The rockefeller university

Mitonuclear discordance has been observed in several shark species. Female philopatry has often been invoked to explain such discordance but has never been explicitly tested. Here, we focus on the white shark, for which female philopatry has been previously proposed, and produced a chromosome-level genome, high-coverage whole-genome autosomal, and uniparental datasets to investigate mitonuclear discordance. We first reconstructed the historical population demography of the species based on autosomal data. We show that this species once comprised a single panmictic population, which experienced a steady decline until recent times when it fragmented into at least three main autosomal genetic groups. Mitochondrial data depict a strikingly different picture, inconsistent with the spatial distribution of autosomal diversity. Using the demographic scenario established from autosomal data, we performed coalescent and forward simulations to test for the occurrence of female philopatry. Coalescent simulations showed that the model can reproduce the autosomal variability, confirming its robustness. A forward simulation framework was further built to explicitly account for a sex-biased reproduction model and track both autosomal and uniparental markers (Y chromosome and mitochondrial DNA). While our model generates data that are consistent with the observed Y chromosome variation, the mitochondrial pattern is never reproduced even under extreme female philopatry (no female migration), strongly suggesting that demography alone cannot explain the mitonuclear discordance. Our framework could, and perhaps should, be extended to other shark species where philopatry has been suggested. It is possible that the proposed widespread occurrence of female philopatry in sharks should be revisited. Significance The mitonuclear discordance seen in sharks is widely attributed to female philopatry but has never been explicitly tested. Herein, we explore the issue in white sharks, for which we assembled a high-resolution genome and reconstructed the demographic history using resequencing data. We used backward and forward simulations to examine the genetic consequences of sex-specific migration patterns using parameter values derived from the demographic analyses of autosomal data. The mitochondrial variability observed in natural populations was never reproduced in any of the simulations-even under extreme female philopatry, suggesting that other forces have contributed to the discordance. The same approach would benefit other species of shark where female philopatry has previously been assumed based on genetic data.

Hyperactivation of the cystic fibrosis transmembrane conductance regulator (CFTR) contributes to secretory diarrhea, a major cause of pediatric mortality worldwide, and autosomal dominant polycystic kidney disease (ADPKD), the most common inherited cause of end-stage renal disease. Selective CFTR inhibition is a potential therapeutic strategy, with (R)-BPO-27 emerging as a promising candidate. Here, we present a cryo-EM structure of CFTR bound to (R)-BPO-27 at an overall resolution of 2.1 & Aring;. Contrary to the previous hypothesis that it inhibits CFTR current by competition with ATP, we demonstrate that (R)-BPO-27 instead directly occludes the chloride-conducting pore while permitting ATP hydrolysis, thus uncoupling the two activities. Furthermore, we find that inhibitor binding requires some degree of NBD separation, as the inhibition rate inversely correlates with the probability NBD dimerization. These findings clarify the compound's mechanism and provide a molecular basis for optimizing its clinical potential.

Congenital anomalies of the kidneys and urinary tract (CAKUT) are developmental disorders that commonly cause pediatric chronic kidney disease and mortality. We examine here rare coding variants in 248 CAKUT trios and 1742 singleton CAKUT cases and compare them to 22,258 controls. Diagnostic and candidate diagnostic variants are detected in 14.1% of cases. We find a significant enrichment of rare damaging variants in constrained genes expressed during kidney development and in genes associated with other developmental disorders, suggesting phenotype expansion. Consistent with these data, 18% of CAKUT patients with diagnostic variants have neurodevelopmental or cardiac phenotypes. We identify 40 candidate genes, including CELSR1, SSBP2, XPO1, NR6A1, and ARID3A. Two are confirmed as CAKUT genes: ARID3A and NR6A1. This study suggests that many yet-unidentified syndromes would be discoverable with larger cohorts and cross-phenotype analysis, leading to clarification of the genetic and phenotypic spectrum of developmental disorders.

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.

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.

Biomolecular condensates are key features of intracellular compartmentalization(1,2). As the most prominent nuclear condensate in eukaryotes, the nucleolus is a multiphase liquid-like structure in which ribosomal RNAs (rRNAs) are transcribed and processed, undergoing multiple maturation steps to form the small (SSU) and large (LSU) ribosomal subunits(3-5). However, how rRNA processing is coupled to the layered organization of the nucleolus is poorly understood owing to a lack of tools to precisely monitor and perturb nucleolar rRNA processing dynamics. Here we developed two complementary approaches to spatiotemporally map rRNA processing and engineer de novo nucleoli. Using sequencing in parallel with imaging, we found that rRNA processing steps are spatially segregated, with sequential maturation of rRNA required for its outward movement through nucleolar phases. By generating synthetic nucleoli in cells using an engineered rDNA plasmid system, we show that defects in SSU processing can alter the ordering of nucleolar phases, resulting in inside-out nucleoli and preventing rRNA outflux, while LSU precursors are necessary to build the outermost layer of the nucleolus. These findings demonstrate how rRNA is both a scaffold and substrate for the nucleolus, with rRNA acting as a programmable blueprint for the multiphase architecture that facilitates assembly of an essential molecular machine.