Approches génomiques des interactions entre l’implantation du microbiote digestif chez le lapereau et la maturation du système immunitaire

La santé digestive du lapin est difficile à maîtriser en élevage, notamment en période de sevrage où des troubles digestifs sont fréquents et peuvent entraîner des pertes importantes. Bien que l’utilisation d’antibiotiques diminue, afin d’éviter l’apparition de problèmes de résistance, des solutions alternatives doivent être trouvées. Nos principaux objectifs étaient : i) d’étudier l’influence de l’incorporation de fibres rapidement fermtentescibles (FRF) dans un aliment distribué précocement dès 15 jours d’âge, en comparaison d’un aliment avec antibiotiques (tiamuline et apramycine) utilisés pour lutter contre l’entéropathie épizootique du lapin ; ii) de déployer des méthodologies en génomique ciblant l’espèce lapin (amélioration et réannotation d’un microarray) et son microbiote digestif (pipeline d’analyse du gène de l’ARNr 16S), pour du phénotypage moléculaire. L’originalité de notre travail réside ainsi dans l’acquisition de données par trois approches complémentaires : phénotypique, transcriptomique, et métagénomique. Au niveau phénotypique, la stimulation de l’activité fermentaire par les FRF est vérifiée avec une hausse de la concentration caecale en AGV (+20%) et une plus forte acidité du biotope caecal. De plus, les FRF tendent à réduire la mortalité en post-sevrage et améliorent l’efficacité alimentaire. Au niveau transcriptomique, nous avons tout d’abord apporté de nouvelles connaissances sur l’immunité du lapin adulte avec la stimulation in vitro de cellules mononuclées du sang périphérique par du LPS et de la PMA-ionomycine. Cette étude confirme l’importance du lapin comme animal modèle pour la recherche biomédicale. Chez le jeune lapin, la nouvelle version du microarray nous a permis de montrer que les fonctions biologiques sont plus rapidement mises en place avec les FRF qu’avec un aliment standard, et que les antibiotiques nivellent l’expression génique. De plus, les FRF contribueraient à limiter l’inflammation intestinale. Au niveau métagénomique et en ciblant l’ARNr 16S, nous montrons qu’une augmentation de l’activité microbienne caecale n’est pas associée à une modification majeure de la composition du microbiote, à l’exception de la stabilisation de l’abondance des Campylobacteraceae, qui limiterait l’inflammation digestive. Nos travaux, encore préliminaires, n’ont pas permis d’identifier des corrélations significatives entre l’expression génique dans le sang et/ou l’iléon, et les profils taxonomiques (OTUs) majoritaires du microbiote digestif. L’ensemble de nos résultats suggère d’une part que les FRF peuvent être proposées comme une nouvelle stratégie nutritionnelle péri-sevrage, avec une amélioration de l’efficacité alimentaire et une protection accrue de la santé digestive, et d’autre part que des approches génomiques sont prometteuses pour qualifier finement les phénotypes d’intérêt chez le lapin. ABSTRACT : Digestive health of rabbits is difficult to manage in breeding systems, especially around weaning where digestive problems are very common and can result in significant losses. The use of antibiotics decreases in order to avoid the emergence of problems with antibiotic resistance, but alternatives must be found. The objectives of this work were: i) to study effects of the incorporation of rapidly fermentable fiber (FRF) in diets fed to rabbits from 15 days of age, and compared to a feed with antibiotics (tiamulin and apramycin), used to fight against epizootic rabbit enteropathy; ii) to deploy genomics methodologies targeting the rabbit species (improvement and re-annotation of a microarray) and the digestive microbiota (pipeline analysis of 16S rRNA gene), for molecular phenotyping. An innovative approach was used in this work through the combination of three complementary approaches: phenotypic, transcriptomic, and metagenomic. At the phenotypic level, the stimulation of the fermentation activity by FRF is verified with higher cecal VFA concentrations (+ 20%) and higher cecal acidity. Moreover, the FRF tend to reduce mortality post-weaning and improve feed efficiency. At the transcriptomic level, we obtained new data on the immunity of the adult rabbit with in vitro stimulation of peripheral blood mononuclear cells with LPS and PMA-ionomycin. This study confirms the importance of the rabbit as an animal model for biomedical research. In young rabbits, the new version of the microarray allowed us to show that biological functions are more quickly implemented with FRF than a standard diet, and that antibiotics level out the gene expression. In addition, FRF help to limit intestinal inflammation. At the metagenomic level, the targeting of the 16S rRNA, we showed that an increase in cecal microbiota activity is not associated with major changes of microbiota composition, with the exception of the stabilization of the relative abundance of Campylobacteraceae, which limits gastrointestinal inflammation. Our preliminary results did not identify significant correlations between gene expression in blood and/or ileum, and principal taxonomic profiles of the digestive microbiota. Our results suggest that FRF can be proposed as a new nutrition strategy peri-weaning, with improved feed efficiency and increased protection of digestive health, and secondly that genomic approaches are promising to describe more precisely phenotypes of interest in rabbits

