Human Gene Research Articles

Elucidation of molecular mechanisms, pathways, and diseases modulated by arsenicals through toxogenomics and multi-omics analysis

Arsenic compounds exist in inorganic and organic forms with inorganic form confirmed as a potent carcinogen. Toxogenomics and multi-omics analysis were used to explore the molecular mechanisms of carcinogenecity induced by arsenicals. Comparative toxogenomics revealed sodium arsenite and arsenate as the most toxic arsenicals to humans, interacting with various genes and altering gene expression through mRNA binding proteins. Both metalloids were classified as Class II toxins by the ProTox II prediction tool, with a lethal dose (LD50) of 149mg/kg body weight. The most frequently interacting genes were HMOX1, CAT, NFE2L2, CASP3, MAPK1, CXCL8, PARP1, TNF, and PYGM. Analysis of TCGA pan-cancer data revealed that 46% of hepatocellular carcinoma patients exhibited alterations in the genes HMOX1, CAT, NFE2L2, CASP3, MAPK1, CXCL8, PARP1, TNF, and PYGM, suggesting their significant role in the development of this disease. The alteration in the gene list decreased the overall patient survival but insignificantly in the Kaplan-Meier curves revealing insignificant role in survival. GSEA suggested significant enrichment of the gene list in pathways involved in the G2M checkpoint, apoptosis, hypoxia, TNFA signaling via NFKB, PI3K AKTMTOR signaling, P53, IFN gamma and inflammatory response pathways revealing the involvement of these pathways. Ten microRNAs (miRNAs) regulated the expressions of the genes involved in the above-mentioned pathways with the significant enrichment in miR-21-3p, miR-206 and mir486a-5p. The relevant pathway and graphical representation of the network of miRNA-target interactions identified by the enrichment analysis along with disease ontologies were predicted. This study will be helpful insight into setting of laboratory experiments.

Gene prioritization-based Active Bio-module identification for Bioinformatics

Massive multi-omics data are being used to research cancer pathogenesis at the molecular level as high-throughput sequencing technology advances. Many present approaches frequently fail to detect strongly coupled modules that are intimately associated with cancer. By combining two forms of omics data, a technique to active bio-module identification known as IdeMod is proposed, which employs gene expression and protein-protein interaction networks. IdeMod is a p-step random walk kernel regression model-based gene activity score algorithm that uses the Pareto optimum consensus (POC) method's dominance connections to generate a prioritised list of genes. IdeMod uses the SA GPROX simulated annealing technique to identify the PPI network's most linked and high-priority bio-modules. The techniques RegMod, LEAN, SigMod, ModFinder, and IdeMod were experimentally tested on real-world cervical and BRCA datasets. These findings show that the IdeMod algorithm may identify a densely linked module containing multiple genes that either promote or hinder tumour growth. The BRCA1 gene increases the likelihood of developing hereditary breast cancer associated with BRCA mutations. As a result, the IdeMod technique can be used in conjunction with other tools to detect bio-modules.

Revealing key proteins in comparison of tumor and para-tumor tissues in stage I esophageal squamous cell carcinoma: A combined gene expression clustering and protein interaction network analysis

Esophageal Squamous Cell Carcinoma (ESCC) which is diagnosed in advanced stages and metastasis to the vital organs, remained the sixth fatal malignancy among other types of cancer-related death. Considering the lack of specific clinical symptoms in the early development of the disease, finding suitable biomarkers and tissue-specific alterations for effective screening of ESCC is targeted in this project. Gene expression profiles of tumor tissue and para-tumors of stage I ESCC patients were retrieved from the Gene Expression Omnibus (GEO) database. The significant tissue-specific differentially expressed genes (DEGs) were identified and studied based on fold change distribution and the functional role of genes in the action map. A total number of 11,483 significant DEGs discriminated the tumor tissue from para-tumor tissue. Clustering analysis of significant DEGs led to identifying 220 DEGs as the final significant genes. Protein interaction network and action map analysis of the final gene list showed matrix metallopeptidase-9 (MMP9) as the critical gene related to the development of ESCC diseases in stage I. Significant expression change of MMP9 in esophageal carcinoma was validated in UALCAN (The University of Alabama at Birmingham Cancer Data Analysis Portal). This study highlighted the pivotal role of MMP9 in combination with SPP1, MMP13, and IL18 as a possible biomarkers panel in studies of tumor invasion and prognosis for stage I ESCC disease.

