Alternating current electric fields of varying frequencies: effects on proliferation and differentiation of porcine neural progenitor cells.

PubMed

Lim, Ji-Hey; McCullen, Seth D; Piedrahita, Jorge A; Loboa, Elizabeth G; Olby, Natasha J

2013-10-01

Application of sinusoidal electric fields (EFs) has been observed to affect cellular processes, including alignment, proliferation, and differentiation. In the present study, we applied low-frequency alternating current (AC) EFs to porcine neural progenitor cells (pNPCs) and investigated the effects on cell patterning, proliferation, and differentiation. pNPCs were grown directly on interdigitated electrodes (IDEs) localizing the EFs to a region accessible visually for fluorescence-based assays. Cultures of pNPCs were exposed to EFs (1 V/cm) of 1 Hz, 10 Hz, and 50 Hz for 3, 7, and 14 days and compared to control cultures. Immunocytochemistry was performed to evaluate the expression of neural markers. pNPCs grew uniformly with no evidence of alignment to the EFs and no change in cell numbers when compared with controls. Nestin expression was shown in all groups at 3 and 7 days, but not at 14 days. NG2 expression was low in all groups. Co-expression of glial fibrillary acidic protein (GFAP) and TUJ1 was significantly higher in the cultures exposed to 10- and 50-Hz EFs than the controls. In summary, sinusoidal AC EFs via IDEs did not alter the alignment and proliferation of pNPCs, but higher frequency stimulation appeared to delay differentiation into mature astrocytes.

Unlike PPAR{gamma}, PPAR{alpha} or PPAR{beta}/{delta} activation does not promote human monocyte differentiation toward alternative macrophages

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

Bouhlel, Mohamed Amine; Inserm U545, F-59000 Lille; UDSL, F-59000 Lille

2009-08-28

Macrophages adapt their response to micro-environmental signals. While Th1 cytokines promote pro-inflammatory M1 macrophages, Th2 cytokines promote an 'alternative' anti-inflammatory M2 macrophage phenotype. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors expressed in macrophages where they control the inflammatory response. It has been shown that PPAR{gamma} promotes the differentiation of monocytes into anti-inflammatory M2 macrophages in humans and mice, while a role for PPAR{beta}/{delta} in this process has been reported only in mice and no data are available for PPAR{alpha}. Here, we show that in contrast to PPAR{gamma}, expression of PPAR{alpha} and PPAR{beta}/{delta} overall does not correlate with the expressionmore » of M2 markers in human atherosclerotic lesions, whereas a positive correlation with genes of lipid metabolism exists. Moreover, unlike PPAR{gamma}, PPAR{alpha} or PPAR{beta}/{delta} activation does not influence human monocyte differentiation into M2 macrophages in vitro. Thus, PPAR{alpha} and PPAR{beta}/{delta} do not appear to modulate the alternative differentiation of human macrophages.« less

Genome-wide dynamics of alternative polyadenylation in rice

PubMed Central

Fu, Haihui; Yang, Dewei; Su, Wenyue; Ma, Liuyin; Shen, Yingjia; Ji, Guoli; Ye, Xinfu; Wu, Xiaohui

2016-01-01

Alternative polyadenylation (APA), in which a transcript uses one of the poly(A) sites to define its 3′-end, is a common regulatory mechanism in eukaryotic gene expression. However, the potential of APA in determining crop agronomic traits remains elusive. This study systematically tallied poly(A) sites of 14 different rice tissues and developmental stages using the poly(A) tag sequencing (PAT-seq) approach. The results indicate significant involvement of APA in developmental and quantitative trait loci (QTL) gene expression. About 48% of all expressed genes use APA to generate transcriptomic and proteomic diversity. Some genes switch APA sites, allowing differentially expressed genes to use alternate 3′ UTRs. Interestingly, APA in mature pollen is distinct where differential expression levels of a set of poly(A) factors and different distributions of APA sites are found, indicating a unique mRNA 3′-end formation regulation during gametophyte development. Equally interesting, statistical analyses showed that QTL tends to use APA for regulation of gene expression of many agronomic traits, suggesting a potential important role of APA in rice production. These results provide thus far the most comprehensive and high-resolution resource for advanced analysis of APA in crops and shed light on how APA is associated with trait formation in eukaryotes. PMID:27733415

Isoflurane is a suitable alternative to ether for anesthetizing rats prior to euthanasia for gene expression analysis.

PubMed

Nakatsu, Noriyuki; Igarashi, Yoshinobu; Aoshi, Taiki; Hamaguchi, Isao; Saito, Masumichi; Mizukami, Takuo; Momose, Haruka; Ishii, Ken J; Yamada, Hiroshi

2017-01-01

Diethyl ether (ether) had been widely used in Japan for anesthesia, despite its explosive properties and toxicity to both humans and animals. We also had used ether as an anesthetic for euthanizing rats for research in the Toxicogenomics Project (TGP). Because the use of ether for these purposes will likely cease, it is required to select an alternative anesthetic which is validated for consistency with existing TGP data acquired under ether anesthesia. We therefore compared two alternative anesthetic candidates, isoflurane and pentobarbital, with ether in terms of hematological findings, serum biochemical parameters, and gene expressions. As a result, few differences among the three agents were observed. In hematological and serum biochemistry analysis, no significant changes were found. In gene expression analysis, four known genes were extracted as differentially expressed genes in the liver of rats anesthetized with ether, isoflurane, or pentobarbital. However, no significant relationships were detected using gene ontology, pathway, or gene enrichment analyses by DAVID and TargetMine. Surprisingly, although it was expected that the lung would be affected by administration via inhalation, only one differentially expressed gene was extracted in the lung. Taken together, our data indicate that there are no significant differences among ether, isoflurane, and pentobarbital with respect to effects on hematological parameters, serum biochemistry parameters, and gene expression. Based on its smallest affect to existing data and its safety profile for humans and animals, we suggest isoflurane as a suitable alternative anesthetic for use in rat euthanasia in toxicogenomics analysis.

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.

Comparing Acquisition of Exchange-Based and Signed Mands with Children with Autism

ERIC Educational Resources Information Center

Barlow, Kathryn E.; Tiger, Jeffrey H.; Slocum, Sarah K.; Miller, Sarah J.

2013-01-01

Therapists and educators frequently teach alternative-communication systems, such as picture exchanges or manual signs, to individuals with developmental disabilities who present with expressive language deficits. Michael (1985) recommended a taxonomy for alternative communication systems that differentiated between selection-based systems in…

Alternative splicing mechanisms orchestrating post-transcriptional gene expression: intron retention and the intron-rich genome of apicomplexan parasites.

PubMed

Lunghi, Matteo; Spano, Furio; Magini, Alessandro; Emiliani, Carla; Carruthers, Vern B; Di Cristina, Manlio

2016-02-01

Apicomplexan parasites including Toxoplasma gondii and Plasmodium species have complex life cycles that include multiple hosts and differentiation through several morphologically distinct stages requiring marked changes in gene expression. This review highlights emerging evidence implicating regulation of mRNA splicing as a mechanism to prime these parasites for rapid gene expression upon differentiation. We summarize the most important insights in alternative splicing including its role in regulating gene expression by decreasing mRNA abundance via 'Regulated Unproductive Splicing and Translation'. As a related but less well-understood mechanism, we discuss also our recent work suggesting a role for intron retention for precluding translation of stage specific isoforms of T. gondii glycolytic enzymes. We additionally provide new evidence that intron retention might be a widespread mechanism during parasite differentiation. Supporting this notion, recent genome-wide analysis of Toxoplasma and Plasmodium suggests intron retention is more pervasive than heretofore thought. These findings parallel recent emergence of intron retention being more prevalent in mammals than previously believed, thereby adding to the established roles in plants, fungi and unicellular eukaryotes. Deeper mechanistic studies of intron retention will provide important insight into its role in regulating gene expression in apicomplexan parasites and more general in eukaryotic organisms.

DIEGO: detection of differential alternative splicing using Aitchison's geometry.

PubMed

Doose, Gero; Bernhart, Stephan H; Wagener, Rabea; Hoffmann, Steve

2018-03-15

Alternative splicing is a biological process of fundamental importance in most eukaryotes. It plays a pivotal role in cell differentiation and gene regulation and has been associated with a number of different diseases. The widespread availability of RNA-Sequencing capacities allows an ever closer investigation of differentially expressed isoforms. However, most tools for differential alternative splicing (DAS) analysis do not take split reads, i.e. the most direct evidence for a splice event, into account. Here, we present DIEGO, a compositional data analysis method able to detect DAS between two sets of RNA-Seq samples based on split reads. The python tool DIEGO works without isoform annotations and is fast enough to analyze large experiments while being robust and accurate. We provide python and perl parsers for common formats. The software is available at: www.bioinf.uni-leipzig.de/Software/DIEGO. steve@bioinf.uni-leipzig.de. Supplementary data are available at Bioinformatics online.

Tissue-Specific 5′ Heterogeneity of PPARα Transcripts and Their Differential Regulation by Leptin

PubMed Central

Garratt, Emma S.; Vickers, Mark H.; Gluckman, Peter D.; Hanson, Mark A.

2013-01-01

The genes encoding nuclear receptors comprise multiple 5′untranslated exons, which give rise to several transcripts encoding the same protein, allowing tissue-specific regulation of expression. Both human and mouse peroxisome proliferator activated receptor (PPAR) α genes have multiple promoters, although their function is unknown. Here we have characterised the rat PPARα promoter region and have identified three alternative PPARα transcripts, which have different transcription start sites owing to the utilisation of distinct first exons. Moreover these alternative PPARα transcripts were differentially expressed between adipose tissue and liver. We show that while the major adipose (P1) and liver (P2) transcripts were both induced by dexamethasone, they were differentially regulated by the PPARα agonist, clofibric acid, and leptin. Leptin had no effect on the adipose-specific P1 transcript, but induced liver-specific P2 promoter activity via a STAT3/Sp1 mechanism. Moreover in Wistar rats, leptin treatment between postnatal day 3–13 led to an increase in P2 but not P1 transcription in adipose tissue which was sustained into adulthood. This suggests that the expression of the alternative PPARα transcripts are in part programmed by early life exposure to leptin leading to persistent change in adipose tissue fatty acid metabolism through specific activation of a quiescent PPARα promoter. Such complexity in the regulation of PPARα may allow the expression of PPARα to be finely regulated in response to environmental factors. PMID:23825665

Transcript expression plasticity as a response to alternative larval host plants in the speciation process of corn and rice strains of Spodoptera frugiperda.

PubMed

Silva-Brandão, Karina Lucas; Horikoshi, Renato Jun; Bernardi, Daniel; Omoto, Celso; Figueira, Antonio; Brandão, Marcelo Mendes

2017-10-16

Our main purpose was to evaluate the expression of plastic and evolved genes involved in ecological speciation in the noctuid moth Spodoptera frugiperda, the fall armyworm (FAW); and to demonstrate how host plants might influence lineage differentiation in this polyphagous insect. FAW is an important pest of several crops worldwide, and it is differentiated into host plant-related strains, corn (CS) and rice strains (RS). RNA-Seq and transcriptome characterization were applied to evaluate unbiased genetic expression differences in larvae from the two strains, fed on primary (corn) and alternative (rice) host plants. We consider that genes that are differently regulated by the same FAW strain, as a response to different hosts, are "plastic". Otherwise, differences in gene expression between the two strains fed on the same host are considered constitutive differences. Individual performance parameters (larval and pupal weight) varied among conditions (strains vs. hosts). A total of 3657 contigs was related to plastic response, and 2395 contigs were differentially regulated in the two strains feeding on preferential and alternative hosts (constitutive contigs). Three molecular functions were present in all comparisons, both down- and up-regulated: oxidoreductase activity, metal-ion binding, and hydrolase activity. Metabolization of foreign chemicals is among the key functions involved in the phenotypic variation of FAW strains. From an agricultural perspective, high plasticity in families of detoxifying genes indicates the capacity for a rapid response to control compounds such as insecticides.

Alternative lengthening of telomeres and loss of ATRX are frequent events in pleomorphic and dedifferentiated liposarcomas.

PubMed

Lee, Jen-Chieh; Jeng, Yung-Ming; Liau, Jau-Yu; Tsai, Jia-Huei; Hsu, Hung-Han; Yang, Ching-Yao

2015-08-01

Telomerase activation and alternative lengthening of telomeres are two major mechanisms of telomere length maintenance. Soft tissue sarcomas appear to use the alternative lengthening of telomeres more frequently. Loss of α-thalassemia/mental retardation syndrome X-linked (ATRX) or death domain-associated protein 6 (DAXX) expression has been implicated in the pathogenesis of alternative telomere lengthening in pancreatic endocrine neoplasm and glioma. The mechanism leading to the alternative lengthening of telomeres in liposarcoma remains unknown. Whereas alternative telomere lengthening was determined to be an indicator of poor prognosis in liposarcomas as a whole, its prognostic power has not been verified in any subtype of liposarcoma. In this study, we characterized the status of alternative telomere lengthening and expression of ATRX and DAXX in 111 liposarcomas (28 well-differentiated, 52 dedifferentiated, 20 myxoid or round cell, and 11 pleomorphic liposarcomas) by telomere fluorescence in situ hybridization and immunohistochemistry, respectively. Alternative lengthening of telomere was observed in 0% (0/16) of well-differentiated, 30% (14/46) of dedifferentiated, 5% (1/19) of myxoid or round cell, and 80% (8/10) of pleomorphic liposarcomas. Eighteen (16%) and one (1%) tumors were negative for ATRX and DAXX immunostaining, respectively. Remarkably, all cases with loss of either ATRX or DAXX expression had alternative lengthening of telomeres, and 83% (19/23) of tumors that had alternative lengthening of telomeres showed loss of either protein. The correlation between loss of either ATRX or DAXX and alternative telomere lengthening was 100% in dedifferentiated liposarcoma. The presence of alternative telomere lengthening in dedifferentiated liposarcoma suggested poor overall survival (hazard ratio=1.954, P=0.077) and was the most significant indicator of short progression-free survival (hazard ratio=3.119, P=0.003). In conclusion, we found that ATRX loss was the most likely mechanism of alternative telomere lengthening in liposarcoma and alternative telomere lengthening was a prognostic factor of poor outcome in dedifferentiated liposarcoma.

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.

System identification principles in studies of forest dynamics.

Treesearch

Rolfe A. Leary

1970-01-01

Shows how it is possible to obtain governing equation parameter estimates on the basis of observed system states. The approach used represents a constructive alternative to regression techniques for models expressed as differential equations. This approach allows scientists to more completely quantify knowledge of forest development processes, to express theories in...

Male-specific expression of Sox9 during gonad development of crocodile and mouse is mediated by alternative splicing of its proline-glutamine-alanine rich domain.

PubMed

Agrawal, Raman; Wessely, Oliver; Anand, Amit; Singh, Lalji; Aggarwal, Ramesh K

2009-08-01

The initial trigger for sexual differentiation is regulated by multiple ways during embryonic development. In vertebrates, chromosome-based mechanisms generally known as genetic sex determination are prevalent; however, some species, such as many reptilians, display temperature-dependent sex determination. The Sry-related transcription factor, Sox9, which is expressed by an evolutionary conserved gene, has been shown to be a key player in the process of sex determination. In the present study, we report the identification and expression of crocodile homolog of Sox9 (cpSox9) from the Indian Mugger, Crocodylus palustris. We show that cpSox9 undergoes extensive alternative splicing around the proline-glutamine-alanine rich transactivation domain that results in cpSox9 variants with presumably impaired or reduced transactivation potential. The multiple isoforms were also detected in various embryonic tissues, with some of them displaying a differential expression profile. With respect to sex differentiation, a putative unspliced full-length cpSox9 could be detected only in the genital ridge-adrenal-mesonephros complex of male, but not female embryos during the temperature-sensitive period. Importantly, we further show that this phenomenon was not restricted to the temperature-dependent sex determination species C. palustris, but was also observed in the mouse, a species exhibiting genetic sex determination. Thus, the present study describes, for the first time, a complete coding locus of Sox9 homolog from a temperature-dependent sex determination species. More importantly, we demonstrate an evolutionarily conserved role of alternative splicing resulting in transcriptional diversity and male-sex specific expression of Sox9 during testis development in vertebrates (i.e. irrespective of their underlying sex-determination mechanisms).

Differential expression of miR-31 between inflammatory bowel disease and microscopic colitis.

PubMed

Zhang, Chen; Zhao, Zijin; Osman, Hany; Watson, Rao; Nalbantoglu, Ilke; Lin, Jingmei

2014-01-01

Idiopathic inflammatory bowel disease (IBD) and microscopic colitis (MC) are distinct entities. However, patients with intermittent episodes of IBD and MC that are encountered in a clinical setting puzzle clinicians and pathologists. This study examined whether microRNA assisted in the classification of IBD and MC. Small RNA was extracted from formalin-fixed, paraffin-embedded (FFPE) colon tissue and qRT-PCR was performed from cohorts of normal control (n=38), ulcerative colitis (n=36), Crohns disease (n=26), collagenous colitis (n=36), lymphocytic colitis (n=30), and patients with intermittent features of IBD and MC (n=6). Differential expression of miR-31 distinguished IBD (ulcerative colitis and Crohns disease) from MC (collagenous colitis and lymphocytic colitis), confirming the specificity of miR-31 expression in IBD (P=0.00001). In addition, expression of miR-31 was increased in collagenous colitis compared to that of lymphocytic colitis (P=0.010). Among 6 patients with alternating episodes of IBD and MC, one patient had matching miR-31 expression in different phases (lymphocytic colitis to ulcerative colitis, and then back to collagenous colitis). The other 5 patients had MC-like expression patterns in both MC and IBD episodes. In summary, IBD and MC have distinct miR-31 expression pattern. Therefore, miR-31 might be used as a biomarker to distinguish between IBD and MC in FFPE colonic tissue. In addition, miR-31 is differentially expressed in colonic tissue between lymphocytic colitis and collagenous colitis, suggesting them of separate disease processes. Finally, patients with alternating IBD and MC episodes represent a diverse group. Among them, the majority demonstrates MC-like miR-31 expression pattern in MC phases, which seems unlikely to support the speculation of MC as an inactive form of IBD. Although the mechanisms deserve further investigation, microRNA is a potentially useful biomarker to differentiate IBD and MC.

The expression analysis of Sfrs10 and Celf4 during mouse retinal development

PubMed Central

Karunakaran, Devi Krishna Priya; Congdon, Sean; Guerrette, Thomas; Banday, Abdul Rouf; Lemoine, Christopher; Chhaya, Nisarg; Kanadia, Rahul

2013-01-01

Processing of mRNAs including, alternative splicing (AS), mRNA transport and translation regulation are crucial to eukaryotic gene expression. For example, >90% of the gene in the human genome are known to undergo alternative splicing thereby expanding the proteome production capacity of a limited number of genes. Similarly, mRNA export and translation regulation plays a vital role in regulating protein production. Thus, it is important to understand how these RNA binding proteins including alternative splicing factors (ASFs) and mRNA transport and translation factors regulate these processes. Here we report the expression of an ASF, Serine-arginine rich splicing factor 10 (Sfrs10) and a mRNA translation regulation factor, CUGBP, elav like family member 4 (Celf4) in the developing mouse retina. Sfrs10 was expressed throughout postnatal (P) retinal development and was observed progressively in newly differentiating neurons. Immunofluorescence (IF) showed Sfrs10 in retinal ganglion cells (RGCs) at P0, followed by amacrine and bipolar cells, and at P8 it was enriched in red/green cone photoreceptor cells. By P22, Sfrs10 was observed in rod photoreceptors in a peri-nuclear pattern. Like Sfrs10, Celf4 was also observed in the developing retina, but with two distinct retinal isoforms. In situ hybridization (ISH) showed progressive expression of Celf4 in differentiating neurons, which was confirmed by IF that showed a dynamic shift in Celf4 localization. Early in development Celf4 expression was restricted to the nuclei of newly differentiating RGCs and later (E16 onwards) it was observed in the initial segments of RGC axons. Later, during postnatal development, Celf4 was observed in amacrine and bipolar cells, but here it was predominantly cytoplasmic and enriched in the two synaptic layers. Specifically, at P14, Celf4 was observed in the synaptic boutons of rod bipolar cells marked by Pkc-α. Thus, Celf4 might be regulating AS early in development besides its known role of regulating mRNA localization/translation. In all, our data suggests an important role for AS and mRNA localization/translation in retinal neuron differentiation. PMID:23932931

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.

Multilevel regulation of gene expression by microRNAs.

PubMed

Makeyev, Eugene V; Maniatis, Tom

2008-03-28

MicroRNAs (miRNAs) are approximately 22-nucleotide-long noncoding RNAs that normally function by suppressing translation and destabilizing messenger RNAs bearing complementary target sequences. Some miRNAs are expressed in a cell- or tissue-specific manner and may contribute to the establishment and/or maintenance of cellular identity. Recent studies indicate that tissue-specific miRNAs may function at multiple hierarchical levels of gene regulatory networks, from targeting hundreds of effector genes incompatible with the differentiated state to controlling the levels of global regulators of transcription and alternative pre-mRNA splicing. This multilevel regulation may allow individual miRNAs to profoundly affect the gene expression program of differentiated cells.

Alternative role of HuD splicing variants in neuronal differentiation.

PubMed

Hayashi, Satoru; Yano, Masato; Igarashi, Mana; Okano, Hirotaka James; Okano, Hideyuki

2015-03-01

HuD is a neuronal RNA-binding protein that plays an important role in neuronal differentiation of the nervous system. HuD has been reported to have three RNA recognition motifs (RRMs) and three splice variants (SVs) that differ in their amino acid sequences between RRM2 and RRM3. This study investigates whether these SVs have specific roles in neuronal differentiation. In primary neural epithelial cells under differentiating conditions, HuD splice variant 1 (HuD-sv1), which is a general form, and HuD-sv2 were expressed at all tested times, whereas HuD-sv4 was transiently expressed at the beginning of differentiation, indicating that HuD-sv4 might play a role compared different from that of HuD-sv1. Indeed, HuD-sv4 did not promote neuronal differentiation in epithelial cells, whereas HuD-sv1 did promote neuronal differentiation. HuD-sv4 overexpression showed less neurite-inducing activity than HuD-sv1 in mouse neuroblastoma N1E-115 cells; however, HuD-sv4 showed stronger growth-arresting activity. HuD-sv1 was localized only in the cytoplasm, whereas HuD-sv4 was localized in both the cytoplasm and the nuclei. The Hu protein has been reported to be involved in translation and alternative splicing in the cytoplasm and nuclei, respectively. Consistent with this observation, HuD-sv1 showed translational activity on p21, which plays a role in growth arrest and neuronal differentiation, whereas HuD-sv4 did not. By contrast, HuD-sv4 showed stronger pre-mRNA splicing activity than did HuD-sv1 on Clasp2, which participates in cell division. Therefore, HuD SVs might play a role in controlling the timing of proliferation/differentiation switching by controlling the translation and alternative splicing of target genes. © 2014 Wiley Periodicals, Inc.

Population differentiation in Pacific salmon: local adaptation, genetic drift, or the environment?

USGS Publications Warehouse

Adkison, Milo D.

1995-01-01

Morphological, behavioral, and life-history differences between Pacific salmon (Oncorhynchus spp.) populations are commonly thought to reflect local adaptation, and it is likewise common to assume that salmon populations separated by small distances are locally adapted. Two alternatives to local adaptation exist: random genetic differentiation owing to genetic drift and founder events, and genetic homogeneity among populations, in which differences reflect differential trait expression in differing environments. Population genetics theory and simulations suggest that both alternatives are possible. With selectively neutral alleles, genetic drift can result in random differentiation despite many strays per generation. Even weak selection can prevent genetic drift in stable populations; however, founder effects can result in random differentiation despite selective pressures. Overlapping generations reduce the potential for random differentiation. Genetic homogeneity can occur despite differences in selective regimes when straying rates are high. In sum, localized differences in selection should not always result in local adaptation. Local adaptation is favored when population sizes are large and stable, selection is consistent over large areas, selective diffeentials are large, and straying rates are neither too high nor too low. Consideration of alternatives to local adaptation would improve both biological research and salmon conservation efforts.

HLA-G Haplotypes Are Differentially Associated with Asthmatic Features.

PubMed

Ribeyre, Camille; Carlini, Federico; René, Céline; Jordier, François; Picard, Christophe; Chiaroni, Jacques; Abi-Rached, Laurent; Gouret, Philippe; Marin, Grégory; Molinari, Nicolas; Chanez, Pascal; Paganini, Julien; Gras, Delphine; Di Cristofaro, Julie

2018-01-01

Human leukocyte antigen (HLA)-G, a HLA class Ib molecule, interacts with receptors on lymphocytes such as T cells, B cells, and natural killer cells to influence immune responses. Unlike classical HLA molecules, HLA-G expression is not found on all somatic cells, but restricted to tissue sites, including human bronchial epithelium cells (HBEC). Individual variation in HLA-G expression is linked to its genetic polymorphism and has been associated with many pathological situations such as asthma, which is characterized by epithelium abnormalities and inflammatory cell activation. Studies reported both higher and equivalent soluble HLA-G (sHLA-G) expression in different cohorts of asthmatic patients. In particular, we recently described impaired local expression of HLA-G and abnormal profiles for alternatively spliced isoforms in HBEC from asthmatic patients. sHLA-G dosage is challenging because of its many levels of polymorphism (dimerization, association with β2-microglobulin, and alternative splicing), thus many clinical studies focused on HLA-G single-nucleotide polymorphisms as predictive biomarkers, but few analyzed HLA-G haplotypes. Here, we aimed to characterize HLA-G haplotypes and describe their association with asthmatic clinical features and sHLA-G peripheral expression and to describe variations in transcription factor (TF) binding sites and alternative splicing sites. HLA - G haplotypes were differentially distributed in 330 healthy and 580 asthmatic individuals. Furthermore, HLA-G haplotypes were associated with asthmatic clinical features showed. However, we did not confirm an association between sHLA-G and genetic, biological, or clinical parameters. HLA-G haplotypes were phylogenetically split into distinct groups, with each group displaying particular variations in TF binding or RNA splicing sites that could reflect differential HLA-G qualitative or quantitative expression, with tissue-dependent specificities. Our results, based on a multicenter cohort, thus support the pertinence of HLA-G haplotypes as predictive genetic markers for asthma.

HLA-G Haplotypes Are Differentially Associated with Asthmatic Features

PubMed Central

Ribeyre, Camille; Carlini, Federico; René, Céline; Jordier, François; Picard, Christophe; Chiaroni, Jacques; Abi-Rached, Laurent; Gouret, Philippe; Marin, Grégory; Molinari, Nicolas; Chanez, Pascal; Paganini, Julien; Gras, Delphine; Di Cristofaro, Julie

2018-01-01

Human leukocyte antigen (HLA)-G, a HLA class Ib molecule, interacts with receptors on lymphocytes such as T cells, B cells, and natural killer cells to influence immune responses. Unlike classical HLA molecules, HLA-G expression is not found on all somatic cells, but restricted to tissue sites, including human bronchial epithelium cells (HBEC). Individual variation in HLA-G expression is linked to its genetic polymorphism and has been associated with many pathological situations such as asthma, which is characterized by epithelium abnormalities and inflammatory cell activation. Studies reported both higher and equivalent soluble HLA-G (sHLA-G) expression in different cohorts of asthmatic patients. In particular, we recently described impaired local expression of HLA-G and abnormal profiles for alternatively spliced isoforms in HBEC from asthmatic patients. sHLA-G dosage is challenging because of its many levels of polymorphism (dimerization, association with β2-microglobulin, and alternative splicing), thus many clinical studies focused on HLA-G single-nucleotide polymorphisms as predictive biomarkers, but few analyzed HLA-G haplotypes. Here, we aimed to characterize HLA-G haplotypes and describe their association with asthmatic clinical features and sHLA-G peripheral expression and to describe variations in transcription factor (TF) binding sites and alternative splicing sites. HLA-G haplotypes were differentially distributed in 330 healthy and 580 asthmatic individuals. Furthermore, HLA-G haplotypes were associated with asthmatic clinical features showed. However, we did not confirm an association between sHLA-G and genetic, biological, or clinical parameters. HLA-G haplotypes were phylogenetically split into distinct groups, with each group displaying particular variations in TF binding or RNA splicing sites that could reflect differential HLA-G qualitative or quantitative expression, with tissue-dependent specificities. Our results, based on a multicenter cohort, thus support the pertinence of HLA-G haplotypes as predictive genetic markers for asthma. PMID:29527207

Osteo-/odontogenic differentiation of induced mesenchymal stem cells generated through epithelial-mesenchyme transition of cultured human keratinocytes.

PubMed

Yi, Jin-Kyu; Mehrazarin, Shebli; Oh, Ju-Eun; Bhalla, Anu; Oo, Jenessa; Chen, Wei; Lee, Min; Kim, Reuben H; Shin, Ki-Hyuk; Park, No-Hee; Kang, Mo K

2014-11-01

Revascularization of necrotic pulp has been successful in the resolution of periradicular inflammation; yet, several case studies suggest the need for cell-based therapies using mesenchymal stem cells (MSCs) as an alternative for de novo pulp regeneration. Because the availability of MSCs may be limited, especially in an aged population, the current study reports an alternative approach in generating MSCs from epidermal keratinocytes through a process called epithelial-mesenchymal transition (EMT). We induced EMT in primary normal human epidermal keratinocytes (NHEKs) by transient transfection of small interfering RNA targeting the p63 gene. The resulting cells were assayed for their mesenchymal marker expression, proliferation capacities as a monolayer and in a 3-dimensional collagen scaffold, and differentiation capacities. Transient transfection of p63 small-interfering RNA successfully abolished the expression of endogenous p63 in NHEKs and induced the expression of mesenchymal markers (eg, vimentin and fibronectin), whereas epithelial markers (eg, E-cadherin and involucrin) were lost. The NHEKs exhibiting the EMT phenotype acquired extended replicative potential and an increased telomere length compared with the control cells. Similar to the established MSCs, the NHEKs with p63 knockdown showed attachment onto the 3-dimensional collagen scaffold and underwent progressive proliferation and differentiation. Upon differentiation, these EMT cells expressed alkaline phosphatase activity, osteocalcin, and osteonectin and readily formed mineralized nodules detected by alizarin S red staining, showing osteo-/odontogenic differentiation. The induction of EMT in primary NHEKs by means of transient p63 knockdown allows the generation of induced MSCs from autologous sources. These cells may be used for tissues engineering purposes, including that of dental pulp. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Involvement of an Alternative Oxidase in Oxidative Stress and Mycelium-to-Yeast Differentiation in Paracoccidioides brasiliensis ▿ †

PubMed Central

Martins, Vicente P.; Dinamarco, Taisa M.; Soriani, Frederico M.; Tudella, Valéria G.; Oliveira, Sergio C.; Goldman, Gustavo H.; Curti, Carlos; Uyemura, Sérgio A.

2011-01-01

Paracoccidioides brasiliensis is a thermodimorphic human pathogenic fungus that causes paracoccidioidomycosis (PCM), which is the most prevalent systemic mycosis in Latin America. Differentiation from the mycelial to the yeast form (M-to-Y) is an essential step for the establishment of PCM. We evaluated the involvement of mitochondria and intracellular oxidative stress in M-to-Y differentiation. M-to-Y transition was delayed by the inhibition of mitochondrial complexes III and IV or alternative oxidase (AOX) and was blocked by the association of AOX with complex III or IV inhibitors. The expression of P. brasiliensis aox (Pbaox) was developmentally regulated through M-to-Y differentiation, wherein the highest levels were achieved in the first 24 h and during the yeast exponential growth phase; Pbaox was upregulated by oxidative stress. Pbaox was cloned, and its heterologous expression conferred cyanide-resistant respiration in Saccharomyces cerevisiae and Escherichia coli and reduced oxidative stress in S. cerevisiae cells. These results reinforce the role of PbAOX in intracellular redox balancing and demonstrate its involvement, as well as that of other components of the mitochondrial respiratory chain complexes, in the early stages of the M-to-Y differentiation of P. brasiliensis. PMID:21183691

Role of the Lipoxygenase Pathway in RSV-induced Alternatively Activated Macrophages Leading to Resolution of Lung Pathology

PubMed Central

Shirey, Kari Ann; Lai, Wendy; Pletneva, Lioubov M.; Karp, Christopher L.; Divanovic, Senad; Blanco, Jorge C. G.; Vogel, Stefanie N.

2013-01-01

Resolution of severe RSV-induced bronchiolitis is mediated by alternatively activated macrophages (AA-Mϕ) that counteract cyclooxygenase (COX)-2-induced lung pathology. Herein, we report that RSV infection of 5-lipoxygenase (LO)−/− and 15-LO−/− macrophages or mice failed to elicit AA-Mϕ differentiation and concomitantly exhibited increased COX-2 expression. Further, RSV infection of 5-LO−/− mice resulted in enhanced lung pathology. Pharmacologic inhibition of 5-LO or 15-LO also blocked differentiation of RSV-induced AA-Mϕ in vitro and, conversely, treatment of 5-LO−/− macrophages with downstream products, lipoxin A4 (LXA4) and resolvin E1 (RvE1), but not leukotriene B4 (LTB4) or LTD4, partially restored expression of AA-Mϕ markers. Indomethacin blockade of COX activity in RSV-infected macrophages increased 5-LO, and 15-LO, as well as arginase-1 mRNA expression. Treatment of RSV-infected mice with indomethacin also resulted not only in enhanced lung arginase-1 mRNA expression and decreased COX-2, but also, decreased lung pathology in RSV-infected 5-LO−/− mice. Treatment of RSV-infected cotton rats with a COX-2-specific inhibitor resulted in enhanced lung 5-LO mRNA and AA-Mϕ marker expression. Together, these data suggest a novel therapeutic approach for RSV that promotes AA-Mϕ differentiation by activating the 5-LO pathway. PMID:24064666

Meta-Analysis of Multiple Sclerosis Microarray Data Reveals Dysregulation in RNA Splicing Regulatory Genes.

PubMed

Paraboschi, Elvezia Maria; Cardamone, Giulia; Rimoldi, Valeria; Gemmati, Donato; Spreafico, Marta; Duga, Stefano; Soldà, Giulia; Asselta, Rosanna

2015-09-30

Abnormalities in RNA metabolism and alternative splicing (AS) are emerging as important players in complex disease phenotypes. In particular, accumulating evidence suggests the existence of pathogenic links between multiple sclerosis (MS) and altered AS, including functional studies showing that an imbalance in alternatively-spliced isoforms may contribute to disease etiology. Here, we tested whether the altered expression of AS-related genes represents a MS-specific signature. A comprehensive comparative analysis of gene expression profiles of publicly-available microarray datasets (190 MS cases, 182 controls), followed by gene-ontology enrichment analysis, highlighted a significant enrichment for differentially-expressed genes involved in RNA metabolism/AS. In detail, a total of 17 genes were found to be differentially expressed in MS in multiple datasets, with CELF1 being dysregulated in five out of seven studies. We confirmed CELF1 downregulation in MS (p=0.0015) by real-time RT-PCRs on RNA extracted from blood cells of 30 cases and 30 controls. As a proof of concept, we experimentally verified the unbalance in alternatively-spliced isoforms in MS of the NFAT5 gene, a putative CELF1 target. In conclusion, for the first time we provide evidence of a consistent dysregulation of splicing-related genes in MS and we discuss its possible implications in modulating specific AS events in MS susceptibility genes.

1-alpha,25-Dihydroxyvitamin D3 up-regulates the expression of 2 types of human intestinal alkaline phosphatase alternative splicing variants in Caco-2 cells and may be an important regulator of their expression in gut homeostasis.

PubMed

Noda, Seiko; Yamada, Asako; Nakaoka, Kanae; Goseki-Sone, Masae

2017-10-01

Vitamin D insufficiency is associated with a greater risk of osteoporosis and also influences skeletal muscle functions, differentiation, and development. The principal function of vitamin D in calcium homeostasis is to increase the absorption of calcium from the intestine, and the level of alkaline phosphatase (ALP) activity, a differentiation marker for intestinal epithelial cells, is regulated by vitamin D. Intestinal-type ALP is expressed at a high concentration in the brush border membrane of intestinal epithelial cells, and is known to be affected by several kinds of nutrients. Recent reviews have highlighted the importance of intestinal-type ALP in gut homeostasis. Intestinal-type ALP controls bacterial endotoxin-induced inflammation by dephosphorylating lipopolysaccharide and is a gut mucosal defense factor. In this study, we investigated the influence of vitamin D on the expression of 2 types of alternative mRNA variants encoding the human alkaline phosphatase, intestinal (ALPI) gene in human Caco-2 cells as an in vitro model of the small intestinal epithelium. After treatment with 1-alpha,25-dihydroxyvitamin D 3 , the biologically active form of vitamin D 3 , there were significant increases in the ALP activities of Caco-2 cells. Inhibitor and thermal inactivation experiments showed that the increased ALP had properties of intestinal-type ALP. Reverse transcription-polymerase chain reaction analysis revealed that expression of the 2 types of alternative mRNA variants from the ALPI gene was markedly enhanced by vitamin D in Caco-2 cells. In conclusion, these findings agree with the hypothesis: vitamin D up-regulated the expression of 2 types of human intestinal alkaline phosphatase alternative splicing variants in Caco-2 cells; vitamin D may be an important regulator of ALPI gene expression in gut homeostasis. Copyright © 2017 Elsevier Inc. All rights reserved.

The Role of CYP3A4 mRNA Transcript with Shortened 3′-Untranslated Region in Hepatocyte Differentiation, Liver Development, and Response to Drug InductionS⃞

PubMed Central

Li, Dan; Gaedigk, Roger; Hart, Steven N.; Leeder, J. Steven

2012-01-01

Cytochrome P450 3A4 (CYP3A4) metabolizes more than 50% of prescribed drugs. The expression of CYP3A4 changes during liver development and may be affected by the administration of some drugs. Alternative mRNA transcripts occur in more than 90% of human genes and are frequently observed in cells responding to developmental and environmental signals. Different mRNA transcripts may encode functionally distinct proteins or contribute to variability of mRNA stability or protein translation efficiency. The purpose of this study was to examine expression of alternative CYP3A4 mRNA transcripts in hepatocytes in response to developmental signals and drugs. cDNA cloning and RNA sequencing (RNA-Seq) were used to identify CYP3A4 mRNA transcripts. Three transcripts were found in HepaRG cells and liver tissues: one represented a canonical mRNA with full-length 3′-untranslated region (UTR), one had a shorter 3′-UTR, and one contained partial intron-6 retention. The alternative mRNA transcripts were validated by either rapid amplification of cDNA 3′-end or endpoint polymerase chain reaction (PCR). Quantification of the transcripts by RNA-Seq and real time quantitative PCR revealed that the CYP3A4 transcript with shorter 3′-UTR was preferentially expressed in developed livers, differentiated hepatocytes, and in rifampicin- and phenobarbital-induced hepatocytes. The CYP3A4 transcript with shorter 3′-UTR was more stable and produced more protein compared with the CYP3A4 transcript with canonical 3′-UTR. We conclude that the 3′-end processing of CYP3A4 contributes to the quantitative regulation of CYP3A4 gene expression through alternative polyadenylation, which may serve as a regulatory mechanism explaining changes of CYP3A4 expression and activity during hepatocyte differentiation and liver development and in response to drug induction. PMID:21998292

Coupling between alternative polyadenylation and alternative splicing is limited to terminal introns.

PubMed

Movassat, Maliheh; Crabb, Tara L; Busch, Anke; Yao, Chengguo; Reynolds, Derrick J; Shi, Yongsheng; Hertel, Klemens J

2016-07-02

Alternative polyadenylation has been implicated as an important regulator of gene expression. In some cases, alternative polyadenylation is known to couple with alternative splicing to influence last intron removal. However, it is unknown whether alternative polyadenylation events influence alternative splicing decisions at upstream exons. Knockdown of the polyadenylation factors CFIm25 or CstF64 in HeLa cells was used as an approach in identifying alternative polyadenylation and alternative splicing events on a genome-wide scale. Although hundreds of alternative splicing events were found to be differentially spliced in the knockdown of CstF64, genes associated with alternative polyadenylation did not exhibit an increased incidence of alternative splicing. These results demonstrate that the coupling between alternative polyadenylation and alternative splicing is usually limited to defining the last exon. The striking influence of CstF64 knockdown on alternative splicing can be explained through its effects on UTR selection of known splicing regulators such as hnRNP A2/B1, thereby indirectly influencing splice site selection. We conclude that changes in the expression of the polyadenylation factor CstF64 influences alternative splicing through indirect effects.

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

PubMed

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

2013-08-01

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

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.

MicroRNA miR-124 Controls the Choice between Neuronal and Astrocyte Differentiation by Fine-tuning Ezh2 Expression*

PubMed Central

Neo, Wen Hao; Yap, Karen; Lee, Suet Hoay; Looi, Liang Sheng; Khandelia, Piyush; Neo, Sheng Xiong; Makeyev, Eugene V.; Su, I-hsin

2014-01-01

Polycomb group protein Ezh2 is a histone H3 Lys-27 histone methyltransferase orchestrating an extensive epigenetic regulatory program. Several nervous system-specific genes are known to be repressed by Ezh2 in stem cells and derepressed during neuronal differentiation. However, the molecular mechanisms underlying this regulation remain poorly understood. Here we show that Ezh2 levels are dampened during neuronal differentiation by brain-enriched microRNA miR-124. Expression of miR-124 in a neuroblastoma cells line was sufficient to up-regulate a significant fraction of nervous system-specific Ezh2 target genes. On the other hand, naturally elevated expression of miR-124 in embryonic carcinoma cells undergoing neuronal differentiation correlated with down-regulation of Ezh2 levels. Importantly, overexpression of Ezh2 mRNA with a 3′-untranslated region (3′-UTR) lacking a functional miR-124 binding site, but not with the wild-type Ezh2 3′-UTR, hampered neuronal and promoted astrocyte-specific differentiation in P19 and embryonic mouse neural stem cells. Overall, our results uncover a molecular mechanism that allows miR-124 to balance the choice between alternative differentiation possibilities through fine-tuning the expression of a critical epigenetic regulator. PMID:24878960

MicroRNA miR-124 controls the choice between neuronal and astrocyte differentiation by fine-tuning Ezh2 expression.

PubMed

Neo, Wen Hao; Yap, Karen; Lee, Suet Hoay; Looi, Liang Sheng; Khandelia, Piyush; Neo, Sheng Xiong; Makeyev, Eugene V; Su, I-hsin

2014-07-25

Polycomb group protein Ezh2 is a histone H3 Lys-27 histone methyltransferase orchestrating an extensive epigenetic regulatory program. Several nervous system-specific genes are known to be repressed by Ezh2 in stem cells and derepressed during neuronal differentiation. However, the molecular mechanisms underlying this regulation remain poorly understood. Here we show that Ezh2 levels are dampened during neuronal differentiation by brain-enriched microRNA miR-124. Expression of miR-124 in a neuroblastoma cells line was sufficient to up-regulate a significant fraction of nervous system-specific Ezh2 target genes. On the other hand, naturally elevated expression of miR-124 in embryonic carcinoma cells undergoing neuronal differentiation correlated with down-regulation of Ezh2 levels. Importantly, overexpression of Ezh2 mRNA with a 3'-untranslated region (3'-UTR) lacking a functional miR-124 binding site, but not with the wild-type Ezh2 3'-UTR, hampered neuronal and promoted astrocyte-specific differentiation in P19 and embryonic mouse neural stem cells. Overall, our results uncover a molecular mechanism that allows miR-124 to balance the choice between alternative differentiation possibilities through fine-tuning the expression of a critical epigenetic regulator.

Regulation of alternative splicing at the single-cell level.

PubMed

Faigenbloom, Lior; Rubinstein, Nimrod D; Kloog, Yoel; Mayrose, Itay; Pupko, Tal; Stein, Reuven

2015-12-28

Alternative splicing is a key cellular mechanism for generating distinct isoforms, whose relative abundances regulate critical cellular processes. It is therefore essential that inclusion levels of alternative exons be tightly regulated. However, how the precision of inclusion levels among individual cells is governed is poorly understood. Using single-cell gene expression, we show that the precision of inclusion levels of alternative exons is determined by the degree of evolutionary conservation at their flanking intronic regions. Moreover, the inclusion levels of alternative exons, as well as the expression levels of the transcripts harboring them, also contribute to this precision. We further show that alternative exons whose inclusion levels are considerably changed during stem cell differentiation are also subject to this regulation. Our results imply that alternative splicing is coordinately regulated to achieve accuracy in relative isoform abundances and that such accuracy may be important in determining cell fate. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.

Suppression subtractive hybridization as a tool to identify anthocyanin metabolism-related genes in apple skin.

PubMed

Ban, Yusuke; Moriguchi, Takaya

2010-01-01

The pigmentation of anthocyanins is one of the important determinants for consumer preference and marketability in horticultural crops such as fruits and flowers. To elucidate the mechanisms underlying the physiological process leading to the pigmentation of anthocyanins, identification of the genes differentially expressed in response to anthocyanin accumulation is a useful strategy. Currently, microarrays have been widely used to isolate differentially expressed genes. However, the use of microarrays is limited by its high cost of special apparatus and materials. Therefore, availability of microarrays is limited and does not come into common use at present. Suppression subtractive hybridization (SSH) is an alternative tool that has been widely used to identify differentially expressed genes due to its easy handling and relatively low cost. This chapter describes the procedures for SSH, including RNA extraction from polysaccharides and polyphenol-rich samples, poly(A)+ RNA purification, evaluation of subtraction efficiency, and differential screening using reverse northern in apple skin.

Splicing regulatory factors, ageing and age-related disease.

PubMed

Latorre, Eva; Harries, Lorna W

2017-07-01

Alternative splicing is a co-transcriptional process, which allows for the production of multiple transcripts from a single gene and is emerging as an important control point for gene expression. Alternatively expressed isoforms often have antagonistic function and differential temporal or spatial expression patterns, yielding enormous plasticity and adaptability to cells and increasing their ability to respond to environmental challenge. The regulation of alternative splicing is critical for numerous cellular functions in both pathological and physiological conditions, and deregulated alternative splicing is a key feature of common chronic diseases. Isoform choice is controlled by a battery of splicing regulatory proteins, which include the serine arginine rich (SRSF) proteins and the heterogeneous ribonucleoprotein (hnRNP) classes of genes. These important splicing regulators have been implicated in age-related disease, and in the ageing process itself. This review will outline the important contribution of splicing regulator proteins to ageing and age-related disease. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterization and comparison of osteoblasts derived from mouse embryonic stem cells and induced pluripotent stem cells.

PubMed

Ma, Ming-San; Kannan, Vishnu; de Vries, Anneriek E; Czepiel, Marcin; Wesseling, Evelyn M; Balasubramaniyan, Veerakumar; Kuijer, Roel; Vissink, Arjan; Copray, Sjef C V M; Raghoebar, Gerry M

2017-01-01

New developments in stem cell biology offer alternatives for the reconstruction of critical-sized bone defects. One of these developments is the use of induced pluripotent stem (iPS) cells. These stem cells are similar to embryonic stem (ES) cells, but can be generated from adult somatic cells and therefore do not raise ethical concerns. Proper characterization of iPS-derived osteoblasts is important for future development of safe clinical applications of these cells. For this reason, we differentiated mouse ES and iPS cells toward osteoblasts using osteogenic medium and compared their functionality. Immunocytochemical analysis showed significant expression of bone markers (osteocalcin and collagen type I) in osteoblasts differentiated from ES and iPS cells on days 7 and 30. An in vitro mineralization assay confirmed the functionality of osteogenically differentiated ES and iPS cells. Gene expression arrays focusing on osteogenic differentiation were performed in order to compare the gene expression pattern in both differentiated and undifferentiated ES cells and iPS cells. We observed a significant upregulation of osteogenesis-related genes such as Runx2, osteopontin, collagen type I, Tnfsf11, Csf1, and alkaline phosphatase upon osteogenic differentiation of the ES and iPS cells. We further validated the expression of key osteogenic genes Runx2, osteopontin, osteocalcin, collagen type I, and osterix in both differentiated and undifferentiated ES and iPS cells by means of quantified real-time polymerase chain reaction. We conclude that ES and iPS cells are similar in their osteogenic differentiation capacities, as well as in their gene expression patterns.

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

PubMed

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

2017-09-22

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

SEASTAR: systematic evaluation of alternative transcription start sites in RNA.

PubMed

Qin, Zhiyi; Stoilov, Peter; Zhang, Xuegong; Xing, Yi

2018-05-04

Alternative first exons diversify the transcriptomes of eukaryotes by producing variants of the 5' Untranslated Regions (5'UTRs) and N-terminal coding sequences. Accurate transcriptome-wide detection of alternative first exons typically requires specialized experimental approaches that are designed to identify the 5' ends of transcripts. We developed a computational pipeline SEASTAR that identifies first exons from RNA-seq data alone then quantifies and compares alternative first exon usage across multiple biological conditions. The exons inferred by SEASTAR coincide with transcription start sites identified directly by CAGE experiments and bear epigenetic hallmarks of active promoters. To determine if differential usage of alternative first exons can yield insights into the mechanism controlling gene expression, we applied SEASTAR to an RNA-seq dataset that tracked the reprogramming of mouse fibroblasts into induced pluripotent stem cells. We observed dynamic temporal changes in the usage of alternative first exons, along with correlated changes in transcription factor expression. Using a combined sequence motif and gene set enrichment analysis we identified N-Myc as a regulator of alternative first exon usage in the pluripotent state. Our results demonstrate that SEASTAR can leverage the available RNA-seq data to gain insights into the control of gene expression and alternative transcript variation in eukaryotic transcriptomes.

Inference for High-dimensional Differential Correlation Matrices.

PubMed

Cai, T Tony; Zhang, Anru

2016-01-01

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

An efficient method to identify differentially expressed genes in microarray experiments

PubMed Central

Qin, Huaizhen; Feng, Tao; Harding, Scott A.; Tsai, Chung-Jui; Zhang, Shuanglin

2013-01-01

Motivation Microarray experiments typically analyze thousands to tens of thousands of genes from small numbers of biological replicates. The fact that genes are normally expressed in functionally relevant patterns suggests that gene-expression data can be stratified and clustered into relatively homogenous groups. Cluster-wise dimensionality reduction should make it feasible to improve screening power while minimizing information loss. Results We propose a powerful and computationally simple method for finding differentially expressed genes in small microarray experiments. The method incorporates a novel stratification-based tight clustering algorithm, principal component analysis and information pooling. Comprehensive simulations show that our method is substantially more powerful than the popular SAM and eBayes approaches. We applied the method to three real microarray datasets: one from a Populus nitrogen stress experiment with 3 biological replicates; and two from public microarray datasets of human cancers with 10 to 40 biological replicates. In all three analyses, our method proved more robust than the popular alternatives for identification of differentially expressed genes. Availability The C++ code to implement the proposed method is available upon request for academic use. PMID:18453554

Differential regulation of oestrogen receptor β isoforms by 5′ untranslated regions in cancer

PubMed Central

Smith, Laura; Brannan, Rebecca A; Hanby, Andrew M; Shaaban, Abeer M; Verghese, Eldo T; Peter, Mark B; Pollock, Steven; Satheesha, Sampoorna; Szynkiewicz, Marcin; Speirs, Valerie; Hughes, Thomas A

2010-01-01

Abstract Oestrogen receptors (ERs) are critical regulators of the behaviour of many cancers. Despite this, the roles and regulation of one of the two known ERs – ERβ– are poorly understood. This is partly because analyses have been confused by discrepancies between ERβ expression at mRNA and proteins levels, and because ERβ is expressed as several functionally distinct isoforms. We investigated human ERβ 5′ untranslated regions (UTRs) and their influences on ERβ expression and function. We demonstrate that two alternative ERβ 5′UTRs have potent and differential influences on expression acting at the level of translation. We show that their influences are modulated by cellular context and in carcinogenesis, and demonstrate the contributions of both upstream open reading frames and RNA secondary structure. These regulatory mechanisms offer explanations for the non-concordance of ERβ mRNA and protein. Importantly, we also demonstrate that 5′UTRs allow the first reported mechanisms for differential regulation of the expression of the ERβ isoforms 1, 2 and 5, and thereby have critical influences on ERβ function. PMID:20920096

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.

Compound-specific effects of diverse neurodevelopmental toxicants on global gene expression in the neural embryonic stem cell test (ESTn)

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

Theunissen, P.T., E-mail: Peter.Theunissen@rivm.nl; Department of Toxicogenomics, Maastricht University, Maastricht; Robinson, J.F.

Alternative assays for developmental toxicity testing are needed to reduce animal use in regulatory toxicology. The in vitro murine neural embryonic stem cell test (ESTn) was designed as an alternative for neurodevelopmental toxicity testing. The integration of toxicogenomic-based approaches may further increase predictivity as well as provide insight into underlying mechanisms of developmental toxicity. In the present study, we investigated concentration-dependent effects of six mechanistically diverse compounds, acetaldehyde (ACE), carbamazepine (CBZ), flusilazole (FLU), monoethylhexyl phthalate (MEHP), penicillin G (PENG) and phenytoin (PHE), on the transcriptome and neural differentiation in the ESTn. All compounds with the exception of PENG altered ESTnmore » morphology (cytotoxicity and neural differentiation) in a concentration-dependent manner. Compound induced gene expression changes and corresponding enriched gene ontology biological processes (GO–BP) were identified after 24 h exposure at equipotent differentiation-inhibiting concentrations of the compounds. Both compound-specific and common gene expression changes were observed between subsets of tested compounds, in terms of significance, magnitude of regulation and functionality. For example, ACE, CBZ and FLU induced robust changes in number of significantly altered genes (≥ 687 genes) as well as a variety of GO–BP, as compared to MEHP, PHE and PENG (≤ 55 genes with no significant changes in GO–BP observed). Genes associated with developmentally related processes (embryonic morphogenesis, neuron differentiation, and Wnt signaling) showed diverse regulation after exposure to ACE, CBZ and FLU. In addition, gene expression and GO–BP enrichment showed concentration dependence, allowing discrimination of non-toxic versus toxic concentrations on the basis of transcriptomics. This information may be used to define adaptive versus toxic responses at the transcriptome level.« less

Cancer-associated variant expression and interaction of CIZ1 with cyclin A1 in differentiating male germ cells.

PubMed

Greaves, Erin A; Copeland, Nikki A; Coverley, Dawn; Ainscough, Justin F X

2012-05-15

CIZ1 is a nuclear-matrix-associated DNA replication factor unique to higher eukaryotes, for which alternatively spliced isoforms have been associated with a range of disorders. In vitro, the CIZ1 N-terminus interacts with cyclin E and cyclin A at distinct sites, enabling functional cooperation with cyclin-A-Cdk2 to promote replication initiation. C-terminal sequences anchor CIZ1 to fixed sites on the nuclear matrix, imposing spatial constraint on cyclin-dependent kinase activity. Here we demonstrate that CIZ1 is predominantly expressed as a predicted full-length product throughout mouse development, consistent with a ubiquitous role in cell and tissue renewal. CIZ1 is expressed in proliferating stem cells of the testis, but is notably downregulated following commitment to differentiation. Significantly, CIZ1 is re-expressed at high levels in non-proliferative spermatocytes before meiotic division. Sequence analysis identifies at least seven alternatively spliced variants, including a dominant cancer-associated form and a set of novel isoforms. Furthermore, we show that in these post-replicative cells, CIZ1 interacts with germ-cell-specific cyclin A1, which has been implicated in the repair of DNA double-strand breaks. Consistent with this role, antibody depletion of CIZ1 reduces the capacity for testis extract to repair digested plasmid DNA in vitro. Together, the data imply post-replicative roles for CIZ1 in germ cell differentiation that might include meiotic recombination - a process intrinsic to genome stability and diversification.

Effect of chitosan conduit under a dynamic culture on the proliferation and neural differentiation of human exfoliated deciduous teeth stem cells.

PubMed

Su, Wen-Ta; Shih, Yi-An; Ko, Chih-Sheng

2016-06-01

Ex vivo engineering of artificial nerve conduit is a suitable alternative clinical treatment for nerve injuries. Stem cells from human exfoliated deciduous teeth (SHEDs) have been considered as alternative sources of adult stem cells because of their potential to differentiate into multiple cell lineages. These cells, when cultured in six-well plates, exhibited a spindle fibroblastic morphology, whereas those under a dynamic culture aggregated into neurosphere-like clusters in the chitosan conduit. In this study, we confirmed that SHEDs efficiently express the neural stem cell marker nestin, the early neural cell marker β-III-tubulin, the late neural marker neuron-specific enolase and the glial cell markers glial fibrillary acidic protein (GFAP) and 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase). The three-dimensional chitosan conduit and dynamic culture system generated fluid shear stress and enhanced nutrient transfer, promoting the differentiation of SHEDs to neural cells. In particular, the gene expressions of GFAP and CNPase increased by 28- and 53-fold, respectively. This study provides evidence for the dynamic culture of SHEDs during ex vivo neural differentiation and demonstrates its potential for cell therapy in neurological diseases. Copyright © 2013 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Evidence that "brain-specific" FOX-1, FOX-2, and nPTB alternatively spliced isoforms are produced in the lens.

PubMed

Bitel, Claudine L; Nathan, Rachel; Wong, Patrick; Kuppasani, Sunil; Matsushita, Masafumi; Kanazawa, Hrioshi; Frederikse, Peter H

2011-04-01

Alternative RNA splicing is essential in development and more rapid physiological processes that include disease mechanisms. Studies over the last 20 years demonstrated that RNA binding protein families, which mediate the alternative splicing of a large percentage of genes in mammals, contain isoforms with mutually exclusive expression in non-neural and neural progenitor cells vs. post-mitotic neurons, and regulate the comprehensive reprogramming of alternative splicing during neurogenesis. Polypyrimidine tract binding (PTB) proteins and Fox-1 proteins also undergo mutually exclusive alternative splicing in neural and non-neural cells that regulates their tissue-specific expression and splicing activities. Over the past 50 years, striking morphological similarities noted between lens fiber cells and neurons suggested that cell biology processes and gene expression profiles may be shared as well. Here, we examined mouse and rat lenses to determine if alternative splicing of neuronal nPTB and Fox-1/Fox-2 isoforms also occurs in lenses. Immunoblot, immunofluorescence, and RT-PCR were used to examine expression and alternative splicing of transcripts in lens and brain. We demonstrated that exon 10 is predominantly included in nPTB transcripts consistent with nPTB protein in lenses, and that alternatively spliced Fox-1/-2 lens transcripts contain exons that have been considered neuron-specific. We identified a 3' alternative Fox-1 exon in lenses that encodes a nuclear localization signal consistent with its protein distribution detected in fiber cells. Neuronal alternative splicing of kinesin KIF1Bβ2 has been associated with PTB/nPTB and Fox-2, and we found that two 'neuron-specific' exons are also included in lenses. The present study provides evidence that alternative neuronal nPTB and Fox-1/Fox-2 isoforms are also produced in lenses. These findings raise questions regarding the extent these factors contribute to a similar reprogramming of alternative splicing during lens differentiation, and the degree that alternative gene transcripts produced during neurogenesis are also expressed in the lens.

Detecting differentially expressed genes in heterogeneous diseases using half Student's t-test.

PubMed

Hsu, Chun-Lun; Lee, Wen-Chung

2010-12-01

Microarray technology provides information about hundreds and thousands of gene-expression data in a single experiment. To search for disease-related genes, researchers test for those genes that are differentially expressed between the case subjects and the control subjects. The authors propose a new test, the 'half Student's t-test', specifically for detecting differentially expressed genes in heterogeneous diseases. Monte-Carlo simulation shows that the test maintains the nominal α level quite well for both normal and non-normal distributions. Power of the half Student's t is higher than that of the conventional 'pooled' Student's t when there is heterogeneity in the disease under study. The power gain by using the half Student's t can reach ∼10% when the standard deviation of the case group is 50% larger than that of the control group. Application to a colon cancer data reveals that when the false discovery rate (FDR) is controlled at 0.05, the half Student's t can detect 344 differentially expressed genes, whereas the pooled Student's t can detect only 65 genes. Or alternatively, if only 50 genes are to be selected, the FDR for the pooled Student's t has to be set at 0.0320 (false positive rate of ∼3%), but for the half Student's t, it can be at as low as 0.0001 (false positive rate of about one per ten thousands). The half Student's t-test is to be recommended for the detection of differentially expressed genes in heterogeneous diseases.

Alternative dominance of the parental genomes in hybrid cells generated through the fusion of mouse embryonic stem cells with fibroblasts.

PubMed

Matveeva, Natalia M; Fishman, Veniamin S; Zakharova, Irina S; Shevchenko, Alexander I; Pristyazhnyuk, Inna E; Menzorov, Aleksei G; Serov, Oleg L

2017-12-22

For the first time, two types of hybrid cells with embryonic stem (ES) cell-like and fibroblast-like phenotypes were produced through the fusion of mouse ES cells with fibroblasts. Transcriptome analysis of 2,848 genes differentially expressed in the parental cells demonstrated that 34-43% of these genes are expressed in hybrid cells, consistent with their phenotypes; 25-29% of these genes display intermediate levels of expression, and 12-16% of these genes maintained expression at the parental cell level, inconsistent with the phenotype of the hybrid cell. Approximately 20% of the analyzed genes displayed unexpected expression patterns that differ from both parents. An unusual phenomenon was observed, namely, the illegitimate activation of Xist expression and the inactivation of one of two X-chromosomes in the near-tetraploid fibroblast-like hybrid cells, whereas both Xs were active before and after in vitro differentiation of the ES cell-like hybrid cells. These results and previous data obtained on heterokaryons suggest that the appearance of hybrid cells with a fibroblast-like phenotype reflects the reprogramming, rather than the induced differentiation, of the ES cell genome under the influence of a somatic partner.

Animal serum-free expansion and differentiation of human mesenchymal stromal cells.

PubMed

Felka, Tino; Schäfer, Richard; De Zwart, Peter; Aicher, Wilhelm K

2010-04-01

Mesenchymal stromal cells (MSC) are attracting increasing interest for possible application in cell therapies. Fetal calf serum (FCS) is widely utilized for cell culture, but its use in the context of clinical applications is associated with too many risks. Therefore we tested FCS-free media for the expansion and differentiation of MSC in compliance with the European good manufacturing practice (GMP) regulations for medicinal products. MSC expansion medium was modified by replacing FCS with human plasma and platelet extract. Cells were characterized according to the defined minimal criteria for multipotent MSC. For chondrogenic differentiation, serum-free micromass cultures were employed. For adipogenic and osteogenic differentiation, the FCS was replaced by human plasma. After 28 days of incubation in differentiation media, cells were analyzed by cytochemical and immunohistochemical staining. Furthermore, mRNA expression of chondrogenic, adipogenic and osteogenic markers was investigated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Expansion and differentiation of MSC under FCS-free conditions yielded cells with chondrogenic, adipogenic and osteogenic phenotypes and a characteristic gene expression. Chondrocytes in micromass pellets revealed an accumulation of proteoglycans and type II collagen as well as a significantly increased mRNA expression of chondrogenic marker genes. The adipocytes displayed Oil red O staining and expressed peroxisome proliferator-activated receptor gamma(2) (ppARgamma2) and lipoprotein lipase (LPL) mRNA. The osteoblasts were positive for von Kossa staining and expressed mRNA of osteogenic marker genes. The results did not indicate any spontaneous differentiation. Human plasma is a suitable FCS replacement for the expansion and differentiation of MSC, providing a feasible alternative for tissue engineering with GMP-compatible protocols.

Alternative Splicing in Breast Cancer and the Potential Development of Therapeutic Tools.

PubMed

Martínez-Montiel, Nancy; Anaya-Ruiz, Maricruz; Pérez-Santos, Martín; Martínez-Contreras, Rebeca D

2017-10-05

Alternative splicing is a key molecular mechanism now considered as a hallmark of cancer that has been associated with the expression of distinct isoforms during the onset and progression of the disease. The leading cause of cancer-related deaths in women worldwide is breast cancer, and even when the role of alternative splicing in this type of cancer has been established, the function of this mechanism in breast cancer biology is not completely decoded. In order to gain a comprehensive view of the role of alternative splicing in breast cancer biology and development, we summarize here recent findings regarding alternative splicing events that have been well documented for breast cancer evolution, considering its prognostic and therapeutic value. Moreover, we analyze how the response to endocrine and chemical therapies could be affected due to alternative splicing and differential expression of variant isoforms. With all this knowledge, it becomes clear that targeting alternative splicing represents an innovative approach for breast cancer therapeutics and the information derived from current studies could guide clinical decisions with a direct impact in the clinical advances for breast cancer patients nowadays.

Alternative splicing of inner-ear-expressed genes.

PubMed

Wang, Yanfei; Liu, Yueyue; Nie, Hongyun; Ma, Xin; Xu, Zhigang

2016-09-01

Alternative splicing plays a fundamental role in the development and physiological function of the inner ear. Inner-ear-specific gene splicing is necessary to establish the identity and maintain the function of the inner ear. For example, exon 68 of Cadherin 23 (Cdh23) gene is subject to inner-ear-specific alternative splicing, and as a result, Cdh23(+ 68) is only expressed in inner ear hair cells. Alternative splicing along the tonotopic axis of the cochlea contributes to frequency tuning, particularly in lower vertebrates, such as chickens and turtles. Differential splicing of Kcnma1, which encodes for the α subunit of the Ca(2+)-activated K(+) channel (BK channel), has been suggested to affect the channel gating properties and is important for frequency tuning. Consequently, deficits in alternative splicing have been shown to cause hearing loss, as we can observe in Bronx Waltzer (bv) mice and Sfswap mutant mice. Despite the advances in this field, the regulation of alternative splicing in the inner ear remains elusive. Further investigation is also needed to clarify the mechanism of hearing loss caused by alternative splicing deficits.

Alternative RNA splicing of leucocyte tissue transglutaminase in coeliac disease.

PubMed

Arbildi, P; Sóñora, C; Del Río, N; Marqués, J M; Hernández, A

2018-05-01

Tissue transglutaminase is a ubiquitous and multifunctional protein that contributes to several processes such as apoptosis/survival, efferocytosis, inflammation and tissue repairing under physiological and pathological conditions. Several activities can be associated with well-established functional domains; in addition, four RNA alternative splice variants have been described, characterized by sequence divergences and residues deletion at the C-terminal domains. Tissue transglutaminase is recognized as the central player in the physiopathology of coeliac disease (CD) mainly through calcium-dependent enzymatic activities. It can be hypothesized that differential regulation of tissue transglutaminase splice variants expression in persons with CD contributes to pathology by altering the protein functionality. We characterized the expression pattern of RNA alternative splice variants by RT-PCR in peripheral cells from patients with CD under free gluten diet adhesion; we considered inflammatory parameters and specific antibodies as markers of the stage of disease. We found significant higher expression of both the full length and the shortest C-truncated splice variants in leucocytes from patients with CD in comparison with healthy individuals. As tissue transglutaminase expression and canonical enzymatic activity are linked to inflammation, we studied the RNA expression of inflammatory cytokines in peripheral leucocytes of persons with CD in relation with splice variants expression; interestingly, we found that recently diagnosed patients showed significant correlation between both the full length and the shortest alternative spliced variants with IL-1 expression. Our results points that regulation of alternative splicing of tissue transglutaminase could account for the complex physiopathology of CD. © 2018 The Foundation for the Scandinavian Journal of Immunology.

Expression of different functional isoforms in haematopoiesis.

PubMed

Grech, Godfrey; Pollacco, Joel; Portelli, Mark; Sacco, Keith; Baldacchino, Shawn; Grixti, Justine; Saliba, Christian

2014-01-01

Haematopoiesis is a complex process regulated at various levels facilitating rapid responses to external factors including stress, modulation of lineage commitment and terminal differentiation of progenitors. Although the transcription program determines the RNA pool of a cell, various mRNA strands can be obtained from the same template, giving rise to multiple protein isoforms. The majority of variants and isoforms co-occur in normal haematopoietic cells or are differentially expressed at various maturity stages of progenitor maturation and cellular differentiation within the same lineage or across lineages. Genetic aberrations or specific cellular states result in the predominant expression of abnormal isoforms leading to deregulation and disease. The presence of upstream open reading frames (uORF) in 5' untranslated regions (UTRs) of a transcript, couples the utilization of start codons with the cellular status and availability of translation initiation factors (eIFs). In addition, tissue-specific and cell lineage-specific alternative promoter use, regulates several transcription factors producing transcript variants with variable 5' exons. In this review, we propose to give a detailed account of the differential isoform formation, causing haematological malignancies.

RNA splicing during terminal erythropoiesis.

PubMed

Conboy, John G

2017-05-01

Erythroid progenitors must accurately and efficiently splice thousands of pre-mRNAs as the cells undergo extensive changes in gene expression and cellular remodeling during terminal erythropoiesis. Alternative splicing choices are governed by interactions between RNA binding proteins and cis-regulatory binding motifs in the RNA. This review will focus on recent studies that define the genome-wide scope of splicing in erythroblasts and discuss what is known about its regulation. RNA-seq analysis of highly purified erythroblast populations has revealed an extensive program of alternative splicing of both exons and introns. During normal erythropoiesis, stage-specific splicing transitions alter the structure and abundance of protein isoforms required for optimized red cell production. Mutation or deficiency of splicing regulators underlies hematopoietic disease in myelopdysplasia syndrome patients via disrupting the splicing program. Erythroid progenitors execute an elaborate alternative splicing program that modulates gene expression posttranscriptionally, ultimately regulating the structure and function of the proteome in a differentiation stage-specific manner during terminal erythropoiesis. This program helps drive differentiation and ensure synthesis of the proper protein isoforms required to produce mechanically stable red cells. Mutation or deficiency of key splicing regulatory proteins disrupts the splicing program to cause disease.

Polarizing the Neuron through Sustained Co-expression of Alternatively Spliced Isoforms.

PubMed

Yap, Karen; Xiao, Yixin; Friedman, Brad A; Je, H Shawn; Makeyev, Eugene V

2016-05-10

Alternative splicing (AS) is an important source of proteome diversity in eukaryotes. However, how this affects protein repertoires at a single-cell level remains an open question. Here, we show that many 3'-terminal exons are persistently co-expressed with their alternatives in mammalian neurons. In an important example of this scenario, cell polarity gene Cdc42, a combination of polypyrimidine tract-binding, protein-dependent, and constitutive splicing mechanisms ensures a halfway switch from the general (E7) to the neuron-specific (E6) alternative 3'-terminal exon during neuronal differentiation. Perturbing the nearly equimolar E6/E7 ratio in neurons results in defects in both axonal and dendritic compartments and suggests that Cdc42E7 is involved in axonogenesis, whereas Cdc42E6 is required for normal development of dendritic spines. Thus, co-expression of a precise blend of functionally distinct splice isoforms rather than a complete switch from one isoform to another underlies proper structural and functional polarization of neurons. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

A Method for the Direct Identification of Differentiating Muscle Cells by a Fluorescent Mitochondrial Dye

PubMed Central

Miyake, Tetsuaki; McDermott, John C.; Gramolini, Anthony O.

2011-01-01

Identification of differentiating muscle cells generally requires fixation, antibodies directed against muscle specific proteins, and lengthy staining processes or, alternatively, transfection of muscle specific reporter genes driving GFP expression. In this study, we examined the possibility of using the robust mitochondrial network seen in maturing muscle cells as a marker of cellular differentiation. The mitochondrial fluorescent tracking dye, MitoTracker, which is a cell-permeable, low toxicity, fluorescent dye, allowed us to distinguish and track living differentiating muscle cells visually by epi-fluorescence microscopy. MitoTracker staining provides a robust and simple detection strategy for living differentiating cells in culture without the need for fixation or biochemical processing. PMID:22174849

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

Bioinspired Nanocomplex for Spatiotemporal Imaging of Sequential mRNA Expression in Differentiating Neural Stem Cells

PubMed Central

2015-01-01

Messenger RNA plays a pivotal role in regulating cellular activities. The expression dynamics of specific mRNA contains substantial information on the intracellular milieu. Unlike the imaging of stationary mRNAs, real-time intracellular imaging of the dynamics of mRNA expression is of great value for investigating mRNA biology and exploring specific cellular cascades. In addition to advanced imaging methods, timely extracellular stimulation is another key factor in regulating the mRNA expression repertoire. The integration of effective stimulation and imaging into a single robust system would significantly improve stimulation efficiency and imaging accuracy, producing fewer unwanted artifacts. In this study, we developed a multifunctional nanocomplex to enable self-activating and spatiotemporal imaging of the dynamics of mRNA sequential expression during the neural stem cell differentiation process. This nanocomplex showed improved enzymatic stability, fast recognition kinetics, and high specificity. With a mechanism regulated by endogenous cell machinery, this nanocomplex realized the successive stimulating motif release and the dynamic imaging of chronological mRNA expression during neural stem cell differentiation without the use of transgenetic manipulation. The dynamic imaging montage of mRNA expression ultimately facilitated genetic heterogeneity analysis. In vivo lateral ventricle injection of this nanocomplex enabled endogenous neural stem cell activation and labeling at their specific differentiation stages. This nanocomplex is highly amenable as an alternative tool to explore the dynamics of intricate mRNA activities in various physiological and pathological conditions. PMID:25494492

Bioinspired nanocomplex for spatiotemporal imaging of sequential mRNA expression in differentiating neural stem cells.

PubMed

Wang, Zhe; Zhang, Ruili; Wang, Zhongliang; Wang, He-Fang; Wang, Yu; Zhao, Jun; Wang, Fu; Li, Weitao; Niu, Gang; Kiesewetter, Dale O; Chen, Xiaoyuan

2014-12-23

Messenger RNA plays a pivotal role in regulating cellular activities. The expression dynamics of specific mRNA contains substantial information on the intracellular milieu. Unlike the imaging of stationary mRNAs, real-time intracellular imaging of the dynamics of mRNA expression is of great value for investigating mRNA biology and exploring specific cellular cascades. In addition to advanced imaging methods, timely extracellular stimulation is another key factor in regulating the mRNA expression repertoire. The integration of effective stimulation and imaging into a single robust system would significantly improve stimulation efficiency and imaging accuracy, producing fewer unwanted artifacts. In this study, we developed a multifunctional nanocomplex to enable self-activating and spatiotemporal imaging of the dynamics of mRNA sequential expression during the neural stem cell differentiation process. This nanocomplex showed improved enzymatic stability, fast recognition kinetics, and high specificity. With a mechanism regulated by endogenous cell machinery, this nanocomplex realized the successive stimulating motif release and the dynamic imaging of chronological mRNA expression during neural stem cell differentiation without the use of transgenetic manipulation. The dynamic imaging montage of mRNA expression ultimately facilitated genetic heterogeneity analysis. In vivo lateral ventricle injection of this nanocomplex enabled endogenous neural stem cell activation and labeling at their specific differentiation stages. This nanocomplex is highly amenable as an alternative tool to explore the dynamics of intricate mRNA activities in various physiological and pathological conditions.

Inference for High-dimensional Differential Correlation Matrices *

PubMed Central

Cai, T. Tony; Zhang, Anru

2015-01-01

Motivated by differential co-expression analysis in genomics, we consider in this paper estimation and testing of high-dimensional differential correlation matrices. An adaptive thresholding procedure is introduced and theoretical guarantees are given. Minimax rate of convergence is established and the proposed estimator is shown to be adaptively rate-optimal over collections of paired correlation matrices with approximately sparse differences. Simulation results show that the procedure significantly outperforms two other natural methods that are based on separate estimation of the individual correlation matrices. The procedure is also illustrated through an analysis of a breast cancer dataset, which provides evidence at the gene co-expression level that several genes, of which a subset has been previously verified, are associated with the breast cancer. Hypothesis testing on the differential correlation matrices is also considered. A test, which is particularly well suited for testing against sparse alternatives, is introduced. In addition, other related problems, including estimation of a single sparse correlation matrix, estimation of the differential covariance matrices, and estimation of the differential cross-correlation matrices, are also discussed. PMID:26500380

Trans-differentiation of the adipose tissue-derived stem cells into neuron-like cells expressing neurotrophins by selegiline.

PubMed

Abdanipour, Alireza; Tiraihi, Taki; Delshad, Alireza

2011-01-01

Adult stem cells (ASC) are undifferentiated cells found throughout the body. These cells are promising tools for cell replacement therapy in neurodegenerative disease. Adipose tissue is the most abundant and accessible source of ASC. This study was conducted to evaluate effect of selegiline on differentiation of adipose-derived stem cells (ADSC) into functional neuron-like cells (NLC), and also level of the neurotrophin expression in differentiated cells. ADSC were transdifferentiated into NLC using selegiline where CD90, CD49d, CD31, CD106 and CD45 were used as markers for ADSC identification. Lipogenic and osteogenic differentiation of ADSC were used to characterize the ADSC. ADSC were treated with selegiline at different concentrations (from 10(-6) to 10(-11) mM) and time points (3, 6, 12, 24 and 48 h). Percentage of viable cells, nestin and neurofilament 68 (NF-68) immunoreactive cells were used as markers for differentiation. The optimal dose for neurotrophin expressions in differentiating cells was evaluated using reverse transcriptase-PCR. NLC function was evaluated by loading and unloading with FM1-43 dye. ADSC were immunoreactive to CD90 (95.67 ± 2.26), CD49d (71.52 ± 6.64) and CD31 (0.6 ± 0.86), but no immunoreactivity was detected for CD106 and CD45. The results of neural differentiation showed the highest percentage of nestin and NF-68 positive cells at 10(-9) mM concentration of selegiline (exposed for 24 h). The differentiated cells expressed synapsin and neurotrophin genes except brain-derived neurotrophic factor. ADSC can be an alternative source in cell-based therapy for neurodegenerative diseases using selegiline to induce ADSC differentiation to neuronal lineage.

STATs shape the active enhancer landscape of T cell populations.

PubMed

Vahedi, Golnaz; Takahashi, Hayato; Nakayamada, Shingo; Sun, Hong-Wei; Sartorelli, Vittorio; Kanno, Yuka; O'Shea, John J

2012-11-21

Signaling pathways are intimately involved in cellular differentiation, allowing cells to respond to their environment by regulating gene expression. Although enhancers are recognized as key elements that regulate selective gene expression, the interplay between signaling pathways and actively used enhancer elements is not clear. Here, we use CD4(+) T cells as a model of differentiation, mapping the activity of cell-type-specific enhancer elements in T helper 1 (Th1) and Th2 cells. Our data establish that STAT proteins have a major impact on the activation of lineage-specific enhancers and the suppression of enhancers associated with alternative cell fates. Transcriptome analysis further supports a functional role for enhancers regulated by STATs. Importantly, expression of lineage-defining master regulators in STAT-deficient cells fails to fully recover the chromatin signature of STAT-dependent enhancers. Thus, these findings point to a critical role of STATs as environmental sensors in dynamically molding the specialized enhancer architecture of differentiating cells. Copyright © 2012 Elsevier Inc. All rights reserved.

STATs Shape the Active Enhancer Landscape of T Cell Populations

PubMed Central

Vahedi, Golnaz; Takahashi, Hayato; Nakayamada, Shingo; Sun, Hong-wei; Sartorelli, Vittorio; Kanno, Yuka; O’Shea, John J.

2012-01-01

SUMMARY Signaling pathways are intimately involved in cellular differentiation, allowing cells to respond to their environment by regulating gene expression. While enhancers are recognized as key elements that regulate selective gene expression, the interplay between signaling pathways and actively used enhancer elements is not clear. Here, we use CD4+ T cells as a model of differentiation, mapping the acquisition of cell-type-specific enhancer elements in T-helper 1 (Th1) and Th2 cells. Our data establish that STAT proteins have a major impact on the acquisition of lineage-specific enhancers and the suppression of enhancers associated with alternative cell fates. Transcriptome analysis further supports a functional role for enhancers regulated by STATs. Importantly, expression of lineage-defining master regulators in STAT-deficient cells fails to fully recover the chromatin signature of STAT-dependent enhancers. Thus, these findings point to a critical role of STATs as environmental sensors in dynamically molding the specialized enhancer architecture of differentiating cells. PMID:23178119

Formation of Cartilage and Synovial Tissue by Human Gingival Stem Cells

PubMed Central

Larjava, Hannu; Loison-Robert, Ludwig-Stanislas; Berbar, Tsouria; Owen, Gethin R.; Berdal, Ariane; Chérifi, Hafida; Gogly, Bruno; Häkkinen, Lari; Fournier, Benjamin P.J.

2014-01-01

Human gingival stem cells (HGSCs) can be easily isolated and manipulated in culture to investigate their multipotency. Osteogenic differentiation of bone-marrow-derived mesenchymal stem/stromal cells has been well documented. HGSCs derive from neural crests, however, and their differentiation capacity has not been fully established. The aim of the present report was to investigate whether HGSCs can be induced to differentiate to osteoblasts and chondrocytes. HGSCs were cultured either in a classical monolayer culture or in three-dimensional floating micromass pellet cultures in specific differentiation media. HGSC differentiation to osteogenic and chondrogenic lineages was determined by protein and gene expression analyses, and also by specific staining of cells and tissue pellets. HGSCs cultured in osteogenic differentiation medium showed induction of Runx2, alkaline phosphatase (ALPL), and osterix expression, and subsequently formed mineralized nodules consistent with osteogenic differentiation. Interestingly, HGSC micromass cultures maintained in chondrogenic differentiation medium showed SOX9-dependent differentiation to both chondrocyte and synoviocyte lineages. Chondrocytes at different stages of differentiation were identified by gene expression profiles and by histochemical and immunohistochemical staining. In 3-week-old cultures, peripheral cells in the micromass cultures organized in layers of cuboidal cells with villous structures facing the medium. These cells were strongly positive for cadherin-11, a marker of synoviocytes. In summary, the findings indicate that HGSCs have the capacity to differentiate to osteogenic, chondrogenic, and synoviocyte lineages. Therefore, HGSCs could serve as an alternative source for stem cell therapies in regenerative medicine for patients with cartilage and joint destructions, such as observed in rheumatoid arthritis. PMID:25003637

Protein Kinase C δ (PKCδ) Splice Variants Modulate Apoptosis Pathway in 3T3L1 Cells during Adipogenesis

PubMed Central

Patel, Rekha; Apostolatos, André; Carter, Gay; Ajmo, Joanne; Gali, Meghanath; Cooper, Denise R.; You, Min; Bisht, Kirpal S.; Patel, Niketa A.

2013-01-01

Increased food intake and lack of physical activity results in excess energy stored in adipocytes, and this imbalance contributes to obesity. New adipocytes are required for storage of energy in the white adipose tissue. This process of adipogenesis is widely studied in differentiating 3T3L1 preadipocytes in vitro. We have identified a key signaling kinase, protein kinase C delta (PKCδ), whose alternative splice variant expression is modulated during adipogenesis. We demonstrate that PKCδII splice variant promotes survival in differentiating 3T3L1 cells through the Bcl2 pathway. Here we demonstrate that resveratrol, a naturally occurring polyphenol, increases apoptosis and inhibits adipogenesis along with disruption of PKCδ alternative splicing during 3T3L1 differentiation. Importantly, we have identified a PKCδII splice variant inhibitor. This inhibitor may be a valuable tool with therapeutic implications in obesity. PMID:23902767

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.

Bladder Smooth Muscle Cells Differentiation from Dental Pulp Stem Cells: Future Potential for Bladder Tissue Engineering

PubMed Central

Song, Bing; Jiang, Wenkai; Alraies, Amr; Liu, Qian; Gudla, Vijay; Oni, Julia; Wei, Xiaoqing; Sloan, Alastair; Ni, Longxing; Agarwal, Meena

2016-01-01

Dental pulp stem cells (DPSCs) are multipotent cells capable of differentiating into multiple cell lines, thus providing an alternative source of cell for tissue engineering. Smooth muscle cell (SMC) regeneration is a crucial step in tissue engineering of the urinary bladder. It is known that DPSCs have the potential to differentiate into a smooth muscle phenotype in vitro with differentiation agents. However, most of these studies are focused on the vascular SMCs. The optimal approaches to induce human DPSCs to differentiate into bladder SMCs are still under investigation. We demonstrate in this study the ability of human DPSCs to differentiate into bladder SMCs in a growth environment containing bladder SMCs-conditioned medium with the addition of the transforming growth factor beta 1 (TGF-β1). After 14 days of exposure to this medium, the gene and protein expression of SMC-specific marker (α-SMA, desmin, and calponin) increased over time. In particular, myosin was present in differentiated cells after 11 days of induction, which indicated that the cells differentiated into the mature SMCs. These data suggested that human DPSCs could be used as an alternative and less invasive source of stem cells for smooth muscle regeneration, a technology that has applications for bladder tissue engineering. PMID:26880982

Effects of ß-TCP scaffolds on neurogenic and osteogenic differentiation of human embryonic stem cells.

PubMed

Arpornmaeklong, Premjit; Pressler, Michael J

2018-01-01

Extracellular matrix (ECM) and adhesion molecules play crucial roles in regulating growth and differentiation of stem cells. The current study aimed to investigate the effects of beta-tricalcium phosphate (ß-TCP) scaffolds on differentiation and expression of ECM and adhesion molecules of human embryonic stem cells (hESCs). Undifferentiated hESCs were seeded on ß-TCP scaffolds and cell culture plates and cultured in growth and osteogenic medium for 21 days. Scanning electron microscopy (SEM) displayed adhesion and growth of hESCs on the porous ß-TCP scaffolds. Histological analysis, immunohistochemical staining and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) demonstrated that the scaffolds supported growth and differentiation of hESCs. Expression levels of neural crest related genes (AP2a, FoxD3, HNK1, P75, Sox1, Sox10) and osteoblast-related genes (Runx2, SPP1 and BGLA) on the scaffolds in osteogenic medium were significantly higher than on the scaffolds in growth and cell culture plates in osteogenic medium, respectively (p<0.05). Polymerase chain reaction array experiments demonstrated increased expression of ECM and adhesion molecule-related genes on the scaffolds. In conclusion, osteoconductive scaffolds such as ß-TCP scaffolds promoted differentiation of hESCs, particularly expression of genes related to neural crest stem cell and osteoblastic differentiations. Beta-TCP scaffolds could be an alternative cell culture substrate for neural crest and osteogenic differentiation of hESCs. Optimization of culture medium may be necessary to enhance lineage restriction of hESCs on the ß-TCP scaffolds. Copyright © 2017 Elsevier GmbH. All rights reserved.

IUTA: a tool for effectively detecting differential isoform usage from RNA-Seq data.

PubMed

Niu, Liang; Huang, Weichun; Umbach, David M; Li, Leping

2014-10-06

Most genes in mammals generate several transcript isoforms that differ in stability and translational efficiency through alternative splicing. Such alternative splicing can be tissue- and developmental stage-specific, and such specificity is sometimes associated with disease. Thus, detecting differential isoform usage for a gene between tissues or cell lines/types (differences in the fraction of total expression of a gene represented by the expression of each of its isoforms) is potentially important for cell and developmental biology. We present a new method IUTA that is designed to test each gene in the genome for differential isoform usage between two groups of samples. IUTA also estimates isoform usage for each gene in each sample as well as averaged across samples within each group. IUTA is the first method to formulate the testing problem as testing for equal means of two probability distributions under the Aitchison geometry, which is widely recognized as the most appropriate geometry for compositional data (vectors that contain the relative amount of each component comprising the whole). Evaluation using simulated data showed that IUTA was able to provide test results for many more genes than was Cuffdiff2 (version 2.2.0, released in Mar. 2014), and IUTA performed better than Cuffdiff2 for the limited number of genes that Cuffdiff2 did analyze. When applied to actual mouse RNA-Seq datasets from six tissues, IUTA identified 2,073 significant genes with clear patterns of differential isoform usage between a pair of tissues. IUTA is implemented as an R package and is available at http://www.niehs.nih.gov/research/resources/software/biostatistics/iuta/index.cfm. Both simulation and real-data results suggest that IUTA accurately detects differential isoform usage. We believe that our analysis of RNA-seq data from six mouse tissues represents the first comprehensive characterization of isoform usage in these tissues. IUTA will be a valuable resource for those who study the roles of alternative transcripts in cell development and disease.

Axial level-specific regulation of neuronal development: lessons from PITX2.

PubMed

Waite, Mindy R; Martin, Donna M

2015-02-01

Transcriptional regulation of gene expression is vital for proper control of proliferation, migration, differentiation, and survival of developing neurons. Pitx2 encodes a homeodomain transcription factor that is highly expressed in the developing and adult mammalian brain. In humans, mutations in PITX2 result in Rieger syndrome, characterized by defects in the development of the eyes, umbilicus, and teeth and variable abnormalities in the brain, including hydrocephalus and cerebellar hypoplasia. Alternative splicing of Pitx2 in the mouse results in three isoforms, Pitx2a, Pitx2b, and Pitx2c, each of which is expressed symmetrically along the left-right axis of the brain throughout development. Here, we review recent evidence for axial and brain region-specific requirements for Pitx2 during neuronal migration and differentiation, highlighting known isoform contributions. © 2014 Wiley Periodicals, Inc.

Combined epigenetic and differentiation-based treatment inhibits neuroblastoma tumor growth and links HIF2α to tumor suppression

PubMed Central

Westerlund, Isabelle; Shi, Yao; Toskas, Konstantinos; Fell, Stuart M.; Li, Shuijie; Surova, Olga; Södersten, Erik; Kogner, Per; Nyman, Ulrika; Schlisio, Susanne; Holmberg, Johan

2017-01-01

Neuroblastoma is a pediatric cancer characterized by variable outcomes ranging from spontaneous regression to life-threatening progression. High-risk neuroblastoma patients receive myeloablative chemotherapy with hematopoietic stem-cell transplant followed by adjuvant retinoid differentiation treatment. However, the overall survival remains low; hence, there is an urgent need for alternative therapeutic approaches. One feature of high-risk neuroblastoma is the high level of DNA methylation of putative tumor suppressors. Combining the reversibility of DNA methylation with the differentiation-promoting activity of retinoic acid (RA) could provide an alternative strategy to treat high-risk neuroblastoma. Here we show that treatment with the DNA-demethylating drug 5-Aza-deoxycytidine (AZA) restores high-risk neuroblastoma sensitivity to RA. Combined systemic distribution of AZA and RA impedes tumor growth and prolongs survival. Genome-wide analysis of treated tumors reveals that this combined treatment rapidly induces a HIF2α-associated hypoxia-like transcriptional response followed by an increase in neuronal gene expression and a decrease in cell-cycle gene expression. A small-molecule inhibitor of HIF2α activity diminishes the tumor response to AZA+RA treatment, indicating that the increase in HIF2α levels is a key component in tumor response to AZA+RA. The link between increased HIF2α levels and inhibited tumor growth is reflected in large neuroblastoma patient datasets. Therein, high levels of HIF2α, but not HIF1α, significantly correlate with expression of neuronal differentiation genes and better prognosis but negatively correlate with key features of high-risk tumors, such as MYCN amplification. Thus, contrary to previous studies, our findings indicate an unanticipated tumor-suppressive role for HIF2α in neuroblastoma. PMID:28696319

Combined epigenetic and differentiation-based treatment inhibits neuroblastoma tumor growth and links HIF2α to tumor suppression.

