RNA Sequencing Analysis Between Ruptured and Un-Ruptured Brain AVM

BackgroundBrain arteriovenous malformation (BAVM) arises as congenital vascular abnormalities with a significant risk for intracerebral hemorrhage (ICH). RNA sequencing technology has been recently used to investigate the mechanism of diseases owing to its ability to identify the gene changes on a transcriptome-wide level. In this study, we aimed to gain insights into the potential mechanism involved in BAVM rupture. MethodsSixty-five BAVM nidus samples were collected among which 28 were ruptured and 37 were un-ruptured. Then next-generation RNA sequencing were performed on all of them to obtain differential expressed genes (DEGs) between these two groups. In addition, bioinformatics analysis was performed to evaluate the involved biological processes and pathways by GO and KEGG analysis. Finally, univariate Cox regression analysis was utilized to obtain the early rupture-prone DEGs. Results: A total of 951 genes were differentially expressed between ruptured and un-ruptured BAVM group, of which 740 genes were up-regulated and 211 genes were down-regulated in ruptured BAVMs. Then bioinformatics analysis showed the biological processes and pathways related to the inflammatory processes and extracellular matrix organization were significantly enriched. Meanwhile, some of down-regulated genes are involved in cell adhesion and genes participating in response to muscle activity, as well as the terms about nervous system development. Finally, one hundred and twenty-five genes, a large number of which were involved in inflammation, were correlated with the early rupture of BAVMs. Conclusions: The up-regulated genes in ruptured BAVM group were involved in inflammatory processes and extracellular matrix organization while some of the down-regulated genes were participating in cell adhesion and myofibril assembly, indicating the role of enhanced inflammation and reduced vessel strength in BAVMs rupture.

Endogenous Retroviruses Provide Protection Against Vaginal HSV-2 Disease

Endogenous retroviruses (ERVs) are genomic sequences that originated from retroviruses and are present in most eukaryotic genomes. Both beneficial and detrimental functions are attributed to ERVs, but whether ERVs contribute to antiviral immunity is not well understood. Here, we used herpes simplex virus type 2 (HSV-2) infection as a model and found that Toll-like receptor 7 (Tlr7-/-) deficient mice that have high systemic levels of infectious ERVs are protected from intravaginal HSV-2 infection and disease, compared to wildtype C57BL/6 mice. We deleted the endogenous ecotropic murine leukemia virus (Emv2) locus on the Tlr7-/- background (Emv2-/-Tlr7-/-) and found that Emv2-/-Tlr7-/- mice lose protection against HSV-2 infection. Intravaginal application of purified ERVs from Tlr7-/- mice prior to HSV-2 infection delays disease in both wildtype and highly susceptible interferon-alpha receptor-deficient (Ifnar1-/-) mice. However, intravaginal ERV treatment did not protect Emv2-/-Tlr7-/- mice from HSV-2 disease, suggesting that the protective mechanism mediated by exogenous ERV treatment may differ from that of constitutively and systemically expressed ERVs in Tlr7-/- mice. We did not observe enhanced type I interferon (IFN-I) signaling in the vaginal tissues from Tlr7-/- mice, and instead found enrichment in genes associated with extracellular matrix organization. Together, our results revealed that constitutive and/or systemic expression of ERVs protect mice against vaginal HSV-2 infection and delay disease.

AEBP1 is Upregulated in Diabetic Nephropathy Biopsies

Background: Worldwide, one in eleven adults have diabetes mellitus and 30% to 40% will develop diabetic kidney disease (DKD). A mechanistic understanding of DKD is crucial to develop treatment strategies. To unravel DKD’s pathogenesis, single cell (scRNA) sequencing has proven a powerful tool, but is limited by a lack of localization. Spatial transcriptomics allows the mapping of scRNA sequencing data back to histology. Methods: Frozen human nephrectomy and biopsy samples were processed according to Visium spatial gene expression protocols, stained with H&E, and imaged. Samples were permeabilized for RNA capture, reverse transcribed and sequenced on an Illumina NovaSeq 6000. Mapping and counting were completed in Space Ranger and data was processed in Seurat. Samples were laser microdissected, protein was isolated, and protein was quantified by HPLC-MS. Results: Clusters from scRNAseq were mapped upon reference and DKD spatial transcriptomic images (N=4 reference, 2 DKD). Differentially expressed genes were identified in diabetic kidneys, including the upregulation of Adipocyte Enhancer Binding Protein (AEBP1). Pathway analysis revealed enrichment of extracellular matrix organization and immune process pathways. To increase the confidence of these findings, glomeruli and the tubulointerstitium were laser microdissected (N=7 diseased, 4 reference) for proteomic analysis. AEBP1 was upregulated in the tubular interstitium of diseased kidneys and selectively upregulated in the glomeruli of Diabetic Nephropathy samples (N=2). AEBP1 localized to the interstitium by spatial transcriptomics and was expressed in highly fibrotic regions. Glomerular expression was not observed due to glomerulosclerosis. Conclusion: AEBP1 upregulation is a marker of interstitial fibrosis, with specific expression in the glomeruli of diabetic nephropathy specimens with glomerulosclerosis. Impact: This is the first study utilizing spatial transcriptomics to define and localize markers of human kidney disease. Confirmatory studies are required in larger sample sizes. AEBP1 is a previously unidentified marker of DKD previously associated with fibrosis in other organ-specific diseases.