DDX3 is critical for female fertility via translational control in oogenesis

AbstractDEAD-box RNA helicase 3 (DDX3) and its homologs play a vital role in translation initiation by unwinding secondary structures of selected mRNAs. The human DDX3 gene is located on the sex chromosomes, so there are DDX3X and DDX3Y. DDX3X is ubiquitously expressed in almost all tissues and critical for embryonic development, whereas DDX3Y is only expressed in the testis and essential for male fertility. Drosophila belle (bel) is the single ortholog of DDX3, and mutations in bel cause male and female infertility. Using Drosophila bel mutants and Ddx3x conditional knockout (cKO) mice, we confirmed the pivotal role of DDX3 in female fertility and ovarian development. Drosophila bel mutants exhibited female infertility and immature egg chambers. Consistently, oocyte-specific Ddx3x knockout in mice resulted in female infertility and impaired oogenesis. We further found that immature egg chambers in Drosophila bel mutants and impaired follicular development in oocyte-specific Ddx3x cKO mice were caused by excessive apoptosis. We also identified a set of DDX3 target genes involved in oocyte meiosis and maturation and demonstrated that DDX3 is involved in their translation in human cells. Our results suggest that DDX3 is critical for female fertility via translational control in oogenesis.

The 30th anniversary of “The History and Geography of Human Genes”

On the occasion of the 30th anniversary of the publication of “The History and Geography of Human Genes”, the story of its origin and motivation is revisited by looking at nearly two decades of work that the book required. By describing details of activities that now have been abandoned thanks to technological advances, we try to communicate the irreplaceable importance of the motivational bases of research. We then look at the remarkable 30 years of the book’s life from its diffusion to the stability and longevity of its citation history, along with its inspirational impact on the scientific inquiries in many different fields.

Tissue-Specific Effects of Aging on Repeat-Mediated Mutation Hotspots In Vivo

Aging constitutes complex and dynamic alterations in molecular and physiological processes and is associated with numerous disorders, in part due to increased genetic instability. The aging population is projected to double by 2050, underscoring the urgent need to better understand the relationships between aging and age-related disorders. Repetitive DNA elements are intrinsic sources of genetic instability and have been found to co-localize with mutation hotspots in human cancer genomes. In this study, we explored the relationship between aging and DNA repeat-mediated genetic instability in vivo using an H-DNA-forming mirror-repeat sequence from the cancer-associated human c-MYC gene. Utilizing a unique mutation-reporter mouse model, we observed tissue-specific effects of aging on H-DNA-induced genetic instability, with mutation frequencies increasing in spleen tissues and remaining unchanged in testis tissues. Analysis of the mutation spectra revealed large deletion mutations as the primary contributor to increasing H-DNA-induced mutations, supported by increased cleavage activity of H-DNA structures in aged spleen tissues. Our findings demonstrate that aging has distinct tissue-specific effects on repeat-mediated, cancer-associated mutations, providing insights into the complex relationship between aging and cancer.

Engineering of Saccharomyces cerevisiae as a platform strain for microbial production of sphingosine-1-phosphate.

Sphingosine-1-phosphate (S1P) is a multifunctional sphingolipid that has been implicated in regulating cellular activities in mammalian cells. Due to its therapeutic potential, there is a growing interest in developing efficient methods for S1P production. To date, the production of S1P has been achieved through chemical synthesis or blood extraction, but these processes have limitations such as complexity and cost. In this study, we generated an S1P-producing Saccharomyces cerevisiae strain by using metabolic engineering and introducing a heterologous sphingolipid biosynthetic pathway to demonstrate the possibility of microbial S1P production. To construct the sphingosine-producing S. cerevisiae strain, both the sphingolipid delta 4 desaturase gene (DES1) and the alkaline ceramidase gene (ACER1) derived from Homo sapiens were introduced into the genome of S. cerevisiae by deleting the dihydrosphingosine phosphate lyase gene (DPL1) and the sphingoid long-chain base kinase gene (LCB5) to prevent S1P degradation and byproduct formation, respectively. The sphingosine-producing strain, DDLA, produced sphingolipids containing sphingosine. In flask fed-batch fermentation, the DDLA strain showed a higher production level of sphingosine under aerobic conditions with high initial cell density. The S1P-producing strain was generated by expressing the human sphingosine kinase gene (SPHK1) under the control of the inducible promoter, while deleting the ORM1 gene involved in the regulation of sphingolipid biosynthesis. The S1P-producing strain, DDLAOgS, exhibited the highest sphingosine production level under fed-batch fermentation in a bioreactor, achieving a 2.6-fold increase compared to flask fermentation. S1P biosynthesis in the DDLAOgS strain was verified by qualitative analysis using electrospray ionization mass spectrometry (ESI-MS). We successfully developed a metabolically engineered S. cerevisiae as a platform strain for microbial production of S1P by introducing an exogenous pathway of sphingolipids metabolism. The engineered yeast strains showed significant capabilities for sphingolipid production, including S1P. To our knowledge, this is the first report demonstrating that engineered S. cerevisiae can be a major platform strain for producing microbial S1P.

