daily activities. If general practitioners (GPs) are unaware of such complaints, this could lead to a mismatch in provided care and needed or expected care. In this qualitative study with community-dwelling older persons we investigated how older persons experience hindering health complaints, how they deal with them, and what they expect from their GP. METHODS:Participants (aged ≥80 years) with pain and/or problems with walking/standing were invited to participate in a (group)interview about hindering health problems and expectations from general practice. Data were analysed using the framework method and results were discussed with a client panel. RESULTS:Participants experienced various hindering health complaints in addition to pain and/or problems with walking/standing. Complaints affecting social activities were experienced as the most hindering. Participants actively tried to remain independent and, to achieve this, GPs were expected to be involved and be easily accessible. However, they did not expect specific help from their GP for pain or problems with walking/standing. Suggestions for improvement of care from GPs included optimisation of accessibility (continuous availability by telephone), a yearly check including medication review, open communication, and empathy and support during major life events. CONCLUSIONS:According to older persons with hindering health complaints, GPs could improve their accessibility/relationship with patients by: 1) continuous telephonic availability, 2) initiating regular contact with medication reviews, and 3) initiating support during major life events. This might lower the reported barriers to contact the GP for hindering health complaints.

The human brain can quickly adapt to changes in the environment. One example is phonetic recalibration: a speech sound is interpreted differently depending on the visual speech and this interpretation persists in the absence of visual information. Here, we examined the mechanisms of phonetic recalibration. Participants categorized the auditory syllables /aba/ and /ada/, which were sometimes preceded by the so-called McGurk stimuli (in which an /aba/ sound, due to visual /aga/ input, is often perceived as ‘ada’). We found that only one trial of exposure to the McGurk illusion was sufficient to induce a recalibration effect, i.e. an auditory /aba/ stimulus was subsequently more often perceived as ‘ada’. Furthermore, phonetic recalibration took place only when auditory and visual inputs were integrated to ‘ada’ (McGurk illusion). Moreover, this recalibration depended on the sensory similarity between the preceding and current auditory stimulus. Finally, signal detection theoretical analysis showed that McGurk-induced phonetic recalibration resulted in both a criterion shift towards /ada/ and a reduced sensitivity to distinguish between /aba/ and /ada/ sounds. The current study shows that phonetic recalibration is dependent on the perceptual integration of audiovisual information and leads to a perceptual shift in phoneme categorization.

We investigated the stability and the directionality of being body bullied and a set of four variables- 1) Body Mass Index (BMI), 2) moderate and vigorous physical activity (MVPA), 3) television time (TV) and 4) video game/computer time (VG)-, termed in the present study as 'health-related state and behaviours (HRSB)'-across adolescence. The Adolescent Nutritional Assessment Longitudinal Study (ELANA) is a cohort study conducted among middle school students from two public and four private schools in Rio de Janeiro-Brazil. We analysed data from 2010 (T1) and 2012 (T2) among 810 adolescents (aged 9-15 at T1). Gender-specific structural equation models (SEM) were estimated, including autoregressive paths for the HRSB and being body bullied over time, correlations at T1 and T2, respectively, and cross-lagged effects. The results presented significant stability coefficients for almost all variables over time in both genders (except for MVPA in boys and girls and TV time among girls). There were positive correlations between BMI and being body bullied, as well as between TV and VG for boys (0.32, p

The scope of this study is to elucidate the physical mechanisms influencing the transient flow behavior of lubricating greases based on biogenic oleochemicals from a polarity point of view. This includes the mutually interacting influence of base oil polarity and thickening agents on the rheologically-measured mechanical structural degradation in transient shear flow. Due to the high temperature dependence of Keesom forces in the background of polar-active bond mechanisms, the analysis of the transient flow response as a function of temperature allows to attribute the observed influences to differences in base oil polarity. In general, clay-thickened greases show a greater tendency to be rheologically influenced by base oil polarities than soap-thickened lubricating greases.

