Serial Analysis Of Gene Expression Research Articles

Genetic Discrimination of Grade 3 and Grade 4 Gliomas by Artificial Neural Network.

Gliomas, including anaplastic gliomas (AG; grade 3) and glioblastomas (GBM; grade 4), are malignant brain tumors associated with poor prognosis and low survival rates. Current classification systems based on histopathology have limitations due to intratumoral heterogeneity. The treatment and prognosis are distinctly different between grade 3 and grade 4 gliomas patients. Therefore, there is a need for molecular markers to differentiate these tumors accurately. In this study, we aimed to identify a gene expression signature using an artificial neural network (ANN) in application to microarray and serial analysis of gene expression (SAGE) data for grade 3 (AG) and grade 4 (GBM) gliomas discrimination. We acquired gene expression data from publicly available datasets on glial tumors of grades 3 and 4-a total of 93 grade 3 gliomas and 224 grade 4 gliomas. To select genes for classification, we implemented an artificial neural network-based method using a combination of self-organized maps (SOM) and perceptron. In general, we implemented a multi-stage procedure that involved multiple runs of a genetic algorithm to identify genes that provided optimal clusterization on the SOM. We performed this procedure multiple times, resulting in different sets of genes each time. Eventually, we selected several genes that appeared most frequently in the reduced sets and performed classification using them. Our analysis identified a set of seven genes (BCAS4, GLUD2, KCNJ10, KCND2, AKR7A2, FOLR1, and KIAA0319). The classification accuracy using this gene set was 87.5%. These findings suggest the potential of this gene set as a molecular marker for distinguishing grade 3 (AG) from grade 4 (GBM) gliomas.

Molecular changes in hippocampal energy metabolism in mice selectively bred for extremes in stress reactivity: relevance of mitochondrial dysfunction for affective disorders.

Affective disorders, such as major depression, are frequently associated with metabolic disturbances involving mitochondria. Although dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is known to alter energy metabolism, the precise mechanisms linking stress and metabolic disturbances are not sufficiently understood. We used a mouse model of affective disorders to investigate the impact of a genetic predisposition for extremes in stress reactivity on behavioural and metabolic phenotypes as well as energy metabolism. Adult males of three independent mouse lines selectively bred for high, intermediate or low HPA axis reactivity were tested for exploratory and locomotor activity as well as stress-coping behaviour. Additionally, basal and stress-induced plasma corticosterone levels, body weight, food intake and body composition were measured. At the molecular level, the hippocampal transcriptome was analysed using microarray, serial analysis of gene expression and qRT-PCR. Finally, mitochondrial DNA copy number, damages and mitochondrial respiration were assessed. We found clear effects of the differential stress reactivity on the behavioural, morphometric and metabolic measures. Remarkably, the hyperactive behavioural and neuroendocrine stress-coping style of High Reactivity mice was associated with significant changes in the expression of an extended list of genes involved in energy metabolism and several mitochondrial functions. Yet, only minor changes were found in mitochondrial DNA copy number, damages and respiration. Thus, our findings support a prominent role of glucocorticoids in shaping the major endophenotypes of the stress reactivity mouse model and contribute towards understanding the important role of HPA axis dysregulation and changes in energy metabolism in the pathophysiology of affective disorders.

MCAM+CD161- Th17 Subset Expressing CD83 Enhances Tc17 Response in Psoriasis.

