Native RNA sequencing in fission yeast reveals frequent alternative splicing isoforms.

The unicellular yeast Schizosaccharomyces pombe (fission yeast) retains many of the splicing features observed in humans and is thus an excellent model to study the basic mechanisms of splicing. Nearly half the genes contain introns, but the impact of alternative splicing in gene regulation and proteome diversification remains largely unexplored. Here we leverage Oxford Nanopore Technologies native RNA sequencing (dRNA), as well as ribosome profiling data, to uncover the full range of polyadenylated transcripts and translated open reading frames. We identify 332 alternative isoforms affecting the coding sequences of 262 different genes, 97 of which occur at frequencies higher than 20%, indicating that functional alternative splicing in S. pombe is more prevalent than previously suspected. Intron retention events make about 80% of the cases; these events may be involved in the regulation of gene expression and, in some cases, generate novel protein isoforms, as supported by ribosome profiling data in 18 of the intron retention isoforms. One example is the rpl22 gene, in which intron retention is associated with the translation of a protein of only 13 amino acids. We also find that lowly expressed transcripts tend to have longer poly(A) tails than highly expressed transcripts, highlighting an interdependence between poly(A) tail length and transcript expression level. Finally, we discover 214 novel transcripts that are not annotated, including 158 antisense transcripts, some of which also show translation evidence. The methodologies described in this work open new opportunities to study the regulation of splicing in a simple eukaryotic model.

Relationship between smoking and mental health in educational settings with high smoking rates: a cohort study among Danish youth.

BACKGROUND: Smoking and poor mental health in youth represent important public health priorities. This study aimed to (i) compare tobacco-related behaviors and mental health in two educational settings with high smoking rates: vocational education and training (VET) schools and preparatory basic education (PBE) schools, and (ii) examine associations between smoking at school start and mental health 5 months later. METHODS: Data were obtained from baseline (N = 1843) and follow-up (N = 1039) assessments conducted as part of a school-based trial in two rounds (baseline in August 2018 and August 2019). Students' characteristics were presented by adjusted prevalences. Logistic regression analyses assessed associations between smoking and measures of mental health: school-related well-being, overall loneliness, and stress. RESULTS: More PBE students than VET students reported daily smoking (40% vs. 27%), nicotine dependence, perceived benefits of smoking (e.g., stress reduction: 41% vs. 33%), low smoking-related self-efficacy (e.g., ability to resist smoking if offered by a friend: 20% vs. 32%), school-related loneliness, and low school connectedness (25% vs. 11%). Daily smokers at VET and PBE schools had lower odds of school-related loneliness (AOR = 0.51, 95% CI: 0.35-0.74) and higher odds of stress (AOR = 2.75, 95% CI: 2.00-3.80). Smoking was associated with better classmate relations in VET schools but not in PBE schools. CONCLUSIONS: Our study suggested that students in PBE schools constitute a more vulnerable group in terms of smoking and mental health compared with students in VET schools. Smoking seemed to prevent loneliness in school but was associated with heightened stress levels.

Behavioral and Neural Dissociation of Social Anxiety and Loneliness.

