In low- and middle-income countries (LMIC) Staphylococcus aureus is regarded as one of the leading bacterial causes of neonatal sepsis, however there is limited knowledge on the species diversity and antimicrobial resistance caused by Gram-positive bacteria (GPB).We characterised GPB isolates from neonatal blood cultures from LMICs in Africa (Ethiopia, Nigeria, Rwanda, and South Africa) and South-Asia (Bangladesh and Pakistan) between 2015-2017. We determined minimum inhibitory concentrations and performed whole genome sequencing (WGS) on Staphylococci isolates recovered and clinical data collected related to the onset of sepsis and the outcome of the neonate up to 60 days of age.From the isolates recovered from blood cultures, Staphylococci species were most frequently identified. Out of 100 S. aureus isolates sequenced, 18 different sequence types (ST) were found which unveiled two small epidemiological clusters caused by methicillin resistant S. aureus (MRSA) in Pakistan (ST8) and South Africa (ST5), both with high mortality (n = 6/17). One-third of S. aureus was MRSA, with methicillin resistance also detected in Staphylococcus epidermidis, Staphylococcus haemolyticus and Mammaliicoccus sciuri. Through additional WGS analysis we report a cluster of M. sciuri in Pakistan identified between July-November 2017.In total we identified 14 different GPB bacterial species, however Staphylococci was dominant. These findings highlight the need of a prospective genomic epidemiology study to comprehensively assess the true burden of GPB neonatal sepsis focusing specifically on mechanisms of resistance and virulence across species and in relation to neonatal outcome.
Early recognition and appropriate management of paediatric sepsis are known to improve outcomes. A previous system's biology investigation of the systemic immune response in neonates to sepsis identified immune and metabolic markers that showed high accuracy for detecting bacterial infection. Further gene expression markers have also been reported previously in the paediatric age group for discriminating sepsis from control cases. More recently, specific gene signatures were identified to discriminate between COVID-19 and its associated inflammatory sequelae. Through the current prospective cohort study, we aim to evaluate immune and metabolic blood markers which discriminate between sepses (including COVID-19) from other acute illnesses in critically unwell children and young persons, up to 18 years of age.
A high-quality sequence assembly of the zebrafish genome reveals the largest gene set of any vertebrate and provides information on key genomic features, and comparison to the human reference genome shows that approximately 70% of human protein-coding genes have at least one clear zebrafish orthologue. The genome of the zebrafish — a key model organism for the study of development and human disease — has now been sequenced and published as a well-annotated reference genome. Zebrafish turns out to have the largest gene set of any vertebrate so far sequenced, and few pseudogenes. Importantly for disease studies, comparison between human and zebrafish sequences reveals that 70% of human genes have at least one obvious zebrafish orthologue. A second paper reports on an ongoing effort to identify and phenotype disruptive mutations in every zebrafish protein-coding gene. Using the reference genome sequence along with high-throughput sequencing and efficient chemical mutagenesis, the project's initial results — covering 38% of all known protein-coding genes — they describe phenotypic consequences of more than 1,000 alleles. The long-term goal is the creation of a knockout allele in every protein-coding gene in the zebrafish genome. All mutant alleles and data are freely available at go.nature.com/en6mos . Zebrafish have become a popular organism for the study of vertebrate gene function1,2. The virtually transparent embryos of this species, and the ability to accelerate genetic studies by gene knockdown or overexpression, have led to the widespread use of zebrafish in the detailed investigation of vertebrate gene function and increasingly, the study of human genetic disease3,4,5. However, for effective modelling of human genetic disease it is important to understand the extent to which zebrafish genes and gene structures are related to orthologous human genes. To examine this, we generated a high-quality sequence assembly of the zebrafish genome, made up of an overlapping set of completely sequenced large-insert clones that were ordered and oriented using a high-resolution high-density meiotic map. Detailed automatic and manual annotation provides evidence of more than 26,000 protein-coding genes6, the largest gene set of any vertebrate so far sequenced. Comparison to the human reference genome shows that approximately 70% of human genes have at least one obvious zebrafish orthologue. In addition, the high quality of this genome assembly provides a clearer understanding of key genomic features such as a unique repeat content, a scarcity of pseudogenes, an enrichment of zebrafish-specific genes on chromosome 4 and chromosomal regions that influence sex determination.
