The rockefeller university

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.

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.

Lipids are essential components of cancer cells due to their structural and signalling roles1. To meet metabolic demands, many cancers take up extracellular lipids2, 3, 4-5; however, how these lipids contribute to cancer growth and progression remains poorly understood. Here, using functional genetic screens, we identify uptake of lipoproteins-the primary mechanism for lipid transport in circulation-as a key determinant of ferroptosis sensitivity in cancer. Lipoprotein supplementation robustly inhibits ferroptosis across diverse cancer types, primarily through the delivery of alpha-tocopherol (alpha-toc), the most abundant form of vitamin E in human lipoproteins. Mechanistically, cancer cells take up lipoproteins through a pathway dependent on sulfated glycosaminoglycans (GAGs) linked to cell-surface proteoglycans. Disrupting GAG biosynthesis or acutely degrading surface GAGs reduces lipoprotein uptake, sensitizes cancer cells to ferroptosis and impairs tumour growth in mice. Notably, human clear cell renal cell carcinomas-a lipid-rich malignancy-exhibit elevated levels of chondroitin sulfate and increased lipoprotein-derived alpha-toc compared with normal kidney tissue. Together, our study establishes lipoprotein uptake as a critical anti-ferroptotic mechanism in cancer and implicates GAG biosynthesis as a therapeutic target.

Retrons are a retroelement class found in diverse prokaryotes that can be adapted to augment CRISPR-Cas9 genome engineering technology to efficiently rewrite short stretches of genetic information in bacteria and yeast. However, efficiency in human cells has been limited by unknown factors. We identified non-coding RNA (ncRNA) instability and impaired Cas9 activity due to 5 ' sgRNA extension as key contributors to low retron editor efficiency in human cells. We re-engineered the Eco1 ncRNA to incorporate an exoribonuclease-resistant RNA pseudoknot from the Zika virus 3 ' UTR and devised an RNA processing strategy using Csy4 ribonuclease to minimize 5 ' sgRNA extension. This strategy increased steady-state ncRNA levels and rescued sgRNA activity, leading to increased templated repair. This work reveals a previously unappreciated role for ncRNA stability in retron editor efficiency in human cells and presents an enhanced Eco1 retron editor capable of precise genome editing in human cells from a single integrated lentivirus and, in the context of the nCas9 H840A nickase, without creating double-strand breaks.