Recent studies have highlighted the pathogenic roles of IL-17-producing CD8+ T cells (T-cytotoxic 17 [Tc17]) in psoriasis. However, the underlying mechanisms of Tc17 induction remain unclear. In this study, we focused on the pathogenic subsets of Th17 and their mechanism of promotion of Tc17 responses. We determined that the pathogenic Th17-enriched fraction expressed melanoma cell adhesion molecule (MCAM) and CCR6, but not CD161, because this subset produced IL-17A abundantly and the presence of these cells in the peripheral blood of patients has been correlated with the severity of psoriasis. Intriguingly, the serial analysis of gene expression revealed that CCR6+MCAM+CD161-CD4+ T cells displayed the gene profile for adaptive immune responses, including CD83, which is an activator for CD8+ T cells. Coculture assay with or without intercellular contact between CD4+ and CD8+ T cells showed that CCR6+MCAM+CD161-CD4+ T cells induced the proliferation of CD8+ T cells in a CD83-dependent manner. However, the production of IL-17A by CD8+ T cells required exogenous IL-17A, suggesting that intercellular contact via CD83 and the production of IL-17A from activated CD4+ T cells elicit Tc17 responses. Intriguingly, the CD83 expression was enhanced in the presence of IL-15, and CD83+ cells stimulated with IL-1β, IL-23, IL-15, and IL-15Rα did not express FOXP3. Furthermore, CCR6+MCAM+CD161-CD4+ T cells expressing CD83 were increased in the peripheral blood of patients, and the CD83+ Th17-type cells accumulated in the lesional skin of psoriasis. In conclusion, pathogenic MCAM+CD161- Th17 cells may be involved in the Tc17 responses via IL-17A and CD83 in psoriasis.

Next generation sequencing of transcribed genes in ruminant γδ T cell populations

Bovine γδ T cells are distinguished by expression of WC1, hybrid pattern recognition receptors and co-receptors to the T cell receptor (TCR), or their absence. WC1 molecules bind pathogens and the ability of γδ T cells to respond to pathogens largely correlates with their expression of particular WC1 genes. Following activation, the TCR and WC1 molecules co-localize and knocking down WC1 abrogates the ability of WC1-expressing γδ T cells to respond to antigen. It is known that these two major populations, WC1+ and WC1-, differ in their TCR gene expression and previous studies showed other differences using semi-quantitative RT-PCR and serial analysis of gene expression. Differences in genes expressed would influence the functional outcome when WC1+ vs. WC1- γδ T cells respond to pathogens. To identify unique aspects of their transcriptome, here we performed RNA-Seq of flow cytometrically sorted bovine WC1+ and WC1- γδ T cells and compared them to all mononuclear cells in blood. The greatest differences in gene expression were found between γδ T cells and other mononuclear cells and included those involved in lymphocyte activation and effector processes. Only minor differences occurred between ex vivo WC1+ vs. WC1- γδ T cells with those gene products being involved in cell adhesion and chemotaxis. After culturing cells from primed animals with Leptospira antigens major difference in the transcriptome was evident, with over 600 genes significantly differentially expressed including those focused on cytokine signaling. Unexpectedly, antigen-responding and non-responding populations of WC1+ γδ T cells had few differences in their transcriptomes outside of cytotoxic factors although they had more WC1–1, WC1–2 and WC1–13 transcripts. Through differential gene expression we were able to define properties of ex vivo and stimulated WC1+ cells which will be useful in understanding their functional biology.

Evaluation of the Effects of Microgravity on Activated Primary Human Hepatic Stellate Cells.

In recent years, research has been conducted to develop new medical treatments by simulating environments existing in space, such as zero-gravity. In this study, we evaluated the cell proliferation and gene expression of activated primary human hepatic stellate cells (HHSteCs) under simulated microgravity (SMG). Under SMG, cell proliferation was slower than in 1 G, and the evaluation of gene expression changes on day 1 of SMG by serial analysis of gene expression revealed the presence of Sirtuin, EIF2 signaling, hippo signaling, and epithelial adherence junction signaling. Moreover, reactive oxygen species were upregulated under SMG, and when N-acetyl-cystein was added, no difference in proliferation between SMG and 1 G was observed, suggesting that the oxidative stress generated by mitochondrial dysfunction caused a decrease in proliferation. Upstream regulators such as smad3, NFkB, and FN were activated, and cell-permeable inhibitors such as Ly294002 and U0126 were inhibited. Immunohistochemistry performed to evaluate cytoskeletal changes showed that more β-actin was localized in the cortical layer under SMG.

Transcriptomics, Epigenetics, and Metabolomics of Primary Aldosteronism.