Haploinsufficiency of Dmxl2, Encoding a Synaptic Protein, Causes Infertility Associated with a Loss of GnRH Neurons in Mouse

International audienceCharacterization of the genetic defects causing gonadotropic deficiency has made a major contribution to elucidation of the fundamental role of Kisspeptins and Neurokinin B in puberty onset and reproduction. The absence of puberty may also reveal neurodevelopmental disorders caused by molecular defects in various cellular pathways. Investigations of these neurodevelopmental disorders may provide information about the neuronal processes controlling puberty onset and reproductive capacity. We describe here a new syndrome observed in three brothers, which involves gonadotropic axis deficiency, central hypothyroidism, peripheral demyelinating sensorimotor polyneuropathy, mental retardation, and profound hypoglycemia, progressing to nonautoimmune insulin-dependent diabetes mellitus. High-throughput sequencing revealed a homozygous in-frame deletion of 15 nucleotides in DMXL2 in all three affected patients. This homozygous deletion was associated with lower DMXL2 mRNA levels in the blood lymphocytes of the patients. DMXL2 encodes the synaptic protein rabconnectin-3a, which has been identified as a putative scaffold protein for Rab3-GAP and Rab3-GEP, two regulators of the GTPase Rab3a. We found that rabconnectin-3a was expressed in exocytosis vesicles in gonadotropin-releasing hormone (GnRH) axonal extremities in the median eminence of the hypothalamus. It was also specifically expressed in cells expressing luteinizing hormone (LH) and follicle-stimulating hormone (FSH) within the pituitary. The conditional heterozygous deletion of Dmxl2 from mouse neurons delayed puberty and resulted in very low fertility. This reproductive phenotype was associated with a lower number of GnRH neurons in the hypothalamus of adult mice. Finally, Dmxl2 knockdown in an insulin-secreting cell line showed that rabconnectin-3a controlled the constitutive and glucose-induced secretion of insulin. In conclusion, this study shows that low levels of DMXL2 expression cause a complex neurological phenotype, with abnormal glucose metabolism and gonadotropic axis deficiency due to a loss of GnRH neurons. Our findings identify rabconectin-3a as a key controller of neuronal and endocrine homeostatic processes

A BBP–Mud2p heterodimer mediates branchpoint recognition and influences splicing substrate abundance in budding yeast

The 3′ end of mammalian introns is marked by the branchpoint binding protein, SF1, and the U2AF65-U2AF35 heterodimer bound at an adjacent sequence. Baker's yeast has equivalent proteins, branchpoint binding protein (BBP) (SF1) and Mud2p (U2AF65), but lacks an obvious U2AF35 homolog, leaving open the question of whether another protein substitutes during spliceosome assembly. Gel filtration, affinity selection and mass spectrometry were used to show that rather than a U2AF65/U2AF35-like heterodimer, Mud2p forms a complex with BBP without a third (U2AF35-like) factor. Using mutants of MUD2 and BBP, we show that the BBP–Mud2p complex bridges partner-specific Prp39p, Mer1p, Clf1p and Smy2p two-hybrid interactions. In addition to inhibiting Mud2p association, the bbpΔ56 mutation impairs splicing, enhances pre-mRNA release from the nucleus, and similar to a mud2::KAN knockout, suppresses a lethal sub2::KAN mutation. Unexpectedly, rather than exacerbating bbpΔ56, the mud2::KAN mutation partially suppresses a pre-mRNA accumulation defect observed with bbpΔ56. We propose that a BBP–Mud2p heterodimer binds as a unit to the branchpoint in vivo and serves as a target for the Sub2p-DExD/H-box ATPase and for other splicing factors during spliceosome assembly. In addition, our results suggest the possibility that the Mud2p may enhance the turnover of pre-mRNA with impaired BBP-branchpoint association

Sterol biosensor reveals LAM-family Ltc1-dependent sterol flow to endosomes upon Arp2/3 inhibition.