Transactivation of the novel 5' cis-acting element of mouse mammary tumor virus (MMTV) by human retroviral transactivators Tat and Tax.

The mouse mammary tumor virus (MMTV) encodes a 5' element crucial for transcription of its genome along with the Rem/Rem-responsive element (RmRE) responsible for nuclear export of this unspliced RNA. Whether the 5' element is Rem-responsive or has any functional interaction with host/viral factors to facilitate MMTV gene expression was tested in this study. Our results reveal that the 5' element is non-responsive to Rem, but can be transactivated by both HIV Tat and HTLV-1 Tax activators. Reciprocally, MMTV could transactivate not only HIV TAR (similar to HTLV Tax), but also its 5' element. Furthermore, we reveal involvement of pTEFb, a general elongation factor associated with transactivation by Tat/Tax. This makes MMTV the first betaretrovirus to encode both Rem/RRE and Tat/TAR-Tax/TRE-like transcription regulatory systems. This study should enhance not only our understanding of retrovirus replication and virally-induced cancers/immunodeficiency syndromes, but also development of improved retroviral vectors for human gene therapy.

Interplay of human ABCC11 transporter gene variants with axillary skin microbiome functional genomics.

The human armpit microbiome is metabolically entangled with skin cell physiology. This "meta-organism" symbiotic mutualism results in sweat either with or without odor (osmidrosis), depending on host ABCC11 gene haplotypes. Apocrine metabolism produces odorless S-glutathione conjugate that is transferred by ABCC11 transporters into secretory vesicles, deglutamylated to S-Cys-Gly-3M3SH thiol, and exuded to skin surface. An anthropogenic clade of skin bacteria then takes up the thiol and bioconverts it to malodorous 3-methyl-3-sulfanylhexan-1-ol (3M3SH). We hypothesized a familial meta-organism association of human ABCC11 gene non-synonymous SNP rs17822931 interplaying with skin microbiome 3M3SH biosynthesis. Subjects were genotyped for ABCC11 SNPs, and their haplotypes were correlated with axilla microbiome DNA sequencing profiles and predicted metagenome functions. A multigeneration family pedigree revealed a Mendelian autosomal recessive pattern: the C allele of ABCC11 correlated with bacterial Cys-S-conjugate β-lyase (PatB) gene known for Staphylococcus hominis biosynthesis of 3M3SH from human precursor; PatB was rescinded in hosts with homozygous TT alleles encoding ABCC11 loss-of-function mutation. We posit that a C allele encoding functional ABCC11 is key to delivering host conjugate precursors that shape heritable skin niche conditions favorable to harboring Staphylococcus having genomics of odor thiol production. This provides existential insights into human evolution and global regional population ancestries.

Abstract A005: APC mutations dysregulate alternative polyadenylation in cancer

Abstract Alternative polyadenylation (APA) affects most human genes and is recurrently dysregulated in cancers. However, the mechanistic origins of this dysregulation are incompletely understood. We completed an unbiased computational analysis of molecular regulators of poly(A) site selection across The Cancer Genome Atlas and identified that colorectal adenocarcinoma is distinct from all other cancer subtypes. We linked this distinction to the frequent presence of loss-of-function APC mutations in colorectal adenocarcinoma, which were strongly associated with expression of long 3′ UTRs relative to tumors lacking APC mutations. APC knockout similarly dysregulated APA in human colon organoids, and reduced APC expression was associated with APA dysregulation in tumor types lacking APC mutations. Building on previous work that identified APC as an RNA-binding protein that preferentially binds 3′ UTRs during mouse neurogenesis, we found that APC binding is most significantly enriched just upstream of proximal poly(A) sites within 3′ UTRs. Our results suggest that APC promotes proximal poly(A) site use and that APC loss contributes to pervasive APA dysregulation in human cancers. Citation Format: Austin M Gabel, Andrea E Belliville, James D Thomas, Jose Mario B Pineda, Robert K Bradley. APC mutations dysregulate alternative polyadenylation in cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A005.

