Differential Coexpression Analysis Reveals Extensive Rewiring of Arabidopsis Gene Coexpression in Response to Pseudomonas syringae Infection

PubMed Central

Jiang, Zhenhong; Dong, Xiaobao; Li, Zhi-Gang; He, Fei; Zhang, Ziding

2016-01-01

Plant defense responses to pathogens involve massive transcriptional reprogramming. Recently, differential coexpression analysis has been developed to study the rewiring of gene networks through microarray data, which is becoming an important complement to traditional differential expression analysis. Using time-series microarray data of Arabidopsis thaliana infected with Pseudomonas syringae, we analyzed Arabidopsis defense responses to P. syringae through differential coexpression analysis. Overall, we found that differential coexpression was a common phenomenon of plant immunity. Genes that were frequently involved in differential coexpression tend to be related to plant immune responses. Importantly, many of those genes have similar average expression levels between normal plant growth and pathogen infection but have different coexpression partners. By integrating the Arabidopsis regulatory network into our analysis, we identified several transcription factors that may be regulators of differential coexpression during plant immune responses. We also observed extensive differential coexpression between genes within the same metabolic pathways. Several metabolic pathways, such as photosynthesis light reactions, exhibited significant changes in expression correlation between normal growth and pathogen infection. Taken together, differential coexpression analysis provides a new strategy for analyzing transcriptional data related to plant defense responses and new insights into the understanding of plant-pathogen interactions. PMID:27721457

Circular RNA and gene expression profiles in gastric cancer based on microarray chip technology.

PubMed

Sui, Weiguo; Shi, Zhoufang; Xue, Wen; Ou, Minglin; Zhu, Ying; Chen, Jiejing; Lin, Hua; Liu, Fuhua; Dai, Yong

2017-03-01

The aim of the present study was to screen gastric cancer (GC) tissue and adjacent tissue for differences in mRNA and circular (circRNA) expression, to analyze the differences in circRNA and mRNA expression, and to investigate the circRNA expression in gastric carcinoma and its mechanism. circRNA and mRNA differential expression profiles generated using Agilent microarray technology were analyzed in the GC tissues and adjacent tissues. qRT-PCR was used to verify the differential expression of circRNAs and mRNAs according to the interactions between circRNAs and miRNAs as well as the possible existence of miRNA and mRNA interactions. We found that: i) the circRNA expression profile revealed 1,285 significant differences in circRNA expression, with circRNA expression downregulated in 594 samples and upregulated in 691 samples via interactions with miRNAs. The qRT-PCR validation experiments showed that hsa_circRNA_400071, hsa_circRNA_000543 and hsa_circRNA_001959 expression was consistent with the microarray analysis results. ii) 29,112 genes were found in the GC tissues and adjacent tissues, including 5,460 differentially expressed genes. Among them, 2,390 differentially expressed genes were upregulated and 3,070 genes were downregulated. Gene Ontology (GO) analysis of the differentially expressed genes revealed these genes involved in biological process classification, cellular component classification and molecular function classification. Pathway analysis of the differentially expressed genes identified 83 significantly enriched genes, including 28 upregulated genes and 55 downregulated genes. iii) 69 differentially expressed circRNAs were found that might adsorb specific miRNAs to regulate the expression of their target gene mRNAs. The conclusions are: i) differentially expressed circRNAs had corresponding miRNA binding sites. These circRNAs regulated the expression of target genes through interactions with miRNAs and might become new molecular biomarkers for GC in the future. ii) Differentially expressed genes may be involved in the occurrence of GC via a variety of mechanisms. iii) CD44, CXXC5, MYH9, MALAT1 and other genes may have important implications for the occurrence and development of GC through the regulation, interaction, and mutual influence of circRNA-miRNA-mRNA via different mechanisms.

Transcriptome Analysis of the Signalling Networks in Coronatine-Induced Secondary Laticifer Differentiation from Vascular Cambia in Rubber Trees

PubMed Central

Wu, Shaohua; Zhang, Shixin; Chao, Jinquan; Deng, Xiaomin; Chen, Yueyi; Shi, Minjing; Tian, Wei-Min

2016-01-01

The secondary laticifer in rubber tree (Hevea brasiliensis Muell. Arg.) is a specific tissue within the secondary phloem. This tissue differentiates from the vascular cambia, and its function is natural rubber biosynthesis and storage. Given that jasmonates play a pivotal role in secondary laticifer differentiation, we established an experimental system with jasmonate (JA) mimic coronatine (COR) for studying the secondary laticifer differentiation: in this system, differentiation occurs within five days of the treatment of epicormic shoots with COR. In the present study, the experimental system was used to perform transcriptome sequencing and gene expression analysis. A total of 67,873 unigenes were assembled, and 50,548 unigenes were mapped at least in one public database. Of these being annotated unigenes, 15,780 unigenes were differentially expressed early after COR treatment, and 19,824 unigenes were differentially expressed late after COR treatment. At the early stage, 8,646 unigenes were up-regulated, while 7,134 unigenes were down-regulated. At the late stage, the numbers of up- and down-regulated unigenes were 7,711 and 12,113, respectively. The annotation data and gene expression analysis of the differentially expressed unigenes suggest that JA-mediated signalling, Ca2+ signal transduction and the CLAVATA-MAPK-WOX signalling pathway may be involved in regulating secondary laticifer differentiation in rubber trees. PMID:27808245

A microarray analysis of potential genes underlying the neurosensitivity of mice to propofol.

PubMed

Lowes, Damon A; Galley, Helen F; Lowe, Peter R; Rikke, Brad A; Johnson, Thomas E; Webster, Nigel R

2005-09-01

Establishing the mechanism of action of general anesthetics at the molecular level is difficult because of the multiple targets with which these drugs are associated. Inbred short sleep (ISS) and long sleep (ILS) mice are differentially sensitive in response to ethanol and other sedative hypnotics and contain a single quantitative trait locus (Lorp1) that accounts for the genetic variance of loss-of-righting reflex in response to propofol (LORP). In this study, we used high-density oligonucleotide microarrays to identify global gene expression and candidate genes differentially expressed within the Lorp1 region that may give insight into the molecular mechanism underlying LORP. Microarray analysis was performed using Affymetrix MG-U74Av2 Genechips and a selection of differentially expressed genes was confirmed by semiquantitative reverse transcription-polymerase chain reaction. Global expression in the brains of ILS and ISS mice revealed 3423 genes that were significantly expressed, of which 139 (4%) were differentially expressed. Analysis of genes located within the Lorp1 region showed that 26 genes were significantly expressed and that just 2 genes (7%) were differentially expressed. These genes encoded for the proteins AWP1 (associated with protein kinase 1) and "BTB (POZ) domain containing 1," whose functions are largely uncharacterized. Genes differentially expressed outside Lorp1 included seven genes with previously characterized neuronal functions and thus stand out as additional candidate genes that may be involved in mediating the neurosensitivity differences between ISS and ILS.

[Cloning and characterization of genes differentially expressed in human dental pulp cells and gingival fibroblasts].

PubMed

Wang, Zhong-dong; Wu, Ji-nan; Zhou, Lin; Ling, Jun-qi; Guo, Xi-min; Xiao, Ming-zhen; Zhu, Feng; Pu, Qin; Chai, Yu-bo; Zhao, Zhong-liang

2007-02-01

To study the biological properties of human dental pulp cells (HDPC) by cloning and analysis of genes differentially expressed in HDPC in comparison with human gingival fibroblasts (HGF). HDPC and HGF were cultured and identified by immunocytochemistry. HPDC and HGF subtractive cDNA library was established by PCR-based modified subtractive hybridization, genes differentially expressed by HPDC were cloned, sequenced and compared to find homogeneous sequence in GenBank by BLAST. Cloning and sequencing analysis indicate 12 genes differentially expressed were obtained, in which two were unknown genes. Among the 10 known genes, 4 were related to signal transduction, 2 were related to trans-membrane transportation (both cell membrane and nuclear membrane), and 2 were related to RNA splicing mechanisms. The biological properties of HPDC are determined by the differential expression of some genes and the growth and differentiation of HPDC are associated to the dynamic protein synthesis and secretion activities of the cell.

Gene expression profiling of bone marrow mesenchymal stem cells from Osteogenesis Imperfecta patients during osteoblast differentiation.

PubMed

Kaneto, Carla Martins; Pereira Lima, Patrícia S; Prata, Karen Lima; Dos Santos, Jane Lima; de Pina Neto, João Monteiro; Panepucci, Rodrigo Alexandre; Noushmehr, Houtan; Covas, Dimas Tadeu; de Paula, Francisco José Alburquerque; Silva, Wilson Araújo

2017-06-01

Mesenchymal stem cells (MSCs) are precursors present in adult bone marrow that are able to differentiate into osteoblasts, adipocytes and chondroblasts that have gained great importance as a source for cell therapy. Recently, a number of studies involving the analysis of gene expression of undifferentiated MSCs and of MSCs in the differentiation into multiple lineage processes were observed but there is no information concerning the gene expression of MSCs from Osteogenesis Imperfecta (OI) patients. Osteogenesis Imperfecta is characterized as a genetic disorder in which a generalized osteopenia leads to excessive bone fragility and severe bone deformities. The aim of this study was to analyze gene expression profile during osteogenic differentiation from BMMSCs (Bone Marrow Mesenchymal Stem Cells) obtained from patients with Osteogenesis Imperfecta and from control subjects. Bone marrow samples were collected from three normal subjects and five patients with OI. Mononuclear cells were isolated for obtaining mesenchymal cells that had been expanded until osteogenic differentiation was induced. RNA was harvested at seven time points during the osteogenic differentiation period (D0, D+1, D+2, D+7, D+12, D+17 and D+21). Gene expression analysis was performed by the microarray technique and identified several differentially expressed genes. Some important genes for osteoblast differentiation had lower expression in OI patients, suggesting a smaller commitment of these patient's MSCs with the osteogenic lineage. Other genes also had their differential expression confirmed by RT-qPCR. An increase in the expression of genes related to adipocytes was observed, suggesting an increase of adipogenic differentiation at the expense osteogenic differentiation. Copyright © 2017. Published by Elsevier Masson SAS.

Robust Principal Component Analysis Regularized by Truncated Nuclear Norm for Identifying Differentially Expressed Genes.

PubMed

Wang, Ya-Xuan; Gao, Ying-Lian; Liu, Jin-Xing; Kong, Xiang-Zhen; Li, Hai-Jun

2017-09-01

Identifying differentially expressed genes from the thousands of genes is a challenging task. Robust principal component analysis (RPCA) is an efficient method in the identification of differentially expressed genes. RPCA method uses nuclear norm to approximate the rank function. However, theoretical studies showed that the nuclear norm minimizes all singular values, so it may not be the best solution to approximate the rank function. The truncated nuclear norm is defined as the sum of some smaller singular values, which may achieve a better approximation of the rank function than nuclear norm. In this paper, a novel method is proposed by replacing nuclear norm of RPCA with the truncated nuclear norm, which is named robust principal component analysis regularized by truncated nuclear norm (TRPCA). The method decomposes the observation matrix of genomic data into a low-rank matrix and a sparse matrix. Because the significant genes can be considered as sparse signals, the differentially expressed genes are viewed as the sparse perturbation signals. Thus, the differentially expressed genes can be identified according to the sparse matrix. The experimental results on The Cancer Genome Atlas data illustrate that the TRPCA method outperforms other state-of-the-art methods in the identification of differentially expressed genes.

Differential expression of pancreatic protein and chemosensing receptor mRNAs in NKCC1-null intestine

PubMed Central

Bradford, Emily M; Vairamani, Kanimozhi; Shull, Gary E

2016-01-01

AIM: To investigate the intestinal functions of the NKCC1 Na+-K+-2Cl cotransporter (SLC12a2 gene), differential mRNA expression changes in NKCC1-null intestine were analyzed. METHODS: Microarray analysis of mRNA from intestines of adult wild-type mice and gene-targeted NKCC1-null mice (n = 6 of each genotype) was performed to identify patterns of differential gene expression changes. Differential expression patterns were further examined by Gene Ontology analysis using the online Gorilla program, and expression changes of selected genes were verified using northern blot analysis and quantitative real time-polymerase chain reaction. Histological staining and immunofluorescence were performed to identify cell types in which upregulated pancreatic digestive enzymes were expressed. RESULTS: Genes typically associated with pancreatic function were upregulated. These included lipase, amylase, elastase, and serine proteases indicative of pancreatic exocrine function, as well as insulin and regenerating islet genes, representative of endocrine function. Northern blot analysis and immunohistochemistry showed that differential expression of exocrine pancreas mRNAs was specific to the duodenum and localized to a subset of goblet cells. In addition, a major pattern of changes involving differential expression of olfactory receptors that function in chemical sensing, as well as other chemosensing G-protein coupled receptors, was observed. These changes in chemosensory receptor expression may be related to the failure of intestinal function and dependency on parenteral nutrition observed in humans with SLC12a2 mutations. CONCLUSION: The results suggest that loss of NKCC1 affects not only secretion, but also goblet cell function and chemosensing of intestinal contents via G-protein coupled chemosensory receptors. PMID:26909237

Differential expression profiles and pathways of genes in sugarcane leaf at elongation stage in response to drought stress

PubMed Central

Li, Changning; Nong, Qian; Solanki, Manoj Kumar; Liang, Qiang; Xie, Jinlan; Liu, Xiaoyan; Li, Yijie; Wang, Weizan; Yang, Litao; Li, Yangrui

2016-01-01

Water stress causes considerable yield losses in sugarcane. To investigate differentially expressed genes under water stress, a pot experiment was performed with the sugarcane variety GT21 at three water-deficit levels (mild, moderate, and severe) during the elongation stage and gene expression was analyzed using microarray technology. Physiological parameters of sugarcane showed significant alterations in response to drought stress. Based on the expression profile of 15,593 sugarcane genes, 1,501 (9.6%) genes were differentially expressed under different water-level treatments; 821 genes were upregulated and 680 genes were downregulated. A gene similarity analysis showed that approximately 62.6% of the differentially expressed genes shared homology with functional proteins. In a Gene Ontology (GO) analysis, 901 differentially expressed genes were assigned to 36 GO categories. Moreover, 325 differentially expressed genes were classified into 101 pathway categories involved in various processes, such as the biosynthesis of secondary metabolites, ribosomes, carbon metabolism, etc. In addition, some unannotated genes were detected; these may provide a basis for studies of water-deficit tolerance. The reliability of the observed expression patterns was confirmed by RT-PCR. The results of this study may help identify useful genes for improving drought tolerance in sugarcane. PMID:27170459

Getting the most out of RNA-seq data analysis.

PubMed

Khang, Tsung Fei; Lau, Ching Yee

2015-01-01

Background. A common research goal in transcriptome projects is to find genes that are differentially expressed in different phenotype classes. Biologists might wish to validate such gene candidates experimentally, or use them for downstream systems biology analysis. Producing a coherent differential gene expression analysis from RNA-seq count data requires an understanding of how numerous sources of variation such as the replicate size, the hypothesized biological effect size, and the specific method for making differential expression calls interact. We believe an explicit demonstration of such interactions in real RNA-seq data sets is of practical interest to biologists. Results. Using two large public RNA-seq data sets-one representing strong, and another mild, biological effect size-we simulated different replicate size scenarios, and tested the performance of several commonly-used methods for calling differentially expressed genes in each of them. We found that, when biological effect size was mild, RNA-seq experiments should focus on experimental validation of differentially expressed gene candidates. Importantly, at least triplicates must be used, and the differentially expressed genes should be called using methods with high positive predictive value (PPV), such as NOISeq or GFOLD. In contrast, when biological effect size was strong, differentially expressed genes mined from unreplicated experiments using NOISeq, ASC and GFOLD had between 30 to 50% mean PPV, an increase of more than 30-fold compared to the cases of mild biological effect size. Among methods with good PPV performance, having triplicates or more substantially improved mean PPV to over 90% for GFOLD, 60% for DESeq2, 50% for NOISeq, and 30% for edgeR. At a replicate size of six, we found DESeq2 and edgeR to be reasonable methods for calling differentially expressed genes at systems level analysis, as their PPV and sensitivity trade-off were superior to the other methods'. Conclusion. When biological effect size is weak, systems level investigation is not possible using RNAseq data, and no meaningful result can be obtained in unreplicated experiments. Nonetheless, NOISeq or GFOLD may yield limited numbers of gene candidates with good validation potential, when triplicates or more are available. When biological effect size is strong, NOISeq and GFOLD are effective tools for detecting differentially expressed genes in unreplicated RNA-seq experiments for qPCR validation. When triplicates or more are available, GFOLD is a sharp tool for identifying high confidence differentially expressed genes for targeted qPCR validation; for downstream systems level analysis, combined results from DESeq2 and edgeR are useful.

Differential co-expression and regulation analyses reveal different mechanisms underlying major depressive disorder and subsyndromal symptomatic depression.

PubMed

Xu, Fan; Yang, Jing; Chen, Jin; Wu, Qingyuan; Gong, Wei; Zhang, Jianguo; Shao, Weihua; Mu, Jun; Yang, Deyu; Yang, Yongtao; Li, Zhiwei; Xie, Peng

2015-04-03

Recent depression research has revealed a growing awareness of how to best classify depression into depressive subtypes. Appropriately subtyping depression can lead to identification of subtypes that are more responsive to current pharmacological treatment and aid in separating out depressed patients in which current antidepressants are not particularly effective. Differential co-expression analysis (DCEA) and differential regulation analysis (DRA) were applied to compare the transcriptomic profiles of peripheral blood lymphocytes from patients with two depressive subtypes: major depressive disorder (MDD) and subsyndromal symptomatic depression (SSD). Six differentially regulated genes (DRGs) (FOSL1, SRF, JUN, TFAP4, SOX9, and HLF) and 16 transcription factor-to-target differentially co-expressed gene links or pairs (TF2target DCLs) appear to be the key differential factors in MDD; in contrast, one DRG (PATZ1) and eight TF2target DCLs appear to be the key differential factors in SSD. There was no overlap between the MDD target genes and SSD target genes. Venlafaxine (Efexor™, Effexor™) appears to have a significant effect on the gene expression profile of MDD patients but no significant effect on the gene expression profile of SSD patients. DCEA and DRA revealed no apparent similarities between the differential regulatory processes underlying MDD and SSD. This bioinformatic analysis may provide novel insights that can support future antidepressant R&D efforts.

Characterizing differential gene expression in polyploid grasses lacking a reference transcriptome

USDA-ARS?s Scientific Manuscript database

Basal transcriptome characterization and differential gene expression in response to varying conditions are often addressed through next generation sequencing (NGS) and data analysis techniques. While these strategies are commonly used, there are countless tools, pipelines, data analysis methods an...

Searching for molecular markers in head and neck squamous cell carcinomas (HNSCC) by statistical and bioinformatic analysis of larynx-derived SAGE libraries

PubMed Central

Silveira, Nelson JF; Varuzza, Leonardo; Machado-Lima, Ariane; Lauretto, Marcelo S; Pinheiro, Daniel G; Rodrigues, Rodrigo V; Severino, Patrícia; Nobrega, Francisco G; Silva, Wilson A; de B Pereira, Carlos A; Tajara, Eloiza H

2008-01-01

Background Head and neck squamous cell carcinoma (HNSCC) is one of the most common malignancies in humans. The average 5-year survival rate is one of the lowest among aggressive cancers, showing no significant improvement in recent years. When detected early, HNSCC has a good prognosis, but most patients present metastatic disease at the time of diagnosis, which significantly reduces survival rate. Despite extensive research, no molecular markers are currently available for diagnostic or prognostic purposes. Methods Aiming to identify differentially-expressed genes involved in laryngeal squamous cell carcinoma (LSCC) development and progression, we generated individual Serial Analysis of Gene Expression (SAGE) libraries from a metastatic and non-metastatic larynx carcinoma, as well as from a normal larynx mucosa sample. Approximately 54,000 unique tags were sequenced in three libraries. Results Statistical data analysis identified a subset of 1,216 differentially expressed tags between tumor and normal libraries, and 894 differentially expressed tags between metastatic and non-metastatic carcinomas. Three genes displaying differential regulation, one down-regulated (KRT31) and two up-regulated (BST2, MFAP2), as well as one with a non-significant differential expression pattern (GNA15) in our SAGE data were selected for real-time polymerase chain reaction (PCR) in a set of HNSCC samples. Consistent with our statistical analysis, quantitative PCR confirmed the upregulation of BST2 and MFAP2 and the downregulation of KRT31 when samples of HNSCC were compared to tumor-free surgical margins. As expected, GNA15 presented a non-significant differential expression pattern when tumor samples were compared to normal tissues. Conclusion To the best of our knowledge, this is the first study reporting SAGE data in head and neck squamous cell tumors. Statistical analysis was effective in identifying differentially expressed genes reportedly involved in cancer development. The differential expression of a subset of genes was confirmed in additional larynx carcinoma samples and in carcinomas from a distinct head and neck subsite. This result suggests the existence of potential common biomarkers for prognosis and targeted-therapy development in this heterogeneous type of tumor. PMID:19014460

Isolation of genes negatively or positively co-expressed with human recombination activating gene 1 (RAG1) by differential display PCR (DD RT-PCR).

PubMed

Verkoczy, L K; Berinstein, N L

1998-10-01

Differential display PCR (DD RT-PCR) has been extensively used for analysis of differential gene expression, but continues to be hampered by technical limitations that impair its effectiveness. In order to isolate novel genes co-expressing with human RAG1, we have developed an effective, multi-tiered screening/purification approach which effectively complements the standard DD RT-PCR methodology. In 'primary' screens, standard DD RT-PCR was used, detecting 22 reproducible differentially expressed amplicons between clonally related cell variants with differential constitutive expression of RAG mRNAs. 'Secondary' screens used differential display (DD) amplicons as probes in low and high stringency northern blotting. Eight of 22 independent DD amplicons detected nine independent differentially expressed transcripts. 'Tertiary' screens used reconfirmed amplicons as probes in northern analysis of multiple RAG-and RAG+sources. Reconfirmed DD amplicons detected six independent RAG co-expressing transcripts. All DD amplicons reconfirmed by northern blot were a heterogeneous mixture of cDNAs, necessitating further purification to isolate single cDNAs prior to subcloning and sequencing. To effectively select the appropriate cDNAs from DD amplicons, we excised and eluted the cDNA(s) directly from regions of prior northern blots in which differentially expressed transcripts were detected. Sequences of six purified cDNA clones specifically detecting RAG co-expressing transcripts included matches to portions of the human RAG2 and BSAP regions and to four novel partial cDNAs (three with homologies to human ESTs). Overall, our results also suggest that even when using clonally related variants from the same cell line in addition to all appropriate internal controls previously reported, further screening and purification steps are still required in order to efficiently and specifically isolate differentially expressed genes by DD RT-PCR.

Differential co-expression analysis of rheumatoid arthritis with microarray data.

PubMed

Wang, Kunpeng; Zhao, Liqiang; Liu, Xuefeng; Hao, Zhenyong; Zhou, Yong; Yang, Chuandong; Li, Hongqiang

2014-11-01

The aim of the present study was to investigate the underlying molecular mechanisms of rheumatoid arthritis (RA) using microarray expression profiles from osteoarthritis and RA patients, to improve diagnosis and treatment strategies for the condition. The gene expression profile of GSE27390 was downloaded from Gene Expression Omnibus, including 19 samples from patients with RA (n=9) or osteoarthritis (n=10). Firstly, the differentially expressed genes (DEGs) were obtained with the thresholds of |logFC|>1.0 and P<0.05, using the t‑test method in LIMMA package. Then, differentially co-expressed genes (DCGs) and differentially co-expressed links (DCLs) were screened with q<0.25 by the differential coexpression analysis and differential regulation analysis of gene expression microarray data package. Secondly, pathway enrichment analysis for DCGs was performed by the Database for Annotation, Visualization and Integrated Discovery and the DCLs associated with RA were selected by comparing the obtained DCLs with known transcription factor (TF)-targets in the TRANSFAC database. Finally, the obtained TFs were mapped to the known TF-targets to construct the network using cytoscape software. A total of 1755 DEGs, 457 DCGs and 101988 DCLs were achieved and there were 20 TFs in the obtained six TF-target relations (STAT3-TNF, PBX1‑PLAU, SOCS3-STAT3, GATA1-ETS2, ETS1-ICAM4 and CEBPE‑GATA1) and 457 DCGs. A number of TF-target relations in the constructed network were not within DCLs when the TF and target gene were DCGs. The identified TFs may have an important role in the pathogenesis of RA and have the potential to be used as biomarkers for the development of novel diagnostic and therapeutic strategies for RA.

Optimal consistency in microRNA expression analysis using reference-gene-based normalization.

PubMed

Wang, Xi; Gardiner, Erin J; Cairns, Murray J

2015-05-01

Normalization of high-throughput molecular expression profiles secures differential expression analysis between samples of different phenotypes or biological conditions, and facilitates comparison between experimental batches. While the same general principles apply to microRNA (miRNA) normalization, there is mounting evidence that global shifts in their expression patterns occur in specific circumstances, which pose a challenge for normalizing miRNA expression data. As an alternative to global normalization, which has the propensity to flatten large trends, normalization against constitutively expressed reference genes presents an advantage through their relative independence. Here we investigated the performance of reference-gene-based (RGB) normalization for differential miRNA expression analysis of microarray expression data, and compared the results with other normalization methods, including: quantile, variance stabilization, robust spline, simple scaling, rank invariant, and Loess regression. The comparative analyses were executed using miRNA expression in tissue samples derived from subjects with schizophrenia and non-psychiatric controls. We proposed a consistency criterion for evaluating methods by examining the overlapping of differentially expressed miRNAs detected using different partitions of the whole data. Based on this criterion, we found that RGB normalization generally outperformed global normalization methods. Thus we recommend the application of RGB normalization for miRNA expression data sets, and believe that this will yield a more consistent and useful readout of differentially expressed miRNAs, particularly in biological conditions characterized by large shifts in miRNA expression.

Principal Angle Enrichment Analysis (PAEA): Dimensionally Reduced Multivariate Gene Set Enrichment Analysis Tool

PubMed Central

Clark, Neil R.; Szymkiewicz, Maciej; Wang, Zichen; Monteiro, Caroline D.; Jones, Matthew R.; Ma’ayan, Avi

2016-01-01

Gene set analysis of differential expression, which identifies collectively differentially expressed gene sets, has become an important tool for biology. The power of this approach lies in its reduction of the dimensionality of the statistical problem and its incorporation of biological interpretation by construction. Many approaches to gene set analysis have been proposed, but benchmarking their performance in the setting of real biological data is difficult due to the lack of a gold standard. In a previously published work we proposed a geometrical approach to differential expression which performed highly in benchmarking tests and compared well to the most popular methods of differential gene expression. As reported, this approach has a natural extension to gene set analysis which we call Principal Angle Enrichment Analysis (PAEA). PAEA employs dimensionality reduction and a multivariate approach for gene set enrichment analysis. However, the performance of this method has not been assessed nor its implementation as a web-based tool. Here we describe new benchmarking protocols for gene set analysis methods and find that PAEA performs highly. The PAEA method is implemented as a user-friendly web-based tool, which contains 70 gene set libraries and is freely available to the community. PMID:26848405

Principal Angle Enrichment Analysis (PAEA): Dimensionally Reduced Multivariate Gene Set Enrichment Analysis Tool.

PubMed

Clark, Neil R; Szymkiewicz, Maciej; Wang, Zichen; Monteiro, Caroline D; Jones, Matthew R; Ma'ayan, Avi

2015-11-01

Gene set analysis of differential expression, which identifies collectively differentially expressed gene sets, has become an important tool for biology. The power of this approach lies in its reduction of the dimensionality of the statistical problem and its incorporation of biological interpretation by construction. Many approaches to gene set analysis have been proposed, but benchmarking their performance in the setting of real biological data is difficult due to the lack of a gold standard. In a previously published work we proposed a geometrical approach to differential expression which performed highly in benchmarking tests and compared well to the most popular methods of differential gene expression. As reported, this approach has a natural extension to gene set analysis which we call Principal Angle Enrichment Analysis (PAEA). PAEA employs dimensionality reduction and a multivariate approach for gene set enrichment analysis. However, the performance of this method has not been assessed nor its implementation as a web-based tool. Here we describe new benchmarking protocols for gene set analysis methods and find that PAEA performs highly. The PAEA method is implemented as a user-friendly web-based tool, which contains 70 gene set libraries and is freely available to the community.

Transcriptome analysis of Schistosoma mansoni larval development using serial analysis of gene expression (SAGE).

PubMed

Taft, A S; Vermeire, J J; Bernier, J; Birkeland, S R; Cipriano, M J; Papa, A R; McArthur, A G; Yoshino, T P

2009-04-01

Infection of the snail, Biomphalaria glabrata, by the free-swimming miracidial stage of the human blood fluke, Schistosoma mansoni, and its subsequent development to the parasitic sporocyst stage is critical to establishment of viable infections and continued human transmission. We performed a genome-wide expression analysis of the S. mansoni miracidia and developing sporocyst using Long Serial Analysis of Gene Expression (LongSAGE). Five cDNA libraries were constructed from miracidia and in vitro cultured 6- and 20-day-old sporocysts maintained in sporocyst medium (SM) or in SM conditioned by previous cultivation with cells of the B. glabrata embryonic (Bge) cell line. We generated 21 440 SAGE tags and mapped 13 381 to the S. mansoni gene predictions (v4.0e) either by estimating theoretical 3' UTR lengths or using existing 3' EST sequence data. Overall, 432 transcripts were found to be differentially expressed amongst all 5 libraries. In total, 172 tags were differentially expressed between miracidia and 6-day conditioned sporocysts and 152 were differentially expressed between miracidia and 6-day unconditioned sporocysts. In addition, 53 and 45 tags, respectively, were differentially expressed in 6-day and 20-day cultured sporocysts, due to the effects of exposure to Bge cell-conditioned medium.

RNA-seq Analysis of Host and Viral Gene Expression Highlights Interaction between Varicella Zoster Virus and Keratinocyte Differentiation

PubMed Central

Singh, Manuraj; Kanda, Ravinder K.; Yee, Michael B.; Kellam, Paul; Hollinshead, Michael; Kinchington, Paul R.; O'Toole, Edel A.; Breuer, Judith

2014-01-01

Varicella zoster virus (VZV) is the etiological agent of chickenpox and shingles, diseases characterized by epidermal skin blistering. Using a calcium-induced keratinocyte differentiation model we investigated the interaction between epidermal differentiation and VZV infection. RNA-seq analysis showed that VZV infection has a profound effect on differentiating keratinocytes, altering the normal process of epidermal gene expression to generate a signature that resembles patterns of gene expression seen in both heritable and acquired skin-blistering disorders. Further investigation by real-time PCR, protein analysis and electron microscopy revealed that VZV specifically reduced expression of specific suprabasal cytokeratins and desmosomal proteins, leading to disruption of epidermal structure and function. These changes were accompanied by an upregulation of kallikreins and serine proteases. Taken together VZV infection promotes blistering and desquamation of the epidermis, both of which are necessary to the viral spread and pathogenesis. At the same time, analysis of the viral transcriptome provided evidence that VZV gene expression was significantly increased following calcium treatment of keratinocytes. Using reporter viruses and immunohistochemistry we confirmed that VZV gene and protein expression in skin is linked with cellular differentiation. These studies highlight the intimate host-pathogen interaction following VZV infection of skin and provide insight into the mechanisms by which VZV remodels the epidermal environment to promote its own replication and spread. PMID:24497829

Unraveling the oral cancer lncRNAome: Identification of novel lncRNAs associated with malignant progression and HPV infection.

PubMed

Nohata, Nijiro; Abba, Martin C; Gutkind, J Silvio

2016-08-01

The role of long non-coding RNA (lncRNA) expression in human head and neck squamous cell carcinoma (HNSCC) is still poorly understood. In this study, we aimed at establishing the onco-lncRNAome profiling of HNSCC and to identify lncRNAs correlating with prognosis and patient survival. The Atlas of Noncoding RNAs in Cancer (TANRIC) database was employed to retrieve the lncRNA expression information generated from The Cancer Genome Atlas (TCGA) HNSCC RNA-sequencing data. RNA-sequencing data from HNSCC cell lines were also considered for this study. Bioinformatics approaches, such as differential gene expression analysis, survival analysis, principal component analysis, and Co-LncRNA enrichment analysis were performed. Using TCGA HNSCC RNA-sequencing data from 426 HNSCC and 42 adjacent normal tissues, we found 728 lncRNA transcripts significantly and differentially expressed in HNSCC. Among the 728 lncRNAs, 55 lncRNAs were significantly associated with poor prognosis, such as overall survival and/or disease-free survival. Next, we found 140 lncRNA transcripts significantly and differentially expressed between Human Papilloma Virus (HPV) positive tumors and HPV negative tumors. Thirty lncRNA transcripts were differentially expressed between TP53 mutated and TP53 wild type tumors. Co-LncRNA analysis suggested that protein-coding genes that are co-expressed with these deregulated lncRNAs might be involved in cancer associated molecular events. With consideration of differential expression of lncRNAs in a HNSCC cell lines panel (n=22), we found several lncRNAs that may represent potential targets for diagnosis, therapy and prevention of HNSCC. LncRNAs profiling could provide novel insights into the potential mechanisms of HNSCC oncogenesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Unraveling the Oral Cancer lncRNAome: Identification of Novel lncRNAs Associated with Malignant Progression and HPV Infection

PubMed Central

Nohata, Nijiro; Abba, Martin C.; Gutkind, J. Silvio

2017-01-01

Objectives The role of long non-coding RNA (lncRNA) expression in human head and neck squamous cell carcinoma (HNSCC) is still poorly understood. In this study, we aimed at establishing the onco-lncRNAome profiling of HNSCC and to identify lncRNAs correlating with prognosis and patient survival. Materials and Methods The Atlas of Noncoding RNAs in Cancer (TANRIC) database was employed to retrieve the lncRNA expression information generated from The Cancer Genome Atlas (TCGA) HNSCC RNA-sequencing data. RNA-sequencing data from HNSCC cell lines were also considered for this study. Bioinformatics approaches, such as differential gene expression analysis, survival analysis, principal component analysis, and Co-LncRNA enrichment analysis were performed. Results Using TCGA HNSCC RNA-sequencing data from 426 HNSCC and 42 adjacent normal tissues, we found 728 lncRNA transcripts significantly and differentially expressed in HNSCC. Among the 728 lncRNAs, 55 lncRNAs were significantly associated with poor prognosis, such as overall survival and/or disease-free survival. Next, we found 140 lncRNA transcripts significantly and differentially expressed between Human Papilloma Virus (HPV) positive tumors and HPV negative tumors. Thirty lncRNA transcripts were differentially expressed between TP53 mutated and TP53 wild type tumors. Co-LncRNA analysis suggested that protein-coding genes that are co-expressed with these deregulated lncRNAs might be involved in cancer associated molecular events. With consideration of differential expression of lncRNAs in a HNSCC cell lines panel (n=22), we found several lncRNAs that may represent potential targets for diagnosis, therapy and prevention of HNSCC. Conclusion LncRNAs profiling could provide novel insights into the potential mechanisms of HNSCC oncogenesis. PMID:27424183

Proteome and Transcriptome Analysis of Ovary, Intersex Gonads, and Testis Reveals Potential Key Sex Reversal/Differentiation Genes and Mechanism in Scallop Chlamys nobilis.

PubMed

Shi, Yu; Liu, Wenguang; He, Maoxian

2018-04-01

Bivalve mollusks exhibit hermaphroditism and sex reversal/differentiation. Studies generally focus on transcriptional profiling and specific genes related to sex determination and differentiation. Few studies on sex reversal/differentiation have been reported. A combination analysis of gonad proteomics and transcriptomics was conducted on Chlamys nobilis to provide a systematic understanding of sex reversal/differentiation in bivalves. We obtained 4258 unique peptides and 93,731 unigenes with good correlation between messenger RNA and protein levels. Candidate genes in sex reversal/differentiation were found: 15 genes differentially expressed between sexes were identified and 12 had obvious sexual functions. Three novel genes (foxl2, β-catenin, and sry) were expressed highly in intersex individuals and were likely involved in the control of gonadal sex in C. nobilis. High expression of foxl2 or β-catenin may inhibit sry and activate 5-HT receptor and vitellogenin to maintain female development. High expression of sry may inhibit foxl2 and β-catenin and activate dmrt2, fem-1, sfp2, sa6, Amy-1, APCP4, and PLK to maintain male function. High expression of sry, foxl2, and β-catenin in C. nobilis may be involved in promoting and maintaining sex reversal/differentiation. The downstream regulator may not be dimorphic expressed genes, but genes expressed in intersex individuals, males and females. Different expression patterns of sex-related genes and gonadal histological characteristics suggested that C. nobilis may change its sex from male to female. These findings suggest highly conserved sex reversal/differentiation with diverged regulatory pathways during C. nobilis evolution. This study provides valuable genetic resources for understanding sex reversal/differentiation (intersex) mechanisms and pathways underlying bivalve reproductive regulation.

Analysis of the differential gene and protein expression profile of the rolled leaf mutant of transgenic rice (Oryza sativa L.).

PubMed

Zhu, Qiuqiang; Yu, Shuguang; Chen, Guanshui; Ke, Lanlan; Pan, Daren

2017-01-01

The importance of leaf rolling in rice (Oryza sativa L.) has been widely recognized. Although several studies have investigated rice leaf rolling and identified some related genes, knowledge of the molecular mechanism underlying rice leaf rolling, especially outward leaf rolling, is limited. Therefore, in this study, differential proteomics and gene expression profiling were used to analyze rolled leaf mutant of transgenic rice in order to investigate differentially expressed genes and proteins related to rice leaf rolling. To this end, 28 differentially expressed proteins related to rolling leaf traits were isolated and identified. Digital expression profiling detected 10 genes related to rice leaf rolling. Some of the proteins and genes detected are involved in lipid metabolism, which is related to the development of bulliform cells, such as phosphoinositide phospholipase C, Mgll gene, and At4g26790 gene. The "omics"-level techniques were useful for simultaneously isolating several proteins and genes related to rice leaf rolling. In addition, the results of the analysis of differentially expressed proteins and genes were closely consistent with those from a corresponding functional analysis of cellular mechanisms; our study findings might form the basis for further research on the molecular mechanisms underlying rice leaf rolling.

Network-based differential gene expression analysis suggests cell cycle related genes regulated by E2F1 underlie the molecular difference between smoker and non-smoker lung adenocarcinoma

PubMed Central

2013-01-01

Background Differential gene expression (DGE) analysis is commonly used to reveal the deregulated molecular mechanisms of complex diseases. However, traditional DGE analysis (e.g., the t test or the rank sum test) tests each gene independently without considering interactions between them. Top-ranked differentially regulated genes prioritized by the analysis may not directly relate to the coherent molecular changes underlying complex diseases. Joint analyses of co-expression and DGE have been applied to reveal the deregulated molecular modules underlying complex diseases. Most of these methods consist of separate steps: first to identify gene-gene relationships under the studied phenotype then to integrate them with gene expression changes for prioritizing signature genes, or vice versa. It is warrant a method that can simultaneously consider gene-gene co-expression strength and corresponding expression level changes so that both types of information can be leveraged optimally. Results In this paper, we develop a gene module based method for differential gene expression analysis, named network-based differential gene expression (nDGE) analysis, a one-step integrative process for prioritizing deregulated genes and grouping them into gene modules. We demonstrate that nDGE outperforms existing methods in prioritizing deregulated genes and discovering deregulated gene modules using simulated data sets. When tested on a series of smoker and non-smoker lung adenocarcinoma data sets, we show that top differentially regulated genes identified by the rank sum test in different sets are not consistent while top ranked genes defined by nDGE in different data sets significantly overlap. nDGE results suggest that a differentially regulated gene module, which is enriched for cell cycle related genes and E2F1 targeted genes, plays a role in the molecular differences between smoker and non-smoker lung adenocarcinoma. Conclusions In this paper, we develop nDGE to prioritize deregulated genes and group them into gene modules by simultaneously considering gene expression level changes and gene-gene co-regulations. When applied to both simulated and empirical data, nDGE outperforms the traditional DGE method. More specifically, when applied to smoker and non-smoker lung cancer sets, nDGE results illustrate the molecular differences between smoker and non-smoker lung cancer. PMID:24341432

Modulation of tumor necrosis factor (TNF) receptor expression during monocytic differentiation by glucocorticoids.

PubMed

Goppelt-Struebe, M; Reiser, C O; Schneider, N; Grell, M

1996-10-01

Regulation of tumor necrosis factor receptors by glucocorticoids was investigated during phorbol ester-induced monocytic differentiation. As model system the human monocytic cell lines U937 and THP-1, which express both types of TNF receptors (TNF-R60 and TNF-R80), were differentiated with tetradecanoyl phorbol-13-acetate (TPA, 5 x 10(-9) M) in the presence or absence of dexamethasone (10(-9) - 10(-6) M). Expression of TNF receptors was determined at the mRNA level by Northern blot analysis and at the protein level by FACS analysis. During differentiation, TNF-R60 mRNA was down-regulated, whereas TNF-R80 mRNA levels were increased. Dexamethasone had no effect on TNF-R60 mRNA expression but attenuated TNF-R80 mRNA expression in both cell lines. Cell surface expression of TNF-R60 protein remained essentially unchanged during differentiation of THP-1 cells, whereas a rapid down-regulation of TNF-R80 was observed that was followed by a slow recovery. Surface expression of TNF-R80 was not affected by dexamethasone, whereas TNF-R60 expression was reduced by about 25%. These results indicate differential regulation of the two types of TNF receptors at the mRNA and protein level during monocytic differentiation. Glucocorticoids interfered with mRNA expression of TNF-R80 and protein expression of TNF-R60, but the rather limited effect leaves the question of its functional relevance open. In contrast to other cytokine systems, TNF receptors do not appear to be major targets of glucocorticoid action.

Male specific genes from dioecious white campion identified by fluorescent differential display.

PubMed

Scutt, Charles P; Jenkins, Tom; Furuya, Masaki; Gilmartin, Philip M

2002-05-01

Fluorescent differential display (FDD) has been used to screen for cDNAs that are differentially up-regulated in male flowers of the dioecious plant Silene latifolia in which an X/Y chromosome system of sex determination operates. To adapt FDD to the cloning of large numbers of differential cDNAs, a novel method of confirming the differential expression of these has been devised. FDD gels were Southern electro-blotted and probed with mixtures of individual cDNA clones derived from different FDD product ligation reactions. These Southern blots were then stripped and re-probed with further mixtures of individual cloned FDD products to identify the maximum number of recombinant clones carrying the true differential amplification products. Of 135 differential bands identified by FDD, 56 differential amplification products were confirmed; these represent 23 unique differentially expressed genes as determined by virtual Northern analysis and two genes expressed at or below the level of detection by virtual Northern analysis. These two low expressed genes show bands of hybridization on genomic Southern blots that are specific to male plants, indicating that they are derived from, or closely related to, Y chromosome genes.

Validation of MIMGO: a method to identify differentially expressed GO terms in a microarray dataset

PubMed Central

2012-01-01

Background We previously proposed an algorithm for the identification of GO terms that commonly annotate genes whose expression is upregulated or downregulated in some microarray data compared with in other microarray data. We call these “differentially expressed GO terms” and have named the algorithm “matrix-assisted identification method of differentially expressed GO terms” (MIMGO). MIMGO can also identify microarray data in which genes annotated with a differentially expressed GO term are upregulated or downregulated. However, MIMGO has not yet been validated on a real microarray dataset using all available GO terms. Findings We combined Gene Set Enrichment Analysis (GSEA) with MIMGO to identify differentially expressed GO terms in a yeast cell cycle microarray dataset. GSEA followed by MIMGO (GSEA + MIMGO) correctly identified (p < 0.05) microarray data in which genes annotated to differentially expressed GO terms are upregulated. We found that GSEA + MIMGO was slightly less effective than, or comparable to, GSEA (Pearson), a method that uses Pearson’s correlation as a metric, at detecting true differentially expressed GO terms. However, unlike other methods including GSEA (Pearson), GSEA + MIMGO can comprehensively identify the microarray data in which genes annotated with a differentially expressed GO term are upregulated or downregulated. Conclusions MIMGO is a reliable method to identify differentially expressed GO terms comprehensively. PMID:23232071

Transcriptome analysis of Petunia axillaris flowers reveals genes involved in morphological differentiation and metabolite transport

PubMed Central

Amano, Ikuko; Kitajima, Sakihito; Suzuki, Hideyuki; Koeduka, Takao

2018-01-01

The biosynthesis of plant secondary metabolites is associated with morphological and metabolic differentiation. As a consequence, gene expression profiles can change drastically, and primary and secondary metabolites, including intermediate and end-products, move dynamically within and between cells. However, little is known about the molecular mechanisms underlying differentiation and transport mechanisms. In this study, we performed a transcriptome analysis of Petunia axillaris subsp. parodii, which produces various volatiles in its corolla limbs and emits metabolites to attract pollinators. RNA-sequencing from leaves, buds, and limbs identified 53,243 unigenes. Analysis of differentially expressed genes, combined with gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses, showed that many biological processes were highly enriched in limbs. These included catabolic processes and signaling pathways of hormones, such as gibberellins, and metabolic pathways, including phenylpropanoids and fatty acids. Moreover, we identified five transporter genes that showed high expression in limbs, and we performed spatiotemporal expression analyses and homology searches to infer their putative functions. Our systematic analysis provides comprehensive transcriptomic information regarding morphological differentiation and metabolite transport in the Petunia flower and lays the foundation for establishing the specific mechanisms that control secondary metabolite biosynthesis in plants. PMID:29902274

Genetic and pharmacological analysis identifies a physiological role for the AHR in epidermal differentiation

PubMed Central

van den Bogaard, Ellen; Podolsky, Michael; Smits, Jos; Cui, Xiao; John, Christian; Gowda, Krishne; Desai, Dhimant; Amin, Shantu; Schalkwijk, Joost; Perdew, Gary H.

2015-01-01

Stimulation of the aryl hydrocarbon receptor (AHR) by xenobiotics is known to affect epidermal differentiation and skin barrier formation. The physiological role of endogenous AHR signaling in keratinocyte differentiation is not known. We used murine and human skin models to address the hypothesis that AHR activation is required for normal keratinocyte differentiation. Using transcriptome analysis of Ahr-/- and Ahr+/+ murine keratinocytes, we found significant enrichment of differentially expressed genes linked to epidermal differentiation. Primary Ahr-/- keratinocytes showed a significant reduction in terminal differentiation gene and protein expression, similar to Ahr+/+ keratinocytes treated with AHR antagonists GNF351 and CH223191, or the selective AHR modulator (SAhRM), SGA360. In vitro keratinocyte differentiation led to increased AHR levels and subsequent nuclear translocation, followed by induced CYP1A1 gene expression. Monolayer cultured primary human keratinocytes treated with AHR antagonists also showed an impaired terminal differentiation program. Inactivation of AHR activity during human skin equivalent development severely impaired epidermal stratification, terminal differentiation protein expression and stratum corneum formation. As disturbed epidermal differentiation is a main feature of many skin diseases, pharmacological agents targeting AHR signaling or future identification of endogenous keratinocyte-derived AHR ligands should be considered as potential new drugs in dermatology. PMID:25602157

Systemic bioinformatics analysis of skeletal muscle gene expression profiles of sepsis

PubMed Central

Yang, Fang; Wang, Yumei

2018-01-01

Sepsis is a type of systemic inflammatory response syndrome with high morbidity and mortality. Skeletal muscle dysfunction is one of the major complications of sepsis that may also influence the outcome of sepsis. The aim of the present study was to explore and identify potential mechanisms and therapeutic targets of sepsis. Systemic bioinformatics analysis of skeletal muscle gene expression profiles from the Gene Expression Omnibus was performed. Differentially expressed genes (DEGs) in samples from patients with sepsis and control samples were screened out using the limma package. Differential co-expression and coregulation (DCE and DCR, respectively) analysis was performed based on the Differential Co-expression Analysis package to identify differences in gene co-expression and coregulation patterns between the control and sepsis groups. Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways of DEGs were identified using the Database for Annotation, Visualization and Integrated Discovery, and inflammatory, cancer and skeletal muscle development-associated biological processes and pathways were identified. DCE and DCR analysis revealed several potential therapeutic targets for sepsis, including genes and transcription factors. The results of the present study may provide a basis for the development of novel therapeutic targets and treatment methods for sepsis. PMID:29805480

ZNF281 inhibits neuronal differentiation and is a prognostic marker for neuroblastoma.

PubMed

Pieraccioli, Marco; Nicolai, Sara; Pitolli, Consuelo; Agostini, Massimiliano; Antonov, Alexey; Malewicz, Michal; Knight, Richard A; Raschellà, Giuseppe; Melino, Gerry

2018-06-25

Derangement of cellular differentiation because of mutation or inappropriate expression of specific genes is a common feature in tumors. Here, we show that the expression of ZNF281, a zinc finger factor involved in several cellular processes, decreases during terminal differentiation of murine cortical neurons and in retinoic acid-induced differentiation of neuroblastoma (NB) cells. The ectopic expression of ZNF281 inhibits the neuronal differentiation of murine cortical neurons and NB cells, whereas its silencing causes the opposite effect. Furthermore, TAp73 inhibits the expression of ZNF281 through miR34a. Conversely, MYCN promotes the expression of ZNF281 at least in part by inhibiting miR34a. These findings imply a functional network that includes p73, MYCN, and ZNF281 in NB cells, where ZNF281 acts by negatively affecting neuronal differentiation. Array analysis of NB cells silenced for ZNF281 expression identified GDNF and NRP2 as two transcriptional targets inhibited by ZNF281. Binding of ZNF281 to the promoters of these genes suggests a direct mechanism of repression. Bioinformatic analysis of NB datasets indicates that ZNF281 expression is higher in aggressive, undifferentiated stage 4 than in localized stage 1 tumors supporting a central role of ZNF281 in affecting the differentiation of NB. Furthermore, patients with NB with high expression of ZNF281 have a poor clinical outcome compared with low-expressors. These observations suggest that ZNF281 is a controller of neuronal differentiation that should be evaluated as a prognostic marker in NB. Copyright © 2018 the Author(s). Published by PNAS.

A powerful nonparametric method for detecting differentially co-expressed genes: distance correlation screening and edge-count test.

PubMed

Zhang, Qingyang

2018-05-16

Differential co-expression analysis, as a complement of differential expression analysis, offers significant insights into the changes in molecular mechanism of different phenotypes. A prevailing approach to detecting differentially co-expressed genes is to compare Pearson's correlation coefficients in two phenotypes. However, due to the limitations of Pearson's correlation measure, this approach lacks the power to detect nonlinear changes in gene co-expression which is common in gene regulatory networks. In this work, a new nonparametric procedure is proposed to search differentially co-expressed gene pairs in different phenotypes from large-scale data. Our computational pipeline consisted of two main steps, a screening step and a testing step. The screening step is to reduce the search space by filtering out all the independent gene pairs using distance correlation measure. In the testing step, we compare the gene co-expression patterns in different phenotypes by a recently developed edge-count test. Both steps are distribution-free and targeting nonlinear relations. We illustrate the promise of the new approach by analyzing the Cancer Genome Atlas data and the METABRIC data for breast cancer subtypes. Compared with some existing methods, the new method is more powerful in detecting nonlinear type of differential co-expressions. The distance correlation screening can greatly improve computational efficiency, facilitating its application to large data sets.

Transcriptome meta-analysis reveals common differential and global gene expression profiles in cystic fibrosis and other respiratory disorders and identifies CFTR regulators.

PubMed

Clarke, Luka A; Botelho, Hugo M; Sousa, Lisete; Falcao, Andre O; Amaral, Margarida D

2015-11-01

A meta-analysis of 13 independent microarray data sets was performed and gene expression profiles from cystic fibrosis (CF), similar disorders (COPD: chronic obstructive pulmonary disease, IPF: idiopathic pulmonary fibrosis, asthma), environmental conditions (smoking, epithelial injury), related cellular processes (epithelial differentiation/regeneration), and non-respiratory "control" conditions (schizophrenia, dieting), were compared. Similarity among differentially expressed (DE) gene lists was assessed using a permutation test, and a clustergram was constructed, identifying common gene markers. Global gene expression values were standardized using a novel approach, revealing that similarities between independent data sets run deeper than shared DE genes. Correlation of gene expression values identified putative gene regulators of the CF transmembrane conductance regulator (CFTR) gene, of potential therapeutic significance. Our study provides a novel perspective on CF epithelial gene expression in the context of other lung disorders and conditions, and highlights the contribution of differentiation/EMT and injury to gene signatures of respiratory disease. Copyright © 2015 Elsevier Inc. All rights reserved.

Comparative transcriptome analysis reveals differentially expressed genes associated with sex expression in garden asparagus (Asparagus officinalis).

PubMed

Li, Shu-Fen; Zhang, Guo-Jun; Zhang, Xue-Jin; Yuan, Jin-Hong; Deng, Chuan-Liang; Gao, Wu-Jun

2017-08-22

Garden asparagus (Asparagus officinalis) is a highly valuable vegetable crop of commercial and nutritional interest. It is also commonly used to investigate the mechanisms of sex determination and differentiation in plants. However, the sex expression mechanisms in asparagus remain poorly understood. De novo transcriptome sequencing via Illumina paired-end sequencing revealed more than 26 billion bases of high-quality sequence data from male and female asparagus flower buds. A total of 72,626 unigenes with an average length of 979 bp were assembled. In comparative transcriptome analysis, 4876 differentially expressed genes (DEGs) were identified in the possible sex-determining stage of female and male/supermale flower buds. Of these DEGs, 433, including 285 male/supermale-biased and 149 female-biased genes, were annotated as flower related. Of the male/supermale-biased flower-related genes, 102 were probably involved in anther development. In addition, 43 DEGs implicated in hormone response and biosynthesis putatively associated with sex expression and reproduction were discovered. Moreover, 128 transcription factor (TF)-related genes belonging to various families were found to be differentially expressed, and this finding implied the essential roles of TF in sex determination or differentiation in asparagus. Correlation analysis indicated that miRNA-DEG pairs were also implicated in asparagus sexual development. Our study identified a large number of DEGs involved in the sex expression and reproduction of asparagus, including known genes participating in plant reproduction, plant hormone signaling, TF encoding, and genes with unclear functions. We also found that miRNAs might be involved in the sex differentiation process. Our study could provide a valuable basis for further investigations on the regulatory networks of sex determination and differentiation in asparagus and facilitate further genetic and genomic studies on this dioecious species.

Gene expression analysis reveals schizophrenia-associated dysregulation of immune pathways in peripheral blood mononuclear cells.

PubMed

Gardiner, Erin J; Cairns, Murray J; Liu, Bing; Beveridge, Natalie J; Carr, Vaughan; Kelly, Brian; Scott, Rodney J; Tooney, Paul A

2013-04-01

Peripheral blood mononuclear cells (PBMCs) represent an accessible tissue source for gene expression profiling in schizophrenia that could provide insight into the molecular basis of the disorder. This study used the Illumina HT_12 microarray platform and quantitative real time PCR (QPCR) to perform mRNA expression profiling on 114 patients with schizophrenia or schizoaffective disorder and 80 non-psychiatric controls from the Australian Schizophrenia Research Bank (ASRB). Differential expression analysis revealed altered expression of 164 genes (59 up-regulated and 105 down-regulated) in the PBMCs from patients with schizophrenia compared to controls. Bioinformatic analysis indicated significant enrichment of differentially expressed genes known to be involved or associated with immune function and regulating the immune response. The differential expression of 6 genes, EIF2C2 (Ago 2), MEF2D, EVL, PI3, S100A12 and DEFA4 was confirmed by QPCR. Genome-wide expression analysis of PBMCs from individuals with schizophrenia was characterized by the alteration of genes with immune system function, supporting the hypothesis that the disorder has a significant immunological component in its etiology. Copyright © 2012 Elsevier Ltd. All rights reserved.

Comparative characterization of microRNAs in Schistosoma japonicum schistosomula from Wistar rats and BALB/c mice.

PubMed

Han, Hongxiao; Peng, Jinbiao; Hong, Yang; Fu, Zhiqiang; Lu, Ke; Li, Hao; Zhu, Chuangang; Zhao, Qiuhua; Lin, Jiaojiao

2015-07-01

More than 40 kinds of mammals in China are known to be naturally infected with Schistosoma japonicum (S. japonicum) (Peng et al. Parasitol Res 106:967-76, 2010). Compared with permissive BALB/c mice, rats are less susceptible to S. japonicum infection and are considered to provide an unsuitable microenvironment for parasite growth and development. MicroRNAs (miRNAs), via the regulation of gene expression at the transcriptional and post-transcriptional levels, may be responsible for developmental differences between schistosomula in these two rodent hosts. Solexa deep-sequencing technology was used to identify differentially expressed miRNAs from schistosomula isolated from Wistar rats and BALB/c mice 10 days post-infection. The deep-sequencing analysis revealed that nearly 40 % of raw reads (10.37 and 10.84 million reads in schistosomula isolated from Wistar rats and BALB/c mice, respectively) can be mapped to selected mirs in miRBase or in species-specific genomes. Further analysis revealed that several miRNAs were differentially expressed in schistosomula isolated from these two rodents; 18 were downregulated (by <2-fold) and 23 were up-regulated (>2-fold) (expression levels in rats compare with those in mice). Additionally, three novel miRNAs were primarily predicted and identified. Among the 41 differentially expressed miRNAs, 4 miRNAs had been identified with specific functions in schistosome development or host-parasite interaction, such as sexual maturation (sja-miR-1, sja-miR-7-5p), embryo development (sja-miR-36-3p) in schistosome, and pathogenesis of schistosomiasis (sja-bantam). Then, the target genes were mapped, filtered, and correlated with a set of genes that were differentially expressed genes in schistosomula isolated from mice and rats, which we identified in a S. japonicum oligonucleotide microarray analysis in a previous study. Gene Ontology (GO) analysis of the predicted target genes of 13 differentially expressed miRNAs revealed that they were involved in some important biological pathways, such as metabolic processes, the regulation of protein catabolic processes, catalytic activity, oxidoreductase activity, and hydrolase activity. The study presented here includes the first identification of differentially expressed miRNAs between schistosomula in mice or rats. Therefore, we hypothesized that the differentially expressed miRNAs may affect the development, growth, and maturation of the schistosome in its life cycle. Our analysis suggested that some differentially expressed miRNAs may impact the survival and development of the parasite within a host. This study increases our understanding of schistosome development and host-parasite interactions.

Automation of fluorescent differential display with digital readout.

PubMed

Meade, Jonathan D; Cho, Yong-Jig; Fisher, Jeffrey S; Walden, Jamie C; Guo, Zhen; Liang, Peng

2006-01-01

Since its invention in 1992, differential display (DD) has become the most commonly used technique for identifying differentially expressed genes because of its many advantages over competing technologies such as DNA microarray, serial analysis of gene expression (SAGE), and subtractive hybridization. Despite the great impact of the method on biomedical research, there has been a lack of automation of DD technology to increase its throughput and accuracy for systematic gene expression analysis. Most of previous DD work has taken a "shot-gun" approach of identifying one gene at a time, with a limited number of polymerase chain reaction (PCR) reactions set up manually, giving DD a low-tech and low-throughput image. We have optimized the DD process with a new platform that incorporates fluorescent digital readout, automated liquid handling, and large-format gels capable of running entire 96-well plates. The resulting streamlined fluorescent DD (FDD) technology offers an unprecedented accuracy, sensitivity, and throughput in comprehensive and quantitative analysis of gene expression. These major improvements will allow researchers to find differentially expressed genes of interest, both known and novel, quickly and easily.

Expression and regulation of long noncoding RNAs during the osteogenic differentiation of periodontal ligament stem cells in the inflammatory microenvironment.

PubMed

Zhang, Qingbin; Chen, Li; Cui, Shiman; Li, Yan; Zhao, Qi; Cao, Wei; Lai, Shixiang; Yin, Sanjun; Zuo, Zhixiang; Ren, Jian

2017-10-25

Although long noncoding RNAs (lncRNAs) have been emerging as critical regulators in various tissues and biological processes, little is known about their expression and regulation during the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) in inflammatory microenvironment. In this study, we have identified 63 lncRNAs that are not annotated in previous database. These novel lncRNAs were not randomly located in the genome but preferentially located near protein-coding genes related to particular functions and diseases, such as stem cell maintenance and differentiation, development disorders and inflammatory diseases. Moreover, we have identified 650 differentially expressed lncRNAs among different subsets of PDLSCs. Pathway enrichment analysis for neighboring protein-coding genes of these differentially expressed lncRNAs revealed stem cell differentiation related functions. Many of these differentially expressed lncRNAs function as competing endogenous RNAs that regulate protein-coding transcripts through competing shared miRNAs.

The effect of chemically modified electrospun silica nanofiber on the mRNA and miRNA expression profile of neural stem cell differentiation.

PubMed

Mercado, Augustus T; Yeh, Jui-Ming; Chin, Ting Yu; Chen, Wen Shuo; Chen-Yang, Yui Whei; Chen, Chung-Yung

2016-11-01

A detailed genomic and epigenomic analyses of neural stem cells (NSCs) differentiation in synthetic microenvironments is essential for the advancement of regenerative medicine and therapeutic treatment of diseases. This study identified the changes in mRNA and miRNA expression profile during NSC differentiation on an artificial matrix. NSCs were grown on a surface-modified, electrospun tetraethyl-orthosilicate nanofiber (designated as SNF-AP) by providing a 3D-environment for cell growth and differentiation. Differentially expressed mRNAs and miRNAs of NSC differentiated in this microenvironment were identified through microarray analysis. The genes and miRNA targets responsible for the differentiation fate of NSCs and neuron development process were determined using Ingenuity Pathway Analysis (IPA). SNF-AP enhanced the expression of genes that activates the proliferation, development, and outgrowth of neurons, differentiation and generation of cells, neuritogenesis, outgrowth of neurites, microtubule dynamics, formation of cellular protrusions, and long-term potentiation during NSC differentiation. On the other hand, PDL inhibited neuritogenesis, microtubule dynamics, and proliferation and differentiation of cells and activated the apoptosis function. Moreover, the nanomaterial promoted the expression of more let-7 miRNAs, which have vital roles in NSC differentiation. Overall, SNF-AP is biocompatible and applicable scaffold for NSC differentiation in the development of neural tissue engineering. These findings are useful in enhancing in vitro NSC differentiation potential for preclinical studies and future clinical applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2730-2743, 2016. © 2016 Wiley Periodicals, Inc.

MURC/cavin-4 Is Co-Expressed with Caveolin-3 in Rhabdomyosarcoma Tumors and Its Silencing Prevents Myogenic Differentiation in the Human Embryonal RD Cell Line

PubMed Central

Faggi, Fiorella; Codenotti, Silvia; Poliani, Pietro Luigi; Cominelli, Manuela; Chiarelli, Nicola; Colombi, Marina; Vezzoli, Marika; Monti, Eugenio; Bono, Federica; Tulipano, Giovanni; Fiorentini, Chiara; Zanola, Alessandra; Lo, Harriet P.; Parton, Robert G.; Keller, Charles; Fanzani, Alessandro

2015-01-01

The purpose of this study was to investigate whether MURC/cavin-4, a plasma membrane and Z-line associated protein exhibiting an overlapping distribution with Caveolin-3 (Cav-3) in heart and muscle tissues, may be expressed and play a role in rhabdomyosarcoma (RMS), an aggressive myogenic tumor affecting childhood. We found MURC/cavin-4 to be expressed, often concurrently with Cav-3, in mouse and human RMS, as demonstrated through in silico analysis of gene datasets and immunohistochemical analysis of tumor samples. In vitro expression studies carried out using human cell lines and primary mouse tumor cultures showed that expression levels of both MURC/cavin-4 and Cav-3, while being low or undetectable during cell proliferation, became robustly increased during myogenic differentiation, as detected via semi-quantitative RT-PCR and immunoblotting analysis. Furthermore, confocal microscopy analysis performed on human RD and RH30 cell lines confirmed that MURC/cavin-4 mostly marks differentiated cell elements, colocalizing at the cell surface with Cav-3 and labeling myosin heavy chain (MHC) expressing cells. Finally, MURC/cavin-4 silencing prevented the differentiation in the RD cell line, leading to morphological cell impairment characterized by depletion of myogenin, Cav-3 and MHC protein levels. Overall, our data suggest that MURC/cavin-4, especially in combination with Cav-3, may play a consistent role in the differentiation process of RMS. PMID:26086601

MURC/cavin-4 Is Co-Expressed with Caveolin-3 in Rhabdomyosarcoma Tumors and Its Silencing Prevents Myogenic Differentiation in the Human Embryonal RD Cell Line.

PubMed

Faggi, Fiorella; Codenotti, Silvia; Poliani, Pietro Luigi; Cominelli, Manuela; Chiarelli, Nicola; Colombi, Marina; Vezzoli, Marika; Monti, Eugenio; Bono, Federica; Tulipano, Giovanni; Fiorentini, Chiara; Zanola, Alessandra; Lo, Harriet P; Parton, Robert G; Keller, Charles; Fanzani, Alessandro

2015-01-01

The purpose of this study was to investigate whether MURC/cavin-4, a plasma membrane and Z-line associated protein exhibiting an overlapping distribution with Caveolin-3 (Cav-3) in heart and muscle tissues, may be expressed and play a role in rhabdomyosarcoma (RMS), an aggressive myogenic tumor affecting childhood. We found MURC/cavin-4 to be expressed, often concurrently with Cav-3, in mouse and human RMS, as demonstrated through in silico analysis of gene datasets and immunohistochemical analysis of tumor samples. In vitro expression studies carried out using human cell lines and primary mouse tumor cultures showed that expression levels of both MURC/cavin-4 and Cav-3, while being low or undetectable during cell proliferation, became robustly increased during myogenic differentiation, as detected via semi-quantitative RT-PCR and immunoblotting analysis. Furthermore, confocal microscopy analysis performed on human RD and RH30 cell lines confirmed that MURC/cavin-4 mostly marks differentiated cell elements, colocalizing at the cell surface with Cav-3 and labeling myosin heavy chain (MHC) expressing cells. Finally, MURC/cavin-4 silencing prevented the differentiation in the RD cell line, leading to morphological cell impairment characterized by depletion of myogenin, Cav-3 and MHC protein levels. Overall, our data suggest that MURC/cavin-4, especially in combination with Cav-3, may play a consistent role in the differentiation process of RMS.

Genome-Wide Analysis of Long Noncoding RNA (lncRNA) Expression in Hepatoblastoma Tissues

PubMed Central

Xue, Ping; Cui, Ximao; Li, Kai; Zheng, Shan; He, Xianghuo; Dong, Kuiran

2014-01-01

Long noncoding RNAs (lncRNAs) have crucial roles in cancer biology. We performed a genome-wide analysis of lncRNA expression in hepatoblastoma tissues to identify novel targets for further study of hepatoblastoma. Hepatoblastoma and normal liver tissue samples were obtained from hepatoblastoma patients. The genome-wide analysis of lncRNA expression in these tissues was performed using a 4×180 K lncRNA microarray and Sureprint G3 Human lncRNA Chips. Quantitative RT-PCR (qRT-PCR) was performed to confirm these results. The differential expressions of lncRNAs and mRNAs were identified through fold-change filtering. Gene Ontology (GO) and pathway analyses were performed using the standard enrichment computation method. Associations between lncRNAs and adjacent protein-coding genes were determined through complex transcriptional loci analysis. We found that 2736 lncRNAs were differentially expressed in hepatoblastoma tissues. Among these, 1757 lncRNAs were upregulated more than two-fold relative to normal tissues and 979 lncRNAs were downregulated. Moreover, in hepatoblastoma there were 420 matched lncRNA-mRNA pairs for 120 differentially expressed lncRNAs, and 167 differentially expressed mRNAs. The co-expression network analysis predicted 252 network nodes and 420 connections between 120 lncRNAs and 132 coding genes. Within this co-expression network, 369 pairs were positive, and 51 pairs were negative. Lastly, qRT-PCR data verified six upregulated and downregulated lncRNAs in hepatoblastoma, plus endothelial cell-specific molecule 1 (ESM1) mRNA. Our results demonstrated that expression of these aberrant lncRNAs could respond to hepatoblastoma development. Further study of these lncRNAs could provide useful insight into hepatoblastoma biology. PMID:24465615

Transcriptomic Analysis and Meta-Analysis of Human Granulosa and Cumulus Cells

PubMed Central

Burnik Papler, Tanja; Vrtacnik Bokal, Eda; Maver, Ales; Kopitar, Andreja Natasa; Lovrečić, Luca

2015-01-01

Specific gene expression in oocytes and its surrounding cumulus (CC) and granulosa (GC) cells is needed for successful folliculogenesis and oocyte maturation. The aim of the present study was to compare genome-wide gene expression and biological functions of human GC and CC. Individual GC and CC were derived from 37 women undergoing IVF procedures. Gene expression analysis was performed using microarrays, followed by a meta-analysis. Results were validated using quantitative real-time PCR. There were 6029 differentially expressed genes (q < 10−4); of which 650 genes had a log2 FC ≥ 2. After the meta-analysis there were 3156 genes differentially expressed. Among these there were genes that have previously not been reported in human somatic follicular cells, like prokineticin 2 (PROK2), higher expressed in GC, and pregnancy up-regulated nonubiquitous CaM kinase (PNCK), higher expressed in CC. Pathways like inflammatory response and angiogenesis were enriched in GC, whereas in CC, cell differentiation and multicellular organismal development were among enriched pathways. In conclusion, transcriptomes of GC and CC as well as biological functions, are distinctive for each cell subpopulation. By describing novel genes like PROK2 and PNCK, expressed in GC and CC, we upgraded the existing data on human follicular biology. PMID:26313571

Successful pod infections by Moniliophthora roreri result in differential Theobroma cacao gene expression depending on the clone's level of tolerance.

PubMed

Ali, Shahin S; Melnick, Rachel L; Crozier, Jayne; Phillips-Mora, Wilberth; Strem, Mary D; Shao, Jonathan; Zhang, Dapeng; Sicher, Richard; Meinhardt, Lyndel; Bailey, Bryan A

2014-09-01

An understanding of the tolerance mechanisms of Theobroma cacao used against Moniliophthora roreri, the causal agent of frosty pod rot, is important for the generation of stable disease-tolerant clones. A comparative view was obtained of transcript populations of infected pods from two susceptible and two tolerant clones using RNA sequence (RNA-Seq) analysis. A total of 3009 transcripts showed differential expression among clones. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis of differentially expressed genes indicated shifts in 152 different metabolic pathways between the tolerant and susceptible clones. Real-time quantitative reverse transcription polymerase chain reaction (real-time qRT-PCR) analyses of 36 genes verified the differential expression. Regression analysis validated a uniform progression in gene expression in association with infection levels and fungal loads in the susceptible clones. Expression patterns observed in the susceptible clones diverged in tolerant clones, with many genes showing higher expression at a low level of infection and fungal load. Principal coordinate analyses of real-time qRT-PCR data separated the gene expression patterns between susceptible and tolerant clones for pods showing malformation. Although some genes were constitutively differentially expressed between clones, most results suggested that defence responses were induced at low fungal load in the tolerant clones. Several elicitor-responsive genes were highly expressed in tolerant clones, suggesting rapid recognition of the pathogen and induction of defence genes. Expression patterns suggested that the jasmonic acid-ethylene- and/or salicylic acid-mediated defence pathways were activated in the tolerant clones, being enhanced by reduced brassinosteroid (BR) biosynthesis and catabolic inactivation of both BR and abscisic acids. Finally, several genes associated with hypersensitive response-like cell death were also induced in tolerant clones. © 2014 BSPP AND JOHN WILEY & SONS LTD.

Differential gene expression of wheat progeny with contrasting levels of transpiration efficiency.

PubMed

Xue, Gang-Ping; McIntyre, C Lynne; Chapman, Scott; Bower, Neil I; Way, Heather; Reverter, Antonio; Clarke, Bryan; Shorter, Ray

2006-08-01

High water use efficiency or transpiration efficiency (TE) in wheat is a desirable physiological trait for increasing grain yield under water-limited environments. The identification of genes associated with this trait would facilitate the selection for genotypes with higher TE using molecular markers. We performed an expression profiling (microarray) analysis of approximately 16,000 unique wheat ESTs to identify genes that were differentially expressed between wheat progeny lines with contrasting TE levels from a cross between Quarrion (high TE) and Genaro 81 (low TE). We also conducted a second microarray analysis to identify genes responsive to drought stress in wheat leaves. Ninety-three genes that were differentially expressed between high and low TE progeny lines were identified. One fifth of these genes were markedly responsive to drought stress. Several potential growth-related regulatory genes, which were down-regulated by drought, were expressed at a higher level in the high TE lines than the low TE lines and are potentially associated with a biomass production component of the Quarrion-derived high TE trait. Eighteen of the TE differentially expressed genes were further analysed using quantitative RT-PCR on a separate set of plant samples from those used for microarray analysis. The expression levels of 11 of the 18 genes were positively correlated with the high TE trait, measured as carbon isotope discrimination (Delta(13)C). These data indicate that some of these TE differentially expressed genes are candidates for investigating processes that underlie the high TE trait or for use as expression quantitative trait loci (eQTLs) for TE.

Quantification of differential gene expression by multiplexed targeted resequencing of cDNA

PubMed Central

Arts, Peer; van der Raadt, Jori; van Gestel, Sebastianus H.C.; Steehouwer, Marloes; Shendure, Jay; Hoischen, Alexander; Albers, Cornelis A.

2017-01-01

Whole-transcriptome or RNA sequencing (RNA-Seq) is a powerful and versatile tool for functional analysis of different types of RNA molecules, but sample reagent and sequencing cost can be prohibitive for hypothesis-driven studies where the aim is to quantify differential expression of a limited number of genes. Here we present an approach for quantification of differential mRNA expression by targeted resequencing of complementary DNA using single-molecule molecular inversion probes (cDNA-smMIPs) that enable highly multiplexed resequencing of cDNA target regions of ∼100 nucleotides and counting of individual molecules. We show that accurate estimates of differential expression can be obtained from molecule counts for hundreds of smMIPs per reaction and that smMIPs are also suitable for quantification of relative gene expression and allele-specific expression. Compared with low-coverage RNA-Seq and a hybridization-based targeted RNA-Seq method, cDNA-smMIPs are a cost-effective high-throughput tool for hypothesis-driven expression analysis in large numbers of genes (10 to 500) and samples (hundreds to thousands). PMID:28474677

5-Hydroxyferulic acid methyl ester isolated from wasabi leaves inhibits 3T3-L1 adipocyte differentiation.

PubMed

Misawa, Naoki; Hosoya, Takahiro; Yoshida, Shuhei; Sugimoto, Osamu; Yamada-Kato, Tomoe; Kumazawa, Shigenori

2018-02-26

To investigate the compounds present in wasabi leaves (Wasabia japonica Matsumura) that inhibit the adipocyte differentiation, activity-guided fractionation was performed on these leaves. 5-Hydroxyferulic acid methyl ester (1: 5-HFA ester), one of the phenylpropanoids, was isolated from wasabi leaves as a compound that inhibits the adipocyte differentiation. Compound 1 suppressed the intracellular lipid accumulation of 3T3-L1 cells without significant cytotoxicity. Gene expression analysis revealed that 1 suppressed the mRNA expression of 2 master regulators of adipocyte differentiation, PPARγ and C/EBPα. Furthermore, 1 downregulated the expression of adipogenesis-related genes, GLUT4, LPL, SREBP-1c, ACC, and FAS. Protein expression analysis revealed that 1 suppressed PPARγ protein expression. Moreover, to investigate the relationship between the structure and activity of inhibiting the adipocyte differentiation, we synthesized 12 kinds of phenylpropanoid analog. Comparison of the activity among 1 and its analogs suggested that the compound containing the substructure that possess a common functional group at the ortho position such as a catechol group exhibits the activity of inhibiting the adipocyte differentiation. Taken together, our findings suggest that 1 from wasabi leaves inhibits adipocyte differentiation via the downregulation of PPARγ. Copyright © 2018 John Wiley & Sons, Ltd.

Analysis of differentially expressed genes between fluoride-sensitive and fluoride-endurable individuals in midgut of silkworm, Bombyx mori.

PubMed

Qian, Heying; Li, Gang; He, Qingling; Zhang, Huaguang; Xu, Anying

2016-08-15

Fluoride tolerance is an economically important trait of silkworm. Near-isogenic lines (NILs) of the dominant endurance to fluoride (Def) gene in Bombyx mori has been constructed before. Here, we analyzed the gene expression profiles of midgut of fluoride-sensitive and fluoride-endurable individuals of Def NILs by using high-throughput Illumina sequencing technology and bioinformatics tools, and identified differentially expressed genes between these individuals. A total of 3,612,399 and 3,567,631 clean tags for the libraries of fluoride-endurable and fluoride-sensitive individuals were obtained, which corresponded to 32,933 and 43,976 distinct clean tags, respectively. Analysis of differentially expressed genes indicates that 241 genes are differentially expressed between the two libraries. Among the 241 genes, 30 are up-regulated and 211 are down-regulated in fluoride-endurable individuals. Pathway enrichment analysis demonstrates that genes related to ribosomes, pancreatic secretion, steroid biosynthesis, glutathione metabolism, steroid biosynthesis, and glycerolipid metabolism are down-regulated in fluoride-endurable individuals. qRT-PCR was conducted to confirm the results of the DGE. The present study analyzed differential expression of related genes and tried to find out whether the crucial genes were related to fluoride detoxification which might elucidate fluoride effect and provide a new way in the fluorosis research. Copyright © 2016 Elsevier B.V. All rights reserved.

High matrix metalloproteinase activity is a hallmark of periapical granulomas.

PubMed

de Paula-Silva, Francisco Wanderley Garcia; D'Silva, Nisha J; da Silva, Léa Assed Bezerra; Kapila, Yvonne Lorraine

2009-09-01

The inability to distinguish periapical cysts from granulomas before performing root canal treatment leads to uncertainty in treatment outcomes because cysts have lower healing rates. Searching for differential expression of molecules within cysts or granulomas could provide information with regard to the identity of the lesion or suggest mechanistic differences that may form the basis for future therapeutic intervention. Thus, we investigated whether granulomas and cysts exhibit differential expression of extracellular matrix (ECM) molecules. Human periapical granulomas, periapical cysts, and healthy periodontal ligament tissues were used to investigate the differential expression of ECM molecules by microarray analysis. Because matrix metalloproteinases (MMP) showed the highest differential expression in the microarray analysis, MMPs were further examined by in situ zymography and immunohistochemistry. Data were analyzed by using one-way analysis of variance followed by the Tukey test. We observed that cysts and granulomas differentially expressed several ECM molecules, especially those from the MMP family. Compared with cysts, granulomas exhibited higher MMP enzymatic activity in areas stained for MMP-9. These areas were composed of polymorphonuclear cells (PMNs) in contrast to cysts. Similarly, MMP-13 was expressed by a greater number of cells in granulomas compared with cysts. Our findings indicate that high enzymatic MMP activity in PMNs together with MMP-9 and MMP-13 stained cells could be a molecular signature of granulomas unlike periapical cysts.

Microarray Analysis of Gene Expression Alteration in Human Middle Ear Epithelial Cells Induced by Asian Sand Dust.

PubMed

Go, Yoon Young; Park, Moo Kyun; Kwon, Jee Young; Seo, Young Rok; Chae, Sung-Won; Song, Jae-Jun

2015-12-01

The primary aim of this study is to evaluate the gene expression profile of Asian sand dust (ASD)-treated human middle ear epithelial cell (HMEEC) using microarray analysis. The HMEEC was treated with ASD (400 µg/mL) and total RNA was extracted for microarray analysis. Molecular pathways among differentially expressed genes were further analyzed. For selected genes, the changes in gene expression were confirmed by real-time polymerase chain reaction. A total of 1,274 genes were differentially expressed by ASD. Among them, 1,138 genes were 2 folds up-regulated, whereas 136 genes were 2 folds down-regulated. Up-regulated genes were mainly involved in cellular processes, including apoptosis, cell differentiation, and cell proliferation. Down-regulated genes affected cellular processes, including apoptosis, cell cycle, cell differentiation, and cell proliferation. The 10 genes including ADM, CCL5, EDN1, EGR1, FOS, GHRL, JUN, SOCS3, TNF, and TNFSF10 were identified as main modulators in up-regulated genes. A total of 11 genes including CSF3, DKK1, FOSL1, FST, TERT, MMP13, PTHLH, SPRY2, TGFBR2, THBS1, and TIMP1 acted as main components of pathway associated with 2-fold down regulated genes. We identified the differentially expressed genes in ASD-treated HMEEC. Our work indicates that air pollutant like ASD, may play an important role in the pathogenesis of otitis media.

Cancer stem cell markers in patterning differentiation and in prognosis of oral squamous cell carcinoma.

PubMed

Mohanta, Simple; Siddappa, Gangotri; Valiyaveedan, Sindhu Govindan; Dodda Thimmasandra Ramanjanappa, Ravindra; Das, Debashish; Pandian, Ramanan; Khora, Samanta Sekhar; Kuriakose, Moni Abraham; Suresh, Amritha

2017-06-01

Differentiation is a major histological parameter determining tumor aggressiveness and prognosis of the patient; cancer stem cells with their slow dividing and undifferentiated nature might be one of the factors determining the same. This study aims to correlate cancer stem cell markers (CD44 and CD147) with tumor differentiation and evaluate their subsequent effect on prognosis. Immunohistochemical analysis in treatment naïve oral cancer patients (n = 53) indicated that the expression of CD147 was associated with poorly differentiated squamous cell carcinoma and moderately differentiated squamous cell carcinoma (p < 0.01). Furthermore, co-expression analysis showed that 45% each of moderately differentiated squamous cell carcinoma and poorly differentiated squamous cell carcinoma patients were CD44 high /CD147 high as compared to only 10% of patients with well-differentiated squamous cell carcinoma. A three-way analysis indicated that differentiation correlated with recurrence and survival (p < 0.05) in only the patients with CD44 high /CD147 high cohort. Subsequently, relevance of these cancer stem cell markers in patterning the differentiation characteristics was evaluated in oral squamous cell carcinoma cell lines originating from different grades of oral cancer. Flowcytometry-based analysis indicated an increase in CD44 + /CD147 + cells in cell lines of poorly differentiated squamous cell carcinoma (94.35 ± 1.14%, p < 0.001) and moderately differentiated squamous cell carcinoma origin (93.49 ± 0.47%, p < 0.001) as compared to cell line of well-differentiated squamous cell carcinoma origin (23.12% ± 0.49%). Expression profiling indicated higher expression of cancer stem cell and epithelial-mesenchymal transition markers in SCC029B (poorly differentiated squamous cell carcinoma originated; p ≤ 0.001), which was further translated into increased spheroid formation, migration, and invasion (p < 0.001) as compared to cell line of well-differentiated squamous cell carcinoma origin. This study suggests that CD44 and CD147 together improve the prognostic efficacy of tumor differentiation; in vitro results further point out that these markers might be determinant of differentiation characteristics, imparting properties of increased self-renewal, migration, and invasion.

microRNA expression profiling in fetal single ventricle malformation identified by deep sequencing.

PubMed

Yu, Zhang-Bin; Han, Shu-Ping; Bai, Yun-Fei; Zhu, Chun; Pan, Ya; Guo, Xi-Rong

2012-01-01

microRNAs (miRNAs) have emerged as key regulators in many biological processes, particularly cardiac growth and development, although the specific miRNA expression profile associated with this process remains to be elucidated. This study aimed to characterize the cellular microRNA profile involved in the development of congenital heart malformation, through the investigation of single ventricle (SV) defects. Comprehensive miRNA profiling in human fetal SV cardiac tissue was performed by deep sequencing. Differential expression of 48 miRNAs was revealed by sequencing by oligonucleotide ligation and detection (SOLiD) analysis. Of these, 38 were down-regulated and 10 were up-regulated in differentiated SV cardiac tissue, compared to control cardiac tissue. This was confirmed by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. Predicted target genes of the 48 differentially expressed miRNAs were analyzed by gene ontology and categorized according to cellular process, regulation of biological process and metabolic process. Pathway-Express analysis identified the WNT and mTOR signaling pathways as the most significant processes putatively affected by the differential expression of these miRNAs. The candidate genes involved in cardiac development were identified as potential targets for these differentially expressed microRNAs and the collaborative network of microRNAs and cardiac development related-mRNAs was constructed. These data provide the basis for future investigation of the mechanism of the occurrence and development of fetal SV malformations.

Microarray Meta-Analysis Identifies Acute Lung Injury Biomarkers in Donor Lungs That Predict Development of Primary Graft Failure in Recipients

PubMed Central

Haitsma, Jack J.; Furmli, Suleiman; Masoom, Hussain; Liu, Mingyao; Imai, Yumiko; Slutsky, Arthur S.; Beyene, Joseph; Greenwood, Celia M. T.; dos Santos, Claudia

2012-01-01

Objectives To perform a meta-analysis of gene expression microarray data from animal studies of lung injury, and to identify an injury-specific gene expression signature capable of predicting the development of lung injury in humans. Methods We performed a microarray meta-analysis using 77 microarray chips across six platforms, two species and different animal lung injury models exposed to lung injury with or/and without mechanical ventilation. Individual gene chips were classified and grouped based on the strategy used to induce lung injury. Effect size (change in gene expression) was calculated between non-injurious and injurious conditions comparing two main strategies to pool chips: (1) one-hit and (2) two-hit lung injury models. A random effects model was used to integrate individual effect sizes calculated from each experiment. Classification models were built using the gene expression signatures generated by the meta-analysis to predict the development of lung injury in human lung transplant recipients. Results Two injury-specific lists of differentially expressed genes generated from our meta-analysis of lung injury models were validated using external data sets and prospective data from animal models of ventilator-induced lung injury (VILI). Pathway analysis of gene sets revealed that both new and previously implicated VILI-related pathways are enriched with differentially regulated genes. Classification model based on gene expression signatures identified in animal models of lung injury predicted development of primary graft failure (PGF) in lung transplant recipients with larger than 80% accuracy based upon injury profiles from transplant donors. We also found that better classifier performance can be achieved by using meta-analysis to identify differentially-expressed genes than using single study-based differential analysis. Conclusion Taken together, our data suggests that microarray analysis of gene expression data allows for the detection of “injury" gene predictors that can classify lung injury samples and identify patients at risk for clinically relevant lung injury complications. PMID:23071521

Transcriptional profiling of murine osteoblast differentiation based on RNA-seq expression analyses.

PubMed

Khayal, Layal Abo; Grünhagen, Johannes; Provazník, Ivo; Mundlos, Stefan; Kornak, Uwe; Robinson, Peter N; Ott, Claus-Eric

2018-04-11

Osteoblastic differentiation is a multistep process characterized by osteogenic induction of mesenchymal stem cells, which then differentiate into proliferative pre-osteoblasts that produce copious amounts of extracellular matrix, followed by stiffening of the extracellular matrix, and matrix mineralization by hydroxylapatite deposition. Although these processes have been well characterized biologically, a detailed transcriptional analysis of murine primary calvaria osteoblast differentiation based on RNA sequencing (RNA-seq) analyses has not previously been reported. Here, we used RNA-seq to obtain expression values of 29,148 genes at four time points as murine primary calvaria osteoblasts differentiate in vitro until onset of mineralization was clearly detectable by microscopic inspection. Expression of marker genes confirmed osteogenic differentiation. We explored differential expression of 1386 protein-coding genes using unsupervised clustering and GO analyses. 100 differentially expressed lncRNAs were investigated by co-expression with protein-coding genes that are localized within the same topologically associated domain. Additionally, we monitored expression of 237 genes that are silent or active at distinct time points and compared differential exon usage. Our data represent an in-depth profiling of murine primary calvaria osteoblast differentiation by RNA-seq and contribute to our understanding of genetic regulation of this key process in osteoblast biology. Copyright © 2018 Elsevier Inc. All rights reserved.

Transcriptome profiling in engrailed-2 mutant mice reveals common molecular pathways associated with autism spectrum disorders.

PubMed

Sgadò, Paola; Provenzano, Giovanni; Dassi, Erik; Adami, Valentina; Zunino, Giulia; Genovesi, Sacha; Casarosa, Simona; Bozzi, Yuri

2013-12-19

Transcriptome analysis has been used in autism spectrum disorder (ASD) to unravel common pathogenic pathways based on the assumption that distinct rare genetic variants or epigenetic modifications affect common biological pathways. To unravel recurrent ASD-related neuropathological mechanisms, we took advantage of the En2-/- mouse model and performed transcriptome profiling on cerebellar and hippocampal adult tissues. Cerebellar and hippocampal tissue samples from three En2-/- and wild type (WT) littermate mice were assessed for differential gene expression using microarray hybridization followed by RankProd analysis. To identify functional categories overrepresented in the differentially expressed genes, we used integrated gene-network analysis, gene ontology enrichment and mouse phenotype ontology analysis. Furthermore, we performed direct enrichment analysis of ASD-associated genes from the SFARI repository in our differentially expressed genes. Given the limited number of animals used in the study, we used permissive criteria and identified 842 differentially expressed genes in En2-/- cerebellum and 862 in the En2-/- hippocampus. Our functional analysis revealed that the molecular signature of En2-/- cerebellum and hippocampus shares convergent pathological pathways with ASD, including abnormal synaptic transmission, altered developmental processes and increased immune response. Furthermore, when directly compared to the repository of the SFARI database, our differentially expressed genes in the hippocampus showed enrichment of ASD-associated genes significantly higher than previously reported. qPCR was performed for representative genes to confirm relative transcript levels compared to those detected in microarrays. Despite the limited number of animals used in the study, our bioinformatic analysis indicates the En2-/- mouse is a valuable tool for investigating molecular alterations related to ASD.

Identification of genes showing differential expression profile associated with growth rate in skeletal muscle tissue of Landrace weanling pig.

PubMed

Komatsu, Yuuta; Sukegawa, Shin; Yamashita, Mai; Katsuda, Naoki; Tong, Bin; Ohta, Takeshi; Kose, Hiroyuki; Yamada, Takahisa

2016-06-01

Suppression subtractive hybridization was used to identify genes showing differential expression profile associated with growth rate in skeletal muscle tissue of Landrace weanling pig. Two subtracted cDNA populations were generated from musculus longissimus muscle tissues of selected pigs with extreme expected breeding values at the age of 100 kg. Three upregulated genes (EEF1A2, TSG101 and TTN) and six downregulated genes (ATP5B, ATP5C1, COQ3, HADHA, MYH1 and MYH7) in pig with genetic propensity for higher growth rate were identified by sequence analysis of 12 differentially expressed clones selected by differential screening following the generation of the subtracted cDNA population. Real-time PCR analysis confirmed difference in expression profiles of the identified genes in musculus longissimus muscle tissues between the two Landrace weanling pig groups with divergent genetic propensity for growth rate. Further, differential expression of the identified genes except for the TTN was validated by Western blot analysis. Additionally, the eight genes other than the ATP5C1 colocalized with the same chromosomal positions as QTLs that have been previously identified for growth rate traits. Finally, the changes of expression predicted from gene function suggested association of upregulation of expression of the EEF1A2, TSG101 and TTN genes and downregulation of the ATP5B, ATP5C1, COQ3, HADHA, MYH1 and MYH7 gene expression with increased growth rate. The identified genes will provide an important insight in understanding the molecular mechanism underlying growth rate in Landrace pig breed.

MiR-133 is Involved in Estrogen Deficiency-Induced Osteoporosis through Modulating Osteogenic Differentiation of Mesenchymal Stem Cells.

PubMed

Lv, Hao; Sun, Yujie; Zhang, Yuchen

2015-05-27

MiR-133 expression is dysregulated in postmenopausal osteoporosis. However, its role in postmenopausal osteoporosis is still not well understood. In the current study, we explore how estrogen deficiency affects miR-133 expression and how miR-133 is involved in osteogenic differentiation of mesenchymal stem cells (MSCs). qRT-PCR analysis was performed to assess miR-133 expression in MSCs isolated from bone marrow of an ovariectomized (OVX) animal model and postmenopausal osteoporosis patients (PMOP) and their corresponding controls. The binding between miR-133 and predicted target SLC39A1 was verified using dual luciferase assay and Western blot analysis. The effect of miR-133 and SLC39A1 on osteogenic differentiation of MSCs was assessed through measuring alkaline phosphatase (ALP), mineralization nodules, and osteoblast-specific genes Runx2 and Osterix expression. miR-133 expression is significantly enhanced as a result of estrogen deficiency. Its overexpression is negatively correlated to osteogenic differentiation of hMSCs. SLC39A1 showed an inverse expression trend to miR-133 during the differentiation. miR-133 can directly target 3'UTR of SLC39A1 and thereby modulate its expression in hMSCs. The miR-133-SLC39A1 axis might play an important role in osteogenic differentiation of hMSCs. SLC39A1 can promote ALP activity and formation of mineralization nodules. In addition, SLC39A1 expression level is also positively correlated with RUNX2 and Osterix. Estrogen deficiency is associated with miR-133 overexpression. MiR-133 can induce postmenopausal osteoporosis by weakening osteogenic differentiation of hMSCs, at least partly through repressing SLC39A1 expression.

MiR-133 is Involved in Estrogen Deficiency-Induced Osteoporosis through Modulating Osteogenic Differentiation of Mesenchymal Stem Cells

PubMed Central

Lv, Hao; Sun, Yujie; Zhang, Yuchen

2015-01-01

Background MiR-133 expression is dysregulated in postmenopausal osteoporosis. However, its role in postmenopausal osteoporosis is still not well understood. In the current study, we explore how estrogen deficiency affects miR-133 expression and how miR-133 is involved in osteogenic differentiation of mesenchymal stem cells (MSCs). Material/Methods qRT-PCR analysis was performed to assess miR-133 expression in MSCs isolated from bone marrow of an ovariectomized (OVX) animal model and postmenopausal osteoporosis patients (PMOP) and their corresponding controls. The binding between miR-133 and predicted target SLC39A1 was verified using dual luciferase assay and Western blot analysis. The effect of miR-133 and SLC39A1 on osteogenic differentiation of MSCs was assessed through measuring alkaline phosphatase (ALP), mineralization nodules, and osteoblast-specific genes Runx2 and Osterix expression. Results miR-133 expression is significantly enhanced as a result of estrogen deficiency. Its overexpression is negatively correlated to osteogenic differentiation of hMSCs. SLC39A1 showed an inverse expression trend to miR-133 during the differentiation. miR-133 can directly target 3′UTR of SLC39A1 and thereby modulate its expression in hMSCs. The miR-133-SLC39A1 axis might play an important role in osteogenic differentiation of hMSCs. SLC39A1 can promote ALP activity and formation of mineralization nodules. In addition, SLC39A1 expression level is also positively correlated with RUNX2 and Osterix. Conclusions Estrogen deficiency is associated with miR-133 overexpression. MiR-133 can induce postmenopausal osteoporosis by weakening osteogenic differentiation of hMSCs, at least partly through repressing SLC39A1 expression. PMID:26013661

Acute hypoxia stress induced abundant differential expression genes and alternative splicing events in heart of tilapia.

PubMed

Xia, Jun Hong; Li, Hong Lian; Li, Bi Jun; Gu, Xiao Hui; Lin, Hao Ran

2018-01-10

Hypoxia is one of the critical environmental stressors for fish in aquatic environments. Although accumulating evidences indicate that gene expression is regulated by hypoxia stress in fish, how genes undergoing differential gene expression and/or alternative splicing (AS) in response to hypoxia stress in heart are not well understood. Using RNA-seq, we surveyed and detected 289 differential expressed genes (DEG) and 103 genes that undergo differential usage of exons and splice junctions events (DUES) in heart of a hypoxia tolerant fish, Nile tilapia, Oreochromis niloticus following 12h hypoxic treatment. The spatio-temporal expression analysis validated the significant association of differential exon usages in two randomly selected DUES genes (fam162a and ndrg2) in 5 tissues (heart, liver, brain, gill and spleen) sampled at three time points (6h, 12h, and 24h) under acute hypoxia treatment. Functional analysis significantly associated the differential expressed genes with the categories related to energy conservation, protein synthesis and immune response. Different enrichment categories were found between the DEG and DUES dataset. The Isomerase activity, Oxidoreductase activity, Glycolysis and Oxidative stress process were significantly enriched for the DEG gene dataset, but the Structural constituent of ribosome and Structural molecule activity, Ribosomal protein and RNA binding protein were significantly enriched only for the DUES genes. Our comparative transcriptomic analysis reveals abundant stress responsive genes and their differential regulation function in the heart tissues of Nile tilapia under acute hypoxia stress. Our findings will facilitate future investigation on transcriptome complexity and AS regulation during hypoxia stress in fish. Copyright © 2017 Elsevier B.V. All rights reserved.