PubMed

Westerlund, Isabelle; Shi, Yao; Toskas, Konstantinos; Fell, Stuart M; Li, Shuijie; Surova, Olga; Södersten, Erik; Kogner, Per; Nyman, Ulrika; Schlisio, Susanne; Holmberg, Johan

2017-07-25

Neuroblastoma is a pediatric cancer characterized by variable outcomes ranging from spontaneous regression to life-threatening progression. High-risk neuroblastoma patients receive myeloablative chemotherapy with hematopoietic stem-cell transplant followed by adjuvant retinoid differentiation treatment. However, the overall survival remains low; hence, there is an urgent need for alternative therapeutic approaches. One feature of high-risk neuroblastoma is the high level of DNA methylation of putative tumor suppressors. Combining the reversibility of DNA methylation with the differentiation-promoting activity of retinoic acid (RA) could provide an alternative strategy to treat high-risk neuroblastoma. Here we show that treatment with the DNA-demethylating drug 5-Aza-deoxycytidine (AZA) restores high-risk neuroblastoma sensitivity to RA. Combined systemic distribution of AZA and RA impedes tumor growth and prolongs survival. Genome-wide analysis of treated tumors reveals that this combined treatment rapidly induces a HIF2α-associated hypoxia-like transcriptional response followed by an increase in neuronal gene expression and a decrease in cell-cycle gene expression. A small-molecule inhibitor of HIF2α activity diminishes the tumor response to AZA+RA treatment, indicating that the increase in HIF2α levels is a key component in tumor response to AZA+RA. The link between increased HIF2α levels and inhibited tumor growth is reflected in large neuroblastoma patient datasets. Therein, high levels of HIF2α, but not HIF1α, significantly correlate with expression of neuronal differentiation genes and better prognosis but negatively correlate with key features of high-risk tumors, such as MYCN amplification. Thus, contrary to previous studies, our findings indicate an unanticipated tumor-suppressive role for HIF2α in neuroblastoma.

New CD20 alternative splice variants: molecular identification and differential expression within hematological B cell malignancies.

PubMed

Gamonet, Clémentine; Bole-Richard, Elodie; Delherme, Aurélia; Aubin, François; Toussirot, Eric; Garnache-Ottou, Francine; Godet, Yann; Ysebaert, Loïc; Tournilhac, Olivier; Caroline, Dartigeas; Larosa, Fabrice; Deconinck, Eric; Saas, Philippe; Borg, Christophe; Deschamps, Marina; Ferrand, Christophe

2015-01-01

CD20 is a B cell lineage-specific marker expressed by normal and leukemic B cells and targeted by several antibody immunotherapies. We have previously shown that the protein from a CD20 mRNA splice variant (D393-CD20) is expressed at various levels in leukemic B cells or lymphoma B cells but not in resting, sorted B cells from the peripheral blood of healthy donors. Western blot (WB) analysis of B malignancy primary samples showed additional CD20 signals. Deep molecular PCR analysis revealed four new sequences corresponding to in-frame CD20 splice variants (D657-CD20, D618-CD20, D480-CD20, and D177-CD20) matching the length of WB signals. We demonstrated that the cell spliceosome machinery can process ex vivo D480-, D657-, and D618-CD20 transcript variants by involving canonical sites associated with cryptic splice sites. Results of specific and quantitative RT-PCR assays showed that these CD20 splice variants are differentially expressed in B malignancies. Moreover, Epstein-Barr virus (EBV) transformation modified the CD20 splicing profile and mainly increased the D393-CD20 variant transcripts. Finally, investigation of three cohorts of chronic lymphocytic leukemia (CLL) patients showed that the total CD20 splice variant expression was higher in a stage B and C sample collection compared to routinely collected CLL samples or relapsed refractory stage A, B, or C CLL. The involvement of these newly discovered alternative CD20 transcript variants in EBV transformation makes them interesting molecular indicators, as does their association with oncogenesis rather than non-oncogenic B cell diseases, differential expression in B cell malignancies, and correlation with CLL stage and some predictive CLL markers. This potential should be investigated in further studies.

C/EBPβ-LAP*/LAP Expression Is Mediated by RSK/eIF4B-Dependent Signalling and Boosted by Increased Protein Stability in Models of Monocytic Differentiation

PubMed Central

Christmann, Martin; Friesenhagen, Judith; Westphal, Andreas; Pietsch, Daniel; Brand, Korbinian

2015-01-01

The transcription factor C/EBPβ plays a key role in monocytic differentiation and inflammation. Its small isoform LIP is associated with proliferation at early premonocytic developmental stages and regulated via mTOR-dependent signalling. During later stages of (pre)monocytic differentiation there is a considerable increase in the large C/EBPβ isoforms LAP*/LAP which inhibit proliferation thus supporting terminal differentiation. Here, we showed in different models of monocytic differentiation that this dramatic increase in the LAP*/LAP protein and LAP/LIP ratio was accompanied by an only modest/retarded mRNA increase suggesting an important role for (post)translational mechanisms. We found that LAP*/LAP formation was induced via MEK/RSK-dependent cascades, whereas mTOR/S6K1 were not involved. Remarkably, LAP*/LAP expression was dependent on phosphorylated eIF4B, an acceleratory protein of RNA helicase eIF4A. PKR inhibition reduced the expression of eIF4B and C/EBPβ in an eIF2α-independent manner. Furthermore, under our conditions a marked stabilisation of LAP*/LAP protein occurred, accompanied by reduced chymotrypsin-like proteasome/calpain activities and increased calpastatin levels. Our study elucidates new signalling pathways inducing LAP*/LAP expression and indicates new alternative PKR functions in monocytes. The switch from mTOR- to RSK-mediated signalling to orchestrate eIF4B-dependent LAP*/LAP translation, accompanied by increased protein stability but only small mRNA changes, may be a prototypical example for the regulation of protein expression during selected processes of differentiation/proliferation. PMID:26646662

The caste- and sex-specific DNA methylome of the termite Zootermopsis nevadensis

PubMed Central

Glastad, Karl M.; Gokhale, Kaustubh; Liebig, Jürgen; Goodisman, Michael A. D.

2016-01-01

Epigenetic inheritance plays an important role in mediating alternative phenotype in highly social species. In order to gain a greater understanding of epigenetic effects in societies, we investigated DNA methylation in the termite Zootermopsis nevadensis. Termites are the most ancient social insects, and developmentally distinct from highly-studied, hymenopteran social insects. We used replicated bisulfite-sequencing to investigate patterns of DNA methylation in both sexes and among castes of Z. nevadensis. We discovered that Z. nevadensis displayed some of the highest levels of DNA methylation found in insects. We also found strong differences in methylation between castes. Methylated genes tended to be uniformly and highly expressed demonstrating the antiquity of associations between intragenic methylation and gene expression. Differentially methylated genes were more likely to be alternatively spliced than not differentially methylated genes, and possessed considerable enrichment for development-associated functions. We further observed strong overrepresentation of multiple transcription factor binding sites and miRNA profiles associated with differential methylation, providing new insights into the possible function of DNA methylation. Overall, our results show that DNA methylation is widespread and associated with caste differences in termites. More generally, this study provides insights into the function of DNA methylation and the success of insect societies. PMID:27848993

Differential gene expression by Moniliophthora roreri while overcoming cacao tolerance in the field.

PubMed

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

2014-09-01

Frosty pod rot (FPR) of Theobroma cacao (cacao) is caused by the hemibiotrophic fungus Moniliophthora roreri. Cacao clones tolerant to FPR are being planted throughout Central America. To determine whether M. roreri shows a differential molecular response during successful infections of tolerant clones, we collected field-infected pods at all stages of symptomatology for two highly susceptible clones (Pound-7 and CATIE-1000) and three tolerant clones (UF-273, CATIE-R7 and CATIE-R4). Metabolite analysis was carried out on clones Pound-7, CATIE-1000, CATIE-R7 and CATIE-R4. As FPR progressed, the concentrations of sugars in pods dropped, whereas the levels of trehalose and mannitol increased. Associations between symptoms and fungal loads and some organic and amino acid concentrations varied depending on the clone. RNA-Seq analysis identified 873 M. roreri genes that were differentially expressed between clones, with the primary difference being whether the clone was susceptible or tolerant. Genes encoding transcription factors, heat shock proteins, transporters, enzymes modifying membranes or cell walls and metabolic enzymes, such as malate synthase and alternative oxidase, were differentially expressed. The differential expression between clones of 43 M. roreri genes was validated by real-time quantitative reverse transcription polymerase chain reaction. The expression profiles of some genes were similar in susceptible and tolerant clones (other than CATIE-R4) and varied with the biotrophic/necrotropic shift. Moniliophthora roreri genes associated with stress metabolism and responses to heat shock and anoxia were induced early in tolerant clones, their expression profiles resembling that of the necrotrophic phase. Moniliophthora roreri stress response genes, induced during the infection of tolerant clones, may benefit the fungus in overcoming cacao defense mechanisms. © 2014 BSPP AND JOHN WILEY & SONS LTD.

Pigment cell interactions and differential xanthophore recruitment underlying zebrafish stripe reiteration and Danio pattern evolution

PubMed Central

Patterson, Larissa B.; Bain, Emily J.; Parichy, David M.

2014-01-01

Fishes have diverse pigment patterns, yet mechanisms of pattern evolution remain poorly understood. In zebrafish, Danio rerio, pigment-cell autonomous interactions generate dark stripes of melanophores that alternate with light interstripes of xanthophores and iridophores. Here, we identify mechanisms underlying the evolution of a uniform pattern in D. albolineatus in which all three pigment cell classes are intermingled. We show that in this species xanthophores differentiate precociously over a wider area, and that cis regulatory evolution has increased expression of xanthogenic Colony Stimulating Factor-1 (Csf1). Expressing Csf1 similarly in D. rerio has cascading effects, driving the intermingling of all three pigment cell classes and resulting in the loss of stripes, as in D. albolineatus. Our results identify novel mechanisms of pattern development and illustrate how pattern diversity can be generated when a core network of pigment-cell autonomous interactions is coupled with changes in pigment cell differentiation. PMID:25374113

Systematically Differentiating Functions for Alternatively Spliced Isoforms through Integrating RNA-seq Data

PubMed Central

Menon, Rajasree; Wen, Yuchen; Omenn, Gilbert S.; Kretzler, Matthias; Guan, Yuanfang

2013-01-01

Integrating large-scale functional genomic data has significantly accelerated our understanding of gene functions. However, no algorithm has been developed to differentiate functions for isoforms of the same gene using high-throughput genomic data. This is because standard supervised learning requires ‘ground-truth’ functional annotations, which are lacking at the isoform level. To address this challenge, we developed a generic framework that interrogates public RNA-seq data at the transcript level to differentiate functions for alternatively spliced isoforms. For a specific function, our algorithm identifies the ‘responsible’ isoform(s) of a gene and generates classifying models at the isoform level instead of at the gene level. Through cross-validation, we demonstrated that our algorithm is effective in assigning functions to genes, especially the ones with multiple isoforms, and robust to gene expression levels and removal of homologous gene pairs. We identified genes in the mouse whose isoforms are predicted to have disparate functionalities and experimentally validated the ‘responsible’ isoforms using data from mammary tissue. With protein structure modeling and experimental evidence, we further validated the predicted isoform functional differences for the genes Cdkn2a and Anxa6. Our generic framework is the first to predict and differentiate functions for alternatively spliced isoforms, instead of genes, using genomic data. It is extendable to any base machine learner and other species with alternatively spliced isoforms, and shifts the current gene-centered function prediction to isoform-level predictions. PMID:24244129

Molecular Characterization of the α-Subunit of Na+/K+ ATPase from the Euryhaline Barnacle Balanus improvisus Reveals Multiple Genes and Differential Expression of Alternative Splice Variants

PubMed Central

Lind, Ulrika; Alm Rosenblad, Magnus; Wrange, Anna-Lisa; Sundell, Kristina S.; Jonsson, Per R.; André, Carl; Havenhand, Jonathan; Blomberg, Anders

2013-01-01

The euryhaline bay barnacle Balanus improvisus has one of the broadest salinity tolerances of any barnacle species. It is able to complete its life cycle in salinities close to freshwater (3 PSU) up to fully marine conditions (35 PSU) and is regarded as one of few truly brackish-water species. Na+/K+ ATPase (NAK) has been shown to be important for osmoregulation when marine organisms are challenged by changing salinities, and we therefore cloned and examined the expression of different NAKs from B. improvisus. We found two main gene variants, NAK1 and NAK2, which were approximately 70% identical at the protein level. The NAK1 mRNA existed in a long and short variant with the encoded proteins differing only by 27 N-terminal amino acids. This N-terminal stretch was coded for by a separate exon, and the two variants of NAK1 mRNAs appeared to be created by alternative splicing. We furthermore showed that the two NAK1 isoforms were differentially expressed in different life stages and in various tissues of adult barnacle, i.e the long isoform was predominant in cyprids and in adult cirri. In barnacle cyprid larvae that were exposed to a combination of different salinities and pCO2 levels, the expression of the long NAK1 mRNA increased relative to the short in low salinities. We suggest that the alternatively spliced long variant of the Nak1 protein might be of importance for osmoregulation in B. improvisus in low salinity conditions. PMID:24130836

Differential regulation of hepatitis B virus core protein expression and genome replication by a small upstream open reading frame and naturally occurring mutations in the precore region.

PubMed

Zong, Li; Qin, Yanli; Jia, Haodi; Ye, Lei; Wang, Yongxiang; Zhang, Jiming; Wands, Jack R; Tong, Shuping; Li, Jisu

2017-05-01

Hepatitis B virus (HBV) transcribes two subsets of 3.5-kb RNAs: precore RNA for hepatitis B e antigen (HBeAg) expression, and pregenomic RNA for core and P protein translation as well as genome replication. HBeAg expression could be prevented by mutations in the precore region, while an upstream open reading frame (uORF) has been proposed as a negative regulator of core protein translation. We employed replication competent HBV DNA constructs and transient transfection experiments in Huh7 cells to verify the uORF effect and to explore the alternative function of precore RNA. Optimized Kozak sequence for the uORF or extra ATG codons as present in some HBV genotypes reduced core protein expression. G1896A nonsense mutation promoted more efficient core protein expression than mutated precore ATG, while a +1 frameshift mutation was ineffective. In conclusion, various HBeAg-negative precore mutations and mutations affecting uORF differentially regulate core protein expression and genome replication. Copyright © 2017 Elsevier Inc. All rights reserved.

Hybrid Sequencing of Full-Length cDNA Transcripts of Stems and Leaves in Dendrobium officinale

PubMed Central

He, Liu; Fu, Shuhua; Xu, Zhichao; Yan, Jun; Xu, Jiang; Zhou, Hong; Zhou, Jianguo; Chen, Xinlian; Li, Ying; Au, Kin Fai; Yao, Hui

2017-01-01

Dendrobium officinale is an extremely valuable orchid used in traditional Chinese medicine, so sought after that it has a higher market value than gold. Although the expression profiles of some genes involved in the polysaccharide synthesis have previously been investigated, little research has been carried out on their alternatively spliced isoforms in D. officinale. In addition, information regarding the translocation of sugars from leaves to stems in D. officinale also remains limited. We analyzed the polysaccharide content of D. officinale leaves and stems, and completed in-depth transcriptome sequencing of these two diverse tissue types using second-generation sequencing (SGS) and single-molecule real-time (SMRT) sequencing technology. The results of this study yielded a digital inventory of gene and mRNA isoform expressions. A comparative analysis of both transcriptomes uncovered a total of 1414 differentially expressed genes, including 844 that were up-regulated and 570 that were down-regulated in stems. Of these genes, one sugars will eventually be exported transporter (SWEET) and one sucrose transporter (SUT) are expressed to a greater extent in D. officinale stems than in leaves. Two glycosyltransferase (GT) and four cellulose synthase (Ces) genes undergo a distinct degree of alternative splicing. In the stems, the content of polysaccharides is twice as much as that in the leaves. The differentially expressed GT and transcription factor (TF) genes will be the focus of further study. The genes DoSWEET4 and DoSUT1 are significantly expressed in the stem, and are likely to be involved in sugar loading in the phloem. PMID:28981454

Dynamic regulation of EZH2 from HPSc to hepatocyte-like cell fate

PubMed Central

Helsen, Nicky; Vanhove, Jolien; Boon, Ruben; Xu, Zhuofei; Ordovas, Laura; Verfaillie, Catherine M.

2017-01-01

Currently, drug metabolization and toxicity studies rely on the use of primary human hepatocytes and hepatoma cell lines, which both have conceivable limitations. Human pluripotent stem cell (hPSC)—derived hepatocyte-like cells (HLCs) are an alternative and valuable source of hepatocytes that can overcome these limitations. EZH2 (enhancer of zeste homolog 2), a transcriptional repressor of the polycomb repressive complex 2 (PRC2), may play an important role in hepatocyte development, but its role during in vitro hPSC-HLC differentiation has not yet been assessed. We here demonstrate dynamic regulation of EZH2 during hepatic differentiation of hPSC. To enhance EZH2 expression, we inducibly overexpressed EZH2 between d0 and d8, demonstrating a significant improvement in definitive endoderm formation, and improved generation of HLCs. Despite induction of EZH2 overexpression until d8, EZH2 transcript and protein levels decreased from d4 onwards, which might be caused by expression of microRNAs predicted to inhibit EZH2 expression. In conclusion, our studies demonstrate that EZH2 plays a role in endoderm formation and hepatocyte differentiation, but its expression is tightly post-transcriptionally regulated during this process. PMID:29091973

DEIsoM: a hierarchical Bayesian model for identifying differentially expressed isoforms using biological replicates

PubMed Central

Peng, Hao; Yang, Yifan; Zhe, Shandian; Wang, Jian; Gribskov, Michael; Qi, Yuan

2017-01-01

Abstract Motivation High-throughput mRNA sequencing (RNA-Seq) is a powerful tool for quantifying gene expression. Identification of transcript isoforms that are differentially expressed in different conditions, such as in patients and healthy subjects, can provide insights into the molecular basis of diseases. Current transcript quantification approaches, however, do not take advantage of the shared information in the biological replicates, potentially decreasing sensitivity and accuracy. Results We present a novel hierarchical Bayesian model called Differentially Expressed Isoform detection from Multiple biological replicates (DEIsoM) for identifying differentially expressed (DE) isoforms from multiple biological replicates representing two conditions, e.g. multiple samples from healthy and diseased subjects. DEIsoM first estimates isoform expression within each condition by (1) capturing common patterns from sample replicates while allowing individual differences, and (2) modeling the uncertainty introduced by ambiguous read mapping in each replicate. Specifically, we introduce a Dirichlet prior distribution to capture the common expression pattern of replicates from the same condition, and treat the isoform expression of individual replicates as samples from this distribution. Ambiguous read mapping is modeled as a multinomial distribution, and ambiguous reads are assigned to the most probable isoform in each replicate. Additionally, DEIsoM couples an efficient variational inference and a post-analysis method to improve the accuracy and speed of identification of DE isoforms over alternative methods. Application of DEIsoM to an hepatocellular carcinoma (HCC) dataset identifies biologically relevant DE isoforms. The relevance of these genes/isoforms to HCC are supported by principal component analysis (PCA), read coverage visualization, and the biological literature. Availability and implementation The software is available at https://github.com/hao-peng/DEIsoM Contact pengh@alumni.purdue.edu Supplementary information Supplementary data are available at Bioinformatics online. PMID:28595376

Volcano plots in analyzing differential expressions with mRNA microarrays.

PubMed

Li, Wentian

2012-12-01

A volcano plot displays unstandardized signal (e.g. log-fold-change) against noise-adjusted/standardized signal (e.g. t-statistic or -log(10)(p-value) from the t-test). We review the basic and interactive use of the volcano plot and its crucial role in understanding the regularized t-statistic. The joint filtering gene selection criterion based on regularized statistics has a curved discriminant line in the volcano plot, as compared to the two perpendicular lines for the "double filtering" criterion. This review attempts to provide a unifying framework for discussions on alternative measures of differential expression, improved methods for estimating variance, and visual display of a microarray analysis result. We also discuss the possibility of applying volcano plots to other fields beyond microarray.

Processing of fish lg heavy chain transcripts diverse splicing patterns and unusual nonsense mediated decay

USDA-ARS?s Scientific Manuscript database

Alternate pathways of RNA processing play an important role in the expression of the secreted (S) and membrane (Mb) forms of immunoglobulin (Ig) heavy (H) chain isotypes in all vertebrates. Interestingly, while the differential splicing mechanism and the splice sites that generate the two forms of I...

Sex Differences and Autism: Brain Function during Verbal Fluency and Mental Rotation

PubMed Central

Minati, Ludovico; Baron-Cohen, Simon; Lombardo, Michael V.; Lai, Meng-Chuan; Walker, Anne; Howard, Dawn; Gray, Marcus A.; Harrison, Neil A.; Critchley, Hugo D.

2012-01-01

Autism spectrum conditions (ASC) affect more males than females. This suggests that the neurobiology of autism: 1) may overlap with mechanisms underlying typical sex-differentiation or 2) alternately reflect sex-specificity in how autism is expressed in males and females. Here we used functional magnetic resonance imaging (fMRI) to test these alternate hypotheses. Fifteen men and fourteen women with Asperger syndrome (AS), and sixteen typically developing men and sixteen typically developing women underwent fMRI during performance of mental rotation and verbal fluency tasks. All groups performed the tasks equally well. On the verbal fluency task, despite equivalent task-performance, both males and females with AS showed enhanced activation of left occipitoparietal and inferior prefrontal activity compared to controls. During mental rotation, there was a significant diagnosis-by-sex interaction across occipital, temporal, parietal, middle frontal regions, with greater activation in AS males and typical females compared to AS females and typical males. These findings suggest a complex relationship between autism and sex that is differentially expressed in verbal and visuospatial domains. PMID:22701630

Sex differences and autism: brain function during verbal fluency and mental rotation.

PubMed

Beacher, Felix D C C; Radulescu, Eugenia; Minati, Ludovico; Baron-Cohen, Simon; Lombardo, Michael V; Lai, Meng-Chuan; Walker, Anne; Howard, Dawn; Gray, Marcus A; Harrison, Neil A; Critchley, Hugo D

2012-01-01

Autism spectrum conditions (ASC) affect more males than females. This suggests that the neurobiology of autism: 1) may overlap with mechanisms underlying typical sex-differentiation or 2) alternately reflect sex-specificity in how autism is expressed in males and females. Here we used functional magnetic resonance imaging (fMRI) to test these alternate hypotheses. Fifteen men and fourteen women with Asperger syndrome (AS), and sixteen typically developing men and sixteen typically developing women underwent fMRI during performance of mental rotation and verbal fluency tasks. All groups performed the tasks equally well. On the verbal fluency task, despite equivalent task-performance, both males and females with AS showed enhanced activation of left occipitoparietal and inferior prefrontal activity compared to controls. During mental rotation, there was a significant diagnosis-by-sex interaction across occipital, temporal, parietal, middle frontal regions, with greater activation in AS males and typical females compared to AS females and typical males. These findings suggest a complex relationship between autism and sex that is differentially expressed in verbal and visuospatial domains.

Smooth Muscle-Like Cells Generated from Human Mesenchymal Stromal Cells Display Marker Gene Expression and Electrophysiological Competence Comparable to Bladder Smooth Muscle Cells.

PubMed

Brun, Juliane; Lutz, Katrin A; Neumayer, Katharina M H; Klein, Gerd; Seeger, Tanja; Uynuk-Ool, Tatiana; Wörgötter, Katharina; Schmid, Sandra; Kraushaar, Udo; Guenther, Elke; Rolauffs, Bernd; Aicher, Wilhelm K; Hart, Melanie L

2015-01-01

The use of mesenchymal stromal cells (MSCs) differentiated toward a smooth muscle cell (SMC) phenotype may provide an alternative for investigators interested in regenerating urinary tract organs such as the bladder where autologous smooth muscle cells cannot be used or are unavailable. In this study we measured the effects of good manufacturing practice (GMP)-compliant expansion followed by myogenic differentiation of human MSCs on the expression of a range of contractile (from early to late) myogenic markers in relation to the electrophysiological parameters to assess the functional role of the differentiated MSCs and found that differentiation of MSCs associated with electrophysiological competence comparable to bladder SMCs. Within 1-2 weeks of myogenic differentiation, differentiating MSCs significantly expressed alpha smooth muscle actin (αSMA; ACTA2), transgelin (TAGLN), calponin (CNN1), and smooth muscle myosin heavy chain (SM-MHC; MYH11) according to qRT-PCR and/or immunofluorescence and Western blot. Voltage-gated Na+ current levels also increased within the same time period following myogenic differentiation. In contrast to undifferentiated MSCs, differentiated MSCs and bladder SMCs exhibited elevated cytosolic Ca2+ transients in response to K+-induced depolarization and contracted in response to K+ indicating functional maturation of differentiated MSCs. Depolarization was suppressed by Cd2+, an inhibitor of voltage-gated Ca2+-channels. The expression of Na+-channels was pharmacologically identified as the Nav1.4 subtype, while the K+ and Ca2+ ion channels were identified by gene expression of KCNMA1, CACNA1C and CACNA1H which encode for the large conductance Ca2+-activated K+ channel BKCa channels, Cav1.2 L-type Ca2+ channels and Cav3.2 T-type Ca2+ channels, respectively. This protocol may be used to differentiate adult MSCs into smooth muscle-like cells with an intermediate-to-late SMC contractile phenotype exhibiting voltage-gated ion channel activity comparable to bladder SMCs which may be important for urological regenerative medicine applications.

Novel isoforms of Dlg are fundamental for neuronal development in Drosophila.

PubMed

Mendoza, Carolina; Olguín, Patricio; Lafferte, Gabriela; Thomas, Ulrich; Ebitsch, Susanne; Gundelfinger, Eckart D; Kukuljan, Manuel; Sierralta, Jimena

2003-03-15

Drosophila discs-large (dlg) mutants exhibit multiple developmental abnormalities, including severe defects in neuronal differentiation and synaptic structure and function. These defects have been ascribed to the loss of a single gene product, Dlg-A, a scaffold protein thought to be expressed in many cell types. Here, we describe that additional isoforms arise as a consequence of different transcription start points and alternative splicing of dlg. At least five different dlg gene products are predicted. We identified a subset of dlg-derived cDNAs that include novel exons encoding a peptide homologous to the N terminus of the mammalian protein SAP97/hDLG (S97N). Dlg isoforms containing the S97N domain are expressed at larval neuromuscular junctions and within the CNS of both embryos and larvae but are not detectable in epithelial tissues. Strong hypomorphic dlg alleles exhibit decreased expression of S97N, which may account for neural-specific aspects of the pleiomorphic dlg mutant phenotype. Selective inhibition of the expression of S97N-containing proteins in embryos by double-strand RNA leads to severe defects in neuronal differentiation and axon guidance, without overt perturbations in epithelia. These results indicate that the differential expression of dlg products correlates with distinct functions in non-neural and neural cells. During embryonic development, proteins that include the S97N domain are essential for proper neuronal differentiation and organization, acting through mechanisms that may include the adequate localization of cell fate determinants.

Genome-Wide Profiling of Plutella xylostella Immunity-Related miRNAs after Isaria fumosorosea Infection.

PubMed

Xu, Jin; Xu, Xiaoxia; Li, Shuzhong; Wang, Shuang; Xu, Xiaojing; Zhou, Xianqiang; Yu, Jialin; Yu, Xiaoqiang; Shakeel, Muhammad; Jin, Fengliang

2017-01-01

The development of resistance by Plutella xylostella to almost all insecticides is of significant concern all over the world. Entomopathogenic fungi such as Isaria fumosorosea have been used as an alternative to insecticides. However, the knowledge of miRNA-regulated reactions against entomopathogenic fungi is still in its infant stage. In the present study, P. xylostella was challenged with I. fumosorosea at four different time points (12, 18, 24, and 36 h) including a control, to build miRNA libraries by Illumina sequencing. The results of differential expression analysis exhibited that 23 miRNAs were differentially expressed, compared to control, in all treatments. It is worth mentioning, of these, some conserved miRNAs such as miR-2, miR-9a, miR-745, miR-7b, and miR-2767, known to play critical roles in host-pathogen interaction, were also identified. Furthermore, differentially expressed miRNAs were validated by RT-qPCR. Our results provide an essential information for further functional studies of the interaction between I. fumosorosea and P. xylostella at the post-transcriptional level.

Genome-Wide Profiling of Plutella xylostella Immunity-Related miRNAs after Isaria fumosorosea Infection

PubMed Central

Xu, Jin; Xu, Xiaoxia; Li, Shuzhong; Wang, Shuang; Xu, Xiaojing; Zhou, Xianqiang; Yu, Jialin; Yu, Xiaoqiang; Shakeel, Muhammad; Jin, Fengliang

2017-01-01

The development of resistance by Plutella xylostella to almost all insecticides is of significant concern all over the world. Entomopathogenic fungi such as Isaria fumosorosea have been used as an alternative to insecticides. However, the knowledge of miRNA-regulated reactions against entomopathogenic fungi is still in its infant stage. In the present study, P. xylostella was challenged with I. fumosorosea at four different time points (12, 18, 24, and 36 h) including a control, to build miRNA libraries by Illumina sequencing. The results of differential expression analysis exhibited that 23 miRNAs were differentially expressed, compared to control, in all treatments. It is worth mentioning, of these, some conserved miRNAs such as miR-2, miR-9a, miR-745, miR-7b, and miR-2767, known to play critical roles in host-pathogen interaction, were also identified. Furthermore, differentially expressed miRNAs were validated by RT-qPCR. Our results provide an essential information for further functional studies of the interaction between I. fumosorosea and P. xylostella at the post-transcriptional level. PMID:29311981

An Orchestrated Intron Retention Program in Meiosis Controls Timely Usage of Transcripts during Germ Cell Differentiation.

PubMed

Naro, Chiara; Jolly, Ariane; Di Persio, Sara; Bielli, Pamela; Setterblad, Niclas; Alberdi, Antonio J; Vicini, Elena; Geremia, Raffaele; De la Grange, Pierre; Sette, Claudio

2017-04-10

Global transcriptome reprogramming during spermatogenesis ensures timely expression of factors in each phase of male germ cell differentiation. Spermatocytes and spermatids require particularly extensive reprogramming of gene expression to switch from mitosis to meiosis and to support gamete morphogenesis. Here, we uncovered an extensive alternative splicing program during this transmeiotic differentiation. Notably, intron retention was largely the most enriched pattern, with spermatocytes showing generally higher levels of retention compared with spermatids. Retained introns are characterized by weak splice sites and are enriched in genes with strong relevance for gamete function. Meiotic intron-retaining transcripts (IRTs) were exclusively localized in the nucleus. However, differently from other developmentally regulated IRTs, they are stable RNAs, showing longer half-life than properly spliced transcripts. Strikingly, fate-mapping experiments revealed that IRTs are recruited onto polyribosomes days after synthesis. These studies reveal an unexpected function for regulated intron retention in modulation of the timely expression of select transcripts during spermatogenesis. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Binary Cell Fate Decisions and Fate Transformation in the Drosophila Larval Eye

PubMed Central

Rister, Jens; Ng, June; Celik, Arzu; Sprecher, Simon G.

2013-01-01

The functionality of sensory neurons is defined by the expression of specific sensory receptor genes. During the development of the Drosophila larval eye, photoreceptor neurons (PRs) make a binary choice to express either the blue-sensitive Rhodopsin 5 (Rh5) or the green-sensitive Rhodopsin 6 (Rh6). Later during metamorphosis, ecdysone signaling induces a cell fate and sensory receptor switch: Rh5-PRs are re-programmed to express Rh6 and become the eyelet, a small group of extraretinal PRs involved in circadian entrainment. However, the genetic and molecular mechanisms of how the binary cell fate decisions are made and switched remain poorly understood. We show that interplay of two transcription factors Senseless (Sens) and Hazy control cell fate decisions, terminal differentiation of the larval eye and its transformation into eyelet. During initial differentiation, a pulse of Sens expression in primary precursors regulates their differentiation into Rh5-PRs and repression of an alternative Rh6-cell fate. Later, during the transformation of the larval eye into the adult eyelet, Sens serves as an anti-apoptotic factor in Rh5-PRs, which helps in promoting survival of Rh5-PRs during metamorphosis and is subsequently required for Rh6 expression. Comparably, during PR differentiation Hazy functions in initiation and maintenance of rhodopsin expression. Hazy represses Sens specifically in the Rh6-PRs, allowing them to die during metamorphosis. Our findings show that the same transcription factors regulate diverse aspects of larval and adult PR development at different stages and in a context-dependent manner. PMID:24385925

Binary cell fate decisions and fate transformation in the Drosophila larval eye.

PubMed

Mishra, Abhishek Kumar; Tsachaki, Maria; Rister, Jens; Ng, June; Celik, Arzu; Sprecher, Simon G

2013-01-01

The functionality of sensory neurons is defined by the expression of specific sensory receptor genes. During the development of the Drosophila larval eye, photoreceptor neurons (PRs) make a binary choice to express either the blue-sensitive Rhodopsin 5 (Rh5) or the green-sensitive Rhodopsin 6 (Rh6). Later during metamorphosis, ecdysone signaling induces a cell fate and sensory receptor switch: Rh5-PRs are re-programmed to express Rh6 and become the eyelet, a small group of extraretinal PRs involved in circadian entrainment. However, the genetic and molecular mechanisms of how the binary cell fate decisions are made and switched remain poorly understood. We show that interplay of two transcription factors Senseless (Sens) and Hazy control cell fate decisions, terminal differentiation of the larval eye and its transformation into eyelet. During initial differentiation, a pulse of Sens expression in primary precursors regulates their differentiation into Rh5-PRs and repression of an alternative Rh6-cell fate. Later, during the transformation of the larval eye into the adult eyelet, Sens serves as an anti-apoptotic factor in Rh5-PRs, which helps in promoting survival of Rh5-PRs during metamorphosis and is subsequently required for Rh6 expression. Comparably, during PR differentiation Hazy functions in initiation and maintenance of rhodopsin expression. Hazy represses Sens specifically in the Rh6-PRs, allowing them to die during metamorphosis. Our findings show that the same transcription factors regulate diverse aspects of larval and adult PR development at different stages and in a context-dependent manner.

Investigation of alternative prodrugs for use with E. coli nitroreductase in 'suicide gene' approaches to cancer therapy.

PubMed

Bailey, S M; Knox, R J; Hobbs, S M; Jenkins, T C; Mauger, A B; Melton, R G; Burke, P J; Connors, T A; Hart, I R

1996-12-01

The most commonly employed 'suicide' gene/prodrug system used in cancer gene therapy is the herpes simplex virus thymidine kinase (HSVtk)/ganciclovir system. We have examined the efficacy of an alternative approach utilising the E. coli nitroreductase B enzyme with CB1954 and a variety of other prodrugs. V79 cells transfected with a nitroreductase expression vector were up to 770-fold more sensitive to CB1954 than control non-expressing cells. In general other prodrugs which were found by HPLC to act as substrates for purified E. coli nitroreductase also exhibited increased cytotoxicity against the nitroreductase-expressing cells, although this correlation was not absolute. In particular nitrofurazone (97-fold) and additional aromatic nitro-compounds (nine- to 50-fold) showed a large differential whereas the quinones and the antimetabolite, B-FU, were less effective (< three-fold). The results support the possibility of using nitroreductase and CB1954 for 'suicide gene' therapy and in addition suggest that alternative prodrugs, such as nitrofurazone, warrant further investigation in this novel approach.

RSRC1 mutation affects intellect and behaviour through aberrant splicing and transcription, downregulating IGFBP3.

PubMed

Perez, Yonatan; Menascu, Shay; Cohen, Idan; Kadir, Rotem; Basha, Omer; Shorer, Zamir; Romi, Hila; Meiri, Gal; Rabinski, Tatiana; Ofir, Rivka; Yeger-Lotem, Esti; Birk, Ohad S

2018-04-01

RSRC1, whose polymorphism is associated with altered brain function in schizophrenia, is a member of the serine and arginine rich-related protein family. Through homozygosity mapping and whole exome sequencing we show that RSRC1 mutation causes an autosomal recessive syndrome of intellectual disability, aberrant behaviour, hypotonia and mild facial dysmorphism with normal brain MRI. Further, we show that RSRC1 is ubiquitously expressed, and that the RSRC1 mutation triggers nonsense-mediated mRNA decay of the RSRC1 transcript in patients' fibroblasts. Short hairpin RNA (shRNA)-mediated lentiviral silencing and overexpression of RSRC1 in SH-SY5Y cells demonstrated that RSRC1 has a role in alternative splicing and transcription regulation. Transcriptome profiling of RSRC1-silenced cells unravelled specific differentially expressed genes previously associated with intellectual disability, hypotonia and schizophrenia, relevant to the disease phenotype. Protein-protein interaction network modelling suggested possible intermediate interactions by which RSRC1 affects gene-specific differential expression. Patient-derived induced pluripotent stem cells, differentiated into neural progenitor cells, showed expression dynamics similar to the RSRC1-silenced SH-SY5Y model. Notably, patient neural progenitor cells had 9.6-fold downregulated expression of IGFBP3, whose brain expression is affected by MECP2, aberrant in Rett syndrome. Interestingly, Igfbp3-null mice have behavioural impairment, abnormal synaptic function and monoaminergic neurotransmission, likely correlating with the disease phenotype.

Characterization of Visceral and Subcutaneous Adipose Tissue Transcriptome and Biological Pathways in Pregnant and Non-Pregnant Women: Evidence for Pregnancy-Related Regional-Specific Differences in Adipose Tissue

PubMed Central

Mazaki-Tovi, Shali; Vaisbuch, Edi; Tarca, Adi L.; Kusanovic, Juan Pedro; Than, Nandor Gabor; Chaiworapongsa, Tinnakorn; Dong, Zhong; Hassan, Sonia S.; Romero, Roberto

2015-01-01

Objective The purpose of this study was to compare the transcriptome of visceral and subcutaneous adipose tissues between pregnant and non-pregnant women. Study Design The transcriptome of paired visceral and abdominal subcutaneous adipose tissues from pregnant women at term and matched non-pregnant women (n = 11) was profiled with the Affymetrix Human Exon 1.0 ST array. Differential expression of selected genes was validated with the use of quantitative reverse transcription–polymerase chain reaction. Results Six hundred forty-four transcripts from 633 known genes were differentially expressed (false discovery rate (FDR) <0.1; fold-change >1.5), while 42 exons from 36 genes showed differential usage (difference in FIRMA scores >2 and FDR<0.1) between the visceral and subcutaneous fat of pregnant women. Fifty-six known genes were differentially expressed between pregnant and non-pregnant subcutaneous fat and three genes in the visceral fat. Enriched biological processes in the subcutaneous adipose tissue of pregnant women were mostly related to inflammation. Conclusion The transcriptome of visceral and subcutaneous fat depots reveals pregnancy-related gene expression and splicing differences in both visceral and subcutaneous adipose tissue. Furthermore, for the first time, alternative splicing in adipose tissue has been associated with regional differences and human parturition. PMID:26636677

Comparison between immunohistochemical expression of Ki-67 and MCM-3 in major salivary gland epithelial tumors in children and adolescents. Preliminary study.

PubMed

Zieliński, Rafał; Kobos, Jozef; Zakrzewska, Anna

While Ki-67 expression is frequently used as an indicator of tumor cell proliferation, alternative markers have also been proposed. Possible alternative indicators of proliferation are the minichromosome maintenance (MCM) proteins, whose levels are inversely associated with tumor cell differentiation. The aim of this preliminary study was to compare the levels of Ki-67 and MCM-3 expression in major salivary gland epithelial tumors in all children and adolescents who underwent surgery in our department in the years 2009-2014. The histopathological diagnosis of the subjects was reviewed, as well as the expression of Ki-67 and MCM-3 in post-op specimens of the tumors. The normality of data was checked with the Shapiro-Wilk test. The t test for independent variables or the U test was used as appropriate to determine statistically significant differences in the expression of Ki-67 and MCM-3. Five cases of pleomorphic adenoma, one of myoepithelioma, one of basal cell adenoma and one of mucoepidermoid carcinoma were identified. Significantly greater MCM-3 than Ki-67 expression was observed in every case. The results of our preliminary study emphasize the need for future research on MCM-3 as a sensitive proliferation marker, providing an alternative to Ki-67, in cases of various major salivary gland epithelial tumors in children and adolescents.

Gene selection for the reconstruction of stem cell differentiation trees: a linear programming approach.

PubMed

Ghadie, Mohamed A; Japkowicz, Nathalie; Perkins, Theodore J

2015-08-15

Stem cell differentiation is largely guided by master transcriptional regulators, but it also depends on the expression of other types of genes, such as cell cycle genes, signaling genes, metabolic genes, trafficking genes, etc. Traditional approaches to understanding gene expression patterns across multiple conditions, such as principal components analysis or K-means clustering, can group cell types based on gene expression, but they do so without knowledge of the differentiation hierarchy. Hierarchical clustering can organize cell types into a tree, but in general this tree is different from the differentiation hierarchy itself. Given the differentiation hierarchy and gene expression data at each node, we construct a weighted Euclidean distance metric such that the minimum spanning tree with respect to that metric is precisely the given differentiation hierarchy. We provide a set of linear constraints that are provably sufficient for the desired construction and a linear programming approach to identify sparse sets of weights, effectively identifying genes that are most relevant for discriminating different parts of the tree. We apply our method to microarray gene expression data describing 38 cell types in the hematopoiesis hierarchy, constructing a weighted Euclidean metric that uses just 175 genes. However, we find that there are many alternative sets of weights that satisfy the linear constraints. Thus, in the style of random-forest training, we also construct metrics based on random subsets of the genes and compare them to the metric of 175 genes. We then report on the selected genes and their biological functions. Our approach offers a new way to identify genes that may have important roles in stem cell differentiation. tperkins@ohri.ca 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.

Human periapical cyst-mesenchymal stem cells differentiate into neuronal cells.

PubMed

Marrelli, M; Paduano, F; Tatullo, M

2015-06-01

It was recently reported that human periapical cysts (hPCys), a commonly occurring odontogenic cystic lesion of inflammatory origin, contain mesenchymal stem cells (MSCs) with the capacity for self-renewal and multilineage differentiation. In this study, periapical inflammatory cysts were compared with dental pulp to determine whether this tissue may be an alternative accessible tissue source of MSCs that retain the potential for neurogenic differentiation. Flow cytometry and immunofluorescence analysis indicated that hPCy-MSCs and dental pulp stem cells spontaneously expressed the neuron-specific protein β-III tubulin and the neural stem-/astrocyte-specific protein glial fibrillary acidic protein (GFAP) in their basal state before differentiation occurs. Furthermore, undifferentiated hPCy-MSCs showed a higher expression of transcripts for neuronal markers (β-III tubulin, NF-M, MAP2) and neural-related transcription factors (MSX-1, Foxa2, En-1) as compared with dental pulp stem cells. After exposure to neurogenic differentiation conditions (neural media containing epidermal growth factor [EGF], basic fibroblast growth factor [bFGF], and retinoic acid), the hPCy-MSCs showed enhanced expression of β-III tubulin and GFAP proteins, as well as increased expression of neurofilaments medium, neurofilaments heavy, and neuron-specific enolase at the transcript level. In addition, neurally differentiated hPCy-MSCs showed upregulated expression of the neural transcription factors Pitx3, Foxa2, Nurr1, and the dopamine-related genes tyrosine hydroxylase and dopamine transporter. The present study demonstrated for the first time that hPCy-MSCs have a predisposition toward the neural phenotype that is increased when exposed to neural differentiation cues, based on upregulation of a comprehensive set of proteins and genes that define neuronal cells. In conclusion, these results provide evidence that hPCy-MSCs might be another optimal source of neural/glial cells for cell-based therapies to treat neurologic diseases. © International & American Associations for Dental Research 2015.

Solid and liquid heat capacities of n-alkyl para-aminobenzoates near the melting point.

PubMed

Neau, S H; Flynn, G L

1990-11-01

The expression that relates the ideal mole fraction solubility of a crystalline compound to physicochemical properties of the compound includes a term involving the difference in the heat capacities of the solid and liquid forms of the solute, delta Cp. There are two alternate conventions which are employed to eliminate this term. The first assumes that the term involving delta Cp, or delta Cp itself, is zero. The alternate assumption assigns the value of the entropy of fusion to the differential heat capacity. The relative validity of these two assumptions was evaluated using the straight-chain alkyl para-aminobenzoates as test compounds. The heat capacities of the solid and liquid forms of each of the para-aminobenzoates, near the respective melting point, were determined by differential scanning calorimetry. The data lead one to conclude that the assumption that the differential heat capacity is not usually negligible and is better approximated by the entropy of fusion.

Melanoma Differentiation-associated Gene 7/IL-24 Exerts Cytotoxic Effects by Altering the Alternative Splicing of Bcl-x Pre-mRNA via the SRC/PKCδ Signaling Axis*

PubMed Central

Shapiro, Brian A.; Vu, Ngoc T.; Shultz, Michael D.; Shultz, Jacqueline C.; Mietla, Jennifer A.; Gouda, Mazen M.; Yacoub, Adly; Dent, Paul; Fisher, Paul B.; Park, Margaret A.; Chalfant, Charles E.

2016-01-01

Melanoma differentiation-associated gene 7 (MDA-7/IL-24) exhibits cytotoxic effects on tumor cells while sparing untransformed cells, and Bcl-x(L) is reported to efficiently block the induction of cell death by MDA-7/IL-24. The expression of Bcl-x(L) is regulated at the level of RNA splicing via alternative 5′ splice site selection within exon 2 to produce either the pro-apoptotic Bcl-x(s) or the anti-apoptotic Bcl-x(L). Our laboratory previously reported that Bcl-x RNA splicing is dysregulated in a large percentage of human non-small cell lung cancer (NSCLC) tumors. Therefore, we investigated whether the alternative RNA splicing of Bcl-x pre-mRNA was modulated by MDA-7/IL-24, which would suggest that specific NSCLC tumors are valid targets for this cytokine therapy. Adenovirus-delivered MDA-7/IL-24 (Ad.mda-7) reduced the viability of NSCLC cells of varying oncogenotypes, which was preceded by a decrease in the ratio of Bcl-x(L)/Bcl-x(s) mRNA and Bcl-x(L) protein expression. Importantly, both the expression of Bcl-x(L) and the loss of cell viability were “rescued” in Ad.mda-7-treated cells incubated with Bcl-x(s) siRNA. In addition, NSCLC cells ectopically expressing Bcl-x(s) exhibited significantly reduced Bcl-x(L) expression, which was again restored by Bcl-x(s) siRNA, suggesting the existence of a novel mechanism by which Bcl-x(s) mRNA restrains the expression of Bcl-x(L). In additional mechanistic studies, inhibition of SRC and PKCδ completely ablated the ability of MDA-7/IL-24 to reduce the Bcl-x(L)/(s) mRNA ratio and cell viability. These findings show that Bcl-x(s) expression is an important mediator of MDA-7/IL-24-induced cytotoxicity requiring the SRC/PKCδ signaling axis in NSCLC cells. PMID:27519412

Intron-mediated alternative splicing of WOOD-ASSOCIATED NAC TRANSCRIPTION FACTOR1B regulates cell wall thickening during fiber development in Populus species.

PubMed

Zhao, Yunjun; Sun, Jiayan; Xu, Peng; Zhang, Rui; Li, Laigeng

2014-02-01

Alternative splicing is an important mechanism involved in regulating the development of multicellular organisms. Although many genes in plants undergo alternative splicing, little is understood of its significance in regulating plant growth and development. In this study, alternative splicing of black cottonwood (Populus trichocarpa) wood-associated NAC domain transcription factor (PtrWNDs), PtrWND1B, is shown to occur exclusively in secondary xylem fiber cells. PtrWND1B is expressed with a normal short-transcript PtrWND1B-s as well as its alternative long-transcript PtrWND1B-l. The intron 2 structure of the PtrWND1B gene was identified as a critical sequence that causes PtrWND1B alternative splicing. Suppression of PtrWND1B expression specifically inhibited fiber cell wall thickening. The two PtrWND1B isoforms play antagonistic roles in regulating cell wall thickening during fiber cell differentiation in Populus spp. PtrWND1B-s overexpression enhanced fiber cell wall thickening, while overexpression of PtrWND1B-l repressed fiber cell wall thickening. Alternative splicing may enable more specific regulation of processes such as fiber cell wall thickening during wood formation.

Intron-Mediated Alternative Splicing of WOOD-ASSOCIATED NAC TRANSCRIPTION FACTOR1B Regulates Cell Wall Thickening during Fiber Development in Populus Species1[W

PubMed Central

Zhao, Yunjun; Sun, Jiayan; Xu, Peng; Zhang, Rui; Li, Laigeng

2014-01-01

Alternative splicing is an important mechanism involved in regulating the development of multicellular organisms. Although many genes in plants undergo alternative splicing, little is understood of its significance in regulating plant growth and development. In this study, alternative splicing of black cottonwood (Populus trichocarpa) wood-associated NAC domain transcription factor (PtrWNDs), PtrWND1B, is shown to occur exclusively in secondary xylem fiber cells. PtrWND1B is expressed with a normal short-transcript PtrWND1B-s as well as its alternative long-transcript PtrWND1B-l. The intron 2 structure of the PtrWND1B gene was identified as a critical sequence that causes PtrWND1B alternative splicing. Suppression of PtrWND1B expression specifically inhibited fiber cell wall thickening. The two PtrWND1B isoforms play antagonistic roles in regulating cell wall thickening during fiber cell differentiation in Populus spp. PtrWND1B-s overexpression enhanced fiber cell wall thickening, while overexpression of PtrWND1B-l repressed fiber cell wall thickening. Alternative splicing may enable more specific regulation of processes such as fiber cell wall thickening during wood formation. PMID:24394777

Megakaryocytic differentiation induced by constitutive activation of mitogen-activated protein kinase kinase.

PubMed Central

Whalen, A M; Galasinski, S C; Shapiro, P S; Nahreini, T S; Ahn, N G

1997-01-01

The K562 erythroleukemia cell line was used to study the molecular mechanisms regulating lineage commitment of hematopoietic stem cells. Phorbol esters, which initiate megakaryocyte differentiation in this cell line, caused a rapid increase in extracellular-signal-regulated kinase (ERK), which remained elevated for 2 h and returned to near-basal levels by 24 h. In the absence of extracellular stimuli, ERK could be activated by expression of constitutively active mutants of mitogen-activated protein (MAP) kinase kinase (MKK), resulting in cell adhesion and spreading, increased cell size, inhibition of cell growth, and induction of the platelet-specific integrin alphaIIb beta3, all hallmarks of megakaryocytic differentiation. In contrast, expression of wild-type MKK had little effect. In addition, constitutively active MKK suppressed the expression of an erythroid marker, alpha-globin, indicating the ability to suppress cellular responses necessary for alternative cell lineages. The MKK inhibitor PD98059 blocked MKK/ERK activation and cellular responses to phorbol ester, demonstrating that activation of MKK is necessary and sufficient to induce a differentiation program along the megakaryocyte lineage. Thus, the MAP kinase cascade, which promotes cell growth and proliferation in many cell types, instead inhibits cell proliferation and initiates lineage-specific differentiation in K562 cells, establishing a model system to investigate the mechanisms by which this signal transduction pathway specifies cell fate and developmental processes. PMID:9121442

Megakaryocytic differentiation induced by constitutive activation of mitogen-activated protein kinase kinase.

PubMed

Whalen, A M; Galasinski, S C; Shapiro, P S; Nahreini, T S; Ahn, N G

1997-04-01

The K562 erythroleukemia cell line was used to study the molecular mechanisms regulating lineage commitment of hematopoietic stem cells. Phorbol esters, which initiate megakaryocyte differentiation in this cell line, caused a rapid increase in extracellular-signal-regulated kinase (ERK), which remained elevated for 2 h and returned to near-basal levels by 24 h. In the absence of extracellular stimuli, ERK could be activated by expression of constitutively active mutants of mitogen-activated protein (MAP) kinase kinase (MKK), resulting in cell adhesion and spreading, increased cell size, inhibition of cell growth, and induction of the platelet-specific integrin alphaIIb beta3, all hallmarks of megakaryocytic differentiation. In contrast, expression of wild-type MKK had little effect. In addition, constitutively active MKK suppressed the expression of an erythroid marker, alpha-globin, indicating the ability to suppress cellular responses necessary for alternative cell lineages. The MKK inhibitor PD98059 blocked MKK/ERK activation and cellular responses to phorbol ester, demonstrating that activation of MKK is necessary and sufficient to induce a differentiation program along the megakaryocyte lineage. Thus, the MAP kinase cascade, which promotes cell growth and proliferation in many cell types, instead inhibits cell proliferation and initiates lineage-specific differentiation in K562 cells, establishing a model system to investigate the mechanisms by which this signal transduction pathway specifies cell fate and developmental processes.

Comprehensive analysis of lncRNAs microarray profile and mRNA-lncRNA co-expression in oncogenic HPV-positive cervical cancer cell lines.

PubMed

Yang, LingYun; Yi, Ke; Wang, HongJing; Zhao, YiQi; Xi, MingRong

2016-08-02

Long non-coding RNAs are emerging to be novel regulators in gene expression. In current study, lncRNAs microarray and lncRNA-mRNA co-expression analysis were performed to explore the alternation and function of lncRNAs in cervical cancer cells. We identified that 4750 lncRNAs (15.52%) were differentially expressed in SiHa (HPV-16 positive) (2127 up-regulated and 2623 down-regulated) compared with C-33A (HPV negative), while 5026 lncRNAs (16.43%) were differentially expressed in HeLa (HPV-18 positive) (2218 up-regulated and 2808 down-regulated) respectively. There were 5008 mRNAs differentially expressed in SiHa and 4993 in HeLa, which were all cataloged by GO terms and KEGG pathway. With the help of mRNA-lncRNA co-expression network, we found that ENST00000503812 was significantly negative correlated with RAD51B and IL-28A expression in SiHa, while ENST00000420168, ENST00000564977 and TCONS_00010232 had significant correlation with FOXQ1 and CASP3 expression in HeLa. Up-regulation of ENST00000503812 may inhibit RAD51B and IL-28A expression and result in deficiency of DNA repair pathway and immune responses in HPV-16 positive cervical cancer cell. Up-regulation of ENST00000420168, ENST00000564977 and down-regulation of TCONS_00010232 might stimulate FOXQ1 expression and suppress CASP3 expression in HPV-18 positive cervical cancer cell, which lead to HPV-induced proliferation and deficiency in apoptosis. These results indicate that changes of lncRNAs and related mRNAs might impact on several cellular pathways and involve in HPV-induced proliferation, which enriches our understanding of lncRNAs and coding transcripts anticipated in HPV oncogenesis of cervical cancer.

Methylglyoxal-bis-guanylhydrazone inhibits osteopontin expression and differentiation in cultured human monocytes.

PubMed

Jin, Xia; Xu, Hua; McGrath, Michael S

2018-01-01

Monocyte activation and polarization play essential roles in many chronic inflammatory diseases. An imbalance of M1 and M2 macrophage activation (pro-inflammatory and alternatively activated, respectively) is believed to be a key aspect in the etiology of these diseases, thus a therapeutic approach that regulates macrophage activation could be of broad clinical relevance. Methylglyoxal-bis-guanylhydrazone (MGBG), a regulator of polyamine metabolism, has recently been shown to be concentrated in monocytes and macrophages, and interfere with HIV integration into the DNA of these cells in vitro. RNA expression analysis of monocytes from HIV+ and control donors with or without MGBG treatment revealed the only gene to be consistently down regulated by MGBG to be osteopontin (OPN). The elevated expression of this pro-inflammatory cytokine and monocyte chemoattractant is associated with various chronic inflammatory diseases. We demonstrate that MGBG is a potent inhibitor of secreted OPN (sOPN) in cultured monocytes with 50% inhibition achieved at 0.1 μM of the drug. Furthermore, inhibition of OPN RNA transcription in monocyte cultures occurs at similar concentrations of the drug. During differentiation of monocytes into macrophages in vitro, monocytes express cell surface CD16 and the cells undergo limited DNA synthesis as measured by uptake of BrdU. MGBG inhibited both activities at similar doses to those regulating OPN expression. In addition, monocyte treatment with MGBG inhibited differentiation into both M1 and M2 classes of macrophages at non-toxic doses. The inhibition of differentiation and anti-OPN effects of MGBG were specific for monocytes in that differentiated macrophages were nearly resistant to MGBG activities. Thus MGBG may have potential therapeutic utility in reducing or normalizing OPN levels and regulating monocyte activation in diseases that involve chronic inflammation.

β-Globin-Expressing Definitive Erythroid Progenitor Cells Generated from Embryonic and Induced Pluripotent Stem Cell-Derived Sacs.

PubMed

Fujita, Atsushi; Uchida, Naoya; Haro-Mora, Juan J; Winkler, Thomas; Tisdale, John

2016-06-01

Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent a potential alternative source for red blood cell transfusion. However, when using traditional methods with embryoid bodies, ES cell-derived erythroid cells predominantly express embryonic type ɛ-globin, with lesser fetal type γ-globin and very little adult type β-globin. Furthermore, no β-globin expression is detected in iPS cell-derived erythroid cells. ES cell-derived sacs (ES sacs) have been recently used to generate functional platelets. Due to its unique structure, we hypothesized that ES sacs serve as hemangioblast-like progenitors capable to generate definitive erythroid cells that express β-globin. With our ES sac-derived erythroid differentiation protocol, we obtained ∼120 erythroid cells per single ES cell. Both primitive (ɛ-globin expressing) and definitive (γ- and β-globin expressing) erythroid cells were generated from not only ES cells but also iPS cells. Primitive erythropoiesis is gradually switched to definitive erythropoiesis during prolonged ES sac maturation, concurrent with the emergence of hematopoietic progenitor cells. Primitive and definitive erythroid progenitor cells were selected on the basis of glycophorin A or CD34 expression from cells within the ES sacs before erythroid differentiation. This selection and differentiation strategy represents an important step toward the development of in vitro erythroid cell production systems from pluripotent stem cells. Further optimization to improve expansion should be required for clinical application. Stem Cells 2016;34:1541-1552. © 2016 AlphaMed Press.

Non-cytotoxic differentiation treatment of renal cell cancer

PubMed Central

Negrotto, Soledad; Hu, Zhenbo; Alcazar, Oscar; Ng, Kwok Peng; Triozzi, Pierre; Lindner, Daniel; Rini, Brian; Saunthararajah, Yogen

2013-01-01

Current drug therapy for metastatic renal cell cancer (RCC) results in temporary disease control but not cure, necessitating continued investigation into alternative mechanistic approaches. Drugs that inhibit chromatin-modifying enzymes involved in transcription repression (chromatin-relaxing drugs) could have a role, by inducing apoptosis, and/or through differentiation pathways. At low doses, the cytosine analogue decitabine can be used to deplete DNA methyl-transferase 1 (DNMT1), modify chromatin and alter differentiation without causing apoptosis (cytotoxicity). Non-cytotoxic regimens of decitabine were evaluated for in vitro and in vivo efficacy against RCC cell lines, including a p53 mutated RCC cell line developed from a patient with treatment refractory metastatic RCC. The cell-division permissive mechanism of action, absence of early apoptosis or DNA damage, increase in expression of HNF4α (a key driver associated with the mesenchymal to epithelial transition), decrease in mesenchymal marker expression, increase in epithelial marker expression, and late increase in cyclin dependent kinase inhibitor CDKN1B (p27) protein, was consistent with differentiation-mediated cell cycle exit. In vivo blood counts and animal weights were consistent with minimal toxicity of therapy. The distinctive mechanism of action of a dose and schedule of decitabine designed for non-cytotoxic depletion of DNMT1 suggests a potential role in treating RCC. PMID:21303982

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

PubMed Central

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

2017-01-01

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

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

PubMed

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

2017-02-28

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

Lipidomic profiling of patient-specific iPSC-derived hepatocyte-like cells

PubMed Central

Viiri, Leena E.; Vihervaara, Terhi; Koistinen, Kaisa M.; Hilvo, Mika; Ekroos, Kim; Käkelä, Reijo; Aalto-Setälä, Katriina

2017-01-01

ABSTRACT Hepatocyte-like cells (HLCs) differentiated from human induced pluripotent stem cells (iPSCs) offer an alternative model to primary human hepatocytes to study lipid aberrations. However, the detailed lipid profile of HLCs is yet unknown. In the current study, functional HLCs were differentiated from iPSCs generated from dermal fibroblasts of three individuals by a three-step protocol through the definitive endoderm (DE) stage. In parallel, detailed lipidomic analyses as well as gene expression profiling of a set of lipid-metabolism-related genes were performed during the entire differentiation process from iPSCs to HLCs. Additionally, fatty acid (FA) composition of the cell culture media at different stages was determined. Our results show that major alterations in the molecular species of lipids occurring during DE and early hepatic differentiation stages mainly mirror the quality and quantity of the FAs supplied in culture medium at each stage. Polyunsaturated phospholipids and sphingolipids with a very long FA were produced in the cells at a later stage of differentiation. This work uncovers the previously unknown lipid composition of iPSC-HLCs and its alterations during the differentiation in conjunction with the expression of key lipid-associated genes. Together with biochemical, functional and gene expression measurements, the lipidomic analyses allowed us to improve our understanding of the concerted influence of the exogenous metabolite supply and cellular biosynthesis essential for iPSC-HLC differentiation and function. Importantly, the study describes in detail a cell model that can be applied in exploring, for example, the lipid metabolism involved in the development of fatty liver disease or atherosclerosis. PMID:28733363

Gene expression profile during testicular development in patients with SRY-negative 46,XX testicular disorder of sex development.

PubMed

Mizuno, Kentaro; Kojima, Yoshiyuki; Kamisawa, Hideyuki; Moritoki, Yoshinobu; Nishio, Hidenori; Kohri, Kenjiro; Hayashi, Yutaro

2013-12-01

To elucidate alternative pathways in testicular development, we attempted to clarify the genetic characteristics of SRY-negative XX testes. We previously reported 5 cases of SRY-negative 46,XX testicular disorders of sex development and demonstrated that coordinated expression of genes such as SOX9, SOX3, and DAX1 was associated with testicular development. We performed a case-control study between the aforementioned boy with 46,XX testicular disorders of sex development and an age-matched patient with hydrocele testis (46,XY). During their consecutive surgeries, testicular biopsy specimens were obtained. Genes with differential expression compared with XY testis were identified using polymerase chain reaction (PCR)-based subtractive hybridization and sequencing. For validation of differential gene expression, real-time RT-PCR was performed using gene-specific primers. The distribution of candidate proteins in the testicular tissue was clarified by immunohistochemistry in human and rodent specimens. Moreover, in vitro inhibitory assays were performed. We identified 13 upregulated and 7 downregulated genes in XX testis. Among the candidate genes, we focused on ROCK1 (Rho-associated, coiled-coil protein kinase 1) in the upregulated gene group, because high expression in XX testis was validated by real-time RT-PCR. ROCK1 protein was detected in germ cells, Leydig cells, and Sertoli cells by immunohistochemistry. Moreover, the addition of specific ROCK1 inhibitor to Sertoli cells decreased SOX9 gene expression. On the basis of in vitro inhibitory assay, it is suggested that ROCK1 phosphorylates and activates SOX9 in Sertoli cells. Testes formation might be initiated by an alternative signaling pathway attributed to ROCK1, not SRY, activation in XX testes. Copyright © 2013 Elsevier Inc. All rights reserved.

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

Galloway, Chad A.; Smith, Harold C., E-mail: harold.smith@rochester.edu

Apolipoprotein B mRNA is edited at cytidine 6666 in the enterocytes lining the small intestine of all mammals; converting a CAA codon to a UAA stop codon. The conversion is {approx}80% efficient in this tissue and leads to the expression of the truncated protein, ApoB48, essential for secretion of dietary lipid as chylomicrons. Caco-2 cell raft cultures have been used as an in vitro model for the induction of editing activity during human small intestinal cell differentiation. This induction of apoB mRNA editing has been ascribed to the expression of APOBEC-1. In agreement our data demonstrated differentiation-dependent induction of expressionmore » of the editing enzyme APOBEC-1 and in addition we show alternative splicing of the essential auxiliary factor ACF. However, transfection of these editing factors in undifferentiated proliferating Caco-2 cells was not sufficient to induce robust apoB mRNA editing activity. Only differentiation of Caco-2 cells could induce more physiological like levels of apoB mRNA editing. The data suggested that additional regulatory mechanism(s) were induced by differentiation that controlled the functional activity of editing factors.« less

Partial wave analysis for folded differential cross sections

NASA Astrophysics Data System (ADS)

Machacek, J. R.; McEachran, R. P.

2018-03-01

The value of modified effective range theory (MERT) and the connection between differential cross sections and phase shifts in low-energy electron scattering has long been recognized. Recent experimental techniques involving magnetically confined beams have introduced the concept of folded differential cross sections (FDCS) where the forward (θ ≤ π/2) and backward scattered (θ ≥ π/2) projectiles are unresolved, that is the value measured at the angle θ is the sum of the signal for particles scattered into the angles θ and π - θ. We have developed an alternative approach to MERT in order to analyse low-energy folded differential cross sections for positrons and electrons. This results in a simplified expression for the FDCS when it is expressed in terms of partial waves and thereby enables one to extract the first few phase shifts from a fit to an experimental FDCS at low energies. Thus, this method predicts forward and backward angle scattering (0 to π) using only experimental FDCS data and can be used to determine the total elastic cross section solely from experimental results at low-energy, which are limited in angular range.

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

PubMed

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

2011-01-01

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

In Vitro Hepatic Trans-Differentiation of Human Mesenchymal Stem Cells Using Sera from Congestive/Ischemic Liver during Cardiac Failure

PubMed Central

Bishi, Dillip Kumar; Mathapati, Santosh; Cherian, Kotturathu Mammen; Guhathakurta, Soma; Verma, Rama Shanker

2014-01-01

Cellular therapy for end-stage liver failures using human mesenchymal stem cells (hMSCs)-derived hepatocytes is a potential alternative to liver transplantation. Hepatic trans-differentiation of hMSCs is routinely accomplished by induction with commercially available recombinant growth factors, which is of limited clinical applications. In the present study, we have evaluated the potential of sera from cardiac-failure-associated congestive/ischemic liver patients for hepatic trans-differentiation of hMSCs. Results from such experiments were confirmed through morphological changes and expression of hepatocyte-specific markers at molecular and cellular level. Furthermore, the process of mesenchymal-to-epithelial transition during hepatic trans-differentiation of hMSCs was confirmed by elevated expression of E-Cadherin and down-regulation of Snail. The functionality of hMSCs-derived hepatocytes was validated by various liver function tests such as albumin synthesis, urea release, glycogen accumulation and presence of a drug inducible cytochrome P450 system. Based on these findings, we conclude that sera from congestive/ischemic liver during cardiac failure support a liver specific microenvironment for effective hepatic trans-differentiation of hMSCs in vitro. PMID:24642599

Neural differentiation promoted by truncated trkC receptors in collaboration with p75(NTR).

PubMed

Hapner, S J; Boeshore, K L; Large, T H; Lefcort, F

1998-09-01

trkC receptors, which serve critical functions during the development of the nervous system, are alternatively spliced to yield isoforms containing the catalytic tyrosine kinase domain (TK+) and truncated isoforms which lack this domain (TK-). To test for potential differences in their roles during early stages of neural development, TK+ and TK- isoforms were ectopically expressed in cultures of neural crest, the stem cell population that gives rise to the vast majority of the peripheral nervous system. NT-3 activation of ectopically expressed trkC TK+ receptors promoted both proliferation of neural crest cells and neuronal differentiation. Strikingly, the trkC TK- isoform was significantly more effective at promoting neuronal differentiation, but had no effect on proliferation. Furthermore, the trkC TK- response was dependent on a conserved receptor cytoplasmic domain and required the participation of the p75(NTR) neurotrophin receptor. Antibody-mediated receptor dimerization of TK+ receptors, but not TK- receptors, was sufficient to stimulate differentiation. These data identify a phenotypic response to activation of the trkC TK- receptor and demonstrate a functional interaction with p75(NTR), indicating there may be multiple trkC receptor-mediated systems guiding neuronal differentiation. Copyright 1998 Academic Press.

Transcriptome analysis of adiposity in domestic ducks by transcriptomic comparison with their wild counterparts.

PubMed

Chen, L; Luo, J; Li, J X; Li, J J; Wang, D Q; Tian, Y; Lu, L Z

2015-06-01

Excessive adiposity is a major problem in the duck industry, but its molecular mechanisms remain unknown. Genetic comparisons between domestic and wild animals have contributed to the exploration of genetic mechanisms responsible for many phenotypic traits. Significant differences in body fat mass have been detected between domestic and wild ducks. In this study, we used the Peking duck and Anas platyrhynchos as the domestic breed and wild counterpart respectively and performed a transcriptomic comparison of abdominal fat between the two breeds to comprehensively analyze the transcriptome basis of adiposity in ducks. We obtained approximately 350 million clean reads; assembled 61 250 transcripts, including 23 699 novel ones; and identified alternative 5' splice sites, alternative 3' splice sites, skipped exons and retained intron as the main alternative splicing events. A differential expression analysis between the two breeds showed that 753 genes exhibited differential expression. In Peking ducks, some lipid metabolism-related genes (IGF2, FABP5, BMP7, etc.) and oncogenes (RRM2, AURKA, CYR61, etc.) were upregulated, whereas genes related to tumor suppression and immunity (TNFRSF19, TNFAIP6, IGSF21, NCF1, etc.) were downregulated, suggesting adiposity might closely associate with tumorigenesis in ducks. Furthermore, 280 576 single-nucleotide variations were found differentiated between the two breeds, including 8641 non-synonymous ones, and some of the non-synonymous ones were found enriched in genes involved in lipid-associated and immune-associated pathways, suggesting abdominal fat of the duck undertakes both a metabolic function and immune-related function. These datasets enlarge our genetic information of ducks and provide valuable resources for analyzing mechanisms underlying adiposity in ducks. © 2015 Stichting International Foundation for Animal Genetics.

Genome-wide DNA methylation reprogramming in response to inorganic arsenic links inhibition of CTCF binding, DNMT expression and cellular transformation

NASA Astrophysics Data System (ADS)

Rea, Matthew; Eckstein, Meredith; Eleazer, Rebekah; Smith, Caroline; Fondufe-Mittendorf, Yvonne N.

2017-02-01

Chronic low dose inorganic arsenic (iAs) exposure leads to changes in gene expression and epithelial-to-mesenchymal transformation. During this transformation, cells adopt a fibroblast-like phenotype accompanied by profound gene expression changes. While many mechanisms have been implicated in this transformation, studies that focus on the role of epigenetic alterations in this process are just emerging. DNA methylation controls gene expression in physiologic and pathologic states. Several studies show alterations in DNA methylation patterns in iAs-mediated pathogenesis, but these studies focused on single genes. We present a comprehensive genome-wide DNA methylation analysis using methyl-sequencing to measure changes between normal and iAs-transformed cells. Additionally, these differential methylation changes correlated positively with changes in gene expression and alternative splicing. Interestingly, most of these differentially methylated genes function in cell adhesion and communication pathways. To gain insight into how genomic DNA methylation patterns are regulated during iAs-mediated carcinogenesis, we show that iAs probably targets CTCF binding at the promoter of DNA methyltransferases, regulating their expression. These findings reveal how CTCF binding regulates DNA methyltransferase to reprogram the methylome in response to an environmental toxin.

Genome-wide DNA methylation reprogramming in response to inorganic arsenic links inhibition of CTCF binding, DNMT expression and cellular transformation

PubMed Central

Rea, Matthew; Eckstein, Meredith; Eleazer, Rebekah; Smith, Caroline; Fondufe-Mittendorf , Yvonne N.

2017-01-01

Chronic low dose inorganic arsenic (iAs) exposure leads to changes in gene expression and epithelial-to-mesenchymal transformation. During this transformation, cells adopt a fibroblast-like phenotype accompanied by profound gene expression changes. While many mechanisms have been implicated in this transformation, studies that focus on the role of epigenetic alterations in this process are just emerging. DNA methylation controls gene expression in physiologic and pathologic states. Several studies show alterations in DNA methylation patterns in iAs-mediated pathogenesis, but these studies focused on single genes. We present a comprehensive genome-wide DNA methylation analysis using methyl-sequencing to measure changes between normal and iAs-transformed cells. Additionally, these differential methylation changes correlated positively with changes in gene expression and alternative splicing. Interestingly, most of these differentially methylated genes function in cell adhesion and communication pathways. To gain insight into how genomic DNA methylation patterns are regulated during iAs-mediated carcinogenesis, we show that iAs probably targets CTCF binding at the promoter of DNA methyltransferases, regulating their expression. These findings reveal how CTCF binding regulates DNA methyltransferase to reprogram the methylome in response to an environmental toxin. PMID:28150704

Stage-specific differential gene expression profiling and functional network analysis during morphogenesis of diphyodont dentition in miniature pigs, Sus Scrofa

PubMed Central

2014-01-01

Background Our current knowledge of tooth development derives mainly from studies in mice, which have only one set of non-replaced teeth, compared with the diphyodont dentition in humans. The miniature pig is also diphyodont, making it a valuable alternative model for understanding human tooth development and replacement. However, little is known about gene expression and function during swine odontogenesis. The goal of this study is to undertake the survey of differential gene expression profiling and functional network analysis during morphogenesis of diphyodont dentition in miniature pigs. The identification of genes related to diphyodont development should lead to a better understanding of morphogenetic patterns and the mechanisms of diphyodont replacement in large animal models and humans. Results The temporal gene expression profiles during early diphyodont development in miniature pigs were detected with the Affymetrix Porcine GeneChip. The gene expression data were further evaluated by ANOVA as well as pathway and STC analyses. A total of 2,053 genes were detected with differential expression. Several signal pathways and 151 genes were then identified through the construction of pathway and signal networks. Conclusions The gene expression profiles indicated that spatio-temporal down-regulation patterns of gene expression were predominant; while, both dynamic activation and inhibition of pathways occurred during the morphogenesis of diphyodont dentition. Our study offers a mechanistic framework for understanding dynamic gene regulation of early diphyodont development and provides a molecular basis for studying teeth development, replacement, and regeneration in miniature pigs. PMID:24498892

Global phenotypic characterisation of human platelet lysate expanded MSCs by high-throughput flow cytometry.

PubMed

Reis, Monica; McDonald, David; Nicholson, Lindsay; Godthardt, Kathrin; Knobel, Sebastian; Dickinson, Anne M; Filby, Andrew; Wang, Xiao-Nong

2018-03-02

Mesenchymal stromal cells (MSCs) are a promising cell source to develop cell therapy for many diseases. Human platelet lysate (PLT) is increasingly used as an alternative to foetal calf serum (FCS) for clinical-scale MSC production. To date, the global surface protein expression of PLT-expended MSCs (MSC-PLT) is not known. To investigate this, paired MSC-PLT and MSC-FCS were analysed in parallel using high-throughput flow cytometry for the expression of 356 cell surface proteins. MSC-PLT showed differential surface protein expression compared to their MSC-FCS counterpart. Higher percentage of positive cells was observed in MSC-PLT for 48 surface proteins, of which 13 were significantly enriched on MSC-PLT. This finding was validated using multiparameter flow cytometry and further confirmed by quantitative staining intensity analysis. The enriched surface proteins are relevant to increased proliferation and migration capacity, as well as enhanced chondrogenic and osteogenic differentiation properties. In silico network analysis revealed that these enriched surface proteins are involved in three distinct networks that are associated with inflammatory responses, carbohydrate metabolism and cellular motility. This is the first study reporting differential cell surface protein expression between MSC-PLT and MSC-FSC. Further studies are required to uncover the impact of those enriched proteins on biological functions of MSC-PLT.

Testis development, fertility, and survival in Ethanolamine kinase 2-deficient mice.

PubMed

Gustin, Sonja E; Western, Patrick S; McClive, Peter J; Harley, Vincent R; Koopman, Peter A; Sinclair, Andrew H

2008-12-01

Ethanolamine kinase 2 (Eki2) was previously isolated from a differential expression screen designed to identify candidate genes involved in testis development and differentiation. In mouse, Eki2 is specifically up-regulated in Sertoli cells of the developing testis at the time of sex determination. Based on this expression profile, Eki2 was considered a good candidate testis-determining gene. To investigate a possible role of Eki2 in testis development, we have generated a mouse with targeted disruption of the Eki2 gene by using an EGFP replacement strategy. No abnormalities were detected in the Eki2-deficient mice with regard to embryonic and adult testis morphology, differentiation, function, or fertility. Furthermore, no significant differences were observed in litter sizes, pup mortality rates, or distribution of the sexes among the offspring. Ethanolamine kinases are involved in the biosynthesis of phosphatidylethanolamine, a major membrane phospholipid. Expression analysis indicates that the absence of an apparent phenotype in the Eki2-deficient mice may be due to compensation by Eki2-family members or the activation of an alternative pathway to generate phosphatidylethanolamine. Expression of EGFP in this mouse model enabled the isolation of gonad cell populations, providing a useful resource from which to obtain relatively pure early steroidogenic cells for further studies.

Alternative Polyadenylation in Human Diseases

PubMed Central

Chang, Jae-Woong; Yeh, Hsin-Sung

2017-01-01

Varying length of messenger RNA (mRNA) 3′-untranslated region is generated by alternating the usage of polyadenylation sites during pre-mRNA processing. It is prevalent through all eukaryotes and has emerged as a key mechanism for controlling gene expression. Alternative polyadenylation (APA) plays an important role for cell growth, proliferation, and differentiation. In this review, we discuss the functions of APA related with various physiological conditions including cellular metabolism, mRNA processing, and protein diversity in a variety of disease models. We also discuss the molecular mechanisms underlying APA regulation, such as variations in the concentration of mRNA processing factors and RNA-binding proteins, as well as global transcriptome changes under cellular signaling pathway. PMID:29271615

MiR-27b Promotes Muscle Development by Inhibiting MDFI Expression.

PubMed

Hou, Lianjie; Xu, Jian; Jiao, Yiren; Li, Huaqin; Pan, Zhicheng; Duan, Junli; Gu, Ting; Hu, Chingyuan; Wang, Chong

2018-01-01

Skeletal muscle plays an essential role in the body movement. However, injuries to the skeletal muscle are common. Lifelong maintenance of skeletal muscle function largely depends on preserving the regenerative capacity of muscle. Muscle satellite cells proliferation, differentiation, and myoblast fusion play an important role in muscle regeneration after injury. Therefore, understanding of the mechanisms associated with muscle development during muscle regeneration is essential for devising the alternative treatments for muscle injury in the future. Edu staining, qRT-PCR and western blot were used to evaluate the miR-27b effects on pig muscle satellite cells (PSCs) proliferation and differentiation in vitro. Then, we used bioinformatics analysis and dual-luciferase reporter assay to predict and confirm the miR-27b target gene. Finally, we elucidate the target gene function on muscle development in vitro and in vivo through Edu staining, qRT-PCR, western blot, H&E staining and morphological observation. miR-27b inhibits PSCs proliferation and promotes PSCs differentiation. And the miR-27b target gene, MDFI, promotes PSCs proliferation and inhibits PSCs differentiation in vitro. Furthermore, interfering MDFI expression promotes mice muscle regeneration after injury. our results conclude that miR-27b promotes PSCs myogenesis by targeting MDFI. These results expand our understanding of muscle development mechanism in which miRNAs and genes work collaboratively in regulating skeletal muscle development. Furthermore, this finding has implications for obtaining the alternative treatments for patients with the muscle injury. © 2018 The Author(s). Published by S. Karger AG, Basel.

Genome-wide analysis of alternative splicing in medulloblastoma identifies splicing patterns characteristic of normal cerebellar development

PubMed Central

Menghi, Francesca; Jacques, Thomas S.; Barenco, Martino; Schwalbe, Ed C.; Clifford, Steven C.; Hubank, Mike; Ham, Jonathan

2011-01-01

Alternative splicing is an important mechanism for the generation of protein diversity at a post-transcriptional level. Modifications in the splicing patterns of several genes have been shown to contribute to the malignant transformation of different tissue types. In this study, we used the Affymetrix Exon arrays to investigate patterns of differential splicing between paediatric medulloblastomas and normal cerebellum on a genome-wide scale. Of the 1262 genes identified as potentially generating tumour-associated splice forms, we selected 14 examples of differential splicing of known cassette exons and successfully validated 11 of them by RT-PCR. The pattern of differential splicing of three validated events was characteristic for the molecular subset of Sonic Hedgehog (Shh)-driven medulloblastomas, suggesting that their unique gene signature includes the expression of distinctive transcript variants. Generally, we observed that tumour and normal fetal cerebellar samples shared significantly lower exon inclusion rates compared to normal adult cerebellum. We investigated whether tumour-associated splice forms were expressed in primary cultures of Shh-dependent mouse cerebellar granule cell precursors (GCPs) and found that Shh caused a decrease in the cassette exon inclusion rate of five out of the seven tested genes. Furthermore, we observed a significant increase in exon inclusion between post-natal days 7 and 14 of mouse cerebellar development, at the time when GCPs mature into post-mitotic neurons. We conclude that inappropriate splicing frequently occurs in human medulloblastomas and may be linked to the activation of developmental signalling pathways and a failure of cerebellar precursor cells to differentiate. PMID:21248070

Effect of autogenous and fresh-frozen bone grafts on osteoblast differentiation.

PubMed

Ferraz, E P; Xavier, S P; Azevedo, F G; de Oliveira, F S; Beloti, M M; Rosa, A L

2015-01-01

Fresh-frozen bone allograft (FFBA) is an alternative to autogenous bone (AB) for reconstructing maxillary bone. Despite the promising clinical results, cell responses to FFBA and AB were not evaluated. Thus, our aim was to compare cells harvested from maxillary reconstructed sites with either AB or FFBA in terms of osteoblast differentiation and to evaluate the effect of culturing cells in contact with FFBA. Cells harvested from three patients submitted to bilateral maxillary reconstruction with AB and FFBA were cultured to evaluate: proliferation, alkaline phosphatase activity, extracellular matrix mineralization and gene expression of osteoblastic markers. The effect of FFBA on osteoblast differentiation was studied by culturing cells harvested from AB in contact with FFBA and evaluating the same parameters. Data were compared using either two-way ANOVA followed by Tukey-b test or Student's t test (p≤0.05). Cell proliferation was higher in cultures from AB grafted sites and extracellular matrix mineralization was higher in cultures derived from FFBA grafted sites. The gene expression of alkaline phosphatase, RUNX2, bone sialoprotein and osteocalcin was higher in cells derived from FFBA compared with cells from AB grafted sites. However, the exposure of cells derived from AB to FFBA particles did not have any remarkable effect on osteoblast differentiation. These results indicate the higher osteogenic activity of cells derived from FFBA compared with AB reconstructed sites, offering an explanation at cellular level of why FFBA could be a suitable alternative to AB for reconstructing maxillary bone defects. Copyright © 2014 Elsevier Ltd. All rights reserved.

N-acetylaspartate (NAA) and N-acetylaspartylglutamate (NAAG) promote growth and inhibit differentiation of glioma stem-like cells.

PubMed

Long, Patrick M; Moffett, John R; Namboodiri, Aryan M A; Viapiano, Mariano S; Lawler, Sean E; Jaworski, Diane M

2013-09-06

Metabolic reprogramming is a pathological feature of cancer and a driver of tumor cell transformation. N-Acetylaspartate (NAA) is one of the most abundant amino acid derivatives in the brain and serves as a source of metabolic acetate for oligodendrocyte myelination and protein/histone acetylation or a precursor for the synthesis of the neurotransmitter N-acetylaspartylglutamate (NAAG). NAA and NAAG as well as aspartoacylase (ASPA), the enzyme responsible for NAA degradation, are significantly reduced in glioma tumors, suggesting a possible role for decreased acetate metabolism in tumorigenesis. This study sought to examine the effects of NAA and NAAG on primary tumor-derived glioma stem-like cells (GSCs) from oligodendroglioma as well as proneural and mesenchymal glioblastoma, relative to oligodendrocyte progenitor cells (Oli-Neu). Although the NAA dicarboxylate transporter NaDC3 is primarily thought to be expressed by astrocytes, all cell lines expressed NaDC3 and, thus, are capable of NAA up-take. Treatment with NAA or NAAG significantly increased GSC growth and suppressed differentiation of Oli-Neu cells and proneural GSCs. Interestingly, ASPA was expressed in both the cytosol and nuclei of GSCs and exhibited greatest nuclear immunoreactivity in differentiation-resistant GSCs. Both NAA and NAAG elicited the expression of a novel immunoreactive ASPA species in select GSC nuclei, suggesting differential ASPA regulation in response to these metabolites. Therefore, this study highlights a potential role for nuclear ASPA expression in GSC malignancy and suggests that the use of NAA or NAAG is not an appropriate therapeutic approach to increase acetate bioavailability in glioma. Thus, an alternative acetate source is required.

Practical applications of insects' sexual development for pest control.

PubMed

Koukidou, M; Alphey, L

2014-01-01

Elucidation of the sex differentiation pathway in insects offers an opportunity to understand key aspects of evolutionary developmental biology. In addition, it provides the understanding necessary to manipulate insects in order to develop new synthetic genetics-based tools for the control of pest insects. Considerable progress has been made in this, especially in improvements to the sterile insect technique (SIT). Large scale sex separation is considered highly desirable or essential for most SIT targets. This separation can be provided by genetic methods based on sex-specific gene expression. Investigation of sex determination by many groups has provided molecular components and methods for this. Though the primary sex determination signal varies considerably, key regulatory genes and mechanisms remain surprisingly similar. In most cases studied so far, a primary signal is transmitted to a basal gene at the bottom of the hierarchy (dsx) through an alternative splicing cascade; dsx is itself differentially spliced in males and females. A sex-specific alternative splicing system therefore offers an attractive route to achieve female-specific expression. Experience has shown that alternative splicing modules can be developed with cross-species function; modularity and standardisation and re-use of parts are key principles of synthetic biology. Both female-killing and sex reversal (XX females to phenotypic males) can in principle also be used as efficient alternatives to sterilisation in SIT-like methods. Sexual maturity is yet another area where understanding of sexual development may be applied to insect control programmes. Further detailed understanding of this crucial aspect of insect biology will undoubtedly continue to underpin innovative practical applications. © 2014 S. Karger AG, Basel.

Differential expression of two scribble isoforms during Drosophila embryogenesis.

PubMed

Li, M; Marhold, J; Gatos, A; Török, I; Mechler, B M

2001-10-01

The tumour suppressor gene scribble (scrib) is required for epithelial polarity and growth control in Drosophila. Here, we report the identification and embryonic expression pattern of two Scrib protein isoforms resulting from alternative splicing during scrib transcription. Both proteins are first ubiquitously expressed during early embryogenesis. Then, during morphogenesis each Scrib protein displays a specific pattern of expression in the central and peripheral nervous systems, CNS and PNS, respectively. During germ band extension, the expression of the longer form Scrib1 occurs predominantly in the neuroblasts derived from the neuro-ectoderm and becomes later restricted to CNS neurones as well as to the pole cells in the gonads. By contrast, the shorter form Scrib2 is strongly expressed in the PNS and a subset of CNS neurones.

[Cytokine-mediated regulation of expression of Gfi1 and U2afll4 genes activated by T-cells with different differentiation status in vitro].

PubMed

Yurova, K A; Sokhonevich, N A; Khaziakhmatova, O G; Litvinova, L S

2016-01-01

The dose-dependent effects of cytokines (IL-2, IL-7, and IL-15), which have a common g-chain, on mRNA expression of U2afll4 and GFi1 genes involved in regulation of alternative splicing of the Ptprc gene, have been investigated in vitro using T-lymphocyte cultures with different degrees of differentiation. IL-2, IL-7, and IL-15 caused a similar unidirectional inhibitory effect of various severity on restimulated CD45RO+ T-cells exposed to an antigen-independent activation; they caused a dose-dependent decrease of the U2af1l4 gene expression, and an increase of Gfi1 gene expression. This may suggest formation of active forms of the CD45 receptor, and also limitation of the formation of low-molecular short splice variants of the CD45RO receptor. Under conditions of antigen-independent stimulation of naive CD45RA+-cells rIL-7 and IL-15 exhibited opposite effects on U2af1l4 and Gfi1 gene expression. The increase of IL-7 concentrations in the incubation medium of naive cells was accompanied by a decrease in expression of both genes. IL-15 IL-7 exhibited opposite effects. Cytokines possessing a common g-chain (IL-2, IL-7, and IL-15) prevented antigen-independent differentiation of naive T-cells, by preventing the formation of polyclonal "surrogate" cells. In general, the study of the molecular mechanisms of genetic control determining homeostatic processes of T-cells in response to exposure to antigenic or non-antigenic treatments may be important for construction of a general model of self-maintenance and differentiation of immune cells.

N-Myc Differentially Regulates Expression of MXI1 Isoforms in Neuroblastoma1

PubMed Central

Armstrong, Michael B; Mody, Rajen J; Ellis, D Christian; Hill, Adam B; Erichsen, David A; Wechsler, Daniel S

2013-01-01

Amplification of the MYCN proto-oncogene is associated with a poor prognosis in patients with metastatic neuroblastoma (NB). MYCN encodes the N-Myc protein, a transcriptional regulator that dimerizes with the Max transcription factor, binds to E-box DNA sequences, and regulates genes involved in cell growth and apoptosis. Overexpression of N-Myc leads to transcriptional activation and an increase in NB cell proliferation. Mxi1, a member of the Myc family of transcriptional regulators, also binds to Max. However, Mxi1 is a transcriptional repressor and inhibits proliferation of NB cells, suggesting that Mxi1 functions as an N-Myc antagonist. Our laboratory previously identified Mxi1-0, an alternatively transcribed Mxi1 isoform. Mxi1-0 has properties distinct from those of Mxi1; in contrast to Mxi1, Mxi1-0 is unable to suppress c-Myc-dependent transcription. We now show that Mxi1-0 expression increases in response to MYCN overexpression in NB cells, with a positive correlation between MYCN and MXI1-0 RNA levels. We also show that N-Myc expression differentially regulates the MXI1 and MXI1-0 promoters: Increased MYCN expression suppresses MXI1 promoter activity while enhancing transcription through the MXI1-0 promoter. Finally, induction of Mxi1-0 leads to increased proliferation, whereas expression of Mxi1 inhibits cell growth, indicating differential roles for these two proteins. These data suggest that N-Myc differentially regulates the expression of MXI1 and MXI1-0 and can alter the balance between the two transcription factors. Furthermore, MXI1-0 appears to be a downstream target of MYCN-dependent signaling pathways and may contribute to N-Myc-dependent cell growth and proliferation. PMID:24403858

Identification and Characterization of Alternative Promoters, Transcripts and Protein Isoforms of Zebrafish R2 Gene

PubMed Central

Shang, Hanqiao; Li, Qing; Feng, Guohui; Cui, Zongbin

2011-01-01

Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the de novo synthesis of deoxyribonucleoside triphosphates. Expression of RNR subunits is closely associated with DNA replication and repair. Mammalian RNR M2 subunit (R2) functions exclusively in DNA replication of normal cells due to its S phase-specific expression and late mitotic degradation. Herein, we demonstrate the control of R2 expression through alternative promoters, splicing and polyadenylation sites in zebrafish. Three functional R2 promoters were identified to generate six transcript variants with distinct 5′ termini. The proximal promoter contains a conserved E2F binding site and two CCAAT boxes, which are crucial for the transcription of R2 gene during cell cycle. Activity of the distal promoter can be induced by DNA damage to generate four transcript variants through alternative splicing. In addition, two novel splice variants were found to encode distinct N-truncated R2 isoforms containing residues for enzymatic activity but no KEN box essential for its proteolysis. These two N-truncated R2 isoforms remained in the cytoplasm and were able to interact with RNR M1 subunit (R1). Thus, our results suggest that multilayered mechanisms control the differential expression and function of zebrafish R2 gene during cell cycle and under genotoxic stress. PMID:21887375

Identification and characterization of alternative promoters, transcripts and protein isoforms of zebrafish R2 gene.