Loneliness is a public health concern with detrimental effects on physical and mental well-being. Given phenotypical overlaps between loneliness and social anxiety (SA), cognitive-behavioral interventions targeting SA might be adopted to reduce loneliness. However, whether SA and loneliness share the same underlying neurocognitive mechanisms is still an elusive question. The current study aimed at investigating to what extent known behavioral and neural correlates of social avoidance in SA are evident in loneliness. We used a prestratified approach involving 42 (21 females) participants with high loneliness (HL) and 40 (20 females) participants with low loneliness (LL) scores. During fMRI, participants completed a social gambling task to measure the subjective value of engaging in social situations and responses to social feedback. Univariate and multivariate analyses of behavioral and neural data replicated known task effects. However, although HL participants showed increased SA, loneliness was associated with a response pattern clearly distinct from SA. Specifically, contrary to expectations based on SA differences, Bayesian analyses revealed moderate evidence for equal subjective values of engaging in social situations and comparable amygdala responses to social decision-making and striatal responses to positive social feedback in both groups. Moreover, while explorative analyses revealed reduced pleasantness ratings, increased striatal activity, and decreased striatal-hippocampal connectivity in response to negative computer feedback in HL participants, these effects were diminished for negative social feedback. Our findings suggest that, unlike SA, loneliness is not associated with withdrawal from social interactions. Thus, established interventions for SA should be adjusted when targeting loneliness.SIGNIFICANCE STATEMENT Loneliness can cause serious health problems. Adapting well-established cognitive-behavioral therapies targeting social anxiety might be promising to reduce chronic loneliness given a close link between both constructs. However, a better understanding of behavioral and neurobiological factors associated with loneliness is needed to identify which specific mechanisms of social anxiety are shared by lonely individuals. We found that lonely individuals show a consistently distinct pattern of behavioral and neural responsiveness to social decision-making and social feedback compared with previous findings for social anxiety. Our results indicate that loneliness is associated with a biased emotional reactivity to negative events rather than social avoidance. Our findings thus emphasize the distinctiveness of loneliness from social anxiety and the need for adjusted psychotherapeutic protocols.

Differential contribution of between and within-brain coupling to movement synchronization.

A fundamental characteristic of the human brain that supports behavior is its capacity to create connections between brain regions. A promising approach holds that during social behavior, brain regions not only create connections with other brain regions within a brain, but also coordinate their activity with other brain regions of an interaction partner. Here we ask whether between-brain and within-brain coupling contribute differentially to movement synchronization. We focused on coupling between the inferior frontal gyrus (IFG), a brain region associated with the observation-execution system, and the dorsomedial prefrontal cortex (dmPFC), a region associated with error-monitoring and prediction. Participants, randomly divided into dyads, were simultaneously scanned with functional near infra-red spectroscopy (fNIRS) while performing an open-ended 3D hand movement task consisting of three conditions: back-to-back movement, free movement, or intentional synchronization. Results show that behavioral synchrony was higher in the intentional synchrony compared with the back-to-back and free movement conditions. Between-brain coupling in the IFG and dmPFC was evident in the free movement and intentional synchrony conditions but not in the back-to-back condition. Importantly, between-brain coupling was found to positively predict intentional synchrony, while within-brain coupling was found to predict synchronization during free movement. These results indicate that during intentional synchronization, brain organization changes such that between-brain networks, but not within-brain connections, contribute to successful communication, pointing to shift from a within-brain feedback loop to a two-brains feedback loop.

Prognostic significance of PI-RADS 5 lesions in patients treated by radical prostatectomy.

PURPOSE: To analyse the pathological features and survival of patients with a PI-RADS 5 lesion on pre-biopsy MRI. METHODS: We extracted from a European multicentre prospectively gathered database the data of patients with a PI-RADS 5 lesion on pre-biopsy MRI, diagnosed using both systematic and targeted biopsies and subsequently treated by radical prostatectomy. The Kaplan-Meier model was used to assess the biochemical-free survival of the whole cohort and univariable and multivariable Cox models were set up to study factors associated with survival. RESULTS: Between 2013 and 2019, 539 consecutive patients with a PI-RADS 5 lesion on pre-biopsy MRI were treated by radical prostatectomy and included in the analysis. Follow-up data were available for 448 patients. Radical prostatectomy and lymph node dissection specimens showed non-organ confined disease in 297/539 (55%), (including 2 patients with a locally staged pT2 lesion and lymph node involvement (LNI)). With a median follow-up of 25 months (12-39), the median biochemical recurrence-free survival was 54% at 2 years (95% CI 45-61) and 28% at 5 years (95% CI 18-39). Among the factors studied, MRI T stage [T3a vs T2 HR 3.57 (95%CI 1.78-7.16); T3b vs T2 HR 6.17 (95% CI 2.99-12.72)] and PSA density (HR 4.47 95% CI 1.55-12.89) were significantly associated with a higher risk of biochemical recurrence in multivariable analysis. CONCLUSION: Patients with a PI-RADS 5 lesion on pre-biopsy MRI have a high risk of early biochemical recurrence after radical prostatectomy. MRI T stage and PSA density can be used to improve patient selection and counselling.

