С����Ƶ

The circadian clock regulates immune responses to microbes and affects pathogen replication, but the underlying molecular mechanisms are not well understood. Here we demonstrate that the circadian components BMAL1 and REV-ERBα influence several steps in the hepatitis C virus (HCV) life cycle, including particle entry into hepatocytes and RNA genome replication. Genetic knock out of Bmal1 and over-expression or activation of REV-ERB with synthetic agonists inhibits the replication of HCV and the related flaviruses dengue and Zika via perturbation of lipid signaling pathways. This study highlights a role for the circadian clock component REV-ERBα in regulating flavivirus replication.

The role of persistent immune activation in postinfectious irritable bowel syndrome (PI-IBS) remains controversial. Here, we prospectively studied healthy subjects traveling to destinations with a high-risk to develop infectious gastroenteritis (IGE) in order to identify immune-mediated mechanisms and risk factors of PI-IBS.

Current molecular biology laboratories rely heavily on the purification and manipulation of nucleic acids. Yet, commonly used centrifuge- and column-based protocols require specialised equipment, often use toxic reagents, and are not economically scalable or practical to use in a high-throughput manner. Although it has been known for some time that magnetic beads can provide an elegant answer to these issues, the development of open-source protocols based on beads has been limited. In this article, we provide step-by-step instructions for an easy synthesis of functionalised magnetic beads, and detailed protocols for their use in the high-throughput purification of plasmids, genomic DNA, RNA and total nucleic acid (TNA) from a range of bacterial, animal, plant, environmental and synthetic sources. We also provide a bead-based protocol for bisulfite conversion and size selection of DNA and RNA fragments. Comparison to other methods highlights the capability, versatility, and extreme cost-effectiveness of using magnetic beads. These open-source protocols and the associated webpage (https://bomb.bio) can serve as a platform for further protocol customisation and community engagement.

Lipids are increasingly recognized as dynamic, critical metabolites affecting human physiology and pathophysiology. LIPID MAPS is a free resource dedicated to serving the lipid research community.

Monitoring of ATG8 proteins by western blotting and immunofluorescence microscopy are the most common methods to monitor the autophagy pathway. However, it has recently been shown that ATG8 proteins can be lipidated to non-autophagosome, single-membrane compartments through a noncanonical autophagy pathway. This is commonly found to occur during macro-endocytic processes such as phagocytosis, where it has been termed LC3-associated phagocytosis, and upon lysosomotropic drug treatment. Therefore, care is required when interpreting data based on ATG8 in order to conclude whether a signal relates to the canonical or noncanonical pathway. Here we provide methods to monitor noncanonical autophagy through fluorescence microscopy.

The inactive X chromosome (Xi) in female mammals adopts an atypical higher-order chromatin structure, manifested as a global loss of local topologically associated domains (TADs), A/B compartments and formation of two mega-domains. Here we demonstrate that the non-canonical SMC family protein, SmcHD1, which is important for gene silencing on Xi, contributes to this unique chromosome architecture. Specifically, allelic mapping of the transcriptome and epigenome in SmcHD1 mutant cells reveals the appearance of sub-megabase domains defined by gene activation, CpG hypermethylation and depletion of Polycomb-mediated H3K27me3. These domains, which correlate with sites of SmcHD1 enrichment on Xi in wild-type cells, additionally adopt features of active X chromosome higher-order chromosome architecture, including A/B compartments and partial restoration of TAD boundaries. Xi chromosome architecture changes also occurred following SmcHD1 knockout in a somatic cell model, but in this case, independent of Xi gene derepression. We conclude that SmcHD1 is a key factor in defining the unique chromosome architecture of Xi.

