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Abstract Plasma RNAemia, delayed antibody responses and inflammation predict COVID-19 outcomes, but the mechanisms underlying these immunovirological patterns are poorly understood. We profile 782 longitudinal plasma samples from 318 hospitalized patients with COVID-19. Integrated analysis using k-means reveals four patient clusters in a discovery cohort: mechanically ventilated critically-ill cases are subdivided into good prognosis and high-fatality clusters (reproduced in a validation cohort), while non-critical survivors segregate into high and low early antibody responders. Only the high-fatality cluster is enriched for transcriptomic signatures associated with COVID-19 severity, and each cluster has distinct RBD-specific antibody elicitation kinetics. Both critical and non-critical clusters with delayed antibody responses exhibit sustained IFN signatures, which negatively correlate with contemporaneous RBD-specific IgG levels and absolute SARS-CoV-2-specific B and CD4+ T cell frequencies. These data suggest that the “Interferon paradox” previously described in murine LCMV models is operative in COVID-19, with excessive IFN signaling delaying development of adaptive virus-specific immunity.

Summary: Background: Despite successful antiretroviral therapy (ART), frequencies and immunological functions of memory CCR6+ Th17-polarised CD4+ T-cells are not fully restored in people with HIV (PWH). Moreover, long-lived Th17 cells contribute to HIV persistence under ART. However, the molecular mechanisms underlying these observations remain understudied. Methods: mRNA-sequencing was performed using Illumina technology on freshly FACS-sorted memory CCR6+CD4+ T-cells from successfully ART-treated (ST), elite controllers (EC), and uninfected donors (HD). Gene expression validation was performed by RT-PCR, flow cytometry, and in vitro functional assays. Findings: Decreased Th17 cell frequencies in STs and ECs versus HDs coincided with reduced Th17-lineage cytokine production in vitro. Accordingly, the RORγt/RORC2 repressor NR1D1 was upregulated, while the RORγt/RORC2 inducer Semaphorin 4D was decreased in memory CCR6+ T-cells of STs and ECs versus HDs. The presence of HIV-DNA in memory CCR6+ T-cells of ST and EC corresponded with the downregulation of HIV restriction factors (SERINC3, KLF3, and RNF125) and HIV inhibitors (tetraspanins), along with increased expression of the HIV-dependency factor MRE11, indicative of higher susceptibility/permissiveness to HIV-1 infection. Furthermore, markers of DNA damage/modification were elevated in memory CCR6+ T-cells of STs and ECs versus HDs, in line with their increased activation (CD38/HLA-DR), senescence/exhaustion phenotype (CTLA-4/PD-1/CD57) and their decreased expression of proliferation marker Ki-67. Interpretation: These results reveal new molecular mechanisms of Th17 cell deficit in ST and EC PWH despite a successful control of HIV-1 replication. This knowledge points to potential therapeutic interventions to limit HIV-1 infection and restore frequencies, effector functions, and senescence/exhaustion in Th17 cells. Funding: This study was funded by the Canadian Institutes of Health Research (CIHR, operating grant MOP 142294, and the Canadian HIV Cure Enterprise [CanCURE 2.0] Team Grant HB2 164064), and in part, by the Réseau SIDA et maladies infectieuses du Fonds de recherche du Québec-Santé (FRQ-S).

Despite its importance, formal education in healthcare training programs on sex- and gender-specific cardiovascular disease (CVD) risk factors, symptoms, treatment, and outcomes is lacking. We completed rapid reviews of the academic and grey literature to describe the current state of women-specific CVD education in medical, nursing, and other healthcare education programs. Second, we analyzed results from a Canada-wide survey of healthcare professional education programs to identify gaps in curricula related to sex- and gender-specific training in CVD. Our academic review yielded only 15 peer-reviewed publications, and our online search only 20 healthcare education programs, that note that they specifically address women, or sex and gender, and CVD in their curricula. Across both searches, the majority of training and education programs were from the USA, varied greatly in length, delivery mode, and content covered, and lacked consistency in evaluation. Of surveys sent to 213 Canadian universities and other entry-to-practice programs, 80 complete responses (37.6%) were received. A total of 47 respondents (59%) reported that their programs included women-specific CVD content. Among those programs without content specific to CVD in women, 69.0% stated that its inclusion would add “quite a bit” or “a great deal” of value to the program. This study highlights the emerging focus on and substantial gaps in women-specific CVD training and education across healthcare education programs. All medical, nursing, and healthcare training programs are implored to incorporate sex- and gender-based CVD content into their regular curricula as part of a consolidated effort to minimize gaps in cardiovascular care. Résumé: Malgré la prévalence des maladies cardiovasculaires (CV), les programmes d’enseignement en santé accordent peu d’attention aux facteurs de risque, aux symptômes, aux traitements et aux issues selon le sexe ou le genre. Premièrement, nous avons fait une revue rapide de la littérature universitaire et la littérature grise pour faire état de la formation sur les maladies CV spécifiques aux femmes dans les programmes d’enseignement en médecine, en soins infirmiers et autres domaines de la santé. Deuxièmement, nous avons analysé les résultats d’une enquête menée à l’échelle du Canada sur des programmes de formation professionnelle pour cerner les lacunes dans les programmes au chapitre de la formation sur les maladies CV en fonction du sexe et du genre. Notre analyse de la littérature universitaire a permis de relever seulement 15 publications révisées par des pairs à ce sujet, et notre recherche en ligne a mis au jour seulement 20 programmes d’enseignement qui comportent un volet portant spécifiquement sur les femmes, ou bien le sexe et le genre, et les maladies CV. Ces deux enquêtes ont révélé que la majorité des programmes de formation et d’enseignement étaient aux États-Unis et qu’ils présentaient une grande diversité sur le plan de la durée, du mode d’enseignement et du contenu abordé. De plus, les méthodes d’évaluation n’étaient pas uniformes. Parmi les sondages envoyés à 213 universités et programmes d’admission à la pratique au Canada, 80 réponses complètes (37,6 %) ont été reçues. Quarante-sept des établissements qui ont répondu (59 %) ont signalé que leurs programmes comprenaient du contenu portant sur les maladies CV spécifiques aux femmes. Parmi les établissements dont les programmes ne comportaient aucun contenu spécifique aux femmes, 69,0 % ont indiqué qu’une telle inclusion ajouterait « beaucoup » ou « énormément » de valeur au programme. Cette étude met en lumière l’attention nouvelle accordée à la formation et à l’enseignement sur les maladies CV spécifiques aux femmes ainsi que les lacunes substantielles observées à cet égard dans les programmes d’enseignement en santé. Les programmes de formation en médecine, en soins infirmiers et en santé sont vivement invités à intégrer du contenu spécifique au sexe et au genre pour ce qui est des maladies CV dans un effort concerté visant à réduire les lacunes dans les soins cardiovasculaires.

