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

Differential global gene expression in red and white skeletal muscle

NASA Technical Reports Server (NTRS)

Campbell, W. G.; Gordon, S. E.; Carlson, C. J.; Pattison, J. S.; Hamilton, M. T.; Booth, F. W.

2001-01-01

The differences in gene expression among the fiber types of skeletal muscle have long fascinated scientists, but for the most part, previous experiments have only reported differences of one or two genes at a time. The evolving technology of global mRNA expression analysis was employed to determine the potential differential expression of approximately 3,000 mRNAs between the white quad (white muscle) and the red soleus muscle (mixed red muscle) of female ICR mice (30-35 g). Microarray analysis identified 49 mRNA sequences that were differentially expressed between white and mixed red skeletal muscle, including newly identified differential expressions between muscle types. For example, the current findings increase the number of known, differentially expressed mRNAs for transcription factors/coregulators by nine and signaling proteins by three. The expanding knowledge of the diversity of mRNA expression between white and mixed red muscle suggests that there could be quite a complex regulation of phenotype between muscles of different fiber types.

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.

Down-regulated RPS3a/nbl expression during retinoid-induced differentiation of HL-60 cells: a close association with diminished susceptibility to actinomycin D-stimulated apoptosis.

PubMed

Russell, L; Naora, H; Naora, H

2000-04-01

The efficacy of anticancer agents significantly depends on the differential susceptibility of undifferentiated cancer cells and differentiated normal cells to undergo apoptosis. We previously found that enhanced expression of RPS3a/nbl, which apparently encodes a ribosomal protein, seems to prime cells for apoptosis, while suppressing such enhanced expression triggers cell death. The present study found that HL-60 cells induced to differentiate by all-trans retinoic acid did not undergo apoptosis following treatment with actinomycin D whereas undifferentiated HL-60 cells were highly apoptosis-susceptible, confirming earlier suggestions that differentiated cells have diminished apoptosis-susceptibility. Undifferentiated HL-60 cells highly expressed RPS3a/nbl whereas all-trans retinoic acid -induced differentiated cells exhibited markedly reduced levels, suggesting that apoptosis-resistance of differentiated cells could be due to low RPS3a/nbl expression. Down-regulation of enhanced RPS3a/nbl expression was also observed in cells induced to differentiate with the retinoid 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl)-1- propenyl]benzoic acid without any significant induction of cell death. While down-regulation of RPS3a/nbl expression during differentiation did not apparently induce apoptosis, RPS3a/nbl antisense oligomers triggered death of undifferentiated HL-60 cells, but not of retinoid-induced differentiated cells. It therefore seems that while down-regulation of enhanced RPS3a/nbl expression can induce apoptosis in undifferentiated cells, down-regulation of enhanced RPS3a/nbl expression during differentiation occurs independently of apoptosis, and could be regarded as reverting the primed condition to the unprimed (low RPS3a/nbl) state.

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.

Induction of hepatocyte-like cells from mouse embryonic stem cells by lentivirus-mediated constitutive expression of Foxa2/Hnf4a.

PubMed

Liu, Tao; Zhang, Shichang; Xiang, Dedong; Wang, Yingjie

2013-11-01

Hepatocytes can be generated from embryonic stem cells (ESCs) using inducers such as chemical compounds and cytokines, but issues related to low differentiation efficiencies remain to be resolved. Recent work has shown that overexpression of lineage-specific transcription factors can directly cause cells phenotypic changes, including differentiation, trans-differentiation, and de-differentiation. We hypothesized that lentivirus-mediated constitutive expression of forkhead box A2 (Foxa2) and hepatocyte nuclear factor 4 alpha (Hnf4a) could promote inducing mouse ESCs to hepatocyte-likes cells. First, ESC lines that stably expressed Foxa2, Hnf4a, or Foxa2/Hnf4a were constructed via lentiviral expression vectors. Second, observations of cell morphology changes were made during the cell culture process, followed by experiments examining teratoma formation. Then, the effects of constitutive expression of Foxa2 and Hnf4a on hepatic differentiation and maturation were determined by measuring the marker gene expression levels of Albumin, α-fetoprotein, Cytokeratin18, and α1-antitrypsin. The results indicate that constitutive expression of Foxa2 and Hnf4a does not affect ESCs culture, teratoma formation, or the expression levels of the specific hepatocyte genes under autonomous differentiation. However, with some assistance from inducing factors, Foxa2 significantly increased the hepatic differentiation of ESCs, whereas the expression of Hnf4a alone or Foxa2/Hnf4a could not. Differentiated CCE-Foxa2 cells were more superior in expressing several liver-specific markers and protein, storing glycogen than differentiated CCE cells. Therefore, our method employing the transduction of Foxa2 would be a valuable tool for the efficient generation of functional hepatocytes derived from ESCs. © 2013 Wiley Periodicals, Inc.

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.

A P-Norm Robust Feature Extraction Method for Identifying Differentially Expressed Genes

PubMed Central

Liu, Jian; Liu, Jin-Xing; Gao, Ying-Lian; Kong, Xiang-Zhen; Wang, Xue-Song; Wang, Dong

2015-01-01

In current molecular biology, it becomes more and more important to identify differentially expressed genes closely correlated with a key biological process from gene expression data. In this paper, based on the Schatten p-norm and Lp-norm, a novel p-norm robust feature extraction method is proposed to identify the differentially expressed genes. In our method, the Schatten p-norm is used as the regularization function to obtain a low-rank matrix and the Lp-norm is taken as the error function to improve the robustness to outliers in the gene expression data. The results on simulation data show that our method can obtain higher identification accuracies than the competitive methods. Numerous experiments on real gene expression data sets demonstrate that our method can identify more differentially expressed genes than the others. Moreover, we confirmed that the identified genes are closely correlated with the corresponding gene expression data. PMID:26201006

A P-Norm Robust Feature Extraction Method for Identifying Differentially Expressed Genes.

PubMed

Liu, Jian; Liu, Jin-Xing; Gao, Ying-Lian; Kong, Xiang-Zhen; Wang, Xue-Song; Wang, Dong

2015-01-01

In current molecular biology, it becomes more and more important to identify differentially expressed genes closely correlated with a key biological process from gene expression data. In this paper, based on the Schatten p-norm and Lp-norm, a novel p-norm robust feature extraction method is proposed to identify the differentially expressed genes. In our method, the Schatten p-norm is used as the regularization function to obtain a low-rank matrix and the Lp-norm is taken as the error function to improve the robustness to outliers in the gene expression data. The results on simulation data show that our method can obtain higher identification accuracies than the competitive methods. Numerous experiments on real gene expression data sets demonstrate that our method can identify more differentially expressed genes than the others. Moreover, we confirmed that the identified genes are closely correlated with the corresponding gene expression data.

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

Inhibition of osteoclast differentiation by overexpression of NDRG2 in monocytes

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

Kang, Kyeongah; Nam, Sorim; Kim, Bomi

N-Myc downstream-regulated gene 2 (NDRG2), a member of the NDRG family of differentiation-related genes, has been characterized as a regulator of dendritic cell differentiation from monocytes, CD34{sup +} progenitor cells, and myelomonocytic leukemic cells. In this study, we show that NDRG2 overexpression inhibits the differentiation of U937 cells into osteoclasts in response to stimulation with a combination of macrophage colony-stimulating factor (M-CSF) and soluble receptor activator of NF-κB ligand (RANKL). U937 cells stably expressing NDRG2 are unable to differentiate into multinucleated osteoclast-like cells and display reduced tartrate-resistant acid phosphatase (TRAP) activity and resorption pit formation. Furthermore, NDRG2 expression significantly suppressesmore » the expression of genes that are crucial for the proliferation, survival, differentiation, and function of osteoclasts, including c-Fos, Atp6v0d2, RANK, and OSCAR. The activation of ERK1/2 and p38 is also inhibited by NDRG2 expression during osteoclastogenesis, and the inhibition of osteoclastogenesis by NDRG2 correlates with the down-regulation of the expression of the transcription factor PU.1. Taken together, our results suggest that the expression of NDRG2 potentially inhibits osteoclast differentiation and plays a role in modulating the signal transduction pathway responsible for osteoclastogenesis. - Highlights: • The expression of NDRG2 significantly impairs osteoclast differentiation. • PU.1 and p38 MAPK inhibitions by NDRG2 are critical for the inhibition of osteoclastogenesis. • Knockdown of NDRG2 rescues the ability of monocytes to differentiate into osteoclasts. • NDRG2 expression in BM and primary macrophages also impairs osteoclast differentiation. • This study implies the potential of NDRG2 expression in the inhibition of osteoclastogenesis.« less

Wheat differential gene expression induced by different races of Puccinia triticina.

PubMed

Neugebauer, Kerri A; Bruce, Myron; Todd, Tim; Trick, Harold N; Fellers, John P

2018-01-01

Puccinia triticina, the causal agent of wheat leaf rust, causes significant losses in wheat yield and quality each year worldwide. During leaf rust infection, the host plant recognizes numerous molecules, some of which trigger host defenses. Although P. triticina reproduces clonally, there is still variation within the population due to a high mutation frequency, host specificity, and environmental adaptation. This study explores how wheat responds on a gene expression level to different P. triticina races. Six P. triticina races were inoculated onto a susceptible wheat variety and samples were taken at six days post inoculation, just prior to pustule eruption. RNA sequence data identified 63 wheat genes differentially expressed between the six races. A time course, conducted over the first seven days post inoculation, was used to examine the expression pattern of 63 genes during infection. Forty-seven wheat genes were verified to have differential expression. Three common expression patterns were identified. In addition, two genes were associated with race specific gene expression. Differential expression of an ER molecular chaperone gene was associated with races from two different P. triticina lineages. Also, differential expression in an alanine glyoxylate aminotransferase gene was associated with races with virulence shifts for leaf rust resistance genes.

DR-nm23 expression affects neuroblastoma cell differentiation, integrin expression, and adhesion characteristics.

PubMed

Amendola, R; Martinez, R; Negroni, A; Venturelli, D; Tanno, B; Calabretta, B; Raschellà, G

2001-01-01

Nm23 gene family has been associated with metastasis suppression and differentiation. We studied DR-nm23 during neuroblastoma cells differentiation. DR-nm23 expression increased after retinoic acid induction of differentiation in human cell lines SK-N-SH and LAN-5. In several cell lines, overexpression of DR-nm23 was associated with more differentiated phenotypes. SK-N-SH cells increased vimentin expression, increased deposition of collagen type IV, modulated integrin expression, and underwent growth arrest; the murine neuroblastoma cell line N1E-115 showed neurite outgrowth and a striking enhancement of beta1 integrin expression. Up-regulation of beta1 integrin was specifically responsible for the increase in the adhesion to collagen type I-coated plates. Finally, cells overexpressing DR-nm23 were unable to growth in soft agar. In conclusion, DR-nm23 expression is directly involved in differentiation of neuroblastoma cells, and its ability to affects the adhesion to extracellular substrates and to inhibit growth in soft agar suggests an involvement in the metastatic potential of neuroblastoma.

Integrated analysis of long noncoding RNA and mRNA expression profile in children with obesity by microarray analysis.

PubMed

Liu, Yuesheng; Ji, Yuqiang; Li, Min; Wang, Min; Yi, Xiaoqing; Yin, Chunyan; Wang, Sisi; Zhang, Meizhen; Zhao, Zhao; Xiao, Yanfeng

2018-06-08

Long noncoding RNAs (lncRNAs) have an important role in adipose tissue function and energy metabolism homeostasis, and abnormalities may lead to obesity. To investigate whether lncRNAs are involved in childhood obesity, we investigated the differential expression profile of lncRNAs in obese children compared with non-obese children. A total number of 1268 differentially expressed lncRNAs and 1085 differentially expressed mRNAs were identified. Gene Ontology (GO) and pathway analysis revealed that these lncRNAs were involved in varied biological processes, including the inflammatory response, lipid metabolic process, osteoclast differentiation and fatty acid metabolism. In addition, the lncRNA-mRNA co-expression network and the protein-protein interaction (PPI) network were constructed to identify hub regulatory lncRNAs and genes based on the microarray expression profiles. This study for the first time identifies an expression profile of differentially expressed lncRNAs in obese children and indicated hub lncRNA RP11-20G13.3 attenuated adipogenesis of preadipocytes, which is conducive to the search for new diagnostic and therapeutic strategies of childhood obesity.

Effect of Wnt-1 inducible signaling pathway protein-2 (WISP-2/CCN5), a downstream protein of Wnt signaling, on adipocyte differentiation

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

Inadera, Hidekuni; Shimomura, Akiko; Tachibana, Shinjiro

2009-02-20

Wnt signaling negatively regulates adipocyte differentiation, and ectopic expression of Wnt-1 in 3T3-L1 cells induces several downstream molecules of Wnt signaling, including Wnt-1 inducible signaling pathway protein (WISP)-2. In this study, we examined the role of WISP-2 in the process of adipocyte differentiation using an in vitro cell culture system. In the differentiation of 3T3-L1 cells, WISP-2 expression was observed in growing cells and declined thereafter. In the mitotic clonal expansion phase of adipocyte differentiation, WISP-2 expression was transiently down-regulated concurrently with up-regulation of CCAAT/enhancer-binding protein {delta} expression. Treatment of 3T3-L1 cells in the differentiation medium with lithium, an activatormore » of Wnt signaling, inhibited the differentiation process with concomitant induction of WISP-2. Treatment of differentiated cells with lithium induced de-differentiation as evidenced by profound reduction of peroxisome proliferator-activator receptor {gamma} expression and concomitant induction of WISP-2. However, de-differentiation of differentiated cells induced by tumor necrosis factor-{alpha} did not induce WISP-2 expression. To directly examine the effect of WISP-2 on adipocyte differentiation, 3T3-L1 cells were infected with a retrovirus carrying WISP-2. Although forced expression of WISP-2 inhibited preadipocyte proliferation, it had no effect on adipocyte differentiation. Thus, although WISP-2 is a downstream protein of Wnt signaling, the role of WISP-2 on adipocyte differentiation may be marginal, at least in this in vitro culture model.« less

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.

Suppression of MicroRNA let-7a Expression by Agmatine Regulates Neural Stem Cell Differentiation

PubMed Central

Song, Juhyun; Oh, Yumi; Kim, Jong Youl; Cho, Kyoung Joo

2016-01-01

Purpose Neural stem cells (NSCs) effectively reverse some severe central nervous system (CNS) disorders, due to their ability to differentiate into neurons. Agmatine, a biogenic amine, has cellular protective effects and contributes to cellular proliferation and differentiation in the CNS. Recent studies have elucidated the function of microRNA let-7a (let-7a) as a regulator of cell differentiation with roles in regulating genes associated with CNS neurogenesis. Materials and Methods This study aimed to investigate whether agmatine modulates the expression of crucial regulators of NSC differentiation including DCX, TLX, c-Myc, and ERK by controlling let-7a expression. Results Our data suggest that high levels of let-7a promoted the expression of TLX and c-Myc, as well as repressed DCX and ERK expression. In addition, agmatine attenuated expression of TLX and increased expression of ERK by negatively regulating let-7a. Conclusion Our study therefore enhances the present understanding of the therapeutic potential of NSCs in CNS disorders. PMID:27593875

Suppression of MicroRNA let-7a Expression by Agmatine Regulates Neural Stem Cell Differentiation.

PubMed

Song, Juhyun; Oh, Yumi; Kim, Jong Youl; Cho, Kyoung Joo; Lee, Jong Eun

2016-11-01

Neural stem cells (NSCs) effectively reverse some severe central nervous system (CNS) disorders, due to their ability to differentiate into neurons. Agmatine, a biogenic amine, has cellular protective effects and contributes to cellular proliferation and differentiation in the CNS. Recent studies have elucidated the function of microRNA let-7a (let-7a) as a regulator of cell differentiation with roles in regulating genes associated with CNS neurogenesis. This study aimed to investigate whether agmatine modulates the expression of crucial regulators of NSC differentiation including DCX, TLX, c-Myc, and ERK by controlling let-7a expression. Our data suggest that high levels of let-7a promoted the expression of TLX and c-Myc, as well as repressed DCX and ERK expression. In addition, agmatine attenuated expression of TLX and increased expression of ERK by negatively regulating let-7a. Our study therefore enhances the present understanding of the therapeutic potential of NSCs in CNS disorders.

Juvenile hormone and colony conditions differentially influence cytochrome P450 gene expression in the termite Reticulitermes flavipes.

PubMed

Zhou, X; Song, C; Grzymala, T L; Oi, F M; Scharf, M E

2006-12-01

In lower termites, the worker caste is a totipotent immature stage that is capable of differentiating into other adult caste phenotypes. We investigated the diversity of family 4 cytochrome P450 (CYP4) genes in Reticulitermes flavipes workers, with the specific goal of identifying P450s potentially involved in regulating caste differentiation. Seven novel CYP4 genes were identified. Quantitative real-time PCR revealed the tissue distribution of expression for the seven CYP4s, as well as temporal expression changes in workers in association with a release from colony influences and during juvenile hormone (JH)-induced soldier caste differentiation. Several fat-body-related CYP4 genes were differentially expressed after JH treatment. Still other genes changed expression in association with removal from colony influences, suggesting that primer pheromones and/or other colony influences impact their expression. These findings add to a growing database of candidate termite caste-regulatory genes, and provide explicit evidence that colony factors influence termite gene expression.

Parallel Gene Expression Differences between Low and High Latitude Populations of Drosophila melanogaster and D. simulans

PubMed Central

Zhao, Li; Wit, Janneke; Svetec, Nicolas; Begun, David J.

2015-01-01

Gene expression variation within species is relatively common, however, the role of natural selection in the maintenance of this variation is poorly understood. Here we investigate low and high latitude populations of Drosophila melanogaster and its sister species, D. simulans, to determine whether the two species show similar patterns of population differentiation, consistent with a role for spatially varying selection in maintaining gene expression variation. We compared at two temperatures the whole male transcriptome of D. melanogaster and D. simulans sampled from Panama City (Panama) and Maine (USA). We observed a significant excess of genes exhibiting differential expression in both species, consistent with parallel adaptation to heterogeneous environments. Moreover, the majority of genes showing parallel expression differentiation showed the same direction of differential expression in the two species and the magnitudes of expression differences between high and low latitude populations were correlated across species, further bolstering the conclusion that parallelism for expression phenotypes results from spatially varying selection. However, the species also exhibited important differences in expression phenotypes. For example, the genomic extent of genotype × environment interaction was much more common in D. melanogaster. Highly differentiated SNPs between low and high latitudes were enriched in the 3’ UTRs and CDS of the geographically differently expressed genes in both species, consistent with an important role for cis-acting variants in driving local adaptation for expression-related phenotypes. PMID:25950438

Parallel Gene Expression Differences between Low and High Latitude Populations of Drosophila melanogaster and D. simulans.

PubMed

Zhao, Li; Wit, Janneke; Svetec, Nicolas; Begun, David J

2015-05-01

Gene expression variation within species is relatively common, however, the role of natural selection in the maintenance of this variation is poorly understood. Here we investigate low and high latitude populations of Drosophila melanogaster and its sister species, D. simulans, to determine whether the two species show similar patterns of population differentiation, consistent with a role for spatially varying selection in maintaining gene expression variation. We compared at two temperatures the whole male transcriptome of D. melanogaster and D. simulans sampled from Panama City (Panama) and Maine (USA). We observed a significant excess of genes exhibiting differential expression in both species, consistent with parallel adaptation to heterogeneous environments. Moreover, the majority of genes showing parallel expression differentiation showed the same direction of differential expression in the two species and the magnitudes of expression differences between high and low latitude populations were correlated across species, further bolstering the conclusion that parallelism for expression phenotypes results from spatially varying selection. However, the species also exhibited important differences in expression phenotypes. For example, the genomic extent of genotype × environment interaction was much more common in D. melanogaster. Highly differentiated SNPs between low and high latitudes were enriched in the 3' UTRs and CDS of the geographically differently expressed genes in both species, consistent with an important role for cis-acting variants in driving local adaptation for expression-related phenotypes.

Relationship of calcitonin mRNA expression to the differentiation state of HL 60 cells.

PubMed

Kiefer, P; Bacher, M; Pflüger, K H

1994-05-01

Raised plasma levels of immunoreactive human calcitonin (ihCT) can be found in patients with myeloid leukemia and seem to indicate a poor prognosis. High levels were found in acute undifferentiated and acute myeloblastic leukemia. To test whether CT expression could be a marker of myeloid differentiation, we used the promyelocytic leukemia cell line HL 60 which also expresses ihCT as a model system for myeloid differentiation. Exponentially growing HL 60 cells as well as differentiation induced HL 60 cells expressed a single 1.0 Kb CT transcript. The induction of HL 60 cell differentiation along the granulocytic lineage by DMSO or HMBA had no effect on the level of CT transcripts. Induction of monocytic/macrophagic differentiation by TPA resulted in a transient, about 10-fold elevated expression of CT steady state mRNA after 24 h. In contrast to TPA, induction of HL 60 cell differentiation along the monocytic pathway by Vit D3 had no detectable effect on the level of the CT in RNA expression at corresponding time points. These findings suggest that the transient induction of CT steady state mRNA expression by TPA is rather a direct effect of the phorbol ester than commitment along the monocytic line of differentiation.

miR-24 and miR-205 expression is dependent on HPV onco-protein expression in keratinocytes

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

McKenna, Declan J., E-mail: dj.mckenna@ulster.ac.uk; Centre for Cancer Research and Cell Biology, School of Medicine, Dentistry and Biomedical Science, Queen's University Belfast, Belfast BT9 7BL; Patel, Daksha, E-mail: d.patel@qub.ac.uk

2014-01-05

A screen of microRNA (miRNA) expression following differentiation in human foreskin keratinocytes (HFKs) identified changes in several miRNAs, including miR-24 and miR-205. We investigated how expression of Human Papilloma Virus Type-16 (HPV16) onco-proteins E6 and E7 affected expression of miR-24 and miR-205 during proliferation and differentiation of HFKs. We show that the induction of both miR-24 and miR-205 observed during differentiation of HFKs is lost in HFKs expressing E6 and E7. We demonstrate that the effect on miR-205 is due to E7 activity, as miR-205 expression is dependent on pRb expression. Finally, we provide evidence that miR-24 effects in themore » cell may be due to targeting of cyclin dependent kinase inhibitor p27. In summary, these results indicate that expression of both miR-24 and miR-205 are impacted by E6 and/or E7 expression, which may be one mechanism by which HPV onco-proteins can disrupt the balance between proliferation and differentiation in keratinocytes. - Highlights: • miR-24 and miR-205 are induced during keratinocyte differentiation. • This induction is lost in keratinocytes expressing HPV onco-proteins E6 and E7. • miR-205 is dependent upon pRb expression. • miR-24 targets p27 in cycling keratinocytes.« less

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.

Induction of neural differentiation by electrically stimulated gene expression of NeuroD2.

PubMed

Mie, Masayasu; Endoh, Tamaki; Yanagida, Yasuko; Kobatake, Eiry; Aizawa, Masuo

2003-02-13

Regulation of cell differentiation is an important assignment for cellular engineering. One of the techniques for regulation is gene transfection into undifferentiated cells. Transient expression of NeuroD2, one of neural bHLH transcription factors, converted mouse N1E-115 neuroblastoma cells into differentiated neurons. The regulation of neural bHLH expression should be a novel strategy for cell differentiation. In this study, we tried to regulate neural differentiation by NeuroD2 gene inserted under the control of heat shock protein-70 (HSP) promoter, which can be activated by electrical stimulation. Mouse neuroblastoma cell line, N1E-115, was stably transfected with expression vector containing mouse NeuroD2 cDNA under HSP promoter. Transfected cells were cultured on the electrode surface and applied electrical stimulation. After stimulation, NeuroD2 expression was induced, and transfected cells adopt a neuronal morphology at 3 days after stimulation. These results suggest that neural differentiation can be induced by electrically stimulated gene expression of NeuroD2.

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.

Long noncoding RNA MALAT1 promotes osterix expression to regulate osteogenic differentiation by targeting miRNA-143 in human bone marrow-derived mesenchymal stem cells.

PubMed

Gao, Yuan; Xiao, Fei; Wang, Chenglong; Wang, Chuandong; Cui, Penglei; Zhang, Xiaoling; Chen, Xiaodong

2018-05-09

Osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) is essential for the human bone formation, and emerging evidence shows that long non-coding RNAs (lncRNAs) play important roles in hBMSC osteogenic differentiation. MALAT1 is often regarded as a tumor-related lncRNA, but its function in mesenchymal stem cell differentiation remains to be defined. In this study, we aimed to investigate whether MALAT1 regulates Osterix (Osx) expression by sponging miR-143 to promote hBMSC osteogenic differentiation. Firstly, we found that the expression of MALAT1 was much lower in hBMSCs from osteoporosis patients and miR-143 was contrarily higher. In addition, MALAT1 expression increased, and miR-143 decreased when hBMSCs were treated with osteogenic induction. Then, we used short hairpin RNAs to knockdown MALAT1, and the results showed that hBMSC osteogenic differentiation decreased significantly, indicating that MALAT1 is a positive regulator of osteogenic differentiation in hBMSCs. Furthermore, by luciferase assays, we found that MALAT1 could directly bind to miR-143 and negatively regulate its expression. Similarly, miR-143 could directly bind to the target site on the Osx 3'-UTR and then inhibit Osx expression. Knockdown of MALAT1 decreased Osx expression, and co-transfection of miR-143 inhibitor could rescue Osx mRNA expression. While Osx expression was increased in MALAT1-overexpressing hBMSCs, it was reversed by the miR-143 mimics. Moreover, Osx silencing decreased ALP, OCN, and OPN mRNA expression induced by the miR-143 inhibitor. Altogether, our findings suggest that MALAT1 acts to regulate Osx expression through targeting miR-143; thus, it is considered as a positive regulator in hBMSC osteogenic differentiation. © 2018 Wiley Periodicals, Inc.

Effect of ECM2 expression on bovine skeletal muscle-derived satellite cell differentiation.

PubMed

Liu, Chang; Tong, Huili; Li, Shufeng; Yan, Yunqin

2018-05-01

Extracellular matrix components have important regulatory functions during cell proliferation and differentiation. In recent study, extracellular matrix were shown to have a strong effect on skeletal muscle differentiation. Here, we aimed to elucidate the effects of extracellular matrix protein 2 (ECM2), an extracellular matrix component, on the differentiation of bovine skeletal muscle-derived satellite cells (MDSCs). Western blot and immunofluorescence analyses were used to elucidate the ECM2 expression pattern in bovine MDSCs during differentiation in vitro. CRISPR/Cas9 technology was used to activate or inhibit ECM2 expression to study its effects on the in vitro differentiation of bovine MDSCs. ECM2 expression was shown to increase gradually during bovine MDSC differentiation, and the levels of this protein were higher in more highly differentiated myotubes. ECM2 activation promoted MDSC differentiation, whereas its suppression inhibited the differentiation of these cells. Here, for the first time, we demonstrated the importance of ECM2 expression during bovine MDSC differentiation; these results could lead to treatments that help to increase beef cattle muscularity. © 2018 International Federation for Cell Biology.

Increased expression of enhancer of Zeste Homolog 2 (EZH2) differentiates squamous cell carcinoma from normal skin and actinic keratosis.

PubMed

Xie, Qiang; Wang, Hongbei; Heilman, Edward R; Walsh, Michael G; Haseeb, M A; Gupta, Raavi

2014-01-01

Enhancer of Zeste Homolog 2 (EZH2) is a polycomb group protein that has been shown to be involved in the progression of multiple human cancers including melanoma. The expression of EZH2 in normal skin and in pre-malignant and malignant cutaneous squamous cell carcinoma (SCC) has not been studied. We examined the expression of EZH2 in normal skin, actinic keratosis (AK), SCC in situ, well-differentiated (SCC-WD), moderately-differentiated (SCC-MD) and poorly-differentiated SCC (SCC-PD) to ascertain whether EZH2 expression differentiates these conditions. Immunohistochemical staining for EZH2 was performed on formalin-fixed paraffin-embedded biopsies and a tissue microarray containing normal skin, AK, SCC in situ, and SCC of different grades. In comparison to the normal skin, EZH2 expression in actinic keratosis was increased (p=0.03). Similarly, EZH2 expression in all of the neoplastic conditions studied (SCC in situ, SCC-WD, SCC-MD and SCC-PD) was greatly increased in comparison to both the normal skin and actinic keratosis (p≤0.001). EZH2 expression increases incrementally from normal skin to AK and further to SCC, suggesting a role for EZH2 in the progression and differentiation of SCC. EZH2 expression may be used as a diagnostic marker for differentiating SCC from AK or normal skin.

[Effect of human oviductal embryotrophic factors on gene expression of mouse preimplantation embryos].

PubMed

Yao, Yuan-Qing; Lee, Kai-Fai; Xu, Jia-Seng; Ho, Pak-Chung; Yeung, Shu-Biu

2007-09-01

To investigate the effect of embryotrophic factors (ETF) from human oviductal cells on gene expression of mouse early developmental embryos and discuss the role of fallopian tube in early development of embryos. ETF was isolated from conditioned medium of human oviductal cell line by sequential liquid chromatographic systems. Mouse embryos were treated by ETF in vitro. Using differential display RT-PCR, the gene expression of embryos treated by ETF was compared with embryos without ETF treatment. The differentially expressed genes were separated, re-amplified, cloned and sequenced. Gene expression profiles of embryos with ETF treatment was different from embryos without this treatment. Eight differentially expressed genes were cloned and sequenced. These genes functioned in RNA degradation, synthesis, splicing, protein trafficking, cellular differentiation and embryo development. Embryotrophic factors from human oviductal cells affect gene expression of early developmental embryos. The human oviductal cells play wide roles in early developmental stages of embryos.

Cells differentiated from mouse embryonic stem cells via embryoid bodies express renal marker molecules.

PubMed

Kramer, Jan; Steinhoff, Jürgen; Klinger, Matthias; Fricke, Lutz; Rohwedel, Jürgen

2006-03-01

Differentiation of mouse embryonic stem (ES) cells via embryoid bodies (EB) is established as a suitable model to study cellular processes of development in vitro. ES cells are known to be pluripotent because of their capability to differentiate into cell types of all three germ layers including germ cells. Here, we show that ES cells differentiate into renal cell types in vitro. We found that genes were expressed during EB cultivation, which have been previously described to be involved in renal development. Marker molecules characteristic for terminally differentiated renal cell types were found to be expressed predominantly during late stages of EB cultivation, while marker molecules involved in the initiation of nephrogenesis were already expressed during early steps of EB development. On the cellular level--using immunostaining--we detected cells expressing podocin, nephrin and wt-1, characteristic for differentiated podocytes and other cells, which expressed Tamm-Horsfall protein, a marker for distal tubule epithelial cells of kidney tissue. Furthermore, the proximal tubule marker molecules renal-specific oxido reductase, kidney androgen-related protein and 25-hydroxyvitamin D3alpha-hydroxylase were found to be expressed in EBs. In particular, we could demonstrate that cells expressing podocyte marker molecules assemble to distinct ring-like structures within the EBs. Because the differentiation efficiency into these cell types is still relatively low, application of fibroblast growth factor (FGF)-2 in combination with leukaemia inhibitory factor was tested for induction, but did not enhance ES cell-derived renal differentiation in vitro.

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.

DCGL v2.0: an R package for unveiling differential regulation from differential co-expression.

PubMed

Yang, Jing; Yu, Hui; Liu, Bao-Hong; Zhao, Zhongming; Liu, Lei; Ma, Liang-Xiao; Li, Yi-Xue; Li, Yuan-Yuan

2013-01-01

Differential co-expression analysis (DCEA) has emerged in recent years as a novel, systematic investigation into gene expression data. While most DCEA studies or tools focus on the co-expression relationships among genes, some are developing a potentially more promising research domain, differential regulation analysis (DRA). In our previously proposed R package DCGL v1.0, we provided functions to facilitate basic differential co-expression analyses; however, the output from DCGL v1.0 could not be translated into differential regulation mechanisms in a straightforward manner. To advance from DCEA to DRA, we upgraded the DCGL package from v1.0 to v2.0. A new module named "Differential Regulation Analysis" (DRA) was designed, which consists of three major functions: DRsort, DRplot, and DRrank. DRsort selects differentially regulated genes (DRGs) and differentially regulated links (DRLs) according to the transcription factor (TF)-to-target information. DRrank prioritizes the TFs in terms of their potential relevance to the phenotype of interest. DRplot graphically visualizes differentially co-expressed links (DCLs) and/or TF-to-target links in a network context. In addition to these new modules, we streamlined the codes from v1.0. The evaluation results proved that our differential regulation analysis is able to capture the regulators relevant to the biological subject. With ample functions to facilitate differential regulation analysis, DCGL v2.0 was upgraded from a DCEA tool to a DRA tool, which may unveil the underlying differential regulation from the observed differential co-expression. DCGL v2.0 can be applied to a wide range of gene expression data in order to systematically identify novel regulators that have not yet been documented as critical. DCGL v2.0 package is available at http://cran.r-project.org/web/packages/DCGL/index.html or at our project home page http://lifecenter.sgst.cn/main/en/dcgl.jsp.

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.

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.

Dynamic and differential expression of the gonadal aromatase during the process of sexual differentiation in a novel transgenic cyp19a1a-eGFP zebrafish line.

PubMed

Hinfray, Nathalie; Sohm, Frédéric; Caulier, Morgane; Chadili, Edith; Piccini, Benjamin; Torchy, Camille; Porcher, Jean-Marc; Guiguen, Yann; Brion, François

2018-05-15

In zebrafish, there exists a clear need for new tools to study sex differentiation dynamic and its perturbation by endocrine disrupting chemicals. In this context, we developed and characterized a novel transgenic zebrafish line expressing green fluorescent protein (GFP) under the control of the zebrafish cyp19a1a (gonadal aromatase) promoter. In most gonochoristic fish species including zebrafish, cyp19a1a, the enzyme responsible for the synthesis of estrogens, has been shown to play a critical role in the processes of reproduction and sexual differentiation. This novel cyp19a1a-eGFP transgenic line allowed a deeper characterization of expression and localization of cyp19a1a gene in zebrafish gonads both at the adult stage and during development. At the adult stage, GFP expression was higher in ovaries than in testis. We showed a perfect co-expression of GFP and endogenous Cyp19a1a protein in gonads that was mainly localized in the cytoplasm of peri-follicular cells in the ovary and of Leydig and germ cells in the testis. During development, GFP was expressed in all immature gonads of 20 dpf-old zebrafish. Then, GFP expression increased in early differentiated female at 30 and 35dpf to reach a high GFP intensity in well-differentiated ovaries at 40dpf. On the contrary, males consistently displayed low GFP expression as compared to female whatever their stage of development, resulting in a clear dimorphic expression between both sexes. Interestingly, fish that undergoes ovary-to-testis transition (35 and 40dpf) presented GFP levels similar to males or intermediate between females and males. In this transgenic line our results confirm that cyp19a1a is expressed early during development, before the histological differentiation of the gonads, and that the down-regulation of cyp19a1a expression is likely responsible for the testicular differentiation. Moreover, we show that although cyp19a1a expression exhibits a clear dimorphic expression pattern in gonads during sexual differentiation, its expression persists whatever the sex suggesting that estradiol synthesis is important for gonadal development of both sexes. Monitoring the expression of GFP in control and exposed-fish will help determine the sensitivity of this transgenic line to EDCs and to refine mechanistic based-assays for the study of EDCs. In fine, this transgenic zebrafish line will be a useful tool to study physiological processes such as reproduction and sexual differentiation, and their perturbations by EDCs. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

Induction of endoplasmic reticulum calcium pump expression during early leukemic B cell differentiation.

PubMed

Aït Ghezali, Lamia; Arbabian, Atousa; Roudot, Hervé; Brouland, Jean-Philippe; Baran-Marszak, Fanny; Salvaris, Evelyn; Boyd, Andrew; Drexler, Hans G; Enyedi, Agnes; Letestu, Remi; Varin-Blank, Nadine; Papp, Bela

2017-06-26

Endoplasmic reticulum (ER) calcium storage and release play important roles in B lymphocyte maturation, survival, antigen-dependent cell activation and immunoglobulin synthesis. Calcium is accumulated in the endoplasmic reticulum (ER) by Sarco/Endoplasmic Reticulum Calcium ATPases (SERCA enzymes). Because lymphocyte function is critically dependent on SERCA activity, it is important to understand qualitative and quantitative changes of SERCA protein expression that occur during B lymphoid differentiation and leukemogenesis. In this work we investigated the modulation of SERCA expression during the pharmacologically induced differentiation of leukemic precursor B lymphoblast cell lines that carry the E2A-PBX1 fusion oncoprotein. Changes of SERCA levels during differentiation were determined and compared to those of established early B lymphoid differentiation markers. SERCA expression of the cells was compared to that of mature B cell lines as well, and the effect of the direct inhibition of SERCA-dependent calcium transport on the differentiation process was investigated. We show that E2A-PBX1 + leukemia cells simultaneously express SERCA2 and SERCA3-type calcium pumps; however, their SERCA3 expression is markedly inferior to that of mature B cells. Activation of protein kinase C enzymes by phorbol ester leads to phenotypic differentiation of the cells, and this is accompanied by the induction of SERCA3 expression. Direct pharmacological inhibition of SERCA-dependent calcium transport during phorbol ester treatment interferes with the differentiation process. These data show that the calcium pump composition of the ER is concurrent with increased SERCA3 expression during the differentiation of precursor B acute lymphoblastic leukemia cells, that a cross-talk exists between SERCA function and the control of differentiation, and that SERCA3 may constitute an interesting new marker for the study of early B cell phenotype.

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

[Expression and Significance of PI-PLCε1 in Colon Cancer].

PubMed

Li, Xiao-Ran; Yang, Kun; Huang, Xiao-Li

2017-11-01

To study the expression and clinical significance of phosphoinositide-specific phospholipase Cε1 (PI-PLCε1) in the pathogenesis of colon cancer. qRT-PCR and immunohistochemistry were used to detect the expression of PI-PLCε1 in the 42 cases of colon cancer tissues and their corresponding adjacent tissues. And the effects of tumor differentiation and tumor site on the expression PI-PLCε1 of colon cancer tissues were compared. The results of qRT-PCR showed that the expression of PI-PLCε1 in colon cancer tissue significantly lower than that in the adjacent tissue ( P <0.05). The expression of PI-PLCε1 gene of colon cancer tissue was not effected by tumor differentiation and tumor site ( P >0.05). The results of immuno-histochemistry showed that the positive expression rate of PI-PLCε1 protein in colon cancer tissue was significantly lower than that in the adjacent tissue ( P <0.05). The positive expression rate of PI-PLCε1 protein was not effected by tumor differentiation ( P >0.05),but the expression was different in tumor site ( P <0.05). Expression of PI-PLCε1 was reduced in colon tissue and barely to tumor differentiation.

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

Identification of embryonic precursor cells that differentiate into thymic epithelial cells expressing autoimmune regulator

PubMed Central

Takizawa, Nobukazu; Miyauchi, Maki; Yanai, Hiromi; Tateishi, Ryosuke; Shinzawa, Miho; Yoshinaga, Riko; Kurihara, Masaaki; Yasuda, Hisataka; Sakamoto, Reiko; Yoshida, Nobuaki

2016-01-01

Medullary thymic epithelial cells (mTECs) expressing autoimmune regulator (Aire) are critical for preventing the onset of autoimmunity. However, the differentiation program of Aire-expressing mTECs (Aire+ mTECs) is unclear. Here, we describe novel embryonic precursors of Aire+ mTECs. We found the candidate precursors of Aire+ mTECs (pMECs) by monitoring the expression of receptor activator of nuclear factor-κB (RANK), which is required for Aire+ mTEC differentiation. pMECs unexpectedly expressed cortical TEC molecules in addition to the mTEC markers UEA-1 ligand and RANK and differentiated into mTECs in reaggregation thymic organ culture. Introduction of pMECs in the embryonic thymus permitted long-term maintenance of Aire+ mTECs and efficiently suppressed the onset of autoimmunity induced by Aire+ mTEC deficiency. Mechanistically, pMECs differentiated into Aire+ mTECs by tumor necrosis factor receptor-associated factor 6-dependent RANK signaling. Moreover, nonclassical nuclear factor-κB activation triggered by RANK and lymphotoxin-β receptor signaling promoted pMEC induction from progenitors exhibiting lower RANK expression and higher CD24 expression. Thus, our findings identified two novel stages in the differentiation program of Aire+ mTECs. PMID:27401343

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.

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.

Fractalkine receptor is expressed in mature ovarian teratomas and required for epidermal lineage differentiation

PubMed Central

2013-01-01

Background The goal of this study was to determine a predominant cell type expressing fractalkine receptor (CX3CR1) in mature ovarian teratomas and to establish functional significance of its expression in cell differentiation. Methods Specimens of ovarian teratoma and human fetal tissues were analyzed by immunohistochemistry for CX3CR1expression. Ovarian teratocarcinoma cell line PA-1 was used as a model for cell differentiation. Results We found that the majority of the specimens contained CX3CR1-positive cells of epidermal lineage. Skin keratinocytes in fetal tissues were also CX3CR1- positive. PA-1 cells with downregulated CX3CR1 failed to express a skin keratinocyte marker cytokeratin 14 when cultured on Matrigel in the presence of a morphogen, bone morphogenic protein 4 (BMP-4), as compared to those expressing scrambled shRNA. Conclusions Here we demonstrate that CX3CR1 is expressed in both normally (fetal skin) and abnormally (ovarian teratoma) differentiated keratinocytes and is required for cell differentiation into epidermal lineage. PMID:23958497

[Regulation effects of liuwei dihuang pill, jingui shenqi pill, jiangu erxian pill containing serums on adipogenic and osteogenic differentiation-related genes expressions in the differentiation process of preadipocytes to osteoblasts].

PubMed

Cheng, Zhi-An; Han, Ling; Wei, Jian-An; Sun, Jing; Duan, Xiao-Dong

2013-02-01

To study the effects of Chinese medical recipes for invigorating Shen on rat bone marrow mesenchymal stem cells (BMSCs)-derived preadipocytes' differentiation to osteoblasts. The BMSCs were cultured using whole bone marrow adherence wall method. The BMSCs were induced to preadipocytes by classic chemical method. The osteogenic differentiation process of preadipocytes was intervened by Liuwei Dihuang Pill (LDP), Jingui Shenqi Pill (JSP), or Jiangu Erxian Pill (JEP)-containing serums (with the concentRation of 10%, on behalf of tonifying Shen yin, tonifying Shen yang, and tonifying Shen essence). Reverse transcription-real time fluorescent quantitative-PCR (RT real time qPCR) was used to detect RUNX2, ALP, BGP, BMP2, BMP4, SPP1, and IGF1 mRNA expressions of osteogenic differentiation-related genes, mRNA expressions of LPL, FABP4, and PPARgamma of adipogenic differentiation-related genes on the 6th, the 12th, and the 18th day. As for the osteogenic differentiation-related gene, when compared with the control group, there was no statistical difference in the gene expression level in the experimental groups on the 6th day (2.0 > Ratio > 0.5). On the 12th day, the mRNA expressions of IGF1 and Runx2 increased more significantly in the JSP group, with their relative quantification (Ratio) being 2.97 and 1.81 respectively. On the 18th day the IGF1 mRNA expression significantly increased, being the Ratio value of 3.74, 12.60, and 8.35, respectively, in the LDP group, the JSP group, and the JEP group. The SPP1 mRNA expression also significantly increased, with the Ratio value of 2.94, 3.18, and 2.62, respectively, in the LDP group, the JSP group, and the JEP group. As for adipogenic differentiation-related genes, on the 6th day, when compared with the control group, FABP4 mRNA expression significantly decreased in the LDP group and the JSP group (with the Ratio value of 0.47 and 0.40 respectively). The expression levels of other genes were all down-regulated, but not significantly. On the 12th day and 18th day, there was no statistical change in the adipogenic differentiation-related genes expressions (2.0 > Ratio > 0.5). Up-regulation of osteogenic differentiation-related genes expression occurred in later time, while down-regulation of adipogenic differentiation-related genes expression occurred in earlier time after treatment by Chinese medical recipes for invigorating Shen. In general, above data indicated that tonifying Shen yang was more effective in promoting osteogenic differentiation and inhibiting adipogenic differentiation of BMSCs.

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.

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.

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.

Residual Expression of the Reprogramming Factors Prevents Differentiation of iPSC Generated from Human Fibroblasts and Cord Blood CD34+ Progenitors

PubMed Central

Ramos-Mejía, Verónica; Montes, Rosa; Bueno, Clara; Ayllón, Verónica; Real, Pedro J.; Rodríguez, René; Menendez, Pablo

2012-01-01

Human induced pluripotent stem cells (hiPSC) have been generated from different tissues, with the age of the donor, tissue source and specific cell type influencing the reprogramming process. Reprogramming hematopoietic progenitors to hiPSC may provide a very useful cellular system for modelling blood diseases. We report the generation and complete characterization of hiPSCs from human neonatal fibroblasts and cord blood (CB)-derived CD34+ hematopoietic progenitors using a single polycistronic lentiviral vector containing an excisable cassette encoding the four reprogramming factors Oct4, Klf4, Sox2 and c-myc (OKSM). The ectopic expression of OKSM was fully silenced upon reprogramming in some hiPSC clones and was not reactivated upon differentiation, whereas other hiPSC clones failed to silence the transgene expression, independently of the cell type/tissue origin. When hiPSC were induced to differentiate towards hematopoietic and neural lineages those hiPSC which had silenced OKSM ectopic expression displayed good hematopoietic and early neuroectoderm differentiation potential. In contrast, those hiPSC which failed to switch off OKSM expression were unable to differentiate towards either lineage, suggesting that the residual expression of the reprogramming factors functions as a developmental brake impairing hiPSC differentiation. Successful adenovirus-based Cre-mediated excision of the provirus OKSM cassette in CB-derived CD34+ hiPSC with residual transgene expression resulted in transgene-free hiPSC clones with significantly improved differentiation capacity. Overall, our findings confirm that residual expression of reprogramming factors impairs hiPSC differentiation. PMID:22545141

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

Correlation between Urothelial Differentiation and Sensory Proteins P2X3, P2X5, TRPV1, and TRPV4 in Normal Urothelium and Papillary Carcinoma of Human Bladder

PubMed Central

Sterle, Igor; Zupančič, Daša; Romih, Rok

2014-01-01

Terminal differentiation of urothelium is a prerequisite for blood-urine barrier formation and enables normal sensory function of the urinary bladder. In this study, urothelial differentiation of normal human urothelium and of low and high grade papillary urothelial carcinomas was correlated with the expression and localization of purinergic receptors (P2X3, and P2X5) and transient receptor potential vanilloid channels (TRPV1, and TRPV4). Western blotting and immunofluorescence of uroplakins together with scanning electron microscopy of urothelial apical surface demonstrated terminal differentiation of normal urothelium, partial differentiation of low grade carcinoma, and poor differentiation of high grade carcinoma. P2X3 was expressed in normal urothelium as well as in low grade carcinoma and in both cases immunolabeling was stronger in the superficial cells. P2X3 expression decreased in high grade carcinoma. P2X5 expression was detected in normal urothelium and in high grade carcinoma, while in low grade carcinoma its expression was diminished. The expression of TRPV1 decreased in low grade and even more in high grade carcinoma when compared with normal urothelium, while TRPV4 expression was unchanged in all samples. Our results suggest that sensory proteins P2X3 and TRPV1 are in correlation with urothelial differentiation, while P2X5 and TRPV4 have unique expression patterns. PMID:24868547

Correlation between urothelial differentiation and sensory proteins P2X3, P2X5, TRPV1, and TRPV4 in normal urothelium and papillary carcinoma of human bladder.

PubMed

Sterle, Igor; Zupančič, Daša; Romih, Rok

2014-01-01

Terminal differentiation of urothelium is a prerequisite for blood-urine barrier formation and enables normal sensory function of the urinary bladder. In this study, urothelial differentiation of normal human urothelium and of low and high grade papillary urothelial carcinomas was correlated with the expression and localization of purinergic receptors (P2X3, and P2X5) and transient receptor potential vanilloid channels (TRPV1, and TRPV4). Western blotting and immunofluorescence of uroplakins together with scanning electron microscopy of urothelial apical surface demonstrated terminal differentiation of normal urothelium, partial differentiation of low grade carcinoma, and poor differentiation of high grade carcinoma. P2X3 was expressed in normal urothelium as well as in low grade carcinoma and in both cases immunolabeling was stronger in the superficial cells. P2X3 expression decreased in high grade carcinoma. P2X5 expression was detected in normal urothelium and in high grade carcinoma, while in low grade carcinoma its expression was diminished. The expression of TRPV1 decreased in low grade and even more in high grade carcinoma when compared with normal urothelium, while TRPV4 expression was unchanged in all samples. Our results suggest that sensory proteins P2X3 and TRPV1 are in correlation with urothelial differentiation, while P2X5 and TRPV4 have unique expression patterns.

Identification of differentially expressed genes in childhood asthma.

PubMed

Zhang, Nian-Zhen; Chen, Xiu-Juan; Mu, Yu-Hua; Wang, Hewen

2018-05-01

Asthma has been the most common chronic disease in children that places a major burden for affected people and their families.An integrated analysis of microarrays studies was performed to identify differentially expressed genes (DEGs) in childhood asthma compared with normal control. We also obtained the differentially methylated genes (DMGs) in childhood asthma according to GEO. The genes that were both differentially expressed and differentially methylated were identified. Functional annotation and protein-protein interaction network construction were performed to interpret biological functions of DEGs. We performed q-RT-PCR to verify the expression of selected DEGs.One DNA methylation and 3 gene expression datasets were obtained. Four hundred forty-one DEGs and 1209 DMGs in childhood asthma were identified. Among which, 16 genes were both differentially expressed and differentially methylated in childhood asthma. Natural killer cell mediated cytotoxicity pathway, Jak-STAT signaling pathway, and Wnt signaling pathway were 3 significantly enriched pathways in childhood asthma according to our KEGG enrichment analysis. The PPI network of top 20 up- and downregulated DEGs consisted of 822 nodes and 904 edges and 2 hub proteins (UBQLN4 and MID2) were identified. The expression of 8 DEGs (GZMB, FGFBP2, CLC, TBX21, ALOX15, IL12RB2, UBQLN4) was verified by qRT-PCR and only the expression of GZMB and FGFBP2 was inconsistent with our integrated analysis.Our finding was helpful to elucidate the underlying mechanism of childhood asthma and develop new potential diagnostic biomarker and provide clues for drug design.

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

Efficient experimental design and analysis strategies for the detection of differential expression using RNA-Sequencing

PubMed Central

2012-01-01

Background RNA sequencing (RNA-Seq) has emerged as a powerful approach for the detection of differential gene expression with both high-throughput and high resolution capabilities possible depending upon the experimental design chosen. Multiplex experimental designs are now readily available, these can be utilised to increase the numbers of samples or replicates profiled at the cost of decreased sequencing depth generated per sample. These strategies impact on the power of the approach to accurately identify differential expression. This study presents a detailed analysis of the power to detect differential expression in a range of scenarios including simulated null and differential expression distributions with varying numbers of biological or technical replicates, sequencing depths and analysis methods. Results Differential and non-differential expression datasets were simulated using a combination of negative binomial and exponential distributions derived from real RNA-Seq data. These datasets were used to evaluate the performance of three commonly used differential expression analysis algorithms and to quantify the changes in power with respect to true and false positive rates when simulating variations in sequencing depth, biological replication and multiplex experimental design choices. Conclusions This work quantitatively explores comparisons between contemporary analysis tools and experimental design choices for the detection of differential expression using RNA-Seq. We found that the DESeq algorithm performs more conservatively than edgeR and NBPSeq. With regard to testing of various experimental designs, this work strongly suggests that greater power is gained through the use of biological replicates relative to library (technical) replicates and sequencing depth. Strikingly, sequencing depth could be reduced as low as 15% without substantial impacts on false positive or true positive rates. PMID:22985019

Efficient experimental design and analysis strategies for the detection of differential expression using RNA-Sequencing.

PubMed

Robles, José A; Qureshi, Sumaira E; Stephen, Stuart J; Wilson, Susan R; Burden, Conrad J; Taylor, Jennifer M

2012-09-17

RNA sequencing (RNA-Seq) has emerged as a powerful approach for the detection of differential gene expression with both high-throughput and high resolution capabilities possible depending upon the experimental design chosen. Multiplex experimental designs are now readily available, these can be utilised to increase the numbers of samples or replicates profiled at the cost of decreased sequencing depth generated per sample. These strategies impact on the power of the approach to accurately identify differential expression. This study presents a detailed analysis of the power to detect differential expression in a range of scenarios including simulated null and differential expression distributions with varying numbers of biological or technical replicates, sequencing depths and analysis methods. Differential and non-differential expression datasets were simulated using a combination of negative binomial and exponential distributions derived from real RNA-Seq data. These datasets were used to evaluate the performance of three commonly used differential expression analysis algorithms and to quantify the changes in power with respect to true and false positive rates when simulating variations in sequencing depth, biological replication and multiplex experimental design choices. This work quantitatively explores comparisons between contemporary analysis tools and experimental design choices for the detection of differential expression using RNA-Seq. We found that the DESeq algorithm performs more conservatively than edgeR and NBPSeq. With regard to testing of various experimental designs, this work strongly suggests that greater power is gained through the use of biological replicates relative to library (technical) replicates and sequencing depth. Strikingly, sequencing depth could be reduced as low as 15% without substantial impacts on false positive or true positive rates.

Role of Ox-PAPCs in the Differentiation of Mesenchymal Stem Cells (MSCs) and Runx2 and PPARγ2 Expression in MSCs-Like of Osteoporotic Patients

PubMed Central

Valenti, Maria Teresa; Garbin, Ulisse; Pasini, Andrea; Zanatta, Mirko; Stranieri, Chiara; Manfro, Stefania; Zucal, Chiara; Dalle Carbonare, Luca

2011-01-01

Background Mesenchymal stem cells (MSCs) can differentiate into osteoblasts and adipocytes and conditions causing bone loss may induce a switch from the osteoblast to adipocyte lineage. In addition, the expression of Runx2 and the PPARγ2 transcription factor genes is essential for cellular commitment to an osteogenic and adipogenic differentiation, respectively. Modified lipoproteins derived from the oxidation of arachidonate-containing phospholipids (ox-PAPCs: POVPC, PGPC and PEIPC) are considered important factors in atherogenesis. Methodology We investigated the effect of ox-PAPCs on osteogenesis and adipogenesis in human mesenchymal stem cells (hMSCs). In particular, we analyzed the transcription factor Runx2 and the PPARγ2 gene expression during osteogenic and adipogenic differentiation in absence and in presence of ox-PAPCs. We also analyzed gene expression level in a panel of osteoblastic and adipogenic differentiation markers. In addition, as circulating blood cells can be used as a “sentinel” that responds to changes in the macro- or micro-environment, we analyzed the Runx2 and the PPARγ2 gene expression in MSCs-like and ox-PAPC levels in serum of osteoporotic patients (OPs). Finally, we examined the effects of sera obtained from OPs in hMSCs comparing the results with age-matched normal donors (NDs). Principal findings Quantitative RT-PCR demonstrated that ox-PAPCs enhanced PPARγ2 and adipogenic gene expression and reduced Runx2 and osteoblast differentiation marker gene expression in differentiating hMSCs. In OPs, ox-PAPC levels and PPARγ2 expression were higher than in NDs, whereas Runx2 was lower than in ND circulant MSCs-like. Conclusions Ox-PAPCs affect the osteogenic differentiation by promoting adipogenic differentiation and this effect may appear involved in bone loss in OPs. PMID:21674037

Am80 induces neuronal differentiation via increased tropomyosin-related kinase B expression in a human neuroblastoma SH-SY5Y cell line.

PubMed

Shiohira, Hideo; Kitaoka, Akira; Enjoji, Munechika; Uno, Tsukasa; Nakashima, Manabu

2012-01-01

Am80, a synthetic retinoid, has been used in differentiation therapy for acute promyelocytic leukemia (APL). All-trans retinoic acid (ATRA) as one of natural retinoid has been also used to treat APL. ATRA treatment causes neuronal differentiation by inducing tropomyosin-related kinase B (TrkB) expression and increasing the sensitivity to brain-derived neurotrophic factor (BDNF), a TrkB ligand. In the present study, we investigated the effects of Am80 on neuronal differentiation, BDNF sensitivity and TrkB expression in human neuroblastoma SH-SY5Y cells. Treatment with Am80 induced morphological differentiation of neurite outgrowth and increased the expression of GAP43 mRNA, a neuronal differentiation marker. Additionally, TrkB protein was also increased, and exogenous BDNF stimulation after treatment with Am80 induced greater neurite outgrowth than without BDNF treatment. These results suggest that Am80 induced neuronal differentiation by increasing TrkB expression and BDNF sensitivity.

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.

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.

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.

Expression of NLRR3 orphan receptor gene is negatively regulated by MYCN and Miz-1, and its downregulation is associated with unfavorable outcome in neuroblastoma.

PubMed

Akter, Jesmin; Takatori, Atsushi; Hossain, Md Shamim; Ozaki, Toshinori; Nakazawa, Atsuko; Ohira, Miki; Suenaga, Yusuke; Nakagawara, Akira

2011-11-01

Our previous study showed that expression of NLRR3 is significantly high in favorable neuroblastomas (NBL), whereas that of NLRR1 is significantly high in unfavorable NBLs. However, the molecular mechanism of transcriptional regulation of NLRR3 remains elusive. This study was undertaken to clarify the transcriptional regulation of NLRR3 and its association with the prognosis of NBL. NLRR3 and MYCN expressions in NBL cell lines were analyzed after induction of cell differentiation, MYCN knockdown, and overexpression. The transcriptional regulation of NLRR3 was analyzed by luciferase reporter and chromatin immunoprecipitation assays. Quantitative PCR was used for examining the expression of NLRR3, Miz-1, or MYCN in 87 primary NBLs. The expression of NLRR3 mRNA was upregulated during differentiation of NBL cells induced by retinoic acid, accompanied with reduced expression of MYCN, suggesting that NLRR3 expression was inversely correlated with MYCN in differentiation. Indeed, knockdown of MYCN induced NLRR3 expression, whereas exogenously expressed MYCN reduced cellular NLRR3 expression. We found that Miz-1 was highly expressed in favorable NBLs and NLRR3 was induced by Miz-1 expression in NBL cells. MYCN and Miz-1 complexes bound to NLRR3 promoter and showed a negative regulation of NLRR3 expression. In addition, a combination of low expression of NLRR3 and high expression of MYCN was highly associated with poor prognosis. NLRR3 is a direct target of MYCN, which associates with Miz-1 and negatively regulates NLRR3 expression. NLRR3 may play a role in NBL differentiation and the survival of NBL patients by inversely correlating with MYCN amplification. ©2011 AACR

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

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.

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

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.

Differential expression analysis of genes involved in high-temperature induced sex differentiation in Nile tilapia.

PubMed

Li, Chun Ge; Wang, Hui; Chen, Hong Ju; Zhao, Yan; Fu, Pei Sheng; Ji, Xiang Shan

2014-01-01

Nowadays, high temperature effects on the molecular pathways during sex differentiation in teleosts need to be deciphered. In this study, a systematic differential expression analysis of genes involved in high temperature-induced sex differentiation was done in the Nile tilapia gonad and brain. Our results showed that high temperature caused significant down-regulation of CYP19A1A in the gonad of both sexes in induction group, and FOXL2 in the ovary of the induction group. The expressions of GTHα, LHβ and ERα were also significantly down-regulated in the brain of both sexes in the induction and recovery groups. On the contrary, the expression of CYP11B2 was significantly up-regulated in the ovary, but not in the testis in both groups. Spearman rank correlation analysis showed that there are significant correlations between the expressions of CYP19A1A, FOXL2, or DMRT1 in the gonads and the expression of some genes in the brain. Another result in this study showed that high temperature up-regulated the expression level of DNMT1 in the testis of the induction group, and DNMT1 and DNMT3A in the female brain of both groups. The expression and correlation analysis of HSPs showed that high temperature action on tilapia HSPs might indirectly induce the expression changes of sex differentiation genes in the gonads. These findings provide new insights on TSD and suggest that sex differentiation related genes, heat shock proteins, and DNA methylation genes are new candidates for studying TSD in fish species. Copyright © 2014 Elsevier Inc. All rights reserved.

Identification and expression patterns of adipokine genes during adipocyte differentiation in the Tibetan goat (Capra hircus).

PubMed

Li, Xueying; Wang, Yan; Guo, Jiazhong; Zhong, Tao; Li, Li; Zhang, Hongping; Wang, Linjie

2018-02-15

Adipokines are secreted by adipose tissue and play an important role in the regulation of lipid metabolism. However, the information regarding adipokines in goats is limited. PPARγ is a key gene in adipocyte differentiation and activates adipokine genes. Rosiglitazone is a PPARγ agonist and can promote the expression of PPARγ to increase the expression of lipogenesis-related genes. Therefore, investigation of the relationship between rosiglitazone and adipokines will help us to better understand the function of PPARγ in lipid metabolism in Tibetan goats. In this study, we cloned the resistin (RETN), apelin (APLN), fibroblast growth factor 21 (FGF21), and visfatin (NAMPT) genes from non-pregnant female Tibetan goat adipose tissue. APLN and NAMPT were predominantly expressed in the kidney, and FGF21 was expressed at the highest levels in the liver in vivo. In fat tissues, the highest expression levels of FGF21 and RETN were detected in omental fat, whereas their expression in perirenal and subcutaneous fat was extremely weak. APLN and NAMPT were abundantly expressed in omental and subcutaneous fat in vivo. In addition, the four adipokines had different expression profiles during goat adipocyte differentiation in vitro. Oil red O staining showed that rosiglitazone could promote adipocyte differentiation and lipid droplet formation. In addition, rosiglitazone significantly increased the expression of FGF21 and RETN (p<0.05) but decreased the expression of APLN and NAMPT (p<0.05). These results suggest that the four adipocytokine genes may have different roles during goat adipocyte differentiation. And PPARγ could regulate the expression of the four adipokines, but the detailed regulatory mechanism still needs to be elucidated. Copyright © 2017 Elsevier B.V. All rights reserved.

Mirna biogenesis pathway is differentially regulated during adipose derived stromal/stem cell differentiation.

PubMed

Martin, E C; Qureshi, A T; Llamas, C B; Burow, M E; King, A G; Lee, O C; Dasa, V; Freitas, M A; Forsberg, J A; Elster, E A; Davis, T A; Gimble, J M

2018-02-07

Stromal/stem cell differentiation is controlled by a vast array of regulatory mechanisms. Included within these are methods of mRNA gene regulation that occur at the level of epigenetic, transcriptional, and/or posttranscriptional modifications. Current studies that evaluate the posttranscriptional regulation of mRNA demonstrate microRNAs (miRNAs) as key mediators of stem cell differentiation through the inhibition of mRNA translation. miRNA expression is enhanced during both adipogenic and osteogenic differentiation; however, the mechanism by which miRNA expression is altered during stem cell differentiation is less understood. Here we demonstrate for the first time that adipose-derived stromal/stem cells (ASCs) induced to an adipogenic or osteogenic lineage have differences in strand preference (-3p and -5p) for miRNAs originating from the same primary transcript. Furthermore, evaluation of miRNA expression in ASCs demonstrates alterations in both miRNA strand preference and 5'seed site heterogeneity. Additionally, we show that during stem cell differentiation there are alterations in expression of genes associated with the miRNA biogenesis pathway. Quantitative RT-PCR demonstrated changes in the Argonautes (AGO1-4), Drosha, and Dicer at intervals of ASC adipogenic and osteogenic differentiation compared to untreated ASCs. Specifically, we demonstrated altered expression of the AGOs occurring during both adipogenesis and osteogenesis, with osteogenesis increasing AGO1-4 expression and adipogenesis decreasing AGO1 gene and protein expression. These data demonstrate changes to components of the miRNA biogenesis pathway during stromal/stem cell differentiation. Identifying regulatory mechanisms for miRNA processing during ASC differentiation may lead to novel mechanisms for the manipulation of lineage differentiation of the ASC through the global regulation of miRNA as opposed to singular regulatory mechanisms.

Mouse ES cells have a potential to differentiate into odontoblast-like cells using hanging drop method.

PubMed

Kawai, R; Ozeki, N; Yamaguchi, H; Tanaka, T; Nakata, K; Mogi, M; Nakamura, H

2014-05-01

We examined whether mouse embryonic stem (ES) cells can differentiate into odontoblast-like cells without epithelial-mesenchymal interaction. Cells were cultured by the 'hanging drop' method using a collagen type-I scaffold (CS) combined with bone morphogenetic protein (BMP)-4 (CS/BMP-4). Expression of odontoblast-related mRNA and protein, and cell proliferation were performed by reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence staining and WST-1 assay, respectively. Cells potently expressed odontoblast-related cell marker mRNAs following induction of odontoblastic differentiation. Dentin sialophosphoprotein, a marker of mature odontoblasts, was strongly expressed in differentiated ES cells. The cells also acquired an odontoblast-like functional phenotype, as evidenced by the appearance of alkaline phosphatase activity and calcification. The cell-surface expression of α2, α6, αV and αVβ3 integrin proteins was rapidly upregulated in differentiated cells. Finally, anti-α2 integrin antibody suppressed the expression of odontoblastic markers in cells grown using this culture system, suggesting that α2 integrin expression in ES cells triggers their differentiation into odontoblast-like cells. Mouse ES cells cultured by the 'hanging drop' method are able to differentiate into cells with odontoblast-specific physiological functions and cell-surface integrin protein expression. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

MERP1: a mammalian ependymin-related protein gene differentially expressed in hematopoietic cells.

