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ObjectivesSystemic lupus erythematosus (SLE), an autoimmune disease with incompletely understood etiology, has a strong genetic component. Although genome-wide association studies (GWAS) have revealed multiple SLE susceptibility loci and associated single nucleotide polymorphisms (SNPs), the precise causal variants, target genes, cell types, tissues, and mechanisms of action remain largely unknown.MethodsHere, we report a comprehensive post-GWAS analysis using extensive annotation, molecular modeling, and integrative functional genomic and epigenomic analyses to optimize fine-mapping. We compile and cross-reference immune cell-specific expression quantitative trait loci (cis- andtrans-eQTLs) with promoter capture Hi-C, allele-specific chromatin accessibility, and massively parallel reporter assay data to define predisposing variants and target genes. To assess our predictions, we experimentally validate a locus using CRISPR/Cas9 genome editing, qPCR, and Western blot.ResultsAnchoring on 452 index SNPs, we selected 9,931 high-linkage disequilibrium (r2>0.8) SNPs and defined 182 independent non-HLA SLE loci. 3,746 SNPs from 143 loci were identified as regulating 564 unique genes. Target genes are enriched in lupus-related tissues and associated with other autoimmune diseases. Of these, 329 SNPs (106 loci) showed significant allele-specific chromatin accessibility or enhancer activity, indicating regulatory potential. Using CRISPR/Cas9, we validated rs57668933 as a functional variant regulating multiple targets, including SLE risk geneELF1, in B-cells.ConclusionWe demonstrate and validate post-GWAS strategies for utilizing multi-dimensional data to prioritize likely causal variants with cognate gene targets underlying SLE pathogenesis. Our results provide a catalog of significantly SLE-associated SNPs and loci, target genes, and likely biochemical mechanisms, to guide experimental characterization.

Genome-wide association studies have identified 2p13.1 as a prominent susceptibility locus for systemic lupus erythematosus (SLE)—a complex, multisystem autoimmune disease. However, the identity of underlying causal variant (s) and molecular mechanisms for increasing disease susceptibility are poorly understood. Using meta-analysis (cases = 10,252, controls = 21,604) followed by conditional analysis, bioinformatic annotation, and eQTL and 3D-chromatin interaction analyses, we computationally prioritized potential functional variants and subsequently experimentally validated their effects. Ethnicity-specific meta-analysis revealed striking allele frequency differences between Asian and European ancestries, but with similar odds ratios. We identified 20 genome-wide significant (p < 5 × 10−8) variants, and conditional analysis pinpointed two potential functional variants, rs6705628 and rs2272165, likely to explain the association. The two SNPs are near DGUOK, mitochondrial deoxyguanosine kinase, and its associated antisense RNA DGUOK-AS1. Using luciferase reporter gene assays, we found significant cell type- and allele-specific promoter activity at rs6705628 and enhancer activity at rs2272165. This is supported by ChIP-qPCR showing allele-specific binding with three histone marks (H3K27ac, H3K4me3, and H3K4me1), RNA polymerase II (Pol II), transcriptional coactivator p300, CCCTC-binding factor (CTCF), and transcription factor ARID3A. Transcriptome data across 28 immune cell types from Asians showed both SNPs are cell-type-specific but only in B-cells. Splicing QTLs showed strong regulation of DGUOK-AS1. Genotype-specific DGOUK protein levels are supported by Western blots. Promoter capture Hi-C data revealed long-range chromatin interactions between rs2272165 and several nearby promoters, including DGUOK. Taken together, we provide mechanistic insights into how two noncoding variants underlie SLE risk at the 2p13.1 locus.

We report a morphometric evaluation of α1,3-galactosyltransferase knockout (GTKO) pig heart and kidney ( n = 9) at the end of one, three and seven months. Organs parameters gradually increased with the age ( p

Porcine heart xenotransplantation is a potential treatment for patients with end-stage heart failure. To understand molecular mechanisms of graft rejection after heart transplantation, we transplanted a 31-day-old alpha-1,3-galactosyltransferase knockout (GTKO) porcine heart to a five-year-old cynomolgus monkey. Histological and transcriptome analyses were conducted on xenografted cardiac tissue at rejection (nine days after transplantation). The recipient monkey&rsquo, s blood parameters were analyzed on days &minus, 7, &minus, 3, 1, 4, and 7. Validation was conducted by quantitative real-time PCR (qPCR) with selected genes. A non-transplanted GTKO porcine heart from an age-matched litter was used as a control. The recipient monkey showed systemic inflammatory responses, and the rejected cardiac graft indicated myocardial infarction and cardiac fibrosis. The transplanted heart exhibited a total of 3748 differentially expressed genes compared to the non-transplanted heart transcriptome, with 2443 upregulated and 1305 downregulated genes. Key biological pathways involved at the terminal stage of graft rejection were cardiomyopathies, extracellular interactions, and ion channel activities. The results of qPCR evaluation were in agreement with the transcriptome data. Transcriptome analysis of porcine cardiac tissue at graft rejection reveals dysregulation of the key molecules and signaling pathways, which play relevant roles on structural and functional integrities of the heart.