Sample preparation is critical to biological electron microscopy (EM), and there have been continuous efforts on optimizing the procedures to best preserve structures of interest in the sample. However, a quantitative characterization of the morphological changes associated with each step in EM sample preparation is currently lacking. Using correlative EM and superresolution microscopy (SRM), we have examined the effects of different drying methods as well as osmium tetroxide (OsO4) post-fixation on cell morphology during scanning electron microscopy (SEM) sample preparation. Here, SRM images of the sample acquired under hydrated conditions were used as a baseline for evaluating morphological changes as the sample went through SEM sample processing. We found that both chemical drying and critical point drying lead to a mild cellular boundary retraction of ~60 nm. Post-fixation by OsO4 causes at least 40 nm additional boundary retraction. We also found that coating coverslips with adhesion molecules such as fibronectin prior to cell plating helps reduce cell distortion from OsO4 post-fixation. These quantitative measurements offer useful information for identifying causes of cell distortions in SEM sample preparation and improving current procedures.

The auxiliary α2δ subunits of voltage-gated calcium channels are extracellular membrane-associated proteins, which are post-translationally cleaved into disulfide-linked polypeptides α2 and δ. We now show, using α2δ constructs containing artificial cleavage sites, that this processing is an essential step permitting voltage-dependent activation of plasma membrane N-type (CaV2.2) calcium channels. Indeed, uncleaved α2δ inhibits native calcium currents in mammalian neurons. By inducing acute cell-surface proteolytic cleavage of α2δ, voltage-dependent activation of channels is promoted, independent from the trafficking role of α2δ. Uncleaved α2δ does not support trafficking of CaV2.2 channel complexes into neuronal processes, and inhibits Ca2+ entry into synaptic boutons, and we can reverse this by controlled intracellular proteolytic cleavage. We propose a model whereby uncleaved α2δ subunits maintain immature calcium channels in an inhibited state. Proteolytic processing of α2δ then permits voltage-dependent activation of the channels, acting as a checkpoint allowing trafficking only of mature calcium channel complexes into neuronal processes.

Epithelial-to-mesenchymal transition (EMT) of retinal pigment epithelial cells is a crucial event in the onset of proliferative vitreoretinopathy (PVR), the most common reason for treatment failure in retinal detachment surgery. We studied alterations in the cell surface glycan expression profile upon EMT of RPE cells and focused on its relevance for the interaction with galectin-3 (Gal-3), a carbohydrate binding protein, which can inhibit attachment and spreading of human RPE cells in a dose- and carbohydrate-dependent manner, and thus bares the potential to counteract PVR-associated cellular events. Lectin blot analysis revealed that EMT of RPE cells in vitro confers a glycomic shift towards an abundance of Thomsen-Friedenreich antigen, poly-N-acetyllactosamine chains, and complex-type branched N-glycans. Using inhibitors of glycosylation we found that both, binding of Gal-3 to the RPE cell surface and Gal-3-mediated inhibition of RPE attachment and spreading, strongly depend on the interaction of Gal-3 with tri- or tetra-antennary complex type N-glycans and sialylation of glycans but not on complex-type O-glycans. Importantly, we found that β1,6 N-acetylglucosaminyltransferase V (Mgat5), the key enzyme catalyzing the synthesis of tetra- or tri-antennary complex type N-glycans, is increased upon EMT of RPE cells. Silencing of Mgat5 by siRNA and CRISPR-Cas9 genome editing resulted in reduced Gal-3 binding. We conclude from these data that binding of recombinant Gal-3 to the RPE cell surface and inhibitory effects on RPE attachment and spreading largely dependent on interaction with Mgat5 modified N-glycans, which are more abundant on dedifferentiated than on the healthy, native RPE cells. Based on these findings we hypothesize that EMT of RPE cells in vitro confers glycomic changes, which account for high affinity binding of recombinant Gal-3, particularly to the cell surface of myofibroblastic RPE. From a future perspective recombinant Gal-3 may disclose a therapeutic option allowing for selectively targeting RPE cells with pathogenic relevance for development of PVR.