The molecular events that contribute to, and result from, the in vivo binding of transcription factors to their cognate DNA sequence motifs in mammalian genomes are poorly understood. We demonstrate that variations within the DNA sequence motifs that bind the transcriptional repressor REST (NRSF) encode in vivo DNA binding affinity hierarchies that contribute to regulatory function during lineage-specific and developmental programs in fundamental ways. First, canonical sequence motifs for REST facilitate strong REST binding and control functional classes of REST targets that are common to all cell types, whilst atypical motifs participate in weak interactions and control those targets, which are cell- or tissue-specific. Second, variations in REST binding relate directly to variations in expression and chromatin configurations of REST's target genes. Third, REST clearance from its binding sites is also associated with variations in the RE1 motif. Finally, and most surprisingly, weak REST binding sites reside in DNA sequences that show the highest levels of constraint through evolution, thus facilitating their roles in maintaining tissue-specific functions. These relationships have never been reported in mammalian systems for any transcription factor.
Genotoxicity models are extremely important to assess retroviral vector biosafety before gene therapy. We have developed an in utero model that demonstrates that hepatocellular carcinoma (HCC) development is restricted to mice receiving nonprimate (np) lentiviral vectors (LV) and does not occur when a primate (p) LV is used regardless of woodchuck post-translation regulatory element (WPRE) mutations to prevent truncated X gene expression. Analysis of 839 npLV and 244 pLV integrations in the liver genomes of vector-treated mice revealed clear differences between vector insertions in gene dense regions and highly expressed genes, suggestive of vector preference for insertion or clonal outgrowth. In npLV-associated clonal tumors, 56% of insertions occurred in oncogenes or genes associated with oncogenesis or tumor suppression and surprisingly, most genes examined (11/12) had reduced expression as compared with control livers and tumors. Two examples of vector-inserted genes were the Park 7 oncogene and Uvrag tumor suppressor gene. Both these genes and their known interactive partners had differential expression profiles. Interactive partners were assigned to networks specific to liver disease and HCC via ingenuity pathway analysis. The fetal mouse model not only exposes the genotoxic potential of vectors intended for gene therapy but can also reveal genes associated with liver oncogenesis.
Abstract Background/Aims Interleukin-27 (IL-27) regulates adaptive immune responses and is critical for the timely resolution of inflammation to restore tissue homeostasis. Studies have highlighted roles for IL-27 in limiting immune cell effector function, stromal cell responses and erosive joint pathology in clinical rheumatoid arthritis and experimental models of the disease. In the murine antigen-induced arthritis (AIA) model, IL-27 receptor-deficient (Il27ra-/-) mice develop severe synovitis associated with increased infiltration of synovial CD4+ T cells, development of synovial ectopic lymphoid-like structures and exacerbated cartilage and bone erosion. Similarly, mice with collagen-induced arthritis (CIA) administered IL-27 show improved joint pathology and reduced serum IFN-γ and IL-17 levels. Identifying mechanisms by which IL-27 regulates arthritis progression is key to understanding its therapeutic potential. Using RNA-sequencing of synovial tissue and joint-infiltrating CD4+ T cells we reveal that IL-27 regulates the magnitude of synovitis and genes in CD4+ T cells central to neutrophil recruitment. Methods Antigen-induced arthritis (AIA) was established in Il27ra-/- and wild-type control mice. RNA-sequencing was performed on whole synovial tissue and joint-infiltrating CD4+ T cells during the peak of joint inflammation and during the resolution of synovitis. Differentially expressed genes were interrogated by molecular pathway analysis and gene set enrichment analysis. CD4+ T cell culture assays, qPCR and ELISA were used to test and validate genes under IL-27 regulation. Results RNA-sequencing of whole synovial tissue revealed that pathways relating to CD4+ T cell and Th17 biology were significantly overrepresented in Il27ra-/- mice with AIA, consistent with a pathogenic role for these cells in inflammatory arthritis. Genes associated with neutrophil effector function (e.g. Elane, Padi4, Prtn3) and recruitment (e.g. Cxcl1, Cxcr2) were also highly expressed