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Monocytes are mononuclear phagocytes that can differentiate to a variety of cell fates under the influence of their microenvironment and hardwired commitment. We found that inhibition of TRPM8 in human blood CD14+ monocytes during a critical 3-h window at the beginning of their differentiation into macrophages led to enhanced survival and LPS-driven TNFα production after 24 h. TRPM8 antagonism also promoted LPS-driven TNFα production in CD14+ monocytes derived from the intestinal mucosa. Macrophages that had been derived for 6 days under blockade of TRPM8 had impaired phagocytic capacity and were transcriptionally distinct. Most of the affected genes were altered in a way that opposed normal monocyte to macrophage differentiation indicating that TRPM8 activity promotes aspects of this differentiation programme. Thus, we reveal a novel role for TRPM8 in regulating human CD14+ monocyte fate and function.

The synthesis of serine from glucose is a key metabolic pathway supporting cellular proliferation in healthy and malignant cells. Despite this, the role that this aspect of metabolism plays in germinal center biology and pathology is not known. Here, we performed a comprehensive characterization of the role of the serine synthesis pathway in germinal center B cells and lymphomas derived from these cells. We demonstrate that upregulation of a functional serine synthesis pathway is a metabolic hallmark of B-cell activation and the germinal center reaction. Inhibition of phosphoglycerate dehydrogenase (PHGDH), the first and rate limiting enzyme in this pathway, leads to defective germinal formation and impaired high-affinity antibody production. In addition, overexpression of enzymes involved in serine synthesis is a characteristic of germinal center B-cell derived lymphomas, with high levels of expression being predictive of reduced overall survival in diffuse large B cell lymphoma. Inhibition of PHGDH induces apoptosis in lymphoma cells reducing disease progression. These findings establish PHGDH as a critical player in humoral immunity and a clinically relevant target in lymphoma.

The germinal center (GC) response is critical for both effective adaptive immunity and establishing peripheral tolerance by limiting autoreactive B cells. Dysfunction in these processes can lead to defective immune responses to infection or contribute to autoimmune disease. To understand the gene regulatory principles underlying the GC response, we generated a single-cell transcriptomic and epigenomic atlas of the human tonsil, a widely studied and representative lymphoid tissue. We characterize diverse immune cell subsets and build a trajectory of dynamic gene expression and transcription factor activity during B cell activation, GC formation, and plasma cell differentiation. We subsequently leverage cell type-specific transcriptomic and epigenomic maps to interpret potential regulatory impact of genetic variants implicated in autoimmunity, revealing that many exhibit their greatest regulatory potential in GC-associated cellular populations. These included gene loci linked with known roles in GC biology (IL21, IL21R, IL4R, BCL6) and transcription factors regulating B cell differentiation (POU2AF1, HHEX). Together, these analyses provide a powerful new cell type-resolved resource for the interpretation of cellular and genetic causes underpinning autoimmune disease.

Haematopoiesis in the bone marrow (BM) maintains blood and immune cell production throughout postnatal life. Haematopoiesis first emerges in human BM at 11–12 weeks after conception1,2, yet almost nothing is known about how fetal BM (FBM) evolves to meet the highly specialized needs of the fetus and newborn. Here we detail the development of FBM, including stroma, using multi-omic assessment of mRNA and multiplexed protein epitope expression. We find that the full blood and immune cell repertoire is established in FBM in a short time window of 6–7 weeks early in the second trimester. FBM promotes rapid and extensive diversification of myeloid cells, with granulocytes, eosinophils and dendritic cell subsets emerging for the first time. The substantial expansion of B lymphocytes in FBM contrasts with fetal liver at the same gestational age. Haematopoietic progenitors from fetal liver, FBM and cord blood exhibit transcriptional and functional differences that contribute to tissue-specific identity and cellular diversification. Endothelial cell types form distinct vascular structures that we show are regionally compartmentalized within FBM. Finally, we reveal selective disruption of B lymphocyte, erythroid and myeloid development owing to a cell-intrinsic differentiation bias as well as extrinsic regulation through an altered microenvironment in Down syndrome (trisomy 21). A single-cell atlas of human fetal bone marrow in healthy fetuses and fetuses with Down syndrome provides insight into developmental haematopoiesis in humans and the transcription and functional differences that occur in Down syndrome.

