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Histone modifications influence the recruitment of reader proteins to chromosomes to regulate events including transcription and cell division. The idea of a histone code, where combinations of modifications specify unique downstream functions, is widely accepted and can be demonstrated in vitro. For example, on synthetic peptides, phosphorylation of Histone H3 at threonine-3 (H3T3ph) prevents the binding of reader proteins that recognize trimethylation of the adjacent lysine-4 (H3K4me3), including the TAF3 component of TFIID. To study these combinatorial effects in cells, we analyzed the genome-wide distribution of H3T3ph and H3K4me2/3 during mitosis. We find that H3T3ph anti-correlates with adjacent H3K4me2/3 in cells, and that the PHD domain of TAF3 can bind H3K4me2/3 in isolated mitotic chromatin despite the presence of H3T3ph. Unlike in vitro, H3K4 readers are still displaced from chromosomes in mitosis in Haspin-depleted cells lacking H3T3ph. H3T3ph is therefore unlikely to be responsible for transcriptional downregulation during cell division. Methyl-phos switches on histones have been shown to regulate reader protein displacement from chromatin. However, in this study the authors find that H3T3ph is not required to remove transcription factors from H3K4me3 in mitosis. This might help to preserve promoter properties during cell division.

Epidemiological evidence suggests that a high intake of resistant starch and NSP protects against colo-rectal cancer. The mechanisms underlying this protection are thought to be mediated by the short-chain fatty acid butyrate, which is present in the colonic lumen in millimolar concentrations as a result of bacterial fermentation of carbohydrates that have resisted digestion in the small intestine. In vitro studies have shown that butyrate displays a host of chemo-preventative properties including increased apoptosis, reduced proliferation, down regulation of angiogenesis, enhanced immunosurveillance and anti-inflammatory effects in colo-rectal cancer cell lines. However, the molecular mechanisms underlying the apparent chemo-preventative actions of butyrate are largely unknown. The evidence supporting the role of butyrate as an anti-cancer agent is reviewed, with particular emphasis on those studies that have attempted to elucidate the mechanism of action of butyrate. Our understanding of the mechanistic action of butyrate and its role in cancer prevention is likely to advance considerably in this post-genomic era with the application of genomic and proteomic technologies. Studies are described that have used gene array and proteomic techniques to investigate the response of colo-rectal cancer cells to butyrate. These pioneering studies illustrate the potential of these technologies to help characterise the molecular responses of the cancer cell to butyrate, and to define the role of butyrate (and other nutrients) in the prevention of colo-rectal cancer.

Analysis of human blood immune cells provides insights into the coordinated response to viral infections such as severe acute respiratory syndrome coronavirus 2, which causes coronavirus disease 2019 (COVID-19). We performed single-cell transcriptome, surface proteome and T and B lymphocyte antigen receptor analyses of over 780,000 peripheral blood mononuclear cells from a cross-sectional cohort of 130 patients with varying severities of COVID-19. We identified expansion of nonclassical monocytes expressing complement transcripts ( CD16 + C1QA/B/C + ) that sequester platelets and were predicted to replenish the alveolar macrophage pool in COVID-19. Early, uncommitted CD34 + hematopoietic stem/progenitor cells were primed toward megakaryopoiesis, accompanied by expanded megakaryocyte-committed progenitors and increased platelet activation. Clonally expanded CD8 + T cells and an increased ratio of CD8 + effector T cells to effector memory T cells characterized severe disease, while circulating follicular helper T cells accompanied mild disease. We observed a relative loss of IgA2 in symptomatic disease despite an overall expansion of plasmablasts and plasma cells. Our study highlights the coordinated immune response that contributes to COVID-19 pathogenesis and reveals discrete cellular components that can be targeted for therapy. Transcriptomic and proteomic profiling of blood samples from individuals with COVID-19 reveals immune cell and hematopoietic progenitor cell alterations that are differentially associated with disease severity.

Histone modifications influence the recruitment of reader proteins to chromosomes to regulate events including transcription and cell division. The idea of a histone code, where particular combinations of modifications specify unique downstream functions, is widely accepted and can be demonstrated in vitro. For example, on synthetic peptides, phosphorylation of Histone H3 at threonine-3 (H3T3ph) prevents the binding of reader proteins that recognise trimethylation of the adjacent lysine-4 (H3K4me3), including the TAF3 component of TFIID. To study these combinatorial effects in cells, we analyzed the genome-wide distribution of H3T3ph and H3K4me3 during mitosis. We find that H3K4me3 hinders adjacent H3T3ph deposition in cells, and that the PHD domain of TAF3 can bind H3K4me3 in mitotic chromatin despite the presence of H3T3ph. Unlike in vitro, H3K4 readers are displaced from chromosomes in mitosis in Haspin-depleted cells lacking H3T3ph. H3T3ph is therefore unlikely to be responsible for transcriptional downregulation during cell division.Competing Interest StatementThe authors have declared no competing interest.