Abstract PR015: Decoding RNA metabolism by RNA-linked CRISPR screening in human cells

Abstract RNAs undergo a complex choreography of metabolic processes in human cells that are regulated by thousands of RNA-associated proteins. While the effects of individual RNA-associated proteins on RNA metabolism have been extensively characterized, the full complement of regulators for most RNA metabolic events remain unknown. Here we present a massively parallel RNA-linked CRISPR (ReLiC) screening approach to measure the responses of diverse RNA metabolic events to knockout of 2,092 human genes encoding all known RNA-associated proteins. ReLiC screens highlight modular interactions between gene networks regulating splicing, translation, and decay of mRNAs. When combined with biochemical fractionation of polysomes, ReLiC reveals striking pathway-specific coupling between growth fitness and mRNA translation. Perturbing different components of the translation and proteostasis machineries have distinct effects on ribosome occupancy, while perturbing mRNA transcription leaves ribosome occupancy largely intact. Isoform-selective ReLiC screens capture differential regulation of intron retention and exon skipping by SF3b complex subunits. Chemogenomic screens using ReLiC decipher translational regulators upstream of mRNA decay and uncover a role for the ribosome collision sensor GCN1 during treatment with the anti-leukemic drug homoharringtonine. Our work demonstrates ReLiC as a versatile platform for discovering and dissecting regulatory principles of human RNA metabolism. Citation Format: Patrick J Nugent, Heungwon Park, Arvind Rasi Subramaniam. Decoding RNA metabolism by RNA-linked CRISPR screening in human cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr PR015.

Abstract A055: Tumor muscle invasion promotes tumor heterogeneity and normal muscle reprogramming

Abstract Aggressive human prostate tumors traverse a contractile smooth muscle pseudo-capsule, in a process termed extracapsular extension (ECE), defining the pT3 pathologic stage associating with increased biochemical recurrence, bone metastases, and cancer-specific mortality. While T3 lesions can be detected by non-invasive mpMRI imaging, how the tumor invades into and through the contractile muscle is unknown. Using a mouse xenograft model system of ECE, we investigated the transcriptomic consequences of human tumor invasion into and through the contractile smooth muscle of the mouse diaphragm. Both human and mouse gene transcripts were documented and analyzed. Tumor cells were intraperitoneally injected into male NSG mice and 6 weeks later, three tumor compartments (pre-invasive, muscle-invasive, and super-invasive cells that had reached the superior diaphragm surface) were analyzed for differential bulk human and mouse gene expression. Whole genome transcriptomic sequencing and GO pathway analysis revealed that approximately 414 human genes were differentially expressed between the non- invasive, muscle-resident, and super-invasive tumor populations and at least 5 enriched pathways were involved. The unique expression gene patterns of muscle-resident tumor cells were reversible when compared to the pre-invasive and super-invasive expression patterns. Significant increases in g-H2AX and nuclear deformation were observed in the muscle-resident tumor clusters as compared to the non-invasive tumor mass. No differences in tumor cell proliferation, as detected by Ki67 staining, were found between the tumor clusters in the three compartments. Immunohistochemistry staining for a damage response cytokeratin (KRT6A) and integrin (CD49f) revealed heterogeneity within the muscle resident tumors as compared to the non- invasive cells. Taken together, these data suggest a hostile contractile muscle environment elicits specific responses by the invading tumor. Single cell analysis within each of the tumor compartments is currently underway to define the spatial heterogeneity of gene expression in invasive tumor cell clusters within the contractile muscle. A dynamic reciprocity of the tumor/muscle microenvironment is suggested by the analysis of the bulk transcriptomic sequencing demonstrating a significant increase in mouse muscle bio-synthetic gene transcription as a consequence of the human tumor penetrating the tissue. Taken together, these data indicate that human tumors, during the act of traversing the contractile muscle layer, respond to this unique microenvironment by transiently altering transcription, while sustaining nuclear damage which results in the reprogramming of the muscle. This new information suggests that novel tumor or muscle biomarkers might indicate early muscle invasion events and assist new high-resolution image analysis for precision diagnostic and/or therapeutic decisions. (Supported in part by P30 CA23074; F30 CA143924, UACC Team Science Award). Citation Format: Kendra D Marr, Beatrice S Knudsen, Rafael Sainz, Kelvin W Pond, Noel E Warfel, Belinda E Sun, Anne E Cress. Tumor muscle invasion promotes tumor heterogeneity and normal muscle reprogramming [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A055.

