The rockefeller university

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection produces B cell responses that continue to evolve for at least a year. During that time, memory B cells express increasingly broad and potent antibodies that are resistant to mutations found in variants of concern(1). As a result, vaccination of coronavirus disease 2019 (COVID-19) convalescent individuals with currently available mRNA vaccines produces high levels of plasma neutralizing activity against all variants tested1,2. Here we examine memory B cell evolution five months after vaccination with either Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) mRNA vaccine in a cohort of SARS-CoV-2-naive individuals. Between prime and boost, memory B cells produce antibodies that evolve increased neutralizing activity, but there is no further increase in potency or breadth thereafter. Instead, memory B cells that emerge five months after vaccination of naive individuals express antibodies that are similar to those that dominate the initial response. While individual memory antibodies selected over time by natural infection have greater potency and breadth than antibodies elicited by vaccination, the overall neutralizing potency of plasma is greater following vaccination. These results suggest that boosting vaccinated individuals with currently available mRNA vaccines will increase plasma neutralizing activity but may not produce antibodies with equivalent breadth to those obtained by vaccinating convalescent individuals.

By combining the use of a small anti-CRISPR protein, AcrIIA23, with direct integration into the host cell's CRISPR locus, the lysogenic phage phi AP1.1 neutralizes CRISPR-mediated anti-phage immunity in Streptococcus pyogenes. CRISPR loci are composed of short DNA repeats separated by sequences, known as spacers, that match the genomes of invaders such as phages and plasmids. Spacers are transcribed and processed to generate RNA guides used by CRISPR-associated nucleases to recognize and destroy the complementary nucleic acids of invaders. To counteract this defence, phages can produce small proteins that inhibit these nucleases, termed anti-CRISPRs (Acrs). Here we demonstrate that the phi AP1.1 temperate phage utilizes an alternative approach to antagonize the type II-A CRISPR response in Streptococcus pyogenes. Immediately after infection, this phage expresses a small anti-CRISPR protein, AcrIIA23, that prevents Cas9 function, allowing phi AP1.1 to integrate into the direct repeats of the CRISPR locus, neutralizing immunity. However, acrIIA23 is not transcribed during lysogeny and phage integration/excision cycles can result in the deletion and/or transduction of spacers, enabling a complex modulation of the type II-A CRISPR immune response. A bioinformatic search identified prophages integrated not only in the CRISPR repeats, but also the cas genes, of diverse bacterial species, suggesting that prophage disruption of the CRISPR-cas locus is a recurrent mechanism to counteract immunity.

The number and variability of the neutralizing epitopes targeted by polyclonal antibodies in individuals who are SARS-CoV-2 convalescent and vaccinated are key determinants of neutralization breadth and the genetic barrier to viral escape(1-4). Using HIV-1 pseudotypes and plasma selection experiments with vesicular stomatitis virus/SARS-CoV-2 chimaeras(5), here we show that multiple neutralizing epitopes, within and outside the receptor-binding domain, are variably targeted by human polyclonal antibodies. Antibody targets coincide with spike sequences that are enriched for diversity in natural SARS-CoV-2 populations. By combining plasma-selected spike substitutions, we generated synthetic 'polymutant' spike protein pseudotypes that resisted polyclonal antibody neutralization to a similar degree as circulating variants of concern. By aggregating variant of concern-associated and antibody-selected spike substitutions into a single polymutant spike protein, we show that 20 naturally occurring mutations in the SARS-CoV-2 spike protein are sufficient to generate pseudotypes with near-complete resistance to the polyclonal neutralizing antibodies generated by individuals who are convalescent or recipients who received an mRNA vaccine. However, plasma from individuals who had been infected and subsequently received mRNA vaccination neutralized pseudotypes bearing this highly resistant SARS-CoV-2 polymutant spike, or diverse sarbecovirus spike proteins. Thus, optimally elicited human polyclonal antibodies against SARS-CoV-2 should be resilient to substantial future SARS-CoV-2 variation and may confer protection against potential future sarbecovirus pandemics.