Novel plant-associated genomoviruses from the Brazilian Cerrado biome.

The discovery rate of new plant viruses has increased due to studies involving high-throughput sequencing (HTS), particularly for single-stranded DNA viruses of the family Genomoviridae. We carried out an HTS-based survey of genomoviruses in a wide range of native and exotic trees grown in the Brazilian Cerrado biome, and the complete genome sequences of two novel members of the family Genomoviridae from two distinct genera were determined. Specific primers were designed to detect these genomoviruses in individual samples. A new gemykolovirus (Tecoma stans associated gemykolovirus) was detected in Tecoma stans, and a new gemykibivirus (Ouratea duparquetiana associated gemykibivirus) was detected in Ouratea duparquetiana. A gemykrogvirus related to Gila monster associated gemykrogvirus (80% pairwise identity) was also detected in foliar samples of Trembleya parviflora. Our pilot study paves the way for a better characterization of this diverse collection of genomoviruses as well as their interactions with the associated tree species.

Changes in Cigarette Smoking and Smokeless Tobacco Use During the Coronavirus Disease 2019 Lockdown Period Among Youth and Young Adults in Denmark.

INTRODUCTION: Cigarettes and smokeless tobacco (snus and nicotine pouches) are prevalent among youth and young adults in Denmark. Here, we examined the extent of changes in the use of cigarettes and smokeless tobacco during the first Coronavirus Disease 2019 (COVID-19) lockdown in March and April 2020 in Denmark as well as reasons for changed behavior. AIMS AND METHODS: This study used data from a nationwide survey conducted among 15- to 29-year-olds from January to March 2021 including 13 530 respondents (response rate = 36.0%). Logistic regression analyses assessed the associations between sociodemographic characteristics and the odds of initiating or increasing as well as trying to stop or decreasing cigarette smoking and smokeless tobacco use. RESULTS: The prevalence of cigarette smoking was 17.8% and 10.5% reported using smokeless tobacco. Around 40% of those currently smoking cigarettes reported smoke on par during the COVID-19 lockdown as before, 24.5% started to smoke or increased their smoking, and 27.4% tried to stop or smoked less. Approximately 37% used smokeless tobacco on the same level as, before the COVID-19 lockdown, 38.8% initiated or used more, and 14.1% tried to stop or used less. Females were more prone to initiate smokeless tobacco use and increase their level of smoking during the lockdown, and younger participants smoked less. More females compared with males changed their smoking behaviors because of their mood, and more younger participants did so because of fewer social gatherings. CONCLUSION: Although most youths and young adults' tobacco behaviors remained the same during the COVID-19 lockdown, many also increased or decreased their behaviors-especially females and younger participants. IMPLICATIONS: This study enables the possibility of detecting new tendencies in smoking and the use of smokeless tobacco among subgroups of the population during the COVID-19 lockdown. This knowledge is crucial for identifying which groups of youths are vulnerable to increasing their tobacco use in other pandemic situations and which groups call for special attention after the lockdown period. Future efforts may focus on vulnerable groups affected by the COVID-19 pandemic, such as females, and there is a need to monitor closely whether youth tobacco use changes as society becomes more normalized.

MAGNETO: Cell type marker panel generator from single-cell transcriptomic data.