BackgroundElectrocardiographic (ECG) features of left bundle branch (LBB) block (LBBB) can be observed in up to 20%–30% of patients suffering from heart failure with reduced ejection fraction. However, predicting which LBBB patients will benefit from cardiac resynchronization therapy (CRT) or conduction system pacing remains challenging. This study aimed to establish a translational model of LBBB to enhance our understanding of its pathophysiology and improve therapeutic approaches.MethodsFourteen male pigs underwent radiofrequency catheter ablation of the proximal LBB under fluoroscopy and ECG guidance. Comprehensive clinical assessments (12-lead ECG, bloodsampling, echocardiography, electroanatomical mapping) were conducted before LBBB induction, after 7, and 21 days. Three pigs received CRT pacemakers 7 days after LBB ablation to assess resynchronization feasibility.ResultsFollowing proximal LBB ablation, ECGs displayed characteristic LBBB features, including QRS widening, slurring in left lateral leads, and QRS axis changes. QRS duration increased from 64.2 ± 4.2 ms to 86.6 ± 12.1 ms, and R wave peak time in V6 extended from 21.3 ± 3.6 ms to 45.7 ± 12.6 ms. Echocardiography confirmed cardiac electromechanical dyssynchrony, with septal flash appearance, prolonged septal-to-posterior-wall motion delay, and extended ventricular electromechanical delays. Electroanatomical mapping revealed a left ventricular breakthrough site shift and significantly prolonged left ventricular activation times. RF-induced LBBB persisted for 3 weeks. CRT reduced QRS duration to 75.9 ± 8.6 ms, demonstrating successful resynchronization.ConclusionThis porcine model accurately replicates the electrical and electromechanical characteristics of LBBB observed in patients. It provides a practical, cost-effective, and reproducible platform to investigate molecular and translational aspects of cardiac electromechanical dyssynchrony in a controlled and clinically relevant setting.

Background Women with preeclampsia have a higher risk of cardiovascular disease. This is partly explained by the worse arterial health after preeclampsia. Central obesity (CO) is a risk factor for both preeclampsia and cardiovascular disease. Whether CO contributes to further worsening of arterial health after preeclampsia remains unclear. Our objective was to evaluate the effect of CO and previous preeclampsia on arterial hemodynamics. Methods and Results We studied 40 women with previous preeclampsia (

Summary: HIV persists in tissues during antiretroviral therapy (ART), but the relative contribution of different anatomical compartments to the viral reservoir in humans remains unknown. We performed an extensive characterization of HIV reservoirs in two men who donated their bodies to HIV cure research and who had been on suppressive ART for years. HIV DNA is detected in all tissues, with large variations across anatomical compartments and between participants. Intact HIV genomes represent 2% and 25% of all proviruses in the two participants and are mainly detected in secondary lymphoid organs, with the spleen and mediastinal lymph nodes harboring intact viral genomes in both individuals. Multiple copies of identical HIV genomes are found in all tissues, indicating that clonal expansions are common in anatomical sites. The majority (>85%) of these expanded clones are shared across multiple tissues. These findings suggest that infected cells expand, migrate, and possibly circulate between anatomical sites.

Throughout the SARS-CoV-2 pandemic, several variants of concern (VOCs) have been identified, many of which share recurrent mutations in the spike glycoprotein’s receptor-binding domain (RBD). This region coincides with known epitopes and can therefore have an impact on immune escape. Protracted infections in immunosuppressed patients have been hypothesized to lead to an enrichment of such mutations and therefore drive evolution towards VOCs. Here, we present the case of an immunosuppressed patient that developed distinct populations with immune escape mutations throughout the course of their infection. Notably, by investigating the co-occurrence of substitutions on individual sequencing reads in the RBD, we found quasispecies harboring mutations that confer resistance to known monoclonal antibodies (mAbs) such as S:E484K and S:E484A. These mutations were acquired without the patient being treated with mAbs nor convalescent sera and without them developing a detectable immune response to the virus. We also provide additional evidence for a viral reservoir based on intra-host phylogenetics, which led to a viral substrain that evolved elsewhere in the patient’s body, colonizing their upper respiratory tract (URT). The presence of SARS-CoV-2 viral reservoirs can shed light on protracted infections interspersed with periods where the virus is undetectable, and potential explanations for long-COVID cases.

Although antiretroviral therapy (ART) is able to successfully suppress plasma viremia in most people living with HIV, ART withdrawal typically results in viral replication and rebound. Authors investigate the effect, in terms of delay in viral replication, and immune cell dynamics in lymphoid tissue, of fingolimod (FTY720) administration at the time of ART initiation in SIV-infected rhesus macaques.

