Transmission of New Bovine Prion to Mice

We previously reported that cattle were affected by a prion disorder that differed from bovine spongiform encephalopathy (BSE) by showing distinct molecular features of disease-associated protease-resistant prion protein (PrPres). We show that intracerebral injection of such isolates into C57BL/6 mice produces a disease with preservation of PrPres molecular features distinct from BSE

An improved method for large scale generation and high-throughput functional characterization of human iPSC-derived microglia

Neuroscience drug discovery has faced significant challenges due to restricted access to relevant human cell models and limited translatability of existing preclinical findings to human pathophysiology. Induced pluripotent stem cells (iPSCs) have emerged as a promising solution, offering the potential to generate patient-specific cell types, including in the recent years, iPSC-derived microglia (iMGL). Current methods rely on complex and time-consuming differentiation procedures, leading to considerable batch-to-batch variability consequently hindering the establishment of standardized and reproducible high-throughput functional screening approaches. Addressing these challenges is critical in ensuring the generation of homogenous iMGL populations with consistent functional properties. In this study we describe an improved high-yield protocol for generating iMGL, which allows for increased reproducibility and flexibility in the execution of high-throughput functional screens. We introduce a two-step process in embryoid bodie (EB) maintenance and a stop point allowing for cryopreservation at the hematopoietic progenitor cell (iHPC) stages. Furthermore, we demonstrate inter-operator robustness of this modified protocol in a range of high-throughput functional assays including phagocytosis, lysosomal acidification, chemotaxis, and cytokine release. Our study underscores the importance of quality control checks at various stages of iPSC-differentiation and functional assay set up, highlighting novel workarounds to the existing challenges such as limited yield, flexibility, and reproducibility, all critical in drug discovery

Memory CD8+ T Cells Balance Pro- and Anti-inflammatory Activity by Reprogramming Cellular Acetate Handling at Sites of Infection.

Serum acetate increases upon systemic infection. Acutely, assimilation of acetate expands the capacity of memory CD8+ T cells to produce IFN-γ. Whether acetate modulates memory CD8+ T cell metabolism and function during pathogen re-encounter remains unexplored. Here we show that at sites of infection, high acetate concentrations are being reached, yet memory CD8+ T cells shut down the acetate assimilating enzymes ACSS1 and ACSS2. Acetate, being thus largely excluded from incorporation into cellular metabolic pathways, now had different effects, namely (1) directly activating glutaminase, thereby augmenting glutaminolysis, cellular respiration, and survival, and (2) suppressing TCR-triggered calcium flux, and consequently cell activation and effector cell function. In vivo, high acetate abundance at sites of infection improved pathogen clearance while reducing immunopathology. This indicates that, during different stages of the immune response, the same metabolite-acetate-induces distinct immunometabolic programs within the same cell type

Invasive non-typhoidal Salmonella from stool samples of healthy human carriers are genetically similar to blood culture isolates: a report from the Democratic Republic of the Congo

Invasive non-typhoidal Salmonella (iNTS) (serotypes Typhimurium and Enteritidis) are major causes of bloodstream infections in sub-Saharan Africa, but their reservoir is unknown. Aiming to demonstrate human carriers as a reservoir, we assessed an iNTS disease endemic rural community (Kikonka health area, Democratic Republic of the Congo) for intestinal carriage of iNTS. After a census, healthy subjects from randomly selected households provided three successive stool samples for Salmonella culture. We next compared the stool isolates for genetic relatedness with time and health area-matched blood culture isolates obtained from hospitalized patients by multiple locus variable-number tandem repeat analysis (MLVA) and performed whole genome sequencing (WGS) on a subset of stool and blood isolates. Among 2,354 eligible subjects, 2,234 (94.9%) consented and provided at least one stool sample, and 2,219 (94.3%) provided three stool samples. The cumulative proportion of Salmonella carriers after 3 days was 4.4% (n = 98). S. Typhimurium and Enteritidis were found in 26 and 3 carriers, respectively, representing 1.3% (29 out of 2,234) of participants living in 6.0% (26 out of 482) of households. MLVA types of all 26 S. Typhimurium stool isolates matched with the corresponding MLVA types of blood isolates. The MLVA type of one out of three Enteritidis stool isolates matched the single MLVA type of the five Enteritidis blood isolates. WGS analysis of S. Typhimurium (n = 20) and S. Enteritidis (n = 4) isolates revealed Typhimurium multilocus sequence type (ST)313 Lineage 2 and Enteritidis ST11 Central/Eastern African and Outlier clades and confirmed the MLVA clustering. More than three-quarters of Typhimurium isolates showed combined multidrug resistance, ceftriaxone resistance, and fluoroquinolone non-susceptibility. In conclusion, the present study demonstrated iNTS carriage among healthy community members, with stool isolates that were genetically similar to blood culture isolates obtained in patients from the same community. These findings contribute to the evidence of a human reservoir of iNTS