Single-cell RNA sequencing experiments produce data useful to identify different cell types, including uncharacterized and rare ones. This enables us to study the specific functional roles of these cells in different microenvironments and contexts. After identifying a (novel) cell type of interest, it is essential to build succinct marker panels, composed of a few genes referring to cell surface proteins and clusters of differentiation molecules, able to discriminate the desired cells from the other cell populations. In this work, we propose a fully-automatic framework called MAGNETO, which can help construct optimal marker panels starting from a single-cell gene expression matrix and a cell type identity for each cell. MAGNETO builds effective marker panels solving a tailored bi-objective optimization problem, where the first objective regards the identification of the genes able to isolate a specific cell type, while the second conflicting objective concerns the minimization of the total number of genes included in the panel. Our results on three public datasets show that MAGNETO can identify marker panels that identify the cell populations of interest better than state-of-the-art approaches. Finally, by fine-tuning MAGNETO, our results demonstrate that it is possible to obtain marker panels with different specificity levels.

Altered activation in the action observation system during synchronization in high loneliness individuals.

Lonely people tend to evaluate social exchanges negatively and to display difficulties in interactions. Interpersonal synchronization is crucial for achieving positive interactions, promoting affinity, closeness, and satisfaction. However, little is known about lonely individuals' ability to synchronize and about their brain activity while synchronizing. Following the screening of 303 participants, we recruited 32 low and 32 high loneliness participants. They were scanned while engaged in movement synchronization, using a novel dyadic interaction paradigm. Results showed that high loneliness individuals exhibited a reduced ability to adapt their movement to their partner's movement. Intriguingly, during movement adaptation periods, high loneliness individuals showed increased activation in the action observation (AO) system, specifically in the inferior frontal gyrus and the inferior parietal lobule. They did not show increased activation in the dorsomedial prefrontal cortex, which in the context of synchronization was suggested to be related to gap-monitoring. Based on these findings, we propose a model according to which lonely people may require stronger activation of their AO system for alignment, to compensate for some deficiency in their synchronization ability. Despite this hyperactivation, they still suffer from reduced synchronization capacity. Consequently, synchronization may be a relevant intervention area for the amelioration of loneliness.

Probabilistic survival modeling in health research: an assessment using cohort data from hospitalized patients with COVID-19 in a Latin American city.

Probabilistic survival methods have been used in health research to analyze risk factors and adverse health outcomes associated with COVID-19. The aim of this study was to employ a probabilistic model selected among three distributions (exponential, Weibull, and lognormal) to investigate the time from hospitalization to death and determine the mortality risks among hospitalized patients with COVID-19. A retrospective cohort study was conducted for patients hospitalized due to COVID-19 within 30 days in Londrina, Brazil, between January 2021 and February 2022, registered in the database for severe acute respiratory infections (SIVEP-Gripe). Graphical and Akaike Information Criterion (AIC) methods were used to compare the efficiency of the three probabilistic models. The results from the final model were presented as hazard and event time ratios. Our study comprised of 7,684 individuals, with an overall case fatality rate of 32.78%. Data suggested that older age, male sex, severe comorbidity score, intensive care unit admission, and invasive ventilation significantly increased risks for in-hospital mortality. Our study highlights the conditions that confer higher risks for adverse clinical outcomes attributed to COVID-19. The step-by-step process for selecting appropriate probabilistic models may be extended to other investigations in health research to provide more reliable evidence on this topic.

Effectiveness of the multi-component intervention 'Focus' on reducing smoking among students in the vocational education setting: a cluster randomized controlled trial.

