The rockefeller university

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.

Leprosy is the second most prevalent mycobacterial disease globally. Despite the existence of an effective therapy, leprosy incidence has consistently remained above 200,000 cases per year since 2010. Numerous host genetic factors have been identified for leprosy that contribute to the persistently high case numbers. In the past decade, genetic epidemiology approaches, including genome-wide association studies (GWAS), identified more than 30 loci contributing to leprosy susceptibility. However, GWAS loci commonly encompass multiple genes, which poses a challenge to define causal candidates for each locus. To address this problem, we hypothesized that genes contributing to leprosy susceptibility differ in their frequencies of rare protein-altering variants between cases and controls. Using deep resequencing we assessed protein-coding variants for 34 genes located in GWAS or linkage loci in 555 Vietnamese leprosy cases and 500 healthy controls. We observed 234 nonsynonymous mutations in the targeted genes. A significant depletion of protein-altering variants was detected for the IL18R1 and BCL10 genes in leprosy cases. The IL18R1 gene is clustered with IL18RAP and IL1RL1 in the leprosy GWAS locus on chromosome 2q12.1. Moreover, in a recent GWAS we identified an HLA-independent signal of association with leprosy on chromosome 6p21. Here, we report amino acid changes in the CDSN and PSORS1C2 genes depleted in leprosy cases, indicating them as candidate genes in the chromosome 6p21 locus. Our results show that deep resequencing can identify leprosy candidate susceptibility genes that had been missed by classic linkage and association approaches.

Quinolone resistance in bacterial pathogens has primarily been associated with mutations in the quinolone resistance-determining regions (QRDRs) of bacterial type-II topoisomerases, which are DNA gyrase and topoisomerase IV. Depending on the position and type of the mutation (s) in the QRDRs, bacteria either become partially or completely resistant to quinolone. QRDR mutations have been identified and characterized in Salmonella enterica isolates from around the globe, particularly during the last decade, and efforts have been made to understand the propensity of different serovars to carry such mutations. Because there is currently no thorough analysis of the available literature on QRDR mutations in different Salmonella serovars, this review aims to provide a comprehensive picture of the mutational diversity in QRDRs of Salmonella serovars, summarizing the literature related to both typhoidal and non-typhoidal Salmonella serovars with a special emphasis on recent findings. This review will also discuss plasmid-mediated quinolone-resistance determinants with respect to their additive or synergistic contributions with QRDR mutations in imparting elevated quinolone resistance. Finally, the review will assess the contribution of membrane transporter-mediated quinolone efflux to quinolone resistance in strains carrying QRDR mutations. This information should be helpful to guide the routine surveillance of foodborne Salmonella serovars, especially with respect to their spread across countries, as well as to improve laboratory diagnosis of quinolone-resistant Salmonella strains.

The post-translational modification of histones by the small ubiquitin-like modifier (SUMO) protein has been associated with gene regulation, centromeric localization, and double-strand break repair in eukaryotes. Although sumoylation of histone H4 was specifically associated with gene repression, this could not be proven due to the challenge of site-specifically sumoylating H4 in cells. Biochemical crosstalk between SUMO and other histone modifications, such as H4 acetylation and H3 methylation, that are associated with active genes also remains unclear. We addressed these challenges in mechanistic studies using an H4 chemically modified at Lys12 by SUMO-3 (H4K12su) and incorporated into mononucleosomes and chromatinized plasmids for functional studies. Mononucleosome-based assays revealed that H4K12su inhibits transcription-activating H4 tail acetylation by the histone acetyltransferase p300, as well as transcription-associated H3K4 methylation by the extended catalytic module of the Set1/COMPASS (complex of proteins associated with Set1) histone methyltransferase complex. Activator- and p300-dependent in vitro transcription assays with chromatinized plasmids revealed that H4K12su inhibits both H4 tail acetylation and RNA polymerase II-mediated transcription. Finally, cell-based assays with a SUMO-H4 fusion that mimics H4 tail sumoylation confirmed the negative crosstalk between histone sumoylation and acetylation/methylation. Thus, our studies establish the key role for histone sumoylation in gene silencing and its negative biochemical crosstalk with active transcription-associated marks in human cells.

Epithelial-to-mesenchymal transition (EMT) is a transcriptionally governed process by which cancer cells establish a front-rear polarity axis that facilitates motility and invasion. Dynamic assembly of focal adhesions and other actin-based cytoskeletal structures on the leading edge of motile cells requires precise spatial and temporal control of protein trafficking. Yet, the way in which EMT-activating transcriptional programs interface with vesicular trafficking networks that effect cell polarity change remains unclear. Here, by utilizing multiple approaches to assess vesicular transport dynamics through endocytic recycling and retrograde trafficking pathways in lung adenocarcinoma cells at distinct positions on the EMT spectrum, we find that the EMT-activating transcription factor ZEB1 accelerates endocytosis and intracellular trafficking of plasma membrane-bound proteins. ZEB1 drives turnover of the MET receptor tyrosine kinase by hastening receptor endocytosis and transport to the lysosomal compartment for degradation. ZEB1 relieves a plus-end-directed microtubule-dependent kinesin motor protein (KIF13A) and a clathrin-associated adaptor protein complex subunit (AP1S2) from microRNA-dependent silencing, thereby accelerating cargo transport through the endocytic recycling and retrograde vesicular pathways, respectively. Depletion of KIF13A or AP1S2 mitigates ZEB1-dependent focal adhesion dynamics, front-rear axis polarization, and cancer cell motility. Thus, ZEB1-dependent transcriptional networks govern vesicular trafficking dynamics to effect cell polarity change. The way in which metastatic tumour cells control endocytic vesicular trafficking networks to establish a front-rear polarity axis that facilitates motility remains unclear. Here, the authors show that the EMT activator ZEB1 influences vesicular trafficking dynamics to execute cell polarity change.