The statistics of identifying differentially expressed genes in Expresso and TM4: a comparison

PubMed Central

Sioson, Allan A; Mane, Shrinivasrao P; Li, Pinghua; Sha, Wei; Heath, Lenwood S; Bohnert, Hans J; Grene, Ruth

2006-01-01

Background Analysis of DNA microarray data takes as input spot intensity measurements from scanner software and returns differential expression of genes between two conditions, together with a statistical significance assessment. This process typically consists of two steps: data normalization and identification of differentially expressed genes through statistical analysis. The Expresso microarray experiment management system implements these steps with a two-stage, log-linear ANOVA mixed model technique, tailored to individual experimental designs. The complement of tools in TM4, on the other hand, is based on a number of preset design choices that limit its flexibility. In the TM4 microarray analysis suite, normalization, filter, and analysis methods form an analysis pipeline. TM4 computes integrated intensity values (IIV) from the average intensities and spot pixel counts returned by the scanner software as input to its normalization steps. By contrast, Expresso can use either IIV data or median intensity values (MIV). Here, we compare Expresso and TM4 analysis of two experiments and assess the results against qRT-PCR data. Results The Expresso analysis using MIV data consistently identifies more genes as differentially expressed, when compared to Expresso analysis with IIV data. The typical TM4 normalization and filtering pipeline corrects systematic intensity-specific bias on a per microarray basis. Subsequent statistical analysis with Expresso or a TM4 t-test can effectively identify differentially expressed genes. The best agreement with qRT-PCR data is obtained through the use of Expresso analysis and MIV data. Conclusion The results of this research are of practical value to biologists who analyze microarray data sets. The TM4 normalization and filtering pipeline corrects microarray-specific systematic bias and complements the normalization stage in Expresso analysis. The results of Expresso using MIV data have the best agreement with qRT-PCR results. In one experiment, MIV is a better choice than IIV as input to data normalization and statistical analysis methods, as it yields as greater number of statistically significant differentially expressed genes; TM4 does not support the choice of MIV input data. Overall, the more flexible and extensive statistical models of Expresso achieve more accurate analytical results, when judged by the yardstick of qRT-PCR data, in the context of an experimental design of modest complexity. PMID:16626497

MiR-27a is Essential for the Shift from Osteogenic Differentiation to Adipogenic Differentiation of Mesenchymal Stem Cells in Postmenopausal Osteoporosis.

PubMed

You, Li; Pan, Ling; Chen, Lin; Gu, Wensha; Chen, Jinyu

2016-01-01

Osteoporosis is a progressive bone disease characterized by a decrease in bone mass and density, which results in an increased risk of fractures. Mesenchymal stem cells (MSCs) are progenitor cells that can differentiate into osteoblasts, osteocytes and adipocytes in bone and fat formation. A reduction in the differentiation of MSCs into osteoblasts contributes to the impaired bone formation observed in osteoporosis. MicroRNAs (miRNAs) play a regulatory role in osteogenesis and MSC differentiation. MiR-27a has been reported to be down-regulated in the development of osteoporosis and during adipogenic differentiation. In this study, a miRNA microarray analysis was used to investigate expression profiles of miRNA in the serum of osteoporotic patients and healthy controls and this data was validated by quantitative real-time PCR (qRT-PCR). MSCs isolated from human and mice with miR-27a inhibition or overexpression were induced to differentiate into osteoblasts or adipocytes. TargetScan and PicTar were used to predict the target gene of miR-27a. The mRNA or protein levels of several specific proteins in MSCs were detected using qRT-PCR or western blot analysis. Ovariectomized mice were used as in vivo model of human postmenopausal osteoporosis for bone mineral density measurement, micro-CT analysis and histomorphometric analysis. Here, we analyzed the role of miR-27a in bone metabolism. Microarray analysis indicated that miR-27a expression was significantly reduced in osteoporotic patients. Analysis on MSCs derived from patients with osteoporosis indicated that osteoblastogenesis was reduced, whereas adipogenesis was increased. MSCs that had undergone osteoblast induction showed a significant increase in miR-27a expression, whereas cells that had undergone adipocyte induction showed a significant decrease in miR-27a expression, indicating that miR-27a was essential for MSC differentiation. We demonstrated that myocyte enhancer factor 2 c (Mef2c), a transcription factor, was the direct target of miR-27a using a dual luciferase assay. An inverse relationship between miR-27a expression and Mef2c expression in osteoporotic patients was shown. Silencing of miR-27a decreased bone formation, confirming the role of miR-27a in bone formation in vivo. In summary, miR-27a was essential for the shift of MSCs from osteogenic differentiation to adipogenic differentiation in osteoporosis by targeting Mef2c. © 2016 S. Karger AG, Basel.

How to normalize metatranscriptomic count data for differential expression analysis.

PubMed

Klingenberg, Heiner; Meinicke, Peter

2017-01-01

Differential expression analysis on the basis of RNA-Seq count data has become a standard tool in transcriptomics. Several studies have shown that prior normalization of the data is crucial for a reliable detection of transcriptional differences. Until now it has not been clear whether and how the transcriptomic approach can be used for differential expression analysis in metatranscriptomics. We propose a model for differential expression in metatranscriptomics that explicitly accounts for variations in the taxonomic composition of transcripts across different samples. As a main consequence the correct normalization of metatranscriptomic count data under this model requires the taxonomic separation of the data into organism-specific bins. Then the taxon-specific scaling of organism profiles yields a valid normalization and allows us to recombine the scaled profiles into a metatranscriptomic count matrix. This matrix can then be analyzed with statistical tools for transcriptomic count data. For taxon-specific scaling and recombination of scaled counts we provide a simple R script. When applying transcriptomic tools for differential expression analysis directly to metatranscriptomic data with an organism-independent (global) scaling of counts the resulting differences may be difficult to interpret. The differences may correspond to changing functional profiles of the contributing organisms but may also result from a variation of taxonomic abundances. Taxon-specific scaling eliminates this variation and therefore the resulting differences actually reflect a different behavior of organisms under changing conditions. In simulation studies we show that the divergence between results from global and taxon-specific scaling can be drastic. In particular, the variation of organism abundances can imply a considerable increase of significant differences with global scaling. Also, on real metatranscriptomic data, the predictions from taxon-specific and global scaling can differ widely. Our studies indicate that in real data applications performed with global scaling it might be impossible to distinguish between differential expression in terms of transcriptomic changes and differential composition in terms of changing taxonomic proportions. As in transcriptomics, a proper normalization of count data is also essential for differential expression analysis in metatranscriptomics. Our model implies a taxon-specific scaling of counts for normalization of the data. The application of taxon-specific scaling consequently removes taxonomic composition variations from functional profiles and therefore provides a clear interpretation of the observed functional differences.

The Small-RNA Profiles of Almond (Prunus dulcis Mill.) Reproductive Tissues in Response to Cold Stress.

PubMed

Karimi, Marzieh; Ghazanfari, Farahnaz; Fadaei, Adeleh; Ahmadi, Laleh; Shiran, Behrouz; Rabei, Mohammad; Fallahi, Hossein

2016-01-01

Spring frost is an important environmental stress that threatens the production of Prunus trees. However, little information is available regarding molecular response of these plants to the frost stress. Using high throughput sequencing, this study was conducted to identify differentially expressed miRNAs, both the conserved and the non-conserved ones, in the reproductive tissues of almond tolerant H genotype under cold stress. Analysis of 50 to 58 million raw reads led to identification of 174 unique conserved and 59 novel microRNAs (miRNAs). Differential expression pattern analysis showed that 50 miRNA families were expressed differentially in one or both of almond reproductive tissues (anther and ovary). Out of these 50 miRNA families, 12 and 15 displayed up-regulation and down-regulation, respectively. The distribution of conserved miRNA families indicated that miR482f harbor the highest number of members. Confirmation of miRNAs expression patterns by quantitative real- time PCR (qPCR) was performed in cold tolerant (H genotype) alongside a sensitive variety (Sh12 genotype). Our analysis revealed differential expression for 9 miRNAs in anther and 3 miRNAs in ovary between these two varieties. Target prediction of miRNAs followed by differential expression analysis resulted in identification of 83 target genes, mostly transcription factors. This study comprehensively catalogued expressed miRNAs under different temperatures in two reproductive tissues (anther and ovary). Results of current study and the previous RNA-seq study, which was conducted in the same tissues by our group, provide a unique opportunity to understand the molecular basis of responses of almond to cold stress. The results can also enhance the possibility for gene manipulation to develop cold tolerant plants.

The Small-RNA Profiles of Almond (Prunus dulcis Mill.) Reproductive Tissues in Response to Cold Stress

PubMed Central

Shiran, Behrouz; Rabei, Mohammad; Fallahi, Hossein

2016-01-01

Spring frost is an important environmental stress that threatens the production of Prunus trees. However, little information is available regarding molecular response of these plants to the frost stress. Using high throughput sequencing, this study was conducted to identify differentially expressed miRNAs, both the conserved and the non-conserved ones, in the reproductive tissues of almond tolerant H genotype under cold stress. Analysis of 50 to 58 million raw reads led to identification of 174 unique conserved and 59 novel microRNAs (miRNAs). Differential expression pattern analysis showed that 50 miRNA families were expressed differentially in one or both of almond reproductive tissues (anther and ovary). Out of these 50 miRNA families, 12 and 15 displayed up-regulation and down-regulation, respectively. The distribution of conserved miRNA families indicated that miR482f harbor the highest number of members. Confirmation of miRNAs expression patterns by quantitative real- time PCR (qPCR) was performed in cold tolerant (H genotype) alongside a sensitive variety (Sh12 genotype). Our analysis revealed differential expression for 9 miRNAs in anther and 3 miRNAs in ovary between these two varieties. Target prediction of miRNAs followed by differential expression analysis resulted in identification of 83 target genes, mostly transcription factors. This study comprehensively catalogued expressed miRNAs under different temperatures in two reproductive tissues (anther and ovary). Results of current study and the previous RNA-seq study, which was conducted in the same tissues by our group, provide a unique opportunity to understand the molecular basis of responses of almond to cold stress. The results can also enhance the possibility for gene manipulation to develop cold tolerant plants. PMID:27253370

Identification of the transcriptional regulators by expression profiling infected with hepatitis B virus.

PubMed

Chai, Xiaoqiang; Han, Yanan; Yang, Jian; Zhao, Xianxian; Liu, Yewang; Hou, Xugang; Tang, Yiheng; Zhao, Shirong; Li, Xiao

2016-02-01

The molecular pathogenesis of infection by hepatitis B virus with human is extremely complex and heterogeneous. To date the molecular information is not clearly defined despite intensive research efforts. Thus, studies aimed at transcription and regulation during virus infection or combined researches of those already known to be beneficial are needed. With the purpose of identifying the transcriptional regulators related to infection of hepatitis B virus in gene level, the gene expression profiles from some normal individuals and hepatitis B patients were analyzed in our study. In this work, the differential expressed genes were selected primarily. The several genes among those were validated in an independent set by qRT-PCR. Then the differentially co-expression analysis was conducted to identify differentially co-expressed links and differential co-expressed genes. Next, the analysis of the regulatory impact factors was performed through mapping the links and regulatory data. In order to give a further insight to these regulators, the co-expression gene modules were identified using a threshold-based hierarchical clustering method. Incidentally, the construction of the regulatory network was generated using the computer software. A total of 137,284 differentially co-expressed links and 780 differential co-expressed genes were identified. These co-expressed genes were significantly enriched inflammatory response. The results of regulatory impact factors revealed several crucial regulators related to hepatocellular carcinoma and other high-rank regulators. Meanwhile, more than one hundred co-expression gene modules were identified using clustering method. In our study, some important transcriptional regulators were identified using a computational method, which may enhance the understanding of disease mechanisms and lead to an improved treatment of hepatitis B. However, further experimental studies are required to confirm these findings. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Gene expression profile in cardiovascular disease and preeclampsia: a meta-analysis of the transcriptome based on raw data from human studies deposited in Gene Expression Omnibus.

PubMed

Sitras, V; Fenton, C; Acharya, G

2015-02-01

Cardiovascular disease (CVD) and preeclampsia (PE) share common clinical features. We aimed to identify common transcriptomic signatures involved in CVD and PE in humans. Meta-analysis of individual raw microarray data deposited in GEO, obtained from blood samples of patients with CVD versus controls and placental samples from women with PE versus healthy women with uncomplicated pregnancies. Annotation of cases versus control samples was taken directly from the microarray documentation. Genes that showed a significant differential expression in the majority of experiments were selected for subsequent analysis. Hypergeometric gene list analysis was performed using Bioconductor GOstats package. Bioinformatic analysis was performed in PANTHER. Seven studies in CVD and 5 studies in PE were eligible for meta-analysis. A total of 181 genes were found to be differentially expressed in microarray studies investigating gene expression in blood samples obtained from patients with CVD compared to controls and 925 genes were differentially expressed between preeclamptic and healthy placentas. Among these differentially expressed genes, 22 were common between CVD and PE. Bioinformatic analysis of these genes revealed oxidative stress, p-53 pathway feedback, inflammation mediated by chemokines and cytokines, interleukin signaling, B-cell activation, PDGF signaling, Wnt signaling, integrin signaling and Alzheimer disease pathways to be involved in the pathophysiology of both CVD and PE. Metabolism, development, response to stimulus, immune response and cell communication were the associated biologic processes in both conditions. Gene set enrichment analysis showed the following overlapping pathways between CVD and PE: TGF-β-signaling, apoptosis, graft-versus-host disease, allograft rejection, chemokine signaling, steroid hormone synthesis, type I and II diabetes mellitus, VEGF signaling, pathways in cancer, GNRH signaling, Huntingtons disease and Notch signaling. CVD and PE share same common traits in their gene expression profile indicating common pathways in their pathophysiology. Copyright © 2014 Elsevier Ltd. All rights reserved.

Gene expression of runx2, Osterix, c-fos, DLX-3, DLX-5, and MSX-2 in dental follicle cells during osteogenic differentiation in vitro.

PubMed

Morsczeck, C

2006-02-01

Recently, osteogenic precursor cells were isolated from human dental follicles, which differentiate into cementoblast- or osteoblast- like cells under in vitro conditions. However, mechanisms for osteogenic differentiation are not known in detail. Dental follicle cell long-term cultures supplemented with dexamethasone or with insulin resulted in mineralized nodules, whereas no mineralization or alkaline phosphatase activity was detected in the control culture without an osteogenic stimulus. A real-time reverse-transcriptase polymerase chain reaction (PCR) analysis was developed to investigate gene expression during osteogenic differentiation in vitro. Expression of the alkaline phosphatase (ALP) gene was detected during differentiation in the control culture and was similar to that in cultures with dexamethasone and insulin. DLX-3, DLX-5, runx2, and MSX-2 are differentially expressed during osteogenic differentiation in bone marrow mesenchymal stem cells. In dental follicle cells, gene expression of runx2, DLX-5, and MSX-2 was unaffected during osteogenic differentiation in vitro. Osteogenic differentiation appeared to be independent of MSX-2 expression; the same was true of runx2 and DLX-5, which were protagonists of osteogenic differentiation and osteocalcin promoter activity in bone marrow mesenchymal stem cells. Like in bone marrow-derived stem cells, DLX-3 gene expression was increased in dental follicle cells during osteogenic differentiation but similar to control cultures. However, gene expression of osterix was not detected in dental follicle cells during osteogenic differentiation; this gene is expressed during osteogenic differentiation in bone marrow stem cells. These real-time PCR results display molecular mechanisms in dental follicle precursor cells during osteogenic differentiation that are different from those in bone marrow-derived mesenchymal stem cells.

Biological mechanism analysis of acute renal allograft rejection: integrated of mRNA and microRNA expression profiles.

PubMed

Huang, Shi-Ming; Zhao, Xia; Zhao, Xue-Mei; Wang, Xiao-Ying; Li, Shan-Shan; Zhu, Yu-Hui

2014-01-01

Renal transplantation is the preferred method for most patients with end-stage renal disease, however, acute renal allograft rejection is still a major risk factor for recipients leading to renal injury. To improve the early diagnosis and treatment of acute rejection, study on the molecular mechanism of it is urgent. MicroRNA (miRNA) expression profile and mRNA expression profile of acute renal allograft rejection and well-functioning allograft downloaded from ArrayExpress database were applied to identify differentially expressed (DE) miRNAs and DE mRNAs. DE miRNAs targets were predicted by combining five algorithm. By overlapping the DE mRNAs and DE miRNAs targets, common genes were obtained. Differentially co-expressed genes (DCGs) were identified by differential co-expression profile (DCp) and differential co-expression enrichment (DCe) methods in Differentially Co-expressed Genes and Links (DCGL) package. Then, co-expression network of DCGs and the cluster analysis were performed. Functional enrichment analysis for DCGs was undergone. A total of 1270 miRNA targets were predicted and 698 DE mRNAs were obtained. While overlapping miRNA targets and DE mRNAs, 59 common genes were gained. We obtained 103 DCGs and 5 transcription factors (TFs) based on regulatory impact factors (RIF), then built the regulation network of miRNA targets and DE mRNAs. By clustering the co-expression network, 5 modules were obtained. Thereinto, module 1 had the highest degree and module 2 showed the most number of DCGs and common genes. TF CEBPB and several common genes, such as RXRA, BASP1 and AKAP10, were mapped on the co-expression network. C1R showed the highest degree in the network. These genes might be associated with human acute renal allograft rejection. We conducted biological analysis on integration of DE mRNA and DE miRNA in acute renal allograft rejection, displayed gene expression patterns and screened out genes and TFs that may be related to acute renal allograft rejection.

Biological mechanism analysis of acute renal allograft rejection: integrated of mRNA and microRNA expression profiles

PubMed Central

Huang, Shi-Ming; Zhao, Xia; Zhao, Xue-Mei; Wang, Xiao-Ying; Li, Shan-Shan; Zhu, Yu-Hui

2014-01-01

Objectives: Renal transplantation is the preferred method for most patients with end-stage renal disease, however, acute renal allograft rejection is still a major risk factor for recipients leading to renal injury. To improve the early diagnosis and treatment of acute rejection, study on the molecular mechanism of it is urgent. Methods: MicroRNA (miRNA) expression profile and mRNA expression profile of acute renal allograft rejection and well-functioning allograft downloaded from ArrayExpress database were applied to identify differentially expressed (DE) miRNAs and DE mRNAs. DE miRNAs targets were predicted by combining five algorithm. By overlapping the DE mRNAs and DE miRNAs targets, common genes were obtained. Differentially co-expressed genes (DCGs) were identified by differential co-expression profile (DCp) and differential co-expression enrichment (DCe) methods in Differentially Co-expressed Genes and Links (DCGL) package. Then, co-expression network of DCGs and the cluster analysis were performed. Functional enrichment analysis for DCGs was undergone. Results: A total of 1270 miRNA targets were predicted and 698 DE mRNAs were obtained. While overlapping miRNA targets and DE mRNAs, 59 common genes were gained. We obtained 103 DCGs and 5 transcription factors (TFs) based on regulatory impact factors (RIF), then built the regulation network of miRNA targets and DE mRNAs. By clustering the co-expression network, 5 modules were obtained. Thereinto, module 1 had the highest degree and module 2 showed the most number of DCGs and common genes. TF CEBPB and several common genes, such as RXRA, BASP1 and AKAP10, were mapped on the co-expression network. C1R showed the highest degree in the network. These genes might be associated with human acute renal allograft rejection. Conclusions: We conducted biological analysis on integration of DE mRNA and DE miRNA in acute renal allograft rejection, displayed gene expression patterns and screened out genes and TFs that may be related to acute renal allograft rejection. PMID:25664019

Order Under Uncertainty: Robust Differential Expression Analysis Using Probabilistic Models for Pseudotime Inference

PubMed Central

Campbell, Kieran R.

2016-01-01

Single cell gene expression profiling can be used to quantify transcriptional dynamics in temporal processes, such as cell differentiation, using computational methods to label each cell with a ‘pseudotime’ where true time series experimentation is too difficult to perform. However, owing to the high variability in gene expression between individual cells, there is an inherent uncertainty in the precise temporal ordering of the cells. Pre-existing methods for pseudotime estimation have predominantly given point estimates precluding a rigorous analysis of the implications of uncertainty. We use probabilistic modelling techniques to quantify pseudotime uncertainty and propagate this into downstream differential expression analysis. We demonstrate that reliance on a point estimate of pseudotime can lead to inflated false discovery rates and that probabilistic approaches provide greater robustness and measures of the temporal resolution that can be obtained from pseudotime inference. PMID:27870852

Polyester: simulating RNA-seq datasets with differential transcript expression.

PubMed

Frazee, Alyssa C; Jaffe, Andrew E; Langmead, Ben; Leek, Jeffrey T

2015-09-01

Statistical methods development for differential expression analysis of RNA sequencing (RNA-seq) requires software tools to assess accuracy and error rate control. Since true differential expression status is often unknown in experimental datasets, artificially constructed datasets must be utilized, either by generating costly spike-in experiments or by simulating RNA-seq data. Polyester is an R package designed to simulate RNA-seq data, beginning with an experimental design and ending with collections of RNA-seq reads. Its main advantage is the ability to simulate reads indicating isoform-level differential expression across biological replicates for a variety of experimental designs. Data generated by Polyester is a reasonable approximation to real RNA-seq data and standard differential expression workflows can recover differential expression set in the simulation by the user. Polyester is freely available from Bioconductor (http://bioconductor.org/). jtleek@gmail.com Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Molecular mechanism of G1 arrest and cellular senescence induced by LEE011, a novel CDK4/CDK6 inhibitor, in leukemia cells.

PubMed

Tao, Yan-Fang; Wang, Na-Na; Xu, Li-Xiao; Li, Zhi-Heng; Li, Xiao-Lu; Xu, Yun-Yun; Fang, Fang; Li, Mei; Qian, Guang-Hui; Li, Yan-Hong; Li, Yi-Ping; Wu, Yi; Ren, Jun-Li; Du, Wei-Wei; Lu, Jun; Feng, Xing; Wang, Jian; He, Wei-Qi; Hu, Shao-Yan; Pan, Jian

2017-01-01

Overexpression of cyclin D1 dependent kinases 4 and 6 (CDK4/6) is a common feature of many human cancers including leukemia. LEE011 is a novel inhibitor of both CDK4 and 6. To date, the molecular function of LEE011 in leukemia remains unclear. Leukemia cell growth and apoptosis following LEE011 treatment was assessed through CCK-8 and annexin V/propidium iodide staining assays. Cell senescence was assessed by β-galactosidase staining and p16 INK4a expression analysis. Gene expression profiles of LEE011 treated HL-60 cells were investigated using an Arraystar Human LncRNA array. Gene ontology and KEGG pathway analysis were then used to analyze the differentially expressed genes from the cluster analysis. Our studies demonstrated that LEE011 inhibited proliferation of leukemia cells and could induce apoptosis. Hoechst 33,342 staining analysis showed DNA fragmentation and distortion of nuclear structures following LEE011 treatment. Cell cycle analysis showed LEE011 significantly induced cell cycle G 1 arrest in seven of eight acute leukemia cells lines, the exception being THP-1 cells. β-Galactosidase staining analysis and p16 INK4a expression analysis showed that LEE011 treatment can induce cell senescence of leukemia cells. LncRNA microarray analysis showed 2083 differentially expressed mRNAs and 3224 differentially expressed lncRNAs in LEE011-treated HL-60 cells compared with controls. Molecular function analysis showed that LEE011 induced senescence in leukemia cells partially through downregulation of the transcriptional expression of MYBL2. We demonstrate for the first time that LEE011 treatment results in inhibition of cell proliferation and induction of G 1 arrest and cellular senescence in leukemia cells. LncRNA microarray analysis showed differentially expressed mRNAs and lncRNAs in LEE011-treated HL-60 cells and we demonstrated that LEE011 induces cellular senescence partially through downregulation of the expression of MYBL2. These results may open new lines of investigation regarding the molecular mechanism of LEE011 induced cellular senescence.

Genome-wide expression profiling of in vivo-derived bloodstream parasite stages and dynamic analysis of mRNA alterations during synchronous differentiation in Trypanosoma brucei

PubMed Central

Kabani, Sarah; Fenn, Katelyn; Ross, Alan; Ivens, Al; Smith, Terry K; Ghazal, Peter; Matthews, Keith

2009-01-01

Background Trypanosomes undergo extensive developmental changes during their complex life cycle. Crucial among these is the transition between slender and stumpy bloodstream forms and, thereafter, the differentiation from stumpy to tsetse-midgut procyclic forms. These developmental events are highly regulated, temporally reproducible and accompanied by expression changes mediated almost exclusively at the post-transcriptional level. Results In this study we have examined, by whole-genome microarray analysis, the mRNA abundance of genes in slender and stumpy forms of T.brucei AnTat1.1 cells, and also during their synchronous differentiation to procyclic forms. In total, five biological replicates representing the differentiation of matched parasite populations derived from five individual mouse infections were assayed, with RNAs being derived at key biological time points during the time course of their synchronous differentiation to procyclic forms. Importantly, the biological context of these mRNA profiles was established by assaying the coincident cellular events in each population (surface antigen exchange, morphological restructuring, cell cycle re-entry), thereby linking the observed gene expression changes to the well-established framework of trypanosome differentiation. Conclusion Using stringent statistical analysis and validation of the derived profiles against experimentally-predicted gene expression and phenotypic changes, we have established the profile of regulated gene expression during these important life-cycle transitions. The highly synchronous nature of differentiation between stumpy and procyclic forms also means that these studies of mRNA profiles are directly relevant to the changes in mRNA abundance within individual cells during this well-characterised developmental transition. PMID:19747379

BFDCA: A Comprehensive Tool of Using Bayes Factor for Differential Co-Expression Analysis.

PubMed

Wang, Duolin; Wang, Juexin; Jiang, Yuexu; Liang, Yanchun; Xu, Dong

2017-02-03

Comparing the gene-expression profiles between biological conditions is useful for understanding gene regulation underlying complex phenotypes. Along this line, analysis of differential co-expression (DC) has gained attention in the recent years, where genes under one condition have different co-expression patterns compared with another. We developed an R package Bayes Factor approach for Differential Co-expression Analysis (BFDCA) for DC analysis. BFDCA is unique in integrating various aspects of DC patterns (including Shift, Cross, and Re-wiring) into one uniform Bayes factor. We tested BFDCA using simulation data and experimental data. Simulation results indicate that BFDCA outperforms existing methods in accuracy and robustness of detecting DC pairs and DC modules. Results of using experimental data suggest that BFDCA can cluster disease-related genes into functional DC subunits and estimate the regulatory impact of disease-related genes well. BFDCA also achieves high accuracy in predicting case-control phenotypes by using significant DC gene pairs as markers. BFDCA is publicly available at http://dx.doi.org/10.17632/jdz4vtvnm3.1. Copyright © 2016 Elsevier Ltd. All rights reserved.

Estimating differential expression from multiple indicators

PubMed Central

Ilmjärv, Sten; Hundahl, Christian Ansgar; Reimets, Riin; Niitsoo, Margus; Kolde, Raivo; Vilo, Jaak; Vasar, Eero; Luuk, Hendrik

2014-01-01

Regardless of the advent of high-throughput sequencing, microarrays remain central in current biomedical research. Conventional microarray analysis pipelines apply data reduction before the estimation of differential expression, which is likely to render the estimates susceptible to noise from signal summarization and reduce statistical power. We present a probe-level framework, which capitalizes on the high number of concurrent measurements to provide more robust differential expression estimates. The framework naturally extends to various experimental designs and target categories (e.g. transcripts, genes, genomic regions) as well as small sample sizes. Benchmarking in relation to popular microarray and RNA-sequencing data-analysis pipelines indicated high and stable performance on the Microarray Quality Control dataset and in a cell-culture model of hypoxia. Experimental-data-exhibiting long-range epigenetic silencing of gene expression was used to demonstrate the efficacy of detecting differential expression of genomic regions, a level of analysis not embraced by conventional workflows. Finally, we designed and conducted an experiment to identify hypothermia-responsive genes in terms of monotonic time-response. As a novel insight, hypothermia-dependent up-regulation of multiple genes of two major antioxidant pathways was identified and verified by quantitative real-time PCR. PMID:24586062

Gene expression profiling in whole blood of patients with coronary artery disease

PubMed Central

Taurino, Chiara; Miller, William H.; McBride, Martin W.; McClure, John D.; Khanin, Raya; Moreno, María U.; Dymott, Jane A.; Delles, Christian; Dominiczak, Anna F.

2010-01-01

Owing to the dynamic nature of the transcriptome, gene expression profiling is a promising tool for discovery of disease-related genes and biological pathways. In the present study, we examined gene expression in whole blood of 12 patients with CAD (coronary artery disease) and 12 healthy control subjects. Furthermore, ten patients with CAD underwent whole-blood gene expression analysis before and after the completion of a cardiac rehabilitation programme following surgical coronary revascularization. mRNA and miRNA (microRNA) were isolated for expression profiling. Gene expression analysis identified 365 differentially expressed genes in patients with CAD compared with healthy controls (175 up- and 190 down-regulated in CAD), and 645 in CAD rehabilitation patients (196 up- and 449 down-regulated post-rehabilitation). Biological pathway analysis identified a number of canonical pathways, including oxidative phosphorylation and mitochondrial function, as being significantly and consistently modulated across the groups. Analysis of miRNA expression revealed a number of differentially expressed miRNAs, including hsa-miR-140-3p (control compared with CAD, P=0.017), hsa-miR-182 (control compared with CAD, P=0.093), hsa-miR-92a and hsa-miR-92b (post- compared with pre-exercise, P<0.01). Global analysis of predicted miRNA targets found significantly reduced expression of genes with target regions compared with those without: hsa-miR-140-3p (P=0.002), hsa-miR-182 (P=0.001), hsa-miR-92a and hsa-miR-92b (P=2.2×10−16). In conclusion, using whole blood as a ‘surrogate tissue’ in patients with CAD, we have identified differentially expressed miRNAs, differentially regulated genes and modulated pathways which warrant further investigation in the setting of cardiovascular function. This approach may represent a novel non-invasive strategy to unravel potentially modifiable pathways and possible therapeutic targets in cardiovascular disease. PMID:20528768

Microarray expression profiling and co-expression network analysis of circulating LncRNAs and mRNAs associated with neurotoxicity induced by BPA.

PubMed

Pang, Wei; Lian, Fu-Zhi; Leng, Xue; Wang, Shu-Min; Li, Yi-Bo; Wang, Zi-Yu; Li, Kai-Ren; Gao, Zhi-Xian; Jiang, Yu-Gang

2018-05-01

A growing body of evidence has shown bisphenol A (BPA), an estrogen-like industrial chemical, has adverse effects on the nervous system. In this study, we investigated the transcriptional behavior of long non-coding RNAs (lncRNAs) and mRNAs to provide the information to explore neurotoxic effects induced by BPA. By microarray expression profiling, we discovered 151 differentially expressed lncRNAs and 794 differentially expressed mRNAs in the BPA intervention group compared with the control group. Gene ontology analysis indicated the differentially expressed mRNAs were mainly involved in fundamental metabolic processes and physiological and pathological conditions, such as development, synaptic transmission, homeostasis, injury, and neuroinflammation responses. In the expression network of the BPA-induced group, a great number of nodes and connections were found in comparison to the control-derived network. We identified lncRNAs that were aberrantly expressed in the BPA group, among which, growth arrest specific 5 (GAS5) might participate in the BPA-induced neurotoxicity by regulating Jun, RAS, and other pathways indirectly through these differentially expressed genes. This study provides the first investigation of genome-wide lncRNA expression and correlation between lncRNA and mRNA expression in the BPA-induced neurotoxicity. Our results suggest that the elevated expression of lncRNAs is a major biomarker in the neurotoxicity induced by BPA.

RNA-seq analysis of the gonadal transcriptome during Alligator mississippiensis temperature-dependent sex determination and differentiation.

PubMed

Yatsu, Ryohei; Miyagawa, Shinichi; Kohno, Satomi; Parrott, Benjamin B; Yamaguchi, Katsushi; Ogino, Yukiko; Miyakawa, Hitoshi; Lowers, Russell H; Shigenobu, Shuji; Guillette, Louis J; Iguchi, Taisen

2016-01-25

The American alligator (Alligator mississippiensis) displays temperature-dependent sex determination (TSD), in which incubation temperature during embryonic development determines the sexual fate of the individual. However, the molecular mechanisms governing this process remain a mystery, including the influence of initial environmental temperature on the comprehensive gonadal gene expression patterns occurring during TSD. Our characterization of transcriptomes during alligator TSD allowed us to identify novel candidate genes involved in TSD initiation. High-throughput RNA sequencing (RNA-seq) was performed on gonads collected from A. mississippiensis embryos incubated at both a male and a female producing temperature (33.5 °C and 30 °C, respectively) in a time series during sexual development. RNA-seq yielded 375.2 million paired-end reads, which were mapped and assembled, and used to characterize differential gene expression. Changes in the transcriptome occurring as a function of both development and sexual differentiation were extensively profiled. Forty-one differentially expressed genes were detected in response to incubation at male producing temperature, and included genes such as Wnt signaling factor WNT11, histone demethylase KDM6B, and transcription factor C/EBPA. Furthermore, comparative analysis of development- and sex-dependent differential gene expression revealed 230 candidate genes involved in alligator sex determination and differentiation, and early details of the suspected male-fate commitment were profiled. We also discovered sexually dimorphic expression of uncharacterized ncRNAs and other novel elements, such as unique expression patterns of HEMGN and ARX. Twenty-five of the differentially expressed genes identified in our analysis were putative transcriptional regulators, among which were MYBL2, MYCL, and HOXC10, in addition to conventional sex differentiation genes such as SOX9, and FOXL2. Inferred gene regulatory network was constructed, and the gene-gene and temperature-gene interactions were predicted. Gonadal global gene expression kinetics during sex determination has been extensively profiled for the first time in a TSD species. These findings provide insights into the genetic framework underlying TSD, and expand our current understanding of the developmental fate pathways during vertebrate sex determination.

A novel method to identify pathways associated with renal cell carcinoma based on a gene co-expression network

PubMed Central

RUAN, XIYUN; LI, HONGYUN; LIU, BO; CHEN, JIE; ZHANG, SHIBAO; SUN, ZEQIANG; LIU, SHUANGQING; SUN, FAHAI; LIU, QINGYONG

2015-01-01

The aim of the present study was to develop a novel method for identifying pathways associated with renal cell carcinoma (RCC) based on a gene co-expression network. A framework was established where a co-expression network was derived from the database as well as various co-expression approaches. First, the backbone of the network based on differentially expressed (DE) genes between RCC patients and normal controls was constructed by the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. The differentially co-expressed links were detected by Pearson’s correlation, the empirical Bayesian (EB) approach and Weighted Gene Co-expression Network Analysis (WGCNA). The co-expressed gene pairs were merged by a rank-based algorithm. We obtained 842; 371; 2,883 and 1,595 co-expressed gene pairs from the co-expression networks of the STRING database, Pearson’s correlation EB method and WGCNA, respectively. Two hundred and eighty-one differentially co-expressed (DC) gene pairs were obtained from the merged network using this novel method. Pathway enrichment analysis based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and the network enrichment analysis (NEA) method were performed to verify feasibility of the merged method. Results of the KEGG and NEA pathway analyses showed that the network was associated with RCC. The suggested method was computationally efficient to identify pathways associated with RCC and has been identified as a useful complement to traditional co-expression analysis. PMID:26058425

Differential Expression of microRNAs in the Ovaries from Letrozole-Induced Rat Model of Polycystic Ovary Syndrome.

PubMed

Li, Dandan; Li, Chunjin; Xu, Ying; Xu, Duo; Li, Hongjiao; Gao, Liwei; Chen, Shuxiong; Fu, Lulu; Xu, Xin; Liu, Yongzheng; Zhang, Xueying; Zhang, Jingshun; Ming, Hao; Zheng, Lianwen

2016-04-01

Polycystic ovary syndrome (PCOS) is a complex and heterogeneous endocrine disorder. To understand the pathogenesis of PCOS, we established rat models of PCOS induced by letrozole and employed deep sequencing to screen the differential expression of microRNAs (miRNAs) in PCOS rats and control rats. We observed vaginal smear and detected ovarian pathological alteration and hormone level changes in PCOS rats. Deep sequencing showed that a total of 129 miRNAs were differentially expressed in the ovaries from letrozole-induced rat model compared with the control, including 49 miRNAs upregulated and 80 miRNAs downregulated. Furthermore, the differential expression of miR-201-5p, miR-34b-5p, miR-141-3p, and miR-200a-3p were confirmed by real-time polymerase chain reaction. Bioinformatic analysis revealed that these four miRNAs were predicted to target a large set of genes with different functions. Pathway analysis supported that the miRNAs regulate oocyte meiosis, mitogen-activated protein kinase (MAPK) signaling, phosphoinositide 3-kinase/Akt (PI3K-Akt) signaling, Rap1 signaling, and Notch signaling. These data indicate that miRNAs are differentially expressed in rat PCOS model and the differentially expressed miRNA are involved in the etiology and pathophysiology of PCOS. Our findings will help identify miRNAs as novel diagnostic markers and therapeutic targets for PCOS.

Cardiomyocyte differentiation of rat bone marrow multipotent progenitor cells is associated with downregulation of Oct-4 expression.

PubMed

Lu, Tiewei; Pelacho, Beatriz; Hao, Hong; Luo, Min; Zhu, Jing; Verfaillie, Catherine M; Tian, Jie; Liu, Zhenguo

2010-10-01

This study was to determine if bone marrow multipotent adult progenitor cells (MAPCs) underwent cardiac specification and Oct-4 expression during their cardiomyocyte differentiation in vitro. MAPCs were isolated from rat bone marrow, treated with 5-azacytidine (5-aza, 1μM) for 24h, and cultured in a serum-free medium for cardiac differentiation for up to 35 days. The cells started to express early cardiac-specific genes Nkx2.5 and GATA-4 with a significant increase in their mRNA level within 24h after 5-aza treatment. Western blotting analysis and immunofluorescence staining revealed that the cardiac-specific proteins connexin-43 and troponin I were expressed in the cells 7 days after 5-aza treatment. Flow cytometry analysis demonstrated that over 37% of the cells were positive for troponin I by 35 days of differentiation, although the cells did not display spontaneous contraction. On the other hand, the undifferentiated MAPCs expressed a significant level of the stem-cell-specific marker Oct-4 that was dramatically decreased in the cells shortly after the initiation of cardiomyocyte differentiation as evaluated using real-time (RT)-polymerase chain reaction, Western blotting, immunofluorescence staining, and flow cytometry. These data indicated that MAPCs were able to effectively differentiate into cardiomyocyte-like cells after 5-aza induction in association with downregulation of Oct-4 expression.

Transcriptome display during tilapia sex determination and differentiation as revealed by RNA-Seq analysis.

PubMed

Tao, Wenjing; Chen, Jinlin; Tan, Dejie; Yang, Jing; Sun, Lina; Wei, Jing; Conte, Matthew A; Kocher, Thomas D; Wang, Deshou

2018-05-15

The factors determining sex in teleosts are diverse. Great efforts have been made to characterize the underlying genetic network in various species. However, only seven master sex-determining genes have been identified in teleosts. While the function of a few genes involved in sex determination and differentiation has been studied, we are far from fully understanding how genes interact to coordinate in this process. To enable systematic insights into fish sexual differentiation, we generated a dynamic co-expression network from tilapia gonadal transcriptomes at 5, 20, 30, 40, 90, and 180 dah (days after hatching), plus 45 and 90 dat (days after treatment) and linked gene expression profiles to both development and sexual differentiation. Transcriptomic profiles of female and male gonads at 5 and 20 dah exhibited high similarities except for a small number of genes that were involved in sex determination, while drastic changes were observed from 90 to 180 dah, with a group of differently expressed genes which were involved in gonadal differentiation and gametogenesis. Weighted gene correlation network analysis identified changes in the expression of Borealin, Gtsf1, tesk1, Zar1, Cdn15, and Rpl that were correlated with the expression of genes previously known to be involved in sex differentiation, such as Foxl2, Cyp19a1a, Gsdf, Dmrt1, and Amh. Global gonadal gene expression kinetics during sex determination and differentiation have been extensively profiled in tilapia. These findings provide insights into the genetic framework underlying sex determination and sexual differentiation, and expand our current understanding of developmental pathways during teleost sex determination.

Characterization of human myoblast differentiation for tissue-engineering purposes by quantitative gene expression analysis.

PubMed

Stern-Straeter, Jens; Bonaterra, Gabriel Alejandro; Kassner, Stefan S; Zügel, Stefanie; Hörmann, Karl; Kinscherf, Ralf; Goessler, Ulrich Reinhart

2011-08-01

Tissue engineering of skeletal muscle is an encouraging possibility for the treatment of muscle loss through the creation of functional muscle tissue in vitro from human stem cells. Currently, the preferred stem cells are primary, non-immunogenic satellite cells ( = myoblasts). The objective of this study was to determine the expression patterns of myogenic markers within the human satellite cell population during their differentiation into multinucleated myotubes for an accurate characterization of stem cell behaviour. Satellite cells were incubated (for 1, 4, 8, 12 or 16 days) with a culture medium containing either a low [ = differentiation medium (DM)] or high [ = growth medium (GM)] concentration of growth factors. Furthermore, we performed a quantitative gene expression analysis of well-defined differentiation makers: myogenic factor 5 (MYF5), myogenin (MYOG), skeletal muscle αactin1 (ACTA1), embryonic (MYH3), perinatal (MYH8) and adult skeletal muscle myosin heavy chain (MYH1). Additionally, the fusion indices of forming myotubes of MYH1, MYH8 and ACTA1 were calculated. We show that satellite cells incubated with DM expressed multiple characteriztic features of mature skeletal muscles, verified by time-dependent upregulation of MYOG, MYH1, MYH3, MYH8 and ACTA1. However, satellite cells incubated with GM did not reveal all morphological aspects of muscle differentiation. Immunocytochemical investigations with antibodies directed against the differentiation markers showed correlations between the gene expression and differentiation. Our data provide information about time-dependent gene expression of differentiation markers in human satellite cells, which can be used for maturation analyses in skeletal muscle tissue-engineering applications. Copyright © 2011 John Wiley & Sons, Ltd.

Proteomics Analysis of Tissue Samples Reveals Changes in Mitochondrial Protein Levels in Parathyroid Hyperplasia over Adenoma

PubMed Central

AKPINAR, GURLER; KASAP, MURAT; CANTURK, NUH ZAFER; ZULFIGAROVA, MEHIN; ISLEK, EYLÜL ECE; GULER, SERTAC ATA; SIMSEK, TURGAY; CANTURK, ZEYNEP

2017-01-01

Background/Aim: To unveil the pathophysiology of primary hyperparathyroidism, molecular details of parathyroid hyperplasia and adenoma have to be revealed. Such details will provide the tools necessary for differentiation of these two look-alike diseases. Therefore, in the present study, a comparative proteomic study using postoperative tissue samples from the parathyroid adenoma and parathyroid hyperplasia patients was performed. Materials and Methods: Protein extracts were prepared from tissue samples (n=8 per group). Protein pools were created for each group and subjected to DIGE and conventional 2DE. Following image analysis, spots representing the differentially regulated proteins were excised from the and used for identification via MALDI-TOF/TOF analysis. Results: The identities of 40 differentially-expressed proteins were revealed. Fourteen of these proteins were over-expressed in the hyperplasia while 26 of them were over-expressed in the adenoma. Conclusion: Most proteins found to be over-expressed in the hyperplasia samples were mitochondrial, underlying the importance of the mitochondrial activity as a potential biomarker for differentiation of parathyroid hyperplasia from adenoma. PMID:28446534

Length bias correction in gene ontology enrichment analysis using logistic regression.

PubMed

Mi, Gu; Di, Yanming; Emerson, Sarah; Cumbie, Jason S; Chang, Jeff H

2012-01-01

When assessing differential gene expression from RNA sequencing data, commonly used statistical tests tend to have greater power to detect differential expression of genes encoding longer transcripts. This phenomenon, called "length bias", will influence subsequent analyses such as Gene Ontology enrichment analysis. In the presence of length bias, Gene Ontology categories that include longer genes are more likely to be identified as enriched. These categories, however, are not necessarily biologically more relevant. We show that one can effectively adjust for length bias in Gene Ontology analysis by including transcript length as a covariate in a logistic regression model. The logistic regression model makes the statistical issue underlying length bias more transparent: transcript length becomes a confounding factor when it correlates with both the Gene Ontology membership and the significance of the differential expression test. The inclusion of the transcript length as a covariate allows one to investigate the direct correlation between the Gene Ontology membership and the significance of testing differential expression, conditional on the transcript length. We present both real and simulated data examples to show that the logistic regression approach is simple, effective, and flexible.

Microarray Meta-Analysis of RNA-Binding Protein Functions in Alternative Polyadenylation

PubMed Central

Hu, Wenchao; Liu, Yuting; Yan, Jun

2014-01-01

Alternative polyadenylation (APA) is a post-transcriptional mechanism to generate diverse mRNA transcripts with different 3′UTRs from the same gene. In this study, we systematically searched for the APA events with differential expression in public mouse microarray data. Hundreds of genes with over-represented differential APA events and the corresponding experiments were identified. We further revealed that global APA differential expression occurred prevalently in tissues such as brain comparing to peripheral tissues, and biological processes such as development, differentiation and immune responses. Interestingly, we also observed widespread differential APA events in RNA-binding protein (RBP) genes such as Rbm3, Eif4e2 and Elavl1. Given the fact that RBPs are considered as the main regulators of differential APA expression, we constructed a co-expression network between APAs and RBPs using the microarray data. Further incorporation of CLIP-seq data of selected RBPs showed that Nova2 represses and Mbnl1 promotes the polyadenylation of closest poly(A) sites respectively. Altogether, our study is the first microarray meta-analysis in a mammal on the regulation of APA by RBPs that integrated massive mRNA expression data under a wide-range of biological conditions. Finally, we present our results as a comprehensive resource in an online website for the research community. PMID:24622240

Transcriptional expression analysis of survivin splice variants reveals differential expression of survivin-3α in breast cancer.

PubMed

Moniri Javadhesari, Solmaz; Gharechahi, Javad; Hosseinpour Feizi, Mohammad Ali; Montazeri, Vahid; Halimi, Monireh

2013-04-01

Survivin, which is a novel member of the inhibitor of apoptosis family proteins, is known to play an important role in the regulation of cell cycle and apoptosis. Differential expression of survivin in tumor tissues introduces it as a new candidate molecular marker for cancer. Here we investigated the expression of survivin and its splice variants in breast tumors, as well as normal adjacent tissues obtained from the same patients. Thirty five tumors and 17 normal adjacent tissues from women diagnosed with breast cancer were explored in this study. Differential expression of different survivin splice variants was detected and semiquantitatively analyzed using reverse transcription-polymerase chain reaction. Results showed that survivin and its splice variants were differentially expressed in tumor specimens compared with normal adjacent tissues. The expression of survivin-3B and survivin-3α was specifically detected in tumor tissues compared with normal adjacent ones (53% in tumor tissues compared to 5% in normal adjacent for survivin-3B and 65% in tumor tissues and 0.0% in normal adjacent tissues for survivin-3α). Statistical analysis showed that survivin and survivin-ΔEx3 were upregulated in benign (90%, p<0.034) and malignant (76%, p<0.042) tumors, respectively. On the other hand, our results showed that survivin-2α (100% of the cases) was the dominant expressed variant of survivin in breast cancer. The data presented here showed that survivin splice variants were differentially expressed in benign and malignant breast cancer tissues, suggesting their potential role in breast cancer development. Differential expression of survivin-2α and survivin-3α splice variants highlights their usefulness as new candidate markers for breast cancer diagnosis and prognosis.

Altered gene expression of the innate immune, neuroendocrine, and nuclear factor-kappa B (NF-κB) systems is associated with posttraumatic stress disorder in military personnel.

PubMed

Guardado, Pedro; Olivera, Anlys; Rusch, Heather L; Roy, Michael; Martin, Christiana; Lejbman, Natasha; Lee, Hwyunhwa; Gill, Jessica M

2016-03-01

Whole transcriptome analysis provides an unbiased examination of biological activity, and likely, unique insight into the mechanisms underlying posttraumatic stress disorder (PTSD) and comorbid depression and traumatic brain injury. This study compared gene-expression profiles in military personnel with PTSD (n=28) and matched controls without PTSD (n=27) using HG-U133 Plus 2.0 microarrays (Affymetrix), which contain 54,675 probe sets representing more than 38,500 genes. Analysis of expression profiles revealed 203 differentially expressed genes in PTSD, of which 72% were upregulated. Using Partek Genomics Suite 6.6, differentially expressed transcription clusters were filtered based on a selection criterion of ≥1.5 relative fold change at a false discovery rate of ≤5%. Ingenuity Pathway Analysis (Qiagen) of the differentially expressed genes indicated a dysregulation of genes associated with the innate immune, neuroendocrine, and NF-κB systems. These findings provide novel insights that may lead to new pharmaceutical agents for PTSD treatments and help mitigate mental and physical comorbidity risk. Copyright © 2016. Published by Elsevier Ltd.

Comparative toxicogenomic analysis of oral Cr(VI) exposure effects in rat and mouse small intestinal epithelia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kopec, Anna K.; Thompson, Chad M.; Kim, Suntae

2012-07-15

Continuous exposure to high concentrations of hexavalent chromium [Cr(VI)] in drinking water results in intestinal tumors in mice but not rats. Concentration-dependent gene expression effects were evaluated in female F344 rat duodenal and jejunal epithelia following 7 and 90 days of exposure to 0.3–520 mg/L (as sodium dichromate dihydrate, SDD) in drinking water. Whole-genome microarrays identified 3269 and 1815 duodenal, and 4557 and 1534 jejunal differentially expressed genes at 8 and 91 days, respectively, with significant overlaps between the intestinal segments. Functional annotation identified gene expression changes associated with oxidative stress, cell cycle, cell death, and immune response that weremore » consistent with reported changes in redox status and histopathology. Comparative analysis with B6C3F1 mouse data from a similarly designed study identified 2790 differentially expressed rat orthologs in the duodenum compared to 5013 mouse orthologs at day 8, and only 1504 rat and 3484 mouse orthologs at day 91. Automated dose–response modeling resulted in similar median EC{sub 50}s in the rodent duodenal and jejunal mucosae. Comparative examination of differentially expressed genes also identified divergently regulated orthologs. Comparable numbers of differentially expressed genes were observed at equivalent Cr concentrations (μg Cr/g duodenum). However, mice accumulated higher Cr levels than rats at ≥ 170 mg/L SDD, resulting in a ∼ 2-fold increase in the number of differentially expressed genes. These qualitative and quantitative differences in differential gene expression, which correlate with differences in tissue dose, likely contribute to the disparate intestinal tumor outcomes. -- Highlights: ► Cr(VI) elicits dose-dependent changes in gene expression in rat intestine. ► Cr(VI) elicits less differential gene expression in rats compared to mice. ► Cr(VI) gene expression can be phenotypically anchored to intestinal changes. ► Species-specific and divergent changes are consistent with species-specific tumors.« less

Comparative Temporal Transcriptome Profiling of Wheat near Isogenic Line Carrying Lr57 under Compatible and Incompatible Interactions

PubMed Central

Yadav, Inderjit S.; Sharma, Amandeep; Kaur, Satinder; Nahar, Natasha; Bhardwaj, Subhash C.; Sharma, Tilak R.; Chhuneja, Parveen

2016-01-01

Leaf rust caused by Puccinia triticina (Pt) is one of the most important diseases of bread wheat globally. Recent advances in sequencing technologies have provided opportunities to analyse the complete transcriptomes of the host as well as pathogen for studying differential gene expression during infection. Pathogen induced differential gene expression was characterized in a near isogenic line carrying leaf rust resistance gene Lr57 and susceptible recipient genotype WL711. RNA samples were collected at five different time points 0, 12, 24, 48, and 72 h post inoculation (HPI) with Pt 77-5. A total of 3020 transcripts were differentially expressed with 1458 and 2692 transcripts in WL711 and WL711+Lr57, respectively. The highest number of differentially expressed transcripts was detected at 12 HPI. Functional categorization using Blast2GO classified the genes into biological processes, molecular function and cellular components. WL711+Lr57 showed much higher number of differentially expressed nucleotide binding and leucine rich repeat genes and expressed more protein kinases and pathogenesis related proteins such as chitinases, glucanases and other PR proteins as compared to susceptible genotype. Pathway annotation with KEGG categorized genes into 13 major classes with carbohydrate metabolism being the most prominent followed by amino acid, secondary metabolites, and nucleotide metabolism. Gene co-expression network analysis identified four and eight clusters of highly correlated genes in WL711 and WL711+Lr57, respectively. Comparative analysis of the differentially expressed transcripts led to the identification of some transcripts which were specifically expressed only in WL711+Lr57. It was apparent from the whole transcriptome sequencing that the resistance gene Lr57 directed the expression of different genes involved in building the resistance response in the host to combat invading pathogen. The RNAseq data and differentially expressed transcripts identified in present study is a genomic resource which can be used for further studying the host pathogen interaction for Lr57 and wheat transcriptome in general. PMID:28066494

Differential gene expression in porcine SK6 cells infected with wild-type and SAP domain-mutant foot-and-mouth disease virus.

PubMed

Ni, Zixin; Yang, Fan; Cao, Weijun; Zhang, Xiangle; Jin, Ye; Mao, Ruoqing; Du, Xiaoli; Li, Weiwei; Guo, Jianhong; Liu, Xiangtao; Zhu, Zixiang; Zheng, Haixue

2016-06-01

Foot-and-mouth disease virus (FMDV) is the causative agent of a highly contagious disease in livestock. The viral proteinase L(pro) of FMDV is involved in pathogenicity, and mutation of the L(pro) SAP domain reduces FMDV pathogenicity in pigs. To determine the gene expression profiles associated with decreased pathogenicity in porcine cells, we performed transcriptome analysis using next-generation sequencing technology and compared differentially expressed genes in SK6 cells infected with FMDV containing L(pro) with either a wild-type or mutated version of the SAP domain. This analysis yielded 1,853 genes that exhibited a ≥ 2-fold change in expression and was validated by real-time quantitative PCR detection of several differentially expressed genes. Many of the differentially expressed genes correlated with antiviral responses corresponded to genes associated with transcription factors, immune regulation, cytokine production, inflammatory response, and apoptosis. Alterations in gene expression profiles may be responsible for the variations in pathogenicity observed between the two FMDV variants. Our results provided genes of interest for the further study of antiviral pathways and pathogenic mechanisms related to FMDV L(pro).

The human disease network in terms of dysfunctional regulatory mechanisms.

PubMed

Yang, Jing; Wu, Su-Juan; Dai, Wen-Tao; Li, Yi-Xue; Li, Yuan-Yuan

2015-10-08

Elucidation of human disease similarities has emerged as an active research area, which is highly relevant to etiology, disease classification, and drug repositioning. In pioneer studies, disease similarity was commonly estimated according to clinical manifestation. Subsequently, scientists started to investigate disease similarity based on gene-phenotype knowledge, which were inevitably biased to well-studied diseases. In recent years, estimating disease similarity according to transcriptomic behavior significantly enhances the probability of finding novel disease relationships, while the currently available studies usually mine expression data through differential expression analysis that has been considered to have little chance of unraveling dysfunctional regulatory relationships, the causal pathogenesis of diseases. We developed a computational approach to measure human disease similarity based on expression data. Differential coexpression analysis, instead of differential expression analysis, was employed to calculate differential coexpression level of every gene for each disease, which was then summarized to the pathway level. Disease similarity was eventually calculated as the partial correlation coefficients of pathways' differential coexpression values between any two diseases. The significance of disease relationships were evaluated by permutation test. Based on mRNA expression data and a differential coexpression analysis based method, we built a human disease network involving 1326 significant Disease-Disease links among 108 diseases. Compared with disease relationships captured by differential expression analysis based method, our disease links shared known disease genes and drugs more significantly. Some novel disease relationships were discovered, for example, Obesity and cancer, Obesity and Psoriasis, lung adenocarcinoma and S. pneumonia, which had been commonly regarded as unrelated to each other, but recently found to share similar molecular mechanisms. Additionally, it was found that both the type of disease and the type of affected tissue influenced the degree of disease similarity. A sub-network including Allergic asthma, Type 2 diabetes and Chronic kidney disease was extracted to demonstrate the exploration of their common pathogenesis. The present study produces a global view of human diseasome for the first time from the viewpoint of regulation mechanisms, which therefore could provide insightful clues to etiology and pathogenesis, and help to perform drug repositioning and design novel therapeutic interventions.

Impaired osteogenic differentiation and enhanced cellular receptor of advanced glycation end products sensitivity in patients with type 2 diabetes.

PubMed

Phimphilai, Mattabhorn; Pothacharoen, Peraphan; Kongtawelert, Prachya; Chattipakorn, Nipon

2017-11-01

Preclinical studies have demonstrated impaired osteoblast differentiation in type 2 diabetes (T2DM), which is related to skeletal accumulation of advanced glycation end products (AGEs). However, the role of AGE in osteoblast differentiation in patients with T2DM is unclear. This cross-sectional study was performed to investigate osteoblast differentiation and its association with serum pentosidine and soluble receptor of AGEs (sRAGE). Twenty-seven patients with T2DM and 15 age-matched controls were included to measure sRAGE and osteogenic differentiation in mononuclear cells derived from peripheral blood. The mononuclear cells isolated from patients with T2DM showed a significantly lower rate of osteogenic differentiation (7.4% vs 86.7%, p < 0.0001) with a lower level of ALPL, COL1A1, and BGLAP expression than those of controls by 11-, 44-, and 15-fold respectively, together with nonvisualized mineralization by alizarin red S staining. The levels of pentosidine and sRAGE were comparable in both groups. AGER expression was significantly higher in the T2DM group. BAX expression was also significantly higher in the T2DM group, and showed a strong correlation with AGER expression (r = 0.86, p < 0.0001). Fasting plasma glucose (FPG) level, AGER expression, and BAX expression showed a strong correlation with osteogenic differentiation defects on univariate analysis. However, only FPG showed a correlation with this defect in a multivariate analysis. In conclusion, patients with T2DM showed impairment of osteoblast differentiation, and FPG was an independent risk factor for this impairment. Moreover, T2DM showed a higher cellular sensitivity for activation of receptor of AGEs and higher cellular apoptosis, which may contribute to the defect in osteoblast differentiation.

Discovery of new candidate genes for rheumatoid arthritis through integration of genetic association data with expression pathway analysis.

PubMed

Shchetynsky, Klementy; Diaz-Gallo, Lina-Marcella; Folkersen, Lasse; Hensvold, Aase Haj; Catrina, Anca Irinel; Berg, Louise; Klareskog, Lars; Padyukov, Leonid

2017-02-02

Here we integrate verified signals from previous genetic association studies with gene expression and pathway analysis for discovery of new candidate genes and signaling networks, relevant for rheumatoid arthritis (RA). RNA-sequencing-(RNA-seq)-based expression analysis of 377 genes from previously verified RA-associated loci was performed in blood cells from 5 newly diagnosed, non-treated patients with RA, 7 patients with treated RA and 12 healthy controls. Differentially expressed genes sharing a similar expression pattern in treated and untreated RA sub-groups were selected for pathway analysis. A set of "connector" genes derived from pathway analysis was tested for differential expression in the initial discovery cohort and validated in blood cells from 73 patients with RA and in 35 healthy controls. There were 11 qualifying genes selected for pathway analysis and these were grouped into two evidence-based functional networks, containing 29 and 27 additional connector molecules. The expression of genes, corresponding to connector molecules was then tested in the initial RNA-seq data. Differences in the expression of ERBB2, TP53 and THOP1 were similar in both treated and non-treated patients with RA and an additional nine genes were differentially expressed in at least one group of patients compared to healthy controls. The ERBB2, TP53. THOP1 expression profile was successfully replicated in RNA-seq data from peripheral blood mononuclear cells from healthy controls and non-treated patients with RA, in an independent collection of samples. Integration of RNA-seq data with findings from association studies, and consequent pathway analysis implicate new candidate genes, ERBB2, TP53 and THOP1 in the pathogenesis of RA.

Secretagogin is a novel marker for neuroendocrine differentiation.

PubMed

Birkenkamp-Demtröder, Karin; Wagner, Ludwig; Brandt Sørensen, Flemming; Bording Astrup, Lone; Gartner, Wolfgang; Scherübl, Hans; Heine, Bernhard; Christiansen, Peer; Ørntoft, Torben Falck

2005-01-01

Our previous microarray-based studies identified secretagogin to be highly expressed in normal colon mucosa compared to basal expression in colon adenocarcinomas. The aim of this study was to analyze the differential expression of secretagogin in normal mucosa, adenocarcinomas, and neuroendocrine tumors. Western blotting, immunohistochemistry, immunofluorescence microscopy and ELISA were applied. Western blot analysis detected a 32-kDa secretagogin band in samples from normal mucosa. Immunohistochemical analyses on tissue specimens showed that secretagogin is exclusively expressed in neuroendocrine cells and nerve cells in normal mucosa of the digestive tract. Tissues adjacent to benign hyperplasic polyps and adenomas showed a decreased number of secretagogin-expressing neuroendocrine cells. Secretagogin co-localized with neuroendocrine markers (chromogranin A, neuron-specific enolase, synaptophysin) in neuroendocrine cells in crypts of normal mucosa, and in tumor cells of carcinoids. Secretagogin was strongly expressed in the cytosol and the nucleus of 19 well-differentiated neuroendocrine carcinoids and carcinoid metastases, as well as in neuroendocrine tumors from the lung, pancreas and adrenal gland. Secretagogin was detected in plasma from carcinoid patients with distant metastasis. Combined immunohistochemical analysis of secretagogin and FK506-binding protein 65, a protein de novo synthesized in adenocarcinomas, distinguished well-differentiated carcinoids, adenocarcinoids and undifferentiated carcinomas. We conclude that secretagogin is a novel marker for neuroendocrine differentiation.

Quantitative Proteomic Analysis of Differentially Expressed Protein Profiles Involved in Pancreatic Ductal Adenocarcinoma

PubMed Central

Kuo, Kung-Kai; Kuo, Chao-Jen; Chiu, Chiang-Yen; Liang, Shih-Shin; Huang, Chun-Hao; Chi, Shu-Wen; Tsai, Kun-Bow; Chen, Chiao-Yun; Hsi, Edward; Cheng, Kuang-Hung; Chiou, Shyh-Horng

2016-01-01

Objectives The aim of this study was to identify differentially expressed proteins among various stages of pancreatic ductal adenocarcinoma (PDAC) by shotgun proteomics using nano-liquid chromatography coupled tandem mass spectrometry and stable isotope dimethyl labeling. Methods Differentially expressed proteins were identified and compared based on the mass spectral differences of their isotope-labeled peptide fragments generated from protease digestion. Results Our quantitative proteomic analysis of the differentially expressed proteins with stable isotope (deuterium/hydrogen ratio, ≥2) identified a total of 353 proteins, with at least 5 protein biomarker proteins that were significantly differentially expressed between cancer and normal mice by at least a 2-fold alteration. These 5 protein biomarker candidates include α-enolase, α-catenin, 14-3-3 β, VDAC1, and calmodulin with high confidence levels. The expression levels were also found to be in agreement with those examined by Western blot and histochemical staining. Conclusions The systematic decrease or increase of these identified marker proteins may potentially reflect the morphological aberrations and diseased stages of pancreas carcinoma throughout progressive developments leading to PDAC. The results would form a firm foundation for future work concerning validation and clinical translation of some identified biomarkers into targeted diagnosis and therapy for various stages of PDAC. PMID:26262590

SPARTA: Simple Program for Automated reference-based bacterial RNA-seq Transcriptome Analysis.

PubMed

Johnson, Benjamin K; Scholz, Matthew B; Teal, Tracy K; Abramovitch, Robert B

2016-02-04

Many tools exist in the analysis of bacterial RNA sequencing (RNA-seq) transcriptional profiling experiments to identify differentially expressed genes between experimental conditions. Generally, the workflow includes quality control of reads, mapping to a reference, counting transcript abundance, and statistical tests for differentially expressed genes. In spite of the numerous tools developed for each component of an RNA-seq analysis workflow, easy-to-use bacterially oriented workflow applications to combine multiple tools and automate the process are lacking. With many tools to choose from for each step, the task of identifying a specific tool, adapting the input/output options to the specific use-case, and integrating the tools into a coherent analysis pipeline is not a trivial endeavor, particularly for microbiologists with limited bioinformatics experience. To make bacterial RNA-seq data analysis more accessible, we developed a Simple Program for Automated reference-based bacterial RNA-seq Transcriptome Analysis (SPARTA). SPARTA is a reference-based bacterial RNA-seq analysis workflow application for single-end Illumina reads. SPARTA is turnkey software that simplifies the process of analyzing RNA-seq data sets, making bacterial RNA-seq analysis a routine process that can be undertaken on a personal computer or in the classroom. The easy-to-install, complete workflow processes whole transcriptome shotgun sequencing data files by trimming reads and removing adapters, mapping reads to a reference, counting gene features, calculating differential gene expression, and, importantly, checking for potential batch effects within the data set. SPARTA outputs quality analysis reports, gene feature counts and differential gene expression tables and scatterplots. SPARTA provides an easy-to-use bacterial RNA-seq transcriptional profiling workflow to identify differentially expressed genes between experimental conditions. This software will enable microbiologists with limited bioinformatics experience to analyze their data and integrate next generation sequencing (NGS) technologies into the classroom. The SPARTA software and tutorial are available at sparta.readthedocs.org.

Regulatory network involving miRNAs and genes in serous ovarian carcinoma

PubMed Central

Zhao, Haiyan; Xu, Hao; Xue, Luchen

2017-01-01

Serous ovarian carcinoma (SOC) is one of the most life-threatening types of gynecological malignancy, but the pathogenesis of SOC remains unknown. Previous studies have indicated that differentially expressed genes and microRNAs (miRNAs) serve important functions in SOC. However, genes and miRNAs are identified in a disperse form, and limited information is known about the regulatory association between miRNAs and genes in SOC. In the present study, three regulatory networks were hierarchically constructed, including a differentially-expressed network, a related network and a global network to reveal associations between each factor. In each network, there were three types of factors, which were genes, miRNAs and transcription factors that interact with each other. Focus was placed on the differentially-expressed network, in which all genes and miRNAs were differentially expressed and therefore may have affected the development of SOC. Following the comparison and analysis between the three networks, a number of signaling pathways which demonstrated differentially expressed elements were highlighted. Subsequently, the upstream and downstream elements of differentially expressed miRNAs and genes were listed, and a number of key elements (differentially expressed miRNAs, genes and TFs predicted using the P-match method) were analyzed. The differentially expressed network partially illuminated the pathogenesis of SOC. It was hypothesized that if there was no differential expression of miRNAs and genes, SOC may be prevented and treatment may be identified. The present study provided a theoretical foundation for gene therapy for SOC. PMID:29113276

Differential gene expression in granulosa cells from polycystic ovary syndrome patients with and without insulin resistance: identification of susceptibility gene sets through network analysis.

PubMed

Kaur, Surleen; Archer, Kellie J; Devi, M Gouri; Kriplani, Alka; Strauss, Jerome F; Singh, Rita

2012-10-01

Polycystic ovary syndrome (PCOS) is a heterogeneous, genetically complex, endocrine disorder of uncertain etiology in women. Our aim was to compare the gene expression profiles in stimulated granulosa cells of PCOS women with and without insulin resistance vs. matched controls. This study included 12 normal ovulatory women (controls), 12 women with PCOS without evidence for insulin resistance (PCOS non-IR), and 16 women with insulin resistance (PCOS-IR) undergoing in vitro fertilization. Granulosa cell gene expression profiling was accomplished using Affymetrix Human Genome-U133 arrays. Differentially expressed genes were classified according to gene ontology using ingenuity pathway analysis tools. Microarray results for selected genes were confirmed by real-time quantitative PCR. A total of 211 genes were differentially expressed in PCOS non-IR and PCOS-IR granulosa cells (fold change≥1.5; P≤0.001) vs. matched controls. Diabetes mellitus and inflammation genes were significantly increased in PCOS-IR patients. Real-time quantitative PCR confirmed higher expression of NCF2 (2.13-fold), TCF7L2 (1.92-fold), and SERPINA1 (5.35-fold). Increased expression of inflammation genes ITGAX (3.68-fold) and TAB2 (1.86-fold) was confirmed in PCOS non-IR. Different cardiometabolic disease genes were differentially expressed in the two groups. Decreased expression of CAV1 (-3.58-fold) in PCOS non-IR and SPARC (-1.88-fold) in PCOS-IR was confirmed. Differential expression of genes involved in TGF-β signaling (IGF2R, increased; and HAS2, decreased), and oxidative stress (TXNIP, increased) was confirmed in both groups. Microarray analysis demonstrated differential expression of genes linked to diabetes mellitus, inflammation, cardiovascular diseases, and infertility in the granulosa cells of PCOS women with and without insulin resistance. Because these dysregulated genes are also involved in oxidative stress, lipid metabolism, and insulin signaling, we hypothesize that these genes may be involved in follicular growth arrest and metabolic disorders associated with the different phenotypes of PCOS.

SCOUP: a probabilistic model based on the Ornstein-Uhlenbeck process to analyze single-cell expression data during differentiation.

PubMed

Matsumoto, Hirotaka; Kiryu, Hisanori

2016-06-08

Single-cell technologies make it possible to quantify the comprehensive states of individual cells, and have the power to shed light on cellular differentiation in particular. Although several methods have been developed to fully analyze the single-cell expression data, there is still room for improvement in the analysis of differentiation. In this paper, we propose a novel method SCOUP to elucidate differentiation process. Unlike previous dimension reduction-based approaches, SCOUP describes the dynamics of gene expression throughout differentiation directly, including the degree of differentiation of a cell (in pseudo-time) and cell fate. SCOUP is superior to previous methods with respect to pseudo-time estimation, especially for single-cell RNA-seq. SCOUP also successfully estimates cell lineage more accurately than previous method, especially for cells at an early stage of bifurcation. In addition, SCOUP can be applied to various downstream analyses. As an example, we propose a novel correlation calculation method for elucidating regulatory relationships among genes. We apply this method to a single-cell RNA-seq data and detect a candidate of key regulator for differentiation and clusters in a correlation network which are not detected with conventional correlation analysis. We develop a stochastic process-based method SCOUP to analyze single-cell expression data throughout differentiation. SCOUP can estimate pseudo-time and cell lineage more accurately than previous methods. We also propose a novel correlation calculation method based on SCOUP. SCOUP is a promising approach for further single-cell analysis and available at https://github.com/hmatsu1226/SCOUP.

Analysis of global gene expression in Brachypodium distachyon reveals extensive network plasticity in response to abiotic stress.

PubMed

Priest, Henry D; Fox, Samuel E; Rowley, Erik R; Murray, Jessica R; Michael, Todd P; Mockler, Todd C

2014-01-01

Brachypodium distachyon is a close relative of many important cereal crops. Abiotic stress tolerance has a significant impact on productivity of agriculturally important food and feedstock crops. Analysis of the transcriptome of Brachypodium after chilling, high-salinity, drought, and heat stresses revealed diverse differential expression of many transcripts. Weighted Gene Co-Expression Network Analysis revealed 22 distinct gene modules with specific profiles of expression under each stress. Promoter analysis implicated short DNA sequences directly upstream of module members in the regulation of 21 of 22 modules. Functional analysis of module members revealed enrichment in functional terms for 10 of 22 network modules. Analysis of condition-specific correlations between differentially expressed gene pairs revealed extensive plasticity in the expression relationships of gene pairs. Photosynthesis, cell cycle, and cell wall expression modules were down-regulated by all abiotic stresses. Modules which were up-regulated by each abiotic stress fell into diverse and unique gene ontology GO categories. This study provides genomics resources and improves our understanding of abiotic stress responses of Brachypodium.

A novel approach to select differential pathways associated with hypertrophic cardiomyopathy based on gene co‑expression analysis.

PubMed

Chen, Xiao-Min; Feng, Ming-Jun; Shen, Cai-Jie; He, Bin; Du, Xian-Feng; Yu, Yi-Bo; Liu, Jing; Chu, Hui-Min

2017-07-01

The present study was designed to develop a novel method for identifying significant pathways associated with human hypertrophic cardiomyopathy (HCM), based on gene co‑expression analysis. The microarray dataset associated with HCM (E‑GEOD‑36961) was obtained from the European Molecular Biology Laboratory‑European Bioinformatics Institute database. Informative pathways were selected based on the Reactome pathway database and screening treatments. An empirical Bayes method was utilized to construct co‑expression networks for informative pathways, and a weight value was assigned to each pathway. Differential pathways were extracted based on weight threshold, which was calculated using a random model. In order to assess whether the co‑expression method was feasible, it was compared with traditional pathway enrichment analysis of differentially expressed genes, which were identified using the significance analysis of microarrays package. A total of 1,074 informative pathways were screened out for subsequent investigations and their weight values were also obtained. According to the threshold of weight value of 0.01057, 447 differential pathways, including folding of actin by chaperonin containing T‑complex protein 1 (CCT)/T‑complex protein 1 ring complex (TRiC), purine ribonucleoside monophosphate biosynthesis and ubiquinol biosynthesis, were obtained. Compared with traditional pathway enrichment analysis, the number of pathways obtained from the co‑expression approach was increased. The results of the present study demonstrated that this method may be useful to predict marker pathways for HCM. The pathways of folding of actin by CCT/TRiC and purine ribonucleoside monophosphate biosynthesis may provide evidence of the underlying molecular mechanisms of HCM, and offer novel therapeutic directions for HCM.

A comparative study of RNA-Seq and microarray data analysis on the two examples of rectal-cancer patients and Burkitt Lymphoma cells.

PubMed

Wolff, Alexander; Bayerlová, Michaela; Gaedcke, Jochen; Kube, Dieter; Beißbarth, Tim

2018-01-01

Pipeline comparisons for gene expression data are highly valuable for applied real data analyses, as they enable the selection of suitable analysis strategies for the dataset at hand. Such pipelines for RNA-Seq data should include mapping of reads, counting and differential gene expression analysis or preprocessing, normalization and differential gene expression in case of microarray analysis, in order to give a global insight into pipeline performances. Four commonly used RNA-Seq pipelines (STAR/HTSeq-Count/edgeR, STAR/RSEM/edgeR, Sailfish/edgeR, TopHat2/Cufflinks/CuffDiff)) were investigated on multiple levels (alignment and counting) and cross-compared with the microarray counterpart on the level of gene expression and gene ontology enrichment. For these comparisons we generated two matched microarray and RNA-Seq datasets: Burkitt Lymphoma cell line data and rectal cancer patient data. The overall mapping rate of STAR was 98.98% for the cell line dataset and 98.49% for the patient dataset. Tophat's overall mapping rate was 97.02% and 96.73%, respectively, while Sailfish had only an overall mapping rate of 84.81% and 54.44%. The correlation of gene expression in microarray and RNA-Seq data was moderately worse for the patient dataset (ρ = 0.67-0.69) than for the cell line dataset (ρ = 0.87-0.88). An exception were the correlation results of Cufflinks, which were substantially lower (ρ = 0.21-0.29 and 0.34-0.53). For both datasets we identified very low numbers of differentially expressed genes using the microarray platform. For RNA-Seq we checked the agreement of differentially expressed genes identified in the different pipelines and of GO-term enrichment results. In conclusion the combination of STAR aligner with HTSeq-Count followed by STAR aligner with RSEM and Sailfish generated differentially expressed genes best suited for the dataset at hand and in agreement with most of the other transcriptomics pipelines.

Microarray‑based bioinformatics analysis of the prospective target gene network of key miRNAs influenced by long non‑coding RNA PVT1 in HCC.

PubMed

Zhang, Yu; Mo, Wei-Jia; Wang, Xiao; Zhang, Tong-Tong; Qin, Yuan; Wang, Han-Lin; Chen, Gang; Wei, Dan-Ming; Dang, Yi-Wu

2018-05-02

The long non‑coding RNA (lncRNA) PVT1 plays vital roles in the tumorigenesis and development of various types of cancer. However, the potential expression profiling, functions and pathways of PVT1 in HCC remain unknown. PVT1 was knocked down in SMMC‑7721 cells, and a miRNA microarray analysis was performed to detect the differentially expressed miRNAs. Twelve target prediction algorithms were used to predict the underlying targets of these differentially expressed miRNAs. Bioinformatics analysis was performed to explore the underlying functions, pathways and networks of the targeted genes. Furthermore, the relationship between PVT1 and the clinical parameters in HCC was confirmed based on the original data in the TCGA database. Among the differentially expressed miRNAs, the top two upregulated and downregulated miRNAs were selected for further analysis based on the false discovery rate (FDR), fold‑change (FC) and P‑values. Based on the TCGA database, PVT1 was obviously highly expressed in HCC, and a statistically higher PVT1 expression was found for sex (male), ethnicity (Asian) and pathological grade (G3+G4) compared to the control groups (P<0.05). Furthermore, Gene Ontology (GO) analysis revealed that the target genes were involved in complex cellular pathways, such as the macromolecule biosynthetic process, compound metabolic process, and transcription. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the MAPK and Wnt signaling pathways may be correlated with the regulation of the four candidate miRNAs. The results therefore provide significant information on the differentially expressed miRNAs associated with PVT1 in HCC, and we hypothesized that PVT1 may play vital roles in HCC by regulating different miRNAs or target gene expression (particularly MAPK8) via the MAPK or Wnt signaling pathways. Thus, further investigation of the molecular mechanism of PVT1 in HCC is needed.

Integrated analysis of miRNA and mRNA expression profiles in tilapia gonads at an early stage of sex differentiation.

PubMed

Tao, Wenjing; Sun, Lina; Shi, Hongjuan; Cheng, Yunying; Jiang, Dongneng; Fu, Beide; Conte, Matthew A; Gammerdinger, William J; Kocher, Thomas D; Wang, Deshou

2016-05-04

MicroRNAs (miRNAs) represent a second regulatory network that has important effects on gene expression and protein translation during biological process. However, the possible role of miRNAs in the early stages of fish sex differentiation is not well understood. In this study, we carried an integrated analysis of miRNA and mRNA expression profiles to explore their possibly regulatory patterns at the critical stage of sex differentiation in tilapia. We identified 279 pre-miRNA genes in tilapia genome, which were highly conserved in other fish species. Based on small RNA library sequencing, we identified 635 mature miRNAs in tilapia gonads, in which 62 and 49 miRNAs showed higher expression in XX and XY gonads, respectively. The predicted targets of these sex-biased miRNAs (e.g., miR-9, miR-21, miR-30a, miR-96, miR-200b, miR-212 and miR-7977) included genes encoding key enzymes in steroidogenic pathways (Cyp11a1, Hsd3b, Cyp19a1a, Hsd11b) and key molecules involved in vertebrate sex differentiation (Foxl2, Amh, Star1, Sf1, Dmrt1, and Gsdf). These genes also showed sex-biased expression in tilapia gonads at 5 dah. Some miRNAs (e.g., miR-96 and miR-737) targeted multiple genes involved in steroid synthesis, suggesting a complex miRNA regulatory network during early sex differentiation in this fish. The sequence and expression patterns of most miRNAs in tilapia are conserved in fishes, indicating the basic functions of vertebrate miRNAs might share a common evolutionary origin. This comprehensive analysis of miRNA and mRNA at the early stage of molecular sex differentiation in tilapia XX and XY gonads lead to the discovery of differentially expressed miRNAs and their putative targets, which will facilitate studies of the regulatory network of molecular sex determination and differentiation in fishes.

Identification of transcript regulatory patterns in cell differentiation.

PubMed

Gusnanto, Arief; Gosling, John Paul; Pope, Christopher

2017-10-15

Studying transcript regulatory patterns in cell differentiation is critical in understanding its complex nature of the formation and function of different cell types. This is done usually by measuring gene expression at different stages of the cell differentiation. However, if the gene expression data available are only from the mature cells, we have some challenges in identifying transcript regulatory patterns that govern the cell differentiation. We propose to exploit the information of the lineage of cell differentiation in terms of correlation structure between cell types. We assume that two different cell types that are close in the lineage will exhibit many common genes that are co-expressed relative to those that are far in the lineage. Current analysis methods tend to ignore this correlation by testing for differential expression assuming some sort of independence between cell types. We employ a Bayesian approach to estimate the posterior distribution of the mean of expression in each cell type, by taking into account the cell formation path in the lineage. This enables us to infer genes that are specific in each cell type, indicating the genes are involved in directing the cell differentiation to that particular cell type. We illustrate the method using gene expression data from a study of haematopoiesis. R codes to perform the analysis are available in http://www1.maths.leeds.ac.uk/∼arief/R/CellDiff/. a.gusnanto@leeds.ac.uk. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Proteomic analysis of cell cycle arrest and differentiation induction caused by ATPR, a derivative of all-trans retinoic acid, in human gastric cancer SGC-7901 cells.

PubMed

Xia, Quan; Zhao, Yingli; Wang, Jiali; Qiao, Wenhao; Zhang, Dongling; Yin, Hao; Xu, Dujuan; Chen, Feihu

2017-07-01

4-amino-2-trifluoromethyl-phenyl retinate (ATPR) was reported to potentially inhibit proliferation and induce differentiation activity in some tumor cells. In this study, a proteomics approach was used to investigate the possible mechanism by screening the differentially expressed protein profiles of SGC-7901 cells before and after ATPR-treatment in vitro. Peptides digested from the total cellular proteins were analyzed by reverse phase LC-MS/MS followed by a label-free quantification analysis. The SEQUEST search engine was used to identify proteins and bioinformatics resources were used to investigate the involved pathways for the differentially expressed proteins. Thirteen down-regulated proteins were identified in the ATPR-treated group. Bioinformatics analysis showed that the effects of ATPR on 14-3-3ε might potentially involve the PI3K-AKT-FOXO pathway and P27Kip1 expression. Western blot and RT-PCR analysis showed that ATPR could inhibit AKT phosphorylation, up-regulate the expression of FOXO1A and P27Kip1 at both the protein and mRNA levels, and down-regulate the cytoplasmic expression of cyclin E and CDK2. ATPR-induced G0/G1 phase arrest and differentiation can be ablated if the P27kip1 gene is silenced with sequence-specific siRNA or in 14-3-3ε overexpression of SGC-7901 cells. ATPR might cause cell cycle arrest and differentiation in SGC-7901 cells by simultaneously inhibiting the phosphorylation of AKT and down-regulating 14-3-3ε. This change would then enhance the inhibition of cyclin E/CDK2 by up-regulating FOXO1A and P27Kip1. Our findings could be of value for finding new drug targets and for developing more effective differentiation inducer. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular pathology of acute kidney injury in a choline-deficient model and fish oil protective effect.

PubMed

Denninghoff, Valeria; Ossani, Georgina; Uceda, Ana; Rugnone, Matias; Fernández, Elmer; Fresno, Cristóbal; González, German; Díaz, Maria Luisa; Avagnina, Alejandra; Elsner, Boris; Monserrat, Alberto

2014-04-01

The aim of this work was to investigate the potential protective effects of fish oil on the basis of kidney transcriptomic data on a nutritional experimental model. Male weanling Wistar rats were divided into four groups and fed choline-deficient (CD) and choline-supplemented (CS) diets with vegetable oil (VO) and menhaden oil (MO): CSVO, CDVO, CSMO and CDMO. Animals were killed after receiving the diets for 6 days. Total RNA was purified from the right kidney and hybridized to Affymetrix GeneChip Rat Gene 1.0 ST Array. Differentially expressed genes were analyzed. All CSVO, CSMO and CDMO rats showed no renal alterations, while all CDVO rats showed renal cortical necrosis. A thorough analysis of the differential expression between groups CSMO and CDMO was carried out. There were no differential genes for p < 0.01. The analysis of the differential expression between groups CSVO and CSMO revealed 32 genes, 11 were over-expressed and 21 were under-expressed in CSMO rats. This work was part of a large set of experiments and was used in a hypothesis-generating manner. The comprehensive analysis of genetic expression allowed confirming that menhaden oil has a protective effect on this nutritional experimental model and identifying 32 genes that could be responsible for that protection, including Gstp1. These results reveal that gene changes could play a role in renal injury.

Analysis of host response to bacterial infection using error model based gene expression microarray experiments

PubMed Central

Stekel, Dov J.; Sarti, Donatella; Trevino, Victor; Zhang, Lihong; Salmon, Mike; Buckley, Chris D.; Stevens, Mark; Pallen, Mark J.; Penn, Charles; Falciani, Francesco

2005-01-01

A key step in the analysis of microarray data is the selection of genes that are differentially expressed. Ideally, such experiments should be properly replicated in order to infer both technical and biological variability, and the data should be subjected to rigorous hypothesis tests to identify the differentially expressed genes. However, in microarray experiments involving the analysis of very large numbers of biological samples, replication is not always practical. Therefore, there is a need for a method to select differentially expressed genes in a rational way from insufficiently replicated data. In this paper, we describe a simple method that uses bootstrapping to generate an error model from a replicated pilot study that can be used to identify differentially expressed genes in subsequent large-scale studies on the same platform, but in which there may be no replicated arrays. The method builds a stratified error model that includes array-to-array variability, feature-to-feature variability and the dependence of error on signal intensity. We apply this model to the characterization of the host response in a model of bacterial infection of human intestinal epithelial cells. We demonstrate the effectiveness of error model based microarray experiments and propose this as a general strategy for a microarray-based screening of large collections of biological samples. PMID:15800204

Molecular Validation of Chondrogenic Differentiation and Hypoxia Responsiveness of Platelet-Lysate Expanded Adipose Tissue-Derived Human Mesenchymal Stromal Cells.

PubMed

Galeano-Garces, Catalina; Camilleri, Emily T; Riester, Scott M; Dudakovic, Amel; Larson, Dirk R; Qu, Wenchun; Smith, Jay; Dietz, Allan B; Im, Hee-Jeong; Krych, Aaron J; Larson, A Noelle; Karperien, Marcel; van Wijnen, Andre J

2017-07-01

To determine the optimal environmental conditions for chondrogenic differentiation of human adipose tissue-derived mesenchymal stromal/stem cells (AMSCs). In this investigation we specifically investigate the role of oxygen tension and 3-dimensional (3D) culture systems. Both AMSCs and primary human chondrocytes were cultured for 21 days in chondrogenic media under normoxic (21% oxygen) or hypoxic (2% oxygen) conditions using 2 distinct 3D culture methods (high-density pellets and poly-ε-caprolactone [PCL] scaffolds). Histologic analysis of chondro-pellets and the expression of chondrocyte-related genes as measured by reverse transcriptase quantitative polymerase chain reaction were used to evaluate the efficiency of differentiation. AMSCs are capable of expressing established cartilage markers including COL2A1, ACAN, and DCN when grown in chondrogenic differentiation media as determined by gene expression and histologic analysis of cartilage markers. Expression of several cartilage-related genes was enhanced by low oxygen tension, including ACAN and HAPLN1. The pellet culture environment also promoted the expression of hypoxia-inducible cartilage markers compared with cells grown on 3D scaffolds. Cell type-specific effects of low oxygen and 3D environments indicate that mesenchymal cell fate and differentiation potential is remarkably sensitive to oxygen. Genetic programming of AMSCs to a chondrocytic phenotype is effective under hypoxic conditions as evidenced by increased expression of cartilage-related biomarkers and biosynthesis of a glycosaminoglycan-positive matrix. Lower local oxygen levels within cartilage pellets may be a significant driver of chondrogenic differentiation.

Combining Shapley value and statistics to the analysis of gene expression data in children exposed to air pollution

PubMed Central

Moretti, Stefano; van Leeuwen, Danitsja; Gmuender, Hans; Bonassi, Stefano; van Delft, Joost; Kleinjans, Jos; Patrone, Fioravante; Merlo, Domenico Franco

2008-01-01

Background In gene expression analysis, statistical tests for differential gene expression provide lists of candidate genes having, individually, a sufficiently low p-value. However, the interpretation of each single p-value within complex systems involving several interacting genes is problematic. In parallel, in the last sixty years, game theory has been applied to political and social problems to assess the power of interacting agents in forcing a decision and, more recently, to represent the relevance of genes in response to certain conditions. Results In this paper we introduce a Bootstrap procedure to test the null hypothesis that each gene has the same relevance between two conditions, where the relevance is represented by the Shapley value of a particular coalitional game defined on a microarray data-set. This method, which is called Comparative Analysis of Shapley value (shortly, CASh), is applied to data concerning the gene expression in children differentially exposed to air pollution. The results provided by CASh are compared with the results from a parametric statistical test for testing differential gene expression. Both lists of genes provided by CASh and t-test are informative enough to discriminate exposed subjects on the basis of their gene expression profiles. While many genes are selected in common by CASh and the parametric test, it turns out that the biological interpretation of the differences between these two selections is more interesting, suggesting a different interpretation of the main biological pathways in gene expression regulation for exposed individuals. A simulation study suggests that CASh offers more power than t-test for the detection of differential gene expression variability. Conclusion CASh is successfully applied to gene expression analysis of a data-set where the joint expression behavior of genes may be critical to characterize the expression response to air pollution. We demonstrate a synergistic effect between coalitional games and statistics that resulted in a selection of genes with a potential impact in the regulation of complex pathways. PMID:18764936

Transcriptome Analysis of Orbital Adipose Tissue in Active Thyroid Eye Disease Using Next Generation RNA Sequencing Technology

PubMed Central

Lee, Bradford W.; Kumar, Virender B.; Biswas, Pooja; Ko, Audrey C.; Alameddine, Ramzi M.; Granet, David B.; Ayyagari, Radha; Kikkawa, Don O.; Korn, Bobby S.

2018-01-01

Objective: This study utilized Next Generation Sequencing (NGS) to identify differentially expressed transcripts in orbital adipose tissue from patients with active Thyroid Eye Disease (TED) versus healthy controls. Method: This prospective, case-control study enrolled three patients with severe, active thyroid eye disease undergoing orbital decompression, and three healthy controls undergoing routine eyelid surgery with removal of orbital fat. RNA Sequencing (RNA-Seq) was performed on freshly obtained orbital adipose tissue from study patients to analyze the transcriptome. Bioinformatics analysis was performed to determine pathways and processes enriched for the differential expression profile. Quantitative Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR) was performed to validate the differential expression of selected genes identified by RNA-Seq. Results: RNA-Seq identified 328 differentially expressed genes associated with active thyroid eye disease, many of which were responsible for mediating inflammation, cytokine signaling, adipogenesis, IGF-1 signaling, and glycosaminoglycan binding. The IL-5 and chemokine signaling pathways were highly enriched, and very-low-density-lipoprotein receptor activity and statin medications were implicated as having a potential role in TED. Conclusion: This study is the first to use RNA-Seq technology to elucidate differential gene expression associated with active, severe TED. This study suggests a transcriptional basis for the role of statins in modulating differentially expressed genes that mediate the pathogenesis of thyroid eye disease. Furthermore, the identification of genes with altered levels of expression in active, severe TED may inform the molecular pathways central to this clinical phenotype and guide the development of novel therapeutic agents. PMID:29760827

Endothelial nitric oxide synthase is dynamically expressed during bone marrow stem cell differentiation into endothelial cells.

PubMed

Liu, Zhenguo; Jiang, Yuehua; Hao, Hong; Gupta, Kalpna; Xu, Jian; Chu, Ling; McFalls, Edward; Zweier, Jay; Verfaillie, Catherine; Bache, Robert J

2007-09-01

This study was designed to investigate the developmental expression of endothelial nitric oxide synthase (eNOS) during stem cell differentiation into endothelial cells and to examine the functional status of the newly differentiated endothelial cells. Mouse adult multipotent progenitor cells (MAPCs) were used as the source of stem cells and were induced to differentiate into endothelial cells with vascular endothelial growth factor (VEGF) in serum-free medium. Expression of eNOS in the cells during differentiation was evaluated with real-time PCR, nitric oxide synthase (NOS) activity, and Western blot analysis. It was found that eNOS, but no other NOS, was present in undifferentiated MAPCs. eNOS expression disappeared in the cells immediately after induction of differentiation. However, eNOS expression reoccurred at day 7 during differentiation. Increasing eNOS mRNA, protein content, and activity were observed in the cells at days 14 and 21 during differentiation. The differentiated endothelial cells formed dense capillary networks on growth factor-reduced Matrigel. VEGF-stimulated phosphorylation of extracellular signal-regulated kinase (ERK)-1 and ERK-2 occurred in these cells, which was inhibited by NOS inhibitor N(G)-nitro-L-arginine methyl ester. In conclusion, these data demonstrate that eNOS is present in MAPCs and is dynamically expressed during the differentiation of MAPCs into endothelial cells in vitro.

Whole-transcriptome, high-throughput RNA sequence analysis of the bovine macrophage response to Mycobacterium bovis infection in vitro.

PubMed

Nalpas, Nicolas C; Park, Stephen D E; Magee, David A; Taraktsoglou, Maria; Browne, John A; Conlon, Kevin M; Rue-Albrecht, Kévin; Killick, Kate E; Hokamp, Karsten; Lohan, Amanda J; Loftus, Brendan J; Gormley, Eamonn; Gordon, Stephen V; MacHugh, David E

2013-04-08

Mycobacterium bovis, the causative agent of bovine tuberculosis, is an intracellular pathogen that can persist inside host macrophages during infection via a diverse range of mechanisms that subvert the host immune response. In the current study, we have analysed and compared the transcriptomes of M. bovis-infected monocyte-derived macrophages (MDM) purified from six Holstein-Friesian females with the transcriptomes of non-infected control MDM from the same animals over a 24 h period using strand-specific RNA sequencing (RNA-seq). In addition, we compare gene expression profiles generated using RNA-seq with those previously generated by us using the high-density Affymetrix® GeneChip® Bovine Genome Array platform from the same MDM-extracted RNA. A mean of 7.2 million reads from each MDM sample mapped uniquely and unambiguously to single Bos taurus reference genome locations. Analysis of these mapped reads showed 2,584 genes (1,392 upregulated; 1,192 downregulated) and 757 putative natural antisense transcripts (558 upregulated; 119 downregulated) that were differentially expressed based on sense and antisense strand data, respectively (adjusted P-value ≤ 0.05). Of the differentially expressed genes, 694 were common to both the sense and antisense data sets, with the direction of expression (i.e. up- or downregulation) positively correlated for 693 genes and negatively correlated for the remaining gene. Gene ontology analysis of the differentially expressed genes revealed an enrichment of immune, apoptotic and cell signalling genes. Notably, the number of differentially expressed genes identified from RNA-seq sense strand analysis was greater than the number of differentially expressed genes detected from microarray analysis (2,584 genes versus 2,015 genes). Furthermore, our data reveal a greater dynamic range in the detection and quantification of gene transcripts for RNA-seq compared to microarray technology. This study highlights the value of RNA-seq in identifying novel immunomodulatory mechanisms that underlie host-mycobacterial pathogen interactions during infection, including possible complex post-transcriptional regulation of host gene expression involving antisense RNA.

Discovery of Transcription Factors Novel to Mouse Cerebellar Granule Cell Development Through Laser-Capture Microdissection.

