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Sample records for alternative differentiation reveals

  1. A global comparison between nuclear and cytosolic transcriptomes reveals differential compartmentalization of alternative transcript isoforms

    PubMed Central

    Chen, Liang

    2010-01-01

    Transcriptome analyses have typically disregarded nucleocytoplasmic differences. This approach has ignored some post-transcriptional regulations and their effect on the ultimate protein expression levels. Despite a longstanding interest in the differences between the nuclear and cytosolic transcriptomes, it is only recently that data have become available to study such differences and their associated features on a genome-wide scale. Here, we compared the nuclear and cytosolic transcriptomes of HepG2 and HeLa cells. HepG2 and HeLa cells vary significantly in the differential compartmentalization of their transcript isoforms, indicating that nucleocytoplasmic compartmentalization is a cell-specific characteristic. The differential compartmentalization is manifested at the transcript isoform level instead of the gene level because alternative isoforms of one gene can display different nucleocytoplasmic distributions. The isoforms enriched in the cytosol tend to have more introns and longer introns in their pre-mRNAs. They have more functional RNA folds and unique exons in the 3′ regions. These isoforms are more conserved than the isoforms enriched in the nucleus. Surprisingly, the presence of microRNAs does not have a significant impact on the nucleocytoplasmic distribution of their target isoforms. In contrast, nonsense-mediated decay is significantly more associated with the isoforms enriched in the nucleus than those enriched in the cytosol. PMID:19969546

  2. 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

  3. Characterization of the interferon genes in homozygous rainbow trout reveals two novel genes, alternate splicing and differential regulation of duplicated genes

    USGS Publications Warehouse

    Purcell, M.K.; Laing, K.J.; Woodson, J.C.; Thorgaard, G.H.; Hansen, J.D.

    2009-01-01

    The genes encoding the type I and type II interferons (IFNs) have previously been identified in rainbow trout and their proteins partially characterized. These previous studies reported a single type II IFN (rtIFN-??) and three rainbow trout type I IFN genes that are classified into either group I (rtIFN1, rtIFN2) or group II (rtIFN3). In this present study, we report the identification of a novel IFN-?? gene (rtIFN-??2) and a novel type I group II IFN (rtIFN4) in homozygous rainbow trout and predict that additional IFN genes or pseudogenes exist in the rainbow trout genome. Additionally, we provide evidence that short and long forms of rtIFN1 are actively and differentially transcribed in homozygous trout, and likely arose due to alternate splicing of the first exon. Quantitative reverse transcriptase PCR (qRT-PCR) assays were developed to systematically profile all of the rainbow trout IFN transcripts, with high specificity at an individual gene level, in na??ve fish and after stimulation with virus or viral-related molecules. Cloned PCR products were used to ensure the specificity of the qRT-PCR assays and as absolute standards to assess transcript abundance of each gene. All IFN genes were modulated in response to Infectious hematopoietic necrosis virus (IHNV), a DNA vaccine based on the IHNV glycoprotein, and poly I:C. The most inducible of the type I IFN genes, by all stimuli tested, were rtIFN3 and the short transcript form of rtIFN1. Gene expression of rtIFN-??1 and rtIFN-??2 was highly up-regulated by IHNV infection and DNA vaccination but rtIFN-??2 was induced to a greater magnitude. The specificity of the qRT-PCR assays reported here will be useful for future studies aimed at identifying which cells produce IFNs at early time points after infection. ?? 2008 Elsevier Ltd.

  4. Histone methylation, alternative splicing and neuronal differentiation.

    PubMed

    Fiszbein, Ana; Kornblihtt, Alberto R

    2016-01-01

    Alternative splicing, as well as chromatin structure, greatly contributes to specific transcriptional programs that promote neuronal differentiation. The activity of G9a, the enzyme responsible for mono- and di-methylation of lysine 9 on histone H3 (H3K9me1 and H3K9me2) in mammalian euchromatin, has been widely implicated in the differentiation of a variety of cell types and tissues. In a recent work from our group (Fiszbein et al., 2016) we have shown that alternative splicing of G9a regulates its nuclear localization and, therefore, the efficiency of H3K9 methylation, which promotes neuronal differentiation. We discuss here our results in the light of a report from other group (Laurent et al. 2015) demonstrating a key role for the alternative splicing of the histone demethylase LSD1 in controlling specific gene expression in neurons. All together, these results illustrate the importance of alternative splicing in the generation of a proper equilibrium between methylation and demethylation of histones for the regulation of neuron-specific transcriptional programs. PMID:27606339

  5. Widespread alternative and aberrant splicing revealed by lariat sequencing

    PubMed Central

    Stepankiw, Nicholas; Raghavan, Madhura; Fogarty, Elizabeth A.; Grimson, Andrew; Pleiss, Jeffrey A.

    2015-01-01

    Alternative splicing is an important and ancient feature of eukaryotic gene structure, the existence of which has likely facilitated eukaryotic proteome expansions. Here, we have used intron lariat sequencing to generate a comprehensive profile of splicing events in Schizosaccharomyces pombe, amongst the simplest organisms that possess mammalian-like splice site degeneracy. We reveal an unprecedented level of alternative splicing, including alternative splice site selection for over half of all annotated introns, hundreds of novel exon-skipping events, and thousands of novel introns. Moreover, the frequency of these events is far higher than previous estimates, with alternative splice sites on average activated at ∼3% the rate of canonical sites. Although a subset of alternative sites are conserved in related species, implying functional potential, the majority are not detectably conserved. Interestingly, the rate of aberrant splicing is inversely related to expression level, with lowly expressed genes more prone to erroneous splicing. Although we validate many events with RNAseq, the proportion of alternative splicing discovered with lariat sequencing is far greater, a difference we attribute to preferential decay of aberrantly spliced transcripts. Together, these data suggest the spliceosome possesses far lower fidelity than previously appreciated, highlighting the potential contributions of alternative splicing in generating novel gene structures. PMID:26261211

  6. Impacts of Alternative Splicing Events on the Differentiation of Adipocytes

    PubMed Central

    Lin, Jung-Chun

    2015-01-01

    Alternative splicing was found to be a common phenomenon after the advent of whole transcriptome analyses or next generation sequencing. Over 90% of human genes were demonstrated to undergo at least one alternative splicing event. Alternative splicing is an effective mechanism to spatiotemporally expand protein diversity, which influences the cell fate and tissue development. The first focus of this review is to highlight recent studies, which demonstrated effects of alternative splicing on the differentiation of adipocytes. Moreover, use of evolving high-throughput approaches, such as transcriptome analyses (RNA sequencing), to profile adipogenic transcriptomes, is also addressed. PMID:26389882

  7. Alternative Splicing of G9a Regulates Neuronal Differentiation.

    PubMed

    Fiszbein, Ana; Giono, Luciana E; Quaglino, Ana; Berardino, Bruno G; Sigaut, Lorena; von Bilderling, Catalina; Schor, Ignacio E; Steinberg, Juliana H Enriqué; Rossi, Mario; Pietrasanta, Lía I; Caramelo, Julio J; Srebrow, Anabella; Kornblihtt, Alberto R

    2016-03-29

    Chromatin modifications are critical for the establishment and maintenance of differentiation programs. G9a, the enzyme responsible for histone H3 lysine 9 dimethylation in mammalian euchromatin, exists as two isoforms with differential inclusion of exon 10 (E10) through alternative splicing. We find that the G9a methyltransferase is required for differentiation of the mouse neuronal cell line N2a and that E10 inclusion increases during neuronal differentiation of cultured cells, as well as in the developing mouse brain. Although E10 inclusion greatly stimulates overall H3K9me2 levels, it does not affect G9a catalytic activity. Instead, E10 increases G9a nuclear localization. We show that the G9a E10(+) isoform is necessary for neuron differentiation and regulates the alternative splicing pattern of its own pre-mRNA, enhancing E10 inclusion. Overall, our findings indicate that by regulating its own alternative splicing, G9a promotes neuron differentiation and creates a positive feedback loop that reinforces cellular commitment to differentiation. PMID:26997278

  8. When Unified Teacher Pay Scales Meet Differential Alternative Returns

    ERIC Educational Resources Information Center

    Walsh, Patrick

    2014-01-01

    This paper quantifies the extent to which unified teacher pay scales and differential alternatives produce opportunity costs that are asymmetric in math and verbal skills. Data from the Baccalaureate and Beyond 1997 and 2003 follow-ups are used to estimate a fully parametric, selection-corrected wage equation for nonteachers, which is then used to…

  9. An Investigation of Differential Reinforcement of Alternative Behavior without Extinction

    ERIC Educational Resources Information Center

    Athens, Elizabeth S.; Vollmer, Timothy R.

    2010-01-01

    We manipulated relative reinforcement for problem behavior and appropriate behavior using differential reinforcement of alternative behavior (DRA) without an extinction component. Seven children with developmental disabilities participated. We manipulated duration (Experiment 1), quality (Experiment 2), delay (Experiment 3), or a combination of…

  10. Dynamic regulation of alternative splicing and chromatin structure in Drosophila gonads revealed by RNA-seq

    PubMed Central

    Gan, Qiang; Chepelev, Iouri; Wei, Gang; Tarayrah, Lama; Cui, Kairong; Zhao, Keji; Chen, Xin

    2010-01-01

    Both transcription and post-transcriptional processes, such as alternative splicing, play crucial roles in controlling developmental programs in metazoans. Recently emerged RNA-seq method has brought our understandings of eukaryotic transcriptomes to a new level, because it can resolve both gene expression level and alternative splicing events simultaneously. To gain a better understanding of cellular differentiation in gonads, we analyzed mRNA profiles from Drosophila testes and ovaries using RNA-seq. We identified a set of genes that have sex-specific isoforms in wild-type (wt) gonads, including several transcription factors. We found that differentiation of sperms from undifferentiated germ cells induced a dramatic down-regulation of RNA splicing factors. Our data confirmed that RNA splicing events are significantly more frequent in the undifferentiated-cell enriched bag of marbles (bam) mutant testis, but down-regulated upon differentiation in wt testis. Consistent with this, we showed that genes required for meiosis and terminal differentiation in wt testis were mainly regulated at the transcriptional level, but not by alternative splicing. Unexpectedly, we observed an increase in expression of all families of chromatin remodeling factors and histone modifying enzymes in the undifferentiated cell-enriched bam testis. More interestingly, chromatin regulators and histone modifying enzymes with opposite enzymatic activities are co-enriched in undifferentiated cells in testis, suggesting these cells may possess dynamic chromatin architecture. Finally, our data revealed many new features of the Drosophila gonadal transcriptomes, and will lead to a more comprehensive understanding of how differential gene expression and splicing regulate gametogenesis in Drosophila. Our data provided a foundation for the systematic study of gene expression and alternative splicing in many interesting areas of germ cell biology in Drosophila, such as the molecular basis for sexual

  11. Transcriptome Reveals Cathepsin K in Periodontal Ligament Differentiation.

    PubMed

    Yamada, S; Ozaki, N; Tsushima, K; Yamaba, S; Fujihara, C; Awata, T; Sakashita, H; Kajikawa, T; Kitagaki, J; Yamashita, M; Yanagita, M; Murakami, S

    2016-08-01

    Periodontal ligaments (PDLs) play an important role in remodeling the alveolar bond and cementum. Characterization of the periodontal tissue transcriptome remains incomplete, and an improved understanding of PDL features could aid in developing new regenerative therapies. Here, we aimed to generate and analyze a large human PDL transcriptome. We obtained PDLs from orthodontic treatment patients, isolated the RNA, and used a vector-capping method to make a complementary DNA library from >20,000 clones. Our results revealed that 58% of the sequences were full length. Furthermore, our analysis showed that genes expressed at the highest frequencies included those for collagen type I, collagen type III, and proteases. We also found 5 genes whose expressions have not been previously reported in human PDL. To access which of the highly expressed genes might be important for PDL cell differentiation, we used real-time polymerase chain reaction to measure their expression in differentiating cells. Among the genes tested, the cysteine protease cathepsin K had the highest upregulation, so we measured its relative expression in several tissues, as well as in osteoclasts, which are known to express high levels of cathepsin K. Our results revealed that PDL cells express cathepsin K at similar levels as osteoclasts, which are both expressed at higher levels than those of the other tissues tested. We also measured cathepsin K protein expression and enzyme activity during cell differentiation and found that both increased during this process. Immunocytochemistry experiments revealed that cathepsin K localizes to the interior of lysosomes. Last, we examined the effect of inhibiting cathepsin K during cell differentiation and found that cathepsin K inhibition stimulated calcified nodule formation and increased the levels of collagen type I and osteocalcin gene expression. Based on these results, cathepsin K seems to regulate collagen fiber accumulation during human PDL cell

  12. Cauliflower mosaic virus Transcriptome Reveals a Complex Alternative Splicing Pattern

    PubMed Central

    Bouton, Clément; Geldreich, Angèle; Ramel, Laëtitia; Ryabova, Lyubov A.; Dimitrova, Maria; Keller, Mario

    2015-01-01

    The plant pararetrovirus Cauliflower mosaic virus (CaMV) uses alternative splic-ing to generate several isoforms from its polycistronic pregenomic 35S RNA. This pro-cess has been shown to be essential for infectivity. Previous works have identified four splice donor sites and a single splice acceptor site in the 35S RNA 5’ region and sug-gested that the main role of CaMV splicing is to downregulate expression of open read-ing frames (ORFs) I and II. In this study, we show that alternative splicing is a conserved process among CaMV isolates. In Cabb B-JI and Cabb-S isolates, splicing frequently leads to different fusion between ORFs, particularly between ORF I and II. The corresponding P1P2 fusion proteins expressed in E. coli interact with viral proteins P2 and P3 in vitro. However, they are detected neither during infection nor upon transient expression in planta, which suggests rapid degradation after synthesis and no important biological role in the CaMV infectious cycle. To gain a better understanding of the functional relevance of 35S RNA alternative splicing in CaMV infectivity, we inactivated the previously described splice sites. All the splicing mutants were as pathogenic as the corresponding wild-type isolate. Through RT-PCR-based analysis we demonstrate that CaMV 35S RNA exhibits a complex splicing pattern, as we identify new splice donor and acceptor sites whose selection leads to more than thirteen 35S RNA isoforms in infected turnip plants. Inactivating splice donor or acceptor sites is not lethal for the virus, since disrupted sites are systematically rescued by the activation of cryptic and/or seldom used splice sites. Taken together, our data depict a conserved, complex and flexible process, involving multiple sites, that ensures splicing of 35S RNA. PMID:26162084

  13. Global Gene Expression Profiling and Alternative Splicing Events during the Chondrogenic Differentiation of Human Cartilage Endplate-Derived Stem Cells

    PubMed Central

    Shang, Jin; Fan, Xin; Shangguan, Lei; Liu, Huan; Zhou, Yue

    2015-01-01

    Low back pain (LBP) is a very prevalent disease and degenerative disc diseases (DDDs) usually account for the LBP. However, the pathogenesis of DDDs is complicated and difficult to elucidate. Alternative splicing is a sophisticated regulatory process which greatly increases cellular complexity and phenotypic diversity of eukaryotic organisms. In addition, the cartilage endplate-derived stem cells have been discovered and identified by our research group. In this paper, we continue to investigate gene expression profiling and alternative splicing events during chondrogenic differentiation of cartilage endplate-derived stem cells. We adopted Affymetrix Human Transcriptome Array 2.0 (HTA 2.0) to compare the transcriptional and splicing changes between the control and differentiated samples. RT-PCR and quantitative PCR are used to validate the microarray results. The GO and KEGG pathway analysis was also performed. After bioinformatics analysis of the data, we detected 1953 differentially expressed genes. In terms of alternative splicing, the Splicing Index algorithm was used to select alternatively spliced genes. We detected 4411 alternatively spliced genes. GO and KEGG pathway analysis also revealed several functionally involved biological processes and signaling pathways. To our knowledge, this is the first study to investigate the alternative splicing mechanisms in chondrogenic differentiation of stem cells on a genome-wide scale. PMID:26649308

  14. Small molecules reveal an alternative mechanism of Bax activation

    PubMed Central

    Brahmbhatt, Hetal; Uehling, David; Al-awar, Rima; Leber, Brian; Andrews, David

    2016-01-01

    The pro-apoptotic protein Bax commits a cell to death by permeabilizing the mitochondrial outer membrane (MOM). To obtain small-molecule probes for elucidating the molecular mechanism(s) of Bax activation, we screened for compounds that induced Bax-mediated liposome permeabilization. We identified five structurally different small molecules that promoted both Bax targeting to and oligomerization at membranes. All five compounds initiated Bax oligomerization in the absence of membranes by a mechanism unlike Bax activation by Bcl-2 homology 3 domain (BH3) proteins. Some of the compounds induced Bax/Bak-dependent apoptosis in cells. Activation of Bax by the most active compound was poorly inhibited by the anti-apoptotic protein Bcl-XL and requires a cysteine residue at position 126 of Bax that is not required for activation by BH3 proteins. Our results reveal a novel pathway for Bax activation independent of pro-apoptotic BH3 proteins that may have important implications for the regulation of Bax activity in cells. PMID:26916338

  15. Small molecules reveal an alternative mechanism of Bax activation.

    PubMed

    Brahmbhatt, Hetal; Uehling, David; Al-Awar, Rima; Leber, Brian; Andrews, David

    2016-04-15

    The pro-apoptotic protein Bax commits a cell to death by permeabilizing the mitochondrial outer membrane (MOM). To obtain small-molecule probes for elucidating the molecular mechanism(s) of Bax activation, we screened for compounds that induced Bax-mediated liposome permeabilization. We identified five structurally different small molecules that promoted both Bax targeting to and oligomerization at membranes. All five compounds initiated Bax oligomerization in the absence of membranes by a mechanism unlike Bax activation by Bcl-2 homology 3 domain (BH3) proteins. Some of the compounds induced Bax/Bak-dependent apoptosis in cells. Activation of Bax by the most active compound was poorly inhibited by the anti-apoptotic protein Bcl-XL and requires a cysteine residue at position 126 of Bax that is not required for activation by BH3 proteins. Our results reveal a novel pathway for Bax activation independent of pro-apoptotic BH3 proteins that may have important implications for the regulation of Bax activity in cells. PMID:26916338

  16. Alternative response training, differential reinforcement of other behavior, and extinction in squirrel monkeys (Saimiri sciureus)1

    PubMed Central

    Mulick, J. A.; Leitenberg, H.; Rawson, R. A.

    1976-01-01

    In Experiment I, (a) extinction, (b) extinction plus reinforcement of a discrete alternative response, and (c) differential reinforcement of other behavior were each correlated with a different stimulus in a three-component multiple schedule. The alternative-response procedure more rapidly and completely suppressed behavior than did differential reinforcement of other behavior. Differential reinforcement of other behavior was slightly more effective than extinction alone. In Experiment II, reinforcement of specific alternative behavior during extinction and differential reinforcement of other behavior were used in two components, while one component continued to provide reinforcement for the original response. Once again, the alternative-response procedure was most effective in reducing responding as long as it remained in effect. However, the responding partially recovered when reinforcement for competing behavior was discontinued. In general, responding was less readily reduced by differential reinforcement of other behavior than by the specific alternative-response procedure. PMID:16811914

  17. On the Lower Alternating Integral of Pontryagin in Linear Differential Games of Pursuit

    NASA Astrophysics Data System (ADS)

    Nikol'skiĭ, M. S.

    1987-02-01

    In this article the concept of the lower alternating integral of Pontryagin, which differs from the analogous concept of A. Azamov (MR 83k: 90142), is introduced. The properties of this object are studied in comparison with those of the alternating integral of Pontryagin. A procedure is given for application of the lower alternating integral of Pontryagin to linear differential games of pursuit, with special consideration for the question of using the lower alternating integral of Pontryagin when there are countably many measurements of the phase vector. The results are illustrated in two known differential games: "the boy and the crocodile" and the "control example of Pontryagin."Bibliography: 18 titles.

  18. Differential Reinforcement of Alternative Behavior in Center-Based Classrooms: Evaluation of Pre-Teaching the Alternative Behavior

    ERIC Educational Resources Information Center

    LeGray, Matthew W.; Dufrene, Brad A.; Mercer, Sterett; Olmi, D. Joe; Sterling, Heather

    2013-01-01

    This study investigated the effectiveness of a differential reinforcement of alternative behavior procedure in decreasing disruptive behavior while simultaneously increasing the appropriate behavior of four children of typical development between the ages of 4 and 6 in center-based classrooms. We began with brief functional analyses for each…

  19. Differential network analysis reveals dysfunctional regulatory networks in gastric carcinogenesis.

    PubMed

    Cao, Mu-Shui; Liu, Bing-Ya; Dai, Wen-Tao; Zhou, Wei-Xin; Li, Yi-Xue; Li, Yuan-Yuan

    2015-01-01

    Gastric Carcinoma is one of the most common cancers in the world. A large number of differentially expressed genes have been identified as being associated with gastric cancer progression, however, little is known about the underlying regulatory mechanisms. To address this problem, we developed a differential networking approach that is characterized by including a nascent methodology, differential coexpression analysis (DCEA), and two novel quantitative methods for differential regulation analysis. We first applied DCEA to a gene expression dataset of gastric normal mucosa, adenoma and carcinoma samples to identify gene interconnection changes during cancer progression, based on which we inferred normal, adenoma, and carcinoma-specific gene regulation networks by using linear regression model. It was observed that cancer genes and drug targets were enriched in each network. To investigate the dynamic changes of gene regulation during carcinogenesis, we then designed two quantitative methods to prioritize differentially regulated genes (DRGs) and gene pairs or links (DRLs) between adjacent stages. It was found that known cancer genes and drug targets are significantly higher ranked. The top 4% normal vs. adenoma DRGs (36 genes) and top 6% adenoma vs. carcinoma DRGs (56 genes) proved to be worthy of further investigation to explore their association with gastric cancer. Out of the 16 DRGs involved in two top-10 DRG lists of normal vs. adenoma and adenoma vs. carcinoma comparisons, 15 have been reported to be gastric cancer or cancer related. Based on our inferred differential networking information and known signaling pathways, we generated testable hypotheses on the roles of GATA6, ESRRG and their signaling pathways in gastric carcinogenesis. Compared with established approaches which build genome-scale GRNs, or sub-networks around differentially expressed genes, the present one proved to be better at enriching cancer genes and drug targets, and prioritizing

  20. Differential network analysis reveals dysfunctional regulatory networks in gastric carcinogenesis

    PubMed Central

    Cao, Mu-Shui; Liu, Bing-Ya; Dai, Wen-Tao; Zhou, Wei-Xin; Li, Yi-Xue; Li, Yuan-Yuan

    2015-01-01

    Gastric Carcinoma is one of the most common cancers in the world. A large number of differentially expressed genes have been identified as being associated with gastric cancer progression, however, little is known about the underlying regulatory mechanisms. To address this problem, we developed a differential networking approach that is characterized by including a nascent methodology, differential coexpression analysis (DCEA), and two novel quantitative methods for differential regulation analysis. We first applied DCEA to a gene expression dataset of gastric normal mucosa, adenoma and carcinoma samples to identify gene interconnection changes during cancer progression, based on which we inferred normal, adenoma, and carcinoma-specific gene regulation networks by using linear regression model. It was observed that cancer genes and drug targets were enriched in each network. To investigate the dynamic changes of gene regulation during carcinogenesis, we then designed two quantitative methods to prioritize differentially regulated genes (DRGs) and gene pairs or links (DRLs) between adjacent stages. It was found that known cancer genes and drug targets are significantly higher ranked. The top 4% normal vs. adenoma DRGs (36 genes) and top 6% adenoma vs. carcinoma DRGs (56 genes) proved to be worthy of further investigation to explore their association with gastric cancer. Out of the 16 DRGs involved in two top-10 DRG lists of normal vs. adenoma and adenoma vs. carcinoma comparisons, 15 have been reported to be gastric cancer or cancer related. Based on our inferred differential networking information and known signaling pathways, we generated testable hypotheses on the roles of GATA6, ESRRG and their signaling pathways in gastric carcinogenesis. Compared with established approaches which build genome-scale GRNs, or sub-networks around differentially expressed genes, the present one proved to be better at enriching cancer genes and drug targets, and prioritizing

  1. Transcriptome Bioinformatical Analysis of Vertebrate Stages of Schistosoma japonicum Reveals Alternative Splicing Events

    PubMed Central

    Wang, Xinye; Xu, Xindong; Lu, Xingyu; Zhang, Yuanbin; Pan, Weiqing

    2015-01-01

    Alternative splicing is a molecular process that contributes greatly to the diversification of proteome and to gene functions. Understanding the mechanisms of stage-specific alternative splicing can provide a better understanding of the development of eukaryotes and the functions of different genes. Schistosoma japonicum is an infectious blood-dwelling trematode with a complex lifecycle that causes the tropical disease schistosomiasis. In this study, we analyzed the transcriptome of Schistosoma japonicum to discover alternative splicing events in this parasite, by applying RNA-seq to cDNA library of adults and schistosomula. Results were validated by RT-PCR and sequencing. We found 11,623 alternative splicing events among 7,099 protein encoding genes and average proportion of alternative splicing events per gene was 42.14%. We showed that exon skip is the most common type of alternative splicing events as found in high eukaryotes, whereas intron retention is the least common alternative splicing type. According to intron boundary analysis, the parasite possesses same intron boundaries as other organisms, namely the classic “GT-AG” rule. And in alternative spliced introns or exons, this rule is less strict. And we have attempted to detect alternative splicing events in genes encoding proteins with signal peptides and transmembrane helices, suggesting that alternative splicing could change subcellular locations of specific gene products. Our results indicate that alternative splicing is prevalent in this parasitic worm, and that the worm is close to its hosts. The revealed secretome involved in alternative splicing implies new perspective into understanding interaction between the parasite and its host. PMID:26407301

  2. Transcriptome Bioinformatical Analysis of Vertebrate Stages of Schistosoma japonicum Reveals Alternative Splicing Events.

    PubMed

    Wang, Xinye; Xu, Xindong; Lu, Xingyu; Zhang, Yuanbin; Pan, Weiqing

    2015-01-01

    Alternative splicing is a molecular process that contributes greatly to the diversification of proteome and to gene functions. Understanding the mechanisms of stage-specific alternative splicing can provide a better understanding of the development of eukaryotes and the functions of different genes. Schistosoma japonicum is an infectious blood-dwelling trematode with a complex lifecycle that causes the tropical disease schistosomiasis. In this study, we analyzed the transcriptome of Schistosoma japonicum to discover alternative splicing events in this parasite, by applying RNA-seq to cDNA library of adults and schistosomula. Results were validated by RT-PCR and sequencing. We found 11,623 alternative splicing events among 7,099 protein encoding genes and average proportion of alternative splicing events per gene was 42.14%. We showed that exon skip is the most common type of alternative splicing events as found in high eukaryotes, whereas intron retention is the least common alternative splicing type. According to intron boundary analysis, the parasite possesses same intron boundaries as other organisms, namely the classic "GT-AG" rule. And in alternative spliced introns or exons, this rule is less strict. And we have attempted to detect alternative splicing events in genes encoding proteins with signal peptides and transmembrane helices, suggesting that alternative splicing could change subcellular locations of specific gene products. Our results indicate that alternative splicing is prevalent in this parasitic worm, and that the worm is close to its hosts. The revealed secretome involved in alternative splicing implies new perspective into understanding interaction between the parasite and its host. PMID:26407301

  3. Planarian Phototactic Assay Reveals Differential Behavioral Responses Based on Wavelength

    PubMed Central

    Paskin, Taylor R.; Jellies, John; Bacher, Jessica; Beane, Wendy S.

    2014-01-01

    Planarians are free-living aquatic flatworms that possess a well-documented photophobic response to light. With a true central nervous system and simple cerebral eyes (ocelli), planarians are an emerging model for regenerative eye research. However, comparatively little is known about the physiology of their photoreception or how their behavior is affected by various wavelengths. Most phototactic studies have examined planarian behavior using white light. Here, we describe a novel planarian behavioral assay to test responses to small ranges of visible wavelengths (red, blue, green), as well as ultraviolet (UV) and infrared (IR) which have not previously been examined. Our data show that planarians display behavioral responses across a range of wavelengths. These responses occur in a hierarchy, with the shortest wavelengths (UV) causing the most intense photophobic responses while longer wavelengths produce no effect (red) or an apparent attraction (IR). In addition, our data reveals that planarian photophobia is comprised of both a general photophobic response (that drives planarians to escape the light source regardless of wavelength) and wavelength-specific responses that encompass specific behavioral reactions to individual wavelengths. Our results serve to improve the understanding of planarian phototaxis and suggest that behavioral studies performed with white light mask a complex behavioral interaction with the environment. PMID:25493551

  4. A genome wide analysis of alternative splicing events during the osteogenic differentiation of human cartilage endplate-derived stem cells.

    PubMed

    Shang, Jin; Wang, Honggang; Fan, Xin; Shangguan, Lei; Liu, Huan

    2016-08-01

    Low back pain is a prevalent disease, which leads to suffering and disabilities in a vast number of individuals. Degenerative disc diseases are usually the underlying causes of low back pain. However, the pathogenesis of degenerative disc diseases is highly complex and difficult to determine. Current therapies for degenerative disc diseases are various. In particular, cell-based therapies have proven to be effective and promising. Our research group has previously isolated and identified the cartilage endplate‑derived stem cells. In addition, alternative splicing is a sophisticated regulatory mechanism, which greatly increases cellular complexity and phenotypic diversity of eukaryotic organisms. The present study continued to investigate alternative splicing events in osteogenic differentiation of cartilage endplate‑derived stem cells. An Affymetrix Human Transcriptome Array 2.0 was used to detect splicing changes between the control and differentiated samples. Additionally, molecular function and pathway analysis were also performed. Following rigorous bioinformatics analysis of the data, 3,802 alternatively spliced genes were identified, and 10 of these were selected for validation by reverse transcription‑polymerase chain reaction. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway analysis also revealed numerous enriched GO terms and signaling pathways. To the best of our knowledge, the present study is the first to investigate alternative splicing mechanisms in osteogenic differentiation of stem cells on a genome‑wide scale. The illumination of molecular mechanisms of stem cell osteogenic differentiation may assist the development novel bioengineered methods to treat degenerative disc diseases. PMID:27278552

  5. Alternative promoter usage and differential expression of multiple transcripts of mouse Prkar1a gene.

    PubMed

    Banday, Abdul Rouf; Azim, Shafquat; Tabish, Mohammad

    2011-11-01

    Prkar1a gene encodes regulatory type 1 alpha subunit (RIα) of cAMP-dependent protein kinase (PKA) in mouse. The role of this gene has been implicated in Carney complex and many cancer types that suggest its involvement in physiological processes like cell cycle regulation, growth and/or proliferation. We have identified and sequenced partial cDNA clones encoding four alternatively spliced transcripts of mouse Prkar1a gene. These transcripts have alternate 5' UTR structure which results from splicing of three exons (designated as E1a, E1b, and E1c) to canonical exon 2. The designated transcripts T1, T2, T3, and T4 contain 5' UTR exons as E1c, E1a + E1b, E1a, and E1b, respectively. The transcript T1 corresponded to earlier reported transcript in GenBank. In silico study of genomic DNA sequence revealed three distinct promoter regions namely, P1, P2, and P3 upstream of the exons E1a, E1b, and E1c, respectively. P1 is non-CpG-related promoter but P2 and P3 are CpG-related promoters; however, all three are TATA less. RT-PCR analysis demonstrated the expression of all four transcripts in late postnatal stages; however, these were differentially regulated in early postnatal stages of 0.5 day, 3 day, and 15 day mice in different tissue types. Variations in expression of Prkar1a gene transcripts suggest their regulation from multiple promoters that respond to a variety of signals arising in or out of the cell in tissue and developmental stage-specific manner. PMID:21638026

  6. Alternative Differential Identification Approaches for 2 Similar Bacilli Commonly Studied in Microbiology.

    ERIC Educational Resources Information Center

    Benathen, Isaiah A.

    1991-01-01

    Alternatives to the traditional unknown tests that permit a clear and unequivocal differential identification decision between Bacillus subtilis and Bacillus megaterium are presented. Plates of Phenylethyl Alcohol agar with Blood (PEAB), slants of Bile Esculin agar and plates of DNA agar are used. The materials, methods, results, and conclusions…

  7. Introducing Differential Equations Students to the Fredholm Alternative--In Staggered Doses

    ERIC Educational Resources Information Center

    Savoye, Philippe

    2011-01-01

    The development, in an introductory differential equations course, of boundary value problems in parallel with initial value problems and the Fredholm Alternative. Examples are provided of pairs of homogeneous and nonhomogeneous boundary value problems for which existence and uniqueness issues are considered jointly. How this heightens students'…

  8. Effects of Treatment Integrity Failures during Differential Reinforcement of Alternative Behavior: A Translational Model

    ERIC Educational Resources Information Center

    Pipkin, Claire St. Peter; Vollmer, Timothy R.; Sloman, Kimberly N.

    2010-01-01

    Differential reinforcement of alternative behavior (DRA) is used frequently as a treatment for problem behavior. Previous studies on treatment integrity failures during DRA suggest that the intervention is robust, but research has not yet investigated the effects of different types of integrity failures. We examined the effects of two types of…

  9. Comparing Main and Collateral Effects of Extinction and Differential Reinforcement of Alternative Behavior

    ERIC Educational Resources Information Center

    Petscher, Erin Seligson; Bailey, Jon S.

    2008-01-01

    This study evaluated the effects and collateral effects of extinction (EXT) and differential reinforcement of alternative behavior (DRA) interventions with inappropriate vocalizations and work refusal. Both interventions have been used frequently to reduce problem behaviors. The benefits of these interventions have been established yet may be…

  10. Alternative 5' exons and differential splicing regulate expression of protein 4.1R isoforms with distinct N-termini.

    PubMed

    Parra, Marilyn K; Gee, Sherry L; Koury, Mark J; Mohandas, Narla; Conboy, John G

    2003-05-15

    Among the alternative pre-mRNA splicing events that characterize protein 4.1R gene expression, one involving exon 2' plays a critical role in regulating translation initiation and N-terminal protein structure. Exon 2' encompasses translation initiation site AUG1 and is located between alternative splice acceptor sites at the 5' end of exon 2; its inclusion or exclusion from mature 4.1R mRNA regulates expression of longer or shorter isoforms of 4.1R protein, respectively. The current study reports unexpected complexity in the 5' region of the 4.1R gene that directly affects alternative splicing of exon 2'. Identified far upstream of exon 2 in both mouse and human genomes were 3 mutually exclusive alternative 5' exons, designated 1A, 1B, and 1C; all 3 are associated with strong transcriptional promoters in the flanking genomic sequence. Importantly, exons 1A and 1B splice differentially with respect to exon 2', generating transcripts with different 5' ends and distinct N-terminal protein coding capacity. Exon 1A-type transcripts splice so as to exclude exon 2' and therefore utilize the downstream AUG2 for translation of 80-kDa 4.1R protein, whereas exon 1B transcripts include exon 2' and initiate at AUG1 to synthesize 135-kDa isoforms. RNA blot analyses revealed that 1A transcripts increase in abundance in late erythroblasts, consistent with the previously demonstrated up-regulation of 80-kDa 4.1R during terminal erythroid differentiation. Together, these results suggest that synthesis of structurally distinct 4.1R protein isoforms in various cell types is regulated by a novel mechanism requiring coordination between upstream transcription initiation events and downstream alternative splicing events. PMID:12522012

  11. Alternative 5' exons and differential splicing regulate expression of protein 4.1R isoforms with distinct n-termini

    SciTech Connect

    Parra, Marilyn K.; Gee, Sherry L.; Koury, Mark J.; Mohandas, Narla; Conboy, John G.

    2003-03-25

    Among the alternative pre-mRNA splicing events that characterize protein 4.1R gene expression, one involving exon 2' plays a critical role in regulating translation initiation and N-terminal protein structure. Exon 2' encompasses translation initiation site AUG1 and is located between alternative splice acceptor sites at the 5' end of exon 2; its inclusion or exclusion from mature 4.1R mRNA regulates expression of longer or shorter isoforms of 4.1R protein, respectively. The current study reports unexpected complexity in the 5' region of the 4.1R gene that directly affects alternative splicing of exon 2'. Three mutually exclusive alternative 5' exons, designated 1A, 1B, and 1C, were identified far upstream of exon 2 in both mouse and human genomes; all three are associated with strong transcriptional promoters in the flanking genomic sequence. Importantly, exons 1A and 1B splice differentially with respect to exon 2', generating transcripts with different 5' ends and distinct N-terminal protein coding capacity. Exon 1A-type transcripts splice so as to exclude exon 2' and therefore utilize the downstream AUG2 for translation of 80kD 4.1R protein, whereas exon 1B transcripts include exon 2' and initiate at AUG1 to synthesize 135kD isoforms. RNA blot analyses revealed that 1A transcripts increase in abundance in late erythroblasts, consistent with the previously demonstrated upregulation of 80kD 4.1R during terminal erythroid differentiation. Together these results suggest that synthesis of structurally distinct 4.1R protein isoforms in various cell types is regulated by a novel mechanism requiring coordination between upstream transcription initiation events and downstream alternative splicing events.

  12. Rbm24 Regulates Alternative Splicing Switch in Embryonic Stem Cell Cardiac Lineage Differentiation.

    PubMed

    Zhang, Tao; Lin, Yu; Liu, Jing; Zhang, Zi Guan; Fu, Wei; Guo, Li Yan; Pan, Lei; Kong, Xu; Zhang, Meng Kai; Lu, Ying Hua; Huang, Zheng Rong; Xie, Qiang; Li, Wei Hua; Xu, Xiu Qin

    2016-07-01

    The transition of embryonic stem cell (ESC) pluripotency to differentiation is accompanied by an expansion of mRNA and proteomic diversity. Post-transcriptional regulation of ESCs is critically governed by cell type-specific splicing. However, little is known about the splicing factors and the molecular mechanisms directing ESC early lineage differentiation. Our study identifies RNA binding motif protein 24 (Rbm24) as a key splicing regulator that plays an essential role in controlling post-transcriptional networks during ESC transition into cardiac differentiation. Using an inducible mouse ESC line in which gene expression could be temporally regulated, we demonstrated that forced expression of Rbm24 in ESCs dramatically induced a switch to cardiac specification. Genome-wide RNA sequencing analysis identified more than 200 Rbm24-regulated alternative splicing events (AS) which occurred in genes essential for the ESC pluripotency or differentiation. Remarkably, AS genes regulated by Rbm24 composed of transcriptional factors, cytoskeleton proteins, and ATPase gene family members which are critical components required for cardiac development and functionality. Furthermore, we show that Rbm24 regulates ESC differentiation by promoting alternative splicing of pluripotency genes. Among the Rbm24-regulated events, Tpm1, an actin filament family gene, was identified to possess ESC/tissue specific isoforms. We demonstrated that these isoforms were functionally distinct and that their exon AS switch was essential for ESC differentiation. Our results suggest that ESC's switching into the differentiation state can be initiated by a tissue-specific splicing regulator, Rbm24. This finding offers a global view on how an RNA binding protein influences ESC lineage differentiation by a splicing-mediated regulatory mechanism. Stem Cells 2016;34:1776-1789. PMID:26990106

  13. Ultrastructural evidence for divergent and alternating differentiations in spindle cell sarcoma xenografts.

    PubMed

    Schmidt, U; Stüben, G; Stuschke, M; Donhuijsen, K

    1997-04-01

    Seven spindle cell sarcomas, 5 poorly differentiated ones and 2 moderately well differentiated ones, were established on nude mice and long term passaging was done. Sarcoma strains were analysed electron microscopically in an attempt to get further insight in spindle cell sarcoma differentiation pathways. Ultrastructurally, the tumours were classified as malignant peripheral nerve sheath tumour (3/7), leiomyosarcoma (2/7), rhabdomyosarcoma (1/7), and spindle cell sarcoma not otherwise classifiable (1/7). Undifferentiated tumour cells including fibroblastoid ones predominated in most xenografts, whereas cells harbouring cytoplasmic specificities tended to be few in number. Nevertheless, divergent differentiations exhibiting unusual double or triple patterns could be documented ultrastructurally in 12/30 xenografts with juxtaposed myomatous as well as nerve sheath-like cells and, in addition, histiocytoid (MFH-like) elements in 3 of the xenografts. Moreover, sarcoma strains alternated fine structural constellations in the course of passaging, whereby different phenotypes, myomatous, nerve sheath-like, unspecific, or mixed ones, succeeded one another. These findings pursue recent immunohistochemical data on multidirectional sarcoma differentiation by means of electron microscopy. They, furthermore, fit well into the concept of multipotential stem cells as progenitors in mesenchymal differentiation and suggest microenvironment to play a modifying role in the expression of cell differentiation. PMID:9165712

  14. Global genome splicing analysis reveals an increased number of alternatively spliced genes with aging.

    PubMed

    Rodríguez, Sofía A; Grochová, Diana; McKenna, Tomás; Borate, Bhavesh; Trivedi, Niraj S; Erdos, Michael R; Eriksson, Maria

    2016-04-01

    Alternative splicing (AS) is a key regulatory mechanism for the development of different tissues; however, not much is known about changes to alternative splicing during aging. Splicing events may become more frequent and widespread genome-wide as tissues age and the splicing machinery stringency decreases. Using skin, skeletal muscle, bone, thymus, and white adipose tissue from wild-type C57BL6/J male mice (4 and 18 months old), we examined the effect of age on splicing by AS analysis of the differential exon usage of the genome. The results identified a considerable number of AS genes in skeletal muscle, thymus, bone, and white adipose tissue between the different age groups (ranging from 27 to 246 AS genes corresponding to 0.3-3.2% of the total number of genes analyzed). For skin, skeletal muscle, and bone, we included a later age group (28 months old) that showed that the number of alternatively spliced genes increased with age in all three tissues (P < 0.01). Analysis of alternatively spliced genes across all tissues by gene ontology and pathway analysis identified 158 genes involved in RNA processing. Additional analysis of AS in a mouse model for the premature aging disease Hutchinson-Gilford progeria syndrome was performed. The results show that expression of the mutant protein, progerin, is associated with an impaired developmental splicing. As progerin accumulates, the number of genes with AS increases compared to in wild-type skin. Our results indicate the existence of a mechanism for increased AS during aging in several tissues, emphasizing that AS has a more important role in the aging process than previously known. PMID:26685868

  15. The Role of Alternative Splicing in the Control of Immune Homeostasis and Cellular Differentiation.

    PubMed

    Yabas, Mehmet; Elliott, Hannah; Hoyne, Gerard F

    2016-01-01

    Alternative splicing of pre-mRNA helps to enhance the genetic diversity within mammalian cells by increasing the number of protein isoforms that can be generated from one gene product. This provides a great deal of flexibility to the host cell to alter protein function, but when dysregulation in splicing occurs this can have important impact on health and disease. Alternative splicing is widely used in the mammalian immune system to control the development and function of antigen specific lymphocytes. In this review we will examine the splicing of pre-mRNAs yielding key proteins in the immune system that regulate apoptosis, lymphocyte differentiation, activation and homeostasis, and discuss how defects in splicing can contribute to diseases. We will describe how disruption to trans-acting factors, such as heterogeneous nuclear ribonucleoproteins (hnRNPs), can impact on cell survival and differentiation in the immune system. PMID:26703587

  16. The Role of Alternative Splicing in the Control of Immune Homeostasis and Cellular Differentiation

    PubMed Central

    Yabas, Mehmet; Elliott, Hannah; Hoyne, Gerard F.

    2015-01-01

    Alternative splicing of pre-mRNA helps to enhance the genetic diversity within mammalian cells by increasing the number of protein isoforms that can be generated from one gene product. This provides a great deal of flexibility to the host cell to alter protein function, but when dysregulation in splicing occurs this can have important impact on health and disease. Alternative splicing is widely used in the mammalian immune system to control the development and function of antigen specific lymphocytes. In this review we will examine the splicing of pre-mRNAs yielding key proteins in the immune system that regulate apoptosis, lymphocyte differentiation, activation and homeostasis, and discuss how defects in splicing can contribute to diseases. We will describe how disruption to trans-acting factors, such as heterogeneous nuclear ribonucleoproteins (hnRNPs), can impact on cell survival and differentiation in the immune system. PMID:26703587

  17. Morphometric Differentiation Among Anastrepha fraterculus (Diptera: Tephritidae) Exploiting Sympatric Alternate Hosts.

    PubMed

    Gómez-Cendra, P V; Paulin, L E; Oroño, L; Ovruski, S M; Vilardi, J C

    2016-04-01

    Anastrepha fraterculus (Wiedemann) is currently considered a complex of cryptic species infesting fruits from Mexico to Argentina and represents an interesting biological model for evolutionary studies. Moreover, detecting and quantifying behavioral, morphological, and genetic differentiation among populations is also relevant to the application of environment-friendly control programs. Here, phenotypic differentiation among individuals coexisting in the wild in a Northern region of Argentina was unveiled and associated with host choice. Six morphometric traits were measured in sympatric flies exploiting three different host species. Phenotypic variation was shown to be host-dependent regardless of geographical or temporal overlap. Flies collected from synchronous alternate hosts (peach and walnut) differed from each other despite the lack of geographical isolation. By contrast, flies emerging from guavas that ripen about two months later than peach and walnut showed no significant differentiation in comparison to flies collected from walnuts, but they differ significantly from flies originating from peaches. This result is consistent with the hypothesis that the same population of flies shifts from walnuts to guavas throughout the year, whereas the population of flies that uses peaches as a host is probably exploiting other alternate hosts when peach availability decreases. Further research is needed to study the underlying mechanism. Results are consistent with previous molecular markers (inter-simple sequence repeat-ISSR) research on flies stemming from the same hosts and the same area, suggesting that differentiation among flies emerging from alternative hosts occurs at both genetic and phenotypic levels. The contribution of host preference in long-term genetic differentiation is discussed. PMID:26787122

  18. 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

  19. Regulation of alternative pre-mRNA splicing during erythroid differentiation.

    PubMed

    Hou, V C; Conboy, J G

    2001-03-01

    Although the mature enucleated erythrocyte is no longer active in nuclear processes such as pre-mRNA splicing, the function of many of its major structural proteins is dependent on alternative splicing choices made during the earlier stages of erythropoiesis. These splicing decisions fundamentally regulate many aspects of protein structure and function by governing the inclusion or exclusion of exons that encode protein interaction domains, regulatory signals, or translation initiation or termination sites. Alternative splicing events may be partially or entirely erythroid-specific, ie, distinct from the splicing patterns imposed on the same transcripts in nonerythroid cells. Moreover, differentiation stage-specific splicing "switches" may alter the structure and function of erythroid proteins in physiologically important ways as the cell is morphologically and functionally remodeled during normal differentiation. Derangements in the splicing of individual mutated pre-mRNAs can produce synthesis of truncated or unstable proteins that are responsible for numerous erythrocyte disorders. This review will summarize the salient features of regulated alternative splicing in general, review existing information concerning the widespread extent of alternative splicing among erythroid genes, and describe recent studies that are beginning to uncover the mechanisms that regulate an erythroid splicing switch in the protein 4.1R gene. PMID:11224680

  20. Dynamic Transcription Factor Activity Profiles Reveal Key Regulatory Interactions During Megakaryocytic and Erythroid Differentiation

    PubMed Central

    Duncan, Mark T.; Shin, Seungjin; Wu, Jia J.; Mays, Zachary; Weng, Stanley; Bagheri, Neda; Miller, William M.; Shea, Lonnie D.

    2014-01-01

    The directed differentiation toward erythroid (E) or megakaryocytic (MK) lineages by the MK-E progenitor (MEP) could enhance the ex vivo generation of red blood cells and platelets for therapeutic transfusions. The lineage choice at the MEP bifurcation is controlled in large part by activity within the intracellular signal transduction network, the output of which determines the activity of transcription factors (TFs) and ultimately gene expression. Although many TFs have been implicated, E or MK differentiation is a complex process requiring multiple days, and the dynamics of TF activities during commitment and terminal maturation are relatively unexplored. Herein, we applied a living cell array for the large-scale, dynamic quantification of TF activities during MEP bifurcation. A panel of hematopoietic TFs (GATA-1, GATA-2, SCL/TAL1, FLI-1, NF-E2, PU.1, c-Myb) was characterized during E and MK differentiation of bipotent K562 cells. Dynamic TF activity profiles associated with differentiation towards each lineage were identified, and validated with previous reports. From these activity profiles, we show that GATA-1 is an important hub during early hemin- and PMA-induced differentiation, and reveal several characteristic TF interactions for E and MK differentiation that confirm regulatory mechanisms documented in the literature. Additionally, we highlight several novel TF interactions at various stages of E and MK differentiation. Furthermore, we investigated the mechanism by which nicotinamide (NIC) promoted terminal MK maturation using an MK-committed cell line, CHRF-288-11 (CHRF). Concomitant with its enhancement of ploidy, NIC strongly enhanced the activity of three TFs with known involvement in terminal MK maturation: FLI-1, NF-E2, and p53. Dynamic profiling of TF activity represents a novel tool to complement traditional assays focused on mRNA and protein expression levels to understand progenitor cell differentiation. PMID:24853077

  1. Dynamic transcription factor activity profiles reveal key regulatory interactions during megakaryocytic and erythroid differentiation.

    PubMed

    Duncan, Mark T; Shin, Seungjin; Wu, Jia J; Mays, Zachary; Weng, Stanley; Bagheri, Neda; Miller, William M; Shea, Lonnie D

    2014-10-01

    The directed differentiation toward erythroid (E) or megakaryocytic (MK) lineages by the MK-E progenitor (MEP) could enhance the ex vivo generation of red blood cells and platelets for therapeutic transfusions. The lineage choice at the MEP bifurcation is controlled in large part by activity within the intracellular signal transduction network, the output of which determines the activity of transcription factors (TFs) and ultimately gene expression. Although many TFs have been implicated, E or MK differentiation is a complex process requiring multiple days, and the dynamics of TF activities during commitment and terminal maturation are relatively unexplored. Herein, we applied a living cell array for the large-scale, dynamic quantification of TF activities during MEP bifurcation. A panel of hematopoietic TFs (GATA-1, GATA-2, SCL/TAL1, FLI-1, NF-E2, PU.1, c-Myb) was characterized during E and MK differentiation of bipotent K562 cells. Dynamic TF activity profiles associated with differentiation towards each lineage were identified, and validated with previous reports. From these activity profiles, we show that GATA-1 is an important hub during early hemin- and PMA-induced differentiation, and reveal several characteristic TF interactions for E and MK differentiation that confirm regulatory mechanisms documented in the literature. Additionally, we highlight several novel TF interactions at various stages of E and MK differentiation. Furthermore, we investigated the mechanism by which nicotinamide (NIC) promoted terminal MK maturation using an MK-committed cell line, CHRF-288-11 (CHRF). Concomitant with its enhancement of ploidy, NIC strongly enhanced the activity of three TFs with known involvement in terminal MK maturation: FLI-1, NF-E2, and p53. Dynamic profiling of TF activity represents a novel tool to complement traditional assays focused on mRNA and protein expression levels to understand progenitor cell differentiation. PMID:24853077

  2. Integrated live imaging and molecular profiling of embryoid bodies reveals a synchronized progression of early differentiation.

    PubMed

    Boxman, Jonathan; Sagy, Naor; Achanta, Sirisha; Vadigepalli, Rajanikanth; Nachman, Iftach

    2016-01-01

    Embryonic stem cells can spontaneously differentiate into cell types of all germ layers within embryoid bodies (EBs) in a highly variable manner. Whether there exists an intrinsic differentiation program common to all EBs is unknown. Here, we present a novel combination of high-throughput live two-photon imaging and gene expression profiling to study early differentiation dynamics spontaneously occurring within developing EBs. Onset timing of Brachyury-GFP was highly variable across EBs, while the spatial patterns as well as the dynamics of mesendodermal progression following onset were remarkably similar. We therefore defined a 'developmental clock' using the Brachyury-GFP signal onset timing. Mapping snapshot gene expression measurements to this clock revealed their temporal trends, indicating that loss of pluripotency, formation of primitive streak and mesodermal lineage progression are synchronized in EBs. Exogenous activation of Wnt or BMP signaling accelerated the intrinsic clock. CHIR down-regulated Wnt3, allowing insights into dependency mechanisms between canonical Wnt signaling and multiple genes. Our findings reveal a developmental clock characteristic of an early differentiation program common to all EBs, further establishing them as an in vitro developmental model. PMID:27530599

  3. Multi-omics maps of cotton fibre reveal epigenetic basis for staged single-cell differentiation.

    PubMed

    Wang, Maojun; Wang, Pengcheng; Tu, Lili; Zhu, Sitao; Zhang, Lin; Li, Zhonghua; Zhang, Qinghua; Yuan, Daojun; Zhang, Xianlong

    2016-05-19

    Epigenetic modifications are highlighted for their great importance in regulating plant development, but their function associated with single-cell differentiation remains undetermined. Here, we used the cotton fibre, which is the epidermal hair on the cotton ovule, as a model to investigate the regulatory role of DNA methylation in cell differentiation. The level of CHH (H = A, T, or C) DNA methylation level was found to increase during fibre development, accompanied by a decrease in RNA-directed DNA methylation (RdDM). Examination of nucleosome positioning revealed a gradual transition from euchromatin to heterochromatin for chromatin dynamics in developing fibres, which could shape the DNA methylation landscape. The observed increase in DNA methylation in fibres, compared with other ovule tissue, was demonstrated to be mediated predominantly by an active H3K9me2-dependent pathway rather than the RdDM pathway, which was inactive. Furthermore, integrated multi-omics analyses revealed that dynamic DNA methylation played a role in the regulation of lipid biosynthesis and spatio-temporal modulation of reactive oxygen species during fibre differentiation. Our study illustrates two divergent pathways mediating a continuous increase of DNA methylation and also sheds further light on the epigenetic basis for single-cell differentiation in plants. These data and analyses are made available to the wider research community through a comprehensive web portal. PMID:27067544

  4. Multi-omics maps of cotton fibre reveal epigenetic basis for staged single-cell differentiation

    PubMed Central

    Wang, Maojun; Wang, Pengcheng; Tu, Lili; Zhu, Sitao; Zhang, Lin; Li, Zhonghua; Zhang, Qinghua; Yuan, Daojun; Zhang, Xianlong

    2016-01-01

    Epigenetic modifications are highlighted for their great importance in regulating plant development, but their function associated with single-cell differentiation remains undetermined. Here, we used the cotton fibre, which is the epidermal hair on the cotton ovule, as a model to investigate the regulatory role of DNA methylation in cell differentiation. The level of CHH (H = A, T, or C) DNA methylation level was found to increase during fibre development, accompanied by a decrease in RNA-directed DNA methylation (RdDM). Examination of nucleosome positioning revealed a gradual transition from euchromatin to heterochromatin for chromatin dynamics in developing fibres, which could shape the DNA methylation landscape. The observed increase in DNA methylation in fibres, compared with other ovule tissue, was demonstrated to be mediated predominantly by an active H3K9me2-dependent pathway rather than the RdDM pathway, which was inactive. Furthermore, integrated multi-omics analyses revealed that dynamic DNA methylation played a role in the regulation of lipid biosynthesis and spatio-temporal modulation of reactive oxygen species during fibre differentiation. Our study illustrates two divergent pathways mediating a continuous increase of DNA methylation and also sheds further light on the epigenetic basis for single-cell differentiation in plants. These data and analyses are made available to the wider research community through a comprehensive web portal. PMID:27067544

  5. Integrated live imaging and molecular profiling of embryoid bodies reveals a synchronized progression of early differentiation

    PubMed Central

    Boxman, Jonathan; Sagy, Naor; Achanta, Sirisha; Vadigepalli, Rajanikanth; Nachman, Iftach

    2016-01-01

    Embryonic stem cells can spontaneously differentiate into cell types of all germ layers within embryoid bodies (EBs) in a highly variable manner. Whether there exists an intrinsic differentiation program common to all EBs is unknown. Here, we present a novel combination of high-throughput live two-photon imaging and gene expression profiling to study early differentiation dynamics spontaneously occurring within developing EBs. Onset timing of Brachyury-GFP was highly variable across EBs, while the spatial patterns as well as the dynamics of mesendodermal progression following onset were remarkably similar. We therefore defined a ‘developmental clock’ using the Brachyury-GFP signal onset timing. Mapping snapshot gene expression measurements to this clock revealed their temporal trends, indicating that loss of pluripotency, formation of primitive streak and mesodermal lineage progression are synchronized in EBs. Exogenous activation of Wnt or BMP signaling accelerated the intrinsic clock. CHIR down-regulated Wnt3, allowing insights into dependency mechanisms between canonical Wnt signaling and multiple genes. Our findings reveal a developmental clock characteristic of an early differentiation program common to all EBs, further establishing them as an in vitro developmental model. PMID:27530599

  6. Switch-like regulation of tissue-specific alternative pre-mRNA processing patterns revealed by customized fluorescence reporters

    PubMed Central

    Kuroyanagi, Hidehito

    2013-01-01

    Alternative processing of precursor mRNAs (pre-mRNAs), including alternative transcription start sites, alternative splicing and alternative polyadenylation, is the major source of protein diversity and plays crucial roles in development, differentiation and diseases in higher eukaryotes. It is estimated from microarray analyses and deep sequencing of mRNAs from synchronized worms that up to 25% of protein-coding genes in Caenorhabditis elegans undergo alternative pre-mRNA processing and that many of them are subject to developmental regulation. Recent progress in visualizing the alternative pre-mRNA processing patterns in living worms with custom-designed fluorescence reporters has enabled genetic analyses of the regulatory mechanisms for alternative processing events of interest in vivo. Expression of the tissue-specific isoforms of actin depolymerising factor (ADF)/cofilin, UNC-60A and UNC-60B, is regulated by a combination of alternative splicing and alternative polyadenylation of pre-mRNA from a single gene unc-60. We recently found that muscle-specific splicing regulators ASD-2 and SUP-12 cooperatively switch the pre-mRNA processing patterns of the unc-60 gene in body wall muscles. Here I summarize the bichromatic fluorescence reporter system utilized for visualizing the tissue-specific alternative processing patterns of the unc-60 pre-mRNA. I also discuss the model for the coordinated regulation of the UNC-60B-type pre-mRNA processing in body wall muscles by ASD-2 and SUP-12. PMID:24778931

  7. Alternative Splicing of Neuronal Differentiation Factor TRF2 Regulated by HNRNPH1/H2.

    PubMed

    Grammatikakis, Ioannis; Zhang, Peisu; Panda, Amaresh C; Kim, Jiyoung; Maudsley, Stuart; Abdelmohsen, Kotb; Yang, Xiaoling; Martindale, Jennifer L; Motiño, Omar; Hutchison, Emmette R; Mattson, Mark P; Gorospe, Myriam

    2016-05-01

    During neuronal differentiation, use of an alternative splice site on the rat telomere repeat-binding factor 2 (TRF2) mRNA generates a short TRF2 protein isoform (TRF2-S) capable of derepressing neuronal genes. However, the RNA-binding proteins (RBPs) controlling this splicing event are unknown. Here, using affinity pull-down analysis, we identified heterogeneous nuclear ribonucleoproteins H1 and H2(HNRNPH) as RBPs specifically capable of interacting with the spliced RNA segment (exon 7) of Trf2 pre-mRNA. HNRNPH proteins prevent the production of the short isoform of Trf2 mRNA, as HNRNPH silencing selectively elevates TRF2-S levels. Accordingly, HNRNPH levels decline while TRF2-S levels increase during neuronal differentiation. In addition, CRISPR/Cas9-mediated deletion of hnRNPH2 selectively accelerates the NGF-triggered differentiation of rat pheochromocytoma cells into neurons. In sum, HNRNPH is a splicing regulator of Trf2 pre-mRNA that prevents the expression of TRF2-S, a factor implicated in neuronal differentiation. PMID:27117401

  8. Immunoproteomics of Brucella abortus reveals differential antibody profiles between S19-vaccinated and naturally infected cattle.

    PubMed

    Pajuaba, Ana C A M; Silva, Deise A O; Almeida, Karine C; Cunha-Junior, Jair P; Pirovani, Carlos P; Camillo, Luciana R; Mineo, José R

    2012-03-01

    Brucella abortus is a Gram-negative intracellular bacterium that causes infectious abortion in food-producing animals and chronic infection in humans. This study aimed to characterize a B. abortus S19 antigen preparation obtained by Triton X-114 (TX-114) extraction through immunoproteomics to differentiate infected from vaccinated cattle. Three groups of bovine sera were studied: GI, 30 naturally infected cows; GII, 30 S19-vaccinated heifers; and GIII, 30 nonvaccinated seronegative cows. One-dimensional (1D) and two-dimensional electrophoretic profiles of TX-114 hydrophilic phase antigen revealed a broad spectrum of polypeptides (10-79 kDa). 1D immunoblot showed widespread seroreactivity profile in GI compared with restricted profile in GII. Three antigenic components (10, 12, 17 kDa) were recognized exclusively by GI sera, representing potential markers of infection and excluding vaccinal response. The proteomic characterization revealed 56 protein spots, 27 of which were antigenic spots showing differential seroreactivity profile between GI and GII, especially polypeptides <20 kDa that were recognized exclusively by GI. MS/MS analysis identified five B. abortus S19 proteins (Invasion protein B, Sod, Dps, Ndk, and Bfr), which were related with antigenicity in naturally infected cattle. In conclusion, immunoproteomics of this new antigen preparation enabled the characterization of proteins that could be used as tools to develop sensitive and specific immunoassays for serodiagnosis of bovine brucellosis, with emphasis on differentiation between S19 vaccinated and infected cattle. PMID:22539433

  9. Ostreococcus tauri ADP-glucose Pyrophosphorylase Reveals Alternative Paths for the Evolution of Subunit Roles*

    PubMed Central

    Kuhn, Misty L.; Falaschetti, Christine A.; Ballicora, Miguel A.

    2009-01-01

    ADP-glucose pyrophosphorylase controls starch synthesis in plants and is an interesting case to study the evolution and differentiation of roles in heteromeric enzymes. It includes two homologous subunits, small (S) and large (L), that originated from a common photosynthetic eukaryotic ancestor. In present day organisms, these subunits became complementary after loss of certain roles in a process described as subfunctionalization. For instance, the potato tuber enzyme has a noncatalytic L subunit that complements an S subunit with suboptimal allosteric properties. To understand the evolution of catalysis and regulation in this family, we artificially synthesized both subunit genes from the unicellular alga Ostreococcus tauri. This is among the most ancient species in the green lineage that diverged from the ancestor of all green plants and algae. After heterologous gene expression, we purified and characterized the proteins. The O. tauri enzyme was not redox-regulated, suggesting that redox regulation of ADP-glucose pyrophosphorylases appeared later in evolution. The S subunit had a typical low apparent affinity for the activator 3-phosphoglycerate, but it was atypically defective in the catalytic efficiency (Vmax/Km) for the substrate Glc-1-P. The L subunit needed the S subunit for soluble expression. In the presence of a mutated S subunit (to avoid interference), the L subunit had a high apparent affinity for 3-phosphoglycerate and substrates suggesting a leading role in catalysis. Therefore, the subfunctionalization of the O. tauri enzyme was different from previously described cases. To the best of our knowledge, this is the first biochemical description of a system with alternative subfunctionalization paths. PMID:19737928

  10. Near-field deformation from the El Mayor-Cucapah earthquake revealed by differential LIDAR.

    PubMed

    Oskin, Michael E; Arrowsmith, J Ramon; Hinojosa Corona, Alejandro; Elliott, Austin J; Fletcher, John M; Fielding, Eric J; Gold, Peter O; Gonzalez Garcia, J Javier; Hudnut, Ken W; Liu-Zeng, Jing; Teran, Orlando J

    2012-02-10

    Large [moment magnitude (M(w)) ≥ 7] continental earthquakes often generate complex, multifault ruptures linked by enigmatic zones of distributed deformation. Here, we report the collection and results of a high-resolution (≥nine returns per square meter) airborne light detection and ranging (LIDAR) topographic survey of the 2010 M(w) 7.2 El Mayor-Cucapah earthquake that produced a 120-kilometer-long multifault rupture through northernmost Baja California, Mexico. This differential LIDAR survey completely captures an earthquake surface rupture in a sparsely vegetated region with pre-earthquake lower-resolution (5-meter-pixel) LIDAR data. The postevent survey reveals numerous surface ruptures, including previously undocumented blind faults within thick sediments of the Colorado River delta. Differential elevation changes show distributed, kilometer-scale bending strains as large as ~10(3) microstrains in response to slip along discontinuous faults cutting crystalline bedrock of the Sierra Cucapah. PMID:22323817

  11. Osteoblastic differentiation and stress response of human mesenchymal stem cells exposed to alternating current electric fields

    PubMed Central

    2011-01-01

    detected in the stimulation group, suggesting a connection with oxidative stress. Both differentiation factors and electrical stimulation improved hMSC differentiation potential to bone based on calcium deposition on day 28. Conclusions Electrical stimulation is a useful tool to improve hMSC osteogenic differentiation, while heat shock proteins may reveal underlying mechanisms, and optical non-invasive imaging may be used to monitor the induced morphological and biochemical changes. PMID:21269490

  12. Molecular cloning and characterization of Izumo1 gene from sheep and cashmere goat reveal alternative splicing.

    PubMed

    Xing, Wan-Jin; Han, Bao-Da; Wu, Qi; Zhao, Li; Bao, Xiao-Hong; Bou, Shorgan

    2011-03-01

    We cloned the cDNA and genomic DNA encoding for Izumo1 of cashmere goat (Capra hircus) and sheep (Ovis aries). Analysis of 4.6 kb Izumo1 genomic sequences in sheep and goat revealed a canonical open reading frame (ORF) of 963 bp spliced by eight exons. Sheep and goat Izumo1 genes share >99% identity at both DNA and protein levels and are also highly homologous to the orthologues in cattle, mouse, rat and human. Extensive cloning and analysis of Izumo1 cDNA revealed three (del 69, del 182 and del 217) and two (del 69 and ins 30) alternative splicing isoforms in goat and sheep, respectively. All of the isoforms are derived from splicing at typical GT-AG sites leading to partial or complete truncation of the immunoglobulin (Ig)-like domain. Bioinformatics analysis showed that caprine and ovine Izumo1 proteins share similar structure with their murine orthologue. There are a signal peptide at the N-terminus (1-22 aa), a transmembrane domain at the C-terminus (302-319 aa), and an extracellular Ig-like region in the middle (161-252 aa) with a putative N-linked glycosylation site (N(205)-N-S). Alignment of Izumo1 protein sequences among 15 mammalian species displayed several highly conserved regions, including LDC and YRC motifs with cysteine residues for potential disulfide bridge formation, CPNKCG motif upstream of the Ig-like domain, GLTDYSFYRVW motif upstream of the putative N-linked glycosylation site, and a number of scattered cysteine residues. These distinctive features are very informative to pinpoint the important gene motifs and functions. The C-terminal regions, however, are more variable across species. Izumo1 cDNA sequences of goat, sheep, and cow were found to be largely homologous, and the molecular phylogenetic analysis is consistent with their morphological taxonomy. This implies the Izumo1 gene evolves from the same ancestor, and the mechanism of sperm-egg fusion in mammals may be under the same principle in which Izumo1 plays an important role. PMID

  13. Differential Network Analysis Reveals Evolutionary Complexity in Secondary Metabolism of Rauvolfia serpentina over Catharanthus roseus.

    PubMed

    Pathania, Shivalika; Bagler, Ganesh; Ahuja, Paramvir S

    2016-01-01

    Comparative co-expression analysis of multiple species using high-throughput data is an integrative approach to determine the uniformity as well as diversification in biological processes. Rauvolfia serpentina and Catharanthus roseus, both members of Apocyanacae family, are reported to have remedial properties against multiple diseases. Despite of sharing upstream of terpenoid indole alkaloid pathway, there is significant diversity in tissue-specific synthesis and accumulation of specialized metabolites in these plants. This led us to implement comparative co-expression network analysis to investigate the modules and genes responsible for differential tissue-specific expression as well as species-specific synthesis of metabolites. Toward these goals differential network analysis was implemented to identify candidate genes responsible for diversification of metabolites profile. Three genes were identified with significant difference in connectivity leading to differential regulatory behavior between these plants. These genes may be responsible for diversification of secondary metabolism, and thereby for species-specific metabolite synthesis. The network robustness of R. serpentina, determined based on topological properties, was also complemented by comparison of gene-metabolite networks of both plants, and may have evolved to have complex metabolic mechanisms as compared to C. roseus under the influence of various stimuli. This study reveals evolution of complexity in secondary metabolism of R. serpentina, and key genes that contribute toward diversification of specific metabolites. PMID:27588023

  14. Diversity of sharp-wave–ripple LFP signatures reveals differentiated brain-wide dynamical events

    PubMed Central

    Ramirez-Villegas, Juan F.; Logothetis, Nikos K.; Besserve, Michel

    2015-01-01

    Sharp-wave–ripple (SPW-R) complexes are believed to mediate memory reactivation, transfer, and consolidation. However, their underlying neuronal dynamics at multiple scales remains poorly understood. Using concurrent hippocampal local field potential (LFP) recordings and functional MRI (fMRI), we study local changes in neuronal activity during SPW-R episodes and their brain-wide correlates. Analysis of the temporal alignment between SPW and ripple components reveals well-differentiated SPW-R subtypes in the CA1 LFP. SPW-R–triggered fMRI maps show that ripples aligned to the positive peak of their SPWs have enhanced neocortical metabolic up-regulation. In contrast, ripples occurring at the trough of their SPWs relate to weaker neocortical up-regulation and absent subcortical down-regulation, indicating differentiated involvement of neuromodulatory pathways in the ripple phenomenon mediated by long-range interactions. To our knowledge, this study provides the first evidence for the existence of SPW-R subtypes with differentiated CA1 activity and metabolic correlates in related brain areas, possibly serving different memory functions. PMID:26540729

  15. Differential Network Analysis Reveals Evolutionary Complexity in Secondary Metabolism of Rauvolfia serpentina over Catharanthus roseus

    PubMed Central

    Pathania, Shivalika; Bagler, Ganesh; Ahuja, Paramvir S.

    2016-01-01

    Comparative co-expression analysis of multiple species using high-throughput data is an integrative approach to determine the uniformity as well as diversification in biological processes. Rauvolfia serpentina and Catharanthus roseus, both members of Apocyanacae family, are reported to have remedial properties against multiple diseases. Despite of sharing upstream of terpenoid indole alkaloid pathway, there is significant diversity in tissue-specific synthesis and accumulation of specialized metabolites in these plants. This led us to implement comparative co-expression network analysis to investigate the modules and genes responsible for differential tissue-specific expression as well as species-specific synthesis of metabolites. Toward these goals differential network analysis was implemented to identify candidate genes responsible for diversification of metabolites profile. Three genes were identified with significant difference in connectivity leading to differential regulatory behavior between these plants. These genes may be responsible for diversification of secondary metabolism, and thereby for species-specific metabolite synthesis. The network robustness of R. serpentina, determined based on topological properties, was also complemented by comparison of gene-metabolite networks of both plants, and may have evolved to have complex metabolic mechanisms as compared to C. roseus under the influence of various stimuli. This study reveals evolution of complexity in secondary metabolism of R. serpentina, and key genes that contribute toward diversification of specific metabolites. PMID:27588023

  16. Diversity of sharp-wave-ripple LFP signatures reveals differentiated brain-wide dynamical events.

    PubMed

    Ramirez-Villegas, Juan F; Logothetis, Nikos K; Besserve, Michel

    2015-11-17

    Sharp-wave-ripple (SPW-R) complexes are believed to mediate memory reactivation, transfer, and consolidation. However, their underlying neuronal dynamics at multiple scales remains poorly understood. Using concurrent hippocampal local field potential (LFP) recordings and functional MRI (fMRI), we study local changes in neuronal activity during SPW-R episodes and their brain-wide correlates. Analysis of the temporal alignment between SPW and ripple components reveals well-differentiated SPW-R subtypes in the CA1 LFP. SPW-R-triggered fMRI maps show that ripples aligned to the positive peak of their SPWs have enhanced neocortical metabolic up-regulation. In contrast, ripples occurring at the trough of their SPWs relate to weaker neocortical up-regulation and absent subcortical down-regulation, indicating differentiated involvement of neuromodulatory pathways in the ripple phenomenon mediated by long-range interactions. To our knowledge, this study provides the first evidence for the existence of SPW-R subtypes with differentiated CA1 activity and metabolic correlates in related brain areas, possibly serving different memory functions. PMID:26540729

  17. Subfunctionalization of duplicate mitf genes associated with differential degeneration of alternative exons in fish.

    PubMed Central

    Altschmied, Joachim; Delfgaauw, Jacqueline; Wilde, Brigitta; Duschl, Jutta; Bouneau, Laurence; Volff, Jean-Nicolas; Schartl, Manfred

    2002-01-01

    The microphthalmia-associated transcription factor (MITF) exists in at least four isoforms. These are generated in higher vertebrates using alternative 5' exons and promoters from a single gene. Two separate genes (mitf-m and mitf-b), however, are present in different teleost fish species including the poeciliid Xiphophorus, the pufferfishes Fugu rubripes and Tetraodon nigroviridis, and the zebrafish Danio rerio. Fish proteins MITF-m and MITF-b correspond at both the structural and the expression levels to one particular bird/mammalian MITF isoform. In the teleost lineage subfunctionalization of mitf genes after duplication at least 100 million years ago is associated with the degeneration of alternative exons and, probably, regulatory elements and promoters. For example, a remnant of the first exon specific for MITF-m is detected within the pufferfish gene encoding MITF-b. Retracing the evolutionary history of mitf genes in vertebrates uncovered the differential recruitment of new introns specific for either the teleost or the bird/mammalian lineage. PMID:12019239

  18. Alternative Splicing in the Differentiation of Human Embryonic Stem Cells into Cardiac Precursors

    PubMed Central

    Salomonis, Nathan; Nelson, Brandon; Vranizan, Karen; Pico, Alexander R.; Hanspers, Kristina; Kuchinsky, Allan; Ta, Linda; Mercola, Mark; Conklin, Bruce R.

    2009-01-01

    The role of alternative splicing in self-renewal, pluripotency and tissue lineage specification of human embryonic stem cells (hESCs) is largely unknown. To better define these regulatory cues, we modified the H9 hESC line to allow selection of pluripotent hESCs by neomycin resistance and cardiac progenitors by puromycin resistance. Exon-level microarray expression data from undifferentiated hESCs and cardiac and neural precursors were used to identify splice isoforms with cardiac-restricted or common cardiac/neural differentiation expression patterns. Splice events for these groups corresponded to the pathways of cytoskeletal remodeling, RNA splicing, muscle specification, and cell cycle checkpoint control as well as genes with serine/threonine kinase and helicase activity. Using a new program named AltAnalyze (http://www.AltAnalyze.org), we identified novel changes in protein domain and microRNA binding site architecture that were predicted to affect protein function and expression. These included an enrichment of splice isoforms that oppose cell-cycle arrest in hESCs and that promote calcium signaling and cardiac development in cardiac precursors. By combining genome-wide predictions of alternative splicing with new functional annotations, our data suggest potential mechanisms that may influence lineage commitment and hESC maintenance at the level of specific splice isoforms and microRNA regulation. PMID:19893621

  19. Differential regulation of alternative 3{prime} splicing of {epsilon} messenger RNA variants

    SciTech Connect

    Diaz-Sanchez, D.; Zhang, K.; Saxon, A.

    1995-08-15

    Alternative 3{prime} splicing of the one active human {epsilon} heavy chain gene results in variants of {epsilon} mRNA encoding distinct IgE proteins. The same relative amounts of these {epsilon} mRNA variants were produced by non-atopic donor B cells when driven in a variety of T-dependent or T-independent systems. The most abundant variants were those for classic secreted {epsilon} and a novel secreted form (CH4-M2{double_prime}). In contrast, cells from subjects with high levels of serum IgE secondary to parasitic infection or atopy spontaneously produced higher relative levels of the CH4-M2{prime} {epsilon} mRNA variant, lower relative amounts of both the membrane and CH4-M2{double_prime} secreted variants, and very low levels of the CH4{prime}-CH5 variant. The existence of and corresponding changes in levels of the CH4-M2{prime}-enclosed secreted protein were demonstrated. IL-10 induced this same differential expression of {epsilon} splice variants in vitro when used to costimulate IL-4 plus CD40-driven B cells and could differentially enhance the production of CH4-M2{prime} protein by established IgE-secreting cell lines. Inhibition of IgE by cross-linking the low affinity IgE receptor (CD23) decreased the levels of {epsilon} mRNA and resulted in a distinct pattern of {epsilon} mRNA characterized by a dramatic decrease in CH4-M2{prime} splice variant. IL-6, IL-2, or IFN-{gamma} did not change the {epsilon} mRNA pattern. Overall, the absolute and relative amounts of the different {epsilon} mRNA splice variants produced appear to be controlled in a differentiation-related fashion.

  20. Comparative proteomics reveals a significant bias toward alternative protein isoforms with conserved structure and function.

    PubMed

    Ezkurdia, Iakes; del Pozo, Angela; Frankish, Adam; Rodriguez, Jose Manuel; Harrow, Jennifer; Ashman, Keith; Valencia, Alfonso; Tress, Michael L

    2012-09-01

    Advances in high-throughput mass spectrometry are making proteomics an increasingly important tool in genome annotation projects. Peptides detected in mass spectrometry experiments can be used to validate gene models and verify the translation of putative coding sequences (CDSs). Here, we have identified peptides that cover 35% of the genes annotated by the GENCODE consortium for the human genome as part of a comprehensive analysis of experimental spectra from two large publicly available mass spectrometry databases. We detected the translation to protein of "novel" and "putative" protein-coding transcripts as well as transcripts annotated as pseudogenes and nonsense-mediated decay targets. We provide a detailed overview of the population of alternatively spliced protein isoforms that are detectable by peptide identification methods. We found that 150 genes expressed multiple alternative protein isoforms. This constitutes the largest set of reliably confirmed alternatively spliced proteins yet discovered. Three groups of genes were highly overrepresented. We detected alternative isoforms for 10 of the 25 possible heterogeneous nuclear ribonucleoproteins, proteins with a key role in the splicing process. Alternative isoforms generated from interchangeable homologous exons and from short indels were also significantly enriched, both in human experiments and in parallel analyses of mouse and Drosophila proteomics experiments. Our results show that a surprisingly high proportion (almost 25%) of the detected alternative isoforms are only subtly different from their constitutive counterparts. Many of the alternative splicing events that give rise to these alternative isoforms are conserved in mouse. It was striking that very few of these conserved splicing events broke Pfam functional domains or would damage globular protein structures. This evidence of a strong bias toward subtle differences in CDS and likely conserved cellular function and structure is remarkable and

  1. Novel Molecular Insights into Classical and Alternative Activation States of Microglia as Revealed by Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-based Proteomics*

    PubMed Central

    Bell-Temin, Harris; Culver-Cochran, Ashley E.; Chaput, Dale; Carlson, Christina M.; Kuehl, Melanie; Burkhardt, Brant R.; Bickford, Paula C.; Liu, Bin; Stevens, Stanley M.

    2015-01-01

    Microglia, the resident immune cells of the brain, have been shown to display a complex spectrum of roles that span from neurotrophic to neurotoxic depending on their activation status. Microglia can be classified into four stages of activation, M1, which most closely matches the classical (pro-inflammatory) activation stage, and the alternative activation stages M2a, M2b, and M2c. The alternative activation stages have not yet been comprehensively analyzed through unbiased, global-scale protein expression profiling. In this study, BV2 mouse immortalized microglial cells were stimulated with agonists specific for each of the four stages and total protein expression for 4644 protein groups was quantified using SILAC-based proteomic analysis. After validating induction of the various stages through a targeted cytokine assay and Western blotting of activation states, the data revealed novel insights into the similarities and differences between the various states. The data identify several protein groups whose expression in the anti-inflammatory, pro-healing activation states are altered presumably to curtail inflammatory activation through differential protein expression, in the M2a state including CD74, LYN, SQST1, TLR2, and CD14. The differential expression of these proteins promotes healing, limits phagocytosis, and limits activation of reactive nitrogen species through toll-like receptor cascades. The M2c state appears to center around the down-regulation of a key member in the formation of actin-rich phagosomes, SLP-76. In addition, the proteomic data identified a novel activation marker, DAB2, which is involved in clathrin-mediated endocytosis and is significantly different between M2a and either M1 or M2b states. Western blot analysis of mouse primary microglia stimulated with the various agonists of the classical and alternative activation states revealed a similar trend of DAB2 expression compared with BV2 cells. PMID:26424600

  2. Novel Molecular Insights into Classical and Alternative Activation States of Microglia as Revealed by Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-based Proteomics.