PubMed

Gregorio-King, Claudia C; McLeod, Janet L; Collier, Fiona McL; Collier, Gregory R; Bolton, Karyn A; Van Der Meer, Gavin J; Apostolopoulos, Jim; Kirkland, Mark A

2002-03-20

We have utilized differential display polymerase chain reaction to investigate the gene expression of hematopoietic progenitor cells from adult bone marrow and umbilical cord blood. A differentially expressed gene was identified in CD34+ hematopoietic progenitor cells, with low expression in CD34- cells. We have obtained the full coding sequence of this gene which we designated human mammalian ependymin-related protein 1 (MERP1). Expression of MERP1 was found in a variety of normal human tissues, and is 4- and 10-fold higher in adult bone marrow and umbilical cord blood CD34+ cells, respectively, compared to CD34- cells. Additionally, MERP1 expression in a hematopoietic stem cell enriched population was down-regulated with proliferation and differentiation. Conceptual translation of the MERP1 open reading frame reveals significant homology to two families of glycoprotein calcium-dependant cell adhesion molecules: ependymins and protocadherins.

Identification of Secretory Odontoblasts Using DMP1-GFP Transgenic Mice

PubMed Central

Balic, Anamaria; Mina, Mina

2011-01-01

Terminal differentiation of odontoblasts from dental papilla is a long process involving several intermediate steps and changes in the transcriptional profile and expression of proteins secreted by cells in the odontoblast lineage. Transgenic mouse lines in which GFP expression is under the control of tissue-and stage specific promoters have provided powerful experimental tools for identification and isolation of cells at specific stages of differentiation along a lineage. Our previous studies showed utilization of pOBCol3.6GFP and pOBCol2.3GFP animals for identification of odontoblasts at early and late stages of polarization respectively. In the present study we used the DMP1-GFP transgenic animal as an experimental model to examine its expression during the differentiation of odontoblasts from progenitor cells in vivo and in vitro. Our observations showed that DMP1-GFP transgene is first activated in secretory/functional odontoblasts engaged in secretion of predentin and then transiently expressed at high levels in newly differentiated odontoblasts. Expression of DMP1-GFP was down-regulated in highly differentiated odontoblasts. The temporal and spatial pattern of expression of DMP1-GFP transgene closely mimics the expression of endogenous DMP1. This transgenic animal will facilitate studies of gene expression and biological functions in secretory/functional odontoblasts. PMID:21172466

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

Selecting antagonistic antibodies that control differentiation through inducible expression in embryonic stem cells

PubMed Central

Melidoni, Anna N.; Dyson, Michael R.; Wormald, Sam; McCafferty, John

2013-01-01

Antibodies that modulate receptor function have great untapped potential in the control of stem cell differentiation. In contrast to many natural ligands, antibodies are stable, exquisitely specific, and are unaffected by the regulatory mechanisms that act on natural ligands. Here we describe an innovative system for identifying such antibodies by introducing and expressing antibody gene populations in ES cells. Following induced antibody expression and secretion, changes in differentiation outcomes of individual antibody-expressing ES clones are monitored using lineage-specific gene expression to identify clones that encode and express signal-modifying antibodies. This in-cell expression and reporting system was exemplified by generating blocking antibodies to FGF4 and its receptor FGFR1β, identified through delayed onset of ES cell differentiation. Functionality of the selected antibodies was confirmed by addition of exogenous antibodies to three different ES reporter cell lines, where retained expression of pluripotency markers Oct4, Nanog, and Rex1 was observed. This work demonstrates the potential for discovery and utility of functional antibodies in stem cell differentiation. This work is also unique in constituting an example of ES cells carrying an inducible antibody that causes a functional protein “knock-down” and allows temporal control of stable signaling components at the protein level. PMID:24082130

MURC, a muscle-restricted coiled-coil protein, is involved in the regulation of skeletal myogenesis.

PubMed

Tagawa, Masashi; Ueyama, Tomomi; Ogata, Takehiro; Takehara, Naofumi; Nakajima, Norio; Isodono, Koji; Asada, Satoshi; Takahashi, Tomosaburo; Matsubara, Hiroaki; Oh, Hidemasa

2008-08-01

Skeletal myogenesis is a multistep process by which multinucleated mature muscle fibers are formed from undifferentiated, mononucleated myoblasts. However, the molecular mechanisms of skeletal myogenesis have not been fully elucidated. Here, we identified muscle-restricted coiled-coil (MURC) protein as a positive regulator of myogenesis. In skeletal muscle, MURC was localized to the cytoplasm with accumulation in the Z-disc of the sarcomere. In C2C12 myoblasts, MURC expression occurred coincidentally with myogenin expression and preceded sarcomeric myosin expression during differentiation into myotubes. RNA interference (RNAi)-mediated knockdown of MURC impaired differentiation in C2C12 myoblasts, which was accompanied by impaired myogenin expression and ERK activation. Overexpression of MURC in C2C12 myoblasts resulted in the promotion of differentiation with enhanced myogenin expression and ERK activation during differentiation. During injury-induced muscle regeneration, MURC expression increased, and a higher abundance of MURC was observed in immature myofibers compared with mature myofibers. In addition, ERK was activated in regenerating tissue, and ERK activation was detected in MURC-expressing immature myofibers. These findings suggest that MURC is involved in the skeletal myogenesis that results from modulation of myogenin expression and ERK activation. MURC may play pivotal roles in the molecular mechanisms of skeletal myogenic differentiation.

The preferentially expressed antigen in melanoma (PRAME) inhibits myeloid differentiation in normal hematopoietic and leukemic progenitor cells

PubMed Central

Guthrie, Katherine A.; Cummings, Carrie L.; Sabo, Kathleen; Wood, Brent L.; Gooley, Ted; Yang, Taimei; Epping, Mirjam T.; Shou, Yaping; Pogosova-Agadjanyan, Era; Ladne, Paula; Stirewalt, Derek L.; Abkowitz, Janis L.; Radich, Jerald P.

2009-01-01

The preferentially expressed antigen in melanoma (PRAME) is expressed in several hematologic malignancies, but either is not expressed or is expressed at only low levels in normal hematopoietic cells, making it a target for cancer therapy. PRAME is a tumor-associated antigen and has been described as a corepressor of retinoic acid signaling in solid tumor cells, but its function in hematopoietic cells is unknown. PRAME mRNA expression increased with chronic myeloid leukemia (CML) disease progression and its detection in late chronic-phase CML patients before tyrosine kinase inhibitor therapy was associated with poorer therapeutic responses and ABL tyrosine kinase domain point mutations. In leukemia cell lines, PRAME protein expression inhibited granulocytic differentiation only in cell lines that differentiate along this lineage after all-trans retinoic acid (ATRA) exposure. Forced PRAME expression in normal hematopoietic progenitors, however, inhibited myeloid differentiation both in the presence and absence of ATRA, and this phenotype was reversed when PRAME was silenced in primary CML progenitors. These observations suggest that PRAME inhibits myeloid differentiation in certain myeloid leukemias, and that its function in these cells is lineage and phenotype dependent. Lastly, these observations suggest that PRAME is a target for both prognostic and therapeutic applications. PMID:19625708

Hepatocyte nuclear factor-4alpha induces transdifferentiation of hematopoietic cells into hepatocytes.

PubMed

Khurana, Satish; Jaiswal, Amit K; Mukhopadhyay, Asok

2010-02-12

Hematopoietic stem cells can directly transdifferentiate into hepatocytes because of cellular plasticity, but the molecular basis of transdifferentiation is not known. Here, we show the molecular basis using lineage-depleted oncostatin M receptor beta-expressing (Lin(-)OSMRbeta(+)) mouse bone marrow cells in a hepatic differentiation culture system. Differentiation of the cells was marked by the expression of albumin. Hepatocyte nuclear factor (HNF)-4alpha was expressed and translocated into the nuclei of the differentiating cells. Suppression of its activation in OSM-neutralized culture medium inhibited cellular differentiation. Ectopic expression of full-length HNF4alpha in 32D myeloid cells resulted in decreased myeloid colony-forming potential and increased expression of hepatocyte-specific genes and proteins. Nevertheless, the neohepatocytes produced in culture expressed active P450 enzyme. The obligatory role of HNF4alpha in hepatic differentiation was confirmed by transfecting Lin(-)OSMRbeta(+) cells with dominant negative HNF4alpha in the differentiation culture because its expression inhibited the transcription of the albumin and tyrosine aminotransferase genes. The loss and gain of functional activities strongly suggested that HNF4alpha plays a central role in the transdifferentiation process. For the first time, this report demonstrates the mechanism of transdifferentiation of hematopoietic cells into hepatocytes, in which HNF4alpha serves as a molecular switch.

Genes and Proteins Differentially Expressed during In Vitro Malignant Transformation of Bovine Pancreatic Duct Cells1

PubMed Central

Jesnowski, R; Zubakov, Dmitri; Faissner, Ralf; Ringel, Jörg; Hoheisel, Jörg D; Lösel, Ralf; Schnölzer, Martina; Löhr, Matthias

2007-01-01

Abstract Pancreatic carcinoma has an extremely bad prognosis due to lack of early diagnostic markers and lack of effective therapeutic strategies. Recently, we have established an in vitro model recapitulating the first steps in the carcinogenesis of the pancreas. SV40 large T antigen-immortalized bovine pancreatic duct cells formed intrapancreatic adenocarcinoma tumors on k-rasmut transfection after orthotopic injection in the nude mouse pancreas. Here we identified genes and proteins differentially expressed in the course of malignant transformation using reciprocal suppression subtractive hybridization and 2D gel electrophoresis and mass spectrometry, respectively. We identified 34 differentially expressed genes, expressed sequence tags, and 15 unique proteins. Differential expression was verified for some of the genes or proteins in samples from pancreatic carcinoma. Among these genes and proteins, the majority had already been described either to be influenced by a mutated ras or to be differentially expressed in pancreatic adenocarcinoma, thus proving the feasibility of our model. Other genes and proteins (e.g., BBC1, GLTSCR2, and rhoGDIα), up to now, have not been implicated in pancreatic tumor development. Thus, we were able to establish an in vitro model of pancreatic carcinogenesis, which enabled us to identify genes and proteins differentially expressed during the early steps of malignant transformation. PMID:17356710

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.

Orphan nuclear receptor chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) protein negatively regulates bone morphogenetic protein 2-induced osteoblast differentiation through suppressing runt-related gene 2 (Runx2) activity.

PubMed

Lee, Kkot-Nim; Jang, Won-Gu; Kim, Eun-Jung; Oh, Sin-Hye; Son, Hye-Ju; Kim, Sun-Hun; Franceschi, Renny; Zhang, Xiao-Kun; Lee, Shee-Eun; Koh, Jeong-Tae

2012-06-01

Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) is an orphan nuclear receptor of the steroid-thyroid hormone receptor superfamily. COUP-TFII is widely expressed in multiple tissues and organs throughout embryonic development and has been shown to regulate cellular growth, differentiation, and organ development. However, the role of COUP-TFII in osteoblast differentiation has not been systematically evaluated. In the present study, COUP-TFII was strongly expressed in multipotential mesenchymal cells, and the endogenous expression level decreased during osteoblast differentiation. Overexpression of COUP-TFII inhibited bone morphogenetic protein 2 (BMP2)-induced osteoblastic gene expression. The results of alkaline phosphatase, Alizarin Red staining, and osteocalcin production assay showed that COUP-TFII overexpression blocks BMP2-induced osteoblast differentiation. In contrast, the down-regulation of COUP-TFII synergistically induced the expression of BMP2-induced osteoblastic genes and osteoblast differentiation. Furthermore, the immunoprecipitation assay showed that COUP-TFII and Runx2 physically interacted and COUP-TFII significantly impaired the Runx2-dependent activation of the osteocalcin promoter. From the ChIP assay, we found that COUP-TFII repressed DNA binding of Runx2 to the osteocalcin gene, whereas Runx2 inhibited COUP-TFII expression via direct binding to the COUP-TFII promoter. Taken together, these findings demonstrate that COUP-TFII negatively regulates osteoblast differentiation via interaction with Runx2, and during the differentiation state, BMP2-induced Runx2 represses COUP-TFII expression and promotes osteoblast differentiation.

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

PubMed

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

2016-02-15

To investigate the intestinal functions of the NKCC1 Na(+)-K(+)-2Cl cotransporter (SLC12a2 gene), differential mRNA expression changes in NKCC1-null intestine were analyzed. 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. 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. 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.

Expression of aromatase in the embryonic brain of the olive ridley sea turtle (Lepidochelys olivacea), and the effect of bisphenol-A in sexually differentiated embryos.

PubMed

Gómez-Picos, Patsy; Sifuentes-Romero, Itzel; Merchant-Larios, Horacio; Hernández-Cornejo, Rubí; Díaz-Hernández, Verónica; García-Gasca, Alejandra

2014-01-01

Brain aromatase participates in several biological processes, such as regulation of the reproductive-endocrine axis, memory, stress, sexual differentiation of the nervous system, male sexual behavior, and brain repair. Here we report the isolation and expression of brain aromatase in olive ridley sea turtle (Lepidochelys olivacea) embryos incubated at male- and female-promoting temperatures (MPT and FPT, respectively), at the thermosensitive period (TSP) and the sex-differentiated period. Also, aromatase expression was assessed in differentiated embryos exposed to bisphenol-A (BPA) during the TSP. BPA is a monomer of polycarbonate plastics and is considered an endocrine-disrupting compound. Normal aromatase expression was measured in both forebrain and hindbrain, showing higher expression levels in the forebrain of differentiated embryos at both incubation temperatures. Although no significant differences were detected in the hindbrain, expression was slightly higher at MPT. BPA did not affect aromatase expression neither in forebrains or hindbrains from embryos incubated at MPT, whereas at FPT an inverted U-shape curve was observed in forebrains with significant differences at lower concentrations, whereas in hindbrains a non-significant increment was observed at higher concentrations. Our data indicate that both incubation temperature and developmental stage are critical factors affecting aromatase expression in the forebrain. Because of the timing and location of aromatase expression in the brain, we suggest that brain aromatase may participate in the imprinting of sexual trends related to reproduction and sexual behavior at the onset of sex differentiation, and BPA exposure may impair aromatase function in the female forebrain.

Expression of multiple slow myosin heavy chain genes reveals a diversity of zebrafish slow twitch muscle fibres with differing requirements for Hedgehog and Prdm1 activity.

PubMed

Elworthy, Stone; Hargrave, Murray; Knight, Robert; Mebus, Katharina; Ingham, Philip W

2008-06-01

The zebrafish embryo develops a series of anatomically distinct slow twitch muscle fibres that characteristically express genes encoding lineage-specific isoforms of sarcomeric proteins such as MyHC and troponin. We show here that different subsets of these slow fibres express distinct members of a tandem array of slow MyHC genes. The first slow twitch muscle fibres to differentiate, which are specified by the activity of the transcription factor Prdm1 (also called Ubo or Blimp1) in response to Hedgehog (Hh) signalling, express the smyhc1 gene. Subsequently, secondary slow twitch fibres differentiate in most cases independently of Hh activity. We find that although some of these later-forming fibres also express smyhc1, others express smyhc2 or smyhc3. We show that the smyhc1-positive fibres express the ubo (prdm1) gene and adopt fast twitch fibre characteristics in the absence of Prdm1 activity, whereas those that do not express smyhc1 can differentiate independently of Prdm1 function. Conversely, some smyhc2-expressing fibres, although independent of Prdm1 function, require Hh activity to form. The adult trunk slow fibres express smyhc2 and smyhc3, but lack smyhc1 expression. The different slow fibres in the craniofacial muscles variously express smyhc1, smyhc2 and smyhc3, and all differentiate independently of Prdm1.

YAP regulates neuronal differentiation through Sonic hedgehog signaling pathway

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

Lin, Yi-Ting; Ding, Jing-Ya; Li, Ming-Yang

2012-09-10

Tight regulation of cell numbers by controlling cell proliferation and apoptosis is important during development. Recently, the Hippo pathway has been shown to regulate tissue growth and organ size in Drosophila. In mammalian cells, it also affects cell proliferation and differentiation in various tissues, including the nervous system. Interplay of several signaling cascades, such as Notch, Wnt, and Sonic Hedgehog (Shh) pathways, control cell proliferation during neuronal differentiation. However, it remains unclear whether the Hippo pathway coordinates with other signaling cascades in regulating neuronal differentiation. Here, we used P19 cells, a mouse embryonic carcinoma cell line, as a model tomore » study roles of YAP, a core component of the Hippo pathway, in neuronal differentiation. P19 cells can be induced to differentiate into neurons by expressing a neural bHLH transcription factor gene Ascl1. Our results showed that YAP promoted cell proliferation and inhibited neuronal differentiation. Expression of Yap activated Shh but not Wnt or Notch signaling activity during neuronal differentiation. Furthermore, expression of Yap increased the expression of Patched homolog 1 (Ptch1), a downstream target of the Shh signaling. Knockdown of Gli2, a transcription factor of the Shh pathway, promoted neuronal differentiation even when Yap was over-expressed. We further demonstrated that over-expression of Yap inhibited neuronal differentiation in primary mouse cortical progenitors and Gli2 knockdown rescued the differentiation defect in Yap over-expressing cells. In conclusion, our study reveals that Shh signaling acts downstream of YAP in regulating neuronal differentiation. -- Highlights: Black-Right-Pointing-Pointer YAP promotes cell proliferation and inhibits neuronal differentiation in P19 cells. Black-Right-Pointing-Pointer YAP promotes Sonic hedgehog signaling activity during neuronal differentiation. Black-Right-Pointing-Pointer Knockdown of Gli2 rescues the Yap-overexpression phenotype in P19 cells. Black-Right-Pointing-Pointer Knockdown of Gli2 rescues the Yap-overexpression phenotype in cortical progenitors.« less

CD36 is required for myoblast fusion during myogenic differentiation

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

Park, Seung-Yoon; Yun, Youngeun; Kim, In-San, E-mail: iskim@knu.ac.kr

2012-11-02

Highlights: Black-Right-Pointing-Pointer CD36 expression was induced during myogenic differentiation. Black-Right-Pointing-Pointer CD36 expression was localized in multinucleated myotubes. Black-Right-Pointing-Pointer The expression of myogenic markers is attenuated in CD36 knockdown C2C12 cells. Black-Right-Pointing-Pointer Knockdown of CD36 significantly inhibited myotube formation during differentiation. -- Abstract: Recently, CD36 has been found to be involved in the cytokine-induced fusion of macrophage. Myoblast fusion to form multinucleated myotubes is required for myogenesis and muscle regeneration. Because a search of gene expression database revealed the attenuation of CD36 expression in the muscles of muscular dystrophy patients, the possibility that CD36 could be required for myoblast fusion wasmore » investigated. CD36 expression was markedly up-regulated during myoblast differentiation and localized in multinucleated myotubes. Knockdown of endogenous CD36 significantly decreased the expression of myogenic markers as well as myotube formation. These results support the notion that CD36 plays an important role in cell fusion during myogenic differentiation. Our finding will aid the elucidation of the common mechanism governing cell-to-cell fusion in various fusion models.« less

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.

Sex-specific differences in transcriptome profiles of brain and muscle tissue of the tropical gar.

PubMed

Cribbin, Kayla M; Quackenbush, Corey R; Taylor, Kyle; Arias-Rodriguez, Lenin; Kelley, Joanna L

2017-04-07

The tropical gar (Atractosteus tropicus) is the southernmost species of the seven extant species of gar fishes in the world. In Mexico and Central America, the species is an important food source due to its nutritional quality and low price. Despite its regional importance and increasing concerns about overexploitation and habitat degradation, basic genetic information on the tropical gar is lacking. Determining genetic information on the tropical gar is important for the sustainable management of wild populations, implementation of best practices in aquaculture settings, evolutionary studies of ancient lineages, and an understanding of sex-specific gene expression. In this study, the transcriptome of the tropical gar was sequenced and assembled de novo using tissues from three males and three females using Illumina sequencing technology. Sex-specific and highly differentially expressed transcripts in brain and muscle tissues between adult males and females were subsequently identified. The transcriptome was assembled de novo resulting in 80,611 transcripts with a contig N50 of 3,355 base pairs and over 168 kilobases in total length. Male muscle, brain, and gonad as well as female muscle and brain were included in the assembly. The assembled transcriptome was annotated to identify the putative function of expressed transcripts using Trinotate and SwissProt, a database of well-annotated proteins. The brain and muscle datasets were then aligned to the assembled transcriptome to identify transcripts that were differentially expressed between males and females. The contrast between male and female brain identified 109 transcripts from 106 genes that were significantly differentially expressed. In the muscle comparison, 82 transcripts from 80 genes were identified with evidence for significant differential expression. Almost all genes identified as differentially expressed were sex-specific. The differentially expressed transcripts were enriched for genes involved in cellular functioning, signaling, immune response, and tissue-specific functions. This study identified differentially expressed transcripts between male and female gar in muscle and brain tissue. The majority of differentially expressed transcripts had sex-specific expression. Expanding on these findings to other developmental stages, populations, and species may lead to the identification of genetic factors contributing to the skewed sex ratio seen in the tropical gar and of sex-specific differences in expression in other species. Finally, the transcriptome assembly will open future research avenues on tropical gar development, cell function, environmental resistance, and evolution in the context of other early vertebrates.

Retinoic acid-induced CHD5 upregulation and neuronal differentiation of neuroblastoma.

PubMed

Higashi, Mayumi; Kolla, Venkatadri; Iyer, Radhika; Naraparaju, Koumudi; Zhuang, Tiangang; Kolla, Sriharsha; Brodeur, Garrett M

2015-08-07

Chromodomain-helicase DNA binding protein 5 (CHD5) is an important tumor suppressor gene deleted from 1p36.31 in neuroblastomas (NBs). High CHD5 expression is associated with a favorable prognosis, but deletion or low expression is frequent in high-risk tumors. We explored the role of CHD5 expression in the neuronal differentiation of NB cell lines. NB cell lines SH-SY5Y (SY5Y), NGP, SK-N-DZ, IMR5, LAN5, SK-N-FI, NB69 and SH-EP were treated with 1-10 μM 13-cis-retinoic acid (13cRA) for 3-12 days. qRT-PCR and Western blot analyses were performed to measure mRNA and protein expression levels, respectively. Morphological differences were examined by both phase contrast and immunofluorescence studies. Treatment of SY5Y cells with 13cRA caused upregulation of CHD5 expression in a time- and dose-dependent manner (1, 5, or 10 μM for 7 or 12 days) and also induced neuronal differentiation. Furthermore, both NGP and SK-N-DZ cells showed CHD5 upregulation and neuronal differentiation after 13cRA treatment. In contrast, 13cRA treatment of IMR5, LAN5, or SK-N-FI induced neither CHD5 expression nor neuronal differentiation. NB69 cells showed two different morphologies (neuronal and substrate adherent) after 12 days treatment with 10 μM of 13cRA. CHD5 expression was high in the neuronal cells, but low/absent in the flat, substrate adherent cells. Finally, NGF treatment caused upregulation of CHD5 expression and neuronal differentiation in SY5Y cells transfected to express TrkA (SY5Y-TrkA) but not in TrkA-null parental SY5Y cells, and both changes were blocked by a pan-TRK inhibitor. Treatment with 13cRA induces neuronal differentiation only in NB cells that upregulate CHD5. In addition, NGF induced CHD5 upregulation and neuronal differentiation only in TrkA expressing cells. Together, these results suggest that CHD5 is downstream of TrkA, and CHD5 expression may be crucial for neuronal differentiation induced by either 13cRA or TrkA/NGF signaling.

The expression and activity of thioredoxin reductase 1 splice variants v1 and v2 regulate the expression of genes associated with differentiation and adhesion

PubMed Central

Nalvarte, Ivan; Damdimopoulos, Anastasios E.; Rüegg, Joëlle; Spyrou, Giannis

2015-01-01

The mammalian redox-active selenoprotein thioredoxin reductase (TrxR1) is a main player in redox homoeostasis. It transfers electrons from NADPH to a large variety of substrates, particularly to those containing redox-active cysteines. Previously, we reported that the classical form of cytosolic TrxR1 (TXNRD1_v1), when overexpressed in human embryonic kidney cells (HEK-293), prompted the cells to undergo differentiation [Nalvarte et al. (2004) J. Biol. Chem. 279, 54510–54517]. In the present study, we show that several genes associated with differentiation and adhesion are differentially expressed in HEK-293 cells stably overexpressing TXNRD1_v1 compared with cells expressing its splice variant TXNRD1_v2. Overexpression of these two splice forms resulted in distinctive effects on various aspects of cellular functions including gene regulation patterns, alteration of growth rate, migration and morphology and susceptibility to selenium-induced toxicity. Furthermore, differentiation of the neuroblastoma cell line SH-SY5Y induced by all-trans retinoic acid (ATRA) increased both TXNRD1_v1 and TXNRD1_v2 expressions along with several of the identified genes associated with differentiation and adhesion. Selenium supplementation in the SH-SY5Y cells also induced a differentiated morphology and changed expression of the adhesion protein fibronectin 1 and the differentiation marker cadherin 11, as well as different temporal expression of the studied TXNRD1 variants. These data suggest that both TXNRD1_v1 and TXNRD1_v2 have distinct roles in differentiation, possibly by altering the expression of the genes associated with differentiation, and further emphasize the importance in distinguishing each unique action of different TrxR1 splice forms, especially when studying the gene silencing or knockout of TrxR1. PMID:26464515

Gene Expression Profile of NF-κB, Nrf2, Glycolytic, and p53 Pathways During the SH-SY5Y Neuronal Differentiation Mediated by Retinoic Acid.

PubMed

de Bittencourt Pasquali, Matheus Augusto; de Ramos, Vitor Miranda; Albanus, Ricardo D Oliveira; Kunzler, Alice; de Souza, Luis Henrinque Trentin; Dalmolin, Rodrigo Juliani Siqueira; Gelain, Daniel Pens; Ribeiro, Leila; Carro, Luigi; Moreira, José Cláudio Fonseca

2016-01-01

SH-SY5Y cells, a neuroblastoma cell line that is a well-established model system to study the initial phases of neuronal differentiation, have been used in studies to elucidate the mechanisms of neuronal differentiation. In the present study, we investigated alterations of gene expression in SH-SY5Y cells during neuronal differentiation mediated by retinoic acid (RA) treatment. We evaluated important pathways involving nuclear factor kappa B (NF-κB), nuclear E2-related factor 2 (Nrf2), glycolytic, and p53 during neuronal differentiation. We also investigated the involvement of reactive oxygen species (ROS) in modulating the gene expression profile of those pathways by antioxidant co-treatment with Trolox®, a hydrophilic analogue of α-tocopherol. We found that RA treatment increases levels of gene expression of NF-κB, glycolytic, and antioxidant pathway genes during neuronal differentiation of SH-SY5Y cells. We also found that ROS production induced by RA treatment in SH-SY5Y cells is involved in gene expression profile alterations, chiefly in NF-κB, and glycolytic pathways. Antioxidant co-treatment with Trolox® reversed the effects mediated by RA NF-κB, and glycolytic pathways gene expression. Interestingly, co-treatment with Trolox® did not reverse the effects in antioxidant gene expression mediated by RA in SH-SY5Y. To confirm neuronal differentiation, we quantified endogenous levels of tyrosine hydroxylase, a recognized marker of neuronal differentiation. Our data suggest that during neuronal differentiation mediated by RA, changes in profile gene expression of important pathways occur. These alterations are in part mediated by ROS production. Therefore, our results reinforce the importance in understanding the mechanism by which RA induces neuronal differentiation in SH-SY5Y cells, principally due this model being commonly used as a neuronal cell model in studies of neuronal pathologies.

Temporal analysis of reciprocal miRNA-mRNA expression patterns predicts regulatory networks during differentiation in human skeletal muscle cells

PubMed Central

Sjögren, Rasmus J. O.; Egan, Brendan; Katayama, Mutsumi; Zierath, Juleen R.

2014-01-01

microRNAs (miRNAs) are short noncoding RNAs that regulate gene expression through posttranscriptional repression of target genes. miRNAs exert a fundamental level of control over many developmental processes, but their role in the differentiation and development of skeletal muscle from myogenic progenitor cells in humans remains incompletely understood. Using primary cultures established from human skeletal muscle satellite cells, we performed microarray profiling of miRNA expression during differentiation of myoblasts (day 0) into myotubes at 48 h intervals (day 2, 4, 6, 8, and 10). Based on a time-course analysis, we identified 44 miRNAs with altered expression [false discovery rate (FDR) < 5%, fold change > ±1.2] during differentiation, including the marked upregulation of the canonical myogenic miRNAs miR-1, miR-133a, miR-133b, and miR-206. Microarray profiling of mRNA expression at day 0, 4, and 10 identified 842 and 949 genes differentially expressed (FDR < 10%) at day 4 and 10, respectively. At day 10, 42% of altered transcripts demonstrated reciprocal expression patterns in relation to the directional change of their in silico predicted regulatory miRNAs based on analysis using Ingenuity Pathway Analysis microRNA Target Filter. Bioinformatic analysis predicted networks of regulation during differentiation including myomiRs miR-1/206 and miR-133a/b, miRNAs previously established in differentiation including miR-26 and miR-30, and novel miRNAs regulated during differentiation of human skeletal muscle cells such as miR-138-5p and miR-20a. These reciprocal expression patterns may represent new regulatory nodes in human skeletal muscle cell differentiation. This analysis serves as a reference point for future studies of human skeletal muscle differentiation and development in healthy and disease states. PMID:25547110

Purification and differentiation of human adipose-derived stem cells by membrane filtration and membrane migration methods

PubMed Central

Lin, Hong Reng; Heish, Chao-Wen; Liu, Cheng-Hui; Muduli, Saradaprasan; Li, Hsing-Fen; Higuchi, Akon; Kumar, S. Suresh; Alarfaj, Abdullah A.; Munusamy, Murugan A.; Hsu, Shih-Tien; Chen, Da-Chung; Benelli, Giovanni; Murugan, Kadarkarai; Cheng, Nai-Chen; Wang, Han-Chow; Wu, Gwo-Jang

2017-01-01

Human adipose-derived stem cells (hADSCs) are easily isolated from fat tissue without ethical concerns, but differ in purity, pluripotency, differentiation ability, and stem cell marker expression, depending on the isolation method. We isolated hADSCs from a primary fat tissue solution using: (1) conventional culture, (2) a membrane filtration method, (3) a membrane migration method where the primary cell solution was permeated through membranes, adhered hADSCs were cultured, and hADSCs migrated out from the membranes. Expression of mesenchymal stem cell markers and pluripotency genes, and osteogenic differentiation were compared for hADSCs isolated by different methods using nylon mesh filter membranes with pore sizes ranging from 11 to 80 μm. hADSCs isolated by the membrane migration method had the highest MSC surface marker expression and efficient differentiation into osteoblasts. Osteogenic differentiation ability of hADSCs and MSC surface marker expression were correlated, but osteogenic differentiation ability and pluripotent gene expression were not. PMID:28071738

[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.

Role of fibroblast growth factor receptors (FGFR) and FGFR like-1 (FGFRL1) in mesenchymal stromal cell differentiation to osteoblasts and adipocytes.

PubMed

Kähkönen, T E; Ivaska, K K; Jiang, M; Büki, K G; Väänänen, H K; Härkönen, P L

2018-02-05

Fibroblast growth factors (FGF) and their receptors (FGFRs) regulate many developmental processes including differentiation of mesenchymal stromal cells (MSC). We developed two MSC lines capable of differentiating to osteoblasts and adipocytes and studied the role of FGFRs in this process. We identified FGFR2 and fibroblast growth factor receptor like-1 (FGFRL1) as possible actors in MSC differentiation with gene microarray and qRT-PCR. FGFR2 and FGFRL1 mRNA expression strongly increased during MSC differentiation to osteoblasts. FGF2 treatment, resulting in downregulation of FGFR2, or silencing FGFR2 expression with siRNAs inhibited osteoblast differentiation. During adipocyte differentiation expression of FGFR1 and FGFRL1 increased and was down-regulated by FGF2. FGFR1 knockdown inhibited adipocyte differentiation. Silencing FGFR2 and FGFR1 in MSCs was associated with decreased FGFRL1 expression in osteoblasts and adipocytes, respectively. Our results suggest that FGFR1 and FGFR2 regulate FGFRL1 expression. FGFRL1 may mediate or modulate FGFR regulation of MSC differentiation together with FGFR2 in osteoblastic and FGFR1 in adipocytic lineage. Copyright © 2017 Elsevier B.V. All rights reserved.

Differentially expressed proteins among normal cervix, cervical intraepithelial neoplasia and cervical squamous cell carcinoma.

PubMed

Zhao, Q; He, Y; Wang, X-L; Zhang, Y-X; Wu, Y-M

2015-08-01

To explore the differentially expressed proteins in normal cervix, cervical intraepithelial neoplasia (CIN) and cervical squamous cell carcinoma (CSCC) tissues by differential proteomics technique. Cervical tissues (including normal cervix, CIN and CSCC) were collected in Department of Gynecologic Oncology of Beijing Obstetrics and Gynecology Hospital. Two-dimensional fluorescence difference in gel electrophoresis (2-D DIGE) and DeCyder software were used to detect the differentially expressed proteins. Matrix-assisted laser desorption/ionization-time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS) was used to identify the differentially expressed proteins. Western blot (WB) and immunohistochemistry (IHC) were performed to validate the expressions of selected proteins among normal cervix, CIN and CSCC. 2-D DIGE images with high resolution and good repeatability were obtained. Forty-six differentially expressed proteins (27 up-regulated and 19 down-regulated) were differentially expressed among the normal cervix, CIN and CSCC. 26 proteins were successfully identified by MALDI-TOF/TOF MS. S100A9 (S100 calcium-binding protein A9) was the most significantly up-regulated protein. Eukaryotic elongation factor 1-alpha-1 (eEF1A1) was the most significantly down-regulated protein. Pyruvate kinase isozymes M2 (PKM2) was both up-regulated and down-regulated. The results of WB showed that with the increase in the severity of cervical lesions, the expression of S100A9 protein was significantly increased among the three groups (P = 0.010). The expression of eEF1A1 was reduced but without significant difference (P = 0.861). The expression of PKM2 was significantly reduced (P = 0.000). IHC showed that protein S100A9 was mainly expressed in the cytoplasm, and its positive expression rate was 20.0 % in normal cervix, 70.0 % in CIN and 100.0 % in CSCC, with a significant difference among them (P = 0.006). eEF1A1 was mainly expressed in the cell plasma, and its positive expression rate was 70.0 % in normal cervix, 73.3 % in CIN and 60.0 % in CSCC tissues, without significant difference among them (P = 0.758). PKM2 was mainly expressed in the cell nuclei, and its positive expression rate was 100.0 % in normal cervix, 93.3 % in CIN and 75.0 % in CSCC tissues, showing a difference close to statistical significance (P = 0.059) among them. There are differentially expressed proteins among normal cervix, CIN and CSCC. S100A9, eEF1A1 and PKM2 may become candidate markers for early diagnosis of cervical cancer and new targets for therapy. It also provides a basis for further studies of the mechanism for CIN developing to CSCC.

UTX regulates mesoderm differentiation of embryonic stem cells independent of H3K27 demethylase activity.

PubMed

Wang, Chaochen; Lee, Ji-Eun; Cho, Young-Wook; Xiao, Ying; Jin, Qihuang; Liu, Chengyu; Ge, Kai

2012-09-18

To investigate the role of histone H3K27 demethylase UTX in embryonic stem (ES) cell differentiation, we have generated UTX knockout (KO) and enzyme-dead knock-in male ES cells. Deletion of the X-chromosome-encoded UTX gene in male ES cells markedly decreases expression of the paralogous UTY gene encoded by Y chromosome, but has no effect on global H3K27me3 level, Hox gene expression, or ES cell self-renewal. However, UTX KO cells show severe defects in mesoderm differentiation and induction of Brachyury, a transcription factor essential for mesoderm development. Surprisingly, UTX regulates mesoderm differentiation and Brachyury expression independent of its enzymatic activity. UTY, which lacks detectable demethylase activity, compensates for the loss of UTX in regulating Brachyury expression. UTX and UTY bind directly to Brachyury promoter and are required for Wnt/β-catenin signaling-induced Brachyury expression in ES cells. Interestingly, male UTX KO embryos express normal levels of UTY and survive until birth. In contrast, female UTX KO mice, which lack the UTY gene, show embryonic lethality before embryonic day 11.5. Female UTX KO embryos show severe defects in both Brachyury expression and embryonic development of mesoderm-derived posterior notochord, cardiac, and hematopoietic tissues. These results indicate that UTX controls mesoderm differentiation and Brachyury expression independent of H3K27 demethylase activity, and suggest that UTX and UTY are functionally redundant in ES cell differentiation and early embryonic development.

Involvement of Retinoblastoma Protein and HBP1 in Histone H10 Gene Expression

PubMed Central

Lemercier, Claudie; Duncliffe, Kym; Boibessot, Isabelle; Zhang, Hui; Verdel, André; Angelov, Dimitar; Khochbin, Saadi

2000-01-01

The histone H10-encoding gene is expressed in vertebrates in differentiating cells during the arrest of proliferation. In the H10 promoter, a specific regulatory element, which we named the H4 box, exhibits features which implicate a role in mediating H10 gene expression in response to both differentiation and cell cycle control signals. For instance, within the linker histone gene family, the H4 box is found only in the promoters of differentiation-associated subtypes, suggesting that it is specifically involved in differentiation-dependent expression of these genes. In addition, an element nearly identical to the H4 box is conserved in the promoters of histone H4-encoding genes and is known to be involved in their cell cycle-dependent expression. The transcription factors interacting with the H10 H4 box were therefore expected to link differentiation-dependent expression of H10 to the cell cycle control machinery. The aim of this work was to identify such transcription factors and to obtain information concerning the regulatory pathway involved. Interestingly, our cloning strategy led to the isolation of a retinoblastoma protein (RB) partner known as HBP1. HBP1, a high-mobility group box transcription factor, interacted specifically with the H10 H4 box and moreover was expressed in a differentiation-dependent manner. We also showed that the HBP1-encoding gene is able to produce different forms of HBP1. Finally, we demonstrated that both HBP1 and RB were involved in the activation of H10 gene expression. We therefore propose that HBP1 mediates a link between the cell cycle control machinery and cell differentiation signals. Through modulating the expression of specific chromatin-associated proteins such as histone H10, HBP1 plays a vital role in chromatin remodeling events during the arrest of cell proliferation in differentiating cells. PMID:10958660

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

DIDO as a Switchboard that Regulates Self-Renewal and Differentiation in Embryonic Stem Cells.

PubMed

Fütterer, Agnes; de Celis, Jésus; Navajas, Rosana; Almonacid, Luis; Gutiérrez, Julio; Talavera-Gutiérrez, Amaia; Pacios-Bras, Cristina; Bernascone, Ilenia; Martin-Belmonte, Fernando; Martinéz-A, Carlos

2017-04-11

Transition from symmetric to asymmetric cell division requires precise coordination of differential gene expression. We show that embryonic stem cells (ESCs) mainly express DIDO3 and that their differentiation after leukemia inhibitory factor withdrawal requires DIDO1 expression. C-terminal truncation of DIDO3 (Dido3ΔCT) impedes ESC differentiation while retaining self-renewal; small hairpin RNA-Dido1 ESCs have the same phenotype. Dido3ΔCT ESC differentiation is rescued by ectopic expression of DIDO3, which binds the Dido locus via H3K4me3 and RNA POL II and induces DIDO1 expression. DIDO1, which is exported to cytoplasm, associates with, and is N-terminally phosphorylated by PKCiota. It binds the E3 ubiquitin ligase WWP2, which contributes to cell fate by OCT4 degradation, to allow expression of primitive endoderm (PE) markers. PE formation also depends on phosphorylated DIDO3 localization to centrosomes, which ensures their correct positioning for PE cell polarization. We propose that DIDO isoforms act as a switchboard that regulates genetic programs for ESC transition from pluripotency maintenance to promotion of differentiation. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

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.

Keratinocyte growth factor expression in human gingival fibroblasts and stimulation of in vitro gene expression by retinoic acid.

PubMed

Mackenzie, I C; Gao, Z

2001-04-01

Keratinocyte growth factor (KGF) is a stromally derived growth factor of the fibroblast growth factor (FGF) family with paracrine effects targeted to influence the growth and differentiation of epithelia. Regional and temporal changes in KGF expression play important roles in the development and maintenance of epithelial structures and in epithelial wound healing. Differing patterns of expression of KGF by fibroblasts in the gingival region could therefore be related to the observed regional variation in the differentiation and behavior of gingival epithelia. The in vitro and in vivo patterns of expression of KGF mRNA in human gingival and periodontal fibroblasts were examined using reverse transcription polymerase chain reactions (RT-PCR) and in situ hybridization with digoxigenin-labeled riboprobes. The patterns observed for human gingiva were compared with those for human skin and for murine tissues. Gingival and periodontal fibroblasts showed expression of KGF transcripts in vitro, and the degree of expression was markedly influenced by the presence of retinoic acid, an agent known to influence patterns of epithelial differentiation. Sections of human and murine gingiva and skin showed regionally variable expression of transcripts with the cells expressing KGF in the subepithelial, rather than the deeper, connective tissues and periodontium. The results point to a role of KGF in the maintenance of normal growth and differentiation of gingival epithelia. A lack of KGF expression by periodontal fibroblasts in vivo is expected to hinder apical epithelial migration and thus stabilize the epithelial attachment. The effects of retinoic acid (RA) on KGF expression in vitro provide an indirect mechanism by which RA may regulate the growth and differentiation of gingival epithelia.

Endoderm development in Caenorhabditis elegans: the synergistic action of ELT-2 and -7 mediates the specification→differentiation transition.

PubMed

Sommermann, Erica M; Strohmaier, Keith R; Maduro, Morris F; Rothman, Joel H

2010-11-01

The transition from specification of cell identity to the differentiation of cells into an appropriate and enduring state is critical to the development of embryos. Transcriptional profiling in Caenorhabditis elegans has revealed a large number of genes that are expressed in the fully differentiated intestine; however, no regulatory factor has been found to be essential to initiate their expression once the endoderm has been specified. These gut-expressed genes possess a preponderance of GATA factor binding sites and one GATA factor, ELT-2, fulfills the expected characteristics of a key regulator of these genes based on its persistent expression exclusively in the developing and differentiated intestine and its ability to bind these regulatory sites. However, a striking characteristic of elt-2(0) knockout mutants is that while they die shortly after hatching owing to an obstructed gut passage, they nevertheless contain a gut that has undergone complete morphological differentiation. We have discovered a second gut-specific GATA factor, ELT-7, that profoundly synergizes with ELT-2 to create a transcriptional switch essential for gut cell differentiation. ELT-7 is first expressed in the early endoderm lineage and, when expressed ectopically, is sufficient to activate gut differentiation in nonendodermal progenitors. elt-7 is transcriptionally activated by the redundant endoderm-specifying factors END-1 and -3, and its product in turn activates both its own expression and that of elt-2, constituting an apparent positive feedback system. While elt-7 loss-of-function mutants lack a discernible phenotype, simultaneous loss of both elt-7 and elt-2 results in a striking all-or-none block to morphological differentiation of groups of gut cells with a region-specific bias, as well as reduced or abolished gut-specific expression of a number of terminal differentiation genes. ELT-2 and -7 synergize not only in activation of gene expression but also in repression of a gene that is normally expressed in the valve cells, which immediately flank the termini of the gut tube. Our results point to a developmental strategy whereby positive feedback and cross-regulatory interactions between two synergistically acting regulatory factors promote a decisive and persistent transition of specified endoderm progenitors into the program of intestinal differentiation. Copyright © 2010 Elsevier Inc. All rights reserved.

Expression of thymidylate synthase and glutathione-s-transferase pi in patients with esophageal squamous cell carcinoma.

PubMed

Huang, Jun-Xing; Li, Feng-Yue; Xiao, Wei; Song, Zheng-Xiang; Qian, Rong-Yu; Chen, Ping; Salminen, Eeva

2009-09-14

To investigate the expression of thymidylate synthase (TS) and glutathione-s-transferase pi (GST-pi) in esophageal squamous cell carcinoma and their association with the clinicopathologic characteristics. Immunohistochemical methods were used to detect the expression of TS and GST-pi in surgically resected formalin-fixed, paraffin-embedded esophageal squamous cell carcinoma (ESCC) tissue sections from 102 patients (median age, 58 years) and in 28 normal esophageal mucosa (NEM) samples. The relationship between TS and GST-pi expression and clinicopathologic factors was examined. The expression of TS and GST-pi was not statistically significantly associated with age of the patients, tumor size, lymph node metastasis, depth of invasion or tumor stage. TS staining was positive in 17.86% of normal esophageal mucosa and in 42.16% of ESCC samples (P < 0.05). The expression level of TS was not only significantly lower in well-differentiated (21.88%) than in poorly-differentiated carcinomas (51.43%, P < 0.05), but was also significantly higher in samples from male patients (46.51%) than from female patients (18.75%, P < 0.05). GST-pi was positively stained in 78.57% of normal esophageal mucosa and in 53.92% of ESCC samples (P < 0.05). The expression level of GST-pi was also significantly higher in well-differentiated carcinomas (65.63%) than in poorly-differentiated carcinomas (35.00%, P < 0.05). The expression of TS and of GST-pi may be used as molecular markers for the characterization of ESCC. Poorly-differentiated cells showed increased expression of TS and reduced expression of GST-pi.

Wnt signaling is involved in human articular chondrocyte de-differentiation in vitro.

PubMed

Sassi, N; Laadhar, L; Allouche, M; Zandieh-Doulabi, B; Hamdoun, M; Klein-Nulend, J; Makni, S; Sellami, S

2014-01-01

Osteoarthritis is the most prevalent form of arthritis in the world. Certain signaling pathways, such as the wnt pathway, are involved in cartilage pathology. Osteoarthritic chondrocytes undergo morphological and biochemical changes that lead to chondrocyte de-differentiation. We investigated whether the Wnt pathway is involved in de-differentiation of human articular chondrocytes in vitro. Human articular chondrocytes were cultured for four passages in the presence or absence of IL-1 in monolayer or micromass culture. Changes in cell morphology were monitored by light microscopy. Protein and gene expression of chondrocyte markers and Wnt pathway components were determined by Western blotting and qPCR after culture. After culturing for four passages, chondrocytes exhibited a fibroblast-like morphology. Collagen type II and aggrecan protein and gene expression decreased, while collagen type I, matrix metalloproteinase 13, and nitric oxide synthase expressions increased. Wnt molecule expression profiles changed; Wnt5a protein expression, the Wnt target gene, c-jun, and in Wnt pathway regulator, sFRP4 increased. Treatment with IL-1 caused chondrocyte morphology to become more filament-like. This change in morphology was accompanied by extinction of col II expression and increased col I, MMP13 and eNOS expression. Changes in expression of the Wnt pathway components also were observed. Wnt7a decreased significantly, while Wnt5a, LRP5, β-catenin and c-jun expressions increased. Culture of human articular chondrocytes with or without IL-1 not only induced chondrocyte de-differentiation, but also changed the expression profiles of Wnt components, which suggests that the Wnt pathway is involved in chondrocyte de-differentiation in vitro.

Long Non-Coding RNAs Differentially Expressed between Normal versus Primary Breast Tumor Tissues Disclose Converse Changes to Breast Cancer-Related Protein-Coding Genes

PubMed Central

Reiche, Kristin; Kasack, Katharina; Schreiber, Stephan; Lüders, Torben; Due, Eldri U.; Naume, Bjørn; Riis, Margit; Kristensen, Vessela N.; Horn, Friedemann; Børresen-Dale, Anne-Lise; Hackermüller, Jörg; Baumbusch, Lars O.

2014-01-01

Breast cancer, the second leading cause of cancer death in women, is a highly heterogeneous disease, characterized by distinct genomic and transcriptomic profiles. Transcriptome analyses prevalently assessed protein-coding genes; however, the majority of the mammalian genome is expressed in numerous non-coding transcripts. Emerging evidence supports that many of these non-coding RNAs are specifically expressed during development, tumorigenesis, and metastasis. The focus of this study was to investigate the expression features and molecular characteristics of long non-coding RNAs (lncRNAs) in breast cancer. We investigated 26 breast tumor and 5 normal tissue samples utilizing a custom expression microarray enclosing probes for mRNAs as well as novel and previously identified lncRNAs. We identified more than 19,000 unique regions significantly differentially expressed between normal versus breast tumor tissue, half of these regions were non-coding without any evidence for functional open reading frames or sequence similarity to known proteins. The identified non-coding regions were primarily located in introns (53%) or in the intergenic space (33%), frequently orientated in antisense-direction of protein-coding genes (14%), and commonly distributed at promoter-, transcription factor binding-, or enhancer-sites. Analyzing the most diverse mRNA breast cancer subtypes Basal-like versus Luminal A and B resulted in 3,025 significantly differentially expressed unique loci, including 682 (23%) for non-coding transcripts. A notable number of differentially expressed protein-coding genes displayed non-synonymous expression changes compared to their nearest differentially expressed lncRNA, including an antisense lncRNA strongly anticorrelated to the mRNA coding for histone deacetylase 3 (HDAC3), which was investigated in more detail. Previously identified chromatin-associated lncRNAs (CARs) were predominantly downregulated in breast tumor samples, including CARs located in the protein-coding genes for CALD1, FTX, and HNRNPH1. In conclusion, a number of differentially expressed lncRNAs have been identified with relation to cancer-related protein-coding genes. PMID:25264628

Long non-coding RNAs differentially expressed between normal versus primary breast tumor tissues disclose converse changes to breast cancer-related protein-coding genes.

PubMed

Reiche, Kristin; Kasack, Katharina; Schreiber, Stephan; Lüders, Torben; Due, Eldri U; Naume, Bjørn; Riis, Margit; Kristensen, Vessela N; Horn, Friedemann; Børresen-Dale, Anne-Lise; Hackermüller, Jörg; Baumbusch, Lars O

2014-01-01

Breast cancer, the second leading cause of cancer death in women, is a highly heterogeneous disease, characterized by distinct genomic and transcriptomic profiles. Transcriptome analyses prevalently assessed protein-coding genes; however, the majority of the mammalian genome is expressed in numerous non-coding transcripts. Emerging evidence supports that many of these non-coding RNAs are specifically expressed during development, tumorigenesis, and metastasis. The focus of this study was to investigate the expression features and molecular characteristics of long non-coding RNAs (lncRNAs) in breast cancer. We investigated 26 breast tumor and 5 normal tissue samples utilizing a custom expression microarray enclosing probes for mRNAs as well as novel and previously identified lncRNAs. We identified more than 19,000 unique regions significantly differentially expressed between normal versus breast tumor tissue, half of these regions were non-coding without any evidence for functional open reading frames or sequence similarity to known proteins. The identified non-coding regions were primarily located in introns (53%) or in the intergenic space (33%), frequently orientated in antisense-direction of protein-coding genes (14%), and commonly distributed at promoter-, transcription factor binding-, or enhancer-sites. Analyzing the most diverse mRNA breast cancer subtypes Basal-like versus Luminal A and B resulted in 3,025 significantly differentially expressed unique loci, including 682 (23%) for non-coding transcripts. A notable number of differentially expressed protein-coding genes displayed non-synonymous expression changes compared to their nearest differentially expressed lncRNA, including an antisense lncRNA strongly anticorrelated to the mRNA coding for histone deacetylase 3 (HDAC3), which was investigated in more detail. Previously identified chromatin-associated lncRNAs (CARs) were predominantly downregulated in breast tumor samples, including CARs located in the protein-coding genes for CALD1, FTX, and HNRNPH1. In conclusion, a number of differentially expressed lncRNAs have been identified with relation to cancer-related protein-coding genes.

Msx homeobox genes inhibit differentiation through upregulation of cyclin D1.

PubMed

Hu, G; Lee, H; Price, S M; Shen, M M; Abate-Shen, C

2001-06-01

During development, patterning and morphogenesis of tissues are intimately coordinated through control of cellular proliferation and differentiation. We describe a mechanism by which vertebrate Msx homeobox genes inhibit cellular differentiation by regulation of the cell cycle. We show that misexpression of Msx1 via retroviral gene transfer inhibits differentiation of multiple mesenchymal and epithelial progenitor cell types in culture. This activity of Msx1 is associated with its ability to upregulate cyclin D1 expression and Cdk4 activity, while Msx1 has minimal effects on cellular proliferation. Transgenic mice that express Msx1 under the control of the mouse mammary tumor virus long terminal repeat (MMTV LTR) display impaired differentiation of the mammary epithelium during pregnancy, which is accompanied by elevated levels of cyclin D1 expression. We propose that Msx1 gene expression maintains cyclin D1 expression and prevents exit from the cell cycle, thereby inhibiting terminal differentiation of progenitor cells. Our model provides a framework for reconciling the mutant phenotypes of Msx and other homeobox genes with their functions as regulators of cellular proliferation and differentiation during embryogenesis.

Desmocollin 2 is a new immunohistochemical marker indicative of squamous differentiation in urothelial carcinoma.

PubMed

Hayashi, Tetsutaro; Sentani, Kazuhiro; Oue, Naohide; Anami, Katsuhiro; Sakamoto, Naoya; Ohara, Shinya; Teishima, Jun; Noguchi, Tsuyoshi; Nakayama, Hirofumi; Taniyama, Kiyomi; Matsubara, Akio; Yasui, Wataru

2011-10-01

Urothelial carcinoma (UC) with squamous differentiation tends to present at higher stages than pure UC. To distinguish UC with squamous differentiation from pure UC, a sensitive and specific marker is needed. Desmocollin 2 (DSC2) is a protein localized in desmosomal junctions of stratified epithelium, but little is known about its biological significance in bladder cancer. We examined the utility of DSC2 as a diagnostic marker. We analysed the immunohistochemical characteristics of DSC2, and studied the relationship of DSC2 expression with the expression of the known markers uroplakin III (UPIII), cytokeratin (CK)7, CK20, epidermal growth factor receptor (EGFR), and p53. DSC2 staining was detected in 24 of 25 (96%) cases of UC with squamous differentiation, but in none of 85 (0%) cases of pure UC. DSC2 staining was detected only in areas of squamous differentiation. DSC2 expression was mutually exclusive of UPIII expression, and was correlated with EGFR expression. Furthermore, DSC2 expression was correlated with higher stage (P = 0.0314) and poor prognosis (P = 0.0477). DSC2 staining offers high sensitivity (96%) and high specificity (100%) for the detection of squamous differentiation in UC. DSC2 is a useful immunohistochemical marker for separation of UC with squamous differentiation from pure UC. 2011 Blackwell Publishing Limited.

CD147 promotes the formation of functional osteoclasts through NFATc1 signalling

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

Nishioku, Tsuyoshi, E-mail: nishiokut@niu.ac.jp; Department of Pharmaceutical Care and Health Sciences, Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka 814-0180; Terasawa, Mariko

CD147, a membrane glycoprotein of the immunoglobulin superfamily, is highly upregulated during dynamic cellular events including tissue remodelling. Elevated CD147 expression is present in the joint of rheumatoid arthritis patients. However, the role of CD147 in bone destruction remains unclear. To determine whether CD147 is involved in osteoclastogenesis, we studied its expression in mouse osteoclasts and its role in osteoclast differentiation and function. CD147 expression was markedly upregulated during osteoclast differentiation. To investigate the role of CD147 in receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis and bone resorption activity, osteoclast precursor cells were transfected with CD147 siRNA. Decreasedmore » CD147 expression inhibited osteoclast formation and bone resorption, inhibited RANKL-induced nuclear translocation of the nuclear factor of activated T cells (NFAT) c1 and decreased the expression of the d2 isoform of vacuolar ATPase Vo domain and cathepsin K. Therefore, CD147 plays a critical role in the differentiation and function of osteoclasts by upregulating NFATc1 through the autoamplification of its expression in osteoclastogenesis. - Highlights: • CD147 expression was markedly upregulated during osteoclast differentiation. • Downregulation of CD147 expression inhibited osteoclastgenesis and bone resorption. • Decreased CD147 expression inhibited RANKL-induced nuclear translocation of NFATc1.« less

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

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.

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.

In vitro differentiation of bone marrow stromal cells into neurons and glial cells and differential protein expression in a two-compartment bone marrow stromal cell/neuron co-culture system.

PubMed

Qi, Xu; Shao, Ming; Peng, Haisheng; Bi, Zhenggang; Su, Zhiqiang; Li, Hulun

2010-07-01

This study was performed to establish a bone marrow stromal cell (BMSC)/neuron two-compartment co-culture model in which differentiation of BMSCs into neurons could occur without direct contact between the two cell types, and to investigate protein expression changes during differentiation of this entirely BMSC-derived population. Cultured BMSCs isolated from Wistar rats were divided into three groups: BMSC culture, BMSC/neuron co-culture and BMSC/neuron two-compartment co-culture. Cells were examined for neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) expression. The electrophysiological behavior of the BMSCs was examined using patch clamping. Proteins that had significantly different expression levels in BMSCs cultured alone and co-cultured with neurons were studied using a protein chip-mass spectroscopy technique. Expression of NSE and GFAP were significantly higher in co-culture cells than in two-compartment co-culture cells, and significantly higher in both co-culture groups than in BMSCs cultured alone. Five proteins showed significant changes in expression during differentiation: TIP39_RAT and CALC_RAT underwent increases, and INSL6_RAT, PNOC_RAT and PCSK1_RAT underwent decreases in expression. We conclude that BMSCs can differentiate into neurons during both contact co-culture with neurons and two-compartment co-culture with neurons. The rate at which BMSCs differentiated into neurons was higher in contact co-culture than in non-contact co-culture.

Changes in Ca(2+) channel expression upon differentiation of SN56 cholinergic cells.

PubMed

Kushmerick, C; Romano-Silva, M A; Gomez, M V; Prado, M A

2001-10-19

The SN56 cell line, a fusion of septal neurons and neuroblastoma cells, has been used as a model for central cholinergic neurons. These cells show increased expression of cholinergic neurochemical features upon differentiation, but little is known about how differentiation affects their electrophysiological properties. We examined the changes in Ca(2+) channel expression that occur as these cells undergo morphological differentiation in response to serum withdrawal and exposure to dibutyryl-cAMP. Undifferentiated cells expressed a T-type current with biophysical and pharmacological properties similar, although not identical, to those reported for the current generated by the alpha(1H) (CaV3.2) Ca(2+) channel subunit. Differentiated cells expressed, in addition to this T-type current, high voltage activated currents which were inhibited 38% by the L-type channel antagonist nifedipine (5 microM), 37% by the N-type channel antagonist omega-conotoxin-GVIA (1 microM), and 15% by the P/Q-type channel antagonist omega-agatoxin-IVA (200 nM). Current resistant to these inhibitors accounted for 15% of the high voltage activated current in differentiated SN56 cells. Our data demonstrate that differentiation increases the expression of neuronal type voltage gated Ca(2+) channels in this cell line, and that the channels expressed are comparable to those reported for native basal forebrain cholinergic neurons. This cell line should thus provide a useful model system to study the relationship between calcium currents and cholinergic function and dysfunction.

CCK processing by pituitary GH3 cells, human teratocarcinoma cells NT2 and hNT differentiated human neuronal cells evidence for a differentiation-induced change in enzyme expression and pro CCK processing.

PubMed

Beinfeld, Margery C; Wang, Wenge

2002-02-01

Human teratocarcinoma Ntera2/c 1.D1 (NT2) cells express very low levels of the prohormone convertase enzyme PC1, moderate levels of PC2 and significant levels of PC5. When infected with an adenovirus which expresses rat CCK mRNA, several glycine-extended forms were secreted that co-eluted with CCK 33, 22 and 12. Amidated CCK is not produced because these cells appear to lack the amidating enzyme. Pituitary GH3 cells express high levels of PC2 and PC5. CCK adenovirus-infected GH3 cells secrete amidated versions of the same peptides as NT2 cells. Differentiation of NT2 cells into hNT cells with retinoic acid and mitotic inhibitors increased expression of PC5 and decreased expression of PCI and PC2. CCK adenovirus-infected differentiated hNT cells also secrete glycine extended CCK products and the major molecular form produced co-eluted with CCK 8 Gly. These experiments demonstrate that the state of differentiation of this neuronal cell line influences its expression of PC 1,2, and 5 and its cleavage of pro CCK and suggests that these cells may make an interesting model to study how differentiation alters prohormone processing. These results also support the hypothesis that PC5 in differentiated neuronal cells is capable of processing pro CCK to glycine-extended CCK 8.

Expression and regulation of aromatase and 17 beta-hydroxysteroid dehydrogenase type 4 in human THP 1 leukemia cells.

PubMed

Jakob, F; Homann, D; Adamski, J

1995-12-01

Estradiol is active in proliferation and differentiation of sex-related tissues like ovary and breast. Glandular steroid metabolism was for a long time believed to dominate the estrogenic milieu around any cell of the organism. Recent reports verified the expression of estrogen receptors in "non-target" tissues as well as the extraglandular expression of steroid metabolizing enzymes. Extraglandular steroid metabolism proved to be important in the brain, skin and in stromal cells of hormone responsive tumors. Aromatase converts testosterone into estradiol and androstenedione into estrone, thereby activating estrogen precursors. The group of 17 beta-hydroxysteroid dehydrogenases catalyzes the oxidation and/or reduction of the forementioned compounds, e.g. estradiol/estrone, thereby either activating or inactivating estradiol. Aromatase is expressed and regulated in the human THP 1 myeloid leukemia cell line after vitamin D/GMCSF-propagated differentiation. Aromatase expression is stimulated by dexamethasone, phorbolesters and granulocyte/macrophage stimulating factor (GMCSF). Exons I.2 and I.4 are expressed in PMA-stimulated cells only, exon I.3 in both PMA- and dexamethasone-stimulated cells. Vitamin D-differentiated THP 1 cells produce a net excess of estradiol in culture supernatants, if testosterone is given as aromatase substrate. In contrast, the 17 beta-hydroxysteroid dehydrogenase type 4 (17 beta-HSD 4) is abundantly expressed in unstimulated THP 1 cells and is further stimulated by glucocorticoids (2-fold). The expression is unchanged after vitamin D/GMCSF-propagated differentiation. 17 beta-HSD 4 expression is not altered by phorbolester treatment in undifferentiated cells but is abolished after vitamin D-propagated differentiation along with downregulation of beta-actin. Protein kinase C activation therefore appears to dissociate the expression of aromatase and 17 beta-HSD 4 in this differentiation stage along the monocyte/phagocyte pathway of THP 1 myeloid cells. The expression of steroid metabolizing enzymes in myeloid cells is able to create a microenvironment which is uncoupled from dominating systemic estrogens. These findings may be relevant in the autocrine, paracrine or iuxtacrine cellular crosstalk of myeloid cells in their respective states of terminal differentiation, e.g. in bone metabolism and inflammation.

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

Effect of TAK1 on osteogenic differentiation of mesenchymal stem cells by regulating BMP-2 via Wnt/β-catenin and MAPK pathway.

PubMed

Yang, Hongpeng; Guo, Yue; Wang, Dawei; Yang, Xiaofei; Ha, Chengzhi

2018-01-02

Mesenchymal stem cells (MSCs) have the ability to differentiate into osteoblasts and chondrocytes. In vitro osteogenic differentiation is critical but the molecular mechanism has yet to be further clarified. The role of TGF-β activated kinase 1 (TAK1) in MSCs osteogenesis differentiation has not been reported. By adding si-TAK1 and rhTAK1, the osteogenic differentiation of MSCs was measured. Expression levels of the osteoblastic marker genes during osteogenic differentiation of MSCs were checked. As well as molecules involved in BMP and Wnt/β-catenin signaling pathways. The phosphorylation of p38 and JNK was also checked. TAK1 is essential for mineralization of MSCs at low concentration, but excessive rhTAK1 inhibits mineralization of MSCs. It up regulates the expression levels of bone sialoprotein (BSP), osteocalcin (OSC), Alkaline phosphatase (ALP), and RUNX2 during osteogenic differentiation of MSCs. It can also promote TGF-β/BMP-2 gene expression and β-catenin expression, and down regulate GSK-3β expression. Meanwhile, TAK1 promotes the phosphorylation of p38 and JNK. Additionally, TAK1 up regulates the expression of BMP-2 at all concentration under the inhibition of p38 and JNK. Our results suggested that TAK1 is essential in MSCs osteogenesis differentiation, and functions as a double-edged sword, probably through regulation of β-catenin and p38/JNK.

Klf10 regulates odontoblast differentiation and mineralization via promoting expression of dentin matrix protein 1 and dentin sialophosphoprotein genes

PubMed Central

Chen, Zhuo; Li, Wentong; Wang, Han; Wan, Chunyan; Luo, Daoshu; Deng, Shuli

2016-01-01

Klf10, a member of the Krüppel-like family of transcription factors, is critical for osteoblast differentiation, bone formation and mineralization. However, whether Klf10 is involved in odontoblastic differentiation and tooth development has not been determined. In this study, we investigate the expression patterns of Klf10 during murine tooth development in vivo and its role in odontoblastic differentiation in vitro. Klf10 protein was expressed in the enamel organ and the underlying mesenchyme, ameloblasts and odontoblasts at early and later stages of murine molar formation. Furthermore, the expression of Klf10, Dmp1, Dspp and Runx2 was significantly elevated during the process of mouse dental papilla mesenchymal differentiation and mineralization. The overexpression of Klf10 induced dental papilla mesenchymal cell differentiation and mineralization as detected by alkaline phosphatase staining and alizarin red S assay. Klf10 additionally up-regulated the expression of odontoblastic differentiation marker genes Dmp1, Dspp and Runx2 in mouse dental papilla mesenchymal cells. The molecular mechanism of Klf10 in controlling Dmp1 and Dspp expression is thus to activate their regulatory regions in a dosage-dependent manner. Our results suggest that Klf10 is involved in tooth development and promotes odontoblastic differentiation via the up-regulation of Dmp1 and Dspp transcription. PMID:26310138

Expression of transcription factors during sodium phenylacetate induced erythroid differentiation in K562 cells.