The differentiation of precursor cells into mature adipocytes (adipogenesis) has been an area of increased focus, spurred by a rise in obesity rates. Though our understanding of adipogenesis and its regulation at the cellular level is growing, many questions remain, especially regarding the regulation of the metabolome. The 3T3-L1 cell line is the most well characterized cellular model of adipogenesis. Using a time course metabolomics approach, we show that the 3T3-L1 preadipocyte metabolome is greatly altered during the first 48 hours of differentiation, where cells go through about two rounds of cell division, a process known as mitotic clonal expansion. Short-chain peptides were among several small molecules that were increased during mitotic clonal expansion. Additional indicators of protein turnover were also increased, including bilirubin, a degradation product of heme-containing proteins, and 3-methylhistidine, a post-translationally modified amino acid that is not reutilized for protein synthesis. To study the origin of the peptides, we treated differentiating preadipocytes with 18O labeled water and found that 18O was incorporated into the short chain peptides, confirming them, at least in part, as products of hydrolysis. Inhibitors of the proteasome or matrix metalloproteinases affected the peptide levels during differentiation, but inhibitors of autophagy or peptidases did not. 18O was also incorporated into several choline metabolites including cytidine 5'-diphosphocholine (CDP-choline), glycerophosphocholine, and several phosphatidylcholine species, indicative of phosphatidylcholine synthesis/degradation and of flux through the CDP-choline cycle, a hallmark of proliferating cells. 18O-Tracer metabolomics further showed metabolic labeling of glutamate, suggestive of glutaminolysis, also characteristic of proliferating cells. Together, these results highlight the utility of 18O isotope labeling in combination with metabolomics to uncover changes in cellular metabolism that are not detectable by time-resolved metabolomics.

Integrated care for older persons with complex care needs is widely advocated. Particularly professionals and policy makers have positive expectations. Care outcome results are ambiguous. Receiver and provider satisfaction is relevant but still poorly understood.During implementation of integrated care in residential homes (The MOVIT project), we compared general satisfaction and satisfaction with specific aspects of General Practitioner (GP) care in older persons and GPs before (cohort I) and after at least 12 months of implementation (cohort II).The general satisfaction score for GP care given by older persons does not change (Cohort I (n = 762) mean score 8.0 (IQR:7.0-9.0) vs. Cohort II (n = 505) mean score 8.0 (IQR:7.0-8.0);P = 0.01). Expressions of general satisfaction in GPs do not show consistent change (Cohort I (n = 87) vs Cohort II (n = 66), percentage satisfied about; role as GP, 56% vs 67%;P = 0.194, ability to provide personal care, 60% vs 67%;P = 0.038, quality of care, 54% vs 62%;P = 0.316). Satisfaction in older persons about some specific aspects of care do show change; GP-patient relationship, points 61.6 vs 63.3;P = 0.001, willingness to talk about mistakes, score 3.47 vs 3.73;P = 0.001, information received about drugs, score 2.79 vs 2.46;P = 0.002. GPs also report changes in specific aspects: percentage satisfied about multidisciplinary meetings; occurrence, 21% vs 53%;P =

The epidemiological importance of Chagas disease vectors largely depends on their spreading ability and adaptation to domestic habitats. Triatoma patagonica is a secondary vector of Chagas disease endemic of Argentina, and it has been found colonizing domiciles and most commonly peridomiciliary structures in several Argentine provinces and morphological variation along its distribution range have been described. To asses if population differentiation represents geographic variants or true biological species, multiple genetic and phenotypic approaches and laboratory cross-breeding were performed in T. patagonica peridomestic populations. Analyses of chromatic variation of forewings, their size and the content of C-heterochromatin on chromosomes revealed that populations are structured following a North-South latitudinal variation. Cytochrome c oxidase I mitochondrial gene (COI) nucleotide analysis showed a mean genetic distance of 5.2% between the most distant populations. The cross-breeding experiments suggest a partial reproductive isolation between some populations with 40% of couples not laying eggs and low hatching efficiency. Our findings reveal phenotypic and genetic variations that suggest an incipient differentiation processes among T. patagonica populations with a pronounced phenotypic and genetic divergence between the most distant populations. The population differentiation here reported is probably related to differential environmental conditions and it could reflect the occurrence of an incipient speciation process in T. patagonica.