Background Early vascular aging (EVA) is associated with higher risk of adverse cardiovascular events and can be estimated noninvasively by assessing arterial hemodynamics. Women with a history of preeclampsia have increased risk of cardiovascular disease, but underlying mechanisms are incompletely understood. We hypothesized that women with a history of preeclampsia display persistent arterial abnormalities and EVA in the postpartum period. Methods and Results We performed a comprehensive, noninvasive arterial hemodynamic evaluation in women with a history of preeclampsia (n=40) and age‐matched controls with previous normotensive pregnancies (n=40). We used validated methods integrating applanation tonometry with transthoracic echocardiography to obtain measures of aortic stiffness, steady and pulsatile arterial load, central blood pressure, and arterial wave reflections. Presence of EVA was defined as aortic stiffness higher than that predicted from reference values based on the participant's age and blood pressure. The association of preeclampsia with arterial hemodynamic variables was assessed with multivariable linear regression, and the association of severe preeclampsia with EVA was assessed with multivariable logistic regression, adjusted for confounders. We found that women with a history of preeclampsia had greater aortic stiffness, steady arterial load, central blood pressure, and arterial wave reflections when compared with controls. We observed a dose–response relationship, with the greatest abnormalities observed in subgroups with severe, preterm, or recurrent preeclampsia. Women with severe preeclampsia had 9.23 times greater odds of having EVA as compared with controls (95% CI, 1.67–51.06, P=0.011) and 7.87 greater odds of EVA as compared with women with nonsevere preeclampsia (95% CI, 1.29–47.77, P=0.025). Conclusions Our study comprehensively characterizes arterial hemodynamic abnormalities after preeclampsia and suggests that specific subgroups of women with a history of preeclampsia exhibit greater alterations in arterial hemodynamics related to arterial health. Our findings have important implications for understanding potential links between preeclampsia and cardiovascular events, and suggest women with severe, preterm, or recurrent preeclampsia as subgroups who may deserve intensification of efforts for prevention and early detection of cardiovascular disease.

Abstract Objectives Identifying biomarkers causing differential SARS‐CoV‐2 infection kinetics associated with severe COVID‐19 is fundamental for effective diagnostics and therapeutic planning. Methods In this work, we applied mathematical modelling to investigate the relationships between patient characteristics, plasma SARS‐CoV‐2 RNA dynamics and COVID‐19 severity. Using a straightforward mathematical model of within‐host viral kinetics, we estimated key model parameters from serial plasma viral RNA (vRNA) samples from 256 hospitalised COVID‐19+ patients. Results Our model predicted that clearance rates distinguish key differences in plasma vRNA kinetics and severe COVID‐19. Moreover, our analyses revealed a strong correlation between plasma vRNA kinetics and plasma receptor for advanced glycation end products (RAGE) concentrations (a plasma biomarker of lung damage), collected in parallel to plasma vRNA from patients in our cohort, suggesting that RAGE can substitute for viral plasma shedding dynamics to prospectively classify seriously ill patients. Conclusion Overall, our study identifies factors of COVID‐19 severity, supports interventions to accelerate viral clearance and underlines the importance of mathematical modelling to better understand COVID‐19.

Introduction There are significant sex differences in the prevalence and severity of cardiac calcifying processes. Women harbour more severe mitral annular calcification (MAC), while men exhibit worse aortic valve (AVC) and coronary artery (CAC) calcification. To better understand these differences, we investigated the correlates of cardiac calcification according to sex.Methods We conducted a cross-sectional study of 406 patients with ≥mild aortic stenosis (AS) defined by an aortic valve area ≤1.5 cm2, a peak aortic jet velocity >2.0 m/s, or a mean transvalvular gradient >15 mm Hg. Doppler-echocardiography and non-contrast multidetector CT were performed concomitantly to assess AS and cardiac calcifications.Results Mean age was 71±11 years and 33% were women. The AS haemodynamics were not significantly different between sexes (all p>0.50), with a mean indexed aortic valve area of 0.59±0.21 cm2/m2, peak aortic jet velocity of 2.78 (2.37–3.68) m/s, and mean gradient of 17.9 (12.8–31.3) mm Hg for the whole cohort. Compared with men, women harboured lower AVC (480 (222–1191) vs 1003 (484–2329) Agatston unit, AU; p

Clinical decisions are influenced by hematocrit values. Centrifugation (reference standard), conductivity, optical and impedance methods are often used interchangeably to measure hematocrit. The effects of albumin, which are known to affect conductivity methods, have not been evaluated for limits of agreement (LOA) between hematocrit assays in small animals. Canine venous blood was collected from 74 clinical cases and measured by centrifugation (n = 72), conductivity (n = 73), impedance (n = 24) and optical (n = 50) methods. Bland-Altman analysis determined bias (± SD) and 95% LOA between methods. There was a statistically significant difference between centrifugation hematocrit values and values obtained via conductivity (p < 0.0001), optical (p < 0.0001), and impedance (p = 0.0082) methods. The conductivity method underestimated hematocrit by 2.1 ± 2.9% (95% LOA −3.54 to 7.88), the optical method by 3.1 ± 3.6% (95% LOA −4.0 to 10.2), and the impedance method by 2.3 ± 3.7% (95% LOA −5 to 9.6) when compared to centrifuged hematocrit values. The hematocrit difference between conductivity and centrifugation methods was statistically different for low (4%, 0–5%), within reference limits (3%, −5 to 8%), and high (2%, −2 to 5%) albumin values, respectively (p = 0.02), with post-hoc analysis demonstrating that the difference occurred between the low and high albumin groups. This study confirms that albumin values outside reference limits can affect the conductivity method and that hematocrit values obtained via conductivity, optical and impedance methods underestimate values obtained via centrifugation. Therefore, the hematocrit methods cannot be used interchangeably. The wide limits of agreement also demonstrates that care must be taken when making clinical decisions with different hematocrit methodologies.