Rare and low-frequency coding variants alter human adult height

Height is a highly heritable, classic polygenic trait with ~700 common associated variants identified so far through genome - wide association studies . Here , we report 83 height - associated coding variants with lower minor allele frequenc ies ( range of 0.1 - 4.8% ) and effects of up to 2 16 cm /allele ( e.g. in IHH , STC2 , AR and CRISPLD2 ) , >10 times the average effect of common variants . In functional follow - up studies, rare height - increasing alleles of STC2 (+1 - 2 cm/allele) compromise d proteolytic inhibition of PAPP - A and increased cleavage of IGFBP - 4 in vitro , resulting in higher bioavailability of insulin - like growth factors . The se 83 height - associated variants overlap genes mutated in monogenic growth disorders and highlight new biological candidates ( e.g. ADAMTS3, IL11RA, NOX4 ) and pathways ( e.g . proteoglycan/ glycosaminoglycan synthesis ) involved in growth . Our results demonstrate that sufficiently large sample sizes can uncover rare and low - frequency variants of moderate to large effect associated with polygenic human phenotypes , and that these variants implicate relevant genes and pathways

The trans-ancestral genomic architecture of glycemic traits

Glycemic traits are used to diagnose and monitor type 2 diabetes and cardiometabolic health. To date, most genetic studies of glycemic traits have focused on individuals of European ancestry. Here we aggregated genome-wide association studies comprising up to 281,416 individuals without diabetes (30% non-European ancestry) for whom fasting glucose, 2-h glucose after an oral glucose challenge, glycated hemoglobin and fasting insulin data were available. Trans-ancestry and single-ancestry meta-analyses identified 242 loci (99 novel; P < 5 x 10(-8)), 80% of which had no significant evidence of between-ancestry heterogeneity. Analyses restricted to individuals of European ancestry with equivalent sample size would have led to 24 fewer new loci. Compared with single-ancestry analyses, equivalent-sized trans-ancestry fine-mapping reduced the number of estimated variants in 99% credible sets by a median of 37.5%. Genomic-feature, gene-expression and gene-set analyses revealed distinct biological signatures for each trait, highlighting different underlying biological pathways. Our results increase our understanding of diabetes pathophysiology by using trans-ancestry studies for improved power and resolution. A trans-ancestry meta-analysis of GWAS of glycemic traits in up to 281,416 individuals identifies 99 novel loci, of which one quarter was found due to the multi-ancestry approach, which also improves fine-mapping of credible variant sets.Peer reviewe

Bi-allelic Loss-of-Function CACNA1B Mutations in Progressive Epilepsy-Dyskinesia.

The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment.MAK is funded by an NIHR Research Professorship and receives funding from the Wellcome Trust, Great Ormond Street Children's Hospital Charity, and Rosetrees Trust. E.M. received funding from the Rosetrees Trust (CD-A53) and Great Ormond Street Hospital Children's Charity. K.G. received funding from Temple Street Foundation. A.M. is funded by Great Ormond Street Hospital, the National Institute for Health Research (NIHR), and Biomedical Research Centre. F.L.R. and D.G. are funded by Cambridge Biomedical Research Centre. K.C. and A.S.J. are funded by NIHR Bioresource for Rare Diseases. The DDD Study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003), a parallel funding partnership between the Wellcome Trust and the Department of Health, and the Wellcome Trust Sanger Institute (grant number WT098051). We acknowledge support from the UK Department of Health via the NIHR comprehensive Biomedical Research Centre award to Guy's and St. Thomas' National Health Service (NHS) Foundation Trust in partnership with King's College London. This research was also supported by the NIHR Great Ormond Street Hospital Biomedical Research Centre. J.H.C. is in receipt of an NIHR Senior Investigator Award. The research team acknowledges the support of the NIHR through the Comprehensive Clinical Research Network. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, Department of Health, or Wellcome Trust. E.R.M. acknowledges support from NIHR Cambridge Biomedical Research Centre, an NIHR Senior Investigator Award, and the University of Cambridge has received salary support in respect of E.R.M. from the NHS in the East of England through the Clinical Academic Reserve. I.E.S. is supported by the National Health and Medical Research Council of Australia (Program Grant and Practitioner Fellowship)