Understanding contributors to mortality during the initial phase of tuberculosis (TB) treatment in patients co-infected with HIV would guide targeted interventions to improve survival. The aim of this study was to ascertain the incidence of death during the initial 2 months (new cases) and 3 months (retreatment cases) of TB treatment and to assess correlates of mortality in HIV co-infected patients.We conducted a hospital-based retrospective cohort study from January 2006 to December 2013 at Yaoundé Central Hospital, Cameroon. We reviewed medical records to identify co-infected TB/HIV inpatients aged 15 years and older who died during TB treatment. Death was defined as any death occurring during TB treatment, as per World Health Organization recommendations. We collected socio-demographic, clinical and laboratory data. We conducted multivariable logistic binary regression analysis to identify factors associated with death during the intensive phase of TB treatment. Magnitudes of associations were expressed by adjusted odds ratio (aOR) with 95% confidence interval. A p value < 0.05 was considered statistically significant.The 99 patients enrolled had a mean age of 39.5 (standard deviation 10.9) years and 53% were male. Patients were followed for 276.3 person-months of observation (PMO). Forty nine patients were died during intensive phase of TB treatment. Death incidence during the intensive phase of TB treatment was 32.2 per 100 PMO. Having a non-AIDS comorbidity (aOR 2.47, 95%CI 1.22-5.02, p = 0.012), having extra-pulmonary TB (aOR 1.89, 95%CI 1.05-3.43, p = 0.035), and one year increase in duration of known HIV infection (aOR 1.23, 95%CI 1.004-1.49) were independently associated with death during the intensive phase of TB treatment.Mortality incidence during intensive phase of TB treatment was high among TB/HIV co-infected patients during TB treatment; and strongly associated with extra pulmonary TB suggesting advanced stage of immunosuppression and non-AIDS comorbidities. Early HIV diagnosis and care and good management of non-comorbidities can reduce this incidence.

Albumin treatment of ischemic stroke was associated with cardiopulmonary adverse events in previous studies and a low incidence of intracranial hemorrhage. We sought to describe the neurological and cardiopulmonary adverse events in the ALIAS Part 2 Multicenter Trial.Ischemic stroke patients, aged 18-83 and a baseline NIHSS ≥ 6, were randomized to treatment with ALB or saline control within 5 hours of stroke onset. Neurological adverse events included symptomatic intracranial hemorrhage, hemicraniectomy, neurological deterioration and neurological death. Cardiopulmonary adverse events included pulmonary edema/congestive heart failure, acute coronary syndromes, atrial fibrillation, pneumonia and pulmonary thromboembolism.Among 830 patients, neurological and cardiopulmonary adverse events were not differentially associated with poor outcome between ALB and saline control subjects. The rate of symptomatic intracranial hemorrhage in the first 24h was low overall (2.9%, 24/830) but more common in the ALB treated subjects (RR = 2.4, CI95 1.01-5.8). The rate of pulmonary edema/CHF in the first 48h was 7.9% (59/830) and was more common among ALB treated subjects (RR = 10.7, CI95 4.3-26.6); this complication was expected and was satisfactorily managed with mandated diuretic administration and intravenous fluid guidelines. Troponin elevations in the first 48h were common, occurring without ECG change or cardiac symptoms in 52 subjects (12.5%).ALB therapy was associated with an increase in symptomatic ICH and pulmonary edema/congestive heart failure but this did not affect final outcomes. Troponin elevation occurs routinely in the first 48 hours after acute ischemic stroke.ClincalTrials.gov NCT00235495.