The COVID-19 pandemic has highlighted the need to identify additional antiviral small molecules to complement existing therapies. Although increasing evidence suggests that metabolites produced by the human microbiome have diverse biological activities, their antiviral properties remain poorly explored. Using a cellbased SARS-CoV-2 infection assay, we screened culture broth extracts from a collection of phylogenetically diverse human-associated bacteria for the production of small molecules with antiviral activity. Bioassay-guided fractionation uncovered three bacterial metabolites capable of inhibiting SARS-CoV-2 infection. This included the nucleoside analogue N-6-(Delta(2)-isopentenyl)adenosine, the 5-hydroxytryptamine receptor agonist tryptamine, and the pyrazine 2,5-bis(3-indolylmethyl)pyrazine. The most potent of these, N-6-(Delta(2)-isopentenyl)adenosine, had a 50% inhibitory concentration (IC50) of 2 mM. These natural antiviral compounds exhibit structural and functional similarities to synthetic drugs that have been clinically examined for use against COVID-19. Our discovery of structurally diverse metabolites with anti-SARS-CoV-2 activity from screening a small fraction of the bacteria reported to be associated with the human microbiome suggests that continued exploration of phylogenetically diverse human-associated bacteria is likely to uncover additional small molecules that inhibit SARS-CoV-2 as well as other viral infections. IMPORTANCE The continued prevalence of COVID-19 and the emergence of new variants has once again put the spotlight on the need for the identification of SARS-CoV-2 antivirals. The human microbiome produces an array of small molecules with bioactivities (e.g., host receptor ligands), but its ability to produce antiviral small molecules is relatively underexplored. Here, using a cell-based screening platform, we describe the isolation of three microbiome-derived metabolites that are able to prevent SARS-CoV-2 infection in vitro. These molecules display structural similarities to synthetic drugs that have been explored for the treatment of COVID-19, and these results suggest that the microbiome may be a fruitful source of the discovery of small molecules with antiviral activities.

Molecular virology tools are critical for basic studies of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and for developing new therapeutics. Experimental systems that do not rely on viruses capable of spread are needed for potential use in lower-containment settings. In this work, we use a yeast-based reverse genetics system to develop spike-deleted SARS-CoV-2 self-replicating RNAs. These noninfectious self-replicating RNAs, or replicons, can be transcomplemented with viral glycoproteins to generate replicon delivery particles for single-cycle delivery into a range of cell types. This SARS-CoV-2 replicon system represents a convenient and versatile platform for antiviral drug screening, neutralization assays, host factor validation, and viral variant characterization.

Frizzleds (FZD(1-10)) are G protein-coupled receptors containing an extracellular cysteine-rich domain (CRD) binding Wingless/Int-1 lipoglycoproteins (WNTs). Despite the role of WNT/FZD signaling in health and disease, our understanding of how WNT binding is translated into receptor activation and transmembrane signaling remains limited. Current hypotheses dispute the roles for conformational dynamics. To clarify how WNT binding to FZD translates into receptor dynamics, we devised conformational FZD-CRD biosensors based on bioluminescence resonance energy transfer (BRET). Using FZD with N-terminal nanoluciferase (Nluc) and fluorescently labeled unnatural amino acids in the linker domain and extracellular loop 3, we show that WNT-3A and WNT-5A induce similar CRD conformational rearrangements despite promoting distinct signaling pathways and that CRD dynamics are not required for WNT/beta-catenin signaling. Thus, these FZD-CRD biosensors provide insights into binding, activation, and signaling processes in FZDs. The sensor design is broadly applicable to explore ligand--induced dynamics also in other membrane receptors.

Whipple's Disease is a rare systemic infectious disease caused by the ubiquitous actinomycetes Tropheryma whipplei (T. whipplei). We report herein a rare case of a cutaneous granulo matosis with hypercalcemia as an unusual presenting feature of Whipple's disease. The diagnosis of the bacteria was obtained from skin and inguinal lymph node biopsy (16 rDNA PCR screening and histological examination using PAS staining). T. whipplei was also identified on saliva and stool specimens, using specific PCR and colonic biopsies. Treatment with hydroxychloroquine and doxycycline allowed a rapid resolution of symptoms with a complete recovery.