PubMed

Rath, A V; Schmahl, G E; Niemeyer, C M

1997-01-01

During 15 days of treatment of K562 cells with sodium phenylacetate, we observed an increase in the cellular hemoglobin concentration with a similar increase in the expression of gamma-globin mRNA. Morphological studies demonstrated characteristic features of erythroid differentiation and maturation. At the same time there was no change in the level of expression of the cell surface antigenes CD33, CD34, CD45, CD71 and glycophorin A. Likewise, the level of expression of the erythroid transcription factors GATA-1, GATA-2, NF-E2, SCL and RBTN2, all expressed in untreated K562 cells, did not increase during sodium phenylacetate induced erythroid differentiation. The expression of the nuclear factors Evi-1 and c-myb, known to inhibit erythroid differentiation, did not decrease. We conclude that sodium phenylacetate treatment of K562 cells increases gamma-globin mRNA and induces cell maturation as judged by morphology without affecting the expression of the erythroid transcription factors, some of which are known to be involved in the regulation of beta-like globin genes.

Analysis of differential gene expression by bead-based fiber-optic array in nonfunctioning pituitary adenomas.

PubMed

Jiang, Z; Gui, S; Zhang, Y

2011-05-01

Nonfunctioning pituitary adenomas (NFPAs) are relatively common, accounting for 30% of all pituitary adenomas; however, their pathogenesis remains enigmatic. To explore the possible pathogenesis of NFPAs, we used fiber-optic BeadArray to examine gene expression in 5 NFPAs compared with 3 normal pituitaries. 4 differentially expressed genes were chosen randomly for validation by reverse transcriptase-real time quantitative polymerase chain reaction (RT-qPCR). We then analyzed the differentially expressed gene profile with Kyoto Encyclopedia of Genes and Genomes (KEGG). The array analysis indentified significant increases in the expression of 1,402 genes and 383 expressed sequence tags (ESTs), and decreases in 1,697 genes and 113 ESTs in the NFPAs. Bioinformatic and pathway analysis showed that the genes HIGD1B, FAM5C, PMAIP1 and the pathway cell-cycle regulation may play an important role in tumorigenesis and progression of NFPAs. Our data suggest fiber-optic BeadArray combined with pathway analysis of differential gene expression profile appears to be a valid approach for investigating the pathogenesis of tumors. © Georg Thieme Verlag KG Stuttgart · New York.

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

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

Loss of c-KIT expression in thyroid cancer cells.

PubMed

Franceschi, Sara; Lessi, Francesca; Panebianco, Federica; Tantillo, Elena; La Ferla, Marco; Menicagli, Michele; Aretini, Paolo; Apollo, Alessandro; Naccarato, Antonio Giuseppe; Marchetti, Ivo; Mazzanti, Chiara Maria

2017-01-01

Papillary thyroid carcinoma is the most frequent histologic type of thyroid tumor. Few studies investigated the role of c-KIT expression in thyroid tumors, suggesting a role for this receptor and its ligand in differentiation and growth control of thyroid epithelium and a receptor loss following malignant transformation. We investigated and correlated c-KIT expression levels and two known markers of thyrocytes differentiation, PAX8 and TTF-1, in malignant and benign cytological thyroid samples. Moreover, we performed functional studies on human papillary thyroid carcinoma cell line to associated c-KIT expression to thyrocytes differentiation and tumor proliferation. c-KIT and PAX8 expression resulted higher in benign samples compared to the malignant ones, and the expression levels of these two genes were significantly correlated to each other. We also observed that c-KIT overexpression led to an increase of PAX8 expression level together with a decrease of proliferation. Furthermore, c-KIT overexpressing cells showed a regression of typical morphological features of malignancy. Taken together these results suggest that c-KIT could be involved in the differentiation of thyroid cells and in tumor progression.

Loss of c-KIT expression in thyroid cancer cells

PubMed Central

Panebianco, Federica; Tantillo, Elena; La Ferla, Marco; Menicagli, Michele; Aretini, Paolo; Apollo, Alessandro; Naccarato, Antonio Giuseppe; Marchetti, Ivo; Mazzanti, Chiara Maria

2017-01-01

Papillary thyroid carcinoma is the most frequent histologic type of thyroid tumor. Few studies investigated the role of c-KIT expression in thyroid tumors, suggesting a role for this receptor and its ligand in differentiation and growth control of thyroid epithelium and a receptor loss following malignant transformation. We investigated and correlated c-KIT expression levels and two known markers of thyrocytes differentiation, PAX8 and TTF-1, in malignant and benign cytological thyroid samples. Moreover, we performed functional studies on human papillary thyroid carcinoma cell line to associated c-KIT expression to thyrocytes differentiation and tumor proliferation. c-KIT and PAX8 expression resulted higher in benign samples compared to the malignant ones, and the expression levels of these two genes were significantly correlated to each other. We also observed that c-KIT overexpression led to an increase of PAX8 expression level together with a decrease of proliferation. Furthermore, c-KIT overexpressing cells showed a regression of typical morphological features of malignancy. Taken together these results suggest that c-KIT could be involved in the differentiation of thyroid cells and in tumor progression. PMID:28301608

Liver-enriched transcription factors uncoupled from expression of hepatic functions in hepatoma cell lines.

PubMed Central

Chaya, D; Fougère-Deschatrette, C; Weiss, M C

1997-01-01

Among the liver-enriched transcription factors identified to date, only expression of hepatocyte nuclear factor 4 (HNF4) and hepatocyte nuclear factor 1 (HNF1) is in strict correlation with hepatic differentiation in cultured rat hepatoma cells. Indeed, differentiated hepatoma cells that stably express an extensive set of adult hepatic functions express liver-enriched transcription factors, while dedifferentiated cells that have lost expression of all these hepatic functions no longer express HNF4 and HNF1. We describe a new heritable phenotype, designated as uncoupled, in which there is a spontaneous dissociation between the expression of these transcription factors and that of the hepatic functions. Cells presenting this phenotype, isolated from differentiated hepatoma cells, cease to accumulate all transcripts coding for hepatic functions but nevertheless maintain expression of HNF4 and HNF1. Transitory transfection experiments indicate that these two factors present in these cells have transcriptional activity similar to that of differentiated hepatoma cells. Characterization of the appropriate intertypic cell hybrids demonstrates that this new phenotype is recessive to the dedifferentiated state and fails to be complemented by differentiated cells. These results indicate the existence of mechanisms that inhibit transcription of genes coding for hepatocyte functions in spite of the presence of functional HNF4 and HNF1. Cells of the uncoupled phenotype present certain properties of oval cells described for pathological states of the liver. PMID:9343392

Liver-enriched transcription factors uncoupled from expression of hepatic functions in hepatoma cell lines.

PubMed

Chaya, D; Fougère-Deschatrette, C; Weiss, M C

1997-11-01

Among the liver-enriched transcription factors identified to date, only expression of hepatocyte nuclear factor 4 (HNF4) and hepatocyte nuclear factor 1 (HNF1) is in strict correlation with hepatic differentiation in cultured rat hepatoma cells. Indeed, differentiated hepatoma cells that stably express an extensive set of adult hepatic functions express liver-enriched transcription factors, while dedifferentiated cells that have lost expression of all these hepatic functions no longer express HNF4 and HNF1. We describe a new heritable phenotype, designated as uncoupled, in which there is a spontaneous dissociation between the expression of these transcription factors and that of the hepatic functions. Cells presenting this phenotype, isolated from differentiated hepatoma cells, cease to accumulate all transcripts coding for hepatic functions but nevertheless maintain expression of HNF4 and HNF1. Transitory transfection experiments indicate that these two factors present in these cells have transcriptional activity similar to that of differentiated hepatoma cells. Characterization of the appropriate intertypic cell hybrids demonstrates that this new phenotype is recessive to the dedifferentiated state and fails to be complemented by differentiated cells. These results indicate the existence of mechanisms that inhibit transcription of genes coding for hepatocyte functions in spite of the presence of functional HNF4 and HNF1. Cells of the uncoupled phenotype present certain properties of oval cells described for pathological states of the liver.

Aberrant Expression of Retinoic Acid Signaling Molecules Influences Patient Survival in Astrocytic Gliomas

PubMed Central

Campos, Benito; Centner, Franz-Simon; Bermejo, Justo Lorenzo; Ali, Ramadan; Dorsch, Katharina; Wan, Feng; Felsberg, Jörg; Ahmadi, Rezvan; Grabe, Niels; Reifenberger, Guido; Unterberg, Andreas; Burhenne, Jürgen; Herold-Mende, Christel

2011-01-01

Undifferentiated cell populations may influence tumor growth in malignant glioma. We investigated potential disruptions in the retinoic acid (RA) differentiation pathway that could lead to a loss of differentiation capacity, influencing patient prognosis. Expression of key molecules belonging to the RA differentiation pathway was analyzed in 283 astrocytic gliomas and was correlated with tumor proliferation, tumor differentiation, and patient survival. In addition, in situ concentrations of retinoids were measured in tumors, and RA signaling events were studied in vitro. Unlike other tumors, in gliomas expression of most RA signaling molecules increased with malignancy and was associated with augmented intratumoral retinoid levels in high-grade gliomas. Aberrantly expressed RA signaling molecules included i) the retinol-binding protein CRBP1, which facilitates cellular retinoid uptake; ii) ALDH1A1, capable of activating RA precursors; iii) the RA-degrading enzyme CYP26B1; and iv) the RA-binding protein FABP5, which can inhibit RA-induced differentiation. In contrast, expression of the RA-binding protein CRABP2, which fosters differentiation, was decreased in high-grade tumors. Moreover, expression of CRBP1 correlated with tumor proliferation, and FABP5 expression correlated with an undifferentiated tumor phenotype. CRBP1 and ALDH1A1 were independent prognostic markers for adverse patient survival. Our data indicate a complex and clinically relevant deregulation of RA signaling, which seems to be a central event in glioma pathogenesis. PMID:21514413

Brca1 regulates in vitro differentiation of mammary epithelial cells.

PubMed

Kubista, Marion; Rosner, Margit; Kubista, Ernst; Bernaschek, Gerhard; Hengstschläger, Markus

2002-07-18

Murine Brca1 is widely expressed during development in different tissues. Why alterations of BRCA1 lead specifically to breast and ovarian cancer is currently not clarified. Here we show that Brca1 protein expression is upregulated during mammary epithelial differentiation of HC11 cells, during differentiation of C2C12 myoblasts into myotubes and during neuronal differentiation of N1E-115 cells. Ectopic overexpression of BRCA1 and downregulation of endogenous Brca1 expression specifically affect the regulation of mammary epithelial cell differentiation. Accelerated mammary epithelial cell differentiation upon high ectopic BRCA1 expression is not a consequence of the anti-proliferative capacity of this tumor suppressor and independent of functional p53. Overexpression of the BRCA1 variant lacking the large central exon 11 has no effects on mammary epithelial cell differentiation. These data provide new insights into the cellular role of Brca1.

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.

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

RNA-binding Protein Quaking Stabilizes Sirt2 mRNA during Oligodendroglial Differentiation*

PubMed Central

Thangaraj, Merlin P.; Furber, Kendra L.; Gan, Jotham K.; Ji, Shaoping; Sobchishin, Larhonda; Doucette, J. Ronald; Nazarali, Adil J.

2017-01-01

Myelination is controlled by timely expression of genes involved in the differentiation of oligodendrocyte precursor cells (OPCs) into myelinating oligodendrocytes (OLs). Sirtuin 2 (SIRT2), a NAD+-dependent deacetylase, plays a critical role in OL differentiation by promoting both arborization and downstream expression of myelin-specific genes. However, the mechanisms involved in regulating SIRT2 expression during OL development are largely unknown. The RNA-binding protein quaking (QKI) plays an important role in myelination by post-transcriptionally regulating the expression of several myelin specific genes. In quaking viable (qkv/qkv) mutant mice, SIRT2 protein is severely reduced; however, it is not known whether these genes interact to regulate OL differentiation. Here, we report for the first time that QKI directly binds to Sirt2 mRNA via a common quaking response element (QRE) located in the 3′ untranslated region (UTR) to control SIRT2 expression in OL lineage cells. This interaction is associated with increased stability and longer half-lives of Sirt2.1 and Sirt2.2 transcripts leading to increased accumulation of Sirt2 transcripts. Consistent with this, overexpression of qkI promoted the expression of Sirt2 mRNA and protein. However, overexpression of the nuclear isoform qkI-5 promoted the expression of Sirt2 mRNA, but not SIRT2 protein, and delayed OL differentiation. These results suggest that the balance in the subcellular distribution and temporal expression of QKI isoforms control the availability of Sirt2 mRNA for translation. Collectively, our study demonstrates that QKI directly plays a crucial role in the post-transcriptional regulation and expression of Sirt2 to facilitate OL differentiation. PMID:28188285

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.

Differentiation of EL4 lymphoma cells by tumoral environment is associated with inappropriate expression of the large chondroitin sulfate proteoglycan PG-M and the tumor-associated antigen HTgp-175.

PubMed

Rottiers, P; Verfaillie, T; Contreras, R; Revets, H; Desmedt, M; Dooms, H; Fiers, W; Grooten, J

1998-11-09

Progression to malignancy of transformed cells involves complex genetic alterations and aberrant gene expression patterns. While aberrant gene expression is often caused by alterations in individual genes, the contribution of the tumoral environment to the triggering of this gene expression is less well established. The stable but heterogeneous expression in cultured EL4/13 cells of a novel tumor-associated antigen, designated as HTgp-175, was chosen for the investigation of gene expression during tumor formation. Homogeneously HTgp-175-negative EL4/13 cells, isolated by cell sorting or obtained by subcloning, acquired HTgp-175 expression as a result of tumor formation. The tumorigenicity of HTgp-175-negative vs. HTgp-175-positive EL4 variants was identical, indicating that induction but not selection accounted for the phenotypic switch from HTgp-175-negative to HTgp-175-positive. Although mutagenesis experiments showed that the protein was not essential for tumor establishment, tumor-derived cells showed increased malignancy, linking HTgp-175 expression with genetic changes accompanying tumor progression. This novel gene expression was not an isolated event, since it was accompanied by ectopic expression of the large chondroitin sulfate proteoglycan PG-M and of normal differentiation antigens. We conclude that signals derived from the tumoral microenvironment contribute significantly to the aberrant gene expression pattern of malignant cells, apparently by fortuitous activation of differentiation processes and cause expression of novel differentiation antigens as well as of inappropriate tumor-associated and ectopic antigens.

Neurogenic transdifferentiation of human adipose-derived stem cells? A critical protocol reevaluation with special emphasis on cell proliferation and cell cycle alterations.

PubMed

Kompisch, Kai Michael; Lange, Claudia; Steinemann, Doris; Skawran, Britta; Schlegelberger, Brigitte; Müller, Reinhard; Schumacher, Udo

2010-11-01

Adipose-derived stem cells (ASCs) are reported to display multilineage differentiation potential, including neuroectodermal pathways. The aim of the present study was to critically re-evaluate the potential neurogenic (trans-)differentiation capacity of ASCs using a neurogenic induction protocol based on the combination of isobutylmethylxanthine (IBMX), indomethacin and insulin. ASCs isolated from lipo-aspirate samples of five healthy female donors were characterized and potential neurogenic (trans-)differentiation was assessed by means of immunohistochemistry and gene expression analyses. Cell proliferation and cell cycle alterations were studied, and the expression of CREB/ATF transcription factors was analyzed. ASCs expressed CD59, CD90 and CD105, and were tested negative for CD34 and CD45. Under neurogenic induction, ASCs adopted a characteristic morphology comparable to neur(on)al progenitors and expressed musashi1, β-III-tubulin and nestin. Gene expression analyses revealed an increased expression of β-III-tubulin, GFAP, vimentin and BDNF, as well as SOX4 in induced ASCs. Cell proliferation was significantly reduced under neurogenic induction; cell cycle analyses showed a G2-cell cycle arrest accompanied by differential expression of key regulators of cell cycle progression. Differential expression of CREB/ATF transcription factors could be observed on neurogenic induction, pointing to a decisive role of the cAMP-CREB/ATF system. Our findings may point to a potential neurogenic (trans-)differentiation of ASCs into early neur(on)al progenitors, but do not present definite evidence for it. Especially, the adoption of a neural progenitor cell-like morphology must not automatically be misinterpreted as a specific characteristic of a respective (trans-)differentiation process, as this may as well be caused by alterations of cell cycle progression.

ErbB2 and EGFR are downmodulated during the differentiation of 3T3-L1 preadipocytes.

PubMed

Pagano, Eleonora; Calvo, Juan Carlos

2003-10-15

The expression of receptors belonging to the epidermal growth factor receptor subfamily has been largely studied these last years in epithelial cells mainly as involved in cell proliferation and malignant progression. Although much work has focused on the role of these growth factor receptors in the differentiation of a variety of tissues, there is little information in regards to normal stromal cells. We investigated erbB2 expression in the murine fibroblast cell line Swiss 3T3L1, which naturally or hormonally induced undergoes adipocyte differentiation. We found that the Swiss 3T3-L1 fibroblasts express erbB2, in addition to EGFR, and in a quantity comparable to or even greater than the breast cancer cell line T47D. Proliferating cells increased erbB2 and EGFR levels when reaching confluence up to 4- and 10-fold, respectively. This expression showed a significant decrease when growth-arrested cells were stimulated to differentiate with dexamethasone and isobutyl-methylxanthine. Differentiated cells presented a decreased expression of both erbB2 and EGFR regardless of whether the cells were hormonally or spontaneously differentiated. EGF stimulation of serum-starved cells increased erbB2 tyrosine phosphorylation and retarded erbB2 migration in SDS-PAGE, suggesting receptor association and activation. Heregulin-alpha1 and -beta1, two EGF related factors, had no effect on erbB2 or EGFR phosphorylation. Although 3T3-L1 cells expressed heregulin, its specific receptors, erbB3 and erbB4, were not found. This is the first time in which erbB2 is reported to be expressed in an adipocytic cell line which does not depend on non EGF family growth factors (thyroid hormone, growth hormone, etc.) to accomplish adipose differentiation. Since erbB2 and EGFR expression were downmodulated as differentiation progressed it is conceivable that a mechanism of switching from a mitogenic to a differentiating signaling pathway may be involved, through regulation of the expression of these growth factor receptors. Copyright 2003 Wiley-Liss, Inc.

Genes are differentially expressed at transcriptional level of Neocaridina denticulata following short-term exposure to nonylphenol.

PubMed

Liu, Chang-Lun; Sung, Hung-Hung

2011-09-01

To assess the toxicity of nonylphenol towards aquatic crustaceans, Neocaridina denticulata were exposed short-term to sublethal concentration (0.001-0.5 mg/L). Following treatment, differentially expressed genes were identified using suppression subtractive hybridization on samples prepared from whole specimens. There were 20 differentially expressed sequence tags that corresponded to known genes and could be divided into six functional classes: defence, translation, metabolism, ribosomal gene expression, respiration, and genes involved in the stress response. Using semi-quantitative RT-PCR, we found that 14 of the differentially expressed sequence tags significantly responded to nonylphenol, including six at a nominal concentration of 0.01 mg/L; among them, 12 genes were down-regulated. These results suggest that under non-lethal concentrations of nonylphenol, the polluted aquatic environment may still present a potential risk to N. denticulata.

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

Role Differentiation in Groups: The Relationship Between Instrumental and Expressive Leadership.

ERIC Educational Resources Information Center

Rees, C. Roger; Segal, Mady Wechsler

1984-01-01

Examined the degree of differentiation between instrumental and expressive leadership roles in two natural groups (N=101). Results showed a relatively high degree of leadership role integration with several members of each group fulfilling both instrumental and expressive leadership roles. (LLL)

[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.

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.

Expression Differentiation Is Constrained to Low-Expression Proteins over Ecological Timescales

PubMed Central

Margres, Mark J.; Wray, Kenneth P.; Seavy, Margaret; McGivern, James J.; Herrera, Nathanael D.; Rokyta, Darin R.

2016-01-01

Protein expression level is one of the strongest predictors of protein sequence evolutionary rate, with high-expression protein sequences evolving at slower rates than low-expression protein sequences largely because of constraints on protein folding and function. Expression evolutionary rates also have been shown to be negatively correlated with expression level across human and mouse orthologs over relatively long divergence times (i.e., ∼100 million years). Long-term evolutionary patterns, however, often cannot be extrapolated to microevolutionary processes (and vice versa), and whether this relationship holds for traits evolving under directional selection within a single species over ecological timescales (i.e., <5000 years) is unknown and not necessarily expected. Expression is a metabolically costly process, and the expression level of a particular protein is predicted to be a tradeoff between the benefit of its function and the costs of its expression. Selection should drive the expression level of all proteins close to values that maximize fitness, particularly for high-expression proteins because of the increased energetic cost of production. Therefore, stabilizing selection may reduce the amount of standing expression variation for high-expression proteins, and in combination with physiological constraints that may place an upper bound on the range of beneficial expression variation, these constraints could severely limit the availability of beneficial expression variants. To determine whether rapid-expression evolution was restricted to low-expression proteins owing to these constraints on highly expressed proteins over ecological timescales, we compared venom protein expression levels across mainland and island populations for three species of pit vipers. We detected significant differentiation in protein expression levels in two of the three species and found that rapid-expression differentiation was restricted to low-expression proteins. Our results suggest that various constraints on high-expression proteins reduce the availability of beneficial expression variants relative to low-expression proteins, enabling low-expression proteins to evolve and potentially lead to more rapid adaptation. PMID:26546003

Divergence between motoneurons: gene expression profiling provides a molecular characterization of functionally discrete somatic and autonomic motoneurons

PubMed Central

Cui, Dapeng; Dougherty, Kimberly J.; Machacek, David W.; Sawchuk, Michael; Hochman, Shawn; Baro, Deborah J.

2009-01-01

Studies in the developing spinal cord suggest that different motoneuron (MN) cell types express very different genetic programs, but the degree to which adult programs differ is unknown. To compare genetic programs between adult MN columnar cell types, we used laser capture micro-dissection (LCM) and Affymetrix microarrays to create expression profiles for three columnar cell types: lateral and medial MNs from lumbar segments and sympathetic preganglionic motoneurons located in the thoracic intermediolateral nucleus. A comparison of the three expression profiles indicated that ~7% (813/11,552) of the genes showed significant differences in their expression levels. The largest differences were observed between sympathetic preganglionic MNs and the lateral motor column, with 6% (706/11,552) of the genes being differentially expressed. Significant differences in expression were observed for 1.8% (207/11,552) of the genes when comparing sympathetic preganglionic MNs with the medial motor column. Lateral and medial MNs showed the least divergence, with 1.3% (150/11,552) of the genes being differentially expressed. These data indicate that the amount of divergence in expression profiles between identified columnar MNs does not strictly correlate with divergence of function as defined by innervation patterns (somatic/muscle vs. autonomic/viscera). Classification of the differentially expressed genes with regard to function showed that they underpin all fundamental cell systems and processes, although most differentially expressed genes encode proteins involved in signal transduction. Mining the expression profiles to examine transcription factors essential for MN development suggested that many of the same transcription factors participatein combinatorial codes in embryonic and adult neurons, but patterns of expression change significantly. PMID:16317082

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.

[Effects of different mechanical stretch conditions on differentiation of rat tendon stem cells].

PubMed

Li, Pao; Gao, Shang; Zhou, Mei; Tang, Hong; Mu, Miduo; Zhang, Jiqiang; Tang, Kanglai

2017-04-01

To investigate the effects of different mechanical stretch conditions on the differentiation of rat tendon stem cells (TSCs), to find the best uniaxial cyclic stretching for TSCs tenogenic differentiation, osteogenic differentiation, and adipogenic differentiation. TSCs were isolated from the Achilles tendons of 8-week-old male Sprague Dawley rats by enzymatic digestion method and cultured. The TSCs at passage 3 were randomly divided into 5 groups: group A (stretch strength of 4% and frequency of 1 Hz), group B (stretch strength of 4% and frequency of 2 Hz), group C (stretch strength of 8% and frequency of 1 Hz), group D (stretch strength of 8% and frequency of 2 Hz), and group E (static culture). At 12, 24, and 48 hours after mechanical stretch, the mRNA expressions of the tenogenic differentiation related genes [Scleraxis (SCX) and Tenascin C (TNC)], the osteogenic differentiation related genes [runt related transcription factor 2 (RUNX2) and distal-less homeobox 5 (DLX5)], and the adipogenic differentiation related genes [CCAAT-enhancer-binding protein-α (CEBPα) and lipoprteinlipase (LPL)] were detected by real-time fluorescent quantitative PCR and the protein expressions of TNC, CEBPα, and RUNX2 were detected by Western blot. The mRNA expressions of SCX and TNC in group B were significantly higher than those in groups A, C, D, and E at 24 hours after mechanical stretch ( P <0.05). The mRNA expressions of CEBPα and LPL in group D were significantly higher than those in groups A, B, C, and E at 48 hours after mechanical stretch ( P <0.05). The mRNA expressions of RUNX2 and DLX5 in group C were significantly higher than those in groups A, B, D, and E at 24 hours after mechanical stretch ( P <0.05). Western blot detection showed that higher protein expression of TNC in group B than group E at each time point after mechanical stretch ( P <0.05), and the protein expression of CEBPα was significantly inhibited when compared with group E at 24 hours after mechanical stretch ( P <0.05). At 24 hours after mechanical stretch, the protein expression of RUNX2 in group C was significantly higher than that in group E ( P <0.05); and the protein expression of TNC was significantly lower than that in group E at 24 and 48 hours after mechanical stretch ( P <0.05). At 48 hours after mechanical stretch, the protein expression of CEBPα was significantly increased and the protein expression of TNC was significantly decreased in group D when compared with group E ( P <0.05), but no significant difference was found in the protein expression of RUNX2 between groups D and E ( P >0.05). The mechanical strain could promote differentiation of TSCs, and different parameter of stretch will lead to different differentiation. The best stretch condition for tenogenic differentiation is 4% strength and 2 Hz frequency for 24 hours; the best stretch condition for osteogenic differentiation is 8% strength and 1 Hz frequency for 24 hours; and the best stretch condition for adipogenic differentiation is 8% strength and 2 Hz frequency for 48 hours.

Long non-coding RNAs regulate effects of β-crystallin B2 on mouse ovary development.

PubMed

Gao, Qian; Ren, Hanxiao; Chen, Mingkun; Niu, Ziguang; Tao, Haibo; Jia, Yin; Zhang, Jianrong; Li, Wenjie

2016-11-01

β-crystallin B2 (CRYBB2) knockout mice exhibit morphological and functional abnormalities in the ovary. Long non‑coding RNAs (lncRNAs) regulate gene transcription and translation, and epigenetic modification of genomic DNA. The present study investigated the role of lncRNAs in mediating the effects of CRYBB2 in the regulation of ovary development in mice. In the current study, ovary tissues from wild‑type (WT) and CRYBB2 knockout mice were subjected to lncRNA and mRNA microarray profiling. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to group the differentially expressed lncRNAs into regulated gene pathways and functions. The correlation matrix method was used to establish a network of lncRNA and mRNA co‑expression. Quantitative reverse transcription-polymerase chain reaction (RT‑qPCR) was used to verify expression of a number of these differentially expressed lncRNAs and mRNAs. There were 157 differentially expressed lncRNAs and 1,085 differentially expressed mRNAs between ovary tissues from WT and CRYBB2 knockout mice. The GO and KEGG analyses indicated that these differentially expressed lncRNAs and mRNAs were important in Ca2+ signaling and ligand and receptor interactions. The correlation matrix method established an lncRNA and mRNA co‑expression network, consisting of 53 lncRNAs and 45 mRNAs with 98 nodes and 75 connections. RT‑qPCR confirmed downregulation of lncRNA A‑30‑P01019163 expression, which further downregulated its downstream gene purinergic receptor P2X, ligand‑gated ion channel, 7 (P2rx7) expression in ovary tissues from CRYBB2 knockout mice. In conclusion, CRYBB2 regulates expression of different lncRNAs to influence ovary development. lncRNA A‑30‑P01019163 may affect ovarian cell cycle and proliferation by regulating P2rx7 expression in the ovary.

Insights into the role of differential gene expression on the ecological adaptation of the snail Littorina saxatilis

PubMed Central

2010-01-01

Background In the past 40 years, there has been increasing acceptance that variation in levels of gene expression represents a major source of evolutionary novelty. Gene expression divergence is therefore likely to be involved in the emergence of incipient species, namely, in a context of adaptive radiation. In this study, a genome-wide expression profiling approach (cDNA-AFLP), validated by quantitative real-time polymerase chain reaction (qPCR) were used to get insights into the role of differential gene expression on the ecological adaptation of the marine snail Littorina saxatilis. This gastropod displays two sympatric ecotypes (RB and SU) which are becoming one of the best studied systems for ecological speciation. Results Among the 99 transcripts shared between ecotypes, 12.12% showed significant differential expression. At least 4% of these transcripts still displayed significant differences after correction for multiple tests, highlighting that gene expression can differ considerably between subpopulations adapted to alternative habitats in the face of gene flow. One of the transcripts identified was Cytochrome c Oxidase subunit I (COI). In addition, 6 possible reference genes were validated to normalize and confirm this result using qPCR. α-Tubulin and histone H3.3 showed the more stable expression levels, being therefore chosen as the best option for normalization. The qPCR analysis confirmed a higher COI expression in SU individuals. Conclusions At least 4% of the transcriptome studied is being differentially expressed between ecotypes living in alternative habitats, even when gene flow is still substantial between ecotypes. We could identify a candidate transcript of such ecotype differentiation: Cytochrome c Oxidase Subunit I (COI), a mitochondrial gene involved in energy metabolism. Quantitative PCR was used to confirm the differences found in COI and its over-expression in the SU ecotype. Interestingly, COI is involved in the oxidative phosphorylation, suggesting an enhanced mitochondrial gene expression (or increased number of mitochondria) to improve energy supply in the ecotype subjected to the strongest wave action. PMID:21087461

Phytohormonal Networks Promote Differentiation of Fiber Initials on Pre-Anthesis Cotton Ovules Grown In Vitro and In Planta

PubMed Central

Kim, Hee Jin; Hinchliffe, Doug J.; Triplett, Barbara A.; Chen, Z. Jeffrey; Stelly, David M.; Yeater, Kathleen M.; Moon, Hong S.; Gilbert, Matthew K.; Thyssen, Gregory N.; Turley, Rickie B.; Fang, David D.

2015-01-01

The number of cotton (Gossypium sp.) ovule epidermal cells differentiating into fiber initials is an important factor affecting cotton yield and fiber quality. Despite extensive efforts in determining the molecular mechanisms regulating fiber initial differentiation, only a few genes responsible for fiber initial differentiation have been discovered. To identify putative genes directly involved in the fiber initiation process, we used a cotton ovule culture technique that controls the timing of fiber initial differentiation by exogenous phytohormone application in combination with comparative expression analyses between wild type and three fiberless mutants. The addition of exogenous auxin and gibberellins to pre-anthesis wild type ovules that did not have visible fiber initials increased the expression of genes affecting auxin, ethylene, ABA and jasmonic acid signaling pathways within 1 h after treatment. Most transcripts expressed differentially by the phytohormone treatment in vitro were also differentially expressed in the ovules of wild type and fiberless mutants that were grown in planta. In addition to MYB25-like, a gene that was previously shown to be associated with the differentiation of fiber initials, several other differentially expressed genes, including auxin/indole-3-acetic acid (AUX/IAA) involved in auxin signaling, ACC oxidase involved in ethylene biosynthesis, and abscisic acid (ABA) 8'-hydroxylase an enzyme that controls the rate of ABA catabolism, were co-regulated in the pre-anthesis ovules of both wild type and fiberless mutants. These results support the hypothesis that phytohormonal signaling networks regulate the temporal expression of genes responsible for differentiation of cotton fiber initials in vitro and in planta. PMID:25927364

Altered gene expression in tree shrew retina and retinal pigment epithelium produced by short periods of minus-lens wear.

PubMed

He, Li; Frost, Michael R; Siegwart, John T; Norton, Thomas T

2018-03-01

Hyperopic refractive error is detected by retinal neurons, which generate GO signals through a direct emmetropization signaling cascade: retinal pigment epithelium (RPE) into choroid and then into sclera, thereby increasing axial elongation. To examine signaling early in this cascade, we measured gene expression in the retina and RPE after short exposure to hyperopia produced by minus-lens wear. Gene expression in each tissue was compared with gene expression in combined retina + RPE. Starting 24 days after normal eye opening, three groups of juvenile tree shrews (n = 7 each) wore a monocular -5 D lens. The untreated fellow eye served as a control. The "6h" group wore the lens for 6 h; the "24h" group wore the lens for 24 h; each group provided separate retina and RPE tissues. Group "24hC" wore the lens for 24 h and provided combined retina + RPE tissue. Quantitative PCR was used to measure the relative differences (treated eye vs. control eye) in mRNA levels for 66 candidate genes. In the retina after 6 h, mRNA levels for seven genes were significantly regulated: EGR1 and FOS (early intermediate genes) were down-regulated in the treated eyes. Genes with secreted protein products, BMP2 and CTGF, were down-regulated, whilst FGF10, IL18, and SST were up-regulated. After 24 h the pattern changed; only one of the seven genes still showed differential expression; BMP2 was still down-regulated. Two new genes with secreted protein products, IGF2 and VIP, were up-regulated. In the RPE, consistent with its role in receiving, processing, and transmitting GO signaling, differential expression was found for genes whose protein products are at the cell surface, intracellular, in the nucleus, and are secreted. After 6 h, mRNA levels for 17 genes were down-regulated in the treated eyes, whilst four genes (GJA1, IGF2R, LRP2, and IL18) were up-regulated. After 24 h the pattern was similar; mRNA levels for 14 of the same genes were still down-regulated; only LRP2 remained up-regulated. mRNA levels for six genes no longer showed differential expression, whilst nine genes, not differentially expressed at 6 h, now showed differential expression. In the combined retina + RPE after 24 h, mRNA levels for only seven genes were differentially regulated despite the differential expression of many genes in the RPE. Four genes showed the same expression in combined tissue as in retina alone, including up-regulation of VIP despite significant VIP down-regulation in RPE. Thus, hyperopia-induced GO signaling, as measured by differential gene expression, differs in the retina and the RPE. Retinal gene expression changed between 6 h and 24 h of treatment, suggesting evolution of the retinal response. Gene expression in the RPE was similar at both time points, suggesting sustained signaling. The combined retina + RPE does not accurately represent gene expression in either retina or, especially, RPE. When gene expression signatures were compared with those in choroid and sclera, GO signaling, as encoded by differential gene expression, differs in each compartment of the direct emmetropization signaling cascade. Copyright © 2018 Elsevier Ltd. All rights reserved.

Discrimination of emotional facial expressions by tufted capuchin monkeys (Sapajus apella).

PubMed

Calcutt, Sarah E; Rubin, Taylor L; Pokorny, Jennifer J; de Waal, Frans B M

2017-02-01

Tufted or brown capuchin monkeys (Sapajus apella) have been shown to recognize conspecific faces as well as categorize them according to group membership. Little is known, though, about their capacity to differentiate between emotionally charged facial expressions or whether facial expressions are processed as a collection of features or configurally (i.e., as a whole). In 3 experiments, we examined whether tufted capuchins (a) differentiate photographs of neutral faces from either affiliative or agonistic expressions, (b) use relevant facial features to make such choices or view the expression as a whole, and (c) demonstrate an inversion effect for facial expressions suggestive of configural processing. Using an oddity paradigm presented on a computer touchscreen, we collected data from 9 adult and subadult monkeys. Subjects discriminated between emotional and neutral expressions with an exceptionally high success rate, including differentiating open-mouth threats from neutral expressions even when the latter contained varying degrees of visible teeth and mouth opening. They also showed an inversion effect for facial expressions, results that may indicate that quickly recognizing expressions does not originate solely from feature-based processing but likely a combination of relational processes. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

Generation of novel pharmacogenomic candidates in the response to methotrexate in juvenile idiopathic arthritis: correlation between gene expression and genotype

PubMed Central

Moncrieffe, Halima; Hinks, Anne; Ursu, Simona; Kassoumeri, Laura; Etheridge, Angela; Hubank, Mike; Martin, Paul; Weiler, Tracey; Glass, David N; Thompson, Susan D.; Thomson, Wendy; Wedderburn, Lucy R

2010-01-01

Objectives Little is known about mechanisms of efficacy of methotrexate (MTX) in childhood arthritis, or genetic influences upon response to MTX. The aims of this study were to use gene expression profiling to identify novel pathways/genes altered by MTX and then investigate these genes for genotype associations with response to MTX treatment. Methods Gene expression profiling before and after MTX treatment was performed on 11 children with juvenile idiopathic arthritis (JIA) treated with MTX, in whom response at 6 months of treatment was defined. Genes showing the most differential gene expression after treatment were selected for SNP genotyping. Genotype frequencies were compared between non-responders and responders (ACR-Ped70). An independent cohort was available for validation. Results Gene expression profiling before and after MTX treatment revealed 1222 differentially expressed probes sets (fold change >1.7, p< 0.05) and 1065 when restricted to full responder cases only. Six highly differentially expressed genes were analysed for genetic association to response to MTX. Three SNPs in the SLC16A7 gene showed significant association with MTX response. One SNP showed validated association in an independent cohort. Conclusions This study is the first, to our knowledge, to evaluate gene expression profiles in children with JIA before and after MTX, and to analyse genetic variation in differentially expressed genes. We have identified a gene which may contribute to genetic variability in MTX response in JIA, and established as proof of principle that genes which are differentially expressed at mRNA level after drug administration may also be good candidates for genetic analysis. PMID:20827233

Bit-1 is an essential regulator of myogenic differentiation

PubMed Central

Griffiths, Genevieve S.; Doe, Jinger; Jijiwa, Mayumi; Van Ry, Pam; Cruz, Vivian; de la Vega, Michelle; Ramos, Joe W.; Burkin, Dean J.; Matter, Michelle L.

2015-01-01

Muscle differentiation requires a complex signaling cascade that leads to the production of multinucleated myofibers. Genes regulating the intrinsic mitochondrial apoptotic pathway also function in controlling cell differentiation. How such signaling pathways are regulated during differentiation is not fully understood. Bit-1 (also known as PTRH2) mutations in humans cause infantile-onset multisystem disease with muscle weakness. We demonstrate here that Bit-1 controls skeletal myogenesis through a caspase-mediated signaling pathway. Bit-1-null mice exhibit a myopathy with hypotrophic myofibers. Bit-1-null myoblasts prematurely express muscle-specific proteins. Similarly, knockdown of Bit-1 expression in C2C12 myoblasts promotes early differentiation, whereas overexpression delays differentiation. In wild-type mice, Bit-1 levels increase during differentiation. Bit-1-null myoblasts exhibited increased levels of caspase 9 and caspase 3 without increased apoptosis. Bit-1 re-expression partially rescued differentiation. In Bit-1-null muscle, Bcl-2 levels are reduced, suggesting that Bcl-2-mediated inhibition of caspase 9 and caspase 3 is decreased. Bcl-2 re-expression rescued Bit-1-mediated early differentiation in Bit-1-null myoblasts and C2C12 cells with knockdown of Bit-1 expression. These results support an unanticipated yet essential role for Bit-1 in controlling myogenesis through regulation of Bcl-2. PMID:25770104

Differential Pre-mRNA Splicing Regulates Nnat Isoforms in the Hypothalamus after Gastric Bypass Surgery in Mice

PubMed Central

Scott, William R.; Gelegen, Cigdem; Chandarana, Keval; Karra, Efthimia; Yousseif, Ahmed; Amouyal, Chloé; Choudhury, Agharul I.; Andreelli, Fabrizio; Withers, Dominic J.; Batterham, Rachel L.

2013-01-01

Background Neuronatin (NNAT) is an endoplasmic reticulum proteolipid implicated in intracellular signalling. Nnat is highly-expressed in the hypothalamus, where it is acutely regulated by nutrients and leptin. Nnat pre-mRNA is differentially spliced to create Nnat-α and -β isoforms. Genetic variation of NNAT is associated with severe obesity. Currently, little is known about the long-term regulation of Nnat. Methods Expression of Nnat isoforms were examined in the hypothalamus of mice in response to acute fast/feed, chronic caloric restriction, diet-induced obesity and modified gastric bypass surgery. Nnat expression was assessed in the central nervous system and gastrointestinal tissues. RTqPCR was used to determine isoform-specific expression of Nnat mRNA. Results Hypothalamic expression of both Nnat isoforms was comparably decreased by overnight and 24-h fasting. Nnat expression was unaltered in diet-induced obesity, or subsequent switch to a calorie restricted diet. Nnat isoforms showed differential expression in the hypothalamus but not brainstem after bypass surgery. Hypothalamic Nnat-β expression was significantly reduced after bypass compared with sham surgery (P = 0.003), and was positively correlated with post-operative weight-loss (R2 = 0.38, P = 0.01). In contrast, Nnat-α expression was not suppressed after bypass surgery (P = 0.19), and expression did not correlate with reduction in weight after surgery (R2 = 0.06, P = 0.34). Hypothalamic expression of Nnat-β correlated weakly with circulating leptin, but neither isoform correlated with fasting gut hormone levels post- surgery. Nnat expression was detected in brainstem, brown-adipose tissue, stomach and small intestine. Conclusions Nnat expression in hypothalamus is regulated by short-term nutrient availability, but unaltered by diet-induced obesity or calorie restriction. While Nnat isoforms in the hypothalamus are co-ordinately regulated by acute nutrient supply, after modified gastric bypass surgery Nnat isoforms show differential expression. These results raise the possibility that in the radically altered nutrient and hormonal milieu created by bypass surgery, resultant differential splicing of Nnat pre-mRNA may contribute to weight-loss. PMID:23527188

Metastatic canine mammary carcinomas can be identified by a gene expression profile that partly overlaps with human breast cancer profiles

PubMed Central

2010-01-01

Background Similar to human breast cancer mammary tumors of the female dog are commonly associated with a fatal outcome due to the development of distant metastases. However, the molecular defects leading to metastasis are largely unknown and the value of canine mammary carcinoma as a model for human breast cancer is unclear. In this study, we analyzed the gene expression signatures associated with mammary tumor metastasis and asked for parallels with the human equivalent. Methods Messenger RNA expression profiles of twenty-seven lymph node metastasis positive or negative canine mammary carcinomas were established by microarray analysis. Differentially expressed genes were functionally characterized and associated with molecular pathways. The findings were also correlated with published data on human breast cancer. Results Metastatic canine mammary carcinomas had 1,011 significantly differentially expressed genes when compared to non-metastatic carcinomas. Metastatic carcinomas had a significant up-regulation of genes associated with cell cycle regulation, matrix modulation, protein folding and proteasomal degradation whereas cell differentiation genes, growth factor pathway genes and regulators of actin organization were significantly down-regulated. Interestingly, 265 of the 1,011 differentially expressed canine genes are also related to human breast cancer and, vice versa, parts of a human prognostic gene signature were identified in the expression profiles of the metastatic canine tumors. Conclusions Metastatic canine mammary carcinomas can be discriminated from non-metastatic carcinomas by their gene expression profiles. More than one third of the differentially expressed genes are also described of relevance for human breast cancer. Many of the differentially expressed genes are linked to functions and pathways which appear to be relevant for the induction and maintenance of metastatic progression and may represent new therapeutic targets. Furthermore, dogs are in some aspects suitable as a translational model for human breast tumors in order to identify prognostic molecular signatures and potential therapeutic targets. PMID:21062462

1,25-dihydroxyvitamin D3 induces CCR10 expression in terminally differentiating human B cells.

PubMed

Shirakawa, Aiko-Konno; Nagakubo, Daisuke; Hieshima, Kunio; Nakayama, Takashi; Jin, Zhe; Yoshie, Osamu

2008-03-01

In the B cell lineage, CCR10 is known to be selectively expressed by plasma cells, especially those secreting IgA. In this study, we examined the regulation of CCR10 expression in terminally differentiating human B cells. As reported previously, IL-21 efficiently induced the differentiation of activated human CD19+ B cells into IgD-CD38+ plasma cells in vitro. A minor proportion of the resulting CD19+IgD-CD38+ cells expressed CCR10 at low levels. 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), the active metabolite of vitamine D3, dramatically increased the proportion of CD19+IgD-CD38+ cells expressing high levels of CCR10. The 1,25-(OH)2D3 also increased the number of CCR10+ cells expressing surface IgA, although the majority of CCR10+ cells remained negative for surface IgA. Thus, 1,25-(OH)2D3 alone may not be sufficient for the induction of IgA expression in terminally differentiating human B cells. To further determine whether 1,25-(OH)2D3 directly induces CCR10 expression in terminally differentiating B cells, we next performed the analysis on the human CCR10 promoter. We identified a proximal Ets-1 site and an upstream potential vitamin D response element to be critical for the inducible expression of CCR10 by 1,25-(OH)2D3. We confirmed the specific binding of Ets-1 and 1,25-(OH)2D3-activated vitamin D receptor to the respective sites. In conclusion, 1,25-(OH)2D3 efficiently induces CCR10 expression in terminally differentiating human B cells in vitro. Furthermore, the human CCR10 promoter is cooperatively activated by Ets-1 and vitamin D receptor in the presence of 1,25-(OH)2D3.

[Differentiation of bone marrow derived from mesenchymal stem cells into cardiomyocyte-like cells induced by co-culture with rat myocardial cells].

PubMed

Zhang, Rong-Li; Jiang, Er-Lie; Wang, Mei; Zhou, Zheng; Zhai, Wen-Jing; Zhai, Wei-Hua; Wang, Hua; Wang, Zhi-Yong; Bao, Yu-Shi; DU, Hong; Han, Ming-Zhe

2008-10-01

The study was purposed to investigate the differentiation ability of mesenchymal stem cells (MSCs) into myocardial cells in vitro. Rat bone marrow-derived MSCs were labeled and co-cultured with neonatal rat cardiomyocytes (CM) for 5 - 7 days. The expression of cell surface antigens was detected by flow cytometry, and the expression of muscle-specific marker myosin and troponin T in labeled cells was detected by immunofluorescence. The results showed that in vitro cultured MSCs expressed CD90, CD44, CD105, CD54, not expressed CD34, CD45, CD31. After co-cultured with neonatal rat CM, labeled MSCs differentiated into cardiomyocyte-like cells expressing myosin and troponin T. It is concluded that MSCs can differentiate into cardiomyocyte-like cells when co-cultured with neonatal myocardial cells in vitro. In co-culture of two kind of cells in ratio of four to one showed obvious efficacy differentiating MSCs into CMs.

Network-based expression analyses and experimental validations revealed high co-expression between Yap1 and stem cell markers compared to differentiated cells.

PubMed

Dehghanian, Fariba; Hojati, Zohreh; Esmaeili, Fariba; Masoudi-Nejad, Ali

2018-05-21

The Hippo signaling pathway is identified as a potential regulatory pathway which plays critical roles in differentiation and stem cell self-renewal. Yap1 is a primary transcriptional effector of this pathway. The importance of Yap1 in embryonic stem cells (ESCs) and differentiation procedure remains a challenging question, since two different observations have been reported. To answer this question we used co-expression network and differential co-expression analyses followed by experimental validations. Our results indicate that Yap1 is highly co-expressed with stem cell markers in ESCs but not in differentiated cells (DCs). The significant Yap1 down-regulation and also translocation of Yap1 into the cytoplasm during P19 differentiation was also detected. Moreover, our results suggest the E2f7, Lin28a and Dppa4 genes as possible regulatory nuclear factors of Hippo pathway in stem cells. The present findings are actively consistent with studies that suggested Yap1 as an essential factor for stem cell self-renewal. Copyright © 2018 Elsevier Inc. All rights reserved.

Enforced expression of KDR receptor promotes proliferation, survival and megakaryocytic differentiation of TF1 progenitor cell line.

PubMed

Coppola, S; Narciso, L; Feccia, T; Bonci, D; Calabrò, L; Morsilli, O; Gabbianelli, M; De Maria, R; Testa, U; Peschle, C

2006-01-01

Vascular endothelial growth factor (VEGF) receptor-2/kinase insert domain-containing receptor (KDR) is expressed in primitive hematopoietic cells, in megakaryocytes and platelets. In primitive hematopoiesis KDR mediates cell survival via autocrine VEGF, while its effect on cell growth and differentiation has not been elucidated. We induced enforced KDR expression in the granulocyte macrophage-colony-stimulating factor (GM-CSF)-dependent TF1 progenitor cell line (TF1-KDR), treated the cells with VEGF and analyzed their response. In GM-CSF-deprived cells, VEGF induces cell proliferation and protection against apoptosis, followed by enhanced expression of megakaryocytic (MK) markers. Combined with GM-CSF, VEGF induces a mild proliferative stimulus, followed by cell adherence, accumulation in G0/G1, massive MK differentiation and Fas-mediated apoptosis. Accordingly, we observed that MK-differentiating cells, derived from hematopoietic progenitors, produce VEGF, express KDR, inhibition of which reduces MK differentiation, indicating a key role of KDR in megakaryopoiesis. In conclusion, TF1-KDR cells provide a reliable model to investigate the biochemical and molecular mechanisms underlying hematopoietic progenitor proliferation, survival and MK differentiation.

Regulation of T-type Ca2+ channel expression by herpes simplex virus-1 infection in sensory-like ND7 cells

PubMed Central

Zhang, Qiaojuan; Hsia, Shao-Chung

2017-01-01

Infection of sensory neurons by herpes simplex virus (HSV)-1 disrupts electrical excitability, altering pain sensory transmission. Because of their low threshold for activation, functional expression of T-type Ca2+ channels regulates various cell functions, including neuronal excitability and neuronal communication. In this study, we have tested the effect of HSV-1 infection on the functional expression of T-type Ca2+ channels in differentiated ND7-23 sensory-like neurons. Voltage-gated Ca2+ currents were measured using whole cell patch clamp recordings in differentiated ND7-23 neurons under various culture conditions. Differentiation of ND7-23 cells evokes a significant increase in T-type Ca2+ current densities. Increased T-type Ca2+ channel expression promotes the morphological differentiation of ND7-23 cells and triggers a rebound depolarization. HSV-1 infection of differentiated ND7-23 cells causes a significant loss of T-type Ca2+ channels from the membrane. HSV-1 evoked reduction in the functional expression of T-type Ca2+ channels is mediated by several factors, including decreased expression of Cav3.2 T-type Ca2+ channel subunits and disruption of endocytic transport. Decreased functional expression of T-type Ca2+ channels by HSV-1 infection requires protein synthesis and viral replication, but occurs independently of Egr-1 expression. These findings suggest that infection of neuron-like cells by HSV-1 causes a significant disruption in the expression of T-type Ca2+ channels, which can results in morphological and functional changes in electrical excitability. PMID:28639215

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.

Interferon regulatory factor-1 binds c-Cbl, enhances mitogen activated protein kinase signaling and promotes retinoic acid-induced differentiation of HL-60 human myelo-monoblastic leukemia cells.

PubMed

Shen, Miaoqing; Bunaciu, Rodica P; Congleton, Johanna; Jensen, Holly A; Sayam, Lavanya G; Varner, Jeffrey D; Yen, Andrew

2011-12-01

All-trans retinoic acid (RA) and interferons (IFNs) have efficacy in treating certain leukemias and lymphomas, respectively, motivating interest in their mechanism of action to improve therapy. Both RA and IFNs induce interferon regulatory factor-1 (IRF-1). We find that in HL-60 myeloblastic leukemia cells which undergo mitogen activated protien kinase (MAPK)-dependent myeloid differentiation in response to RA, IRF-1 propels differentiation. RA induces MAPK-dependent expression of IRF-1. IRF-1 binds c-Cbl, a MAPK related adaptor. Ectopic IRF-1 expression causes CD38 expression and activation of the Raf/MEK/ERK axis, and enhances RA-induced differentiation by augmenting CD38, CD11b, respiratory burst and G0 arrest. Ectopic IRF-1 expression also decreases the activity of aldehyde dehydrogenase 1, a stem cell marker, and enhances RA-induced ALDH1 down-regulation. Interestingly, expression of aryl hydrocarbon receptor (AhR), which is RA-induced and known to down-regulate Oct4 and drive RA-induced differentiation, also enhances IRF-1 expression. The data are consistent with a model whereby IRF-1 acts downstream of RA and AhR to enhance Raf/MEK/ERK activation and propel differentiation.

Expression of growth differentiation factor 6 in the human developing fetal spine retreats from vertebral ossifying regions and is restricted to cartilaginous tissues.

PubMed

Wei, Aiqun; Shen, Bojiang; Williams, Lisa A; Bhargav, Divya; Gulati, Twishi; Fang, Zhimin; Pathmanandavel, Sarennya; Diwan, Ashish D

2016-02-01

During embryogenesis vertebral segmentation is initiated by sclerotomal cell migration and condensation around the notochord, forming anlagen of vertebral bodies and intervertebral discs. The factors that govern the segmentation are not clear. Previous research demonstrated that mutations in growth differentiation factor 6 resulted in congenital vertebral fusion, suggesting this factor plays a role in development of vertebral column. In this study, we detected expression and localization of growth differentiation factor 6 in human fetal spinal column, especially in the period of early ossification of vertebrae and the developing intervertebral discs. The extracellular matrix proteins were also examined. Results showed that high levels of growth differentiation factor 6 were expressed in the nucleus pulposus of intervertebral discs and the hypertrophic chondrocytes adjacent to the ossification centre in vertebral bodies, where strong expression of proteoglycan and collagens was also detected. As fetal age increased, the expression of growth differentiation factor 6 was decreased correspondingly with the progress of ossification in vertebral bodies and restricted to cartilaginous regions. This expression pattern and the genetic link to vertebral fusion suggest that growth differentiation factor 6 may play an important role in suppression of ossification to ensure proper vertebral segmentation during spinal development. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Vesicular glutamate transporters play a role in neuronal differentiation of cultured SVZ-derived neural precursor cells

PubMed Central

Sánchez-Mendoza, Eduardo H.; Bellver-Landete, Victor; Arce, Carmen; Doeppner, Thorsten R.; Hermann, Dirk M.

2017-01-01

The role of glutamate in the regulation of neurogenesis is well-established, but the role of vesicular glutamate transporters (VGLUTs) and excitatory amino acid transporters (EAATs) in controlling adult neurogenesis is unknown. Here we investigated the implication of VGLUTs in the differentiation of subventricular zone (SVZ)-derived neural precursor cells (NPCs). Our results show that NPCs express VGLUT1-3 and EAAT1-3 both at the mRNA and protein level. Their expression increases during differentiation closely associated with the expression of marker genes. In expression analyses we show that VGLUT1 and VGLUT2 are preferentially expressed by cultured SVZ-derived doublecortin+ neuroblasts, while VGLUT3 is found on GFAP+ glial cells. In cultured NPCs, inhibition of VGLUT by Evans Blue increased the mRNA level of neuronal markers doublecortin, B3T and MAP2, elevated the number of NPCs expressing doublecortin protein and promoted the number of cells with morphological appearance of branched neurons, suggesting that VGLUT function prevents neuronal differentiation of NPCs. This survival- and differentiation-promoting effect of Evans blue was corroborated by increased AKT phosphorylation and reduced MAPK phosphorylation. Thus, under physiological conditions, VGLUT1-3 inhibition, and thus decreased glutamate exocytosis, may promote neuronal differentiation of NPCs. PMID:28493916

Impaired detection and differentiation of briefly presented facial emotions in adults with high-functioning autism and asperger syndrome.

PubMed

Frank, R; Schulze, L; Hellweg, R; Koehne, S; Roepke, S

2018-05-01

Although deficits in the recognition of emotional facial expressions are considered a hallmark of autism spectrum disorder (ASD), characterization of abnormalities in the differentiation of emotional expressions (e.g., sad vs. angry) has been rather inconsistent, especially in adults without intellectual impairments who may compensate for their deficits. In addition, previous research neglected the ability to detect emotional expressions (e.g., angry vs. neutral). The present study used a backward masking paradigm to investigate, a) the detection of emotional expressions, and b) the differentiation of emotional expressions in adults diagnosed with high functioning autism or Asperger syndrome (n = 23) compared to neurotypical controls (n = 25). Compensatory strategies were prevented by shortening the stimulus presentation time (33, 67, and 100 ms). In general, participants with ASD were significantly less accurate in detecting and differentiating emotional expressions compared to the control group. In the emotion differentiation task, individuals with ASD profited significantly less from an increase in presentation time. These results reinforce theoretical models that individuals with ASD have deficits in emotion recognition under time constraints. Furthermore, first evidence was provided that emotion detection and emotion differentiation are impaired in ASD. Copyright © 2018 Elsevier Ltd. All rights reserved.

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.

Integrating microRNA and mRNA expression profiles of acute promyelocytic leukemia cells to explore the occurrence mechanisms of differentiation syndrome

PubMed Central

Ge, Fei; Cao, Fenglin; Li, Haitao; Wang, Ping; Xu, Mengyuan; Song, Peng; Li, Xiaoxia; Wang, Shuye; Li, Jinmei; Han, Xueying; Zhao, Yanhong; Su, Yanhua; Li, Yinghua; Fan, Shengjin; Li, Limin; Zhou, Jin

2016-01-01

The pathogenesis of therapy-induced differentiation syndrome (DS) in patients with acute promyelocytic leukemia (APL) remains unclear. In this study, mRNA and microRNA (miRNA) expression profiling of peripheral blood APL cells from patients complicated with vs. without DS were integratively analyzed to explore the mechanisms underlying arsenic trioxide treatment-associated DS. By integrating the differentially expressed data with the data of differentially expressed microRNAs and their computationally predicted target genes, as well as the data of transcription factors and differentially expressed target microRNAs obtained from a literature search, a DS-related genetic regulatory network was constructed. Then using an EAGLE algorithm in clusterViz, the network was subdivided into 10 modules. Using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database the modules were annotated functionally, and three functionally active modules were recognized. The further in-depth analyses on the annotated functions of the three modules and the expression and roles of the related genes revealed that proliferation, differentiation, apoptosis and infiltration capability of APL cells might play important roles in the DS pathogenesis. The results could improve our understanding of DS pathogenesis from a more overall perspective, and could provide new clues for future research. PMID:27634874

Ascl1-induced neuronal differentiation of P19 cells requires expression of a specific inhibitor protein of cAMP-dependent protein kinase

PubMed Central

Huang, Holly S.; Turner, David L.; Thompson, Robert C.; Uhler, Michael D.

2011-01-01

cAMP-dependent protein kinase (PKA) plays a critical role in nervous system development by modulating sonic hedgehog and bone morphogenetic protein signaling. In the current studies, P19 embryonic carcinoma cells were neuronally differentiated by expression of the proneural basic helix-loop-helix transcription factor Ascl1. After expression of Ascl1, but prior to expression of neuronal markers such as microtubule associated protein 2 and neuronal β-tubulin, P19 cells demonstrated a large, transient increase in both mRNA and protein for the endogenous protein kinase inhibitor (PKI)β. PKIβ-targeted shRNA constructs both reduced the levels of PKIβ expression and blocked the neuronal differentiation of P19 cells. This inhibition of differentiation was rescued by transfection of a shRNA-resistant expression vector for the PKIβ protein, and this rescue required the PKA-specific inhibitory sequence of the PKIβprotein. PKIβ played a very specific role in the Ascl1-mediated differentiation process since other PKI isoforms were unable to rescue the deficit conferred by shRNA-mediated knockdown of PKIβ. Our results define a novel requirement for PKIβ and its inhibition of PKA during neuronal differentiation of P19 cells. PMID:21623794

Expression of c-Fes protein isoforms correlates with differentiation in myeloid leukemias.

PubMed

Carlson, Anne; Berkowitz, Jeanne McAdara; Browning, Damaris; Slamon, Dennis J; Gasson, Judith C; Yates, Karen E

2005-05-01

The cellular fes gene encodes a 93-kilodalton protein-tyrosine kinase (p93) that is expressed in both normal and neoplastic myeloid cells. Increased c-Fes expression is associated with differentiation in normal myeloid cells and cell lines. Our hypothesis was that primary leukemia cells would show a similar pattern of increased expression in more differentiated cells. Therefore, we compared c-Fes expression in cells with an undifferentiated, blast phenotype (acute myelogenous leukemia--AML) to cells with a differentiated phenotype (chronic myelogenous leukemia--CML). Instead of differences in p93 expression levels, we found complex patterns of c-Fes immunoreactive proteins that corresponded with differentiation in normal and leukemic myeloid cells. The "blast" pattern consisted of c-Fes immunoreactive proteins p93, p74, and p70; the "differentiated" pattern showed two additional c-Fes immunoreactive proteins, p67 and p62. Using mRNA from mouse and human cell lines, we found deletion of one or more exons in the c-fes mRNA. Those deletions predicted truncation of conserved domains (CDC15/FCH and SH2) involved in protein-protein interactions. No deletions were found, however, within the kinase domain. We infer that alternative splicing generates a family of c-Fes proteins. This may be a mechanism to direct the c-Fes kinase domain to different subcellular locations and/or substrates at specific stages of myeloid cell differentiation.

Macrophage differentiation increases expression of the ascorbate transporter (SVCT2)

PubMed Central

Qiao, Huan; May, James M.

2013-01-01

To determine whether macrophage differentiation involves increased uptake of vitamin C, or ascorbic acid, we assessed the expression and function of its transporter SVCT2 during phorbol ester-induced differentiation of human-derived THP-1 monocytes. Induction of THP-1 monocyte differentiation by phorbol 12-myristate 13-acetate (PMA) markedly increased SVCT2 mRNA, protein, and function. When ascorbate was present during PMA-induced differentiation, the increase in SVCT2 protein expression was inhibited, but differentiation was enhanced. PMA-induced SVCT2 protein expression was blocked by inhibitors of protein kinase C (PKC), with most of the affect due to the PKCβI and βII isoforms. Activation of MEK/ERK was sustained up to 48 h after PMA treatment, and the inhibitors completely blocked PMA-stimulated SVCT2 protein expression, indicating an exclusive role for the classical MAP kinase pathway. However, inhibitors of NF-κB activation, NADPH oxidase inhibitors, and several antioxidants also partially prevented SVCT2 induction, suggesting diverse distal routes for control of SVCT2 transcription. Both known promoters for the SVCT2 were involved in these effects. In conclusion, PMA-induced monocyte-macrophage differentiation is enhanced by ascorbate and associated with increased expression and function of the SVCT2 protein through a pathway involving sustained activation of PKCβI/II, MAP kinase, NADPH oxidase, and NF-κB. PMID:19232538

Isolation and characterization of porcine adipose tissue-derived adult stem cells.

PubMed

Williams, Kellie J; Picou, Alicia A; Kish, Sharon L; Giraldo, Angelica M; Godke, Robert A; Bondioli, Kenneth R

2008-01-01

Stem cell characteristics such as self-renewal, differentiation and expression of CD34 and CD44 stem cell markers have not been identified in porcine adipose tissue-derived adult stem (ADAS) cells. The objective of this study was to develop a protocol for the isolation and culture of porcine adipose tissue-derived cells and to determine stem cell-like characteristics. Primary cultures were established and cell cultures were maintained. Cloning capacity was determined using a ring cloning procedure. Primary cultures and clones were differentiated and stained for multiple differentiated phenotypes. CD34 and CD44 messenger ribonucleic acid (mRNA) was isolated and reverse transcriptase polymerase chain reaction was used to compare expression profiles. An average of 2,700,000 nucleated cells/ml was isolated; 26% were adherent, and cells completed a cell cycle approximately every 3.3 days. Ring cloning identified 19 colonies. Primary cultures and clones were determined to differentiate along osteogenic, adipogenic and chondrogenic tissue lineages. The mRNA expression profiles showed CD34 expression was higher for undifferentiated ADAS cells versus differentiated cell types and the CD34 expression level was lower than that of CD44 among differentiated cells. Improved culture conditions and defined cellular characteristics of these porcine ADAS cells have been identified. Porcine ADAS can self-renew, can differentiate into multiple tissue lineages and they express CD34. Copyright 2008 S. Karger AG, Basel.

Effect of The Receptor Activator of Nuclear Factor кB and RANK Ligand on In Vitro Differentiation of Cord Blood CD133(+) Hematopoietic Stem Cells to Osteoclasts.

PubMed

Kalantari, Nasim; Abroun, Saeid; Soleimani, Masoud; Kaviani, Saeid; Azad, Mehdi; Eskandari, Fatemeh; Habibi, Hossein

2016-01-01

Receptor activator of nuclear factor-kappa B ligand (RANKL) appears to be an osteoclast-activating factor, bearing an important role in the pathogenesis of multiple myeloma. Some studies demonstrated that U-266 myeloma cell line and primary myeloma cells expressed RANK and RANKL. It had been reported that the expression of myeloid and monocytoid markers was increased by co-culturing myeloma cells with hematopoietic stem cells (HSCs). This study also attempted to show the molecular mechanism of RANK and RANKL on differentiation capability of human cord blood HSC to osteoclast, as well as expression of calcitonin receptor (CTR) on cord blood HSC surface. In this experimental study, CD133(+) hematopoietic stem cells were isolated from umbilical cord blood and cultured in the presence of macrophage colony-stimulating factor (M-CSF) and RANKL. Osteoclast differentiation was characterized by using tartrate-resistant acid phosphatase (TRAP) staining, giemsa staining, immunophenotyping, and reverse transcription-polymerase chain reaction (RT-PCR) assay for specific genes. Hematopoietic stem cells expressed RANK before and after differentiation into osteoclast. Compared to control group, flow cytometric results showed an increased expression of RANK after differentiation. Expression of CTR mRNA showed TRAP reaction was positive in some differentiated cells, including osteoclast cells. Presence of RANKL and M-CSF in bone marrow could induce HSCs differentiation into osteoclast.