PubMed

Zhang, Peter G Y; Yeung, Joanna; Gupta, Ishita; Ramirez, Miguel; Ha, Thomas; Swanson, Douglas J; Nagao-Sato, Sayaka; Itoh, Masayoshi; Kawaji, Hideya; Lassmann, Timo; Daub, Carsten O; Arner, Erik; de Hoon, Michiel; Carninci, Piero; Forrest, Alistair R R; Hayashizaki, Yoshihide; Goldowitz, Dan

2018-06-01

Laser-capture microdissection was used to isolate external germinal layer tissue from three developmental periods of mouse cerebellar development: embryonic days 13, 15, and 18. The cerebellar granule cell-enriched mRNA library was generated with next-generation sequencing using the Helicos technology. Our objective was to discover transcriptional regulators that could be important for the development of cerebellar granule cells-the most numerous neuron in the central nervous system. Through differential expression analysis, we have identified 82 differentially expressed transcription factors (TFs) from a total of 1311 differentially expressed genes. In addition, with TF-binding sequence analysis, we have identified 46 TF candidates that could be key regulators responsible for the variation in the granule cell transcriptome between developmental stages. Altogether, we identified 125 potential TFs (82 from differential expression analysis, 46 from motif analysis with 3 overlaps in the two sets). From this gene set, 37 TFs are considered novel due to the lack of previous knowledge about their roles in cerebellar development. The results from transcriptome-wide analyses were validated with existing online databases, qRT-PCR, and in situ hybridization. This study provides an initial insight into the TFs of cerebellar granule cells that might be important for development and provide valuable information for further functional studies on these transcriptional regulators.

Microarray analysis of differentially expressed genes engaged in fruit development between Prunus mume and Prunus armeniaca.

PubMed

Li, Xiaoying; Korir, Nicholas Kibet; Liu, Lili; Shangguan, Lingfei; Wang, Yuzhu; Han, Jian; Chen, Ming; Fang, Jinggui

2012-11-15

Microarray analysis is a technique that can be employed to provide expression profiles of single genes and new insights to elucidate the biological mechanisms responsible for fruit development. To evaluate expression of genes mostly engaged in fruit development between Prunus mume and Prunus armeniaca, we first identified differentially expressed transcripts along the entire fruit life cycle by using microarrays spotted with 10,641 ESTs collected from P. mume and other Prunus EST sequences. A total of 1418 ESTs were selected after quality control of microarray spots and analysis for differential gene expression patterns during fruit development of P. mume and P. Armeniaca. From these, 707 up-regulated and 711 down-regulated genes showing more than two-fold differences in expression level were annotated by GO based on biological processes, molecular functions and cellular components. These differentially expressed genes were found to be involved in several important pathways of carbohydrate, galactose, and starch and sucrose metabolism as well as in biosynthesis of other secondary metabolites via KEGG. This could provide detailed information on the fruit quality differences during development and ripening of these two species. With the results obtained, we provide a practical database for comprehensive understanding of molecular events during fruit development and also lay a theoretical foundation for the cloning of genes regulating in a series of important rate-limiting enzymes involved in vital metabolic pathways during fruit development. Copyright © 2012 Elsevier GmbH. All rights reserved.

Comparative proteomic analysis in children with idiopathic short stature (ISS) before and after short-term recombinant human growth hormone (rhGH) therapy.

PubMed

Heo, Sun Hee; Choi, Jin-Ho; Kim, Yoo-Mi; Jung, Chang-Woo; Lee, Jin; Jin, Hye Young; Kim, Gu-Hwan; Lee, Beom Hee; Shin, Choong Ho; Yoo, Han-Wook

2013-04-01

This study was undertaken to identify growth hormone (GH) responsive proteins and protein expression patterns by short-term recombinant human growth hormone (rhGH) therapy in patients with idiopathic short stature (ISS) using proteomic analysis. Seventeen children (14 males and three females) with ISS were included. They were treated with rhGH at a dose of 0.31 ± 0.078 mg/kg/week for 3 months. Immunodepletion of six highly-abundant serum proteins followed by 2D DIGE analysis, and subsequent MALDI TOF MS, were employed to generate a panel of proteins differentially expressed after short-term rhGH therapy and verify the differences in serum levels of specific proteins by rhGH therapy. Fourteen spots were differentially expressed after rhGH treatment. Among them, apo E and apo L-1 expression were consistently enhanced, whereas serum amyloid A was reduced after rhGH therapy. The differential expressions of these proteins were subsequently verified by Western blot analysis using sera of the before and after rhGH treatment. This study suggests that rhGH therapy influences lipoprotein metabolism and enhances apo L-1 protein expression in ISS patients. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Using Peptide-Level Proteomics Data for Detecting Differentially Expressed Proteins.

PubMed

Suomi, Tomi; Corthals, Garry L; Nevalainen, Olli S; Elo, Laura L

2015-11-06

The expression of proteins can be quantified in high-throughput means using different types of mass spectrometers. In recent years, there have emerged label-free methods for determining protein abundance. Although the expression is initially measured at the peptide level, a common approach is to combine the peptide-level measurements into protein-level values before differential expression analysis. However, this simple combination is prone to inconsistencies between peptides and may lose valuable information. To this end, we introduce here a method for detecting differentially expressed proteins by combining peptide-level expression-change statistics. Using controlled spike-in experiments, we show that the approach of averaging peptide-level expression changes yields more accurate lists of differentially expressed proteins than does the conventional protein-level approach. This is particularly true when there are only few replicate samples or the differences between the sample groups are small. The proposed technique is implemented in the Bioconductor package PECA, and it can be downloaded from http://www.bioconductor.org.

ProbFAST: Probabilistic functional analysis system tool.

PubMed

Silva, Israel T; Vêncio, Ricardo Z N; Oliveira, Thiago Y K; Molfetta, Greice A; Silva, Wilson A

2010-03-30

The post-genomic era has brought new challenges regarding the understanding of the organization and function of the human genome. Many of these challenges are centered on the meaning of differential gene regulation under distinct biological conditions and can be performed by analyzing the Multiple Differential Expression (MDE) of genes associated with normal and abnormal biological processes. Currently MDE analyses are limited to usual methods of differential expression initially designed for paired analysis. We proposed a web platform named ProbFAST for MDE analysis which uses Bayesian inference to identify key genes that are intuitively prioritized by means of probabilities. A simulated study revealed that our method gives a better performance when compared to other approaches and when applied to public expression data, we demonstrated its flexibility to obtain relevant genes biologically associated with normal and abnormal biological processes. ProbFAST is a free accessible web-based application that enables MDE analysis on a global scale. It offers an efficient methodological approach for MDE analysis of a set of genes that are turned on and off related to functional information during the evolution of a tumor or tissue differentiation. ProbFAST server can be accessed at http://gdm.fmrp.usp.br/probfast.

ProbFAST: Probabilistic Functional Analysis System Tool

PubMed Central

2010-01-01

Background The post-genomic era has brought new challenges regarding the understanding of the organization and function of the human genome. Many of these challenges are centered on the meaning of differential gene regulation under distinct biological conditions and can be performed by analyzing the Multiple Differential Expression (MDE) of genes associated with normal and abnormal biological processes. Currently MDE analyses are limited to usual methods of differential expression initially designed for paired analysis. Results We proposed a web platform named ProbFAST for MDE analysis which uses Bayesian inference to identify key genes that are intuitively prioritized by means of probabilities. A simulated study revealed that our method gives a better performance when compared to other approaches and when applied to public expression data, we demonstrated its flexibility to obtain relevant genes biologically associated with normal and abnormal biological processes. Conclusions ProbFAST is a free accessible web-based application that enables MDE analysis on a global scale. It offers an efficient methodological approach for MDE analysis of a set of genes that are turned on and off related to functional information during the evolution of a tumor or tissue differentiation. ProbFAST server can be accessed at http://gdm.fmrp.usp.br/probfast. PMID:20353576

Pathway Inspector: a pathway based web application for RNAseq analysis of model and non-model organisms.

PubMed

Bianco, Luca; Riccadonna, Samantha; Lavezzo, Enrico; Falda, Marco; Formentin, Elide; Cavalieri, Duccio; Toppo, Stefano; Fontana, Paolo

2017-02-01

Pathway Inspector is an easy-to-use web application helping researchers to find patterns of expression in complex RNAseq experiments. The tool combines two standard approaches for RNAseq analysis: the identification of differentially expressed genes and a topology-based analysis of enriched pathways. Pathway Inspector is equipped with ad hoc interactive graphical interfaces simplifying the discovery of modulated pathways and the integration of the differentially expressed genes in the corresponding pathway topology. Pathway Inspector is available at the website http://admiral.fmach.it/PI and has been developed in Python, making use of the Django Web Framework. Contact:paolo.fontana@fmach.it

Gene expression profiles in whole blood and associations with metabolic dysregulation in obesity.

PubMed

Cox, Amanda J; Zhang, Ping; Evans, Tiffany J; Scott, Rodney J; Cripps, Allan W; West, Nicholas P

Gene expression data provides one tool to gain further insight into the complex biological interactions linking obesity and metabolic disease. This study examined associations between blood gene expression profiles and metabolic disease in obesity. Whole blood gene expression profiles, performed using the Illumina HT-12v4 Human Expression Beadchip, were compared between (i) individuals with obesity (O) or lean (L) individuals (n=21 each), (ii) individuals with (M) or without (H) Metabolic Syndrome (n=11 each) matched on age and gender. Enrichment of differentially expressed genes (DEG) into biological pathways was assessed using Ingenuity Pathway Analysis. Association between sets of genes from biological pathways considered functionally relevant and Metabolic Syndrome were further assessed using an area under the curve (AUC) and cross-validated classification rate (CR). For OvL, only 50 genes were significantly differentially expressed based on the selected differential expression threshold (1.2-fold, p<0.05). For MvH, 582 genes were significantly differentially expressed (1.2-fold, p<0.05) and pathway analysis revealed enrichment of DEG into a diverse set of pathways including immune/inflammatory control, insulin signalling and mitochondrial function pathways. Gene sets from the mTOR signalling pathways demonstrated the strongest association with Metabolic Syndrome (p=8.1×10 -8 ; AUC: 0.909, CR: 72.7%). These results support the use of expression profiling in whole blood in the absence of more specific tissue types for investigations of metabolic disease. Using a pathway analysis approach it was possible to identify an enrichment of DEG into biological pathways that could be targeted for in vitro follow-up. Copyright © 2017 Asia Oceania Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.

Identification of microRNAs differentially expressed involved in male flower development.

PubMed

Wang, Zhengjia; Huang, Jianqin; Sun, Zhichao; Zheng, Bingsong

2015-03-01

Hickory (Carya cathayensis Sarg.) is one of the most economically important woody trees in eastern China, but its long flowering phase delays yield. Our understanding of the regulatory roles of microRNAs (miRNAs) in male flower development in hickory remains poor. Using high-throughput sequencing technology, we have pyrosequenced two small RNA libraries from two male flower differentiation stages in hickory. Analysis of the sequencing data identified 114 conserved miRNAs that belonged to 23 miRNA families, five novel miRNAs including their corresponding miRNA*s, and 22 plausible miRNA candidates. Differential expression analysis revealed 12 miRNA sequences that were upregulated in the later (reproductive) stage of male flower development. Quantitative real-time PCR showed similar expression trends as that of the deep sequencing. Novel miRNAs and plausible miRNA candidates were predicted using bioinformatic analysis methods. The miRNAs newly identified in this study have increased the number of known miRNAs in hickory, and the identification of differentially expressed miRNAs will provide new avenues for studies into miRNAs involved in the process of male flower development in hickory and other related trees.

Global Analysis of Differentially Expressed Genes and Proteins in the Wheat Callus Infected by Agrobacterium tumefaciens

PubMed Central

Zhou, Xiaohong; Wang, Ke; Lv, Dongwen; Wu, Chengjun; Li, Jiarui; Zhao, Pei; Lin, Zhishan; Du, Lipu; Yan, Yueming; Ye, Xingguo

2013-01-01

Agrobacterium-mediated plant transformation is an extremely complex and evolved process involving genetic determinants of both the bacteria and the host plant cells. However, the mechanism of the determinants remains obscure, especially in some cereal crops such as wheat, which is recalcitrant for Agrobacterium-mediated transformation. In this study, differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were analyzed in wheat callus cells co-cultured with Agrobacterium by using RNA sequencing (RNA-seq) and two-dimensional electrophoresis (2-DE) in conjunction with mass spectrometry (MS). A set of 4,889 DEGs and 90 DEPs were identified, respectively. Most of them are related to metabolism, chromatin assembly or disassembly and immune defense. After comparative analysis, 24 of the 90 DEPs were detected in RNA-seq and proteomics datasets simultaneously. In addition, real-time RT-PCR experiments were performed to check the differential expression of the 24 genes, and the results were consistent with the RNA-seq data. According to gene ontology (GO) analysis, we found that a big part of these differentially expressed genes were related to the process of stress or immunity response. Several putative determinants and candidate effectors responsive to Agrobacterium mediated transformation of wheat cells were discussed. We speculate that some of these genes are possibly related to Agrobacterium infection. Our results will help to understand the interaction between Agrobacterium and host cells, and may facilitate developing efficient transformation strategies in cereal crops. PMID:24278131

QPROT: Statistical method for testing differential expression using protein-level intensity data in label-free quantitative proteomics.

PubMed

Choi, Hyungwon; Kim, Sinae; Fermin, Damian; Tsou, Chih-Chiang; Nesvizhskii, Alexey I

2015-11-03

We introduce QPROT, a statistical framework and computational tool for differential protein expression analysis using protein intensity data. QPROT is an extension of the QSPEC suite, originally developed for spectral count data, adapted for the analysis using continuously measured protein-level intensity data. QPROT offers a new intensity normalization procedure and model-based differential expression analysis, both of which account for missing data. Determination of differential expression of each protein is based on the standardized Z-statistic based on the posterior distribution of the log fold change parameter, guided by the false discovery rate estimated by a well-known Empirical Bayes method. We evaluated the classification performance of QPROT using the quantification calibration data from the clinical proteomic technology assessment for cancer (CPTAC) study and a recently published Escherichia coli benchmark dataset, with evaluation of FDR accuracy in the latter. QPROT is a statistical framework with computational software tool for comparative quantitative proteomics analysis. It features various extensions of QSPEC method originally built for spectral count data analysis, including probabilistic treatment of missing values in protein intensity data. With the increasing popularity of label-free quantitative proteomics data, the proposed method and accompanying software suite will be immediately useful for many proteomics laboratories. This article is part of a Special Issue entitled: Computational Proteomics. Copyright © 2015 Elsevier B.V. All rights reserved.

Integrated analysis of long non-coding RNAs in human gastric cancer: An in silico study.

PubMed

Han, Weiwei; Zhang, Zhenyu; He, Bangshun; Xu, Yijun; Zhang, Jun; Cao, Weijun

2017-01-01

Accumulating evidence highlights the important role of long non-coding RNAs (lncRNAs) in a large number of biological processes. However, the knowledge of genome scale expression of lncRNAs and their potential biological function in gastric cancer is still lacking. Using RNA-seq data from 420 gastric cancer patients in The Cancer Genome Atlas (TCGA), we identified 1,294 lncRNAs differentially expressed in gastric cancer compared with adjacent normal tissues. We also found 247 lncRNAs differentially expressed between intestinal subtype and diffuse subtype. Survival analysis revealed 33 lncRNAs independently associated with patient overall survival, of which 6 lncRNAs were validated in the internal validation set. There were 181 differentially expressed lncRNAs located in the recurrent somatic copy number alterations (SCNAs) regions and their correlations between copy number and RNA expression level were also analyzed. In addition, we inferred the function of lncRNAs by construction of a co-expression network for mRNAs and lncRNAs. Together, this study presented an integrative analysis of lncRNAs in gastric cancer and provided a valuable resource for further functional research of lncRNAs in gastric cancer.

Regulation of c- and N-myc expression during induced differentiation of murine neuroblastoma cells.

PubMed

Larcher, J C; Vayssière, J L; Lossouarn, L; Gros, F; Croizat, B

1991-04-01

Using clones N1E-115 and N1A-103 from mouse neuroblastoma C1300, a comparative analysis of c- and N-myc gene expression was undertaken both in proliferating cells and in cultures exposed to conditions which induce differentiation. Under the latter conditions, while N1E-115 cells extend abundant neurites and express many biochemical features of mature neurons, clone N1A-103 stops dividing and expresses certain neurospecific markers but is unable to differentiate morphologically. In both clones, chemical agents, i.e. 1-methyl cyclohexane carboxylic acid (CCA) or dimethyl sulfoxide (DMSO), induce a decrease in c-myc expression. Similar results were found for N-myc gene in N1E-115 cells, but in contrast, in clone N1A-103, N-myc expression is increased with CCA and not modified with DMSO. Globally, this study favours the hypothesis that changes in c-myc expression would correspond to cell division blockade and differentiation, while modulations in N-myc are more closely related to an early phase of terminal differentiation.

High Matrix Metalloproteinase Activity is a Hallmark of Periapical Granulomas

PubMed Central

de Paula e Silva, Francisco Wanderley Garcia; D'Silva, Nisha J.; da Silva, Léa Assed Bezerra; Kapila, Yvonne Lorraine

2009-01-01

Introduction Inability to distinguish periapical cysts from granulomas prior to performing root canal treatment leads to uncertainty in treatment outcomes, because cysts have lower healing rates. Searching for differential expression of molecules within cysts or granulomas could provide information with regard to the identity of the lesion or suggest mechanistic differences that may form the basis for future therapeutic intervention. Thus, we investigated whether granulomas and cysts exhibit differential expression of extracellular matrix (ECM) molecules. Methods Human periapical granulomas, periapical cysts, and healthy periodontal ligament tissues were used to investigate the differential expression of ECM molecules by microarray analysis. Since matrix metalloproteinases (MMP) showed the highest differential expression in the microarray analysis, MMPs were further examined by in situ zymography and immunohistochemistry. Data were analyzed using one-way ANOVA followed by Tukey test. Results We observed that cysts and granulomas differentially expressed several ECM molecules, especially those from the matrix metalloproteinase (MMP) family. Compared to cysts, granulomas exhibited higher MMP enzymatic activity in areas stained for MMP-9. These areas were composed of polymorphonuclear cells (PMNs), in contrast to cysts. Similarly, MMP-13 was expressed by a greater number of cells in granulomas compared to cysts. Conclusion Our findings indicate that high enzymatic MMP activity in PMNs together with MMP-9 and MMP-13 stained cells could be a molecular signature of granulomas, unlike periapical cysts. PMID:19720222

Gene expression profile analysis of rat cerebellum under acute alcohol intoxication.

PubMed

Zhang, Yu; Wei, Guangkuan; Wang, Yuehong; Jing, Ling; Zhao, Qingjie

2015-02-25

Acute alcohol intoxication, a common disease causing damage to the central nervous system (CNS) has been primarily studied on the aspects of alcohol addiction and chronic alcohol exposure. The understanding of gene expression change in the CNS during acute alcohol intoxication is still lacking. We established a model for acute alcohol intoxication in SD rats by oral gavage. A rat cDNA microarray was used to profile mRNA expression in the cerebella of alcohol-intoxicated rats (experimental group) and saline-treated rats (control group). A total of 251 differentially expressed genes were identified in response to acute alcohol intoxication, in which 208 of them were up-regulated and 43 were down-regulated. Gene ontology (GO) term enrichment analysis and pathway analysis revealed that the genes involved in the biological processes of immune response and endothelial integrity are among the most severely affected in response to acute alcohol intoxication. We discovered five transcription factors whose consensus binding motifs are overrepresented in the promoter region of differentially expressed genes. Additionally, we identified 20 highly connected hub genes by co-expression analysis, and validated the differential expression of these genes by real-time quantitative PCR. By determining novel biological pathways and transcription factors that have functional implication to acute alcohol intoxication, our study substantially contributes to the understanding of the molecular mechanism underlying the pathology of acute alcoholism. Copyright © 2014 Elsevier B.V. All rights reserved.

RNA‑sequencing analysis of aberrantly expressed long non‑coding RNAs and mRNAs in a mouse model of ventilator‑induced lung injury.

PubMed

Xu, Bo; Wang, Yizhou; Li, Xiujuan; Mao, Yanfei; Deng, Xiaoming

2018-05-17

Long non-coding RNAs (lncRNAs) are closely associated with the regulation of various biological processes and are involved in the pathogenesis of numerous diseases. However, to the best of our knowledge, the role of lncRNAs in ventilator‑induced lung injury (VILI) has yet to be evaluated. In the present study, high‑throughput sequencing was applied to investigate differentially expressed lncRNAs and mRNAs (fold change >2; false discovery rate <0.05). Bioinformatics analysis was employed to predict the functions of differentially expressed lncRNAs. A total of 104 lncRNAs (74 upregulated and 30 downregulated) and 809 mRNAs (521 upregulated and 288 downregulated) were differentially expressed in lung tissues from the VILI group. Gene ontology analysis demonstrated that the differentially expressed lncRNAs and mRNAs were mainly associated with biological functions, including apoptosis, angiogenesis, neutrophil chemotaxis and skeletal muscle cell differentiation. The top four enriched pathways were the tumor necrosis factor (TNF) signaling pathway, P53 signaling pathway, neuroactive ligand‑receptor interaction and the forkhead box O signaling pathway. Several lncRNAs were predicted to serve a vital role in VILI. Subsequently, three lncRNAs [mitogen‑activated protein kinase kinase 3, opposite strand (Map2k3os), dynamin 3, opposite strand and abhydrolase domain containing 11, opposite strand] and three mRNAs (growth arrest and DNA damage‑inducible α, claudin 4 and thromboxane A2 receptor) were measured by reverse transcription‑quantitative polymerase chain reaction, in order to confirm the veracity of RNA‑sequencing analysis. In addition, Map2k3os small interfering RNA transfection inhibited the expression of stretch‑induced cytokines [TNF‑α, interleukin (IL)‑1β and IL‑6] in MLE12 cells. In conclusion, the results of the present study provided a profile of differentially expressed lncRNAs in VILI. Several important lncRNAs may be involved in the pathological process of VILI, which may be useful to guide further investigation into the pathogenesis for this disease.

The Effect of Recombinant Tyrosine Hydroxylase Expression on the Neurogenic Differentiation Potency of Mesenchymal Stem Cells

PubMed Central

Duruksu, Gokhan; Karaoz, Erdal

2018-01-01

Objective Tyrosine hydroxylase (TH) is a rate-limiting enzyme in dopamine synthesis, making the enhancement of its activity a target for ensuring sufficient dopamine levels. Rat bone marrow mesenchymal stem cells (rBM-MSCs) are known to synthesize TH after differentiating into neuronal cells through chemical induction, but the effect of its ectopic expression on these cells has not yet been determined. This study investigated the effects of ectopic recombinant TH expression on the stemness characteristics of rBM-MSCs. Methods After cloning, a cell line with stable TH expression was maintained, and the proliferation, the gene expression profile, and differentiation potential of rBM-MSCs were analyzed. Analysis of the cells showed an increment in the proliferation rate that could be reversed by the neutralization of TH. Results The constitutive expression of TH in rBM-MSCs was successfully implemented, without significantly affecting their osteogenic and adipogenic differentiation potential. TH expression improved the expression of other neuronal markers, such as glial fibrillary acidic protein, β-tubulin, nestin, and c-Fos, confirming the neurogenic differentiation capacity of the stem cells. The expression of brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) significantly increased after the chemical induction of neurogenic differentiation. Conclusion In this study, the expression of recombinant TH improved the neuroprotective effect of MSCs by upregulating the expression of BDNF and CNTF. Although the neuronal markers were upregulated, the expression of recombinant TH alone in rBM-MSCs was not sufficient for MSCs to differentiate into neurogenic cell lines. PMID:29656620

RPL13A and EEF1A1 Are Suitable Reference Genes for qPCR during Adipocyte Differentiation of Vascular Stromal Cells from Patients with Different BMI and HOMA-IR.

PubMed

Gentile, Adriana-Mariel; Lhamyani, Said; Coín-Aragüez, Leticia; Oliva-Olivera, Wilfredo; Zayed, Hatem; Vega-Rioja, Antonio; Monteseirin, Javier; Romero-Zerbo, Silvana-Yanina; Tinahones, Francisco-José; Bermúdez-Silva, Francisco-Javier; El Bekay, Rajaa

2016-01-01

Real-time or quantitative PCR (qPCR) is a useful technique that requires reliable reference genes for data normalization in gene expression analysis. Adipogenesis is among the biological processes suitable for this technique. The selection of adequate reference genes is essential for qPCR gene expression analysis of human Vascular Stromal Cells (hVSCs) during their differentiation into adipocytes. To the best of our knowledge, there are no studies validating reference genes for the analyses of visceral and subcutaneous adipose tissue hVSCs from subjects with different Body Mass Index (BMI) and Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) index. The present study was undertaken to analyze this question. We first analyzed the stability of expression of five potential reference genes: CYC, GAPDH, RPL13A, EEF1A1, and 18S ribosomal RNA, during in vitro adipogenic differentiation, in samples from these types of patients. The expression of RPL13A and EEF1A1 was not affected by differentiation, thus being these genes the most stable candidates, while CYC, GAPDH, and 18S were not suitable for this sort of analysis. This work highlights that RPL13A and EEF1A1 are good candidates as reference genes for qPCR analysis of hVSCs differentiation into adipocytes from subjects with different BMI and HOMA-IR.

RPL13A and EEF1A1 Are Suitable Reference Genes for qPCR during Adipocyte Differentiation of Vascular Stromal Cells from Patients with Different BMI and HOMA-IR

PubMed Central

Gentile, Adriana-Mariel; Lhamyani, Said; Coín-Aragüez, Leticia; Oliva-Olivera, Wilfredo; Zayed, Hatem; Vega-Rioja, Antonio; Monteseirin, Javier; Romero-Zerbo, Silvana-Yanina; Tinahones, Francisco-José; Bermúdez-Silva, Francisco-Javier; El Bekay, Rajaa

2016-01-01

Real-time or quantitative PCR (qPCR) is a useful technique that requires reliable reference genes for data normalization in gene expression analysis. Adipogenesis is among the biological processes suitable for this technique. The selection of adequate reference genes is essential for qPCR gene expression analysis of human Vascular Stromal Cells (hVSCs) during their differentiation into adipocytes. To the best of our knowledge, there are no studies validating reference genes for the analyses of visceral and subcutaneous adipose tissue hVSCs from subjects with different Body Mass Index (BMI) and Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) index. The present study was undertaken to analyze this question. We first analyzed the stability of expression of five potential reference genes: CYC, GAPDH, RPL13A, EEF1A1, and 18S ribosomal RNA, during in vitro adipogenic differentiation, in samples from these types of patients. The expression of RPL13A and EEF1A1 was not affected by differentiation, thus being these genes the most stable candidates, while CYC, GAPDH, and 18S were not suitable for this sort of analysis. This work highlights that RPL13A and EEF1A1 are good candidates as reference genes for qPCR analysis of hVSCs differentiation into adipocytes from subjects with different BMI and HOMA-IR. PMID:27304673

Gene expression analysis of a porcine hepatocyte/bile duct in vitro differentiaion model

USDA-ARS?s Scientific Manuscript database

A serum-free, feeder-cell-dependent, inductive differentiation culture system of porcine hepatocytes and bile ductules was analyzed for differential gene expression on a porcine genome microarray. Primary cultures of baby pig hepatocytes (BPH) were matured in culture as a monolayer of hepatocytes w...

DigOut: viewing differential expression genes as outliers.

PubMed

Yu, Hui; Tu, Kang; Xie, Lu; Li, Yuan-Yuan

2010-12-01

With regards to well-replicated two-conditional microarray datasets, the selection of differentially expressed (DE) genes is a well-studied computational topic, but for multi-conditional microarray datasets with limited or no replication, the same task is not properly addressed by previous studies. This paper adopts multivariate outlier analysis to analyze replication-lacking multi-conditional microarray datasets, finding that it performs significantly better than the widely used limit fold change (LFC) model in a simulated comparative experiment. Compared with the LFC model, the multivariate outlier analysis also demonstrates improved stability against sample variations in a series of manipulated real expression datasets. The reanalysis of a real non-replicated multi-conditional expression dataset series leads to satisfactory results. In conclusion, a multivariate outlier analysis algorithm, like DigOut, is particularly useful for selecting DE genes from non-replicated multi-conditional gene expression dataset.

Microarray Analysis Gene Expression Profiles in Laryngeal Muscle After Recurrent Laryngeal Nerve Injury.

PubMed

Bijangi-Vishehsaraei, Khadijeh; Blum, Kevin; Zhang, Hongji; Safa, Ahmad R; Halum, Stacey L

2016-03-01

The pathophysiology of recurrent laryngeal nerve (RLN) transection injury is rare in that it is characteristically followed by a high degree of spontaneous reinnervation, with reinnervation of the laryngeal adductor complex (AC) preceding that of the abducting posterior cricoarytenoid (PCA) muscle. Here, we aim to elucidate the differentially expressed myogenic factors following RLN injury that may be at least partially responsible for the spontaneous reinnervation. F344 male rats underwent RLN injury (n = 12) or sham surgery (n = 12). One week after RLN injury, larynges were harvested following euthanasia. The mRNA was extracted from PCA and AC muscles bilaterally, and microarray analysis was performed using a full rat genome array. Microarray analysis of denervated AC and PCA muscles demonstrated dramatic differences in gene expression profiles, with 205 individual probes that were differentially expressed between the denervated AC and PCA muscles and only 14 genes with similar expression patterns. The differential expression patterns of the AC and PCA suggest different mechanisms of reinnervation. The PCA showed the gene patterns of Wallerian degeneration, while the AC expressed the gene patterns of reinnervation by adjacent axonal sprouting. This finding may reveal important therapeutic targets applicable to RLN and other peripheral nerve injuries. © The Author(s) 2015.

The effects of dexamethasone, ascorbic acid, and β-glycerophosphate on osteoblastic differentiation by regulating estrogen receptor and osteopontin expression.

PubMed

Park, Jun-Beom

2012-03-01

Ascorbic acid (AA), β-glycerophosphate (GP), and dexamethasone (DEX) are the compounds known to favor the expression of the osteoblastic phenotype in several bone cell systems. In this report, the combination effects of differentiation agents on osteoprecursor cells were evaluated. The effect on cell proliferation was determined by a cell viability test with morphologic analysis. Differentiation and mineralization were evaluated using an alkaline phosphatase activity test and alizarin red-S staining. Protein expressions related to bone formation, such as transforming growth factor-beta (TGF-β), estrogen receptor-alpha (ER-α), and osteopontin (OPN) were evaluated by using a Western blot analysis. AA and GP provided an inductive effect for differentiation of osteoprecusor cells, while short-term application of DEX seemed to lead to a dose-dependent increase of cellular differentiation. Long-term use of DEX seemed to reduce mineralization. These effects may seem to be regulated by the expression of ER-α, OPN, and TGF-β. Further studies related to this mechanism within the in vivo model may be necessary to ascertain greater detail. Copyright © 2012 Elsevier Inc. All rights reserved.

PodNet, a protein-protein interaction network of the podocyte.

PubMed

Warsow, Gregor; Endlich, Nicole; Schordan, Eric; Schordan, Sandra; Chilukoti, Ravi K; Homuth, Georg; Moeller, Marcus J; Fuellen, Georg; Endlich, Karlhans

2013-07-01

Interactions between proteins crucially determine cellular structure and function. Differential analysis of the interactome may help elucidate molecular mechanisms during disease development; however, this analysis necessitates mapping of expression data on protein-protein interaction networks. These networks do not exist for the podocyte; therefore, we built PodNet, a literature-based mouse podocyte network in Cytoscape format. Using database protein-protein interactions, we expanded PodNet to XPodNet with enhanced connectivity. In order to test the performance of XPodNet in differential interactome analysis, we examined podocyte developmental differentiation and the effect of cell culture. Transcriptomes of podocytes in 10 different states were mapped on XPodNet and analyzed with the Cytoscape plugin ExprEssence, based on the law of mass action. Interactions between slit diaphragm proteins are most significantly upregulated during podocyte development and most significantly downregulated in culture. On the other hand, our analysis revealed that interactions lost during podocyte differentiation are not regained in culture, suggesting a loss rather than a reversal of differentiation for podocytes in culture. Thus, we have developed PodNet as a valuable tool for differential interactome analysis in podocytes, and we have identified established and unexplored regulated interactions in developing and cultured podocytes.

Differential gene expression profiles of peripheral blood mononuclear cells in childhood asthma.

PubMed

Kong, Qian; Li, Wen-Jing; Huang, Hua-Rong; Zhong, Ying-Qiang; Fang, Jian-Pei

2015-05-01

Asthma is a common childhood disease with strong genetic components. This study compared whole-genome expression differences between asthmatic young children and healthy controls to identify gene signatures of childhood asthma. Total RNA extracted from peripheral blood mononuclear cells (PBMC) was subjected to microarray analysis. QRT-PCR was performed to verify the microarray results. Classification and functional characterization of differential genes were illustrated by hierarchical clustering and gene ontology analysis. Multiple logistic regression (MLR) analysis, receiver operating characteristic (ROC) curve analysis, and discriminate power were used to scan asthma-specific diagnostic markers. For fold-change>2 and p < 0.05, there were 758 named differential genes. The results of QRT-PCR confirmed successfully the array data. Hierarchical clustering divided 29 highly possible genes into seven categories and the genes in the same cluster were likely to possess similar expression patterns or functions. Gene ontology analysis presented that differential genes primarily enriched in immune response, response to stress or stimulus, and regulation of apoptosis in biological process. MLR and ROC curve analysis revealed that the combination of ADAM33, Smad7, and LIGHT possessed excellent discriminating power. The combination of ADAM33, Smad7, and LIGHT would be a reliable and useful childhood asthma model for prediction and diagnosis.

Regional and temporal differences in gene expression of LH(BETA)T(AG) retinoblastoma tumors.

PubMed

Houston, Samuel K; Pina, Yolanda; Clarke, Jennifer; Koru-Sengul, Tulay; Scott, William K; Nathanson, Lubov; Schefler, Amy C; Murray, Timothy G

2011-07-23

The purpose of this study was to evaluate by microarray the hypothesis that LH(BETA)T(AG) retinoblastoma tumors exhibit regional and temporal variations in gene expression. LH(BETA)T(AG) mice aged 12, 16, and 20 weeks were euthanatized (n = 9). Specimens were taken from five tumor areas (apex, anterior lateral, center, base, and posterior lateral). Samples were hybridized to gene microarrays. The data were preprocessed and analyzed, and genes with a P < 0.01, according to the ANOVA models, and a log(2)-fold change >2.5 were considered to be differentially expressed. Differentially expressed genes were analyzed for overlap with known networks by using pathway analysis tools. There were significant temporal (P < 10(-8)) and regional differences in gene expression for LH(BETA)T(AG) retinoblastoma tumors. At P < 0.01 and log(2)-fold change >2.5, there were significant changes in gene expression of 190 genes apically, 84 genes anterolaterally, 126 genes posteriorly, 56 genes centrally, and 134 genes at the base. Differentially expressed genes overlapped with known networks, with significant involvement in regulation of cellular proliferation and growth, response to oxygen levels and hypoxia, regulation of cellular processes, cellular signaling cascades, and angiogenesis. There are significant temporal and regional variations in the LH(BETA)T(AG) retinoblastoma model. Differentially expressed genes overlap with key pathways that may play pivotal roles in murine retinoblastoma development. These findings suggest the mechanisms involved in tumor growth and progression in murine retinoblastoma tumors and identify pathways for analysis at a functional level, to determine significance in human retinoblastoma. Microarray analysis of LH(BETA)T(AG) retinal tumors showed significant regional and temporal variations in gene expression, including dysregulation of genes involved in hypoxic responses and angiogenesis.

Reconstruction of the genome-scale co-expression network for the Hippo signaling pathway in colorectal cancer.

PubMed

Dehghanian, Fariba; Hojati, Zohreh; Hosseinkhan, Nazanin; Mousavian, Zaynab; Masoudi-Nejad, Ali

2018-05-26

The Hippo signaling pathway (HSP) has been identified as an essential and complex signaling pathway for tumor suppression that coordinates proliferation, differentiation, cell death, cell growth and stemness. In the present study, we conducted a genome-scale co-expression analysis to reconstruct the HSP in colorectal cancer (CRC). Five key modules were detected through network clustering, and a detailed discussion of two modules containing respectively 18 and 13 over and down-regulated members of HSP was provided. Our results suggest new potential regulatory factors in the HSP. The detected modules also suggest novel genes contributing to CRC. Moreover, differential expression analysis confirmed the differential expression pattern of HSP members and new suggested regulatory factors between tumor and normal samples. These findings can further reveal the importance of HSP in CRC. Copyright © 2018 Elsevier Ltd. All rights reserved.

SigEMD: A powerful method for differential gene expression analysis in single-cell RNA sequencing data.

PubMed

Wang, Tianyu; Nabavi, Sheida

2018-04-24

Differential gene expression analysis is one of the significant efforts in single cell RNA sequencing (scRNAseq) analysis to discover the specific changes in expression levels of individual cell types. Since scRNAseq exhibits multimodality, large amounts of zero counts, and sparsity, it is different from the traditional bulk RNA sequencing (RNAseq) data. The new challenges of scRNAseq data promote the development of new methods for identifying differentially expressed (DE) genes. In this study, we proposed a new method, SigEMD, that combines a data imputation approach, a logistic regression model and a nonparametric method based on the Earth Mover's Distance, to precisely and efficiently identify DE genes in scRNAseq data. The regression model and data imputation are used to reduce the impact of large amounts of zero counts, and the nonparametric method is used to improve the sensitivity of detecting DE genes from multimodal scRNAseq data. By additionally employing gene interaction network information to adjust the final states of DE genes, we further reduce the false positives of calling DE genes. We used simulated datasets and real datasets to evaluate the detection accuracy of the proposed method and to compare its performance with those of other differential expression analysis methods. Results indicate that the proposed method has an overall powerful performance in terms of precision in detection, sensitivity, and specificity. Copyright © 2018 Elsevier Inc. All rights reserved.

Comparison of normalization methods for differential gene expression analysis in RNA-Seq experiments

PubMed Central

Maza, Elie; Frasse, Pierre; Senin, Pavel; Bouzayen, Mondher; Zouine, Mohamed

2013-01-01

In recent years, RNA-Seq technologies became a powerful tool for transcriptome studies. However, computational methods dedicated to the analysis of high-throughput sequencing data are yet to be standardized. In particular, it is known that the choice of a normalization procedure leads to a great variability in results of differential gene expression analysis. The present study compares the most widespread normalization procedures and proposes a novel one aiming at removing an inherent bias of studied transcriptomes related to their relative size. Comparisons of the normalization procedures are performed on real and simulated data sets. Real RNA-Seq data sets analyses, performed with all the different normalization methods, show that only 50% of significantly differentially expressed genes are common. This result highlights the influence of the normalization step on the differential expression analysis. Real and simulated data sets analyses give similar results showing 3 different groups of procedures having the same behavior. The group including the novel method named “Median Ratio Normalization” (MRN) gives the lower number of false discoveries. Within this group the MRN method is less sensitive to the modification of parameters related to the relative size of transcriptomes such as the number of down- and upregulated genes and the gene expression levels. The newly proposed MRN method efficiently deals with intrinsic bias resulting from relative size of studied transcriptomes. Validation with real and simulated data sets confirmed that MRN is more consistent and robust than existing methods. PMID:26442135

Differentially-Expressed Pseudogenes in HIV-1 Infection.

PubMed

Gupta, Aditi; Brown, C Titus; Zheng, Yong-Hui; Adami, Christoph

2015-09-29

Not all pseudogenes are transcriptionally silent as previously thought. Pseudogene transcripts, although not translated, contribute to the non-coding RNA pool of the cell that regulates the expression of other genes. Pseudogene transcripts can also directly compete with the parent gene transcripts for mRNA stability and other cell factors, modulating their expression levels. Tissue-specific and cancer-specific differential expression of these "functional" pseudogenes has been reported. To ascertain potential pseudogene:gene interactions in HIV-1 infection, we analyzed transcriptomes from infected and uninfected T-cells and found that 21 pseudogenes are differentially expressed in HIV-1 infection. This is interesting because parent genes of one-third of these differentially-expressed pseudogenes are implicated in HIV-1 life cycle, and parent genes of half of these pseudogenes are involved in different viral infections. Our bioinformatics analysis identifies candidate pseudogene:gene interactions that may be of significance in HIV-1 infection. Experimental validation of these interactions would establish that retroviruses exploit this newly-discovered layer of host gene expression regulation for their own benefit.

Identification of a role for the nuclear receptor EAR-2 in the maintenance of clonogenic status within the leukemia cell hierarchy

PubMed Central

Ichim, CV; Atkins, HL; Iscove, NN; Wells, RA

2016-01-01

Identification of genes that regulate clonogenicity of acute myelogenous leukemia (AML) cells is hindered by the difficulty of isolating pure populations of cells with defined proliferative abilities. By analyzing the growth of clonal siblings in low passage cultures of the cell line OCI/AML4 we resolved this heterogeneous population into strata of distinct clonogenic potential, permitting analysis of the transcriptional signature of single cells with defined proliferative abilities. By microarray analysis we showed that the expression of the orphan nuclear receptor EAR-2 (NR2F6) is greater in leukemia cells with extensive proliferative capacity than in those that have lost proliferative ability. EAR-2 is expressed highly in long-term hematopoietic stem cells, relative to short-term hematopoietic stem and progenitor cells, and is downregulated in AML cells after induction of differentiation. Exogenous expression of EAR-2 increased the growth of U937 cells and prevented the proliferative arrest associated with terminal differentiation, and blocked differentiation of U937 and 32Dcl3 cells. Conversely, silencing of EAR-2 by short-hairpin RNA initiated terminal differentiation of these cell lines. These data identify EAR-2 as an important factor in the regulation of clonogenicity and differentiation, and establish that analysis of clonal siblings allows the elucidation of differences in gene expression within the AML hierarchy. PMID:21637284

Regulation of osteogenesis by long noncoding RNAs: An epigenetic mechanism contributing to bone formation.

PubMed

Tye, Coralee E; Boyd, Joseph R; Page, Natalie A; Falcone, Michelle M; Stein, Janet L; Stein, Gary S; Lian, Jane B

2018-12-01

Long noncoding RNAs (lncRNAs) have recently emerged as novel regulators of lineage commitment, differentiation, development, viability, and disease progression. Few studies have examined their role in osteogenesis; however, given their critical and wide-ranging roles in other tissues, lncRNAs are most likely vital regulators of osteogenesis. In this study, we extensively characterized lncRNA expression in mesenchymal cells during commitment and differentiation to the osteoblast lineage using a whole transcriptome sequencing approach (RNA-Seq). Using mouse primary mesenchymal stromal cells (mMSC), we identified 1438 annotated lncRNAs expressed during MSC differentiation, 462 of which are differentially expressed. We performed guilt-by-association analysis using lncRNA and mRNA expression profiles to identify lncRNAs influencing MSC commitment and differentiation. These findings open novel dimensions for exploring lncRNAs in regulating normal bone formation and in skeletal disorders.

Differential Gene Expression (DEX) and Alternative Splicing Events (ASE) for Temporal Dynamic Processes Using HMMs and Hierarchical Bayesian Modeling Approaches.

PubMed

Oh, Sunghee; Song, Seongho

2017-01-01

In gene expression profile, data analysis pipeline is categorized into four levels, major downstream tasks, i.e., (1) identification of differential expression; (2) clustering co-expression patterns; (3) classification of subtypes of samples; and (4) detection of genetic regulatory networks, are performed posterior to preprocessing procedure such as normalization techniques. To be more specific, temporal dynamic gene expression data has its inherent feature, namely, two neighboring time points (previous and current state) are highly correlated with each other, compared to static expression data which samples are assumed as independent individuals. In this chapter, we demonstrate how HMMs and hierarchical Bayesian modeling methods capture the horizontal time dependency structures in time series expression profiles by focusing on the identification of differential expression. In addition, those differential expression genes and transcript variant isoforms over time detected in core prerequisite steps can be generally further applied in detection of genetic regulatory networks to comprehensively uncover dynamic repertoires in the aspects of system biology as the coupled framework.

Integrator complex plays an essential role in adipose differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Otani, Yuichiro; Nakatsu, Yusuke; Sakoda, Hideyuki

2013-05-03

Highlights: •IntS6 and IntS11 are subunits of the Integrator complex. •Expression levels of IntS6 and IntS11 were very low in 3T3-L1 fibroblast. •IntS6 and IntS11 were upregulated during adipose differentiation. •Suppression of IntS6 or IntS11 expression inhibited adipose differentiation. -- Abstract: The dynamic process of adipose differentiation involves stepwise expressions of transcription factors and proteins specific to the mature fat cell phenotype. In this study, it was revealed that expression levels of IntS6 and IntS11, subunits of the Integrator complex, were increased in 3T3-L1 cells in the period when the cells reached confluence and differentiated into adipocytes, while being reducedmore » to basal levels after the completion of differentiation. Suppression of IntS6 or IntS11 expression using siRNAs in 3T3-L1 preadipocytes markedly inhibited differentiation into mature adipocytes, based on morphological findings as well as mRNA analysis of adipocyte-specific genes such as Glut4, perilipin and Fabp4. Although Pparγ2 protein expression was suppressed in IntS6 or IntS11-siRNA treated cells, adenoviral forced expression of Pparγ2 failed to restore the capacity for differentiation into mature adipocytes. Taken together, these findings demonstrate that increased expression of Integrator complex subunits is an indispensable event in adipose differentiation. Although further study is necessary to elucidate the underlying mechanism, the processing of U1, U2 small nuclear RNAs may be involved in cell differentiation steps.« less

Identification of candidate genes in osteoporosis by integrated microarray analysis.

PubMed

Li, J J; Wang, B Q; Fei, Q; Yang, Y; Li, D

2016-12-01

In order to screen the altered gene expression profile in peripheral blood mononuclear cells of patients with osteoporosis, we performed an integrated analysis of the online microarray studies of osteoporosis. We searched the Gene Expression Omnibus (GEO) database for microarray studies of peripheral blood mononuclear cells in patients with osteoporosis. Subsequently, we integrated gene expression data sets from multiple microarray studies to obtain differentially expressed genes (DEGs) between patients with osteoporosis and normal controls. Gene function analysis was performed to uncover the functions of identified DEGs. A total of three microarray studies were selected for integrated analysis. In all, 1125 genes were found to be significantly differentially expressed between osteoporosis patients and normal controls, with 373 upregulated and 752 downregulated genes. Positive regulation of the cellular amino metabolic process (gene ontology (GO): 0033240, false discovery rate (FDR) = 1.00E + 00) was significantly enriched under the GO category for biological processes, while for molecular functions, flavin adenine dinucleotide binding (GO: 0050660, FDR = 3.66E-01) and androgen receptor binding (GO: 0050681, FDR = 6.35E-01) were significantly enriched. DEGs were enriched in many osteoporosis-related signalling pathways, including those of mitogen-activated protein kinase (MAPK) and calcium. Protein-protein interaction (PPI) network analysis showed that the significant hub proteins contained ubiquitin specific peptidase 9, X-linked (Degree = 99), ubiquitin specific peptidase 19 (Degree = 57) and ubiquitin conjugating enzyme E2 B (Degree = 57). Analysis of gene function of identified differentially expressed genes may expand our understanding of fundamental mechanisms leading to osteoporosis. Moreover, significantly enriched pathways, such as MAPK and calcium, may involve in osteoporosis through osteoblastic differentiation and bone formation.Cite this article: J. J. Li, B. Q. Wang, Q. Fei, Y. Yang, D. Li. Identification of candidate genes in osteoporosis by integrated microarray analysis. Bone Joint Res 2016;5:594-601. DOI: 10.1302/2046-3758.512.BJR-2016-0073.R1. © 2016 Fei et al.

Differentiating Human Multipotent Mesenchymal Stromal Cells Regulate microRNAs: Prediction of microRNA Regulation by PDGF During Osteogenesis

PubMed Central

Goff, Loyal A.; Boucher, Shayne; Ricupero, Christopher L.; Fenstermacher, Sara; Swerdel, Mavis; Chase, Lucas; Adams, Christopher; Chesnut, Jonathan; Lakshmipathy, Uma; Hart, Ronald P.

2009-01-01

Objective Human multipotent mesenchymal stromal cells (MSC) have the potential to differentiate into multiple cell types, although little is known about factors that control their fate. Differentiation-specific microRNAs may play a key role in stem cell self renewal and differentiation. We propose that specific intracellular signalling pathways modulate gene expression during differentiation by regulating microRNA expression. Methods Illumina mRNA and NCode microRNA expression analyses were performed on MSC and their differentiated progeny. A combination of bioinformatic prediction and pathway inhibition was used to identify microRNAs associated with PDGF signalling. Results The pattern of microRNA expression in MSC is distinct from that in pluripotent stem cells such as human embryonic stem cells. Specific populations of microRNAs are regulated in MSC during differentiation targeted towards specific cell types. Complementary mRNA expression analysis increases the pool of markers characteristic of MSC or differentiated progeny. To identify microRNA expression patterns affected by signalling pathways, we examined the PDGF pathway found to be regulated during osteogenesis by microarray studies. A set of microRNAs bioinformatically predicted to respond to PDGF signalling was experimentally confirmed by direct PDGF inhibition. Conclusion Our results demonstrate that a subset of microRNAs regulated during osteogenic differentiation of MSCs is responsive to perturbation of the PDGF pathway. This approach not only identifies characteristic classes of differentiation-specific mRNAs and microRNAs, but begins to link regulated molecules with specific cellular pathways. PMID:18657893

Transcriptome study of differential expression in schizophrenia

PubMed Central

Sanders, Alan R.; Göring, Harald H. H.; Duan, Jubao; Drigalenko, Eugene I.; Moy, Winton; Freda, Jessica; He, Deli; Shi, Jianxin; Gejman, Pablo V.

2013-01-01

Schizophrenia genome-wide association studies (GWAS) have identified common SNPs, rare copy number variants (CNVs) and a large polygenic contribution to illness risk, but biological mechanisms remain unclear. Bioinformatic analyses of significantly associated genetic variants point to a large role for regulatory variants. To identify gene expression abnormalities in schizophrenia, we generated whole-genome gene expression profiles using microarrays on lymphoblastoid cell lines (LCLs) from 413 cases and 446 controls. Regression analysis identified 95 transcripts differentially expressed by affection status at a genome-wide false discovery rate (FDR) of 0.05, while simultaneously controlling for confounding effects. These transcripts represented 89 genes with functions such as neurotransmission, gene regulation, cell cycle progression, differentiation, apoptosis, microRNA (miRNA) processing and immunity. This functional diversity is consistent with schizophrenia's likely significant pathophysiological heterogeneity. The overall enrichment of immune-related genes among those differentially expressed by affection status is consistent with hypothesized immune contributions to schizophrenia risk. The observed differential expression of extended major histocompatibility complex (xMHC) region histones (HIST1H2BD, HIST1H2BC, HIST1H2BH, HIST1H2BG and HIST1H4K) converges with the genetic evidence from GWAS, which find the xMHC to be the most significant susceptibility locus. Among the differentially expressed immune-related genes, B3GNT2 is implicated in autoimmune disorders previously tied to schizophrenia risk (rheumatoid arthritis and Graves’ disease), and DICER1 is pivotal in miRNA processing potentially linking to miRNA alterations in schizophrenia (e.g. MIR137, the second strongest GWAS finding). Our analysis provides novel candidate genes for further study to assess their potential contribution to schizophrenia. PMID:23904455

Sex genes for genomic analysis in human brain: internal controls for comparison of probe level data extraction.

PubMed Central

Galfalvy, Hanga C; Erraji-Benchekroun, Loubna; Smyrniotopoulos, Peggy; Pavlidis, Paul; Ellis, Steven P; Mann, J John; Sibille, Etienne; Arango, Victoria

2003-01-01

Background Genomic studies of complex tissues pose unique analytical challenges for assessment of data quality, performance of statistical methods used for data extraction, and detection of differentially expressed genes. Ideally, to assess the accuracy of gene expression analysis methods, one needs a set of genes which are known to be differentially expressed in the samples and which can be used as a "gold standard". We introduce the idea of using sex-chromosome genes as an alternative to spiked-in control genes or simulations for assessment of microarray data and analysis methods. Results Expression of sex-chromosome genes were used as true internal biological controls to compare alternate probe-level data extraction algorithms (Microarray Suite 5.0 [MAS5.0], Model Based Expression Index [MBEI] and Robust Multi-array Average [RMA]), to assess microarray data quality and to establish some statistical guidelines for analyzing large-scale gene expression. These approaches were implemented on a large new dataset of human brain samples. RMA-generated gene expression values were markedly less variable and more reliable than MAS5.0 and MBEI-derived values. A statistical technique controlling the false discovery rate was applied to adjust for multiple testing, as an alternative to the Bonferroni method, and showed no evidence of false negative results. Fourteen probesets, representing nine Y- and two X-chromosome linked genes, displayed significant sex differences in brain prefrontal cortex gene expression. Conclusion In this study, we have demonstrated the use of sex genes as true biological internal controls for genomic analysis of complex tissues, and suggested analytical guidelines for testing alternate oligonucleotide microarray data extraction protocols and for adjusting multiple statistical analysis of differentially expressed genes. Our results also provided evidence for sex differences in gene expression in the brain prefrontal cortex, supporting the notion of a putative direct role of sex-chromosome genes in differentiation and maintenance of sexual dimorphism of the central nervous system. Importantly, these analytical approaches are applicable to all microarray studies that include male and female human or animal subjects. PMID:12962547

Sex genes for genomic analysis in human brain: internal controls for comparison of probe level data extraction.

PubMed

Galfalvy, Hanga C; Erraji-Benchekroun, Loubna; Smyrniotopoulos, Peggy; Pavlidis, Paul; Ellis, Steven P; Mann, J John; Sibille, Etienne; Arango, Victoria

2003-09-08

Genomic studies of complex tissues pose unique analytical challenges for assessment of data quality, performance of statistical methods used for data extraction, and detection of differentially expressed genes. Ideally, to assess the accuracy of gene expression analysis methods, one needs a set of genes which are known to be differentially expressed in the samples and which can be used as a "gold standard". We introduce the idea of using sex-chromosome genes as an alternative to spiked-in control genes or simulations for assessment of microarray data and analysis methods. Expression of sex-chromosome genes were used as true internal biological controls to compare alternate probe-level data extraction algorithms (Microarray Suite 5.0 [MAS5.0], Model Based Expression Index [MBEI] and Robust Multi-array Average [RMA]), to assess microarray data quality and to establish some statistical guidelines for analyzing large-scale gene expression. These approaches were implemented on a large new dataset of human brain samples. RMA-generated gene expression values were markedly less variable and more reliable than MAS5.0 and MBEI-derived values. A statistical technique controlling the false discovery rate was applied to adjust for multiple testing, as an alternative to the Bonferroni method, and showed no evidence of false negative results. Fourteen probesets, representing nine Y- and two X-chromosome linked genes, displayed significant sex differences in brain prefrontal cortex gene expression. In this study, we have demonstrated the use of sex genes as true biological internal controls for genomic analysis of complex tissues, and suggested analytical guidelines for testing alternate oligonucleotide microarray data extraction protocols and for adjusting multiple statistical analysis of differentially expressed genes. Our results also provided evidence for sex differences in gene expression in the brain prefrontal cortex, supporting the notion of a putative direct role of sex-chromosome genes in differentiation and maintenance of sexual dimorphism of the central nervous system. Importantly, these analytical approaches are applicable to all microarray studies that include male and female human or animal subjects.

Whole Transcriptome Analysis of Pre-invasive and Invasive Early Squamous Lung Carcinoma in Archival Laser Microdissected Samples.

PubMed

Koper, Andre; Zeef, Leo A H; Joseph, Leena; Kerr, Keith; Gosney, John; Lindsay, Mark A; Booton, Richard

2017-01-10

Preinvasive squamous cell cancer (PSCC) are local transformations of bronchial epithelia that are frequently observed in current or former smokers. Their different grades and sizes suggest a continuum of dysplastic change with increasing severity, which may culminate in invasive squamous cell carcinoma (ISCC). As a consequence of the difficulty in isolating cancerous cells from biopsies, the molecular pathology that underlies their histological variability remains largely unknown. To address this issue, we have employed microdissection to isolate normal bronchial epithelia and cancerous cells from low- and high-grade PSCC and ISCC, from paraffin embedded (FFPE) biopsies and determined gene expression using Affymetric Human Exon 1.0 ST arrays. Tests for differential gene expression were performed using the Bioconductor package limma followed by functional analyses of differentially expressed genes in IPA. Examination of differential gene expression showed small differences between low- and high-grade PSCC but substantial changes between PSCC and ISCC samples (184 vs 1200 p-value <0.05, fc ±1.75). However, the majority of the differentially expressed PSCC genes (142 genes: 77%) were shared with those in ISCC samples. Pathway analysis showed that these shared genes are associated with DNA damage response, DNA/RNA metabolism and inflammation as major biological themes. Cluster analysis identified 12 distinct patterns of gene expression including progressive up or down-regulation across PSCC and ISCC. Pathway analysis of incrementally up-regulated genes revealed again significant enrichment of terms related to DNA damage response, DNA/RNA metabolism, inflammation, survival and proliferation. Altered expression of selected genes was confirmed using RT-PCR, as well as immunohistochemistry in an independent set of 45 ISCCs. Gene expression profiles in PSCC and ISCC differ greatly in terms of numbers of genes with altered transcriptional activity. However, altered gene expression in PSCC affects canonical pathways and cellular and biological processes, such as inflammation and DNA damage response, which are highly consistent with hallmarks of cancer.

Exploring Regulatory Mechanisms of Atrial Myocyte Hypertrophy of Mitral Regurgitation through Gene Expression Profiling Analysis: Role of NFAT in Cardiac Hypertrophy

PubMed Central

Chang, Tzu-Hao; Chen, Mien-Cheng; Chang, Jen-Ping; Huang, Hsien-Da; Ho, Wan-Chun; Lin, Yu-Sheng; Pan, Kuo-Li; Huang, Yao-Kuang; Liu, Wen-Hao; Wu, Chia-Chen

2016-01-01

Background Left atrial enlargement in mitral regurgitation (MR) predicts a poor prognosis. The regulatory mechanisms of atrial myocyte hypertrophy of MR patients remain unknown. Methods and Results This study comprised 14 patients with MR, 7 patients with aortic valve disease (AVD), and 6 purchased samples from normal subjects (NC). We used microarrays, enrichment analysis and quantitative RT-PCR to study the gene expression profiles in the left atria. Microarray results showed that 112 genes were differentially up-regulated and 132 genes were differentially down-regulated in the left atria between MR patients and NC. Enrichment analysis of differentially expressed genes demonstrated that “NFAT in cardiac hypertrophy” pathway was not only one of the significant associated canonical pathways, but also the only one predicted with a non-zero score of 1.34 (i.e. activated) through Ingenuity Pathway Analysis molecule activity predictor. Ingenuity Pathway Analysis Global Molecular Network analysis exhibited that the highest score network also showed high association with cardiac related pathways and functions. Therefore, 5 NFAT associated genes (PPP3R1, PPP3CB, CAMK1, MEF2C, PLCE1) were studies for validation. The mRNA expressions of PPP3CB and MEF2C were significantly up-regulated, and CAMK1 and PPP3R1 were significantly down-regulated in MR patients compared to NC. Moreover, MR patients had significantly increased mRNA levels of PPP3CB, MEF2C and PLCE1 compared to AVD patients. The atrial myocyte size of MR patients significantly exceeded that of the AVD patients and NC. Conclusions Differentially expressed genes in the “NFAT in cardiac hypertrophy” pathway may play a critical role in the atrial myocyte hypertrophy of MR patients. PMID:27907007

Comparative study of ROR2 and WNT5a expression in squamous/adenosquamous carcinoma and adenocarcinoma of the gallbladder

PubMed Central

Wu, Zheng-Chun; Xiong, Li; Wang, Ling-Xiang; Miao, Xiong-Ying; Liu, Zi-Ru; Li, Dai-Qiang; Zou, Qiong; Liu, Kui-Jie; Zhao, Hua; Yang, Zhu-Lin

2017-01-01

AIM To investigate the expression and clinical pathological significance of ROR2 and WNT5a in gallbladder squamous/adenosquamous carcinoma (SC/ASC) and adenocarcinoma (AC). METHODS EnVision immunohistochemistry was used to stain for ROR2 and WNT5a in 46 SC/ASC patients and 80 AC patients. RESULTS Poorly differentiated AC among AC patients aged > 45 years were significantly more frequent compared with SC/ASC patients, while tumors with a maximal diameter > 3 cm in the SC/ASC group were significantly more frequent compared with the AC group. Positive ROR2 and WNT5a expression was significantly lower in SC/ASC or AC with a maximal mass diameter ≤ 3 cm, a TNM stage of I + II, no lymph node metastasis, no surrounding invasion, and radical resection than in patients with a maximal mass diameter > 3 cm, TNM stage IV, lymph node metastasis, surrounding invasion, and no resection. Positive ROR2 expression in patients with highly differentiated SC/ASC was significantly lower than in patients with poorly differentiated SC/ASC. Positive ROR2 and WNT5a expression levels in highly differentiated AC were significantly lower than in poorly differentiated AC. Kaplan-Meier survival analysis showed that differentiation degree, maximal mass diameter, TNM stage, lymph node metastasis, surrounding invasion, surgical procedure and the ROR2 and WNT5a expression levels were closely related to average survival of SC/ASC or AC. The survival of SC/ASC or AC patients with positive expression of ROR2 and WNT5a was significantly shorter than that of patients with negative expression results. Cox multivariate analysis revealed that poor differentiation, a maximal diameter of the mass ≥ 3 cm, TNM stage III or IV, lymph node metastasis, surrounding invasion, unresected surgery and positive ROR2 or WNT5a expression in the SC/ASC or AC patients were negatively correlated with the postoperative survival rate and positively correlated with mortality, which are risk factors and independent prognostic predictors. CONCLUSION SC/ASC or AC patients with positive ROR2 or WNT5a expression generally have a poor prognosis. PMID:28465645

High resolution array CGH and gene expression profiling of alveolar soft part sarcoma

PubMed Central

Selvarajah, Shamini; Pyne, Saumyadipta; Chen, Eleanor; Sompallae, Ramakrishna; Ligon, Azra H.; Nielsen, Gunnlaugur P.; Dranoff, Glenn; Stack, Edward; Loda, Massimo; Flavin, Richard

2014-01-01

Purpose Alveolar soft part sarcoma (ASPS) is a soft tissue sarcoma with poor prognosis, and little molecular evidence for its origin, initiation and progression. The aim of this study was to elucidate candidate molecular pathways involved in tumor pathogenesis. Experimental Design We employed high-throughput array comparative genomic hybridization and cDNA-Mediated Annealing, Selection, Ligation, and Extension Assay to profile the genomic and expression signatures of primary and metastatic ASPS from 17 tumors derived from 11 patients. We used an integrative bioinformatics approach to elucidate the molecular pathways associated with ASPS progression. Fluorescence in situ hybridization was performed to validate the presence of the t(X;17)(p11.2;q25) ASPL-TFE3 fusion and hence confirm the aCGH observations. Results FISH analysis identified the ASPL-TFE3 fusion in all cases. ArrayCGH revealed a higher number of numerical aberrations in metastatic tumors relative to primaries, but failed to identify consistent alterations in either group. Gene expression analysis highlighted 1,063 genes which were differentially expressed between the two groups. Gene set enrichment analysis identified 16 enriched gene sets (p < 0.1) associated with differentially expressed genes. Notable among these were several stem cell gene expression signatures and pathways related to differentiation. In particular, the paired box transcription factor PAX6 was up-regulated in the primary tumors, along with several genes whose mouse orthologs have previously been implicated in Pax6-DNA binding during neural stem cell differentiation. Conclusion In addition to suggesting a tentative neural line of differentiation for ASPS, these results implicate transcriptional deregulation from fusion genes in the pathogenesis of ASPS. PMID:24493828

In silico identification and comparative analysis of differentially expressed genes in human and mouse tissues

PubMed Central

Pao, Sheng-Ying; Lin, Win-Li; Hwang, Ming-Jing

2006-01-01

Background Screening for differentially expressed genes on the genomic scale and comparative analysis of the expression profiles of orthologous genes between species to study gene function and regulation are becoming increasingly feasible. Expressed sequence tags (ESTs) are an excellent source of data for such studies using bioinformatic approaches because of the rich libraries and tremendous amount of data now available in the public domain. However, any large-scale EST-based bioinformatics analysis must deal with the heterogeneous, and often ambiguous, tissue and organ terms used to describe EST libraries. Results To deal with the issue of tissue source, in this work, we carefully screened and organized more than 8 million human and mouse ESTs into 157 human and 108 mouse tissue/organ categories, to which we applied an established statistic test using different thresholds of the p value to identify genes differentially expressed in different tissues. Further analysis of the tissue distribution and level of expression of human and mouse orthologous genes showed that tissue-specific orthologs tended to have more similar expression patterns than those lacking significant tissue specificity. On the other hand, a number of orthologs were found to have significant disparity in their expression profiles, hinting at novel functions, divergent regulation, or new ortholog relationships. Conclusion Comprehensive statistics on the tissue-specific expression of human and mouse genes were obtained in this very large-scale, EST-based analysis. These statistical results have been organized into a database, freely accessible at our website , for easy searching of human and mouse tissue-specific genes and for investigating gene expression profiles in the context of comparative genomics. Comparative analysis showed that, although highly tissue-specific genes tend to exhibit similar expression profiles in human and mouse, there are significant exceptions, indicating that orthologous genes, while sharing basic genomic properties, could result in distinct phenotypes. PMID:16626500

Suppression subtractive hybridization and comparative expression analysis to identify developmentally regulated genes in filamentous fungi.

PubMed

Gesing, Stefan; Schindler, Daniel; Nowrousian, Minou

2013-09-01

Ascomycetes differentiate four major morphological types of fruiting bodies (apothecia, perithecia, pseudothecia and cleistothecia) that are derived from an ancestral fruiting body. Thus, fruiting body differentiation is most likely controlled by a set of common core genes. One way to identify such genes is to search for genes with evolutionary conserved expression patterns. Using suppression subtractive hybridization (SSH), we selected differentially expressed transcripts in Pyronema confluens (Pezizales) by comparing two cDNA libraries specific for sexual and for vegetative development, respectively. The expression patterns of selected genes from both libraries were verified by quantitative real time PCR. Expression of several corresponding homologous genes was found to be conserved in two members of the Sordariales (Sordaria macrospora and Neurospora crassa), a derived group of ascomycetes that is only distantly related to the Pezizales. Knockout studies with N. crassa orthologues of differentially regulated genes revealed a functional role during fruiting body development for the gene NCU05079, encoding a putative MFS peptide transporter. These data indicate conserved gene expression patterns and a functional role of the corresponding genes during fruiting body development; such genes are candidates of choice for further functional analysis. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Differential proteome analysis of the cell differentiation regulated by BCC, CRH, CXCR4, GnRH, GPCR, IL1 signaling pathways in Chinese fire-bellied newt limb regeneration.

PubMed

Geng, Xiaofang; Xu, Tiantian; Niu, Zhipeng; Zhou, Xiaochun; Zhao, Lijun; Xie, Zhaohui; Xue, Deming; Zhang, Fuchun; Xu, Cunshuan

2014-01-01

Following amputation, the newt has the remarkable ability to regenerate its limb, and this process involves dedifferentiation, proliferation and differentiation. To investigate the potential proteome during a dynamic network of Chinese fire-bellied newt limb regeneration (CNLR), two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mass spectrum (MS) were applied to examine changes in the proteome that occurred at 11 time points after amputation. Meanwhile, several proteins were selected to validate their expression levels by Western blot. The results revealed that 1476 proteins had significantly changed as compared to the control group. Gene Ontology annotation and protein network analysis by Ingenuity Pathway Analysis 9.0 (IPA) software suggested that the differentially expressed proteins were involved in 33 kinds of physiological activities including signal transduction, cell proliferation, cell differentiation, etc. Among these proteins, 407 proteins participated in cell differentiation with 212 proteins in the differentiation of skin cell, myocyte, neurocyte, chondrocyte and osteocyte, and 37 proteins participated in signaling pathways of BCC, CRH, CXCR4, GnRH, GPCR and IL1 which regulated cell differentiation and redifferentiation. On the other hand, the signal transduction activity and cell differentiation activity were analyzed by IPA based on the changes in the expression of these proteins. The results showed that BCC, CRH, CXCR4, GnRH, GPCR and IL1 signaling pathways played an important role in regulating the differentiation of skin cell, myocyte, neurocyte, chondrocyte and osteocyte during CNLR. Copyright © 2014 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Characterization of distinct classes of differential gene expression in osteoblast cultures from non-syndromic craniosynostosis bone.

PubMed

Rojas-Peña, Monica L; Olivares-Navarrete, Rene; Hyzy, Sharon; Arafat, Dalia; Schwartz, Zvi; Boyan, Barbara D; Williams, Joseph; Gibson, Greg

2014-01-01

Craniosynostosis, the premature fusion of one or more skull sutures, occurs in approximately 1 in 2500 infants, with the majority of cases non-syndromic and of unknown etiology. Two common reasons proposed for premature suture fusion are abnormal compression forces on the skull and rare genetic abnormalities. Our goal was to evaluate whether different sub-classes of disease can be identified based on total gene expression profiles. RNA-Seq data were obtained from 31 human osteoblast cultures derived from bone biopsy samples collected between 2009 and 2011, representing 23 craniosynostosis fusions and 8 normal cranial bones or long bones. No differentiation between regions of the skull was detected, but variance component analysis of gene expression patterns nevertheless supports transcriptome-based classification of craniosynostosis. Cluster analysis showed 4 distinct groups of samples; 1 predominantly normal and 3 craniosynostosis subtypes. Similar constellations of sub-types were also observed upon re-analysis of a similar dataset of 199 calvarial osteoblast cultures. Annotation of gene function of differentially expressed transcripts strongly implicates physiological differences with respect to cell cycle and cell death, stromal cell differentiation, extracellular matrix (ECM) components, and ribosomal activity. Based on these results, we propose non-syndromic craniosynostosis cases can be classified by differences in their gene expression patterns and that these may provide targets for future clinical intervention.

Characterization of Distinct Classes of Differential Gene Expression in Osteoblast Cultures from Non-Syndromic Craniosynostosis Bone

PubMed Central

Rojas-Peña, Monica L.; Olivares-Navarrete, Rene; Hyzy, Sharon; Arafat, Dalia; Schwartz, Zvi; Boyan, Barbara D.; Williams, Joseph; Gibson, Greg

2014-01-01

Craniosynostosis, the premature fusion of one or more skull sutures, occurs in approximately 1 in 2500 infants, with the majority of cases non-syndromic and of unknown etiology. Two common reasons proposed for premature suture fusion are abnormal compression forces on the skull and rare genetic abnormalities. Our goal was to evaluate whether different sub-classes of disease can be identified based on total gene expression profiles. RNA-Seq data were obtained from 31 human osteoblast cultures derived from bone biopsy samples collected between 2009 and 2011, representing 23 craniosynostosis fusions and 8 normal cranial bones or long bones. No differentiation between regions of the skull was detected, but variance component analysis of gene expression patterns nevertheless supports transcriptome-based classification of craniosynostosis. Cluster analysis showed 4 distinct groups of samples; 1 predominantly normal and 3 craniosynostosis subtypes. Similar constellations of sub-types were also observed upon re-analysis of a similar dataset of 199 calvarial osteoblast cultures. Annotation of gene function of differentially expressed transcripts strongly implicates physiological differences with respect to cell cycle and cell death, stromal cell differentiation, extracellular matrix (ECM) components, and ribosomal activity. Based on these results, we propose non-syndromic craniosynostosis cases can be classified by differences in their gene expression patterns and that these may provide targets for future clinical intervention. PMID:25184005

Transcriptome Analysis of CD4+ T Cells in Coeliac Disease Reveals Imprint of BACH2 and IFNγ Regulation

PubMed Central

Molloy, Ben; Dominguez Castro, Patricia; Cormican, Paul; Trimble, Valerie; Mahmud, Nasir; McManus, Ross

2015-01-01

Genetic studies have to date identified 43 genome wide significant coeliac disease susceptibility (CD) loci comprising over 70 candidate genes. However, how altered regulation of such disease associated genes contributes to CD pathogenesis remains to be elucidated. Recently there has been considerable emphasis on characterising cell type specific and stimulus dependent genetic variants. Therefore in this study we used RNA sequencing to profile over 70 transcriptomes of CD4+ T cells, a cell type crucial for CD pathogenesis, in both stimulated and resting samples from individuals with CD and unaffected controls. We identified extensive transcriptional changes across all conditions, with the previously established CD gene IFNy the most strongly up-regulated gene (log2 fold change 4.6; Padjusted = 2.40x10-11) in CD4+ T cells from CD patients compared to controls. We show a significant correlation of differentially expressed genes with genetic studies of the disease to date (Padjusted = 0.002), and 21 CD candidate susceptibility genes are differentially expressed under one or more of the conditions used in this study. Pathway analysis revealed significant enrichment of immune related processes. Co-expression network analysis identified several modules of coordinately expressed CD genes. Two modules were particularly highly enriched for differentially expressed genes (P<2.2x10-16) and highlighted IFNy and the genetically associated transcription factor BACH2 which showed significantly reduced expression in coeliac samples (log2FC -1.75; Padjusted = 3.6x10-3) as key regulatory genes in CD. Genes regulated by BACH2 were very significantly over-represented among our differentially expressed genes (P<2.2x10-16) indicating that reduced expression of this master regulator of T cell differentiation promotes a pro-inflammatory response and strongly corroborates genetic evidence that BACH2 plays an important role in CD pathogenesis. PMID:26444573

Pathway Inspector: a pathway based web application for RNAseq analysis of model and non-model organisms

PubMed Central

Bianco, Luca; Riccadonna, Samantha; Lavezzo, Enrico; Falda, Marco; Formentin, Elide; Cavalieri, Duccio; Toppo, Stefano

2017-01-01

Abstract Summary: Pathway Inspector is an easy-to-use web application helping researchers to find patterns of expression in complex RNAseq experiments. The tool combines two standard approaches for RNAseq analysis: the identification of differentially expressed genes and a topology-based analysis of enriched pathways. Pathway Inspector is equipped with ad hoc interactive graphical interfaces simplifying the discovery of modulated pathways and the integration of the differentially expressed genes in the corresponding pathway topology. Availability and Implementation: Pathway Inspector is available at the website http://admiral.fmach.it/PI and has been developed in Python, making use of the Django Web Framework. Contact: paolo.fontana@fmach.it PMID:28158604

DEApp: an interactive web interface for differential expression analysis of next generation sequence data.

PubMed

Li, Yan; Andrade, Jorge

2017-01-01

A growing trend in the biomedical community is the use of Next Generation Sequencing (NGS) technologies in genomics research. The complexity of downstream differential expression (DE) analysis is however still challenging, as it requires sufficient computer programing and command-line knowledge. Furthermore, researchers often need to evaluate and visualize interactively the effect of using differential statistical and error models, assess the impact of selecting different parameters and cutoffs, and finally explore the overlapping consensus of cross-validated results obtained with different methods. This represents a bottleneck that slows down or impedes the adoption of NGS technologies in many labs. We developed DEApp, an interactive and dynamic web application for differential expression analysis of count based NGS data. This application enables models selection, parameter tuning, cross validation and visualization of results in a user-friendly interface. DEApp enables labs with no access to full time bioinformaticians to exploit the advantages of NGS applications in biomedical research. This application is freely available at https://yanli.shinyapps.io/DEAppand https://gallery.shinyapps.io/DEApp.

Differentially expressed genes of Coptotermes formosanus (Isoptera: Rhinotermitidae) challenged by chemical insecticides.

PubMed

Zhang, Yi; Zhao, Yuanyuan; Qiu, Xuehong; Han, Richou

2013-08-01

Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae) termites are harmful social insects to wood constructions. The current control methods heavily depend on the chemical insecticides with increasing resistance. Analysis of the differentially expressed genes mediated by chemical insecticides will contribute to the understanding of the termite resistance to chemicals and to the establishment of alternative control measures. In the present article, a full-length cDNA library was constructed from the termites induced by a mixture of commonly used insecticides (0.01% sulfluramid and 0.01% triflumuron) for 24 h, by using the RNA ligase-mediated Rapid Amplification cDNA End method. Fifty-eight differentially expressed clones were obtained by polymerase chain reaction and confirmed by dot-blot hybridization. Forty-six known sequences were obtained, which clustered into 33 unique sequences grouped in 6 contigs and 27 singlets. Sixty-seven percent (22) of the sequences had counterpart genes from other organisms, whereas 33% (11) were undescribed. A Gene Ontology analysis classified 33 unique sequences into different functional categories. In general, most of the differential expression genes were involved in binding and catalytic activity.

Comparative 2D-DIGE proteomic analysis of bovine mammary epithelial cells during lactation reveals protein signatures for lactation persistency and milk yield.

PubMed

Janjanam, Jagadeesh; Singh, Surender; Jena, Manoj K; Varshney, Nishant; Kola, Srujana; Kumar, Sudarshan; Kaushik, Jai K; Grover, Sunita; Dang, Ajay K; Mukesh, Manishi; Prakash, B S; Mohanty, Ashok K

2014-01-01

Mammary gland is made up of a branching network of ducts that end with alveoli which surrounds the lumen. These alveolar mammary epithelial cells (MEC) reflect the milk producing ability of farm animals. In this study, we have used 2D-DIGE and mass spectrometry to identify the protein changes in MEC during immediate early, peak and late stages of lactation and also compared differentially expressed proteins in MEC isolated from milk of high and low milk producing cows. We have identified 41 differentially expressed proteins during lactation stages and 22 proteins in high and low milk yielding cows. Bioinformatics analysis showed that a majority of the differentially expressed proteins are associated in metabolic process, catalytic and binding activity. The differentially expressed proteins were mapped to the available biological pathways and networks involved in lactation. The proteins up-regulated during late stage of lactation are associated with NF-κB stress induced signaling pathways and whereas Akt, PI3K and p38/MAPK signaling pathways are associated with high milk production mediated through insulin hormone signaling.

Cloning of Trametes versicolar genes induced by nitrogen starvation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Trudel, P.; Courchesne, D.; Roy, C.

1988-06-01

We have screened a genomic library of Trametes versicolar for genes whose expression is associated with nitrogen starvation, which has been shown to induce ligninolytic activity. Using two different approaches based on differential expression, we isolated 29 clones. These were shown by restriction mapping and cross-hybridization to code for 11 distinct differentially expressed genes. Northern analysis of the kinetics of expression of these genes revealed that at least four of them have kinetics of induction that parallel kinetics of induction of ligninolytic activity.

A Scalable Approach for Discovering Conserved Active Subnetworks across Species

PubMed Central

Verfaillie, Catherine M.; Hu, Wei-Shou; Myers, Chad L.

2010-01-01

Overlaying differential changes in gene expression on protein interaction networks has proven to be a useful approach to interpreting the cell's dynamic response to a changing environment. Despite successes in finding active subnetworks in the context of a single species, the idea of overlaying lists of differentially expressed genes on networks has not yet been extended to support the analysis of multiple species' interaction networks. To address this problem, we designed a scalable, cross-species network search algorithm, neXus (Network - cross(X)-species - Search), that discovers conserved, active subnetworks based on parallel differential expression studies in multiple species. Our approach leverages functional linkage networks, which provide more comprehensive coverage of functional relationships than physical interaction networks by combining heterogeneous types of genomic data. We applied our cross-species approach to identify conserved modules that are differentially active in stem cells relative to differentiated cells based on parallel gene expression studies and functional linkage networks from mouse and human. We find hundreds of conserved active subnetworks enriched for stem cell-associated functions such as cell cycle, DNA repair, and chromatin modification processes. Using a variation of this approach, we also find a number of species-specific networks, which likely reflect mechanisms of stem cell function that have diverged between mouse and human. We assess the statistical significance of the subnetworks by comparing them with subnetworks discovered on random permutations of the differential expression data. We also describe several case examples that illustrate the utility of comparative analysis of active subnetworks. PMID:21170309

Analytical workflow profiling gene expression in murine macrophages

PubMed Central

Nixon, Scott E.; González-Peña, Dianelys; Lawson, Marcus A.; McCusker, Robert H.; Hernandez, Alvaro G.; O’Connor, Jason C.; Dantzer, Robert; Kelley, Keith W.

2015-01-01

Comprehensive and simultaneous analysis of all genes in a biological sample is a capability of RNA-Seq technology. Analysis of the entire transcriptome benefits from summarization of genes at the functional level. As a cellular response of interest not previously explored with RNA-Seq, peritoneal macrophages from mice under two conditions (control and immunologically challenged) were analyzed for gene expression differences. Quantification of individual transcripts modeled RNA-Seq read distribution and uncertainty (using a Beta Negative Binomial distribution), then tested for differential transcript expression (False Discovery Rate-adjusted p-value < 0.05). Enrichment of functional categories utilized the list of differentially expressed genes. A total of 2079 differentially expressed transcripts representing 1884 genes were detected. Enrichment of 92 categories from Gene Ontology Biological Processes and Molecular Functions, and KEGG pathways were grouped into 6 clusters. Clusters included defense and inflammatory response (Enrichment Score = 11.24) and ribosomal activity (Enrichment Score = 17.89). Our work provides a context to the fine detail of individual gene expression differences in murine peritoneal macrophages during immunological challenge with high throughput RNA-Seq. PMID:25708305

ECM1 and TMPRSS4 Are Diagnostic Markers of Malignant Thyroid Neoplasms and Improve the Accuracy of Fine Needle Aspiration Biopsy

PubMed Central

Kebebew, Electron; Peng, Miao; Reiff, Emily; Duh, Quan-Yang; Clark, Orlo H.; McMillan, Alex

2005-01-01

Objective: The objective of this study was to determine whether genes that regulate cellular invasion and metastasis are differentially expressed and could serve as diagnostic markers of malignant thyroid nodules. Summary and Background Data: Patients whose thyroid nodules have indeterminate or suspicious cytologic features on fine needle aspiration (FNA) biopsy require thyroidectomy because of a 20% to 30% risk of thyroid cancer. Cell invasion and metastasis is a hallmark of malignant phenotype; therefore, genes that regulate these processes might be differentially expressed and could serve as diagnostic markers of malignancy. Methods: Differentially expressed genes (2-fold higher or lower) in malignant versus benign thyroid neoplasms were identified by extracellular matrix and adhesion molecule cDNA array analysis and confirmed by real-time quantitative polymerase chain reaction (PCR). The area under the receiver operating characteristic (AUC) curve was calculated to determine diagnostic accuracy of gene expression level cutoffs established by logistic regression analysis. Results: By cDNA array analysis, ADAMTS8, ECM1, MMP8, PLAU, SELP, and TMPRSS4 were upregulated, and by quantitative PCR, ECM1, SELP, and TMPRSS4 mRNA expression was higher in malignant (n = 57) than in benign (n = 38) thyroid neoplasms (P< 0.002). ECM1 and TMPRSS4 mRNA expression levels were independent predictors of a malignant thyroid neoplasm (P < 0.003). The AUC was 0.956 for ECM1 and 0.926 for TMPRSS4. Combining both markers improved their diagnostic use (AUC 0.985; sensitivity, 91.7%; specificity, 89.8%; positive predictive value, 85.7%; negative predictive value, 82.8%). ECM1 and TMPRSS4 expression analysis improved the diagnostic accuracy of FNA biopsy in 35 of 38 indeterminate or suspicious results. The level of ECM1 mRNA expression was higher in TNM stage I differentiated thyroid cancers than in stage II and III tumors (P ≤ 0.031). Conclusions: ECM1 and TMPRSS4 are excellent diagnostic markers of malignant thyroid nodules and may be used to improve the diagnostic accuracy of FNA biopsy. ECM1 is also a marker of the extent of disease in differentiated thyroid cancers. PMID:16135921

[Differential gene expression in incompatible interaction between Lilium regale Wilson and Fusarium oxysporum f. sp. lilii revealed by combined SSH and microarray analysis].

PubMed

Rao, J; Liu, D; Zhang, N; He, H; Ge, F; Chen, C

2014-01-01

Fusarium wilt, caused by a soilborne pathogen Fusarium oxysporum f. sp. lilii, is the major disease of lily (Lilium L.). In order to isolate the genes differentially expressed in a resistant reaction to F. oxysporum in L. regale Wilson, a cDNA library was constructed with L. regale root during F. oxysporum infection using the suppression subtractive hybridization (SSH), and a total of 585 unique expressed sequence tags (ESTs) were obtained. Furthermore, the gene expression profiles in the incompatible interaction between L. regale and F. oxysporum were revealed by oligonucleotide microarray analysis of 585 unique ESTs comparison to the compatible interaction between a susceptible Lilium Oriental Hybrid 'Siberia' and F. oxysporum. The result of expression profile analysis indicated that the genes encoding pathogenesis-related proteins (PRs), antioxidative stress enzymes, secondary metabolism enzymes, transcription factors, signal transduction proteins as well as a large number of unknown genes were involved in early defense response of L. regale to F. oxysporum infection. Moreover, the following quantitative reverse transcription PCR (QRT-PCR) analysis confirmed reliability of the oligonucleotide microarray data. In the present study, isolation of differentially expressed genes in L. regale during response to F. oxysporum helped to uncover the molecular mechanism associated with the resistance of L. regale against F. oxysporum.

Identification of novel mRNAs and lncRNAs associated with mouse experimental colitis and human inflammatory bowel disease.

PubMed

Rankin, Carl Robert; Theodorou, Evangelos; Law, Ivy Ka Man; Rowe, Lorraine; Kokkotou, Efi; Pekow, Joel; Wang, Jiafang; Martin, Martin G; Pothoulakis, Charalabos; Padua, David Miguel

2018-06-28

Inflammatory bowel disease (IBD) is a complex disorder that is associated with significant morbidity. While many recent advances have been made with new diagnostic and therapeutic tools, a deeper understanding of its basic pathophysiology is needed to continue this trend towards improving treatments. By utilizing an unbiased, high-throughput transcriptomic analysis of two well-established mouse models of colitis, we set out to uncover novel coding and non-coding RNAs that are differentially expressed in the setting of colonic inflammation. RNA-seq analysis was performed using colonic tissue from two mouse models of colitis, a dextran sodium sulfate induced model and a genetic-induced model in mice lacking IL-10. We identified 81 coding RNAs that were commonly altered in both experimental models. Of these coding RNAs, 12 of the human orthologs were differentially expressed in a transcriptomic analysis of IBD patients. Interestingly, 5 of the 12 of human differentially expressed genes have not been previously identified as IBD-associated genes, including ubiquitin D. Our analysis also identified 15 non-coding RNAs that were differentially expressed in either mouse model. Surprisingly, only three non-coding RNAs were commonly dysregulated in both of these models. The discovery of these new coding and non-coding RNAs expands our transcriptional knowledge of mouse models of IBD and offers additional targets to deepen our understanding of the pathophysiology of IBD.

Functional network analysis of genes differentially expressed during xylogenesis in soc1ful woody Arabidopsis plants.

PubMed

Davin, Nicolas; Edger, Patrick P; Hefer, Charles A; Mizrachi, Eshchar; Schuetz, Mathias; Smets, Erik; Myburg, Alexander A; Douglas, Carl J; Schranz, Michael E; Lens, Frederic

2016-06-01

Many plant genes are known to be involved in the development of cambium and wood, but how the expression and functional interaction of these genes determine the unique biology of wood remains largely unknown. We used the soc1ful loss of function mutant - the woodiest genotype known in the otherwise herbaceous model plant Arabidopsis - to investigate the expression and interactions of genes involved in secondary growth (wood formation). Detailed anatomical observations of the stem in combination with mRNA sequencing were used to assess transcriptome remodeling during xylogenesis in wild-type and woody soc1ful plants. To interpret the transcriptome changes, we constructed functional gene association networks of differentially expressed genes using the STRING database. This analysis revealed functionally enriched gene association hubs that are differentially expressed in herbaceous and woody tissues. In particular, we observed the differential expression of genes related to mechanical stress and jasmonate biosynthesis/signaling during wood formation in soc1ful plants that may be an effect of greater tension within woody tissues. Our results suggest that habit shifts from herbaceous to woody life forms observed in many angiosperm lineages could have evolved convergently by genetic changes that modulate the gene expression and interaction network, and thereby redeploy the conserved wood developmental program. © 2016 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.

Distributional fold change test – a statistical approach for detecting differential expression in microarray experiments

PubMed Central

2012-01-01

Background Because of the large volume of data and the intrinsic variation of data intensity observed in microarray experiments, different statistical methods have been used to systematically extract biological information and to quantify the associated uncertainty. The simplest method to identify differentially expressed genes is to evaluate the ratio of average intensities in two different conditions and consider all genes that differ by more than an arbitrary cut-off value to be differentially expressed. This filtering approach is not a statistical test and there is no associated value that can indicate the level of confidence in the designation of genes as differentially expressed or not differentially expressed. At the same time the fold change by itself provide valuable information and it is important to find unambiguous ways of using this information in expression data treatment. Results A new method of finding differentially expressed genes, called distributional fold change (DFC) test is introduced. The method is based on an analysis of the intensity distribution of all microarray probe sets mapped to a three dimensional feature space composed of average expression level, average difference of gene expression and total variance. The proposed method allows one to rank each feature based on the signal-to-noise ratio and to ascertain for each feature the confidence level and power for being differentially expressed. The performance of the new method was evaluated using the total and partial area under receiver operating curves and tested on 11 data sets from Gene Omnibus Database with independently verified differentially expressed genes and compared with the t-test and shrinkage t-test. Overall the DFC test performed the best – on average it had higher sensitivity and partial AUC and its elevation was most prominent in the low range of differentially expressed features, typical for formalin-fixed paraffin-embedded sample sets. Conclusions The distributional fold change test is an effective method for finding and ranking differentially expressed probesets on microarrays. The application of this test is advantageous to data sets using formalin-fixed paraffin-embedded samples or other systems where degradation effects diminish the applicability of correlation adjusted methods to the whole feature set. PMID:23122055

Identification of the key molecules involved in chronic copper exposure-aggravated memory impairment in transgenic mice of Alzheimer's disease using proteomic analysis.

PubMed

Yu, Jun; Luo, Xiaobin; Xu, Hua; Ma, Quan; Yuan, Jianhui; Li, Xuling; Chang, Raymond Chuen-Chung; Qu, Zhongsen; Huang, Xinfeng; Zhuang, Zhixiong; Liu, Jianjun; Yang, Xifei

2015-01-01

Alzheimer's disease (AD) is the most common neurodegenerative disease characterized by a progressive impairment of cognitive functions including spatial learning and memory. Excess copper exposure accelerates the development of AD; however, the potential mechanisms by which copper exacerbates the symptoms of AD remain unknown. In this study, we explored the effects of chronic copper exposure on cognitive function by treating 6 month-old triple AD transgenic (3xTg-AD) mice with 250 ppm copper sulfate in drinking water for 6 months, and identified several potential key molecules involved in the effects of chronic copper exposure on memory by proteomic analysis. The behavioral test showed that chronic copper exposure aggravated memory impairment of 3xTg-AD mice. Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry revealed a total of 44 differentially expressed proteins (18 upregulated and 26 down-regulated) in hippocampus between the wild-type (WT) mice and non-exposed 3xTg-AD mice. A total of 40 differentially expressed proteins were revealed (20 upregulated and 20 down-regulated) in hippocampus between copper exposed and non-exposed 3xTg-AD mice. Among these differentially expressed proteins, complexin-1 and complexin-2, two memory associated proteins, were significantly decreased in hippocampus of 3xTg-AD mice compared with the WT mice. Furthermore, the expression of these two proteins was further down-regulated in 3xTg-AD mice when exposed to copper. The abnormal expression of complexin-1 and complexin-2 identified by proteomic analysis was verified by western blot analysis. Taken together, our data showed that chronic copper exposure accelerated memory impairment and altered the expression of proteins in hippocampus in 3xTg-AD mice. The functional analysis on the differentially expressed proteins suggested that complexin-1 and complexin-2 may be the key molecules involved in chronic copper exposure-aggravated memory impairment in AD.

Protein expression profiling in head fragments during planarian regeneration after amputation.

PubMed

Chen, Xiaoguang; Xu, Cunshuan

2015-04-01

Following amputation, a planarian tail fragment can regrow into a complete organism including a well-organized brain within about 2-3 weeks, thus restoring the structure and function to presurgical levels. Despite the enormous potential of these animals for regenerative medicine, our understanding of the exact mechanism of planarian regeneration is incomplete. To better understand the molecular nature of planarian head regeneration, we applied two-dimensional electrophoresis (2-DE)/matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)/time-of-flight mass spectrometry (TOF MS) technique to analyze the dynamic proteomic expression profiles over the course of 6 to 168 h post-decapitation. This approach identified a total of 141 differentially expressed proteins, 47 of which exhibited exceptionally high fold changes (≥3-fold change). Of these, Rx protein, an important regulator of head and brain development, was considered to be closely related to planarian head regeneration because of its exceptional high expression almost throughout the time course of regeneration process. Functional annotation analysis classified the 141 proteins into eight categories: (1) signaling, (2) Ca(2+) binding and translocation, (3) transcription and translation, (4) cytoskeleton, (5) metabolism, (6) cell protection, (7) tissue differentiation, and (8) cell cycle. Signaling pathway analysis indicated that Wnt1/Ca(2+) signaling pathway was activated during head regeneration. Integrating the analyses of proteome expression profiling, functional annotation, and signaling pathway, amputation-induced head reformation requires some mechanisms to promote cell proliferation and differentiation, including differential regulation of proapoptotic and antiapoptotic proteins, and the regulation of proliferation and differentiation-related proteins. Importantly, Wnt1/Ca(2+) signaling pathway upregulates Rx expression, finally facilitating the differentiation of neoblasts into various cell types. Taken together, our study demonstrated that proteomic analysis approach used by us is a powerful tool in understanding molecular process related to head regeneration of planarian.

A Key Gene, PLIN1, Can Affect Porcine Intramuscular Fat Content Based on Transcriptome Analysis

PubMed Central

Li, Bojiang; Weng, Qiannan; Dong, Chao; Zhang, Zengkai; Li, Rongyang; Liu, Jingge; Jiang, Aiwen; Li, Qifa; Jia, Chao; Wu, Wangjun; Liu, Honglin

2018-01-01

Intramuscular fat (IMF) content is an important indicator for meat quality evaluation. However, the key genes and molecular regulatory mechanisms affecting IMF deposition remain unclear. In the present study, we identified 75 differentially expressed genes (DEGs) between the higher (H) and lower (L) IMF content of pigs using transcriptome analysis, of which 27 were upregulated and 48 were downregulated. Notably, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that the DEG perilipin-1 (PLIN1) was significantly enriched in the fat metabolism-related peroxisome proliferator-activated receptor (PPAR) signaling pathway. Furthermore, we determined the expression patterns and functional role of porcine PLIN1. Our results indicate that PLIN1 was highly expressed in porcine adipose tissue, and its expression level was significantly higher in the H IMF content group when compared with the L IMF content group, and expression was increased during adipocyte differentiation. Additionally, our results confirm that PLIN1 knockdown decreases the triglyceride (TG) level and lipid droplet (LD) size in porcine adipocytes. Overall, our data identify novel candidate genes affecting IMF content and provide new insight into PLIN1 in porcine IMF deposition and adipocyte differentiation. PMID:29617344

A Key Gene, PLIN1, Can Affect Porcine Intramuscular Fat Content Based on Transcriptome Analysis.