BRAF is a cytoplasmic protein kinase, which activates the MEK-ERK signalling pathway. Deregulation of the pathway is associated with the presence of mutations in human cancer, the most common being , although structural rearrangements, which remove N-terminal regulatory sequences, have also been reported. RAF-MEK-ERK signalling is normally thought to occur in the cytoplasm of the cell. However, in an investigation of BRAF localisation using fluorescence microscopy combined with subcellular fractionation of Green Fluorescent Protein (GFP)-tagged proteins expressed in NIH3T3 cells, surprisingly, we detected N-terminally truncated BRAF (ΔBRAF) in both nuclear and cytoplasmic compartments. In contrast, ΔCRAF and full-length, wild-type BRAF (BRAF) were detected at lower levels in the nucleus while full-length BRAF was virtually excluded from this compartment. Similar results were obtained using ΔBRAF tagged with the hormone-binding domain of the oestrogen receptor (hbER) and with the KIAA1549-ΔBRAF translocation mutant found in human pilocytic astrocytomas. Here we show that GFP-ΔBRAF nuclear translocation does not involve a canonical Nuclear Localisation Signal (NLS), but is suppressed by N-terminal sequences. Nuclear GFP-ΔBRAF retains MEK/ERK activating potential and is associated with the accumulation of phosphorylated MEK and ERK in the nucleus. In contrast, full-length GFP-BRAF and GFP-BRAF are associated with the accumulation of phosphorylated ERK but not phosphorylated MEK in the nucleus. These data have implications for cancers bearing single nucleotide variants or N-terminal deleted structural variants of .

The temporal order of DNA replication (replication timing [RT]) is highly coupled with genome architecture, but cis-elements regulating either remain elusive. We created a series of CRISPR-mediated deletions and inversions of a pluripotency-associated topologically associating domain (TAD) in mouse ESCs. CTCF-associated domain boundaries were dispensable for RT. CTCF protein depletion weakened most TAD boundaries but had no effect on RT or A/B compartmentalization genome-wide. By contrast, deletion of three intra-TAD CTCF-independent 3D contact sites caused a domain-wide early-to-late RT shift, an A-to-B compartment switch, weakening of TAD architecture, and loss of transcription. The dispensability of TAD boundaries and the necessity of these "early replication control elements" (ERCEs) was validated by deletions and inversions at additional domains. Our results demonstrate that discrete cis-regulatory elements orchestrate domain-wide RT, A/B compartmentalization, TAD architecture, and transcription, revealing fundamental principles linking genome structure and function.

Inflammatory activation of CD8 T cells can, when left unchecked, drive severe immunopathology. Hyperstimulation of CD8 T cells through a broad set of triggering signals can precipitate hemophagocytic lymphohistiocytosis (HLH), a life-threatening systemic inflammatory disorder.

The original version of this Article contained errors in the author affiliations.Martin R. Bennett was incorrectly associated with Nuclear Dynamics Programme, С����Ƶ, С����Ƶ Research Campus, Cambridge, CB22 3AT, UK. This has now been corrected in both the PDF and HTML versions of the Article. Furthermore, Phoebe Oldach was incorrectly associated with Centre for Molecular Informatics, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK.This has now been corrected in the HTML version of the Article. The PDF version of the Article was correct at the time of publication.

is a key immunological transcription factor, with knockout mice studies identifying functional roles in multiple immune cell types. Despite the importance of NFIL3, little is known about its function in humans.

The role of somatic variants in diseases beyond cancer is increasingly being recognized, with potential roles in autoinflammatory and autoimmune diseases. However, as mutation rates and allele fractions are lower, studies in these diseases are substantially less tolerant of false positives and bio-informatics algorithms require high replication rates. We developed a pipeline combining two variant callers, MuTect2 and VarScan2, with technical filtering and prioritization. Our pipeline detects somatic variants with allele fractions as low as 0.5% and achieves a replication rate >55%. Validation in an independent dataset demonstrates excellent performance (sensitivity >57%, specificity >98%, replication rate >80%). We applied this pipeline to the autoimmune disease multiple sclerosis (MS) as a proof-of-principle. We demonstrate that 60% of MS patients carry 2-10 exonic somatic variants in their peripheral blood T and B cells, with the vast majority (80%) occurring in T cells and variants persisting over time. Synonymous variants significantly co-occur with nonsynonymous variants. Systematic characterization indicates somatic variants are enriched for being novel or very rare in public databases of germline variants and trend towards being more damaging and conserved, as reflected by higher CADD and GERP scores. Our pipeline and proof-of-principle now warrant further investigation of common somatic genetic variation on top of inherited genetic variation in the context of autoimmune disease, where it may offer subtle survival advantages to immune cells and contribute to the capacity of these cells to participate in the autoimmune reaction.

