The rockefeller university

BackgroundArthropod-borne viruses (arboviruses) such as dengue virus (DENV) and Zika virus (ZIKV) pose a significant threat to global health. Novel approaches to control the spread of arboviruses focus on harnessing the antiviral immune system of their primary vector, the Aedes aegypti mosquito. In arthropods, genes of the Vago family are often presented as analogs of mammalian cytokines with potential antiviral functions, but the role of Vago genes upon virus infection in Ae. aegypti is largely unknown.ResultsWe conducted a phylogenetic analysis of the Vago gene family in Diptera, which led us to focus on a Vago-like gene that we named VLG-1. Using CRISPR/Cas9-mediated gene editing, we generated a VLG-1 mutant line of Ae. aegypti, which revealed a broad impact of VLG-1 on the mosquito transcriptome, affecting several biological processes potentially related to viral replication, including the oxidative stress response. Surprisingly, experimental viral challenge of the VLG-1 mutant line indicated a modest proviral role for this gene during DENV and ZIKV infections in vivo. In the absence of VLG-1, virus dissemination throughout the mosquito's body was slightly impaired, albeit not altering virus transmission rates.ConclusionsOur results challenge the conventional understanding of Vago-like genes as antiviral factors and underscore the need for further in vivo research to elucidate the molecular mechanisms underlying mosquito-arbovirus interactions.

Mitotic onset is a critical transition for eukaryotic cell proliferation. The commonly held view of mitotic control is that the master regulator, cyclin-dependent kinase (CDK), is first activated in the cytoplasm, at the centrosome, initiating mitosis1, 2-3. Bistability in CDK activation ensures that the transition is irreversible, but how this unfolds in a spatially compartmentalized cell is unknown4, 5, 6, 7-8. Here, using fission yeast, we show that CDK is first activated in the nucleus, and that the bistable responses differ markedly between the nucleus and the cytoplasm, with a stronger response in the nucleus driving mitotic signal propagation from there to the cytoplasm. Abolishing cyclin-CDK localization to the centrosome led to activation occurring only in the nucleus, spatially uncoupling the nucleus and cytoplasm mitotically, suggesting that centrosomal cyclin-CDK acts as a 'signal relayer'. We propose that the key mitotic regulatory system operates in the nucleus in proximity to DNA, which enables incomplete DNA replication and DNA damage to be effectively monitored to preserve genome integrity and to integrate ploidy within the CDK control network. This spatiotemporal regulatory framework establishes core principles for control of the onset of mitosis and highlights that the CDK control system operates within distinct regulatory domains in the nucleus and cytoplasm.

Loss-of-function (LOF) variants in IL6ST, encoding GP130, can cause hyper-IgE syndrome (HIES). Monoallelic LOF variants in IL6ST lead to HIES when located in the intracellular domain downstream of box 1/2 and upstream of the STAT3 phosphorylation sites and the recycling motif, due to their dominant negative (DN) activity. In this region, 2 previously unreported IL6ST variants, p.K702Sfs7* and p.Y759Wfs26*, were identified in 2 families with autosomal dominant (AD) HIES. Both variants were LOF and exhibited DN effects, leading to the accumulation of mutant GP130 on the cell surface. The p.K702Sfs7* mutation was the most upstream N-terminal mutation linked to HIES caused by heterozygous IL6STvariants. Comprehensive screening of IL6ST mutants revealed that most premature terminations downstream of amino acid F641, at the end of the transmembrane domain, resulted in LOF and DN effects via GP130 accumulation on the cell surface. The absence of the recycling motif (positions 782-787) in surface-expressed LOF GP130 led to its accumulation, contributing to the DN effect. The importance of intracellular truncating IL6STvariants can possibly be predicted based on the location of the premature stop codon. GP130 accumulation on the cell surface is a characteristic and potentially diagnostic finding in patients with HIES with heterozygous IL6STvariants.