    PubMed

    Bell-Temin, Harris; Culver-Cochran, Ashley E; Chaput, Dale; Carlson, Christina M; Kuehl, Melanie; Burkhardt, Brant R; Bickford, Paula C; Liu, Bin; Stevens, Stanley M

    2015-12-01

    Microglia, the resident immune cells of the brain, have been shown to display a complex spectrum of roles that span from neurotrophic to neurotoxic depending on their activation status. Microglia can be classified into four stages of activation, M1, which most closely matches the classical (pro-inflammatory) activation stage, and the alternative activation stages M2a, M2b, and M2c. The alternative activation stages have not yet been comprehensively analyzed through unbiased, global-scale protein expression profiling. In this study, BV2 mouse immortalized microglial cells were stimulated with agonists specific for each of the four stages and total protein expression for 4644 protein groups was quantified using SILAC-based proteomic analysis. After validating induction of the various stages through a targeted cytokine assay and Western blotting of activation states, the data revealed novel insights into the similarities and differences between the various states. The data identify several protein groups whose expression in the anti-inflammatory, pro-healing activation states are altered presumably to curtail inflammatory activation through differential protein expression, in the M2a state including CD74, LYN, SQST1, TLR2, and CD14. The differential expression of these proteins promotes healing, limits phagocytosis, and limits activation of reactive nitrogen species through toll-like receptor cascades. The M2c state appears to center around the down-regulation of a key member in the formation of actin-rich phagosomes, SLP-76. In addition, the proteomic data identified a novel activation marker, DAB2, which is involved in clathrin-mediated endocytosis and is significantly different between M2a and either M1 or M2b states. Western blot analysis of mouse primary microglia stimulated with the various agonists of the classical and alternative activation states revealed a similar trend of DAB2 expression compared with BV2 cells. PMID:26424600

  3. Single-Cell RNA-Sequencing Reveals a Continuous Spectrum of Differentiation in Hematopoietic Cells

    PubMed Central

    Macaulay, Iain C.; Svensson, Valentine; Labalette, Charlotte; Ferreira, Lauren; Hamey, Fiona; Voet, Thierry; Teichmann, Sarah A.; Cvejic, Ana

    2016-01-01

    Summary The transcriptional programs that govern hematopoiesis have been investigated primarily by population-level analysis of hematopoietic stem and progenitor cells, which cannot reveal the continuous nature of the differentiation process. Here we applied single-cell RNA-sequencing to a population of hematopoietic cells in zebrafish as they undergo thrombocyte lineage commitment. By reconstructing their developmental chronology computationally, we were able to place each cell along a continuum from stem cell to mature cell, refining the traditional lineage tree. The progression of cells along this continuum is characterized by a highly coordinated transcriptional program, displaying simultaneous suppression of genes involved in cell proliferation and ribosomal biogenesis as the expression of lineage specific genes increases. Within this program, there is substantial heterogeneity in the expression of the key lineage regulators. Overall, the total number of genes expressed, as well as the total mRNA content of the cell, decreases as the cells undergo lineage commitment. PMID:26804912

  4. Hierarchical Clustering of Breast Cancer Methylomes Revealed Differentially Methylated and Expressed Breast Cancer Genes

    PubMed Central

    Lin, I-Hsuan; Chen, Dow-Tien; Chang, Yi-Feng; Lee, Yu-Ling; Su, Chia-Hsin; Cheng, Ching; Tsai, Yi-Chien; Ng, Swee-Chuan; Chen, Hsiao-Tan; Lee, Mei-Chen; Chen, Hong-Wei; Suen, Shih-Hui; Chen, Yu-Cheng; Liu, Tze-Tze; Chang, Chuan-Hsiung; Hsu, Ming-Ta

    2015-01-01

    Oncogenic transformation of normal cells often involves epigenetic alterations, including histone modification and DNA methylation. We conducted whole-genome bisulfite sequencing to determine the DNA methylomes of normal breast, fibroadenoma, invasive ductal carcinomas and MCF7. The emergence, disappearance, expansion and contraction of kilobase-sized hypomethylated regions (HMRs) and the hypomethylation of the megabase-sized partially methylated domains (PMDs) are the major forms of methylation changes observed in breast tumor samples. Hierarchical clustering of HMR revealed tumor-specific hypermethylated clusters and differential methylated enhancers specific to normal or breast cancer cell lines. Joint analysis of gene expression and DNA methylation data of normal breast and breast cancer cells identified differentially methylated and expressed genes associated with breast and/or ovarian cancers in cancer-specific HMR clusters. Furthermore, aberrant patterns of X-chromosome inactivation (XCI) was found in breast cancer cell lines as well as breast tumor samples in the TCGA BRCA (breast invasive carcinoma) dataset. They were characterized with differentially hypermethylated XIST promoter, reduced expression of XIST, and over-expression of hypomethylated X-linked genes. High expressions of these genes were significantly associated with lower survival rates in breast cancer patients. Comprehensive analysis of the normal and breast tumor methylomes suggests selective targeting of DNA methylation changes during breast cancer progression. The weak causal relationship between DNA methylation and gene expression observed in this study is evident of more complex role of DNA methylation in the regulation of gene expression in human epigenetics that deserves further investigation. PMID:25706888

  5. Multivoxel patterns reveal functionally differentiated networks underlying auditory feedback processing of speech.

    PubMed

    Zheng, Zane Z; Vicente-Grabovetsky, Alejandro; MacDonald, Ewen N; Munhall, Kevin G; Cusack, Rhodri; Johnsrude, Ingrid S

    2013-03-01

    The everyday act of speaking involves the complex processes of speech motor control. An important component of control is monitoring, detection, and processing of errors when auditory feedback does not correspond to the intended motor gesture. Here we show, using fMRI and converging operations within a multivoxel pattern analysis framework, that this sensorimotor process is supported by functionally differentiated brain networks. During scanning, a real-time speech-tracking system was used to deliver two acoustically different types of distorted auditory feedback or unaltered feedback while human participants were vocalizing monosyllabic words, and to present the same auditory stimuli while participants were passively listening. Whole-brain analysis of neural-pattern similarity revealed three functional networks that were differentially sensitive to distorted auditory feedback during vocalization, compared with during passive listening. One network of regions appears to encode an "error signal" regardless of acoustic features of the error: this network, including right angular gyrus, right supplementary motor area, and bilateral cerebellum, yielded consistent neural patterns across acoustically different, distorted feedback types, only during articulation (not during passive listening). In contrast, a frontotemporal network appears sensitive to the speech features of auditory stimuli during passive listening; this preference for speech features was diminished when the same stimuli were presented as auditory concomitants of vocalization. A third network, showing a distinct functional pattern from the other two, appears to capture aspects of both neural response profiles. Together, our findings suggest that auditory feedback processing during speech motor control may rely on multiple, interactive, functionally differentiated neural systems. PMID:23467350

  6. Differential lysine acetylation profiles of Erwinia amylovora strains revealed by proteomics.

    PubMed

    Wu, Xia; Vellaichamy, Adaikkalam; Wang, Dongping; Zamdborg, Leonid; Kelleher, Neil L; Huber, Steven C; Zhao, Youfu

    2013-02-21

    Protein lysine acetylation (LysAc) has recently been demonstrated to be widespread in E. coli and Salmonella, and to broadly regulate bacterial physiology and metabolism. However, LysAc in plant pathogenic bacteria is largely unknown. Here we first report the lysine acetylome of Erwinia amylovora, an enterobacterium causing serious fire blight disease of apples and pears. Immunoblots using generic anti-lysine acetylation antibodies demonstrated that growth conditions strongly affected the LysAc profiles in E. amylovora. Differential LysAc profiles were also observed for two E. amylovora strains, known to have differential virulence in plants, indicating translational modification of proteins may be important in determining virulence of bacterial strains. Proteomic analysis of LysAc in two E. amylovora strains identified 141 LysAc sites in 96 proteins that function in a wide range of biological pathways. Consistent with previous reports, 44% of the proteins are involved in metabolic processes, including central metabolism, lipopolysaccharide, nucleotide and amino acid metabolism. Interestingly, for the first time, several proteins involved in E. amylovora virulence, including exopolysaccharide amylovoran biosynthesis- and type III secretion-associated proteins, were found to be lysine acetylated, suggesting that LysAc may play a major role in bacterial virulence. Comparative analysis of LysAc sites in E. amylovora and E. coli further revealed the sequence and structural commonality for LysAc in the two organisms. Collectively, these results reinforce the notion that LysAc of proteins is widespread in bacterial metabolism and virulence. PMID:23234799

  7. 2D gel blood serum biomarkers reveal differential clinical proteomics of the neurodegenerative diseases.

    PubMed

    Sheta, Essam A; Appel, Stanley H; Goldknopf, Ira L

    2006-02-01

    This review addresses the challenges of neuroproteomics and recent progress in biomarkers and tests for neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. The review will discuss how the application of quantitative 2D gel electrophoresis, combined with appropriate single-variable and multivariate biostatistics, allows for selection of disease-specific serum biomarkers. It will also address how the use of large cohorts of specifically targeted patient blood serum samples and complimentary age-matched controls, in parallel with the use of selected panels of these biomarkers, are being applied to the development of blood tests to specifically address unmet pressing needs in the differential diagnosis of these diseases, and to provide potential avenues for mechanism-based drug targeting and treatment monitoring. While exploring recent findings in this area, the review discusses differences in critical pathways of immune/inflammation and amyloid formation between Parkinson's disease and amyotrophic lateral sclerosis, as well as discernable synergistic relationships between these pathways that are revealed by this approach. The potential for pathway measurement in blood tests for differential diagnosis, disease burden and therapeutic monitoring is also outlined. PMID:16445350

  8. Differential lysine acetylation profiles of Erwinia amylovora strains revealed by proteomics

    PubMed Central

    Wu, Xia; Vellaichamy, Adaikkalam; Wang, Dongping; Zamdborg, Leonid; Kelleher, Neil L.; Huber, Steven C.; Zhao, Youfu

    2015-01-01

    Protein lysine acetylation (LysAc) has recently been demonstrated to be widespread in E. coli and Salmonella, and to broadly regulate bacterial physiology and metabolism. However, LysAc in plant pathogenic bacteria is largely unknown. Here we first report the lysine acetylome of Erwinia amylovora, an enterobacterium causing serious fire blight disease of apples and pears. Immunoblots using generic anti-lysine acetylation antibodies demonstrated that growth conditions strongly affected the LysAc profiles in E. amylovora. Differential LysAc profiles were also observed for two E. amylovora strains, known to have differential virulence in plants, indicating translational modification of proteins may be important in determining virulence of bacterial strains. Proteomic analysis of LysAc in two E. amylovora strains identified 141 LysAc sites in 96 proteins that function in a wide range of biological pathways. Consistent with previous reports, 44% of the proteins are involved in metabolic processes, including central metabolism, lipopolysaccharide, nucleotide and amino acid metabolism. Interestingly, for the first time, several proteins involved in E. amylovora virulence, including exopolysaccharide amylovoran biosynthesis- and type III secretion-associated proteins, were found to be lysine acetylated, suggesting that LysAc may play a major role in bacterial virulence. Comparative analysis of LysAc sites in E. amylovora and E. coli further revealed the sequence and structural commonality for LysAc in the two organisms. Collectively, these results reinforce the notion that LysAc of proteins is widespread in bacterial metabolism and virulence. PMID:23234799

  9. Structures of HSF2 Reveal Mechanisms for Differential Regulation of Human Heat Shock Factors

    PubMed Central

    Jaeger, Alex M.; Pemble, Charles W.; Sistonen, Lea; Thiele, Dennis J.

    2016-01-01

    Heat Shock Transcription Factor (HSF) family members function in stress protection and in human disease including proteopathies, neurodegeneration and cancer. The mechanisms that drive distinct post-translational modifications, co-factor recruitment and target gene activation for specific HSF paralogs are unknown. We present high-resolution crystal structures of the human HSF2 DNA-binding domain (DBD) bound to DNA, revealing an unprecedented view of HSFs that provides insights into their unique biology. The HSF2 DBD structures resolve a novel carboxyl-terminal helix that directs the coiled-coil domain to wrap around DNA, exposing paralog-specific sequences of the DBD surface, for differential post-translational modifications and co-factor interactions. We further demonstrate a direct interaction between HSF1 and HSF2 through their coiled-coil domains. Together, these features provide a new model for HSF structure as the basis for differential and combinatorial regulation to influence the transcriptional response to cellular stress. PMID:26727490

  10. Genetic Tagging During Human Mesoderm Differentiation Reveals Tripotent Lateral Plate Mesodermal Progenitors.

    PubMed

    Chin, Chee Jia; Cooper, Aaron R; Lill, Georgia R; Evseenko, Denis; Zhu, Yuhua; He, Chong Bin; Casero, David; Pellegrini, Matteo; Kohn, Donald B; Crooks, Gay M

    2016-05-01

    Although clonal studies of lineage potential have been extensively applied to organ specific stem and progenitor cells, much less is known about the clonal origins of lineages formed from the germ layers in early embryogenesis. We applied lentiviral tagging followed by vector integration site analysis (VISA) with high-throughput sequencing to investigate the ontogeny of the hematopoietic, endothelial and mesenchymal lineages as they emerge from human embryonic mesoderm. In contrast to studies that have used VISA to track differentiation of self-renewing stem cell clones that amplify significantly over time, we focused on a population of progenitor clones with limited self-renewal capability. Our analyses uncovered the critical influence of sampling on the interpretation of lentiviral tag sharing, particularly among complex populations with minimal clonal duplication. By applying a quantitative framework to estimate the degree of undersampling we revealed the existence of tripotent mesodermal progenitors derived from pluripotent stem cells, and the subsequent bifurcation of their differentiation into bipotent endothelial/hematopoietic or endothelial/mesenchymal progenitors. Stem Cells 2016;34:1239-1250. PMID:26934332

  11. Exome Analysis Reveals Differentially Mutated Gene Signatures of Stage, Grade and Subtype in Breast Cancers

    PubMed Central

    Li, You; Wang, Xiaosheng; Vural, Suleyman; Mishra, Nitish K.; Cowan, Kenneth H.; Guda, Chittibabu

    2015-01-01

    Breast cancers exhibit highly heterogeneous molecular profiles. Although gene expression profiles have been used to predict the risks and prognostic outcomes of breast cancers, the high variability of gene expression limits its clinical application. In contrast, genetic mutation profiles would be more advantageous than gene expression profiles because genetic mutations can be stably detected and the mutational heterogeneity widely exists in breast cancer genomes. We analyzed 98 breast cancer whole exome samples that were sorted into three subtypes, two grades and two stages. The sum deleterious effect of all mutations in each gene was scored to identify differentially mutated genes (DMGs) for this case-control study. DMGs were corroborated using extensive published knowledge. Functional consequences of deleterious SNVs on protein structure and function were also investigated. Genes such as ERBB2, ESP8, PPP2R4, KIAA0922, SP4, CENPJ, PRCP and SELP that have been experimentally or clinically verified to be tightly associated with breast cancer prognosis are among the DMGs identified in this study. We also identified some genes such as ARL6IP5, RAET1E, and ANO7 that could be crucial for breast cancer development and prognosis. Further, SNVs such as rs1058808, rs2480452, rs61751507, rs79167802, rs11540666, and rs2229437 that potentially influence protein functions are observed at significantly different frequencies in different comparison groups. Protein structure modeling revealed that many non-synonymous SNVs have a deleterious effect on protein stability, structure and function. Mutational profiling at gene- and SNV-level revealed differential patterns within each breast cancer comparison group, and the gene signatures correlate with expected prognostic characteristics of breast cancer classes. Some of the genes and SNVs identified in this study show high promise and are worthy of further investigation by experimental studies. PMID:25803781

  12. Multiple Differential Networks Strategy Reveals Carboplatin and Melphalan-Induced Dynamic Module Changes in Retinoblastoma.

    PubMed

    Chen, Cui; Ma, Feng-Wei; Du, Cui-Yun; Wang, Ping

    2016-01-01

    BACKGROUND Retinoblastoma (RB) is the most common malignant tumor of the eye in childhood. The objective of this paper was to investigate carboplatin (CAR)- and melphalan (MEL)-induced dynamic module changes in RB based on multiple (M) differential networks, and to generate systems-level insights into RB progression. MATERIAL AND METHODS To achieve this goal, we constructed M-differential co-expression networks (DCNs), assigned a weight to each edge, and identified seed genes in M DCNs by ranking genes based on their topological features. Starting with seed genes, a module search was performed to explore candidate modules in CAR and MEL condition. M-DMs were detected according to significance evaluations of M-modules, which originated from refinement of candidate modules. Further, we revealed dynamic changes in M-DM activity and connectivity on the basis of significance of Module Connectivity Dynamic Score (MCDS). RESULTS In the present study, M=2, a total of 21 seed genes were obtained. By assessing module search, refinement, and evaluation, we gained 18 2-DMs. Moreover, 3 significant 2-DMs (Module 1, Module 2, and Module 3) with dynamic changes across CAR and MEL condition were determined, and we denoted them as dynamic modules. Module 1 had 27 nodes of which 6 were seed genes and 56 edges. Module 2 was composed of 28 nodes and 54 edges. A total of 28 nodes interacted with 45 edges presented in Module 3. CONCLUSIONS We have identified 3 dynamic modules with changes induced by CAR and MEL in RB, which might give insights in revealing molecular mechanism for RB therapy. PMID:27144687

  13. Differential Gene Expression Reveals Candidate Genes for Drought Stress Response in Abies alba (Pinaceae)

    PubMed Central

    Ziegenhagen, Birgit; Liepelt, Sascha

    2015-01-01

    Increasing drought periods as a result of global climate change pose a threat to many tree species by possibly outpacing their adaptive capabilities. Revealing the genetic basis of drought stress response is therefore implemental for future conservation strategies and risk assessment. Access to informative genomic regions is however challenging, especially for conifers, partially due to their large genomes, which puts constraints on the feasibility of whole genome scans. Candidate genes offer a valuable tool to reduce the complexity of the analysis and the amount of sequencing work and costs. For this study we combined an improved drought stress phenotyping of needles via a novel terahertz water monitoring technique with Massive Analysis of cDNA Ends to identify candidate genes for drought stress response in European silver fir (Abies alba Mill.). A pooled cDNA library was constructed from the cotyledons of six drought stressed and six well-watered silver fir seedlings, respectively. Differential expression analyses of these libraries revealed 296 candidate genes for drought stress response in silver fir (247 up- and 49 down-regulated) of which a subset was validated by RT-qPCR of the twelve individual cotyledons. A majority of these genes code for currently uncharacterized proteins and hint on new genomic resources to be explored in conifers. Furthermore, we could show that some traditional reference genes from model plant species (GAPDH and eIF4A2) are not suitable for differential analysis and we propose a new reference gene, TPC1, for drought stress expression profiling in needles of conifer seedlings. PMID:25924061

  14. Evidence for differential alternative splicing in blood of young boys with autism spectrum disorders

    PubMed Central

    2013-01-01

    Background Since RNA expression differences have been reported in autism spectrum disorder (ASD) for blood and brain, and differential alternative splicing (DAS) has been reported in ASD brains, we determined if there was DAS in blood mRNA of ASD subjects compared to typically developing (TD) controls, as well as in ASD subgroups related to cerebral volume. Methods RNA from blood was processed on whole genome exon arrays for 2-4–year-old ASD and TD boys. An ANCOVA with age and batch as covariates was used to predict DAS for ALL ASD (n=30), ASD with normal total cerebral volumes (NTCV), and ASD with large total cerebral volumes (LTCV) compared to TD controls (n=20). Results A total of 53 genes were predicted to have DAS for ALL ASD versus TD, 169 genes for ASD_NTCV versus TD, 1 gene for ASD_LTCV versus TD, and 27 genes for ASD_LTCV versus ASD_NTCV. These differences were significant at P <0.05 after false discovery rate corrections for multiple comparisons (FDR <5% false positives). A number of the genes predicted to have DAS in ASD are known to regulate DAS (SFPQ, SRPK1, SRSF11, SRSF2IP, FUS, LSM14A). In addition, a number of genes with predicted DAS are involved in pathways implicated in previous ASD studies, such as ROS monocyte/macrophage, Natural Killer Cell, mTOR, and NGF signaling. The only pathways significant after multiple comparison corrections (FDR <0.05) were the Nrf2-mediated reactive oxygen species (ROS) oxidative response (superoxide dismutase 2, catalase, peroxiredoxin 1, PIK3C3, DNAJC17, microsomal glutathione S-transferase 3) and superoxide radical degradation (SOD2, CAT). Conclusions These data support differences in alternative splicing of mRNA in blood of ASD subjects compared to TD controls that differ related to head size. The findings are preliminary, need to be replicated in independent cohorts, and predicted alternative splicing differences need to be confirmed using direct analytical methods. PMID:24007566

  15. Differential spectral power alteration following acupuncture at different designated places revealed by magnetoencephalography

    NASA Astrophysics Data System (ADS)

    You, Youbo; Bai, Lijun; Dai, Ruwei; Xue, Ting; Zhong, Chongguang; Liu, Zhenyu; Wang, Hu; Feng, Yuanyuan; Wei, Wenjuan; Tian, Jie

    2012-03-01

    As an ancient therapeutic technique in Traditional Chinese Medicine, acupuncture has been used increasingly in modern society to treat a range of clinical conditions as an alternative and complementary therapy. However, acupoint specificity, lying at the core of acupuncture, still faces many controversies. Considering previous neuroimaging studies on acupuncture have mainly employed functional magnetic resonance imaging, which only measures the secondary effect of neural activity on cerebral metabolism and hemodynamics, in the current study, we adopted an electrophysiological measurement technique named magnetoencephalography (MEG) to measure the direct neural activity. 28 healthy college students were recruited in this study. We filtered MEG data into 5 consecutive frequency bands (delta, theta, alpha, beta and gamma band) and grouped 140 sensors into 10 main brain regions (left/right frontal, central, temporal, parietal and occipital regions). Fast Fourier Transformation (FFT) based spectral analysis approach was further performed to explore the differential band-limited power change patterns of acupuncture at Stomach Meridian 36 (ST36) using a nearby nonacupoint (NAP) as control condition. Significantly increased delta power and decreased alpha as well as beta power in bilateral frontal ROIs were observed following stimulation at ST36. Compared with ST36, decreased alpha power in left and right central, right parietal as well as right temporal ROIs were detected in NAP group. Our research results may provide additional evidence for acupoint specificity.

  16. Teacher Implementation of Trial-Based Functional Analysis and Differential Reinforcement of Alternative Behavior for Students with Challenging Behavior

    ERIC Educational Resources Information Center

    Flynn, Susan D.; Lo, Ya-yu

    2016-01-01

    The purpose of this study was to examine the effects of a training package on three middle school special education teachers' accurate implementation of trial-based functional analysis (TBFA) and differential reinforcement of alternative behavior (DRA) with their students with autism spectrum disorders or emotional and behavioral disorders in the…

  17. Differential Reinforcement of Alternative Behavior Increases Resistance to Extinction: Clinical Demonstration, Animal Modeling, and Clinical Test of One Solution

    ERIC Educational Resources Information Center

    Mace, F. Charles; McComas, Jennifer J.; Mauro, Benjamin C.; Progar, Patrick R.; Taylor, Bridget; Ervin, Ruth; Zangrillo, Amanda N.

    2010-01-01

    Basic research with pigeons on behavioral momentum suggests that differential reinforcement of alternative behavior (DRA) can increase the resistance of target behavior to change. This finding suggests that clinical applications of DRA may inadvertently increase the persistence of target behavior even as it decreases its frequency. We conducted…

  18. Sister Dehalobacter Genomes Reveal Specialization in Organohalide Respiration and Recent Strain Differentiation Likely Driven by Chlorinated Substrates.

    PubMed

    Tang, Shuiquan; Wang, Po Hsiang; Higgins, Steven A; Löffler, Frank E; Edwards, Elizabeth A

    2016-01-01

    The genomes of two closely related Dehalobacter strains (strain CF and strain DCA) were assembled from the metagenome of an anaerobic enrichment culture that reductively dechlorinates chloroform (CF), 1,1,1-trichloroethane (1,1,1-TCA) and 1,1-dichloroethane (1,1-DCA). The 3.1 Mbp genomes of strain CF (that dechlorinates CF and 1,1,1-TCA) and strain DCA (that dechlorinates 1,1-DCA) each contain 17 putative reductive dehalogenase homologous (rdh) genes. These two genomes were systematically compared to three other available organohalide-respiring Dehalobacter genomes (Dehalobacter restrictus strain PER-K23, Dehalobacter sp. strain E1 and Dehalobacter sp. strain UNSWDHB), and to the genomes of Dehalococcoides mccartyi strain 195 and Desulfitobacterium hafniense strain Y51. This analysis compared 42 different metabolic and physiological categories. The genomes of strains CF and DCA share 90% overall average nucleotide identity and >99.8% identity over a 2.9 Mbp alignment that excludes large insertions, indicating that these genomes differentiated from a close common ancestor. This differentiation was likely driven by selection pressures around two orthologous reductive dehalogenase genes, cfrA and dcrA, that code for the enzymes that reduce CF or 1,1,1-TCA and 1,1-DCA. The many reductive dehalogenase genes found in the five Dehalobacter genomes cluster into two small conserved regions and were often associated with Crp/Fnr transcriptional regulators. Specialization is on-going on a strain-specific basis, as some strains but not others have lost essential genes in the Wood-Ljungdahl (strain E1) and corrinoid biosynthesis pathways (strains E1 and PER-K23). The gene encoding phosphoserine phosphatase, which catalyzes the last step of serine biosynthesis, is missing from all five Dehalobacter genomes, yet D. restrictus can grow without serine, suggesting an alternative or unrecognized biosynthesis route exists. In contrast to D. mccartyi, a complete heme biosynthesis

  19. Sister Dehalobacter Genomes Reveal Specialization in Organohalide Respiration and Recent Strain Differentiation Likely Driven by Chlorinated Substrates

    DOE PAGESBeta

    Tang, Shuiquan; Wang, Po Hsiang; Higgins, Steven A.; Löffler, Frank E.; Edwards, Elizabeth A.

    2016-02-12

    Here we report that the genomes of two closely related Dehalobacter strains (strain CF and strain DCA) were assembled from the metagenome of an anaerobic enrichment culture that reductively dechlorinates chloroform (CF), 1,1,1-trichloroethane (1,1,1-TCA) and 1,1-dichloroethane (1,1-DCA). The 3.1 Mbp genomes of strain CF (that dechlorinates CF and 1,1,1-TCA) and strain DCA (that dechlorinates 1,1-DCA) each contain 17 putative reductive dehalogenase homologous (rdh) genes. These two genomes were systematically compared to three other available organohalide-respiring Dehalobacter genomes (Dehalobacter restrictus strain PER-K23, Dehalobacter sp. strain E1 and Dehalobacter sp. strain UNSWDHB), and to the genomes of Dehalococcoides mccartyi strain 195 andmore » Desulfitobacterium hafniense strain Y51. This analysis compared 42 different metabolic and physiological categories. The genomes of strains CF and DCA share 90% overall average nucleotide identity and >99.8% identity over a 2.9 Mbp alignment that excludes large insertions, indicating that these genomes differentiated from a close common ancestor. This differentiation was likely driven by selection pressures around two orthologous reductive dehalogenase genes, cfrA and dcrA, that code for the enzymes that reduce CF or 1,1,1-TCA and 1,1-DCA. The many reductive dehalogenase genes found in the five Dehalobacter genomes cluster into two small conserved regions and were often associated with Crp/Fnr transcriptional regulators. Specialization is on-going on a strain-specific basis, as some strains but not others have lost essential genes in the Wood-Ljungdahl (strain E1) and corrinoid biosynthesis pathways (strains E1 and PER-K23). The gene encoding phosphoserine phosphatase, which catalyzes the last step of serine biosynthesis, is missing from all five Dehalobacter genomes, yet D. restrictus can grow without serine, suggesting an alternative or unrecognized biosynthesis route exists. In contrast to D. mccartyi, a

  20. Sister Dehalobacter Genomes Reveal Specialization in Organohalide Respiration and Recent Strain Differentiation Likely Driven by Chlorinated Substrates

    PubMed Central

    Tang, Shuiquan; Wang, Po Hsiang; Higgins, Steven A.; Löffler, Frank E.; Edwards, Elizabeth A.

    2016-01-01

    The genomes of two closely related Dehalobacter strains (strain CF and strain DCA) were assembled from the metagenome of an anaerobic enrichment culture that reductively dechlorinates chloroform (CF), 1,1,1-trichloroethane (1,1,1-TCA) and 1,1-dichloroethane (1,1-DCA). The 3.1 Mbp genomes of strain CF (that dechlorinates CF and 1,1,1-TCA) and strain DCA (that dechlorinates 1,1-DCA) each contain 17 putative reductive dehalogenase homologous (rdh) genes. These two genomes were systematically compared to three other available organohalide-respiring Dehalobacter genomes (Dehalobacter restrictus strain PER-K23, Dehalobacter sp. strain E1 and Dehalobacter sp. strain UNSWDHB), and to the genomes of Dehalococcoides mccartyi strain 195 and Desulfitobacterium hafniense strain Y51. This analysis compared 42 different metabolic and physiological categories. The genomes of strains CF and DCA share 90% overall average nucleotide identity and >99.8% identity over a 2.9 Mbp alignment that excludes large insertions, indicating that these genomes differentiated from a close common ancestor. This differentiation was likely driven by selection pressures around two orthologous reductive dehalogenase genes, cfrA and dcrA, that code for the enzymes that reduce CF or 1,1,1-TCA and 1,1-DCA. The many reductive dehalogenase genes found in the five Dehalobacter genomes cluster into two small conserved regions and were often associated with Crp/Fnr transcriptional regulators. Specialization is on-going on a strain-specific basis, as some strains but not others have lost essential genes in the Wood-Ljungdahl (strain E1) and corrinoid biosynthesis pathways (strains E1 and PER-K23). The gene encoding phosphoserine phosphatase, which catalyzes the last step of serine biosynthesis, is missing from all five Dehalobacter genomes, yet D. restrictus can grow without serine, suggesting an alternative or unrecognized biosynthesis route exists. In contrast to D. mccartyi, a complete heme biosynthesis

  1. Morphology and genetics reveal an intriguing pattern of differentiation at a very small geographic scale in a bird species, the forest thrush Turdus lherminieri

    PubMed Central

    Arnoux, E; Eraud, C; Navarro, N; Tougard, C; Thomas, A; Cavallo, F; Vetter, N; Faivre, B; Garnier, S

    2014-01-01

    Mobile organisms are expected to show population differentiation only over fairly large geographical distances. However, there is growing evidence of discrepancy between dispersal potential and realized gene flow. Here we report an intriguing pattern of differentiation at a very small spatial scale in the forest thrush (Turdus lherminieri), a bird species endemic to the Lesser Antilles. Analysis of 331 individuals from 17 sampling sites distributed over three islands revealed a clear morphological and genetic differentiation between these islands isolated by 40–50 km. More surprisingly, we found that the phenotypic divergence between the two geographic zones of the island of Guadeloupe was associated with a very strong genetic differentiation (Fst from 0.073–0.153), making this pattern a remarkable case in birds given the very small spatial scale considered. Molecular data (mitochondrial control region sequences and microsatellite genotypes) suggest that this strong differentiation could have occurred in situ, although alternative hypotheses cannot be fully discarded. This study suggests that the ongoing habitat fragmentation, especially in tropical forests, may have a deeper impact than previously thought on avian populations. PMID:24984605

  2. Histone modification profiling reveals differential signatures associated with human embryonic stem cell self-renewal and differentiation.

    PubMed

    Bhanu, Natarajan V; Sidoli, Simone; Garcia, Benjamin A

    2016-02-01

    In this study, we trace developmental stages using epigenome changes in human embryonic stem cells (hESCs) treated with drugs modulating either self-renewal or differentiation. Based on microscopy, qPCR and flow cytometry, we classified the treatment outcome as inducing pluripotency (hESC, flurbiprofen and gatifloxacin), mesendoderm (sinomenine), differentiation (cyamarin, digoxin, digitoxin, selegeline and theanine) and lineage-commitment (RA). When we analyzed histone PTMs that imprinted these gene and protein expressions, the above classification was reassorted. Hyperacetylation at H3K4, 9, 14, 18, 56 and 122 as well as H4K5, 8, 12 and 16 emerged as the pluripotency signature of hESCs. Methylations especially of H3 at K9, K20, K27 and K36 characterized differentiation initiation as seen in no-drug control and fluribiprofen. Sinomenine-treated cells clustered close to "differentiation initiators", consistent with flow cytometry where it induced mesendoderm, along with cyamarin and possibly selegnine. Neurectoderm, induced by RA and theanine manifested methylations on H3 shifts to H3.3. By both flow cytometry and histone PTM clustering, it appears that cells treated with gatifloxacin, flurbiprofen, digitoxin and digoxin were not yet lineage-committed or mixed cell types. Taken together, our moderate-throughput histone PTM profiling approach highlighted subtle epigenetic signatures that permitted us to predict divergent lineage progression even in differentiating cells with similar phenotype and gene expression. PMID:26631989

  3. Histone modification profiling reveals differential signatures associated with human embryonic stem cell self-renewal and differentiation

    PubMed Central

    Bhanu, Natarajan V.; Sidoli, Simone; Garcia, Benjamin A.

    2016-01-01

    In this study, we trace developmental stages using epigenome changes in human embryonic stem cells (hESCs) treated with drugs modulating either self-renewal or differentiation. Based on microscopy, qPCR and flow cytometry, we classified the treatment outcome as inducing pluripotency (hESC, flurbiprofen and gatifloxacin), mesendoderm (sinomenine), differentiation (cyamarin, digoxin, digitoxin, selegeline and theanine) and lineage-commitment (RA). When we analyzed histone PTMs that imprinted these gene and protein expressions, the above classification was reassorted. Hyperacetylation at H3K4, 9, 14, 18, 56 and 122 as well as H4K5, 8, 12 and 16 emerged as the pluripotency signature of hESCs. Methylations especially of H3 at K9, K20, K27 and K36 characterized differentiation initiation as seen in no-drug control and fluribiprofen. Sinomenine-treated cells clustered close to “differentiation initiators”, consistent with flow cytometry where it induced mesendoderm, along with cyamarin and possibly selegnine. Neurectoderm, induced by RA and theanine manifested methylations on H3 shifts to H3.3. By both flow cytometry and histone PTM clustering, it appears that cells treated with gatifloxacin, flurbiprofen, digitoxin and digoxin were not yet lineage-committed or mixed cell types. Taken together, our moderate-throughput histone PTM profiling approach highlighted subtle epigenetic signatures that permitted us to predict divergent lineage progression even in differentiating cells with similar phenotype and gene expression. PMID:26631989

  4. Alternative splicing of spleen tyrosine kinase differentially regulates colorectal cancer progression

    PubMed Central

    Ni, Beibei; Hu, Jun; Chen, Dianke; Li, Li; Chen, Daici; Wang, Jianping; Wang, Lei

    2016-01-01

    Spleen tyrosine kinase (SYK) has been reported as a potential tumor suppressor in colorectal cancer (CRC). However, the role of alternative splicing of SYK in carcinogenesis remains unclear. In the present study, SYK isoforms were overexpressed in the human CRC HCT 116 cell line using lentiviral expression vectors to investigate the biological functions of full length SYK [SYK(L)] and short form SYK [SYK(S)] in CRC. Real-time cellular analysis and the 5-ethynyl-2-deoxyuridine assay were used to detect the effects of SYK(L) and SYK(S) on cell proliferation. Cell cycle progression and migration were assessed via flow cytometry and Transwell assays, respectively. The results revealed that the recombinant lentivirus with SYK(L) overexpression significantly suppressed the proliferation and metastasis of CRC cells, while SYK(S) overexpression did not. In addition, MTS assays demonstrated that SYK(L) and SYK(S) increased the cellular sensitivity to 5-fluorouracil (5-FU), suggesting that SYK(L) and 5-FU produce a significant synergistic effect on CRC cell proliferation, while SYK(S) has an effect on modulating CRC 5-FU sensitivity. Furthermore, quantitative polymerase chain reaction results revealed that SYK(L) was downregulated in 69% of 26 pairs of CRC and adjacent non-cancerous tissues, whereas SYK(S) exhibited no significant differences between tumor and normal tissues. Overall, the present data provides evidence that SYK(L) is a tumor suppressor in CRC, and both SYK(L) and SYK(S) may serve as important predictors in the chemotherapeutic treatment of CRC. PMID:27602108

  5. Prognostic microRNAs in high-grade glioma reveal a link to oligodendrocyte precursor differentiation

    PubMed Central

    Hayes, Josie; Thygesen, Helene; Droop, Alastair; Hughes, Thomas A.; Westhead, David; Lawler, Sean E.; Wurdak, Heiko; Short, Susan C.

    2015-01-01

    MicroRNA expression can be exploited to define tumor prognosis and stratification for precision medicine. It remains unclear whether prognostic microRNA signatures are exclusively tumor grade and/or molecular subtype-specific, or whether common signatures of aggressive clinical behavior can be identified. Here, we defined microRNAs that are associated with good and poor prognosis in grade III and IV gliomas using data from The Cancer Genome Atlas. Pathway analysis of microRNA targets that are differentially expressed in good and poor prognosis glioma identified a link to oligodendrocyte development. Notably, a microRNA expression profile that is characteristic of a specific oligodendrocyte precursor cell type (OP1) correlates with microRNA expression from 597 of these tumors and is consistently associated with poor patient outcome in grade III and IV gliomas. Our study reveals grade-independent and subtype-independent prognostic molecular signatures in high-grade glioma and provides a framework for investigating the mechanisms of brain tumor aggressiveness. PMID:25897422

  6. The Brain-to-Pancreatic Islet Neuronal Map Reveals Differential Glucose Regulation From Distinct Hypothalamic Regions.

    PubMed

    Rosario, Wilfredo; Singh, Inderroop; Wautlet, Arnaud; Patterson, Christa; Flak, Jonathan; Becker, Thomas C; Ali, Almas; Tamarina, Natalia; Philipson, Louis H; Enquist, Lynn W; Myers, Martin G; Rhodes, Christopher J

    2016-09-01

    The brain influences glucose homeostasis, partly by supplemental control over insulin and glucagon secretion. Without this central regulation, diabetes and its complications can ensue. Yet, the neuronal network linking to pancreatic islets has never been fully mapped. Here, we refine this map using pseudorabies virus (PRV) retrograde tracing, indicating that the pancreatic islets are innervated by efferent circuits that emanate from the hypothalamus. We found that the hypothalamic arcuate nucleus (ARC), ventromedial nucleus (VMN), and lateral hypothalamic area (LHA) significantly overlap PRV and the physiological glucose-sensing enzyme glucokinase. Then, experimentally lowering glucose sensing, specifically in the ARC, resulted in glucose intolerance due to deficient insulin secretion and no significant effect in the VMN, but in the LHA it resulted in a lowering of the glucose threshold that improved glucose tolerance and/or improved insulin sensitivity, with an exaggerated counter-regulatory response for glucagon secretion. No significant effect on insulin sensitivity or metabolic homeostasis was noted. Thus, these data reveal novel direct neuronal effects on pancreatic islets and also render a functional validation of the brain-to-islet neuronal map. They also demonstrate that distinct regions of the hypothalamus differentially control insulin and glucagon secretion, potentially in partnership to help maintain glucose homeostasis and guard against hypoglycemia. PMID:27207534

  7. Transcriptome sequencing of purple petal spot region in tree peony reveals differentially expressed anthocyanin structural genes

    PubMed Central

    Zhang, Yanzhao; Cheng, Yanwei; Ya, Huiyuan; Xu, Shuzhen; Han, Jianming

    2015-01-01

    The pigmented cells in defined region of a petal constitute the petal spots. Petal spots attract pollinators and are found in many angiosperm families. Several cultivars of tree peony contain a single red or purple spot at the base of petal that makes the flower more attractive for the ornamental market. So far, the understanding of the molecular mechanism of spot formation is inadequate. In this study, we sequenced the transcriptome of the purple spot and the white non-spot of tree peony flower. We assembled and annotated 67,892 unigenes. Comparative analyses of the two transcriptomes showed 1,573 differentially expressed genes, among which 933 were up-regulated, and 640 were down-regulated in the purple spot. Subsequently, we examined four anthocyanin structural genes, including PsCHS, PsF3′H, PsDFR, and PsANS, which expressed at a significantly higher level in the purple spot than in the white non-spot. We further validated the digital expression data using quantitative real-time PCR. Our result uncovered transcriptome variance between the spot and non-spot of tree peony flower, and revealed that the co-expression of four anthocyanin structural genes was responsible for spot pigment in tree peony. The data will further help to unravel the genetic mechanism of peony flower spot formation. PMID:26583029

  8. Transcriptional Profiling Reveals Differential Gene Expression of Amur Ide (Leuciscus waleckii) during Spawning Migration

    PubMed Central

    Cui, Jun; Xu, Jian; Zhang, Songhao; Wang, Kai; Jiang, Yanliang; Mahboob, Shahid; Al-Ghanim, Khalid A.; Xu, Peng

    2015-01-01

    Amur ide (Leuciscus waleckii), an important aquaculture species, inhabits neutral freshwater but can tolerate high salinity or alkalinity. As an extreme example, the population in Dali Nor lake inhabits alkalized soda water permanently, and migrates from alkaline water to neutral freshwater to spawn. In this study, we performed comparative transcriptome profiling study on the livers of Amur ide to interrogate the expression differences between the population that permanently inhabit freshwater in Ganggeng Nor lake (FW) and the spawning population that recently migrated from alkaline water into freshwater (SM). A total of 637,234,880 reads were generated, resulting in 53,440 assembled contigs that were used as reference sequences. Comparisons of these transcriptome files revealed 444 unigenes with significant differential expression (p-value ≤ 0.01, fold-change ≥ 2), including 246 genes that were up-regulated in SM and 198 genes that were up-regulated in FW. The gene ontology (GO) enrichment analysis and KEGG pathway analysis indicated that the mTOR signaling pathway, Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway, and oxidative phosphorylation were highly likely to affect physiological changes during spawning migration. Overall, this study demonstrates that transcriptome changes played a role in Amur ide spawning migration. These results provide a foundation for further analyses on the physiological and molecular mechanisms underlying Amur ide spawning migration. PMID:26096003

  9. Species versus guild level differentiation revealed across the annual cycle by isotopic niche examination.

    PubMed

    Bodey, Thomas W; Ward, Eric J; Phillips, Richard A; McGill, Rona A R; Bearhop, Stuart

    2014-03-01

    Interspecific competitive interactions typically result in niche differentiation to alleviate competition through mechanisms including character displacement. However, competition is not the sole constraint on resource partitioning, and its effects are mediated by factors including the environmental context in which species coexist. Colonial seabirds provide an excellent opportunity to investigate the importance of competition in shaping realized niche widths because their life histories lead to variation in intra- and interspecific competition across the annual cycle. Dense breeding aggregations result in intense competition for prey in surrounding waters, whereas non-breeding dispersal to larger geographical areas produces lower densities of competitors. Bayesian hierarchical models of the isotopic niche, closely aligned to the trophic niche, reveal the degree of segregation between species and functional groups during both time periods. Surprisingly, species explained far more of the variance in the isotopic niche during the non-breeding than the breeding period. Our results underline the key role of non-breeding dynamics in alleviating competition and promoting distinctions between species through the facilitation of resource partitioning. Such situations may be common in a diverse range of communities sustained by ephemeral but abundant food items. This highlights how consideration of the hierarchical grouping of competitive interactions alongside consideration of abiotic constraints across the complete annual cycle allows a full understanding of the role of competition in driving patterns of character displacement. PMID:24215391

  10. Computational analysis of translational readthrough proteins in Drosophila and yeast reveals parallels to alternative splicing

    PubMed Central

    Pancsa, Rita; Macossay-Castillo, Mauricio; Kosol, Simone; Tompa, Peter

    2016-01-01

    In translational readthrough (TR) the ribosome continues extending the nascent protein beyond the first in-frame termination codon. Due to the lack of dedicated analyses of eukaryotic TR cases, the associated functional-evolutionary advantages are still unclear. Here, based on a variety of computational methods, we describe the structural and functional properties of previously proposed D. melanogaster and S. cerevisiae TR proteins and extensions. We found that in D. melanogaster TR affects long proteins in mainly regulatory roles. Their TR-extensions are structurally disordered and rich in binding motifs, which, together with their cell-type- and developmental stage-dependent inclusion, suggest that similarly to alternatively spliced exons they rewire cellular interaction networks in a temporally and spatially controlled manner. In contrast, yeast TR proteins are rather short and fulfil mainly housekeeping functions, like translation. Yeast extensions usually lack disorder and linear motifs, which precludes elucidating their functional relevance with sufficient confidence. Therefore we propose that by being much more restricted and by lacking clear functional hallmarks in yeast as opposed to fruit fly, TR shows remarkable parallels with alternative splicing. Additionally, the lack of conservation of TR extensions among orthologous TR proteins suggests that TR-mediated functions may be generally specific to lower taxonomic levels. PMID:27561673

  11. Computational analysis of translational readthrough proteins in Drosophila and yeast reveals parallels to alternative splicing.

    PubMed

    Pancsa, Rita; Macossay-Castillo, Mauricio; Kosol, Simone; Tompa, Peter

    2016-01-01

    In translational readthrough (TR) the ribosome continues extending the nascent protein beyond the first in-frame termination codon. Due to the lack of dedicated analyses of eukaryotic TR cases, the associated functional-evolutionary advantages are still unclear. Here, based on a variety of computational methods, we describe the structural and functional properties of previously proposed D. melanogaster and S. cerevisiae TR proteins and extensions. We found that in D. melanogaster TR affects long proteins in mainly regulatory roles. Their TR-extensions are structurally disordered and rich in binding motifs, which, together with their cell-type- and developmental stage-dependent inclusion, suggest that similarly to alternatively spliced exons they rewire cellular interaction networks in a temporally and spatially controlled manner. In contrast, yeast TR proteins are rather short and fulfil mainly housekeeping functions, like translation. Yeast extensions usually lack disorder and linear motifs, which precludes elucidating their functional relevance with sufficient confidence. Therefore we propose that by being much more restricted and by lacking clear functional hallmarks in yeast as opposed to fruit fly, TR shows remarkable parallels with alternative splicing. Additionally, the lack of conservation of TR extensions among orthologous TR proteins suggests that TR-mediated functions may be generally specific to lower taxonomic levels. PMID:27561673

  12. MicroRNA-222 regulates muscle alternative splicing through Rbm24 during differentiation of skeletal muscle cells.

    PubMed

    Cardinali, B; Cappella, M; Provenzano, C; Garcia-Manteiga, J M; Lazarevic, D; Cittaro, D; Martelli, F; Falcone, G

    2016-01-01

    A number of microRNAs have been shown to regulate skeletal muscle development and differentiation. MicroRNA-222 is downregulated during myogenic differentiation and its overexpression leads to alteration of muscle differentiation process and specialized structures. By using RNA-induced silencing complex (RISC) pulldown followed by RNA sequencing, combined with in silico microRNA target prediction, we have identified two new targets of microRNA-222 involved in the regulation of myogenic differentiation, Ahnak and Rbm24. Specifically, the RNA-binding protein Rbm24 is a major regulator of muscle-specific alternative splicing and its downregulation by microRNA-222 results in defective exon inclusion impairing the production of muscle-specific isoforms of Coro6, Fxr1 and NACA transcripts. Reconstitution of normal levels of Rbm24 in cells overexpressing microRNA-222 rescues muscle-specific splicing. In conclusion, we have identified a new function of microRNA-222 leading to alteration of myogenic differentiation at the level of alternative splicing, and we provide evidence that this effect is mediated by Rbm24 protein. PMID:26844700

  13. MicroRNA-222 regulates muscle alternative splicing through Rbm24 during differentiation of skeletal muscle cells

    PubMed Central

    Cardinali, B; Cappella, M; Provenzano, C; Garcia-Manteiga, J M; Lazarevic, D; Cittaro, D; Martelli, F; Falcone, G

    2016-01-01

    A number of microRNAs have been shown to regulate skeletal muscle development and differentiation. MicroRNA-222 is downregulated during myogenic differentiation and its overexpression leads to alteration of muscle differentiation process and specialized structures. By using RNA-induced silencing complex (RISC) pulldown followed by RNA sequencing, combined with in silico microRNA target prediction, we have identified two new targets of microRNA-222 involved in the regulation of myogenic differentiation, Ahnak and Rbm24. Specifically, the RNA-binding protein Rbm24 is a major regulator of muscle-specific alternative splicing and its downregulation by microRNA-222 results in defective exon inclusion impairing the production of muscle-specific isoforms of Coro6, Fxr1 and NACA transcripts. Reconstitution of normal levels of Rbm24 in cells overexpressing microRNA-222 rescues muscle-specific splicing. In conclusion, we have identified a new function of microRNA-222 leading to alteration of myogenic differentiation at the level of alternative splicing, and we provide evidence that this effect is mediated by Rbm24 protein. PMID:26844700

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

    PubMed Central

    2014-01-01

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

  15. ROS play a critical role in the differentiation of alternatively activated macrophages and the occurrence of tumor-associated macrophages

    PubMed Central

    Zhang, Yan; Choksi, Swati; Chen, Kun; Pobezinskaya, Yelena; Linnoila, Ilona; Liu, Zheng-Gang

    2013-01-01

    Differentiation to different types of macrophages determines their distinct functions. Tumor-associated macrophages (TAMs) promote tumorigenesis owing to their proangiogenic and immune-suppressive functions similar to those of alternatively activated (M2) macrophages. We report that reactive oxygen species (ROS) production is critical for macrophage differentiation and that inhibition of superoxide (O2−) production specifically blocks the differentiation of M2 macrophages. We found that when monocytes are triggered to differentiate, O2− is generated and is needed for the biphasic ERK activation, which is critical for macrophage differentiation. We demonstrated that ROS elimination by butylated hydroxyanisole (BHA) and other ROS inhibitors blocks macrophage differentiation. However, the inhibitory effect of ROS elimination on macrophage differentiation is overcome when cells are polarized to classically activated (M1), but not M2, macrophages. More importantly, the continuous administration of the ROS inhibitor BHA efficiently blocked the occurrence of TAMs and markedly suppressed tumorigenesis in mouse cancer models. Targeting TAMs by blocking ROS can be a potentially effective method for cancer treatment. PMID:23752925

  16. Transcriptional profiling in response to terminal drought stress reveals differential responses along the wheat genome

    PubMed Central

    Aprile, Alessio; Mastrangelo, Anna M; De Leonardis, Anna M; Galiba, Gabor; Roncaglia, Enrica; Ferrari, Francesco; De Bellis, Luigi; Turchi, Luana; Giuliano, Giovanni; Cattivelli, Luigi

    2009-01-01

    Background Water stress during grain filling has a marked effect on grain yield, leading to a reduced endosperm cell number and thus sink capacity to accumulate dry matter. The bread wheat cultivar Chinese Spring (CS), a Chinese Spring terminal deletion line (CS_5AL-10) and the durum wheat cultivar Creso were subjected to transcriptional profiling after exposure to mild and severe drought stress at the grain filling stage to find evidences of differential stress responses associated to different wheat genome regions. Results The transcriptome analysis of Creso, CS and its deletion line revealed 8,552 non redundant probe sets with different expression levels, mainly due to the comparisons between the two species. The drought treatments modified the expression of 3,056 probe sets. Besides a set of genes showing a similar drought response in Creso and CS, cluster analysis revealed several drought response features that can be associated to the different genomic structure of Creso, CS and CS_5AL-10. Some drought-related genes were expressed at lower level (or not expressed) in Creso (which lacks the D genome) or in the CS_5AL-10 deletion line compared to CS. The chromosome location of a set of these genes was confirmed by PCR-based mapping on the D genome (or the 5AL-10 region). Many clusters were characterized by different level of expression in Creso, CS and CS_AL-10, suggesting that the different genome organization of the three genotypes may affect plant adaptation to stress. Clusters with similar expression trend were grouped and functional classified to mine the biological mean of their activation or repression. Genes involved in ABA, proline, glycine-betaine and sorbitol pathways were found up-regulated by drought stress. Furthermore, the enhanced expression of a set of transposons and retrotransposons was detected in CS_5AL-10. Conclusion Bread and durum wheat genotypes were characterized by a different physiological reaction to water stress and by a

  17. Differential Molecular Responses of Rapeseed Cotyledons to Light and Dark Reveal Metabolic Adaptations toward Autotrophy Establishment

    PubMed Central

    He, Dongli; Damaris, Rebecca N.; Fu, Jinlei; Tu, Jinxing; Fu, Tingdong; Xi, Chen; Yi, Bin; Yang, Pingfang

    2016-01-01

    Photosynthesis competent autotrophy is established during the postgerminative stage of plant growth. Among the multiple factors, light plays a decisive role in the switch from heterotrophic to autotrophic growth. Under dark conditions, the rapeseed hypocotyl extends quickly with an apical hook, and the cotyledon is yellow and folded, and maintains high levels of the isocitrate lyase (ICL). By contrast, in the light, the hypocotyl extends slowly, the cotyledon unfolds and turns green, the ICL content changes in parallel with cotyledon greening. To reveal metabolic adaptations during the establishment of postgerminative autotrophy in rapeseed, we conducted comparative proteomic and metabolomic analyses of the cotyledons of seedlings grown under light versus dark conditions. Under both conditions, the increase in proteases, fatty acid β-oxidation and glyoxylate-cycle related proteins was accompanied by rapid degradation of the stored proteins and lipids with an accumulation of the amino acids. While light condition partially retarded these conversions. Light significantly induced the expression of chlorophyll-binding and photorespiration related proteins, resulting in an increase in reducing-sugars. However, the levels of some chlorophyllide conversion, Calvin-cycle and photorespiration related proteins also accumulated in dark grown cotyledons, implying that the transition from heterotrophy to autotrophy is programmed in the seed rather than induced by light. Various anti-stress systems, e.g., redox related proteins, salicylic acid, proline and chaperones, were employed to decrease oxidative stress, which was mainly derived from lipid oxidation or photorespiration, under both conditions. This study provides a comprehensive understanding of the differential molecular responses of rapeseed cotyledons to light and dark conditions, which will facilitate further study on the complex mechanism underlying the transition from heterotrophy to autotrophy. PMID:27471506

  18. Comorbid Analysis of Genes Associated with Autism Spectrum Disorders Reveals Differential Evolutionary Constraints.

    PubMed

    David, Maude M; Enard, David; Ozturk, Alp; Daniels, Jena; Jung, Jae-Yoon; Diaz-Beltran, Leticia; Wall, Dennis P

    2016-01-01

    The burden of comorbidity in Autism Spectrum Disorder (ASD) is substantial. The symptoms of autism overlap with many other human conditions, reflecting common molecular pathologies suggesting that cross-disorder analysis will help prioritize autism gene candidates. Genes in the intersection between autism and related conditions may represent nonspecific indicators of dysregulation while genes unique to autism may play a more causal role. Thorough literature review allowed us to extract 125 ICD-9 codes comorbid to ASD that we mapped to 30 specific human disorders. In the present work, we performed an automated extraction of genes associated with ASD and its comorbid disorders, and found 1031 genes involved in ASD, among which 262 are involved in ASD only, with the remaining 779 involved in ASD and at least one comorbid disorder. A pathway analysis revealed 13 pathways not involved in any other comorbid disorders and therefore unique to ASD, all associated with basal cellular functions. These pathways differ from the pathways associated with both ASD and its comorbid conditions, with the latter being more specific to neural function. To determine whether the sequence of these genes have been subjected to differential evolutionary constraints, we studied long term constraints by looking into Genomic Evolutionary Rate Profiling, and showed that genes involved in several comorbid disorders seem to have undergone more purifying selection than the genes involved in ASD only. This result was corroborated by a higher dN/dS ratio for genes unique to ASD as compare to those that are shared between ASD and its comorbid disorders. Short-term evolutionary constraints showed the same trend as the pN/pS ratio indicates that genes unique to ASD were under significantly less evolutionary constraint than the genes associated with all other disorders. PMID:27414027

  19. Comorbid Analysis of Genes Associated with Autism Spectrum Disorders Reveals Differential Evolutionary Constraints

    PubMed Central

    David, Maude M.; Enard, David; Ozturk, Alp; Daniels, Jena; Jung, Jae-Yoon; Diaz-Beltran, Leticia; Wall, Dennis. P.

    2016-01-01

    The burden of comorbidity in Autism Spectrum Disorder (ASD) is substantial. The symptoms of autism overlap with many other human conditions, reflecting common molecular pathologies suggesting that cross-disorder analysis will help prioritize autism gene candidates. Genes in the intersection between autism and related conditions may represent nonspecific indicators of dysregulation while genes unique to autism may play a more causal role. Thorough literature review allowed us to extract 125 ICD-9 codes comorbid to ASD that we mapped to 30 specific human disorders. In the present work, we performed an automated extraction of genes associated with ASD and its comorbid disorders, and found 1031 genes involved in ASD, among which 262 are involved in ASD only, with the remaining 779 involved in ASD and at least one comorbid disorder. A pathway analysis revealed 13 pathways not involved in any other comorbid disorders and therefore unique to ASD, all associated with basal cellular functions. These pathways differ from the pathways associated with both ASD and its comorbid conditions, with the latter being more specific to neural function. To determine whether the sequence of these genes have been subjected to differential evolutionary constraints, we studied long term constraints by looking into Genomic Evolutionary Rate Profiling, and showed that genes involved in several comorbid disorders seem to have undergone more purifying selection than the genes involved in ASD only. This result was corroborated by a higher dN/dS ratio for genes unique to ASD as compare to those that are shared between ASD and its comorbid disorders. Short-term evolutionary constraints showed the same trend as the pN/pS ratio indicates that genes unique to ASD were under significantly less evolutionary constraint than the genes associated with all other disorders. PMID:27414027

  20. Differential Molecular Responses of Rapeseed Cotyledons to Light and Dark Reveal Metabolic Adaptations toward Autotrophy Establishment.

    PubMed

    He, Dongli; Damaris, Rebecca N; Fu, Jinlei; Tu, Jinxing; Fu, Tingdong; Xi, Chen; Yi, Bin; Yang, Pingfang

    2016-01-01

    Photosynthesis competent autotrophy is established during the postgerminative stage of plant growth. Among the multiple factors, light plays a decisive role in the switch from heterotrophic to autotrophic growth. Under dark conditions, the rapeseed hypocotyl extends quickly with an apical hook, and the cotyledon is yellow and folded, and maintains high levels of the isocitrate lyase (ICL). By contrast, in the light, the hypocotyl extends slowly, the cotyledon unfolds and turns green, the ICL content changes in parallel with cotyledon greening. To reveal metabolic adaptations during the establishment of postgerminative autotrophy in rapeseed, we conducted comparative proteomic and metabolomic analyses of the cotyledons of seedlings grown under light versus dark conditions. Under both conditions, the increase in proteases, fatty acid β-oxidation and glyoxylate-cycle related proteins was accompanied by rapid degradation of the stored proteins and lipids with an accumulation of the amino acids. While light condition partially retarded these conversions. Light significantly induced the expression of chlorophyll-binding and photorespiration related proteins, resulting in an increase in reducing-sugars. However, the levels of some chlorophyllide conversion, Calvin-cycle and photorespiration related proteins also accumulated in dark grown cotyledons, implying that the transition from heterotrophy to autotrophy is programmed in the seed rather than induced by light. Various anti-stress systems, e.g., redox related proteins, salicylic acid, proline and chaperones, were employed to decrease oxidative stress, which was mainly derived from lipid oxidation or photorespiration, under both conditions. This study provides a comprehensive understanding of the differential molecular responses of rapeseed cotyledons to light and dark conditions, which will facilitate further study on the complex mechanism underlying the transition from heterotrophy to autotrophy. PMID:27471506

  1. Endophenotyping reveals differential phenotype-genotype correlations between myopia-associated polymorphisms and eye biometric parameters

    PubMed Central

    Chen, Jian Huan; Chen, Haoyu; Huang, Shulan; Lin, Jianwei; Zheng, Yuqian; Xie, Mingliang; Lin, Wenjie; Pang, Chi Pui

    2012-01-01

    Purpose To investigate the association with ocular biometric parameters in myopia-associated single nucleotide polymorphisms (SNPs) of the gap junction protein delta 2 (GJD2), insulin-like growth factor-1 (IGF1) and hepatocyte growth factor (HGF) genes in two geographically different Chinese cohorts. Methods In 814 unrelated Han Chinese individuals aged above 50 years including 362 inland residents and 432 island dwellers, comprehensive ophthalmic examinations were performed. Three SNPs, including GJD2 rs634990, IGF1 rs6214, and HGF rs3735520, were genotyped. Genetic association with ocular biometric parameters was analyzed in individual cohorts, using linear regression controlled for sex and age. Common associations shared by the two cohorts were revealed by meta-analysis. Results Meta-analysis showed that GJD2 rs634990 alone was not associated with any biometric parameters (adjusted p>0.645). The T allele of IGF1 rs6214 was specifically associated with thicker lens (β±SE=0.055±0.022, adjusted p=0.034). The A allele of HGF rs3735520 was associated with longer vitreous chamber depth (β±SE=0.143±0.060, adjusted p=0.050). Significant interaction between HGF rs3735520 and GJD2 rs634990 was found in association with axial length and vitreous chamber depth (adjusted p=0.003 and 0.033, respectively), and possibly with spherical error (adjusted p=0.056). Conclusions Our endophenotyping analysis showed differential association between selected myopia-associated genes and ocular biometric parameters in our Chinese cohorts, which may underline substantial but diversified effects of these genes and their interaction on the development of eye structure and etiology of myopia. PMID:22509107

  2. Molecular cloning and functional characterization of a mouse gene upregulated by lipopolysaccharide treatment reveals alternative splicing

    SciTech Connect

    Du, Kejun; Chen, Yaoming; Dai, Zongming; Bi, Yuan; Cai, Tongjian; Hou, Lichao; Chai, Yubo; Song, Qinghe; Chen, Sumin; Luo, Wenjing; Chen, Jingyuan

    2010-01-01

    Treatment of mouse cells with lipopolysaccharide (LPS) potently initiates an inflammatory response, but the underlying mechanisms are unclear. We therefore sought to characterize cDNA sequences of a new mouse LPS-responsive gene, and to evaluate the effects of MLrg. Full-length cDNAs were obtained from LPS-treated NIH3T3 cells. We report that the MLrg gene produces two alternative splice products (GenBank Accession Nos. (DQ316984) and (DQ320011)), respectively, encoding MLrgW and MLrgS polypeptides. Both proteins contain zinc finger and leucine zipper domains and are thus potential regulators of transcription. Expression of MLrgW and MLrgS were robustly upregulated following LPS treatment, and the proteins were localized predominantly in the nuclear membrane and cytoplasm. In stable transfectants over-expressing MLrgW the proportion of cells in G1 phase was significantly reduced, while in cells over-expressing MLrgS the proportion of cells in G2 was significantly increased; both proteins are thus potential regulators of cell cycle progression. Upregulation of MLrgW and MLrgS may be an important component of the LPS inflammatory pathway and of the host response to infection with GNB.

  3. 'Living stones' reveal alternative petal identity programs within the core eudicots.

    PubMed

    Brockington, Samuel F; Rudall, Paula J; Frohlich, Michael W; Oppenheimer, David G; Soltis, Pamela S; Soltis, Douglas E

    2012-01-01

    Petals, defined as the showy laminar floral organs in the second floral whorl, have been shown to be under similar genetic control in distantly related core eudicot model organisms. On the basis of these findings, it is commonly assumed that the petal identity program regulated by B-class MADS-box gene homologs is invariant across the core eudicot clade. However, the core eudicots, which comprise >70% of angiosperm species, exhibit numerous instances of petal and sepal loss, transference of petal function between floral whorls, and recurrent petal evolution. In the face of these complex patterns of perianth evolution, the concept of a core eudicot petal identity program has not been tested. We therefore examined the petal identity program in the Caryophyllales, a core eudicot clade in which perianth differentiation into sepals and petals has evolved multiple times. Specifically, we analyzed the expression patterns of B- and C-class MADS-box homologs for evidence of a conserved petal identity program between sepal-derived and stamen-derived petaloid organs in the 'living stone' family Aizoaceae. We found that neither sepal-derived nor stamen-derived petaloid organs exhibit gene expression patterns consistent with the core eudicot petal identity program. B-class gene homologs are not expressed during the development of sepal-derived petals and are not implicated in petal identity in stamen-derived petals, as their transient expression coincides with early expression of the C-class homolog. We therefore provide evidence for petal development that is independent of B-class genes and suggest that different genetic control of petal identity has evolved within this lineage of core eudicots. These findings call for a more comprehensive understanding of perianth variation and its genetic causes within the core eudicots--an endeavor that will have broader implications for the interpretation of perianth evolution across angiosperms. PMID:21951031

  4. Functional Cross-Talking between Differentially Expressed and Alternatively Spliced Genes in Human Liver Cancer Cells Treated with Berberine

    PubMed Central

    Sheng, Zhen; Sun, Yi; Zhu, Ruixin; Jiao, Na; Tang, Kailin; Cao, Zhiwei; Ma, Chao

    2015-01-01

    Berberine has been identified with anti-proliferative effects on various cancer cells. Many researchers have been trying to elucidate the anti-cancer mechanisms of berberine based on differentially expressed genes. However, differentially alternative splicing genes induced by berberine might also contribute to its pharmacological actions and have not been reported yet. Moreover, the potential functional cross-talking between the two sets of genes deserves further exploration. In this study, RNA-seq technology was used to detect the differentially expressed genes and differentially alternative spliced genes in BEL-7402 cancer cells induced by berberine. Functional enrichment analysis indicated that these genes were mainly enriched in the p53 and cell cycle signalling pathway. In addition, it was statistically proven that the two sets of genes were locally co-enriched along chromosomes, closely connected to each other based on protein-protein interaction and functionally similar on Gene Ontology tree. These results suggested that the two sets of genes regulated by berberine might be functionally cross-talked and jointly contribute to its cell cycle arresting effect. It has provided new clues for further researches on the pharmacological mechanisms of berberine as well as the other botanical drugs. PMID:26606055

  5. Quantitative Proteomics Reveals a Role for Epigenetic Reprogramming During Human Monocyte Differentiation*

    PubMed Central

    Nicholas, Dequina; Tang, Hui; Zhang, Qiongyi; Rudra, Jai; Xu, Feng; Langridge, William; Zhang, Kangling

    2015-01-01

    The differentiation of monocytes into macrophages and dendritic cells involves mechanisms for activation of the innate immune system in response to inflammatory stimuli, such as pathogen infection and environmental cues. Epigenetic reprogramming is thought to play an important role during monocyte differentiation. Complementary to cell surface markers, the characterization of monocytic cell lineages by mass spectrometry based protein/histone expression profiling opens a new avenue for studying immune cell differentiation. Here, we report the application of mass spectrometry and bioinformatics to identify changes in human monocytes during their differentiation into macrophages and dendritic cells. Our data show that linker histone H1 proteins are significantly down-regulated during monocyte differentiation. Although highly enriched H3K9-methyl/S10-phos/K14-acetyl tri-modification forms of histone H3 were identified in monocytes and macrophages, they were dramatically reduced in dendritic cells. In contrast, histone H4 K16 acetylation was found to be markedly higher in dendritic cells than in monocytes and macrophages. We also found that global hyperacetylation generated by the nonspecific histone deacetylase HDAC inhibitor Apicidin induces monocyte differentiation. Together, our data suggest that specific regulation of inter- and intra-histone modifications including H3 K9 methylation, H3 S10 phosphorylation, H3 K14 acetylation, and H4 K16 acetylation must occur in concert with chromatin remodeling by linker histones for cell cycle progression and differentiation of human myeloid cells into macrophages and dendritic cells. PMID:25316709

  6. Quantitative proteomics reveals a role for epigenetic reprogramming during human monocyte differentiation.

    PubMed

    Nicholas, Dequina; Tang, Hui; Zhang, Qiongyi; Rudra, Jai; Xu, Feng; Langridge, William; Zhang, Kangling

    2015-01-01

    The differentiation of monocytes into macrophages and dendritic cells involves mechanisms for activation of the innate immune system in response to inflammatory stimuli, such as pathogen infection and environmental cues. Epigenetic reprogramming is thought to play an important role during monocyte differentiation. Complementary to cell surface markers, the characterization of monocytic cell lineages by mass spectrometry based protein/histone expression profiling opens a new avenue for studying immune cell differentiation. Here, we report the application of mass spectrometry and bioinformatics to identify changes in human monocytes during their differentiation into macrophages and dendritic cells. Our data show that linker histone H1 proteins are significantly down-regulated during monocyte differentiation. Although highly enriched H3K9-methyl/S10-phos/K14-acetyl tri-modification forms of histone H3 were identified in monocytes and macrophages, they were dramatically reduced in dendritic cells. In contrast, histone H4 K16 acetylation was found to be markedly higher in dendritic cells than in monocytes and macrophages. We also found that global hyperacetylation generated by the nonspecific histone deacetylase HDAC inhibitor Apicidin induces monocyte differentiation. Together, our data suggest that specific regulation of inter- and intra-histone modifications including H3 K9 methylation, H3 S10 phosphorylation, H3 K14 acetylation, and H4 K16 acetylation must occur in concert with chromatin remodeling by linker histones for cell cycle progression and differentiation of human myeloid cells into macrophages and dendritic cells. PMID:25316709

  7. Alternative to the Kohn-Sham equations: The Pauli potential differential equation

    NASA Astrophysics Data System (ADS)

    Levämäki, H.; Nagy, Á.; Kokko, K.; Vitos, L.

    2015-12-01

    A recently developed theoretical framework of performing self-consistent orbital-free (OF) density functional theory (DFT) calculations at Kohn-Sham DFT level accuracy is tested in practice. The framework is valid for spherically symmetric systems. Numerical results for the Beryllium atom are presented and compared to accurate Kohn-Sham data. These calculations make use of a differential equation that we have developed for the so called Pauli potential, a key quantity in OF-DFT. The Pauli potential differential equation and the OF Euler equation form a system of two coupled differential equations, which have to be solved simultaneously within the DFT self-consistent loop.

  8. Geologically Controlled Isotope-Time Patterns Reveal Early Differentiation and Crust Formation Processes

    NASA Astrophysics Data System (ADS)

    Bennett, V. C.; Nutman, A. P.

    2014-12-01

    The mechanisms of continental crust production and evolution in the early Earth remain controversial, as are questions of the relative roles of early differentiation versus subsequent tectonic procssing in creating Earth's chemical signatures. Here we present geologic observations integrated with whole rock major, trace element and Sm-Nd isotopic signatures and combined with U-Pb and Lu-Hf isotopic compositions of zircon populations from the same rocks, from the most extensive early rock record comprising the 3.9 Ga to 3.6 Ga terranes of southwest Greenland. These data reveal repeated patterns of formation of juvenile TTG crust and associated mafic and ultramafic rocks in convergent margin settings followed by formation of more evolved granites [1]. Our new zircon Lu-Hf data from rare 3.6-3.7 Ga tonalites within the Itsaq Gneiss Complex, obtained from single component, non-migmatitic gneisses with simple zircon populations, limited within sample Hf isotopic variability and accurate U-Pb ages, now document extraction of juvenile tonalites from a near chondritic mantle source between 3.9 Ga and 3.6 Ga. The more evolved, granitic rocks in each area show slightly negative initial ɛHf in accord with crustal reworking of the older (3.8-3.9 Ga) gniesses. There is no evidence for Hadean material in the sources of the granitoids. The Hf isotope-time patterns are consistent with juvenile crust production from a mantle source that experienced only modest amounts of prior crustal extraction. They are distinct from those predicted by reprocessing of an enriched Hadean mafic crust, as has been proposed for this region [2] and for the source of the Hadean Jack Hills zircons [3]. The well-documented, time decreasing, positive 142Nd anomalies [e.g., 4] from these rocks are further evidence of crustal derivation from a convecting mantle source, rather than reworking of an enriched mafic lithosphere. The 143Nd isotopic -time patterns are more complex, reflecting the interplay

  9. Differential gene expression and alternative splicing between diploid and tetraploid watermelon lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Synthetic tetraploid plants have been used for production of seedless triploid watermelon lines being pollinated with diploid plants. When compared to their diploid or triploid counterparts, the tetraploid exhibit wide phenotypic differences. Though many factors, including alternative splicing (AS),...

  10. Characterization of Differentiated SH-SY5Y as Neuronal Screening Model Reveals Increased Oxidative Vulnerability

    PubMed Central

    Forster, J. I.; Köglsberger, S.; Trefois, C.; Boyd, O.; Baumuratov, A. S.; Buck, L.; Balling, R.; Antony, P. M. A.

    2016-01-01

    The immortalized and proliferative cell line SH-SY5Y is one of the most commonly used cell lines in neuroscience and neuroblastoma research. However, undifferentiated SH-SY5Y cells share few properties with mature neurons. In this study, we present an optimized neuronal differentiation protocol for SH-SY5Y that requires only two work steps and 6 days. After differentiation, the cells present increased levels of ATP and plasma membrane activity but reduced expression of energetic stress response genes. Differentiation results in reduced mitochondrial membrane potential and decreased robustness toward perturbations with 6-hydroxydopamine. We are convinced that the presented differentiation method will leverage genetic and chemical high-throughput screening projects targeting pathways that are involved in the selective vulnerability of neurons with high energetic stress levels. PMID:26738520

  11. Planetesimal differentiation revealed by the Hf-W systematics of ureilites

    NASA Astrophysics Data System (ADS)

    Budde, Gerrit; Kruijer, Thomas S.; Fischer-Gödde, Mario; Irving, Anthony J.; Kleine, Thorsten

    2015-11-01

    Determining the timescales of the accretion and chemical differentiation of meteorite parent bodies provides some of the most direct constraints on the formation of planetesimals and the earliest stages of planet formation. We present high-precision Hf-W isotope data for a comprehensive set of ureilites, ultramafic mantle restites derived from a partially melted and incompletely differentiated asteroid. All samples are characterized by strong 182W deficits, indicating that silicate melt extraction on the ureilite parent body at 3.3 ± 0.7 Ma after CAI formation postdated core formation in iron meteorite parent bodies by ∼2-3 Ma. Thermal modeling of planetesimal heating by 26Al-decay combined with the new Hf-W data indicates that the ureilite parent body accreted at ∼1.6 Ma after CAI formation and, therefore, more than ∼1 Ma later than iron meteorite parent bodies, but more than ∼0.5 Ma earlier that most chondrite parent bodies. Due to its relatively 'late' accretion, the ureilite parent body contained too little 26Al to cause complete melting and, therefore, would have probably remained incompletely differentiated even without exhaustion of 26Al by silicate melt segregation. Our results show that both in terms of degree of differentiation and accretion timescale the ureilite parent body is intermediate between fully differentiated and undifferentiated bodies, implying that there is an inverse correlation between extent of melting and metal-silicate separation versus time of accretion and differentiation.

  12. rMATS: robust and flexible detection of differential alternative splicing from replicate RNA-Seq data.

    PubMed

    Shen, Shihao; Park, Juw Won; Lu, Zhi-xiang; Lin, Lan; Henry, Michael D; Wu, Ying Nian; Zhou, Qing; Xing, Yi

    2014-12-23

    Ultra-deep RNA sequencing (RNA-Seq) has become a powerful approach for genome-wide analysis of pre-mRNA alternative splicing. We previously developed multivariate analysis of transcript splicing (MATS), a statistical method for detecting differential alternative splicing between two RNA-Seq samples. Here we describe a new statistical model and computer program, replicate MATS (rMATS), designed for detection of differential alternative splicing from replicate RNA-Seq data. rMATS uses a hierarchical model to simultaneously account for sampling uncertainty in individual replicates and variability among replicates. In addition to the analysis of unpaired replicates, rMATS also includes a model specifically designed for paired replicates between sample groups. The hypothesis-testing framework of rMATS is flexible and can assess the statistical significance over any user-defined magnitude of splicing change. The performance of rMATS is evaluated by the analysis of simulated and real RNA-Seq data. rMATS outperformed two existing methods for replicate RNA-Seq data in all simulation settings, and RT-PCR yielded a high validation rate (94%) in an RNA-Seq dataset of prostate cancer cell lines. Our data also provide guiding principles for designing RNA-Seq studies of alternative splicing. We demonstrate that it is essential to incorporate biological replicates in the study design. Of note, pooling RNAs or merging RNA-Seq data from multiple replicates is not an effective approach to account for variability, and the result is particularly sensitive to outliers. The rMATS source code is freely available at rnaseq-mats.sourceforge.net/. As the popularity of RNA-Seq continues to grow, we expect rMATS will be useful for studies of alternative splicing in diverse RNA-Seq projects. PMID:25480548

  13. Boolean Modeling Reveals the Necessity of Transcriptional Regulation for Bistability in PC12 Cell Differentiation

    PubMed Central

    Offermann, Barbara; Knauer, Steffen; Singh, Amit; Fernández-Cachón, María L.; Klose, Martin; Kowar, Silke; Busch, Hauke; Boerries, Melanie

    2016-01-01

    The nerve growth factor NGF has been shown to cause cell fate decisions toward either differentiation or proliferation depending on the relative activity of downstream pERK, pAKT, or pJNK signaling. However, how these protein signals are translated into and fed back from transcriptional activity to complete cellular differentiation over a time span of hours to days is still an open question. Comparing the time-resolved transcriptome response of NGF- or EGF-stimulated PC12 cells over 24 h in combination with protein and phenotype data we inferred a dynamic Boolean model capturing the temporal sequence of protein signaling, transcriptional response and subsequent autocrine feedback. Network topology was optimized by fitting the model to time-resolved transcriptome data under MEK, PI3K, or JNK inhibition. The integrated model confirmed the parallel use of MAPK/ERK, PI3K/AKT, and JNK/JUN for PC12 cell differentiation. Redundancy of cell signaling is demonstrated from the inhibition of the different MAPK pathways. As suggested in silico and confirmed in vitro, differentiation was substantially suppressed under JNK inhibition, yet delayed only under MEK/ERK inhibition. Most importantly, we found that positive transcriptional feedback induces bistability in the cell fate switch. De novo gene expression was necessary to activate autocrine feedback that caused Urokinase-Type Plasminogen Activator (uPA) Receptor signaling to perpetuate the MAPK activity, finally resulting in the expression of late, differentiation related genes. Thus, the cellular decision toward differentiation depends on the establishment of a transcriptome-induced positive feedback between protein signaling and gene expression thereby constituting a robust control between proliferation and differentiation. PMID:27148350

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

    PubMed Central

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

    2016-01-01

    Stress adaptation plays a pivotal role in biological processes and requires tight regulation of gene expression. In this study, we explored the effect of cellular stress on mRNA polyadenylation and investigated the implications of regulated polyadenylation site usage on mammalian gene expression. High-confidence polyadenylation site mapping combined with global pre-mRNA and mRNA expression profiling revealed that stress induces an accumulation of genes with differentially expressed polyadenylated mRNA isoforms in human cells. Specifically, stress provokes a global trend in polyadenylation site usage toward decreased utilization of promoter-proximal poly(A) sites in introns or ORFs and increased utilization of promoter-distal polyadenylation sites in intergenic regions. This extensively affects gene expression beyond regulating mRNA abundance by changing mRNA length and by altering the configuration of open reading frames. Our study highlights the impact of post-transcriptional mechanisms on stress-dependent gene regulation and reveals the differential expression of alternatively polyadenylated transcripts as a common stress-induced mechanism in mammalian cells. PMID:27407180

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

    PubMed

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

    2016-09-01

    Stress adaptation plays a pivotal role in biological processes and requires tight regulation of gene expression. In this study, we explored the effect of cellular stress on mRNA polyadenylation and investigated the implications of regulated polyadenylation site usage on mammalian gene expression. High-confidence polyadenylation site mapping combined with global pre-mRNA and mRNA expression profiling revealed that stress induces an accumulation of genes with differentially expressed polyadenylated mRNA isoforms in human cells. Specifically, stress provokes a global trend in polyadenylation site usage toward decreased utilization of promoter-proximal poly(A) sites in introns or ORFs and increased utilization of promoter-distal polyadenylation sites in intergenic regions. This extensively affects gene expression beyond regulating mRNA abundance by changing mRNA length and by altering the configuration of open reading frames. Our study highlights the impact of post-transcriptional mechanisms on stress-dependent gene regulation and reveals the differential expression of alternatively polyadenylated transcripts as a common stress-induced mechanism in mammalian cells. PMID:27407180

  16. Whole-Genome Analysis Revealed the Positively Selected Genes during the Differentiation of indica and Temperate japonica Rice

    PubMed Central

    Sun, Xinli; Jia, Qi; Guo, Yuchun; Zheng, Xiujuan; Liang, Kangjing

    2015-01-01

    To investigate the selective pressures acting on the protein-coding genes during the differentiation of indica and japonica, all of the possible orthologous genes between the Nipponbare and 93–11 genomes were identified and compared with each other. Among these genes, 8,530 pairs had identical sequences, and 27,384 pairs shared more than 90% sequence identity. Only 2,678 pairs of genes displaying a Ka/Ks ratio significantly greater than one were revealed, and most of these genes contained only nonsynonymous sites. The genes without synonymous site were further analyzed with the SNP data of 1529 O. sativa and O. rufipogon accessions, and 1068 genes were identified to be under positive selection during the differentiation of indica and temperate japonica. The positively selected genes (PSGs) are unevenly distributed on 12 chromosomes, and the proteins encoded by the PSGs are dominant with binding, transferase and hydrolase activities, and especially enriched in the plant responses to stimuli, biological regulations, and transport processes. Meanwhile, the most PSGs of the known function and/or expression were involved in the regulation of biotic/abiotic stresses. The evidence of pervasive positive selection suggested that many factors drove the differentiation of indica and japonica, which has already started in wild rice but is much lower than in cultivated rice. Lower differentiation and less PSGs revealed between the Or-It and Or-IIIt wild rice groups implied that artificial selection provides greater contribution on the differentiation than natural selection. In addition, the phylogenetic tree constructed with positively selected sites showed that the japonica varieties exhibited more diversity than indica on differentiation, and Or-III of O. rufipogon exhibited more than Or-I. PMID:25774680

  17. Aptitude Testing: A Critical Examination of the Differential Aptitude Tests, Alternative Batteries, and Problems in Prediction.

    ERIC Educational Resources Information Center

    Toronto Board of Education (Ontario). Research Dept.