The development of humoral autoimmunity following organ transplantation is increasingly recognised, but of uncertain significance. We examine whether autoimmunity contributes independently to allograft rejection. In a MHC class II-mismatched murine model of chronic humoral rejection, we report that effector antinuclear autoantibody responses were initiated upon graft-versus-host allorecognition of recipient B cells by donor CD4 T-cells transferred within heart allografts. Consequently, grafts were rejected more rapidly, and with markedly augmented autoantibody responses, upon transplantation of hearts from donors previously primed against recipient. Nevertheless, rejection was dependent upon recipient T follicular helper (T) cell differentiation and provision of cognate (peptide-specific) help for maintenance as long-lived GC reactions, which diversified to encompass responses against vimentin autoantigen. Heart grafts transplanted into stable donor/recipient mixed haematopoietic chimeras, or from parental strain donors into F1 recipients (neither of which can trigger host adaptive alloimmune responses), nevertheless provoked GC autoimmunity and were rejected chronically, with rejection similarly dependent upon host T cell differentiation. Thus, autoantibody responses contribute independently of host adaptive alloimmunity to graft rejection, but require host T cell differentiation to maintain long-lived GC responses. The demonstration that one population of helper CD4 T-cells initiates humoral autoimmunity, but that a second population of T cells is required for its maintenance as a GC reaction, has important implications for how autoimmune-related phenomena manifest.

Members of the ZFP36 family of RNA-binding proteins regulate gene expression post-transcriptionally by binding to AU-rich elements in the 3'UTR of mRNA and stimulating mRNA degradation. The proteins within this family target different transcripts in different tissues. In particular, ZFP36 targets myogenic transcripts and may have a role in adult muscle stem cell quiescence. Our study examined the requirement of ZFP36L1 and ZFP36L2 in adult muscle cell fate regulation.

Replication-dependent (RD) core histone mRNA produced during S-phase is the only known metazoan protein-coding mRNA presenting a 3' stem-loop instead of the otherwise universal polyA tail. A metallo β-lactamase (MBL) fold enzyme, cleavage and polyadenylation specificity factor 73 (CPSF73), is proposed to be the sole endonuclease responsible for 3' end processing of both mRNA classes. We report cellular, genetic, biochemical, substrate selectivity, and crystallographic studies providing evidence that an additional endoribonuclease, MBL domain containing protein 1 (MBLAC1), is selective for 3' processing of RD histone pre-mRNA during the S-phase of the cell cycle. Depletion of MBLAC1 in cells significantly affects cell cycle progression thus identifying MBLAC1 as a new type of S-phase-specific cancer target.

Metabolism is one of the attributes of life and supplies energy and building blocks to organisms. Therefore, understanding metabolism is crucial for the understanding of complex biological phenomena. Despite having been in the focus of research for centuries, our picture of metabolism is still incomplete. Metabolomics, the systematic analysis of all small molecules in a biological system, aims to close this gap. In order to facilitate such investigations a blueprint of the metabolic network is required. Recently, several metabolic network reconstructions for the model organism have been published, each having unique features. We have established the WormJam Community to merge and reconcile these (and other unpublished models) into a single consensus metabolic reconstruction. In a series of workshops and annotation seminars this model was refined with manual correction of incorrect assignments, metabolite structure and identifier curation as well as addition of new pathways. The WormJam consensus metabolic reconstruction represents a rich data source not only for network-based approaches like flux balance analysis, but also for metabolomics, as it includes a database of metabolites present in , which can be used for annotation. Here we present the process of model merging, correction and curation and give a detailed overview of the model. In the future it is intended to expand the model toward different tissues and put special emphasizes on lipid metabolism and secondary metabolism including ascaroside metabolism in accordance to their central role in physiology.

The placenta is the extraembryonic organ that supports the fetus during intrauterine life. Although placental dysfunction results in major disorders of pregnancy with immediate and lifelong consequences for the mother and child, our knowledge of the human placenta is limited owing to a lack of functional experimental models. After implantation, the trophectoderm of the blastocyst rapidly proliferates and generates the trophoblast, the unique cell type of the placenta. In vivo, proliferative villous cytotrophoblast cells differentiate into two main sub-populations: syncytiotrophoblast, the multinucleated epithelium of the villi responsible for nutrient exchange and hormone production, and extravillous trophoblast cells, which anchor the placenta to the maternal decidua and transform the maternal spiral arteries. Here we describe the generation of long-term, genetically stable organoid cultures of trophoblast that can differentiate into both syncytiotrophoblast and extravillous trophoblast. We used human leukocyte antigen (HLA) typing to confirm that the organoids were derived from the fetus, and verified their identities against four trophoblast-specific criteria. The cultures organize into villous-like structures, and we detected the secretion of placental-specific peptides and hormones, including human chorionic gonadotropin (hCG), growth differentiation factor 15 (GDF15) and pregnancy-specific glycoprotein (PSG) by mass spectrometry. The organoids also differentiate into HLA-G extravillous trophoblast cells, which vigorously invade in three-dimensional cultures. Analysis of the methylome reveals that the organoids closely resemble normal first trimester placentas. This organoid model will be transformative for studying human placental development and for investigating trophoblast interactions with the local and systemic maternal environment.