Funder: Medical Research Council, The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. Here, to comprehensively map cell lineages, we use single-cell RNA sequencing and antigen receptor analysis of almost half a million cells from up to 5 anatomical regions in the developing and up to 11 distinct anatomical regions in the healthy paediatric and adult human gut. This reveals the existence of transcriptionally distinct BEST4 epithelial cells throughout the human intestinal tract. Furthermore, we implicate IgG sensing as a function of intestinal tuft cells. We describe neural cell populations in the developing enteric nervous system, and predict cell-type-specific expression of genes associated with Hirschsprung's disease. Finally, using a systems approach, we identify key cell players that drive the formation of secondary lymphoid tissue in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. This catalogue of intestinal cells will provide new insights into cellular programs in development, homeostasis and disease.

Throughout postnatal life, haematopoiesis in the bone marrow (BM) maintains blood and immune cell production. Haematopoiesis first emerges in human BM at 12 post conception weeks while fetal liver (FL) haematopoiesis is still expanding. Yet, almost nothing is known about how fetal BM evolves to meet the highly specialised needs of the fetus and newborn infant. Here, we detail the development of fetal BM including stroma using single cell RNA-sequencing. We find that the full blood and immune cell repertoire is established in fetal BM in a short time window of 6-7 weeks early in the second trimester. Fetal BM promotes rapid and extensive diversification of myeloid cells, with granulocytes, eosinophils and dendritic cell (DC) subsets emerging for the first time. B-lymphocyte expansion occurs, in contrast with erythroid predominance in FL at the same gestational age. We identify transcriptional and functional differences that underlie tissue-specific identity and cellular diversification in fetal BM and FL. Finally, we reveal selective disruption of B-lymphocyte, erythroid and myeloid development due to cell intrinsic differentiation bias as well as extrinsic regulation through an altered microenvironment in the fetal BM from constitutional chromosome anomaly Down syndrome during this crucial developmental time window.

Chen et al. describe how B-cell clones observed in the gut of many different individuals (recurrent or "public" clonotypes) are shaped by the combined influences of common microbial antigens and underlying genomic recombination biases.

Despite their crucial role in health and disease, our knowledge of immune cells within human tissues, in contrast to those circulating in the blood, remains limited. Here, we surveyed the immune compartment of lymphoid and non-lymphoid tissues of six adult donors by single-cell RNA sequencing, including alpha beta T-cell receptor (αβ TCR), gamma delta (γδ) TCR and B-cell receptor (BCR) variable regions. To aid systematic cell type identification we developed CellTypist, a tool for automated and accurate cell type annotation. Using this approach combined with manual curation, we determined the tissue distribution of finely phenotyped immune cell types and cell states. This revealed tissue-specific features within cell subsets, such as a subtype of activated dendritic cells in the airways expressing CSF2RA, GPR157, CRLF2, ITGAD-expressing γδ T cells in spleen and liver, and ITGAX+ splenic memory B cells. Single cell paired chain TCR analysis revealed cell type-specific biases in VDJ usage, and BCR analysis revealed characteristic patterns of somatic hypermutation and isotype usage in plasma and memory B cell subsets. In summary, our multi-tissue approach lays the foundation for identifying highly resolved immune cell types by leveraging a common reference dataset, tissue-integrated expression analysis and antigen receptor sequencing.

The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. To comprehensively map cell lineages in the healthy developing, pediatric and adult human gut from ten distinct anatomical regions, as well as draining lymph nodes, we used singlecell RNA-seq and VDJ analysis of roughly one third of a million cells. This reveals the presence of BEST4+ absorptive cells throughout the human intestinal tract, demonstrating the existence of this cell type beyond the colon for the first time. Furthermore, we implicate IgG sensing as a novel function of intestinal tuft cells, and link these cells to the pathogenesis of inflammatory bowel disease. We define novel glial and neuronal cell populations in the developing enteric nervous system, and predict cell-type specific expression of Hirschsprung’s disease-associated genes. Finally, using a systems approach, we identify key cell players across multiple cell lineages driving secondary lymphoid tissue formation in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. These data provide an unprecedented catalogue of intestinal cells, and new insights into cellular programs in development, homeostasis and disease.