PubMed

Li, Bojiang; Weng, Qiannan; Dong, Chao; Zhang, Zengkai; Li, Rongyang; Liu, Jingge; Jiang, Aiwen; Li, Qifa; Jia, Chao; Wu, Wangjun; Liu, Honglin

2018-04-04

Intramuscular fat (IMF) content is an important indicator for meat quality evaluation. However, the key genes and molecular regulatory mechanisms affecting IMF deposition remain unclear. In the present study, we identified 75 differentially expressed genes (DEGs) between the higher (H) and lower (L) IMF content of pigs using transcriptome analysis, of which 27 were upregulated and 48 were downregulated. Notably, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that the DEG perilipin-1 ( PLIN1 ) was significantly enriched in the fat metabolism-related peroxisome proliferator-activated receptor (PPAR) signaling pathway. Furthermore, we determined the expression patterns and functional role of porcine PLIN1. Our results indicate that PLIN1 was highly expressed in porcine adipose tissue, and its expression level was significantly higher in the H IMF content group when compared with the L IMF content group, and expression was increased during adipocyte differentiation. Additionally, our results confirm that PLIN1 knockdown decreases the triglyceride (TG) level and lipid droplet (LD) size in porcine adipocytes. Overall, our data identify novel candidate genes affecting IMF content and provide new insight into PLIN1 in porcine IMF deposition and adipocyte differentiation.

Changes in the expression profiles of claudins during gonocyte differentiation and in seminomas.

PubMed

Manku, G; Hueso, A; Brimo, F; Chan, P; Gonzalez-Peramato, P; Jabado, N; Gayden, T; Bourgey, M; Riazalhosseini, Y; Culty, M

2016-01-01

Testicular germ cell tumors (TGCTs) are the most common type of cancer in young men and their incidence has been steadily increasing for the past decades. TGCTs and their precursor carcinoma in situ (CIS) are thought to arise from the deficient differentiation of gonocytes, precursors of spermatogonial stem cells. However, the mechanisms relating failed gonocyte differentiation to CIS formation remain unknown. The goal of this study was to uncover genes regulated during gonocyte development that would show abnormal patterns of expression in testicular tumors, as prospective links between failed gonocyte development and TGCT. To identify common gene and protein signatures between gonocytes and seminomas, we first performed gene expression analyses of transitional rat gonocytes, spermatogonia, human normal testicular, and TGCT specimens. Gene expression arrays, pathway analysis, and quantitative real-time PCR analysis identified cell adhesion molecules as a functional gene category including genes downregulated during gonocyte differentiation and highly expressed in seminomas. In particular, the mRNA and protein expressions of claudins 6 and 7 were found to decrease during gonocyte transition to spermatogonia, and to be abnormally elevated in seminomas. The dynamic changes in these genes suggest that they may play important physiological roles during gonocyte development. Moreover, our findings support the idea that TGCTs arise from a disruption of gonocyte differentiation, and position claudins as interesting genes to further study in relation to testicular cancer. © 2015 American Society of Andrology and European Academy of Andrology.

Differential Gene Expression in Liver, Gill, and Olfactory Rosettes of Coho Salmon (Oncorhynchus kisutch) After Acclimation to Salinity.

PubMed

Maryoung, Lindley A; Lavado, Ramon; Bammler, Theo K; Gallagher, Evan P; Stapleton, Patricia L; Beyer, Richard P; Farin, Federico M; Hardiman, Gary; Schlenk, Daniel

2015-12-01

Most Pacific salmonids undergo smoltification and transition from freshwater to saltwater, making various adjustments in metabolism, catabolism, osmotic, and ion regulation. The molecular mechanisms underlying this transition are largely unknown. In the present study, we acclimated coho salmon (Oncorhynchus kisutch) to four different salinities and assessed gene expression through microarray analysis of gills, liver, and olfactory rosettes. Gills are involved in osmotic regulation, liver plays a role in energetics, and olfactory rosettes are involved in behavior. Between all salinity treatments, liver had the highest number of differentially expressed genes at 1616, gills had 1074, and olfactory rosettes had 924, using a 1.5-fold cutoff and a false discovery rate of 0.5. Higher responsiveness of liver to metabolic changes after salinity acclimation to provide energy for other osmoregulatory tissues such as the gills may explain the differences in number of differentially expressed genes. Differentially expressed genes were tissue- and salinity-dependent. There were no known genes differentially expressed that were common to all salinity treatments and all tissues. Gene ontology term analysis revealed biological processes, molecular functions, and cellular components that were significantly affected by salinity, a majority of which were tissue-dependent. For liver, oxygen binding and transport terms were highlighted. For gills, muscle, and cytoskeleton-related terms predominated and for olfactory rosettes, immune response-related genes were accentuated. Interaction networks were examined in combination with GO terms and determined similarities between tissues for potential osmosensors, signal transduction cascades, and transcription factors.

Gene Expression Profiling of Gastric Cancer

PubMed Central

Marimuthu, Arivusudar; Jacob, Harrys K.C.; Jakharia, Aniruddha; Subbannayya, Yashwanth; Keerthikumar, Shivakumar; Kashyap, Manoj Kumar; Goel, Renu; Balakrishnan, Lavanya; Dwivedi, Sutopa; Pathare, Swapnali; Dikshit, Jyoti Bajpai; Maharudraiah, Jagadeesha; Singh, Sujay; Sameer Kumar, Ghantasala S; Vijayakumar, M.; Veerendra Kumar, Kariyanakatte Veeraiah; Premalatha, Chennagiri Shrinivasamurthy; Tata, Pramila; Hariharan, Ramesh; Roa, Juan Carlos; Prasad, T.S.K; Chaerkady, Raghothama; Kumar, Rekha Vijay; Pandey, Akhilesh

2015-01-01

Gastric cancer is the second leading cause of cancer death worldwide, both in men and women. A genomewide gene expression analysis was carried out to identify differentially expressed genes in gastric adenocarcinoma tissues as compared to adjacent normal tissues. We used Agilent’s whole human genome oligonucleotide microarray platform representing ~41,000 genes to carry out gene expression analysis. Two-color microarray analysis was employed to directly compare the expression of genes between tumor and normal tissues. Through this approach, we identified several previously known candidate genes along with a number of novel candidate genes in gastric cancer. Testican-1 (SPOCK1) was one of the novel molecules that was 10-fold upregulated in tumors. Using tissue microarrays, we validated the expression of testican-1 by immunohistochemical staining. It was overexpressed in 56% (160/282) of the cases tested. Pathway analysis led to the identification of several networks in which SPOCK1 was among the topmost networks of interacting genes. By gene enrichment analysis, we identified several genes involved in cell adhesion and cell proliferation to be significantly upregulated while those corresponding to metabolic pathways were significantly downregulated. The differentially expressed genes identified in this study are candidate biomarkers for gastric adenoacarcinoma. PMID:27030788

Differential roles of HIC-5 isoforms in the regulation of cell death and myotube formation during myogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao Zhengliang; Deblis, Ryan; Glenn, Honor

2007-11-15

Hic-5 is a LIM-Only member of the paxillin superfamily of focal adhesion proteins. It has been shown to regulate a range of biological processes including: senescence, tumorigenesis, steroid hormone action, integrin signaling, differentiation, and apoptosis. To better understand the roles of Hic-5 during development, we initiated a detailed analysis of Hic-5 expression and function in C{sub 2}C{sub 12} myoblasts, a well-established model for myogenesis. We have found that: (1) myoblasts express at least 6 distinct Hic-5 isoforms; (2) the two predominant isoforms, Hic-5{alpha} and Hic-5{beta}, are differentially expressed during myogenesis; (3) any experimentally induced change in Hic-5 expression results inmore » a substantial increase in apoptosis during differentiation; (4) ectopic expression of Hic-5{alpha} is permissive to differentiation while expression of either Hic-5{beta} or antisense Hic-5 blocks myoblast fusion but not chemodifferentiation; (5) Hic-5 localizes to focal adhesions in C{sub 2}C{sub 12} myoblasts and perturbation of Hic-5 leads to defects in cell spreading; (6) alterations in Hic-5 expression interfere with the normal dynamics of laminin expression; and (7) ectopic laminin, but not fibronectin, can rescue the Hic-5-induced blockade of myoblast survival and differentiation. Our data demonstrate differential roles for individual Hic-5 isoforms during myogenesis and support the hypothesis that Hic-5 mediates these effects via integrin signaling.« less

Differential transcriptome analysis reveals genes related to cold tolerance in seabuckthorn carpenter moth, Eogystia hippophaecolus

PubMed Central

Hu, Ping; Wang, Tao; Tao, Jing; Zong, Shixiang

2017-01-01

Seabuckthorn carpenter moth, Eogystia hippophaecolus (Lepidoptera: Cossidae), is an important pest of sea buckthorn (Hippophae rhamnoides), which is a shrub that has significant ecological and economic value in China. E. hippophaecolus is highly cold tolerant, but limited studies have been conducted to elucidate the molecular mechanisms underlying its cold resistance. Here we sequenced the E. hippophaecolus transcriptome using RNA-Seq technology and performed de novo assembly from the short paired-end reads. We investigated the larval response to cold stress by comparing gene expression profiles between treatments. We obtained 118,034 unigenes, of which 22,161 were annotated with gene descriptions, conserved domains, gene ontology terms, and metabolic pathways. These resulted in 57 GO terms and 193 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. By comparing transcriptome profiles for differential gene expression, we identified many differentially expressed proteins and genes, including heat shock proteins and cuticular proteins which have previously been reported to be involved in cold resistance of insects. This study provides a global transcriptome analysis and an assessment of differential gene expression in E. hippophaecolus under cold stress. We found seven differential expressed genes in common between developmental stages, which were verified with qPCR. Our findings facilitate future genomic studies aimed at improving our understanding of the molecular mechanisms underlying the response of insects to low temperatures. PMID:29131867

Differential transcriptome analysis reveals genes related to cold tolerance in seabuckthorn carpenter moth, Eogystia hippophaecolus.

PubMed

Cui, Mingming; Hu, Ping; Wang, Tao; Tao, Jing; Zong, Shixiang

2017-01-01

Seabuckthorn carpenter moth, Eogystia hippophaecolus (Lepidoptera: Cossidae), is an important pest of sea buckthorn (Hippophae rhamnoides), which is a shrub that has significant ecological and economic value in China. E. hippophaecolus is highly cold tolerant, but limited studies have been conducted to elucidate the molecular mechanisms underlying its cold resistance. Here we sequenced the E. hippophaecolus transcriptome using RNA-Seq technology and performed de novo assembly from the short paired-end reads. We investigated the larval response to cold stress by comparing gene expression profiles between treatments. We obtained 118,034 unigenes, of which 22,161 were annotated with gene descriptions, conserved domains, gene ontology terms, and metabolic pathways. These resulted in 57 GO terms and 193 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. By comparing transcriptome profiles for differential gene expression, we identified many differentially expressed proteins and genes, including heat shock proteins and cuticular proteins which have previously been reported to be involved in cold resistance of insects. This study provides a global transcriptome analysis and an assessment of differential gene expression in E. hippophaecolus under cold stress. We found seven differential expressed genes in common between developmental stages, which were verified with qPCR. Our findings facilitate future genomic studies aimed at improving our understanding of the molecular mechanisms underlying the response of insects to low temperatures.

Identification of ELF3 as an early transcriptional regulator of human urothelium.

PubMed

Böck, Matthias; Hinley, Jennifer; Schmitt, Constanze; Wahlicht, Tom; Kramer, Stefan; Southgate, Jennifer

2014-02-15

Despite major advances in high-throughput and computational modelling techniques, understanding of the mechanisms regulating tissue specification and differentiation in higher eukaryotes, particularly man, remains limited. Microarray technology has been explored exhaustively in recent years and several standard approaches have been established to analyse the resultant datasets on a genome-wide scale. Gene expression time series offer a valuable opportunity to define temporal hierarchies and gain insight into the regulatory relationships of biological processes. However, unless datasets are exactly synchronous, time points cannot be compared directly. Here we present a data-driven analysis of regulatory elements from a microarray time series that tracked the differentiation of non-immortalised normal human urothelial (NHU) cells grown in culture. The datasets were obtained by harvesting differentiating and control cultures from finite bladder- and ureter-derived NHU cell lines at different time points using two previously validated, independent differentiation-inducing protocols. Due to the asynchronous nature of the data, a novel ranking analysis approach was adopted whereby we compared changes in the amplitude of experiment and control time series to identify common regulatory elements. Our approach offers a simple, fast and effective ranking method for genes that can be applied to other time series. The analysis identified ELF3 as a candidate transcriptional regulator involved in human urothelial cytodifferentiation. Differentiation-associated expression of ELF3 was confirmed in cell culture experiments and by immunohistochemical demonstration in situ. The importance of ELF3 in urothelial differentiation was verified by knockdown in NHU cells, which led to reduced expression of FOXA1 and GRHL3 transcription factors in response to PPARγ activation. The consequences of this were seen in the repressed expression of late/terminal differentiation-associated uroplakin 3a gene expression and in the compromised development and regeneration of urothelial barrier function. Copyright © 2014 Elsevier Inc. All rights reserved.

Comparative Proteomics Analysis Reveals L-Arginine Activates Ethanol Degradation Pathways in HepG2 Cells.

PubMed

Yan, Guokai; Lestari, Retno; Long, Baisheng; Fan, Qiwen; Wang, Zhichang; Guo, Xiaozhen; Yu, Jie; Hu, Jun; Yang, Xingya; Chen, Changqing; Liu, Lu; Li, Xiuzhi; Purnomoadi, Agung; Achmadi, Joelal; Yan, Xianghua

2016-03-17

L-Arginine (Arg) is a versatile amino acid that plays crucial roles in a wide range of physiological and pathological processes. In this study, to investigate the alteration induced by Arg supplementation in proteome scale, isobaric tags for relative and absolute quantification (iTRAQ) based proteomic approach was employed to comparatively characterize the differentially expressed proteins between Arg deprivation (Ctrl) and Arg supplementation (+Arg) treated human liver hepatocellular carcinoma (HepG2) cells. A total of 21 proteins were identified as differentially expressed proteins and these 21 proteins were all up-regulated by Arg supplementation. Six amino acid metabolism-related proteins, mostly metabolic enzymes, showed differential expressions. Intriguingly, Ingenuity Pathway Analysis (IPA) based pathway analysis suggested that the three ethanol degradation pathways were significantly altered between Ctrl and +Arg. Western blotting and enzymatic activity assays validated that the key enzymes ADH1C, ALDH1A1, and ALDH2, which are mainly involved in ethanol degradation pathways, were highly differentially expressed, and activated between Ctrl and +Arg in HepG2 cells. Furthermore, 10 mM Arg significantly attenuated the cytotoxicity induced by 100 mM ethanol treatment (P < 0.0001). This study is the first time to reveal that Arg activates ethanol degradation pathways in HepG2 cells.

Partial least squares based gene expression analysis in estrogen receptor positive and negative breast tumors.

PubMed

Ma, W; Zhang, T-F; Lu, P; Lu, S H

2014-01-01

Breast cancer is categorized into two broad groups: estrogen receptor positive (ER+) and ER negative (ER-) groups. Previous study proposed that under trastuzumab-based neoadjuvant chemotherapy, tumor initiating cell (TIC) featured ER- tumors response better than ER+ tumors. Exploration of the molecular difference of these two groups may help developing new therapeutic strategies, especially for ER- patients. With gene expression profile from the Gene Expression Omnibus (GEO) database, we performed partial least squares (PLS) based analysis, which is more sensitive than common variance/regression analysis. We acquired 512 differentially expressed genes. Four pathways were found to be enriched with differentially expressed genes, involving immune system, metabolism and genetic information processing process. Network analysis identified five hub genes with degrees higher than 10, including APP, ESR1, SMAD3, HDAC2, and PRKAA1. Our findings provide new understanding for the molecular difference between TIC featured ER- and ER+ breast tumors with the hope offer supports for therapeutic studies.

The expression of native and cultured human retinal pigment epithelial cells grown in different culture conditions.

PubMed

Tian, J; Ishibashi, K; Honda, S; Boylan, S A; Hjelmeland, L M; Handa, J T

2005-11-01

To determine the transcriptional proximity of retinal pigment epithelium (RPE) cells grown under different culture conditions and native RPE. ARPE-19 cells were grown under five conditions in 10% CO(2): "subconfluent" in DMEM/F12+10% FBS, "confluent" in serum and serum withdrawn, and "differentiated" for 2.5 months in serum and serum withdrawn medium. Native RPE was laser microdissected. Total RNA was extracted, reverse transcribed, and radiolabelled probes were hybridised to an array containing 5,353 genes. Arrays were evaluated by hierarchical cluster analysis and significance analysis of microarrays. 78% of genes were expressed by native RPE while 45.3--47.7% were expressed by ARPE-19 cells, depending on culture condition. While the most abundant genes were expressed by native and cultured cells, significant differences in low abundance genes were seen. Hierarchical cluster analysis showed that confluent and differentiated, serum withdrawn cultures clustered closest to native RPE, and that serum segregated cultured cells from native RPE. The number of differentially expressed genes and their function, and profile of expressed and unexpressed genes, demonstrate differences between native and cultured cells. While ARPE-19 cells have significant value for studying RPE behaviour, investigators must be aware of how culture conditions can influence the mRNA phenotype of the cell.

Proteomic analysis associated with coronary artery dilatation caused by Kawasaki disease using serum exosomes.

PubMed

Zhang, Li; Wang, Wei; Bai, Jun; Xu, Yu-Fen; Li, Lai-Qing; Hua, Liang; Deng, Li; Jia, Hong-Ling

2016-05-01

The aim of this study was to investigate the serum exosome proteome profile of coronary artery dilatation (CAD) caused by Kawasaki disease (KD). Two-dimensional electrophoresis was implemented on proteins of serum exosomes obtained from children with CAD caused by KD and from healthy controls. Differentially expressed proteins were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry analysis. We identified 38 differentially expressed proteins (13 up-regulated and 25 down-regulated) from serum exosomes of patients with CAD caused by KD compared with healthy controls. Expression levels of three differentially expressed proteins (leucine-rich alpha-2-glycoprotein, sex hormone-binding globulin, and serotransferrin) were validated using western blot analysis. Classification and protein-protein network analysis showed that they are associated with multiple functional groups involved in the acute inflammatory response, defense response, complement activation, humoral immune response, and response to wounding. The majority of the proteins are involved in the inflammation and coagulation cascades. These findings establish a comprehensive proteome profile of CAD caused by KD and increase our knowledge of scientific insight into its mechanisms. Copyright © 2016 Sociedade Portuguesa de Cardiologia. Published by Elsevier España. All rights reserved.

[Differentially expressed genes of cell signal transduction associated with benzene poisoning by cDNA microarray].

PubMed

Wang, Hong; Bi, Yongyi; Tao, Ning; Wang, Chunhong

2005-08-01

To detect the differential expression of cell signal transduction genes associated with benzene poisoning, and to explore the pathogenic mechanisms of blood system damage induced by benzene. Peripheral white blood cell gene expression profile of 7 benzene poisoning patients, including one aplastic anemia, was determined by cDNA microarray. Seven chips from normal workers were served as controls. Cluster analysis of gene expression profile was performed. Among the 4265 target genes, 176 genes associated with cell signal transduction were differentially expressed. 35 up-regulated genes including PTPRC, STAT4, IFITM1 etc were found in at least 6 pieces of microarray; 45 down-regulated genes including ARHB, PPP3CB, CDC37 etc were found in at least 5 pieces of microarray. cDNA microarray technology is an effective technique for screening the differentially expressed genes of cell signal transduction. Disorder in cell signal transduction may play certain role in the pathogenic mechanism of benzene poisoning.

Genome-wide analysis of miRNAs in the ovaries of Jining Grey and Laiwu Black goats to explore the regulation of fecundity.

PubMed

Miao, Xiangyang; Luo, Qingmiao; Zhao, Huijing; Qin, Xiaoyu

2016-11-29

Goat fecundity is important for agriculture and varies depending on the genetic background of the goat. Two excellent domestic breeds in China, the Jining Grey and Laiwu Black goats, have different fecundity and prolificacies. To explore the potential miRNAs that regulate the expression of the genes involved in these prolific differences and to potentially discover new miRNAs, we performed a genome-wide analysis of the miRNAs in the ovaries from these two goats using RNA-Seq technology. Thirty miRNAs were differentially expressed between the Jining Grey and Laiwu Black goats. Gene Ontology and KEGG pathway analyses revealed that the target genes of the differentially expressed miRNAs were significantly enriched in several biological processes and pathways. A protein-protein interaction analysis indicated that the miRNAs and their target genes were related to the reproduction complex regulation network. The differential miRNA expression profiles found in the ovaries between the two distinctive breeds of goats studied here provide a unique resource for addressing fecundity differences in goats.

Paracrine Pathways in Uterine Leiomyoma Stem Cells Involve Insulinlike Growth Factor 2 and Insulin Receptor A.

PubMed

Moravek, Molly B; Yin, Ping; Coon, John S; Ono, Masanori; Druschitz, Stacy A; Malpani, Saurabh S; Dyson, Matthew T; Rademaker, Alfred W; Robins, Jared C; Wei, Jian-Jun; Kim, J Julie; Bulun, Serdar E

2017-05-01

Uterine leiomyomas (fibroids) are the most common benign tumors in women. Recently, three populations of leiomyoma cells were discovered on the basis of CD34 and CD49b expression, but molecular differences between these populations remain unknown. To define differential gene expression and signaling pathways in leiomyoma cell populations. Cells from human leiomyoma tissue were sorted by flow cytometry into three populations: CD34+/CD49b+, CD34+/CD49b-, and CD34-/CD49b-. Microarray gene expression profiling and pathway analysis were performed. To investigate the insulinlike growth factor (IGF) pathway, real-time quantitative polymerase chain reaction, immunoblotting, and 5-ethynyl-2'-deoxyuridine incorporation studies were performed in cells isolated from fresh leiomyoma. Research laboratory. Eight African American women. None. Gene expression patterns, cell proliferation, and differentiation. A total of 1164 genes were differentially expressed in the three leiomyoma cell populations, suggesting a hierarchical differentiation order whereby CD34+/CD49b+ stem cells differentiate to CD34+/CD49b- intermediary cells, which then terminally differentiate to CD34-/CD49b- cells. Pathway analysis revealed differential expression of several IGF signaling pathway genes. IGF2 was overexpressed in CD34+/CD49b- vs CD34-/CD49b- cells (83-fold; P < 0.05). Insulin receptor A (IR-A) expression was higher and IGF1 receptor lower in CD34+/CD49b+ vs CD34-/CD49b- cells (15-fold and 0.35-fold, respectively; P < 0.05). IGF2 significantly increased cell number (1.4-fold; P < 0.001), proliferation indices, and extracellular signal-regulated kinase (ERK) phosphorylation. ERK inhibition decreased IGF2-stimulated cell proliferation. IGF2 and IR-A are important for leiomyoma stem cell proliferation and may represent paracrine signaling between leiomyoma cell types. Therapies targeting the IGF pathway should be investigated for both treatment and prevention of leiomyomas. Copyright © 2017 by the Endocrine Society

Integrated Analyses of Gene Expression Profiles Digs out Common Markers for Rheumatic Diseases

PubMed Central

Wang, Lan; Wu, Long-Fei; Lu, Xin; Mo, Xing-Bo; Tang, Zai-Xiang; Lei, Shu-Feng; Deng, Fei-Yan

2015-01-01

Objective Rheumatic diseases have some common symptoms. Extensive gene expression studies, accumulated thus far, have successfully identified signature molecules for each rheumatic disease, individually. However, whether there exist shared factors across rheumatic diseases has yet to be tested. Methods We collected and utilized 6 public microarray datasets covering 4 types of representative rheumatic diseases including rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, and osteoarthritis. Then we detected overlaps of differentially expressed genes across datasets and performed a meta-analysis aiming at identifying common differentially expressed genes that discriminate between pathological cases and normal controls. To further gain insights into the functions of the identified common differentially expressed genes, we conducted gene ontology enrichment analysis and protein-protein interaction analysis. Results We identified a total of eight differentially expressed genes (TNFSF10, CX3CR1, LY96, TLR5, TXN, TIA1, PRKCH, PRF1), each associated with at least 3 of the 4 studied rheumatic diseases. Meta-analysis warranted the significance of the eight genes and highlighted the general significance of four genes (CX3CR1, LY96, TLR5, and PRF1). Protein-protein interaction and gene ontology enrichment analyses indicated that the eight genes interact with each other to exert functions related to immune response and immune regulation. Conclusion The findings support that there exist common factors underlying rheumatic diseases. For rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis and osteoarthritis diseases, those common factors include TNFSF10, CX3CR1, LY96, TLR5, TXN, TIA1, PRKCH, and PRF1. In-depth studies on these common factors may provide keys to understanding the pathogenesis and developing intervention strategies for rheumatic diseases. PMID:26352601

[Study on action mechanism and material base of compound Danshen dripping pills in treatment of carotid atherosclerosis based on techniques of gene expression profile and molecular fingerprint].

PubMed

Zhou, Wei; Song, Xiang-gang; Chen, Chao; Wang, Shu-mei; Liang, Sheng-wang

2015-08-01

Action mechanism and material base of compound Danshen dripping pills in treatment of carotid atherosclerosis were discussed based on gene expression profile and molecular fingerprint in this paper. First, gene expression profiles of atherosclerotic carotid artery tissues and histologically normal tissues in human body were collected, and were screened using significance analysis of microarray (SAM) to screen out differential gene expressions; then differential genes were analyzed by Gene Ontology (GO) analysis and KEGG pathway analysis; to avoid some genes with non-outstanding differential expression but biologically importance, Gene Set Enrichment Analysis (GSEA) were performed, and 7 chemical ingredients with higher negative enrichment score were obtained by Cmap method, implying that they could reversely regulate the gene expression profiles of pathological tissues; and last, based on the hypotheses that similar structures have similar activities, 336 ingredients of compound Danshen dripping pills were compared with 7 drug molecules in 2D molecular fingerprints method. The results showed that 147 differential genes including 60 up-regulated genes and 87 down regulated genes were screened out by SAM. And in GO analysis, Biological Process ( BP) is mainly concerned with biological adhesion, response to wounding and inflammatory response; Cellular Component (CC) is mainly concerned with extracellular region, extracellular space and plasma membrane; while Molecular Function (MF) is mainly concerned with antigen binding, metalloendopeptidase activity and peptide binding. KEGG pathway analysis is mainly concerned with JAK-STAT, RIG-I like receptor and PPAR signaling pathway. There were 10 compounds, such as hexadecane, with Tanimoto coefficients greater than 0.85, which implied that they may be the active ingredients (AIs) of compound Danshen dripping pills in treatment of carotid atherosclerosis (CAs). The present method can be applied to the research on material base and molecular action mechanism of TCM.

Differential gene expression in Varroa jacobsoni mites following a host shift to European honey bees (Apis mellifera).

PubMed

Andino, Gladys K; Gribskov, Michael; Anderson, Denis L; Evans, Jay D; Hunt, Greg J

2016-11-16

Varroa mites are widely considered the biggest honey bee health problem worldwide. Until recently, Varroa jacobsoni has been found to live and reproduce only in Asian honey bee (Apis cerana) colonies, while V. destructor successfully reproduces in both A. cerana and A. mellifera colonies. However, we have identified an island population of V. jacobsoni that is highly destructive to A. mellifera, the primary species used for pollination and honey production. The ability of these populations of mites to cross the host species boundary potentially represents an enormous threat to apiculture, and is presumably due to genetic variation that exists among populations of V. jacobsoni that influences gene expression and reproductive status. In this work, we investigate differences in gene expression between populations of V. jacobsoni reproducing on A. cerana and those either reproducing or not capable of reproducing on A. mellifera, in order to gain insight into differences that allow V. jacobsoni to overcome its normal species tropism. We sequenced and assembled a de novo transcriptome of V. jacobsoni. We also performed a differential gene expression analysis contrasting biological replicates of V. jacobsoni populations that differ in their ability to reproduce on A. mellifera. Using the edgeR, EBSeq and DESeq R packages for differential gene expression analysis, we found 287 differentially expressed genes (FDR ≤ 0.05), of which 91% were up regulated in mites reproducing on A. mellifera. In addition, mites found reproducing on A. mellifera showed substantially more variation in expression among replicates. We searched for orthologous genes in public databases and were able to associate 100 of these 287 differentially expressed genes with a functional description. There is differential gene expression between the two mite groups, with more variation in gene expression among mites that were able to reproduce on A. mellifera. A small set of genes showed reduced expression in mites on the A. mellifera host, including putative transcription factors and digestive tract developmental genes. The vast majority of differentially expressed genes were up-regulated in this host. This gene set showed enrichment for genes associated with mitochondrial respiratory function and apoptosis, suggesting that mites on this host may be experiencing higher stress, and may be less optimally adapted to parasitize it. Some genes involved in reproduction and oogenesis were also overexpressed, which should be further studied in regards to this host shift.

[Plasma proteomic analysis in children with infectious mononucleosis].

PubMed

Ran, Zhi-Ling; Xiao, Bin; Liu, Hong-Rui; Liu, You-Ping; Sheng, Qiao-Ni

2015-03-01

To explore the abnormal expression of plasma proteins by analysis of proteomic expression profile in children with infectious mononucleosis (IM). Two dimensional gel electrophoresis (2-DE) followed by the mass spectrometry was used to examine important protein spots with different expression levels between children with IM and normal controls. Seven differential proteins were obtained: hemopexin, vitamin D binding protein, fetuin A, C-reactive protein, apolipoprotein A, haptoglobin and transthyretin. Compared with the control group, haptoglobin showed a higher expression level in children with IM, and the expression levels of the other proteins were obviously down-regulated. The expression changes of differential proteins identified in this study are all related with the liver acute injury, suggesting that children with IM are associated with acute liver injury. Further studies on the characteristics of above proteins will contribute to the diagnosis and treatment of pediatric IM.

[Preliminary analysis of retinal gene expression profile of diabetic rat].

PubMed

Mei, Yan; Zhou, Hong-ying; Xiang, Tao; Lu, You-guang; Li, Ai-dong; Tang, En-jie; Yang, Hui-jun

2005-10-01

Establishing the retinal gene expression profiles of non-diabetic rat and diabetic rat and comparing the profiles in order to analyze the possible genes related with diabetic retinopathy. The whole retinal transcriptional fragments of non-diabetic rat and 8-week diabetic rat were obtained by restriction fragments differential display-PCR (RFDD-PCR). Bioinformatic analysis of retinal gene expression was performed using soft wares, including Fragment Analysis. After comparison of the expression profiles, the related gene fragments of diabetic retinopathy were initially selected as the target gene of further approach. A total of 3639 significant fragments were obtained. By means of more than 3-fold contrast of fluorescent intensity as the differential expression standard, the authors got 840 differential fragments, accounting for 23.08% of the expressed numbers and including 5 visual related genes, 13 excitatory neruotransmitter genes and 3 inhibitory neurotransmitter genes. At the 8th week, the expression of Rhodopsin kinase, beta-arrestin, Phosducinìrod photoreceptor cGMP-gated channel and Rpe65 as well as iGlu R1-4 were down-regulated. mGluRs and GABA-Rs were all up-regulated, whereas the expression of GlyR was unchanged. These results prompt again that the changes in retinal nervous layer of rat have occurred at an early stage of diabetes. The genes expression pattern of visual related genes and excitatory and inhibitory neurotransmitters in rat diabetic retina have been involved in neuro-dysfunctions of diabetic retina.

A whole-blood transcriptome meta-analysis identifies gene expression signatures of cigarette smoking

PubMed Central

Huan, Tianxiao; Joehanes, Roby; Schurmann, Claudia; Schramm, Katharina; Pilling, Luke C.; Peters, Marjolein J.; Mägi, Reedik; DeMeo, Dawn; O'Connor, George T.; Ferrucci, Luigi; Teumer, Alexander; Homuth, Georg; Biffar, Reiner; Völker, Uwe; Herder, Christian; Waldenberger, Melanie; Peters, Annette; Zeilinger, Sonja; Metspalu, Andres; Hofman, Albert; Uitterlinden, André G.; Hernandez, Dena G.; Singleton, Andrew B.; Bandinelli, Stefania; Munson, Peter J.; Lin, Honghuang; Benjamin, Emelia J.; Esko, Tõnu; Grabe, Hans J.; Prokisch, Holger; van Meurs, Joyce B.J.; Melzer, David; Levy, Daniel

2016-01-01

Abstract Cigarette smoking is a leading modifiable cause of death worldwide. We hypothesized that cigarette smoking induces extensive transcriptomic changes that lead to target-organ damage and smoking-related diseases. We performed a meta-analysis of transcriptome-wide gene expression using whole blood-derived RNA from 10,233 participants of European ancestry in six cohorts (including 1421 current and 3955 former smokers) to identify associations between smoking and altered gene expression levels. At a false discovery rate (FDR) <0.1, we identified 1270 differentially expressed genes in current vs. never smokers, and 39 genes in former vs. never smokers. Expression levels of 12 genes remained elevated up to 30 years after smoking cessation, suggesting that the molecular consequence of smoking may persist for decades. Gene ontology analysis revealed enrichment of smoking-related genes for activation of platelets and lymphocytes, immune response, and apoptosis. Many of the top smoking-related differentially expressed genes, including LRRN3 and GPR15, have DNA methylation loci in promoter regions that were recently reported to be hypomethylated among smokers. By linking differential gene expression with smoking-related disease phenotypes, we demonstrated that stroke and pulmonary function show enrichment for smoking-related gene expression signatures. Mediation analysis revealed the expression of several genes (e.g. ALAS2) to be putative mediators of the associations between smoking and inflammatory biomarkers (IL6 and C-reactive protein levels). Our transcriptomic study provides potential insights into the effects of cigarette smoking on gene expression in whole blood and their relations to smoking-related diseases. The results of such analyses may highlight attractive targets for treating or preventing smoking-related health effects. PMID:28158590

Whole Genome Gene Expression Meta-Analysis of Inflammatory Bowel Disease Colon Mucosa Demonstrates Lack of Major Differences between Crohn's Disease and Ulcerative Colitis

PubMed Central

Østvik, Ann E.; Drozdov, Ignat; Gustafsson, Bjørn I.; Kidd, Mark; Beisvag, Vidar; Torp, Sverre H.; Waldum, Helge L.; Martinsen, Tom Christian; Damås, Jan Kristian; Espevik, Terje; Sandvik, Arne K.

2013-01-01

Background In inflammatory bowel disease (IBD), genetic susceptibility together with environmental factors disturbs gut homeostasis producing chronic inflammation. The two main IBD subtypes are Ulcerative colitis (UC) and Crohn’s disease (CD). We present the to-date largest microarray gene expression study on IBD encompassing both inflamed and un-inflamed colonic tissue. A meta-analysis including all available, comparable data was used to explore important aspects of IBD inflammation, thereby validating consistent gene expression patterns. Methods Colon pinch biopsies from IBD patients were analysed using Illumina whole genome gene expression technology. Differential expression (DE) was identified using LIMMA linear model in the R statistical computing environment. Results were enriched for gene ontology (GO) categories. Sets of genes encoding antimicrobial proteins (AMP) and proteins involved in T helper (Th) cell differentiation were used in the interpretation of the results. All available data sets were analysed using the same methods, and results were compared on a global and focused level as t-scores. Results Gene expression in inflamed mucosa from UC and CD are remarkably similar. The meta-analysis confirmed this. The patterns of AMP and Th cell-related gene expression were also very similar, except for IL23A which was consistently higher expressed in UC than in CD. Un-inflamed tissue from patients demonstrated minimal differences from healthy controls. Conclusions There is no difference in the Th subgroup involvement between UC and CD. Th1/Th17 related expression, with little Th2 differentiation, dominated both diseases. The different IL23A expression between UC and CD suggests an IBD subtype specific role. AMPs, previously little studied, are strongly overexpressed in IBD. The presented meta-analysis provides a sound background for further research on IBD pathobiology. PMID:23468882

Whole genome gene expression meta-analysis of inflammatory bowel disease colon mucosa demonstrates lack of major differences between Crohn's disease and ulcerative colitis.

PubMed

Granlund, Atle van Beelen; Flatberg, Arnar; Østvik, Ann E; Drozdov, Ignat; Gustafsson, Bjørn I; Kidd, Mark; Beisvag, Vidar; Torp, Sverre H; Waldum, Helge L; Martinsen, Tom Christian; Damås, Jan Kristian; Espevik, Terje; Sandvik, Arne K

2013-01-01

In inflammatory bowel disease (IBD), genetic susceptibility together with environmental factors disturbs gut homeostasis producing chronic inflammation. The two main IBD subtypes are Ulcerative colitis (UC) and Crohn's disease (CD). We present the to-date largest microarray gene expression study on IBD encompassing both inflamed and un-inflamed colonic tissue. A meta-analysis including all available, comparable data was used to explore important aspects of IBD inflammation, thereby validating consistent gene expression patterns. Colon pinch biopsies from IBD patients were analysed using Illumina whole genome gene expression technology. Differential expression (DE) was identified using LIMMA linear model in the R statistical computing environment. Results were enriched for gene ontology (GO) categories. Sets of genes encoding antimicrobial proteins (AMP) and proteins involved in T helper (Th) cell differentiation were used in the interpretation of the results. All available data sets were analysed using the same methods, and results were compared on a global and focused level as t-scores. Gene expression in inflamed mucosa from UC and CD are remarkably similar. The meta-analysis confirmed this. The patterns of AMP and Th cell-related gene expression were also very similar, except for IL23A which was consistently higher expressed in UC than in CD. Un-inflamed tissue from patients demonstrated minimal differences from healthy controls. There is no difference in the Th subgroup involvement between UC and CD. Th1/Th17 related expression, with little Th2 differentiation, dominated both diseases. The different IL23A expression between UC and CD suggests an IBD subtype specific role. AMPs, previously little studied, are strongly overexpressed in IBD. The presented meta-analysis provides a sound background for further research on IBD pathobiology.

Gene expression profile of mouse prostate tumors reveals dysregulations in major biological processes and identifies potential murine targets for preclinical development of human prostate cancer therapy.

PubMed

Haram, Kerstyn M; Peltier, Heidi J; Lu, Bin; Bhasin, Manoj; Otu, Hasan H; Choy, Bob; Regan, Meredith; Libermann, Towia A; Latham, Gary J; Sanda, Martin G; Arredouani, Mohamed S

2008-10-01

Translation of preclinical studies into effective human cancer therapy is hampered by the lack of defined molecular expression patterns in mouse models that correspond to the human counterpart. We sought to generate an open source TRAMP mouse microarray dataset and to use this array to identify differentially expressed genes from human prostate cancer (PCa) that have concordant expression in TRAMP tumors, and thereby represent lead targets for preclinical therapy development. We performed microarrays on total RNA extracted and amplified from eight TRAMP tumors and nine normal prostates. A subset of differentially expressed genes was validated by QRT-PCR. Differentially expressed TRAMP genes were analyzed for concordant expression in publicly available human prostate array datasets and a subset of resulting genes was analyzed by QRT-PCR. Cross-referencing differentially expressed TRAMP genes to public human prostate array datasets revealed 66 genes with concordant expression in mouse and human PCa; 56 between metastases and normal and 10 between primary tumor and normal tissues. Of these 10 genes, two, Sox4 and Tubb2a, were validated by QRT-PCR. Our analysis also revealed various dysregulations in major biologic pathways in the TRAMP prostates. We report a TRAMP microarray dataset of which a gene subset was validated by QRT-PCR with expression patterns consistent with previous gene-specific TRAMP studies. Concordance analysis between TRAMP and human PCa associated genes supports the utility of the model and suggests several novel molecular targets for preclinical therapy.

Gene expression signatures differentiate ovarian/peritoneal serous carcinoma from breast carcinoma in effusions

PubMed Central

Davidson, Ben; Stavnes, Helene Tuft; Holth, Arild; Chen, Xu; Yang, Yanqin; Shih, Ie-Ming; Wang, Tian-Li

2011-01-01

Abstract Ovarian/primary peritoneal carcinoma and breast carcinoma are the gynaecological cancers that most frequently involve the serosal cavities. With the objective of improving on the limited diagnostic panel currently available for the differential diagnosis of these two malignancies, as well as to define tumour-specific biological targets, we compared their global gene expression patterns. Gene expression profiles of 10 serous ovarian/peritoneal and eight ductal breast carcinoma effusions were analysed using the HumanRef-8 BeadChip from Illumina. Differentially expressed candidate genes were validated using quantitative real-time PCR and immunohistochemistry. Unsupervised hierarchical clustering using all 54,675 genes in the array separated ovarian from breast carcinoma samples. We identified 288 unique probes that were significantly differentially expressed in the two cancers by greater than 3.5-fold, of which 81 and 207 were overexpressed in breast and ovarian/peritoneal carcinoma, respectively. SAM analysis identified 1078 differentially expressed probes with false discovery rate less than 0.05. Genes overexpressed in breast carcinoma included TFF1, TFF3, FOXA1, CA12, GATA3, SDC1, PITX1, TH, EHFD1, EFEMP1, TOB1 and KLF2. Genes overexpressed in ovarian/peritoneal carcinoma included SPON1, RBP1, MFGE8, TM4SF12, MMP7, KLK5/6/7, FOLR1/3, PAX8, APOL2 and NRCAM. The differential expression of 14 genes was validated by quantitative real-time PCR, and differences in 5 gene products were confirmed by immunohistochemistry. Expression profiling distinguishes ovarian/peritoneal carcinoma from breast carcinoma and identifies genes that are differentially expressed in these two tumour types. The molecular signatures unique to these cancers may facilitate their differential diagnosis and may provide a molecular basis for therapeutic target discovery. PMID:20132413

Bovine mammary gene expression profiling during the onset of lactation.

PubMed

Gao, Yuanyuan; Lin, Xueyan; Shi, Kerong; Yan, Zhengui; Wang, Zhonghua

2013-01-01

Lactogenesis includes two stages. Stage I begins a few weeks before parturition. Stage II is initiated around the time of parturition and extends for several days afterwards. To better understand the molecular events underlying these changes, genome-wide gene expression profiling was conducted using digital gene expression (DGE) on bovine mammary tissue at three time points (on approximately day 35 before parturition (-35 d), day 7 before parturition (-7 d) and day 3 after parturition (+3 d)). Approximately 6.2 million (M), 5.8 million (M) and 6.1 million (M) 21-nt cDNA tags were sequenced in the three cDNA libraries (-35 d, -7 d and +3 d), respectively. After aligning to the reference sequences, the three cDNA libraries included 8,662, 8,363 and 8,359 genes, respectively. With a fold change cutoff criteria of ≥ 2 or ≤-2 and a false discovery rate (FDR) of ≤ 0.001, a total of 812 genes were significantly differentially expressed at -7 d compared with -35 d (stage I). Gene ontology analysis showed that those significantly differentially expressed genes were mainly associated with cell cycle, lipid metabolism, immune response and biological adhesion. A total of 1,189 genes were significantly differentially expressed at +3 d compared with -7 d (stage II), and these genes were mainly associated with the immune response and cell cycle. Moreover, there were 1,672 genes significantly differentially expressed at +3 d compared with -35 d. Gene ontology analysis showed that the main differentially expressed genes were those associated with metabolic processes. The results suggest that the mammary gland begins to lactate not only by a gain of function but also by a broad suppression of function to effectively push most of the cell's resources towards lactation.

Comparative proteomic study on Brassica hexaploid and its parents provides new insights into the effects of polyploidization.

PubMed

Shen, Yanyue; Zhang, Yu; Zou, Jun; Meng, Jinling; Wang, Jianbo

2015-01-01

Polyploidy has played an important role in promoting plant evolution through genomic merging and doubling. Although genomic and transcriptomic changes have been observed in polyploids, the effects of polyploidization on proteomic divergence are poorly understood. In this study, we reported quantitative analysis of proteomic changes in leaves of Brassica hexaploid and its parents using isobaric tags for relative and absolute quantitation (iTRAQ) coupled with mass spectrometry. A total of 2044 reproducible proteins were quantified by at least two unique peptides. We detected 452 proteins differentially expressed between Brassica hexaploid and its parents, and 100 proteins were non-additively expressed in Brassica hexaploid, which suggested a trend of non-additive protein regulation following genomic merger and doubling. Functional categories of cellular component biogenesis, immune system process, and response to stimulus, were significantly enriched in non-additive proteins, probably providing a driving force for variation and adaptation in allopolyploids. In particular, majority of the total 452 differentially expressed proteins showed expression level dominance of one parental expression, and there was an expression level dominance bias toward the tetraploid progenitor. In addition, the percentage of differentially expressed proteins that matched previously reported differentially genes were relatively low. This study aimed to get new insights into the effects of polyploidization on proteomic divergence. Using iTRAQ LC-MS/MS technology, we identified 452 differentially expressed proteins between allopolyploid and its parents which involved in response to stimulus, multi-organism process, and immune system process, much more than previous studies using 2-DE coupled with mass spectrometry technology. Therefore, our manuscript represents the most comprehensive analysis of protein profiles in allopolyploid and its parents, which will lead to a better understanding of novelty and plasticity of the allopolyploid genomes. Copyright © 2014 Elsevier B.V. All rights reserved.

Genome-wide Analysis of the H3K4 Histone Demethylase RBP2 Reveals a Transcriptional Program Controlling Differentiation

PubMed Central

Lopez-Bigas, Nuria; Kisiel, Tomasz A.; DeWaal, Dannielle C.; Holmes, Katie B.; Volkert, Tom L.; Gupta, Sumeet; Love, Jennifer; Murray, Heather L.; Young, Richard A.; Benevolenskaya, Elizaveta V.

2010-01-01

SUMMARY Retinoblastoma protein (pRB) mediates cell-cycle withdrawal and differentiation by interacting with a variety of proteins. RB-Binding Protein 2 (RBP2) has been shown to be a key effector. We sought to determine transcriptional regulation by RBP2 genome-wide by using location analysis and gene expression profiling experiments. We describe that RBP2 shows high correlation with the presence of H3K4me3 and its target genes are separated into two functionally distinct classes: differentiation-independent and differentiation-dependent genes. The former class is enriched by genes that encode mitochondrial proteins, while the latter is represented by cell-cycle genes. We demonstrate the role of RBP2 in mitochondrial biogenesis, which involves regulation of H3K4me3-modified nucleosomes. Analysis of expression changes upon RBP2 depletion depicted genes with a signature of differentiation control, analogous to the changes seen upon reintroduction of pRB. We conclude that, during differentiation, RBP2 exerts inhibitory effects on multiple genes through direct interaction with their promoters. PMID:18722178

Differential co-expression analysis of a microarray gene expression profiles of pulmonary adenocarcinoma.

PubMed

Fu, Shijie; Pan, Xufeng; Fang, Wentao

2014-08-01

Lung cancer severely reduces the quality of life worldwide and causes high socioeconomic burdens. However, key genes leading to the generation of pulmonary adenocarcinoma remain elusive despite intensive research efforts. The present study aimed to identify the potential associations between transcription factors (TFs) and differentially co‑expressed genes (DCGs) in the regulation of transcription in pulmonary adenocarcinoma. Gene expression profiles of pulmonary adenocarcinoma were downloaded from the Gene Expression Omnibus, and gene expression was analyzed using a computational method. A total of 37,094 differentially co‑expressed links (DCLs) and 251 DCGs were identified, which were significantly enriched in 10 pathways. The construction of the regulatory network and the analysis of the regulatory impact factors revealed eight crucial TFs in the regulatory network. These TFs regulated the expression of DCGs by promoting or inhibiting their expression. In addition, certain TFs and target genes associated with DCGs did not appear in the DCLs, which indicated that those TFs could be synergistic with other factors. This is likely to provide novel insights for research into pulmonary adenocarcinoma. In conclusion, the present study may enhance the understanding of disease mechanisms and lead to an improved diagnosis of lung cancer. However, further studies are required to confirm these observations.

Proteomic analysis of Herbaspirillum seropedicae cultivated in the presence of sugar cane extract.

PubMed

Cordeiro, Fabio Aparecido; Tadra-Sfeir, Michelle Zibetti; Huergo, Luciano Fernandes; de Oliveira Pedrosa, Fábio; Monteiro, Rose Adele; de Souza, Emanuel Maltempi

2013-03-01

Bacterial endophytes of the genus Herbaspirillum colonize sugar cane and can promote plant growth. The molecular mechanisms that mediate plant- H. seropedicae interaction are poorly understood. In this work, we used 2D-PAGE electrophoresis to identify H. seropedicae proteins differentially expressed at the log growth phase in the presence of sugar cane extract. The differentially expressed proteins were validated by RT qPCR. A total of 16 differential spots (1 exclusively expressed, 7 absent, 5 up- and 3 down-regulated) in the presence of 5% sugar cane extract were identified; thus the host extract is able to induce and repress specific genes of H. seropedicae. The differentially expressed proteins suggest that exposure to sugar cane extract induced metabolic changes and adaptations in H. seropedicae presumably in preparation to establish interaction with the plant.

Advanced glycation end product-induced astrocytic differentiation of cultured neurospheres through inhibition of Notch-Hes1 pathway-mediated neurogenesis.

PubMed

Guo, Yijing; Wang, Pin; Sun, Haixia; Cai, Rongrong; Xia, Wenqing; Wang, Shaohua

2013-12-23

This study aims to investigate the roles of the Notch-Hes1 pathway in the advanced glycation end product (AGE)-mediated differentiation of neural stem cells (NSCs). We prepared pLentiLox3.7 lentiviral vectors that express short hairpin RNA (shRNA) against Notch1 and transfected it into NSCs. Cell differentiation was analyzed under confocal laser-scanning microscopy. The percentage of neurons and astrocytes was quantified by normalizing the total number of TUJ1+ (Neuron-specific class III β-tubulin) and GFAP+ (Glial fibrillary acidic protein) cells to the total number of Hoechst 33342-labeled cell nuclei. The protein and gene expression of Notch-Hes1 pathway components was examined via western blot analysis and real-time PCR. After 1 week of incubation, we found that AGE-bovine serum albumin (BSA) (400 μg/mL) induced the astrocytic differentiation of cultured neurospheres and inhibited neuronal formation. The expression of Notch-Hes1 pathway components was upregulated in the cells in the AGE-BSA culture medium. Immunoblot analysis indicated that shRNA silencing of Notch1 expression in NSCs significantly increases neurogenesis and suppresses astrocytic differentiation in NSCs incubated with AGE-BSA. AGEs promote the astrocytic differentiation of cultured neurospheres by inhibiting neurogenesis through the Notch-Hes1 pathway, providing a potential therapeutic target for hyperglycemia-related cognitive deficits.

Isoform-level gene expression patterns in single-cell RNA-sequencing data.

PubMed

Vu, Trung Nghia; Wills, Quin F; Kalari, Krishna R; Niu, Nifang; Wang, Liewei; Pawitan, Yudi; Rantalainen, Mattias

2018-02-27

RNA sequencing of single cells enables characterization of transcriptional heterogeneity in seemingly homogeneous cell populations. Single-cell sequencing has been applied in a wide range of researches fields. However, few studies have focus on characterization of isoform-level expression patterns at the single-cell level. In this study we propose and apply a novel method, ISOform-Patterns (ISOP), based on mixture modeling, to characterize the expression patterns of isoform pairs from the same gene in single-cell isoform-level expression data. We define six principal patterns of isoform expression relationships and describe a method for differential-pattern analysis. We demonstrate ISOP through analysis of single-cell RNA-sequencing data from a breast cancer cell line, with replication in three independent datasets. We assigned the pattern types to each of 16,562 isoform-pairs from 4,929 genes. Among those, 26% of the discovered patterns were significant (p<0.05), while remaining patterns are possibly effects of transcriptional bursting, drop-out and stochastic biological heterogeneity. Furthermore, 32% of genes discovered through differential-pattern analysis were not detected by differential-expression analysis. The effect of drop-out events, mean expression level, and properties of the expression distribution on the performances of ISOP were also investigated through simulated datasets. To conclude, ISOP provides a novel approach for characterization of isoformlevel preference, commitment and heterogeneity in single-cell RNA-sequencing data. The ISOP method has been implemented as a R package and is available at https://github.com/nghiavtr/ISOP under a GPL-3 license. mattias.rantalainen@ki.se. Supplementary data are available at Bioinformatics online.

Global analysis of glycoproteins identifies markers of endotoxin tolerant monocytes and GPR84 as a modulator of TNFα expression.

PubMed

Müller, Mario M; Lehmann, Roland; Klassert, Tilman E; Reifenstein, Stella; Conrad, Theresia; Moore, Christoph; Kuhn, Anna; Behnert, Andrea; Guthke, Reinhard; Driesch, Dominik; Slevogt, Hortense

2017-04-12

Exposure of human monocytes to lipopolysaccharide (LPS) induces a temporary insensitivity to subsequent LPS challenges, a cellular state called endotoxin tolerance. In this study, we investigated the LPS-induced global glycoprotein expression changes of tolerant human monocytes and THP-1 cells to identify markers and glycoprotein targets capable to modulate the immunosuppressive state. Using hydrazide chemistry and LC-MS/MS analysis, we analyzed glycoprotein expression changes during a 48 h LPS time course. The cellular snapshots at different time points identified 1491 glycoproteins expressed by monocytes and THP-1 cells. Label-free quantitative analysis revealed transient or long-lasting LPS-induced expression changes of secreted or membrane-anchored glycoproteins derived from intracellular membrane coated organelles or from the plasma membrane. Monocytes and THP-1 cells demonstrated marked differences in glycoproteins differentially expressed in the tolerant state. Among the shared differentially expressed glycoproteins G protein-coupled receptor 84 (GPR84) was identified as being capable of modulating pro-inflammatory TNFα mRNA expression in the tolerant cell state when activated with its ligand Decanoic acid.

A Functional Genomic Meta-Analysis of Clinical Trials in Systemic Sclerosis: Toward Precision Medicine and Combination Therapy.

PubMed

Taroni, Jaclyn N; Martyanov, Viktor; Mahoney, J Matthew; Whitfield, Michael L

2017-05-01

Systemic sclerosis is an orphan, systemic autoimmune disease with no FDA-approved treatments. Its heterogeneity and rarity often result in underpowered clinical trials making the analysis and interpretation of associated molecular data challenging. We performed a meta-analysis of gene expression data from skin biopsies of patients with systemic sclerosis treated with five therapies: mycophenolate mofetil, rituximab, abatacept, nilotinib, and fresolimumab. A common clinical improvement criterion of -20% or -5 modified Rodnan skin score was applied to each study. We applied a machine learning approach that captured features beyond differential expression and was better at identifying targets of therapies than the differential expression alone. Regardless of treatment mechanism, abrogation of inflammatory pathways accompanied clinical improvement in multiple studies suggesting that high expression of immune-related genes indicates active and targetable disease. Our framework allowed us to compare different trials and ask if patients who failed one therapy would likely improve on a different therapy, based on changes in gene expression. Genes with high expression at baseline in fresolimumab nonimprovers were downregulated in mycophenolate mofetil improvers, suggesting that immunomodulatory or combination therapy may have benefitted these patients. This approach can be broadly applied to increase tissue specificity and sensitivity of differential expression results. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Overexpression of hTERT increases stem-like properties and decreases spontaneous differentiation in human mesenchymal stem cell lines

PubMed Central

2010-01-01

To overcome loss of stem-like properties and spontaneous differentiation those hinder the expansion and application of human mesenchymal stem cells (hMSCs), we have clonally isolated permanent and stable human MSC lines by ectopic overexpression of primary cell cultures of hMSCs with HPV 16 E6E7 and human telomerase reverse transcriptase (hTERT) genes. These cells were found to have a differentiation potential far beyond the ordinary hMSCs. They expressed trophoectoderm and germline specific markers upon differentiation with BMP4 and retinoic acid, respectively. Furthermore, they displayed higher osteogenic and neural differentiation efficiency than primary hMSCs or hMSCs expressed HPV16 E6E7 alone with a decrease in methylation level as proven by a global CpG island methylation profile analysis. Notably, the demethylated CpG islands were highly associated with development and differentiation associated genes. Principal component analysis further pointed out the expression profile of the cells converged toward embryonic stem cells. These data demonstrate these cells not only are a useful tool for the studies of cell differentiation both for the mesenchymal and neurogenic lineages, but also provide a valuable source of cells for cell therapy studies in animal models of skeletal and neurological disorders. PMID:20670406

An Integrated Analysis of MicroRNA and mRNA Expression Profiles to Identify RNA Expression Signatures in Lambskin Hair Follicles in Hu Sheep

PubMed Central

Lv, Xiaoyang; Sun, Wei; Yin, Jinfeng; Ni, Rong; Su, Rui; Wang, Qingzeng; Gao, Wen; Bao, Jianjun; Yu, Jiarui; Wang, Lihong; Chen, Ling

2016-01-01

Wave patterns in lambskin hair follicles are an important factor determining the quality of sheep’s wool. Hair follicles in lambskin from Hu sheep, a breed unique to China, have 3 types of waves, designated as large, medium, and small. The quality of wool from small wave follicles is excellent, while the quality of large waves is considered poor. Because no molecular and biological studies on hair follicles of these sheep have been conducted to date, the molecular mechanisms underlying the formation of different wave patterns is currently unknown. The aim of this article was to screen the candidate microRNAs (miRNA) and genes for the development of hair follicles in Hu sheep. Two-day-old Hu lambs were selected from full-sib individuals that showed large, medium, and small waves. Integrated analysis of microRNA and mRNA expression profiles employed high-throughout sequencing technology. Approximately 13, 24, and 18 differentially expressed miRNAs were found between small and large waves, small and medium waves, and medium and large waves, respectively. A total of 54, 190, and 81 differentially expressed genes were found between small and large waves, small and medium waves, and medium and large waves, respectively, by RNA sequencing (RNA-seq) analysis. Differentially expressed genes were classified using gene ontology and pathway analyses. They were found to be mainly involved in cell differentiation, proliferation, apoptosis, growth, immune response, and ion transport, and were associated with MAPK and the Notch signaling pathway. Reverse transcription-polymerase chain reaction (RT-PCR) analyses of differentially-expressed miRNA and genes were consistent with sequencing results. Integrated analysis of miRNA and mRNA expression indicated that, compared to small waves, large waves included 4 downregulated miRNAs that had regulatory effects on 8 upregulated genes and 3 upregulated miRNAs, which in turn influenced 13 downregulated genes. Compared to small waves, medium waves included 13 downregulated miRNAs that had regulatory effects on 64 upregulated genes and 4 upregulated miRNAs, which in turn had regulatory effects on 22 downregulated genes. Compared to medium waves, large waves consisted of 13 upregulated miRNAs that had regulatory effects on 48 downregulated genes. These differentially expressed miRNAs and genes may play a significant role in forming different patterns, and provide evidence for the molecular mechanisms underlying the formation of hair follicles of varying patterns. PMID:27404636

Altered Pathway Analyzer: A gene expression dataset analysis tool for identification and prioritization of differentially regulated and network rewired pathways

PubMed Central

Kaushik, Abhinav; Ali, Shakir; Gupta, Dinesh

2017-01-01

Gene connection rewiring is an essential feature of gene network dynamics. Apart from its normal functional role, it may also lead to dysregulated functional states by disturbing pathway homeostasis. Very few computational tools measure rewiring within gene co-expression and its corresponding regulatory networks in order to identify and prioritize altered pathways which may or may not be differentially regulated. We have developed Altered Pathway Analyzer (APA), a microarray dataset analysis tool for identification and prioritization of altered pathways, including those which are differentially regulated by TFs, by quantifying rewired sub-network topology. Moreover, APA also helps in re-prioritization of APA shortlisted altered pathways enriched with context-specific genes. We performed APA analysis of simulated datasets and p53 status NCI-60 cell line microarray data to demonstrate potential of APA for identification of several case-specific altered pathways. APA analysis reveals several altered pathways not detected by other tools evaluated by us. APA analysis of unrelated prostate cancer datasets identifies sample-specific as well as conserved altered biological processes, mainly associated with lipid metabolism, cellular differentiation and proliferation. APA is designed as a cross platform tool which may be transparently customized to perform pathway analysis in different gene expression datasets. APA is freely available at http://bioinfo.icgeb.res.in/APA. PMID:28084397

Discovering high-resolution patterns of differential DNA methylation that correlate with gene expression changes

PubMed Central

VanderKraats, Nathan D.; Hiken, Jeffrey F.; Decker, Keith F.; Edwards, John R.

2013-01-01

Methylation of the CpG-rich region (CpG island) overlapping a gene’s promoter is a generally accepted mechanism for silencing expression. While recent technological advances have enabled measurement of DNA methylation and expression changes genome-wide, only modest correlations between differential methylation at gene promoters and expression have been found. We hypothesize that stronger associations are not observed because existing analysis methods oversimplify their representation of the data and do not capture the diversity of existing methylation patterns. Recently, other patterns such as CpG island shore methylation and long partially hypomethylated domains have also been linked with gene silencing. Here, we detail a new approach for discovering differential methylation patterns associated with expression change using genome-wide high-resolution methylation data: we represent differential methylation as an interpolated curve, or signature, and then identify groups of genes with similarly shaped signatures and corresponding expression changes. Our technique uncovers a diverse set of patterns that are conserved across embryonic stem cell and cancer data sets. Overall, we find strong associations between these methylation patterns and expression. We further show that an extension of our method also outperforms other approaches by generating a longer list of genes with higher quality associations between differential methylation and expression. PMID:23748561

Proteomic analysis of acute responses to copper sulfate stress in larvae of the brine shrimp, Artemia sinica

NASA Astrophysics Data System (ADS)

Zhou, Qian; Wu, Changgong; Dong, Bo; Li, Fuhua; Liu, Fengqi; Xiang, Jianhai

2010-03-01

Proteomics was used to reveal the differential protein expression profiles of acute responses to copper sulfate exposure in larvae of Artemia sinica. Fourteen differentially displayed protein spots were detected and seven of them were identified. Three spots were up-expressed and identified: actin, heat shock protein 70, and chaperone subunit 1; three down-regulated proteins were identified: arginine kinase, elongation factor-2, and glycine-rich protein; and a newly expressed protein was identified as peroxiredoxin. The study indicates the involvement of all the differentially expressed proteins in the early responses of protein expression, and in the survival of A. sinica in the presence of copper and other heavy metals; the findings improve understanding of the organism’s adaptive responses and resistance.

Differentially-Expressed Pseudogenes in HIV-1 Infection

PubMed Central

Gupta, Aditi; Brown, C. Titus; Zheng, Yong-Hui; Adami, Christoph

2015-01-01

Not all pseudogenes are transcriptionally silent as previously thought. Pseudogene transcripts, although not translated, contribute to the non-coding RNA pool of the cell that regulates the expression of other genes. Pseudogene transcripts can also directly compete with the parent gene transcripts for mRNA stability and other cell factors, modulating their expression levels. Tissue-specific and cancer-specific differential expression of these “functional” pseudogenes has been reported. To ascertain potential pseudogene:gene interactions in HIV-1 infection, we analyzed transcriptomes from infected and uninfected T-cells and found that 21 pseudogenes are differentially expressed in HIV-1 infection. This is interesting because parent genes of one-third of these differentially-expressed pseudogenes are implicated in HIV-1 life cycle, and parent genes of half of these pseudogenes are involved in different viral infections. Our bioinformatics analysis identifies candidate pseudogene:gene interactions that may be of significance in HIV-1 infection. Experimental validation of these interactions would establish that retroviruses exploit this newly-discovered layer of host gene expression regulation for their own benefit. PMID:26426037

Suppression subtractive hybridization identified differentially expressed genes in lung adenocarcinoma: ERGIC3 as a novel lung cancer-related gene

PubMed Central

2013-01-01

Background To understand the carcinogenesis caused by accumulated genetic and epigenetic alterations and seek novel biomarkers for various cancers, studying differentially expressed genes between cancerous and normal tissues is crucial. In the study, two cDNA libraries of lung cancer were constructed and screened for identification of differentially expressed genes. Methods Two cDNA libraries of differentially expressed genes were constructed using lung adenocarcinoma tissue and adjacent nonmalignant lung tissue by suppression subtractive hybridization. The data of the cDNA libraries were then analyzed and compared using bioinformatics analysis. Levels of mRNA and protein were measured by quantitative real-time polymerase chain reaction (q-RT-PCR) and western blot respectively, as well as expression and localization of proteins were determined by immunostaining. Gene functions were investigated using proliferation and migration assays after gene silencing and gene over-expression. Results Two libraries of differentially expressed genes were obtained. The forward-subtracted library (FSL) and the reverse-subtracted library (RSL) contained 177 and 59 genes, respectively. Bioinformatic analysis demonstrated that these genes were involved in a wide range of cellular functions. The vast majority of these genes were newly identified to be abnormally expressed in lung cancer. In the first stage of the screening for 16 genes, we compared lung cancer tissues with their adjacent non-malignant tissues at the mRNA level, and found six genes (ERGIC3, DDR1, HSP90B1, SDC1, RPSA, and LPCAT1) from the FSL were significantly up-regulated while two genes (GPX3 and TIMP3) from the RSL were significantly down-regulated (P < 0.05). The ERGIC3 protein was also over-expressed in lung cancer tissues and cultured cells, and expression of ERGIC3 was correlated with the differentiated degree and histological type of lung cancer. The up-regulation of ERGIC3 could promote cellular migration and proliferation in vitro. Conclusions The two libraries of differentially expressed genes may provide the basis for new insights or clues for finding novel lung cancer-related genes; several genes were newly found in lung cancer with ERGIC3 seeming a novel lung cancer-related gene. ERGIC3 may play an active role in the development and progression of lung cancer. PMID:23374247

Pairwise comparisons of ten porcine tissues identify differential transcriptional regulation at the gene, isoform, promoter and transcription start site level

DOE Office of Scientific and Technical Information (OSTI.GOV)

Farajzadeh, Leila; Hornshøj, Henrik; Momeni, Jamal

Highlights: •Transcriptome sequencing yielded 223 mill porcine RNA-seq reads, and 59,000 transcribed locations. •Establishment of unique transcription profiles for ten porcine tissues including four brain tissues. •Comparison of transcription profiles at gene, isoform, promoter and transcription start site level. •Highlights a high level of regulation of neuro-related genes at both gene, isoform, and TSS level. •Our results emphasize the pig as a valuable animal model with respect to human biological issues. -- Abstract: The transcriptome is the absolute set of transcripts in a tissue or cell at the time of sampling. In this study RNA-Seq is employed to enable themore » differential analysis of the transcriptome profile for ten porcine tissues in order to evaluate differences between the tissues at the gene and isoform expression level, together with an analysis of variation in transcription start sites, promoter usage, and splicing. Totally, 223 million RNA fragments were sequenced leading to the identification of 59,930 transcribed gene locations and 290,936 transcript variants using Cufflinks with similarity to approximately 13,899 annotated human genes. Pairwise analysis of tissues for differential expression at the gene level showed that the smallest differences were between tissues originating from the porcine brain. Interestingly, the relative level of differential expression at the isoform level did generally not vary between tissue contrasts. Furthermore, analysis of differential promoter usage between tissues, revealed a proportionally higher variation between cerebellum (CBE) versus frontal cortex and cerebellum versus hypothalamus (HYP) than in the remaining comparisons. In addition, the comparison of differential transcription start sites showed that the number of these sites is generally increased in comparisons including hypothalamus in contrast to other pairwise assessments. A comprehensive analysis of one of the tissue contrasts, i.e. cerebellum versus heart for differential variation at the gene, isoform, and transcription start site (TSS), and promoter level showed that several of the genes differed at all four levels. Interestingly, these genes were mainly annotated to the “electron transport chain” and neuronal differentiation, emphasizing that “tissue important” genes are regulated at several levels. Furthermore, our analysis shows that the “across tissue approach” has a promising potential when screening for possible explanations for variations, such as those observed at the gene expression levels.« less

A comprehensive comparison of RNA-Seq-based transcriptome analysis from reads to differential gene expression and cross-comparison with microarrays: a case study in Saccharomyces cerevisiae

PubMed Central

Nookaew, Intawat; Papini, Marta; Pornputtapong, Natapol; Scalcinati, Gionata; Fagerberg, Linn; Uhlén, Matthias; Nielsen, Jens

2012-01-01

RNA-seq, has recently become an attractive method of choice in the studies of transcriptomes, promising several advantages compared with microarrays. In this study, we sought to assess the contribution of the different analytical steps involved in the analysis of RNA-seq data generated with the Illumina platform, and to perform a cross-platform comparison based on the results obtained through Affymetrix microarray. As a case study for our work we, used the Saccharomyces cerevisiae strain CEN.PK 113-7D, grown under two different conditions (batch and chemostat). Here, we asses the influence of genetic variation on the estimation of gene expression level using three different aligners for read-mapping (Gsnap, Stampy and TopHat) on S288c genome, the capabilities of five different statistical methods to detect differential gene expression (baySeq, Cuffdiff, DESeq, edgeR and NOISeq) and we explored the consistency between RNA-seq analysis using reference genome and de novo assembly approach. High reproducibility among biological replicates (correlation ≥0.99) and high consistency between the two platforms for analysis of gene expression levels (correlation ≥0.91) are reported. The results from differential gene expression identification derived from the different statistical methods, as well as their integrated analysis results based on gene ontology annotation are in good agreement. Overall, our study provides a useful and comprehensive comparison between the two platforms (RNA-seq and microrrays) for gene expression analysis and addresses the contribution of the different steps involved in the analysis of RNA-seq data. PMID:22965124

Parallel human genome analysis: microarray-based expression monitoring of 1000 genes.

PubMed Central

Schena, M; Shalon, D; Heller, R; Chai, A; Brown, P O; Davis, R W

1996-01-01

Microarrays containing 1046 human cDNAs of unknown sequence were printed on glass with high-speed robotics. These 1.0-cm2 DNA "chips" were used to quantitatively monitor differential expression of the cognate human genes using a highly sensitive two-color hybridization assay. Array elements that displayed differential expression patterns under given experimental conditions were characterized by sequencing. The identification of known and novel heat shock and phorbol ester-regulated genes in human T cells demonstrates the sensitivity of the assay. Parallel gene analysis with microarrays provides a rapid and efficient method for large-scale human gene discovery. Images Fig. 1 Fig. 2 Fig. 3 PMID:8855227

Identification of differentially expressed microRNA in the stems and leaves during sugar accumulation in sweet sorghum.

PubMed

Yu, Huilin; Cong, Ling; Zhu, Zhenxing; Wang, Chunyu; Zou, Jianqiu; Tao, Chengguang; Shi, Zhensheng; Lu, Xiaochun

2015-10-25

MicroRNAs (miRNAs) have been shown to play important roles in plant development, growth and stress response. Sweet sorghum [Sorghum bicolor (L.) Moench] is an important source of bioenergy due to the high sugar content in its stems. However, it is not clear how the miRNA is involved in sugar accumulation in sorghum stems. In order to identify the miRNAs in the stems and the leaves of sweet sorghum, we extracted RNAs of the stems and leaves of sweet sorghum (Rio) and grain sorghum (BTx623) at the heading and dough stages for high-throughput sequencing. A total of 179279048 reads were obtained from Illumina-based sequencing. Further analysis identified nine known miRNAs and twelve novel miRNAs that showed significantly and specifically differentially expressed in the stems of sweet sorghum. The target genes of the differentially expressed novel miRNAs include the transcription factor, glucosyltransferase, protein kinase, cytochrome P450, transporters etc. GO enrichment analysis showed that the predicted targets of these differentially expressed miRNAs participated in diverse physiological and metabolic processes. We performed RT-qRCR analysis on these miRNAs across eight different libraries to validate the miRNAs. Finally, we screened stem-specifically expressed novel miRNA and a leaf-specifically expressed novel miRNA in sweet sorghum comparing with grain sorghum. Our results provide a basis for further investigation of the potential role of these individual miRNAs in sugar accumulation. Copyright © 2015 Elsevier B.V. All rights reserved.

Dissecting Embryonic Stem Cell Self-Renewal and Differentiation Commitment from Quantitative Models.

PubMed

Hu, Rong; Dai, Xianhua; Dai, Zhiming; Xiang, Qian; Cai, Yanning

2016-10-01

To model quantitatively embryonic stem cell (ESC) self-renewal and differentiation by computational approaches, we developed a unified mathematical model for gene expression involved in cell fate choices. Our quantitative model comprised ESC master regulators and lineage-specific pivotal genes. It took the factors of multiple pathways as input and computed expression as a function of intrinsic transcription factors, extrinsic cues, epigenetic modifications, and antagonism between ESC master regulators and lineage-specific pivotal genes. In the model, the differential equations of expression of genes involved in cell fate choices from regulation relationship were established according to the transcription and degradation rates. We applied this model to the Murine ESC self-renewal and differentiation commitment and found that it modeled the expression patterns with good accuracy. Our model analysis revealed that Murine ESC was an attractor state in culture and differentiation was predominantly caused by antagonism between ESC master regulators and lineage-specific pivotal genes. Moreover, antagonism among lineages played a critical role in lineage reprogramming. Our results also uncovered that the ordered expression alteration of ESC master regulators over time had a central role in ESC differentiation fates. Our computational framework was generally applicable to most cell-type maintenance and lineage reprogramming.

Identification of differential pathways in papillary thyroid carcinoma utilizing pathway co-expression analysis.

PubMed

Qiu, Wei-Hai; Chen, Gui-Yan; Cui, Lu; Zhang, Ting-Ming; Wei, Feng; Yang, Yong

2016-01-01

To identify differential pathways between papillary thyroid carcinoma (PTC) patients and normal controls utilizing a novel method which combined pathway with co-expression network. The proposed method included three steps. In the first step, we conducted pretreatments for background pathways and gained representative pathways in PTC. Subsequently, a co-expression network for representative pathways was constructed using empirical Bayes (EB) approach to assign a weight value for each pathway. Finally, random model was extracted to set the thresholds of identifying differential pathways. We obtained 1267 representative pathways and their weight values based on the co-expressed pathway network, and then by meeting the criterion (Weight > 0.0296), 87 differential pathways in total across PTC patients and normal controls were identified. The top three ranked differential pathways were CREB phosphorylation, attachment of GPI anchor to urokinase plasminogen activator receptor (uPAR) and loss of function of SMAD2/3 in cancer. In conclusion, we successfully identified differential pathways (such as CREB phosphorylation, attachment of GPI anchor to uPAR and post-translational modification: synthesis of GPI-anchored proteins) for PTC using the proposed pathway co-expression method, and these pathways might be potential biomarkers for target therapy and detection of PTC.

[Differential expression genes of bone tissues surrounding implants in diabetic rats by gene chip].

PubMed

Wang, Xin-xin; Ma, Yue; Li, Qing; Jiang, Bao-qi; Lan, Jing

2012-10-01

To compare mRNA expression profiles of bone tissues surrounding implants between normal rats and rats with diabetes using microarray technology. Six Wistar rats were randomly selected and divided into normal model group and diabetic group. Diabetic model condition was established by injecting Streptozotocin into peritoneal space. Titanium implants were implanted into the epiphyseal end of the rats' tibia. Bone tissues surrounding implant were harvested and sampled after 3 months to perform comprehensive RNA gene expression profiling, including 17983 for genome-wide association study.GO analysis was used to compare different gene expression and real-time PCR was used to confirm the results on core samples. The results indicated that there were 1084 differential gene expression. In the diabetic model, there were 352 enhanced expression genes, 732 suppressed expression genes. GO analysis involved 1154 different functional type. Osteoblast related gene expressions in bone tissue samples of diabetic rats were decreased, and lipid metabolism pathway related gene expression was increased.

Dynamic Organization of lncRNA and Circular RNA Regulators Collectively Controlled Cardiac Differentiation in Humans.

PubMed

Li, Yongsheng; Zhang, Jinwen; Huo, Caiqin; Ding, Na; Li, Junyi; Xiao, Jun; Lin, Xiaoyu; Cai, Benzhi; Zhang, Yunpeng; Xu, Juan

2017-10-01

Advances in developmental cardiology have increased our understanding of the early aspects of heart differentiation. However, understanding noncoding RNA (ncRNA) transcription and regulation during this process remains elusive. Here, we constructed transcriptomes for both long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) in four important developmental stages ranging from early embryonic to cardiomyocyte based on high-throughput sequencing datasets, which indicate the high stage-specific expression patterns of two ncRNA types. Additionally, higher similarities of samples within each stage were found, highlighting the divergence of samples collected from distinct cardiac developmental stages. Next, we developed a method to identify numerous lncRNA and circRNA regulators whose expression was significantly stage-specific and shifted gradually and continuously during heart differentiation. We inferred that these ncRNAs are important for the stages of cardiac differentiation. Moreover, transcriptional regulation analysis revealed that the expression of stage-specific lncRNAs is controlled by known key stage-specific transcription factors (TFs). In addition, circRNAs exhibited dynamic expression patterns independent from their host genes. Functional enrichment analysis revealed that lncRNAs and circRNAs play critical roles in pathways that are activated specifically during heart differentiation. We further identified candidate TF-ncRNA-gene network modules for each differentiation stage, suggesting the dynamic organization of lncRNAs and circRNAs collectively controlled cardiac differentiation, which may cause heart-related diseases when defective. Our study provides a foundation for understanding the dynamic regulation of ncRNA transcriptomes during heart differentiation and identifies the dynamic organization of novel key lncRNAs and circRNAs to collectively control cardiac differentiation. Copyright © 2017. Published by Elsevier B.V.

Serial analysis of gene expression reveals differential expression between endometriosis and normal endometrium. Possible roles for AXL and SHC1 in the pathogenesis of endometriosis

PubMed Central

Honda, Hiroshi; Barrueto, Fermin F; Gogusev, Jean; Im, Dwight D; Morin, Patrice J

2008-01-01

Background Endometriosis is a clinical condition that affects up to 10% of the women of reproductive age. Endometriosis is characterized by the presence of endometrial tissues outside the uterine cavity and can lead to chronic pelvic pain, infertility and, in some cases, to ovarian cancer. Methods In order to better understand the pathogenesis of endometriosis, we have used Serial Analysis of Gene Expression (SAGE) to identify genes differentially in this disease by studying three endometriotic tissues and a normal endometrium sample. Promising candidates (AXL, SHC1, ACTN4, PI3KCA, p-AKT, p-mTOR, and p-ERK) were independently validated by immunohistochemistry in additional normal and endometriotic tissues. Results We identified several genes differentially expressed between endometriosis and normal endometrium. IGF2, ACTN4, AXL, and SHC1 were among the most upregulated genes. Comparison of the endometriosis gene expression profiles with the gene expression patterns observed in normal human tissues allowed the identification of endometriosis-specific genes, which included several members of the MMP family (MMP1,2,3,10,11,14). Immunohistochemical analysis of several candidates confirmed the SAGE findings, and suggested the involvement of the PI3K-Akt and MAPK signaling pathways in endometriosis. Conclusion In human endometriosis, the PI3K-Akt and MAPK signaling pathways may be activated via overexpression of AXL and SHC1, respectively. These genes, as well as others identified as differentially expressed in this study, may be useful for the development of novel strategies for the detection and/or therapy of endometriosis. PMID:19055724

Toward an Understanding of Divergent Compound Eye Development in Drones and Workers of the Honeybee (Apis mellifera L.): A Correlative Analysis of Morphology and Gene Expression.

PubMed

Marco Antonio, David S; Hartfelder, Klaus

2017-01-01

Eye development in insects is best understood in Drosophila melanogaster, but little is known for other holometabolous insects. Combining a morphological with a gene expression analysis, we investigated eye development in the honeybee, putting emphasis on the sex-specific differences in eye size. Optic lobe development starts from an optic lobe anlage in the larval brain, which sequentially gives rise to the lobula, medulla, and lamina. The lamina differentiates in the last larval instar, when it receives optic nerve projections from the developing retina. The expression analysis focused on seven genes important for Drosophila eye development: eyes absent, sine oculis, embryonic lethal abnormal vision, minibrain, small optic lobes, epidermal growth factor receptor, and roughest. All except small optic lobes were more highly expressed in third-instar drone larvae, but then, in the fourth and fifth instar, their expression was sex-specifically modulated, showing shifts in temporal dynamics. The clearest differences were seen for small optic lobes, which is highly expressed in the developing eye of workers, and minibrain and roughest, which showed a strong expression peak coinciding with retina differentiation. A microarray analysis for optic lobe/retina complexes revealed the differential expression of several metabolism-related genes, as well as of two micro-RNAs. While we could not see major morphological differences in the developing eye structures before the pupal stage, the expression differences observed for the seven candidate genes and in the transcriptional microarray profiles indicate that molecular signatures underlying sex-specific optic lobe and retina development become established throughout the larval stages. © 2016 Wiley Periodicals, Inc.

Releasing Ski-Smad4 mediated suppression is essential to license Th17 differentiation

PubMed Central

Zhang, Song; Takaku, Motoki; Zou, Liyun; Gu, Ai-di; Chou, Wei-chun; Zhang, Ge; Wu, Bing; Kong, Qing; Thomas, Seddon Y.; Serody, Jonathan S.; Chen, Xian; Xu, Xiaojiang; Wade, Paul A.; Cook, Donald N.; Ting, Jenny P.; Wan, Yisong Y.

2017-01-01

Th17 cells are critically involved in host defense, inflammation, and autoimmunity1–5. TGF-β is instrumental in Th17 differentiation by cooperating with IL-66,7. Yet, the mechanism of how TGF-β enables Th17 differentiation remains elusive. Here we reveal that TGF-β licenses Th17 differentiation by releasing Ski-Smad4-complex suppressed RORγt expression. We found serendipitously that, unlike wild-type T cells, Smad4-deficient T cells differentiated into Th17 cells in the absence of TGF-β signaling in a RORγt-dependent manner. Ectopic Smad4 expression suppressed the RORγt expression and Th17 differentiation of Smad4-deficient T cells. Unexpectedly however, TGF-β neutralized Smad4 mediated suppression without affecting Smad4 binding to Rorc locus. Proteomic analysis revealed that Smad4 interacted with Ski, a transcriptional repressor degraded upon TGF-β stimulation. Ski controlled the histone acetylation/de-acetylation of Rorc locus and Th17 differentiation via Smad4 because ectopic Ski expression inhibited H3K9Ac of Rorc locus, Rorc expression and Th17 differentiation in a Smad4-dependent manner. Therefore, TGF-β-induced disruption of Ski releases Ski-Smad4 complex imposed suppression of RORγt to license Th17 differentiation. This study reveals a critical mechanism by which TGF-β controls Th17 differentiation and uncovers Ski-Smad4 axis as a potential therapeutic target for treating Th17 related diseases. PMID:29072299

Transcriptome interrogation of human myometrium identifies differentially expressed sense-antisense pairs of protein-coding and long non-coding RNA genes in spontaneous labor at term.

PubMed

Romero, Roberto; Tarca, Adi L; Chaemsaithong, Piya; Miranda, Jezid; Chaiworapongsa, Tinnakorn; Jia, Hui; Hassan, Sonia S; Kalita, Cynthia A; Cai, Juan; Yeo, Lami; Lipovich, Leonard

2014-09-01

To identify differentially expressed long non-coding RNA (lncRNA) genes in human myometrium in women with spontaneous labor at term. Myometrium was obtained from women undergoing cesarean deliveries who were not in labor (n = 19) and women in spontaneous labor at term (n = 20). RNA was extracted and profiled using an Illumina® microarray platform. We have used computational approaches to bound the extent of long non-coding RNA representation on this platform, and to identify co-differentially expressed and correlated pairs of long non-coding RNA genes and protein-coding genes sharing the same genomic loci. We identified co-differential expression and correlation at two genomic loci that contain coding-lncRNA gene pairs: SOCS2-AK054607 and LMCD1-NR_024065 in women in spontaneous labor at term. This co-differential expression and correlation was validated by qRT-PCR, an experimental method completely independent of the microarray analysis. Intriguingly, one of the two lncRNA genes differentially expressed in term labor had a key genomic structure element, a splice site, that lacked evolutionary conservation beyond primates. We provide, for the first time, evidence for coordinated differential expression and correlation of cis-encoded antisense lncRNAs and protein-coding genes with known as well as novel roles in pregnancy in the myometrium of women in spontaneous labor at term.

De novo transcriptome assembly and analysis of differential gene expression in response to drought in European beech

PubMed Central

Seifert, Sarah; Lübbe, Torben; Leuschner, Christoph; Finkeldey, Reiner

2017-01-01

Despite the ecological and economic importance of European beech (Fagus sylvatica L.) genomic resources of this species are still limited. This hampers an understanding of the molecular basis of adaptation to stress. Since beech will most likely be threatened by the consequences of climate change, an understanding of adaptive processes to climate change-related drought stress is of major importance. Here, we used RNA-seq to provide the first drought stress-related transcriptome of beech. In a drought stress trial with beech saplings, 50 samples were taken for RNA extraction at five points in time during a soil desiccation experiment. De novo transcriptome assembly and analysis of differential gene expression revealed 44,335 contigs, and 662 differentially expressed genes between the stress and normally watered control group. Gene expression was specific to the different time points, and only five genes were significantly differentially expressed between the stress and control group on all five sampling days. GO term enrichment showed that mostly genes involved in lipid- and homeostasis-related processes were upregulated, whereas genes involved in oxidative stress response were downregulated in the stressed seedlings. This study gives first insights into the genomic drought stress response of European beech, and provides new genetic resources for adaptation research in this species. PMID:28873454

Proteomic and Transcriptomic Analysis of Aspergillus fumigatus on Exposure to Amphotericin B▿ †

PubMed Central

Gautam, Poonam; Shankar, Jata; Madan, Taruna; Sirdeshmukh, Ravi; Sundaram, Curam Sreenivasacharlu; Gade, Wasudev Namdeo; Basir, Seemi Farhat; Sarma, Puranam Usha

2008-01-01

Amphotericin B (AMB) is the most widely used polyene antifungal drug for the treatment of systemic fungal infections, including invasive aspergillosis. It has been our aim to understand the molecular targets of AMB in Aspergillus fumigatus by genomic and proteomic approaches. In transcriptomic analysis, a total of 295 genes were found to be differentially expressed (165 upregulated and 130 downregulated), including many involving the ergosterol pathway, cell stress proteins, cell wall proteins, transport proteins, and hypothetical proteins. Proteomic profiles of A. fumigatus alone or A. fumigatus treated with AMB showed differential expression levels for 85 proteins (76 upregulated and 9 downregulated). Forty-eight of them were identified with high confidence and belonged to the above-mentioned categories. Differential expression levels for Rho-GDP dissociation inhibitor (Rho-GDI), secretory-pathway GDI, clathrin, Sec 31 (a subunit of the exocyst complex), and RAB GTPase Ypt51 in response to an antifungal drug are reported here for the first time and may represent a specific response of A. fumigatus to AMB. The expression of some of these genes was validated by real-time reverse transcription-PCR. The AMB responsive genes/proteins observed to be differentially expressed in A. fumigatus may be further explored for novel drug development. PMID:18838595

Proteomic and transcriptomic analysis of Aspergillus fumigatus on exposure to amphotericin B.

PubMed

Gautam, Poonam; Shankar, Jata; Madan, Taruna; Sirdeshmukh, Ravi; Sundaram, Curam Sreenivasacharlu; Gade, Wasudev Namdeo; Basir, Seemi Farhat; Sarma, Puranam Usha

2008-12-01

Amphotericin B (AMB) is the most widely used polyene antifungal drug for the treatment of systemic fungal infections, including invasive aspergillosis. It has been our aim to understand the molecular targets of AMB in Aspergillus fumigatus by genomic and proteomic approaches. In transcriptomic analysis, a total of 295 genes were found to be differentially expressed (165 upregulated and 130 downregulated), including many involving the ergosterol pathway, cell stress proteins, cell wall proteins, transport proteins, and hypothetical proteins. Proteomic profiles of A. fumigatus alone or A. fumigatus treated with AMB showed differential expression levels for 85 proteins (76 upregulated and 9 downregulated). Forty-eight of them were identified with high confidence and belonged to the above-mentioned categories. Differential expression levels for Rho-GDP dissociation inhibitor (Rho-GDI), secretory-pathway GDI, clathrin, Sec 31 (a subunit of the exocyst complex), and RAB GTPase Ypt51 in response to an antifungal drug are reported here for the first time and may represent a specific response of A. fumigatus to AMB. The expression of some of these genes was validated by real-time reverse transcription-PCR. The AMB responsive genes/proteins observed to be differentially expressed in A. fumigatus may be further explored for novel drug development.

New Statistics for Testing Differential Expression of Pathways from Microarray Data

NASA Astrophysics Data System (ADS)

Siu, Hoicheong; Dong, Hua; Jin, Li; Xiong, Momiao

Exploring biological meaning from microarray data is very important but remains a great challenge. Here, we developed three new statistics: linear combination test, quadratic test and de-correlation test to identify differentially expressed pathways from gene expression profile. We apply our statistics to two rheumatoid arthritis datasets. Notably, our results reveal three significant pathways and 275 genes in common in two datasets. The pathways we found are meaningful to uncover the disease mechanisms of rheumatoid arthritis, which implies that our statistics are a powerful tool in functional analysis of gene expression data.

Synchrotron radiation based STXM analysis and micro-XRF mapping of differential expression of extracellular thiol groups by Acidithiobacillus ferrooxidans grown on Fe(2+) and S(0).

PubMed

Xia, Jin-Lan; Liu, Hong-Chang; Nie, Zhen-Yuan; Peng, An-An; Zhen, Xiang-Jun; Yang, Yun; Zhang, Xiu-Li

2013-09-01

The differential expression of extracellular thiol groups by Acidithiobacillus ferrooxidans grown on substrates Fe(2+) and S(0) was investigated by using synchrotron radiation based scanning transmission X-ray microscopy (STXM) imaging and microbeam X-ray fluorescence (μ-XRF) mapping. The extracellular thiol groups (SH) were first alkylated by iodoacetic acid forming Protein-SCH2COOH and then the P-SCH2COOH was marked by calcium ions forming P-SCH2COOCa. The STXM imaging and μ-XRF mapping of SH were based on analysis of SCH2COO-bonded Ca(2+). The results indicated that the thiol group content of A. ferrooxidans grown on S(0) is 3.88 times to that on Fe(2+). Combined with selective labeling of SH by Ca(2+), the STXM imaging and μ-XRF mapping provided an in situ and rapid analysis of differential expression of extracellular thiol groups. © 2013.

Comparison of software packages for detecting differential expression in RNA-seq studies

PubMed Central

Seyednasrollah, Fatemeh; Laiho, Asta

2015-01-01

RNA-sequencing (RNA-seq) has rapidly become a popular tool to characterize transcriptomes. A fundamental research problem in many RNA-seq studies is the identification of reliable molecular markers that show differential expression between distinct sample groups. Together with the growing popularity of RNA-seq, a number of data analysis methods and pipelines have already been developed for this task. Currently, however, there is no clear consensus about the best practices yet, which makes the choice of an appropriate method a daunting task especially for a basic user without a strong statistical or computational background. To assist the choice, we perform here a systematic comparison of eight widely used software packages and pipelines for detecting differential expression between sample groups in a practical research setting and provide general guidelines for choosing a robust pipeline. In general, our results demonstrate how the data analysis tool utilized can markedly affect the outcome of the data analysis, highlighting the importance of this choice. PMID:24300110

Comparison of software packages for detecting differential expression in RNA-seq studies.

PubMed

Seyednasrollah, Fatemeh; Laiho, Asta; Elo, Laura L

2015-01-01

RNA-sequencing (RNA-seq) has rapidly become a popular tool to characterize transcriptomes. A fundamental research problem in many RNA-seq studies is the identification of reliable molecular markers that show differential expression between distinct sample groups. Together with the growing popularity of RNA-seq, a number of data analysis methods and pipelines have already been developed for this task. Currently, however, there is no clear consensus about the best practices yet, which makes the choice of an appropriate method a daunting task especially for a basic user without a strong statistical or computational background. To assist the choice, we perform here a systematic comparison of eight widely used software packages and pipelines for detecting differential expression between sample groups in a practical research setting and provide general guidelines for choosing a robust pipeline. In general, our results demonstrate how the data analysis tool utilized can markedly affect the outcome of the data analysis, highlighting the importance of this choice. © The Author 2013. Published by Oxford University Press.

miRNAome expression profiles in the gonads of adult Melopsittacus undulatus

PubMed Central

Jiang, Lan; Wang, Qingqing; Yu, Jue; Gowda, Vinita; Johnson, Gabriel; Yang, Jianke

2018-01-01

The budgerigar (Melopsittacus undulatus) is one of the most widely studied parrot species, serving as an excellent animal model for behavior and neuroscience research. Until recently, it was unknown how sexual differences in the behavior, physiology, and development of organisms are regulated by differential gene expression. MicroRNAs (miRNAs) are endogenous short non-coding RNA molecules that can post-transcriptionally regulate gene expression and play a critical role in gonadal differentiation as well as early development of animals. However, very little is known about the role gonadal miRNAs play in the early development of birds. Research on the sex-biased expression of miRNAs in avian gonads are limited, and little is known about M. undulatus. In the current study, we sequenced two small non-coding RNA libraries made from the gonads of adult male and female budgerigars using Illumina paired-end sequencing technology. We obtained 254 known and 141 novel miRNAs, and randomly validated five miRNAs. Of these, three miRNAs were differentially expressed miRNAs and 18 miRNAs involved in sexual differentiation as determined by functional analysis with GO annotation and KEGG pathway analysis. In conclusion, this work is the first report of sex-biased miRNAs expression in the budgerigar, and provides additional sequences to the avian miRNAome database which will foster further functional genomic research. PMID:29666766

Transcriptional profiling of host gene expression in chicken embryo lung cells infected with laryngotracheitis virus

PubMed Central

2010-01-01

Background Infection by infectious laryngotracheitis virus (ILTV; gallid herpesvirus 1) causes acute respiratory diseases in chickens often with high mortality. To better understand host-ILTV interactions at the host transcriptional level, a microarray analysis was performed using 4 × 44 K Agilent chicken custom oligo microarrays. Results Microarrays were hybridized using the two color hybridization method with total RNA extracted from ILTV infected chicken embryo lung cells at 0, 1, 3, 5, and 7 days post infection (dpi). Results showed that 789 genes were differentially expressed in response to ILTV infection that include genes involved in the immune system (cytokines, chemokines, MHC, and NF-κB), cell cycle regulation (cyclin B2, CDK1, and CKI3), matrix metalloproteinases (MMPs) and cellular metabolism. Differential expression for 20 out of 789 genes were confirmed by quantitative reverse transcription-PCR (qRT-PCR). A bioinformatics tool (Ingenuity Pathway Analysis) used to analyze biological functions and pathways on the group of 789 differentially expressed genes revealed that 21 possible gene networks with intermolecular connections among 275 functionally identified genes. These 275 genes were classified into a number of functional groups that included cancer, genetic disorder, cellular growth and proliferation, and cell death. Conclusion The results of this study provide comprehensive knowledge on global gene expression, and biological functionalities of differentially expressed genes in chicken embryo lung cells in response to ILTV infections. PMID:20663125

Transcriptome analysis of PCOS arrested 2-cell embryos.

PubMed

Lu, Cuiling; Chi, Hongbin; Wang, Yapeng; Feng, Xue; Wang, Lina; Huang, Shuo; Yan, Liying; Lin, Shengli; Liu, Ping; Qiao, Jie

2018-06-18

In an attempt to explore the early developmental arrest in embryos from polycystic ovarian syndrome (PCOS) patients, we sequenced the transcriptome profiles of PCOS arrested 2-cell embryos, non-PCOS arrested 2-cell embryos and non-arrested 2-cell embryos using single-cell RNA-Seq technique. Differential expression analysis was performed using the DEGSeq R package. Gene Ontology (GO) enrichment was analyzed using the GOseq R package. Data revealed 62 differentially expressed genes between non-PCOS arrested and PCOS arrested embryos and 2217 differentially expressed genes between PCOS arrested and non-arrested 2-cell embryos. A total of 49 differently expressed genes (DEGs) were annotated with GO terms in the up-regulated genes between PCOS arrested and non-PCOS arrested embryos after GO enrichment. A total of 29 DEGs were annotated with GO terms in the down-regulated genes between PCOS arrested and non-arrested 2-cell embryos after GO enrichment. These data can provide a reference for screening specific genes involved in the arrest of PCOS embryos.

ITRAQ-based quantitative proteomic analysis of Cynops orientalis limb regeneration.