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

Effective myotube formation in human adipose tissue-derived stem cells expressing dystrophin and myosin heavy chain by cellular fusion with mouse C2C12 myoblasts

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

Eom, Young Woo; Biomedical Research Institute, Lifeliver Co., Ltd., Suwon; Lee, Jong Eun

2011-04-29

Highlights: {yields} hASCs were differentiated into skeletal muscle cells by treatment with 5-azacytidine, FGF-2, and the supernatant of cultured hASCs. {yields} Dystrophin and MyHC were expressed in late differentiation step by treatment with the supernatant of cultured hASCs. {yields} hASCs expressing dystrophin and MyHC contributed to myotube formation during co-culture with mouse myoblast C2C12 cells. -- Abstract: Stem cell therapy for muscular dystrophies requires stem cells that are able to participate in the formation of new muscle fibers. However, the differentiation steps that are the most critical for this process are not clear. We investigated the myogenic phases of humanmore » adipose tissue-derived stem cells (hASCs) step by step and the capability of myotube formation according to the differentiation phase by cellular fusion with mouse myoblast C2C12 cells. In hASCs treated with 5-azacytidine and fibroblast growth factor-2 (FGF-2) for 1 day, the early differentiation step to express MyoD and myogenin was induced by FGF-2 treatment for 6 days. Dystrophin and myosin heavy chain (MyHC) expression was induced by hASC conditioned medium in the late differentiation step. Myotubes were observed only in hASCs undergoing the late differentiation step by cellular fusion with C2C12 cells. In contrast, hASCs that were normal or in the early stage were not involved in myotube formation. Our results indicate that stem cells expressing dystrophin and MyHC are more suitable for myotube formation by co-culture with myoblasts than normal or early differentiated stem cells expressing MyoD and myogenin.« less

Differential expression of human lysyl hydroxylase genes, lysine hydroxylation, and cross-linking of type I collagen during osteoblastic differentiation in vitro

NASA Technical Reports Server (NTRS)

Uzawa, K.; Grzesik, W. J.; Nishiura, T.; Kuznetsov, S. A.; Robey, P. G.; Brenner, D. A.; Yamauchi, M.

1999-01-01

The pattern of lysyl hydroxylation in the nontriple helical domains of collagen is critical in determining the cross-linking pathways that are tissue specific. We hypothesized that the tissue specificity of type I collagen cross-linking is, in part, due to the differential expression of lysyl hydroxylase genes (Procollagen-lysine,2-oxyglutarate,5-dioxygenase 1, 2, and 3 [PLOD1, PLOD2, and PLOD3]). In this study, we have examined the expression patterns of these three genes during the course of in vitro differentiation of human osteoprogenitor cells (bone marrow stromal cells [BMSCs]) and normal skin fibroblasts (NSFs). In addition, using the medium and cell layer/matrix fractions in these cultures, lysine hydroxylation of type I collagen alpha chains and collagen cross-linking chemistries have been characterized. High levels of PLOD1 and PLOD3 genes were expressed in both BMSCs and NSFs, and the expression levels did not change in the course of differentiation. In contrast to the PLOD1 and PLOD3 genes, both cell types showed low PLOD2 gene expression in undifferentiated and early differentiated conditions. However, fully differentiated BMSCs, but not NSFs, exhibited a significantly elevated level (6-fold increase) of PLOD2 mRNA. This increase coincided with the onset of matrix mineralization and with the increase in lysyl hydroxylation in the nontriple helical domains of alpha chains of type I collagen molecule. Furthermore, the collagen cross-links that are derived from the nontriple helical hydroxylysine-aldehyde were found only in fully differentiated BMSC cultures. The data suggests that PLOD2 expression is associated with lysine hydroxylation in the nontriple helical domains of collagen and, thus, could be partially responsible for the tissue-specific collagen cross-linking pattern.

Expression of polysialylated neural cell adhesion molecules on adult stem cells after neuronal differentiation of inner ear spiral ganglion neurons

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

Park, Kyoung Ho; Yeo, Sang Won, E-mail: swyeo@catholic.ac.kr; Troy, Frederic A., E-mail: fatroy@ucdavis.edu

Highlights: • PolySia expressed on neurons primarily during early stages of neuronal development. • PolySia–NCAM is expressed on neural stem cells from adult guinea pig spiral ganglion. • PolySia is a biomarker that modulates neuronal differentiation in inner ear stem cells. - Abstract: During brain development, polysialylated (polySia) neural cell adhesion molecules (polySia–NCAMs) modulate cell–cell adhesive interactions involved in synaptogenesis, neural plasticity, myelination, and neural stem cell (NSC) proliferation and differentiation. Our findings show that polySia–NCAM is expressed on NSC isolated from adult guinea pig spiral ganglion (GPSG), and in neurons and Schwann cells after differentiation of the NSC withmore » epidermal, glia, fibroblast growth factors (GFs) and neurotrophins. These differentiated cells were immunoreactive with mAb’s to polySia, NCAM, β-III tubulin, nestin, S-100 and stained with BrdU. NSC could regenerate and be differentiated into neurons and Schwann cells. We conclude: (1) polySia is expressed on NSC isolated from adult GPSG and on neurons and Schwann cells differentiated from these NSC; (2) polySia is expressed on neurons primarily during the early stage of neuronal development and is expressed on Schwann cells at points of cell–cell contact; (3) polySia is a functional biomarker that modulates neuronal differentiation in inner ear stem cells. These new findings suggest that replacement of defective cells in the inner ear of hearing impaired patients using adult spiral ganglion neurons may offer potential hope to improve the quality of life for patients with auditory dysfunction and impaired hearing disorders.« less

Expression Variations of miRNAs and mRNAs in Rice (Oryza sativa)

PubMed Central

Wen, Ming; Xie, Munan; He, Lian; Wang, Yushuai; Shi, Suhua; Tang, Tian

2016-01-01

Differences in expression levels are an important source of phenotypic variation within and between populations. MicroRNAs (miRNAs) are key players in post-transcriptional gene regulation that are important for plant development and stress responses. We surveyed expression variation of miRNAs and mRNAs of six accessions from two rice subspecies Oryza sativa L. ssp. indica and Oryza sativa L. ssp. japonica using deep sequencing. While more than half (53.7%) of the mature miRNAs exhibit differential expression between grains and seedlings of rice, only 11.0% show expression differences between subspecies, with an additional 2.2% differentiated for the development-by-subspecies interaction. Expression variation is greater for lowly conserved miRNAs than highly conserved miRNAs, whereas the latter show stronger negative correlation with their targets in expression changes between subspecies. Using a permutation test, we identified 51 miRNA–mRNA pairs that correlate negatively or positively in expression level among cultivated rice. Genes involved in various metabolic processes and stress responses are enriched in the differentially expressed genes between rice indica and japonica subspecies. Our results indicate that stabilizing selection is the major force governing miRNA expression in cultivated rice, albeit positive selection may be responsible for much of the between-subspecies expression divergence. PMID:27797952

Analysis of differential gene expression by bead-based fiber-optic array in growth-hormone-secreting pituitary adenomas.

PubMed

Jiang, Zhiquan; Gui, Songbo; Zhang, Yazhuo

2010-09-01

Growth-hormone-secreting pituitary adenomas (GHomas) account for approximately 20% of all pituitary neoplasms. However, the pathogenesis of GHomas remains to be elucidated. To explore the possible pathogenesis of GHomas, we used bead-based fiber-optic arrays to examine the gene expression in five GHomas and compared them to three healthy pituitaries. Four differentially expressed genes were chosen randomly for validation by quantitative real-time reverse transcription-polymerase chain reaction. We then performed pathway analysis on the identified differentially expressed genes using the Kyoto Encyclopedia of Genes and Genomes. Array analysis showed significant increases in the expression of 353 genes and 206 expressed sequence tags (ESTs) and decreases in 565 genes and 29 ESTs. Bioinformatic analysis showed that the genes HIGD1B, HOXB2, ANGPT2, HPGD and BTG2 may play an important role in the tumorigenesis and progression of GHomas. Pathway analysis showed that the wingless-type signaling pathway and extracellular-matrix receptor interactions may play a key role in the tumorigenesis and progression of GHomas. Our data suggested that there are numerous aberrantly expressed genes and pathways involved in the pathogenesis of GHomas. Bead-based fiber-optic arrays combined with pathway analysis of differentially expressed genes appear to be a valid method for investigating the pathogenesis of tumors.

Analysis of differential gene expression by bead-based fiber-optic array in growth-hormone-secreting pituitary adenomas

PubMed Central

JIANG, ZHIQUAN; GUI, SONGBO; ZHANG, YAZHUO

2010-01-01

Growth-hormone-secreting pituitary adenomas (GHomas) account for approximately 20% of all pituitary neoplasms. However, the pathogenesis of GHomas remains to be elucidated. To explore the possible pathogenesis of GHomas, we used bead-based fiber-optic arrays to examine the gene expression in five GHomas and compared them to three healthy pituitaries. Four differentially expressed genes were chosen randomly for validation by quantitative real-time reverse transcription-polymerase chain reaction. We then performed pathway analysis on the identified differentially expressed genes using the Kyoto Encyclopedia of Genes and Genomes. Array analysis showed significant increases in the expression of 353 genes and 206 expressed sequence tags (ESTs) and decreases in 565 genes and 29 ESTs. Bioinformatic analysis showed that the genes HIGD1B, HOXB2, ANGPT2, HPGD and BTG2 may play an important role in the tumorigenesis and progression of GHomas. Pathway analysis showed that the wingless-type signaling pathway and extracellular-matrix receptor interactions may play a key role in the tumorigenesis and progression of GHomas. Our data suggested that there are numerous aberrantly expressed genes and pathways involved in the pathogenesis of GHomas. Bead-based fiber-optic arrays combined with pathway analysis of differentially expressed genes appear to be a valid method for investigating the pathogenesis of tumors. PMID:22993617

Tissue-specific NETs alter genome organization and regulation even in a heterologous system.

PubMed

de Las Heras, Jose I; Zuleger, Nikolaj; Batrakou, Dzmitry G; Czapiewski, Rafal; Kerr, Alastair R W; Schirmer, Eric C

2017-01-02

Different cell types exhibit distinct patterns of 3D genome organization that correlate with changes in gene expression in tissue and differentiation systems. Several tissue-specific nuclear envelope transmembrane proteins (NETs) have been found to influence the spatial positioning of genes and chromosomes that normally occurs during tissue differentiation. Here we study 3 such NETs: NET29, NET39, and NET47, which are expressed preferentially in fat, muscle and liver, respectively. We found that even when exogenously expressed in a heterologous system they can specify particular genome organization patterns and alter gene expression. Each NET affected largely different subsets of genes. Notably, the liver-specific NET47 upregulated many genes in HT1080 fibroblast cells that are normally upregulated in hepatogenesis, showing that tissue-specific NETs can favor expression patterns associated with the tissue where the NET is normally expressed. Similarly, global profiling of peripheral chromatin after exogenous expression of these NETs using lamin B1 DamID revealed that each NET affected the nuclear positioning of distinct sets of genomic regions with a significant tissue-specific component. Thus NET influences on genome organization can contribute to gene expression changes associated with differentiation even in the absence of other factors and overt cellular differentiation changes.

Identification and comparison of long non-conding RNA in Jinhua and Landrace pigs.

PubMed

Miao, Zhiguo; Wang, Shan; Zhang, Jinzhou; Wei, Panpeng; Guo, Liping; Liu, Dongyang; Wang, Yimin; Shi, Mingyan

2018-06-23

The regulatory role of long non-coding RNAs (lncRNAs) in various biological functions has been demonstrated. However, their role in fat deposition and lipid metabolism in pigs remains less understood. To explore the expression profile of lncRNAs in Jinhua and Landrace pigs, we investigated the expression levels of lncRNAs in intramuscular adipose tissues obtained from these pigs. Results showed that the expression levels of lncRNAs in these pig breeds significantly (Fold Change ≥ 2.0, FDR < 0.05) differed. A total of 4910 lncRNAs were identified, and 119 of these lncRNAs were differentially expressed. Of these differentially expressed lncRNAs, 60 and 59 were up- and down-regulated, respectively. The differentially expressed lncRNAs are involved in mitogen-activated protein kinase (MAPK) signaling pathway, Ras signaling pathway, PI3k-Akt signaling pathway. We then compared these differentially expressed lncRNAs with mRNAs and found that six of the co-expressed lncRNAs were implicated in pathways related to fat deposition and lipid metabolism. Overall, our results revealed a remarkable difference in fat metabolism in intramuscular adipose tissues of pigs, and provide a basis for subsequent research on fat deposition. Copyright © 2018. Published by Elsevier Inc.

Anti-differentiation non-coding RNA, ANCR, is differentially expressed in different types of brain tumors.

PubMed

Malakootian, Mahshid; Mirzadeh Azad, Fatemeh; Fouani, Youssef; Taheri Bajgan, Elham; Saberi, Hooshang; Mowla, Seyed Javad

2018-06-01

Long non-coding RNAs (lncRNAs) are important modulators of various cellular and molecular events, including cancer-associated pathways. The Anti-differentiation ncRNA (ANCR) is a key regulator of keratinocyte differentiation, where its expression is necessary to maintain epidermal progenitor's cells. Herein, we investigated the expression pattern of ANCR in the course of neural differentiation. Moreover, we used published RNAseq data and clinical samples to evaluate the alteration of ANCR expression in different cell types and brain tumors. Furthermore, we manipulated ANCR expression in glioma cell lines to clarify a potential functional role for ANCR in tumorigenesis. Our qRT-PCR results revealed a significant upregulation of ANCR in more malignant and less differentiated types of brain tumors (P = 0.03). This data was in accordance with down regulation of ANCR during neural differentiation. ANCR suppression caused an elevation in apoptosis rate, as well as a G1 cell cycle arrest in glioblastoma cell line. Altogether, our data demonstrated that ANCR may play a role in glioma genesis and that it could be considered as a potential diagnostic and therapeutic target to combat brain cancers.

The Changing Integrin Expression and a Role for Integrin β8 in the Chondrogenic Differentiation of Mesenchymal Stem Cells

PubMed Central

LaPointe, Vanessa L. S.; Verpoorte, Amanda; Stevens, Molly M.

2013-01-01

Many cartilage tissue engineering approaches aim to differentiate human mesenchymal stem cells (hMSCs) into chondrocytes and develop cartilage in vitro by targeting cell-matrix interactions. We sought to better inform the design of cartilage tissue engineering scaffolds by understanding how integrin expression changes during chondrogenic differentiation. In three models of in vitro chondrogenesis, we studied the temporal change of cartilage phenotype markers and integrin subunits during the differentiation of hMSCs. We found that transcript expression of most subunits was conserved across the chondrogenesis models, but was significantly affected by the time-course of differentiation. In particular, ITGB8 was up-regulated and its importance in chondrogenesis was further established by a knockdown of integrin β8, which resulted in a non-hyaline cartilage phenotype, with no COL2A1 expression detected. In conclusion, we performed a systematic study of the temporal changes of integrin expression during chondrogenic differentiation in multiple chondrogenesis models, and revealed a role for integrin β8 in chondrogenesis. This work enhances our understanding of the changing adhesion requirements of hMSCs during chondrogenic differentiation and underlines the importance of integrins in establishing a cartilage phenotype. PMID:24312400

Retinoic acid induces expression of SLP-76: expression with c-FMS enhances ERK activation and retinoic acid-induced differentiation/G0 arrest of HL-60 cells.

PubMed

Yen, Andrew; Varvayanis, Susi; Smith, James L; Lamkin, Thomas J

2006-02-01

Retinoic acid (RA) is known to cause MAPK signaling which propels G0 arrest and myeloid differentiation of HL-60 human myeloblastic leukemia cells. The present studies show that RA up-regulated expression of SLP-76 (Src-homology 2 domain-containing leukocyte-specific phospho-protein of 76 kDa), which became a prominent tyrosine-phosphorylated protein in RA-treated cells. SLP-76 is a known adaptor molecule associated with T-cell receptor and MAPK signaling. To characterize functional effects of SLP-76 expression in RA-induced differentiation and G0 arrest, HL-60 cells were stably transfected with SLP-76. Expression of SLP-76 had no discernable effect on RA-induced ERK activation, subsequent functional differentiation, or the rate of RA-induced G0 arrest. To determine the effects of SLP-76 in the presence of a RA-regulated receptor, SLP-76 was stably transfected into HL-60 cells already overexpressing the colony stimulating factor-1 (CSF-1) receptor, c-FMS, from a previous stable transfection. SLP-76 now enhanced RA-induced ERK activation, compared to parental c-FMS transfectants. It also enhanced RA-induced differentiation, evidenced by enhanced paxillin expression, inducible oxidative metabolism and superoxide production. RA-induced RB tumor suppressor protein hypophosphorylation was also enhanced, as was RA-induced G0 cell cycle arrest. A triple Y to F mutant SLP-76 known to be a dominant negative in T-cell receptor signaling failed to enhance RA-induced paxillin expression, but enhanced RA-induced ERK activation, differentiation and G0 arrest essentially as well as wild-type SLP-76. Thus, SLP-76 overexpression in the presence of c-FMS, a RA-induced receptor, had the effect of enhancing RA-induced cell differentiation. This is the first indication to our knowledge that RA induces the expression of an adapter molecule to facilitate induced differentiation via co-operation between c-FMS and SLP-76.

Gene expression during ovarian differentiation in parasitic and non-parasitic lampreys: implications for fecundity and life history types.

PubMed

Spice, Erin K; Whyard, Steven; Docker, Margaret F

2014-11-01

Lampreys diverged from the jawed vertebrate lineage approximately 500million years ago. Lampreys undergo sex differentiation much later than most other vertebrates, and ovarian differentiation occurs several years before testicular differentiation. The genetic basis of lamprey sex differentiation is of particular interest both because of the phylogenetic importance of lampreys and because of their unusual pattern of sex differentiation. As well, differences between parasitic and non-parasitic lampreys may first become evident at ovarian differentiation. However, nothing is known about the genetic basis of ovarian differentiation in lampreys. This study examined potential differences in gene expression before, during, and after ovarian differentiation in parasitic chestnut lamprey Ichthyomyzon castaneus and non-parasitic northern brook lamprey Ichthyomyzonfossor. Eight target genes (17β-hydroxysteroid dehydrogenase, germ cell-less, estrogen receptor β, insulin-like growth factor 1 receptor, daz-associated protein 1, cytochrome c oxidase subunit III, Wilms' tumour suppressor protein 1, and dehydrocholesterol reductase 7) were examined. Northern brook lamprey displayed higher expression of cytochrome c oxidase subunit III, whereas chestnut lamprey displayed higher expression of insulin-like growth factor 1 receptor; these genes may be involved in apoptosis and oocyte growth, respectively. Presumptive male larvae had higher expression of Wilms' tumour suppressor protein 1, which may be involved in the undifferentiated gonad and/or later testicular development. Differentiated females had higher expression of 17β hydroxysteroid dehydrogenase and daz-associated protein 1, which may be involved in female development. This study is the first to identify genes that may be involved in ovarian differentiation and fecundity in lampreys. Copyright © 2014 Elsevier Inc. All rights reserved.

Genome-wide gene expression effects in B6C3F1 mouse intestinal epithelia following 7 and 90 days of exposure to hexavalent chromium in drinking water

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

Kopec, Anna K.; Kim, Suntae; Forgacs, Agnes L.

2012-02-15

Chronic administration of high doses of hexavalent chromium [Cr(VI)] as sodium dichromate dihydrate (SDD) elicits alimentary cancers in mice. To further elucidate key events underlying tumor formation, a 90-day drinking water study was conducted in B6C3F1 mice. Differential gene expression was examined in duodenal and jejunal epithelial samples following 7 or 90 days of exposure to 0, 0.3, 4, 14, 60, 170 or 520 mg/L SDD in drinking water. Genome-wide microarray analyses identified 6562 duodenal and 4448 jejunal unique differentially expressed genes at day 8, and 4630 and 4845 unique changes, respectively, in the duodenum and jejunum at day 91.more » Comparative analysis identified significant overlap in duodenal and jejunal differential gene expression. Automated dose–response modeling identified > 80% of the differentially expressed genes exhibited sigmoidal dose–response curves with EC{sub 50} values ranging from 10 to 100 mg/L SDD. Only 16 genes satisfying the dose-dependent differential expression criteria had EC{sub 50} values < 10 mg/L SDD, 3 of which were regulated by Nrf2, suggesting oxidative stress in response to SDD at low concentrations. Analyses of differentially expressed genes identified over-represented functions associated with oxidative stress, cell cycle, lipid metabolism, and immune responses consistent with the reported effects on redox status and histopathology at corresponding SDD drinking water concentrations. Collectively, these data are consistent with a mode of action involving oxidative stress and cytotoxicity as early key events. This suggests that the tumorigenic effects of chronic Cr(VI) oral exposure likely require chronic tissue damage and compensatory epithelial cell proliferation. Highlights: ► Mouse small intestine gene expression is highly responsive to hexavalent chromium [Cr(VI)]. ► Cr(VI) elicits more differential gene expression after 7 days of exposure than 90 days of exposure. ► Oral exposure to Cr(VI) leads to oxidative stress, cell cycle, lipid and immune dysregulation. ► Cr(VI) elicits dose-dependent changes in gene expression with an overall median EC{sub 50} of 47 mg/L SDD.« less

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

Differential Language Functioning of Monolinguals and Bilinguals on Positive-Negative Emotional Expression

ERIC Educational Resources Information Center

Kheirzadeh, Shiela; Hajiabed, Mohammadreza

2016-01-01

The present interdisciplinary research investigates the differential emotional expression between Persian monolinguals and Persian-English bilinguals. In other words, the article was an attempt to answer the questions whether bilinguals and monolinguals differ in the expression of positive and negative emotions elicited through sad and happy…

Using differential gene expression to study Entamoeba histolytica pathogenesis

PubMed Central

Gilchrist, Carol A.; Petri, William A.

2010-01-01

The release of the Entamoeba histolytica genome has facilitated the development of techniques to survey rapidly and to relate gene expression with biology. The association and potential contribution of differential gene expression to the life cycle and the virulence of this protozoan parasite of humans are reviewed here. PMID:19217826

Gender-Specific Gene Expression in Post-Mortem Human Brain: Localization to Sex Chromosomes

PubMed Central

Vawter, Marquis P; Evans, Simon; Choudary, Prabhakara; Tomita, Hiroaki; Meador-Woodruff, Jim; Molnar, Margherita; Li, Jun; Lopez, Juan F; Myers, Rick; Cox, David; Watson, Stanley J; Akil, Huda; Jones, Edward G; Bunney, William E

2011-01-01

Gender differences in brain development and in the prevalence of neuropsychiatric disorders such as depression have been reported. Gender differences in human brain might be related to patterns of gene expression. Microarray technology is one useful method for investigation of gene expression in brain. We investigated gene expression, cell types, and regional expression patterns of differentially expressed sex chromosome genes in brain. We profiled gene expression in male and female dorsolateral prefrontal cortex, anterior cingulate cortex, and cerebellum using the Affymetrix oligonucleotide microarray platform. Differentially expressed genes between males and females on the Y chromosome (DBY, SMCY, UTY, RPS4Y, and USP9Y) and X chromosome (XIST) were confirmed using real-time PCR measurements. In situ hybridization confirmed the differential expression of gender-specific genes and neuronal expression of XIST, RPS4Y, SMCY, and UTY in three brain regions examined. The XIST gene, which silences gene expression on regions of the X chromosome, is expressed in a subset of neurons. Since a subset of neurons express gender-specific genes, neural subpopulations may exhibit a subtle sexual dimorphism at the level of differences in gene regulation and function. The distinctive pattern of neuronal expression of XIST, RPS4Y, SMCY, and UTY and other sex chromosome genes in neuronal subpopulations may possibly contribute to gender differences in prevalence noted for some neuropsychiatric disorders. Studies of the protein expression of these sex- chromosome-linked genes in brain tissue are required to address the functional consequences of the observed gene expression differences. PMID:14583743

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.

Crosstalk between HIF-1 and ROCK pathways in neuronal differentiation of mesenchymal stem cells, neurospheres and in PC12 neurite outgrowth.

PubMed

Pacary, Emilie; Tixier, Emmanuelle; Coulet, Florence; Roussel, Simon; Petit, Edwige; Bernaudin, Myriam

2007-07-01

This study demonstrates that the Rho-kinase (ROCK) inhibitor, Y-27632, potentiates not only the effect of cobalt chloride (CoCl(2)) but also that of deferoxamine, another HIF-1 inducer, on mesenchymal stem cell (MSC) neuronal differentiation. HIF-1 is essential for CoCl(2)+/-Y-27632-induced MSC neuronal differentiation, since agents inhibiting HIF-1 abolish the changes of morphology and cell cycle arrest-related gene or protein expressions (p21, cyclin D1) and the increase of neuronal marker expressions (Tuj1, NSE). Y-27632 potentiates the CoCl(2)-induced decrease of cyclin D1 and nestin expressions, the increase of HIF-1 activation and EPO expression, and decreases pVHL expression. Interestingly, CoCl(2) decreases RhoA expression, an effect potentiated by Y-27632, revealing crosstalk between HIF-1 and RhoA/ROCK pathways. Moreover, we demonstrate a synergistic effect of CoCl(2) and Y-27632 on neurosphere differentiation into neurons and PC12 neurite outgrowth underlining that a co-treatment targeting both HIF-1 and ROCK pathways might be relevant to differentiate stem cells into neurons.

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.

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.

HSP27 regulates TGF-β mediated lung fibroblast differentiation through the Smad3 and ERK pathways.

PubMed

Wang, Gang; Jiao, Hao; Zheng, Jun-Nian; Sun, Xia

2017-01-01

Idiopathic pulmonary fibrosis (IPF) is a chronic lethal interstitial lung disease with unknown etiology. Recent studies have indicated that heat-shock protein 27 (HSP27) contributes to the pathogenesis of IPF through the regulation of epithelial-mesenchymal transition (EMT). However, the expression and role of HSP27 in fibroblasts during pulmonary fibrogenesis has not been investigated to date, at least to the best of our knowledge. In this study, we examined the expression of HSP27 in fibrotic lung tissue and fibroblasts from bleomycin (BLM)-challenged mice and human lung fibroblasts treated with transforming growth factor-β (TGF-β). The results revealed that the expression of HSP27 was significantly increased in fibrotic lung tissue and fibroblasts from BLM-challenged mice. In vitro, TGF-β stimulated HSP27 expression in and the differentiation of human lung fibroblasts. The knockdown of Smad3 expression or nuclear factor-κB p65 subunit attenuated the TGF-β-induced increase in HSP27 expression and the differentiation of human lung fibroblasts. In addition, the knockdown of HSP27 expression attenuated the TGF-β-induced activation of ERK and Smad3, and inhibited the differentiation of human lung fibroblasts. On the whole, the findings of our study demonstrate that HSP27 expression is upregulated in lung fibroblasts during pulmonary fibrosis, and subsequently, HSP27 modulates lung fibroblast differentiation through the Smad3 and ERK pathways.

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.

BAG3 is involved in neuronal differentiation and migration.

PubMed

Santoro, Antonietta; Nicolin, Vanessa; Florenzano, Fulvio; Rosati, Alessandra; Capunzo, Mario; Nori, Stefania L

2017-05-01

Bcl2-associated athanogene 3 (BAG3) protein belongs to the family of co-chaperones interacting with several heat shock proteins. It plays a key role in protein quality control and mediates the clearance of misfolded proteins. Little is known about the expression and cellular localization of BAG3 during nervous system development and differentiation. Therefore, we analyze the subcellular distribution and expression of BAG3 in nerve-growth-factor-induced neurite outgrowth in PC12 cells and in developing and adult cortex of mouse brain. In differentiated PC12 cells, BAG3 was localized mainly in the neuritic domain rather than the cell body, whereas in control cells, it appeared to be confined to the cytoplasm near the nuclear membrane. Interestingly, the change of BAG3 localization during neuronal differentiation was associated only with a slight increase in total BAG3 expression. These data were coroborated by transmission electron microscopy showing that BAG3 was confined mainly within large dense-core vesicles of the axon in differentiated PC12 cells. In mouse developing cortex, BAG3 appeared to be intensely expressed in cellular processes of migrating cells, whereas in adult brain, a diffuse expression of low to medium intensity was detected in neuronal cell bodies. These findings suggest that BAG3 expression is required for neuronal differentiation and migration and that its role is linked to a change in its distribution pattern rather than to an increase in its protein expression levels.

Differential in vivo gene expression of major Leptospira proteins in resistant or susceptible animal models.

PubMed

Matsui, Mariko; Soupé, Marie-Estelle; Becam, Jérôme; Goarant, Cyrille

2012-09-01

Transcripts of Leptospira 16S rRNA, FlaB, LigB, LipL21, LipL32, LipL36, LipL41, and OmpL37 were quantified in the blood of susceptible (hamsters) and resistant (mice) animal models of leptospirosis. We first validated adequate reference genes and then evaluated expression patterns in vivo compared to in vitro cultures. LipL32 expression was downregulated in vivo and differentially regulated in resistant and susceptible animals. FlaB expression was also repressed in mice but not in hamsters. In contrast, LigB and OmpL37 were upregulated in vivo. Thus, we demonstrated that a virulent strain of Leptospira differentially adapts its gene expression in the blood of infected animals.

Hepatic differentiation capability of rat bone marrow-derived mesenchymal stem cells and hematopoietic stem cells.

PubMed

Shu, Sai-Nan; Wei, Lai; Wang, Jiang-Hua; Zhan, Yu-Tao; Chen, Hong-Song; Wang, Yu

2004-10-01

To investigate the different effects of mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) on hepatic differentiation. MSCs from rat bone marrow were isolated and cultured by standard methods. HSCs from rat bone marrow were isolated and purified by magnetic activated cell sorting. Both cell subsets were induced. Morphology, RT-PCR and immunocytochemistry were used to identify the hepatic differentiation grade. MSCs exhibited round in shape after differentiation, instead of fibroblast-like morphology before differentiation. Albumin mRNA and protein were expressed positively in MSCs, without detection of alpha-fetoprotein (AFP). HSCs were polygonal in shape after differentiation. The expression of albumin signal decreased and AFP signal increased. The expression of CK18 was continuous in MSCs and HSCs both before and after induction. Both MSCs and HSCs have hepatic differentiation capabilities. However, their capabilities are not the same. MSCs can differentiate into mature hepatocyte-like cells, never expressing early hepatic specific genes, while Thy-1.1(+) cells are inclined to differentiate into hepatic stem cell-like cells, with an increasing AFP expression and a decreasing albumin signal. CK18 mRNA is positive in Thy-1.1(+) cells and MSCs, negative in Thy-1.1(-) cells. It seems that CK18 has some relationship with Thy-1.1 antigen, and CK18 may be a predictive marker of hepatic differentiation capability.

Role of C/EBPβ-LAP and C/EBPβ-LIP in early adipogenic differentiation of human white adipose-derived progenitors and at later stages in immature adipocytes.

PubMed

Lechner, Stefan; Mitterberger, Maria C; Mattesich, Monika; Zwerschke, Werner

2013-01-01

We investigated the role of the major isoforms of CCAAT enhancer binding protein β (C/EBPβ), C/EBPβ-LAP and C/EBPβ-LIP, in adipogenesis of human white adipose-derived stromal/progenitor cells (ASC). C/EBPβ gene expression was transiently induced early in adipogenesis. At later stages, in immature adipocytes, the C/EBPβ mRNA and protein levels declined. The C/EBPβ-LIP protein steady-state level decreased considerably stronger than the C/EBPβ-LAP level and the C/EBPβ-LIP half-life was significantly shorter than the C/EBPβ-LAP half-life. The turn-over of both C/EBPβ-isoforms was regulated by ubiquitin/proteasome-dependent degradation. These data suggest that the protein stability of the C/EBPβ-isoforms is differentially regulated in the course of adipogenesis and in immature adipocytes. Constitutive overexpression of C/EBPβ-LIP had antiadipogenic activity in human ASC. C/EBPβ-LAP, which promotes adipogenesis in mouse 3T3-L1 preadipocytes by directly activating expression of the adipogenic keyregulator PPARγ2, induced the expression of PPARγ2 and of the adipocyte differentiation gene product FABP4 in confluent ASC in the absence of adipogenic hormones. At later stages after hormone cocktail-induced adipogenesis, in immature adipocytes, constitutive overexpression of C/EBPβ-LAP led to reduced expression of PPARγ2 and FABP4, C/EBPα expression was downregulated and the expression of the adipocyte differentiation gene products adiponectin and leptin was impaired. These findings suggest that constitutive overexpression of C/EBPβ-LAP induces adipogenesis in human ASC and negatively regulates the expression of adipogenic regulators and certain adipocyte differentiation gene products in immature adipocytes. We conclude the regulation of both C/EBPβ gene expression and C/EBPβ-LIP and C/EBPβ-LAP protein turn-over plays an important role for the expression of adipogenic regulators and/or adipocyte differentiation genes in early adipogenic differentiation of human ASC and at later stages in human immature adipocytes. Copyright © 2012 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Circulating Long Noncoding RNAs as Potential Biomarkers of Sepsis: A Preliminary Study.

PubMed

Dai, Yu; Liang, Zhixin; Li, Yulin; Li, Chunsun; Chen, Liangan

2017-11-01

Long noncoding RNAs (lncRNAs) are becoming promising biomarker candidates in various diseases as assessed via sequencing technologies. Sepsis is a life-threatening disease without ideal biomarkers. The aim of this study was to investigate the expression profile of lncRNAs in the peripheral blood of sepsis patients and to find potential biomarkers of sepsis. A lncRNA expression profile was performed using peripheral blood from three sepsis patients and three healthy volunteers using microarray screening. The differentially expressed lncRNAs were validated by real-time quantitative polymerase chain reaction (qRT-PCR) in a further set of 22 sepsis patients and 22 healthy volunteers. Among 1316 differentially expressed lncRNAs, 771 were downregulated and 545 were upregulated. Results of the qRT-PCR were consistent with the microarray data. lncRNA ENST00000452391.1, uc001vji.1, and uc021zxw.1 were significantly differentially expressed between sepsis patients and healthy volunteers. Moreover, lncRNA ENST00000504301.1 and ENST00000452391.1 were significantly differentially expressed between sepsis survivors and nonsurvivors. The lncRNA expression profile in the peripheral blood of sepsis patients significantly differed from that of healthy volunteers. Circulating lncRNAs may be good candidates for sepsis biomarkers.

Identification and characterization of novel and differentially expressed microRNAs in peripheral blood from healthy and mastitis Holstein cattle by deep sequencing.

PubMed

Li, Zhixiong; Wang, Hongliang; Chen, Ling; Wang, Lijun; Liu, Xiaolin; Ru, Caixia; Song, Ailong

2014-02-01

MicroRNA (miRNA) mediates post-transcriptional gene regulation and plays an important role in regulating the development of immune cells and in modulating innate and adaptive immune responses in mammals, including cattle. In the present study, we identified novel and differentially expressed miRNAs in peripheral blood from healthy and mastitis Holstein cattle by Solexa sequencing and bioinformatics. In total, 608 precursor hairpins (pre-miRNAs) encoding for 753 mature miRNAs were detected. Statistically, 173 unique miRNAs (of 753, 22.98%) were identified that had significant differential expression between healthy and mastitis Holstein cattle (P < 0.001). Most differentially expressed miRNAs (118 of 173, 68.21%) belonged to the chemokine signaling pathway involved in the immune responses. This study expands the number of miRNAs known to be expressed in cattle. The patterns of miRNAs expression differed significantly between the peripheral blood from healthy and mastitis Holstein cattle, which provide important information on mastitis in miRNAs expression. Diverse miRNAs may play an important role in the treatment of mastitis in Holstein cattle. © 2013 Stichting International Foundation for Animal Genetics.

Differential expression of non-coding RNAs and continuous evolution of the X chromosome in testicular transcriptome of two mouse species.

PubMed

Homolka, David; Ivanek, Robert; Forejt, Jiri; Jansa, Petr

2011-02-14

Tight regulation of testicular gene expression is a prerequisite for male reproductive success, while differentiation of gene activity in spermatogenesis is important during speciation. Thus, comparison of testicular transcriptomes between closely related species can reveal unique regulatory patterns and shed light on evolutionary constraints separating the species. Here, we compared testicular transcriptomes of two closely related mouse species, Mus musculus and Mus spretus, which diverged more than one million years ago. We analyzed testicular expression using tiling arrays overlapping Chromosomes 2, X, Y and mitochondrial genome. An excess of differentially regulated non-coding RNAs was found on Chromosome 2 including the intronic antisense RNAs, intergenic RNAs and premature forms of Piwi-interacting RNAs (piRNAs). Moreover, striking difference was found in the expression of X-linked G6pdx gene, the parental gene of the autosomal retrogene G6pd2. The prevalence of non-coding RNAs among differentially expressed transcripts indicates their role in species-specific regulation of spermatogenesis. The postmeiotic expression of G6pdx in Mus spretus points towards the continuous evolution of X-chromosome silencing and provides an example of expression change accompanying the out-of-the X-chromosomal retroposition.

Detergents with different chemical properties induce variable degree of cytotoxicity and mRNA expression of lipid-metabolizing enzymes and differentiation markers in cultured keratinocytes.

PubMed

Wei, Tianling; Geijer, Sophia; Lindberg, Magnus; Berne, Berit; Törmä, Hans

2006-12-01

The knowledge how detergents with different chemical properties influence epidermal keratinocytes is sparse. In the present study, the effects of five detergents were examined with respect to cell-toxicity and mRNA expression of key-enzymes in barrier lipid production and keratinocyte differentiation markers. First, the LD(50) for each detergent were determined. Secondly, keratinocytes were exposed to sub-toxic concentrations and the mRNA expression was analysed by real-time PCR after 24 h exposure to the detergents. SLS and CAPB induced a concentration-dependent increase in the expression of enzymes producing cholesterol and ceramides, while transcripts of enzymes producing fatty acids were unaffected. SLES and cocoglucoside increased the expression of certain enzymes involved in cholesterol and fatty acid synthesis while sodium cocoamphoacetate (SCAA) stimulated expression of transcripts involved in fatty acid synthesis. The expression of differentiation markers were increased by SLS, SLES and CAPB, while SCAA and cocoglucoside exhibited no effect. The present findings show that detergents have variable effects on lipid synthesis and keratinocyte differentiation, which could partly explain their barrier destruction potential in vivo.

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).

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.

Differential expression of homeobox-containing genes Msx-1 and Msx-2 and homeoprotein Msx-2 expression during chick craniofacial development.

PubMed

Nishikawa, K; Nakanishi, T; Aoki, C; Hattori, T; Takahashi, K; Taniguchi, S

1994-03-01

The expression pattern of chick Msx-1 and Msx-2 homeobox genes in craniofacial primordia was examined by in situ hybridization using cRNA probes. Both genes were expressed in the distal region of the facial primordia, where the distribution of Msx-2 expression was restricted distally within the Msx-1 expression domain. On the contrary, Msx-2 expression in the lateral choroid plexus and cranial skull was broader and more intensive than Msx-1 expression. Our findings suggest that these two genes cooperate to play differential roles in craniofacial development. Msx-2 protein was detected immunohistochemically, and its localization essentially corresponded to the mRNA expression pattern, substantiating the involvement of Msx-2 protein as a transcriptional regulator in developing limb and face.

The caspase-1 inhibitor CARD18 is specifically expressed during late differentiation of keratinocytes and its expression is lost in lichen planus.

PubMed

Qin, Haihong; Jin, Jiang; Fischer, Heinz; Mildner, Michael; Gschwandtner, Maria; Mlitz, Veronika; Eckhart, Leopold; Tschachler, Erwin

2017-08-01

CARD18 contains a caspase recruitment domain (CARD) via which it binds to caspase-1 and thereby inhibits caspase-1-mediated activation of the pro-inflammatory cytokine interleukin (IL)-1β. To determine the expression profile and the role of CARD18 during differentiation of keratinocytes and to compare the expression of CARD18 in normal skin and in inflammatory skin diseases. Human keratinocytes were induced to differentiate in monolayer and in 3D skin equivalent cultures. In some experiments, CARD18-specific siRNAs were used to knock down expression of CARD18. CARD18 mRNA levels were determined by quantitative real-time PCR, and CARD18 protein was detected by Western blot and immunofluorescence analyses. In situ expression was analyzed in skin biopsies obtained from healthy donors and patients with psoriasis and lichen planus. CARD18 mRNA was expressed in the epidermis at more than 100-fold higher levels than in any other human tissue. Within the epidermis, CARD18 was specifically expressed in the granular layer. In vitro CARD18 was strongly upregulated at both mRNA and protein levels in keratinocytes undergoing terminal differentiation. In skin equivalent cultures the expression of CARD18 was efficiently suppressed by siRNAs without impairing stratum corneum formation. Epidermal expression of CARD18 was increased after ultraviolet (UV)B irradiation of skin explants. In skin biopsies of patients with psoriasis no consistent regulation of CARD18 expression was observed, however, in lesional epidermis of patients with lichen planus, CARD18 expression was either greatly diminished or entirely absent whereas in non-lesional areas expression was comparable to normal skin. Our results identify CARD18 as a differentiation-associated keratinocyte protein that is altered in abundance by UV stress. Its downregulation in lichen planus indicates a potential role in inflammatory reactions of the epidermis in this disease. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

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

Decreased expression of the stress protein HSP70 is an early event in murine erythroleukemic cell differentiation.

PubMed Central

Hensold, J O; Housman, D E

1988-01-01

Two-dimensional protein gels were used to systematically assess changes in gene expression in Friend erythroleukemia cells after exposure to inducers of differentiation. A rapid decrease in expression of the stress protein HSP70 was observed after exposure to inducers. The kinetics of this change suggest that it may be related to the cellular events that regulate the onset of differentiation. Images PMID:3164440

Functional studies of TcRjl, a novel GTPase of Trypanosoma cruzi, reveals phenotypes related with MAPK activation during parasite differentiation and after heterologous expression in Drosophila model system

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

Reis Monteiro dos-Santos, Guilherme Rodrigo; Fontenele, Marcio Ribeiro; Dias, Felipe de Almeida

The life cycle of the protozoan parasite Trypanosoma cruzi comprises rounds of proliferative cycles and differentiation in distinct host environments. Ras GTPases are molecular switches that play pivotal regulatory functions in cell fate. Rjl is a novel GTPase with unknown function. Herein we show that TcRjl blocks in vivo cell differentiation. The forced expression of TcRjl leads to changes in the overall tyrosine protein phosphorylation profile of parasites. TcRjl expressing parasites sustained DNA synthesis regardless the external stimuli for differentiation. Heterologous expression in the Drosophila melanogaster genetic system strongly suggests a role from TcRjl protein in RTK-dependent pathways and MAPK activation.

The zebrafish gene cloche acts upstream of a flk-1 homologue to regulate endothelial cell differentiation.

PubMed

Liao, W; Bisgrove, B W; Sawyer, H; Hug, B; Bell, B; Peters, K; Grunwald, D J; Stainier, D Y

1997-01-01

The zebrafish cloche mutation affects both the endothelial and hematopoietic lineages at a very early stage (Stainier, D. Y. R., Weinstein, B. M., Detrich, H. W., Zon, L. I. and Fishman, M. C. (1995). Development 121, 3141-3150). The most striking vascular phenotype is the absence of endocardial cells from the heart. Microscopic examination of mutant embryos reveals the presence of endothelial-like cells in the lower trunk and tail regions while head vessels appear to be missing, indicating a molecular diversification of the endothelial lineage. Cell transplantation experiments show that cloche acts cell-autonomously within the endothelial lineage. To analyze further the role of cloche in regulating endothelial cell differentiation, we have examined the expression of flk-1 and tie, two receptor tyrosine kinase genes expressed early and sequentially in the endothelial lineage. In wild-type fish, flk-1-positive cells are found throughout the embryo and differentiate to form the nascent vasculature. In cloche mutants, flk-1-positive cells are found only in the lower trunk and tail regions, and this expression is delayed as compared to wild-type. Unlike the flk-1-positive cells in wild-type embryos, those in cloche mutants do not go on to express tie, suggesting that their differentiation is halted at an early stage. We also find that the cloche mutation is not linked to flk-1. These data indicate that cloche affects the differentiation of all endothelial cells and that it acts at a very early stage, either by directly regulating flk-1 expression or by controlling the differentiation of cells that normally develop to express flk-1. cloche mutants also have a blood deficit and their hematopoietic tissues show no expression of the hematopoietic transcription factor genes GATA-1 or GATA-2 at early stages. Because the appearance of distinct levels of flk-1 expression is delayed in cloche mutants, we examined GATA-1 expression at late embryonic stages and found some blood cell differentiation that appears to be limited to the region lined by the flk-1-expressing cells. The spatial restriction of blood in the ventroposterior-most region of cloche mutant embryos may be indicative of a ventral source of signal(s) controlling hematopoietic differentiation. In addition, the restricted colocalization of blood and endothelium in cloche mutants suggests that important interactions occur between these two lineages during normal development.

Transcriptome sequencing of Eucalyptus camaldulensis seedlings subjected to water stress reveals functional single nucleotide polymorphisms and genes under selection

PubMed Central

2012-01-01

Background Water stress limits plant survival and production in many parts of the world. Identification of genes and alleles responding to water stress conditions is important in breeding plants better adapted to drought. Currently there are no studies examining the transcriptome wide gene and allelic expression patterns under water stress conditions. We used RNA sequencing (RNA-seq) to identify the candidate genes and alleles and to explore the evolutionary signatures of selection. Results We studied the effect of water stress on gene expression in Eucalyptus camaldulensis seedlings derived from three natural populations. We used reference-guided transcriptome mapping to study gene expression. Several genes showed differential expression between control and stress conditions. Gene ontology (GO) enrichment tests revealed up-regulation of 140 stress-related gene categories and down-regulation of 35 metabolic and cell wall organisation gene categories. More than 190,000 single nucleotide polymorphisms (SNPs) were detected and 2737 of these showed differential allelic expression. Allelic expression of 52% of these variants was correlated with differential gene expression. Signatures of selection patterns were studied by estimating the proportion of nonsynonymous to synonymous substitution rates (Ka/Ks). The average Ka/Ks ratio among the 13,719 genes was 0.39 indicating that most of the genes are under purifying selection. Among the positively selected genes (Ka/Ks > 1.5) apoptosis and cell death categories were enriched. Of the 287 positively selected genes, ninety genes showed differential expression and 27 SNPs from 17 positively selected genes showed differential allelic expression between treatments. Conclusions Correlation of allelic expression of several SNPs with total gene expression indicates that these variants may be the cis-acting variants or in linkage disequilibrium with such variants. Enrichment of apoptosis and cell death gene categories among the positively selected genes reveals the past selection pressures experienced by the populations used in this study. PMID:22853646

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

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.

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.

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

Shim, Ki Shuk; Department of Neonatology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna; Rosner, Margit

Bach1 and Bach2 are evolutionarily related members of the BTB-basic region leucine zipper transcription factor family. We found that Bach2 downregulates cell proliferation of N1E-115 cells and negatively affects their potential to differentiate. Nuclear localization of the cyclin-dependent kinase inhibitor p21 is known to arrest cell cycle progression, and cytoplasmic p21 has been shown to promote neuronal differentiation of N1E-115 cells. We found that ectopic Bach2 causes upregulation of p21 expression in the nucleus and in the cytoplasm in undifferentiated N1E-115 cells. In differentiated cells, Bach2 specifically triggers upregulation of cytoplasmic p21. Our data suggest that Bach2 expression could representmore » a switch during the process of neuronal differentiation. Bach2 is not expressed in neuronal precursor cells. It would have negative effects on proliferation and differentiation of these cells. In differentiated neuronal cells Bach2 expression is upregulated, which could allow Bach2 to function as a gatekeeper of the differentiated status.« less

Differential Expression of Hox and Notch Genes in Larval and Adult Stages of Echinococcus granulosus.

PubMed

Dezaki, Ebrahim Saedi; Yaghoobi, Mohammad Mehdi; Taheri, Elham; Almani, Pooya Ghaseminejad; Tohidi, Farideh; Gottstein, Bruno; Harandi, Majid Fasihi

2016-10-01

This investigation aimed to evaluate the differential expression of HoxB7 and notch genes in different developmental stages of Echinococcus granulosus sensu stricto. The expression of HoxB7 gene was observed at all developmental stages. Nevertheless, significant fold differences in the expression level was documented in the juvenile worm with 3 or more proglottids, the germinal layer from infected sheep, and the adult worm from an experimentally infected dog. The notch gene was expressed at all developmental stages of E. granulosus ; however, the fold difference was significantly increased at the microcysts in monophasic culture medium and the germinal layer of infected sheep in comparison with other stages. The findings demonstrated that the 2 aforementioned genes evaluated in the present study were differentially expressed at different developmental stages of the parasite and may contribute to some important biological processes of E. granulosus .

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.

4E-BP1 regulates the differentiation of white adipose tissue.

PubMed

Tsukiyama-Kohara, Kyoko; Katsume, Asao; Kimura, Kazuhiro; Saito, Masayuki; Kohara, Michinori

2013-07-01

4E Binding protein 1 (4E-BP1) suppresses translation initiation. The absence of 4E-BP1 drastically reduces the amount of adipose tissue in mice. To address the role of 4E-BP1 in adipocyte differentiation, we characterized 4E-BP1(-/-) mice in this study. The lack of 4E-BP1 decreased the amount of white adipose tissue and increased the amount of brown adipose tissue. In 4E-BP1(-/-) MEF cells, PPARγ coactivator 1 alpha (PGC-1α) expression increased and exogenous 4E-BP1 expression suppressed PGC-1α expression. The level of 4E-BP1 expression was higher in white adipocytes than in brown adipocytes and showed significantly greater up-regulation in white adipocytes than in brown adipocytes during preadipocyte differentiation into mature adipocytes. The amount of PGC-1α was consistently higher in HB cells (a brown preadipocyte cell line) than in HW cells (a white preadipocyte cell line) during differentiation. Moreover, the ectopic over-expression of 4E-BP1 suppressed PGC-1α expression in white adipocytes, but not in brown adipocytes. Thus, the results of our study indicate that 4E-BP1 may suppress brown adipocyte differentiation and PGC-1α expression in white adipose tissues. © 2013 The Authors Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

Eicosapentaenoic acid and arachidonic acid differentially regulate adipogenesis, acquisition of a brite phenotype and mitochondrial function in primary human adipocytes.

PubMed

Fleckenstein-Elsen, Manuela; Dinnies, Daniela; Jelenik, Tomas; Roden, Michael; Romacho, Tania; Eckel, Jürgen

2016-09-01

n-3 and n-6 PUFAs have several opposing biological effects and influence white adipose tissue (WAT) function. The recent discovery of thermogenic UCP1-expressing brite adipocytes within WAT raised the question whether n-3 and n-6 PUFAs exert differential effects on brite adipocyte formation and mitochondrial function. Primary human preadipocytes were treated with n-3 PUFAs (eicosapentaenoic acid, EPA; docosahexaenoic acid, DHA) or n-6 PUFA (arachidonic acid, ARA) during differentiation, and adipogenesis, white and brite gene expression markers, mitochondrial content and function were analyzed at day 12 of differentiation. Adipogenesis was equally increased by n-3 and n-6 PUFAs. The n-6 PUFA ARA increased lipid droplet size and expression of the white-specific marker TCF21 while decreased mitochondrial protein expression and respiratory function. In contrast, EPA increased expression of the brown adipocyte-related genes UCP1 and CPT1B, and improved mitochondrial function of adipocytes. The opposing effects of EPA and ARA on gene expression and mitochondrial function were also observed in cells treated from day 8 to 12 of adipocyte differentiation. EPA promotes brite adipogenesis and improves parameters of mitochondrial function, such as increased expression of CPTB1, citrate synthase activity and higher maximal respiratory capacity, while ARA reduced mitochondrial spare respiratory capacity in vitro. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

The expression of MUC mucin in cholangiocarcinoma.

PubMed

Mall, Anwar S; Tyler, Marilyn G; Ho, Sam B; Krige, Jake E J; Kahn, Delawir; Spearman, Wendy; Myer, Landon; Govender, Dhirendra

2010-12-15

Cholangiocarcinoma (CC) is a highly malignant epithelial cancer of the biliary tract, the cellular and molecular pathogenesis of which remains unclear. Malignant transformation of glandular epithelial cells is associated with the altered expression of mucin. We investigated the type of mucins expressed in CC. Twenty-six patients with histologically confirmed CC were included in this study. The expression of mucin was studied by immunohistochemistry using antibodies to MUC1, MUC1 core, MUC2, MUC3, MUC4, MUC5AC, and MUC6. There was extensive (>50%) expression of mucin, mainly MUC1 in 11/25 and MUC5AC in 12/26 cases. In the case of MUC3, 6/26 cases expressed mucin extensively, whilst only 1/26 had MUC2, MUC4, and MUC6 expression. Well-differentiated tumors significantly expressed MUC3 extensively compared to poor or moderately differentiated tumors (p=0.003). Fifteen of 25 cases had metastatic disease. MUC1 was extensively expressed in 9/15 cases with metastatic disease. In contrast, MUC1 expression was present in 2/10 cases where metastases were absent. Hilar lesions were less likely to express MUC1, but this was not statistically significant. Fifteen of 25 cases had metastatic disease. Extensive MUC3 expression was significantly associated with well-differentiated tumors, whilst there was an approaching significance between the extensive expression of MUC1 and metastasis in cholangiocarcinoma. Copyright © 2010 Elsevier GmbH. All rights reserved.

Voltage-gated sodium channel expression and action potential generation in differentiated NG108-15 cells.

PubMed

Liu, Jinxu; Tu, Huiyin; Zhang, Dongze; Zheng, Hong; Li, Yu-Long

2012-10-25

The generation of action potential is required for stimulus-evoked neurotransmitter release in most neurons. Although various voltage-gated ion channels are involved in action potential production, the initiation of the action potential is mainly mediated by voltage-gated Na+ channels. In the present study, differentiation-induced changes of mRNA and protein expression of Na+ channels, Na+ currents, and cell membrane excitability were investigated in NG108-15 cells. Whole-cell patch-clamp results showed that differentiation (9 days) didn't change cell membrane excitability, compared to undifferentiated state. But differentiation (21 days) induced the action potential generation in 45.5% of NG108-15 cells (25/55 cells). In 9-day-differentiated cells, Na+ currents were mildly increased, which was also found in 21-day differentiated cells without action potential. In 21-day differentiated cells with action potential, Na+ currents were significantly enhanced. Western blot data showed that the expression of Na+ channels was increased with differentiated-time dependent manner. Single-cell real-time PCR data demonstrated that the expression of Na+ channel mRNA was increased by 21 days of differentiation in NG108-15 cells. More importantly, the mRNA level of Na+ channels in cells with action potential was higher than that in cells without action potential. Differentiation induces expression of voltage-gated Na+ channels and action potential generation in NG108-15 cells. A high level of the Na+ channel density is required for differentiation-triggered action potential generation.

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

Trithorax complex component Menin controls differentiation and maintenance of T helper 17 cells

PubMed Central

Watanabe, Yukiko; Onodera, Atsushi; Kanai, Urara; Ichikawa, Tomomi; Obata-Ninomiya, Kazushige; Wada, Tomoko; Kiuchi, Masahiro; Iwamura, Chiaki; Tumes, Damon J.; Shinoda, Kenta; Yagi, Ryoji; Motohashi, Shinichiro; Hirahara, Kiyoshi; Nakayama, Toshinori

2014-01-01

Epigenetic modifications, such as posttranslational modifications of histones, play an important role in gene expression and regulation. These modifications are in part mediated by the Trithorax group (TrxG) complex and the Polycomb group (PcG) complex, which activate and repress transcription, respectively. We herein investigate the role of Menin, a component of the TrxG complex in T helper (Th) cell differentiation and show a critical role for Menin in differentiation and maintenance of Th17 cells. Menin−/− T cells do not efficiently differentiate into Th17 cells, leaving Th1 and Th2 cell differentiation intact in in vitro cultures. Menin deficiency resulted in the attenuation of Th17-induced airway inflammation. In differentiating Th17 cells, Menin directly bound to the Il17a gene locus and was required for the deposition of permissive histone modifications and recruitment of the RNA polymerase II transcriptional complex. Interestingly, although Menin bound to the Rorc locus, Menin was dispensable for the induction of Rorc expression and permissive histone modifications in differentiating Th17 cells. In contrast, Menin was required to maintain expression of Rorc in differentiated Th17 cells, indicating that Menin is essential to stabilize expression of the Rorc gene. Thus, Menin orchestrates Th17 cell differentiation and function by regulating both the induction and maintenance of target gene expression. PMID:25136117

Cyclooxygenases in human and mouse skin and cultured human keratinocytes: association of COX-2 expression with human keratinocyte differentiation

NASA Technical Reports Server (NTRS)

Leong, J.; Hughes-Fulford, M.; Rakhlin, N.; Habib, A.; Maclouf, J.; Goldyne, M. E.

1996-01-01

Epidermal expression of the two isoforms of the prostaglandin H-generating cyclooxygenase (COX-1 and COX-2) was evaluated both by immunohistochemistry performed on human and mouse skin biopsy sections and by Western blotting of protein extracts from cultured human neonatal foreskin keratinocytes. In normal human skin, COX-1 immunostaining is observed throughout the epidermis whereas COX-2 immunostaining increases in the more differentiated, suprabasilar keratinocytes. Basal cell carcinomas express little if any COX-1 or COX-2 immunostaining whereas both isozymes are strongly expressed in squamous cell carcinomas deriving from a more differentiated layer of the epidermis. In human keratinocyte cultures, raising the extracellular calcium concentration, a recognized stimulus for keratinocyte differentiation, leads to an increased expression of both COX-2 protein and mRNA; expression of COX-1 protein, however, shows no significant alteration in response to calcium. Because of a recent report that failed to show COX-2 in normal mouse epidermis, we also looked for COX-1 and COX-2 immunostaining in sections of normal and acetone-treated mouse skin. In agreement with a previous report, some COX-1, but no COX-2, immunostaining is seen in normal murine epidermis. However, following acetone treatment, there is a marked increase in COX-1 expression as well as the appearance of significant COX-2 immunostaining in the basal layer. These data suggest that in human epidermis as well as in human keratinocyte cultures, the expression of COX-2 occurs as a part of normal keratinocyte differentiation whereas in murine epidermis, its constitutive expression is absent, but inducible as previously published.

Three members of the iodothyronine deiodinase family, dio1, dio2 and dio3, are expressed in spatially and temporally specific patterns during metamorphosis of the flounder, Paralichthys olivaceus.

PubMed

Itoh, Kae; Watanabe, Kohei; Wu, Xiaoming; Suzuki, Tohru

2010-07-01

Flounder metamorphosis, marked by eye migration, lateralized pigmentation, and tissue differentiation in the stomach and skeletal muscle, is stimulated by thyroid hormone (TH). It is known that tri-iodothyronine (T3) produced by iodothyronine deiodinase type-1 (Dio1) from thyroxine (T4) enters the blood, whereas T3 produced by Dio2 penetrates into the nucleus of the Dio2-expressing cells, and then Dio3 inactivates both T4 and T3. To better understand the distinct functions of these three deiodinases in T3 regulation during flounder metamorphosis, we examined the tissue expression patterns of dio1, dio2, and dio3 in larvae of the Japanese flounder, Paralichthys olivaceus, by section in situ hybridization (SISH). We found that each deiodinase is expressed in a spatially and temporally specific pattern. dio1 is expressed in liver parenchymal cells from pro-metamorphosis to early climax, while dio2 is expressed in limited regions of the eyes, tectum, and skeletal muscles from pro-metamorphosis to post-climax. Considering these findings together with reports on other vertebrates, we predict that the liver cells expressing dio1 supply T3 to the blood, and that this systemic T3 synchronizes metamorphosis of differentiating tissues throughout the larval body, whereas the eyes, tectum, and skeletal muscles autonomously produce additional T3 for local tissue differentiation. Finally, dio3 expression is detected in skeletal muscle and gastric gland blastemas, which both undergo marked tissue differentiation at metamorphic climax. We hypothesize that dio3 expression protects these tissues from basal T3 levels early in metamorphosis, ensuring, together with the T3 surge from the liver, the synchronization of tissue differentiation at metamorphic climax.

Fibronectin-mediation cell adhesion is required for induction of 92-kDa type IV collagenase/gelatinase (MMP-9) gene expression during macrophage differentiation : the signaling role of protein kinase C-{beta}.

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

Xie, B.; Laouar, A.; Huberman, E.

1998-05-08

Induction of the 92-kDa gelatinase (MMP-9) gene expression is associated with macrophage differentiation. In this study, we explored the regulatory mechanisms underlying this differentiation-associated MMP-9 gene expression in human HL-60 myeloid leukemia cells and human peripheral blood monocytes. Phorbol 12-myristate 13-acetate (PMA) markedly induced MMP-9 gene expression in HL-60 cells; the induction closely paralleled the timing and extent of PMA-induced cell adhesion and spreading, a hallmark of macrophage differentiation. Similarly, treatment with PMA or macrophage-colony stimulating factor stimulated adherence and spreading of blood monocytes with a concurrent 7- or 5-fold increase in MMP-9 production, respectively. In protein kinase C (PKC)-betamore » -deficient HL-60 variant cells (HL-525), PMA failed to induce cell adhesion and MMP-9 gene expression. Transfecting HL-525 cells with a PKC-beta expression plasmid restored PKC-beta levels and PMA inducibility of cell adhesion and spreading as well as MMP-9 gene expression. Induction of cell adhesion and MMP-9 gene expression in HL-60 cells and blood monocytes was strongly inhibited by neutralizing monoclonal antibodies to fibronectin (FN) and its receptor {alpha}5{beta}1 integrin. HL-525 cells, which constitutively display high levels of surface {alpha}5{beta}1 integrin, adhered and spread on immobilized FN with concomitant induction of MMP-9 gene expression. Cytochalasins B and D were each a potent inhibitor of MMP-9 production. Our results suggest that {alpha}5{beta}1 integrin-mediated interaction of immature hematopoietic cells with FN plays a critical role in modulating matrix-degrading activities during macrophage differentiation.« less

LncRNA MEG3 inhibited osteogenic differentiation of bone marrow mesenchymal stem cells from postmenopausal osteoporosis by targeting miR-133a-3p.

PubMed

Wang, Qiujun; Li, Ying; Zhang, Yuanxia; Ma, Lan; Lin, Lin; Meng, Jia; Jiang, Lihong; Wang, Liping; Zhou, Ping; Zhang, Yina

2017-05-01

Long non-coding RNA (lncRNA) MEG3 has proven to be an important regulator involved in the pathogenesis and development of various human diseases. However, the functional involvement of MEG3 in postmenopausal osteoporosis (PMOP) and its mechanism is still unclear. Bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured from mouse pathologic models and patients with PMOP, respectively. The expression of MEG3 and miR-133a-3p in BMSCs was detected using qRT-PCR. The recombinant expression vector was constructed and transfected into BMSCs to regulate the endogenous expression of MEG3 and miR-133a-3p. The mineralized nodules formation, alkaline phosphatase (ALP) activity and Runx2, OCN, OPN expressions were used as specific markers for the differentiation of osteoblasts. The expressions of MEG3 and miR-133a-3p in BMSCs from PMOP were increased, and there was a positive correlation between MEG3 and miR-133a-3p expression in BMSCs. In the differentiation process from BMSCs to osteoblasts, the expressions of MEG3 and miR-133a-3p were markedly decreased, and MEG3 overexpression reversed the osteogenic induction-mediated downregulation of miR-133a-3p, which was accompanied by significant decline in SLC39A1 expression. Furthermore, miR-133a-3p silencing or upregulation eliminated the effects of MEG3 on the osteogenic differentiation of BMSCs through direct binding. The research indicated that MEG3 regulated the expression of miR-133a-3p, and inhibited the osteogenic differentiation of BMSCs induced PMOP. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

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.

Effect of TCEA3 on the differentiation of bovine skeletal muscle satellite cells

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

Zhu, Yue; Tong, Hui-Li; Li, Shu-Feng

Bovine muscle-derived satellite cells (MDSCs) are important for animal growth. In this study, the effect of transcription elongation factor A3 (TCEA3) on bovine MDSC differentiation was investigated. Western blotting, immunofluorescence assays, and cytoplasmic and nuclear protein isolation and purification techniques were used to determine the expression pattern and protein localization of TCEA3 in bovine MDSCs during in vitro differentiation. TCEA3 expression was upregulated using the CRISPR/Cas9 technique to study its effects on MDSC differentiation in vitro. TCEA3 expression gradually increased during the in vitro differentiation of bovine MDSCs and peaked on the 5th day of differentiation. TCEA3 was mainly localized in the cytoplasmmore » of bovine MDSCs, and its expression was not detected in the nucleus. The level of TCEA3 was relatively higher in myotubes at a higher degree of differentiation than during early differentiation. After transfection with a TCEA3-activating plasmid vector (TCEA3 overexpression) for 24 h, the myotube fusion rate, number of myotubes, and expression levels of the muscle differentiation-related loci myogenin (MYOG) and myosin heavy chain 3 (MYH3) increased significantly during the in vitro differentiation of bovine MDSCs. After transfection with a TCEA3-inhibiting plasmid vector for 24 h, the myotube fusion rate, number of myotubes, and expression levels of MYOG and MYH3 decreased significantly. Our results indicated, for the first time, that TCEA3 promotes the differentiation of bovine MDSCs and have implications for meat production and animal rearing. - Highlights: • Muscle-derived satellite cell differentiation is promoted by TCEA3. • TCEA3 protein was localized in the cytoplasm, but not nuclei of bovine MDSCs. • TCEA3 levels increased as myotube differentiation increased. • TCEA3 affected myotube fusion, myotube counts, and MYOG and MYH3 levels.« less

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.

Expression and localisation of aquaporin water channels in human urothelium in situ and in vitro.

