Key pathways and genes that are altered during treatment with hyperbaric oxygen in patients with sepsis due to necrotizing soft tissue infection (HBOmic study).

BACKGROUND: For decades, the basic treatment strategies of necrotizing soft tissue infections (NSTI) have remained unchanged, primarily relying on aggressive surgical removal of infected tissue, broad-spectrum antibiotics, and supportive intensive care. One treatment strategy that has been proposed as an adjunctive measure to improve patient outcomes is hyperbaric oxygen (HBO2) treatment. HBO2 treatment has been linked to several immune modulatory effects; however, investigating these effects is complicated due to the disease's acute life-threatening nature, metabolic and cell homeostasis dependent variability in treatment effects, and heterogeneity with respect to both patient characteristics and involved pathogens. To embrace this complexity, we aimed to explore the underlying biological mechanisms of HBO2 treatment in patients with NSTI on the gene expression level. METHODS: We conducted an observational cohort study on prospective collected data, including 85 patients admitted to the intensive care unit (ICU) for NSTI. All patients were treated with one or two HBO2 treatments and had one blood sample taken before and after the intervention. Total RNAs from blood samples were extracted and mRNA purified with rRNA depletion, followed by whole-transcriptome RNA sequencing with a targeted sequencing depth of 20 million reads. A model for differentially expressed genes (DEGs) was fitted, and the functional aspects of the obtained set of genes was predicted with GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of genes and Genomes) enrichment analyses. All analyses were corrected for multiple testing with FDR. RESULTS: After sequential steps of quality control, a final of 160 biological replicates were included in the present study. We found 394 protein coding genes that were significantly DEGs between the two conditions with FDR < 0.01, of which 205 were upregulated and 189 were downregulated. The enrichment analysis of these DEGs revealed 20 GO terms in biological processes and 12 KEGG pathways that were significantly overrepresented in the upregulated DEGs, of which the term; "adaptive immune response" (GO:0002250) (FDR = 9.88E-13) and "T cell receptor signaling pathway" (hsa04660) (FDR = 1.20E-07) were the most significant. Among the downregulated DEGs two biological processes were significantly enriched, of which the GO term "apoptotic process" (GO:0006915) was the most significant (FDR = 0.001), followed by "Positive regulation of T helper 1 cell cytokine production" (GO:2000556), and "NF-kappa B signaling pathway" (hsa04064) was the only KEGG pathway that was significantly overrepresented (FDR = 0.001). CONCLUSIONS: When one or two sessions of HBO2 treatment were administered to patients with a dysregulated immune response and systemic inflammation due to NSTI, the important genes that were regulated during the intervention were involved in activation of T helper cells and downregulation of the disease-induced highly inflammatory pathway NF-κB, which was associated with a decrease in the mRNA level of pro-inflammatory factors. TRIAL REGISTRATION: Biological material was collected during the INFECT study, registered at ClinicalTrials.gov (NCT01790698).

Investigating the Effects of Hyperbaric Oxygen Treatment in Necrotizing Soft Tissue Infection With Transcriptomics and Machine Learning (the HBOmic Study): Protocol for a Prospective Cohort Study With Data Validation.

BACKGROUND: Necrotizing soft tissue infections (NSTIs) are complex multifactorial diseases characterized by rapid bacterial proliferation and progressive tissue death. Treatment is multidisciplinary, including surgery, broad-spectrum antibiotics, and intensive care; adjunctive treatment with hyperbaric oxygen (HBO2) may also be applied. Recent advances in molecular technology and biological computation have given rise to new approaches to infectious diseases based on identifying target groups defined by activated pathophysiological mechanisms. OBJECTIVE: We aim to capture NSTI disease signatures and mechanisms and responses to treatment in patients that receive the highest standard of care; therefore, we set out to investigate genome-wide transcriptional responses to HBO2 treatment during NSTI in the host and bacteria. METHODS: The Effects of Hyperbaric Oxygen Treatment Studied with Omics (HBOmic) study is a prospective cohort study including 95 patients admitted for NSTI at the intensive care unit of Copenhagen University Hospital (Rigshospitalet), Denmark, between January 2013 and June 2017. All participants were treated according to a local protocol for management of NSTI, and biological samples were obtained and stored according to a standard operational procedure. In the proposed study, we will generate genome-wide expression profiles of whole-blood samples and samples of infected tissue taken before and after HBO2 treatment administered during the initial acute phase of infection, and we will analyze the profiles with unsupervised hierarchical clustering and machine learning. Differential gene expression will be compared in samples taken before and after HBO2 treatment (N=85), and integration of profiles from blood and tissue samples will be performed. Furthermore, findings will be compared to NSTI patients who did not receive HBO2 treatment (N=10). Transcriptomic data will be integrated with clinical data to investigate associations and predictors. RESULTS: The first participant was enrolled on July 27, 2021, and data analysis is expected to begin during autumn 2022, with publication of results immediately thereafter. CONCLUSIONS: The HBOmic study will provide new insights into personalized patient management in NSTIs. TRIAL REGISTRATION: ClinicalTrials.gov NCT01790698; https://clinicaltrials.gov/ct2/show/NCT01790698. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/39252.