Over 500 primary immunodeficiency diseases (PID) have been described, but immunological assessment after a severe infection is not routine. We aimed to evaluate the feasibility of a PID screening protocol and calculate PID prevalence in children admitted for severe infection in a pediatric intensive care unit (PICU). This monocentric retrospective study evaluated the feasibility of a PID monitoring protocol after severe infection in children aged 1 month to 16 years-old hospitalized in the Montpellier University Hospital from January 2018 to December 2020. Follow-up consultations at 3 and 12 months included the three main PID screening scores, comprehensive immunological and genetic screenings. Among 1125 children admitted to the PICU, 46 had severe infections and caused by bacterial (48%), viral (39%) or fungal (2%) pathogens. Before infection, none had completed any screening score recommended by dedicated societies (Jeffrey Modell Foundation, German Patients' Organization for Primary Immunodeficiencies, French Reference Center for Hereditary Immunodeficiencies). At 3 months, three patients had a PID diagnosis (6.5% prevalence, 95% CI 1.4-17.9). These were associated with a deletion of chromosomal region 22q11.21 (DiGeorge syndrome), ELANE mutation (Elastase deficiency or Severe Congenital Neutropenia 1), and C5 deficiency Forty children (87%) presented immunological anomalies without a formal PID diagnosis. These persisted in only 4/17 children tested at 12 months. The most frequent abnormalities were low NK lymphocytes (41.18%), and abnormal B lymphocyte population distribution (25%). The observed PID prevalence post-severe infection matches previous reports, even with a high rate of viral infections, often overlooked. Systematic PID investigation after severe infection, regardless of the pathogen, should be implemented to improve early detection and treatment.

To produce a murine monoclonal antibody (mAb) that binds to glycoprotein IIb/IIIa (alpha IIb beta 3) and inhibits clot retraction (CR), we immunized mice with human platelets and tested hybridoma supernatants for their ability to bind to alpha IIb beta 3 and inhibit CR. The immunoglobulin G1 (IgG1) mAb R6H8 completely inhibited CR at 20 mu g/mL. Paradoxically, at 5 mu g/mL, R6H8 initiated platelet aggregation and induced P-selectin expression, fibrinogen binding, and PAC-1 binding. At 20 mu g/mL, however, R6H8 completely inhibited aggregation induced by thrombin PAR-1 receptor activating peptide SFLLRN (T6; 25 mu g/mL) and T6-induced fibrinogen and PAC-1 binding to platelets. Platelet aggregation induced by R6H8 was inhibited by mAb IV.3, which blocks the Fc gamma IIa receptor (Fc gamma RIIa), and the Fab fragment of R6H8 did not induce platelet aggregation, suggesting that R6H8 binds to both alpha IIb beta 3 and Fc gamma RIIa. Cryogenic electron microscopy analysis of the R6H8 Fab-alpha IIb beta 3 complex revealed that R6H8 (1) binds to the alpha IIb beta 3 RGD binding pocket via an Arg-Tyr-Asp (RYD) sequence in its heavy chain complementarity-determining region 3; (2) interacts with beta 3 Asp126, producing a reorientation of Asp126 and loss of the adjacent to metal ion-dependent adhesion site Ca2+; and (3) initiates swing-out of the beta 3 hybrid domain. We conclude that R6H8 is an alpha IIb beta 3 ligand-mimetic mAb that activates platelets via Fc gamma RIIa at low concentrations and potently inhibits platelet aggregation and CR at high concentrations. R6H8 simulates the actions of a number of pathological antibodies, including platelet-activating antibodies developed after therapy with alpha IIb beta 3 inhibitors and platelet-activating antibodies in heparin-induced thrombocytopenia and vaccine-induced immune thrombotic thrombocytopenia. As such, it may be a valuable reagent for better understanding these disorders and identifying potential therapies.

Proper fuelling of the brain is critical to sustain cognitive function, but the role of fatty acid (FA) combustion in this process has been elusive. Here we show that acute block of a neuron-specific triglyceride lipase, DDHD2 (a genetic driver of complex hereditary spastic paraplegia), or of the mitochondrial lipid transporter CPT1 leads to rapid onset of torpor in adult male mice. These data indicate that in vivo neurons are probably constantly fluxing FAs derived from lipid droplets (LDs) through beta-oxidation to support neuronal bioenergetics. We show that in dissociated neurons, electrical silencing or blocking of DDHD2 leads to accumulation of neuronal LDs, including at nerve terminals, and that FAs derived from axonal LDs enter mitochondria in an activity-dependent fashion to drive local mitochondrial ATP production. These data demonstrate that nerve terminals can make use of LDs during electrical activity to provide metabolic support and probably have a critical role in supporting neuron function in vivo.