The role of systemic autoimmunity in human traumatic brain injury (TBI) and other forms of brain injuries is recognized but not well understood. In this study, a systematic investigation was performed to identify serum autoantibody responses to brain-specific proteins after TBI in humans. TBI autoantibodies showed predominant immunoreactivity against a cluster of bands from 38-50 kDa on human brain immunoblots, which were identified as GFAP and GFAP breakdown products. GFAP autoantibody levels increased by 7 days after injury, and were of the IgG subtype predominantly. Results from in vitro tests and rat TBI experiments also indicated that calpain was responsible for removing the amino and carboxyl termini of GFAP to yield a 38 kDa fragment. Additionally, TBI autoantibody staining co-localized with GFAP in injured rat brain and in primary rat astrocytes. These results suggest that GFAP breakdown products persist within degenerating astrocytes in the brain. Anti-GFAP autoantibody also can enter living astroglia cells in culture and its presence appears to compromise glial cell health. TBI patients showed an average 3.77 fold increase in anti-GFAP autoantibody levels from early (0-1 days) to late (7-10 days) times post injury. Changes in autoantibody levels were negatively correlated with outcome as measured by GOS-E score at 6 months, suggesting that TBI patients with greater anti-GFAP immune-responses had worse outcomes. Due to the long lasting nature of IgG, a test to detect anti-GFAP autoantibodies is likely to prolong the temporal window for assessment of brain damage in human patients.

Hyaluronidases are enzymes that degrade hyaluronan an important constituent of the extracellular matrix. They have been used as a spreading agent, improving the absorption of drugs and facilitating the subcutaneous infusion of fluids. Here, we investigated the influence of bovine testes hyaluronidase (HYAL) during cutaneous wound healing in in vitro and in vivo assays. We demonstrated in the wound scratch assay that HYAL increased the migration and proliferation of fibroblasts in vitro at low concentration, e.g. 0.1 U HYAL enhanced the cell number by 20%. HYAL presented faster and higher reepithelialization in in vivo full-thickness excisional wounds generated on adult Wistar rats back skin already in the early phase at 2nd day post operatory compared to vehicle-control group. Wound closured area observed in the 16 U and 32 U HYAL treated rats reached 38% and 46% compared to 19% in the controls, respectively. Histological and biochemical analyses supported the clinical observations and showed that HYAL treated wounds exhibited increased granulation tissue, diminished edema formation and regulated the inflammatory response by modulating the release of pro and anti-inflammatory cytokines, growth factor and eicosanoids mediators. Moreover, HYAL increased gene expression of peroxisome proliferator-activated receptors (PPAR) γ and PPAR β/δ, the collagen content in the early stages of healing processes as well as angiogenesis. Altogether these data revealed that HYAL accelerates wound healing processes and might be beneficial for treating wound disorders.

Contributors to fatal outcomes in TB/HIV co-infected patients actively undergoing TB treatment are poorly characterized. The aim was to assess factors associated with death in TB/HIV co-infected patients during the initial 6 months of TB treatment.We conducted a hospital-based retrospective cohort study from January 2006 to December 2013 at the Yaoundé Central Hospital, Cameroon. We reviewed medical records to identify hospitalized co-infected TB/HIV patients aged 15 years and older. Death was defined as any death occurring during TB treatment, as per the World Health Organization's recommendations. We conducted logistic regression analysis to identify factors associated with a fatal outcome. Magnitudes of associations were expressed by adjusted odds ratio (aOR) with 95% confidence interval.The 337 patients enrolled had a mean age of 39.3 (standard deviation 10.3) years and 54.3% were female. TB treatment outcomes were distributed as follows: 205 (60.8%) treatment success, 99 (29.4%) deaths, 18 (5.3%) not evaluated, 14 (4.2%) lost to follow-up, and 1 (0.3%) failed. After exclusion of patients lost to follow-up and not evaluated, death in TB/HIV co-infected patients during TB treatment was associated with a TB diagnosis made before 2010 (aOR = 2.50 [1.31-4.78]; p = 0.006), the presence of other AIDS-defining diseases (aOR = 2.73 [1.27-5.86]; p = 0.010), non-AIDS comorbidities (aOR = 3.35 [1.37-8.21]; p = 0.008), not receiving cotrimoxazole prophylaxis (aOR = 3.61 [1.71-7.63]; p = 0.001), not receiving antiretroviral therapy (aOR = 2.45 [1.18-5.08]; p = 0.016), and CD4 cells count