    In addition to a review of the Differential Aptitude Tests (DAT), a number of other aptitude tests are examined. They are: (1) Flanagan Aptitude Classification Tests, (2) Holzinger-Crowder Uni-Factor Tests, (3) Employee Aptitude Survey, (4) Revised Minnesota Paper Form Board Test, (5) Minnesota Clerical Test, and (6) Turse Clerical Aptitudes Test.…

  18. Implications to Postsecondary Faculty of Alternative Calculation Methods of Gender-Based Wage Differentials.

    ERIC Educational Resources Information Center

    Hagedorn, Linda Serra

    1998-01-01

    A study explored two distinct methods of calculating a precise measure of gender-based wage differentials among college faculty. The first estimation considered wage differences using a formula based on human capital; the second included compensation for past discriminatory practices. Both measures were used to predict three specific aspects of…

  19. Cost Differentials and the Treatment of Equipment Assets: An Analysis of Alternatives.

    ERIC Educational Resources Information Center

    Frohreich, Lloyd E.

    This paper is a discussion of alternative state approaches to aiding and costing capital outlay programs, particularly equipment purchases for vocational programs. Equipment costs for vocational programs tend to be a larger proportion of the total costs than in other programs. The paper includes a discussion of such topics as the magnitude of…

  20. Modified Multiple-Choice Items for Alternate Assessments: Reliability, Difficulty, and Differential Boost

    ERIC Educational Resources Information Center

    Kettler, Ryan J.; Rodriguez, Michael C.; Bolt, Daniel M.; Elliott, Stephen N.; Beddow, Peter A.; Kurz, Alexander

    2011-01-01

    Federal policy on alternate assessment based on modified academic achievement standards (AA-MAS) inspired this research. Specifically, an experimental study was conducted to determine whether tests composed of modified items would have the same level of reliability as tests composed of original items, and whether these modified items helped reduce…

  1. Computational Modeling Reveals that a Combination of Chemotaxis and Differential Adhesion Leads to Robust Cell Sorting during Tissue Patterning

    PubMed Central

    Tan, Rui Zhen; Chiam, Keng-Hwee

    2014-01-01

    Robust tissue patterning is crucial to many processes during development. The "French Flag" model of patterning, whereby naïve cells in a gradient of diffusible morphogen signal adopt different fates due to exposure to different amounts of morphogen concentration, has been the most widely proposed model for tissue patterning. However, recently, using time-lapse experiments, cell sorting has been found to be an alternative model for tissue patterning in the zebrafish neural tube. But it remains unclear what the sorting mechanism is. In this article, we used computational modeling to show that two mechanisms, chemotaxis and differential adhesion, are needed for robust cell sorting. We assessed the performance of each of the two mechanisms by quantifying the fraction of correct sorting, the fraction of stable clusters formed after correct sorting, the time needed to achieve correct sorting, and the size variations of the cells having different fates. We found that chemotaxis and differential adhesion confer different advantages to the sorting process. Chemotaxis leads to high fraction of correct sorting as individual cells will either migrate towards or away from the source depending on its cell type. However after the cells have sorted correctly, there is no interaction among cells of the same type to stabilize the sorted boundaries, leading to cell clusters that are unstable. On the other hand, differential adhesion results in low fraction of correct clusters that are more stable. In the absence of morphogen gradient noise, a combination of both chemotaxis and differential adhesion yields cell sorting that is both accurate and robust. However, in the presence of gradient noise, the simple combination of chemotaxis and differential adhesion is insufficient for cell sorting; instead, chemotaxis coupled with delayed differential adhesion is required to yield optimal sorting. PMID:25302949

  2. 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

  3. Structure of the voltage-gated K⁺ channel Eag1 reveals an alternative voltage sensing mechanism.

    PubMed

    Whicher, Jonathan R; MacKinnon, Roderick

    2016-08-12

    Voltage-gated potassium (K(v)) channels are gated by the movement of the transmembrane voltage sensor, which is coupled, through the helical S4-S5 linker, to the potassium pore. We determined the single-particle cryo-electron microscopy structure of mammalian K(v)10.1, or Eag1, bound to the channel inhibitor calmodulin, at 3.78 angstrom resolution. Unlike previous K(v) structures, the S4-S5 linker of Eag1 is a five-residue loop and the transmembrane segments are not domain swapped, which suggest an alternative mechanism of voltage-dependent gating. Additionally, the structure and position of the S4-S5 linker allow calmodulin to bind to the intracellular domains and to close the potassium pore, independent of voltage-sensor position. The structure reveals an alternative gating mechanism for K(v) channels and provides a template to further understand the gating properties of Eag1 and related channels. PMID:27516594

  4. Ehrlichia chaffeensis Transcriptome in Mammalian and Arthropod Hosts Reveals Differential Gene Expression and Post Transcriptional Regulation

    PubMed Central

    Kuriakose, Jeeba A.; Miyashiro, Simone; Luo, Tian; Zhu, Bing; McBride, Jere W.

    2011-01-01

    Background Human monocytotropic ehrlichiosis is an emerging life-threatening zoonosis caused by obligately intracellular bacterium, Ehrlichia chaffeensis. E. chaffeensis is transmitted by the lone star tick, Amblyomma americanum, and replicates in mononuclear phagocytes in mammalian hosts. Differences in the E. chaffeensis transcriptome in mammalian and arthropod hosts are unknown. Thus, we determined host-specific E. chaffeensis gene expression in human monocyte (THP-1) and in Amblyomma and Ixodes tick cell lines (AAE2 and ISE6) using a whole genome microarray. Methodology/Principal Findings The majority (∼80%) of E. chaffeensis genes were expressed during infection in human and tick cells. There were few differences observed in E. chaffeensis gene expression between the vector Amblyomma and non-vector Ixodes tick cells, but extensive host-specific and differential gene expression profiles were detected between human and tick cells, including higher transcriptional activity in tick cells and identification of gene subsets that were differentially expressed in the two hosts. Differentially and host-specifically expressed ehrlichial genes encoded major immunoreactive tandem repeat proteins (TRP), the outer membrane protein (OMP-1) family, and hypothetical proteins that were 30–80 amino acids in length. Consistent with previous observations, high expression of p28 and OMP-1B genes was detected in human and tick cells, respectively. Notably, E. chaffeensis genes encoding TRP32 and TRP47 were highly upregulated in the human monocytes and expressed as proteins; however, although TRP transcripts were expressed in tick cells, the proteins were not detected in whole cell lysates demonstrating that TRP expression was post transcriptionally regulated. Conclusions/Significance Ehrlichia gene expression is highly active in tick cells, and differential gene expression among a wide variety of host-pathogen associated genes occurs. Furthermore, we demonstrate that genes

  5. Substratum-induced differentiation of human pluripotent stem cells reveals the coactivator YAP is a potent regulator of neuronal specification

    PubMed Central

    Musah, Samira; Wrighton, Paul J.; Zaltsman, Yefim; Zhong, Xiaofen; Zorn, Stefan; Parlato, Matthew B.; Hsiao, Cheston; Palecek, Sean P.; Chang, Qiang; Murphy, William L.; Kiessling, Laura L.

    2014-01-01

    Physical stimuli can act in either a synergistic or antagonistic manner to regulate cell fate decisions, but it is less clear whether insoluble signals alone can direct human pluripotent stem (hPS) cell differentiation into specialized cell types. We previously reported that stiff materials promote nuclear localization of the Yes-associated protein (YAP) transcriptional coactivator and support long-term self-renewal of hPS cells. Here, we show that even in the presence of soluble pluripotency factors, compliant substrata inhibit the nuclear localization of YAP and promote highly efficient differentiation of hPS cells into postmitotic neurons. In the absence of neurogenic factors, the effective substrata produce neurons rapidly (2 wk) and more efficiently (>75%) than conventional differentiation methods. The neurons derived from substrate induction express mature markers and possess action potentials. The hPS differentiation observed on compliant surfaces could be recapitulated on stiff surfaces by adding small-molecule inhibitors of F-actin polymerization or by depleting YAP. These studies reveal that the matrix alone can mediate differentiation of hPS cells into a mature cell type, independent of soluble inductive factors. That mechanical cues can override soluble signals suggests that their contributions to early tissue development and lineage commitment are profound. PMID:25201954

  6. Single-Cell Analyses of ESCs Reveal Alternative Pluripotent Cell States and Molecular Mechanisms that Control Self-Renewal

    PubMed Central

    Papatsenko, Dmitri; Darr, Henia; Kulakovskiy, Ivan V.; Waghray, Avinash; Makeev, Vsevolod J.; MacArthur, Ben D.; Lemischka, Ihor R.

    2015-01-01

    Summary Analyses of gene expression in single mouse embryonic stem cells (mESCs) cultured in serum and LIF revealed the presence of two distinct cell subpopulations with individual gene expression signatures. Comparisons with published data revealed that cells in the first subpopulation are phenotypically similar to cells isolated from the inner cell mass (ICM). In contrast, cells in the second subpopulation appear to be more mature. Pluripotency Gene Regulatory Network (PGRN) reconstruction based on single-cell data and published data suggested antagonistic roles for Oct4 and Nanog in the maintenance of pluripotency states. Integrated analyses of published genomic binding (ChIP) data strongly supported this observation. Certain target genes alternatively regulated by OCT4 and NANOG, such as Sall4 and Zscan10, feed back into the top hierarchical regulator Oct4. Analyses of such incoherent feedforward loops with feedback (iFFL-FB) suggest a dynamic model for the maintenance of mESC pluripotency and self-renewal. PMID:26267829

  7. An RNAi screen reveals intestinal regulators of branching morphogenesis, differentiation, and stem cell proliferation in planarians

    PubMed Central

    Forsthoefel, David J.; James, Noelle P.; Escobar, David J.; Stary, Joel M.; Vieira, Ana P.; Waters, Forrest A.; Newmark, Phillip A.

    2012-01-01

    SUMMARY Planarians grow and regenerate organs by coordinating proliferation and differentiation of pluripotent stem cells with remodeling of post-mitotic tissues. Understanding how these processes are orchestrated requires characterizing cell type-specific gene expression programs and their regulation during regeneration and homeostasis. To this end, we analyzed the expression profile of planarian intestinal phagocytes, cells responsible for digestion and nutrient storage/distribution. Utilizing RNA interference, we identified cytoskeletal regulators required for intestinal branching morphogenesis, and a modulator of bioactive sphingolipid metabolism, ceramide synthase, required for the production of functional phagocytes. Additionally, we found that a gut-enriched homeobox transcription factor, nkx-2.2, is required for somatic stem cell proliferation, suggesting a niche-like role for phagocytes. Identification of evolutionarily conserved regulators of intestinal branching, differentiation, and stem cell dynamics demonstrates the utility of the planarian digestive system as a model for elucidating the mechanisms controlling post-embryonic organogenesis. PMID:23079596

  8. RNAi Screen Reveals Potentially Novel Roles of Cytokines in Myoblast Differentiation

    PubMed Central

    Ge, Yejing; Waldemer, Rachel J.; Nalluri, Ramakrishna; Nuzzi, Paul D.; Chen, Jie

    2013-01-01

    Cytokines are cell-secreted signaling molecules that modulate various cellular functions, with the best-characterized roles in immune responses. The expression of numerous cytokines in skeletal muscle tissues and muscle cells has been reported, but their function in skeletal myogenesis, the formation of skeletal muscle, has been largely underexplored. To systematically examine the potential roles of cytokines in skeletal myogenesis, we undertook an RNAi screen of 134 mouse cytokine genes for their involvement in the differentiation of C2C12 myoblasts. Our results have uncovered 29 cytokines as strong candidates for novel myogenic regulators, potentially conferring positive and negative regulation at distinct stages of myogenesis. These candidates represent a diverse collection of cytokine families, including interleukins, TNF-related factors, and chemokines. Our findings suggest the fundamental importance of cytokines in the cell-autonomous regulation of myoblast differentiation, and may facilitate future identification of novel therapeutic targets for improving muscle regeneration and growth in health and diseases. PMID:23844157

  9. An alternative approach to estimating rainfall rate by radar using propagation differential phase shift

    NASA Technical Reports Server (NTRS)

    Jameson, A. R.

    1994-01-01

    In this work it is shown that for frequencies from 3 to 13 GHz, the ratio of the specific propagation differential phase shift phi(sub DP) to the rainfall rate can be specified essentially independently of the form of the drop size distribution by a function only of the mass-weighted mean drop size D(sub m). This significantly reduces one source of substantial bias errors common to most other techniques for measuring rain by radar. For frequencies 9 GHz and greater, the coefficient can be well estimated from the ratio of the specific differential attenuation to phi(sub DP), while at nonattenuating frequencies such as 3 GHz, the coefficient can be well estimated using the differential reflectivity. In practice it appears that this approach yields better estimates of the rainfall rate than any other current technique. The best results are most likely at 13.80 GHz, followed by those at 2.80 GHz. An optimum radar system for measuring rain should probably include components at a both frequencies so that when signals at 13.8 GHz are lost because of attenuation, good measurements are still possible at the lower frequency.

  10. Adaptive Epigenetic Differentiation between Upland and Lowland Rice Ecotypes Revealed by Methylation-Sensitive Amplified Polymorphism

    PubMed Central

    Xiong, Jie; Tao, Tao; Zheng, Xiaoguo; Wei, Haibin; Yue, Yunxia; Chen, Liang; Luo, Lijun

    2016-01-01

    The stress-induced epimutations could be inherited over generations and play important roles in plant adaption to stressful environments. Upland rice has been domesticated in water-limited environments for thousands of years and accumulated drought-induced epimutations of DNA methylation, making it epigenetically differentiated from lowland rice. To study the epigenetic differentiation between upland and lowland rice ecotypes on their drought-resistances, the epigenetic variation was investigated in 180 rice landraces under both normal and osmotic conditions via methylation-sensitive amplified polymorphism (MSAP) technique. Great alterations (52.9~54.3% of total individual-locus combinations) of DNA methylation are recorded when rice encountering the osmotic stress. Although the general level of epigenetic differentiation was very low, considerable level of ΦST (0.134~0.187) was detected on the highly divergent epiloci (HDE). The HDE detected in normal condition tended to stay at low levels in upland rice, particularly the ones de-methylated in responses to osmotic stress. Three out of four selected HDE genes differentially expressed between upland and lowland rice under normal or stressed conditions. Moreover, once a gene at HDE was up-/down-regulated in responses to the osmotic stress, its expression under the normal condition was higher/lower in upland rice. This result suggested expressions of genes at the HDE in upland rice might be more adaptive to the osmotic stress. The epigenetic divergence and its influence on the gene expression should contribute to the higher drought-resistance in upland rice as it is domesticated in the water-limited environment. PMID:27380174

  11. Novel Polymorphic Microsatellite Markers Reveal Genetic Differentiation between Two Sympatric Types of Galaxea fascicularis

    PubMed Central

    Nakajima, Yuichi; Shinzato, Chuya; Satoh, Noriyuki; Mitarai, Satoshi

    2015-01-01

    The reef-building, scleractinian coral, Galaxea fascicularis, is classified into soft and hard types, based on nematocyst morphology. This character is correlated with the length of the mitochondrial non-coding region (mt-Long: soft colony type, and nematocysts with wide capsules and long shafts; mt-Short: hard colony type, and nematocysts with thin capsules and short shafts). We isolated and characterized novel polymorphic microsatellite markers for G. fascicularis using next-generation sequencing. Based upon the mitochondrial non-coding region, 53 of the 97 colonies collected were mt-Long (mt-L) and 44 were mt-Short (mt-S). Among the 53 mt-L colonies, 27 loci were identified as amplifiable, polymorphic microsatellite loci, devoid of somatic mutations and free of scoring errors. Eleven of those 27 loci were also amplifiable and polymorphic in the 44 mt-S colonies; these 11 are cross-type microsatellite loci. The other 16 loci were considered useful only for mt-L colonies. These 27 loci identified 10 multilocus lineages (MLLs) among the 53 mt-L colonies (NMLL/N = 0.189), and the 11 cross-type loci identified 7 MLLs in 44 mt-S colonies (NMLL/N = 0.159). Significant genetic differentiation between the two types was detected based on the genetic differentiation index (FST = 0.080, P = 0.001). Bayesian clustering also indicated that these two types are genetically isolated. While nuclear microsatellite genotypes also showed genetic differentiation between mitochondrial types, the mechanism of divergence is not yet clear. These markers will be useful to estimate genetic diversity, differentiation, and connectivity among populations, and to understand evolutionary processes, including divergence of types in G. fascicularis. PMID:26147677

  12. Genotypic diversity and differentiation among populations of two benthic freshwater diatoms as revealed by microsatellites.

    PubMed

    Vanormelingen, Pieter; Evans, Katharine M; Mann, David G; Lance, Stacey; Debeer, Ann-Eline; D'Hondt, Sofie; Verstraete, Tine; De Meester, Luc; Vyverman, Wim

    2015-09-01

    Given their large population sizes and presumed high dispersal capacity, protists are expected to exhibit homogeneous population structure over large spatial scales. On the other hand, the fragmented and short-lived nature of the lentic freshwater habitats that many protists inhabit promotes strong population differentiation. We used microsatellites in two benthic freshwater diatoms, Eunotia bilunaris 'robust' and Sellaphora capitata, sampled from within a pond and connected ponds, through isolated ponds from the same region to western Europe to determine the spatial scale at which differentiation appears. Because periods of low genotypic diversity contribute to population differentiation, we also assessed genotypic diversity. While genotypic diversity was very high to maximal in most samples of both species, some had a markedly lower diversity, with up to half (Eunotia) and over 90% (Sellaphora) of the strains having the same multilocus genotype. Population differentiation showed an isolation-by-distance pattern with very low standardized FST values between samples from the same or connected ponds but high values between isolated ponds, even when situated in the same region. Partial rbcL sequences in Eunotia were consistent with this pattern as isolated ponds in the same region could differ widely in haplotype composition. Populations identified by Structure corresponded to the source ponds, confirming that 'pond' is the main factor structuring these populations. We conclude that freshwater benthic diatom populations are highly fragmented on a regional scale, reflecting either less dispersal than is often assumed or reduced establishment success of immigrants, so that dispersal does not translate into gene flow. PMID:26227512

  13. Molecular Mechanisms of Fiber Differential Development between G. barbadense and G. hirsutum Revealed by Genetical Genomics

    PubMed Central

    Chen, Xiangdong; Guo, Wangzhen; Liu, Bingliang; Zhang, Yuanming; Song, Xianliang; Cheng, Yu; Zhang, Lili; Zhang, Tianzhen

    2012-01-01

    Cotton fiber qualities including length, strength and fineness are known to be controlled by genes affecting cell elongation and secondary cell wall (SCW) biosynthesis, but the molecular mechanisms that govern development of fiber traits are largely unknown. Here, we evaluated an interspecific backcrossed population from G. barbadense cv. Hai7124 and G. hirsutum acc. TM-1 for fiber characteristics in four-year environments under field conditions, and detected 12 quantitative trait loci (QTL) and QTL-by-environment interactions by multi-QTL joint analysis. Further analysis of fiber growth and gene expression between TM-1 and Hai7124 showed greater differences at 10 and 25 days post-anthesis (DPA). In this two period important for fiber performances, we integrated genome-wide expression profiling with linkage analysis using the same genetic materials and identified in total 916 expression QTL (eQTL) significantly (P<0.05) affecting the expression of 394 differential genes. Many positional cis-/trans-acting eQTL and eQTL hotspots were detected across the genome. By comparative mapping of eQTL and fiber QTL, a dataset of candidate genes affecting fiber qualities was generated. Real-time quantitative RT-PCR (qRT-PCR) analysis confirmed the major differential genes regulating fiber cell elongation or SCW synthesis. These data collectively support molecular mechanism for G. hirsutum and G. barbadense through differential gene regulation causing difference of fiber qualities. The down-regulated expression of abscisic acid (ABA) and ethylene signaling pathway genes and high-level and long-term expression of positive regulators including auxin and cell wall enzyme genes for fiber cell elongation at the fiber developmental transition stage may account for superior fiber qualities. PMID:22253876

  14. Molecular mechanisms of fiber differential development between G. barbadense and G. hirsutum revealed by genetical genomics.

    PubMed

    Chen, Xiangdong; Guo, Wangzhen; Liu, Bingliang; Zhang, Yuanming; Song, Xianliang; Cheng, Yu; Zhang, Lili; Zhang, Tianzhen

    2012-01-01

    Cotton fiber qualities including length, strength and fineness are known to be controlled by genes affecting cell elongation and secondary cell wall (SCW) biosynthesis, but the molecular mechanisms that govern development of fiber traits are largely unknown. Here, we evaluated an interspecific backcrossed population from G. barbadense cv. Hai7124 and G. hirsutum acc. TM-1 for fiber characteristics in four-year environments under field conditions, and detected 12 quantitative trait loci (QTL) and QTL-by-environment interactions by multi-QTL joint analysis. Further analysis of fiber growth and gene expression between TM-1 and Hai7124 showed greater differences at 10 and 25 days post-anthesis (DPA). In this two period important for fiber performances, we integrated genome-wide expression profiling with linkage analysis using the same genetic materials and identified in total 916 expression QTL (eQTL) significantly (P<0.05) affecting the expression of 394 differential genes. Many positional cis-/trans-acting eQTL and eQTL hotspots were detected across the genome. By comparative mapping of eQTL and fiber QTL, a dataset of candidate genes affecting fiber qualities was generated. Real-time quantitative RT-PCR (qRT-PCR) analysis confirmed the major differential genes regulating fiber cell elongation or SCW synthesis. These data collectively support molecular mechanism for G. hirsutum and G. barbadense through differential gene regulation causing difference of fiber qualities. The down-regulated expression of abscisic acid (ABA) and ethylene signaling pathway genes and high-level and long-term expression of positive regulators including auxin and cell wall enzyme genes for fiber cell elongation at the fiber developmental transition stage may account for superior fiber qualities. PMID:22253876

  15. Targeted deletion of Atg5 reveals differential roles of autophagy in keratin K5-expressing epithelia

    SciTech Connect

    Sukseree, Supawadee; Rossiter, Heidemarie; Mildner, Michael; Pammer, Johannes; Buchberger, Maria; Gruber, Florian; Watanapokasin, Ramida; Tschachler, Erwin; Eckhart, Leopold

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer We generated mice lacking Atg5 and autophagy in keratin K5-positive epithelia. Black-Right-Pointing-Pointer Suppression of autophagy in thymic epithelium was not associated with signs of autoimmunity. Black-Right-Pointing-Pointer Autophagy was required for normal terminal differentiation of preputial gland cells. Black-Right-Pointing-Pointer Autophagy-deficient cells of the preputial glands degraded nuclear DNA prematurely. -- Abstract: Autophagy contributes to the homeostasis of many tissues, yet its role in epithelia is incompletely understood. A recent report proposed that Atg5-dependent autophagy in thymic epithelial cells is essential for their function in the negative selection of self-reactive T-cells and, thus, for the suppression of tissue inflammation. Here we crossed mice carrying floxed alleles of the Atg5 gene with mice expressing the Cre recombinase under the control of the keratin K5 promoter to suppress autophagy in all K5-positive epithelia. The efficiency of autophagy abrogation was confirmed by immunoanalyses of LC3, which was converted to the autophagy-associated LC3-II form in normal but not Atg5-deficient cells, and of p62, which accumulated in Atg5-deficient cells. Mice carrying the epithelium-specific deletion of Atg5 showed normal weight gain, absence of tissue inflammation, and a normal morphology of the thymic epithelium. By contrast, autophagy-deficient epithelial cells of the preputial gland showed aberrant eosinophilic staining in histology and premature degradation of nuclear DNA during terminal differentiation. Taken together, the results of this study suggest that autophagy is dispensable for the suppression of autoimmunity by thymic epithelial cells but essential for normal differentiation of the preputial gland in mice.

  16. Genome-wide view of genetic diversity reveals paths of selection and cultivar differentiation in peach domestication.

    PubMed

    Akagi, Takashi; Hanada, Toshio; Yaegaki, Hideaki; Gradziel, Thomas M; Tao, Ryutaro

    2016-06-01

    Domestication and cultivar differentiation are requisite processes for establishing cultivated crops. These processes inherently involve substantial changes in population structure, including those from artificial selection of key genes. In this study, accessions of peach (Prunus persica) and its wild relatives were analysed genome-wide to identify changes in genetic structures and gene selections associated with their differentiation. Analysis of genome-wide informative single-nucleotide polymorphism loci revealed distinct changes in genetic structures and delineations among domesticated peach and its wild relatives and among peach landraces and modern fruit (F) and modern ornamental (O-A) cultivars. Indications of distinct changes in linkage disequilibrium extension/decay and of strong population bottlenecks or inbreeding were identified. Site frequency spectrum- and extended haplotype homozygosity-based evaluation of genome-wide genetic diversities supported selective sweeps distinguishing the domesticated peach from its wild relatives and each F/O-A cluster from the landrace clusters. The regions with strong selective sweeps harboured promising candidates for genes subjected to selection. Further sequence-based evaluation further defined the candidates and revealed their characteristics. All results suggest opportunities for identifying critical genes associated with each differentiation by analysing genome-wide genetic diversity in currently established populations. This approach obviates the special development of genetic populations, which is particularly difficult for long-lived tree crops. PMID:27085183

  17. Genome-wide view of genetic diversity reveals paths of selection and cultivar differentiation in peach domestication

    PubMed Central

    Akagi, Takashi; Hanada, Toshio; Yaegaki, Hideaki; Gradziel, Thomas M.; Tao, Ryutaro

    2016-01-01

    Domestication and cultivar differentiation are requisite processes for establishing cultivated crops. These processes inherently involve substantial changes in population structure, including those from artificial selection of key genes. In this study, accessions of peach (Prunus persica) and its wild relatives were analysed genome-wide to identify changes in genetic structures and gene selections associated with their differentiation. Analysis of genome-wide informative single-nucleotide polymorphism loci revealed distinct changes in genetic structures and delineations among domesticated peach and its wild relatives and among peach landraces and modern fruit (F) and modern ornamental (O-A) cultivars. Indications of distinct changes in linkage disequilibrium extension/decay and of strong population bottlenecks or inbreeding were identified. Site frequency spectrum- and extended haplotype homozygosity-based evaluation of genome-wide genetic diversities supported selective sweeps distinguishing the domesticated peach from its wild relatives and each F/O-A cluster from the landrace clusters. The regions with strong selective sweeps harboured promising candidates for genes subjected to selection. Further sequence-based evaluation further defined the candidates and revealed their characteristics. All results suggest opportunities for identifying critical genes associated with each differentiation by analysing genome-wide genetic diversity in currently established populations. This approach obviates the special development of genetic populations, which is particularly difficult for long-lived tree crops. PMID:27085183

  18. Alternating Current Electric Fields of Varying Frequencies: Effects on Proliferation and Differentiation of Porcine Neural Progenitor Cells

    PubMed Central

    Lim, Ji-Hey; McCullen, Seth D.; Piedrahita, Jorge A.

    2013-01-01

    Abstract 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. PMID:23961767

  19. Differential reinforcement of alternative behavior increases resistance to extinction: clinical demonstration, animal modeling, and clinical test of one solution.

    PubMed

    Mace, F Charles; McComas, Jennifer J; Mauro, Benjamin C; Progar, Patrick R; Taylor, Bridget; Ervin, Ruth; Zangrillo, Amanda N

    2010-05-01

    Basic research with pigeons on behavioral momentum suggests that differential reinforcement of alternative behavior (DRA) can increase the resistance of target behavior to change. This finding suggests that clinical applications of DRA may inadvertently increase the persistence of target behavior even as it decreases its frequency. We conducted three coordinated experiments to test whether DRA has persistence-strengthening effects on clinically significant target behavior and then tested the effectiveness of a possible solution to this problem in both a nonhuman and clinical study. Experiment 1 compared resistance to extinction following baseline rates of reinforcement versus higher DRA rates of reinforcement in a clinical study. Resistance to extinction was substantially greater following DRA. Experiment 2 tested a rat model of a possible solution to this problem. Training an alternative response in a context without reinforcement of the target response circumvented the persistence-strengthening effects of DRA. Experiment 3 translated the rat model into a novel clinical application of DRA. Training an alternative response with DRA in a separate context resulted in lower resistance to extinction than employing DRA in the context correlated with reinforcement of target behavior. The value of coordinated bidirectional translational research is discussed. PMID:21119850

  20. Differential Reinforcement of Alternative Behavior Increases Resistance to Extinction: Clinical Demonstration, Animal Modeling, and Clinical Test of One Solution

    PubMed Central

    Mace, F. Charles; McComas, Jennifer J; Mauro, Benjamin C; Progar, Patrick R; Taylor, Bridget; Ervin, Ruth; Zangrillo, Amanda N

    2010-01-01

    Basic research with pigeons on behavioral momentum suggests that differential reinforcement of alternative behavior (DRA) can increase the resistance of target behavior to change. This finding suggests that clinical applications of DRA may inadvertently increase the persistence of target behavior even as it decreases its frequency. We conducted three coordinated experiments to test whether DRA has persistence-strengthening effects on clinically significant target behavior and then tested the effectiveness of a possible solution to this problem in both a nonhuman and clinical study. Experiment 1 compared resistance to extinction following baseline rates of reinforcement versus higher DRA rates of reinforcement in a clinical study. Resistance to extinction was substantially greater following DRA. Experiment 2 tested a rat model of a possible solution to this problem. Training an alternative response in a context without reinforcement of the target response circumvented the persistence-strengthening effects of DRA. Experiment 3 translated the rat model into a novel clinical application of DRA. Training an alternative response with DRA in a separate context resulted in lower resistance to extinction than employing DRA in the context correlated with reinforcement of target behavior. The value of coordinated bidirectional translational research is discussed. PMID:21119850

  1. An assessment of alternative soft magnetic materials in rotary variable differential transformers

    SciTech Connect

    Midgley, G.W.; Howe, D.; Mellor, P.H.

    1997-04-01

    Position sensors are a key technology for controlled actuation systems, which are required to meet increasingly exacting dynamic performance specifications. Of the various sensing technologies, variable differential transformers are capable of satisfying stringent performance criteria, in terms of resolution, repeatability, and stability of output, while operating in the harshest of environments. They utilize the variation of mutual inductance which occurs between a primary and two secondary coils as a ferromagnetic core is moved by the object whose position is to be measured. The article is concerned with rotary variable differential transformers, which currently use high permeability magnetic alloys, such as nickel{endash}iron, either solid or laminated. However, since they are being required to operate at increased excitation frequencies, up to 5 kHz, there is interest in the use of powder composite magnetic materials, which, although having a lower permeability, have a higher electrical resistivity, and hence reduced eddy current effects. The potential for such materials is investigated by steady-state ac finite element analysis, and shown to be promising. {copyright} {ital 1997 American Institute of Physics.}

  2. Establishment of Two Mouse Models for CEDNIK Syndrome Reveals the Pivotal Role of SNAP29 in Epidermal Differentiation.

    PubMed

    Schiller, Stina A; Seebode, Christina; Wieser, Georg L; Goebbels, Sandra; Möbius, Wiebke; Horowitz, Mia; Sarig, Ofer; Sprecher, Eli; Emmert, Steffen

    2016-03-01

    Loss-of-function mutations in the synaptosomal-associated protein 29 (SNAP29) gene cause the cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma syndrome. In this study, we created total (Snap29(-/-)) as well as keratinocyte-specific (Snap29(fl/fl)/K14-Cre) Snap29 knockout mice. Both mutant mice exhibited a congenital distinct ichthyotic phenotype resulting in neonatal lethality. Mutant mice revealed acanthosis and hyperkeratosis as well as abnormal keratinocyte differentiation and increased proliferation. In addition, the epidermal barrier was severely impaired. These results indicate an essential role of SNAP29 in epidermal differentiation and barrier formation. Markedly decreased deposition of lamellar body contents in mutant mice epidermis and the observation of malformed lamellar bodies indicate severe impairments in lamellar body function due to the Snap29 knockout. We also found increased microtubule associated protein-1 light chain 3, isoform B-II levels, unchanged p62/SQSTM1 protein amounts, and strong induction of the endoplasmic reticulum stress marker C/EBP homologous protein in mutant mice. This emphasizes a role of SNAP29 in autophagy and endoplasmic reticulum stress. Our murine models serve as powerful tools for investigating keratinocyte differentiation processes and provide insights into the essential contribution of SNAP29 to epidermal differentiation. PMID:26747696

  3. Label-Free Quantitative Mass Spectrometry Reveals a Panel of Differentially Expressed Proteins in Colorectal Cancer

    PubMed Central

    Fan, Nai-Jun; Gao, Jiang-Ling; Liu, Yan; Song, Wei; Zhang, Zhan-Yang; Gao, Chun-Fang

    2015-01-01

    To identify potential biomarkers involved in CRC, a shotgun proteomic method was applied to identify soluble proteins in three CRCs and matched normal mucosal tissues using high-performance liquid chromatography and mass spectrometry. Label-free protein profiling of three CRCs and matched normal mucosal tissues were then conducted to quantify and compare proteins. Results showed that 67 of the 784 identified proteins were linked to CRC (28 upregulated and 39 downregulated). Gene Ontology and DAVID databases were searched to identify the location and function of differential proteins that were related to the biological processes of binding, cell structure, signal transduction, cell adhesion, and so on. Among the differentially expressed proteins, tropomyosin-3 (TPM3), endoplasmic reticulum resident protein 29 (ERp29), 18 kDa cationic antimicrobial protein (CAMP), and heat shock 70 kDa protein 8 (HSPA8) were verified to be upregulated in CRC tissue and seven cell lines through western blot analysis. Furthermore, the upregulation of TPM3, ERp29, CAMP, and HSPA8 was validated in 69 CRCs byimmunohistochemistry (IHC) analysis. Combination of TPM3, ERp29, CAMP, and HSPA8 can identify CRC from matched normal mucosal achieving an accuracy of 73.2% using IHC score. These results suggest that TPM3, ERp29, CAMP, and HSPA8 are great potential IHC diagnostic biomarkers for CRC. PMID:25699276

  4. Genomic Analysis of Differentiation between Soil Types Reveals Candidate Genes for Local Adaptation in Arabidopsis lyrata

    PubMed Central

    Turner, Thomas L.; von Wettberg, Eric J.; Nuzhdin, Sergey V.

    2008-01-01

    Serpentine soil, which is naturally high in heavy metal content and has low calcium to magnesium ratios, comprises a difficult environment for most plants. An impressive number of species are endemic to serpentine, and a wide range of non-endemic plant taxa have been shown to be locally adapted to these soils. Locating genomic polymorphisms which are differentiated between serpentine and non-serpentine populations would provide candidate loci for serpentine adaptation. We have used the Arabidopsis thaliana tiling array, which has 2.85 million probes throughout the genome, to measure genetic differentiation between populations of Arabidopsis lyrata growing on granitic soils and those growing on serpentinic soils. The significant overrepresentation of genes involved in ion transport and other functions provides a starting point for investigating the molecular basis of adaptation to soil ion content, water retention, and other ecologically and economically important variables. One gene in particular, calcium-exchanger 7, appears to be an excellent candidate gene for adaptation to low Ca∶Mg ratio in A. lyrata. PMID:18784841

  5. Transcriptional profiles reveal a stepwise developmental program of memory CD8(+) T cell differentiation.

    PubMed

    Roychoudhuri, Rahul; Lefebvre, Francois; Honda, Mitsuo; Pan, Li; Ji, Yun; Klebanoff, Christopher A; Nichols, Carmen N; Fourati, Slim; Hegazy, Ahmed N; Goulet, Jean-Philippe; Gattinoni, Luca; Nabel, Gary J; Gilliet, Michel; Cameron, Mark; Restifo, Nicholas P; Sékaly, Rafick P; Flatz, Lukas

    2015-02-11

    The generation of CD8(+) T-cell memory is a major aim of vaccination. While distinct subsets of CD8(+) T-cells are generated following immunization that differ in their ability to confer long-term immunity against infection, the transcriptional profiles of these subsets within endogenous vaccine-induced CD8(+) T cell responses have not been resolved. Here, we measure global transcriptional profiles of endogenous effector (TEFF), effector memory (TEM) and central memory (TCM) CD8(+) T-cells arising from immunization with three distinct prime-boost vaccine regimens. While a proportion of transcripts were uniquely regulated within distinct CD8(+) T cell populations, we observed progressive up- or down-regulation in the expression of a majority of differentially expressed transcripts when subsets were compared in the order TN>TCM>TEM>TEFF. Strikingly, when we compared global differences in gene expression between TN, TCM, TEM and TEFF cells with known transcriptional changes that result when CD8(+) T cells repetitively encounter antigen, our analysis overwhelmingly favored a model whereby cumulative antigen stimulation drives differentiation specifically from TN>TCM>TEM>TEFF and this was common to all vaccines tested. These findings provide insight into the molecular basis of immunological memory and identify potential biomarkers for characterization of vaccine-induced responses and prediction of vaccine efficacy. PMID:25446821

  6. Genetic diversity and differentiation of the Ryukyu endemic frog Babina holsti as revealed by mitochondrial DNA.

    PubMed

    Tominaga, Atsushi; Matsui, Masafumi; Nakata, Katsushi

    2014-02-01

    We surveyed the genetic diversity and genetic differentiation of an endangered frog, Babina holsti, endemic to Okinawajima and Tokashikijima Islands of the Ryukyus, to elucidate its divergence history and obtain basic data for its conservation. Genetic differentiation between the two island lineages is moderate (3.1% p-distance in the cyt b gene). This result suggests that the two island lineages have been isolated between the late Pliocene and the middle Pleistocene and have never migrated between the current northern part of Okinawajima and Tokashikijima Islands, which were once connected in the late Pleistocene glacial age. On Okinawajima Island, the southernmost sample was constituted by a unique haplotype, without considerable genetic distance from haplotypes detected from northern samples. This unique haplotype composition in the southernmost sample would have resulted from the restricted gene flow between the southernmost population and the other populations in Okinawajima Island. Furthermore, the absence of genetic diversity within the southernmost sample indicates that this population has recently experienced population size reduction, possibly by predation pressure from an introduced mongoose, which is more abundant in the southern part than in the northern part of the island. Lower genetic diversity in the Tokashikijima sample implies a small effective population size for mitochondrial DNA (mtDNA) in B. holsti on the island. Immediate conservation measures should be taken for the populations from the southernmost range in Okinawajima and Tokashikijima. PMID:24521314

  7. Dynamic BRG1 Recruitment during T Helper Differentiation and Activation Reveals Distal Regulatory Elements▿§

    PubMed Central

    De, Supriyo; Wurster, Andrea L.; Precht, Patricia; Wood, William H.; Becker, Kevin G.; Pazin, Michael J.

    2011-01-01

    T helper cell differentiation and activation require specific transcriptional programs accompanied by changes in chromatin structure. However, little is known about the chromatin remodeling enzymes responsible. We performed genome-wide analysis to determine the general principles of BRG1 binding, followed by analysis of specific genes to determine whether these general rules were typical of key T cell genes. We found that binding of the remodeling protein BRG1 was programmed by both lineage and activation signals. BRG1 binding positively correlated with gene activity at protein-coding and microRNA (miRNA) genes. BRG1 binding was found at promoters and distal regions, including both novel and previously validated distal regulatory elements. Distal BRG1 binding correlated with expression, and novel distal sites in the Gata3 locus possessed enhancer-like activity, suggesting a general role for BRG1 in long-distance gene regulation. BRG1 recruitment to distal sites in Gata3 was impaired in cells lacking STAT6, a transcription factor that regulates lineage-specific genes. Together, these findings suggest that BRG1 interprets both differentiation and activation signals and plays a causal role in gene regulation, chromatin structure, and cell fate. Our findings suggest that BRG1 binding is a useful marker for identifying active cis-regulatory regions in protein-coding and miRNA genes. PMID:21262765

  8. Metaproteomics reveals differential modes of metabolic coupling among ubiquitous oxygen minimum zone microbes

    SciTech Connect

    Hawley, Alyse K.; Brewer, Heather M.; Norbeck, Angela D.; Pasa-Tolic, Ljiljana; Hallam, Steven J.

    2014-08-05

    Oxygen minimum zones (OMZs) are intrinsic water column features arising from respiratory oxygen demand during organic matter degradation in stratified marine waters. Currently OMZs are expanding due to global climate change. This expansion alters marine ecosystem function and the productivity of fisheries due to habitat compression and changes in biogeochemical cycling leading to fixed nitrogen loss and greenhouse gas production. Here we use metaproteomics to chart spatial and temporal patterns of gene expression along defined redox gradients in a seasonally anoxic fjord, Saanich Inlet to better understand microbial community responses to OMZ expansion. The expression of metabolic pathway components for nitrification, anaerobic ammonium oxidation (anammox), denitrification and inorganic carbon fixation predominantly co-varied with abundance and distribution patterns of Thaumarchaeota, Nitrospira, Planctomycetes and SUP05/ARCTIC96BD-19 Gammaproteobacteria. Within these groups, pathways mediating inorganic carbon fixation and nitrogen and sulfur transformations were differentially expressed across the redoxcline. Nitrification and inorganic carbon fixation pathways affiliated with Thaumarchaeota dominated dysoxic waters and denitrification, sulfur-oxidation and inorganic carbon fixation pathways affiliated with SUP05 dominated suboxic and anoxic waters. Nitrite-oxidation and anammox pathways affiliated with Nitrospina and Planctomycetes respectively, also exhibited redox partitioning between dysoxic and suboxic waters. The differential expression of these pathways under changing water column redox conditions has quantitative implications for coupled biogeochemical cycling linking different modes of inorganic carbon fixation with distributed nitrogen and sulfur-based energy metabolism extensible to coastal and open ocean OMZs.

  9. Heterochromatin dynamics during the differentiation process revealed by the DNA methylation reporter mouse, MethylRO.

    PubMed

    Ueda, Jun; Maehara, Kazumitsu; Mashiko, Daisuke; Ichinose, Takako; Yao, Tatsuma; Hori, Mayuko; Sato, Yuko; Kimura, Hiroshi; Ohkawa, Yasuyuki; Yamagata, Kazuo

    2014-06-01

    In mammals, DNA is methylated at CpG sites, which play pivotal roles in gene silencing and chromatin organization. Furthermore, DNA methylation undergoes dynamic changes during development, differentiation, and in pathological processes. The conventional methods represent snapshots; therefore, the dynamics of this marker within living organisms remains unclear. To track this dynamics, we made a knockin mouse that expresses a red fluorescent protein (RFP)-fused methyl-CpG-binding domain (MBD) protein from the ROSA26 locus ubiquitously; we named it MethylRO (methylation probe in ROSA26 locus). Using this mouse, we performed RFP-mediated methylated DNA immunoprecipitation sequencing (MeDIP-seq), whole-body section analysis, and live-cell imaging. We discovered that mobility and pattern of heterochromatin as well as DNA methylation signal intensity inside the nuclei can be markers for cellular differentiation status. Thus, the MethylRO mouse represents a powerful bioresource and technique for DNA methylation dynamics studies in developmental biology, stem cell biology, as well as in disease states. PMID:24936475

  10. Roles of ERα during mouse trophectoderm lineage differentiation: revealed by antagonist and agonist of ERα.

    PubMed

    Cheng, Xiaoxiang; Xu, Songhua; Song, Chanchan; He, Lin; Lian, Xiuli; Liu, Yue; Wei, Jianen; Pang, Lili; Wang, Shie

    2016-04-01

    During mouse early embryogenesis, blastomeres increase in number by the morula stage. Among them, the outer cells are polarized and differentiated into trophectoderm (TE), while the inner cells remain unpolarized and give rise to inner cell mass (ICM). TE provides an important liquid environment for ICM development. In spite of extensive research, the molecular mechanisms underlying TE formation are still obscure. In order to investigate the roles of estrogen receptor α (ERα) in this course, mouse 8-cell embryos were collected and cultured in media containing ERα specific antagonist MPP and/or agonist PPT. The results indicated that MPP treatment inhibits blastocyst formation in a dose-dependent manner, while PPT, at proper concentration, promotes the cavitation ratio of mouse embryos. Immunofluorescence staining results showed that MPP significantly decreased the nuclear expression of CDX2 in morula, but no significant changes of OCT4 were observed. Moreover, after MPP treatment, the expression levels of the genes related to TE specification, Tead4, Gata3 and Cdx2, were significantly reduced. Overall, these results indicated that ERα might affect mouse embryo cavitation by regulating TE lineage differentiation. PMID:27037955

  11. Differential proteome and gene expression reveal response to carbon ion irradiation in pubertal mice testes.

    PubMed

    Li, Hongyan; He, Yuxuan; Zhang, Hong; Miao, Guoying

    2014-03-21

    Heavy ion radiation, a high linear energy transfer (LET) radiation, has been shown to have adverse effects on reproduction in male mice. The aim of this study was to profile and investigate the differentially expressed proteins in pubertal male mice testes following carbon ion radiation (CIR). Male mice underwent whole-body irradiation with CIR (1 and 4 Gy), and MALDI-TOF/TOF analysis was used to investigate the alteration in protein expression in 2-DE (two-dimensional gel electrophoresis) gels of testes caused by irradiation after 14 days. 8 differentially expressed proteins were identified and these proteins were mainly involved in energy supply, the endoplasmic reticulum, cell proliferation, cell cycle, antioxidant capacity and mitochondrial respiration, which play important roles in the inhibition of testicular function in response to CIR. Furthermore, we confirmed the relationship between transcription of mRNA and the abundance of proteins. Our results indicated that these proteins may lead to new insights into the molecular mechanism of CIR toxicity, and suggested that the gene expression response to CIR involves diverse regulatory mechanisms from transcription of mRNA to the formation of functional proteins. PMID:24440814

  12. Heterochromatin Dynamics during the Differentiation Process Revealed by the DNA Methylation Reporter Mouse, MethylRO

    PubMed Central

    Ueda, Jun; Maehara, Kazumitsu; Mashiko, Daisuke; Ichinose, Takako; Yao, Tatsuma; Hori, Mayuko; Sato, Yuko; Kimura, Hiroshi; Ohkawa, Yasuyuki; Yamagata, Kazuo

    2014-01-01

    Summary In mammals, DNA is methylated at CpG sites, which play pivotal roles in gene silencing and chromatin organization. Furthermore, DNA methylation undergoes dynamic changes during development, differentiation, and in pathological processes. The conventional methods represent snapshots; therefore, the dynamics of this marker within living organisms remains unclear. To track this dynamics, we made a knockin mouse that expresses a red fluorescent protein (RFP)-fused methyl-CpG-binding domain (MBD) protein from the ROSA26 locus ubiquitously; we named it MethylRO (methylation probe in ROSA26 locus). Using this mouse, we performed RFP-mediated methylated DNA immunoprecipitation sequencing (MeDIP-seq), whole-body section analysis, and live-cell imaging. We discovered that mobility and pattern of heterochromatin as well as DNA methylation signal intensity inside the nuclei can be markers for cellular differentiation status. Thus, the MethylRO mouse represents a powerful bioresource and technique for DNA methylation dynamics studies in developmental biology, stem cell biology, as well as in disease states. PMID:24936475

  13. Comparative metagenomics reveals microbial community differentiation in a biological heap leaching system.

    PubMed

    Hu, Qi; Guo, Xue; Liang, Yili; Hao, Xiaodong; Ma, Liyuan; Yin, Huaqun; Liu, Xueduan

    2015-01-01

    The microbial community in a biological heap leaching (BHL) system is crucial for the decomposition of ores. However, the microbial community structure and functional differentiation in different parts of a biological heap leaching system are still unknown. In this study, metagenomic sequencing was used to fully illuminate the microbial community differentiation in the pregnant leach solution (PLS) and leaching heap (LH) of a BHL system. Long-read sequences (1.3 million) were obtained for the two samples, and the MG_RAST server was used to perform further analysis. The taxa analysis results indicated that the dominant genera of PLS is autotrophic bacterium Acidithiobacillus, but heterotrophic bacterium Acidiphilium is predominant in LH. Furthermore, functional annotation and hierarchical comparison with different reference samples showed that the abundant presence of genes was involved in transposition, DNA repair and heavy metal transport. The sequences related to transposase, which is important for the survival of the organism in the hostile environment, were both mainly classified into Acidiphilium for PLS and LH. These results indicated that not only autotrophic bacteria such as Acidithiobacillus, but also heterotrophic bacteria such as Acidiphilium, were essential participants in the bioleaching process. This new meta-view research will further facilitate the effective application of bioleaching. PMID:26117598

  14. Chemical regulators of epithelial plasticity reveal a nuclear receptor pathway controlling myofibroblast differentiation

    PubMed Central

    Carthy, Jon M.; Stöter, Martin; Bellomo, Claudia; Vanlandewijck, Michael; Heldin, Angelos; Morén, Anita; Kardassis, Dimitris; Gahman, Timothy C.; Shiau, Andrew K.; Bickle, Marc; Zerial, Marino; Heldin, Carl-Henrik; Moustakas, Aristidis

    2016-01-01

    Plasticity in epithelial tissues relates to processes of embryonic development, tissue fibrosis and cancer progression. Pharmacological modulation of epithelial transitions during disease progression may thus be clinically useful. Using human keratinocytes and a robotic high-content imaging platform, we screened for chemical compounds that reverse transforming growth factor β (TGF-β)-induced epithelial-mesenchymal transition. In addition to TGF-β receptor kinase inhibitors, we identified small molecule epithelial plasticity modulators including a naturally occurring hydroxysterol agonist of the liver X receptors (LXRs), members of the nuclear receptor transcription factor family. Endogenous and synthetic LXR agonists tested in diverse cell models blocked α-smooth muscle actin expression, myofibroblast differentiation and function. Agonist-dependent LXR activity or LXR overexpression in the absence of ligand counteracted TGF-β-mediated myofibroblast terminal differentiation and collagen contraction. The protective effect of LXR agonists against TGF-β-induced pro-fibrotic activity raises the possibility that anti-lipidogenic therapy may be relevant in fibrotic disorders and advanced cancer. PMID:27430378

  15. Chemical regulators of epithelial plasticity reveal a nuclear receptor pathway controlling myofibroblast differentiation.

    PubMed

    Carthy, Jon M; Stöter, Martin; Bellomo, Claudia; Vanlandewijck, Michael; Heldin, Angelos; Morén, Anita; Kardassis, Dimitris; Gahman, Timothy C; Shiau, Andrew K; Bickle, Marc; Zerial, Marino; Heldin, Carl-Henrik; Moustakas, Aristidis

    2016-01-01

    Plasticity in epithelial tissues relates to processes of embryonic development, tissue fibrosis and cancer progression. Pharmacological modulation of epithelial transitions during disease progression may thus be clinically useful. Using human keratinocytes and a robotic high-content imaging platform, we screened for chemical compounds that reverse transforming growth factor β (TGF-β)-induced epithelial-mesenchymal transition. In addition to TGF-β receptor kinase inhibitors, we identified small molecule epithelial plasticity modulators including a naturally occurring hydroxysterol agonist of the liver X receptors (LXRs), members of the nuclear receptor transcription factor family. Endogenous and synthetic LXR agonists tested in diverse cell models blocked α-smooth muscle actin expression, myofibroblast differentiation and function. Agonist-dependent LXR activity or LXR overexpression in the absence of ligand counteracted TGF-β-mediated myofibroblast terminal differentiation and collagen contraction. The protective effect of LXR agonists against TGF-β-induced pro-fibrotic activity raises the possibility that anti-lipidogenic therapy may be relevant in fibrotic disorders and advanced cancer. PMID:27430378

  16. Quantitative Analysis of Global Proteome and Lysine Acetylome Reveal the Differential Impacts of VPA and SAHA on HL60 Cells.

    PubMed

    Zhu, Xiaoyu; Liu, Xin; Cheng, Zhongyi; Zhu, Jun; Xu, Lei; Wang, Fengsong; Qi, Wulin; Yan, Jiawei; Liu, Ning; Sun, Zimin; Liu, Huilan; Peng, Xiaojun; Hao, Yingchan; Zheng, Nan; Wu, Quan

    2016-01-01

    Valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) are both HDAC inhibitors (HDACi). Previous studies indicated that both inhibitors show therapeutic effects on acute myeloid leukaemia (AML), while the differential impacts of the two different HDACi on AML treatment still remains elusive. In this study, using 3-plex SILAC based quantitative proteomics technique, anti-acetyllysine antibody based affinity enrichment, high resolution LC-MS/MS and intensive bioinformatic analysis, the quantitative proteome and acetylome in SAHA and VPA treated AML HL60 cells were extensively studied. In total, 5,775 proteins and 1,124 lysine acetylation sites were successfully obtained in response to VAP and SAHA treatment. It is found that VPA and SAHA treatment differently induced proteome and acetylome profiling in AML HL60 cells. This study revealed the differential impacts of VPA and SAHA on proteome/acetylome in AML cells, deepening our understanding of HDAC inhibitor mediated AML therapeutics. PMID:26822725

  17. Quantitative Analysis of Global Proteome and Lysine Acetylome Reveal the Differential Impacts of VPA and SAHA on HL60 Cells

    PubMed Central

    Zhu, Xiaoyu; Liu, Xin; Cheng, Zhongyi; Zhu, Jun; Xu, Lei; Wang, Fengsong; Qi, Wulin; Yan, Jiawei; Liu, Ning; Sun, Zimin; Liu, Huilan; Peng, Xiaojun; Hao, Yingchan; Zheng, Nan; Wu, Quan

    2016-01-01

    Valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) are both HDAC inhibitors (HDACi). Previous studies indicated that both inhibitors show therapeutic effects on acute myeloid leukaemia (AML), while the differential impacts of the two different HDACi on AML treatment still remains elusive. In this study, using 3-plex SILAC based quantitative proteomics technique, anti-acetyllysine antibody based affinity enrichment, high resolution LC-MS/MS and intensive bioinformatic analysis, the quantitative proteome and acetylome in SAHA and VPA treated AML HL60 cells were extensively studied. In total, 5,775 proteins and 1,124 lysine acetylation sites were successfully obtained in response to VAP and SAHA treatment. It is found that VPA and SAHA treatment differently induced proteome and acetylome profiling in AML HL60 cells. This study revealed the differential impacts of VPA and SAHA on proteome/acetylome in AML cells, deepening our understanding of HDAC inhibitor mediated AML therapeutics. PMID:26822725

  18. Mature adipocyte proteome reveals differentially altered protein abundances between lean, overweight and morbidly obese human subjects.

    PubMed

    Benabdelkamel, Hicham; Masood, Afshan; Almidani, Ghaith M; Alsadhan, Abdulmajeed A; Bassas, Abdulelah F; Duncan, Mark W; Alfadda, Assim A

    2015-02-01

    Overweight (OW) and obese individuals are considered to be graded parts of the scale having increasing weight as a common feature. They may not, however, be part of the same continuum and may differ metabolically. In this study we applied an untargeted proteomic approach to compare protein abundances in mature adipocytes derived from the subcutaneous adipose tissue of overweight and morbidly obese female subjects to those of lean age matched controls. Mature adipocytes were isolated from liposuction samples of abdominal subcutaneous adipose tissue collected from both lean (L; n = 7, 23.3 ± 0.4 kg/m(2); mean BMI ± SD), overweight (OW; n = 8, 27.9 ± 0.6 kg/m(2); mean BMI ± SD) and morbidly obese (MOB; n = 7, 44.8 ± 3.8 kg/m(2); mean BMI ± SD) individuals. Total protein extracts were then compared by two-dimensional difference in gel electrophoresis (2D DIGE). One hundred and ten differentially expressed protein spots (i.e., fitting the statistical criteria ANOVA test, p < 0.05; fold-change ≥1.5) were detected, and of these, 89 were identified by MALDI-TOF mass spectrometry. Of these, 66 protein spots were common to both groups whereas 23 were unique to the MOB group. Significant differences were evident in the abundances of key proteins involved in glucose and lipid metabolism, energy regulation, cytoskeletal structure and redox control signaling pathways. Differences in the abundance of some chaperones were also evident. The differentially abundant proteins were investigated using Ingenuity Pathway Analysis (IPA) to establish their associations with known biological functions. The network identified in the OW group with the highest score relates to-: cell-to-cell signaling and interaction; in contrast, in the MOB group the major interacting pathways are associated with lipid metabolism, small molecule biochemistry and cancer. The differences in abundance of the differentially regulated proteins were validated by

  19. 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. PMID:26382298

  20. Proteomic analysis reveals the differential histone programs between male germline cells and vegetative cells in Lilium davidii.

    PubMed

    Yang, Hao; Yang, Ning; Wang, Tai

    2016-03-01

    In flowering plants, male germline fate is determined after asymmetric division of the haploid microspore. Daughter cells have distinct fates: the generative cell (GC) undergoes further mitosis to generate sperm cells (SCs), and the vegetative cell (VC) terminally differentiates. However, our understanding of the mechanisms underlying germline development remains limited. Histone variants and modifications define chromatin states, and contribute to establishing and maintaining cell identities by affecting gene expression. Here, we constructed a lily protein database, then extracted and detailed histone entries into a comprehensive lily histone database. We isolated large amounts of nuclei from VCs, GCs and SCs from lily, and profiled histone variants of all five histone families in all three cell types using proteomics approaches. We revealed 92 identities representing 32 histone variants: six for H1, 11 for H2A, eight for H2B, five for H3 and two for H4. Nine variants, including five H1, two H2B, one H3 and one H4 variant, specifically accumulated in GCs and SCs. We also detected H3 modification patterns in the three cell types. GCs and SCs had almost identical histone profiles and similar H3 modification patterns, which were significantly different from those of VCs. Our study also revealed the presence of multiple isoforms, and differential expression patterns between isoforms of a variant. The results suggest that differential histone programs between the germline and companion VCs may be established following the asymmetric division, and are important for identity establishment and differentiation of the male germline as well as the VC. PMID:26846354

  1. Whole Genome Expression Analysis Reveals Differential Effects of TiO2 Nanotubes on Vascular Cells

    PubMed Central

    Peng, Lily; Barczak, Andrea J.; Barbeau, Rebecca A.; Xiao, Yuanyuan; LaTempa, Thomas J.; Grimes, Craig A.; Desai, Tejal A.

    2010-01-01

    The response of primary human endothelial (ECs) and vascular smooth muscle cells (VSMCs) to TiO2 nanotube arrays is studied through gene expression analysis. Microarrays revealed that nanotubes enhanced EC proliferation and motility, decreased VSMC proliferation, and decreased expression of molecules involved in inflammation and coagulation in both cell types. Networks generated from significantly affected genes suggest that cells may be sensing nanotopographical cues via pathways previously implicated in sensing shear stress. PMID:20030358

  2. By Ounce or By Calorie: The Differential Effects of Alternative Sugar-Sweetened Beverage Tax Strategies

    PubMed Central

    Zhen, Chen; Brissette, Ian F.; Ruff, Ryan R.

    2014-01-01

    The obesity epidemic and excessive consumption of sugar-sweetened beverages have led to proposals of economics-based interventions to promote healthy eating in the United States. Targeted food and beverage taxes and subsidies are prominent examples of such potential intervention strategies. This paper examines the differential effects of taxing sugar-sweetened beverages by calories and by ounces on beverage demand. To properly measure the extent of substitution and complementarity between beverage products, we developed a fully modified distance metric model of differentiated product demand that endogenizes the cross-price effects. We illustrated the proposed methodology in a linear approximate almost ideal demand system, although other flexible demand systems can also be used. In the empirical application using supermarket scanner data, the product-level demand model consists of 178 beverage products with combined market share of over 90%. The novel demand model outperformed the conventional distance metric model in non-nested model comparison tests and in terms of the economic significance of model predictions. In the fully modified model, a calorie-based beverage tax was estimated to cost $1.40 less in compensating variation than an ounce-based tax per 3,500 beverage calories reduced. This difference in welfare cost estimates between two tax strategies is more than three times as much as the difference estimated by the conventional distance metric model. If applied to products purchased from all sources, a 0.04-cent per kcal tax on sugar-sweetened beverages is predicted to reduce annual per capita beverage intake by 5,800 kcal. PMID:25414517

  3. RNA-Seq Reveals Different mRNA Abundance of Transporters and Their Alternative Transcript Isoforms During Liver Development

    PubMed Central

    Cui, Julia Yue; Gunewardena, Sumedha S.; Yoo, Byunggil; Liu, Jie; Renaud, Helen J.; Lu, Hong; Zhong, Xiao-bo; Klaassen, Curtis D.

    2012-01-01

    During development, the maturation of liver transporters is essential for chemical elimination in newborns and children. One cannot compare the real abundance of transcripts by conventional messenger RNA (mRNA) profiling methods; in comparison, RNA-Seq provides a “true quantification” of transcript counts and an unbiased detection of novel transcripts. The purpose of this study was to compare the mRNA abundance of liver transporters and seek their novel transcripts during liver development. Livers from male C57BL/6J mice were collected at 12 ages from prenatal to adulthood. The transcriptome was determined by RNA-Seq, with transcript abundance estimated by Cufflinks. Among 498 known transporters, the ontogeny of 62 known critical xenobiotic transporters was examined in detail. The cumulative mRNAs of the uptake transporters increased more than the efflux transporters in livers after birth. A heatmap revealed three ontogenic patterns of these transporters, namely perinatal (reaching maximal expression before birth), adolescent (about 20 days), and adult enriched (about 60 days of age). Before birth, equilibrative nucleoside transporter 1 was the transporter with highest expression in liver (29%), followed by breast cancer resistance protein (Bcrp) (26%). Within 1 day after birth, the mRNAs of these two transporters decreased markedly, and Ntcp became the transporter with highest expression (52%). In adult liver, the transporters with highest expression were organic cation transporter 1 and Ntcp (23% and 22%, respectively). Three isoforms of Bcrp with alternate leading exons were identified (E1a, E1b, and E1c), with E1b being the major isoform. In conclusion, this study reveals the mRNA abundance of transporters in liver and demonstrates that the expression of liver transporters is both age and isoform specific. PMID:22454430

  4. Integrative Multi-omic Analysis of Human Platelet eQTLs Reveals Alternative Start Site in Mitofusin 2.

    PubMed

    Simon, Lukas M; Chen, Edward S; Edelstein, Leonard C; Kong, Xianguo; Bhatlekar, Seema; Rigoutsos, Isidore; Bray, Paul F; Shaw, Chad A

    2016-05-01

    Platelets play a central role in ischemic cardiovascular events. Cardiovascular disease (CVD) is a major cause of death worldwide. Numerous genome-wide association studies (GWASs) have identified loci associated with CVD risk. However, our understanding of how these variants contribute to disease is limited. Using data from the platelet RNA and expression 1 (PRAX1) study, we analyzed cis expression quantitative trait loci (eQTLs) in platelets from 154 normal human subjects. We confirmed these results in silico by performing allele-specific expression (ASE) analysis, which demonstrated that the allelic directionality of eQTLs and ASE patterns correlate significantly. Comparison of platelet eQTLs with data from the Genotype-Tissue Expression (GTEx) project revealed that a number of platelet eQTLs are platelet specific and that platelet eQTL peaks localize to the gene body at a higher rate than eQTLs from other tissues. Upon integration with data from previously published GWASs, we found that the trait-associated variant rs1474868 coincides with the eQTL peak for mitofusin 2 (MFN2). Additional experimental and computational analyses revealed that this eQTL is linked to an unannotated alternate MFN2 start site preferentially expressed in platelets. Integration of phenotype data from the PRAX1 study showed that MFN2 expression levels were significantly associated with platelet count. This study links the variant rs1474868 to a platelet-specific regulatory role for MFN2 and demonstrates the utility of integrating multi-omic data with eQTL analysis in disease-relevant tissues for interpreting GWAS results. PMID:27132591

  5. Complexity of indica-japonica varietal differentiation in Bangladesh rice landraces revealed by microsatellite markers

    PubMed Central

    Wang, Mumu; Zhu, Zuofeng; Tan, Lubin; Liu, Fengxia; Fu, Yongcai; Sun, Chuanqing; Cai, Hongwei

    2013-01-01

    To understand the genetic diversity and indica-japonica differentiation in Bangladesh rice varieties, a total of 151 accessions of rice varieties mostly Bangladesh traditional varieties including Aus, Boro, broadcast Aman, transplant Aman and Rayada varietal groups were genotyped using 47 rice nuclear SSRs. As a result, three distinct groups were detected by cluster analysis, corresponding to indica, Aus and japonica rice. Among deepwater rice varieties analyzed some having particular morphological features that mainly corresponded to the japonica varietal group. Some small seeded and aromatic varieties from Bangladesh also corresponded to the japonica varietal group. This research for the first time establishes that the japonica varietal group is a prominent component of traditional varieties in Bangladesh, particularly in deepwater areas. PMID:23853518

  6. Transient Exposure to Ethanol during Zebrafish Embryogenesis Results in Defects in Neuronal Differentiation: An Alternative Model System to Study FASD

    PubMed Central

    Joya, Xavier; Garcia-Algar, Oscar; Vall, Oriol; Pujades, Cristina

    2014-01-01

    Background The exposure of the human embryo to ethanol results in a spectrum of disorders involving multiple organ systems, including the impairment of the development of the central nervous system (CNS). In spite of the importance for human health, the molecular basis of prenatal ethanol exposure remains poorly understood, mainly to the difficulty of sample collection. Zebrafish is now emerging as a powerful organism for the modeling and the study of human diseases. In this work, we have assessed the sensitivity of specific subsets of neurons to ethanol exposure during embryogenesis and we have visualized the sensitive embryonic developmental periods for specific neuronal groups by the use of different transgenic zebrafish lines. Methodology/Principal Findings In order to evaluate the teratogenic effects of acute ethanol exposure, we exposed zebrafish embryos to ethanol in a given time window and analyzed the effects in neurogenesis, neuronal differentiation and brain patterning. Zebrafish larvae exposed to ethanol displayed small eyes and/or a reduction of the body length, phenotypical features similar to the observed in children with prenatal exposure to ethanol. When neuronal populations were analyzed, we observed a clear reduction in the number of differentiated neurons in the spinal cord upon ethanol exposure. There was a decrease in the population of sensory neurons mainly due to a decrease in cell proliferation and subsequent apoptosis during neuronal differentiation, with no effect in motoneuron specification. Conclusion Our investigation highlights that transient exposure to ethanol during early embryonic development affects neuronal differentiation although does not result in defects in early neurogenesis. These results establish the use of zebrafish embryos as an alternative research model to elucidate the molecular mechanism(s) of ethanol-induced developmental toxicity at very early stages of embryonic development. PMID:25383948

  7. Whole genome sequencing of two North American Drosophila melanogaster populations reveals genetic differentiation and positive selection

    PubMed Central

    Campo, D; Lehmann, K; Fjeldsted, C; Souaiaia, T; Kao, J; Nuzhdin, SV

    2013-01-01

    The prevailing demographic model for Drosophila melanogaster suggests that the colonization of North America occurred very recently from a subset of European flies that rapidly expanded across the continent. This model implies a sudden population growth and range expansion consistent with very low or no population subdivision. As flies adapt to new environments, local adaptation events may be expected. In order to describe demographic and selective events during North American colonization, we have generated a dataset of 35 individual whole genome sequences from inbred lines of D. melanogaster from a west coast US population (Winters, California, USA) and compared them with a public genome dataset from Raleigh (Raleigh, North Carolina, USA). We analyzed nuclear and mitochondrial genomes and describe levels of variation and divergence within and between these two North American D. melanogaster populations. Both populations exhibit negative values of Tajima’s D across the genome, a common signature of demographic expansion. We also detected a low but significant level of genome-wide differentiation between the two populations, as well as multiple allele surfing events, which can be the result of gene drift in local subpopulations on the edge of an expansion wave. In contrast to this genome-wide pattern, we uncovered a 50 kilobases segment in chromosome arm 3L that showed all the hallmarks of a soft selective sweep in both populations. A comparison of allele frequencies within this divergent region among six populations from three continents allowed us to cluster these populations in two differentiated groups, providing evidence for the action of natural selection on a global scale. PMID:24102956

  8. Alternative substrates reveal catalytic cycle and key binding events in the reaction catalysed by anthranilate phosphoribosyltransferase from Mycobacterium tuberculosis.

    PubMed

    Cookson, Tammie V M; Castell, Alina; Bulloch, Esther M M; Evans, Genevieve L; Short, Francesca L; Baker, Edward N; Lott, J Shaun; Parker, Emily J

    2014-07-01

    AnPRT (anthranilate phosphoribosyltransferase), required for the biosynthesis of tryptophan, is essential for the virulence of Mycobacterium tuberculosis (Mtb). AnPRT catalyses the Mg2+-dependent transfer of a phosphoribosyl group from PRPP (5'-phosphoribosyl-1'-pyrophosphate) to anthranilate to form PRA (5'-phosphoribosyl anthranilate). Mtb-AnPRT was shown to catalyse a sequential reaction and significant substrate inhibition by anthranilate was observed. Antimycobacterial fluoroanthranilates and methyl-substituted analogues were shown to act as alternative substrates for Mtb-AnPRT, producing the corresponding substituted PRA products. Structures of the enzyme complexed with anthranilate analogues reveal two distinct binding sites for anthranilate. One site is located over 8 Å (1 Å=0.1 nm) from PRPP at the entrance to a tunnel leading to the active site, whereas in the second, inner, site anthranilate is adjacent to PRPP, in a catalytically relevant position. Soaking the analogues for variable periods of time provides evidence for anthranilate located at transient positions during transfer from the outer site to the inner catalytic site. PRPP and Mg2+ binding have been shown to be associated with the rearrangement of two flexible loops, which is required to complete the inner anthranilate-binding site. It is proposed that anthranilate first binds to the outer site, providing an unusual mechanism for substrate capture and efficient transfer to the catalytic site following the binding of PRPP. PMID:24712732

  9. Differentiation processes in FeO-rich asteroids revealed by the achondrite Lewis Cliff 88763

    NASA Astrophysics Data System (ADS)

    Day, James M. D.; Corder, Christopher A.; Rumble, Douglas; Assayag, Nelly; Cartigny, Pierre; Taylor, Lawrence A.

    2015-10-01

    Olivine-dominated (70-80 modal %) achondrite meteorite Lewis Cliff (LEW) 88763 originated from metamorphism and limited partial melting of a FeO-rich parent body. The meteorite experienced some alteration on Earth, evident from subchondritic Re/Os, and redistribution of rhenium within the sample. LEW 88763 is texturally similar to winonaites, has a Δ17O value of -1.19 ± 0.10‰, and low bulk-rock Mg/(Mg+Fe) (0.39), similar to the FeO-rich cumulate achondrite Northwest Africa (NWA) 6693. The similar bulk-rock major-, minor-, and trace-element abundances of LEW 88763, relative to some carbonaceous chondrites, including ratios of Pd/Os, Pt/Os, Ir/Os, and 187Os/188Os (0.1262), implies a FeO- and volatile-rich precursor composition. Lack of fractionation of the rare earth elements, but a factor of approximately two lower highly siderophile element abundances in LEW 88763, compared with chondrites, implies limited loss of Fe-Ni-S melts during metamorphism and anatexis. These results support the generation of high Fe/Mg, sulfide, and/or metal-rich partial melts from FeO-rich parent bodies during partial melting. In detail, however, LEW 88763 cannot be a parent composition to any other meteorite sample, due to highly limited silicate melt loss (0 to <<5%). As such, LEW 88763 represents the least-modified FeO-rich achondrite source composition recognized to date and is distinct from all other meteorites. LEW 88763 should be reclassified as an anomalous achondrite that experienced limited Fe,Ni-FeS melt loss. Lewis Cliff 88763, combined with a growing collection of FeO-rich meteorites, such as brachinites, brachinite-like achondrites, the Graves Nunataks (GRA) 06128/9 meteorites, NWA 6693, and Tafassasset, has important implications for understanding the initiation of planetary differentiation. Specifically, regardless of precursor compositions, partial melting and differentiation processes appear to be similar on asteroidal bodies spanning a range of initial oxidation

  10. Comparative Circadian Metabolomics Reveal Differential Effects of Nutritional Challenge in the Serum and Liver.

    PubMed

    Abbondante, Serena; Eckel-Mahan, Kristin L; Ceglia, Nicholas J; Baldi, Pierre; Sassone-Corsi, Paolo

    2016-02-01

    Diagnosis and therapeutic interventions in pathological conditions rely upon clinical monitoring of key metabolites in the serum. Recent studies show that a wide range of metabolic pathways are controlled by circadian rhythms whose oscillation is affected by nutritional challenges, underscoring the importance of assessing a temporal window for clinical testing and thereby questioning the accuracy of the reading of critical pathological markers in circulation. We have been interested in studying the communication between peripheral tissues under metabolic homeostasis perturbation. Here we present a comparative circadian metabolomic analysis on serum and liver in mice under high fat diet. Our data reveal that the nutritional challenge induces a loss of serum metabolite rhythmicity compared with liver, indicating a circadian misalignment between the tissues analyzed. Importantly, our results show that the levels of serum metabolites do not reflect the circadian liver metabolic signature or the effect of nutritional challenge. This notion reveals the possibility that misleading reads of metabolites in circulation may result in misdiagnosis and improper treatments. Our findings also demonstrate a tissue-specific and time-dependent disruption of metabolic homeostasis in response to altered nutrition. PMID:26644470

  11. Cerebral hemovelocity reveals differential resource allocation strategies for extraverts and introverts during vigilance.

    PubMed

    Shaw, Tyler H; Nguyen, Cynthia; Satterfield, Kelly; Ramirez, Raul; McKnight, Patrick E

    2016-02-01

    Extraversion--one of the Big 5 personality factors--correlates negatively with vigilance, but most studies focus on performance outcomes and not the performance process. Previous research has shown that transcranial Doppler sonography (TCD), which measures cerebral blood flow velocity (CBFV), can be used to examine resource allocation strategies during vigilance performance. Hence, this study was designed to assess the attentional resource allocation strategies of introverts and extraverts using the CBFV measure. Twelve extroverts and 13 introverts monitored a 60-min vigilance task for a critical signal--the absence of a line on a five-circle array. The results revealed an overall performance decrement that was not modulated by extraversion. We observed an interaction between extraversion and time; CBFV declined in the introversion group, but not in the extraversion group. Additionally, an interaction between cerebral hemisphere and personality revealed that extraverts were recruiting resources from both the left and right cerebral hemispheres, while introverts only recruited resources from the right hemisphere. The results suggest that extraverts can allocate compensatory effort to mask performance differences. We discuss the theoretical and practical implications of these findings and offer future research directions that may help us understand these effects. PMID:26563163

  12. Differential gene expression and alternative splicing between diploid and tetraploid watermelon.

    PubMed

    Saminathan, Thangasamy; Nimmakayala, Padma; Manohar, Sumanth; Malkaram, Sridhar; Almeida, Aldo; Cantrell, Robert; Tomason, Yan; Abburi, Lavanya; Rahman, Mohammad A; Vajja, Venkata G; Khachane, Amit; Kumar, Brajendra; Rajasimha, Harsha K; Levi, Amnon; Wehner, Todd; Reddy, Umesh K

    2015-03-01

    The exploitation of synthetic polyploids for producing seedless fruits is well known in watermelon. Tetraploid progenitors of triploid watermelon plants, compared with their diploid counterparts, exhibit wide phenotypic differences. Although many factors modulate alternative splicing (AS) in plants, the effects of autopolyploidization on AS are still unknown. In this study, we used tissues of leaf, stem, and fruit of diploid and tetraploid sweet watermelon to understand changes in gene expression and the occurrence of AS. RNA-sequencing analysis was performed along with reverse transcription quantitative PCR and rapid amplification of cDNA ends (RACE)-PCR to demonstrate changes in expression and splicing. All vegetative tissues except fruit showed an increased level of AS in the tetraploid watermelon throughout the growth period. The ploidy levels of diploids and the tetraploid were confirmed using a ploidy analyser. We identified 5362 and 1288 genes that were up- and downregulated, respectively, in tetraploid as compared with diploid plants. We further confirmed that 22 genes underwent AS events across tissues, indicating possibilities of generating different protein isoforms with altered functions of important transcription factors and transporters. Arginine biosynthesis, chlorophyllide synthesis, GDP mannose biosynthesis, trehalose biosynthesis, and starch and sucrose degradation pathways were upregulated in autotetraploids. Phloem protein 2, chloroplastic PGR5-like protein, zinc-finger protein, fructokinase-like 2, MYB transcription factor, and nodulin MtN21 showed AS in fruit tissues. These results should help in developing high-quality seedless watermelon and provide additional transcriptomic information related to other cucurbits. PMID:25520388

  13. Differential gene expression and alternative splicing between diploid and tetraploid watermelon

    PubMed Central

    Saminathan, Thangasamy; Nimmakayala, Padma; Manohar, Sumanth; Malkaram, Sridhar; Almeida, Aldo; Cantrell, Robert; Tomason, Yan; Abburi, Lavanya; Rahman, Mohammad A.; Vajja, Venkata G.; Khachane, Amit; Kumar, Brajendra; Rajasimha, Harsha K.; Levi, Amnon; Wehner, Todd; Reddy, Umesh K.

    2015-01-01

    The exploitation of synthetic polyploids for producing seedless fruits is well known in watermelon. Tetraploid progenitors of triploid watermelon plants, compared with their diploid counterparts, exhibit wide phenotypic differences. Although many factors modulate alternative splicing (AS) in plants, the effects of autopolyploidization on AS are still unknown. In this study, we used tissues of leaf, stem, and fruit of diploid and tetraploid sweet watermelon to understand changes in gene expression and the occurrence of AS. RNA-sequencing analysis was performed along with reverse transcription quantitative PCR and rapid amplification of cDNA ends (RACE)-PCR to demonstrate changes in expression and splicing. All vegetative tissues except fruit showed an increased level of AS in the tetraploid watermelon throughout the growth period. The ploidy levels of diploids and the tetraploid were confirmed using a ploidy analyser. We identified 5362 and 1288 genes that were up- and downregulated, respectively, in tetraploid as compared with diploid plants. We further confirmed that 22 genes underwent AS events across tissues, indicating possibilities of generating different protein isoforms with altered functions of important transcription factors and transporters. Arginine biosynthesis, chlorophyllide synthesis, GDP mannose biosynthesis, trehalose biosynthesis, and starch and sucrose degradation pathways were upregulated in autotetraploids. Phloem protein 2, chloroplastic PGR5-like protein, zinc-finger protein, fructokinase-like 2, MYB transcription factor, and nodulin MtN21 showed AS in fruit tissues. These results should help in developing high-quality seedless watermelon and provide additional transcriptomic information related to other cucurbits. PMID:25520388

  14. Differential DNA methylation analysis of breast cancer reveals the impact of immune signaling in radiation therapy

    PubMed Central

    Halvorsen, Ann Rita; Helland, Åslaug; Fleischer, Thomas; Haug, Karen Marie; Grenaker Alnæs, Grethe Irene; Nebdal, Daniel; Syljuåsen, Randi G; Touleimat, Nizar; Busato, Florence; Tost, Jörg; Sætersdal, Anna B; Børresen-Dale, Anne-Lise; Kristensen, Vessela; Edvardsen, Hege

    2014-01-01

    Radiotherapy (RT) is a central treatment modality for breast cancer patients. The purpose of our study was to investigate the DNA methylation changes in tumors following RT, and to identify epigenetic markers predicting treatment outcome. Paired biopsies from patients with inoperable breast cancer were collected both before irradiation (n = 20) and after receiving 10–24 Gray (Gy) (n = 19). DNA methylation analysis was performed by using Illumina Infinium 27K arrays. Fourteen genes were selected for technical validation by pyrosequencing. Eighty-two differentially methylated genes were identified in irradiated (n = 11) versus nonirradiated (n = 19) samples (false discovery rate, FDR = 1.1%). Methylation levels in pathways belonging to the immune system were most altered after RT. Based on methylation levels before irradiation, a panel of five genes (H2AFY, CTSA, LTC4S, IL5RA and RB1) were significantly associated with clinical response (p = 0.041). Furthermore, the degree of methylation changes for 2,516 probes correlated with the given radiation dose. Within the 2,516 probes, an enrichment for pathways involved in cellular immune response, proliferation and apoptosis was identified (FDR < 5%). Here, we observed clear differences in methylation levels induced by radiation, some associated with response to treatment. Our study adds knowledge on the molecular mechanisms behind radiation response. PMID:24658971

  15. Decoding regulatory landscape of somatic embryogenesis reveals differential regulatory networks between japonica and indica rice subspecies

    PubMed Central

    Indoliya, Yuvraj; Tiwari, Poonam; Chauhan, Abhisekh Singh; Goel, Ridhi; Shri, Manju; Bag, Sumit Kumar; Chakrabarty, Debasis

    2016-01-01

    Somatic embryogenesis is a unique process in plants and has considerable interest for biotechnological application. Compare to japonica, indica rice has been less responsive to in vitro culture. We used Illumina Hiseq 2000 sequencing platform for comparative transcriptome analysis between two rice subspecies at six different developmental stages combined with a tag-based digital gene expression profiling. Global gene expression among different samples showed greater complexity in japonica rice compared to indica which may be due to polyphyletic origin of two rice subspecies. Expression pattern in initial stage indicate major differences in proembryogenic callus induction phase that may serve as key regulator to observe differences between both subspecies. Our data suggests that phytohormone signaling pathways consist of elaborate networks with frequent crosstalk, thereby allowing plants to regulate somatic embryogenesis pathway. However, this crosstalk varies between the two rice subspecies. Down regulation of positive regulators of meristem development (i.e. KNOX, OsARF5) and up regulation of its counterparts (OsRRs, MYB, GA20ox1/GA3ox2) in japonica may be responsible for its better regeneration and differentiation of somatic embryos. Comprehensive gene expression information in the present experiment may also facilitate to understand the monocot specific meristem regulation for dedifferentiation of somatic cell to embryogenic cells. PMID:26973288

  16. Neonatal thymectomy reveals differentiation and plasticity within human naive T cells

    PubMed Central

    van den Broek, Theo; Delemarre, Eveline M.; Janssen, Willemijn J.M.; Nievelstein, Rutger A.J.; Broen, Jasper C.; Tesselaar, Kiki; Borghans, Jose A.M.; Nieuwenhuis, Edward E.S.; Prakken, Berent J.; Mokry, Michal; Jansen, Nicolaas J.G.