Chronic HIV infection is characterized by persistent inflammation despite antiretroviral therapy (ART). Cannabinoids may help reduce systemic inflammation in people with HIV (PWH). To assess the effects of oral cannabinoids during HIV, ten PWH on ART were randomized (n = 5/group) to increasing doses of oral Δ9-tetrahydrocannabinol (THC): cannabidiol (CBD) combination (2.5:2.5–15:15 mg/day) capsules or CBD-only (200–800 mg/day) capsules for 12 weeks. Blood specimens were collected prospectively 7–21 days prior to treatment initiation and at weeks 0 to 14. Plasma cytokine levels were determined via Luminex and ELISA. Immune cell subsets were characterized by flow cytometry. HIV DNA/RNA were measured in circulating CD4 T-cells and sperm by ultra-sensitive qPCR. Results from both arms were combined for statistical analysis. Plasma levels of IFN-γ, IL-1β, sTNFRII, and REG-3α were significantly reduced at the end of treatment (p ˂ 0.05). A significant decrease in frequencies of PD1+ memory CD4 T-cells, CD73+ regulatory CD4 T-cells, and M-DC8+ intermediate monocytes was also observed (p ˂ 0.05), along with a transient decrease in CD28–CD57+ senescent CD4 and CD8 T-cells. Ki-67+ CD4 T-cells, CCR2+ non-classical monocytes, and myeloid dendritic cells increased over time (p ˂ 0.05). There were no significant changes in other inflammatory markers or HIV DNA/RNA levels. These findings can guide future large clinical trials investigating cannabinoid anti-inflammatory properties.

Abstract Background We previously reported the results of CRISPR/Cas9 knock-out (KO) of type-I and type-III vitellogenins (Vtgs) in zebrafish, which provided the first experimental evidence on essentiality and disparate functioning of Vtgs at different stages during early development. However, the specific contributions of different types of Vtg to major cellular processes remained to be investigated. The present study employed liquid chromatography and tandem mass spectrometry (LC-MS/MS) to meet this deficit. Proteomic profiles of zebrafish eggs lacking three type-I Vtgs simultaneously (vtg1-KO), or lacking only type III Vtg (vtg3-KO) were compared to those of wild type (Wt) eggs. Obtained spectra were searched against a zebrafish proteome database and identified proteins were quantified based on normalized spectral counts. Results The vtg-KO caused severe changes in the proteome of 1-cell stage zebrafish eggs. These changes were disclosed by molecular signatures that highly resembled the proteomic phenotype of poor quality zebrafish eggs reported in our prior studies. Proteomic profiles of vtg-KO eggs and perturbations in abundances of hundreds of proteins revealed unique, noncompensable contributions of multiple Vtgs to protein and in energy homeostasis. The lack of this contribution appears to have a significant impact on endoplasmic reticulum and mitochondrial functions, and thus embryonic development, even after zygotic genome activation. Increased endoplasmic reticulum stress, Redox/Detox activities, glycolysis/gluconeogenesis, enrichment in cellular proliferation and in human neurodegenerative disease related activities in both vtg1- and vtg3-KO eggs were found to be indicators of the aforementioned conditions. Distinctive increase in apoptosis and Parkinson disease pathways, as well as the decrease in lipid metabolism related activities in vtg3-KO eggs implies compelling roles of Vtg3, the least abundant form of Vtgs in vertebrate eggs, in mitochondrial activities. Several differentially abundant proteins representing the altered molecular mechanisms have been identified as strong candidate markers for studying the details of these mechanisms during early embryonic development in zebrafish and possibly other vertebrates. Conclusions These findings indicate that the global egg proteome is subject to extensive modification depending on the presence or absence of specific Vtgs and that these modifications can have a major impact on developmental competence.

Teaching Point: Benign granulomatous prostatitis mimics prostate cancer on MRI. Peculiar urological history of a patient undergoing prostate MRI helps sorting out that differential, such as a treatment with topic Bacilli Calmette-Guerin instillations.

The cause of clonal expansions in the HIV reservoir remains unclear. Here, Gantner et al. perform single-cell TCR sequencing on longitudinal samples from eight individuals on antiretroviral therapy and find that antigens inducing clonal expansions of memory cells are major contributors to the HIV reservoir.

Abstract Background The breeding time of many species has changed over the past 2–3 decades in response to climate change. Yet it is a key reproductive trait that affects individual's parturition time and reproductive success, and thereby population dynamics. In order to predict how climate change will affect species’ viability, it is crucial to understand how species base their reproductive efforts on environmental cues. Results By using long-term datasets of mating behaviours and copulation dates recorded since 1996 on a semi-domesticated reindeer population, we showed that mating time occurred earlier in response to weather conditions at different key periods in their annual breeding cycle. The mating time occurred earlier following a reducing snow cover in early spring, colder minimum temperatures in the last 2 weeks of July and less precipitation in August-September. Conclusions The mediated effect of a reduced snow cover in early spring on improving individuals’ pre-rut body weight through a better availability of late winter food and reduced costs of locomotion on snow would explain that mating time has occurred earlier overtime. A lower level of insect harassment caused by colder maximum temperatures in July might have caused an advance in mating time. Less precipitation in August-September also caused the mating time to occur earlier, although the direct effects of the last two weather variables were not mediated through the pre-rut body weight of individuals. As such, the causal effects of weather conditions on seasonal timing of animals are still unclear and other mechanisms than just body weight might be involved (e.g. socio-biological factors). The plastic response of reindeer mating time to climatic variability, despite supplemental feeding occurring in late April, demonstrated that environmental factors may have a greater influence on reproductive outputs than previously thought in reindeer.