PubMed

Shang, Hanqiao; Li, Qing; Feng, Guohui; Cui, Zongbin

2011-01-01

Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the de novo synthesis of deoxyribonucleoside triphosphates. Expression of RNR subunits is closely associated with DNA replication and repair. Mammalian RNR M2 subunit (R2) functions exclusively in DNA replication of normal cells due to its S phase-specific expression and late mitotic degradation. Herein, we demonstrate the control of R2 expression through alternative promoters, splicing and polyadenylation sites in zebrafish. Three functional R2 promoters were identified to generate six transcript variants with distinct 5' termini. The proximal promoter contains a conserved E2F binding site and two CCAAT boxes, which are crucial for the transcription of R2 gene during cell cycle. Activity of the distal promoter can be induced by DNA damage to generate four transcript variants through alternative splicing. In addition, two novel splice variants were found to encode distinct N-truncated R2 isoforms containing residues for enzymatic activity but no KEN box essential for its proteolysis. These two N-truncated R2 isoforms remained in the cytoplasm and were able to interact with RNR M1 subunit (R1). Thus, our results suggest that multilayered mechanisms control the differential expression and function of zebrafish R2 gene during cell cycle and under genotoxic stress.

Alternative Splicing Studies of the Reactive Oxygen Species Gene Network in Populus Reveal Two Isoforms of High-Isoelectric-Point Superoxide Dismutase1[C][W

PubMed Central

Srivastava, Vaibhav; Srivastava, Manoj Kumar; Chibani, Kamel; Nilsson, Robert; Rouhier, Nicolas; Melzer, Michael; Wingsle, Gunnar

2009-01-01

Recent evidence has shown that alternative splicing (AS) is widely involved in the regulation of gene expression, substantially extending the diversity of numerous proteins. In this study, a subset of expressed sequence tags representing members of the reactive oxygen species gene network was selected from the PopulusDB database to investigate AS mechanisms in Populus. Examples of all known types of AS were detected, but intron retention was the most common. Interestingly, the closest Arabidopsis (Arabidopsis thaliana) homologs of half of the AS genes identified in Populus are not reportedly alternatively spliced. Two genes encoding the protein of most interest in our study (high-isoelectric-point superoxide dismutase [hipI-SOD]) have been found in black cottonwood (Populus trichocarpa), designated PthipI-SODC1 and PthipI-SODC2. Analysis of the expressed sequence tag libraries has indicated the presence of two transcripts of PthipI-SODC1 (hipI-SODC1b and hipI-SODC1s). Alignment of these sequences with the PthipI-SODC1 gene showed that hipI-SODC1b was 69 bp longer than hipI-SODC1s due to an AS event involving the use of an alternative donor splice site in the sixth intron. Transcript analysis showed that the splice variant hipI-SODC1b was differentially expressed, being clearly expressed in cambial and xylem, but not phloem, regions. In addition, immunolocalization and mass spectrometric data confirmed the presence of hipI-SOD proteins in vascular tissue. The functionalities of the spliced gene products were assessed by expressing recombinant hipI-SOD proteins and in vitro SOD activity assays. PMID:19176719

Alternative splicing studies of the reactive oxygen species gene network in Populus reveal two isoforms of high-isoelectric-point superoxide dismutase.

PubMed

Srivastava, Vaibhav; Srivastava, Manoj Kumar; Chibani, Kamel; Nilsson, Robert; Rouhier, Nicolas; Melzer, Michael; Wingsle, Gunnar

2009-04-01

Recent evidence has shown that alternative splicing (AS) is widely involved in the regulation of gene expression, substantially extending the diversity of numerous proteins. In this study, a subset of expressed sequence tags representing members of the reactive oxygen species gene network was selected from the PopulusDB database to investigate AS mechanisms in Populus. Examples of all known types of AS were detected, but intron retention was the most common. Interestingly, the closest Arabidopsis (Arabidopsis thaliana) homologs of half of the AS genes identified in Populus are not reportedly alternatively spliced. Two genes encoding the protein of most interest in our study (high-isoelectric-point superoxide dismutase [hipI-SOD]) have been found in black cottonwood (Populus trichocarpa), designated PthipI-SODC1 and PthipI-SODC2. Analysis of the expressed sequence tag libraries has indicated the presence of two transcripts of PthipI-SODC1 (hipI-SODC1b and hipI-SODC1s). Alignment of these sequences with the PthipI-SODC1 gene showed that hipI-SODC1b was 69 bp longer than hipI-SODC1s due to an AS event involving the use of an alternative donor splice site in the sixth intron. Transcript analysis showed that the splice variant hipI-SODC1b was differentially expressed, being clearly expressed in cambial and xylem, but not phloem, regions. In addition, immunolocalization and mass spectrometric data confirmed the presence of hipI-SOD proteins in vascular tissue. The functionalities of the spliced gene products were assessed by expressing recombinant hipI-SOD proteins and in vitro SOD activity assays.

Distinct Properties of Human M-CSF and GM-CSF Monocyte-Derived Macrophages to Simulate Pathological Lung Conditions In Vitro: Application to Systemic and Inflammatory Disorders with Pulmonary Involvement.

PubMed

Lescoat, Alain; Ballerie, Alice; Augagneur, Yu; Morzadec, Claudie; Vernhet, Laurent; Fardel, Olivier; Jégo, Patrick; Jouneau, Stéphane; Lecureur, Valérie

2018-03-17

Macrophages play a central role in the pathogenesis of inflammatory and fibrotic lung diseases. However, alveolar macrophages (AM) are poorly available in humans to perform in vitro studies due to a limited access to broncho-alveolar lavage (BAL). In this study, to identify the best alternative in vitro model for human AM, we compared the phenotype of AM obtained from BAL of patients suffering from three lung diseases (lung cancers, sarcoidosis and Systemic Sclerosis (SSc)-associated interstitial lung disease) to human blood monocyte-derived macrophages (MDMs) differentiated with M-CSF or GM-CSF. The expression of eight membrane markers was evaluated by flow cytometry. Globally, AM phenotype was closer to GM-CSF MDMs. However, the expression levels of CD163, CD169, CD204, CD64 and CD36 were significantly higher in SSc-ILD than in lung cancers. Considering the expression of CD204 and CD36, the phenotype of SSc-AM was closer to MDMs, from healthy donors or SSc patients, differentiated by M-CSF rather than GM-CSF. The comparative secretion of IL-6 by SSc-MDMs and SSc-AM is concordant with these phenotypic considerations. Altogether, these results support the M-CSF MDM model as a relevant in vitro alternative to simulate AM in fibrotic disorders such as SSc.

ΔN-P63α and TA-P63α exhibit intrinsic differences in transactivation specificities that depend on distinct features of DNA target sites

PubMed Central

Foggetti, Giorgia; Raimondi, Ivan; Campomenosi, Paola; Menichini, Paola

2014-01-01

TP63 is a member of the TP53 gene family that encodes for up to ten different TA and ΔN isoforms through alternative promoter usage and alternative splicing. Besides being a master regulator of gene expression for squamous epithelial proliferation, differentiation and maintenance, P63, through differential expression of its isoforms, plays important roles in tumorigenesis. All P63 isoforms share an immunoglobulin-like folded DNA binding domain responsible for binding to sequence-specific response elements (REs), whose overall consensus sequence is similar to that of the canonical p53 RE. Using a defined assay in yeast, where P63 isoforms and RE sequences are the only variables, and gene expression assays in human cell lines, we demonstrated that human TA- and ΔN-P63α proteins exhibited differences in transactivation specificity not observed with the corresponding P73 or P53 protein isoforms. These differences 1) were dependent on specific features of the RE sequence, 2) could be related to intrinsic differences in their oligomeric state and cooperative DNA binding, and 3) appeared to be conserved in evolution. Since genotoxic stress can change relative ratio of TA- and ΔN-P63α protein levels, the different transactivation specificity of each P63 isoform could potentially influence cellular responses to specific stresses. PMID:24926492

Male and Female Homosexuality: A Review of Differential Factors in Etiology and Implications for Treatment.

ERIC Educational Resources Information Center

Strautin, Astra Lydia

Today's society, in large part, has granted public acknowledgment of the practice of homosexuality as an "alternative lifestyle" and as a valid expression of sexuality and self. However, there are men and women who, having engaged in an active homosexual lifestyle for varying lengths of time, feel regretful about their homosexuality and want to…

Bidirectional regulation of neurite elaboration by alternatively spliced metabotropic glutamate receptor 5 (mGluR5) isoforms.

PubMed

Mion, S; Corti, C; Neki, A; Shigemoto, R; Corsi, M; Fumagalli, G; Ferraguti, F

2001-06-01

Alternative splicing in the mGluR5 gene generates two different receptor isoforms, of which expression is developmentally regulated. However, little is known about the functional significance of mGluR5 splice variants. We have examined the functional coupling, subcellular targeting, and effect on neuronal differentiation of epitope-tagged mGluR5 isoforms by expression in neuroblastoma NG108-15 cells. We found that both mGluR5 splice variants give rise to comparable [Ca2+]i transients and have similar pharmacological profile. Tagged receptors were shown by immunofluorescence to be inserted in the plasma membrane. In undifferentiated cells the subcellular localization of the two mGluR5 isoforms was partially segregated, whereas in differentiated cells the labeling largely redistributed to the newly formed neurites. Interestingly, we demonstrate that mGluR5 splice variants dramatically influence the formation and maturation of neurites; mGluR5a hinders the acquisition of mature neuronal traits and mGluR5b fosters the elaboration and extension of neurites. These effects are partly inhibited by MPEP. Copyright 2001 Academic Press.

Misregulation of Gene Expression and Sterility in Interspecies Hybrids: Causal Links and Alternative Hypotheses.

PubMed

Civetta, Alberto

2016-05-01

Understanding the origin of species is of interest to biologist in general and evolutionary biologist in particular. Hybrid male sterility (HMS) has been a focus in studies of speciation because sterility imposes a barrier to free gene flow between organisms, thus effectively isolating them as distinct species. In this review, I focus on the role of differential gene expression in HMS and speciation. Microarray and qPCR assays have established associations between misregulation of gene expression and sterility in hybrids between closely related species. These studies originally proposed disrupted expression of spermatogenesis genes as a causative of sterility. Alternatively, rapid genetic divergence of regulatory elements, particularly as they relate to the male sex (fast-male evolution), can drive the misregulation of sperm developmental genes in the absence of sterility. The use of fertile hybrids (both backcross and F1 progeny) as controls has lent support to this alternative explanation. Differences in gene expression between fertile and sterile hybrids can also be influenced by a pattern of faster evolution of the sex chromosome (fast-X evolution) than autosomes. In particular, it would be desirable to establish whether known X-chromosome sterility factors can act as trans-regulatory drivers of genome-wide patterns of misregulation. Genome-wide expression studies coupled with assays of proxies of sterility in F1 and BC progeny have identified candidate HMS genes but functional assays, and a better phenotypic characterization of sterility phenotypes, are needed to rigorously test how these genes might contribute to HMS.

Differentiation of Mesenchymal Stem Cells Towards Nephrogenic Lineage and Their Enhanced Resistance to Oxygen Peroxide-induced Oxidative Stress.

PubMed

Tayyeb, Asima; Shahzad, Naveed; Ali, Gibran

2017-07-01

Mesenchymal stem cells (MSCs) have been publicized to ameliorate kidney injury both in vitro and in vivo. However, very less is known if MSCs can be differentiated towards renal lineages and their further application potential in kidney injuries. The present study developed a conditioning system of growth factors fibroblast growth factor 2, transforming growth factor-β2, and leukemia inhibitory factor for in vitro differentiation of MSCs isolated from different sources towards nephrogenic lineage. Less invasively isolated adipose-derived MSCs were also compared to bone marrow-derived MSCs for their differentiation potential to induce renal cell. Differentiated MSCs were further evaluated for their resistance to oxidative stress induced by oxygen peroxide. A combination of growth factors successfully induced differentiation of MSCs. Both types of differentiated cells showed significant expression of pronephrogenic markers (Wnt4, Wt1, and Pax2) and renal epithelial markers (Ecad and ZO1). In contrast, expression of mesenchymal stem cells marker Oct4 and Vim were downregulated. Furthermore, differentiated adipose-derived MSCs and bone marrow-derived MSCs showed enhanced and comparable resistance to oxygen peroxide-induced oxidative stress. Adipose-derived MSC provides a promising alternative to bone marrow-derived MSC as a source of autologous stem cells in human kidney injuries. In addition, differentiated MSCs with further in vivo investigations may serve as a cell source for tissue engineering or cell therapy in different renal ailments.

Alternate Bearing in Citrus: Changes in the Expression of Flowering Control Genes and in Global Gene Expression in ON- versus OFF-Crop Trees

PubMed Central

Shalom, Liron; Samuels, Sivan; Zur, Naftali; Shlizerman, Lyudmila; Zemach, Hanita; Weissberg, Mira; Ophir, Ron; Blumwald, Eduardo; Sadka, Avi

2012-01-01

Alternate bearing (AB) is the process in fruit trees by which cycles of heavy yield (ON crop) one year are followed by a light yield (OFF crop) the next. Heavy yield usually reduces flowering intensity the following year. Despite its agricultural importance, how the developing crop influences the following year's return bloom and yield is not fully understood. It might be assumed that an ‘AB signal’ is generated in the fruit, or in another organ that senses fruit presence, and moves into the bud to determine its fate—flowering or vegetative growth. The bud then responds to fruit presence by altering regulatory and metabolic pathways. Determining these pathways, and when they are altered, might indicate the nature of this putative AB signal. We studied bud morphology, the expression of flowering control genes, and global gene expression in ON- and OFF-crop buds. In May, shortly after flowering and fruit set, OFF-crop buds were already significantly longer than ON-crop buds. The number of differentially expressed genes was higher in May than at the other tested time points. Processes differentially expressed between ON- and OFF-crop trees included key metabolic and regulatory pathways, such as photosynthesis and secondary metabolism. The expression of genes of trehalose metabolism and flavonoid metabolism was validated by nCounter technology, and the latter was confirmed by metabolomic analysis. Among genes induced in OFF-crop trees was one homologous to SQUAMOSA PROMOTER BINDING-LIKE (SPL), which controls juvenile-to-adult and annual phase transitions, regulated by miR156. The expression pattern of SPL-like, miR156 and other flowering control genes suggested that fruit load affects bud fate, and therefore development and metabolism, a relatively long time before the flowering induction period. Results shed light on some of the metabolic and regulatory processes that are altered in ON and OFF buds. PMID:23071667

Alternate bearing in citrus: changes in the expression of flowering control genes and in global gene expression in ON- versus OFF-crop trees.

PubMed

Shalom, Liron; Samuels, Sivan; Zur, Naftali; Shlizerman, Lyudmila; Zemach, Hanita; Weissberg, Mira; Ophir, Ron; Blumwald, Eduardo; Sadka, Avi

2012-01-01

Alternate bearing (AB) is the process in fruit trees by which cycles of heavy yield (ON crop) one year are followed by a light yield (OFF crop) the next. Heavy yield usually reduces flowering intensity the following year. Despite its agricultural importance, how the developing crop influences the following year's return bloom and yield is not fully understood. It might be assumed that an 'AB signal' is generated in the fruit, or in another organ that senses fruit presence, and moves into the bud to determine its fate-flowering or vegetative growth. The bud then responds to fruit presence by altering regulatory and metabolic pathways. Determining these pathways, and when they are altered, might indicate the nature of this putative AB signal. We studied bud morphology, the expression of flowering control genes, and global gene expression in ON- and OFF-crop buds. In May, shortly after flowering and fruit set, OFF-crop buds were already significantly longer than ON-crop buds. The number of differentially expressed genes was higher in May than at the other tested time points. Processes differentially expressed between ON- and OFF-crop trees included key metabolic and regulatory pathways, such as photosynthesis and secondary metabolism. The expression of genes of trehalose metabolism and flavonoid metabolism was validated by nCounter technology, and the latter was confirmed by metabolomic analysis. Among genes induced in OFF-crop trees was one homologous to SQUAMOSA PROMOTER BINDING-LIKE (SPL), which controls juvenile-to-adult and annual phase transitions, regulated by miR156. The expression pattern of SPL-like, miR156 and other flowering control genes suggested that fruit load affects bud fate, and therefore development and metabolism, a relatively long time before the flowering induction period. Results shed light on some of the metabolic and regulatory processes that are altered in ON and OFF buds.

Proglucagons in vertebrates: Expression and processing of multiple genes in a bony fish.

PubMed

Busby, Ellen R; Mommsen, Thomas P

2016-09-01

In contrast to mammals, where a single proglucagon (PG) gene encodes three peptides: glucagon, glucagon-like peptide 1 and glucagon-like peptide 2 (GLP-1; GLP-2), many non-mammalian vertebrates carry multiple PG genes. Here, we investigate proglucagon mRNA sequences, their tissue expression and processing in a diploid bony fish. Copper rockfish (Sebastes caurinus) express two independent genes coding for distinct proglucagon sequences (PG I, PG II), with PG II lacking the GLP-2 sequence. These genes are differentially transcribed in the endocrine pancreas, the brain, and the gastrointestinal tract. Alternative splicing identified in rockfish is only one part of this complex regulation of the PG transcripts: the system has the potential to produce two glucagons, four GLP-1s and a single GLP-2, or any combination of these peptides. Mass spectrometric analysis of partially purified PG-derived peptides in endocrine pancreas confirms translation of both PG transcripts and differential processing of the resulting peptides. The complex differential regulation of the two PG genes and their continued presence in this extant teleostean fish strongly suggests unique and, as yet largely unidentified, roles for the peptide products encoded in each gene. Copyright © 2016 Elsevier Inc. All rights reserved.

Dendrimer-driven neurotrophin expression differs in temporal patterns between rodent and human stem cells.

PubMed

Shakhbazau, Antos; Shcharbin, Dzmitry; Seviaryn, Ihar; Goncharova, Natalya; Kosmacheva, Svetlana; Potapnev, Mihail; Bryszewska, Maria; Kumar, Ranjan; Biernaskie, Jeffrey; Midha, Rajiv

2012-05-07

This study reports the use of a nonviral expression system based on polyamidoamine dendrimers for time-restricted neurotrophin overproduction in mesenchymal stem cells and skin precursor-derived Schwann cells. The dendrimers were used to deliver plasmids for brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3) expression in both rodent and human stem cells, and the timelines of expression were studied. We have found that, despite the fact that transfection efficiencies and protein expression levels were comparable, dendrimer-driven expression in human mesenchymal stem cells was characterized by a more rapid decline compared to rodent cells. Transient expression systems can be beneficial for some neurotrophins, which were earlier reported to cause unwanted side effects in virus-based long-term expression models. Nonviral neurotrophin expression is a biologically safe and accessible alternative to increase the therapeutic potential of autologous adult stem cells and stem cell-derived functional differentiated cells.

The In Vitro Differentiation of GDNF Gene-Engineered Amniotic Fluid-Derived Stem Cells into Renal Tubular Epithelial-Like Cells.

PubMed

Lu, Ying; Wang, Zhuojun; Chen, Lu; Wang, Jia; Li, Shulin; Liu, Caixia; Sun, Dong

2018-05-01

Amniotic fluid is an alternative source of stem cells, and human amniotic fluid-derived stem cells (AFSCs) obtained from a small amount of amniotic fluid collected during the second trimester represent a novel source for use in regenerative medicine. These AFSCs are characterized by lower diversity, a higher proliferation rate, and a wider differentiation capability than adult mesenchymal stem cells. AFSCs are selected based on the cell surface marker c-kit receptor (CD117) using immunomagnetic sorting. Glial cell line-derived neurotrophic factor (GDNF) is expressed during early kidney development and regulates the proliferation and differentiation of stem cells in vitro. In this study, c-kit-sorted AFSCs were induced toward osteogenic or adipogenic differentiation. AFSCs engineered via the insertion of GDNF were cocultured with mouse renal tubular epithelial cells (mRTECs), which were preconditioned by hypoxia-reoxygenation in vitro. After coculture for 8 days, AFSCs differentiation into epithelial-like cells was evaluated by performing immunofluorescence, flow cytometry, and quantitative real-time polymerase chain reaction to identify cells expressing the renal epithelial markers, cytokeratin 18 (CK18), E-cadherin, aquaporin-1 (AQP1), and paired box 2 gene (Pax2). The GDNF gene enhanced AFSCs differentiation into RTECs. AFSCs possess self-renewal ability and multiple differentiation potential and thus represent a new source of stem cells.

Rewiring of the inferred protein interactome during blood development studied with the tool PPICompare.

PubMed

Will, Thorsten; Helms, Volkhard

2017-04-04

Differential analysis of cellular conditions is a key approach towards understanding the consequences and driving causes behind biological processes such as developmental transitions or diseases. The progress of whole-genome expression profiling enabled to conveniently capture the state of a cell's transcriptome and to detect the characteristic features that distinguish cells in specific conditions. In contrast, mapping the physical protein interactome for many samples is experimentally infeasible at the moment. For the understanding of the whole system, however, it is equally important how the interactions of proteins are rewired between cellular states. To overcome this deficiency, we recently showed how condition-specific protein interaction networks that even consider alternative splicing can be inferred from transcript expression data. Here, we present the differential network analysis tool PPICompare that was specifically designed for isoform-sensitive protein interaction networks. Besides detecting significant rewiring events between the interactomes of grouped samples, PPICompare infers which alterations to the transcriptome caused each rewiring event and what is the minimal set of alterations necessary to explain all between-group changes. When applied to the development of blood cells, we verified that a reasonable amount of rewiring events were reported by the tool and found that differential gene expression was the major determinant of cellular adjustments to the interactome. Alternative splicing events were consistently necessary in each developmental step to explain all significant alterations and were especially important for rewiring in the context of transcriptional control. Applying PPICompare enabled us to investigate the dynamics of the human protein interactome during developmental transitions. A platform-independent implementation of the tool PPICompare is available at https://sourceforge.net/projects/ppicompare/ .

Disturbed expression of type 1 iodothyronine deiodinase splice variants in human renal cancer.

PubMed

Piekielko-Witkowska, Agnieszka; Master, Adam; Wojcicka, Anna; Boguslawska, Joanna; Brozda, Izabela; Tanski, Zbigniew; Nauman, Alicja

2009-10-01

Alternative splicing, one of the sources of protein diversity, is often disturbed in cancer. Type 1 iodothyronine deiodinase (DIO1) catalyzes deiodination of thyroxine generating triiodothyronine, an important regulator of cell proliferation and differentiation. The expression of DIO1 is disturbed in different types of cancer. The aim of the study was to analyze the alternative splicing of DIO1 and its possible disturbance in renal cancer. Using real-time PCR, we analyzed 19 tissue samples (T) of renal cancer and 19 matched control samples (C) of the opposite pole of the kidney, not infiltrated by tumor, and 6 control samples (N) (nonneoplastic kidney abnormalities). Cloning of DIO1 mRNA isoforms revealed 11 different transcripts, among them 7 new splice variants, not previously reported. The expression of all variants of DIO1 was dramatically (>90%) and significantly (p < or = 0.0003) lowered in samples T compared to control samples C. The ratio of mRNA isoforms encoding DIO1 protein variants possessing or lacking the active center was lowered in samples T compared with control samples C, suggesting disturbed alternative splicing of DIO1. The expression of mRNA of splicing factors SF2/ASF (splicing factor-2/alternative-splicing factor) and hnRNPA1 (heterogeneous ribonucleoprotein A1), regulating 5'-splice site selection, was significantly but not proportionally lowered in samples T compared to samples C. The mRNA ratio of splicing factors SF2/ASF and hnRNPA1 correlated with the ratio of mRNA isoforms encoding DIO1 protein variants possessing or lacking the active center in controls C but not in samples T. Our results show that the expression and alternative splicing of DIO1 mRNA is disturbed in renal cancer, possibly due to changes in expression of splicing factors SF2/ASF and hnRNPA1.

Role of miRNAs and alternative mRNA 3'-end cleavage and polyadenylation of their mRNA targets in cardiomyocyte hypertrophy.

PubMed

Soetanto, R; Hynes, C J; Patel, H R; Humphreys, D T; Evers, M; Duan, G; Parker, B J; Archer, S K; Clancy, J L; Graham, R M; Beilharz, T H; Smith, N J; Preiss, T

2016-05-01

miRNAs play critical roles in heart disease. In addition to differential miRNA expression, miRNA-mediated control is also affected by variable miRNA processing or alternative 3'-end cleavage and polyadenylation (APA) of their mRNA targets. To what extent these phenomena play a role in the heart remains unclear. We sought to explore miRNA processing and mRNA APA in cardiomyocytes, and whether these change during cardiac hypertrophy. Thoracic aortic constriction (TAC) was performed to induce hypertrophy in C57BL/6J mice. RNA extracted from cardiomyocytes of sham-treated, pre-hypertrophic (2 days post-TAC), and hypertrophic (7 days post-TAC) mice was subjected to small RNA- and poly(A)-test sequencing (PAT-Seq). Differential expression analysis matched expectations; nevertheless we identified ~400 mRNAs and hundreds of noncoding RNA loci as altered with hypertrophy for the first time. Although multiple processing variants were observed for many miRNAs, there was little change in their relative proportions during hypertrophy. PAT-Seq mapped ~48,000 mRNA 3'-ends, identifying novel 3' untranslated regions (3'UTRs) for over 7000 genes. Importantly, hypertrophy was associated with marked changes in APA with a net shift from distal to more proximal mRNA 3'-ends, which is predicted to decrease overall miRNA repression strength. We independently validated several examples of 3'UTR proportion change and showed that alternative 3'UTRs associate with differences in mRNA translation. Our work suggests that APA contributes to altered gene expression with the development of cardiomyocyte hypertrophy and provides a rich resource for a systems-level understanding of miRNA-mediated regulation in physiological and pathological states of the heart. Copyright © 2016 Elsevier B.V. All rights reserved.

A rat RNA-Seq transcriptomic BodyMap across 11 organs and 4 developmental stages

PubMed Central

Yu, Ying; Fuscoe, James C.; Zhao, Chen; Guo, Chao; Jia, Meiwen; Qing, Tao; Bannon, Desmond I.; Lancashire, Lee; Bao, Wenjun; Du, Tingting; Luo, Heng; Su, Zhenqiang; Jones, Wendell D.; Moland, Carrie L.; Branham, William S.; Qian, Feng; Ning, Baitang; Li, Yan; Hong, Huixiao; Guo, Lei; Mei, Nan; Shi, Tieliu; Wang, Kevin Y.; Wolfinger, Russell D.; Nikolsky, Yuri; Walker, Stephen J.; Duerksen-Hughes, Penelope; Mason, Christopher E.; Tong, Weida; Thierry-Mieg, Jean; Thierry-Mieg, Danielle; Shi, Leming; Wang, Charles

2014-01-01

The rat has been used extensively as a model for evaluating chemical toxicities and for understanding drug mechanisms. However, its transcriptome across multiple organs, or developmental stages, has not yet been reported. Here we show, as part of the SEQC consortium efforts, a comprehensive rat transcriptomic BodyMap created by performing RNA-Seq on 320 samples from 11 organs of both sexes of juvenile, adolescent, adult and aged Fischer 344 rats. We catalogue the expression profiles of 40,064 genes, 65,167 transcripts, 31,909 alternatively spliced transcript variants and 2,367 non-coding genes/non-coding RNAs (ncRNAs) annotated in AceView. We find that organ-enriched, differentially expressed genes reflect the known organ-specific biological activities. A large number of transcripts show organ-specific, age-dependent or sex-specific differential expression patterns. We create a web-based, open-access rat BodyMap database of expression profiles with crosslinks to other widely used databases, anticipating that it will serve as a primary resource for biomedical research using the rat model. PMID:24510058

Transcriptomic profile analysis of mouse neural tube development by RNA-Seq.

PubMed

Yu, Juan; Mu, Jianbing; Guo, Qian; Yang, Lihong; Zhang, Juan; Liu, Zhizhen; Yu, Baofeng; Zhang, Ting; Xie, Jun

2017-09-01

The neural tube is the primordium of the central nervous system (CNS) in which its development is not entirely clear. Understanding the cellular and molecular basis of neural tube development could, therefore, provide vital clues to the mechanism of neural tube defects (NTDs). Here, we investigated the gene expression profiles of three different time points (embryonic day (E) 8.5, 9.5 and 10.5) of mouse neural tube by using RNA-seq approach. About 391 differentially expressed genes (DEGs) were screened during mouse neural tube development, including 45 DEGs involved in CNS development, among which Bmp2, Ascl1, Olig2, Lhx1, Wnt7b and Eomes might play the important roles. Of 45 DEGs, Foxp2, Eomes, Hoxb3, Gpr56, Hap1, Nkx2-1, Sez6l2, Wnt7b, Tbx20, Nfib, Cntn1 and Dcx had different isoforms, and the opposite expression pattern of different isoforms was observed for Gpr56, Nkx2-1 and Sez6l2. In addition, alternative splicing, such as mutually exclusive exon, retained intron, skipped exon and alternative 3' splice site was identified in 10 neural related differentially splicing genes, including Ngrn, Ddr1, Dctn1, Dnmt3b, Ect2, Map2, Mbnl1, Meis2, Vcan and App. Moreover, seven neural splicing factors, such as Nova1/2, nSR100/Srrm4, Elavl3/4, Celf3 and Rbfox1 were differentially expressed during mouse neural tube development. Interestingly, nine DEGs identified above were dysregulated in retinoic acid-induced NTDs model, indicating the possible important role of these genes in NTDs. Taken together, our study provides more comprehensive information on mouse neural tube development, which might provide new insights on NTDs occurrence. © 2017 IUBMB Life, 69(9):706-719, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

Involvement of alternative oxidase (AOX) in adventitious rooting of Olea europaea L. microshoots is linked to adaptive phenylpropanoid and lignin metabolism.

PubMed

Santos Macedo, E; Sircar, D; Cardoso, H G; Peixe, A; Arnholdt-Schmitt, B

2012-09-01

Alternative oxidase (AOX) has been proposed as a functional marker candidate in a number of events involving cell differentiation, including rooting efficiency in semi-hardwood shoot cuttings of olive (Olea europaea L.). To ascertain the general importance of AOX in olive rooting, the auxin-induced rooting process was studied in an in vitro system for microshoot propagation. Inhibition of AOX by salicylhydroxamic acid (SHAM) significantly reduced rooting efficiency. However, the inhibitor failed to exhibit any effect on the preceding calli stage. This makes the system appropriate for distinguishing dedifferentiation and de novo differentiation during root induction. Metabolite analyses of microshoots showed that total phenolics, total flavonoids and lignin contents were significantly reduced upon SHAM treatment. It was concluded that the influence of alternative respiration on root formation was associated to adaptive phenylpropanoid and lignin metabolism. Transcript profiles of two olive AOX genes (OeAOX1a and OeAOX2) were examined during the process of auxin-induced root induction. Both genes displayed stable transcript accumulation in semi-quantitative RT-PCR analysis during all experimental stages. In contrary, when the reverse primer for OeAOX2 was designed from the 3'-UTR instead of the ORF, differential transcript accumulation was observed suggesting posttranscriptional regulation of OeAOX2 during metabolic acclimation. This result confirms former observations in olive semi-hardwood shoot cuttings on differential OeAOX2 expression during root induction. It further points to the importance of future studies on the functional role of sequence and length polymorphisms in the 3'-UTR of this gene. The manuscript reports the general importance of AOX in olive adventitious rooting and the association of alternative respiration to adaptive phenylpropanoid and lignin metabolism.

Expression of REST4 in human gliomas in vivo and influence of pioglitazone on REST in vitro

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

Ren, Huan; Institute of Clinical Pharmacology, Central South University, Hunan Key Laboratory of Pharmacogenetics, Changsha 410078; Gao, Zhangfeng

The repressor element-1 (RE1) silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) has an irreplaceable role during the differentiation of neurons. REST has multiple splice variants which link to various types of cancer. Previous work had highlighted the role of REST in glioma, where the expression of REST is enhanced. But whether alternative splicing of REST is expressed in glioma has not been described. Here, we show that a specific isoform REST4 is expressed in glioma specimens, and will influence the mRNA level of REST in vivo. Peroxisome proliferator-activated receptor-γ (PPARγ) agonists have a role of antineoplastic in various tumor cells, which includingmore » glioma cells. Moreover, study indicated that PPARγ agonist pioglitazone can promote alternative splicing of REST pre-mRNA. In this study, we selected pioglitazone as a tool drug to explore whether the role of pioglitazone in anti-glioma is mediated by regulating REST expression or promoting alternative splicing of REST in glioma cells. Results show that pioglitazone can inhibit proliferation and induce apoptosis of glioma cell in vitro, which may be mediated by down-regulating REST mRNA level but not by inducing alternative splicing of REST pre-mRNA. Our study firstly reports the expression of REST4 in glioma tissue samples. And we recommend that pioglitazone, which can reduce the expression level of REST, represents a promising drug for therapy of glioma. - Highlights: • A specific isoform REST4 is expressed in glioma specimens in vivo. • REST4 will influence the mRNA level of REST in vivo. • Pioglitazone can inhibit proliferation and induce apoptosis of glioma cells. • The role of pioglitazone in anti-glioma may be mediated by down-regulating REST.« less

A Natural Light/Dark Cycle Regulation of Carbon-Nitrogen Metabolism and Gene Expression in Rice Shoots.

PubMed

Li, Haixing; Liang, Zhijun; Ding, Guangda; Shi, Lei; Xu, Fangsen; Cai, Hongmei

2016-01-01

Light and temperature are two particularly important environmental cues for plant survival. Carbon and nitrogen are two essential macronutrients required for plant growth and development, and cellular carbon and nitrogen metabolism must be tightly coordinated. In order to understand how the natural light/dark cycle regulates carbon and nitrogen metabolism in rice plants, we analyzed the photosynthesis, key carbon-nitrogen metabolites, and enzyme activities, and differentially expressed genes and miRNAs involved in the carbon and nitrogen metabolic pathway in rice shoots at the following times: 2:00, 6:00, 10:00, 14:00, 18:00, and 22:00. Our results indicated that more CO2 was fixed into carbohydrates by a high net photosynthetic rate, respiratory rate, and stomatal conductance in the daytime. Although high levels of the nitrate reductase activity, free ammonium and carbohydrates were exhibited in the daytime, the protein synthesis was not significantly facilitated by the light and temperature. In mRNA sequencing, the carbon and nitrogen metabolism-related differentially expressed genes were obtained, which could be divided into eight groups: photosynthesis, TCA cycle, sugar transport, sugar metabolism, nitrogen transport, nitrogen reduction, amino acid metabolism, and nitrogen regulation. Additionally, a total of 78,306 alternative splicing events have been identified, which primarily belong to alternative 5' donor sites, alternative 3' acceptor sites, intron retention, and exon skipping. In sRNA sequencing, four carbon and nitrogen metabolism-related miRNAs (osa-miR1440b, osa-miR2876-5p, osa-miR1877 and osa-miR5799) were determined to be regulated by natural light/dark cycle. The expression level analysis showed that the four carbon and nitrogen metabolism-related miRNAs negatively regulated their target genes. These results may provide a good strategy to study how natural light/dark cycle regulates carbon and nitrogen metabolism to ensure plant growth and development.

Increased phospho-AKT is associated with loss of the androgen receptor during the progression of N-methyl-N-nitrosourea-induced prostate carcinogenesis in rats.

PubMed

Liao, Zhiming; Wang, Shihua; Boileau, Thomas W-M; Erdman, John W; Clinton, Steven K

2005-07-01

Characterization of molecular events during N-methyl-N-nitrosourea (MNU)-induced rat prostate carcinogenesis enhances the utility of this model for the preclinical assessment of preventive strategies. Androgen independence is typical of advanced human prostate cancer and may occur through multiple mechanisms including the loss of androgen receptor (AR) expression and the activation of alternative signaling pathways. We examined the interrelationships between AR and p-AKT expression by immunohistochemical staining during MNU-androgen-induced prostate carcinogenesis in male Wistar-Unilever rats. Histone nuclear staining and image analysis was employed to assess parallel changes in chromatin and nuclear structure. The percentage of AR positive nuclei decreased (P < 0.01) as carcinogenesis progressed: hyperplasia (92%), atypical hyperplasia (92%), well-differentiated adenocarcinoma (57%), moderately-differentiated adenocarcinoma (19%), and poorly-differentiated adenocarcinoma (10%). Conversely, p-AKT staining increased significantly during carcinogenesis. Sparse staining was observed in normal tissues (0.2% of epithelial area) and hyperplastic lesions (0.1%), while expression increased significantly (P < 0.001) in atypical hyperplasia (7.6%), well-differentiated adenocarcinoma (16.7%), moderately-differentiated adenocarcinoma (19.6%), and poorly-differentiated adenocarcinoma (17.4%). In parallel, nuclear morphometry revealed increased nuclear size, greater irregularity, and lower DNA compactness as cancers became more poorly differentiated. In the MNU model, the progressive evolution of dominant tumor cell populations showing an increase in p-AKT in parallel with a decline in AR staining suggests that activation of AKT signaling may be one of several mechanisms contributing to androgen insensitivity during prostate cancer progression. Our observations mimic findings suggested by human studies and support the relevance of the MNU model in preclinical studies of preventive strategies. (c) 2005 Wiley-Liss, Inc.

Organotins Are Potent Activators of PPARγ and Adipocyte Differentiation in Bone Marrow Multipotent Mesenchymal Stromal Cells

PubMed Central

Yanik, Susan C.; Baker, Amelia H.; Mann, Koren K.; Schlezinger, Jennifer J.

2011-01-01

Adipocyte differentiation in bone marrow is potentially deleterious to both bone integrity and lymphopoiesis. Here, we examine the hypothesis that organotins, common environmental contaminants that are dual ligands for peroxisome proliferator–activated receptor (PPAR) γ and its heterodimerization partner retinoid X receptor (RXR), are potent activators of bone marrow adipogenesis. A C57Bl/6-derived bone marrow multipotent mesenchymal stromal cell (MSC) line, BMS2, was treated with rosiglitazone, a PPARγ agonist, bexarotene, an RXR agonist, or a series of organotins. Rosiglitazone and bexarotene potently activated adipocyte differentiation; however, bexarotene had a maximal efficacy of only 20% of that induced by rosiglitazone. Organotins (tributyltin [TBT], triphenyltin, and dibutyltin) also stimulated adipocyte differentiation (EC50 of 10–20nM) but with submaximal, structure-dependent efficacy. In coexposures, both bexarotene and TBT enhanced rosiglitazone-induced adipogenesis. To investigate the contribution of PPARγ to TBT-induced adipogenesis, we examined expression of PPARγ2, as well as its transcriptional target FABP4. TBT-induced PPARγ2 and FABP4 protein expression with an efficacy intermediate between rosiglitazone and bexarotene, similar to lipid accumulation. A PPARγ antagonist and PPARγ-specific small hairpin RNA suppressed TBT-induced differentiation, although to a lesser extent than rosiglitazone-induced differentiation, suggesting that TBT may engage alternate pathways. TBT and bexarotene, but not rosiglitazone, also induced the expression of TGM2 (an RXR target) and ABCA1 (a liver X receptor target). The results show that an environmental contaminant, acting with the same potency as a therapeutic drug, induces PPARγ-dependent adipocyte differentiation in bone marrow MSCs. Activation of multiple nuclear receptor pathways by organotins may have significant implications for bone physiology. PMID:21622945

A Plastid Terminal Oxidase Associated with Carotenoid Desaturation during Chromoplast Differentiation1

PubMed Central

Josse, Eve-Marie; Simkin, Andrew J.; Gaffé, Joël; Labouré, Anne-Marie; Kuntz, Marcel; Carol, Pierre

2000-01-01

The Arabidopsis IMMUTANS gene encodes a plastid homolog of the mitochondrial alternative oxidase, which is associated with phytoene desaturation. Upon expression in Escherichia coli, this protein confers a detectable cyanide-resistant electron transport to isolated membranes. In this assay this activity is sensitive to n-propyl-gallate, an inhibitor of the alternative oxidase. This protein appears to be a plastid terminal oxidase (PTOX) that is functionally equivalent to a quinol:oxygen oxidoreductase. This protein was immunodetected in achlorophyllous pepper (Capsicum annuum) chromoplast membranes, and a corresponding cDNA was cloned from pepper and tomato (Lycopersicum esculentum) fruits. Genomic analysis suggests the presence of a single gene in these organisms, the expression of which parallels phytoene desaturase and ζ-carotene desaturase gene expression during fruit ripening. Furthermore, this PTOX gene is impaired in the tomato ghost mutant, which accumulates phytoene in leaves and fruits. These data show that PTOX also participates in carotenoid desaturation in chromoplasts in addition to its role during early chloroplast development. PMID:10938359

Monoamine oxidase A (MAO-A): a signature marker of alternatively activated monocytes/macrophages

PubMed Central

Cathcart, Martha K.; Bhattacharjee, Ashish

2015-01-01

Monocytes/macrophages are versatile cells centrally involved in host defense and immunity. Th1 cytokines induce a classical activation program in monocytes/macrophages leading to a proinflammatory M1 macrophage phenotype while Th2 cytokines IL-4 and IL-13 promote monocyte differentiation into an alternatively activated, anti-inflammatory M2 macrophage phenotype. Although monoamine oxidase A (MAO-A) is primarily known for its action in the nervous system, several recent studies have identified MAO-A as a signature marker of alternative activation of monocytes/macrophages. In this brief review we explore the signaling pathways/molecules that regulate MAO-A expression in alternatively activated monocytes/macrophages. We further discuss the contribution of MAO-A to the resolution of inflammation and identify potential therapeutic targets for controlling inflammation. Altogether this review provides deeper insight into the role of MAO-A in alternative activation of monocytes/macrophages and their participation in the inflammatory response. PMID:26052543

Monoamine oxidase A (MAO-A): a signature marker of alternatively activated monocytes/macrophages.

PubMed

Cathcart, Martha K; Bhattacharjee, Ashish

Monocytes/macrophages are versatile cells centrally involved in host defense and immunity. Th1 cytokines induce a classical activation program in monocytes/macrophages leading to a proinflammatory M1 macrophage phenotype while Th2 cytokines IL-4 and IL-13 promote monocyte differentiation into an alternatively activated, anti-inflammatory M2 macrophage phenotype. Although monoamine oxidase A (MAO-A) is primarily known for its action in the nervous system, several recent studies have identified MAO-A as a signature marker of alternative activation of monocytes/macrophages. In this brief review we explore the signaling pathways/molecules that regulate MAO-A expression in alternatively activated monocytes/macrophages. We further discuss the contribution of MAO-A to the resolution of inflammation and identify potential therapeutic targets for controlling inflammation. Altogether this review provides deeper insight into the role of MAO-A in alternative activation of monocytes/macrophages and their participation in the inflammatory response.

Nutrition metabolism plays an important role in the alternate bearing of the olive tree (Olea europaea L.).

PubMed

Turktas, Mine; Inal, Behcet; Okay, Sezer; Erkilic, Emine Gulden; Dundar, Ekrem; Hernandez, Pilar; Dorado, Gabriel; Unver, Turgay

2013-01-01

The olive tree (Olea europaea L.) is widely known for its strong tendency for alternate bearing, which severely affects the fruit yield from year to year. Microarray based gene expression analysis using RNA from olive samples (on-off years leaves and ripe-unripe fruits) are particularly useful to understand the molecular mechanisms influencing the periodicity in the olive tree. Thus, we carried out genome wide transcriptome analyses involving different organs and temporal stages of the olive tree using the NimbleGen Array containing 136,628 oligonucleotide probe sets. Cluster analyses of the genes showed that cDNAs originated from different organs could be sorted into separate groups. The nutritional control had a particularly remarkable impact on the alternate bearing of olive, as shown by the differential expression of transcripts under different temporal phases and organs. Additionally, hormonal control and flowering processes also played important roles in this phenomenon. Our analyses provide further insights into the transcript changes between "on year" and "off year" leaves along with the changes from unrpipe to ripe fruits, which shed light on the molecular mechanisms underlying the olive tree alternate bearing. These findings have important implications for the breeding and agriculture of the olive tree and other crops showing periodicity. To our knowledge, this is the first study reporting the development and use of an olive array to document the gene expression profiling associated with the alternate bearing in olive tree.

Profiling the array of Ca(v)3.1 variants from the human T-type calcium channel gene CACNA1G: alternative structures, developmental expression, and biophysical variations.

PubMed

Emerick, Mark C; Stein, Rebecca; Kunze, Robin; McNulty, Megan M; Regan, Melissa R; Hanck, Dorothy A; Agnew, William S

2006-08-01

We describe the regulated transcriptome of CACNA1G, a human gene for T-type Ca(v)3.1 calcium channels that is subject to extensive alternative RNA splicing. Fifteen sites of transcript variation include 2 alternative 5'-UTR promoter sites, 2 alternative 3'-UTR polyadenylation sites, and 11 sites of alternative splicing within the open reading frame. A survey of 1580 fetal and adult human brain full-length complementary DNAs reveals a family of 30 distinct transcripts, including multiple functional forms that vary in expression with development. Statistical analyses of fetal and adult transcript populations reveal patterns of linkages among intramolecular splice site configurations that change dramatically with development. A shift from nearly independent, biased splicing in fetal transcripts to strongly concerted splicing in adult transcripts suggests progressive activation of multiple "programs" of splicing regulation that reorganize molecular structures in differentiating cells. Patch-clamp studies of nine selected variants help relate splicing regulation to permutations of the gating parameters most likely to modify T-channel physiology in expressing neurons. Gating behavior reflects combinatorial interactions between variable domains so that molecular phenotype depends on ensembles of coselected domains, consistent with the observed emergence of concerted splicing during development. We conclude that the structural gene and networks of splicing regulatory factors define an integrated system for the phenotypic variation of Ca(v)3.1 biophysics during nervous system development. Copyright 2006 Wiley-Liss, Inc.

Epigenetic control of alternative mRNA processing at the imprinted Herc3/Nap1l5 locus

PubMed Central

Cowley, Michael; Wood, Andrew J.; Böhm, Sabrina; Schulz, Reiner; Oakey, Rebecca J.

2012-01-01

Alternative polyadenylation increases transcriptome diversity by generating multiple transcript isoforms from a single gene. It is thought that this process can be subject to epigenetic regulation, but few specific examples of this have been reported. We previously showed that the Mcts2/H13 locus is subject to genomic imprinting and that alternative polyadenylation of H13 transcripts occurs in an allele-specific manner, regulated by epigenetic mechanisms. Here, we demonstrate that allele-specific polyadenylation occurs at another imprinted locus with similar features. Nap1l5 is a retrogene expressed from the paternally inherited allele, is situated within an intron of a ‘host’ gene Herc3, and overlaps a CpG island that is differentially methylated between the parental alleles. In mouse brain, internal Herc3 polyadenylation sites upstream of Nap1l5 are used on the paternally derived chromosome, from which Nap1l5 is expressed, whereas a downstream site is used more frequently on the maternally derived chromosome. Ablating DNA methylation on the maternal allele at the Nap1l5 promoter increases the use of an internal Herc3 polyadenylation site and alters exon splicing. These changes demonstrate the influence of epigenetic mechanisms in regulating Herc3 alternative mRNA processing. Internal Herc3 polyadenylation correlates with expression levels of Nap1l5, suggesting a possible role for transcriptional interference. Similar mechanisms may regulate alternative polyadenylation elsewhere in the genome. PMID:22790983

Nutrition Metabolism Plays an Important Role in the Alternate Bearing of the Olive Tree (Olea europaea L.)

PubMed Central

Turktas, Mine; Inal, Behcet; Okay, Sezer; Erkilic, Emine Gulden; Dundar, Ekrem; Hernandez, Pilar; Dorado, Gabriel; Unver, Turgay

2013-01-01

The olive tree (Olea europaea L.) is widely known for its strong tendency for alternate bearing, which severely affects the fruit yield from year to year. Microarray based gene expression analysis using RNA from olive samples (on-off years leaves and ripe-unripe fruits) are particularly useful to understand the molecular mechanisms influencing the periodicity in the olive tree. Thus, we carried out genome wide transcriptome analyses involving different organs and temporal stages of the olive tree using the NimbleGen Array containing 136,628 oligonucleotide probe sets. Cluster analyses of the genes showed that cDNAs originated from different organs could be sorted into separate groups. The nutritional control had a particularly remarkable impact on the alternate bearing of olive, as shown by the differential expression of transcripts under different temporal phases and organs. Additionally, hormonal control and flowering processes also played important roles in this phenomenon. Our analyses provide further insights into the transcript changes between ”on year” and “off year” leaves along with the changes from unrpipe to ripe fruits, which shed light on the molecular mechanisms underlying the olive tree alternate bearing. These findings have important implications for the breeding and agriculture of the olive tree and other crops showing periodicity. To our knowledge, this is the first study reporting the development and use of an olive array to document the gene expression profiling associated with the alternate bearing in olive tree. PMID:23555820

Inhibition of Sirt1 promotes neural progenitors toward motoneuron differentiation from human embryonic stem cells

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

Zhang, Yun; Wang, Jing; Clinical Stem Cell Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191

2011-01-14

Research highlights: {yields} Nicotinamide inhibit Sirt1. {yields} MASH1 and Ngn2 activation. {yields} Increase the expression of HB9. {yields} Motoneurons formation increases significantly. -- Abstract: Several protocols direct human embryonic stem cells (hESCs) toward differentiation into functional motoneurons, but the efficiency of motoneuron generation varies based on the human ESC line used. We aimed to develop a novel protocol to increase the formation of motoneurons from human ESCs. In this study, we tested a nuclear histone deacetylase protein, Sirt1, to promote neural precursor cell (NPC) development during differentiation of human ESCs into motoneurons. A specific inhibitor of Sirt1, nicotinamide, dramatically increasedmore » motoneuron formation. We found that about 60% of the cells from the total NPCs expressed HB9 and {beta}III-tubulin, commonly used motoneuronal markers found in neurons derived from ESCs following nicotinamide treatment. Motoneurons derived from ESC expressed choline acetyltransferase (ChAT), a positive marker of mature motoneuron. Moreover, we also examined the transcript levels of Mash1, Ngn2, and HB9 mRNA in the differentiated NPCs treated with the Sirt1 activator resveratrol (50 {mu}M) or inhibitor nicotinamide (100 {mu}M). The levels of Mash1, Ngn2, and HB9 mRNA were significantly increased after nicotinamide treatment compared with control groups, which used the traditional protocol. These results suggested that increasing Mash1 and Ngn2 levels by inhibiting Sirt1 could elevate HB9 expression, which promotes motoneuron differentiation. This study provides an alternative method for the production of transplantable motoneurons, a key requirement in the development of hESC-based cell therapy in motoneuron disease.« less

Gene-expression signatures of Atlantic salmon's plastic life cycle.

PubMed

Aubin-Horth, Nadia; Letcher, Benjamin H; Hofmann, Hans A

2009-09-15

How genomic expression differs as a function of life history variation is largely unknown. Atlantic salmon exhibits extreme alternative life histories. We defined the gene-expression signatures of wild-caught salmon at two different life stages by comparing the brain expression profiles of mature sneaker males and immature males, and early migrants and late migrants. In addition to life-stage-specific signatures, we discovered a surprisingly large gene set that was differentially regulated-at similar magnitudes, yet in opposite direction-in both life history transitions. We suggest that this co-variation is not a consequence of many independent cellular and molecular switches in the same direction but rather represents the molecular equivalent of a physiological shift orchestrated by one or very few master regulators.

Differential HFE Gene Expression Is Regulated by Alternative Splicing in Human Tissues

PubMed Central

Proença, Daniela; Faustino, Paula

2011-01-01

Background The pathophysiology of HFE-derived Hereditary Hemochromatosis and the function of HFE protein in iron homeostasis remain uncertain. Also, the role of alternative splicing in HFE gene expression regulation and the possible function of the corresponding protein isoforms are still unknown. The aim of this study was to gain insights into the physiological significance of these alternative HFE variants. Methodology/Principal Findings Alternatively spliced HFE transcripts in diverse human tissues were identified by RT-PCR, cloning and sequencing. Total HFE transcripts, as well as two alternative splicing transcripts were quantified using a real-time PCR methodology. Intracellular localization, trafficking and protein association of GFP-tagged HFE protein variants were analysed in transiently transfected HepG2 cells by immunoprecipitation and immunofluorescence assays. Alternatively spliced HFE transcripts present both level- and tissue-specificity. Concerning the exon 2 skipping and intron 4 inclusion transcripts, the liver presents the lowest relative level, while duodenum presents one of the highest amounts. The protein resulting from exon 2 skipping transcript is unable to associate with β2M and TfR1 and reveals an ER retention. Conversely, the intron 4 inclusion transcript gives rise to a truncated, soluble protein (sHFE) that is mostly secreted by cells to the medium in association with β2M. Conclusions/Significance HFE gene post-transcriptional regulation is clearly affected by a tissue-dependent alternative splicing mechanism. Among the corresponding proteins, a sHFE isoform stands out, which upon being secreted into the bloodstream, may act in remote tissues. It could be either an agonist or antagonist of the full length HFE, through hepcidin expression regulation in the liver or by controlling dietary iron absorption in the duodenum. PMID:21407826

Differential HFE gene expression is regulated by alternative splicing in human tissues.

PubMed

Martins, Rute; Silva, Bruno; Proença, Daniela; Faustino, Paula

2011-03-03

The pathophysiology of HFE-derived Hereditary Hemochromatosis and the function of HFE protein in iron homeostasis remain uncertain. Also, the role of alternative splicing in HFE gene expression regulation and the possible function of the corresponding protein isoforms are still unknown. The aim of this study was to gain insights into the physiological significance of these alternative HFE variants. Alternatively spliced HFE transcripts in diverse human tissues were identified by RT-PCR, cloning and sequencing. Total HFE transcripts, as well as two alternative splicing transcripts were quantified using a real-time PCR methodology. Intracellular localization, trafficking and protein association of GFP-tagged HFE protein variants were analysed in transiently transfected HepG2 cells by immunoprecipitation and immunofluorescence assays. Alternatively spliced HFE transcripts present both level- and tissue-specificity. Concerning the exon 2 skipping and intron 4 inclusion transcripts, the liver presents the lowest relative level, while duodenum presents one of the highest amounts. The protein resulting from exon 2 skipping transcript is unable to associate with β2M and TfR1 and reveals an ER retention. Conversely, the intron 4 inclusion transcript gives rise to a truncated, soluble protein (sHFE) that is mostly secreted by cells to the medium in association with β2M. HFE gene post-transcriptional regulation is clearly affected by a tissue-dependent alternative splicing mechanism. Among the corresponding proteins, a sHFE isoform stands out, which upon being secreted into the bloodstream, may act in remote tissues. It could be either an agonist or antagonist of the full length HFE, through hepcidin expression regulation in the liver or by controlling dietary iron absorption in the duodenum.

Prognostic value of decreased expression of RBM4 in human gastric cancer.

PubMed

Yong, Hongmei; Zhu, Huijun; Zhang, Shu; Zhao, Wei; Wang, Wei; Chen, Chen; Ding, Guipeng; Zhu, Lun; Zhu, Ziyuan; Liu, Huaidong; Zhang, Yongjie; Wen, Jinbo; Kang, Xing; Zhu, Jin; Feng, Zhenqing; Liu, Baorui

2016-06-21

RNA-binding motif 4 (RBM4) is a multifunctional protein that participates in regulating alternative splicing and mRNA translation. Its reduced expression has been associated with poor overall survival in lung cancer, breast cancer and ovarian cancer. We assessed RBM4 protein expression levels with immunohistochemistry in tissue microarrays containing malignant gastric cancer tissues and benign tissues from 813 patients. We also examined the expression levels of RBM4 mRNA in twenty-five paired gastric cancer samples and adjacent noncancerous tissues. Both RBM4 protein and mRNA expression levels were significantly lower in gastric cancer tissues compared with the adjacent noncancerous tissues. There was a significant association between reduced RBM4 protein expression and differentiation (P < 0.001), lymph node metastasis (P = 0.026), TNM state (P = 0.014) and distant metastasis (P = 0.036). Patients with reduced RBM4 expression (P < 0.001, CI = 0.315-0.710) and TNM stage III and IV (P < 0.001, CI = 4.757-11.166) had a poor overall survival. These findings suggest that RBM4 is a new biomarker in gastric cancer, as the reduced expression of this protein is correlated with poor differentiation, lymph node status and distant metastasis. Further, lower RBM4 expression is an independent prognostic marker for gastric cancer.

IL-4 and IL-13 employ discrete signaling pathways for target gene expression in alternatively activated monocytes/macrophages.

PubMed

Bhattacharjee, Ashish; Shukla, Meenakshi; Yakubenko, Valentin P; Mulya, Anny; Kundu, Suman; Cathcart, Martha K

2013-01-01

Monocytes/macrophages are innate immune cells that play a crucial role in the resolution of inflammation. In the presence of the Th2 cytokines interleukin-4 (IL-4) and interleukin-13 (IL-13), they display an anti-inflammatory profile and this activation pathway is known as alternative activation. In this study we compare and differentiate pathways mediated by IL-4 and IL-13 activation of human monocytes/macrophages. Here we report differential regulation of IL-4 and IL-13 signaling in monocytes/macrophages starting from IL-4/IL-13 cytokine receptors to Jak/Stat-mediated signaling pathways that ultimately control expression of several inflammatory genes. Our data demonstrate that although the receptor-associated tyrosine kinases Jak2 and Tyk2 are activated after the recruitment of IL-13 to its receptor (containing IL-4Rα and IL-13Rα1), IL-4 stimulates Jak1 activation. We further show that Jak2 is upstream of Stat3 activation and Tyk2 controls Stat1 and Stat6 activation in response to IL-13 stimulation. In contrast, Jak1 regulates Stat3 and Stat6 activation in IL-4-induced monocytes. Our results further reveal that although IL-13 utilizes both IL-4Rα/Jak2/Stat3 and IL-13Rα1/Tyk2/Stat1/Stat6 signaling pathways, IL-4 can use only the IL-4Rα/Jak1/Stat3/Stat6 cascade to regulate the expression of some critical inflammatory genes, including 15-lipoxygenase, monoamine oxidase A (MAO-A), and the scavenger receptor CD36. Moreover, we demonstrate here that IL-13 and IL-4 can uniquely affect the expression of particular genes such as dual-specificity phosphatase 1 and tissue inhibitor of metalloprotease-3 and do so through different Jaks. As evidence of differential regulation of gene function by IL-4 and IL-13, we further report that MAO-A-mediated reactive oxygen species generation is influenced by different Jaks. Collectively, these results have major implications for understanding the mechanism and function of alternatively activated monocytes/macrophages by IL-4 and IL-13 and add novel insights into the pathogenesis and potential treatment of various inflammatory diseases. Copyright © 2012 Elsevier Inc. All rights reserved.

Characterization and profiling of immunomodulatory genes of equine mesenchymal stromal cells from non-invasive sources

PubMed Central

2014-01-01

Introduction Mesenchymal stromal cells (MSCs) have been extensively studied for their promising capabilities in regenerative medicine. Although bone marrow is the best-known source for isolating equine MSCs, non-invasive alternative sources such as umbilical cord blood (UCB), umbilical cord matrix (UCM), and peripheral blood (PB) have also been reported. Methods Equine MSCs from three non-invasive alternative sources were isolated from six individual mares (PB) and their foals (UCB and UCM) at parturition. To minimize inter-horse variability, the samples from the three sources were matched within the same mare and for UCB and UCM even within the same foal from that specific mare. The following parameters were analyzed: (i) success rate of isolation, (ii) proliferation capacity, (iii) tri-lineage differentiation ability, (iv) immunophenotypical protein, and (v) immunomodulatory mRNA profiles. Linear regression models were fit to determine the association between the source of MSCs (UCB, UCM, PB) and (i) the moment of first observation, (ii) the moment of first passage, (iii) cell proliferation data, (iv) the expression of markers related to cell immunogenicity, and (v) the mRNA profile of immunomodulatory factors, except for hepatocyte growth factor (HGF) as no normal distribution could be obtained for the latter variable. To evaluate the association between the source of MSCs and the mRNA expression of HGF, the non-parametric Kruskal-Wallis test was performed instead. Results While equine MSCs could be isolated from all the UCB and PB samples, isolation from UCM was successful in only two samples because of contamination issues. Proliferation data showed that equine MSCs from all three sources could be easily expanded, although UCB-derived MSCs appeared significantly faster in culture than PB- or UCM-derived MSCs. Equine MSCs from both UCB and PB could be differentiated toward the osteo-, chondro-, and adipogenic lineage, in contrast to UCM-derived MSCs in which only chondro- and adipogenic differentiation could be confirmed. Regardless of the source, equine MSCs expressed the immunomodulatory genes CD40, CD80, HGF, and transforming growth factor-beta (TGFβ). In contrast, no mRNA expression was found for CD86, indoleamine 2,3-dioxygenase (IDO), and tumor necrosis factor-alpha (TNFα). Conclusions Whereas UCM seems less feasible because of the high contamination risks and low isolation success rates, UCB seems a promising alternative MSC source, especially when considering allogeneic MSC use. PMID:24418262

In vitro hepatic differentiation of human endometrial stromal stem cells.

PubMed

Yang, Xin-yuan; Wang, Wei; Li, Xu

2014-02-01

Human endometrial stromal stem cells (hESSCs) can differentiate into mesodermal and ectodermal cellular lineages in the endometrium. However, whether hESSCs can differentiate into functional hepatic-like cells is unknown. In this study, we developed a multiple-step induction protocol to differentiate hESSCs into functional hepatic-like cells in vitro. Endometrial stromal cells were isolated by magnetic affinity sorting using anti-epithelial cell adhesion molecule-coated Dynabeads. The enriched hESSCs were analyzed by flow cytometry and were able to differentiate into osteoblasts or adipocytes under proper induction media. To differentiate into hepatic-like cells, hESSCs were cultured in a stepwise system containing hepatocyte growth factor, fibroblast growth factor-4, oncostatin M, and trichostatin A for a total of 24 d. The hepatic-like cell differentiation was analyzed by confocal microscopy and immunocytochemical staining. Glycogen storage, cellular urea synthesis, and ammonia concentrations were measured. Hepatic-like cells were successfully generated from hESSCs and were identified by their epithelial-like shape characteristics and expression of specific biomarkers albumin and cytokeratin 8 accompanied with a reduction of alpha-fetoprotein and alpha-smooth muscle actin expression. The hepatic-like cells generated were functional as evidenced by urea synthesis and glycogen storage. Our study demonstrated that hESSCs were able to differentiate into hepatic-like cells in vitro. Thus, endometrial stromal cells may be used as an easily accessible alternative source of stem cells for potential therapeutic applications in liver disease.

Distinct regulation of alternative polyadenylation and gene expression by nuclear poly(A) polymerases

PubMed Central

Li, Wencheng; Laishram, Rakesh S.; Hoque, Mainul; Ji, Zhe

2017-01-01

Abstract Polyadenylation of nascent RNA by poly(A) polymerase (PAP) is important for 3′ end maturation of almost all eukaryotic mRNAs. Most mammalian genes harbor multiple polyadenylation sites (PASs), leading to expression of alternative polyadenylation (APA) isoforms with distinct functions. How poly(A) polymerases may regulate PAS usage and hence gene expression is poorly understood. Here, we show that the nuclear canonical (PAPα and PAPγ) and non-canonical (Star-PAP) PAPs play diverse roles in PAS selection and gene expression. Deficiencies in the PAPs resulted in perturbations of gene expression, with Star-PAP impacting lowly expressed mRNAs and long-noncoding RNAs to the greatest extent. Importantly, different PASs of a gene are distinctly regulated by different PAPs, leading to widespread relative expression changes of APA isoforms. The location and surrounding sequence motifs of a PAS appear to differentiate its regulation by the PAPs. We show Star-PAP-specific PAS usage regulates the expression of the eukaryotic translation initiation factor EIF4A1, the tumor suppressor gene PTEN and the long non-coding RNA NEAT1. The Star-PAP-mediated APA of PTEN is essential for DNA damage-induced increase of PTEN protein levels. Together, our results reveal a PAS-guided and PAP-mediated paradigm for gene expression in response to cellular signaling cues. PMID:28911096

Forced expression of the Ikaros 6 isoform in human placental blood CD34(+) cells impairs their ability to differentiate toward the B-lymphoid lineage.

PubMed

Tonnelle, C; Bardin, F; Maroc, C; Imbert, A M; Campa, F; Dalloul, A; Schmitt, C; Chabannon, C

2001-11-01

Studies in mice suggest that the Ikaros (Ik) gene encodes several isoforms and is a critical regulator of hematolymphoid differentiation. Little is known on the role of Ikaros in human stem cell differentiation. Herein, the biological consequences of the forced expression of Ikaros 6 (Ik6) in human placental blood CD34(+) progenitors are evaluated. Ik6 is one of the isoforms produced from the Ikaros premessenger RNA by alternative splicing and is thought to behave as a dominant negative isoform of the gene product because it lacks the DNA binding domain present in transcriptionally active isoforms. The results demonstrate that human cord blood CD34(+) cells that express high levels of Ik6 as a result of retrovirally mediated gene transfer have a reduced capacity to produce lymphoid B cells in 2 independent assays: (1) in vitro reinitiation of human hematopoiesis during coculture with the MS-5 murine stromal cell line and (2) xenotransplantation in nonobese diabetic-severe combined immunodeficient mice. These results suggest that Ikaros plays an important role in stem cell commitment in humans and that the balance between the different isoforms is a key element of this regulatory system; they support the hypothesis that posttranscriptional events can participate in the control of human hematopoietic differentiation.

N-Acetylaspartate (NAA) and N-Acetylaspartylglutamate (NAAG) Promote Growth and Inhibit Differentiation of Glioma Stem-like Cells*

PubMed Central

Long, Patrick M.; Moffett, John R.; Namboodiri, Aryan M. A.; Viapiano, Mariano S.; Lawler, Sean E.; Jaworski, Diane M.

2013-01-01

Metabolic reprogramming is a pathological feature of cancer and a driver of tumor cell transformation. N-Acetylaspartate (NAA) is one of the most abundant amino acid derivatives in the brain and serves as a source of metabolic acetate for oligodendrocyte myelination and protein/histone acetylation or a precursor for the synthesis of the neurotransmitter N-acetylaspartylglutamate (NAAG). NAA and NAAG as well as aspartoacylase (ASPA), the enzyme responsible for NAA degradation, are significantly reduced in glioma tumors, suggesting a possible role for decreased acetate metabolism in tumorigenesis. This study sought to examine the effects of NAA and NAAG on primary tumor-derived glioma stem-like cells (GSCs) from oligodendroglioma as well as proneural and mesenchymal glioblastoma, relative to oligodendrocyte progenitor cells (Oli-Neu). Although the NAA dicarboxylate transporter NaDC3 is primarily thought to be expressed by astrocytes, all cell lines expressed NaDC3 and, thus, are capable of NAA up-take. Treatment with NAA or NAAG significantly increased GSC growth and suppressed differentiation of Oli-Neu cells and proneural GSCs. Interestingly, ASPA was expressed in both the cytosol and nuclei of GSCs and exhibited greatest nuclear immunoreactivity in differentiation-resistant GSCs. Both NAA and NAAG elicited the expression of a novel immunoreactive ASPA species in select GSC nuclei, suggesting differential ASPA regulation in response to these metabolites. Therefore, this study highlights a potential role for nuclear ASPA expression in GSC malignancy and suggests that the use of NAA or NAAG is not an appropriate therapeutic approach to increase acetate bioavailability in glioma. Thus, an alternative acetate source is required. PMID:23884408

Identification of potential tumor-educated platelets RNA biomarkers in non-small-cell lung cancer by integrated bioinformatical analysis.

PubMed

Xue, Linlin; Xie, Li; Song, Xingguo; Song, Xianrang

2018-04-17

Platelets have emerged as key players in tumorigenesis and tumor progression. Tumor-educated platelet (TEP) RNA profile has the potential to diagnose non-small-cell lung cancer (NSCLC). The objective of this study was to identify potential TEP RNA biomarkers for the diagnosis of NSCLC and to explore the mechanisms in alternations of TEP RNA profile. The RNA-seq datasets GSE68086 and GSE89843 were downloaded from Gene Expression Omnibus DataSets (GEO DataSets). Then, the functional enrichment of the differentially expressed mRNAs was analyzed by the Database for Annotation Visualization and Integrated Discovery (DAVID). The miRNAs which regulated the differential mRNAs and the target mRNAs of miRNAs were identified by miRanda and miRDB. Then, the miRNA-mRNA regulatory network was visualized via Cytoscape software. Twenty consistently altered mRNAs (2 up-regulated and 18 down-regulated) were identified from the two GSE datasets, and they were significantly enriched in several biological processes, including transport and establishment of localization. Twenty identical miRNAs were found between exosomal miRNA-seq dataset and 229 miRNAs that regulated 20 consistently differential mRNAs in platelets. We also analyzed 13 spliceosomal mRNAs and their miRNA predictions; there were 27 common miRNAs between 206 differential exosomal miRNAs and 338 miRNAs that regulated 13 distinct spliceosomal mRNAs. This study identified 20 potential TEP RNA biomarkers in NSCLC for diagnosis by integrated bioinformatical analysis, and alternations in TEP RNA profile may be related to the post-transcriptional regulation and the splicing metabolisms of spliceosome. © 2018 Wiley Periodicals, Inc.

Osteogenic Differentiation of Human and Ovine Bone Marrow Stromal Cells in response to β-Glycerophosphate and Monosodium Phosphate.

PubMed

Bottagisio, Marta; Lovati, Arianna B; Lopa, Silvia; Moretti, Matteo

2015-08-01

Bone defects are severe burdens in clinics, and thus cell therapy offers an alternative strategy exploiting the features of bone marrow stromal cells (BMSCs). Sheep are a suitable orthopedic preclinical model for similarities with humans. This study compares the influence of two phosphate sources combined with bone morphogenetic protein-2 (BMP-2) on the osteogenic potential of human and ovine BMSCs. β-Glycerophosphate (β-GlyP) and monosodium phosphate (NaH2PO4) were used as organic and inorganic phosphate sources. Osteogenic differentiation of the BMSCs was assessed by calcified matrix, alkaline phosphatase (ALP) activity, and gene expression analysis. A higher calcified matrix deposition was detected in BMSCs cultured with NaH2PO4. Although no significant differences were detected among media for human BMSCs, β-GlyP with or without BMP-2 determined a positive trend in ALP levels compared to NaH2PO4. In contrast, NaH2PO4 had a positive effect on ALP levels in ovine BMSCs. β-GlyP better supported the expression of COL1A1 in human BMSCs, whereas all media enhanced RUNX2 and SPARC expression. Ovine BMSCs responded poorly to any media for RUNX2, COL1A1, and SPARC expression. NaH2PO4 improved calcified matrix deposition without upregulating the transcriptional expression of osteogenic markers. A further optimization of differentiation protocols needs to be performed to translate the procedures from preclinical to clinical models.

Two-Stage, In Silico Deconvolution of the Lymphocyte Compartment of the Peripheral Whole Blood Transcriptome in the Context of Acute Kidney Allograft Rejection

PubMed Central

Shannon, Casey P.; Balshaw, Robert; Ng, Raymond T.; Wilson-McManus, Janet E.; Keown, Paul; McMaster, Robert; McManus, Bruce M.; Landsberg, David; Isbel, Nicole M.; Knoll, Greg; Tebbutt, Scott J.

2014-01-01

Acute rejection is a major complication of solid organ transplantation that prevents the long-term assimilation of the allograft. Various populations of lymphocytes are principal mediators of this process, infiltrating graft tissues and driving cell-mediated cytotoxicity. Understanding the lymphocyte-specific biology associated with rejection is therefore critical. Measuring genome-wide changes in transcript abundance in peripheral whole blood cells can deliver a comprehensive view of the status of the immune system. The heterogeneous nature of the tissue significantly affects the sensitivity and interpretability of traditional analyses, however. Experimental separation of cell types is an obvious solution, but is often impractical and, more worrying, may affect expression, leading to spurious results. Statistical deconvolution of the cell type-specific signal is an attractive alternative, but existing approaches still present some challenges, particularly in a clinical research setting. Obtaining time-matched sample composition to biologically interesting, phenotypically homogeneous cell sub-populations is costly and adds significant complexity to study design. We used a two-stage, in silico deconvolution approach that first predicts sample composition to biologically meaningful and homogeneous leukocyte sub-populations, and then performs cell type-specific differential expression analysis in these same sub-populations, from peripheral whole blood expression data. We applied this approach to a peripheral whole blood expression study of kidney allograft rejection. The patterns of differential composition uncovered are consistent with previous studies carried out using flow cytometry and provide a relevant biological context when interpreting cell type-specific differential expression results. We identified cell type-specific differential expression in a variety of leukocyte sub-populations at the time of rejection. The tissue-specificity of these differentially expressed probe-set lists is consistent with the originating tissue and their functional enrichment consistent with allograft rejection. Finally, we demonstrate that the strategy described here can be used to derive useful hypotheses by validating a cell type-specific ratio in an independent cohort using the nanoString nCounter assay. PMID:24733377

The developmental dynamics of the Populus stem transcriptome.

PubMed

Chao, Qing; Gao, Zhi-Fang; Zhang, Dong; Zhao, Biligen-Gaowa; Dong, Feng-Qin; Fu, Chun-Xiang; Liu, Li-Jun; Wang, Bai-Chen

2018-05-31

The Populus shoot undergoes primary growth (longitudinal growth) followed by secondary growth (radial growth), which produces biomass that is an important source of energy worldwide. We adopted joint PacBio Iso-Seq and RNA-seq analysis to identify differentially expressed transcripts along a developmental gradient from the shoot apex to the fifth internode of Populus Nanlin895. We obtained 87,150 full-length transcripts, including 2,081 new isoforms and 62,058 new alternatively spliced isoforms, most of which were produced by intron retention, that were used to update the Populus annotation. Among these novel isoforms, there are 1,187 long noncoding RNAs and 356 fusion genes. Using this annotation, we found 15,838 differentially expressed transcripts along the shoot developmental gradient, of which 1,216 were transcription factors (TFs). Only a few of these genes were reported previously. The differential expression of these TFs suggests that they may play important roles in primary and secondary growth. AP2, ARF, YABBY and GRF TFs are highly expressed in the apex, whereas NAC, bZIP, PLATZ and HSF TFs are likely to be important for secondary growth. Overall, our findings provide evidence that long-read sequencing can complement short-read sequencing for cataloging and quantifying eukaryotic transcripts and increase our understanding of the vital and dynamic process of shoot development. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Maximum-entropy probability distributions under Lp-norm constraints

NASA Technical Reports Server (NTRS)

Dolinar, S.

1991-01-01

Continuous probability density functions and discrete probability mass functions are tabulated which maximize the differential entropy or absolute entropy, respectively, among all probability distributions with a given L sub p norm (i.e., a given pth absolute moment when p is a finite integer) and unconstrained or constrained value set. Expressions for the maximum entropy are evaluated as functions of the L sub p norm. The most interesting results are obtained and plotted for unconstrained (real valued) continuous random variables and for integer valued discrete random variables. The maximum entropy expressions are obtained in closed form for unconstrained continuous random variables, and in this case there is a simple straight line relationship between the maximum differential entropy and the logarithm of the L sub p norm. Corresponding expressions for arbitrary discrete and constrained continuous random variables are given parametrically; closed form expressions are available only for special cases. However, simpler alternative bounds on the maximum entropy of integer valued discrete random variables are obtained by applying the differential entropy results to continuous random variables which approximate the integer valued random variables in a natural manner. All the results are presented in an integrated framework that includes continuous and discrete random variables, constraints on the permissible value set, and all possible values of p. Understanding such as this is useful in evaluating the performance of data compression schemes.

Global isoform-specific transcript alterations and deregulated networks in clear cell renal cell carcinoma

PubMed Central

Hamilton, Michael J.; Girke, Thomas; Martinez, Ernest

2018-01-01

Extensive genome-wide analyses of deregulated gene expression have now been performed for many types of cancer. However, most studies have focused on deregulation at the gene-level, which may overlook the alterations of specific transcripts for a given gene. Clear cell renal cell carcinoma (ccRCC) is one of the best-characterized and most pervasive renal cancers, and ccRCCs are well-documented to have aberrant RNA processing. In the present study, we examine the extent of aberrant isoform-specific RNA expression by reporting a comprehensive transcript-level analysis, using the new kallisto-sleuth-RATs pipeline, investigating coding and non-coding differential transcript expression in ccRCC. We analyzed 50 ccRCC tumors and their matched normal samples from The Cancer Genome Altas datasets. We identified 7,339 differentially expressed transcripts and 94 genes exhibiting differential transcript isoform usage in ccRCC. Additionally, transcript-level coexpression network analyses identified vasculature development and the tricarboxylic acid cycle as the most significantly deregulated networks correlating with ccRCC progression. These analyses uncovered several uncharacterized transcripts, including lncRNAs FGD5-AS1 and AL035661.1, as potential regulators of the tricarboxylic acid cycle associated with ccRCC progression. As ccRCC still presents treatment challenges, our results provide a new resource of potential therapeutics targets and highlight the importance of exploring alternative methodologies in transcriptome-wide studies.

Ibandronate promotes osteogenic differentiation of periodontal ligament stem cells by regulating the expression of microRNAs

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

Zhou, Qiang; Zhao, Zhi-Ning; Cheng, Jing-Tao

2011-01-07

Research highlights: {yields} Ibandronate significantly promote the proliferation of PDLSC cells. {yields} Ibandronate enhanced the expression of ALP, COL-1, OPG, OCN, Runx2. {yields} The expression of a class of miRNAs, e.g., miR-18a, miR-133a, miR-141 and miR-19a, was significantly modified in PDLSC cells cultured with ibandronate. {yields} Ibandronate regulates the expression of diverse bone formation-related genes via miRNAs in PDLSCs. {yields} Ibandronate can suppress the activity of osteoclast while promoting the proliferation of osteoblast by regulating the expression of microRNAs. -- Abstract: Bisphosphonates (BPs) have a profound effect on bone resorption and are widely used to treat osteoclast-mediated bone diseases. Theymore » suppress bone resorption by inhibiting the activity of mature osteoclasts and/or the formation of new osteoclasts. Osteoblasts may be an alternative target for BPs. Periodontal ligament stem cells (PDLSCs) exhibit osteoblast-like features and are capable of differentiating into osteoblasts or cementoblasts. This study aimed to determine the effects of ibandronate, a nitrogen-containing BP, on the proliferation and the differentiation of PDLSCs and to identify the microRNAs (miRNAs) that mediate these effects. The PDLSCs were treated with ibandronate, and cell proliferation was measured using the MTT (3-dimethylthiazol-2,5-diphenyltetrazolium bromide) assay. The expression of genes and miRNAs involved in osteoblastic differentiation was assayed using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). Ibandronate promoted the proliferation of PDLSCs and enhanced the expression of alkaline phosphatase (ALP), type I collagen (COL-1), osteoprotegerin (OPG), osteocalcin (OCN), and Runx2. The expression of miRNAs, including miR-18a, miR-133a, miR-141 and miR-19a, was significantly altered in the PDLSCs cultured with ibandronate. In PDLSCs, ibandronate regulates the expression of diverse bone formation-related genes via miRNAs. The exact mechanism underlying the role of ibandronate in osteoblasts has not been completely understood. Ibandronate may suppress the activity of osteoclasts while promoting the proliferation of osteoblasts by regulating the expression of miRNAs.« less

Involvement of Alternative Splicing in Barley Seed Germination

PubMed Central

Zhang, Qisen; Zhang, Xiaoqi; Wang, Songbo; Tan, Cong; Zhou, Gaofeng; Li, Chengdao

2016-01-01

Seed germination activates many new biological processes including DNA, membrane and mitochondrial repairs and requires active protein synthesis and sufficient energy supply. Alternative splicing (AS) regulates many cellular processes including cell differentiation and environmental adaptations. However, limited information is available on the regulation of seed germination at post-transcriptional levels. We have conducted RNA-sequencing experiments to dissect AS events in barley seed germination. We identified between 552 and 669 common AS transcripts in germinating barley embryos from four barley varieties (Hordeum vulgare L. Bass, Baudin, Harrington and Stirling). Alternative 3’ splicing (34%-45%), intron retention (32%-34%) and alternative 5’ splicing (16%-21%) were three major AS events in germinating embryos. The AS transcripts were predominantly mapped onto ribosome, RNA transport machineries, spliceosome, plant hormone signal transduction, glycolysis, sugar and carbon metabolism pathways. Transcripts of these genes were also very abundant in the early stage of seed germination. Correlation analysis of gene expression showed that AS hormone responsive transcripts could also be co-expressed with genes responsible for protein biosynthesis and sugar metabolisms. Our RNA-sequencing data revealed that AS could play important roles in barley seed germination. PMID:27031341

The transcriptional landscape of seasonal coat colour moult in the snowshoe hare.

PubMed

Ferreira, Mafalda S; Alves, Paulo C; Callahan, Colin M; Marques, João P; Mills, L Scott; Good, Jeffrey M; Melo-Ferreira, José

2017-08-01

Seasonal coat colour change is an important adaptation to seasonally changing environments but the evolution of this and other circannual traits remains poorly understood. In this study, we use gene expression to understand seasonal coat colour moulting in wild snowshoe hares (Lepus americanus). We used hair colour to follow the progression of the moult, simultaneously sampling skin from three moulting stages in hares collected during the peak of the spring moult from white winter to brown summer pelage. Using RNA sequencing, we tested whether patterns of expression were consistent with predictions based on the established phases of the hair growth cycle. We found functionally consistent clustering across skin types, with 766 genes differentially expressed between moult stages. "White" pelage showed more differentially expressed genes that were upregulated relative to other skin types, involved in the transition between late telogen (quiescent stage) and the onset of anagen (proliferative stage). Skin samples from transitional "intermediate" and "brown" pelage were transcriptionally similar and resembled the regressive transition to catagen (regressive stage). We also detected differential expression of several key circadian clock and pigmentation genes, providing important means to dissect the bases of alternate seasonal colour morphs. Our results reveal that pelage colour is a useful biomarker for seasonal change but that there is a consistent lag between the main gene expression waves and change in visible coat colour. These experiments establish that developmental sampling from natural populations of nonmodel organisms can provide a crucial resource to dissect the genetic basis and evolution of complex seasonally changing traits. © 2017 John Wiley & Sons Ltd.

Hepatogenic and neurogenic differentiation of bone marrow mesenchymal stem cells from abattoir-derived bovine fetuses

PubMed Central

2014-01-01

Background Mesenchymal stem cells (MSC) are multipotent progenitor cells characterized by their ability to both self-renew and differentiate into tissues of mesodermal origin. The plasticity or transdifferentiation potential of MSC is not limited to mesodermal derivatives, since under appropriate cell culture conditions and stimulation by bioactive factors, MSC have also been differentiated into endodermal (hepatocytes) and neuroectodermal (neurons) cells. The potential of MSC for hepatogenic and neurogenic differentiation has been well documented in different animal models; however, few reports are currently available on large animal models. In the present study we sought to characterize the hepatogenic and neurogenic differentiation and multipotent potential of bovine MSC (bMSC) isolated from bone marrow (BM) of abattoir-derived fetuses. Results Plastic-adherent bMSC isolated from fetal BM maintained a fibroblast-like morphology under monolayer culture conditions. Flow cytometric analysis demonstrated that bMSC populations were positive for MSC markers CD29 and CD73 and pluripotency markers OCT4 and NANOG; whereas, were negative for hematopoietic markers CD34 and CD45. Levels of mRNA of hepatic genes α-fetoprotein (AFP), albumin (ALB), alpha1 antitrypsin (α1AT), connexin 32 (CNX32), tyrosine aminotransferase (TAT) and cytochrome P450 (CYP3A4) were up-regulated in bMSC during a 28-Day period of hepatogenic differentiation. Functional analyses in differentiated bMSC cultures evidenced an increase (P < 0.05) in albumin and urea production and glycogen storage. bMSC cultured under neurogenic conditions expressed NESTIN and MAP2 proteins at 24 h of culture; whereas, at 144 h also expressed TRKA and PrPC. Levels of MAP2 and TRKA mRNA were up-regulated at the end of the differentiation period. Conversely, bMSC expressed lower levels of NANOG mRNA during both hepatogenic and neurogenic differentiation processes. Conclusion The expression patterns of linage-specific markers and the production of functional metabolites support the potential for hepatogenic and neurogenic differentiation of bMSC isolated from BM of abattoir-derived fetuses. The simplicity of isolation and the potential to differentiate into a wide variety of cell lineages lays the foundation for bMSC as an interesting alternative for investigation in MSC biology and eventual applications for regenerative therapy in veterinary medicine. PMID:25011474

Computational identification and validation of alternative splicing in ZSF1 rat RNA-seq data, a preclinical model for type 2 diabetic nephropathy.

PubMed

Zhang, Chi; Dower, Ken; Zhang, Baohong; Martinez, Robert V; Lin, Lih-Ling; Zhao, Shanrong

2018-05-16

Obese ZSF1 rats exhibit spontaneous time-dependent diabetic nephropathy and are considered to be a highly relevant animal model of progressive human diabetic kidney disease. We previously identified gene expression changes between disease and control animals across six time points from 12 to 41 weeks. In this study, the same data were analysed at the isoform and exon levels to reveal additional disease mechanisms that may be governed by alternative splicing. Our analyses identified alternative splicing patterns in genes that may be implicated in disease pathogenesis (such as Shc1, Serpinc1, Epb4.1l5, and Il-33), which would have been overlooked in standard gene-level analysis. The alternatively spliced genes were enriched in pathways related to cell adhesion, cell-cell interactions/junctions, and cytoskeleton signalling, whereas the differentially expressed genes were enriched in pathways related to immune response, G protein-coupled receptor, and cAMP signalling. Our findings indicate that additional mechanistic insights can be gained from exon- and isoform-level data analyses over standard gene-level analysis. Considering alternative splicing is poorly conserved between rodents and humans, it is noted that this work is not translational, but the point holds true that additional insights can be gained from alternative splicing analysis of RNA-seq data.

Informatics for RNA Sequencing: A Web Resource for Analysis on the Cloud

PubMed Central

Griffith, Malachi; Walker, Jason R.; Spies, Nicholas C.; Ainscough, Benjamin J.; Griffith, Obi L.

2015-01-01

Massively parallel RNA sequencing (RNA-seq) has rapidly become the assay of choice for interrogating RNA transcript abundance and diversity. This article provides a detailed introduction to fundamental RNA-seq molecular biology and informatics concepts. We make available open-access RNA-seq tutorials that cover cloud computing, tool installation, relevant file formats, reference genomes, transcriptome annotations, quality-control strategies, expression, differential expression, and alternative splicing analysis methods. These tutorials and additional training resources are accompanied by complete analysis pipelines and test datasets made available without encumbrance at www.rnaseq.wiki. PMID:26248053

Large Field Visualization with Demand-Driven Calculation

NASA Technical Reports Server (NTRS)

Moran, Patrick J.; Henze, Chris

1999-01-01

We present a system designed for the interactive definition and visualization of fields derived from large data sets: the Demand-Driven Visualizer (DDV). The system allows the user to write arbitrary expressions to define new fields, and then apply a variety of visualization techniques to the result. Expressions can include differential operators and numerous other built-in functions, ail of which are evaluated at specific field locations completely on demand. The payoff of following a demand-driven design philosophy throughout becomes particularly evident when working with large time-series data, where the costs of eager evaluation alternatives can be prohibitive.

Alternative Splicing in Neurogenesis and Brain Development.

PubMed

Su, Chun-Hao; D, Dhananjaya; Tarn, Woan-Yuh

2018-01-01

Alternative splicing of precursor mRNA is an important mechanism that increases transcriptomic and proteomic diversity and also post-transcriptionally regulates mRNA levels. Alternative splicing occurs at high frequency in brain tissues and contributes to every step of nervous system development, including cell-fate decisions, neuronal migration, axon guidance, and synaptogenesis. Genetic manipulation and RNA sequencing have provided insights into the molecular mechanisms underlying the effects of alternative splicing in stem cell self-renewal and neuronal fate specification. Timely expression and perhaps post-translational modification of neuron-specific splicing regulators play important roles in neuronal development. Alternative splicing of many key transcription regulators or epigenetic factors reprograms the transcriptome and hence contributes to stem cell fate determination. During neuronal differentiation, alternative splicing also modulates signaling activity, centriolar dynamics, and metabolic pathways. Moreover, alternative splicing impacts cortical lamination and neuronal development and function. In this review, we focus on recent progress toward understanding the contributions of alternative splicing to neurogenesis and brain development, which has shed light on how splicing defects may cause brain disorders and diseases.

RNA-Seq analysis identifies aberrant RNA splicing of TRIP12 in acute myeloid leukemia patients at remission.

PubMed

Gao, Panke; Jin, Zhen; Cheng, Yingying; Cao, Xiangshan

2014-10-01

Aberrant splicing events play important roles in the pathogenesis of acute myeloid leukemia (AML). To investigate the aberrant splicing events in AML during treatment, we carried out RNA sequencing in peripheral mononuclear cell samples from a patient with complete remission. In addition to the sequencing samples, selected splicing events were confirmed and validated with real-time quantitative RT-PCR in another seven pairs of samples. A total of 4.05 and 3.39 GB clean data of the AML and remission sample were generated, respectively, and 2,223 differentially expressed genes (DEGs) were identified. Integrated with gene expression profiling on T cells from AML patients compared with healthy donors, 82 DEGs were also differentially expressed in AML CD4 T cells and CD8 T cells. Twenty-three alternative splicing events were considered to be confidential, and they were involved in many biological processes, such as RNA processing, cellular macromolecule catabolic process, and DNA binding process. An exon3-skipping event in TRIP12 was detected in patients at remission and further validated in another three independent samples. TRIP12 is an ubiquitin ligase of ARF, which suppresses aberrant cell growth by activating p53 responses. The exon3-skipping isoform of TRIP12 increased significantly after treatment. Our results may provide new understanding of AML, and the confirmed alternative splicing event of TRIP12 may be used as potential target for future investigations.