Although tropism of SARS-CoV-2 for respiratory tract epithelial cells is well established, an open question is whether the conjunctival epithelium is also a target for SARS-CoV-2. Conjunctival epithelial cells, which express viral entry receptors ACE2 and TMPRSS2, constitute the largest exposed epithelium of the ocular surface tissue, and may represent a relevant viral entry route. To address this question, we generated an organotypic air-liquid-interface model of conjunctival epithelium, composed of progenitor, basal and superficial epithelial cells and fibroblasts, which could be maintained successfully up to day 75 of differentiation. Using single-cell RNA Seq, with complementary imaging and virological assays, we observed that while all conjunctival cell types were permissive to SARS-CoV-2 genome expression, a productive infection did not ensue. The early innate immune response to SARS-CoV-2 infection in conjunctival cells was characterised by a robust autocrine and paracrine NF-Kβ activity, without activation of antiviral interferon signalling. Collectively, these data enrich our understanding of SARS-CoV-2 infection at the human ocular surface, with potential implications for the design of preventive strategies and conjunctival transplants.

To study the development and composition of human ocular surface, we performed single cell (sc) RNA-Seq at key embryonic, fetal and adult stages and generated the first atlas of the corneal cell types from development to adulthood. Our data indicate that during development, the conjunctival epithelium is the first to be specified from the ocular surface epithelium, followed by the corneal epithelium and the establishment of proliferative epithelial progenitors, which predate the formation of limbal niche by a few weeks. Bioinformatic comparison of adult cell clusters identified GPHA2, a novel cell-surface marker for quiescent limbal stem cells (qLSCs), whose function is to maintain qLSCs self-renewal. Combining scRNA- and ATAC-Seq analysis, we identified multiple upstream regulators for qLSCs and transit amplifying (TA) cells and demonstrated a close interaction between the immune cells and epithelial stem and progenitor cells in the cornea. RNA-Seq analysis indicated loss of qLSCs and acquisition of proliferative limbal basal epithelial progenitor markers during ex vivo limbal epithelial cell expansion, independently of the culture method used. Extending the single cell analyses to keratoconus, we were able to reveal activation of collagenase in the corneal stroma and a reduced pool of TA cells in the limbal epithelium as two key changes underlying the disease phenotype. Our scRNA-and ATAC-Seq data of developing and adult cornea in steady state and disease conditions provide a unique resource for defining pathways/genes that can lead to improvement in ex vivo expansion and differentiation methods for cell based replacement therapies and better understanding and treatment of ocular surface disorders. Competing Interest Statement The authors have declared no competing interest.

The nasal epithelium is a plausible entry point for SARS-CoV-2, a site of pathogenesis and transmission, and may initiate the host response to SARS-CoV-2. Antiviral interferon (IFN) responses are critical to outcome of SARS-CoV-2. Yet little is known about the interaction between SARS-CoV-2 and innate immunity in this tissue. Here we apply single-cell RNA sequencing and proteomics to a primary cell model of human nasal epithelium differentiated at air-liquid interface. SARS-CoV-2 demonstrates widespread tropism for nasal epithelial cell types. The host response is dominated by type I and III IFNs and interferon-stimulated gene products. This response is notably delayed in onset relative to viral gene expression and compared to other respiratory viruses. Nevertheless, once established, the paracrine IFN response begins to impact on SARS-CoV-2 replication. When provided prior to infection, recombinant IFNβ or IFNλ1 induces an efficient antiviral state that potently restricts SARS-CoV-2 viral replication, preserving epithelial barrier integrity. These data imply that the IFN-I/III response to SARS-CoV-2 initiates in the nasal airway and suggest nasal delivery of recombinant IFNs to be a potential chemoprophylactic strategy. The innate immune response in epithelial cells after SARS-CoV-2 infection is not fully understood. Here the authors use human air-liquid interface culture and show single cell transcription changes and delayed type I Interferon responses after SARS-CoV-2 infection compared with other respiratory viruses.

Age‐related macular degeneration (AMD) is a leading cause of blindness. Vision loss is caused by the retinal pigment epithelium (RPE) and photoreceptors atrophy and/or retinal and choroidal angiogenesis. Here we use AMD patient‐specific RPE cells with the Complement Factor H Y402H high‐risk polymorphism to perform a comprehensive analysis of extracellular vesicles (EVs), their cargo and role in disease pathology. We show that AMD RPE is characterised by enhanced polarised EV secretion. Multi‐omics analyses demonstrate that AMD RPE EVs carry RNA, proteins and lipids, which mediate key AMD features including oxidative stress, cytoskeletal dysfunction, angiogenesis and drusen accumulation. Moreover, AMD RPE EVs induce amyloid fibril formation, revealing their role in drusen formation. We demonstrate that exposure of control RPE to AMD RPE apical EVs leads to the acquisition of AMD features such as stress vacuoles, cytoskeletal destabilization and abnormalities in the morphology of the nucleus. Retinal organoid treatment with apical AMD RPE EVs leads to disrupted neuroepithelium and the appearance of cytoprotective alpha B crystallin immunopositive cells, with some co‐expressing retinal progenitor cell markers Pax6/Vsx2, suggesting injury‐induced regenerative pathways activation. These findings indicate that AMD RPE EVs are potent inducers of AMD phenotype in the neighbouring RPE and retinal cells.