Background Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. However, underlying molecular mechanisms are insufficiently understood. Previous studies suggested that microRNA (miRNA) dependent gene regulation plays an important role in the initiation and maintenance of AF. The 2‐pore‐domain potassium channel TASK‐1 (tandem of P domains in a weak inward rectifying K+ channel–related acid sensitive K+ channel 1) is an atrial‐specific ion channel that is upregulated in AF. Inhibition of TASK‐1 current prolongs the atrial action potential duration to similar levels as in patients with sinus rhythm. Here, we hypothesize that miRNAs might be responsible for the regulation of KCNK3 that encodes for TASK‐1. Methods and Results We selected miRNAs potentially regulating KCNK3 and studied their expression in atrial tissue samples obtained from patients with sinus rhythm, paroxysmal AF, or permanent/chronic AF. MiRNAs differentially expressed in AF were further investigated for their ability to regulate KCNK3 mRNA and TASK‐1 protein expression in human induced pluripotent stem cells, transfected with miRNA mimics or inhibitors. Thereby, we observed that miR‐34a increases TASK‐1 expression and current and further decreases the resting membrane potential of Xenopus laevis oocytes, heterologously expressing hTASK‐1. Finally, we investigated associations between miRNA expression in atrial tissues and clinical parameters of our patient cohort. A cluster containing AF stage, left ventricular end‐diastolic diameter, left ventricular end‐systolic diameter, left atrial diameter, atrial COL1A2 (collagen alpha‐2(I) chain), and TASK‐1 protein level was associated with increased expression of miR‐25, miR‐21, miR‐34a, miR‐23a, miR‐124, miR‐1, and miR‐29b as well as decreased expression of miR‐9 and miR‐485. Conclusions These results suggest an important pathophysiological involvement of miRNAs in the regulation of atrial expression of the TASK‐1 potassium channel in patients with atrial cardiomyopathy.

ObjectivesThe aim of this study was to describe the seroprevalence, presenting complaint, clinicopathological changes, co-morbidities and outcomes of feline leukemia virus positive cats presented to a specialty referral center in Florida, USA.MethodsIn this retrospective study, medical records of 8050 cats presented to a private referral center from August 2008 to September 2019 were reviewed. Inclusion criteria required was a positive result for feline leukemia virus by point-of-care antigen testing or immunofluorescence assay.ResultsForty-one cases met the inclusion criteria. Of 2002 cats that were tested, 41 cats (2%) met the inclusion criteria. One cat had a negative point of care antigen test result and positive bone marrow IFA result. The mean age at diagnosis was 9 years. The main reasons for presentation were abnormal complete blood cell count results (35%), followed by pleural effusion (18%), and anorexia (15%). The most common laboratory abnormalities included anaemia (71%), of which 74% had a nonregenerative anemia, thrombocytopenia (52%), elevated aspartate aminotransferase (50%), hyperbilirubinemia (35%), and hypokalemia (35%). Seven percent of cats (3/41) were also positive for feline immunodeficiency virus. The most common diagnoses were neoplasia (76%) and bone marrow disorders (12%). Cats with neoplasia were significantly younger. Survival to discharge was 88%.Conclusion and relevanceResults of this study show that feline leukemia virus is uncommon in secondary referral center, even if this represents a population of unhealthy cats. The most common associated diagnosis was neoplasia, which was more likely to be seen in younger cats (< 4 years of age). The mean age of cats positive for feline leukemia virus was also older than previously published data. These findings support the current guidelines which indicate that cats presented with clinical illness should be tested for FeLV at the time of presentation.

Introduction The prognosis of first episode psychosis (FEP), which is a severe disorder, can be notably impaired by patients’ disengagement from healthcare providers. Coordinated specialty care with case management is now considered as the gold standard in this population, but there are still challenges for engagement with subsequent functional impairments. Youth-friendly and patient-centred clinical approaches are sought to improve engagement in patients with FEP. Mobile applications are widely used by young people, including patients with FEP, and can increase the youth friendliness of clinical tools. We hypothesise that a co-designed mobile application used during case management can improve functioning in patients with FEP as compared with usual case management practices.Methods and analysis A mobile case management application for planning and monitoring individualised care objectives will be co-designed with patients, caregivers and health professionals in a recovery-oriented approach. The application will be compared with usual case management practices in a multicentre, two-arm and parallel groups clinical trial. Patients will be recruited by specialised FEP teams. Impact on functioning will be assessed using the Personal and Social Performance Scale; the variation between baseline and 12 months in each group (control and active) will be the primary outcome.Ethics and dissemination This study has been approved by the Inserm Institutional Review Board IRB00003888 (Comité d’évaluation éthique de l’INSERM, reference number 20-647). The results of the study will be published in a peer-reviewed journal and presented at national and international conferences. We will also communicate the results to patients and family representatives’ associations. An optimised version of the application will be then disseminated through the French FEP network (Transition Network).Trial registration number ClinicalTrials.gov: NCT04657380

Clinical outcomes are inferior for individuals with HIV having suboptimal CD4 T-cell recovery during antiretroviral therapy (ART). We investigated if the levels of infection and the response to homeostatic cytokines of CD4 T-cell subsets contributed to divergent CD4 T-cell recovery and HIV reservoir during ART by studying virologically-suppressed immunologic responders (IR, achieving a CD4 cell count >500 cells/μL on or before two years after ART initiation), and virologically-suppressed suboptimal responders (ISR, did not achieve a CD4 cell count >500 cells/μL in the first two years after ART initiation). Compared to IR, ISR demonstrated higher levels of HIV-DNA in naïve, central (CM), transitional (TM), and effector (EM) memory CD4 T-cells in blood, both pre- and on-ART, and specifically in CM CD4 T-cells in LN on-ART. Furthermore, ISR had higher pre-ART plasma levels of IL-7 and IL-15, cytokines regulating T-cell homeostasis. Notably, pre-ART PD-1 and TIGIT expression levels were higher in blood CM and TM CD4 T-cells for ISR; this was associated with a significantly lower fold-changes in HIV-DNA levels between pre- and on-ART time points exclusively on CM and TM T-cell subsets, but not naïve or EM T-cells. Finally, the frequency of CM CD4 T-cells expressing PD-1 or TIGIT pre-ART as well as plasma levels of IL-7 and IL-15 predicted HIV-DNA content on-ART. Our results establish the association between infection, T-cell homeostasis, and expression of PD-1 and TIGIT in long-lived CD4 T-cell subsets prior to ART with CD4 T-cell recovery and HIV persistence on-ART.