Transcriptional diversity during lineage commitment of human blood progenitors.

PubMed

Chen, Lu; Kostadima, Myrto; Martens, Joost H A; Canu, Giovanni; Garcia, Sara P; Turro, Ernest; Downes, Kate; Macaulay, Iain C; Bielczyk-Maczynska, Ewa; Coe, Sophia; Farrow, Samantha; Poudel, Pawan; Burden, Frances; Jansen, Sjoert B G; Astle, William J; Attwood, Antony; Bariana, Tadbir; de Bono, Bernard; Breschi, Alessandra; Chambers, John C; Consortium, Bridge; Choudry, Fizzah A; Clarke, Laura; Coupland, Paul; van der Ent, Martijn; Erber, Wendy N; Jansen, Joop H; Favier, Rémi; Fenech, Matthew E; Foad, Nicola; Freson, Kathleen; van Geet, Chris; Gomez, Keith; Guigo, Roderic; Hampshire, Daniel; Kelly, Anne M; Kerstens, Hindrik H D; Kooner, Jaspal S; Laffan, Michael; Lentaigne, Claire; Labalette, Charlotte; Martin, Tiphaine; Meacham, Stuart; Mumford, Andrew; Nürnberg, Sylvia; Palumbo, Emilio; van der Reijden, Bert A; Richardson, David; Sammut, Stephen J; Slodkowicz, Greg; Tamuri, Asif U; Vasquez, Louella; Voss, Katrin; Watt, Stephen; Westbury, Sarah; Flicek, Paul; Loos, Remco; Goldman, Nick; Bertone, Paul; Read, Randy J; Richardson, Sylvia; Cvejic, Ana; Soranzo, Nicole; Ouwehand, Willem H; Stunnenberg, Hendrik G; Frontini, Mattia; Rendon, Augusto

2014-09-26

Blood cells derive from hematopoietic stem cells through stepwise fating events. To characterize gene expression programs driving lineage choice, we sequenced RNA from eight primary human hematopoietic progenitor populations representing the major myeloid commitment stages and the main lymphoid stage. We identified extensive cell type-specific expression changes: 6711 genes and 10,724 transcripts, enriched in non-protein-coding elements at early stages of differentiation. In addition, we found 7881 novel splice junctions and 2301 differentially used alternative splicing events, enriched in genes involved in regulatory processes. We demonstrated experimentally cell-specific isoform usage, identifying nuclear factor I/B (NFIB) as a regulator of megakaryocyte maturation-the platelet precursor. Our data highlight the complexity of fating events in closely related progenitor populations, the understanding of which is essential for the advancement of transplantation and regenerative medicine. Copyright © 2014, American Association for the Advancement of Science.

Constraint reasoning in deep biomedical models.

PubMed

Cruz, Jorge; Barahona, Pedro

2005-05-01

Deep biomedical models are often expressed by means of differential equations. Despite their expressive power, they are difficult to reason about and make decisions, given their non-linearity and the important effects that the uncertainty on data may cause. The objective of this work is to propose a constraint reasoning framework to support safe decisions based on deep biomedical models. The methods used in our approach include the generic constraint propagation techniques for reducing the bounds of uncertainty of the numerical variables complemented with new constraint reasoning techniques that we developed to handle differential equations. The results of our approach are illustrated in biomedical models for the diagnosis of diabetes, tuning of drug design and epidemiology where it was a valuable decision-supporting tool notwithstanding the uncertainty on data. The main conclusion that follows from the results is that, in biomedical decision support, constraint reasoning may be a worthwhile alternative to traditional simulation methods, especially when safe decisions are required.

Functionalized α-Helical Peptide Hydrogels for Neural Tissue Engineering

PubMed Central

2015-01-01

Trauma to the central and peripheral nervous systems often lead to serious morbidity. Current surgical methods for repairing or replacing such damage have limitations. Tissue engineering offers a potential alternative. Here we show that functionalized α-helical-peptide hydrogels can be used to induce attachment, migration, proliferation and differentiation of murine embryonic neural stem cells (NSCs). Specifically, compared with undecorated gels, those functionalized with Arg-Gly-Asp-Ser (RGDS) peptides increase the proliferative activity of NSCs; promote their directional migration; induce differentiation, with increased expression of microtubule-associated protein-2, and a low expression of glial fibrillary acidic protein; and lead to the formation of larger neurospheres. Electrophysiological measurements from NSCs grown in RGDS-decorated gels indicate developmental progress toward mature neuron-like behavior. Our data indicate that these functional peptide hydrogels may go some way toward overcoming the limitations of current approaches to nerve-tissue repair. PMID:26240838

VEGF induces differentiation of functional endothelium from human embryonic stem cells: implications for tissue engineering

PubMed Central

Nourse, Marilyn B.; Halpin, Daniel E.; Scatena, Marta; Mortisen, Derek J.; Tulloch, Nathaniel L.; Hauch, Kip D.; Torok-Storb, Beverly; Ratner, Buddy D.; Pabon, Lil; Murry, Charles E.

2010-01-01

Objective Human embryonic stem cells (hESCs) offer a sustainable source of endothelial cells for therapeutic vascularization and tissue engineering, but current techniques for generating these cells remain inefficient. We endeavored to induce and isolate functional endothelial cells from differentiating hESCs. Methods and Results To enhance endothelial cell differentiation above a baseline of ∼2% in embryoid body (EB) spontaneous differentiation, three alternate culture conditions were compared. Vascular endothelial growth factor (VEGF) treatment of EBs showed the best induction, with markedly increased expression of endothelial cell proteins CD31, VE-Cadherin, and von Willebrand Factor, but not the hematopoietic cell marker CD45. CD31 expression peaked around days 10-14. Continuous VEGF treatment resulted in a four- to five-fold enrichment of CD31+ cells but did not increase endothelial proliferation rates, suggesting a primary effect on differentiation. CD31+ cells purified from differentiating EBs upregulated ICAM-1 and VCAM-1 in response to TNFα, confirming their ability to function as endothelial cells. These cells also expressed multiple endothelial genes and formed lumenized vessels when seeded onto porous poly(2-hydroxyethyl methacrylate) scaffolds and implanted in vivo subcutaneously in athymic rats. Collagen gel constructs containing hESC-derived endothelial cells and implanted into infarcted nude rat hearts formed robust networks of patent vessels filled with host blood cells. Conclusions VEGF induces functional endothelial cells from hESCs independent of endothelial cell proliferation. These enrichment methods increase endothelial cell yield, enabling applications for revascularization as well as basic studies of human endothelial biology. We demonstrate the ability of hESC-derived endothelial cells to facilitate vascularization of tissue-engineered implants. PMID:19875721

Whole-transcriptome brain expression and exon-usage profiling in major depression and suicide: evidence for altered glial, endothelial and ATPase activity

PubMed Central

Pantazatos, Spiro P.; Huang, Yung-yu; Rosoklija, Gorazd B.; Dwork, Andrew J.; Arango, Victoria; Mann, J. John

2016-01-01

Brain gene expression profiling studies of suicide and depression using oligonucleotide microarrays have often failed to distinguish these two phenotypes. Moreover, next generation sequencing (NGS) approaches are more accurate in quantifying gene expression and can detect alternative splicing. Using RNA-seq, we examined whole-exome gene and exon expression in non-psychiatric controls (CON, N=29), DSM-IV major depressive disorder suicides (MDD-S, N=21) and MDD non-suicides (MDD, N=9) in dorsal lateral prefrontal cortex (Brodmann Area 9) of sudden-death medication-free individuals postmortem. Using small RNA-seq, we also examined miRNA expression (9 samples per group). DeSeq2 identified thirty-five genes differentially expressed between groups and surviving adjustment for false discovery rate (adjusted p<0.1). In depression, altered genes include humanin like-8 (MTRNRL8), interleukin-8 (IL8), and serpin peptidase inhibitor, clade H (SERPINH1) and chemokine ligand 4 (CCL4), while exploratory gene ontology (GO) analyses revealed lower expression of immune-related pathways such as chemokine receptor activity, chemotaxis and cytokine biosynthesis, and angiogenesis and vascular development in (adjusted p<0.1). Hypothesis-driven GO analysis suggests lower expression of genes involved in oligodendrocyte differentiation, regulation of glutamatergic neurotransmission, and oxytocin receptor expression in both suicide and depression, and provisional evidence for altered DNA-dependent ATPase expression in suicide only. DEXSEq analysis identified differential exon usage in ATPase, class II, type 9B (adjusted p<0.1) in depression. Differences in miRNA expression or structural gene variants were not detected. Results lend further support for models in which deficits in microglial, endothelial (blood-brain barrier), ATPase activity and astrocytic cell functions contribute to MDD and suicide, and identify putative pathways and mechanisms for further study in these disorders. PMID:27528462

Whole-transcriptome brain expression and exon-usage profiling in major depression and suicide: evidence for altered glial, endothelial and ATPase activity.

PubMed

Pantazatos, S P; Huang, Y-Y; Rosoklija, G B; Dwork, A J; Arango, V; Mann, J J

2017-05-01

Brain gene expression profiling studies of suicide and depression using oligonucleotide microarrays have often failed to distinguish these two phenotypes. Moreover, next generation sequencing approaches are more accurate in quantifying gene expression and can detect alternative splicing. Using RNA-seq, we examined whole-exome gene and exon expression in non-psychiatric controls (CON, N=29), DSM-IV major depressive disorder suicides (MDD-S, N=21) and MDD non-suicides (MDD, N=9) in the dorsal lateral prefrontal cortex (Brodmann Area 9) of sudden death medication-free individuals post mortem. Using small RNA-seq, we also examined miRNA expression (nine samples per group). DeSeq2 identified 35 genes differentially expressed between groups and surviving adjustment for false discovery rate (adjusted P<0.1). In depression, altered genes include humanin-like-8 (MTRNRL8), interleukin-8 (IL8), and serpin peptidase inhibitor, clade H (SERPINH1) and chemokine ligand 4 (CCL4), while exploratory gene ontology (GO) analyses revealed lower expression of immune-related pathways such as chemokine receptor activity, chemotaxis and cytokine biosynthesis, and angiogenesis and vascular development in (adjusted P<0.1). Hypothesis-driven GO analysis suggests lower expression of genes involved in oligodendrocyte differentiation, regulation of glutamatergic neurotransmission, and oxytocin receptor expression in both suicide and depression, and provisional evidence for altered DNA-dependent ATPase expression in suicide only. DEXSEq analysis identified differential exon usage in ATPase, class II, type 9B (adjusted P<0.1) in depression. Differences in miRNA expression or structural gene variants were not detected. Results lend further support for models in which deficits in microglial, endothelial (blood-brain barrier), ATPase activity and astrocytic cell functions contribute to MDD and suicide, and identify putative pathways and mechanisms for further study in these disorders.

IL-34 is associated with obesity, chronic inflammation, and insulin resistance.

PubMed

Chang, Eun-Ju; Lee, Seul Ki; Song, Young Sook; Jang, Yeon Jin; Park, Hye Soon; Hong, Joon Pio; Ko, A Ra; Kim, Dae Yeon; Kim, Jong-Hyeok; Lee, Yeon Ji; Heo, Yoon-Suk

2014-07-01

IL-34 is a recently identified alternative ligand for colony-stimulating factor-1 (CSF-1) receptor. IL-34 and CSF-1 are regulators of differentiation, proliferation, and survival in mononuclear phagocytes. Here, we investigated the IL-34 serum concentration and expression in human adipose tissues and any associations with insulin resistance. We recruited 19 nondiabetic obese women, 9 type 2 diabetic women, and 27 normal-weight women. Metabolic parameters, abdominal fat distribution, serum IL-34 concentration, and IL-34 mRNA expression were measured in abdominal sc adipose tissue (SAT) and visceral adipose tissue (VAT). In addition, the expression/secretion and putative effects of IL-34 were assessed in human differentiated adipocytes. Serum IL-34 concentration was measured before and 5 to 9 months after laparoscopic Roux-en-Y gastric bypass surgery was performed on the 20 obese patients. Regardless of diabetes status, obese patients demonstrated significantly higher serum IL-34 concentrations than controls. Serum IL-34 was significantly and positively correlated with insulin resistance-related metabolic parameters. IL-34 mRNA was significantly higher in VAT than SAT. IL-34 was expressed in adipocytes as well as nonadipocytes, and expression was significantly higher during adipogenesis. In differentiated adipocytes, the expression/secretion of IL-34 was enhanced by TNFα and IL-1β. In addition, IL-34 augmented fat accumulation and inhibited the stimulatory effects of insulin on glucose transport. Moreover, serum IL-34 was significantly decreased after Roux-en-Y gastric bypass-induced weight loss. The present study demonstrates, for the first time, that IL-34 is expressed in human adipose tissues and the circulating concentration is significantly elevated in obese patients. This suggests that IL-34 is associated with insulin resistance.

Emotional facial expressions differentially influence predictions and performance for face recognition.

PubMed

Nomi, Jason S; Rhodes, Matthew G; Cleary, Anne M

2013-01-01

This study examined how participants' predictions of future memory performance are influenced by emotional facial expressions. Participants made judgements of learning (JOLs) predicting the likelihood that they would correctly identify a face displaying a happy, angry, or neutral emotional expression in a future two-alternative forced-choice recognition test of identity (i.e., recognition that a person's face was seen before). JOLs were higher for studied faces with happy and angry emotional expressions than for neutral faces. However, neutral test faces with studied neutral expressions had significantly higher identity recognition rates than neutral test faces studied with happy or angry expressions. Thus, these data are the first to demonstrate that people believe happy and angry emotional expressions will lead to better identity recognition in the future relative to neutral expressions. This occurred despite the fact that neutral expressions elicited better identity recognition than happy and angry expressions. These findings contribute to the growing literature examining the interaction of cognition and emotion.

Improved osteogenesis and upregulated immunogenicity in human placenta-derived mesenchymal stem cells primed with osteogenic induction medium.

PubMed

Fu, Xuejie; Yang, Huilin; Zhang, Hui; Wang, Guichao; Liu, Ke; Gu, Qiaoli; Tao, Yunxia; Chen, Guangcun; Jiang, Xiaohua; Li, Gang; Gu, Yanzheng; Shi, Qin

2016-09-20

Mesenchymal stem cells (MSCs) are widely used in cell-based therapy owing to their multilineage potential and low immunogenicity. However, low differentiation efficiency and unpredictable immunogenicity of allogeneic MSCs in vivo limit their success in therapeutic treatment. Herein, we evaluated the differentiation potential and immunogenicity of human placenta-derived MSCs manipulated with osteogenic priming and dedifferentiation process. MSCs from human placentas were subjected to osteogenic induction and then cultivated in osteogenic factor-free media; the obtained cell population was termed dedifferentiated mesenchymal stem cells (De-MSCs). De-MSCs were induced into osteo-, chondro- and adipo-differentiation in vitro. Cell proliferation was quantified by a Cell-Counting Kit-8 or tritiated thymidine ([(3)H]-TdR) incorporation. Meanwhile, the osteogenesis of De-MSCs in vivo was assayed by real-time PCR and histological staining. The expressions of stem cell markers and co-stimulatory molecules on De-MSCs and lymphocytes from primed BALB/c mouse with De-MSCs were determined by flow cytometry. De-MSCs exhibited some properties similar to MSCs including multiple differentiation potential and hypoimmunogenicity. Upon re-osteogenic induction, De-MSCs exhibited higher differentiation capability than MSCs both in vitro and in vivo. Of note, De-MSCs had upregulated immunogenicity in association with their osteogenesis, reflected by the alternated expressions of co-stimulatory molecules on the surface and decreased suppression on T cell activation. Functionally, De-MSC-derived osteoblasts could prime lymphocytes of peripheral blood and spleen in BALB/c mice in vivo. These data are of great significance for the potential application of De-MSCs as an alternative resource for regenerative medicine and tissue engineering. In order to avoid being rejected by the host during allogeneic De-MSC therapy, we suggest that immune intervention should be considered to boost the immune acceptance and integration because of the upregulated immunogenicity of De-MSCs with redifferentiation in clinical applications.

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

Gao, Xiugong, E-mail: xiugong.gao@fda.hhs.gov; Sprando, Robert L.; Yourick, Jeffrey J.

Developmental toxicity testing has traditionally relied on animal models which are costly, time consuming, and require the sacrifice of large numbers of animals. In addition, there are significant disparities between human beings and animals in their responses to chemicals. Thalidomide is a species-specific developmental toxicant that causes severe limb malformations in humans but not in mice. Here, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on differentiation of mouse embryonic stem cells (mESCs). C57BL/6 mESCs were allowed to differentiate spontaneously and RNA was collected at 24, 48, and 72 h after exposuremore » to 0.25 mM thalidomide. Global gene expression analysis using microarrays revealed hundreds of differentially expressed genes upon thalidomide exposure that were enriched in gene ontology (GO) terms and canonical pathways associated with embryonic development and differentiation. In addition, many genes were found to be involved in small GTPases-mediated signal transduction, heart development, and inflammatory responses, which coincide with clinical evidences and may represent critical embryotoxicities of thalidomide. These results demonstrate that transcriptomics in combination with mouse embryonic stem cell differentiation is a promising alternative model for developmental toxicity assessment. - Highlights: • Studied genomic changes in mouse embryonic stem cells upon thalidomide exposure • Identified gene expression changes that may represent thalidomide embryotoxicity • The toxicogenomic changes coincide well with known thalidomide clinical outcomes. • The mouse embryonic stem cell model is suitable for developmental toxicity testing. • The model has the potential for high-throughput screening of a multitude of compounds.« less

Relating Androgen Receptor Conformation to Function in Prostate Cancer Cells

DTIC Science & Technology

2005-01-01

line development , but have made progress towards resolving these issues and in development of alternate strategies. Task 1. Development of AR and...conformation. Task 2. Development of LNCaP Cells to Express Human AR mutants. We experienced unexpected difficulties in Task 2. We transfected the TET...Coactivators in AR Transactivation Summary Androgens drive sex differentiation, bone and muscle development , and promote growth of hormone dependent cancers

Gene-expression signatures of Atlantic salmon's plastic life cycle

USGS Publications Warehouse

Aubin-Horth, N.; Letcher, B.H.; Hofmann, H.A.

2009-01-01

How genomic expression differs as a function of life history variation is largely unknown. Atlantic salmon exhibits extreme alternative life histories. We defined the gene-expression signatures of wild-caught salmon at two different life stages by comparing the brain expression profiles of mature sneaker males and immature males, and early migrants and late migrants. In addition to life-stage-specific signatures, we discovered a surprisingly large gene set that was differentially regulated-at similar magnitudes, yet in opposite direction-in both life history transitions. We suggest that this co-variation is not a consequence of many independent cellular and molecular switches in the same direction but rather represents the molecular equivalent of a physiological shift orchestrated by one or very few master regulators. ?? 2009 Elsevier Inc. All rights reserved.

Gene-expression signatures of Atlantic salmon’s plastic life cycle

PubMed Central

Aubin-Horth, Nadia; Letcher, Benjamin H.; Hofmann, Hans A.

2009-01-01

How genomic expression differs as a function of life history variation is largely unknown. Atlantic salmon exhibits extreme alternative life histories. We defined the gene-expression signatures of wild-caught salmon at two different life stages by comparing the brain expression profiles of mature sneaker males and immature males, and early migrants and late migrants. In addition to life-stage-specific signatures, we discovered a surprisingly large gene set that was differentially regulated - at similar magnitudes, yet in opposite direction - in both life history transitions. We suggest that this co-variation is not a consequence of many independent cellular and molecular switches in the same direction but rather represents the molecular equivalent of a physiological shift orchestrated by one or very few master regulators. PMID:19401203

Computational Identification of Tissue-Specific Splicing Regulatory Elements in Human Genes from RNA-Seq Data.

PubMed

Badr, Eman; ElHefnawi, Mahmoud; Heath, Lenwood S

2016-01-01

Alternative splicing is a vital process for regulating gene expression and promoting proteomic diversity. It plays a key role in tissue-specific expressed genes. This specificity is mainly regulated by splicing factors that bind to specific sequences called splicing regulatory elements (SREs). Here, we report a genome-wide analysis to study alternative splicing on multiple tissues, including brain, heart, liver, and muscle. We propose a pipeline to identify differential exons across tissues and hence tissue-specific SREs. In our pipeline, we utilize the DEXSeq package along with our previously reported algorithms. Utilizing the publicly available RNA-Seq data set from the Human BodyMap project, we identified 28,100 differentially used exons across the four tissues. We identified tissue-specific exonic splicing enhancers that overlap with various previously published experimental and computational databases. A complicated exonic enhancer regulatory network was revealed, where multiple exonic enhancers were found across multiple tissues while some were found only in specific tissues. Putative combinatorial exonic enhancers and silencers were discovered as well, which may be responsible for exon inclusion or exclusion across tissues. Some of the exonic enhancers are found to be co-occurring with multiple exonic silencers and vice versa, which demonstrates a complicated relationship between tissue-specific exonic enhancers and silencers.

Characterisation of the macrophage transcriptome in glomerulonephritis-susceptible and -resistant rat strains

PubMed Central

Maratou, Klio; Behmoaras, Jacques; Fewings, Chris; Srivastava, Prashant; D’Souza, Zelpha; Smith, Jennifer; Game, Laurence; Cook, Terence; Aitman, Tim

2010-01-01

Crescentic glomerulonephritis (CRGN) is a major cause of rapidly progressive renal failure for which the underlying genetic basis is unknown. WKY rats show marked susceptibility to CRGN, while Lewis rats are resistant. Glomerular injury and crescent formation are macrophage-dependent and mainly explained by seven quantitative trait loci (Crgn1-7). Here, we used microarray analysis in basal and lipopolysaccharide (LPS)-stimulated macrophages to identify genes that reside on pathways predisposing WKY rats to CRGN. We detected 97 novel positional candidates for the uncharacterised Crgn3-7. We identified 10 additional secondary effector genes with profound differences in expression between the two strains (>5-fold change, <1% False Discovery Rate) for basal and LPS-stimulated macrophages. Moreover, we identified 8 genes with differentially expressed alternatively spliced isoforms, by using an in depth analysis at probe-level that allowed us to discard false positives due to polymorphisms between the two rat strains. Pathway analysis identified several common linked pathways, enriched for differentially expressed genes, which affect macrophage activation. In summary, our results identify distinct macrophage transcriptome profiles between two rat strains that differ in susceptibility to glomerulonephritis, provide novel positional candidates for Crgn3-7, and define groups of genes that play a significant role in differential regulation of macrophage activity. PMID:21179115

Striated muscle preferentially expressed genes alpha and beta are two serine/threonine protein kinases derived from the same gene as the aortic preferentially expressed gene-1.

PubMed

Hsieh, C M; Fukumoto, S; Layne, M D; Maemura, K; Charles, H; Patel, A; Perrella, M A; Lee, M E

2000-11-24

Aortic preferentially expressed gene (APEG)-1 is a 1.4-kilobase pair (kb) mRNA expressed in vascular smooth muscle cells and is down-regulated by vascular injury. An APEG-1 5'-end cDNA probe identified three additional isoforms. The 9-kb striated preferentially expressed gene (SPEG)alpha and the 11-kb SPEGbeta were found in skeletal muscle and heart. The 4-kb brain preferentially expressed gene was detected in the brain and aorta. We report here cloning of the 11-kb SPEGbeta cDNA. SPEGbeta encodes a 355-kDa protein that contains two serine/threonine kinase domains and is homologous to proteins of the myosin light chain kinase family. At least one kinase domain is active and capable of autophosphorylation. In the genome, all four isoforms share the middle three of the five exons of APEG-1, and they differ from each other by using different 5'- and 3'-ends and alternative splicing. We show that the expression of SPEGalpha and SPEGbeta is developmentally regulated in the striated muscle during C2C12 myoblast to myotube differentiation in vitro and cardiomyocyte maturation in vivo. This developmental regulation suggests that both SPEGalpha and SPEGbeta can serve as sensitive markers for striated muscle differentiation and that they may be important for adult striated muscle function.

Expansion of Genes Encoding piRNA-Associated Argonaute Proteins in the Pea Aphid: Diversification of Expression Profiles in Different Plastic Morphs

PubMed Central

Lu, Hsiao-ling; Tanguy, Sylvie; Rispe, Claude; Gauthier, Jean-Pierre; Walsh, Tom; Gordon, Karl; Edwards, Owain; Tagu, Denis; Chang, Chun-che; Jaubert-Possamai, Stéphanie

2011-01-01

Piwi-interacting RNAs (piRNAs) are known to regulate transposon activity in germ cells of several animal models that propagate sexually. However, the role of piRNAs during asexual reproduction remains almost unknown. Aphids that can alternate sexual and asexual reproduction cycles in response to seasonal changes of photoperiod provide a unique opportunity to study piRNAs and the piRNA pathway in both reproductive modes. Taking advantage of the recently sequenced genome of the pea aphid Acyrthosiphon pisum, we found an unusually large lineage-specific expansion of genes encoding the Piwi sub-clade of Argonaute proteins. In situ hybridisation showed differential expressions between the duplicated piwi copies: while Api-piwi2 and Api-piwi6 are “specialised” in germ cells their most closely related copy, respectively Api-piwi5 and Api-piwi3, are expressed in the somatic cells. The differential expression was also identified in duplicated ago3: Api-ago3a in germ cells and Api-ago3b in somatic cells. Moreover, analyses of expression profiles of the expanded piwi and ago3 genes by semi-quantitative RT-PCR showed that expressions varied according to the reproductive types. These specific expression patterns suggest that expanded aphid piwi and ago3 genes have distinct roles in asexual and sexual reproduction. PMID:22162754

The Transcriptomes of Two Heritable Cell Types Illuminate the Circuit Governing Their Differentiation

PubMed Central

Homann, Oliver R.; Hernday, Aaron D.; Monighetti, Cinna K.; De La Vega, Francisco M.; Johnson, Alexander D.

2010-01-01

The differentiation of cells into distinct cell types, each of which is heritable for many generations, underlies many biological phenomena. White and opaque cells of the fungal pathogen Candida albicans are two such heritable cell types, each thought to be adapted to unique niches within their human host. To systematically investigate their differences, we performed strand-specific, massively-parallel sequencing of RNA from C. albicans white and opaque cells. With these data we first annotated the C. albicans transcriptome, finding hundreds of novel differentially-expressed transcripts. Using the new annotation, we compared differences in transcript abundance between the two cell types with the genomic regions bound by a master regulator of the white-opaque switch (Wor1). We found that the revised transcriptional landscape considerably alters our understanding of the circuit governing differentiation. In particular, we can now resolve the poor concordance between binding of a master regulator and the differential expression of adjacent genes, a discrepancy observed in several other studies of cell differentiation. More than one third of the Wor1-bound differentially-expressed transcripts were previously unannotated, which explains the formerly puzzling presence of Wor1 at these positions along the genome. Many of these newly identified Wor1-regulated genes are non-coding and transcribed antisense to coding transcripts. We also find that 5′ and 3′ UTRs of mRNAs in the circuit are unusually long and that 5′ UTRs often differ in length between cell-types, suggesting UTRs encode important regulatory information and that use of alternative promoters is widespread. Further analysis revealed that the revised Wor1 circuit bears several striking similarities to the Oct4 circuit that specifies the pluripotency of mammalian embryonic stem cells. Additional characteristics shared with the Oct4 circuit suggest a set of general hallmarks characteristic of heritable differentiation states in eukaryotes. PMID:20808890

Effects of airborne particulate matter on alternative pre-mRNA splicing in colon cancer cells.

PubMed

Buggiano, Valeria; Petrillo, Ezequiel; Alló, Mariano; Lafaille, Celina; Redal, María Ana; Alghamdi, Mansour A; Khoder, Mamdouh I; Shamy, Magdy; Muñoz, Manuel J; Kornblihtt, Alberto R

2015-07-01

Alternative pre-mRNA splicing plays key roles in determining tissue- and species-specific cell differentiation as well as in the onset of hereditary disease and cancer, being controlled by multiple post- and co-transcriptional regulatory mechanisms. We report here that airborne particulate matter, resulting from industrial pollution, inhibits expression and specifically affects alternative splicing at the 5' untranslated region of the mRNA encoding the bone morphogenetic protein BMP4 in human colon cells in culture. These effects are consistent with a previously reported role for BMP4 in preventing colon cancer development, suggesting that ingestion of particulate matter could contribute to the onset of colon cell proliferation. We also show that the underlying mechanism might involve changes in transcriptional elongation. This is the first study to demonstrate that particulate matter causes non-pleiotropic changes in alternative splicing. Copyright © 2015 Elsevier Inc. All rights reserved.

Differentiation of Spermatogonia Stem Cells into Functional Mature Neurons Characterized with Differential Gene Expression.

PubMed

Bojnordi, Maryam Nazm; Azizi, Hossein; Skutella, Thomas; Movahedin, Mansoureh; Pourabdolhossein, Fereshteh; Shojaei, Amir; Hamidabadi, Hatef Ghasemi

2017-09-01

Transplantation of embryonic stem cells (ESCs) is a promising therapeutic approach for the treatment of neurodegenerative diseases. However, ESCs are not usable clinically due to immunological and ethical limitations. The identification of an alternative safe cell source opens novel options via autologous transplantation in neuro-regeneration circumventing these problems. Here, we examined the neurogenic capacity of embryonic stem-like cells (ES-like cells) derived from the testis using neural growth factor inducers and utilized them to generate functional mature neurons. The neuronal differentiation of ES-like cells is induced in three stages. Stage 1 is related to embryoid body (EB) formation. To induce neuroprogenitor cells, EBs were cultured in the presence of retinoic acid, N 2 supplement and fibroblast growth factor followed by culturing in a neurobasal medium containing B 27 , N 2 supplements for additional 10 days, to allow the maturation and development of neuronal progenitor cells. The neurogenic differentiation was confirmed by immunostaining for markers of mature neurons. The differentiated neurons were positive for Tuj1 and Tau1. Real-time PCR dates indicated the expression of Nestin and Neuro D (neuroprogenitor markers) in induced cells at the second stage of the differentiation protocol. The differentiated mature neurons exhibited the specific neuron markers Map2 and β-tubulin. The functional maturity of neurons was confirmed by an electrophysiological analysis of passive and active neural membrane properties. These findings indicated a differentiation capacity of ES-like cells derived from the testis to functionally mature neurons, which proposes them as a novel cell source for neuroregenerative medicine.

The IL-4/STAT6 signaling axis establishes a conserved microRNA signature in human and mouse macrophages regulating cell survival via miR-342-3p.

PubMed

Czimmerer, Zsolt; Varga, Tamas; Kiss, Mate; Vázquez, Cesaré Ovando; Doan-Xuan, Quang Minh; Rückerl, Dominik; Tattikota, Sudhir Gopal; Yan, Xin; Nagy, Zsuzsanna S; Daniel, Bence; Poliska, Szilard; Horvath, Attila; Nagy, Gergely; Varallyay, Eva; Poy, Matthew N; Allen, Judith E; Bacso, Zsolt; Abreu-Goodger, Cei; Nagy, Laszlo

2016-05-31

IL-4-driven alternative macrophage activation and proliferation are characteristic features of both antihelminthic immune responses and wound healing in contrast to classical macrophage activation, which primarily occurs during inflammatory responses. The signaling pathways defining the genome-wide microRNA expression profile as well as the cellular functions controlled by microRNAs during alternative macrophage activation are largely unknown. Hence, in the current work we examined the regulation and function of IL-4-regulated microRNAs in human and mouse alternative macrophage activation. We utilized microarray-based microRNA profiling to detect the dynamic expression changes during human monocyte-macrophage differentiation and IL-4-mediated alternative macrophage activation. The expression changes and upstream regulatory pathways of selected microRNAs were further investigated in human and mouse in vitro and in vivo models of alternative macrophage activation by integrating small RNA-seq, ChIP-seq, ChIP-quantitative PCR, and gene expression data. MicroRNA-controlled gene networks and corresponding functions were identified using a combination of transcriptomic, bioinformatic, and functional approaches. The IL-4-controlled microRNA expression pattern was identified in models of human and mouse alternative macrophage activation. IL-4-dependent induction of miR-342-3p and repression of miR-99b along with miR-125a-5p occurred in both human and murine macrophages in vitro. In addition, a similar expression pattern was observed in peritoneal macrophages of Brugia malayi nematode-implanted mice in vivo. By using IL4Rα- and STAT6-deficient macrophages, we were able to show that IL-4-dependent regulation of miR-342-3p, miR-99b, and miR-125a-5p is mediated by the IL-4Rα-STAT6 signaling pathway. The combination of gene expression studies and chromatin immunoprecipitation experiments demonstrated that both miR-342-3p and its host gene, EVL, are coregulated directly by STAT6. Finally, we found that miR-342-3p is capable of controlling macrophage survival through targeting an anti-apoptotic gene network including Bcl2l1. Our findings identify a conserved IL-4/STAT6-regulated microRNA signature in alternatively activated human and mouse macrophages. Moreover, our study indicates that miR-342-3p likely plays a pro-apoptotic role in such cells, thereby providing a negative feedback arm to IL-4-dependent macrophage proliferation.

Shrinkage estimation of effect sizes as an alternative to hypothesis testing followed by estimation in high-dimensional biology: applications to differential gene expression.

PubMed

Montazeri, Zahra; Yanofsky, Corey M; Bickel, David R

2010-01-01

Research on analyzing microarray data has focused on the problem of identifying differentially expressed genes to the neglect of the problem of how to integrate evidence that a gene is differentially expressed with information on the extent of its differential expression. Consequently, researchers currently prioritize genes for further study either on the basis of volcano plots or, more commonly, according to simple estimates of the fold change after filtering the genes with an arbitrary statistical significance threshold. While the subjective and informal nature of the former practice precludes quantification of its reliability, the latter practice is equivalent to using a hard-threshold estimator of the expression ratio that is not known to perform well in terms of mean-squared error, the sum of estimator variance and squared estimator bias. On the basis of two distinct simulation studies and data from different microarray studies, we systematically compared the performance of several estimators representing both current practice and shrinkage. We find that the threshold-based estimators usually perform worse than the maximum-likelihood estimator (MLE) and they often perform far worse as quantified by estimated mean-squared risk. By contrast, the shrinkage estimators tend to perform as well as or better than the MLE and never much worse than the MLE, as expected from what is known about shrinkage. However, a Bayesian measure of performance based on the prior information that few genes are differentially expressed indicates that hard-threshold estimators perform about as well as the local false discovery rate (FDR), the best of the shrinkage estimators studied. Based on the ability of the latter to leverage information across genes, we conclude that the use of the local-FDR estimator of the fold change instead of informal or threshold-based combinations of statistical tests and non-shrinkage estimators can be expected to substantially improve the reliability of gene prioritization at very little risk of doing so less reliably. Since the proposed replacement of post-selection estimates with shrunken estimates applies as well to other types of high-dimensional data, it could also improve the analysis of SNP data from genome-wide association studies.

A double-labeling immunohistochemical study of tau exon 10 in Alzheimer's disease, progressive supranuclear palsy and Pick's disease.

PubMed

Ishizawa, K; Ksiezak-Reding, H; Davies, P; Delacourte, A; Tiseo, P; Yen, S H; Dickson, D W

2000-09-01

Neurofibrillary tangles (NFT), one of the histopathological hallmarks of Alzheimer's disease (AD) and progressive supranuclear palsy (PSP), and Pick bodies in Pick's disease (PiD) are composed of microtubule-associated protein tau, which is the product of alternative splicing of a gene on chromosome 17. Alternative expression of exon 10 leads to formation of three- or four-repeat tau isoforms. To study the differential expression of exon 10, we performed double-labeling immunohistochemistry of the hippocampal formation in nine AD, four PSP and three PiD cases. Cryostat sections were processed with and without formic acid (FA) treatment, and double-stained with anti-tau (Alz-50 or PHF-1) or anti-amyloid P component antibodies and one of two specific anti-exon 10 antibodies (E-10). The effect of proteinase-K treatment was also evaluated. The results suggest the following. First, in AD, E-10 immunoreactivity is present in most intracellular NFT, but not in most dystrophic neurites and neuropil threads, suggesting differential expression of tau isoforms in specific cellular domains. Second, in AD, E-10 immunoreactivity is lost or blocked in most extracellular NFT, possibly due to proteolysis. Third, in PSP, E-10 immunoreactivity is hidden or blocked in NFT and tau-positive glial inclusions, but FA treatment exposes the epitope consistent with the hypothesis that PSP inclusions contain four-repeat tau. Fourth, E-10 immunoreactivity is present in dentate fascia NFT in AD and PSP, but not in Pick bodies in the dentate fascia or other areas. The results suggest that expression of exon 10 in tau is specific for cellular domains in a disease-specific manner.

Redundant roles of the phosphatidate phosphatase family in triacylglycerol synthesis in human adipocytes.

PubMed

Temprano, Ana; Sembongi, Hiroshi; Han, Gil-Soo; Sebastián, David; Capellades, Jordi; Moreno, Cristóbal; Guardiola, Juan; Wabitsch, Martin; Richart, Cristóbal; Yanes, Oscar; Zorzano, Antonio; Carman, George M; Siniossoglou, Symeon; Miranda, Merce

2016-09-01

In mammals, the evolutionary conserved family of Mg(2+)-dependent phosphatidate phosphatases (PAP1), involved in phospholipid and triacylglycerol synthesis, consists of lipin-1, lipin-2 and lipin-3. While mutations in the murine Lpin1 gene cause lipodystrophy and its knockdown in mouse 3T3-L1 cells impairs adipogenesis, deleterious mutations of human LPIN1 do not affect adipose tissue distribution. However, reduced LPIN1 and PAP1 activity has been described in participants with type 2 diabetes. We aimed to characterise the roles of all lipin family members in human adipose tissue and adipogenesis. The expression of the lipin family was analysed in adipose tissue in a cross-sectional study. Moreover, the effects of lipin small interfering RNA (siRNA)-mediated depletion on in vitro human adipogenesis were assessed. Adipose tissue gene expression of the lipin family is altered in type 2 diabetes. Depletion of every lipin family member in a human Simpson-Golabi-Behmel syndrome (SGBS) pre-adipocyte cell line, alters expression levels of adipogenic transcription factors and lipid biosynthesis genes in early stages of differentiation. Lipin-1 knockdown alone causes a 95% depletion of PAP1 activity. Despite the reduced PAP1 activity and alterations in early adipogenesis, lipin-silenced cells differentiate and accumulate neutral lipids. Even combinatorial knockdown of lipins shows mild effects on triacylglycerol accumulation in mature adipocytes. Overall, our data support the hypothesis of alternative pathways for triacylglycerol synthesis in human adipocytes under conditions of repressed lipin expression. We propose that induction of alternative lipid phosphate phosphatases, along with the inhibition of lipid hydrolysis, contributes to the maintenance of triacylglycerol content to near normal levels.

A Leveraged Signal-to-Noise Ratio (LSTNR) Method to Extract Differentially Expressed Genes and Multivariate Patterns of Expression From Noisy and Low-Replication RNAseq Data

PubMed Central

Lozoya, Oswaldo A.; Santos, Janine H.; Woychik, Richard P.

2018-01-01

To life scientists, one important feature offered by RNAseq, a next-generation sequencing tool used to estimate changes in gene expression levels, lies in its unprecedented resolution. It can score countable differences in transcript numbers among thousands of genes and between experimental groups, all at once. However, its high cost limits experimental designs to very small sample sizes, usually N = 3, which often results in statistically underpowered analysis and poor reproducibility. All these issues are compounded by the presence of experimental noise, which is harder to distinguish from instrumental error when sample sizes are limiting (e.g., small-budget pilot tests), experimental populations exhibit biologically heterogeneous or diffuse expression phenotypes (e.g., patient samples), or when discriminating among transcriptional signatures of closely related experimental conditions (e.g., toxicological modes of action, or MOAs). Here, we present a leveraged signal-to-noise ratio (LSTNR) thresholding method, founded on generalized linear modeling (GLM) of aligned read detection limits to extract differentially expressed genes (DEGs) from noisy low-replication RNAseq data. The LSTNR method uses an agnostic independent filtering strategy to define the dynamic range of detected aggregate read counts per gene, and assigns statistical weights that prioritize genes with better sequencing resolution in differential expression analyses. To assess its performance, we implemented the LSTNR method to analyze three separate datasets: first, using a systematically noisy in silico dataset, we demonstrated that LSTNR can extract pre-designed patterns of expression and discriminate between “noise” and “true” differentially expressed pseudogenes at a 100% success rate; then, we illustrated how the LSTNR method can assign patient-derived breast cancer specimens correctly to one out of their four reported molecular subtypes (luminal A, luminal B, Her2-enriched and basal-like); and last, we showed the ability to retrieve five different modes of action (MOA) elicited in livers of rats exposed to three toxicants under three nutritional routes by using the LSTNR method. By combining differential measurements with resolving power to detect DEGs, the LSTNR method offers an alternative approach to interrogate noisy and low-replication RNAseq datasets, which handles multiple biological conditions at once, and defines benchmarks to validate RNAseq experiments with standard benchtop assays. PMID:29868123

Characterization of visceral and subcutaneous adipose tissue transcriptome in pregnant women with and without spontaneous labor at term: Implication of alternative splicing in the metabolic adaptations of adipose tissue to parturition

PubMed Central

Mazaki-Tovi, Shali; Tarca, Adi L.; Vaisbuch, Edi; Kusanovic, Juan Pedro; Than, Nandor Gabor; Chaiworapongsa, Tinnakorn; Dong, Zhong; Hassan, Sonia S; Romero, Roberto

2018-01-01

OBJECTIVE The aim of this study was to determine gene expression and splicing changes associated with parturition and regions (visceral vs subcutaneous) of the adipose tissue of pregnant women. STUDY DESIGN The transcriptome of visceral and abdominal subcutaneous adipose tissue from pregnant women at term with (n=15) and without (n=25) spontaneous labor was profiled with Affymetrix GeneChip Human Exon 1.0 ST array. Overall gene expression changes and differential exon usage rate were compared between patient groups and adipose tissue regions (paired analyses). Selected genes were tested by quantitative reverse transcription–polymerase chain reaction. RESULTS Four hundred eighty-two genes were differentially expressed between visceral and subcutaneous fat of pregnant women with spontaneous labor at term (q-value <0.1; fold change >1.5). Biological processes enriched in this comparison included tissue and vasculature development, inflammatory and metabolic pathways. Differential splicing was found for 42 genes (q-value <0.1; difference FIRMA scores >2) between adipose tissue regions of women not in labor. Differential exon usage associated with parturition was found for three genes (LIMS1, HSPA5 and GSTK1) in subcutaneous tissues. CONCLUSION We show for the first time evidence of implication of mRNA splicing and processing machinery in the subcutaneous adipose tissue of women in labor compared to those without labor. PMID:26994472

Characterization of visceral and subcutaneous adipose tissue transcriptome in pregnant women with and without spontaneous labor at term: implication of alternative splicing in the metabolic adaptations of adipose tissue to parturition.

PubMed

Mazaki-Tovi, Shali; Tarca, Adi L; Vaisbuch, Edi; Kusanovic, Juan Pedro; Than, Nandor Gabor; Chaiworapongsa, Tinnakorn; Dong, Zhong; Hassan, Sonia S; Romero, Roberto

2016-10-01

The aim of this study was to determine gene expression and splicing changes associated with parturition and regions (visceral vs. subcutaneous) of the adipose tissue of pregnant women. The transcriptome of visceral and abdominal subcutaneous adipose tissue from pregnant women at term with (n=15) and without (n=25) spontaneous labor was profiled with the Affymetrix GeneChip Human Exon 1.0 ST array. Overall gene expression changes and the differential exon usage rate were compared between patient groups (unpaired analyses) and adipose tissue regions (paired analyses). Selected genes were tested by quantitative reverse transcription-polymerase chain reaction. Four hundred and eighty-two genes were differentially expressed between visceral and subcutaneous fat of pregnant women with spontaneous labor at term (q-value <0.1; fold change >1.5). Biological processes enriched in this comparison included tissue and vasculature development as well as inflammatory and metabolic pathways. Differential splicing was found for 42 genes [q-value <0.1; differences in Finding Isoforms using Robust Multichip Analysis scores >2] between adipose tissue regions of women not in labor. Differential exon usage associated with parturition was found for three genes (LIMS1, HSPA5, and GSTK1) in subcutaneous tissues. We show for the first time evidence of implication of mRNA splicing and processing machinery in the subcutaneous adipose tissue of women in labor compared to those without labor.

Selective expression of a splice variant of decay-accelerating factor in c-erbB-2-positive mammary carcinoma cells showing increased transendothelial invasiveness

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

Brandt, Burkhard; Mikesch, Jan-Hendrik; Simon, Ronald

2005-04-01

By differential-display-PCR a subclone of the SK-BR-3 cell line with high in vitro transendothelial invasiveness was identified to express increased levels of a new alternative splice variant of decay-accelerating factor (DAF). DAF seems to play an important role in some malignant tumours since on the one hand the expression of complement inhibitors on the surface of tumour cells prevents the accumulation of complement factors and in consequence cell lysis. On the other hand, DAF has been identified as a ligand for the CD97 surface receptor which induces cell migration. Immunofluorescence procedures, Western blot analyses, and cDNA clone sequencing were employedmore » to confirm the expression of DAF restricted to invasive tumour cells. Using a radioactive RNA-in situ hybridisation on freshly frozen tissue microarrays and RT-PCR on native tumour tissue, the expression of alternative spliced DAF mRNA was demonstrated in invasive breast cancer. Due to the fact that it could thereby not be detected in normal mammary tissues, it has to be confirmed in larger studies that the DAF splice variant might be a specific tumour marker for invasive breast cancer.« less

Alternative life histories shape brain gene expression profiles in males of the same population

USGS Publications Warehouse

Aubin-Horth, N.; Landry, C.R.; Letcher, B.H.; Hofmann, H.A.

2005-01-01

Atlantic salmon (Salmo salar) undergo spectacular marine migrations before homing to spawn in natal rivers. However, males that grow fastest early in life can adopt an alternative 'sneaker' tactic by maturing earlier at greatly reduced size without leaving freshwater. While the ultimate evolutionary causes have been well studied, virtually nothing is known about the molecular bases of this developmental plasticity. We investigate the nature and extent of coordinated molecular changes that accompany such a fundamental transformation by comparing the brain transcription profiles of wild mature sneaker males to age-matched immature males (future large anadromous males) and immature females. Of the ca. 3000 genes surveyed, 15% are differentially expressed in the brains of the two male types. These genes are involved in a wide range of processes, including growth, reproduction and neural plasticity. Interestingly, despite the potential for wide variation in gene expression profiles among individuals sampled in nature, consistent patterns of gene expression were found for individuals of the same reproductive tactic. Notably, gene expression patterns in immature males were different both from immature females and sneakers, indicating that delayed maturation and sea migration by immature males, the 'default' life cycle, may actually result from an active inhibition of development into a sneaker. ?? 2005 The Royal Society.

Alternative life histories shape brain gene expression profiles in males of the same population

PubMed Central

Aubin-Horth, Nadia; Landry, Christian R; Letcher, Benjamin H; Hofmann, Hans A

2005-01-01

Atlantic salmon (Salmo salar) undergo spectacular marine migrations before homing to spawn in natal rivers. However, males that grow fastest early in life can adopt an alternative ‘sneaker’ tactic by maturing earlier at greatly reduced size without leaving freshwater. While the ultimate evolutionary causes have been well studied, virtually nothing is known about the molecular bases of this developmental plasticity. We investigate the nature and extent of coordinated molecular changes that accompany such a fundamental transformation by comparing the brain transcription profiles of wild mature sneaker males to age-matched immature males (future large anadromous males) and immature females. Of the ca. 3000 genes surveyed, 15% are differentially expressed in the brains of the two male types. These genes are involved in a wide range of processes, including growth, reproduction and neural plasticity. Interestingly, despite the potential for wide variation in gene expression profiles among individuals sampled in nature, consistent patterns of gene expression were found for individuals of the same reproductive tactic. Notably, gene expression patterns in immature males were different both from immature females and sneakers, indicating that delayed maturation and sea migration by immature males, the ‘default’ life cycle, may actually result from an active inhibition of development into a sneaker. PMID:16087419

Alternative life histories shape brain gene expression profiles in males of the same population.

PubMed

Aubin-Horth, Nadia; Landry, Christian R; Letcher, Benjamin H; Hofmann, Hans A

2005-08-22

Atlantic salmon (Salmo salar) undergo spectacular marine migrations before homing to spawn in natal rivers. However, males that grow fastest early in life can adopt an alternative 'sneaker' tactic by maturing earlier at greatly reduced size without leaving freshwater. While the ultimate evolutionary causes have been well studied, virtually nothing is known about the molecular bases of this developmental plasticity. We investigate the nature and extent of coordinated molecular changes that accompany such a fundamental transformation by comparing the brain transcription profiles of wild mature sneaker males to age-matched immature males (future large anadromous males) and immature females. Of the ca. 3000 genes surveyed, 15% are differentially expressed in the brains of the two male types. These genes are involved in a wide range of processes, including growth, reproduction and neural plasticity. Interestingly, despite the potential for wide variation in gene expression profiles among individuals sampled in nature, consistent patterns of gene expression were found for individuals of the same reproductive tactic. Notably, gene expression patterns in immature males were different both from immature females and sneakers, indicating that delayed maturation and sea migration by immature males, the 'default' life cycle, may actually result from an active inhibition of development into a sneaker.

Data-driven information retrieval in heterogeneous collections of transcriptomics data links SIM2s to malignant pleural mesothelioma.

PubMed

Caldas, José; Gehlenborg, Nils; Kettunen, Eeva; Faisal, Ali; Rönty, Mikko; Nicholson, Andrew G; Knuutila, Sakari; Brazma, Alvis; Kaski, Samuel

2012-01-15

Genome-wide measurement of transcript levels is an ubiquitous tool in biomedical research. As experimental data continues to be deposited in public databases, it is becoming important to develop search engines that enable the retrieval of relevant studies given a query study. While retrieval systems based on meta-data already exist, data-driven approaches that retrieve studies based on similarities in the expression data itself have a greater potential of uncovering novel biological insights. We propose an information retrieval method based on differential expression. Our method deals with arbitrary experimental designs and performs competitively with alternative approaches, while making the search results interpretable in terms of differential expression patterns. We show that our model yields meaningful connections between biological conditions from different studies. Finally, we validate a previously unknown connection between malignant pleural mesothelioma and SIM2s suggested by our method, via real-time polymerase chain reaction in an independent set of mesothelioma samples. Supplementary data and source code are available from http://www.ebi.ac.uk/fg/research/rex.

Spliceosome Protein (SRp) Regulation of Glucocorticoid Receptor Isoforms and Glucocorticoid Response in Human Trabecular Meshwork Cells

PubMed Central

Jain, Ankur; Wordinger, Robert J.; Yorio, Thomas; Clark, Abbot F.

2012-01-01

Purpose. Glaucoma is a leading cause of visual impairment and blindness, with elevated intraocular pressure (IOP) as a major causative risk factor. Glucocorticoid (GC) therapy causes morphologic and biochemical changes in the trabecular meshwork (TM), an ocular tissue involved in regulating IOP, which can lead to the development of glaucoma in susceptible individuals (steroid responders). Steroid responders comprise 40% of the general population and are at higher risk of developing glaucoma. In addition, a majority of glaucoma patients are steroid responders. Differential distribution of various isoforms of GC receptor (GR) may be responsible for this heterogeneity in the steroid response. The alternatively spliced GRβ isoform acts as dominant negative regulator of classical GRα transcriptional activity. mRNA splicing is mediated by spliceosomes, which include serine-arginine rich proteins (SRps). The purpose of this study was to determine whether specific SRps regulate levels of these isoforms and thereby GC response in TM cells. Methods. Quantitative RT-PCR, Western blot analysis, and immunocytochemistry were used to determine the differential expression of different SRps (SRp20, 30c, and 40) in human normal and glaucomatous TM cell strains. Bioinformatics was used to find putative binding sites for SRp20 and SRp40 on exon 9 of the GR gene. A peptide modulator of splicing (bombesin) and SRp expression vectors were used to modulate SRp levels and determine their effects on GRα/GRβ ratios as well as dexamethasone (DEX) responsiveness via GRE- luciferase reporter activity, fibronectin, and myocilin induction in TM cells. Results. SRp20, SRp30c, and SRp40 regulate GR splicing and the GC response in TM cells. Modulation of SRp levels altered the GRβ/α ratio that correlated with DEX responsiveness. Bombesin decreased SRp20; increased SRp30c, SRp40 levels, and GRβ/α ratio, and suppressed DEX response in TM cells. Conclusions. Relative levels of SRp20, SRp30c, and SRp40 in TM cells control differential expression of the two alternatively spliced isoforms of the GR and thereby regulate GC responsiveness. Different levels and/or activities of these SRps may account for differential GC sensitivity among the normal and glaucoma populations. PMID:22205602

Fox-2 Splicing Factor Binds to a Conserved Intron Motif to PromoteInclusion of Protein 4.1R Alternative Exon 16

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

Ponthier, Julie L.; Schluepen, Christina; Chen, Weiguo

Activation of protein 4.1R exon 16 (E16) inclusion during erythropoiesis represents a physiologically important splicing switch that increases 4.1R affinity for spectrin and actin. Previous studies showed that negative regulation of E16 splicing is mediated by the binding of hnRNP A/B proteins to silencer elements in the exon and that downregulation of hnRNP A/B proteins in erythroblasts leads to activation of E16 inclusion. This paper demonstrates that positive regulation of E16 splicing can be mediated by Fox-2 or Fox-1, two closely related splicing factors that possess identical RNA recognition motifs. SELEX experiments with human Fox-1 revealed highly selective binding tomore » the hexamer UGCAUG. Both Fox-1 and Fox-2 were able to bind the conserved UGCAUG elements in the proximal intron downstream of E16, and both could activate E16 splicing in HeLa cell co-transfection assays in a UGCAUG-dependent manner. Conversely, knockdown of Fox-2 expression, achieved with two different siRNA sequences resulted in decreased E16 splicing. Moreover, immunoblot experiments demonstrate mouse erythroblasts express Fox-2, but not Fox-1. These findings suggest that Fox-2 is a physiological activator of E16 splicing in differentiating erythroid cells in vivo. Recent experiments show that UGCAUG is present in the proximal intron sequence of many tissue-specific alternative exons, and we propose that the Fox family of splicing enhancers plays an important role in alternative splicing switches during differentiation in metazoan organisms.« less

Quaking and PTB control overlapping splicing regulatory networks during muscle cell differentiation

PubMed Central

Hall, Megan P.; Nagel, Roland J.; Fagg, W. Samuel; Shiue, Lily; Cline, Melissa S.; Perriman, Rhonda J.; Donohue, John Paul; Ares, Manuel

2013-01-01

Alternative splicing contributes to muscle development, but a complete set of muscle-splicing factors and their combinatorial interactions are unknown. Previous work identified ACUAA (“STAR” motif) as an enriched intron sequence near muscle-specific alternative exons such as Capzb exon 9. Mass spectrometry of myoblast proteins selected by the Capzb exon 9 intron via RNA affinity chromatography identifies Quaking (QK), a protein known to regulate mRNA function through ACUAA motifs in 3′ UTRs. We find that QK promotes inclusion of Capzb exon 9 in opposition to repression by polypyrimidine tract-binding protein (PTB). QK depletion alters inclusion of 406 cassette exons whose adjacent intron sequences are also enriched in ACUAA motifs. During differentiation of myoblasts to myotubes, QK levels increase two- to threefold, suggesting a mechanism for QK-responsive exon regulation. Combined analysis of the PTB- and QK-splicing regulatory networks during myogenesis suggests that 39% of regulated exons are under the control of one or both of these splicing factors. This work provides the first evidence that QK is a global regulator of splicing during muscle development in vertebrates and shows how overlapping splicing regulatory networks contribute to gene expression programs during differentiation. PMID:23525800

MicroRNA Profiling as Tool for In Vitro Developmental Neurotoxicity Testing: The Case of Sodium Valproate

PubMed Central

Smirnova, Lena; Block, Katharina; Sittka, Alexandra; Oelgeschläger, Michael; Seiler, Andrea E. M.; Luch, Andreas

2014-01-01

Studying chemical disturbances during neural differentiation of murine embryonic stem cells (mESCs) has been established as an alternative in vitro testing approach for the identification of developmental neurotoxicants. miRNAs represent a class of small non-coding RNA molecules involved in the regulation of neural development and ESC differentiation and specification. Thus, neural differentiation of mESCs in vitro allows investigating the role of miRNAs in chemical-mediated developmental toxicity. We analyzed changes in miRNome and transcriptome during neural differentiation of mESCs exposed to the developmental neurotoxicant sodium valproate (VPA). A total of 110 miRNAs and 377 mRNAs were identified differently expressed in neurally differentiating mESCs upon VPA treatment. Based on miRNA profiling we observed that VPA shifts the lineage specification from neural to myogenic differentiation (upregulation of muscle-abundant miRNAs, mir-206, mir-133a and mir-10a, and downregulation of neural-specific mir-124a, mir-128 and mir-137). These findings were confirmed on the mRNA level and via immunochemistry. Particularly, the expression of myogenic regulatory factors (MRFs) as well as muscle-specific genes (Actc1, calponin, myosin light chain, asporin, decorin) were found elevated, while genes involved in neurogenesis (e.g. Otx1, 2, and Zic3, 4, 5) were repressed. These results were specific for valproate treatment and―based on the following two observations―most likely due to the inhibition of histone deacetylase (HDAC) activity: (i) we did not observe any induction of muscle-specific miRNAs in neurally differentiating mESCs exposed to the unrelated developmental neurotoxicant sodium arsenite; and (ii) the expression of muscle-abundant mir-206 and mir-10a was similarly increased in cells exposed to the structurally different HDAC inhibitor trichostatin A (TSA). Based on our results we conclude that miRNA expression profiling is a suitable molecular endpoint for developmental neurotoxicity. The observed lineage shift into myogenesis, where miRNAs may play an important role, could be one of the developmental neurotoxic mechanisms of VPA. PMID:24896083

Conditioned medium as a strategy for human stem cells chondrogenic differentiation.

PubMed

Alves da Silva, M L; Costa-Pinto, A R; Martins, A; Correlo, V M; Sol, P; Bhattacharya, M; Faria, S; Reis, R L; Neves, Nuno M

2015-06-01

Paracrine signalling from chondrocytes has been reported to increase the synthesis and expression of cartilage extracellular matrix (ECM) by stem cells. The use of conditioned medium obtained from chondrocytes for stimulating stem cells chondrogenic differentiation may be a very interesting alternative for moving into the clinical application of these cells, as chondrocytes could be partially replaced by stem cells for this type of application. In the present study we aimed to achieve chondrogenic differentiation of two different sources of stem cells using conditioned medium, without adding growth factors. We tested both human bone marrow-derived mesenchymal stem cells (hBSMCs) and human Wharton's jelly-derived stem cells (hWJSCs). Conditioned medium obtained from a culture of human articular chondrocytes was used to feed the cells during the experiment. Cultures were performed in previously produced three-dimensional (3D) scaffolds, composed of a blend of 50:50 chitosan:poly(butylene succinate). Both types of stem cells were able to undergo chondrogenic differentiation without the addition of growth factors. Cultures using hWJSCs showed significantly higher GAGs accumulation and expression of cartilage-related genes (aggrecan, Sox9 and collagen type II) when compared to hBMSCs cultures. Conditioned medium obtained from articular chondrocytes induced the chondrogenic differentiation of MSCs and ECM formation. Obtained results showed that this new strategy is very interesting and should be further explored for clinical applications. Copyright © 2013 John Wiley & Sons, Ltd.

Loss of end-differentiated β-cell phenotype following pancreatic islet transplantation.

PubMed

Anderson, S J; White, M G; Armour, S L; Maheshwari, R; Tiniakos, D; Muller, Y D; Berishvili, E; Berney, T; Shaw, J A M

2018-03-01

Replacement of pancreatic β-cells through deceased donor islet transplantation is a proven therapy for preventing recurrent life-threatening hypoglycemia in type 1 diabetes. Although near-normal glucose levels and insulin independence can be maintained for many years following successful islet transplantation, restoration of normal functional β-cell mass has remained elusive. It has recently been proposed that dedifferentiation/plasticity towards other endocrine phenotypes may play an important role in stress-induced β-cell dysfunction in type 2 diabetes. Here we report loss of end-differentiated β-cell phenotype in 2 intraportal islet allotransplant recipients. Despite excellent graft function and sustained insulin independence, all examined insulin-positive cells had lost expression of the end-differentiation marker, urocortin-3, or appeared to co-express the α-cell marker, glucagon. In contrast, no insulin + /urocortin-3 - cells were seen in nondiabetic deceased donor control pancreatic islets. Loss of end-differentiated phenotype may facilitate β-cell survival during the stresses associated with islet isolation and culture, in addition to sustained hypoxia following engraftment. As further refinements in islet isolation and culture are made in parallel with exploration of alternative β-cell sources, graft sites, and ultimately fully vascularized bioengineered insulin-secreting microtissues, differentiation status immunostaining provides a novel tool to assess whether fully mature β-cell phenotype has been maintained. © 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.

MicroRNAs Associated with Caste Determination and Differentiation in a Primitively Eusocial Insect

PubMed Central

Collins, David H.; Mohorianu, Irina; Beckers, Matthew; Moulton, Vincent; Dalmay, Tamas; Bourke, Andrew F. G.

2017-01-01

In eusocial Hymenoptera (ants, bees and wasps), queen and worker adult castes typically arise via environmental influences. A fundamental challenge is to understand how a single genome can thereby produce alternative phenotypes. A powerful approach is to compare the molecular basis of caste determination and differentiation along the evolutionary trajectory between primitively and advanced eusocial species, which have, respectively, relatively undifferentiated and strongly differentiated adult castes. In the advanced eusocial honeybee, Apis mellifera, studies suggest that microRNAs (miRNAs) play an important role in the molecular basis of caste determination and differentiation. To investigate how miRNAs affect caste in eusocial evolution, we used deep sequencing and Northern blots to isolate caste-associated miRNAs in the primitively eusocial bumblebee Bombus terrestris. We found that the miRNAs Bte-miR-6001-5p and -3p are more highly expressed in queen- than in worker-destined late-instar larvae. These are the first caste-associated miRNAs from outside advanced eusocial Hymenoptera, so providing evidence for caste-associated miRNAs occurring relatively early in eusocial evolution. Moreover, we found little evidence that miRNAs previously shown to be associated with caste in A. mellifera were differentially expressed across caste pathways in B. terrestris, suggesting that, in eusocial evolution, the caste-associated role of individual miRNAs is not conserved. PMID:28361900

Human mesenchymal stem cell osteoblast differentiation, ECM deposition, and biomineralization on PAH/PAA polyelectrolyte multilayers.

PubMed

Pattabhi, Sudhakara Rao; Lehaf, Ali M; Schlenoff, Joseph B; Keller, Thomas C S

2015-05-01

Polyelectrolyte multilayer (PEMU) coatings built layer by layer with alternating pairs of polyelectrolytes can be tuned to improve cell interactions with surfaces and may be useful as biocompatible coatings to improve fixation between implants and tissues. Here, we show that human mesenchymal stromal cells (hMSCs) induced with bone differentiation medium (BDM) to become osteoblasts biomineralize crosslinked PEMUs built with the polycation poly(allylamine hydrochloride) (PAH) and the polyanion poly(acrylic acid) (PAA). Degrees of hMSC osteoblast differentiation and surface biomineralization on the smooth PAH-terminated PEMUs (PAH-PEMUs) and microstructured PAA-terminated PEMUs (PAA-PEMUs) reflect differences in cell-deposited extracellular matrix (ECM). BDM-induced hMSCs expressed higher levels of the early osteoblast differentiation marker alkaline phosphatase and collagen 1 (COL1) sooner on PAA-PEMUs than on PAH-PEMUs. Cells on both types of PEMUs proceeded to express the later stage osteoblast differentiation marker bone sialoprotein (BSP), but the BDM-induced cells organized a more amorphous Collagen I and denser BSP localization on PAA-PEMUs than on PAH-PEMUs. These ECM properties correlated with greater biomineralization on the PAA-PEMUs than on PAH-PEMUs. Together, these results confirm the suitability of PAH/PAA PEMUs as a substrate for hMSC osteogenesis and highlight the importance of substrate effects on ECM organization and BSP presentation on biomineralization. © 2014 Wiley Periodicals, Inc.

Robust extraction of functional signals from gene set analysis using a generalized threshold free scoring function

PubMed Central

2009-01-01

Background A central task in contemporary biosciences is the identification of biological processes showing response in genome-wide differential gene expression experiments. Two types of analysis are common. Either, one generates an ordered list based on the differential expression values of the probed genes and examines the tail areas of the list for over-representation of various functional classes. Alternatively, one monitors the average differential expression level of genes belonging to a given functional class. So far these two types of method have not been combined. Results We introduce a scoring function, Gene Set Z-score (GSZ), for the analysis of functional class over-representation that combines two previous analysis methods. GSZ encompasses popular functions such as correlation, hypergeometric test, Max-Mean and Random Sets as limiting cases. GSZ is stable against changes in class size as well as across different positions of the analysed gene list in tests with randomized data. GSZ shows the best overall performance in a detailed comparison to popular functions using artificial data. Likewise, GSZ stands out in a cross-validation of methods using split real data. A comparison of empirical p-values further shows a strong difference in favour of GSZ, which clearly reports better p-values for top classes than the other methods. Furthermore, GSZ detects relevant biological themes that are missed by the other methods. These observations also hold when comparing GSZ with popular program packages. Conclusion GSZ and improved versions of earlier methods are a useful contribution to the analysis of differential gene expression. The methods and supplementary material are available from the website http://ekhidna.biocenter.helsinki.fi/users/petri/public/GSZ/GSZscore.html. PMID:19775443

Differential expression of a novel gene during seed triacylglycerol accumulation in lupin species ( Lupinus angustifolius L. and L. mutabilis L.).

PubMed

Francki, Michael G; Whitaker, Peta; Smith, Penelope M; Atkins, Craig A

2002-11-01

Seed triacylglycerols (TAGs) are stored as energy reserves and extracted for various end-product uses. In lupins, seed oil content varies from 16% in Lupinus mutabilisto 8% in L. angustifolius. We have shown that TAGs rapidly accumulate during mid-stages of seed development in L. mutabilis compared to the lower seed oil species, L. angustifolius. In this study, we have targeted the key enzymes of the lipid biosynthetic pathway, acetyl-CoA carboxylase (ACCase) and diacylglycerol acyltransferase (DAGAT), to determine factors regulating TAG accumulation between two lupin species. A twofold increase in ACCase activity was observed in L. mutabilis relative to L. angustifolius and correlated with rapid TAG accumulation. No difference in DAGAT activity was detected. We have identified, cloned and partially characterised a novel gene differentially expressed during TAG accumulation between L. angustifolius and L. mutabilis. The gene has some identity to the glucose dehydrogenase family previously described in barley and bacteria and the significance of its expression levels during seed development in relation to TAG accumulation is discussed. DNA sequence analysis of the promoter in both L. angustifolius and L. mutabilis identified putative matrix attachment regions and recognition sequences for transcription binding sites similar to those found in the Adh1 gene from Arabidopsis. The identical promoter regions between species indicate that differential gene expression is controlled by alternative transcription factors, accessibility to binding sites or a combination of both.

SPIB and BATF provide alternate determinants of IRF4 occupancy in diffuse large B-cell lymphoma linked to disease heterogeneity

PubMed Central

Care, Matthew A.; Cocco, Mario; Laye, Jon P.; Barnes, Nicholas; Huang, Yuanxue; Wang, Ming; Barrans, Sharon; Du, Ming; Jack, Andrew; Westhead, David R.; Doody, Gina M.; Tooze, Reuben M.

2014-01-01

Interferon regulatory factor 4 (IRF4) is central to the transcriptional network of activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL), an aggressive lymphoma subgroup defined by gene expression profiling. Since cofactor association modifies transcriptional regulatory input by IRF4, we assessed genome occupancy by IRF4 and endogenous cofactors in ABC-DLBCL cell lines. IRF4 partners with SPIB, PU.1 and BATF genome-wide, but SPIB provides the dominant IRF4 partner in this context. Upon SPIB knockdown IRF4 occupancy is depleted and neither PU.1 nor BATF acutely compensates. Integration with ENCODE data from lymphoblastoid cell line GM12878, demonstrates that IRF4 adopts either SPIB- or BATF-centric genome-wide distributions in related states of post-germinal centre B-cell transformation. In primary DLBCL high-SPIB and low-BATF or the reciprocal low-SPIB and high-BATF mRNA expression links to differential gene expression profiles across nine data sets, identifying distinct associations with SPIB occupancy, signatures of B-cell differentiation stage and potential pathogenetic mechanisms. In a population-based patient cohort, SPIBhigh/BATFlow-ABC-DLBCL is enriched for mutation of MYD88, and SPIBhigh/BATFlow-ABC-DLBCL with MYD88-L265P mutation identifies a small subgroup of patients among this otherwise aggressive disease subgroup with distinct favourable outcome. We conclude that differential expression of IRF4 cofactors SPIB and BATF identifies biologically and clinically significant heterogeneity among ABC-DLBCL. PMID:24875472

Effects of airborne particulate matter on alternative pre-mRNA splicing in colon cancer cells

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

Buggiano, Valeria; Petrillo, Ezequiel; Alló, Mariano

2015-07-15

Alternative pre-mRNA splicing plays key roles in determining tissue- and species-specific cell differentiation as well as in the onset of hereditary disease and cancer, being controlled by multiple post- and co-transcriptional regulatory mechanisms. We report here that airborne particulate matter, resulting from industrial pollution, inhibits expression and specifically affects alternative splicing at the 5′ untranslated region of the mRNA encoding the bone morphogenetic protein BMP4 in human colon cells in culture. These effects are consistent with a previously reported role for BMP4 in preventing colon cancer development, suggesting that ingestion of particulate matter could contribute to the onset of colonmore » cell proliferation. We also show that the underlying mechanism might involve changes in transcriptional elongation. This is the first study to demonstrate that particulate matter causes non-pleiotropic changes in alternative splicing. - Highlights: • Airborne particulate matter (PM10) affects alternative splicing in colon cells. • PM10 upregulates one of the two mRNA variants of the growth factor BMP-4. • This variant has a longer 5′ unstranslated region and introduces an upstream AUG. • By regulating BMP-4 mRNA splicing PM10 inhibits total expression of BMP-4 protein. • BMP-4 downregulation was previously reported to be associated to colon cancer.« less

Increase of Alternatively Activated Antigen Presenting Cells in Active Experimental Autoimmune Encephalomyelitis.

PubMed

Wasser, Beatrice; Pramanik, Gautam; Hess, Moritz; Klein, Matthias; Luessi, Felix; Dornmair, Klaus; Bopp, Tobias; Zipp, Frauke; Witsch, Esther

2016-12-01

The importance of CD11c + antigen-presenting cells (APCs) in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) is well accepted and the gate keeper function of perivascular CD11c + APCs has been demonstrated. CD11c can be expressed by APCs from external sources or by central nervous system (CNS) resident APCs such as microglia. Yet, changes in the gene expression pattern of CNS CD11c + APCs during disease are still unclear and differentially expressed genes might play a decisive role in EAE progression. Due to their low numbers in the diseased brain and due to the absence of considerable numbers in the healthy CNS, analysis of CNS CD11c + cells is technically difficult. To ask whether the CD11c + APC population contributes to remission of EAE disease, we used Illumina deep mRNA sequencing (RNA-Seq) and quantitative real time polymerase chain reaction (qRT-PCR) analyses to identify the transcriptome of CD11c + APCs during disease course. We identified a battery of genes that were significantly regulated during the exacerbation of the disease compared to remission and relapse. Three of these genes, Arginase-1, Chi3l3 and Ms4a8a, showed a higher expression at the exacerbation than at later time points during the disease, both in SJL/J and in C57BL/6 mice, and could be attributed to alternatively activated APCs. Expression of Arginase-1, Chi3l3 and Ms4a8a genes was linked to the disease phase of EAE rather than to disease score. Expression of these genes suggested that APCs resembling alternatively activated macrophages are involved during the first wave of neuroinflammation and can be directly associated with the disease progress.

Differential expression of Oct4 variants and pseudogenes in normal urothelium and urothelial cancer.

PubMed

Wezel, Felix; Pearson, Joanna; Kirkwood, Lisa A; Southgate, Jennifer

2013-10-01

The transcription factor octamer-binding protein 4 (Oct4; encoded by POU5F1) has a key role in maintaining embryonic stem cell pluripotency during early embryonic development and it is required for generation of induced pluripotent stem cells. Controversy exists concerning Oct4 expression in somatic tissues, with reports that Oct4 is expressed in normal and in neoplastic urothelium carrying implications for a bladder cancer stem cell phenotype. Here, we show that the pluripotency-associated Oct4A transcript was absent from cultures of highly regenerative normal human urothelial cells and from low-grade to high-grade urothelial carcinoma cell lines, whereas alternatively spliced variants and transcribed pseudogenes were expressed in abundance. Immunolabeling and immunoblotting studies confirmed the absence of Oct4A in normal and neoplastic urothelial cells and tissues, but indicated the presence of alternative isoforms or potentially translated pseudogenes. The stable forced expression of Oct4A in normal human urothelial cells in vitro profoundly inhibited growth and affected morphology, but protein expression was rapidly down-regulated. Our findings demonstrate that pluripotency-associated isoform Oct4A is not expressed by normal or malignant human urothelium and therefore is unlikely to play a role in a cancer stem cell phenotype. However, our findings also indicate that urothelium expresses a variety of other Oct4 splice-variant isoforms and transcribed pseudogenes that warrant further study. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Postnatal Expression of V2 Vasopressin Receptor Splice Variants in the Rat Cerebellum

PubMed Central

Vargas, Karina J.; Sarmiento, José M.; Ehrenfeld, Pamela; Añazco, Carolina C.; Villanueva, Carolina I.; Carmona, Pamela L.; Brenet, Marianne; Navarro, Javier; Müller-Esterl, Werner; Figueroa, Carlos D.; González, Carlos B.

2010-01-01

The V2 vasopressin receptor gene contains an alternative splice site in exon-3, which leads to the generation of two splice variants (V2a and V2b) first identified in the kidney. The open reading frame of the alternatively spliced V2b transcripten codes a truncated receptor, showing the same amino acid sequence as the canonical V2a receptor up to the 6th transmembrane segment, but displaying a distinct sequence to the corresponding 7th transmembrane segment and C-terminal domain relative to the V2a receptor. Here, we demonstrate the postnatal expression of V2a and V2b variants in the rat cerebellum. Most importantly, we showed by in situ hybridization and immunocytochemistry that both V2 splice variants were preferentially expressed in Purkinje cells, from early to late postnatal development. In addition, both variants were transiently expressed in the neuroblastic external granule cells and Bergmann fibers. These results indicate that the cellular distributions of both splice variants are developmentally regulated, and suggest that the transient expression of the V2 receptor is involved in the mechanisms of cerebellar cytodifferentiation by AVP. Finally, transfected CHO-K1 .expressing similar amounts of both V2 splice variants, as that found in the cerebellum, showed a significant reduction in the surface expression of V2a receptors, suggesting that the differential expression of the V2 splice variants regulate the vasopressin signaling in the cerebellum. PMID:19281786

Differentiation of hepatocytes from induced pluripotent stem cells derived from human hair follicle mesenchymal stem cells.

PubMed

Shi, Xu; Lv, Shuang; He, Xia; Liu, Xiaomei; Sun, Meiyu; Li, Meiying; Chi, Guangfan; Li, Yulin

2016-10-01

Due to the limitations of organ donors and immune rejection in severe liver diseases, stem cell-based therapy presents a promising application for tissue repair and regeneration. As a novel cell source, mesenchymal stem cells separated from human hair follicles (HF-MSCs) are convenient to obtain and have no age limit. To date, the differentiation of HF-MSCs into hepatocytes has not been reported. In this study, we explored whether HF-MSCs and HF-MSC-derived-induced pluripotent stem cells (HF-iPS) could differentiate into hepatocytes in vitro. Flow cytometry, Oil Red O stain and Alizarin Red stain were used to identify the characteristics of HF-MSCs. The expression of liver-specific gene was detected by immunofluorescence and Quantitative Polymerase Chain Reaction. Periodic Acid-Schiff stain, Indocyanine Green stain and Low-Density Lipoprotein stain were performed to evaluate the functions of induced hepatocyte-like cells (HLCs). HF-MSCs were unable to differentiate into HLCs using previously reported procedures for MSCs from other tissues. However, HF-iPS efficiently induced the generation of HLCs that expressed hepatocyte markers and drug metabolism-related genes. HF-iPS can be used as novel and alternative cellular tools for inducing hepatocytes in vitro, simultaneously benefiting from utilizing HF-MSCs as a noninvasive and convenient cell source for reprogramming.

mRNA changes in nucleus accumbens related to methamphetamine addiction in mice

NASA Astrophysics Data System (ADS)

Zhu, Li; Li, Jiaqi; Dong, Nan; Guan, Fanglin; Liu, Yufeng; Ma, Dongliang; Goh, Eyleen L. K.; Chen, Teng

2016-11-01

Methamphetamine (METH) is a highly addictive psychostimulant that elicits aberrant changes in the expression of microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) in the nucleus accumbens of mice, indicating a potential role of METH in post-transcriptional regulations. To decipher the potential consequences of these post-transcriptional regulations in response to METH, we performed strand-specific RNA sequencing (ssRNA-Seq) to identify alterations in mRNA expression and their alternative splicing in the nucleus accumbens of mice following exposure to METH. METH-mediated changes in mRNAs were analyzed and correlated with previously reported changes in non-coding RNAs (miRNAs and lncRNAs) to determine the potential functions of these mRNA changes observed here and how non-coding RNAs are involved. A total of 2171 mRNAs were differentially expressed in response to METH with functions involved in synaptic plasticity, mitochondrial energy metabolism and immune response. 309 and 589 of these mRNAs are potential targets of miRNAs and lncRNAs respectively. In addition, METH treatment decreases mRNA alternative splicing, and there are 818 METH-specific events not observed in saline-treated mice. Our results suggest that METH-mediated addiction could be attributed by changes in miRNAs and lncRNAs and consequently, changes in mRNA alternative splicing and expression. In conclusion, our study reported a methamphetamine-modified nucleus accumbens transcriptome and provided non-coding RNA-mRNA interaction networks possibly involved in METH addiction.

Chondrogenic induction of mesenchymal stromal/stem cells from Wharton's jelly embedded in alginate hydrogel and without added growth factor: an alternative stem cell source for cartilage tissue engineering.

PubMed

Reppel, Loïc; Schiavi, Jessica; Charif, Naceur; Leger, Léonore; Yu, Hao; Pinzano, Astrid; Henrionnet, Christel; Stoltz, Jean-François; Bensoussan, Danièle; Huselstein, Céline

2015-12-30

Due to their intrinsic properties, stem cells are promising tools for new developments in tissue engineering and particularly for cartilage tissue regeneration. Although mesenchymal stromal/stem cells from bone marrow (BM-MSC) have long been the most used stem cell source in cartilage tissue engineering, they have certain limits. Thanks to their properties such as low immunogenicity and particularly chondrogenic differentiation potential, mesenchymal stromal/stem cells from Wharton's jelly (WJ-MSC) promise to be an interesting source of MSC for cartilage tissue engineering. In this study, we propose to evaluate chondrogenic potential of WJ-MSC embedded in alginate/hyaluronic acid hydrogel over 28 days. Hydrogels were constructed by the original spraying method. Our main objective was to evaluate chondrogenic differentiation of WJ-MSC on three-dimensional scaffolds, without adding growth factors, at transcript and protein levels. We compared the results to those obtained from standard BM-MSC. After 3 days of culture, WJ-MSC seemed to be adapted to their new three-dimensional environment without any detectable damage. From day 14 and up to 28 days, the proportion of WJ-MSC CD73(+), CD90(+), CD105(+) and CD166(+) decreased significantly compared to monolayer marker expression. Moreover, WJ-MSC and BM-MSC showed different phenotype profiles. After 28 days of scaffold culture, our results showed strong upregulation of cartilage-specific transcript expression. WJ-MSC exhibited greater type II collagen synthesis than BM-MSC at both transcript and protein levels. Furthermore, our work highlighted a relevant result showing that WJ-MSC expressed Runx2 and type X collagen at lower levels than BM-MSC. Once seeded in the hydrogel scaffold, WJ-MSC and BM-MSC have different profiles of chondrogenic differentiation at both the phenotypic level and matrix synthesis. After 4 weeks, WJ-MSC, embedded in a three-dimensional environment, were able to adapt to their environment and express specific cartilage-related genes and matrix proteins. Today, WJ-MSC represent a real alternative source of stem cells for cartilage tissue engineering.

Chitin synthase genes in Manduca sexta: characterization of a gut-specific transcript and differential tissue expression of alternately spliced mRNAs during development.

PubMed

Hogenkamp, David G; Arakane, Yasuyuki; Zimoch, Lars; Merzendorfer, Hans; Kramer, Karl J; Beeman, Richard W; Kanost, Michael R; Specht, Charles A; Muthukrishnan, Subbaratnam

2005-06-01

Chitin, the linear homopolymer of beta-1,4-linked N-acetylglucosamine, is produced by the enzyme chitin synthase (CHS). In general, this insoluble polysaccharide is found in two major extracellular structures in insects, the cuticle that overlays the epidermis and the peritrophic membrane (PM) that lines the midgut. Based on amino acid sequence similarities, insect CHSs are divided into two classes, A and B, and to date no more than two CHS genes have been identified in any single insect species. In species where both CHSs have been identified, one class A CHS and one class B CHS are always present. This finding suggests that these two genes may encode enzymes that synthesize chitin in different epithelial tissues. In our laboratory, we previously characterized transcripts for a class A CHS gene (MsCHS1) from the tobacco hornworm, Manduca sexta. We observed the expression of this gene in the larval epidermis, suggesting that the encoded enzyme functions to synthesize cuticular chitin. In this paper, we characterize a second chitin synthase gene (MsCHS2) belonging to class B and its cDNA from Manduca and show that it is expressed only in the midgut. This cDNA contains an open reading frame of 4575 nucleotides, which encodes a conceptual protein that is 1524 amino acids in length and is predicted to contain 16 transmembrane spans. Northern blot analysis of RNA isolated from anterior, medial, and posterior sections of the midgut from feeding larvae indicate that MsCHS2 is primarily expressed in the anterior midgut, with transcript levels tapering off in the medial and posterior midgut. Analysis of the MsCHS2 gene sequence indicates the absence of an alternate exon in contrast to the MsCHS1 gene, which yields two transcripts, MsCHS1a and MsCHS1b. RT-PCR analysis of the differential expression of these alternately spliced transcripts reveals that both splice variants are present in the epidermis. However, the ratio of the two alternately spliced transcripts varies during development, with MsCHS1a being generally more predominant. Southern blot analysis using a probe specific for CHS indicated that Manduca has only two CHS genes, akin to other insect species. Results from an analysis of expression of both genes in different tissues and developmental times indicate that the MsCHS1 enzyme is used for the synthesis of chitin in the cuticle and tracheae, whereas MsCHS2 is utilized exclusively for the synthesis of PM-associated chitin in the midgut.

Genome-wide characterization of differential transcript usage in Arabidopsis thaliana.

PubMed

Vaneechoutte, Dries; Estrada, April R; Lin, Ying-Chen; Loraine, Ann E; Vandepoele, Klaas

2017-12-01

Alternative splicing and the usage of alternate transcription start- or stop sites allows a single gene to produce multiple transcript isoforms. Most plant genes express certain isoforms at a significantly higher level than others, but under specific conditions this expression dominance can change, resulting in a different set of dominant isoforms. These events of differential transcript usage (DTU) have been observed for thousands of Arabidopsis thaliana, Zea mays and Vitis vinifera genes, and have been linked to development and stress response. However, neither the characteristics of these genes, nor the implications of DTU on their protein coding sequences or functions, are currently well understood. Here we present a dataset of isoform dominance and DTU for all genes in the AtRTD2 reference transcriptome based on a protocol that was benchmarked on simulated data and validated through comparison with a published reverse transciptase-polymerase chain reaction panel. We report DTU events for 8148 genes across 206 public RNA-Seq samples, and find that protein sequences are affected in 22% of the cases. The observed DTU events show high consistency across replicates, and reveal reproducible patterns in response to treatment and development. We also demonstrate that genes with different evolutionary ages, expression breadths and functions show large differences in the frequency at which they undergo DTU, and in the effect that these events have on their protein sequences. Finally, we showcase how the generated dataset can be used to explore DTU events for genes of interest or to find genes with specific DTU in samples of interest. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Transcriptome Sequencing Revealed Significant Alteration of Cortical Promoter Usage and Splicing in Schizophrenia

PubMed Central

Wu, Jing Qin; Wang, Xi; Beveridge, Natalie J.; Tooney, Paul A.; Scott, Rodney J.; Carr, Vaughan J.; Cairns, Murray J.

2012-01-01

Background While hybridization based analysis of the cortical transcriptome has provided important insight into the neuropathology of schizophrenia, it represents a restricted view of disease-associated gene activity based on predetermined probes. By contrast, sequencing technology can provide un-biased analysis of transcription at nucleotide resolution. Here we use this approach to investigate schizophrenia-associated cortical gene expression. Methodology/Principal Findings The data was generated from 76 bp reads of RNA-Seq, aligned to the reference genome and assembled into transcripts for quantification of exons, splice variants and alternative promoters in postmortem superior temporal gyrus (STG/BA22) from 9 male subjects with schizophrenia and 9 matched non-psychiatric controls. Differentially expressed genes were then subjected to further sequence and functional group analysis. The output, amounting to more than 38 Gb of sequence, revealed significant alteration of gene expression including many previously shown to be associated with schizophrenia. Gene ontology enrichment analysis followed by functional map construction identified three functional clusters highly relevant to schizophrenia including neurotransmission related functions, synaptic vesicle trafficking, and neural development. Significantly, more than 2000 genes displayed schizophrenia-associated alternative promoter usage and more than 1000 genes showed differential splicing (FDR<0.05). Both types of transcriptional isoforms were exemplified by reads aligned to the neurodevelopmentally significant doublecortin-like kinase 1 (DCLK1) gene. Conclusions This study provided the first deep and un-biased analysis of schizophrenia-associated transcriptional diversity within the STG, and revealed variants with important implications for the complex pathophysiology of schizophrenia. PMID:22558445

Multiple Scenarios of Transition to Chaos in the Alternative Splicing Model

NASA Astrophysics Data System (ADS)

Kogai, Vladislav V.; Likhoshvai, Vitaly A.; Fadeev, Stanislav I.; Khlebodarova, Tamara M.

We have investigated the scenarios of transition to chaos in the mathematical model of a genetic system constituted by a single transcription factor-encoding gene, the expression of which is self-regulated by a feedback loop that involves protein isoforms. Alternative splicing results in the synthesis of protein isoforms providing opposite regulatory outcomes — activation or repression. The model is represented by a differential equation with two delayed arguments. The possibility of transition to chaos dynamics via all classical scenarios: a cascade of period-doubling bifurcations, quasiperiodicity and type-I, type-II and type-III intermittencies, has been numerically demonstrated. The parametric features of each type of transition to chaos have been described.

Novel developmental biology-based protocol of embryonic stem cell differentiation to morphologically sound and functional yet immature hepatocytes.

PubMed

Bukong, Terence N; Lo, Tracie; Szabo, Gyongyi; Dolganiuc, Angela

2012-05-01

Liver diseases are common in the United States and often require liver transplantation; however, donated organs are limited and thus alternative sources for liver cells are in high demand. Embryonic stem cells (ESC) can provide a continuous and readily available source of liver cells. ESC differentiation to liver cells is yet to be fully understood and comprehensive differentiation protocols are yet to be defined. Here, we aimed to achieve human (h)ESC differentiation into mature hepatocytes using defined recombinant differentiation factors and metabolites. Embryonic stem cell H1 line was sub-cultured on feeder layer. We induced hESCs into endodermal differentiation succeeded by early/late hepatic specification and finally into hepatocyte maturation using step combinations of Activin A and fibroblast growth factor (FGF)-2 for 7 days; followed by FGF-4 and bone morphogenic protein 2 (BMP2) for 7 days, succeeded by FGF-10 + hepatocyte growth factor 4 + epidermal growth factor for 14 days. Specific inhibitors/stimulators were added sequentially throughout differentiation. Cells were analysed by PCR, flow cytometry, microscopy or functional assays. Our hESC differentiation protocol resulted in viable cells with hepatocyte shape and morphology. We observed gradual changes in cell transcriptome, including up-regulation of differentiation-promoting GATA4, GATA6, POU5F1 and HNF4 transcription factors, steady levels of stemness-promoting SOX-2 and low levels of Nanog, as defined by PCR. The hESC-derived hepatocytes expressed alpha-antitrypsin, CD81, cytokeratin 8 and low density lipoprotein (LDL) receptor. The levels of alpha-fetoprotein and proliferation marker Ki-67 in hESC-derived hepatocytes remained elevated. Unlike stem cells, the hESC-derived hepatocytes performed LDL uptake, produced albumin and alanine aminotransferase and had functional alcohol dehydrogenase. We report a novel protocol for hESC differentiation into morphological and functional yet immature hepatocytes as an alternative method for hepatocyte generation. © 2012 John Wiley & Sons A/S.

Identification of a Sjögren's syndrome susceptibility locus at OAS1 that influences isoform switching, protein expression, and responsiveness to type I interferons

PubMed Central

Li, He; Reksten, Tove Ragna; Ice, John A.; Kelly, Jennifer A.; Adrianto, Indra; Wang, Shaofeng; He, Bo; Grundahl, Kiely M.; Glenn, Stuart B.; Miceli-Richard, Corinne; Bowman, Simon; Lester, Sue; Eriksson, Per; Brun, Johan G.; Gøransson, Lasse G.; Harboe, Erna; Guthridge, Joel M.; Patel, Ketan; Adler, Adam J.; Farris, A. Darise; Brennan, Michael T.; Chodosh, James; Gopalakrishnan, Rajaram; Weisman, Michael H.; Venuturupalli, Swamy; Wallace, Daniel J.; Hefner, Kimberly S.; Houston, Glen D.; Hughes, Pamela J.; Lewis, David M.; Radfar, Lida; Vista, Evan S.; Rohrer, Michael D.; Stone, Donald U.; Vyse, Timothy J.; Harley, John B.; James, Judith A.; Turner, Sean; Alevizos, Ilias; Anaya, Juan-Manuel; Rhodus, Nelson L.; Segal, Barbara M.; Montgomery, Courtney G.; Scofield, R. Hal; Kovats, Susan; Mariette, Xavier; Witte, Torsten; Rischmueller, Maureen; Omdal, Roald; Lessard, Christopher J.; Sivils, Kathy L.