Le rôle d'une sérine-thréonine phosphatase de type 2C parasitaire (TgPP2C) dans l'interaction Toxoplasma gondii - cellule hôte : Identification des modules interactifs au sein du tricomplexe actine-toxofiline-TgPP2C et étude de ses propriétés fonctionnelles dans le parasite et la cellule infectée

Toxoplasma gondii, le protozoaire intracellulaire obligatoire responsable de la toxoplasmose, fait partie du phylum des Apicomplexa. Comme les autres membres de ce phylum, il a développé des propriétés mobiles particulières qui lui permettent de se déplacer et d'envahir très rapidement la cellule dans laquelle il se multiplie. Ces processus de locomotion et d'invasion sont dépendants du cytosquelette d'actine du parasite. Une protéine, la toxofiline, identifiée uniquement chez T. gondii, a été caractérisée au laboratoire comme étant capable de réguler la dynamique du cytosquelette d'actine. Plus précisément, la toxofiline séquestre l'actine monomérique, empêchant ainsi sa polymérisation. L'affinité de la toxofiline pour l'actine est modulée par phosphorylation/déphosphorylation par une caséine kinase 2 et une sérine-thréonine phosphatase de type 2C (TgPP2C). Mon projet de thèse a porté sur l'étude du rôle de la TgPP2C dans l'interaction entre T. gondii et sa cellule hôte. Le premier objectif de mon travail a été de disséquer les interactions moléculaires entre les trois membres du complexe actine G - toxofiline - TgPP2C. J'ai ainsi identifié, par différentes approches biochimiques, les régions et les acides aminés de la toxofiline, critiques pour sa liaison avec ses deux partenaires. Tout d'abord, la toxofiline possède un domaine suffisant pour lier et séquestrer l'actine G dans des tests de polymérisation. Ce domaine, qui a été appelé CC1A, correspond à une région de 9 kDa comprenant un domaine en super hélice. Trois séries de peptides chevauchants, présents dans CC1A, capables de lier l'actine G, ont été plus précisément détectés. Nous avons également identifié d'autres régions de la toxofiline ayant des propriétés de liaison différentes à l'actine, en particulier dans le domaine N-terminal (NoCC). Le premier domaine en super hélice, et en position C-terminale de CC1A, lie la TgPP2C. Une autre séquence de plusieurs acides aminés, dans la région N-terminale et contenant la sérine 53, substrat de la TgPP2C, interagit avec la phosphatase. Le deuxième objectif de mon projet de thèse a porté sur l'analyse des propriétés fonctionnelles de la TgPP2C dans le tachyzoïte. Par une stratégie de surexpression dans les parasites, nous avons tout d'abord montré que la TgPP2C jouait un rôle dans la multiplication du parasite. En effet, des tachyzoïtes qui surexpriment la forme active de la TgPP2C ne se divisent pas normalement dans la vacuole parasitophore et finissent par dégénérer. On observe des parasites anormaux avec un noyau géant, qui suggère un défaut dans la division nucléaire ou l'individualisation des cellules filles. Nous avons également mis en évidence que la TgPP2C est sécrétée par le parasite dans le cytoplasme et le noyau de sa cellule hôte. Cette sécrétion est dépendante du domaine N-terminal unique de 18 acides aminés de cette phosphatase. Enfin, le troisième objectif de mon travail a consisté à étudier le rôle de la TgPP2C dans la cellule infectée. Par une approche d'expression ectopique de la phosphatase dans des cellules de mammifère, nous avons mis en évidence que la TgPP2C agissait au niveau du cycle de ces cellules. En effet, les cellules qui expriment la phosphatase parasitaire sont bloquées en phase G2/M. Certaines cellules en division n'achèvent pas la cytocinèse et restent reliées par un long pont cytoplasmique contenant de l'ADN et la TgPP2C. A un temps plus tardif, elles présentent les signes caractéristiques d'une apoptose. D'autre part, nous avons réalisé un crible double hybride en levure afin d'identifier les partenaires hôtes de la TgPP2C. Plusieurs protéines candidates on ainsi été obtenues, dont la protéine Spire qui possède des propriétés de nucléation de l'actine in vitro et dont nous avons confirmé l'interaction avec la TgPP2C. La poursuite de cette étude permettra de caractériser l'effet de la TgPP2C sur Spire, et le rôle de cette dernière dans le développement des tachyzoïtes