Simple SummaryImprovement in the understanding of the development of primary aldosteronism, the most common cause of endocrine hypertension and mainly caused by aldosterone producing adenomas or hyperplasia, has been continuously accomplished over the past several years. Herein, we summarize the major milestones in the field, including utilization of the newest available molecular techniques to not only shed light on the mechanisms involved in disease development but also to assist in the identification of disease subtypes with distinct laboratory and molecular findings, enabling the personalized treatment of the patients.Introduction: Primary aldosteronism (PA) is the most common cause of endocrine hypertension, mainly caused by aldosterone-producing adenomas or hyperplasia; understanding its pathophysiological background is important in order to provide ameliorative treatment strategies. Over the past several years, significant progress has been documented in this field, in particular in the clarification of the genetic and molecular mechanisms responsible for the pathogenesis of aldosterone-producing adenomas (APAs). Methods: Systematic searches of the PubMed and Cochrane databases were performed for all human studies applying transcriptomic, epigenetic or metabolomic analyses to PA subjects. Studies involving serial analysis of gene expression and microarray, epigenetic studies with methylome analyses and micro-RNA expression profiles, and metabolomic studies focused on improving understanding of the regulation of autonomous aldosterone production in PA were all included. Results: In this review we summarize the main findings in this area and analyze the interplay between primary aldosteronism and several signaling pathways with differential regulation of the RNA and protein expression of several factors involved in, among others, steroidogenesis, calcium signaling, and nuclear, membrane and G-coupled protein receptors. Distinct transcriptomic and metabolomic patterns are also presented herein, depending on the mutational status of APAs. In particular, two partially opposite transcriptional and steroidogenic profiles appear to distinguish APAs carrying a KCNJ5 mutation from all other APAs, which carry different mutations. Conclusions: These findings can substantially contribute to the development of personalized treatment in patients with PA.

Analysis of the P53N a Novel Protein Encoded on Chromosome 22q12.1-12.3 in Glioblastomas and Ependymomas Specimens

The P53N gene maps precisely to human chromosome sub-band 22q12.1-12.3, a region where loss of heterozygosity has been reported in 30% of astrocytic tumors and associated with progression to anaplasia. Moreover, a putative tumor suppressor gene has been indicated on 22q11 region involved in pathogenesis of ependymal tumors. Our objectives to examine the expression level of novel membrane-associated protein (termed P53N) encoded by a novel human gene on chromosome 22q12.1-12.3 in glioblastomas and ependymomas. Serial analysis of gene expression (SAGE) and immunofluorescence analysis of the P53N in the brain tumor tissues were performed. Our analysis revealed that there was high expression of the P53N mRNA in brain ependymoma and brain well-differentiated astrocytoma libraries. The P53N protein. P53N protein contains a high mobility group (HMG) domain at amino acid positions 301 to 360 expressed highly in glioblastoma and ependymoma specimens. Anti-P53N carboxyl-terminal peptide antibody localized the P53N protein to the cytoplasmic membranes of protoplasmic astrocytes in the glioblastoma and ependymoma specimens. These results are in good agreement with the SAGE analysis and the predicted transmembrane topology for the P53N protein and support a possible transmembrane model in which the P53N contains a predicted transmembrane region with its amino terminus localized to the inside of the cytoplasmic membrane.

A broad introduction to RNA-Seq

RNA-Seq, named as an abbreviation of "RNA sequencing" and sometimes spelled RNA-seq, RNAseq, or RNASeq, uses next-generation sequencing (NGS) to reveal the presence and quantity of ribonucleic acid (RNA) in a biological sample at a given moment.[1][2] RNA-Seq is used to analyze the continuously changing cellular transcriptome (Figure 1). Specifically, RNA-Seq facilitates the ability to look at alternative gene spliced transcripts, post-transcriptional modifications, gene fusion, mutations/single nucleotide polymorphisms (SNPs) and changes in gene expression over time, or differences in gene expression in different groups or treatments.[3] In addition to messenger RNA (mRNA) transcripts, RNA-Seq can look at different populations of RNA to include total RNA, small RNA, such as microRNA (miRNA), transfer RNA (tRNA), and ribosomal profiling.[4] RNA-Seq can also be used to determine exon/intron boundaries and verify or amend previously annotated 5' and 3' gene boundaries. Recent advances in RNA-Seq include single cell sequencing, in situ sequencing of fixed tissue, and native RNA molecule sequencing with single-molecule real-time sequencing.[5] Prior to RNA-Seq, gene expression studies were done with hybridization-based microarrays. Issues with microarrays include cross-hybridization artifacts, poor quantification of lowly and highly expressed genes, and needing to know the sequence a priori.[6] Because of these technical issues, transcriptomics transitioned to sequencing-based methods. These progressed from Sanger sequencing of Expressed Sequence Tag libraries, to chemical tag-based methods (e.g., serial analysis of gene expression), and finally to the current technology, next-gen sequencing of complementary DNA ( cDNA), notably RNA-Seq.