BACKGROUND: Previous findings suggest that the loss of a family member is associated with health and mortality. The purpose of this study was to investigate the association between bereavement experiences and BMI, and whether there are socio-demographic differences in this association. OBJECTIVE: To investigate the association between bereavement experiences and BMI, and whether there are socio-demographic differences in this association. METHODS: We used cross-sectional data with retrospective questions from the Swedish Level of Living Survey (LNU) of 2000, including 5,142 individuals. The bereavement experiences examined in the study include the loss of a sibling, a parent or a spouse, and time since the death of a parent. BMI (kg/m2) was calculated using self-reported measurements of weight and height. The association between bereavement and BMI was evaluated through linear regressions. RESULTS: After controlling for possible confounders, most of the models detected an association between bereavement and BMI. The fully-adjusted model showed that loss of parents was associated with a 0.45 increase in BMI (SE = 0.20). The effect also seemed to be dependent on time since the loss and social class position. CONCLUSIONS: The present study is the first to examine associations between different types of familial losses and BMI. We find an association between the death of a family member and BMI, but it appears to be related to time since the death, type of bereavement experience and social class.

p53 is a central tumor suppressor protein and its inhibition is believed to be a prerequisite for cancer development. In approximately 50% of all malignancies this is achieved by inactivating mutations in the p53 gene. However, in several cancer entities, including melanoma, p53 mutations are rare. It has been recently proposed that tyrosinase related protein 2 (TRP2), a protein involved in melanin synthesis, may act as suppressor of the p53 pathway in melanoma. To scrutinize this notion we analyzed p53 and TRP2 expression by immunohistochemistry in 172 melanoma tissues and did not find any correlation. Furthermore, we applied three different TRP2 shRNAs to five melanoma cell lines and could not observe a target specific effect of the TRP2 knockdown on either p53 expression nor p53 reporter gene activity. Likewise, ectopic expression of TRP2 in a TRP2 negative melanoma cell line had no impact on p53 expression. In conclusion our data suggest that p53 repression critically controlled by TRP2 is not a general event in melanoma.

Several arenaviruses can cause severe hemorrhagic fever (HF) in humans, representing a public health threat in endemic areas of Africa and South America. The present study characterizes the potent virucidal activity of the carboxamide-derivatized aromatic disulfide NSC4492, an antiretroviral zinc finger-reactive compound, against Junín virus (JUNV), the causative agent of Argentine HF. The compound was able to inactivate JUNV in a time and temperature-dependent manner, producing more than 99 % reduction in virus titer upon incubation with virions at 37 °C for 90 min. The ability of NSC4492-treated JUNV to go through different steps of the multiplication cycle was then evaluated. Inactivated virions were able to bind and enter into the host cell with similar efficiency as control infectious particles. In contrast, treatment with NSC4492 impaired the capacity of JUNV to drive viral RNA synthesis, as measured by quantitative RT-PCR, and blocked viral protein expression, as determined by indirect immunofluorescence. These results suggest that the disulfide NSC4492 targets on the arenavirus replication complex leading to impairment in viral RNA synthesis. Additionally, analysis of VLP produced in NSC4492-treated cells expressing JUNV matrix Z protein revealed that the compound may interact with Z resulting in an altered aggregation behavior of this protein, but without affecting its intrinsic self-budding properties. The potential perspectives of NSC4492 as an inactivating vaccinal compound for pathogenic arenaviruses are discussed.