Effect of PI3-kinase inhibitors on DNA double strand break repair pathways: observations using a site specific DSB induction system.

We established two types of site-specific DNA double strand breaks (DSB) induction systems to elucidate factors which affect the efficiency or quality of DSB repair. For mutation assays, a site specific DSB was generated by the transient expression of a zinc finger nuclease which targets the human HPRT1 gene. A cell line in which time and site specific DSBs can be generated in a HR reporter construct was used for homology-directed repair (HR) analysis. By using these two systems, we investigated the effects of PI3-kinase inhibitors on the efficiency and quality of DSB repair. Ataxia telangiectasia mutated (ATM) kinase inhibition resulted a decrease in mutant frequency and a slight increase in deletion-type mutations accompanied by microhomology. Furthermore, the HR frequency increased significantly when ATM kinase activity was inhibited. Thus, ATM kinase activity might be involved in the suppression of DSB end resection, and this may promote DSB repair through canonical non-homologous end joining.

RNA-Sequencing Identification of Genes Supporting HepG2 as a Model Cell Line for Hepatocellular Carcinoma or Hepatocytes

Background/Objectives: Cell lines do not faithfully replicate the authentic transcriptomic condition of the disease under study. The HepG2 cell line is widely used for studying hepatocellular carcinoma (HCC), but not all biological processes and genes exhibit congruent expression patterns between cell lines and the actual disease. The objective of this study is to perform a comparative transcriptomic analysis of the HepG2 cell line, HCC, and primary hepatocytes (PH) in order to identify genes suitable for research in HepG2 as a model for PH or HCC research. Methods: We conducted a differential expression analysis between publicly available data from HCC patients, PH, and HepG2. We examined specific overlaps of differentially expressed genes (DEGs) in a pairwise manner between groups in order to obtain a valuable gene list for studying HCC or PH using different parameter filtering. We looked into the function and druggability of these genes. Conclusions: In total, we identified 397 genes for HepG2 as a valuable HCC model and 421 genes for HepG2 as a valuable PH model, and with more stringent criteria, we derived a smaller list of 40 and 21 genes, respectively. The majority of genes identified as a valuable set for the HCC model are involved in DNA repair and protein degradation mechanisms. This research aims to provide detailed guidance on gene selection for studying diseases like hepatocellular carcinoma, primary hepatocytes, or others using cell lines.

12-Lipoxygenase inhibition delays onset of autoimmune diabetes in human gene replacement mice.

Type 1 diabetes (T1D) is characterized by the autoimmune destruction of insulin-producing beta cells and involves an interplay between beta cells and cells of the innate and adaptive immune systems. We investigated the therapeutic potential of targeting 12-lipoxygenase (12-LOX), an enzyme implicated in inflammatory pathways in beta cells and macrophages, using a mouse model in which the endogenous mouse Alox15 gene is replaced by the human ALOX12 gene. Our finding demonstrated that VLX-1005, a potent 12-LOX inhibitor, effectively delayed the onset of autoimmune diabetes in human gene replacement non-obese diabetic mice. By spatial proteomics analysis, VLX-1005 treatment resulted in marked reductions in infiltrating T and B cells and macrophages with accompanying increases in immune checkpoint molecule PD-L1, suggesting a shift towards an immune-suppressive microenvironment. RNA sequencing analysis of isolated islets and polarized proinflammatory macrophages revealed significant alteration of cytokine-responsive pathways and a reduction in interferon response after VLX-1005 treatment. Our studies demonstrated that the ALOX12 human replacement gene mouse provides a platform for the preclinical evaluation of LOX inhibitors and supports VLX-1005 as an inhibitor of human 12-LOX that engages the enzymatic target and alters the inflammatory phenotypes of islets and macrophages to promote the delay of autoimmune diabetes.