BACKGROUND: Social inequality in smoking remains an important public health issue. Upper secondary schools offering vocational education and training (VET) comprise more students from lower socioeconomic backgrounds and have higher smoking prevalence than general high schools. This study examined the effects of a school-based multi-component intervention on students' smoking. METHODS: A cluster randomized controlled trial. Eligible participants were schools offering VET basic courses or preparatory basic education in Denmark, and their students. Schools were stratified by subject area and eight schools were randomly allocated to intervention (1,160 invited students; 844 analyzed) and six schools to control (1,093 invited students; 815 analyzed). The intervention program comprised smoke-free school hours, class-based activities, and access to smoking cessation support. The control group was encouraged to continue with normal practice. Primary outcomes were daily cigarette consumption and daily smoking status at student level. Secondary outcomes were determinants expected to impact smoking behavior. Outcomes were assessed in students at five-month follow-up. Analyses were by intention-to-treat and per protocol (i.e., whether the intervention was delivered as intended), adjusted for covariates measured at baseline. Moreover, subgroup analyses defined by school type, gender, age, and smoking status at baseline were performed. Multilevel regression models were used to account for the cluster design. Missing data were imputed using multiple imputations. Participants and the research team were not blinded to allocation. RESULTS: Intention-to-treat analyses showed no intervention effect on daily cigarette consumption and daily smoking. Pre-planned subgroup analyses showed statistically significant reduction in daily smoking among girls compared with their counterparts in the control group (OR = 0.39, 95% CI: 0.16, 0.98). Per-protocol analysis suggested that schools with full intervention had higher benefits compared with the control group (daily smoking: OR = 0.44, 95% CI: 0.19, 1.02), while no marked differences were seen among schools with partial intervention. CONCLUSION: This study was among the first to test whether a complex, multicomponent intervention could reduce smoking in schools with high smoking risk. Results showed no overall effects. There is a great need to develop programs for this target group and it is important that they are fully implemented if an effect is to be achieved. TRIAL REGISTRATION: https://www.isrctn.com/ISRCTN16455577 , date of registration 14/06/2018.

Response of Soybean Cultivars to Oxidative Stress caused by Meloidogyne javanica.

The present study aimed to investigate the response of soybean cultivars with different susceptibility levels to the root-knot nematode Meloidogyne javanica at varied time intervals by analyzing the initial plant-nematode interaction using antioxidant enzymes as oxidative stress markers. A 4 × 4 × 2 factorial method with 5 repetitions was used to analyze 4 soybean cultivars at 4 different collection times-6, 12, 24, and 48 h-with and without M. javanica inoculation. The parameters evaluated were the activities of antioxidant enzymes phenol peroxidase (POX) and ascorbate peroxidase (APX); the concentrations of hydrogen peroxide (H2O2) and malondialdehyde (MDA); and the number of M. javanica juveniles penetrated into each plant. H2O2 concentration varied among the cultivars with and without inoculation and at different collection times as indicated by MDA concentration and POX and APX activities, demonstrating a rapid response of the host to an infection by M. javanica. Oxidative stress caused by M. javanica did not vary among the soybean cultivars regardless of their susceptibility level; however, the antioxidant enzymes POX and APX responded according to the susceptibility level of the cultivars.

Context matters in smoking prevention: evaluating smoke-free school hours in Danish vocational schools.

Youth is a crucial period for smoking preventive interventions. School-based interventions targeting the policy level and the sociocultural processes of smoking show promising effects in reducing smoking uptake and prevalence. This study presents findings from the qualitative process evaluation of a smoking preventive intervention, Focus, in the vocational school (VET) setting. Specifically, the study focused on contextual factors affecting the implementation of smoke-free school hours (SFSH). Participant observations and focus groups were conducted in four VETs during the implementation period October-December 2018. The data encompass participant observation field notes (n = 21 school days), student focus groups (n = 8) (aged 16-20), teacher focus groups (n = 5) and semi-structured individual interviews with VET leaders (n = 3). The study found that SFSH was not clearly communicated to students due to the educational structure and chaotic rhythm of the school days, ambivalent attitudes among teachers toward enforcement of smoking rules and lack of clear managerial support. The interplay of these factors counteracted the implementation of SFSH in the VET context. The presented contextual factors are important when interpreting the effectiveness of the Focus intervention and for informing future preventive efforts aiming to reduce smoking among youth in high risk of smoking cigarettes.