2017-01-01

Sjögren’s syndrome (SS) is a common, autoimmune exocrinopathy distinguished by keratoconjunctivitis sicca and xerostomia. Patients frequently develop serious complications including lymphoma, pulmonary dysfunction, neuropathy, vasculitis, and debilitating fatigue. Dysregulation of type I interferon (IFN) pathway is a prominent feature of SS and is correlated with increased autoantibody titers and disease severity. To identify genetic determinants of IFN pathway dysregulation in SS, we performed cis-expression quantitative trait locus (eQTL) analyses focusing on differentially expressed type I IFN-inducible transcripts identified through a transcriptome profiling study. Multiple cis-eQTLs were associated with transcript levels of 2'-5'-oligoadenylate synthetase 1 (OAS1) peaking at rs10774671 (PeQTL = 6.05 × 10−14). Association of rs10774671 with SS susceptibility was identified and confirmed through meta-analysis of two independent cohorts (Pmeta = 2.59 × 10−9; odds ratio = 0.75; 95% confidence interval = 0.66–0.86). The risk allele of rs10774671 shifts splicing of OAS1 from production of the p46 isoform to multiple alternative transcripts, including p42, p48, and p44. We found that the isoforms were differentially expressed within each genotype in controls and patients with and without autoantibodies. Furthermore, our results showed that the three alternatively spliced isoforms lacked translational response to type I IFN stimulation. The p48 and p44 isoforms also had impaired protein expression governed by the 3' end of the transcripts. The SS risk allele of rs10774671 has been shown by others to be associated with reduced OAS1 enzymatic activity and ability to clear viral infections, as well as reduced responsiveness to IFN treatment. Our results establish OAS1 as a risk locus for SS and support a potential role for defective viral clearance due to altered IFN response as a genetic pathophysiological basis of this complex autoimmune disease. PMID:28640813

Crosstalk between Wnt Signaling and RNA Processing in Colorectal Cancer.

PubMed

Bordonaro, Michael

2013-01-01

RNA processing involves a variety of processes affecting gene expression, including the removal of introns through RNA splicing, as well as 3' end processing (cleavage and polyadenylation). Alternative RNA processing is fundamentally important for gene regulation, and aberrant processing is associated with the initiation and progression of cancer. Deregulated Wnt signaling, which is the initiating event in the development of most cases of human colorectal cancer (CRC), has been linked to modified RNA processing, which may contribute to Wnt-mediated colonic carcinogenesis. Crosstalk between Wnt signaling and alternative RNA splicing with relevance to CRC includes effects on the expression of Rac1b, an alternatively spliced gene associated with tumorigenesis, which exhibits alternative RNA splicing that is influenced by Wnt activity. In addition, Tcf4, a crucial component of Wnt signaling, also exhibits alternative splicing, which is likely involved in colonic tumorigenesis. Modulation of 3' end formation, including of the Wnt target gene COX-2, also can influence the neoplastic process, with implications for CRC. While many human genes are dependent on introns and splicing for normal levels of gene expression, naturally intronless genes exist with a unique metabolism that allows for intron-independent gene expression. Effects of Wnt activity on the RNA metabolism of the intronless Wnt-target gene c-jun is a likely contributor to cancer development. Further, butyrate, a breakdown product of dietary fiber and a histone deacetylase inhibitor, upregulates Wnt activity in CRC cells, and also modulates RNA processing; therefore, the interplay between Wnt activity, the modulation of this activity by butyrate, and differential RNA metabolism in colonic cells can significantly influence tumorigenesis. Determining the role played by altered RNA processing in Wnt-mediated neoplasia may lead to novel interventions aimed at restoring normal RNA metabolism for therapeutic benefit. Therefore, this minireview presents a brief overview of several aspects of RNA processing of relevance to cancer, which potentially influence, or are influenced by, Wnt signaling activity.

6-Formylindolo(3,2-b)Carbazole (FICZ) Modulates the Signalsome Responsible for RA-Induced Differentiation of HL-60 Myeloblastic Leukemia Cells

PubMed Central

Bunaciu, Rodica P.; LaTocha, Dorian H.; Varner, Jeffrey D.; Yen, Andrew

2015-01-01

6-Formylindolo(3,2-b)carbazole (FICZ) is a photoproduct of tryptophan and an endogenous high affinity ligand for aryl hydrocarbon receptor (AhR). It was previously reported that, in patient-derived HL-60 myeloblastic leukemia cells, retinoic acid (RA)-induced differentiation is driven by a signalsome containing c-Cbl and AhR. FICZ enhances RA-induced differentiation, assessed by expression of the membrane differentiation markers CD38 and CD11b, cell cycle arrest and the functional differentiation marker, inducible oxidative metabolism. Moreover, FICZ augments the expression of a number of the members of the RA-induced signalsome, such as c-Cbl, Vav1, Slp76, PI3K, and the Src family kinases Fgr and Lyn. Pursuing the molecular signaling responsible for RA-induced differentiation, we characterized, using FRET and clustering analysis, associations of key molecules thought to drive differentiation. Here we report that, assayed by FRET, AhR interacts with c-Cbl upon FICZ plus RA-induced differentiation, whereas AhR constitutively interacts with Cbl-b. Moreover, correlation analysis based on the flow cytometric assessment of differentiation markers and western blot detection of signaling factors reveal that Cbl-b, p-p38α and pT390-GSK3β, are not correlated with other known RA-induced signaling components or with a phenotypic outcome. We note that FICZ plus RA elicited signaling responses that were not typical of RA alone, but may represent alternative differentiation-driving pathways. In clusters of signaling molecules seminal to cell differentiation, FICZ co-administered with RA augments type and intensity of the dynamic changes induced by RA. Our data suggest relevance for FICZ in differentiation-induction therapy. The mechanism of action includes modulation of a SFK and MAPK centered signalsome and c-Cbl-AhR association. PMID:26287494

Alternative Mating Tactics in Male Chameleons (Chamaeleo chamaeleon) Are Evident in Both Long-Term Body Color and Short-Term Courtship Pattern

PubMed Central

Keren-Rotem, Tammy; Levy, Noga; Wolf, Lior; Bouskila, Amos; Geffen, Eli

2016-01-01

Alternative mating tactics in males of various taxa are associated with body color, body size, and social status. Chameleons are known for their ability to change body color following immediate environmental or social stimuli. In this study, we examined whether the differential appearance of male common chameleon during the breeding season is indeed an expression of alternative mating tactics. We documented body color of males and used computer vision techniques to classify images of individuals into discrete color patterns associated with seasons, individual characteristics, and social contexts. Our findings revealed no differences in body color and color patterns among males during the non-breeding season. However, during the breeding season males appeared in several color displays, which reflected body size, social status, and behavioral patterns. Furthermore, smaller and younger males resembled the appearance of small females. Consequently, we suggest that long-term color change in males during the breeding season reflects male alternative mating tactics. Upon encounter with a receptive female, males rapidly alter their appearance to that of a specific brief courtship display, which reflects their social status. The females, however, copulated indiscriminately in respect to male color patterns. Thus, we suggest that the differential color patterns displayed by males during the breeding season are largely aimed at inter-male signaling. PMID:27409771

Human palatine tonsil: a new potential tissue source of multipotent mesenchymal progenitor cells

PubMed Central

Janjanin, Sasa; Djouad, Farida; Shanti, Rabie M; Baksh, Dolores; Gollapudi, Kiran; Prgomet, Drago; Rackwitz, Lars; Joshi, Arjun S; Tuan, Rocky S

2008-01-01

Introduction Mesenchymal progenitor cells (MPCs) are multipotent progenitor cells in adult tissues, for example, bone marrow (BM). Current challenges of clinical application of BM-derived MPCs include donor site morbidity and pain as well as low cell yields associated with an age-related decrease in cell number and differentiation potential, underscoring the need to identify alternative sources of MPCs. Recently, MPC sources have diversified; examples include adipose, placenta, umbilicus, trabecular bone, cartilage, and synovial tissue. In the present work, we report the presence of MPCs in human tonsillar tissue. Methods We performed comparative and quantitative analyses of BM-MPCs with a subpopulation of adherent cells isolated from this lymphoid tissue, termed tonsil-derived MPCs (T-MPCs). The expression of surface markers was assessed by fluorescent-activated cell sorting analysis. Differentiation potential of T-MPCs was analyzed histochemically and by reverse transcription-polymerase chain reaction for the expression of lineage-related marker genes. The immunosuppressive properties of MPCs were determined in vitro in mixed lymphocyte reactions. Results Surface epitope analysis revealed that T-MPCs were negative for CD14, CD31, CD34, and CD45 expression and positive for CD29, CD44, CD90, and CD105 expression, a characteristic phenotype of BM-MPCs. Similar to BM-MPCs, T-MPCs could be induced to undergo adipogenic differentiation and, to a lesser extent, osteogenic and chondrogenic differentiation. T-MPCs did not express class II major histocompatibility (MHC) antigens, and in a similar but less pronounced manner compared with BM-MPCs, T-MPCs were immunosuppressive, inhibiting the proliferation of T cells stimulated by allogeneic T cells or by non-specific mitogenic stimuli via an indoleamine 2,3-dioxygenase-dependent mechanism. Conclusion Human palatine T-MPCs represent a new source of progenitor cells, potentially applicable for cell-based therapies. PMID:18662393

Identification of multiple dmrt1s in catfish: localization, dimorphic expression pattern, changes during testicular cycle and after methyltestosterone treatment.

PubMed

Raghuveer, K; Senthilkumaran, B

2009-05-01

The double sex and mab-3 related (DM) transcription factor 1 (dmrt1) plays an important role in testicular differentiation. Here, we report cloning of multiple dmrt1s, a full-length and two alternative spliced forms from adult catfish (Clarias gariepinus) testis, which encode predicted proteins of 287 (dmrt1a), 253 (dmrt1b) and 233 (dmrt1c) amino acid residues respectively. Interestingly, dmrt1c lacks the majority of the DM domain. Multiple dmrt1s (dmrt1a and dmrt1c) were obtained from Clarias batrachus as well. Tissue distribution (transcript and protein) of catfish dmrt1 revealed exclusive expression in testis. Semi-quantitative RT-PCR revealed the presence of multiple dmrt1s with high levels of dmrt1a in adult testis but not in ovary. Real-time RT-PCR analysis during testicular cycle showed higher levels of dmrt1 transcripts in preparatory and pre-spawning when compared with spawning and post-spawning phases. Immunocytochemical and immunofluorescence localization revealed the presence of catfish Dmrt1 protein in spermatogonia and spermatocytes, which indicates plausible role in spermatogenesis. Histological analysis indicated initiation of gonadal sex differentiation in catfish around 40-50 days after hatching. The potential role for dmrt1 in testicular differentiation is evident from its stage-dependent elevated expression in developing testis. Furthermore, dimorphic expressions of dmrt1s were evident at different stages of gonadal development or recrudescence in catfish. Treatment of methyl testosterone (MT) during early stages of gonadal sex differentiation resulted in adult males. Interestingly, we also obtained MT-treated fishes having ova-testis gonads. Analysis of dmrt1, sox9a, foxl2 and cyp19a1 expression patterns in MT-treated gonads revealed tissue-specific pattern. These results together suggest that multiple dmrt1s are testis-specific markers in catfish.

Validation and Interrogation of Differentially Expressed and Alternatively Spliced Genes in African-American Prostate Cancer

DTIC Science & Technology

2015-10-01

Epigenetic Regulation of Tyrosine Receptor Kinases and Childhood Respiratory Disease (Murphy – Duke site PI) Time Commitment: 5% 0.6 calendar...children to the risk of childhood asthma and lower lung function when exposed to prenatal tobacco smoke. Specific Aims: Specific aims are: (1) to...epigenetic and/or genetic variation in the TAM genes are independently or jointly associated with childhood asthma and 24 with attained lung function at

A study of alternative splicing in the pig

PubMed Central

2010-01-01

Background Since at least half of the genes in mammalian genomes are subjected to alternative splicing, alternative pre-mRNA splicing plays an important contribution to the complexity of the mammalian proteome. Expressed sequence tags (ESTs) provide evidence of a great number of possible alternative isoforms. With the EST resource for the domestic pig now containing more than one million porcine ESTs, it is possible to identify alternative splice forms of the individual transcripts in this species from the EST data with some confidence. Results The pig EST data generated by the Sino-Danish Pig Genome project has been assembled with publicly available ESTs and made available in the PigEST database. Using the Distiller package 2,515 EST clusters with candidate alternative isoforms were identified in the EST data with high confidence. In agreement with general observations in human and mouse, we find putative splice variants in about 30% of the contigs with more than 50 ESTs. Based on the criteria that a minimum of two EST sequences confirmed each splice event, a list of 100 genes with the most distinct tissue-specific alternative splice events was generated from the list of candidates. To confirm the tissue specificity of the splice events, 10 genes with functional annotation were randomly selected from which 16 individual splice events were chosen for experimental verification by quantitative PCR (qPCR). Six genes were shown to have tissue specific alternatively spliced transcripts with expression patterns matching those of the EST data. The remaining four genes had tissue-restricted expression of alternative spliced transcripts. Five out of the 16 splice events that were experimentally verified were found to be putative pig specific. Conclusions In accordance with human and rodent studies we estimate that approximately 30% of the porcine genes undergo alternative splicing. We found a good correlation between EST predicted tissue-specificity and experimentally validated splice events in different porcine tissue. This study indicates that a cluster size of around 50 ESTs is optimal for in silico detection of alternative splicing. Although based on a limited number of splice events, the study supports the notion that alternative splicing could have an important impact on species differentiation since 31% of the splice events studied appears to be species specific. PMID:20444244

Screening of biomarkers for prediction of response to and prognosis after chemotherapy for breast cancers.

PubMed

Bing, Feng; Zhao, Yu

2016-01-01

To screen the biomarkers having the ability to predict prognosis after chemotherapy for breast cancers. Three microarray data of breast cancer patients undergoing chemotherapy were collected from Gene Expression Omnibus database. After preprocessing, data in GSE41112 were analyzed using significance analysis of microarrays to screen the differentially expressed genes (DEGs). The DEGs were further analyzed by Differentially Coexpressed Genes and Links to construct a function module, the prognosis efficacy of which was verified by the other two datasets (GSE22226 and GSE58644) using Kaplan-Meier plots. The involved genes in function module were subjected to a univariate Cox regression analysis to confirm whether the expression of each prognostic gene was associated with survival. A total of 511 DEGs between breast cancer patients who received chemotherapy or not were obtained, consisting of 421 upregulated and 90 downregulated genes. Using the Differentially Coexpressed Genes and Links package, 1,244 differentially coexpressed genes (DCGs) were identified, among which 36 DCGs were regulated by the transcription factor complex NFY (NFYA, NFYB, NFYC). These 39 genes constructed a gene module to classify the samples in GSE22226 and GSE58644 into three subtypes and these subtypes exhibited significantly different survival rates. Furthermore, several genes of the 39 DCGs were shown to be significantly associated with good (such as CDC20) and poor (such as ARID4A) prognoses following chemotherapy. Our present study provided a serial of biomarkers for predicting the prognosis of chemotherapy or targets for development of alternative treatment (ie, CDC20 and ARID4A) in breast cancer patients.

Serine/Arginine-Rich Splicing Factor 3 and Heterogeneous Nuclear Ribonucleoprotein A1 Regulate Alternative RNA Splicing and Gene Expression of Human Papillomavirus 18 through Two Functionally Distinguishable cis Elements.

PubMed

Ajiro, Masahiko; Tang, Shuang; Doorbar, John; Zheng, Zhi-Ming

2016-10-15

Human papillomavirus 18 (HPV18) is the second most common oncogenic HPV type associated with cervical, anogenital, and oropharyngeal cancers. Like other oncogenic HPVs, HPV18 encodes two major (one early and one late) polycistronic pre-mRNAs that are regulated by alternative RNA splicing to produce a repertoire of viral transcripts for the expression of individual viral genes. However, RNA cis-regulatory elements and trans-acting factors contributing to HPV18 alternative RNA splicing remain unknown. In this study, an exonic splicing enhancer (ESE) in the nucleotide (nt) 3520 to 3550 region in the HPV18 genome was identified and characterized for promotion of HPV18 929^3434 splicing and E1^E4 production through interaction with SRSF3, a host oncogenic splicing factor differentially expressed in epithelial cells and keratinocytes. Introduction of point mutations in the SRSF3-binding site or knockdown of SRSF3 expression in cells reduces 929^3434 splicing and E1^E4 production but activates other, minor 929^3465 and 929^3506 splicing. Knockdown of SRSF3 expression also enhances the expression of E2 and L1 mRNAs. An exonic splicing silencer (ESS) in the HPV18 nt 612 to 639 region was identified as being inhibitory to the 233^416 splicing of HPV18 E6E7 pre-mRNAs via binding to hnRNP A1, a well-characterized, abundantly and ubiquitously expressed RNA-binding protein. Introduction of point mutations into the hnRNP A1-binding site or knockdown of hnRNP A1 expression promoted 233^416 splicing and reduced E6 expression. These data provide the first evidence that the alternative RNA splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host trans-acting splicing factors. Expression of HPV18 genes is regulated by alternative RNA splicing of viral polycistronic pre-mRNAs to produce a repertoire of viral early and late transcripts. RNA cis elements and trans-acting factors contributing to HPV18 alternative RNA splicing have been discovered in this study for the first time. The identified ESS at the E7 open reading frame (ORF) prevents HPV18 233^416 splicing in the E6 ORF through interaction with a host splicing factor, hnRNP A1, and regulates E6 and E7 expression of the early E6E7 polycistronic pre-mRNA. The identified ESE at the E1^E4 ORF promotes HPV18 929^3434 splicing of both viral early and late pre-mRNAs and E1^E4 production through interaction with SRSF3. This study provides important observations on how alternative RNA splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host splicing factors and offers potential therapeutic targets to overcome HPV-related cancer. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Serine/Arginine-Rich Splicing Factor 3 and Heterogeneous Nuclear Ribonucleoprotein A1 Regulate Alternative RNA Splicing and Gene Expression of Human Papillomavirus 18 through Two Functionally Distinguishable cis Elements

PubMed Central

Ajiro, Masahiko; Tang, Shuang; Doorbar, John

2016-01-01

ABSTRACT Human papillomavirus 18 (HPV18) is the second most common oncogenic HPV type associated with cervical, anogenital, and oropharyngeal cancers. Like other oncogenic HPVs, HPV18 encodes two major (one early and one late) polycistronic pre-mRNAs that are regulated by alternative RNA splicing to produce a repertoire of viral transcripts for the expression of individual viral genes. However, RNA cis-regulatory elements and trans-acting factors contributing to HPV18 alternative RNA splicing remain unknown. In this study, an exonic splicing enhancer (ESE) in the nucleotide (nt) 3520 to 3550 region in the HPV18 genome was identified and characterized for promotion of HPV18 929^3434 splicing and E1^E4 production through interaction with SRSF3, a host oncogenic splicing factor differentially expressed in epithelial cells and keratinocytes. Introduction of point mutations in the SRSF3-binding site or knockdown of SRSF3 expression in cells reduces 929^3434 splicing and E1^E4 production but activates other, minor 929^3465 and 929^3506 splicing. Knockdown of SRSF3 expression also enhances the expression of E2 and L1 mRNAs. An exonic splicing silencer (ESS) in the HPV18 nt 612 to 639 region was identified as being inhibitory to the 233^416 splicing of HPV18 E6E7 pre-mRNAs via binding to hnRNP A1, a well-characterized, abundantly and ubiquitously expressed RNA-binding protein. Introduction of point mutations into the hnRNP A1-binding site or knockdown of hnRNP A1 expression promoted 233^416 splicing and reduced E6 expression. These data provide the first evidence that the alternative RNA splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host trans-acting splicing factors. IMPORTANCE Expression of HPV18 genes is regulated by alternative RNA splicing of viral polycistronic pre-mRNAs to produce a repertoire of viral early and late transcripts. RNA cis elements and trans-acting factors contributing to HPV18 alternative RNA splicing have been discovered in this study for the first time. The identified ESS at the E7 open reading frame (ORF) prevents HPV18 233^416 splicing in the E6 ORF through interaction with a host splicing factor, hnRNP A1, and regulates E6 and E7 expression of the early E6E7 polycistronic pre-mRNA. The identified ESE at the E1^E4 ORF promotes HPV18 929^3434 splicing of both viral early and late pre-mRNAs and E1^E4 production through interaction with SRSF3. This study provides important observations on how alternative RNA splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host splicing factors and offers potential therapeutic targets to overcome HPV-related cancer. PMID:27489271

The mouse Gtl2 gene is differentially expressed during embryonic development, encodes multiple alternatively spliced transcripts, and may act as an RNA.

PubMed

Schuster-Gossler, K; Bilinski, P; Sado, T; Ferguson-Smith, A; Gossler, A

1998-06-01

We have isolated a novel mouse gene (Gtl2) from the site of a gene trap integration (Gtl2lacZ) that gave rise to developmentally regulated lacZ expression, and a dominant parental-origin-dependent phenotype. Heterozygous Gtl2lacZ mice that inherited the transgene from the father showed a proportionate dwarfism phenotype, whereas the penetrance and expressivity of the phenotype was strongly reduced in Gtl2lacZ mice that inherited the transgene from the mother. Gtl2 expression is highly similar to the beta-galactosidase staining pattern, and is down-regulated but not abolished in mice carrying the Gtl2lacZ insertion. In early postimplantation embryos, Gtl2 is expressed in the visceral yolk sac and embryonic ectoderm. During subsequent development and organogenesis, Gtl2 transcripts are abundant in the paraxial mesoderm closely correlated with myogenic differentiation, in parts of the central nervous system, and in the epithelial ducts of developing excretory organs. The Gtl2 gene gives rise to various differentially spliced transcripts, which contain multiple small open reading frames (ORF). However, none of the ATG codons of these ORFs is in the context of a strong Kozak consensus sequence for initiation of translation, suggesting that Gtl2 might function as an RNA. Nuclear Gtl2 RNA was detected in a temporally and spatially regulated manner, and partially processed Gtl2 transcripts were readily detected in Northern blot hybridizations of polyadenylated RNA, suggesting that primary Gtl2 transcripts are differently processed in various cell types during development. Gtl2 transcript levels are present in parthenogenic embryos but may be reduced, consistent with the pattern of inheritance of the Gtl2lacZ phenotype.

Axed MUC4 (MUC4/X) aggravates pancreatic malignant phenotype by activating integrin-β1/FAK/ERK pathway.

PubMed

Jahan, Rahat; Macha, Muzafar A; Rachagani, Satyanarayana; Das, Srustidhar; Smith, Lynette M; Kaur, Sukhwinder; Batra, Surinder K

2018-08-01

Alternative splicing is evolving as an eminent player of oncogenic signaling for tumor development and progression. Mucin 4 (MUC4), a type I membrane-bound mucin, is differentially expressed in pancreatic cancer (PC) and plays a critical role in its progression and metastasis. However, the molecular implications of MUC4 splice variants during disease pathogenesis remain obscure. The present study delineates the pathological and molecular significance of a unique splice variant of MUC4, MUC4/X, which lacks the largest exon 2, along with exon 3. Exon 2 encodes for the highly glycosylated tandem repeat (TR) domain of MUC4 and its absence creates MUC4/X, which is devoid of TR. Expression analysis from PC clinical samples revealed significant upregulation of MUC4/X in PC tissues with most differential expression in poorly differentiated tumors. In vitro studies suggest that overexpression of MUC4/X in wild-type-MUC4 (WT-MUC4) null PC cell lines markedly enhanced PC cell proliferation, invasion, and adhesion to extracellular matrix (ECM) proteins. Furthermore, MUC4/X overexpression leads to an increase in the tumorigenic potential of PC cells in orthotopic transplantation studies. In line with these findings, doxycycline-induced expression of MUC4/X in an endogenous WT-MUC4 expressing PC cell line (Capan-1) also displayed enhanced cell proliferation, invasion, and adhesion to ECM, compared to WT-MUC4 alone, emphasizing its direct involvement in the aggressive behavior of PC cells. Investigation into the molecular mechanism suggested that MUC4/X facilitated PC tumorigenesis via integrin-β1/FAK/ERK signaling pathway. Overall, these findings revealed the novel role of MUC4/X in promoting and sustaining the oncogenic features of PC. Copyright © 2018 Elsevier B.V. All rights reserved.

Integrative topological analysis of mass spectrometry data reveals molecular features with clinical relevance in esophageal squamous cell carcinoma

PubMed Central

Gao, She-Gan; Liu, Rui-Min; Zhao, Yun-Gang; Wang, Pei; Ward, Douglas G.; Wang, Guang-Chao; Guo, Xiang-Qian; Gu, Juan; Niu, Wan-Bin; Zhang, Tian; Martin, Ashley; Guo, Zhi-Peng; Feng, Xiao-Shan; Qi, Yi-Jun; Ma, Yuan-Fang

2016-01-01

Combining MS-based proteomic data with network and topological features of such network would identify more clinically relevant molecules and meaningfully expand the repertoire of proteins derived from MS analysis. The integrative topological indexes representing 95.96% information of seven individual topological measures of node proteins were calculated within a protein-protein interaction (PPI) network, built using 244 differentially expressed proteins (DEPs) identified by iTRAQ 2D-LC-MS/MS. Compared with DEPs, differentially expressed genes (DEGs) and comprehensive features (CFs), structurally dominant nodes (SDNs) based on integrative topological index distribution produced comparable classification performance in three different clinical settings using five independent gene expression data sets. The signature molecules of SDN-based classifier for distinction of early from late clinical TNM stages were enriched in biological traits of protein synthesis, intracellular localization and ribosome biogenesis, which suggests that ribosome biogenesis represents a promising therapeutic target for treating ESCC. In addition, ITGB1 expression selected exclusively by integrative topological measures correlated with clinical stages and prognosis, which was further validated with two independent cohorts of ESCC samples. Thus the integrative topological analysis of PPI networks proposed in this study provides an alternative approach to identify potential biomarkers and therapeutic targets from MS/MS data with functional insights in ESCC. PMID:26898710

Molecular characterization and gene expression patterns of retinoid receptors, in normal and regenerating tissues of the sea cucumber, Holothuria glaberrima.

PubMed

Viera-Vera, Jorge; García-Arrarás, José E

2018-05-15

Retinoic acid receptors (RAR) and retinoid X receptors (RXR) are ligand-mediated transcription factors that synchronize intricate signaling networks in metazoans. Dimer formation between these two nuclear receptors mediates the recruitment of co-regulatory complexes coordinating the progression of signaling cascades during developmental and regenerative events. In the present study we identified and characterized the receptors for retinoic acid in the sea cucumber Holothuria glaberrima; a model system capable of regenerative organogenesis during adulthood. Molecular characterizations revealed the presence of three isoforms of RAR and two of RXR as a consequence of alternative splicing events. Various analyses including: primary structure sequencing, phylogenetic analysis, protein domain prediction, and multiple sequence alignment further confirmed their identity. Semiquantitative reverse transcription PCR analysis of each receptor isoform herein identified showed that the retinoid receptors are expressed in all tissues sampled: the mesenteries, respiratory trees, muscles, gonads, and the digestive tract. During regenerative organogenesis two of the receptors (RAR-L and RXR-T) showed differential expression in the posterior segment while RAR-S is differentially expressed in the anterior segment of the intestine. This work presents the first description of the components relaying the signaling for retinoic acid within this model system. Copyright © 2018 Elsevier B.V. All rights reserved.

PDZ-containing proteins: alternative splicing as a source of functional diversity.

PubMed

Sierralta, Jimena; Mendoza, Carolina

2004-12-01

Scaffold proteins allow specific protein complexes to be assembled in particular regions of the cell at which they organize subcellular structures and signal transduction complexes. This characteristic is especially important for neurons, which are highly polarized cells. Among the domains contained by scaffold proteins, the PSD-95, Discs-large, ZO-1 (PDZ) domains are of particular relevance in signal transduction processes and maintenance of neuronal and epithelial polarity. These domains are specialized in the binding of the carboxyl termini of proteins allowing membrane proteins to be localized by the anchoring to the cytoskeleton mediated by PDZ-containing scaffold proteins. In vivo studies carried out in Drosophila have taught that the role of many scaffold proteins is not limited to a single process; thus, in many cases the same genes are expressed in different tissues and participate in apparently very diverse processes. In addition to the differential expression of interactors of scaffold proteins, the expression of variants of these molecular scaffolds as the result of the alternative processing of the genes that encode them is proving to be a very important source of variability and complexity on a main theme. Alternative splicing in the nervous system is well documented, where specific isoforms play roles in neurotransmission, ion channel function, neuronal cell recognition, and are developmentally regulated making it a major mechanism of functional diversity. Here we review the current state of knowledge about the diversity and the known function of PDZ-containing proteins in Drosophila with emphasis in the role played by alternatively processed forms in the diversity of functions attributed to this family of proteins.

Short- and long-term memory are modulated by multiple isoforms of the fragile X mental retardation protein.

PubMed

Banerjee, Paromita; Schoenfeld, Brian P; Bell, Aaron J; Choi, Catherine H; Bradley, Michael P; Hinchey, Paul; Kollaros, Maria; Park, Jae H; McBride, Sean M J; Dockendorff, Thomas C

2010-05-12

The diversity of protein isoforms arising from alternative splicing is thought to modulate fine-tuning of synaptic plasticity. Fragile X mental retardation protein (FMRP), a neuronal RNA binding protein, exists in isoforms as a result of alternative splicing, but the contribution of these isoforms to neural plasticity are not well understood. We show that two isoforms of Drosophila melanogaster FMRP (dFMR1) have differential roles in mediating neural development and behavior functions conferred by the dfmr1 gene. These isoforms differ in the presence of a protein interaction module that is related to prion domains and is functionally conserved between FMRPs. Expression of both isoforms is necessary for optimal performance in tests of short- and long-term memory of courtship training. The presence or absence of the protein interaction domain may govern the types of ribonucleoprotein (RNP) complexes dFMR1 assembles into, with different RNPs regulating gene expression in a manner necessary for establishing distinct phases of memory formation.

Brain Transcriptional Profiles of Male Alternative Reproductive Tactics and Females in Bluegill Sunfish

PubMed Central

Partridge, Charlyn G.; MacManes, Matthew D.; Knapp, Rosemary; Neff, Bryan D.

2016-01-01

Bluegill sunfish (Lepomis macrochirus) are one of the classic systems for studying male alternative reproductive tactics (ARTs) in teleost fishes. In this species, there are two distinct life histories: parental and cuckolder, encompassing three reproductive tactics, parental, satellite, and sneaker. The parental life history is fixed, whereas individuals who enter the cuckolder life history transition from sneaker to satellite tactic as they grow. For this study, we used RNAseq to characterize the brain transcriptome of the three male tactics and females during spawning to identify gene ontology (GO) categories and potential candidate genes associated with each tactic. We found that sneaker males had higher levels of gene expression differentiation compared to the other two male tactics. Sneaker males also had higher expression in ionotropic glutamate receptor genes, specifically AMPA receptors, compared to other males, which may be important for increased spatial working memory while attempting to cuckold parental males at their nests. Larger differences in gene expression also occurred among male tactics than between males and females. We found significant expression differences in several candidate genes that were previously identified in other species with ARTs and suggest a previously undescribed role for cAMP-responsive element modulator (crem) in influencing parental male behaviors during spawning. PMID:27907106

Brain Transcriptional Profiles of Male Alternative Reproductive Tactics and Females in Bluegill Sunfish.

PubMed

Partridge, Charlyn G; MacManes, Matthew D; Knapp, Rosemary; Neff, Bryan D

2016-01-01

Bluegill sunfish (Lepomis macrochirus) are one of the classic systems for studying male alternative reproductive tactics (ARTs) in teleost fishes. In this species, there are two distinct life histories: parental and cuckolder, encompassing three reproductive tactics, parental, satellite, and sneaker. The parental life history is fixed, whereas individuals who enter the cuckolder life history transition from sneaker to satellite tactic as they grow. For this study, we used RNAseq to characterize the brain transcriptome of the three male tactics and females during spawning to identify gene ontology (GO) categories and potential candidate genes associated with each tactic. We found that sneaker males had higher levels of gene expression differentiation compared to the other two male tactics. Sneaker males also had higher expression in ionotropic glutamate receptor genes, specifically AMPA receptors, compared to other males, which may be important for increased spatial working memory while attempting to cuckold parental males at their nests. Larger differences in gene expression also occurred among male tactics than between males and females. We found significant expression differences in several candidate genes that were previously identified in other species with ARTs and suggest a previously undescribed role for cAMP-responsive element modulator (crem) in influencing parental male behaviors during spawning.

Detection of high CD44 expression in oral cancers using the novel monoclonal antibody, C44Mab-5.

PubMed

Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Yanaka, Miyuki; Kaneko, Mika K; Kato, Yukinari

2018-07-01

CD44 is a transmembrane glycoprotein that regulates a variety of genes related to cell-adhesion, migration, proliferation, differentiation, and survival. A large number of alternative splicing isoforms of CD44, containing various combinations of alternative exons, have been reported. CD44 standard (CD44s), which lacks variant exons, is widely expressed on the surface of most tissues and all hematopoietic cells. In contrast, CD44 variant isoforms show tissue-specific expression patterns and have been extensively studied as both prognostic markers and therapeutic targets in cancer and other diseases. In this study, we immunized mice with CHO-K1 cell lines overexpressing CD44v3-10 to obtain novel anti-CD44 mAbs. One of the clones, C 44 Mab-5 (IgG 1 , kappa), recognized both CD44s and CD44v3-10. C 44 Mab-5 also reacted with oral cancer cells such as Ca9-22, HO-1-u-1, SAS, HSC-2, HSC-3, and HSC-4 using flow cytometry. Moreover, immunohistochemical analysis revealed that C 44 Mab-5 detected 166/182 (91.2%) of oral cancers. These results suggest that the C 44 Mab-5 antibody may be useful for investigating the expression and function of CD44 in various cancers.

Transcriptome Profiling of a Multiple Recurrent Muscle-Invasive Urothelial Carcinoma of the Bladder by Deep Sequencing

PubMed Central

Zhang, Shufang; Liu, Yanxuan; Liu, Zhenxiang; Zhang, Chong; Cao, Hui; Ye, Yongqing; Wang, Shunlan; Zhang, Ying'ai; Xiao, Sifang; Yang, Peng; Li, Jindong; Bai, Zhiming

2014-01-01

Urothelial carcinoma of the bladder (UCB) is one of the commonly diagnosed cancers in the world. The UCB has the highest rate of recurrence of any malignancy. A genome-wide screening of transcriptome dysregulation between cancer and normal tissue would provide insight into the molecular basis of UCB recurrence and is a key step to discovering biomarkers for diagnosis and therapeutic targets. Compared with microarray technology, which is commonly used to identify expression level changes, the recently developed RNA-seq technique has the ability to detect other abnormal regulations in the cancer transcriptome, such as alternative splicing. In this study, we performed high-throughput transcriptome sequencing at ∼50× coverage on a recurrent muscle-invasive cisplatin-resistance UCB tissue and the adjacent non-tumor tissue. The results revealed cancer-specific differentially expressed genes between the tumor and non-tumor tissue enriched in the cell adhesion molecules, focal adhesion and ECM-receptor interaction pathway. Five dysregulated genes, including CDH1, VEGFA, PTPRF, CLDN7, and MMP2 were confirmed by Real time qPCR in the sequencing samples and the additional eleven samples. Our data revealed that more than three hundred genes showed differential splicing patterns between tumor tissue and non-tumor tissue. Among these genes, we filtered 24 cancer-associated alternative splicing genes with differential exon usage. The findings from RNA-Seq were validated by Real time qPCR for CD44, PDGFA, NUMB, and LPHN2. This study provides a comprehensive survey of the UCB transcriptome, which provides better insight into the complexity of regulatory changes during recurrence and metastasis. PMID:24622401

Histologic and immunohistochemical characteristics of cutaneous cysts in Goltz-Gorlin syndrome: clues for differentiation of nonsyndromic cysts.

PubMed

Tirado, Mariantonieta; Ständer, Sonja; Metze, Dieter

2014-11-01

Goltz-Gorlin syndrome presents with multiple basal cell carcinomas, odontogenic keratocysts, and cutaneous cysts, among other manifestations. The cutaneous cysts have been described as both epidermoid cysts and keratocysts but were not further characterized. Light microscopic examinations were made on 23 cutaneous cysts in 4 patients associated with Goltz-Gorlin syndrome located on extremities, face, trunk, palms, and soles and compared with nonsyndromic vellus hair cysts, steatocystomas, and hybrid cysts. Twenty-one of the syndromic cysts revealed alternating infundibular-like and steatocystoma-like squamous epitheliums in varying proportions. The cysts were lined by both smooth and corrugated squamous epithelium. The horny layer was composed by alternating areas of thin, lamellate, and compact eosinophilic keratin. Only 2 cases showed an exclusive steatocystoma-like type of epithelium very similar to odontogenic keratocysts. Sebaceous glands and follicular structures were absent. There were no differences between palmar and plantar cysts and other anatomic locations. The ultrastructural findings in syndromatic cysts confirmed variable expression of keratohyalin granules. Only 3 of 6 cases of nonsyndromic hybrid cysts showed overlapping features with syndromic cysts. Immunohistochemical profiling of keratin, involucrin, filaggrin, loricrin, and BCL-2 expression in syndromatic cysts showed exclusive positivity of K19 and continuous staining for BCL-2. In summary, 2 types of cutaneous cysts are characteristic of Goltz-Gorlin, irrelevant of their anatomic location, namely steatocystoma-like and more frequently hybrid-like. The diagnosis of syndromic hybrid-like cysts should be considered whenever infundibular and steatocystoma differentiation alternate and overlap. Altogether, these findings in epithelial cysts may raise the suspicion of Goltz-Gorlin as an underlying cause.

Alternative splice isoforms of small conductance calcium-activated SK2 channels differ in molecular interactions and surface levels

PubMed Central

Scholl, Elizabeth Storer; Pirone, Antonella; Cox, Daniel H; Duncan, R Keith; Jacob, Michele H

2014-01-01

Small conductance Ca2+-sensitive potassium (SK2) channels are voltage-independent, Ca2+-activated ion channels that conduct potassium cations and thereby modulate the intrinsic excitability and synaptic transmission of neurons and sensory hair cells. In the cochlea, SK2 channels are functionally coupled to the highly Ca2+ permeant α9/10-nicotinic acetylcholine receptors (nAChRs) at olivocochlear postsynaptic sites. SK2 activation leads to outer hair cell hyperpolarization and frequency-selective suppression of afferent sound transmission. These inhibitory responses are essential for normal regulation of sound sensitivity, frequency selectivity, and suppression of background noise. However, little is known about the molecular interactions of these key functional channels. Here we show that SK2 channels co-precipitate with α9/10-nAChRs and with the actin-binding protein α-actinin-1. SK2 alternative splicing, resulting in a 3 amino acid insertion in the intracellular 3′ terminus, modulates these interactions. Further, relative abundance of the SK2 splice variants changes during developmental stages of synapse maturation in both the avian cochlea and the mammalian forebrain. Using heterologous cell expression to separately study the 2 distinct isoforms, we show that the variants differ in protein interactions and surface expression levels, and that Ca2+ and Ca2+-bound calmodulin differentially regulate their protein interactions. Our findings suggest that the SK2 isoforms may be distinctly modulated by activity-induced Ca2+ influx. Alternative splicing of SK2 may serve as a novel mechanism to differentially regulate the maturation and function of olivocochlear and neuronal synapses. PMID:24394769

Cardiomyogenic differentiation of human sternal bone marrow mesenchymal stem cells using a combination of basic fibroblast growth factor and hydrocortisone.

PubMed

Hafez, Pezhman; Jose, Shinsmon; Chowdhury, Shiplu R; Ng, Min Hwei; Ruszymah, B H I; Abdul Rahman Mohd, Ramzisham

2016-01-01

The alarming rate of increase in myocardial infarction and marginal success in efforts to regenerate the damaged myocardium through conventional treatments creates an exceptional avenue for cell-based therapy. Adult bone marrow mesenchymal stem cells (MSCs) can be differentiated into cardiomyocytes, by treatment with 5-azacytidine, thus, have been anticipated as a therapeutic tool for myocardial infarction treatment. In this study, we investigated the ability of basic fibroblastic growth factor (bFGF) and hydrocortisone as a combined treatment to stimulate the differentiation of MSCs into cardiomyocytes. MSCs were isolated from sternal marrow of patients undergoing heart surgery (CABG). The isolated cells were initially monitored for the growth pattern, followed by characterization using ISCT recommendations. Cells were then differentiated using a combination of bFGF and hydrocortisone and evaluated for the expression of characteristic cardiac markers such as CTnI, CTnC, and Cnx43 at protein level using immunocytochemistry and flow cytometry, and CTnC and CTnT at mRNA level. The expression levels and pattern of the cardiac markers upon analysis with ICC and qRT-PCR were similar to that of 5-azacytidine induced cells and cultured primary human cardiomyocytes. However, flow cytometric evaluation revealed that induction with bFGF and hydrocortisone drives MSC differentiation to cardiomyocytes with a marginally higher efficiency. These results indicate that combination treatment of bFGF and hydrocortisone can be used as an alternative induction method for cardiomyogenic differentiation of MSCs for future clinical applications. © 2015 International Federation for Cell Biology.

Cigarette smoke differentially affects IL-13-induced gene expression in human airway epithelial cells.

PubMed

Mertens, Tinne C J; van der Does, Anne M; Kistemaker, Loes E; Ninaber, Dennis K; Taube, Christian; Hiemstra, Pieter S

2017-07-01

Allergic airways inflammation in asthma is characterized by an airway epithelial gene signature composed of POSTN , CLCA1 , and SERPINB2 This Th2 gene signature is proposed as a tool to classify patients with asthma into Th2-high and Th2-low phenotypes. However, many asthmatics smoke and the effects of cigarette smoke exposure on the epithelial Th2 gene signature are largely unknown. Therefore, we investigated the combined effect of IL-13 and whole cigarette smoke (CS) on the Th2 gene signature and the mucin-related genes MUC5AC and SPDEF in air-liquid interface differentiated human bronchial (ALI-PBEC) and tracheal epithelial cells (ALI-PTEC). Cultures were exposed to IL-13 for 14 days followed by 5 days of IL-13 with CS exposure. Alternatively, cultures were exposed once daily to CS for 14 days, followed by 5 days CS with IL-13. POSTN , SERPINB2 , and CLCA1 expression were measured 24 h after the last exposure to CS and IL-13. In both models POSTN , SERPINB2 , and CLCA1 expression were increased by IL-13. CS markedly affected the IL-13-induced Th2 gene signature as indicated by a reduced POSTN , CLCA1 , and MUC5AC expression in both models. In contrast, IL-13-induced SERPINB2 expression remained unaffected by CS, whereas SPDEF expression was additively increased. Importantly, cessation of CS exposure failed to restore IL-13-induced POSTN and CLCA1 expression. We show for the first time that CS differentially affects the IL-13-induced gene signature for Th2-high asthma. These findings provide novel insights into the interaction between Th2 inflammation and cigarette smoke that is important for asthma pathogenesis and biomarker-guided therapy in asthma. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

RNA-sequence analysis of gene expression from honeybees (Apis mellifera) infected with Nosema ceranae

PubMed Central

Fougeroux, André; Petit, Fabien; Anselmo, Anna; Gorni, Chiara; Cucurachi, Marco; Cersini, Antonella; Granato, Anna; Cardeti, Giusy; Formato, Giovanni; Mutinelli, Franco; Giuffra, Elisabetta; Williams, John L.; Botti, Sara

2017-01-01

Honeybees (Apis mellifera) are constantly subjected to many biotic stressors including parasites. This study examined honeybees infected with Nosema ceranae (N. ceranae). N. ceranae infection increases the bees energy requirements and may contribute to their decreased survival. RNA-seq was used to investigate gene expression at days 5, 10 and 15 Post Infection (P.I) with N. ceranae. The expression levels of genes, isoforms, alternative transcription start sites (TSS) and differential promoter usage revealed a complex pattern of transcriptional and post-transcriptional gene regulation suggesting that bees use a range of tactics to cope with the stress of N. ceranae infection. N. ceranae infection may cause reduced immune function in the bees by: (i)disturbing the host amino acids metabolism (ii) down-regulating expression of antimicrobial peptides (iii) down-regulation of cuticle coatings and (iv) down-regulation of odorant binding proteins. PMID:28350872

Chondrogenic differentiation of human mesenchymal stem cells on fish scale collagen.

PubMed

Hsu, Han-Hsiu; Uemura, Toshimasa; Yamaguchi, Isamu; Ikoma, Toshiyuki; Tanaka, Junzo

2016-08-01

Fish collagen has recently been reported to be a novel biomaterial for cell and tissue culture as an alternative to conventional mammalian collagens such as bovine and porcine collagens. Fish collagen could overcome the risk of zoonosis, such as from bovine spongiform encephalopathy. Among fish collagens, tilapia collagen, the denaturing temperature of which is near 37°C, is appropriate for cell and tissue culture. In this study, we investigated chondrogenic differentiation of human mesenchymal stem cells (hMSCs) cultured on tilapia scale collagen fibrils compared with porcine collagen and non-coated dishes. The collagen fibrils were observed using a scanning electronic microscope. Safranin O staining, glycosaminoglycans (GAG) expression, and real-time PCR were examined to evaluate chondrogenesis of hMSCs on each type of collagen fibril. The results showed that hMSCs cultured on tilapia scale collagen showed stronger Safranin O staining and higher GAG expression at day 6. Results of real-time PCR indicated that hMSCs cultured on tilapia collagen showed earlier SOX9 expression on day 4 and higher AGGRECAN and COLLAGEN II expression on day 6 compared with on porcine collagen and non-coated dishes. Furthermore, low mRNA levels of bone gamma-carboxyglutamate, a specific marker of osteogenesis, showed that tilapia collagen fibrils specifically enhanced chondrogenic differentiation of hMSCs in chondrogenic medium, as well as porcine collagen. Accordingly, tilapia scale collagen may provide an appropriate collagen source for hMSC chondrogenesis in vitro. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Role of KCa3.1 Channels in Macrophage Polarization and Its Relevance in Atherosclerotic Plaque Instability.

PubMed

Xu, Rende; Li, Chenguang; Wu, Yizhe; Shen, Li; Ma, Jianying; Qian, Juying; Ge, Junbo

2017-02-01

Emerging evidence indicates that proinflammatory macrophage polarization imbalance plays a key role in atherosclerotic plaque progression and instability. The calcium-activated potassium channel KCa3.1 is critically involved in macrophage activation and function. However, the role of KCa3.1 in macrophage polarization is unknown. This study investigates the potential role of KCa3.1 in transcriptional regulation in macrophage polarization and its relationship to plaque instability. Human monocytes were differentiated into macrophages using macrophage colony-stimulating factor. Macrophages were then polarized into proinflammatory M1 cells by interferon-γ and lipopolysaccharide and into alternative M2 macrophages by interleukin-4. A model for plaque instability was induced by combined partial ligation of the left renal artery and left common carotid artery in apolipoprotein E knockout mice. Significant upregulation of KCa3.1 expression was observed during the differentiation of human monocytes into macrophages. Blocking KCa3.1 significantly reduced the expression of proinflammatory genes during macrophages polarization. Further mechanistic studies indicated that blocking KCa3.1 inhibited macrophage differentiation toward the M1 phenotype by downregulating signal transducer and activator of transcription-1 phosphorylation. In animal models, KCa3.1 blockade therapy strikingly reduced the incidence of plaque rupture and luminal thrombus in carotid arteries, decreased the expression of markers associated with M1 macrophage polarization, and enhanced the expression of M2 markers within atherosclerotic lesions. These results suggest that blocking KCa3.1 suppresses plaque instability in advanced stages of atherosclerosis by inhibiting macrophage polarization toward an M1 phenotype. © 2016 American Heart Association, Inc.

Extensive transcriptional response associated with seasonal plasticity of butterfly wing patterns.

PubMed

Daniels, Emily V; Murad, Rabi; Mortazavi, Ali; Reed, Robert D

2014-12-01

In the eastern United States, the buckeye butterfly, Junonia coenia, shows seasonal wing colour plasticity where adults emerging in the spring are tan, while those emerging in the autumn are dark red. This variation can be artificially induced in laboratory colonies, thus making J. coenia a useful model system to examine the mechanistic basis of plasticity. To better understand the developmental basis of seasonal plasticity, we used RNA-seq to quantify transcription profiles associated with development of alternative seasonal wing morphs. Depending on the developmental stage, between 547 and 1420 transfrags were significantly differentially expressed between morphs. These extensive differences in gene expression stand in contrast to the much smaller numbers of differentially expressed transcripts identified in previous studies of genetic wing pattern variation in other species and suggest that environmentally induced phenotypic shifts arise from very broad systemic processes. Analyses of candidate endocrine and pigmentation transcripts revealed notable genes upregulated in the red morph, including several ecdysone-associated genes, and cinnabar, an ommochrome pigmentation gene implicated in colour pattern variation in other butterflies. We also found multiple melanin-related transcripts strongly upregulated in the red morph, including tan and yellow-family genes, leading us to speculate that dark red pigmentation in autumn J. coenia may involve nonommochrome pigments. While we identified several endocrine and pigmentation genes as obvious candidates for seasonal colour morph differentiation, we speculate that the majority of observed expression differences were due to thermal stress response. The buckeye transcriptome provides a basis for further developmental studies of phenotypic plasticity. © 2014 John Wiley & Sons Ltd.

Extensive alternative splicing and dual promoter usage generate Tcf-1 protein isoforms with differential transcription control properties.

PubMed Central

Van de Wetering, M; Castrop, J; Korinek, V; Clevers, H

1996-01-01

Previously, we reported the isolation of cDNA clones representing four alternative splice forms of TCF-1, a T-cell-specific transcription factor. In the present study, Western blotting (immunoblotting) yielded a multitude of TCF-1 proteins ranging from 25-55 kDa, a pattern not simply explained from the known splice alternatives. Subsequent cDNA cloning, PCR amplification, and analysis by rapid amplification of 5' cDNA ends revealed (i) the presence of an alternative upstream promoter, which extended the known N terminus by 116 amino acids, (ii) the presence of four alternative exons, and (iii) the existence of a second reading frame in the last exon encoding an extended C terminus. Inclusion of the extended N terminus into the originally reported protein resulted in a striking similarity to the lymphoid factor Lef-1. Several of the TCF-1 isoforms, although less potent, mimicked Lef-1 in transactivating transcription through the T-cell receptor alpha-chain (TCR-alpha) enhancer. These data provide a molecular basis for the complexity of the expressed TCF-1 proteins and establish the existence of functional differences between these isoforms. Furthermore, the functional redundancy between Tcf-1 and Lef-1 explains the apparently normal TCR-alpha expression in single Tcf-1 or Lef-1 knockout mice despite the firm in vitro evidence for the importance of the Tcf/Lef site in the TCR-alpha enhancer. PMID:8622675

Drosophila Hey is a target of Notch in asymmetric divisions during embryonic and larval neurogenesis.

PubMed

Monastirioti, Maria; Giagtzoglou, Nikolaos; Koumbanakis, Konstantinos A; Zacharioudaki, Evanthia; Deligiannaki, Myrto; Wech, Irmgard; Almeida, Mara; Preiss, Anette; Bray, Sarah; Delidakis, Christos

2010-01-01

bHLH-O proteins are a subfamily of the basic-helix-loop-helix transcription factors characterized by an 'Orange' protein-protein interaction domain. Typical members are the Hairy/E(spl), or Hes, proteins, well studied in their ability, among others, to suppress neuronal differentiation in both invertebrates and vertebrates. Hes proteins are often effectors of Notch signalling. In vertebrates, another bHLH-O protein group, the Hey proteins, have also been shown to be Notch targets and to interact with Hes. We have studied the single Drosophila Hey orthologue. We show that it is primarily expressed in a subset of newly born neurons, which receive Notch signalling during their birth. Unlike in vertebrates, however, Hey is not expressed in precursor cells and does not block neuronal differentiation. It rather promotes one of two alternative fates that sibling neurons adopt at birth. Although in the majority of cases Hey is a Notch target, it is also expressed independently of Notch in some lineages, most notably the larval mushroom body. The availability of Hey as a Notch readout has allowed us to study Notch signalling during the genesis of secondary neurons in the larval central nervous system.

Gene position in a long operon governs motility development in Bacillus subtilis

PubMed Central

Cozy, Loralyn M.; Kearns, Daniel B.

2010-01-01

Growing cultures of Bacillus subtilis bifurcate into subpopulations of motile individuals and non-motile chains of cells that are differentiated at the level of gene expression. The motile cells are ON and the chaining cells are OFF for transcription that depends on RNA polymerase and the alternative sigma factor σD. Here we show that chaining cells were OFF for σD-dependent gene expression because σD levels fell below a threshold, and σD activity was inhibited by the anti-sigma factor FlgM. The probability that σD exceeded the threshold was governed by the position of the sigD genes. The proportion of ON cells increased when sigD was artificially moved forward in the 27kb fla/che operon. In addition, we identified a new σD-dependent promoter that increases sigD expression and may provide positive feedback to stabilize the ON state. Finally, we demonstrate that ON/OFF motility states in B. subtilis are a form of development because mosaics of stable and differentiated epigenotypes were evident when the normally dispersed bacteria were forced to grow in one dimension. PMID:20233303

Sodium chloride-induced filamentation and alternative gene expression of fts, murZ, and gnd in Listeria monocytogenes 08-5923 on vacuum-packaged ham.

PubMed

Liu, Xiaoji; Miller, Petr; Basu, Urmila; McMullen, Lynn M

2014-11-01

The aim of this study was to examine the filament formation and differential gene expression of Listeria monocytogenes 08-5923 grown on refrigerated vacuum-packaged ham products with various NaCl concentrations. Filament formation of L. monocytogenes was observed on ham products with 1.35% and 2.35% NaCl, which was monitored using flow cytometry by measuring forward light scatter. Quantitative real-time PCR was used to study the differential expression of genes in filamented cells of L. monocytogenes grown on hams following 2 or 3 months of storage at 4 °C. The genes involved in cell division (ftsX/lmo2506), cell wall synthesis (murZ/lmo2552), and NADPH production (gnd/lmo1376) were significantly downregulated in filamented cells of L. monocytogenes grown on ham with 2.35% NaCl stored at 4 °C. To our knowledge, this study reports the first evidence of filament formation of Listeria grown on meat products, which could impact the food safety risk and tolerance levels of L. monocytogenes set by regulatory agencies. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

The parathyroid hormone-regulated transcriptome in osteocytes: parallel actions with 1,25-dihydroxyvitamin D3 to oppose gene expression changes during differentiation and to promote mature cell function.

PubMed

St John, Hillary C; Meyer, Mark B; Benkusky, Nancy A; Carlson, Alex H; Prideaux, Mathew; Bonewald, Lynda F; Pike, J Wesley

2015-03-01

Although localized to the mineralized matrix of bone, osteocytes are able to respond to systemic factors such as the calciotropic hormones 1,25(OH)2D3 and PTH. In the present studies, we examined the transcriptomic response to PTH in an osteocyte cell model and found that this hormone regulated an extensive panel of genes. Surprisingly, PTH uniquely modulated two cohorts of genes, one that was expressed and associated with the osteoblast to osteocyte transition and the other a cohort that was expressed only in the mature osteocyte. Interestingly, PTH's effects were largely to oppose the expression of differentiation-related genes in the former cohort, while potentiating the expression of osteocyte-specific genes in the latter cohort. A comparison of the transcriptional effects of PTH with those obtained previously with 1,25(OH)2D3 revealed a subset of genes that was strongly overlapping. While 1,25(OH)2D3 potentiated the expression of osteocyte-specific genes similar to that seen with PTH, the overlap between the two hormones was more limited. Additional experiments identified the PKA-activated phospho-CREB (pCREB) cistrome, revealing that while many of the differentiation-related PTH regulated genes were apparent targets of a PKA-mediated signaling pathway, a reduction in pCREB binding at sites associated with osteocyte-specific PTH targets appeared to involve alternative PTH activation pathways. That pCREB binding activities positioned near important hormone-regulated gene cohorts were localized to control regions of genes was reinforced by the presence of epigenetic enhancer signatures exemplified by unique modifications at histones H3 and H4. These studies suggest that both PTH and 1,25(OH)2D3 may play important and perhaps cooperative roles in limiting osteocyte differentiation from its precursors while simultaneously exerting distinct roles in regulating mature osteocyte function. Our results provide new insight into transcription factor-associated mechanisms through which PTH and 1,25(OH)2D3 regulate a plethora of genes important to the osteoblast/osteocyte lineage. Copyright © 2014 Elsevier Inc. All rights reserved.

RNA-Seq transcriptome analysis of Amaranthus palmeri with differential tolerance to glufosinate herbicide

PubMed Central

Salas-Perez, Reiofeli A.; Saski, Christopher A.; Noorai, Rooksana E.; Srivastava, Subodh K.; Lawton-Rauh, Amy L.; Nichols, Robert L.

2018-01-01

Amaranthus palmeri (Amaranthaceae) is a noxious weed in several agroecosystems and in some cases seriously threatens the sustainability of crop production in North America. Glyphosate-resistant Amaranthus species are widespread, prompting the use of alternatives to glyphosate such as glufosinate, in conjunction with glufosinate-resistant crop cultivars, to help control glyphosate-resistant weeds. An experiment was conducted to analyze the transcriptome of A. palmeri plants that survived exposure to 0.55 kg ha-1 glufosinate. Since there was no record of glufosinate use at the collection site, survival of plants within the population are likely due to genetic expression that pre-dates selection; in the formal parlance of weed science this is described as natural tolerance. Leaf tissues from glufosinate-treated and non-treated seedlings were harvested 24 h after treatment (HAT) for RNA-Seq analysis. Global gene expression was measured using Illumina DNA sequence reads from non-treated and treated surviving (presumably tolerant, T) and susceptible (S) plants. The same plants were used to determine the mechanisms conferring differential tolerance to glufosinate. The S plants accumulated twice as much ammonia as did the T plants, 24 HAT. The relative copy number of the glufosinate target gene GS2 did not differ between T and S plants, with 1 to 3 GS2 copies in both biotypes. A reference cDNA transcriptome consisting of 72,780 contigs was assembled, with 65,282 sequences putatively annotated. Sequences of GS2 from the transcriptome assembly did not have polymorphisms unique to the tolerant plants. Five hundred sixty-seven genes were differentially expressed between treated T and S plants. Of the upregulated genes in treated T plants, 210 were more highly induced than were the upregulated genes in the treated S plants. Glufosinate-tolerant plants had greater induction of ABC transporter, glutathione S-transferase (GST), NAC transcription factor, nitronate monooxygenase (NMO), chitin elicitor receptor kinase (CERK1), heat shock protein 83, ethylene transcription factor, heat stress transcription factor, NADH-ubiquinone oxidoreductase, ABA 8’-hydroxylase, and cytochrome P450 genes (CYP72A, CYP94A1). Seven candidate genes were selected for validation using quantitative real time-PCR. While GST was upregulated in treated tolerant plants in at least one population, CYP72A219 was consistently highly expressed in all treated tolerant biotypes. These genes are candidates for contributing tolerance to glufosinate. Taken together, these results show that differential induction of stress-protection genes in a population can enable some individuals to survive herbicide application. Elevated expression of detoxification-related genes can get fixed in a population with sustained selection pressure, leading to evolution of resistance. Alternatively, sustained selection pressure could select for mutation(s) in the GS2 gene with the same consequence. PMID:29672568

TDP-43 regulates the microprocessor complex activity during in vitro neuronal differentiation.

PubMed

Di Carlo, Valerio; Grossi, Elena; Laneve, Pietro; Morlando, Mariangela; Dini Modigliani, Stefano; Ballarino, Monica; Bozzoni, Irene; Caffarelli, Elisa

2013-12-01

TDP-43 (TAR DNA-binding protein 43) is an RNA-binding protein implicated in RNA metabolism at several levels. Even if ubiquitously expressed, it is considered as a neuronal activity-responsive factor and a major signature for neurological pathologies, making the comprehension of its activity in the nervous system a very challenging issue. TDP-43 has also been described as an accessory component of the Drosha-DGCR8 (DiGeorge syndrome critical region gene 8) microprocessor complex, which is crucially involved in basal and tissue-specific RNA processing events. In the present study, we exploited in vitro neuronal differentiation systems to investigate the TDP-43 demand for the microprocessor function, focusing on both its canonical microRNA biosynthetic activity and its alternative role as a post-transcriptional regulator of gene expression. Our findings reveal a novel role for TDP-43 as an essential factor that controls the stability of Drosha protein during neuronal differentiation, thus globally affecting the production of microRNAs. We also demonstrate that TDP-43 is required for the Drosha-mediated regulation of Neurogenin 2, a master gene orchestrating neurogenesis, whereas post-transcriptional control of Dgcr8, another Drosha target, resulted to be TDP-43-independent. These results implicate a previously uncovered contribution of TDP-43 in regulating the abundance and the substrate specificity of the microprocessor complex and provide new insights into TDP-43 as a key player in neuronal differentiation.

Cell cycle activation in p21 dependent pathway: An alternative mechanism of organophosphate induced dopaminergic neurodegeneration.

PubMed

Wani, Willayat Yousuf; Kandimalla, Ramesh J L; Sharma, Deep Raj; Kaushal, Alka; Ruban, Anand; Sunkaria, Aditya; Vallamkondu, Jayalakshmi; Chiarugi, Alberto; Reddy, P Hemachandra; Gill, Kiran Dip

2017-07-01

In the previous study, we demonstrated that dichlorvos induces oxidative stress in dopaminergic neuronal cells and subsequent caspase activation mediates apoptosis. In the present study, we evaluated the effect and mechanism of dichlorvos induced oxidative stress on cell cycle activation in NGF-differentiated PC12 cells. Dichlorvos exposure resulted in oxidative DNA damage along with activation of cell cycle machinery in differentiated PC12 cells. Dichlorvos exposed cells exhibited an increased expression of p53, cyclin-D1, pRb and decreased expression of p21suggesting a re-entry of differentiated cells into the cell cycle. Cell cycle analysis of dichlorvos exposed cells revealed a reduction of cells in the G 0 /G 1 phase of the cell cycle (25%), and a concomitant increase of cells in S phase (30%) and G2/M phase (43.3%) compared to control PC12 cells. Further, immunoblotting of cytochrome c, Bax, Bcl-2 and cleaved caspase-3 revealed that dichlorvos induces a caspase-dependent cell death in PC12 cells. These results suggest that Dichlorvos exposure has the potential to generate oxidative stress which evokes activation of cell cycle machinery leading to apoptotic cell death via cytochrome c release from mitochondria and subsequent caspase-3 activation in differentiated PC12 cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Validation and Interrogation of Differentially Expressed and Alternatively Spliced Genes in African American Prostate Cancer

DTIC Science & Technology

2016-10-01

These analyses have led to two submitted manuscripts. The first manuscript, “Variants of stemness -related genes predicted to regulate RNA splicing...and Table 1-3 at the end of this progress report. The second manuscript, “Single nucleotide polymorphisms of stemness pathway genes predicted to...cancer and support a contribution of the stemness pathway to prostate cancer patient outcome. Please see Figure 5-7 and Table 4-6 at the end of this

[Current view and critic of alternatives to transurethral surgery of prostatic benign prostate].

PubMed

Rodríguez, José Vicente

2003-11-01

Critical update of transurethral surgery options based on the last decade most relevant bibliography. Comparative study between Incision/TUR of the prostate and alternative techniques, accordingly to data from 30 randomized studies and 28 clinical studies. We evaluate efficiency, retreatment index, morbidity, post operative number of days with catheter, anesthetic requirements, and cost. Data are expressed as percentages resulting from a differential formula in randomized studies and simple percentages or numeric expression for relevant clinical data. Efficiency: all alternative treatments show a symptomatic improvement (> 50%) similar to that achieved by transurethral surgery; post treatment flowmetry percentage increase is inferior in all alternatives except vaporization, holmium laser and prosthesis. Re-Treatment requirements: they were higher in all alternatives except the ablative ones (vaporization and holmium laser). Morbidity: all of them had an operative estimated blood loss inferior to TUR and similar to prostatic incision; contact laser and vaporization had more irritative symptoms than incision, and VLAP and TUMT more than TUR; TUMT and interstitial laser have a higher rate of post operative infection; all alternatives except the ablative ones had lower percentages of urethral stenosis and retrograde ejaculation. Number of days of catheter post operative: it was comparatively longer after vaporization and very long after VLAP, interstitial laser and TUMT. All treatments except TUNA and TUMT require the same anesthesia than transurethral surgery. The cost/benefit has not been sufficiently evaluated, but it is superior with holmium laser, contact laser and vaporization than with transurethral surgery. Based on study data we can accept holmium laser as a real alternative, TUMT in cases when surgery is questioned and intraprostatic prosthesis when it is not possible.

Alternative Polyadenylation of mRNAs: 3′-Untranslated Region Matters in Gene Expression

PubMed Central

Yeh, Hsin-Sung; Yong, Jeongsik

2016-01-01

Almost all of eukaryotic mRNAs are subjected to polyadenylation during mRNA processing. Recent discoveries showed that many of these mRNAs contain more than one polyadenylation sites in their 3′ untranslated regions (UTR) and that alternative polyadenylation (APA) is prevalent among these genes. Many biological processes such as differentiation, proliferation, and tumorigenesis have been correlated to global APA events in the 3′ UTR of mRNAs, suggesting that these APA events are tightly regulated and may play important physiological roles. In this review, recent discoveries in the physiological roles of APA events, as well as the known and proposed mechanisms are summarized. Perspective for future directions is also discussed. PMID:26912084

Vitamin D Receptor gene (VDR) transcripts in bone, cartilage, muscles and blood and microarray analysis of vitamin D responsive genes expression in paravertebral muscles of Juvenile and Adolescent Idiopathic Scoliosis patients

PubMed Central

2012-01-01

Background VDR may be considered as a candidate gene potentially related to Idiopathic Scoliosis susceptibility and natural history. Transcriptional profile of VDR mRNA isoforms might be changed in the structural tissues of the scoliotic spine and potentially influence the expression of VDR responsive genes. The purpose of the study was to determine differences in mRNA abundance of VDR isoforms in bone, cartilage and paravertebral muscles between tissues from curve concavity and convexity, between JIS and AIS and to identify VDR responsive genes differentiating Juvenile and Adolescent Idiopathic Scoliosis in paravertebral muscles. Methods In a group of 29 patients with JIS and AIS, specimens of bone, cartilage, paravertebral muscles were harvested at the both sides of the curve apex together with peripheral blood samples. Extracted total RNA served as a matrix for VDRs and VDRl mRNA quantification by QRT PCR. Subsequent microarray analysis of paravertebral muscular tissue samples was performed with HG U133A chips (Affymetrix). Quantitative data were compared by a nonparametric Mann Whitney U test. Microarray results were analyzed with GeneSpring 11GX application. Matrix plot of normalized log-intensities visualized the degree of differentiation between muscular tissue transcriptomes of JIS and AIS group. Fold Change Analysis with cutoff of Fold Change ≥2 identified differentially expressed VDR responsive genes in paravertebral muscles of JIS and AIS. Results No significant differences in transcript abundance of VDR isoforms between tissues of the curve concavity and convexity were found. Statistically significant difference between JIS and AIS group in mRNA abundance of VDRl isoform was found in paravertebral muscles of curve concavity. Higher degree of muscular transcriptome differentiation between curve concavity and convexity was visualized in JIS group. In paravertebral muscles Tob2 and MED13 were selected as genes differentially expressed in JIS and AIS group. Conclusions In Idiopathic Scolioses transcriptional activity and alternative splicing of VDR mRNA in osseous, cartilaginous, and paravertebral muscular tissues are tissue specific and equal on both sides of the curve. The number of mRNA copies of VDRl izoform in concave paravertebral muscles might be one of the factors differentiating JIS and AIS. In paravertebral muscles Tob2 and Med13 genes differentiate Adolescent and Juvenile type of Idiopathic Scoliosis. PMID:23259508

DiffSplice: the genome-wide detection of differential splicing events with RNA-seq

PubMed Central

Hu, Yin; Huang, Yan; Du, Ying; Orellana, Christian F.; Singh, Darshan; Johnson, Amy R.; Monroy, Anaïs; Kuan, Pei-Fen; Hammond, Scott M.; Makowski, Liza; Randell, Scott H.; Chiang, Derek Y.; Hayes, D. Neil; Jones, Corbin; Liu, Yufeng; Prins, Jan F.; Liu, Jinze

2013-01-01

The RNA transcriptome varies in response to cellular differentiation as well as environmental factors, and can be characterized by the diversity and abundance of transcript isoforms. Differential transcription analysis, the detection of differences between the transcriptomes of different cells, may improve understanding of cell differentiation and development and enable the identification of biomarkers that classify disease types. The availability of high-throughput short-read RNA sequencing technologies provides in-depth sampling of the transcriptome, making it possible to accurately detect the differences between transcriptomes. In this article, we present a new method for the detection and visualization of differential transcription. Our approach does not depend on transcript or gene annotations. It also circumvents the need for full transcript inference and quantification, which is a challenging problem because of short read lengths, as well as various sampling biases. Instead, our method takes a divide-and-conquer approach to localize the difference between transcriptomes in the form of alternative splicing modules (ASMs), where transcript isoforms diverge. Our approach starts with the identification of ASMs from the splice graph, constructed directly from the exons and introns predicted from RNA-seq read alignments. The abundance of alternative splicing isoforms residing in each ASM is estimated for each sample and is compared across sample groups. A non-parametric statistical test is applied to each ASM to detect significant differential transcription with a controlled false discovery rate. The sensitivity and specificity of the method have been assessed using simulated data sets and compared with other state-of-the-art approaches. Experimental validation using qRT-PCR confirmed a selected set of genes that are differentially expressed in a lung differentiation study and a breast cancer data set, demonstrating the utility of the approach applied on experimental biological data sets. The software of DiffSplice is available at http://www.netlab.uky.edu/p/bioinfo/DiffSplice. PMID:23155066

Cap-proximal nucleotides via differential eIF4E binding and alternative promoter usage mediate translational response to energy stress.

PubMed

Tamarkin-Ben-Harush, Ana; Vasseur, Jean-Jacques; Debart, Françoise; Ulitsky, Igor; Dikstein, Rivka

2017-02-08

Transcription start-site (TSS) selection and alternative promoter (AP) usage contribute to gene expression complexity but little is known about their impact on translation. Here we performed TSS mapping of the translatome following energy stress. Assessing the contribution of cap-proximal TSS nucleotides, we found dramatic effect on translation only upon stress. As eIF4E levels were reduced, we determined its binding to capped-RNAs with different initiating nucleotides and found the lowest affinity to 5'cytidine in correlation with the translational stress-response. In addition, the number of differentially translated APs was elevated following stress. These include novel glucose starvation-induced downstream transcripts for the translation regulators eIF4A and Pabp, which are also translationally-induced despite general translational inhibition. The resultant eIF4A protein is N-terminally truncated and acts as eIF4A inhibitor. The induced Pabp isoform has shorter 5'UTR removing an auto-inhibitory element. Our findings uncovered several levels of coordination of transcription and translation responses to energy stress.

Genome-Wide Posttranscriptional Dysregulation by MicroRNAs in Human Asthma as Revealed by Frac-seq.

PubMed

Martinez-Nunez, Rocio T; Rupani, Hitasha; Platé, Manuela; Niranjan, Mahesan; Chambers, Rachel C; Howarth, Peter H; Sanchez-Elsner, Tilman

2018-05-16

MicroRNAs are small noncoding RNAs that inhibit gene expression posttranscriptionally, implicated in virtually all biological processes. Although the effect of individual microRNAs is generally studied, the genome-wide role of multiple microRNAs is less investigated. We assessed paired genome-wide expression of microRNAs with total (cytoplasmic) and translational (polyribosome-bound) mRNA levels employing subcellular fractionation and RNA sequencing (Frac-seq) in human primary bronchoepithelium from healthy controls and severe asthmatics. Severe asthma is a chronic inflammatory disease of the airways characterized by poor response to therapy. We found genes (i.e., isoforms of a gene) and mRNA isoforms differentially expressed in asthma, with novel inflammatory and structural pathophysiological mechanisms related to bronchoepithelium disclosed solely by polyribosome-bound mRNAs (e.g., IL1A and LTB genes or ITGA6 and ITGA2 alternatively spliced isoforms). Gene expression (i.e., isoforms of a gene) and mRNA expression analysis revealed different molecular candidates and biological pathways, with differentially expressed polyribosome-bound and total mRNAs also showing little overlap. We reveal a hub of six dysregulated microRNAs accounting for ∼90% of all microRNA targeting, displaying preference for polyribosome-bound mRNAs. Transfection of this hub in bronchial epithelial cells from healthy donors mimicked asthma characteristics. Our work demonstrates extensive posttranscriptional gene dysregulation in human asthma, in which microRNAs play a central role, illustrating the feasibility and importance of assessing posttranscriptional gene expression when investigating human disease. Copyright © 2018 by The American Association of Immunologists, Inc.

Deciphering the Developmental Dynamics of the Mouse Liver Transcriptome

PubMed Central

Gunewardena, Sumedha S.; Yoo, Byunggil; Peng, Lai; Lu, Hong; Zhong, Xiaobo; Klaassen, Curtis D.; Cui, Julia Yue

2015-01-01

During development, liver undergoes a rapid transition from a hematopoietic organ to a major organ for drug metabolism and nutrient homeostasis. However, little is known on a transcriptome level of the genes and RNA-splicing variants that are differentially regulated with age, and which up-stream regulators orchestrate age-specific biological functions in liver. We used RNA-Seq to interrogate the developmental dynamics of the liver transcriptome in mice at 12 ages from late embryonic stage (2-days before birth) to maturity (60-days after birth). Among 21,889 unique NCBI RefSeq-annotated genes, 9,641 were significantly expressed in at least one age, 7,289 were differently regulated with age, and 859 had multiple (> = 2) RNA splicing-variants. Factor analysis showed that the dynamics of hepatic genes fall into six distinct groups based on their temporal expression. The average expression of cytokines, ion channels, kinases, phosphatases, transcription regulators and translation regulators decreased with age, whereas the average expression of peptidases, enzymes and transmembrane receptors increased with age. The average expression of growth factors peak between Day-3 and Day-10, and decrease thereafter. We identified critical biological functions, upstream regulators, and putative transcription modules that seem to govern age-specific gene expression. We also observed differential ontogenic expression of known splicing variants of certain genes, and 1,455 novel splicing isoform candidates. In conclusion, the hepatic ontogeny of the transcriptome ontogeny has unveiled critical networks and up-stream regulators that orchestrate age-specific biological functions in liver, and suggest that age contributes to the complexity of the alternative splicing landscape of the hepatic transcriptome. PMID:26496202

Deciphering the Developmental Dynamics of the Mouse Liver Transcriptome.

PubMed

Gunewardena, Sumedha S; Yoo, Byunggil; Peng, Lai; Lu, Hong; Zhong, Xiaobo; Klaassen, Curtis D; Cui, Julia Yue

2015-01-01

During development, liver undergoes a rapid transition from a hematopoietic organ to a major organ for drug metabolism and nutrient homeostasis. However, little is known on a transcriptome level of the genes and RNA-splicing variants that are differentially regulated with age, and which up-stream regulators orchestrate age-specific biological functions in liver. We used RNA-Seq to interrogate the developmental dynamics of the liver transcriptome in mice at 12 ages from late embryonic stage (2-days before birth) to maturity (60-days after birth). Among 21,889 unique NCBI RefSeq-annotated genes, 9,641 were significantly expressed in at least one age, 7,289 were differently regulated with age, and 859 had multiple (> = 2) RNA splicing-variants. Factor analysis showed that the dynamics of hepatic genes fall into six distinct groups based on their temporal expression. The average expression of cytokines, ion channels, kinases, phosphatases, transcription regulators and translation regulators decreased with age, whereas the average expression of peptidases, enzymes and transmembrane receptors increased with age. The average expression of growth factors peak between Day-3 and Day-10, and decrease thereafter. We identified critical biological functions, upstream regulators, and putative transcription modules that seem to govern age-specific gene expression. We also observed differential ontogenic expression of known splicing variants of certain genes, and 1,455 novel splicing isoform candidates. In conclusion, the hepatic ontogeny of the transcriptome ontogeny has unveiled critical networks and up-stream regulators that orchestrate age-specific biological functions in liver, and suggest that age contributes to the complexity of the alternative splicing landscape of the hepatic transcriptome.

Translation inhibition of the developmental cycle protein HctA by the small RNA IhtA is conserved across Chlamydia.

PubMed

Tattersall, Jeremiah; Rao, Geeta Vittal; Runac, Justin; Hackstadt, Ted; Grieshaber, Scott S; Grieshaber, Nicole A

2012-01-01

The developmental cycle of the obligate intracellular pathogen Chlamydia trachomatis serovar L2 is controlled in part by the small non-coding RNA (sRNA), IhtA. All Chlamydia alternate in a regulated fashion between the infectious elementary body (EB) and the replicative reticulate body (RB) which asynchronously re-differentiates back to the terminal EB form at the end of the cycle. The histone like protein HctA is central to RB:EB differentiation late in the cycle as it binds to and occludes the genome, thereby repressing transcription and translation. The sRNA IhtA is a critical component of this regulatory loop as it represses translation of hctA until late in infection at which point IhtA transcription decreases, allowing HctA expression to occur and RB to EB differentiation to proceed. It has been reported that IhtA is expressed during infection by the human pathogens C. trachomatis serovars L2, D and L2b and C. pneumoniae. We show in this work that IhtA is also expressed by the animal pathogens C. caviae and C. muridarum. Expression of HctA in E. coli is lethal and co-expression of IhtA relieves this phenotype. To determine if regulation of HctA by IhtA is a conserved mechanism across pathogenic chlamydial species, we cloned hctA and ihtA from C. trachomatis serovar D, C. muridarum, C. caviae and C. pneumoniae and assayed for rescue of growth repression in E. coli co-expression studies. In each case, co-expression of ihtA with the cognate hctA resulted in relief of growth repression. In addition, expression of each chlamydial species IhtA rescued the lethal phenotype of C. trachomatis serovar L2 HctA expression. As biolayer interferometry studies indicate that IhtA interacts directly with hctA message for all species tested, we predict that conserved sequences of IhtA are necessary for function and/or binding.

A short treatise concerning a musical approach for the interpretation of gene expression data

PubMed Central

Staege, Martin S.

2015-01-01

Recent technical developments allow the genome-wide and near-complete analysis of gene expression in a given sample, e.g. by usage of high-density DNA microarrays or next generation sequencing. The generated data structure is usually multi-dimensional and requires extensive processing not only for analysis but also for presentation of the results. Today, such data are usually presented graphically, e.g. in the form of heat maps. In the present paper, we propose an alternative form of analysis and presentation which is based on the transformation of gene expression data into sounds that are characterized by their frequency (pitch) and tone duration. Using DNA microarray data from a panel of neuroblastoma and Ewing sarcoma cell lines as well as from Hodgkin’s lymphoma cell lines and normal B cells, we demonstrate that this Gene Expression Music Algorithm (GEMusicA) can be used for discrimination between samples with different biology and for the characterization of differentially expressed genes. PMID:26472273

CD33: increased inclusion of exon 2 implicates the Ig V-set domain in Alzheimer's disease susceptibility

PubMed Central

Raj, Towfique; Ryan, Katie J.; Replogle, Joseph M.; Chibnik, Lori B.; Rosenkrantz, Laura; Tang, Anna; Rothamel, Katie; Stranger, Barbara E.; Bennett, David A.; Evans, Denis A.; De Jager, Philip L.; Bradshaw, Elizabeth M.

2014-01-01

We previously demonstrated that the Alzheimer's disease (AD) associated risk allele, rs3865444C, results in a higher surface density of CD33 on monocytes. Here, we find alternative splicing of exon 2 to be the primary mechanism of the genetically driven differential expression of CD33 protein. We report that the risk allele, rs3865444C, is associated with greater cell surface expression of CD33 in both subjects of European and African–American ancestry and that there is a single haplotype influencing CD33 surface expression. A meta-analysis of the two populations narrowed the number of significant SNPs in high linkage disequilibrium (LD) (r2 > 0.8) with rs3865444 to just five putative causal variants associated with increased protein expression. Using gene expression data from flow-sorted CD14+CD16− monocytes from 398 healthy subjects of three populations, we show that the rs3865444C risk allele is strongly associated with greater expression of CD33 exon 2 (pMETA = 2.36 × 10−60). Western blotting confirms increased protein expression of the full-length CD33 isoform containing exon 2 relative to the rs3865444C allele (P < 0.0001). Of the variants in strong LD with rs3865444, rs12459419, which is located in a putative SRSF2 splice site of exon 2, is the most likely candidate to mediate the altered alternative splicing of CD33's Immunoglobulin V-set domain 2 and ultimately influence AD susceptibility. PMID:24381305

Alternative Polyadenylation Allows Differential Negative Feedback of Human miRNA miR-579 on Its Host Gene ZFR

PubMed Central

Hinske, Ludwig Christian; Galante, Pedro A. F.; Limbeck, Elisabeth; Möhnle, Patrick; Parmigiani, Raphael B.; Ohno-Machado, Lucila; Camargo, Anamaria A.; Kreth, Simone

2015-01-01

About half of the known miRNA genes are located within protein-coding host genes, and are thus subject to co-transcription. Accumulating data indicate that this coupling may be an intrinsic mechanism to directly regulate the host gene’s expression, constituting a negative feedback loop. Inevitably, the cell requires a yet largely unknown repertoire of methods to regulate this control mechanism. We propose APA as one possible mechanism by which negative feedback of intronic miRNA on their host genes might be regulated. Using in-silico analyses, we found that host genes that contain seed matching sites for their intronic miRNAs yield longer 32UTRs with more polyadenylation sites. Additionally, the distribution of polyadenylation signals differed significantly between these host genes and host genes of miRNAs that do not contain potential miRNA binding sites. We then transferred these in-silico results to a biological example and investigated the relationship between ZFR and its intronic miRNA miR-579 in a U87 cell line model. We found that ZFR is targeted by its intronic miRNA miR-579 and that alternative polyadenylation allows differential targeting. We additionally used bioinformatics analyses and RNA-Seq to evaluate a potential cross-talk between intronic miRNAs and alternative polyadenylation. CPSF2, a gene previously associated with alternative polyadenylation signal recognition, might be linked to intronic miRNA negative feedback by altering polyadenylation signal utilization. PMID:25799583

Simultaneous Quantification of Multiple Alternatively Spliced mRNA Transcripts Using Droplet Digital PCR.

PubMed

Sun, Bing; Zheng, Yun-Ling

2018-01-01

Currently there is no sensitive, precise, and reproducible method to quantitate alternative splicing of mRNA transcripts. Droplet digital™ PCR (ddPCR™) analysis allows for accurate digital counting for quantification of gene expression. Human telomerase reverse transcriptase (hTERT) is one of the essential components required for telomerase activity and for the maintenance of telomeres. Several alternatively spliced forms of hTERT mRNA in human primary and tumor cells have been reported in the literature. Using one pair of primers and two probes for hTERT, four alternatively spliced forms of hTERT (α-/β+, α+/β- single deletions, α-/β- double deletion, and nondeletion α+/β+) were accurately quantified through a novel analysis method via data collected from a single ddPCR reaction. In this chapter, we describe this ddPCR method that enables direct quantitative comparison of four alternatively spliced forms of the hTERT messenger RNA without the need for internal standards or multiple pairs of primers specific for each variant, eliminating the technical variation due to differential PCR amplification efficiency for different amplicons and the challenges of quantification using standard curves. This simple and straightforward method should have general utility for quantifying alternatively spliced gene transcripts.

The Transcription Factor BcLTF1 Regulates Virulence and Light Responses in the Necrotrophic Plant Pathogen Botrytis cinerea

PubMed Central

Schumacher, Julia; Simon, Adeline; Cohrs, Kim Christopher; Viaud, Muriel; Tudzynski, Paul

2014-01-01

Botrytis cinerea is the causal agent of gray mold diseases in a range of dicotyledonous plant species. The fungus can reproduce asexually by forming macroconidia for dispersal and sclerotia for survival; the latter also participate in sexual reproduction by bearing the apothecia after fertilization by microconidia. Light induces the differentiation of conidia and apothecia, while sclerotia are exclusively formed in the absence of light. The relevance of light for virulence of the fungus is not obvious, but infections are observed under natural illumination as well as in constant darkness. By a random mutagenesis approach, we identified a novel virulence-related gene encoding a GATA transcription factor (BcLTF1 for light-responsive TF1) with characterized homologues in Aspergillus nidulans (NsdD) and Neurospora crassa (SUB-1). By deletion and over-expression of bcltf1, we confirmed the predicted role of the transcription factor in virulence, and discovered furthermore its functions in regulation of light-dependent differentiation, the equilibrium between production and scavenging of reactive oxygen species (ROS), and secondary metabolism. Microarray analyses revealed 293 light-responsive genes, and that the expression levels of the majority of these genes (66%) are modulated by BcLTF1. In addition, the deletion of bcltf1 affects the expression of 1,539 genes irrespective of the light conditions, including the overexpression of known and so far uncharacterized secondary metabolism-related genes. Increased expression of genes encoding alternative respiration enzymes, such as the alternative oxidase (AOX), suggest a mitochondrial dysfunction in the absence of bcltf1. The hypersensitivity of Δbctlf1 mutants to exogenously applied oxidative stress - even in the absence of light - and the restoration of virulence and growth rates in continuous light by antioxidants, indicate that BcLTF1 is required to cope with oxidative stress that is caused either by exposure to light or arising during host infection. PMID:24415947

Transcriptome Profiling of Pediatric Core Binding Factor AML

PubMed Central

Hsu, Chih-Hao; Nguyen, Cu; Yan, Chunhua; Ries, Rhonda E.; Chen, Qing-Rong; Hu, Ying; Ostronoff, Fabiana; Stirewalt, Derek L.; Komatsoulis, George; Levy, Shawn

2015-01-01

The t(8;21) and Inv(16) translocations disrupt the normal function of core binding factors alpha (CBFA) and beta (CBFB), respectively. These translocations represent two of the most common genomic abnormalities in acute myeloid leukemia (AML) patients, occurring in approximately 25% pediatric and 15% of adult with this malignancy. Both translocations are associated with favorable clinical outcomes after intensive chemotherapy, and given the perceived mechanistic similarities, patients with these translocations are frequently referred to as having CBF-AML. It remains uncertain as to whether, collectively, these translocations are mechanistically the same or impact different pathways in subtle ways that have both biological and clinical significance. Therefore, we used transcriptome sequencing (RNA-seq) to investigate the similarities and differences in genes and pathways between these subtypes of pediatric AMLs. Diagnostic RNA from patients with t(8;21) (N = 17), Inv(16) (N = 14), and normal karyotype (NK, N = 33) were subjected to RNA-seq. Analyses compared the transcriptomes across these three cytogenetic subtypes, using the NK cohort as the control. A total of 1291 genes in t(8;21) and 474 genes in Inv(16) were differentially expressed relative to the NK controls, with 198 genes differentially expressed in both subtypes. The majority of these genes (175/198; binomial test p-value < 10−30) are consistent in expression changes among the two subtypes suggesting the expression profiles are more similar between the CBF cohorts than in the NK cohort. Our analysis also revealed alternative splicing events (ASEs) differentially expressed across subtypes, with 337 t(8;21)-specific and 407 Inv(16)-specific ASEs detected, the majority of which were acetylated proteins (p = 1.5x10-51 and p = 1.8x10-54 for the two subsets). In addition to known fusions, we identified and verified 16 de novo fusions in 43 patients, including three fusions involving NUP98 in six patients. Clustering of differentially expressed genes indicated that the homeobox (HOX) gene family, including two transcription factors (MEIS1 and NKX2-3) were down-regulated in CBF compared to NK samples. This finding supports existing data that the dysregulation of HOX genes play a central role in biology CBF-AML hematopoiesis. These data provide comprehensive transcriptome profiling of CBF-AML and delineate genes and pathways that are differentially expressed, providing insights into the shared biology as well as differences in the two CBF subsets. PMID:26397705

Integration of Genome-Wide Computation DRE Search, AhR ChIP-chip and Gene Expression Analyses of TCDD-Elicited Responses in the Mouse Liver

PubMed Central

2011-01-01

Background The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor (TF) that mediates responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Integration of TCDD-induced genome-wide AhR enrichment, differential gene expression and computational dioxin response element (DRE) analyses further elucidate the hepatic AhR regulatory network. Results Global ChIP-chip and gene expression analyses were performed on hepatic tissue from immature ovariectomized mice orally gavaged with 30 μg/kg TCDD. ChIP-chip analysis identified 14,446 and 974 AhR enriched regions (1% false discovery rate) at 2 and 24 hrs, respectively. Enrichment density was greatest in the proximal promoter, and more specifically, within ± 1.5 kb of a transcriptional start site (TSS). AhR enrichment also occurred distal to a TSS (e.g. intergenic DNA and 3' UTR), extending the potential gene expression regulatory roles of the AhR. Although TF binding site analyses identified over-represented DRE sequences within enriched regions, approximately 50% of all AhR enriched regions lacked a DRE core (5'-GCGTG-3'). Microarray analysis identified 1,896 number of TCDD-responsive genes (|fold change| ≥ 1.5, P1(t) > 0.999). Integrating this gene expression data with our ChIP-chip and DRE analyses only identified 625 differentially expressed genes that involved an AhR interaction at a DRE. Functional annotation analysis of differentially regulated genes associated with AhR enrichment identified overrepresented processes related to fatty acid and lipid metabolism and transport, and xenobiotic metabolism, which are consistent with TCDD-elicited steatosis in the mouse liver. Conclusions Details of the AhR regulatory network have been expanded to include AhR-DNA interactions within intragenic and intergenic genomic regions. Moreover, the AhR can interact with DNA independent of a DRE core suggesting there are alternative mechanisms of AhR-mediated gene regulation. PMID:21762485

Screening of biomarkers for prediction of response to and prognosis after chemotherapy for breast cancers

PubMed Central

Bing, Feng; Zhao, Yu

2016-01-01

Objective To screen the biomarkers having the ability to predict prognosis after chemotherapy for breast cancers. Methods Three microarray data of breast cancer patients undergoing chemotherapy were collected from Gene Expression Omnibus database. After preprocessing, data in GSE41112 were analyzed using significance analysis of microarrays to screen the differentially expressed genes (DEGs). The DEGs were further analyzed by Differentially Coexpressed Genes and Links to construct a function module, the prognosis efficacy of which was verified by the other two datasets (GSE22226 and GSE58644) using Kaplan–Meier plots. The involved genes in function module were subjected to a univariate Cox regression analysis to confirm whether the expression of each prognostic gene was associated with survival. Results A total of 511 DEGs between breast cancer patients who received chemotherapy or not were obtained, consisting of 421 upregulated and 90 downregulated genes. Using the Differentially Coexpressed Genes and Links package, 1,244 differentially coexpressed genes (DCGs) were identified, among which 36 DCGs were regulated by the transcription factor complex NFY (NFYA, NFYB, NFYC). These 39 genes constructed a gene module to classify the samples in GSE22226 and GSE58644 into three subtypes and these subtypes exhibited significantly different survival rates. Furthermore, several genes of the 39 DCGs were shown to be significantly associated with good (such as CDC20) and poor (such as ARID4A) prognoses following chemotherapy. Conclusion Our present study provided a serial of biomarkers for predicting the prognosis of chemotherapy or targets for development of alternative treatment (ie, CDC20 and ARID4A) in breast cancer patients. PMID:27217777

Distinctive features and differential regulation of the DRTS genes of Arabidopsis thaliana

PubMed Central

Maniga, Antonio; Ghisaura, Stefania; Perrotta, Lara; Marche, Maria Giovanna; Cella, Rino

2017-01-01

In plants and protists, dihydrofolate reductase (DHFR) and thymidylate synthase (TS) are part of a bifunctional enzyme (DRTS) that allows efficient recycling of the dihydrofolate resulting from TS activity. Arabidopsis thaliana possesses three DRTS genes, called AtDRTS1, AtDRTS2 and AtDRTS3, that are located downstream of three members of the sec14-like SFH gene family. In this study, a characterization of the AtDRTS genes identified alternatively spliced transcripts coding for AtDRTS isoforms which may account for monofunctional DHFR enzymes supporting pathways unrelated to DNA synthesis. Moreover, we discovered a complex differential regulation of the AtDRTS genes that confirms the expected involvement of the AtDRTS genes in cell proliferation and endoreduplication, but indicates also functions related to other cellular activities. AtDRTS1 is widely expressed in both meristematic and differentiated tissues, whereas AtDRTS2 expression is almost exclusively limited to the apical meristems and AtDRTS3 is preferentially expressed in the shoot apex, in stipules and in root cap cells. The differential regulation of the AtDRTS genes is associated to distinctive promoter architectures and the expression of AtDRTS1 in the apical meristems is strictly dependent on the presence of an intragenic region that includes the second intron of the gene. Upon activation of cell proliferation in germinating seeds, the activity of the AtDRTS1 and AtDRTS2 promoters in meristematic cells appears to be maximal at the G1/S phase of the cell cycle. In addition, the promoters of AtDRTS2 and AtDRTS3 are negatively regulated through E2F cis-acting elements and both genes, but not AtDRTS1, are downregulated in plants overexpressing the AtE2Fa factor. Our study provides new information concerning the function and the regulation of plant DRTS genes and opens the way to further investigations addressing the importance of folate synthesis with respect to specific cellular activities. PMID:28594957

Isolation and clonal characterization of hematopoietic and liver stem cells.

