The rockefeller university

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.

Murine astrovirus 2 (MuAstV2) is a novel murine astrovirus recently identified in laboratory and wild mice. MuAstV2 readily transmits between immunocompetent mice yet fails to transmit to highly immunocompromised mouse strains-a unique characteristic when contrasted with other murine viruses including other astroviruses. We characterized the viral shedding kinetics and tissue tropism of MuAstV2 in immunocompetent C57BL/6NCrl mice and evaluated the apparent Temporal patterns of viral shedding were determined by serially measuring fecal viral RNA. Tissue tropism and viral load were characterized and quantified by using in-situ hybridization (ISH) targeting viral RNA. Cellular tropism was characterized by evaluating fluorescent colocalization of viral ISH with various immunohistochemical markers. We found a rapid increase of fecal viral RNA in B6 mice, which peaked at 5 d after inoculation (dpi) followed by cessation of shedding by 168 dpi. The small intestine had the highest percentage of hybridization (3.09% of tissue area) of all tissues in which hybridization occurred at 5 dpi. The thymus displayed the next highest degree of hybridization (2.3%) at 7 dpi, indicating extraintestinal viral spread. MuAstV2 RNA hybridization was found to colocalize with only 3 of the markers evaluated: CD3 (T cells), Iba1 (macrophages), and cytokeratin (enterocytes). A higher percentage of CD3 cells and Iba1 cells hybridized with MuAstV2 as compared with cytokeratin at 2 dpi (CD3, 59%; Iba1, 46%; cytokeratin, 6%) and 35 dpi (CD3, 14%; Iba1, 55%; cytokeratin, 3%). Neither fecal viral RNA nor viral hybridization was noted in NCG mice at the time points examined. In addition, mice of mixed genetic background were inoculated, and only those with a functioning Il2rg gene shed MuAstV2. Results from this study suggest that infection of, or interaction with, the immune system is required for infection by or replication of MuAstV2.

eLife digest At birth, the mammalian brain contains tens of billions of neurons. Although the number does not increase much as the animal grows, there are many dramatic changes to their size and structure. These changes allow the neurons to communicate with one another, develop into networks, and learn the tasks of the adult brain. One way that these changes occur is by the accumulation of chemical marks on each neuron's DNA that help dictate which genes switch on, and which turn off. One of the most common ways that DNA can be marked is through the addition of a chemical group called a methyl group to one of the four DNA bases, cytosine. This process is called methylation. When methylation occurs, cytosine becomes 5-methylcytosine, or 5mC for short. In 2009, researchers found another modification present in the DNA in the brain: 5-hydroxymethylcytosine, or 5hmC. This modification appears when a group of proteins called the Tet hydroxylases turn 5mC into 5hmC. Converting 5mC to 5hmC normally helps cells remove marks on their DNA before they divide and expand. This is important because the newly generated cells need to be able to accumulate their own methylation marks to perform their roles properly. However, neurons in the brain accumulate 5hmC after birth, when the cells are no longer dividing, indicating that 5hmC may be required for the neurons to mature. Stoyanova et al. set out to determine whether mouse neurons need 5hmC to get their adult characteristics by tracking the chemical changes that occur in DNA from birth to adulthood. Some of the mice they tested produced 5hmC normally, while others lacked the genes necessary to make the Tet proteins in a specific class of neurons, preventing them from converting 5mC to 5hmC as they differentiate. The results reveal that neurons do not mature properly if 5hmC is not produced continuously following the first week of life. This is because neurons need to have the right genes switched on and off to differentiate correctly, and this only happens when 5hmC accumulates in some genes, while 5hmC and 5mC are removed from others. The data highlight the role of the Tet proteins, which convert 5mC into 5hmC, in preparing the marks for removal and demonstrate that active removal of these marks is essential for neuronal differentiation. Given the role of 5hmC in the development of neurons, it is possible that problems in this system could contribute to brain disorders. Further studies aimed at understanding how cells control 5hmC levels could lead to new ways to improve brain health. Research has also shown that if dividing cells lose the ability to make 5hmC, they can become cancerous. Future work could explain more about how and why this happens. Although high levels of 5-hydroxymethylcytosine (5hmC) accumulate in mammalian neurons, our knowledge of its roles in terminal differentiation or as an intermediate in active DNA demethylation is incomplete. We report high-resolution mapping of DNA methylation and hydroxymethylation, chromatin accessibility, and histone marks in developing postmitotic Purkinje cells (PCs) in Mus musculus. Our data reveal new relationships between PC transcriptional and epigenetic programs, and identify a class of genes that lose both 5-methylcytosine (5mC) and 5hmC during terminal differentiation. Deletion of the 5hmC writers Tet1, Tet2, and Tet3 from postmitotic PCs prevents loss of 5mC and 5hmC in regulatory domains and gene bodies, and hinders transcriptional and epigenetic developmental transitions. Our data demonstrate that Tet-mediated active DNA demethylation occurs in vivo, and that acquisition of the precise molecular properties of adult PCs require continued oxidation of 5mC to 5hmC during the final phases of differentiation.