PubMed

Rubenwolf, Peter C; Georgopoulos, Nikolaos T; Clements, Lisa A; Feather, Sally; Holland, Philip; Thomas, David F M; Southgate, Jennifer

2009-12-01

Urothelium is generally considered to be impermeable to water and constituents of urine. The possibility that human urothelium expresses aquaporin (AQP) water channels as the basis for water and solute transport has not previously been investigated. To investigate the expression of AQP water channels by human urothelium in situ, in proliferating urothelial cell cultures and in differentiated tissue constructs. AQP expression by human urothelium in situ and cultured urothelial cells was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunolabelling. Expression screening was carried out on samples of freshly isolated urothelia from multiple surgical (bladder and ureteric) specimens and on proliferating and differentiated normal human urothelial (NHU) cells in culture. Urothelial tissue constructs were established and investigated for expression of urothelial differentiation markers and AQPs. Qualitative study. Transcripts for AQP3, AQP4, AQP7, AQP9, and AQP11 were expressed consistently by freshly isolated urothelia as well as by cultured NHU cells. AQP0, AQP1, AQP2, AQP5, AQP6, AQP8, AQP10, and AQP12 were not expressed. Immunochemistry confirmed expression of AQP3, AQP4, AQP7, and AQP9 at the protein level. AQP3 was shown to be intensely expressed at cell borders in the basal and intermediate layers in both urothelium in situ and differentiated tissue constructs in vitro. This is the first study to demonstrate that AQPs are expressed by human urothelium, suggesting a potential role in transurothelial water and solute transport. Our findings challenge the traditional concept of the urinary tract as an impermeable transit and storage unit and provide a versatile platform for further investigations into the biological and clinical relevance of AQPs in human urothelium.

Influence of white spot syndrome virus infection on hepatopancreas gene expression of `Huanghai No. 2' shrimp ( Fenneropenaeus chinensis)

NASA Astrophysics Data System (ADS)

Meng, Xianhong; Shi, Xiaoli; Kong, Jie; Luan, Sheng; Luo, Kun; Cao, Baoxiang; Liu, Ning; Lu, Xia; Li, Xupeng; Deng, Kangyu; Cao, Jiawang; Zhang, Yingxue; Zhang, Hengheng

2017-10-01

To elucidate the molecular response of shrimp hepatopancreas to white spot syndrome virus (WSSV) infection, microarray was applied to investigate the differentially expressed genes in the hepatopancreas of `Huanghai No. 2' ( Fenneropenaeus chinensis). A total of 59137 unigenes were designed onto a custom-made 60K Agilent chip. After infection, the gene expression profiles in the hepatopancreas of the shrimp with a lower viral load at early (48-96 h), peak (168-192 h) and late (264-288 h) infection phases were analyzed. Of 18704 differentially expressed genes, 6412 were annotated. In total, 5453 differentially expressed genes (1916 annotated) expressed at all three phases, and most of the annotated were either up- or down-regulated continuously. These genes function diversely in, for example, immune response, cytoskeletal system, signal transduction, stress resistance, protein synthesis and processing, metabolism among others. Some of the immune-related genes, including antilipopolysaccharide factor, Kazal-type proteinase inhibitor, C-type lectin and serine protease encoding genes, were up-regulated after WSSV infection. These genes have been reported to be involved in the anti-WSSV responses. The expression of genes related to the cytoskeletal system, including β-actin and myosin but without tubulin genes, were down-regulated after WSSV infection. Astakine was found for the first time in the WSSV-infected F. chinensis. To further confirm the expression of differentially expressed genes, quantitative real-time PCR was performed to test the expression of eight randomly selected genes and verified the reliability and accuracy of the microarray expression analysis. The data will provide valuable information to understanding the immune mechanism of shrimp's response to WSSV.

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.

Lamin A/C Is Required for ChAT-Dependent Neuroblastoma Differentiation.

PubMed

Guglielmi, Loredana; Nardella, Marta; Musa, Carla; Iannetti, Ilaria; Arisi, Ivan; D'Onofrio, Mara; Storti, Andrea; Valentini, Alessandra; Cacci, Emanuele; Biagioni, Stefano; Augusti-Tocco, Gabriella; D'Agnano, Igea; Felsani, Armando

2017-07-01

The mouse neuroblastoma N18TG2 clone is unable to differentiate and is defective for the enzymes of the biosynthesis of neurotransmitters. The forced expression of choline acetyltransferase (ChAT) in these cells results in the synthesis and release of acetylcholine (Ach) and hence in the expression of neurospecific features and markers. To understand how the expression of ChAT triggered neuronal differentiation, we studied the differences in genome-wide transcription profiles between the N18TG2 parental cells and its ChAT-expressing 2/4 derived clone. The engagement of the 2/4 cells in the neuronal developmental program was confirmed by the increase of the expression level of several differentiation-related genes and by the reduction of the amount of transcripts of cell cycle genes. At the same time, we observed a massive reorganization of cytoskeletal proteins in terms of gene expression, with the accumulation of the nucleoskeletal lamina component Lamin A/C in differentiating cells. The increase of the Lmna transcripts induced by ChAT expression in 2/4 cells was mimicked treating the parental N18TG2 cells with the acetylcholine receptor agonist carbachol, thus demonstrating the direct role played by this receptor in neuron nuclei maturation. Conversely, a treatment of 2/4 cells with the muscarinic receptor antagonist atropine resulted in the reduction of the amount of Lmna RNA. Finally, the hypothesis that Lmna gene product might play a crucial role in the ChAT-dependent molecular differentiation cascade was strongly supported by Lmna knockdown in 2/4 cells leading to the downregulation of genes involved in differentiation and cytoskeleton formation and to the upregulation of genes known to regulate self-renewal and stemness.

Gene expression studies of developing bovine longissimus muscle from two different beef cattle breeds

PubMed Central

Lehnert, Sigrid A; Reverter, Antonio; Byrne, Keren A; Wang, Yonghong; Nattrass, Greg S; Hudson, Nicholas J; Greenwood, Paul L

2007-01-01

Background The muscle fiber number and fiber composition of muscle is largely determined during prenatal development. In order to discover genes that are involved in determining adult muscle phenotypes, we studied the gene expression profile of developing fetal bovine longissimus muscle from animals with two different genetic backgrounds using a bovine cDNA microarray. Fetal longissimus muscle was sampled at 4 stages of myogenesis and muscle maturation: primary myogenesis (d 60), secondary myogenesis (d 135), as well as beginning (d 195) and final stages (birth) of functional differentiation of muscle fibers. All fetuses and newborns (total n = 24) were from Hereford dams and crossed with either Wagyu (high intramuscular fat) or Piedmontese (GDF8 mutant) sires, genotypes that vary markedly in muscle and compositional characteristics later in postnatal life. Results We obtained expression profiles of three individuals for each time point and genotype to allow comparisons across time and between sire breeds. Quantitative reverse transcription-PCR analysis of RNA from developing longissimus muscle was able to validate the differential expression patterns observed for a selection of differentially expressed genes, with one exception. We detected large-scale changes in temporal gene expression between the four developmental stages in genes coding for extracellular matrix and for muscle fiber structural and metabolic proteins. FSTL1 and IGFBP5 were two genes implicated in growth and differentiation that showed developmentally regulated expression levels in fetal muscle. An abundantly expressed gene with no functional annotation was found to be developmentally regulated in the same manner as muscle structural proteins. We also observed differences in gene expression profiles between the two different sire breeds. Wagyu-sired calves showed higher expression of fatty acid binding protein 5 (FABP5) RNA at birth. The developing longissimus muscle of fetuses carrying the Piedmontese mutation shows an emphasis on glycolytic muscle biochemistry and a large-scale up-regulation of the translational machinery at birth. We also document evidence for timing differences in differentiation events between the two breeds. Conclusion Taken together, these findings provide a detailed description of molecular events accompanying skeletal muscle differentiation in the bovine, as well as gene expression differences that may underpin the phenotype differences between the two breeds. In addition, this study has highlighted a non-coding RNA, which is abundantly expressed and developmentally regulated in bovine fetal muscle. PMID:17697390

Influence of 1α, 25-dihydroxyvitamin D3 [1, 25(OH)2D3] on the expression of Sox 9 and the transient receptor potential vanilloid 5/6 ion channels in equine articular chondrocytes.

PubMed

Hdud, Ismail M; Loughna, Paul T

2014-01-01

Sox 9 is a major marker of chondrocyte differentiation. When chondrocytes are cultured in vitro they progressively de-differentiate and this is associated with a decline in Sox 9 expression. The active form of vitamin D, 1, 25 (OH)2D3 has been shown to be protective of cartilage in both humans and animals. In this study equine articular chondrocytes were grown in culture and the effects of 1, 25 (OH)2D3 upon Sox 9 expression examined. The expression of the transient receptor potential vanilloid (TRPV) ion channels 5 and 6 in equine chondrocytes in vitro, we have previously shown, is inversely correlated with de-differentiation. The expression of these channels in response to 1, 25 (OH)2D3 administration was therefore also examined. The active form of vitamin D (1, 25 (OH)2D3) when administered to cultured equine chondrocytes at two different concentrations significantly increased the expression of Sox 9 at both. In contrast 1, 25 (OH)2D3 had no significant effect upon the expression of either TRPV 5 or 6 at either the protein or the mRNA level. The increased expression of Sox 9, in equine articular chondrocytes in vitro, in response to the active form of vitamin D suggests that this compound could be utilized to inhibit the progressive de-differentiation that is normally observed in these cells. It is also supportive of previous studies indicating that 1α, 25-dihydroxyvitamin D3 can have a protective effect upon cartilage in animals in vivo. The previously observed correlation between the degree of differentiation and the expression levels of TRPV 5/6 had suggested that these ion channels may have a direct involvement in, or be modulated by, the differentiation process in vitro. The data in the present study do not support this.

A Genome-Wide Screen Indicates Correlation between Differentiation and Expression of Metabolism Related Genes

PubMed Central

Shende, Akhilesh; Singh, Anupama; Meena, Anil; Ghosal, Ritika; Ranganathan, Madhav; Bandyopadhyay, Amitabha

2013-01-01

Differentiated tissues may be considered as materials with distinct properties. The differentiation program of a given tissue ensures that it acquires material properties commensurate with its function. It may be hypothesized that some of these properties are acquired through production of tissue-specific metabolites synthesized by metabolic enzymes. To establish correlation between metabolism and organogenesis we have carried out a genome-wide expression study of metabolism related genes by RNA in-situ hybridization. 23% of the metabolism related genes studied are expressed in a tissue-restricted but not tissue-exclusive manner. We have conducted the screen on whole mount chicken (Gallus gallus) embryos from four distinct developmental stages to correlate dynamic changes in expression patterns of metabolic enzymes with spatio-temporally unique developmental events. Our data strongly suggests that unique combinations of metabolism related genes, and not specific metabolic pathways, are upregulated during differentiation. Further, expression of metabolism related genes in well established signaling centers that regulate different aspects of morphogenesis indicates developmental roles of some of the metabolism related genes. The database of tissue-restricted expression patterns of metabolism related genes, generated in this study, should serve as a resource for systematic identification of these genes with tissue-specific functions during development. Finally, comprehensive understanding of differentiation is not possible unless the downstream genes of a differentiation cascade are identified. We propose, metabolic enzymes constitute a significant portion of these downstream target genes. Thus our study should help elucidate different aspects of tissue differentiation. PMID:23717462

A genome-wide screen indicates correlation between differentiation and expression of metabolism related genes.

PubMed

Roy, Priti; Kumar, Brijesh; Shende, Akhilesh; Singh, Anupama; Meena, Anil; Ghosal, Ritika; Ranganathan, Madhav; Bandyopadhyay, Amitabha

2013-01-01

Differentiated tissues may be considered as materials with distinct properties. The differentiation program of a given tissue ensures that it acquires material properties commensurate with its function. It may be hypothesized that some of these properties are acquired through production of tissue-specific metabolites synthesized by metabolic enzymes. To establish correlation between metabolism and organogenesis we have carried out a genome-wide expression study of metabolism related genes by RNA in-situ hybridization. 23% of the metabolism related genes studied are expressed in a tissue-restricted but not tissue-exclusive manner. We have conducted the screen on whole mount chicken (Gallus gallus) embryos from four distinct developmental stages to correlate dynamic changes in expression patterns of metabolic enzymes with spatio-temporally unique developmental events. Our data strongly suggests that unique combinations of metabolism related genes, and not specific metabolic pathways, are upregulated during differentiation. Further, expression of metabolism related genes in well established signaling centers that regulate different aspects of morphogenesis indicates developmental roles of some of the metabolism related genes. The database of tissue-restricted expression patterns of metabolism related genes, generated in this study, should serve as a resource for systematic identification of these genes with tissue-specific functions during development. Finally, comprehensive understanding of differentiation is not possible unless the downstream genes of a differentiation cascade are identified. We propose, metabolic enzymes constitute a significant portion of these downstream target genes. Thus our study should help elucidate different aspects of tissue differentiation.

Differential In Vivo Gene Expression of Major Leptospira Proteins in Resistant or Susceptible Animal Models

PubMed Central

Matsui, Mariko; Soupé, Marie-Estelle; Becam, Jérôme

2012-01-01

Transcripts of Leptospira 16S rRNA, FlaB, LigB, LipL21, LipL32, LipL36, LipL41, and OmpL37 were quantified in the blood of susceptible (hamsters) and resistant (mice) animal models of leptospirosis. We first validated adequate reference genes and then evaluated expression patterns in vivo compared to in vitro cultures. LipL32 expression was downregulated in vivo and differentially regulated in resistant and susceptible animals. FlaB expression was also repressed in mice but not in hamsters. In contrast, LigB and OmpL37 were upregulated in vivo. Thus, we demonstrated that a virulent strain of Leptospira differentially adapts its gene expression in the blood of infected animals. PMID:22729538

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.

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.

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.

DD-RT-PCR identifies 7-dehydrocholesterol reductase as a key marker of early Leydig cell steroidogenesis.

PubMed

Anbalagan, M; Yashwanth, R; Jagannadha Rao, A

2004-04-30

Postnatal Leydig cell development in rat involves an initial phase of proliferation of progenitor Leydig cells (PLCs) and subsequent differentiation of these cells into immature Leydig cells (ILCs) and adult Leydig cells (ALCs). With an objective to identify the molecular changes associated with Leydig cell differentiation, the mRNA population in PLCs and ILCs were analyzed by the technique of differential display reverse transcription polymerase chain reaction (DD-RT-PCR). Results revealed differential expression of several transcripts in PLCs and ILCs. Of the several differentially expressed transcripts, the expression of transcripts corresponding to collagen IV alpha6 (Col IV alpha6) and ribosomal protein L 41 (RpL41) decreased during the differentiation of PLC to ILC. Also there was an increase in the expression of transcripts encoding enzymes such as microsomal glutathione-S-transferase (mGST 1) and 7-dehydrocholesterol reductase (7-DHCR) during this process. While Col IV alpha6 and RpL41 are known to be involved in cellular proliferation, mGST 1 and 7-DHCR are essential for normal Leydig cell steroidogenesis. A detailed study on 7-DHCR expression in Leydig cells revealed that this enzyme plays a crucial role in steroidogenesis. Interestingly expression of this enzyme is not under acute regulation by Luteinizing hormone (LH). Copyright 2004 Elsevier Ireland Ltd.

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.

Differential expression of conserved and novel microRNAs during tail regeneration in the lizard Anolis carolinensis.

PubMed

Hutchins, Elizabeth D; Eckalbar, Walter L; Wolter, Justin M; Mangone, Marco; Kusumi, Kenro

2016-05-05

Lizards are evolutionarily the most closely related vertebrates to humans that can lose and regrow an entire appendage. Regeneration in lizards involves differential expression of hundreds of genes that regulate wound healing, musculoskeletal development, hormonal response, and embryonic morphogenesis. While microRNAs are able to regulate large groups of genes, their role in lizard regeneration has not been investigated. MicroRNA sequencing of green anole lizard (Anolis carolinensis) regenerating tail and associated tissues revealed 350 putative novel and 196 known microRNA precursors. Eleven microRNAs were differentially expressed between the regenerating tail tip and base during maximum outgrowth (25 days post autotomy), including miR-133a, miR-133b, and miR-206, which have been reported to regulate regeneration and stem cell proliferation in other model systems. Three putative novel differentially expressed microRNAs were identified in the regenerating tail tip. Differentially expressed microRNAs were identified in the regenerating lizard tail, including known regulators of stem cell proliferation. The identification of 3 putative novel microRNAs suggests that regulatory networks, either conserved in vertebrates and previously uncharacterized or specific to lizards, are involved in regeneration. These findings suggest that differential regulation of microRNAs may play a role in coordinating the timing and expression of hundreds of genes involved in regeneration.

Aberrant epithelial differentiation by cigarette smoke dysregulates respiratory host defence.

PubMed

Amatngalim, Gimano D; Schrumpf, Jasmijn A; Dishchekenian, Fernanda; Mertens, Tinne C J; Ninaber, Dennis K; van der Linden, Abraham C; Pilette, Charles; Taube, Christian; Hiemstra, Pieter S; van der Does, Anne M

2018-04-01

It is currently unknown how cigarette smoke-induced airway remodelling affects highly expressed respiratory epithelial defence proteins and thereby mucosal host defence.Localisation of a selected set of highly expressed respiratory epithelial host defence proteins was assessed in well-differentiated primary bronchial epithelial cell (PBEC) cultures. Next, PBEC were cultured at the air-liquid interface, and during differentiation for 2-3 weeks exposed daily to whole cigarette smoke. Gene expression, protein levels and epithelial cell markers were subsequently assessed. In addition, functional activities and persistence of the cigarette smoke-induced effects upon cessation were determined.Expression of the polymeric immunoglobulin receptor, secretory leukocyte protease inhibitor and long and short PLUNC (palate, lung and nasal epithelium clone protein) was restricted to luminal cells and exposure of differentiating PBECs to cigarette smoke resulted in a selective reduction of the expression of these luminal cell-restricted respiratory host defence proteins compared to controls. This reduced expression was a consequence of cigarette smoke-impaired end-stage differentiation of epithelial cells, and accompanied by a significant decreased transepithelial transport of IgA and bacterial killing.These findings shed new light on the importance of airway epithelial cell differentiation in respiratory host defence and could provide an additional explanation for the increased susceptibility of smokers and patients with chronic obstructive pulmonary disease to respiratory infections. Copyright ©ERS 2018.

Expression Variations of miRNAs and mRNAs in Rice (Oryza sativa).

PubMed

Wen, Ming; Xie, Munan; He, Lian; Wang, Yushuai; Shi, Suhua; Tang, Tian

2016-12-31

Differences in expression levels are an important source of phenotypic variation within and between populations. MicroRNAs (miRNAs) are key players in post-transcriptional gene regulation that are important for plant development and stress responses. We surveyed expression variation of miRNAs and mRNAs of six accessions from two rice subspecies Oryza sativa L. ssp. indica and Oryza sativa L. ssp. japonica using deep sequencing. While more than half (53.7%) of the mature miRNAs exhibit differential expression between grains and seedlings of rice, only 11.0% show expression differences between subspecies, with an additional 2.2% differentiated for the development-by-subspecies interaction. Expression variation is greater for lowly conserved miRNAs than highly conserved miRNAs, whereas the latter show stronger negative correlation with their targets in expression changes between subspecies. Using a permutation test, we identified 51 miRNA-mRNA pairs that correlate negatively or positively in expression level among cultivated rice. Genes involved in various metabolic processes and stress responses are enriched in the differentially expressed genes between rice indica and japonica subspecies. Our results indicate that stabilizing selection is the major force governing miRNA expression in cultivated rice, albeit positive selection may be responsible for much of the between-subspecies expression divergence. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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.

Expression patterns of nestin and dentin sialoprotein during dentinogenesis in mice.

PubMed

Quispe-Salcedo, Angela; Ida-Yonemochi, Hiroko; Nakatomi, Mitsushiro; Ohshima, Hayato

2012-04-01

Differentiated odontoblasts could not be identified by one unique phenotypic marker, but the combination of expression of dentin phosphoprotein (Dpp), dentin sialoprotein (Dsp), dentin matrix protein 1 (Dmp1), and nestin may be valuable for the assessment of these cells. However, the findings using these proteins remain controversial. This study aimed to compare two odontoblast differentiation markers: nestin and Dsp in the process of dentinogenesis in mice. We performed immunohistochemistry and/or in situ hybridization technique for nestin and Dsp using 3-week-old incisors as well as postnatal 1-day- to 8-week-old molars. Preodontoblasts began to express nestin and Dsp proteins and Dsp mRNA, which increased in their intensity according to the progress of odontoblast differentiation in both incisors and developing molars. Nestin was consistently expressed in the differentiated odontoblasts even after the completion of dentin matrix deposition. The expression of Dsp mRNA coincided with the odontoblast secretory activity for dentin matrix deposition. In contrast, other pulpal cells, predentin matrix and dentinal tubules also showed a positive reaction for Dsp protein in addition to differentiated odontoblasts. In conclusion, nestin is valuable as a differentiation marker for odontoblasts, whereas Dsp mRNA is a functional marker for their secretory activity.

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 of Zinc Transporters in Prostate Epithelia of Racial Groups

DTIC Science & Technology

2012-09-01

of significant genes or proteins in the prostate cancers taken from AAs versus those from European Americans (EAs)?” Because there is a well...may be differentially expressed in AAs and EAs. A study of the genes and proteins which influence the expression of any gene confirmed to be...type of TMA, based on long term clinical follow up was used to address the question of whether hZIP gene and protein expression is associated with

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

Zhang, Long; Shi, Songting; Zhang, Juan

Highlights: Black-Right-Pointing-Pointer Expression of Id3 but not Id1 is induced by Wnt3a stimulation in C2C12 cells. Black-Right-Pointing-Pointer Wnt3a induces Id3 expression via canonical Wnt/{beta}-catenin pathway. Black-Right-Pointing-Pointer Wnt3a-induced Id3 expression does not depend on BMP signaling activation. Black-Right-Pointing-Pointer Induction of Id3 expression is critical determinant in Wnt3a-induced cell proliferation and differentiation. -- Abstract: Canonical Wnt signaling plays important roles in regulating cell proliferation and differentiation. In this study, we report that inhibitor of differentiation (Id)3 is a Wnt-inducible gene in mouse C2C12 myoblasts. Wnt3a induced Id3 expression in a {beta}-catenin-dependent manner. Bone morphogenetic protein (BMP) also potently induced Id3 expression. However,more » Wnt-induced Id3 expression occurred independent of the BMP/Smad pathway. Functional studies showed that Id3 depletion in C2C12 cells impaired Wnt3a-induced cell proliferation and alkaline phosphatase activity, an early marker of osteoblast cells. Id3 depletion elevated myogenin induction during myogenic differentiation and partially impaired Wnt3a suppressed myogenin expression in C2C12 cells. These results suggest that Id3 is an important Wnt/{beta}-catenin induced gene in myoblast cell fate determination.« less

Differential Protein Expressions in Virus-Infected and Uninfected Trichomonas vaginalis.

PubMed

He, Ding; Pengtao, Gong; Ju, Yang; Jianhua, Li; He, Li; Guocai, Zhang; Xichen, Zhang

2017-04-01

Protozoan viruses may influence the function and pathogenicity of the protozoa. Trichomonas vaginalis is a parasitic protozoan that could contain a double stranded RNA (dsRNA) virus, T. vaginalis virus (TVV). However, there are few reports on the properties of the virus. To further determine variations in protein expression of T. vaginalis , we detected 2 strains of T. vaginalis ; the virus-infected (V + ) and uninfected (V - ) isolates to examine differentially expressed proteins upon TVV infection. Using a stable isotope N-terminal labeling strategy (iTRAQ) on soluble fractions to analyze proteomes, we identified 293 proteins, of which 50 were altered in V + compared with V - isolates. The results showed that the expression of 29 proteins was increased, and 21 proteins decreased in V + isolates. These differentially expressed proteins can be classified into 4 categories: ribosomal proteins, metabolic enzymes, heat shock proteins, and putative uncharacterized proteins. Quantitative PCR was used to detect 4 metabolic processes proteins: glycogen phosphorylase, malate dehydrogenase, triosephosphate isomerase, and glucose-6-phosphate isomerase, which were differentially expressed in V + and V - isolates. Our findings suggest that mRNA levels of these genes were consistent with protein expression levels. This study was the first which analyzed protein expression variations upon TVV infection. These observations will provide a basis for future studies concerning the possible roles of these proteins in host-parasite interactions.

Bach2 is involved in neuronal differentiation of N1E-115 neuroblastoma cells.

PubMed

Shim, Ki Shuk; Rosner, Margit; Freilinger, Angelika; Lubec, Gert; Hengstschläger, Markus

2006-07-15

Bach1 and Bach2 are evolutionarily related members of the BTB-basic region leucine zipper transcription factor family. We found that Bach2 downregulates cell proliferation of N1E-115 cells and negatively affects their potential to differentiate. Nuclear localization of the cyclin-dependent kinase inhibitor p21 is known to arrest cell cycle progression, and cytoplasmic p21 has been shown to promote neuronal differentiation of N1E-115 cells. We found that ectopic Bach2 causes upregulation of p21 expression in the nucleus and in the cytoplasm in undifferentiated N1E-115 cells. In differentiated cells, Bach2 specifically triggers upregulation of cytoplasmic p21. Our data suggest that Bach2 expression could represent a switch during the process of neuronal differentiation. Bach2 is not expressed in neuronal precursor cells. It would have negative effects on proliferation and differentiation of these cells. In differentiated neuronal cells Bach2 expression is upregulated, which could allow Bach2 to function as a gatekeeper of the differentiated status.

Loss of Anterior Gradient 2 (Agr2) Expression Results in Hyperplasia and Defective Lineage Maturation in the Murine Stomach*

PubMed Central

Gupta, Aparna; Wodziak, Dariusz; Tun, May; Bouley, Donna M.; Lowe, Anson W.

2013-01-01

Recent studies of epithelial tissues have revealed the presence of tissue-specific stem cells that are able to establish multiple cell lineages within an organ. The stem cells give rise to progenitors that replicate before differentiating into specific cell lineages. The mechanism by which homeostasis is established between proliferating stem or progenitor cells and terminally differentiated cells is unclear. This study demonstrates that Agr2 expression by mucous neck cells in the stomach promotes the differentiation of multiple cell lineages while also inhibiting the proliferation of stem or progenitor cells. When Agr2 expression is absent, gastric mucous neck cells increased in number as does the number of proliferating cells. Agr2 expression loss also resulted in the decline of terminally differentiated cells, which was supplanted by cells that exhibited nuclear SOX9 labeling. Sox9 expression has been associated with progenitor and stem cells. Similar effects of the Agr2 null on cell proliferation in the intestine were also observed. Agr2 consequently serves to maintain the balance between proliferating and differentiated epithelial cells. PMID:23209296

Tissue Inhibitor of Metalloproteinase-2 (TIMP-2) expression is regulated by multiple neural differentiation signals

PubMed Central

Jaworski, Diane M.; Pérez-Martínez, Leonor

2010-01-01

Neuronal differentiation requires exquisitely timed cell cycle arrest for progenitors to acquire an appropriate neuronal cell fate and is achieved by communication between soluble signals, such as growth factors and extracellular matrix molecules. Here we report that the expression of TIMP-2, a matrix metalloproteinase inhibitor, is up-regulated by signals that control proliferation (bFGF and EGF) and differentiation (retinoic acid and NGF) in neural progenitor and neuroblastoma cell lines. TIMP-2 expression coincides with the appearance of neurofilament-positive neurons, indicating that TIMP-2 may play a role in neurogenesis. The up-regulation of TIMP-2 expression by proliferative signals suggests a role in the transition from proliferation to neuronal differentiation. Live labeling experiments demonstrate TIMP-2 expression only on α3 integrin-positive cells. Thus, TIMP-2 function may be mediated via interaction with integrin receptor(s). We propose that TIMP-2 represents a component of the neurogenic signaling cascade induced by mitogenic stimuli that may withdraw progenitor cells from the cell cycle permitting their terminal neuronal differentiation. PMID:16805810

Threshold-dependent cooperativity of Pdx1 and Oc1 in pancreatic progenitors establishes competency for endocrine differentiation and β-cell function

PubMed Central

Wright, Christopher V.E.; Won, Kyoung-Jae

2016-01-01

Summary Pdx1 and Oc1 are co-expressed in multipotent pancreatic progenitors and regulate the pro-endocrine gene Neurog3. Their expression diverges in later organogenesis, with Oc1 absent from hormone+ cells and Pdx1 maintained in mature β cells. In a classical genetic test for cooperative functional interactions, we derived mice with combined Pdx1 and Oc1 heterozygosity. Endocrine development in double-heterozygous pancreata was normal at embryonic day (e)13.5, but defects in specification and differentiation were apparent at e15.5, the height of the second wave of differentiation. Pancreata from double heterozygotes showed alterations in the expression of genes crucial for β-cell development and function, decreased numbers and altered allocation of Neurog3-expressing endocrine progenitors, and defective endocrine differentiation. Defects in islet gene expression and β-cell function persisted in double heterozygous neonates. These results suggest that Oc1 and Pdx1 cooperate prior to their divergence, in pancreatic progenitors, to allow for proper differentiation and functional maturation of β cells. PMID:27292642

Transcriptomic investigation of meat tenderness in two Italian cattle breeds.

PubMed

Bongiorni, S; Gruber, C E M; Bueno, S; Chillemi, G; Ferrè, F; Failla, S; Moioli, B; Valentini, A

2016-06-01

Our objectives for this study were to understand the biological basis of meat tenderness and to provide an overview of the gene expression profiles related to meat quality as a tool for selection. Through deep mRNA sequencing, we analyzed gene expression in muscle tissues of two Italian cattle breeds: Maremmana and Chianina. We uncovered several differentially expressed genes that encode for proteins belonging to a family of tripartite motif proteins, which are involved in growth, cell differentiation and apoptosis, such as TRIM45, or play an essential role in regulating skeletal muscle differentiation and the regeneration of adult skeletal muscle, such as TRIM32. Other differentially expressed genes (SCN2B, SLC9A7 and KCNK3) emphasize the involvement of potassium-sodium pumps in tender meat. By mapping splice junctions in RNA-Seq reads, we found significant differences in gene isoform expression levels. The PRKAG3 gene, which is involved in the regulation of energy metabolism, showed four isoforms that were differentially expressed. This distinct pattern of PRKAG3 gene expression could indicate impaired glycogen storage in skeletal muscle, and consequently, this gene very likely has a role in the tenderization process. Furthermore, with this deep RNA-sequencing, we captured a high number of expressed SNPs, for example, we found 1462 homozygous SNPs showing the alternative allele with a 100% frequency when comparing tender and tough meat. SNPs were then classified into categories by their position and also by their effect on gene coding (174 non-synonymous polymorphisms) based on the available UMD_3.1 annotations. © 2016 Stichting International Foundation for Animal Genetics.

[Identification of differential proteins of serum in the patients suffering from coal-burning arsenism].

PubMed

Han, Bing; Yang, Qin; Luo, Xin-hua; He, Xiao-fei; Wu, Jun; Cheng, Ming-liang

2009-06-02

To compare and analyze the differential expression of proteins between coal-burning arsenism serum and normal human serum and identify the proteins related with arseniasis caused by coal-burning. Serum samples were collected from 6 normal subjects and 6 patients suffering from coal-burning arsenism. 2-DE was performed to separate serum proteins. After silver staining, the differential expression of proteins was analyzed and then identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). There were an average of 779 +/- 35 spots and 865 +/- 30 spots on 2-DE matching of two groups and the matching rate was 90.1% between two groups. From these two groups, 60 different protein spots were identified. Up-regulated expression was observed in 25 proteins and down-regulated expression in 35 proteins in the patient serum group. Among which 35 with differential expression above three times were singled out and MALDI-TOF-MS analysis was carried out on them. Thirteen proteins were identified, including keratin 10, apolipoprotein A-V, transferrin, alpha-1-antitrypsin, human zinc-alpha-2-glycoprotein, mitogen-activated protein kinase 3, vacuolar protein sorting 33A, O-linked GlcNAc transferase and etc. Up-regulated expression was observed in 5 proteins and down-regulated expression in 8 proteins in the patient serum group. The well-resolved and reproducible 2-DE serum patterns of patients suffering from coal-burning arsenism were established and some differentially expressed proteins characterized. These data will be used to screen the biomarker and to further study arseniasis caused by coal-burning.

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.

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

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.

Characterization of lipid metabolism in insulin-sensitive adipocytes differentiated from immortalized human mesenchymal stem cells

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

Prawitt, Janne; Niemeier, Andreas; Kassem, Moustapha

2008-02-15

There is a great demand for cell models to study human adipocyte function. Here we describe the adipogenic differentiation of a telomerase-immortalized human mesenchymal stem cell line (hMSC-Tert) that maintains numerous features of terminally differentiated adipocytes even after prolonged withdrawal of the peroxisome proliferator activated receptor {gamma} (PPAR{gamma}) agonist rosiglitazone. Differentiated hMSC-Tert developed the characteristic monolocular phenotype of mature adipocytes. The expression of adipocyte specific markers was highly increased during differentiation. Most importantly, the presence of the PPAR{gamma} agonist rosiglitazone was not required for the stable expression of lipoprotein lipase, adipocyte fatty acid binding protein and perilipin on mRNA andmore » protein levels. Adiponectin expression was post-transcriptionally down-regulated in the absence of rosiglitazone. Insulin sensitivity as measured by insulin-induced phosphorylation of Akt and S6 ribosomal protein was also independent of rosiglitazone. In addition to commonly used adipogenic markers, we investigated further PPAR{gamma}-stimulated proteins with a role in lipid metabolism. We observed an increase of lipoprotein receptor (VLDLR, LRP1) and apolipoprotein E expression during differentiation. Despite this increased expression, the receptor-mediated endocytosis of lipoproteins was decreased in differentiated adipocytes, suggesting that these proteins may have an additional function in adipose tissue beyond lipoprotein uptake.« less

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

Tu Qisheng; Valverde, Paloma; Chen, Jake

Osterix (Osx) is a zinc-finger-containing transcription factor that is expressed in osteoblasts of all endochondral and membranous bones. In Osx null mice osteoblast differentiation is impaired and bone formation is absent. In this study, we hypothesized that overexpression of Osx in murine bone marrow stromal cells (BMSC) would be able to enhance their osteoblastic differentiation and mineralization in vitro. Retroviral transduction of Osx in BMSC cultured in non-differentiating medium did not affect expression of Runx2/Cbfa1, another key transcription factor of osteoblast differentiation, but induced an increase in the expression of other markers associated with the osteoblastic lineage including alkaline phosphatase,more » bone sialoprotein, osteocalcin, and osteopontin. Retroviral transduction of Osx in BMSC also increased their proliferation, alkaline phosphatase activity, and ability to form bone nodules. These events occurred without significant changes in the expression of {alpha}1(II) procollagen or lipoprotein lipase, which are markers of chondrogenic and adipogenic differentiation, respectively.« less

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.

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.

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

miR-21 promotes the differentiation of hair follicle-derived neural crest stem cells into Schwann cells

PubMed Central

Ni, Yuxin; Zhang, Kaizhi; Liu, Xuejuan; Yang, Tingting; Wang, Baixiang; Fu, Li; A, Lan; Zhou, Yanmin

2014-01-01

Hair follicle-derived neural crest stem cells can be induced to differentiate into Schwann cells in vivo and in vitro. However, the underlying regulatory mechanism during cell differentiation remains poorly understood. This study isolated neural crest stem cells from human hair follicles and induced them to differentiate into Schwann cells. Quantitative RT-PCR showed that microRNA (miR)-21 expression was gradually increased during the differentiation of neural crest stem cells into Schwann cells. After transfection with the miR-21 agonist (agomir-21), the differentiation capacity of neural crest stem cells was enhanced. By contrast, after transfection with the miR-21 antagonist (antagomir-21), the differentiation capacity was attenuated. Further study results showed that SOX-2 was an effective target of miR-21. Without compromising SOX2 mRNA expression, miR-21 can down-regulate SOX protein expression by binding to the 3′-UTR of miR-21 mRNA. Knocking out the SOX2 gene from the neural crest stem cells significantly reversed the antagomir-21 inhibition of neural crest stem cells differentiating into Schwann cells. The results suggest that miR-21 expression was increased during the differentiation of neural crest stem cells into Schwann cells and miR-21 promoted the differentiation through down-regulating SOX protein expression by binding to the 3′-UTR of SOX2 mRNA. PMID:25206896

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.

Identification of genes differentially expressed in ectomycorrhizal roots during the Pinus pinaster-Laccaria bicolor interaction.

PubMed

Flores-Monterroso, Aranzazu; Canales, Javier; de la Torre, Fernando; Ávila, Concepción; Cánovas, Francisco M

2013-06-01

Ectomycorrhizal associations are of major ecological importance in temperate and boreal forests. The development of a functional ectomycorrhiza requires many genetic and biochemical changes. In this study, suppressive subtraction hybridization was used to identify differentially expressed genes in the roots of maritime pine (Pinus pinaster Aiton) inoculated with Laccaria bicolor, a mycorrhizal fungus. A total number of 200 unigenes were identified as being differentially regulated in maritime pine roots during the development of mycorrhiza. These unigenes were classified into 10 categories according to the function of their homologues in the GenBank database. Approximately, 40 % of the differentially expressed transcripts were genes that coded for unknown proteins in the databases or that had no homology to known genes. A group of these differentially expressed genes was selected to validate the results using quantitative real-time PCR. The transcript levels of the representative genes were compared between the non-inoculated and inoculated plants at 1, 5, 15 and 30 days after inoculation. The observed expression patterns indicate (1) changes in the composition of the wall cell, (2) tight regulation of defence genes during the development of mycorrhiza and (3) changes in carbon and nitrogen metabolism. Ammonium excess or deficiency dramatically affected the stability of ectomycorrhiza and altered gene expression in maritime pine roots.

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.

Smad 1/5 and Smad 4 Expression Are Important for Osteoclast Differentiation

PubMed Central

Tasca, Amy; Stemig, Melissa; Broege, Aaron; Huang, Brandon; Davydova, Julia; Zwijsen, An; Umans, Lieve; Jensen, Eric D.; Gopalakrishnan, Raj; Mansky, Kim C.

2015-01-01

To investigate the necessity of the canonical BMP pathway during osteoclast differentiation, we created osteoclasts with a conditional gene deletion for Smad1 and Smad5 (SMAD1/5), or Smad4 using adenovirus expressing CRE recombinase (Ad-CRE). Reduction of either Smad4 or Smad1/5 expression resulted in fewer and smaller multinuclear cells compared to control cells. We also detected changes in osteoclast enriched genes, demonstrated by decreased Dc-stamp and cathepsin K expression in both Smad4 and Smad1/5 Ad-CRE osteoclasts, and changes in c-fos and Nfatc1 expression in only Smad4 Ad-CRE cells. Lastly we also detected a significant decrease in resorption pits and area resorbed in both the Smad4 and Smad1/5 Ad-CRE osteoclasts. Because we inhibited osteoclast differentiation with loss of either Smad4 or Smad1/5 expression, we assessed whether BMPs affected osteoclast activity in addition to BMP’s effects on differentiation. Therefore, we treated mature osteoclasts with BMP2 or with dorsomorphin, a chemical inhibitor that selectively suppresses canonical BMP signaling. We demonstrated that BMP2 stimulated resorption in mature osteoclasts whereas treatment with dorsomorphin blocks osteoclast resorption. These results indicate that the BMP canonical signaling pathway is important for osteoclast differentiation and activity. PMID:25711193

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

Global identification of microRNAs associated with chlorantraniliprole resistance in diamondback moth Plutella xylostella (L.)

PubMed Central

Zhu, Bin; Li, Xiuxia; Liu, Ying; Gao, Xiwu; Liang, Pei

2017-01-01

The diamondback moth (DBM), Plutella xylostella (L.), is one of the most serious cruciferous pests and has developed high resistance to most insecticides, including chlorantraniliprole. Previous studies have reported several protein-coding genes that involved in chlorantraniliprole resistance, but research on resistance mechanisms at the post-transcription level is still limited. In this study, a global screen of microRNAs (miRNAs) associated with chlorantraniliprole resistance in P. xylostella was performed. The small RNA libraries for a susceptible (CHS) and two chlorantraniliprole resistant strains (CHR, ZZ) were constructed and sequenced, and a total of 199 known and 30 novel miRNAs were identified. Among them, 23 miRNAs were differentially expressed between CHR and CHS, and 90 miRNAs were differentially expressed between ZZ and CHS, of which 11 differentially expressed miRNAs were identified in both CHR and ZZ. Using miRanda and RNAhybrid, a total of 1,411 target mRNAs from 102 differentially expressed miRNAs were predicted, including mRNAs in several groups of detoxification enzymes. The expression of several differentially expressed miRNAs and their potential targets was validated by qRT-PCR. The results may provide important clues for further study of the mechanisms of miRNA-mediated chlorantraniliprole resistance in DBM and other target insects. PMID:28098189

Induced miR-99a expression represses Mtor cooperatively with miR-150 to promote regulatory T-cell differentiation

PubMed Central

Warth, Sebastian C; Hoefig, Kai P; Hiekel, Anian; Schallenberg, Sonja; Jovanovic, Ksenija; Klein, Ludger; Kretschmer, Karsten; Ansel, K Mark; Heissmeyer, Vigo

2015-01-01

Peripheral induction of regulatory T (Treg) cells provides essential protection from inappropriate immune responses. CD4+ T cells that lack endogenous miRNAs are impaired to differentiate into Treg cells, but the relevant miRNAs are unknown. We performed an overexpression screen with T-cell-expressed miRNAs in naive mouse CD4+ T cells undergoing Treg differentiation. Among 130 candidates, the screen identified 29 miRNAs with a negative and 10 miRNAs with a positive effect. Testing reciprocal Th17 differentiation revealed specific functions for miR-100, miR-99a and miR-10b, since all of these promoted the Treg and inhibited the Th17 program without impacting on viability, proliferation and activation. miR-99a cooperated with miR-150 to repress the expression of the Th17-promoting factor mTOR. The comparably low expression of miR-99a was strongly increased by the Treg cell inducer “retinoic acid”, and the abundantly expressed miR-150 could only repress Mtor in the presence of miR-99a. Our data suggest that induction of Treg cell differentiation is regulated by a miRNA network, which involves cooperation of constitutively expressed as well as inducible miRNAs. PMID:25712478

Single-Cell RNA-Sequencing: Assessment of Differential Expression Analysis Methods.

PubMed

Dal Molin, Alessandra; Baruzzo, Giacomo; Di Camillo, Barbara

2017-01-01

The sequencing of the transcriptomes of single-cells, or single-cell RNA-sequencing, has now become the dominant technology for the identification of novel cell types and for the study of stochastic gene expression. In recent years, various tools for analyzing single-cell RNA-sequencing data have been proposed, many of them with the purpose of performing differentially expression analysis. In this work, we compare four different tools for single-cell RNA-sequencing differential expression, together with two popular methods originally developed for the analysis of bulk RNA-sequencing data, but largely applied to single-cell data. We discuss results obtained on two real and one synthetic dataset, along with considerations about the perspectives of single-cell differential expression analysis. In particular, we explore the methods performance in four different scenarios, mimicking different unimodal or bimodal distributions of the data, as characteristic of single-cell transcriptomics. We observed marked differences between the selected methods in terms of precision and recall, the number of detected differentially expressed genes and the overall performance. Globally, the results obtained in our study suggest that is difficult to identify a best performing tool and that efforts are needed to improve the methodologies for single-cell RNA-sequencing data analysis and gain better accuracy of results.

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

Global gene expression analyses of hematopoietic stem cell-like cell lines with inducible Lhx2 expression

PubMed Central

Richter, Karin; Wirta, Valtteri; Dahl, Lina; Bruce, Sara; Lundeberg, Joakim; Carlsson, Leif; Williams, Cecilia

2006-01-01

Background Expression of the LIM-homeobox gene Lhx2 in murine hematopoietic cells allows for the generation of hematopoietic stem cell (HSC)-like cell lines. To address the molecular basis of Lhx2 function, we generated HSC-like cell lines where Lhx2 expression is regulated by a tet-on system and hence dependent on the presence of doxycyclin (dox). These cell lines efficiently down-regulate Lhx2 expression upon dox withdrawal leading to a rapid differentiation into various myeloid cell types. Results Global gene expression of these cell lines cultured in dox was compared to different time points after dox withdrawal using microarray technology. We identified 267 differentially expressed genes. The majority of the genes overlapping with HSC-specific databases were those down-regulated after turning off Lhx2 expression and a majority of the genes overlapping with those defined as late progenitor-specific genes were the up-regulated genes, suggesting that these cell lines represent a relevant model system for normal HSCs also at the level of global gene expression. Moreover, in situ hybridisations of several genes down-regulated after dox withdrawal showed overlapping expression patterns with Lhx2 in various tissues during embryonic development. Conclusion Global gene expression analysis of HSC-like cell lines with inducible Lhx2 expression has identified genes putatively linked to self-renewal / differentiation of HSCs, and function of Lhx2 in organ development and stem / progenitor cells of non-hematopoietic origin. PMID:16600034

Transforming growth factor-β-induced gene product-h3 inhibits odontoblastic differentiation of dental pulp cells.

PubMed

Serita, Suguru; Tomokiyo, Atsushi; Hasegawa, Daigaku; Hamano, Sayuri; Sugii, Hideki; Yoshida, Shinichiro; Mizumachi, Hiroyuki; Mitarai, Hiromi; Monnouchi, Satoshi; Wada, Naohisa; Maeda, Hidefumi

2017-06-01

The aim of this study was to investigate transforming growth factor-β-induced gene product-h3 (βig-h3) expression in dental pulp tissue and its effects on odontoblastic differentiation of dental pulp cells (DPCs). A rat direct pulp capping model was prepared using perforated rat upper first molars capped with mineral trioxide aggregate cement. Human DPCs (HDPCs) were isolated from extracted teeth. βig-h3 expression in rat dental pulp tissue and HDPCs was assessed by immunostaining. Mineralization of HDPCs was assessed by Alizarin red-S staining. Odontoblast-related gene expression in HDPCs was analyzed by quantitative RT-PCR. Expression of βig-h3 was detected in rat dental pulp tissue, and attenuated by direct pulp capping, while expression of interleukin-1β and tumor necrosis factor-α was increased in exposed pulp tissue. βig-h3 expression was also detected in HDPCs, with reduced expression during odontoblastic differentiation. The above cytokines reduced βig-h3 expression in HDPCs, and promoted their mineralization. Recombinant βig-h3 inhibited the expression of odontoblast-related genes and mineralization of HDPCs, while knockdown of βig-h3 gene expression promoted the expression of odontoblast-related genes in HDPCs. The present findings suggest that βig-h3 in DPCs may be involved in reparative dentin formation and that its expression is likely to negatively regulate this process. Copyright © 2017 Elsevier Ltd. All rights reserved.

[Role of CD2-associated protein in podocyte differentiation.].

PubMed

Jiang, Hua-Jun; Chang, Ying; Zhu, Zhong-Hua; Liu, Jian-She; Deng, An-Guo; Zhang, Chun

2008-02-25

To study the cellular changes and the potential role of CD2-associated protein (CD2AP) in podocyte differentiation, conditionally immortalized murine podocyte cell line was cultured in RPMI 1640 medium under permissive condition at 33 °C. After transfection with CD2AP small interfering RNA (siRNA) the cells were shifted to non-permissive condition at 37 °C. Simultaneously, untransfected cells were taken as differentiation control. The podocyte proliferation rate was determined by MTT method. The expressions of CD2AP, WT1, synaptopodin and nephrin mRNAs were examined by RT-PCR. CD2AP, WT1 and nephrin protein expressions were examined by Western blot. The distribution of CD2AP, nephrin, F-actin and tubulin in differentiated and undifferentiated podocytes was detected by laser scanning confocal microscopy. The results showed: (1) CD2AP, WT1 and nephrin were stably expressed in differentiated and undifferentiated podocytes while synaptopodin was only expressed in differentiated podocytes. (2) CD2AP and nephrin mRNA and protein expressions were up-regulated during podocyte differentiation (P<0.05). (3) CD2AP and tubulin were distributed in the cytoplasm and perinulcear region in undifferentiated podocytes, and F-actin was predominantly localized to a cortical belt and paralleled to the cell axis. Under differentiation condition, CD2AP distribution profile was presented as peripheral accumulation, tubulin took on fascicular style and F-actin extended into foot processes in podocytes. CD2AP colocalized with nephrin and F-actin in undifferentiated podocytes. (4) After transfection with CD2AP siRNA, the expression of CD2AP was partially inhibited and cell growth was arrested; Synaptopodin, the differentiation podocyte marker, was apparently down-regulated; The differentiation of podocytes was delayed. The results demonstrate that podocyte differentiation is accompanied by cytoskeleton rearrangement and cell morphology change. CD2AP might play an essential role in podocyte differentiation.

All-Trans-Retinoic Acid Stimulates Overexpression of Tumor Protein D52 (TPD52, Isoform 3) and Neuronal Differentiation of IMR-32 Cells.

PubMed

Kotapalli, Sudha Sravanti; Dasari, Chandrashekhar; Duscharla, Divya; Kami Reddy, Karthik Reddy; Kasula, Manjula; Ummanni, Ramesh

2017-12-01

Tumor protein D52 (TPD52), a proto-oncogene is overexpressed in a variety of epithelial carcinomas and plays an important role in cell proliferation, migration, and cell death. In the present study we found that the treatment of IMR-32 neuroblastoma (NB) cells with retinoic acid (RA) stimulates an increase in expression of TPD52. TPD52 expression is detectable after 72 h, can be maintained till differentiation of NB cells suggesting that TPD52 is involved in differentiation. Here, we demonstrate that TPD52 is essential for RA to promote differentiation of NB cells. Our results show that exogenous expression of EGFP-TPD52 in IMR-32 cells resulted cell differentiation even without RA. RA by itself and with overexpression of TPD52 can increase the ability of NB cells differentiation. Interestingly, transfection of IMR-32 cells with a specific small hairpin RNA for efficient knockdown of TPD52 attenuated RA induced NB cells differentiation. Transcriptional and translational level expression of neurotropic (BDNF, NGF, Nestin) and differentiation (β III tubulin, NSE, TH) factors in NB cells with altered TPD52 expression and/or RA treatment confirmed essential function of TPD52 in cellular differentiation. Furthermore, we show that TPD52 protects cells from apoptosis and arrest cell proliferation by varying expression of p27Kip1, activation of Akt and ERK1/2 thus promoting cell differentiation. Additionally, inhibition of STAT3 activation by its specific inhibitor arrested NB cells differentiation by EGFP-TPD52 overexpression with or without RA. Taken together, our data reveal that TPD52 act through activation of JAK/STAT signaling pathway to undertake NB cells differentiation induced by RA. J. Cell. Biochem. 118: 4358-4369, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Differential Effects of Language Attrition in the Domains of Verb Placement and Object Expression

ERIC Educational Resources Information Center

Flores, Cristina

2012-01-01

This study investigates the differential effects of language attrition in two diverse linguistic domains: verb placement and object expression. Linguistic phenomena at the syntax--discourse interface, such as object expression, have been shown to be more vulnerable to attrition than narrow syntax properties, such as verb placement. This study aims…

Adult human pancreas-derived cells expressing stage-specific embryonic antigen 4 differentiate into Sox9-expressing and Ngn3-expressing pancreatic ducts in vivo.

PubMed

Lee, Song; Lee, Chan Mi; Kim, Song Cheol

2016-11-11

Tissue-specific stem/progenitor cells are found in various adult tissues and may have the capacity for lineage-specific differentiation, facilitating applications in autologous transplantation. Stage-specific embryonic antigen 4 (SSEA-4), an early embryonic glycolipid antigen, is expressed in cells derived from adult human pancreas exocrine tissue. Here, we examined the characteristics and lineage-specific differentiation capacity of SSEA-4 + cells. Human adult partial pancreas tissues were obtained from different donors and cultured in vitro. SSEA-4 + and CA19-9 + cells were isolated from adult human pancreas exocrine cells using magnetic-activated cell sorting, and gene expression was validated by quantitative polymerase chain reaction. To confirm in-vivo differentiation, SSEA-4 + and CA19-9 + cells were transplanted into the dorsal subcutaneous region of mice. Finally, morphological features of differentiated areas were confirmed by immunostaining and morphometric analysis. SSEA-4-expressing cells were detected in isolated pancreas exocrine cells from adult humans. These SSEA-4 + cells exhibited coexpression of CA19-9, a marker of pancreatic duct cells, but not amylase expression, as shown by immunostaining and flow cytometry. SSEA-4 + cells exhibited higher relative expression of Oct4, Nanog, Klf4, Sox2, and c-Myc mRNAs than CA19-9 + cells. Pancreatic intralobular ducts (PIDs) were generated from SSEA-4 + or CA19-9 + cells in vivo at 5 weeks after transplantation. However, newly formed PIDs from CA19-9 + cells were less abundant and showed an incomplete PID morphology. In contrast, newly formed PIDs from SSEA-4 + cells were abundant in the transplanted area and showed a crowded morphology, typical of PIDs. Sox9 and Ngn3, key transcription factors associated with pancreatic development and regeneration, were expressed in PIDs from SSEA-4 + cells. SSEA-4-expressing cells in the adult human pancreas may have the potential for regeneration of the pancreas and may be used as a source of stem/progenitor cells for pancreatic cell lineage-specific differentiation.

An in vitro ES cell imprinting model shows that imprinted expression of the Igf2r gene arises from an allele-specific expression bias

PubMed Central

Latos, Paulina A.; Stricker, Stefan H.; Steenpass, Laura; Pauler, Florian M.; Huang, Ru; Senergin, Basak H.; Regha, Kakkad; Koerner, Martha V.; Warczok, Katarzyna E.; Unger, Christine; Barlow, Denise P.

2010-01-01

Genomic imprinting is an epigenetic process that results in parental-specific gene expression. Advances in understanding the mechanism that regulates imprinted gene expression in mammals have largely depended on generating targeted manipulations in embryonic stem (ES) cells that are analysed in vivo in mice. However, genomic imprinting consists of distinct developmental steps, some of which occur in post-implantation embryos, indicating that they could be studied in vitro in ES cells. The mouse Igf2r gene shows imprinted expression only in post-implantation stages, when repression of the paternal allele has been shown to require cis-expression of the Airn non-coding (nc) RNA and to correlate with gain of DNA methylation and repressive histone modifications. Here we follow the gain of imprinted expression of Igf2r during in vitro ES cell differentiation and show that it coincides with the onset of paternal-specific expression of the Airn ncRNA. Notably, although Airn ncRNA expression leads, as predicted, to gain of repressive epigenetic marks on the paternal Igf2r promoter, we unexpectedly find that the paternal Igf2r promoter is expressed at similar low levels throughout ES cell differentiation. Our results further show that the maternal and paternal Igf2r promoters are expressed equally in undifferentiated ES cells, but during differentiation expression of the maternal Igf2r promoter increases up to 10-fold, while expression from the paternal Igf2r promoter remains constant. This indicates, contrary to expectation, that the Airn ncRNA induces imprinted Igf2r expression not by silencing the paternal Igf2r promoter, but by generating an expression bias between the two parental alleles. PMID:19141673

Expression of myeloid differentiation antigens on normal and malignant myeloid cells.

PubMed Central

Griffin, J D; Ritz, J; Nadler, L M; Schlossman, S F

1981-01-01

A series of monoclonal antibodies have been characterized that define four surface antigens (MY3, MY4, MY7, and MY8) of human myeloid cells. They were derived from a fusion of the NS-1 plasmacytoma cell line with splenocytes from a mouse immunized with human acute myelomonocytic leukemia cells. MY3 and MY4 are expressed by normal monocytes and by greater than 90% of patients with acute monocytic leukemia or acute myelomonocytic leukemia, but are detected much less often on other types of myeloid leukemia. MY7 is expressed by granulocytes, monocytes, and 5% of normal bone marrow cells. 79% of all acute myeloblastic leukemia (AML) patients tested (72 patients) express MY7 without preferential expression by any AML subtype. MY8 is expressed by normal monocytes, granulocytes, all peroxidase-positive bone marrow cells, and 50% of AML patients. MY3, MY4, and MY8 define myeloid differentiation antigens in that they are not detected on myeloid precursor cells and appear at discrete stages of differentiation. These antigens are not expressed by lymphocytes, erythrocytes, platelets, or lymphoid malignancies. The monoclonal antisera defining these antigens have been used to study differentiation of normal myeloid cells and malignant cell lines. Images PMID:6945311

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.

Differential Expression of Non-Coding RNAs and Continuous Evolution of the X Chromosome in Testicular Transcriptome of Two Mouse Species

PubMed Central

Homolka, David; Ivanek, Robert; Forejt, Jiri; Jansa, Petr

2011-01-01

Background Tight regulation of testicular gene expression is a prerequisite for male reproductive success, while differentiation of gene activity in spermatogenesis is important during speciation. Thus, comparison of testicular transcriptomes between closely related species can reveal unique regulatory patterns and shed light on evolutionary constraints separating the species. Methodology/Principal Findings Here, we compared testicular transcriptomes of two closely related mouse species, Mus musculus and Mus spretus, which diverged more than one million years ago. We analyzed testicular expression using tiling arrays overlapping Chromosomes 2, X, Y and mitochondrial genome. An excess of differentially regulated non-coding RNAs was found on Chromosome 2 including the intronic antisense RNAs, intergenic RNAs and premature forms of Piwi-interacting RNAs (piRNAs). Moreover, striking difference was found in the expression of X-linked G6pdx gene, the parental gene of the autosomal retrogene G6pd2. Conclusions/Significance The prevalence of non-coding RNAs among differentially expressed transcripts indicates their role in species-specific regulation of spermatogenesis. The postmeiotic expression of G6pdx in Mus spretus points towards the continuous evolution of X-chromosome silencing and provides an example of expression change accompanying the out-of-the X-chromosomal retroposition. PMID:21347268

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.

Changes in rat spinal cord gene expression after inflammatory hyperalgesia of the joint and manual therapy.

PubMed

Ruhlen, Rachel L; Singh, Vineet K; Pazdernik, Vanessa K; Towns, Lex C; Snider, Eric J; Sargentini, Neil J; Degenhardt, Brian F

2014-10-01

Mobilization of a joint affects local tissue directly but may also have other effects that are mediated through the central nervous system. To identify differential gene expression in the spinal cords of rats with or without inflammatory joint injury after manual therapy or no treatment. Rats were randomly assigned to 1 of 4 treatment groups: no injury and no touch (NI/NT), injury and no touch (I/NT), no injury and manual therapy (NI/MT), and injury and manual therapy (I/MT). We induced acute inflammatory joint injury in the rats by injecting carrageenan into an ankle. Rats in the no-injury groups did not receive carrageenan injection. One day after injury, rats received manual therapy to the knee of the injured limb. Rats in the no-touch groups were anesthetized without receiving manual therapy. Spinal cords were harvested 30 minutes after therapy or no touch, and spinal cord gene expression was analyzed by microarray for 3 comparisons: NI/NT vs I/NT, I/MT vs I/NT, and NI/NT vs NI/MT. Three rats were assigned to each group. Of 38,875 expressed sequence tags, 755 were differentially expressed in the NI/NT vs I/NT comparison. For the other comparisons, no expressed sequence tags were differentially expressed. Cluster analysis revealed that the differentially expressed sequence tags were over-represented in several categories, including ion homeostasis (enrichment score, 2.29), transmembrane (enrichment score, 1.55), and disulfide bond (enrichment score, 2.04). An inflammatory injury to the ankle of rats caused differential expression of genes in the spinal cord. Consistent with other studies, genes involved in ion transport were among those affected. However, manual therapy to the knees of injured limbs or to rats without injury did not alter gene expression in the spinal cord. Thus, evidence for central nervous system mediation of manual therapy was not observed. © 2014 The American Osteopathic Association.

Sex-based differences in gene expression in hippocampus following postnatal lead exposure

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

Schneider, J.S., E-mail: jay.schneider@jefferson.edu; Anderson, D.W.; Sonnenahalli, H.

The influence of sex as an effect modifier of childhood lead poisoning has received little systematic attention. Considering the paucity of information available concerning the interactive effects of lead and sex on the brain, the current study examined the interactive effects of lead and sex on gene expression patterns in the hippocampus, a structure involved in learning and memory. Male or female rats were fed either 1500 ppm lead-containing chow or control chow for 30 days beginning at weaning.Blood lead levels were 26.7 {+-} 2.1 {mu}g/dl and 27.1 {+-} 1.7 {mu}g/dl for females and males, respectively. The expression of 175more » unique genes was differentially regulated between control male and female rats. A total of 167 unique genes were differentially expressed in response to lead in either males or females. Lead exposure had a significant effect without a significant difference between male and female responses in 77 of these genes. In another set of 71 genes, there were significant differences in male vs. female response. A third set of 30 genes was differentially expressed in opposite directions in males vs. females, with the majority of genes expressed at a lower level in females than in males. Highly differentially expressed genes in males and females following lead exposure were associated with diverse biological pathways and functions. These results show that a brief exposure to lead produced significant changes in expression of a variety of genes in the hippocampus and that the response of the brain to a given lead exposure may vary depending on sex. - Highlights: > Postnatal lead exposure has a significant effect on hippocampal gene expression patterns. > At least one set of genes was affected in opposite directions in males and females. > Differentially expressed genes were associated with diverse biological pathways.« less

Explicit expressions for meromorphic solutions of autonomous nonlinear ordinary differential equations

NASA Astrophysics Data System (ADS)

Demina, Maria V.; Kudryashov, Nikolay A.

2011-03-01

Meromorphic solutions of autonomous nonlinear ordinary differential equations are studied. An algorithm for constructing meromorphic solutions in explicit form is presented. General expressions for meromorphic solutions (including rational, periodic, elliptic) are found for a wide class of autonomous nonlinear ordinary differential equations.

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.

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.

Soluble soy protein peptic hydrolysate stimulates adipocyte differentiation in 3T3-L1 cells.

PubMed

Goto, Tsuyoshi; Mori, Ayaka; Nagaoka, Satoshi

2013-08-01

The molecular mechanisms underlying the potential health benefit effects of soybean proteins on obesity-associated metabolic disorders have not been fully clarified. In this study, we investigated the effects of soluble soybean protein peptic hydrolysate (SPH) on adipocyte differentiation by using 3T3-L1 murine preadipocytes. The addition of SPH increased lipid accumulation during adipocyte differentiation. SPH increased the mRNA expression levels of an adipogenic marker gene and decreased that of a preadipocyte marker gene, suggesting that SPH promotes adipocyte differentiation. SPH induced antidiabetic and antiatherogenic adiponectin mRNA expression and secretion. Moreover, SPH increased the mRNA expression levels of insulin-responsive glucose transporter 4 and insulin-stimulated glucose uptake. The expression levels of peroxisome proliferator-activated receptor γ (PPARγ), a key regulator of adipocyte differentiation, during adipocyte differentiation were up-regulated in 3T3-L1 cells treated with SPH, and lipid accumulation during adipocyte differentiation induced by SPH was inhibited in the presence of a PPARγ antagonist. However, SPH did not exhibit PPARγ ligand activity. These findings indicate that SPH stimulates adipocyte differentiation, at least in part, via the up-regulation of PPARγ expression levels. These effects of SPH might be important for the health benefit effects of soybean proteins on obesity-associated metabolic disorders. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Transforming growth factor (TGF)beta, fibroblast growth factor (FGF) and retinoid signalling pathways promote pancreatic exocrine gene expression in mouse embryonic stem cells.

PubMed Central

Skoudy, Anouchka; Rovira, Meritxell; Savatier, Pierre; Martin, Franz; León-Quinto, Trinidad; Soria, Bernat; Real, Francisco X

2004-01-01

Extracellular signalling cues play a major role in the activation of differentiation programmes. Mouse embryonic stem (ES) cells are pluripotent and can differentiate into a wide variety of specialized cells. Recently, protocols designed to induce endocrine pancreatic differentiation in vitro have been designed but little information is currently available concerning the potential of ES cells to differentiate into acinar pancreatic cells. By using conditioned media of cultured foetal pancreatic rudiments, we demonstrate that ES cells can respond in vitro to signalling pathways involved in exocrine development and differentiation. In particular, modulation of the hedgehog, transforming growth factor beta, retinoid, and fibroblast growth factor pathways in ES cell-derived embryoid bodies (EB) resulted in increased levels of transcripts encoding pancreatic transcription factors and cytodifferentiation markers, as demonstrated by RT-PCR. In EB undergoing spontaneous differentiation, expression of the majority of the acinar genes (i.e. amylase, carboxypeptidase A and elastase) was induced after the expression of endocrine genes, as occurs in vivo during development. These data indicate that ES cells can undergo exocrine pancreatic differentiation with a kinetic pattern of expression reminiscent of pancreas development in vivo and that ES cells can be coaxed to express an acinar phenotype by activation of signalling pathways known to play a role in pancreatic development and differentiation. PMID:14733613

Ret function in muscle stem cells points to tyrosine kinase inhibitor therapy for facioscapulohumeral muscular dystrophy.

PubMed

Moyle, Louise A; Blanc, Eric; Jaka, Oihane; Prueller, Johanna; Banerji, Christopher Rs; Tedesco, Francesco Saverio; Harridge, Stephen Dr; Knight, Robert D; Zammit, Peter S

2016-11-14

Facioscapulohumeral muscular dystrophy (FSHD) involves sporadic expression of DUX4, which inhibits myogenesis and is pro-apoptotic. To identify target genes, we over-expressed DUX4 in myoblasts and found that the receptor tyrosine kinase Ret was significantly up-regulated, suggesting a role in FSHD. RET is dynamically expressed during myogenic progression in mouse and human myoblasts. Constitutive expression of either RET9 or RET51 increased myoblast proliferation, whereas siRNA-mediated knockdown of Ret induced myogenic differentiation. Suppressing RET activity using Sunitinib, a clinically-approved tyrosine kinase inhibitor, rescued differentiation in both DUX4-expressing murine myoblasts and in FSHD patient-derived myoblasts. Importantly, Sunitinib also increased engraftment and differentiation of FSHD myoblasts in regenerating mouse muscle. Thus, DUX4-mediated activation of Ret prevents myogenic differentiation and could contribute to FSHD pathology by preventing satellite cell-mediated repair. Rescue of DUX4-induced pathology by Sunitinib highlights the therapeutic potential of tyrosine kinase inhibitors for treatment of FSHD.