Abstract 4134551: Outstanding Research Award in Pediatric Cardiology: Cardiomyopathy-Associated Pathogenic Variants in Pediatric Myocarditis: A Study from the Pediatric Cardiomyopathy Registry

Background: Recent studies have demonstrated that patients with myocarditis (MC) may have a higher burden of cardiomyopathy (CM)-associated gene variants than the general population. However, data in children is limited. The Pediatric Cardiomyopathy Registry (PCMR), a large multi-center registry of children with CM, includes patients with dilated CM (DCM) secondary to myocarditis (MC). Hypothesis: We hypothesized patients with DCM secondary to MC in the PCMR have a higher burden of CM-associated gene variants than healthy controls, and clinical genetic testing in this population is rare. Aims: We aimed to: (1) calculate the prevalence of pathogenic (P) or likely pathogenic (LP) CM-associated variants in patients with DCM secondary to MC and compare to the prevalence in heart-healthy controls and (2) report the rate of clinical genetic testing in this population. Methods: The PCMR was queried to identify patients with DCM secondary to MC who underwent exome sequencing as part of a prior prospective study at 14 sites in North America. Controls from the Indiana University Biobank were matched 4:1 with cases based on genomic similarity. A curated gene list was comprised of 46 genes associated with CM. A second gene list included 102 genes associated with CM or MC in the ClinVar database. The prevalence of variants was compared using Chi-Square or Fisher’s exact tests. Results: A total of 32 patients were identified with DCM secondary to MC. Median enrollment age was 1.5 years (IQR 0.4-10.3) and 53% were female. There was no family history in 88%. The prevalence of P/LP variants from the curated list was 34.4% in cases compared to 4.7% in controls (p<0.001). The prevalence of P/LP variants from the ClinVar list was 31.3% in cases compared to 6.3% in controls (p<0.001). Variants were identified in 8 genes: ABCC9, ANKRD1, MYH6, MYH7, MYPN, PLN, TPM1 and TTN. No P/LP variants were found in desmosomal genes. The prevalence of variants was higher but not significantly different in older children compared to infants (45.0% vs 16.6%, p=0.1). The prevalence of variants in MC was similar to previously reported rates in all causes of DCM (34.4% vs 28.8%, p=0.5). Only 22% of cases received clinical genetic testing. Conclusion: Approximately 1 in 3 children with DCM secondary to MC carry a CM-associated P/LP variant, which is significantly higher than the prevalence in healthy controls. Given the high prevalence, routine genetic testing should be considered in this population.

Integration of genomic and transcriptomic layers in RNA-Seq data leads to protein interaction modules with improved Alzheimer's disease associations.

Alzheimer's disease (AD) is the most common neurodegenerative disease, and it is currently untreatable. RNA sequencing (RNA-Seq) is commonly used in the literature to identify AD-associated molecular mechanisms by analysing changes in gene expression. RNA-Seq data can also be used to detect genomic variants, enabling the identification of the genes with a higher load of deleterious variants in patients compared with controls. Here, we analysed AD RNA-Seq datasets to obtain differentially expressed genes and genes with a higher load of pathogenic variants in AD, and we combined them in a single list. We mapped these genes on a human protein-protein interaction network to discover subnetworks perturbed by AD. Our results show that utilizing gene pathogenicity information from RNA-Seq data positively contributes to the disclosure of AD-related mechanisms. Moreover, dividing the discovered subnetworks into highly connected modules reveals a clearer picture of altered molecular pathways that, otherwise, would not be captured. Repeating the whole pipeline with human metabolic network genes led to results confirming the positive contribution of gene pathogenicity information and enabled a more detailed identification of altered metabolic pathways in AD.