Adaptive strategies used by cells to scavenge and recycle essential nutrients are important for survival in nutrient-depleted environments such as cancer tissues. Autophagy and macropinocytosis are two major mechanisms that promote nutrient recycling and scavenging, which share considerable, yet poorly understood, cross-regulation. Here we review recent findings that connect these starvation response mechanisms and discuss the implications of their crosstalk.

This article is part of the Theo Murphy meeting issue ‘Macropinocytosis’.

Tumour cells exhibit altered lipid metabolism compared to normal cells. Cell signalling kinases are important for regulating lipid synthesis and energy storage. How upstream kinases regulate lipid content, versus direct targeting of lipid metabolising enzymes, is currently unexplored. We evaluated intracellular lipid concentrations in prostate and breast tumour spheroids, treated with drugs directly inhibiting metabolic enzymes FASN, ACC, DGAT and PDHK, or cell signalling kinase enzymes PI3K, AKT and mTOR with lipidomic analysis. We assessed whether baseline lipid profiles corresponded to inhibitors' effectiveness in modulating lipid profiles in 3D-growth, and their relationship to therapeutic activity. Inhibitors against PI3K, AKT and mTOR significantly inhibited MDA-MB-468 and PC3 cell growth in 2D and 3D spheroid growth, while moderately altering lipid content. Conversely, metabolism inhibitors against FASN and DGAT altered lipid content most effectively, while only moderately inhibiting growth compared to kinase inhibitors. The FASN and ACC inhibitors' effectiveness in MDA-MB-468, versus PC3, suggested the former depended more on synthesis whereas the latter may salvage lipids. Although baseline lipid profiles didn't predict growth effects, lipid changes on therapy matched the growth effects of FASN and DGAT inhibitors. Several phospholipids, including phosphatidylcholine, were also upregulated following treatment, possibly via the Kennedy pathway. As this promotes tumour growth, combination studies should include drugs targeting it. Two-dimensional drug screening may miss important metabolism inhibitors or underestimate their potency. Clinical studies should consider serial measurements of tumour lipids to prove target modulation. Pre-therapy tumour classification by de novo lipid synthesis versus uptake may help demonstrate efficacy.

Dietary, pharmacological and genetic interventions can extend health- and lifespan in diverse mammalian species. DNA methylation has been implicated in mediating the beneficial effects of these interventions; methylation patterns deteriorate during ageing, and this is prevented by lifespan-extending interventions. However, whether these interventions also actively shape the epigenome, and whether such epigenetic reprogramming contributes to improved health at old age, remains underexplored. We analysed published, whole-genome, BS-seq data sets from mouse liver to explore DNA methylation patterns in aged mice in response to three lifespan-extending interventions: dietary restriction (DR), reduced TOR signaling (rapamycin), and reduced growth (Ames dwarf mice). Dwarf mice show enhanced DNA hypermethylation in the body of key genes in lipid biosynthesis, cell proliferation and somatotropic signaling, which strongly correlates with the pattern of transcriptional repression. Remarkably, DR causes a similar hypermethylation in lipid biosynthesis genes, while rapamycin treatment increases methylation signatures in genes coding for growth factor and growth hormone receptors. Shared changes of DNA methylation were restricted to hypermethylated regions, and they were not merely a consequence of slowed ageing, thus suggesting an active mechanism driving their formation. By comparing the overlap in ageing-independent hypermethylated patterns between all three interventions, we identified four regions, which, independent of genetic background or gender, may serve as novel biomarkers for longevity-extending interventions. In summary, we identified gene body hypermethylation as a novel and partly conserved signature of lifespan-extending interventions in mouse, highlighting epigenetic reprogramming as a possible intervention to improve health at old age.