Enterovirus (EV) infection rarely results in life-threatening infection of the central nervous system. We report two unrelated children with EV30 and EV71 rhombencephalitis. One patient carries compound heterozygous TLR3 variants (loss-of-function F322fs2* and hypomorphic D280N), and the other is homozygous for an IFIH1 variant (loss-of-function c.1641+1G>C). Their fibroblasts respond poorly to extracellular (TLR3) or intracellular (MDA5) poly(I:C) stimulation. The baseline (TLR3) and EV-responsive (MDA5) levels of IFN-beta in the patients' fibroblasts are low. EV growth is enhanced at early and late time points of infection in TLR3- and MDA5-deficient fibroblasts, respectively. Treatment with exogenous IFN-alpha 2b before infection renders both cell lines resistant to EV30 and EV71, whereas post-infection treatment with IFN-alpha 2b rescues viral susceptibility fully only in MDA5-deficient fibroblasts. Finally, the poly(I:C) and viral phenotypes of fibroblasts are rescued by the expression of WT TLR3 or MDA5. Human TLR3 and MDA5 are critical for cell-intrinsic immunity to EV, via the control of baseline and virus-induced type I IFN production, respectively.

Interferons (IFNs) are one of the hallmarks of host antiviral immunity. IFNs exert their antiviral activities through the induction of IFN-stimulated genes (ISGs) and antiviral proteins; however, the mechanism by which ISGs inhibit adenovirus (Ad) replication is not clearly understood. IFNs repress Ad immediate early gene expression and, consequently, all subsequent aspects of the viral life cycle. In this study, we found that IFN-induced protein with tetratricopeptide repeats 3, IFIT3 (ISG60), restricts Ad replication. IFIT3 repressed Ad E1A immediate early gene expression but did not alter Ad genome entry into the nucleus. Expression of IFIT3 led to phosphorylation of TBK1, IRF3, and STAT1; increased expression of IFN beta and ISGs; and required IFIT1 and IFIT2 partner proteins. During RNA virus infections, it is known that IFIT3 stimulates IFN production through mitochondrial antiviral signaling (MAVS)-mediated activation of TBK1 which synergizes activation of IRF3 and NF-kappa B. MAVS or TBK1 depletion in cells expressing IFIT3 blocked IFN signaling and reversed the Ad replication restriction. In addition, STING depletion phenocopied the effect suggesting that IFIT3 activates the STING pathway with cross talk to the MAVS pathway. This occurs independently of viral pathogen-associated molecular patterns (PAMPs). These results demonstrate that the expression of a single ISG, IFIT3, activates IFN signaling and establishes a cellular antiviral state independent of viral PAMPs. IMPORTANCE IFITs belong to a family of IFN-induced proteins that have broad antiviral functions, primarily studied with RNA viruses leaving a gap of knowledge on the effects of these proteins on DNA viruses. In this study we show that IFIT3, with its partner proteins IFIT1 and IFIT2, specifically restricts replication of human Ad, a DNA virus, by stimulating IFN beta production via the STING and MAVS pathways. This effect enhanced the IFN response and is independent of viral PAMPs. These results reveal a novel mechanism of activation of IFN signaling to enhance cellular antiviral responses.