Chromatin modifier HUSH co-operates with RNA decay factor NEXT to restrict transposable element expression.

Transposable elements (TEs) are widespread genetic parasites known to be kept under tight transcriptional control. Here, we describe a functional connection between the mouse-orthologous "nuclear exosome targeting" (NEXT) and "human silencing hub" (HUSH) complexes, involved in nuclear RNA decay and the epigenetic silencing of TEs, respectively. Knocking out the NEXT component ZCCHC8 in embryonic stem cells results in elevated TE RNA levels. We identify a physical interaction between ZCCHC8 and the MPP8 protein of HUSH and establish that HUSH recruits NEXT to chromatin at MPP8-bound TE loci. However, while NEXT and HUSH both dampen TE RNA expression, their activities predominantly affect shorter non-polyadenylated and full-length polyadenylated transcripts, respectively. Indeed, our data suggest that the repressive action of HUSH promotes a condition favoring NEXT RNA decay activity. In this way, transcriptional and post-transcriptional machineries synergize to suppress the genotoxic potential of TE RNAs.

Mesenchymal-epithelial crosstalk shapes intestinal regionalisation via Wnt and Shh signalling.

Organs are anatomically compartmentalised to cater for specialised functions. In the small intestine (SI), regionalisation enables sequential processing of food and nutrient absorption. While several studies indicate the critical importance of non-epithelial cells during development and homeostasis, the extent to which these cells contribute to regionalisation during morphogenesis remains unexplored. Here, we identify a mesenchymal-epithelial crosstalk that shapes the developing SI during late morphogenesis. We find that subepithelial mesenchymal cells are characterised by gradients of factors supporting Wnt signalling and stimulate epithelial growth in vitro. Such a gradient impacts epithelial gene expression and regional villus formation along the anterior-posterior axis of the SI. Notably, we further provide evidence that Wnt signalling directly regulates epithelial expression of Sonic Hedgehog (SHH), which, in turn, acts on mesenchymal cells to drive villi formation. Taken together our results uncover a mechanistic link between Wnt and Hedgehog signalling across different cellular compartments that is central for anterior-posterior regionalisation and correct formation of the SI.

Photochemotherapy Induces Interferon Type III Expression via STING Pathway.

DNA-damaging cancer therapies induce interferon expression and stimulate the immune system, promoting therapy responses. The immune-activating STING (Stimulator of Interferon Genes) pathway is induced when DNA or double-stranded RNA (dsRNA) is detected in the cell cytoplasm, which can be caused by viral infection or by DNA damage following chemo- or radiotherapy. Here, we investigated the responses of cutaneous T-cell lymphoma (CTCL) cells to the clinically applied DNA crosslinking photochemotherapy (combination of 8-methoxypsoralen and UVA light; 8-MOP + UVA). We showed that this treatment evokes interferon expression and that the type III interferon IFNL1 is the major cytokine induced. IFNL1 upregulation is dependent on STING and on the cytoplasmic DNA sensor cyclic GMP-AMP synthase (cGAS). Furthermore, 8-MOP + UVA treatment induced the expression of genes in pathways involved in response to the tumor necrosis factor, innate immune system and acute inflammatory response. Notably, a subset of these genes was under control of the STING-IFNL1 pathway. In conclusion, our data connected DNA damage with immune system activation via the STING pathway and contributed to a better understanding of the effectiveness of photochemotherapy.

A Two-Layered Targeting Mechanism Underlies Nuclear RNA Sorting by the Human Exosome.