PubMed

Tang, Jie; Yu, Yuan; Zheng, Hanxue; Yin, Lu; Sun, Mei; Wang, Wenjun; Cui, Jihong; Liu, Wenguang; Xie, Xin; Chen, Fulin

2017-09-22

Salamanders regenerate their limbs after amputation. However, the molecular mechanism of this unique regeneration remains unclear. In this study, isobaric tags for relative and absolute quantification (iTRAQ) coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS) was employed to quantitatively identify differentially expressed proteins in regenerating limbs 3, 7, 14, 30 and 42 days post amputation (dpa). Of 2636 proteins detected in total, 253 proteins were differentially expressed during different regeneration stages. Among these proteins, Asporin, Cadherin-13, Keratin, Collagen alpha-1(XI) and Titin were down-regulated. CAPG, Coronin-1A, AnnexinA1, Cathepsin B were up-regulated compared with the control. The identified proteins were further analyzed to obtain information about their expression patterns and functions in limb regeneration. Functional analysis indicated that the differentially expressed proteins were associated with wound healing, immune response, cellular process, metabolism and binding. This work indicated that significant proteome alternations occurred during salamander limb regeneration. The results may provide fundamental knowledge to understand the mechanism of limb regeneration.

Assessing technical performance in differential gene expression experiments with external spike-in RNA control ratio mixtures.

PubMed

Munro, Sarah A; Lund, Steven P; Pine, P Scott; Binder, Hans; Clevert, Djork-Arné; Conesa, Ana; Dopazo, Joaquin; Fasold, Mario; Hochreiter, Sepp; Hong, Huixiao; Jafari, Nadereh; Kreil, David P; Łabaj, Paweł P; Li, Sheng; Liao, Yang; Lin, Simon M; Meehan, Joseph; Mason, Christopher E; Santoyo-Lopez, Javier; Setterquist, Robert A; Shi, Leming; Shi, Wei; Smyth, Gordon K; Stralis-Pavese, Nancy; Su, Zhenqiang; Tong, Weida; Wang, Charles; Wang, Jian; Xu, Joshua; Ye, Zhan; Yang, Yong; Yu, Ying; Salit, Marc

2014-09-25

There is a critical need for standard approaches to assess, report and compare the technical performance of genome-scale differential gene expression experiments. Here we assess technical performance with a proposed standard 'dashboard' of metrics derived from analysis of external spike-in RNA control ratio mixtures. These control ratio mixtures with defined abundance ratios enable assessment of diagnostic performance of differentially expressed transcript lists, limit of detection of ratio (LODR) estimates and expression ratio variability and measurement bias. The performance metrics suite is applicable to analysis of a typical experiment, and here we also apply these metrics to evaluate technical performance among laboratories. An interlaboratory study using identical samples shared among 12 laboratories with three different measurement processes demonstrates generally consistent diagnostic power across 11 laboratories. Ratio measurement variability and bias are also comparable among laboratories for the same measurement process. We observe different biases for measurement processes using different mRNA-enrichment protocols.

Whole transcriptome analysis of the fasting and fed Burmese python heart: insights into extreme physiological cardiac adaptation.

PubMed

Wall, Christopher E; Cozza, Steven; Riquelme, Cecilia A; McCombie, W Richard; Heimiller, Joseph K; Marr, Thomas G; Leinwand, Leslie A

2011-01-01

The infrequently feeding Burmese python (Python molurus) experiences significant and rapid postprandial cardiac hypertrophy followed by regression as digestion is completed. To begin to explore the molecular mechanisms of this response, we have sequenced and assembled the fasted and postfed Burmese python heart transcriptomes with Illumina technology using the chicken (Gallus gallus) genome as a reference. In addition, we have used RNA-seq analysis to identify differences in the expression of biological processes and signaling pathways between fasted, 1 day postfed (DPF), and 3 DPF hearts. Out of a combined transcriptome of ∼2,800 mRNAs, 464 genes were differentially expressed. Genes showing differential expression at 1 DPF compared with fasted were enriched for biological processes involved in metabolism and energetics, while genes showing differential expression at 3 DPF compared with fasted were enriched for processes involved in biogenesis, structural remodeling, and organization. Moreover, we present evidence for the activation of physiological and not pathological signaling pathways in this rapid, novel model of cardiac growth in pythons. Together, our data provide the first comprehensive gene expression profile for a reptile heart.

Identification of differentially expressed genes in cucumber (Cucumis sativus L.) root under waterlogging stress by digital gene expression profile.

PubMed

Qi, Xiao-Hua; Xu, Xue-Wen; Lin, Xiao-Jian; Zhang, Wen-Jie; Chen, Xue-Hao

2012-03-01

High-throughput tag-sequencing (Tag-seq) analysis based on the Solexa Genome Analyzer platform was applied to analyze the gene expression profiling of cucumber plant at 5 time points over a 24h period of waterlogging treatment. Approximately 5.8 million total clean sequence tags per library were obtained with 143013 distinct clean tag sequences. Approximately 23.69%-29.61% of the distinct clean tags were mapped unambiguously to the unigene database, and 53.78%-60.66% of the distinct clean tags were mapped to the cucumber genome database. Analysis of the differentially expressed genes revealed that most of the genes were down-regulated in the waterlogging stages, and the differentially expressed genes mainly linked to carbon metabolism, photosynthesis, reactive oxygen species generation/scavenging, and hormone synthesis/signaling. Finally, quantitative real-time polymerase chain reaction using nine genes independently verified the tag-mapped results. This present study reveals the comprehensive mechanisms of waterlogging-responsive transcription in cucumber. Copyright © 2011 Elsevier Inc. All rights reserved.

Global analysis of gene expression in mineralizing fish vertebra-derived cell lines: new insights into anti-mineralogenic effect of vanadate

PubMed Central

2011-01-01

Background Fish has been deemed suitable to study the complex mechanisms of vertebrate skeletogenesis and gilthead seabream (Sparus aurata), a marine teleost with acellular bone, has been successfully used in recent years to study the function and regulation of bone and cartilage related genes during development and in adult animals. Tools recently developed for gilthead seabream, e.g. mineralogenic cell lines and a 4 × 44K Agilent oligo-array, were used to identify molecular determinants of in vitro mineralization and genes involved in anti-mineralogenic action of vanadate. Results Global analysis of gene expression identified 4,223 and 4,147 genes differentially expressed (fold change - FC > 1.5) during in vitro mineralization of VSa13 (pre-chondrocyte) and VSa16 (pre-osteoblast) cells, respectively. Comparative analysis indicated that nearly 45% of these genes are common to both cell lines and gene ontology (GO) classification is also similar for both cell types. Up-regulated genes (FC > 10) were mainly associated with transport, matrix/membrane, metabolism and signaling, while down-regulated genes were mainly associated with metabolism, calcium binding, transport and signaling. Analysis of gene expression in proliferative and mineralizing cells exposed to vanadate revealed 1,779 and 1,136 differentially expressed genes, respectively. Of these genes, 67 exhibited reverse patterns of expression upon vanadate treatment during proliferation or mineralization. Conclusions Comparative analysis of expression data from fish and data available in the literature for mammalian cell systems (bone-derived cells undergoing differentiation) indicate that the same type of genes, and in some cases the same orthologs, are involved in mechanisms of in vitro mineralization, suggesting their conservation throughout vertebrate evolution and across cell types. Array technology also allowed identification of genes differentially expressed upon exposure of fish cell lines to vanadate and likely involved in its anti-mineralogenic activity. Many were found to be unknown or they were never associated to bone homeostasis previously, thus providing a set of potential candidates whose study will likely bring insights into the complex mechanisms of tissue mineralization and bone formation. PMID:21668972

Production of stable GFP-expressing neural cells from P19 embryonal carcinoma stem cells.

PubMed

Shirzad, Hedayatollah; Esmaeili, Fariba; Bakhshalizadeh, Shabnam; Ebrahimie, Marzieh; Ebrahimie, Esmaeil

2017-04-01

Murine P19 embryonal carcinoma (EC) cells are convenient to differentiate into all germ layer derivatives. One of the advantages of P19 cells is that the exogenous DNA can be easily inserted into them. Here, at the first part of this study, we generated stable GFP-expressing P19 cells (P19-GFP + ). FACS and western-blot analysis confirmed stable expression of GFP in the cells. We previously demonstrated the efficient induction of neuronal differentiation from mouse ES and EC cells by application of a neuroprotective drug, selegiline In the second part of this study selegiline was used to induce differentiation of P19-GFP + into stable GFP-expressing neuron-like cells. Cresyl violet staining confirmed neuronal morphology of the differentiated cells. Furthermore, real-time PCR and immunoflourescence approved the expression of neuron specific markers. P19-GFP + cells were able to survive, migrate and integrated into host tissues when transplanted to developing chick embryo CNS. The obtained live GFP-expressing cells can be used as an abundant source of developmentally pluripotent material for transplantation studies, investigating the cellular and molecular aspects of early differentiation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Forced expression of Hnf4a induces hepatic gene activation through directed differentiation.

PubMed

Yahoo, Neda; Pournasr, Behshad; Rostamzadeh, Jalal; Fathi, Fardin

2016-08-05

Embryonic stem (ES) cells are capable of unlimited self-renewal and have a diverse differentiation potential. These unique features make ES cells as an attractive source for developmental biology studies. Having the mature hepatocyte in the lab with functional activities is valuable in drug discovery studies. Overexpression of hepatocyte lineage-specific transcription factors (TFs) becomes a promising approach in pluripotent cell differentiation toward liver cells. Many studies generate transgenic ES cell lines to examine the effects of specific TFs overexpression in cell differentiation. In the present report, we have addressed whether a suspension or adherent model of differentiation is an appropriate way to study the role of Hnf4a overexpression. We generated ES cells that carried a doxycycline (Dox)-inducible Hnf4a using lentiviral vectors. The transduced cells were subjected to induced Hnf4a overexpression through both spontaneous and directed differentiation methods. Gene expression analysis showed substantially increased expression of hepatic gene markers, particularly Ttr and endogenous Hnf4a, in transduced cells differentiated by the directed approach. These results demonstrated that forced expression of TFs during directed differentiation would be an appropriate way to study relevant gene activation and the effects of overexpression in the context of hepatic differentiation. Copyright © 2016 Elsevier Inc. All rights reserved.

Modulation of Mitochondrial DNA Copy Number to Induce Hepatocytic Differentiation of Human Amniotic Epithelial Cells.

PubMed

Vaghjiani, Vijesh; Cain, Jason E; Lee, William; Vaithilingam, Vijayaganapathy; Tuch, Bernard E; St John, Justin C

2017-10-15

Mitochondrial deoxyribonucleic acid (mtDNA) copy number is tightly regulated during pluripotency and differentiation. There is increased demand of cellular adenosine triphosphate (ATP) during differentiation for energy-intensive cell types such as hepatocytes and neurons to meet the cell's functional requirements. During hepatocyte differentiation, mtDNA copy number should be synchronously increased to generate sufficient ATP through oxidative phosphorylation. Unlike bone marrow mesenchymal cells, mtDNA copy number failed to increase by 28 days of differentiation of human amniotic epithelial cells (hAEC) into hepatocyte-like cells (HLC) despite their expression of some end-stage hepatic markers. This was due to higher levels of DNA methylation at exon 2 of POLGA, the mtDNA-specific replication factor. Treatment with a DNA demethylation agent, 5-azacytidine, resulted in increased mtDNA copy number, reduced DNA methylation at exon 2 of POLGA, and reduced hepatic gene expression. Depletion of mtDNA followed by subsequent differentiation did not increase mtDNA copy number, but reduced DNA methylation at exon 2 of POLGA and increased expression of hepatic and pluripotency genes. We encapsulated hAEC in barium alginate microcapsules and subsequently differentiated them into HLC. Encapsulation resulted in no net increase of mtDNA copy number but a significant reduction in DNA methylation of POLGA. RNAseq analysis showed that differentiated HLC express hepatocyte-specific genes but also increased expression of inflammatory interferon genes. Differentiation in encapsulated cells showed suppression of inflammatory genes as well as increased expression of genes associated with hepatocyte function pathways and networks. This study demonstrates that an increase in classical hepatic gene expression can be achieved in HLC through encapsulation, although they fail to effectively regulate mtDNA copy number.

Normal uniform mixture differential gene expression detection for cDNA microarrays

PubMed Central

Dean, Nema; Raftery, Adrian E

2005-01-01

Background One of the primary tasks in analysing gene expression data is finding genes that are differentially expressed in different samples. Multiple testing issues due to the thousands of tests run make some of the more popular methods for doing this problematic. Results We propose a simple method, Normal Uniform Differential Gene Expression (NUDGE) detection for finding differentially expressed genes in cDNA microarrays. The method uses a simple univariate normal-uniform mixture model, in combination with new normalization methods for spread as well as mean that extend the lowess normalization of Dudoit, Yang, Callow and Speed (2002) [1]. It takes account of multiple testing, and gives probabilities of differential expression as part of its output. It can be applied to either single-slide or replicated experiments, and it is very fast. Three datasets are analyzed using NUDGE, and the results are compared to those given by other popular methods: unadjusted and Bonferroni-adjusted t tests, Significance Analysis of Microarrays (SAM), and Empirical Bayes for microarrays (EBarrays) with both Gamma-Gamma and Lognormal-Normal models. Conclusion The method gives a high probability of differential expression to genes known/suspected a priori to be differentially expressed and a low probability to the others. In terms of known false positives and false negatives, the method outperforms all multiple-replicate methods except for the Gamma-Gamma EBarrays method to which it offers comparable results with the added advantages of greater simplicity, speed, fewer assumptions and applicability to the single replicate case. An R package called nudge to implement the methods in this paper will be made available soon at . PMID:16011807

Vaginal Gene Expression During Treatment With Aromatase Inhibitors.

PubMed

Kallak, Theodora Kunovac; Baumgart, Juliane; Nilsson, Kerstin; Åkerud, Helena; Poromaa, Inger Sundström; Stavreus-Evers, Anneli

2015-12-01

Aromatase inhibitor (AI) treatment suppresses estrogen biosynthesis and causes genitourinary symptoms of menopause such as vaginal symptoms, ultimately affecting the quality of life for many postmenopausal women with breast cancer. Thus, the aim of this study was to examine vaginal gene expression in women during treatment with AIs compared with estrogen-treated women. The secondary aim was to study the presence and localization of vaginal aromatase. Vaginal biopsies were collected from postmenopausal women treated with AIs and from age-matched control women treated with vaginal estrogen therapy. Differential gene expression was studied with the Affymetrix Gene Chip Gene 1.0 ST Array (Affymetrix Inc, Santa Clara, CA) system, Ingenuity pathway analysis, quantitative real-time polymerase chain reaction, and immunohistochemistry. The expression of 279 genes differed between the 2 groups; AI-treated women had low expression of genes involved in cell differentiation, proliferation, and cell adhesion. Some differentially expressed genes were found to interact indirectly with the estrogen receptor alpha. In addition, aromatase protein staining was evident in the basal and the intermediate vaginal epithelium layers, and also in stromal cells with a slightly stronger staining intensity found in AI-treated women. In this study, we demonstrated that genes involved in cell differentiation, proliferation, and cell adhesion are differentially expressed in AI-treated women. The expression of vaginal aromatase suggests that this could be the result of local and systemic inhibition of aromatase. Our results emphasize the role of estrogen for vaginal cell differentiation and proliferation and future drug candidates should be aimed at improving cell differentiation and proliferation. Copyright © 2015 Elsevier Inc. All rights reserved.

Multiway modeling and analysis in stem cell systems biology

PubMed Central

2008-01-01

Background Systems biology refers to multidisciplinary approaches designed to uncover emergent properties of biological systems. Stem cells are an attractive target for this analysis, due to their broad therapeutic potential. A central theme of systems biology is the use of computational modeling to reconstruct complex systems from a wealth of reductionist, molecular data (e.g., gene/protein expression, signal transduction activity, metabolic activity, etc.). A number of deterministic, probabilistic, and statistical learning models are used to understand sophisticated cellular behaviors such as protein expression during cellular differentiation and the activity of signaling networks. However, many of these models are bimodal i.e., they only consider row-column relationships. In contrast, multiway modeling techniques (also known as tensor models) can analyze multimodal data, which capture much more information about complex behaviors such as cell differentiation. In particular, tensors can be very powerful tools for modeling the dynamic activity of biological networks over time. Here, we review the application of systems biology to stem cells and illustrate application of tensor analysis to model collagen-induced osteogenic differentiation of human mesenchymal stem cells. Results We applied Tucker1, Tucker3, and Parallel Factor Analysis (PARAFAC) models to identify protein/gene expression patterns during extracellular matrix-induced osteogenic differentiation of human mesenchymal stem cells. In one case, we organized our data into a tensor of type protein/gene locus link × gene ontology category × osteogenic stimulant, and found that our cells expressed two distinct, stimulus-dependent sets of functionally related genes as they underwent osteogenic differentiation. In a second case, we organized DNA microarray data in a three-way tensor of gene IDs × osteogenic stimulus × replicates, and found that application of tensile strain to a collagen I substrate accelerated the osteogenic differentiation induced by a static collagen I substrate. Conclusion Our results suggest gene- and protein-level models whereby stem cells undergo transdifferentiation to osteoblasts, and lay the foundation for mechanistic, hypothesis-driven studies. Our analysis methods are applicable to a wide range of stem cell differentiation models. PMID:18625054

puma: a Bioconductor package for propagating uncertainty in microarray analysis.

PubMed

Pearson, Richard D; Liu, Xuejun; Sanguinetti, Guido; Milo, Marta; Lawrence, Neil D; Rattray, Magnus

2009-07-09

Most analyses of microarray data are based on point estimates of expression levels and ignore the uncertainty of such estimates. By determining uncertainties from Affymetrix GeneChip data and propagating these uncertainties to downstream analyses it has been shown that we can improve results of differential expression detection, principal component analysis and clustering. Previously, implementations of these uncertainty propagation methods have only been available as separate packages, written in different languages. Previous implementations have also suffered from being very costly to compute, and in the case of differential expression detection, have been limited in the experimental designs to which they can be applied. puma is a Bioconductor package incorporating a suite of analysis methods for use on Affymetrix GeneChip data. puma extends the differential expression detection methods of previous work from the 2-class case to the multi-factorial case. puma can be used to automatically create design and contrast matrices for typical experimental designs, which can be used both within the package itself but also in other Bioconductor packages. The implementation of differential expression detection methods has been parallelised leading to significant decreases in processing time on a range of computer architectures. puma incorporates the first R implementation of an uncertainty propagation version of principal component analysis, and an implementation of a clustering method based on uncertainty propagation. All of these techniques are brought together in a single, easy-to-use package with clear, task-based documentation. For the first time, the puma package makes a suite of uncertainty propagation methods available to a general audience. These methods can be used to improve results from more traditional analyses of microarray data. puma also offers improvements in terms of scope and speed of execution over previously available methods. puma is recommended for anyone working with the Affymetrix GeneChip platform for gene expression analysis and can also be applied more generally.

Accounting for technical noise in differential expression analysis of single-cell RNA sequencing data.

PubMed

Jia, Cheng; Hu, Yu; Kelly, Derek; Kim, Junhyong; Li, Mingyao; Zhang, Nancy R

2017-11-02

Recent technological breakthroughs have made it possible to measure RNA expression at the single-cell level, thus paving the way for exploring expression heterogeneity among individual cells. Current single-cell RNA sequencing (scRNA-seq) protocols are complex and introduce technical biases that vary across cells, which can bias downstream analysis without proper adjustment. To account for cell-to-cell technical differences, we propose a statistical framework, TASC (Toolkit for Analysis of Single Cell RNA-seq), an empirical Bayes approach to reliably model the cell-specific dropout rates and amplification bias by use of external RNA spike-ins. TASC incorporates the technical parameters, which reflect cell-to-cell batch effects, into a hierarchical mixture model to estimate the biological variance of a gene and detect differentially expressed genes. More importantly, TASC is able to adjust for covariates to further eliminate confounding that may originate from cell size and cell cycle differences. In simulation and real scRNA-seq data, TASC achieves accurate Type I error control and displays competitive sensitivity and improved robustness to batch effects in differential expression analysis, compared to existing methods. TASC is programmed to be computationally efficient, taking advantage of multi-threaded parallelization. We believe that TASC will provide a robust platform for researchers to leverage the power of scRNA-seq. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Accounting for technical noise in differential expression analysis of single-cell RNA sequencing data

PubMed Central

Jia, Cheng; Hu, Yu; Kelly, Derek; Kim, Junhyong

2017-01-01

Abstract Recent technological breakthroughs have made it possible to measure RNA expression at the single-cell level, thus paving the way for exploring expression heterogeneity among individual cells. Current single-cell RNA sequencing (scRNA-seq) protocols are complex and introduce technical biases that vary across cells, which can bias downstream analysis without proper adjustment. To account for cell-to-cell technical differences, we propose a statistical framework, TASC (Toolkit for Analysis of Single Cell RNA-seq), an empirical Bayes approach to reliably model the cell-specific dropout rates and amplification bias by use of external RNA spike-ins. TASC incorporates the technical parameters, which reflect cell-to-cell batch effects, into a hierarchical mixture model to estimate the biological variance of a gene and detect differentially expressed genes. More importantly, TASC is able to adjust for covariates to further eliminate confounding that may originate from cell size and cell cycle differences. In simulation and real scRNA-seq data, TASC achieves accurate Type I error control and displays competitive sensitivity and improved robustness to batch effects in differential expression analysis, compared to existing methods. TASC is programmed to be computationally efficient, taking advantage of multi-threaded parallelization. We believe that TASC will provide a robust platform for researchers to leverage the power of scRNA-seq. PMID:29036714

Gene expression analysis of bud and leaf color in tea.

PubMed

Wei, Kang; Zhang, Yazhen; Wu, Liyun; Li, Hailin; Ruan, Li; Bai, Peixian; Zhang, Chengcai; Zhang, Fen; Xu, Liyi; Wang, Liyuan; Cheng, Hao

2016-10-01

Purple shoot tea attributing to the high anthocyanin accumulation is of great interest for its wide health benefits. To better understand potential mechanisms involved in purple buds and leaves formation in tea plants, we performed transcriptome analysis of six green or purple shoot tea individuals from a F1 population using the Illumina sequencing method. Totally 292 million RNA-Seq reads were obtained and assembled into 112,233 unigenes, with an average length of 759 bp and an N50 of 1081 bp. Moreover, totally 2193 unigenes showed significant differences in expression levels between green and purple tea samples, with 1143 up- and 1050 down-regulated in the purple teas. Further real time PCR analysis confirmed RNA-Seq results. Our study identified 28 differentially expressed transcriptional factors and A CsMYB gene was found to be highly similar to AtPAP1 in Arabidopsis. Further analysis of differentially expressed genes involved in anthocyanin biosynthesis and transportation showed that the late biosynthetic genes and genes involved in anthocyanin transportation were largely affected but the early biosynthetic genes were less or none affected. Overall, the identification of a large number of differentially expressed genes offers a global view of the potential mechanisms associated with purple buds and leaves formation, which will facilitate molecular breeding in tea plants. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Machine Learning–Based Differential Network Analysis: A Study of Stress-Responsive Transcriptomes in Arabidopsis[W

PubMed Central

Ma, Chuang; Xin, Mingming; Feldmann, Kenneth A.; Wang, Xiangfeng

2014-01-01

Machine learning (ML) is an intelligent data mining technique that builds a prediction model based on the learning of prior knowledge to recognize patterns in large-scale data sets. We present an ML-based methodology for transcriptome analysis via comparison of gene coexpression networks, implemented as an R package called machine learning–based differential network analysis (mlDNA) and apply this method to reanalyze a set of abiotic stress expression data in Arabidopsis thaliana. The mlDNA first used a ML-based filtering process to remove nonexpressed, constitutively expressed, or non-stress-responsive “noninformative” genes prior to network construction, through learning the patterns of 32 expression characteristics of known stress-related genes. The retained “informative” genes were subsequently analyzed by ML-based network comparison to predict candidate stress-related genes showing expression and network differences between control and stress networks, based on 33 network topological characteristics. Comparative evaluation of the network-centric and gene-centric analytic methods showed that mlDNA substantially outperformed traditional statistical testing–based differential expression analysis at identifying stress-related genes, with markedly improved prediction accuracy. To experimentally validate the mlDNA predictions, we selected 89 candidates out of the 1784 predicted salt stress–related genes with available SALK T-DNA mutagenesis lines for phenotypic screening and identified two previously unreported genes, mutants of which showed salt-sensitive phenotypes. PMID:24520154

Chilling Affects Phytohormone and Post-Embryonic Development Pathways during Bud Break and Fruit Set in Apple (Malus domestica Borkh.)

PubMed Central

Kumar, Gulshan; Gupta, Khushboo; Pathania, Shivalika; Swarnkar, Mohit Kumar; Rattan, Usha Kumari; Singh, Gagandeep; Sharma, Ram Kumar; Singh, Anil Kumar

2017-01-01

The availability of sufficient chilling during bud dormancy plays an important role in the subsequent yield and quality of apple fruit, whereas, insufficient chilling availability negatively impacts the apple production. The transcriptome profiling during bud dormancy release and initial fruit set under low and high chill conditions was performed using RNA-seq. The comparative high number of differentially expressed genes during bud break and fruit set under high chill condition indicates that chilling availability was associated with transcriptional reorganization. The comparative analysis reveals the differential expression of genes involved in phytohormone metabolism, particularly for Abscisic acid, gibberellic acid, ethylene, auxin and cytokinin. The expression of Dormancy Associated MADS-box, Flowering Locus C-like, Flowering Locus T-like and Terminal Flower 1-like genes was found to be modulated under differential chilling. The co-expression network analysis indentified two high chill specific modules that were found to be enriched for “post-embryonic development” GO terms. The network analysis also identified hub genes including Early flowering 7, RAF10, ZEP4 and F-box, which may be involved in regulating chilling-mediated dormancy release and fruit set. The results of transcriptome and co-expression network analysis indicate that chilling availability majorly regulates phytohormone-related pathways and post-embryonic development during bud break. PMID:28198417

Differential Gene Expression in Liver, Gill, and Olfactory Rosettes of Coho Salmon (Oncorhynchus kisutch) After Acclimation to Salinity

PubMed Central

Lavado, Ramon; Bammler, Theo K.; Gallagher, Evan P.; Stapleton, Patricia L.; Beyer, Richard P.; Farin, Federico M.; Hardiman, Gary; Schlenk, Daniel

2015-01-01

Most Pacific salmonids undergo smoltification and transition from freshwater to saltwater, making various adjustments in metabolism, catabolism, osmotic, and ion regulation. The molecular mechanisms underlying this transition are largely unknown. In the present study, we acclimated coho salmon (Oncorhynchus kisutch) to four different salinities and assessed gene expression through microarray analysis of gills, liver, and olfactory rosettes. Gills are involved in osmotic regulation, liver plays a role in energetics, and olfactory rosettes are involved in behavior. Between all salinity treatments, liver had the highest number of differentially expressed genes at 1616, gills had 1074, and olfactory rosettes had 924, using a 1.5-fold cutoff and a false discovery rate of 0.5. Higher responsiveness of liver to metabolic changes after salinity acclimation to provide energy for other osmoregulatory tissues such as the gills may explain the differences in number of differentially expressed genes. Differentially expressed genes were tissue- and salinity-dependent. There were no known genes differentially expressed that were common to all salinity treatments and all tissues. Gene ontology term analysis revealed biological processes, molecular functions, and cellular components that were significantly affected by salinity, a majority of which were tissue-dependent. For liver, oxygen binding and transport terms were highlighted. For gills, muscle, and cytoskeleton-related terms predominated and for olfactory rosettes, immune response-related genes were accentuated. Interaction networks were examined in combination with GO terms and determined similarities between tissues for potential osmosensors, signal transduction cascades, and transcription factors. PMID:26260986

The Effect of Agmatine on Expression of IL-1β and TLX Which Promotes Neuronal Differentiation in Lipopolysaccharide-Treated Neural Progenitors.

PubMed

Song, Juhyun; Kumar, Bokara Kiran; Kang, Somang; Park, Kyung Ah; Lee, Won Taek; Lee, Jong Eun

2013-12-01

Differentiation of neural progenitor cells (NPCs) is important for protecting neural cells and brain tissue during inflammation. Interleukin-1 beta (IL-1β) is the most common pro- inflammatory cytokine in brain inflammation, and increased IL-1β levels can decrease the proliferation of NPCs. We aimed to investigate whether agmatine (Agm), a primary polyamine that protects neural cells, could trigger differentiation of NPCs by activating IL-1β in vitro. The cortex of ICR mouse embryos (E14) was dissociated to culture NPCs. NPCs were stimulated by lipopolysaccharide (LPS). After 6 days, protein expression of stem cell markers and differentiation signal factors was confirmed by using western blot analysis. Also, immunocytochemistry was used to confirm the cell fate. Agm treatment activated NPC differentiation significantly more than in the control group, which was evident by the increased expression of a neuronal marker, MAP2, in the LPS-induced, Agm-treated group. Differentiation of LPS-induced, Agm-treated NPCs was regulated by the MAPK pathway and is thought to be related to IL-1β activation and decreased expression of TLX, a transcription factor that regulates NPC differentiation. Our results reveal that Agm can promote NPC differentiation to neural stem cells by modulating IL-1β expression under inflammatory condition, and they suggest that Agm may be a novel therapeutic strategy for neuroinflammatory diseases.

Inference for High-dimensional Differential Correlation Matrices.

PubMed

Cai, T Tony; Zhang, Anru

2016-01-01

Motivated by differential co-expression analysis in genomics, we consider in this paper estimation and testing of high-dimensional differential correlation matrices. An adaptive thresholding procedure is introduced and theoretical guarantees are given. Minimax rate of convergence is established and the proposed estimator is shown to be adaptively rate-optimal over collections of paired correlation matrices with approximately sparse differences. Simulation results show that the procedure significantly outperforms two other natural methods that are based on separate estimation of the individual correlation matrices. The procedure is also illustrated through an analysis of a breast cancer dataset, which provides evidence at the gene co-expression level that several genes, of which a subset has been previously verified, are associated with the breast cancer. Hypothesis testing on the differential correlation matrices is also considered. A test, which is particularly well suited for testing against sparse alternatives, is introduced. In addition, other related problems, including estimation of a single sparse correlation matrix, estimation of the differential covariance matrices, and estimation of the differential cross-correlation matrices, are also discussed.

Meta-Analysis of Maternal and Fetal Transcriptomic Data Elucidates the Role of Adaptive and Innate Immunity in Preterm Birth

PubMed Central

Vora, Bianca; Wang, Aolin; Kosti, Idit; Huang, Hongtai; Paranjpe, Ishan; Woodruff, Tracey J.; MacKenzie, Tippi; Sirota, Marina

2018-01-01

Preterm birth (PTB) is the leading cause of newborn deaths around the world. Spontaneous preterm birth (sPTB) accounts for two-thirds of all PTBs; however, there remains an unmet need of detecting and preventing sPTB. Although the dysregulation of the immune system has been implicated in various studies, small sizes and irreproducibility of results have limited identification of its role. Here, we present a cross-study meta-analysis to evaluate genome-wide differential gene expression signals in sPTB. A comprehensive search of the NIH genomic database for studies related to sPTB with maternal whole blood samples resulted in data from three separate studies consisting of 339 samples. After aggregating and normalizing these transcriptomic datasets and performing a meta-analysis, we identified 210 genes that were differentially expressed in sPTB relative to term birth. These genes were enriched in immune-related pathways, showing upregulation of innate immunity and downregulation of adaptive immunity in women who delivered preterm. An additional analysis found several of these differentially expressed at mid-gestation, suggesting their potential to be clinically relevant biomarkers. Furthermore, a complementary analysis identified 473 genes differentially expressed in preterm cord blood samples. However, these genes demonstrated downregulation of the innate immune system, a stark contrast to findings using maternal blood samples. These immune-related findings were further confirmed by cell deconvolution as well as upstream transcription and cytokine regulation analyses. Overall, this study identified a strong immune signature related to sPTB as well as several potential biomarkers that could be translated to clinical use.

Comparative 2D-DIGE Proteomic Analysis of Bovine Mammary Epithelial Cells during Lactation Reveals Protein Signatures for Lactation Persistency and Milk Yield

PubMed Central

Janjanam, Jagadeesh; Singh, Surender; Jena, Manoj K.; Varshney, Nishant; Kola, Srujana; Kumar, Sudarshan; Kaushik, Jai K.; Grover, Sunita; Dang, Ajay K.; Mukesh, Manishi; Prakash, B. S.; Mohanty, Ashok K.

2014-01-01

Mammary gland is made up of a branching network of ducts that end with alveoli which surrounds the lumen. These alveolar mammary epithelial cells (MEC) reflect the milk producing ability of farm animals. In this study, we have used 2D-DIGE and mass spectrometry to identify the protein changes in MEC during immediate early, peak and late stages of lactation and also compared differentially expressed proteins in MEC isolated from milk of high and low milk producing cows. We have identified 41 differentially expressed proteins during lactation stages and 22 proteins in high and low milk yielding cows. Bioinformatics analysis showed that a majority of the differentially expressed proteins are associated in metabolic process, catalytic and binding activity. The differentially expressed proteins were mapped to the available biological pathways and networks involved in lactation. The proteins up-regulated during late stage of lactation are associated with NF-κB stress induced signaling pathways and whereas Akt, PI3K and p38/MAPK signaling pathways are associated with high milk production mediated through insulin hormone signaling. PMID:25111801

Computational genomic analysis of PARK7 interactome reveals high BBS1 gene expression as a prognostic factor favoring survival in malignant pleural mesothelioma.

PubMed

Vavougios, Georgios D; Solenov, Evgeniy I; Hatzoglou, Chrissi; Baturina, Galina S; Katkova, Liubov E; Molyvdas, Paschalis Adam; Gourgoulianis, Konstantinos I; Zarogiannis, Sotirios G

2015-10-01

The aim of our study was to assess the differential gene expression of Parkinson protein 7 (PARK7) interactome in malignant pleural mesothelioma (MPM) using data mining techniques to identify novel candidate genes that may play a role in the pathogenicity of MPM. We constructed the PARK7 interactome using the ConsensusPathDB database. We then interrogated the Oncomine Cancer Microarray database using the Gordon Mesothelioma Study, for differential gene expression of the PARK7 interactome. In ConsensusPathDB, 38 protein interactors of PARK7 were identified. In the Gordon Mesothelioma Study, 34 of them were assessed out of which SUMO1, UBC3, KIAA0101, HDAC2, DAXX, RBBP4, BBS1, NONO, RBBP7, HTRA2, and STUB1 were significantly overexpressed whereas TRAF6 and MTA2 were significantly underexpressed in MPM patients (network 2). Furthermore, Kaplan-Meier analysis revealed that MPM patients with high BBS1 expression had a median overall survival of 16.5 vs. 8.7 mo of those that had low expression. For validation purposes, we performed a meta-analysis in Oncomine database in five sarcoma datasets. Eight network 2 genes (KIAA0101, HDAC2, SUMO1, RBBP4, NONO, RBBP7, HTRA2, and MTA2) were significantly differentially expressed in an array of 18 different sarcoma types. Finally, Gene Ontology annotation enrichment analysis revealed significant roles of the PARK7 interactome in NuRD, CHD, and SWI/SNF protein complexes. In conclusion, we identified 13 novel genes differentially expressed in MPM, never reported before. Among them, BBS1 emerged as a novel predictor of overall survival in MPM. Finally, we identified that PARK7 interactome is involved in novel pathways pertinent in MPM disease. Copyright © 2015 the American Physiological Society.

A microRNA-mRNA expression network during oral siphon regeneration in Ciona.

PubMed

Spina, Elijah J; Guzman, Elmer; Zhou, Hongjun; Kosik, Kenneth S; Smith, William C

2017-05-15

Here we present a parallel study of mRNA and microRNA expression during oral siphon (OS) regeneration in Ciona robusta , and the derived network of their interactions. In the process of identifying 248 mRNAs and 15 microRNAs as differentially expressed, we also identified 57 novel microRNAs, several of which are among the most highly differentially expressed. Analysis of functional categories identified enriched transcripts related to stress responses and apoptosis at the wound healing stage, signaling pathways including Wnt and TGFβ during early regrowth, and negative regulation of extracellular proteases in late stage regeneration. Consistent with the expression results, we found that inhibition of TGFβ signaling blocked OS regeneration. A correlation network was subsequently inferred for all predicted microRNA-mRNA target pairs expressed during regeneration. Network-based clustering associated transcripts into 22 non-overlapping groups, the functional analysis of which showed enrichment of stress response, signaling pathway and extracellular protease categories that could be related to specific microRNAs. Predicted targets of the miR-9 cluster suggest a role in regulating differentiation and the proliferative state of neural progenitors through regulation of the cytoskeleton and cell cycle. © 2017. Published by The Company of Biologists Ltd.

A Study of the Differential Effects of Eicosapentaenoic Acid (EPA) and Docosahexaenoic Acid (DHA) on Gene Expression Profiles of Stimulated Thp-1 Macrophages.

PubMed

Allam-Ndoul, Bénédicte; Guénard, Frédéric; Barbier, Olivier; Vohl, Marie-Claude

2017-04-25

Background: An appropriate intake of omega-3 ( n -3) fatty acids (FAs) such as eicosapentaenoic and docosahexaenoic acid (EPA/DHA) from marine sources is known to have anti-inflammatory effects. However, molecular mechanisms underlying their beneficial effects on health are not fully understood. The aim of the present study was to characterize gene expression profiles of THP-1 macrophages, incubated in either EPA or DHA and stimulated with lipopolysaccharide (LPS), a pro-inflammatory agent. Methods: THP-1 macrophages were incubated into 10, 50 and 75 µM of EPA or DHA for 24 h, and 100 nM of LPS was added to the culture media for 18 h. Total mRNA was extracted and gene expression examined by microarray analysis using Illumina Human HT-12 expression beadchips (Illumina). Results: Pathway analysis revealed that EPA and DHA regulate genes involved in cell cycle regulation, apoptosis, immune response and inflammation, oxidative stress and cancer pathways in a differential and dose-dependent manner. Conclusions: EPA and DHA appear to exert differential effects on gene expression in THP-1 macrophages. Specific effects of n -3 FAs on gene expression levels are also dose-dependent.

Gene expression analysis of colorectal cancer by bioinformatics strategy.

PubMed

Cui, Meng; Yuan, Junhua; Li, Jun; Sun, Bing; Li, Tao; Li, Yuantao; Wu, Guoliang

2014-10-01

We used bioinformatics technology to analyze gene expression profiles involved in colorectal cancer tissue samples and healthy controls. In this paper, we downloaded the gene expression profile GSE4107 from Gene Expression Omnibus (GEO) database, in which a total of 22 chips were available, including normal colonic mucosa tissue from normal healthy donors (n=10), colorectal cancer tissue samples from colorectal patients (n=33). To further understand the biological functions of the screened DGEs, the KEGG pathway enrichment analysis were conducted. Then we built a transcriptome network to study differentially co-expressed links. A total of 3151 DEGs of CRC were selected. Besides, total 164 DCGs (Differentially Coexpressed Gene, DCG) and 29279 DCLs (Differentially Co-expressed Link, DCL) were obtained. Furthermore, the significantly enriched KEGG pathways were Endocytosis, Calcium signaling pathway, Vascular smooth muscle contraction, Linoleic acid metabolism, Arginine and proline metabolism, Inositol phosphate metabolism and MAPK signaling pathway. Our results show that the generation of CRC involves multiple genes, TFs and pathways. Several signal and immune pathways are linked to CRC and give us more clues in the process of CRC. Hence, our work would pave ways for novel diagnosis of CRC, and provided theoretical guidance into cancer therapy.

A microRNA-mRNA expression network during oral siphon regeneration in Ciona

PubMed Central

Spina, Elijah J.; Guzman, Elmer; Zhou, Hongjun; Kosik, Kenneth S.

2017-01-01

Here we present a parallel study of mRNA and microRNA expression during oral siphon (OS) regeneration in Ciona robusta, and the derived network of their interactions. In the process of identifying 248 mRNAs and 15 microRNAs as differentially expressed, we also identified 57 novel microRNAs, several of which are among the most highly differentially expressed. Analysis of functional categories identified enriched transcripts related to stress responses and apoptosis at the wound healing stage, signaling pathways including Wnt and TGFβ during early regrowth, and negative regulation of extracellular proteases in late stage regeneration. Consistent with the expression results, we found that inhibition of TGFβ signaling blocked OS regeneration. A correlation network was subsequently inferred for all predicted microRNA-mRNA target pairs expressed during regeneration. Network-based clustering associated transcripts into 22 non-overlapping groups, the functional analysis of which showed enrichment of stress response, signaling pathway and extracellular protease categories that could be related to specific microRNAs. Predicted targets of the miR-9 cluster suggest a role in regulating differentiation and the proliferative state of neural progenitors through regulation of the cytoskeleton and cell cycle. PMID:28432214

Gene expression pattern recognition algorithm inferences to classify samples exposed to chemical agents

NASA Astrophysics Data System (ADS)

Bushel, Pierre R.; Bennett, Lee; Hamadeh, Hisham; Green, James; Ableson, Alan; Misener, Steve; Paules, Richard; Afshari, Cynthia

2002-06-01

We present an analysis of pattern recognition procedures used to predict the classes of samples exposed to pharmacologic agents by comparing gene expression patterns from samples treated with two classes of compounds. Rat liver mRNA samples following exposure for 24 hours with phenobarbital or peroxisome proliferators were analyzed using a 1700 rat cDNA microarray platform. Sets of genes that were consistently differentially expressed in the rat liver samples following treatment were stored in the MicroArray Project System (MAPS) database. MAPS identified 238 genes in common that possessed a low probability (P < 0.01) of being randomly detected as differentially expressed at the 95% confidence level. Hierarchical cluster analysis on the 238 genes clustered specific gene expression profiles that separated samples based on exposure to a particular class of compound.

Weighted gene co-expression network analysis of expression data of monozygotic twins identifies specific modules and hub genes related to BMI.

PubMed

Wang, Weijing; Jiang, Wenjie; Hou, Lin; Duan, Haiping; Wu, Yili; Xu, Chunsheng; Tan, Qihua; Li, Shuxia; Zhang, Dongfeng

2017-11-13

The therapeutic management of obesity is challenging, hence further elucidating the underlying mechanisms of obesity development and identifying new diagnostic biomarkers and therapeutic targets are urgent and necessary. Here, we performed differential gene expression analysis and weighted gene co-expression network analysis (WGCNA) to identify significant genes and specific modules related to BMI based on gene expression profile data of 7 discordant monozygotic twins. In the differential gene expression analysis, it appeared that 32 differentially expressed genes (DEGs) were with a trend of up-regulation in twins with higher BMI when compared to their siblings. Categories of positive regulation of nitric-oxide synthase biosynthetic process, positive regulation of NF-kappa B import into nucleus, and peroxidase activity were significantly enriched within GO database and NF-kappa B signaling pathway within KEGG database. DEGs of NAMPT, TLR9, PTGS2, HBD, and PCSK1N might be associated with obesity. In the WGCNA, among the total 20 distinct co-expression modules identified, coral1 module (68 genes) had the strongest positive correlation with BMI (r = 0.56, P = 0.04) and disease status (r = 0.56, P = 0.04). Categories of positive regulation of phospholipase activity, high-density lipoprotein particle clearance, chylomicron remnant clearance, reverse cholesterol transport, intermediate-density lipoprotein particle, chylomicron, low-density lipoprotein particle, very-low-density lipoprotein particle, voltage-gated potassium channel complex, cholesterol transporter activity, and neuropeptide hormone activity were significantly enriched within GO database for this module. And alcoholism and cell adhesion molecules pathways were significantly enriched within KEGG database. Several hub genes, such as GAL, ASB9, NPPB, TBX2, IL17C, APOE, ABCG4, and APOC2 were also identified. The module eigengene of saddlebrown module (212 genes) was also significantly correlated with BMI (r = 0.56, P = 0.04), and hub genes of KCNN1 and AQP10 were differentially expressed. We identified significant genes and specific modules potentially related to BMI based on the gene expression profile data of monozygotic twins. The findings may help further elucidate the underlying mechanisms of obesity development and provide novel insights to research potential gene biomarkers and signaling pathways for obesity treatment. Further analysis and validation of the findings reported here are important and necessary when more sample size is acquired.

Composite transcriptome assembly of RNA-seq data in a sheep model for delayed bone healing.

PubMed

Jäger, Marten; Ott, Claus-Eric; Grünhagen, Johannes; Hecht, Jochen; Schell, Hanna; Mundlos, Stefan; Duda, Georg N; Robinson, Peter N; Lienau, Jasmin

2011-03-24

The sheep is an important model organism for many types of medically relevant research, but molecular genetic experiments in the sheep have been limited by the lack of knowledge about ovine gene sequences. Prior to our study, mRNA sequences for only 1,556 partial or complete ovine genes were publicly available. Therefore, we developed a composite de novo transcriptome assembly method for next-generation sequence data to combine known ovine mRNA and EST sequences, mRNA sequences from mouse and cow, and sequences assembled de novo from short read RNA-Seq data into a composite reference transcriptome, and identified transcripts from over 12 thousand previously undescribed ovine genes. Gene expression analysis based on these data revealed substantially different expression profiles in standard versus delayed bone healing in an ovine tibial osteotomy model. Hundreds of transcripts were differentially expressed between standard and delayed healing and between the time points of the standard and delayed healing groups. We used the sheep sequences to design quantitative RT-PCR assays with which we validated the differential expression of 26 genes that had been identified by RNA-seq analysis. A number of clusters of characteristic expression profiles could be identified, some of which showed striking differences between the standard and delayed healing groups. Gene Ontology (GO) analysis showed that the differentially expressed genes were enriched in terms including extracellular matrix, cartilage development, contractile fiber, and chemokine activity. Our results provide a first atlas of gene expression profiles and differentially expressed genes in standard and delayed bone healing in a large-animal model and provide a number of clues as to the shifts in gene expression that underlie delayed bone healing. In the course of our study, we identified transcripts of 13,987 ovine genes, including 12,431 genes for which no sequence information was previously available. This information will provide a basis for future molecular research involving the sheep as a model organism.

Composite transcriptome assembly of RNA-seq data in a sheep model for delayed bone healing

PubMed Central

2011-01-01

Background The sheep is an important model organism for many types of medically relevant research, but molecular genetic experiments in the sheep have been limited by the lack of knowledge about ovine gene sequences. Results Prior to our study, mRNA sequences for only 1,556 partial or complete ovine genes were publicly available. Therefore, we developed a composite de novo transcriptome assembly method for next-generation sequence data to combine known ovine mRNA and EST sequences, mRNA sequences from mouse and cow, and sequences assembled de novo from short read RNA-Seq data into a composite reference transcriptome, and identified transcripts from over 12 thousand previously undescribed ovine genes. Gene expression analysis based on these data revealed substantially different expression profiles in standard versus delayed bone healing in an ovine tibial osteotomy model. Hundreds of transcripts were differentially expressed between standard and delayed healing and between the time points of the standard and delayed healing groups. We used the sheep sequences to design quantitative RT-PCR assays with which we validated the differential expression of 26 genes that had been identified by RNA-seq analysis. A number of clusters of characteristic expression profiles could be identified, some of which showed striking differences between the standard and delayed healing groups. Gene Ontology (GO) analysis showed that the differentially expressed genes were enriched in terms including extracellular matrix, cartilage development, contractile fiber, and chemokine activity. Conclusions Our results provide a first atlas of gene expression profiles and differentially expressed genes in standard and delayed bone healing in a large-animal model and provide a number of clues as to the shifts in gene expression that underlie delayed bone healing. In the course of our study, we identified transcripts of 13,987 ovine genes, including 12,431 genes for which no sequence information was previously available. This information will provide a basis for future molecular research involving the sheep as a model organism. PMID:21435219

Cell adhesion molecules expression pattern indicates that somatic cells arbitrate gonadal sex of differentiating bipotential fetal mouse gonad.

PubMed

Piprek, Rafal P; Kolasa, Michal; Podkowa, Dagmara; Kloc, Malgorzata; Kubiak, Jacek Z

2017-10-01

Unlike other organ anlagens, the primordial gonad is sexually bipotential in all animals. In mouse, the bipotential gonad differentiates into testis or ovary depending on the genetic sex (XY or XX) of the fetus. During gonad development cells segregate, depending on genetic sex, into distinct compartments: testis cords and interstitium form in XY gonad, and germ cell cysts and stroma in XX gonad. However, our knowledge of mechanisms governing gonadal sex differentiation remains very vague. Because it is known that adhesion molecules (CAMs) play a key role in organogenesis, we suspected that diversified expression of CAMs should also play a crucial role in gonad development. Using microarray analysis we identified 129 CAMs and factors regulating cell adhesion during sexual differentiation of mouse gonad. To identify genes expressed differentially in three cell lines in XY and XX gonads: i) supporting (Sertoli or follicular cells), ii) interstitial or stromal cells, and iii) germ cells, we used transgenic mice expressing EGFP reporter gene and FACS cell sorting. Although a large number of CAMs expressed ubiquitously, expression of certain genes was cell line- and genetic sex-specific. The sets of CAMs differentially expressed in supporting versus interstitial/stromal cells may be responsible for segregation of these two cell lines during gonadal development. There was also a significant difference in CAMs expression pattern between XY supporting (Sertoli) and XX supporting (follicular) cells but not between XY and XX germ cells. This indicates that differential CAMs expression pattern in the somatic cells but not in the germ line arbitrates structural organization of gonadal anlagen into testis or ovary. Copyright © 2017 Elsevier B.V. All rights reserved.

Genome-wide analysis of miRNA and mRNA transcriptomes during amelogenesis.

PubMed

Yin, Kaifeng; Hacia, Joseph G; Zhong, Zhe; Paine, Michael L

2014-11-19

In the rodent incisor during amelogenesis, as ameloblast cells transition from secretory stage to maturation stage, their morphology and transcriptome profiles change dramatically. Prior whole genome transcriptome analysis has given a broad picture of the molecular activities dominating both stages of amelogenesis, but this type of analysis has not included miRNA transcript profiling. In this study, we set out to document which miRNAs and corresponding target genes change significantly as ameloblasts transition from secretory- to maturation-stage amelogenesis. Total RNA samples from both secretory- and maturation-stage rat enamel organs were subjected to genome-wide miRNA and mRNA transcript profiling. We identified 59 miRNAs that were differentially expressed at the maturation stage relative to the secretory stage of enamel development (False Discovery Rate (FDR)<0.05, fold change (FC)≥1.8). In parallel, transcriptome profiling experiments identified 1,729 mRNA transcripts that were differentially expressed in the maturation stage compared to the secretory stage (FDR<0.05, FC≥1.8). Based on bioinformatics analyses, 5.8% (629 total) of these differentially expressed genes (DEGS) were highlighted as being the potential targets of 59 miRNAs that were differentially expressed in the opposite direction, in the same tissue samples. Although the number of predicted target DEGs was not higher than baseline expectations generated by examination of stably expressed miRNAs, Gene Ontology (GO) analysis showed that these 629 DEGS were enriched for ion transport, pH regulation, calcium handling, endocytotic, and apoptotic activities. Seven differentially expressed miRNAs (miR-21, miR-31, miR-488, miR-153, miR-135b, miR-135a and miR298) in secretory- and/or maturation-stage enamel organs were confirmed by in situ hybridization. Further, we used luciferase reporter assays to provide evidence that two of these differentially expressed miRNAs, miR-153 and miR-31, are potential regulators for their predicated target mRNAs, Lamp1 (miR-153) and Tfrc (miR-31). In conclusion, these data indicate that miRNAs exhibit a dynamic expression pattern during the transition from secretory-stage to maturation-stage tooth enamel formation. Although they represent only one of numerous mechanisms influencing gene activities, miRNAs specific to the maturation stage could be involved in regulating several key processes of enamel maturation by influencing mRNA stability and translation.

contamDE: differential expression analysis of RNA-seq data for contaminated tumor samples.

PubMed

Shen, Qi; Hu, Jiyuan; Jiang, Ning; Hu, Xiaohua; Luo, Zewei; Zhang, Hong

2016-03-01

Accurate detection of differentially expressed genes between tumor and normal samples is a primary approach of cancer-related biomarker identification. Due to the infiltration of tumor surrounding normal cells, the expression data derived from tumor samples would always be contaminated with normal cells. Ignoring such cellular contamination would deflate the power of detecting DE genes and further confound the biological interpretation of the analysis results. For the time being, there does not exists any differential expression analysis approach for RNA-seq data in literature that can properly account for the contamination of tumor samples. Without appealing to any extra information, we develop a new method 'contamDE' based on a novel statistical model that associates RNA-seq expression levels with cell types. It is demonstrated through simulation studies that contamDE could be much more powerful than the existing methods that ignore the contamination. In the application to two cancer studies, contamDE uniquely found several potential therapy and prognostic biomarkers of prostate cancer and non-small cell lung cancer. An R package contamDE is freely available at http://homepage.fudan.edu.cn/zhangh/softwares/ zhanghfd@fudan.edu.cn Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Transcription profile analysis of Lycopersicum esculentum leaves, unravels volatile emissions and gene expression under salinity stress.

PubMed

Zhang, Jihong; Zeng, Li; Chen, Shaoyang; Sun, Helong; Ma, Shuang

2018-05-01

Salinity stress can impede development and plant growth adversely. However, there is very little molecular information on NaCl resistance and volatile emissions in Lycopersicum esculentum. In order to investigate the effects of salt stress on the release of volatile compounds, we quantified and compared transcriptome changes by RNA-Seq analysis and volatile constituents with gas chromatography/mass spectrometry (GC/MS) coupled with solid-phase microextraction (SPME) after exposure to continuous salt stress. Chemical analysis by GC-MS analysis revealed that NaCl stress had changed species and quantity of volatile compounds released. In this research, 21,578 unigenes that represented 44,714 assembled unique transcripts were separated from tomato leaves exposed to NaCl stress based on de novo transcriptome assembly. The total number of differentially expressed genes was 7210 after exposure to NaCl, including 6200 down-regulated and 1208 up-regulated genes. Among these differentially expressed genes (DEGs), there were eighteen differentially expressed genes associated with volatile biosynthesis. Of the unigenes, 3454 were mapped to 131 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, mainly those are involved in RNA transport, plant-pathogen interactions, and plant hormone signal transduction. qRT-PCR analysis showed that NaCl exposure affected the expression profiles of the biosynthesis genes for eight volatile compounds (IPI, GPS, and TPS, etc.), which corresponded well with the RNA-Seq analysis and GC-MS results. Our results suggest that NaCl stress affects the emission of volatile substances from L. esculentum leaves by regulating the expression of genes that are involved in volatile organic compounds' biosynthesis. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Comparative analysis of human protein-coding and noncoding RNAs between brain and 10 mixed cell lines by RNA-Seq.

PubMed

Chen, Geng; Yin, Kangping; Shi, Leming; Fang, Yuanzhang; Qi, Ya; Li, Peng; Luo, Jian; He, Bing; Liu, Mingyao; Shi, Tieliu

2011-01-01

In their expression process, different genes can generate diverse functional products, including various protein-coding or noncoding RNAs. Here, we investigated the protein-coding capacities and the expression levels of their isoforms for human known genes, the conservation and disease association of long noncoding RNAs (ncRNAs) with two transcriptome sequencing datasets from human brain tissues and 10 mixed cell lines. Comparative analysis revealed that about two-thirds of the genes expressed between brain and cell lines are the same, but less than one-third of their isoforms are identical. Besides those genes specially expressed in brain and cell lines, about 66% of genes expressed in common encoded different isoforms. Moreover, most genes dominantly expressed one isoform and some genes only generated protein-coding (or noncoding) RNAs in one sample but not in another. We found 282 human genes could encode both protein-coding and noncoding RNAs through alternative splicing in the two samples. We also identified more than 1,000 long ncRNAs, and most of those long ncRNAs contain conserved elements across either 46 vertebrates or 33 placental mammals or 10 primates. Further analysis showed that some long ncRNAs differentially expressed in human breast cancer or lung cancer, several of those differentially expressed long ncRNAs were validated by RT-PCR. In addition, those validated differentially expressed long ncRNAs were found significantly correlated with certain breast cancer or lung cancer related genes, indicating the important biological relevance between long ncRNAs and human cancers. Our findings reveal that the differences of gene expression profile between samples mainly result from the expressed gene isoforms, and highlight the importance of studying genes at the isoform level for completely illustrating the intricate transcriptome.

Knockdown of the schizophrenia susceptibility gene TCF4 alters gene expression and proliferation of progenitor cells from the developing human neocortex.

PubMed

Hill, Matthew J; Killick, Richard; Navarrete, Katherinne; Maruszak, Aleksandra; McLaughlin, Gemma M; Williams, Brenda P; Bray, Nicholas J

2017-05-01

Common variants in the TCF4 gene are among the most robustly supported genetic risk factors for schizophrenia. Rare TCF4 deletions and loss-of-function point mutations cause Pitt-Hopkins syndrome, a developmental disorder associated with severe intellectual disability. To explore molecular and cellular mechanisms by which TCF4 perturbation could interfere with human cortical development, we experimentally reduced the endogenous expression of TCF4 in a neural progenitor cell line derived from the developing human cerebral cortex using RNA interference. Effects on genome-wide gene expression were assessed by microarray, followed by Gene Ontology and pathway analysis of differentially expressed genes. We tested for genetic association between the set of differentially expressed genes and schizophrenia using genome-wide association study data from the Psychiatric Genomics Consortium and competitive gene set analysis (MAGMA). Effects on cell proliferation were assessed using high content imaging. Genes that were differentially expressed following TCF4 knockdown were highly enriched for involvement in the cell cycle. There was a nonsignificant trend for genetic association between the differentially expressed gene set and schizophrenia. Consistent with the gene expression data, TCF4 knockdown was associated with reduced proliferation of cortical progenitor cells in vitro. A detailed mechanistic explanation of how TCF4 knockdown alters human neural progenitor cell proliferation is not provided by this study. Our data indicate effects of TCF4 perturbation on human cortical progenitor cell proliferation, a process that could contribute to cognitive deficits in individuals with Pitt-Hopkins syndrome and risk for schizophrenia.

Metabolic Disturbances in Adult-Onset Still's Disease Evaluated Using Liquid Chromatography/Mass Spectrometry-Based Metabolomic Analysis.

PubMed

Chen, Der-Yuan; Chen, Yi-Ming; Chien, Han-Ju; Lin, Chi-Chen; Hsieh, Chia-Wei; Chen, Hsin-Hua; Hung, Wei-Ting; Lai, Chien-Chen

2016-01-01

Liquid chromatography/mass spectrometry (LC/MS)-based comprehensive analysis of metabolic profiles with metabolomics approach has potential diagnostic and predictive implications. However, no metabolomics data have been reported in adult-onset Still's disease (AOSD). This study investigated the metabolomic profiles in AOSD patients and examined their association with clinical characteristics and disease outcome. Serum metabolite profiles were determined on 32 AOSD patients and 30 healthy controls (HC) using ultra-performance liquid chromatography (UPLC)/MS analysis, and the differentially expressed metabolites were quantified using multiple reactions monitoring (MRM)/MS analysis in 44 patients and 42 HC. Pure standards were utilized to confirm the presence of the differentially expressed metabolites. Eighteen differentially expressed metabolites were identified in AOSD patents using LC/MS-based analysis, of which 13 metabolites were validated by MRM/MS analysis. Among them, serum levels of lysoPC(18:2), urocanic acid and indole were significantly lower, and L-phenylalanine levels were significantly higher in AOSD patients compared with HC. Moreover, serum levels of lysoPC(18:2), PhePhe, uridine, taurine, L-threonine, and (R)-3-Hydroxy-hexadecanoic acid were significantly correlated with disease activity scores (all p<0.05) in AOSD patients. A different clustering of metabolites was associated with a different disease outcome, with significantly lower levels of isovalerylsarcosine observed in patients with chronic articular pattern (median, 77.0AU/ml) compared with monocyclic (341.5AU/ml, p<0.01) or polycyclic systemic pattern (168.0AU/ml, p<0.05). Thirteen differentially expressed metabolites identified and validated in AOSD patients were shown to be involved in five metabolic pathways. Significant associations of metabolic profiles with disease activity and outcome of AOSD suggest their involvement in AOSD pathogenesis.

Blockade of LGR4 inhibits proliferation and odonto/osteogenic differentiation of stem cells from apical papillae.

PubMed

Zhou, Meng; Guo, Shuyu; Yuan, Lichan; Zhang, Yuxin; Zhang, Mengnan; Chen, Huimin; Lu, Mengting; Yang, Jianrong; Ma, Junqing

2017-12-01

During tooth root development, stem cells from apical papillae (SCAPs) are indispensable, and their abilities of proliferation, migration and odontoblast differentiation are linked to root formation. Leucine-rich repeat-containing GPCR 4 (LGR4) modulates the biological processes of proliferation and differentiation in multiple stem cells. In this study, we showed that LGR4 is expressed in all odontoblast cell lineage cells and Hertwig's epithelial root sheath (HERS) during the mouse root formation in vivo. In vitro we determined that LGR4 is involved in the Wnt/β-catenin signaling pathway regulating proliferation and odonto/osteogenic differentiation of SCAPs. Quantitative reverse-transcription PCR (qRT-PCR) confirmed that LGR4 is expressed during odontogenic differentiation of SCAPs. CCK8 assays and in vitro scratch tests, together with cell cycle flow cytometric analysis, demonstrated that downregulation of LGR4 inhibited SCAPs proliferation, delayed migration and arrested cell cycle progression at the S and G2/M phases. ALP staining revealed that blockade of LGR4 decreased ALP activity. QRT-PCR and Western blot analysis demonstrated that LGR4 silencing reduced the expression of odonto/osteogenic markers (RUNX2, OSX, OPN, OCN and DSPP). Further Western blot and immunofluorescence studies clarified that inhibition of LGR4 disrupted β-catenin stabilization. Taken together, downregulation of LGR4 gene expression inhibited SCAPs proliferation, migration and odonto/osteogenic differentiation by blocking the Wnt/β-catenin signaling pathway. These results indicate that LGR4 might play a vital role in SCAPs proliferation and odontoblastic differentiation.

Cassava (Manihot esculenta Krantz) genome harbors KNOX genes differentially expressed during storage root development.

PubMed

Guo, D; Li, H L; Tang, X; Peng, S Q

2014-12-18

In plants, homeodomain proteins play a critical role in regulating various aspects of plant growth and development. KNOX proteins are members of the homeodomain protein family. The KNOX transcription factors have been reported from Arabidopsis, rice, and other higher plants. The recent publication of the draft genome sequence of cassava (Manihot esculenta Krantz) has allowed a genome-wide search for M. esculenta KNOX (MeKNOX) transcription factors and the comparison of these positively identified proteins with their homologs in model plants. In the present study, we identified 12 MeKNOX genes in the cassava genome and grouped them into two distinct subfamilies based on their domain composition and phylogenetic analysis. Furthermore, semi-quantitative reverse transcription polymerase chain reaction analysis was performed to elucidate the expression profiles of these genes in different tissues and during various stages of root development. The analysis of MeKNOX expression profiles of indicated that 12 MeKNOX genes display differential expressions either in their transcript abundance or expression patterns.

Coordination of Myeloid Differentiation with Reduced Cell Cycle Progression by PU.1 Induction of MicroRNAs Targeting Cell Cycle Regulators and Lipid Anabolism.

PubMed

Solomon, Lauren A; Podder, Shreya; He, Jessica; Jackson-Chornenki, Nicholas L; Gibson, Kristen; Ziliotto, Rachel G; Rhee, Jess; DeKoter, Rodney P

2017-05-15

During macrophage development, myeloid progenitor cells undergo terminal differentiation coordinated with reduced cell cycle progression. Differentiation of macrophages from myeloid progenitors is accompanied by increased expression of the E26 transformation-specific transcription factor PU.1. Reduced PU.1 expression leads to increased proliferation and impaired differentiation of myeloid progenitor cells. It is not understood how PU.1 coordinates macrophage differentiation with reduced cell cycle progression. In this study, we utilized cultured PU.1-inducible myeloid cells to perform genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) analysis coupled with gene expression analysis to determine targets of PU.1 that may be involved in regulating cell cycle progression. We found that genes encoding cell cycle regulators and enzymes involved in lipid anabolism were directly and inducibly bound by PU.1 although their steady-state mRNA transcript levels were reduced. Inhibition of lipid anabolism was sufficient to reduce cell cycle progression in these cells. Induction of PU.1 reduced expression of E2f1 , an important activator of genes involved in cell cycle and lipid anabolism, indirectly through microRNA 223. Next-generation sequencing identified microRNAs validated as targeting cell cycle and lipid anabolism for downregulation. These results suggest that PU.1 coordinates cell cycle progression with differentiation through induction of microRNAs targeting cell cycle regulators and lipid anabolism. Copyright © 2017 American Society for Microbiology.

Altered gene expression in dry age-related macular degeneration suggests early loss of choroidal endothelial cells.

PubMed

Whitmore, S Scott; Braun, Terry A; Skeie, Jessica M; Haas, Christine M; Sohn, Elliott H; Stone, Edwin M; Scheetz, Todd E; Mullins, Robert F

2013-01-01

Age-related macular degeneration (AMD) is a major cause of blindness in developed countries. The molecular pathogenesis of early events in AMD is poorly understood. We investigated differential gene expression in samples of human retinal pigment epithelium (RPE) and choroid from early AMD and control maculas with exon-based arrays. Gene expression levels in nine human donor eyes with early AMD and nine control human donor eyes were assessed using Affymetrix Human Exon ST 1.0 arrays. Two controls did not pass quality control and were removed. Differentially expressed genes were annotated using the Database for Annotation, Visualization and Integrated Discovery (DAVID), and gene set enrichment analysis (GSEA) was performed on RPE-specific and endothelium-associated gene sets. The complement factor H (CFH) genotype was also assessed, and differential expression was analyzed regarding high AMD risk (YH/HH) and low AMD risk (YY) genotypes. Seventy-five genes were identified as differentially expressed (raw p value <0.01; ≥50% fold change, mean log2 expression level in AMD or control ≥ median of all average gene expression values); however, no genes were significant (adj. p value <0.01) after correction for multiple hypothesis testing. Of 52 genes with decreased expression in AMD (fold change <0.5; raw p value <0.01), 18 genes were identified by DAVID analysis as associated with vision or neurologic processes. The GSEA of the RPE-associated and endothelium-associated genes revealed a significant decrease in genes typically expressed by endothelial cells in the early AMD group compared to controls, consistent with previous histologic and proteomic studies. Analysis of the CFH genotype indicated decreased expression of ADAMTS9 in eyes with high-risk genotypes (fold change = -2.61; raw p value=0.0008). GSEA results suggest that RPE transcripts are preserved or elevated in early AMD, concomitant with loss of endothelial cell marker expression. These results are consistent with the notion that choroidal endothelial cell dropout or dedifferentiation occurs early in the pathogenesis of AMD.

Expression and clinical significance of PIWIL2 in hilar cholangiocarcinoma tissues and cell lines.

PubMed

Chen, Y J; Xiong, X F; Wen, S Q; Tian, L; Cheng, W L; Qi, Y Q

2015-06-26

The objective of this study was to explore the relationship between PIWI-like protein 2 (PIWIL2) and clinicopathological charac-teristics and prognosis after radical resection. To accomplish this, we analyzed PIWIL2 expression in hilar cholangiocarcinoma tissues and cell lines. PIWIL2 expression was detected by immunohistochemistry in 41 hilar cholangiocarcinoma samples and 10 control tissues. Western blotting and immunocytofluorescence were used to investigate PIWIL2 expression in the cholangiocarcinoma cell line QBC939 and the bile duct epithelial cell line HIBEpic. Univariate and multivariate surviv-al analyses were performed using the Kaplan-Meier method for hilar cholangiocarcinoma patients who underwent radical resection. PIWIL2 expression was significantly higher in the hilar cholangiocarcinoma tissues and QBC939 cells than in control tissues and HIBEpic cells, respectively (P < 0.05). Poorly and moderately differentiated cholan-giocarcinoma tissues had significantly higher PIWIL2 expression than well-differentiated tissues (P < 0.05). Univariate analysis demonstrated that high PIWIL2 expression was associated with shorter survival time after radical resection (P < 0.05). Multivariate analysis showed that PI-WIL2 expression was an independent prognostic factor after radical re-section of hilar cholangiocarcinoma (P < 0.05). PIWIL2 expression was also associated with tumor-node-metastasis stage and differentiation. PIWIL2 was an independent prognostic factor after radical resection of hilar cholangiocarcinoma.

Integrative transcriptome network analysis of iPSC-derived neurons from schizophrenia and schizoaffective disorder patients with 22q11.2 deletion.

PubMed

Lin, Mingyan; Pedrosa, Erika; Hrabovsky, Anastasia; Chen, Jian; Puliafito, Benjamin R; Gilbert, Stephanie R; Zheng, Deyou; Lachman, Herbert M

2016-11-15

Individuals with 22q11.2 Deletion Syndrome (22q11.2 DS) are a specific high-risk group for developing schizophrenia (SZ), schizoaffective disorder (SAD) and autism spectrum disorders (ASD). Several genes in the deleted region have been implicated in the development of SZ, e.g., PRODH and DGCR8. However, the mechanistic connection between these genes and the neuropsychiatric phenotype remains unclear. To elucidate the molecular consequences of 22q11.2 deletion in early neural development, we carried out RNA-seq analysis to investigate gene expression in early differentiating human neurons derived from induced pluripotent stem cells (iPSCs) of 22q11.2 DS SZ and SAD patients. Eight cases (ten iPSC-neuron samples in total including duplicate clones) and seven controls (nine in total including duplicate clones) were subjected to RNA sequencing. Using a systems level analysis, differentially expressed genes/gene-modules and pathway of interests were identified. Lastly, we related our findings from in vitro neuronal cultures to brain development by mapping differentially expressed genes to BrainSpan transcriptomes. We observed ~2-fold reduction in expression of almost all genes in the 22q11.2 region in SZ (37 genes reached p-value < 0.05, 36 of which reached a false discovery rate < 0.05). Outside of the deleted region, 745 genes showed significant differences in expression between SZ and control neurons (p < 0.05). Function enrichment and network analysis of the differentially expressed genes uncovered converging evidence on abnormal expression in key functional pathways, such as apoptosis, cell cycle and survival, and MAPK signaling in the SZ and SAD samples. By leveraging transcriptome profiles of normal human brain tissues across human development into adulthood, we showed that the differentially expressed genes converge on a sub-network mediated by CDC45 and the cell cycle, which would be disrupted by the 22q11.2 deletion during embryonic brain development, and another sub-network modulated by PRODH, which could contribute to disruption of brain function during adolescence. This study has provided evidence for disruption of potential molecular events in SZ patient with 22q11.2 deletion and related our findings from in vitro neuronal cultures to functional perturbations that can occur during brain development in SZ.

Cell population structure prior to bifurcation predicts efficiency of directed differentiation in human induced pluripotent cells

PubMed Central

Bargaje, Rhishikesh; Trachana, Kalliopi; Shelton, Martin N.; McGinnis, Christopher S.; Zhou, Joseph X.; Chadick, Cora; Cook, Savannah; Cavanaugh, Christopher; Huang, Sui; Hood, Leroy

2017-01-01

Steering the differentiation of induced pluripotent stem cells (iPSCs) toward specific cell types is crucial for patient-specific disease modeling and drug testing. This effort requires the capacity to predict and control when and how multipotent progenitor cells commit to the desired cell fate. Cell fate commitment represents a critical state transition or “tipping point” at which complex systems undergo a sudden qualitative shift. To characterize such transitions during iPSC to cardiomyocyte differentiation, we analyzed the gene expression patterns of 96 developmental genes at single-cell resolution. We identified a bifurcation event early in the trajectory when a primitive streak-like cell population segregated into the mesodermal and endodermal lineages. Before this branching point, we could detect the signature of an imminent critical transition: increase in cell heterogeneity and coordination of gene expression. Correlation analysis of gene expression profiles at the tipping point indicates transcription factors that drive the state transition toward each alternative cell fate and their relationships with specific phenotypic readouts. The latter helps us to facilitate small molecule screening for differentiation efficiency. To this end, we set up an analysis of cell population structure at the tipping point after systematic variation of the protocol to bias the differentiation toward mesodermal or endodermal cell lineage. We were able to predict the proportion of cardiomyocytes many days before cells manifest the differentiated phenotype. The analysis of cell populations undergoing a critical state transition thus affords a tool to forecast cell fate outcomes and can be used to optimize differentiation protocols to obtain desired cell populations. PMID:28167799

Cell population structure prior to bifurcation predicts efficiency of directed differentiation in human induced pluripotent cells.

PubMed

Bargaje, Rhishikesh; Trachana, Kalliopi; Shelton, Martin N; McGinnis, Christopher S; Zhou, Joseph X; Chadick, Cora; Cook, Savannah; Cavanaugh, Christopher; Huang, Sui; Hood, Leroy

2017-02-28

Steering the differentiation of induced pluripotent stem cells (iPSCs) toward specific cell types is crucial for patient-specific disease modeling and drug testing. This effort requires the capacity to predict and control when and how multipotent progenitor cells commit to the desired cell fate. Cell fate commitment represents a critical state transition or "tipping point" at which complex systems undergo a sudden qualitative shift. To characterize such transitions during iPSC to cardiomyocyte differentiation, we analyzed the gene expression patterns of 96 developmental genes at single-cell resolution. We identified a bifurcation event early in the trajectory when a primitive streak-like cell population segregated into the mesodermal and endodermal lineages. Before this branching point, we could detect the signature of an imminent critical transition: increase in cell heterogeneity and coordination of gene expression. Correlation analysis of gene expression profiles at the tipping point indicates transcription factors that drive the state transition toward each alternative cell fate and their relationships with specific phenotypic readouts. The latter helps us to facilitate small molecule screening for differentiation efficiency. To this end, we set up an analysis of cell population structure at the tipping point after systematic variation of the protocol to bias the differentiation toward mesodermal or endodermal cell lineage. We were able to predict the proportion of cardiomyocytes many days before cells manifest the differentiated phenotype. The analysis of cell populations undergoing a critical state transition thus affords a tool to forecast cell fate outcomes and can be used to optimize differentiation protocols to obtain desired cell populations.

Gene expression profiling of human mesenchymal stem cells derived from bone marrow during expansion and osteoblast differentiation.

PubMed

Kulterer, Birgit; Friedl, Gerald; Jandrositz, Anita; Sanchez-Cabo, Fatima; Prokesch, Andreas; Paar, Christine; Scheideler, Marcel; Windhager, Reinhard; Preisegger, Karl-Heinz; Trajanoski, Zlatko

2007-03-12

Human mesenchymal stem cells (MSC) with the capacity to differentiate into osteoblasts provide potential for the development of novel treatment strategies, such as improved healing of large bone defects. However, their low frequency in bone marrow necessitate ex vivo expansion for further clinical application. In this study we asked if MSC are developing in an aberrant or unwanted way during ex vivo long-term cultivation and if artificial cultivation conditions exert any influence on their stem cell maintenance. To address this question we first developed human oligonucleotide microarrays with 30.000 elements and then performed large-scale expression profiling of long-term expanded MSC and MSC during differentiation into osteoblasts. The results showed that MSC did not alter their osteogenic differentiation capacity, surface marker profile, and the expression profiles of MSC during expansion. Microarray analysis of MSC during osteogenic differentiation identified three candidate genes for further examination and functional analysis: ID4, CRYAB, and SORT1. Additionally, we were able to reconstruct the three developmental phases during osteoblast differentiation: proliferation, matrix maturation, and mineralization, and illustrate the activation of the SMAD signaling pathways by TGF-beta2 and BMPs. With a variety of assays we could show that MSC represent a cell population which can be expanded for therapeutic applications.

Exosomes from bulk and stem cells from human prostate cancer have a differential microRNA content that contributes cooperatively over local and pre-metastatic niche.

PubMed

Sánchez, Catherine A; Andahur, Eliana I; Valenzuela, Rodrigo; Castellón, Enrique A; Fullá, Juan A; Ramos, Christian G; Triviño, Juan C

2016-01-26

The different prostate cancer (PCa) cell populations (bulk and cancer stem cells, CSCs) release exosomes that contain miRNAs that could modify the local or premetastatic niche. The analysis of the differential expression of miRNAs in exosomes allows evaluating the differential biological effect of both populations on the niche, and the identification of potential biomarkers and therapeutic targets. Five PCa primary cell cultures were established to originate bulk and CSCs cultures. From them, exosomes were purified by precipitation for miRNAs extraction to perform a comparative profile of miRNAs by next generation sequencing in an Illumina platform. 1839 miRNAs were identified in the exosomes. Of these 990 were known miRNAs, from which only 19 were significantly differentially expressed: 6 were overexpressed in CSCs and 13 in bulk cells exosomes. miR-100-5p and miR-21-5p were the most abundant miRNAs. Bioinformatics analysis indicated that differentially expressed miRNAs are highly related with PCa carcinogenesis, fibroblast proliferation, differentiation and migration, and angiogenesis. Besides, miRNAs from bulk cells affects osteoblast differentiation. Later, their effect was evaluated in normal prostate fibroblasts (WPMY-1) where transfection with miR-100-5p, miR-21-5p and miR-139-5p increased the expression of metalloproteinases (MMPs) -2, -9 and -13 and RANKL and fibroblast migration. The higher effect was achieved with miR21 transfection. As conclusion, miRNAs have a differential pattern between PCa bulk and CSCs exosomes that act collaboratively in PCa progression and metastasis. The most abundant miRNAs in PCa exosomes are interesting potential biomarkers and therapeutic targets.

MicroRNA-363 negatively regulates the left ventricular determining transcription factor HAND1 in human embryonic stem cell-derived cardiomyocytes.

PubMed

Wagh, Vilas; Pomorski, Alexander; Wilschut, Karlijn J; Piombo, Sebastian; Bernstein, Harold S

2014-06-06

Posttranscriptional control of mRNA by microRNA (miRNA) has been implicated in the regulation of diverse biologic processes from directed differentiation of stem cells through organism development. We describe a unique pathway by which miRNA regulates the specialized differentiation of cardiomyocyte (CM) subtypes. We differentiated human embryonic stem cells (hESCs) to cardiac progenitor cells and functional CMs, and characterized the regulated expression of specific miRNAs that target transcriptional regulators of left/right ventricular-subtype specification. From >900 known human miRNAs in hESC-derived cardiac progenitor cells and functional CMs, a subset of differentially expressed cardiac miRNAs was identified, and in silico analysis predicted highly conserved binding sites in the 3'-untranslated regions (3'UTRs) of Hand-and-neural-crest-derivative-expressed (HAND) genes 1 and 2 that are involved in left and right ventricular development. We studied the temporal and spatial expression patterns of four miRNAs in differentiating hESCs, and found that expression of miRNA (miR)-363, miR-367, miR-181a, and miR-181c was specific for stage and site. Further analysis showed that miR-363 overexpression resulted in downregulation of HAND1 mRNA and protein levels. A dual luciferase reporter assay demonstrated functional interaction of miR-363 with the full-length 3'UTR of HAND1. Expression of anti-miR-363 in-vitro resulted in enrichment for HAND1-expressing CM subtype populations. We also showed that BMP4 treatment induced the expression of HAND2 with less effect on HAND1, whereas miR-363 overexpression selectively inhibited HAND1. These data show that miR-363 negatively regulates the expression of HAND1 and suggest that suppression of miR-363 could provide a novel strategy for generating functional left-ventricular CMs.

Variable DAXX gene methylation is a common feature of placental trophoblast differentiation, preeclampsia, and response to hypoxia.

PubMed

Novakovic, Boris; Evain-Brion, Danièle; Murthi, Padma; Fournier, Thiery; Saffery, Richard

2017-06-01

Placental functioning relies on the appropriate differentiation of progenitor villous cytotrophoblasts (CTBs) into extravillous cytotrophoblasts (EVCTs), including invasive EVCTs, and the multinucleated syncytiotrophoblast (ST) layer. This is accompanied by a general move away from a proliferative, immature phenotype. Genome-scale expression studies have provided valuable insight into genes that are associated with the shift to both an invasive EVCT and ST phenotype, whereas genome-scale DNA methylation analysis has shown that differentiation to ST involves widespread methylation shifts, which are counteracted by low oxygen. In the current study, we sought to identify DNA methylation variation that is associated with transition from CTB to ST in vitro and from a noninvasive to invasive EVCT phenotype after culture on Matrigel. Of the several hundred differentially methylated regions that were identified in each comparison, the majority showed a loss of methylation with differentiation. This included a large differentially methylated region (DMR) in the gene body of death domain-associated protein 6 ( DAXX ), which lost methylation during both CTB syncytialization to ST and EVCT differentiation to invasive EVCT. Comparison to publicly available methylation array data identified the same DMR as among the most consistently differentially methylated genes in placental samples from preeclampsia pregnancies. Of interest, in vitro culture of CTB or ST in low oxygen increases methylation in the same region, which correlates with delayed differentiation. Analysis of combined epigenomics signatures confirmed DAXX DMR as a likely regulatory element, and direct gene expression analysis identified a positive association between methylation at this site and DAXX expression levels. The widespread dynamic nature of DAXX methylation in association with trophoblast differentiation and placenta-associated pathologies is consistent with an important role for this gene in proper placental development and function.-Novakovic, B., Evain-Brion, D., Murthi, P., Fournier, T., Saffery, R. Variable DAXX gene methylation is a common feature of placental trophoblast differentiation, preeclampsia, and response to hypoxia. © FASEB.

Manganese-Induced Neurotoxicity and Alterations in Gene Expression in Human Neuroblastoma SH-SY5Y Cells.

PubMed

Gandhi, Deepa; Sivanesan, Saravanadevi; Kannan, Krishnamurthi

2018-06-01

Manganese (Mn) is an essential trace element required for many physiological functions including proper biochemical and cellular functioning of the central nervous system (CNS). However, exposure to excess level of Mn through occupational settings or from environmental sources has been associated with neurotoxicity. The cellular and molecular mechanism of Mn-induced neurotoxicity remains unclear. In the current study, we investigated the effects of 30-day exposure to a sub-lethal concentration of Mn (100 μM) in human neuroblastoma cells (SH-SY5Y) using transcriptomic approach. Microarray analysis revealed differential expression of 1057 transcripts in Mn-exposed SH-SY5Y cells as compared to control cells. Gene functional annotation cluster analysis exhibited that the differentially expressed genes were associated with several biological pathways. Specifically, genes involved in neuronal pathways including neuron differentiation and development, regulation of neurogenesis, synaptic transmission, and neuronal cell death (apoptosis) were found to be significantly altered. KEGG pathway analysis showed upregulation of p53 signaling pathways and neuroactive ligand-receptor interaction pathways, and downregulation of neurotrophin signaling pathway. On the basis of the gene expression profile, possible molecular mechanisms underlying Mn-induced neuronal toxicity were predicted.

Transcriptome Analysis of Thermal Parthenogenesis of the Domesticated Silkworm.

PubMed

Liu, Peigang; Wang, Yongqiang; Du, Xin; Yao, Lusong; Li, Fengbo; Meng, Zhiqi

2015-01-01

Thermal induction of parthenogenesis (also known as thermal parthenogenesis) in silkworms is an important technique that has been used in artificial insemination, expansion of hybridization, transgenesis and sericultural production; however, the exact mechanisms of this induction remain unclear. This study aimed to investigate the gene expression profile in silkworms undergoing thermal parthenogenesis using RNA-seq analysis. The transcriptome profiles indicated that in non-induced and induced eggs, the numbers of differentially expressed genes (DEGs) for the parthenogenetic line (PL) and amphigenetic line (AL) were 538 and 545, respectively, as determined by fold-change ≥ 2. Gene ontology (GO) analysis showed that DEGs between two lines were mainly involved in reproduction, formation of chorion, female gamete generation and cell development pathways. Upregulation of many chorion genes in AL suggests that the maturation rate of AL eggs was slower than PL eggs. Some DEGs related to reactive oxygen species removal, DNA repair and heat shock response were differentially expressed between the two lines, such as MPV-17, REV1 and HSP68. These results supported the view that a large fraction of genes are differentially expressed between PL and AL, which offers a new approach to identifying the molecular mechanism of silkworm thermal parthenogenesis.

Transcriptome Analysis of Thermal Parthenogenesis of the Domesticated Silkworm

PubMed Central

Du, Xin; Yao, Lusong; Li, Fengbo; Meng, Zhiqi

2015-01-01

Thermal induction of parthenogenesis (also known as thermal parthenogenesis) in silkworms is an important technique that has been used in artificial insemination, expansion of hybridization, transgenesis and sericultural production; however, the exact mechanisms of this induction remain unclear. This study aimed to investigate the gene expression profile in silkworms undergoing thermal parthenogenesis using RNA-seq analysis. The transcriptome profiles indicated that in non-induced and induced eggs, the numbers of differentially expressed genes (DEGs) for the parthenogenetic line (PL) and amphigenetic line (AL) were 538 and 545, respectively, as determined by fold-change ≥ 2. Gene ontology (GO) analysis showed that DEGs between two lines were mainly involved in reproduction, formation of chorion, female gamete generation and cell development pathways. Upregulation of many chorion genes in AL suggests that the maturation rate of AL eggs was slower than PL eggs. Some DEGs related to reactive oxygen species removal, DNA repair and heat shock response were differentially expressed between the two lines, such as MPV-17, REV1 and HSP68. These results supported the view that a large fraction of genes are differentially expressed between PL and AL, which offers a new approach to identifying the molecular mechanism of silkworm thermal parthenogenesis. PMID:26274803

Transcriptomic data analysis and differential gene expression of antioxidant pathways in king penguin juveniles (Aptenodytes patagonicus) before and after acclimatization to marine life.

PubMed

Rey, Benjamin; Dégletagne, Cyril; Duchamp, Claude

2016-12-01

In this article, we present differentially expressed gene profiles in the pectoralis muscle of wild juvenile king penguins that were either naturally acclimated to cold marine environment or experimentally immersed in cold water as compared with penguin juveniles that never experienced cold water immersion. Transcriptomic data were obtained by hybridizing penguins total cDNA on Affymetrix GeneChip Chicken Genome arrays and analyzed using maxRS algorithm , " Transcriptome analysis in non-model species: a new method for the analysis of heterologous hybridization on microarrays " (Dégletagne et al., 2010) [1] . We focused on genes involved in multiple antioxidant pathways. For better clarity, these differentially expressed genes were clustered into six functional groups according to their role in controlling redox homeostasis. The data are related to a comprehensive research study on the ontogeny of antioxidant functions in king penguins, "Hormetic response triggers multifaceted anti-oxidant strategies in immature king penguins (Aptenodytes patagonicus)" (Rey et al., 2016) [2] . The raw microarray dataset supporting the present analyses has been deposited at the Gene Expression Omnibus (GEO) repository under accessions GEO: GSE17725 and GEO: GSE82344.

An analysis of gene expression in PTSD implicates genes involved in the glucocorticoid receptor pathway and neural responses to stress

PubMed Central

Logue, Mark W.; Smith, Alicia K.; Baldwin, Clinton; Wolf, Erika J.; Guffanti, Guia; Ratanatharathorn, Andrew; Stone, Annjanette; Schichman, Steven A.; Humphries, Donald; Binder, Elisabeth B.; Arloth, Janine; Menke, Andreas; Uddin, Monica; Wildman, Derek; Galea, Sandro; Aiello, Allison E.; Koenen, Karestan C.; Miller, Mark W.

2015-01-01

We examined the association between posttraumatic stress disorder (PTSD) and gene expression using whole blood samples from a cohort of trauma-exposed white non-Hispanic male veterans (115 cases and 28 controls). 10,264 probes of genes and gene transcripts were analyzed. We found 41 that were differentially expressed in PTSD cases versus controls (multiple-testing corrected p<0.05). The most significant was DSCAM, a neurological gene expressed widely in the developing brain and in the amygdala and hippocampus of the adult brain. We then examined the 41 differentially expressed genes in a meta-analysis using two replication cohorts and found significant associations with PTSD for 7 of the 41 (p<0.05), one of which (ATP6AP1L) survived multiple-testing correction. There was also broad evidence of overlap across the discovery and replication samples for the entire set of genes implicated in the discovery data based on the direction of effect and an enrichment of p<0.05 significant probes beyond what would be expected under the null. Finally, we found that the set of differentially expressed genes from the discovery sample was enriched for genes responsive to glucocorticoid signaling with most showing reduced expression in PTSD cases compared to controls. PMID:25867994

EMMPRIN expression in oral squamous cell carcinomas: correlation with tumor proliferation and patient survival.

PubMed

Monteiro, Luís Silva; Delgado, Maria Leonor; Ricardo, Sara; Garcez, Fernanda; do Amaral, Barbas; Pacheco, José Júlio; Lopes, Carlos; Bousbaa, Hassan

2014-01-01

The aim of our study was to explore the clinicopathological and prognostic significance of extracellular matrix metalloproteinase inducer (EMMPRIN) expression in oral squamous cell carcinomas (OSCC), and its relation with the proliferative tumor status of OSCC. We examined EMMPRIN and Ki-67 proteins expression by immunohistochemistry in 74 cases with OSCC. Statistical analysis was conducted to examine their clinicopathological and prognostic significance in OSCC. EMMPRIN membrane expression was observed in all cases, with both membrane and cytoplasmic tumor expression in 61 cases (82.4%). EMMPRIN overexpression was observed in 56 cases (75.7%). Moderately or poorly differentiated tumors showed EMMPRIN overexpression more frequently than well-differentiated tumors (P = 0.002). Overexpression of EMMPRIN was correlated with high Ki-67 expression (P = 0.004). In the multivariate analysis, EMMPRIN overexpression reveals an adverse independent prognostic value for cancer-specific survival (CSS) (P = 0.034). Our results reveal that EMMPRIN protein is overexpressed in more than two-thirds of OSCC cases, especially in high proliferative and less differentiated tumors. The independent value of EMMPRIN overexpression in CSS suggests that this protein could be used as an important biological prognostic marker for patients with OSCC. Moreover, the high expression of EMMPRIN makes it a possible therapeutic target in OSCC patients.

Altered Molecular Expression of the TLR4/NF-κB Signaling Pathway in Mammary Tissue of Chinese Holstein Cattle with Mastitis

PubMed Central

Wu, Jie; Li, Lian; Sun, Yu; Huang, Shuai; Tang, Juan; Yu, Pan; Wang, Genlin

2015-01-01

Toll-like receptor 4 (TLR4) mediated activation of the nuclear transcription factor κB (NF-κB) signaling pathway by mastitis initiates expression of genes associated with inflammation and the innate immune response. In this study, the profile of mastitis-induced differential gene expression in the mammary tissue of Chinese Holstein cattle was investigated by Gene-Chip microarray and bioinformatics. The microarray results revealed that 79 genes associated with the TLR4/NF-κB signaling pathway were differentially expressed. Of these genes, 19 were up-regulated and 29 were down-regulated in mastitis tissue compared to normal, healthy tissue. Statistical analysis of transcript and protein level expression changes indicated that 10 genes, namely TLR4, MyD88, IL-6, and IL-10, were up-regulated, while, CD14, TNF-α, MD-2, IL-β, NF-κB, and IL-12 were significantly down-regulated in mastitis tissue in comparison with normal tissue. Analyses using bioinformatics database resources, such as the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and the Gene Ontology Consortium (GO) for term enrichment analysis, suggested that these differently expressed genes implicate different regulatory pathways for immune function in the mammary gland. In conclusion, our study provides new evidence for better understanding the differential expression and mechanisms of the TLR4 /NF-κB signaling pathway in Chinese Holstein cattle with mastitis. PMID:25706977

Altered molecular expression of the TLR4/NF-κB signaling pathway in mammary tissue of Chinese Holstein cattle with mastitis.

PubMed

Wu, Jie; Li, Lian; Sun, Yu; Huang, Shuai; Tang, Juan; Yu, Pan; Wang, Genlin

2015-01-01

Toll-like receptor 4 (TLR4) mediated activation of the nuclear transcription factor κB (NF-κB) signaling pathway by mastitis initiates expression of genes associated with inflammation and the innate immune response. In this study, the profile of mastitis-induced differential gene expression in the mammary tissue of Chinese Holstein cattle was investigated by Gene-Chip microarray and bioinformatics. The microarray results revealed that 79 genes associated with the TLR4/NF-κB signaling pathway were differentially expressed. Of these genes, 19 were up-regulated and 29 were down-regulated in mastitis tissue compared to normal, healthy tissue. Statistical analysis of transcript and protein level expression changes indicated that 10 genes, namely TLR4, MyD88, IL-6, and IL-10, were up-regulated, while, CD14, TNF-α, MD-2, IL-β, NF-κB, and IL-12 were significantly down-regulated in mastitis tissue in comparison with normal tissue. Analyses using bioinformatics database resources, such as the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and the Gene Ontology Consortium (GO) for term enrichment analysis, suggested that these differently expressed genes implicate different regulatory pathways for immune function in the mammary gland. In conclusion, our study provides new evidence for better understanding the differential expression and mechanisms of the TLR4 /NF-κB signaling pathway in Chinese Holstein cattle with mastitis.

Bioinformatic analysis of the effects and mechanisms of decitabine and cytarabine on acute myeloid leukemia

PubMed Central

Zhou, Shiyong; Liu, Pengfei; Zhang, Huilai

2017-01-01

Acute myeloid leukemia (AML) is a frequently occurring malignant disease of the blood and may result from a variety of genetic disorders. The present study aimed to identify the underlying mechanisms associated with the therapeutic effects of decitabine and cytarabine on AML, using microarray analysis. The microarray datasets GSE40442 and GSE40870 were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) and differentially methylated sites were identified in AML cells treated with decitabine compared with those treated with cytarabine via the Linear Models for Microarray Data package, following data pre-processing. Gene Ontology (GO) analysis of DEGs was performed using the Database for Annotation, Visualization and Integrated Analysis Discovery. Genes corresponding to the differentially methylated sites were obtained using the annotation package of the methylation microarray platform. The overlapping genes were identified, which exhibited the opposite variation trend between gene expression and DNA methylation. Important transcription factor (TF)-gene pairs were screened out, and a regulated network subsequently constructed. A total of 190 DEGs and 540 differentially methylated sites were identified in AML cells treated with decitabine compared with those treated with cytarabine. A total of 36 GO terms of DEGs were enriched, including nucleosomes, protein-DNA complexes and the nucleosome assembly. The 540 differentially methylated sites were located on 240 genes, including the acid-repeat containing protein (ACRC) gene that was additionally differentially expressed. In addition, 60 TF pairs and overlapped methylated sites, and 140 TF-pairs and DEGs were screened out. The regulated network included 68 nodes and 140 TF-gene pairs. The present study identified various genes including ACRC and proliferating cell nuclear antigen, in addition to various TFs, including TATA-box binding protein associated factor 1 and CCCTC-binding factor, which may be potential therapeutic targets of AML. PMID:28498449

Bioinformatic analysis of the effects and mechanisms of decitabine and cytarabine on acute myeloid leukemia.

PubMed

Zhou, Shiyong; Liu, Pengfei; Zhang, Huilai

2017-07-01

Acute myeloid leukemia (AML) is a frequently occurring malignant disease of the blood and may result from a variety of genetic disorders. The present study aimed to identify the underlying mechanisms associated with the therapeutic effects of decitabine and cytarabine on AML, using microarray analysis. The microarray datasets GSE40442 and GSE40870 were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) and differentially methylated sites were identified in AML cells treated with decitabine compared with those treated with cytarabine via the Linear Models for Microarray Data package, following data pre‑processing. Gene Ontology (GO) analysis of DEGs was performed using the Database for Annotation, Visualization and Integrated Analysis Discovery. Genes corresponding to the differentially methylated sites were obtained using the annotation package of the methylation microarray platform. The overlapping genes were identified, which exhibited the opposite variation trend between gene expression and DNA methylation. Important transcription factor (TF)‑gene pairs were screened out, and a regulated network subsequently constructed. A total of 190 DEGs and 540 differentially methylated sites were identified in AML cells treated with decitabine compared with those treated with cytarabine. A total of 36 GO terms of DEGs were enriched, including nucleosomes, protein‑DNA complexes and the nucleosome assembly. The 540 differentially methylated sites were located on 240 genes, including the acid‑repeat containing protein (ACRC) gene that was additionally differentially expressed. In addition, 60 TF pairs and overlapped methylated sites, and 140 TF‑pairs and DEGs were screened out. The regulated network included 68 nodes and 140 TF‑gene pairs. The present study identified various genes including ACRC and proliferating cell nuclear antigen, in addition to various TFs, including TATA‑box binding protein associated factor 1 and CCCTC‑binding factor, which may be potential therapeutic targets of AML.