The germinal center (GC) response is critical for both effective adaptive immunity and establishing peripheral tolerance by limiting auto-reactive B cells. Dysfunction in these processes can lead to defects in immune response to pathogens or contribute to autoimmune disease. To understand the gene regulatory principles underlying the GC response, we generated a single-cell transcriptomic and epigenomic atlas of the human tonsil, a widely studied and representative lymphoid tissue. We characterize diverse immune cell subsets and build a trajectory of dynamic gene expression and transcription factor activity during B cell activation, GC formation, and plasma cell differentiation. We subsequently leverage cell type-specific transcriptomic and epigenomic maps to interpret potential regulatory impact of genetic variants implicated in autoimmunity, revealing that many exhibit their greatest regulatory potential in GC cell populations. Together, these analyses provide a powerful new cell type-resolved resource for the interpretation of cellular and genetic causes underpinning autoimmune disease.One sentence summarySingle-cell chromatin accessibility landscapes of immune cell subsets reveal regulatory potential of autoimmune-associated genetic variants during the germinal center response.

The COVID-19 pandemic, caused by SARS coronavirus 2 (SARS-CoV-2), has resulted in excess morbidity and mortality as well as economic decline. To characterise the systemic host immune response to SARS-CoV-2, we performed single-cell RNA-sequencing coupled with analysis of cell surface proteins, providing molecular profiling of over 800,000 peripheral blood mononuclear cells from a cohort of 130 patients with COVID-19. Our cohort, from three UK centres, spans the spectrum of clinical presentations and disease severities ranging from asymptomatic to critical. Three control groups were included: healthy volunteers, patients suffering from a non-COVID-19 severe respiratory illness and healthy individuals administered with intravenous lipopolysaccharide to model an acute inflammatory response. Full single cell transcriptomes coupled with quantification of 188 cell surface proteins, and T and B lymphocyte antigen receptor repertoires have provided several insights into COVID-19: 1. a new non-classical monocyte state that sequesters platelets and replenishes the alveolar macrophage pool; 2. platelet activation accompanied by early priming towards megakaryopoiesis in immature haematopoietic stem/progenitor cells and expansion of megakaryocyte-primed progenitors; 3. increased clonally expanded CD8+ effector:effector memory T cells, and proliferating CD4+ and CD8+ T cells in patients with more severe disease; and 4. relative increase of IgA plasmablasts in asymptomatic stages that switches to expansion of IgG plasmablasts and plasma cells, accompanied with higher incidence of BCR sharing, as disease severity increases. All data and analysis results are available for interrogation and data mining through an intuitive web portal. Together, these data detail the cellular processes present in peripheral blood during an acute immune response to COVID-19, and serve as a template for multi-omic single cell data integration across multiple centers to rapidly build powerful resources to help combat diseases such as COVID-19.

The spatial organization of cell types in tissues fundamentally shapes cellular interactions and function, but the high-throughput spatial mapping of complex tissues remains a challenge. We present сell2location, a principled and versatile Bayesian model that integrates single-cell and spatial transcriptomics to map cell types in situ in a comprehensive manner. We show that сell2location outperforms existing tools in accuracy and comprehensiveness and we demonstrate its utility by mapping two complex tissues. In the mouse brain, we use a new paired single nucleus and spatial RNA-sequencing dataset to map dozens of cell types and identify tissue regions in an automated manner. We discover novel regional astrocyte subtypes including fine subpopulations in the thalamus and hypothalamus. In the human lymph node, we resolve spatially interlaced immune cell states and identify co-located groups of cells underlying tissue organisation. We spatially map a rare pre-germinal centre B-cell population and predict putative cellular interactions relevant to the interferon response. Collectively our results demonstrate how сell2location can serve as a versatile first-line analysis tool to map tissue architectures in a high-throughput manner.

Spatial transcriptomic technologies promise to resolve cellular wiring diagrams of tissues in health and disease, but comprehensive mapping of cell types in situ remains a challenge. Here we present сell2location, a Bayesian model that can resolve fine-grained cell types in spatial transcriptomic data and create comprehensive cellular maps of diverse tissues. Cell2location accounts for technical sources of variation and borrows statistical strength across locations, thereby enabling the integration of single-cell and spatial transcriptomics with higher sensitivity and resolution than existing tools. We assessed cell2location in three different tissues and show improved mapping of fine-grained cell types. In the mouse brain, we discovered fine regional astrocyte subtypes across the thalamus and hypothalamus. In the human lymph node, we spatially mapped a rare pre-germinal center B cell population. In the human gut, we resolved fine immune cell populations in lymphoid follicles. Collectively, our results present сell2location as a versatile analysis tool for mapping tissue architectures in a comprehensive manner.