    2016-01-01

    The generation of naive T cells is dependent on thymic output, but in adults, the naive T cell pool is primarily maintained by peripheral proliferation. Naive T cells have long been regarded as relatively quiescent cells; however, it was recently shown that IL-8 production is a signatory effector function of naive T cells, at least in newborns. How this functional signature relates to naive T cell dynamics and aging is unknown. Using a cohort of children and adolescents who underwent neonatal thymectomy, we demonstrate that the naive CD4+ T cell compartment in healthy humans is functionally heterogeneous and that this functional diversity is lost after neonatal thymectomy. Thymic tissue regeneration later in life resulted in functional restoration of the naive T cell compartment, implicating the thymus as having functional regenerative capacity. Together, these data shed further light on functional differentiation within the naive T cell compartment and the importance of the thymus in human naive T cell homeostasis and premature aging. In addition, these results affect and alter our current understanding on the identification of truly naive T cells and recent thymic emigrants. PMID:26901814

  17. A Quantitative Proteomic Analysis of Hemogenic Endothelium Reveals Differential Regulation of Hematopoiesis by SOX17.

    PubMed

    Clarke, Raedun L; Robitaille, Aaron M; Moon, Randall T; Keller, Gordon

    2015-08-11

    The in vitro derivation of hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) is complicated by the existence of multiple overlapping embryonic blood cell programs called primitive, erythromyeloid progenitor (EMP), and definitive. As HSCs are only generated during the definitive stage of hematopoiesis, deciphering the regulatory pathways that control the emergence of this program and identifying markers that distinguish it from the other programs are essential. To identify definitive specific pathways and marker sets, we used label-free proteomics to determine the proteome of embryo-derived and mouse embryonic stem cell-derived VE-CADHERIN(+)CD45(-) definitive hematopoietic progenitors. With this approach, we identified Stat1 as a marker that distinguishes the definitive erythroid lineage from the primitive- and EMP-derived lineages. Additionally, we provide evidence that the generation of the Stat1(+) definitive lineage is dependent on Sox17. These findings establish an approach for monitoring the emergence of definitive hematopoiesis in the PSC differentiation cultures. PMID:26267830

  18. Metaproteomics reveals differential modes of metabolic coupling among ubiquitous oxygen minimum zone microbes

    PubMed Central

    Hawley, Alyse K.; Brewer, Heather M.; Norbeck, Angela D.; Paša-Tolić, Ljiljana; Hallam, Steven J.

    2014-01-01

    Marine oxygen minimum zones (OMZs) are intrinsic water column features arising from respiratory oxygen demand during organic matter degradation in stratified waters. Currently OMZs are expanding due to global climate change with resulting feedback on marine ecosystem function. Here we use metaproteomics to chart spatial and temporal patterns of gene expression along defined redox gradients in a seasonally stratified fjord to better understand microbial community responses to OMZ expansion. The expression of metabolic pathway components for nitrification, anaerobic ammonium oxidation (anammox), denitrification, and inorganic carbon fixation were differentially expressed across the redoxcline and covaried with distribution patterns of ubiquitous OMZ microbes including Thaumarchaeota, Nitrospina, Nitrospira, Planctomycetes, and SUP05/ARCTIC96BD-19 Gammaproteobacteria. Nitrification and inorganic carbon fixation pathways affiliated with Thaumarchaeota dominated dysoxic waters, and denitrification, sulfur oxidation, and inorganic carbon fixation pathways affiliated with the SUP05 group of nitrate-reducing sulfur oxidizers dominated suboxic and anoxic waters. Nitrifier nitrite oxidation and anammox pathways affiliated with Nirospina, Nitrospira, and Planctomycetes, respectively, also exhibited redox partitioning between dysoxic and suboxic waters. The numerical abundance of SUP05 proteins mediating inorganic carbon fixation under anoxic conditions suggests that SUP05 will become increasingly important in global ocean carbon and nutrient cycling as OMZs expand. PMID:25053816

  19. Decoding regulatory landscape of somatic embryogenesis reveals differential regulatory networks between japonica and indica rice subspecies.

    PubMed

    Indoliya, Yuvraj; Tiwari, Poonam; Chauhan, Abhisekh Singh; Goel, Ridhi; Shri, Manju; Bag, Sumit Kumar; Chakrabarty, Debasis

    2016-01-01

    Somatic embryogenesis is a unique process in plants and has considerable interest for biotechnological application. Compare to japonica, indica rice has been less responsive to in vitro culture. We used Illumina Hiseq 2000 sequencing platform for comparative transcriptome analysis between two rice subspecies at six different developmental stages combined with a tag-based digital gene expression profiling. Global gene expression among different samples showed greater complexity in japonica rice compared to indica which may be due to polyphyletic origin of two rice subspecies. Expression pattern in initial stage indicate major differences in proembryogenic callus induction phase that may serve as key regulator to observe differences between both subspecies. Our data suggests that phytohormone signaling pathways consist of elaborate networks with frequent crosstalk, thereby allowing plants to regulate somatic embryogenesis pathway. However, this crosstalk varies between the two rice subspecies. Down regulation of positive regulators of meristem development (i.e. KNOX, OsARF5) and up regulation of its counterparts (OsRRs, MYB, GA20ox1/GA3ox2) in japonica may be responsible for its better regeneration and differentiation of somatic embryos. Comprehensive gene expression information in the present experiment may also facilitate to understand the monocot specific meristem regulation for dedifferentiation of somatic cell to embryogenic cells. PMID:26973288

  20. Transcriptional Analysis of Deinococcus radiodurans Reveals Novel Small RNAs That Are Differentially Expressed under Ionizing Radiation

    PubMed Central

    Tsai, Chen-Hsun; Liao, Rick; Chou, Brendan

    2014-01-01

    Small noncoding RNAs (sRNAs) are posttranscriptional regulators that have been identified in multiple species and shown to play essential roles in responsive mechanisms to environmental stresses. The natural ability of specific bacteria to resist high levels of radiation has been of high interest to mechanistic studies of DNA repair and biomolecular protection. Deinococcus radiodurans is a model extremophile for radiation studies that can survive doses of ionizing radiation of >12,000 Gy, 3,000 times higher than for most vertebrates. Few studies have investigated posttranscriptional regulatory mechanisms of this organism that could be relevant in its general gene regulatory patterns. In this study, we identified 199 potential sRNA candidates in D. radiodurans by whole-transcriptome deep sequencing analysis and confirmed the expression of 41 sRNAs by Northern blotting and reverse transcriptase PCR (RT-PCR). A total of 8 confirmed sRNAs showed differential expression during recovery after acute ionizing radiation (15 kGy). We have also found and confirmed 7 sRNAs in Deinococcus geothermalis, a closely related radioresistant species. The identification of several novel sRNAs in Deinococcus bacteria raises important questions about the evolution and nature of global gene regulation in radioresistance. PMID:25548054

  1. Neonatal thymectomy reveals differentiation and plasticity within human naive T cells.

    PubMed

    van den Broek, Theo; Delemarre, Eveline M; Janssen, Willemijn J M; Nievelstein, Rutger A J; Broen, Jasper C; Tesselaar, Kiki; Borghans, Jose A M; Nieuwenhuis, Edward E S; Prakken, Berent J; Mokry, Michal; Jansen, Nicolaas J G; van Wijk, Femke

    2016-03-01

    The generation of naive T cells is dependent on thymic output, but in adults, the naive T cell pool is primarily maintained by peripheral proliferation. Naive T cells have long been regarded as relatively quiescent cells; however, it was recently shown that IL-8 production is a signatory effector function of naive T cells, at least in newborns. How this functional signature relates to naive T cell dynamics and aging is unknown. Using a cohort of children and adolescents who underwent neonatal thymectomy, we demonstrate that the naive CD4+ T cell compartment in healthy humans is functionally heterogeneous and that this functional diversity is lost after neonatal thymectomy. Thymic tissue regeneration later in life resulted in functional restoration of the naive T cell compartment, implicating the thymus as having functional regenerative capacity. Together, these data shed further light on functional differentiation within the naive T cell compartment and the importance of the thymus in human naive T cell homeostasis and premature aging. In addition, these results affect and alter our current understanding on the identification of truly naive T cells and recent thymic emigrants. PMID:26901814

  2. Mechanistic Studies of Bismuth(V)-Mediated Thioglycoside Activation Reveal Differential Reactivity of Anomers.

    PubMed

    Goswami, Manibarsha; Ashley, Daniel C; Baik, Mu-Hyun; Pohl, Nicola L B

    2016-07-15

    The mechanism of bismuth(V)-mediated thioglycoside activation was examined using reaction kinetics and quantum chemical reaction models. NMR experiments show an unusual nonlinear growth/decay curve for the glycosylation reaction. Further studies suggest an anomeric inversion of the β-glycoside donor to the α-donor during its activation, even in the presence of a neighboring 2-position acetate. Interestingly, in situ anomerization was not observed in the activation of an α-glycoside donor, and this anomer also showed faster reaction times and higher product diastereoselectivites. Density functional theory calculations identify the structure of the promoter triphenyl bismuth ditriflate, [Ph3Bi(OTf)2, 1], in solution and map out the energetics of its interactions with the two thioglycoside anomers. These calculations suggest that 1 must bind the thiopropyl arm to induce triflate loss. The computational analyses also show that, unlike most O-glycosides, the β- and α-donor S-glycosides are similar in energy. One energetically reasonable anomerization pathway of the donors is an SN1-like mechanism promoted by forming a bismuth-sulfonium adduct with the Lewis acidic Bi(V) for the formation of an oxacarbenium intermediate. Finally, the computed energy compensations needed to form these α vs β Bi adducts is a possible explanation for the differential reactivity of these donors. PMID:27295299

  3. Differential proteomic profiles from distinct Toxoplasma gondii strains revealed by 2D-difference gel electrophoresis.

    PubMed

    Zhou, Huaiyu; Zhao, Qunli; Das Singla, Lachhman; Min, Juan; He, Shenyi; Cong, Hua; Li, Ying; Su, Chunlei

    2013-04-01

    Toxoplasma gondii is an obligate intracellular protozoan that infects mammals and birds. Human infection during pregnancy may cause severe damage to the fetus. Reactivation of latent infection in immunocompromised patients can cause life-threatening encephalitis. T. gondii strains are highly diverse but only a few lineages (Type I, II and III) are widely spread. In mouse model, Type I strains are highly virulent, whereas Type II and III strains are intermediately or non virulent. It is not clear how much quantitative difference exists in proteomic profiles among these distinct T. gondii lineages. In the present study, the proteomic profiles of T. gondii tachyzoites from these lineages were investigated by two dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS) technologies. A total of 2321 protein spots were detected. Overall, the GT1 strain of Type I lineage and the strain PTG of Type II lineage have highly similar proteomic profiles and both are different from that of the CTG strain of Type III lineage. Eighty-four protein spots were differentially expressed by greater than 1.5-fold in relative abundance and 10 of them were identified to 7 T. gondii proteins in existing database. Investigation of the quantitative differences in proteomics among distinct T. gondii strains should facilitate our understanding of difference in biological processes and pathogenesis of distinct T. gondii genotypes, which will provide basic information to determine treatment regimen for different manifestation of toxoplasmosis. PMID:23340323

  4. Differential Proteomics of Urinary Exovesicles from Classical Galactosemic Patients Reveals Subclinical Kidney Insufficiency.

    PubMed

    Staubach, Simon; Pekmez, Murat; Hanisch, Franz-Georg

    2016-06-01

    Classical galactosemia is caused by a nearly complete deficiency of galactose-1-phosphate uridyltransferase (GALT; EC 2.7.712), resulting in a severely impaired galactose metabolism with galactose-1-phosphate and galactitol accumulation. Even on a galactose-restricted diet, patients develop serious long-term complications of the central nervous system and ovaries that may result from chronic cell-toxic effects exerted by endogenous galactose. To address the question of whether disease-associated cellular perturbations could affect the kidney function of the patients, we performed differential proteomics of detergent-resistant membranes from urinary exovesicles. Galactosemic samples (showing drastic shifts from high-mannose to complex-type N-glycosylation on exosomal N-glycoproteins) and healthy, sex-matched controls were analyzed in quadruplex iTRAQ experiments performed in biological and technical replicates. Particularly in the female patient group, the most striking finding was a drastic increase of abundant serum (glyco)proteins, like albumin, leucine-rich α-2-glycoprotein, fetuin, immunoglobulins, prostaglandin H2 d-isomerase, and α-1-microglobulin protein (AMBP), pointing to a subclinical failure of kidney filter function in galactosemic patients and resulting in a heavy overload of exosomal membranes with adsorbed serum (glyco)proteins. Several of these proteins are connected to TBMN and IgAN, proteinuria, and renal damage. The impairment of renal protein filtration was also indicated by increased protein contents derived from extracellular matrices and lysosomes. PMID:27103203

  5. Transcriptional analysis of Deinococcus radiodurans reveals novel small RNAs that are differentially expressed under ionizing radiation.

    PubMed

    Tsai, Chen-Hsun; Liao, Rick; Chou, Brendan; Contreras, Lydia M

    2015-03-01

    Small noncoding RNAs (sRNAs) are posttranscriptional regulators that have been identified in multiple species and shown to play essential roles in responsive mechanisms to environmental stresses. The natural ability of specific bacteria to resist high levels of radiation has been of high interest to mechanistic studies of DNA repair and biomolecular protection. Deinococcus radiodurans is a model extremophile for radiation studies that can survive doses of ionizing radiation of >12,000 Gy, 3,000 times higher than for most vertebrates. Few studies have investigated posttranscriptional regulatory mechanisms of this organism that could be relevant in its general gene regulatory patterns. In this study, we identified 199 potential sRNA candidates in D. radiodurans by whole-transcriptome deep sequencing analysis and confirmed the expression of 41 sRNAs by Northern blotting and reverse transcriptase PCR (RT-PCR). A total of 8 confirmed sRNAs showed differential expression during recovery after acute ionizing radiation (15 kGy). We have also found and confirmed 7 sRNAs in Deinococcus geothermalis, a closely related radioresistant species. The identification of several novel sRNAs in Deinococcus bacteria raises important questions about the evolution and nature of global gene regulation in radioresistance. PMID:25548054

  6. Metaproteomics reveals differential modes of metabolic coupling among ubiquitous oxygen minimum zone microbes.

    PubMed

    Hawley, Alyse K; Brewer, Heather M; Norbeck, Angela D; Paša-Tolić, Ljiljana; Hallam, Steven J

    2014-08-01

    Marine oxygen minimum zones (OMZs) are intrinsic water column features arising from respiratory oxygen demand during organic matter degradation in stratified waters. Currently OMZs are expanding due to global climate change with resulting feedback on marine ecosystem function. Here we use metaproteomics to chart spatial and temporal patterns of gene expression along defined redox gradients in a seasonally stratified fjord to better understand microbial community responses to OMZ expansion. The expression of metabolic pathway components for nitrification, anaerobic ammonium oxidation (anammox), denitrification, and inorganic carbon fixation were differentially expressed across the redoxcline and covaried with distribution patterns of ubiquitous OMZ microbes including Thaumarchaeota, Nitrospina, Nitrospira, Planctomycetes, and SUP05/ARCTIC96BD-19 Gammaproteobacteria. Nitrification and inorganic carbon fixation pathways affiliated with Thaumarchaeota dominated dysoxic waters, and denitrification, sulfur oxidation, and inorganic carbon fixation pathways affiliated with the SUP05 group of nitrate-reducing sulfur oxidizers dominated suboxic and anoxic waters. Nitrifier nitrite oxidation and anammox pathways affiliated with Nirospina, Nitrospira, and Planctomycetes, respectively, also exhibited redox partitioning between dysoxic and suboxic waters. The numerical abundance of SUP05 proteins mediating inorganic carbon fixation under anoxic conditions suggests that SUP05 will become increasingly important in global ocean carbon and nutrient cycling as OMZs expand. PMID:25053816

  7. Differential introgression in a mosaic hybrid zone reveals candidate barrier genes.

    PubMed

    Larson, Erica L; Andrés, Jose A; Bogdanowicz, Steven M; Harrison, Richard G

    2013-12-01

    Hybrid zones act as genomic sieves. Although globally advantageous alleles will spread throughout the zone and neutral alleles can be freely exchanged between species, introgression will be restricted for genes that contribute to reproductive barriers or local adaptation. Seminal fluid proteins (SFPs) are known to contribute to reproductive barriers in insects and have been proposed as candidate barrier genes in the hybridizing field crickets Gryllus pennsylvanicus and Gryllus firmus. Here, we have used 125 single nucleotide polymorphisms to characterize patterns of differential introgression and to identify genes that may contribute to prezygotic barriers between these species. Using a transcriptome scan of the male cricket accessory gland (the site of SFP synthesis), we identified genes with major allele frequency differences between the species. We then compared patterns of introgression for genes encoding SFPs with patterns for genes expressed in the same tissue that do not encode SFPs. We find no evidence that SFPs have reduced gene exchange across the cricket hybrid zone. However, a number of genes exhibit dramatically reduced introgression, and many of these genes encode proteins with functional roles consistent with known barriers. PMID:24299416

  8. Differentially methylated CpG island within human XIST mediates alternative P2 transcription and YY1 binding

    PubMed Central

    2014-01-01

    Background X-chromosome inactivation silences one X chromosome in females to achieve dosage compensation with the single X chromosome in males. While most genes are silenced on the inactive X chromosome, the gene for the long non-coding RNA XIST is silenced on the active X chromosome and expressed from the inactive X chromosome with which the XIST RNA associates, triggering silencing of the chromosome. In mouse, an alternative Xist promoter, P2 is also the site of YY1 binding, which has been shown to serve as a tether between the Xist RNA and the DNA of the chromosome. In humans there are many differences from the initial events of mouse Xist activation, including absence of a functional antisense regulator Tsix, and absence of strictly paternal inactivation in extraembryonic tissues, prompting us to examine regulatory regions for the human XIST gene. Results We demonstrate that the female-specific DNase hypersensitivity site within XIST is specific to the inactive X chromosome and correlates with transcription from an internal P2 promoter. P2 is located within a CpG island that is differentially methylated between males and females and overlaps conserved YY1 binding sites that are only bound on the inactive X chromosome where the sites are unmethylated. However, YY1 binding is insufficient to drive P2 expression or establish the DHS, which may require a development-specific factor. Furthermore, reduction of YY1 reduces XIST transcription in addition to causing delocalization of XIST. Conclusions The differentially methylated DNase hypersensitive site within XIST marks the location of an alternative promoter, P2, that generates a transcript of unknown function as it lacks the A repeats that are critical for silencing. In addition, this region binds YY1 on the unmethylated inactive X chromosome, and depletion of YY1 untethers the XIST RNA as well as decreasing transcription of XIST. PMID:25200388

  9. Cilium transition zone proteome reveals compartmentalization and differential dynamics of ciliopathy complexes.

    PubMed

    Dean, Samuel; Moreira-Leite, Flavia; Varga, Vladimir; Gull, Keith

    2016-08-30

    The transition zone (TZ) of eukaryotic cilia and flagella is a structural intermediate between the basal body and the axoneme that regulates ciliary traffic. Mutations in genes encoding TZ proteins (TZPs) cause human inherited diseases (ciliopathies). Here, we use the trypanosome to identify TZ components and localize them to TZ subdomains, showing that the Bardet-Biedl syndrome complex (BBSome) is more distal in the TZ than the Meckel syndrome (MKS) complex. Several of the TZPs identified here have human orthologs. Functional analysis shows essential roles for TZPs in motility, in building the axoneme central pair apparatus and in flagellum biogenesis. Analysis using RNAi and HaloTag fusion protein approaches reveals that most TZPs (including the MKS ciliopathy complex) show long-term stable association with the TZ, whereas the BBSome is dynamic. We propose that some Bardet-Biedl syndrome and MKS pleiotropy may be caused by mutations that impact TZP complex dynamics. PMID:27519801

  10. Differential Proteomics in Dequeened Honeybee Colonies Reveals Lower Viral Load in Hemolymph of Fertile Worker Bees

    PubMed Central

    Cardoen, Dries; Ernst, Ulrich R.; Van Vaerenbergh, Matthias; Boerjan, Bart; de Graaf, Dirk C.; Wenseleers, Tom; Schoofs, Liliane; Verleyen, Peter

    2011-01-01

    The eusocial societies of honeybees, where the queen is the only fertile female among tens of thousands sterile worker bees, have intrigued scientists for centuries. The proximate factors, which cause the inhibition of worker bee ovaries, remain largely unknown; as are the factors which cause the activation of worker ovaries upon the loss of queen and brood in the colony. In an attempt to reveal key players in the regulatory network, we made a proteomic comparison of hemolymph profiles of workers with completely activated ovaries vs. rudimentary ovaries. An unexpected finding of this study is the correlation between age matched worker sterility and the enrichment of Picorna-like virus proteins. Fertile workers, on the other hand, show the upregulation of potential components of the immune system. It remains to be investigated whether viral infections contribute to worker sterility directly or are the result of a weaker immune system of sterile workers. PMID:21698281

  11. Coseismic fault zone deformation revealed with differential lidar: Examples from Japanese Mw ∼7 intraplate earthquakes

    NASA Astrophysics Data System (ADS)

    Nissen, Edwin; Maruyama, Tadashi; Ramon Arrowsmith, J.; Elliott, John R.; Krishnan, Aravindhan K.; Oskin, Michael E.; Saripalli, Srikanth

    2014-11-01

    We use two recent Japanese earthquakes to demonstrate the rich potential, as well as some of the challenges, of differencing repeat airborne Light Detection and Ranging (lidar) topographic data to measure coseismic fault zone deformation. We focus on densely-vegetated sections of the 14 June 2008 Iwate-Miyagi (Mw 6.9) and 11 April 2011 Fukushima-Hamadori (Mw 7.1) earthquake ruptures, each covered by 2 m-resolution pre-event and 1 m-resolution post-event bare Earth digital terrain models (DTMs) obtained from commercial lidar providers. Three-dimensional displacements and rotations were extracted from these datasets using an adaptation of the Iterative Closest Point (ICP) algorithm. These displacements remain coherent close to surface fault breaks, as well as within dense forest, despite intervals of ∼2 years (Iwate-Miyagi) and ∼4 years (Fukushima-Hamadori) encompassed by the lidar scenes. Differential lidar analysis is thus complementary to Interferometric Synthetic Aperture Radar (InSAR) and sub-pixel correlation techniques which often break down under conditions of long time intervals, dense vegetation or steep displacement gradients. Although the ICP displacements are much noisier than overlapping InSAR line-of-sight displacements, they still provide powerful constraints on near-surface fault slip. In the Fukushima-Hamadori case, near-fault displacements and rotations are consistent with decreased primary fault slip at very shallow depths of a few tens of meters, helping to account for the large, along-strike heterogeneity in surface offsets observed in the field. This displacement field also captures long-wavelength deformation resulting from the 11 March 2011 Tohoku great earthquake.

  12. Alternative catalytic anions differentially modulate human alpha-amylase activity and specificity.

    PubMed

    Maurus, Robert; Begum, Anjuman; Williams, Leslie K; Fredriksen, Jason R; Zhang, Ran; Withers, Stephen G; Brayer, Gary D

    2008-03-18

    A mechanistic study of the essential allosteric activation of human pancreatic alpha-amylase by chloride ion has been conducted by exploring a wide range of anion substitutions through kinetic and structural experiments. Surprisingly, kinetic studies indicate that the majority of these alternative anions can induce some level of enzymatic activity despite very different atomic geometries, sizes, and polyatomic natures. These data and subsequent structural studies attest to the remarkable plasticity of the chloride binding site, even though earlier structural studies of wild-type human pancreatic alpha-amylase suggested this site would likely be restricted to chloride binding. Notably, no apparent relationship is observed between anion binding affinity and relative activity, emphasizing the complexity of the relationship between chloride binding parameters and the activation mechanism that facilitates catalysis. Of the anions studied, particularly intriguing in terms of observed trends in substrate kinetics and their novel atomic compositions were the nitrite, nitrate, and azide anions, the latter of which was found to enhance the relative activity of human pancreatic alpha-amylase by nearly 5-fold. Structural studies have provided considerable insight into the nature of the interactions formed in the chloride binding site by the nitrite and nitrate anions. To probe the role such interactions play in allosteric activation, further structural analyses were conducted in the presence of acarbose, which served as a sensitive reporter molecule of the catalytic ability of these modified enzymes to carry out its expected rearrangement by human pancreatic alpha-amylase. These studies show that the largest anion of this group, nitrate, can comfortably fit in the chloride binding pocket, making all the necessary hydrogen bonds. Further, this anion has nearly the same ability to activate human pancreatic alpha-amylase and leads to the production of the same acarbose product

  13. Lachnospiraceae and Bacteroidales Alternative Fecal Indicators Reveal Chronic Human Sewage Contamination in an Urban Harbor▿†

    PubMed Central

    Newton, Ryan J.; VandeWalle, Jessica L.; Borchardt, Mark A.; Gorelick, Marc H.; McLellan, Sandra L.

    2011-01-01

    The complexity of fecal microbial communities and overlap among human and other animal sources have made it difficult to identify source-specific fecal indicator bacteria. However, the advent of next-generation sequencing technologies now provides increased sequencing power to resolve microbial community composition within and among environments. These data can be mined for information on source-specific phylotypes and/or assemblages of phylotypes (i.e., microbial signatures). We report the development of a new genetic marker for human fecal contamination identified through microbial pyrotag sequence analysis of the V6 region of the 16S rRNA gene. Sequence analysis of 37 sewage samples and comparison with database sequences revealed a human-associated phylotype within the Lachnospiraceae family, which was closely related to the genus Blautia. This phylotype, termed Lachno2, was on average the second most abundant fecal bacterial phylotype in sewage influent samples from Milwaukee, WI. We developed a quantitative PCR (qPCR) assay for Lachno2 and used it along with the qPCR-based assays for human Bacteroidales (based on the HF183 genetic marker), total Bacteroidales spp., and enterococci and the conventional Escherichia coli and enterococci plate count assays to examine the prevalence of fecal and human fecal pollution in Milwaukee's harbor. Both the conventional fecal indicators and the human-associated indicators revealed chronic fecal pollution in the harbor, with significant increases following heavy rain events and combined sewer overflows. The two human-associated genetic marker abundances were tightly correlated in the harbor, a strong indication they target the same source (i.e., human sewage). Human adenoviruses were routinely detected under all conditions in the harbor, and the probability of their occurrence increased by 154% for every 10-fold increase in the human indicator concentration. Both Lachno2 and human Bacteroidales increased specificity to

  14. Physiological and Transcriptional Analyses Reveal Differential Phytohormone Responses to Boron Deficiency in Brassica napus Genotypes

    PubMed Central

    Zhou, Ting; Hua, Yingpeng; Huang, Yupu; Ding, Guangda; Shi, Lei; Xu, Fangsen

    2016-01-01

    Phytohormones play pivotal roles in the response of plants to various biotic and abiotic stresses. Boron (B) is an essential microelement for plants, and Brassica napus (B. napus) is hypersensitive to B deficiency. However, how auxin responds to B deficiency remained a dilemma for many years and little is known about how other phytohormones respond to B deficiency. The identification of B-efficient/inefficient B. napus indicates that breeding might overcome these constraints in the agriculture production. Here, we seek to identify phytohormone-related processes underlying B-deficiency tolerance in B. napus at the physiological and gene expression levels. Our study indicated low-B reduced indole-3-acetic acid (IAA) concentration in both the shoots and roots of B. napus, and affected the expression of the auxin biosynthesis gene BnNIT1 and the efflux gene BnPIN1 in a time-dependent manner. Low-B increased the jasmonates (JAs) and abscisic acid (ABA) concentrations and induced the expression of the ABA biosynthesis gene BnNCED3 and the ABA sensor gene BnPYL4 in the shoot. In two contrasting genotypes, the auxin concentration decreased more drastically in the B-inefficient genotype ‘W10,’ and together the expression of BnNIT1 and BnPIN1 also decreased more significantly in ‘W10’ under long-term B deficiency. While the JAs concentration was considerably higher in this genotype, and the ABA concentration was induced in ‘W10’ compared with the B-efficient genotype ‘QY10.’ Digital gene expression (DGE) profiling confirmed the differential expression of the phytohormone-related genes, indicating more other phyohormone differences involving in gene regulation between ‘QY10’ and ‘W10’ under low-B stress. Additionally, the activity of DR5:GFP was reduced in the root under low-B in Arabidopsis, and the application of exogenous IAA could partly restore the B-defective phenotype in ‘W10.’ Overall, our data suggested that low-B disturbed phytohormone

  15. Comparative materials differences revealed in engineered bone as a function of cell-specific differentiation

    NASA Astrophysics Data System (ADS)

    Gentleman, Eileen; Swain, Robin J.; Evans, Nicholas D.; Boonrungsiman, Suwimon; Jell, Gavin; Ball, Michael D.; Shean, Tamaryn A. V.; Oyen, Michelle L.; Porter, Alexandra; Stevens, Molly M.

    2009-09-01

    An important aim of regenerative medicine is to restore tissue function with implantable, laboratory-grown constructs that contain tissue-specific cells that replicate the function of their counterparts in the healthy native tissue. It remains unclear, however, whether cells used in bone regeneration applications produce a material that mimics the structural and compositional complexity of native bone. By applying multivariate analysis techniques to micro-Raman spectra of mineralized nodules formed in vitro, we reveal cell-source-dependent differences in interactions between multiple bone-like mineral environments. Although osteoblasts and adult stem cells exhibited bone-specific biological activities and created a material with many of the hallmarks of native bone, the `bone nodules' formed from embryonic stem cells were an order of magnitude less stiff, and lacked the distinctive nanolevel architecture and complex biomolecular and mineral composition noted in the native tissue. Understanding the biological mechanisms of bone formation in vitro that contribute to cell-source-specific materials differences may facilitate the development of clinically successful engineered bone.

  16. Thermodynamics imprinting reveals differential binding of metals to {alpha}-synuclein: Relevance to parkinson's disease

    SciTech Connect

    Bharathi; Rao, K.S.J. . E-mail: kjr5n@yahoo.co.in

    2007-07-20

    The aggregation of {alpha}-synuclein is a hallmark feature of Parkinson's disease (PD) and other synucleinopathies. Metals are the significant etiological factors in PD, and their interaction with {alpha}-synuclein affect dramatically the kinetics of fibrillation in vitro and are proposed to play an important and potential neurodegenerative role in vivo. In the present study, we investigated the stoichiometry of binding of copper [Cu (II)] and iron [Fe (III)] with {alpha}-synuclein (wild recombinant type and A30P, A53T, E46K mutant forms) using isothermal titration calorimetry (ITC). {alpha}-Synuclein monomer (wild and mutant forms) titrated by Cu (II), showed two binding sites, with an apparent K {sub B} of 10{sup 5} M and 10{sup 4} M, respectively. But, {alpha}-synuclein (wild type and mutant forms) titrated with Fe (III) revealed a K {sub B} of 10{sup 5} M with single binding site. The present investigation uncovers the detailed binding propensities between metals and {alpha}-synuclein and has biological implications in PD.

  17. Differential Chromosome Conformations as Hallmarks of Cellular Identity Revealed by Mathematical Polymer Modeling

    PubMed Central

    Goiffon, Isabelle; Tanguy-le-Gac, Nicolas; Bystricky, Kerstin

    2015-01-01

    Inherently dynamic, chromosomes adopt many different conformations in response to DNA metabolism. Models of chromosome organization in the yeast nucleus obtained from genome-wide chromosome conformation data or biophysical simulations provide important insights into the average behavior but fail to reveal features from dynamic or transient events that are only visible in a fraction of cells at any given moment. We developed a method to determine chromosome conformation from relative positions of three fluorescently tagged DNA in living cells imaged in 3D. Cell type specific chromosome folding properties could be assigned based on positional combinations between three loci on yeast chromosome 3. We determined that the shorter left arm of chromosome 3 is extended in MATα cells, but can be crumpled in MATa cells. Furthermore, we implemented a new mathematical model that provides for the first time an estimate of the relative physical constraint of three linked loci related to cellular identity. Variations in this estimate allowed us to predict functional consequences from chromatin structural alterations in asf1 and recombination enhancer deletion mutant cells. The computational method is applicable to identify and characterize dynamic chromosome conformations in any cell type. PMID:26030148

  18. Differential proteomic analysis of STAT6 knockout mice reveals new regulatory function in liver lipid homeostasis.

    PubMed

    Iff, Joël; Wang, Wei; Sajic, Tatjana; Oudry, Nathalie; Gueneau, Estelle; Hopfgartner, Gérard; Varesio, Emmanuel; Szanto, Ildiko

    2009-10-01

    Increased inflammatory signaling is a key feature of metabolic disorders. In this context, the role of increased pro-inflammatory signals has been extensively studied. By contrast, no efforts have been dedicated to study the contrasting scenario: the attenuation of anti-inflammatory signals and their role in metabolic homeostasis. IL-4 and IL-13 are anti-inflammatory cytokines signaling through the Signal Transducer and Activator of Transcription 6 (STAT6). Our study was aimed at evaluating the lack of STAT6 signaling on liver homeostasis. To this end we analyzed the liver proteome of wild type and STAT6 knock-out mice using 2D nanoscale LC-MS/MS with iTRAQ labeling technique. The coordinated changes in proteins identified by this quantitative proteome analysis indicated disturbed lipid homeostasis and a state of hepatocellular stress. Most significantly, the expression of the liver fatty acid binding protein (FABP1) was increased in the knock-out mice. In line with the elevated FABP1 expression we found latent liver lipid accumulation in the STAT6-deficient mice which was further aggravated when mice were challenged by a high fat diet. In conclusion, our study revealed a so far uncharacterized role for STAT6 in regulating liver lipid homeostasis and demonstrates the importance of anti-inflammatory signaling in the defense against the development of liver steatosis. PMID:19663508

  19. Differential contributions of connexin37 and connexin43 to oogenesis revealed in chimeric reaggregated mouse ovaries.

    PubMed

    Gittens, Joanne E I; Kidder, Gerald M

    2005-11-01

    The gap junction proteins connexin37 and connexin43 are required for ovarian folliculogenesis in the mouse. To define their respective roles in oogenesis, chimeric ovaries containing either null mutant oocytes and wild-type granulosa cells or the reverse combination were grafted to the renal capsules of immunodeficient female mice. After three weeks, the oocytes were tested for meiotic competence and fertilizability in vitro. Ovaries composed of connexin43-deficient oocytes and wild-type granulosa cells produced antral follicles enclosing oocytes that could develop to at least the two-cell stage, demonstrating that oocytes need not express connexin43 to reach maturity. Conversely, both follicle development and oocyte maturation were impaired in ovaries containing either wild-type oocytes and connexin43-deficient granulosa cells or connexin37-deficient oocytes and wild-type granulosa cells. Thus absence of connexin43 from granulosa cells or connexin37 from oocytes is sufficient to compromise both oocyte and follicle development. Wild-type oocytes paired with connexin37-deficient granulosa cells generated antral follicles containing oocytes that developed to at least the two-cell stage. Therefore, connexin37 absence from granulosa cells need not impair fertility in mice. Dye transfer experiments revealed persistent oocyte-granulosa cell coupling in those follicles, indicating functional compensation by another connexin. The results indicate that mouse oocytes do not need to express connexin43 in order to develop into meiotically competent, fertilizable gametes, but must express connexin37 for communication with granulosa cells, a requirement for oogenesis. PMID:16254245

  20. Single-cell imaging of inflammatory caspase dimerization reveals differential recruitment to inflammasomes.

    PubMed

    Sanders, M G; Parsons, M J; Howard, A G A; Liu, J; Fassio, S R; Martinez, J A; Bouchier-Hayes, L

    2015-01-01

    The human inflammatory caspases, including caspase-1, -4, -5 and -12, are considered as key regulators of innate immunity protecting from sepsis and numerous inflammatory diseases. Caspase-1 is activated by proximity-induced dimerization following recruitment to inflammasomes but the roles of the remaining inflammatory caspases in inflammasome assembly are unclear. Here, we use caspase bimolecular fluorescence complementation to visualize the assembly of inflammasomes and dimerization of inflammatory caspases in single cells. We observed caspase-1 dimerization induced by the coexpression of a range of inflammasome proteins and by lipospolysaccharide (LPS) treatment in primary macrophages. Caspase-4 and -5 were only dimerized by select inflammasome proteins, whereas caspase-12 dimerization was not detected by any investigated treatment. Strikingly, we determined that certain inflammasome proteins could induce heterodimerization of caspase-1 with caspase-4 or -5. Caspase-5 homodimerization and caspase-1/-5 heterodimerization was also detected in LPS-primed primary macrophages in response to cholera toxin subunit B. The subcellular localization and organization of the inflammasome complexes varied markedly depending on the upstream trigger and on which caspase or combination of caspases were recruited. Three-dimensional imaging of the ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain)/caspase-1 complexes revealed a large spherical complex of ASC with caspase-1 dimerized on the outer surface. In contrast, NALP1 (NACHT leucine-rich repeat protein 1)/caspase-1 complexes formed large filamentous structures. These results argue that caspase-1, -4 or -5 can be recruited to inflammasomes under specific circumstances, often leading to distinctly organized and localized complexes that may impact the functions of these proteases. PMID:26158519

  1. Single-cell imaging of inflammatory caspase dimerization reveals differential recruitment to inflammasomes

    PubMed Central

    Sanders, M G; Parsons, M J; Howard, A G A; Liu, J; Fassio, S R; Martinez, J A; Bouchier-Hayes, L

    2015-01-01

    The human inflammatory caspases, including caspase-1, -4, -5 and -12, are considered as key regulators of innate immunity protecting from sepsis and numerous inflammatory diseases. Caspase-1 is activated by proximity-induced dimerization following recruitment to inflammasomes but the roles of the remaining inflammatory caspases in inflammasome assembly are unclear. Here, we use caspase bimolecular fluorescence complementation to visualize the assembly of inflammasomes and dimerization of inflammatory caspases in single cells. We observed caspase-1 dimerization induced by the coexpression of a range of inflammasome proteins and by lipospolysaccharide (LPS) treatment in primary macrophages. Caspase-4 and -5 were only dimerized by select inflammasome proteins, whereas caspase-12 dimerization was not detected by any investigated treatment. Strikingly, we determined that certain inflammasome proteins could induce heterodimerization of caspase-1 with caspase-4 or -5. Caspase-5 homodimerization and caspase-1/-5 heterodimerization was also detected in LPS-primed primary macrophages in response to cholera toxin subunit B. The subcellular localization and organization of the inflammasome complexes varied markedly depending on the upstream trigger and on which caspase or combination of caspases were recruited. Three-dimensional imaging of the ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain)/caspase-1 complexes revealed a large spherical complex of ASC with caspase-1 dimerized on the outer surface. In contrast, NALP1 (NACHT leucine-rich repeat protein 1)/caspase-1 complexes formed large filamentous structures. These results argue that caspase-1, -4 or -5 can be recruited to inflammasomes under specific circumstances, often leading to distinctly organized and localized complexes that may impact the functions of these proteases. PMID:26158519

  2. Whole Hillslope Irrigation Reveals Differential Interflow Behavior of Dye Tracers, Conservative Solutes and Nutrients

    NASA Astrophysics Data System (ADS)

    Jackson, C. R.; Du, E.; Klaus, J.; Griffiths, N. A.; McDonnell, J. J.; Blake, J. I.

    2012-12-01

    Previous investigations of perching and interflow behavior in low angle hillslopes in the SC Coastal Plain have suggested a high threshold for interflow occurrence. Here we report a new irrigation experiment designed to quantify interflow thresholds and reveal subsurface mixing processes during steady state flow conditions over a 12m x 16.5m plot draining to an interflow interception trench. Dye tracers were applied on surface transects prior to irrigation, and bromide (conservative tracer), nitrate, ammonium, and phosphorus (reactive tracers) were added at constant concentrations to the irrigation water drawn from a deep aquifer with a distinct isotopic signature. 417mm of water were applied over 51 hours, and drainage conditions were monitored for a week following irrigation. Interflow in the two drains commenced after 131 and 178mm, and flow rates diminished immediately after irrigation ceased, although interflow continued for four more days. Over the experiment, 199mm of water (49% of applied water) appeared as interflow. Dye tracers moved rapidly with the wetting front, with peak concentrations measured shortly after flow commencement, suggesting saturated topsoil conductivities of 0.5 to 1.5 m/hr. No preferential flow was observed during this experiment or previously during rainfall events at the trench face. Bromide concentrations and the new water fraction rose steadily throughout irrigation, peaking about 16 hours after irrigation ceased. Ammonium and phosphorus concentrations at the trench face were low, suggesting rapid uptake or sorption, while nitrate concentrations were higher, suggesting more conservative transport. Our two collection drains showed identical temporal variation in bromide concentrations but consistently different new/old water fractions, indicating differences in flow paths and storages within the plot. These data suggest that tightly bound soil water exchanged with new water throughout the experiment, and that a significant portion

  3. Feather isotope analysis reveals differential patterns of habitat and resource use in populations of white-winged doves

    USGS Publications Warehouse

    Carleton, Scott A.; Martinez Del Rio, Carlos; Robinson, Timothy J.

    2015-01-01

    The white-winged dove (Zenaida asiatica) serves an important ecological role as a diurnal pollinator of the saguaro cactus in the Sonoran desert and an economic role as a highly sought after game bird in North America. White-winged doves are intimately linked to anthropogenic changes on the landscape and because of this, have experienced dramatic population fluctuations over the last 75 years in response, both positively and negatively, to anthropogenic changes on the landscape. To understand the factors driving population growth and decline of migratory species like the white-winged dove, it is imperative we study resource use on both their breeding and wintering grounds. To understand how populations are distributed on the wintering grounds, we tested an alternative to band recovery approaches by using stable isotope analysis. Before we could use isotope analysis to link breeding and wintering locations for this species, we first needed to determine if hydrogen (δ2H) and carbon (δ13C) stable isotopes in feather tissue (δ2Hf and δ13Cf, respectively) could differentiate among populations of white-winged doves across their breeding range in Texas, New Mexico, and Arizona. δ2Hf and δ13Cf not only differentiated between populations of white-winged doves that breed in the United States, but δ2Hf also provided further differentiation in white-winged doves that breed in native Sonoran Desert and agricultural habitats in the western portion of their range. Ecological processes associated with desert resources and anthropogenic influences, specifically saguaro cacti and irrigated crops, largely determined δ2Hf in some white-winged doves in Arizona whereas δ2H of precipitation (δ2Hp) largely determined δ2Hfof doves in New Mexico and Texas. This study highlights the usefulness of stable isotope analysis to differentiate populations of animals across the landscape and the insight isotopes can provide into habitat and resource use. Published 2015. This article

  4. Successful In Vitro Expansion and Differentiation of Cord Blood Derived CD34+ Cells into Early Endothelial Progenitor Cells Reveals Highly Differential Gene Expression

    PubMed Central

    Topcic, Denijal; Haviv, Izhak; Merivirta, Ruusu-Maaria; Agrotis, Alexander; Leitner, Ephraem; Jowett, Jeremy B.; Bode, Christoph; Lappas, Martha; Peter, Karlheinz

    2011-01-01

    Endothelial progenitor cells (EPCs) can be purified from peripheral blood, bone marrow or cord blood and are typically defined by a limited number of cell surface markers and a few functional tests. A detailed in vitro characterization is often restricted by the low cell numbers of circulating EPCs. Therefore in vitro culturing and expansion methods are applied, which allow at least distinguishing two different types of EPCs, early and late EPCs. Herein, we describe an in vitro culture technique with the aim to generate high numbers of phenotypically, functionally and genetically defined early EPCs from human cord blood. Characterization of EPCs was done by flow cytometry, immunofluorescence microscopy, colony forming unit (CFU) assay and endothelial tube formation assay. There was an average 48-fold increase in EPC numbers. EPCs expressed VEGFR-2, CD144, CD18, and CD61, and were positive for acetylated LDL uptake and ulex lectin binding. The cells stimulated endothelial tube formation only in co-cultures with mature endothelial cells and formed CFUs. Microarray analysis revealed highly up-regulated genes, including LL-37 (CAMP), PDK4, and alpha-2-macroglobulin. In addition, genes known to be associated with cardioprotective (GDF15) or pro-angiogenic (galectin-3) properties were also significantly up-regulated after a 72 h differentiation period on fibronectin. We present a novel method that allows to generate high numbers of phenotypically, functionally and genetically characterized early EPCs. Furthermore, we identified several genes newly linked to EPC differentiation, among them LL-37 (CAMP) was the most up-regulated gene. PMID:21858032

  5. Becoming Irreplaceable: How Comparisons to the Partner’s Alternatives Differentially Affect Low and High Self-Esteem People

    PubMed Central

    Murray, Sandra L.; Leder, Sadie; McClellan, Jennifer C. D.; Holmes, John G.; Pinkus, Rebecca T.; Harris, Brianna

    2009-01-01

    It is proposed that people are motivated to feel hard to replace in romantic relationships because feeling irreplaceable fosters trust in a partner’s continued responsiveness. By contrast, feeling replaceable motivates compensatory behavior aimed at strengthening the partner’s commitment to the relationship. A correlational study of dating couples and 2 experiments examined how satiating/thwarting the goal of feeling irreplaceable differentially affects relationship perception and behavior for low and high self-esteem people. The results revealed that satiating the goal of feeling irreplaceable increases trust for people low in self-esteem. In contrast, thwarting the goal of feeling irreplaceable increases compensatory behaviors meant to prove one’s indispensability for people high in self-esteem. PMID:20161401

  6. Transcriptional profiling of cortical versus cancellous bone from mechanically-loaded murine tibiae reveals differential gene expression.

    PubMed

    Kelly, Natalie H; Schimenti, John C; Ross, F Patrick; van der Meulen, Marjolein C H

    2016-05-01

    Mechanical loading is an anabolic stimulus that increases bone mass, and thus a promising method to counteract osteoporosis-related bone loss. The mechanism of this anabolism remains unclear, and needs to be established for both cortical and cancellous envelopes individually. We hypothesized that cortical and cancellous bone display different gene expression profiles at baseline and in response to mechanical loading. To test this hypothesis, the left tibiae of 10-week-old female C57Bl/6 mice were subjected to one session of axial tibial compression (9N, 1200cycles, 4Hz triangle waveform) and euthanized 3 and 24h following loading. The right limb served as the contralateral control. We performed RNA-seq on marrow-free metaphyseal samples from the cortical shell and the cancellous core to determine differential gene expression at baseline (control limb) and in response to load. Differential expression was verified with qPCR. Cortical and cancellous bone exhibited distinctly different transcriptional profiles basally and in response to mechanical loading. More genes were differentially expressed with loading at 24h with more genes downregulated at 24h than at 3h in both tissues. Enhanced Wnt signaling dominated the response in cortical bone at 3 and 24h, but in cancellous bone only at 3h. In cancellous bone at 24h many muscle-related genes were downregulated. These findings reveal key differences between cortical and cancellous genetic regulation in response to mechanical loading. Future studies at different time points and multiple loading sessions will add to our knowledge of cortical and cancellous mechanotransduction with the potential to identify new targets for mouse genetic knockout studies and drugs to treat osteoporosis. PMID:26876048

  7. Thermal stability and molecular microstructure of heat-induced cereal grains, revealed with Raman molecular microspectroscopy and differential scanning calorimetry.

    PubMed

    Khan, Md Majibur Rahman; Yu, Peiqiang

    2013-07-01

    The objectives of the present study were to use Raman molecular microspectroscopy and differential scanning calorimetry (DSC) to reveal molecular thermal stability and thermal degradation behavior of heat-induced cereal grains and reveal the molecular chemistry of the protein structures of cereal grain tissues affected by heat processing and to quantify the protein secondary structures using multicomponent peak modeling Gaussian and Lorentzian methods. Hierarchical cluster analysis (CLA) and principal components analysis (PCA) were also conducted to identify molecular differences in the Raman spectra. Three cereal grain seeds, wheat, triticale, and corn, were used as the model for feed protein in the experiment. The specimens were autoclaved (moist heating) and dry-heated (roasted) at 121 °C for 80 min, respectively. Raman spectroscopy results revealed that there are marked differences in the secondary structures of the proteins subjected to various heating treatments of different cereals. The sensitivity of cereals to moist heating was much higher than the sensitivity to dry heating. The multivariate analyses (CLA and PCA) showed that heat treatment was significantly isolated between the different Raman raw spectra. The DSC study revealed that the thermal degradation behavior of cereals was significantly changed after moist- and dry-heat treatments. The position of the major endothermic peak of dry-heated cereals shifted toward a higher temperature, from 131.7 to 134.0 °C, suggesting the high thermal stability of dry-heated cereals. In contrast, the endothermic peak position was slightly decreased to 132.1 °C in the case of moist autoclaved heating. The digestive behavior and nutritive value of rumen-undegradable protein in animals may be related to the changes of the protein secondary molecular structure and thermal stability of the cereal grain materials, which is attributed by Raman microspectroscopy and DSC endotherm profiles. PMID:23724957

  8. The Protein Architecture of Human Secretory Vesicles Reveals Differential Regulation of Signaling Molecule Secretion by Protein Kinases

    PubMed Central

    Taupenot, Laurent; Ziegler, Michael; O'Connor, Daniel T.; Ma, Qi; Smoot, Michael; Ideker, Trey; Hook, Vivian

    2012-01-01

    Secretory vesicles are required for release of chemical messengers to mediate intercellular signaling among human biological systems. It is necessary to define the organization of the protein architecture of the ‘human’ dense core secretory vesicles (DCSV) to understand mechanisms for secretion of signaling molecules essential for cellular regulatory processes. This study, therefore, conducted extensive quantitative proteomics and systems biology analyses of human DCSV purified from human pheochromocytoma. Over 600 human DCSV proteins were identified with quantitative evaluation of over 300 proteins, revealing that most proteins participate in producing peptide hormones and neurotransmitters, enzymes, and the secretory machinery. Systems biology analyses provided a model of interacting DCSV proteins, generating hypotheses for differential intracellular protein kinases A and C signaling pathways. Activation of cellular PKA and PKC pathways resulted in differential secretion of neuropeptides, catecholamines, and β-amyloid of Alzheimer's disease for mediating cell-cell communication. This is the first study to define a model of the protein architecture of human DCSV for human disease and health. PMID:22916103

  9. Differential Gene Expression in Sugarcane in Response to Challenge by Fungal Pathogen Ustilago scitaminea Revealed by cDNA-AFLP

    PubMed Central

    You-Xiong, Que; Jian-Wei, Lin; Xian-Xian, Song; Li-Ping, Xu; Ru-Kai, Chen

    2011-01-01

    Differential gene expression in sugarcane during sugarcane-Ustilago scitaminea interaction was conducted in a smut-resistant genotype. Using cDNA-AFLP along with silver staining, a total of 136 transcript-derived fragments (TDFs) were found to be differentially expressed in response to challenge by U. scitaminea. Forty TDFs, 34 newly induced plus six with obvious upregulated expression after infection, were sequenced and validated by RT-PCR analysis. These results demonstrated that the expression of 37 out of these TDFs in RT-PCR analysis was consistent with that in cDNA-AFLP analysis. Based on BlastX in NCBI, 28 TDFs were assumed to function in sugarcane under U. scitaminea stress. Analysis of expression profile of three TDFs revealed that they responded differently after infection with U. scitaminea, and the transcription was significantly enhanced. The response of two TDFs, SUC06 and SUC09, occurred before that of SUC10. This study enriches our knowledge of the molecular basis for sugarcane response to U. scitaminea infection. PMID:21792273

  10. Negative differential mobility for negative carriers as revealed by space charge measurements on crosslinked polyethylene insulated model cables

    NASA Astrophysics Data System (ADS)

    Teyssedre, G.; Vu, T. T. N.; Laurent, C.

    2015-12-01

    Among features observed in polyethylene materials under relatively high field, space charge packets, consisting in a pulse of net charge that remains in the form of a pulse as it crosses the insulation, are repeatedly observed but without complete theory explaining their formation and propagation. Positive charge packets are more often reported, and the models based on negative differential mobility(NDM) for the transport of holes could account for some charge packets phenomenology. Conversely, NDM for electrons transport has never been reported so far. The present contribution reports space charge measurements by pulsed electroacoustic method on miniature cables that are model of HVDC cables. The measurements were realized at room temperature or with a temperature gradient of 10 °C through the insulation under DC fields on the order 30-60 kV/mm. Space charge results reveal systematic occurrence of a negative front of charges generated at the inner electrode that moves toward the outer electrode at the beginning of the polarization step. It is observed that the transit time of the front of negative charge increases, and therefore the mobility decreases, with the applied voltage. Further, the estimated mobility, in the range 10-14-10-13 m2 V-1 s-1 for the present results, increases when the temperature increases for the same condition of applied voltage. The features substantiate the hypothesis of negative differential mobility used for modelling space charge packets.

  11. Comparative Genomic and Transcriptomic Analyses Reveal Habitat Differentiation and Different Transcriptional Responses during Pectin Metabolism in Alishewanella Species

    PubMed Central

    Jung, Jaejoon

    2013-01-01

    Alishewanella species are expected to have high adaptability to diverse environments because they are isolated from different natural habitats. To investigate how the evolutionary history of Alishewanella species is reflected in their genomes, we performed comparative genomic and transcriptomic analyses of A. jeotgali, A. aestuarii, and A. agri, which were isolated from fermented seafood, tidal flat sediment, and soil, respectively. Genomic islands with variable GC contents indicated that invasion of prophage and transposition events occurred in A. jeotgali and A. agri but not in A. aestuarii. Habitat differentiation of A. agri from a marine environment to a terrestrial environment was proposed because the species-specific genes of A. agri were similar to those of soil bacteria, whereas those of A. jeotgali and A. aestuarii were more closely related to marine bacteria. Comparative transcriptomic analysis with pectin as a sole carbon source revealed different transcriptional responses in Alishewanella species, especially in oxidative stress-, methylglyoxal detoxification-, membrane maintenance-, and protease/chaperone activity-related genes. Transcriptomic and experimental data demonstrated that A. agri had a higher pectin degradation rate and more resistance to oxidative stress under pectin-amended conditions than the other 2 Alishewanella species. However, expression patterns of genes in the pectin metabolic pathway and of glyoxylate bypass genes were similar among all 3 Alishewanella species. Our comparative genomic and transcriptomic data revealed that Alishewanella species have evolved through horizontal gene transfer and habitat differentiation and that pectin degradation pathways in Alishewanella species are highly conserved, although stress responses of each Alishewanella species differed under pectin culture conditions. PMID:23934491

  12. RNA-Seq analysis reveals new gene models and alternative splicing in the fungal pathogen Fusarium graminearum

    PubMed Central

    2013-01-01

    Background The genome of Fusarium graminearum has been sequenced and annotated previously, but correct gene annotation remains a challenge. In addition, posttranscriptional regulations, such as alternative splicing and RNA editing, are poorly understood in F. graminearum. Here we took advantage of RNA-Seq to improve gene annotations and to identify alternative splicing and RNA editing in F. graminearum. Results We identified and revised 655 incorrectly predicted gene models, including revisions of intron predictions, intron splice sites and prediction of novel introns. 231 genes were identified with two or more alternative splice variants, mostly due to intron retention. Interestingly, the expression ratios between different transcript isoforms appeared to be developmentally regulated. Surprisingly, no RNA editing was identified in F. graminearum. Moreover, 2459 novel transcriptionally active regions (nTARs) were identified and our analysis indicates that many of these could be missed genes. Finally, we identified the 5′ UTR and/or 3′ UTR sequences of 7666 genes. A number of representative novel gene models and alternatively spliced genes were validated by reverse transcription polymerase chain reaction and sequencing of the generated amplicons. Conclusions We have developed novel and efficient strategies to identify alternatively spliced genes and incorrect gene models based on RNA-Seq data. Our study identified hundreds of alternatively spliced genes in F. graminearum and for the first time indicated that alternative splicing is developmentally regulated in filamentous fungi. In addition, hundreds of incorrect predicted gene models were identified and revised and thousands of nTARs were discovered in our study, which will be helpful for the future genomic and transcriptomic studies in F. graminearum. PMID:23324402

  13. The alternate AP-1 adaptor subunit Apm2 interacts with the Mil1 regulatory protein and confers differential cargo sorting

    PubMed Central

    Whitfield, Shawn T.; Burston, Helen E.; Bean, Björn D. M.; Raghuram, Nandini; Maldonado-Báez, Lymarie; Davey, Michael; Wendland, Beverly; Conibear, Elizabeth

    2016-01-01

    Heterotetrameric adaptor protein complexes are important mediators of cargo protein sorting in clathrin-coated vesicles. The cell type–specific expression of alternate μ chains creates distinct forms of AP-1 with altered cargo sorting, but how these subunits confer differential function is unclear. Whereas some studies suggest the μ subunits specify localization to different cellular compartments, others find that the two forms of AP-1 are present in the same vesicle but recognize different cargo. Yeast have two forms of AP-1, which differ only in the μ chain. Here we show that the variant μ chain Apm2 confers distinct cargo-sorting functions. Loss of Apm2, but not of Apm1, increases cell surface levels of the v-SNARE Snc1. However, Apm2 is unable to replace Apm1 in sorting Chs3, which requires a dileucine motif recognized by the γ/σ subunits common to both complexes. Apm2 and Apm1 colocalize at Golgi/early endosomes, suggesting that they do not associate with distinct compartments. We identified a novel, conserved regulatory protein that is required for Apm2-dependent sorting events. Mil1 is a predicted lipase that binds Apm2 but not Apm1 and contributes to its membrane recruitment. Interactions with specific regulatory factors may provide a general mechanism to diversify the functional repertoire of clathrin adaptor complexes. PMID:26658609

  14. Measuring Differential Beliefs in Complementary Therapy Research: An Exploration of the Complementary and Alternative Medicine Beliefs Inventory (CAMBI)

    PubMed Central

    Grzywacz, Joseph G.; Neiberg, Rebecca; Quandt, Sara A.; Lang, Wei; Bell, Ronny A.; Arcury, Thomas A.

    2011-01-01

    The Complementary and Alternative Medicine Beliefs Inventory (CAMBI) was developed to provide a comprehensive measure of beliefs believed to differentiate complementary therapy (CT) users from nonusers. The initial evaluation of the CAMBI was based on a relatively homogeneous sample of CT users, which raises questions about its applicability in more generalized samples. This study uses data from a community-based sample of older adults (N=200) to evaluate the utility of the CAMBI in more diverse samples. Results indicated substantial variation in responses to items with each of a-priori belief domains (i.e., perceived value of natural treatments, preference for participation in treatments, and orientation toward holistic health) and modest inter-correlation among items within each belief domain. Confirmatory factor analysis results indicated the a-priori measurement structure provided a poor fit to obtained data. Post-hoc analyses indicated that African Americans and those with less education had less consistent responses to items within each belief domain. Revision and additional development of the CAMBI is needed to enable its use in more diverse research samples. PMID:22305249

  15. Targeted disruption of the LAMA3 gene in mice reveals abnormalities in survival and late stage differentiation of epithelial cells.

    PubMed

    Ryan, M C; Lee, K; Miyashita, Y; Carter, W G

    1999-06-14

    Laminin 5 regulates anchorage and motility of epithelial cells through integrins alpha6beta4 and alpha3beta1, respectively. We used targeted disruption of the LAMA3 gene, which encodes the alpha3 subunit of laminin 5 and other isoforms, to examine developmental functions that are regulated by adhesion to the basement membrane (BM). In homozygous null animals, profound epithelial abnormalities were detected that resulted in neonatal lethality, consistent with removal of all alpha3-laminin isoforms from epithelial BMs. Alterations in three different cellular functions were identified. First, using a novel tissue adhesion assay, we found that the mutant BM could not induce stable adhesion by integrin alpha6beta4, consistent with the presence of junctional blisters and abnormal hemidesmosomes. In the absence of laminin 5 function, we were able to detect a new ligand for integrin alpha3beta1 in the epidermal BM, suggesting that basal keratinocytes can utilize integrin alpha3beta1 to interact with an alternative ligand. Second, we identified a survival defect in mutant epithelial cells that could be rescued by exogenous laminin 5, collagen, or an antibody against integrin alpha6beta4, suggesting that signaling through beta1 or beta4 integrins is sufficient for survival. Third, we detected abnormalities in ameloblast differentiation in developing mutant incisors indicating that events downstream of adhesion are affected in mutant animals. These results indicate that laminin 5 has an important role in regulating tissue organization, gene expression, and survival of epithelium. PMID:10366601

  16. Ability or Access-Ability: Differential Item Functioning of Items on Alternate Performance-Based Assessment Tests for Students with Visual Impairments

    ERIC Educational Resources Information Center

    Zebehazy, Kim T.; Zigmond, Naomi; Zimmerman, George J.

    2012-01-01

    Introduction: This study investigated differential item functioning (DIF) of test items on Pennsylvania's Alternate System of Assessment (PASA) for students with visual impairments and severe cognitive disabilities and what the reasons for the differences may be. Methods: The Wilcoxon signed ranks test was used to analyze differences in the scores…

  17. Structure and expression of the human L-myc gene reveal a complex pattern of alternative mRNA processing

    SciTech Connect

    Kaye, F.; Battey, J.; Nau, M.; Brooks, B.; Seifter, E.; De Greve, J.; Birrer, M.; Sausville, E.; Minna, J.

    1988-01-01

    The authors' analyzed in detail the structure of the L-myc gene isolated from human placental DNA and characterized its expression in several small-cell lung cancer cell lines. The gene is composed of three exons and two introns spanning 6.6 kilobases in human DNA. Several distinct mRNA species are produced in all small-cell lung cancer cell lines that express L-myc. These transcripts are generated from a single gene by alternative splicing of introns 1 and 2 and by use of alternative polyadenylation signals. In some mRNAs that is a long open reading frame with a predicted translated protein of 364 residues. Amino acid sequence comparison with c-myc and N-myc demonstrated multiple discrete regions with extensive homology. In contrast, other mRNA transcripts, generated by alternative processing, could encode a truncated protein with a novel carboxy-terminal end.

  18. The transcriptomes of dormant leafy spurge seeds under alternating temperature are differentially affected by a germination-enhancing pretreatment.

    PubMed

    Foley, Michael E; Chao, Wun S; Horvath, David P; Doğramaci, Münevver; Anderson, James V

    2013-04-15

    Seed dormancy is an important stage in the life cycle of many non-domesticated plants, often characterized by the temporary failure to germinate under conditions that normally favor the process. Pre-treating dormant imbibed seeds at a constant temperate accelerated germination of leafy spurge seeds under alternating temperatures. However, dormant seeds will also germinate without a pre-treatment, albeit at a much slower rate, which gives rise to longer periods of imbibition before germination. Transcriptome analyses on seeds exposed to prolonged imbibition highlighted pathways associated with phenylpropanoid biosynthesis and interacting networks of genes involved in plant defense. In addition to the many pathways associated with phenylpropanoid biosynthesis enriched with down-regulated genes upon germination, there were also numerous pathways enriched with up-regulated genes associated with energy metabolism, such as glycolysis. Transcriptome data further suggest that metabolism and signaling by the plant hormones ethylene, gibberellin, and abscisic acid are involved in the developmental transition from dormancy to germination. More specifically, sub-network enrichment analysis identified ABI3 as a central hub of a sub-network at germination including several down-regulated genes such as DELLA (i.e., RGL2), which represses gibberellin signaling processes required for germination. The 595-fold increase in the expression of ACC oxidase (ACO4) at germination also suggests an important role for ethylene biosynthesis in germinating leafy surge seeds. Furthermore, the 10-578-fold difference in expression of many genes such as HY5 and Histone H3 between two populations at germination, which were treated with and without a constant temperature germination-enhancing pretreatment, revealed disparate impacts on various biosynthetic, growth, signaling, and response processes. Overall, our results indicate a constant temperature pretreatment (20°C for 21d) is not required for

  19. Vascular Cell Induction Culture System Using Arabidopsis Leaves (VISUAL) Reveals the Sequential Differentiation of Sieve Element-Like Cells.

    PubMed

    Kondo, Yuki; Nurani, Alif Meem; Saito, Chieko; Ichihashi, Yasunori; Saito, Masato; Yamazaki, Kyoko; Mitsuda, Nobutaka; Ohme-Takagi, Masaru; Fukuda, Hiroo

    2016-06-01

    Cell differentiation is a complex process involving multiple steps, from initial cell fate specification to final differentiation. Procambial/cambial cells, which act as vascular stem cells, differentiate into both xylem and phloem cells during vascular development. Recent studies have identified regulatory cascades for xylem differentiation. However, the molecular mechanism underlying phloem differentiation is largely unexplored due to technical challenges. Here, we established an ectopic induction system for phloem differentiation named Vascular Cell Induction Culture System Using Arabidopsis Leaves (VISUAL). Our results verified similarities between VISUAL-induced Arabidopsis thaliana phloem cells and in vivo sieve elements. We performed network analysis using transcriptome data with VISUAL to dissect the processes underlying phloem differentiation, eventually identifying a factor involved in the regulation of the master transcription factor gene APL Thus, our culture system opens up new avenues not only for genetic studies of phloem differentiation, but also for future investigations of multidirectional differentiation from vascular stem cells. PMID:27194709

  20. Communication is key: Reducing DEK1 activity reveals a link between cell-cell contacts and epidermal cell differentiation status.

    PubMed

    Galletti, Roberta; Ingram, Gwyneth C

    2015-01-01

    Plant epidermis development requires not only the initial acquisition of tissue identity, but also the ability to differentiate specific cell types over time and to maintain these differentiated states throughout the plant life. To set-up and maintain differentiation, plants activate specific transcriptional programs. Interfering with these programs can prevent differentiation and/or force differentiated cells to lose their identity and re-enter a proliferative state. We have recently shown that the Arabidopsis Defective Kernel 1 (DEK1) protein is required both for the differentiation of epidermal cells and for the maintenance of their fully differentiated state. Defects in DEK1 activity lead to a deregulation of the expression of epidermis-specific differentiation-promoting HD-ZIP IV transcription factors. Here we propose a working model in which DEK1, by maintaining cell-cell contacts, and thus communication between neighboring cells, influences HD-ZIP IV gene expression and epidermis differentiation. PMID:27064205

  1. Microsatellites reveal substantial among-population genetic differentiation and strong inbreeding in the relict fern Dryopteris aemula

    PubMed Central

    Jiménez, Ares; Holderegger, Rolf; Csencsics, Daniela; Quintanilla, Luis G.

    2010-01-01

    Background and Aims A previous study detected no allozyme diversity in Iberian populations of the buckler-fern Dryopteris aemula. The use of a more sensitive marker, such as microsatellites, was thus needed to reveal the genetic diversity, breeding system and spatial genetic structure of this species in natural populations. Methods Eight microsatellite loci for D. aemula were developed and their cross-amplification with other ferns was tested. Five polymorphic loci were used to characterize the amount and distribution of genetic diversity of D. aemula in three populations from the Iberian Peninsula and one population from the Azores. Key Results Most microsatellite markers developed were transferable to taxa close to D. aemula. Overall genetic variation was low (HT = 0·447), but was higher in the Azorean population than in the Iberian populations of this species. Among-population genetic differentiation was high (FST = 0·520). All loci strongly departed from Hardy–Weinberg equilibrium. In the population where genetic structure was studied, no spatial autocorrelation was found in any distance class. Conclusions The higher genetic diversity observed in the Azorean population studied suggested a possible refugium in this region from which mainland Europe has been recolonized after the Pleistocene glaciations. High among-population genetic differentiation indicated restricted gene flow (i.e. lack of spore exchange) across the highly fragmented area occupied by D. aemula. The deviations from Hardy–Weinberg equilibrium reflected strong inbreeding in D. aemula, a trait rarely observed in homosporous ferns. The absence of spatial genetic structure indicated effective spore dispersal over short distances. Additionally, the cross-amplification of some D. aemula microsatellites makes them suitable for use in other Dryopteris taxa. PMID:20495199

  2. Extracellular matrix of adipogenically differentiated mesenchymal stem cells reveals a network of collagen filaments, mostly interwoven by hexagonal structural units.

    PubMed

    Ullah, Mujib; Sittinger, Michael; Ringe, Jochen

    2013-01-01

    Extracellular matrix (ECM) is the non-cellular component of tissues, which not only provides biological shelter but also takes part in the cellular decisions for diverse functions. Every tissue has an ECM with unique composition and topology that governs the process of determination, differentiation, proliferation, migration and regeneration of cells. Little is known about the structural organization of matrix especially of MSC-derived adipogenic ECM. Here, we particularly focus on the composition and architecture of the fat ECM to understand the cellular behavior on functional bases. Thus, mesenchymal stem cells (MSC) were adipogenically differentiated, then, were transferred to adipogenic propagation medium, whereas they started the release of lipid droplets leaving bare network of ECM. Microarray analysis was performed, to indentify the molecular machinery of matrix. Adipogenesis was verified by Oil Red O staining of lipid droplets and by qPCR of adipogenic marker genes PPARG and FABP4. Antibody staining demonstrated the presence of collagen type I, II and IV filaments, while alkaline phosphatase activity verified the ossified nature of these filaments. In the adipogenic matrix, the hexagonal structures were abundant followed by octagonal structures, whereas they interwoven in a crisscross manner. Regarding molecular machinery of adipogenic ECM, the bioinformatics analysis revealed the upregulated expression of COL4A1, ITGA7, ITGA7, SDC2, ICAM3, ADAMTS9, TIMP4, GPC1, GPC4 and downregulated expression of COL14A1, ADAMTS5, TIMP2, TIMP3, BGN, LAMA3, ITGA2, ITGA4, ITGB1, ITGB8, CLDN11. Moreover, genes associated with integrins, glycoproteins, laminins, fibronectins, cadherins, selectins and linked signaling pathways were found. Knowledge of the interactive-language between cells and matrix could be beneficial for the artificial designing of biomaterials and bioscaffolds. PMID:23851162

  3. Fluorescently Tagged pUL47 of Marek's Disease Virus Reveals Differential Tissue Expression of the Tegument Protein In Vivo

    PubMed Central

    Arndt, Sina; Kaufer, Benedikt B.; Osterrieder, Nikolaus

    2012-01-01

    Marek's disease virus (MDV), a lymphotropic alphaherpesvirus, causes Marek's disease (MD) in chickens. MD is characterized by neurological signs, chronic wasting, and T cell lymphomas that predominate in the visceral organs. MDV replicates in a highly cell-associated manner in vitro and in vivo, with infectious virus particles being released only from feather follicle epithelial (FFE) cells in the skin. Virus produced and shed from FFE cells allows transmission of MDV from infected to naïve chickens, but the mechanisms or roles of differential virus gene expression have remained elusive. Here, we generated recombinant MDV in which we fused enhanced green fluorescent protein (EGFP) to the C terminus of the tegument protein pUL47 (vUL47-EGFP) or pUL49 (vUL49-EGFP). While vUL49-EGFP was highly attenuated in vitro and in vivo, vUL47-EGFP showed unaltered pathogenic potential and stable production of pUL47-EGFP, which facilitated direct analysis of pUL47 expression in cells and tissues. Our studies revealed that pUL47-EGFP is expressed at low levels and localizes to the nucleus during lytic replication in vitro and in lymphocytes in the spleen in vivo, while it is undetectable in tumors. In contrast, pUL47-EGFP is highly abundant and localizes predominantly in the cytoplasm in FFE cells in the skin, where MDV is shed into the environment. We concluded that differential expression and localization of MDV pUL47-EGFP tegument protein is potentially important for the unique cell-associated nature of MDV in vitro and in lymphocytes in vivo, as well as production of free virus in FFE cells. PMID:22190714

  4. Fluorescently tagged pUL47 of Marek's disease virus reveals differential tissue expression of the tegument protein in vivo.

    PubMed

    Jarosinski, Keith W; Arndt, Sina; Kaufer, Benedikt B; Osterrieder, Nikolaus

    2012-03-01

    Marek's disease virus (MDV), a lymphotropic alphaherpesvirus, causes Marek's disease (MD) in chickens. MD is characterized by neurological signs, chronic wasting, and T cell lymphomas that predominate in the visceral organs. MDV replicates in a highly cell-associated manner in vitro and in vivo, with infectious virus particles being released only from feather follicle epithelial (FFE) cells in the skin. Virus produced and shed from FFE cells allows transmission of MDV from infected to naïve chickens, but the mechanisms or roles of differential virus gene expression have remained elusive. Here, we generated recombinant MDV in which we fused enhanced green fluorescent protein (EGFP) to the C terminus of the tegument protein pUL47 (vUL47-EGFP) or pUL49 (vUL49-EGFP). While vUL49-EGFP was highly attenuated in vitro and in vivo, vUL47-EGFP showed unaltered pathogenic potential and stable production of pUL47-EGFP, which facilitated direct analysis of pUL47 expression in cells and tissues. Our studies revealed that pUL47-EGFP is expressed at low levels and localizes to the nucleus during lytic replication in vitro and in lymphocytes in the spleen in vivo, while it is undetectable in tumors. In contrast, pUL47-EGFP is highly abundant and localizes predominantly in the cytoplasm in FFE cells in the skin, where MDV is shed into the environment. We concluded that differential expression and localization of MDV pUL47-EGFP tegument protein is potentially important for the unique cell-associated nature of MDV in vitro and in lymphocytes in vivo, as well as production of free virus in FFE cells. PMID:22190714

  5. Negative differential mobility for negative carriers as revealed by space charge measurements on crosslinked polyethylene insulated model cables

    SciTech Connect

    Teyssedre, G. Laurent, C.; Vu, T. T. N.

    2015-12-21

    Among features observed in polyethylene materials under relatively high field, space charge packets, consisting in a pulse of net charge that remains in the form of a pulse as it crosses the insulation, are repeatedly observed but without complete theory explaining their formation and propagation. Positive charge packets are more often reported, and the models based on negative differential mobility(NDM) for the transport of holes could account for some charge packets phenomenology. Conversely, NDM for electrons transport has never been reported so far. The present contribution reports space charge measurements by pulsed electroacoustic method on miniature cables that are model of HVDC cables. The measurements were realized at room temperature or with a temperature gradient of 10 °C through the insulation under DC fields on the order 30–60 kV/mm. Space charge results reveal systematic occurrence of a negative front of charges generated at the inner electrode that moves toward the outer electrode at the beginning of the polarization step. It is observed that the transit time of the front of negative charge increases, and therefore the mobility decreases, with the applied voltage. Further, the estimated mobility, in the range 10{sup −14}–10{sup −13} m{sup 2} V{sup −1} s{sup −1} for the present results, increases when the temperature increases for the same condition of applied voltage. The features substantiate the hypothesis of negative differential mobility used for modelling space charge packets.

  6. Comparative genomics in acid mine drainage biofilm communities reveals metabolic and structural differentiation of co-occurring archaea

    PubMed Central

    2013-01-01

    Background Metal sulfide mineral dissolution during bioleaching and acid mine drainage (AMD) formation creates an environment that is inhospitable to most life. Despite dominance by a small number of bacteria, AMD microbial biofilm communities contain a notable variety of coexisting and closely related Euryarchaea, most of which have defied cultivation efforts. For this reason, we used metagenomics to analyze variation in gene content that may contribute to niche differentiation among co-occurring AMD archaea. Our analyses targeted members of the Thermoplasmatales and related archaea. These results greatly expand genomic information available for this archaeal order. Results We reconstructed near-complete genomes for uncultivated, relatively low abundance organisms A-, E-, and Gplasma, members of Thermoplasmatales order, and for a novel organism, Iplasma. Genomic analyses of these organisms, as well as Ferroplasma type I and II, reveal that all are facultative aerobic heterotrophs with the ability to use many of the same carbon substrates, including methanol. Most of the genomes share genes for toxic metal resistance and surface-layer production. Only Aplasma and Eplasma have a full suite of flagellar genes whereas all but the Ferroplasma spp. have genes for pili production. Cryogenic-electron microscopy (cryo-EM) and tomography (cryo-ET) strengthen these metagenomics-based ultrastructural predictions. Notably, only Aplasma, Gplasma and the Ferroplasma spp. have predicted iron oxidation genes and Eplasma and Iplasma lack most genes for cobalamin, valine, (iso)leucine and histidine synthesis. Conclusion The Thermoplasmatales AMD archaea share a large number of metabolic capabilities. All of the uncultivated organisms studied here (A-, E-, G-, and Iplasma) are metabolically very similar to characterized Ferroplasma spp., differentiating themselves mainly in their genetic capabilities for biosynthesis, motility, and possibly iron oxidation. These results indicate that

  7. Estradiol differentially induces progesterone receptor isoforms expression through alternative promoter regulation in a mouse embryonic hypothalamic cell line.

    PubMed

    Vázquez-Martínez, Edgar Ricardo; Camacho-Arroyo, Ignacio; Zarain-Herzberg, Angel; Rodríguez, María Carmen; Mendoza-Garcés, Luciano; Ostrosky-Wegman, Patricia; Cerbón, Marco

    2016-06-01

    Progesterone receptor (PR) presents two main isoforms (PR-A and PR-B) that are regulated by two specific promoters and transcribed from alternative transcriptional start sites. The molecular regulation of PR isoforms expression in embryonic hypothalamus is poorly understood. The aim of the present study was to assess estradiol regulation of PR isoforms in a mouse embryonic hypothalamic cell line (mHypoE-N42), as well as the transcriptional status of their promoters. MHypoE-N42 cells were treated with estradiol for 6 and 12 h. Then, Western blot, real-time quantitative reverse transcription polymerase chain reaction, and chromatin and DNA immunoprecipitation experiments were performed. PR-B expression was transiently induced by estradiol after 6 h of treatment in an estrogen receptor alpha (ERα)-dependent manner. This induction was associated with an increase in ERα phosphorylation (serine 118) and its recruitment to PR-B promoter. After 12 h of estradiol exposure, a downregulation of this PR isoform was associated with a decrease of specific protein 1, histone 3 lysine 4 trimethylation, and RNA polymerase II occupancy on PR-B promoter, without changes in DNA methylation and hydroxymethylation. In contrast, there were no estradiol-dependent changes in PR-A expression that could be related with the epigenetic marks or the transcription factors evaluated. We demonstrate that PR isoforms are differentially regulated by estradiol and that the induction of PR-B expression is associated to specific transcription factors interactions and epigenetic changes in its promoter in embryonic hypothalamic cells. PMID:26676302

  8. Gene expression profiling reveals differentially expressed genes in ovarian cancer of the hen: support for oviductal origin?

    PubMed

    Treviño, Lindsey S; Giles, James R; Wang, Wei; Urick, Mary Ellen; Johnson, Patricia Ann

    2010-08-01

    Ovarian cancer has a high mortality rate due, in part, to the lack of early detection and incomplete understanding of the origin of the disease. The hen is the only spontaneous model of ovarian cancer and can therefore aid in the identification and testing of early detection strategies and therapeutics. Our aim was to combine the use of the hen animal model and microarray technology to identify differentially expressed genes in ovarian tissue from normal hens compared with hens with ovarian cancer. We found that the transcripts up-regulated in chicken ovarian tumors were enriched for oviduct-related genes. Quantitative real-time PCR and immunohistochemistry confirmed expression of oviduct-related genes in normal oviduct and in ovaries from hens with early- and late-stage ovarian tumors, but not in normal ovarian surface epithelium. In addition, one of the oviduct-related genes identified in our analysis, paired box 2 has been implicated in human ovarian cancer and may serve as a marker of the disease. Furthermore, estrogen receptor 1 mRNA is over-expressed in early-stage tumors, suggesting that expression of the oviduct-related genes may be regulated by estrogen. We have also identified oviduct-related genes that encode secreted proteins that could represent putative serum biomarkers. The expression of oviduct-related genes in early-stage tumors is similar to what is seen in human ovarian cancer, with tumors resembling normal Müllerian epithelium. These data suggest that chicken ovarian tumors may arise from alternative sites, including the oviduct. PMID:21761365

  9. HEB-deficient T-cell precursors lose T-cell potential and adopt an alternative pathway of differentiation.

    PubMed

    Braunstein, Marsela; Anderson, Michele K

    2011-03-01

    Early thymocytes possess multilineage potential, which is progressively restricted as cells transit through the double-negative stages of T-cell development. DN1 cells retain the ability to become natural killer cells, dendritic cells, B cells, and myeloid cells as well as T cells, but these options are lost by the DN3 stage. The Notch1 signaling pathway is indispensable for initiation of the T-cell lineage and inhibitory for the B-cell lineage, but the regulatory mechanisms by which the T-cell fate is locked in are largely undefined. Previously, we discovered that the E-protein transcription factor HEBAlt promoted T-cell specification. Here, we report that HEB(-/-) T-cell precursors have compromised Notch1 function and lose T-cell potential. Moreover, reconstituting HEB(-/-) precursors with Notch1 activity enforced fidelity to the T-cell fate. However, instead of becoming B cells, HEB(-/-) DN3 cells adopted a DN1-like phenotype and could be induced to differentiate into thymic NK cells. HEB(-/-) DN1-like cells retained GATA3 and Id2 expression but had lower levels of the Bcl11b gene, a Notch target gene. Therefore, our studies have revealed a new set of interactions between HEB, Notch1, and GATA3 that regulate the T-cell fate choice in developing thymocytes. PMID:21189289

  10. Pronounced genetic differentiation and recent secondary contact in the mangrove tree Lumnitzera racemosa revealed by population genomic analyses

    PubMed Central

    Li, Jianfang; Yang, Yuchen; Chen, Qipian; Fang, Lu; He, Ziwen; Guo, Wuxia; Qiao, Sitan; Wang, Zhengzhen; Guo, Miaomiao; Zhong, Cairong; Zhou, Renchao; Shi, Suhua

    2016-01-01

    Systematically investigating the impacts of Pleistocene sea-level fluctuations on mangrove plants may provide a better understanding of their demographic history and useful information for their conservation. Therefore, we conducted population genomic analyses of 88 nuclear genes to explore the population dynamics of a mangrove tree Lumnitzera racemosa across the Indo-West Pacific region. Our results revealed pronounced genetic differentiation in this species between the populations from the Indian Ocean and the Pacific Ocean, which may be attributable to the long-term isolation between the western and eastern coasts of the Malay Peninsula during sea-level drops in the Pleistocene glacial periods. The mixing of haplotypes from the two highly divergent groups was identified in a Cambodian population at almost all 88 nuclear genes, suggesting genetic admixture of the two lineages at the boundary region. Similar genetic admixture was also found in other populations from Southeast Asia based on the Bayesian clustering analysis of six nuclear genes, which suggests extensive and recent secondary contact of the two divergent lineages in Southeast Asia. Computer simulations indicated substantial migration from the Indian Ocean towards the South China Sea, which likely results in the genetic admixture in Southeast Asia. PMID:27380895

  11. Proteome profiling of the growth phases of Leishmania pifanoi promastigotes in axenic culture reveals differential abundance of immunostimulatory proteins.