Sterols are crucial components of biological membranes, which are synthetized in the ER and accumulate in the plasma membrane (PM). Here, by applying a genetically encoded sterol biosensor (D4H), we visualize a sterol flow between PM and endosomes in the fission yeast Schizosaccharomyces pombe. Using time-lapse and correlative light-electron microscopy, we found that inhibition of Arp2/3-dependent F-actin assembly promotes the reversible relocalization of D4H from the PM to internal sterol-rich compartments (STRIC) labeled by synaptobrevin Syb1. Retrograde sterol internalization to STRIC is independent of endocytosis or an intact Golgi, but depends on Ltc1, a LAM/StARkin-family protein localized to ER-PM contact sites. The PM in ltc1Δ cells over-accumulates sterols and upon Arp2/3 inhibition forms extended ER-interacting invaginations, indicating that sterol transfer contributes to PM size homeostasis. Anterograde sterol movement from STRIC is independent of canonical vesicular trafficking but requires Arp2/3, suggesting a novel role for this complex. Thus, transfer routes orthogonal to vesicular trafficking govern the flow of sterols in the cell

Effect of Hydrocortisone on Mortality and Organ Support in Patients With Severe COVID-19: The REMAP-CAP COVID-19 Corticosteroid Domain Randomized Clinical Trial.

Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited. Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19. Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020. Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (n = 143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (n = 152), or no hydrocortisone (n = 108). Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%). Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (n = 137), shock-dependent (n = 146), and no (n = 101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively. Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions. Trial Registration: ClinicalTrials.gov Identifier: NCT02735707

Extracorporeal Membrane Oxygenation for Severe Acute Respiratory Distress Syndrome associated with COVID-19: An Emulated Target Trial Analysis.

RATIONALE: Whether COVID patients may benefit from extracorporeal membrane oxygenation (ECMO) compared with conventional invasive mechanical ventilation (IMV) remains unknown. OBJECTIVES: To estimate the effect of ECMO on 90-Day mortality vs IMV only Methods: Among 4,244 critically ill adult patients with COVID-19 included in a multicenter cohort study, we emulated a target trial comparing the treatment strategies of initiating ECMO vs. no ECMO within 7 days of IMV in patients with severe acute respiratory distress syndrome (PaO2/FiO2 <80 or PaCO2 ≥60 mmHg). We controlled for confounding using a multivariable Cox model based on predefined variables. MAIN RESULTS: 1,235 patients met the full eligibility criteria for the emulated trial, among whom 164 patients initiated ECMO. The ECMO strategy had a higher survival probability at Day-7 from the onset of eligibility criteria (87% vs 83%, risk difference: 4%, 95% CI 0;9%) which decreased during follow-up (survival at Day-90: 63% vs 65%, risk difference: -2%, 95% CI -10;5%). However, ECMO was associated with higher survival when performed in high-volume ECMO centers or in regions where a specific ECMO network organization was set up to handle high demand, and when initiated within the first 4 days of MV and in profoundly hypoxemic patients. CONCLUSIONS: In an emulated trial based on a nationwide COVID-19 cohort, we found differential survival over time of an ECMO compared with a no-ECMO strategy. However, ECMO was consistently associated with better outcomes when performed in high-volume centers and in regions with ECMO capacities specifically organized to handle high demand. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Genomic approaches of the interactions between microbiota and immunity in the young rabbit