Annals of surgical treatment and research 100(3), 127-136 (2021). doi:10.4174/astr.2021.100.3.127, Published by Korean Surgical Society, Seoul

Cancer informatics 19, 117693512096969 (2020). doi:10.1177/1176935120969696, Published by Sage Publications, Thousand Oaks, CA

We report a morphometric evaluation of

BACKGROUND: Intestinal organoids have evolved as potential molecular tools that could be used to study host-microbiome interactions, nutrient uptake, and drug screening. Gut epithelial barrier functions play a crucial role in health and diseases, especially in autoimmune diseases, such as inflammatory bowel diseases (IBDs), because they disrupt the epithelial mucosa and impair barrier function. METHODS: In this study, we generated an in vitro IBD model based on dextran sodium sulfate (DSS) and intestinal organoids that could potentially be used to assess barrier integrity. Intestinal organoids were long-term cultivated and characterized with several specific markers, and the key functionality of paracellular permeability was determined using FITC-dextran 4 kDa. Intestinal organoids that had been treated with 2 µM DSS for 3 h were developed and the intestinal epithelial barrier function was sequentially evaluated. RESULTS: The results indicated that the paracellular permeability represented epithelial characteristics and their barrier function had declined when they were exposed to FITC-dextran 4 kDa after DSS treatment. In addition, we analyzed the endogenous mRNA expression of pro-inflammatory cytokines and their downstream effector genes. The results demonstrated that the inflammatory cytokines genes significantly increased in inflamed organoids compared to the control, leading to epithelial barrier damage and dysfunction. CONCLUSION: The collective results showed that in vitro 3D organoids mimic in vivo tissue topology and functionality with minor limitations, and hence are helpful for testing disease models. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13770-020-00278-0) contains supplementary material, which is available to authorized users.

Phytocompounds are gaining popularity, due to lesser toxicity, greater bioavailability and high chemodiversity. Phytocompounds are evolving as new leads for the development of a novel drug. Phytocompounds exhibit a wide range of biological properties, and are being used as antioxidants, immunomodulatory, antimicrobial, cardiovascular, and anticancer drugs. However, their identification is still relatively limited. The major hurdle in the discovery of an efficient phytocompound is a complete dependence on time-consuming in vitro and in vivo screening systems. Alternatively, the computational drug discovery approach of using an online database and bioinformatics tools would be a cost-effective and time-saving option. Recent advancements in computational and structural biology have attributed the three-dimensional (3D) structure to many natural drug-like compounds and disease-related target macromolecules, and have been stored into renowned databases like Protein Data Bank (RCSB-PDB), DrugBank, and ZINC. The drug discovery filed is swiftly advancing with bioinformatics applications, and the availability of digitalized molecular data is paving new avenues in Computer-aided drug discovery (CADD) area. Virtual library screening (VLS), a widely recognized technique due to it being cost-effective, time-saving, and less laborious, evaluates drug candidates using computational tools, such as AutoDock Vina, GOLD, and Glide. This chapter provides comprehensive information about the available biological databases and bioinformatics tools that are useful for the analysis of plant-derived bioactive compounds and their molecular interactions in diseases. Applications of advanced bioinformatics tools and methods for designing, optimization, and high-throughput screening of phytocompounds are detailed. In addition, it emphasizes the advantages, limitations, challenges, and future perspectives of the computational approaches in analyzing phytocompound interactions.

Diseases of bowl wall mucosa stemming from sudden mutation lead to the development of colorectal cancer (CRC) cells by the transformation of normal epithelial cells into neoplastic lesions. CRC is considered to be a global burden; hence, its incidence rate is expeditiously increased up to ten-fold higher, worldwide. The epidemiological report pinpointed CRC as the utmost third common malignancy in men and second in women. Because of greater efficacy, the synthetic drugs are unsatisfactory due to higher toxic effects to the normal cells, and a chance of developing multidrug resistance by tumor cells. Therefore, dietary flavonoids with potent anticarcinogenic effects have been focused on recent investigations. Pelargonidin (PD), a bioactive molecule classified under anthocyanidin is present in red and pink pigmented berries. PD efficiently modulates intercellular antioxidant status, thereby reducing oxidative DNA damage, cellular proliferation, differentiation, apoptosis, angiogenesis, and reverse drug resistance of metastatic cells, and potentially induces cell cycle arrest, thereby interfering in colorectal carcinogenesis. PD scavenges and normalizes the intracellular reactive oxygen species (ROS), which results in gene mutation and induction of colon carcinogenesis. Therefore, the proliferation of tumor cells would be affected or blocked potentially due to disturbance in cell cycle protein by these ROS. Considering the wide pharmacological benefits of PD, this chapter deliberately reviews the cumulative research data from in vitro human colon cancer cell line studies on chemoprotective property of PD against CRC, and also summarizes the underlying mechanism in experimental models.

In this study, we attempted to upgrade GT(−MCP/−MCP) pig genetically to express MCP at a higher level and additionally thrombomodulin (TBM), which have respective roles as a complement regulatory protein and a coagulation inhibitor. We constructed a dicistronic cassette consisting of codon-optimized MCP (mMCP) and TBM (m-pI2), designed for ubiquitous expression of MCP and endothelium specific expression of TBM. The cassette was confirmed to allow extremely increased MCP expression compared with non-modified MCP, and an endothelial-specific TBM expression. We thus transfected m-pI2 into ear-skin fibroblasts isolated from a GT(−MCP/−MCP) pig. By twice selection using magnetically activated cell sorting (MACS), and single-cell culture, we were able to obtain clones over 90% expressing MCP. The cells of a clone were provided as a donor for nuclear transfer resulting in the generation of a GT(−MCP/−MCP)/mMCP/TBM pig, which was confirmed to be carrying cells expressing MCP and functioning as an inhibitor against the cytotoxic effect of normal monkey serum, comparable with donor cells. Collectively, these results demonstrated an effective approach for upgrading transgenic pig, and we assumed that upgraded pig would increase graft survival. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-020-2091-z) contains supplementary material, which is available to authorized users.