    PubMed

    Alcolea, Pedro J; Alonso, Ana; García-Tabares, Francisco; Mena, María del Carmen; Ciordia, Sergio; Larraga, Vicente

    2016-06-01

    Leishmaniasis is a term that encompasses a compendium of neglected tropical diseases caused by dimorphic and digenetic protozoan parasites from the genus Leishmania (Kinetoplastida: Trypanosomatidae). The clinical manifestations of neotropical cutaneous leishmaniasis (NCL) caused by Leishmania pifanoi and other species of the "Leishmania mexicana complex" mainly correspond to anergic diffuse cutaneous leishmaniasis (ADCL), which is the origin of considerable morbidity. Despite the outstanding advances in the characterization of the trypanosomatid genomes and proteomes, the biology of this species has been scarcely explored. However, the close relation of L. pifanoi to the sequenced species L. mexicana and others included in the "L. mexicana complex" allowed us to perform a two-dimension electrophoresis (2DE) approach to the promastigote proteome at the differential expression level. Protein identifications were performed by matrix-assisted laser desorption-ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF). This insight has revealed similarities and differences between L. pifanoi and other species responsible for cutaneous and visceral leishmaniasis. Interestingly, certain proteins that were previously described as immunostimulatory (elongation factor 1β, trypanothione peroxidase, heat shock protein 70, enolase, GDP-forming succinyl-CoA and aldehyde dehydrogenase) are more abundant in the final growth stages of promastigotes (late-logarithmic and/or stationary phase) in the case of L. pifanoi. PMID:26992294

  12. Transcriptome analysis reveals regulatory networks underlying differential susceptibility to Botrytis cinerea in response to nitrogen availability in Solanum lycopersicum

    PubMed Central

    Vega, Andrea; Canessa, Paulo; Hoppe, Gustavo; Retamal, Ignacio; Moyano, Tomas C.; Canales, Javier; Gutiérrez, Rodrigo A.; Rubilar, Joselyn

    2015-01-01

    Nitrogen (N) is one of the main limiting nutrients for plant growth and crop yield. It is well documented that changes in nitrate availability, the main N source found in agricultural soils, influences a myriad of developmental programs and processes including the plant defense response. Indeed, many agronomical reports indicate that the plant N nutritional status influences their ability to respond effectively when challenged by different pathogens. However, the molecular mechanisms involved in N-modulation of plant susceptibility to pathogens are poorly characterized. In this work, we show that Solanum lycopersicum defense response to the necrotrophic fungus Botrytis cinerea is affected by plant N availability, with higher susceptibility in nitrate-limiting conditions. Global gene expression responses of tomato against B. cinerea under contrasting nitrate conditions reveals that plant primary metabolism is affected by the fungal infection regardless of N regimes. This result suggests that differential susceptibility to pathogen attack under contrasting N conditions is not only explained by a metabolic alteration. We used a systems biology approach to identify the transcriptional regulatory network implicated in plant response to the fungus infection under contrasting nitrate conditions. Interestingly, hub genes in this network are known key transcription factors involved in ethylene and jasmonic acid signaling. This result positions these hormones as key integrators of nitrate and defense against B. cinerea in tomato plants. Our results provide insights into potential crosstalk mechanisms between necrotrophic defense response and N status in plants. PMID:26583019

  13. Pronounced genetic differentiation and recent secondary contact in the mangrove tree Lumnitzera racemosa revealed by population genomic analyses.

    PubMed

    Li, Jianfang; Yang, Yuchen; Chen, Qipian; Fang, Lu; He, Ziwen; Guo, Wuxia; Qiao, Sitan; Wang, Zhengzhen; Guo, Miaomiao; Zhong, Cairong; Zhou, Renchao; Shi, Suhua

    2016-01-01

    Systematically investigating the impacts of Pleistocene sea-level fluctuations on mangrove plants may provide a better understanding of their demographic history and useful information for their conservation. Therefore, we conducted population genomic analyses of 88 nuclear genes to explore the population dynamics of a mangrove tree Lumnitzera racemosa across the Indo-West Pacific region. Our results revealed pronounced genetic differentiation in this species between the populations from the Indian Ocean and the Pacific Ocean, which may be attributable to the long-term isolation between the western and eastern coasts of the Malay Peninsula during sea-level drops in the Pleistocene glacial periods. The mixing of haplotypes from the two highly divergent groups was identified in a Cambodian population at almost all 88 nuclear genes, suggesting genetic admixture of the two lineages at the boundary region. Similar genetic admixture was also found in other populations from Southeast Asia based on the Bayesian clustering analysis of six nuclear genes, which suggests extensive and recent secondary contact of the two divergent lineages in Southeast Asia. Computer simulations indicated substantial migration from the Indian Ocean towards the South China Sea, which likely results in the genetic admixture in Southeast Asia. PMID:27380895

  14. Differential Features between Chronic Skin Inflammatory Diseases Revealed in Skin-Humanized Psoriasis and Atopic Dermatitis Mouse Models.

    PubMed

    Carretero, Marta; Guerrero-Aspizua, Sara; Illera, Nuria; Galvez, Victoria; Navarro, Manuel; García-García, Francisco; Dopazo, Joaquin; Jorcano, Jose Luis; Larcher, Fernando; del Rio, Marcela

    2016-01-01

    Psoriasis and atopic dermatitis are chronic and relapsing inflammatory diseases of the skin affecting a large number of patients worldwide. Psoriasis is characterized by a T helper type 1 and/or T helper type 17 immunological response, whereas acute atopic dermatitis lesions exhibit T helper type 2-dominant inflammation. Current single gene and signaling pathways-based models of inflammatory skin diseases are incomplete. Previous work allowed us to model psoriasis in skin-humanized mice through proper combinations of inflammatory cell components and disruption of barrier function. Herein, we describe and characterize an animal model for atopic dermatitis using similar bioengineered-based approaches, by intradermal injection of human T helper type 2 lymphocytes in regenerated human skin after partial removal of stratum corneum. In this work, we have extensively compared this model with the previous and an improved version of the psoriasis model, in which T helper type 1 and/or T helper type 17 lymphocytes replace exogenous cytokines. Comparative expression analyses revealed marked differences in specific epidermal proliferation and differentiation markers and immune-related molecules, including antimicrobial peptides. Likewise, the composition of the dermal inflammatory infiltrate presented important differences. The availability of accurate and reliable animal models for these diseases will contribute to the understanding of the pathogenesis and provide valuable tools for drug development and testing. PMID:26763433

  15. Transcriptome analysis reveals regulatory networks underlying differential susceptibility to Botrytis cinerea in response to nitrogen availability in Solanum lycopersicum.

    PubMed

    Vega, Andrea; Canessa, Paulo; Hoppe, Gustavo; Retamal, Ignacio; Moyano, Tomas C; Canales, Javier; Gutiérrez, Rodrigo A; Rubilar, Joselyn

    2015-01-01

    Nitrogen (N) is one of the main limiting nutrients for plant growth and crop yield. It is well documented that changes in nitrate availability, the main N source found in agricultural soils, influences a myriad of developmental programs and processes including the plant defense response. Indeed, many agronomical reports indicate that the plant N nutritional status influences their ability to respond effectively when challenged by different pathogens. However, the molecular mechanisms involved in N-modulation of plant susceptibility to pathogens are poorly characterized. In this work, we show that Solanum lycopersicum defense response to the necrotrophic fungus Botrytis cinerea is affected by plant N availability, with higher susceptibility in nitrate-limiting conditions. Global gene expression responses of tomato against B. cinerea under contrasting nitrate conditions reveals that plant primary metabolism is affected by the fungal infection regardless of N regimes. This result suggests that differential susceptibility to pathogen attack under contrasting N conditions is not only explained by a metabolic alteration. We used a systems biology approach to identify the transcriptional regulatory network implicated in plant response to the fungus infection under contrasting nitrate conditions. Interestingly, hub genes in this network are known key transcription factors involved in ethylene and jasmonic acid signaling. This result positions these hormones as key integrators of nitrate and defense against B. cinerea in tomato plants. Our results provide insights into potential crosstalk mechanisms between necrotrophic defense response and N status in plants. PMID:26583019

  16. Host and non-host roots in rice: cellular and molecular approaches reveal differential responses to arbuscular mycorrhizal fungi

    PubMed Central

    Fiorilli, Valentina; Vallino, Marta; Biselli, Chiara; Faccio, Antonella; Bagnaresi, Paolo; Bonfante, Paola

    2015-01-01

    Oryza sativa, a model plant for Arbuscular Mycorrhizal (AM) symbiosis, has both host and non-host roots. Large lateral (LLR) and fine lateral (FLR) roots display opposite responses: LLR support AM colonization, but FLR do not. Our research aimed to study the molecular, morphological and physiological aspects related to the non-host behavior of FLR. RNA-seq analysis revealed that LLR and FLR displayed divergent expression profiles, including changes in many metabolic pathways. Compared with LLR, FLR showed down-regulation of genes instrumental for AM establishment and gibberellin signaling, and a higher expression of nutrient transporters. Consistent with the transcriptomic data, FLR had higher phosphorus content. Light and electron microscopy demonstrated that, surprisingly, in the Selenio cultivar, FLR have a two-layered cortex, which is theoretically compatible with AM colonization. According to RNA-seq, a gibberellin inhibitor treatment increased anticlinal divisions leading to a higher number of cortex cells in FLR. We propose that some of the differentially regulated genes that lead to the anatomical and physiological properties of the two root types also function as genetic factors regulating fungal colonization. The rice root apparatus offers a unique tool to study AM symbiosis, allowing direct comparisons of host and non-host roots in the same individual plant. PMID:26322072

  17. Poly(A) code analyses reveal key determinants for tissue-specific mRNA alternative polyadenylation.

    PubMed

    Weng, Lingjie; Li, Yi; Xie, Xiaohui; Shi, Yongsheng

    2016-06-01

    mRNA alternative polyadenylation (APA) is a critical mechanism for post-transcriptional gene regulation and is often regulated in a tissue- and/or developmental stage-specific manner. An ultimate goal for the APA field has been to be able to computationally predict APA profiles under different physiological or pathological conditions. As a first step toward this goal, we have assembled a poly(A) code for predicting tissue-specific poly(A) sites (PASs). Based on a compendium of over 600 features that have known or potential roles in PAS selection, we have generated and refined a machine-learning algorithm using multiple high-throughput sequencing-based data sets of tissue-specific and constitutive PASs. This code can predict tissue-specific PASs with >85% accuracy. Importantly, by analyzing the prediction performance based on different RNA features, we found that PAS context, including the distance between alternative PASs and the relative position of a PAS within the gene, is a key feature for determining the susceptibility of a PAS to tissue-specific regulation. Our poly(A) code provides a useful tool for not only predicting tissue-specific APA regulation, but also for studying its underlying molecular mechanisms. PMID:27095026

  18. Distinct proteins encoded by alternative transcripts of the PURG gene, located contrapodal to WRN on chromosome 8, determined by differential termination/polyadenylation.

    PubMed

    Liu, Hong; Johnson, Edward M

    2002-06-01

    A gene encoding a new member of the Pur protein family, Purgamma, has been detected upstream of, and contrapodal to, the gene encoding the Werner syndrome helicase, Wrn, at human chromosome band 8p11-12. Both the PURG and WRN genes initiate transcription at multiple sites, the major clusters of which are approximately 90 bp apart. A segment containing this region strongly promotes transcription of a reporter gene in both directions. Both promoters are TATA-less and CAAT-less and both are positively regulated by Sp1 elements. While promoter elements for the two genes are interleaved, in the contrapodal direction, certain elements critical for each gene are distinct. Sequencing of cDNAs for Purgamma mRNA reveals that two alternative coding sequences are generated from a single gene, resulting in different Purgamma C-termini. PURG-A mRNA consists of a single intronless transcript of approximately 3 kb. PURG-B mRNA results from transcription through the PURG-A polyadenylation site and splicing out of an intron of >30 kb. In this unique example of a switch, splicing of a single intron either occurs or does not occur depending upon differential termination/polyadenylation. PURG-B is the primary PURG transcript detected in testis, but it is undetectable in all members of a normal adult tissue cDNA panel. PURG-A levels are low or undetectable in the normal tissue panel, but they are greatly elevated in all members of a tumor tissue panel. PURG-B is detected in several tumor panel members. PMID:12034829

  19. Mobility and age of black carbon in two temperate grassland soils revealed by differential scanning calorimetry and radiocarbon dating

    NASA Astrophysics Data System (ADS)

    Leifeld, Jens; Feng, Xiaojuan; Eglinton, Timothy; Wacker, Lukas

    2015-04-01

    Black carbon (BC) is a natural component of soil organic matter (SOM) and abundant in many ecosystems. Its stability, due to its relative resistance to microbial decomposition, means it plays an important role in soil C sequestration. A recent review suggests that BC may be mobile in soil; hence, its contribution to a stable SOM pool may change over time due to its lateral or vertical reallocation (Rumpel et al. 2014). However, direct evidence of the mobility of BC, particularly with reference to its vertical mobility, is scarce. We studied the amount of BC in two temperate grassland fields (eutric clayey Camibsol,) that were established in 2001 on former cropland. Volumetric soil samples (0-50 cm, 5 cm increments) were taken at 10 spots in each field in 2001, 2006 and 2011. One of the fields was ploughed in 2007 and the sward was re-sown. BC content was measured by differential scanning calorimetry for a total number of c. 500 samples. The mean BC/OC ratio was 0.10 (±0.05) and reached 0.25 in some samples. Radiocarbon measurements from 24 bulk soil samples revealed relatively small 14C contents in 2001 (92±2.7 pMC) which increased over time (2006: 99.0±1.1 pMC; 2011: 99.1±1.1 pMC). Thermal fractionation of BC by DSC revealed calibrated BC ages of 400 to 1000 years (pMC 87-94), suggesting that BC originates from medieval and post-medieval fire clearings. The change in soil signature may have been caused by a preferential transport of old BC down the soil profile, leading to a selective enrichment of younger soil C over time. In line with this interpretation the DSC measurements suggest that in both fields, BC concentrations significantly decreased for most layers between 2001 and 2006. However, between 2006 and 2011, no further vertical reallocation was observed in the continuous grassland, whereas BC contents of the field ploughed in 2007 significantly increased in the top layers. Together, these data suggest that ploughing in 2001 triggered subsequent

  20. Vasculature-specific MRI reveals differential anti-angiogenic effects of a biomimetic peptide in an orthotopic breast cancer model.

    PubMed

    Kim, Eugene; Lee, Esak; Plummer, Charlesa; Gil, Stacy; Popel, Aleksander S; Pathak, Arvind P

    2015-04-01

    Translational vasculature-specific MRI biomarkers were used to measure the effects of a novel anti-angiogenic biomimetic peptide in an orthotopic MDA-MB-231 human triple-negative breast cancer model at an early growth stage. In vivo diffusion-weighted and steady-state susceptibility contrast (SSC) MRI was performed pre-treatment and 2 weeks post-treatment in tumor volume-matched treatment and control groups (n = 5/group). Treatment response was measured by changes in tumor volume; baseline transverse relaxation time (T2); apparent diffusion coefficient (ADC); and SSC-MRI metrics of blood volume, vessel size, and vessel density. These vasculature-specific SSC-MRI biomarkers were compared to the more conventional, non-vascular biomarkers (tumor growth, ADC, and T2) in terms of their sensitivity to anti-angiogenic treatment response. After 2 weeks of peptide treatment, tumor growth inhibition was evident but not yet significant, and the changes in ADC or T2 were not significantly different between treated and control groups. In contrast, the vascular MRI biomarkers revealed a significant anti-angiogenic response to the peptide after 2 weeks—blood volume and vessel size decreased, and vessel density increased in treated tumors; the opposite was seen in control tumors. The MRI results were validated with histology—H&E staining showed no difference in tumor viability between groups, while peptide-treated tumors exhibited decreased vascularity. These results indicate that translational SSC-MRI biomarkers are able to detect the differential effects of anti-angiogenic therapy on the tumor vasculature before significant tumor growth inhibition or changes in tumor viability. PMID:25408417

  1. Terminal differentiation of osteogenic cells in the embryonic chick tibia is revealed by a monoclonal antibody against osteocytes.

    PubMed

    Bruder, S P; Caplan, A I

    1990-01-01

    Monoclonal antibodies against the surface of embryonic osteogenic cells have been used to characterize the sequence of transitions involved in the osteoblastic cell lineage. These previous data identified distinct cell stages within the osteogenic lineage, but were incomplete. To further refine and extend these observations, additional monoclonal antibodies were generated against the surface of osteogenic cells by immunizing mice with a heterogeneous population of chick embryonic bone cells. Supernatants from growing hybridoma colonies were immunohistochemically screened against frozen sections of stage 35 (day 9.5) chick tibiae. One cell line, SB-5, which secretes an antibody against the surface of osteogenic cells was successfully cloned, stabilized, and immortalized. Studies on the developmental progression of osteogenesis in the embryonic chick tibia reveal that cells within the lineage stages from Pre-Osteoblast to Secretory Osteoblast were never observed to react with antibody SB-5 at any time. By contrast, strong cell surface immunoreactivity was present on mature osteoblastic cells as they became Osteocytes. Furthermore, in cultures of osteogenic cells derived from embryonic calvaria or tibiae, cells possessing the SB-5 antigen on their surface displayed a morphology remarkably similar to that of Osteocytes found in situ. Double immunofluorescent staining of developing chick tibiae with SB-5 and SB-2, a monoclonal antibody directed against the surface of Secretory Osteoblasts, indicates that these cells proceed through an intermediate lineage step before becoming terminally differentiated Osteocytes. This transitory cell state is characterized by the simultaneous cell surface binding of antibodies SB-2 and SB-5, and is referred to as the Osteocytic Osteoblast stage.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2202356

  2. Systems biology of tissue-specific response to Anaplasma phagocytophilum reveals differentiated apoptosis in the tick vector Ixodes scapularis.

    PubMed

    Ayllón, Nieves; Villar, Margarita; Galindo, Ruth C; Kocan, Katherine M; Šíma, Radek; López, Juan A; Vázquez, Jesús; Alberdi, Pilar; Cabezas-Cruz, Alejandro; Kopáček, Petr; de la Fuente, José

    2015-03-01

    Anaplasma phagocytophilum is an emerging pathogen that causes human granulocytic anaplasmosis. Infection with this zoonotic pathogen affects cell function in both vertebrate host and the tick vector, Ixodes scapularis. Global tissue-specific response and apoptosis signaling pathways were characterized in I. scapularis nymphs and adult female midguts and salivary glands infected with A. phagocytophilum using a systems biology approach combining transcriptomics and proteomics. Apoptosis was selected for pathway-focused analysis due to its role in bacterial infection of tick cells. The results showed tissue-specific differences in tick response to infection and revealed differentiated regulation of apoptosis pathways. The impact of bacterial infection was more pronounced in tick nymphs and midguts than in salivary glands, probably reflecting bacterial developmental cycle. All apoptosis pathways described in other organisms were identified in I. scapularis, except for the absence of the Perforin ortholog. Functional characterization using RNA interference showed that Porin knockdown significantly increases tick colonization by A. phagocytophilum. Infection with A. phagocytophilum produced complex tissue-specific alterations in transcript and protein levels. In tick nymphs, the results suggested a possible effect of bacterial infection on the inhibition of tick immune response. In tick midguts, the results suggested that A. phagocytophilum infection inhibited cell apoptosis to facilitate and establish infection through up-regulation of the JAK/STAT pathway. Bacterial infection inhibited the intrinsic apoptosis pathway in tick salivary glands by down-regulating Porin expression that resulted in the inhibition of Cytochrome c release as the anti-apoptotic mechanism to facilitate bacterial infection. However, tick salivary glands may promote apoptosis to limit bacterial infection through induction of the extrinsic apoptosis pathway. These dynamic changes in response to A

  3. Multilocus sequence analysis of Thermoanaerobacter isolates reveals recombining, but differentiated, populations from geothermal springs of the Uzon Caldera, Kamchatka, Russia

    PubMed Central

    Wagner, Isaac D.; Varghese, Litty B.; Hemme, Christopher L.; Wiegel, Juergen

    2013-01-01

    Thermal environments have island-like characteristics and provide a unique opportunity to study population structure and diversity patterns of microbial taxa inhabiting these sites. Strains having ≥98% 16S rRNA gene sequence similarity to the obligately anaerobic Firmicutes Thermoanaerobacter uzonensis were isolated from seven geothermal springs, separated by up to 1600 m, within the Uzon Caldera (Kamchatka, Russian Far East). The intraspecies variation and spatial patterns of diversity for this taxon were assessed by multilocus sequence analysis (MLSA) of 106 strains. Analysis of eight protein-coding loci (gyrB, lepA, leuS, pyrG, recA, recG, rplB, and rpoB) revealed that all loci were polymorphic and that nucleotide substitutions were mostly synonymous. There were 148 variable nucleotide sites across 8003 bp concatenates of the protein-coding loci. While pairwise FST values indicated a small but significant level of genetic differentiation between most subpopulations, there was a negligible relationship between genetic divergence and spatial separation. Strains with the same allelic profile were only isolated from the same hot spring, occasionally from consecutive years, and single locus variant (SLV) sequence types were usually derived from the same spring. While recombination occurred, there was an “epidemic” population structure in which a particular T. uzonensis sequence type rose in frequency relative to the rest of the population. These results demonstrate spatial diversity patterns for an anaerobic bacterial species in a relative small geographic location and reinforce the view that terrestrial geothermal springs are excellent places to look for biogeographic diversity patterns regardless of the involved distances. PMID:23801987

  4. Molecular and iridescent feather reflectance data reveal recent genetic diversification and phenotypic differentiation in a cloud forest hummingbird.

    PubMed

    Ornelas, Juan Francisco; González, Clementina; Hernández-Baños, Blanca E; García-Moreno, Jaime

    2016-02-01

    The present day distribution and spatial genetic diversity of Mesoamerican biota reflects a long history of responses to habitat change. The hummingbird Lampornis amethystinus is distributed in northern Mesoamerica, with geographically disjunct populations. Based on sampling across the species range using mitochondrial DNA (mtDNA) sequences and nuclear microsatellites jointly analysed with phenotypic and climatic data, we (1) test whether the fragmented distribution is correlated with main evolutionary lineages, (2) assess body size and plumage color differentiation of populations in geographic isolation, and (3) evaluate a set of divergence scenarios and demographic patterns of the hummingbird populations. Analysis of genetic variation revealed four main groups: blue-throated populations (Sierra Madre del Sur); two groups of amethyst-throated populations (Trans-Mexican Volcanic Belt and Sierra Madre Oriental); and populations east of the Isthmus of Tehuantepec (IT) with males showing an amethyst throat. The most basal split is estimated to have originated in the Pleistocene, 2.39-0.57 million years ago (MYA), and corresponded to groups of populations separated by the IT. However, the estimated recent divergence time between blue- and amethyst-throated populations does not correspond to the 2-MY needed to be in isolation for substantial plumage divergence, likely because structurally iridescent colors are more malleable than others. Results of species distribution modeling and Approximate Bayesian Computation analysis fit a model of lineage divergence west of the Isthmus after the Last Glacial Maximum (LGM), and that the species' suitable habitat was disjunct during past and current conditions. These results challenge the generality of the contraction/expansion glacial model to cloud forest-interior species and urges management of cloud forest, a highly vulnerable ecosystem to climate change and currently facing destruction, to prevent further loss of genetic

  5. A Method of Accounting for Enzyme Costs in Flux Balance Analysis Reveals Alternative Pathways and Metabolite Stores in an Illuminated Arabidopsis Leaf.

    PubMed

    Cheung, C Y Maurice; Ratcliffe, R George; Sweetlove, Lee J

    2015-11-01

    Flux balance analysis of plant metabolism is an established method for predicting metabolic flux phenotypes and for exploring the way in which the plant metabolic network delivers specific outcomes in different cell types, tissues, and temporal phases. A recurring theme is the need to explore the flexibility of the network in meeting its objectives and, in particular, to establish the extent to which alternative pathways can contribute to achieving specific outcomes. Unfortunately, predictions from conventional flux balance analysis minimize the simultaneous operation of alternative pathways, but by introducing flux-weighting factors to allow for the variable intrinsic cost of supporting each flux, it is possible to activate different pathways in individual simulations and, thus, to explore alternative pathways by averaging thousands of simulations. This new method has been applied to a diel genome-scale model of Arabidopsis (Arabidopsis thaliana) leaf metabolism to explore the flexibility of the network in meeting the metabolic requirements of the leaf in the light. This identified alternative flux modes in the Calvin-Benson cycle revealed the potential for alternative transitory carbon stores in leaves and led to predictions about the light-dependent contribution of alternative electron flow pathways and futile cycles in energy rebalancing. Notable features of the analysis include the light-dependent tradeoff between the use of carbohydrates and four-carbon organic acids as transitory storage forms and the way in which multiple pathways for the consumption of ATP and NADPH can contribute to the balancing of the requirements of photosynthetic metabolism with the energy available from photon capture. PMID:26265776

  6. Systematic discovery of regulated and conserved alternative exons in the mammalian brain reveals NMD modulating chromatin regulators.

    PubMed

    Yan, Qinghong; Weyn-Vanhentenryck, Sebastien M; Wu, Jie; Sloan, Steven A; Zhang, Ye; Chen, Kenian; Wu, Jia Qian; Barres, Ben A; Zhang, Chaolin

    2015-03-17

    Alternative splicing (AS) dramatically expands the complexity of the mammalian brain transcriptome, but its atlas remains incomplete. Here we performed deep mRNA sequencing of mouse cortex to discover and characterize alternative exons with potential functional significance. Our analysis expands the list of AS events over 10-fold compared with previous annotations, demonstrating that 72% of multiexon genes express multiple splice variants in this single tissue. To evaluate functionality of the newly discovered AS events, we conducted comprehensive analyses on central nervous system (CNS) cell type-specific splicing, targets of tissue- or cell type-specific RNA binding proteins (RBPs), evolutionary selection pressure, and coupling of AS with nonsense-mediated decay (AS-NMD). We show that newly discovered events account for 23-42% of all cassette exons under tissue- or cell type-specific regulation. Furthermore, over 7,000 cassette exons are under evolutionary selection for regulated AS in mammals, 70% of which are new. Among these are 3,058 highly conserved cassette exons, including 1,014 NMD exons that may function directly to control gene expression levels. These NMD exons are particularly enriched in RBPs including splicing factors and interestingly also regulators for other steps of RNA metabolism. Unexpectedly, a second group of NMD exons reside in genes encoding chromatin regulators. Although the conservation of NMD exons in RBPs frequently extends into lower vertebrates, NMD exons in chromatin regulators are introduced later into the mammalian lineage, implying the emergence of a novel mechanism coupling AS and epigenetics. Our results highlight previously uncharacterized complexity and evolution in the mammalian brain transcriptome. PMID:25737549

  7. High-fidelity promoter profiling reveals widespread alternative promoter usage and transposon-driven developmental gene expression

    PubMed Central

    Batut, Philippe; Dobin, Alexander; Plessy, Charles; Carninci, Piero; Gingeras, Thomas R.

    2013-01-01

    Many eukaryotic genes possess multiple alternative promoters with distinct expression specificities. Therefore, comprehensively annotating promoters and deciphering their individual regulatory dynamics is critical for gene expression profiling applications and for our understanding of regulatory complexity. We introduce RAMPAGE, a novel promoter activity profiling approach that combines extremely specific 5′-complete cDNA sequencing with an integrated data analysis workflow, to address the limitations of current techniques. RAMPAGE features a streamlined protocol for fast and easy generation of highly multiplexed sequencing libraries, offers very high transcription start site specificity, generates accurate and reproducible promoter expression measurements, and yields extensive transcript connectivity information through paired-end cDNA sequencing. We used RAMPAGE in a genome-wide study of promoter activity throughout 36 stages of the life cycle of Drosophila melanogaster, and describe here a comprehensive data set that represents the first available developmental time-course of promoter usage. We found that >40% of developmentally expressed genes have at least two promoters and that alternative promoters generally implement distinct regulatory programs. Transposable elements, long proposed to play a central role in the evolution of their host genomes through their ability to regulate gene expression, contribute at least 1300 promoters shaping the developmental transcriptome of D. melanogaster. Hundreds of these promoters drive the expression of annotated genes, and transposons often impart their own expression specificity upon the genes they regulate. These observations provide support for the theory that transposons may drive regulatory innovation through the distribution of stereotyped cis-regulatory modules throughout their host genomes. PMID:22936248

  8. Spike-Interval Triggered Averaging Reveals a Quasi-Periodic Spiking Alternative for Stochastic Resonance in Catfish Electroreceptors

    PubMed Central

    Lankheet, Martin J. M.; Klink, P. Christiaan; Borghuis, Bart G.; Noest, André J.

    2012-01-01

    Catfish detect and identify invisible prey by sensing their ultra-weak electric fields with electroreceptors. Any neuron that deals with small-amplitude input has to overcome sensitivity limitations arising from inherent threshold non-linearities in spike-generation mechanisms. Many sensory cells solve this issue with stochastic resonance, in which a moderate amount of intrinsic noise causes irregular spontaneous spiking activity with a probability that is modulated by the input signal. Here we show that catfish electroreceptors have adopted a fundamentally different strategy. Using a reverse correlation technique in which we take spike interval durations into account, we show that the electroreceptors generate a supra-threshold bias current that results in quasi-periodically produced spikes. In this regime stimuli modulate the interval between successive spikes rather than the instantaneous probability for a spike. This alternative for stochastic resonance combines threshold-free sensitivity for weak stimuli with similar sensitivity for excitations and inhibitions based on single interspike intervals. PMID:22403709

  9. Flux balance analysis reveals acetate metabolism modulates cyclic electron flow and alternative glycolytic pathways in Chlamydomonas reinhardtii.

    PubMed

    Chapman, Stephen P; Paget, Caroline M; Johnson, Giles N; Schwartz, Jean-Marc

    2015-01-01

    Cells of the green alga Chlamydomonas reinhardtii cultured in the presence of acetate perform mixotrophic growth, involving both photosynthesis and organic carbon assimilation. Under such conditions, cells exhibit a reduced capacity for photosynthesis but a higher growth rate, compared to phototrophic cultures. Better understanding of the down regulation of photosynthesis would enable more efficient conversion of carbon into valuable products like biofuels. In this study, Flux Balance Analysis (FBA) and Flux Variability Analysis (FVA) have been used with a genome scale model of C. reinhardtii to examine changes in intracellular flux distribution in order to explain their changing physiology. Additionally, a reaction essentiality analysis was performed to identify which reaction subsets are essential for a given growth condition. Our results suggest that exogenous acetate feeds into a modified tricarboxylic acid (TCA) cycle, which bypasses the CO2 evolution steps, explaining increases in biomass, consistent with experimental data. In addition, reactions of the oxidative pentose phosphate and glycolysis pathways, inactive under phototrophic conditions, show substantial flux under mixotrophic conditions. Importantly, acetate addition leads to an increased flux through cyclic electron flow (CEF), but results in a repression of CO2 fixation via Rubisco, explaining the down regulation of photosynthesis. However, although CEF enhances growth on acetate, it is not essential-impairment of CEF results in alternative metabolic pathways being increased. We have demonstrated how the reactions of photosynthesis interconnect with carbon metabolism on a global scale, and how systems approaches play a viable tool in understanding complex relationships at the scale of the organism. PMID:26175742

  10. Structural Insight into Archaic and Alternative Chaperone-Usher Pathways Reveals a Novel Mechanism of Pilus Biogenesis

    PubMed Central

    Pakharukova, Natalia; Garnett, James A.; Tuittila, Minna; Paavilainen, Sari; Diallo, Mamou; Xu, Yingqi; Matthews, Steve J.; Zavialov, Anton V.

    2015-01-01

    Gram-negative pathogens express fibrous adhesive organelles that mediate targeting to sites of infection. The major class of these organelles is assembled via the classical, alternative and archaic chaperone-usher pathways. Although non-classical systems share a wider phylogenetic distribution and are associated with a range of diseases, little is known about their assembly mechanisms. Here we report atomic-resolution insight into the structure and biogenesis of Acinetobacter baumannii Csu and Escherichia coli ECP biofilm-mediating pili. We show that the two non-classical systems are structurally related, but their assembly mechanism is strikingly different from the classical assembly pathway. Non-classical chaperones, unlike their classical counterparts, maintain subunits in a substantially disordered conformational state, akin to a molten globule. This is achieved by a unique binding mechanism involving the register-shifted donor strand complementation and a different subunit carboxylate anchor. The subunit lacks the classical pre-folded initiation site for donor strand exchange, suggesting that recognition of its exposed hydrophobic core starts the assembly process and provides fresh inspiration for the design of inhibitors targeting chaperone-usher systems. PMID:26587649

  11. Feedback-Controlled Transcranial Alternating Current Stimulation Reveals a Functional Role of Sleep Spindles in Motor Memory Consolidation.

    PubMed

    Lustenberger, Caroline; Boyle, Michael R; Alagapan, Sankaraleengam; Mellin, Juliann M; Vaughn, Bradley V; Fröhlich, Flavio

    2016-08-22

    Transient episodes of brain oscillations are a common feature of both the waking and the sleeping brain. Sleep spindles represent a prominent example of a poorly understood transient brain oscillation that is impaired in disorders such as Alzheimer's disease and schizophrenia. However, the causal role of these bouts of thalamo-cortical oscillations remains unknown. Demonstrating a functional role of sleep spindles in cognitive processes has, so far, been hindered by the lack of a tool to target transient brain oscillations in real time. Here, we show, for the first time, selective enhancement of sleep spindles with non-invasive brain stimulation in humans. We developed a system that detects sleep spindles in real time and applies oscillatory stimulation. Our stimulation selectively enhanced spindle activity as determined by increased sigma activity after transcranial alternating current stimulation (tACS) application. This targeted modulation caused significant enhancement of motor memory consolidation that correlated with the stimulation-induced change in fast spindle activity. Strikingly, we found a similar correlation between motor memory and spindle characteristics during the sham night for the same spindle frequencies and electrode locations. Therefore, our results directly demonstrate a functional relationship between oscillatory spindle activity and cognition. PMID:27476602

  12. Spike-interval triggered averaging reveals a quasi-periodic spiking alternative for stochastic resonance in catfish electroreceptors.

    PubMed

    Lankheet, Martin J M; Klink, P Christiaan; Borghuis, Bart G; Noest, André J

    2012-01-01

    Catfish detect and identify invisible prey by sensing their ultra-weak electric fields with electroreceptors. Any neuron that deals with small-amplitude input has to overcome sensitivity limitations arising from inherent threshold non-linearities in spike-generation mechanisms. Many sensory cells solve this issue with stochastic resonance, in which a moderate amount of intrinsic noise causes irregular spontaneous spiking activity with a probability that is modulated by the input signal. Here we show that catfish electroreceptors have adopted a fundamentally different strategy. Using a reverse correlation technique in which we take spike interval durations into account, we show that the electroreceptors generate a supra-threshold bias current that results in quasi-periodically produced spikes. In this regime stimuli modulate the interval between successive spikes rather than the instantaneous probability for a spike. This alternative for stochastic resonance combines threshold-free sensitivity for weak stimuli with similar sensitivity for excitations and inhibitions based on single interspike intervals. PMID:22403709

  13. Flux balance analysis reveals acetate metabolism modulates cyclic electron flow and alternative glycolytic pathways in Chlamydomonas reinhardtii

    PubMed Central

    Chapman, Stephen P.; Paget, Caroline M.; Johnson, Giles N.; Schwartz, Jean-Marc

    2015-01-01

    Cells of the green alga Chlamydomonas reinhardtii cultured in the presence of acetate perform mixotrophic growth, involving both photosynthesis and organic carbon assimilation. Under such conditions, cells exhibit a reduced capacity for photosynthesis but a higher growth rate, compared to phototrophic cultures. Better understanding of the down regulation of photosynthesis would enable more efficient conversion of carbon into valuable products like biofuels. In this study, Flux Balance Analysis (FBA) and Flux Variability Analysis (FVA) have been used with a genome scale model of C. reinhardtii to examine changes in intracellular flux distribution in order to explain their changing physiology. Additionally, a reaction essentiality analysis was performed to identify which reaction subsets are essential for a given growth condition. Our results suggest that exogenous acetate feeds into a modified tricarboxylic acid (TCA) cycle, which bypasses the CO2 evolution steps, explaining increases in biomass, consistent with experimental data. In addition, reactions of the oxidative pentose phosphate and glycolysis pathways, inactive under phototrophic conditions, show substantial flux under mixotrophic conditions. Importantly, acetate addition leads to an increased flux through cyclic electron flow (CEF), but results in a repression of CO2 fixation via Rubisco, explaining the down regulation of photosynthesis. However, although CEF enhances growth on acetate, it is not essential—impairment of CEF results in alternative metabolic pathways being increased. We have demonstrated how the reactions of photosynthesis interconnect with carbon metabolism on a global scale, and how systems approaches play a viable tool in understanding complex relationships at the scale of the organism. PMID:26175742

  14. Structural Insight into Archaic and Alternative Chaperone-Usher Pathways Reveals a Novel Mechanism of Pilus Biogenesis.

    PubMed

    Pakharukova, Natalia; Garnett, James A; Tuittila, Minna; Paavilainen, Sari; Diallo, Mamou; Xu, Yingqi; Matthews, Steve J; Zavialov, Anton V

    2015-11-01

    Gram-negative pathogens express fibrous adhesive organelles that mediate targeting to sites of infection. The major class of these organelles is assembled via the classical, alternative and archaic chaperone-usher pathways. Although non-classical systems share a wider phylogenetic distribution and are associated with a range of diseases, little is known about their assembly mechanisms. Here we report atomic-resolution insight into the structure and biogenesis of Acinetobacter baumannii Csu and Escherichia coli ECP biofilm-mediating pili. We show that the two non-classical systems are structurally related, but their assembly mechanism is strikingly different from the classical assembly pathway. Non-classical chaperones, unlike their classical counterparts, maintain subunits in a substantially disordered conformational state, akin to a molten globule. This is achieved by a unique binding mechanism involving the register-shifted donor strand complementation and a different subunit carboxylate anchor. The subunit lacks the classical pre-folded initiation site for donor strand exchange, suggesting that recognition of its exposed hydrophobic core starts the assembly process and provides fresh inspiration for the design of inhibitors targeting chaperone-usher systems. PMID:26587649

  15. Changes in localization of human discs large (hDlg) during keratinocyte differentiation is associated with expression of alternatively spliced hDlg variants

    SciTech Connect

    Roberts, S. . E-mail: s.roberts@bham.ac.uk; Calautti, E.; Vanderweil, S.; Nguyen, H.O.; Foley, A.; Baden, H.P.; Viel, A.

    2007-07-15

    Alternative spliced variants of the human discs large (hDlg) tumour suppressor are characterized by combinations of insertions. Here, using insertions I2- and I3-specific antibodies, we show that I2 and I3 variants have distinct distributions in epidermal and cervical epithelia. In skin and cervix, I3 variants are found in the cytoplasm. Cytoplasmic localization of I3 variants decreases as cervical keratinocytes differentiate, concomitant with relocalization to the cell periphery. I2 variants are found at the cell periphery of differentiated epidermal and cervical keratinocytes. Nuclear localization of I2 variants was evident in both tissues, with concentration of nuclear I2 variants in basal and parabasal cervical keratinocytes. A prominent nuclear localization of hDlg in cells of hyperproliferative layers of psoriatic lesions, but not in mature differentiated keratinocytes, together with I2 redistribution in differentiating keratinocytes, suggests that nuclear hDlg functions may be pertinent to growth of undifferentiated cells. Supporting our findings in squamous tissues, a decrease of nuclear hDlg and an increase of membrane-bound and cytoplasmic hDlg upon calcium-induced keratinocyte differentiation were not concomitant processes. Furthermore, we confirm that the exit of I2 variants from the nucleus is linked to stimulation of epithelial differentiation. The dynamic redistribution of hDlg also correlated with a marked increase in the expression of I3 variants while the level of I2 variants showed only a moderate decrease. Because changes in the intracellular distribution of hDlg splice variants, and in their expression levels, correlate with changes in differentiation state we hypothesize that the different hDlg isoforms play distinct roles at various stages of epithelial differentiation.

  16. Synchrotron FTIR microspectroscopy reveals early adipogenic differentiation of human mesenchymal stem cells at single-cell level.

    PubMed

    Liu, Zhixiao; Tang, Yuzhao; Chen, Feng; Liu, Xia; Liu, Zhaojian; Zhong, Jiajia; Hu, Jun; Lü, Junhong

    2016-09-23

    Human mesenchymal stem cells (hMSCs) have been used as an ideal in vitro model to study human adipogenesis. However, little knowledge of the early stage differentiation greatly hinders our understanding on the mechanism of the adipogenesis processes. In this study, synchrotron radiation-based Fourier transform infrared (SR-FTIR) microspectroscopy was applied to track the global structural and compositional changes of lipids, proteins and nucleic acids inside individual hMSCs along the time course. The multivariate analysis of the SR-FTIR spectra distinguished the dynamic and significant changes of the lipids and nucleic acid at early differentiation stage. Importantly, changes of lipid structure during early days (Day 1-3) of differentiation might serve as a potential biomarker in identifying the state in early differentiation at single cell level. These results proved that SR-FTIR is a powerful tool to study the stem cell fate determination and early lipogenesis events. PMID:27553281

  17. Metabolite Profiling Reveals YihU as a Novel Hydroxybutyrate Dehydrogenase for Alternative Succinic Semialdehyde Metabolism in Escherichia coli*

    PubMed Central

    Saito, Natsumi; Robert, Martin; Kochi, Hayataro; Matsuo, Goh; Kakazu, Yuji; Soga, Tomoyoshi; Tomita, Masaru

    2009-01-01

    The search for novel enzymes and enzymatic activities is important to map out all metabolic activities and reveal cellular metabolic processes in a more exhaustive manner. Here we present biochemical and physiological evidence for the function of the uncharacterized protein YihU in Escherichia coli using metabolite profiling by capillary electrophoresis time-of-flight mass spectrometry. To detect enzymatic activity and simultaneously identify possible substrates and products of the putative enzyme, we profiled a complex mixture of metabolites in the presence or absence of YihU. In this manner, succinic semialdehyde was identified as a substrate for YihU. The purified YihU protein catalyzed in vitro the NADH-dependent reduction of succinic semialdehyde to γ-hydroxybutyrate. Moreover, a yihU deletion mutant displayed reduced tolerance to the cytotoxic effects of exogenous addition of succinic semialdehyde. Profiling of intracellular metabolites following treatment of E. coli with succinic semialdehyde supports the existence of a YihU-catalyzed reduction of succinic semialdehyde to γ-hydroxybutyrate in addition to its known oxidation to succinate and through the tricarboxylic acid cycle. These findings suggest that YihU is a novel γ-hydroxybutyrate dehydrogenase involved in the metabolism of succinic semialdehyde, and other potentially toxic intermediates that may accumulate under stress conditions in E. coli. PMID:19372223

  18. Aromatase knockout mice reveal an impact of estrogen on drug-induced alternation of murine electrocardiography parameters.

    PubMed

    Kurokawa, Junko; Sasano, Tetsuo; Kodama, Masami; Li, Min; Ebana, Yusuke; Harada, Nobuhiro; Honda, Shin-ichiro; Nakaya, Haruaki; Furukawa, Tetsushi

    2015-06-01

    Our in vitro characterization showed that physiological concentrations of estrogen partially suppressed the I(Kr) channel current in guinea pig ventricular myocytes and the human ether-a-go-go-related gene (hERG) channel currents in CHO-K1 cells regardless of estrogen receptor signaling and revealed that the partially suppressed hERG currents enhanced the sensitivity to the hERG blocker E-4031. To obtain in vivo proof-of-concept data to support the effects of estrogen on cardiac electrophysiology, we here employed an aromatase knockout mouse as an in vivo estrogen-null model and compared the acute effects of E-4031 on cardiac electrophysiological parameters with those in wild-type mice (C57/BL6J) by recording surface electrocardiogram (ECG). The ablation of circulating estrogens blunted the effects of E-4031 on heart rate and QT interval in mice under a denervation condition. Our result provides in vivo proof of principle and demonstrates that endogenous estrogens increase the sensitivity of E-4031 to cardiac electrophysiology. PMID:25972195

  19. Novel splice variants in the 5'UTR of Gtf2i expressed in the rat brain: alternative 5'UTRs and differential expression in the neuronal dendrites.

    PubMed

    Shirai, Yoshinori; Watanabe, Masahiko; Sakagami, Hiroyuki; Suzuki, Tatsuo

    2015-08-01

    General transcription factor II-I (Gtf2i) is a transcription factor and one of the genes implicated in Willams-Beuren syndrome, an autism spectrum disorder. In this study, we investigated splice variants of the Gtf2i gene in both the 5'untranslated region (5'UTR) and the coding region. To search for novel 5'UTRs of Gtf2i, we utilized the cap analysis gene expression database of the mouse. We identified seven novel Gtf2i transcripts with alternatively spliced 5'UTRs in the rat brain. We also identified four novel splice variants in the coding sequence of Gtf2i. Furthermore, we identified a selective usage of certain types of 5'UTR by coding variants. In situ hybridization demonstrated a differential pattern of expression of Gtf2i mRNAs with alternatively spliced 5'UTRs among neuronal cells, and the localization of one of the variants in neuronal dendrites in the rat brain. Immunohistochemistry also demonstrated a distribution of Gtf2i-immunoreactivity in the dendrites. These results suggest multiple pathways of expression of Gtf2i gene in the brain. The expression patterns may be under the control of alternative promoters coupled to the alternative splicing in the coding region. Differential localization of mRNA to neuronal dendrites suggests spatiotemporal-specific translation at the post-synaptic sites that is involved in transfer of synaptic activity to expression of specific sets of genes in the nucleus. Gtf2i is a transcription factor and implicated in Willams-Beuren syndrome. We identified seven novel Gtf2i transcripts with alternatively spliced 5'UTRs in the rat brain. In situ hybridization demonstrated a differential expression of Gtf2i mRNAs with different 5'UTRs in somas and dendrites of neuronal cells. Differential localization of mRNA to neuronal dendrites suggests spatiotemporal-specific translation at the postsynaptic sites. The scheme shows genomic structure showing the positions of the potential transcription start tags (rDEC695, rDEC3D7, rDEC1D3, r

  20. Analysis of Zfhx1a mutant mice reveals palatal shelf contact independent medial edge epithelial differentiation during palate fusion

    PubMed Central

    Jin, Jiu-Zhen; Li, Qun; Higashi, Yujiro; Darling, Douglas S.; Ding, Jixiang

    2008-01-01

    Cleft palate is a common birth defect that involves disruptions in multiple developmental steps such as growth, differentiation, elevation and fusion. Medial edge epithelial (MEE) differentiation is essential for palate fusion. An important question is that the MEE differentiation during fusion is induced by palate shelf contact or is programmed intrinsically by the palate shelf itself. Here, we report that the loss of Zfhx1a function in mice leads to a cleft palate phenotype that is mainly due to a delay in palate elevation. Zfhx1a encodes a transcription regulatory protein that modulates several signaling pathways including those activated by members of the TGF-β superfamily. Loss of Zfhx1a function in mice leads to a complete cleft palate with 100% penetrance. Zfhx1a mutant palatal shelves display normal cell differentiation and proliferation and are able to fuse in an in vitro culture system. The only defect detected was a 24–48 hour delay in palatal shelf elevation. Using the Zfhx1a mutant as a model, we studied the relationship between medial edge epithelial differentiation and palate contact/adhesion. We found that down regulation of Jag2 expression in the medial edge epithelial cells, a key differentiation event establishing palate fusion competence is independent of palate contact/adhesion. Moreover, the expression of several key factors essential for fusion, such as TGF-β3 and MMP13, are also down regulated at stage E16.5 in a contact independent manner, suggesting that differentiation of the medial edge epithelium is largely programmed through an intrinsic mechanism within the palate shelf. PMID:18470539

  1. New insights in osteogenic differentiation revealed by mass spectrometric assessment of phosphorylated substrates in murine skin mesenchymal cells

    PubMed Central

    2013-01-01

    Background Bone fractures and loss represent significant costs for the public health system and often affect the patients quality of life, therefore, understanding the molecular basis for bone regeneration is essential. Cytokines, such as IL-6, IL-10 and TNFα, secreted by inflammatory cells at the lesion site, at the very beginning of the repair process, act as chemotactic factors for mesenchymal stem cells, which proliferate and differentiate into osteoblasts through the autocrine and paracrine action of bone morphogenetic proteins (BMPs), mainly BMP-2. Although it is known that BMP-2 binds to ActRI/BMPR and activates the SMAD 1/5/8 downstream effectors, little is known about the intracellular mechanisms participating in osteoblastic differentiation. We assessed differences in the phosphorylation status of different cellular proteins upon BMP-2 osteogenic induction of isolated murine skin mesenchymal stem cells using Triplex Stable Isotope Dimethyl Labeling coupled with LC/MS. Results From 150 μg of starting material, 2,264 proteins were identified and quantified at five different time points, 235 of which are differentially phosphorylated. Kinase motif analysis showed that several substrates display phosphorylation sites for Casein Kinase, p38, CDK and JNK. Gene ontology analysis showed an increase in biological processes related with signaling and differentiation at early time points after BMP2 induction. Moreover, proteins involved in cytoskeleton rearrangement, Wnt and Ras pathways were found to be differentially phosphorylated during all timepoints studied. Conclusions Taken together, these data, allow new insights on the intracellular substrates which are phosphorylated early on during differentiation to BMP2-driven osteoblastic differentiation of skin-derived mesenchymal stem cells. PMID:24148232

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

    SciTech Connect

    Bouhlel, Mohamed Amine; Brozek, John; Derudas, Bruno; Zawadzki, Christophe; Jude, Brigitte; Staels, Bart; Chinetti-Gbaguidi, Giulia

    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 expression 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.

  3. Functional screen reveals essential roles of miR-27a/24 in differentiation of embryonic stem cells

    PubMed Central

    Ma, Yanni; Yao, Nan; Liu, Guang; Dong, Lei; Liu, Yufang; Zhang, Meili; Wang, Fang; Wang, Bin; Wei, Xueju; Dong, He; Wang, Lanlan; Ji, Shaowei; Zhang, Junwu; Wang, Yangming; Huang, Yue; Yu, Jia

    2015-01-01

    MicroRNAs play important roles in controlling the embryonic stem cell (ESC) state. Although much is known about microRNAs maintaining ESC state, microRNAs that are responsible for promoting ESC differentiation are less reported. Here, by screening 40 microRNAs pre-selected by their expression patterns and predicted targets in Dgcr8-null ESCs, we identify 14 novel differentiation-associated microRNAs. Among them, miR-27a and miR-24, restrained by c-Myc in ESC, exert their roles of silencing self-renewal through directly targeting several important pluripotency-associated factors, such as Oct4, Foxo1 and Smads. CRISPR/Cas9-mediated knockout of all miR-27/24 in ESCs leads to serious deficiency in ESC differentiation in vitro and in vivo. Moreover, depleting of them in mouse embryonic fibroblasts can evidently promote somatic cell reprogramming. Altogether, our findings uncover the essential role of miR-27 and miR-24 in ESC differentiation and also demonstrate novel microRNAs responsible for ESC differentiation. PMID:25519956

  4. Endogenous Optical Signals Reveal Changes of Elastin and Collagen Organization During Differentiation of Mouse Embryonic Stem Cells.

    PubMed

    Thimm, Terra N; Squirrell, Jayne M; Liu, Yuming; Eliceiri, Kevin W; Ogle, Brenda M

    2015-10-01

    Components of the extracellular matrix (ECM) have recently been shown to influence stem cell specification. However, it has been challenging to assess the spatial and temporal dynamics of stem cell-ECM interactions because most methodologies utilized to date require sample destruction or fixation. We examined the efficacy of utilizing the endogenous optical signals of two important ECM proteins, elastin (Eln), through autofluorescence, and type I collagen (ColI), through second harmonic generation (SHG), during mouse embryonic stem cell differentiation. After finding favorable overlap between antibody labeling and the endogenous fluorescent signal of Eln, we used this endogenous signal to map temporal changes in Eln and ColI during murine embryoid body differentiation and found that Eln increases until day 9 and then decreases slightly by day 12, while Col1 steadily increases over the 12-day period. Furthermore, we combined endogenous fluorescence imaging and SHG with antibody labeling of cardiomyocytes to examine the spatial relationship between Eln and ColI accumulation and cardiomyocyte differentiation. Eln was ubiquitously present, with enrichment in regions with cardiomyocyte differentiation, while there was an inverse correlation between ColI and cardiomyocyte differentiation. This work provides an important first step for utilizing endogenous optical signals, which can be visualized in living cells, to understand the relationship between the ECM and cardiomyocyte development and sets the stage for future studies of stem cell-ECM interactions and dynamics relevant to stem cells as well as other cell and tissue types. PMID:25923353

  5. Differential Roles of Interleukin 15 mRNA Isoforms Generated by Alternative Splicing in Immune Responses in Vivo

    PubMed Central

    Nishimura, Hitoshi; Yajima, Toshiki; Naiki, Yoshikazu; Tsunobuchi, Hironaka; Umemura, Masayuki; Itano, Keiko; Matsuguchi, Tetsuya; Suzuki, Misao; Ohashi, Pamela S.; Yoshikai, Yasunobu

    2000-01-01

    At least two types of interleukin (IL)-15 mRNA isoforms are generated by alternative splicing at the 5′ upstream of exon 5 in mice. To elucidate the potential roles of IL-15 isoforms in immune responses in vivo, we constructed two groups of transgenic mice using originally described IL-15 cDNA with a normal exon 5 (normal IL-15 transgenic [Tg] mice) and IL-15 cDNA with an alternative exon 5 (alternative IL-15 Tg mice) under the control of an MHC class I promoter. Normal IL-15 Tg mice constitutionally produced a significant level of IL-15 protein and had markedly increased numbers of memory type (CD44high Ly6C+) of CD8+ T cells in the LN. These mice showed resistance to Salmonella infection accompanied by the enhanced interferon (IFN)-γ production, but depletion of CD8+ T cells exaggerated the bacterial growth, suggesting that the IL-15–dependent CD8+ T cells with a memory phenotype may serve to protect against Salmonella infection in normal IL-15 Tg mice. On the other hand, a large amount of intracellular IL-15 protein was detected but hardly secreted extracellularly in alternative IL-15 Tg mice. Although most of the T cells developed normally in the alternative IL-15 Tg mice, they showed impaired IFN-γ production upon TCR engagement. The alternative IL-15 transgenic mice were susceptible to Salmonella accompanied by impaired production of endogenous IL-15 and IFN-γ. Thus, two groups of IL-15 Tg mice may provide information concerning the different roles of IL-15 isoforms in the immune system in vivo. PMID:10620614

  6. Dynamic Proteomic Analysis of Pancreatic Mesenchyme Reveals Novel Factors That Enhance Human Embryonic Stem Cell to Pancreatic Cell Differentiation

    PubMed Central

    Russ, Holger A.; Landsman, Limor; Moss, Christopher L.; Higdon, Roger; Greer, Renee L.; Kaihara, Kelly; Salamon, Randy; Kolker, Eugene; Hebrok, Matthias

    2016-01-01

    Current approaches in human embryonic stem cell (hESC) to pancreatic beta cell differentiation have largely been based on knowledge gained from developmental studies of the epithelial pancreas, while the potential roles of other supporting tissue compartments have not been fully explored. One such tissue is the pancreatic mesenchyme that supports epithelial organogenesis throughout embryogenesis. We hypothesized that detailed characterization of the pancreatic mesenchyme might result in the identification of novel factors not used in current differentiation protocols. Supplementing existing hESC differentiation conditions with such factors might create a more comprehensive simulation of normal development in cell culture. To validate our hypothesis, we took advantage of a novel transgenic mouse model to isolate the pancreatic mesenchyme at distinct embryonic and postnatal stages for subsequent proteomic analysis. Refined sample preparation and analysis conditions across four embryonic and prenatal time points resulted in the identification of 21,498 peptides with high-confidence mapping to 1,502 proteins. Expression analysis of pancreata confirmed the presence of three potentially important factors in cell differentiation: Galectin-1 (LGALS1), Neuroplastin (NPTN), and the Laminin α-2 subunit (LAMA2). Two of the three factors (LGALS1 and LAMA2) increased expression of pancreatic progenitor transcript levels in a published hESC to beta cell differentiation protocol. In addition, LAMA2 partially blocks cell culture induced beta cell dedifferentiation. Summarily, we provide evidence that proteomic analysis of supporting tissues such as the pancreatic mesenchyme allows for the identification of potentially important factors guiding hESC to pancreas differentiation. PMID:26681951

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

    PubMed

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

    2015-11-01

    A meta-analysis of 13 independent microarray data sets was performed and gene expression profiles from cystic fibrosis (CF), similar disorders (COPD: chronic obstructive pulmonary disease, IPF: idiopathic pulmonary fibrosis, asthma), environmental conditions (smoking, epithelial injury), related cellular processes (epithelial differentiation/regeneration), and non-respiratory "control" conditions (schizophrenia, dieting), were compared. Similarity among differentially expressed (DE) gene lists was assessed using a permutation test, and a clustergram was constructed, identifying common gene markers. Global gene expression values were standardized using a novel approach, revealing that similarities between independent data sets run deeper than shared DE genes. Correlation of gene expression values identified putative gene regulators of the CF transmembrane conductance regulator (CFTR) gene, of potential therapeutic significance. Our study provides a novel perspective on CF epithelial gene expression in the context of other lung disorders and conditions, and highlights the contribution of differentiation/EMT and injury to gene signatures of respiratory disease. PMID:26225835

  8. Genetic variability, differentiation, and founder effect in golden jackals (Canis aureus) from Serbia as revealed by mitochondrial DNA and nuclear microsatellite loci.

    PubMed

    Zachos, Frank E; Cirovic, Dusko; Kirschning, Julia; Otto, Marthe; Hartl, Günther B; Petersen, Britt; Honnen, Ann-Christin

    2009-04-01

    We analyzed 121 golden jackals (Canis aureus) from six sample sites in Serbia with regard to genetic variability and differentiation as revealed by mitochondrial control region sequences and eight nuclear microsatellite loci. There was no variation at all in the mtDNA sequences, and nuclear variability was very low (average observed and expected heterozygosity of 0.29 and 0.34, respectively). This is in line with the considerable recent range expansion of this species in the Balkans and indicates a strong founder effect in the recently established Serbian population. We did not find evidence of differentiation between the northeastern jackals and those from the plain of Srem or those in between. F-statistics and Bayesian Structure analyses, however, were indicative of a low degree of overall differentiation in the Serbian population. A vagrant Austrian jackal that was also analyzed was genetically indistinguishable from its Serbian conspecifics. PMID:19169806

  9. Differential reinforcement of alternative behavior and demand fading in the treatment of escape-maintained destructive behavior.

    PubMed Central

    Piazza, C C; Moes, D R; Fisher, W W

    1996-01-01

    The escape-maintained destructive behavior of a boy with autism was reduced during instructional sequences with differential reinforcement of compliance (DRA), escape extinction without physical guidance, and demand fading. The procedure decreased destructive behaviors to near-zero levels and greatly increased compliance. PMID:8995837

  10. Genomic Data Reveal Toxoplasma gondii Differentiation Mutants Are Also Impaired with Respect to Switching into a Novel Extracellular Tachyzoite State

    PubMed Central

    Lescault, Pamela J.; Thompson, Ann B.; Patil, Veerupaxagouda; Lirussi, Dario; Burton, Amanda; Margarit, Juan; Bond, Jeffrey; Matrajt, Mariana

    2010-01-01

    Toxoplasma gondii pathogenesis includes the invasion of host cells by extracellular parasites, replication of intracellular tachyzoites, and differentiation to a latent bradyzoite stage. We present the analysis of seven novel T. gondii insertional mutants that do not undergo normal differentiation to bradyzoites. Microarray quantification of the variation in genome-wide RNA levels for each parasite line and times after induction allowed us to describe states in the normal differentiation process, to analyze mutant lines in the context of these states, and to identify genes that may have roles in initiating the transition from tachyzoite to bradyzoite. Gene expression patterns in wild-type parasites undergoing differentiation suggest a novel extracellular state within the tachyzoite stage. All mutant lines exhibit aberrant regulation of bradyzoite gene expression and notably some of the mutant lines appear to exhibit high proportions of the intracellular tachyzoite state regardless of whether they are intracellular or extracellular. In addition to the genes identified by the insertional mutagenesis screen, mixture model analysis allowed us to identify a small number of genes, in mutants, for which expression patterns could not be accounted for using the three parasite states – genes that may play a mechanistic role in switching from the tachyzoite to bradyzoite stage. PMID:21209930

  11. Systems Chemo-Biology and Transcriptomic Meta-Analysis Reveal the Molecular Roles of Bioactive Lipids in Cardiomyocyte Differentiation.

    PubMed

    de Faria Poloni, Joice; Bonatto, Diego

    2015-09-01

    Lipids, which are essential constituents of biological membranes, play structural and functional roles in the cell. In recent years, certain lipids have been identified as regulatory signaling molecules and have been termed "bioactive lipids". Subsequently, the importance of bioactive lipids in stem cell differentiation and cardiogenesis has gained increasing recognition. Therefore, the aim of this study was to identify the biological processes underlying murine cardiac differentiation and the mechanisms by which bioactive lipids affect these processes. For this purpose, a transcriptomic meta-analysis of microarray and RNA-seq data from murine stem cells undergoing cardiogenic differentiation was performed. The differentially expressed genes identified via this meta-analysis, as well as bioactive lipids, were evaluated using systems chemo-biology tools. These data indicated that bioactive lipids are associated with the regulation of cell motility, cell adhesion, cytoskeletal rearrangement, and gene expression. Moreover, bioactive lipids integrate the signaling pathways involved in cell migration, the secretion and remodeling of extracellular matrix components, and the establishment of the cardiac phenotype. In conclusion, this study provides new insights into the contribution of bioactive lipids to the induction of cellular responses to various stimuli, which may originate from the extracellular environment and morphogens, and the manner in which this contribution directly affects murine heart morphogenesis. PMID:25752681

  12. Single-molecule Force Spectroscopy Reveals the Calcium Dependence of the Alternative Conformations in the Native State of a βγ-Crystallin Protein.

    PubMed

    Scholl, Zackary N; Li, Qing; Yang, Weitao; Marszalek, Piotr E

    2016-08-26

    Although multidomain proteins predominate the proteome of all organisms and are expected to display complex folding behaviors and significantly greater structural dynamics as compared with single-domain proteins, their conformational heterogeneity and its impact on their interaction with ligands are poorly understood due to a lack of experimental techniques. The multidomain calcium-binding βγ-crystallin proteins are particularly important because their deterioration and misfolding/aggregation are associated with melanoma tumors and cataracts. Here we investigate the mechanical stability and conformational dynamics of a model calcium-binding βγ-crystallin protein, Protein S, and elaborate on its interactions with calcium. We ask whether domain interactions and calcium binding affect Protein S folding and potential structural heterogeneity. Our results from single-molecule force spectroscopy show that the N-terminal (but not the C-terminal) domain is in equilibrium with an alternative conformation in the absence of Ca(2+), which is mechanically stable in contrast to other proteins that were observed to sample a molten globule under similar conditions. Mutagenesis experiments and computer simulations reveal that the alternative conformation of the N-terminal domain is caused by structural instability produced by the high charge density of a calcium binding site. We find that this alternative conformation in the N-terminal domain is diminished in the presence of calcium and can also be partially eliminated with a hitherto unrecognized compensatory mechanism that uses the interaction of the C-terminal domain to neutralize the electronegative site. We find that up to 1% of all identified multidomain calcium-binding proteins contain a similarly highly charged site and therefore may exploit a similar compensatory mechanism to prevent structural instability in the absence of ligand. PMID:27378818

  13. Longitudinal Transcriptome Analysis Reveals a Sustained Differential Gene Expression Signature in Patients Treated for Acute Lyme Disease

    PubMed Central

    Bouquet, Jerome; Soloski, Mark J.; Swei, Andrea; Cheadle, Chris; Federman, Scot; Billaud, Jean-Noel; Rebman, Alison W.; Kabre, Beniwende; Halpert, Richard; Boorgula, Meher

    2016-01-01

    ABSTRACT Lyme disease is a tick-borne illness caused by the bacterium Borrelia burgdorferi, and approximately 10 to 20% of patients report persistent symptoms lasting months to years despite appropriate treatment with antibiotics. To gain insights into the molecular basis of acute Lyme disease and the ensuing development of post-treatment symptoms, we conducted a longitudinal transcriptome study of 29 Lyme disease patients (and 13 matched controls) enrolled at the time of diagnosis and followed for up to 6 months. The differential gene expression signature of Lyme disease following the acute phase of infection persisted for at least 3 weeks and had fewer than 44% differentially expressed genes (DEGs) in common with other infectious or noninfectious syndromes. Early Lyme disease prior to antibiotic therapy was characterized by marked upregulation of Toll-like receptor signaling but lack of activation of the inflammatory T-cell apoptotic and B-cell developmental pathways seen in other acute infectious syndromes. Six months after completion of therapy, Lyme disease patients were found to have 31 to 60% of their pathways in common with three different immune-mediated chronic diseases. No differential gene expression signature was observed between Lyme disease patients with resolved illness to those with persistent symptoms at 6 months post-treatment. The identification of a sustained differential gene expression signature in Lyme disease suggests that a panel of selected human host-based biomarkers may address the need for sensitive clinical diagnostics during the “window period” of infection prior to the appearance of a detectable antibody response and may also inform the development of new therapeutic targets. PMID:26873097

  14. Combinatorial Analysis of Growth Factors Reveals the Contribution of Bone Morphogenetic Proteins to Chondrogenic Differentiation of Human Periosteal Cells.

    PubMed

    Mendes, Luis Filipe; Tam, Wai Long; Chai, Yoke Chin; Geris, Liesbet; Luyten, Frank P; Roberts, Scott J

    2016-05-01

    Successful application of cell-based strategies in cartilage and bone tissue engineering has been hampered by the lack of robust protocols to efficiently differentiate mesenchymal stem cells into the chondrogenic lineage. The development of chemically defined culture media supplemented with growth factors (GFs) has been proposed as a way to overcome this limitation. In this work, we applied a fractional design of experiment (DoE) strategy to screen the effect of multiple GFs (BMP2, BMP6, GDF5, TGF-β1, and FGF2) on chondrogenic differentiation of human periosteum-derived mesenchymal stem cells (hPDCs) in vitro. In a micromass culture (μMass) system, BMP2 had a positive effect on glycosaminoglycan deposition at day 7 (p < 0.001), which in combination with BMP6 synergistically enhanced cartilage-like tissue formation that displayed in vitro mineralization capacity at day 14 (p < 0.001). Gene expression of μMasses cultured for 7 days with a medium formulation supplemented with 100 ng/mL of BMP2 and BMP6 and a low concentration of GDF5, TGF-β1, and FGF2 showed increased expression of Sox9 (1.7-fold) and the matrix molecules aggrecan (7-fold increase) and COL2A1 (40-fold increase) compared to nonstimulated control μMasses. The DoE analysis indicated that in GF combinations, BMP2 was the strongest effector for chondrogenic differentiation of hPDCs. When transplanted ectopically in nude mice, the in vitro-differentiated μMasses showed maintenance of the cartilaginous phenotype after 4 weeks in vivo. This study indicates the power of using the DoE approach for the creation of new medium formulations for skeletal tissue engineering approaches. PMID:27018617

  15. Gender-Differentiated Parenting Revisited: Meta-Analysis Reveals Very Few Differences in Parental Control of Boys and Girls

    PubMed Central

    Endendijk, Joyce J.; Groeneveld, Marleen G.; Bakermans-Kranenburg, Marian J.; Mesman, Judi

    2016-01-01

    Although various theories describe mechanisms leading to differential parenting of boys and girls, there is no consensus about the extent to which parents do treat their sons and daughters differently. The last meta-analyses on the subject were conducted more than fifteen years ago, and changes in gender-specific child rearing in the past decade are quite plausible. In the current set of meta-analyses, based on 126 observational studies (15,034 families), we examined mothers’ and fathers’ differential use of autonomy-supportive and controlling strategies with boys and girls, and the role of moderators related to the decade in which the study was conducted, the observational context, and sample characteristics. Databases of Web of Science, ERIC, PsychInfo, Online Contents, Picarta, and Proquest were searched for studies examining differences in observed parental control of boys and girls between the ages of 0 and 18 years. Few differences were found in parents’ use of control with boys and girls. Parents were slightly more controlling with boys than with girls, but the effect size was negligible (d = 0.08). The effect was larger, but still small, in normative groups and in samples with younger children. No overall effect for gender-differentiated autonomy-supportive strategies was found (d = 0.03). A significant effect of time emerged: studies published in the 1970s and 1980s reported more autonomy-supportive strategies with boys than toward girls, but from 1990 onwards parents showed somewhat more autonomy-supportive strategies with girls than toward boys. Taking into account parents’ gender stereotypes might uncover subgroups of families where gender-differentiated control is salient, but based on our systematic review of the currently available large data base we conclude that in general the differences between parenting of boys versus girls are minimal. PMID:27416099

  16. High genetic differentiation of Aegla longirostri (Crustacea, Decapoda, Anomura) populations in southern Brazil revealed by multi-loci microsatellite analysis.

    PubMed

    Bartholomei-Santos, M L; Roratto, P A; Santos, S

    2011-01-01

    Species with a broad distribution rarely have the same genetic make-up throughout their entire range. In some cases, they may constitute a cryptic complex consisting of a few species, each with a narrow distribution, instead of a single-, widely distributed species. These differences can have profound impacts for biodiversity conservation planning. The genetic differentiation of four populations of Aegla longirostri, a freshwater crab found in two geographically isolated basins in Rio Grande do Sul State, Brazil, was investigated by analyzing pentanucleotide multi-loci microsatellites in a heteroduplex assay. Although no morphological differences were evident, we found significant genetic differentiation among the four populations, based on F(ST) values and clustering analysis. This high level of differentiation may be indicative of cryptic species in these populations. If this hypothesis is correct, then the species occurring in the Ibicuí-Mirim River, at the southern limit of the Atlantic Rain Forest, would be under threat, considering its very restricted distribution. PMID:22179994

  17. Key developmental transitions in human germinal center B cells are revealed by differential CD45RB expression.

    PubMed

    Jackson, Stephen M; Harp, Natessa; Patel, Darshna; Wulf, Jordan; Spaeth, Erich D; Dike, Uzoamaka K; James, Judith A; Capra, J Donald

    2009-04-23

    We previously reported that RO(+) expression correlated with increased mutation, activation, and selection among human germinal center (GC) B cells. Here, we subdivided human tonsillar B cells, including IgD(-)CD38(+) GC B cells, into different fractions based on RB expression. Although each subset contained RB(+) cells, when used as an intrasubset marker, differential RB expression effectively discriminated between phenotypically distinct cells. For example, RB(+) GC B cells were enriched for activated cells with lower AID expression. RB inversely correlated with mutation frequency, demonstrating a key difference between RB- and RO-expressing GC B cells. Reduced RB expression during the transition from pre-GC (IgM(+)IgD(+)CD38(+)CD27(-)) to GCB cells was followed by a dramatic increase during the GC-to-plasmablast (IgD(-)CD38(++)CD27(+)) and memory (IgD(-)CD38(-)CD27(+)) transition. Interestingly, RB(+) GC B cells showed increased signs of terminal differentiation toward CD27(+) post-GC early plasmablast (increased CD38 and RO) or early memory (decreased CD38 and RO) B cells. We propose that as in T cells, differential RB expression directly correlates with development- and function-based transitions in tonsillar B cells. Application of this RB:RO system should advance our understanding of normal B-cell development and facilitate the isolation of more discrete B-cell populations with potentially different propensities in disease pathogenesis. PMID:19059880

  18. Comparative genomic analysis of transgenic poplar dwarf mutant reveals numerous differentially expressed genes involved in energy flow.

    PubMed

    Chen, Su; Bai, Shuang; Liu, Guifeng; Li, Huiyu; Jiang, Jing

    2014-01-01

    In our previous research, the Tamarix androssowii LEA gene (Tamarix androssowii late embryogenesis abundant protein Mrna, GenBank ID: DQ663481) was transferred into Populus simonii × Populus nigra. Among the eleven transgenic lines, one exhibited a dwarf phenotype compared to the wild type and other transgenic lines, named dwf1. To uncover the mechanisms underlying this phenotype, digital gene expression libraries were produced from dwf1, wild-type, and other normal transgenic lines, XL-5 and XL-6. Gene expression profile analysis indicated that dwf1 had a unique gene expression pattern in comparison to the other two transgenic lines. Finally, a total of 1246 dwf1-unique differentially expressed genes were identified. These genes were further subjected to gene ontology and pathway analysis. Results indicated that photosynthesis and carbohydrate metabolism related genes were significantly affected. In addition, many transcription factors genes were also differentially expressed in dwf1. These various differentially expressed genes may be critical for dwarf mutant formation; thus, the findings presented here might provide insight for our understanding of the mechanisms of tree growth and development. PMID:25192286

  19. Quantitative gene expression profiling of mouse brain regions reveals differential transcripts conserved in human and affected in disease models.

    PubMed

    Brochier, Camille; Gaillard, Marie-Claude; Diguet, Elsa; Caudy, Nicolas; Dossat, Carole; Ségurens, Béatrice; Wincker, Patrick; Roze, Emmanuel; Caboche, Jocelyne; Hantraye, Philippe; Brouillet, Emmanuel; Elalouf, Jean-Marc; de Chaldée, Michel

    2008-04-22

    Using serial analysis of gene expression, we collected quantitative transcriptome data in 11 regions of the adult wild-type mouse brain: the orbital, prelimbic, cingulate, motor, somatosensory, and entorhinal cortices, the caudate-putamen, the nucleus accumbens, the thalamus, the substantia nigra, and the ventral tegmental area. With >1.2 million cDNA tags sequenced, this database is a powerful resource to explore brain functions and disorders. As an illustration, we performed interregional comparisons and found 315 differential transcripts. Most of them are poorly characterized and 20% lack functional annotation. For 78 differential transcripts, we provide independent expression level measurements in mouse brain regions by real-time quantitative RT-PCR. We also show examples where we used in situ hybridization to achieve infrastructural resolution. For 30 transcripts, we next demonstrated that regional enrichment is conserved in the human brain. We then quantified the expression levels of region-enriched transcripts in the R6/2 mouse model of Huntington disease and the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson disease and observed significant alterations in the striatum, cerebral cortex, thalamus and substantia nigra of R6/2 mice and in the striatum of MPTP-treated mice. These results show that the gene expression data provided here for the mouse brain can be used to explore pathophysiological models and disclose transcripts differentially expressed in human brain regions. PMID:18252803

  20. Differential Gene Expression for Curvularia eragrostidis Pathogenic Incidence in Crabgrass (Digitaria sanguinalis) Revealed by cDNA-AFLP Analysis

    PubMed Central

    Wang, Jianshu; Wang, Xuemin; Yuan, Bohua; Qiang, Sheng

    2013-01-01

    Gene expression profiles of Digitaria sanguinalis infected by Curvularia eragrostidis strain QZ-2000 at two concentrations of conidia and two dew durations were analyzed by cDNA amplified fragment length polymorphisms (cDNA-AFLP). Inoculum strength was more determinant of gene expression than dew duration. A total of 256 primer combinations were used for selective amplification and 1214 transcript-derived fragments (TDFs) were selected for their differential expression. Of these, 518 up-regulated differentially expressed TDFs were identified. Forty-six differential cDNA fragments were chosen to be cloned and 35 of them were successfully cloned and sequenced, of which 25 were homologous to genes of known function according to the GenBank database. Only 6 genes were up-regulated in Curvularia eragrostidis-inoculated D. sanguinalis, with functions involved in signal transduction, energy metabolism, cell growth and development, stress responses, abscisic acid biosynthesis and response. It appears that a few pathways may be important parts of the pathogenic strategy of C. eragrostidis strain QZ-2000 on D. sanguinalis. Our study provides the fundamentals to further study the pathogenic mechanism, screen for optimal C. eragrostidis strains as potential mycoherbicide and apply this product to control D. sanguinalis. PMID:24116044

  1. Differential Proteomic Analysis Using iTRAQ Reveals Alterations in Hull Development in Rice (Oryza sativa L.)

    PubMed Central

    Xiao, Wenfei; Yang, Changdeng; Xin, Ya; Qiu, Jieren; Hu, Weimin; Ying, Wu; Fu, Yaping; Tong, Jianxin; Hu, Guocheng; Chen, Zhongzhong; Fang, Xianping; Yu, Hong; Lai, Wenguo; Ruan, Songlin; Ma, Huasheng

    2015-01-01

    Rice hull, the outer cover of the rice grain, determines grain shape and size. Changes in the rice hull proteome in different growth stages may reflect the underlying mechanisms involved in grain development. To better understand these changes, isobaric tags for relative and absolute quantitative (iTRAQ) MS/MS was used to detect statistically significant changes in the rice hull proteome in the booting, flowering, and milk-ripe growth stages. Differentially expressed proteins were analyzed to predict their potential functions during development. Gene ontology (GO) terms and pathways were used to evaluate the biological mechanisms involved in rice hull at the three growth stages. In total, 5,268 proteins were detected and characterized, of which 563 were differentially expressed across the development stages. The results showed that the flowering and milk-ripe stage proteomes were more similar to each other (r=0.61) than either was to the booting stage proteome. A GO enrichment analysis of the differentially expressed proteins was used to predict their roles during rice hull development. The potential functions of 25 significantly differentially expressed proteins were used to evaluate their possible roles at various growth stages. Among these proteins, an unannotated protein (Q7X8A1) was found to be overexpressed especially in the flowering stage, while a putative uncharacterized protein (B8BF94) and an aldehyde dehydrogenase (Q9FPK6) were overexpressed only in the milk-ripe stage. Pathways regulated by differentially expressed proteins were also analyzed. Magnesium-protoporphyrin IX monomethyl ester [oxidative] cyclase (Q9SDJ2), and two magnesium-chelatase subunits, ChlD (Q6ATS0), and ChlI (Q53RM0), were associated with chlorophyll biosynthesis at different developmental stages. The expression of Q9SDJ2 in the flowering and milk-ripe stages was validated by qRT-PCR. The 25 candidate proteins may be pivotal markers for controlling rice hull development at various

  2. Multiple cis elements regulate an alternative splicing event at 4.1R pre-mRNA during erythroid differentiation.

    PubMed

    Deguillien, M; Huang, S C; Morinière, M; Dreumont, N; Benz, E J; Baklouti, F

    2001-12-15

    The inclusion of exon 16 in the mature protein 4.1R messenger RNA (mRNA) is a critical event in red blood cell membrane biogenesis. It occurs during late erythroid development and results in inclusion of the 10-kd domain needed for stabilization of the spectrin/actin lattice. In this study, an experimental model was established in murine erythroleukemia cells that reproduces the endogenous exon 16 splicing patterns from a transfected minigene. Exon 16 was excluded in predifferentiated and predominantly included after induction. This suggests that the minigene contained exon and abutting intronic sequences sufficient for splicing regulation. A systematic analysis of the cis-acting regulatory sequences that reside within the exon and flanking introns was performed. Results showed that (1) the upstream intron of 4.1R pre-mRNA is required for exon recognition and it displays 2 enhancer elements, a distal element acting in differentiating cells and a proximal constitutive enhancer that resides within the 25 nucleotides preceding the acceptor site; (2) the exon itself contains a strong constitutive splicing silencer; (3) the exon has a weak 5' splice site; and (4) the downstream intron contains at least 2 splicing enhancer elements acting in differentiating cells, a proximal element at the vicinity of the 5' splice site, and a distal element containing 3 copies of the UGCAUG motif. These results suggest that the interplay between negative and positive elements may determine the inclusion or exclusion of exon 16. The activation of the enhancer elements in late erythroid differentiation may play an important role in the retention of exon 16. PMID:11739190

  3. Second-order non-iterative ADI solution of non-linear partial differential equations. [Alternating Direction Implicit scheme

    NASA Technical Reports Server (NTRS)

    Wolfshtein, M.; Hirsh, R. S.; Pitts, B. H.

    1975-01-01

    A new method for the solution of non-linear partial differential equations by an ADI procedure is described. Although the method is second order accurate in time, it does not require either iterations or predictor corrector methods to overcome the nonlinearity of the equations. Thus the computational effort required for the solution of the non-linear problem becomes similar to that required for the linear case. The method is applied to a two-dimensional 'extended Burgers equation'. Linear stability is studied, and some numerical solutions obtained. The improved accuracy obtained by the 2nd order truncation error is clearly manifested.