Youth represents a crucial period for smoking prevention. School-based interventions show promising effects in this respect. This study presents findings from the qualitative process evaluation of a smoking preventive intervention, Focus, in the vocational school (VET) setting. The aim was to examine the role of context in the implementation process. The data consist of participant observation field notes (n = 21 school days), focus groups (n = 8 with students and n =  5 with teachers) and semi-structured individual interviews with VET leaders (n = 3). The study found that smoke-free school hours was not clearly communicated to students due to several contextual factors, namely an unclear structure and purpose of the school day, ambivalent attitudes among teachers toward smoking rules and lack of managerial support. These factors are important when interpreting the effectiveness of the Focus intervention and for informing future smoking preventive efforts among youth in high risk of smoking cigarettes.

Evaluation of postprandial glycemic response in rats (Wistar) fed with different starch sources.

The consumption of readily digestible starch sources, such as corn, can negatively impact endocrine disorders related to hyperglycaemia in predisposing animals. In this sense, starch sources containing slowly digestible and resistant fractions can assist glycemic control. The present study analyzed the postprandial glycemic response and blood variables of rats fed with four extruded diets containing corn, brown rice, sweet potato and pea as the main starch source. Thirty-two male Wistar rats (90 days old) were divided into groups of eight animals each. The rats received one of the experimental diets for 30 days according to a completely randomised design. The glycemia was measured on the 29th and 30th days. The glycemia measured on the 29th day was analyzed at 0, 30, 60, 120 and 240 min after oral administration of 50% glucose solution. On the 30th day, the same protocol was repeated after providing 3 g of the experimental diet for each animal to obtain the glycemic curve. After the euthanasia on the 30th day, 7 ml of blood was collected via cardiac puncture for glycated haemoglobin (HBA1c), triglycerides, cholesterol, and aspartate and alanine aminotransferases analysis. Diets with pea and sweet potato provided lower glycemic index, average and maximum glycemia, and glycemic increment in relation to the other starch sources (p < 0.05). Animals fed with the corn diet had higher serum concentrations of triglycerides and HBA1c than the other treatments (p < 0.05). Results demonstrated that pea and sweet potato are interesting starch sources for the control of metabolic disorders related to glycemia.

Fine cassava fibre utilization as a dietary fibre source for dogs: Effects on kibble characteristics, diet digestibility and palatability, faecal metabolites and microbiota.

The objective was to evaluate through three experiments the effects of a fine cassava fibre (CA: 106 µm) on kibble characteristics, coefficients of total tract apparent digestibility (CTTAD) of macronutrients, diet palatability and faecal metabolites and microbiota of dogs. Dietary treatments consisted of a control diet (CO), without an added fibre source and with 4.3% total dietary fibre (TDF), and a diet with 9.6% CA (106 µm), with 8.4% TDF. Experiment I evaluated the physical characteristics of the kibbles. The palatability test was evaluated in experiment II, which compared the diets CO versus CA. In experiment III, 12 adult dogs were randomly assigned to one of the two dietary treatments for 15 days, totalling six replicates/treatment, to assess the CTTAD of macronutrients; faecal characteristics, faecal metabolites and microbiota. The expansion index, kibble size and friability of diets with CA were higher than the CO (p < 0.05). Additionally, the CA diet presented higher palatability than the CO (p < 0.05) but did not affect CTTAD except for those of fibre (p > 0.05). Moreover, a greater faecal concentration of acetate, butyrate and total short-chain fatty acids (SCFA) and a lower faecal concentration of phenol, indole and isobutyrate were observed in dogs fed the CA diet (p < 0.05). Dogs fed with the CA diet presented a greater bacterial diversity and richness and a greater abundance of genera considered to be beneficial for gut health, such as Blautia, Faecalibacterium and Fusobacterium when compared to the CO group (p < 0.05). The inclusion of 9.6% of a fine CA improves the expansion of kibbles and diet palatability without affecting most of the CTTAD of nutrients. Besides, it improves the production of some SCFA and modulates the faecal microbiota of dogs.

Translation, cross-cultural adaptation, reliability, and construct validity of the Brazilian Portuguese version of the Basic Psychological Needs in Exercise Scale.