Gastrointestinal microbiota and immune cells interact closely and display regional specificity, but little is known about how these communities differ with location. Here, we simultaneously assess microbiota and single immune cells across the healthy, adult human colon, with paired characterisation of immune cells in the mesenteric lymph nodes, to delineate colonic immune niches at steady-state. We describe distinct T helper cell activation and migration profiles along the colon and characterise the transcriptional adaptation trajectory of T regulatory cells between lymphoid tissue and colon. Finally, we show increasing B cell accumulation, clonal expansion and mutational frequency from caecum to sigmoid colon, and link this to the increasing number of reactive bacterial species.

In response to antigen challenge, B cells clonally expand, undergo selection and differentiate to produce mature B cell subsets and high affinity antibodies. However, the interplay between dynamic B cell states and their antibody-based selection is challenging to decipher in primary human tissue. We have applied an integrated analysis of bulk and single-cell antibody repertoires paired with single-cell transcriptomics of human B cells undergoing affinity maturation. We define unique gene expression and antibody repertoires of known and novel B cell states, including a pre-germinal centre state primed to undergo class switch recombination. We dissect antibody class-dependent gene expression of germinal centre and memory B cells to find that class switching prior to germinal centre entry dictates the capacity of B cells to undergo antibody-based selection and differentiate. Together, our analyses provide unprecedented resolution into the gene expression and selection dynamics that shape B cell-mediated immunity.

Epigenetic modifications on chromatin play important roles in regulating gene expression. While chromatin states are often governed by multi-layered structure, how individual pathways contribute to gene expression remains poorly understood. For example, DNA methylation is known to regulate transcription factor binding but also to recruit methyl-CpG binding proteins that affect chromatin structure through the activity of histone deacetylase complexes (HDACs). Both of these mechanisms can potentially affect gene expression, but the importance of each, and whether these activities are integrated to achieve appropriate gene regulation, remains largely unknown. To address this important question, we measured gene expression, chromatin accessibility, and transcription factor occupancy in wild-type or DNA methylation-deficient mouse embryonic stem cells following HDAC inhibition. Interestingly, we observe widespread increases in chromatin accessibility at repeat elements when HDACs are inhibited, and this is magnified when cells also lack DNA methylation. A subset of these elements have elevated binding of the YY1 and GABPA transcription factors and increased expression. The pronounced additive effect of HDAC inhibition in DNA methylation deficient cells demonstrate that DNA methylation and histone deacetylation act largely independently to suppress transcription factor binding and gene expression.