Identification of CCN1 transferred by extracellular vesicles derived from breast cancer cells responsible for doxorubicin-resistance.

e12623 Background: The anthracycline drug, doxorubicin, is one of the most frequently used chemotherapeutic drugs for breast cancer. However, not all patients respond to doxorubicin-containing combination chemotherapy due to chemo-resistance (natural or acquired). Preceding researches have shown that extracellular vesicles (EVs) can be readily harvested from peripheral blood for further biological analysis and thus perform an attractive source of tumor derived materials for identification of chemo-resistance factors. This study aims to provide a theoretical basis to use CCN1-containing EVs as new biomarkers and therapeutic targets for doxorubicin-resistance in breast cancer. Methods: A strategy combining RNA-sequencing technique and bioinformatic analysis was used to determine the level of differential expressed mRNAs in EVs samples from cellular supernatant (n = 4) and blood serum (n = 8) from chemo-resistant and chemo-sensitive patients. Serial gene expression analysis in patients (n = 203) who treated with neoadjuvant (GSE32603) were also employed to identified drug-resistance-related proteins. The expression of CCN1 was detected in doxorubicin-resistant cell lines (MCF-7/DOX),MCF-7 and their EVs by RT-qPCR and western blot. Then, we used cell apoptosis and immunofluorescence stain to confirm the ability of CCN1-containing EVs in conferring drug resistance. Immunohistochemistry stain and RT-qPCR were used to prove the expression level of CCN1 in tumor tissues from breast cancer patients (n = 80) and in serum EVs from patients treated with the whole neoadjuvant chemotherapy. Results: We combined the differentially expressed mRNAs from three datasets and found identified CCN1 as a drug-resistance-related factor in breast cancer. The expression level of CCN1 is much higher in MCF-7/DOX cells and their EVs compared with MCF-7 cells and their EVs. The addition of CCN1-containing EVs has been found significantly increase the resistance to doxorubicin in sensitive cells. Moreover, a higher expression level of CCN1 was detected in the progressive disease (PD)/stable disease (SD) group than in the partial response (PR)/complete response (CR) group both in tissues and serum EVs. Conclusions: All These findings demonstrate that CCN1-containing EVs appear to be essential in conferring resistance to doxorubicin. More importantly, we exhibit the potential applications of CCN1-containing EVs for predicting the efficiency and outcome of patients treated with doxorubicin-based chemotherapy.

Analysis of P78: A Novel Cytoplasmic Membrane-Associated Protein Encoded on Chromosome 19q13.3 in Glioma Specimens.

Allelic losses of the q13.3 region of chromosome 19 have been documented in all major types of diffuse gliomas, strongly suggesting the presence of a 19q13.3 tumor suppressor gene responsible for these malignancies. The P78 gene precisely maps to 19q13.3, the glioma candidate region, and encodes a recently identified novel protein (P78). The purpose of this study was to determine P78 protein expression in gliomas. Serial analysis of gene expression (SAGE) reveals P78 mRNA expression to be significantly reduced in high-grade gliomas such as glioblastoma (GB), as compared with the low-grade tumors including astrocytomas, oligodendrogliomas, and ependymomas. We observed the distribution of staining of P78 protein was concentrated on the cell membranes of the luminal epithelial cells, not cytoplasm. In contrast, the pre-immune serum controls demonstrated no staining. These results demonstrate that P78 protein is highly expressed in the cytoplasmic membranes of low but not high-grade astrocytomas, and correlates with grade of malignancy. In these double immunostaining experiments, the anti-Map-2 and anti-NeuN antibodies did not stain round cells that were stained with the anti-P78 carboxyl-terminal peptide antibodies, demonstrating that these round cells were not neurons, and likely protoplasmic astrocytes. Current results also suggest that the astrocytes stained with the anti-P78 carboxyl-terminal peptide antibody are likely protoplasmic astrocytes. We also observed preincubation of anti-P78 carboxyl-terminal antibodies with immunizing peptides abolished immunostaining in gliomas. These results suggest a role for the P78 protein in the process of abnormal growth in glial tumors.