PubMed

Nakauchi, Hiromitsu

2004-11-01

Prospective isolation of stem cells is essential to understanding the mechanisms that control their proliferation and differentiation. Using 9 monoclonal antibodies and fluorescence-activated cell sorting (FACS), we have succeeded in prospectively identifying hematopoietic stem cells (HSCs) in adult mouse bone marrow. Mouse HSCs were exclusively enriched in CD34 negative, c-Kit Sca-1 Lineage Marker (CD34 KSL) cells representing 0.004% of bone marrow (BM) mononuclear cells. When single CD34-KSL cells were transplanted individually into a lethally irradiated mouse, 25% of the recipient mice survived and showed long-term reconstitution of the BM, providing evidence for multipotency and a self-renewal capacity of HSCs. Using a similar approach, we also prospectively identified hepatic stem cells with multilineage differentiation potential and self-renewal capability in the c-Met CD49f c-Kit CD45 Ter119 fraction of cells isolated from day 13.5 fetal mouse liver. On cell transplantation, these cells differentiated into hepatocytes and cholangiocytes. As an alternative to the antibody based stem cell isolation, Hoechst33342 staining is useful. To understand the mechanism responsible for SP phenotype, we performed an expression cloning and identified bcrp-1/ABCG2 gene, a member of ATP binding-cassette (ABC) transporter family. Bcrp-1 is almost exclusively expressed in CD34 KSL cells among blood cells; however their expression in other tissue specific stem cells remains to be studied. With the use of FACS and monoclonal antibodies, hematopoietic and liver stem cells were prospectively isolated and characterized. HSCs could also be purified by Hoechst 33342 staining. By expression cloning, we identify bcrp-1/ABCG2 transporter as a molecule responsible for SP phenotype. Elucidation of the physiological role of bcrp-1/ABCG2 in HSCs may provide us with clues to understand the molecular mechanisms of stem cell self-renewal and differentiation.

Immunogenicity and immunomodulatory properties of hepatocyte-like cells derived from human amniotic epithelial cells.

PubMed

Tee, Jing Yang; Vaghjiani, Vijesh; Liu, Yu Han; Murthi, Padma; Chan, James; Manuelpillai, Ursula

2013-01-01

Hepatocyte transplantation is being trialled as an alternative to whole organ transplant for patients with acute liver failure and liver specific metabolic diseases. Due to the scarcity of human hepatocytes, hepatocyte-like cells (HLC) generated from stem cells may become a viable alternative to hepatocyte transplantation. Human amniotic epithelial cells (hAEC) from the placenta have stem cell-like properties and can be differentiated into HLC. Naïve hAEC have low immunogenicity and exert immunomodulatory effects that may facilitate allogeneic transplantation. However, whether the immunogenicity and immunomodulatory properties alter with differentiation into HLC are unknown. We further characterized HLC generated from hAEC, examined changes in human leucocyte antigens (HLA) and co-stimulatory molecules and effects exerted by the HLC on human peripheral blood mononuclear cells (PBMC). HLC derived from hAEC expressed proteins found in hepatocytes, had CYP3A4 drug metabolizing enzyme activity and secreted urea. IFN-γ treatment increased HLA Class IA, Class II and co-stimulatory molecule CD40 expression in the HLC. IFN-γ treated HLC stimulated proliferation of PBMC in one-way mixed lymphocyte reactions and were more immunogenic than undifferentiated hAEC. However, the HLC showed immunomodulatory properties and inhibited mitogen induced PBMC proliferation in vitro. PBMC proliferation may have been inhibited by IL-6, TGF-β1, PGE2 and HLA-G secreted by the HLC. The retention of immunomodulatory properties may enable HLC grafts to survive for longer periods despite the immunogenicity of the HLC.

Stemness and angiogenic gene expression changes of serial-passage human amnion mesenchymal cells.

PubMed

Fatimah, Simat Siti; Tan, Geok Chin; Chua, Kienhui; Fariha, Mohd Manzor Nur; Tan, Ay Eeng; Hayati, Abdul Rahman

2013-03-01

Particular attention has been directed towards human amnion mesenchymal stem cells (HAMCs) due to their accessibility, availability and immunomodulatory properties. Therefore, the aim of the present study was to determine the temporal changes of stemness and angiogenic gene expressions of serial-passage HAMCs. HAMCs were isolated from human term placenta and cultured in serial passages in culture medium supplemented with 10% fetal bovine serum. Morphological analysis, growth kinetic and CFU-F assay of HAMCs were assessed. In vitro differentiation and the immunophenotype of HAMCs at P5 were also analyzed. Quantitative PCR was used to determine the stemness, angiogenic and endothelial gene expression of cultured HAMCs after serial passage. Cultured HAMCs displayed intermediate epitheloid-fibroblastoid morphology at an initial culture and the fibroblastoid features became more pronounced in later passages. They showed high clonogenic activity and faster proliferation at later passages with colony forming efficiency of 0.88%. HAMCs were successfully differentiated into adipocytes, osteocytes and neuron-like cells. Most HAMCs expressed CD9, CD44, CD73, CD90 and HLA-A,B,C but negligibly expressed CD31, CD34, CD45, CD117 and HLA-DR,DP,DQ. After serial passage, stemness genes Oct-3/4, Sox-2, Nanog3, Rex-1, FGF-4 and FZD-9 expressions significantly decreased. Of the angiogenic genes PECAM-1, bFGF, eNOS, VEGFR-2, VEGF, and vWF expressions also decreased significantly except angiopoietin-1 which significantly increased. No significant differences were observed in ABCG-2, BST-1, nestin, PGF and HGF expressions after serial passage. These results suggested that cultured HAMCs could be an alternative source of stem cells and may have the potential for angiogenesis and hence its use in stem-cell based therapy. Copyright © 2012 Elsevier Inc. All rights reserved.

Investigating the Role of Surface Materials and Three Dimensional Architecture on In Vitro Differentiation of Porcine Monocyte-Derived Dendritic Cells

PubMed Central

Hartmann, Sofie Bruun; Mohanty, Soumyaranjan; Skovgaard, Kerstin; Brogaard, Louise; Flagstad, Frederikke Bjergvang; Emnéus, Jenny; Wolff, Anders; Summerfield, Artur; Jungersen, Gregers

2016-01-01

In vitro generation of dendritic-like cells through differentiation of peripheral blood monocytes is typically done using two-dimensional polystyrene culture plates. In the process of optimising cell culture techniques, engineers have developed fluidic micro-devises usually manufactured in materials other than polystyrene and applying three-dimensional structures more similar to the in vivo environment. Polydimethylsiloxane (PDMS) is an often used polymer for lab-on-a-chip devices but not much is known about the effect of changing the culture surface material from polystyrene to PDMS. In the present study the differentiation of porcine monocytes to monocyte-derived dendritic cells (moDCs) was investigated using CD172apos pig blood monocytes stimulated with GM-CSF and IL-4. Monocytes were cultured on surfaces made of two- and three-dimensional polystyrene as well as two- and three-dimensional PDMS and carbonised three-dimensional PDMS. Cells cultured conventionally (on two-dimensional polystyrene) differentiated into moDCs as expected. Interestingly, gene expression of a wide range of cytokines, chemokines, and pattern recognition receptors was influenced by culture surface material and architecture. Distinct clustering of cells, based on similar expression patterns of 46 genes of interest, was seen for cells isolated from two- and three-dimensional polystyrene as well as two- and three-dimensional PDMS. Changing the material from polystyrene to PDMS resulted in cells with expression patterns usually associated with macrophage expression (upregulation of CD163 and downregulation of CD1a, FLT3, LAMP3 and BATF3). However, this was purely based on gene expression level, and no functional assays were included in this study which would be necessary in order to classify the cells as being macrophages. When changing to three-dimensional culture the cells became increasingly activated in terms of IL6, IL8, IL10 and CCR5 gene expression. Further stimulation with LPS resulted in a slight increase in the expression of maturation markers (SLA-DRB1, CD86 and CD40) as well as cytokines (IL6, IL8, IL10 and IL23A) but the influence of the surfaces was unchanged. These findings highlights future challenges of combining and comparing data generated from microfluidic cell culture-devices made using alternative materials to data generated using conventional polystyrene plates used by most laboratories today. PMID:27362493

Investigating the Role of Surface Materials and Three Dimensional Architecture on In Vitro Differentiation of Porcine Monocyte-Derived Dendritic Cells.

PubMed

Hartmann, Sofie Bruun; Mohanty, Soumyaranjan; Skovgaard, Kerstin; Brogaard, Louise; Flagstad, Frederikke Bjergvang; Emnéus, Jenny; Wolff, Anders; Summerfield, Artur; Jungersen, Gregers

2016-01-01

In vitro generation of dendritic-like cells through differentiation of peripheral blood monocytes is typically done using two-dimensional polystyrene culture plates. In the process of optimising cell culture techniques, engineers have developed fluidic micro-devises usually manufactured in materials other than polystyrene and applying three-dimensional structures more similar to the in vivo environment. Polydimethylsiloxane (PDMS) is an often used polymer for lab-on-a-chip devices but not much is known about the effect of changing the culture surface material from polystyrene to PDMS. In the present study the differentiation of porcine monocytes to monocyte-derived dendritic cells (moDCs) was investigated using CD172apos pig blood monocytes stimulated with GM-CSF and IL-4. Monocytes were cultured on surfaces made of two- and three-dimensional polystyrene as well as two- and three-dimensional PDMS and carbonised three-dimensional PDMS. Cells cultured conventionally (on two-dimensional polystyrene) differentiated into moDCs as expected. Interestingly, gene expression of a wide range of cytokines, chemokines, and pattern recognition receptors was influenced by culture surface material and architecture. Distinct clustering of cells, based on similar expression patterns of 46 genes of interest, was seen for cells isolated from two- and three-dimensional polystyrene as well as two- and three-dimensional PDMS. Changing the material from polystyrene to PDMS resulted in cells with expression patterns usually associated with macrophage expression (upregulation of CD163 and downregulation of CD1a, FLT3, LAMP3 and BATF3). However, this was purely based on gene expression level, and no functional assays were included in this study which would be necessary in order to classify the cells as being macrophages. When changing to three-dimensional culture the cells became increasingly activated in terms of IL6, IL8, IL10 and CCR5 gene expression. Further stimulation with LPS resulted in a slight increase in the expression of maturation markers (SLA-DRB1, CD86 and CD40) as well as cytokines (IL6, IL8, IL10 and IL23A) but the influence of the surfaces was unchanged. These findings highlights future challenges of combining and comparing data generated from microfluidic cell culture-devices made using alternative materials to data generated using conventional polystyrene plates used by most laboratories today.

Modulation of Differentiation Processes in Murine Embryonic Stem Cells Exposed to Parabolic Flight-Induced Acute Hypergravity and Microgravity.

PubMed

Acharya, Aviseka; Brungs, Sonja; Henry, Margit; Rotshteyn, Tamara; Singh Yaduvanshi, Nirmala; Wegener, Lucia; Jentzsch, Simon; Hescheler, Jürgen; Hemmersbach, Ruth; Boeuf, Helene; Sachinidis, Agapios

2018-06-15

Embryonic developmental studies under microgravity conditions in space are very limited. To study the effects of short-term altered gravity on embryonic development processes, we exposed mouse embryonic stem cells (mESCs) to phases of hypergravity and microgravity and studied the differentiation potential of the cells using wide-genome microarray analysis. During the 64th European Space Agency's parabolic flight campaign, mESCs were exposed to 31 parabolas. Each parabola comprised phases lasting 22 s of hypergravity, microgravity, and a repeat of hypergravity. On different parabolas, RNA was isolated for microarray analysis. After exposure to 31 parabolas, mESCs (P31 mESCs) were further differentiated under normal gravity (1 g) conditions for 12 days, producing P31 12-day embryoid bodies (EBs). After analysis of the microarrays, the differentially expressed genes were analyzed using different bioinformatic tools to identify developmental and nondevelopmental biological processes affected by conditions on the parabolic flight experiment. Our results demonstrated that several genes belonging to GOs associated with cell cycle and proliferation were downregulated in undifferentiated mESCs exposed to gravity changes. However, several genes belonging to developmental processes, such as vasculature development, kidney development, skin development, and to the TGF-β signaling pathway, were upregulated. Interestingly, similar enriched and suppressed GOs were obtained in P31 12-day EBs compared with ground control 12-day EBs. Our results show that undifferentiated mESCs exposed to alternate hypergravity and microgravity phases expressed several genes associated with developmental/differentiation and cell cycle processes, suggesting a transition from the undifferentiated pluripotent to a more differentiated stage of mESCs.

Decoding the Regulatory Landscape of Ageing in Musculoskeletal Engineered Tissues Using Genome-Wide DNA Methylation and RNASeq

PubMed Central

Peffers, Mandy Jayne; Goljanek-Whysall, Katarzyna; Collins, John; Fang, Yongxiang; Rushton, Michael; Loughlin, John; Proctor, Carole; Clegg, Peter David

2016-01-01

Mesenchymal stem cells (MSC) are capable of multipotent differentiation into connective tissues and as such are an attractive source for autologous cell-based regenerative medicine and tissue engineering. Epigenetic mechanisms, like DNA methylation, contribute to the changes in gene expression in ageing. However there was a lack of sufficient knowledge of the role that differential methylation plays during chondrogenic, osteogenic and tenogenic differentiation from ageing MSCs. This study undertook genome level determination of the effects of DNA methylation on expression in engineered tissues from chronologically aged MSCs. We compiled unique DNA methylation signatures from chondrogenic, osteogenic, and tenogenic engineered tissues derived from young; n = 4 (21.8 years ± 2.4 SD) and old; n = 4 (65.5 years±8.3SD) human MSCs donors using the Illumina HumanMethylation 450 Beadchip arrays and compared these to gene expression by RNA sequencing. Unique and common signatures of global DNA methylation were identified. There were 201, 67 and 32 chondrogenic, osteogenic and tenogenic age-related DE protein-coding genes respectively. Findings inferred the nature of the transcript networks was predominantly for ‘cell death and survival’, ‘cell morphology’, and ‘cell growth and proliferation’. Further studies are required to validate if this gene expression effect translates to cell events. Alternative splicing (AS) was dysregulated in ageing with 119, 21 and 9 differential splicing events identified in chondrogenic, osteogenic and tenogenic respectively, and enrichment in genes associated principally with metabolic processes. Gene ontology analysis of differentially methylated loci indicated age-related enrichment for all engineered tissue types in ‘skeletal system morphogenesis’, ‘regulation of cell proliferation’ and ‘regulation of transcription’ suggesting that dynamic epigenetic modifications may occur in genes associated with shared and distinct pathways dependent upon engineered tissue type. An altered phenotype in engineered tissues was observed with ageing at numerous levels. These changes represent novel insights into the ageing process, with implications for stem cell therapies in older patients. In addition we have identified a number of tissue-dependant pathways, which warrant further studies. PMID:27533049

Fruit regulates seasonal expression of flowering genes in alternate-bearing ‘Moncada’ mandarin

PubMed Central

Muñoz-Fambuena, Natalia; Mesejo, Carlos; Carmen González-Mas, M.; Primo-Millo, Eduardo; Agustí, Manuel; Iglesias, Domingo J.

2011-01-01

Background and Aims The presence of fruit has been widely reported to act as an inhibitor of flowering in fruit trees. This study is an investigation into the effect of fruit load on flowering of ‘Moncada’ mandarin and on the expression of putative orthologues of genes involved in flowering pathways to provide insight into the molecular mechanisms underlying alternate bearing in citrus. Methods The relationship between fruit load and flowering intensity was examined first. Defruiting experiments were further conducted to demonstrate the causal effect of fruit removal upon flowering. Finally, the activity of flowering-related genes was investigated to determine the extent to which their seasonal expression is affected by fruit yield. Key Results First observations and defruiting experiments indicated a significant inverse relationship between preceding fruit load and flowering intensity. Moreover, data indicated that when fruit remained on the tree from November onwards, a dramatic inhibition of flowering occurred the following spring. The study of the expression pattern of flowering-genes of on (fully loaded) and off (without fruits) trees revealed that homologues of FLOWERING LOCUS T (FT), SUPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), APETALA1 (AP1) and LEAFY (LFY) were negatively affected by fruit load. Thus, CiFT expression showed a progressive increase in leaves from off trees through the study period, the highest differences found from December onwards (10-fold). Whereas differences in the relative expression of SOC1 only reached significance from September to mid-December, CsAP1 expression was constantly higher in those trees through the whole study period. Significant variations in CsLFY expression only were found in late February (close to 20 %). On the other hand, the expression of the homologues of TERMINAL FLOWER 1 (TFL1) and FLOWERING LOCUS C (FLC) did not appear to be related to fruit load. Conclusions These results suggest for the first time that fruit inhibits flowering by repressing CiFT and SOC1 expression in leaves of alternate-bearing citrus. Fruit also reduces CsAP1 expression in leaves, and the significant increase in leaf CsLFY expression from off trees in late February was associated with the onset of floral differentiation. PMID:21856639

Molecular characterization and expression profiles of four transformer-2 isoforms in the Chinese mitten crab Eriocheir sinensis

NASA Astrophysics Data System (ADS)

Luo, Danli; Liu, Yuan; Hui, Min; Song, Chengwen; Liu, Hourong; Cui, Zhaoxia

2017-07-01

The transformer-2 ( tra-2) gene plays a key role in the regulatory hierarchy of sexual differentiation in somatic tissues and in the germline of Drosophila melanogaster. In this study, sequences and expression profiles of tra-2 in the Chinese mitten crab Eriocheir sinensis were characterized. Four tra-2 isoforms, designated as Estra-2a, Estra-2b, Estra-2c, and Estra-2d, were isolated. They all contained an RNA-recognition motif (RRM) and a linker region, which shared high similarity with other reported tra-2s. Sequence analysis revealed that Estra-2a, Estra-2b and Estra-2c are encoded by the same genomic locus and are generated by alternative splicing of the pre-mRNA. Compared with the other three isoforms, Estra-2d lacks the RS2 domain. Quantitative real-time PCR showed that all four isoforms were highly expressed in the fertilized egg, and in the 2-4 cell and blastula stages compared with larval stages ( P≤0.01), suggesting their maternal origin in early embryonic developmental stages. Notably, Estra-2a was highly expressed in male somatic tissues, while Estra-2c was significantly highly expressed in the ovary. These results suggest that Estra-2c is involved in sexual differentiation of the Chinese mitten crab. Our findings provide basic information for further functional studies of the tra-2 gene/protein in this species.

The effect of shock wave therapy on gene expression in human osteoblasts isolated from hypertrophic fracture non-unions

NASA Astrophysics Data System (ADS)

Hofmann, A.; Ritz, U.; Rompe, J.-D.; Tresch, A.; Rommens, P. M.

2015-01-01

Shock wave therapy has been increasingly evaluated as a non-invasive alternative for the treatment of delayed fracture healing and non-unions. Although several clinical studies showed a beneficial effect especially for the hypertrophic type of non-union, little is known about the biological mechanism of its osteogenic effect. To identify the molecular background for the positive effect of shock waves on healing of fracture non-unions, we have analyzed the changes of the global gene expression in human osteoblasts after exposure to shock waves of different energy flux densities. Human osteoblasts were isolated from five patients at non-union sites, treated with 500 impulses of energy flux densities of 0.06 and , and cultured for 96 h. HG-U133A microarrays were used for the analysis of the shock wave-regulated mRNA-transcripts. Differential gene expression was verified by reverse transcriptase polymerase chain reactions. We identified 47 transcripts that showed differential expression after and 45 transcripts after energy treatment. Most intriguing was the up-regulation of neprilysin, calmegin, osteoglycin, asporin, and interleukin-13 receptor-. Eighteen identified genes were previously described to fulfill an important function in bone growth and metabolism. Our study provides the first molecular profile of shock wave-induced gene expression changes in human osteoblasts from patients with hypertrophic fracture non-unions, and it offers a possible molecular explanation for the positive effects of shock waves in patients ridden with this disease.

Comparison of alternative approaches for analysing multi-level RNA-seq data

PubMed Central

Mohorianu, Irina; Bretman, Amanda; Smith, Damian T.; Fowler, Emily K.; Dalmay, Tamas

2017-01-01

RNA sequencing (RNA-seq) is widely used for RNA quantification in the environmental, biological and medical sciences. It enables the description of genome-wide patterns of expression and the identification of regulatory interactions and networks. The aim of RNA-seq data analyses is to achieve rigorous quantification of genes/transcripts to allow a reliable prediction of differential expression (DE), despite variation in levels of noise and inherent biases in sequencing data. This can be especially challenging for datasets in which gene expression differences are subtle, as in the behavioural transcriptomics test dataset from D. melanogaster that we used here. We investigated the power of existing approaches for quality checking mRNA-seq data and explored additional, quantitative quality checks. To accommodate nested, multi-level experimental designs, we incorporated sample layout into our analyses. We employed a subsampling without replacement-based normalization and an identification of DE that accounted for the hierarchy and amplitude of effect sizes within samples, then evaluated the resulting differential expression call in comparison to existing approaches. In a final step to test for broader applicability, we applied our approaches to a published set of H. sapiens mRNA-seq samples, The dataset-tailored methods improved sample comparability and delivered a robust prediction of subtle gene expression changes. The proposed approaches have the potential to improve key steps in the analysis of RNA-seq data by incorporating the structure and characteristics of biological experiments. PMID:28792517

Thermal plasticity of growth and development varies adaptively among alternative developmental pathways.

PubMed

Kivelä, Sami M; Svensson, Beatrice; Tiwe, Alma; Gotthard, Karl

2015-09-01

Polyphenism, the expression of discrete alternative phenotypes, is often a consequence of a developmental switch. Physiological changes induced by a developmental switch potentially affect reaction norms, but the evolution and existence of alternative reaction norms remains poorly understood. Here, we demonstrate that, in the butterfly Pieris napi (Lepidoptera: Pieridae), thermal reaction norms of several life history traits vary adaptively among switch-induced alternative developmental pathways of diapause and direct development. The switch was affected both by photoperiod and temperature, ambient temperature during late development having the potential to override earlier photoperiodic cues. Directly developing larvae had higher development and growth rates than diapausing ones across the studied thermal gradient. Reaction norm shapes also differed between the alternative developmental pathways, indicating pathway-specific selection on thermal sensitivity. Relative mass increments decreased linearly with increasing temperature and were higher under direct development than diapause. Contrary to predictions, population phenology did not explain trait variation or thermal sensitivity, but our experimental design probably lacks power for finding subtle phenology effects. We demonstrate adaptive differentiation in thermal reaction norms among alternative phenotypes, and suggest that the consequences of an environmentally dependent developmental switch primarily drive the evolution of alternative thermal reaction norms in P. napi. © 2015 The Author(s). Evolution © 2015 The Society for the Study of Evolution.

Multilineage potential and proteomic profiling of human dental stem cells derived from a single donor

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

Patil, Rajreddy; Kumar, B. Mohana; Lee, Won-Jae

Dental tissues provide an alternative autologous source of mesenchymal stem cells (MSCs) for regenerative medicine. In this study, we isolated human dental MSCs of follicle, pulp and papilla tissue from a single donor tooth after impacted third molar extraction by excluding the individual differences. We then compared the morphology, proliferation rate, expression of MSC-specific and pluripotency markers, and in vitro differentiation ability into osteoblasts, adipocytes, chondrocytes and functional hepatocyte-like cells (HLCs). Finally, we analyzed the protein expression profiles of undifferentiated dental MSCs using 2DE coupled with MALDI-TOF-MS. Three types of dental MSCs largely shared similar morphology, proliferation potential, expression ofmore » surface markers and pluripotent transcription factors, and differentiation ability into osteoblasts, adipocytes, and chondrocytes. Upon hepatogenic induction, all MSCs were transdifferentiated into functional HLCs, and acquired hepatocyte functions by showing their ability for glycogen storage and urea production. Based on the proteome profiling results, we identified nineteen proteins either found commonly or differentially expressed among the three types of dental MSCs. In conclusion, three kinds of dental MSCs from a single donor tooth possessed largely similar cellular properties and multilineage potential. Further, these dental MSCs had similar proteomic profiles, suggesting their interchangeable applications for basic research and call therapy. - Highlights: • Isolated and characterized three types of human dental MSCs from a single donor. • MSCs of dental follicle, pulp and papilla had largely similar biological properties. • All MSCs were capable of transdifferentiating into functional hepatocyte-like cells. • 2DE proteomics with MALDI-TOF/MS identified 19 proteins in three types of MSCs. • Similar proteomic profiles suggest interchangeable applications of dental MSCs.« less

Integrative functional analyses using rainbow trout selected for tolerance to plant diets reveal nutrigenomic signatures for soy utilization without the concurrence of enteritis.

PubMed

Abernathy, Jason; Brezas, Andreas; Snekvik, Kevin R; Hardy, Ronald W; Overturf, Ken

2017-01-01

Finding suitable alternative protein sources for diets of carnivorous fish species remains a major concern for sustainable aquaculture. Through genetic selection, we created a strain of rainbow trout that outperforms parental lines in utilizing an all-plant protein diet and does not develop enteritis in the distal intestine, as is typical with salmonids on long-term plant protein-based feeds. By incorporating this strain into functional analyses, we set out to determine which genes are critical to plant protein utilization in the absence of gut inflammation. After a 12-week feeding trial with our selected strain and a control trout strain fed either a fishmeal-based diet or an all-plant protein diet, high-throughput RNA sequencing was completed on both liver and muscle tissues. Differential gene expression analyses, weighted correlation network analyses and further functional characterization were performed. A strain-by-diet design revealed differential expression ranging from a few dozen to over one thousand genes among the various comparisons and tissues. Major gene ontology groups identified between comparisons included those encompassing central, intermediary and foreign molecule metabolism, associated biosynthetic pathways as well as immunity. A systems approach indicated that genes involved in purine metabolism were highly perturbed. Systems analysis among the tissues tested further suggests the interplay between selection for growth, dietary utilization and protein tolerance may also have implications for nonspecific immunity. By combining data from differential gene expression and co-expression networks using selected trout, along with ontology and pathway analyses, a set of 63 candidate genes for plant diet tolerance was found. Risk loci in human inflammatory bowel diseases were also found in our datasets, indicating rainbow trout selected for plant-diet tolerance may have added utility as a potential biomedical model.

Neuronal differentiation of stem cells isolated from adult muscle.

PubMed

Romero-Ramos, Marina; Vourc'h, Patrick; Young, Henry E; Lucas, Paul A; Wu, Young; Chivatakarn, Onanong; Zaman, Rumina; Dunkelman, Noushin; el-Kalay, Mohammad A; Chesselet, Marie-Françoise

2002-09-15

Lineage uncommitted pluripotent stem cells reside in the connective tissue of skeletal muscle. The present study was carried out with pluripotent stem cells (PPSCs) isolated from 6-month old rat muscle. Before differentiation, these cells were vimentin+, CD90+, CD45-, and varied in their expression of CD34. The PPSCs were expanded as non-adherent aggregates under similar conditions to those used to generate neurospheres from embryonic or neural stem cells. The PPSC-derived neurospheres were positive for nestin, an early marker present in neuronal precursors, and expressed the two alternative mRNA forms of the neuroectodermal marker Pax-6, as well as mRNA for Oct-4, a gene related to the pluripotentiality of stem cells. To confirm their neural potential, PPSC-derived neurospheres were plated on coated coverslips under varying conditions: Neurobasal medium with N2 or B27, and either NT3 or BDNF. After 4-6 days the cells expressed neuronal (Tuj1+, NF68), astrocytic (GFAP) and oligodendrocytic (MOSP+, MBP+) markers, both by immunocytochemistry and RT-PCR. In addition, PPSCs were cultured as monolayers under adherent conditions, exposed to growth factors and defined differentiating conditions for 5 hr, and subsequently kept for 2 days in a maturation medium. At this point they gave rise to a mixed population of early neural progenitors (Nestin+ or NG2+), immature and mature neurons (Tuj1+ and NF145+) and myelin producing oligodendrocytes (CNPase + and MOSP+). Our study shows that PPSCs present in adult muscle can overcome germ lineage restrictions and express the molecular characteristics of brain cells. Therefore, PPSCs isolated from adult muscle could provide a novel source for autologous cell replacement in neurodegenerative and demyelinating diseases. Copyright 2002 Wiley-Liss, Inc.

Failure of in vitro-differentiated mesenchymal stem cells from the synovial membrane to form ectopic stable cartilage in vivo.

PubMed

De Bari, Cosimo; Dell'Accio, Francesco; Luyten, Frank P

2004-01-01

We previously reported the identification in a nude mouse assay of molecular markers predictive of the capacity of articular cartilage-derived cells (ACDCs) to form ectopic stable cartilage that is resistant to vascular invasion and endochondral ossification. In the present study, we investigated whether in vitro-differentiated mesenchymal stem cells (MSCs) from the synovial membrane (SM) express the stable-chondrocyte markers and form ectopic stable cartilage in vivo. Chondrogenesis was induced in micromass culture with the addition of transforming growth factor beta1 (TGFbeta1). After acquisition of the cartilage phenotype, micromasses were implanted subcutaneously into nude mice. Alternatively, cells were released enzymatically and either replated in monolayer or injected intramuscularly into nude mice. Marker analysis was performed by quantitative reverse transcription-polymerase chain reaction. Cell death was detected with TUNEL assay. Cartilage-like micromasses and released cells expressed the stable-chondrocyte markers at levels comparable with those expressed by stable ACDCs. The released cells lost chondrocyte marker expression by 24 hours in monolayer and failed to form cartilage when injected intramuscularly into nude mice. Instead, myogenic differentiation was detected. When intact TGFbeta1-treated micromasses were implanted subcutaneously, they partially lost their cartilage phenotype and underwent cell death and neoangiogenesis within 1 week. At later time points (15-40 days), we retrieved neither cartilage nor bone, and human cells were not detectable. The chondrocyte-like phenotype of human SM MSCs, induced in vitro under specific conditions, appears to be unstable and is not sufficient to obtain ectopic formation of stable cartilage in vivo. Studies in animal models of joint surface defect repair are necessary to evaluate the stability of the SM MSC chondrocyte-like phenotype within the joint environment.

Effects of Pleiotrophin on endothelial and inflammatory cells: Pro-angiogenic and anti-inflammatory properties and potential role for vascular bio-prosthesis endothelialization.

PubMed

Palmieri, Daniela; Mura, Marzia; Mambrini, Simone; Palombo, Domenico

2015-09-01

One of the limitations emerged with both synthetic and degradable vascular grafts is the lack of endothelialization after implantation that is known to be the main reason leading to unfavourable outcomes. It emerges the need to find new strategies to promote a rapid endothelialization of the scaffold. Pleiotrophin is a growth/differentiation cytokine for various cell type. We here evaluated the effect of Pleiotrophin on endothelial cells (EC), monocytes and macrophages that have been shown as key cells promoting neovascularization. EA.hy926 endothelial cells, THP-1 monocytes and PMA-differentiated macrophages were treated with Pleiotrophin (10 and 100ng/ml). VEGF, Flk-1, Nrp-1, COX-2, ICAM-1 and TGFβ expression were detected by Western Blot, IL-10, MCP-1 and TNFα levels by ELISA. Chemotaxis was performed in Boyden chambers. Wound healing was performed by scratch wound assay. Pleiotrophin induces in EC the expression of VEGF and its receptors Flk-1 and Nrp-1 and improves the migratory capacity. In THP-1 monocytes, Pleiotrophin induces the expression of VEGF and its receptor Nrp-1 and decreases the levels of COX-2 and TNFα. In PMA-differentiated macrophages COX-2 expression was significantly reduced by Pleiotrophin, while IL-10 and TGFβ were increased. Pleiotrophin acts as an angiogenesis 'driver' by promoting the creation of a pro-angiogenic environment, a migratory behaviour in EC and a pro-regenerative alternative phenotype in macrophages. Our results suggest that Pleiotrophin might be considered for vascular prosthesis engineering. Copyright © 2015 Medical University of Bialystok. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Genetic and epigenetic mechanisms of gene regulation during lens development

PubMed Central

Cvekl, Ales; Duncan, Melinda K.

2007-01-01

Recent studies demonstrated a number of links between chromatin structure, gene expression, extracellular signaling and cellular differentiation during lens development. Lens progenitor cells originate from a pool of common progenitor cells, the pre-placodal region (PPR) which is formed due to a complex exchange of extracellular signals between the neural plate, naïve ectoderm and mesendoderm. A specific commitment to the lens program over alternate choices such as the formation of olfactory epithelium or the anterior pituitary is manifested by the formation of a thickened surface ectoderm, the lens placode. Mouse lens progenitor cells are characterized by the expression of a complement of lens lineage-specific transcription factors including Pax6, Six3 and Sox2, controlled by FGF and BMP signaling, followed later by c-Maf, Mab21like1, Prox1 and FoxE3. Proliferation of lens progenitors together with their morphogenetic movements results in the formation of the lens vesicle. This transient structure, comprised of lens precursor cells, is polarized with its anterior cells retaining their epithelial morphology and proliferative capacity, whereas the posterior lens precursor cells initiate terminal differentiation forming the primary lens fibers. Lens differentiation is marked by expression and accumulation of crystallins and other structural proteins. The transcriptional control of crystallin genes is characterized by the reiterative use of transcription factors required for the establishment of lens precursors in combination with more ubiquitously expressed factors (e.g. AP-1, AP-2α, CREB and USF) and recruitment of histone acetyltransferases (HATs) CBP and p300, and chromatin remodeling complexes SWI/SNF and ISWI. These studies have poised the study of lens development at the forefront of efforts to understand the connections between development, cell signaling, gene transcription and chromatin remodeling. PMID:17905638

Bridging the Synaptic Gap: Neuroligins and Neurexin I in Apis mellifera

PubMed Central

Biswas, Sunita; Russell, Robyn J.; Jackson, Colin J.; Vidovic, Maria; Ganeshina, Olga; Oakeshott, John G.; Claudianos, Charles

2008-01-01

Vertebrate studies show neuroligins and neurexins are binding partners in a trans-synaptic cell adhesion complex, implicated in human autism and mental retardation disorders. Here we report a genetic analysis of homologous proteins in the honey bee. As in humans, the honeybee has five large (31–246 kb, up to 12 exons each) neuroligin genes, three of which are tightly clustered. RNA analysis of the neuroligin-3 gene reveals five alternatively spliced transcripts, generated through alternative use of exons encoding the cholinesterase-like domain. Whereas vertebrates have three neurexins the bee has just one gene named neurexin I (400 kb, 28 exons). However alternative isoforms of bee neurexin I are generated by differential use of 12 splice sites, mostly located in regions encoding LNS subdomains. Some of the splice variants of bee neurexin I resemble the vertebrate α- and β-neurexins, albeit in vertebrates these forms are generated by alternative promoters. Novel splicing variations in the 3′ region generate transcripts encoding alternative trans-membrane and PDZ domains. Another 3′ splicing variation predicts soluble neurexin I isoforms. Neurexin I and neuroligin expression was found in brain tissue, with expression present throughout development, and in most cases significantly up-regulated in adults. Transcripts of neurexin I and one neuroligin tested were abundant in mushroom bodies, a higher order processing centre in the bee brain. We show neuroligins and neurexins comprise a highly conserved molecular system with likely similar functional roles in insects as vertebrates, and with scope in the honeybee to generate substantial functional diversity through alternative splicing. Our study provides important prerequisite data for using the bee as a model for vertebrate synaptic development. PMID:18974885

Nematogalectin, a nematocyst protein with GlyXY and galectin domains, demonstrates nematocyte-specific alternative splicing in Hydra

PubMed Central

Hwang, Jung Shan; Takaku, Yasuharu; Momose, Tsuyoshi; Adamczyk, Patrizia; Özbek, Suat; Ikeo, Kazuho; Khalturin, Konstantin; Hemmrich, Georg; Bosch, Thomas C. G.; Holstein, Thomas W.; David, Charles N.; Gojobori, Takashi

2010-01-01

Taxonomically restricted genes or lineage-specific genes contribute to morphological diversification in metazoans and provide unique functions for particular taxa in adapting to specific environments. To understand how such genes arise and participate in morphological evolution, we have investigated a gene called nematogalectin in Hydra, which has a structural role in the formation of nematocysts, stinging organelles that are unique to the phylum Cnidaria. Nematogalectin is a 28-kDa protein with an N-terminal GlyXY domain (glycine followed by two hydrophobic amino acids), which can form a collagen triple helix, followed by a galactose-binding lectin domain. Alternative splicing of the nematogalectin transcript allows the gene to encode two proteins, nematogalectin A and nematogalectin B. We demonstrate that expression of nematogalectin A and B is mutually exclusive in different nematocyst types: Desmonemes express nematogalectin B, whereas stenoteles and isorhizas express nematogalectin B early in differentiation, followed by nematogalectin A. Like Hydra, the marine hydrozoan Clytia also has two nematogalectin transcripts, which are expressed in different nematocyte types. By comparison, anthozoans have only one nematogalectin gene. Gene phylogeny indicates that tandem duplication of nematogalectin B exons gave rise to nematogalectin A before the divergence of Anthozoa and Medusozoa and that nematogalectin A was subsequently lost in Anthozoa. The emergence of nematogalectin A may have played a role in the morphological diversification of nematocysts in the medusozoan lineage. PMID:20937891

Peri‐prosthetic tissue cells show osteogenic capacity to differentiate into the osteoblastic lineage

PubMed Central

Schoeman, Monique A.E.; Oostlander, Angela E.; Rooij, Karien Ede; Valstar, Edward R.

2017-01-01

ABSTRACT During the process of aseptic loosening of prostheses, particulate wear debris induces a continuous inflammatory‐like response resulting in the formation of a layer of fibrous peri‐prosthetic tissue at the bone‐prosthesis interface. The current treatment for loosening is revision surgery which is associated with a high‐morbidity rate, especially in old patients. Therefore, less invasive alternatives are necessary. One approach could be to re‐establish osseointegration of the prosthesis by inducing osteoblast differentiation in the peri‐prosthetic tissue. Therefore, the aim of this study was to investigate the capacity of peri‐prosthetic tissue cells to differentiate into the osteoblast lineage. Cells isolated from peri‐prosthetic tissue samples (n = 22)−obtained during revision surgeries−were cultured under normal and several osteogenic culture conditions. Osteogenic differentiation was assessed by measurement of Alkaline Phosphatse (ALP), mineralization of the matrix and expression of several osteogenic genes. Cells cultured in osteogenic medium showed a significant increase in ALP staining (p = 0.024), mineralization of the matrix (p < 0.001) and ALP gene expression (p = 0.014) compared to normal culture medium. Addition of bone morphogenetic proteins (BMPs), a specific GSK3β inhibitor (GIN) or a combination of BMP and GIN to osteogenic medium could not increase ALP staining, mineralization, and ALP gene expression. In one donor, addition of GIN was required to induce mineralization of the matrix. Overall, we observed a high‐inter‐donor variability in response to osteogenic stimuli. In conclusion, peri‐prosthetic tissue cells, cultured under osteogenic conditions, can produce alkaline phosphatase and mineralized matrix, and therefore show characteristics of differentiation into the osteoblastic lineage. © 2016 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 35:1732–1742, 2017. PMID:27714894

Galectin-3 is essential for proper bone cell differentiation and activity, bone remodeling and biomechanical competence in mice.

PubMed

Iacobini, Carla; Fantauzzi, Claudia Blasetti; Bedini, Rossella; Pecci, Raffaella; Bartolazzi, Armando; Amadio, Bruno; Pesce, Carlo; Pugliese, Giuseppe; Menini, Stefano

2018-02-09

Galectin-3 is constitutively expressed in bone cells and was recently shown to modulate osteogenic transdifferentiation of vascular smooth muscle cells and atherosclerotic calcification. However, the role of galectin-3 in bone physiology is largely undefined. To address this issue, we analyzed (1) the skeletal features of 1-, 3- and 6-month-old galectin-3 null (Lgals3 -/- ) and wild type (WT) mice and (2) the differentiation and function of osteoblasts and osteoclasts derived from these animals. Long bone phenotype, gene expression profile, and remodeling were investigated by micro-computed tomography, real time-PCR, static and dynamic histomorphometry, and assessment of biochemical markers of bone resorption and formation. Bone competence was also evaluated by biomechanical testing at 3 months. In vitro, the effects of galectin-3 deficiency on bone cell differentiation and function were investigated by assessing (a) gene expression of osteoblast markers, alkaline phosphatase activity, mineralization assay, and WNT/β-catenin signaling (of which galectin-3 is a known regulator) in osteoblasts; and (b) tartrate-resistant acid phosphatase activity and bone resorption activity in osteoclasts. Lgals3 -/- mice revealed a wide range of age-dependent alterations including lower bone formation and higher bone resorption, accelerated age-dependent trabecular bone loss (p < 0.01 vs. WT at 3 months) and reduced bone strength (p < 0.01 vs. WT at 3 months). These abnormalities were accompanied by a steady inflammatory state, as revealed by higher bone expression of the pro-inflammatory cytokines interleukin (IL)-1β and IL-6 (p < 0.001 vs. WT at 3 months), increased content of osteal macrophages (p < 0.01 vs. WT at 3 months), and reduced expression of markers of alternative (M2) macrophage activation. Lgals3 -/- osteoblasts and osteoclasts showed impaired terminal differentiation, reduced mineralization capacity (p < 0.01 vs. WT cells) and resorption activity (p < 0.01 vs. WT cells). Mechanistically, impaired differentiation and function of Lgals3 -/- osteoblasts was associated with altered WNT/β-catenin signaling (p < 0.01 vs. WT cells). These data provide evidence for a contribution of galectin-3 to bone cell maturation and function, bone remodeling, and biomechanical competence, thus identifying galectin-3 as a promising therapeutic target for age-related disorders of bone remodeling. Copyright © 2018. Published by Elsevier Inc.

Isolation and characterization of alternatively spliced variants of the mouse sigma1 receptor gene, Sigmar1.

PubMed

Pan, Ling; Pasternak, David A; Xu, Jin; Xu, Mingming; Lu, Zhigang; Pasternak, Gavril W; Pan, Ying-Xian

2017-01-01

The sigma1 receptor acts as a chaperone at the endoplasmic reticulum, associates with multiple proteins in various cellular systems, and involves in a number of diseases, such as addiction, pain, cancer and psychiatric disorders. The sigma1 receptor is encoded by the single copy SIGMAR1 gene. The current study identifies five alternatively spliced variants of the mouse sigma1 receptor gene using a polymerase chain reaction cloning approach. All the splice variants are generated by exon skipping or alternative 3' or 5' splicing, producing the truncated sigma1 receptor. Similar alternative splicing has been observed in the human SIGMAR1 gene based on the molecular cloning or genome sequence prediction, suggesting conservation of alternative splicing of SIGMAR1 gene. Using quantitative polymerase chain reactions, we demonstrate differential expression of several splice variants in mouse tissues and brain regions. When expressed in HEK293 cells, all the splice variants fail to bind sigma ligands, implicating that each truncated region in these splice variants is important for ligand binding. However, co-immunoprecipitation (Co-IP) study in HEK293 cells co-transfected with tagged constructs reveals that all the splice variants maintain their ability to physically associate with a mu opioid receptor (mMOR-1), providing useful information to correlate the motifs/sequences necessary for their physical association. Furthermore, a competition Co-IP study showed that all the variants can disrupt in a dose-dependent manner the dimerization of the original sigma1 receptor with mMOR-1, suggesting a potential dominant negative function and providing significant insights into their function.

PlantAPA: A Portal for Visualization and Analysis of Alternative Polyadenylation in Plants

PubMed Central

Wu, Xiaohui; Zhang, Yumin; Li, Qingshun Q.

2016-01-01

Alternative polyadenylation (APA) is an important layer of gene regulation that produces mRNAs that have different 3′ ends and/or encode diverse protein isoforms. Up to 70% of annotated genes in plants undergo APA. Increasing numbers of poly(A) sites collected in various plant species demand new methods and tools to access and mine these data. We have created an open-access web service called PlantAPA (http://bmi.xmu.edu.cn/plantapa) to visualize and analyze genome-wide poly(A) sites in plants. PlantAPA provides various interactive and dynamic graphics and seamlessly integrates a genome browser that can profile heterogeneous cleavage sites and quantify expression patterns of poly(A) sites across different conditions. Particularly, through PlantAPA, users can analyze poly(A) sites in extended 3′ UTR regions, intergenic regions, and ambiguous regions owing to alternative transcription or RNA processing. In addition, it also provides tools for analyzing poly(A) site selections, 3′ UTR lengthening or shortening, non-canonical APA site switching, and differential gene expression between conditions, making it more powerful for the study of APA-mediated gene expression regulation. More importantly, PlantAPA offers a bioinformatics pipeline that allows users to upload their own short reads or ESTs for poly(A) site extraction, enabling users to further explore poly(A) site selection using stored PlantAPA poly(A) sites together with their own poly(A) site datasets. To date, PlantAPA hosts the largest database of APA sites in plants, including Oryza sativa, Arabidopsis thaliana, Medicago truncatula, and Chlamydomonas reinhardtii. As a user-friendly web service, PlantAPA will be a valuable addition to the community of biologists studying APA mechanisms and gene expression regulation in plants. PMID:27446120

Qualitative research of alternatively splice variants of fibronectin in different development stage of mice heart.

PubMed

Lu, Feng; Ma, Fang-Fang; Zhang, Wei; Li, Ying; Wei, Fei-Yu; Zhou, Lei

2015-12-01

Fibronectin (FN) plays vital roles in cell adhesion, differentiation, proliferation and migration. It is involved in the process of embryonic development and is highly conserved during evolution. The EIIIA and EIIIB of FN show a very high degree of homology among vertebrates. Embryos deleting both EIIIA and EIIIB displayed multiple embryonic cardiovascular defects, implying their crucial role during embryogenesis. The correlation of spliced EIIIB, EIIIA, and IIICS of FN to heart development was studied by observing their chronological expression in mice heart. C57 mice embryos at E11.5, E12.5, E13.5, E14.5, E15.5, E16.5, E17.5, E18.5, E19.5 days, postnatal day 1 (P1d), and adult male mice (3 months) were used. For each alternatively spliced FN1 domain (EIIIB, EIIIA and IIICS), primer pairs were designed for specific amplification. Total RNA was extracted from the heart tissue, reverse transcripted to cDNA, followed by RT-PCR with specific primers. The PCR amplification was verified by agarose gel electrophoresis, showing specific fragments of the expected sizes. In adult mice heart, only alternatively splice variants of EIIIA-, EIIIB-, IIICS+ were expressed. While in embryonic mice, spliced variant of EIIIA+/-, EIIIB+/-, IIICS+ were observed. The expression of EIIIA and EIIIB changed during heart development. FN is crucial for the normal development of the embryonic heart by modulating cardiac neural crest (CNC) proliferation and survival, and maintenance of CNC cells. FN1 gene seems to play a significant role by expression of highly conserved EIIIA and EIIIB in embryonic heart development.

Deep Sequencing Reveals Uncharted Isoform Heterogeneity of the Protein-Coding Transcriptome in Cerebral Ischemia.

PubMed

Bhattarai, Sunil; Aly, Ahmed; Garcia, Kristy; Ruiz, Diandra; Pontarelli, Fabrizio; Dharap, Ashutosh

2018-06-03

Gene expression in cerebral ischemia has been a subject of intense investigations for several years. Studies utilizing probe-based high-throughput methodologies such as microarrays have contributed significantly to our existing knowledge but lacked the capacity to dissect the transcriptome in detail. Genome-wide RNA-sequencing (RNA-seq) enables comprehensive examinations of transcriptomes for attributes such as strandedness, alternative splicing, alternative transcription start/stop sites, and sequence composition, thus providing a very detailed account of gene expression. Leveraging this capability, we conducted an in-depth, genome-wide evaluation of the protein-coding transcriptome of the adult mouse cortex after transient focal ischemia at 6, 12, or 24 h of reperfusion using RNA-seq. We identified a total of 1007 transcripts at 6 h, 1878 transcripts at 12 h, and 1618 transcripts at 24 h of reperfusion that were significantly altered as compared to sham controls. With isoform-level resolution, we identified 23 splice variants arising from 23 genes that were novel mRNA isoforms. For a subset of genes, we detected reperfusion time-point-dependent splice isoform switching, indicating an expression and/or functional switch for these genes. Finally, for 286 genes across all three reperfusion time-points, we discovered multiple, distinct, simultaneously expressed and differentially altered isoforms per gene that were generated via alternative transcription start/stop sites. Of these, 165 isoforms derived from 109 genes were novel mRNAs. Together, our data unravel the protein-coding transcriptome of the cerebral cortex at an unprecedented depth to provide several new insights into the flexibility and complexity of stroke-related gene transcription and transcript organization.

Alternative functional in vitro models of human intestinal epithelia

PubMed Central

Kauffman, Amanda L.; Gyurdieva, Alexandra V.; Mabus, John R.; Ferguson, Chrissa; Yan, Zhengyin; Hornby, Pamela J.

2013-01-01

Physiologically relevant sources of absorptive intestinal epithelial cells are crucial for human drug transport studies. Human adenocarcinoma-derived intestinal cell lines, such as Caco-2, offer conveniences of easy culture maintenance and scalability, but do not fully recapitulate in vivo intestinal phenotypes. Additional sources of renewable physiologically relevant human intestinal cells would provide a much needed tool for drug discovery and intestinal physiology. We compared two alternative sources of human intestinal cells, commercially available primary human intestinal epithelial cells (hInEpCs) and induced pluripotent stem cell (iPSC)-derived intestinal cells to Caco-2, for use in in vitro transwell monolayer intestinal transport assays. To achieve this for iPSC-derived cells, intestinal organogenesis was adapted to transwell differentiation. Intestinal cells were assessed by marker expression through immunocytochemical and mRNA expression analyses, monolayer integrity through Transepithelial Electrical Resistance (TEER) measurements and molecule permeability, and functionality by taking advantage the well-characterized intestinal transport mechanisms. In most cases, marker expression for primary hInEpCs and iPSC-derived cells appeared to be as good as or better than Caco-2. Furthermore, transwell monolayers exhibited high TEER with low permeability. Primary hInEpCs showed molecule efflux indicative of P-glycoprotein (Pgp) transport. Primary hInEpCs and iPSC-derived cells also showed neonatal Fc receptor-dependent binding of immunoglobulin G variants. Primary hInEpCs and iPSC-derived intestinal cells exhibit expected marker expression and demonstrate basic functional monolayer formation, similar to or better than Caco-2. These cells could offer an alternative source of human intestinal cells for understanding normal intestinal epithelial physiology and drug transport. PMID:23847534

Alternative functional in vitro models of human intestinal epithelia.

PubMed

Kauffman, Amanda L; Gyurdieva, Alexandra V; Mabus, John R; Ferguson, Chrissa; Yan, Zhengyin; Hornby, Pamela J

2013-01-01

Physiologically relevant sources of absorptive intestinal epithelial cells are crucial for human drug transport studies. Human adenocarcinoma-derived intestinal cell lines, such as Caco-2, offer conveniences of easy culture maintenance and scalability, but do not fully recapitulate in vivo intestinal phenotypes. Additional sources of renewable physiologically relevant human intestinal cells would provide a much needed tool for drug discovery and intestinal physiology. We compared two alternative sources of human intestinal cells, commercially available primary human intestinal epithelial cells (hInEpCs) and induced pluripotent stem cell (iPSC)-derived intestinal cells to Caco-2, for use in in vitro transwell monolayer intestinal transport assays. To achieve this for iPSC-derived cells, intestinal organogenesis was adapted to transwell differentiation. Intestinal cells were assessed by marker expression through immunocytochemical and mRNA expression analyses, monolayer integrity through Transepithelial Electrical Resistance (TEER) measurements and molecule permeability, and functionality by taking advantage the well-characterized intestinal transport mechanisms. In most cases, marker expression for primary hInEpCs and iPSC-derived cells appeared to be as good as or better than Caco-2. Furthermore, transwell monolayers exhibited high TEER with low permeability. Primary hInEpCs showed molecule efflux indicative of P-glycoprotein (Pgp) transport. Primary hInEpCs and iPSC-derived cells also showed neonatal Fc receptor-dependent binding of immunoglobulin G variants. Primary hInEpCs and iPSC-derived intestinal cells exhibit expected marker expression and demonstrate basic functional monolayer formation, similar to or better than Caco-2. These cells could offer an alternative source of human intestinal cells for understanding normal intestinal epithelial physiology and drug transport.

Transcriptional analysis of the Arabidopsis ovule by massively parallel signature sequencing

PubMed Central

Sánchez-León, Nidia; Arteaga-Vázquez, Mario; Alvarez-Mejía, César; Mendiola-Soto, Javier; Durán-Figueroa, Noé; Rodríguez-Leal, Daniel; Rodríguez-Arévalo, Isaac; García-Campayo, Vicenta; García-Aguilar, Marcelina; Olmedo-Monfil, Vianey; Arteaga-Sánchez, Mario; Martínez de la Vega, Octavio; Nobuta, Kan; Vemaraju, Kalyan; Meyers, Blake C.; Vielle-Calzada, Jean-Philippe

2012-01-01

The life cycle of flowering plants alternates between a predominant sporophytic (diploid) and an ephemeral gametophytic (haploid) generation that only occurs in reproductive organs. In Arabidopsis thaliana, the female gametophyte is deeply embedded within the ovule, complicating the study of the genetic and molecular interactions involved in the sporophytic to gametophytic transition. Massively parallel signature sequencing (MPSS) was used to conduct a quantitative large-scale transcriptional analysis of the fully differentiated Arabidopsis ovule prior to fertilization. The expression of 9775 genes was quantified in wild-type ovules, additionally detecting >2200 new transcripts mapping to antisense or intergenic regions. A quantitative comparison of global expression in wild-type and sporocyteless (spl) individuals resulted in 1301 genes showing 25-fold reduced or null activity in ovules lacking a female gametophyte, including those encoding 92 signalling proteins, 75 transcription factors, and 72 RNA-binding proteins not reported in previous studies based on microarray profiling. A combination of independent genetic and molecular strategies confirmed the differential expression of 28 of them, showing that they are either preferentially active in the female gametophyte, or dependent on the presence of a female gametophyte to be expressed in sporophytic cells of the ovule. Among 18 genes encoding pentatricopeptide-repeat proteins (PPRs) that show transcriptional activity in wild-type but not spl ovules, CIHUATEOTL (At4g38150) is specifically expressed in the female gametophyte and necessary for female gametogenesis. These results expand the nature of the transcriptional universe present in the ovule of Arabidopsis, and offer a large-scale quantitative reference of global expression for future genomic and developmental studies. PMID:22442422

Transcriptional analysis of the Arabidopsis ovule by massively parallel signature sequencing.

PubMed

Sánchez-León, Nidia; Arteaga-Vázquez, Mario; Alvarez-Mejía, César; Mendiola-Soto, Javier; Durán-Figueroa, Noé; Rodríguez-Leal, Daniel; Rodríguez-Arévalo, Isaac; García-Campayo, Vicenta; García-Aguilar, Marcelina; Olmedo-Monfil, Vianey; Arteaga-Sánchez, Mario; de la Vega, Octavio Martínez; Nobuta, Kan; Vemaraju, Kalyan; Meyers, Blake C; Vielle-Calzada, Jean-Philippe

2012-06-01

The life cycle of flowering plants alternates between a predominant sporophytic (diploid) and an ephemeral gametophytic (haploid) generation that only occurs in reproductive organs. In Arabidopsis thaliana, the female gametophyte is deeply embedded within the ovule, complicating the study of the genetic and molecular interactions involved in the sporophytic to gametophytic transition. Massively parallel signature sequencing (MPSS) was used to conduct a quantitative large-scale transcriptional analysis of the fully differentiated Arabidopsis ovule prior to fertilization. The expression of 9775 genes was quantified in wild-type ovules, additionally detecting >2200 new transcripts mapping to antisense or intergenic regions. A quantitative comparison of global expression in wild-type and sporocyteless (spl) individuals resulted in 1301 genes showing 25-fold reduced or null activity in ovules lacking a female gametophyte, including those encoding 92 signalling proteins, 75 transcription factors, and 72 RNA-binding proteins not reported in previous studies based on microarray profiling. A combination of independent genetic and molecular strategies confirmed the differential expression of 28 of them, showing that they are either preferentially active in the female gametophyte, or dependent on the presence of a female gametophyte to be expressed in sporophytic cells of the ovule. Among 18 genes encoding pentatricopeptide-repeat proteins (PPRs) that show transcriptional activity in wild-type but not spl ovules, CIHUATEOTL (At4g38150) is specifically expressed in the female gametophyte and necessary for female gametogenesis. These results expand the nature of the transcriptional universe present in the ovule of Arabidopsis, and offer a large-scale quantitative reference of global expression for future genomic and developmental studies.

Switch-like reprogramming of gene expression after fusion of multinucleate plasmodial cells of two Physarum polycephalum sporulation mutants

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

Walter, Pauline; Hoffmann, Xenia-Katharina; Ebeling, Britta

2013-05-24

Highlights: •We investigate reprogramming of gene expression in multinucleate single cells. •Cells of two differentiation control mutants are fused. •Fused cells proceed to alternative gene expression patterns. •The population of nuclei damps stochastic fluctuations in gene expression. •Dynamic processes of cellular reprogramming can be observed by repeated sampling of a cell. -- Abstract: Nonlinear dynamic processes involving the differential regulation of transcription factors are considered to impact the reprogramming of stem cells, germ cells, and somatic cells. Here, we fused two multinucleate plasmodial cells of Physarum polycephalum mutants defective in different sporulation control genes while being in different physiological states.more » The resulting heterokaryons established one of two significantly different expression patterns of marker genes while the plasmodial halves that were fused to each other synchronized spontaneously. Spontaneous synchronization suggests that switch-like control mechanisms spread over and finally control the entire plasmodium as a result of cytoplasmic mixing. Regulatory molecules due to the large volume of the vigorously streaming cytoplasm will define concentrations in acting on the population of nuclei and in the global setting of switches. Mixing of a large cytoplasmic volume is expected to damp stochasticity when individual nuclei deliver certain RNAs at low copy number into the cytoplasm. We conclude that spontaneous synchronization, the damping of molecular noise in gene expression by the large cytoplasmic volume, and the option to take multiple macroscopic samples from the same plasmodium provide unique options for studying the dynamics of cellular reprogramming at the single cell level.« less

Differential involvement of Hedgehog signaling in butterfly wing and eyespot development.

PubMed

Tong, Xiaoling; Lindemann, Anna; Monteiro, Antónia

2012-01-01

Butterfly eyespots may have evolved from the recruitment of pre-existent gene circuits or regulatory networks into novel locations on the wing. Gene expression data suggests one such circuit, the Hedgehog (Hh) signaling pathway and its target gene engrailed (en), was recruited from a role in patterning the anterior-posterior insect wing axis to a role patterning butterfly eyespots. However, while Junonia coenia expresses hh and en both in the posterior compartment of the wing and in eyespot centers, Bicyclus anynana lacks hh eyespot-specific expression. This suggests that Hh signaling may not be functioning in eyespot development in either species or that it functions in J. coenia but not in B. anynana. In order to test these hypotheses, we performed functional tests of Hh signaling in these species. We investigated the effects of Hh protein sequestration during the larval stage on en expression levels, and on wing size and eyespot size in adults. Hh sequestration led to significantly reduced en expression and to significantly smaller wings and eyespots in both species. But while eyespot size in B. anynana was reduced proportionately to wing size, in J. coenia, eyespots were reduced disproportionately, indicating an independent role of Hh signaling in eyespot development in J. coenia. We conclude that while Hh signaling retains a conserved role in promoting wing growth across nymphalid butterflies, it plays an additional role in eyespot development in some, but not all, lineages of nymphalid butterflies. We discuss our findings in the context of alternative evolutionary scenarios that led to the differential expression of hh and other Hh pathway signaling members across nymphalid species.

TCC: an R package for comparing tag count data with robust normalization strategies

PubMed Central

2013-01-01

Background Differential expression analysis based on “next-generation” sequencing technologies is a fundamental means of studying RNA expression. We recently developed a multi-step normalization method (called TbT) for two-group RNA-seq data with replicates and demonstrated that the statistical methods available in four R packages (edgeR, DESeq, baySeq, and NBPSeq) together with TbT can produce a well-ranked gene list in which true differentially expressed genes (DEGs) are top-ranked and non-DEGs are bottom ranked. However, the advantages of the current TbT method come at the cost of a huge computation time. Moreover, the R packages did not have normalization methods based on such a multi-step strategy. Results TCC (an acronym for Tag Count Comparison) is an R package that provides a series of functions for differential expression analysis of tag count data. The package incorporates multi-step normalization methods, whose strategy is to remove potential DEGs before performing the data normalization. The normalization function based on this DEG elimination strategy (DEGES) includes (i) the original TbT method based on DEGES for two-group data with or without replicates, (ii) much faster methods for two-group data with or without replicates, and (iii) methods for multi-group comparison. TCC provides a simple unified interface to perform such analyses with combinations of functions provided by edgeR, DESeq, and baySeq. Additionally, a function for generating simulation data under various conditions and alternative DEGES procedures consisting of functions in the existing packages are provided. Bioinformatics scientists can use TCC to evaluate their methods, and biologists familiar with other R packages can easily learn what is done in TCC. Conclusion DEGES in TCC is essential for accurate normalization of tag count data, especially when up- and down-regulated DEGs in one of the samples are extremely biased in their number. TCC is useful for analyzing tag count data in various scenarios ranging from unbiased to extremely biased differential expression. TCC is available at http://www.iu.a.u-tokyo.ac.jp/~kadota/TCC/ and will appear in Bioconductor (http://bioconductor.org/) from ver. 2.13. PMID:23837715

Sequencing of mRNA identifies re-expression of fetal splice variants in cardiac hypertrophy

PubMed Central

Ames, EG; Lawson, MJ; Mackey, AJ; Holmes, JW

2013-01-01

Cardiac hypertrophy has been well-characterized at the level of transcription. During cardiac hypertrophy, genes normally expressed primarily during fetal heart development are reexpressed, and this fetal gene program is believed to be a critical component of the hypertrophic process. Recently, alternative splicing of mRNA transcripts has been shown to be temporally regulated during heart development, leading us to consider whether fetal patterns of splicing also reappear during hypertrophy. We hypothesized that patterns of alternative splicing occurring during heart development are recapitulated during cardiac hypertrophy. Here we present a study of isoform expression during pressure-overload cardiac hypertrophy induced by 10 days of transverse aortic constriction (TAC) in rats and in developing fetal rat hearts compared to sham-operated adult rat hearts, using high-throughput sequencing of poly(A) tail mRNA. We find a striking degree of overlap between the isoforms expressed differentially in fetal and pressure-overloaded hearts compared to control: forty-four percent of the isoforms with significantly altered expression in TAC hearts are also expressed at significantly different levels in fetal hearts compared to control (P < 0.001). The isoforms that are shared between hypertrophy and fetal heart development are significantly enriched for genes involved in cytoskeletal organization, RNA processing, developmental processes, and metabolic enzymes. Our data strongly support the concept that mRNA splicing patterns normally associated with heart development recur as part of the hypertrophic response to pressure overload. These findings suggest that cardiac hypertrophy shares post-transcriptional as well as transcriptional regulatory mechanisms with fetal heart development. PMID:23688780

Deciphering Transcriptome and Complex Alternative Splicing Transcripts in Mammary Gland Tissues from Cows Naturally Infected with Staphylococcus aureus Mastitis

PubMed Central

Jiang, Qiang; Yang, Chun Hong; Zhang, Yan; Sun, Yan; Li, Rong Ling; Wang, Chang Fa; Zhong, Ji Feng; Huang, Jin Ming

2016-01-01

Alternative splicing (AS) contributes to the complexity of the mammalian proteome and plays an important role in diseases, including infectious diseases. The differential AS patterns of these transcript sequences between the healthy (HS3A) and mastitic (HS8A) cows naturally infected by Staphylococcus aureus were compared to understand the molecular mechanisms underlying mastitis resistance and susceptibility. In this study, using the Illumina paired-end RNA sequencing method, 1352 differentially expressed genes (DEGs) with higher than twofold changes were found in the HS3A and HS8A mammary gland tissues. Gene ontology and KEGG pathway analyses revealed that the cytokine–cytokine receptor interaction pathway is the most significantly enriched pathway. Approximately 16k annotated unigenes were respectively identified in two libraries, based on the bovine Bos taurus UMD3.1 sequence assembly and search. A total of 52.62% and 51.24% annotated unigenes were alternatively spliced in term of exon skipping, intron retention, alternative 5′ splicing and alternative 3ʹ splicing. Additionally, 1,317 AS unigenes were HS3A-specific, whereas 1,093 AS unigenes were HS8A-specific. Some immune-related genes, such as ITGB6, MYD88, ADA, ACKR1, and TNFRSF1B, and their potential relationships with mastitis were highlighted. From Chromosome 2, 4, 6, 7, 10, 13, 14, 17, and 20, 3.66% (HS3A) and 5.4% (HS8A) novel transcripts, which harbor known quantitative trait locus associated with clinical mastitis, were identified. Many DEGs in the healthy and mastitic mammary glands are involved in immune, defense, and inflammation responses. These DEGs, which exhibit diverse and specific splicing patterns and events, can endow dairy cattle with the potential complex genetic resistance against mastitis. PMID:27459697

CD40 signaling synergizes with TLR-2 in the BCR independent activation of resting B cells.

PubMed

Jain, Shweta; Chodisetti, Sathi Babu; Agrewala, Javed N

2011-01-01

Conventionally, signaling through BCR initiates sequence of events necessary for activation and differentiation of B cells. We report an alternative approach, independent of BCR, for stimulating resting B (RB) cells, by involving TLR-2 and CD40--molecules crucial for innate and adaptive immunity. CD40 triggering of TLR-2 stimulated RB cells significantly augments their activation, proliferation and differentiation. It also substantially ameliorates the calcium flux, antigen uptake capacity and ability of B cells to activate T cells. The survival of RB cells was improved and it increases the number of cells expressing activation induced deaminase (AID), signifying class switch recombination (CSR). Further, we also observed increased activation rate and decreased threshold period required for optimum stimulation of RB cells. These results corroborate well with microarray gene expression data. This study provides novel insights into coordination between the molecules of innate and adaptive immunity in activating B cells, in a BCR independent manner. This strategy can be exploited to design vaccines to bolster B cell activation and antigen presenting efficiency, leading to faster and better immune response.

A population of adult satellite-like cells in Drosophila is maintained through a switch in RNA-isoforms

PubMed Central

Boukhatmi, Hadi

2018-01-01

Adult stem cells are important for tissue maintenance and repair. One key question is how such cells are specified and then protected from differentiation for a prolonged period. Investigating the maintenance of Drosophila muscle progenitors (MPs) we demonstrate that it involves a switch in zfh1/ZEB1 RNA-isoforms. Differentiation into functional muscles is accompanied by expression of miR-8/miR-200, which targets the major zfh1-long RNA isoform and decreases Zfh1 protein. Through activity of the Notch pathway, a subset of MPs produce an alternate zfh1-short isoform, which lacks the miR-8 seed site. Zfh1 protein is thus maintained in these cells, enabling them to escape differentiation and persist as MPs in the adult. There, like mammalian satellite cells, they contribute to muscle homeostasis. Such preferential regulation of a specific RNA isoform, with differential sensitivity to miRs, is a powerful mechanism for maintaining a population of poised progenitors and may be of widespread significance. PMID:29629869

Fip1 regulates mRNA alternative polyadenylation to promote stem cell self-renewal

PubMed Central

Lackford, Brad; Yao, Chengguo; Charles, Georgette M; Weng, Lingjie; Zheng, Xiaofeng; Choi, Eun-A; Xie, Xiaohui; Wan, Ji; Xing, Yi; Freudenberg, Johannes M; Yang, Pengyi; Jothi, Raja; Hu, Guang; Shi, Yongsheng

2014-01-01

mRNA alternative polyadenylation (APA) plays a critical role in post-transcriptional gene control and is highly regulated during development and disease. However, the regulatory mechanisms and functional consequences of APA remain poorly understood. Here, we show that an mRNA 3′ processing factor, Fip1, is essential for embryonic stem cell (ESC) self-renewal and somatic cell reprogramming. Fip1 promotes stem cell maintenance, in part, by activating the ESC-specific APA profiles to ensure the optimal expression of a specific set of genes, including critical self-renewal factors. Fip1 expression and the Fip1-dependent APA program change during ESC differentiation and are restored to an ESC-like state during somatic reprogramming. Mechanistically, we provide evidence that the specificity of Fip1-mediated APA regulation depends on multiple factors, including Fip1-RNA interactions and the distance between APA sites. Together, our data highlight the role for post-transcriptional control in stem cell self-renewal, provide mechanistic insight on APA regulation in development, and establish an important function for APA in cell fate specification. PMID:24596251

Alternative Splicing of MBD2 Supports Self-Renewal in Human Pluripotent Stem Cells

PubMed Central

Lu, Yu; Loh, Yuin-Han; Li, Hu; Cesana, Marcella; Ficarro, Scott B.; Parikh, Jignesh R.; Salomonis, Nathan; Toh, Cheng-Xu Delon; Andreadis, Stelios T.; Luckey, C. John; Collins, James J.; Daley, George Q.; Marto, Jarrod A.

2014-01-01

Summary Alternative RNA splicing (AS) regulates proteome diversity, including isoform-specific expression of several pluripotency genes. Here, we integrated global gene expression and proteomic analyses and identified a molecular signature suggesting a central role for AS in maintaining human pluripotent stem cell (hPSC) self-renewal. We demonstrate the splicing factor SFRS2 is an OCT4 target gene required for pluripotency. SFRS2 regulates AS of the methyl-CpG-binding protein MBD2, whose isoforms play opposing roles in maintenance of, and reprogramming to, pluripotency. While both MDB2a and MBD2c are enriched at the OCT4 and NANOG promoters, MBD2a preferentially interacts with repressive NuRD chromatin remodeling factors and promotes hPSC differentiation, whereas overexpression of MBD2c enhances reprogramming of fibroblasts to pluripotency. The miR-301 and miR-302 families provide additional regulation by targeting SFRS2 and MDB2a. These data suggest that OCT4, SFRS2, and MBD2 participate in a positive feedback loop, regulating proteome diversity complexity in support of hPSC self-renewal and reprogramming. PMID:24813856

In vitro effects of three blood derivatives on human corneal epithelial cells.

PubMed

Freire, Vanesa; Andollo, Noelia; Etxebarria, Jaime; Durán, Juan A; Morales, María-Celia

2012-08-15

We compared the effects of three blood derivatives, autologous serum (AS), platelet-rich plasma (PRP), and serum derived from plasma rich in growth factors (PRGF), on a human corneal epithelial (HCE) cell line to evaluate their potential as an effective treatment for corneal epithelial disorders. The concentrations of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and fibronectin were quantified by ELISA. The proliferation and viability of HCE cells were measured by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay. Cell morphology was assessed by phase-contrast microscopy. The patterns of expression of several connexin, involucrin, and integrin α6 genes were analyzed by real-time RT-PCR. We found significantly higher levels of EGF in PRGF compared to AS and PRP. However, AS and PRGF induced robust proliferation of HCE cells. In addition, PRGF cultured cells grew as heterogeneous colonies, exhibiting differentiated and non-differentiated cell phenotypes, whereas AS- and PRP-treated cultures exhibited quite homogeneous colonies. Finally, PRGF upregulated the expression of several genes associated with communication and cell differentiation, in comparison to AS or PRP. PRGF promotes biological processes required for corneal epithelialization, such as proliferation and differentiation. Since PRGF effects are similar to those associated with routinely used blood derivatives, the present findings warrant further research on PRGF as a novel alternative treatment for ocular surface diseases.

Anti-stress and neuronal cell differentiation induction effects of Rosmarinus officinalis L. essential oil.

PubMed

Villareal, Myra O; Ikeya, Ayumi; Sasaki, Kazunori; Arfa, Abdelkarim Ben; Neffati, Mohamed; Isoda, Hiroko

2017-12-22

Mood disorder accounts for 13 % of global disease burden. And while therapeutic agents are available, usually orally administered, most have unwanted side effects, and thus making the inhalation of essential oils (EOs) an attractive alternative therapy. Rosmarinus officinalis EO (ROEO), Mediterranean ROEO reported to improve cognition, mood, and memory, the effect on stress of which has not yet been determined. Here, the anti-stress effect of ROEO on stress was evaluated in vivo and in vitro. Six-week-old male ICR mice were made to inhale ROEO and subjected to tail suspension test (TST). To determine the neuronal differentiation effect of ROEO in vitro, induction of ROEO-treated PC12 cells differentiation was observed. Intracellular acetylcholine and choline, as well as the Gap43 gene expression levels were also determined. Inhalation of ROEO significantly decreased the immobility time of ICR mice and serum corticosterone level, accompanied by increased brain dopamine level. Determination of the underlying mechanism in vitro revealed a PC12 differentiation-induction effect through the modulation of intracellular acetylcholine, choline, and Gap43 gene expression levels. ROEO activates the stress response system through the NGF pathway and the hypothalamus-pituitary-adrenal axis, promoting dopamine production and secretion. The effect of ROEO may be attributed to its bioactive components, specifically to α-pinene, one of its major compounds that has anxiolytic property. The results of this study suggest that ROEO inhalation has therapeutic potential against stress-related psychiatric disorders.

Cutting Edge: Differential Regulation of PTEN by TCR, Akt, and FoxO1 Controls CD4+ T Cell Fate Decisions.

PubMed

Hawse, William F; Sheehan, Robert P; Miskov-Zivanov, Natasa; Menk, Ashley V; Kane, Lawrence P; Faeder, James R; Morel, Penelope A

2015-05-15

Signaling via the Akt/mammalian target of rapamycin pathway influences CD4(+) T cell differentiation; low levels favor regulatory T cell induction and high levels favor Th induction. Although the lipid phosphatase phosphatase and tensin homolog (PTEN) suppresses Akt activity, the control of PTEN activity is poorly studied in T cells. In this study, we identify multiple mechanisms that regulate PTEN expression. During Th induction, PTEN function is suppressed via lower mRNA levels, lower protein levels, and an increase in C-terminal phosphorylation. Conversely, during regulatory T cell induction, PTEN function is maintained through the stabilization of PTEN mRNA transcription and sustained protein levels. We demonstrate that differential Akt/mammalian target of rapamycin signaling regulates PTEN transcription via the FoxO1 transcription factor. A mathematical model that includes multiple modes of PTEN regulation recapitulates our experimental findings and demonstrates how several feedback loops determine differentiation outcomes. Collectively, this work provides novel mechanistic insights into how differential regulation of PTEN controls alternate CD4(+) T cell fate outcomes. Copyright © 2015 by The American Association of Immunologists, Inc.

Chm-1 gene-modified bone marrow mesenchymal stem cells maintain the chondrogenic phenotype of tissue-engineered cartilage.

PubMed

Chen, Zhuoyue; Wei, Jing; Zhu, Jun; Liu, Wei; Cui, Jihong; Li, Hongmin; Chen, Fulin

2016-05-05

Marrow mesenchymal stem cells (MSCs) can differentiate into specific phenotypes, including chondrocytes, and have been widely used for cartilage tissue engineering. However, cartilage grafts from MSCs exhibit phenotypic alternations after implantation, including matrix calcification and vascular ingrowth. We compared chondromodulin-1 (Chm-1) expression between chondrocytes and MSCs. We found that chondrocytes expressed a high level of Chm-1. We then adenovirally transduced MSCs with Chm-1 and applied modified cells to engineer cartilage in vivo. A gross inspection and histological observation indicated that the chondrogenic phenotype of the tissue-engineered cartilage graft was well maintained, and the stable expression of Chm-1 was detected by immunohistological staining in the cartilage graft derived from the Chm-1 gene-modified MSCs. Our findings defined an essential role for Chm-1 in maintaining chondrogenic phenotype and demonstrated that Chm-1 gene-modified MSCs may be used in cartilage tissue engineering.

Role of the DNA Damage Response in Human Papillomavirus RNA Splicing and Polyadenylation.

PubMed

Nilsson, Kersti; Wu, Chengjun; Schwartz, Stefan

2018-06-12

Human papillomaviruses (HPVs) have evolved to use the DNA repair machinery to replicate its DNA genome in differentiated cells. HPV activates the DNA damage response (DDR) in infected cells. Cellular DDR factors are recruited to the HPV DNA genome and position the cellular DNA polymerase on the HPV DNA and progeny genomes are synthesized. Following HPV DNA replication, HPV late gene expression is activated. Recent research has shown that the DDR factors also interact with RNA binding proteins and affects RNA processing. DDR factors activated by DNA damage and that associate with HPV DNA can recruit splicing factors and RNA binding proteins to the HPV DNA and induce HPV late gene expression. This induction is the result of altered alternative polyadenylation and splicing of HPV messenger RNA (mRNA). HPV uses the DDR machinery to replicate its DNA genome and to activate HPV late gene expression at the level of RNA processing.

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

PubMed Central

2012-01-01

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

Characterization of cDNAs encoding the chick retinoic acid receptor gamma 2 and preferential distribution of retinoic acid receptor gamma transcripts during chick skin development.

PubMed

Michaille, J J; Blanchet, S; Kanzler, B; Garnier, J M; Dhouailly, D

1994-12-01

Retinoic acid receptors alpha, beta and gamma (RAR alpha, beta and gamma) are ligand-inductible transcriptional activators which belong to the steroid/thyroid hormone receptor superfamily. At least two major isoforms (1 and 2) of each RAR arise by differential use of two promoters and alternative splicing. In mouse, the three RAR genes are expressed in stage- and tissue-specific patterns during embryonic development. In order to understand the role of the different RARs in chick, RAR gamma 2 cDNAs were isolated from an 8.5-day (stage 35 of Hamburger and Hamilton) chick embryo skin library. The deduced chick RAR gamma 2 amino acid sequence displays uncommon features such as 21 specific amino acid replacements, 12 of them being clustered in the amino-terminal region (domains A2 and B), and a truncated acidic carboxy-terminal region (F domain). However, the pattern of RAR gamma expression in chick embryo resembles that reported in mouse, particularly in skin where RAR gamma expression occurs in both the dermal and epidermal layers at the beginning of feather formation, and is subsequently restricted to the differentiating epidermal cells. Northern blot analysis suggests that different RAR gamma isoforms could be successively required during chick development.

Oxidative phosphorylation in Debaryomyces hansenii: physiological uncoupling at different growth phases.

PubMed

Cabrera-Orefice, Alfredo; Guerrero-Castillo, Sergio; Díaz-Ruíz, Rodrigo; Uribe-Carvajal, Salvador

2014-07-01

Physiological uncoupling of mitochondrial oxidative phosphorylation (OxPhos) was studied in Debaryomyces hansenii. In other species, such as Yarrowia lipolytica and Saccharomyces cerevisiae, OxPhos can be uncoupled through differential expression of branched respiratory chain enzymes or by opening of a mitochondrial unspecific channel (ScMUC), respectively. However D. hansenii mitochondria, which contain both a branched respiratory chain and a mitochondrial unspecific channel (DhMUC), selectively uncouple complex I-dependent rate of oxygen consumption in the stationary growth phase. The uncoupled complex I-dependent respiration was only 20% of the original activity. Inhibition was not due to inactivation of complex I, lack of protein expression or to differential expression of alternative oxidoreductases. Furthermore, all other respiratory chain activities were normal. Decrease of complex I-dependent respiration was due to NAD(+) loss from the matrix, probably through an open of DhMUC. When NAD(+) was added back, coupled complex I-activity was recovered. NAD(+) re-uptake was independent of DhMUC opening and seemed to be catalyzed by a NAD(+)-specific transporter, which was sensitive to bathophenanthroline, bromocresol purple or pyridoxal-5'-phosphate as described for S. cerevisiae mitochondrial NAD(+) transporters. Loss of NAD(+) from the matrix through an open MUC is proposed as an additional mechanism to uncouple OxPhos. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

In vitro growth of Vibrio cholerae in cholera stool fluid leads to differential expression of virulence factors.

PubMed

Sánchez, J; Castillo, G; Medrano, A I; Martinez-Palomo, A; Rodríguez, M H

1995-01-01

We report on the physiological response of Vibrio cholerae upon growth on bacteria-free intestinal fluids prepared from feces of individuals in the acute phase of cholera. Sterilized stool fluids supported growth of V. cholerae to reach 0.3-0.4 O.D. units (600 nm) at 37 degrees C. Scanning electron microscopy showed vibrios to be slender and elongated as compared to bacteria in synthetic media. Growth in stool fluid apparently induced expression of several immunoreactive proteins using cholera convalescent sera. Supernatants of fluid-grown vibrios had undetectable cholera toxin (CT) concentrations. Soluble hemagglutinins and soluble proteases were much less reduced when compared to cultures in Syncase or AKI media while cell-associated mannose-sensitive hemagglutinin (MSHA) was expressed at good levels. Lack of production of CT in fluid devoid of tissue may be due to absence of stimulating elements in intact intestine. Alternatively, culturing V. cholerae in stool fluid might resemble a late proliferation stage where downregulation of toxin might occur. Irrespectively, concomitant production of other virulence factors represents a phenomenon of differential regulation by fluid. Efforts are now underway to determine if this response depends upon factors in stool fluid acting through known genetic regulatory cascades or other. Attempts are also geared to identify fluid-induced proteins and their genes.

IBTK Differently Modulates Gene Expression and RNA Splicing in HeLa and K562 Cells.

PubMed

Fiume, Giuseppe; Scialdone, Annarita; Rizzo, Francesca; De Filippo, Maria Rosaria; Laudanna, Carmelo; Albano, Francesco; Golino, Gaetanina; Vecchio, Eleonora; Pontoriero, Marilena; Mimmi, Selena; Ceglia, Simona; Pisano, Antonio; Iaccino, Enrico; Palmieri, Camillo; Paduano, Sergio; Viglietto, Giuseppe; Weisz, Alessandro; Scala, Giuseppe; Quinto, Ileana

2016-11-07

The IBTK gene encodes the major protein isoform IBTKα that was recently characterized as substrate receptor of Cul3-dependent E3 ligase, regulating ubiquitination coupled to proteasomal degradation of Pdcd4, an inhibitor of translation. Due to the presence of Ankyrin-BTB-RCC1 domains that mediate several protein-protein interactions, IBTKα could exert expanded regulatory roles, including interaction with transcription regulators. To verify the effects of IBTKα on gene expression, we analyzed HeLa and K562 cell transcriptomes by RNA-Sequencing before and after IBTK knock-down by shRNA transduction. In HeLa cells, 1285 (2.03%) of 63,128 mapped transcripts were differentially expressed in IBTK -shRNA-transduced cells, as compared to cells treated with control-shRNA, with 587 upregulated (45.7%) and 698 downregulated (54.3%) RNAs. In K562 cells, 1959 (3.1%) of 63128 mapped RNAs were differentially expressed in IBTK -shRNA-transduced cells, including 1053 upregulated (53.7%) and 906 downregulated (46.3%). Only 137 transcripts (0.22%) were commonly deregulated by IBTK silencing in both HeLa and K562 cells, indicating that most IBTKα effects on gene expression are cell type-specific. Based on gene ontology classification, the genes responsive to IBTK are involved in different biological processes, including in particular chromatin and nucleosomal organization, gene expression regulation, and cellular traffic and migration. In addition, IBTK RNA interference affected RNA maturation in both cell lines, as shown by the evidence of alternative 3'- and 5'-splicing, mutually exclusive exons, retained introns, and skipped exons. Altogether, these results indicate that IBTK differently modulates gene expression and RNA splicing in HeLa and K562 cells, demonstrating a novel biological role of this protein.

Differential expression of GAP-43 and neurofilament during peripheral nerve regeneration through bio-artificial conduits.

PubMed

Carriel, Víctor; Garzón, Ingrid; Campos, Antonio; Cornelissen, Maria; Alaminos, Miguel

2017-02-01

Nerve conduits are promising alternatives for repairing nerve gaps; they provide a close microenvironment that supports nerve regeneration. In this sense, histological analysis of axonal growth is a determinant to achieve successful nerve regeneration. To evaluate this process, the most-used immunohistochemical markers are neurofilament (NF), β-III tubulin and, infrequently, GAP-43. However, GAP-43 expression in long-term nerve regeneration models is still poorly understood. In this study we analysed GAP-43 expression and its correlation with NF and S-100, using three tissue-engineering approaches with different regeneration profiles. A 10 mm gap was created in the sciatic nerve of 12 rats and repaired using collagen conduits or collagen conduits filled with fibrin-agarose hydrogels or with hydrogels containing autologous adipose-derived mesenchymal stem cells (ADMSCs). After 12 weeks the conduits were harvested for histological analysis. Our results confirm the long-term expression of GAP-43 in all groups. The expression of GAP-43 and NF was significantly higher in the group with ADMSCs. Interestingly, GAP-43 was observed in immature, newly formed axons and NF in thicker and mature axons. These proteins were not co-expressed, demonstrating their differential expression in newly formed nerve fascicles. Our descriptive and quantitative histological analysis of GAP-43 and NFL allowed us to determine, with high accuracy, the heterogenic population of axons at different stages of maturation in three tissue-engineering approaches. Finally, to perform a complete assessment of axonal regeneration, the quantitative immunohistochemical evaluation of both GAP-43 and NF could be a useful quality control in tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

IBTK Differently Modulates Gene Expression and RNA Splicing in HeLa and K562 Cells

PubMed Central

Fiume, Giuseppe; Scialdone, Annarita; Rizzo, Francesca; De Filippo, Maria Rosaria; Laudanna, Carmelo; Albano, Francesco; Golino, Gaetanina; Vecchio, Eleonora; Pontoriero, Marilena; Mimmi, Selena; Ceglia, Simona; Pisano, Antonio; Iaccino, Enrico; Palmieri, Camillo; Paduano, Sergio; Viglietto, Giuseppe; Weisz, Alessandro; Scala, Giuseppe; Quinto, Ileana

2016-01-01

The IBTK gene encodes the major protein isoform IBTKα that was recently characterized as substrate receptor of Cul3-dependent E3 ligase, regulating ubiquitination coupled to proteasomal degradation of Pdcd4, an inhibitor of translation. Due to the presence of Ankyrin-BTB-RCC1 domains that mediate several protein-protein interactions, IBTKα could exert expanded regulatory roles, including interaction with transcription regulators. To verify the effects of IBTKα on gene expression, we analyzed HeLa and K562 cell transcriptomes by RNA-Sequencing before and after IBTK knock-down by shRNA transduction. In HeLa cells, 1285 (2.03%) of 63,128 mapped transcripts were differentially expressed in IBTK-shRNA-transduced cells, as compared to cells treated with control-shRNA, with 587 upregulated (45.7%) and 698 downregulated (54.3%) RNAs. In K562 cells, 1959 (3.1%) of 63128 mapped RNAs were differentially expressed in IBTK-shRNA-transduced cells, including 1053 upregulated (53.7%) and 906 downregulated (46.3%). Only 137 transcripts (0.22%) were commonly deregulated by IBTK silencing in both HeLa and K562 cells, indicating that most IBTKα effects on gene expression are cell type-specific. Based on gene ontology classification, the genes responsive to IBTK are involved in different biological processes, including in particular chromatin and nucleosomal organization, gene expression regulation, and cellular traffic and migration. In addition, IBTK RNA interference affected RNA maturation in both cell lines, as shown by the evidence of alternative 3′- and 5′-splicing, mutually exclusive exons, retained introns, and skipped exons. Altogether, these results indicate that IBTK differently modulates gene expression and RNA splicing in HeLa and K562 cells, demonstrating a novel biological role of this protein. PMID:27827994

p53 Mediates Vast Gene Expression Changes That Contribute to Poor Chemotherapeutic Response in a Mouse Model of Breast Cancer.