Protein half-life determines expression of proteostatic networks in podocyte differentiation.

PubMed

Schroeter, Christina B; Koehler, Sybille; Kann, Martin; Schermer, Bernhard; Benzing, Thomas; Brinkkoetter, Paul T; Rinschen, Markus M

2018-04-25

Podocytes are highly specialized, epithelial, postmitotic cells, which maintain the renal filtration barrier. When adapting to considerable metabolic and mechanical stress, podocytes need to accurately maintain their proteome. Immortalized podocyte cell lines are a widely used model for studying podocyte biology in health and disease in vitro. In this study, we performed a comprehensive proteomic analysis of the cultured human podocyte proteome in both proliferative and differentiated conditions at a depth of >7000 proteins. Similar to mouse podocytes, human podocyte differentiation involved a shift in proteostasis: undifferentiated podocytes have high expression of proteasomal proteins, whereas differentiated podocytes have high expression of lysosomal proteins. Additional analyses with pulsed stable-isotope labeling by amino acids in cell culture and protein degradation assays determined protein dynamics and half-lives. These studies unraveled a globally increased stability of proteins in differentiated podocytes. Mitochondrial, cytoskeletal, and membrane proteins were stabilized, particularly in differentiated podocytes. Importantly, protein half-lives strongly contributed to protein abundance in each state. These data suggest that regulation of protein turnover of particular cellular functions determines podocyte differentiation, a paradigm involving mitophagy and, potentially, of importance in conditions of increased podocyte stress and damage.-Schroeter, C. B., Koehler, S., Kann, M., Schermer, B., Benzing, T., Brinkkoetter, P. T., Rinschen, M. M. Protein half-life determines expression of proteostatic networks in podocyte differentiation.

Surprisal analysis of genome-wide transcript profiling identifies differentially expressed genes and pathways associated with four growth conditions in the microalga Chlamydomonas.

PubMed

Bogaert, Kenny A; Manoharan-Basil, Sheeba S; Perez, Emilie; Levine, Raphael D; Remacle, Francoise; Remacle, Claire

2018-01-01

The usual cultivation mode of the green microalga Chlamydomonas is liquid medium and light. However, the microalga can also be grown on agar plates and in darkness. Our aim is to analyze and compare gene expression of cells cultivated in these different conditions. For that purpose, RNA-seq data are obtained from Chlamydomonas samples of two different labs grown in four environmental conditions (agar@light, agar@dark, liquid@light, liquid@dark). The RNA seq data are analyzed by surprisal analysis, which allows the simultaneous meta-analysis of all the samples. First we identify a balance state, which defines a state where the expression levels are similar in all the samples irrespectively of their growth conditions, or lab origin. In addition our analysis identifies additional constraints needed to quantify the deviation with respect to the balance state. The first constraint differentiates the agar samples versus the liquid ones; the second constraint the dark samples versus the light ones. The two constraints are almost of equal importance. Pathways involved in stress responses are found in the agar phenotype while the liquid phenotype comprises ATP and NADH production pathways. Remodeling of membrane is suggested in the dark phenotype while photosynthetic pathways characterize the light phenotype. The same trends are also present when performing purely statistical analysis such as K-means clustering and differentially expressed genes.

Flow cytometric analysis of immunoglobulin heavy chain expression in B-cell lymphoma and reactive lymphoid hyperplasia

PubMed Central

Grier, David D; Al-Quran, Samer Z; Cardona, Diana M; Li, Ying; Braylan, Raul C

2012-01-01

The diagnosis of B-cell lymphoma (BCL) is often dependent on the detection of clonal immunoglobulin (Ig) light chain expression. In some BCLs, the determination of clonality based on Ig light chain restriction may be difficult. The aim of our study was to assess the utility of flow cytometric analysis of surface Ig heavy chain (HC) expression in lymphoid tissues in distinguishing lymphoid hyperplasias from BCLs, and also differentiating various BCL subtypes. HC expression on B-cells varied among different types of hyperplasias. In follicular hyperplasia, IgM and IgD expression was high in mantle cells while germinal center cells showed poor HC expression. In other hyperplasias, B cell compartments were blurred but generally showed high IgD and IgM expression. Compared to hyperplasias, BCLs varied in IgM expression. Small lymphocytic lymphomas had lower IgM expression than mantle cell lymphomas. Of importance, IgD expression was significantly lower in BCLs than in hyperplasias, a finding that can be useful in differentiating lymphoma from reactive processes. PMID:22400070

Expression profiling of snoRNAs in normal hematopoiesis and AML

PubMed Central

Warner, Wayne A.; Spencer, David H.; Trissal, Maria; White, Brian S.; Helton, Nichole; Ley, Timothy J.

2018-01-01

Small nucleolar RNAs (snoRNAs) are noncoding RNAs that contribute to ribosome biogenesis and RNA splicing by modifying ribosomal RNA and spliceosome RNAs, respectively. We optimized a next-generation sequencing approach and a custom analysis pipeline to identify and quantify expression of snoRNAs in acute myeloid leukemia (AML) and normal hematopoietic cell populations. We show that snoRNAs are expressed in a lineage- and development-specific fashion during hematopoiesis. The most striking examples involve snoRNAs located in 2 imprinted loci, which are highly expressed in hematopoietic progenitors and downregulated during myeloid differentiation. Although most snoRNAs are expressed at similar levels in AML cells compared with CD34+, a subset of snoRNAs showed consistent differential expression, with the great majority of these being decreased in the AML samples. Analysis of host gene expression, splicing patterns, and whole-genome sequence data for mutational events did not identify transcriptional patterns or genetic alterations that account for these expression differences. These data provide a comprehensive analysis of the snoRNA transcriptome in normal and leukemic cells and should be helpful in the design of studies to define the contribution of snoRNAs to normal and malignant hematopoiesis. PMID:29365324

Macrophage differentiation induced by PMA is mediated by activation of RhoA/ROCK signaling.

PubMed

Yang, Lifeng; Dai, Fan; Tang, Lian; Le, Yulan; Yao, Wenjuan

2017-01-01

In order to investigate the effects of RhoA/ROCK signaling in macrophage differentiation, we used 100 ng/mL PMA to induce macrophage differentiation from U937 cells in vitro. The observation of cell morphology and the expression of CD68 and SR-A were performed to confirm the differentiation induced by PMA. Western blot analysis showed that the expression of ROCK1 and ROCK2 and the phosphorylation of MYPT1 were significantly increased after PMA treatment. Pulldown assay showed that the activation of RhoA was obviously enhanced when U937 cells were treated with PMA. In order to further demonstrate whether RhoA/ROCK signaling could mediate the macrophage differentiation induced by PMA, we successfully suppressed the expression of RhoA, ROCK1 and ROCK2 by performing siRNA technology in U937 cells, respectively. The macrophage differentiation and the expression of CD68 and SR-A were significantly inhibited by the suppression of RhoA, ROCK1 or ROCK2 in PMA-induced U937 cells, indicating that the macrophage differentiation induced by PMA is associated with RhoA/ROCK signaling pathway. In addition, we pretreated U937 cells with Y27632 (ROCK inhibitor, 20 μM) for 30 min and then observed the macrophage differentiation induced by PMA. The result illustrated that Y27632 pretreatment obviously inhibited PMA-induced differentiation and the expression of CD68 and SR-A. In conclusion, the activation of RhoA/ROCK signaling is responsible for the macrophage differentiation induced by PMA.

Distinct profiles of expressed sequence tags during intestinal regeneration in the sea cucumber Holothuria glaberrima

PubMed Central

Rojas-Cartagena, Carmencita; Ortíz-Pineda, Pablo; Ramírez-Gómez, Francisco; Suárez-Castillo, Edna C.; Matos-Cruz, Vanessa; Rodríguez, Carlos; Ortíz-Zuazaga, Humberto; García-Arrarás, José E.

2010-01-01

Repair and regeneration are key processes for tissue maintenance, and their disruption may lead to disease states. Little is known about the molecular mechanisms that underline the repair and regeneration of the digestive tract. The sea cucumber Holothuria glaberrima represents an excellent model to dissect and characterize the molecular events during intestinal regeneration. To study the gene expression profile, cDNA libraries were constructed from normal, 3-day, and 7-day regenerating intestines of H. glaberrima. Clones were randomly sequenced and queried against the nonredundant protein database at the National Center for Biotechnology Information. RT-PCR analyses were made of several genes to determine their expression profile during intestinal regeneration. A total of 5,173 sequences from three cDNA libraries were obtained. About 46.2, 35.6, and 26.2% of the sequences for the normal, 3-days, and 7-days cDNA libraries, respectively, shared significant similarity with known sequences in the protein database of GenBank but only present 10% of similarity among them. Analysis of the libraries in terms of functional processes, protein domains, and most common sequences suggests that a differential expression profile is taking place during the regeneration process. Further examination of the expressed sequence tag dataset revealed that 12 putative genes are differentially expressed at significant level (R > 6). Experimental validation by RT-PCR analysis reveals that at least three genes (unknown C-4677-1, melanotransferrin, and centaurin) present a differential expression during regeneration. These findings strongly suggest that the gene expression profile varies among regeneration stages and provide evidence for the existence of differential gene expression. PMID:17579180

Transgenic Expression of Osteoactivin/gpnmb Enhances Bone Formation In Vivo and Osteoprogenitor Differentiation Ex Vivo.

PubMed

Frara, Nagat; Abdelmagid, Samir M; Sondag, Gregory R; Moussa, Fouad M; Yingling, Vanessa R; Owen, Thomas A; Popoff, Steven N; Barbe, Mary F; Safadi, Fayez F

2016-01-01

Initial identification of osteoactivin (OA)/glycoprotein non-melanoma clone B (gpnmb) was demonstrated in an osteopetrotic rat model, where OA expression was increased threefold in mutant bones, compared to normal. OA mRNA and protein expression increase during active bone regeneration post-fracture, and primary rat osteoblasts show increased OA expression during differentiation in vitro. To further examine OA/gpnmb as an osteoinductive agent, we characterized the skeletal phenotype of transgenic mouse overexpressing OA/gpnmb under the CMV-promoter (OA-Tg). Western blot analysis showed increased OA/gpnmb in OA-Tg osteoblasts, compared to wild-type (WT). In OA-Tg mouse femurs versus WT littermates, micro-CT analysis showed increased trabecular bone volume and thickness, and cortical bone thickness; histomorphometry showed increased osteoblast numbers, bone formation and mineral apposition rates in OA-Tg mice; and biomechanical testing showed higher peak moment and stiffness. Given that OA/gpnmb is also over-expressed in osteoclasts in OA-Tg mice, we evaluated bone resorption by ELISA and histomorphometry, and observed decreased serum CTX-1 and RANK-L, and decreased osteoclast numbers in OA-Tg, compared to WT mice, indicating decreased bone remodeling in OA-Tg mice. The proliferation rate of OA-Tg osteoblasts in vitro was higher, compared to WT, as was alkaline phosphatase staining and activity, the latter indicating enhanced differentiation of OA-Tg osteoprogenitors. Quantitative RT-PCR analysis showed increased TGF-β1 and TGF-β receptors I and II expression in OA-Tg osteoblasts, compared to WT. Together, these data suggest that OA overexpression has an osteoinductive effect on bone mass in vivo and stimulates osteoprogenitor differentiation ex vivo. © 2015 Wiley Periodicals, Inc.

Gene expression changes in the retina following subretinal injection of human neural progenitor cells into a rodent model for retinal degeneration.

PubMed

Jones, Melissa K; Lu, Bin; Saghizadeh, Mehrnoosh; Wang, Shaomei

2016-01-01

Retinal degenerative diseases (RDDs) affect millions of people and are the leading cause of vision loss. Although treatment options for RDDs are limited, stem and progenitor cell-based therapies have great potential to halt or slow the progression of vision loss. Our previous studies have shown that a single subretinal injection of human forebrain derived neural progenitor cells (hNPCs) into the Royal College of Surgeons (RCS) retinal degenerate rat offers long-term preservation of photoreceptors and visual function. Furthermore, neural progenitor cells are currently in clinical trials for treating age-related macular degeneration; however, the molecular mechanisms of stem cell-based therapies are largely unknown. This is the first study to analyze gene expression changes in the retina of RCS rats following subretinal injection of hNPCs using high-throughput sequencing. RNA-seq data of retinas from RCS rats injected with hNPCs (RCS(hNPCs)) were compared to sham surgery in RCS (RCS(sham)) and wild-type Long Evans (LE(sham)) rats. Differential gene expression patterns were determined with in silico analysis and confirmed with qRT-PCR. Function, biologic, cellular component, and pathway analyses were performed on differentially expressed genes and investigated with immunofluorescent staining experiments. Analysis of the gene expression data sets identified 1,215 genes that were differentially expressed between RCS(sham) and LE(sham) samples. Additionally, 283 genes were differentially expressed between the RCS(hNPCs) and RCS(sham) samples. Comparison of these two gene sets identified 68 genes with inverse expression (termed rescue genes), including Pdc, Rp1, and Cdc42ep5. Functional, biologic, and cellular component analyses indicate that the immune response is enhanced in RCS(sham). Pathway analysis of the differential expression gene sets identified three affected pathways in RCS(hNPCs), which all play roles in phagocytosis signaling. Immunofluorescent staining detected the increased presence of macrophages and microglia in RCS(sham) retinas, which decreased in RCS(hNPCs) retinas similar to the patterns detected in LE(sham). The results from this study provide evidence of the gene expression changes that occur following treatment with hNPCs in the degenerating retina. This information can be used in future studies to potentially enhance or predict responses to hNPC and other stem cell therapies for retinal degenerative diseases.

Gonad Transcriptome Analysis of the Pacific Oyster Crassostrea gigas Identifies Potential Genes Regulating the Sex Determination and Differentiation Process.

PubMed

Yue, Chenyang; Li, Qi; Yu, Hong

2018-04-01

The Pacific oyster Crassostrea gigas is a commercially important bivalve in aquaculture worldwide. C. gigas has a fascinating sexual reproduction system consisting of dioecism, sex change, and occasional hermaphroditism, while knowledge of the molecular mechanisms of sex determination and differentiation is still limited. In this study, the transcriptomes of male and female gonads at different gametogenesis stages were characterized by RNA-seq. Hierarchical clustering based on genes differentially expressed revealed that 1269 genes were expressed specifically in female gonads and 817 genes were expressed increasingly over the course of spermatogenesis. Besides, we identified two and one gene modules related to female and male gonad development, respectively, using weighted gene correlation network analysis (WGCNA). Interestingly, GO and KEGG enrichment analysis showed that neurotransmitter-related terms were significantly enriched in genes related to ovary development, suggesting that the neurotransmitters were likely to regulate female sex differentiation. In addition, two hub genes related to testis development, lncRNA LOC105321313 and Cg-Sh3kbp1, and one hub gene related to ovary development, Cg-Malrd1-like, were firstly investigated. This study points out the role of neurotransmitter and non-coding RNA regulation during gonad development and produces lists of novel relevant candidate genes for further studies. All of these provided valuable information to understand the molecular mechanisms of C. gigas sex determination and differentiation.

Ectopic expression of necdin induces differentiation of mouse neuroblastoma cells.

PubMed

Kobayashi, Masakatsu; Taniura, Hideo; Yoshikawa, Kazuaki

2002-11-01

Necdin is expressed predominantly in postmitotic neurons, and ectopic expression of this protein strongly suppresses cell growth. Necdin has been implicated in the pathogenesis of Prader-Willi syndrome, a human neurodevelopmental disorder associated with genomic imprinting. Here we demonstrate that ectopic expression of necdin induces a neuronal phenotype in neuroblastoma cells. Necdin was undetectable in mouse neuroblastoma N1E-115 cells under undifferentiated and differentiated conditions. N1E-115 cells transfected with necdin cDNA showed morphological differentiation such as neurite outgrowth and expression of the synaptic marker proteins synaptotagmin and synaptophysin. In addition, Western blot analysis of the retinoblastoma protein (Rb) family members Rb, p130, and p107 revealed that necdin cDNA transfectants contained an increased level of p130 and a reduced level of p107, a pattern seen in differentiated G(0) cells. The transcription factors E2F1 and E2F4 physically interacted with necdin via their carboxyl-terminal transactivation domains, but only E2F1 abrogated necdin-induced growth arrest and neurite outgrowth of neuroblastoma cells. Overexpression of E2F1 in differentiated N1E-115 cells induced apoptosis, which was antagonized by co-expression of necdin. These results suggest that necdin promotes the differentiation and survival of neurons through its antagonistic interactions with E2F1.

RNA-Seq Profiling Reveals Novel Hepatic Gene Expression Pattern in Aflatoxin B1 Treated Rats

PubMed Central

Merrick, B. Alex; Phadke, Dhiral P.; Auerbach, Scott S.; Mav, Deepak; Stiegelmeyer, Suzy M.; Shah, Ruchir R.; Tice, Raymond R.

2013-01-01

Deep sequencing was used to investigate the subchronic effects of 1 ppm aflatoxin B1 (AFB1), a potent hepatocarcinogen, on the male rat liver transcriptome prior to onset of histopathological lesions or tumors. We hypothesized RNA-Seq would reveal more differentially expressed genes (DEG) than microarray analysis, including low copy and novel transcripts related to AFB1’s carcinogenic activity compared to feed controls (CTRL). Paired-end reads were mapped to the rat genome (Rn4) with TopHat and further analyzed by DESeq and Cufflinks-Cuffdiff pipelines to identify differentially expressed transcripts, new exons and unannotated transcripts. PCA and cluster analysis of DEGs showed clear separation between AFB1 and CTRL treatments and concordance among group replicates. qPCR of eight high and medium DEGs and three low DEGs showed good comparability among RNA-Seq and microarray transcripts. DESeq analysis identified 1,026 differentially expressed transcripts at greater than two-fold change (p<0.005) compared to 626 transcripts by microarray due to base pair resolution of transcripts by RNA-Seq, probe placement within transcripts or an absence of probes to detect novel transcripts, splice variants and exons. Pathway analysis among DEGs revealed signaling of Ahr, Nrf2, GSH, xenobiotic, cell cycle, extracellular matrix, and cell differentiation networks consistent with pathways leading to AFB1 carcinogenesis, including almost 200 upregulated transcripts controlled by E2f1-related pathways related to kinetochore structure, mitotic spindle assembly and tissue remodeling. We report 49 novel, differentially-expressed transcripts including confirmation by PCR-cloning of two unique, unannotated, hepatic AFB1-responsive transcripts (HAfT’s) on chromosomes 1.q55 and 15.q11, overexpressed by 10 to 25-fold. Several potentially novel exons were found and exon refinements were made including AFB1 exon-specific induction of homologous family members, Ugt1a6 and Ugt1a7c. We find the rat transcriptome contains many previously unidentified, AFB1-responsive exons and transcripts supporting RNA-Seq’s capabilities to provide new insights into AFB1-mediated gene expression leading to hepatocellular carcinoma. PMID:23630614

RNA-Seq profiling reveals novel hepatic gene expression pattern in aflatoxin B1 treated rats.

PubMed

Merrick, B Alex; Phadke, Dhiral P; Auerbach, Scott S; Mav, Deepak; Stiegelmeyer, Suzy M; Shah, Ruchir R; Tice, Raymond R

2013-01-01

Deep sequencing was used to investigate the subchronic effects of 1 ppm aflatoxin B1 (AFB1), a potent hepatocarcinogen, on the male rat liver transcriptome prior to onset of histopathological lesions or tumors. We hypothesized RNA-Seq would reveal more differentially expressed genes (DEG) than microarray analysis, including low copy and novel transcripts related to AFB1's carcinogenic activity compared to feed controls (CTRL). Paired-end reads were mapped to the rat genome (Rn4) with TopHat and further analyzed by DESeq and Cufflinks-Cuffdiff pipelines to identify differentially expressed transcripts, new exons and unannotated transcripts. PCA and cluster analysis of DEGs showed clear separation between AFB1 and CTRL treatments and concordance among group replicates. qPCR of eight high and medium DEGs and three low DEGs showed good comparability among RNA-Seq and microarray transcripts. DESeq analysis identified 1,026 differentially expressed transcripts at greater than two-fold change (p<0.005) compared to 626 transcripts by microarray due to base pair resolution of transcripts by RNA-Seq, probe placement within transcripts or an absence of probes to detect novel transcripts, splice variants and exons. Pathway analysis among DEGs revealed signaling of Ahr, Nrf2, GSH, xenobiotic, cell cycle, extracellular matrix, and cell differentiation networks consistent with pathways leading to AFB1 carcinogenesis, including almost 200 upregulated transcripts controlled by E2f1-related pathways related to kinetochore structure, mitotic spindle assembly and tissue remodeling. We report 49 novel, differentially-expressed transcripts including confirmation by PCR-cloning of two unique, unannotated, hepatic AFB1-responsive transcripts (HAfT's) on chromosomes 1.q55 and 15.q11, overexpressed by 10 to 25-fold. Several potentially novel exons were found and exon refinements were made including AFB1 exon-specific induction of homologous family members, Ugt1a6 and Ugt1a7c. We find the rat transcriptome contains many previously unidentified, AFB1-responsive exons and transcripts supporting RNA-Seq's capabilities to provide new insights into AFB1-mediated gene expression leading to hepatocellular carcinoma.

Characterization of transformation related genes in oral cancer cells.

PubMed

Chang, D D; Park, N H; Denny, C T; Nelson, S F; Pe, M

1998-04-16

A cDNA representational difference analysis (cDNA-RDA) and an arrayed filter technique were used to characterize transformation-related genes in oral cancer. From an initial comparison of normal oral epithelial cells and a human papilloma virus (HPV)-immortalized oral epithelial cell line, we obtained 384 differentially expressed gene fragments and arrayed them on a filter. Two hundred and twelve redundant clones were identified by three rounds of back hybridization. Sequence analysis of the remaining clones revealed 99 unique clones corresponding to 69 genes. The expression of these transformation related gene fragments in three nontumorigenic HPV-immortalized oral epithelial cell lines and three oral cancer cell lines were simultaneously monitored using a cDNA array hybridization. Although there was a considerable cell line-to-cell line variability in the expression of these clones, a reliable prediction of their expression could be made from the cDNA array hybridization. Our study demonstrates the utility of combining cDNA-RDA and arrayed filters in high-throughput gene expression difference analysis. The differentially expressed genes identified in this study should be informative in studying oral epithelial cell carcinogenesis.

The HMX/NKX homeodomain protein MLS-2 specifies the identity of the AWC sensory neuron type via regulation of the ceh-36 Otx gene in C. elegans

PubMed Central

Kim, Kyuhyung; Kim, Rinho; Sengupta, Piali

2010-01-01

The differentiated features of postmitotic neurons are dictated by the expression of specific transcription factors. The mechanisms by which the precise spatiotemporal expression patterns of these factors are regulated are poorly understood. In C. elegans, the ceh-36 Otx homeobox gene is expressed in the AWC sensory neurons throughout postembryonic development, and regulates terminal differentiation of this neuronal subtype. Here, we show that the HMX/NKX homeodomain protein MLS-2 regulates ceh-36 expression specifically in the AWC neurons. Consequently, the AWC neurons fail to express neuron type-specific characteristics in mls-2 mutants. mls-2 is expressed transiently in postmitotic AWC neurons, and directly initiates ceh-36 expression. CEH-36 subsequently interacts with a distinct site in its cis-regulatory sequences to maintain its own expression, and also directly regulates the expression of AWC-specific terminal differentiation genes. We also show that MLS-2 acts in additional neuron types to regulate their development and differentiation. Our analysis describes a transcription factor cascade that defines the unique postmitotic characteristics of a sensory neuron subtype, and provides insights into the spatiotemporal regulatory mechanisms that generate functional diversity in the sensory nervous system. PMID:20150279

MicroRNA-205 targets tight junction-related proteins during urothelial cellular differentiation.

PubMed

Chung, Pei-Jung Katy; Chi, Lang-Ming; Chen, Chien-Lun; Liang, Chih-Lung; Lin, Chung-Tzu; Chang, Yu-Xun; Chen, Chun-Hsien; Chang, Yu-Sun

2014-09-01

The mammalian bladder urothelium classified as basal, intermediate, and terminally differentiated umbrella cells offers one of the most effective permeability barrier functions known to exist in nature because of the formation of apical uroplakin plaques and tight junctions. To improve our understanding of urothelial differentiation, we analyzed the microRNA (miRNA) expression profiles of mouse urinary tissues and by TaqMan miRNA analysis of microdissected urothelial layers and in situ miRNA-specific hybridization to determine the dependence of these miRNAs on the differentiation stage. Our in situ hybridization studies revealed that miR-205 was enriched in the undifferentiated basal and intermediate cell layers. We then used a quantitative proteomics approach to identify miR-205 target genes in primary cultured urothelial cells subjected to antagomir-mediated knockdown of specific miRNAs. Twenty-four genes were reproducibly regulated by miR-205; eleven of them were annotated as cell junction- and tight junction-related molecules. Western blot analysis demonstrated that antagomir-induced silencing of miR-205 in primary cultured urothelial cells elevated the expression levels of Tjp1, Cgnl1, and Cdc42. Ectopic expression of miR-205 in MDCK cells inhibited the expression of tight junction proteins and the formation of tight junctions. miR-205- knockdown urothelial cells showed alterations in keratin synthesis and increases of uroplakin Ia and Ib, which are the urothelial differentiation products. These results suggest that miR-205 may contribute a role in regulation of urothelial differentiation by modulating the expression of tight junction-related molecules. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Immunohistochemical analysis of human milk fat globulin expression in extramammary Paget's disease.

PubMed

Ohnishi, T; Watanabe, S

2001-03-01

Primary extramammary Paget's disease is thought to be an intraepidermal carcinoma indicating apocrine secretory differentiation. In addition to expression in breast tissue, human milk fat globulin (HMFG) is expressed in the normal apocrine glands and tumours with apocrine differentiation. In this study HMFG expression in extramammary Paget's disease was analysed immunohistochemically in 18 cases of primary extramammary Paget's disease and two cases of secondary extramammary Paget's disease. The proportion and staining pattern of positive tumour cells with the anti-HMFG antibody was variable in each case. Cytoplasmic staining was observed frequently in dermal invasion and metastasis of Paget cells. The variabilities were thought to be due to modulation of the cellular localization of the cell surface component, HMFG, according to changes in cellular differentiation or malignant potency.

Paracrine Pathways in Uterine Leiomyoma Stem Cells Involve Insulinlike Growth Factor 2 and Insulin Receptor A

PubMed Central

Moravek, Molly B.; Yin, Ping; Coon, John S.; Ono, Masanori; Druschitz, Stacy A.; Malpani, Saurabh S.; Dyson, Matthew T.; Rademaker, Alfred W.; Robins, Jared C.; Wei, Jian-Jun; Kim, J. Julie

2017-01-01

Context: Uterine leiomyomas (fibroids) are the most common benign tumors in women. Recently, three populations of leiomyoma cells were discovered on the basis of CD34 and CD49b expression, but molecular differences between these populations remain unknown. Objective: To define differential gene expression and signaling pathways in leiomyoma cell populations. Design: Cells from human leiomyoma tissue were sorted by flow cytometry into three populations: CD34+/CD49b+, CD34+/CD49b−, and CD34−/CD49b−. Microarray gene expression profiling and pathway analysis were performed. To investigate the insulinlike growth factor (IGF) pathway, real-time quantitative polymerase chain reaction, immunoblotting, and 5-ethynyl-2′-deoxyuridine incorporation studies were performed in cells isolated from fresh leiomyoma. Setting: Research laboratory. Patients: Eight African American women. Interventions: None Main Outcomes Measures: Gene expression patterns, cell proliferation, and differentiation. Results: A total of 1164 genes were differentially expressed in the three leiomyoma cell populations, suggesting a hierarchical differentiation order whereby CD34+/CD49b+ stem cells differentiate to CD34+/CD49b− intermediary cells, which then terminally differentiate to CD34−/CD49b− cells. Pathway analysis revealed differential expression of several IGF signaling pathway genes. IGF2 was overexpressed in CD34+/CD49b− vs CD34−/CD49b− cells (83-fold; P < 0.05). Insulin receptor A (IR-A) expression was higher and IGF1 receptor lower in CD34+/CD49b+ vs CD34−/CD49b− cells (15-fold and 0.35-fold, respectively; P < 0.05). IGF2 significantly increased cell number (1.4-fold; P < 0.001), proliferation indices, and extracellular signal-regulated kinase (ERK) phosphorylation. ERK inhibition decreased IGF2-stimulated cell proliferation. Conclusions: IGF2 and IR-A are important for leiomyoma stem cell proliferation and may represent paracrine signaling between leiomyoma cell types. Therapies targeting the IGF pathway should be investigated for both treatment and prevention of leiomyomas. PMID:28324020

Bioinformatics and expressional analysis of cDNA clones from floral buds

NASA Astrophysics Data System (ADS)

Pawełkowicz, Magdalena Ewa; Skarzyńska, Agnieszka; Cebula, Justyna; Hincha, Dirck; ZiÄ bska, Karolina; PlÄ der, Wojciech; Przybecki, Zbigniew

2017-08-01

The application of genomic approaches may serve as an initial step in understanding the complexity of biochemical network and cellular processes responsible for regulation and execution of many developmental tasks. The molecular mechanism of sex expression in cucumber is still not elucidated. A study of differential expression was conducted to identify genes involved in sex determination and floral organ morphogenesis. Herein, we present generation of expression sequence tags (EST) obtained by differential hybridization (DH) and subtraction technique (cDNA-DSC) and their characteristic features such as molecular function, involvement in biology processes, expression and mapping position on the genome.

Profiling of differential gene expression in the hypothalamus of broiler-type Taiwan country chickens in response to acute heat stress.

PubMed

Tu, Wei-Lin; Cheng, Chuen-Yu; Wang, Shih-Han; Tang, Pin-Chi; Chen, Chih-Feng; Chen, Hsin-Hsin; Lee, Yen-Pai; Chen, Shuen-Ei; Huang, San-Yuan

2016-02-01

Acute heat stress severely impacts poultry production. The hypothalamus acts as a crucial center to regulate body temperature, detect temperature changes, and modulate the autonomic nervous system and endocrine loop for heat retention and dissipation. The purpose of this study was to investigate global gene expression in the hypothalamus of broiler-type B strain Taiwan country chickens after acute heat stress. Twelve 30-week-old hens were allocated to four groups. Three heat-stressed groups were subjected to acute heat stress at 38 °C for 2 hours without recovery (H2R0), with 2 hours of recovery (H2R2), and with 6 hours of recovery (H2R6). The control hens were maintained at 25 °C. At the end, hypothalamus samples were collected for gene expression analysis. The results showed that 24, 11, and 25 genes were upregulated and 41, 15, and 42 genes were downregulated in H2R0, H2R2, and H2R6 treatments, respectively. The expressions of gonadotropin-releasing hormone 1 (GNRH1), heat shock 27-kDa protein 1 (HSPB1), neuropeptide Y (NPY), and heat shock protein 25 (HSP25) were upregulated at all recovery times after heat exposure. Conversely, the expression of TPH2 was downregulated at all recovery times. A gene ontology analysis showed that most of the differentially expressed genes were involved in biological processes including cellular processes, metabolic processes, localization, multicellular organismal processes, developmental processes, and biological regulation. A functional annotation analysis showed that the differentially expressed genes were related to the gene networks of responses to stress and reproductive functions. These differentially expressed genes might be essential and unique key factors in the heat stress response of the hypothalamus in chickens. Copyright © 2016 Elsevier Inc. All rights reserved.

Activation of dynamin I gene expression by Sp1 and Sp3 is required for neuronal differentiation of N1E-115 cells.

PubMed

Yoo, Jiyun; Jeong, Moon-Jin; Kwon, Byoung-Mog; Hur, Man-Wook; Park, Young-Mee; Han, Mi Young

2002-04-05

Dynamin I is a key molecule required for the recycling of synaptic vesicles in neurons, and it has been known that dynamin I gene expression is induced during neuronal differentiation. Our previous studies established that neuronal restriction of dynamin I gene expression is controlled by Sp1 and nuclear factor-kappaB-like element-1. Here, using a series of deletion constructs and site-directed mutation, we found that transcription of dynamin I gene during neuronal differentiation of N1E-115 cells is controlled primarily by the Sp1 element located between -13 to -4 bp of the dynamin I promoter. Gel shift analysis demonstrated that in addition to Sp1, Sp3 could interact with this Sp1 element. The requirement for Sp family transcription factors in dynamin I gene expression was confirmed by using mithramycin, an inhibitor of Sp1/Sp3 binding. Mithramycin repressed dynamin I gene expression and resulted in blocking of neuronal differentiation of N1E-115 cells. The localization of the dynamin I protein was also restricted in the peripheral region of the nucleus by the mithramycin treatment. Thus, all of our results suggest that induction of dynamin I gene expression during N1E-115 cell differentiation is modulated by Sp1/Sp3 interactions with the dynamin I promoter, and its expression is important for neuronal differentiation of the N1E-115 cells.

Hypoxia-inducible factor 1–mediated human GATA1 induction promotes erythroid differentiation under hypoxic conditions

PubMed Central

Zhang, Feng-Lin; Shen, Guo-Min; Liu, Xiao-Ling; Wang, Fang; Zhao, Ying-Ze; Zhang, Jun-Wu

2012-01-01

Abstract Hypoxia-inducible factor promotes erythropoiesis through coordinated cell type–specific hypoxia responses. GATA1 is essential to normal erythropoiesis and plays a crucial role in erythroid differentiation. In this study, we show that hypoxia-induced GATA1 expression is mediated by HIF1 in erythroid cells. Under hypoxic conditions, significantly increased GATA1 mRNA and protein levels were detected in K562 cells and erythroid induction cultures of CD34+ haematopoietic stem/progenitor cells. Enforced HIF1α expression increased GATA1 expression, while HIF1α knockdown by RNA interference decreased GATA1 expression. In silico analysis revealed one potential hypoxia response element (HRE). The results from reporter gene and mutation analysis suggested that this element is necessary for hypoxic response. Chromatin immunoprecipitation (ChIP)-PCR showed that the putative HRE was recognized and bound by HIF1 in vivo. These results demonstrate that the up-regulation of GATA1 during hypoxia is directly mediated by HIF1.The mRNA expression of some erythroid differentiation markers was increased under hypoxic conditions, but decreased with RNA interference of HIF1α or GATA1. Flow cytometry analysis also indicated that hypoxia, desferrioxamine or CoCl2 induced expression of erythroid surface markers CD71 and CD235a, while expression repression of HIF1α or GATA1 by RNA interference led to a decreased expression of CD235a. These results suggested that HIF1-mediated GATA1 up-regulation promotes erythropoiesis in order to satisfy the needs of an organism under hypoxic conditions. PMID:22050843

Analysis of global gene expression profiles to identify differentially expressed genes critical for embryo development in Brassica rapa.

PubMed

Zhang, Yu; Peng, Lifang; Wu, Ya; Shen, Yanyue; Wu, Xiaoming; Wang, Jianbo

2014-11-01

Embryo development represents a crucial developmental period in the life cycle of flowering plants. To gain insights into the genetic programs that control embryo development in Brassica rapa L., RNA sequencing technology was used to perform transcriptome profiling analysis of B. rapa developing embryos. The results generated 42,906,229 sequence reads aligned with 32,941 genes. In total, 27,760, 28,871, 28,384, and 25,653 genes were identified from embryos at globular, heart, early cotyledon, and mature developmental stages, respectively, and analysis between stages revealed a subset of stage-specific genes. We next investigated 9,884 differentially expressed genes with more than fivefold changes in expression and false discovery rate ≤ 0.001 from three adjacent-stage comparisons; 1,514, 3,831, and 6,633 genes were detected between globular and heart stage embryo libraries, heart stage and early cotyledon stage, and early cotyledon and mature stage, respectively. Large numbers of genes related to cellular process, metabolism process, response to stimulus, and biological process were expressed during the early and middle stages of embryo development. Fatty acid biosynthesis, biosynthesis of secondary metabolites, and photosynthesis-related genes were expressed predominantly in embryos at the middle stage. Genes for lipid metabolism and storage proteins were highly expressed in the middle and late stages of embryo development. We also identified 911 transcription factor genes that show differential expression across embryo developmental stages. These results increase our understanding of the complex molecular and cellular events during embryo development in B. rapa and provide a foundation for future studies on other oilseed crops.

Expression of the transforming growth factor alpha protooncogene in differentiating human promyelocytic leukemia (HL-60) cells.

PubMed

Walz, T M; Malm, C; Wasteson, A

1993-01-01

The process of myeloid differentiation in human promyelocytic leukemia cells (HL-60) is accompanied by the coordinate expression of numerous protooncogenes. To investigate the expression of transforming growth factor alpha (TGF-alpha) in myeloid differentiation, HL-60 cells were induced to differentiate into granulocytes with 1.25% dimethyl sulfoxide, 0.2 microM all-trans retinoic acid, or 500 microM N6,O2-dibutyryladenosine-3'5'-cyclic monophosphate or differentiated along the monocyte/macrophage pathway with 0.1 microM phorbol-12-myristate-13-acetate. Using Northern blot analyses, TGF-alpha transcripts were detected within 24 h of treatment in cells differentiating toward granulocytes; maximal levels of gene expression were reached after 3 days or later and remained essentially constant throughout the observation period. These cells released TGF-alpha protein, as demonstrated by analysis of the incubation medium. In contrast, no TGF-alpha RNA or protein was detectable in HL-60 cell cultures when induced with phorbol-12-myristate-13-acetate. Epidermal growth factor receptor transcripts could not be detected either in undifferentiated or in differentiated HL-60 cells; therefore it appears as if an autocrine loop involving TGF-alpha in HL-60 cells is unlikely. In conclusion, the results demonstrate, for the first time, the expression of TGF-alpha in human granulocyte precursor cells. Our findings may indicate novel regulatory pathways in hematopoiesis.

Differentially Coexpressed Disease Gene Identification Based on Gene Coexpression Network.

PubMed

Jiang, Xue; Zhang, Han; Quan, Xiongwen

2016-01-01

Screening disease-related genes by analyzing gene expression data has become a popular theme. Traditional disease-related gene selection methods always focus on identifying differentially expressed gene between case samples and a control group. These traditional methods may not fully consider the changes of interactions between genes at different cell states and the dynamic processes of gene expression levels during the disease progression. However, in order to understand the mechanism of disease, it is important to explore the dynamic changes of interactions between genes in biological networks at different cell states. In this study, we designed a novel framework to identify disease-related genes and developed a differentially coexpressed disease-related gene identification method based on gene coexpression network (DCGN) to screen differentially coexpressed genes. We firstly constructed phase-specific gene coexpression network using time-series gene expression data and defined the conception of differential coexpression of genes in coexpression network. Then, we designed two metrics to measure the value of gene differential coexpression according to the change of local topological structures between different phase-specific networks. Finally, we conducted meta-analysis of gene differential coexpression based on the rank-product method. Experimental results demonstrated the feasibility and effectiveness of DCGN and the superior performance of DCGN over other popular disease-related gene selection methods through real-world gene expression data sets.

T-cell differentiation of multipotent hematopoietic cell line EML in the OP9-DL1 coculture system

PubMed Central

Kutleša, Snježana; Zayas, Jennifer; Valle, Alexandra; Levy, Robert B.; Jurecic, Roland

2011-01-01

Objective Multipotent hematopoietic cell line EML can differentiate into myeloid, erythroid, megakaryocytic, and B-lymphoid lineages, but it remained unknown whether EML cells have T-cell developmental potential as well. The goal of this study was to determine whether the coculture with OP9 stromal cells expressing Notch ligand Delta-like 1 (OP9-DL1) could induce differentiation of EML cells into T-cell lineage. Materials and Methods EML cells were cocultured with control OP9 or OP9-DL1 stromal cells in the presence of cytokines (stem cell factor, interleukin-7, and Fms-like tyrosine kinase 3 ligand). Their T-cell lineage differentiation was assessed through flow cytometry and reverse transcription polymerase chain reaction expression analysis of cell surface markers and genes characterizing and associated with specific stages of T-cell development. Results The phenotypic, molecular, and functional analysis has revealed that in EML/OP9-DL1 cocultures with cytokines, but not in control EML/OP9 cocultures, EML cell line undergoes T-cell lineage commitment and differentiation. In OP9-DL1 cocultures, EML cell line has differentiated into cells that 1) resembled double-negative, double-positive, and single-positive stages of T-cell development; 2) initiated expression of GATA-3, Pre-Tα, RAG-1, and T-cell receptor – Vβ genes; and 3) produced interferon-γ in response to T-cell receptor stimulation. Conclusions These results support the notion that EML cell line has the capacity for T-cell differentiation. Remarkably, induction of T-lineage gene expression and differentiation of EML cells into distinct stages of T-cell development were very similar to previously described T-cell differentiation of adult hematopoietic stem cells and progenitors in OP9-DL1 cocultures. Thus, EML/OP9-DL1 coculture could be a useful experimental system to study the role of particular genes in T-cell lineage specification, commitment, and differentiation. PMID:19447159

T-cell differentiation of multipotent hematopoietic cell line EML in the OP9-DL1 coculture system.

PubMed

Kutlesa, Snjezana; Zayas, Jennifer; Valle, Alexandra; Levy, Robert B; Jurecic, Roland

2009-08-01

Multipotent hematopoietic cell line EML can differentiate into myeloid, erythroid, megakaryocytic, and B-lymphoid lineages, but it remained unknown whether EML cells have T-cell developmental potential as well. The goal of this study was to determine whether the coculture with OP9 stromal cells expressing Notch ligand Delta-like 1 (OP9-DL1) could induce differentiation of EML cells into T-cell lineage. EML cells were cocultured with control OP9 or OP9-DL1 stromal cells in the presence of cytokines (stem cell factor, interleukin-7, and Fms-like tyrosine kinase 3 ligand). Their T-cell lineage differentiation was assessed through flow cytometry and reverse transcription polymerase chain reaction expression analysis of cell surface markers and genes characterizing and associated with specific stages of T-cell development. The phenotypic, molecular, and functional analysis has revealed that in EML/OP9-DL1 cocultures with cytokines, but not in control EML/OP9 cocultures, EML cell line undergoes T-cell lineage commitment and differentiation. In OP9-DL1 cocultures, EML cell line has differentiated into cells that 1) resembled double-negative, double-positive, and single-positive stages of T-cell development; 2) initiated expression of GATA-3, Pre-Talpha, RAG-1, and T-cell receptor-Vbeta genes; and 3) produced interferon-gamma in response to T-cell receptor stimulation. These results support the notion that EML cell line has the capacity for T-cell differentiation. Remarkably, induction of T-lineage gene expression and differentiation of EML cells into distinct stages of T-cell development were very similar to previously described T-cell differentiation of adult hematopoietic stem cells and progenitors in OP9-DL1 cocultures. Thus, EML/OP9-DL1 coculture could be a useful experimental system to study the role of particular genes in T-cell lineage specification, commitment, and differentiation.

Social Concepts and Judgments: A Semantic Differential Analysis of the Concepts Feminist, Man, and Woman

ERIC Educational Resources Information Center

Pierce, W. David; Sydie, R. A.; Stratkotter, Rainer

2003-01-01

Male and female participants (N = 274) made judgments about the social concepts of "feminist," "man," and "woman" on 63 semantic differential items. Factor analysis identified three basic dimensions termed evaluative, potency, and activity as well as two secondary factors called expressiveness and sexuality. Results for the evaluative dimension…

Generation of Mast Cells from Mouse Fetus: Analysis of Differentiation and Functionality, and Transcriptome Profiling Using Next Generation Sequencer

PubMed Central

Fukuishi, Nobuyuki; Igawa, Yuusuke; Kunimi, Tomoyo; Hamano, Hirofumi; Toyota, Masao; Takahashi, Hironobu; Kenmoku, Hiromichi; Yagi, Yasuyuki; Matsui, Nobuaki; Akagi, Masaaki

2013-01-01

While gene knockout technology can reveal the roles of proteins in cellular functions, including in mast cells, fetal death due to gene manipulation frequently interrupts experimental analysis. We generated mast cells from mouse fetal liver (FLMC), and compared the fundamental functions of FLMC with those of bone marrow-derived mouse mast cells (BMMC). Under electron microscopy, numerous small and electron-dense granules were observed in FLMC. In FLMC, the expression levels of a subunit of the FcεRI receptor and degranulation by IgE cross-linking were comparable with BMMC. By flow cytometry we observed surface expression of c-Kit prior to that of FcεRI on FLMC, although on BMMC the expression of c-Kit came after FcεRI. The surface expression levels of Sca-1 and c-Kit, a marker of putative mast cell precursors, were slightly different between bone marrow cells and fetal liver cells, suggesting that differentiation stage or cell type are not necessarily equivalent between both lineages. Moreover, this indicates that phenotypically similar mast cells may not have undergone an identical process of differentiation. By comprehensive analysis using the next generation sequencer, the same frequency of gene expression was observed for 98.6% of all transcripts in both cell types. These results indicate that FLMC could represent a new and useful tool for exploring mast cell differentiation, and may help to elucidate the roles of individual proteins in the function of mast cells where gene manipulation can induce embryonic lethality in the mid to late stages of pregnancy. PMID:23573287

microRNA Profiling of Amniotic Fluid: Evidence of Synergy of microRNAs in Fetal Development.

PubMed

Sun, Tingting; Li, Weiyun; Li, Tianpeng; Ling, Shucai

2016-01-01

Amniotic fluid (AF) continuously exchanges molecules with the fetus, playing critical roles in fetal development especially via its complex components. Among these components, microRNAs are thought to be transferred between cells loaded in microvesicles. However, the functions of AF microRNAs remain unknown. To date, few studies have examined microRNAs in amniotic fluid. In this study, we employed miRCURY Locked Nucleotide Acid arrays to profile the dynamic expression of microRNAs in AF from mice on embryonic days E13, E15, and E17. At these times, 233 microRNAs were differentially expressed (p< 0.01), accounting for 23% of the total Mus musculus microRNAs. These differentially-expressed microRNAs were divided into two distinct groups based on their expression patterns. Gene ontology analysis showed that the intersectional target genes of these differentially-expressed microRNAs were mainly distributed in synapse, synaptosome, cell projection, and cytoskeleton. Pathway analysis revealed that the target genes of the two groups of microRNAs were synergistically enriched in axon guidance, focal adhesion, and MAPK signaling pathways. MicroRNA-mRNA network analysis and gene- mapping showed that these microRNAs synergistically regulated cell motility, cell proliferation and differentiation, and especially the axon guidance process. Cancer pathways associated with growth and proliferation were also enriched in AF. Taken together, the results of this study are the first to show the functions of microRNAs in AF during fetal development, providing novel insights into interpreting the roles of AF microRNAs in fetal development.

Changes in Global Transcriptional Profiling of Women Following Obesity Surgery Bypass.

PubMed

Pinhel, Marcela Augusta de Souza; Noronha, Natalia Yumi; Nicoletti, Carolina Ferreira; de Oliveira, Bruno Affonso Parente; Cortes-Oliveira, Cristiana; Pinhanelli, Vitor Caressato; Salgado Junior, Wilson; Machry, Ana Julia; da Silva Junior, Wilson Araújo; Souza, Dorotéia Rossi Silva; Marchini, Júlio Sérgio; Nonino, Carla Barbosa

2018-01-01

Differential gene expression in peripheral blood mononuclear cells (PBMCs) after Roux-en-Y gastric bypass (RYGB) is poorly characterized. Markers of these processes may provide a deeper understanding of the mechanisms that underlie these events. The main goal of this study was to identify changes in PBMC gene expression in women with obesity before and 6 months after RYGB-induced weight loss. The ribonucleic acid (RNA) of PBMCs from 13 obese women was analyzed before and 6 months after RYGB; the RNA of PBMCs from nine healthy women served as control. The gene expression levels were determined by microarray analysis. Significant differences in gene expression were validated by real-time quantitative polymerase chain reaction (RT-qPCR). Microarray analysis for comparison of the pre- and postoperative periods showed that 1366 genes were differentially expressed genes (DEGs). The main pathways were related to gene transcription; lipid, energy, and glycide metabolism; inflammatory and immunological response; cell differentiation; oxidative stress regulation; response to endogenous and exogenous stimuli; substrate oxidation; mTOR signaling pathway; interferon signaling; mitogen-activated protein kinases (MAPK), cAMP response element binding protein (CREB1), heat shock factor 1 (HSF1), and sterol regulatory element binding protein 1c (SREBP-1c) gene expression; adipocyte differentiation; and methylation. Six months after bariatric surgery and significant weight loss, many molecular pathways involved in obesity and metabolic diseases change. These findings are an important tool to identify potential targets for therapeutic intervention and clinical practice of nutritional genomics in obesity.

Gene expression profiling analysis of the effects of low-intensity pulsed ultrasound on induced pluripotent stem cell-derived neural crest stem cells.

PubMed

Xia, Bin; Zou, Yang; Xu, Zhiling; Lv, Yonggang

2017-11-01

Low-intensity pulsed ultrasound (LIPUS) is a noninvasive technique that has been shown to affect cell proliferation, migration, and differentiation and promote the regeneration of damaged peripheral nerve. Our previous studies had proved that LIPUS can significantly promote the neural differentiation of induced pluripotent stem cell-derived neural crest stem cells (iPSCs-NCSCs) and enhance the repair of rat-transected sciatic nerve. To further explore the underlying mechanisms of LIPUS treatment of iPSCs-NCSCs, this study reported the gene expression profiling analysis of iPSCs-NCSCs before and after LIPUS treatment using the RNA-sequencing (RNA-Seq) method. It was found that expression of 76 genes of iPSCs-NCSCs cultured in a serum-free neural induction medium and expression of 21 genes of iPSCs-NCSCs cultured in a neuronal differentiation medium were significantly changed by LIPUS treatment. The differentially expressed genes are related to angiogenesis, nervous system activity and functions, cell activities, and so on. The RNA-seq results were further verified by a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). High correlation was observed between the results obtained from qRT-PCR and RNA-Seq. This study presented new information on the global gene expression patterns of iPSCs-NCSCs after LIPUS treatment and may expand the understanding of the complex molecular mechanism of LIPUS treatment of iPSCs-NCSCs. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

Gene Expression Analysis of Mouse Embryonic Stem Cells Following Levitation in an Ultrasound Standing Wave Trap

PubMed Central

Bazou, Despina; Kearney, Roisin; Mansergh, Fiona; Bourdon, Celine; Farrar, Jane; Wride, Michael

2011-01-01

In the present paper, gene expression analysis of mouse embryonic stem (ES) cells levitated in a novel ultrasound standing wave trap (USWT) (Bazou et al. 2005a) at variable acoustic pressures (0.08–0.85 MPa) and times (5–60 min) was performed. Our results showed that levitation of ES cells at the highest employed acoustic pressure for 60 min does not modify gene expression and cells maintain their pluripotency. Embryoid bodies (EBs) also expressed the early and late neural differentiation markers, which were also unaffected by the acoustic field. Our results suggest that the ultrasound trap microenvironment is minimally invasive as the biologic consequences of ES cell replication and EB differentiation proceed without significantly affecting gene expression. The technique holds great promise in safe cell manipulation techniques for a variety of applications including tissue engineering and regenerative medicine. (E-mail: Bazoud@tcd.ie) PMID:21208732

Immunophenotypic and Molecular Analysis of Human Dental Pulp Stem Cells Potential for Neurogenic Differentiation

PubMed Central

Fatima, Nikhat; Khan, Aleem A.; Vishwakarma, Sandeep K.

2017-01-01

Background: Growing evidence shows that dental pulp (DP) tissues could be a potential source of adult stem cells for the treatment of devastating neurological diseases and several other conditions. Aims: Exploration of the expression profile of several key molecular markers to evaluate the molecular dynamics in undifferentiated and differentiated DP-derived stem cells (DPSCs) in vitro. Settings and Design: The characteristics and multilineage differentiation ability of DPSCs were determined by cellular and molecular kinetics. DPSCs were further induced to form adherent (ADH) and non-ADH (NADH) neurospheres under serum-free condition which was further induced into neurogenic lineage cells and characterized for their molecular and cellular diversity at each stage. Statistical Analysis Used: Statistical analysis used one-way analysis of variance, Student's t-test, Livak method for relative quantification, and R programming. Results: Immunophenotypic analysis of DPSCs revealed >80% cells positive for mesenchymal markers CD90 and CD105, >70% positive for transferring receptor (CD71), and >30% for chemotactic factor (CXCR3). These cells showed mesodermal differentiation also and confirmed by specific staining and molecular analysis. Activation of neuronal lineage markers and neurogenic growth factors was observed during lineage differentiation of cells derived from NADH and ADH spheroids. Greater than 80% of cells were found to express β-tubulin III in both differentiation conditions. Conclusions: The present study reported a cascade of immunophenotypic and molecular markers to characterize neurogenic differentiation of DPSCs under serum-free condition. These findings trigger the future analyses for clinical applicability of DP-derived cells in regenerative applications. PMID:28566856

Genome-wide methylation and gene expression changes in newborn rats following maternal protein restriction and reversal by folic acid.

PubMed

Altobelli, Gioia; Bogdarina, Irina G; Stupka, Elia; Clark, Adrian J L; Langley-Evans, Simon

2013-01-01

A large body of evidence from human and animal studies demonstrates that the maternal diet during pregnancy can programme physiological and metabolic functions in the developing fetus, effectively determining susceptibility to later disease. The mechanistic basis of such programming is unclear but may involve resetting of epigenetic marks and fetal gene expression. The aim of this study was to evaluate genome-wide DNA methylation and gene expression in the livers of newborn rats exposed to maternal protein restriction. On day one postnatally, there were 618 differentially expressed genes and 1183 differentially methylated regions (FDR 5%). The functional analysis of differentially expressed genes indicated a significant effect on DNA repair/cycle/maintenance functions and of lipid, amino acid metabolism and circadian functions. Enrichment for known biological functions was found to be associated with differentially methylated regions. Moreover, these epigenetically altered regions overlapped genetic loci associated with metabolic and cardiovascular diseases. Both expression changes and DNA methylation changes were largely reversed by supplementing the protein restricted diet with folic acid. Although the epigenetic and gene expression signatures appeared to underpin largely different biological processes, the gene expression profile of DNA methyl transferases was altered, providing a potential link between the two molecular signatures. The data showed that maternal protein restriction is associated with widespread differential gene expression and DNA methylation across the genome, and that folic acid is able to reset both molecular signatures.

Influence of age, sex, and strength training on human muscle gene expression determined by microarray

PubMed Central

ROTH, STEPHEN M.; FERRELL, ROBERT E.; PETERS, DAVID G.; METTER, E. JEFFREY; HURLEY, BEN F.; ROGERS, MARC A.

2010-01-01

The purpose of this study was to determine the influence of age, sex, and strength training (ST) on large-scale gene expression patterns in vastus lateralis muscle biopsies using high-density cDNA microarrays and quantitative PCR. Muscle samples from sedentary young (20–30 yr) and older (65–75 yr) men and women (5 per group) were obtained before and after a 9-wk unilateral heavy resistance ST program. RNA was hybridized to cDNA filter microarrays representing ~4,000 known human genes and comparisons were made among arrays to determine differential gene expression as a result of age and sex differences, and/or response to ST. Sex had the strongest influence on muscle gene expression, with differential expression (>1.7-fold) observed for ~200 genes between men and women (~75% with higher expression in men). Age contributed to differential expression as well, as ~50 genes were identified as differentially expressed (>1.7-fold) in relation to age, representing structural, metabolic, and regulatory gene classes. Sixty-nine genes were identified as being differentially expressed (>1.7-fold) in all groups in response to ST, and the majority of these were downregulated. Quantitative PCR was employed to validate expression levels for caldesmon, SWI/SNF (BAF60b), and four-and-a-half LIM domains 1. These significant differences suggest that in the analysis of skeletal muscle gene expression issues of sex, age, and habitual physical activity must be addressed, with sex being the most critical variable. PMID:12209020

Altered expression of miRNAs in the uterus from a letrozole-induced rat PCOS model.

PubMed

Li, Chunjin; Chen, Lu; Zhao, Yun; Chen, Shuxiong; Fu, Lulu; Jiang, Yanwen; Gao, Shan; Liu, Zhuo; Wang, Fengge; Zhu, Xiaoling; Rao, Jiahui; Zhang, Jing; Zhou, Xu

2017-01-20

Polycystic ovary syndrome (PCOS) causes female subfertility with ovarian disorders and may be associated with increased rate of early-pregnancy failure. Rat PCOS models were established using letrozole to understand the uterine pathogenesis of PCOS. The differential expression of microRNAs (miRNAs) was observed in rat uterus with PCOS. After estrous cycles were disrupted, significantly abnormal ovarian morphology and hormone level were observed in rats with PCOS. A total of 148 miRNAs differentially expressed were identified in the uterus from the letrozole-induced rat model compared with the control. These miRNAs included 111 upregulated miRNAs and 37 downregulated miRNAs. The differential expression of miR-484, miR-375-3p, miR-324-5p, and miR-223-3p was further confirmed by quantitative reverse transcription polymerase chain reaction. Bioinformatic analysis showed that these four miRNAs were predicted to regulate a large number of genes with different functions. Pathway analysis supported that target genes of miRNAs were involved in insulin secretion and signaling pathways, such as wnt, AMPK, PI3K-Akt, and Ras. These data indicated that miRNAs differentially expressed in rat uterus with PCOS may be associated with PCOS pathogenesis in the uterus. Our findings can help clarify the mechanism of uterine defects in PCOS. Copyright © 2016. Published by Elsevier B.V.

GC-Content Normalization for RNA-Seq Data

PubMed Central

2011-01-01

Background Transcriptome sequencing (RNA-Seq) has become the assay of choice for high-throughput studies of gene expression. However, as is the case with microarrays, major technology-related artifacts and biases affect the resulting expression measures. Normalization is therefore essential to ensure accurate inference of expression levels and subsequent analyses thereof. Results We focus on biases related to GC-content and demonstrate the existence of strong sample-specific GC-content effects on RNA-Seq read counts, which can substantially bias differential expression analysis. We propose three simple within-lane gene-level GC-content normalization approaches and assess their performance on two different RNA-Seq datasets, involving different species and experimental designs. Our methods are compared to state-of-the-art normalization procedures in terms of bias and mean squared error for expression fold-change estimation and in terms of Type I error and p-value distributions for tests of differential expression. The exploratory data analysis and normalization methods proposed in this article are implemented in the open-source Bioconductor R package EDASeq. Conclusions Our within-lane normalization procedures, followed by between-lane normalization, reduce GC-content bias and lead to more accurate estimates of expression fold-changes and tests of differential expression. Such results are crucial for the biological interpretation of RNA-Seq experiments, where downstream analyses can be sensitive to the supplied lists of genes. PMID:22177264

Differentiation and activation of equine monocyte-derived dendritic cells are not correlated with CD206 or CD83 expression

PubMed Central

Moyo, Nathifa A; Marchi, Emanuele; Steinbach, Falko

2013-01-01

Dendritic cells (DC) are the main immune mediators inducing primary immune responses. DC generated from monocytes (MoDC) are a model system to study the biology of DC in vitro, as they represent inflammatory DC in vivo. Previous studies on the generation of MoDC in horses indicated that there was no distinct difference between immature and mature DC and that the expression profile was distinctly different from humans, where CD206 is expressed on immature MoDC whereas CD83 is expressed on mature MoDC. Here we describe the kinetics of equine MoDC differentiation and activation, analysing both phenotypic and functional characteristics. Blood monocytes were first differentiated with equine granulocyte–macrophage colony-stimulating factor and interleukin-4 generating immature DC (iMoDC). These cells were further activated with a cocktail of cytokines including interferon-γ) but not CD40 ligand to obtain mature DC (mMoDC). To determine the expression of a broad range of markers for which no monoclonal antibodies were available to analyse the protein expression, microarray and quantitative PCR analysis were performed to carry out gene expression analysis. This study demonstrates that equine iMoDC and mMoDC can be distinguished both phenotypically and functionally but the expression pattern of some markers including CD206 and CD83 is dissimilar to the human system. PMID:23461413

Cell recognition molecule L1 promotes embryonic stem cell differentiation through the regulation of cell surface glycosylation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Ying; Department of Clinical Laboratory, Second Affiliated Hospital of Dalian Medical University, Dalian 116023; Huang, Xiaohua

2013-10-25

Highlights: •Down-regulating FUT9 and ST3Gal4 expression blocks L1-induced neuronal differentiation of ESCs. •Up-regulating FUT9 and ST3Gal4 expression in L1-ESCs depends on the activation of PLCγ. •L1 promotes ESCs to differentiate into neuron through regulating cell surface glycosylation. -- Abstract: Cell recognition molecule L1 (CD171) plays an important role in neuronal survival, migration, differentiation, neurite outgrowth, myelination, synaptic plasticity and regeneration after injury. Our previous study has demonstrated that overexpressing L1 enhances cell survival and proliferation of mouse embryonic stem cells (ESCs) through promoting the expression of FUT9 and ST3Gal4, which upregulates cell surface sialylation and fucosylation. In the present study,more » we examined whether sialylation and fucosylation are involved in ESC differentiation through L1 signaling. RNA interference analysis showed that L1 enhanced differentiation of ESCs into neurons through the upregulation of FUT9 and ST3Gal4. Furthermore, blocking the phospholipase Cγ (PLCγ) signaling pathway with either a specific PLCγ inhibitor or knockdown PLCγ reduced the expression levels of both FUT9 and ST3Gal4 mRNAs and inhibited L1-mediated neuronal differentiation. These results demonstrate that L1 promotes neuronal differentiation from ESCs through the L1-mediated enhancement of FUT9 and ST3Gal4 expression.« less

Identification of Differentially Expressed Genes in Blood Cells of Narcolepsy Patients

PubMed Central

Tanaka, Susumu; Honda, Yutaka; Honda, Makoto

2007-01-01

Study Objective: A close association between the human leukocyte antigen (HLA)-DRB1*1501/DQB1*0602 and abnormalities in some inflammatory cytokines have been demonstrated in narcolepsy. Specific alterations in the immune system have been suggested to occur in this disorder. We attempted to identify alterations in gene expression underlying the abnormalities in the blood cells of narcoleptic patients. Designs: Total RNA from 12 narcolepsy-cataplexy patients and from 12 age- and sex-matched healthy controls were pooled. The pooled samples were initially screened for candidate genes for narcolepsy by differential display analysis using annealing control primers (ACP). The second screening of the samples was carried out by semiquantitative PCR using gene-specific primers. Finally, the expression levels of the candidate genes were further confirmed by quantitative real-time PCR using a new set of samples (20 narcolepsy-cataplexy patients and 20 healthy controls). Results: The second screening revealed differential expression of 4 candidate genes. Among them, MX2 was confirmed as a significantly down-regulated gene in the white blood cells of narcoleptic patients by quantitative real-time PCR. Conclusion: We found the MX2 gene to be significantly less expressed in comparison with normal subjects in the white blood cells of narcoleptic patients. This gene is relevant to the immune system. Although differential display analysis using ACP technology has a limitation in that it does not help in determining the functional mechanism underlying sleep/wakefulness dysregulation, it is useful for identifying novel genetic factors related to narcolepsy, such as HLA molecules. Further studies are required to explore the functional relationship between the MX2 gene and narcolepsy pathophysiology. Citation: Tanaka S; Honda Y; Honda M. Identification of differentially expressed genes in blood cells of narcolepsy patients. SLEEP 2007;30(8):974-979. PMID:17702266

An Adult Mouse Thyroid Side Population Cell Line that Exhibits Enriched Epithelial–Mesenchymal Transition

PubMed Central

Murata, Tsubasa; Iwadate, Manabu; Takizawa, Yoshinori; Miyakoshi, Masaaki; Hayase, Suguru; Yang, Wenjing; Cai, Yan; Yokoyama, Shigetoshi; Nagashima, Kunio; Wakabayashi, Yoshiyuki; Zhu, Jun

2017-01-01

Background: Studies of thyroid stem/progenitor cells have been hampered due to the small organ size and lack of tissue, which limits the yield of these cells. A continuous source that allows the study and characterization of thyroid stem/progenitor cells is desired to push the field forward. Method: A cell line was established from Hoechst-resistant side population cells derived from mouse thyroid that were previously shown to contain stem/progenitor-like cells. Characterization of these cells were carried out by using in vitro two- and three-dimensional cultures and in vivo reconstitution of mice after orthotopic or intravenous injection, in conjunction with quantitative reverse transcription polymerase chain reaction, Western blotting, immunohisto(cyto)chemistry/immunofluorescence, and RNA seq analysis. Results: These cells were named SPTL (side population cell-derived thyroid cell line). Under low serum culturing conditions, SPTL cells expressed the thyroid differentiation marker NKX2-1, a transcription factor critical for thyroid differentiation and function, while no expression of other thyroid differentiation marker genes were observed. SPTL cells formed follicle-like structures in Matrigel® cultures, which did not express thyroid differentiation marker genes. In mouse models of orthotopic and intravenous injection, the latter following partial thyroidectomy, a few SPTL cells were found in part of the follicles, most of which expressed NKX2-1. SPTL cells highly express genes involved in epithelial–mesenchymal transition, as demonstrated by RNA seq analysis, and exhibit a gene-expression pattern similar to anaplastic thyroid carcinoma. Conclusion: These results demonstrate that SPTL cells have the capacity to differentiate into thyroid to a limited degree. SPTL cells may provide an excellent tool to study stem cells, including cancer stem cells of the thyroid. PMID:28125936

Differential gene expression by Moniliophthora roreri while overcoming cacao tolerance in the field.

PubMed

Bailey, Bryan A; Melnick, Rachel L; Strem, Mary D; Crozier, Jayne; Shao, Jonathan; Sicher, Richard; Phillips-Mora, Wilberth; Ali, Shahin S; Zhang, Dapeng; Meinhardt, Lyndel

2014-09-01

Frosty pod rot (FPR) of Theobroma cacao (cacao) is caused by the hemibiotrophic fungus Moniliophthora roreri. Cacao clones tolerant to FPR are being planted throughout Central America. To determine whether M. roreri shows a differential molecular response during successful infections of tolerant clones, we collected field-infected pods at all stages of symptomatology for two highly susceptible clones (Pound-7 and CATIE-1000) and three tolerant clones (UF-273, CATIE-R7 and CATIE-R4). Metabolite analysis was carried out on clones Pound-7, CATIE-1000, CATIE-R7 and CATIE-R4. As FPR progressed, the concentrations of sugars in pods dropped, whereas the levels of trehalose and mannitol increased. Associations between symptoms and fungal loads and some organic and amino acid concentrations varied depending on the clone. RNA-Seq analysis identified 873 M. roreri genes that were differentially expressed between clones, with the primary difference being whether the clone was susceptible or tolerant. Genes encoding transcription factors, heat shock proteins, transporters, enzymes modifying membranes or cell walls and metabolic enzymes, such as malate synthase and alternative oxidase, were differentially expressed. The differential expression between clones of 43 M. roreri genes was validated by real-time quantitative reverse transcription polymerase chain reaction. The expression profiles of some genes were similar in susceptible and tolerant clones (other than CATIE-R4) and varied with the biotrophic/necrotropic shift. Moniliophthora roreri genes associated with stress metabolism and responses to heat shock and anoxia were induced early in tolerant clones, their expression profiles resembling that of the necrotrophic phase. Moniliophthora roreri stress response genes, induced during the infection of tolerant clones, may benefit the fungus in overcoming cacao defense mechanisms. © 2014 BSPP AND JOHN WILEY & SONS LTD.

Differential DNA methylation profile of key genes in malignant prostate epithelial cells transformed by inorganic arsenic or cadmium.

PubMed

Pelch, Katherine E; Tokar, Erik J; Merrick, B Alex; Waalkes, Michael P

2015-08-01

Previous work shows altered methylation patterns in inorganic arsenic (iAs)- or cadmium (Cd)-transformed epithelial cells. Here, the methylation status near the transcriptional start site was assessed in the normal human prostate epithelial cell line (RWPE-1) that was malignantly transformed by 10μM Cd for 11weeks (CTPE) or 5μM iAs for 29weeks (CAsE-PE), at which time cells showed multiple markers of acquired cancer phenotype. Next generation sequencing of the transcriptome of CAsE-PE cells identified multiple dysregulated genes. Of the most highly dysregulated genes, five genes that can be relevant to the carcinogenic process (S100P, HYAL1, NTM, NES, ALDH1A1) were chosen for an in-depth analysis of the DNA methylation profile. DNA was isolated, bisulfite converted, and combined bisulfite restriction analysis was used to identify differentially methylated CpG sites, which was confirmed with bisulfite sequencing. Four of the five genes showed differential methylation in transformants relative to control cells that was inversely related to altered gene expression. Increased expression of HYAL1 (>25-fold) and S100P (>40-fold) in transformants was correlated with hypomethylation near the transcriptional start site. Decreased expression of NES (>15-fold) and NTM (>1000-fold) in transformants was correlated with hypermethylation near the transcriptional start site. ALDH1A1 expression was differentially expressed in transformed cells but was not differentially methylated relative to control. In conclusion, altered gene expression observed in Cd and iAs transformed cells may result from altered DNA methylation status. Published by Elsevier Inc.

Molecular Insights on Post-chemotherapy Retinoblastoma by Microarray Gene Expression Analysis

PubMed Central

Nalini, Venkatesan; Segu, Ramya; Deepa, Perinkulam Ravi; Khetan, Vikas; Vasudevan, Madavan; Krishnakumar, Subramanian

2013-01-01

Purpose Management of Retinoblastoma (RB), a pediatric ocular cancer is limited by drug-resistance and drug-dosage related side effects during chemotherapy. Molecular de-regulation in post-chemotherapy RB tumors was investigated. Materials and Methods cDNA microarray analysis of two post-chemotherapy and one pre-chemotherapy RB tumor tissues was performed, followed by Principle Component Analysis, Gene ontology, Pathway Enrichment analysis and Biological Analysis Network (BAN) modeling. The drug modulation role of two significantly up-regulated genes (p≤0.05) − Ect2 (Epithelial-cell-transforming-sequence-2), and PRAME (preferentially-expressed-Antigen-in-Melanoma) was assessed by qRT-PCR, immunohistochemistry and cell viability assays. Results Differential up-regulation of 1672 genes and down-regulation of 2538 genes was observed in RB tissues (relative to normal adult retina), while 1419 genes were commonly de-regulated between pre-chemotherapy and post- chemotherapy RB. Twenty one key gene ontology categories, pathways, biomarkers and phenotype groups harboring 250 differentially expressed genes were dys-regulated (EZH2, NCoR1, MYBL2, RB1, STAMN1, SYK, JAK1/2, STAT1/2, PLK2/4, BIRC5, LAMN1, Ect2, PRAME and ABCC4). Differential molecular expressions of PRAME and Ect2 in RB tumors with and without chemotherapy were analyzed. There was neither up- regulation of MRP1, nor any significant shift in chemotherapeutic IC50, in PRAME over-expressed versus non-transfected RB cells. Conclusion Cell cycle regulatory genes were dys-regulated post-chemotherapy. Ect2 gene was expressed in response to chemotherapy-induced stress. PRAME does not contribute to drug resistance in RB, yet its nuclear localization and BAN information, points to its possible regulatory role in RB. PMID:24092970

Altered expression of four miRNA (miR-1238-3p, miR-202-3p, miR-630 and miR-766-3p) and their potential targets in peripheral blood from vitiligo patients.

PubMed

Shang, Zhiwei; Li, Hongwen

2017-10-01

Vitiligo is an acquired skin disease with pigmentary disorder. Autoimmune destruction of melanocytes is thought to be major factor in the etiology of vitiligo. miRNA-based regulators of gene expression have been reported to play crucial roles in autoimmune disease. Therefore, we attempt to profile the miRNA expressions and predict their potential targets, assessing the biological functions of differentially expressed miRNA. Total RNA was extracted from peripheral blood of vitiligo (experimental group, n = 5) and non-vitiligo (control group, n = 5) age-matched patients. Samples were hybridized to a miRNA array. Box, scatter and principal component analysis plots were performed, followed by unsupervised hierarchical clustering analysis to classify the samples. Quantitative reverse transcription polymerase chain reaction (RT-PCR) was conducted for validation of microarray data. Three different databases, TargetScan, PITA and microRNA.org, were used to predict the potential target genes. Gene ontology (GO) annotation and pathway analysis were performed to assess the potential functions of predicted genes of identified miRNA. A total of 100 (29 upregulated and 71 downregulated) miRNA were filtered by volcano plot analysis. Four miRNA were validated by quantitative RT-PCR as significantly downregulated in the vitiligo group. The functions of predicted target genes associated with differentially expressed miRNA were assessed by GO analysis, showing that the GO term with most significantly enriched target genes was axon guidance, and that the axon guidance pathway was most significantly correlated with these miRNA. In conclusion, we identified four downregulated miRNA in vitiligo and assessed the potential functions of target genes related to these differentially expressed miRNA. © 2017 Japanese Dermatological Association.

Immunohistochemical Analysis of P63 Expression in Odontogenic Lesions

PubMed Central

Atarbashi Moghadam, Saede; Atarbashi Moghadam, Fazele; Eini, Ebrahim

2013-01-01

P63 may have a role in tumorigenesis and cytodifferentiation of odontogenic lesions. We investigated the immunohistochemical expression of P63 in a total of 30 cases of odontogenic cysts and tumors. The percentage of positive cells was calculated in the lining of odontogenic cysts and islands of ameloblastoma. P63 expression was evident in all types of odontogenic lesions. P63 was expressed throughout the lining epithelium of odontogenic keratocyst except surface parakeratinized layer. In addition, calcifying odontogenic cyst showed P63 expression in all layers. In almost all radicular and dentigerous cysts, the basal and parabasal layers were immunoreactive. Peripheral cells of ameloblastoma expressed P63; however, stellate reticulum had weaker immunostaining. No significant difference in P63 expression was observed between studied lesions (P = 0.86). Expression of P63 in odontogenic lesions suggests that this protein is important in differentiation and proliferation of odontogenic epithelial cells. However, it seems that it could not be a useful marker to differentiate between aggressive and nonaggressive lesions. P63 also represents a progenitor or basal cell marker, and it is not expressed in mature differentiated cells. PMID:24350278

Differentially expressed genes in the silk gland of silkworm (Bombyx mori) treated with TiO2 NPs.

PubMed

Xue, Bin; Li, Fanchi; Hu, Jingsheng; Tian, Jianghai; Li, Jinxin; Cheng, Xiaoyu; Hu, Jiahuan; Li, Bing

2017-05-05

Silk gland is a silkworm organ where silk proteins are synthesized and secreted. Dietary supplement of TiO 2 nanoparticles (NPs) promotes silk protein synthesis in silkworms. In this study, digital gene expression (DGE) tag was used to analyze the gene expression profile of the posterior silk gland of silkworms that were fed with TiO 2 NPs. In total, 5,702,823 and 6,150,719 clean tags, 55,096 and 74,715 distinct tags were detected in TiO 2 NPs treated and control groups, respectively. Compared with the control, TiO 2 NPs treated silkworms showed 306 differentially expressed genes, including 137 upregulated genes and 169 downregulated genes. Of these differentially expressed genes, 106 genes were related to silk protein synthesis, among which 97 genes were upregulated and 9 genes were downregulated. Pathway mapping using the Kyoto Encyclopedia of Genes and Genomes (KEGG) showed that 20 pathways were significantly enriched in TiO 2 NPs treated silkworms, and the metabolic pathway-related genes were the most significantly enriched. The DGE results were verified by qRT-PCR analysis of eight differentially expressed genes. The DGE and qRT-PCR results were consistent for all three upregulated genes and three of the five downregulated genes, but the expression trends of the remaining two genes were different between qRT-PCR and DGE analysis. This study enhances our understanding of the mechanism of TiO 2 NPs promoted silk protein synthesis. Copyright © 2017 Elsevier B.V. All rights reserved.

Differences in expression of retinal pigment epithelium mRNA between normal canines

PubMed Central

2004-01-01

Abstract A reference database of differences in mRNA expression in normal healthy canine retinal pigment epithelium (RPE) has been established. This database identifies non-informative differences in mRNA expression that can be used in screening canine RPE for mutations associated with clinical effects on vision. Complementary DNA (cDNA) pools were prepared from mRNA harvested from RPE, amplified by PCR, and used in a subtractive hybridization protocol (representational differential analysis) to identify differences in RPE mRNA expression between canines. The effect of relatedness of the test canines on the frequency of occurrence of differences was evaluated by using 2 unrelated canines for comparison with 2 female sibling canines of blue heeler/bull terrier lineage. Differentially expressed cDNA species were cloned, sequenced, and identified by comparison to public database entries. The most frequently observed differentially expressed sequence from the unrelated canine comparison was cDNA with 21 base pairs (bp) identical to the human epithelial membrane protein 1 gene (present in 8 of 20 clones). Different clones from the same-sex sibling RPE contained repetitions of several short sequence motifs including the human epithelial membrane protein 1 (4 of 25 clones). Other prevalent differences between sibling RPE included sequences similar to a chicken genetic marker sequence motif (5 of 25), and 6 clones with homology to porcine major histocompatibility loci. In addition to identifying several repetitively occurring, noninformative, differentially expressed RPE mRNA species, the findings confirm that fewer differences occurred between siblings, highlighting the importance of using closely related subjects in representational difference analysis studies. PMID:15352545

Distinctive gene expression profiles characterize donor biopsies from HCV-positive kidney donors.

PubMed

Mas, Valeria R; Archer, Kellie J; Suh, Lacey; Scian, Mariano; Posner, Marc P; Maluf, Daniel G

2010-12-15

Because of the shortage of organs for transplantation, procurement of kidneys from extended criteria donors is inevitable. Frequently, donors infected with hepatitis C virus (HCV) are used. To elucidate an initial compromise of molecular pathways in HCV graft, gene expression profiles were evaluated. Twenty-four donor allograft biopsies (n=12 HCV positive (+) and n=12 HCV negative (-)) were collected at preimplantation time and profiled using microarrays. Donors were age, race, gender, and cold and warm ischemia time matched between groups. Probe level data were read into the R programming environment using the affy Bioconductor package, and the robust multiarray average method was used to obtain probe set expression summaries. To identify probe sets exhibiting differential expression, a two sample t test was performed. Molecular and biologic functions were analyzed using Interaction Networks and Functional Analysis. Fifty-eight probe sets were differentially expressed between HCV (+) versus HCV (-) donors (P<0.001). The molecular functions associated with the two top scored networks from the analysis of the differentially expressed genes were connective tissue development and function and tissue morphology (score 34), cell death, cell signaling, cellular assembly, and organization (score 32). Among the differentially affected top canonical pathways, we found the role of RIG1-like receptors in antiviral innate immunity (P<0.001), natural killer cell signaling (P=0.007), interleukin-8 signaling (P=0.048), interferon signaling (P=0.0 11; INFA21, INFGR1, and MED14), ILK signaling (P=0.001), and apoptosis signaling. A unique gene expression pattern was identified in HCV (+) kidney grafts. Innate immune system and inflammatory pathways were the most affected.

Differential gene expression between African American and European American colorectal cancer patients.

PubMed

Jovov, Biljana; Araujo-Perez, Felix; Sigel, Carlie S; Stratford, Jeran K; McCoy, Amber N; Yeh, Jen Jen; Keku, Temitope

2012-01-01

The incidence and mortality of colorectal cancer (CRC) is higher in African Americans (AAs) than other ethnic groups in the U. S., but reasons for the disparities are unknown. We performed gene expression profiling of sporadic CRCs from AAs vs. European Americans (EAs) to assess the contribution to CRC disparities. We evaluated the gene expression of 43 AA and 43 EA CRC tumors matched by stage and 40 matching normal colorectal tissues using the Agilent human whole genome 4x44K cDNA arrays. Gene and pathway analyses were performed using Significance Analysis of Microarrays (SAM), Ten-fold cross validation, and Ingenuity Pathway Analysis (IPA). SAM revealed that 95 genes were differentially expressed between AA and EA patients at a false discovery rate of ≤5%. Using IPA we determined that most prominent disease and pathway associations of differentially expressed genes were related to inflammation and immune response. Ten-fold cross validation demonstrated that following 10 genes can predict ethnicity with an accuracy of 94%: CRYBB2, PSPH, ADAL, VSIG10L, C17orf81, ANKRD36B, ZNF835, ARHGAP6, TRNT1 and WDR8. Expression of these 10 genes was validated by qRT-PCR in an independent test set of 28 patients (10 AA, 18 EA). Our results are the first to implicate differential gene expression in CRC racial disparities and indicate prominent difference in CRC inflammation between AA and EA patients. Differences in susceptibility to inflammation support the existence of distinct tumor microenvironments in these two patient populations.

Differential Gene Expression between African American and European American Colorectal Cancer Patients

PubMed Central

Jovov, Biljana; Araujo-Perez, Felix; Sigel, Carlie S.; Stratford, Jeran K.; McCoy, Amber N.; Yeh, Jen Jen; Keku, Temitope

2012-01-01

The incidence and mortality of colorectal cancer (CRC) is higher in African Americans (AAs) than other ethnic groups in the U. S., but reasons for the disparities are unknown. We performed gene expression profiling of sporadic CRCs from AAs vs. European Americans (EAs) to assess the contribution to CRC disparities. We evaluated the gene expression of 43 AA and 43 EA CRC tumors matched by stage and 40 matching normal colorectal tissues using the Agilent human whole genome 4x44K cDNA arrays. Gene and pathway analyses were performed using Significance Analysis of Microarrays (SAM), Ten-fold cross validation, and Ingenuity Pathway Analysis (IPA). SAM revealed that 95 genes were differentially expressed between AA and EA patients at a false discovery rate of ≤5%. Using IPA we determined that most prominent disease and pathway associations of differentially expressed genes were related to inflammation and immune response. Ten-fold cross validation demonstrated that following 10 genes can predict ethnicity with an accuracy of 94%: CRYBB2, PSPH, ADAL, VSIG10L, C17orf81, ANKRD36B, ZNF835, ARHGAP6, TRNT1 and WDR8. Expression of these 10 genes was validated by qRT-PCR in an independent test set of 28 patients (10 AA, 18 EA). Our results are the first to implicate differential gene expression in CRC racial disparities and indicate prominent difference in CRC inflammation between AA and EA patients. Differences in susceptibility to inflammation support the existence of distinct tumor microenvironments in these two patient populations. PMID:22276153

Gene function in early mouse embryonic stem cell differentiation

PubMed Central

Sene, Kagnew Hailesellasse; Porter, Christopher J; Palidwor, Gareth; Perez-Iratxeta, Carolina; Muro, Enrique M; Campbell, Pearl A; Rudnicki, Michael A; Andrade-Navarro, Miguel A

2007-01-01

Background Little is known about the genes that drive embryonic stem cell differentiation. However, such knowledge is necessary if we are to exploit the therapeutic potential of stem cells. To uncover the genetic determinants of mouse embryonic stem cell (mESC) differentiation, we have generated and analyzed 11-point time-series of DNA microarray data for three biologically equivalent but genetically distinct mESC lines (R1, J1, and V6.5) undergoing undirected differentiation into embryoid bodies (EBs) over a period of two weeks. Results We identified the initial 12 hour period as reflecting the early stages of mESC differentiation and studied probe sets showing consistent changes of gene expression in that period. Gene function analysis indicated significant up-regulation of genes related to regulation of transcription and mRNA splicing, and down-regulation of genes related to intracellular signaling. Phylogenetic analysis indicated that the genes showing the largest expression changes were more likely to have originated in metazoans. The probe sets with the most consistent gene changes in the three cell lines represented 24 down-regulated and 12 up-regulated genes, all with closely related human homologues. Whereas some of these genes are known to be involved in embryonic developmental processes (e.g. Klf4, Otx2, Smn1, Socs3, Tagln, Tdgf1), our analysis points to others (such as transcription factor Phf21a, extracellular matrix related Lama1 and Cyr61, or endoplasmic reticulum related Sc4mol and Scd2) that have not been previously related to mESC function. The majority of identified functions were related to transcriptional regulation, intracellular signaling, and cytoskeleton. Genes involved in other cellular functions important in ESC differentiation such as chromatin remodeling and transmembrane receptors were not observed in this set. Conclusion Our analysis profiles for the first time gene expression at a very early stage of mESC differentiation, and identifies a functional and phylogenetic signature for the genes involved. The data generated constitute a valuable resource for further studies. All DNA microarray data used in this study are available in the StemBase database of stem cell gene expression data [1] and in the NCBI's GEO database. PMID:17394647

Novel candidate genes of the PARK7 interactome as mediators of apoptosis and acetylation in multiple sclerosis: An in silico analysis.

PubMed

Vavougios, George D; Zarogiannis, Sotirios G; Krogfelt, Karen Angeliki; Gourgoulianis, Konstantinos; Mitsikostas, Dimos Dimitrios; Hadjigeorgiou, Georgios

2018-01-01

currently only 4 studies have explored the potential role of PARK7's dysregulation in MS pathophysiology Currently, no study has evaluated the potential role of the PARK7 interactome in MS. The aim of our study was to assess the differential expression of PARK7 mRNA in peripheral blood mononuclears (PBMCs) donated from MS versus healthy patients using data mining techniques. The PARK7 interactome data from the GDS3920 profile were scrutinized for differentially expressed genes (DEGs); Gene Enrichment Analysis (GEA) was used to detect significantly enriched biological functions. 27 differentially expressed genes in the MS dataset were detected; 12 of these (NDUFA4, UBA2, TDP2, NPM1, NDUFS3, SUMO1, PIAS2, KIAA0101, RBBP4, NONO, RBBP7 AND HSPA4) are reported for the first time in MS. Stepwise Linear Discriminant Function Analysis constructed a predictive model (Wilk's λ = 0.176, χ 2 = 45.204, p = 1.5275e -10 ) with 2 variables (TIDP2, RBBP4) that achieved 96.6% accuracy when discriminating between patients and controls. Gene Enrichment Analysis revealed that induction and regulation of programmed / intrinsic cell death represented the most salient Gene Ontology annotations. Cross-validation on systemic lupus erythematosus and ischemic stroke datasets revealed that these functions are unique to the MS dataset. Based on our results, novel potential target genes are revealed; these differentially expressed genes regulate epigenetic and apoptotic pathways that may further elucidate underlying mechanisms of autorreactivity in MS. Copyright © 2017 Elsevier B.V. All rights reserved.

Insight into Genes Regulating Postharvest Aflatoxin Contamination of Tetraploid Peanut from Transcriptional Profiling.

PubMed

Korani, Walid; Chu, Ye; Holbrook, C Corley; Ozias-Akins, Peggy

2018-05-01

Postharvest aflatoxin contamination is a challenging issue that affects peanut quality. Aflatoxin is produced by fungi belonging to the Aspergilli group, and is known as an acutely toxic, carcinogenic, and immune-suppressing class of mycotoxins. Evidence for several host genetic factors that may impact aflatoxin contamination has been reported, e.g. , genes for lipoxygenase (PnLOX1 and PnLOX2/PnLOX3 that showed either positive or negative regulation with Aspergillus infection), reactive oxygen species, and WRKY (highly associated with or differentially expressed upon infection of maize with Aspergillus flavus ); however, their roles remain unclear. Therefore, we conducted an RNA-sequencing experiment to differentiate gene response to the infection by A. flavus between resistant (ICG 1471) and susceptible (Florida-07) cultivated peanut genotypes. The gene expression profiling analysis was designed to reveal differentially expressed genes in response to the infection (infected vs. mock-treated seeds). In addition, the differential expression of the fungal genes was profiled. The study revealed the complexity of the interaction between the fungus and peanut seeds as the expression of a large number of genes was altered, including some in the process of plant defense to aflatoxin accumulation. Analysis of the experimental data with "keggseq," a novel designed tool for Kyoto Encyclopedia of Genes and Genomes enrichment analysis, showed the importance of α-linolenic acid metabolism, protein processing in the endoplasmic reticulum, spliceosome, and carbon fixation and metabolism pathways in conditioning resistance to aflatoxin accumulation. In addition, coexpression network analysis was carried out to reveal the correlation of gene expression among peanut and fungal genes. The results showed the importance of WRKY, toll/Interleukin1 receptor-nucleotide binding site leucine-rich repeat (TIR-NBS-LRR), ethylene, and heat shock proteins in the resistance mechanism. Copyright © 2018 by the Genetics Society of America.

Proteome analysis reveals that de novo regenerated mucosa over fibula flap-reconstructed mandibles resembles mature keratinized oral mucosa.

PubMed

Kumar, Vinay V; James, Bonney L; Ruß, Manuela; Mikkat, Stefan; Suresh, Amritha; Kämmerer, Peer W; Glocker, Michael O

2018-03-01

The aim of this study was to determine whether intra-oral de novo regenerated mucosa (D) that grew over free fibula flap reconstructed-mandibles resembled the donor tissue i.e. external skin (S) of the lateral leg, or the recipient site tissue, i.e. keratinized oral mucosa (K). Differential proteome analysis was performed with ten tissue samples from each of the three groups: de novo regenerated mucosa (D), external skin (S), and keratinized oral mucosa (K). Expression differences of cornulin and involucrin were validated by Western blot analysis and their spatial distributions in the respective tissues were ascertained by immunohistochemistry. From all three investigated tissue types a total of 1188 proteins were identified, 930 of which were reproducibly and robustly quantified by proteome analysis. The best differentiating proteins were assembled in an oral mucosa proteome signature that encompasses 56 differentially expressed proteins. Principal component analysis of both, the 930 quantifiable proteins and the 56 oral mucosa signature proteins revealed that the de novo regenerated mucosa resembles keratinized oral mucosa much closer than extra-oral skin. Differentially expressed cornification-related proteins comprise proteins from all subclasses of the cornified cell envelope. Prominently expressed in intra-oral mucosa tissues were (i) cornifin-A, cornifin-B, SPRR3, and involucrin from the cornified-cell-envelope precursor group, (ii) S100A9, S100A8 and S100A2 from the S100 group, and (iii) cornulin which belongs to the fused-gene-protein group. According to its proteome signature de novo regenerated mucosa over the free fibula flap not only presents a passive structural surface layer but has adopted active tissue function. Copyright © 2018 Elsevier Ltd. All rights reserved.

Robust extraction of functional signals from gene set analysis using a generalized threshold free scoring function

PubMed Central

2009-01-01

Background A central task in contemporary biosciences is the identification of biological processes showing response in genome-wide differential gene expression experiments. Two types of analysis are common. Either, one generates an ordered list based on the differential expression values of the probed genes and examines the tail areas of the list for over-representation of various functional classes. Alternatively, one monitors the average differential expression level of genes belonging to a given functional class. So far these two types of method have not been combined. Results We introduce a scoring function, Gene Set Z-score (GSZ), for the analysis of functional class over-representation that combines two previous analysis methods. GSZ encompasses popular functions such as correlation, hypergeometric test, Max-Mean and Random Sets as limiting cases. GSZ is stable against changes in class size as well as across different positions of the analysed gene list in tests with randomized data. GSZ shows the best overall performance in a detailed comparison to popular functions using artificial data. Likewise, GSZ stands out in a cross-validation of methods using split real data. A comparison of empirical p-values further shows a strong difference in favour of GSZ, which clearly reports better p-values for top classes than the other methods. Furthermore, GSZ detects relevant biological themes that are missed by the other methods. These observations also hold when comparing GSZ with popular program packages. Conclusion GSZ and improved versions of earlier methods are a useful contribution to the analysis of differential gene expression. The methods and supplementary material are available from the website http://ekhidna.biocenter.helsinki.fi/users/petri/public/GSZ/GSZscore.html. PMID:19775443

Comprehensive transcriptome-based characterization of differentially expressed genes involved in microsporogenesis of radish CMS line and its maintainer.

PubMed

Xie, Yang; Zhang, Wei; Wang, Yan; Xu, Liang; Zhu, Xianwen; Muleke, Everlyne M; Liu, Liwang

2016-09-01

Microsporogenesis is an indispensable period for investigating microspore development and cytoplasmic male sterility (CMS) occurrence. Radish CMS line plays a critical role in elite F1 hybrid seed production and heterosis utilization. However, the molecular mechanisms of microspore development and CMS occurrence have not been thoroughly uncovered in radish. In this study, a comparative analysis of radish floral buds from a CMS line (NAU-WA) and its maintainer (NAU-WB) was conducted using next generation sequencing (NGS) technology. Digital gene expression (DGE) profiling revealed that 3504 genes were significantly differentially expressed between NAU-WA and NAU-WB library, among which 1910 were upregulated and 1594 were downregulated. Gene ontology (GO) analysis showed that these differentially expressed genes (DEGs) were mainly enriched in extracellular region, catalytic activity, and response to stimulus. KEGG enrichment analysis revealed that the DEGs were predominantly associated with flavonoid biosynthesis, glycolysis, and biosynthesis of secondary metabolites. Real-time quantitative PCR analysis showed that the expression profiles of 13 randomly selected DEGs were in high agreement with results from Illumina sequencing. Several candidate genes encoding ATP synthase, auxin response factor (ARF), transcription factors (TFs), chalcone synthase (CHS), and male sterility (MS) were responsible for microsporogenesis. Furthermore, a schematic diagram for functional interaction of DEGs from NAU-WA vs. NAU-WB library in radish plants was proposed. These results could provide new information on the dissection of the molecular mechanisms underlying microspore development and CMS occurrence in radish.

Quantitative proteomic analysis of whey proteins in the colostrum and mature milk of yak (Bos grunniens).

PubMed

Yang, Yongxin; Zhao, Xiaowei; Yu, Shumin; Cao, Suizhong

2015-02-01

Yak (Bos grunniens) is an important natural resource in mountainous regions. To date, few studies have addressed the differences in the protein profiles of yak colostrum and milk. We used quantitative proteomics to compare the protein profiles of whey from yak colostrum and milk. Milk samples were collected from 21 yaks after calving (1 and 28 d). Whey protein profiles were generated through isobaric tag for relative and absolute quantification (iTRAQ)-labelled proteomics. We identified 183 proteins in milk whey; of these, the expression levels of 86 proteins differed significantly between the whey from colostrum and milk. Haemoglobin expression showed the greatest change; its levels were significantly higher in the whey from colostrum than in mature milk whey. Functional analysis revealed that many of the differentially expressed proteins were associated with biological regulation and response to stimuli. Further, eight differentially expressed proteins involved in the complement and coagulation cascade pathway were enriched in milk whey. These findings add to the general understanding of the protein composition of yak milk, suggest potential functions of the differentially expressed proteins, and provide novel information on the role of colostral components in calf survival. © 2014 Society of Chemical Industry.

Differential co-expression analysis reveals a novel prognostic gene module in ovarian cancer.

PubMed

Gov, Esra; Arga, Kazim Yalcin

2017-07-10

Ovarian cancer is one of the most significant disease among gynecological disorders that women suffered from over the centuries. However, disease-specific and effective biomarkers were still not available, since studies have focused on individual genes associated with ovarian cancer, ignoring the interactions and associations among the gene products. Here, ovarian cancer differential co-expression networks were reconstructed via meta-analysis of gene expression data and co-expressed gene modules were identified in epithelial cells from ovarian tumor and healthy ovarian surface epithelial samples to propose ovarian cancer associated genes and their interactions. We propose a novel, highly interconnected, differentially co-expressed, and co-regulated gene module in ovarian cancer consisting of 84 prognostic genes. Furthermore, the specificity of the module to ovarian cancer was shown through analyses of datasets in nine other cancers. These observations underscore the importance of transcriptome based systems biomarkers research in deciphering the elusive pathophysiology of ovarian cancer, and here, we present reciprocal interplay between candidate ovarian cancer genes and their transcriptional regulatory dynamics. The corresponding gene module might provide new insights on ovarian cancer prognosis and treatment strategies that continue to place a significant burden on global health.