Integrative Analysis of Siglec-15 mRNA in Human Cancers Based on Data Mining.

Objective: Cancer is expected to be the leading cause of death worldwide within the 21st century and is the single most important obstacle to extending life expectancy. Unfortunately, the most effective approach to combating cancers remains a complex and unsolved problem. Siglec-15 is a member of the Siglec family and plays a conserved regulatory role in the immune system of vertebrates. Previous studies on Siglec-15 have focused on its function in osteoclast regulation. The purpose of this study was to explore the significance of Siglec-15 mRNA in human cancer mainly based on information obtained from online databases.Method: Data were collected from several online databases. Serial analysis of gene expression (SAGE) and Virtual Northern, UALCAN Database Analysis, Catalog of Somatic Mutations in Cancer (COSMIC) analysis, the cBio cancer genomics portal, Cancer Regulome tools and data, Kaplan-Meier Plotter Analysis and the UCSC Xena website were used to analyze the data.Results: Compared with normal tissues, Siglec-15 up-regulation was widely observed in tuomrs. Differences in Siglec-15 expression were associated with different prognoses. Siglec-15 mutations are widely observed in tumors and interact with different genes in different cancer types.Conclusion: Siglec-15 is a potential target for the expansion of cancer immunotherapy.

Prognostic Significance of Prostaglandin-Endoperoxide Synthase-2 Expressions in Human Breast Carcinoma: A Multiomic Approach.

Prostaglandin-endoperoxide synthase-2 (PTGS2) plays a pivotal role in inflammation and carcinogenesis in human breast cancer. Our aim of the study is to find the prognostic value of PTGS2 in breast cancer. We conducted a multiomic analysis to determine whether PTGS2 functions as a prognostic biomarker in human breast cancer. We explored PTGS2 mRNA expressions using different public bioinformatics portals. Oncomine, Serial Analysis of Gene Expression (SAGE), GEPIA, ULCAN, PrognoScan database, Kaplan-Meier Plotter, bc-GenExMiner, USC XENA, and Cytoscape/STRING DB were used to identify the prognostic roles of PTGS2 in breast cancer. Based on the clinicopathological analysis, decreased PTGS2 expressions correlated positively with older age, lymph node status, the human epidermal growth factor receptor 2 (HER2) status (P < .0001), estrogen receptor (ER+) expression (P < .0001) Luminal A (P < .0001), and Luminal B (P < .0001). Interestingly, progesterone receptor (PR) (P < .0001) negative showed a high expression of PTGS2. Prostaglandin-endoperoxide synthase-2 was downregulated in breast cancer tissues than in normal tissues. In the PrognoScan database and, Kaplan-Meier Scanner, downregulated expressions of PTGS2 associated with poor overall survival (OS), relapse-free survival (RFS), and distant metastasis-free survival. The methylation levels were significantly higher in the Luminal B subtype. Through oncomine coexpressed gene analysis, we found a positive correlation between PTGS2 and interleukin-6 (IL-6) expression in breast cancer tissues. These results indicate that downregulated expressions of PTGS2 can be used as a promising prognostic biomarker and Luminal B hyper methylation may play an important role in the development of breast cancers. However, to clarify our results, extensive study is required.

Dynamic Regulation of SARS-CoV-2 Binding and Cell Entry Mechanisms in Remodeled Human Ventricular Myocardium

