The rockefeller university

Biallelic pathogenic variants in the essential DNA repair gene BRCA2 cause Fanconi anemia complementation group D1. Patients in this group are highly prone to develop embryonal tumors, most commonly medulloblastoma arising from the cerebellar granule cell progenitors (GCPs). GCPs undergo high proliferation in the postnatal cerebellum under Sonic Hedgehog (SHH) activation, but the type of DNA lesions that require the function of the BRCA2 to prevent tumorigenesis remains unknown. To identify such lesions, we assessed both GCP neurodevelopment and tumor formation using a mouse model with deletion of exons three and four of Brca2 in the central nervous system, coupled with global Trp53 loss. Brca2 Delta ex3-4;Trp53-/- animals developed SHH subgroup medulloblastomas with complete penetrance. Whole-genome sequencing of the tumors identified structural variants with breakpoints enriched in areas overlapping putative G-quadruplexes (G4s). Brca2-deficient GCPs exhibited decreased replication speed in the presence of the G4-stabilizer pyridostatin. Pif1 helicase, which resolves G4s during replication, was highly upregulated in tumors, and Pif1 knockout in primary medulloblastoma tumor cells resulted in increased genome instability upon pyridostatin treatment. These data suggest that G4s may represent sites prone to replication stalling in highly proliferative GCPs and without BRCA2, G4s become a source of genome instability. Tumor cells upregulate G4-resolving helicases to facilitate rapid proliferation through G4s highlighting PIF1 helicase as a potential therapeutic target for treatment of BRCA2-deficient medulloblastomas.

A search for flavor-changing neutral current interactions of the top quark (t) and the Higgs boson (H) is presented. The search is based on proton-proton collision data collected in 2016-2018 at a center-of-mass energy of 13 TeV with the CMS detector at the LHC, and corresponding to an integrated luminosity of 138 fb(-1). Events containing a pair of leptons with the same-sign electric charge and at least one jet are considered. The results are used to constrain the branching fraction (B) of the top quark decaying to a Higgs boson and an up (u) or charm (c) quark. No significant excess above the estimated background was found. The observed (expected) upper limits at a 95% confidence level are found to be 0.072% (0.059%) for B(t -> Hu) and 0.043% (0.062%) for B(t -> Hc). These results are combined with two other searches performed by the CMS Collaboration for flavor-changing neutral current interactions of top quarks and Higgs bosons in final states where the Higgs boson decays to either a pair of photons or a pair of bottom quarks. The resulting observed (expected) upper limits at the 95% confidence level are 0.019% (0.027%) for B(t -> Hu) and 0.037% (0.035%) for B(t -> Hc).

Effective vaccines elicit B cell clonal expansion in germinal centers (GCs) that produce memory B cells and antibody-secreting plasma cells. In mice, memory B cells rarely re-enter GCs upon boosting and instead differentiate into plasma cells. However, mouse circulating memory constitutes only 1%-2% of B cells, compared to 30%-50% in primates. We examine memory and GC B cell responses in rhesus macaques immunized and boosted ipsilaterally or contralaterally with an mRNA vaccine encoding the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. The neutralizing activity of antibodies cloned from the memory compartment, as well as the size of the compartment, was independent of the site of boosting. We show that memory B cells enter and undergo iterative expansion in newly developing GCs when boosting is at a site distal to the site of priming. Thus, in primates, high-affinity memory B cells constitute a reservoir that actively participates in further development of immunity irrespective of the anatomical site of vaccine boosting.

Molecular diagnoses of Entamoeba histolytica and Strongyloides stercoralis in human samples are becoming increasingly common. To contribute to the ongoing standardization of molecular diagnostic approaches targeting these parasites, we compared three published E. histolytica- and S. stercoralis-specific real-time PCR assays in test comparisons without a reference standard. Latent class analysis (LCA) was used to calculate diagnostic accuracy estimations for the three compared assays per parameter. The comparison was conducted using stool samples from Ghanaian individuals. In the course of the assessment of 873 stool samples, the number of detected positive PCR results ranged from 10 to 15 for S. stercoralis and from 4 to 54 for E. histolytica depending on the applied assay. Diagnostic accuracy estimates of real-time PCR sensitivity for S. stercoralis and E. histolytica ranged from 89% to 100% and from 75% to 100%, respectively; diagnostic estimates of specificity ranged from 99% to 100% and from 94% to 100%, respectively. Diagnostic accuracy-adjusted prevalence estimates were 1.2% for S. stercoralis and 0.5% for E. histolytica. High cycle threshold values of real-time PCR > 35 showed a particularly reduced likeliness of reproducibility when applying competitor real-time PCR assays. There were no clear-cut differences in terms of diagnostic accuracy favoring either small-subunit ribosomal ribonucleic acid (SSU rRNA) gene sequences or the S. stercoralis dispersed repetitive sequence for S. stercoralis PCR. The same applied to the comparison of real-time PCRs targeting SSU rRNA gene sequences and the SSU rRNA episomal repeat sequence (SREPH) of E. histolytica. In conclusion, interchangeability of the compared real-time PCR assays was higher for the assessed S. stercoralis assays compared with the assessed E. histolytica assays. Regional diagnostic accuracy testing seems advisable before literature-adapted assays for rare tropical pathogens like S. stercoralis and E. histolytica are applied in different study regions.

