The rockefeller university

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.

A critical component of the function of IgG antibodies is their capacity to engage specialized cellular receptors, Fc gamma receptors (Fc gamma Rs), expressed on effector leukocytes. Highlighting the importance of Fc gamma R-mediated signaling in the regulation of the fate, activation, and differentiation status of leukocytes, Fc gamma Rs are ubiquitously expressed by nearly all leukocyte populations. Here, we report that while at steady state, T cells are negative for all classes of Fc gamma Rs, CD8 T cells specifically induce the expression of the activating Fc gamma R, Fc gamma RIIIa, in response to viral infection in cohorts of COVID-19 and dengue patients, as well as in virus infection models using Fc gamma R humanized mouse strains. In in vivo mechanistic studies, we demonstrate that induction of Fc gamma RIIIa expression on effector CD8 T cells follows a well-defined trajectory that closely tracks the course and magnitude of the immune response, while immune resolution is characterized by receptor downregulation. Uniquely to these CD8 T cells, Fc gamma RIIIa crosslinking alone is paradoxically insufficient to elicit T cell activation and cytotoxicity. However, when coupled with T cell receptor (TCR) stimulation, it results in synergistic cellular activation and, compensates for the downregulation of canonical costimulatory molecules on terminal effector CD8 T cells. These results reveal a previously unappreciated role for Fc gamma RIIIa as a unique costimulatory molecule that synergizes with TCR signaling to lower the effective threshold required for CD8 T cell activation, highlighting the role of virally induced antibodies in modulating CD8 effector cell responses.

Clinical and animal studies suggest that multiple brain systems are involved in mediating reward-motivated and related emotional behavior including the consumption of commonly used drugs and palatable food, and there is evidence that the repeated ingestion of or exposure to these rewarding substances may in turn stimulate these brain systems to produce an overconsumption of these substances along with co-occurring emotional disturbances. To understand this positive feedback loop, this review focuses on a specific population of hypothalamic peptide neurons expressing melanin-concentrating hormone (MCH), which are positively related to dopamine reward and project to forebrain areas that mediate this behavior. It also examines neurons expressing the peptide hypocretin/orexin (HCRT) that are anatomically and functionally linked to MCH neurons and the molecular systems within these peptide neurons that stimulate their development and ultimately affect behavior. This report first describes evidence in animals that exposure in adults and during adolescence to rewarding substances, such as the drugs alcohol, nicotine and cocaine and palatable fat-rich food, stimulates the expression of MCH as well as HCRT and their intracellular molecular systems. It also increases reward-seeking and emotional behavior, leading to excess consumption and abuse of these substances and neurological conditions, completing this positive feedback loop. Next, this review focuses on the model involving embryonic exposure to these rewarding substances. In addition to revealing a similar positive feedback circuit, this model greatly advances our understanding of the diverse changes that occur in these neuropeptide/molecular systems in the embryo and how they relate, perhaps causally, to the disturbances in behavior early in life that predict a later increased risk of developing substance use disorders. Studies using this model demonstrate in animals that embryonic exposure to these rewarding substances, in addition to stimulating the expression of peptide neurons, increases the intracellular molecular systems in neuroprogenitor cells that promote their development. It also alters the morphology, migration, location and neurochemical profile of the peptide neurons and causes them to develop aberrant neuronal projections to forebrain structures. Moreover, it produces disturbances in behavior at a young age, which are sex-dependent and occur in females more than in males, that can be directly linked to the neuropeptide/molecular changes in the embryo and predict the development of behavioral disorders later in life. These results supporting the close relationship between the brain and behavior are consistent with clinical studies, showing females to be more vulnerable than males to developing substance use disorders with co-occurring emotional conditions and female offspring to respond more adversely than male offspring to prenatal exposure to rewarding substances. It is concluded that the continued consumption of or exposure to rewarding substances at any stage of life can, through such peptide brain systems, significantly increase an individual's vulnerability to developing neurological disorders such as substance use disorders, anxiety, depression, or cognitive impairments.

While human and mouse memory B cells (MBCs) can express the transcription factor T-bet, its role in regulating MBC function remains unclear. We characterized multiple transcriptionally distinct clusters of mature, somatically mutated nucleoprotein (NP)-specific MBCs in lymph nodes (LNs) and lungs of influenza-infected mice. Although none of the MBCs expressed the plasma cell (PC) lineage commitment factor Blimp1, one cluster was enriched for Tbx21+ cells. Similar to the previously described human T-bet + effector MBC (eMBC) population, Tbx21+ mouse MBCs upregulated gene networks associated with effector metabolism, protein synthesis, and the unfolded protein response. Constitutive and inducible ablation of T-bet in murine B cells showed that T-bet expression by MBCs was required for persistence of LN and lung eMBCs with rapid in vitro and in vivo PC differentiation potential. Thus, T-bet marks NP + eMBCs that are poised to differentiate, and it regulates maintenance of lung-resident MBCs and local PC responses following virus re-exposure.

Replicative helicases are assembled on chromosomes by helicase loaders before the initiation of DNA replication. Here, we investigate the mechanisms employed by the bacterial Vibrio cholerae (Vc) DnaB replicative helicase and the DciA helicase loader. Structural analysis of the ATP gamma S form of the VcDnaB-ssDNA complex reveals a configuration distinct from that observed with GDP center dot AlF4. With ATP gamma S, the amino-terminal domain (NTD) tier, previously found as an open spiral in the GDP center dot AlF4 complex, adopts a closed planar arrangement. Furthermore, the DnaB subunit at the top of the carboxy-terminal domain (CTD) spiral is displaced by approximately 25 & Aring; between the two forms. We suggest that remodeling the NTD layer between closed planar and open spiral configurations, along with the migration of two distinct CTDs to the top of the DnaB spiral, repeated three times, mediates hand-over-hand translocation. Biochemical analysis indicates that VcDciA utilizes its Lasso domain to interact with DnaB near its Docking-Helix Linker-Helix interface. Up to three copies of VcDciA bind to VcDnaB, suppressing its ATPase activity during loading onto physiological DNA substrates. Our data suggest that DciA loads DnaB onto DNA using the ring-opening mechanism.