Abstract Background Egg quality can be defined as the egg ability to be fertilized and subsequently develop into a normal embryo. Previous research has shed light on factors that can influence egg quality. Large gaps however remain including a comprehensive view of what makes a bad egg. Initial development of the embryo relies on maternally-inherited molecules, such as transcripts, deposited in the egg during its formation. Bad egg quality is therefore susceptible to be associated with alteration or dysregulation of maternally-inherited transcripts. We performed transcriptome analysis on a large number (N = 136) of zebrafish egg clutches, each clutch being split to monitor developmental success and perform transcriptome analysis in parallel. We aimed at drawing a molecular portrait of the egg in order to characterize the relation between egg transcriptome and developmental success and to subsequently identify new candidate genes involved in fertility. Results We identified 66 transcript that were differentially abundant in eggs of contrasted phenotype (low or high developmental success). Statistical modeling using partial least squares regression and genetics algorithm demonstrated that gene signatures from transcriptomic data can be used to predict developmental success. The identity and function of differentially expressed genes indicate a major dysregulation of genes of the translational machinery in poor quality eggs. Two genes, otulina and slc29a1a, predominantly expressed in the ovary and dysregulated in poor quality eggs were further investigated using CRISPR/Cas9 mediated genome editing. Mutants of each gene revealed remarkable subfertility whereby the majority of their eggs were unfertilizable. The Wnt pathway appeared to be dysregulated in the otulina mutant-derived eggs. Conclusions Here we show that egg transcriptome contains molecular signatures, which can be used to predict developmental success. Our results also indicate that poor egg quality in zebrafish is associated with a dysregulation of (i) the translational machinery genes and (ii) novel fertility genes, otulina and slc29a1a, playing an important role for fertilization. Together, our observations highlight the diversity of the possible causes of egg quality defects and reveal mechanisms of maternal origin behind the lack of fertilization and early embryonic failures that can occur under normal reproduction conditions.

Pulmonary dysbiosis may predispose people living with HIV (PLWH) to chronic lung disease. Herein, we assessed whether intrapulmonary HIV reservoir size and immune disruption are associated with reduced bacterial lung diversity in PLWH. Bacterial DNA was extracted and PCR-amplified from cell-free bronchoalveolar lavage (BAL) fluid from 28 PLWH and 9 HIV-negative controls. Amplicon sequence variant (ASV) relative abundances and taxonomic identities were analyzed using joint species distribution modeling. HIV-DNA was quantified from blood and pulmonary CD4+ T-cells using ultra-sensitive qPCR. Immunophenotyping of BAL T-cells was performed using flow cytometry. Lung microbiome diversity was lower in smokers than non-smokers and microbiome composition was more variable in PLWH than HIV-negative individuals. Frequencies of effector memory BAL CD4+ and CD8+ T-cells positively correlated with abundance of several bacterial families while frequencies of BAL activated CD4+ T-cells negatively correlated with abundance of most lung bacterial families. Higher HIV-DNA levels in blood, but not in BAL, as well as frequencies of senescent CD4+ T-cells were associated with reduced bacterial diversity. These findings suggest that HIV infection may weaken the relationship between the lung microbiome and smoking status. Viral reservoir and immune activation levels may impact the lung microbiome, predisposing PLWH to pulmonary comorbidities.

Summary: Background: Early antiretroviral therapy (ART) initiation (ie, within 3 months of infection) limits establishment of the HIV reservoir. However, the effect of early ART initiation on the long-term dynamics of the pool of infected cells remains unclear. Methods: In this longitudinal analysis, we included cisgender men who have sex with men (MSM) and transgender women (aged 18–54 years) at high risk for HIV infection, enrolled in the ongoing longitudinal MERLIN study in Peru between Oct 28, 2014, and Nov 8, 2018. Participants were eligible if they had been infected with HIV less than 90 days before enrolment, and if they had cryopreserved peripheral blood mononuclear cell (PBMC) samples. Participants were stratified into three groups on the basis of whether they initiated ART at 30 days or less (acute group), at 31–90 days (early group), or more than 24 weeks (deferred group) after the estimated date of detectable infection. PBMC samples were collected before ART initiation and longitudinally for up to 4 years on ART. The main outcomes were to establish the size of the HIV reservoir before ART initiation and to assess the effect of the timing of ART initiation on the decay of the HIV reservoir over 4 years follow-up. We quantified viral load, and isolated CD4 cells to quantify total HIV DNA, integrated HIV DNA and 2-long terminal repeat circles. Longitudinal analysis of active and inducible HIV reservoirs were measured by quantifying the frequency of CD4 cells producing multiply-spliced HIV RNA ex vivo and after in-vitro stimulation with a tat/rev induced limiting dilution assay (TILDA). A mixed-effects model from the time of ART initiation was used to measure longitudinal decays in viral loads and each HIV reservoir measure in each of the three groups. Findings: We included 56 participants in this analysis, all of whom were MSM: 15 were in the acute group, 19 were in the early group, and 22 were in the deferred group. Participants in all three groups had similar levels of all HIV reservoir markers before ART initiation. All participants, including those in the acute group, had a pool of transcriptionally silent HIV-infected cells before ART initiation, as indicated by a substantial increase in TILDA measures upon stimulation. Longitudinal analysis over 4 years of ART revealed a biphasic decay of all HIV persistence markers, with a rapid initial decline followed by a slower decay in all participants. During the first-phase decay, the half-lives of both total and integrated HIV DNA and TILDA measures were significantly shorter in the acute group than in the early and deferred groups. During the second-phase decay, HIV reservoir markers continued to decline only in participants in the acute group. Interpretation: Participants who initiated ART within 30 days or less of HIV infection showed a steeper and more sustained decay in HIV reservoir measures, suggesting long-term benefit of acute ART initiation on reservoir clearance. Funding: The US National Institutes of Health and the Canadian Institutes for Health Research.