  4. Investigations of TGF-β signaling in preantral follicles of female mice reveal differential roles for bone morphogenetic protein 15.

    PubMed

    Fenwick, Mark A; Mora, Jocelyn M; Mansour, Yosef T; Baithun, Christina; Franks, Stephen; Hardy, Kate

    2013-09-01

    Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are 2 closely related TGF-β ligands implicated as key regulators of follicle development and fertility. Animals harboring mutations of these factors often exhibit a blockage in follicle development beyond the primary stage and therefore little is known about the role of these ligands during subsequent (preantral) stages. Preantral follicles isolated from immature mice were cultured with combinations of BMP15, GDF9, and activin receptor-like kinase (ALK) inhibitors. Individually, GDF9 and BMP15 promoted follicle growth during the first 24 hours, whereas BMP15 subsequently (48-72 h) caused follicle shrinkage and atresia with increased granulosa cell apoptosis. Inhibition of ALK6 prevented the BMP15-induced reduction in follicle size and under basal conditions promoted a rapid increase in granulosa cell proliferation, suggesting BMP15 signals through ALK6, which in turn acts to restrain follicle growth. In the presence of GDF9, BMP15 no longer promoted atresia and in fact follicle growth was increased significantly more than with either ligand alone. This cooperative effect was accompanied by differential expression of Id1-3, Smad6-7, and Has2 and was blocked by the same ALK5 inhibitor used to block GDF9 signaling. Immunostaining for SMAD2/3 and SMAD1/5/8, representing the 2 main branches of TGF-β signaling, supported the fact that both canonical pathways have the potential to be active in growing follicles, whereas primordial follicles only express SMAD2/3. Overall results highlight differential effects of the 2 main TGF-β signaling pathways during preantral follicle growth. PMID:23782946

  5. RNA-Seq Profiling of a Defective Seed Coat Mutation in Glycine max Reveals Differential Expression of Proline-Rich and Other Cell Wall Protein Transcripts

    PubMed Central

    Kour, Anupreet; Boone, Anne M.; Vodkin, Lila O.

    2014-01-01

    The plant cell wall performs a number of essential functions including providing shape to many different cell types and serving as a defense against potential pathogens. The net pattern mutation creates breaks in the seed coat of soybean (Glycine max) because of ruptured cell walls. Using RNA-Seq, we examined the seed coat transcriptome from three stages of immature seed development in two pairs of isolines with normal or defective seed coat phenotypes due to the net pattern. The genome-wide comparative study of the transcript profiles of these isolines revealed 364 differentially expressed genes in common between the two varieties that were further divided into different broad functional categories. Genes related to cell wall processes accounted for 19% of the differentially expressed genes in the middle developmental stage of 100–200 mg seed weight. Within this class, the cell wall proline-rich and glycine-rich protein genes were highly differentially expressed in both genetic backgrounds. Other genes that showed significant expression changes in each of the isoline pairs at the 100–200 mg seed weight stage were xylem serine proteinase, fasciclin-related genes, auxin and stress response related genes, TRANSPARENT TESTA 1 (TT1) and other transcription factors. The mutant appears to shift the timing of either the increase or decrease in the levels of some of the transcripts. The analysis of these data sets reveals the physiological changes that the seed coat undergoes during the formation of the breaks in the cell wall. PMID:24828743

  6. Secreted protein gene derived-single nucleotide polymorphisms (SP-SNPs) reveal population diversity and differentiation of Puccinia striiformis f. sp. tritici in the United States.

    PubMed

    Xia, Chongjing; Wan, Anmin; Wang, Meinan; Jiwan, Derick A; See, Deven R; Chen, Xianming

    2016-05-01

    Single nucleotide polymorphism (SNP) is a powerful molecular marker technique that has been widely used in population genetics and molecular mapping studies for various organisms. However, the technique has not been used for studying Puccinia striiformis f. sp. tritici (Pst), the wheat stripe rust pathogen. In this study, we developed over a hundred secreted protein gene-derived SNP (SP-SNP) markers and used 92 markers to study the population structure of Pst. From 352 isolates collected in the United States, we identified 242 multi-locus genotypes. The SP-SNP genotypes had a moderate, but significant correlation with the virulence phenotype data. Clustering of the multi-locus genotypes was consistent by various analyses, revealing distinct genetic groups. Analysis of molecular variance detected significant differences between the eastern and western US Pst populations. High heterozygosity was found in the US population with significant differences identified among epidemiological regions. Analysis of population differentiation revealed that populations between the eastern and western US were highly differentiated while moderate differentiation was found in populations within the western or eastern US. Isolates from the western US were more diverse than isolates from the eastern US. The information is useful for guiding the disease management in different epidemiological regions. PMID:27109369

  7. Whole-Exome Sequencing Identifies Loci Associated with Blood Cell Traits and Reveals a Role for Alternative GFI1B Splice Variants in Human Hematopoiesis.

    PubMed

    Polfus, Linda M; Khajuria, Rajiv K; Schick, Ursula M; Pankratz, Nathan; Pazoki, Raha; Brody, Jennifer A; Chen, Ming-Huei; Auer, Paul L; Floyd, James S; Huang, Jie; Lange, Leslie; van Rooij, Frank J A; Gibbs, Richard A; Metcalf, Ginger; Muzny, Donna; Veeraraghavan, Narayanan; Walter, Klaudia; Chen, Lu; Yanek, Lisa; Becker, Lewis C; Peloso, Gina M; Wakabayashi, Aoi; Kals, Mart; Metspalu, Andres; Esko, Tõnu; Fox, Keolu; Wallace, Robert; Franceshini, Nora; Matijevic, Nena; Rice, Kenneth M; Bartz, Traci M; Lyytikäinen, Leo-Pekka; Kähönen, Mika; Lehtimäki, Terho; Raitakari, Olli T; Li-Gao, Ruifang; Mook-Kanamori, Dennis O; Lettre, Guillaume; van Duijn, Cornelia M; Franco, Oscar H; Rich, Stephen S; Rivadeneira, Fernando; Hofman, Albert; Uitterlinden, André G; Wilson, James G; Psaty, Bruce M; Soranzo, Nicole; Dehghan, Abbas; Boerwinkle, Eric; Zhang, Xiaoling; Johnson, Andrew D; O'Donnell, Christopher J; Johnsen, Jill M; Reiner, Alexander P; Ganesh, Santhi K; Sankaran, Vijay G

    2016-08-01

    Circulating blood cell counts and indices are important indicators of hematopoietic function and a number of clinical parameters, such as blood oxygen-carrying capacity, inflammation, and hemostasis. By performing whole-exome sequence association analyses of hematologic quantitative traits in 15,459 community-dwelling individuals, followed by in silico replication in up to 52,024 independent samples, we identified two previously undescribed coding variants associated with lower platelet count: a common missense variant in CPS1 (rs1047891, MAF = 0.33, discovery + replication p = 6.38 × 10(-10)) and a rare synonymous variant in GFI1B (rs150813342, MAF = 0.009, discovery + replication p = 1.79 × 10(-27)). By performing CRISPR/Cas9 genome editing in hematopoietic cell lines and follow-up targeted knockdown experiments in primary human hematopoietic stem and progenitor cells, we demonstrate an alternative splicing mechanism by which the GFI1B rs150813342 variant suppresses formation of a GFI1B isoform that preferentially promotes megakaryocyte differentiation and platelet production. These results demonstrate how unbiased studies of natural variation in blood cell traits can provide insight into the regulation of human hematopoiesis. PMID:27486782

  8. Comprehensive Transcriptome Profiling Reveals Long Noncoding RNA Expression and Alternative Splicing Regulation during Fruit Development and Ripening in Kiwifruit (Actinidia chinensis).

    PubMed

    Tang, Wei; Zheng, Yi; Dong, Jing; Yu, Jia; Yue, Junyang; Liu, Fangfang; Guo, Xiuhong; Huang, Shengxiong; Wisniewski, Michael; Sun, Jiaqi; Niu, Xiangli; Ding, Jian; Liu, Jia; Fei, Zhangjun; Liu, Yongsheng

    2016-01-01

    Genomic and transcriptomic data on kiwifruit (Actinidia chinensis) in public databases are very limited despite its nutritional and economic value. Previously, we have constructed and sequenced nine fruit RNA-Seq libraries of A. chinensis "Hongyang" at immature, mature, and postharvest ripening stages of fruit development, and generated over 66.2 million paired-end and 24.4 million single-end reads. From this dataset, here we have identified 7051 long noncoding RNAs (lncRNAs), 29,327 alternative splicing (AS) events and 2980 novel protein-coding genes that were not annotated in the draft genome of "Hongyang." AS events were demonstrated in genes involved in the synthesis of nutritional metabolites in fruit, such as ascorbic acids, carotenoids, anthocyanins, and chlorophylls, and also in genes in the ethylene signaling pathway, which plays an indispensable role in fruit ripening. Additionally, transcriptome profiles and the contents of sugars, organic and main amino acids were compared between immature, mature, and postharvest ripening stages in kiwifruits. A total of 5931 differentially expressed genes were identified, including those associated with the metabolism of sugar, organic acid, and main amino acids. The data generated in this study provide a foundation for further studies of fruit development and ripening in kiwifruit, and identify candidate genes and regulatory elements that could serve as targets for improving important agronomic traits through marker assisted breeding and biotechnology. PMID:27594858

  9. Comprehensive Transcriptome Profiling Reveals Long Noncoding RNA Expression and Alternative Splicing Regulation during Fruit Development and Ripening in Kiwifruit (Actinidia chinensis)

    PubMed Central

    Tang, Wei; Zheng, Yi; Dong, Jing; Yu, Jia; Yue, Junyang; Liu, Fangfang; Guo, Xiuhong; Huang, Shengxiong; Wisniewski, Michael; Sun, Jiaqi; Niu, Xiangli; Ding, Jian; Liu, Jia; Fei, Zhangjun; Liu, Yongsheng

    2016-01-01

    Genomic and transcriptomic data on kiwifruit (Actinidia chinensis) in public databases are very limited despite its nutritional and economic value. Previously, we have constructed and sequenced nine fruit RNA-Seq libraries of A. chinensis “Hongyang” at immature, mature, and postharvest ripening stages of fruit development, and generated over 66.2 million paired-end and 24.4 million single-end reads. From this dataset, here we have identified 7051 long noncoding RNAs (lncRNAs), 29,327 alternative splicing (AS) events and 2980 novel protein-coding genes that were not annotated in the draft genome of “Hongyang.” AS events were demonstrated in genes involved in the synthesis of nutritional metabolites in fruit, such as ascorbic acids, carotenoids, anthocyanins, and chlorophylls, and also in genes in the ethylene signaling pathway, which plays an indispensable role in fruit ripening. Additionally, transcriptome profiles and the contents of sugars, organic and main amino acids were compared between immature, mature, and postharvest ripening stages in kiwifruits. A total of 5931 differentially expressed genes were identified, including those associated with the metabolism of sugar, organic acid, and main amino acids. The data generated in this study provide a foundation for further studies of fruit development and ripening in kiwifruit, and identify candidate genes and regulatory elements that could serve as targets for improving important agronomic traits through marker assisted breeding and biotechnology.

  10. 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.

  11. Systems level approach reveals the correlation of endoderm differentiation of mouse embryonic stem cells with specific microstructural cues of fibrin gels

    PubMed Central

    Task, Keith; D'Amore, Antonio; Singh, Satish; Candiello, Joe; Jaramillo, Maria; Wagner, William R.; Kumta, Prashant; Banerjee, Ipsita

    2014-01-01

    Stem cells receive numerous cues from their associated substrate that help to govern their behaviour. However, identification of influential substrate characteristics poses difficulties because of their complex nature. In this study, we developed an integrated experimental and systems level modelling approach to investigate and identify specific substrate features influencing differentiation of mouse embryonic stem cells (mESCs) on a model fibrous substrate, fibrin. We synthesized a range of fibrin gels by varying fibrinogen and thrombin concentrations, which led to a range of substrate stiffness and microstructure. mESCs were cultured on each of these gels, and characterization of the differentiated cells revealed a strong influence of substrate modulation on gene expression patterning. To identify specific substrate features influencing differentiation, the substrate microstructure was quantified by image analysis and correlated with stem cell gene expression patterns using a statistical model. Significant correlations were observed between differentiation and microstructure features, specifically fibre alignment. Furthermore, this relationship occurred in a lineage-specific manner towards endoderm. This systems level approach allows for identification of specific substrate features from a complex material which are influential to cellular behaviour. Such analysis may be effective in guiding the design of scaffolds with specific properties for tissue engineering applications. PMID:24718448

  12. Proteomic Analysis of Human Brain Microvascular Endothelial Cells Reveals Differential Protein Expression in Response to Enterovirus 71 Infection

    PubMed Central

    Luo, Wenying; Zhong, Jiayu; Zhao, Wei; Liu, Jianjun; Zhang, Renli; Peng, Liang; Hong, Wenxu; Huang, Sheng He; Cao, Hong

    2015-01-01

    2D DIGE technology was employed on proteins prepared from human brain microvascular endothelial cells (HBMEC), to study the differentially expressed proteins in cells at 0 h, 1 h, 16 h, and 24 h after infection. Proteins found to be differentially expressed were identified with matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDITOF/TOF MS) analysis. We identified 43 spots showing changes of at least 2.5 fold up- or downregulated expressions in EV71-infected cells at different time when comparing to control, and 28 proteins could be successfully identified by MALDI TOF/TOF mass spectrometry analysis. 4 proteins were significantly upregulated, and 6 proteins were downregulated, another 18 proteins were different expression at different incubation time. We identified changes in the expression of 12 cellular metabolism-related proteins, 5 molecules involved in cytoskeleton, 3 molecules involved in energy metabolism, 2 molecules involved in signal transduction, 1 molecule involved in the ubiquitin-proteasome pathway, 1 molecule involved in cell cycle, 1 molecule involved in apoptosis-related protein, 1 molecular chaperone, and 2 unknown proteins. These findings build up a comprehensive profile of the HBMEC proteome and provide a useful basis for further analysis of the pathogenic mechanism that underlies EV71 infections to induce severe neural complications. PMID:25821824

  13. A systematic method to identify modulation of transcriptional regulation via chromatin activity reveals regulatory network during mESC differentiation.

    PubMed

    Duren, Zhana; Wang, Yong

    2016-01-01

    Chromatin regulators (CRs) are crucial for connecting the chromatin level and transcriptome level by modulating chromatin structures, establishing, and maintaining epigenetic modifications. We present a systematic method to identify MOdulation of transcriptional regulation via CHromatin Activity (MOCHA) from gene expression data and demonstrate its advantage in associating CRs to their chromatin localization and understand CRs' function. We first re-construct the CRs modulation network by integrating the correlation and conditional correlation concepts. Then we quantify the chromatin activity as hidden variable in network by integrating the upstream and downstream information. We applied MOCHA to systematically explore the interplay of CRs, TFs, and target genes in mouse embryonic stem cells (ESC). As a result, MOCHA identified 420 chromatin regulators with modulation preference, including Pou5f1 and Eed. We found that BAF complex, NuRD complex, and polycomb-group proteins, regulate the delicate balance between pluripotency and differentiation by modulating key TFs including Klf4, Tcf3, and Max; NuRD complex members Mbd3 and Hdac1 may modulate Klf4 to achieve its dual functional roles in pluripotent and differentiation stages;Imprinted gene H19 and Igf2 are modulated by DNA methylation, histone acetylation, and insulator CTCF. Finally, we analyzed CR's combinational modulation pattern by constructing a CR-CR interaction network. PMID:26949222

  14. A systematic method to identify modulation of transcriptional regulation via chromatin activity reveals regulatory network during mESC differentiation

    PubMed Central

    Duren, Zhana; Wang, Yong

    2016-01-01

    Chromatin regulators (CRs) are crucial for connecting the chromatin level and transcriptome level by modulating chromatin structures, establishing, and maintaining epigenetic modifications. We present a systematic method to identify MOdulation of transcriptional regulation via CHromatin Activity (MOCHA) from gene expression data and demonstrate its advantage in associating CRs to their chromatin localization and understand CRs’ function. We first re-construct the CRs modulation network by integrating the correlation and conditional correlation concepts. Then we quantify the chromatin activity as hidden variable in network by integrating the upstream and downstream information. We applied MOCHA to systematically explore the interplay of CRs, TFs, and target genes in mouse embryonic stem cells (ESC). As a result, MOCHA identified 420 chromatin regulators with modulation preference, including Pou5f1 and Eed. We found that BAF complex, NuRD complex, and polycomb-group proteins, regulate the delicate balance between pluripotency and differentiation by modulating key TFs including Klf4, Tcf3, and Max; NuRD complex members Mbd3 and Hdac1 may modulate Klf4 to achieve its dual functional roles in pluripotent and differentiation stages;Imprinted gene H19 and Igf2 are modulated by DNA methylation, histone acetylation, and insulator CTCF. Finally, we analyzed CR’s combinational modulation pattern by constructing a CR-CR interaction network. PMID:26949222

  15. Transcriptome analysis in Coffea eugenioides, an Arabica coffee ancestor, reveals differentially expressed genes in leaves and fruits.

    PubMed

    Yuyama, Priscila Mary; Reis Júnior, Osvaldo; Ivamoto, Suzana Tiemi; Domingues, Douglas Silva; Carazzolle, Marcelo Falsarella; Pereira, Gonçalo Amarante Guimarães; Charmetant, Pierre; Leroy, Thierry; Pereira, Luiz Filipe Protasio

    2016-02-01

    Studies in diploid parental species of polyploid plants are important to understand their contributions to the formation of plant and species evolution. Coffea eugenioides is a diploid species that is considered to be an ancestor of allopolyploid Coffea arabica together with Coffea canephora. Despite its importance in the evolutionary history of the main economic species of coffee, no study has focused on C. eugenioides molecular genetics. RNA-seq creates the possibility to generate reference transcriptomes and identify coding genes and potential candidates related to important agronomic traits. Therefore, the main objectives were to obtain a global overview of transcriptionally active genes in this species using next-generation sequencing and to analyze specific genes that were highly expressed in leaves and fruits with potential exploratory characteristics for breeding and understanding the evolutionary biology of coffee. A de novo assembly generated 36,935 contigs that were annotated using eight databases. We observed a total of ~5000 differentially expressed genes between leaves and fruits. Several genes exclusively expressed in fruits did not exhibit similarities with sequences in any database. We selected ten differentially expressed unigenes in leaves and fruits to evaluate transcriptional profiles using qPCR. Our study provides the first gene catalog for C. eugenioides and enhances the knowledge concerning the mechanisms involved in the C. arabica homeologous. Furthermore, this work will open new avenues for studies into specific genes and pathways in this species, especially related to fruit, and our data have potential value in assisted breeding applications. PMID:26334613

  16. Ultrastructure of the innermost surface of differentiating normal and compression wood tracheids as revealed by field emission scanning electron microscopy.

    PubMed

    Kim, Jong Sik; Awano, Tatsuya; Yoshinaga, Arata; Takabe, Keiji

    2012-06-01

    The ultrastructure of the innermost surface of Cryptomeria japonica differentiating normal wood (NW) and compression wood (CW) was comparatively investigated by field emission electron microscopy (FE-SEM) combined with enzymatic degradation of hemicelluloses. Cellulose microfibril (CMF) bundles were readily observed in NW tracheids in the early stage of secondary cell wall formation, but not in CW tracheids because of the heavy accumulation of amorphous materials composed mainly of galactans and lignin. This result suggests that the ultrastructural deposition of cell wall components in the tracheid cell wall differ between NW and CW from the early stage of secondary cell wall formation. Delignified NW and CW tracheids showed similar structural changes during differentiating stages after xylanase or β-mannanase treatment, whereas they exhibited clear differences in ultrastructure in mature stages. Although thin CMF bundles were exposed in both delignified mature NW and CW tracheids by xylanase treatment, ultrastructural changes following β-mannanase treatment were only observed in CW tracheids. CW tracheids also showed different degradation patterns between xylanase and β-mannanase. CMF bundles showed a smooth surface in delignified mature CW tracheids treated with xylanase, whereas they had an uneven surface in delignified mature CW tracheids treated with β-mannanase, indicating that the uneven surface of CMF bundles was related to xylans. The present results suggest that ultrastructural deposition and organization of lignin and hemicelluloses in CW tracheids may differ from those of NW tracheids. PMID:22173277

  17. High-Throughput Sequencing Reveals Differential Expression of miRNAs in Intestine from Sea Cucumber during Aestivation

    PubMed Central

    Chen, Muyan; Zhang, Xiumei; Liu, Jianning; Storey, Kenneth B.

    2013-01-01

    The regulatory role of miRNA in gene expression is an emerging hot new topic in the control of hypometabolism. Sea cucumber aestivation is a complicated physiological process that includes obvious hypometabolism as evidenced by a decrease in the rates of oxygen consumption and ammonia nitrogen excretion, as well as a serious degeneration of the intestine into a very tiny filament. To determine whether miRNAs play regulatory roles in this process, the present study analyzed profiles of miRNA expression in the intestine of the sea cucumber (Apostichopus japonicus), using Solexa deep sequencing technology. We identified 308 sea cucumber miRNAs, including 18 novel miRNAs specific to sea cucumber. Animals sampled during deep aestivation (DA) after at least 15 days of continuous torpor, were compared with animals from a non-aestivation (NA) state (animals that had passed through aestivation and returned to the active state). We identified 42 differentially expressed miRNAs [RPM (reads per million) >10, |FC| (|fold change|) ≥1, FDR (false discovery rate) <0.01] during aestivation, which were validated by two other miRNA profiling methods: miRNA microarray and real-time PCR. Among the most prominent miRNA species, miR-200-3p, miR-2004, miR-2010, miR-22, miR-252a, miR-252a-3p and miR-92 were significantly over-expressed during deep aestivation compared with non-aestivation animals. Preliminary analyses of their putative target genes and GO analysis suggest that these miRNAs could play important roles in global transcriptional depression and cell differentiation during aestivation. High-throughput sequencing data and microarray data have been submitted to GEO database. PMID:24143179

  18. High-throughput sequencing reveals differential expression of miRNAs in intestine from sea cucumber during aestivation.

    PubMed

    Chen, Muyan; Zhang, Xiumei; Liu, Jianning; Storey, Kenneth B

    2013-01-01

    The regulatory role of miRNA in gene expression is an emerging hot new topic in the control of hypometabolism. Sea cucumber aestivation is a complicated physiological process that includes obvious hypometabolism as evidenced by a decrease in the rates of oxygen consumption and ammonia nitrogen excretion, as well as a serious degeneration of the intestine into a very tiny filament. To determine whether miRNAs play regulatory roles in this process, the present study analyzed profiles of miRNA expression in the intestine of the sea cucumber (Apostichopus japonicus), using Solexa deep sequencing technology. We identified 308 sea cucumber miRNAs, including 18 novel miRNAs specific to sea cucumber. Animals sampled during deep aestivation (DA) after at least 15 days of continuous torpor, were compared with animals from a non-aestivation (NA) state (animals that had passed through aestivation and returned to the active state). We identified 42 differentially expressed miRNAs [RPM (reads per million) >10, |FC| (|fold change|) ≥ 1, FDR (false discovery rate) <0.01] during aestivation, which were validated by two other miRNA profiling methods: miRNA microarray and real-time PCR. Among the most prominent miRNA species, miR-200-3p, miR-2004, miR-2010, miR-22, miR-252a, miR-252a-3p and miR-92 were significantly over-expressed during deep aestivation compared with non-aestivation animals. Preliminary analyses of their putative target genes and GO analysis suggest that these miRNAs could play important roles in global transcriptional depression and cell differentiation during aestivation. High-throughput sequencing data and microarray data have been submitted to GEO database. PMID:24143179

  19. Differential origin of reticulospinal drive to motoneurons innervating trunk and hindlimb muscles in the mouse revealed by optical recording

    PubMed Central

    Szokol, Karolina; Glover, Joel C; Perreault, Marie-Claude

    2008-01-01

    To better understand how the brainstem reticular formation controls and coordinates trunk and hindlimb muscle activity, we used optical recording to characterize the functional connections between medullary reticulospinal neurons and lumbar motoneurons of the L2 segment in the neonatal mouse. In an isolated brainstem–spinal cord preparation, synaptically induced calcium transients were visualized in individual MNs of the ipsilateral and contralateral medial and lateral motor columns (MMC, LMC) following focal electrical stimulation of the medullary reticular formation (MRF). Stimulation of the MRF elicited differential responses in MMC and LMC, according to a specific spatial organization. Stimulation of the medial MRF elicited responses predominantly in the LMC whereas stimulation of the lateral MRF elicited responses predominantly in the MMC. This reciprocal response pattern was observed on both the ipsilateral and contralateral sides of the spinal cord. To ascertain whether the regions stimulated contained reticulospinal neurons, we retrogradely labelled MRF neurons with axons coursing in different spinal funiculi, and compared the distributions of the labelled neurons to the stimulation sites. We found a large number of retrogradely labelled neurons within regions of the gigantocellularis reticular nucleus (including its pars ventralis and alpha) where most stimulation sites were located. The existence of a mediolateral organization within the MRF, whereby distinct populations of reticulospinal neurons predominantly influence medial or lateral motoneurons, provides an anatomical substrate for the differential control of trunk and hindlimb muscles. Such an organization introduces flexibility in the initiation and coordination of activity in the two sets of muscles that would satisfy many of the functional requirements that arise during postural and non-postural motor control in mammals. PMID:18772205

  20. Disturbed Dreaming and the Instability of Sleep: Altered Nonrapid Eye Movement Sleep Microstructure in Individuals with Frequent Nightmares as Revealed by the Cyclic Alternating Pattern

    PubMed Central

    Simor, Péter; Bódizs, Róbert; Horváth, Klára; Ferri, Raffaele

    2013-01-01

    Study Objectives: Nightmares are disturbing mental experiences during sleep that usually result in abrupt awakenings. Frequent nightmares are associated with poor subjective sleep quality, and recent polysomnographic data suggest that nightmare sufferers exhibit impaired sleep continuity during nonrapid eye movement (NREM) sleep. Because disrupted sleep might be related to abnormal arousal processes, the goal of this study was to examine polysomnographic arousal-related activities in a group of nightmare sufferers and a healthy control group. Design: Sleep microstructure analysis was carried out by scoring the cyclic alternating pattern (CAP) in NREM sleep and the arousal index in rapid eye movement (REM) sleep on the second night of the polysomnographic examination. Setting: Hospital-based sleep research laboratory. Participants: There were 17 in the nightmare (NMs) group and 23 in the healthy control (CTLs) group. Interventions: N/A. Measurements and Results: The NMs group exhibited reduced amounts of CAP A1 subtype and increased CAP A2 and A3 subtypes, as well as longer duration of CAP A phases in comparison with CTLs. Moreover, these differences remained significant after controlling for the confounding factors of anxious and depressive symptoms. The absolute number and frequency of REM arousals did not differ significantly between the two groups. Conclusions: The results of our study indicate that NREM sleep microstructure is altered during nonsymptomatic nights of nightmares. Disrupted sleep in the NMs group seems to be related to abnormal arousal processes, specifically an imbalance in sleep-promoting and arousing mechanisms during sleep. Citation: Simor P; Bódizs R; Horváth K; Ferri R. Disturbed dreaming and the instability of sleep: altered nonrapid eye movement sleep microstructure in individuals with frequent nightmares as revealed by the cyclic alternating pattern. SLEEP 2013;36(3):413-419. PMID:23449753

  1. Differential proteomic profiling reveals regulatory proteins and novel links between primary metabolism and spinosad production in Saccharopolyspora spinosa

    PubMed Central

    2014-01-01

    Background Saccharopolyspora spinosa is an important producer of antibiotic spinosad with clarified biosynthesis pathway but its complex regulation networks associated with primary metabolism and secondary metabolites production almost have never been concerned or studied before. The proteomic analysis of a novel Saccharopolyspora spinosa CCTCC M206084 was performed and aimed to provide a global profile of regulatory proteins. Results Two-dimensional-liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 1090, 1166, 701, and 509 proteins from four phases respectively, i.e., the logarithmic growth phase (T1), early stationary phase (T2), late stationary phase (T3), and decline phase (T4). Among the identified proteins, 1579 were unique to the S. spinosa proteome, including almost all the enzymes for spinosad biosynthesis. Trends in protein expression over the various time phases were deduced from using the modified protein abundance index (PAI), revealed the importance of stress pathway proteins and other global regulatory network proteins during spinosad biosynthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis followed by one-dimensional LC-MS/MS identification revealed similar trend of protein expression from four phases with the results of semi-quantification by PAI. qRT-PCR analysis revealed that 6 different expressed genes showed a positive correlation between changes at translational and transcriptional expression level. Expression of three proteins that likely promote spinosad biosynthesis, namely, 5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase (MHSM), glutamine synthetase (GS) and cyclic nucleotide-binding domain-containing protein (CNDP) was validated by western blot, which confirmed the results of proteomic analysis. Conclusions This study is the first systematic analysis of the S. spinosa proteome during fermentation and its valuable proteomic data of regulatory proteins may be used to enhance

  2. The proteome of cytosolic lipid droplets isolated from differentiated Caco-2/TC7 enterocytes reveals cell-specific characteristics

    PubMed Central

    Bouchoux, Julien; Beilstein, Frauke; Pauquai, Thomas; Guerrera, I. Chiara; Chateau, Danielle; Ly, Nathalie; Alqub, Malik; Klein, Christophe; Chambaz, Jean; Rousset, Monique; Lacorte, Jean-Marc; Morel, Etienne; Demignot, Sylvie

    2011-01-01

    Background information. Intestinal absorption of alimentary lipids is a complex process ensured by enterocytes and leading to TRL [TAG (triacylglycerol)-rich lipoprotein] assembly and secretion. The accumulation of circulating intestine-derived TRL is associated with atherosclerosis, stressing the importance of the control of postprandial hypertriglyceridaemia. During the postprandial period, TAGs are also transiently stored as CLDs (cytosolic lipid droplets) in enterocytes. As a first step for determining whether CLDs could play a role in the control of enterocyte TRL secretion, we analysed the protein endowment of CLDs isolated by sucrose-gradient centrifugation from differentiated Caco-2/TC7 enterocytes, the only human model able to secrete TRL in culture and to store transiently TAGs as CLDs when supplied with lipids. Cells were analysed after a 24 h incubation with lipid micelles and thus in a state of CLD-associated TAG mobilization. Results. Among the 105 proteins identified in the CLD fraction by LC-MS/MS (liquid chromatography coupled with tandem MS), 27 were directly involved in lipid metabolism pathways potentially relevant to enterocyte-specific functions. The transient feature of CLDs was consistent with the presence of proteins necessary for fatty acid activation (acyl-CoA synthetases) and for TAG hydrolysis. In differentiated Caco-2/TC7 enterocytes, we identified for the first time LPCAT2 (lysophosphatidylcholine acyltransferase 2), involved in PC (phosphatidylcholine) synthesis, and 3BHS1 (3-β-hydroxysteroid dehydrogenase 1), involved in steroid metabolism, and confirmed their partial CLD localization by immunofluorescence. In enterocytes, LPCAT2 may provide an economical source of PC, necessary for membrane synthesis and lipoprotein assembly, from the lysoPC present in the intestinal lumen. We also identified proteins involved in lipoprotein metabolism, such as ApoA-IV (apolipoprotein A-IV), which is specifically expressed by enterocytes and has

  3. High-throughput sequencing reveals differential expression of miRNAs in prehierarchal follicles of laying and brooding geese

    PubMed Central

    Yu, Jing; He, Ke; Ren, Ting; Lou, Yaping

    2016-01-01

    Broodiness is the primary factor influencing egg production in geese, in which several genes and miRNAs participate. Detailed spatiotemporal profiles of miRNAs encompassing follicle development levels, however, are lacking. In this study, we collected preovulatory follicles (classified as small white follicles, large white follicles, and small yellow follicles) from brooding and laying geese and aimed to analyze microRNA (miRNA or miR) during folliculogenesis. High-throughput sequencing and bioinformatics analysis were used to identify the miRNAs involved in follicle development. The let7 family, miR-10 family, and miR-143 family were abundant in these libraries, and they have been suggested to play a housekeeping role during folliculogenesis. Joint comparisons revealed 23 upregulated and 21 downregulated miRNAs (in at least two comparisons of follicles during brooding and laying, P < 0.1) in the laying stage. Unlike reproduction pathways reported for ovaries, GO and KEGG analysis suggested pathways for cell apoptosis and proliferation, such as the regulation of actin cytoskeleton, endocytosis, axon guidance, pathways in cancer, tight junctions, focal adhesion, the MAPK signaling pathway, cytokine-cytokine receptor interactions, and the Wnt signaling pathway in folliculogenesis. This study revealed the miRNAs that were directly involved in follicular atresia, and our results added to the understanding of the functional involvement of miRNAs during specific stages of follicle development. PMID:27199452

  4. Nanoscale analysis of pyritized microfossils reveals differential heterotrophic consumption in the ∼1.9-Ga Gunflint chert

    PubMed Central

    Wacey, David; McLoughlin, Nicola; Kilburn, Matt R.; Saunders, Martin; Cliff, John B.; Kong, Charlie; Barley, Mark E.; Brasier, Martin D.

    2013-01-01

    The 1.88-Ga Gunflint biota is one of the most famous Precambrian microfossil lagerstätten and provides a key record of the biosphere at a time of changing oceanic redox structure and chemistry. Here, we report on pyritized replicas of the iconic autotrophic Gunflintia–Huroniospora microfossil assemblage from the Schreiber Locality, Canada, that help capture a view through multiple trophic levels in a Paleoproterozoic ecosystem. Nanoscale analysis of pyritic Gunflintia (sheaths) and Huroniospora (cysts) reveals differing relic carbon and nitrogen distributions caused by contrasting spectra of decay and pyritization between taxa, reflecting in part their primary organic compositions. In situ sulfur isotope measurements from individual microfossils (δ34SV-CDT +6.7‰ to +21.5‰) show that pyritization was mediated by sulfate-reducing microbes within sediment pore waters whose sulfate ion concentrations rapidly became depleted, owing to occlusion of pore space by coeval silicification. Three-dimensional nanotomography reveals additional pyritized biomaterial, including hollow, cellular epibionts and extracellular polymeric substances, showing a preference for attachment to Gunflintia over Huroniospora and interpreted as components of a saprophytic heterotrophic, decomposing community. This work also extends the record of remarkable biological preservation in pyrite back to the Paleoproterozoic and provides criteria to assess the authenticity of even older pyritized microstructures that may represent some of the earliest evidence for life on our planet. PMID:23630257

  5. High-throughput sequencing reveals differential expression of miRNAs in prehierarchal follicles of laying and brooding geese.

    PubMed

    Yu, Jing; He, Ke; Ren, Ting; Lou, Yaping; Zhao, Ayong

    2016-07-01

    Broodiness is the primary factor influencing egg production in geese, in which several genes and miRNAs participate. Detailed spatiotemporal profiles of miRNAs encompassing follicle development levels, however, are lacking. In this study, we collected preovulatory follicles (classified as small white follicles, large white follicles, and small yellow follicles) from brooding and laying geese and aimed to analyze microRNA (miRNA or miR) during folliculogenesis. High-throughput sequencing and bioinformatics analysis were used to identify the miRNAs involved in follicle development. The let7 family, miR-10 family, and miR-143 family were abundant in these libraries, and they have been suggested to play a housekeeping role during folliculogenesis. Joint comparisons revealed 23 upregulated and 21 downregulated miRNAs (in at least two comparisons of follicles during brooding and laying, P < 0.1) in the laying stage. Unlike reproduction pathways reported for ovaries, GO and KEGG analysis suggested pathways for cell apoptosis and proliferation, such as the regulation of actin cytoskeleton, endocytosis, axon guidance, pathways in cancer, tight junctions, focal adhesion, the MAPK signaling pathway, cytokine-cytokine receptor interactions, and the Wnt signaling pathway in folliculogenesis. This study revealed the miRNAs that were directly involved in follicular atresia, and our results added to the understanding of the functional involvement of miRNAs during specific stages of follicle development. PMID:27199452

  6. Nanoscale analysis of pyritized microfossils reveals differential heterotrophic consumption in the ~1.9-Ga Gunflint chert.

    PubMed

    Wacey, David; McLoughlin, Nicola; Kilburn, Matt R; Saunders, Martin; Cliff, John B; Kong, Charlie; Barley, Mark E; Brasier, Martin D

    2013-05-14

    The 1.88-Ga Gunflint biota is one of the most famous Precambrian microfossil lagerstätten and provides a key record of the biosphere at a time of changing oceanic redox structure and chemistry. Here, we report on pyritized replicas of the iconic autotrophic Gunflintia-Huroniospora microfossil assemblage from the Schreiber Locality, Canada, that help capture a view through multiple trophic levels in a Paleoproterozoic ecosystem. Nanoscale analysis of pyritic Gunflintia (sheaths) and Huroniospora (cysts) reveals differing relic carbon and nitrogen distributions caused by contrasting spectra of decay and pyritization between taxa, reflecting in part their primary organic compositions. In situ sulfur isotope measurements from individual microfossils (δ(34)S(V-CDT) +6.7‰ to +21.5‰) show that pyritization was mediated by sulfate-reducing microbes within sediment pore waters whose sulfate ion concentrations rapidly became depleted, owing to occlusion of pore space by coeval silicification. Three-dimensional nanotomography reveals additional pyritized biomaterial, including hollow, cellular epibionts and extracellular polymeric substances, showing a preference for attachment to Gunflintia over Huroniospora and interpreted as components of a saprophytic heterotrophic, decomposing community. This work also extends the record of remarkable biological preservation in pyrite back to the Paleoproterozoic and provides criteria to assess the authenticity of even older pyritized microstructures that may represent some of the earliest evidence for life on our planet. PMID:23630257

  7. Differential regulation of translation and endocytosis of alternatively spliced forms of the type II bone morphogenetic protein (BMP) receptor

    PubMed Central

    Amsalem, Ayelet R.; Marom, Barak; Shapira, Keren E.; Hirschhorn, Tal; Preisler, Livia; Paarmann, Pia; Knaus, Petra; Henis, Yoav I.; Ehrlich, Marcelo

    2016-01-01

    The expression and function of transforming growth factor-β superfamily receptors are regulated by multiple molecular mechanisms. The type II BMP receptor (BMPRII) is expressed as two alternatively spliced forms, a long and a short form (BMPRII-LF and –SF, respectively), which differ by an ∼500 amino acid C-terminal extension, unique among TGF-β superfamily receptors. Whereas this extension was proposed to modulate BMPRII signaling output, its contribution to the regulation of receptor expression was not addressed. To map regulatory determinants of BMPRII expression, we compared synthesis, degradation, distribution, and endocytic trafficking of BMPRII isoforms and mutants. We identified translational regulation of BMPRII expression and the contribution of a 3’ terminal coding sequence to this process. BMPRII-LF and -SF differed also in their steady-state levels, kinetics of degradation, intracellular distribution, and internalization rates. A single dileucine signal in the C-terminal extension of BMPRII-LF accounted for its faster clathrin-mediated endocytosis relative to BMPRII-SF, accompanied by mildly faster degradation. Higher expression of BMPRII-SF at the plasma membrane resulted in enhanced activation of Smad signaling, stressing the potential importance of the multilayered regulation of BMPRII expression at the plasma membrane. PMID:26739752

  8. Differential regulation of translation and endocytosis of alternatively spliced forms of the type II bone morphogenetic protein (BMP) receptor.

    PubMed

    Amsalem, Ayelet R; Marom, Barak; Shapira, Keren E; Hirschhorn, Tal; Preisler, Livia; Paarmann, Pia; Knaus, Petra; Henis, Yoav I; Ehrlich, Marcelo

    2016-02-15

    The expression and function of transforming growth factor-β superfamily receptors are regulated by multiple molecular mechanisms. The type II BMP receptor (BMPRII) is expressed as two alternatively spliced forms, a long and a short form (BMPRII-LF and -SF, respectively), which differ by an ∼500 amino acid C-terminal extension, unique among TGF-β superfamily receptors. Whereas this extension was proposed to modulate BMPRII signaling output, its contribution to the regulation of receptor expression was not addressed. To map regulatory determinants of BMPRII expression, we compared synthesis, degradation, distribution, and endocytic trafficking of BMPRII isoforms and mutants. We identified translational regulation of BMPRII expression and the contribution of a 3' terminal coding sequence to this process. BMPRII-LF and -SF differed also in their steady-state levels, kinetics of degradation, intracellular distribution, and internalization rates. A single dileucine signal in the C-terminal extension of BMPRII-LF accounted for its faster clathrin-mediated endocytosis relative to BMPRII-SF, accompanied by mildly faster degradation. Higher expression of BMPRII-SF at the plasma membrane resulted in enhanced activation of Smad signaling, stressing the potential importance of the multilayered regulation of BMPRII expression at the plasma membrane. PMID:26739752

  9. Alternate Immersion in an External Glucose Solution Differentially Affects Blood Sugar Values in Older Versus Younger Zebrafish Adults.

    PubMed

    Connaughton, Victoria P; Baker, Cassandra; Fonde, Lauren; Gerardi, Emily; Slack, Carly

    2016-04-01

    Recently, zebrafish have been used to examine hyperglycemia-induced complications (retinopathy and neuropathy), as would occur in individuals with diabetes. Current models to induce hyperglycemia in zebrafish include glucose immersion and streptozotocin injections. Both are effective, although neither is reported to elevate blood sugar values for more than 1 month. In this article, we report differences in hyperglycemia induction and maintenance in young (4-11 months) versus old (1-3 years) zebrafish adults. In particular, older fish immersed in an alternating constant external glucose solution (2%) for 2 months displayed elevated blood glucose levels for the entire experimental duration. In contrast, younger adults displayed only transient hyperglycemia, suggesting the fish were acclimating to the glucose exposure protocol. However, modifying the immersion protocol to include a stepwise increasing glucose concentration (from 1% → 2%→3%) resulted in maintained hyperglycemia in younger zebrafish adults for up to 2 months. Glucose-exposed younger fish collected after 8 weeks of exposure also displayed a significant decrease in wet weight. Taken together, these data suggest different susceptibilities to hyperglycemia in older and younger fish and that stepwise increasing glucose concentrations of 1% are required for maintenance of hyperglycemia in younger adults, with higher concentrations of glucose resulting in greater increases in blood sugar values. PMID:26771444

  10. Introns, alternative splicing, spliced leader trans-splicing and differential expression of pcna and cyclin in Perkinsus marinus.

    PubMed

    Zhang, Huan; Dungan, Christopher F; Lin, Senjie

    2011-01-01

    To gain understanding on the structure and regulation of growth-related genes of the parasitic alveolatePerkinsus marinus, we analyzed genes encoding proliferating cell nuclear antigen (pcna) and cyclins (cyclin). Comparison of the full-length cDNAs with the corresponding genomic sequences revealedtrans-splicing of the mRNAs of these genes with a conserved 21-22 nt spliced leader. Over 10 copies ofpcnawere detected, with identical gene structures and similar nucleotide (nt) sequences (88-99%), encoding largely identical amino acid sequences (aa). Two distinct types ofcyclin(Pmacyclin1 andPmacyclin2) were identified, with 66-69% nt and 81-85% aa similarities.Pmacyclin2 was organized in tandem repeats, and was alternatively spliced, giving rise to five subtypes of transcripts. For bothpcnaandcyclingenes, 6-10 introns were found. Quantitative RT-PCR assays showed thatpcnaandPmacyclin2 expression levels were low with small variations during a 28-h time course, whereasPmacyclin1 transcript abundance was 10-100 times higher, and increased markedly during active cell division, suggesting that it is a mitoticcyclinand can be a useful growth marker for this species. The gene structure and expression features along with phylogenetic results position this organism between dinoflagellates and apicomplexans, but its definitive affiliation among alveolates requires further studies. PMID:20650682

  11. Gentamicin differentially alters cellular metabolism of cochlear hair cells as revealed by NAD(P)H fluorescence lifetime imaging

    PubMed Central

    Zholudeva, Lyandysha V.; Ward, Kristina G.; Nichols, Michael G.; Smith, Heather Jensen

    2015-01-01

    Abstract. Aminoglycoside antibiotics are implicated as culprits of hearing loss in more than 120,000 individuals annually. Research has shown that the sensory cells, but not supporting cells, of the cochlea are readily damaged and/or lost after use of such antibiotics. High-frequency outer hair cells (OHCs) show a greater sensitivity to antibiotics than high- and low-frequency inner hair cells (IHCs). We hypothesize that variations in mitochondrial metabolism account for differences in susceptibility. Fluorescence lifetime microscopy was used to quantify changes in NAD(P)H in sensory and supporting cells from explanted murine cochleae exposed to mitochondrial uncouplers, inhibitors, and an ototoxic antibiotic, gentamicin (GM). Changes in metabolic state resulted in a redistribution of NAD(P)H between subcellular fluorescence lifetime pools. Supporting cells had a significantly longer lifetime than sensory cells. Pretreatment with GM increased NAD(P)H intensity in high-frequency sensory cells, as well as the NAD(P)H lifetime within IHCs. GM specifically increased NAD(P)H concentration in high-frequency OHCs, but not in IHCs or pillar cells. Variations in NAD(P)H intensity in response to mitochondrial toxins and GM were greatest in high-frequency OHCs. These results demonstrate that GM rapidly alters mitochondrial metabolism, differentially modulates cell metabolism, and provides evidence that GM-induced changes in metabolism are significant and greatest in high-frequency OHCs. PMID:25688541

  12. High-throughput sequencing reveals differential regulation of miRNAs in fenoxaprop-P-ethyl-resistant Beckmannia syzigachne

    PubMed Central

    Pan, Lang; Wang, Zhaoyun; Cai, Jia; Gao, Haitao; Zhao, Hongwei; Dong, Liyao

    2016-01-01

    Non-target site resistance (NTSR) to herbicides is an increasing concern for weed control. The majority of previous studies have focused on metabolic resistance mechanisms of NTSR, but no research exists on gene regulation mechanisms behind herbicide resistance, such as microRNA (miRNA). Here, we identified 3 American sloughgrass (Beckmannia syzigachne Steud.) populations containing fenoxaprop-P-ethyl-resistant plants. We then constructed small RNA libraries and subjected them to deep sequencing and bioinformatics analyses. Forty known and 36 potentially novel, predicted miRNAs were successfully identified. Of these, we identified 3 conserved, predicted candidate NTSR-determinant miRNAs and their potential corresponding target genes, as well as 4 novel potential miRNAs with high count. Target gene prediction and annotation indicated that these 7 differentially expressed miRNAs potentially play a role in regulating specific stress-responsive genes, very likely related to herbicide resistance. Expression profiles were determined with quantitative real-time PCR. The present study is a novel, large-scale characterization of weed miRNAs. The results should further our understanding of miRNA expression profiles associated with herbicide resistance, allowing for the development of more effective weed management strategies. PMID:27353151

  13. Comparative Proteomic Analysis Reveals Differential Root Proteins in Medicago sativa and Medicago truncatula in Response to Salt Stress

    PubMed Central

    Long, Ruicai; Li, Mingna; Zhang, Tiejun; Kang, Junmei; Sun, Yan; Cong, Lili; Gao, Yanli; Liu, Fengqi; Yang, Qingchuan

    2016-01-01

    Salt stress is an important abiotic stress that causes decreased crop yields. Root growth and plant activities are affected by salt stress through the actions of specific genes that help roots adapt to adverse environmental conditions. For a more comprehensive understanding of proteins affected by salinity, we used two-dimensional gel electrophoresis and mass spectrometry to characterize the proteome-level changes associated with salt stress response in Medicago sativa cv. Zhongmu-1 and Medicago truncatula cv. Jemalong A17 roots. Our physiological and phenotypic observations indicated that Zhongmu-1 was more salt tolerant than Jemalong A17. We identified 93 and 30 proteins whose abundance was significantly affected by salt stress in Zhongmu-1 and Jemalong A17 roots, respectively. The tandem mass spectrometry analysis of the differentially accumulated proteins resulted in the identification of 60 and 26 proteins in Zhongmu-1 and Jemalong A17 roots, respectively. Function analyses indicated molecule binding and catalytic activity were the two primary functional categories. These proteins have known functions in various molecular processes, including defense against oxidative stress, metabolism, photosynthesis, protein synthesis and processing, and signal transduction. The transcript levels of four identified proteins were determined by quantitative reverse transcription polymerase chain reaction. Our results indicate that some of the identified proteins may play key roles in salt stress tolerance. PMID:27066057

  14. EPG Recordings Reveal Differential Feeding Behaviors in Sogatella furcifera in Response to Plant Virus Infection and Transmission Success.

    PubMed

    Lei, Wenbin; Li, Pei; Han, Yongqiang; Gong, Shaolong; Yang, Lang; Hou, Maolin

    2016-01-01

    Plant viruses are primarily transmitted by insect vectors and virus infection may influence on the vectors' feeding behaviors. Using an electrical penetration graph, we detected that infection with the Southern rice black-streaked dwarf virus (SRBSDV) in the white-backed planthopper (WBPH) and in rice plants both altered the vector's feeding behavior. When viruliferous WBPH (carrying SRBSDV) were fed on uninfected plants, they spent more time in salivation and phloem sap ingestion than non-viruliferous insects. In comparison with uninfected plants, infected plants showed an arrestant effect on non-viruliferous WBPH for phloem sap ingestion. Differential feeding behaviors were also detected between the WBPH that inoculated or acquired SRBSDV and those that failed to. The WBPH that inoculated SRBSDV exhibited more probing bouts, salivation events and phloem sap ingestion events and longer salivation than those that failed to. The WBPH that acquired SRBSDV were quicker to reach phloem and spent more time in phloem sap ingestion than those that failed to. These behavior alterations in the vector may have adaptive advantages for SRBSDV transmission and spread success because greater salivation by viruliferous vectors on uninfected hosts will promote virus inoculation, whereas more sap ingestion by non-viruliferous vectors on infected hosts will promote virus acquisition. PMID:27492995

  15. Genetic differentiation of Octopus minor (Mollusca, Cephalopoda) off the northern coast of China as revealed by amplified fragment length polymorphisms.

    PubMed

    Yang, J M; Sun, G H; Zheng, X D; Ren, L H; Wang, W J; Li, G R; Sun, B C

    2015-01-01

    Octopus minor (Sasaki, 1920) is an economically important cephalopod that is found in the northern coastal waters of China. In this study, we investigated genetic differentiation in fishery populations using amplified fragment length polymorphisms (AFLPs). A total of 150 individuals were collected from five locations: Dalian (DL), Yan-tai (YT), Qingdao (QD), Lianyungang (LY), and Zhoushan (ZS), and 243 reproducible bands were amplified using five AFLP primer combinations. The percentage of polymorphic bands ranged from 53.33 to 76.08%. Nei's genetic identity ranged from 0.9139 to 0.9713, and the genetic distance ranged from 0.0291 to 0.0900. A phylogenetic tree was constructed using the unweighted pair group method with arithmetic mean, based on the genetic distance. The DL and YT populations originated from one clade, while the QD, LY, and ZS populations originated from another. The results indicate that the O. minor stock consisted of two genetic populations with an overall significantly analogous FST value (0.1088, P < 0.05). Most of the variance was within populations. These findings will be important for more sustainable octopus fisheries, so that this marine resource can be conserved for its long-term utilization. PMID:26634529

  16. High-throughput sequencing reveals differential regulation of miRNAs in fenoxaprop-P-ethyl-resistant Beckmannia syzigachne.

    PubMed

    Pan, Lang; Wang, Zhaoyun; Cai, Jia; Gao, Haitao; Zhao, Hongwei; Dong, Liyao

    2016-01-01

    Non-target site resistance (NTSR) to herbicides is an increasing concern for weed control. The majority of previous studies have focused on metabolic resistance mechanisms of NTSR, but no research exists on gene regulation mechanisms behind herbicide resistance, such as microRNA (miRNA). Here, we identified 3 American sloughgrass (Beckmannia syzigachne Steud.) populations containing fenoxaprop-P-ethyl-resistant plants. We then constructed small RNA libraries and subjected them to deep sequencing and bioinformatics analyses. Forty known and 36 potentially novel, predicted miRNAs were successfully identified. Of these, we identified 3 conserved, predicted candidate NTSR-determinant miRNAs and their potential corresponding target genes, as well as 4 novel potential miRNAs with high count. Target gene prediction and annotation indicated that these 7 differentially expressed miRNAs potentially play a role in regulating specific stress-responsive genes, very likely related to herbicide resistance. Expression profiles were determined with quantitative real-time PCR. The present study is a novel, large-scale characterization of weed miRNAs. The results should further our understanding of miRNA expression profiles associated with herbicide resistance, allowing for the development of more effective weed management strategies. PMID:27353151

  17. Comparative gene expression analysis of genital tubercle development reveals a putative appendicular Wnt7 network for the epidermal differentiation

    PubMed Central

    Chiu, Han Sheng; Szucsik, John C.; Georgas, Kylie M.; Jones, Julia L.; Rumballe, Bree A.; Tang, Dave; Grimmond, Sean M.; Lewis, Alfor G.; Aronow, Bruce J.; Lessard, James L.; Little, Melissa H.

    2010-01-01

    Here we describe the first detailed catalogue of gene expression in the developing lower urinary tract (LUT), including epithelial and mesenchymal portions of the developing bladder, urogenital sinus, urethra and genital tubercle (GT) at E13 and E14. Top compartment-specific genes implicated by the microarray data were validated using wholemount in situ hybridization (ISH) over the entire LUT. To demonstrate the potential of this resource to implicate developmentally critical features, we focused on gene expression patterns and pathways in the sexually indeterminate, androgen-independent GT. GT expression patterns reinforced the proposed similarities between development of GT, limb and craniofacial prominences. Comparison of spatial expression patterns predicted a network of Wnt7a-associated GT-enriched epithelial genes, including Gjb2, Dsc3, Krt5 and Sostdc1. Known from other contexts, these genes are associated with normal epidermal differentiation, with disruptions in Dsc3 and Gjb2 showing palmo-plantar keratoderma in the limb. We propose that this gene network contributes to normal foreskin, scrotum and labial development. As several of these are known regulated by, or contain cis elements responsive to retinoic acid, estrogen, or androgen, this implicates this pathway in the later androgen-dependent development of the GT. PMID:20510229

  18. EPG Recordings Reveal Differential Feeding Behaviors in Sogatella furcifera in Response to Plant Virus Infection and Transmission Success

    PubMed Central

    Lei, Wenbin; Li, Pei; Han, Yongqiang; Gong, Shaolong; Yang, Lang; Hou, Maolin

    2016-01-01

    Plant viruses are primarily transmitted by insect vectors and virus infection may influence on the vectors’ feeding behaviors. Using an electrical penetration graph, we detected that infection with the Southern rice black-streaked dwarf virus (SRBSDV) in the white-backed planthopper (WBPH) and in rice plants both altered the vector’s feeding behavior. When viruliferous WBPH (carrying SRBSDV) were fed on uninfected plants, they spent more time in salivation and phloem sap ingestion than non-viruliferous insects. In comparison with uninfected plants, infected plants showed an arrestant effect on non-viruliferous WBPH for phloem sap ingestion. Differential feeding behaviors were also detected between the WBPH that inoculated or acquired SRBSDV and those that failed to. The WBPH that inoculated SRBSDV exhibited more probing bouts, salivation events and phloem sap ingestion events and longer salivation than those that failed to. The WBPH that acquired SRBSDV were quicker to reach phloem and spent more time in phloem sap ingestion than those that failed to. These behavior alterations in the vector may have adaptive advantages for SRBSDV transmission and spread success because greater salivation by viruliferous vectors on uninfected hosts will promote virus inoculation, whereas more sap ingestion by non-viruliferous vectors on infected hosts will promote virus acquisition. PMID:27492995

  19. Gentamicin differentially alters cellular metabolism of cochlear hair cells as revealed by NAD(P)H fluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

    Zholudeva, Lyandysha V.; Ward, Kristina G.; Nichols, Michael G.; Smith, Heather Jensen

    2015-05-01

    Aminoglycoside antibiotics are implicated as culprits of hearing loss in more than 120,000 individuals annually. Research has shown that the sensory cells, but not supporting cells, of the cochlea are readily damaged and/or lost after use of such antibiotics. High-frequency outer hair cells (OHCs) show a greater sensitivity to antibiotics than high- and low-frequency inner hair cells (IHCs). We hypothesize that variations in mitochondrial metabolism account for differences in susceptibility. Fluorescence lifetime microscopy was used to quantify changes in NAD(P)H in sensory and supporting cells from explanted murine cochleae exposed to mitochondrial uncouplers, inhibitors, and an ototoxic antibiotic, gentamicin (GM). Changes in metabolic state resulted in a redistribution of NAD(P)H between subcellular fluorescence lifetime pools. Supporting cells had a significantly longer lifetime than sensory cells. Pretreatment with GM increased NAD(P)H intensity in high-frequency sensory cells, as well as the NAD(P)H lifetime within IHCs. GM specifically increased NAD(P)H concentration in high-frequency OHCs, but not in IHCs or pillar cells. Variations in NAD(P)H intensity in response to mitochondrial toxins and GM were greatest in high-frequency OHCs. These results demonstrate that GM rapidly alters mitochondrial metabolism, differentially modulates cell metabolism, and provides evidence that GM-induced changes in metabolism are significant and greatest in high-frequency OHCs.

  20. Profiling of Human Acquired Immunity Against the Salivary Proteins of Phlebotomus papatasi Reveals Clusters of Differential Immunoreactivity

    PubMed Central

    Geraci, Nicholas S.; Mukbel, Rami M.; Kemp, Michael T.; Wadsworth, Mariha N.; Lesho, Emil; Stayback, Gwen M.; Champion, Matthew M.; Bernard, Megan A.; Abo-Shehada, Mahmoud; Coutinho-Abreu, Iliano V.; Ramalho-Ortigão, Marcelo; Hanafi, Hanafi A.; Fawaz, Emadeldin Y.; El-Hossary, Shabaan S.; Wortmann, Glenn; Hoel, David F.; McDowell, Mary Ann

    2014-01-01

    Phlebotomus papatasi sand flies are among the primary vectors of Leishmania major parasites from Morocco to the Indian subcontinent and from southern Europe to central and eastern Africa. Antibody-based immunity to sand fly salivary gland proteins in human populations remains a complex contextual problem that is not yet fully understood. We profiled the immunoreactivities of plasma antibodies to sand fly salivary gland sonicates (SGSs) from 229 human blood donors residing in different regions of sand fly endemicity throughout Jordan and Egypt as well as 69 US military personnel, who were differentially exposed to P. papatasi bites and L. major infections in Iraq. Compared with plasma from control region donors, antibodies were significantly immunoreactive to five salivary proteins (12, 26, 30, 38, and 44 kDa) among Jordanian and Egyptian donors, with immunoglobulin G4 being the dominant anti-SGS isotype. US personnel were significantly immunoreactive to only two salivary proteins (38 and 14 kDa). Using k-means clustering, donors were segregated into four clusters distinguished by unique immunoreactivity profiles to varying combinations of the significantly immunogenic salivary proteins. SGS-induced cellular proliferation was diminished among donors residing in sand fly-endemic regions. These data provide a clearer picture of human immune responses to sand fly vector salivary constituents. PMID:24615125

  1. Proteomic responses reveal the differential effects induced by cadmium in mussels Mytilus galloprovincialis at early life stages.

    PubMed

    Xu, Lanlan; Peng, Xiao; Yu, Deliang; Ji, Chenglong; Zhao, Jianmin; Wu, Huifeng

    2016-08-01

    Cadmium (Cd) has become an important metal contaminant and posed severe risk on the organisms in the coastal environments of the Bohai Sea. Marine mussel Mytilus galloprovincialis is widely distributed along the Bohai coast and consumed as seafood by local residents. Evidences indicate that the early stages of marine organisms are more sensitive to metal contaminants. In this study, we applied two-dimensional electrophoresis-based proteomics to characterize the biological effects of Cd (50 μg L(-1)) in the early life stages (D-shape larval and juvenile) of mussels. The different proteomic responses demonstrated the differential responsive mechanisms to Cd exposure in these two early life stages of mussels. In details, results indicated that Cd mainly induced immune and oxidative stresses in both D-shape larval and juvenile mussels via different pathways. In addition, the significant up-regulation of triosephosphate isomerase and metallothionein confirmed the enhanced energy demand and mobilized detoxification mechanism in D-shape larval mussels exposed to Cd. In juvenile mussels, Cd exposure also induced clear apoptosis. Overall, this work suggests that Cd is a potential immune toxicant to mussel M. galloprovincialis at early life stages. PMID:27302865

  2. Systematic Profiling of Poly(A)+ Transcripts Modulated by Core 3’ End Processing and Splicing Factors Reveals Regulatory Rules of Alternative Cleavage and Polyadenylation

    PubMed Central

    Li, Wencheng; You, Bei; Hoque, Mainul; Zheng, Dinghai; Luo, Wenting; Ji, Zhe; Park, Ji Yeon; Gunderson, Samuel I.; Kalsotra, Auinash; Manley, James L.; Tian, Bin

    2015-01-01

    Alternative cleavage and polyadenylation (APA) results in mRNA isoforms containing different 3’ untranslated regions (3’UTRs) and/or coding sequences. How core cleavage/polyadenylation (C/P) factors regulate APA is not well understood. Using siRNA knockdown coupled with deep sequencing, we found that several C/P factors can play significant roles in 3’UTR-APA. Whereas Pcf11 and Fip1 enhance usage of proximal poly(A) sites (pAs), CFI-25/68, PABPN1 and PABPC1 promote usage of distal pAs. Strong cis element biases were found for pAs regulated by CFI-25/68 or Fip1, and the distance between pAs plays an important role in APA regulation. In addition, intronic pAs are substantially regulated by splicing factors, with U1 mostly inhibiting C/P events in introns near the 5’ end of gene and U2 suppressing those in introns with features for efficient splicing. Furthermore, PABPN1 inhibits expression of transcripts with pAs near the transcription start site (TSS), a property possibly related to its role in RNA degradation. Finally, we found that groups of APA events regulated by C/P factors are also modulated in cell differentiation and development with distinct trends. Together, our results support an APA code where an APA event in a given cellular context is regulated by a number of parameters, including relative location to the TSS, splicing context, distance between competing pAs, surrounding cis elements and concentrations of core C/P factors. PMID:25906188

  3. Genome-wide Medicago truncatula small RNA analysis revealed novel microRNAs and isoforms differentially regulated in roots and nodules.

    PubMed

    Lelandais-Brière, Christine; Naya, Loreto; Sallet, Erika; Calenge, Fanny; Frugier, Florian; Hartmann, Caroline; Gouzy, Jérome; Crespi, Martin

    2009-09-01

    Posttranscriptional regulation of a variety of mRNAs by small 21- to 24-nucleotide RNAs, notably the microRNAs (miRNAs), is emerging as a novel developmental mechanism. In legumes like the model Medicago truncatula, roots are able to develop a de novo meristem through the symbiotic interaction with nitrogen-fixing rhizobia. We used deep sequencing of small RNAs from root apexes and nodules of M. truncatula to identify 100 novel candidate miRNAs encoded by 265 hairpin precursors. New atypical precursor classes producing only specific 21- and 24-nucleotide small RNAs were found. Statistical analysis on sequencing reads abundance revealed specific miRNA isoforms in a same family showing contrasting expression patterns between nodules and root apexes. The differentially expressed conserved and nonconserved miRNAs may target a large variety of mRNAs. In root nodules, which show diverse cell types ranging from a persistent meristem to a fully differentiated central region, we discovered miRNAs spatially enriched in nodule meristematic tissues, vascular bundles, and bacterial infection zones using in situ hybridization. Spatial regulation of miRNAs may determine specialization of regulatory RNA networks in plant differentiation processes, such as root nodule formation. PMID:19767456

  4. Representational Difference Analysis (RDA) reveals differential expression of conserved as well as novel genes during caste-specific development of the honey bee (Apis mellifera L.) ovary.

    PubMed

    Humann, Fernanda C; Hartfelder, Klaus

    2011-08-01

    In highly eusocial insects, such as the honey bee, Apis mellifera, the reproductive bias has become embedded in morphological caste differences. These are most expressively denoted in ovary size, with adult queens having large ovaries consisting of 150-200 ovarioles each, while workers typically have only 1-20 ovarioles per ovary. This morphological differentiation is a result of hormonal signals triggered by the diet change in the third larval instar, which eventually generate caste-specific gene expression patterns. To reveal these we produced differential gene expression libraries by Representational Difference Analysis (RDA) for queen and worker ovaries in a developmental stage when cell death is a prominent feature in the ovarioles of workers, whereas all ovarioles are maintained and extend in length in queens. In the queen library, 48% of the gene set represented homologs of known Drosophila genes, whereas in the worker ovary, the largest set (59%) were ESTs evidencing novel genes, not even computationally predicted in the honey bee genome. Differential expression was confirmed by quantitative RT-PCR for a selected gene set, denoting major differences for two queen and two worker library genes. These included two unpredicted genes located in chromosome 11 (Group11.35 and Group11.31, respectively) possibly representing long non-coding RNAs. Being candidates as modulators of ovary development, their expression and functional analysis should be a focal point for future studies. PMID:21477651

  5. Transcriptome Analysis of Blunt Snout Bream (Megalobrama amblycephala) Reveals Putative Differential Expression Genes Related to Growth and Hypoxia

    PubMed Central

    Li, Fu-Gui; Chen, Jie; Jiang, Xia-Yun; Zou, Shu-Ming

    2015-01-01

    The blunt snout bream (Megalobrama amblycephala) is an important freshwater aquaculture species, but it is sensitive to hypoxia. No transcriptome data related to growth and hypoxia response are available for this species. In this study, we performed de novo transcriptome sequencing for the liver and gills of the fast-growth family and slow-growth family derived from ‘Pujiang No.1’ F10 blunt snout bream that were under hypoxic stress and normoxia, respectively. The fish were divided into the following 4 groups: fast-growth family under hypoxic stress, FH; slow-growth family under hypoxic stress, SH; fast-growth family under normoxia, FN; and slow-growth family under normoxia, SN. A total of 185 million high-quality reads were obtained from the normalized cDNA of the pooled samples, which were assembled into 465,582 contigs and 237,172 transcripts. A total of 31,338 transcripts from the same locus (unigenes) were annotated and assigned to 104 functional groups, and 23,103 unigenes were classified into seven main categories, including 45 secondary KEGG pathways. A total of 22,255 (71%) known putative unigenes were found to be shared across the genomes of five model fish species and mammals, and a substantial number (9.4%) of potentially novel genes were identified. When 6,639 unigenes were used in the analysis of differential expression (DE) genes, the number of putative DE genes related to growth pathways in FH, SH, SN and FN was 159, 118, 92 and 65 in both the liver and gills, respectively, and the number of DE genes related to hypoxic response was 57, 33, 23 and 21 in FH, FN, SH and SN, respectively. Our results suggest that growth performance of the fast-growth family should be due to complex mutual gene regulatory mechanisms of these putative DE genes between growth and hypoxia. PMID:26554582

  6. Comparative cell-specific transcriptomics reveals differentiation of C4 photosynthesis pathways in switchgrass and other C4 lineages

    PubMed Central

    Rao, Xiaolan; Lu, Nan; Li, Guifen; Nakashima, Jin; Tang, Yuhong; Dixon, Richard A.

    2016-01-01

    Almost all C4 plants require the co-ordination of the adjacent and fully differentiated cell types, mesophyll (M) and bundle sheath (BS). The C4 photosynthetic pathway operates through two distinct subtypes based on how malate is decarboxylated in BS cells; through NAD-malic enzyme (NAD-ME) or NADP-malic enzyme (NADP-ME). The diverse or unique cell-specific molecular features of M and BS cells from separate C4 subtypes of independent lineages remain to be determined. We here provide an M/BS cell type-specific transcriptome data set from the monocot NAD-ME subtype switchgrass (Panicum virgatum). A comparative transcriptomics approach was then applied to compare the M/BS mRNA profiles of switchgrass, monocot NADP-ME subtype C4 plants maize and Setaria viridis, and dicot NAD-ME subtype Cleome gynandra. We evaluated the convergence in the transcript abundance of core components in C4 photosynthesis and transcription factors to establish Kranz anatomy, as well as gene distribution of biological functions, in these four independent C4 lineages. We also estimated the divergence between NAD-ME and NADP-ME subtypes of C4 photosynthesis in the two cell types within C4 species, including differences in genes encoding decarboxylating enzymes, aminotransferases, and metabolite transporters, and differences in the cell-specific functional enrichment of RNA regulation and protein biogenesis/homeostasis. We suggest that C4 plants of independent lineages in both monocots and dicots underwent convergent evolution to establish C4 photosynthesis, while distinct C4 subtypes also underwent divergent processes for the optimization of M and BS cell co-ordination. The comprehensive data sets in our study provide a basis for further research on evolution of C4 species. PMID:26896851

  7. Transcript Quantification by RNA-Seq Reveals Differentially Expressed Genes in the Red and Yellow Fruits of Fragaria vesca

    PubMed Central

    Zhang, Junxiang; Jiang, Guihua; Miao, Lixiang; Han, Guofen; Liu, Yuexue; Li, He; Zhang, Zhihong

    2015-01-01

    Fragaria vesca (2n = 2x = 14), the woodland strawberry, is a perennial herbaceous plant with a small sequenced genome (240 Mb). It is commonly used as a genetic model plant for the Fragaria genus and the Rosaceae family. Fruit skin color is one of the most important traits for both the commercial and esthetic value of strawberry. Anthocyanins are the most prominent pigments in strawberry that bring red, pink, white, and yellow hues to the fruits in which they accumulate. In this study, we conducted a de novo assembly of the fruit transcriptome of woodland strawberry and compared the gene expression profiles with yellow (Yellow Wonder, YW) and red (Ruegen, RG) fruits. De novo assembly yielded 75,426 unigenes, 21.3% of which were longer than 1,000 bp. Among the high-quality unique sequences, 45,387 (60.2%) had at least one significant match to an existing gene model. A total of 595 genes, representing 0.79% of total unigenes, were differentially expressed in YW and RG. Among them, 224 genes were up-regulated and 371 genes were down-regulated in the fruit of YW. Particularly, some flavonoid biosynthetic pathway genes, including C4H, CHS, CHI, F3H, DFR and ANS, as well as some transcription factors (TFs), including MYB (putative MYB86 and MYB39), WDR and MADS, were down-regulated in YW fruit, concurrent with a reduction in anthocyanin accumulation in the yellow pigment phenotype, whereas a putative transcription repressor MYB1R was up-regulated in YW fruit. The altered expression levels of the genes encoding flavonoid biosynthetic enzymes and TFs were confirmed by quantitative RT-PCR. Our study provides important insights into the molecular mechanisms underlying the yellow pigment phenotype in F. vesca. PMID:26636322

  8. Transcript Quantification by RNA-Seq Reveals Differentially Expressed Genes in the Red and Yellow Fruits of Fragaria vesca.

    PubMed

    Zhang, Yuchao; Li, Weijia; Dou, Yujuan; Zhang, Junxiang; Jiang, Guihua; Miao, Lixiang; Han, Guofen; Liu, Yuexue; Li, He; Zhang, Zhihong

    2015-01-01

    Fragaria vesca (2n = 2x = 14), the woodland strawberry, is a perennial herbaceous plant with a small sequenced genome (240 Mb). It is commonly used as a genetic model plant for the Fragaria genus and the Rosaceae family. Fruit skin color is one of the most important traits for both the commercial and esthetic value of strawberry. Anthocyanins are the most prominent pigments in strawberry that bring red, pink, white, and yellow hues to the fruits in which they accumulate. In this study, we conducted a de novo assembly of the fruit transcriptome of woodland strawberry and compared the gene expression profiles with yellow (Yellow Wonder, YW) and red (Ruegen, RG) fruits. De novo assembly yielded 75,426 unigenes, 21.3% of which were longer than 1,000 bp. Among the high-quality unique sequences, 45,387 (60.2%) had at least one significant match to an existing gene model. A total of 595 genes, representing 0.79% of total unigenes, were differentially expressed in YW and RG. Among them, 224 genes were up-regulated and 371 genes were down-regulated in the fruit of YW. Particularly, some flavonoid biosynthetic pathway genes, including C4H, CHS, CHI, F3H, DFR and ANS, as well as some transcription factors (TFs), including MYB (putative MYB86 and MYB39), WDR and MADS, were down-regulated in YW fruit, concurrent with a reduction in anthocyanin accumulation in the yellow pigment phenotype, whereas a putative transcription repressor MYB1R was up-regulated in YW fruit. The altered expression levels of the genes encoding flavonoid biosynthetic enzymes and TFs were confirmed by quantitative RT-PCR. Our study provides important insights into the molecular mechanisms underlying the yellow pigment phenotype in F. vesca. PMID:26636322

  9. In situ detection of Mycobacterium tuberculosis transcripts in human lung granulomas reveals differential gene expression in necrotic lesions.

    PubMed

    Fenhalls, Gael; Stevens, Liesel; Moses, Lorraine; Bezuidenhout, Juanita; Betts, Joanna C; Helden Pv, Paul van; Lukey, Pauline T; Duncan, Ken

    2002-11-01

    We have used RNA-RNA in situ hybridization to detect the expression of several Mycobacterium tuberculosis genes in tuberculous granulomas in lung tissue sections from tuberculosis patients. The M. tuberculosis genes chosen fall into two classes. Four genes (icl, narX, and Rv2557 and Rv2558) have been implicated in the persistence of the bacterium in the host, and two genes (iniB and kasA) are upregulated in response to isoniazid exposure. Both necrotic and nonnecrotic granulomas were identified in all of the patients. Necrotic granulomas were divided into three zones: an outer lymphocyte cuff containing lymphocytes and macrophages, a transition zone consisting of necrotic material interspersed with macrophages, and a central acellular necrotic region. Transcripts of all of the genes studied were found in nonnecrotic granulomas and in the lymphocyte cuff of necrotic granulomas. Mycobacterial gene expression was associated with CD68-positive myeloid cells. Rv2557 and/or its homologue Rv2558, kasA, and iniB were expressed within the transition zone of necrotic granulomas, whereas icl and narX transcripts were absent from this area. There was no evidence of transcription of any of the genes examined in the central necrotic region, although mycobacterial DNA was present. The differential expression of genes within granulomas demonstrates that M. tuberculosis exists in a variety of metabolic states and may be indicative of the response to different microenvironments. These observations confirm that genes identified in models of persistence or in response to drug treatment in vitro are expressed in the human host. PMID:12379712

  10. Phylogeography of the rare Balkan endemic Martino's vole, Dinaromys bogdanovi, reveals strong differentiation within the western Balkan Peninsula.

    PubMed

    Krystufek, Boris; Buzan, Elena V; Hutchinson, William F; Hänfling, Bernd

    2007-03-01

    The spatial genetic structure of Martino's vole, a rare palaeoendemic species of the western Balkans, was investigated using DNA isolated from archived museum samples. The study was based on partial sequencing (555 bp) of the mitochondrial cytochrome b gene for 63 specimens from 20 different localities throughout the species' range. Three highly divergent allopatric phylogenetic lineages (Northwestern, Central and Southeastern) were recognized among 47 haplotypes, suggesting three independent glacial differentiation centres within the western Balkans. The Northwestern lineage, which showed the highest divergence from all other samples (mean sequence divergence of 6.64% +/- 1.10), comprised samples collected from northwest of the Neretva River in Croatia, western Bosnia and Herzegovina. The Central and Southeastern lineages were separated by an average sequence divergence of 2.95% +/- 0.66 and were geographically divided by the Drim River (the Kosovo Basin in Serbia). Overall, haplotype diversity decreased from the Northwestern lineage to the Central and subsequently the Southeastern lineage, in a geographical pattern consistent with a stepping stone colonization. The observed distribution indicates a gradual southerly expansion with subsequent allopatry across the Neretva River and Drim River approximately 1 and 0.3 million years ago, respectively. Such a scenario is concordant with palaeontological evidence. Several highly divergent sublineages within the Northwestern and Central lineages showed no significant geographical structuring, suggesting secondary contact of allopatrically evolved lineages. We hypothesize that the topographical complexity of the Balkans promoted allopatry and isolation on a small geographical scale during interglacial periods, with secondary contact during glacial maxima. Furthermore, the three main lineages should be regarded as evolutionary significant units with important implications for conservation. Ecological data show that the

  11. Differential proteomic analysis of grapevine leaves by iTRAQ reveals responses to heat stress and subsequent recovery

    PubMed Central

    2014-01-01

    Background High temperature is a major environmental factor limiting grape yield and affecting berry quality. Thermotolerance includes the direct response to heat stress and the ability to recover from heat stress. To better understand the mechanism of the thermotolerance of Vitis, we combined a physiological analysis with iTRAQ-based proteomics of Vitis vinifera cv Cabernet Sauvignon, subjected to 43°C for 6 h, and then followed by recovery at 25/18°C. Results High temperature increased the concentrations of TBARS and inhibited electronic transport in photosynthesis apparatus, indicating that grape leaves were damaged by heat stress. However, these physiological changes rapidly returned to control levels during the subsequent recovery phase from heat stress. One hundred and seventy-four proteins were differentially expressed under heat stress and/or during the recovery phase, in comparison to unstressed controls, respectively. Stress and recovery conditions shared 42 proteins, while 113 and 103 proteins were respectively identified under heat stress and recovery conditions alone. Based on MapMan ontology, functional categories for these dysregulated proteins included mainly photosynthesis (about 20%), proteins (13%), and stress (8%). The subcellular localization using TargetP showed most proteins were located in the chloroplasts (34%), secretory pathways (8%) and mitochondrion (3%). Conclusion On the basis of these findings, we proposed that some proteins related to electron transport chain of photosynthesis, antioxidant enzymes, HSPs and other stress response proteins, and glycolysis may play key roles in enhancing grapevine adaptation to and recovery capacity from heat stress. These results provide a better understanding of the proteins involved in, and mechanisms of thermotolerance in grapevines. PMID:24774513

  12. Quantitative Western ligand blotting reveals common patterns and differential features of IGFBP-fingerprints in domestic ruminant breeds and species.

    PubMed

    Wirthgen, Elisa; Höflich, Christine; Spitschak, Marion; Helmer, Carina; Brand, Bodo; Langbein, Jan; Metzger, Friedrich; Hoeflich, Andreas

    2016-02-01

    The insulin-like growth factor binding proteins (IGFBPs) are determinants of local IGF-effects and thus have an impact on growth and metabolism in vertebrate species. In farm animals, IGFBPs are associated with traits such as growth rate, body composition, milk production, or fertility. It may be assumed, that selective breeding and characteristic phenotypes of breeds are related to differential expression of IGFBPs. Therefore, the aim of the present study was to investigate the effects of selective breeding on blood IGFBP concentrations of farm animals. Breeds of the sheep, goat, and cattle species were investigated. IGFBP-3, -2, and -4 were analyzed with quantitative Western ligand blotting (qWLB), enabling comprehensive monitoring of intact IGFBPs with IGF-binding capacity. We show that in sera of all species and breeds investigated, IGFBP-3, -2, and -4 were simultaneously detectable by qWLB analysis. IGFBP-3 and the total amount of IGFBPs were significantly increased (P<0.05) in Cameroon sheep, if compared to 3 of 4 other sheep breeds, as well as in Dwarf goats versus Toggenburg and Boer goats (P<0.01). IGFBP-2 was elevated in Cameroon sheep and Boer goats, if compared to other breeds of these species (P<0.01), respectively. Holstein Friesian dairy cows had higher levels of IGFBP-4 (P<0.05), if compared to conventional crossbreeds of beef cattle. In Dwarf goats the ratio of IGFBP-3/IGFBP-2 was about 3-fold higher than in other goat breeds (P<0.001). The total IGFBP amount of Toggenburg goats was reduced (P<0.05), compared to the other goat breeds. In conclusion, our data indicate that common and specific features of IGFBP fingerprints are found in different ruminant species and breeds. Our findings may introduce quantitative Western ligand blotting as an attractive tool for biomarker development and molecular phenotyping in farm animal breeds. PMID:26597140

  13. Comparative cell-specific transcriptomics reveals differentiation of C4 photosynthesis pathways in switchgrass and other C4 lineages.

    PubMed

    Rao, Xiaolan; Lu, Nan; Li, Guifen; Nakashima, Jin; Tang, Yuhong; Dixon, Richard A

    2016-04-01

    Almost all C4 plants require the co-ordination of the adjacent and fully differentiated cell types, mesophyll (M) and bundle sheath (BS). The C4 photosynthetic pathway operates through two distinct subtypes based on how malate is decarboxylated in BS cells; through NAD-malic enzyme (NAD-ME) or NADP-malic enzyme (NADP-ME). The diverse or unique cell-specific molecular features of M and BS cells from separate C4 subtypes of independent lineages remain to be determined. We here provide an M/BS cell type-specific transcriptome data set from the monocot NAD-ME subtype switchgrass (Panicum virgatum). A comparative transcriptomics approach was then applied to compare the M/BS mRNA profiles of switchgrass, monocot NADP-ME subtype C4 plants maize and Setaria viridis, and dicot NAD-ME subtype Cleome gynandra. We evaluated the convergence in the transcript abundance of core components in C4 photosynthesis and transcription factors to establish Kranz anatomy, as well as gene distribution of biological functions, in these four independent C4 lineages. We also estimated the divergence between NAD-ME and NADP-ME subtypes of C4 photosynthesis in the two cell types within C4 species, including differences in genes encoding decarboxylating enzymes, aminotransferases, and metabolite transporters, and differences in the cell-specific functional enrichment of RNA regulation and protein biogenesis/homeostasis. We suggest that C4 plants of independent lineages in both monocots and dicots underwent convergent evolution to establish C4 photosynthesis, while distinct C4 subtypes also underwent divergent processes for the optimization of M and BS cell co-ordination. The comprehensive data sets in our study provide a basis for further research on evolution of C4 species. PMID:26896851

  14. Genome-wide analysis reveals conserved transcriptional responses downstream of resting potential change in Xenopus embryos, axolotl regeneration, and human mesenchymal cell differentiation.