Metabolic reprogramming contributes to cancer development and progression. Here, the authors show the utility of a metabolic drug library to uncover metabolic vulnerabilities and obtain functional insights into myeloid leukemia biology. Interrogation of cellular metabolism with high-throughput screening approaches can unravel contextual biology and identify cancer-specific metabolic vulnerabilities. To systematically study the consequences of distinct metabolic perturbations, we assemble a comprehensive metabolic drug library (CeMM Library of Metabolic Drugs; CLIMET) covering 243 compounds. We, next, characterize it phenotypically in a diverse panel of myeloid leukemia cell lines and primary patient cells. Analysis of the drug response profiles reveals that 77 drugs affect cell viability, with the top effective compounds targeting nucleic acid synthesis, oxidative stress, and the PI3K/mTOR pathway. Clustering of individual drug response profiles stratifies the cell lines into five functional groups, which link to specific molecular and metabolic features. Mechanistic characterization of selective responses to the PI3K inhibitor pictilisib, the fatty acid synthase inhibitor GSK2194069, and the SLC16A1 inhibitor AZD3965, bring forth biomarkers of drug response. Phenotypic screening using CLIMET represents a valuable tool to probe cellular metabolism and identify metabolic dependencies at large.

Animals encounter microorganisms in their habitats, adapting physiology and behavior accordingly. The nematode Caenorhabditis elegans is found in microbe-rich environments; however, its responses to fungi are not extensively studied. Here, we describe interactions of C. elegans and Penicillium brevicompactum, an ecologically relevant mold. Transcriptome studies reveal that co-culture upregulates stress response genes, including xenobiotic-metabolizing enzymes (XMEs), in C. elegans intestine and AMsh glial cells. The nuclear hormone receptors (NHRs) NHR-45 and NHR-156 are induction regulators, and mutants that cannot induce XMEs in the intestine when exposed to P. brevicompactum experience mitochondrial stress and exhibit developmental defects. Different C. elegans wild isolates harbor sequence polymorphisms in nhr156, resulting in phenotypic diversity in AMsh glia responses to microbe exposure. We propose that P. brevicompactum mitochondria-targeting mycotoxins are deactivated by intestinal detoxification, allowing tolerance to moldy environments. Our studies support the idea that C. elegans NHRs may be regulated by environmental cues.

BACKGROUND: Extracellular RNAs (exRNAs) in biofluids are amenable to quantitative analysis and proposed as noninvasive biomarkers for monitoring organ function. Cell-lineage-specific microRNAs (miRNAs) are present in plasma as soluble ribonucleoproteins or enclosed in exRNA carriers and transported through the vasculature. However, more extensive studies of healthy individuals are needed to gain insights into the variability of plasma miRNA abundance and composition. METHODS: The exRNA composition of platelet-depleted plasma collected twice from 236 healthy individuals was characterized by small RNA sequencing. Plasma of pregnant women featuring dramatically increased placental miRNAs and samples from subject P12 with noticeably increased epithelial- and neuroendocrine-origin miRNAs were included for comparison. The miRNA content of 10 000g and 100 000g pellet fractions of plasma generated by ultracentrifugation was also determined. Data analysis methods included Pearson correlation, differential gene expression, and unsupervised clustering. RESULTS: The abundance changes for more variable miRNAs in plasma of normal individuals correlated between coexpressed cell-lineage-specific miRNAs of the liver, neuroendocrine organs, epithelial cells, and muscle. ExRNA of pellet fractions contained <2% of total plasma miRNA with modest enrichment of lineage-specific and variable miRNAs compared to supernatant. The abundance fold changes of miRNAs observed in pregnancy and P12 compared to normal exceeded interquartile variability of healthy individuals. The neuroendocrine miRNA signature of P12 persisted for more than 4 years and was absent in other individuals. CONCLUSIONS: This study defines the framework and effect size for screening of extensive plasma collections for miRNA phenotypes and biomarker discovery.

The brain generates internal representations that translate sensory stimuli into appropriate behavior. In the taste system, different tastes activate distinct populations of sensory neurons. We investigated the temporal properties of taste responses in Drosophila and discovered that different types of taste sensory neurons show striking differences in their response dynamics. Strong responses to stimulus onset (ON responses) and offset (OFF responses) were observed in bitter-sensing neurons in the labellum, whereas bitter neurons in the leg and other classes of labellar taste neurons showed only an ON response. Individual labellar bitter neurons generate both ON and OFF responses through a cell-intrinsic mechanism that requires canonical bitter receptors. A single receptor complex likely generates both ON and OFF responses to a given bitter ligand. These ON and OFF responses in the periphery are propagated to dopaminergic neurons that mediate aversive learning, and the presence of the OFF response impacts synaptic plasticity when bitter is used as a reinforcement cue. These studies reveal previously unknown features of taste responses that impact neural circuit function and may be important for behavior. Moreover, these studies show that OFF responses can dramatically influence timing-based synaptic plasticity, which is thought to underlie associative learning.