We have investigated the effect of different maturation stimuli on the ability of mature dendritic cells (DCs) to cross-present newly acquired particulate antigens. Cross-presentation was impaired in DCs matured by treatment with TNF-α, CpG and LPS, but was less affected upon CD40L-induced maturation. The difference could not be explained by decreased antigen uptake or translocation into the cytosol, but decreased cross-presentation ability did correlate with increased phagosomal/lysosomal acidification. Nevertheless, intra-phagosomal degradation of OVA was not increased in matured samples, suggesting that decreasing phagosomal pH may also regulate cross-presentation by a mechanism other than enhancing degradation.

Proliferative vitreoretinopathy (PVR) is a blinding disease frequently occurring after retinal detachment surgery. Adhesion, migration and matrix remodeling of dedifferentiated retinal pigment epithelial (RPE) cells characterize the onset of the disease. Treatment options are still restrained and identification of factors responsible for the abnormal behavior of the RPE cells will facilitate the development of novel therapeutics. Galectin-3, a carbohydrate-binding protein, was previously found to inhibit attachment and spreading of retinal pigment epithelial cells, and thus bares the potential to counteract PVR-associated cellular events. However, the identities of the corresponding cell surface glycoprotein receptor proteins on RPE cells are not known. Here we characterize RPE-specific Gal-3 containing glycoprotein complexes using a proteomic approach. Integrin-β1, integrin-α3 and CD147/EMMPRIN, a transmembrane glycoprotein implicated in regulating matrix metalloproteinase induction, were identified as potential Gal-3 interactors on RPE cell surfaces. In reciprocal immunoprecipitation experiments we confirmed that Gal-3 associated with CD147 and integrin-β1, but not with integrin-α3. Additionally, association of Gal-3 with CD147 and integrin-β1 was observed in co-localization analyses, while integrin-α3 only partially co-localized with Gal-3. Blocking of CD147 and integrin-β1 on RPE cell surfaces inhibited binding of Gal-3, whereas blocking of integrin-α3 failed to do so, suggesting that integrin-α3 is rather an indirect interactor. Importantly, Gal-3 binding promoted pronounced clustering and co-localization of CD147 and integrin-β1, with only partial association of integrin-α3. Finally, we show that RPE derived CD147 and integrin-β1, but not integrin-α3, carry predominantly β-1,6-N-actyl-D-glucosamine-branched glycans, which are high-affinity ligands for Gal-3. We conclude from these data that extracellular Gal-3 triggers clustering of CD147 and integrin-β1 via interaction with β1,6-branched N-glycans on RPE cells and hypothesize that Gal-3 acts as a positive regulator for CD147/integrin-β1 clustering and therefore modifies RPE cell behavior contributing to the pathogenesis of PVR. Further investigations at this pathway may aid in the development of specific therapies for PVR.

Pine Wilt Disease (PWD) is a complex disease integrating three major agents: the pathogenic agent, the pinewood nematode Bursaphelenchus xylophilus; the insect-vector Monochamus spp.; and the host pine tree, Pinus sp. Since the early 80's, the notion that another pathogenic agent, namely bacteria, may play a role in PWD has been gaining traction, however the role of bacteria in PWD is still unknown. The present work supports the possibility that some B. xylophilus-associated bacteria may play a significant role in the development of this disease. This is inferred as a consequence of: (i) the phenotypic characterization of a collection of 35 isolates of B. xylophilus-associated bacteria, in different tests broadly used to test plant pathogenic and plant growth promoting bacteria, and (ii) greenhouse experiments that infer the pathogenicity of these bacteria in maritime pine, Pinus pinaster. The results illustrate the presence of a heterogeneous microbial community associated with B. xylophilus and the traits exhibited by at least, some of these bacteria, appear to be related to PWD symptoms. The inoculation of four specific B. xylophilus-associated bacteria isolates in P. pinaster seedlings resulted in the development of some PWD symptoms suggesting that these bacteria likely play an active role with B. xylophilus in PWD.