    PubMed

    Pai, Vaibhav P; Martyniuk, Christopher J; Echeverri, Karen; Sundelacruz, Sarah; Kaplan, David L; Levin, Michael

    2016-02-01

    Endogenous bioelectric signaling via changes in cellular resting potential (V mem) is a key regulator of patterning during regeneration and embryogenesis in numerous model systems. Depolarization of V mem has been functionally implicated in dedifferentiation, tumorigenesis, anatomical re-specification, and appendage regeneration. However, no unbiased analyses have been performed to understand genome-wide transcriptional responses to V mem change in vivo. Moreover, it is unknown which genes or gene networks represent conserved targets of bioelectrical signaling across different patterning contexts and species. Here, we use microarray analysis to comparatively analyze transcriptional responses to V mem depolarization. We compare the response of the transcriptome during embryogenesis (Xenopus development), regeneration (axolotl regeneration), and stem cell differentiation (human mesenchymal stem cells in culture) to identify common networks across model species that are associated with depolarization. Both subnetwork enrichment and PANTHER analyses identified a number of key genetic modules as targets of V mem change, and also revealed important (well-conserved) commonalities in bioelectric signal transduction, despite highly diverse experimental contexts and species. Depolarization regulates specific transcriptional networks across all three germ layers (ectoderm, mesoderm, and endoderm) such as cell differentiation and apoptosis, and this information will be used for developing mechanistic models of bioelectric regulation of patterning. Moreover, our analysis reveals that V mem change regulates transcripts related to important disease pathways such as cancer and neurodegeneration, which may represent novel targets for emerging electroceutical therapies. PMID:27499876

  15. Genome‐wide analysis reveals conserved transcriptional responses downstream of resting potential change in Xenopus embryos, axolotl regeneration, and human mesenchymal cell differentiation

    PubMed Central

    Pai, Vaibhav P.; Martyniuk, Christopher J.; Echeverri, Karen; Sundelacruz, Sarah; Kaplan, David L.

    2015-01-01

    Abstract Endogenous bioelectric signaling via changes in cellular resting potential (V mem) is a key regulator of patterning during regeneration and embryogenesis in numerous model systems. Depolarization of V mem has been functionally implicated in dedifferentiation, tumorigenesis, anatomical re‐specification, and appendage regeneration. However, no unbiased analyses have been performed to understand genome‐wide transcriptional responses to V mem change in vivo. Moreover, it is unknown which genes or gene networks represent conserved targets of bioelectrical signaling across different patterning contexts and species. Here, we use microarray analysis to comparatively analyze transcriptional responses to V mem depolarization. We compare the response of the transcriptome during embryogenesis (Xenopus development), regeneration (axolotl regeneration), and stem cell differentiation (human mesenchymal stem cells in culture) to identify common networks across model species that are associated with depolarization. Both subnetwork enrichment and PANTHER analyses identified a number of key genetic modules as targets of V mem change, and also revealed important (well‐conserved) commonalities in bioelectric signal transduction, despite highly diverse experimental contexts and species. Depolarization regulates specific transcriptional networks across all three germ layers (ectoderm, mesoderm, and endoderm) such as cell differentiation and apoptosis, and this information will be used for developing mechanistic models of bioelectric regulation of patterning. Moreover, our analysis reveals that V mem change regulates transcripts related to important disease pathways such as cancer and neurodegeneration, which may represent novel targets for emerging electroceutical therapies. PMID:27499876

  16. The Polymyxin Ceftazidime Oxford Medium as an alternative selective and differential medium for isolation of Listeria monocytogenes from raw or unpasteurized food.

    PubMed

    Martínez-Gonzáles, N E; Martínez-Chávez, L; Martínez-Cárdenas, C; Cabrera-Díaz, E; Castillo, A

    2014-04-01

    The Polymyxin Ceftazidime Oxford Medium (PCOM) was developed to recover Listeria monocytogenes from raw or unpasteurized foods. It contains esculin-ferric ammonium citrate as indicator system for Listeria growth, and ceftazidime and polymyxin B as selective agents, which are available in several Latin American countries. Comparison of PCOM, Modified Oxford Medium (MOX) and Tryptic Soy agar with 0.6% yeast extract (TSAYE) indicated that both selective media were equally effective at recovering four individual strains of L. monocytogenes (Scott A, V7, California and broccoli), and a mixture of these strains (LMM) (P > 0.05). The ability of PCOM, MOX, TSAYE and TSAYE supplemented with 4% NaCl to recover heat, acid and freeze-damaged LMM was similar for all media (P > 0.05). The PCOM proved to be effective at isolating colonies of LMM from inoculated raw beef chunks, unpasteurized orange juice, cabbage, and Mexican-style cheese by direct plating and by the US Department of Agriculture's Food Safety and Inspection Service enrichment method. Differentiation of L. monocytogenes colonies was easier on PCOM than on MOX for foods with high levels of background microbiota. Based on the evaluations performed on foods naturally contaminated with L. monocytogenes, PCOM was a more economical alternative than MOX for selective and differential isolation of Listeria from raw or unpasteurized foods. PMID:24290624

  17. Genetically null mice reveal a central role for epidermal growth factor receptor in the differentiation of the hair follicle and normal hair development.

    PubMed Central

    Hansen, L. A.; Alexander, N.; Hogan, M. E.; Sundberg, J. P.; Dlugosz, A.; Threadgill, D. W.; Magnuson, T.; Yuspa, S. H.

    1997-01-01

    Mice harboring a targeted disruption of the epidermal growth factor receptor (EGFR) allele exhibit a severely disorganized hair follicle phenotype, fuzzy coat, and systemic disease resulting in death before 3 weeks. This skin phenotype was reproduced in whole skin grafts and in grafts of EGFR null hair follicle buds onto nude mice, providing a model to evaluate the natural evolution of skin lacking the EGFR. Hair follicles in grafts of null skin did not progress from anagen to telogen and scanning electron micrografts revealed wavy, flattened hair fibers with cuticular abnormalities. Many of the EGFR null hair follicles in the grafted skin were consumed by an inflammatory reaction resulting in complete hair loss in 67% of the grafts by 10 weeks. Localization of follicular differentiation markers including keratin 6, transglutaminase, and the hair keratins mHa2 and hacl-1 revealed a pattern of premature differentiation within the null hair follicles. In intact EGFR null mice, proliferation in the interfollicular epidermis, but not hair follicles, was greatly decreased in the absence of EGFR. In contrast, grafting of EGFR null skin resulted in a hyperplastic response in the epidermis that did not resolve even after 10 weeks, although the wound-induced hyperplasia in EGFR wild-type grafts had resolved within 3 to 4 weeks. Thus, epithelial expression of the EGFR has complex functions in the skin. It is important in delaying follicular differentiation, may serve to protect the hair follicle from immunological reactions, and modifies both normal and wound-induced epidermal proliferation but seems dispensable for follicular proliferation. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:9176390

  18. Differential Responses to a Visual Self-Motion Signal in Human Medial Cortical Regions Revealed by Wide-View Stimulation

    PubMed Central

    Wada, Atsushi; Sakano, Yuichi; Ando, Hiroshi

    2016-01-01

    Vision is important for estimating self-motion, which is thought to involve optic-flow processing. Here, we investigated the fMRI response profiles in visual area V6, the precuneus motion area (PcM), and the cingulate sulcus visual area (CSv)—three medial brain regions recently shown to be sensitive to optic-flow. We used wide-view stereoscopic stimulation to induce robust self-motion processing. Stimuli included static, randomly moving, and coherently moving dots (simulating forward self-motion). We varied the stimulus size and the presence of stereoscopic information. A combination of univariate and multi-voxel pattern analyses (MVPA) revealed that fMRI responses in the three regions differed from each other. The univariate analysis identified optic-flow selectivity and an effect of stimulus size in V6, PcM, and CSv, among which only CSv showed a significantly lower response to random motion stimuli compared with static conditions. Furthermore, MVPA revealed an optic-flow specific multi-voxel pattern in the PcM and CSv, where the discrimination of coherent motion from both random motion and static conditions showed above-chance prediction accuracy, but that of random motion from static conditions did not. Additionally, while area V6 successfully classified different stimulus sizes regardless of motion pattern, this classification was only partial in PcM and was absent in CSv. This may reflect the known retinotopic representation in V6 and the absence of such clear visuospatial representation in CSv. We also found significant correlations between the strength of subjective self-motion and univariate activation in all examined regions except for primary visual cortex (V1). This neuro-perceptual correlation was significantly higher for V6, PcM, and CSv when compared with V1, and higher for CSv when compared with the visual motion area hMT+. Our convergent results suggest the significant involvement of CSv in self-motion processing, which may give rise to its

  19. Alternative binding modes identified for growth and differentiation factor-associated serum protein (GASP) family antagonism of myostatin.

    PubMed

    Walker, Ryan G; Angerman, Elizabeth B; Kattamuri, Chandramohan; Lee, Yun-Sil; Lee, Se-Jin; Thompson, Thomas B

    2015-03-20

    Myostatin, a member of the TGF-β family of ligands, is a strong negative regulator of muscle growth. As such, it is a prime therapeutic target for muscle wasting disorders. Similar to other TGF-β family ligands, myostatin is neutralized by binding one of a number of structurally diverse antagonists. Included are the antagonists GASP-1 and GASP-2, which are unique in that they specifically antagonize myostatin. However, little is known from a structural standpoint describing the interactions of GASP antagonists with myostatin. Here, we present the First low resolution solution structure of myostatin-free and myostatin-bound states of GASP-1 and GASP-2. Our studies have revealed GASP-1, which is 100 times more potent than GASP-2, preferentially binds myostatin in an asymmetrical 1:1 complex, whereas GASP-2 binds in a symmetrical 2:1 complex. Additionally, C-terminal truncations of GASP-1 result in less potent myostatin inhibitors that form a 2:1 complex, suggesting that the C-terminal domains of GASP-1 are the primary mediators for asymmetric complex formation. Overall, this study provides a new perspective on TGF-β antagonism, where closely related antagonists can utilize different ligand-binding strategies. PMID:25657005

  20. Alternative Binding Modes Identified for Growth and Differentiation Factor-associated Serum Protein (GASP) Family Antagonism of Myostatin*

    PubMed Central

    Walker, Ryan G.; Angerman, Elizabeth B.; Kattamuri, Chandramohan; Lee, Yun-Sil; Lee, Se-Jin; Thompson, Thomas B.

    2015-01-01

    Myostatin, a member of the TGF-β family of ligands, is a strong negative regulator of muscle growth. As such, it is a prime therapeutic target for muscle wasting disorders. Similar to other TGF-β family ligands, myostatin is neutralized by binding one of a number of structurally diverse antagonists. Included are the antagonists GASP-1 and GASP-2, which are unique in that they specifically antagonize myostatin. However, little is known from a structural standpoint describing the interactions of GASP antagonists with myostatin. Here, we present the First low resolution solution structure of myostatin-free and myostatin-bound states of GASP-1 and GASP-2. Our studies have revealed GASP-1, which is 100 times more potent than GASP-2, preferentially binds myostatin in an asymmetrical 1:1 complex, whereas GASP-2 binds in a symmetrical 2:1 complex. Additionally, C-terminal truncations of GASP-1 result in less potent myostatin inhibitors that form a 2:1 complex, suggesting that the C-terminal domains of GASP-1 are the primary mediators for asymmetric complex formation. Overall, this study provides a new perspective on TGF-β antagonism, where closely related antagonists can utilize different ligand-binding strategies. PMID:25657005

  1. Timing of late Pleistocene glaciation in Mongolia: Surface exposure dating reveals a differentiated pattern of glacial forcing

    NASA Astrophysics Data System (ADS)

    Pötsch, Steffen; Rother, Henrik; Lorenz, Sebastian; Walther, Michael; Lehmkuhl, Frank

    2015-04-01

    of atmospheric circulation and its significance for controlling regional precipitation results in a more differentiated pattern of late Pleistocene glaciation in Mongolia than previously recognized. Compared to other glacial records from High Asia, the observed patterns of past glaciations in Mongolia show similar results (i.e. ice maxima during interstadial wet phases) compared to monsoon influenced regions in southern Central Asia and NE-Tibet, while major expansion during insolation minima (MIS-4 and MIS-2) are more in tune with glacier responses known from western Central Asia and Siberia.

  2. Suppression Substractive Hybridization and NGS Reveal Differential Transcriptome Expression Profiles in Wayfaring Tree (Viburnum lantana L.) Treated with Ozone.

    PubMed

    Gottardini, Elena; Cristofori, Antonella; Pellegrini, Elisa; La Porta, Nicola; Nali, Cristina; Baldi, Paolo; Sablok, Gaurav

    2016-01-01

    Tropospheric ozone (O3) is a global air pollutant that causes high economic damages by decreasing plant productivity. It enters the leaves through the stomata, generates reactive oxygen species, which subsequent decrease in photosynthesis, plant growth, and biomass accumulation. In order to identify genes that are important for conferring O3 tolerance or sensitivity to plants, a suppression subtractive hybridization analysis was performed on the very sensitive woody shrub, Viburnum lantana, exposed to chronic O3 treatment (60 ppb, 5 h d(-1) for 45 consecutive days). Transcript profiling and relative expression assessment were carried out in asymptomatic leaves, after 15 days of O3 exposure. At the end of the experiment symptoms were observed on all treated leaves and plants, with an injured leaf area per plant accounting for 16.7% of the total surface. Cloned genes were sequenced by 454-pyrosequencing and transcript profiling and relative expression assessment were carried out on sequenced reads. A total of 38,800 and 12,495 high quality reads obtained in control and O3-treated libraries, respectively (average length of 319 ± 156.7 and 255 ± 107.4 bp). The Ensembl transcriptome yielded a total of 1241 unigenes with a total sequence length of 389,126 bp and an average length size of 389 bp (guanine-cytosine content = 49.9%). mRNA abundance was measured by reads per kilobase per million and 41 and 37 ensembl unigenes showed up- and down-regulation respectively. Unigenes functionally associated to photosynthesis and carbon utilization were repressed, demonstrating the deleterious effect of O3 exposure. Unigenes functionally associated to heat-shock proteins and glutathione were concurrently induced, suggesting the role of thylakoid-localized proteins and antioxidant-detoxification pathways as an effective strategy for responding to O3. Gene Ontology analysis documented a differential expression of co-regulated transcripts for several functional categories, including

  3. Suppression Substractive Hybridization and NGS Reveal Differential Transcriptome Expression Profiles in Wayfaring Tree (Viburnum lantana L.) Treated with Ozone

    PubMed Central

    Gottardini, Elena; Cristofori, Antonella; Pellegrini, Elisa; La Porta, Nicola; Nali, Cristina; Baldi, Paolo; Sablok, Gaurav

    2016-01-01

    Tropospheric ozone (O3) is a global air pollutant that causes high economic damages by decreasing plant productivity. It enters the leaves through the stomata, generates reactive oxygen species, which subsequent decrease in photosynthesis, plant growth, and biomass accumulation. In order to identify genes that are important for conferring O3 tolerance or sensitivity to plants, a suppression subtractive hybridization analysis was performed on the very sensitive woody shrub, Viburnum lantana, exposed to chronic O3 treatment (60 ppb, 5 h d−1 for 45 consecutive days). Transcript profiling and relative expression assessment were carried out in asymptomatic leaves, after 15 days of O3 exposure. At the end of the experiment symptoms were observed on all treated leaves and plants, with an injured leaf area per plant accounting for 16.7% of the total surface. Cloned genes were sequenced by 454-pyrosequencing and transcript profiling and relative expression assessment were carried out on sequenced reads. A total of 38,800 and 12,495 high quality reads obtained in control and O3-treated libraries, respectively (average length of 319 ± 156.7 and 255 ± 107.4 bp). The Ensembl transcriptome yielded a total of 1241 unigenes with a total sequence length of 389,126 bp and an average length size of 389 bp (guanine-cytosine content = 49.9%). mRNA abundance was measured by reads per kilobase per million and 41 and 37 ensembl unigenes showed up- and down-regulation respectively. Unigenes functionally associated to photosynthesis and carbon utilization were repressed, demonstrating the deleterious effect of O3 exposure. Unigenes functionally associated to heat-shock proteins and glutathione were concurrently induced, suggesting the role of thylakoid-localized proteins and antioxidant-detoxification pathways as an effective strategy for responding to O3. Gene Ontology analysis documented a differential expression of co-regulated transcripts for several functional categories, including

  4. Differential Dopamine Release Dynamics in the Nucleus Accumbens Core and Shell Reveal Complementary Signals for Error Prediction and Incentive Motivation

    PubMed Central

    Cacciapaglia, Fabio; Wightman, R. Mark; Carelli, Regina M.

    2015-01-01

    Mesolimbic dopamine (DA) is phasically released during appetitive behaviors, though there is substantive disagreement about the specific purpose of these DA signals. For example, prediction error (PE) models suggest a role of learning, while incentive salience (IS) models argue that the DA signal imbues stimuli with value and thereby stimulates motivated behavior. However, within the nucleus accumbens (NAc) patterns of DA release can strikingly differ between subregions, and as such, it is possible that these patterns differentially contribute to aspects of PE and IS. To assess this, we measured DA release in subregions of the NAc during a behavioral task that spatiotemporally separated sequential goal-directed stimuli. Electrochemical methods were used to measure subsecond NAc dopamine release in the core and shell during a well learned instrumental chain schedule in which rats were trained to press one lever (seeking; SL) to gain access to a second lever (taking; TL) linked with food delivery, and again during extinction. In the core, phasic DA release was greatest following initial SL presentation, but minimal for the subsequent TL and reward events. In contrast, phasic shell DA showed robust release at all task events. Signaling decreased between the beginning and end of sessions in the shell, but not core. During extinction, peak DA release in the core showed a graded decrease for the SL and pauses in release during omitted expected rewards, whereas shell DA release decreased predominantly during the TL. These release dynamics suggest parallel DA signals capable of supporting distinct theories of appetitive behavior. SIGNIFICANCE STATEMENT Dopamine signaling in the brain is important for a variety of cognitive functions, such as learning and motivation. Typically, it is assumed that a single dopamine signal is sufficient to support these cognitive functions, though competing theories disagree on how dopamine contributes to reward-based behaviors. Here, we have

  5. Label-free quantitative proteomics reveals differentially regulated proteins in the latex of sticky diseased Carica papaya L. plants.

    PubMed

    Rodrigues, Silas P; Ventura, José A; Aguilar, Clemente; Nakayasu, Ernesto S; Choi, HyungWon; Sobreira, Tiago J P; Nohara, Lilian L; Wermelinger, Luciana S; Almeida, Igor C; Zingali, Russolina B; Fernandes, Patricia M B

    2012-06-18

    Papaya meleira virus (PMeV) is so far the only described laticifer-infecting virus, the causal agent of papaya (Carica papaya L.) sticky disease. The effects of PMeV on the laticifers' regulatory network were addressed here through the proteomic analysis of papaya latex. Using both 1-DE- and 1D-LC-ESI-MS/MS, 160 unique papaya latex proteins were identified, representing 122 new proteins in the latex of this plant. Quantitative analysis by normalized spectral counting revealed 10 down-regulated proteins in the latex of diseased plants, 9 cysteine proteases (chymopapain) and 1 latex serine proteinase inhibitor. A repression of papaya latex proteolytic activity during PMeV infection was hypothesized. This was further confirmed by enzymatic assays that showed a reduction of cysteine-protease-associated proteolytic activity in the diseased papaya latex. These findings are discussed in the context of plant responses against pathogens and may greatly contribute to understand the roles of laticifers in plant stress responses. PMID:22465191

  6. The hand in motion of liberals and conservatives reveals the differential processing of positive and negative information.

    PubMed

    Carraro, Luciana; Castelli, Luigi; Negri, Paolo

    2016-07-01

    Recent research revealed that political conservatives and liberals differ in the processing of valenced information. In particular, conservatives (vs. liberals) tend to weigh negative information more than positive information in their perception of the physical and social world. In the present work, we further investigated the ideology-based asymmetries in the processing of negative and positive information examining both the attention-grabbing power of negative information and the trajectories of the movements performed by respondents when required to categorize positive and negative stimuli. To this end we employed a modified version of the Mouse-Tracking procedure (Freeman & Ambady, 2010), recording hand movements during the execution of categorization tasks. Results showed that conservatives were indeed slower to start and execute response actions to negative stimuli, and, more specifically, the trajectories of their movements signaled avoidance tendencies aimed at increasing the distance from negative stimuli. In addition, this pattern of findings emerged both when participants were asked to categorize the stimuli according to their valence and when the same stimuli had to be categorized on the basis of irrelevant perceptual features. Overall, results demonstrate that conservatives and liberals process valenced information differently, perform different spontaneous movements when exposed to them, and that such asymmetries are largely independent from current processing goals. PMID:27160061

  7. Millennial-scale faunal record reveals differential resilience of European large mammals to human impacts across the Holocene

    PubMed Central

    Crees, Jennifer J.; Carbone, Chris; Sommer, Robert S.; Benecke, Norbert; Turvey, Samuel T.

    2016-01-01

    The use of short-term indicators for understanding patterns and processes of biodiversity loss can mask longer-term faunal responses to human pressures. We use an extensive database of approximately 18 700 mammalian zooarchaeological records for the last 11 700 years across Europe to reconstruct spatio-temporal dynamics of Holocene range change for 15 large-bodied mammal species. European mammals experienced protracted, non-congruent range losses, with significant declines starting in some species approximately 3000 years ago and continuing to the present, and with the timing, duration and magnitude of declines varying individually between species. Some European mammals became globally extinct during the Holocene, whereas others experienced limited or no significant range change. These findings demonstrate the relatively early onset of prehistoric human impacts on postglacial biodiversity, and mirror species-specific patterns of mammalian extinction during the Late Pleistocene. Herbivores experienced significantly greater declines than carnivores, revealing an important historical extinction filter that informs our understanding of relative resilience and vulnerability to human pressures for different taxa. We highlight the importance of large-scale, long-term datasets for understanding complex protracted extinction processes, although the dynamic pattern of progressive faunal depletion of European mammal assemblages across the Holocene challenges easy identification of ‘static’ past baselines to inform current-day environmental management and restoration. PMID:27009229

  8. Pyrosequencing-Based Transcriptome Analysis of the Asian Rice Gall Midge Reveals Differential Response during Compatible and Incompatible Interaction

    PubMed Central

    Sinha, Deepak Kumar; Nagaraju, Javaregowda; Tomar, Archana; Bentur, Jagadish S.; Nair, Suresh

    2012-01-01

    The Asian rice gall midge (Orseolia oryzae) is a major pest responsible for immense loss in rice productivity. Currently, very little knowledge exists with regard to this insect at the molecular level. The present study was initiated with the aim of developing molecular resources as well as identifying alterations at the transcriptome level in the gall midge maggots that are in a compatible (SH) or in an incompatible interaction (RH) with their rice host. Roche 454 pyrosequencing strategy was used to develop both transcriptomics and genomics resources that led to the identification of 79,028 and 85,395 EST sequences from gall midge biotype 4 (GMB4) maggots feeding on a susceptible and resistant rice variety, TN1 (SH) and Suraksha (RH), respectively. Comparative transcriptome analysis of the maggots in SH and RH revealed over-representation of transcripts from proteolysis and protein phosphorylation in maggots from RH. In contrast, over-representation of transcripts for translation, regulation of transcription and transcripts involved in electron transport chain were observed in maggots from SH. This investigation, besides unveiling various mechanisms underlying insect-plant interactions, will also lead to a better understanding of strategies adopted by insects in general, and the Asian rice gall midge in particular, to overcome host defense. PMID:23202939

  9. Label-free quantitative proteomics reveals differentially regulated proteins in the latex of sticky diseased Carica papaya L. plants

    PubMed Central

    Rodrigues, Silas P.; Ventura, José A.; Aguilar, Clemente; Nakayasu, Ernesto S.; Choi, HyungWon; Sobreira, Tiago J. P.; Nohara, Lilian L.; Wermelinger, Luciana S.; Almeida, Igor C.; Zingali, Russolina B.; Fernandes, Patricia M. B.

    2012-01-01

    Papaya meleira virus (PMeV) is so far the only described laticifer-infecting virus, the causal agent of papaya (Carica papaya L.) sticky disease. The effects of PMeV on the laticifers’ regulatory network were addressed here through the proteomic analysis of papaya latex. Using both 1-DE- and 1D-LC-ESI-MS/MS, 160 unique papaya latex proteins were identified, representing 122 new proteins in the latex of this plant. Quantitative analysis by normalized spectral counting revealed 10 down-regulated proteins in the latex of diseased plants, 9 cysteine proteases (chymopapain) and 1 latex serine proteinase inhibitor. A repression of papaya latex proteolytic activity during PMeV infection was hypothesized. This was further confirmed by enzymatic assays that showed a reduction of cysteine-protease-associated proteolytic activity in the diseased papaya latex. These findings are discussed in the context of plant responses against pathogens and may greatly contribute to understand the roles of laticifers in plant stress responses. PMID:22465191

  10. Label-free quantitative phosphoproteomic analysis reveals differentially regulated proteins and pathway in PRRSV-infected pulmonary alveolar macrophages.

    PubMed

    Luo, Rui; Fang, Liurong; Jin, Hui; Wang, Dang; An, Kang; Xu, Ningzhi; Chen, Huanchun; Xiao, Shaobo

    2014-03-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen of swine worldwide and causes significant economic losses. Through regulating the host proteins phosphorylation, PRRSV was found to manipulate the activities of several signaling molecules to regulate innate immune responses. However, the role of protein phosphorylation during PRRSV infection and the signal pathways responsible for it are relatively unknown. Here liquid chromatography-tandem mass spectrometry for label-free quantitative phosphoproteomics was applied to systematically investigate the global phosphorylation events in PRRSV-infected pulmonary alveolar macrophages. In total, we identified 2125 unique phosphosites, of which the phosphorylation level of 292 phosphosites on 242 proteins and 373 phosphosites on 249 proteins was significantly altered at 12 and 36 h pi, respectively. The phosphoproteomics data were analyzed using ingenuity pathways analysis to identify defined canonical pathways and functional networks. Pathway analysis revealed that PRRSV-induced inflammatory cytokines production was probably due to the activation of mitogen-activated protein kinase and NF-κB signal pathway, which were regulated by several protein kinases during virus infection. Interacting network analysis indicated that altered phosphoproteins were involved in cellular assembly and organization, protein synthesis, molecular transport, and signal transduction in PRRSV infected cells. These pathways and functional networks analysis could provide direct insights into the biological significance of phosphorylation events modulated by PRRSV and may help us elucidate the pathogenic mechanisms of PRRSV infection. PMID:24533505

  11. A transcriptomic study reveals differentially expressed genes and pathways respond to simulated acid rain in Arabidopsis thaliana.

    PubMed

    Liu, Ting-Wu; Niu, Li; Fu, Bin; Chen, Juan; Wu, Fei-Hua; Chen, Juan; Wang, Wen-Hua; Hu, Wen-Jun; He, Jun-Xian; Zheng, Hai-Lei

    2013-01-01

    Acid rain, as a worldwide environmental issue, can cause serious damage to plants. In this study, we provided the first case study on the systematic responses of arabidopsis (Arabidopsis thaliana (L.) Heynh.) to simulated acid rain (SiAR) by transcriptome approach. Transcriptomic analysis revealed that the expression of a set of genes related to primary metabolisms, including nitrogen, sulfur, amino acid, photosynthesis, and reactive oxygen species metabolism, were altered under SiAR. In addition, transport and signal transduction related pathways, especially calcium-related signaling pathways, were found to play important roles in the response of arabidopsis to SiAR stress. Further, we compared our data set with previously published data sets on arabidopsis transcriptome subjected to various stresses, including wound, salt, light, heavy metal, karrikin, temperature, osmosis, etc. The results showed that many genes were overlapped in several stresses, suggesting that plant response to SiAR is a complex process, which may require the participation of multiple defense-signaling pathways. The results of this study will help us gain further insights into the response mechanisms of plants to acid rain stress. PMID:23379338

  12. Millennial-scale faunal record reveals differential resilience of European large mammals to human impacts across the Holocene.

    PubMed

    Crees, Jennifer J; Carbone, Chris; Sommer, Robert S; Benecke, Norbert; Turvey, Samuel T

    2016-03-30

    The use of short-term indicators for understanding patterns and processes of biodiversity loss can mask longer-term faunal responses to human pressures. We use an extensive database of approximately 18 700 mammalian zooarchaeological records for the last 11 700 years across Europe to reconstruct spatio-temporal dynamics of Holocene range change for 15 large-bodied mammal species. European mammals experienced protracted, non-congruent range losses, with significant declines starting in some species approximately 3000 years ago and continuing to the present, and with the timing, duration and magnitude of declines varying individually between species. Some European mammals became globally extinct during the Holocene, whereas others experienced limited or no significant range change. These findings demonstrate the relatively early onset of prehistoric human impacts on postglacial biodiversity, and mirror species-specific patterns of mammalian extinction during the Late Pleistocene. Herbivores experienced significantly greater declines than carnivores, revealing an important historical extinction filter that informs our understanding of relative resilience and vulnerability to human pressures for different taxa. We highlight the importance of large-scale, long-term datasets for understanding complex protracted extinction processes, although the dynamic pattern of progressive faunal depletion of European mammal assemblages across the Holocene challenges easy identification of 'static' past baselines to inform current-day environmental management and restoration. PMID:27009229

  13. Disturbed local auxin homeostasis enhances cellular anisotropy and reveals alternative wiring of auxin-ethylene crosstalk in Brachypodium distachyon seminal roots.

    PubMed

    Pacheco-Villalobos, David; Sankar, Martial; Ljung, Karin; Hardtke, Christian S

    2013-06-01

    cell elongation, as suggested by our observations. Thus, our results reveal a delicate homeostasis of local auxin and ethylene activity to control cell elongation in Brachypodium roots and suggest alternative wiring of auxin-ethylene crosstalk as compared to Arabidopsis. PMID:23840182

  14. A conserved alternative form of the purple sea urchin HEB/E2-2/E2A transcription factor mediates a switch in E-protein regulatory state in differentiating immune cells.

    PubMed

    Schrankel, Catherine S; Solek, Cynthia M; Buckley, Katherine M; Anderson, Michele K; Rast, Jonathan P

    2016-08-01

    E-proteins are basic helix-loop-helix (bHLH) transcription factors with essential roles in animal development. In mammals, these are encoded by three loci: E2-2 (ITF-2/ME2/SEF2/TCF4), E2A (TCF3), and HEB (ME1/REB/TCF12). The HEB and E2-2 paralogs are expressed as alternative (Alt) isoforms with distinct N-terminal sequences encoded by unique exons under separate regulatory control. Expression of these alternative transcripts is restricted relative to the longer (Can) forms, suggesting distinct regulatory roles, although the functions of the Alt proteins remain poorly understood. Here, we characterize the single sea urchin E-protein ortholog (SpE-protein). The organization of the SpE-protein gene closely resembles that of the extended HEB/E2-2 vertebrate loci, including a transcript that initiates at a homologous alternative transcription start site (SpE-Alt). The existence of an Alt form in the sea urchin indicates that this feature predates the emergence of the vertebrates. We present additional evidence indicating that this transcript was present in the common bilaterian ancestor. In contrast to the widely expressed canonical form (SpE-Can), SpE-Alt expression is tightly restricted. SpE-Alt is expressed in two phases: first in aboral non-skeletogenic mesenchyme (NSM) cells and then in oral NSM cells preceding their differentiation and ingression into the blastocoel. Derivatives of these cells mediate immune response in the larval stage. Inhibition of SpE-Alt activity interferes with these events. Notably, although the two isoforms are initially co-expressed, as these cells differentiate, SpE-Can is excluded from the SpE-Alt(+) cell population. This mutually exclusive expression is dependent on SpE-Alt function, which reveals a previously undescribed negative regulatory linkage between the two E-protein forms. Collectively, these findings reorient our understanding of the evolution of this transcription factor family and highlight fundamental properties of E

  15. Differential expression of the alternatively-spliced OPRM1 isoform MOR-1K in HIV-infected subjects

    PubMed Central

    Dever, Seth M.; Costin, Blair N.; Xu, Ruqiang; El-Hage, Nazira; Balinang, Joyce; Samoshkin, Alexander; O’Brien, Megan A.; McRae, MaryPeace; Diatchenko, Luda; Knapp, Pamela E.; Hauser, Kurt F.

    2014-01-01

    Objective We previously examined the expression of specific C-terminal μ-opioid receptor (MOR) splice variants in human central nervous system cell types and HIV-infected brain tissue from subjects with neurocognitive impairment ± HIV encephalitis (HIVE). In the present study, we examined the N-terminal splice variant MOR-1K which mediates excitatory cellular signaling. Methods and Results We found segregation of expression ranging from undetectable to seemingly exclusive across nervous system cell types compared to the pool of C-terminal MOR splice variants using RT-PCR. Expression of MOR-1K mRNA was also increased in HIV-infected subjects with combined neurocognitive impairment and HIVE compared to the other groups. MOR-1K expression correlated with the level of subject neurocognitive impairment whereas the pool of C-terminal MOR splice variants did not. HIVE was also associated with increased expression of the inflammatory mediators MCP-1, MCP-2, and RANTES, but not the host HIV co-receptors CXCR4 and CCR5 or the CD4 receptor, using qRT-PCR. Network analysis of microarray data from these same subjects revealed filamin A (FLNA) as a possible interaction partner with MOR-1K, and FLNA gene expression was also found to be upregulated in HIVE using qRT-PCR. Overexpression of filamin A in HEK293 cells redistributed MOR-1K from intracellular compartments to the cell surface. Conclusion These results suggest that HIVE, and neurocognitive impairment depending on its severity, are associated with enhanced MOR-1K signaling through both increased expression and trafficking to the cell surface, which may alter the contribution of MOR receptor isoforms and exacerbate the effects of MOR activation in neuroAIDS. PMID:24413261

  16. Spontaneous Chronic Pain After Experimental Thoracotomy Revealed by Conditioned Place Preference: Morphine Differentiates Tactile Evoked Pain From Spontaneous Pain.

    PubMed

    Hung, Ching-Hsia; Wang, Jeffrey Chi-Fei; Strichartz, Gary R

    2015-09-01

    Chronic pain after surgery limits social activity, interferes with work, and causes emotional suffering. A major component of such pain is reported as resting or spontaneous pain with no apparent external stimulus. Although experimental animal models can simulate the stimulus-evoked chronic pain that occurs after surgery, there have been no studies of spontaneous chronic pain in such models. Here the conditioned place preference (CPP) paradigm was used to reveal resting pain after experimental thoracotomy. Male Sprague Dawley rats received a thoracotomy with 1-hour rib retraction, resulting in evoked tactile hypersensitivity, previously shown to last for at least 9 weeks. Intraperitoneal injections of morphine (2.5 mg/kg) or gabapentin (40 mg/kg) gave equivalent 2- to 3-hour-long relief of tactile hypersensitivity when tested 12 to 14 days postoperatively. In separate experiments, single trial CPP was conducted 1 week before thoracotomy and then 12 days (gabapentin) or 14 days (morphine) after surgery, followed the next day by 1 conditioning session with morphine or gabapentin, both versus saline. The gabapentin-conditioned but not the morphine-conditioned rats showed a significant preference for the analgesia-paired chamber, despite the equivalent effect of the 2 agents in relieving tactile allodynia. These results show that experimental thoracotomy in rats causes spontaneous pain and that some analgesics, such as morphine, that reduce evoked pain do not also relieve resting pain, suggesting that pathophysiological mechanisms differ between these 2 aspects of long-term postoperative pain. Perspective: Spontaneous pain, a hallmark of chronic postoperative pain, is demonstrated here in a rat model of experimental postthoracotomy pain, further validating the use of this model for the development of analgesics to treat such symptoms. Although stimulus-evoked pain was sensitive to systemic morphine, spontaneous pain was not, suggesting different mechanistic

  17. Molecular Diagnostic Tools for Detection and Differentiation of Phytoplasmas Based on Chaperonin-60 Reveal Differences in Host Plant Infection Patterns

    PubMed Central

    Dumonceaux, Tim J.; Green, Margaret; Hammond, Christine; Perez, Edel; Olivier, Chrystel

    2014-01-01

    Phytoplasmas (‘Candidatus Phytoplasma’ spp.) are insect-vectored bacteria that infect a wide variety of plants, including many agriculturally important species. The infections can cause devastating yield losses by inducing morphological changes that dramatically alter inflorescence development. Detection of phytoplasma infection typically utilizes sequences located within the 16S–23S rRNA-encoding locus, and these sequences are necessary for strain identification by currently accepted standards for phytoplasma classification. However, these methods can generate PCR products >1400 bp that are less divergent in sequence than protein-encoding genes, limiting strain resolution in certain cases. We describe a method for accessing the chaperonin-60 (cpn60) gene sequence from a diverse array of ‘Ca.Phytoplasma’ spp. Two degenerate primer sets were designed based on the known sequence diversity of cpn60 from ‘Ca.Phytoplasma’ spp. and used to amplify cpn60 gene fragments from various reference samples and infected plant tissues. Forty three cpn60 sequences were thereby determined. The cpn60 PCR-gel electrophoresis method was highly sensitive compared to 16S-23S-targeted PCR-gel electrophoresis. The topology of a phylogenetic tree generated using cpn60 sequences was congruent with that reported for 16S rRNA-encoding genes. The cpn60 sequences were used to design a hybridization array using oligonucleotide-coupled fluorescent microspheres, providing rapid diagnosis and typing of phytoplasma infections. The oligonucleotide-coupled fluorescent microsphere assay revealed samples that were infected simultaneously with two subtypes of phytoplasma. These tools were applied to show that two host plants, Brassica napus and Camelina sativa, displayed different phytoplasma infection patterns. PMID:25551224

  18. Repeated exposure to immobilization or two different footshock intensities reveals differential adaptation of the hypothalamic-pituitary-adrenal axis.

    PubMed

    Rabasa, Cristina; Muñoz-Abellán, Cristina; Daviu, Núria; Nadal, Roser; Armario, Antonio

    2011-05-01

    Factors involved in adaptation to repeated stress are not well-characterized. For instance, acute footshock (FS) of high intensity appears to be less severe than immobilization (IMO) in light of the speed of post-stress recovery of the hypothalamic-pituitary-adrenal (HPA) axis and other physiological variables. However, repeated exposure to IMO consistently resulted in reduction of the HPA response to the same stressor (adaptation), whereas failure to adapt has been usually reported after FS. Thus, in the present work we directly compared the activation of HPA axis and other physiological changes in response to both acute and repeated exposure to IMO and two intensities of FS (medium and high) in adult male rats. Control rats were exposed to the FS boxes but they did not receive shocks. Daily repeated exposure to IMO resulted in significant adaptation of the overall ACTH and corticosterone responses to the stressor. Such a reduction was also observed with repeated exposure to FS boxes and FS-medium, whereas repeated exposure to FS-high only resulted in a small reduction of the corticosterone response during the post-stress period. This suggests that some properties of FS-high make adaptation to it difficult. Interestingly, overall changes in food intake and body weight gain throughout the week of exposure to the stressors reveal a greater impact of IMO than FS-high, indicating that factors other than the intensity of a stressor, at least when evaluated in function of the above physiological variables, can influence HPA adaptation. Since FS exposure is likely to cause more pain than IMO, activation of nociceptive signals above a certain level may negatively affect HPA adaptation to repeated stressors. PMID:21352836

  19. Global transcriptional profiling of Burkholderia pseudomallei under salt stress reveals differential effects on the Bsa type III secretion system

    PubMed Central

    2010-01-01

    Background Burkholderia pseudomallei is the causative agent of melioidosis where the highest reported incidence world wide is in the Northeast of Thailand, where saline soil and water are prevalent. Moreover, recent reports indicate a potential pathogenic role for B. pseudomallei in cystic fibrosis lung disease, where an increased sodium chloride (NaCl) concentration in airway surface liquid has been proposed. These observations raise the possibility that high salinity may represent a favorable niche for B. pseudomallei. We therefore investigated the global transcriptional response of B. pseudomallei to increased salinity using microarray analysis. Results Transcriptome analysis of B. pseudomallei under salt stress revealed several genes significantly up-regulated in the presence of 320 mM NaCl including genes associated with the bsa-derived Type III secretion system (T3SS). Microarray data were verified by reverse transcriptase-polymerase chain reactions (RT-PCR). Western blot analysis confirmed the increased expression and secretion of the invasion-associated type III secreted proteins BipD and BopE in B. pseudomallei cultures at 170 and 320 mM NaCl relative to salt-free medium. Furthermore, salt-treated B. pseudomallei exhibited greater invasion efficiency into the lung epithelial cell line A549 in a manner partly dependent on a functional Bsa system. Conclusions B. pseudomallei responds to salt stress by modulating the transcription of a relatively small set of genes, among which is the bsa locus associated with invasion and virulence. Expression and secretion of Bsa-secreted proteins was elevated in the presence of exogenous salt and the invasion efficiency was enhanced. Our data indicate that salinity has the potential to influence the virulence of B. pseudomallei. PMID:20540813

  20. Integrating nap and night-time sleep into sleep patterns reveals differential links to health-relevant outcomes.

    PubMed

    Devine, Jaime K; Wolf, Jutta M

    2016-04-01

    Both night-time sleep and nap behaviour have been linked consistently to health outcomes. Although reasons for napping are usually tied to night-time sleep, the majority of studies assess their effects independently. The current study thus aimed to examine the health relevance of patterns of sleep behaviour that take into account both night-time and daytime sleep habits. Night-time sleep, recorded during 7 days via actigraphy from 313 participants (aged 34-82 years) of the Midlife in the United States II Biomarker study, was assessed. Blood and urine specimens were assayed for noradrenaline, interleukin-6 and C-reactive protein. Participants self-reported nap behaviour, depressive symptoms, perceived chronic stress and the presence of medical symptoms and conditions. Overall, nappers (n = 208) showed elevated waist-hip ratios, C-reactive protein and interleukin-6 levels compared to non-nappers and reported more physiological symptoms and conditions (all P ≤ 0.019). Within nappers, cluster analysis revealed three patterns of sleep behaviour-infrequent nappers with good night-time sleep, frequent nappers with good night-time sleep and nappers with poor night-time sleep. Nappers with poor night-time sleep thereby exhibited elevated noradrenaline levels, depressive symptoms and perceived stress scores compared to other groups (all P ≤ 0.041). These findings support the idea that nap-health relationships are complex, in that frequency of napping and accumulation of nap sleep is not related linearly to health consequences. Assessing nap behaviour in conjunction with night-time sleep behaviour appeared crucial to elucidate further the health relevance of napping, particularly in terms of psychological health outcomes, including chronic stress and depressive symptoms. PMID:26718988

  1. Molecular diagnostic tools for detection and differentiation of phytoplasmas based on chaperonin-60 reveal differences in host plant infection patterns.

    PubMed

    Dumonceaux, Tim J; Green, Margaret; Hammond, Christine; Perez, Edel; Olivier, Chrystel

    2014-01-01

    Phytoplasmas ('Candidatus Phytoplasma' spp.) are insect-vectored bacteria that infect a wide variety of plants, including many agriculturally important species. The infections can cause devastating yield losses by inducing morphological changes that dramatically alter inflorescence development. Detection of phytoplasma infection typically utilizes sequences located within the 16S-23S rRNA-encoding locus, and these sequences are necessary for strain identification by currently accepted standards for phytoplasma classification. However, these methods can generate PCR products >1400 bp that are less divergent in sequence than protein-encoding genes, limiting strain resolution in certain cases. We describe a method for accessing the chaperonin-60 (cpn60) gene sequence from a diverse array of 'Ca.Phytoplasma' spp. Two degenerate primer sets were designed based on the known sequence diversity of cpn60 from 'Ca.Phytoplasma' spp. and used to amplify cpn60 gene fragments from various reference samples and infected plant tissues. Forty three cpn60 sequences were thereby determined. The cpn60 PCR-gel electrophoresis method was highly sensitive compared to 16S-23S-targeted PCR-gel electrophoresis. The topology of a phylogenetic tree generated using cpn60 sequences was congruent with that reported for 16S rRNA-encoding genes. The cpn60 sequences were used to design a hybridization array using oligonucleotide-coupled fluorescent microspheres, providing rapid diagnosis and typing of phytoplasma infections. The oligonucleotide-coupled fluorescent microsphere assay revealed samples that were infected simultaneously with two subtypes of phytoplasma. These tools were applied to show that two host plants, Brassica napus and Camelina sativa, displayed different phytoplasma infection patterns. PMID:25551224

  2. Differential Interaction Kinetics of a Bipolar Structure-Specific Endonuclease with DNA Flaps Revealed by Single-Molecule Imaging

    PubMed Central

    Rezgui, Rachid; Lestini, Roxane; Kühn, Joëlle; Fave, Xenia; McLeod, Lauren; Myllykallio, Hannu; Alexandrou, Antigoni; Bouzigues, Cedric

    2014-01-01

    As DNA repair enzymes are essential for preserving genome integrity, understanding their substrate interaction dynamics and the regulation of their catalytic mechanisms is crucial. Using single-molecule imaging, we investigated the association and dissociation kinetics of the bipolar endonuclease NucS from Pyrococcus abyssi (Pab) on 5′ and 3′-flap structures under various experimental conditions. We show that association of the PabNucS with ssDNA flaps is largely controlled by diffusion in the NucS-DNA energy landscape and does not require a free 5′ or 3′ extremity. On the other hand, NucS dissociation is independent of the flap length and thus independent of sliding on the single-stranded portion of the flapped DNA substrates. Our kinetic measurements have revealed previously unnoticed asymmetry in dissociation kinetics from these substrates that is markedly modulated by the replication clamp PCNA. We propose that the replication clamp PCNA enhances the cleavage specificity of NucS proteins by accelerating NucS loading at the ssDNA/dsDNA junctions and by minimizing the nuclease interaction time with its DNA substrate. Our data are also consistent with marked reorganization of ssDNA and nuclease domains occurring during NucS catalysis, and indicate that NucS binds its substrate directly at the ssDNA-dsDNA junction and then threads the ssDNA extremity into the catalytic site. The powerful techniques used here for probing the dynamics of DNA-enzyme binding at the single-molecule have provided new insight regarding substrate specificity of NucS nucleases. PMID:25412080

  3. Inducible Knockdown of MONOGALACTOSYLDIACYLGLYCEROL SYNTHASE1 Reveals Roles of Galactolipids in Organelle Differentiation in Arabidopsis Cotyledons1[W][OPEN

    PubMed Central

    Fujii, Sho; Kobayashi, Koichi; Nakamura, Yuki; Wada, Hajime

    2014-01-01

    Monogalactosyldiacylglycerol (MGDG) is the major lipid constituent of thylakoid membranes and is essential for chloroplast biogenesis in plants. In Arabidopsis (Arabidopsis thaliana), MGDG is predominantly synthesized by inner envelope-localized MONOGALACTOSYLDIACYLGLYCEROL SYNTHASE1 (MGD1); its knockout causes albino seedlings. Because of the lethal phenotype of the null MGD1 mutant, functional details of MGDG synthesis at seedling development have remained elusive. In this study, we used an inducible gene-suppression system to investigate the impact of MGDG synthesis on cotyledon development. We created transgenic Arabidopsis lines that express an artificial microRNA targeting MGD1 (amiR-MGD1) under the control of a dexamethasone-inducible promoter. The induction of amiR-MGD1 resulted in up to 75% suppression of MGD1 expression, although the resulting phenotypes related to chloroplast development were diverse, even within a line. The strong MGD1 suppression by continuous dexamethasone treatment caused substantial decreases in galactolipid content in cotyledons, leading to severe defects in the formation of thylakoid membranes and impaired photosynthetic electron transport. Time-course analyses of the MGD1 suppression during seedling germination revealed that MGDG synthesis at the very early germination stage is particularly important for chloroplast biogenesis. The MGD1 suppression down-regulated genes associated with the photorespiratory pathway in peroxisomes and mitochondria as well as those responsible for photosynthesis in chloroplasts and caused high expression of genes for the glyoxylate cycle. MGD1 function may link galactolipid synthesis with the coordinated transcriptional regulation of chloroplasts and other organelles during cotyledon greening. PMID:25253888

  4. 1H NMR-Based Profiling Reveals Differential Immune-Metabolic Networks during Influenza Virus Infection in Obese Mice

    PubMed Central

    Milner, J. Justin; Wang, Jue; Sheridan, Patricia A.; Ebbels, Tim; Beck, Melinda A.; Saric, Jasmina

    2014-01-01

    Obese individuals are at greater risk for death from influenza virus infection. Paralleling human evidence, obese mice are also more susceptible to influenza infection mortality. However, the underlying mechanisms driving greater influenza severity in the obese remain unclear. Metabolic profiling has been utilized in infectious disease models to enhance prognostic or diagnostic methods, and to gain insight into disease pathogenesis by providing a more global picture of dynamic infection responses. Herein, metabolic profiling was used to develop a deeper understanding of the complex processes contributing to impaired influenza protection in obese mice and to facilitate generation of new explanatory hypotheses. Diet-induced obese and lean mice were infected with influenza A/Puerto Rico/8/34. 1H nuclear magnetic resonance-based metabolic profiling of urine, feces, lung, liver, mesenteric white adipose tissue, bronchoalveolar lavage fluid and serum revealed distinct metabolic signatures in infected obese mice, including perturbations in nucleotide, vitamin, ketone body, amino acid, carbohydrate, choline and lipid metabolic pathways. Further, metabolic data was integrated with immune analyses to obtain a more comprehensive understanding of potential immune-metabolic interactions. Of interest, uncovered metabolic signatures in urine and feces allowed for discrimination of infection status in both lean and obese mice at an early influenza time point, which holds prognostic and diagnostic implications for this methodology. These results confirm that obesity causes distinct metabolic perturbations during influenza infection and provide a basis for generation of new hypotheses and use of this methodology in detection of putative biomarkers and metabolic patterns to predict influenza infection outcome. PMID:24844920

  5. Novel fluorescence resonance energy transfer-based reporter reveals differential calcineurin activation in neonatal and adult cardiomyocytes.

    PubMed

    Bazzazi, Hojjat; Sang, Lingjie; Dick, Ivy E; Joshi-Mukherjee, Rosy; Yang, Wanjun; Yue, David T

    2015-09-01

    Novel fluorescence resonance energy transfer-based genetically encoded reporters of calcineurin are constructed by fusing the two subunits of calcineurin with P2A-based linkers retaining the expected native conformation of calcineurin. Calcineurin reporters display robust responses to calcium transients in HEK293 cells. The sensor responses are correlated with NFATc1 translocation dynamics in HEK293 cells. The sensors are uniformly distributed in neonatal myocytes and respond efficiently to single electrically evoked calcium transients and show cumulative activation at frequencies of 0.5 and 1 Hz. In adult myocytes, the calcineurin sensors appear to be localized to the cardiac z-lines, and respond to cumulative calcium transients at frequencies of 0.5 and 1 Hz. The phosphatase calcineurin is a central component of many calcium signalling pathways, relaying calcium signals from the plasma membrane to the nucleus. It has critical functions in a multitude of systems, including immune, cardiac and neuronal. Given the widespread importance of calcineurin in both normal and pathological conditions, new tools that elucidate the spatiotemporal dynamics of calcineurin activity would be invaluable. Here we develop two separate genetically encoded fluorescence resonance energy transfer (FRET)-based sensors of calcineurin activation, DuoCaN and UniCaN. Both sensors showcase a large dynamic range and rapid response kinetics, differing primarily in the linker structure between the FRET pairs. Both sensors were calibrated in HEK293 cells and their responses correlated well with NFAT translocation to the nucleus, validating the biological relevance of the sensor readout. The sensors were subsequently expressed in neonatal rat ventricular myocytes and acutely isolated adult guinea pig ventricular myocytes. Both sensors demonstrated robust responses in myocytes and revealed kinetic differences in calcineurin activation during changes in pacing rate for neonatal versus adult myocytes

  6. Integrative genomic analysis in K562 chronic myelogenous leukemia cells reveals that proximal NCOR1 binding positively regulates genes that govern erythroid differentiation and Imatinib sensitivity

    PubMed Central

    Long, Mark D.; van den Berg, Patrick R.; Russell, James L.; Singh, Prashant K.; Battaglia, Sebastiano; Campbell, Moray J.

    2015-01-01

    To define the functions of NCOR1 we developed an integrative analysis that combined ENCODE and NCI-60 data, followed by in vitro validation. NCOR1 and H3K9me3 ChIP-Seq, FAIRE-seq and DNA CpG methylation interactions were related to gene expression using bootstrapping approaches. Most NCOR1 combinations (24/44) were associated with significantly elevated level expression of protein coding genes and only very few combinations related to gene repression. DAVID's biological process annotation revealed that elevated gene expression was uniquely associated with acetylation and ETS binding. A matrix of gene and drug interactions built on NCI-60 data identified that Imatinib significantly targeted the NCOR1 governed transcriptome. Stable knockdown of NCOR1 in K562 cells slowed growth and significantly repressed genes associated with NCOR1 cistrome, again, with the GO terms acetylation and ETS binding, and significantly dampened sensitivity to Imatinib-induced erythroid differentiation. Mining public microarray data revealed that NCOR1-targeted genes were significantly enriched in Imatinib response gene signatures in cell lines and chronic myelogenous leukemia (CML) patients. These approaches integrated cistrome, transcriptome and drug sensitivity relationships to reveal that NCOR1 function is surprisingly most associated with elevated gene expression, and that these targets, both in CML cell lines and patients, associate with sensitivity to Imatinib. PMID:26117541

  7. Crystal Structures of Wild-type and Mutant Methicillin-resistant Staphylococcus aureus Dihydrofolate Reductase Reveal an Alternative Conformation of NADPH that may be Linked to Trimethoprim Resistance

    PubMed Central

    Frey, Kathleen M.; Liu, Jieying; Lombardo, Michael N.; Bolstad, David B.; Wright, Dennis L.; Anderson, Amy C.

    2009-01-01

    SUMMARY Both hospital- and community-acquired Staphylococcus aureus infections have become major health concerns in terms of morbidity, suffering and cost. Trimethoprim-sulfamethoxazole (TMP-SMZ) is an alternative treatment for methicillin-resistant S. aureus (MRSA) infections. However, TMP-resistant strains have arisen with point mutations in dihydrofolate reductase (DHFR), the target for TMP. A single point mutation, F98Y, has been shown biochemically to confer the majority of this resistance to TMP. Using a structure-based approach, we have designed a series of novel propargyl-linked DHFR inhibitors that are active against several trimethoprim-resistant enzymes. We screened this series against wild-type and mutant (F98Y) S. aureus DHFR and found that several are active against both enzymes and specifically that the meta-biphenyl class of these inhibitors is the most potent. In order to understand the structural basis of this potency, we determined eight high-resolution crystal structures: four each of the wild-type and mutant DHFR enzymes bound to various propargyl-linked DHFR inhibitors. In addition to explaining the structure-activity relationships, several of the structures reveal a novel conformation for the cofactor, NADPH. In this new conformation that is predominantly associated with the mutant enzyme, the nicotinamide ring is displaced from its conserved location and three water molecules complete a network of hydrogen bonds between the nicotinamide ring and the protein. In this new position, NADPH has reduced interactions with the inhibitor. An equilibrium between the two conformations of NADPH, implied by their occupancies in the eight crystal structures, is influenced both by the ligand and the F98Y mutation. The mutation induced equilibrium between two NADPH binding conformations may contribute to decrease TMP binding and thus may be responsible for TMP resistance. PMID:19249312

  8. Crystal Structures of Wild-type and Mutant Methicillin-resistant Staphylococcus aureus Dihydrofolate Reductase Reveal an Alternative Conformation of NADPH that may be Linked to Trimethoprim Resistance

    SciTech Connect

    Frey, K.; Liu, J; Lombardo, M; Bolstad, D; Wright, D; Anderson, A

    2009-01-01

    Both hospital- and community-acquired Staphylococcus aureus infections have become major health concerns in terms of morbidity, suffering and cost. Trimethoprim-sulfamethoxazole (TMP-SMZ) is an alternative treatment for methicillin-resistant S. aureus (MRSA) infections. However, TMP-resistant strains have arisen with point mutations in dihydrofolate reductase (DHFR), the target for TMP. A single point mutation, F98Y, has been shown biochemically to confer the majority of this resistance to TMP. Using a structure-based approach, we have designed a series of novel propargyl-linked DHFR inhibitors that are active against several trimethoprim-resistant enzymes. We screened this series against wild-type and mutant (F98Y) S. aureus DHFR and found that several are active against both enzymes and specifically that the meta-biphenyl class of these inhibitors is the most potent. In order to understand the structural basis of this potency, we determined eight high-resolution crystal structures: four each of the wild-type and mutant DHFR enzymes bound to various propargyl-linked DHFR inhibitors. In addition to explaining the structure-activity relationships, several of the structures reveal a novel conformation for the cofactor, NADPH. In this new conformation that is predominantly associated with the mutant enzyme, the nicotinamide ring is displaced from its conserved location and three water molecules complete a network of hydrogen bonds between the nicotinamide ring and the protein. In this new position, NADPH has reduced interactions with the inhibitor. An equilibrium between the two conformations of NADPH, implied by their occupancies in the eight crystal structures, is influenced both by the ligand and the F98Y mutation. The mutation induced equilibrium between two NADPH-binding conformations may contribute to decrease TMP binding and thus may be responsible for TMP resistance.

  9. Comparative analyses across cattle genders and breeds reveal the pitfalls caused by false positive and lineage-differential copy number variations.

    PubMed

    Zhou, Yang; Utsunomiya, Yuri T; Xu, Lingyang; Hay, El Hamidi Abdel; Bickhart, Derek M; Sonstegard, Tad S; Van Tassell, Curtis P; Garcia, Jose Fernando; Liu, George E

    2016-01-01

    We compared CNV region (CNVR) results derived from 1,682 Nellore cattle with equivalent results derived from our previous analysis of Bovine HapMap samples. By comparing CNV segment frequencies between different genders and groups, we identified 9 frequent, false positive CNVRs with a total length of 0.8 Mbp that were likely caused by assembly errors. Although there was a paucity of lineage specific events, we did find one 54 kb deletion on chr5 significantly enriched in Nellore cattle. A few highly frequent CNVRs present in both datasets were detected within genomic regions containing olfactory receptor, ATP-binding cassette, and major histocompatibility complex genes. We further evaluated their impacts on downstream bioinformatics and CNV association analyses. Our results revealed pitfalls caused by false positive and lineage-differential copy number variations and will increase the accuracy of future CNV studies in both taurine and indicine cattle. PMID:27381368

  10. Comparative analyses across cattle genders and breeds reveal the pitfalls caused by false positive and lineage-differential copy number variations

    PubMed Central

    Zhou, Yang; Utsunomiya, Yuri T.; Xu, Lingyang; Hay, El Hamidi abdel; Bickhart, Derek M.; Sonstegard, Tad S.; Van Tassell, Curtis P.; Garcia, Jose Fernando; Liu, George E.

    2016-01-01

    We compared CNV region (CNVR) results derived from 1,682 Nellore cattle with equivalent results derived from our previous analysis of Bovine HapMap samples. By comparing CNV segment frequencies between different genders and groups, we identified 9 frequent, false positive CNVRs with a total length of 0.8 Mbp that were likely caused by assembly errors. Although there was a paucity of lineage specific events, we did find one 54 kb deletion on chr5 significantly enriched in Nellore cattle. A few highly frequent CNVRs present in both datasets were detected within genomic regions containing olfactory receptor, ATP-binding cassette, and major histocompatibility complex genes. We further evaluated their impacts on downstream bioinformatics and CNV association analyses. Our results revealed pitfalls caused by false positive and lineage-differential copy number variations and will increase the accuracy of future CNV studies in both taurine and indicine cattle. PMID:27381368

  11. Genetic variation and population differentiation in a medical herb Houttuynia cordata in China revealed by inter-simple sequence repeats (ISSRs).

    PubMed

    Wei, Lin; Wu, Xian-Jin

    2012-01-01

    Houttuynia cordata is an important traditional Chinese herb with unresolved genetics and taxonomy, which lead to potential problems in the conservation and utilization of the resource. Inter-simple sequence repeat (ISSR) markers were used to assess the level and distribution of genetic diversity in 226 individuals from 15 populations of H. cordata in China. ISSR analysis revealed low genetic variations within populations but high genetic differentiations among populations. This genetic structure probably mainly reflects the historical association among populations. Genetic cluster analysis showed that the basal clade is composed of populations from Southwest China, and the other populations have continuous and eastward distributions. The structure of genetic diversity in H. cordata demonstrated that this species might have survived in Southwest China during the glacial age, and subsequently experienced an eastern postglacial expansion. Based on the results of genetic analysis, it was proposed that as many as possible targeted populations for conservation be included. PMID:22942696

  12. Extensive sampling of polar bears (Ursus maritimus) in the Northwest Passage (Canadian Arctic Archipelago) reveals population differentiation across multiple spatial and temporal scales.

    PubMed

    Campagna, Leonardo; Van Coeverden de Groot, Peter J; Saunders, Brenda L; Atkinson, Stephen N; Weber, Diana S; Dyck, Markus G; Boag, Peter T; Lougheed, Stephen C

    2013-09-01

    As global warming accelerates the melting of Arctic sea ice, polar bears (Ursus maritimus) must adapt to a rapidly changing landscape. This process will necessarily alter the species distribution together with population dynamics and structure. Detailed knowledge of these changes is crucial to delineating conservation priorities. Here, we sampled 361 polar bears from across the center of the Canadian Arctic Archipelago spanning the Gulf of Boothia (GB) and M'Clintock Channel (MC). We use DNA microsatellites and mitochondrial control region sequences to quantify genetic differentiation, estimate gene flow, and infer population history. Two populations, roughly coincident with GB and MC, are significantly differentiated at both nuclear (F ST = 0.01) and mitochondrial (ΦST = 0.47; F ST = 0.29) loci, allowing Bayesian clustering analyses to assign individuals to either group. Our data imply that the causes of the mitochondrial and nuclear genetic patterns differ. Analysis of mtDNA reveals the matrilineal structure dates at least to the Holocene, and is common to individuals throughout the species' range. These mtDNA differences probably reflect both genetic drift and historical colonization dynamics. In contrast, the differentiation inferred from microsatellites is only on the scale of hundreds of years, possibly reflecting contemporary impediments to gene flow. Taken together, our data suggest that gene flow is insufficient to homogenize the GB and MC populations and support the designation of GB and MC as separate polar bear conservation units. Our study also provide a striking example of how nuclear DNA and mtDNA capture different aspects of a species demographic history. PMID:24102001

  13. Cellular Heterogeneity During Embryonic Stem Cell Differentiation to Epiblast Stem Cells is Revealed by the ShcD/RaLP Adaptor Protein

    PubMed Central

    Turco, Margherita Y; Furia, Laura; Dietze, Anja; Fernandez Diaz, Luis; Ronzoni, Simona; Sciullo, Anna; Simeone, Antonio; Constam, Daniel; Faretta, Mario; Lanfrancone, Luisa

    2012-01-01

    The Shc family of adaptor proteins are crucial mediators of a plethora of receptors such as the tyrosine kinase receptors, cytokine receptors, and integrins that drive signaling pathways governing proliferation, differentiation, and migration. Here, we report the role of the newly identified family member, ShcD/RaLP, whose expression in vitro and in vivo suggests a function in embryonic stem cell (ESC) to epiblast stem cells (EpiSCs) transition. The transition from the naïve (ESC) to the primed (EpiSC) pluripotent state is the initial important step for ESCs to commit to differentiation and the mechanisms underlying this process are still largely unknown. Using a novel approach to simultaneously assess pluripotency, apoptosis, and proliferation by multiparameter flow cytometry, we show that ESC to EpiSC transition is a process involving a tight coordination between the modulation of the Oct4 expression, cell cycle progression, and cell death. We also describe, by high-content immunofluorescence analysis and time-lapse microscopy, the emergence of cells expressing caudal-related homeobox 2 (Cdx2) transcription factor during ESC to EpiSC transition. The use of the ShcD knockout ESCs allowed the unmasking of this process as they presented deregulated Oct4 modulation and an enrichment in Oct4-negative Cdx2-positive cells with increased MAPK/extracellular-regulated kinases 1/2 activation, within the differentiating population. Collectively, our data reveal ShcD as an important modulator in the switch of key pathway(s) involved in determining EpiSC identity. Stem Cells2012;30:2423–2436 PMID:22948967

  14. MicroRNA screen of human embryonic stem cell differentiation reveals miR-105 as an enhancer of megakaryopoiesis from adult CD34+ cells.

    PubMed

    Kamat, Viraj; Paluru, Prasuna; Myint, Melissa; French, Deborah L; Gadue, Paul; Diamond, Scott L

    2014-05-01

    MicroRNAs (miRNAs) can control stem cell differentiation by targeting mRNAs. Using 96-well plate electroporation, we screened 466 human miRNA mimics by four-color flow cytometry to explore differentiation of common myeloid progenitors (CMP) derived from human embryonic stem cells (hESCs). The transfected cells were then cultured in a cytokine cocktail that supported multiple hematopoietic lineages. At 4-5 days post-transfection, flow cytometry of erythroid (CD235(+)CD41(-)), megakaryocyte (CD41(+)CD42(+)), and myeloid (CD18(+)CD235(-)) lineages revealed miR-105 as a novel enhancer of megakaryocyte production during in vitro primitive hematopoiesis. In hESC-derived CMPs, miR-105 caused a sixfold enhancement in megakaryocyte production. miR-513a, miR-571, and miR-195 were found to be less potent megakaryocyte enhancers. We confirmed the relevance of miR-105 in adult megakaryopoiesis by demonstrating increased megakaryocyte yield and megakaryocyte colony forming potential in human adult CD34(+) cells derived from peripheral blood. In addition, adult CD34(+) cells express endogenous miR-105 during megakaryocyte differentiation. siRNA knockdown of the hematopoietic transcription factor c-Myb caused a similar enhancement of megakaryocyte production as miR-105. Finally, a luciferase/c-Myb-3'UTR construct and Western blot analysis demonstrated that the hematopoietic transcription factor c-Myb mRNA was a target of miR-105. We report a novel hESC-based miR screening platform and demonstrate that miR-105 is an enhancer of megakaryopoiesis in both primitive and definitive hematopoiesis. PMID:24446170

  15. Genetic Differentiation and Genetic Diversity of Castanopsis (Fagaceae), the Dominant Tree Species in Japanese Broadleaved Evergreen Forests, Revealed by Analysis of EST-Associated Microsatellites

    PubMed Central

    Aoki, Kyoko; Ueno, Saneyoshi; Kamijo, Takashi; Setoguchi, Hiroaki; Murakami, Noriaki; Kato, Makoto; Tsumura, Yoshihiko

    2014-01-01

    The broadleaved evergreen forests of the East Asian warm temperate zone are characterised by their high biodiversity and endemism, and there is therefore a need to extend our understanding of its genetic diversity and phylogeographic patterns. Castanopsis (Fagaceae) is one of the dominant tree species in the broadleaved evergreen forests of Japan. In this study we investigate the genetic diversity, genetic structure and leaf epidermal morphology of 63 natural populations of C. sieboldii and C. cuspidata, using 32 Expressed Sequence Tag associated microsatellites. The overall genetic differentiation between populations was low (GST = 0.069 in C. sieboldii and GST = 0.057 in C. cuspidata). Neighbor-joining tree and Bayesian clustering analyses revealed that the populations of C. sieboldii and C. cuspidata were genetically clearly differentiated, a result which is consistent with the morphology of their epidermal cell layers. This suggests that C. sieboldii and C. cuspidata should be treated as independent species, although intermediate morphologies are often observed, especially at sites where the two species coexist. The higher level of genetic diversity observed in the Kyushu region (for both species) and the Ryukyu Islands (for C. sieboldii) is consistent with the available fossil pollen data for Castanopsis-type broadleaved evergreen trees during the Last Glacial Maximum and suggests the existence of refugia for Castanopsis forests in southern Japan. Within the C. sieboldii populations, Bayesian clustering analyses detected three clusters, in the western and eastern parts of the main islands and in the Ryukyu Islands. The west-east genetic differentiation observed for this species in the main islands, a pattern which is also found in several plant and animal species inhabiting Castanopsis forests in Japan, suggests that they have been isolated from each other in the western and eastern populations for an extended period of time, and may imply the

  16. Genetic differentiation and genetic diversity of Castanopsis (Fagaceae), the dominant tree species in Japanese broadleaved evergreen forests, revealed by analysis of EST-associated microsatellites.

    PubMed

    Aoki, Kyoko; Ueno, Saneyoshi; Kamijo, Takashi; Setoguchi, Hiroaki; Murakami, Noriaki; Kato, Makoto; Tsumura, Yoshihiko

    2014-01-01

    The broadleaved evergreen forests of the East Asian warm temperate zone are characterised by their high biodiversity and endemism, and there is therefore a need to extend our understanding of its genetic diversity and phylogeographic patterns. Castanopsis (Fagaceae) is one of the dominant tree species in the broadleaved evergreen forests of Japan. In this study we investigate the genetic diversity, genetic structure and leaf epidermal morphology of 63 natural populations of C. sieboldii and C. cuspidata, using 32 Expressed Sequence Tag associated microsatellites. The overall genetic differentiation between populations was low (GST = 0.069 in C. sieboldii and GST = 0.057 in C. cuspidata). Neighbor-joining tree and Bayesian clustering analyses revealed that the populations of C. sieboldii and C. cuspidata were genetically clearly differentiated, a result which is consistent with the morphology of their epidermal cell layers. This suggests that C. sieboldii and C. cuspidata should be treated as independent species, although intermediate morphologies are often observed, especially at sites where the two species coexist. The higher level of genetic diversity observed in the Kyushu region (for both species) and the Ryukyu Islands (for C. sieboldii) is consistent with the available fossil pollen data for Castanopsis-type broadleaved evergreen trees during the Last Glacial Maximum and suggests the existence of refugia for Castanopsis forests in southern Japan. Within the C. sieboldii populations, Bayesian clustering analyses detected three clusters, in the western and eastern parts of the main islands and in the Ryukyu Islands. The west-east genetic differentiation observed for this species in the main islands, a pattern which is also found in several plant and animal species inhabiting Castanopsis forests in Japan, suggests that they have been isolated from each other in the western and eastern populations for an extended period of time, and may imply the

  17. MALDI Mass Spectrometry Imaging Reveals Decreased CK5 Levels in Vulvar Squamous Cell Carcinomas Compared to the Precursor Lesion Differentiated Vulvar Intraepithelial Neoplasia

    PubMed Central

    Zhang, Chao; Arentz, Georgia; Winderbaum, Lyron; Lokman, Noor A.; Klingler-Hoffmann, Manuela; Mittal, Parul; Carter, Christopher; Oehler, Martin K.; Hoffmann, Peter

    2016-01-01

    Vulvar cancer is the fourth most common gynecological cancer worldwide. However, limited studies have been completed on the molecular characterization of vulvar squamous cell carcinoma resulting in a poor understanding of the disease initiation and progression. Analysis and early detection of the precursor lesion of HPV-independent vulvar squamous cell carcinoma (VSCC), differentiated vulvar intraepithelial neoplasia (dVIN), is of great importance given dVIN lesions have a high level of malignant potential. Here we present an examination of adjacent normal vulvar epithelium, dVIN, and VSCC from six patients by peptide Matrix-assisted laser desorption/ionization Mass Spectrometry Imaging (MALDI-MSI). The results reveal the differential expression of multiple peptides from the protein cytokeratin 5 (CK5) across the three vulvar tissue types. The difference observed in the relative abundance of CK5 by MALDI-MSI between the healthy epithelium, dVIN, and VSCC was further analyzed by immunohistochemistry (IHC) in tissue from eight VSCC patients. A decrease in CK5 immunostaining was observed in the VSCC compared to the healthy epithelium and dVIN. These results provide an insight into the molecular fingerprint of the vulvar intraepithelial neoplasia that appears to be more closely related to the healthy epithelium than the VSCC. PMID:27399691

  18. Genome-wide analysis of histone methylation reveals chromatin state-based complex regulation of differential gene transcription and function of CD8 memory T cells

    PubMed Central

    Araki, Yasuto; Wang, Zhibin; Zang, Chongzhi; Wood, William H.; Schones, Dustin; Cui, Kairong; Roh, Tae-Young; Lhotsky, Brad; Wersto, Robert P.; Peng, Weiqun; Becker, Kevin G.; Zhao, Keji; Weng, Nan-ping

    2009-01-01

    Summary Memory lymphocytes are characterized by their ability to exhibit a rapid response to the recall antigen, in which differential transcription plays a significant role, yet the underlying mechanism is not understood. We report here a genome-wide analysis of histone methylation on two histone H3 lysine residues (H3K4me3 and H3K27me3) and gene expression profiles in naïve and memory CD8 T cells. We found that a general correlation exists between the levels of gene expression and the levels of H3K4me3 (positive correlation) and H3K27me3 (negative correlation) across the gene body. These correlations display four distinct modes: repressive, active, poised, and bivalent, reflecting different functions of these genes. Furthermore, a permissive chromatin state of each gene is established by a combination of different histone modifications. Our findings reveal a complex regulation by histone methylation in differential gene expression and suggest that histone methylation may be responsible for memory CD8 T cell function. PMID:19523850

  19. MALDI Mass Spectrometry Imaging Reveals Decreased CK5 Levels in Vulvar Squamous Cell Carcinomas Compared to the Precursor Lesion Differentiated Vulvar Intraepithelial Neoplasia.

    PubMed

    Zhang, Chao; Arentz, Georgia; Winderbaum, Lyron; Lokman, Noor A; Klingler-Hoffmann, Manuela; Mittal, Parul; Carter, Christopher; Oehler, Martin K; Hoffmann, Peter

    2016-01-01

    Vulvar cancer is the fourth most common gynecological cancer worldwide. However, limited studies have been completed on the molecular characterization of vulvar squamous cell carcinoma resulting in a poor understanding of the disease initiation and progression. Analysis and early detection of the precursor lesion of HPV-independent vulvar squamous cell carcinoma (VSCC), differentiated vulvar intraepithelial neoplasia (dVIN), is of great importance given dVIN lesions have a high level of malignant potential. Here we present an examination of adjacent normal vulvar epithelium, dVIN, and VSCC from six patients by peptide Matrix-assisted laser desorption/ionization Mass Spectrometry Imaging (MALDI-MSI). The results reveal the differential expression of multiple peptides from the protein cytokeratin 5 (CK5) across the three vulvar tissue types. The difference observed in the relative abundance of CK5 by MALDI-MSI between the healthy epithelium, dVIN, and VSCC was further analyzed by immunohistochemistry (IHC) in tissue from eight VSCC patients. A decrease in CK5 immunostaining was observed in the VSCC compared to the healthy epithelium and dVIN. These results provide an insight into the molecular fingerprint of the vulvar intraepithelial neoplasia that appears to be more closely related to the healthy epithelium than the VSCC. PMID:27399691

  20. Variation of pathways and network profiles reveals the differential pharmacological mechanisms of each effective component to treat middle cerebral artery ischemia-reperfusion mice.

    PubMed

    Dang, HaiXia; Li, KangNing; Yu, YaNan; Zhang, YingYing; Liu, Jun; Wang, PengQian; Li, Bing; Wang, HaiNan; Li, Haixia; Wang, Zhong; Wang, YongYan

    2016-01-01

    Using a system pharmacology strategy, this study evaluated the unique pharmacological characteristics of three different neuroprotective compounds for the treatment of cerebral ischemia-reperfusion. A microarray including 374 brain ischemia-related genes was used to identify the differentially expressed genes among five treatment groups: baicalin, jasminoidin, ursodeoxycholic acid, sham, and vehicle, and MetaCore analysis software was applied to identify the significantly altered pathways, processes and interaction network parameters. At pathway level, 46, 25, and 31 pathways were activated in the baicalin, jasminoidin, and ursodeoxycholic acid groups, respectively. Thirteen pathways mainly related with apoptosis and development were commonly altered in the three groups. Additionally, baicalin also targeted pathways related with development, neurophysiologic process and cytoskeleton remodeling, while jasminoidin targeted pathways related with cell cycle and ursodeoxycholic acid targeted those related with apoptosis and development. At process level, three processes were commonly regulated by the three groups in the top 10 processes. Further interaction network analysis revealed that baicalin, jasminoidin, and ursodeoxycholic acid displayed unique features either on network topological parameters or network structure. Additional overlapping analysis demonstrated that compared with ursodeoxycholic acid, the pharmacological mechanism of baicalin was more similar with that of jasminoidin in treating brain ischemia. The data presented in this study may contribute toward the understanding of the common and differential pharmacological mechanisms of these three compounds. PMID:26168995

  1. De novo transcriptome assembly and analysis of differentially expressed genes of two barley genotypes reveal root-zone-specific responses to salt exposure.

    PubMed

    Hill, Camilla Beate; Cassin, Andrew; Keeble-Gagnère, Gabriel; Doblin, Monika S; Bacic, Antony; Roessner, Ute

    2016-01-01

    Plant roots are the first organs sensing and responding to salinity stress, manifested differentially between different root types, and also at the individual tissue and cellular level. High genetic diversity and the current lack of an assembled map-based sequence of the barley genome severely limit barley research potential. We used over 580 and 600 million paired-end reads, respectively, to create two de novo assemblies of a barley landrace (Sahara) and a malting cultivar (Clipper) with known contrasting responses to salinity. Generalized linear models were used to statistically access spatial, treatment-related, and genotype-specific responses. This revealed a spatial gene expression gradient along the barley root, with more differentially expressed transcripts detected between different root zones than between treatments. The root transcriptome also showed a gradual transition from transcripts related to sugar-mediated signaling at the root meristematic zone to those involved in cell wall metabolism in the elongation zone, and defense response-related pathways toward the maturation zone, with significant differences between the two genotypes. The availability of these additional transcriptome reference sets will serve as a valuable resource to the cereal research community, and may identify valuable traits to assist in breeding programmes. PMID:27527578

  2. Ancient DNA of the Extinct Lava Shearwater (Puffinus olsoni) from the Canary Islands Reveals Incipient Differentiation within the P. puffinus Complex

    PubMed Central

    Ramirez, Oscar; Illera, Juan Carlos; Rando, Juan Carlos; Gonzalez-Solis, Jacob; Alcover, Josep Antoni; Lalueza-Fox, Carles

    2010-01-01

    Background The loss of species during the Holocene was, dramatically more important on islands than on continents. Seabirds from islands are very vulnerable to human-induced alterations such as habitat destruction, hunting and exotic predators. For example, in the genus Puffinus (family Procellariidae) the extinction of at least five species has been recorded during the Holocene, two of them coming from the Canary Islands. Methodology/Principal Findings We used bones of the two extinct Canary shearwaters (P. olsoni and P. holeae) to obtain genetic data, for use in providing insights into the differentiation process within the genus Puffinus. Although mitochondrial DNA (mtDNA) cytochrome b sequences were successfully retrieved from four Holocene specimens of the extinct Lava shearwater (P. olsoni) from Fuerteventura (Canary Islands), the P. holeae specimens yielded no DNA. Only one haplotype was detected in P. olsoni, suggesting a low genetic diversity within this species. Conclusions The phylogenetic analyses based on the DNA data reveal that: (i) the “Puffinus puffinus complex”, an assemblage of species defined using osteological characteristics (P. puffinus, P. olsoni, P. mauretanicus, P. yelkouan and probably P. holeae), shows unresolved phylogenetic relationships; (ii) despite the differences in body size and proportions, P. olsoni and the extant P. puffinus are sister species. Several hypotheses can be considered to explain the incipient differentiation between P. olsoni and P. puffinus. PMID:21209838

  3. De novo transcriptome assembly and analysis of differentially expressed genes of two barley genotypes reveal root-zone-specific responses to salt exposure

    PubMed Central

    Hill, Camilla Beate; Cassin, Andrew; Keeble-Gagnère, Gabriel; Doblin, Monika S.; Bacic, Antony; Roessner, Ute

    2016-01-01

    Plant roots are the first organs sensing and responding to salinity stress, manifested differentially between different root types, and also at the individual tissue and cellular level. High genetic diversity and the current lack of an assembled map-based sequence of the barley genome severely limit barley research potential. We used over 580 and 600 million paired-end reads, respectively, to create two de novo assemblies of a barley landrace (Sahara) and a malting cultivar (Clipper) with known contrasting responses to salinity. Generalized linear models were used to statistically access spatial, treatment-related, and genotype-specific responses. This revealed a spatial gene expression gradient along the barley root, with more differentially expressed transcripts detected between different root zones than between treatments. The root transcriptome also showed a gradual transition from transcripts related to sugar-mediated signaling at the root meristematic zone to those involved in cell wall metabolism in the elongation zone, and defense response-related pathways toward the maturation zone, with significant differences between the two genotypes. The availability of these additional transcriptome reference sets will serve as a valuable resource to the cereal research community, and may identify valuable traits to assist in breeding programmes. PMID:27527578

  4. Comparative Proteomic Analysis of saccharopolyspora spinosa SP06081 and PR2 strains reveals the differentially expressed proteins correlated with the increase of spinosad yield

    PubMed Central

    2011-01-01

    Background Saccharopolyspora spinosa produces the environment-friendly biopesticide spinosad, a mixture of two polyketide-derived macrolide active ingredients called spinosyns A and D. Therefore considerable interest is in the improvement of spinosad production because of its low yield in wild-type S. spinosa. Recently, a spinosad-hyperproducing PR2 strain with stable heredity was obtained from protoplast regeneration of the wild-type S. spinosa SP06081 strain. A comparative proteomic analysis was performed on the two strains during the first rapid growth phase (RG1) in seed medium (SM) by using label-free quantitative proteomics to investigate the underlying mechanism leading to the enhancement of spinosad yield. Results In total, 224 proteins from the SP06081 strain and 204 proteins from the PR2 strain were unambiguously identified by liquid chromatography-tandem mass spectrometry analysis, sharing 140 proteins. A total of 12 proteins directly related to spinosad biosynthesis were identified from the two strains in RG1. Comparative analysis of the shared proteins revealed that approximately 31% of them changed their abundance significantly and fell in all of the functional groups, such as tricarboxylic acid cycles, glycolysis, biosynthetic processes, catabolic processes, transcription, translation, oxidation and reduction. Several key enzymes involved in the synthesis of primary metabolic intermediates used as precursors for spinosad production, energy supply, polyketide chain assembly, deoxysugar methylation, and antioxidative stress were differentially expressed in the same pattern of facilitating spinosad production by the PR2 strain. Real-time reverse transcriptase polymerase chain reaction analysis revealed that four of five selected genes showed a positive correlation between changes at the translational and transcriptional expression level, which further confirmed the proteomic analysis. Conclusions The present study is the first comprehensive and

  5. N-glycosylation Profiling of Colorectal Cancer Cell Lines Reveals Association of Fucosylation with Differentiation and Caudal Type Homebox 1 (CDX1)/Villin mRNA Expression.