Effect of NK-5962 on Gene Expression Profiling of Retina in a Rat Model of Retinitis Pigmentosa

Purpose: NK-5962 is a key component of photoelectric dye-coupled polyethylene film, designated Okayama University type-retinal prosthesis (OUReP™). Previously, we found that NK-5962 solution could reduce the number of apoptotic photoreceptors in the eyes of the Royal College of Surgeons (RCS) rats by intravitreal injection under a 12 h light/dark cycle. This study aimed to explore possible molecular mechanisms underlying the anti-apoptotic effect of NK-5962 in the retina of RCS rats. Methods: RCS rats received intravitreal injections of NK-5962 solution in the left eye at the age of 3 and 4 weeks, before the age of 5 weeks when the speed in the apoptotic degeneration of photoreceptors reaches its peak. The vehicle-treated right eyes served as controls. All rats were housed under a 12 h light/dark cycle, and the retinas were dissected out at the age of 5 weeks for RNA sequence (RNA-seq) analysis. For the functional annotation of differentially expressed genes (DEGs), the Metascape and DAVID databases were used. Results: In total, 55 up-regulated DEGs, and one down-regulated gene (LYVE1) were found to be common among samples treated with NK-5962. These DEGs were analyzed using Gene Ontology (GO) term enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome pathway analyses. We focused on the up-regulated DEGs that were enriched in extracellular matrix organization, extracellular exosome, and PI3K–Akt signaling pathways. These terms and pathways may relate to mechanisms to protect photoreceptor cells. Moreover, our analyses suggest that SERPINF1, which encodes pigment epithelium-derived factor (PEDF), is one of the key regulatory genes involved in the anti-apoptotic effect of NK-5962 in RCS rat retinas. Conclusions: Our findings suggest that photoelectric dye NK-5962 may delay apoptotic death of photoreceptor cells in RCS rats by up-regulating genes related to extracellular matrix organization, extracellular exosome, and PI3K–Akt signaling pathways. Overall, our RNA-seq and bioinformatics analyses provide insights in the transcriptome responses in the dystrophic RCS rat retinas that were induced by NK-5962 intravitreal injection and offer potential target genes for developing new therapeutic strategies for patients with retinitis pigmentosa.

Integrative epigenomics in Sjögren´s syndrome reveals novel pathways and a strong interaction between the HLA, autoantibodies and the interferon signature

Primary Sjögren’s syndrome (SS) is a systemic autoimmune disease characterized by lymphocytic infiltration and damage of exocrine salivary and lacrimal glands. The etiology of SS is complex with environmental triggers and genetic factors involved. By conducting an integrated multi-omics study, we confirmed a vast coordinated hypomethylation and overexpression effects in IFN-related genes, what is known as the IFN signature. Stratified and conditional analyses suggest a strong interaction between SS-associated HLA genetic variation and the presence of Anti-Ro/SSA autoantibodies in driving the IFN epigenetic signature and determining SS. We report a novel epigenetic signature characterized by increased DNA methylation levels in a large number of genes enriched in pathways such as collagen metabolism and extracellular matrix organization. We identified potential new genetic variants associated with SS that might mediate their risk by altering DNA methylation or gene expression patterns, as well as disease-interacting genetic variants that exhibit regulatory function only in the SS population. Our study sheds new light on the interaction between genetics, autoantibody profiles, DNA methylation and gene expression in SS, and contributes to elucidate the genetic architecture of gene regulation in an autoimmune population.

Proteomic Analysis of Mesenchymal Stromal Cell-Derived Extracellular Vesicles and Reconstructed Membrane Particles

Extracellular vesicles (EV) derived from mesenchymal stromal cells (MSC) are a potential therapy for immunological and degenerative diseases. However, large-scale production of EV free from contamination by soluble proteins is a major challenge. The generation of particles from isolated membranes of MSC, membrane particles (MP), may be an alternative to EV. In the present study we generated MP from the membranes of lysed MSC after removal of the nuclei. The yield of MP per MSC was 1 × 105 times higher than EV derived from the same number of MSC. To compare the proteome of MP and EV, proteomic analysis of MP and EV was performed. MP contained over 20 times more proteins than EV. The proteins present in MP evidenced a multi-organelle origin of MP. The projected function of the proteins in EV and MP was very different. Whilst proteins in EV mainly play a role in extracellular matrix organization, proteins in MP were interconnected in diverse molecular pathways, including protein synthesis and degradation pathways and demonstrated enzymatic activity. Treatment of MSC with IFNγ led to a profound effect on the protein make up of EV and MP, demonstrating the possibility to modify the phenotype of EV and MP through modification of parent MSC. These results demonstrate that MP are an attractive alternative to EV for the development of potential therapies. Functional studies will have to demonstrate therapeutic efficacy of MP in preclinical disease models.