The fields of cancer- and immuno-metabolism have re-emerged as areas of significant translational potential. Even though the upregulation of glycolysis by proliferating lymphocytes is the basis for widely used clinical tests such as FDG-PET, little is known about which metabolic pathways are involved in the utilization of glucose to support B-cell proliferation. The synthesis of serine from glucose has been demonstrated to be a key metabolic pathway supporting cellular proliferation in some healthy and malignant cell types. Importantly, this pathway is regulated by MYC, which is known to be essential for germinal centre formation and is commonly dysregulated in lymphoma. Despite this, the role that the serine synthesis pathway (SSP) plays in germinal center biology and pathology has not been previously investigated. We performed a comprehensive characterization of the role of the SSP in germinal center B cells and lymphomas derived from these cells. We demonstrate that upregulation of a functional SSP is a metabolic hallmark of B-cell activation and the germinal center reaction. We show that both human and murine resting naïve B cells lack expression of the first two enzymes in this pathway, phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT1) enzymes. However, B-cell activation, predominantly through the B-cell receptor, robustly induces the expression of these enzymes in vitro, resulting in an acquired ability to synthesize serine, glycine and the purine nucleotides adenosine and guanosine from glucose. This is reflected in striking expression of PHGDH and PSAT1 within germinal centers but not in marginal zones confirming that this upregulation is occurring in germinal B cells activated in vivo. We then proceeded to investigate the impact of inhibiting PHGDH on germinal center formation and high-affinity antibody production in vivo. This was done both genetically, using a conditional B-cell knockout mouse model, and pharmacologically using a specific inhibitor of PHGDH, PH-755. Importantly, we show that PHGDH inhibition impairs germinal center formation with a resultant reduction in high-affinity antibody production. Mechanistic experiments demonstrate that PHGDH inhibition effectively blocks cells from synthesising serine and glycine from glucose, making them unable to proliferate in environments that lack these amino acids. We then investigated role of PHDGH and PSAT1 in Burkitt Lymphoma (BL), Diffuse Large B Cell Lymphoma (DLBCL) and Chronic Lymphocytic Leukemia (CLL). Notably, very high expression of these two proteins was observed in BL, with intermediate-to-high expression in DLBCL and relatively low expression in CLL, where expression was restricted to proliferation centers. Given the heterogeneity of expression in DLBCL patients, we next interrogated a published GSE database (Lenz et al. NEJM 2008) to investigate the impact on outcome. Notably high expression of PSAT1 was significantly associated with a poorer overall survival rate in DLBCL. We then investigated whether the SSP could be a therapeutic target in lymphoma. We demonstrate that PHGDH inhibition effectively inhibits de novo serine, glycine and purine nucleotide synthesis from glucose, resulting in impaired proliferation and increased apoptosis in a panel of human BL cell lines in vitro. We then analyzed the impact of PHGDH inhibition on lymphoma development in vivo using Eµ-myc mice, which harbour Myc coupled to the IgH enhancer characteristic of BL. Importantly we confirm the role of MYC by demonstrating that Eµ-Myc B-cells show significantly higher expression of PHGDH and PSAT1 expression resulting in increased serine and glycine synthesis when compared to control cells. We demonstrate that pharmacological inhibition of PHGDH using PH-755 impairs lymphoma progression in this model. We confirm the importance of PHGDH by showing that genetic ablation of Phgdh in Eµ-myc cells in a tamoxifen inducible system (using Eµ-myc/+;Rosa26-CreER T2/+;Phgdh fl/fl mice) also results in a significant reduction in lymphoma progression. Taken together, this work represents the first report of the role of the SSP in the biology of the germinal centre response and lymphomas derived from these cells. These findings establish PHGDH as a critical player in humoral immunity and a clinically relevant target in MYC-driven lymphoma, which is an area of significant unmet need. Disclosures Gribben: Abbvie: Honoraria; AZ: Honoraria, Research Funding; BMS: Honoraria; Gilead/Kite: Honoraria; Janssen: Honoraria, Research Funding; Morphosys: Honoraria; Novartis: Honoraria; Takeda: Honoraria; TG Therapeutis: Honoraria. Calado: Myricx Pharma: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Patents & Royalties: Cancer Treatments. WO patent WO 2020/128475 A1 (2020).

Polycomb group (PcG) proteins are transcriptional repressors that play important roles in regulating gene expression during animal development. In vitro experiments have shown that PcG protein complexes can compact chromatin to limit the activity of chromatin remodeling enzymes and access of the transcriptional machinery to DNA. In fitting with these ideas, gene promoters associated with PcG proteins have been reported to be less accessible than other gene promoters. However, it remains largely untested in vivo whether PcG proteins define chromatin accessibility or other chromatin features. To address this important question, we examine the chromatin accessibility and nucleosome landscape at PcG protein-bound promoters in mouse embryonic stem cells using the assay for transposase accessible chromatin (ATAC)-seq. Combined with genetic ablation strategies, we unexpectedly discover that although PcG protein-occupied gene promoters exhibit reduced accessibility, this does not rely on PcG proteins. Instead, the Polycomb repressive complex 1 (PRC1) appears to play a unique role in driving elevated nucleosome occupancy and decreased nucleosomal spacing in Polycomb chromatin domains. Our new genome-scale observations argue, in contrast to the prevailing view, that PcG proteins do not significantly affect chromatin accessibility and highlight an underappreciated complexity in the relationship between chromatin accessibility, the nucleosome landscape, and PcG-mediated transcriptional repression.