Effects of Glutamine or Glucose Deprivation on Inflammation and Tight Junction Disruption in Yak Rumen Epithelial Cells

Yak is a special free-ranging cattle breed in the plateau areas of Qinghai and Tibet. Pasture withering in cold-season pastures results in energy deficiency in yaks, which undermines the rumen epithelial barrier. However, the leading factor causing rumen epithelial injury remains unknown. Glutamine (Gln), a conditionally essential amino acid, is insufficient under pathological conditions. Glucose (GLU) is an important energy source. Thus, we explored the effects of Gln or GLU deprivation on the barrier function of yak rumen epithelial cells and investigated the underlying mechanisms, as well as the differences in rumen epithelial barrier function between Gln deprivation (Gln-D) and GLU deprivation (GLU-D). In previous work, we constructed the yak rumen epithelial cells (YRECs) line by transferring the human telomerase reverse transcriptase gene (hTERT) and simian virus 40 large T antigen (SV40T) into primary YRECs. The YRECs were exposed to normal, Gln-D, GLU-D, and serum replacement (SR) media for 6, 12, and 24 h. Our data displayed that cell viability and tight junction protein expression in the SR group were not significantly changed compared to the normal group. Whereas, compared with the SR group, Gln-D treated for more than 12 h reduced cell viability and proliferation, and GLU-D treated for more than 12 h damaged the cell morphology and reduced cell viability and proliferation. The cell proliferation and cell viability were decreased more in GLU-D than in Gln-D. In addition, Gln-D treated for more than 12 h disrupted YREC cellular partially tight junctions by inducing oxidative stress and inflammation, and GLU-D treated for more than 12 h disrupted YREC cellular tight junctions by inducing apoptosis, oxidative stress, and inflammation. Compared with Gln-D, GLU-D more significantly induced cell injury and reduced tight junction protein levels. Our results provided evidence that GLU-D induced damage through the p38 mitogen-activated protein kinase (p38 MAPK)/c-junN-terminal kinase (JNK) signaling pathway, which was more serious than Gln-D treated for more than 12 h.

CYP2C gene polymorphisms in North African populations.

Cytochrome P450 is a superfamily of genes generating hemoproteins that metabolize foreign chemicals as well as endogenous compounds, such as steroids. The human CYP2C genes (CYP2C8, CYP2C9, CYP2C18, CYP2C19) cluster on chromosome 10 and metabolize many clinically useful drugs. CYP2C19 and CYP2C9 have been the most studied while CYP2C8 has been studied less frequently. CYP2C18 has been relatively ignored until recently but its importance has begun to be recognized. We studied the genotypes of 7 pharmacogenetic markers in 3 CYP2C genes: CYP2C19 (rs12248560), CYP2C9 (rs4918758, rs1799853), and CYP2C8 (rs10509681, rs11572103, rs1058930, rs11572080), in one Libyan population and 7 Tunisian populations. Five of the 7 SNPs are in exons and have functional consequences while one intronic SNP is considered to be in close proximity to a regulatory region because of the many studies that report associations with metabolic effects. We carried out principal component analysis (PCA) on the North African populations and 83 other populations from the 1000 Genomes Project and Kidd Laboratory. The geographic clustering observed via PCA was more pronounced when considering multi-SNP haplotype frequencies. This studyreveals the intermediate position of North Africans between Europeans and Asians and the varied dissimilarities with other world regions. The genetic variation observed within and between geographic regions have implications for drug metabolism and adverse individual responses to medical treatments.

Effect of Culture Supernatant of Clostridium butyricum TO-A on Human DNA-Repair-Factor-Encoding Gene Promoters

In this study, Clostridium butyricum TO-A culture supernatant (CBCS) or butyric acid was added to a culture medium of human cervical carcinoma HeLa S3 cells, and changes in DNA-repair-related gene promoter activities were investigated. The HeLa S3 cells were transfected with a luciferase (Luc) expression vector containing approximately 500 bp of the 5′-upstream region of several human DNA-repair-related genes and cultured with a medium containing the CBCS (10%) or butyric acid (2.5 mM). The cells were harvested after 19 to 42 h of incubation. A Luc assay revealed that the human ATM, PARG, PARP1, and RB1 gene promoter activities were significantly increased. A Western blot analysis showed that the amounts of the proteins encoded by these genes markedly increased. Furthermore, 8, 24, and 48 h after the addition of the CBCS (10%), total RNA was extracted and subjected to RNAseq analysis. The results showed that the expression of several inflammation- and DNA-replication/repair-related genes, including NFKB and the MCM gene groups, decreased markedly after 8 h. However, the expression of the histone genes increased after 24 h. Elucidation of the mechanism by which the CBCS and butyrate affect the expression of genes that encode DNA-repair-associated proteins may contribute to the prevention of carcinogenesis, the risk of which rises in accordance with aging.