From reads to genes to pathways: differential expression analysis of RNA-Seq experiments using Rsubread and the edgeR quasi-likelihood pipeline.

PubMed

Chen, Yunshun; Lun, Aaron T L; Smyth, Gordon K

2016-01-01

In recent years, RNA sequencing (RNA-seq) has become a very widely used technology for profiling gene expression. One of the most common aims of RNA-seq profiling is to identify genes or molecular pathways that are differentially expressed (DE) between two or more biological conditions. This article demonstrates a computational workflow for the detection of DE genes and pathways from RNA-seq data by providing a complete analysis of an RNA-seq experiment profiling epithelial cell subsets in the mouse mammary gland. The workflow uses R software packages from the open-source Bioconductor project and covers all steps of the analysis pipeline, including alignment of read sequences, data exploration, differential expression analysis, visualization and pathway analysis. Read alignment and count quantification is conducted using the Rsubread package and the statistical analyses are performed using the edgeR package. The differential expression analysis uses the quasi-likelihood functionality of edgeR.

EPConDB: a web resource for gene expression related to pancreatic development, beta-cell function and diabetes.

PubMed

Mazzarelli, Joan M; Brestelli, John; Gorski, Regina K; Liu, Junmin; Manduchi, Elisabetta; Pinney, Deborah F; Schug, Jonathan; White, Peter; Kaestner, Klaus H; Stoeckert, Christian J

2007-01-01

EPConDB (http://www.cbil.upenn.edu/EPConDB) is a public web site that supports research in diabetes, pancreatic development and beta-cell function by providing information about genes expressed in cells of the pancreas. EPConDB displays expression profiles for individual genes and information about transcripts, promoter elements and transcription factor binding sites. Gene expression results are obtained from studies examining tissue expression, pancreatic development and growth, differentiation of insulin-producing cells, islet or beta-cell injury, and genetic models of impaired beta-cell function. The expression datasets are derived using different microarray platforms, including the BCBC PancChips and Affymetrix gene expression arrays. Other datasets include semi-quantitative RT-PCR and MPSS expression studies. For selected microarray studies, lists of differentially expressed genes, derived from PaGE analysis, are displayed on the site. EPConDB provides database queries and tools to examine the relationship between a gene, its transcriptional regulation, protein function and expression in pancreatic tissues.

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.

Postmitotic Expression of SOD1G93A Gene Affects the Identity of Myogenic Cells and Inhibits Myoblasts Differentiation

PubMed Central

Martini, Martina; Dobrowolny, Gabriella; Aucello, Michela; Musarò, Antonio

2015-01-01

To determine the role of mutant SOD1 gene (SOD1G93A) on muscle cell differentiation, we derived C2C12 muscle cell lines carrying a stably transfected SOD1G93A gene under the control of a myosin light chain (MLC) promoter-enhancer cassette. Expression of MLC/SOD1G93A in C2C12 cells resulted in dramatic inhibition of myoblast differentiation. Transfected SOD1G93A gene expression in postmitotic skeletal myocytes downregulated the expression of relevant markers of committed and differentiated myoblasts such as MyoD, Myogenin, MRF4, and the muscle specific miRNA expression. The inhibitory effects of SOD1G93A gene on myogenic program perturbed Akt/p70 and MAPK signaling pathways which promote differentiation cascade. Of note, the inhibition of the myogenic program, by transfected SOD1G93A gene expression, impinged also the identity of myogenic cells. Expression of MLC/SOD1G93A in C2C12 myogenic cells promoted a fibro-adipogenic progenitors (FAPs) phenotype, upregulating HDAC4 protein and preventing the myogenic commitment complex BAF60C-SWI/SNF. We thus identified potential molecular mediators of the inhibitory effects of SOD1G93A on myogenic program and disclosed potential signaling, activated by SOD1G93A, that affect the identity of the myogenic cell population. PMID:26491230

Postmitotic Expression of SOD1(G93A) Gene Affects the Identity of Myogenic Cells and Inhibits Myoblasts Differentiation.

PubMed

Martini, Martina; Dobrowolny, Gabriella; Aucello, Michela; Musarò, Antonio

2015-01-01

To determine the role of mutant SOD1 gene (SOD1(G93A)) on muscle cell differentiation, we derived C2C12 muscle cell lines carrying a stably transfected SOD1(G93A) gene under the control of a myosin light chain (MLC) promoter-enhancer cassette. Expression of MLC/SOD1(G93A) in C2C12 cells resulted in dramatic inhibition of myoblast differentiation. Transfected SOD1(G93A) gene expression in postmitotic skeletal myocytes downregulated the expression of relevant markers of committed and differentiated myoblasts such as MyoD, Myogenin, MRF4, and the muscle specific miRNA expression. The inhibitory effects of SOD1(G93A) gene on myogenic program perturbed Akt/p70 and MAPK signaling pathways which promote differentiation cascade. Of note, the inhibition of the myogenic program, by transfected SOD1(G93A) gene expression, impinged also the identity of myogenic cells. Expression of MLC/SOD1(G93A) in C2C12 myogenic cells promoted a fibro-adipogenic progenitors (FAPs) phenotype, upregulating HDAC4 protein and preventing the myogenic commitment complex BAF60C-SWI/SNF. We thus identified potential molecular mediators of the inhibitory effects of SOD1(G93A) on myogenic program and disclosed potential signaling, activated by SOD1(G93A), that affect the identity of the myogenic cell population.

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.

Sex hormone-binding globulin b expression in the rainbow trout ovary prior to sex differentiation.

PubMed

Pérez, Claudio; Araneda, Cristian; Estay, Francisco; Díaz, Nelson F; Vizziano-Cantonnet, Denise

2018-04-01

Salmonids have two sex hormone-binding globulin (Shbg) paralogs. Shbga is mainly expressed in the liver, while Shbgb is secreted by the granulosa cells of the rainbow trout ovary. Coexpression of shbgb and the gonadal aromatase cyp19a1a mRNAs been observed in granulosa cells, suggesting a physiological coordination between Shbgb expression and estrogen synthesis. As estrogens are essential for female sex determination in the fish ovary, we propose that Shbgb participates in early ovarian differentiation, either by binding with estrogen or through another mechanism that remains to be discovered. To elucidate this potential role, monosex populations of female trout were studied during the molecular ovarian differentiation period (28-56 dpf). shbgb mRNA expression was measured using qPCR and compared with expression of genes for other ovarian markers (cyp19a1a, foxl2, follistatin, and estrogen receptors). shbgb transcript expression was detected during the final stages of embryonic development (21-26 dpf) and during molecular ovarian differentiation (32-52 dpf) after hatching (which occurred at 31 dpf). In situ hybridization localized shbgb transcription to the undifferentiated ovary at 42 dpf, and shbgb and cyp19a1a mRNA showed similar expression patterns. These results suggest that Shbgb is involved in early ovarian differentiation, supporting an important role for the salmonid shbgb gene in sex determination. Copyright © 2017 Elsevier Inc. All rights reserved.

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

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.

The differential expression of alternatively polyadenylated transcripts is a common stress-induced response mechanism that modulates mammalian mRNA expression in a quantitative and qualitative fashion.

PubMed

Hollerer, Ina; Curk, Tomaz; Haase, Bettina; Benes, Vladimir; Hauer, Christian; Neu-Yilik, Gabriele; Bhuvanagiri, Madhuri; Hentze, Matthias W; Kulozik, Andreas E

2016-09-01

Stress adaptation plays a pivotal role in biological processes and requires tight regulation of gene expression. In this study, we explored the effect of cellular stress on mRNA polyadenylation and investigated the implications of regulated polyadenylation site usage on mammalian gene expression. High-confidence polyadenylation site mapping combined with global pre-mRNA and mRNA expression profiling revealed that stress induces an accumulation of genes with differentially expressed polyadenylated mRNA isoforms in human cells. Specifically, stress provokes a global trend in polyadenylation site usage toward decreased utilization of promoter-proximal poly(A) sites in introns or ORFs and increased utilization of promoter-distal polyadenylation sites in intergenic regions. This extensively affects gene expression beyond regulating mRNA abundance by changing mRNA length and by altering the configuration of open reading frames. Our study highlights the impact of post-transcriptional mechanisms on stress-dependent gene regulation and reveals the differential expression of alternatively polyadenylated transcripts as a common stress-induced mechanism in mammalian cells. © 2016 Hollerer et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

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.

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

Differential expression of extracellular-signal-regulated kinase 5 (ERK5) in normal and degenerated human nucleus pulposus tissues and cells

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

Liang, Weiguo, E-mail: liangweiguo@tom.com; Fang, Dejian; Ye, Dongping

2014-07-11

Highlights: • ERK5 involved in NP cells. • ERK5 involved in NP tissue. • It was important modulator. - Abstract: Extracellular-signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase (MAPK) family and regulates a wide variety of cellular processes such as proliferation, differentiation, necrosis, apoptosis and degeneration. However, the expression of ERK5 and its role in degenerated human nucleus pulposus (NP) is hitherto unknown. In this study, we observed the differential expression of ERK5 in normal and degenerated human nucleus pulposus tissues by using immunohistochemical staining and Western blot. Treatment of NP cells with Pro-inflammatory cytokine, TNF-αmore » decreased ERK5 gene expression as well as NP marker gene expression; including the type II collagen and aggrecan. Suppression of ERK5 gene expression in NP cells by ERK5 siRNA resulted in decreased gene expression of type II collagen and aggrecan. Furthermore, inhibition of ERK5 activation by BIX02188 (5 μM) decreased the gene expression of type II collagen and aggrecan in NP cells. Our results document the expression of ERK5 in degenerated nucleus pulposus tissues, and suggest a potential involvement of ERK5 in human degenerated nucleus pulposus.« less

Immunohistochemical analysis of cytokeratin and human milk fat globulin expression in mucinous carcinoma of the skin.

PubMed

Ohnishi, Takamitsu; Takizawa, Hajime; Watanabe, Shinichi

2002-01-01

Mucinous carcinoma of the skin (MCS) is a rare epithelial tumor which arises primarily in the skin. Metastatic MC from extracutaneous sites, especially breast or colon, mimics MCS and cannot be differentiated from MCS by routine histology alone. Nine cases of MCS were analyzed immunohistochemically using monoclonal antibodies against cytokeratins (CKs) and human milk fat globulin 1 (HMFG) in order to clarify their nature and compare the immunophenotypes with those of other MCs studied in the literature. Expression of simple epithelial CKs in most of the tumor cells of all cases studied and co-expression of simple and stratified epithelial CKs in some tumor cells of two cases were recognized. CK 20 expression could not detected in any tumor cells. Focal HMFG expression in the luminal or outer surface of the nests was observed in three cases. From CKs expression, MCS was speculated to differentiated mainly toward the secretory cells of the sweat glands, and some tumor cells toward the transient portion between the dermal duct and the secretory portion. Focal HMFG expression suggested either a consequence of malignant transformation or apocrine differentiation. No expression of CK 20 in MCS suggests that we may exclude the diagnosis of metastatic colorectal MC which expressed CK 20.

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.

Plasticity for axolotl lens regeneration is associated with age‐related changes in gene expression

PubMed Central

Sousounis, Konstantinos; Athippozhy, Antony T.; Voss, S. Randal

2014-01-01

Abstract Mexican axolotls lose potential for lens regeneration 2 weeks after hatching. We used microarrays to identify differently expressed genes before and after this critical time, using RNA isolated from iris. Over 3700 genes were identified as differentially expressed in response to lentectomy between young (7 days post‐hatching) and old (3 months post‐hatching) axolotl larvae. Strikingly, many of the genes were only expressed in the early or late iris. Genes that were highly expressed in young iris significantly enriched electron transport chain, transcription, metabolism, and cell cycle gene ontologies, all of which are associated with lens regeneration. In contrast, genes associated with cellular differentiation and tissue maturation were uniquely expressed in old iris. Many of these expression differences strongly suggest that young and old iris samples were collected before and after the spleen became developmentally competent to produce and secrete cells with humoral and innate immunity functions. Our study establishes the axolotl as a powerful model to investigate age‐related cellular differentiation and immune system ontogeny within the context of tissue regeneration. PMID:27499863

Transcription Factor ZBED6 Mediates IGF2 Gene Expression by Regulating Promoter Activity and DNA Methylation in Myoblasts

NASA Astrophysics Data System (ADS)

Huang, Yong-Zhen; Zhang, Liang-Zhi; Lai, Xin-Sheng; Li, Ming-Xun; Sun, Yu-Jia; Li, Cong-Jun; Lan, Xian-Yong; Lei, Chu-Zhao; Zhang, Chun-Lei; Zhao, Xin; Chen, Hong

2014-04-01

Zinc finger, BED-type containing 6 (ZBED6) is an important transcription factor in placental mammals, affecting development, cell proliferation and growth. In this study, we found that the expression of the ZBED6 and IGF2 were upregulated during C2C12 differentiation. The IGF2 expression levels were negatively associated with the methylation status in beef cattle (P < 0.05). A luciferase assay for the IGF2 intron 3 and P3 promoter showed that the mutant-type 439 A-SNP-pGL3 in driving reporter gene transcription is significantly higher than that of the wild-type 439 G-SNP-pGL3 construct (P < 0.05). An over-expression assay revealed that ZBED6 regulate IGF2 expression and promote myoblast differentiation. Furthermore, knockdown of ZBED6 led to IGF2 expression change in vitro. Taken together, these results suggest that ZBED6 inhibits IGF2 activity and expression via a G to A transition disrupts the interaction. Thus, we propose that ZBED6 plays a critical role in myogenic differentiation.

Terminal epidermal differentiation is regulated by the interaction of Fra-2/AP-1 with Ezh2 and ERK1/2

PubMed Central

Wurm, Stefanie; Zhang, Jisheng; Guinea-Viniegra, Juan; García, Fernando; Muñoz, Javier; Bakiri, Latifa; Ezhkova, Elena

2015-01-01

Altered epidermal differentiation characterizes numerous skin diseases affecting >25% of the human population. Here we identified Fra-2/AP-1 as a key regulator of terminal epidermal differentiation. Epithelial-restricted, ectopic expression of Fra-2 induced expression of epidermal differentiation genes located within the epidermal differentiation complex (EDC). Moreover, in a papilloma-prone background, a reduced tumor burden was observed due to precocious keratinocyte differentiation by Fra-2 expression. Importantly, loss of Fra-2 in suprabasal keratinocytes is sufficient to cause skin barrier defects due to reduced expression of differentiation genes. Mechanistically, Fra-2 binds and transcriptionally regulates EDC gene promoters, which are co-occupied by the transcriptional repressor Ezh2. Fra-2 remains transcriptionally inactive in nondifferentiated keratinocytes, where it was found monomethylated and dimethylated on Lys104 and interacted with Ezh2. Upon keratinocyte differentiation, Fra-2 is C-terminally phosphorylated on Ser320 and Thr322 by ERK1/2, leading to transcriptional activation. Thus, the induction of epidermal differentiation by Fra-2 is controlled by a dual mechanism involving Ezh2-dependent methylation and activation by ERK1/2-dependent phosphorylation. PMID:25547114

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.

MMP‐2 and MMP‐14 Silencing Inhibits VEGFR2 Cleavage and Induces the Differentiation of Porcine Adipose‐Derived Mesenchymal Stem Cells to Endothelial Cells

PubMed Central

Almalki, Sami G.; Llamas Valle, Yovani

2017-01-01

Abstract The molecular mechanisms that control the ability of adipose‐derived mesenchymal stem cells (AMSCs) to remodel three‐dimensional extracellular matrix barriers during differentiation are not clearly understood. Herein, we studied the expression of matrix metalloproteinases (MMPs) during the differentiation of AMSCs to endothelial cells (ECs) in vitro. MSCs were isolated from porcine abdominal adipose tissue, and characterized by immunopositivity to CD44, CD90, CD105, and immunonegativity to CD14 and CD45. Plasticity of AMSCs was confirmed by multilineage differentiation. The mRNA transcripts for MMPs and Tissue Inhibitor of Metalloproteinases (TIMPs), and protein expression of EC markers were analyzed. The enzyme activity and protein expression were analyzed by gelatin zymography, enzyme‐linked immunosorbent assay (ELISA), and Western blot. The differentiation of AMSCs to ECs was confirmed by mRNA and protein expressions of the endothelial markers. The mRNA transcripts for MMP‐2 and MMP‐14 were significantly increased during the differentiation of MSCs into ECs. Findings revealed an elevated MMP‐14 and MMP‐2 expression, and MMP2 enzyme activity. Silencing of MMP‐2 and MMP‐14 significantly increased the expression of EC markers, formation of capillary tubes, and acetylated‐low‐density lipoprotein uptake, and decreased the cleavage of vascular endothelial growth factor receptor type 2 (VEGFR2). Inhibition of VEGFR2 significantly decreased the expression of EC markers. These novel findings demonstrate that the upregulation of MMP2 and MMP14 has an inhibitory effect on the differentiation of AMSCs to ECs, and silencing these MMPs inhibit the cleavage of VEGFR2 and stimulate the differentiation of AMSCs to ECs. These findings provide a potential mechanism for the regulatory role of MMP‐2 and MMP‐14 in the re‐endothelialization of coronary arteries following intervention. Stem Cells Translational Medicine 2017;6:1385–1398 PMID:28213979

Effects of Leucine Supplementation and Serum Withdrawal on Branched-Chain Amino Acid Pathway Gene and Protein Expression in Mouse Adipocytes

PubMed Central

Vivar, Juan C.; Knight, Megan S.; Pointer, Mildred A.; Gwathmey, Judith K.; Ghosh, Sujoy

2014-01-01

The essential branched-chain amino acids (BCAA), leucine, valine and isoleucine, are traditionally associated with skeletal muscle growth and maintenance, energy production, and generation of neurotransmitter and gluconeogenic precursors. Recent evidence from human and animal model studies has established an additional link between BCAA levels and obesity. However, details of the mechanism of regulation of BCAA metabolism during adipogenesis are largely unknown. We interrogated whether the expression of genes and proteins involved in BCAA metabolism are sensitive to the adipocyte differentiation process, and responsive to nutrient stress from starvation or BCAA excess. Murine 3T3-L1 preadipocytes were differentiated to adipocytes under control conditions and under conditions of L-leucine supplementation or serum withdrawal. RNA and proteins were isolated at days 0, 4 and 10 of differentiation to represent pre-differentiation, early differentiation and late differentiation stages. Expression of 16 BCAA metabolism genes was quantified by quantitative real-time PCR. Expression of the protein levels of branched-chain amino acid transaminase 2 (Bcat2) and branched-chain alpha keto acid dehydrogenase (Bckdha) was quantified by immunoblotting. Under control conditions, all genes displayed induction of gene expression during early adipogenesis (Day 4) compared to Day 0. Leucine supplementation resulted in an induction of Bcat2 and Bckdha genes during early and late differentiation. Western blot analysis demonstrated condition-specific concordance between gene and protein expression. Serum withdrawal resulted in undetectable Bcat2 and Bckdha protein levels at all timepoints. These results demonstrate that the expression of genes related to BCAA metabolism are regulated during adipocyte differentiation and influenced by nutrient levels. These results provide additional insights on how BCAA metabolism is associated with adipose tissue function and extends our understanding of the transcriptomic response of this pathway to variations in nutrient availability. PMID:25050624

SVEP1 is a novel marker of activated pre-determined skeletal muscle satellite cells.

PubMed

Shefer, Gabi; Benayahu, Dafna

2010-03-01

In this study we explored the expression pattern of SVEP1, a novel cell adhesion molecule (CAM), in bona fide satellite cells and their immediate progeny. We show that SVEP1 is expressed in activated satellite cells prior to their determination to the myogenic lineage. SVEP1 was also expressed during early phases of myogenic differentiation through the initial stage of myoblast fusion and myotube formation. The expression of SVEP1 was shown by immunostaining two cell culture systems: freshly isolated myofibers and primary myoblasts. Pax7 was used to pinpoint satellite cells situated in their niche on myofibers, and activated satellite cells were determined based on BrdU incorporation (Pax7(+)/BrdU(+)cells). MyoD marked satellite cells fated to undergo myogenesis as well as proliferating and differentiating myoblasts. Differentiating myoblasts and myotubes were identified based on their sarcomeric myosin expression. We showed that SVEP1 was specifically expressed in pre-determined activated satellite cells (Pax7(+)/ BrdU(+) /MyoD(-)) accounting for about 24% of total satellite cells. On the other hand, SVEP1 expression was absent in quiescent satellite cells (Pax7(+)/BrdU(-)/MyoD(-)). Moreover, based on MyoD/sarcomeric myosin co-expression SVEP1 was shown to be expressed throughout the early phases of myogenesis up until myoblast fusion and myotube formation. A decline in SVEP1 expression occurred upon myotube maturation. We suggest SVEP1 as a potential biomarker for pre-fated satellite cells. The impact of this finding is that it may allow scrutinizing signals that affect differentiation commitment. Thus, holds a therapeutic potential for maladies that involve deregulated stem cell fate-decision.

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

The expression of the class 1 glucose transporter isoforms in human embryonic stem cells, and the potential use of GLUT2 as a marker for pancreatic progenitor enrichment.

PubMed

Segev, Hana; Fishman, Betina; Schulman, Rita; Itskovitz-Eldor, Joseph

2012-07-01

Even before the first appearance of the developing pancreas, glucose is the major substrate in the growing embryo. The transport of glucose across cell membranes is facilitated by a family of membranal glucose transporters (GLUT). We analyzed changes in expression of class 1 glucose transporters (GLUT1-4) during human embryonic stem cell (hESC) and human induced pluripotent stem cell (hiPSC) differentiation, from undifferentiated cells to 28-day-old embryoid bodies (EBs). We also examined the potential use of GLUT2 as a marker for differentiating pancreatic progenitor cells. Using quantitative real time polymerase chain reaction (qPCR), western blot, and immunofluorescence, we observed enhanced expression of GLUT1 and GLUT2 during differentiation, but only minor change in GLUT3 expression. GLUT4 expression was found to be very low both at the RNA and in the protein levels. Expression of the early pancreatic transcription factor, pancreatic duodenal homeobox gene 1 (PDX1), correlated with GLUT2 expression, suggesting the potential use of GLUT2 as a surface marker for tracking pancreatic precursor cells. After sorting EBs according to their membranal GLUT2 expression, GLUT2 and PDX1 expression were found elevated, as was expression of other endodermal markers such as PAX4, NGN3, CXCR4, and SOX17. This simple method may be used to differentiate embryonic stem cells and to isolate from them, using GLUT2 as a surface marker, an enriched pancreatic progenitor cell population in order to achieve insulin-producing cells. The sorted GLUT2 cells may potentially be used in the future as insulin-producing cells for beta cell therapies.

Interleukin-24 is correlated with differentiation and lymph node numbers in rectal cancer

PubMed Central

Choi, Youngmin; Roh, Mee-Sook; Hong, Young-Seoub; Lee, Hyung-Sik; Hur, Won-Joo

2011-01-01

AIM: To assess the significance of interleukin (IL)-24 and vascular endothelial growth factor (VEGF) expression in lymph-node-positive rectal cancer. METHODS: Between 1998 and 2005, 90 rectal adenocarcinoma patients with lymph node involvement were enrolled. All patients received radical surgery and postoperative pelvic chemoradiotherapy of 50.4-54.0 Gy. Chemotherapy of 5-fluorouracil and leucovorin or levamisole was given intravenously during the first and last week of radiotherapy, and then monthly for about 6 mo. Expression of IL-24 and VEGF was evaluated by immunohistochemical staining of surgical specimens, and their relations with patient characteristics and survival were analyzed. The median follow-up of surviving patients was 73 mo (range: 52-122 mo). RESULTS: IL-24 expression was found in 81 out of 90 patients; 31 showed weak intensity and 50 showed strong intensity. VEGF expression was found in 64 out of 90 patients. Negative and weak intensities of IL-24 expression were classified as negative expression for analysis. IL-24 expression was significantly reduced in poorly differentiated tumors in comparison with well or moderately differentiated tumors (P = 0.004), N2b to earlier N stages (P = 0.016), and stage IIIc to stage IIIa or IIIb (P = 0.028). The number of involved lymph nodes was also significantly reduced in IL-24-positive patients in comparison with IL-24-negative ones.There was no correlation between VEGF expression and patient characteristics. Expression of IL-24 and VEGF was not correlated with survival, but N stage and stages were significantly correlated with survival. CONCLUSION: IL-24 expression was significantly correlated with histological differentiation, and inversely correlated with the degree of lymph node involvement in stage III rectal cancer. PMID:21448421

Blood-stage malaria of Plasmodium chabaudi induces differential Tlr expression in the liver of susceptible and vaccination-protected Balb/c mice.

PubMed

Al-Quraishy, Saleh; Dkhil, Mohamed A; Alomar, Suliman; Abdel-Baki, Abdel Azeem S; Delic, Denis; Wunderlich, Frank; Araúzo-Bravo, Marcos J

2016-05-01

Protective vaccination induces self-healing of otherwise lethal blood-stage infections of Plasmodium chabaudi malaria. Here, we investigate mRNA expression patterns of all 12 members of the Toll-like receptor (Tlr) gene family in the liver, a major effector organ against blood-stage malaria, during lethal and vaccination-induced self-healing infections of P. chabaudi in female Balb/c mice. Gene expression microarrays reveal that all 12 Tlr genes are constitutively expressed, though at varying levels, and specifically respond to infection. Protective vaccination does not affect constitutive expression of any of the 12 Tlr genes but leads to differential expression (p < 0.05) of seven Tlrs (1, 2, 4, 7, 8, 12, and 13) in response to malaria. Quantitative PCR substantiates differential expression at p < 0.01. There is an increased expression of Tlr2 by approximately five-fold on day 1 post-infection (p.i.) and Tlr1 by approximately threefold on day 4 p.i.. At peak parasitemia on day 8 p.i., none of the 12 Tlrs display any differential expression. After peak parasitemia, towards the end of the crisis phase on day 11 p.i., expression of Tlrs 1, 4, and 12 is increased by approximately four-, two-, and three-fold, respectively, and that of Tlr7 is decreased by approximately two-fold. Collectively, our data suggest that though all 12 members of the Tlr gene family are specifically responsive to malaria in the liver, not only Tlr2 at the early stage of infection but also the Tlrs 1, 4, 7, and 12 towards the end of crisis phase are critical for vaccination-induced resolution and survival of otherwise lethal blood-stage malaria.

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.

Naringin promotes differentiation of bone marrow stem cells into osteoblasts by upregulating the expression levels of microRNA-20a and downregulating the expression levels of PPARγ.

PubMed

Fan, Jifeng; Li, Jie; Fan, Qinbo

2015-09-01

Naringin is a dihydrotestosterone flavonoid compound that significantly inhibits bone loss, improves bone density, and enhances biomechanical anti‑compression performance. Previous studies have demonstrated that naringin improves the activity levels of osteocalcin (OC) and alkaline phosphatase (ALP) in MC3T3‑E1 osteoblast precursor cells. The present study investigated the effects of naringin on osteoblastic differentiation and inhibition of adipocyte formation in bone marrow stem cells (BMSCs). The levels of osteogenesis were modulated via upregulation of the expression levels of microRNA (miR)‑20a, and downregulation of the expression levels of peroxisome proliferator‑activated receptor γ (PPARγ). The results indicated that naringin significantly enhanced BMSC proliferation in a dose‑dependent manner. In addition, naringin significantly increased the mRNA expression levels of OC, ALP, and collagen type I. Furthermore, naringin decreased the protein expression levels of PPARγ, and increased the expression levels of miR‑20a in the BMSCs. These results suggested that miR‑20a may regulate the expression of PPARγ in BMSCs. To our knowledge, this is the first study to report naringin‑induced osteogenesis via upregulation of the expression levels of miR‑20a, and downregulation of the expression levels of PPARγ. These results indicated the important role of naringin in BMSC differentiation.

Kynurenine promotes the goblet cell differentiation of HT-29 colon carcinoma cells by modulating Wnt, Notch and AhR signals.

PubMed

Park, Joo-Hung; Lee, Jeong-Min; Lee, Eun-Jin; Kim, Da-Jeong; Hwang, Won-Bhin

2018-04-01

Various amino acids regulate cell growth and differentiation. In the present study, we examined the ability of HT-29 cells to differentiate into goblet cells in RPMI and DMEM which are largely different in the amounts of numerous amino acids. Most of the HT-29 cells differentiated into goblet cells downregulating the stem cell marker Lgr5 when cultured in DMEM, but remained undifferentiated in RPMI. The goblet cell differentiation in DMEM was inhibited by 1-methyl-tryptophan (1-MT), an inhibitor of indoleamine 2,3 dioxygenase-1 which is the initial enzyme in tryptophan metabolism along the kynurenine (KN) pathway, whereas tryptophan and KN induced goblet cell differentiation in RPMI. The levels of Notch1 and its activation product Notch intracytoplasmic domain in HT-29 cells were lower in DMEM than those in RPMI and were increased by 1-MT in both media. HT-29 cells grown in both media expressed β-catenin at the same level on day 2 when goblet cell differentiation was not observed. β-catenin expression, which was increased by 1-MT in both media, was decreased by KN. DMEM reduced Hes1 expression while enhancing Hath1 expression. Finally, aryl hydrocarbon receptor (AhR) activation moderately induced goblet cell differentiation. Our results suggest that KN promotes goblet cell differentiation by regulating Wnt, Notch, and AhR signals and expression of Hes1 and Hath1.

CTCF-Mediated and Pax6-Associated Gene Expression in Corneal Epithelial Cell-Specific Differentiation

PubMed Central

Tsui, Shanli; Wang, Jie; Wang, Ling; Dai, Wei; Lu, Luo

2016-01-01

Background The purpose of the study is to elicit the epigenetic mechanism involving CCCTC binding factor (CTCF)-mediated chromatin remodeling that regulates PAX6 gene interaction with differentiation-associated genes to control corneal epithelial differentiation. Methods Cell cycle progression and specific keratin expressions were measured to monitor changes of differentiation-induced primary human limbal stem/progenitor (HLS/P), human corneal epithelial (HCE) and human telomerase-immortalized corneal epithelial (HTCE) cells. PAX6-interactive and differentiation-associated genes in chromatin remodeling mediated by the epigenetic factor CTCF were detected by circular chromosome conformation capture (4C) and ChIP (Chromatin immunoprecipitation)-on-chip approaches, and verified by FISH (Fluorescent in situ hybridization). Furthermore, CTCF activities were altered by CTCF-shRNA to study the effect of CTCF on mediating interaction of Pax6 and differentiation-associated genes in corneal epithelial cell fate. Results Our results demonstrated that differentiation-induced human corneal epithelial cells expressed typical corneal epithelial characteristics including morphological changes, increased keratin12 expression and G0/G1 accumulations. Expressions of CTCF and PAX6 were suppressed and elevated following the process of differentiation, respectively. During corneal epithelial cell differentiation, differentiation-induced RCN1 and ADAM17 were found interacting with PAX6 in the process of CTCF-mediated chromatin remodeling detected by 4C and verified by ChIP-on-chip and FISH. Diminished CTCF mRNA with CTCF-shRNA in HTCE cells weakened the interaction of PAX6 gene in controlling RCN1/ADAM17 and enhanced early onset of the genes in cell differentiation. Conclusion Our results explain how epigenetic factor CTCF-mediated chromatin remodeling regulates interactions between eye-specific PAX6 and those genes that are induced/associated with cell differentiation to modulate corneal epithelial cell-specific differentiation. PMID:27583466

Involvement of the DNA mismatch repair system in cisplatin sensitivity of testicular germ cell tumours.

PubMed

Rudolph, Christiane; Melau, Cecilie; Nielsen, John E; Vile Jensen, Kristina; Liu, Dekang; Pena-Diaz, Javier; Rajpert-De Meyts, Ewa; Rasmussen, Lene Juel; Jørgensen, Anne

2017-08-01

Testicular germ cell tumours (TGCT) are highly sensitive to cisplatin-based chemotherapy, but patients with tumours containing differentiated teratoma components are less responsive to this treatment. The cisplatin sensitivity in TGCT has previously been linked to the embryonic phenotype in the majority of tumours, although the underlying mechanism largely remains to be elucidated. The aim of this study was to investigate the role of the DNA mismatch repair (MMR) system in the cisplatin sensitivity of TGCT. The expression pattern of key MMR proteins, including MSH2, MSH6, MLH1 and PMS2, were investigated during testis development and in the pathogenesis of TGCT, including germ cell neoplasia in situ (GCNIS). The TGCT-derived cell line NTera2 was differentiated using retinoic acid (10 μM, 6 days) after which MMR protein expression and activity, as well as cisplatin sensitivity, were investigated in both undifferentiated and differentiated cells. Finally, the expression of MSH2 was knocked down by siRNA in NTera2 cells after which the effect on cisplatin sensitivity was examined. MMR proteins were expressed in proliferating cells in the testes, while in malignant germ cells MMR protein expression was found to coincide with the expression of the pluripotency factor OCT4, with no or low expression in the more differentiated yolk sac tumours, choriocarcinomas and teratomas. In differentiated NTera2 cells we found a significantly (p < 0.05) lower expression of the MMR and pluripotency factors, as well as a reduced MMR activity and cisplatin sensitivity, compared to undifferentiated NTera2 cells. Also, we found that partial knockdown of MSH2 expression in undifferentiated NTera2 cells resulted in a significantly (p < 0.001) reduced cisplatin sensitivity. This study reports, for the first time, expression of the MMR system in fetal gonocytes, from which GCNIS cells are derived. Our findings in primary TGCT specimens and TGCT-derived cells suggest that a reduced sensitivity to cisplatin in differentiated TGCT components could result from a reduced expression of MMR proteins, in particular MSH2 and MLH1, which are involved in the recognition of cisplatin adducts and in activation of the DNA damage response pathway to initiate apoptosis.

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.

Bone morphogenic protein-2 regulates the myogenic differentiation of PMVECs in CBDL rat serum-induced pulmonary microvascular remodeling

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

Liu, Chang; Chen, Lin; Zeng, Jing

Hepatopulmonary syndrome (HPS) is characterized by an arterial oxygenation defect induced by intrapulmonary vasodilation (IPVD) that increases morbidity and mortality. In our previous study, it was determined that both the proliferation and the myogenic differentiation of pulmonary microvascular endothelial cells (PMVECs) play a key role in the development of IPVD. However, the molecular mechanism underlying the relationship between IPVD and the myogenic differentiation of PMVECs remains unknown. Additionally, it has been shown that bone morphogenic protein-2 (BMP2), via the control of protein expression, may regulate cell differentiation including cardiomyocyte differentiation, neuronal differentiation and odontoblastic differentiation. In this study, we observedmore » that common bile duct ligation (CBDL)-rat serum induced the upregulation of the expression of several myogenic proteins (SM-α-actin, calponin, SM-MHC) and enhanced the expression levels of BMP2 mRNA and protein in PMVECs. We also observed that both the expression levels of Smad1/5 and the activation of phosphorylated Smad1/5 were significantly elevated in PMVECs following exposure to CBDL-rat serum, which was accompanied by the down-regulation of Smurf1. The blockage of the BMP2/Smad signaling pathway with Noggin inhibited the myogenic differentiation of PMVECs, a process that was associated with relatively low expression levels of both SM-α-actin and calponin in the setting of CBDL-rat serum exposure, although SM-MHC expression was not affected. These findings suggested that the BMP2/Smad signaling pathway is involved in the myogenic differentiation of the PMVECs. In conclusion, our data highlight the pivotal role of BMP2 in the CBDL-rat serum-induced myogenic differentiation of PMVECs via the activation of both Smad1 and Smad5 and the down-regulation of Smurf1, which may represent a potential therapy for HPS-induced pulmonary vascular remodeling. - Highlights: • CBDL-rat serum promotes the myogenic differentiaton and expression of BMP2 in PMVECs. • CBDL-rat serum activates the BMP2/smad signaling pathway. • The downregulation of Smurf1 stimulates the accumulation of Smad1/5 in PMVECs. • Noggin reverses partially the myogenic differentiaton in PMVECs.« less

Prostaglandin E2 inhibits Tr1 cell differentiation through suppression of c-Maf

PubMed Central

Hooper, Kirsten Mary; Kong, Weimin

2017-01-01

Prostaglandin E2 (PGE2), a major lipid mediator abundant at inflammatory sites, acts as a proinflammatory agent in models of inflammatory/autoimmune diseases by promoting CD4 Th1/Th17 differentiation. Regulatory T cells, including the IL-10 producing Tr1 cells counterbalance the proinflammatory activity of effector Th1/Th17 cells. Tr1 cell differentiation and function are induced by IL-27, and depend primarily on sustained expression of c-Maf in addition to AhR and Blimp-1. In agreement with the in vivo proinflammatory role of PGE2, here we report for the first time that PGE2 inhibits IL-27-induced differentiation and IL-10 production of murine CD4+CD49b+LAG-3+Foxp3- Tr1 cells. The inhibitory effect of PGE2 was mediated through EP4 receptors and induction of cAMP, leading to a significant reduction in c-Maf expression. Although PGE2 reduced IL-21 production in differentiating Tr1 cells, its inhibitory effect on Tr1 differentiation and c-Maf expression also occurred independent of IL-21 signaling. PGE2 did not affect STAT1/3 activation, AhR expression and only marginally reduced Egr-2/Blimp-1 expression. The effect of PGE2 on CD4+CD49b+LAG-3+ Tr1 differentiation was not associated with either induction of Foxp3 or IL-17 production, suggesting a lack of transdifferentiation into Foxp3+ Treg or effector Th17 cells. We recently reported that PGE2 inhibits the expression and production of IL-27 from activated conventional dendritic cells (cDC) in vivo and in vitro. The present study indicates that PGE2 also reduces murine Tr1 differentiation and function directly by acting on IL-27-differentiating Tr1 cells. Together, the ability of PGE2 to inhibit IL-27 production by cDC, and the direct inhibitory effect on Tr1 differentiation mediated through reduction in c-Maf expression, represent a new mechanistic perspective for the proinflammatory activity of PGE2. PMID:28604806

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

Biased gene expression in early honeybee larval development

PubMed Central

2013-01-01

Background Female larvae of the honeybee (Apis mellifera) develop into either queens or workers depending on nutrition. This nutritional stimulus triggers different developmental trajectories, resulting in adults that differ from each other in physiology, behaviour and life span. Results To understand how these trajectories are established we have generated a comprehensive atlas of gene expression throughout larval development. We found substantial differences in gene expression between worker and queen-destined larvae at 6 hours after hatching. Some of these early changes in gene expression are maintained throughout larval development, indicating that caste-specific developmental trajectories are established much earlier than previously thought. Within our gene expression data we identified processes that potentially underlie caste differentiation. Queen-destined larvae have higher expression of genes involved in transcription, translation and protein folding early in development with a later switch to genes involved in energy generation. Using RNA interference, we were able to demonstrate that one of these genes, hexamerin 70b, has a role in caste differentiation. Both queen and worker developmental trajectories are associated with the expression of genes that have alternative splice variants, although only a single variant of a gene tends to be differentially expressed in a given caste. Conclusions Our data, based on the biases in gene expression early in development together with published data, supports the idea that caste development in the honeybee consists of two phases; an initial biased phase of development, where larvae can still switch to the other caste by differential feeding, followed by commitment to a particular developmental trajectory. PMID:24350621

Differential effect of 1{alpha},25-dihydroxyvitamin D{sub 3} on Hsp28 and PKC{beta} gene expression in the phorbol ester-resistant human myeloid HL-525 leukemic cells

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

Lee, Yong J.; Galoforo, S.S.; Berns, C.M.

We investigated the effect of 1{alpha},25-dihydroxyvitamin D{sub 3} [1,25-(OH){sub 2}D{sub 3}] on the expression of the 28-kDa heat shock protein gene (hsp28) and the protein kinase C beta gene (PKC{beta}) in the human myeloid HL-60 leukemic cell variant HL-525, which is resistance to phorbol ester-induced macrophage differentiation. Northern and Western blot analysis showed little or no hsp28 gene expression in the HL-60 cell variant, HL-205, which is susceptible to such differentiation, while a relatively high basal level of hps28 gene expression was observed in the HL-525 cells. However, both cell lines demonstrated heat shock-induced expression of this gene. During treatmentmore » with 50-300 nM 1,25-(OH){sub 2}D{sub 3}, a marked reduction of hsp28 gene expression was not associated with heat shock transcription factor-heat shock element (HSF-HSE) binding activity. Our results suggest that the differential effect of 1,25-(OH){sub 2}D{sub 3} on hsp28 and PKC{beta} gene expression is due to the different sequence composition of the vitamin D response element in the in the promoter region as well as an accessory factor for each gene or that 1,25-(OH){sub 2}D{sub 3} increases PKC{beta} gene expression, which in turn negatively regulates the expression of the hsp28 gene, or vice versa.« less

Vaccine-induced modulation of gene expression in turbot peritoneal cells. A microarray approach.

PubMed

Fontenla, Francisco; Blanco-Abad, Verónica; Pardo, Belén G; Folgueira, Iria; Noia, Manuel; Gómez-Tato, Antonio; Martínez, Paulino; Leiro, José M; Lamas, Jesús

2016-07-01

We used a microarray approach to examine changes in gene expression in turbot peritoneal cells after injection of the fish with vaccines containing the ciliate parasite Philasterides dicentrarchi as antigen and one of the following adjuvants: chitosan-PVMMA microspheres, Freund́s complete adjuvant, aluminium hydroxide gel or Matrix-Q (Isconova, Sweden). We identified 374 genes that were differentially expressed in all groups of fish. Forty-two genes related to tight junctions and focal adhesions and/or actin cytoskeleton were differentially expressed in free peritoneal cells. The profound changes in gene expression related to cell adherence and cytoskeleton may be associated with cell migration and also with the formation of cell-vaccine masses and their attachment to the peritoneal wall. Thirty-five genes related to apoptosis were differentially expressed. Although most of the proteins coded by these genes have a proapoptotic effect, others are antiapoptotic, indicating that both types of signals occur in peritoneal leukocytes of vaccinated fish. Interestingly, many of the genes related to lymphocytes and lymphocyte activity were downregulated in the groups injected with vaccine. We also observed decreased expression of genes related to antigen presentation, suggesting that macrophages (which were abundant in the peritoneal cavity after vaccination) did not express these during the early inflammatory response in the peritoneal cavity. Finally, several genes that participate in the inflammatory response were differentially expressed, and most participated in resolution of inflammation, indicating that an M2 macrophage response is generated in the peritoneal cavity of fish one day post vaccination. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

Drosophila E-Cadherin Functions in Hematopoietic Progenitors to Maintain Multipotency and Block Differentiation

PubMed Central

Gao, Hongjuan; Wu, Xiaorong; Fossett, Nancy

2013-01-01

A fundamental question in stem cell biology concerns the regulatory strategies that control the choice between multipotency and differentiation. Drosophila blood progenitors or prohemocytes exhibit key stem cell characteristics, including multipotency, quiescence, and niche dependence. As a result, studies of Drosophila hematopoiesis have provided important insights into the molecular mechanisms that control these processes. Here, we show that E-cadherin is an important regulator of prohemocyte fate choice, maintaining prohemocyte multipotency and blocking differentiation. These functions are reminiscent of the role of E-cadherin in mammalian embryonic stem cells. We also show that mis-expression of E-cadherin in differentiating hemocytes disrupts the boundary between these cells and undifferentiated prohemocytes. Additionally, upregulation of E-cadherin in differentiating hemocytes increases the number of intermediate cell types expressing the prohemocyte marker, Patched. Furthermore, our studies indicate that the Drosophila GATA transcriptional co-factor, U-shaped, is required for E-cadherin expression. Consequently, E-cadherin is a downstream target of U-shaped in the maintenance of prohemocyte multipotency. In contrast, we showed that forced expression of the U-shaped GATA-binding partner, Serpent, repressed E-cadherin expression and promoted lamellocyte differentiation. Thus, U-shaped may maintain E-cadherin expression by blocking the inhibitory activity of Serpent. Collectively, these observations suggest that GATA:FOG complex formation regulates E-cadherin levels and, thereby, the choice between multipotency and differentiation. The work presented in this report further defines the molecular basis of prohemocyte cell fate choice, which will provide important insights into the mechanisms that govern stem cell biology. PMID:24040319

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

Psoriatic T cells reduce epidermal turnover time and affect cell proliferation contributed from differential gene expression.

PubMed

Li, Junqin; Li, Xinhua; Hou, Ruixia; Liu, Ruifeng; Zhao, Xincheng; Dong, Feng; Wang, Chunfang; Yin, Guohua; Zhang, Kaiming

2015-09-01

Psoriasis is mediated primarily by T cells, which reduce epidermal turnover time and affect keratinocyte proliferation. We aimed to identify differentially expressed genes (DEG) in T cells from normal, five pairs of monozygotic twins concordant or discordant for psoriasis, to determine whether these DEG may account for the influence to epidermal turnover time and keratinocyte proliferation. The impact of T cells on keratinocyte proliferation and epidermal turnover time were investigated separately by immunohistochemistry and cultured with (3) H-TdR. mRNA expression patterns were investigated by RNA sequencing and verified by real-time reverse transcription polymerase chain reaction. After co-culture with psoriatic T cells, the expression of Ki-67, c-Myc and p53 increased, while expression of Bcl-2 and epidermal turnover time decreased. There were 14 DEG which were found to participate in the regulation of cell proliferation or differentiation. Psoriatic T cells exhibited the ability to decrease epidermal turnover time and affect keratinocyte proliferation because of the differential expression of PPIL1, HSPH1, SENP3, NUP54, FABP5, PLEKHG3, SLC9A9 and CHCHD4. © 2015 Japanese Dermatological Association.

Expression of Mutant Human DISC1 in Mice Supports Abnormalities in Differentiation of Oligodendrocytes

PubMed Central

Katsel, Pavel; Tan, Weilun; Abazyan, Bagrat; Davis, Kenneth L; Ross, Christopher; Pletnikov, Mikhail V; Haroutunian, Vahram

2011-01-01

Abnormalities in oligodendrocyte (OLG) differentiation and OLG gene expression deficit have been described in schizophrenia (SZ). Recent studies revealed a critical requirement for Disrupted-in-Schizophrenia 1 (DISC1) in neural development. Transgenic mice with forebrain restricted expression of mutant human DISC1 (ΔhDISC1) are characterized by neuroanatomical and behavioral abnormalities reminiscent of some features of SZ. We sought to determine whether the expression of ΔhDISC1 may influence the development of OLGs in this mouse model. OLG- and cell cycle-associated gene and protein expression were characterized in the forebrain of ΔhDISC1 mice during different stages of neurodevelopment (E15 and P1 days) and in adulthood. The results suggest that the expression of ΔhDISC1 exerts a significant influence on oligodendrocyte differentiation and function, evidenced by premature OLG differentiation and increased proliferation of their progenitors. Additional findings showed that neuregulin 1 and its receptors may be contributing factors to the observed upregulation of OLG genes. Thus, OLG function may be perturbed by mutant hDISC1 in a model system that provides new avenues for studying aspects of the pathogenesis of SZ. PMID:21605958

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.

The expression of PTEN in the development of mouse cochlear lateral wall.

PubMed

Dong, Y; Sui, L; Yamaguchi, F; Kamitori, K; Hirata, Y; Hossain, A; Noguchi, C; Katagi, A; Nishio, M; Suzuki, A; Lou, X; Tokuda, M

2014-01-31

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor gene that regulates various cell processes including proliferation, growth, synaptogenesis, neural and glioma stem/progenitor cell renewal. In addition, PTEN can regulate sensory cell proliferation and differentiation of hair bundles in the mammalian cochlea. In this study we use immunofluorescence, Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) to reveal the expression of PTEN in the developing cochlear lateral wall, which is crucial for regulating K(+) homeostasis. Relatively high levels of PTEN are initially expressed in the marginal cells (MCs) of the lateral wall at embryonic day (E) 17.5 when they start to differentiate. Similarly high levels are subsequently expressed in differentiating root cells (RCs) at postnatal day (P) 3 and then in spiral ligament fibrocytes (SLFs) at P 10. In the mature cochlea, PTEN expression is low or undetectable in MCs and SLFs but it remains high in RCs and their processes. The expression pattern for PTEN in the developing lateral wall suggests that it plays a critical role in the differentiation of the cellular pathways that regulate K(+) homeostasis in the cochlea. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

Differential transcriptome expression in human nucleus accumbens as a function of loneliness

PubMed Central

Canli, Turhan; Wen, Ruofeng; Wang, Xuefeng; Mikhailik, Anatoly; Yu, Lei; Fleischman, Debra; Wilson, Robert S.; Bennett, David A.

2017-01-01

Loneliness is associated with impaired mental and physical health. Studies of lonely individuals reported differential expression of inflammatory genes in peripheral leukocytes and diminished activation in brain reward regions such as nucleus accumbens, but could not address gene expression in the human brain. Here, we examined genome-wide RNA expression in postmortem nucleus accumbens from donors (N = 26) with known loneliness measures. Loneliness was associated with 1 710 differentially expressed transcripts from 1 599 genes (DEGs; FDR p < 0.05, fold-change ≥ |2|, controlling for confounds) previously associated with behavioral processes, neurological disease, psychological disorders, cancer, organismal injury, and skeletal and muscular disorders, as well as networks of upstream RNA regulators. Furthermore, a number of DEGs were associated with Alzheimer’s disease genes (which was correlated with loneliness in this sample, although gene expression analyses controlled for AD diagnosis). These results identify novel targets for future mechanistic studies of gene networks in nucleus accumbens and gene regulatory mechanisms across a variety of diseases exacerbated by loneliness. PMID:27801889

Characterization of three human cell line models for high-throughput neuronal cytotoxicity screening.

PubMed

Tong, Zhi-Bin; Hogberg, Helena; Kuo, David; Sakamuru, Srilatha; Xia, Menghang; Smirnova, Lena; Hartung, Thomas; Gerhold, David

2017-02-01

More than 75 000 man-made chemicals contaminate the environment; many of these have not been tested for toxicities. These chemicals demand quantitative high-throughput screening assays to assess them for causative roles in neurotoxicities, including Parkinson's disease and other neurodegenerative disorders. To facilitate high throughput screening for cytotoxicity to neurons, three human neuronal cellular models were compared: SH-SY5Y neuroblastoma cells, LUHMES conditionally-immortalized dopaminergic neurons, and Neural Stem Cells (NSC) derived from human fetal brain. These three cell lines were evaluated for rapidity and degree of differentiation, and sensitivity to 32 known or candidate neurotoxicants. First, expression of neural differentiation genes was assayed during a 7-day differentiation period. Of the three cell lines, LUHMES showed the highest gene expression of neuronal markers after differentiation. Both in the undifferentiated state and after 7 days of neuronal differentiation, LUHMES cells exhibited greater cytotoxic sensitivity to most of 32 suspected or known neurotoxicants than SH-SY5Y or NSCs. LUHMES cells were also unique in being more susceptible to several compounds in the differentiating state than in the undifferentiated state; including known neurotoxicants colchicine, methyl-mercury (II), and vincristine. Gene expression results suggest that differentiating LUHMES cells may be susceptible to apoptosis because they express low levels of anti-apoptotic genes BCL2 and BIRC5/survivin, whereas SH-SY5Y cells may be resistant to apoptosis because they express high levels of BCL2, BIRC5/survivin, and BIRC3 genes. Thus, LUHMES cells exhibited favorable characteristics for neuro-cytotoxicity screening: rapid differentiation into neurons that exhibit high level expression neuronal marker genes, and marked sensitivity of LUHMES cells to known neurotoxicants. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Differentiation of monkey embryonic stem cells to hepatocytes by feeder-free dispersion culture and expression analyses of cytochrome p450 enzymes responsible for drug metabolism.

PubMed

Maruyama, Junya; Matsunaga, Tamihide; Yamaori, Satoshi; Sakamoto, Sakae; Kamada, Noboru; Nakamura, Katsunori; Kikuchi, Shinji; Ohmori, Shigeru

2013-01-01

We reported previously that monkey embryonic stem cells (ESCs) were differentiated into hepatocytes by formation of embryoid bodies (EBs). However, this EB formation method is not always efficient for assays using a large number of samples simultaneously. A dispersion culture system, one of the differentiation methods without EB formation, is able to more efficiently provide a large number of feeder-free undifferentiated cells. A previous study demonstrated the effectiveness of the Rho-associated kinase inhibitor Y-27632 for feeder-free dispersion culture and induction of differentiation of monkey ESCs into neural cells. In the present study, the induction of differentiation of cynomolgus monkey ESCs (cmESCs) into hepatocytes was performed by the dispersion culture method, and the expression and drug inducibility of cytochrome P450 (CYP) enzymes in these hepatocytes were examined. The cmESCs were successfully differentiated into hepatocytes under feeder-free dispersion culture conditions supplemented with Y-27632. The hepatocytes differentiated from cmESCs expressed the mRNAs for three hepatocyte marker genes (α-fetoprotein, albumin, CYP7A1) and several CYP enzymes, as measured by real-time polymerase chain reaction. In particular, the basal expression of cmCYP3A4 (3A8) in these hepatocytes was detected at mRNA and enzyme activity (testosterone 6β-hydroxylation) levels. Furthermore, the expression and activity of cmCYP3A4 (3A8) were significantly upregulated by rifampicin. These results indicated the effectiveness of Y-27632 supplementation for feeder-free dispersed culture and induction of differentiation into hepatocytes, and the expression of functional CYP enzyme(s) in cmESC-derived hepatic cells.

Gene expression profiling of mesenteric lymph nodes from sheep with natural scrapie

PubMed Central

2014-01-01

Background Prion diseases are characterized by the accumulation of the pathogenic PrPSc protein, mainly in the brain and the lymphoreticular system. Although prions multiply/accumulate in the lymph nodes without any detectable pathology, transcriptional changes in this tissue may reflect biological processes that contribute to the molecular pathogenesis of prion diseases. Little is known about the molecular processes that occur in the lymphoreticular system in early and late stages of prion disease. We performed a microarray-based study to identify genes that are differentially expressed at different disease stages in the mesenteric lymph node of sheep naturally infected with scrapie. Oligo DNA microarrays were used to identify gene-expression profiles in the early/middle (preclinical) and late (clinical) stages of the disease. Results In the clinical stage of the disease, we detected 105 genes that were differentially expressed (≥2-fold change in expression). Of these, 43 were upregulated and 62 downregulated as compared with age-matched negative controls. Fewer genes (50) were differentially expressed in the preclinical stage of the disease. Gene Ontology enrichment analysis revealed that the differentially expressed genes were largely associated with the following terms: glycoprotein, extracellular region, disulfide bond, cell cycle and extracellular matrix. Moreover, some of the annotated genes could be grouped into 3 specific signaling pathways: focal adhesion, PPAR signaling and ECM-receptor interaction. We discuss the relationship between the observed gene expression profiles and PrPSc deposition and the potential involvement in the pathogenesis of scrapie of 7 specific differentially expressed genes whose expression levels were confirmed by real time-PCR. Conclusions The present findings identify new genes that may be involved in the pathogenesis of natural scrapie infection in the lymphoreticular system, and confirm previous reports describing scrapie-induced alterations in the expression of genes involved in protein misfolding, angiogenesis and the oxidative stress response. Further studies will be necessary to determine the role of these genes in prion replication, dissemination and in the response of the organism to this disease. PMID:24450868

Gene expression profiling data of Schizosaccharomyces pombe under nitrosative stress using differential display.

PubMed

Biswas, Pranjal; Majumdar, Uddalak; Ghosh, Sanjay

2016-03-01

Excess production of nitric oxide (NO) and reactive nitrogen intermediates (RNIs) causes nitrosative stress on cells. Schizosaccharomyces pombe was used as a model to study nitrosative stress response. In the present data article, we have used differential display to identify the differentially expressed genes in the fission yeast under nitrosative stress conditions. We have used pure NO donor compound detaNONOate at final concentrations of 0.1 mM and 1 mM to treat the cells for 15 min alongside control before studying their gene expression profiles. At both the treated conditions, we identified genes which were commonly repressed while several genes were induced upon both 0.1 mM and 1 mM treatments. The differentially expressed genes were further analyzed in DAVID and categorized into several different pathways.

Effector Regulatory T Cell Differentiation and Immune Homeostasis Depend on the Transcription Factor Myb.

PubMed

Dias, Sheila; D'Amico, Angela; Cretney, Erika; Liao, Yang; Tellier, Julie; Bruggeman, Christine; Almeida, Francisca F; Leahy, Jamie; Belz, Gabrielle T; Smyth, Gordon K; Shi, Wei; Nutt, Stephen L

2017-01-17

FoxP3-expressing regulatory T (Treg) cells are essential for maintaining immune homeostasis. Activated Treg cells undergo further differentiation into an effector state that highly expresses genes critical for Treg cell function, although how this process is coordinated on a transcriptional level is poorly understood. Here, we demonstrate that mice lacking the transcription factor Myb in Treg cells succumbed to a multi-organ inflammatory disease. Myb was specifically expressed in, and required for the differentiation of, thymus-derived effector Treg cells. The combination of transcriptome and genomic footprint analyses revealed that Myb directly regulated a large proportion of the gene expression specific to effector Treg cells, identifying Myb as a critical component of the gene regulatory network controlling effector Treg cell differentiation and function. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

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.

snoU6 and 5S RNAs are not reliable miRNA reference genes in neuronal differentiation.

PubMed

Lim, Q E; Zhou, L; Ho, Y K; Wan, G; Too, H P

2011-12-29

Accurate profiling of microRNAs (miRNAs) is an essential step for understanding the functional significance of these small RNAs in both physiological and pathological processes. Quantitative real-time PCR (qPCR) has gained acceptance as a robust and reliable transcriptomic method to profile subtle changes in miRNA levels and requires reference genes for accurate normalization of gene expression. 5S and snoU6 RNAs are commonly used as reference genes in microRNA quantification. It is currently unknown if these small RNAs are stably expressed during neuronal differentiation. Panels of miRNAs have been suggested as alternative reference genes to 5S and snoU6 in various physiological contexts. To test the hypothesis that miRNAs may serve as stable references during neuronal differentiation, the expressions of eight miRNAs, 5S and snoU6 RNAs in five differentiating neuronal cell types were analyzed using qPCR. The stabilities of the expressions were evaluated using two complementary statistical approaches (geNorm and Normfinder). Expressions of 5S and snoU6 RNAs were stable under some but not all conditions of neuronal differentiation and thus are not suitable reference genes. In contrast, a combination of three miRNAs (miR-103, miR-106b and miR-26b) allowed accurate expression normalization across different models of neuronal differentiation. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Differential gene expression of two extreme honey bee (Apis mellifera) colonies showing varroa tolerance and susceptibility.

PubMed

Jiang, S; Robertson, T; Mostajeran, M; Robertson, A J; Qiu, X

2016-06-01

Varroa destructor, an ectoparasitic mite of honey bees (Apis mellifera), is the most serious pest threatening the apiculture industry. In our honey bee breeding programme, two honey bee colonies showing extreme phenotypes for varroa tolerance/resistance (S88) and susceptibility (G4) were identified by natural selection from a large gene pool over a 6-year period. To investigate potential defence mechanisms for honey bee tolerance to varroa infestation, we employed DNA microarray and real time quantitative (PCR) analyses to identify differentially expressed genes in the tolerant and susceptible colonies at pupa and adult stages. Our results showed that more differentially expressed genes were identified in the tolerant bees than in bees from the susceptible colony, indicating that the tolerant colony showed an increased genetic capacity to respond to varroa mite infestation. In both colonies, there were more differentially expressed genes identified at the pupa stage than at the adult stage, indicating that pupa bees are more responsive to varroa infestation than adult bees. Genes showing differential expression in the colony phenotypes were categorized into several groups based on their molecular functions, such as olfactory signalling, detoxification processes, exoskeleton formation, protein degradation and long-chain fatty acid metabolism, suggesting that these biological processes play roles in conferring varroa tolerance to naturally selected colonies. Identification of differentially expressed genes between the two colony phenotypes provides potential molecular markers for selecting and breeding varroa-tolerant honey bees. © 2016 The Royal Entomological Society.

Knock-in strategy at 3'-end of Crx gene by CRISPR/Cas9 system shows the gene expression profiles during human photoreceptor differentiation.

PubMed

Homma, Kohei; Usui, Sumiko; Kaneda, Makoto

2017-03-01

Fluorescent reporter gene knock-in induced pluripotent stem cell (iPSC) lines have been used to evaluate the efficiency of differentiation into specific cell lineages. Here, we report a knock-in strategy for the generation of human iPSC reporter lines in which a 2A peptide sequence and a red fluorescent protein (E2-Crimson) gene were inserted at the termination codon of the cone-rod homeobox (Crx) gene, a photoreceptor-specific transcriptional factor gene. The knock-in iPSC lines were differentiated into fluorescence-expressing cells in 3D retinal differentiation culture, and the fluorescent cells also expressed Crx specifically in the nucleus. We found that the fluorescence intensity was positively correlated with the expression levels of Crx mRNA and that fluorescent cells expressed rod photoreceptor-specific genes in the later stage of differentiation. Finally, we treated the fluorescent cells with DAPT, a Notch inhibitor, and found that DAPT-enhanced retinal differentiation was associated with up-regulation of Crx, Otx2 and NeuroD1, and down-regulation of Hes5 and Ngn2. These suggest that this knock-in strategy at the 3'-end of the target gene, combined with the 2A peptide linked to fluorescent proteins, offers a useful tool for labeling specific cell lineages or monitoring expression of any marker genes without affecting the function of the target gene. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Expression of toll-like receptors 2 and 4 and CD14 during differentiation of HL-60 cells induced by phorbol 12-myristate 13-acetate and 1 alpha, 25-dihydroxy-vitamin D(3).

PubMed

Li, Changlin; Wang, Yibing; Gao, Li; Zhang, Jingsong; Shao, Jie; Wang, Shengnian; Feng, Weiguo; Wang, Xingyu; Li, Minglie; Chang, Zongliang

2002-01-01

Macrophages form a crucial bridge between the innate and adaptive immune response. One of their most important functions is to recognize infectious microorganisms. Toll-like receptors (TLRs) are key elements in pathogen recognition, and among them, TLR2 and TLR4 are most discussed. However, expression patterns of TLRs during myeloid cell differentiation to macrophage are unknown. In this study, we examined differentiation in the model human myeloid cell line, HL-60, treated with phorbol 12-myristate 13-acetate (PMA) or VitD(3). Expression of TLR2, TLR4, and CD14 were measured by reverse transcription-PCR, RNase protection assay, and fluorescence-activated cell sorter assays. After treatment by PMA (1, 10, and 100 nM) for 12, 24, and 48 h, expression of TLR2 and CD14 mRNA was increased in a time- and dose-dependent manner. However, VitD(3) only induced expression of CD14 but not TLR2 in HL-60 cells. TLR4 was expressed constitutively before differentiation and increased slightly after that. Thus, PMA-mediated differentiation of HL-60 cells to macrophages is associated largely with TLR2 expression and, to a much lesser extent, with TLR4. Furthermore, up-regulation of TLR2 and CD14 mRNA expression by PMA was abrogated by a protein kinase C inhibitor, Calphostine C, suggesting the up-regulation of TLR2 and CD14 mRNA is dependent on the activation of protein kinase C. Coexpression of CD14/TLR2 and/or CD14/TLR4 may be essential but not sufficient for the production of tumor necrosis factor-alpha in response to lipopolysaccharide in our system.

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.

Identification of stable reference genes in differentiating human pluripotent stem cells.

PubMed

Holmgren, Gustav; Ghosheh, Nidal; Zeng, Xianmin; Bogestål, Yalda; Sartipy, Peter; Synnergren, Jane

2015-06-01

Reference genes, often referred to as housekeeping genes (HKGs), are frequently used to normalize gene expression data based on the assumption that they are expressed at a constant level in the cells. However, several studies have shown that there may be a large variability in the gene expression levels of HKGs in various cell types. In a previous study, employing human embryonic stem cells (hESCs) subjected to spontaneous differentiation, we observed that the expression of commonly used HKG varied to a degree that rendered them inappropriate to use as reference genes under those experimental settings. Here we present a substantially extended study of the HKG signature in human pluripotent stem cells (hPSC), including nine global gene expression datasets from both hESC and human induced pluripotent stem cells, obtained during directed differentiation toward endoderm-, mesoderm-, and ectoderm derivatives. Sets of stably expressed genes were compiled, and a handful of genes (e.g., EID2, ZNF324B, CAPN10, and RABEP2) were identified as generally applicable reference genes in hPSCs across all cell lines and experimental conditions. The stability in gene expression profiles was confirmed by reverse transcription quantitative PCR analysis. Taken together, the current results suggest that differentiating hPSCs have a distinct HKG signature, which in some aspects is different from somatic cell types, and underscore the necessity to validate the stability of reference genes under the actual experimental setup used. In addition, the novel putative HKGs identified in this study can preferentially be used for normalization of gene expression data obtained from differentiating hPSCs. Copyright © 2015 the American Physiological Society.

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

Exploring of the molecular mechanism of rhinitis via bioinformatics methods

PubMed Central

Song, Yufen; Yan, Zhaohui

2018-01-01

The aim of this study was to analyze gene expression profiles for exploring the function and regulatory network of differentially expressed genes (DEGs) in pathogenesis of rhinitis by a bioinformatics method. The gene expression profile of GSE43523 was downloaded from the Gene Expression Omnibus database. The dataset contained 7 seasonal allergic rhinitis samples and 5 non-allergic normal samples. DEGs between rhinitis samples and normal samples were identified via the limma package of R. The webGestal database was used to identify enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the DEGs. The differentially co-expressed pairs of the DEGs were identified via the DCGL package in R, and the differential co-expression network was constructed based on these pairs. A protein-protein interaction (PPI) network of the DEGs was constructed based on the Search Tool for the Retrieval of Interacting Genes database. A total of 263 DEGs were identified in rhinitis samples compared with normal samples, including 125 downregulated ones and 138 upregulated ones. The DEGs were enriched in 7 KEGG pathways. 308 differential co-expression gene pairs were obtained. A differential co-expression network was constructed, containing 212 nodes. In total, 148 PPI pairs of the DEGs were identified, and a PPI network was constructed based on these pairs. Bioinformatics methods could help us identify significant genes and pathways related to the pathogenesis of rhinitis. Steroid biosynthesis pathway and metabolic pathways might play important roles in the development of allergic rhinitis (AR). Genes such as CDC42 effector protein 5, solute carrier family 39 member A11 and PR/SET domain 10 might be also associated with the pathogenesis of AR, which provided references for the molecular mechanisms of AR. PMID:29257233

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

Differentiation of Human Dental Stem Cells Reveal a Role for microRNA-218

PubMed Central

Gay, Isabel; Cavender, Adriana; Peto, David; Sun, Zhao; Speer, Aline; Cao, Huojun; Amendt, Brad A.