Degradation of transcripts in human nuclei is primarily facilitated by the RNA exosome. To obtain substrate specificity, the exosome is aided by adaptors; in the nucleoplasm, those adaptors are the nuclear exosome-targeting (NEXT) complex and the poly(A) (pA) exosome-targeting (PAXT) connection. How these adaptors guide exosome targeting remains enigmatic. Employing high-resolution 3' end sequencing, we demonstrate that NEXT substrates arise from heterogenous and predominantly pA- 3' ends often covering kilobase-wide genomic regions. In contrast, PAXT targets harbor well-defined pA+ 3' ends defined by canonical pA site use. Irrespective of this clear division, NEXT and PAXT act redundantly in two ways: (1) regional redundancy, where the majority of exosome-targeted transcription units produce NEXT- and PAXT-sensitive RNA isoforms, and (2) isoform redundancy, where the PAXT connection ensures fail-safe decay of post-transcriptionally polyadenylated NEXT targets. In conjunction, this provides a two-layered targeting mechanism for efficient nuclear sorting of the human transcriptome.

A Functional Link between Nuclear RNA Decay and Transcriptional Control Mediated by the Polycomb Repressive Complex 2.

Pluripotent embryonic stem cells (ESCs) constitute an essential cellular niche sustained by epigenomic and transcriptional regulation. Any role of post-transcriptional processes remains less explored. Here, we identify a link between nuclear RNA levels, regulated by the poly(A) RNA exosome targeting (PAXT) connection, and transcriptional control by the polycomb repressive complex 2 (PRC2). Knockout of the PAXT component ZFC3H1 impairs mouse ESC differentiation. In addition to the upregulation of bona fide PAXT substrates, Zfc3h1-/- cells abnormally express developmental genes usually repressed by PRC2. Such de-repression is paralleled by decreased PRC2 binding to chromatin and low PRC2-directed H3K27 methylation. PRC2 complex stability is compromised in Zfc3h1-/- cells with elevated levels of unspecific RNA bound to PRC2 components. We propose that excess RNA hampers PRC2 function through its sequestration from DNA. Our results highlight the importance of balancing nuclear RNA levels and demonstrate the capacity of bulk RNA to regulate chromatin-associated proteins.

Global network of computational biology communities: ISCB's Regional Student Groups breaking barriers.

Regional Student Groups (RSGs) of the International Society for Computational Biology Student Council (ISCB-SC) have been instrumental to connect computational biologists globally and to create more awareness about bioinformatics education. This article highlights the initiatives carried out by the RSGs both nationally and internationally to strengthen the present and future of the bioinformatics community. Moreover, we discuss the future directions the organization will take and the challenges to advance further in the ISCB-SC main mission: "Nurture the new generation of computational biologists".

Differential regulation of RNA polymerase III genes during liver regeneration.

Mouse liver regeneration after partial hepatectomy involves cells in the remaining tissue synchronously entering the cell division cycle. We have used this system and H3K4me3, Pol II and Pol III profiling to characterize adaptations in Pol III transcription. Our results broadly define a class of genes close to H3K4me3 and Pol II peaks, whose Pol III occupancy is high and stable, and another class, distant from Pol II peaks, whose Pol III occupancy strongly increases after partial hepatectomy. Pol III regulation in the liver thus entails both highly expressed housekeeping genes and genes whose expression can adapt to increased demand.

Rapid Recapitulation of Nonalcoholic Steatohepatitis upon Loss of Host Cell Factor 1 Function in Mouse Hepatocytes

Host-cell factor 1 (HCF-1), encoded by the ubiquitously expressed X-linked gene Hcfc1, is an epigenetic coregulator important for mouse development and cell proliferation, including during liver regeneration. We used a hepatocyte-specific inducible Hcfc1 knock-out allele (called Hcfc1hepKO), to induce HCF-1 loss in hepatocytes of hemizygous Hcfc1hepKO/Y males by four days. In heterozygous Hcfc1hepKO/+ females, owing to random X-chromosome inactivation, upon Hcfc1hepKO allele induction, a 50/50 mix of HCF-1 positive and negative hepatocyte clusters is engineered. The livers with Hcfc1hepKO/Y hepatocytes displayed a 21-24-day terminal non-alcoholic fatty liver (NAFL) followed by non-alcoholic steatohepatitis (NASH) disease progression typical of severe NAFL disease (NAFLD). In contrast, in livers with heterozygous Hcfc1hepKO/+ hepatocytes, HCF-1-positive hepatocytes replaced HCF-1-negative hepatocytes and revealed only mild-NAFL development. Loss of HCF-1 led to loss of PGC1α protein, probably owing to its destabilization, and deregulation of gene expression particularly of genes involved in mitochondrial structure and function, likely explaining the severe Hcfc1 hepKO/Y liver pathology. Thus, HCF-1 is essential for hepatocyte function, likely playing both transcriptional and non-transcriptional roles. These genetically-engineered loss-of-HCF-1 mice can be used to study NASH as well as NAFLD resolution.