Differential Network Analysis Reveals Evolutionary Complexity in Secondary Metabolism of Rauvolfia serpentina over Catharanthus roseus

PubMed Central

Pathania, Shivalika; Bagler, Ganesh; Ahuja, Paramvir S.

2016-01-01

Comparative co-expression analysis of multiple species using high-throughput data is an integrative approach to determine the uniformity as well as diversification in biological processes. Rauvolfia serpentina and Catharanthus roseus, both members of Apocyanacae family, are reported to have remedial properties against multiple diseases. Despite of sharing upstream of terpenoid indole alkaloid pathway, there is significant diversity in tissue-specific synthesis and accumulation of specialized metabolites in these plants. This led us to implement comparative co-expression network analysis to investigate the modules and genes responsible for differential tissue-specific expression as well as species-specific synthesis of metabolites. Toward these goals differential network analysis was implemented to identify candidate genes responsible for diversification of metabolites profile. Three genes were identified with significant difference in connectivity leading to differential regulatory behavior between these plants. These genes may be responsible for diversification of secondary metabolism, and thereby for species-specific metabolite synthesis. The network robustness of R. serpentina, determined based on topological properties, was also complemented by comparison of gene-metabolite networks of both plants, and may have evolved to have complex metabolic mechanisms as compared to C. roseus under the influence of various stimuli. This study reveals evolution of complexity in secondary metabolism of R. serpentina, and key genes that contribute toward diversification of specific metabolites. PMID:27588023

Differential Network Analysis Reveals Evolutionary Complexity in Secondary Metabolism of Rauvolfia serpentina over Catharanthus roseus.

PubMed

Pathania, Shivalika; Bagler, Ganesh; Ahuja, Paramvir S

2016-01-01

Comparative co-expression analysis of multiple species using high-throughput data is an integrative approach to determine the uniformity as well as diversification in biological processes. Rauvolfia serpentina and Catharanthus roseus, both members of Apocyanacae family, are reported to have remedial properties against multiple diseases. Despite of sharing upstream of terpenoid indole alkaloid pathway, there is significant diversity in tissue-specific synthesis and accumulation of specialized metabolites in these plants. This led us to implement comparative co-expression network analysis to investigate the modules and genes responsible for differential tissue-specific expression as well as species-specific synthesis of metabolites. Toward these goals differential network analysis was implemented to identify candidate genes responsible for diversification of metabolites profile. Three genes were identified with significant difference in connectivity leading to differential regulatory behavior between these plants. These genes may be responsible for diversification of secondary metabolism, and thereby for species-specific metabolite synthesis. The network robustness of R. serpentina, determined based on topological properties, was also complemented by comparison of gene-metabolite networks of both plants, and may have evolved to have complex metabolic mechanisms as compared to C. roseus under the influence of various stimuli. This study reveals evolution of complexity in secondary metabolism of R. serpentina, and key genes that contribute toward diversification of specific metabolites.

Label free quantitative proteomics analysis on the cisplatin resistance in ovarian cancer cells.

PubMed

Wang, F; Zhu, Y; Fang, S; Li, S; Liu, S

2017-05-20

Quantitative proteomics has been made great progress in recent years. Label free quantitative proteomics analysis based on the mass spectrometry is widely used. Using this technique, we determined the differentially expressed proteins in the cisplatin-sensitive ovarian cancer cells COC1 and cisplatin-resistant cells COC1/DDP before and after the application of cisplatin. Using the GO analysis, we classified those proteins into different subgroups bases on their cellular component, biological process, and molecular function. We also used KEGG pathway analysis to determine the key signal pathways that those proteins were involved in. There are 710 differential proteins between COC1 and COC1/DDP cells, 783 between COC1 and COC1/DDP cells treated with cisplatin, 917 between the COC1/DDP cells and COC1/DDP cells treated with LaCl3, 775 between COC1/DDP cells treated with cisplatin and COC1/DDP cells treated with cisplatin and LaCl3. Among the same 411 differentially expressed proteins in cisplatin-sensitive COC1 cells and cisplain-resistant COC1/DDP cells before and after cisplatin treatment, 14% of them were localized on the cell membrane. According to the KEGG results, differentially expressed proteins were classified into 21 groups. The most abundant proteins were involved in spliceosome. This study lays a foundation for deciphering the mechanism for drug resistance in ovarian tumor.

Mutation spectrum and differential gene expression in cystic and solid vestibular schwannoma.

PubMed

Zhang, Zhihua; Wang, Zhaoyan; Sun, Lianhua; Li, Xiaohua; Huang, Qi; Yang, Tao; Wu, Hao

2014-03-01

We sought to characterize the mutation spectrum of NF2 and the differential gene expression in cystic and solid vestibular schwannomas. We collected tumor tissue and blood samples of 31 cystic vestibular schwannomas and 114 solid vestibular schwannomas. Mutation screening of NF2 was performed in both tumor and blood DNA samples of all patients. cDNA microarray was used to analyze the differential gene expression between 11 cystic vestibular schwannomas and 6 solid vestibular schwannomas. Expression levels of top candidate genes were verified by quantitative reverse transcription PCR. NF2 mutations were identified in 34.5% of sporadic vestibular schwannomas, with all mutations being exclusively somatic. No significant difference was found between the mutation detection rates of cystic vestibular schwannoma (35.5%) and solid vestibular schwannoma (34.2%). cDNA microarray analysis detected a total of 46 differentially expressed genes between the cystic vestibular schwannoma and solid vestibular schwannoma samples. The significantly decreased expression of four top candidate genes, C1orf130, CNTF, COL4A3, and COL4A4, was verified by quantitative reverse transcription PCR. NF2 mutations are not directly involved in the cystic formation of vestibular schwannoma. In addition, the differential gene expression of cystic vestibular schwannoma reported in our study may provide useful insights into the molecular mechanism underlying this process.

Enhanced identification and biological validation of differential gene expression via Illumina whole-genome expression arrays through the use of the model-based background correction methodology

PubMed Central

Ding, Liang-Hao; Xie, Yang; Park, Seongmi; Xiao, Guanghua; Story, Michael D.

2008-01-01

Despite the tremendous growth of microarray usage in scientific studies, there is a lack of standards for background correction methodologies, especially in single-color microarray platforms. Traditional background subtraction methods often generate negative signals and thus cause large amounts of data loss. Hence, some researchers prefer to avoid background corrections, which typically result in the underestimation of differential expression. Here, by utilizing nonspecific negative control features integrated into Illumina whole genome expression arrays, we have developed a method of model-based background correction for BeadArrays (MBCB). We compared the MBCB with a method adapted from the Affymetrix robust multi-array analysis algorithm and with no background subtraction, using a mouse acute myeloid leukemia (AML) dataset. We demonstrated that differential expression ratios obtained by using the MBCB had the best correlation with quantitative RT–PCR. MBCB also achieved better sensitivity in detecting differentially expressed genes with biological significance. For example, we demonstrated that the differential regulation of Tnfr2, Ikk and NF-kappaB, the death receptor pathway, in the AML samples, could only be detected by using data after MBCB implementation. We conclude that MBCB is a robust background correction method that will lead to more precise determination of gene expression and better biological interpretation of Illumina BeadArray data. PMID:18450815

Microarray Analysis of Long Noncoding RNAs in Female Diabetic Peripheral Neuropathy Patients.

PubMed

Luo, Lin; Ji, Lin-Dan; Cai, Jiang-Jia; Feng, Mei; Zhou, Mi; Hu, Su-Pei; Xu, Jin; Zhou, Wen-Hua

2018-01-01

Diabetic peripheral neuropathy (DPN) is the most common complication of diabetes mellitus (DM). Because of its controversial pathogenesis, DPN is still not diagnosed or managed properly in most patients. In this study, human lncRNA microarrays were used to identify the differentially expressed lncRNAs in DM and DPN patients, and some of the discovered lncRNAs were further validated in additional 78 samples by quantitative realtime PCR (qRT-PCR). The microarray analysis identified 446 and 1327 differentially expressed lncRNAs in DM and DPN, respectively. The KEGG pathway analysis further revealed that the differentially expressed lncRNA-coexpressed mRNAs between DPN and DM groups were significantly enriched in the MAPK signaling pathway. The lncRNA/mRNA coexpression network indicated that BDNF and TRAF2 correlated with 6 lncRNAs. The qRT-PCR confirmed the initial microarray results. These findings demonstrated that the interplay between lncRNAs and mRNA may be involved in the pathogenesis of DPN, especially the neurotrophin-MAPK signaling pathway, thus providing relevant information for future studies. © 2018 The Author(s). Published by S. Karger AG, Basel.

Identification of Potential Biomarkers for Rhegmatogenous Retinal Detachment Associated with Choroidal Detachment by Vitreous iTRAQ-Based Proteomic Profiling

PubMed Central

Wu, Zhifeng; Ding, Nannan; Yu, Mengxi; Wang, Ke; Luo, Shasha; Zou, Wenjun; Zhou, Ying; Yan, Biao; Jiang, Qin

2016-01-01

Rhegmatogenous retinal detachment associated with choroidal detachment (RRDCD) is a complicated and serious type of rhegmatogenous retinal detachment (RRD). In this study, we identified differentially expressed proteins in the vitreous humors of RRDCD and RRD using isobaric tags for relative and absolute quantitation (iTRAQ) combined with nano-liquid chromatography-electrospray ion trap-mass spectrometry-mass spectrometry (nano-LC-ESI-MS/MS) and bioinformatic analysis. Our result shows that 103 differentially expressed proteins, including 54 up-regulated and 49 down-regulated proteins were identified in RRDCD. Gene ontology (GO) analysis suggested that most of the differentially expressed proteins were extracellular．The Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis suggested that proteins related to complement and coagulation cascades were significantly enriched. iTRAQ-based proteomic profiling reveals that complement and coagulation cascades and inflammation may play important roles in the pathogenesis of RRDCD. This study may provide novel insights into the pathogenesis of RRDCD and offer potential opportunities for the diagnosis and treatment of RRDCD. PMID:27941623

Monoethylhexyl Phthalate Elicits an Inflammatory Response in Adipocytes Characterized by Alterations in Lipid and Cytokine Pathways.

PubMed

Manteiga, Sara; Lee, Kyongbum

2017-04-01

A growing body of evidence links endocrine-disrupting chemicals (EDCs) with obesity-related metabolic diseases. While it has been shown that EDCs can predispose individuals toward adiposity by affecting developmental processes, little is known about the chemicals' effects on adult adipose tissue. Our aim was to study the effects of low, physiologically relevant doses of EDCs on differentiated murine adipocytes. We combined metabolomics, proteomics, and gene expression analysis to characterize the effects of mono-ethylhexyl phthalate (MEHP) in differentiated adipocytes. Repeated exposure to MEHP over several days led to changes in metabolite and enzyme levels indicating elevated lipogenesis and lipid oxidation. The chemical exposure also increased expression of major inflammatory cytokines, including chemotactic factors. Proteomic and gene expression analysis revealed significant alterations in pathways regulated by peroxisome proliferator activated receptor-γ (PPARγ). Inhibiting the nuclear receptor's activity using a chemical antagonist abrogated not only the alterations in PPARγ-regulated metabolic pathways, but also the increases in cytokine expression. Our results show that MEHP can induce a pro-inflammatory state in differentiated adipocytes. This effect is at least partially mediated PPARγ.

DEIsoM: a hierarchical Bayesian model for identifying differentially expressed isoforms using biological replicates

PubMed Central

Peng, Hao; Yang, Yifan; Zhe, Shandian; Wang, Jian; Gribskov, Michael; Qi, Yuan

2017-01-01

Abstract Motivation High-throughput mRNA sequencing (RNA-Seq) is a powerful tool for quantifying gene expression. Identification of transcript isoforms that are differentially expressed in different conditions, such as in patients and healthy subjects, can provide insights into the molecular basis of diseases. Current transcript quantification approaches, however, do not take advantage of the shared information in the biological replicates, potentially decreasing sensitivity and accuracy. Results We present a novel hierarchical Bayesian model called Differentially Expressed Isoform detection from Multiple biological replicates (DEIsoM) for identifying differentially expressed (DE) isoforms from multiple biological replicates representing two conditions, e.g. multiple samples from healthy and diseased subjects. DEIsoM first estimates isoform expression within each condition by (1) capturing common patterns from sample replicates while allowing individual differences, and (2) modeling the uncertainty introduced by ambiguous read mapping in each replicate. Specifically, we introduce a Dirichlet prior distribution to capture the common expression pattern of replicates from the same condition, and treat the isoform expression of individual replicates as samples from this distribution. Ambiguous read mapping is modeled as a multinomial distribution, and ambiguous reads are assigned to the most probable isoform in each replicate. Additionally, DEIsoM couples an efficient variational inference and a post-analysis method to improve the accuracy and speed of identification of DE isoforms over alternative methods. Application of DEIsoM to an hepatocellular carcinoma (HCC) dataset identifies biologically relevant DE isoforms. The relevance of these genes/isoforms to HCC are supported by principal component analysis (PCA), read coverage visualization, and the biological literature. Availability and implementation The software is available at https://github.com/hao-peng/DEIsoM Contact pengh@alumni.purdue.edu Supplementary information Supplementary data are available at Bioinformatics online. PMID:28595376

Expression of Master Regulators of T-cell, Helper T-cell and Follicular Helper T-cell Differentiation in Angioimmunoblastic T-cell Lymphoma.

PubMed

Matsumoto, Yosuke; Nagoshi, Hisao; Yoshida, Mihoko; Kato, Seiichi; Kuroda, Junya; Shimura, Kazuho; Kaneko, Hiroto; Horiike, Shigeo; Nakamura, Shigeo; Taniwaki, Masafumi

2017-11-01

Objective It has been postulated that the normal counterpart of angioimmunoblastic T-cell lymphoma (AITL) is the follicular helper T-cell (TFH). Recent immunological studies have identified several transcription factors responsible for T-cell differentiation. The master regulators associated with T-cell, helper T-cell (Th), and TFH differentiation are reportedly BCL11B, Th-POK, and BCL6, respectively. We explored the postulated normal counterpart of AITL with respect to the expression of the master regulators of T-cell differentiation. Methods We performed an immunohistochemical analysis in 15 AITL patients to determine the expression of the master regulators and several surface markers associated with T-cell differentiation. Results BCL11B was detected in 10 patients (67%), and the surface marker of T-cells (CD3) was detected in all patients. Only 2 patients (13%) expressed the marker of naïve T-cells (CD45RA), but all patients expressed the marker of effector T-cells (CD45RO). Nine patients expressed Th-POK (60%), and 7 (47%) expressed a set of surface antigens of Th (CD4-positive and CD8-negative). In addition, BCL6 and the surface markers of TFH (CXCL13, PD-1, and SAP) were detected in 11 (73%), 8 (53%), 14 (93%), and all patients, respectively. Th-POK-positive/BCL6-negative patients showed a significantly shorter overall survival (OS) than the other patients (median OS: 33.0 months vs. 74.0 months, p=0.020; log-rank test). Conclusion Many of the AITL patients analyzed in this study expressed the master regulators of T-cell differentiation. The clarification of the diagnostic significance and pathophysiology based on the expression of these master regulators in AITL is expected in the future.

Identification of differentially expressed genes of brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss) in response to Tetracapsuloides bryosalmonae (Myxozoa).

PubMed

Kumar, Gokhlesh; Abd-Elfattah, Ahmed; El-Matbouli, Mansour

2015-03-01

Tetracapsuloides bryosalmonae Canning et al., 1999 (Myxozoa) is the causative agent of proliferative kidney disease in various species of salmonids in Europe and North America. We have shown previously that the development and distribution of the European strain of T. bryosalmonae differs in the kidney of brown trout (Salmo trutta) Linnaeus, 1758 and rainbow trout (Oncorhynchus mykiss) Walbaum, 1792, and that intra-luminal sporogonic stages were found in brown trout but not in rainbow trout. We have now compared transcriptomes from kidneys of brown trout and rainbow trout infected with T. bryosalmonae using suppressive subtractive hybridization (SSH). The differentially expressed transcripts produced by SSH were cloned, transformed, and tested by colony PCR. Differential expression screening of PCR products was validated using dot blot, and positive clones having different signal intensities were sequenced. Differential screening and a subsequent NCBI-BLAST analysis of expressed sequence tags revealed nine clones expressed differently between both fish species. These differentially expressed genes were validated by quantitative real-time PCR of kidney samples from both fish species at different time points of infection. Expression of anti-inflammatory (TSC22 domain family protein 3) and cell proliferation (Prothymin alpha) genes were upregulated significantly in brown trout but downregulated in rainbow trout. The expression of humoral immune response (immunoglobulin mu) and endocytic pathway (Ras-related protein Rab-11b) genes were significantly upregulated in rainbow trout but downregulated in brown trout. This study suggests that differential expression of host anti-inflammatory, humoral immune and endocytic pathway responses, cell proliferation, and cell growth processes do not inhibit the development of intra-luminal sporogonic stages of the European strain of T. bryosalmonae in brown trout but may suppress it in rainbow trout.

cDNA-AFLP analysis reveals differential gene expression in response to salt stress in foxtail millet (Setaria italica L.).

PubMed

Jayaraman, Ananthi; Puranik, Swati; Rai, Neeraj Kumar; Vidapu, Sudhakar; Sahu, Pranav Pankaj; Lata, Charu; Prasad, Manoj

2008-11-01

Plant growth and productivity are affected by various abiotic stresses such as heat, drought, cold, salinity, etc. The mechanism of salt tolerance is one of the most important subjects in plant science as salt stress decreases worldwide agricultural production. In our present study we used cDNA-AFLP technique to compare gene expression profiles of a salt tolerant and a salt-sensitive cultivar of foxtail millet (Seteria italica) in response to salt stress to identify early responsive differentially expressed transcripts accumulated upon salt stress and validate the obtained result through quantitative real-time PCR (qRT-PCR). The expression profile was compared between a salt tolerant (Prasad) and susceptible variety (Lepakshi) of foxtail millet in both control condition (L0 and P0) and after 1 h (L1 and P1) of salt stress. We identified 90 transcript-derived fragments (TDFs) that are differentially expressed, out of which 86 TDFs were classified on the basis of their either complete presence or absence (qualitative variants) and 4 on differential expression pattern levels (quantitative variants) in the two varieties. Finally, we identified 27 non-redundant differentially expressed cDNAs that are unique to salt tolerant variety which represent different groups of genes involved in metabolism, cellular transport, cell signaling, transcriptional regulation, mRNA splicing, seed development and storage, etc. The expression patterns of seven out of nine such genes showed a significant increase of differential expression in tolerant variety after 1 h of salt stress in comparison to salt-sensitive variety as analyzed by qRT-PCR. The direct and indirect relationship of identified TDFs with salinity tolerance mechanism is discussed.

Development of a targeted transgenesis strategy in highly differentiated cells: a powerful tool for functional genomic analysis.

PubMed

Puttini, Stefania; Ouvrard-Pascaud, Antoine; Palais, Gael; Beggah, Ahmed T; Gascard, Philippe; Cohen-Tannoudji, Michel; Babinet, Charles; Blot-Chabaud, Marcel; Jaisser, Frederic

2005-03-16

Functional genomic analysis is a challenging step in the so-called post-genomic field. Identification of potential targets using large-scale gene expression analysis requires functional validation to identify those that are physiologically relevant. Genetically modified cell models are often used for this purpose allowing up- or down-expression of selected targets in a well-defined and if possible highly differentiated cell type. However, the generation of such models remains time-consuming and expensive. In order to alleviate this step, we developed a strategy aimed at the rapid and efficient generation of genetically modified cell lines with conditional, inducible expression of various target genes. Efficient knock-in of various constructs, called targeted transgenesis, in a locus selected for its permissibility to the tet inducible system, was obtained through the stimulation of site-specific homologous recombination by the meganuclease I-SceI. Our results demonstrate that targeted transgenesis in a reference inducible locus greatly facilitated the functional analysis of the selected recombinant cells. The efficient screening strategy we have designed makes possible automation of the transfection and selection steps. Furthermore, this strategy could be applied to a variety of highly differentiated cells.

Gene Network Construction from Microarray Data Identifies a Key Network Module and Several Candidate Hub Genes in Age-Associated Spatial Learning Impairment

PubMed Central

Uddin, Raihan; Singh, Shiva M.

2017-01-01

As humans age many suffer from a decrease in normal brain functions including spatial learning impairments. This study aimed to better understand the molecular mechanisms in age-associated spatial learning impairment (ASLI). We used a mathematical modeling approach implemented in Weighted Gene Co-expression Network Analysis (WGCNA) to create and compare gene network models of young (learning unimpaired) and aged (predominantly learning impaired) brains from a set of exploratory datasets in rats in the context of ASLI. The major goal was to overcome some of the limitations previously observed in the traditional meta- and pathway analysis using these data, and identify novel ASLI related genes and their networks based on co-expression relationship of genes. This analysis identified a set of network modules in the young, each of which is highly enriched with genes functioning in broad but distinct GO functional categories or biological pathways. Interestingly, the analysis pointed to a single module that was highly enriched with genes functioning in “learning and memory” related functions and pathways. Subsequent differential network analysis of this “learning and memory” module in the aged (predominantly learning impaired) rats compared to the young learning unimpaired rats allowed us to identify a set of novel ASLI candidate hub genes. Some of these genes show significant repeatability in networks generated from independent young and aged validation datasets. These hub genes are highly co-expressed with other genes in the network, which not only show differential expression but also differential co-expression and differential connectivity across age and learning impairment. The known function of these hub genes indicate that they play key roles in critical pathways, including kinase and phosphatase signaling, in functions related to various ion channels, and in maintaining neuronal integrity relating to synaptic plasticity and memory formation. Taken together, they provide a new insight and generate new hypotheses into the molecular mechanisms responsible for age associated learning impairment, including spatial learning. PMID:29066959

Gene Network Construction from Microarray Data Identifies a Key Network Module and Several Candidate Hub Genes in Age-Associated Spatial Learning Impairment.

PubMed

Uddin, Raihan; Singh, Shiva M

2017-01-01

As humans age many suffer from a decrease in normal brain functions including spatial learning impairments. This study aimed to better understand the molecular mechanisms in age-associated spatial learning impairment (ASLI). We used a mathematical modeling approach implemented in Weighted Gene Co-expression Network Analysis (WGCNA) to create and compare gene network models of young (learning unimpaired) and aged (predominantly learning impaired) brains from a set of exploratory datasets in rats in the context of ASLI. The major goal was to overcome some of the limitations previously observed in the traditional meta- and pathway analysis using these data, and identify novel ASLI related genes and their networks based on co-expression relationship of genes. This analysis identified a set of network modules in the young, each of which is highly enriched with genes functioning in broad but distinct GO functional categories or biological pathways. Interestingly, the analysis pointed to a single module that was highly enriched with genes functioning in "learning and memory" related functions and pathways. Subsequent differential network analysis of this "learning and memory" module in the aged (predominantly learning impaired) rats compared to the young learning unimpaired rats allowed us to identify a set of novel ASLI candidate hub genes. Some of these genes show significant repeatability in networks generated from independent young and aged validation datasets. These hub genes are highly co-expressed with other genes in the network, which not only show differential expression but also differential co-expression and differential connectivity across age and learning impairment. The known function of these hub genes indicate that they play key roles in critical pathways, including kinase and phosphatase signaling, in functions related to various ion channels, and in maintaining neuronal integrity relating to synaptic plasticity and memory formation. Taken together, they provide a new insight and generate new hypotheses into the molecular mechanisms responsible for age associated learning impairment, including spatial learning.

Genome-wide expression analysis of human in vivo irritated epidermis: differential profiles induced by sodium lauryl sulfate and nonanoic acid.

PubMed

Clemmensen, Anders; Andersen, Klaus E; Clemmensen, Ole; Tan, Qihua; Petersen, Thomas K; Kruse, Torben A; Thomassen, Mads

2010-09-01

The pathogenesis of irritant contact dermatitis (ICD) is poorly understood, and genes participating in the epidermal response to chemical irritants are only partly known. It is commonly accepted that different irritants have different mechanisms of action in the development of ICD. To define the differential molecular events induced in the epidermis by different irritants, we collected sequential biopsies ((1/2), 4, and 24 hours after a single exposure and at day 11 after repeated exposure) from human volunteers exposed to either sodium lauryl sulfate (SLS) or nonanoic acid (NON). Gene expression analysis using high-density oligonucleotide microarrays (representing 47,000 transcripts) revealed essentially different pathway responses (1/2)hours after exposure: NON transiently induced the IL-6 pathway as well as a number of mitogen-activated signaling cascades including extracellular signal-regulated kinase and growth factor receptor signaling, whereas SLS transiently downregulated cellular energy metabolism pathways. Differential expression of the cyclooxygenase-2 and matrix metalloproteinase 3 transcripts was confirmed immunohistochemically. After cumulative exposure, 883 genes were differentially expressed, whereas we identified 23 suggested common biomarkers for ICD. In conclusion, we bring new insights into two hitherto less well-elucidated phases of skin irritancy: the very initial as well as the late phase after single and cumulative mild exposures, respectively.

Identification of host genes leading to West Nile virus encephalitis in mice brain using RNA-seq analysis

PubMed Central

Kumar, Mukesh; Belcaid, Mahdi; Nerurkar, Vivek R.

2016-01-01

Differential host responses may be critical determinants of distinct pathologies of West Nile virus (WNV) NY99 (pathogenic) and WNV Eg101 (non-pathogenic) strains. We employed RNA-seq technology to analyze global differential gene expression in WNV-infected mice brain and to identify the host cellular factors leading to lethal encephalitis. We identified 1,400 and 278 transcripts, which were differentially expressed after WNV NY99 and WNV Eg101 infections, respectively, and 147 genes were common to infection with both the viruses. Genes that were up-regulated in infection with both the viruses were mainly associated with interferon signaling. Genes associated with inflammation and cell death/apoptosis were only expressed after WNV NY99 infection. We demonstrate that differences in the activation of key pattern recognition receptors resulted in the induction of unique innate immune profiles, which corresponded with the induction of interferon and inflammatory responses. Pathway analysis of differentially expressed genes indicated that after WNV NY99 infection, TREM-1 mediated activation of toll-like receptors leads to the high inflammatory response. In conclusion, we have identified both common and specific responses to WNV NY99 and WNV Eg101 infections as well as genes linked to potential resistance to infection that may be targets for therapeutics. PMID:27211830

[Endoplasmic reticulum stress in INS-1-3 cell associated with the expression changes of MODY gene pathway].

PubMed

Liu, Y T; Li, S R; Wang, Z; Xiao, J Z

2016-09-13

Objective: To profile the gene expression changes associated with endoplasmic reticulum stress in INS-1-3 cells induced by thapsigargin (TG) and tunicamycin (TM). Methods: Normal cultured INS-1-3 cells were used as a control. TG and TM were used to induce endoplasmic reticulum stress in INS-1-3 cells. Digital gene expression profiling technique was used to detect differentially expressed gene. The changes of gene expression were detected by expression pattern clustering analysis, gene ontology (GO) function and pathway enrichment analysis. Real time polymerase chain reaction (RT-PCR) was used to verify the key changes of gene expression. Results: Compared with the control group, there were 57 (45 up-regulated, 12 down-regulated) and 135 (99 up-regulated, 36 down-regulated) differentially expressed genes in TG and TM group, respectively. GO function enrichment analyses indicated that the main enrichment was in the endoplasmic reticulum. In signaling pathway analysis, the identified pathways were related with endoplasmic reticulum stress, antigen processing and presentation, protein export, and most of all, the maturity onset diabetes of the young (MODY) pathway. Conclusion: Under the condition of endoplasmic reticulum stress, the related expression changes of transcriptional factors in MODY signaling pathway may be related with the impaired function in islet beta cells.

A Study of the Differential Effects of Eicosapentaenoic Acid (EPA) and Docosahexaenoic Acid (DHA) on Gene Expression Profiles of Stimulated Thp-1 Macrophages

PubMed Central

Allam-Ndoul, Bénédicte; Guénard, Frédéric; Barbier, Olivier; Vohl, Marie-Claude

2017-01-01

Background: An appropriate intake of omega-3 (n-3) fatty acids (FAs) such as eicosapentaenoic and docosahexaenoic acid (EPA/DHA) from marine sources is known to have anti-inflammatory effects. However, molecular mechanisms underlying their beneficial effects on health are not fully understood. The aim of the present study was to characterize gene expression profiles of THP-1 macrophages, incubated in either EPA or DHA and stimulated with lipopolysaccharide (LPS), a pro-inflammatory agent. Methods: THP-1 macrophages were incubated into 10, 50 and 75 µM of EPA or DHA for 24 h, and 100 nM of LPS was added to the culture media for 18 h. Total mRNA was extracted and gene expression examined by microarray analysis using Illumina Human HT-12 expression beadchips (Illumina). Results: Pathway analysis revealed that EPA and DHA regulate genes involved in cell cycle regulation, apoptosis, immune response and inflammation, oxidative stress and cancer pathways in a differential and dose-dependent manner. Conclusions: EPA and DHA appear to exert differential effects on gene expression in THP-1 macrophages. Specific effects of n-3 FAs on gene expression levels are also dose-dependent. PMID:28441337

Transcriptomic Analysis Reveals Mechanisms of Sterile and Fertile Flower Differentiation and Development in Viburnum macrocephalum f. keteleeri

PubMed Central

Lu, Zhaogeng; Xu, Jing; Li, Weixing; Zhang, Li; Cui, Jiawen; He, Qingsong; Wang, Li; Jin, Biao

2017-01-01

Sterile and fertile flowers are an important evolutionary developmental (evo-devo) phenotype in angiosperm flowers, playing important roles in pollinator attraction and sexual reproductive success. However, the gene regulatory mechanisms underlying fertile and sterile flower differentiation and development remain largely unknown. Viburnum macrocephalum f. keteleeri, which possesses fertile and sterile flowers in a single inflorescence, is a useful candidate species for investigating the regulatory networks in differentiation and development. We developed a de novo-assembled flower reference transcriptome. Using RNA sequencing (RNA-seq), we compared the expression patterns of fertile and sterile flowers isolated from the same inflorescence over its rapid developmental stages. The flower reference transcriptome consisted of 105,683 non-redundant transcripts, of which 5,675 transcripts showed significant differential expression between fertile and sterile flowers. Combined with morphological and cytological changes between fertile and sterile flowers, we identified expression changes of many genes potentially involved in reproductive processes, phytohormone signaling, and cell proliferation and expansion using RNA-seq and qRT-PCR. In particular, many transcription factors (TFs), including MADS-box family members and ABCDE-class genes, were identified, and expression changes in TFs involved in multiple functions were analyzed and highlighted to determine their roles in regulating fertile and sterile flower differentiation and development. Our large-scale transcriptional analysis of fertile and sterile flowers revealed the dynamics of transcriptional networks and potentially key components in regulating differentiation and development of fertile and sterile flowers in Viburnum macrocephalum f. keteleeri. Our data provide a useful resource for Viburnum transcriptional research and offer insights into gene regulation of differentiation of diverse evo-devo processes in flowers. PMID:28298915

Assessment of myoblast circular RNA dynamics and its correlation with miRNA during myogenic differentiation.

PubMed

Zhang, Pengpeng; Xu, Haixia; Li, Rui; Wu, Wei; Chao, Zhe; Li, Cencen; Xia, Wei; Wang, Lei; Yang, Jinzeng; Xu, Yongjie

2018-06-01

Myoblast differentiation is a highly complex process that is regulated by proteins as well as by non-coding RNAs. Circular RNAs have been identified as an emerging new class of non-coding RNA in the modulation of skeletal muscle development, whereas their expression profiles and functional regulation in myoblast differentiation remain unknown. In the present study, we performed deep RNA-sequencing of C2C12 myoblasts during cell differentiation and uncovered 37,751 unique circular RNAs derived from 6943 hosting genes. The ensuing qRT-PCR and RNA fluorescence in situ hybridization verification were carried out to confirm the RNA-sequencing results. An unbiased analysis demonstrated dynamic circular RNA expression changes in the process of myoblast differentiation, and the circular RNA abundances were independent from their cognate linear RNAs. Gene ontology analysis showed that many down-regulated circular RNAs were exclusive to cell division and the cell cycle, whereas up-regulated circular RNAs were related to the cell development process. Furthermore, interaction networks of circular RNA-microRNA were constructed. Several microRNAs well-known for myoblast regulation, such as miR-133, miR-24 and miR-23a, were in this network. In summary, this study showed that circular RNA expression dynamics changed during myoblast differentiation. Circular RNAs play a role in regulating the myoblast cell cycle and development by acting as microRNA binding sites to facilitate their regulation of gene expression during myoblast differentiation. These findings open a new avenue for future investigation of this emerging RNA class in skeletal muscle growth and development. Copyright © 2018 Elsevier Ltd. All rights reserved.

It's DE-licious: A Recipe for Differential Expression Analyses of RNA-seq Experiments Using Quasi-Likelihood Methods in edgeR.

PubMed

Lun, Aaron T L; Chen, Yunshun; Smyth, Gordon K

2016-01-01

RNA sequencing (RNA-seq) is widely used to profile transcriptional activity in biological systems. Here we present an analysis pipeline for differential expression analysis of RNA-seq experiments using the Rsubread and edgeR software packages. The basic pipeline includes read alignment and counting, filtering and normalization, modelling of biological variability and hypothesis testing. For hypothesis testing, we describe particularly the quasi-likelihood features of edgeR. Some more advanced downstream analysis steps are also covered, including complex comparisons, gene ontology enrichment analyses and gene set testing. The code required to run each step is described, along with an outline of the underlying theory. The chapter includes a case study in which the pipeline is used to study the expression profiles of mammary gland cells in virgin, pregnant and lactating mice.

Characterization of microRNAs of Beta macrocarpa and their responses to Beet necrotic yellow vein virus infection.

PubMed

Liu, Jun-Ying; Fan, Hui-Yan; Wang, Ying; Zhang, Yong-Liang; Li, Da-Wei; Yu, Jia-Lin; Han, Cheng-Gui

2017-01-01

Plant microRNAs (miRNAs) are a class of non-coding RNAs that play important roles in plant development, defense, and symptom development. Here, 547 known miRNAs representing 129 miRNA families, and 282 potential novel miRNAs were identified in Beta macrocarpa using small RNA deep sequencing. A phylogenetic analysis was performed, and 8 Beta lineage-specific miRNAs were identified. Through a differential expression analysis, miRNAs associated with Beet necrotic yellow vein virus (BNYVV) infection were identified and confirmed using a microarray analysis and stem-loop RT-qPCR. In total, 103 known miRNAs representing 38 miRNA families, and 45 potential novel miRNAs were differentially regulated, with at least a two-fold change, in BNYVV-infected plants compared with that of the mock-inoculated control. Targets of these differentially expressed miRNAs were also predicted by degradome sequencing. These differentially expressed miRNAs were involved in hormone biosynthesis and signal transduction pathways, and enhanced axillary bud development and plant defenses. This work is the first to describe miRNAs of the plant genus Beta and may offer a reference for miRNA research in other species in the genus. It provides valuable information on the pathogenicity mechanisms of BNYVV.

Knock down of GCN5 histone acetyltransferase by siRNA decreases ethanol-induced histone acetylation and affects differential expression of genes in human hepatoma cells.

PubMed

Choudhury, Mahua; Pandey, Ravi S; Clemens, Dahn L; Davis, Justin Wade; Lim, Robert W; Shukla, Shivendra D

2011-06-01

We have investigated whether Gcn5, a histone acetyltransferase (HAT), is involved in ethanol-induced acetylation of histone H3 at lysine 9 (H3AcK9) and has any effect on the gene expression. Human hepatoma HepG2 cells transfected with ethanol-metabolizing enzyme alcohol dehydrogenase 1 (VA 13 cells) were used. Knock down of Gcn5 by siRNA silencing decreased mRNA and protein levels of general control nondepressible 5 (GCN5), HAT activity, and also attenuated ethanol-induced H3AcK9 in VA13 cells. Illumina gene microarray analysis using total RNA showed 940 transcripts affected by GCN5 silencing or ethanol. Silencing caused differential expression of 891 transcripts (≥1.5-fold upregulated or downregulated). Among these, 492 transcripts were upregulated and 399 were downregulated compared with their respective controls. Using a more stringent threshold (≥2.5-fold), the array data from GCN5-silenced samples showed 57 genes differentially expressed (39 upregulated and 18 downregulated). Likewise, ethanol caused differential regulation of 57 transcripts with ≥1.5-fold change (35 gene upregulated and 22 downregulated). Further analysis showed that eight genes were differentially regulated that were common for both ethanol treatment and GCN5 silencing. Among these, SLC44A2 (a putative choline transporter) was strikingly upregulated by ethanol (three fold), and GCN5 silencing downregulated it (1.5-fold). The quantitative real-time polymerase chain reaction profile corroborated the array findings. This report demonstrates for the first time that (1) GCN5 differentially affects expression of multiple genes, (2) ethanol-induced histone H3-lysine 9 acetylation is mediated via GCN5, and (3) GCN5 is involved in ethanol-induced expression of the putative choline transporter SLC44A2. Copyright © 2011 Elsevier Inc. All rights reserved.

OP17MICRORNA PROFILING USING SMALL RNA-SEQ IN PAEDIATRIC LOW GRADE GLIOMAS

PubMed Central

Jeyapalan, Jennie N.; Jones, Tania A.; Tatevossian, Ruth G.; Qaddoumi, Ibrahim; Ellison, David W.; Sheer, Denise

2014-01-01

INTRODUCTION: MicroRNAs regulate gene expression by targeting mRNAs for translational repression or degradation at the post-transcriptional level. In paediatric low-grade gliomas a few key genetic mutations have been identified, including BRAF fusions, FGFR1 duplications and MYB rearrangements. Our aim in the current study is to profile aberrant microRNA expression in paediatric low-grade gliomas and determine the role of epigenetic changes in the aetiology and behaviour of these tumours. METHOD: MicroRNA profiling of tumour samples (6 pilocytic, 2 diffuse, 2 pilomyxoid astrocytomas) and normal brain controls (4 adult normal brain samples and a primary glial progenitor cell-line) was performed using small RNA sequencing. Bioinformatic analysis included sequence alignment, analysis of the number of reads (CPM, counts per million) and differential expression. RESULTS: Sequence alignment identified 695 microRNAs, whose expression was compared in tumours v. normal brain. PCA and hierarchical clustering showed separate groups for tumours and normal brain. Computational analysis identified approximately 400 differentially expressed microRNAs in the tumours compared to matched location controls. Our findings will then be validated and integrated with extensive genetic and epigenetic information we have previously obtained for the full tumour cohort. CONCLUSION: We have identified microRNAs that are differentially expressed in paediatric low-grade gliomas. As microRNAs are known to target genes involved in the initiation and progression of cancer, they provide critical information on tumour pathogenesis and are an important class of biomarkers.

Whole-genome transcription and DNA methylation analysis of peripheral blood mononuclear cells identified aberrant gene regulation pathways in systemic lupus erythematosus.

PubMed

Zhu, Honglin; Mi, Wentao; Luo, Hui; Chen, Tao; Liu, Shengxi; Raman, Indu; Zuo, Xiaoxia; Li, Quan-Zhen

2016-07-13

Recent achievement in genetics and epigenetics has led to the exploration of the pathogenesis of systemic lupus erythematosus (SLE). Identification of differentially expressed genes and their regulatory mechanism(s) at whole-genome level will provide a comprehensive understanding of the development of SLE and its devastating complications, lupus nephritis (LN). We performed whole-genome transcription and DNA methylation analysis in PBMC of 30 SLE patients, including 15 with LN (SLE LN(+)) and 15 without LN (SLE LN(-)), and 25 normal controls (NC) using HumanHT-12 Beadchips and Illumina Human Methy450 chips. The serum proinflammatory cytokines were quantified using Bio-plex Human Cytokine 27-plex assay. Differentially expressed genes and differentially methylated CpG were analyzed with GenomeStudio, R, and SAM software. The association between DNA methylation and gene expression were tested. Gene interaction pathways of the differentially expressed genes were analyzed by IPA software. We identified 552 upregulated genes and 550 downregulated genes in PBMC of SLE. Integration of DNA methylation and gene expression profiling showed that 334 upregulated genes were hypomethylated, and 479 downregulated genes were hypermethylated. Pathway analysis on the differential genes in SLE revealed significant enrichment in interferon (IFN) signaling and toll-like receptor (TLR) signaling pathways. Nine IFN- and seven TLR-related genes were identified and displayed step-wise increase in SLE LN(-) and SLE LN(+). Hypomethylated CpG sites were detected on these genes. The gene expressions for MX1, GPR84, and E2F2 were increased in SLE LN(+) as compared to SLE LN(-) patients. The serum levels of inflammatory cytokines, including IL17A, IP-10, bFGF, TNF-α, IL-6, IL-15, GM-CSF, IL-1RA, IL-5, and IL-12p70, were significantly elevated in SLE compared with NC. The levels of IL-15 and IL1RA correlated with their mRNA expression. The upregulation of IL-15 may be regulated by hypomethylated CpG sites in the promotor region of the gene. Our study has demonstrated that significant number of differential genes in SLE were involved in IFN, TLR signaling pathways, and inflammatory cytokines. The enrichment of differential genes has been associated with aberrant DNA methylation, which may be relevant to the pathogenesis of SLE. Our observations have laid the groundwork for further diagnostic and mechanistic studies of SLE and LN.

Inference for High-dimensional Differential Correlation Matrices *

PubMed Central

Cai, T. Tony; Zhang, Anru

2015-01-01

Motivated by differential co-expression analysis in genomics, we consider in this paper estimation and testing of high-dimensional differential correlation matrices. An adaptive thresholding procedure is introduced and theoretical guarantees are given. Minimax rate of convergence is established and the proposed estimator is shown to be adaptively rate-optimal over collections of paired correlation matrices with approximately sparse differences. Simulation results show that the procedure significantly outperforms two other natural methods that are based on separate estimation of the individual correlation matrices. The procedure is also illustrated through an analysis of a breast cancer dataset, which provides evidence at the gene co-expression level that several genes, of which a subset has been previously verified, are associated with the breast cancer. Hypothesis testing on the differential correlation matrices is also considered. A test, which is particularly well suited for testing against sparse alternatives, is introduced. In addition, other related problems, including estimation of a single sparse correlation matrix, estimation of the differential covariance matrices, and estimation of the differential cross-correlation matrices, are also discussed. PMID:26500380

PRELP (proline/arginine-rich end leucine-rich repeat protein) promotes osteoblastic differentiation of preosteoblastic MC3T3-E1 cells by regulating the β-catenin pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Haiying; Cui, Yazhou; Luan, Jing

Proline/arginine-rich end leucine-rich repeat protein (PRELP) is a collagen-binding proteoglycan highly expressed in the developing bones. Recent studies indicated that PRELP could inhibit osteoclastogenesis as a NF-κB inhibitor. However, its role during osteoblast differentiation is still unclear. In this study, we confirmed that the expression of PRELP increased with the osteogenesis induction of preosteoblastic MC3T3-E1 cells. Down-regulation of PRELP expression by shRNA reduced ALP activity, mineralization and expression of osteogenic marker gene Runx2. Our microarray analysis data suggested that β-catenin may act as a hub gene in the PRELP-mediated gene network. We validated furtherly that PRELP knockdown could inhibit themore » level of connexin43, a key regulator of osteoblast differentiation by affecting β-catenin protein expression, and its nuclear translocation in MC3T3-E1 preosteoblasts. Therefore, this study established a new role of PRELP in modulating β-catenin/connexin43 pathway and osteoblast differentiation.« less

Differential gene expression detection and sample classification using penalized linear regression models.

PubMed

Wu, Baolin

2006-02-15

Differential gene expression detection and sample classification using microarray data have received much research interest recently. Owing to the large number of genes p and small number of samples n (p > n), microarray data analysis poses big challenges for statistical analysis. An obvious problem owing to the 'large p small n' is over-fitting. Just by chance, we are likely to find some non-differentially expressed genes that can classify the samples very well. The idea of shrinkage is to regularize the model parameters to reduce the effects of noise and produce reliable inferences. Shrinkage has been successfully applied in the microarray data analysis. The SAM statistics proposed by Tusher et al. and the 'nearest shrunken centroid' proposed by Tibshirani et al. are ad hoc shrinkage methods. Both methods are simple, intuitive and prove to be useful in empirical studies. Recently Wu proposed the penalized t/F-statistics with shrinkage by formally using the (1) penalized linear regression models for two-class microarray data, showing good performance. In this paper we systematically discussed the use of penalized regression models for analyzing microarray data. We generalize the two-class penalized t/F-statistics proposed by Wu to multi-class microarray data. We formally derive the ad hoc shrunken centroid used by Tibshirani et al. using the (1) penalized regression models. And we show that the penalized linear regression models provide a rigorous and unified statistical framework for sample classification and differential gene expression detection.

Development, Characterization, and Pluripotency Analysis of Buffalo (Bubalus bubalis) Embryonic Stem Cell Lines Derived from In Vitro–Fertilized, Hand-Guided Cloned, and Parthenogenetic Embryos

PubMed Central

Shah, Syed Mohmad; Saini, Neha; Ashraf, Syma; Zandi, Mohammad; Manik, Radhey Sham; Singla, Suresh Kumar; Palta, Prabhat

2015-01-01

Abstract We present the derivation, characterization, and pluripotency analysis of three buffalo embryonic stem cell (buESC) lines, from in vitro–fertilized, somatic cell nuclear–transferred, and parthenogenetic blastocysts. These cell lines were developed for later differentiation into germ lineage cells and elucidation of the signaling pathways involved. The cell lines were established from inner cell masses (ICMs) that were isolated manually from the in vitro–produced blastocysts. Most of the ICMs (45–55%) resulted in formation of primary colonies that were subcultured after 8–10 days, leading subsequently to the formation of three buESC lines, one from each blastocyst type. All the cell lines expressed stem cell markers, such as Alkaline Phosphatase, OCT4, NANOG, SSEA1, SSEA4, TRA-1-60, TRA-1-81, SOX2, REX1, CD-90, STAT3, and TELOMERASE. They differentiated into all three germ layers as determined by ectodermal, mesodermal, and endodermal RNA and protein markers. All of the cell lines showed equal expression of pluripotency markers as well as equivalent differentiation potential into all the three germ layers. The static suspension culture–derived embryoid bodies (EBs) showed greater expression of all the three germ layer markers as compared to hanging drop culture–derived EBs. When analyzed for germ layer marker expression, EBs derived from 15% fetal bovine serum (FBS)-based spontaneous differentiation medium showed greater differentiation across all the three germ layers as compared to those derived from Knock-Out Serum Replacement (KoSR)-based differentiation medium. PMID:26168169

Gene expression in the rectus abdominus muscle of patients with and without pelvic organ prolapse.

PubMed

Hundley, Andrew F; Yuan, Lingwen; Visco, Anthony G

2008-02-01

The objective of the study was to compare gene expression in a group of actin and myosin-related proteins in the rectus muscle of 15 patients with pelvic organ prolapse and 13 controls. Six genes previously identified by microarray GeneChip analysis were examined using real-time quantitative reverse transcriptase-polymerase chain reaction analysis, including 2 genes showing differential expression in pubococcygeus muscle. Samples and controls were run in triplicate in multiplexed wells, and levels of gene expression were analyzed using the comparative critical threshold method. One gene, MYH3, was 3.2 times overexpressed in patients with prolapse (P = .032), but no significant differences in expression were seen for the other genes examined. An age-matched subset of 9 patients and controls showed that MYH3 gene expression was no longer significantly different (P = .058). Differential messenger ribonucleic acid levels of actin and myosin-related genes in patients with pelvic organ prolapse and controls may be limited to skeletal muscle from the pelvic floor.

miR-214 promotes periodontal ligament stem cell osteoblastic differentiation by modulating Wnt/β-catenin signaling

PubMed Central

Cao, Fengdi; Zhan, Jialin; Chen, Xufeng; Zhang, Kai; Lai, Renfa; Feng, Zhiqiang

2017-01-01

The canonical Wnt/β-catenin signaling is important in the differentiation of human mesenchymal stem cells into osteoblasts. Accumulating evidence suggests that the expression of β-catenin is, in part, regulated by specific microRNAs (miRNAs). The aim of the present study was to investigate the putative roles of miRNAs in osteoblast differentiation. Polymerase chain reaction (PCR) arrays were used to identify miRNAs that were differentially expressed between differentiated and non-differentiated periodontal ligament stem cells (PDLSCs), and reverse transcription-quantitative PCR (RT-qPCR) was used for validation. Since miR-214 was revealed to be significantly downregulated during PDLSC differentiation, its function was further investigated via silencing and overexpression. In addition, osteogenic differentiation of PDLSCs was evaluated at 10 and 21 days following induction, using Alizarin red staining and RT-qPCR analysis for mRNA expression levels of the osteogenic differentiation markers alkaline phosphatase (ALP), osteocalcin and bone sialoprotein. Furthermore, the potential target genes of miR-214 were investigated using a dual-luciferase reporter assay, RT-qPCR and western blot analysis, whereas a TOPflash/FOPflash reporter plasmid system followed by a luciferase assay was used to examine the effects of miR-214 on Wnt/β-catenin signaling. The present results demonstrated that miR-214 was significantly downregulated during the osteoblastic differentiation of PDLSCs. Notably, its overexpression inhibited PDLSC differentiation, whereas its knockdown promoted PDLSC differentiation, as revealed by alterations in mRNA expression of osteoblast-specific genes and ALP. In addition, miR-214 was demonstrated to directly interact with the 3′-untranslated region of the β-catenin gene CTNNB1, and suppressed Wnt/β-catenin signaling through the inhibition of β-catenin. The results of the present study suggested that miR-214 may participate in the regulation of the Wnt/β-catenin signaling pathway, and may have potential as a candidate target for the development of preventive or therapeutic agents for the treatment of patients with osteogenic disorders. PMID:29152645

miR-214 promotes periodontal ligament stem cell osteoblastic differentiation by modulating Wnt/β‑catenin signaling.

PubMed

Cao, Fengdi; Zhan, Jialin; Chen, Xufeng; Zhang, Kai; Lai, Renfa; Feng, Zhiqiang

2017-12-01

The canonical Wnt/β‑catenin signaling is important in the differentiation of human mesenchymal stem cells into osteoblasts. Accumulating evidence suggests that the expression of β‑catenin is, in part, regulated by specific microRNAs (miRNAs). The aim of the present study was to investigate the putative roles of miRNAs in osteoblast differentiation. Polymerase chain reaction (PCR) arrays were used to identify miRNAs that were differentially expressed between differentiated and non‑differentiated periodontal ligament stem cells (PDLSCs), and reverse transcription‑quantitative PCR (RT‑qPCR) was used for validation. Since miR‑214 was revealed to be significantly downregulated during PDLSC differentiation, its function was further investigated via silencing and overexpression. In addition, osteogenic differentiation of PDLSCs was evaluated at 10 and 21 days following induction, using Alizarin red staining and RT‑qPCR analysis for mRNA expression levels of the osteogenic differentiation markers alkaline phosphatase (ALP), osteocalcin and bone sialoprotein. Furthermore, the potential target genes of miR‑214 were investigated using a dual‑luciferase reporter assay, RT‑qPCR and western blot analysis, whereas a TOPflash/FOPflash reporter plasmid system followed by a luciferase assay was used to examine the effects of miR‑214 on Wnt/β‑catenin signaling. The present results demonstrated that miR‑214 was significantly downregulated during the osteoblastic differentiation of PDLSCs. Notably, its overexpression inhibited PDLSC differentiation, whereas its knockdown promoted PDLSC differentiation, as revealed by alterations in mRNA expression of osteoblast‑specific genes and ALP. In addition, miR‑214 was demonstrated to directly interact with the 3'‑untranslated region of the β‑catenin gene CTNNB1, and suppressed Wnt/β‑catenin signaling through the inhibition of β‑catenin. The results of the present study suggested that miR‑214 may participate in the regulation of the Wnt/β‑catenin signaling pathway, and may have potential as a candidate target for the development of preventive or therapeutic agents for the treatment of patients with osteogenic disorders.

Differential Gene Expression from Midguts of Refractory and Susceptible Lines of the Mosquito, Aedes aegypti, Infected with Dengue-2 Virus

PubMed Central

Barón, Olga L.; Ursic-Bedoya, Raul J.; Lowenberger, Carl A.; Ocampo, Clara B.

2010-01-01

Suppressive subtractive hybridization was used to evaluate the differential expression of midgut genes of feral populations of Aedes aegypti (Diptera: Culicidae) from Colombia that are naturally refractory or susceptible to Dengue-2 virus infection. A total of 165 differentially expressed sequence tags (ESTs) were identified in the subtracted libraries. The analysis showed a higher number of differentially expressed genes in the susceptible Ae. aegypti individuals than the refractory mosquitoes. The functional annotation of ESTs revealed a broad response in the susceptible library that included immune molecules, metabolic molecules and transcription factors. In the refractory strain, there was the presence of a trypsin inhibitor gene, which could play a role in the infection. These results serve as a template for more detailed studies aiming to characterize the genetic components of refractoriness, which in turn can be used to devise new approaches to combat transmission of dengue fever. PMID:20572793

Identification of a mouse synaptic glycoprotein gene in cultured neurons.

PubMed

Yu, Albert Cheung-Hoi; Sun, Chun Xiao; Li, Qiang; Liu, Hua Dong; Wang, Chen Ran; Zhao, Guo Ping; Jin, Meilei; Lau, Lok Ting; Fung, Yin-Wan Wendy; Liu, Shuang

2005-10-01

Neuronal differentiation and aging are known to involve many genes, which may also be differentially expressed during these developmental processes. From primary cultured cerebral cortical neurons, we have previously identified various differentially expressed gene transcripts from cultured cortical neurons using the technique of arbitrarily primed PCR (RAP-PCR). Among these transcripts, clone 0-2 was found to have high homology to rat and human synaptic glycoprotein. By in silico analysis using an EST database and the FACTURA software, the full-length sequence of 0-2 was assembled and the clone was named as mouse synaptic glycoprotein homolog 2 (mSC2). DNA sequencing revealed transcript size of mSC2 being smaller than the human and rat homologs. RT-PCR indicated that mSC2 was expressed differentially at various culture days. The mSC2 gene was located in various tissues with higher expression in brain, lung, and liver. Functions of mSC2 in neurons and other tissues remain elusive and will require more investigation.

Characteristics and Expression Profile of KRT71 Screened by Suppression Subtractive Hybridization cDNA Library in Curly Fleece Chinese Tan Sheep.

PubMed

Kang, Xiaolong; Liu, Yufang; Zhang, Jibin; Xu, Qinqin; Liu, Chengkun; Fang, Meiying

2017-07-01

As an important commercial trait for sheep, curly fleece has a great economic impact on production costs and efficiency in sheep industry. To identify genes that are important for curly fleece formation in mammals, a suppression subtractive hybridization analysis was performed on the shoulder skin tissues exposed to two different growth stages of Chinese Tan sheep with different phenotypes (curly fleece and noncurling fleece). BLAST analysis identified 67 differentially expressed genes, of which 31 were expressed lower and 36 were expressed higher in lambs than in adult sheep. Differential expressions of seven randomly selected genes were verified using quantitative real-time polymerase chain reaction (qRT-PCR). KRT71 gene was selected for further study due to its high correlation with the curly hair phenotype in various mammal species. Semi-qPCR showed distinctively high expression of KRT71 in skin tissues. Moreover, qPCR result showed a significantly higher expression of KRT71 in curly fleece than noncurling Tan sheep. The luciferase assay and electrophoresis mobility shift assay showed that there were transcription factor binding sites in the promoter region of KRT71 related to the differential expression of KRT71 at the two growth stages of Tan sheep. Online bioinformation tools predicted MFZ1 as a transcriptional factor that regulates the expression of KRT71. These studies on KRT71 gene revealed some mechanisms underlying the relationship between the KRT71 gene and the curly fleece phenotype of Tan sheep.

Induction of dystrophin Dp71 expression during neuronal differentiation: opposite roles of Sp1 and AP2alpha in Dp71 promoter activity.

PubMed

Morales-Lázaro, Sara Luz; González-Ramírez, Ricardo; Gómez, Pablo; Tapia-Ramírez, Victor; de León, Mario Bermúdez; Cisneros, Bulmaro

2010-01-01

In this study, we delineated the molecular mechanisms that modulate Dp71 expression during neuronal differentiation, using the N1E-115 cell line. We demonstrated that Dp71 expression is up-regulated in response to cAMP-mediated neuronal differentiation of these cells, and that this induction is controlled at promoter level. Functional deletion analysis of the Dp71 promoter revealed that a 5'-flanking 159-bp DNA fragment that contains Sp1 and AP2 binding sites is necessary and sufficient for basal expression of this TATA-less promoter, as well as for its induction during neuronal differentiation. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that Sp1 and AP2alpha bind to their respective DNA elements within the Dp71 basal promoter. Overall, mutagenesis assays on the Sp1 and AP2 binding sites, over-expression of Sp1 and AP2alpha, as well as knock-down experiments on Sp1 and AP2alpha gene expression established that Dp71 basal expression is controlled by the combined action of Sp1 and AP2alpha, which act as activator and repressor, respectively. Furthermore, we demonstrated that induction of Dp71 expression in differentiated cells is the result of the maintenance of positive regulation exerted by Sp1, as well as of the loss of AP2alpha binding, which ultimately releases the promoter from repression.

Shift of microRNA profile upon orthotopic xenografting of glioblastoma spheroid cultures.

PubMed

Halle, Bo; Thomassen, Mads; Venkatesan, Ranga; Kaimal, Vivek; Marcusson, Eric G; Munthe, Sune; Sørensen, Mia D; Aaberg-Jessen, Charlotte; Jensen, Stine S; Meyer, Morten; Kruse, Torben A; Christiansen, Helle; Schmidt, Steffen; Mollenhauer, Jan; Schulz, Mette K; Andersen, Claus; Kristensen, Bjarne W

2016-07-01

Glioblastomas always recur despite surgery, radiotherapy and chemotherapy. A key player in the therapeutic resistance may be immature tumor cells with stem-like properties (TSCs) escaping conventional treatment. A group of promising molecular targets are microRNAs (miRs). miRs are small non-coding RNAs exerting post-transcriptional regulation of gene expression. In this study we aimed to identify over-expressed TSC-related miRs potentially amenable for therapeutic targeting. We used non-differentiated glioblastoma spheroid cultures (GSCs) containing TSCs and compared these to xenografts using a NanoString nCounter platform. This revealed 19 over-expressed miRs in the non-differentiated GSCs. Additionally, non-differentiated GSCs were compared to neural stem cells (NSCs) using a microarray platform. This revealed four significantly over-expressed miRs in the non-differentiated GSCs in comparison to the NSCs. The three most over-expressed miRs in the non-differentiated GSCs compared to xenografts were miR-126, -137 and -128. KEGG pathway analysis suggested the main biological function of these over-expressed miRs to be cell-cycle arrest and diminished proliferation. To functionally validate the profiling results suggesting association of these miRs with stem-like properties, experimental over-expression of miR-128 was performed. A consecutive limiting dilution assay confirmed a significantly elevated spheroid formation in the miR-128 over-expressing cells. This may provide potential therapeutic targets for anti-miRs to identify novel treatment options for GBM patients.

Reversing SKI-SMAD4-mediated suppression is essential for TH17 cell differentiation.

PubMed

Zhang, Song; Takaku, Motoki; Zou, Liyun; Gu, Ai-di; Chou, Wei-Chun; Zhang, Ge; Wu, Bing; Kong, Qing; Thomas, Seddon Y; Serody, Jonathan S; Chen, Xian; Xu, Xiaojiang; Wade, Paul A; Cook, Donald N; Ting, Jenny P Y; Wan, Yisong Y

2017-11-02

T helper 17 (T H 17) cells are critically involved in host defence, inflammation, and autoimmunity. Transforming growth factor β (TGFβ) is instrumental in T H 17 cell differentiation by cooperating with interleukin-6 (refs 6, 7). Yet, the mechanism by which TGFβ enables T H 17 cell differentiation remains elusive. Here we reveal that TGFβ enables T H 17 cell differentiation by reversing SKI-SMAD4-mediated suppression of the expression of the retinoic acid receptor (RAR)-related orphan receptor γt (RORγt). We found that, unlike wild-type T cells, SMAD4-deficient T cells differentiate into T H 17 cells in the absence of TGFβ signalling in a RORγt-dependent manner. Ectopic SMAD4 expression suppresses RORγt expression and T H 17 cell differentiation of SMAD4-deficient T cells. However, TGFβ neutralizes SMAD4-mediated suppression without affecting SMAD4 binding to the Rorc locus. Proteomic analysis revealed that SMAD4 interacts with SKI, a transcriptional repressor that is degraded upon TGFβ stimulation. SKI controls histone acetylation and deacetylation of the Rorc locus and T H 17 cell differentiation via SMAD4: ectopic SKI expression inhibits H3K9 acetylation of the Rorc locus, Rorc expression, and T H 17 cell differentiation in a SMAD4-dependent manner. Therefore, TGFβ-induced disruption of SKI reverses SKI-SMAD4-mediated suppression of RORγt to enable T H 17 cell differentiation. This study reveals a critical mechanism by which TGFβ controls T H 17 cell differentiation and uncovers the SKI-SMAD4 axis as a potential therapeutic target for treating T H 17-related diseases.

Therapeutic activities of intravenous immunoglobulins in multiple sclerosis involve modulation of chemokine expression.

PubMed

Pigard, Nadine; Elovaara, Irina; Kuusisto, Hanna; Paalavuo, Raija; Dastidar, Prasun; Zimmermann, Klaus; Schwarz, Hans-Peter; Reipert, Birgit

2009-04-30

The objective of this study was to identify genes that are differentially expressed in peripheral T cells of patients with MS exacerbation receiving treatment with IVIG. Using microarray analysis, we identified 360 genes that were at least two-fold up- or down-regulated. The expression of four representative genes (PTGER4, CXCL5, IL11 and CASP2) was confirmed by quantitative PCR. Four of the differentially expressed genes encode chemokines (CXCL3, CXCL5, CCL13 and XCL2) that are involved in directing leukocyte migration. We suggest that the modulation of chemokine expression in peripheral T cells contributes to the beneficial activity of IVIG in patients with MS exacerbation.

Aberrant expression of long noncoding RNAs in cumulus cells isolated from PCOS patients.

PubMed

Huang, Xin; Hao, Cuifang; Bao, Hongchu; Wang, Meimei; Dai, Huangguan

2016-01-01

To describe the long noncoding RNA (lncRNA) profiles in cumulus cells isolated from polycystic ovary syndrome (PCOS) patients by employing a microarray and in-depth bioinformatics analysis. This information will help us understand the occurrence and development of PCOS. In this study, we used a microarray to describe lncRNA profiles in cumulus cells isolated from ten patients (five PCOS and five normal women). Several differentially expressed lncRNAs were chosen to validate the microarray results by quantitative RT-PCR (qRT-PCR). Then, the differentially expressed lncRNAs were classified into three subgroups (HOX loci lncRNA, enhancer-like lncRNA, and lincRNA) to deduce their potential features. Furthermore, a lncRNA/mRNA co-expression network was constructed by using the Cytoscape software (V2.8.3, http://www.cytoscape.org/ ). We observed that 623 lncRNAs and 260 messenger RNAs (mRNAs) were significantly up- or down-regulated (≥2-fold change), and these differences could be used to discriminate cumulus cells of PCOS from those of normal patients. Five differentially expressed lncRNAs (XLOC_011402, ENST00000454271, ENST00000433673, ENST00000450294, and ENST00000432431) were selected to validate the microarray results using quantitative RT-PCR (qRT-PCR). The qRT-PCR results were consistent with the microarray data. Further analysis indicated that many differentially expressed lncRNAs were transcribed from chromosome 2 and may act as enhancers to regulate their neighboring protein-coding genes. Forty-three lncRNAs and 29 mRNAs were used to construct the coding-non-coding gene co-expression network. Most pairs positively correlated, and one mRNA correlated with one or more lncRNAs. Our study is the first to determine genome-wide lncRNA expression patterns in cumulus cells isolated from PCOS patients by microarray. The results show that clusters of lncRNAs were aberrantly expressed in cumulus cells of PCOS patients compared with those of normal women, which revealed that lncRNAs differentially expressed in PCOS and normal women may contribute to the occurrence of PCOS and affect oocyte development.

Identification of suitable reference genes for quantitative gene expression analysis in rat adipose stromal cells induced to trilineage differentiation.

PubMed

Santos, Bruno Paiva Dos; da Costa Diesel, Luciana Fraga; da Silva Meirelles, Lindolfo; Nardi, Nance Beyer; Camassola, Melissa

2016-12-15

This study was designed to (i) identify stable reference genes for the analysis of gene expression during in vitro differentiation of rat adipose stromal cells (rASCs), (ii) recommend stable genes for individual treatment conditions, and (iii) validate these genes by comparison with normalization results from stable and unstable reference genes. On the basis of a literature review, eight genes were selected: Actb, B2m, Hprt1, Ppia, Rplp0, Rpl13a, Rpl5, and Ywhaz. Genes were ranked according to their stability under different culture conditions as assessed using GenNorm, NormFinder, and RefFinder algorithms. Although the employed algorithms returned different rankings, the most frequently top-ranked genes were: B2m and/or Ppia for all 28day treatments (ALL28); Ppia and Hprt1 (adipogenic differentiation; A28), B2m (chondrogenic differentiation; C28), Rpl5 (controls maintained in complete culture medium; CCM), Rplp0 (osteogenic differentiation for 3days; O3), Rpl13a and Actb (osteogenic differentiation for 7days; O7), Rplp0 and Ppia (osteogenic differentiation for 14days; O14), Hprt1 and Ppia (osteogenic differentiation for 28days; O28), as well as Actb (all osteogenesis time points combined; ALLOSTEO). The obtained results indicate that the performance of reference genes depends on the differentiation protocol and on the analysis time, thus providing valuable information for the design of RT-PCR experiments. Copyright © 2016. Published by Elsevier B.V.

The symbolic computation and automatic analysis of trajectories

NASA Technical Reports Server (NTRS)

Grossman, Robert

1991-01-01

Research was generally done on computation of trajectories of dynamical systems, especially control systems. Algorithms were further developed for rewriting expressions involving differential operators. The differential operators involved arise in the local analysis of nonlinear control systems. An initial design was completed of the system architecture for software to analyze nonlinear control systems using data base computing.

Glutathione S-transferase expression and isoenzyme composition during cell differentiation of Caco-2 cells.

PubMed

Scharmach, E; Hessel, S; Niemann, B; Lampen, A

2009-11-30

The human colon adenocarcinoma cell line Caco-2 is frequently used to study human intestinal metabolism and transport of xenobiotica. Previous studies have shown that both Caco-2 cells and human colon cells constitutively express the multigene family of detoxifying enzymes glutathione S-transferases (GSTs), particularly GST alpha and GST pi. GSTs may play a fundamental role in the molecular interplay between phase I, II enzymes and ABC-transporters. The gut fermentation product, butyrate, can modulate the potential for detoxification. The aim of this study was to investigate the basal expression of further cytosolic GSTs in Caco-2 cells during cell differentiation. In addition, a comparison was made with expression levels in MCF-7 and HepG2, two other cell types with barrier functions. Finally, the butyrate-mediated modulation of gene and protein expression was determined by real time PCR and western blot analysis. In Caco-2, gene and protein expression levels of GST alpha increased during cell differentiation. High levels of GSTO1 and GSTP1 were constantly expressed. No expression of GSTM5 and GSTT1 was detected. HepG2 expressed GSTO1 and MCF-7 GSTZ1 most intensively. No expression of GSTA5, GSTM5, or GSTP1 was detected in either cell. Incubation of Caco-2 cells with butyrate (5 mM) significantly induced GSTA1 and GSTM2 in proliferating Caco-2 cells. In differentiated cells, butyrate tended to increase GSTO1 and GSTP1. The results of this study show that a differentiation-dependent expression of GSTs in Caco-2 cells may reflect the in vivo situation and indicate the potential of butyrate to modify intestinal metabolism. GSTA1-A4 have been identified as good markers for cell differentiation. The Caco-2 cell line is a useful model for assessing the potential of food-related substances to modulate the GST expression pattern.

Proteins differentially expressed during limonene biotransformation by Penicillium digitatum DSM 62840 were examined using iTRAQ labeling coupled with 2D-LC-MS/MS.

PubMed

Zhang, Lu-Lu; Zhang, Yan; Ren, Jing-Nan; Liu, Yan-Long; Li, Jia-Jia; Tai, Ya-Nan; Yang, Shu-Zhen; Pan, Si-Yi; Fan, Gang

2016-10-01

This study focused on the differences in protein expression at various periods during limonene biotransformation by Penicillium digitatum DSM 62840. A total of 3644 protein-species were quantified by iTRAQ during limonene biotransformation (0 and 12 h). A total of 643 proteins were differentially expressed, 316 proteins were significantly up-regulated and 327 proteins were markedly down-regulated. GO, COG, and pathway enrichment analysis showed that the differentially expressed proteins possessed catalytic and binding functions and were involved in a variety of cellular and metabolic process. Furthermore, the enzymes involved in limonene transformation might be related to cytochrome P-450. This study provided a powerful platform for further exploration of biotransformation, and the identified proteins provided insight into the mechanism of limonene transformation.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zeadin, Melec G.; Butcher, Martin K.; Shaughnessy, Stephen G.

Highlights: Black-Right-Pointing-Pointer Leptin promotes osteoblast differentiation of primary smooth muscle cells. Black-Right-Pointing-Pointer Leptin regulates the expression of genes involved in osteoblast differentiation. Black-Right-Pointing-Pointer Constitutively active GSK-3{beta} attenuates leptin-induced osteoblast differentiation. Black-Right-Pointing-Pointer This suggests that leptin signals through GSK-3{beta} to promote osteoblast differentiation. -- Abstract: In this study, we begin to investigate the underlying mechanism of leptin-induced vascular calcification. We found that treatment of cultured bovine aortic smooth muscle cells (BASMCs) with leptin (0.5-4 {mu}g/ml) induced osteoblast differentiation in a dose-dependent manner. Furthermore, we found that leptin significantly increased the mRNA expression of osteopontin and bone sialoprotein, while down-regulating matrix glamore » protein (MGP) expression in BASMCs. Key factors implicated in osteoblast differentiation, including members of the Wnt signaling pathway, were examined. Exposure to leptin enhanced phosphorylation of GSK-3{beta} on serine-9 thereby inhibiting activity and promoting the nuclear accumulation of {beta}-catenin. Transfection of BASMCs with an adenovirus that expressed constitutively active GSK-3{beta} (Ad-GSK-3{beta} S9A) resulted in a >2-fold increase in GSK-3{beta} activity and a significant decrease in leptin-induced alkaline phosphatase (ALP) activity. In addition, qRT-PCR analysis showed that GSK-3{beta} activation resulted in a significant decrease in the expression of osteopontin and bone sialoprotein, but a marked increase in MGP mRNA expression. When taken together, our results suggest a mechanism by which leptin promotes osteoblast differentiation and vascular calcification in vivo.« less

Epigenetic Control of Skeletal Development by the Histone Methyltransferase Ezh2*

PubMed Central

Dudakovic, Amel; Camilleri, Emily T.; Xu, Fuhua; Riester, Scott M.; McGee-Lawrence, Meghan E.; Bradley, Elizabeth W.; Paradise, Christopher R.; Lewallen, Eric A.; Thaler, Roman; Deyle, David R.; Larson, A. Noelle; Lewallen, David G.; Dietz, Allan B.; Stein, Gary S.; Montecino, Martin A.; Westendorf, Jennifer J.; van Wijnen, Andre J.

2015-01-01

Epigenetic control of gene expression is critical for normal fetal development. However, chromatin-related mechanisms that activate bone-specific programs during osteogenesis have remained underexplored. Therefore, we investigated the expression profiles of a large cohort of epigenetic regulators (>300) during osteogenic differentiation of human mesenchymal cells derived from the stromal vascular fraction of adipose tissue (AMSCs). Molecular analyses establish that the polycomb group protein EZH2 (enhancer of zeste homolog 2) is down-regulated during osteoblastic differentiation of AMSCs. Chemical inhibitor and siRNA knockdown studies show that EZH2, a histone methyltransferase that catalyzes trimethylation of histone 3 lysine 27 (H3K27me3), suppresses osteogenic differentiation. Blocking EZH2 activity promotes osteoblast differentiation and suppresses adipogenic differentiation of AMSCs. High throughput RNA sequence (mRNASeq) analysis reveals that EZH2 inhibition stimulates cell cycle inhibitory proteins and enhances the production of extracellular matrix proteins. Conditional genetic loss of Ezh2 in uncommitted mesenchymal cells (Prrx1-Cre) results in multiple defects in skeletal patterning and bone formation, including shortened forelimbs, craniosynostosis, and clinodactyly. Histological analysis and mRNASeq profiling suggest that these effects are attributable to growth plate abnormalities and premature cranial suture closure because of precocious maturation of osteoblasts. We conclude that the epigenetic activity of EZH2 is required for skeletal patterning and development, but EZH2 expression declines during terminal osteoblast differentiation and matrix production. PMID:26424790

Persistent organic pollutants alter DNA methylation during human adipocyte differentiation.

PubMed

van den Dungen, Myrthe W; Murk, Albertinka J; Kok, Dieuwertje E; Steegenga, Wilma T

2017-04-01

Ubiquitous persistent organic pollutants (POPs) can accumulate in humans where they might influence differentiation of adipocytes. The aim of this study was to investigate whether DNA methylation is one of the underlying mechanisms by which POPs affect adipocyte differentiation, and to what extent DNA methylation can be related to gene transcription. Adipocyte differentiation was induced in two human cell models with continuous exposure to different POPs throughout differentiation. From the seven tested POPs, perfluorooctanesulfonic acid (PFOS) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) decreased lipid accumulation, while tributyltin (TBT) increased lipid accumulation. In human mesenchymal stem cells (hMSCs), TCDD and TBT induced opposite gene expression profiles, whereas after PFOS exposure gene expression remained relatively stable. Genome-wide DNA methylation analysis showed that all three POPs affected DNA methylation patterns in adipogenic and other genes, possibly related to the phenotypic outcome, but without concomitant gene expression changes. Differential methylation was predominantly detected in intergenic regions, where the biological relevance of alterations in DNA methylation is unclear. This study demonstrates that POPs, at environmentally relevant levels, are able to induce differential DNA methylation in human differentiating adipocytes. Copyright © 2017 Wageningen University. Published by Elsevier Ltd.. All rights reserved.

Achaete-Scute Homolog 1 Expression Controls Cellular Differentiation of Neuroblastoma

PubMed Central

Kasim, Mumtaz; Heß, Vicky; Scholz, Holger; Persson, Pontus B.; Fähling, Michael

2016-01-01

Neuroblastoma, the major cause of infant cancer deaths, results from fast proliferation of undifferentiated neuroblasts. Treatment of high-risk neuroblastoma includes differentiation with retinoic acid (RA); however, the resistance of many of these tumors to RA-induced differentiation poses a considerable challenge. Human achaete-scute homolog 1 (hASH1) is a proneural basic helix-loop-helix transcription factor essential for neurogenesis and is often upregulated in neuroblastoma. Here, we identified a novel function for hASH1 in regulating the differentiation phenotype of neuroblastoma cells. Global analysis of 986 human neuroblastoma datasets revealed a negative correlation between hASH1 and neuron differentiation that was independent of the N-myc (MYCN) oncogene. Using RA to induce neuron differentiation in two neuroblastoma cell lines displaying high and low levels of hASH1 expression, we confirmed the link between hASH1 expression and the differentiation defective phenotype, which was reversed by silencing hASH1 or by hypoxic preconditioning. We further show that hASH1 suppresses neuronal differentiation by inhibiting transcription at the RA receptor element. Collectively, our data indicate hASH1 to be key for understanding neuroblastoma resistance to differentiation therapy and pave the way for hASH1-targeted therapies for augmenting the response of neuroblastoma to differentiation therapy. PMID:28066180

Transcriptome changes during fruit development and ripening of sweet orange (Citrus sinensis).

PubMed

Yu, Keqin; Xu, Qiang; Da, Xinlei; Guo, Fei; Ding, Yuduan; Deng, Xiuxin

2012-01-10

The transcriptome of the fruit pulp of the sweet orange variety Anliu (WT) and that of its red fleshed mutant Hong Anliu (MT) were compared to understand the dynamics and differential expression of genes expressed during fruit development and ripening. The transcriptomes of WT and MT were sampled at four developmental stages using an Illumina sequencing platform. A total of 19,440 and 18,829 genes were detected in MT and WT, respectively. Hierarchical clustering analysis revealed 24 expression patterns for the set of all genes detected, of which 20 were in common between MT and WT. Over 89% of the genes showed differential expression during fruit development and ripening in the WT. Functional categorization of the differentially expressed genes revealed that cell wall biosynthesis, carbohydrate and citric acid metabolism, carotenoid metabolism, and the response to stress were the most differentially regulated processes occurring during fruit development and ripening. A description of the transcriptomic changes occurring during fruit development and ripening was obtained in sweet orange, along with a dynamic view of the gene expression differences between the wild type and a red fleshed mutant. © 2012 Yu et al; licensee BioMed Central Ltd.

Identification and expression analysis of cold and freezing stress responsive genes of Brassica oleracea.

PubMed

Ahmed, Nasar Uddin; Jung, Hee-Jeong; Park, Jong-In; Cho, Yong-Gu; Hur, Yoonkang; Nou, Ill-Sup

2015-01-10

Cold and freezing stress is a major environmental constraint to the production of Brassica crops. Enhancement of tolerance by exploiting cold and freezing tolerance related genes offers the most efficient approach to address this problem. Cold-induced transcriptional profiling is a promising approach to the identification of potential genes related to cold and freezing stress tolerance. In this study, 99 highly expressed genes were identified from a whole genome microarray dataset of Brassica rapa. Blast search analysis of the Brassica oleracea database revealed the corresponding homologous genes. To validate their expression, pre-selected cold tolerant and susceptible cabbage lines were analyzed. Out of 99 BoCRGs, 43 were differentially expressed in response to varying degrees of cold and freezing stress in the contrasting cabbage lines. Among the differentially expressed genes, 18 were highly up-regulated in the tolerant lines, which is consistent with their microarray expression. Additionally, 12 BoCRGs were expressed differentially after cold stress treatment in two contrasting cabbage lines, and BoCRG54, 56, 59, 62, 70, 72 and 99 were predicted to be involved in cold regulatory pathways. Taken together, the cold-responsive genes identified in this study provide additional direction for elucidating the regulatory network of low temperature stress tolerance and developing cold and freezing stress resistant Brassica crops. Copyright © 2014 Elsevier B.V. All rights reserved.

Modeling genome-wide dynamic regulatory network in mouse lungs with influenza infection using high-dimensional ordinary differential equations.

PubMed

Wu, Shuang; Liu, Zhi-Ping; Qiu, Xing; Wu, Hulin

2014-01-01

The immune response to viral infection is regulated by an intricate network of many genes and their products. The reverse engineering of gene regulatory networks (GRNs) using mathematical models from time course gene expression data collected after influenza infection is key to our understanding of the mechanisms involved in controlling influenza infection within a host. A five-step pipeline: detection of temporally differentially expressed genes, clustering genes into co-expressed modules, identification of network structure, parameter estimate refinement, and functional enrichment analysis, is developed for reconstructing high-dimensional dynamic GRNs from genome-wide time course gene expression data. Applying the pipeline to the time course gene expression data from influenza-infected mouse lungs, we have identified 20 distinct temporal expression patterns in the differentially expressed genes and constructed a module-based dynamic network using a linear ODE model. Both intra-module and inter-module annotations and regulatory relationships of our inferred network show some interesting findings and are highly consistent with existing knowledge about the immune response in mice after influenza infection. The proposed method is a computationally efficient, data-driven pipeline bridging experimental data, mathematical modeling, and statistical analysis. The application to the influenza infection data elucidates the potentials of our pipeline in providing valuable insights into systematic modeling of complicated biological processes.

Dynamic Proteomic Analysis of Pancreatic Mesenchyme Reveals Novel Factors That Enhance Human Embryonic Stem Cell to Pancreatic Cell Differentiation.

PubMed

Russ, Holger A; Landsman, Limor; Moss, Christopher L; Higdon, Roger; Greer, Renee L; Kaihara, Kelly; Salamon, Randy; Kolker, Eugene; Hebrok, Matthias

2016-01-01

Current approaches in human embryonic stem cell (hESC) to pancreatic beta cell differentiation have largely been based on knowledge gained from developmental studies of the epithelial pancreas, while the potential roles of other supporting tissue compartments have not been fully explored. One such tissue is the pancreatic mesenchyme that supports epithelial organogenesis throughout embryogenesis. We hypothesized that detailed characterization of the pancreatic mesenchyme might result in the identification of novel factors not used in current differentiation protocols. Supplementing existing hESC differentiation conditions with such factors might create a more comprehensive simulation of normal development in cell culture. To validate our hypothesis, we took advantage of a novel transgenic mouse model to isolate the pancreatic mesenchyme at distinct embryonic and postnatal stages for subsequent proteomic analysis. Refined sample preparation and analysis conditions across four embryonic and prenatal time points resulted in the identification of 21,498 peptides with high-confidence mapping to 1,502 proteins. Expression analysis of pancreata confirmed the presence of three potentially important factors in cell differentiation: Galectin-1 (LGALS1), Neuroplastin (NPTN), and the Laminin α-2 subunit (LAMA2). Two of the three factors (LGALS1 and LAMA2) increased expression of pancreatic progenitor transcript levels in a published hESC to beta cell differentiation protocol. In addition, LAMA2 partially blocks cell culture induced beta cell dedifferentiation. Summarily, we provide evidence that proteomic analysis of supporting tissues such as the pancreatic mesenchyme allows for the identification of potentially important factors guiding hESC to pancreas differentiation.

Dynamic Proteomic Analysis of Pancreatic Mesenchyme Reveals Novel Factors That Enhance Human Embryonic Stem Cell to Pancreatic Cell Differentiation

PubMed Central

Russ, Holger A.; Landsman, Limor; Moss, Christopher L.; Higdon, Roger; Greer, Renee L.; Kaihara, Kelly; Salamon, Randy; Kolker, Eugene; Hebrok, Matthias

2016-01-01

Current approaches in human embryonic stem cell (hESC) to pancreatic beta cell differentiation have largely been based on knowledge gained from developmental studies of the epithelial pancreas, while the potential roles of other supporting tissue compartments have not been fully explored. One such tissue is the pancreatic mesenchyme that supports epithelial organogenesis throughout embryogenesis. We hypothesized that detailed characterization of the pancreatic mesenchyme might result in the identification of novel factors not used in current differentiation protocols. Supplementing existing hESC differentiation conditions with such factors might create a more comprehensive simulation of normal development in cell culture. To validate our hypothesis, we took advantage of a novel transgenic mouse model to isolate the pancreatic mesenchyme at distinct embryonic and postnatal stages for subsequent proteomic analysis. Refined sample preparation and analysis conditions across four embryonic and prenatal time points resulted in the identification of 21,498 peptides with high-confidence mapping to 1,502 proteins. Expression analysis of pancreata confirmed the presence of three potentially important factors in cell differentiation: Galectin-1 (LGALS1), Neuroplastin (NPTN), and the Laminin α-2 subunit (LAMA2). Two of the three factors (LGALS1 and LAMA2) increased expression of pancreatic progenitor transcript levels in a published hESC to beta cell differentiation protocol. In addition, LAMA2 partially blocks cell culture induced beta cell dedifferentiation. Summarily, we provide evidence that proteomic analysis of supporting tissues such as the pancreatic mesenchyme allows for the identification of potentially important factors guiding hESC to pancreas differentiation. PMID:26681951

Identification and integrated analysis of differentially expressed lncRNAs and circRNAs reveal the potential ceRNA networks during PDLSC osteogenic differentiation.

PubMed

Gu, Xiuge; Li, Mengying; Jin, Ye; Liu, Dongxu; Wei, Fulan

2017-12-02

Researchers have been exploring the molecular mechanisms underlying the control of periodontal ligament stem cell (PDLSC) osteogenic differentiation. Recently, long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) were shown to function as competitive endogenous RNAs (ceRNAs) to regulate the effect of microRNAs (miRNAs) on their target genes during cell differentiation. However, comprehensive identification and integrated analysis of lncRNAs and circRNAs acting as ceRNAs during PDLSC osteogenic differentiation have not been performed. PDLSCs were derived from healthy human periodontal ligament and cultured separately with osteogenic induction and normal media for 7 days. Cultured PDLSCs were positive for STRO-1 and CD146 and negative for CD31 and CD45. Osteo-induced PDLSCs showed increased ALP (alkaline phosphatase) activity and up-regulated expression levels of the osteogenesis-related markers ALP, Runt-related transcription factor 2 and osteocalcin. Then, a total of 960 lncRNAs and 1456 circRNAs were found to be differentially expressed by RNA sequencing. The expression profiles of eight lncRNAs and eight circRNAs were measured with quantitative real-time polymerase chain reaction and were shown to agree with the RNA-seq results. Furthermore, the potential functions of lncRNAs and circRNAs as ceRNAs were predicted based on miRanda and were investigated using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis. In total, 147 lncRNAs and 1382 circRNAs were predicted to combine with 148 common miRNAs and compete for miRNA binding sites with 744 messenger RNAs. These mRNAs were predicted to significantly participate in osteoblast differentiation, the MAPK pathway, the Wnt pathway and the signaling pathways regulating pluripotency of stem cells. Among them, lncRNAs coded as TCONS_00212979 and TCONS_00212984, as well as circRNA BANP and circRNA ITCH, might interact with miRNA34a and miRNA146a to regulate PDLSC osteogenic differentiation via the MAPK pathway. This study comprehensively identified lncRNAs/circRNAs and first integrated their potential ceRNA function during PDLSC osteogenic differentiation. These findings suggest that specific lncRNAs and circRNAs might function as ceRNAs to promote PDLSC osteogenic differentiation and periodontal regeneration.

Molecular Profile of Peripheral Blood Mononuclear Cells from Patients with Rheumatoid Arthritis

PubMed Central

Edwards, Christopher J; Feldman, Jeffrey L; Beech, Jonathan; Shields, Kathleen M; Stover, Jennifer A; Trepicchio, William L; Larsen, Glenn; Foxwell, Brian MJ; Brennan, Fionula M; Feldmann, Marc; Pittman, Debra D

2007-01-01

Rheumatoid arthritis (RA) is a chronic inflammatory arthritis. Currently, diagnosis of RA may take several weeks, and factors used to predict a poor prognosis are not always reliable. Gene expression in RA may consist of a unique signature. Gene expression analysis has been applied to synovial tissue to define molecularly distinct forms of RA; however, expression analysis of tissue taken from a synovial joint is invasive and clinically impractical. Recent studies have demonstrated that unique gene expression changes can be identified in peripheral blood mononuclear cells (PBMCs) from patients with cancer, multiple sclerosis, and lupus. To identify RA disease-related genes, we performed a global gene expression analysis. RNA from PBMCs of 9 RA patients and 13 normal volunteers was analyzed on an oligonucleotide array. Compared with normal PBMCs, 330 transcripts were differentially expressed in RA. The differentially regulated genes belong to diverse functional classes and include genes involved in calcium binding, chaperones, cytokines, transcription, translation, signal transduction, extracellular matrix, integral to plasma membrane, integral to intracellular membrane, mitochondrial, ribosomal, structural, enzymes, and proteases. A k-nearest neighbor analysis identified 29 transcripts that were preferentially expressed in RA. Ten genes with increased expression in RA PBMCs compared with controls mapped to a RA susceptibility locus, 6p21.3. These results suggest that analysis of RA PBMCs at the molecular level may provide a set of candidate genes that could yield an easily accessible gene signature to aid in early diagnosis and treatment. PMID:17515956

Synthetic data sets for the identification of key ingredients for RNA-seq differential analysis.

PubMed

Rigaill, Guillem; Balzergue, Sandrine; Brunaud, Véronique; Blondet, Eddy; Rau, Andrea; Rogier, Odile; Caius, José; Maugis-Rabusseau, Cathy; Soubigou-Taconnat, Ludivine; Aubourg, Sébastien; Lurin, Claire; Martin-Magniette, Marie-Laure; Delannoy, Etienne

2018-01-01

Numerous statistical pipelines are now available for the differential analysis of gene expression measured with RNA-sequencing technology. Most of them are based on similar statistical frameworks after normalization, differing primarily in the choice of data distribution, mean and variance estimation strategy and data filtering. We propose an evaluation of the impact of these choices when few biological replicates are available through the use of synthetic data sets. This framework is based on real data sets and allows the exploration of various scenarios differing in the proportion of non-differentially expressed genes. Hence, it provides an evaluation of the key ingredients of the differential analysis, free of the biases associated with the simulation of data using parametric models. Our results show the relevance of a proper modeling of the mean by using linear or generalized linear modeling. Once the mean is properly modeled, the impact of the other parameters on the performance of the test is much less important. Finally, we propose to use the simple visualization of the raw P-value histogram as a practical evaluation criterion of the performance of differential analysis methods on real data sets. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Transcriptome profiling analysis reveals biomarkers in colon cancer samples of various differentiation

PubMed Central

Yu, Tonghu; Zhang, Huaping; Qi, Hong

2018-01-01

The aim of the present study was to investigate more colon cancer-related genes in different stages. Gene expression profile E-GEOD-62932 was extracted for differentially expressed gene (DEG) screening. Series test of cluster analysis was used to obtain significant trending models. Based on the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases, functional and pathway enrichment analysis were processed and a pathway relation network was constructed. Gene co-expression network and gene signal network were constructed for common DEGs. The DEGs with the same trend were clustered and in total, 16 clusters with statistical significance were obtained. The screened DEGs were enriched into small molecule metabolic process and metabolic pathways. The pathway relation network was constructed with 57 nodes. A total of 328 common DEGs were obtained. Gene signal network was constructed with 71 nodes. Gene co-expression network was constructed with 161 nodes and 211 edges. ABCD3, CPT2, AGL and JAM2 are potential biomarkers for the diagnosis of colon cancer. PMID:29928385

Transcriptome and gene expression analysis of DHA producer Aurantiochytrium under low temperature conditions

PubMed Central

Ma, Zengxin; Tan, Yanzhen; Cui, Guzhen; Feng, Yingang; Cui, Qiu; Song, Xiaojin

2015-01-01

Aurantiochytrium is a promising docosahexaenoic acid (DHA) production candidate due to its fast growth rate and high proportions of lipid and DHA content. In this study, high-throughput RNA sequencing technology was employed to explore the acclimatization of this DHA producer under cold stress at the transcriptional level. The overall de novo assembly of the cDNA sequence data generated 29,783 unigenes, with an average length of 1,200 bp. In total, 13,245 unigenes were annotated in at least one database. A comparative genomic analysis between normal conditions and cold stress revealed that 2,013 genes were differentially expressed during the growth stage, while 2,071 genes were differentially expressed during the lipid accumulation stage. Further functional categorization and analyses showed some differentially expressed genes were involved in processes crucial to cold acclimation, such as signal transduction, cellular component biogenesis, and carbohydrate and lipid metabolism. A brief survey of the transcripts obtained in response to cold stress underlines the survival strategy of Aurantiochytrium; of these transcripts, many directly or indirectly influence the lipid composition. This is the first study to perform a transcriptomic analysis of the Aurantiochytrium under low temperature conditions. Our results will help to enhance DHA production by Aurantiochytrium in the future. PMID:26403200

Transcriptome and gene expression analysis of DHA producer Aurantiochytrium under low temperature conditions.

PubMed

Ma, Zengxin; Tan, Yanzhen; Cui, Guzhen; Feng, Yingang; Cui, Qiu; Song, Xiaojin

2015-09-25

Aurantiochytrium is a promising docosahexaenoic acid (DHA) production candidate due to its fast growth rate and high proportions of lipid and DHA content. In this study, high-throughput RNA sequencing technology was employed to explore the acclimatization of this DHA producer under cold stress at the transcriptional level. The overall de novo assembly of the cDNA sequence data generated 29,783 unigenes, with an average length of 1,200 bp. In total, 13,245 unigenes were annotated in at least one database. A comparative genomic analysis between normal conditions and cold stress revealed that 2,013 genes were differentially expressed during the growth stage, while 2,071 genes were differentially expressed during the lipid accumulation stage. Further functional categorization and analyses showed some differentially expressed genes were involved in processes crucial to cold acclimation, such as signal transduction, cellular component biogenesis, and carbohydrate and lipid metabolism. A brief survey of the transcripts obtained in response to cold stress underlines the survival strategy of Aurantiochytrium; of these transcripts, many directly or indirectly influence the lipid composition. This is the first study to perform a transcriptomic analysis of the Aurantiochytrium under low temperature conditions. Our results will help to enhance DHA production by Aurantiochytrium in the future.

AhR-mediated gene expression in the developing mouse telencephalon.

PubMed

Gohlke, Julia M; Stockton, Pat S; Sieber, Stella; Foley, Julie; Portier, Christopher J

2009-11-01

We hypothesize that TCDD-induced developmental neurotoxicity is modulated through an AhR-dependent interaction with key regulatory neuronal differentiation pathways during telencephalon development. To test this hypothesis we examined global gene expression in both dorsal and ventral telencephalon tissues in E13.5 AhR-/- and wildtype mice exposed to TCDD or vehicle. Consistent with previous biochemical, pathological and behavioral studies, our results suggest TCDD initiated changes in gene expression in the developing telencephalon are primarily AhR-dependent, as no statistically significant gene expression changes are evident after TCDD exposure in AhR-/- mice. Based on a gene regulatory network for neuronal specification in the developing telencephalon, the present analysis suggests differentiation of GABAergic neurons in the ventral telencephalon is compromised in TCDD exposed and AhR-/- mice. In addition, our analysis suggests Sox11 may be directly regulated by AhR based on gene expression and comparative genomics analyses. In conclusion, this analysis supports the hypothesis that AhR has a specific role in the normal development of the telencephalon and provides a mechanistic framework for neurodevelopmental toxicity of chemicals that perturb AhR signaling.

Effects of keratinocyte growth factor on skin epithelial differentiation of human amnion epithelial cells.

PubMed

Fatimah, Simat Siti; Tan, Geok Chin; Chua, Kienhui; Tan, Ay Eeng; Nur Azurah, Abdul Ghani; Hayati, Abdul Rahman

2013-08-01

The aim of the present study was to determine the effects of KGF on the differentiation of cultured human amnion epithelial cells (HAECs) towards skin keratinocyte. HAECs at passage 1 were cultured in medium HAM's F12: Dulbecco's Modified Eagles Medium (1:1) supplemented with different concentrations of KGF (0, 5, 10, 20, 30 and 50 ng/ml KGF). Dose-response of KGF on HAECs was determined by morphological assessment; growth kinetic evaluation; immunocytochemical analysis; stemness and epithelial gene expression quantification with two step real time RT-PCR. KGF promotes the proliferation of HAECs with maximal effect observed at 10 ng/ml KGF. However, KGF decreased the stemness genes expression: Oct-3/4, Sox-2, Nanog3, Rex-1, FGF-4, FZD-9 and BST-1. KGF also down-regulates epithelial genes expression: CK3, CK18, CK19, Integrin-β1, p63 and involucrin in cultured HAECs. No significant difference on the gene expression was detected for each Nestin, ABCG-2, CK1 and CK14 in KGF-treated HAECs. Immunocytochemical analysis for both control and KGF-treated HAECs demonstrated positive staining against CK14 and CK18 but negative staining against involucrin. The results suggested that KGF stimulates an early differentiation of HAECs towards epidermal cells. Differentiation of KGF-treated HAECs to corneal lineage is unfavourable. Therefore, further studies are needed to elucidate the roles of KGF in the differentiation of HAECs towards skin keratinocytes. Copyright © 2012 Elsevier Ltd and ISBI. All rights reserved.