2013-01-01

Background Regeneration of the lost periodontium is the ultimate goal of periodontal therapy. Advances in tissue engineering have demonstrated the multilineage potential and plasticity of adult stem cells located in the periodontal apparatus. However, it remains unclear how epigenetic mechanisms controlling signals determine tissue specification and cell lineage decisions. To date, no data is available on micro-RNAs (miRNAs) activity behind human-derived dental stem cells. Methods In this study, we isolated periodontal ligament stem cells (PDLSCs), dental pulp stem cells (DPSCs), and gingival stem cells (GSCs) from extracted third molars; human bone marrow stem cells (BMSCs) were used as a positive control. The expression of OCT4A and NANOG was confirmed in these undifferentiated cells. All cells were cultured under osteogenic inductive conditions and RUNX2 expression was analyzed as a marker of mineralized tissue differentiation. A miRNA expression profile was obtained at baseline and after osteogenic induction in all cell types. Results RUNX2 expression demonstrated the successful osteogenic induction of all cell types, which was confirmed by alizarin red stain. The analysis of 765 miRNAs demonstrated a shift in miRNA expression occurred in all four stem cell types, including a decrease in hsa-mir-218 across all differentiated cell populations. Hsa-mir-218 targets RUNX2 and decreases RUNX2 expression in undifferentiated human dental stem cells (DSCs). DSC mineralized tissue type differentiation is associated with a decrease in hsa-mir-218 expression. Conclusions These data reveal a miRNA regulated pathway for the differentiation of human DSCs and a select network of human microRNAs that control DSC osteogenic differentiation. PMID:23662917

Transcriptome profiling and cataloging differential gene expression in floral buds of fertile and sterile lines of cotton (Gossypium hirsutum L.).

PubMed

Hamid, Rasmieh; Tomar, Rukam S; Marashi, Hassan; Shafaroudi, Saeid Malekzadeh; Golakiya, Balaji A; Mohsenpour, Motahhareh

2018-06-20

Cytoplasmic Male Sterility is maternally inherited trait in plants, characterized by failure to produce functional pollen during anther development. Anther development is modulated through the interaction of nuclear and mitochondrial genes. In the present study, differential gene expression of floral buds at the sporogenous stage (SS) and microsporocyte stage (MS) between CGMS and its fertile maintainer line of cotton plants was studied. A total of 320 significantly differentially expressed genes, including 20 down-regulated and 37 up-regulated in CGMS comparing with its maintainer line at the SS stage, as well as and 89 down-regulated and 4 up-regulated in CGMS compared to the fertile line at MS stage. Comparing the two stages in the same line, there were 6 down-regulated differentially expressed genes only induced in CGMS and 9 up-regulated differentially expressed gene only induced in its maintainer. GO analysis revealed essential genes responsible for pollen development, and cytoskeleton category show differential expression between the fertile and CGMS lines. Validation studies by qRT-PCR shows concordance with RNA-seq result. A set of novel SSRs identified in this study can be used in evaluating genetic relationships among cultivars, QTL mapping, and marker-assisted breeding. We reported aberrant expression of genes related to pollen exine formation, and synthesis of pectin lyase, myosine heavy chain, tubulin, actin-beta, heat shock protein and myeloblastosis (MYB) protein as targets for CMS in cotton. The results of this study contribute to basic information for future screening of genes and identification of molecular portraits responsible for CMS as well as to elucidate molecular mechanisms that lead to CMS in cotton. Copyright © 2018 Elsevier B.V. All rights reserved.

Lysyl Oxidase-like-2 (LOXL2) Is a Major Isoform in Chondrocytes and Is Critically Required for Differentiation*

PubMed Central

Iftikhar, Mussadiq; Hurtado, Paola; Bais, Manish V.; Wigner, Nate; Stephens, Danielle N.; Gerstenfeld, Louis C.; Trackman, Philip C.

2011-01-01

The lysyl oxidase family is made up of five members: lysyl oxidase (LOX) and lysyl oxidase-like 1–4 (LOXL1-LOXL4). All members share conserved C-terminal catalytic domains that provide for lysyl oxidase or lysyl oxidase-like enzyme activity; and more divergent propeptide regions. LOX family enzyme activities catalyze the final enzymatic conversion required for the formation of normal biosynthetic collagen and elastin cross-links. The importance of lysyl oxidase enzyme activity to normal bone development has long been appreciated, but regulation and roles for specific LOX isoforms in bone formation in vivo is largely unexplored. Fracture healing recapitulates aspects of endochondral bone development. The present study first investigated the expression of all LOX isoforms in fracture healing. A remarkable coincidence of LOXL2 expression with the chondrogenic phase of fracture healing was found, prompting more detailed analyses of LOXL2 expression in normal growth plates, and LOXL2 expression and function in developing ATDC5 chondrogenic cells. Data show that LOXL2 is expressed by pre-hypertrophic and hypertrophic chondrocytes in vivo, and that LOXL2 expression is regulated in vitro as a function of chondrocyte differentiation. Moreover, LOXL2 knockdown studies in vitro show that LOXL2 expression is required for ATDC5 chondrocyte cell line differentiation through regulation of SNAIL and SOX9, important transcription factors that control chondrocyte differentiation. Taken together, data provide evidence that LOXL2, like LOX, is a multifunctional protein. LOXL2 promotes chondrocyte differentiation by mechanisms that are likely to include roles as both a regulator and an effector of chondrocyte differentiation. PMID:21071451

Whole Blood Gene Expression Profile Associated with Spontaneous Preterm Birth in Women with Threatened Preterm Labor

PubMed Central

Heng, Yujing Jan; Pennell, Craig Edward; Chua, Hon Nian; Perkins, Jonathan Edward; Lye, Stephen James

2014-01-01

Threatened preterm labor (TPTL) is defined as persistent premature uterine contractions between 20 and 37 weeks of gestation and is the most common condition that requires hospitalization during pregnancy. Most of these TPTL women continue their pregnancies to term while only an estimated 5% will deliver a premature baby within ten days. The aim of this work was to study differential whole blood gene expression associated with spontaneous preterm birth (sPTB) within 48 hours of hospital admission. Peripheral blood was collected at point of hospital admission from 154 women with TPTL before any medical treatment. Microarrays were utilized to investigate differential whole blood gene expression between TPTL women who did (n = 48) or did not have a sPTB (n = 106) within 48 hours of admission. Total leukocyte and neutrophil counts were significantly higher (35% and 41% respectively) in women who had sPTB than women who did not deliver within 48 hours (p<0.001). Fetal fibronectin (fFN) test was performed on 62 women. There was no difference in the urine, vaginal and placental microbiology and histopathology reports between the two groups of women. There were 469 significant differentially expressed genes (FDR<0.05); 28 differentially expressed genes were chosen for microarray validation using qRT-PCR and 20 out of 28 genes were successfully validated (p<0.05). An optimal random forest classifier model to predict sPTB was achieved using the top nine differentially expressed genes coupled with peripheral clinical blood data (sensitivity 70.8%, specificity 75.5%). These differentially expressed genes may further elucidate the underlying mechanisms of sPTB and pave the way for future systems biology studies to predict sPTB. PMID:24828675

Reversible effects of oxygen partial pressure on genes associated with placental angiogenesis and differentiation in primary-term cytotrophoblast cell culture.

PubMed

Debiève, F; Depoix, C; Gruson, D; Hubinont, C

2013-09-01

Timely regulated changes in oxygen partial pressure are important for placental formation. Disturbances could be responsible for pregnancy-related diseases like preeclampsia and intrauterine growth restriction. We aimed to (i) determine the effect of oxygen partial pressure on cytotrophoblast differentiation; (ii) measure mRNA expression and protein secretion from genes associated with placental angiogenesis; and (iii) determine the reversibility of these effects at different oxygen partial pressures. Term cytotrophoblasts were incubated at 21% and 2.5% O2 for 96 hr, or were switched between the two oxygen concentrations after 48 hr. Real-time PCR and enzyme-linked immunosorbent assays (ELISAs) were used to evaluate cell fusion and differentiation, measuring transcript levels for those genes involved in cell fusion and placental angiogenesis, including VEGF, PlGF, VEGFR1, sVEGFR1, sENG, INHA, and GCM1. Cytotrophoblasts underwent fusion and differentiation in 2.5% O2 . PlGF expression was inhibited while sVEGFR1 expression increased. VEGF and sENG mRNA expressions increased in 2.5% compared to 21% O2 , but no protein was detected in the cell supernatants. Finally, GCM1 mRNA expression increased during trophoblast differentiation at 21% O2 , but was inhibited at 2.5% O2 . These mRNA expression effects were reversed by returning the cells to 21% O2 . Thus, low-oxygen partial pressure does not inhibit term-cytotrophoblast cell fusion and differentiation in vitro. Lowering the oxygen partial pressure from 21% to 2.5% caused normal-term trophoblasts to reversibly modify their expression of genes associated with placental angiogenesis. This suggests that modifications observed in pregnancy diseases such as preeclampsia or growth retardation are probably due to an extrinsic effect on trophoblasts. Copyright © 2013 Wiley Periodicals, Inc.

Fos Promotes Early Stage Teno-Lineage Differentiation of Tendon Stem/Progenitor Cells in Tendon.

PubMed

Chen, Jialin; Zhang, Erchen; Zhang, Wei; Liu, Zeyu; Lu, Ping; Zhu, Ting; Yin, Zi; Backman, Ludvig J; Liu, Huanhuan; Chen, Xiao; Ouyang, Hongwei

2017-11-01

Stem cells have been widely used in tendon tissue engineering. The lack of refined and controlled differentiation strategy hampers the tendon repair and regeneration. This study aimed to find new effective differentiation factors for stepwise tenogenic differentiation. By microarray screening, the transcript factor Fos was found to be expressed in significantly higher amounts in postnatal Achilles tendon tissue derived from 1 day as compared with 7-days-old rats. It was further confirmed that expression of Fos decreased with time in postnatal rat Achilles tendon, which was accompanied with the decreased expression of multiply tendon markers. The expression of Fos also declined during regular in vitro cell culture, which corresponded to the loss of tendon phenotype. In a cell-sheet and a three-dimensional cell culture model, the expression of Fos was upregulated as compared with in regular cell culture, together with the recovery of tendon phenotype. In addition, significant higher expression of tendon markers was found in Fos-overexpressed tendon stem/progenitor cells (TSPCs), and Fos knock-down gave opposite results. In situ rat tendon repair experiments found more normal tendon-like tissue formed and higher tendon markers expression at 4 weeks postimplantation of Fos-overexpressed TSPCs derived nonscaffold engineering tendon (cell-sheet), as compared with the control group. This study identifies Fos as a new marker and functional driver in the early stage teno-lineage differentiation of tendon, which paves the way for effective stepwise tendon differentiation and future tendon regeneration. Stem Cells Translational Medicine 2017;6:2009-2019. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Redox-mediated enrichment of self-renewing adult human pancreatic cells that possess endocrine differentiation potential.

PubMed

Linning, Katrina D; Tai, Mei-Hui; Madhukar, Burra V; Chang, C C; Reed, Donald N; Ferber, Sarah; Trosko, James E; Olson, L Karl

2004-10-01

The limited availability of transplantable human islets has stimulated the development of methods needed to isolate adult pancreatic stem/progenitor cells capable of self-renewal and endocrine differentiation. The objective of this study was to determine whether modulation of intracellular redox state with N-acetyl-L-cysteine (NAC) would allow for the propagation of pancreatic stem/progenitor cells from adult human pancreatic tissue. Cells were propagated from human pancreatic tissue using a serum-free, low-calcium medium supplemented with NAC and tested for their ability to differentiate when cultured under different growth conditions. Human pancreatic cell (HPC) cultures coexpressed alpha-amylase, albumin, vimentin, and nestin. The HPC cultures, however, did not express other genes associated with differentiated pancreatic exocrine, duct, or endocrine cells. A number of transcription factors involved in endocrine cell development including Beta 2, Islet-1, Nkx6.1, Pax4, and Pax6 were expressed at variable levels in HPC cultures. In contrast, pancreatic duodenal homeobox factor 1 (Pdx-1) expression was extremely low and at times undetectable. Overexpression of Pdx-1 in HPC cultures stimulated somatostatin, glucagon, and carbonic anhydrase expression but had no effect on insulin gene expression. HPC cultures could form 3-dimensional islet-like cell aggregates, and this was associated with expression of somatostatin and glucagon but not insulin. Cultivation of HPCs in a differentiation medium supplemented with nicotinamide, exendin-4, and/or LY294002, an inhibitor of phosphatidylinositol-3 kinase, stimulated expression of insulin mRNA and protein. These data support the use of intracellular redox modulation for the enrichment of pancreatic stem/progenitor cells capable of self-renewal and endocrine differentiation.

Genome-wide gene expression effects in B6C3F1 mouse intestinal epithelia following 7 and 90days of exposure to hexavalent chromium in drinking water.

PubMed

Kopec, Anna K; Kim, Suntae; Forgacs, Agnes L; Zacharewski, Timothy R; Proctor, Deborah M; Harris, Mark A; Haws, Laurie C; Thompson, Chad M

2012-02-15

Chronic administration of high doses of hexavalent chromium [Cr(VI)] as sodium dichromate dihydrate (SDD) elicits alimentary cancers in mice. To further elucidate key events underlying tumor formation, a 90-day drinking water study was conducted in B6C3F1 mice. Differential gene expression was examined in duodenal and jejunal epithelial samples following 7 or 90days of exposure to 0, 0.3, 4, 14, 60, 170 or 520mg/L SDD in drinking water. Genome-wide microarray analyses identified 6562 duodenal and 4448 jejunal unique differentially expressed genes at day 8, and 4630 and 4845 unique changes, respectively, in the duodenum and jejunum at day 91. Comparative analysis identified significant overlap in duodenal and jejunal differential gene expression. Automated dose-response modeling identified >80% of the differentially expressed genes exhibited sigmoidal dose-response curves with EC(50) values ranging from 10 to 100mg/L SDD. Only 16 genes satisfying the dose-dependent differential expression criteria had EC(50) values <10mg/L SDD, 3 of which were regulated by Nrf2, suggesting oxidative stress in response to SDD at low concentrations. Analyses of differentially expressed genes identified over-represented functions associated with oxidative stress, cell cycle, lipid metabolism, and immune responses consistent with the reported effects on redox status and histopathology at corresponding SDD drinking water concentrations. Collectively, these data are consistent with a mode of action involving oxidative stress and cytotoxicity as early key events. This suggests that the tumorigenic effects of chronic Cr(VI) oral exposure likely require chronic tissue damage and compensatory epithelial cell proliferation. Copyright © 2011 Elsevier Inc. All rights reserved.

Altered MENIN expression disrupts the MAFA differentiation pathway in insulinoma.

PubMed

Hamze, Z; Vercherat, C; Bernigaud-Lacheretz, A; Bazzi, W; Bonnavion, R; Lu, J; Calender, A; Pouponnot, C; Bertolino, P; Roche, C; Stein, R; Scoazec, J Y; Zhang, C X; Cordier-Bussat, M

2013-12-01

The protein MENIN is the product of the multiple endocrine neoplasia type I (MEN1) gene. Altered MENIN expression is one of the few events that are clearly associated with foregut neuroendocrine tumours (NETs), classical oncogenes or tumour suppressors being not involved. One of the current challenges is to understand how alteration of MENIN expression contributes to the development of these tumours. We hypothesised that MENIN might regulate factors maintaining endocrine-differentiated functions. We chose the insulinoma model, a paradigmatic example of well-differentiated pancreatic NETs, to study whether MENIN interferes with the expression of v-MAF musculoaponeurotic fibrosarcoma oncogene homologue A (MAFA), a master glucose-dependent transcription factor in differentiated β-cells. Immunohistochemical analysis of a series of human insulinomas revealed a correlated decrease in both MENIN and MAFA. Decreased MAFA expression resulting from targeted Men1 ablation was also consistently observed in mouse insulinomas. In vitro analyses using insulinoma cell lines showed that MENIN regulated MAFA protein and mRNA levels, and bound to Mafa promoter sequences. MENIN knockdown concomitantly decreased mRNA expression of both Mafa and β-cell differentiation markers (Ins1/2, Gck, Slc2a2 and Pdx1) and, in parallel, increased the proliferation rate of tumours as measured by bromodeoxyuridine incorporation. Interestingly, MAFA knockdown alone also increased proliferation rate but did not affect the expression of candidate proliferation genes regulated by MENIN. Finally, MENIN variants with missense mutations detected in patients with MEN1 lost the WT MENIN properties to regulate MAFA. Together, our findings unveil a previously unsuspected MENIN/MAFA connection regarding control of the β-cell differentiation/proliferation balance, which could contribute to tumorigenesis.

Transcription factors SOHLH1 and SOHLH2 coordinate oocyte differentiation without affecting meiosis I.

PubMed

Shin, Yong-Hyun; Ren, Yu; Suzuki, Hitomi; Golnoski, Kayla J; Ahn, Hyo Won; Mico, Vasil; Rajkovic, Aleksandar

2017-06-01

Following migration of primordial germ cells to the genital ridge, oogonia undergo several rounds of mitotic division and enter meiosis at approximately E13.5. Most oocytes arrest in the dictyate (diplotene) stage of meiosis circa E18.5. The genes necessary to drive oocyte differentiation in parallel with meiosis are unknown. Here, we have investigated whether expression of spermatogenesis and oogenesis bHLH transcription factor 1 (Sohlh1) and Sohlh2 coordinates oocyte differentiation within the embryonic ovary. We found that SOHLH2 protein was expressed in the mouse germline as early as E12.5 and preceded SOHLH1 protein expression, which occurred circa E15.5. SOHLH1 protein appearance at E15.5 correlated with SOHLH2 translocation from the cytoplasm into the nucleus and was dependent on SOHLH1 expression. NOBOX oogenesis homeobox (NOBOX) and LIM homeobox protein 8 (LHX8), two important regulators of postnatal oogenesis, were coexpressed with SOHLH1. Single deficiency of Sohlh1 or Sohlh2 disrupted the expression of LHX8 and NOBOX in the embryonic gonad without affecting meiosis. Sohlh1-KO infertility was rescued by conditional expression of the Sohlh1 transgene after the onset of meiosis. However, Sohlh1 or Sohlh2 transgene expression could not rescue Sohlh2-KO infertility due to a lack of Sohlh1 or Sohlh2 expression in rescued mice. Our results indicate that Sohlh1 and Sohlh2 are essential regulators of oocyte differentiation but do not affect meiosis I.

A small population of resident limb bud mesenchymal cells express few MSC-associated markers, but the expression of these markers is increased immediately after cell culture.

PubMed

Marín-Llera, Jessica Cristina; Chimal-Monroy, Jesús

2018-05-01

Skeletal progenitors are derived from resident limb bud mesenchymal cells of the vertebrate embryos. However, it remains poorly understood if they represent stem cells, progenitors, or multipotent mesenchymal stromal cells (MSC). Derived-MSC of different adult tissues under in vitro experimental conditions can differentiate into the same cellular lineages that are present in the limb. Here, comparing non-cultured versus cultured mesenchymal limb bud cells, we determined the expression of MSC-associated markers, the in vitro differentiation capacity and their gene expression profile. Results showed that in freshly isolated limb bud mesenchymal cells, the proportion of cells expressing Sca1, CD44, CD105, CD90, and CD73 is very low and a low expression of lineage-specific genes was observed. However, recently seeded limb bud mesenchymal cells acquired Sca1 and CD44 markers and the expression of the key differentiation genes Runx2 and Sox9, while Scx and Pparg genes decreased. Also, their chondrogenic differentiation capacity decreased through cellular passages while the osteogenic increased. Our findings suggest that the modification of the cell adhesion process through the in vitro method changed the limb mesenchymal cell immunophenotype leading to the expression and maintenance of common MSC-associated markers. These findings could have a significant impact on MSC study and isolation strategy because they could explain common variations observed in the MSC immunophenotype in different tissues. © 2018 International Federation for Cell Biology.

Role of neurotransmitter Substance P in progression of oral squamous cell carcinoma.

PubMed

Mehboob, Riffat; Tanvir, Imrana; Warraich, Riaz Ahmad; Perveen, Shahida; Yasmeen, Sehrish; Ahmad, Fridoon Jawad

2015-03-01

Oral squamous cell carcinoma (OSCC) is the most frequent type of head and neck cancers. In the present study, we evaluated the expression and distribution of Substance P (SP) in different grades of OSCC and role of SP in its proliferation and progression. Forty OSCC biopsies were immunohistochemically analyzed by using SP antibody, including 29 male and 11 female cases. 35% were well differentiated, 35% moderately differentiated and 30% poorly differentiated OSCC. The majority of patients were in the age range of 41-80 years. 62% of the cases were positive for SP. SP positivity was expressed in the cytoplasm of the tumor cells. Most of the positive cases were from the tongue region. 93% of moderately differentiated, 92% of poorly differentiated and 8% of well-differentiated carcinomas were SP-positive, but SP expression intensity was highest in poorly differentiated cases (+3). More positive patients were males (68.96% of all male patients) with moderately and poorly differentiated OSCC. Among all positive cases, 48% were poorly differentiated, 48% moderately differentiated and 4% well differentiated. Strong expression of SP in poorly and moderately differentiated cases suggests a role of SP in the progression and development of tumor. Expression of SP in the current study increased as the proliferation of cells increased. Prevalence of oral cancer in males may be due to the fact that they smoke and use pan, chewing gum, beetle nut etc. in this region. SP antagonists can help in the reduction and inhibition of oral cancer. SP has a diagnostic value with sensitivity of 92.5% and specificity of 93.7%. The positive predictive value is 96.2% and the negative predictive value 88.2%. Copyright © 2014 Elsevier GmbH. All rights reserved.

Expressed Sequence Reference Standards for Evaluating Stage-specific Gene Expression in Southern Green Lacewings, Chrysoperla rufilabris

USDA-ARS?s Scientific Manuscript database

Five developmental stages of Chrysoperla rufilabris were tested using nine primer pairs. Three sequences were highly expressed at all life stages and six were differentially expressed. These primer pairs may be used as standards to quantitate functional gene expression associated with physiological ...

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.

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

Effect of environmentally-relevant concentrations of nonylphenol on sexual differentiation in zebrafish: a multi-generational study

NASA Astrophysics Data System (ADS)

Sun, Dong; Chen, Qi; He, Ning; Diao, Pan-Pan; Jia, Li-Xing; Duan, Shun-Shan

2017-02-01

Nonylphenol (NP) is a persistent environmental chemical that can disrupt the organism’s endocrine system, and is detected in the surface water and sea. In this study, we investigated whether NP can alter transcriptional expression of sexual differentiation-related genes. Three generations of zebrafish were exposed to 0, 2, 20 and 200 μg·L-1 of NP, and transcriptional expression of sexual differentiation genes were assessed in 10, 20 and 40 dpf in the F1 and F2 generations. Growth of zebrafish exposed to 200 μg·L-1 of NP was inhibited at 125 dpf in the F1 generation. 20 μg·L-1 of NP resulted in 80% females in the F1 generation, but had no effect on the F2 generation. In terms of the sexual differentiation genes, the transcriptional expression of cyp19a1a and esr1 genes were upregulated in 20 μg·L-1 of NP in the F1 generation. But expression of the sexual differentiation genes were not affected in the F2 generation. Overall, NP could affect sexual differentiation and gene transcriptional expression in the F1 generation. The tolerance of contaminant in the offsprings was improved at low concentration.

Microgravity Induction of TRAIL Expression in Preosteoclast Cells Enhances Osteoclast Differentiation

NASA Astrophysics Data System (ADS)

Sambandam, Yuvaraj; Baird, Kelsey L.; Stroebel, Maxwell; Kowal, Emily; Balasubramanian, Sundaravadivel; Reddy, Sakamuri V.

2016-05-01

Evidence indicates that astronauts experience significant bone loss in space. We previously showed that simulated microgravity (μXg) using the NASA developed rotary cell culture system (RCCS) enhanced bone resorbing osteoclast (OCL) differentiation. However, the mechanism by which μXg increases OCL formation is unclear. RANK/RANKL signaling pathway is critical for OCL differentiation. Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) has been shown to increase osteoclastogenesis. We hypothesize that TRAIL may play an important role in μXg enhanced OCL differentiation. In this study, we identified by RT profiler PCR array screening that μXg induces high levels of TRAIL expression in murine preosteoclast cells in the absence of RANKL stimulation compared to ground based (Xg) cultures. We further identified that μXg elevated the adaptor protein TRAF-6 and fusion genes OC-STAMP and DC-STAMP expression in preosteoclast cells. Interestingly, neutralizing antibody against TRAIL significantly reduced μXg induced OCL formation. We further identified that over-expression of pTRAIL in RAW 264.7 cells enhanced OCL differentiation. These results indicate that TRAIL signaling plays an important role in the μXg increased OCL differentiation. Therefore, inhibition of TRAIL expression could be an effective countermeasure for μXg induced bone loss.

Transcriptome analyses and differential gene expression in a non-model fish species with alternative mating tactics

PubMed Central

2014-01-01

Background Social dominance is important for the reproductive success of males in many species. In the black-faced blenny (Tripterygion delaisi) during the reproductive season, some males change color and invest in nest making and defending a territory, whereas others do not change color and ‘sneak’ reproductions when females lay their eggs. Using RNAseq, we profiled differential gene expression between the brains of territorial males, sneaker males, and females to study the molecular signatures of male dimorphism. Results We found that more genes were differentially expressed between the two male phenotypes than between males and females, suggesting that during the reproductive period phenotypic plasticity is a more important factor in differential gene expression than sexual dimorphism. The territorial male overexpresses genes related to synaptic plasticity and the sneaker male overexpresses genes involved in differentiation and development. Conclusions Previously suggested candidate genes for social dominance in the context of alternative mating strategies seem to be predominantly species-specific. We present a list of novel genes which are differentially expressed in Tripterygion delaisi. This is the first genome-wide study for a molecular non-model species in the context of alternative mating strategies and provides essential information for further studies investigating the molecular basis of social dominance. PMID:24581002

Decline in c-myc mRNA expression but not the induction of c-fos mRNA expression is associated with differentiation of SH-SY5Y human neuroblastoma cells

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

Jalava, A.M.; Heikkilae, J.E.; Akerman, K.E.O.

1988-11-01

The induction of differentiation in SH-SY5Y human neuroblastoma cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is accompanied by a rapid and a transient expression of c-fos mRNA and a down-regulation of c-myc RNA. The TPA-induced expression of c-fos mRNA was inhibited by H-7, a specific inhibitor of protein kinase C (PK-C). Dioctanoylglycerol (DiC{sub 8}) failed to induce differentiation of SH-SY5Y cells or to down-regulate c-myc mRNA but it did induce the expression of c-fos mRNA. Treatment of IMR-32 human neuroblastoma cells with TPA did not cause differentiation although c-fos mRNA was induced. Since PK-C in SH-SY5Y cells was activated by both TPA andmore » DiC{sub 8} it is suggested that the activation of PK-C alone is not sufficient to induce differentiation in SH-SY5Y cells. The down-regulation of c-myc mRNA rather than the induction of c-fos mRNA seems to be associated with differentiation process in SH-SY5Y cells.« less

Transcriptome analyses and differential gene expression in a non-model fish species with alternative mating tactics.

PubMed

Schunter, Celia; Vollmer, Steven V; Macpherson, Enrique; Pascual, Marta

2014-02-28

Social dominance is important for the reproductive success of males in many species. In the black-faced blenny (Tripterygion delaisi) during the reproductive season, some males change color and invest in nest making and defending a territory, whereas others do not change color and 'sneak' reproductions when females lay their eggs. Using RNAseq, we profiled differential gene expression between the brains of territorial males, sneaker males, and females to study the molecular signatures of male dimorphism. We found that more genes were differentially expressed between the two male phenotypes than between males and females, suggesting that during the reproductive period phenotypic plasticity is a more important factor in differential gene expression than sexual dimorphism. The territorial male overexpresses genes related to synaptic plasticity and the sneaker male overexpresses genes involved in differentiation and development. Previously suggested candidate genes for social dominance in the context of alternative mating strategies seem to be predominantly species-specific. We present a list of novel genes which are differentially expressed in Tripterygion delaisi. This is the first genome-wide study for a molecular non-model species in the context of alternative mating strategies and provides essential information for further studies investigating the molecular basis of social dominance.

Differentiation of Odontoblast-Like Cells From Mouse Induced Pluripotent Stem Cells by Pax9 and Bmp4 Transfection.

PubMed

Seki, Daisuke; Takeshita, Nobuo; Oyanagi, Toshihito; Sasaki, Shutaro; Takano, Ikuko; Hasegawa, Masakazu; Takano-Yamamoto, Teruko

2015-09-01

The field of tooth regeneration has progressed in recent years, and human tooth regeneration could become viable in the future. Because induced pluripotent stem (iPS) cells can differentiate into odontogenic cells given appropriate conditions, iPS cells are a potential cell source for tooth regeneration. However, a definitive method to induce iPS cell-derived odontogenic cells has not been established. We describe a novel method of odontoblast differentiation from iPS cells using gene transfection. We generated mouse iPS cell-derived neural crest-like cells (iNCLCs), which exhibited neural crest markers. Next, we differentiated iNCLCs into odontoblast-like cells by transfection of Pax9 and Bmp4 expression plasmids. Exogenous Pax9 upregulated expression of Msx1 and dentin matrix protein 1 (Dmp1) in iNCLCs but not bone morphogenetic protein 4 (Bmp4) or dentin sialophosphoprotein (Dspp). Exogenous Bmp4 upregulated expression of Msx1, Dmp1, and Dspp in iNCLCs, but not Pax9. Moreover, cotransfection of Pax9 and Bmp4 plasmids in iNCLCs revealed a higher expression of Pax9 than when Pax9 plasmid was used alone. In contrast, exogenous Pax9 downregulated Bmp4 overexpression. Cotransfection of Pax9 and Bmp4 synergistically upregulated Dmp1 expression; however, Pax9 overexpression downregulated exogenous Bmp4-induced Dspp expression. Together, these findings suggest that an interaction between exogenous Pax9- and Bmp4-induced signaling modulated Dmp1 and Dspp expression. In conclusion, transfection of Pax9 and Bmp4 expression plasmids in iNCLCs induced gene expression associated with odontoblast differentiation, suggesting that iNCLCs differentiated into odontoblast-like cells. The iPS cell-derived odontoblast-like cells could be a useful cell source for tooth regeneration. It has been reported that induced pluripotent stem (iPS) cells differentiate into odontogenic cells by administration of recombinant growth factors and coculture with odontogenic cells. Therefore, they can be potential cell sources for tooth regeneration. However, these previous methods still have problems, such as usage of other cell types, heterogeneity of differentiated cells, and tumorigenicity. In the present study, a novel method to differentiate iPS cells into odontoblast-like cells without tumorigenicity using gene transfection was established. It is an important advance in the establishment of efficient methods to generate homogeneous functional odontogenic cells derived from iPS cells. ©AlphaMed Press.

Ectopic Expression of Nolz-1 in Neural Progenitors Promotes Cell Cycle Exit/Premature Neuronal Differentiation Accompanying with Abnormal Apoptosis in the Developing Mouse Telencephalon

PubMed Central

Chang, Sunny Li-Yun; Chen, Shih-Yun; Huang, Huai-Huei; Ko, Hsin-An; Liu, Pei-Tsen; Liu, Ya-Chi; Chen, Ping-Hau; Liu, Fu-Chin

2013-01-01

Nolz-1, as a murine member of the NET zinc-finger protein family, is expressed in post-mitotic differentiating neurons of striatum during development. To explore the function of Nolz-1 in regulating the neurogenesis of forebrain, we studied the effects of ectopic expression of Nolz-1 in neural progenitors. We generated the Cre-loxP dependent conditional transgenic mice in which Nolz-1 was ectopically expressed in proliferative neural progenitors. Ectopic expression of Nolz-1 in neural progenitors by intercrossing the Nolz-1 conditional transgenic mice with the nestin-Cre mice resulted in hypoplasia of telencephalon in double transgenic mice. Decreased proliferation of neural progenitor cells were found in the telencephalon, as evidenced by the reduction of BrdU−, Ki67− and phospho-histone 3-positive cells in E11.5–12.5 germinal zone of telencephalon. Transgenic Nolz-1 also promoted cell cycle exit and as a consequence might facilitate premature differentiation of progenitors, because TuJ1-positive neurons were ectopically found in the ventricular zone and there was a general increase of TuJ1 immunoreactivity in the telencephalon. Moreover, clusters of strong TuJ1-expressing neurons were present in E12.5 germinal zone. Some of these strong TuJ1-positive clusters, however, contained apoptotic condensed DNA, suggesting that inappropriate premature differentiation may lead to abnormal apoptosis in some progenitor cells. Consistent with the transgenic mouse analysis in vivo, similar effects of Nozl-1 over-expression in induction of apoptosis, inhibition of cell proliferation and promotion of neuronal differentiation were also observed in three different N18, ST14A and N2A neural cell lines in vitro. Taken together, our study indicates that ectopic expression of Nolz-1 in neural progenitors promotes cell cycle exit/premature neuronal differentiation and induces abnormal apoptosis in the developing telencephalon. PMID:24073229

Ectopic expression of nolz-1 in neural progenitors promotes cell cycle exit/premature neuronal differentiation accompanying with abnormal apoptosis in the developing mouse telencephalon.

PubMed

Chang, Sunny Li-Yun; Chen, Shih-Yun; Huang, Huai-Huei; Ko, Hsin-An; Liu, Pei-Tsen; Liu, Ya-Chi; Chen, Ping-Hau; Liu, Fu-Chin

2013-01-01

Nolz-1, as a murine member of the NET zinc-finger protein family, is expressed in post-mitotic differentiating neurons of striatum during development. To explore the function of Nolz-1 in regulating the neurogenesis of forebrain, we studied the effects of ectopic expression of Nolz-1 in neural progenitors. We generated the Cre-loxP dependent conditional transgenic mice in which Nolz-1 was ectopically expressed in proliferative neural progenitors. Ectopic expression of Nolz-1 in neural progenitors by intercrossing the Nolz-1 conditional transgenic mice with the nestin-Cre mice resulted in hypoplasia of telencephalon in double transgenic mice. Decreased proliferation of neural progenitor cells were found in the telencephalon, as evidenced by the reduction of BrdU-, Ki67- and phospho-histone 3-positive cells in E11.5-12.5 germinal zone of telencephalon. Transgenic Nolz-1 also promoted cell cycle exit and as a consequence might facilitate premature differentiation of progenitors, because TuJ1-positive neurons were ectopically found in the ventricular zone and there was a general increase of TuJ1 immunoreactivity in the telencephalon. Moreover, clusters of strong TuJ1-expressing neurons were present in E12.5 germinal zone. Some of these strong TuJ1-positive clusters, however, contained apoptotic condensed DNA, suggesting that inappropriate premature differentiation may lead to abnormal apoptosis in some progenitor cells. Consistent with the transgenic mouse analysis in vivo, similar effects of Nozl-1 over-expression in induction of apoptosis, inhibition of cell proliferation and promotion of neuronal differentiation were also observed in three different N18, ST14A and N2A neural cell lines in vitro. Taken together, our study indicates that ectopic expression of Nolz-1 in neural progenitors promotes cell cycle exit/premature neuronal differentiation and induces abnormal apoptosis in the developing telencephalon.

Microglia Transcriptome Changes in a Model of Depressive Behavior after Immune Challenge

PubMed Central

Gonzalez-Pena, Dianelys; Nixon, Scott E.; O’Connor, Jason C.; Southey, Bruce R.; Lawson, Marcus A.; McCusker, Robert H.; Borras, Tania; Machuca, Debbie; Hernandez, Alvaro G.; Dantzer, Robert; Kelley, Keith W.; Rodriguez-Zas, Sandra L.

2016-01-01

Depression symptoms following immune response to a challenge have been reported after the recovery from sickness. A RNA-Seq study of the dysregulation of the microglia transcriptome in a model of inflammation-associated depressive behavior was undertaken. The transcriptome of microglia from mice at day 7 after Bacille Calmette Guérin (BCG) challenge was compared to that from unchallenged Control mice and to the transcriptome from peripheral macrophages from the same mice. Among the 562 and 3,851 genes differentially expressed between BCG-challenged and Control mice in microglia and macrophages respectively, 353 genes overlapped between these cells types. Among the most differentially expressed genes in the microglia, serum amyloid A3 (Saa3) and cell adhesion molecule 3 (Cadm3) were over-expressed and coiled-coil domain containing 162 (Ccdc162) and titin-cap (Tcap) were under-expressed in BCG-challenged relative to Control. Many of the differentially expressed genes between BCG-challenged and Control mice were associated with neurological disorders encompassing depression symptoms. Across cell types, S100 calcium binding protein A9 (S100A9), interleukin 1 beta (Il1b) and kynurenine 3-monooxygenase (Kmo) were differentially expressed between challenged and control mice. Immune response, chemotaxis, and chemokine activity were among the functional categories enriched by the differentially expressed genes. Functional categories enriched among the 9,117 genes differentially expressed between cell types included leukocyte regulation and activation, chemokine and cytokine activities, MAP kinase activity, and apoptosis. More than 200 genes exhibited alternative splicing events between cell types including WNK lysine deficient protein kinase 1 (Wnk1) and microtubule-actin crosslinking factor 1(Macf1). Network visualization revealed the capability of microglia to exhibit transcriptome dysregulation in response to immune challenge still after resolution of sickness symptoms, albeit lower than that observed in macrophages. The persistent transcriptome dysregulation in the microglia shared patterns with neurological disorders indicating that the associated persistent depressive symptoms share a common transcriptome basis. PMID:26959683

Microglia Transcriptome Changes in a Model of Depressive Behavior after Immune Challenge.

PubMed

Gonzalez-Pena, Dianelys; Nixon, Scott E; O'Connor, Jason C; Southey, Bruce R; Lawson, Marcus A; McCusker, Robert H; Borras, Tania; Machuca, Debbie; Hernandez, Alvaro G; Dantzer, Robert; Kelley, Keith W; Rodriguez-Zas, Sandra L

2016-01-01

Depression symptoms following immune response to a challenge have been reported after the recovery from sickness. A RNA-Seq study of the dysregulation of the microglia transcriptome in a model of inflammation-associated depressive behavior was undertaken. The transcriptome of microglia from mice at day 7 after Bacille Calmette Guérin (BCG) challenge was compared to that from unchallenged Control mice and to the transcriptome from peripheral macrophages from the same mice. Among the 562 and 3,851 genes differentially expressed between BCG-challenged and Control mice in microglia and macrophages respectively, 353 genes overlapped between these cells types. Among the most differentially expressed genes in the microglia, serum amyloid A3 (Saa3) and cell adhesion molecule 3 (Cadm3) were over-expressed and coiled-coil domain containing 162 (Ccdc162) and titin-cap (Tcap) were under-expressed in BCG-challenged relative to Control. Many of the differentially expressed genes between BCG-challenged and Control mice were associated with neurological disorders encompassing depression symptoms. Across cell types, S100 calcium binding protein A9 (S100A9), interleukin 1 beta (Il1b) and kynurenine 3-monooxygenase (Kmo) were differentially expressed between challenged and control mice. Immune response, chemotaxis, and chemokine activity were among the functional categories enriched by the differentially expressed genes. Functional categories enriched among the 9,117 genes differentially expressed between cell types included leukocyte regulation and activation, chemokine and cytokine activities, MAP kinase activity, and apoptosis. More than 200 genes exhibited alternative splicing events between cell types including WNK lysine deficient protein kinase 1 (Wnk1) and microtubule-actin crosslinking factor 1(Macf1). Network visualization revealed the capability of microglia to exhibit transcriptome dysregulation in response to immune challenge still after resolution of sickness symptoms, albeit lower than that observed in macrophages. The persistent transcriptome dysregulation in the microglia shared patterns with neurological disorders indicating that the associated persistent depressive symptoms share a common transcriptome basis.

Peroxisome proliferator-activated receptor-β/δ inhibits human neuroblastoma cell tumorigenesis by inducing p53- and SOX2-mediated cell differentiation.

PubMed

Yao, Pei-Li; Chen, Liping; Dobrzański, Tomasz P; Zhu, Bokai; Kang, Boo-Hyon; Müller, Rolf; Gonzalez, Frank J; Peters, Jeffrey M

2017-05-01

Neuroblastoma is a common childhood cancer typically treated by inducing differentiation with retinoic acid (RA). Peroxisome proliferator-activated receptor-β/δ, (PPARβ/δ) is known to promote terminal differentiation of many cell types. In the present study, PPARβ/δ was over-expressed in three human neuroblastoma cell lines, NGP, SK-N-BE(2), and IMR-32, that exhibit high, medium, and low sensitivity, respectively, to retinoic acid-induced differentiation to determine if PPARβ/δ and retinoic acid receptors (RARs) could be jointly targeted to increase the efficacy of treatment. All-trans-RA (atRA) decreased expression of SRY (sex determining region Y)-box 2 (SOX2), a stem cell regulator and marker of de-differentiation, in NGP and SK-N-BE(2) cells with inactive or mutant tumor suppressor p53, respectively. However, atRA did not suppress SOX2 expression in IMR-32 cells carrying wild-type p53. Over-expression and/or ligand activation of PPARβ/δ reduced the average volume and weight of ectopic tumor xenografts from NGP, SK-N-BE(2), or IMR-32 cells compared to controls. Compared with that found with atRA, PPARβ/δ suppressed SOX2 expression in NGP and SK-N-BE(2) cells and ectopic xenografts, and was also effective in suppressing SOX2 expression in IMR-32 cells that exhibit higher p53 expression compared to the former cell lines. Combined, these observations demonstrate that activating or over-expressing PPARβ/δ induces cell differentiation through p53- and SOX2-dependent signaling pathways in neuroblastoma cells and tumors. This suggests that combinatorial activation of both RARα and PPARβ/δ may be suitable as an alternative therapeutic approach for RA-resistant neuroblastoma patients. Published [2016]. This article is a U.S. Government work and is in the public domain in the USA.

Subcellular proteomic analysis of host-pathogen interactions using human monocytes exposed to Yersinia pestis and Yersinia pseudotuberculosis

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

Zhang, C G; Gonzales, A D; Choi, M W

2004-05-20

Yersinia pestis, the etiological agent of plague, is of concern to human health both from an infectious disease and a civilian biodefense perspective. While Y. pestis and Y. pseudotuberculosis share more than 90% DNA homology, they have significantly different clinical manifestations. Plague is often fatal if untreated, yet Y. pseudotuberculosis causes severe intestinal distress and is rarely fatal. A better understanding of host response to these closely related pathogens may help explain the different mechanisms of virulence and pathogenesis that result in such different clinical outcomes. The aim of this study was to characterize host protein expression changes in humanmore » monocyte-like U937 cells after exposure to Y. pestis and Y. pseudotuberculosis. In order to gain global proteomic coverage of host response, proteins from cytoplasmic, nuclear and membrane fractions of host cells were studied by 2-dimensional differential gel electrophoresis (2-D DIGE) and relative protein expression differences were quantitated. Differentially expressed proteins, with at least 1.5 fold expression changes and p values of 0.01 or less, were identified by MALDI-MS or LC/MS/MS. With these criteria, differential expression was detected in 16 human proteins after Y. pestis exposure and 13 human proteins after Y. pseudotuberculosis exposure, of which only two of the differentially expressed proteins identified were shared between the two exposures. Proteins identified in this study are reported to be involved in a wide spectrum of cellular functions and host defense mechanisms including apoptosis, cytoskeletal rearrangement, protein synthesis and degradation, DNA replication and transcription, metabolism, protein folding, and cell signaling. Notably, the differential expression patterns observed can distinguish the two pathogen exposures from each other and from unexposed host cells. The functions of the differentially expressed proteins identified provide insight on the different virulence and pathogenic mechanisms of Y. pestis and Y. pseudotuberculosis.« less

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.

Effects of insulin, triiodothyronine and fat soluble vitamins on adipocyte differentiation and LPL gene expression in the stromal-vascular cells of red sea bream, Pagrus major.

PubMed

Oku, Hiromi; Tokuda, Masaharu; Okumura, Takuji; Umino, Tetsuya

2006-07-01

Various kinds of hormones including insulin, triiodothyronine (T(3)) and fat-soluble vitamins have been proposed as mediators of adipocyte differentiation in mammals. To investigate the factors which are responsible for fish adipocyte differentiation, we developed a serum-free culture system of stromal-vascular cells of red sea bream adipose tissue and examined the effects of bovine insulin, T(3), and fat-soluble vitamins (all-trans retinoic acid, retinyl acetate and 1,25-dihydroxyvitamin D(3)) on the differentiation-linked expression of the lipoprotein lipase (LPL) gene. As assessed by the increase in LPL gene expression after 3 day cultivation, like in mammalian adipocytes, insulin enhanced the adipocyte differentiation in a concentration-dependent manner. During 2 week cultivation, bovine insulin promoted lipid accumulation in differentiating adipocytes concentration-dependently until the terminal differentiation. These results indicate that the differentiation of fish adipocytes is inducible by insulin alone. T(3) alone had no effect but enhanced the differentiation-linked LPL gene expression in the presence of insulin. Fat-soluble vitamins, unlike in mammalian adipocytes, did not show any significant effects. The method developed in this study should be of interest for the characterization of factors involved in fish adipocyte differentiation.

A combined gene expression and functional study reveals the crosstalk between N-Myc and differentiation-inducing microRNAs in neuroblastoma cells

PubMed Central

Zhao, Zhenze; Ma, Xiuye; Shelton, Spencer D.; Sung, Derek C.; Li, Monica; Hernandez, Daniel; Zhang, Maggie; Losiewicz, Michael D.; Chen, Yidong; Pertsemlidis, Alexander; Yu, Xiaojie; Liu, Yuanhang; Du, Liqin

2016-01-01

MYCN amplification is the most common genetic alteration in neuroblastoma and plays a critical role in neuroblastoma tumorigenesis. MYCN regulates neuroblastoma cell differentiation, which is one of the mechanisms underlying its oncogenic function. We recently identified a group of differentiation-inducing microRNAs. Given the demonstrated inter-regulation between MYCN and microRNAs, we speculated that MYCN and the differentiation-inducing microRNAs might form an interaction network to control the differentiation of neuroblastoma cells. In this study, we found that eight of the thirteen differentiation-inducing microRNAs, miR-506-3p, miR-124-3p, miR-449a, miR-34a-5p, miR-449b-5p, miR-103a-3p, miR-2110 and miR-34b-5p, inhibit N-Myc expression by either directly targeting the MYCN 3′UTR or through indirect regulations. Further investigation showed that both MYCN-dependent and MYCN-independent pathways play roles in mediating the differentiation-inducing function of miR-506-3p and miR-449a, two microRNAs that dramatically down-regulate MYCN expression. On the other hand, we found that N-Myc inhibits the expression of multiple differentiation-inducing microRNAs, suggesting that these miRNAs play a role in mediating the function of MYCN. In examining the published dataset collected from clinical neuroblastoma specimens, we found that expressions of two miRNAs, miR-137 and miR-2110, were significantly anti-correlated with MYCN mRNA levels, suggesting their interactions with MYCN play a clinically-relevant role in maintaining the MYCN and miRNA expression levels in neuroblastoma. Our findings altogether suggest that MYCN and differentiation-inducing miRNAs form an interaction network that play an important role in neuroblastoma tumorigenesis through regulating cell differentiation. PMID:27764804

A combined gene expression and functional study reveals the crosstalk between N-Myc and differentiation-inducing microRNAs in neuroblastoma cells.

PubMed

Zhao, Zhenze; Ma, Xiuye; Shelton, Spencer D; Sung, Derek C; Li, Monica; Hernandez, Daniel; Zhang, Maggie; Losiewicz, Michael D; Chen, Yidong; Pertsemlidis, Alexander; Yu, Xiaojie; Liu, Yuanhang; Du, Liqin

2016-11-29

MYCN amplification is the most common genetic alteration in neuroblastoma and plays a critical role in neuroblastoma tumorigenesis. MYCN regulates neuroblastoma cell differentiation, which is one of the mechanisms underlying its oncogenic function. We recently identified a group of differentiation-inducing microRNAs. Given the demonstrated inter-regulation between MYCN and microRNAs, we speculated that MYCN and the differentiation-inducing microRNAs might form an interaction network to control the differentiation of neuroblastoma cells. In this study, we found that eight of the thirteen differentiation-inducing microRNAs, miR-506-3p, miR-124-3p, miR-449a, miR-34a-5p, miR-449b-5p, miR-103a-3p, miR-2110 and miR-34b-5p, inhibit N-Myc expression by either directly targeting the MYCN 3'UTR or through indirect regulations. Further investigation showed that both MYCN-dependent and MYCN-independent pathways play roles in mediating the differentiation-inducing function of miR-506-3p and miR-449a, two microRNAs that dramatically down-regulate MYCN expression. On the other hand, we found that N-Myc inhibits the expression of multiple differentiation-inducing microRNAs, suggesting that these miRNAs play a role in mediating the function of MYCN. In examining the published dataset collected from clinical neuroblastoma specimens, we found that expressions of two miRNAs, miR-137 and miR-2110, were significantly anti-correlated with MYCN mRNA levels, suggesting their interactions with MYCN play a clinically-relevant role in maintaining the MYCN and miRNA expression levels in neuroblastoma. Our findings altogether suggest that MYCN and differentiation-inducing miRNAs form an interaction network that play an important role in neuroblastoma tumorigenesis through regulating cell differentiation.

Dexamethasone facilitates erythropoiesis in murine embryonic stem cells differentiating into hematopoietic cells in vitro

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

Srivastava, Anand S.; Kaushal, Sharmeela; Mishra, Rangnath

2006-07-28

Differentiating embryonic stem (ES) cells are increasingly emerging as an important source of hematopoietic progenitors with a potential to be useful for both basic and clinical research applications. It has been suggested that dexamethasone facilitates differentiation of ES cells towards erythrocytes but the mechanism responsible for sequential expression of genes regulating this process are not well-understood. Therefore, we in vitro induced differentiation of murine ES cells towards erythropoiesis and studied the sequential expression of a set of genes during the process. We hypothesized that dexamethasone-activates its cognate nuclear receptors inducing up-regulation of erythropoietic genes such as GATA-1, Flk-1, Epo-R, andmore » direct ES cells towards erythropoietic differentiation. ES cells were cultured in primary hematopoietic differentiation media containing methyl-cellulose, IMDM, IL-3, IL-6, and SCF to promote embryoid body (EB) formation. Total RNA of day 3, 5, and 9-old EBs was isolated for gene expression studies using RT-PCR. Cells from day 9 EBs were subjected to secondary differentiation using three different cytokines and growth factors combinations: (1) SCF, EPO, dexamethasone, and IGF; (2) SCF, IL-3, IL-6, and TPO; and (3) SCF IL-3, IL-6, TPO, and EPO. Total RNA from day 12 of secondary differentiated ES cells was isolated to study the gene expression pattern during this process. Our results demonstrate an up-regulation of GATA-1, Flk-1, HoxB-4, Epo-R, and globin genes ({alpha}-globin, {beta}H-1 globin, {beta}-major globin, {epsilon} -globin, and {zeta}-globin) in the 9-day-old EBs, whereas, RNA from 5-day-old EBs showed expression of HoxB-4, {epsilon}-globin, {gamma}-globin, {beta}H1-globin, and Flk-1. Three-day-old EBs showed only HoxB-4 and Flk-1 gene expression and lacked expression of all globin genes. These findings indicate that erythropoiesis-specific genes are activated later in the course of differentiation. Gene expression studies on the ES cells of secondary EB origin cultured in media containing dexamethasone showed a down-regulation of GATA-3 and an up-regulation of GATA-1, Flk-1, and Epo-R in comparison to the two other cytokines and growth factor combinations containing media. The secondary differentiation also showed an enhanced production of erythrocytic precursors in dexamethasone containing media in comparison to that in the control media. Our results indicate that dexamethasone can prove to be an effective agent which can be employed to enhance differentiation towards erythrocytic progenitors from ES cells.« less

Fidelity and enhanced sensitivity of differential transcription profiles following linear amplification of nanogram amounts of endothelial mRNA

NASA Technical Reports Server (NTRS)

Polacek, Denise C.; Passerini, Anthony G.; Shi, Congzhu; Francesco, Nadeene M.; Manduchi, Elisabetta; Grant, Gregory R.; Powell, Steven; Bischof, Helen; Winkler, Hans; Stoeckert, Christian J Jr;

[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.

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.

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.

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.

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

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

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.

Brain Natriuretic Peptide Stimulates Lipid Metabolism through Its Receptor NPR1 and the Glycerolipid Metabolism Pathway in Chicken Adipocytes.

PubMed

Huang, H Y; Zhao, G P; Liu, R R; Li, Q H; Zheng, M Q; Li, S F; Liang, Z; Zhao, Z H; Wen, J

2015-11-03

Brain natriuretic peptide (BNP) is related to lipid metabolism in mammals, but its effect and the molecular mechanisms underlying it in chickens are incompletely understood. We found that the level of natriuretic peptide precursor B (NPPB, which encodes BNP) mRNA expression in high-abdominal-fat chicken groups was significantly higher than that of low-abdominal-fat groups. Partial correlations indicated that changes in the weight of abdominal fat were positively correlated with NPPB mRNA expression level. In vitro, compared with the control group, preadipocytes with NPPB interference showed reduced levels of proliferation, differentiation, and glycerin in media. Treatments of cells with BNP led to enhanced proliferation and differentiation of cells and glycerin concentration, and mRNA expression of its receptor natriuretic peptide receptor 1 (NPR1) was upregulated significantly. In cells exposed to BNP, 482 differentially expressed genes were identified compared with controls without BNP. Four genes known to be related to lipid metabolism (diacylglycerol kinase; lipase, endothelial; 1-acylglycerol-3-phosphate O-acyltransferase 1; and 1-acylglycerol-3-phosphate O-acyltransferase 2) were enriched in the glycerolipid metabolism pathway and expressed differentially. In conclusion, BNP stimulates the proliferation, differentiation, and lipolysis of preadipocytes through upregulation of the levels of expression of its receptor NPR1 and key genes enriched in the glycerolipid metabolic pathway.

The Gem GTP-binding protein promotes morphological differentiation in neuroblastoma.

PubMed

Leone, A; Mitsiades, N; Ward, Y; Spinelli, B; Poulaki, V; Tsokos, M; Kelly, K

2001-05-31

Gem is a small GTP-binding protein within the Ras superfamily whose function has not been determined. We report here that ectopic Gem expression is sufficient to stimulate cell flattening and neurite extension in N1E-115 and SH-SY5Y neuroblastoma cells, suggesting a role for Gem in cytoskeletal rearrangement and/or morphological differentiation of neurons. Consistent with this potential function, in clinical samples of neuroblastoma, Gem protein was most highly expressed within cells which had differentiated to express ganglionic morphology. Gem was also observed in developing trigeminal nerve ganglia in 12.5 day mouse embryos, demonstrating that Gem expression is a property of normal ganglionic development. Although Gem expression is rare in epithelial and hematopoietic cancer cell lines, constitutive Gem levels were detected in several neuroblastoma cell lines and could be further induced as much as 10-fold following treatment with PMA or the acetylcholine muscarinic agonist, carbachol.

Discovering causal signaling pathways through gene-expression patterns

PubMed Central

Parikh, Jignesh R.; Klinger, Bertram; Xia, Yu; Marto, Jarrod A.; Blüthgen, Nils

2010-01-01

High-throughput gene-expression studies result in lists of differentially expressed genes. Most current meta-analyses of these gene lists include searching for significant membership of the translated proteins in various signaling pathways. However, such membership enrichment algorithms do not provide insight into which pathways caused the genes to be differentially expressed in the first place. Here, we present an intuitive approach for discovering upstream signaling pathways responsible for regulating these differentially expressed genes. We identify consistently regulated signature genes specific for signal transduction pathways from a panel of single-pathway perturbation experiments. An algorithm that detects overrepresentation of these signature genes in a gene group of interest is used to infer the signaling pathway responsible for regulation. We expose our novel resource and algorithm through a web server called SPEED: Signaling Pathway Enrichment using Experimental Data sets. SPEED can be freely accessed at http://speed.sys-bio.net/. PMID:20494976

Changes in expression and secretion patterns of fibroblast growth factor 8 and Sonic Hedgehog signaling pathway molecules during murine neural stem/progenitor cell differentiation in vitro☆

PubMed Central

Lu, Jiang; Lu, Kehuan; Li, Dongsheng

2012-01-01

In the present study, we investigated the dynamic expression of fibroblast growth factor 8 and Sonic Hedgehog signaling pathway related factors in the process of in vitro hippocampal neural stem/progenitor cell differentiation from embryonic Sprague-Dawley rats or embryonic Kunming species mice, using fluorescent quantitative reverse transcription-PCR and western blot analyses. Results demonstrated that the dynamic expression of fibroblast growth factor 8 was similar to fibroblast growth factor receptor 1 expression but not to other fibroblast growth factor receptors. Enzyme-linked immunosorbent assay demonstrated that fibroblast growth factor 8 and Sonic Hedgehog signaling pathway protein factors were secreted by neural cells into the intercellular niche. Our experimental findings indicate that fibroblast growth factor 8 and Sonic Hedgehog expression may be related to the differentiation of neural stem/progenitor cells. PMID:25624789

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

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.

Nuclear factor of activated T-cells 5 increases intestinal goblet cell differentiation through an mTOR/Notch signaling pathway

PubMed Central

Zhou, Yuning; Wang, Qingding; Weiss, Heidi L.; Evers, B. Mark

2014-01-01

The intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis that is regulated by multiple signaling pathways. Previously, we have shown that the nuclear factor of activated T-cells 5 (NFAT5) is involved in the regulation of intestinal enterocyte differentiation. Here we show that treatment with sodium chloride (NaCl), which activates NFAT5 signaling, increased mTORC1 repressor regulated in development and DNA damage response 1 (REDD1) protein expression and inhibited mTOR signaling; these alterations were attenuated by knockdown of NFAT5. Knockdown of NFAT5 activated mammalian target of rapamycin (mTOR) signaling and significantly inhibited REDD1 mRNA expression and protein expression. Consistently, overexpression of NFAT5 increased REDD1 expression. In addition, knockdown of REDD1 activated mTOR and Notch signaling, whereas treatment with mTOR inhibitor rapamycin repressed Notch signaling and increased the expression of the goblet cell differentiation marker mucin 2 (MUC2). Moreover, knockdown of NFAT5 activated Notch signaling and decreased MUC2 expression, while overexpression of NFAT5 inhibited Notch signaling and increased MUC2 expression. Our results demonstrate a role for NFAT5 in the regulation of mTOR signaling in intestinal cells. Importantly, these data suggest that NFAT5 participates in the regulation of intestinal homeostasis via the suppression of mTORC1/Notch signaling pathway. PMID:25057011

Hidden among the crowd: differential DNA methylation-expression correlations in cancer occur at important oncogenic pathways

PubMed Central

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. PMID:26029238

Expression of root-related transcription factors associated with flooding tolerance of soybean (Glycine max).

PubMed

Valliyodan, Babu; Van Toai, Tara T; Alves, Jose Donizeti; de Fátima P Goulart, Patricia; Lee, Jeong Dong; Fritschi, Felix B; Rahman, Mohammed Atiqur; Islam, Rafiq; Shannon, J Grover; Nguyen, Henry T

2014-09-29

Much research has been conducted on the changes in gene expression of the model plant Arabidopsis to low-oxygen stress. Flooding results in a low oxygen environment in the root zone. However, there is ample evidence that tolerance to soil flooding is more than tolerance to low oxygen alone. In this study, we investigated the physiological response and differential expression of root-related transcription factors (TFs) associated with the tolerance of soybean plants to soil flooding. Differential responses of PI408105A and S99-2281 plants to ten days of soil flooding were evaluated at physiological, morphological and anatomical levels. Gene expression underlying the tolerance response was investigated using qRT-PCR of root-related TFs, known anaerobic genes, and housekeeping genes. Biomass of flood-sensitive S99-2281 roots remained unchanged during the entire 10 days of flooding. Flood-tolerant PI408105A plants exhibited recovery of root growth after 3 days of flooding. Flooding induced the development of aerenchyma and adventitious roots more rapidly in the flood-tolerant than the flood-sensitive genotype. Roots of tolerant plants also contained more ATP than roots of sensitive plants at the 7th and 10th days of flooding. Quantitative transcript analysis identified 132 genes differentially expressed between the two genotypes at one or more time points of flooding. Expression of genes related to the ethylene biosynthesis pathway and formation of adventitious roots was induced earlier and to higher levels in roots of the flood-tolerant genotype. Three potential flood-tolerance TFs which were differentially expressed between the two genotypes during the entire 10-day flooding duration were identified. This study confirmed the expression of anaerobic genes in response to soil flooding. Additionally, the differential expression of TFs associated with soil flooding tolerance was not qualitative but quantitative and temporal. Functional analyses of these genes will be necessary to reveal their potential to enhance flooding tolerance of soybean cultivars.

Expression of Root-Related Transcription Factors Associated with Flooding Tolerance of Soybean (Glycine max)

PubMed Central

Valliyodan, Babu; Van Toai, Tara T.; Alves, Jose Donizeti; de Fátima P. Goulart, Patricia; Lee, Jeong Dong; Fritschi, Felix B.; Rahman, Mohammed Atiqur; Islam, Rafiq; Shannon, J. Grover; Nguyen, Henry T.

2014-01-01

Much research has been conducted on the changes in gene expression of the model plant Arabidopsis to low-oxygen stress. Flooding results in a low oxygen environment in the root zone. However, there is ample evidence that tolerance to soil flooding is more than tolerance to low oxygen alone. In this study, we investigated the physiological response and differential expression of root-related transcription factors (TFs) associated with the tolerance of soybean plants to soil flooding. Differential responses of PI408105A and S99-2281 plants to ten days of soil flooding were evaluated at physiological, morphological and anatomical levels. Gene expression underlying the tolerance response was investigated using qRT-PCR of root-related TFs, known anaerobic genes, and housekeeping genes. Biomass of flood-sensitive S99-2281 roots remained unchanged during the entire 10 days of flooding. Flood-tolerant PI408105A plants exhibited recovery of root growth after 3 days of flooding. Flooding induced the development of aerenchyma and adventitious roots more rapidly in the flood-tolerant than the flood-sensitive genotype. Roots of tolerant plants also contained more ATP than roots of sensitive plants at the 7th and 10th days of flooding. Quantitative transcript analysis identified 132 genes differentially expressed between the two genotypes at one or more time points of flooding. Expression of genes related to the ethylene biosynthesis pathway and formation of adventitious roots was induced earlier and to higher levels in roots of the flood-tolerant genotype. Three potential flood-tolerance TFs which were differentially expressed between the two genotypes during the entire 10-day flooding duration were identified. This study confirmed the expression of anaerobic genes in response to soil flooding. Additionally, the differential expression of TFs associated with soil flooding tolerance was not qualitative but quantitative and temporal. Functional analyses of these genes will be necessary to reveal their potential to enhance flooding tolerance of soybean cultivars. PMID:25268626

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.

Human Keratinocytes That Express hTERT and Also Bypass a p16INK4a-Enforced Mechanism That Limits Life Span Become Immortal yet Retain Normal Growth and Differentiation Characteristics

PubMed Central

Dickson, Mark A.; Hahn, William C.; Ino, Yasushi; Ronfard, Vincent; Wu, Jenny Y.; Weinberg, Robert A.; Louis, David N.; Li, Frederick P.; Rheinwald, James G.

2000-01-01

Normal human cells exhibit a limited replicative life span in culture, eventually arresting growth by a process termed senescence. Progressive telomere shortening appears to trigger senescence in normal human fibroblasts and retinal pigment epithelial cells, as ectopic expression of the telomerase catalytic subunit, hTERT, immortalizes these cell types directly. Telomerase expression alone is insufficient to enable certain other cell types to evade senescence, however. Such cells, including keratinocytes and mammary epithelial cells, appear to require loss of the pRB/p16INK4a cell cycle control mechanism in addition to hTERT expression to achieve immortality. To investigate the relationships among telomerase activity, cell cycle control, senescence, and differentiation, we expressed hTERT in two epithelial cell types, keratinocytes and mesothelial cells, and determined the effect on proliferation potential and on the function of cell-type-specific growth control and differentiation systems. Ectopic hTERT expression immortalized normal mesothelial cells and a premalignant, p16INK4a-negative keratinocyte line. In contrast, when four keratinocyte strains cultured from normal tissue were transduced to express hTERT, they were incompletely rescued from senescence. After reaching the population doubling limit of their parent cell strains, hTERT+ keratinocytes entered a slow growth phase of indefinite length, from which rare, rapidly dividing immortal cells emerged. These immortal cell lines frequently had sustained deletions of the CDK2NA/INK4A locus or otherwise were deficient in p16INK4a expression. They nevertheless typically retained other keratinocyte growth controls and differentiated normally in culture and in xenografts. Thus, keratinocyte replicative potential is limited by a p16INK4a-dependent mechanism, the activation of which can occur independent of telomere length. Abrogation of this mechanism together with telomerase expression immortalizes keratinocytes without affecting other major growth control or differentiation systems. PMID:10648628

RNA-Seq workflow: gene-level exploratory analysis and differential expression

PubMed Central

Love, Michael I.; Anders, Simon; Kim, Vladislav; Huber, Wolfgang

2015-01-01

Here we walk through an end-to-end gene-level RNA-Seq differential expression workflow using Bioconductor packages. We will start from the FASTQ files, show how these were aligned to the reference genome, and prepare a count matrix which tallies the number of RNA-seq reads/fragments within each gene for each sample. We will perform exploratory data analysis (EDA) for quality assessment and to explore the relationship between samples, perform differential gene expression analysis, and visually explore the results. PMID:26674615

DR-nm23 gene expression in neuroblastoma cells: relationship to integrin expression, adhesion characteristics, and differentiation.