Objectives: In intact Nonfailing (NF) and failing/remodeled left ventricles (LVs) in patients with nonischemic dilated cardiomyopathies (F/NDC), measure gene expression of elements required for SARS-CoV-2 cell binding and entry, to determine: 1) whether the CoV-2 binding cell membrane receptor ACE2 is up-regulated; 2) if host cell proteases required for CoV-2 cell entry are expressed and/or up-regulated; and 3) whether ACE2 interacting integrins that may play a role in viral internalization are present and upregulated.Background: Cardiac involvement in COVID-19 infection appears to worsen outcomes, but to date there is no histopathologic evidence of cardiac myocyte CoV-2 involvement and no information on whether myocardial cells contain the biologic machinery for CoV-2 binding and cell entry.Methods: We measured serial analysis of gene expression in 46 F/NDC patients, using serial endomyocardial biopsies for RNA extraction and mRNA measurements by microarray and RNA-sequencing at baseline and after institution of beta-blocker therapy to effect reverse remodeling. Baseline measurements were compared to 4 NF controls.Results: ACE2 mRNA was upregulated vs. controls by nearly two-fold at baseline and then downregulated with reverse remodeling, five proteases previously shown to participate in SARS-Co-V or CoV-2 cell membrane fusion were expressed but not regulated with remodeling, and multiple integrins known to participate in viral endocytosis were expressed, with one (ITGA5) that binds to ACE2 and contains a CoV-2 binding motif upregulated in remodeling.Conclusions: Failing, remodeled human ventricular myocardium contains the biologic elements necessary for CoV-2 cell binding and entry, some of which are enhanced in remodeling.Funding: This work was supported by NHLBI grants 2R01 HL48913 (awarded to MRB), 1R01 HL71118 (awarded to MRB and BDL), K23 HL068875 (awarded to BDL), K08 HL125725 (awarded to DPK), AHA grant 16SFRN31420008 (awarded to PMB and MRB), and an AHA COVID-19 Rapid Response grant awarded to MRB.Conflict of Interest: No author has any conflict of interest with any aspect of the data or its interpretation.Ethical Approval: All patients signed written consent for this multi-center study conducted at the University of Colorado Anschutz Medical Campus and the University of Utah Medical Center. The study included a Data and Safety Monitoring Board and was approved by the Institutional Review Boards at both sites.

Neutral endopeptidase inhibitors blunt kidney fibrosis by reducing myofibroblast formation.

Kidney fibrosis is the common pathophysiological mechanism in end-stage renal disease characterized by excessive accumulation of myofibroblast-derived extracellular matrix. Natriuretic peptides have been demonstrated to have cyclic guanosine monophosphate (cGMP)-dependent anti-fibrotic properties likely due to interference with pro-fibrotic tissue growth factor β (TGF-β) signaling. However, in vivo, natriuretic peptides are rapidly degraded by neutral endopeptidases (NEP). In a unilateral ureteral obstruction (UUO) mouse model for kidney fibrosis we assessed the anti-fibrotic effects of SOL1, an orally active compound that inhibits NEP and endothelin-converting enzyme (ECE). Mice (n=10 per group) subjected to UUO were treated for 1 week with either solvent, NEP-/ECE-inhibitor SOL1 (two doses), reference NEP-inhibitor candoxatril or the angiotensin II receptor type 1 (AT1)-antagonist losartan. While NEP-inhibitors had no significant effect on blood pressure, they did increase urinary cGMP levels as well as endothelin-1 (ET-1) levels. Immunohistochemical staining revealed a marked decrease in renal collagen (∼55% reduction, P<0.05) and α-smooth muscle actin (α-SMA; ∼40% reduction, P<0.05). Moreover, the number of α-SMA positive cells in the kidneys of SOL1-treated groups inversely correlated with cGMP levels consistent with a NEP-dependent anti-fibrotic effect. To dissect the molecular mechanisms associated with the anti-fibrotic effects of NEP inhibition, we performed a 'deep serial analysis of gene expression (Deep SAGE)' transcriptome and targeted metabolomics analysis of total kidneys of all treatment groups. Pathway analyses linked increased cGMP and ET-1 levels with decreased nuclear receptor signaling (peroxisome proliferator-activated receptor [PPAR] and liver X receptor/retinoid X receptor [LXR/RXR] signaling) and actin cytoskeleton organization. Taken together, although our transcriptome and metabolome data indicate metabolic dysregulation, our data support the therapeutic potential of NEP inhibition in the treatment of kidney fibrosis via cGMP elevation and reduced myofibroblast formation.

Identification of IFITM3 and MGAT1 as novel interaction partners of BRI3 by yeast two-hybrid screening.