PubMed

Tonnessen-Murray, Crystal; Ungerleider, Nathan A; Rao, Sonia G; Wasylishen, Amanda R; Frey, Wesley D; Jackson, James G

2018-05-28

p53 is a transcription factor that regulates expression of genes involved in cell cycle arrest, senescence, and apoptosis. TP53 harbors mutations that inactivate its transcriptional activity in roughly 30% of breast cancers, and these tumors are much more likely to undergo a pathological complete response to chemotherapy. Thus, the gene expression program activated by wild-type p53 contributes to a poor response. We used an in vivo genetic model system to comprehensively define the p53- and p21-dependent genes and pathways modulated in tumors following doxorubicin treatment. We identified genes differentially expressed in spontaneous mammary tumors harvested from treated MMTV-Wnt1 mice that respond poorly (Trp53+/+) or favorably (Trp53-null) and those that lack the critical senescence/arrest p53 target gene Cdkn1a. Trp53 wild-type tumors differentially expressed nearly 10-fold more genes than Trp53-null tumors after treatment. Pathway analyses showed that genes involved in cell cycle, senescence, and inflammation were enriched in treated Trp53 wild-type tumors; however, no genes/pathways were identified that adequately explain the superior cell death/tumor regression observed in Trp53-null tumors. Cdkn1a-null tumors that retained arrest capacity (responded poorly) and those that proliferated (responded well) after treatment had remarkably different gene regulation. For instance, Cdkn1a-null tumors that arrested upregulated Cdkn2a (p16), suggesting an alternative, p21-independent route to arrest. Live animal imaging of longitudinal gene expression of a senescence/inflammation gene reporter in Trp53+/+ tumors showed induction during and after chemotherapy treatment, while tumors were arrested, but expression rapidly diminished immediately upon relapse. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Genome-wide analysis of alternative splicing during human heart development

NASA Astrophysics Data System (ADS)

Wang, He; Chen, Yanmei; Li, Xinzhong; Chen, Guojun; Zhong, Lintao; Chen, Gangbing; Liao, Yulin; Liao, Wangjun; Bin, Jianping

2016-10-01

Alternative splicing (AS) drives determinative changes during mouse heart development. Recent high-throughput technological advancements have facilitated genome-wide AS, while its analysis in human foetal heart transition to the adult stage has not been reported. Here, we present a high-resolution global analysis of AS transitions between human foetal and adult hearts. RNA-sequencing data showed extensive AS transitions occurred between human foetal and adult hearts, and AS events occurred more frequently in protein-coding genes than in long non-coding RNA (lncRNA). A significant difference of AS patterns was found between foetal and adult hearts. The predicted difference in AS events was further confirmed using quantitative reverse transcription-polymerase chain reaction analysis of human heart samples. Functional foetal-specific AS event analysis showed enrichment associated with cell proliferation-related pathways including cell cycle, whereas adult-specific AS events were associated with protein synthesis. Furthermore, 42.6% of foetal-specific AS events showed significant changes in gene expression levels between foetal and adult hearts. Genes exhibiting both foetal-specific AS and differential expression were highly enriched in cell cycle-associated functions. In conclusion, we provided a genome-wide profiling of AS transitions between foetal and adult hearts and proposed that AS transitions and deferential gene expression may play determinative roles in human heart development.

Epigenetic Basis of Neuronal and Synaptic Plasticity.

PubMed

Karpova, Nina N; Sales, Amanda J; Joca, Samia R

2017-01-01

Neuronal network and plasticity change as a function of experience. Altered neural connectivity leads to distinct transcriptional programs of neuronal plasticity-related genes. The environmental challenges throughout life may promote long-lasting reprogramming of gene expression and the development of brain disorders. The modifications in neuronal epigenome mediate gene-environmental interactions and are required for activity-dependent regulation of neuronal differentiation, maturation and plasticity. Here, we highlight the latest advances in understanding the role of the main players of epigenetic machinery (DNA methylation and demethylation, histone modifications, chromatin-remodeling enzymes, transposons, and non-coding RNAs) in activity-dependent and long- term neural and synaptic plasticity. The review focuses on both the transcriptional and post-transcriptional regulation of gene expression levels, including the processes of promoter activation, alternative splicing, regulation of stability of gene transcripts by natural antisense RNAs, and alternative polyadenylation. Further, we discuss the epigenetic aspects of impaired neuronal plasticity and the pathogenesis of neurodevelopmental (Rett syndrome, Fragile X Syndrome, genomic imprinting disorders, schizophrenia, and others), stressrelated (mood disorders) and neurodegenerative Alzheimer's, Parkinson's and Huntington's disorders. The review also highlights the pharmacological compounds that modulate epigenetic programming of gene expression, the potential treatment strategies of discussed brain disorders, and the questions that should be addressed during the development of effective and safe approaches for the treatment of brain disorders.

The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements

PubMed Central

2012-01-01

Over the last decade, the introduction of microarray technology has had a profound impact on gene expression research. The publication of studies with dissimilar or altogether contradictory results, obtained using different microarray platforms to analyze identical RNA samples, has raised concerns about the reliability of this technology. The MicroArray Quality Control (MAQC) project was initiated to address these concerns, as well as other performance and data analysis issues. Expression data on four titration pools from two distinct reference RNA samples were generated at multiple test sites using a variety of microarray-based and alternative technology platforms. Here we describe the experimental design and probe mapping efforts behind the MAQC project. We show intraplatform consistency across test sites as well as a high level of interplatform concordance in terms of genes identified as differentially expressed. This study provides a resource that represents an important first step toward establishing a framework for the use of microarrays in clinical and regulatory settings. PMID:16964229

FoxK1 splice variants show developmental stage-specific plasticity of expression with temperature in the tiger pufferfish.

PubMed

Fernandes, Jorge M O; MacKenzie, Matthew G; Kinghorn, James R; Johnston, Ian A

2007-10-01

FoxK1 is a member of the highly conserved forkhead/winged helix (Fox) family of transcription factors and it is known to play a key role in mammalian muscle development and myogenic stem cell function. The tiger pufferfish (Takifugu rubripes) orthologue of mammalian FoxK1 (TFoxK1) has seven exons and is located in a region of conserved synteny between pufferfish and mouse. TFoxK1 is expressed as three alternative transcripts: TFoxK1-alpha, TFoxK1-gamma and TFoxK1-delta. TFoxK1-alpha is the orthologue of mouse FoxK1-alpha, coding for a putative protein of 558 residues that contains the forkhead and forkhead-associated domains typical of Fox proteins and shares 53% global identity with its mammalian homologue. TFoxK1-gamma and TFoxK1-delta arise from intron retention events and these transcripts translate into the same 344-amino acid protein with a truncated forkhead domain. Neither are orthologues of mouse FoxK1-beta. In adult fish, the TFoxK1 splice variants were differentially expressed between fast and slow myotomal muscle, as well as other tissues, and the FoxK1-alpha protein was expressed in myogenic progenitor cells of fast myotomal muscle. During embryonic development, TFoxK1 was transiently expressed in the developing somites, heart, brain and eye. The relative expression of TFoxK1-alpha and the other two alternative transcripts varied with the incubation temperature regime for equivalent embryonic stages and the differences were particularly marked at later developmental stages. The developmental expression pattern of TFoxK1 and its localisation to mononuclear myogenic progenitor cells in adult fast muscle indicate that it may play an essential role in myogenesis in T. rubripes.

Comparative transcriptomics of 5 high-altitude vertebrates and their low-altitude relatives.

PubMed

Tang, Qianzi; Gu, Yiren; Zhou, Xuming; Jin, Long; Guan, Jiuqiang; Liu, Rui; Li, Jing; Long, Kereng; Tian, Shilin; Che, Tiandong; Hu, Silu; Liang, Yan; Yang, Xuemei; Tao, Xuan; Zhong, Zhijun; Wang, Guosong; Chen, Xiaohui; Li, Diyan; Ma, Jideng; Wang, Xun; Mai, Miaomiao; Jiang, An'an; Luo, Xiaolin; Lv, Xuebin; Gladyshev, Vadim N; Li, Xuewei; Li, Mingzhou

2017-12-01

Species living at high altitude are subject to strong selective pressures due to inhospitable environments (e.g., hypoxia, low temperature, high solar radiation, and lack of biological production), making these species valuable models for comparative analyses of local adaptation. Studies that have examined high-altitude adaptation have identified a vast array of rapidly evolving genes that characterize the dramatic phenotypic changes in high-altitude animals. However, how high-altitude environment shapes gene expression programs remains largely unknown. We generated a total of 910 Gb of high-quality RNA-seq data for 180 samples derived from 6 tissues of 5 agriculturally important high-altitude vertebrates (Tibetan chicken, Tibetan pig, Tibetan sheep, Tibetan goat, and yak) and their cross-fertile relatives living in geographically neighboring low-altitude regions. Of these, ∼75% reads could be aligned to their respective reference genomes, and on average ∼60% of annotated protein coding genes in each organism showed FPKM expression values greater than 0.5. We observed a general concordance in topological relationships between the nucleotide alignments and gene expression-based trees. Tissue and species accounted for markedly more variance than altitude based on either the expression or the alternative splicing patterns. Cross-species clustering analyses showed a tissue-dominated pattern of gene expression and a species-dominated pattern for alternative splicing. We also identified numerous differentially expressed genes that could potentially be involved in phenotypic divergence shaped by high-altitude adaptation. These data serve as a valuable resource for examining the convergence and divergence of gene expression changes between species as they adapt or acclimatize to high-altitude environments. © The Authors 2017. Published by Oxford University Press.

A differential neural response to threatening and non-threatening negative facial expressions in paranoid and non-paranoid schizophrenics.

PubMed

Phillips, M L; Williams, L; Senior, C; Bullmore, E T; Brammer, M J; Andrew, C; Williams, S C; David, A S

1999-11-08

Several studies have demonstrated impaired facial expression recognition in schizophrenia. Few have examined the neural basis for this; none have compared the neural correlates of facial expression perception in different schizophrenic patient subgroups. We compared neural responses to facial expressions in 10 right-handed schizophrenic patients (five paranoid and five non-paranoid) and five normal volunteers using functional Magnetic Resonance Imaging (fMRI). In three 5-min experiments, subjects viewed alternating 30-s blocks of black-and-white facial expressions of either fear, anger or disgust contrasted with expressions of mild happiness. After scanning, subjects categorised each expression. All patients were less accurate in identifying expressions, and showed less activation to these stimuli than normals. Non-paranoids performed poorly in the identification task and failed to activate neural regions that are normally linked with perception of these stimuli. They categorised disgust as either anger or fear more frequently than paranoids, and demonstrated in response to disgust expressions activation in the amygdala, a region associated with perception of fearful faces. Paranoids were more accurate in recognising expressions, and demonstrated greater activation than non-paranoids to most stimuli. We provide the first evidence for a distinction between two schizophrenic patient subgroups on the basis of recognition of and neural response to different negative facial expressions.

Term amniotic fluid: an unexploited reserve of mesenchymal stromal cells for reprogramming and potential cell therapy applications.

PubMed

Moraghebi, Roksana; Kirkeby, Agnete; Chaves, Patricia; Rönn, Roger E; Sitnicka, Ewa; Parmar, Malin; Larsson, Marcus; Herbst, Andreas; Woods, Niels-Bjarne

2017-08-25

Mesenchymal stromal cells (MSCs) are currently being evaluated in numerous pre-clinical and clinical cell-based therapy studies. Furthermore, there is an increasing interest in exploring alternative uses of these cells in disease modelling, pharmaceutical screening, and regenerative medicine by applying reprogramming technologies. However, the limited availability of MSCs from various sources restricts their use. Term amniotic fluid has been proposed as an alternative source of MSCs. Previously, only low volumes of term fluid and its cellular constituents have been collected, and current knowledge of the MSCs derived from this fluid is limited. In this study, we collected amniotic fluid at term using a novel collection system and evaluated amniotic fluid MSC content and their characteristics, including their feasibility to undergo cellular reprogramming. Amniotic fluid was collected at term caesarean section deliveries using a closed catheter-based system. Following fluid processing, amniotic fluid was assessed for cellularity, MSC frequency, in-vitro proliferation, surface phenotype, differentiation, and gene expression characteristics. Cells were also reprogrammed to the pluripotent stem cell state and differentiated towards neural and haematopoietic lineages. The average volume of term amniotic fluid collected was approximately 0.4 litres per donor, containing an average of 7 million viable mononuclear cells per litre, and a CFU-F content of 15 per 100,000 MNCs. Expanded CFU-F cultures showed similar surface phenotype, differentiation potential, and gene expression characteristics to MSCs isolated from traditional sources, and showed extensive expansion potential and rapid doubling times. Given the high proliferation rates of these neonatal source cells, we assessed them in a reprogramming application, where the derived induced pluripotent stem cells showed multigerm layer lineage differentiation potential. The potentially large donor base from caesarean section deliveries, the high yield of term amniotic fluid MSCs obtainable, the properties of the MSCs identified, and the suitability of the cells to be reprogrammed into the pluripotent state demonstrated these cells to be a promising and plentiful resource for further evaluation in bio-banking, cell therapy, disease modelling, and regenerative medicine applications.

MicroRNA-29a ameliorates glucocorticoid-induced suppression of osteoblast differentiation by regulating β-catenin acetylation.

PubMed

Ko, Jih-Yang; Chuang, Pei-Chin; Chen, Ming-Wen; Ke, Huei-Ching; Wu, Shin-Long; Chang, Yu-Hsuan; Chen, Yu-Shan; Wang, Feng-Sheng

2013-12-01

Excess glucocorticoid treatment induces loss of osteoblast differentiation. Post-translational modification of β-catenin reportedly regulates osteogenic activities in bone cells. This study was undertaken to test whether miR-29a signaling regulates the acetylation status of β-catenin in the glucocorticoid-mediated osteoblast dysfunction. Murine osteoblast cultures were incubated under osteogenic conditions with or without supraphysiological glucocorticoid, miR-29a precursor, antisense oligonucleotides or histone deacetylase 4 (HDAC4) RNA interferences. Osteoblast differentiation was determined by alkaline phosphatase activity, calcium deposition, and von Kossa stain. β-Catenin acetylation and miR-29a transcription were detected by immunoblotting, chromatin immunoprecipitation and quantitative PCR. Protein interaction was detected by fluorescence protein ligation assay. Supraphysiological glucocorticoid treatment repressed osteoblast differentiation and induced loss of miR-29a expression and acetylated β-catenin levels in osteoblast cultures. Gain of miR-29a function attenuated the deleterious effects of glucocorticoid on osteogenic gene expression and mineralized nodule formation, whereas knockdown of miR-29a signaling accelerated loss of osteoblast differentiation capacity. miR-29a reduced HDAC4 signaling and attenuated the glucocorticoid-mediated β-catenin deacetylation and ubiquitination and restored nuclear β-catenin levels. Glucocorticoid-induced loss of miR-29a signaling occurred through transcriptional and translational regulation. Interruption of HDAC4 signaling attenuated the glucocorticoid-induced hypoacetylation of histone H3 at lysine 9 (H3K9Ac) and restored the enrichment of H3K9Ac in miR-29a proximal promoter region and miR-29a transcription in cell cultures. Taken together, excess glucocorticoid-induced loss of miR-29a signaling accelerates β-catenin deacetylation and ubiquitination that impairs osteogenic activities of osteoblast cultures. miR-29a and HDAC4 reciprocal regulation of H3K9 acetylation contributes to the acetylation status of β-catenin and miR-29a expression. Enhancement of miR-29a signaling is an alternative strategy for protecting against the adverse actions of excess glucocorticoid on differentiation capacity of osteogenic cells. © 2013.

Mouse androgenetic embryonic stem cells differentiated to multiple cell lineages in three embryonic germ layers in vitro.

PubMed

Teramura, Takeshi; Onodera, Yuta; Murakami, Hideki; Ito, Syunsuke; Mihara, Toshihiro; Takehara, Toshiyuki; Kato, Hiromi; Mitani, Tasuku; Anzai, Masayuki; Matsumoto, Kazuya; Saeki, Kazuhiro; Fukuda, Kanji; Sagawa, Norimasa; Osoi, Yoshihiko

2009-06-01

The embryos of some rodents and primates can precede early development without the process of fertilization; however, they cease to develop after implantation because of restricted expressions of imprinting genes. Asexually developed embryos are classified into parthenote/gynogenote and androgenote by their genomic origins. Embryonic stem cells (ESCs) derived from asexual origins have also been reported. To date, ESCs derived from parthenogenetic embryos (PgESCs) have been established in some species, including humans, and the possibility to be alternative sources for autologous cell transplantation in regenerative medicine has been proposed. However, some developmental characteristics, which might be important for therapeutic applications, such as multiple differentiation capacity and transplantability of the ESCs of androgenetic origin (AgESCs) are uncertain. Here, we induced differentiation of mouse AgESCs and observed derivation of neural cells, cardiomyocytes and hepatocytes in vitro. Following differentiated embryoid body (EB) transplantation in various mouse strains including the strain of origin, we found that the EBs could engraft in theoretically MHC-matched strains. Our results indicate that AgESCs possess at least two important characteristics, multiple differentiation properties in vitro and transplantability after differentiation, and suggest that they can also serve as a source of histocompatible tissues for transplantation.

iTRAQ-Based Quantitative Proteomics Analysis of Black Rice Grain Development Reveals Metabolic Pathways Associated with Anthocyanin Biosynthesis.

PubMed

Chen, Linghua; Huang, Yining; Xu, Ming; Cheng, Zuxin; Zhang, Dasheng; Zheng, Jingui

2016-01-01

Black rice (Oryza sativa L.), whose pericarp is rich in anthocyanins (ACNs), is considered as a healthier alternative to white rice. Molecular species of ACNs in black rice have been well documented in previous studies; however, information about the metabolic mechanisms underlying ACN biosynthesis during black rice grain development is unclear. The aim of the present study was to determine changes in the metabolic pathways that are involved in the dynamic grain proteome during the development of black rice indica cultivar, (Oryza sativa L. indica var. SSP). Isobaric tags for relative and absolute quantification (iTRAQ) MS/MS were employed to identify statistically significant alterations in the grain proteome. Approximately 928 proteins were detected, of which 230 were differentially expressed throughout 5 successive developmental stages, starting from 3 to 20 days after flowering (DAF). The greatest number of differentially expressed proteins was observed on 7 and 10 DAF, including 76 proteins that were upregulated and 39 that were downregulated. The biological process analysis of gene ontology revealed that the 230 differentially expressed proteins could be sorted into 14 functional groups. Proteins in the largest group were related to metabolic process, which could be integrated into multiple biochemical pathways. Specifically, proteins with a role in ACN biosynthesis, sugar synthesis, and the regulation of gene expression were upregulated, particularly from the onset of black rice grain development and during development. In contrast, the expression of proteins related to signal transduction, redox homeostasis, photosynthesis and N-metabolism decreased during grain maturation. Finally, 8 representative genes encoding different metabolic proteins were verified via quantitative real-time polymerase chain reaction (qRT-PCR) analysis, these genes had differed in transcriptional and translational expression during grain development. Expression analyses of metabolism-related protein groups belonging to different functional categories and subcategories indicated that significantly upregulated proteins were related to flavonoid and starch synthesis. On the other hand, the downregulated proteins were determined to be related to nitrogen metabolism, as well as other functional categories and subcategories, including photosynthesis, redox homeostasis, tocopherol biosynthetic, and signal transduction. The results provide valuable new insights into the characterization and understanding of ACN pigment production in black rice.

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

PubMed

Morsczeck, C

2006-02-01

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

Clinical Application of Pluripotent Stem Cells: An Alternative Cell-Based Therapy for Treating Liver Diseases?

PubMed

Tolosa, Laia; Pareja, Eugenia; Gómez-Lechón, Maria José

2016-12-01

The worldwide shortage of donor livers for organ and hepatocyte transplantation has prompted the search for alternative therapies for intractable liver diseases. Cell-based therapy is envisaged as a useful therapeutic option to recover and stabilize the lost metabolic function for acute liver failure, end-stage and congenital liver diseases, or for those patients who are not considered eligible for organ transplantation. In recent years, research to identify alternative and reliable cell sources for transplantation that can be derived by reproducible methods has been encouraged. Human pluripotent stem cells (PSCs), which comprise both embryonic and induced PSCs, may offer many advantages as an alternative to hepatocytes for liver cell therapy. Their capacity for expansion, hepatic differentiation and self-renewal make them a promising source of unlimited numbers of hepatocyte-like cells for treating and repairing damaged livers. Immunogenicity and tumorigenicity of human PSCs remain the bottleneck for successful clinical application. However, recent advances made to develop disease-corrected hepatocyte-like cells from patients' human-induced PSCs by gene editing have opened up many potential gateways for the autologous treatment of hereditary liver diseases, which may likely reduce the risk of rejection and the need for lifelong immunosuppression. Well-defined methods to reduce the expression of oncogenic genes in induced PSCs, including protocols for their complete and safe hepatic differentiation, should be established to minimize the tumorigenicity of transplanted cells. On top of this, such new strategies are currently being rigorously tested and validated in preclinical studies before they can be safely transferred to clinical practice with patients.

Thousands of exon skipping events differentiate among splicing patterns in sixteen human tissues.

PubMed

Florea, Liliana; Song, Li; Salzberg, Steven L

2013-01-01

Alternative splicing is widely recognized for its roles in regulating genes and creating gene diversity. However, despite many efforts, the repertoire of gene splicing variation is still incompletely characterized, even in humans. Here we describe a new computational system, ASprofile, and its application to RNA-seq data from Illumina's Human Body Map project (>2.5 billion reads).  Using the system, we identified putative alternative splicing events in 16 different human tissues, which provide a dynamic picture of splicing variation across the tissues. We detected 26,989 potential exon skipping events representing differences in splicing patterns among the tissues. A large proportion of the events (>60%) were novel, involving new exons (~3000), new introns (~16000), or both. When tracing these events across the sixteen tissues, only a small number (4-7%) appeared to be differentially expressed ('switched') between two tissues, while 30-45% showed little variation, and the remaining 50-65% were not present in one or both tissues compared.  Novel exon skipping events appeared to be slightly less variable than known events, but were more tissue-specific. Our study represents the first effort to build a comprehensive catalog of alternative splicing in normal human tissues from RNA-seq data, while providing insights into the role of alternative splicing in shaping tissue transcriptome differences. The catalog of events and the ASprofile software are freely available from the Zenodo repository ( http://zenodo.org/record/7068; doi: 10.5281/zenodo.7068) and from our web site http://ccb.jhu.edu/software/ASprofile.

Pluripotency, Differentiation, and Reprogramming: A Gene Expression Dynamics Model with Epigenetic Feedback Regulation

PubMed Central

Miyamoto, Tadashi; Furusawa, Chikara; Kaneko, Kunihiko

2015-01-01

Embryonic stem cells exhibit pluripotency: they can differentiate into all types of somatic cells. Pluripotent genes such as Oct4 and Nanog are activated in the pluripotent state, and their expression decreases during cell differentiation. Inversely, expression of differentiation genes such as Gata6 and Gata4 is promoted during differentiation. The gene regulatory network controlling the expression of these genes has been described, and slower-scale epigenetic modifications have been uncovered. Although the differentiation of pluripotent stem cells is normally irreversible, reprogramming of cells can be experimentally manipulated to regain pluripotency via overexpression of certain genes. Despite these experimental advances, the dynamics and mechanisms of differentiation and reprogramming are not yet fully understood. Based on recent experimental findings, we constructed a simple gene regulatory network including pluripotent and differentiation genes, and we demonstrated the existence of pluripotent and differentiated states from the resultant dynamical-systems model. Two differentiation mechanisms, interaction-induced switching from an expression oscillatory state and noise-assisted transition between bistable stationary states, were tested in the model. The former was found to be relevant to the differentiation process. We also introduced variables representing epigenetic modifications, which controlled the threshold for gene expression. By assuming positive feedback between expression levels and the epigenetic variables, we observed differentiation in expression dynamics. Additionally, with numerical reprogramming experiments for differentiated cells, we showed that pluripotency was recovered in cells by imposing overexpression of two pluripotent genes and external factors to control expression of differentiation genes. Interestingly, these factors were consistent with the four Yamanaka factors, Oct4, Sox2, Klf4, and Myc, which were necessary for the establishment of induced pluripotent stem cells. These results, based on a gene regulatory network and expression dynamics, contribute to our wider understanding of pluripotency, differentiation, and reprogramming of cells, and they provide a fresh viewpoint on robustness and control during development. PMID:26308610

Differential 3’ processing of specific transcripts expands regulatory and protein diversity across neuronal cell types

PubMed Central

Jereb, Saša; Hwang, Hun-Way; Van Otterloo, Eric; Govek, Eve-Ellen; Fak, John J; Yuan, Yuan; Hatten, Mary E

2018-01-01

Alternative polyadenylation (APA) regulates mRNA translation, stability, and protein localization. However, it is unclear to what extent APA regulates these processes uniquely in specific cell types. Using a new technique, cTag-PAPERCLIP, we discovered significant differences in APA between the principal types of mouse cerebellar neurons, the Purkinje and granule cells, as well as between proliferating and differentiated granule cells. Transcripts that differed in APA in these comparisons were enriched in key neuronal functions and many differed in coding sequence in addition to 3’UTR length. We characterize Memo1, a transcript that shifted from expressing a short 3’UTR isoform to a longer one during granule cell differentiation. We show that Memo1 regulates granule cell precursor proliferation and that its long 3’UTR isoform is targeted by miR-124, contributing to its downregulation during development. Our findings provide insight into roles for APA in specific cell types and establish a platform for further functional studies. PMID:29578408

Methacrylated gelatin/hyaluronan-based hydrogels for soft tissue engineering

PubMed Central

Kessler, Lukas; Gehrke, Sandra; Winnefeld, Marc; Huber, Birgit; Hoch, Eva; Walter, Torsten; Wyrwa, Ralf; Schnabelrauch, Matthias; Schmidt, Malte; Kückelhaus, Maximilian; Lehnhardt, Marcus; Hirsch, Tobias; Jacobsen, Frank

2017-01-01

In vitro–generated soft tissue could provide alternate therapies for soft tissue defects. The aim of this study was to evaluate methacrylated gelatin/hyaluronan as scaffolds for soft tissue engineering and their interaction with human adipose–derived stem cells (hASCs). ASCs were incorporated into methacrylated gelatin/hyaluronan hydrogels. The gels were photocrosslinked with a lithium phenyl-2,4,6-trimethylbenzoylphosphinate photoinitiator and analyzed for cell viability and adipogenic differentiation of ASCs over a period of 30 days. Additionally, an angiogenesis assay was performed to assess their angiogenic potential. After 24 h, ASCs showed increased viability on composite hydrogels. These results were consistent over 21 days of culture. By induction of adipogenic differentiation, the mature adipocytes were observed after 7 days of culture, their number significantly increased until day 28 as well as expression of fatty acid binding protein 4 and adiponectin. Our scaffolds are promising as building blocks for adipose tissue engineering and allowed long viability, proliferation, and differentiation of ASCs. PMID:29318000

Selective Modulators of PPAR-γ Activity: Molecular Aspects Related to Obesity and Side-Effects

PubMed Central

Zhang, Fang; Lavan, Brian E.; Gregoire, Francine M.

2007-01-01

Peroxisome proliferator-activated receptor γ (PPAR-γ) is a key regulator of lipid metabolism and energy balance implicated in the development of insulin resistance and obesity. The identification of putative natural and synthetic ligands and activators of PPAR-γ has helped to unravel the molecular basis of its function, including molecular details regarding ligand binding, conformational changes of the receptor, and cofactor binding, leading to the emergence of the concept of selective PPAR-γ modulators (SPPARγMs). SPPARγMs bind in distinct manners to the ligand-binding pocket of PPAR-γ, leading to alternative receptor conformations, differential cofactor recruitment/displacement, differential gene expression, and ultimately differential biological responses. Based on this concept, new and improved antidiabetic agents for the treatment of diabetes are in development. This review summarizes the current knowledge on the mechanism of action and biological effects of recently characterized SPPARγMs, including metaglidasen/halofenate, PA-082, and the angiotensin receptor antagonists, recently characterized as a new class of SPPARγMs. PMID:17389769

Amino-termini isoforms of the Slack K+ channel, regulated by alternative promoters, differentially modulate rhythmic firing and adaptation.

PubMed

Brown, Maile R; Kronengold, Jack; Gazula, Valeswara-Rao; Spilianakis, Charalampos G; Flavell, Richard A; von Hehn, Christian A A; Bhattacharjee, Arin; Kaczmarek, Leonard K

2008-11-01

The rates of activation and unitary properties of Na+-activated K+ (K(Na)) currents have been found to vary substantially in different types of neurones. One class of K(Na) channels is encoded by the Slack gene. We have now determined that alternative RNA splicing gives rise to at least five different transcripts for Slack, which produce Slack channels that differ in their predicted cytoplasmic amino-termini and in their kinetic properties. Two of these, termed Slack-A channels, contain an amino-terminus domain closely resembling that of another class of K(Na) channels encoded by the Slick gene. Neuronal expression of Slack-A channels and of the previously described Slack isoform, now called Slack-B, are driven by independent promoters. Slack-A mRNAs were enriched in the brainstem and olfactory bulb and detected at significant levels in four different brain regions. When expressed in CHO cells, Slack-A channels activate rapidly upon depolarization and, in single channel recordings in Xenopus oocytes, are characterized by multiple subconductance states with only brief transient openings to the fully open state. In contrast, Slack-B channels activate slowly over hundreds of milliseconds, with openings to the fully open state that are approximately 6-fold longer than those for Slack-A channels. In numerical simulations, neurones in which outward currents are dominated by a Slack-A-like conductance adapt very rapidly to repeated or maintained stimulation over a wide range of stimulus strengths. In contrast, Slack-B currents promote rhythmic firing during maintained stimulation, and allow adaptation rate to vary with stimulus strength. Using an antibody that recognizes all amino-termini isoforms of Slack, Slack immunoreactivity is present at locations that have no Slack-B-specific staining, including olfactory bulb glomeruli and the dendrites of hippocampal neurones, suggesting that Slack channels with alternate amino-termini such as Slack-A channels are present at these locations. Our data suggest that alternative promoters of the Slack gene differentially modulate the properties of neurones.

Altered expression of CD45 isoforms in differentiation of acute myeloid leukemia.

PubMed

Miyachi, H; Tanaka, Y; Gondo, K; Kawada, T; Kato, S; Sasao, T; Hotta, T; Oshima, S; Ando, Y

1999-11-01

Specific expression of different CD45 isoforms can be seen in various stages of differentiation of normal nucleated hematopoietic cells. Association of membrane expression of CD45 isoforms and differential levels of leukemia cells was studied in 91 cases with de novo acute myeloid leukemia (AML). Membrane expression of CD45RA and CD45RO was analyzed by flow cytometry and their expression patterns were compared with AML subtypes classified according to the French-American-British (FAB) classification. CD45RA was essentially expressed in all of the FAB myelocytic subtypes (M0-M3). Its expression in percentage was lower in the most differentiated subtype of AML (M3) when compared with other myelocytic subtypes. CD45RO expression was rarely observed in cases with myelocytic subtypes (1/56 cases of M0, M1, M2, and M3) except for the minimally differentiated myelocytic subtype (M0) or those with potential for differentiation to T-cell lineage where three of 12 cases showed CD45RO expression. When leukemia cells of an M3 case were differentiated to mature granulocytes by treatment of all-trans-retinoic acid, they showed increasing expression of CD45RO. In subtypes with a monocytic component (M4 and M5), both of CD45RA and CD45RO expression were observed and mutually exclusive. When 10 cases of M5 were subdivided by the differential level into undifferentiated (M5a) and differentiated monocytic leukemia (M5b), expression of CD45RA and CD45RO was strictly restricted to cases with M5a and M5b, respectively. These results suggest that CD45 isoform expression in AML characterizes differential levels both in myelocytic and monocytic lineages and specifically disturbed in each subtype. The assessment of CD45 isoform expression appears to provide an insight on biological characteristics and a useful supplementary test for differential diagnosis of AML subtypes. Copyright 1999 Wiley-Liss, Inc.

PRAPI: post-transcriptional regulation analysis pipeline for Iso-Seq.

PubMed

Gao, Yubang; Wang, Huiyuan; Zhang, Hangxiao; Wang, Yongsheng; Chen, Jinfeng; Gu, Lianfeng

2018-05-01

The single-molecule real-time (SMRT) isoform sequencing (Iso-Seq) based on Pacific Bioscience (PacBio) platform has received increasing attention for its ability to explore full-length isoforms. Thus, comprehensive tools for Iso-Seq bioinformatics analysis are extremely useful. Here, we present a one-stop solution for Iso-Seq analysis, called PRAPI to analyze alternative transcription initiation (ATI), alternative splicing (AS), alternative cleavage and polyadenylation (APA), natural antisense transcripts (NAT), and circular RNAs (circRNAs) comprehensively. PRAPI is capable of combining Iso-Seq full-length isoforms with short read data, such as RNA-Seq or polyadenylation site sequencing (PAS-seq) for differential expression analysis of NAT, AS, APA and circRNAs. Furthermore, PRAPI can annotate new genes and correct mis-annotated genes when gene annotation is available. Finally, PRAPI generates high-quality vector graphics to visualize and highlight the Iso-Seq results. The Dockerfile of PRAPI is available at http://www.bioinfor.org/tool/PRAPI. lfgu@fafu.edu.cn.

Proteomic Profiling for Identification of Novel Biomarkers Differentially Expressed in Human Ovaries from Polycystic Ovary Syndrome Patients

PubMed Central

Li, Li; Zhang, Jiangyu; Deng, Qingshan; Li, Jieming; Li, Zhengfen; Xiao, Yao; Hu, Shuiwang; Li, Tiantian; Tan, Qiuxiao; Li, Xiaofang; Luo, Bingshu; Mo, Hui

2016-01-01

Objectives To identify differential protein expression pattern associated with polycystic ovary syndrome (PCOS). Methods Twenty women were recruited for the study, ten with PCOS as a test group and ten without PCOS as a control group. Differential in-gel electrophoresis (DIGE) analysis and mass spectroscopy were employed to identify proteins that were differentially expressed between the PCOS and normal ovaries. The differentially expressed proteins were further validated by western blot (WB) and immunohistochemistry (IHC). Results DIGE analysis revealed eighteen differentially expressed proteins in the PCOS ovaries of which thirteen were upregulated, and five downregulated. WB and IHC confirmed the differential expression of membrane-associated progesterone receptor component 1 (PGRMC1), retinol-binding protein 1 (RBP1), heat shock protein 90B1, calmodulin 1, annexin A6, and tropomyosin 2. Also, WB analysis revealed significantly (P<0.05) higher expression of PGRMC1 and RBP1 in PCOS ovaries as compared to the normal ovaries. The differential expression of the proteins was also validated by IHC. Conclusions The present study identified novel differentially expressed proteins in the ovarian tissues of women with PCOS that can serve as potential biomarkers for the diagnosis and development of novel therapeutics for the treatment of PCOS using molecular interventions. PMID:27846214

Proteomic Profiling for Identification of Novel Biomarkers Differentially Expressed in Human Ovaries from Polycystic Ovary Syndrome Patients.

PubMed

Li, Li; Zhang, Jiangyu; Deng, Qingshan; Li, Jieming; Li, Zhengfen; Xiao, Yao; Hu, Shuiwang; Li, Tiantian; Tan, Qiuxiao; Li, Xiaofang; Luo, Bingshu; Mo, Hui

2016-01-01

To identify differential protein expression pattern associated with polycystic ovary syndrome (PCOS). Twenty women were recruited for the study, ten with PCOS as a test group and ten without PCOS as a control group. Differential in-gel electrophoresis (DIGE) analysis and mass spectroscopy were employed to identify proteins that were differentially expressed between the PCOS and normal ovaries. The differentially expressed proteins were further validated by western blot (WB) and immunohistochemistry (IHC). DIGE analysis revealed eighteen differentially expressed proteins in the PCOS ovaries of which thirteen were upregulated, and five downregulated. WB and IHC confirmed the differential expression of membrane-associated progesterone receptor component 1 (PGRMC1), retinol-binding protein 1 (RBP1), heat shock protein 90B1, calmodulin 1, annexin A6, and tropomyosin 2. Also, WB analysis revealed significantly (P<0.05) higher expression of PGRMC1 and RBP1 in PCOS ovaries as compared to the normal ovaries. The differential expression of the proteins was also validated by IHC. The present study identified novel differentially expressed proteins in the ovarian tissues of women with PCOS that can serve as potential biomarkers for the diagnosis and development of novel therapeutics for the treatment of PCOS using molecular interventions.

Generation of enhanced definitive endoderm from human embryonic stem cells under an albumin/insulin-free and chemically defined condition.

PubMed

Qu, Su; Yan, Liang; Fang, Bo; Ye, Shoudong; Li, Ping; Ge, Shengyang; Wu, Jian; Qu, Di; Song, Houyan

2017-04-15

To enhance survival and generation of definitive endoderm cells from human embryonic stem cells in a simple and reproducible system. Definitive endoderm (DE) differentiation from human embryonic stem cells (hESCs) was induced under a chemical-defined condition withdrawn insulin supplement and serum albumin. We dissected influence of "alternative growth factors", WNT3A, BMP4 and bFGF in activin A-driven differentiation by detection of DE-associated genes expression and cell viability. Expression of DE-associated SOX17 and FOXA2 genes was analyzed by real time reverse transcription polymerase chain reaction (RT-PCR) and Western blot assays. Quantitative evaluation of DE efficiency was performed by flow cytometry analysis of CXCR4-expressed cell population. Cell viability during DE differentiation was analyzed by an Annexin V/PI double staining test. Supplementation with WNT3A, BMP4 or bFGF promoted DE generation in a dose- and time-dependent manner. Cell apoptosis elicited by activin A was significantly ameliorated by a cocktail with WNT3A, BMP4 and bFGF. This allowed for sustained cell viability without insulin-containing supplements, thereby indirectly improving the efficiency of DE generation. Therefore, the cocktail containing is optimal for efficient DE generation in the presence of activin A and an insulin/albumin-free condition. This optimal condition facilitates the balance between the productivity and the viability maintenance, and could be valuable for mass production of DE with minimal variation. Copyright © 2017 Elsevier Inc. All rights reserved.

Graphene oxide enrichment of collagen membranes improves DPSCs differentiation and controls inflammation occurrence.

PubMed

Radunovic, Milena; De Colli, Marianna; De Marco, Patrizia; Di Nisio, Chiara; Fontana, Antonella; Piattelli, Adriano; Cataldi, Amelia; Zara, Susi

2017-08-01

Collagen membranes are used in oral surgery for bone defects treatment acting as a barrier that does not allow the invasion of soft tissue into the growing bone. To improve biocompatibility collagen membranes were coated with graphene oxide (GO), a graphene derivative. The aim of this study was to investigate the biocompatibility of GO coated collagen membranes on human dental pulp stem cells (DPSCs) focusing on biomaterial cytotoxicity, ability to promote DPSCs differentiation process and to control inflammation event induction. DPSCs were cultured on uncoated membranes and on both 2 and 10 μg mL -1 GO coated membranes up to 28 days. Alamar blue and LDH cytotocicity assay, PGE2 ELISA assay, real time RT-PCR for RUNX2, BMP2, SP7, TNFα and COX2 genes expression were performed. Proliferation is higher on GO coated membranes at days 14 and 28. LDH assay evidences no cytotoxicity. BMP2 and RUNX2 expression is higher on coated membranes, BMP2 at early and RUNX2 and SP7 at late experimental times. PGE2 levels are lower on GO coated membranes at days 14 and 28, both TNFα and COX2 expression is significantly decreased when GO is applied. GO coated membranes are not toxic for DPSCs, induce a faster DPSCs differentiation into odontoblasts/osteoblasts and may represent good alternative to conventional membranes thus ensuring more efficient bone formation and improving the clinical performance. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2312-2320, 2017. © 2017 Wiley Periodicals, Inc.

Gene expression and splicing alterations analyzed by high throughput RNA sequencing of chronic lymphocytic leukemia specimens.

PubMed

Liao, Wei; Jordaan, Gwen; Nham, Phillipp; Phan, Ryan T; Pelegrini, Matteo; Sharma, Sanjai

2015-10-16

To determine differentially expressed and spliced RNA transcripts in chronic lymphocytic leukemia specimens a high throughput RNA-sequencing (HTS RNA-seq) analysis was performed. Ten CLL specimens and five normal peripheral blood CD19+ B cells were analyzed by HTS RNA-seq. The library preparation was performed with Illumina TrueSeq RNA kit and analyzed by Illumina HiSeq 2000 sequencing system. An average of 48.5 million reads for B cells, and 50.6 million reads for CLL specimens were obtained with 10396 and 10448 assembled transcripts for normal B cells and primary CLL specimens respectively. With the Cuffdiff analysis, 2091 differentially expressed genes (DEG) between B cells and CLL specimens based on FPKM (fragments per kilobase of transcript per million reads and false discovery rate, FDR q < 0.05, fold change >2) were identified. Expression of selected DEGs (n = 32) with up regulated and down regulated expression in CLL from RNA-seq data were also analyzed by qRT-PCR in a test cohort of CLL specimens. Even though there was a variation in fold expression of DEG genes between RNA-seq and qRT-PCR; more than 90 % of analyzed genes were validated by qRT-PCR analysis. Analysis of RNA-seq data for splicing alterations in CLL and B cells was performed by Multivariate Analysis of Transcript Splicing (MATS analysis). Skipped exon was the most frequent splicing alteration in CLL specimens with 128 significant events (P-value <0.05, minimum inclusion level difference >0.1). The RNA-seq analysis of CLL specimens identifies novel DEG and alternatively spliced genes that are potential prognostic markers and therapeutic targets. High level of validation by qRT-PCR for a number of DEG genes supports the accuracy of this analysis. Global comparison of transcriptomes of B cells, IGVH non-mutated CLL (U-CLL) and mutated CLL specimens (M-CLL) with multidimensional scaling analysis was able to segregate CLL and B cell transcriptomes but the M-CLL and U-CLL transcriptomes were indistinguishable. The analysis of HTS RNA-seq data to identify alternative splicing events and other genetic abnormalities specific to CLL is an added advantage of RNA-seq that is not feasible with other genome wide analysis.

Stem cells as a good tool to investigate dysregulated biological systems in autism spectrum disorders.

PubMed

Griesi-Oliveira, Karina; Sunaga, Daniele Yumi; Alvizi, Lucas; Vadasz, Estevão; Passos-Bueno, Maria Rita

2013-10-01

Identification of the causes of autism spectrum disorders (ASDs) is hampered by their genetic heterogeneity; however, the different genetic alterations leading to ASD seem to be implicated in the disturbance of common molecular pathways or biological processes. In this scenario, the search for differentially expressed genes (DEGs) between ASD patients and controls is a good alternative to identify the molecular etiology of such disorders. Here, we employed genome-wide expression analysis to compare the transcriptome of stem cells of human exfoliated deciduous teeth (SHEDs) of idiopathic autistic patients (n = 7) and control samples (n = 6). Nearly half of the 683 identified DEGs are expressed in the brain (P = 0.003), and a significant number of them are involved in mechanisms previously associated with ASD such as protein synthesis, cytoskeleton regulation, cellular adhesion and alternative splicing, which validate the use of SHEDs to disentangle the causes of autism. Autistic patients also presented overexpression of genes regulated by androgen receptor (AR), and AR itself, which in turn interacts with CHD8 (chromodomain helicase DNA binding protein 8), a gene recently shown to be associated with the cause of autism and found to be upregulated in some patients tested here. These data provide a rationale for the mechanisms through which CHD8 leads to these diseases. In summary, our results suggest that ASD share deregulated pathways and revealed that SHEDs represent an alternative cell source to be used in the understanding of the biological mechanisms involved in the etiology of ASD. © 2013 International Society for Autism Research, Wiley Periodicals, Inc.

Alternative splicing regulated by butyrate in bovine epithelial cells.

PubMed

Wu, Sitao; Li, Congjun; Huang, Wen; Li, Weizhong; Li, Robert W

2012-01-01

As a signaling molecule and an inhibitor of histone deacetylases (HDACs), butyrate exerts its impact on a broad range of biological processes, such as apoptosis and cell proliferation, in addition to its critical role in energy metabolism in ruminants. This study examined the effect of butyrate on alternative splicing in bovine epithelial cells using RNA-seq technology. Junction reads account for 11.28 and 12.32% of total mapped reads between the butyrate-treated (BT) and control (CT) groups. 201,326 potential splicing junctions detected were supported by ≥ 3 junction reads. Approximately 94% of these junctions conformed to the consensus sequence (GT/AG) while ~3% were GC/AG junctions. No AT/AC junctions were observed. A total of 2,834 exon skipping events, supported by a minimum of 3 junction reads, were detected. At least 7 genes, their mRNA expression significantly affected by butyrate, also had exon skipping events differentially regulated by butyrate. Furthermore, COL5A3, which was induced 310-fold by butyrate (FDR <0.001) at the gene level, had a significantly higher number of junction reads mapped to Exon#8 (Donor) and Exon#11 (Acceptor) in BT. This event had the potential to result in the formation of a COL5A3 mRNA isoform with 2 of the 69 exons missing. In addition, 216 differentially expressed transcript isoforms regulated by butyrate were detected. For example, Isoform 1 of ORC1 was strongly repressed by butyrate while Isoform 2 remained unchanged. Butyrate physically binds to and inhibits all zinc-dependent HDACs except HDAC6 and HDAC10. Our results provided evidence that butyrate also regulated deacetylase activities of classical HDACs via its transcriptional control. Moreover, thirteen gene fusion events differentially affected by butyrate were identified. Our results provided a snapshot into complex transcriptome dynamics regulated by butyrate, which will facilitate our understanding of the biological effects of butyrate and other HDAC inhibitors.

An Alternative Splice Product of IκB Kinase (IKKγ), IKKγ-Δ, Differentially Mediates Cytokine and Human T-Cell Leukemia Virus Type 1 Tax-Induced NF-κB Activation

PubMed Central

Hai, Tao; Yeung, Man-Lung; Wood, Thomas G.; Wei, Yuanfen; Yamaoka, Shoji; Gatalica, Zoran; Jeang, Kuan-Teh; Brasier, Allan R.

2006-01-01

NF-κB is an inducible transcription factor mediating innate immune responses whose activity is controlled by the multiprotein IκB kinase (IKK) “signalsome”. The core IKK consists of two catalytic serine kinases, IKKα and IKKβ, and a noncatalytic subunit, IKKγ. IKKγ is required for IKK activity by mediating kinase oligomerization and serving to couple the core catalytic subunits to upstream mitogen-activated protein 3-kinase cascades. We have discovered an alternatively spliced IKKγ mRNA isoform, encoding an in-frame deletion of exon 5, termed IKKγ-Δ. Using a specific reverse transcription-PCR assay, we find that IKKγ-Δ is widely expressed in cultured human cells and normal human tissues. Because IKKγ-Δ protein is lacking a critical coiled-coil domain important in protein-protein interactions, we sought to determine its signaling properties by examining its ability to self associate, couple to activators of the canonical pathway, and mediate human T-cell leukemia virus type 1 (HTLV-1) Tax-induced NF-κB activity. Coimmunoprecipitation and confocal colocalization assays indicate IKKγ-Δ has strong homo- and heterotypic association with wild-type (WT) IKKγ and, like IKKγ WT, associates with the IKKβ kinase. Similarly, IKKγ-Δ mediates IKK kinase activity and downstream NF-κB-dependent transcription in response to tumor necrosis factor (TNF) and the NF-κB-inducing kinase-IKKα signaling pathway. Surprisingly, however, in contrast to IKKγ WT, IKKγ-Δ is not able to mediate HTLV-1 Tax-induced NF-κB-dependent transcription, even though IKKγ-Δ binds and colocalizes with Tax. These observations suggest that IKKγ-Δ is a functionally distinct alternatively spliced mRNA product differentially mediating TNF-induced, but not Tax-induced, signals converging on the IKK signalsome. Differing levels of IKKγ-Δ expression, therefore, may affect signal transduction cascades coupling to IKK. PMID:16611882

Biphasic Scaffolds from Marine Collagens for Regeneration of Osteochondral Defects.

PubMed

Bernhardt, Anne; Paul, Birgit; Gelinsky, Michael

2018-03-13

Collagens of marine origin are applied increasingly as alternatives to mammalian collagens in tissue engineering. The aim of the present study was to develop a biphasic scaffold from exclusively marine collagens supporting both osteogenic and chondrogenic differentiation and to find a suitable setup for in vitro chondrogenic and osteogenic differentiation of human mesenchymal stroma cells (hMSC). Biphasic scaffolds from biomimetically mineralized salmon collagen and fibrillized jellyfish collagen were fabricated by joint freeze-drying and crosslinking. Different experiments were performed to analyze the influence of cell density and TGF-β on osteogenic differentiation of the cells in the scaffolds. Gene expression analysis and analysis of cartilage extracellular matrix components were performed and activity of alkaline phosphatase was determined. Furthermore, histological sections of differentiated cells in the biphasic scaffolds were analyzed. Stable biphasic scaffolds from two different marine collagens were prepared. An in vitro setup for osteochondral differentiation was developed involving (1) different seeding densities in the phases; (2) additional application of alginate hydrogel in the chondral part; (3) pre-differentiation and sequential seeding of the scaffolds and (4) osteochondral medium. Spatially separated osteogenic and chondrogenic differentiation of hMSC was achieved in this setup, while osteochondral medium in combination with the biphasic scaffolds alone was not sufficient to reach this ambition. Biphasic, but monolithic scaffolds from exclusively marine collagens are suitable for the development of osteochondral constructs.

Dynamic changes in copper homeostasis and post-transcriptional regulation of Atp7a during myogenic differentiation.

PubMed

Vest, Katherine E; Paskavitz, Amanda L; Lee, Joseph B; Padilla-Benavides, Teresita

2018-02-21

Copper (Cu) is an essential metal required for activity of a number of redox active enzymes that participate in critical cellular pathways such as metabolism and cell signaling. Because it is also a toxic metal, Cu must be tightly controlled by a series of transporters and chaperone proteins that regulate Cu homeostasis. The critical nature of Cu is highlighted by the fact that mutations in Cu homeostasis genes cause pathologic conditions such as Menkes and Wilson diseases. While Cu homeostasis in highly affected tissues like the liver and brain is well understood, no study has probed the role of Cu in development of skeletal muscle, another tissue that often shows pathology in these conditions. Here, we found an increase in whole cell Cu content during differentiation of cultured immortalized or primary myoblasts derived from mouse satellite cells. We demonstrate that Cu is required for both proliferation and differentiation of primary myoblasts. We also show that a key Cu homeostasis gene, Atp7a, undergoes dynamic changes in expression during myogenic differentiation. Alternative polyadenylation and stability of Atp7a mRNA fluctuates with differentiation stage of the myoblasts, indicating post-transcriptional regulation of Atp7a that depends on the differentiation state. This is the first report of a requirement for Cu during myogenic differentiation and provides the basis for understanding the network of Cu transport associated with myogenesis.

Menstrual blood-derived mesenchymal stem cells differentiate into functional hepatocyte-like cells*

PubMed Central

Mou, Xiao-zhou; Lin, Jian; Chen, Jin-yang; Li, Yi-fei; Wu, Xiao-xing; Xiang, Bing-yu; Li, Cai-yun; Ma, Ju-ming; Xiang, Charlie

2013-01-01

Orthotopic liver transplantation (OLT) is the only proven effective treatment for both end-stage and metabolic liver diseases. Hepatocyte transplantation is a promising alternative for OLT, but the lack of available donor livers has hampered its clinical application. Hepatocyte-like cells (HLCs) differentiated from many multi-potential stem cells can help repair damaged liver tissue. Yet almost suitable cells currently identified for human use are difficult to harvest and involve invasive procedures. Recently, a novel mesenchymal stem cell derived from human menstrual blood (MenSC) has been discovered and obtained easily and repeatedly. In this study, we examined whether the MenSCs are able to differentiate into functional HLCs in vitro. After three weeks of incubation in hepatogenic differentiation medium containing hepatocyte growth factor (HGF), fibroblast growth factor-4 (FGF-4), and oncostain M (OSM), cuboidal HLCs were observed, and cells also expressed hepatocyte-specific marker genes including albumin (ALB), α-fetoprotein (AFP), cytokeratin 18/19 (CK18/19), and cytochrome P450 1A1/3A4 (CYP1A1/3A4). Differentiated cells further demonstrated in vitro mature hepatocyte functions such as urea synthesis, glycogen storage, and indocyanine green (ICG) uptake. After intrasplenic transplantation into mice with 2/3 partial hepatectomy, the MenSC-derived HLCs were detected in recipient livers and expressed human ALB protein. We also showed that MenSC-derived HLC transplantation could restore the serum ALB level and significantly suppressed transaminase activity of liver injury animals. In conclusion, MenSCs may serve as an ideal, easily accessible source of material for tissue engineering and cell therapy of liver tissues. PMID:24190442

Genome-wide genetic variation and comparison of fruit-associated traits between kumquat (Citrus japonica) and Clementine mandarin (Citrus clementina).

PubMed

Liu, Tian-Jia; Li, Yong-Ping; Zhou, Jing-Jing; Hu, Chun-Gen; Zhang, Jin-Zhi

2018-03-01

The comprehensive genetic variation of two citrus species were analyzed at genome and transcriptome level. A total of 1090 differentially expressed genes were found during fruit development by RNA-sequencing. Fruit size (fruit equatorial diameter) and weight (fresh weight) are the two most important components determining yield and consumer acceptability for many horticultural crops. However, little is known about the genetic control of these traits. Here, we performed whole-genome resequencing to reveal the comprehensive genetic variation of the fruit development between kumquat (Citrus japonica) and Clementine mandarin (Citrus clementina). In total, 5,865,235 single-nucleotide polymorphisms (SNPs) and 414,447 insertions/deletions (InDels) were identified in the two citrus species. Based on integrative analysis of genome and transcriptome of fruit, 640,801 SNPs and 20,733 InDels were identified. The features, genomic distribution, functional effect, and other characteristics of these genetic variations were explored. RNA-sequencing identified 1090 differentially expressed genes (DEGs) during fruit development of kumquat and Clementine mandarin. Gene Ontology revealed that these genes were involved in various molecular functional and biological processes. In addition, the genetic variation of 939 DEGs and 74 multiple fruit development pathway genes from previous reports were also identified. A global survey identified 24,237 specific alternative splicing events in the two citrus species and showed that intron retention is the most prevalent pattern of alternative splicing. These genome variation data provide a foundation for further exploration of citrus diversity and gene-phenotype relationships and for future research on molecular breeding to improve kumquat, Clementine mandarin and related species.

Ectopic expression of homeobox gene NKX2-1 in diffuse large B-cell lymphoma is mediated by aberrant chromatin modifications.

PubMed

Nagel, Stefan; Ehrentraut, Stefan; Tomasch, Jürgen; Quentmeier, Hilmar; Meyer, Corinna; Kaufmann, Maren; Drexler, Hans G; MacLeod, Roderick A F

2013-01-01

Homeobox genes encode transcription factors ubiquitously involved in basic developmental processes, deregulation of which promotes cell transformation in multiple cancers including hematopoietic malignancies. In particular, NKL-family homeobox genes TLX1, TLX3 and NKX2-5 are ectopically activated by chromosomal rearrangements in T-cell neoplasias. Here, using transcriptional microarray profiling and RQ-PCR we identified ectopic expression of NKL-family member NKX2-1, in a diffuse large B-cell lymphoma (DLBCL) cell line SU-DHL-5. Moreover, in silico analysis demonstrated NKX2-1 overexpression in 5% of examined DLBCL patient samples. NKX2-1 is physiologically expressed in lung and thyroid tissues where it regulates differentiation. Chromosomal and genomic analyses excluded rearrangements at the NKX2-1 locus in SU-DHL-5, implying alternative activation. Comparative expression profiling implicated several candidate genes in NKX2-1 regulation, variously encoding transcription factors, chromatin modifiers and signaling components. Accordingly, siRNA-mediated knockdown and overexpression studies confirmed involvement of transcription factor HEY1, histone methyltransferase MLL and ubiquitinated histone H2B in NKX2-1 deregulation. Chromosomal aberrations targeting MLL at 11q23 and the histone gene cluster HIST1 at 6p22 which we observed in SU-DHL-5 may, therefore, represent fundamental mutations mediating an aberrant chromatin structure at NKX2-1. Taken together, we identified ectopic expression of NKX2-1 in DLBCL cells, representing the central player in an oncogenic regulative network compromising B-cell differentiation. Thus, our data extend the paradigm of NKL homeobox gene deregulation in lymphoid malignancies.

GOexpress: an R/Bioconductor package for the identification and visualisation of robust gene ontology signatures through supervised learning of gene expression data.

PubMed

Rue-Albrecht, Kévin; McGettigan, Paul A; Hernández, Belinda; Nalpas, Nicolas C; Magee, David A; Parnell, Andrew C; Gordon, Stephen V; MacHugh, David E

2016-03-11

Identification of gene expression profiles that differentiate experimental groups is critical for discovery and analysis of key molecular pathways and also for selection of robust diagnostic or prognostic biomarkers. While integration of differential expression statistics has been used to refine gene set enrichment analyses, such approaches are typically limited to single gene lists resulting from simple two-group comparisons or time-series analyses. In contrast, functional class scoring and machine learning approaches provide powerful alternative methods to leverage molecular measurements for pathway analyses, and to compare continuous and multi-level categorical factors. We introduce GOexpress, a software package for scoring and summarising the capacity of gene ontology features to simultaneously classify samples from multiple experimental groups. GOexpress integrates normalised gene expression data (e.g., from microarray and RNA-seq experiments) and phenotypic information of individual samples with gene ontology annotations to derive a ranking of genes and gene ontology terms using a supervised learning approach. The default random forest algorithm allows interactions between all experimental factors, and competitive scoring of expressed genes to evaluate their relative importance in classifying predefined groups of samples. GOexpress enables rapid identification and visualisation of ontology-related gene panels that robustly classify groups of samples and supports both categorical (e.g., infection status, treatment) and continuous (e.g., time-series, drug concentrations) experimental factors. The use of standard Bioconductor extension packages and publicly available gene ontology annotations facilitates straightforward integration of GOexpress within existing computational biology pipelines.

Differential gene expression analysis in glioblastoma cells and normal human brain cells based on GEO database.

PubMed

Wang, Anping; Zhang, Guibin

2017-11-01

The differentially expressed genes between glioblastoma (GBM) cells and normal human brain cells were investigated to performed pathway analysis and protein interaction network analysis for the differentially expressed genes. GSE12657 and GSE42656 gene chips, which contain gene expression profile of GBM were obtained from Gene Expression Omniub (GEO) database of National Center for Biotechnology Information (NCBI). The 'limma' data packet in 'R' software was used to analyze the differentially expressed genes in the two gene chips, and gene integration was performed using 'RobustRankAggreg' package. Finally, pheatmap software was used for heatmap analysis and Cytoscape, DAVID, STRING and KOBAS were used for protein-protein interaction, Gene Ontology (GO) and KEGG analyses. As results: i) 702 differentially expressed genes were identified in GSE12657, among those genes, 548 were significantly upregulated and 154 were significantly downregulated (p<0.01, fold-change >1), and 1,854 differentially expressed genes were identified in GSE42656, among the genes, 1,068 were significantly upregulated and 786 were significantly downregulated (p<0.01, fold-change >1). A total of 167 differentially expressed genes including 100 upregulated genes and 67 downregulated genes were identified after gene integration, and the genes showed significantly different expression levels in GBM compared with normal human brain cells (p<0.05). ii) Interactions between the protein products of 101 differentially expressed genes were identified using STRING and expression network was established. A key gene, called CALM3, was identified by Cytoscape software. iii) GO enrichment analysis showed that differentially expressed genes were mainly enriched in 'neurotransmitter:sodium symporter activity' and 'neurotransmitter transporter activity', which can affect the activity of neurotransmitter transportation. KEGG pathway analysis showed that the differentially expressed genes were mainly enriched in 'protein processing in endoplasmic reticulum', which can affect protein processing in endoplasmic reticulum. The results showed that: i) 167 differentially expressed genes were identified from two gene chips after integration; and ii) protein interaction network was established, and GO and KEGG pathway analyses were successfully performed to identify and annotate the key gene, which provide new insights for the studies on GBN at gene level.

Colorectal tumor molecular phenotype and miRNA: expression profiles and prognosis.

PubMed

Slattery, Martha L; Herrick, Jennifer S; Mullany, Lila E; Wolff, Erica; Hoffman, Michael D; Pellatt, Daniel F; Stevens, John R; Wolff, Roger K

2016-08-01

MiRNAs regulate gene expression by post-transcriptionally suppressing mRNA translation or by causing mRNA degradation. It has been proposed that unique miRNAs influence specific tumor molecular phenotype. In this paper, we test the hypotheses that miRNA expression differs by tumor molecular phenotype and that those differences may influence prognosis. Data come from population-based studies of colorectal cancer conducted in Utah and the Northern California Kaiser Permanente Medical Care Program. A total of 1893 carcinoma samples were run on the Agilent Human miRNA Microarray V19.0 containing 2006 miRNAs. We assessed differences in miRNA expression between TP53-mutated and non-mutated, KRAS-mutated and non-mutated, BRAF-mutated and non-mutated, CpG island methylator phenotype (CIMP) high and CIMP low, and microsatellite instability (MSI) and microsatellite stable (MSS) colon and rectal tumors. Using a Cox proportional hazard model we evaluated if those miRNAs differentially expressed by tumor phenotype influenced survival after adjusting for age, sex, and AJCC stage. There were 22 differentially expressed miRNAs for TP53-mutated colon tumors and 5 for TP53-mutated rectal tumors with a fold change of >1.49 (or <0.67). Additionally, 13 miRNAS were differentially expressed for KRAS-mutated rectal tumors, 8 differentially expressed miRNAs for colon CIMP high tumors, and 2 differentially expressed miRNAs for BRAF-mutated colon tumors. The majority of differentially expressed miRNAS were observed between MSI and MSS tumors (94 differentially expressed miRNAs for colon; 41 differentially expressed miRNAs for rectal tumors). Of these miRNAs differentially expressed between MSI and MSS tumors, the majority were downregulated. Ten of the differentially expressed miRNAs were associated with survival; after adjustment for MSI status, five miRNAS, miR-196b-5p, miR-31-5p, miR-99b-5p, miR-636, and miR-192-3p, were significantly associated with survival. In summary, it appears that the majority of miRNAs that are differentially expressed by tumor molecular phenotype are MSI tumors. However, these miRNAs appear to have minimal effect on prognosis.

Purinergic signaling during macrophage differentiation results in M2 alternative activated macrophages.

PubMed

Barberà-Cremades, Maria; Baroja-Mazo, Alberto; Pelegrín, Pablo

2016-02-01

Macrophages represent a highly heterogenic cell population of the innate immune system, with important roles in the initiation and resolution of the inflammatory response. Purinergic signaling regulates both M1 and M2 macrophage function at different levels by controlling the secretion of cytokines, phagocytosis, and the production of reactive oxygen species. We found that extracellular nucleotides arrest macrophage differentiation from bone marrow precursors via adenosine and P2 receptors. This results in a mature macrophage with increased expression of M2, but not M1, genes. Similar to adenosine and ATP, macrophage growth arrested with LPS treatment resulted in an increase of the M2-related marker Ym1. Recombinant Ym1 was able to affect macrophage proliferation and could, potentially, be involved in the arrest of macrophage growth during hematopoiesis. © Society for Leukocyte Biology.

Effect of isodillapiole on the expression of the insecticide resistance genes GSTE7 and CYP6N12 in Aedes aegypti from central Amazonia.

PubMed

Lima, V S; Pinto, A C; Rafael, M S

2015-12-11

The yellow fever mosquito Aedes (Stegomyia) aegypti is the main vector of dengue arbovirus and other arboviruses. Dengue prevention measures for the control of A. aegypti involve mainly the use of synthetic insecticides. The constant use of insecticides has caused resistance in this mosquito. Alternative studies on plant extracts and their products have been conducted with the aim of controlling the spread of the mosquito. Dillapiole is a compound found in essential oils of the plant Piper aduncum (Piperaceae) which has been effective as a biopesticide against A. aegypti. Isodillapiole is a semisynthetic substance obtained by the isomerization of dillapiole. In the present study, isodillapiole was evaluated for its potential to induce differential expression of insecticide resistance genes (GSTE7 and CYP6N12) in 3rd instar larvae of A. aegypti. These larvae were exposed to this compound at two concentrations (20 and 40 μg/mL) for 4 h during four generations (G1, G2, G3, and G4). Quantitative RT-PCR was used to assess the expression of GSTE7 and CYP6N12 genes. GSTE7 and CYP6N12 relative expression levels were higher at 20 than at 40 μg/mL and varied among generations. The decrease in GSTE7 and CYP6N12 expression levels at the highest isodillapiole concentration suggests that larvae may have suffered from metabolic stress, revealing a potential alternative product in the control of A. aegypti.

Expression analysis of a heat-inducible, Myo-inositol-1-phosphate synthase (MIPS) gene from wheat and the alternatively spliced variants of rice and Arabidopsis.

PubMed

Khurana, Neetika; Chauhan, Harsh; Khurana, Paramjit

2012-01-01

Molecular dissection and a deeper analysis of the heat stress response mechanism in wheat have been poorly understood so far. This study delves into the molecular basis of action of TaMIPS, a heat stress-inducible enzyme that was identified through PCR-select subtraction technology, which is named here as TaMIPS2. MIPS (L-Myo-inositol-phosphate synthase) is important for the normal growth and development in plants. Expression profiling showed that TaMIPS2 is expressed during different developing seed stages upon heat stress. Also, the transcript levels increase in unfertilized ovaries and significant amounts are present during the recovery period providing evidence that MIPS is crucial for its role in heat stress recovery and flower development. Alternatively spliced forms from rice and Arabidopsis were also identified and their expression analysis revealed that apart from heat stress, some of the spliced variants were also inducible by drought, NaCl, Cold, ABA, BR, SA and mannitol. In silico promoter analysis revealed various cis-elements that could contribute for the differential regulation of MIPS in different plant systems. Phylogenetic analysis indicated that MIPS are highly conserved among monocots and dicots and TaMIPS2 grouped specifically with monocots. Comparative analyses was undertaken by different experimental approaches, i.e., semi-quantitative RT-PCR, quantitative RT-PCR, Genevestigator as a reference expression tool and motif analysis to predict the possible function of TaMIPS2 in regulating the different aspects of plant development under abiotic stress in wheat.

Mapping gene expression patterns during myeloid differentiation using the EML hematopoietic progenitor cell line.

PubMed

Du, Yang; Campbell, Janee L; Nalbant, Demet; Youn, Hyewon; Bass, Ann C Hughes; Cobos, Everardo; Tsai, Schickwann; Keller, Jonathan R; Williams, Simon C

2002-07-01

The detailed examination of the molecular events that control the early stages of myeloid differentiation has been hampered by the relative scarcity of hematopoietic stem cells and the lack of suitable cell line models. In this study, we examined the expression of several myeloid and nonmyeloid genes in the murine EML hematopoietic stem cell line. Expression patterns for 19 different genes were examined by Northern blotting and RT-PCR in RNA samples from EML, a variety of other immortalized cell lines, and purified murine hematopoietic stem cells. Representational difference analysis (RDA) was performed to identify differentially expressed genes in EML. Expression patterns of genes encoding transcription factors (four members of the C/EBP family, GATA-1, GATA-2, PU.1, CBFbeta, SCL, and c-myb) in EML were examined and were consistent with the proposed functions of these proteins in hematopoietic differentiation. Expression levels of three markers of terminal myeloid differentiation (neutrophil elastase, proteinase 3, and Mac-1) were highest in EML cells at the later stages of differentiation. In a search for genes that were differentially expressed in EML cells during myeloid differentiation, six cDNAs were isolated. These included three known genes (lysozyme, histidine decarboxylase, and tryptophan hydroxylase) and three novel genes. Expression patterns of known genes in differentiating EML cells accurately reflected their expected expression patterns based on previous studies. The identification of three novel genes, two of which encode proteins that may act as regulators of hematopoietic differentiation, suggests that EML is a useful model system for the molecular analysis of hematopoietic differentiation.

Proteogenomic analysis reveals alternative splicing and translation as part of the abscisic acid response in Arabidopsis seedlings.

PubMed

Zhu, Fu-Yuan; Chen, Mo-Xian; Ye, Neng-Hui; Shi, Lu; Ma, Kai-Long; Yang, Jing-Fang; Cao, Yun-Ying; Zhang, Youjun; Yoshida, Takuya; Fernie, Alisdair R; Fan, Guang-Yi; Wen, Bo; Zhou, Ruo; Liu, Tie-Yuan; Fan, Tao; Gao, Bei; Zhang, Di; Hao, Ge-Fei; Xiao, Shi; Liu, Ying-Gao; Zhang, Jianhua

2017-08-01

In eukaryotes, mechanisms such as alternative splicing (AS) and alternative translation initiation (ATI) contribute to organismal protein diversity. Specifically, splicing factors play crucial roles in responses to environment and development cues; however, the underlying mechanisms are not well investigated in plants. Here, we report the parallel employment of short-read RNA sequencing, single molecule long-read sequencing and proteomic identification to unravel AS isoforms and previously unannotated proteins in response to abscisic acid (ABA) treatment. Combining the data from the two sequencing methods, approximately 83.4% of intron-containing genes were alternatively spliced. Two AS types, which are referred to as alternative first exon (AFE) and alternative last exon (ALE), were more abundant than intron retention (IR); however, by contrast to AS events detected under normal conditions, differentially expressed AS isoforms were more likely to be translated. ABA extensively affects the AS pattern, indicated by the increasing number of non-conventional splicing sites. This work also identified thousands of unannotated peptides and proteins by ATI based on mass spectrometry and a virtual peptide library deduced from both strands of coding regions within the Arabidopsis genome. The results enhance our understanding of AS and alternative translation mechanisms under normal conditions, and in response to ABA treatment. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Isolation and characterization of alternatively spliced variants of the mouse sigma1 receptor gene, Sigmar1

PubMed Central

Pan, Ling; Pasternak, David A.; Xu, Jin; Xu, Mingming; Lu, Zhigang; Pasternak, Gavril W.

2017-01-01

The sigma1 receptor acts as a chaperone at the endoplasmic reticulum, associates with multiple proteins in various cellular systems, and involves in a number of diseases, such as addiction, pain, cancer and psychiatric disorders. The sigma1 receptor is encoded by the single copy SIGMAR1 gene. The current study identifies five alternatively spliced variants of the mouse sigma1 receptor gene using a polymerase chain reaction cloning approach. All the splice variants are generated by exon skipping or alternative 3’ or 5’ splicing, producing the truncated sigma1 receptor. Similar alternative splicing has been observed in the human SIGMAR1 gene based on the molecular cloning or genome sequence prediction, suggesting conservation of alternative splicing of SIGMAR1 gene. Using quantitative polymerase chain reactions, we demonstrate differential expression of several splice variants in mouse tissues and brain regions. When expressed in HEK293 cells, all the splice variants fail to bind sigma ligands, implicating that each truncated region in these splice variants is important for ligand binding. However, co-immunoprecipitation (Co-IP) study in HEK293 cells co-transfected with tagged constructs reveals that all the splice variants maintain their ability to physically associate with a mu opioid receptor (mMOR-1), providing useful information to correlate the motifs/sequences necessary for their physical association. Furthermore, a competition Co-IP study showed that all the variants can disrupt in a dose-dependent manner the dimerization of the original sigma1 receptor with mMOR-1, suggesting a potential dominant negative function and providing significant insights into their function. PMID:28350844

Metatranscriptomic profiles of Eastern subterranean termites, Reticulitermes flavipes (Kollar) fed on second generation feedstocks.

PubMed

Rajarapu, Swapna Priya; Shreve, Jacob T; Bhide, Ketaki P; Thimmapuram, Jyothi; Scharf, Michael E

2015-04-22

Second generation lignocellulosic feedstocks are being considered as an alternative to first generation biofuels that are derived from grain starches and sugars. However, the current pre-treatment methods for second generation biofuel production are inefficient and expensive due to the recalcitrant nature of lignocellulose. In this study, we used the lower termite Reticulitermes flavipes (Kollar), as a model to identify potential pretreatment genes/enzymes specifically adapted for use against agricultural feedstocks. Metatranscriptomic profiling was performed on worker termite guts after feeding on corn stover (CS), soybean residue (SR), or 98% pure cellulose (paper) to identify (i) microbial community, (ii) pathway level and (iii) gene-level responses. Microbial community profiles after CS and SR feeding were different from the paper feeding profile, and protist symbiont abundance decreased significantly in termites feeding on SR and CS relative to paper. Functional profiles after CS feeding were similar to paper and SR; whereas paper and SR showed different profiles. Amino acid and carbohydrate metabolism pathways were downregulated in termites feeding on SR relative to paper and CS. Gene expression analyses showed more significant down regulation of genes after SR feeding relative to paper and CS. Stereotypical lignocellulase genes/enzymes were not differentially expressed, but rather were among the most abundant/constitutively-expressed genes. These results suggest that the effect of CS and SR feeding on termite gut lignocellulase composition is minimal and thus, the most abundantly expressed enzymes appear to encode the best candidate catalysts for use in saccharification of these and related second-generation feedstocks. Further, based on these findings we hypothesize that the most abundantly expressed lignocellulases, rather than those that are differentially expressed have the best potential as pretreatment enzymes for CS and SR feedstocks.

Comparative transcriptomics reveals different strategies of Trichoderma mycoparasitism

PubMed Central

2013-01-01

Background Trichoderma is a genus of mycotrophic filamentous fungi (teleomorph Hypocrea) which possess a bright variety of biotrophic and saprotrophic lifestyles. The ability to parasitize and/or kill other fungi (mycoparasitism) is used in plant protection against soil-borne fungal diseases (biological control, or biocontrol). To investigate mechanisms of mycoparasitism, we compared the transcriptional responses of cosmopolitan opportunistic species and powerful biocontrol agents Trichoderma atroviride and T. virens with tropical ecologically restricted species T. reesei during confrontations with a plant pathogenic fungus Rhizoctonia solani. Results The three Trichoderma spp. exhibited a strikingly different transcriptomic response already before physical contact with alien hyphae. T. atroviride expressed an array of genes involved in production of secondary metabolites, GH16 ß-glucanases, various proteases and small secreted cysteine rich proteins. T. virens, on the other hand, expressed mainly the genes for biosynthesis of gliotoxin, respective precursors and also glutathione, which is necessary for gliotoxin biosynthesis. In contrast, T. reesei increased the expression of genes encoding cellulases and hemicellulases, and of the genes involved in solute transport. The majority of differentially regulated genes were orthologues present in all three species or both in T. atroviride and T. virens, indicating that the regulation of expression of these genes is different in the three Trichoderma spp. The genes expressed in all three fungi exhibited a nonrandom genomic distribution, indicating a possibility for their regulation via chromatin modification. Conclusion This genome-wide expression study demonstrates that the initial Trichoderma mycotrophy has differentiated into several alternative ecological strategies ranging from parasitism to predation and saprotrophy. It provides first insights into the mechanisms of interactions between Trichoderma and other fungi that may be exploited for further development of biofungicides. PMID:23432824

Differential splicing and glycosylation of Apoer2 alters synaptic plasticity and fear learning.

PubMed

Wasser, Catherine R; Masiulis, Irene; Durakoglugil, Murat S; Lane-Donovan, Courtney; Xian, Xunde; Beffert, Uwe; Agarwala, Anandita; Hammer, Robert E; Herz, Joachim

2014-11-25

Apoer2 is an essential receptor in the central nervous system that binds to the apolipoprotein ApoE. Various splice variants of Apoer2 are produced. We showed that Apoer2 lacking exon 16, which encodes the O-linked sugar (OLS) domain, altered the proteolytic processing and abundance of Apoer2 in cells and synapse number and function in mice. In cultured cells expressing this splice variant, extracellular cleavage of OLS-deficient Apoer2 was reduced, consequently preventing γ-secretase-dependent release of the intracellular domain of Apoer2. Mice expressing Apoer2 lacking the OLS domain had increased Apoer2 abundance in the brain, hippocampal spine density, and glutamate receptor abundance, but decreased synaptic efficacy. Mice expressing a form of Apoer2 lacking the OLS domain and containing an alternatively spliced cytoplasmic tail region that promotes glutamate receptor signaling showed enhanced hippocampal long-term potentiation (LTP), a phenomenon associated with learning and memory. However, these mice did not display enhanced spatial learning in the Morris water maze, and cued fear conditioning was reduced. Reducing the expression of the mutant Apoer2 allele so that the abundance of the protein was similar to that of Apoer2 in wild-type mice normalized spine density, hippocampal LTP, and cued fear learning. These findings demonstrated a role for ApoE receptors as regulators of synaptic glutamate receptor activity and established differential receptor glycosylation as a potential regulator of synaptic function and memory. Copyright © 2014, American Association for the Advancement of Science.

Differential splicing and glycosylation of Apoer2 alters synaptic plasticity and fear learning

PubMed Central

Wasser, Catherine R.; Masiulis, Irene; Durakoglugil, Murat S.; Lane-Donovan, Courtney; Xian, Xunde; Beffert, Uwe; Agarwala, Anandita; Hammer, Robert E.; Herz, Joachim

2015-01-01

Apoer2 is an essential receptor in the central nervous system that binds to the apolipoprotein ApoE. Various splice variants of Apoer2 are produced. We showed that Apoer2 lacking exon 16, which encodes the O-linked sugar (OLS) domain, altered the proteolytic processing and abundance of Apoer2 in cells and synapse number and function in mice. In cultured cells expressing this splice variant, extracellular cleavage of OLS-deficient Apoer2 was reduced, consequently preventing γ-secretase-dependent release of the intracellular domain of Apoer2. Mice expressing Apoer2 lacking the OLS domain had increased Apoer2 abundance in the brain, hippocampal spine density, and glutamate receptor abundance, but decreased synaptic efficacy. Mice expressing a form of Apoer2 lacking the OLS domain and containing an alternatively spliced cytoplasmic tail region that promotes glutamate receptor signaling showed enhanced hippocampal long-term potentiation (LTP), a phenomenon associated with learning and memory. However, these mice did not display enhanced spatial learning in the Morris water maze, and cued fear conditioning was reduced. Reducing the expression of the mutant Apoer2 allele so that the abundance of the protein was similar to that of Apoer2 in wild-type mice normalized spine density, hippocampal LTP, and cued fear learning. These findings demonstrated a role for ApoE receptors as regulators of synaptic glutamate receptor activity and established differential receptor glycosylation as a potential regulator of synaptic function and memory. PMID:25429077

MYCN controls an alternative RNA splicing program in high-risk metastatic neuroblastoma

PubMed Central

Zhang, Shile; Wei, Jun S.; Li, Samuel Q.; Badgett, Tom C.; Song, Young K.; Agarwal, Saurabh; Coarfa, Cristian; Tolman, Catherine; Hurd, Laura; Liao, Hongling; He, Jianbin; Wen, Xinyu; Liu, Zhihui; Thiele, Carol J.; Westermann, Frank; Asgharzadeh, Shahab; Seeger, Robert C.; Maris, John M.; Auvil, Jamie M Guidry; Smith, Malcolm A; Kolaczyk, Eric D; Shohet, Jason; Khan, Javed

2016-01-01

The molecular mechanisms underlying the aggressive behavior of MYCN driven neuroblastoma (NBL) is under intense investigation; however, little is known about the impact of this family of transcription factors on the splicing program. Here we used high-throughput RNA sequencing to systematically study the expression of RNA isoforms in stage 4 MYCN-amplified NBL, an aggressive subtype of metastatic NBL. We show that MYCN-amplified NBL tumors display a distinct gene splicing pattern affecting multiple cancer hallmark functions. Six splicing factors displayed unique differential expression patterns in MYCN-amplified tumors and cell lines, and the binding motifs for some of these splicing factors are significantly enriched in differentially-spliced genes. Direct binding of MYCN to promoter regions of the splicing factors PTBP1 and HNRNPA1 detected by ChIP-seq demonstrates MYCN controls the splicing pattern by direct regulation of the expression of these key splicing factors. Furthermore, high expression of PTBP1 and HNRNPA1 was significantly associated with poor overall survival of stage4 NBL patients (p≤0.05). Knocking down PTBP1, HNRNPA1 and their downstream target PKM2, an isoform of pro-tumor-growth, result in repressed growth of NBL cells. Therefore, our study reveals a novel role of MYCN in controlling global splicing program through regulation of splicing factors in addition to its well-known role in the transcription program. These findings suggest a therapeutically potential to target the key splicing factors or gene isoforms in high-risk NBL with MYCN-amplification. PMID:26683771

Genetic and Functional Investigation of Zn2Cys6 Transcription Factors RSE2 and RSE3 in Podospora anserina

PubMed Central

Bovier, Elodie; Sellem, Carole H.; Humbert, Adeline

2014-01-01

In Podospora anserina, the two zinc cluster proteins RSE2 and RSE3 are essential for the expression of the gene encoding the alternative oxidase (aox) when the mitochondrial electron transport chain is impaired. In parallel, they activated the expression of gluconeogenic genes encoding phosphoenolpyruvate carboxykinase (pck) and fructose-1,6-biphosphatase (fbp). Orthologues of these transcription factors are present in a wide range of filamentous fungi, and no other role than the regulation of these three genes has been evidenced so far. In order to better understand the function and the organization of RSE2 and RSE3, we conducted a saturated genetic screen based on the constitutive expression of the aox gene. We identified 10 independent mutations in 9 positions in rse2 and 11 mutations in 5 positions in rse3. Deletions were generated at some of these positions and the effects analyzed. This analysis suggests the presence of central regulatory domains and a C-terminal activation domain in both proteins. Microarray analysis revealed 598 genes that were differentially expressed in the strains containing gain- or loss-of-function mutations in rse2 or rse3. It showed that in addition to aox, fbp, and pck, RSE2 and RSE3 regulate the expression of genes encoding the alternative NADH dehydrogenase, a Zn2Cys6 transcription factor, a flavohemoglobin, and various hydrolases. As a complement to expression data, a metabolome profiling approach revealed that both an rse2 gain-of-function mutation and growth on antimycin result in similar metabolic alterations in amino acids, fatty acids, and α-ketoglutarate pools. PMID:24186951

Defining the Human Macula Transcriptome and Candidate Retinal Disease Genes UsingEyeSAGE

PubMed Central

Rickman, Catherine Bowes; Ebright, Jessica N.; Zavodni, Zachary J.; Yu, Ling; Wang, Tianyuan; Daiger, Stephen P.; Wistow, Graeme; Boon, Kathy; Hauser, Michael A.

2009-01-01

Purpose To develop large-scale, high-throughput annotation of the human macula transcriptome and to identify and prioritize candidate genes for inherited retinal dystrophies, based on ocular-expression profiles using serial analysis of gene expression (SAGE). Methods Two human retina and two retinal pigment epithelium (RPE)/choroid SAGE libraries made from matched macula or midperipheral retina and adjacent RPE/choroid of morphologically normal 28- to 66-year-old donors and a human central retina longSAGE library made from 41- to 66-year-old donors were generated. Their transcription profiles were entered into a relational database, EyeSAGE, including microarray expression profiles of retina and publicly available normal human tissue SAGE libraries. EyeSAGE was used to identify retina- and RPE-specific and -associated genes, and candidate genes for retina and RPE disease loci. Differential and/or cell-type specific expression was validated by quantitative and single-cell RT-PCR. Results Cone photoreceptor-associated gene expression was elevated in the macula transcription profiles. Analysis of the longSAGE retina tags enhanced tag-to-gene mapping and revealed alternatively spliced genes. Analysis of candidate gene expression tables for the identified Bardet-Biedl syndrome disease gene (BBS5) in the BBS5 disease region table yielded BBS5 as the top candidate. Compelling candidates for inherited retina diseases were identified. Conclusions The EyeSAGE database, combining three different gene-profiling platforms including the authors’ multidonor-derived retina/RPE SAGE libraries and existing single-donor retina/RPE libraries, is a powerful resource for definition of the retina and RPE transcriptomes. It can be used to identify retina-specific genes, including alternatively spliced transcripts and to prioritize candidate genes within mapped retinal disease regions. PMID:16723438

Defining the human macula transcriptome and candidate retinal disease genes using EyeSAGE.

PubMed

Bowes Rickman, Catherine; Ebright, Jessica N; Zavodni, Zachary J; Yu, Ling; Wang, Tianyuan; Daiger, Stephen P; Wistow, Graeme; Boon, Kathy; Hauser, Michael A

2006-06-01

To develop large-scale, high-throughput annotation of the human macula transcriptome and to identify and prioritize candidate genes for inherited retinal dystrophies, based on ocular-expression profiles using serial analysis of gene expression (SAGE). Two human retina and two retinal pigment epithelium (RPE)/choroid SAGE libraries made from matched macula or midperipheral retina and adjacent RPE/choroid of morphologically normal 28- to 66-year-old donors and a human central retina longSAGE library made from 41- to 66-year-old donors were generated. Their transcription profiles were entered into a relational database, EyeSAGE, including microarray expression profiles of retina and publicly available normal human tissue SAGE libraries. EyeSAGE was used to identify retina- and RPE-specific and -associated genes, and candidate genes for retina and RPE disease loci. Differential and/or cell-type specific expression was validated by quantitative and single-cell RT-PCR. Cone photoreceptor-associated gene expression was elevated in the macula transcription profiles. Analysis of the longSAGE retina tags enhanced tag-to-gene mapping and revealed alternatively spliced genes. Analysis of candidate gene expression tables for the identified Bardet-Biedl syndrome disease gene (BBS5) in the BBS5 disease region table yielded BBS5 as the top candidate. Compelling candidates for inherited retina diseases were identified. The EyeSAGE database, combining three different gene-profiling platforms including the authors' multidonor-derived retina/RPE SAGE libraries and existing single-donor retina/RPE libraries, is a powerful resource for definition of the retina and RPE transcriptomes. It can be used to identify retina-specific genes, including alternatively spliced transcripts and to prioritize candidate genes within mapped retinal disease regions.

Comparative transcriptomics of 5 high-altitude vertebrates and their low-altitude relatives

PubMed Central

Tang, Qianzi; Zhou, Xuming; Jin, Long; Guan, Jiuqiang; Liu, Rui; Li, Jing; Long, Kereng; Tian, Shilin; Che, Tiandong; Hu, Silu; Liang, Yan; Yang, Xuemei; Tao, Xuan; Zhong, Zhijun; Wang, Guosong; Chen, Xiaohui; Li, Diyan; Ma, Jideng; Wang, Xun; Mai, Miaomiao; Jiang, An’an; Luo, Xiaolin; Lv, Xuebin; Gladyshev, Vadim N; Li, Xuewei

2017-01-01

Abstract Background Species living at high altitude are subject to strong selective pressures due to inhospitable environments (e.g., hypoxia, low temperature, high solar radiation, and lack of biological production), making these species valuable models for comparative analyses of local adaptation. Studies that have examined high-altitude adaptation have identified a vast array of rapidly evolving genes that characterize the dramatic phenotypic changes in high-altitude animals. However, how high-altitude environment shapes gene expression programs remains largely unknown. Findings We generated a total of 910 Gb of high-quality RNA-seq data for 180 samples derived from 6 tissues of 5 agriculturally important high-altitude vertebrates (Tibetan chicken, Tibetan pig, Tibetan sheep, Tibetan goat, and yak) and their cross-fertile relatives living in geographically neighboring low-altitude regions. Of these, ∼75% reads could be aligned to their respective reference genomes, and on average ∼60% of annotated protein coding genes in each organism showed FPKM expression values greater than 0.5. We observed a general concordance in topological relationships between the nucleotide alignments and gene expression–based trees. Tissue and species accounted for markedly more variance than altitude based on either the expression or the alternative splicing patterns. Cross-species clustering analyses showed a tissue-dominated pattern of gene expression and a species-dominated pattern for alternative splicing. We also identified numerous differentially expressed genes that could potentially be involved in phenotypic divergence shaped by high-altitude adaptation. Conclusions These data serve as a valuable resource for examining the convergence and divergence of gene expression changes between species as they adapt or acclimatize to high-altitude environments. PMID:29149296

Alternative Mechanism by which IFN-γ Enhances Tumor Recognition: Active Release of Heat Shock Protein 721

PubMed Central

Bausero, Maria A.; Gastpar, Robert; Multhoff, Gabriele; Asea, Alexzander

2006-01-01

IFN-γ exhibits differential effects depending on the target and can induce cellular activation and enhance survival or mediate cell death via activation of apoptotic pathways. In this study, we demonstrate an alternative mechanism by which IFN-γ enhances tumor recognition, mediated by the active release of Hsp72. We demonstrate that stimulation of 4T1 breast adenocarcinoma cells and K562 erythroleukemic cells with IFN-γ triggers the cellular stress response, which results in the enhanced expression of total Hsp72 expression without a significant increase in cell death. Intracellular expression of Hsp72 was abrogated in cells stably transfected with a mutant hsf-1 gene. IFN-γ-induced Hsp72 expression correlated with enhanced surface expression and consequent release of Hsp72 into the culture medium. Pretreatment of tumors with compounds known to the block the classical protein transport pathway, including monensin, brefeldin A, tunicamycin, and thapsigargin, did not significantly block Hsp72 release. However, pretreatment with intracellular calcium chelator BAPTA-AM or disruption of lipid rafts using methyl β-cyclodextrin completely abrogated IFN-γ-induced Hsp72 release. Biochemical characterization revealed that Hsp72 is released within exosomes and has the ability to up-regulate CD83 expression and stimulate IL-12 release by naive dendritic cells. Pretreatment with neutralizing mAb or depletion of Hsp72 completely abrogated its chaperokine function. Taken together, these findings are indicative of an additional previously unknown mechanism by which IFN-γ promotes tumor surveillance and furthers our understanding of the central role of extracellular Hsp72 as an endogenous adjuvant and danger signal. PMID:16116176

Alternative mechanism by which IFN-gamma enhances tumor recognition: active release of heat shock protein 72.

PubMed

Bausero, Maria A; Gastpar, Robert; Multhoff, Gabriele; Asea, Alexzander

2005-09-01

IFN-gamma exhibits differential effects depending on the target and can induce cellular activation and enhance survival or mediate cell death via activation of apoptotic pathways. In this study, we demonstrate an alternative mechanism by which IFN-gamma enhances tumor recognition, mediated by the active release of Hsp72. We demonstrate that stimulation of 4T1 breast adenocarcinoma cells and K562 erythroleukemic cells with IFN-gamma triggers the cellular stress response, which results in the enhanced expression of total Hsp72 expression without a significant increase in cell death. Intracellular expression of Hsp72 was abrogated in cells stably transfected with a mutant hsf-1 gene. IFN-gamma-induced Hsp72 expression correlated with enhanced surface expression and consequent release of Hsp72 into the culture medium. Pretreatment of tumors with compounds known to the block the classical protein transport pathway, including monensin, brefeldin A, tunicamycin, and thapsigargin, did not significantly block Hsp72 release. However, pretreatment with intracellular calcium chelator BAPTA-AM or disruption of lipid rafts using methyl beta-cyclodextrin completely abrogated IFN-gamma-induced Hsp72 release. Biochemical characterization revealed that Hsp72 is released within exosomes and has the ability to up-regulate CD83 expression and stimulate IL-12 release by naive dendritic cells. Pretreatment with neutralizing mAb or depletion of Hsp72 completely abrogated its chaperokine function. Taken together, these findings are indicative of an additional previously unknown mechanism by which IFN-gamma promotes tumor surveillance and furthers our understanding of the central role of extracellular Hsp72 as an endogenous adjuvant and danger signal.