Polycomb group (PcG) proteins are transcriptional repressors that play important roles regulating gene expression during animal development. In vitro experiments have shown that PcG protein complexes can compact chromatin limiting the activity of chromatin remodelling enzymes and access of the transcriptional machinery to DNA. In fitting with these ideas, gene promoters associated with PcG proteins have been reported to be less accessible than other gene promotors. However, it remains largely untested in vivo whether PcG proteins define chromatin accessibility or other chromatin features. To address this important question, we measured chromatin accessibility and examined the nucleosome landscape at PcG protein-bound promoters in mouse embryonic stem cells using the assay for transposase accessible chromatin (ATAC)-seq. Combined with genetic ablation strategies, we unexpectedly discover that although PcG protein-occupied gene promoters exhibit reduced accessibility, this does not rely on PcG proteins. Instead, the Polycomb repressive complex 1 (PRC1) appears to play a unique role in driving elevated nucleosome occupancy and decreased nucleosomal spacing in Polycomb chromatin domains. Our new genome-scale observations argue, in contrast to the prevailing view, that PcG proteins and Polycomb chromatin domains do not significantly affect chromatin accessibility and highlight an underappreciated complexity in the relationship between chromatin accessibility, the nucleosome landscape and PcG-mediated transcriptional repression.

Exosomes are membrane-bound vesicles of endosomal origin, present in a wide range of biological fluids, including blood and urine. They range between 30 and 100 nm in diameter, and consist of a limiting lipid bilayer, transmembrane proteins and a hydrophilic core containing proteins, mRNAs and microRNAs (miRNA). Exosomes can act as extracellular vehicles by which cells communicate, through the delivery of their functional cargo to recipient cells, with many important biological, physiological and pathological implications. The exosome release pathway contributes towards protein secretion, antigen presentation, pathogen transfer and cancer progression. Exosomes and exosome-mediated signalling have been implicated in disease processes such as atherosclerosis, calcification and kidney diseases. Circulating levels of exosomes and extracellular vesicles can be influenced by the progression of renal disease. Advances in methods for purification and analysis of exosomes are leading to potential diagnostic and therapeutic avenues for kidney diseases. This review will focus on biophysical properties and biogenesis of exosomes, their pathophysiological roles and their potential as biomarkers and therapeutics in kidney diseases.

Polycomb group (PcG) proteins function as chromatin-based transcriptional repressors that are essential for normal gene regulation during development. However, how these systems function to achieve transcriptional regulation remains very poorly understood. Here, we discover that the histone H2AK119 E3 ubiquitin ligase activity of Polycomb repressive complex 1 (PRC1) is defined by the composition of its catalytic subunits and is highly regulated by RYBP/YAF2-dependent stimulation. In mouse embryonic stem cells, RYBP plays a central role in shaping H2AK119 mono-ubiquitylation at PcG targets and underpins an activity-based communication between PRC1 and Polycomb repressive complex 2 (PRC2) which is required for normal histone H3 lysine 27 trimethylation (H3K27me3). Without normal histone modification-dependent communication between PRC1 and PRC2, repressive Polycomb chromatin domains can erode, rendering target genes susceptible to inappropriate gene expression signals. This suggests that activity-based communication and histone modification-dependent thresholds create a localized form of epigenetic memory required for normal PcG chromatin domain function in gene regulation. DOI: http://dx.doi.org/10.7554/eLife.18591.001

Fish oils are rich in omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA), predominantly 20:5n-3 and 22:6n-3, whereas vegetable oils contain abundant C(18)-PUFA, predominantly 18:3n-3 or 18:2n-6. We hypothesized that replacement of fish oils with vegetable oils would increase the oxidative stability of fish lipids. Here we have used the long established and easily cultivated FHM cell line derived from the freshwater fish species fathead minnow (Pimephales promelas) to test this hypothesis. The FHM cells were readily able to synthesize 20:5n-3 and 24:6n-3 from 18:3n-3 but 22:6n-3 synthesis was negligible. Also, they were readily able to synthesize 20:3n-6 from 18:2n-6 but 20:4n-6 synthesis was negligible. Mitochondrial β-oxidation was greatest for 18:3n-3 and 20:5n-3 and the rates for 16:0, 18:2n-6, 22:6n-3 and 18:1n-9 were significantly lower. Fatty acid incorporation was predominantly into phospholipids (79-97%) with very little incorporation into neutral lipids. Increasing the fatty acid concentration in the growth medium substantially increased the concentrations of 18:3n-3 and 18:2n-6 in the cell phospholipids but this was not the case for 20:5n-3 or 22:6n-3. When they were subjected to oxidative stress, the FHM cells supplemented with either 20:5n-3 or 22:6n-3 (as compared with 18:3n-3 or saturated fatty acids) exhibited significantly higher levels of thiobarbituric reactive substances (TBARS) indicating higher levels of lipid peroxidation. The results are discussed in relation to the effects of fatty acid unsaturation on the oxidative stability of cellular lipids and the implications for sustainable aquaculture.