BRI3 (brain protein I3) is one of the Wnt/β-catenin pathway target genes as indicated by the results of serial analysis of gene expression (SAGE) and microarray analyses performed in our laboratory. The Wnt/β-catenin signaling pathway is an evolutionarily conserved pathway, which has important functions in early vertebrate development, axis formation, cellular proliferation, and morphogenesis. Previous studies showed that BRI3 expression is upregulated at both mRNA and protein levels upon β-catenin activation by various approaches, such as lithium treatment and overexpression of Wnt ligands in Huh7 (hepatocellular carcinoma) cell lines. Moreover, with regard to the previous literature, BRI3 was found to have a very important role in the TNFα-mediated cell death pathway. In this study, we screened a human liver cDNA library by yeast two-hybrid assay using BRI3 protein as bait, with the aim of finding novel interaction partners of BRI3. Library screening by yeast mating resulted in the identification of three candidate positive clones. Among these, IFITM3 and MGAT1 proteins were confirmed as interaction partners by using cotransformation in yeast cells and coimmunoprecipitation from mammalian cell lines. Considering the poor functional characterization of BRI3 to date, identification of novel BRI3-interacting proteins is an essential first step in determining the action mechanism of BRI3 with respect to the Wnt/β-catenin pathway.

Quantification of gene expression while taking into account RNA alternative splicing

Gene expression has been widely used in functional genomics research; however, the gene expressions quantified with different methods have been frequently inconsistent, thus challenging the conclusions from such research. Here we have addressed this issue, while taking into account RNA alternative splicing. We found that when a gene was subjected to RNA alternative splicing, it was impossible or difficult to properly quantify the expression of a transcript of the gene or its overall expression using quantitative real-time PCR (qPCR), Northern hybridization, microarray, or serial analysis of gene expression. Shot-gun RNA-seq was the most proper to quantify the expression of a transcript or a gene in such cases. Moreover, the expressions of individual transcripts quantified by shot-gun RNA-seq were highly reproducible (r = 0.90–0.98) between individuals. Therefore, shot-gun or full-length RNA-seq should be the method of choice to properly quantify the expression of a transcript or a gene.

Exploring the molecular pathogenesis and biomarkers of high risk oral premalignant lesions on the basis of long noncoding RNA expression profiling by serial analysis of gene expression.

Oral premalignant lesions (OPLs) have malignant transformation potential, with no reliable markers available. This study aimed to assess molecular events to identify biomarkers that can reflect high-risk lesions as predictive factors to tailor clinical decision for patients on the basis of long noncoding RNAs (lncRNA) expression profiling by serial analysis of gene expression. The GSE31021 and GSE8127 datasets were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) and lncRNAs were identified using the LIMMA package in R language. The genes targeted by lncRNAs were predicted among screened DEGs using Pearson’s correlation. Gene ontology function and Kyoto Encyclopedia of Genes and Genomes pathway analyses were carried out for genes targeted by lncRNAs using the Database for Annotation, Visualization, and Integrated Discovery online tool. A total of 674 DEGs and differentially expressed lncRNAs were screened. Thirty-two interactions of 10 lncRNAs and 524 target genes were predicted. The lncRNA NEAT1 was among the top 10 lncRNAs. The coregulated target genes RP4-684O24, RP11-283I3, and RP11-350G8 were significantly enriched in the immune response and mannosyl-oligosaccharide mannosidase activity. The target genes coregulated by LINC00665 and MIR378D2 were significantly enriched in the ubiquitin-dependent protein catabolic process, ubiquitin-protein ligase activity, and neurotrophin signaling. The lncRNA NEAT1 may play an important role in high-risk lesions. The novel lncRNAs and DEGs identified in OPLs may mediate the immune response and neurotrophin signaling and show ubiquitin ligase activity. These results improve our understanding of the molecular pathogenesis of OPLs and identify some potential targets for early diagnosis of high risk OPLs.