RESULTS: 59 patients were included (61% with COPD and 39% with ILD). BPNES factor scores were not significantly different between raters' assessments (p > 0.05). The internal consistency was 0.70 for autonomy, 0.76 for competence, and 0.80 for relatedness. Inter-rater and test-retest reliability were good to very good for autonomy (ICC = 0.78, 95%CI 0.62-0.87; ICC = 0.75, 95%CI 0.57-0.86, respectively), competence (ICC = 0.81, 95%CI 0.68-0.89; ICC = 0.65, 95%CI 0.43-0.80, respectively), and relatedness (ICC = 0.79, 95%CI 0.65-0.88; ICC = 0.70, 95%CI 0.50-0.83, respectively). Significant correlations were observed between BPNES factors and quality of life, anxiety, depression, and functional status. In conclusion, this study confirmed the reliability and construct validity of the Brazilian Portuguese version of the BPNES in patients with COPD and ILD.

Alterations in CD39/CD73 axis of T cells associated with COVID-19 severity.

Purinergic signaling modulates immune function and is involved in the immunopathogenesis of several viral infections. This study aimed to investigate alterations in purinergic pathways in coronavirus disease 2019 (COVID-19) patients. Mild and severe COVID-19 patients had lower extracellular adenosine triphosphate and adenosine levels, and higher cytokines than healthy controls. Mild COVID-19 patients presented lower frequencies of CD4+ CD25+ CD39+ (activated/memory regulatory T cell [mTreg]) and increased frequencies of high-differentiated (CD27- CD28- ) CD8+ T cells compared with healthy controls. Severe COVID-19 patients also showed higher frequencies of CD4+ CD39+ , CD4+ CD25- CD39+ (memory T effector cell), and high-differentiated CD8+ T cells (CD27- CD28- ), and diminished frequencies of CD4+ CD73+ , CD4+ CD25+ CD39+ mTreg cell, CD8+ CD73+ , and low-differentiated CD8+ T cells (CD27+ CD28+ ) in the blood in relation to mild COVID-19 patients and controls. Moreover, severe COVID-19 patients presented higher expression of PD-1 on low-differentiated CD8+ T cells. Both severe and mild COVID-19 patients presented higher frequencies of CD4+ Annexin-V+ and CD8+ Annexin-V+ T cells, indicating increased T-cell apoptosis. Plasma samples collected from severe COVID-19 patients were able to decrease the expression of CD73 on CD4+ and CD8+ T cells of a healthy donor. Interestingly, the in vitro incubation of peripheral blood mononuclear cell from severe COVID-19 patients with adenosine reduced the nuclear factor-κB activation in T cells and monocytes. Together, these data add new knowledge to the COVID-19 immunopathology through purinergic regulation.

Higher expression of proline dehydrogenase altered mitochondrial function and increased Trypanosoma cruzi differentiation in vitro and in the insect vector.

The pathogenic protist Trypanosoma cruzi uses kissing bugs as invertebrate hosts that vectorize the infection among mammals. This parasite oxidizes proline to glutamate through two enzymatic steps and one nonenzymatic step. In insect vectors, T. cruzi differentiates from a noninfective replicating form to nonproliferative infective forms. Proline sustains this differentiation, but to date, a link between proline metabolism and differentiation has not been established. In T. cruzi, the enzymatic steps of the proline-glutamate oxidation pathway are catalyzed exclusively by the mitochondrial enzymes proline dehydrogenase [TcPRODH, EC: 1.5.5.2] and Δ1-pyrroline-5-carboxylate dehydrogenase [TcP5CDH, EC: 1.2.1.88]. Both enzymatic steps produce reducing equivalents that are able to directly feed the mitochondrial electron transport chain (ETC) and thus produce ATP. In this study, we demonstrate the contribution of each enzyme of the proline-glutamate pathway to ATP production. In addition, we show that parasites overexpressing these enzymes produce increased levels of H2O2, but only those overexpressing TcP5CDH produce increased levels of superoxide anion. We show that parasites overexpressing TcPRODH, but not parasites overexpressing TcP5CDH, exhibit a higher rate of differentiation into metacyclic trypomastigotes in vitro. Finally, insect hosts infected with parasites overexpressing TcPRODH showed a diminished parasitic load but a higher percent of metacyclic trypomastigotes, when compared with controls. Our data show that parasites overexpressing both, PRODH and P5CDH had increased mitochondrial functions that orchestrated different oxygen signaling, resulting in different outcomes in relation to the efficiency of parasitic differentiation in the invertebrate host.