Background: Chronic inflammation and residual HIV transcription persist in people living with HIV (PLWH) receiving antiretroviral therapy (ART), thus increasing the risk of developing non-AIDS co-morbidities. The mechanistic target of rapamycin (mTOR) is a key regulator of cellular metabolism and HIV transcription, and therefore represents an interesting novel therapeutic target. Methods: The LILAC pilot clinical trial, performed on non-diabetic ART-treated PLWH with CD4+/CD8+ T-cell ratios

Objective and Design: A randomized, open-label pilot study in individuals treated with antiretroviral therapy (ART) since acute HIV infection (AHI) with a regimen including a histone deacetylase inhibitor to induce HIV from latency and control HIV replication during subsequent treatment interruption (TI). Methods: Fifteen participants who initiated ART at AHI were randomized to vorinostat/hydroxychloroquine/maraviroc (VHM) plus ART (n ​= ​10) or ART alone (n ​= ​5). The VHM arm received three 14-day vorinostat cycles within 10 weeks before TI. ART was resumed for plasma viral load (VL) ​> ​1,000 HIV RNA copies/mL. Primary outcome was proportion of participants on VHM ​+ ​ART versus ART only with VL ​

Atrial fibrillation (AF) is an arrhythmia associated with an increased stroke risk and mortality rate. Current treatment options leave unmet needs in AF therapy. Recently, doxapram has been introduced as a possible new option for AF treatment in a porcine animal model. To better understand its pharmacokinetics, three German Landrace pigs were treated with intravenous doxapram (1 mg/kg). Plasma and brain tissue samples were collected. For the analysis of these samples, an ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay for the simultaneous measurement of doxapram and its active metabolite 2-ketodoxapram was developed and validated. The assay had a lower limit of quantification (LLOQ) of 10 pg/mL for plasma and 1 pg/sample for brain tissue. In pigs, doxapram pharmacokinetics were biphasic with a terminal elimination half-life (t1/2) of 1.38 ± 0.22 h and a maximal plasma concentration (cmax) of 1780 ± 275 ng/mL. Its active metabolite 2-ketodoxapram had a t1/2 of 2.42 ± 0.04 h and cmax of 32.3 ± 5.5 h after administration of doxapram. Protein binding was 95.5 ± 0.9% for doxapram and 98.4 ± 0.3% for 2-ketodoxapram with a brain-to-plasma ratio of 0.58 ± 0.24 for doxapram and 0.12 ± 0.02 for 2-ketodoxapram. In conclusion, the developed assay was successfully applied to the creation of pharmacokinetic data for doxapram, possibly improving the safety of its usage.

Summary: Latent proviruses persist in central (TCM), transitional (TTM), and effector (TEM) memory cells. We measured the levels of cellular factors involved in HIV gene expression in these subsets. The highest levels of acetylated H4, active nuclear factor κB (NF-κB), and active positive transcription elongation factor b (P-TEFb) were measured in TEM, TCM, and TTM cells, respectively. Vorinostat and romidepsin display opposite abilities to induce H4 acetylation across subsets. Protein kinase C (PKC) agonists are more efficient at inducing NF-κB phosphorylation in TCM cells but more potent at activating PTEF-b in the TEM subset. We selected the most efficient latency-reversing agents (LRAs) and measured their ability to reverse latency in each subset. While ingenol alone has modest activities in the three subsets, its combination with a histone deacetylase inhibitor (HDACi) dramatically increases latency reversal in TCM cells. Altogether, these results indicate that cellular HIV reservoirs are differentially responsive to common LRAs and suggest that combination of compounds will be required to achieve latency reversal in all subsets. : Pardons et al. measured the levels of cellular factors regulating HIV production in different memory CD4+ T cell subsets and their responsiveness to latency-reversing agents. Their results reveal that the HIV reservoir is composed of heterogenous pools of latently infected cells that differentially respond to latency-reversing agents. Keywords: HIV reservoir, latency, LRA, latency-reversing agent, memory subsets, HIV-Flow, histone acetylation, NF-κB, P-TEFb, CD4 T cells

The phenotypic characterization of the cells in which HIV persists during antiretroviral therapy (ART) remains technically challenging. We developed a simple flow cytometry-based assay to quantify and characterize infected cells producing HIV proteins during untreated and treated HIV infection. By combining two antibodies targeting the HIV capsid in a standard intracellular staining protocol, we demonstrate that p24-producing cells can be detected with high specificity and sensitivity in the blood from people living with HIV. In untreated individuals, the frequency of productively infected cells strongly correlated with plasma viral load. Infected cells preferentially displayed a transitional memory phenotype and were enriched in Th17, peripheral Tfh and regulatory T cells subsets. These cells also preferentially expressed activation markers (CD25, HLA-DR, Ki67), immune checkpoint molecules (PD-1, LAG-3, TIGIT, Tim-3) as well as the integrins α4β7 and α4β1. In virally suppressed individuals on ART, p24-producing cells were only detected upon stimulation (median frequency of 4.3 p24+ cells/106 cells). These measures correlated with other assays assessing the size of the persistent reservoir including total and integrated HIV DNA, Tat/rev Induced Limiting Dilution Assay (TILDA) and quantitative viral outgrowth assay (QVOA). In ART-suppressed individuals, p24-producing cells preferentially displayed a transitional and effector memory phenotype, and expressed immune checkpoint molecules (PD-1, TIGIT) as well as the integrin α4β1. Remarkably, α4β1 was expressed by more than 70% of infected cells both in untreated and ART-suppressed individuals. Altogether, these results highlight a broad diversity in the phenotypes of HIV-infected cells in treated and untreated infection and suggest that strategies targeting multiple and phenotypically distinct cellular reservoirs will be needed to exert a significant impact on the size of the reservoir.