Salient aspects of the commissioning, calibration, and performance of the CMS silicon strip tracker are discussed, drawing on experience during operation with proton-proton collisions delivered by the CERN LHC. The data were obtained with a variety of luminosities. The operating temperature of the strip tracker was changed several times during this period and results are shown as a function of temperature in several cases. Details of the system performance are presented, including occupancy, signal-to-noise ratio, Lorentz angle, and single-hit spatial resolution. Saturation effects in the APV25 readout chip preamplifier observed during early Run 2 are presented, showing the effect on various observables and the subsequent remedy. Studies of radiation effects on the strip tracker are presented both for the optical readout links and the silicon sensors. The observed effects are compared to simulation, where available, and they generally agree well with expectations.

Uncovering the role of upstream open reading frames (uORFs) challenges conventional views of one protein per messenger RNA and reveals the capacity of some uORFs to encode microproteins that contribute to cellular biology and physiology. This study explores the functional role of a recently identified mitochondrial microprotein, SLC35A4-MP, in the brown adipose tissue of mice. Our findings reveal dynamic regulation of SLC35A4-MP expression during primary brown adipocyte differentiation in vitro and during cold exposure or high-fat diet (HFD)-induced obesity in mice. Using a knockout mouse model, we show that loss of SLC35A4-MP disrupts mitochondrial lipid composition, decreasing cardiolipins and phosphatidylethanolamine in brown adipose tissue from HFD-fed mice. SLC35A4-MP deficiency also impairs mitochondrial activity, alters mitochondrial number and morphology, and promotes inflammation. Knockout mice accumulate acylcarnitines during cold exposure, indicating defective fatty acid oxidation. These findings reveal SLC35A4-MP as a previously unrecognized microprotein in regulating mitochondrial function and tissue lipid metabolism, adding to the growing list of functional endogenous microproteins.

The regulation of gene expression is crucial for the functional integration of evolutionarily young genes, particularly those that emerge de novo. However, the regulatory programmes governing the expression of de novo genes remain unknown. To address this, we applied computational methods to single-cell RNA sequencing data, identifying key transcription factors probably instrumental in regulating de novo genes. We found that transcription factors do not have the same propensity for regulating de novo genes; some transcription factors regulate more de novo genes than others. Leveraging genetic and genomic tools in Drosophila, we further examined the role of two key transcription factors, achintya and vismay, and the regulatory architecture of new genes. Our findings identify key transcription factors associated with the expression of de novo genes and highlight how transcription factors, and possibly their duplications, are linked to the expressional regulation of de novo genes.

A primary goal in the development of an AIDS vaccine is the elicitation of broadly neutralizing antibodies (bNAbs) that protect against diverse HIV-1 strains. To this aim, germline-targeting immunogens have been developed to activate bNAb precursors and initiate the induction of bNAbs. While most preclinical germline-targeting HIV-1 vaccine candidates only include a single bNAb precursor epitope, an effective HIV-1 vaccine will likely require bNAbs that target multiple epitopes on Env. Here, we report a newly designed germline-targeting Env SOSIP trimer, named 3nv.2, that presents three bNAb epitopes on Env: the CD4bs, V3, and V2 epitopes. 3nv.2 forms a stable trimeric Env and binds to bNAb precursors from each of the desired epitopes. Immunization experiments in rhesus macaques and mice demonstrate 3nv.2 elicits the combined effects of its parent immunogens. Our results provide proof of concept for using a germline-targeting immunogen presenting three or more bNAb epitopes and a framework to develop improved next-generation HIV-1 vaccine candidates.

Heightened activity in the orbitofrontal cortex (OFC), a brain region that contributes to motivation, emotion, and reward-related decision-making, is a key clinical feature of major depressive disorder (MDD). However, the cellular and molecular substrates underlying this dysfunction remain unclear. Here, we performed cell-type-specific profiling of human OFC and unexpectedly mapped MDD-linked epigenomic features (including genetic risk variants) to non-neuronal cells, revealing significant glial dysregulation in this region. Characterization of MDD-specific chromatin loci further identified ZBTB7A-a transcriptional regulator of astrocyte reactivity-as an important mediator of MDD-related alterations. In rodent models, we found that Zbtb7a induction in astrocytes is both necessary and sufficient to drive stress-mediated behavioral deficits, cell-type-specific transcriptional/epigenomic signatures, and aberrant OFC astrocyte-neuronal communication in male mice-an established MDD risk factor. These findings thus highlight essential roles for astrocytes in OFC-mediated stress susceptibility and identify ZBTB7A as a critical and therapeutically relevant regulator of MDD-related OFC dysfunction.