Evaluation of the effects of ascorbic acid on metabolism of human mesenchymal stem cells

BackgroundMesenchymal stem cells (MSCs) are multipotent cells holding much promise for applications in regenerative medicine. However, with problems such as aging, increases in heteroploid cells, genomic instability, and reduced maintenance of stemness, more stable culturing methods and the production of MSCs with an improved therapeutic effect are desired. Ascorbic acid (AsA), which is a cofactor for a variety of enzymes and has an antioxidant effect, cannot be synthesized by certain animals, including humans. Nevertheless, little attention has been paid to AsA when culturing MSCs.MethodsWe analyzed the effect of adding AsA to the culture medium on the proliferation and metabolism of human MSCs by serial analysis of gene expression and metabolome analysis.ResultsWe found that AsA promotes MSC proliferation, and is particularly useful when expanding MSCs isolated from bone marrow. Serial analysis of gene expression and metabolome analysis suggested that, due to HIF1α accumulation caused by decreased activity of the enzymes that use AsA as a coenzyme in cultures without AsA, genes downstream of HIF1α are expressed and there is a conversion to a hypoxia-mimetic metabolism. AsA promotes HIF1α breakdown and activates mitochondria, affecting cell proliferation and metabolism. Comprehensive evaluation of the effects of AsA on various metabolic products in MSCs revealed that AsA increases HIF1α hydroxylase activity, suppressing HIF1a transcription and leading to mitochondrial activation.ConclusionsAdding AsA during MSC expansion leads to more efficient preparation of cells. These are expected to be important findings for the future application of MSCs in regenerative medicine.

SuperSAGE digital expression analysis of differential growth rate in a European sea bass population

One of the goals of the aquaculture industry is to understand and control growth associated traits through selective breeding. In the present study the molecular basis of growth heterogeneity in the European sea bass (Dicentrarchus labrax) was addressed. To establish growth heterogeneity in a group of hatchery bred sea bass individuals were tagged and their specific growth rates (SGR) determined at monthly intervals. Gene expression in the brain, liver and white muscle from fish with the most divergent sustained SGR (6 individuals of the first and last quartile) was assessed using SuperSAGE (Serial Analysis Gene Expression) combined with next generation SOLiD4 sequencing. A total of approx. 11 million edited tags (26 bp), on average 2 million tags per SAGE library, that represented 47.071 unique transcripts were identified. Comparison of transcripts in fish with high and low SGR yielded 344, 698 and 601 differently expressed tags (0.01% false discovery rate and 4-fold change) in brain, liver and muscle, respectively. The tags were mapped onto the sea bass genome and approximately one third of the tags could be assigned to annotated genes. Pathway enrichment analysis revealed in liver, muscle and brain intricate gene expression changes in endocrine regulatory pathways involved in growth, metabolic and the stress axis, underlying divergent SGR in sea bass.

Endogenous Selenoprotein P, a Liver-Derived Secretory Protein, Mediates Myocardial Ischemia/Reperfusion Injury in Mice.

Selenoprotein P (SeP), a liver-derived secretory protein, functions as a selenium supply protein in the body. SeP has been reported to be associated with insulin resistance in humans through serial analysis of gene expression. Recently, SeP has been found to inhibit vascular endothelial growth factor-stimulated cell proliferation in human umbilical vein endothelial cells, and impair angiogenesis in a mouse hind limb model. In this study, the role of SeP in ischemia/reperfusion (I/R) injury has been investigated. SeP knockout (KO) and littermate wild-type (WT) mice were subjected to 30 min of myocardial ischemia followed by 24 h of reperfusion. The myocardial infarct area/area at risk (IA/AAR), evaluated using Evans blue (EB) and 2,3,5-triphenyltetrazolium chloride (TTC) staining, was significantly smaller in SeP KO mice than in WT mice. The number of terminal de-oxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive nuclei was significantly lower in SeP KO mice than in WT mice. In addition, caspase-3 activation was reduced in SeP KO mice compared to that in WT mice. Furthermore, phosphoinositide 3-kinase/Akt and Erk levels were examined for the reperfusion injury salvage kinase (RISK) pathway. Interestingly, SeP KO significantly increased the phosphorylation of IGF-1, Akt, and Erk compared to that in WT mice after I/R. Finally, I/R-induced myocardial IA/AAR was significantly increased in SeP KO mice overexpressing SeP in the liver compared to other SeP KO mice. These results, together, suggest that inhibition of SeP protects the heart from I/R injury through upregulation of the RISK pathway.