Extensive tracts of the mammalian genome that lack protein-coding function are still transcribed into long noncoding RNA. While these lncRNAs are generally short lived, length restricted, and non-polyadenylated, how their expression is distinguished from protein-coding genes remains enigmatic. Surprisingly, depletion of the ubiquitous Pol-II-associated transcription elongation factor SPT6 promotes a redistribution of H3K36me3 histone marks from active protein coding to lncRNA genes, which correlates with increased lncRNA transcription. SPT6 knockdown also impairs the recruitment of the Integrator complex to chromatin, which results in a transcriptional termination defect for lncRNA genes. This leads to the formation of extended, polyadenylated lncRNAs that are both chromatin restricted and form increased levels of RNA:DNA hybrid (R-loops) that are associated with DNA damage. Additionally, these deregulated lncRNAs overlap with DNA replication origins leading to localized DNA replication stress and a cellular senescence phenotype. Overall, our results underline the importance of restricting lncRNA expression.

Class IA PI3Ks have many roles in health and disease. The rules that govern intersubunit and receptor associations, however, remain unclear. We engineered mouse lines in which individual endogenous class IA PI3K subunits were C-terminally tagged with 17aa that could be biotinylated in vivo. Using these tools we quantified PI3K subunits in streptavidin or PDGFR pull-downs and cell lysates. This revealed that p85α and β bound equivalently to p110α or p110β but p85α bound preferentially to p110δ. p85s were found in molar-excess over p110s in a number of contexts including MEFs (p85β, 20%) and liver (p85α, 30%). In serum-starved MEFs, p110-free-p85s were preferentially, compared with heterodimeric p85s, bound to PDGFRs, consistent with in vitro assays that demonstrated they bound PDGFR-based tyrosine-phosphorylated peptides with higher affinity and co-operativity; suggesting they may act to tune a PI3K activation threshold. p110α-heterodimers were recruited 5-6× more efficiently than p110β-heterodimers to activated PDGFRs in MEFs or to PDGFR-based tyrosine-phosphorylated peptides in MEF-lysates. This suggests that PI3Kα has a higher affinity for relevant tyrosine-phosphorylated motifs than PI3Kβ. Nevertheless, PI3Kβ contributes substantially to acute PDGF-stimulation of PIP and PKB in MEFs because it is synergistically, and possibly sequentially, activated by receptor-recruitment and small GTPases (Rac/CDC42) via its RBD, whereas parallel activation of PI3Kα is independent of its RBD. These results begin to provide molecular clarity to the rules of engagement between class IA PI3K subunits in vivo and past work describing "excess p85," p85α as a tumor suppressor, and differential receptor activation of PI3Kα and PI3Kβ.

Protein misfolding is linked to a wide array of human disorders, including Alzheimer's disease, Parkinson's disease and type II diabetes. Protective cellular protein quality control (PQC) mechanisms have evolved to selectively recognize misfolded proteins and limit their toxic effects, thus contributing to the maintenance of the proteome (proteostasis). Here we examine how molecular chaperones and the ubiquitin-proteasome system cooperate to recognize and promote the clearance of soluble misfolded proteins. Using a panel of PQC substrates with distinct characteristics and localizations, we define distinct chaperone and ubiquitination circuitries that execute quality control in the cytoplasm and nucleus. In the cytoplasm, proteasomal degradation of misfolded proteins requires tagging with mixed lysine 48 (K48)- and lysine 11 (K11)-linked ubiquitin chains. A distinct combination of E3 ubiquitin ligases and specific chaperones is required to achieve each type of linkage-specific ubiquitination. In the nucleus, however, proteasomal degradation of misfolded proteins requires only K48-linked ubiquitin chains, and is thus independent of K11-specific ligases and chaperones. The distinct ubiquitin codes for nuclear and cytoplasmic PQC appear to be linked to the function of the ubiquilin protein Dsk2, which is specifically required to clear nuclear misfolded proteins. Our work defines the principles of cytoplasmic and nuclear PQC as distinct, involving combinatorial recognition by defined sets of cooperating chaperones and E3 ligases. A better understanding of how these organelle-specific PQC requirements implement proteome integrity has implications for our understanding of diseases linked to impaired protein clearance and proteostasis dysfunction.