The family of forkhead box (Fox) transcription factors regulates gonadogenesis and embryogenesis, but the role of foxr1 in reproduction is unknown. Evolutionary history of foxr1 in vertebrates was examined and the gene was found to exist in most vertebrates, including mammals, ray-finned fish, amphibians, and sauropsids. By quantitative PCR and RNA-seq, we found that foxr1 had an ovarian-specific expression in zebrafish, a common feature of maternal-effect genes. In addition, it was demonstrated using in situ hybridization that foxr1 was a maternally-inherited transcript that was highly expressed even in early-stage oocytes and accumulated in the developing eggs during oogenesis. We also analyzed the function of foxr1 in female reproduction using a zebrafish CRISPR/cas9 knockout model. It was observed that embryos from the foxr1-deficient females had a significantly lower survival rate whereby they either failed to undergo cell division or underwent abnormal division that culminated in growth arrest at around the mid-blastula transition and early death. These mutant-derived eggs contained dramatically increased levels of p21, a cell cycle inhibitor, and reduced rictor, a component of mTOR and regulator of cell survival, which were in line with the observed growth arrest phenotype. Our study shows for the first time that foxr1 is an essential maternal-effect gene and may be required for proper cell division and survival via the p21 and mTOR pathways. These novel findings will broaden our knowledge on the functions of specific maternal factors stored in the developing egg and the underlying mechanisms that contribute to reproductive success.

In a context of climate change, a mismatch has been shown to occur between some species' reproductive phenology and their environment. So far, few studies have either documented temporal trends in calving phenology or assessed which climatic variables influence the calving phenology in ungulate species, yet the phenology of ungulates' births affects offspring survival and population's recruitment rate. Using a long-term dataset (45 years) of birth dates of a semi-domesticated reindeer population in Kaamanen, North Finland, we show that calving season has advanced by ~ 7 days between 1970 and 2016. Advanced birth dates were associated with lower precipitation and a reduced snow cover in April and warmer temperatures in April-May. Improved females' physical condition in late gestation due to warmer temperatures in April-May and reduced snow conditions in April probably accounted for such advance in calving date. On the other hand, a lengthening of the calving season was reported following a warmer temperature in January, a higher number of days when mean temperature exceeds 0°C in October-November and a decreasing snow cover from October to November. By affecting the inter-individual heterogeneity in the plastic response of females' calving date to better climatic conditions in fall and winter, climatic variability contributed to weaken the calving synchrony in this herd. Whether variability in climatic conditions form environmental cues for the adaptation of calving phenology by females to climate change is however uncertain, but it is likely. As such this study enhances our understanding on how reproductive phenology of ungulate species would be affected by climate change.

HIV persists because a reservoir of latently infected CD4 T cells do not express viral proteins and are indistinguishable from uninfected cells. One approach to HIV cure suggests that reactivating HIV will activate cytotoxic pathways; yet when tested in vivo, reactivating cells do not die sufficiently to reduce cell-associated HIV DNA levels. We recently showed that following reactivation from latency, HIV infected cells generate the HIV specific cytotoxic protein Casp8p41 which is produced by HIV protease cleaving procaspase 8. However, cell death is prevented, possibly due to low procaspase 8 expression. Here, we tested whether increasing procaspase 8 levels in CD4 T cells will produce more Casp8p41 following HIV reactivation, causing more reactivated cells to die. Screening 1277 FDA approved drugs identified 168 that increased procaspase 8 expression by at least 1.7-fold. Of these 30 were tested for anti-HIV effects in an acute HIVIIIb infection model, and 9 drugs at physiologic relevant levels significantly reduced cell-associated HIV DNA. Primary CD4 T cells from ART suppressed HIV patients were treated with one of these 9 drugs and reactivated with αCD3/αCD28. Four drugs significantly increased Casp8p41 levels following HIV reactivation, and decreased total cell associated HIV DNA levels (flurbiprofen: p = 0.014; doxycycline: p = 0.044; indomethacin: p = 0.025; bezafibrate: P = 0.018) without effecting the viability of uninfected cells. Thus procaspase 8 levels can be increased pharmacologically and, in the context of HIV reactivation, increase Casp8p41 causing death of reactivating cells and decreased HIV DNA levels. Future studies will be required to define the clinical utility of this or similar approaches.

Egg quality is a complex biological trait and a major determinant of reproductive fitness in all animals. This study delivered the first proteomic portraits of egg quality in zebrafish, a leading biomedical model for early development. Egg batches of good and poor quality, evidenced by embryo survival for 24 h, were sampled immediately after spawning and used to create pooled or replicated sample sets whose protein extracts were subjected to different levels of fractionation before liquid chromatography and tandem mass spectrometry. Obtained spectra were searched against a zebrafish proteome database and detected proteins were annotated, categorized and quantified based on normalized spectral counts. Manually curated and automated enrichment analyses revealed poor quality eggs to be deficient of proteins involved in protein synthesis and energy and lipid metabolism, and of some vitellogenin products and lectins, and to have a surfeit of proteins involved in endo-lysosomal activities, autophagy, and apoptosis, and of some oncogene products, lectins and egg envelope proteins. Results of pathway and network analyses suggest that this aberrant proteomic profile results from failure of oocytes giving rise to poor quality eggs to properly transit through final maturation, and implicated Wnt signaling in the etiology of this defect. Quantitative comparisons of abundant proteins in good versus poor quality eggs revealed 17 candidate egg quality markers. Thus, the zebrafish egg proteome is clearly linked to embryo developmental potential, a phenomenon that begs further investigation to elucidate the root causes of poor egg quality, presently a serious and intractable problem in livestock and human reproductive medicine.