BACKGROUND: Inactivation of the p53 pathway that controls cell cycle progression, apoptosis and senescence, has been proposed to occur in virtually all human tumors and p53 is the protein most frequently mutated in human cancer. However, the mutational status of p53 in melanoma is still controversial; to clarify this notion we analysed the largest series of melanoma samples reported to date. METHODOLOGY/PRINCIPAL FINDINGS: Immunohistochemical analysis of more than 180 melanoma specimens demonstrated that high levels of p53 are expressed in the vast majority of cases. Subsequent sequencing of the p53 exons 5-8, however, revealed only in one case the presence of a mutation. Nevertheless, by means of two different p53 reporter constructs we demonstrate transcriptional inactivity of wild type p53 in 6 out of 10 melanoma cell lines; the 4 other p53 wild type melanoma cell lines exhibit p53 reporter gene activity, which can be blocked by shRNA knock down of p53. CONCLUSIONS/SIGNIFICANCE: In melanomas expressing high levels of wild type p53 this tumor suppressor is frequently inactivated at transcriptional level.

BACKGROUND:Malignant catarrhal fever (MCF) is a lethal disease of cattle, characterized by vasculitis, necrosis, and accumulation of activated, dysregulated cytotoxic lymphocytes in various tissues. Ovine gamma herpesvirus 2 (OvHV-2) is a causative agent of MCF, which may trigger the disease through immunopathogenic pathways. Lymphocytes are the main target of the virus. However, the pathogenic basis of the disease is still mysterious. METHODS/FINDINGS:We hypothesized that the gene expression patterns of OvHV-2 and the relative abundances of host cell transcripts in lymphnodes may be used to identify pathways that help to explain the pathogenesis of MCF. Therefore, viral and host cell gene expression patterns in lymph nodes of animals with MCF and healthy controls were analyzed by microarray. Two regions on the viral genome were transcriptionally active, one encoding an orthologue to the latency-associated nuclear antigen (ORF73) of other gamma herpesviruses, the other with no predicted open reading frame. A vast number of transcripts related to inflammatory processes, lymphocyte activation, cell proliferation and apoptosis were detected at different abundances. However, the IL-2 transcript was eminent among the transcripts, which were, compared to healthy controls, less abundant in animals with MCF. The ratio between CD4- and CD8-positive T-lymphocytes was decreased in the lymphnodes of animals with MCF compared to healthy controls. In contrast, the same ratio was stable, when peripheral blood lymphocytes were analyzed. CONCLUSIONS/SIGNIFICANCE:The phenotype of mice with a deficient IL-2-system almost perfectly matches the clinical signs observed in cattle with MCF, which feature a significantly decreased IL-2 transcript abundance, compared to healthy cattle. This supports the hypothesis that immunopathogenic events are linked to the pathogenesis of MCF. IL-2-deficiency may play an important role in the process. Therefore, this work opens new avenues for research on MCF.

Abstract Assessing localization of the transient receptor potential vanilloid-1 (TRPV1) in skin nerve fibers is crucial for understanding its role in peripheral neuropathy and pain. However, information on the specificity and sensitivity of TRPV1 antibodies used for immunofluorescence (IF) on human skin is currently lacking. To find a reliable TRPV1 antibody and IF protocol, we explored antibody candidates from different manufacturers, used rat DRG sections and human skin samples for screening and human TRPV1-expressing HEK293 cells for further validation. Final specificity assessment was done on human skin samples. Additionally, we developed two automated image analysis methods: a Python-based deep-learning approach and a Fiji-based machine-learning approach. These methods involve training a model or classifier for nerve fibers based on pre-annotations and utilize a nerve fiber mask to filter and count TRPV1 immunoreactive puncta and TRPV1 fluorescence intensity on nerve fibers. Both automated analysis methods effectively distinguished TRPV1 signals on nerve fibers from those in keratinocytes, demonstrating high reliability as evidenced by excellent intraclass correlation coefficient (ICC) values exceeding 0.75. This method holds the potential to uncover alterations in TRPV1 associated with neuropathic pain conditions, using a minimally invasive approach.