Isocitrate dehydrogenase 1/2 (IDH) mutations are early initiating events in acute myeloid leukemia (AML). The complex clonal architecture and cellular heterogeneity in IDH-mutant AML underlies the heterogeneous clinical presentation and outcomes. Integrating single-cell genotyping and transcriptomics, we demonstrate a stem-like and inflammatory phenotype of IDH-mutant AML and identify clone-specific programs associated with NPM1, NRAS, and SRSF2 co-mutations. Furthermore, these clones had distinct responses to treatment with combination IDH inhibitors and chemotherapy, including elimination, reconstitution of myeloid differentiation, or retention within progenitor populations. At relapse after IDH inhibitor monotherapy, we identify up-regulated stemness, inflammation, mitochondrial metabolism, and anti-apoptotic factors, as well as down-regulated major histocompatibility complex (MHC) class II antigen presentation. At the pre-leukemic stage, we observe upregulation of IDH2-associated pathways, including inflammation. We deliver a detailed phenotyping of IDH-mutant AML and a framework for dissecting contributions of recurrently mutated genes in AML at diagnosis and following therapy, with implications for precision medicine.

Fanconi anemia (FA) is a rare genetic disease characterized by loss-of-function variants in any of the 22 previously identified genes (FANCA-FANCW) that encode proteins participating in the repair of DNA interstrand crosslinks (ICLs). Patient phenotypes are variable but may include developmental abnormalities, early-onset pancytopenia, and a predisposition to hematologic and solid tumors. Here, we describe 2 unrelated families with multiple pregnancy losses and offspring presenting with severe developmental and hematologic abnormalities leading to death in utero or in early life. Homozygous loss-of-function variants in FAAP100 were identified in affected children of both families. The FAAP100 protein associates with FANCB and FANCL, the E3 ubiquitin ligase responsible for the monoubiquitination of FANCD2 and FANCI, which is necessary for FA pathway function. Patient-derived cells exhibited phenotypes consistent with FA. Expression of the WT FAAP100 cDNA, but not the patient-derived variants, rescued the observed cellular phenotypes. This establishes FAAP100 deficiency as a cause of FA, with FAAP100 gaining an alias as FANCX. The extensive developmental malformations of individuals with FAAP100 loss-of-function variants are among the most severe across previously described FA phenotypes, indicating that the FA pathway is essential for human development.

Nanodiscs, small discoidal membrane patches stabilized by membrane-scaffold proteins (MSPs), are popular tools to stabilize integral membrane proteins (IMPs) for structural studies by cryogenic electron microscopy (cryo-EM). While nanodiscs provide a near-native membrane environment for the incorporated IMPs, they do not reproduce all characteristics of a native membrane. Also, IMPs must first be purified in detergent before they can be reconstituted into MSP-based nanodiscs, a problem that has been overcome by newer approaches, such as copolymer-based native nanodiscs and cell-derived vesicles. In this review, we argue that despite these advances, MSP-based nanodiscs remain a unique tool for the structural interrogation of IMPs.

Despite waning of virus-neutralizing antibodies, protection against severe SARS-CoV-2 in the majority of immune individuals remains high, but the underlying immune mechanisms are incompletely understood. Here, rhesus macaques with pre-existing immunity from Novavax WA-1 and/or P.1 vaccines and WA-1 or P.1 infection are immunized with a bivalent WA-1/Omicron BA.5 Novavax vaccine ten months after the last exposure. The boost vaccination primarily increases the frequency of cross-reactive spike (S)-specific antibodies and B cells instead of inducing de novo BA.5-specific responses. Reinfection with heterologous Omicron XBB.1.5 six months after the boost vaccination results in low levels of virus replication in the respiratory tract compared with virus-na & iuml;ve results from other studies. Whereas systemic S-specific immunity remains largely unchanged in all animals, the animals with complete protection from infection exhibit a stronger influx of S-specific IgG, monocytes, B cells and T cells into the bronchioalveolar space combined with expansion of CD69+CD103+ lung tissue-resident, S-specific CD8 T cells compared to actively infected animals. Our results underscore the importance of localized respiratory immune responses in mediating protection from Omicron reinfection and provide guidance for future vaccine development.