Reprogramming human umbilical cord mesenchymal stromal cells to islet-like cells with the use of in vitro-synthesized pancreatic-duodenal homebox 1 messenger RNA.

PubMed

Wang, Xiao Li; Hu, Pei; Guo, Xing Rong; Yan, Ding; Yuan, Yahong; Yan, Shi Rong; Li, Dong Sheng

2014-11-01

Human umbilical cord mesenchymal stromal cells (hUC-MSCs) hold great potential as a therapeutic candidate to treat diabetes, owing to their unlimited source and ready availability. In this study, we differentiated hUC-MSCs with in vitro-synthesized pancreatic-duodenal homebox 1 (PDX1) messenger (m)RNA into islet-like cell clusters. hUC-MSCs were confirmed by both biomarker detection and functional differentiation. In vitro-synthesized PDX1 messenger RNA can be transfected into hUC-MSCs efficiently. The upregulated expression of PDX1 protein can be detected 4 h after transfection and remains detectable for 36 h. The induction of islet-like structures was confirmed by means of morphology and dithizone staining. Reverse transcriptase-polymerase chain reaction results revealed the expression of some key pancreatic transcription factors, such as PDX1, NeuroD, NKX6.1, Glut-2 and insulin in islet-like cell clusters. Immunofluorescence analysis showed that differentiated cells express both insulin and C-peptide. Enzyme-linked immunosorbent assay analysis validated the insulin secretion of islet-like cell clusters in response to the glucose stimulation. Our results demonstrate the use of in vitro-synthesized PDX1 messenger RNA to differentiate hUC-MSCs into islet-like cells and pave the way toward the development of reprogramming and directed-differentiation methods for the expression of encoded proteins. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Mining, Validation, and Clinical Significance of Colorectal Cancer (CRC)-Associated lncRNAs.

PubMed

Sun, Xiangwei; Hu, Yingying; Zhang, Liang; Hu, Changyuan; Guo, Gangqiang; Mao, Chenchen; Xu, Jianfeng; Ye, Sisi; Huang, Guanli; Xue, Xiangyang; Guo, Aizhen; Shen, Xian

2016-01-01

Colorectal cancer (CRC) is one of the deadliest tumours, but its pathogenesis remains unclear. The involvement of differentially expressed long non-coding RNAs (lncRNAs) in CRC tumorigenesis makes them suitable tumour biomarkers. Here, we screened 150 cases of CRC and 85 cases of paracancerous tissues in the GEO database for differentially expressed lncRNAs. The levels of lncRNA candidates in 84 CRC and paracancerous tissue samples were validated by qRT-PCR and their clinical significance was analyzed. We identified 15 lncRNAs with differential expression in CRC tumours; among them, AK098081 was significantly up-regulated, whereas AK025209, BC040303, BC037331, AK026659, and CR749831 were down-regulated in CRC. In a receiver operating characteristic curve analysis, the area under the curve for the six lncRNAs was 0.914. High expression of AK098081 and low expression of BC040303, CR749831, and BC037331 indicated poor CRC differentiation. CRC patients with lymph node metastasis had lower expression of BC037331. In addition, the group with high AK098081 expression presented significantly lower overall survival and disease-free survival rates than the low-expression group, confirming AK098081 as an independent risk factor for CRC patients. In conclusion, we have identified multiple CRC-associated lncRNAs from microarray expression profiles that can serve as novel biomarkers for the diagnosis and prognosis of CRC.

Pleiotrophin Expression during Odontogenesis

PubMed Central

Ames, Jennifer E.; Tamkenath, Amena; Mamaeva, Olga; Stidham, Katherine; Wilson, Mary E.; Perez-Pinera, Pablo; Deuel, Thomas F.; MacDougall, Mary

2012-01-01

Pleiotrophin (PTN) is an extracellular matrix–associated growth factor and chemokine expressed in mesodermal and ectodermal cells. It plays an important role in osteoblast recruitment and differentiation. There is limited information currently available about PTN expression during odontoblast differentiation and tooth formation, and thus the authors aimed to establish the spatiotemporal expression pattern of PTN during mouse odontogenesis. Immortalized mouse dental pulp (MD10-D3, MD10-A11) and odontoblast-like (M06-G3) and ameloblast-like (EOE-3M) cell lines were grown and samples prepared for immunocytochemistry, Western blot, and conventional and quantitative PCR analysis. Effects of BMP2, BMP4, and BMP7 treatment on PTN expression in odontoblast-like M06-G3 cells were tested by quantitative PCR. Finally, immunohistochemistry of sectioned mice mandibles and maxillaries at developmental stages E16, E18, P1, P6, P10, and P28 was performed. The experiments showed that PTN, at both the mRNA and protein level, was expressed in all tested epithelial and mesenchymal dental cell lines and that the level of PTN mRNA was influenced differentially by the bone morphogenetic proteins. The authors observed initial expression of PTN in the inner enamel epithelium with prolonged expression in the ameloblasts and odontoblasts throughout their stages of maturation and strong expression in the terminally differentiated and enamel matrix–secreting ameloblasts and odontoblasts of the adult mouse incisors and molars. PMID:22382872

Quality Evaluation of Human Bone Marrow Mesenchymal Stem Cells for Cartilage Repair

PubMed Central

Shiraishi, Katsunori; Takeuchi, Shunsuke; Yanada, Shinobu; Mera, Hisashi; Wakitani, Shigeyuki; Adachi, Nobuo

2017-01-01

Quality evaluation of mesenchymal stem cells (MSCs) based on efficacy would be helpful for their clinical application. In this study, we aimed to find the factors of human bone marrow MSCs relating to cartilage repair. The expression profiles of humoral factors, messenger RNAs (mRNAs), and microRNAs (miRNAs) were analyzed in human bone marrow MSCs from five different donors. We investigated the correlations of these expression profiles with the capacity of the MSCs for proliferation, chondrogenic differentiation, and cartilage repair in vivo. The mRNA expression of MYBL1 was positively correlated with proliferation and cartilage differentiation. By contrast, the mRNA expression of RCAN2 and the protein expression of TIMP-1 and VEGF were negatively correlated with proliferation and cartilage differentiation. However, MSCs from all five donors had the capacity to promote cartilage repair in vivo regardless of their capacity for proliferation and cartilage differentiation. The mRNA expression of HLA-DRB1 was positively correlated with cartilage repair in vivo. Meanwhile, the mRNA expression of TMEM155 and expression of miR-486-3p, miR-148b, miR-93, and miR-320B were negatively correlated with cartilage repair. The expression analysis of these factors might help to predict the ability of bone marrow MSCs to promote cartilage repair. PMID:28835756

Evolution of the Chordate Regeneration Blastema: Differential Gene Expression and Conserved Role of Notch Signaling During Siphon Regeneration in the Ascidian Ciona

PubMed Central

Hamada, Mayuko; Goricki, Spela; Byerly, Mardi S.; Satoh, Noriyuki; Jeffery, William R.

2015-01-01

The regeneration of the oral siphon (OS) and other distal structures in the ascidian Ciona intestinalis occurs by epimorphosis involving the formation of a blastema of proliferating cells. Despite the longstanding use of Ciona as a model in molecular developmental biology, regeneration in this system has not been previously explored by molecular analysis. Here we have employed microarray analysis and quantitative real time RT-PCR to identify genes with differential expression profiles during OS regeneration. The majority of differentially expressed genes were downregulated during OS regeneration, suggesting roles in normal growth and homeostasis. However, a subset of differentially expressed genes was upregulated in the regenerating OS, suggesting functional roles during regeneration. Among the upregulated genes were key members of the Notch signaling pathway, including those encoding the delta and jagged ligands, two fringe modulators, and to a lesser extent the notch receptor. In situ hybridization showed a complementary pattern of delta1 and notch gene expression in the blastema of the regenerating OS. Chemical inhibition of the Notch signaling pathway reduced the levels of cell proliferation in the branchial sac, a stem cell niche that contributes progenitor cells to the regenerating OS, and in the OS regeneration blastema, where siphon muscle fibers eventually re-differentiate. Chemical inhibition also prevented the replacement of oral siphon pigment organs, sensory receptors rimming the entrance of the OS, and siphon muscle fibers, but had no effects on the formation of the wound epidermis. Since Notch signaling is involved in the maintenance of proliferative activity in both the Ciona and vertebrate regeneration blastema, the results suggest a conserved evolutionary role of this signaling pathway in chordate regeneration. The genes identified in this investigation provide the foundation for future molecular analysis of OS regeneration. PMID:26206613

[Significance and mechanism of MSCT perfusion scan on differentiation of NSCLC].

PubMed

Liu, Jin-Kang; Hu, Cheng-Ping; Zhou, Mo-Ling; Zhou, Hui; Xiong, Zeng; Xia, Yu; Chen, Wei

2009-06-01

To determine the significance of MSCT perfusion scan on differentiation of NSCLC and to investigate its possible mechanisms. Forty four NSCLC patients underwent CT perfusion scan by MSCT. Among them, 22 cases were selected to detected the two-dimensional tumor microvascular architecture phenotype (2D-TMAP), the relationships between CT perfusion parameters (BF, BV, PEI, TIP), and the differentiation of NSCLC were analysed by using the correlation analysis and trend test. Spearman correlation analysis was used to study the relationships between CT perfusion parameters, differentiation, and 2D-TMAP. The total BF, BV and PEI decreased with decreasing differentiation of NSCLC (P<0.05). The total PEI showed a positive correlation with the total MVD (P<0.05). There were negative correlations between the surrounding area BF, the total BF, BV, and PEI, the uncomplete lumen of the surrounding area MVD, and expression of PCNA, respectively (P<0.05). There were positive correlations between degree of differentiation and the uncomplete lumen of the surrounding area MVD (P<0.05). It was the same as degree of differentiation and expression of PCNA, VEGF, respectively. There were positive correlations between the uncomplete lumen of the surrounding area MVD and expression of VEGF, ephrinB2, EphB4, and PCNA, respectively (P<0.05). Perfusion parameters reflect the difference of density of vassels with mature functional lumen. Careful evaluation of the differences of blood flow pattern in pulmonary space-occupying lesions by MSCT perfusion scan can be used to identify the degree of NSCLC differentiation.

Comparative transcriptome analysis to investigate the potential role of miRNAs in milk protein/fat quality.

PubMed

Wang, Xuehui; Zhang, Li; Jin, Jing; Xia, Anting; Wang, Chunmei; Cui, Yingjun; Qu, Bo; Li, Qingzhang; Sheng, Chunyan

2018-04-19

miRNAs play an important role in the processes of cell differentiation, biological development, and physiology. Here we investigated the molecular mechanisms regulating milk secretion and quality in dairy cows via transcriptome analyses of mammary gland tissues from dairy cows during the high-protein/high-fat, low-protein/low-fat or dry periods. To characterize the important roles of miRNAs and mRNAs in milk quality and to elucidate their regulatory networks in relation to milk secretion and quality, an integrated analysis was performed. A total of 25 core miRNAs were found to be differentially expressed (DE) during lactation compared to non-lactation, and these miRNAs were involved in epithelial cell terminal differentiation and mammary gland development. In addition, comprehensive analysis of mRNA and miRNA expression between high-protein/high-fat group and low-protein/low-fat groups indicated that, 38 miRNAs and 944 mRNAs were differentially expressed between them. Furthermore, 38 DE miRNAs putatively negatively regulated 253 DE mRNAs. The putative genes (253 DE mRNAs) were enriched in lipid biosynthetic process and amino acid transmembrane transporter activity. Moreover, putative DE genes were significantly enriched in fatty acid (FA) metabolism, biosynthesis of amino acids, synthesis and degradation of ketone bodies and biosynthesis of unsaturated FAs. Our results suggest that DE miRNAs might play roles as regulators of milk quality and milk secretion during mammary gland differentiation.

Proteomic Profiling and Differential Messenger RNA Expression Correlate HSP27 and Serpin Family B Member 1 to Apical Periodontitis Outcomes.

PubMed

Cavalla, Franco; Biguetti, Claudia; Jain, Sameer; Johnson, Cleverick; Letra, Ariadne; Garlet, Gustavo Pompermaier; Silva, Renato Menezes

2017-09-01

Understanding protein expression profiles of apical periodontitis may contribute to the discovery of novel diagnostic or therapeutic molecular targets. Periapical tissue samples (n = 5) of patients with lesions characterized as nonhealing were submitted for proteomic analysis. Two differentially expressed proteins (heat shock protein 27 [HSP27] and serpin family B member 1 [SERPINB1]) were selected for characterization, localization by immunofluorescence, and association with known biomarkers of acute inflammatory response in human apical periodontitis (n = 110) and healthy periodontal ligaments (n = 26). Apical periodontitis samples were categorized as stable/inactive (n = 70) or progressive/active (n = 40) based on the ratio of expression of receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG). Next, the expression of HSP27, SERPINB1, C-X-C motif Chemokine Receptor 1 (CXCR1), matrix metalloproteinase 8 (MMP8), myeloperoxidase (MPO), and cathepsin G (CTSG) messenger RNA was evaluated using real-time polymerase chain reaction. Data analysis was performed using the Shapiro-Wilk test, analysis of variance, and the Pearson test. P values <.05 were considered statistically significant. Proteomic analysis revealed 48 proteins as differentially expressed in apical periodontitis compared with a healthy periodontium, with 30 of these proteins found to be expressed in all 4 lesions. The expression of HSP27 and SERPINB1 was ∼2-fold higher in apical periodontitis. Next, an increased expression of HSP27 was detected in epithelial cells, whereas SERPINB1 expression was noted in neutrophils and epithelial cells. HSP27 and SERPINB1 transcripts were highly expressed in stable/inactive lesions (P < .05). Significant negative correlations were found between the expression of HSP27 and SERPINB1 with biomarkers of acute inflammation including CXCR1, MPO, and CTSG. Our data suggest HSP27 and SERPINB1 as potential regulators of the inflammatory response in apical periodontitis. Additional functional studies should be performed to further characterize the role of these molecules during the development/progression of apical periodontitis. Copyright © 2017 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Characterizing the Grape Transcriptome. Analysis of Expressed Sequence Tags from Multiple Vitis Species and Development of a Compendium of Gene Expression during Berry Development1[w

PubMed Central

Silva, Francisco Goes da; Iandolino, Alberto; Al-Kayal, Fadi; Bohlmann, Marlene C.; Cushman, Mary Ann; Lim, Hyunju; Ergul, Ali; Figueroa, Rubi; Kabuloglu, Elif K.; Osborne, Craig; Rowe, Joan; Tattersall, Elizabeth; Leslie, Anna; Xu, Jane; Baek, JongMin; Cramer, Grant R.; Cushman, John C.; Cook, Douglas R.

2005-01-01

We report the analysis and annotation of 146,075 expressed sequence tags from Vitis species. The majority of these sequences were derived from different cultivars of Vitis vinifera, comprising an estimated 25,746 unique contig and singleton sequences that survey transcription in various tissues and developmental stages and during biotic and abiotic stress. Putatively homologous proteins were identified for over 17,752 of the transcripts, with 1,962 transcripts further subdivided into one or more Gene Ontology categories. A simple structured vocabulary, with modules for plant genotype, plant development, and stress, was developed to describe the relationship between individual expressed sequence tags and cDNA libraries; the resulting vocabulary provides query terms to facilitate data mining within the context of a relational database. As a measure of the extent to which characterized metabolic pathways were encompassed by the data set, we searched for homologs of the enzymes leading from glycolysis, through the oxidative/nonoxidative pentose phosphate pathway, and into the general phenylpropanoid pathway. Homologs were identified for 65 of these 77 enzymes, with 86% of enzymatic steps represented by paralogous genes. Differentially expressed transcripts were identified by means of a stringent believability index cutoff of ≥98.4%. Correlation analysis and two-dimensional hierarchical clustering grouped these transcripts according to similarity of expression. In the broadest analysis, 665 differentially expressed transcripts were identified across 29 cDNA libraries, representing a range of developmental and stress conditions. The groupings revealed expected associations between plant developmental stages and tissue types, with the notable exception of abiotic stress treatments. A more focused analysis of flower and berry development identified 87 differentially expressed transcripts and provides the basis for a compendium that relates gene expression and annotation to previously characterized aspects of berry development and physiology. Comparison with published results for select genes, as well as correlation analysis between independent data sets, suggests that the inferred in silico patterns of expression are likely to be an accurate representation of transcript abundance for the conditions surveyed. Thus, the combined data set reveals the in silico expression patterns for hundreds of genes in V. vinifera, the majority of which have not been previously studied within this species. PMID:16219919

Identification of differentially expressed proteins in Ostrinia furnacalis adults after exposure to ultraviolet A.

PubMed

Zhang, Changyu; Meng, Jianyu

2018-06-23

Ultraviolet A (UVA), the major component of solar UV irradiation, is an important environmental factor inducing damage to insects including cell death, photoreceptor damage, and oxidative stress. In order to improve understanding of the adaptation mechanisms of insect after UVA exposure, a comparative proteomic analysis was carried out to reveal differential protein expression in Ostrinia furnacalis. Three-day-old adults were treated with UVA for 1 h. Total proteins of control and UVA-treated insects were examined using two-dimensional electrophoresis (2-DE). 2-DE analysis demonstrated that 19 proteins were increased and 18 proteins were decreased significantly in O. furnacalis after UVA exposure, respectively. Thirty differentially expressed proteins were successfully identified by mass spectrometry. The identified proteins were involved in diverse biological processes, such as signal transduction, transport processing, cellular stress, metabolisms, and cytoskeleton organization. Our results reveal that the response patterns of O. furnacalis to UVA irradiation are complex and provide novel insights into the adaptation response to UVA irradiation stress.

Quantitative proteomics and systems analysis of cultured H9C2 cardiomyoblasts during differentiation over time supports a 'function follows form' model of differentiation.

PubMed

Kankeu, Cynthia; Clarke, Kylie; Van Haver, Delphi; Gevaert, Kris; Impens, Francis; Dittrich, Anna; Roderick, H Llewelyn; Passante, Egle; Huber, Heinrich J

2018-05-17

The rat cardiomyoblast cell line H9C2 has emerged as a valuable tool for studying cardiac development, mechanisms of disease and toxicology. We present here a rigorous proteomic analysis that monitored the changes in protein expression during differentiation of H9C2 cells into cardiomyocyte-like cells over time. Quantitative mass spectrometry followed by gene ontology (GO) enrichment analysis revealed that early changes in H9C2 differentiation are related to protein pathways of cardiac muscle morphogenesis and sphingolipid synthesis. These changes in the proteome were followed later in the differentiation time-course by alterations in the expression of proteins involved in cation transport and beta-oxidation. Studying the temporal profile of the H9C2 proteome during differentiation in further detail revealed eight clusters of co-regulated proteins that can be associated with early, late, continuous and transient up- and downregulation. Subsequent reactome pathway analysis based on these eight clusters further corroborated and detailed the results of the GO analysis. Specifically, this analysis confirmed that proteins related to pathways in muscle contraction are upregulated early and transiently, and proteins relevant to extracellular matrix organization are downregulated early. In contrast, upregulation of proteins related to cardiac metabolism occurs at later time points. Finally, independent validation of the proteomics results by immunoblotting confirmed hereto unknown regulators of cardiac structure and ionic metabolism. Our results are consistent with a 'function follows form' model of differentiation, whereby early and transient alterations of structural proteins enable subsequent changes that are relevant to the characteristic physiology of cardiomyocytes.

Towards decrypting cryptobiosis--analyzing anhydrobiosis in the tardigrade Milnesium tardigradum using transcriptome sequencing.

PubMed

Wang, Chong; Grohme, Markus A; Mali, Brahim; Schill, Ralph O; Frohme, Marcus

2014-01-01

Many tardigrade species are capable of anhydrobiosis; however, mechanisms underlying their extreme desiccation resistance remain elusive. This study attempts to quantify the anhydrobiotic transcriptome of the limno-terrestrial tardigrade Milnesium tardigradum. A prerequisite for differential gene expression analysis was the generation of a reference hybrid transcriptome atlas by assembly of Sanger, 454 and Illumina sequence data. The final assembly yielded 79,064 contigs (>100 bp) after removal of ribosomal RNAs. Around 50% of them could be annotated by SwissProt and NCBI non-redundant protein sequences. Analysis using CEGMA predicted 232 (93.5%) out of the 248 highly conserved eukaryotic genes in the assembly. We used this reference transcriptome for mapping and quantifying the expression of transcripts regulated under anhdydrobiosis in a time-series during dehydration and rehydration. 834 of the transcripts were found to be differentially expressed in a single stage (dehydration/inactive tun/rehydration) and 184 were overlapping in two stages while 74 were differentially expressed in all three stages. We have found interesting patterns of differentially expressed transcripts that are in concordance with a common hypothesis of metabolic shutdown during anhydrobiosis. This included down-regulation of several proteins of the DNA replication and translational machinery and protein degradation. Among others, heat shock proteins Hsp27 and Hsp30c were up-regulated in response to dehydration and rehydration. In addition, we observed up-regulation of ployubiquitin-B upon rehydration together with a higher expression level of several DNA repair proteins during rehydration than in the dehydration stage. Most of the transcripts identified to be differentially expressed had distinct cellular function. Our data suggest a concerted molecular adaptation in M. tardigradum that permits extreme forms of ametabolic states such as anhydrobiosis. It is temping to surmise that the desiccation tolerance of tradigrades can be achieved by a constitutive cellular protection system, probably in conjunction with other mechanisms such as rehydration-induced cellular repair.

Towards Decrypting Cryptobiosis—Analyzing Anhydrobiosis in the Tardigrade Milnesium tardigradum Using Transcriptome Sequencing

PubMed Central

Wang, Chong; Grohme, Markus A.; Mali, Brahim; Schill, Ralph O.; Frohme, Marcus

2014-01-01

Background Many tardigrade species are capable of anhydrobiosis; however, mechanisms underlying their extreme desiccation resistance remain elusive. This study attempts to quantify the anhydrobiotic transcriptome of the limno-terrestrial tardigrade Milnesium tardigradum. Results A prerequisite for differential gene expression analysis was the generation of a reference hybrid transcriptome atlas by assembly of Sanger, 454 and Illumina sequence data. The final assembly yielded 79,064 contigs (>100 bp) after removal of ribosomal RNAs. Around 50% of them could be annotated by SwissProt and NCBI non-redundant protein sequences. Analysis using CEGMA predicted 232 (93.5%) out of the 248 highly conserved eukaryotic genes in the assembly. We used this reference transcriptome for mapping and quantifying the expression of transcripts regulated under anhdydrobiosis in a time-series during dehydration and rehydration. 834 of the transcripts were found to be differentially expressed in a single stage (dehydration/inactive tun/rehydration) and 184 were overlapping in two stages while 74 were differentially expressed in all three stages. We have found interesting patterns of differentially expressed transcripts that are in concordance with a common hypothesis of metabolic shutdown during anhydrobiosis. This included down-regulation of several proteins of the DNA replication and translational machinery and protein degradation. Among others, heat shock proteins Hsp27 and Hsp30c were up-regulated in response to dehydration and rehydration. In addition, we observed up-regulation of ployubiquitin-B upon rehydration together with a higher expression level of several DNA repair proteins during rehydration than in the dehydration stage. Conclusions Most of the transcripts identified to be differentially expressed had distinct cellular function. Our data suggest a concerted molecular adaptation in M. tardigradum that permits extreme forms of ametabolic states such as anhydrobiosis. It is temping to surmise that the desiccation tolerance of tradigrades can be achieved by a constitutive cellular protection system, probably in conjunction with other mechanisms such as rehydration-induced cellular repair. PMID:24651535

Transient Downregulation of Nanog and Oct4 Induced by DETA/NO Exposure in Mouse Embryonic Stem Cells Leads to Mesodermal/Endodermal Lineage Differentiation

PubMed Central

Mora-Castilla, Sergio; Tejedo, Juan R.; Díaz, Irene; Hitos, Ana B.; Cahuana, Gladys M.; Hmadcha, Abdelkrim; Martín, Franz; Soria, Bernat

2014-01-01

The function of pluripotency genes in differentiation is a matter of investigation. We report here that Nanog and Oct4 are reexpressed in two mouse embryonic stem cell (mESC) lines following exposure to the differentiating agent DETA/NO. Both cell lines express a battery of both endoderm and mesoderm markers following induction of differentiation with DETA/NO-based protocols. Confocal analysis of cells undergoing directed differentiation shows that the majority of cells expressing Nanog express also endoderm genes such as Gata4 and FoxA2 (75.4% and 96.2%, resp.). Simultaneously, mRNA of mesodermal markers Flk1 and Mef2c are also regulated by the treatment. Acetylated histone H3 occupancy at the promoter of Nanog is involved in the process of reexpression. Furthermore, Nanog binding to the promoter of Brachyury leads to repression of this gene, thus disrupting mesendoderm transition. PMID:25544848

Discovery of a stem-like multipotent cell fate.

PubMed

Paffhausen, Emily S; Alowais, Yasir; Chao, Cara W; Callihan, Evan C; Creswell, Karen; Bracht, John R

2018-01-01

Adipose derived stem cells (ASCs) can be obtained from lipoaspirates and induced in vitro to differentiate into bone, cartilage, and fat. Using this powerful model system we show that after in vitro adipose differentiation a population of cells retain stem-like qualities including multipotency. They are lipid (-), retain the ability to propagate, express two known stem cell markers, and maintain the capacity for trilineage differentiation into chondrocytes, adipocytes, and osteoblasts. However, these cells are not traditional stem cells because gene expression analysis showed an overall expression profile similar to that of adipocytes. In addition to broadening our understanding of cellular multipotency, our work may be particularly relevant to obesity-associated metabolic disorders. The adipose expandability hypothesis proposes that inability to differentiate new adipocytes is a primary cause of metabolic syndrome in obesity, including diabetes and cardiovascular disease. Here we have defined a differentiation-resistant stem-like multipotent cell population that may be involved in regulation of adipose expandability in vivo and may therefore play key roles in the comorbidities of obesity.

Specificity protein 1 regulates topoisomerase IIβ expression in SH-SY5Y cells during neuronal differentiation.

PubMed

Guo, Hui; Cao, Cuili; Chi, Xueqian; Zhao, Junxia; Liu, Xia; Zhou, Najing; Han, Shuo; Yan, Yongxin; Wang, Yanling; Xu, Yannan; Yan, Yunli; Cui, Huixian; Sun, Hongxia

2014-10-01

Topoisomerase IIβ (top IIβ) is a nuclear enzyme with an essential role in neural development. The regulation of top IIβ gene expression during neural differentiation is poorly understood. Functional analysis of top IIβ gene structure displayed a GC box sequence in its transcription promoter, which binds the nuclear transcription factor specificity protein 1 (Sp1). Sp1 regulates gene expression via multiple mechanisms and is essential for early embryonic development. This study seeks to determine whether Sp1 regulates top IIβ gene expression during neuronal differentiation. For this purpose, human neuroblastoma SH-SY5Y cells were induced to neuronal differentiation in the presence of all-trans retinoic acid (RA) for 5 days. After incubation with 10 μM RA for 3-5 days, a majority of the cells exited the cell cycle to become postmitotic neurons, characterized by the presence of longer neurite outgrowths and expression of the neuronal marker microtubule-associated protein-2 (MAP2). Elevated Sp1 and top IIβ mRNA and protein levels were detected and found to be positively correlated with the differentiation stage. Chromatin immunoprecipitation assay demonstrated an increased recruitment of Sp1 to the top IIβ promoter after RA treatment. Mithramycin A, a compound that interferes with Sp1 binding to GC-rich DNA sequences, downregulated the expression of top IIβ, resulting in reduced expression of MAP2 and decreased neurite length compared with the control group. Our results indicate that Sp1 regulates top IIβ expression by binding to the GC box of the gene promoter during neuronal differentiation in SH-SY5Y cells. © 2014 Wiley Periodicals, Inc.

Comparison of Osteogenic and Chondrogenic Differentiation Ability of Buccal Fat Pad Derived Mesenchymal Stem Cells and Gingival Derived Cells.

PubMed

Ghaderi, Hamid; Razmkhah, Mahboobeh; Kiany, Farin; Chenari, Nooshafarin; Haghshenas, Mohammad Reza; Ghaderi, Abbas

2018-06-01

One major goal of tissue engineering and regenerative medicine is to find an appropriate source of mesenchymal stem cells (MSCs) with higher differentiation ability. In this experimental study, the osteogenic and chondrogenic differentiation ability of buccal fat pad derived MSCs (BFP-MSCs) with gingival derived cells (GDCs) were compared. BFP-MSCs and GDCs were cultured enzymatically and expanded. The expanded cells were analyzed for membrane-associated markers, using flow cytometry. Then the ability of these cells to differentiate into osteocyte and chondrocyte was assessed morphologically and by mRNA expression of collagen I (COLL), BGLA and bone morphogenetic protein 2 (BMP2) using qRT-PCR. Flow cytometry analysis showed that both BFP-MSCs and GDCs expressed the characteristic stem cell markers such as CD73, CD44, and CD90, whereas they did not express hematopoietic markers. Mineralized calcium deposition was observed apparently in BFP-MSCs cultured in osteogenic medium but GDCs showed fewer mineralized nodules. The mRNA expression levels of BGLA and BMP2 showed 7×105 and 733-fold more mRNA expression in BFP-MSCs treated with differentiation media compared to the control group. In chondrogenic differentiation, BFP-MSCs transformed from a spindle to a cuboidal shape while GDCs showed only a slight transformation. In addition, mRNA expression of COLL showed 282-fold higher expression in BFP-MSCs in comparison to the control group. Such significant difference in mRNA expression of BGLA, BMP2, and COLL was not observed in GDCs compared to their corresponding controls. Based on the present results, BFP yields a greater proportion of stem cells compared to gingiva. Therefore, this tissue can be introduced as an easily available source for the treatment of periodontal defects and other maxillofacial injuries.

Alcohol-induced suppression of KDM6B dysregulates the mineralization potential in dental pulp stem cells

PubMed Central

Hoang, Michael; Kim, Jeffrey J.; Kim, Yiyoung; Tong, Elizabeth; Trammell, Benjamin; Liu, Yao; Shi, Songtao; Lee, Chang-Ryul; Hong, Christine; Wang, Cun-Yu; Kim, Yong

2016-01-01

Epigenetic changes, such as alteration of DNA methylation patterns, have been proposed as a molecular mechanism underlying the effect of alcohol on the maintenance of adult stem cells. We have performed genome-wide gene expression microarray and DNA methylome analysis to identify molecular alterations via DNA methylation changes associated with exposure of human dental pulp stem cells (DPSCs) to ethanol (EtOH). By combined analysis of the gene expression and DNA methylation, we have found a significant number of genes that are potentially regulated by EtOH-induced DNA methylation. As a focused approach, we have also performed a pathway-focused RT-PCR array analysis to examine potential molecular effects of EtOH on genes involved in epigenetic chromatin modification enzymes, fibroblastic markers, and stress and toxicity pathways in DPSCs. We have identified and verified that lysine specific demethylase 6B (KDM6B) was significantly dysregulated in DPSCs upon EtOH exposure. EtOH treatment during odontogenic/osteogenic differentiation of DPSCs suppressed the induction of KDM6B with alterations in the expression of differentiation markers. Knockdown of KDM6B resulted in a marked decrease in mineralization from implanted DPSCs in vivo. Furthermore, an ectopic expression of KDM6B in EtOH-treated DPSCs restored the expression of differentiation-related genes. Our study has demonstrated that EtOH-induced inhibition of KDM6B plays a role in the dysregulation of odontogenic/osteogenic differentiation in the DPSC model. This suggests a potential molecular mechanism for cellular insults of heavy alcohol consumption that can lead to decreased mineral deposition potentially associated with abnormalities in dental development and also osteopenia/osteoporosis, hallmark features of fetal alcohol spectrum disorders. PMID:27286573

Transcriptome Analysis of the Differentially Expressed Genes in the Male and Female Shrub Willows (Salix suchowensis)

PubMed Central

Liu, Jingjing; Yin, Tongming; Ye, Ning; Chen, Yingnan; Yin, Tingting; Liu, Min; Hassani, Danial

2013-01-01

Background The dioecious system is relatively rare in plants. Shrub willow is an annual flowering dioecious woody plant, and possesses many characteristics that lend it as a great model for tracking the missing pieces of sex determination evolution. To gain a global view of the genes differentially expressed in the male and female shrub willows and to develop a database for further studies, we performed a large-scale transcriptome sequencing of flower buds which were separately collected from two types of sexes. Results Totally, 1,201,931 high quality reads were obtained, with an average length of 389 bp and a total length of 467.96 Mb. The ESTs were assembled into 29,048 contigs, and 132,709 singletons. These unigenes were further functionally annotated by comparing their sequences to different proteins and functional domain databases and assigned with Gene Ontology (GO) terms. A biochemical pathway database containing 291 predicted pathways was also created based on the annotations of the unigenes. Digital expression analysis identified 806 differentially expressed genes between the male and female flower buds. And 33 of them located on the incipient sex chromosome of Salicaceae, among which, 12 genes might involve in plant sex determination empirically. These genes were worthy of special notification in future studies. Conclusions In this study, a large number of EST sequences were generated from the flower buds of a male and a female shrub willow. We also reported the differentially expressed genes between the two sex-type flowers. This work provides valuable information and sequence resources for uncovering the sex determining genes and for future functional genomics analysis of Salicaceae spp. PMID:23560075

Transcriptome from circulating cells suggests dysregulated pathways associated with long-term recurrent events following first-time myocardial infarction.

PubMed

Suresh, Rahul; Li, Xing; Chiriac, Anca; Goel, Kashish; Terzic, Andre; Perez-Terzic, Carmen; Nelson, Timothy J

2014-09-01

Whole-genome gene expression analysis has been successfully utilized to diagnose, prognosticate, and identify potential therapeutic targets for high-risk cardiovascular diseases. However, the feasibility of this approach to identify outcome-related genes and dysregulated pathways following first-time myocardial infarction (AMI) remains unknown and may offer a novel strategy to detect affected expressome networks that predict long-term outcome. Whole-genome expression microarray on blood samples from normal cardiac function controls (n=21) and first-time AMI patients (n=31) within 48-hours post-MI revealed expected differential gene expression profiles enriched for inflammation and immune-response pathways. To determine molecular signatures at the time of AMI associated with long-term outcomes, transcriptional profiles from sub-groups of AMI patients with (n=5) or without (n=22) any recurrent events over an 18-month follow-up were compared. This analysis identified 559 differentially-expressed genes. Bioinformatic analysis of this differential gene-set for associated pathways revealed 1) increasing disease severity in AMI patients is associated with a decreased expression of genes involved in the developmental epithelial-to-mesenchymal transition pathway, and 2) modulation of cholesterol transport genes that include ABCA1, CETP, APOA1, and LDLR is associated with clinical outcome. Differentially regulated genes and modulated pathways were identified that were associated with recurrent cardiovascular outcomes in first-time AMI patients. This cell-based approach for risk stratification in AMI could represent a novel, non-invasive platform to anticipate modifiable pathways and therapeutic targets to optimize long-term outcome for AMI patients and warrants further study to determine the role of metabolic remodeling and regenerative processes required for optimal outcomes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Characterization and comparison of osteoblasts derived from mouse embryonic stem cells and induced pluripotent stem cells.

PubMed

Ma, Ming-San; Kannan, Vishnu; de Vries, Anneriek E; Czepiel, Marcin; Wesseling, Evelyn M; Balasubramaniyan, Veerakumar; Kuijer, Roel; Vissink, Arjan; Copray, Sjef C V M; Raghoebar, Gerry M

2017-01-01

New developments in stem cell biology offer alternatives for the reconstruction of critical-sized bone defects. One of these developments is the use of induced pluripotent stem (iPS) cells. These stem cells are similar to embryonic stem (ES) cells, but can be generated from adult somatic cells and therefore do not raise ethical concerns. Proper characterization of iPS-derived osteoblasts is important for future development of safe clinical applications of these cells. For this reason, we differentiated mouse ES and iPS cells toward osteoblasts using osteogenic medium and compared their functionality. Immunocytochemical analysis showed significant expression of bone markers (osteocalcin and collagen type I) in osteoblasts differentiated from ES and iPS cells on days 7 and 30. An in vitro mineralization assay confirmed the functionality of osteogenically differentiated ES and iPS cells. Gene expression arrays focusing on osteogenic differentiation were performed in order to compare the gene expression pattern in both differentiated and undifferentiated ES cells and iPS cells. We observed a significant upregulation of osteogenesis-related genes such as Runx2, osteopontin, collagen type I, Tnfsf11, Csf1, and alkaline phosphatase upon osteogenic differentiation of the ES and iPS cells. We further validated the expression of key osteogenic genes Runx2, osteopontin, osteocalcin, collagen type I, and osterix in both differentiated and undifferentiated ES and iPS cells by means of quantified real-time polymerase chain reaction. We conclude that ES and iPS cells are similar in their osteogenic differentiation capacities, as well as in their gene expression patterns.

DOE Office of Scientific and Technical Information (OSTI.GOV)

He, Yonghan; Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3; State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223

Highlights: •Radicicol suppressed intracellular fat accumulation in 3T3-L1 adipocytes. •Radicicol inhibited the expression of FAS and FABP4. •Radicicol blocked cell cycle at the G1-S phase during cell differentiation. •Radicicol inhibited the PDK1/Akt pathway in adipocyte differentiation. -- Abstract: Heat shock protein 90 (Hsp90) is involved in various cellular processes, such as cell proliferation, differentiation and apoptosis. As adipocyte differentiation plays a critical role in obesity development, the present study investigated the effect of an Hsp90 inhibitor radicicol on the differentiation of 3T3-L1 preadipocytes and potential mechanisms. The cells were treated with different concentrations of radicicol during the first 8 daysmore » of cell differentiation. Adipogenesis, the expression of adipogenic transcriptional factors, differentiation makers and cell cycle were determined. It was found that radicicol dose-dependently decreased intracellular fat accumulation through down-regulating the expression of peroxisome proliferator-activated receptor γ (PPAR{sub γ}) and CCAAT element binding protein α (C/EBP{sub α}), fatty acid synthase (FAS) and fatty acid-binding protein 4 (FABP4). Flow cytometry analysis revealed that radicicol blocked cell cycle at G1-S phase. Radicicol redcued the phosphorylation of Akt while showing no effect on β-catenin expression. Radicicol decreased the phosphorylation of phosphoinositide-dependent kinase 1 (PDK1). The results suggest that radicicol inhibited 3T3-L1 preadipocyte differentiation through affecting the PDK1/Akt pathway and subsequent inhibition of mitotic clonal expansion and the expression/activity of adipogenic transcriptional factors and their downstream adipogenic proteins.« less

Cloning, sequencing and expression in MEL cells of a cDNA encoding the mouse ribosomal protein S5.

PubMed

Vanegas, N; Castañeda, V; Santamaría, D; Hernández, P; Schvartzman, J B; Krimer, D B

1997-06-05

We describe the isolation and characterization of a cDNA encoding the mouse S5 ribosomal protein. It was isolated from a MEL (murine erythroleukemia) cell cDNA library by differential hybridization as a down regulated sequence during HMBA-induced differentiation. Northern series analysis showed that S5 mRNA expression is reduced 5-fold throughout the differentiation process. The mouse S5 mRNA is 760 bp long and encodes for a 204 amino acid protein with 94% homology with the human and rat S5.

Hidden among the crowd: differential DNA methylation-expression correlations in cancer occur at important oncogenic pathways.

PubMed

Mosquera Orgueira, Adrián

2015-01-01

DNA methylation is a frequent epigenetic mechanism that participates in transcriptional repression. Variations in DNA methylation with respect to gene expression are constant, and, for unknown reasons, some genes with highly methylated promoters are sometimes overexpressed. In this study we have analyzed the expression and methylation patterns of thousands of genes in five groups of cancer and normal tissue samples in order to determine local and genome-wide differences. We observed significant changes in global methylation-expression correlation in all the neoplasms, which suggests that differential correlation events are frequent in cancer. A focused analysis in the breast cancer cohort identified 1662 genes whose correlation varies significantly between normal and cancerous breast, but whose DNA methylation and gene expression patterns do not change substantially. These genes were enriched in cancer-related pathways and repressive chromatin features across various model cell lines, such as PRC2 binding and H3K27me3 marks. Substantial changes in methylation-expression correlation indicate that these genes are subject to epigenetic remodeling, where the differential activity of other factors break the expected relationship between both variables. Our findings suggest a complex regulatory landscape where a redistribution of local and large-scale chromatin repressive domains at differentially correlated genes (DCGs) creates epigenetic hotspots that modulate cancer-specific gene expression.

Amniotic fluid RNA gene expression profiling provides insights into the phenotype of Turner syndrome.

PubMed

Massingham, Lauren J; Johnson, Kirby L; Scholl, Thomas M; Slonim, Donna K; Wick, Heather C; Bianchi, Diana W

2014-09-01

Turner syndrome is a sex chromosome aneuploidy with characteristic malformations. Amniotic fluid, a complex biological material, could contribute to the understanding of Turner syndrome pathogenesis. In this pilot study, global gene expression analysis of cell-free RNA in amniotic fluid supernatant was utilized to identify specific genes/organ systems that may play a role in Turner syndrome pathophysiology. Cell-free RNA from amniotic fluid of five mid-trimester Turner syndrome fetuses and five euploid female fetuses matched for gestational age was extracted, amplified, and hybridized onto Affymetrix(®) U133 Plus 2.0 arrays. Significantly differentially regulated genes were identified using paired t tests. Biological interpretation was performed using Ingenuity Pathway Analysis and BioGPS gene expression atlas. There were 470 statistically significantly differentially expressed genes identified. They were widely distributed across the genome. XIST was significantly down-regulated (p < 0.0001); SHOX was not differentially expressed. One of the most highly represented organ systems was the hematologic/immune system, distinguishing the Turner syndrome transcriptome from other aneuploidies we previously studied. Manual curation of the differentially expressed gene list identified genes of possible pathologic significance, including NFATC3, IGFBP5, and LDLR. Transcriptomic differences in the amniotic fluid of Turner syndrome fetuses are due to genome-wide dysregulation. The hematologic/immune system differences may play a role in early-onset autoimmune dysfunction. Other genes identified with possible pathologic significance are associated with cardiac and skeletal systems, which are known to be affected in females with Turner syndrome. The discovery-driven approach described here may be useful in elucidating novel mechanisms of disease in Turner syndrome.

Optimization of cDNA microarrays procedures using criteria that do not rely on external standards.

PubMed

Bruland, Torunn; Anderssen, Endre; Doseth, Berit; Bergum, Hallgeir; Beisvag, Vidar; Laegreid, Astrid

2007-10-18

The measurement of gene expression using microarray technology is a complicated process in which a large number of factors can be varied. Due to the lack of standard calibration samples such as are used in traditional chemical analysis it may be a problem to evaluate whether changes done to the microarray procedure actually improve the identification of truly differentially expressed genes. The purpose of the present work is to report the optimization of several steps in the microarray process both in laboratory practices and in data processing using criteria that do not rely on external standards. We performed a cDNA microarry experiment including RNA from samples with high expected differential gene expression termed "high contrasts" (rat cell lines AR42J and NRK52E) compared to self-self hybridization, and optimized a pipeline to maximize the number of genes found to be differentially expressed in the "high contrasts" RNA samples by estimating the false discovery rate (FDR) using a null distribution obtained from the self-self experiment. The proposed high-contrast versus self-self method (HCSSM) requires only four microarrays per evaluation. The effects of blocking reagent dose, filtering, and background corrections methodologies were investigated. In our experiments a dose of 250 ng LNA (locked nucleic acid) dT blocker, no background correction and weight based filtering gave the largest number of differentially expressed genes. The choice of background correction method had a stronger impact on the estimated number of differentially expressed genes than the choice of filtering method. Cross platform microarray (Illumina) analysis was used to validate that the increase in the number of differentially expressed genes found by HCSSM was real. The results show that HCSSM can be a useful and simple approach to optimize microarray procedures without including external standards. Our optimizing method is highly applicable to both long oligo-probe microarrays which have become commonly used for well characterized organisms such as man, mouse and rat, as well as to cDNA microarrays which are still of importance for organisms with incomplete genome sequence information such as many bacteria, plants and fish.

Optimization of cDNA microarrays procedures using criteria that do not rely on external standards

PubMed Central

Bruland, Torunn; Anderssen, Endre; Doseth, Berit; Bergum, Hallgeir; Beisvag, Vidar; Lægreid, Astrid

2007-01-01

Background The measurement of gene expression using microarray technology is a complicated process in which a large number of factors can be varied. Due to the lack of standard calibration samples such as are used in traditional chemical analysis it may be a problem to evaluate whether changes done to the microarray procedure actually improve the identification of truly differentially expressed genes. The purpose of the present work is to report the optimization of several steps in the microarray process both in laboratory practices and in data processing using criteria that do not rely on external standards. Results We performed a cDNA microarry experiment including RNA from samples with high expected differential gene expression termed "high contrasts" (rat cell lines AR42J and NRK52E) compared to self-self hybridization, and optimized a pipeline to maximize the number of genes found to be differentially expressed in the "high contrasts" RNA samples by estimating the false discovery rate (FDR) using a null distribution obtained from the self-self experiment. The proposed high-contrast versus self-self method (HCSSM) requires only four microarrays per evaluation. The effects of blocking reagent dose, filtering, and background corrections methodologies were investigated. In our experiments a dose of 250 ng LNA (locked nucleic acid) dT blocker, no background correction and weight based filtering gave the largest number of differentially expressed genes. The choice of background correction method had a stronger impact on the estimated number of differentially expressed genes than the choice of filtering method. Cross platform microarray (Illumina) analysis was used to validate that the increase in the number of differentially expressed genes found by HCSSM was real. Conclusion The results show that HCSSM can be a useful and simple approach to optimize microarray procedures without including external standards. Our optimizing method is highly applicable to both long oligo-probe microarrays which have become commonly used for well characterized organisms such as man, mouse and rat, as well as to cDNA microarrays which are still of importance for organisms with incomplete genome sequence information such as many bacteria, plants and fish. PMID:17949480

SARTools: A DESeq2- and EdgeR-Based R Pipeline for Comprehensive Differential Analysis of RNA-Seq Data.

PubMed

Varet, Hugo; Brillet-Guéguen, Loraine; Coppée, Jean-Yves; Dillies, Marie-Agnès

2016-01-01

Several R packages exist for the detection of differentially expressed genes from RNA-Seq data. The analysis process includes three main steps, namely normalization, dispersion estimation and test for differential expression. Quality control steps along this process are recommended but not mandatory, and failing to check the characteristics of the dataset may lead to spurious results. In addition, normalization methods and statistical models are not exchangeable across the packages without adequate transformations the users are often not aware of. Thus, dedicated analysis pipelines are needed to include systematic quality control steps and prevent errors from misusing the proposed methods. SARTools is an R pipeline for differential analysis of RNA-Seq count data. It can handle designs involving two or more conditions of a single biological factor with or without a blocking factor (such as a batch effect or a sample pairing). It is based on DESeq2 and edgeR and is composed of an R package and two R script templates (for DESeq2 and edgeR respectively). Tuning a small number of parameters and executing one of the R scripts, users have access to the full results of the analysis, including lists of differentially expressed genes and a HTML report that (i) displays diagnostic plots for quality control and model hypotheses checking and (ii) keeps track of the whole analysis process, parameter values and versions of the R packages used. SARTools provides systematic quality controls of the dataset as well as diagnostic plots that help to tune the model parameters. It gives access to the main parameters of DESeq2 and edgeR and prevents untrained users from misusing some functionalities of both packages. By keeping track of all the parameters of the analysis process it fits the requirements of reproducible research.

Transcriptome-wide analysis supports environmental adaptations of two Pinus pinaster populations from contrasting habitats.

PubMed

Cañas, Rafael A; Feito, Isabel; Fuente-Maqueda, José Francisco; Ávila, Concepción; Majada, Juan; Cánovas, Francisco M

2015-11-06

Maritime pine (Pinus pinaster Aiton) grows in a range of different climates in the southwestern Mediterranean region and the existence of a variety of latitudinal ecotypes or provenances is well established. In this study, we have conducted a deep analysis of the transcriptome in needles from two P. pinaster provenances, Leiria (Portugal) and Tamrabta (Morocco), which were grown in northern Spain under the same conditions. An oligonucleotide microarray (PINARRAY3) and RNA-Seq were used for whole-transcriptome analyses, and we found that 90.95% of the data were concordant between the two platforms. Furthermore, the two methods identified very similar percentages of differentially expressed genes with values of 5.5% for PINARRAY3 and 5.7% for RNA-Seq. In total, 6,023 transcripts were shared and 88 differentially expressed genes overlapped in the two platforms. Among the differentially expressed genes, all transport related genes except aquaporins were expressed at higher levels in Tamrabta than in Leiria. In contrast, genes involved in secondary metabolism were expressed at higher levels in Tamrabta, and photosynthesis-related genes were expressed more highly in Leiria. The genes involved in light sensing in plants were well represented in the differentially expressed groups of genes. In addition, increased levels of hormones such as abscisic acid, gibberellins, jasmonic and salicylic acid were observed in Leiria. Both transcriptome platforms have proven to be useful resources, showing complementary and reliable results. The results presented here highlight the different abilities of the two maritime pine populations to sense environmental conditions and reveal one type of regulation that can be ascribed to different genetic and epigenetic backgrounds.

Genome-Wide Identification of the PHD-Finger Family Genes and Their Responses to Environmental Stresses in Oryza sativa L.

PubMed Central

Sun, Mingzhe; Yang, Junkai; Cui, Na; Zhu, Yanming

2017-01-01

The PHD-finger family has been demonstrated to be involved in regulating plant growth and development. However, little information is given for its role in environmental stress responses. Here, we identified a total of 59 PHD family genes in the rice genome. These OsPHDs genes were located on eleven chromosomes and synteny analysis only revealed nine duplicated pairs within the rice PHD family. Phylogenetic analysis of all OsPHDs and PHDs from other species revealed that they could be grouped into two major clusters. Furthermore, OsPHDs were clustered into eight groups and members from different groups displayed a great divergence in terms of gene structure, functional domains and conserved motifs. We also found that with the exception of OsPHD6, all OsPHDs were expressed in at least one of the ten tested tissues and OsPHDs from certain groups were expressed in specific tissues. Moreover, our results also uncovered differential responses of OsPHDs expression to environmental stresses, including ABA (abscisic acid), water deficit, cold and high Cd. By using quantitative real-time PCR, we further confirmed the differential expression of OsPHDs under these stresses. OsPHD1/7/8/13/33 were differentially expressed under water deficit and Cd stresses, while OsPHD5/17 showed altered expression under water deficit and cold stresses. Moreover, OsPHD3/44/28 displayed differential expression under ABA and Cd stresses. In conclusion, our results provide valuable information on the rice PHD family in plant responses to environmental stress, which will be helpful for further characterizing their biological roles in responding to environmental stresses.

Four-miRNA signature as a prognostic tool for lung adenocarcinoma.

PubMed

Lin, Yan; Lv, Yufeng; Liang, Rong; Yuan, Chunling; Zhang, Jinyan; He, Dan; Zheng, Xiaowen; Zhang, Jianfeng

2018-01-01

The aim of this study was to generate a novel miRNA expression signature to accurately predict prognosis for patients with lung adenocarcinoma (LUAD). Using expression profiles downloaded from The Cancer Genome Atlas database, we identified multiple miRNAs with differential expression between LUAD and paired healthy tissues. We then evaluated the prognostic values of the differentially expressed miRNAs using univariate/multivariate Cox regression analysis. This analysis was ultimately used to construct a four-miRNA signature that effectively predicted patient survival. Finally, we analyzed potential functional roles of the target genes for these four miRNAs using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. Based on our cutoff criteria ( P <0.05 and |log2FC| >1.0), we identified a total of 187 differentially expressed miRNAs, including 148 that were upregulated in LUAD tissues and 39 that were downregulated. Four miRNAs (miR-148a-5p, miR-31-5p, miR-548v, and miR-550a-5p) were independently associated with survival based on Kaplan-Meier analysis. We generated a signature index based on the expression of these four miRNAs and stratified patients into low- and high-risk groups. Patients in the high-risk group had significantly shorter survival times than those in the low-risk group ( P =0.002). A functional enrichment analysis suggested that the target genes of these four miRNAs were involved in protein phosphorylation and the Hippo and sphingolipid signaling pathways. Taken together, our results suggest that our four-miRNA signature can be used as a prognostic tool for patients with LUAD.

Comparative Profiling of Primary Colorectal Carcinomas and Liver Metastases Identifies LEF1 as a Prognostic Biomarker

PubMed Central

Lin, Albert Y.; Chua, Mei-Sze; Choi, Yoon-La; Yeh, William; Kim, Young H.; Azzi, Raymond; Adams, Gregg A.; Sainani, Kristin; van de Rijn, Matt; So, Samuel K.; Pollack, Jonathan R.

2011-01-01

Purpose We sought to identify genes of clinical significance to predict survival and the risk for colorectal liver metastasis (CLM), the most common site of metastasis from colorectal cancer (CRC). Patients and Methods We profiled gene expression in 31 specimens from primary CRC and 32 unmatched specimens of CLM, and performed Significance Analysis of Microarrays (SAM) to identify genes differentially expressed between these two groups. To characterize the clinical relevance of two highly-ranked differentially-expressed genes, we analyzed the expression of secreted phosphoprotein 1 (SPP1 or osteopontin) and lymphoid enhancer factor-1 (LEF1) by immunohistochemistry using a tissue microarray (TMA) representing an independent set of 154 patients with primary CRC. Results Supervised analysis using SAM identified 963 genes with significantly higher expression in CLM compared to primary CRC, with a false discovery rate of <0.5%. TMA analysis showed SPP1 and LEF1 protein overexpression in 60% and 44% of CRC cases, respectively. Subsequent occurrence of CLM was significantly correlated with the overexpression of LEF1 (chi-square p = 0.042), but not SPP1 (p = 0.14). Kaplan Meier analysis revealed significantly worse survival in patients with overexpression of LEF1 (p<0.01), but not SPP1 (p = 0.11). Both univariate and multivariate analyses identified stage (p<0.0001) and LEF1 overexpression (p<0.05) as important prognostic markers, but not tumor grade or SPP1. Conclusion Among genes differentially expressed between CLM and primary CRC, we demonstrate overexpression of LEF1 in primary CRC to be a prognostic factor for poor survival and increased risk for liver metastasis. PMID:21383983

Comprehensive processing of high-throughput small RNA sequencing data including quality checking, normalization, and differential expression analysis using the UEA sRNA Workbench

PubMed Central

Beckers, Matthew; Mohorianu, Irina; Stocks, Matthew; Applegate, Christopher; Dalmay, Tamas; Moulton, Vincent

2017-01-01

Recently, high-throughput sequencing (HTS) has revealed compelling details about the small RNA (sRNA) population in eukaryotes. These 20 to 25 nt noncoding RNAs can influence gene expression by acting as guides for the sequence-specific regulatory mechanism known as RNA silencing. The increase in sequencing depth and number of samples per project enables a better understanding of the role sRNAs play by facilitating the study of expression patterns. However, the intricacy of the biological hypotheses coupled with a lack of appropriate tools often leads to inadequate mining of the available data and thus, an incomplete description of the biological mechanisms involved. To enable a comprehensive study of differential expression in sRNA data sets, we present a new interactive pipeline that guides researchers through the various stages of data preprocessing and analysis. This includes various tools, some of which we specifically developed for sRNA analysis, for quality checking and normalization of sRNA samples as well as tools for the detection of differentially expressed sRNAs and identification of the resulting expression patterns. The pipeline is available within the UEA sRNA Workbench, a user-friendly software package for the processing of sRNA data sets. We demonstrate the use of the pipeline on a H. sapiens data set; additional examples on a B. terrestris data set and on an A. thaliana data set are described in the Supplemental Information. A comparison with existing approaches is also included, which exemplifies some of the issues that need to be addressed for sRNA analysis and how the new pipeline may be used to do this. PMID:28289155

Comparative Analysis of AhR-Mediated TCDD-Elicited Gene Expression in Human Liver Adult Stem Cells

PubMed Central

Kim, Suntae; Dere, Edward; Burgoon, Lyle D.; Chang, Chia-Cheng; Zacharewski, Timothy R.

2009-01-01

Time course and dose-response studies were conducted in HL1-1 cells, a human liver cell line with stem cell–like characteristics, to assess the differential gene expression elicited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) compared with other established models. Cells were treated with 0.001, 0.01, 0.1, 1, 10, or 100nM TCDD or dimethyl sulfoxide vehicle control for 12 h for the dose-response study, or with 10nM TCDD or vehicle for 1, 2, 4, 8, 12, 24, or 48 h for the time course study. Elicited changes were monitored using a human cDNA microarray with 6995 represented genes. Empirical Bayes analysis identified 144 genes differentially expressed at one or more time points following treatment. Most genes exhibited dose-dependent responses including CYP1A1, CYP1B1, ALDH1A3, and SLC7A5 genes. Comparative analysis of HL1-1 differential gene expression to human HepG2 data identified 74 genes with comparable temporal expression profiles including 12 putative primary responses. HL1-1–specific changes were related to lipid metabolism and immune responses, consistent with effects elicited in vivo. Furthermore, comparative analysis of HL1-1 cells with mouse Hepa1c1c7 hepatoma cell lines and C57BL/6 hepatic tissue identified 18 and 32 commonly regulated orthologous genes, respectively, with functions associated with signal transduction, transcriptional regulation, metabolism and transport. Although some common pathways are affected, the results suggest that TCDD elicits species- and model-specific gene expression profiles. PMID:19684285

Searching for non-transposable targets of planarian nuclear PIWI in pluripotent stem cells and differentiated cells.

PubMed

Kashima, Makoto; Agata, Kiyokazu; Shibata, Norito

2018-06-01

Nuclear PIWIs together with their guide RNAs (piRNAs) epigenetically silence various genes including transposons in many organisms. In planarians, the nuclear piwi family gene, DjpiwiB is specifically transcribed in adult pluripotent stem cells (adult PSC, neoblast), but not in differentiated cells. However, the protein accumulates in the nuclei of both neoblasts and their descendant differentiated cells. Interestingly, PIWI(DjPiwiB)-piRNA complexes are indispensable for the repression of transposable genes at the onset of differentiation from neoblasts. Here, we conducted a comparative transcriptome analysis between control and DjpiwiB(RNAi) animals to identify non-transposable target genes of the DjPiwiB-piRNA complexes. Using bioinformatic analyses and RNAi we demonstrate that DjPiwiB-piRNA complexes are required for the proper expression of Djmcm2 and Djhistone h4 in neoblasts and that DjPiwiB-piRNA complexes regulate the transient expression of Djcalu during neoblast differentiation. Thus, DjPiwiB-piRNA complexes regulate the correct expression patterns during neoblast self-renewal and differentiation. © 2018 Japanese Society of Developmental Biologists.

Thyroid paraganglioma. Report of 3 cases and description of an immunohistochemical profile useful in the differential diagnosis with medullary thyroid carcinoma, based on complementary DNA array results.

PubMed

Castelblanco, Esmeralda; Gallel, Pilar; Ros, Susana; Gatius, Sonia; Valls, Joan; De-Cubas, Aguirre A; Maliszewska, Agnieszka; Yebra-Pimentel, M Teresa; Menarguez, Javier; Gamallo, Carlos; Opocher, Giuseppe; Robledo, Mercedes; Matias-Guiu, Xavier

2012-07-01

Thyroid paraganglioma is a rare disorder that sometimes poses problems in differential diagnosis with medullary thyroid carcinoma. So far, differential diagnosis is solved with the help of some markers that are frequently expressed in medullary thyroid carcinoma (thyroid transcription factor 1, calcitonin, and carcinoembryonic antigen). However, some of these markers are not absolutely specific of medullary thyroid carcinoma and may be expressed in other tumors. Here we report 3 new cases of thyroid paraganglioma and describe our strategy to design a diagnostic immunohistochemical battery. First, we performed a comparative analysis of the expression profile of head and neck paragangliomas and medullary thyroid carcinoma, obtained after complementary DNA array analysis of 2 series of fresh-frozen samples of paragangliomas and medullary thyroid carcinoma, respectively. Seven biomarkers showing differential expression were selected (nicotinamide adenine dinucleotide dehydrogenase 1 alpha subcomplex, 4-like 2, NDUFA4L2; cytochrome c oxidase subunit IV isoform 2; vesicular monoamine transporter 2; calcitonin gene-related protein/calcitonin; carcinoembryonic antigen; and thyroid transcription factor 1) for immunohistochemical analysis. Two tissue microarrays were constructed from 2 different series of paraffin-embedded samples of paragangliomas and medullary thyroid carcinoma. We provide a classifying rule for differential diagnosis that combines negativity or low staining for calcitonin gene-related protein (histologic score, <10) or calcitonin (histologic score, <50) together with positivity of any of NADH dehydrogenase 1 alpha subcomplex, 4-like 2; cytochrome c oxidase subunit IV isoform 2; or vesicular monoamine transporter 2 to predict paragangliomas, showing a prediction error of 0%. Finally, the immunohistochemical battery was checked in paraffin-embedded blocks from 4 examples of thyroid paraganglioma (1 previously reported case and 3 new cases), showing also a prediction error of 0%. Our results suggest that the comparative expression profile, obtained by complementary DNA arrays, seems to be a good tool to design immunohistochemical batteries used in differential diagnosis. Copyright © 2012 Elsevier Inc. All rights reserved.

Integrated Phloem Sap mRNA and Protein Expression Analysis Reveals Phytoplasma-infection Responses in Mulberry.

PubMed

Gai, Yingping; Yuan, Shuo-Shuo; Liu, Zhao-Yang; Zhao, Huai-Ning; Liu, Qi; Qin, Yong-Li; Fang, Li-Jing; Ji, Xian-Ling

2018-05-30

To gain insight into the response of mulberry to phytoplasma-infection, the expression profiles of mRNAs and proteins in mulberry phloem sap were examined. A total of 955 unigenes and 136 proteins were found to be differentially expressed between the healthy and infected phloem sap. These differentially expressed mRNAs and proteins are involved in signalling, hormone metabolism, stress responses, etc. Interestingly, we found that both the mRNA and protein levels of the major latex protein-like 329 ( MuMLPL329 ) gene were increased in the infected phloem saps. Expression of the MuMLPL329 gene was induced by pathogen inoculation and was responsive to jasmonic acid. Ectopic expression of MuMLPL329 in Arabidopsis enhances transgenic plant resistance to Botrytis cinerea, Pseudomonas syringae pv tomato DC3000 ( Pst. DC3000 ) and phytoplasma. Further analysis revealed that MuMLPL329 can enhance the expression of some defense genes and might be involved in altering flavonoid content resulting in increased resistance of plants to pathogen infection. Finally, the roles of the differentially expressed mRNAs and proteins and the potential molecular mechanisms of their changes were discussed. It was likely that the phytoplasma-responsive mRNAs and proteins in the phloem saps were involved in multiple pathways of mulberry responses to phytoplasma-infection, and their changes may be partially responsible for some symptoms in the phytoplasma infected plants. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Integrated Analysis of Alzheimer's Disease and Schizophrenia Dataset Revealed Different Expression Pattern in Learning and Memory.

PubMed

Li, Wen-Xing; Dai, Shao-Xing; Liu, Jia-Qian; Wang, Qian; Li, Gong-Hua; Huang, Jing-Fei

2016-01-01

Alzheimer's disease (AD) and schizophrenia (SZ) are both accompanied by impaired learning and memory functions. This study aims to explore the expression profiles of learning or memory genes between AD and SZ. We downloaded 10 AD and 10 SZ datasets from GEO-NCBI for integrated analysis. These datasets were processed using RMA algorithm and a global renormalization for all studies. Then Empirical Bayes algorithm was used to find the differentially expressed genes between patients and controls. The results showed that most of the differentially expressed genes were related to AD whereas the gene expression profile was little affected in the SZ. Furthermore, in the aspects of the number of differentially expressed genes, the fold change and the brain region, there was a great difference in the expression of learning or memory related genes between AD and SZ. In AD, the CALB1, GABRA5, and TAC1 were significantly downregulated in whole brain, frontal lobe, temporal lobe, and hippocampus. However, in SZ, only two genes CRHBP and CX3CR1 were downregulated in hippocampus, and other brain regions were not affected. The effect of these genes on learning or memory impairment has been widely studied. It was suggested that these genes may play a crucial role in AD or SZ pathogenesis. The different gene expression patterns between AD and SZ on learning and memory functions in different brain regions revealed in our study may help to understand the different mechanism between two diseases.

Environmental history impacts gene expression during diapause development in the alfalfa leafcutting bee, Megachile rotundata.

PubMed

Yocum, George D; Childers, Anna K; Rinehart, Joseph P; Rajamohan, Arun; Pitts-Singer, Theresa L; Greenlee, Kendra J; Bowsher, Julia H

2018-05-10

Our understanding of the mechanisms controlling insect diapause has increased dramatically with the introduction of global gene expression techniques, such as RNA-seq. However, little attention has been given to how ecologically relevant field conditions may affect gene expression during diapause development because previous studies have focused on laboratory reared and maintained insects. To determine whether gene expression differs between laboratory and field conditions, prepupae of the alfalfa leafcutting bee, Megachile rotundata , entering diapause early or late in the growing season were collected. These two groups were further subdivided in early autumn into laboratory and field maintained groups, resulting in four experimental treatments of diapausing prepupae: early and late field, and early and late laboratory. RNA-seq and differential expression analyses were performed on bees from the four treatment groups in November, January, March and May. The number of treatment-specific differentially expressed genes (97 to 1249) outnumbered the number of differentially regulated genes common to all four treatments (14 to 229), indicating that exposure to laboratory or field conditions had a major impact on gene expression during diapause development. Principle component analysis and hierarchical cluster analysis yielded similar grouping of treatments, confirming that the treatments form distinct clusters. Our results support the conclusion that gene expression during the course of diapause development is not a simple ordered sequence, but rather a highly plastic response determined primarily by the environmental history of the individual insect. © 2018. Published by The Company of Biologists Ltd.

Profound Effect of Profiling Platform and Normalization Strategy on Detection of Differentially Expressed MicroRNAs – A Comparative Study

PubMed Central

Meyer, Swanhild U.; Kaiser, Sebastian; Wagner, Carola; Thirion, Christian; Pfaffl, Michael W.

2012-01-01

Background Adequate normalization minimizes the effects of systematic technical variations and is a prerequisite for getting meaningful biological changes. However, there is inconsistency about miRNA normalization performances and recommendations. Thus, we investigated the impact of seven different normalization methods (reference gene index, global geometric mean, quantile, invariant selection, loess, loessM, and generalized procrustes analysis) on intra- and inter-platform performance of two distinct and commonly used miRNA profiling platforms. Methodology/Principal Findings We included data from miRNA profiling analyses derived from a hybridization-based platform (Agilent Technologies) and an RT-qPCR platform (Applied Biosystems). Furthermore, we validated a subset of miRNAs by individual RT-qPCR assays. Our analyses incorporated data from the effect of differentiation and tumor necrosis factor alpha treatment on primary human skeletal muscle cells and a murine skeletal muscle cell line. Distinct normalization methods differed in their impact on (i) standard deviations, (ii) the area under the receiver operating characteristic (ROC) curve, (iii) the similarity of differential expression. Loess, loessM, and quantile analysis were most effective in minimizing standard deviations on the Agilent and TLDA platform. Moreover, loess, loessM, invariant selection and generalized procrustes analysis increased the area under the ROC curve, a measure for the statistical performance of a test. The Jaccard index revealed that inter-platform concordance of differential expression tended to be increased by loess, loessM, quantile, and GPA normalization of AGL and TLDA data as well as RGI normalization of TLDA data. Conclusions/Significance We recommend the application of loess, or loessM, and GPA normalization for miRNA Agilent arrays and qPCR cards as these normalization approaches showed to (i) effectively reduce standard deviations, (ii) increase sensitivity and accuracy of differential miRNA expression detection as well as (iii) increase inter-platform concordance. Results showed the successful adoption of loessM and generalized procrustes analysis to one-color miRNA profiling experiments. PMID:22723911

MUC4, a novel immunohistochemical marker identified by gene expression profiling, differentiates pleural sarcomatoid mesothelioma from lung sarcomatoid carcinoma.

PubMed

Amatya, Vishwa Jeet; Kushitani, Kei; Mawas, Amany Sayed; Miyata, Yoshihiro; Okada, Morihito; Kishimoto, Takumi; Inai, Kouki; Takeshima, Yukio

2017-05-01

Sarcomatoid mesothelioma, a histological subtype of malignant pleural mesothelioma, is a very aggressive tumor with a poor prognosis. Histological diagnosis of sarcomatoid mesothelioma largely depends on the histomorphological feature of spindled tumor cells with immunohistochemical reactivity to cytokeratins. Diagnosis also requires clinico-radiological and/or macroscopic evidence of an extrapulmonary location to differentiate it from lung sarcomatoid carcinoma. Although there are promising immunohistochemical antibody panels to differentiate mesothelioma from lung carcinoma, a consensus on the immunohistochemical markers that distinguish sarcomatoid mesothelioma from lung sarcomatoid carcinoma has not been reached and requires further study. We performed whole gene expression analysis of formalin-fixed paraffin-embedded tissue from sarcomatoid mesothelioma and lung sarcomatoid carcinoma and observed significant differences in the expression of MUC4 and other genes between sarcomatoid mesothelioma and lung sarcomatoid carcinoma. Immunohistochemistry demonstrated that MUC4 was expressed in the spindled tumor cells of lung sarcomatoid carcinoma (21/29, 72%) but was not expressed in any sarcomatoid mesothelioma (0/31, 0%). To differentiate sarcomatoid mesothelioma from lung sarcomatoid carcinoma, negative MUC4 expression showed 100% sensitivity and 72% specificity and accuracy rate of 87%, which is higher than immunohistochemical markers such as calretinin, D2-40 and Claudin-4. Therefore, we recommend to include MUC4 as a novel and useful negative immunohistochemical marker for differentiating sarcomatoid mesothelioma from lung sarcomatoid carcinoma.

Notch signaling pathways in human thoracic ossification of the ligamentum flavum.

PubMed

Qu, Xiaochen; Chen, Zhongqiang; Fan, Dongwei; Sun, Chuiguo; Zeng, Yan; Hou, Xiaofei; Ning, Shanglong

2016-08-01

This study investigated the pathological process of Notch signaling in the osteogenesis of ligamentum flavum tissues and cells, and the associated regulatory mechanisms. Notch receptors, ligands, and target genes were identified by quantitative polymerase chain reaction (qPCR) in ligamentum flavum cells and immunohistochemistry in ligamentum flavum sections from ossification of the ligamentum flavum (OLF) patients and controls. The temporospatial expression patterns of JAG1/Notch2/HES1 in human ligamentum flavum cells during osteogenic differentiation were determined by qPCR. Lentiviral vectors for Notch2 overexpression and knockdown were constructed and transfected into ligamentum flavum cells before osteogenic differentiation to examine the function of Notch signaling pathways in the osteogenic differentiation of ligamentum flavum cells. Alkaline phosphatase, Runx2, Osterix, osteocalcin, and osteopontin mRNA levels, alkaline phosphatase activity, and Alizarin Red staining were used as indicators of osteogenic differentiation. JAG1/Notch2/HES1 mRNA levels were up-regulated in ligamentum flavum cells from OLF patients, which increased during osteogenic differentiation. Immunohistochemical analysis suggested positive Notch2 expression at the ossification front. Down-regulation of Notch2 expression decelerated osteogenic differentiation of ligamentum flavum cells, and Notch2 overexpression promoted osteogenic differentiation of ligamentum flavum cells. Expression of Runx2 and Osterix increased in a manner similar to that of Notch2 during osteogenic differentiation of ligamentum flavum cells, and Notch2 knockdown and overexpression influenced their expression levels. Notch signaling plays an important role in OLF, and Notch may affect the osteogenic differentiation of ligamentum flavum cells via interactions with Runx2 and Osterix.© 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1481-1491, 2016. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Constitutive Uncoupling of Pathways of Gene Expression That Control Growth and Differentiation in Myeloid Leukemia: A Model for the Origin and Progression of Malignancy

NASA Astrophysics Data System (ADS)

Sachs, Leo

1980-10-01

Chemical carcinogens and tumor promoters have pleiotropic effects. Tumor initiators can produce a variety of mutations and tumor promoters can regulate a variety of physiological molecules that control growth and differentiation. The appropriate mutation and the regulation of the appropriate molecules to induce cell growth can initiate and promote the sequence of changes required for transformation of normal cells into malignant cells. After this sequence of changes, some tumors can still be induced to revert with a high frequency from a malignant phenotype to a nonmalignant phenotype. Results obtained from analysis of regulation of growth and differentiation in normal and leukemic myeloid cells, the phenotypic reversion of malignancy by induction of normal differentiation in myeloid leukemia, and the blocks in differentiation-defective leukemic cell mutants have been used to propose a general model for the origin and progression of malignancy. The model states that malignancy originates by changing specific pathways of gene expression required for growth from inducible to constitutive in cells that can still be induced to differentiate normally by the physiological inducer of differentiation. The malignant cells, unlike the normal cells, then no longer require the physiological inducer for growth. This changes the requirements for growth and uncouples growth from differentiation. Constitutive expression of other specific pathways can uncouple other controls, which then causes blocks in differentiation and the further progression of malignancy. The existence of specific constitutive pathways of gene expression that uncouple controls in malignant cells can also explain the expression of fetal proteins, hormones, and some other specialized products of normal development in various types of tumors.

Malignant pleural mesothelioma and mesothelial hyperplasia: A new molecular tool for the differential diagnosis.

PubMed

Bruno, Rossella; Alì, Greta; Giannini, Riccardo; Proietti, Agnese; Lucchi, Marco; Chella, Antonio; Melfi, Franca; Mussi, Alfredo; Fontanini, Gabriella

2017-01-10

Malignant pleural mesothelioma (MPM) is a rare asbestos related cancer, aggressive and unresponsive to therapies. Histological examination of pleural lesions is the gold standard of MPM diagnosis, although it is sometimes hard to discriminate the epithelioid type of MPM from benign mesothelial hyperplasia (MH).This work aims to define a new molecular tool for the differential diagnosis of MPM, using the expression profile of 117 genes deregulated in this tumour.The gene expression analysis was performed by nanoString System on tumour tissues from 36 epithelioid MPM and 17 MH patients, and on 14 mesothelial pleural samples analysed in a blind way. Data analysis included raw nanoString data normalization, unsupervised cluster analysis by Pearson correlation, non-parametric Mann Whitney U-test and molecular classification by the Uncorrelated Shrunken Centroid (USC) Algorithm.The Mann-Whitney U-test found 35 genes upregulated and 31 downregulated in MPM. The unsupervised cluster analysis revealed two clusters, one composed only of MPM and one only of MH samples, thus revealing class-specific gene profiles. The Uncorrelated Shrunken Centroid algorithm identified two classifiers, one including 22 genes and the other 40 genes, able to properly classify all the samples as benign or malignant using gene expression data; both classifiers were also able to correctly determine, in a blind analysis, the diagnostic categories of all the 14 unknown samples.In conclusion we delineated a diagnostic tool combining molecular data (gene expression) and computational analysis (USC algorithm), which can be applied in the clinical practice for the differential diagnosis of MPM.

Alternative splicing and differential gene expression in colon cancer detected by a whole genome exon array

PubMed Central

Gardina, Paul J; Clark, Tyson A; Shimada, Brian; Staples, Michelle K; Yang, Qing; Veitch, James; Schweitzer, Anthony; Awad, Tarif; Sugnet, Charles; Dee, Suzanne; Davies, Christopher; Williams, Alan; Turpaz, Yaron

2006-01-01

Background Alternative splicing is a mechanism for increasing protein diversity by excluding or including exons during post-transcriptional processing. Alternatively spliced proteins are particularly relevant in oncology since they may contribute to the etiology of cancer, provide selective drug targets, or serve as a marker set for cancer diagnosis. While conventional identification of splice variants generally targets individual genes, we present here a new exon-centric array (GeneChip Human Exon 1.0 ST) that allows genome-wide identification of differential splice variation, and concurrently provides a flexible and inclusive analysis of gene expression. Results We analyzed 20 paired tumor-normal colon cancer samples using a microarray designed to detect over one million putative exons that can be virtually assembled into potential gene-level transcripts according to various levels of prior supporting evidence. Analysis of high confidence (empirically supported) transcripts identified 160 differentially expressed genes, with 42 genes occupying a network impacting cell proliferation and another twenty nine genes with unknown functions. A more speculative analysis, including transcripts based solely on computational prediction, produced another 160 differentially expressed genes, three-fourths of which have no previous annotation. We also present a comparison of gene signal estimations from the Exon 1.0 ST and the U133 Plus 2.0 arrays. Novel splicing events were predicted by experimental algorithms that compare the relative contribution of each exon to the cognate transcript intensity in each tissue. The resulting candidate splice variants were validated with RT-PCR. We found nine genes that were differentially spliced between colon tumors and normal colon tissues, several of which have not been previously implicated in cancer. Top scoring candidates from our analysis were also found to substantially overlap with EST-based bioinformatic predictions of alternative splicing in cancer. Conclusion Differential expression of high confidence transcripts correlated extremely well with known cancer genes and pathways, suggesting that the more speculative transcripts, largely based solely on computational prediction and mostly with no previous annotation, might be novel targets in colon cancer. Five of the identified splicing events affect mediators of cytoskeletal organization (ACTN1, VCL, CALD1, CTTN, TPM1), two affect extracellular matrix proteins (FN1, COL6A3) and another participates in integrin signaling (SLC3A2). Altogether they form a pattern of colon-cancer specific alterations that may particularly impact cell motility. PMID:17192196

Genome-Wide Identification and Expression Analysis of the Mitogen-Activated Protein Kinase Gene Family in Cassava

PubMed Central

Yan, Yan; Wang, Lianzhe; Ding, Zehong; Tie, Weiwei; Ding, Xupo; Zeng, Changying; Wei, Yunxie; Zhao, Hongliang; Peng, Ming; Hu, Wei

2016-01-01

Mitogen-activated protein kinases (MAPKs) play central roles in plant developmental processes, hormone signaling transduction, and responses to abiotic stress. However, no data are currently available about the MAPK family in cassava, an important tropical crop. Herein, 21 MeMAPK genes were identified from cassava. Phylogenetic analysis indicated that MeMAPKs could be classified into four subfamilies. Gene structure analysis demonstrated that the number of introns in MeMAPK genes ranged from 1 to 10, suggesting large variation among cassava MAPK genes. Conserved motif analysis indicated that all MeMAPKs had typical protein kinase domains. Transcriptomic analysis suggested that MeMAPK genes showed differential expression patterns in distinct tissues and in response to drought stress between wild subspecies and cultivated varieties. Interaction networks and co-expression analyses revealed that crucial pathways controlled by MeMAPK networks may be involved in the differential response to drought stress in different accessions of cassava. Expression of nine selected MAPK genes showed that these genes could comprehensively respond to osmotic, salt, cold, oxidative stressors, and abscisic acid (ABA) signaling. These findings yield new insights into the transcriptional control of MAPK gene expression, provide an improved understanding of abiotic stress responses and signaling transduction in cassava, and lead to potential applications in the genetic improvement of cassava cultivars. PMID:27625666

Computations involving differential operators and their actions on functions

NASA Technical Reports Server (NTRS)

Crouch, Peter E.; Grossman, Robert; Larson, Richard

1991-01-01

The algorithms derived by Grossmann and Larson (1989) are further developed for rewriting expressions involving differential operators. The differential operators involved arise in the local analysis of nonlinear dynamical systems. These algorithms are extended in two different directions: the algorithms are generalized so that they apply to differential operators on groups and the data structures and algorithms are developed to compute symbolically the action of differential operators on functions. Both of these generalizations are needed for applications.

Sexual dimorphic floral development in dioecious plants revealed by transcriptome, phytohormone, and DNA methylation analysis in Populus tomentosa.

PubMed

Song, Yuepeng; Ma, Kaifeng; Ci, Dong; Chen, Qingqing; Tian, Jiaxing; Zhang, Deqiang

2013-12-01

Dioecious plants have evolved sex-specific floral development mechanisms. However, the precise gene expression patterns in dioecious plant flower development remain unclear. Here, we used andromonoecious poplar, an exceptional model system, to eliminate the confounding effects of genetic background of dioecious plants. Comparative transcriptome and physiological analysis allowed us to characterize sex-specific development of female and male flowers. Transcriptome analysis identified genes significantly differentially expressed between the sexes, including genes related to floral development, phytohormone synthesis and metabolism, and DNA methylation. Correlation analysis revealed a significant correlation between phytohormone signaling and gene expression, identifying specific phytohormone-responsive genes and their cis-regulatory elements. Two genes related to DNA methylation, METHYLTRANSFERASE1 (MET1) and DECREASED DNA METHYLATION 1 (DDM1), which are located in the sex determination region of Chromosome XIX, have differential expression between female and male flowers. A time-course analysis revealed that MET1 and DDM1 expression may produce different DNA methylation levels in female and male flowers. Understanding the interactions of phytohormone signaling, DNA methylation and target gene expression should lead to a better understanding of sexual differences in floral development. Thus, this study identifies a set of candidate genes for further studies of poplar sexual dimorphism and relates sex-specific floral development to physiological and epigenetic changes.

Gene expression profiling in multipotent DFAT cells derived from mature adipocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ono, Hiromasa; Database Center for Life Science; Oki, Yoshinao

2011-04-15

Highlights: {yields} Adipocyte dedifferentiation is evident in a significant decrease in typical genes. {yields} Cell proliferation is strongly related to adipocyte dedifferentiation. {yields} Dedifferentiated adipocytes express several lineage-specific genes. {yields} Comparative analyses using publicly available datasets boost the interpretation. -- Abstract: Cellular dedifferentiation signifies the withdrawal of cells from a specific differentiated state to a stem cell-like undifferentiated state. However, the mechanism of dedifferentiation remains obscure. Here we performed comparative transcriptome analyses during dedifferentiation in mature adipocytes (MAs) to identify the transcriptional signatures of multipotent dedifferentiated fat (DFAT) cells derived from MAs. Using microarray systems, we explored similarly expressed asmore » well as significantly differentially expressed genes in MAs during dedifferentiation. This analysis revealed significant changes in gene expression during this process, including a significant reduction in expression of genes for lipid metabolism concomitantly with a significant increase in expression of genes for cell movement, cell migration, tissue developmental processes, cell growth, cell proliferation, cell morphogenesis, altered cell shape, and cell differentiation. Our observations indicate that the transcriptional signatures of DFAT cells derived from MAs are summarized in terms of a significant decrease in functional phenotype-related genes and a parallel increase in cell proliferation, altered cell morphology, and regulation of the differentiation of related genes. A better understanding of the mechanisms involved in dedifferentiation may enable scientists to control and possibly alter the plasticity of the differentiated state, which may lead to benefits not only in stem cell research but also in regenerative medicine.« less

Interleukin-17A Differentially Induces Inflammatory and Metabolic Gene Expression in the Adipose Tissues of Lean and Obese Mice

PubMed Central

Qu, Yine; Zhang, Qiuyang; Ma, Siqi; Liu, Sen; Chen, Zhiquan; Mo, Zhongfu; You, Zongbing

2016-01-01

The functions of interleukin-17A (IL-17A) in adipose tissues and adipocytes have not been well understood. In the present study, male mice were fed with a regular diet (n = 6, lean mice) or a high-fat diet (n = 6, obese mice) for 30 weeks. Subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) were analyzed for IL-17A levels. SAT and VAT were treated with IL-17A and analyzed for inflammatory and metabolic gene expression. Mouse 3T3-L1 pre-adipocytes were differentiated into adipocytes, followed with IL-17A treatment and analysis for inflammatory and metabolic gene expression. We found that IL-17A levels were higher in obese SAT than lean SAT; the basal expression of inflammatory and metabolic genes was different between SAT and VAT and between lean and obese adipose tissues. IL-17A differentially induced expression of inflammatory and metabolic genes, such as tumor necrosis factor α, Il-6, Il-1β, leptin, and glucose transporter 4, in adipose tissues of lean and obese mice. IL-17A also differentially induced expression of inflammatory and metabolic genes in pre-adipocytes and adipocytes, and IL-17A selectively activated signaling pathways in adipose tissues and adipocytes. These findings suggest that IL-17A differentially induces inflammatory and metabolic gene expression in the adipose tissues of lean and obese mice. PMID:27070576

Transcriptome analyses of the Giardia lamblia life cycle

PubMed Central

Birkeland, Shanda R.; Preheim, Sarah P.; Davids, Barbara J.; Cipriano, Michael J.; Palm, Daniel; Reiner, David S.; Svärd, Staffan G.; Gillin, Frances D.; McArthur, Andrew G.

2010-01-01

We quantified mRNA abundance from 10 stages in the Giardia lamblia life cycle in vitro using Serial Analysis of Gene Expression (SAGE). 163 abundant transcripts were expressed constitutively. 71 transcripts were upregulated specifically during excystation and 42 during encystation. Nonetheless, the transcriptomes of cysts and trophozoites showed major differences. SAGE detected co-expressed clusters of 284 transcripts differentially expressed in cysts and excyzoites and 287 transcripts in vegetative trophozoites and encysting cells. All clusters included known genes and pathways as well as proteins unique to Giardia or diplomonads. SAGE analysis of the Giardia life cycle identified a number of kinases, phosphatases, and DNA replication proteins involved in excystation and encystation, which could be important for examining the roles of cell signaling in giardial differentiation. Overall, these data pave the way for directed gene discovery and a better understanding of the biology of Giardia lamblia. PMID:20570699

Expression Profiling Analysis Reveals Key MicroRNA-mRNA Interactions in Early Retinal Degeneration in Retinitis Pigmentosa.

PubMed

Anasagasti, Ander; Ezquerra-Inchausti, Maitane; Barandika, Olatz; Muñoz-Culla, Maider; Caffarel, María M; Otaegui, David; López de Munain, Adolfo; Ruiz-Ederra, Javier

2018-05-01

The aim of this study was to identify differentially expressed microRNAs (miRNAs) that might play an important role in the etiology of retinal degeneration in a genetic mouse model of retinitis pigmentosa (rd10 mice) at initial stages of the disease. miRNAs-mRNA interaction networks were generated for analysis of biological pathways involved in retinal degeneration. Of more than 1900 miRNAs analyzed, we selected 19 miRNAs on the basis of (1) a significant differential expression in rd10 retinas compared with control samples and (2) an inverse expression relationship with predicted mRNA targets involved in biological pathways relevant to retinal biology and/or degeneration. Seven of the selected miRNAs have been associated with retinal dystrophies, whereas, to our knowledge, nine have not been previously linked to any disease. This study contributes to our understanding of the etiology and progression of retinal degeneration.

Comparative analysis of TCDD-induced AhR-mediated gene expression in human, mouse and rat primary B cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kovalova, Natalia, E-mail: kovalova@msu.edu

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental pollutant that activates the aryl hydrocarbon receptor (AhR) resulting in altered gene expression. In vivo, in vitro, and ex vivo studies have demonstrated that B cells are directly impaired by TCDD, and are a sensitive target as evidenced by suppression of antibody responses. The window of sensitivity to TCDD-induced suppression of IgM secretion among mouse, rat and human B cells is similar. Specifically, TCDD must be present within the initial 12 h post B cell stimulation, indicating that TCDD disrupts early signaling network(s) necessary for B lymphocyte activation and differentiation. Therefore, we hypothesized thatmore » TCDD treatment across three different species (mouse, rat and human) triggers a conserved, B cell-specific mechanism that is involved in TCDD-induced immunosuppression. RNA sequencing (RNA-Seq) was used to identify B cell-specific orthologous genes that are differentially expressed in response to TCDD in primary mouse, rat and human B cells. Time course studies identified TCDD-elicited differential expression of 515 human, 2371 mouse and 712 rat orthologous genes over the 24-h period. 28 orthologs were differentially expressed in response to TCDD in all three species. Overrepresented pathways enriched in all three species included cytokine-cytokine receptor interaction, ECM-receptor interaction, focal adhesion, regulation of actin cytoskeleton and pathways in cancer. Differentially expressed genes functionally associated with cell-cell signaling in humans, immune response in mice, and oxidation reduction in rats. Overall, these results suggest that despite the conservation of the AhR and its signaling mechanism, TCDD elicits species-specific gene expression changes. - Highlights: • Kovalova TAAP Highlights Nov. 2016 • RNA-Seq identified TCDD-induced gene expression in PWM-activated primary B cells. • TCDD elicited differential expression of 515 human, 2371 mouse and 712 rat orthologs. • 28 orthologs were differentially expressed in response to TCDD in all three species. • TCDD elicits mostly species-specific gene expression changes in activated B cells.« less

GeneMesh: a web-based microarray analysis tool for relating differentially expressed genes to MeSH terms.

PubMed

Jani, Saurin D; Argraves, Gary L; Barth, Jeremy L; Argraves, W Scott

2010-04-01

An important objective of DNA microarray-based gene expression experimentation is determining inter-relationships that exist between differentially expressed genes and biological processes, molecular functions, cellular components, signaling pathways, physiologic processes and diseases. Here we describe GeneMesh, a web-based program that facilitates analysis of DNA microarray gene expression data. GeneMesh relates genes in a query set to categories available in the Medical Subject Headings (MeSH) hierarchical index. The interface enables hypothesis driven relational analysis to a specific MeSH subcategory (e.g., Cardiovascular System, Genetic Processes, Immune System Diseases etc.) or unbiased relational analysis to broader MeSH categories (e.g., Anatomy, Biological Sciences, Disease etc.). Genes found associated with a given MeSH category are dynamically linked to facilitate tabular and graphical depiction of Entrez Gene information, Gene Ontology information, KEGG metabolic pathway diagrams and intermolecular interaction information. Expression intensity values of groups of genes that cluster in relation to a given MeSH category, gene ontology or pathway can be displayed as heat maps of Z score-normalized values. GeneMesh operates on gene expression data derived from a number of commercial microarray platforms including Affymetrix, Agilent and Illumina. GeneMesh is a versatile web-based tool for testing and developing new hypotheses through relating genes in a query set (e.g., differentially expressed genes from a DNA microarray experiment) to descriptors making up the hierarchical structure of the National Library of Medicine controlled vocabulary thesaurus, MeSH. The system further enhances the discovery process by providing links between sets of genes associated with a given MeSH category to a rich set of html linked tabular and graphic information including Entrez Gene summaries, gene ontologies, intermolecular interactions, overlays of genes onto KEGG pathway diagrams and heatmaps of expression intensity values. GeneMesh is freely available online at http://proteogenomics.musc.edu/genemesh/.

A data analysis framework for biomedical big data: Application on mesoderm differentiation of human pluripotent stem cells

PubMed Central

Karlsson, Alexander; Riveiro, Maria; Améen, Caroline; Åkesson, Karolina; Andersson, Christian X.; Sartipy, Peter; Synnergren, Jane

2017-01-01

The development of high-throughput biomolecular technologies has resulted in generation of vast omics data at an unprecedented rate. This is transforming biomedical research into a big data discipline, where the main challenges relate to the analysis and interpretation of data into new biological knowledge. The aim of this study was to develop a framework for biomedical big data analytics, and apply it for analyzing transcriptomics time series data from early differentiation of human pluripotent stem cells towards the mesoderm and cardiac lineages. To this end, transcriptome profiling by microarray was performed on differentiating human pluripotent stem cells sampled at eleven consecutive days. The gene expression data was analyzed using the five-stage analysis framework proposed in this study, including data preparation, exploratory data analysis, confirmatory analysis, biological knowledge discovery, and visualization of the results. Clustering analysis revealed several distinct expression profiles during differentiation. Genes with an early transient response were strongly related to embryonic- and mesendoderm development, for example CER1 and NODAL. Pluripotency genes, such as NANOG and SOX2, exhibited substantial downregulation shortly after onset of differentiation. Rapid induction of genes related to metal ion response, cardiac tissue development, and muscle contraction were observed around day five and six. Several transcription factors were identified as potential regulators of these processes, e.g. POU1F1, TCF4 and TBP for muscle contraction genes. Pathway analysis revealed temporal activity of several signaling pathways, for example the inhibition of WNT signaling on day 2 and its reactivation on day 4. This study provides a comprehensive characterization of biological events and key regulators of the early differentiation of human pluripotent stem cells towards the mesoderm and cardiac lineages. The proposed analysis framework can be used to structure data analysis in future research, both in stem cell differentiation, and more generally, in biomedical big data analytics. PMID:28654683

A data analysis framework for biomedical big data: Application on mesoderm differentiation of human pluripotent stem cells.

PubMed

Ulfenborg, Benjamin; Karlsson, Alexander; Riveiro, Maria; Améen, Caroline; Åkesson, Karolina; Andersson, Christian X; Sartipy, Peter; Synnergren, Jane

2017-01-01

The development of high-throughput biomolecular technologies has resulted in generation of vast omics data at an unprecedented rate. This is transforming biomedical research into a big data discipline, where the main challenges relate to the analysis and interpretation of data into new biological knowledge. The aim of this study was to develop a framework for biomedical big data analytics, and apply it for analyzing transcriptomics time series data from early differentiation of human pluripotent stem cells towards the mesoderm and cardiac lineages. To this end, transcriptome profiling by microarray was performed on differentiating human pluripotent stem cells sampled at eleven consecutive days. The gene expression data was analyzed using the five-stage analysis framework proposed in this study, including data preparation, exploratory data analysis, confirmatory analysis, biological knowledge discovery, and visualization of the results. Clustering analysis revealed several distinct expression profiles during differentiation. Genes with an early transient response were strongly related to embryonic- and mesendoderm development, for example CER1 and NODAL. Pluripotency genes, such as NANOG and SOX2, exhibited substantial downregulation shortly after onset of differentiation. Rapid induction of genes related to metal ion response, cardiac tissue development, and muscle contraction were observed around day five and six. Several transcription factors were identified as potential regulators of these processes, e.g. POU1F1, TCF4 and TBP for muscle contraction genes. Pathway analysis revealed temporal activity of several signaling pathways, for example the inhibition of WNT signaling on day 2 and its reactivation on day 4. This study provides a comprehensive characterization of biological events and key regulators of the early differentiation of human pluripotent stem cells towards the mesoderm and cardiac lineages. The proposed analysis framework can be used to structure data analysis in future research, both in stem cell differentiation, and more generally, in biomedical big data analytics.

An Effective Model of the Retinoic Acid Induced HL-60 Differentiation Program.

PubMed

Tasseff, Ryan; Jensen, Holly A; Congleton, Johanna; Dai, David; Rogers, Katharine V; Sagar, Adithya; Bunaciu, Rodica P; Yen, Andrew; Varner, Jeffrey D

2017-10-30

In this study, we present an effective model All-Trans Retinoic Acid (ATRA)-induced differentiation of HL-60 cells. The model describes reinforcing feedback between an ATRA-inducible signalsome complex involving many proteins including Vav1, a guanine nucleotide exchange factor, and the activation of the mitogen activated protein kinase (MAPK) cascade. We decomposed the effective model into three modules; a signal initiation module that sensed and transformed an ATRA signal into program activation signals; a signal integration module that controlled the expression of upstream transcription factors; and a phenotype module which encoded the expression of functional differentiation markers from the ATRA-inducible transcription factors. We identified an ensemble of effective model parameters using measurements taken from ATRA-induced HL-60 cells. Using these parameters, model analysis predicted that MAPK activation was bistable as a function of ATRA exposure. Conformational experiments supported ATRA-induced bistability. Additionally, the model captured intermediate and phenotypic gene expression data. Knockout analysis suggested Gfi-1 and PPARg were critical to the ATRAinduced differentiation program. These findings, combined with other literature evidence, suggested that reinforcing feedback is central to hyperactive signaling in a diversity of cell fate programs.