Summary Chromatin modifying activities inherent to polycomb repressive complexes PRC1 and PRC2 play an essential role in gene regulation, cellular differentiation, and development. However, the mechanisms by which these complexes recognize their target sites and function together to form repressive chromatin domains remain poorly understood. Recruitment of PRC1 to target sites has been proposed to occur through a hierarchical process, dependent on prior nucleation of PRC2 and placement of H3K27me3. Here, using a de novo targeting assay in mouse embryonic stem cells we unexpectedly discover that PRC1-dependent H2AK119ub1 leads to recruitment of PRC2 and H3K27me3 to effectively initiate a polycomb domain. This activity is restricted to variant PRC1 complexes, and genetic ablation experiments reveal that targeting of the variant PCGF1/PRC1 complex by KDM2B to CpG islands is required for normal polycomb domain formation and mouse development. These observations provide a surprising PRC1-dependent logic for PRC2 occupancy at target sites in vivo., Graphical Abstract, Highlights • Variant PRC1 complex-dependent H2AK119ub1 leads to binding of PRC2 and H3K27me3 • Canonical PRC1 complexes fail to efficiently deposit H2AK119ub1 and recruit PRC2 • A variant KDM2B/PCGF1/PRC1 complex is required for polycomb domain formation at CGIs • Failure to target KDM2B/PCGF1/PRC1 causes polycomb phenotypes and lethality in mice, Formation of repressive polycomb domains depends on histone ubiquitination catalyzed by variant PRC1 complexes at unmethylated CpG islands followed by PRC2 recruitment and methylation, rather than exclusively through initial association of PRC2.

Exosomes are membrane-bound vesicles of endosomal origin, present in a wide range of biological fluids, including blood and urine. They range between 30 and 100 nm in diameter, and consist of a limiting lipid bilayer, transmembrane proteins and a hydrophilic core containing proteins, mRNAs and microRNAs (miRNA). Exosomes can act as extracellular vehicles by which cells communicate, through the delivery of their functional cargo to recipient cells, with many important biological, physiological and pathological implications. The exosome release pathway contributes towards protein secretion, antigen presentation, pathogen transfer and cancer progression. Exosomes and exosome-mediated signalling have been implicated in disease processes such as atherosclerosis, calcification and kidney diseases. Circulating levels of exosomes and extracellular vesicles can be influenced by the progression of renal disease. Advances in methods for purification and analysis of exosomes are leading to potential diagnostic and therapeutic avenues for kidney diseases. This review will focus on biophysical properties and biogenesis of exosomes, their pathophysiological roles and their potential as biomarkers and therapeutics in kidney diseases.

CpG islands (CGIs) are associated with the majority of mammalian gene promoters and function to recruit chromatin modifying enzymes. It has therefore been proposed that CGIs regulate gene expression through chromatin-based mechanisms, however in most cases this has not been directly tested. Here, we reveal that the histone H3 lysine 36 (H3K36) demethylase activity of the CGI-binding KDM2 proteins contributes only modestly to the H3K36me2-depleted state at CGI-associated gene promoters and is dispensable for normal gene expression. Instead, we discover that KDM2 proteins play a widespread and demethylase-independent role in constraining gene expression from CGI-associated gene promoters. We further show that KDM2 proteins shape RNA Polymerase II occupancy but not chromatin accessibility at CGI-associated promoters. Together this reveals a demethylase-independent role for KDM2 proteins in transcriptional repression and uncovers a new function for CGIs in constraining gene expression.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited., Background Exosomes are nanovesicles secreted by tumour cells which have roles in paracrine signalling during tumour progression, including tumour-stromal interactions, activation of proliferative pathways and bestowing immunosuppression. Hypoxia is an important feature of solid tumours which promotes tumour progression, angiogenesis and metastasis, potentially through exosome-mediated signalling. Methods Breast cancer cell lines were cultured under either moderate (1% O2) or severe (0.1% O2) hypoxia. Exosomes were isolated from conditioned media and quantitated by nanoparticle tracking analysis (NTA) and immunoblotting for the exosomal protein CD63 in order to assess the impact of hypoxia on exosome release. Hypoxic exosome fractions were assayed for miR-210 by real-time reverse transcription polymerase chain reaction and normalised to exogenous and endogenous control genes. Statistical significance was determined using the Student T test with a P value of