La santé digestive du lapin est difficile à maîtriser en élevage, notamment en période de sevrage où des troubles digestifs sont fréquents et peuvent entraîner des pertes importantes. Bien que l’utilisation d’antibiotiques diminue, afin d’éviter l’apparition de problèmes de résistance, des solutions alternatives doivent être trouvées. Nos principaux objectifs étaient : i) d’étudier l’influence de l’incorporation de fibres rapidement fermtentescibles (FRF) dans un aliment distribué précocement dès 15 jours d’âge, en comparaison d’un aliment avec antibiotiques (tiamuline et apramycine) utilisés pour lutter contre l’entéropathie épizootique du lapin ; ii) de déployer des méthodologies en génomique ciblant l’espèce lapin (amélioration et réannotation d’un microarray) et son microbiote digestif (pipeline d’analyse du gène de l’ARNr 16S), pour du phénotypage moléculaire. L’originalité de notre travail réside ainsi dans l’acquisition de données par trois approches complémentaires : phénotypique, transcriptomique, et métagénomique. Au niveau phénotypique, la stimulation de l’activité fermentaire par les FRF est vérifiée avec une hausse de la concentration caecale en AGV (+20%) et une plus forte acidité du biotope caecal. De plus, les FRF tendent à réduire la mortalité en post-sevrage et améliorent l’efficacité alimentaire. Au niveau transcriptomique, nous avons tout d’abord apporté de nouvelles connaissances sur l’immunité du lapin adulte avec la stimulation in vitro de cellules mononuclées du sang périphérique par du LPS et de la PMA-ionomycine. Cette étude confirme l’importance du lapin comme animal modèle pour la recherche biomédicale. Chez le jeune lapin, la nouvelle version du microarray nous a permis de montrer que les fonctions biologiques sont plus rapidement mises en place avec les FRF qu’avec un aliment standard, et que les antibiotiques nivellent l’expression génique. De plus, les FRF contribueraient à limiter l’inflammation intestinale. Au niveau métagénomique et en ciblant l’ARNr 16S, nous montrons qu’une augmentation de l’activité microbienne caecale n’est pas associée à une modification majeure de la composition du microbiote, à l’exception de la stabilisation de l’abondance des Campylobacteraceae, qui limiterait l’inflammation digestive. Nos travaux, encore préliminaires, n’ont pas permis d’identifier des corrélations significatives entre l’expression génique dans le sang et/ou l’iléon, et les profils taxonomiques (OTUs) majoritaires du microbiote digestif. L’ensemble de nos résultats suggère d’une part que les FRF peuvent être proposées comme une nouvelle stratégie nutritionnelle péri-sevrage, avec une amélioration de l’efficacité alimentaire et une protection accrue de la santé digestive, et d’autre part que des approches génomiques sont prometteuses pour qualifier finement les phénotypes d’intérêt chez le lapin.Digestive health of rabbits is difficult to manage in breeding systems, especially around weaning where digestive problems are very common and can result in significant losses. The use of antibiotics decreases in order to avoid the emergence of problems with antibiotic resistance, but alternatives must be found. The objectives of this work were: i) to study effects of the incorporation of rapidly fermentable fiber (FRF) in diets fed to rabbits from 15 days of age, and compared to a feed with antibiotics (tiamulin and apramycin), used to fight against epizootic rabbit enteropathy; ii) to deploy genomics methodologies targeting the rabbit species (improvement and re-annotation of a microarray) and the digestive microbiota (pipeline analysis of 16S rRNA gene), for molecular phenotyping. An innovative approach was used in this work through the combination of three complementary approaches: phenotypic, transcriptomic, and metagenomic. At the phenotypic level, the stimulation of the fermentation activity by FRF is verified with higher cecal VFA concentrations (+ 20%) and higher cecal acidity. Moreover, the FRF tend to reduce mortality post-weaning and improve feed efficiency. At the transcriptomic level, we obtained new data on the immunity of the adult rabbit with in vitro stimulation of peripheral blood mononuclear cells with LPS and PMA-ionomycin. This study confirms the importance of the rabbit as an animal model for biomedical research. In young rabbits, the new version of the microarray allowed us to show that biological functions are more quickly implemented with FRF than a standard diet, and that antibiotics level out the gene expression. In addition, FRF help to limit intestinal inflammation. At the metagenomic level, the targeting of the 16S rRNA, we showed that an increase in cecal microbiota activity is not associated with major changes of microbiota composition, with the exception of the stabilization of the relative abundance of Campylobacteraceae, which limits gastrointestinal inflammation. Our preliminary results did not identify significant correlations between gene expression in blood and/or ileum, and principal taxonomic profiles of the digestive microbiota. Our results suggest that FRF can be proposed as a new nutrition strategy peri-weaning, with improved feed efficiency and increased protection of digestive health, and secondly that genomic approaches are promising to describe more precisely phenotypes of interest in rabbits