Cycles of gene expression and genome response during mammalian tissue regeneration.

BACKGROUND: Compensatory liver hyperplasia-or regeneration-induced by two-thirds partial hepatectomy (PH) permits the study of synchronized activation of mammalian gene expression, particularly in relation to cell proliferation. Here, we measured genomic transcriptional responses and mRNA accumulation changes after PH and sham surgeries. RESULTS: During the first 10-20 h, the PH- and sham-surgery responses were very similar, including parallel early activation of cell-division-cycle genes. After 20 h, however, whereas post-PH livers continued with a robust and coordinate cell-division-cycle gene-expression response before returning to the resting state by 1 week, sham-surgery livers returned directly to a resting gene-expression state. Localization of RNA polymerase II (Pol II), and trimethylated histone H3 lysine 4 (H3K4me3) and 36 (H3K36me3) on genes dormant in the resting liver and activated during the PH response revealed a general de novo promoter Pol II recruitment and H3K4me3 increase during the early 10-20 h phase followed by Pol II elongation and H3K36me3 accumulation in gene bodies during the later proliferation phase. H3K36me3, generally appearing at the first internal exon, was preceded 5' by H3K36me2; 3' of the first internal exon, in about half of genes H3K36me3 predominated and in the other half H3K36me2 and H3K36me3 co-existed. Further, we observed some unusual gene profiles with abundant Pol II but little evident H3K4me3 or H3K36me3 modification, indicating that these modifications are neither universal nor essential partners to Pol II transcription. CONCLUSIONS: PH and sham surgical procedures on mice reveal striking early post-operatory gene expression similarities followed by synchronized mRNA accumulation and epigenetic histone mark changes specific to PH.

Segregated hepatocyte proliferation and metabolic states within the regenerating mouse liver.

Mammalian partial hepatectomy (PH) induces an orchestrated compensatory hyperplasia, or regeneration, in remaining tissue to restore liver mass; during this process, liver functions are maintained. We probed this process in mice with feeding- and light/dark-entrained animals subjected to sham or PH surgery. Early on (i.e., 10 hours), irrespective of sham or PH surgery, hepatocytes equidistant from the portal and central veins (i.e., midlobular) accumulated the G1-phase cell-division-cycle marker cyclin D1. By 24 hours, however, cyclin D1 disappeared absent PH but was reinforced in midlobular hepatocytes after PH. At 48 hours after PH and 2 hours fasting, synchronously mitotic hepatocytes possessed less glycogen than surrounding nonproliferating hepatocytes. The differential glycogen content generated a conspicuous entangled pattern of proliferating midlobular and nonproliferating periportal and pericentral hepatocytes. The nonproliferating hepatocytes maintained aspects of normal liver properties. Conclusion: In the post-PH regenerating mouse liver, a binary switch segregates midlobular cells to proliferate side-by-side with nonproliferating periportal and pericentral cells, which maintain metabolic functions. Our results also indicate that mechanisms of liver regeneration display evolutionary flexibility. (Hepatology Communications 2017;1:871-885).

A new gene-based association test for genome-wide association studies.

Genome-wide association studies are widely used today to discover genetic factors that modify the risk of complex diseases. Usually, these methods work in a SNP-by-SNP fashion. We present a gene-based test that can be applied in the context of genome-wide association studies. We compare both strategies, SNP-based and gene-based, in a sample of cases and controls for rheumatoid arthritis.We obtained different results using each strategy. The SNP-based test found the PTPN22 gene while the gene-based test found the PHF19-TRAF1-C5 region. That suggests that no single strategy performs better than another in all cases and that a certain underlying genetic architecture can be delineated more easily with one strategy rather than with another.