Analyses of chondrogenic induction of adipose mesenchymal stem cells by combined co-stimulation mediated by adenoviral gene transfer

PubMed Central

2013-01-01

Introduction Adipose-derived stem cells (ASCs) have the potential to differentiate into cartilage under stimulation with some reported growth and transcriptional factors, which may constitute an alternative for cartilage replacement approaches. In this study, we analyzed the in vitro chondrogenesis of ASCs transduced with adenoviral vectors encoding insulin-like growth factor-1 (IGF-1), transforming growth factor beta-1 (TGF-β1), fibroblast growth factor-2 (FGF-2), and sex-determining region Y-box 9 (SOX9) either alone or in combinations. Methods Aggregate cultures of characterized ovine ASCs were transduced with 100 multiplicity of infections of Ad.IGF-1, Ad.TGF-β1, Ad.FGF-2, and Ad.SOX9 alone or in combination. These were harvested at various time points for detection of cartilage-specific genes expression by quantitative real-time PCR or after 14 and 28 days for histologic and biochemical analyses detecting proteoglycans, collagens (II, I and X), and total sulfated glycosaminoglycan and collagen content, respectively. Results Expression analyses showed that co-expression of IGF-1 and FGF-2 resulted in higher significant expression levels of aggrecan, biglycan, cartilage matrix, proteoglycan, and collagen II (all P ≤0.001 at 28 days). Aggregates co-transduced with Ad.IGF-1/Ad.FGF-2 showed a selective expression of proteoglycans and collagen II, with limited expression of collagens I and × demonstrated by histological analyses, and had significantly greater glycosaminoglycan and collagen production than the positive control (P ≤0.001). Western blot analyses for this combination also demonstrated increased expression of collagen II, while expression of collagens I and × was undetectable and limited, respectively. Conclusion Combined overexpression of IGF-1/FGF-2 within ASCs enhances their chondrogenic differentiation inducing the expression of chondrogenic markers, suggesting that this combination is more beneficial than the other factors tested for the development of cell-based therapies for cartilage repair. PMID:23899094

Growth differentiation factor 9 and its spatiotemporal expression and regulation in the zebrafish ovary.

PubMed

Liu, Lin; Ge, Wei

2007-02-01

Growth differentiation factor 9 (GDF9) is a member of the transforming growth factor beta (TGFB) superfamily. As an oocyte-specific growth factor, GDF9 plays critical roles in controlling folliculogenesis in mammals. In the present study, we cloned a 2.1-kb cDNA of the zebrafish GDF9 homolog (Gdf9, gdf9), which shares approximately 60% homology with that of mammals in the mature region. RT-PCR analysis showed that zebrafish gdf9 expression was present only in the gonads and Northern blot analysis revealed a single transcript of about 2.0 kb in the ovary. Real-time RT-PCR analysis revealed that gdf9 expression was highest in primary growth (PG, stage I) follicles and gradually decreased during follicular development, with the lowest level being found in fully grown (FG) follicles. The expression of gdf9 was maintained through fertilization and early embryonic development until gastrulation, at which point the expression level dramatically decreased. Expression was barely detectable after the late gastrula stage. Within the follicle, gdf9 mRNA was localized exclusively in the oocytes, as demonstrated by RT-PCR of denuded oocytes and freshly isolated follicle layers as well as by in situ hybridization. Interestingly, when amplified for high numbers of cycles, the expression of gdf9 was detected in cultured zebrafish follicular cells that were free of oocytes. The expression of gdf9 was downregulated by hCG in both ovarian fragments and isolated follicles in dose- and time-dependent manners, and this inhibition appeared to be stage-dependent, with the strongest inhibition observed for the FG follicles and no effect seen for the PG follicles. This correlates well with the expression profile of the LH receptor (lhcgr) in zebrafish follicles. In conclusion, as an oocyte-derived growth factor, GDF9 is highly conserved across vertebrates. With its biological advantages, zebrafish provides an alternative model for studying gene function and regulation.

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.

Two alternatively spliced GPR39 transcripts in seabream: molecular cloning, genomic organization, and regulation of gene expression by metabolic signals.

PubMed

Zhang, Yong; Liu, Yun; Huang, Xigui; Liu, Xiaochun; Jiao, Baowei; Meng, Zining; Zhu, Pei; Li, Shuisheng; Lin, Haoran; Cheng, Christopher H K

2008-12-01

Two GPR39 transcripts, designated as sbGPR39-1a and sbGPR39-1b, were identified in black seabream (Acanthopagrus schlegeli). The deduced amino acid (aa) sequence of sbGPR39-1a contains 423 residues with seven putative transmembrane (TM) domains. On the other hand, sbGPR39-1b contains 284 aa residues with only five putative TM domains. Northern blot analysis confirmed the presence of two GPR39 transcripts in the seabream intestine, stomach, and liver. Apart from seabream, the presence of two GPR39 transcripts was also found to exist in a number of teleosts (zebrafish and pufferfish) and mammals (human and mouse). Analysis of the GPR39 gene structure in different species suggests that the two GPR39 transcripts are generated by alternative splicing. When the seabream receptors were expressed in cultured HEK293 cells, Zn(2)(+) could trigger sbGPR39-1a signaling through the serum response element pathway, but no such functionality could be detected for the sbGPR39-1b receptor. The two receptors were found to be differentially expressed in seabream tissues. sbGPR39-1a is predominantly expressed in the gastrointestinal tract. On the other hand, sbGPR39-1b is widely expressed in most central and peripheral tissues except muscle and ovary. The expression of sbGPR39-1a in the intestine and the expression of sbGPR39-1b in the hypothalamus were decreased significantly during food deprivation in seabream. On the contrary, the expression of the GH secretagogue receptors (sbGHSR-1a and sbGHSR-1b) was significantly increased in the hypothalamus of the food-deprived seabream. The reciprocal regulatory patterns of expression of these two genes suggest that both of them are involved in controlling the physiological response of the organism during starvation.

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

Microarray Detection Call Methodology as a Means to Identify and Compare Transcripts Expressed within Syncytial Cells from Soybean (Glycine max) Roots Undergoing Resistant and Susceptible Reactions to the Soybean Cyst Nematode (Heterodera glycines)

PubMed Central

Klink, Vincent P.; Overall, Christopher C.; Alkharouf, Nadim W.; MacDonald, Margaret H.; Matthews, Benjamin F.

2010-01-01

Background. A comparative microarray investigation was done using detection call methodology (DCM) and differential expression analyses. The goal was to identify genes found in specific cell populations that were eliminated by differential expression analysis due to the nature of differential expression methods. Laser capture microdissection (LCM) was used to isolate nearly homogeneous populations of plant root cells. Results. The analyses identified the presence of 13,291 transcripts between the 4 different sample types. The transcripts filtered down into a total of 6,267 that were detected as being present in one or more sample types. A comparative analysis of DCM and differential expression methods showed a group of genes that were not differentially expressed, but were expressed at detectable amounts within specific cell types. Conclusion. The DCM has identified patterns of gene expression not shown by differential expression analyses. DCM has identified genes that are possibly cell-type specific and/or involved in important aspects of plant nematode interactions during the resistance response, revealing the uniqueness of a particular cell population at a particular point during its differentiation process. PMID:20508855

Expression of POEM, a positive regulator of osteoblast differentiation, is suppressed by TNF-{alpha}

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

Tsukasaki, Masayuki; Yamada, Atsushi, E-mail: yamadaa@dent.showa-u.ac.jp; Suzuki, Dai

2011-07-15

Highlights: {yields} TNF-{alpha} inhibits POEM gene expression. {yields} Inhibition of POEM gene expression is caused by NF-{kappa}B activation by TNF-{alpha}. {yields} Over-expression of POEM recovers inhibition of osteoblast differentiation by TNF-{alpha}. -- Abstract: POEM, also known as nephronectin, is an extracellular matrix protein considered to be a positive regulator of osteoblast differentiation. In the present study, we found that tumor necrosis factor-{alpha} (TNF-{alpha}), a key regulator of bone matrix properties and composition that also inhibits terminal osteoblast differentiation, strongly inhibited POEM expression in the mouse osteoblastic cell line MC3T3-E1. TNF-{alpha}-induced down-regulation of POEM gene expression occurred in both time- andmore » dose-dependent manners through the nuclear factor kappa B (NF-{kappa}B) pathway. In addition, expressions of marker genes in differentiated osteoblasts were down-regulated by TNF-{alpha} in a manner consistent with our findings for POEM, while over-expression of POEM recovered TNF-{alpha}-induced inhibition of osteoblast differentiation. These results suggest that TNF-{alpha} inhibits POEM expression through the NF-{kappa}B signaling pathway and down-regulation of POEM influences the inhibition of osteoblast differentiation by TNF-{alpha}.« less

Random forests-based differential analysis of gene sets for gene expression data.

PubMed

Hsueh, Huey-Miin; Zhou, Da-Wei; Tsai, Chen-An

2013-04-10

In DNA microarray studies, gene-set analysis (GSA) has become the focus of gene expression data analysis. GSA utilizes the gene expression profiles of functionally related gene sets in Gene Ontology (GO) categories or priori-defined biological classes to assess the significance of gene sets associated with clinical outcomes or phenotypes. Many statistical approaches have been proposed to determine whether such functionally related gene sets express differentially (enrichment and/or deletion) in variations of phenotypes. However, little attention has been given to the discriminatory power of gene sets and classification of patients. In this study, we propose a method of gene set analysis, in which gene sets are used to develop classifications of patients based on the Random Forest (RF) algorithm. The corresponding empirical p-value of an observed out-of-bag (OOB) error rate of the classifier is introduced to identify differentially expressed gene sets using an adequate resampling method. In addition, we discuss the impacts and correlations of genes within each gene set based on the measures of variable importance in the RF algorithm. Significant classifications are reported and visualized together with the underlying gene sets and their contribution to the phenotypes of interest. Numerical studies using both synthesized data and a series of publicly available gene expression data sets are conducted to evaluate the performance of the proposed methods. Compared with other hypothesis testing approaches, our proposed methods are reliable and successful in identifying enriched gene sets and in discovering the contributions of genes within a gene set. The classification results of identified gene sets can provide an valuable alternative to gene set testing to reveal the unknown, biologically relevant classes of samples or patients. In summary, our proposed method allows one to simultaneously assess the discriminatory ability of gene sets and the importance of genes for interpretation of data in complex biological systems. The classifications of biologically defined gene sets can reveal the underlying interactions of gene sets associated with the phenotypes, and provide an insightful complement to conventional gene set analyses. Copyright © 2012 Elsevier B.V. All rights reserved.

Differential impacts of juvenile hormone, soldier head extract and alternate caste phenotypes on host and symbiont transcriptome composition in the gut of the termite Reticulitermes flavipes.

PubMed

Sen, Ruchira; Raychoudhury, Rhitoban; Cai, Yunpeng; Sun, Yijun; Lietze, Verena-Ulrike; Boucias, Drion G; Scharf, Michael E

2013-07-19

Termites are highly eusocial insects and show a division of labor whereby morphologically distinct individuals specialize in distinct tasks. In the lower termite Reticulitermes flavipes (Rhinotermitidae), non-reproducing individuals form the worker and soldier castes, which specialize in helping (e.g., brood care, cleaning, foraging) and defense behaviors, respectively. Workers are totipotent juveniles that can either undergo status quo molts or develop into soldiers or neotenic reproductives. This caste differentiation can be regulated by juvenile hormone (JH) and primer pheromones contained in soldier head extracts (SHE). Here we offered worker termites a cellulose diet treated with JH or SHE for 24-hr, or held them with live soldiers (LS) or live neotenic reproductives (LR). We then determined gene expression profiles of the host termite gut and protozoan symbionts concurrently using custom cDNA oligo-microarrays containing 10,990 individual ESTs. JH was the most influential treatment (501 total ESTs affected), followed by LS (24 ESTs), LR (12 ESTs) and SHE treatments (6 ESTs). The majority of JH up- and downregulated ESTs were of host and symbiont origin, respectively; in contrast, SHE, LR and LS treatments had more uniform impacts on host and symbiont gene expression. Repeat "follow-up" bioassays investigating combined JH + SHE impacts in relation to individual JH and SHE treatments on a subset of array-positive genes revealed (i) JH and SHE treatments had opposite impacts on gene expression and (ii) JH + SHE impacts on gene expression were generally intermediate between JH and SHE. Our results show that JH impacts hundreds of termite and symbiont genes within 24-hr, strongly suggesting a role for the termite gut in JH-dependent caste determination. Additionally, differential impacts of SHE and LS treatments were observed that are in strong agreement with previous studies that specifically investigated soldier caste regulation. However, it is likely that gene expression outside the gut may be of equal or greater importance than gut gene expression.

Differential impacts of juvenile hormone, soldier head extract and alternate caste phenotypes on host and symbiont transcriptome composition in the gut of the termite Reticulitermes flavipes

PubMed Central

2013-01-01

Background Termites are highly eusocial insects and show a division of labor whereby morphologically distinct individuals specialize in distinct tasks. In the lower termite Reticulitermes flavipes (Rhinotermitidae), non-reproducing individuals form the worker and soldier castes, which specialize in helping (e.g., brood care, cleaning, foraging) and defense behaviors, respectively. Workers are totipotent juveniles that can either undergo status quo molts or develop into soldiers or neotenic reproductives. This caste differentiation can be regulated by juvenile hormone (JH) and primer pheromones contained in soldier head extracts (SHE). Here we offered worker termites a cellulose diet treated with JH or SHE for 24-hr, or held them with live soldiers (LS) or live neotenic reproductives (LR). We then determined gene expression profiles of the host termite gut and protozoan symbionts concurrently using custom cDNA oligo-microarrays containing 10,990 individual ESTs. Results JH was the most influential treatment (501 total ESTs affected), followed by LS (24 ESTs), LR (12 ESTs) and SHE treatments (6 ESTs). The majority of JH up- and downregulated ESTs were of host and symbiont origin, respectively; in contrast, SHE, LR and LS treatments had more uniform impacts on host and symbiont gene expression. Repeat “follow-up” bioassays investigating combined JH + SHE impacts in relation to individual JH and SHE treatments on a subset of array-positive genes revealed (i) JH and SHE treatments had opposite impacts on gene expression and (ii) JH + SHE impacts on gene expression were generally intermediate between JH and SHE. Conclusions Our results show that JH impacts hundreds of termite and symbiont genes within 24-hr, strongly suggesting a role for the termite gut in JH-dependent caste determination. Additionally, differential impacts of SHE and LS treatments were observed that are in strong agreement with previous studies that specifically investigated soldier caste regulation. However, it is likely that gene expression outside the gut may be of equal or greater importance than gut gene expression. PMID:23870282

Isolation and cellular properties of mesenchymal cells derived from the decidua of human term placenta.

PubMed

Kanematsu, Daisuke; Shofuda, Tomoko; Yamamoto, Atsuyo; Ban, Chiaki; Ueda, Takafumi; Yamasaki, Mami; Kanemura, Yonehiro

2011-09-01

The clinical promise of cell-based therapies is generally recognized, and has driven an intense search for good cell sources. In this study, we isolated plastic-adherent cells from human term decidua vera, called decidua-derived-mesenchymal cells (DMCs), and compared their properties with those of bone marrow-derived-mesenchymal stem cells (BM-MSCs). The DMCs strongly expressed the mesenchymal cell marker vimentin, but not cytokeratin 19 or HLA-G, and had a high proliferative potential. That is, they exhibited a typical fibroblast-like morphology for over 30 population doublings. Cells phenotypically identical to the DMCs were identified in the decidua vera, and genotyping confirmed that the DMCs were derived from the maternal components of the fetal adnexa. Flow cytometry analysis showed that the expression pattern of CD antigens on the DMCs was almost identical to that on BM-MSCs, but some DMCs expressed the CD45 antigen, and over 50% of them also expressed anti-fibroblast antigen. In vitro, the DMCs showed good differentiation into chondrocytes and moderate differentiation into adipocytes, but scant evidence of osteogenesis, compared with the BM-MSCs. Gene expression analysis showed that, compared with BM-MSCs, the DMCs expressed higher levels of TWIST2 and RUNX2 (which are associated with early mesenchymal development and/or proliferative capacity), several matrix metalloproteinases (MMP1, 3, 10, and 12), and cytokines (BMP2 and TGFB2), and lower levels of MSX2, interleukin 26, and HGF. Although DMCs did not show the full multipotency of BM-MSCs, their higher proliferative ability indicates that their cultivation would require less maintenance. Furthermore, the use of DMCs avoids the ethical concerns associated with the use of embryonic tissues, because they are derived from the maternal portion of the placenta, which is otherwise discarded. Thus, the unique properties of DMCs give them several advantages for clinical use, making them an interesting and attractive alternative to MSCs for regenerative medicine. 2011 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

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.

Induction of human embryonic and induced pluripotent stem cells into urothelium.

PubMed

Osborn, Stephanie L; Thangappan, Ravikumar; Luria, Ayala; Lee, Justin H; Nolta, Jan; Kurzrock, Eric A

2014-05-01

In vitro generation of human urothelium from stem cells would be a major advancement in the regenerative medicine field, providing alternate nonurologic and/or nonautologous tissue sources for bladder grafts. Such a model would also help decipher the mechanisms of urothelial differentiation and would facilitate investigation of deviated differentiation of normal progenitors into urothelial cancer stem cells, perhaps elucidating areas of intervention for improved treatments. Thus far, in vitro derivation of urothelium from human embryonic stem cells (hESCs) or human induced pluripotent stem (hiPS) cells has not been reported. The goal of this work was to develop an efficient in vitro protocol for the induction of hESCs into urothelium through an intermediary definitive endoderm step and free of matrices and cell contact. During directed differentiation in a urothelial-specific medium ("Uromedium"), hESCs produced up to 60% urothelium, as determined by uroplakin expression; subsequent propagation selected for 90% urothelium. Alteration of the epithelial and mesenchymal cell signaling contribution through noncell contact coculture or conditioned media did not enhance the production of urothelium. Temporospatial evaluation of transcription factors known to be involved in urothelial specification showed association of IRF1, GET1, and GATA4 with uroplakin expression. Additional hESC and hiPS cell lines could also be induced into urothelium using this in vitro system. These results demonstrate that derivation and propagation of urothelium from hESCs and hiPS cells can be efficiently accomplished in vitro in the absence of matrices, cell contact, or adult cell signaling and that the induction process appears to mimic normal differentiation.

Induction of Human Embryonic and Induced Pluripotent Stem Cells Into Urothelium

PubMed Central

Osborn, Stephanie L.; Thangappan, Ravikumar; Luria, Ayala; Lee, Justin H.; Nolta, Jan

2014-01-01

In vitro generation of human urothelium from stem cells would be a major advancement in the regenerative medicine field, providing alternate nonurologic and/or nonautologous tissue sources for bladder grafts. Such a model would also help decipher the mechanisms of urothelial differentiation and would facilitate investigation of deviated differentiation of normal progenitors into urothelial cancer stem cells, perhaps elucidating areas of intervention for improved treatments. Thus far, in vitro derivation of urothelium from human embryonic stem cells (hESCs) or human induced pluripotent stem (hiPS) cells has not been reported. The goal of this work was to develop an efficient in vitro protocol for the induction of hESCs into urothelium through an intermediary definitive endoderm step and free of matrices and cell contact. During directed differentiation in a urothelial-specific medium (“Uromedium”), hESCs produced up to 60% urothelium, as determined by uroplakin expression; subsequent propagation selected for 90% urothelium. Alteration of the epithelial and mesenchymal cell signaling contribution through noncell contact coculture or conditioned media did not enhance the production of urothelium. Temporospatial evaluation of transcription factors known to be involved in urothelial specification showed association of IRF1, GET1, and GATA4 with uroplakin expression. Additional hESC and hiPS cell lines could also be induced into urothelium using this in vitro system. These results demonstrate that derivation and propagation of urothelium from hESCs and hiPS cells can be efficiently accomplished in vitro in the absence of matrices, cell contact, or adult cell signaling and that the induction process appears to mimic normal differentiation. PMID:24657961

Effect of curcumin on aged Drosophila melanogaster: a pathway prediction analysis.

PubMed

Zhang, Zhi-guo; Niu, Xu-yan; Lu, Ai-ping; Xiao, Gary Guishan

2015-02-01

To re-analyze the data published in order to explore plausible biological pathways that can be used to explain the anti-aging effect of curcumin. Microarray data generated from other study aiming to investigate effect of curcumin on extending lifespan of Drosophila melanogaster were further used for pathway prediction analysis. The differentially expressed genes were identified by using GeneSpring GX with a criterion of 3.0-fold change. Two Cytoscape plugins including BisoGenet and molecular complex detection (MCODE) were used to establish the protein-protein interaction (PPI) network based upon differential genes in order to detect highly connected regions. The function annotation clustering tool of Database for Annotation, Visualization and Integrated Discovery (DAVID) was used for pathway analysis. A total of 87 genes expressed differentially in D. melanogaster melanogaster treated with curcumin were identified, among which 50 were up-regulated significantly and 37 were remarkably down-regulated in D. melanogaster melanogaster treated with curcumin. Based upon these differential genes, PPI network was constructed with 1,082 nodes and 2,412 edges. Five highly connected regions in PPI networks were detected by MCODE algorithm, suggesting anti-aging effect of curcumin may be underlined through five different pathways including Notch signaling pathway, basal transcription factors, cell cycle regulation, ribosome, Wnt signaling pathway, and p53 pathway. Genes and their associated pathways in D. melanogaster melanogaster treated with anti-aging agent curcumin were identified using PPI network and MCODE algorithm, suggesting that curcumin may be developed as an alternative therapeutic medicine for treating aging-associated diseases.

Isolation and Characterization of Human Mesenchymal Stem Cells From Facet Joints and Interspinous Ligaments.

PubMed

Kristjánsson, Baldur; Limthongkul, Worawat; Yingsakmongkol, Wicharn; Thantiworasit, Pattarawat; Jirathanathornnukul, Napaphat; Honsawek, Sittisak

2016-01-01

A descriptive in vitro study on isolation and differentiation of human mesenchymal stem cells (MSCs) derived from the facet joints and interspinous ligaments. To isolate cells from the facet joints and interspinous ligaments and investigate their surface marker profile and differentiation potentials. Lumbar spinal canal stenosis and ossification of the posterior longitudinal ligament are progressive conditions characterized by the hypertrophy and ossification of ligaments and joints within the spinal canal. MSCs are believed to play a role in the advancement of these diseases and the existence of MSCs has been demonstrated within the ligamentum flavum and posterior longitudinal ligament. The aim of this study was to investigate whether these cells could also be found within facet joints and interspinous ligaments. Samples were harvested from 10 patients undergoing spinal surgery. The MSCs from facet joints and interspinous ligaments were isolated using direct tissue explant technique. Cell surface antigen profilings were performed via flow cytometry. Their lineage differentiation potentials were analyzed. The facet joints and interspinous ligaments-derived MSCs have the tri-lineage potential to be differentiated into osteogenic, adipogenic, and chondrogenic cells under appropriate inductions. Flow cytometry analysis revealed both cell lines expressed MSCs markers. Both facet joints and interspinous ligaments-derived MSCs expressed marker genes for osteoblasts, adipocytes, and chondrocytes. The facet joints and interspinous ligaments may provide alternative sources of MSCs for tissue engineering applications. The facet joints and interspinous ligaments-derived MSCs are part of the microenvironment of the human ligaments of the spinal column and might play a crucial role in the development and progression of degenerative spine conditions.

Searching for microbial protein over-expression in a complex matrix using automated high throughput MS-based proteomics tools.

PubMed

Akeroyd, Michiel; Olsthoorn, Maurien; Gerritsma, Jort; Gutker-Vermaas, Diana; Ekkelkamp, Laurens; van Rij, Tjeerd; Klaassen, Paul; Plugge, Wim; Smit, Ed; Strupat, Kerstin; Wenzel, Thibaut; van Tilborg, Marcel; van der Hoeven, Rob

2013-03-10

In the discovery of new enzymes genomic and cDNA expression libraries containing thousands of differential clones are generated to obtain biodiversity. These libraries need to be screened for the activity of interest. Removing so-called empty and redundant clones significantly reduces the size of these expression libraries and therefore speeds up new enzyme discovery. Here, we present a sensitive, generic workflow for high throughput screening of successful microbial protein over-expression in microtiter plates containing a complex matrix based on mass spectrometry techniques. MALDI-LTQ-Orbitrap screening followed by principal component analysis and peptide mass fingerprinting was developed to obtain a throughput of ∼12,000 samples per week. Alternatively, a UHPLC-MS(2) approach including MS(2) protein identification was developed for microorganisms with a complex protein secretome with a throughput of ∼2000 samples per week. TCA-induced protein precipitation enhanced by addition of bovine serum albumin is used for protein purification prior to MS detection. We show that this generic workflow can effectively reduce large expression libraries from fungi and bacteria to their minimal size by detection of successful protein over-expression using MS. Copyright © 2012 Elsevier B.V. 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.

Tissue Elasticity Regulated Tumor Gene Expression: Implication for Diagnostic Biomarkers of Primitive Neuroectodermal Tumor

PubMed Central

Vu, Long T.; Keschrumrus, Vic; Zhang, Xi; Zhong, Jiang F.; Su, Qingning; Kabeer, Mustafa H.; Loudon, William G.; Li, Shengwen Calvin

2015-01-01

Background The tumor microenvironment consists of both physical and chemical factors. Tissue elasticity is one physical factor contributing to the microenvironment of tumor cells. To test the importance of tissue elasticity in cell culture, primitive neuroectodermal tumor (PNET) stem cells were cultured on soft polyacrylamide (PAA) hydrogel plates that mimics the elasticity of brain tissue compared with PNET on standard polystyrene (PS) plates. We report the molecular profiles of PNET grown on either PAA or PS. Methodology/Principal Findings A whole-genome microarray profile of transcriptional expression between the two culture conditions was performed as a way to probe effects of substrate on cell behavior in culture. The results showed more genes downregulated on PAA compared to PS. This led us to propose microRNA (miRNA) silencing as a potential mechanism for downregulation. Bioinformatic analysis predicted a greater number of miRNA binding sites from the 3' UTR of downregulated genes and identified as specific miRNA binding sites that were enriched when cells were grown on PAA—this supports the hypothesis that tissue elasticity plays a role in influencing miRNA expression. Thus, Dicer was examined to determine if miRNA processing was affected by tissue elasticity. Dicer genes were downregulated on PAA and had multiple predicted miRNA binding sites in its 3' UTR that matched the miRNA binding sites found enriched on PAA. Many differentially regulated genes were found to be present on PS but downregulated on PAA were mapped onto intron sequences. This suggests expression of alternative polyadenylation sites within intron regions that provide alternative 3' UTRs and alternative miRNA binding sites. This results in tissue specific transcriptional downregulation of mRNA in humans by miRNA. We propose a mechanism, driven by the physical characteristics of the microenvironment by which downregulation of genes occur. We found that tissue elasticity-mediated cytokines (TGFβ2 and TNFα) signaling affect expression of ECM proteins. Conclusions Our results suggest that tissue elasticity plays important roles in miRNA expression, which, in turn, regulate tumor growth or tumorigenicity. PMID:25774514

MCM2: An alternative to Ki-67 for measuring breast cancer cell proliferation.

PubMed

Yousef, Einas M; Furrer, Daniela; Laperriere, David L; Tahir, Muhammad R; Mader, Sylvie; Diorio, Caroline; Gaboury, Louis A

2017-05-01

Breast cancer is a heterogeneous disease comprising a diversity of tumor subtypes that manifest themselves in a wide variety of clinical, pathological, and molecular features. One important subset, luminal breast cancers, comprises two clinically distinct subtypes luminal A and B each of them endowed with its own genetic program of differentiation and proliferation. Luminal breast cancers were operationally defined as follows: Luminal A: ER+, PR+, HER2-, Ki-67<14% and Luminal B: ER+ and/or PR+, HER2-,Ki-67≥14% or, alternatively ER+ and/or PR+, HER2+, any Ki-67. There is currently a need for a clinically robust and validated immunohistochemical assay that can help distinguish between luminal A and B breast cancer. MCM2 is a family member of the minichromosome maintenance protein complex whose role in DNA replication and cell proliferation is firmly established. As MCM2 appears to be an attractive alternative to Ki-67, we sought to study the expression of MCM2 and Ki-67 in different histological grades and molecular subtypes of breast cancer focusing primarily on ER-positive tumors. MCM2 and Ki-67 mRNA expression were studied using in silico analysis of available DNA microarray and RNA-sequencing data of human breast cancer. We next used immunohistochemistry to evaluate protein expression of MCM2 and Ki-67 on tissue microarrays of invasive breast carcinoma. We found that MCM2 and Ki-67 are highly expressed in breast tumors of high histological grades, comprising clinically aggressive tumors such as triple-negative, HER2-positive and luminal B subtypes. MCM2 expression was detected at higher levels than that of Ki-67 in normal breast tissues and in breast cancers. The bimodal distribution of MCM2 scores in ER+/HER2- breast tumors led to the identification of two distinct subgroups with different relapse-free survival rates. In conclusion, MCM2 expression can help sorting out two clinically important subsets of luminal breast cancer whose treatment and clinical outcomes are likely to diverge.

alpha2-chimaerin, a Cdc42/Rac1 regulator, is selectively expressed in the rat embryonic nervous system and is involved in neuritogenesis in N1E-115 neuroblastoma cells.

PubMed

Hall, C; Michael, G J; Cann, N; Ferrari, G; Teo, M; Jacobs, T; Monfries, C; Lim, L

2001-07-15

Neuronal differentiation involves Rac and Cdc42 GTPases. alpha-Chimaerin, a Rac/Cdc42 regulator, occurs as alpha1- and alternatively spliced Src homology 2 (SH2) domain-containing alpha2-isoforms. alpha2-chimaerin mRNA was highly expressed in the rat embryonic nervous system, especially in early postmitotic neurons. alpha1-chimaerin mRNA was undetectable before embryonic day 16.5. Adult alpha2-chimaerin mRNA was restricted to neurons within specific brain regions, with highest expression in the entorhinal cortex. alpha2-chimaerin protein localized to neuronal perikarya, dendrites, and axons. The overall pattern of alpha2-chimaerin mRNA expression resembles that of cyclin-dependent kinase regulator p35 (CDK5/p35) which participates in neuronal differentiation and with which chimaerin interacts. To determine whether alpha2-chimaerin may have a role in neuronal differentiation and the relevance of the SH2 domain, the morphological effects of both chimaerin isoforms were investigated in N1E-115 neuroblastoma cells. When plated on poly-lysine, transient alpha2-chimaerin but not alpha1-chimaerin transfectants formed neurites. Permanent alpha2-chimaerin transfectants generated neurites whether or not they were stimulated by serum starvation, and many cells were enlarged. Permanent alpha1-chimaerin transfectants displayed numerous microspikes and contained F-actin clusters, a Cdc42-phenotype, but generated few neurites. In neuroblastoma cells, alpha2-chimaerin was predominantly soluble with some being membrane-associated, whereas alpha1-chimaerin was absent from the cytosol, being membrane- and cytoskeleton-associated, paralleling their subcellular distribution in brain. Transient transfection with alpha2-chimaerin mutated in the SH2 domain (N94H) generated an alpha1-chimaerin-like phenotype, protein partitioned in the particulate fraction, and in NGF-stimulated pheochromocytoma cell line 12 (PC12) cells, neurite formation was inhibited. These results indicate a role for alpha2-chimaerin in morphological differentiation for which its SH2 domain is vital.

Regulatory network involving miRNAs and genes in serous ovarian carcinoma

PubMed Central

Zhao, Haiyan; Xu, Hao; Xue, Luchen

2017-01-01

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

An efficient technique for higher order fractional differential equation.

PubMed

Ali, Ayyaz; Iqbal, Muhammad Asad; Ul-Hassan, Qazi Mahmood; Ahmad, Jamshad; Mohyud-Din, Syed Tauseef

2016-01-01

In this study, we establish exact solutions of fractional Kawahara equation by using the idea of [Formula: see text]-expansion method. The results of different studies show that the method is very effective and can be used as an alternative for finding exact solutions of nonlinear evolution equations (NLEEs) in mathematical physics. The solitary wave solutions are expressed by the hyperbolic, trigonometric, exponential and rational functions. Graphical representations along with the numerical data reinforce the efficacy of the used procedure. The specified idea is very effective, expedient for fractional PDEs, and could be extended to other physical problems.

Histone acetylation is associated with differential gene expression in the rapid and robust memory CD8+ T-cell response

PubMed Central

Fann, Monchou; Godlove, Jason M.; Catalfamo, Marta; Wood, William H.; Chrest, Francis J.; Chun, Nicholas; Granger, Larry; Wersto, Robert; Madara, Karen; Becker, Kevin; Henkart, Pierre A.; Weng, Nan-ping

2006-01-01

To understand the molecular basis for the rapid and robust memory T-cell responses, we examined gene expression and chromatin modification by histone H3 lysine 9 (H3K9) acetylation in resting and activated human naive and memory CD8+ T cells. We found that, although overall gene expression patterns were similar, a number of genes are differentially expressed in either memory or naive cells in their resting and activated states. To further elucidate the basis for differential gene expression, we assessed the role of histone H3K9 acetylation in differential gene expression. Strikingly, higher H3K9 acetylation levels were detected in resting memory cells, prior to their activation, for those genes that were differentially expressed following activation, indicating that hyperacetylation of histone H3K9 may play a role in selective and rapid gene expression of memory CD8+ T cells. Consistent with this model, we showed that inducing high levels of H3K9 acetylation resulted in an increased expression in naive cells of those genes that are normally expressed differentially in memory cells. Together, these findings suggest that differential gene expression mediated at least in part by histone H3K9 hyperacetylation may be responsible for the rapid and robust memory CD8+ T-cell response. PMID:16868257

Trend of telomerase activity change during human iPSC self-renewal and differentiation revealed by a quartz crystal microbalance based assay

NASA Astrophysics Data System (ADS)

Zhou, Yitian; Zhou, Ping; Xin, Yinqiang; Wang, Jie; Zhu, Zhiqiang; Hu, Ji; Wei, Shicheng; Ma, Hongwei

2014-11-01

Telomerase plays an important role in governing the life span of cells for its capacity to extend telomeres. As high activity of telomerase has been found in stem cells and cancer cells specifically, various methods have been developed for the evaluation of telomerase activity. To overcome the time-consuming procedures and complicated manipulations of existing methods, we developed a novel method named Telomeric Repeat Elongation Assay based on Quartz crystal microbalance (TREAQ) to monitor telomerase activity during the self-renewal and differentiation of human induced pluripotent stem cells (hiPSCs). TREAQ results indicated hiPSCs possess invariable telomerase activity for 11 passages on Matrigel and a steady decline of telomerase activity when differentiated for different periods, which is confirmed with existing golden standard method. The pluripotency of hiPSCs during differentiation could be estimated through monitoring telomerase activity and compared with the expression levels of markers of pluripotency gene via quantitative real time PCR. Regular assessment for factors associated with pluripotency or stemness was expensive and requires excessive sample consuming, thus TREAQ could be a promising alternative technology for routine monitoring of telomerase activity and estimate the pluripotency of stem cells.

Differential effects of miR-34c-3p and miR-34c-5p on SiHa cells proliferation apoptosis, migration and invasion.

PubMed

López, Jesús Adrián; Alvarez-Salas, Luis Marat

2011-06-10

MicroRNAs (miRNA) regulate expression of several genes associated with human cancer. Here, we analyzed the function of miR-34c, an effector of p53, in cervical carcinoma cells. Expression of either miR-34c-3p or miR-34c-5p mimics caused inhibition of cell proliferation in the HPV-containing SiHa cells but not in other cervical cells irrespective of tumorigenicity and HPV content. These results suggest that SiHa cells may lack of regulatory mechanisms for miR-34c. Monolayer proliferation results showed that miR-34c-3p produced a more pronounced inhibitory effect although both miRNAs caused inhibition of anchorage independent growth at similar extent. However, ectopic expression of pre-miR-34c-3p, but not pre-miR-34c-5p, caused S-phase arrest in SiHa cells triggering a strong dose-dependent apoptosis. A significant inhibition was observed only for miR-34c-3p on SiHa cells migration and invasion, therefore implying alternative regulatory pathways and targets. These results suggest differential tumor suppressor roles for miR-34c-3p and miR-34c-5p and provide new insights in the understanding of miRNA biology. Copyright © 2011 Elsevier Inc. All rights reserved.

Time-Dependent Effect of Encapsulating Alginate Hydrogel on Neurogenic Potential

PubMed Central

Razavi, Shahnaz; Khosravizadeh, Zahra; Bahramian, Hamid; Kazemi, Mohammad

2015-01-01

Objective Due to the restricted potential of neural stem cells for regeneration of central nervous system (CNS) after injury, providing an alternative source for neural stem cells is essential. Adipose derived stem cells (ADSCs) are multipotent cells with properties suitable for tissue engineering. In addition, alginate hydrogel is a biocompatible polysaccharide polymer that has been used to encapsulate many types of cells. The aim of this study was to assess the proliferation rate and level of expression of neural markers; NESTIN, glial fibrillary acidic protein (GFAP) and microtubule-associated protein 2 (MAP2) in encapsulated human ADSCs (hADSCs) 10 and14 days after neural induction. Materials and Methods In this experimental study, ADSCs isolated from human were cultured in neural induction media and seeded into alginate hydrogel. The rate of proliferation and differentiation of encapsulated cells were evaluated by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay, immunocytoflourescent and realtime reverse transcriptase polymerase chain reaction (RT-PCR) analyzes 10 and 14 days after induction. Results The rate of proliferation of encapsulated cells was not significantly changed with time passage. The expression of NESTIN and GFAP significantly decreased on day 14 relative to day 10 (P<0.001) but MAP2 expression was increased. Conclusion Alginate hydrogel can promote the neural differentiation of encapsulated hADSCs with time passage. PMID:26199909

Transcriptomic Analysis Reveals Selective Metabolic Adaptation of Streptococcus suis to Porcine Blood and Cerebrospinal Fluid

PubMed Central

Koczula, Anna; Jarek, Michael; Visscher, Christian; Valentin-Weigand, Peter; Goethe, Ralph; Willenborg, Jörg

2017-01-01

Streptococcus suis is a zoonotic pathogen that can cause severe pathologies such as septicemia and meningitis in its natural porcine host as well as in humans. Establishment of disease requires not only virulence of the infecting strain but also an appropriate metabolic activity of the pathogen in its host environment. However, it is yet largely unknown how the streptococcal metabolism adapts to the different host niches encountered during infection. Our previous isotopologue profiling studies on S. suis grown in porcine blood and cerebrospinal fluid (CSF) revealed conserved activities of central carbon metabolism in both body fluids. On the other hand, they suggested differences in the de novo amino acid biosynthesis. This prompted us to further dissect S. suis adaptation to porcine blood and CSF by RNA deep sequencing (RNA-seq). In blood, the majority of differentially expressed genes were associated with transport of alternative carbohydrate sources and the carbohydrate metabolism (pentose phosphate pathway, glycogen metabolism). In CSF, predominantly genes involved in the biosynthesis of branched-chain and aromatic amino acids were differentially expressed. Especially, isoleucine biosynthesis seems to be of major importance for S. suis in CSF because several related biosynthetic genes were more highly expressed. In conclusion, our data revealed niche-specific metabolic gene activity which emphasizes a selective adaptation of S. suis to host environments. PMID:28212285

Transcriptomic Analysis Reveals Selective Metabolic Adaptation of Streptococcus suis to Porcine Blood and Cerebrospinal Fluid.

PubMed

Koczula, Anna; Jarek, Michael; Visscher, Christian; Valentin-Weigand, Peter; Goethe, Ralph; Willenborg, Jörg

2017-02-15

Streptococcus suis is a zoonotic pathogen that can cause severe pathologies such as septicemia and meningitis in its natural porcine host as well as in humans. Establishment of disease requires not only virulence of the infecting strain but also an appropriate metabolic activity of the pathogen in its host environment. However, it is yet largely unknown how the streptococcal metabolism adapts to the different host niches encountered during infection. Our previous isotopologue profiling studies on S. suis grown in porcine blood and cerebrospinal fluid (CSF) revealed conserved activities of central carbon metabolism in both body fluids. On the other hand, they suggested differences in the de novo amino acid biosynthesis. This prompted us to further dissect S. suis adaptation to porcine blood and CSF by RNA deep sequencing (RNA-seq). In blood, the majority of differentially expressed genes were associated with transport of alternative carbohydrate sources and the carbohydrate metabolism (pentose phosphate pathway, glycogen metabolism). In CSF, predominantly genes involved in the biosynthesis of branched-chain and aromatic amino acids were differentially expressed. Especially, isoleucine biosynthesis seems to be of major importance for S. suis in CSF because several related biosynthetic genes were more highly expressed. In conclusion, our data revealed niche-specific metabolic gene activity which emphasizes a selective adaptation of S. suis to host environments.

Computational selection of antibody-drug conjugate targets for breast cancer

PubMed Central

Fauteux, François; Hill, Jennifer J.; Jaramillo, Maria L.; Pan, Youlian; Phan, Sieu; Famili, Fazel; O'Connor-McCourt, Maureen

2016-01-01

The selection of therapeutic targets is a critical aspect of antibody-drug conjugate research and development. In this study, we applied computational methods to select candidate targets overexpressed in three major breast cancer subtypes as compared with a range of vital organs and tissues. Microarray data corresponding to over 8,000 tissue samples were collected from the public domain. Breast cancer samples were classified into molecular subtypes using an iterative ensemble approach combining six classification algorithms and three feature selection techniques, including a novel kernel density-based method. This feature selection method was used in conjunction with differential expression and subcellular localization information to assemble a primary list of targets. A total of 50 cell membrane targets were identified, including one target for which an antibody-drug conjugate is in clinical use, and six targets for which antibody-drug conjugates are in clinical trials for the treatment of breast cancer and other solid tumors. In addition, 50 extracellular proteins were identified as potential targets for non-internalizing strategies and alternative modalities. Candidate targets linked with the epithelial-to-mesenchymal transition were identified by analyzing differential gene expression in epithelial and mesenchymal tumor-derived cell lines. Overall, these results show that mining human gene expression data has the power to select and prioritize breast cancer antibody-drug conjugate targets, and the potential to lead to new and more effective cancer therapeutics. PMID:26700623

Identification of ovarian cancer-associated proteins in symptomatic women: A novel method for semi-quantitative plasma proteomics.

PubMed

Shield-Artin, Kristy L; Bailey, Mark J; Oliva, Karen; Liovic, Ana K; Barker, Gillian; Dellios, Nicole L; Reisman, Simone; Ayhan, Mustafa; Rice, Gregory E

2012-04-01

To evaluate the utility of an enhanced biomarker discovery approach in order to identify potential biomarkers relevant to ovarian cancer detection. We combined immuno-depletion, liquid-phase IEF, 1D-DIGE, MALDI-TOF/MS and LC-MS/MS to identify differentially expressed proteins in the plasma of symptomatic ovarian cancer patients, stratified by stage, compared to samples obtained from normal subjects. We demonstrate that this approach is a practical alternative to traditional 2D gel techniques and that it has some advantages, most notably increased protein capacity. Proteins were identified in all 76 bands excised from the gels in this project and confirmed the cancer-associated expression of several well-established biomarkers of ovarian cancer. These included C-reactive protein (CRP), haptoglobin, alpha-2 macroglobulin and A1A2. We also identified new ovarian cancer candidate biomarkers, Protein S100-A9 (S100A9) and multimerin-2. The cancer-associated differential expression of CRP and S100A9 was further confirmed by Western blot and ELISA. The methods developed in this study allow for the increased loading of plasma proteins into the analytical stream when compared to traditional 2D-DIGE. This increased protein identification sensitivity allowed us to identify new putative ovarian cancer biomarkers. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Variable expression of podocyte-related markers in the glomeruloid bodies in Wilms tumor.

PubMed

Kanemoto, Katsuyoshi; Takahashi, Shori; Shu, Yujing; Usui, Joichi; Tomari, Shinsuke; Yan, Kunimasa; Hamazaki, Yutaka; Nagata, Michio

2003-09-01

Several podocyte-related markers are organized to express in glomerular differentiation. However, whether expression of them is virtually synchronized and a reliable indicator of the state of differentiation is unknown. The present study investigated, by immunohistochemistry, the divergent expression of several podocyte markers in the improperly differentiated glomeruloid bodies from four cases of Wilms tumors. The glomeruloid bodies were classified into immature (IGB) or mature forms (MGB) based on morphology and epithelial features. Podocytes in IGB expressed WT1, synaptopodin, podocalyxin, and nephrin, and their expression was stronger in MGB. In contrast, Pax2 was strong in IGB and diminished in MGB. p27 was first expressed in MGB. The expression pattern in each molecule mimics normal glomerulogenesis. Podocytes in MGB showed persistent expression of bcl-2 and cytokeratin with synaptopodin, podocalyxin, and nephrin by serial section, a finding unusual for normal glomerulogenesis. Moreover, parietal cells in MGB also occasionally expressed these podocyte markers. The ultrastructure revealed that podocytes in MGB showed tight junctions without foot process formations, which indicated incomplete differentiation. These results suggest that a set of podocyte differentiation markers are occasionally diversely expressed, and raise the possibility that expression of these markers is insufficient to determine the state of terminal differentiation in podocytes.

Engineering bone tissue substitutes from human induced pluripotent stem cells.

PubMed

de Peppo, Giuseppe Maria; Marcos-Campos, Iván; Kahler, David John; Alsalman, Dana; Shang, Linshan; Vunjak-Novakovic, Gordana; Marolt, Darja

2013-05-21

Congenital defects, trauma, and disease can compromise the integrity and functionality of the skeletal system to the extent requiring implantation of bone grafts. Engineering of viable bone substitutes that can be personalized to meet specific clinical needs represents a promising therapeutic alternative. The aim of our study was to evaluate the utility of human-induced pluripotent stem cells (hiPSCs) for bone tissue engineering. We first induced three hiPSC lines with different tissue and reprogramming backgrounds into the mesenchymal lineages and used a combination of differentiation assays, surface antigen profiling, and global gene expression analysis to identify the lines exhibiting strong osteogenic differentiation potential. We then engineered functional bone substitutes by culturing hiPSC-derived mesenchymal progenitors on osteoconductive scaffolds in perfusion bioreactors and confirmed their phenotype stability in a subcutaneous implantation model for 12 wk. Molecular analysis confirmed that the maturation of bone substitutes in perfusion bioreactors results in global repression of cell proliferation and an increased expression of lineage-specific genes. These results pave the way for growing patient-specific bone substitutes for reconstructive treatments of the skeletal system and for constructing qualified experimental models of development and disease.

Phenotypic Heterogeneity and the Evolution of Bacterial Life Cycles.

PubMed

van Gestel, Jordi; Nowak, Martin A

2016-02-01

Most bacteria live in colonies, where they often express different cell types. The ecological significance of these cell types and their evolutionary origin are often unknown. Here, we study the evolution of cell differentiation in the context of surface colonization. We particularly focus on the evolution of a 'sticky' cell type that is required for surface attachment, but is costly to express. The sticky cells not only facilitate their own attachment, but also that of non-sticky cells. Using individual-based simulations, we show that surface colonization rapidly evolves and in most cases leads to phenotypic heterogeneity, in which sticky and non-sticky cells occur side by side on the surface. In the presence of regulation, cell differentiation leads to a remarkable set of bacterial life cycles, in which cells alternate between living in the liquid and living on the surface. The dominant life stage is formed by the surface-attached colony that shows many complex features: colonies reproduce via fission and by producing migratory propagules; cells inside the colony divide labour; and colonies can produce filaments to facilitate expansion. Overall, our model illustrates how the evolution of an adhesive cell type goes hand in hand with the evolution of complex bacterial life cycles.

Gankyrin is a predictive and oncogenic factor in well-differentiated and dedifferentiated liposarcoma.

PubMed

Hwang, Ju-Ae; Yang, Heung-Mo; Hong, Doo-Pyo; Joo, Sung-Yeon; Choi, Yoon-La; Park, Joo-Hung; Lazar, Alexander J; Pollock, Raphael E; Lev, Dina; Kim, Sung Joo

2014-10-15

Liposarcoma is one of the most common histologic types of soft tissue sarcoma and is frequently an aggressive cancer with poor outcome. Hence, alternative approaches other than surgical excision are necessary to improve treatment of well-differentiated/dedifferentiated liposarcoma (WDLPS/DDLPS). For this reason, we performed a two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry/mass spectrometry (MALDI-TOF/MS) analysis to identify new factors for WDLPS and DDLPS. Among the selected candidate proteins, gankyrin, known to be an oncoprotein, showed a significantly high level of expression pattern and inversely low expression of p53/p21 in WDLPS and DDLPS tissues, suggesting possible utility as a new predictive factor. Moreover, inhibition of gankyrin not only led to reduction of in vitro cell growth ability including cell proliferation, colony-formation, and migration, but also in vivo DDLPS cell tumorigenesis, perhaps via downregulation of the p53 tumor suppressor gene and its p21 target and also reduction of AKT/mTOR signal activation. This study identifies gankyrin, for the first time, as new potential predictive and oncogenic factor of WDLPS and DDLPS, suggesting the potential for service as a future LPS therapeutic approach.

Differential transcriptional regulation by alternatively designed mechanisms: A mathematical modeling approach.

PubMed

Yildirim, Necmettin; Aktas, Mehmet Emin; Ozcan, Seyma Nur; Akbas, Esra; Ay, Ahmet

2017-01-01

Cells maintain cellular homeostasis employing different regulatory mechanisms to respond external stimuli. We study two groups of signal-dependent transcriptional regulatory mechanisms. In the first group, we assume that repressor and activator proteins compete for binding to the same regulatory site on DNA (competitive mechanisms). In the second group, they can bind to different regulatory regions in a noncompetitive fashion (noncompetitive mechanisms). For both competitive and noncompetitive mechanisms, we studied the gene expression dynamics by increasing the repressor or decreasing the activator abundance (inhibition mechanisms), or by decreasing the repressor or increasing the activator abundance (activation mechanisms). We employed delay differential equation models. Our simulation results show that the competitive and noncompetitive inhibition mechanisms exhibit comparable repression effectiveness. However, response time is fastest in the noncompetitive inhibition mechanism due to increased repressor abundance, and slowest in the competitive inhibition mechanism by increased repressor level. The competitive and noncompetitive inhibition mechanisms through decreased activator abundance show comparable and moderate response times, while the competitive and noncompetitive activation mechanisms by increased activator protein level display more effective and faster response. Our study exemplifies the importance of mathematical modeling and computer simulation in the analysis of gene expression dynamics.

Endophenotype Network Models: Common Core of Complex Diseases

PubMed Central

Ghiassian, Susan Dina; Menche, Jörg; Chasman, Daniel I.; Giulianini, Franco; Wang, Ruisheng; Ricchiuto, Piero; Aikawa, Masanori; Iwata, Hiroshi; Müller, Christian; Zeller, Tania; Sharma, Amitabh; Wild, Philipp; Lackner, Karl; Singh, Sasha; Ridker, Paul M.; Blankenberg, Stefan; Barabási, Albert-László; Loscalzo, Joseph

2016-01-01

Historically, human diseases have been differentiated and categorized based on the organ system in which they primarily manifest. Recently, an alternative view is emerging that emphasizes that different diseases often have common underlying mechanisms and shared intermediate pathophenotypes, or endo(pheno)types. Within this framework, a specific disease’s expression is a consequence of the interplay between the relevant endophenotypes and their local, organ-based environment. Important examples of such endophenotypes are inflammation, fibrosis, and thrombosis and their essential roles in many developing diseases. In this study, we construct endophenotype network models and explore their relation to different diseases in general and to cardiovascular diseases in particular. We identify the local neighborhoods (module) within the interconnected map of molecular components, i.e., the subnetworks of the human interactome that represent the inflammasome, thrombosome, and fibrosome. We find that these neighborhoods are highly overlapping and significantly enriched with disease-associated genes. In particular they are also enriched with differentially expressed genes linked to cardiovascular disease (risk). Finally, using proteomic data, we explore how macrophage activation contributes to our understanding of inflammatory processes and responses. The results of our analysis show that inflammatory responses initiate from within the cross-talk of the three identified endophenotypic modules. PMID:27278246

Endophenotype Network Models: Common Core of Complex Diseases

NASA Astrophysics Data System (ADS)

Ghiassian, Susan Dina; Menche, Jörg; Chasman, Daniel I.; Giulianini, Franco; Wang, Ruisheng; Ricchiuto, Piero; Aikawa, Masanori; Iwata, Hiroshi; Müller, Christian; Zeller, Tania; Sharma, Amitabh; Wild, Philipp; Lackner, Karl; Singh, Sasha; Ridker, Paul M.; Blankenberg, Stefan; Barabási, Albert-László; Loscalzo, Joseph

2016-06-01

Historically, human diseases have been differentiated and categorized based on the organ system in which they primarily manifest. Recently, an alternative view is emerging that emphasizes that different diseases often have common underlying mechanisms and shared intermediate pathophenotypes, or endo(pheno)types. Within this framework, a specific disease’s expression is a consequence of the interplay between the relevant endophenotypes and their local, organ-based environment. Important examples of such endophenotypes are inflammation, fibrosis, and thrombosis and their essential roles in many developing diseases. In this study, we construct endophenotype network models and explore their relation to different diseases in general and to cardiovascular diseases in particular. We identify the local neighborhoods (module) within the interconnected map of molecular components, i.e., the subnetworks of the human interactome that represent the inflammasome, thrombosome, and fibrosome. We find that these neighborhoods are highly overlapping and significantly enriched with disease-associated genes. In particular they are also enriched with differentially expressed genes linked to cardiovascular disease (risk). Finally, using proteomic data, we explore how macrophage activation contributes to our understanding of inflammatory processes and responses. The results of our analysis show that inflammatory responses initiate from within the cross-talk of the three identified endophenotypic modules.

A chromatin link to caste identity in the carpenter ant Camponotus floridanus

PubMed Central

Simola, Daniel F.; Ye, Chaoyang; Mutti, Navdeep S.; Dolezal, Kelly; Bonasio, Roberto; Liebig, Jürgen; Reinberg, Danny; Berger, Shelley L.

2013-01-01

In many ant species, sibling larvae follow alternative ontogenetic trajectories that generate striking variation in morphology and behavior among adults. These organism-level outcomes are often determined by environmental rather than genetic factors. Therefore, epigenetic mechanisms may mediate the expression of adult polyphenisms. We produced the first genome-wide maps of chromatin structure in a eusocial insect and found that gene-proximal changes in histone modifications, notably H3K27 acetylation, discriminate two female worker and male castes in Camponotus floridanus ants and partially explain differential gene expression between castes. Genes showing coordinated changes in H3K27ac and RNA implicate muscle development, neuronal regulation, and sensory responses in modulating caste identity. Binding sites of the acetyltransferase CBP harbor the greatest caste variation in H3K27ac, are enriched with motifs for conserved transcription factors, and show evolutionary expansion near developmental and neuronal genes. These results suggest that environmental effects on caste identity may be mediated by differential recruitment of CBP to chromatin. We propose that epigenetic mechanisms that modify chromatin structure may help orchestrate the generation and maintenance of polyphenic caste morphology and social behavior in ants. PMID:23212948

Biological characterization of metanephric mesenchymal stem cells from the Beijing duck.

PubMed

Chen, Jia; Pu, Yabin; Sun, Yujiao; Zhang, Ping; Li, Qian; Wang, Kunfu; Wang, Wenjie; Ma, Yuehui; Guan, Weijun

2016-02-01

Mesenchymal stem cells (MSCs) possess self-proliferation and multi-directional differentiation abilities. Previous studies on MSCs have mostly focused on the bone marrow, lungs, pancreas and umbilical cord blood, with few studies on metanephric tissues in ducks. For the present study, the Beijing duck was selected as an experimental animal. Duck embryo metanephric mesenchymal stem cells (MMSCs) were studied. MMSC isolation culture, analysis of biological characteristics, induced differentiation and identification were performed in preliminary experiments. In the current study, surface antigens and gene expression patterns were detected using immunofluorescence, reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry. The induced cells, adipocytes, hepatocytes, epithelial cells and islet cells were identified by oil red O staining, periodic acid-Schiff staining, immunofluorescence and dithizone staining, respectively. RT-PCR was performed for detection of specific marker genes. The results suggested that the biological characteristics of MMSCs were similar to those of the MSCs previously analyzed. Primary MMSCs were sub-cultured to passage 21. The induced cells exhibit typical staining and immunofluorescence indicating the expression of specific genes. This demonstrates that MMSCs may be a novel alternative source of MSCs for experimental and clinical applications.

A Comprehensive Analysis of Alternative Splicing in Paleopolyploid Maize.

PubMed

Mei, Wenbin; Liu, Sanzhen; Schnable, James C; Yeh, Cheng-Ting; Springer, Nathan M; Schnable, Patrick S; Barbazuk, William B

2017-01-01

Identifying and characterizing alternative splicing (AS) enables our understanding of the biological role of transcript isoform diversity. This study describes the use of publicly available RNA-Seq data to identify and characterize the global diversity of AS isoforms in maize using the inbred lines B73 and Mo17, and a related species, sorghum. Identification and characterization of AS within maize tissues revealed that genes expressed in seed exhibit the largest differential AS relative to other tissues examined. Additionally, differences in AS between the two genotypes B73 and Mo17 are greatest within genes expressed in seed. We demonstrate that changes in the level of alternatively spliced transcripts (intron retention and exon skipping) do not solely reflect differences in total transcript abundance, and we present evidence that intron retention may act to fine-tune gene expression across seed development stages. Furthermore, we have identified temperature sensitive AS in maize and demonstrate that drought-induced changes in AS involve distinct sets of genes in reproductive and vegetative tissues. Examining our identified AS isoforms within B73 × Mo17 recombinant inbred lines (RILs) identified splicing QTL (sQTL). The 43.3% of cis- sQTL regulated junctions are actually identified as alternatively spliced junctions in our analysis, while 10 Mb windows on each side of 48.2% of trans -sQTLs overlap with splicing related genes. Using sorghum as an out-group enabled direct examination of loss or conservation of AS between homeologous genes representing the two subgenomes of maize. We identify several instances where AS isoforms that are conserved between one maize homeolog and its sorghum ortholog are absent from the second maize homeolog, suggesting that these AS isoforms may have been lost after the maize whole genome duplication event. This comprehensive analysis provides new insights into the complexity of AS in maize.

Relaxed selection is a precursor to the evolution of phenotypic plasticity.

PubMed

Hunt, Brendan G; Ometto, Lino; Wurm, Yannick; Shoemaker, DeWayne; Yi, Soojin V; Keller, Laurent; Goodisman, Michael A D

2011-09-20

Phenotypic plasticity allows organisms to produce alternative phenotypes under different conditions and represents one of the most important ways by which organisms adaptively respond to the environment. However, the relationship between phenotypic plasticity and molecular evolution remains poorly understood. We addressed this issue by investigating the evolution of genes associated with phenotypically plastic castes, sexes, and developmental stages of the fire ant Solenopsis invicta. We first determined if genes associated with phenotypic plasticity in S. invicta evolved at a rapid rate, as predicted under theoretical models. We found that genes differentially expressed between S. invicta castes, sexes, and developmental stages all exhibited elevated rates of evolution compared with ubiquitously expressed genes. We next investigated the evolutionary history of genes associated with the production of castes. Surprisingly, we found that orthologs of caste-biased genes in S. invicta and the social bee Apis mellifera evolved rapidly in lineages without castes. Thus, in contrast to some theoretical predictions, our results suggest that rapid rates of molecular evolution may not arise primarily as a consequence of phenotypic plasticity. Instead, genes evolving under relaxed purifying selection may more readily adopt new forms of biased expression during the evolution of alternate phenotypes. These results suggest that relaxed selective constraint on protein-coding genes is an important and underappreciated element in the evolutionary origin of phenotypic plasticity.

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.

Noninvasive Monitoring of Three-Dimensional Chondrogenic Constructs Using Molecular Beacon Nanosensors.

PubMed

Tay, Li Min; Wiraja, Christian; Yeo, David C; Wu, Yingnan; Yang, Zheng; Chuah, Yon Jin; Lee, Eng Hin; Kang, Yuejun; Xu, Chenjie

2017-01-01

Chondrogenic differentiation of human mesenchymal stem cells (MSCs) in three-dimensional hydrogel holds promise as a method for repairing injured articular cartilage. Given MSC plasticity (its potential to mature into alternative lineages), nondestructive monitoring is critical for the optimization of chondrogenic differentiation conditions and the evaluation of the final product. However, conventional validation/assessments of the differentiation process (i.e., quantitative reverse transcription polymerase chain reaction [qRT-PCR] and histology) are end-point assays requiring disruption of the sample. This report introduces molecular beacon (MB)-based nanosensors to achieve noninvasive monitoring of chondrogenic differentiation. These nanosensors consist of biodegradable poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) encapsulating MBs to detect Type II Collagen (Col2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs that serve as lineage-specific and housekeeping biomarkers, respectively. The sustainable release of MBs from MB-NPs allows longitudinal monitoring of MSCs undergoing chondrogenic differentiation over a period of 28 days. Dual-colored MB loading ensures accurate assessment of Col2 mRNA expression level, where potential heterogeneity in nanosensor uptake and retention by MSCs are taken into account. When normalized nanosensor signal was compared against qRT-PCR result, a tight correlation was observed (R 2  = 0.9301). Finally, nanosensor usage was compatible with MSC potency with minimal influence on chondrogenic, adipogenic, and osteogenic differentiation.

Iron uptake controls the generation of Leishmania infective forms through regulation of ROS levels

PubMed Central

Mittra, Bidyottam; Cortez, Mauro; Haydock, Andrew; Ramasamy, Gowthaman; Myler, Peter J.

2013-01-01

During its life cycle, Leishmania undergoes extreme environmental changes, alternating between insect vectors and vertebrate hosts. Elevated temperature and decreased pH, conditions encountered after macrophage invasion, can induce axenic differentiation of avirulent promastigotes into virulent amastigotes. Here we show that iron uptake is a major trigger for the differentiation of Leishmania amazonensis amastigotes, independently of temperature and pH changes. We found that iron depletion from the culture medium triggered expression of the ferrous iron transporter LIT1 (Leishmania iron transporter 1), an increase in iron content of the parasites, growth arrest, and differentiation of wild-type (WT) promastigotes into infective amastigotes. In contrast, LIT1-null promastigotes showed reduced intracellular iron content and sustained growth in iron-poor media, followed by cell death. LIT1 up-regulation also increased iron superoxide dismutase (FeSOD) activity in WT but not in LIT1-null parasites. Notably, the superoxide-generating drug menadione or H2O2 was sufficient to trigger differentiation of WT promastigotes into fully infective amastigotes. LIT1-null promastigotes accumulated superoxide radicals and initiated amastigote differentiation after exposure to H2O2 but not to menadione. Our results reveal a novel role for FeSOD activity and reactive oxygen species in orchestrating the differentiation of virulent Leishmania amastigotes in a process regulated by iron availability. PMID:23382545

Burkitt lymphoma beyond MYC translocation: N-MYC and DNA methyltransferases dysregulation.

PubMed

De Falco, Giulia; Ambrosio, Maria Raffaella; Fuligni, Fabio; Onnis, Anna; Bellan, Cristiana; Rocca, Bruno Jim; Navari, Mohsen; Etebari, Maryam; Mundo, Lucia; Gazaneo, Sara; Facchetti, Fabio; Pileri, Stefano A; Leoncini, Lorenzo; Piccaluga, Pier Paolo

2015-10-09

The oncogenic transcription factor MYC is pathologically activated in many human malignancies. A paradigm for MYC dysregulation is offered by Burkitt lymphoma, where chromosomal translocations leading to Immunoglobulin gene-MYC fusion are the crucial initiating oncogenic events. However, Burkitt lymphoma cases with no detectable MYC rearrangement but maintaining MYC expression have been identified and alternative mechanisms can be involved in MYC dysregulation in these cases. We studied the microRNA profile of MYC translocation-positive and MYC translocation-negative Burkitt lymphoma cases in order to uncover possible differences at the molecular level. Data was validated at the mRNA and protein level by quantitative Real-Time polymerase chain reaction and immunohistochemistry, respectively. We identified four microRNAs differentially expressed between the two groups. The impact of these microRNAs on the expression of selected genes was then investigated. Interestingly, in MYC translocation-negative cases we found over-expression of DNA-methyl transferase family members, consistent to hypo-expression of the hsa-miR-29 family. This finding suggests an alternative way for the activation of lymphomagenesis in these cases, based on global changes in methylation landscape, aberrant DNA hypermethylation, lack of epigenetic control on transcription of targeted genes, and increase of genomic instability. In addition, we observed an over-expression of another MYC family gene member, MYCN that may therefore represent a cooperating mechanism of MYC in driving the malignant transformation in those cases lacking an identifiable MYC translocation but expressing the gene at the mRNA and protein levels. Collectively, our results showed that MYC translocation-positive and MYC translocation-negative Burkitt lymphoma cases are slightly different in terms of microRNA and gene expression. MYC translocation-negative Burkitt lymphoma, similarly to other aggressive B-cell non Hodgkin's lymphomas, may represent a model to understand the intricate molecular pathway responsible for MYC dysregulation in cancer.

Deregulation of Genes Associated with Alternate Drug Resistance Mechanisms in Mycobacterium tuberculosis.

PubMed

Sriraman, Kalpana; Nilgiriwala, Kayzad; Saranath, Dhananjaya; Chatterjee, Anirvan; Mistry, Nerges

2018-04-01

Alternate mechanisms of drug resistance involving intrinsic defense pathways play an important role in development of drug resistance. Deregulation of drug efflux, cellular metabolism, and DNA repair have been indicated to have effect on drug tolerance and persistence. Here we chose eight genes from these pathways to investigate their association with development of multidrug resistance (MDR). We generated mono drug resistant and MDR strains of rifampicin and isoniazid and examined the differential expression of genes belonging to efflux, DNA repair and cell wall lipid synthesis pathways. Rv1687c, recB, ppsD and embC genes showed significant (P <0.05) upregulation in mono-resistant (both rifampicin and isoniazid) as well as MDR strains. mmr showed significant upregulation with rifampicin resistance while Rv1457c showed significant upregulation only with mono-resistant strains. Highest expression change was observed with Rv1687c and ppsD. The study identified potential key genes that are significantly associated with development of drug resistance in vitro. These genes may help identify clinical strains predisposed to acquiring drug resistance in patients during the course of treatment or help in management of MDR forms of tuberculosis.

PAX6 maintains β cell identity by repressing genes of alternative islet cell types.

PubMed

Swisa, Avital; Avrahami, Dana; Eden, Noa; Zhang, Jia; Feleke, Eseye; Dahan, Tehila; Cohen-Tayar, Yamit; Stolovich-Rain, Miri; Kaestner, Klaus H; Glaser, Benjamin; Ashery-Padan, Ruth; Dor, Yuval

2017-01-03

Type 2 diabetes is thought to involve a compromised β cell differentiation state, but the mechanisms underlying this dysfunction remain unclear. Here, we report a key role for the TF PAX6 in the maintenance of adult β cell identity and function. PAX6 was downregulated in β cells of diabetic db/db mice and in WT mice treated with an insulin receptor antagonist, revealing metabolic control of expression. Deletion of Pax6 in β cells of adult mice led to lethal hyperglycemia and ketosis that were attributed to loss of β cell function and expansion of α cells. Lineage-tracing, transcriptome, and chromatin analyses showed that PAX6 is a direct activator of β cell genes, thus maintaining mature β cell function and identity. In parallel, we found that PAX6 binds promoters and enhancers to repress alternative islet cell genes including ghrelin, glucagon, and somatostatin. Chromatin analysis and shRNA-mediated gene suppression experiments indicated a similar function of PAX6 in human β cells. We conclude that reduced expression of PAX6 in metabolically stressed β cells may contribute to β cell failure and α cell dysfunction in diabetes.

PAX6 maintains β cell identity by repressing genes of alternative islet cell types

PubMed Central

Swisa, Avital; Avrahami, Dana; Eden, Noa; Zhang, Jia; Feleke, Eseye; Dahan, Tehila; Cohen-Tayar, Yamit; Stolovich-Rain, Miri; Kaestner, Klaus H.; Glaser, Benjamin; Ashery-Padan, Ruth

2016-01-01

Type 2 diabetes is thought to involve a compromised β cell differentiation state, but the mechanisms underlying this dysfunction remain unclear. Here, we report a key role for the TF PAX6 in the maintenance of adult β cell identity and function. PAX6 was downregulated in β cells of diabetic db/db mice and in WT mice treated with an insulin receptor antagonist, revealing metabolic control of expression. Deletion of Pax6 in β cells of adult mice led to lethal hyperglycemia and ketosis that were attributed to loss of β cell function and expansion of α cells. Lineage-tracing, transcriptome, and chromatin analyses showed that PAX6 is a direct activator of β cell genes, thus maintaining mature β cell function and identity. In parallel, we found that PAX6 binds promoters and enhancers to repress alternative islet cell genes including ghrelin, glucagon, and somatostatin. Chromatin analysis and shRNA-mediated gene suppression experiments indicated a similar function of PAX6 in human β cells. We conclude that reduced expression of PAX6 in metabolically stressed β cells may contribute to β cell failure and α cell dysfunction in diabetes. PMID:27941241

Jacalin Lectin At5g28520 Is Regulated By ABA and miR846

PubMed Central

Jia, Fan; Rock, Christopher D.

2013-01-01

Plant microRNAs (miRNAs) are important regulators of development and stress responses and are oftentimes under transcriptional regulation by stresses and plant hormones. We recently showed that polycistronic MIR842 and MIR846 are expressed from the same primary transcript which is subject to alternative splicing. ABA treatment affects the alternative splicing of the primary cistronic transcript which results in differential expression of the two miRNAs that are predicted to target the same family of jacalin lectin genes. One variant of miR846 in roots can direct the cleavage of AT5G28520, which is also highly upregulated by ABA in roots. In this addendum, we present additional results further supporting the regulation of AT5G28520 by MIR846 using a T-DNA insertion line mapping upstream of MIR842 and MIR846. We also show that AT5G28520 is transcriptionally induced by ABA and this induction is subject to ABA signaling effectors in seedlings. Based on previous results and data presented in this paper, we propose an interaction loop between MIR846, AT5G28520 and ABA in roots. PMID:23603955

Role of Bmznf-2, a Bombyx mori CCCH zinc finger gene, in masculinisation and differential splicing of Bmtra-2.

PubMed

Gopinath, Gajula; Arunkumar, Kallare P; Mita, Kazuei; Nagaraju, Javaregowda

2016-08-01

Deciphering the regulatory factors involved in Bombyx mori sex determination has been a puzzle, challenging researchers for nearly a century now. The pre-mRNA of B. mori doublesex (Bmdsx), a master regulator gene of sexual differentiation, is differentially spliced, producing Bmdsxm and Bmdsxf transcripts in males and females respectively. The putative proteins encoded by these differential transcripts orchestrate antagonistic functions, which lead to sexual differentiation. A recent study in B. mori illustrated the role of a W-derived fem piRNA in conferring femaleness. In females, the fem piRNA was shown to suppress the activity of a Z-linked CCCH type zinc finger (znf) gene, Masculiniser (masc), which indirectly promotes the Bmdsxm type of splicing. In this study, we report a novel autosomal (Chr 25) CCCH type znf motif encoding gene Bmznf-2 as one of the potential factors in the Bmdsx sex specific differential splicing, and we also provide insights into its role in the alternative splicing of Bmtra2 by using ovary derived BmN cells. Over-expression of Bmznf-2 induced Bmdsxm type of splicing (masculinisation) with a correspondingly reduced expression of Bmdsxf type isoform in BmN cells. Further, the site-directed mutational studies targeting the tandem CCCH znf motifs revealed their indispensability in the observed phenotype of masculinisation. Additionally, the dual luciferase assays in BmN cells using 5' UTR region of the Bmznf-2 strongly implied the existence of a translational repression over this gene. From these findings, we propose Bmznf-2 to be one of the potential factors of masculinisation similar to Masc. From the growing number of Bmdsx splicing regulators, we assume that the sex determination cascade of B. mori is quite intricate in nature; hence, it has to be further investigated for its comprehensive understanding. Copyright © 2016 Elsevier Ltd. All rights reserved.

Regulation of collagenase-3 and osteocalcin gene expression by collagen and osteopontin in differentiating MC3T3-E1 cells

NASA Technical Reports Server (NTRS)

D'Alonzo, Richard C.; Kowalski, Aaron J.; Denhardt, David T.; Nickols, G. Allen; Partridge, Nicola C.

2002-01-01

Both collagenase-3 and osteocalcin mRNAs are expressed maximally during the later stages of osteoblast differentiation. Here, we demonstrate that collagenase-3 mRNA expression in differentiating MC3T3-E1 cells is dependent upon the presence of ascorbic acid, is inhibited in the presence of the collagen synthesis inhibitor, 3,4-dehydroproline, and is stimulated by growth on collagen in the absence of ascorbic acid. Transient transfection studies show that collagenase-3 promoter activity increases during cell differentiation and requires the presence of ascorbic acid. Additionally, we show that, in differentiating MC3T3-E1 cells, collagenase-3 gene expression increases in the presence of an anti-osteopontin monoclonal antibody that binds near the RGD motif of this protein, whereas osteocalcin expression is inhibited. Furthermore, an RGD peptidomimetic compound, designed to block interaction of ligands to the alpha(v) integrin subunit, increases osteocalcin expression and inhibits collagenase-3 expression, suggesting that the RGD peptidomimetic initiates certain alpha(v) integrin signaling in osteoblastic cells. Overall, these studies demonstrate that stimulation of collagenase-3 expression during osteoblast differentiation requires synthesis of a collagenous matrix and that osteopontin and alpha(v) integrins exert divergent regulation of collagenase-3 and osteocalcin expression during osteoblast differentiation.

Integration of genetic, genomic and transcriptomic information identifies putative regulators of adventitious root formation in Populus

DOE PAGES

Ribeiro, Cintia L.; Silva, Cynthia M.; Drost, Derek R.; ...

2016-03-16

In this study, adventitious roots (AR) develop from tissues other than the primary root, in a process physiologically regulated by phytohormones. Adventitious roots provide structural support and contribute to water and nutrient absorption, and are critical for commercial vegetative propagation of several crops. Here we quantified the number of AR, root architectural traits and root biomass in cuttings from a pseudo-backcross population of Populus deltoides and Populus trichocarpa. Quantitative trait loci (QTL) mapping and whole-transcriptome analysis of individuals with alternative QTL alleles for AR number were used to identify putative regulators of AR development. As a result, parental individuals andmore » progeny showed extensive segregation for AR developmental traits. Quantitative trait loci for number of AR mapped consistently in the same interval of linkage group (LG) II and LG XIV, explaining 7–10 % of the phenotypic variation. A time series transcriptome analysis identified 26,121 genes differentially expressed during AR development, particularly during the first 24 h after cuttings were harvested. Of those, 1929 genes were differentially regulated between individuals carrying alternative alleles for the two QTL for number of AR, in one or more time point. Eighty-one of these genes were physically located within the QTL intervals for number of AR, including putative homologs of the Arabidopsis genes SUPERROOT2 (SUR2) and TRYPTOPHAN SYNTHASE ALPHA CHAIN (TSA1), both of which are involved in the auxin indole-3-acetic acid (IAA) biosynthesis pathway. In conclusion, this study suggests the involvement of two genes of the tryptophan-dependent auxin biosynthesis pathway, SUR2 and TSA1, in the regulation of a critical trait for the clonal propagation of woody species. A possible model for this regulation is that poplar individuals that have poor AR formation synthesize auxin indole-3-acetic acid (IAA) primarily through the tryptophan (Trp) pathway. Much of the Trp pathway flux appears to be directed to the synthesis of indole glucosinolates (IG), as suggested by the over-expression of SUR2. Individuals that are efficient in AR formation may utilize alternative (non-Trp) pathways to synthesize IAA, based on the observation that they down-regulate the expression of TSA1, one of the critical steps in the synthesis of tryptophan.« less

Cost analysis of Navy acquisition alternatives for the NAVSTAR Global Positioning System

NASA Astrophysics Data System (ADS)

Darcy, T. F.; Smith, G. P.

1982-12-01

This research analyzes the life cycle cost (LCC) of the Navy's current and two hypothetical procurement alternatives for NAVSTAR Global Positioning System (GPS) user equipment. Costs are derived by the ARINC Research Corporation ACBEN cost estimating system. Data presentation is in a comparative format describing individual alternative LCC and differential costs between alternatives. Sensitivity analysis explores the impact receiver-processor unit (RPU) first unit production cost has on individual alternative LCC, as well as cost differentials between each alternative. Several benefits are discussed that might provide sufficient cost savings and/or system effectiveness improvements to warrant a procurement strategy other than the existing proposal.

Isolation and characterization of equine endometrial mesenchymal stromal cells.

PubMed

Rink, B Elisabeth; Amilon, Karin R; Esteves, Cristina L; French, Hilari M; Watson, Elaine; Aurich, Christine; Donadeu, F Xavier

2017-07-12

Equine mesenchymal stromal/stem cells (MSCs) are most commonly harvested from bone marrow (BM) or adipose tissue, requiring the use of surgical procedures. By contrast, the uterus can be accessed nonsurgically, and may provide a more readily available cell source. While human endometrium is known to harbor mesenchymal precursor cells, MSCs have not been identified in equine endometrium. This study reports the isolation, culture, and characterization of MSCs from equine endometrium. The presence of MSC and pericyte markers in endometrial sections was determined using immunohistochemistry. Stromal cells were harvested and cultured after separation of epithelial cells from endometrial fragments using Mucin-1-bound beads. For comparison, MSCs were also harvested from BM. The expression of surface markers in endometrial and BM-derived MSCs was characterized using flow cytometry and quantitative polymerase chain reaction. MSCs were differentiated in vitro into adipogenic, chondrogenic, osteogenic, and smooth muscle lineages. Typical markers of MSCs (CD29, CD44, CD90, and CD105) and pericytes (NG2 and CD146) were localized in the equine endometrium. Both endometrial and BM MSCs grew clonally and robustly expressed MSC and pericyte markers in culture while showing greatly reduced or negligible expression of hematopoietic markers (CD45, CD34) and MHC-II. Additionally, both endometrial and BM MSCs differentiated into adipogenic, osteogenic, and chondrogenic lineages in vitro, and endometrial MSCs had a distinct ability to undergo smooth muscle differentiation. We have demonstrated for the first time the presence of cells in equine endometrium that fulfill the definition of MSCs. The equine endometrium may provide an alternative, easily accessible source of MSCs, not only for therapeutic regeneration of the uterus, but also for other tissues where MSCs from other sources are currently being used therapeutically.

mapDIA: Preprocessing and statistical analysis of quantitative proteomics data from data independent acquisition mass spectrometry.

PubMed

Teo, Guoshou; Kim, Sinae; Tsou, Chih-Chiang; Collins, Ben; Gingras, Anne-Claude; Nesvizhskii, Alexey I; Choi, Hyungwon

2015-11-03

Data independent acquisition (DIA) mass spectrometry is an emerging technique that offers more complete detection and quantification of peptides and proteins across multiple samples. DIA allows fragment-level quantification, which can be considered as repeated measurements of the abundance of the corresponding peptides and proteins in the downstream statistical analysis. However, few statistical approaches are available for aggregating these complex fragment-level data into peptide- or protein-level statistical summaries. In this work, we describe a software package, mapDIA, for statistical analysis of differential protein expression using DIA fragment-level intensities. The workflow consists of three major steps: intensity normalization, peptide/fragment selection, and statistical analysis. First, mapDIA offers normalization of fragment-level intensities by total intensity sums as well as a novel alternative normalization by local intensity sums in retention time space. Second, mapDIA removes outlier observations and selects peptides/fragments that preserve the major quantitative patterns across all samples for each protein. Last, using the selected fragments and peptides, mapDIA performs model-based statistical significance analysis of protein-level differential expression between specified groups of samples. Using a comprehensive set of simulation datasets, we show that mapDIA detects differentially expressed proteins with accurate control of the false discovery rates. We also describe the analysis procedure in detail using two recently published DIA datasets generated for 14-3-3β dynamic interaction network and prostate cancer glycoproteome. The software was written in C++ language and the source code is available for free through SourceForge website http://sourceforge.net/projects/mapdia/.This article is part of a Special Issue entitled: Computational Proteomics. Copyright © 2015 Elsevier B.V. All rights reserved.

Bone marrow mesenchymal stem cells could acquire the phenotypes of epithelial cells and accelerate vaginal reconstruction combined with small intestinal submucosa.

PubMed

Li, Yanan; Liu, Fangfang; Zhang, Zhiqiang; Zhang, Mingle; Cao, Shanjin; Li, Yachai; Zhang, Lin; Huang, Xianghua; Xu, Yanfang

2015-11-01

Grafting material for vaginal reconstruction commonly includes the bowel, peritoneum, skin, and amniotic membrane. Bone marrow mesenchymal stem cells (MSCs) have the potential of multilineage differentiation into a variety of cells and have been widely explored in tissue engineering. In the current study, we examined whether MSCs could be differentiated to vaginal epithelial cells (VECs) upon co-culturing with VECs. We also examined whether Wnt/β-catenin signaling pathway is implicated in such differentiation. Co-culture of MSCs with VECs using a transwell insert system (with no direct contact) induced the expression of VECs marker AE1/AE3 in MSCs. MSCs combined with small intestinal submucosa (SIS) scaffold were implanted in place of the native vagina in rats to observe the implications for vaginal reconstruction in vivo. Anatomic repair of neovagina was assessed by histological staining for H/E and Masson's Trichrome. GSK-3β and β-catenin, main members of Wnt/β-catenin signaling pathway, in MSCs were increased upon co-culturing with VECs. Exposure of co-cultured MSCs to a Wnt/β-catenin signaling activator, lithium chloride (LiCl, 20 µM) increased phosphorylated GSK-3β and β-catenin and enhanced expression of AE1/AE3. In vivo-grafted cells displayed significant matrix infiltration and expressed epithelial markers in neovagina. These findings suggest that MSCs could acquire the phenotype of VECs when co-cultured with VECs, possibly via activation of Wnt/β-catenin signaling. MSCs provide an alternative cell source for potential use in vaginal tissue engineering. © 2015 International Federation for Cell Biology.

Role of Chondroitin Sulfate (CS) Modification in the Regulation of Protein-tyrosine Phosphatase Receptor Type Z (PTPRZ) Activity: PLEIOTROPHIN-PTPRZ-A SIGNALING IS INVOLVED IN OLIGODENDROCYTE DIFFERENTIATION.

PubMed

Kuboyama, Kazuya; Fujikawa, Akihiro; Suzuki, Ryoko; Tanga, Naomi; Noda, Masaharu

2016-08-26

Protein-tyrosine phosphatase receptor type Z (PTPRZ) is predominantly expressed in the developing brain as a CS proteoglycan. PTPRZ has long (PTPRZ-A) and short type (PTPRZ-B) receptor forms by alternative splicing. The extracellular CS moiety of PTPRZ is required for high-affinity binding to inhibitory ligands, such as pleiotrophin (PTN), midkine, and interleukin-34; however, its functional significance in regulating PTPRZ activity remains obscure. We herein found that protein expression of CS-modified PTPRZ-A began earlier, peaking at approximately postnatal days 5-10 (P5-P10), and then that of PTN peaked at P10 at the developmental stage corresponding to myelination onset in the mouse brain. Ptn-deficient mice consistently showed a later onset of the expression of myelin basic protein, a major component of the myelin sheath, than wild-type mice. Upon ligand application, PTPRZ-A/B in cultured oligodendrocyte precursor cells exhibited punctate localization on the cell surface instead of diffuse distribution, causing the inactivation of PTPRZ and oligodendrocyte differentiation. The same effect was observed with the removal of CS chains with chondroitinase ABC but not polyclonal antibodies against the extracellular domain of PTPRZ. These results indicate that the negatively charged CS moiety prevents PTPRZ from spontaneously clustering and that the positively charged ligand PTN induces PTPRZ clustering, potentially by neutralizing electrostatic repulsion between CS chains. Taken altogether, these data indicate that PTN-PTPRZ-A signaling controls the timing of oligodendrocyte precursor cell differentiation in vivo, in which the CS moiety of PTPRZ receptors maintains them in a monomeric active state until its ligand binding. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

BmTGIF, a Bombyx mori Homolog of Drosophila DmTGIF, Regulates Progression of Spermatogenesis

PubMed Central

Sheng, Jie; Xue, Renyu; Gong, Chengliang

2012-01-01

TG-interacting factor (TGIF) in Drosophila consists of two tandemly-repeated genes, achintya (Dmachi) and vismay (Dmvis), which act as transcriptional activators in Drosophila spermatogenesis. In contrast, TGIF in humans is a transcriptional repressor that binds directly to DNA or interacts with corepressors to repress the transcription of target genes. In this study, we investigated the characteristics and functions of BmTGIF, a Bombyx mori homolog of DmTGIF. Like DmTGIF, BmTGIF is predominantly expressed in the testes and ovaries. Four alternatively spliced isoforms could be isolated from testes, and two isoforms from ovaries. Quantitative polymerase chain reaction indicated BmTGIF was abundantly expressed in the testis of 3rd instar larvae, when the testis is almost full of primary spermatocytes. The results of luciferase assays indicated that BmTGIF contains two adjacent acidic domains that activate the transcription of reporter genes. Immunofluorescence assay in BmN cells showed that the BmTGIF protein was located mainly in the nucleus, and paraffin sections of testis showed BmTGIF was grossly expressed in primary spermatocytes and mature sperms. Consistent with the role of DmVis in Drosophila development, BmTGIF significantly affected spermatid differentiation, as indicated by hematoxylin-eosin staining of paraffin sections of testis from BmTGIF-small interfering RNA (siRNA)-injected male silkworms. Co-immunoprecipitation experiments suggested that BmTGIF interacted with BmAly, and that they may recruit other factors to form a complex to regulate the genes required for meiotic divisions and spermatid differentiation. The results of this analysis of BmTGIF will improve our understanding of the mechanism of spermatid differentiation in B. mori, with potential applications for pest control. PMID:23152760

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

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.

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

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

PubMed

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

2017-03-01

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

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.

The Brassica oleracea genome reveals the asymmetrical evolution of polyploid genomes

PubMed Central

Liu, Shengyi; Liu, Yumei; Yang, Xinhua; Tong, Chaobo; Edwards, David; Parkin, Isobel A. P.; Zhao, Meixia; Ma, Jianxin; Yu, Jingyin; Huang, Shunmou; Wang, Xiyin; Wang, Junyi; Lu, Kun; Fang, Zhiyuan; Bancroft, Ian; Yang, Tae-Jin; Hu, Qiong; Wang, Xinfa; Yue, Zhen; Li, Haojie; Yang, Linfeng; Wu, Jian; Zhou, Qing; Wang, Wanxin; King, Graham J; Pires, J. Chris; Lu, Changxin; Wu, Zhangyan; Sampath, Perumal; Wang, Zhuo; Guo, Hui; Pan, Shengkai; Yang, Limei; Min, Jiumeng; Zhang, Dong; Jin, Dianchuan; Li, Wanshun; Belcram, Harry; Tu, Jinxing; Guan, Mei; Qi, Cunkou; Du, Dezhi; Li, Jiana; Jiang, Liangcai; Batley, Jacqueline; Sharpe, Andrew G; Park, Beom-Seok; Ruperao, Pradeep; Cheng, Feng; Waminal, Nomar Espinosa; Huang, Yin; Dong, Caihua; Wang, Li; Li, Jingping; Hu, Zhiyong; Zhuang, Mu; Huang, Yi; Huang, Junyan; Shi, Jiaqin; Mei, Desheng; Liu, Jing; Lee, Tae-Ho; Wang, Jinpeng; Jin, Huizhe; Li, Zaiyun; Li, Xun; Zhang, Jiefu; Xiao, Lu; Zhou, Yongming; Liu, Zhongsong; Liu, Xuequn; Qin, Rui; Tang, Xu; Liu, Wenbin; Wang, Yupeng; Zhang, Yangyong; Lee, Jonghoon; Kim, Hyun Hee; Denoeud, France; Xu, Xun; Liang, Xinming; Hua, Wei; Wang, Xiaowu; Wang, Jun; Chalhoub, Boulos; Paterson, Andrew H

2014-01-01

Polyploidization has provided much genetic variation for plant adaptive evolution, but the mechanisms by which the molecular evolution of polyploid genomes establishes genetic architecture underlying species differentiation are unclear. Brassica is an ideal model to increase knowledge of polyploid evolution. Here we describe a draft genome sequence of Brassica oleracea, comparing it with that of its sister species B. rapa to reveal numerous chromosome rearrangements and asymmetrical gene loss in duplicated genomic blocks, asymmetrical amplification of transposable elements, differential gene co-retention for specific pathways and variation in gene expression, including alternative splicing, among a large number of paralogous and orthologous genes. Genes related to the production of anticancer phytochemicals and morphological variations illustrate consequences of genome duplication and gene divergence, imparting biochemical and morphological variation to B. oleracea. This study provides insights into Brassica genome evolution and will underpin research into the many important crops in this genus. PMID:24852848

The up and down of sleep: From molecules to electrophysiology.

PubMed

Navarro-Lobato, Irene; Genzel, Lisa

2018-03-12

Alternations of up and down can be seen across many different levels during sleep. Neural firing-rates, synaptic markers, molecular pathways, and gene expression all show differential up and down regulation across brain areas and sleep stages. And also the hallmarks of sleep - sleep stage specific oscillations - are characterized themselves by up and down as seen within the slow oscillation or theta cycles. In this review, we summarize the up and down of sleep covering molecules to electrophysiology and present different theories how this up and down could be regulated by the up and down of sleep oscillations. Further, we propose a tentative theory how this differential up and down could contribute to various outcomes of sleep related memory consolidation: enhancement of hippocampal representations of very novel memories and cortical consolidation of memories congruent with previous knowledge-networks. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Differential gene expression in queen–worker caste determination in bumble-bees

PubMed Central

Pereboom, Jeffrey J. M; Jordan, William C; Sumner, Seirian; Hammond, Robert L; Bourke, Andrew F. G

2005-01-01

Investigating how differential gene expression underlies caste determination in the social Hymenoptera is central to understanding how variation in gene expression underlies adaptive phenotypic diversity. We investigated for the first time the association between differential gene expression and queen–worker caste determination in the bumble-bee Bombus terrestris. Using suppression subtractive hybridization we isolated 12 genes that were differentially expressed in queen- and worker-destined larvae. We found that the sets of genes underlying caste differences in larvae and adults failed to overlap greatly. We also found that B. terrestris shares some of the genes whose differential expression is associated with caste determination in the honeybee, Apis mellifera, but their expression patterns were not identical. Instead, we found B. terrestris to exhibit a novel pattern, whereby most genes upregulated (i.e. showing relatively higher levels of expression) in queen-destined larvae early in development were upregulated in worker-destined larvae late in development. Overall, our results suggest that caste determination in B. terrestris involves a difference not so much in the identity of genes expressed by queen- and worker-destined larvae, but primarily in the relative timing of their expression. This conclusion is of potential importance in the further study of phenotypic diversification via differential gene expression. PMID:16024376