Toward an Integrated View of Operational Tolerance in Human Renal Transplantation: A Systems Biology Perspective.

Operational tolerance (OT) is the phenomenon occurring in human renal and liver transplantation in which the body does not reject the organ after discontinuing immunosuppression for at least a year. We revisited the data generated by The Brazilian Multicenter Study on Operational Tolerance involving different conceptual fields - antigen-specific cytokine response, immune cell numbers and repertoire, signaling pathways, and epigenetics. We integrated our data to pave the way to systems biology thinking and harness debate on potential mechanisms in OT. We present original data on systems biology in OT, connecting potential mechanistic players. Using bioinformatics, we identified three dominant features that discriminate OT from its opposing clinical outcome, chronic rejection (CR). The OT-CR discriminative molecules were FOXP3, GATA3 and STAT6, each corresponding to a differential profile: (1) In FOXP3, OT presents preserved regulatory T cell (Treg) numbers but decreased numbers in CR; (2) in GATA3, increased expression is seen in OT; and (3) in STAT6, decreased monocyte activation is seen in OT. With these variables, we built molecular networks to identify interactions related to OT versus CR. Our first systems biology endeavor gave rise to novel potentially relevant interconnected players in OT mechanisms: FOXP3 connecting to interleukin-9 (IL-9) and IL-35 signaling, suggesting their immunoregulatory involvement in OT. Likewise, GATA3/FOXP3 interactions incrementing/stabilizing FOXP3 transcription suggest participation in keeping healthy FOXP3+ Tregs in OT. We envision that systems biology thinking will greatly contribute to advancing knowledge in human transplantation tolerance in an interactive perspective.

Late complications of robot-assisted radical cystectomy with totally intracorporeal urinary diversion.

INTRODUCTION AND OBJECTIVES: To evaluate late complications in a large cohort of patients undergoing robot-assisted radical cystectomy (RARC) with totally intracorporeal urinary diversion (ICUD). MATERIALS AND METHODS: We prospectively enrolled patients who underwent RARC and ICUD between August 2012 and June 2019. We excluded patients with Ejection fraction < 36%, retinal vasculopathy, ventriculoperitoneal shunts, and those treated without curative intent. All complications and their onset date have been recorded, defined, and graded according to Clavien classification adapted for radical cystectomy. RESULTS: 210 patients were included, 76% of whom were men, with a mean age of 62 years. Urinary diversions used were Padua Ileal Bladder (PIB) in 80% of cases, and ileal conduit (IC) in 20% of patients (generally older and with more comorbidity). The mean follow-up was 30 ± 22 months. The stenosis rate of uretero-ileal anastomosis was 14%, while a reduction in eGFR (≥ 20%) was observed in about half of the cases. UTIs occurred in 37% of the patients, especially in the first 12 months. Only 2% of patients had bowel occlusion, whereas incisional hernia, lymphocele, and systemic events (metabolic acidosis and major cardiovascular events) occurred respectively in 20%, 10%, and 1% of cases. CONCLUSIONS: Our study evaluates first late complications in a cohort of patients who underwent RARC with ICUD. These data are encouraging and in line with findings from a historical series of open radical cystectomy (ORC). This study is a further step in supporting RARC as a safe and effective surgical option for the treatment of muscle-invasive bladder cancer (MIBC) in tertiary referral centers.