PubMed

Amendola, R; Martinez, R; Negroni, A; Venturelli, D; Tanno, B; Calabretta, B; Raschellà, G

1997-09-03

Neuroblastoma, a childhood tumor originating from cells of the embryonic neural crest, retains the ability to differentiate, yielding cells with epithelial-Schwann-like, neuronal, or melanocytic characteristics. Since nm23 gene family members have been proposed to play a role in cellular differentiation, as well as in metastasis suppression, we investigated whether and how DR-nm23, a recently identified third member of the human nm23 gene family, might be involved in neuroblastoma differentiation. Three neuroblastoma cell lines (human LAN-5, human SK-N-SH, and murine N1E-115) were used in these experiments; cells from two of the lines (SK-N-SH and N1E-115) were also studied after being stably transfected with a plasmid containing a full-length DR-nm23 complementary DNA. Cellular expression of specific messenger RNAs and proteins was assessed by use of standard techniques. Cellular adhesion to a variety of protein substrates was also evaluated. DR-nm23 messenger RNA levels in nontransfected LAN-5 and SK-N-SH cells generally increased with time after exposure to differentiation-inducing conditions; levels of the other two human nm23 messenger RNAs (nm23-H1 and nm23-H2) remained essentially constant. Transfected SK-N-SH cells overexpressing DR-nm23 exhibited some characteristics of differentiated cells (increased vimentin and collagen type IV expression) even in the absence of differentiation-inducing conditions. Compared with control cells, DR-nm23-transfected cells exposed to differentiation-inducing conditions showed a greater degree of growth arrest (SK-N-SH cells) and greater increases in integrin protein expression, especially of integrin beta1 (N1E-115 cells). DR-nm23-transfected N1E-115 cells also showed a marked increase in adhesion to collagen type I-coated tissue culture plates that was inhibited by preincubation with an anti-integrin beta1 antibody. DR-nm23 gene expression appears to be associated with differentiation in neuroblastoma cells and may affect cellular adhesion through regulation of integrin protein expression.

Differential Expression of Glutamate Receptors in Avian Neural Pathways for Learned Vocalization

PubMed Central

WADA, KAZUHIRO; SAKAGUCHI, HIRONOBU; JARVIS, ERICH D.; HAGIWARA, MASATOSHI

2008-01-01

Learned vocalization, the substrate for human language, is a rare trait. It is found in three distantly related groups of birds—parrots, hummingbirds, and songbirds. These three groups contain cerebral vocal nuclei for learned vocalization not found in their more closely related vocal nonlearning relatives. Here, we cloned 21 receptor subunits/subtypes of all four glutamate receptor families (AMPA, kainate, NMDA, and metabotropic) and examined their expression in vocal nuclei of songbirds. We also examined expression of a subset of these receptors in vocal nuclei of hummingbirds and parrots, as well as in the brains of dove species as examples of close vocal nonlearning relatives. Among the 21 subunits/subtypes, 19 showed higher and/or lower prominent differential expression in songbird vocal nuclei relative to the surrounding brain subdivisions in which the vocal nuclei are located. This included relatively lower levels of all four AMPA subunits in lMAN, strikingly higher levels of the kainite subunit GluR5 in the robust nucleus of the arcopallium (RA), higher and lower levels respectively of the NMDA subunits NR2A and NR2B in most vocal nuclei and lower levels of the metabotropic group I subtypes (mGluR1 and -5) in most vocal nuclei and the group II subtype (mGluR2), showing a unique expression pattern of very low levels in RA and very high levels in HVC. The splice variants of AMPA subunits showed further differential expression in vocal nuclei. Some of the receptor subunits/subtypes also showed differential expression in hummingbird and parrot vocal nuclei. The magnitude of differential expression in vocal nuclei of all three vocal learners was unique compared with the smaller magnitude of differences found for nonvocal areas of vocal learners and vocal nonlearners. Our results suggest that evolution of vocal learning was accompanied by differential expression of a conserved gene family for synaptic transmission and plasticity in vocal nuclei. They also suggest that neural activity and signal transduction in vocal nuclei of vocal learners will be different relative to the surrounding brain areas. PMID:15236466

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

[Differential gene expression profile in ischemic myocardium of Wistar rats with acute myocardial infarction: the study on gene construction, identification and function].

PubMed

Guo, Chun Yu; Yin, Hui Jun; Jiang, Yue Rong; Xue, Mei; Zhang, Lu; Shi, Da Zhuo

2008-06-18

To construct the differential genes expressed profile in the ischemic myocardium tissue reduced from acute myocardial infarction(AMI), and determine the biological functions of target genes. AMI model was generated by ligation of the left anterior descending coronary artery in Wistar rats. Total RNA was extracted from the normal and the ischemic heart tissues under the ligation point 7 days after the operation. Differential gene expression profiles of the two samples were constructed using Long Serial Analysis of Gene Expression(LongSAGE). Real time fluorescence quantitative PCR was used to verify gene expression profile and to identify the expression of 2 functional genes. The activities of enzymes from functional genes were determined by histochemistry. A total of 15,966 tags were screened from the normal and the ischemic LongSAGE maps. The similarities of the sequences were compared using the BLAST algebra in NCBI and 7,665 novel tags were found. In the ischemic tissue 142 genes were significantly changed compared with those in the normal tissue (P<0.05). These differentially expressed genes represented the proteins which might play important roles in the pathways of oxidation and phosphorylation, ATP synthesis and glycolysis. The partial genes identified by LongSAGE were confirmed using real time fluorescence quantitative PCR. Two genes related to energy metabolism, COX5a and ATP5e, were screened and quantified. Expression of two functional genes down-regulated at their mRNA levels and the activities of correlative functional enzymes decreased compared with those in the normal tissue. AMI causes a series of changes in gene expression, in which the abnormal expression of genes related to energy metabolism could be one of the molecular mechanisms of AMI. The intervention of the expressions of COX5a and ATP5e may be a new target for AMI therapy.

Differential gene expression patterns between smokers and non‐smokers: cause or consequence?

PubMed Central

Jansen, Rick; Brooks, Andy; Willemsen, Gonneke; van Grootheest, Gerard; de Geus, Eco; Smit, Jan H.; Penninx, Brenda W.; Boomsma, Dorret I.

2015-01-01

Abstract The molecular mechanisms causing smoking‐induced health decline are largely unknown. To elucidate the molecular pathways involved in cause and consequences of smoking behavior, we conducted a genome‐wide gene expression study in peripheral blood samples targeting 18 238 genes. Data of 743 smokers, 1686 never smokers and 890 ex‐smokers were available from two population‐based cohorts from the Netherlands. In addition, data of 56 monozygotic twin pairs discordant for ever smoking were used. One hundred thirty‐two genes were differentially expressed between current smokers and never smokers (P < 1.2 × 10−6, Bonferroni correction). The most significant genes were G protein‐coupled receptor 15 (P < 1 × 10−150) and leucine‐rich repeat neuronal 3 (P < 1 × 10−44). The smoking‐related genes were enriched for immune system, blood coagulation, natural killer cell and cancer pathways. By taking the data of ex‐smokers into account, expression of these 132 genes was classified into reversible (94 genes), slowly reversible (31 genes), irreversible (6 genes) or inconclusive (1 gene). Expression of 6 of the 132 genes (three reversible and three slowly reversible) was confirmed to be reactive to smoking as they were differentially expressed in monozygotic pairs discordant for smoking. Cis‐expression quantitative trait loci for GPR56 and RARRES3 (downregulated in smokers) were associated with increased number of cigarettes smoked per day in a large genome‐wide association meta‐analysis, suggesting a causative effect of GPR56 and RARRES3 expression on smoking behavior. In conclusion, differential gene expression patterns in smokers are extensive and cluster in several underlying disease pathways. Gene expression differences seem mainly direct consequences of smoking, and largely reversible after smoking cessation. However, we also identified DNA variants that may influence smoking behavior via the mediating gene expression. PMID:26594007

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

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.

Runx2- and histone deacetylase 3-mediated repression is relieved in differentiating human osteoblast cells to allow high bone sialoprotein expression.

PubMed

Lamour, Virginie; Detry, Cédric; Sanchez, Christelle; Henrotin, Yves; Castronovo, Vincent; Bellahcène, Akeila

2007-12-14

Bone sialoprotein (BSP) is a bone matrix glycoprotein whose expression coincides with terminal osteoblastic differentiation and the onset of mineralization. In this study we show that BSP expression is considerably increased in confluent Saos-2 human osteosarcoma cells and in differentiating normal human osteoblasts, concomitantly with the decrease of Runx2, a key transcription factor controlling bone formation. Therefore, we investigated the role of Runx2 in the regulation of BSP expression in Saos-2 cells. Using a mobility shift assay, we demonstrated that Runx2 binds to the BSP promoter only in preconfluent cells. Histone deacetylase 3 (HDAC3) has been recently shown to act as a Runx2 co-repressor. Chromatin immunoprecipitation assays demonstrated that both Runx2 and HDAC3 are detectable at the BSP promoter in preconfluent Saos-2 cells but not when they are confluent and overexpress BSP. Consistently, nuclear Runx2 protein level is down-regulated, whereas Saos-2 cells became increasingly confluent. Finally, the suppression of HDAC3, Runx2, or both by RNA interference induced the expression of BSP at both mRNA and protein levels in Saos-2 cells. Our data demonstrate that Runx2 and HDAC3 repress BSP gene expression and that this repression is suspended upon osteoblastic cell differentiation. Both the nuclear disappearance of Runx2 and the non-recruitment of HDAC3 represent new means to relieve Runx2-mediated suppression of BSP expression, thus allowing the acquisition of a fully differentiated and mineralization-competent phenotype by osteoblast cells.

A subset of replication-dependent histone mRNAs are expressed as polyadenylated RNAs in terminally differentiated tissues.

PubMed

Lyons, Shawn M; Cunningham, Clark H; Welch, Joshua D; Groh, Beezly; Guo, Andrew Y; Wei, Bruce; Whitfield, Michael L; Xiong, Yue; Marzluff, William F

2016-11-02

Histone proteins are synthesized in large amounts during S-phase to package the newly replicated DNA, and are among the most stable proteins in the cell. The replication-dependent (RD)-histone mRNAs expressed during S-phase end in a conserved stem-loop rather than a polyA tail. In addition, there are replication-independent (RI)-histone genes that encode histone variants as polyadenylated mRNAs. Most variants have specific functions in chromatin, but H3.3 also serves as a replacement histone for damaged histones in long-lived terminally differentiated cells. There are no reported replacement histone genes for histones H2A, H2B or H4. We report that a subset of RD-histone genes are expressed in terminally differentiated tissues as polyadenylated mRNAs, likely serving as replacement histone genes in long-lived non-dividing cells. Expression of two genes, HIST2H2AA3 and HIST1H2BC, is conserved in mammals. They are expressed as polyadenylated mRNAs in fibroblasts differentiated in vitro, but not in serum starved fibroblasts, suggesting that their expression is part of the terminal differentiation program. There are two histone H4 genes and an H3 gene that encode mRNAs that are polyadenylated and expressed at 5- to 10-fold lower levels than the mRNAs from H2A and H2B genes, which may be replacement genes for the H3.1 and H4 proteins. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Transcriptome architecture across tissues in the pig

PubMed Central

Ferraz, André LJ; Ojeda, Ana; López-Béjar, Manel; Fernandes, Lana T; Castelló, Anna; Folch, Josep M; Pérez-Enciso, Miguel

2008-01-01

Background Artificial selection has resulted in animal breeds with extreme phenotypes. As an organism is made up of many different tissues and organs, each with its own genetic programme, it is pertinent to ask: How relevant is tissue in terms of total transcriptome variability? Which are the genes most distinctly expressed between tissues? Does breed or sex equally affect the transcriptome across tissues? Results In order to gain insight on these issues, we conducted microarray expression profiling of 16 different tissues from four animals of two extreme pig breeds, Large White and Iberian, two males and two females. Mixed model analysis and neighbor – joining trees showed that tissues with similar developmental origin clustered closer than those with different embryonic origins. Often a sound biological interpretation was possible for overrepresented gene ontology categories within differentially expressed genes between groups of tissues. For instance, an excess of nervous system or muscle development genes were found among tissues of ectoderm or mesoderm origins, respectively. Tissue accounted for ~11 times more variability than sex or breed. Nevertheless, we were able to confidently identify genes with differential expression across tissues between breeds (33 genes) and between sexes (19 genes). The genes primarily affected by sex were overall different than those affected by breed or tissue. Interaction with tissue can be important for differentially expressed genes between breeds but not so much for genes whose expression differ between sexes. Conclusion Embryonic development leaves an enduring footprint on the transcriptome. The interaction in gene × tissue for differentially expressed genes between breeds suggests that animal breeding has targeted differentially each tissue's transcriptome. PMID:18416811

Fndc5 knockdown induced suppression of mitochondrial integrity and significantly decreased cardiac differentiation of mouse embryonic stem cells.

PubMed

Nazem, Shima; Rabiee, Farzaneh; Ghaedi, Kamran; Babashah, Sadegh; Sadeghizadeh, Majid; Nasr-Esfahani, Mohammad Hossein

2018-06-01

Fibronectin type III domain-containing 5 protein (Fndc5) is a glycosylated protein with elevated expression in high energy demanded tissues as heart, brain, and muscle. It has been shown that upregulation of Fndc5 is regulated by peroxisome proliferator-activated receptor-γ coactivator-1 alpha (PGC-1α), which is known as a master regulator of mitochondrial function and biogenesis. Also, our group indicated that Fndc5 expression increases gradually during cardiac differentiation of mouse embryonic stem cells (mESCs). In this paper, to clarify the importance of Fndc5 in cardiac differentiation, we south to knock down Fndc5 expression by generation a stably transduced mESC line that derives the expression of a short hairpin RNA (shRNA) against Fndc5 gene following doxycycline (Dox) induction. Knock-down of Fndc5 demonstrated a considerable decrease in expression of cardiac progenitor and cardiomyocyte markers. Considering the fact that mitochondria play a crucial role in cardiac differentiation of ESCs, we investigated the role of Fndc5, as a downstream target of PGC1-α, on mitochondrial indices. Results showed that expression of nuclear encoded mitochondrial genes including PGC1-α, Atp5b, Ndufb5, and SOD2 significantly decreased. Moreover, mitochondrial membrane potential (ΔΨm) and relative ATP content of cardiomyocytes decreased markedly with relative ROS level increase. Together, our results suggest that Fndc5 attenuates process of cardiac differentiation of mESCs which is associated with modulation of mitochondrial function and gene expression. © 2017 Wiley Periodicals, Inc.

Developmental Progression in the Coral Acropora digitifera Is Controlled by Differential Expression of Distinct Regulatory Gene Networks

PubMed Central

Reyes-Bermudez, Alejandro; Villar-Briones, Alejandro; Ramirez-Portilla, Catalina; Hidaka, Michio; Mikheyev, Alexander S.

2016-01-01

Corals belong to the most basal class of the Phylum Cnidaria, which is considered the sister group of bilaterian animals, and thus have become an emerging model to study the evolution of developmental mechanisms. Although cell renewal, differentiation, and maintenance of pluripotency are cellular events shared by multicellular animals, the cellular basis of these fundamental biological processes are still poorly understood. To understand how changes in gene expression regulate morphogenetic transitions at the base of the eumetazoa, we performed quantitative RNA-seq analysis during Acropora digitifera’s development. We collected embryonic, larval, and adult samples to characterize stage-specific transcription profiles, as well as broad expression patterns. Transcription profiles reconstructed development revealing two main expression clusters. The first cluster grouped blastula and gastrula and the second grouped subsequent developmental time points. Consistently, we observed clear differences in gene expression between early and late developmental transitions, with higher numbers of differentially expressed genes and fold changes around gastrulation. Furthermore, we identified three coexpression clusters that represented discrete gene expression patterns. During early transitions, transcriptional networks seemed to regulate cellular fate and morphogenesis of the larval body. In late transitions, these networks seemed to play important roles preparing planulae for switch in lifestyle and regulation of adult processes. Although developmental progression in A. digitifera is regulated to some extent by differential coexpression of well-defined gene networks, stage-specific transcription profiles appear to be independent entities. While negative regulation of transcription is predominant in early development, cell differentiation was upregulated in larval and adult stages. PMID:26941230

Adipose Gene Expression Prior to Weight Loss Can Differentiate and Weakly Predict Dietary Responders

PubMed Central

Mutch, David M.; Temanni, M. Ramzi; Henegar, Corneliu; Combes, Florence; Pelloux, Véronique; Holst, Claus; Sørensen, Thorkild I. A.; Astrup, Arne; Martinez, J. Alfredo; Saris, Wim H. M.; Viguerie, Nathalie; Langin, Dominique; Zucker, Jean-Daniel; Clément, Karine

2007-01-01

Background The ability to identify obese individuals who will successfully lose weight in response to dietary intervention will revolutionize disease management. Therefore, we asked whether it is possible to identify subjects who will lose weight during dietary intervention using only a single gene expression snapshot. Methodology/Principal Findings The present study involved 54 female subjects from the Nutrient-Gene Interactions in Human Obesity-Implications for Dietary Guidelines (NUGENOB) trial to determine whether subcutaneous adipose tissue gene expression could be used to predict weight loss prior to the 10-week consumption of a low-fat hypocaloric diet. Using several statistical tests revealed that the gene expression profiles of responders (8–12 kgs weight loss) could always be differentiated from non-responders (<4 kgs weight loss). We also assessed whether this differentiation was sufficient for prediction. Using a bottom-up (i.e. black-box) approach, standard class prediction algorithms were able to predict dietary responders with up to 61.1%±8.1% accuracy. Using a top-down approach (i.e. using differentially expressed genes to build a classifier) improved prediction accuracy to 80.9%±2.2%. Conclusion Adipose gene expression profiling prior to the consumption of a low-fat diet is able to differentiate responders from non-responders as well as serve as a weak predictor of subjects destined to lose weight. While the degree of prediction accuracy currently achieved with a gene expression snapshot is perhaps insufficient for clinical use, this work reveals that the comprehensive molecular signature of adipose tissue paves the way for the future of personalized nutrition. PMID:18094752

Effects of MicroRNA-23a on Differentiation and Gene Expression Profiles in 3T3-L1 Adipocytes

PubMed Central

Huang, Yong; Huang, Jinxiu; Qi, Renli; Wang, Qi; Wu, Yongjiang; Wang, Jing

2016-01-01

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate growth, development, and programmed death of cells. A newly-published study has shown that miRNA-23a could regulate 3T3-L1 adipocyte differentiation. Here, we identified miRNA-23a as a negative regulator of 3T3-L1 adipocyte differentiation again. Over-expression of miRNA-23a inhibited differentiation and decreased lipogenesis as well as down-regulated mRNA and protein expression of both peroxisome proliferator-activated receptor (PPAR) γ and fatty acid binding protein (FABP) 4, whereas knock down of miRNA-23a showed the opposite effects on differentiation as well as increasing the number of apoptotic cells. Additionally, digital gene expression profiling sequencing (DGE-Seq) was used to assay changes in gene expression profiles following alterations in the level of miR-23a. In total, over-expression or knock down of miRNA-23a significantly changed the expression of 313 and 425 genes, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that these genes were mainly involved in the stress response, immune system, metabolism, cell cycle, among other pathways. Additionally, the signal transducer and activator of transcription 1 (Stat1) was shown to be a target of miRNA-23a by computational and dual-luciferase reporter assays that indicated Janus Kinase (Jak)-Stat signal pathway was implicated in regulating adipogenesis mediated by miRNA-23a in adipocytes. PMID:27783036

Transcriptional profiling of root-knot nematode induced feeding sites in cowpea (Vigna unguiculata L. Walp.) using a soybean genome array.

PubMed

Das, Sayan; Ehlers, Jeffrey D; Close, Timothy J; Roberts, Philip A

2010-08-19

The locus Rk confers resistance against several species of root-knot nematodes (Meloidogyne spp., RKN) in cowpea (Vigna unguiculata). Based on histological and reactive oxygen species (ROS) profiles, Rk confers a delayed but strong resistance mechanism without a hypersensitive reaction-mediated cell death process, which allows nematode development but blocks reproduction. Responses to M. incognita infection in roots of resistant genotype CB46 and a susceptible near-isogenic line (null-Rk) were investigated using a soybean Affymetrix GeneChip expression array at 3 and 9 days post-inoculation (dpi). At 9 dpi 552 genes were differentially expressed in incompatible interactions (infected resistant tissue compared with non-infected resistant tissue) and 1,060 genes were differentially expressed in compatible interactions (infected susceptible tissue compared with non-infected susceptible tissue). At 3 dpi the differentially expressed genes were 746 for the incompatible and 623 for the compatible interactions. When expression between infected resistant and susceptible genotypes was compared, 638 and 197 genes were differentially expressed at 9 and 3 dpi, respectively. In comparing the differentially expressed genes in response to nematode infection, a greater number and proportion of genes were down-regulated in the resistant than in the susceptible genotype, whereas more genes were up-regulated in the susceptible than in the resistant genotype. Gene ontology based functional categorization revealed that the typical defense response was partially suppressed in resistant roots, even at 9 dpi, allowing nematode juvenile development. Differences in ROS concentrations, induction of toxins and other defense related genes seem to play a role in this unique resistance mechanism.

A feedback regulatory loop involving microRNA-9 and nuclear receptor TLX in neural stem cell fate determination.

PubMed

Zhao, Chunnian; Sun, GuoQiang; Li, Shengxiu; Shi, Yanhong

2009-04-01

MicroRNAs have been implicated as having important roles in stem cell biology. MicroRNA-9 (miR-9) is expressed specifically in neurogenic areas of the brain and may be involved in neural stem cell self-renewal and differentiation. We showed previously that the nuclear receptor TLX is an essential regulator of neural stem cell self-renewal. Here we show that miR-9 suppresses TLX expression to negatively regulate neural stem cell proliferation and accelerate neural differentiation. Introducing a TLX expression vector that is not prone to miR-9 regulation rescued miR-9-induced proliferation deficiency and inhibited precocious differentiation. In utero electroporation of miR-9 in embryonic brains led to premature differentiation and outward migration of the transfected neural stem cells. Moreover, TLX represses expression of the miR-9 pri-miRNA. By forming a negative regulatory loop with TLX, miR-9 provides a model for controlling the balance between neural stem cell proliferation and differentiation.

A feedback regulatory loop involving microRNA-9 and nuclear receptor TLX in neural stem cell fate determination

PubMed Central

Zhao, Chunnian; Sun, GuoQiang; Li, Shengxiu; Shi, Yanhong

2009-01-01

Summary MicroRNAs are important players in stem cell biology. Among them, microRNA-9 (miR-9) is expressed specifically in neurogenic areas of the brain. Whether miR-9 plays a role in neural stem cell self-renewal and differentiation is unknown. We showed previously that nuclear receptor TLX is an essential regulator of neural stem cell self-renewal. Here we show that miR-9 suppresses TLX expression to negatively regulate neural stem cell proliferation and accelerate neural differentiation. Introducing a TLX expression vector lacking the miR-9 recognition site rescued miR-9-induced proliferation deficiency and inhibited precocious differentiation. In utero electroporation of miR-9 in embryonic brains led to premature differentiation and outward migration of the transfected neural stem cells. Moreover, TLX represses miR-9 pri-miRNA expression. MiR-9, by forming a negative regulatory loop with TLX, establishes a model for controlling the balance between neural stem cell proliferation and differentiation. PMID:19330006

Immunohistochemical analysis of the role and relationship between Notch-1 and Oct-4 expression in urinary bladder carcinoma.

PubMed

Abdou, Asmaa Gaber; El-Wahed, Moshira Mohammed Abd; Kandil, Mona Abd-Elhalim; Samaka, Rehab Monir; Elkady, Noha

2013-10-01

Most tumors contain a minor population of cancer stem cells that are responsible for tumor heterogeneity, resistance to therapy and recurrence. Oct-4 is a transcription factor responsible for self-renewal of stem cells, whereas the Notch family of receptors and ligands may play a pivotal role in the regulation of stem cell maintenance and differentiation. This study aimed at an evaluation of Oct-4 and Notch-1 expression in both carcinoma and stromal cells of 83 cases of primary bladder carcinoma and to study the relationship between them. Notch-1 was expressed in carcinoma and stromal cells of all malignant cases, where expression in both cell types was correlated with parameters indicating differentiation, such as low grade (p < 0.05) and less proliferation (p < 0.05). However, Notch-1 expression in stromal cells was associated with nodal metastasis (p = 0.016) and advanced stage (p = 0.030). 56.6 and 75.9% of carcinoma and stromal cells of malignant cases showed Oct-4 expression, respectively. Oct-4 expression in carcinoma cells or stromal cells was associated with aggressive features of bladder carcinoma, such as poor differentiation (p = 0.001), high proliferation (p < 0.001, 0.030), and liability for recurrence (p = 0.010, p < 0.001). There was an inverse relationship between Notch-1 and Oct-4 expression in carcinoma cells (p = 0.002), but stromal expression of Notch-1 was found to be associated with a nuclear pattern of Oct-4 expression in carcinoma cells (p = 0.030). Oct-4 as a stem cell marker is expressed in carcinoma cells and in stromal cells of bladder carcinoma, where they may cooperate in the progression of bladder carcinoma by acquiring aggressive features, such as a liability for recurrence and dissemination. Notch-1 is also expressed in both carcinoma cells and stromal cells of bladder carcinoma. Although they could share in enhancing differentiation, stromal expression of Notch-1 may have a bad impact, possibly through up-regulation of the active nuclear form of Oct-4 in carcinoma cells. © 2013 APMIS Published by Blackwell Publishing Ltd.

Demonstration of intermediate cells during human prostate epithelial differentiation in situ and in vitro using triple-staining confocal scanning microscopy.

PubMed

van Leenders, G; Dijkman, H; Hulsbergen-van de Kaa, C; Ruiter, D; Schalken, J

2000-08-01

In human prostate epithelium, morphologically basal and luminal cells can be discriminated. The basal cell layer that putatively contains progenitor cells of the secretory epithelium is characterized by the expression of keratins (K) 5 and 14. Luminal cells represent the secretory compartment of the epithelium and express K8 and 18. We developed a technique for the simultaneous analysis of K5, 14, and 18 to identify intermediate cell stages in the prostate epithelium and to study the dynamic aspects of its differentiation in vitro. Nonmalignant prostate tissue and primary epithelial cultures were immunohistochemically characterized using triple staining with antibodies for K5, K14, and K18. Antibodies for K18 and K5 were conjugated directly with fluorochromes Alexa 488 and 546. K14 was visualized indirectly with streptavidin-Cy5. Keratin expression was analyzed by confocal scanning microscopy. The occurrence of exocrine and neuroendocrine differentiation in culture was determined via antibodies to prostate-specific antigen (PSA), chromogranin A, and serotonin. We found that basal cells expressed either K5(++)/14(++)/18+ or K5(++)/18+. The majority of luminal cells expressed K18(++), but colocalization of K5+/18(++) were recognized. Epithelial monolayer cultures predominantly revealed the basal cell phenotype K5(++)/14(++)/18+, whereas intermediate subpopulations expressing K5+/14+/18(++) and K5+/18(++) were also identified. On confluence, differentiation was induced as multicellular gland-like buds, and extensions became evident on top of the monolayer. These structures were composed of K18(++)- and K5+/18(+)-positive cell clusters surrounded by phenotypically basal cells. Few multicellular structures and cells in the monolayer showed exocrine differentiation (PSA+), but expression of chromogranin A and serotonin was absent. We conclude that simultaneous evaluation of keratin expression is useful for analyzing epithelial differentiation in the prostate. During this process, putative stem cells phenotypically resembling K5(++)/14(++)/18+ differentiate toward luminal cells (K18(++)) via intermediate cell stages, as identified by up-regulation of K18 and down-regulation of K5 and 14.

Oligo-dT anchored cDNA-SCoT: a novel differential display method for analyzing differential gene expression in response to several stress treatments in mango (Mangifera indica L.).

PubMed

Luo, Cong; He, Xin-Hua; Hu, Ying; Yu, Hai-xia; Ou, Shi-Jin; Fang, Zhong-Bin

2014-09-15

Differential display is a powerful technique for analyzing differences in gene expression. Oligo-dT cDNAstart codon targeted marker (cDNA-SCoT) technique is a novel, simple, cheap, rapid, and efficient method for differential gene expression research. In the present study, the oligo-dT anchored cDNA-SCoT technique was exploited to identify differentially expressed genes during several stress treatments in mango. A total of 37 primers combined with oligo-dT anchor primers 3side amplified approximately 150 fragments of 150 bp to 1500 bp in length. Up to 100 fragments were differentially expressed among the stress treatments and control samples, among which 92 were obtained and sequenced. Out of the 92 transcript derived fragments (TDFs), 70% were highly homologous to known genes, and 30% encoded unclassified proteins with unknown functions. The expression pattern of nine genes with known functions involved in several abiotic stresses in other species was confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) under cold (4 °C), salinity (NaCl), polyethylene glycol (PEG, MW 6000), and heavy metal treatments in leaves and stems at different time points (0, 24, 48, and 72 h). The expression patterns of the genes (TDF4, TDF7, TDF23, TDF45, TDF49, TDF50, TDF57, TDF91 and TDF92) that had direct or indirect relationships with cold, salinity, drought and heavy metal stress response were analyzed through qRT-PCR. The possible roles of these genes are discussed. This study suggests that the oligo-dT anchored cDNA-SCoT differential display method is a useful tool to serve as an initial step for characterizing transcriptional changes induced by abiotic stresses and provide gene information for further study and application in genetic improvement and breeding in mango. Copyright © 2014 Elsevier B.V. All rights reserved.

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

Dynamic changes in gene expression during human trophoblast differentiation.

PubMed

Handwerger, Stuart; Aronow, Bruce

2003-01-01

The genetic program that directs human placental differentiation is poorly understood. In a recent study, we used DNA microarray analyses to determine genes that are dynamically regulated during human placental development in an in vitro model system in which highly purified cytotrophoblast cells aggregate spontaneously and fuse to form a multinucleated syncytium that expresses placental lactogen, human chorionic gonadotropin, and other proteins normally expressed by fully differentiated syncytiotrophoblast cells. Of the 6918 genes present on the Incyte Human GEM V microarray that we analyzed over a 9-day period, 141 were induced and 256 were downregulated by more than 2-fold. The dynamically regulated genes fell into nine distinct kinetic patterns of induction or repression, as detected by the K-means algorithm. Classifying the genes according to functional characteristics, the regulated genes could be divided into six overall categories: cell and tissue structural dynamics, cell cycle and apoptosis, intercellular communication, metabolism, regulation of gene expression, and expressed sequence tags and function unknown. Gene expression changes within key functional categories were tightly coupled to the morphological changes that occurred during trophoblast differentiation. Within several key gene categories (e.g., cell and tissue structure), many genes were strongly activated, while others with related function were strongly repressed. These findings suggest that trophoblast differentiation is augmented by "categorical reprogramming" in which the ability of induced genes to function is enhanced by diminished synthesis of other genes within the same category. We also observed categorical reprogramming in human decidual fibroblasts decidualized in vitro in response to progesterone, estradiol, and cyclic AMP. While there was little overlap between genes that are dynamically regulated during trophoblast differentiation versus decidualization, many of the categories in which genes were strongly activated also contained genes whose expression was strongly diminished. Taken together, these findings point to a fundamental role for simultaneous induction and repression of mRNAs that encode functionally related proteins during the differentiation process.

IDH1R132H in Neural Stem Cells: Differentiation Impaired by Increased Apoptosis

PubMed Central

Rosiak, Kamila; Smolarz, Maciej; Stec, Wojciech J.; Peciak, Joanna; Grzela, Dawid; Winiecka-Klimek, Marta; Stoczynska-Fidelus, Ewelina; Krynska, Barbara; Piaskowski, Sylwester; Rieske, Piotr

2016-01-01

Background The high frequency of mutations in the isocitrate dehydrogenase 1 (IDH1) gene in diffuse gliomas indicates its importance in the process of gliomagenesis. These mutations result in loss of the normal function and acquisition of the neomorphic activity converting α-ketoglutarate to 2-hydroxyglutarate. This potential oncometabolite may induce the epigenetic changes, resulting in the deregulated expression of numerous genes, including those related to the differentiation process or cell survivability. Methods Neural stem cells were derived from human induced pluripotent stem cells following embryoid body formation. Neural stem cells transduced with mutant IDH1R132H, empty vector, non-transduced and overexpressing IDH1WT controls were differentiated into astrocytes and neurons in culture. The neuronal and astrocytic differentiation was determined by morphology and expression of lineage specific markers (MAP2, Synapsin I and GFAP) as determined by real-time PCR and immunocytochemical staining. Apoptosis was evaluated by real-time observation of Caspase-3 activation and measurement of PARP cleavage by Western Blot. Results Compared with control groups, cells expressing IDH1R132H retained an undifferentiated state and lacked morphological changes following stimulated differentiation. The significant inhibitory effect of IDH1R132H on neuronal and astrocytic differentiation was confirmed by immunocytochemical staining for markers of neural stem cells. Additionally, real-time PCR indicated suppressed expression of lineage markers. High percentage of apoptotic cells was detected within IDH1R132H-positive neural stem cells population and their derivatives, if compared to normal neural stem cells and their derivatives. The analysis of PARP and Caspase-3 activity confirmed apoptosis sensitivity in mutant protein-expressing neural cells. Conclusions Our study demonstrates that expression of IDH1R132H increases apoptosis susceptibility of neural stem cells and their derivatives. Robust apoptosis causes differentiation deficiency of IDH1R132H-expressing cells. PMID:27145078

Global miRNA expression profile reveals novel molecular players in aneurysmal subarachnoid haemorrhage.

PubMed

Lopes, Katia de Paiva; Vinasco-Sandoval, Tatiana; Vialle, Ricardo Assunção; Paschoal, Fernando Mendes; Bastos, Vanessa Albuquerque P Aviz; Bor-Seng-Shu, Edson; Teixeira, Manoel Jacobsen; Yamada, Elizabeth Sumi; Pinto, Pablo; Vidal, Amanda Ferreira; Ribeiro-Dos-Santos, Arthur; Moreira, Fabiano; Santos, Sidney; Paschoal, Eric Homero Albuquerque; Ribeiro-Dos-Santos, Ândrea

2018-06-08

The molecular mechanisms behind aneurysmal subarachnoid haemorrhage (aSAH) are still poorly understood. Expression patterns of miRNAs may help elucidate the post-transcriptional gene expression in aSAH. Here, we evaluate the global miRNAs expression profile (miRnome) of patients with aSAH to identify potential biomarkers. We collected 33 peripheral blood samples (27 patients with cerebral aneurysm, collected 7 to 10 days after the haemorrhage, when usually is the cerebral vasospasm risk peak, and six controls). Then, were performed small RNA sequencing using an Illumina Next Generation Sequencing (NGS) platform. Differential expression analysis identified eight differentially expressed miRNAs. Among them, three were identified being up-regulated, and five down-regulated. miR-486-5p was the most abundant expressed and is associated with poor neurological admission status. In silico miRNA gene target prediction showed 148 genes associated with at least two differentially expressed miRNAs. Among these, THBS1 and VEGFA, known to be related to thrombospondin and vascular endothelial growth factor. Moreover, MYC gene was found to be regulated by four miRNAs, suggesting an important role in aneurysmal subarachnoid haemorrhage. Additionally, 15 novel miRNAs were predicted being expressed only in aSAH, suggesting possible involvement in aneurysm pathogenesis. These findings may help the identification of novel biomarkers of clinical interest.

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.

A novel approach for discovering condition-specific correlations of gene expressions within biological pathways by using cloud computing technology.

PubMed

Chang, Tzu-Hao; Wu, Shih-Lin; Wang, Wei-Jen; Horng, Jorng-Tzong; Chang, Cheng-Wei

2014-01-01

Microarrays are widely used to assess gene expressions. Most microarray studies focus primarily on identifying differential gene expressions between conditions (e.g., cancer versus normal cells), for discovering the major factors that cause diseases. Because previous studies have not identified the correlations of differential gene expression between conditions, crucial but abnormal regulations that cause diseases might have been disregarded. This paper proposes an approach for discovering the condition-specific correlations of gene expressions within biological pathways. Because analyzing gene expression correlations is time consuming, an Apache Hadoop cloud computing platform was implemented. Three microarray data sets of breast cancer were collected from the Gene Expression Omnibus, and pathway information from the Kyoto Encyclopedia of Genes and Genomes was applied for discovering meaningful biological correlations. The results showed that adopting the Hadoop platform considerably decreased the computation time. Several correlations of differential gene expressions were discovered between the relapse and nonrelapse breast cancer samples, and most of them were involved in cancer regulation and cancer-related pathways. The results showed that breast cancer recurrence might be highly associated with the abnormal regulations of these gene pairs, rather than with their individual expression levels. The proposed method was computationally efficient and reliable, and stable results were obtained when different data sets were used. The proposed method is effective in identifying meaningful biological regulation patterns between conditions.

RNASeq analysis of differentiated keratinocytes reveals a massive response to late events during human papillomavirus type 16 infection, including loss of epithelial barrier function.

PubMed

Klymenko, T; Gu, Q; Herbert, I; Stevenson, A; Iliev, V; Watkins, G; Pollock, C; Bhatia, R; Cuschieri, K; Herzyk, P; Gatherer, D; Graham, S V

2017-10-11

The human papillomavirus (HPV) replication cycle is tightly linked to epithelial cell differentiation. To examine HPV-associated changes in the keratinocyte transcriptome, RNAs isolated from undifferentiated and differentiated cell populations of normal, spontaneously immortalised, keratinocytes (NIKS), and NIKS stably transfected with HPV16 episomal genomes (NIKS16), were compared using RNASeq. HPV16 infection altered expression of 2862 cellular genes. Next, to elucidate the role of keratinocyte gene expression in late events during the viral life cycle, RNASeq was carried out on triplicate differentiated populations of NIKS (uninfected) and NIKS16 (infected). Of the top 966 genes altered (>log 2 = 1.8, 3.5-fold change) 670 genes were downregulated and 296 genes were up-regulated. HPV down-regulated many genes involved in epithelial barrier function that involves structural resistance to the environment and immunity to infectious agents. For example, HPV infection repressed expression of the differentiated keratinocyte-specific pattern recognition receptor TLR7, the Langerhans cell chemoattractant, CCL20, and proinflammatory cytokines, IL1A and IL1B. However, IRF1, IFNκ and viral restriction factors (IFIT1, 2, 3, 5, OASL, CD74, RTP4) were up-regulated. HPV infection abrogated gene expression associated with the physical epithelial barrier, including keratinocyte cytoskeleton, intercellular junctions and cell adhesion. qPCR and western blotting confirmed changes in expression of seven of the most significantly altered mRNAs. Expression of three genes showed statistically significant changes during cervical disease progression in clinical samples. Taken together, the data indicate that HPV infection manipulates the differentiating keratinocyte transcriptome to create an environment conducive to productive viral replication and egress. IMPORTANCE Human papillomavirus (HPV) genome amplification and capsid formation takes place in differentiated keratinocytes. The viral life cycle is intimately associated with host cell differentiation. Deep sequencing (RNASeq) of RNA from undifferentiated and differentiated uninfected and HPV16-positive keratinocytes showed that almost 3000 genes were differentially expressed in keratinocyte due to HPV16 infection. Strikingly, the epithelial barrier function of differentiated keratinocytes, comprising keratinocyte immune function and cellular structure, was found to be disrupted. These data provide new insights into virus-host interaction crucial for production of infectious virus and reveal that HPV infection remodels keratinocytes for completion of the virus replication cycle. Copyright © 2017 American Society for Microbiology.

Different Subcellular Localization of ALCAM Molecules in Neuroblastoma: Association with Relapse

PubMed Central

Corrias, Maria Valeria; Gambini, Claudio; Gregorio, Andrea; Croce, Michela; Barisione, Gaia; Cossu, Claudia; Rossello, Armando; Ferrini, Silvano; Fabbi, Marina

2010-01-01

Background: The Activated Leukocyte Cell Adhesion Molecule (ALCAM/CD), involved in nervous system development, has been linked to tumor progression and metastasis in several tumors. No information is available on ALCAM expression in neuroblastoma, a childhood neoplasia originating from the sympathetic nervous system. Methods: ALCAM expression was analysed by immunofluorescence and immunohistochemistry on differentiated neuroblastoma cell lines and on archival specimens of stroma-poor, not MYCN amplified, resectable neuroblastoma tumors, respectively. Results: ALCAM is variously expressed in neuroblastoma cell lines, is shed by metalloproteases and is cleaved by ADAM17/TACE in vitro. ALCAM is expressed in neuroblastoma primary tumors with diverse patterns of subcellular localization and is highly expressed in the neuropil area in a subgroup of cases. Tumor specimens showing high expression of ALCAM at the membrane of the neuroblast body or low levels in the neuropil area are associated with relapse (P = 0.044 and P < 0.0001, respectively). In vitro differentiated neuroblastoma cells show strong ALCAM expression on neurites, suggesting that ALCAM expression in the neuropil is related to a differentiated phenotype. Conclusions: Assessment of ALCAM localization by immunohistochemistry may help to identify patients who, in the absence of negative prognostic factors, are at risk of relapse and require a more careful follow-up. PMID:20208136

Oestrogen receptor-mediated expression of Olfactomedin 4 regulates the progression of endometrial adenocarcinoma

PubMed Central

Duan, Chao; Liu, Xubin; Liang, Shuang; Yang, Zheng; Xia, Meng; Wang, Liantang; Chen, Shangwu; Yu, Li

2014-01-01

Endometrial adenocarcinoma is the most common tumour of the female genital tract in developed countries, and oestrogen receptor (ER) signalling plays a pivotal role in its pathogenesis. When we used bioinformatics tools to search for the genes contributing to gynecological cancers, the expression of Olfactomedin 4 (OLFM4) was found by digital differential display to be associated with differentiation of endometrial adenocarcinoma. Aberrant expression of OLFM4 has been primarily reported in tumours of the digestive system. The mechanism of OLFM4 in tumuorigenesis is elusive. We investigated OLFM4 expression in endometrium, analysed the association of OLFM4 with ER signalling in endometrial adenocarcinoma, and examined the roles of OLFM4 in endometrial adenocarcinoma. Expression of OLFM4 was increased during endometrial carcinogenesis, linked to the differentiation of endometrioid adenocarcinoma, and positively related to the expression of oestrogen receptor-α (ERα) and progesterone receptor. Moreover, ERα-mediated signalling regulated expression of OLFM4, and knockdown of OLFM4 enhanced proliferation, migration and invasion of endometrial carcinoma cells. Down-regulation of OLFM4 was associated with decreased cumulative survival rate of patients with endometrioid adenocarcinoma. Our data suggested that impairment of ERα signal-mediated OLFM4 expression promoted the malignant progression of endometrioid adenocarcinoma, which may have significance for the therapy of this carcinoma. PMID:24495253

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

Extremely High Expression of Antisense RNA for Wilms' Tumor 1 in Active Osteoclasts: Suppression of Wilms' Tumor 1 Protein Expression during Osteoclastogenesis.

PubMed

Li, Yin-Ji; Kukita, Akiko; Kyumoto-Nakamura, Yukari; Kukita, Toshio

2016-09-01

Wilms' tumor 1 (WT1), a zinc-finger transcription regulator of the early growth response family, identified as the product of a tumor suppressor gene of Wilms' tumors, bears potential ability to induce macrophage differentiation in blood cell differentiation. Herein, we examined the involvement of WT1 in the regulation of osteoclastogenesis. We detected a high level of WT1 protein expression in osteoclast precursors; however, WT1 expression was markedly suppressed during osteoclastogenesis. We examined expression of WT1 transcripts in bone tissue by RNA in situ hybridization. We found a high level of antisense transcripts in osteoclasts actively resorbing bone in mandible of newborn rats. Expression of antisense WT1 RNA in mandible was also confirmed by Northern blot analysis and strand-specific RT-PCR. Overexpression of antisense WT1 RNA in RAW-D cells, an osteoclast precursor cell line, resulted in a marked enhancement of osteoclastogenesis, suggesting that antisense WT1 RNA functions to suppress expression of WT1 protein in osteoclastogenesis. High level expression of antisense WT1 RNA may contribute to commitment to osteoclastogenesis, and may allow osteoclasts to maintain or stabilize their differentiation state. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Differentially expressed JAK-STAT signaling pathway genes and target microRNAs in the spleen of necrotic enteritis-afflicted chicken lines

USDA-ARS?s Scientific Manuscript database

The JAK signal transducer and STAT signaling pathway is an important regulator of cell proliferation, differentiation, survival, motility, apoptosis, immune response, and development. In this study, we used RNA-Sequencing, qRT-PCR, and bioinformatics tools to investigate the differential expression ...

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

Dose-related gene expression changes in forebrain following acute, low-level chlorpyrifos exposure in neonatal rats

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

Ray, Anamika; Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078; Liu Jing

2010-10-15

Chlorpyrifos (CPF) is a widely used organophosphorus insecticide (OP) and putative developmental neurotoxicant in humans. The acute toxicity of CPF is elicited by acetylcholinesterase (AChE) inhibition. We characterized dose-related (0.1, 0.5, 1 and 2 mg/kg) gene expression profiles and changes in cell signaling pathways 24 h following acute CPF exposure in 7-day-old rats. Microarray experiments indicated that approximately 9% of the 44,000 genes were differentially expressed following either one of the four CPF dosages studied (546, 505, 522, and 3,066 genes with 0.1, 0.5, 1.0 and 2.0 mg/kg CPF). Genes were grouped according to dose-related expression patterns using K-means clusteringmore » while gene networks and canonical pathways were evaluated using Ingenuity Pathway Analysis (registered) . Twenty clusters were identified and differential expression of selected genes was verified by RT-PCR. The four largest clusters (each containing from 276 to 905 genes) constituted over 50% of all differentially expressed genes and exhibited up-regulation following exposure to the highest dosage (2 mg/kg CPF). The total number of gene networks affected by CPF also rose sharply with the highest dosage of CPF (18, 16, 18 and 50 with 0.1, 0.5, 1 and 2 mg/kg CPF). Forebrain cholinesterase (ChE) activity was significantly reduced (26%) only in the highest dosage group. Based on magnitude of dose-related changes in differentially expressed genes, relative numbers of gene clusters and signaling networks affected, and forebrain ChE inhibition only at 2 mg/kg CPF, we focused subsequent analyses on this treatment group. Six canonical pathways were identified that were significantly affected by 2 mg/kg CPF (MAPK, oxidative stress, NF{Kappa}B, mitochondrial dysfunction, arylhydrocarbon receptor and adrenergic receptor signaling). Evaluation of different cellular functions of the differentially expressed genes suggested changes related to olfactory receptors, cell adhesion/migration, synapse/synaptic transmission and transcription/translation. Nine genes were differentially affected in all four CPF dosing groups. We conclude that the most robust, consistent changes in differential gene expression in neonatal forebrain across a range of acute CPF dosages occurred at an exposure level associated with the classical marker of OP toxicity, AChE inhibition. Disruption of multiple cellular pathways, in particular cell adhesion, may contribute to the developmental neurotoxicity potential of this pesticide.« less

Prox1 and fibroblast growth factor receptors form a novel regulatory loop controlling lens fiber differentiation and gene expression

PubMed Central

Audette, Dylan S.; Anand, Deepti; So, Tammy; Rubenstein, Troy B.; Lachke, Salil A.; Lovicu, Frank J.; Duncan, Melinda K.

2016-01-01

Lens epithelial cells differentiate into lens fibers (LFs) in response to a fibroblast growth factor (FGF) gradient. This cell fate decision requires the transcription factor Prox1, which has been hypothesized to promote cell cycle exit in differentiating LF cells. However, we find that conditional deletion of Prox1 from mouse lenses results in a failure in LF differentiation despite maintenance of normal cell cycle exit. Instead, RNA-seq demonstrated that Prox1 functions as a global regulator of LF cell gene expression. Intriguingly, Prox1 also controls the expression of fibroblast growth factor receptors (FGFRs) and can bind to their promoters, correlating with decreased downstream signaling through MAPK and AKT in Prox1 mutant lenses. Further, culturing rat lens explants in FGF increased their expression of Prox1, and this was attenuated by the addition of inhibitors of MAPK. Together, these results describe a novel feedback loop required for lens differentiation and morphogenesis, whereby Prox1 and FGFR signaling interact to mediate LF differentiation in response to FGF. PMID:26657765

Sexual dimorphic expression of DMRT1 and Sox9a during gonadal differentiation and hormone-induced sex reversal in the teleost fish Nile tilapia (Oreochromis niloticus).

PubMed

Kobayashi, Tohru; Kajiura-Kobayashi, Hiroko; Guan, Guijun; Nagahama, Yoshitaka

2008-01-01

We examined the expression profiles of tDMRT1 and Sox9a during gonadal sex differentiation and hormone-induced sex reversal. tDMRT1 was detected in the gonial germ-cell-surrounding cells in XY fry specifically before the appearance of any signs of morphological sex differentiation, that is, sex differences in germ cell number and histogenesis, such as differentiation into intratesticular efferent duct or ovarian cavity. The signals became localized in the Sertoli and epithelial cells comprising the efferent duct during gonadal differentiation. After the induction of XY sex reversal with estrogen, tDMRT1 decreased and then disappeared completely. In contrast, tDMRT1 was expressed in the germ-cell-surrounding cells in XX sex reversal with androgen. On the other hand, Sox9a did not show sexual dimorphism before the appearance of sex differences in histogenesis and was not expressed in the efferent duct in the testis. These results suggest that tDMRT1 is a superior testicular differentiation marker in tilapia.

Prox1 and fibroblast growth factor receptors form a novel regulatory loop controlling lens fiber differentiation and gene expression.

PubMed

Audette, Dylan S; Anand, Deepti; So, Tammy; Rubenstein, Troy B; Lachke, Salil A; Lovicu, Frank J; Duncan, Melinda K

2016-01-15

Lens epithelial cells differentiate into lens fibers (LFs) in response to a fibroblast growth factor (FGF) gradient. This cell fate decision requires the transcription factor Prox1, which has been hypothesized to promote cell cycle exit in differentiating LF cells. However, we find that conditional deletion of Prox1 from mouse lenses results in a failure in LF differentiation despite maintenance of normal cell cycle exit. Instead, RNA-seq demonstrated that Prox1 functions as a global regulator of LF cell gene expression. Intriguingly, Prox1 also controls the expression of fibroblast growth factor receptors (FGFRs) and can bind to their promoters, correlating with decreased downstream signaling through MAPK and AKT in Prox1 mutant lenses. Further, culturing rat lens explants in FGF increased their expression of Prox1, and this was attenuated by the addition of inhibitors of MAPK. Together, these results describe a novel feedback loop required for lens differentiation and morphogenesis, whereby Prox1 and FGFR signaling interact to mediate LF differentiation in response to FGF. © 2016. Published by The Company of Biologists Ltd.

Differentially displayed expressed sequence tags in Melipona scutellaris (Hymenoptera, Apidae, Meliponini) development.

PubMed

Santana, Flávia A; Nunes, Francis M F; Vieira, Carlos U; Machado, Maria Alice M S; Kerr, Warwick E; Silva, Wilson A; Bonetti, Ana Maria

2006-03-01

We have compared gene expression, using the Differential Display Reverse Transcriptase-Polymerase Chain Reaction (DDRT-PCR) technique, by means of mRNA profile in Melipona scutellaris during ontogenetic postembryonic development, in adult worker, and in both Natural and Juvenile Hormone III-induced adult queen. Six, out of the nine ESTs described here, presented differentially expressed in the phases L1 or L2, or even in both of them, suggesting that key mechanisms to the development of Melipona scutellaris are regulated in these stages. The combination HT11G-AP05 revealed in L1 and L2 a product which matches to thioredoxin reductase protein domain in the Clostridium sporogenes, an important protein during cellular oxidoreduction processes. This study represents the first molecular evidence of differential gene expression profiles toward a description of the genetic developmental traits in the genus Melipona.

[Study on screening differentially expressed genes in mice livers by silver staining DD-PCR].

PubMed

Luan, Xin-Hong; Hu, Zhong-Ming; Liu, Wei-Quan; Jiang, Yu; Wang, Kai; Wu, Yong-Kui; Li, Qian-Xue

2005-08-01

To screen swimming-fatigue related genes in mice and lay theoretic basis for researching the molecular mechanism of fatigue. 30 male BALB/c mice (20 +/- 2g) were divided into control group, dipping in water group and swimming-fatigue group respectively. After fatigue for swimming in swimming-fatigue group, with control group and dipping in water group, liver tissues in mice were collected. With improved silver staining mRNA differential display method, the differentially expressed genes in mice livers were screened and evaluated by reversed Northern blot. The positive segments were analyzed homology by BLAST. 7 of DD-ESTs were gained. Two of them only expressed in swimming-fatigue group, two down-regulated expressed, and three up-regulated. One of them was a novel gene and was accepted by GenBank, AY615302. Seven DD-ESTs in swimming-fatigue mice were gained by silver staining mRNA differential display method.

Differentiation Affects the Release of Exosomes from Colon Cancer Cells and Their Ability to Modulate the Behavior of Recipient Cells.

PubMed

Lucchetti, Donatella; Calapà, Federica; Palmieri, Valentina; Fanali, Caterina; Carbone, Federica; Papa, Alfredo; De Maria, Ruggero; De Spirito, Marco; Sgambato, Alessandro

2017-07-01

Exosomes are involved in intercellular communication. We previously reported that sodium butyrate-induced differentiation of HT29 colon cancer cells is associated with a reduced CD133 expression. Herein, we analyzed the role of exosomes in the differentiation of HT29 cells. Exosomes were prepared using ultracentrifugation. Gene expression levels were evaluated by real-time PCR. The cell proliferation rate was assessed by MTT assay and with the electric cell-substrate impedance sensing system, whereas cell motility was assessed using the scratch test and confocal microscopy. Sodium butyrate-induced differentiation of HT29 and Caco-2 cells increased the levels of released exosomes and their expression of CD133. Cell differentiation and the decrease of cellular CD133 expression levels were prevented by blocking multivesicular body maturation. Exosomes released by HT29 differentiating cells carried increased levels of miRNAs, induced an increased proliferation and motility of both colon cancer cells and normal fibroblasts, increased the colony-forming efficiency of cancer cells, and reduced the sodium butyrate-induced differentiation of HT29 cells. Such effects were associated with an increased phosphorylation level of both Src and extracellular signal regulated kinase proteins and with an increased expression of epithelial-to-mesenchymal transition-related genes. Release of exosomes is affected by differentiation of colon cancer cells; exosomes might be used by differentiating cells to get rid of components that are no longer necessary but might continue to exert their effects on recipient cells. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

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

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.

HES6 reverses nuclear reprogramming of insulin-producing cells following cell fusion

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

Ball, Andrew J.; Abrahamsson, Annelie E.; Tyrberg, Bjoern

2007-04-06

To examine the mechanism by which growth-stimulated pancreatic {beta}-cells dedifferentiate, somatic cell fusions were performed between MIN6, a highly differentiated mouse insulinoma, and {beta}lox5, a cell line derived from human {beta}-cells which progressively dedifferentiated in culture. MIN6/{beta}lox5 somatic cells hybrids underwent silencing of insulin expression and a marked decline in PDX1, NeuroD, and MafA, indicating that {beta}lox5 expresses a dominant transacting factor(s) that represses {beta}-cell differentiation. Expression of Hes1, which inhibits endocrine differentiation was higher in hybrid cells than in parental MIN6 cells. Hes6, a repressor of Hes1, was highly expressed in primary {beta}-cells as well as MIN6, but wasmore » repressed in hybrids. Hes6 overexpression using a retroviral vector led to a decrease in Hes1 levels, an increase in {beta}-cell transcription factors and partial restoration of insulin expression. We conclude that the balance of Notch activators and inhibitors may play an important role in maintaining the {beta}-cell differentiated state.« less

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

Expression of PD-1 and PD-L1 in poorly differentiated neuroendocrine carcinomas of the digestive system: a potential target for anti-PD-1/PD-L1 therapy.

PubMed

Roberts, Jordan A; Gonzalez, Raul S; Das, Satya; Berlin, Jordan; Shi, Chanjuan

2017-12-01

Poorly differentiated neuroendocrine carcinoma of the digestive system has a dismal prognosis with limited treatment options. This study aimed to investigate expression of the PD-1/PD-L1 pathway in these tumors. Thirty-seven patients with a poorly differentiated neuroendocrine carcinoma of the digestive system were identified. Their electronic medical records, pathology reports, and pathology slides were reviewed for demographics, clinical history, and pathologic features. Tumor sections were immunohistochemically labeled for PD-1 and PD-L1, and expression of PD-1 and PD-L1 on tumor and tumor-associated immune cells was analyzed and compared between small cell and large cell neuroendocrine carcinomas. The mean age of patients was 61 years old with 18 men and 19 women. The colorectum (n=20) was the most common primary site; other primary sites included the pancreaticobiliary system, esophagus, stomach, duodenum, and ampulla. Expression of PD-1 was detected on tumor cells (n=6, 16%) as well as on tumor-associated immune cells (n=23, 63%). The 6 cases with PD-1 expression on tumor cells also had the expression on immune cells. Expression of PD-L1 was visualized on tumor cells in 5 cases (14%) and on tumor-associated immune cells in 10 cases (27%). There was no difference in PD-1 and PD-L1 expression between small cell and large cell neuroendocrine carcinomas. In conclusion, PD-1/PD-L1 expression is a frequent occurrence in poorly differentiated neuroendocrine carcinomas of the digestive system. Checkpoint blockade targeting the PD-1/PD-L1 pathway may have a potential role in treating patients with this disease. Copyright © 2017 Elsevier Inc. All rights reserved.

EWS Knockdown and Taxifolin Treatment Induced Differentiation and Removed DNA Methylation from p53 Promoter to Promote Expression of Puma and Noxa for Apoptosis in Ewing’s Sarcoma

PubMed Central

Hossain, Mohammad Motarab; Ray, Swapan Kumar

2016-01-01

Ewing’s sarcoma is a pediatric tumor that mainly occurs in soft tissues and bones. Malignant characteristics of Ewing’s sarcoma are correlated with expression of EWS oncogene. We achieved knockdown of EWS expression using a plasmid vector encoding EWS short hairpin RNA (shRNA) to increase anti-tumor mechanisms of taxifolin (TFL), a new flavonoid, in human Ewing’s sarcoma cells in culture and animal models. Immunofluorescence microscopy and flow cytometric analysis showed high expression of EWS in human Ewing’s sarcoma SK-N-MC and RD-ES cell lines. EWS shRNA plus TFL inhibited 80% cell viability and caused the highest decreases in EWS expression at mRNA and protein levels in both cell lines. Knockdown of EWS expression induced morphological features of differentiation. EWS shRNA plus TFL caused more alterations in molecular markers of differentiation than either agent alone. EWS shRNA plus TFL caused the highest decreases in cell migration with inhibition of survival, angiogenic and invasive factors. Knockdown of EWS expression was associated with removal of DNA methylation from p53 promoter, promoting expression of p53, Puma, and Noxa. EWS shRNA plus TFL induced the highest amounts of apoptosis with activation of extrinsic and intrinsic pathways in both cell lines in culture. EWS shRNA plus TFL also inhibited growth of Ewing’s sarcoma tumors in animal models due to inhibition of differentiation inhibitors and angiogenic and invasive factors and also induction of activation of caspase-3 for apoptosis. Collectively, knockdown of EWS expression increased various anti-tumor mechanisms of TFL in human Ewing’s sarcoma in cell culture and animal models. PMID:27547487

EWS Knockdown and Taxifolin Treatment Induced Differentiation and Removed DNA Methylation from p53 Promoter to Promote Expression of Puma and Noxa for Apoptosis in Ewing's Sarcoma.

PubMed

Hossain, Mohammad Motarab; Ray, Swapan Kumar

2014-10-01

Ewing's sarcoma is a pediatric tumor that mainly occurs in soft tissues and bones. Malignant characteristics of Ewing's sarcoma are correlated with expression of EWS oncogene. We achieved knockdown of EWS expression using a plasmid vector encoding EWS short hairpin RNA (shRNA) to increase anti-tumor mechanisms of taxifolin (TFL), a new flavonoid, in human Ewing's sarcoma cells in culture and animal models. Immunofluorescence microscopy and flow cytometric analysis showed high expression of EWS in human Ewing's sarcoma SK-N-MC and RD-ES cell lines. EWS shRNA plus TFL inhibited 80% cell viability and caused the highest decreases in EWS expression at mRNA and protein levels in both cell lines. Knockdown of EWS expression induced morphological features of differentiation. EWS shRNA plus TFL caused more alterations in molecular markers of differentiation than either agent alone. EWS shRNA plus TFL caused the highest decreases in cell migration with inhibition of survival, angiogenic and invasive factors. Knockdown of EWS expression was associated with removal of DNA methylation from p53 promoter, promoting expression of p53, Puma, and Noxa. EWS shRNA plus TFL induced the highest amounts of apoptosis with activation of extrinsic and intrinsic pathways in both cell lines in culture. EWS shRNA plus TFL also inhibited growth of Ewing's sarcoma tumors in animal models due to inhibition of differentiation inhibitors and angiogenic and invasive factors and also induction of activation of caspase-3 for apoptosis. Collectively, knockdown of EWS expression increased various anti-tumor mechanisms of TFL in human Ewing's sarcoma in cell culture and animal models.

Decreased expression of glutathione S-transferase pi correlates with poorly differentiated grade in patients with oral squamous cell carcinoma.

PubMed

Ma, Hai-long; Yu, Cong; Liu, Ying; Tan, Yi-ran; Qiao, Jin-ke; Yang, Xi; Wang, Li-zhen; Li, Jiang; Chen, Qiong; Chen, Fu-xiang; Zhang, Zhi-yuan; Zhong, Lai-ping

2015-03-01

Glutathione S transferase pi (GSTP1) is a member of phase II detoxification enzymes as a major regulator of cell signaling in response to stress, hypoxia, growth factors, and other stimuli. The clinical role of GSTP1 in cancer is still unclear. The aim of this study was to investigate the serum GSTP1 level in patients with oral squamous cell carcinoma (OSCC) and the GSTP1 expression in tissue samples from patients with OSCC and OSCC lines. One hundred and sixty-six patients with OSCC and 120 normal persons were used to screen potential serum peptide biomarkers using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Serum GSTP1 concentration was detected in 18 patients with OSCC and 18 normal persons using ELISA. Immunohistochemistry was used to detect GSTP1 expression in tissue samples from twenty-eight OSCC patients. Western blot and real-time PCR were used to detect GSTP1 expression in nine OSCC lines. Decreased GSTP1 concentration was found in the patients with OSCC compared with the normal persons by MALDI-TOF-MS, which was then confirmed by ELISA (P = 0.019). Decreased GSTP1 mRNA level and protein expression were also found in the OSCC lines. Decreased GSTP1 expression was found correlating with pathological differentiation grade in the tissue samples from OSCC patients, a lower GSTP1 expression indicating a poorer pathological differentiation grade (P = 0.041). These results suggest that decreased GSTP1 expression in patients with OSCC and a lower GSTP1 expression indicating a poorer pathological differentiation grade in OSCC tissue samples. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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.

Hypothermia reduces VEGF-165 expression, but not osteogenic differentiation of human adipose stem cells under hypoxia

PubMed Central

Bakker, Astrid D.; Hogervorst, Jolanda M. A.; Nolte, Peter A.; Klein-Nulend, Jenneke

2017-01-01

Cryotherapy is successfully used in the clinic to reduce pain and inflammation after musculoskeletal damage, and might prevent secondary tissue damage under the prevalent hypoxic conditions. Whether cryotherapy reduces mesenchymal stem cell (MSC) number and differentiation under hypoxic conditions, causing impaired callus formation is unknown. We aimed to determine whether hypothermia modulates proliferation, apoptosis, nitric oxide production, VEGF gene and protein expression, and osteogenic/chondrogenic differentiation of human MSCs under hypoxia. Human adipose MSCs were cultured under hypoxia (37°C, 1% O2), hypothermia and hypoxia (30°C, 1% O2), or control conditions (37°C, 20% O2). Total DNA, protein, nitric oxide production, alkaline phosphatase activity, gene expression, and VEGF protein concentration were measured up to day 8. Hypoxia enhanced KI67 expression at day 4. The combination of hypothermia and hypoxia further enhanced KI67 gene expression compared to hypoxia alone, but was unable to prevent the 1.2-fold reduction in DNA amount caused by hypoxia at day 4. Addition of hypothermia to hypoxic cells did not alter the effect of hypoxia alone on BAX-to-BCL-2 ratio, alkaline phosphatase activity, gene expression of SOX9, COL1, or osteocalcin, or nitric oxide production. Hypothermia decreased the stimulating effect of hypoxia on VEGF-165 gene expression by 6-fold at day 4 and by 2-fold at day 8. Hypothermia also decreased VEGF protein expression under hypoxia by 2.9-fold at day 8. In conclusion, hypothermia decreased VEGF-165 gene and protein expression, but did not affect differentiation, or apoptosis of MSCs cultured under hypoxia. These in vitro results implicate that hypothermia treatment in vivo, applied to alleviate pain and inflammation, is not likely to harm early stages of callus formation. PMID:28166273