Development of Prognostic Model Based on Five-gene Related to Tumor Mutation Burden in Hepatocellular Carcinoma

BackgroundHepatocellular carcinoma (HCC) , the incidence rate ranks sixth and regards as the second leading cause of cancer-related death in the world. Tumor mutation burden (TMB) , a novel biomarker featured with microsatellite instability, has been thought to be closely related to tumor microenvironment and immunotherapy.MethodsIn this study, the 357 HCC samples were download from The Cancer Genome Atlas (TCGA) and analyzed.ResultsSingle nucleotide polymorphism (SNP) and C > T variation had the most missense mutations, and we also observed the higher mutation frequency in TP53, TTN, CTNNB1. With an optimal cut-off value of 4.61, the high level of TMB presented a worse prognosis. Functional analysis of the differentially expressed genes (DEGs) showed they were enriched in the pathway of the formation of extracellular matrix organization and sulfur compound metabolic. A 5-gene-based (including SFRP4, IL7R, FBLN2, COLEC10 and CHGA) model has been constructed to explore the independent prognosis capacity of each HCC patient.ConclusionsThe establishment of 5-gene-based prognosis model functions well in predicting the median of 1- , 3- and 5-year outcome of the individual patient and which will be beneficial for pre-clinical diagnosis and therapeutic decision-making.

Distinct DNA Methylation Patterns of Subependymal Giant Cell Astrocytomas in Tuberous Sclerosis Complex

Tuberous sclerosis complex (TSC) is a monogenic disorder caused by mutations in either the TSC1 or TSC2 gene, two key regulators of the mechanistic target of the rapamycin complex pathway. Phenotypically, this leads to growth and formation of hamartomas in several organs, including the brain. Subependymal giant cell astrocytomas (SEGAs) are low-grade brain tumors commonly associated with TSC. Recently, gene expression studies provided evidence that the immune system, the MAPK pathway and extracellular matrix organization play an important role in SEGA development. However, the precise mechanisms behind the gene expression changes in SEGA are still largely unknown, providing a potential role for DNA methylation. We investigated the methylation profile of SEGAs using the Illumina Infinium HumanMethylation450 BeadChip (SEGAs n = 42, periventricular control n = 8). The SEGA methylation profile was enriched for the adaptive immune system, T cell activation, leukocyte mediated immunity, extracellular structure organization and the ERK1 & ERK2 cascade. More interestingly, we identified two subgroups in the SEGA methylation data and show that the differentially expressed genes between the two subgroups are related to the MAPK cascade and adaptive immune response. Overall, this study shows that the immune system, the MAPK pathway and extracellular matrix organization are also affected on DNA methylation level, suggesting that therapeutic intervention on DNA level could be useful for these specific pathways in SEGA. Moreover, we identified two subgroups in SEGA that seem to be driven by changes in the adaptive immune response and MAPK pathway and could potentially hold predictive information on target treatment response.

Genome Profiling of H3k4me3 Histone Modification in Human Adipose Tissue during Obesity and Insulin Resistance

Background: Adipose tissue (AT) dysfunction is involved in obesity-related comorbidities. Epigenetic alterations have been recently associated with AT deterioration in obesity conditions. In this work, we profiled the H3K4me3 histone mark in human AT, with special emphasis on the changes in the pattern of histone modification in obesity and insulin resistance (IR). Visceral AT (VAT) was collected and subjected to chromatin immunoprecipitation (ChIP) using anti-H3K4me3 antibody and then sequenced to obtain the H3K4me3 genome profile. Results: We found that most of the H3K4me3 enriched regions were located in gene promoters of pathways related to AT biology and function. H3K4me3 enrichment at gene promoters was strongly related to higher mRNA levels. Differentially expressed genes in AT of patients classified as non-obese, obese with low IR, and obese with high IR could be regulated by differentially enriched H3K4me3; these genes encoded for pathways that could in part explain AT functioning during obesity and insulin resistance (e.g., extracellular matrix organization, PPARG signaling or inflammation). Conclusions: In conclusion, we emphasize the importance of the epigenetic mark H3K4me3 in VAT dysfunction in obesity and IR. The understanding of such mechanisms could give rise to the development of new epigenetic-based pharmacological strategies to ameliorate obesity-related comorbidities.