in Il27ra-/- synovium and was reflected in the increased number of Ly6G+CD11b+ neutrophils recovered from the joints of Il27ra-/- mice. In contrast to the transcriptomic analysis of inflamed synovial tissue, surprisingly RNA-sequencing of joint-infiltrating CD4+ T cells revealed that the expression of signature Th17 genes (e.g. Il17a, Il17f, Rorc) was comparable between WT and Il27ra-/- T cells. This implies that IL-27 predominantly regulates the magnitude of the joint CD4+ T cell infiltrate more than the effector characteristics of the infiltrating T cells. Differential gene expression analysis revealed heightened Cxcl1 expression in both whole synovial tissue and joint-infiltrating CD4+ T cells recovered from Il27ra-/- mice with AIA. CD4+ T cell differentiation cultures confirmed upregulation of Cxcl1 following T cell receptor activation and under Th1 polarizing conditions. Here, IL-27 suppressed the expression of CXCL1 at the mRNA and protein level. Conclusion Together, our data reveals a role for IL-27 in limiting CXCL1 expression in activated and joint-infiltrating CD4+ T cells, highlighting a novel mechanism by which IL-27 may integrate innate and adaptive arms of the immune response to regulate arthritis progression. Disclosure I. Burridge: None. S. Eastham: None. D.G. Hill: None. R. Andrews: None. B. Szomolay: None. N. Williams: None. S.A. Jones: None. G.W. Jones: None.
Abstract Oxylipins are potent biological mediators requiring strict control, but how they are removed en masse during infection and inflammation is unknown. Here we show that lipopolysaccharide (LPS) dynamically enhances oxylipin removal via mitochondrial β-oxidation. Specifically, genetic or pharmacological targeting of carnitine palmitoyl transferase 1 (CPT1), a mitochondrial importer of fatty acids, reveal that many oxylipins are removed by this protein during inflammation in vitro and in vivo. Using stable isotope-tracing lipidomics, we find secretion-reuptake recycling for 12-HETE and its intermediate metabolites. Meanwhile, oxylipin β-oxidation is uncoupled from oxidative phosphorylation, thus not contributing to energy generation. Testing for genetic control checkpoints, transcriptional interrogation of human neonatal sepsis finds upregulation of many genes involved in mitochondrial removal of long-chain fatty acyls, such as ACSL1,3,4, ACADVL, CPT1B, CPT2 and HADHB . Also, ACSL1/Acsl1 upregulation is consistently observed following the treatment of human/murine macrophages with LPS and IFN-γ. Last, dampening oxylipin levels by β-oxidation is suggested to impact on their regulation of leukocyte functions. In summary, we propose mitochondrial β-oxidation as a regulatory metabolic checkpoint for oxylipins during inflammation.
Postnatal kidney growth is substantial and involves expansion in kidney tubules without growth of new nephrons, which are the functional units of the kidney. Proliferation and differentiation pathways underpinning nephron elongation are not well defined. To address this, we performed sequential characterization of mouse kidney transcriptomics at the single cell level. Single nuclear RNA sequencing (snRNA-seq) was performed on kidney tissue from male and female mice at 1, 2, 4 and 12 weeks of age using the 10x Chromium platform. Unbiased clustering was performed on 68,775 nuclei from 16 animals. 31 discrete cellular clusters were seen, which were identified through comparison of their gene expression profiles to canonical markers of kidney cell populations. High levels of proliferation were evident at early time points in some cell types, especially tubular cells, but not in other cell types, for example podocytes. Proliferation was especially evident in Proximal Tubular Cells (PTCs) which are the most abundant cell type in the adult kidney. Uniquely when compared to other kidney cell types, PTCs demonstrated sex-specific expression profiles at late, but not early, time points. Mapping of PTC differentiation pathways using techniques including trajectory and RNA Velocity analyses delineated increasing PTC specialization and sex-specific phenotype specification. Our single-cell transcriptomics data characterise cellular states observed during kidney growth. We have identified PTC differentiation pathways that lead to sex-specific tubular cell phenotypes. Tubular proliferative responses are of central importance in postnatal kidney growth and have also been linked to kidney recovery versus fibrosis following injury. Our unbiased and comprehensive dataset of tubular cell development can be used to identify candidate pathways for therapeutic targeting.
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