    PubMed

    Holst, Stephanie; Deuss, Anna J M; van Pelt, Gabi W; van Vliet, Sandra J; Garcia-Vallejo, Juan J; Koeleman, Carolien A M; Deelder, André M; Mesker, Wilma E; Tollenaar, Rob A; Rombouts, Yoann; Wuhrer, Manfred

    2016-01-01

    Various cancers such as colorectal cancer (CRC) are associated with alterations in protein glycosylation. CRC cell lines are frequently used to study these (glyco)biological changes and their mechanisms. However, differences between CRC cell lines with regard to their glycosylation have hitherto been largely neglected. Here, we comprehensively characterized the N-glycan profiles of 25 different CRC cell lines, derived from primary tumors and metastatic sites, in order to investigate their potential as glycobiological tumor model systems and to reveal glycans associated with cell line phenotypes. We applied an optimized, high-throughput membrane-based enzymatic glycan release for small sample amounts. Released glycans were derivatized to stabilize and differentiate between α2,3- and α2,6-linked N-acetylneuraminic acids, followed by N-glycosylation analysis by MALDI-TOF(/TOF)-MS. Our results showed pronounced differences between the N-glycosylation patterns of CRC cell lines. CRC cell line profiles differed from tissue-derived N-glycan profiles with regard to their high-mannose N-glycan content but showed a large overlap for complex type N-glycans, supporting their use as a glycobiological cancer model system. Importantly, we could show that the high-mannose N-glycans did not only occur as intracellular precursors but were also present at the cell surface. The obtained CRC cell line N-glycan features were not clearly correlated with mRNA expression levels of glycosyltransferases, demonstrating the usefulness of performing the structural analysis of glycans. Finally, correlation of CRC cell line glycosylation features with cancer cell markers and phenotypes revealed an association between highly fucosylated glycans and CDX1 and/or villin mRNA expression that both correlate with cell differentiation. Together, our findings provide new insights into CRC-associated glycan changes and setting the basis for more in-depth experiments on glycan function and regulation

  6. Evidence for an anterior-posterior differentiation in the human hippocampal formation revealed by meta-analytic parcellation of fMRI coordinate maps: Focus on the subiculum

    PubMed Central

    Chase, Henry W.; Clos, Mareike; Dibble, Sofia; Fox, Peter; Grace, Anthony A.; Phillips, Mary L.; Eickhoff, Simon B.

    2015-01-01

    Previous studies, predominantly in experimental animals, have suggested the presence of a differentiation of function across the hippocampal formation. In rodents, ventral regions are thought to be involved in emotional behavior while dorsal regions mediate cognitive or spatial processes. Using a combination of modeling the co-occurrence of significant activations across thousands of neuroimaging experiments and subsequent data-driven clustering of these data we were able to provide evidence of distinct subregions within a region corresponding to the human subiculum, a critical hub within the hippocampal formation. This connectivity-based model consists of a bilateral anterior region, as well as separate posterior and intermediate regions on each hemisphere. Functional connectivity assessed both by meta-analytic and resting fMRI approaches revealed that more anterior regions were more strongly connected to the default mode network, and more posterior regions were more strongly connected to ‘task positive’ regions. In addition, our analysis revealed that the anterior subregion was functionally connected to the ventral striatum, midbrain and amygdala, a circuit that is central to models of stress and motivated behavior. Analysis of a behavioral taxonomy provided evidence for a role for each subregion in mnemonic processing, as well as implication of the anterior subregion in emotional and visual processing and the right posterior subregion in reward processing. These findings lend support to models which posit anterior-posterior differentiation of function within the human hippocampal formation and complement other early steps toward a comparative (cross-species) model of the region. PMID:25776219

  7. N-glycosylation Profiling of Colorectal Cancer Cell Lines Reveals Association of Fucosylation with Differentiation and Caudal Type Homebox 1 (CDX1)/Villin mRNA Expression*

    PubMed Central

    Holst, Stephanie; Deuss, Anna J. M.; van Pelt, Gabi W.; van Vliet, Sandra J.; Garcia-Vallejo, Juan J.; Koeleman, Carolien A. M.; Deelder, André M.; Mesker, Wilma E.; Tollenaar, Rob A.; Rombouts, Yoann; Wuhrer, Manfred

    2016-01-01

    Various cancers such as colorectal cancer (CRC) are associated with alterations in protein glycosylation. CRC cell lines are frequently used to study these (glyco)biological changes and their mechanisms. However, differences between CRC cell lines with regard to their glycosylation have hitherto been largely neglected. Here, we comprehensively characterized the N-glycan profiles of 25 different CRC cell lines, derived from primary tumors and metastatic sites, in order to investigate their potential as glycobiological tumor model systems and to reveal glycans associated with cell line phenotypes. We applied an optimized, high-throughput membrane-based enzymatic glycan release for small sample amounts. Released glycans were derivatized to stabilize and differentiate between α2,3- and α2,6-linked N-acetylneuraminic acids, followed by N-glycosylation analysis by MALDI-TOF(/TOF)-MS. Our results showed pronounced differences between the N-glycosylation patterns of CRC cell lines. CRC cell line profiles differed from tissue-derived N-glycan profiles with regard to their high-mannose N-glycan content but showed a large overlap for complex type N-glycans, supporting their use as a glycobiological cancer model system. Importantly, we could show that the high-mannose N-glycans did not only occur as intracellular precursors but were also present at the cell surface. The obtained CRC cell line N-glycan features were not clearly correlated with mRNA expression levels of glycosyltransferases, demonstrating the usefulness of performing the structural analysis of glycans. Finally, correlation of CRC cell line glycosylation features with cancer cell markers and phenotypes revealed an association between highly fucosylated glycans and CDX1 and/or villin mRNA expression that both correlate with cell differentiation. Together, our findings provide new insights into CRC-associated glycan changes and setting the basis for more in-depth experiments on glycan function and regulation

  8. Genome-wide mapping of Sox6 binding sites in skeletal muscle reveals both direct and indirect regulation of muscle terminal differentiation by Sox6

    PubMed Central

    2011-01-01

    Background Sox6 is a multi-faceted transcription factor involved in the terminal differentiation of many different cell types in vertebrates. It has been suggested that in mice as well as in zebrafish Sox6 plays a role in the terminal differentiation of skeletal muscle by suppressing transcription of slow fiber specific genes. In order to understand how Sox6 coordinately regulates the transcription of multiple fiber type specific genes during muscle development, we have performed ChIP-seq analyses to identify Sox6 target genes in mouse fetal myotubes and generated muscle-specific Sox6 knockout (KO) mice to determine the Sox6 null muscle phenotype in adult mice. Results We have identified 1,066 Sox6 binding sites using mouse fetal myotubes. The Sox6 binding sites were found to be associated with slow fiber-specific, cardiac, and embryonic isoform genes that are expressed in the sarcomere as well as transcription factor genes known to play roles in muscle development. The concurrently performed RNA polymerase II (Pol II) ChIP-seq analysis revealed that 84% of the Sox6 peak-associated genes exhibited little to no binding of Pol II, suggesting that the majority of the Sox6 target genes are transcriptionally inactive. These results indicate that Sox6 directly regulates terminal differentiation of muscle by affecting the expression of sarcomere protein genes as well as indirectly through influencing the expression of transcription factors relevant to muscle development. Gene expression profiling of Sox6 KO skeletal and cardiac muscle revealed a significant increase in the expression of the genes associated with Sox6 binding. In the absence of the Sox6 gene, there was dramatic upregulation of slow fiber-specific, cardiac, and embryonic isoform gene expression in Sox6 KO skeletal muscle and fetal isoform gene expression in Sox6 KO cardiac muscle, thus confirming the role Sox6 plays as a transcriptional suppressor in muscle development. Conclusions Our present data indicate

  9. A constitutive region is responsible for nuclear targeting of 4.1R: modulation by alternative sequences results in differential intracellular localization.

    PubMed

    Luque, C M; Correas, I

    2000-07-01

    Red blood cell protein 4.1, 4.1R, is an extreme variation on the theme of isoform multiplicity. The diverse 4.1R isoforms, mainly generated by alternative pre-mRNA splicing, are localized at different intracellular sites, including the nucleus. To characterize nonerythroid 4.1 proteins lacking the most upstream translation initiation site, analyze their intracellular localization and define specific domains involved in differential intracellular targeting of 4.1R, we cloned 4.1 cDNAs lacking that translation initiation site. Seven different 4.1R cDNAs were isolated. Four of these encoded 4.1R proteins localized predominantly to the nucleus and the other three localized to the cytoplasm. Three of the nuclear 4.1R isoforms did not contain the nuclear localization signal previously identified in the alternative exon 16. A comparative analysis of the exon composition of the naturally occurring 4.1R cDNAs cloned and of appropriate composite cDNA constructs, with the subcellular distribution of their respective products, demonstrated that a region encoded by constitutive exons, which is therefore common to all 4.1R isoforms and has been termed 'core region', had the capacity of localizing to the nucleus. This region was able to confer nuclear targeting to a cytosolic reporter. In protein 4.1R isoforms, the nuclear targeting of the core region is modulated by the expression of alternative exons. Thus, exon 5-encoded sequences eclipsed nuclear entry of the core region, resulting in 4.1R isoforms that predominantly distributed to the cytoplasm. Exon 5 was also able to confer cytoplasmic localization to a nuclear reporter. In protein 4.1R isoforms, when exons 5 and 16 were both expressed the nuclear targeting effect of exon 16 was dominant to the inhibitory effect observed by the expression of exon 5, yielding proteins that predominantly localized to the nucleus. Taken together, these results indicate that all 4.1R molecules contain a conserved region that is sufficient to

  10. Genome-specific differential gene expressions in resynthesized Brassica allotetraploids from pair-wise crosses of three cultivated diploids revealed by RNA-seq

    PubMed Central

    Zhang, Dawei; Pan, Qi; Cui, Cheng; Tan, Chen; Ge, Xianhong; Shao, Yujiao; Li, Zaiyun

    2015-01-01

    Polyploidy is popular for the speciation of angiosperms but the initial stage of allopolyploidization resulting from interspecific hybridization and genome duplication is associated with different extents of changes in genome structure and gene expressions. Herein, the transcriptomes detected by RNA-seq in resynthesized Brassica allotetraploids (Brassica juncea, AABB; B. napus, AACC; B. carinata, BBCC) from the pair-wise crosses of the same three diploids (B. rapa, AA; B. nigra, BB; B. oleracea, CC) were compared to reveal the patterns of gene expressions from progenitor genomes and the effects of different types of genome combinations and cytoplasm, upon the genome merger and duplication. From transcriptomic analyses for leaves and silique walls, extensive expression alterations were revealed in these resynthesized allotetraploids relative to their diploid progenitors, as well as during the transition from vegetative to reproductive development, for differential and transgressive gene expressions were variable in numbers and functions. Genes involved in glucosinolates and DNA methylation were transgressively up-regulated among most samples, suggesting that gene expression regulation was immediately established after allopolyploidization. The expression of ribosomal protein genes was also tissue-specific and showed a similar expression hierarchy of rRNA genes. The balance between the co-up and co-down regulation was observed between reciprocal B. napus with different types of the cytoplasm. Our results suggested that gene expression changes occurred after initial genome merger and such profound alterations might enhance the growth vigor and adaptability of Brassica allotetraploids. PMID:26583027

  11. Genome-specific differential gene expressions in resynthesized Brassica allotetraploids from pair-wise crosses of three cultivated diploids revealed by RNA-seq.

    PubMed

    Zhang, Dawei; Pan, Qi; Cui, Cheng; Tan, Chen; Ge, Xianhong; Shao, Yujiao; Li, Zaiyun

    2015-01-01

    Polyploidy is popular for the speciation of angiosperms but the initial stage of allopolyploidization resulting from interspecific hybridization and genome duplication is associated with different extents of changes in genome structure and gene expressions. Herein, the transcriptomes detected by RNA-seq in resynthesized Brassica allotetraploids (Brassica juncea, AABB; B. napus, AACC; B. carinata, BBCC) from the pair-wise crosses of the same three diploids (B. rapa, AA; B. nigra, BB; B. oleracea, CC) were compared to reveal the patterns of gene expressions from progenitor genomes and the effects of different types of genome combinations and cytoplasm, upon the genome merger and duplication. From transcriptomic analyses for leaves and silique walls, extensive expression alterations were revealed in these resynthesized allotetraploids relative to their diploid progenitors, as well as during the transition from vegetative to reproductive development, for differential and transgressive gene expressions were variable in numbers and functions. Genes involved in glucosinolates and DNA methylation were transgressively up-regulated among most samples, suggesting that gene expression regulation was immediately established after allopolyploidization. The expression of ribosomal protein genes was also tissue-specific and showed a similar expression hierarchy of rRNA genes. The balance between the co-up and co-down regulation was observed between reciprocal B. napus with different types of the cytoplasm. Our results suggested that gene expression changes occurred after initial genome merger and such profound alterations might enhance the growth vigor and adaptability of Brassica allotetraploids. PMID:26583027

  12. Comparative Transcriptomics in East African Cichlids Reveals Sex- and Species-Specific Expression and New Candidates for Sex Differentiation in Fishes

    PubMed Central

    Böhne, Astrid; Sengstag, Thierry; Salzburger, Walter

    2014-01-01

    Males and females of the same species differ largely in gene expression, which accounts for most of the morphological and physiological differences and sex-specific phenotypes. Here, we analyzed sex-specific gene expression in the brain and the gonads of cichlid fishes from Lake Tanganyika belonging to four different lineages, so-called tribes (Eretmodini, Ectodini, Haplochromini, and Lamprologini), using the outgroup Nile tilapia (Oreochromis niloticus) as reference. The comparison between male and female brains revealed few differences between the sexes, consistent in all investigated species. The gonads, on the other hand, showed a large fraction of differentially expressed transcripts with the majority of them showing the same direction of expression in all four species. All here-studied cichlids, especially the three investigated mouth-breeding species, showed a trend toward more male- than female-biased transcripts. Transcripts, which were female-biased in expression in all four species, were overrepresented on linkage group (LG)1 in the reference genome and common male-biased transcripts showed accumulation on LG23, the presumable sex chromosomes of the Nile tilapia. Sex-specific transcripts contained candidate genes for sex determination and differentiation in fishes, especially members of the transforming growth factor-β-superfamily and the Wnt-pathway and also prominent members of the sox-, dm-domain-, and high mobility group-box families. We further confirmed our previous finding on species/lineage-specific gene expression shifts in the sex steroid pathway, including synthesizing enzymes as the aromatase cyp19a1 and estrogen and androgen receptors. PMID:25364805

  13. Differential expression and interaction specificity of the heterotrimeric G-protein family in Brassica nigra reveal their developmental- and condition-specific roles.

    PubMed

    Kumar, Roshan; Arya, Gulab C; Bisht, Naveen C

    2014-11-01

    Heterotrimeric G-proteins, comprised of α, β and γ subunits, are important signal transducers across phyla. The G-proteins are well characterized in the model plants Arabidopsis and rice, and their inventories are possible from a few other plant species; however, information about the roles played by G-proteins in regulating various growth and developmental traits particularly from polyploid crops is still awaited. In this study, we have isolated one Gα (BniB.Gα1), three Gβ (BniB.Gβ1-BniB.Gβ3) and four Gγ (BniB.Gγ1-BniB.Gγ4) coding sequences from the paleopolyploid Brassica nigra, a major condiment crop of the Brassicaceae family. Sequence and phylogenetic analysis revealed that whole-genome triplication events in the Brassica lineage had proportionally increased the inventory of the Gβ subunit, but not of the Gα and Gγ subunits in B. nigra. Real-time quantitative reverse transcription-PCR (qRT-PCR) analysis showed that members of the G-protein subunit genes have distinct temporal and spatial expression patterns and were differentially altered in response to various stress and phytohormone treatments, thereby suggesting differential transcriptional regulation of G-protein genes in B. nigra. Interestingly, specific members of G-protein subunits were co-expressed across plant developmental stages, and in response to different elicitor treatments. Yeast-based interaction screens further predicted that the B. nigra G-protein subunits interacted in most of the possible combinations, although showing a high degree of interaction specificity between different G-protein subunits. Our data on physical interactions coupled with the co-expression pattern of the multiple G-protein subunit genes suggested that tissue- and condition-specific functional combinations of Gαβγ heterotrimers may exist in paleopolyploid B. nigra, to control diverse growth and development processes. PMID:25231958

  14. Integration of High-Resolution Physical and Genetic Map Reveals Differential Recombination Frequency between Chromosomes and the Genome Assembling Quality in Cucumber

    PubMed Central

    Cheng, Chunyan; Zhang, Zhonghua; Li, Ji; Huang, Sanwen; Chen, Jinfeng

    2013-01-01

    Cucumber is an important model crop and the first species sequenced in Cucurbitaceae family. Compared to the fast increasing genetic and genomics resources, the molecular cytogenetic researches in cucumber are still very limited, which results in directly the shortage of relation between plenty of physical sequences or genetic data and chromosome structure. We mapped twenty-three fosmids anchored by SSR markers from LG-3, the longest linkage group, and LG-4, the shortest linkage group on pachytene chromosomes 3 and 4, using uorescence in situ hybridization (FISH). Integrated molecular cytogenetic maps of chromosomes 3 and 4 were constructed. Except for three SSR markers located on heterochromatin region, the cytological order of markers was concordant with those on the linkage maps. Distinct structural differences between chromosomes 3 and 4 were revealed by the high resolution pachytene chromosomes. The extreme difference of genetic length between LG-3 and LG-4 was mainly attributed to the difference of overall recombination frequency. The significant differentiation of heterochromatin contents in chromosomes 3 and 4 might have a direct correlation with recombination frequency. Meanwhile, the uneven distribution of recombination frequency along chromosome 4 was observed, and recombination frequency of the long arm was nearly 3.5 times higher than that of the short arm. The severe suppression of recombination was exhibited in centromeric and heterochromatin domains of chromosome 4. Whereas a close correlation between the gene density and recombination frequency was observed in chromosome 4, no significant correlation was observed between them along chromosome 3. The comparison between cytogenetic and sequence maps revealed a large gap on the pericentromeric heterochromatin region of sequence map of chromosome 4. These results showed that integrated molecular cytogenetic maps can provide important information for the study of genetic and genomics in cucumber. PMID

  15. Strong differential regulation of serum and mucosal IgA responses as revealed in CD28-deficient mice using cholera toxin adjuvant.

    PubMed

    Gärdby, Eva; Wrammert, Jens; Schön, Karin; Ekman, Lena; Leanderson, Tomas; Lycke, Nils

    2003-01-01

    In this study, we show that costimulation required for mucosal IgA responses is strikingly different from that needed for systemic responses, including serum IgA. Following oral immunization with cholera toxin (CT) adjuvant we found that whereas CTLA4-H1 transgenic mice largely failed to respond, CD28-/- mice developed near normal gut mucosal IgA responses but poor serum Ab responses. The local IgA response was functional in that strong antitoxic protection developed in CT-immunized CD28-/- mice. This was in spite of the fact that no germinal centers (GC) were observed in the Peyer's patches, spleen, or other peripheral lymph nodes. Moreover, significant somatic hypermutation was found in isolated IgA plasma cells from gut lamina propria of CD28-/- mice. Thus, differentiation to functional gut mucosal IgA responses against T cell-dependent Ags does not require signaling through CD28 and can be independent of GC formations and isotype-switching in Peyer's patches. By contrast, serum IgA responses, similar to IgG-responses, are dependent on GC and CD28. However, both local and systemic responses are impaired in CTLA4-Hgamma1 transgenic mice, indicating that mucosal IgA responses are dependent on the B7-family ligands, but require signaling via CTLA4 or more likely a third related receptor. Therefore, T-B cell interactions leading to mucosal as opposed to serum IgA responses are uniquely regulated and appear to represent separate events. Although CT is known to strongly up-regulate B7-molecules, we have demonstrated that it acts as a potent mucosal adjuvant in the absence of CD28, suggesting that alternative costimulatory pathways are involved. PMID:12496383

  16. The allele frequency spectrum in genome-wide human variation data reveals signals of differential demographic history in three large world populations.

    PubMed Central

    Marth, Gabor T; Czabarka, Eva; Murvai, Janos; Sherry, Stephen T

    2004-01-01

    We have studied a genome-wide set of single-nucleotide polymorphism (SNP) allele frequency measures for African-American, East Asian, and European-American samples. For this analysis we derived a simple, closed mathematical formulation for the spectrum of expected allele frequencies when the sampled populations have experienced nonstationary demographic histories. The direct calculation generates the spectrum orders of magnitude faster than coalescent simulations do and allows us to generate spectra for a large number of alternative histories on a multidimensional parameter grid. Model-fitting experiments using this grid reveal significant population-specific differences among the demographic histories that best describe the observed allele frequency spectra. European and Asian spectra show a bottleneck-shaped history: a reduction of effective population size in the past followed by a recent phase of size recovery. In contrast, the African-American spectrum shows a history of moderate but uninterrupted population expansion. These differences are expected to have profound consequences for the design of medical association studies. The analytical methods developed for this study, i.e., a closed mathematical formulation for the allele frequency spectrum, correcting the ascertainment bias introduced by shallow SNP sampling, and dealing with variable sample sizes provide a general framework for the analysis of public variation data. PMID:15020430

  17. High-throughput sequencing of partially edited trypanosome mRNAs reveals barriers to editing progression and evidence for alternative editing.

    PubMed

    Simpson, Rachel M; Bruno, Andrew E; Bard, Jonathan E; Buck, Michael J; Read, Laurie K

    2016-05-01

    Uridine insertion/deletion RNA editing in kinetoplastids entails the addition and deletion of uridine residues throughout the length of mitochondrial transcripts to generate translatable mRNAs. This complex process requires the coordinated use of several multiprotein complexes as well as the sequential use of noncoding template RNAs called guide RNAs. The majority of steady-state mitochondrial mRNAs are partially edited and often contain regions of mis-editing, termed junctions, whose role is unclear. Here, we report a novel method for sequencing entire populations of pre-edited partially edited, and fully edited RNAs and analyzing editing characteristics across populations using a new bioinformatics tool, the Trypanosome RNA Editing Alignment Tool (TREAT). Using TREAT, we examined populations of two transcripts, RPS12 and ND7-5', in wild-typeTrypanosoma brucei We provide evidence that the majority of partially edited sequences contain junctions, that intrinsic pause sites arise during the progression of editing, and that the mechanisms that mediate pausing in the generation of canonical fully edited sequences are distinct from those that mediate the ends of junction regions. Furthermore, we identify alternatively edited sequences that constitute plausible alternative open reading frames and identify substantial variability in the 5' UTRs of both canonical and alternatively edited sequences. This work is the first to use high-throughput sequencing to examine full-length sequences of whole populations of partially edited transcripts. Our method is highly applicable to current questions in the RNA editing field, including defining mechanisms of action for editing factors and identifying potential alternatively edited sequences. PMID:26908922

  18. 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

  19. Next-Generation Sequencing Analysis Reveals Differential Expression Profiles of MiRNA-mRNA Target Pairs in KSHV-Infected Cells

    PubMed Central

    Viollet, Coralie; Davis, David A.; Reczko, Martin; Ziegelbauer, Joseph M.; Pezzella, Francesco

    2015-01-01

    Kaposi’s sarcoma associated herpesvirus (KSHV) causes several tumors, including primary effusion lymphoma (PEL) and Kaposi’s sarcoma (KS). Cellular and viral microRNAs (miRNAs) have been shown to play important roles in regulating gene expression. A better knowledge of the miRNA-mediated pathways affected by KSHV infection is therefore important for understanding viral infection and tumor pathogenesis. In this study, we used deep sequencing to analyze miRNA and cellular mRNA expression in a cell line with latent KSHV infection (SLKK) as compared to the uninfected SLK line. This approach revealed 153 differentially expressed human miRNAs, eight of which were independently confirmed by qRT-PCR. KSHV infection led to the dysregulation of ~15% of the human miRNA pool and most of these cellular miRNAs were down-regulated, including nearly all members of the 14q32 miRNA cluster, a genomic locus linked to cancer and that is deleted in a number of PEL cell lines. Furthermore, we identified 48 miRNAs that were associated with a total of 1,117 predicted or experimentally validated target mRNAs; of these mRNAs, a majority (73%) were inversely correlated to expression changes of their respective miRNAs, suggesting miRNA-mediated silencing mechanisms were involved in a number of these alterations. Several dysregulated miRNA-mRNA pairs may facilitate KSHV infection or tumor formation, such as up-regulated miR-708-5p, associated with a decrease in pro-apoptotic caspase-2 and leukemia inhibitory factor LIF, or down-regulated miR-409-5p, associated with an increase in the p53-inhibitor MDM2. Transfection of miRNA mimics provided further evidence that changes in miRNAs are driving some observed mRNA changes. Using filtered datasets, we also identified several canonical pathways that were significantly enriched in differentially expressed miRNA-mRNA pairs, such as the epithelial-to-mesenchymal transition and the interleukin-8 signaling pathways. Overall, our data provide a more

  20. Fetal heart rate variability reveals differential dynamics in the intrauterine development of the sympathetic and parasympathetic branches of the autonomic nervous system.

    PubMed

    Schneider, U; Schleussner, E; Fiedler, A; Jaekel, S; Liehr, M; Haueisen, J; Hoyer, D

    2009-02-01

    The aim of this study was to investigate the hypothesis that fetal beat-to-beat heart rate variability (fHRV) displays the different time scales of sympatho-vagal development prior to and after 32 weeks of gestation (wks GA). Ninety-two magnetocardiograms of singletons with normal courses of pregnancy between 24 + 1 and 41 + 6 wks GA were studied. Heart rate patterns were either quiet/non-accelerative (fHRP I) or active/accelerative (fHRP II) and recording quality sufficient for fHRV. The sample was divided into the GA groups <32 wks GA/>32 wks GA. Linear parameters of fHRV were calculated: mean heart rate (mHR), SDNN and RMSSD of normal-to-normal interbeat intervals, power in the low (0.04-0.15 Hz) and high frequency range (0.15-0.4 Hz) and the ratios SDNN/RMSSD and LF/HF as markers for sympatho-vagal balance. fHRP I is characterized by decreasing SDNN/RMSSD, LF/HF and mHR. The decrease is more pronounced <32 wks GA. Beyond that GA SDNN/RMSSD is predominantly determined by RMSSD during fHRP I and by SDNN during fHRP II. In contrast to fHRP I, during fHRP II, mHR is positively correlated to SDNN/RMSSD instead of SDNN >32 wks GA. LF/HF increases in fHRP II during the first half of the third trimester. Non-accelerative fHRP are indicative of parasympathetic dominance >32 wks GA. In contrast, the sympathetic accentuation during accelerative fHRP is displayed in the interrelations between mHR, SDNN and SDNN/RMSSD. Prior to 32 wks GA, fHRV reveals the increasing activity of the respective branches of the autonomic nervous system differentiating the types of fHRP. PMID:19179746

  1. SSU rDNA sequence diversity and seasonally differentiated distribution of nanoplanktonic ciliates in neritic Bohai and Yellow Seas as revealed by T-RFLP.

    PubMed

    Dong, Jun; Shi, Fei; Li, Han; Zhang, Xiaoming; Hu, Xiaozhong; Gong, Jun

    2014-01-01

    Nanociliates have been frequently found to be important players in the marine microbial loop, however, little is known about their diversity and distribution in coastal ecosystems. We investigated the molecular diversity and distribution patterns of nanoplanktonic oligotrich and choreotrich (OC) ciliates in surface water of three neritic basins of northern China, the South Yellow Sea (SYS), North Yellow Sea (NYS), and Bohai Sea (BS) in June and November 2011. SSU rRNA gene clone libraries generated from three summertime samples (sites B38, B4 and H8) were analyzed and revealed a large novel ribotype diversity, of which many were low-abundant phylotypes belonging to the subclass Oligotrichia, but divergent from described morphospecies. Based on the data of terminal-restriction fragment length polymorphism (T-RFLP) analysis of all 35 samples, we found that the T-RF richness was generally higher in the SYS than in the BS, and negatively correlated with the molar ratio of P to Si. Overall, multidimensional scaling and permutational multivariate analysis of variance of the community turnover demonstrated a distinct seasonal pattern but no basin-to-basin differentiation across all samples. Nevertheless, significant community differences among basins were recognized in the winter dataset. Mantel tests showed that the environmental factors, P:Si ratio, water temperature and concentration of dissolved oxygen (DO), determined the community across all samples. However, both biogeographic distance and environment shaped the community in winter, with DO being the most important physicochemical factor. Our results indicate that the stoichiometric ratio of P:Si is a key factor, through which the phytoplankton community may be shaped, resulting in a cascade effect on the diversity and community composition of OC nanociliates in the N-rich, Si-limited coastal surface waters, and that the Yellow Sea Warm Current drives the nanociliate community, and possibly the microbial food webs

  2. Early events in plastid protein degradation in stay-green Arabidopsis reveal differential regulation beyond the retention of LHCII and chlorophyll.

    PubMed

    Grassl, Julia; Pružinská, Adriana; Hörtensteiner, Stefan; Taylor, Nicolas L; Millar, A Harvey

    2012-11-01

    An individually darkened leaf model was used to study protein changes in the Arabidopsis mutant stay-green1 (sgr1) to partially mimic the process of leaf covering senescence that occurs naturally in the shaded rosettes of Arabidopsis plants. Utilizing this controlled and predictable induced senescence model has allowed the direct comparison of sgr1 with Col-0 during the developmental period preceding the retention of chlorophyll and light harvesting complex II (LHCII) in sgr1 and the induction of senescence in Col-0. Quantitative proteomic analysis of soluble leaf proteins from sgr1 and Col-0 before the initiation of senescence has revealed a range of differences in plastid soluble protein abundance in sgr1 when compared to Col-0. Changes were also observed in membrane located machinery for photosystem II (PSII), in Calvin cycle components, proteins involved in redox control of the stromal compartment and ammonia assimilation that differentiated sgr1 during the early stages of the senescence process. The changes in PSII abundance were accompanied with a lower capacity of photosynthetic CO(2) assimilation in sgr1 than Col-0 after return of plants to lighted conditions following 3 and 5 days of darkness. A light-harvesting chlorophyll-a/b binding protein (LHCB2) was retained during the later stages of senescence in sgr1 but this was accompanied by an enhanced loss of oxygen evolving complex (OEC) subunits from PSII, which was confirmed by Western blotting, and an enhanced stability of PSII repair proteins in sgr1, compared to Col-0. Together these data provide insights into the significant differences in the steady-state proteome in sgr1 and its response to senescence, showing this cosmetic stay-green mutant is in fact significantly different to wild-type plants both before and during leaf senescence. PMID:23025280

  3. cDNA-AFLP analysis reveals differential gene expression in incompatible interaction between infected non-heading Chinese cabbage and Hyaloperonospora parasitica.

    PubMed

    Xiao, Dong; Liu, Shi-Tuo; Wei, Yan-Ping; Zhou, Dao-Yun; Hou, Xi-Lin; Li, Ying; Hu, Chun-Mei

    2016-01-01

    Non-heading Chinese cabbage (Brassica rapa ssp. chinensis) is one of the main green leafy vegetables in the world, especially in China, with significant economic value. Hyaloperonospora parasitica is a fungal pathogen responsible for causing downy mildew disease in Chinese cabbage, which greatly affects its production. The objective of this study was to identify transcriptionally regulated genes during incompatible interactions between non-heading Chinese cabbage and H. parasitica using complementary DNA-amplified fragment length polymorphism (cDNA-AFLP). We obtained 129 reliable differential transcript-derived fragments (TDFs) in a resistant line 'Suzhou Qing'. Among them, 121 upregulated TDFs displayed an expression peak at 24-48 h post inoculation (h.p.i.). Fifteen genes were further selected for validation of cDNA-AFLP expression patterns using quantitative reverse transcription PCR. Results confirmed the altered expression patterns of 13 genes (86.7%) revealed by the cDNA-AFLP. We identified four TDFs related to fungal resistance among the 15 TDFs. Furthermore, comparative analysis of four TDFs between resistant line 'Suzhou Qing' and susceptible line 'Aijiao Huang' showed that transcript levels of TDF14 (BcLIK1_A01) peaked at 48 h.p.i. and 25.1-fold increased in the resistant line compared with the susceptible line. Similarly, transcript levels of the other three genes, TDF42 (BcCAT3_A07), TDF75 (BcAAE3_A06) and TDF88 (BcAMT2_A05) peaked at 24, 48 and 24 h.p.i. with 25.1-, 100- and 15.8-fold increases, respectively. The results suggested that the resistance genes tended to transcribe at higher levels in the resistance line than in the susceptible line, which may provide resistance against pathogen infections. The present study might facilitate elucidating the molecular basis of the infection process and identifying candidate genes for resistance improvement of susceptible cultivars. PMID:27602230

  4. Genetic differentiation in the soil-feeding termite Cubitermes sp. affinis subarquatus: occurrence of cryptic species revealed by nuclear and mitochondrial markers

    PubMed Central

    Roy, Virginie; Demanche, Christine; Livet, Alexandre; Harry, Myriam

    2006-01-01

    Background Soil-feeding termites are particularly interesting models for studying the effects of fragmentation, a natural or anthropic phenomenon described as promoting genetic differentiation. However, studying the link between fragmentation and genetics requires a method for identifying species unambiguously, especially when morphological diagnostic characters are lacking. In humivorous termites, which contribute to the fertility of tropical soils, molecular taxonomy and phylogenetic relationships are rarely studied, though mitochondrial and nuclear molecular markers are widely used in studies of pest termites. Here, we attempt to clarify the taxonomy of soil-feeding colonies collected throughout the naturally fragmented Lopé Reserve area (Gabon) and morphologically affiliated to Cubitermes sp. affinis subarquatus. The mitochondrial gene of cytochrome oxidase II (COII), the second nuclear rDNA internal transcribed spacer (ITS2) and five microsatellites were analyzed in 19 colonies. Results Bayesian Inference, Maximum Likelihood and Maximum Parsimony phylogenetic analyses, which were applied to the COII and ITS2 sequences, and Neighbor-Joining reconstructions, applied to the microsatellite data, reveal four major lineages in the Cubitermes sp. affinis subarquatus colonies. The concordant genealogical pattern of these unlinked markers strongly supports the existence of four cryptic species. Three are sympatric in the Reserve and are probably able to disperse within a mosaic of forests of variable ages and savannahs. One is limited to a very restricted gallery forest patch located in the North, outside the Reserve. Conclusion Our survey highlights the value of combined mitochondrial and nuclear markers for exploring unknown groups such as soil-feeding termites, and their relevance for resolving the taxonomy of organisms with ambiguous morphological diagnostic characters. PMID:17123444

  5. Identification and Comparative Analysis of Differential Gene Expression in Soybean Leaf Tissue under Drought and Flooding Stress Revealed by RNA-Seq.

    PubMed

    Chen, Wei; Yao, Qiuming; Patil, Gunvant B; Agarwal, Gaurav; Deshmukh, Rupesh K; Lin, Li; Wang, Biao; Wang, Yongqin; Prince, Silvas J; Song, Li; Xu, Dong; An, Yongqiang C; Valliyodan, Babu; Varshney, Rajeev K; Nguyen, Henry T

    2016-01-01

    Drought and flooding are two major causes of severe yield loss in soybean worldwide. A lack of knowledge of the molecular mechanisms involved in drought and flood stress has been a limiting factor for the effective management of soybeans; therefore, it is imperative to assess the expression of genes involved in response to flood and drought stress. In this study, differentially expressed genes (DEGs) under drought and flooding conditions were investigated using Illumina RNA-Seq transcriptome profiling. A total of 2724 and 3498 DEGs were identified under drought and flooding treatments, respectively. These genes comprise 289 Transcription Factors (TFs) representing Basic Helix-loop Helix (bHLH), Ethylene Response Factors (ERFs), myeloblastosis (MYB), No apical meristem (NAC), and WRKY amino acid motif (WRKY) type major families known to be involved in the mechanism of stress tolerance. The expression of photosynthesis and chlorophyll synthesis related genes were significantly reduced under both types of stresses, which limit the metabolic processes and thus help prolong survival under extreme conditions. However, cell wall synthesis related genes were up-regulated under drought stress and down-regulated under flooding stress. Transcript profiles involved in the starch and sugar metabolism pathways were also affected under both stress conditions. The changes in expression of genes involved in regulating the flux of cell wall precursors and starch/sugar content can serve as an adaptive mechanism for soybean survival under stress conditions. This study has revealed the involvement of TFs, transporters, and photosynthetic genes, and has also given a glimpse of hormonal cross talk under the extreme water regimes, which will aid as an important resource for soybean crop improvement. PMID:27486466

  6. Identification and Comparative Analysis of Differential Gene Expression in Soybean Leaf Tissue under Drought and Flooding Stress Revealed by RNA-Seq

    PubMed Central

    Chen, Wei; Yao, Qiuming; Patil, Gunvant B.; Agarwal, Gaurav; Deshmukh, Rupesh K.; Lin, Li; Wang, Biao; Wang, Yongqin; Prince, Silvas J.; Song, Li; Xu, Dong; An, Yongqiang C.; Valliyodan, Babu; Varshney, Rajeev K.; Nguyen, Henry T.

    2016-01-01

    Drought and flooding are two major causes of severe yield loss in soybean worldwide. A lack of knowledge of the molecular mechanisms involved in drought and flood stress has been a limiting factor for the effective management of soybeans; therefore, it is imperative to assess the expression of genes involved in response to flood and drought stress. In this study, differentially expressed genes (DEGs) under drought and flooding conditions were investigated using Illumina RNA-Seq transcriptome profiling. A total of 2724 and 3498 DEGs were identified under drought and flooding treatments, respectively. These genes comprise 289 Transcription Factors (TFs) representing Basic Helix-loop Helix (bHLH), Ethylene Response Factors (ERFs), myeloblastosis (MYB), No apical meristem (NAC), and WRKY amino acid motif (WRKY) type major families known to be involved in the mechanism of stress tolerance. The expression of photosynthesis and chlorophyll synthesis related genes were significantly reduced under both types of stresses, which limit the metabolic processes and thus help prolong survival under extreme conditions. However, cell wall synthesis related genes were up-regulated under drought stress and down-regulated under flooding stress. Transcript profiles involved in the starch and sugar metabolism pathways were also affected under both stress conditions. The changes in expression of genes involved in regulating the flux of cell wall precursors and starch/sugar content can serve as an adaptive mechanism for soybean survival under stress conditions. This study has revealed the involvement of TFs, transporters, and photosynthetic genes, and has also given a glimpse of hormonal cross talk under the extreme water regimes, which will aid as an important resource for soybean crop improvement. PMID:27486466

  7. Comparative proteomics reveals novel components at the plasma membrane of differentiated HepaRG cells and different distribution in hepatocyte- and biliary-like cells.

    PubMed

    Petrareanu, Catalina; Macovei, Alina; Sokolowska, Izabela; Woods, Alisa G; Lazar, Catalin; Radu, Gabriel L; Darie, Costel C; Branza-Nichita, Norica

    2013-01-01

    Hepatitis B virus (HBV) is a human pathogen causing severe liver disease and eventually death. Despite important progress in deciphering HBV internalization, the early virus-cell interactions leading to infection are not known. HepaRG is a human bipotent liver cell line bearing the unique ability to differentiate towards a mixture of hepatocyte- and biliary-like cells. In addition to expressing metabolic functions normally found in liver, differentiated HepaRG cells support HBV infection in vitro, thus resembling cultured primary hepatocytes more than other hepatoma cells. Therefore, extensive characterization of the plasma membrane proteome from HepaRG cells would allow the identification of new cellular factors potentially involved in infection. Here we analyzed the plasma membranes of non-differentiated and differentiated HepaRG cells using nanoliquid chromatography-tandem mass spectrometry to identify the differences between the proteomes and the changes that lead to differentiation of these cells. We followed up on differentially-regulated proteins in hepatocytes- and biliary-like cells, focusing on Cathepsins D and K, Cyclophilin A, Annexin 1/A1, PDI and PDI A4/ERp72. Major differences between the two proteomes were found, including differentially regulated proteins, protein-protein interactions and intracellular localizations following differentiation. The results advance our current understanding of HepaRG differentiation and the unique properties of these cells. PMID:23977166

  8. Single-target high-throughput transcription analyses reveal high levels of alternative splicing present in the FPPS/GGPPS from Plasmodium falciparum.

    PubMed

    Gabriel, Heloisa B; de Azevedo, Mauro F; Palmisano, Giuseppe; Wunderlich, Gerhard; Kimura, Emília A; Katzin, Alejandro M; Alves, João M P

    2015-01-01

    Malaria is a tropical disease with significant morbidity and mortality. A better understanding of the metabolism of its most important etiological agent, Plasmodium falciparum, is paramount to the development of better treatment and other mitigation measures. Farnesyldiphosphate synthase/geranylgeranyldiphosphate synthase (FPPS/GGPPS) is a key enzyme in the synthesis of isoprenic chains present in many essential structures. In P. falciparum, as well as a handful of other organisms, FPPS/GGPPS has been shown to be a bifunctional enzyme. By genetic tagging and microscopy, we observed a changing localization of FPPS/GGPPS in blood stage parasites. Given the great importance of alternative splicing and other transcriptional phenomena in gene regulation and the generation of protein diversity, we have investigated the processing of the FPPS/GGPPS transcript in P. falciparum by high-throughput sequencing methods in four time-points along the intraerythrocytic cycle of P. falciparum. We have identified levels of transcript diversity an order of magnitude higher than previously observed in this organism, as well as a few stage-specific splicing events. Our data suggest that alternative splicing in P. falciparum is an important feature for gene regulation and the generation of protein diversity. PMID:26688062

  9. Single-target high-throughput transcription analyses reveal high levels of alternative splicing present in the FPPS/GGPPS from Plasmodium falciparum

    PubMed Central

    Gabriel, Heloisa B.; de Azevedo, Mauro F.; Palmisano, Giuseppe; Wunderlich, Gerhard; Kimura, Emília A.; Katzin, Alejandro M.; Alves, João M. P.

    2015-01-01

    Malaria is a tropical disease with significant morbidity and mortality. A better understanding of the metabolism of its most important etiological agent, Plasmodium falciparum, is paramount to the development of better treatment and other mitigation measures. Farnesyldiphosphate synthase/geranylgeranyldiphosphate synthase (FPPS/GGPPS) is a key enzyme in the synthesis of isoprenic chains present in many essential structures. In P. falciparum, as well as a handful of other organisms, FPPS/GGPPS has been shown to be a bifunctional enzyme. By genetic tagging and microscopy, we observed a changing localization of FPPS/GGPPS in blood stage parasites. Given the great importance of alternative splicing and other transcriptional phenomena in gene regulation and the generation of protein diversity, we have investigated the processing of the FPPS/GGPPS transcript in P. falciparum by high-throughput sequencing methods in four time-points along the intraerythrocytic cycle of P. falciparum. We have identified levels of transcript diversity an order of magnitude higher than previously observed in this organism, as well as a few stage-specific splicing events. Our data suggest that alternative splicing in P. falciparum is an important feature for gene regulation and the generation of protein diversity. PMID:26688062

  10. Molecular cloning and functional expression of two monocyte chemoattractant protein 1 receptors reveals alternative splicing of the carboxyl-terminal tails.

    PubMed Central

    Charo, I F; Myers, S J; Herman, A; Franci, C; Connolly, A J; Coughlin, S R

    1994-01-01

    Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine family of cytokines that mediate leukocyte chemotaxis. The potent and specific activation of monocytes by MCP-1 may mediate the monocytic infiltration of tissues in atherosclerosis and other inflammatory diseases. We have isolated cDNAs that encode two MCP-1-specific receptors with alternatively spliced carboxyl tails. Expression of the receptors in Xenopus oocytes conferred robust mobilization of intracellular calcium in response to nanomolar concentrations of MCP-1 but not to related chemokines. The MCP-1 receptors are most closely related to the receptor for the chemokines macrophage inflammatory protein 1 alpha and RANTES (regulated on activation, normal T expressed and secreted). The identification of the MCP-1 receptor and cloning of two distinct isoforms provide powerful tools for understanding the specificity and signaling mechanisms of this important chemokine. Images PMID:8146186

  11. MicroRNA expression profiling of human bone marrow mesenchymal stem cells during osteogenic differentiation reveals Osterix regulation by miR-31.

    PubMed

    Baglìo, Serena Rubina; Devescovi, Valentina; Granchi, Donatella; Baldini, Nicola

    2013-09-15

    Osteogenesis is the result of a complex sequence of events that involve the differentiation of mesenchymal stem cells (MSC) into osteoblasts. MSCs are multipotent adult stem cells that can give rise to different cell types of the mesenchymal germ layer. The differentiation fate of MSCs depends on the microenvironmental signals received by these cells and is tightly regulated by multiple pathways that lead to the activation of specific transcription factors. Among the transcription factors involved in osteogenic differentiation Osterix (Sp7) plays a key role and has been shown to be fundamental for bone homeostasis. However, the molecular events governing the expression of this transcription factor are not fully understood. In this study we set out to investigate the changes in the microRNA (miRNA) expression that occur during the osteogenic differentiation of bone marrow-derived MSCs. To this purpose, we analyzed the miRNA expression profile of MSCs deriving from 3 donors during the differentiation and mineralization processes by microarray. 29 miRNAs were significantly and consistently modulated during the osteogenic differentiation and 5 during the mineralization process. Interestingly, most of the differentially expressed miRNAs have been reported to be implicated in stemness maintenance, differentiation and/or oncogenesis. Subsequently, we focused our attention on the regulation of Osterix by miRNAs and demonstrated that one of the miRNAs differentially modulated during osteogenic differentiation, miR-31, controls Osterix expression through association to the 3' untranslated region of this transcription factor. By analyzing miR-31 and Osterix expression levels we found an inverse miRNA-target expression trend during osteogenic differentiation and in osteosarcoma cell lines. Moreover, the inhibition of the microRNA activity led to an increase in the endogenous expression of Osterix. Our results define a miRNA signature characterizing the osteogenic

  12. Unabridged Analysis of Human Histone H3 by Differential Top-Down Mass Spectrometry Reveals Hypermethylated Proteoforms from MMSET/NSD2 Overexpression.

    PubMed

    Zheng, Yupeng; Fornelli, Luca; Compton, Philip D; Sharma, Seema; Canterbury, Jesse; Mullen, Christopher; Zabrouskov, Vlad; Fellers, Ryan T; Thomas, Paul M; Licht, Jonathan D; Senko, Michael W; Kelleher, Neil L

    2016-03-01

    Histones, and their modifications, are critical components of cellular programming and epigenetic inheritance. Recently, cancer genome sequencing has uncovered driver mutations in chromatin modifying enzymes spurring high interest how such mutations change histone modification patterns. Here, we applied Top-Down mass spectrometry for the characterization of combinatorial modifications (i.e. methylation and acetylation) on full length histone H3 from human cell lines derived from multiple myeloma patients with overexpression of the histone methyltransferase MMSET as the result of a t(4;14) chromosomal translocation. Using the latest in Orbitrap-based technology for clean isolation of isobaric proteoforms containing up to 10 methylations and/or up to two acetylations, we provide extensive characterization of histone H3.1 and H3.3 proteoforms. Differential analysis of modifications by electron-based dissociation recapitulated antagonistic crosstalk between K27 and K36 methylation in H3.1, validating that full-length histone H3 (15 kDa) can be analyzed with site-specific assignments for multiple modifications. It also revealed K36 methylation in H3.3 was affected less by the overexpression of MMSET because of its higher methylation levels in control cells. The co-occurrence of acetylation with a minimum of three methyl groups in H3K9 and H3K27 suggested a hierarchy in the addition of certain modifications. Comparative analysis showed that high levels of MMSET in the myeloma-like cells drove the formation of hypermethyled proteoforms containing H3K36me2 co-existent with the repressive marks H3K9me2/3 and H3K27me2/3. Unique histone proteoforms with such "trivalent hypermethylation" (K9me2/3-K27me2/3-K36me2) were not discovered when H3.1 peptides were analyzed by Bottom-Up. Such disease-correlated proteoforms could link tightly to aberrant transcription programs driving cellular proliferation, and their precise description demonstrates that Top-Down mass spectrometry can

  13. cDNA-AFLP analysis reveals differential gene expression in incompatible interaction between infected non-heading Chinese cabbage and Hyaloperonospora parasitica

    PubMed Central

    Xiao, Dong; Liu, Shi-Tuo; Wei, Yan-Ping; Zhou, Dao-Yun; Hou, Xi-Lin; Li, Ying; Hu, Chun-Mei

    2016-01-01

    Non-heading Chinese cabbage (Brassica rapa ssp. chinensis) is one of the main green leafy vegetables in the world, especially in China, with significant economic value. Hyaloperonospora parasitica is a fungal pathogen responsible for causing downy mildew disease in Chinese cabbage, which greatly affects its production. The objective of this study was to identify transcriptionally regulated genes during incompatible interactions between non-heading Chinese cabbage and H. parasitica using complementary DNA-amplified fragment length polymorphism (cDNA-AFLP). We obtained 129 reliable differential transcript-derived fragments (TDFs) in a resistant line ‘Suzhou Qing’. Among them, 121 upregulated TDFs displayed an expression peak at 24–48 h post inoculation (h.p.i.). Fifteen genes were further selected for validation of cDNA-AFLP expression patterns using quantitative reverse transcription PCR. Results confirmed the altered expression patterns of 13 genes (86.7%) revealed by the cDNA-AFLP. We identified four TDFs related to fungal resistance among the 15 TDFs. Furthermore, comparative analysis of four TDFs between resistant line ‘Suzhou Qing’ and susceptible line ‘Aijiao Huang’ showed that transcript levels of TDF14 (BcLIK1_A01) peaked at 48 h.p.i. and 25.1-fold increased in the resistant line compared with the susceptible line. Similarly, transcript levels of the other three genes, TDF42 (BcCAT3_A07), TDF75 (BcAAE3_A06) and TDF88 (BcAMT2_A05) peaked at 24, 48 and 24 h.p.i. with 25.1-, 100- and 15.8-fold increases, respectively. The results suggested that the resistance genes tended to transcribe at higher levels in the resistance line than in the susceptible line, which may provide resistance against pathogen infections. The present study might facilitate elucidating the molecular basis of the infection process and identifying candidate genes for resistance improvement of susceptible cultivars. PMID:27602230

  14. Analysis of rice ER-resident J-proteins reveals diversity and functional differentiation of the ER-resident Hsp70 system in plants

    PubMed Central

    Takaiwa, Fumio

    2013-01-01

    The heat shock protein 70 (Hsp70) chaperone system participates in protein folding and quality control of unfolded proteins. To examine the roles of co-chaperones in the rice Hsp70 chaperone system in the endoplasmic reticulum (ER), the functions of six ER-resident J-proteins (OsP58A, OsP58B, OsERdj2, OsERdj3A, OsERdj3B, and OsERdj7) in rice were investigated. The expression of OsP58B, OsERdj3A, and OsERdj3B was predominantly up-regulated in roots subjected to ER stress. This response was mediated by signalling through ATF6 orthologues such as OsbZIP39 and OsbZIP60, but not through the IRE1/OsbZIP50 pathway. A co-immunoprecipitation assay demonstrated that OsP58A, OsP58B, and OsERdj3B preferentially interact with the major OsBiP, OsBiP1, while OsERdj3A interacts preferentially with OsBiP5, suggesting that there are different affinities between OsBiPs and J-proteins. In the endosperm tissue, OsP58A, OsP58B, and OsERdj2 were mainly localized in the ER, whereas OsERdj2 was localized around the outer surfaces of ER-derived protein bodies (PB-Is). Furthermore, OsERdj3A was not expressed in wild-type seeds but was up-regulated in transgenic seeds accumulating human interleukin-7 (hIL-7). Since ERdj3A–green fluorescent protein (GFP) was also detected in vacuoles of callus cells under ER stress conditions, OsERdj3A is a bona fide vacuole-localized protein. OsP58A, OsP58B and OsERdj3A were differentially accumulated in transgenic plants expressing various recombinant proteins. These results reveal the functional diversity of the rice ER-resident Hsp70 system. PMID:24153418

  15. Alternative strategies to manipulate fibrocyte involvement in the fibrotic tissue response: pharmacokinetic inhibition and the feasibility of directed-adipogenic differentiation

    PubMed Central

    Baker, David W; Tsai, Yi-Ting; Weng, Hong; Tang, Liping

    2014-01-01

    Fibrocytes have previously been identified as important mediators in several inflammatory and fibrotic diseases. However, there is no effective treatment thus far to reduce fibrotic tissue responses without affecting wound healing reactions. Here we investigate two strategies to alleviate fibrocyte interactions at the biomaterial interface reducing collagen production and scar tissue formation. First, in an indirect approach, TGF-β inhibitor-SB431542 and IL-1β/TNF-α inhibitor SB203580 were locally released from scaffold implants to block their respective signaling pathways. We show that inhibition of IL-1β/TNF-α has no influence on overall fibrotic tissue reactions to the implants. However, the reduction of localized TGF-β significantly decreases the fibrocyte accumulation and myofibroblast activation while reducing the fibrotic tissue formation. Since fibrocytes can be differentiated into non-fibrotic cell types, such as adipocytes, we further sought a more direct approach to reduce fibrocyte responses by directing fibrocyte differentiation into adipocytes. Interestingly, by initiating fibrocyte-to-adipocyte differentiation through sustained differentiation cocktail release, we find that adipogentic differentiation forces incoming fibrocytes away from the traditional myofibroblast lineage leading to a substantial reduction in the collagen formation and fibrotic response. Our results support a novel and effective strategy to improve implant safety by reducing implant-associated fibrotic tissue reactions via directing non-fibrotic differentiation of fibrocytes. PMID:24657674

  16. Alternative strategies to manipulate fibrocyte involvement in the fibrotic tissue response: pharmacokinetic inhibition and the feasibility of directed-adipogenic differentiation.

    PubMed

    Baker, David W; Tsai, Yi-Ting; Weng, Hong; Tang, Liping

    2014-07-01

    Fibrocytes have previously been identified as important mediators in several inflammatory and fibrotic diseases. However, there is no effective treatment thus far to reduce fibrotic tissue responses without affecting wound healing reactions. Here we investigate two strategies to alleviate fibrocyte interactions at the biomaterial interface, reducing collagen production and scar tissue formation. First, in an indirect approach, TGF-β inhibitor-SB431542 and IL-1β/TNF-α inhibitor SB203580 were locally released from scaffold implants to block their respective signaling pathways. We show that the inhibition of IL-1β/TNF-α has no influence on overall fibrotic tissue reactions to the implants. However, the reduction of localized TGF-β significantly decreases the fibrocyte accumulation and myofibroblast activation while reducing the fibrotic tissue formation. Since fibrocytes can be differentiated into non-fibrotic cell types, such as adipocytes, we further sought a more direct approach to reduce fibrocyte responses by directing fibrocyte differentiation into adipocytes. Interestingly, by initiating fibrocyte-to-adipocyte differentiation through sustained differentiation cocktail release, we find that adipogenic differentiation forces incoming fibrocytes away from the traditional myofibroblast lineage, leading to a substantial reduction in the collagen formation and fibrotic response. Our results support a novel and effective strategy to improve implant safety by reducing implant-associated fibrotic tissue reactions via directing non-fibrotic differentiation of fibrocytes. PMID:24657674

  17. Systems-Wide Prediction of Enzyme Promiscuity Reveals a New Underground Alternative Route for Pyridoxal 5’-Phosphate Production in E. coli

    DOE PAGESBeta

    Oberhardt, Matthew A.; Zarecki, Raphy; Reshef, Leah; Xia, Fangfang; Duran-Frigola, Miquel; Schreiber, Rachel; Henry, Christopher S.; Ben-Tal, Nir; Dwyer, Daniel J.; Gophna, Uri; et al

    2016-01-28

    Recent insights suggest that non-specific and/or promiscuous enzymes are common and active across life. Understanding the role of such enzymes is an important open question in biology. Here we develop a genome-wide method, PROPER, that uses a permissive PSI-BLAST approach to predict promiscuous activities of metabolic genes. Enzyme promiscuity is typically studied experimentally using multicopy suppression, in which over-expression of a promiscuous ‘replacer’ gene rescues lethality caused by inactivation of a ‘target’ gene. We use PROPER to predict multicopy suppression in Escherichia coli, achieving highly significant overlap with published cases (hypergeometric p = 4.4e-13). We then validate three novel predictedmore » target-replacer gene pairs in new multicopy suppression experiments. We next go beyond PROPER and develop a network-based approach, GEM-PROPER, that integrates PROPER with genome-scale metabolic modeling to predict promiscuous replacements via alternative metabolic pathways. GEM-PROPER predicts a new indirect replacer (thiG) for an essential enzyme (pdxB) in production of pyridoxal 5’-phosphate (the active form of Vitamin B6), which we validate experimentally via multicopy suppression. Here, we perform a structural analysis of thiG to determine its potential promiscuous active site, which we validate experimentally by inactivating the pertaining residues and showing a loss of replacer activity. Thus, this study is a successful example where a computational investigation leads to a network-based identification of an indirect promiscuous replacement of a key metabolic enzyme, which would have been extremely difficult to identify directly.« less

  18. Systems-Wide Prediction of Enzyme Promiscuity Reveals a New Underground Alternative Route for Pyridoxal 5’-Phosphate Production in E. coli

    PubMed Central

    Reshef, Leah; Xia, Fangfang; Duran-Frigola, Miquel; Schreiber, Rachel; Henry, Christopher S.; Ben-Tal, Nir; Dwyer, Daniel J.; Gophna, Uri; Ruppin, Eytan

    2016-01-01

    Recent insights suggest that non-specific and/or promiscuous enzymes are common and active across life. Understanding the role of such enzymes is an important open question in biology. Here we develop a genome-wide method, PROPER, that uses a permissive PSI-BLAST approach to predict promiscuous activities of metabolic genes. Enzyme promiscuity is typically studied experimentally using multicopy suppression, in which over-expression of a promiscuous ‘replacer’ gene rescues lethality caused by inactivation of a ‘target’ gene. We use PROPER to predict multicopy suppression in Escherichia coli, achieving highly significant overlap with published cases (hypergeometric p = 4.4e-13). We then validate three novel predicted target-replacer gene pairs in new multicopy suppression experiments. We next go beyond PROPER and develop a network-based approach, GEM-PROPER, that integrates PROPER with genome-scale metabolic modeling to predict promiscuous replacements via alternative metabolic pathways. GEM-PROPER predicts a new indirect replacer (thiG) for an essential enzyme (pdxB) in production of pyridoxal 5’-phosphate (the active form of Vitamin B6), which we validate experimentally via multicopy suppression. We perform a structural analysis of thiG to determine its potential promiscuous active site, which we validate experimentally by inactivating the pertaining residues and showing a loss of replacer activity. Thus, this study is a successful example where a computational investigation leads to a network-based identification of an indirect promiscuous replacement of a key metabolic enzyme, which would have been extremely difficult to identify directly. PMID:26821166

  19. Systems-Wide Prediction of Enzyme Promiscuity Reveals a New Underground Alternative Route for Pyridoxal 5'-Phosphate Production in E. coli.

    PubMed

    Oberhardt, Matthew A; Zarecki, Raphy; Reshef, Leah; Xia, Fangfang; Duran-Frigola, Miquel; Schreiber, Rachel; Henry, Christopher S; Ben-Tal, Nir; Dwyer, Daniel J; Gophna, Uri; Ruppin, Eytan

    2016-01-01

    Recent insights suggest that non-specific and/or promiscuous enzymes are common and active across life. Understanding the role of such enzymes is an important open question in biology. Here we develop a genome-wide method, PROPER, that uses a permissive PSI-BLAST approach to predict promiscuous activities of metabolic genes. Enzyme promiscuity is typically studied experimentally using multicopy suppression, in which over-expression of a promiscuous 'replacer' gene rescues lethality caused by inactivation of a 'target' gene. We use PROPER to predict multicopy suppression in Escherichia coli, achieving highly significant overlap with published cases (hypergeometric p = 4.4e-13). We then validate three novel predicted target-replacer gene pairs in new multicopy suppression experiments. We next go beyond PROPER and develop a network-based approach, GEM-PROPER, that integrates PROPER with genome-scale metabolic modeling to predict promiscuous replacements via alternative metabolic pathways. GEM-PROPER predicts a new indirect replacer (thiG) for an essential enzyme (pdxB) in production of pyridoxal 5'-phosphate (the active form of Vitamin B6), which we validate experimentally via multicopy suppression. We perform a structural analysis of thiG to determine its potential promiscuous active site, which we validate experimentally by inactivating the pertaining residues and showing a loss of replacer activity. Thus, this study is a successful example where a computational investigation leads to a network-based identification of an indirect promiscuous replacement of a key metabolic enzyme, which would have been extremely difficult to identify directly. PMID:26821166

  20. Matched miRNA and mRNA signatures from an hESC-based in vitro model of pancreatic differentiation reveal novel regulatory interactions.

    PubMed

    Liao, Xiaoyan; Xue, Haipeng; Wang, Yu-Chieh; Nazor, Kristopher L; Guo, Shuren; Trivedi, Neha; Peterson, Suzanne E; Liu, Ying; Loring, Jeanne F; Laurent, Louise C

    2013-09-01

    The differentiation of human pluripotent stem cells (hPSCs) to insulin-expressing beta islet-like cells is a promising in vitro model system for studying the molecular signaling pathways underlying beta cell differentiation, as well as a potential source of cells for the treatment of type 1 diabetes. MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate many biological processes, including cellular differentiation. We studied the miRNA and mRNA expression profiles of hPSCs at five stages of in vitro differentiation along the pancreatic beta cell lineage (definitive endoderm, primitive gut tube, posterior foregut, pancreatic progenitor and hormone-expressing endocrine cells) in the context of samples of primary human fetal pancreas and purified adult islet cells using microarray analysis. Bioinformatic analysis of the resulting data identified a unique miRNA signature in differentiated beta islet cells, and predicted the effects of key miRNAs on mRNA expression. Many of the predicted miRNA-mRNA interactions involved mRNAs known to play key roles in the epithelial-mesenchymal transition process and pancreatic differentiation. We validated a subset of the predictions using qRT-PCR, luciferase reporter assays and western blotting, including the known interaction between miR-200 and ZEB2 (involved in epithelial-mesenchymal transition) and the novel interaction between miR-200 and SOX17 (a key transcription factor in specification of definitive endoderm). In addition, we found that miR-30d and let-7e, two miRNAs induced during differentiation, regulated the expression of RFX6, a transcription factor that directs pancreatic islet formation. These findings suggest that precise control of target mRNA expression by miRNAs ensures proper lineage specification during pancreatic development. PMID:23813959

  1. Integrative Analysis of MicroRNA and mRNA Data Reveals an Orchestrated Function of MicroRNAs in Skeletal Myocyte Differentiation in Response to TNF-α or IGF1

    PubMed Central

    Meyer, Swanhild U.; Sass, Steffen; Mueller, Nikola S.; Krebs, Stefan; Bauersachs, Stefan; Kaiser, Sebastian; Blum, Helmut; Thirion, Christian; Krause, Sabine; Theis, Fabian J.; Pfaffl, Michael W.

    2015-01-01

    Introduction Skeletal muscle cell differentiation is impaired by elevated levels of the inflammatory cytokine tumor necrosis factor-α (TNF-α) with pathological significance in chronic diseases or inherited muscle disorders. Insulin like growth factor-1 (IGF1) positively regulates muscle cell differentiation. Both, TNF-α and IGF1 affect gene and microRNA (miRNA) expression in this process. However, computational prediction of miRNA-mRNA relations is challenged by false positives and targets which might be irrelevant in the respective cellular transcriptome context. Thus, this study is focused on functional information about miRNA affected target transcripts by integrating miRNA and mRNA expression profiling data. Methodology/Principal Findings Murine skeletal myocytes PMI28 were differentiated for 24 hours with concomitant TNF-α or IGF1 treatment. Both, mRNA and miRNA expression profiling was performed. The data-driven integration of target prediction and paired mRNA/miRNA expression profiling data revealed that i) the quantity of predicted miRNA-mRNA relations was reduced, ii) miRNA targets with a function in cell cycle and axon guidance were enriched, iii) differential regulation of anti-differentiation miR-155-5p and miR-29b-3p as well as pro-differentiation miR-335-3p, miR-335-5p, miR-322-3p, and miR-322-5p seemed to be of primary importance during skeletal myoblast differentiation compared to the other miRNAs, iv) the abundance of targets and affected biological processes was miRNA specific, and v) subsets of miRNAs may collectively regulate gene expression. Conclusions Joint analysis of mRNA and miRNA profiling data increased the process-specificity and quality of predicted relations by statistically selecting miRNA-target interactions. Moreover, this study revealed miRNA-specific predominant biological implications in skeletal muscle cell differentiation and in response to TNF-α or IGF1 treatment. Furthermore, myoblast differentiation-associated mi

  2. Cross-species functional analyses reveal shared and separate roles for Sox11 in frog primary neurogenesis and mouse cortical neuronal differentiation

    PubMed Central

    Chen, Chao; Jin, Jing; Lee, Garrett A.; Silva, Elena; Donoghue, Maria

    2016-01-01

    ABSTRACT A well-functioning brain requires production of the correct number and types of cells during development; cascades of transcription factors are essential for cellular coordination. Sox proteins are transcription factors that affect various processes in the development of the nervous system. Sox11, a member of the SoxC family, is expressed in differentiated neurons and supports neuronal differentiation in several systems. To understand how generalizable the actions of Sox11 are across phylogeny, its function in the development of the frog nervous system and the mouse cerebral cortex were compared. Expression of Sox11 is largely conserved between these species; in the developing frog, Sox11 is expressed in the neural plate, neural tube and throughout the segmented brain, while in the mouse cerebral cortex, Sox11 is expressed in differentiated zones, including the preplate, subplate, marginal zone and cortical plate. In both frog and mouse, data demonstrate that Sox11 supports a role in promoting neuronal differentiation, with Sox11-positive cells expressing pan-neural markers and becoming morphologically complex. However, frog and mouse Sox11 cannot substitute for one another; a functional difference likely reflected in sequence divergence. Thus, Sox11 appears to act similarly in subserving neuronal differentiation but is species-specific in frog neural development and mouse corticogenesis. PMID:26962049

  3. Quantitative Phosphoproteomic Study Reveals that Protein Kinase A Regulates Neural Stem Cell Differentiation Through Phosphorylation of Catenin Beta-1 and Glycogen Synthase Kinase 3β.

    PubMed

    Wang, Shuxin; Li, Zheyi; Shen, Hongyan; Zhang, Zhong; Yin, Yuxin; Wang, Qingsong; Zhao, Xuyang; Ji, Jianguo

    2016-08-01

    Protein phosphorylation is central to the understanding of multiple cellular signaling pathways responsible for regulating the self-renewal and differentiation of neural stem cells (NSCs). Here we performed a large-scale phosphoproteomic analysis of rat fetal NSCs using strong cation exchange chromatography prefractionation and citric acid-assisted two-step enrichment with TiO2 strategy followed by nanoLC-MS/MS analysis. Totally we identified 32,546 phosphosites on 5,091 phosphoproteins, among which 23,945 were class I phosphosites, and quantified 16,000 sites during NSC differentiation. More than 65% of class I phosphosites were novel when compared with PhosphoSitePlus database. Quantification results showed that the early and late stage of NSC differentiation differ greatly. We mapped 69 changed phosphosites on 20 proteins involved in Wnt signaling pathway, including S552 on catenin beta-1 (Ctnnb1) and S9 on glycogen synthase kinase 3β (Gsk3β). Western blotting and real-time PCR results proved that Wnt signaling pathway plays critical roles in NSC fate determination. Furthermore, inhibition and activation of PKA dramatically affected the phosphorylation state of Ctnnb1 and Gsk3β, which regulates the differentiation of NSCs. Our data provides a valuable resource for studying the self-renewal and differentiation of NSCs. Stem Cells 2016;34:2090-2101. PMID:27097102

  4. Alternative Mammalian Target of Rapamycin (mTOR) Signal Activation in Sorafenib-resistant Hepatocellular Carcinoma Cells Revealed by Array-based Pathway Profiling*

    PubMed Central

    Masuda, Mari; Chen, Wei-Yu; Miyanaga, Akihiko; Nakamura, Yuka; Kawasaki, Kumiko; Sakuma, Tomohiro; Ono, Masaya; Chen, Chi-Long; Honda, Kazufumi; Yamada, Tesshi

    2014-01-01

    Sorafenib is a multi-kinase inhibitor that has been proven effective for the treatment of unresectable hepatocellular carcinoma (HCC). However, its precise mechanisms of action and resistance have not been well established. We have developed high-density fluorescence reverse-phase protein arrays and used them to determine the status of 180 phosphorylation sites of signaling molecules in the 120 pathways registered in the NCI-Nature curated database in 23 HCC cell lines. Among the 180 signaling nodes, we found that the level of ribosomal protein S6 phosphorylated at serine residue 235/236 (p-RPS6 S235/236) was most significantly correlated with the resistance of HCC cells to sorafenib. The high expression of p-RPS6 S235/236 was confirmed immunohistochemically in biopsy samples obtained from HCC patients who responded poorly to sorafenib. Sorafenib-resistant HCC cells showed constitutive activation of the mammalian target of rapamycin (mTOR) pathway, but whole-exon sequencing of kinase genes revealed no evident alteration in the pathway. p-RPS6 S235/236 is a potential biomarker that predicts unresponsiveness of HCC to sorafenib. The use of mTOR inhibitors may be considered for the treatment of such tumors. PMID:24643969

  5. An alternative approach to systems of second-order ordinary differential equations with maximal symmetry. Realizations of sl(n + 2 , R) by special functions

    NASA Astrophysics Data System (ADS)

    Campoamor-Stursberg, R.

    2016-08-01

    Using the general solution of the differential equation x¨(t) +g1(t) x˙ +g2(t) x = 0 , a generic basis of the point-symmetry algebra sl(3 , R) is constructed. Deriving the equation from a time-dependent Lagrangian, the basis elements corresponding to Noether symmetries are deduced. The generalized Lewis invariant is constructed explicitly using a linear combination of Noether symmetries. The procedure is generalized to the case of systems of second-order ordinary differential equations with maximal sl(n + 2 , R) -symmetry, and its possible adaptation to the inhomogeneous non-linear case illustrated by an example.

  6. Teacher Learning through Self-Regulation: An Exploratory Study of Alternatively Prepared Teachers' Ability to Plan Differentiated Instruction in an Urban Elementary School

    ERIC Educational Resources Information Center

    Tricarico, Katie; Yendol-Hoppey, Diane

    2012-01-01

    Differentiated Instruction (DI) is an approach that recognizes the strengths and weaknesses of diverse learners and requires the teacher to base instructional accommodations on student strengths and weaknesses. Specifically, teachers use DI strategies to adjust the content, process, or product of instruction depending on student needs. Given the…

  7. Differential Item Functioning Comparisons on a Performance-Based Alternate Assessment for Students with Severe Cognitive Impairments, Autism and Orthopedic Impairments

    ERIC Educational Resources Information Center

    Laitusis, Cara Cahalan; Maneckshana, Behroz; Monfils, Lora; Ahlgrim-Delzell, Lynn

    2009-01-01

    The purpose of this study was to examine Differential Item Functioning (DIF) by disability groups on an on-demand performance assessment for students with severe cognitive impairments. Researchers examined the presence of DIF for two comparisons. One comparison involved students with severe cognitive impairments who served as the reference group…

  8. Differential fruit gene expression in two strawberry cultivars in response to elevated CO2 during storage revealed by a heterologous fruit microarray approach

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The use of a heterologous fruit microarray system to identify differentially expressed genes between strawberry cultivars with different responses to 20kPa CO2 (balance air) during storage has been evaluated. Specifically, a tomato cDNA microarray was hybridized with strawberry cDNA populations to c...

  9. Secreted protein gene derived-single nucleotide polymorphisms (SP-SNPs) reveal population diversity and differentiation of Puccinia striiformis f. sp. tritici in the United States

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In population genetic studies, growing awareness has been raised on non-neutral markers which can provide more estimates of evolutionary differentiation caused by different selections among populations, even though neutral genetic markers have provided unbiased estimates of divergence time and the a...

  10. Genome-wide analysis of gene expression during adipogenesis in human adipose-derived stromal cells reveals novel patterns of gene expression during adipocyte differentiation.

    PubMed

    Ambele, Melvin Anyasi; Dessels, Carla; Durandt, Chrisna; Pepper, Michael Sean

    2016-05-01

    We have undertaken an in-depth transcriptome analysis of adipogenesis in human adipose-derived stromal cells (ASCs) induced to differentiate into adipocytes in vitro. Gene expression was assessed on days 1, 7, 14 and 21 post-induction and genes differentially expressed numbered 128, 218, 253 and 240 respectively. Up-regulated genes were associated with blood vessel development, leukocyte migration, as well as tumor growth, invasion and metastasis. They also shared common pathways with certain obesity-related pathophysiological conditions. Down-regulated genes were enriched for immune response processes. KLF15, LMO3, FOXO1 and ZBTB16 transcription factors were up-regulated throughout the differentiation process. CEBPA, PPARG, ZNF117, MLXIPL, MMP3 and RORB were up-regulated only on days 14 and 21, which coincide with the maturation of adipocytes and could possibly serve as candidates for controlling fat accumulation and the size of mature adipocytes. In summary, we have identified genes that were up-regulated only on days 1 and 7 or days 14 and 21 that could serve as potential early and late-stage differentiation markers. PMID:27108396

  11. Integrated analysis of miRNAs and transcriptomes in Aedes albopictus midgut reveals the differential expression profiles of immune-related genes during dengue virus serotype-2 infection.

    PubMed

    Liu, Yan-Xia; Li, Fen-Xiang; Liu, Zhuan-Zhuan; Jia, Zhi-Rong; Zhou, Yan-He; Zhang, Hao; Yan, Hui; Zhou, Xian-Qiang; Chen, Xiao-Guang

    2016-06-01

    Mosquito microRNAs (miRNAs) are involved in host-virus interaction, and have been reported to be altered by dengue virus (DENV) infection in Aedes albopictus (Diptera: Culicidae). However, little is known about the molecular mechanisms of Aedes albopictus midgut-the first organ to interact with DENV-involved in its resistance to DENV. Here we used high-throughput sequencing to characterize miRNA and messenger RNA (mRNA) expression patterns in Aedes albopictus midgut in response to dengue virus serotype 2. A total of three miRNAs and 777 mRNAs were identified to be differentially expressed upon DENV infection. For the mRNAs, we identified 198 immune-related genes and 31 of them were differentially expressed. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses also showed that the differentially expressed immune-related genes were involved in immune response. Then the differential expression patterns of six immune-related genes and three miRNAs were confirmed by real-time reverse transcription polymerase chain reaction. Furthermore, seven known miRNA-mRNA interaction pairs were identified by aligning our two datasets. These analyses of miRNA and mRNA transcriptomes provide valuable information for uncovering the DENV response genes and provide a basis for future study of the resistance mechanisms in Aedes albopictus midgut. PMID:27029517

  12. Alternate Polypurine Tracts (PPTs) Affect the Rous Sarcoma Virus RNase H Cleavage Specificity and Reveal a Preferential Cleavage following a GA Dinucleotide Sequence at the PPT-U3 Junction

    PubMed Central

    Chang, Kevin W.; Julias, John G.; Alvord, W. Gregory; Oh, Jangsuk; Hughes, Stephen H.

    2005-01-01

    Retroviral polypurine tracts (PPTs) serve as primers for plus-strand DNA synthesis during reverse transcription. The generation and removal of the PPT primer requires specific cleavages by the RNase H activity of reverse transcriptases; removal of the PPT primer defines the left end of the linear viral DNA. We replaced the endogenous PPT from RSVP(A)Z, a replication-competent shuttle vector based on Rous sarcoma virus (RSV), with alternate retroviral PPTs and the duck hepatitis B virus “PPT.” Viruses in which the endogenous RSV PPT was replaced with alternate PPTs had lower relative titers than the wild-type virus. 2-LTR circle junction analysis showed that the alternate PPTs caused significant decreases in the fraction of viral DNAs with complete (consensus) ends and significant increases in the insertion of part or all of the PPT at the 2-LTR circle junctions. The last two nucleotides in the 3′ end of the RSV PPT are GA. Examination of the (mis)cleavages of the alternate PPTs revealed preferential cleavages after GA dinucleotide sequences. Replacement of the terminal 3′ A of the RSV PPT with G caused a preferential miscleavage at a GA sequence spanning the PPT-U3 boundary, resulting in the deletion of the terminal adenine normally present at the 5′ end of the U3. A reciprocal G-to-A substitution at the 3′ end of the murine leukemia virus PPT increased the relative titer of the chimeric RSV-based virus and the fraction of consensus 2-LTR circle junctions. PMID:16227289

  13. RNA sequencing reveals sexually dimorphic gene expression before gonadal differentiation in chicken and allows comprehensive annotation of the W-chromosome

    PubMed Central

    2013-01-01

    Background Birds have a ZZ male: ZW female sex chromosome system and while the Z-linked DMRT1 gene is necessary for testis development, the exact mechanism of sex determination in birds remains unsolved. This is partly due to the poor annotation of the W chromosome, which is speculated to carry a female determinant. Few genes have been mapped to the W and little is known of their expression. Results We used RNA-seq to produce a comprehensive profile of gene expression in chicken blastoderms and embryonic gonads prior to sexual differentiation. We found robust sexually dimorphic gene expression in both tissues pre-dating gonadogenesis, including sex-linked and autosomal genes. This supports the hypothesis that sexual differentiation at the molecular level is at least partly cell autonomous in birds. Different sets of genes were sexually dimorphic in the two tissues, indicating that molecular sexual differentiation is tissue specific. Further analyses allowed the assembly of full-length transcripts for 26 W chromosome genes, providing a view of the W transcriptome in embryonic tissues. This is the first extensive analysis of W-linked genes and their expression profiles in early avian embryos. Conclusion Sexual differentiation at the molecular level is established in chicken early in embryogenesis, before gonadal sex differentiation. We find that the W chromosome is more transcriptionally active than previously thought, expand the number of known genes to 26 and present complete coding sequences for these W genes. This includes two novel W-linked sequences and three small RNAs reassigned to the W from the Un_Random chromosome. PMID:23531366

  14. A favorable role of prolactin in human breast cancer reveals novel pathway-based gene signatures indicative of tumor differentiation and favorable patient outcome.

    PubMed

    Hachim, Ibrahim Y; Shams, Anwar; Lebrun, Jean-Jacques; Ali, Suhad

    2016-07-01

    Prolactin (PRL) hormone is known to play a key role in mammary gland development allowing for successful lactation. The role of this hormone in breast tumorigenesis is still controversial. Here, we evaluated PRL protein and gene expression levels in human breast cancer using tissue microarray of 100 breast cancer cases, as well as different publically available human breast cancer gene profiling databases. Interestingly, our results showed a significant downregulation of PRL expression in breast cancer compared to normal adjacent tissue. Moreover, expression of PRL was associated with more differentiated tumors, early stage, smaller tumor size and absence of distant metastasis. Importantly, our results indicate that higher PRL mRNA levels are significantly associated with prolonged relapse-free survival (RFS) in breast cancer patients (P=3.7 x 10(-9)). Additionally, examining expression of PRL pathway-based gene signature composed of PRL, PRLR, Jak2 and Stat5a showed a significant association with more differentiated tumors (P<.00001), prolonged RFS (P=1.8 x 10(-6)) as well as overall survival (OS) (P=.0026). As well, our results indicate that PRL-directed differentiation program in mammary epithelial cells offer good prognosis in human breast cancer. Indeed, expression of a gene signature composed of PRL-upregulated genes showed a significant association with well-differentiated tumors (P<.00001). Whereas expression of a gene signature composed of PRL-downregulated genes showed a significant association with shortened distant metastasis-free survival (DMFS) (P=.0086). Altogether our results highlight that PRL hormone and its signaling pathway may play an important role in maintaining tumor differentiation state and in turn better patient outcome. PMID:26980025

  15. Alternative security

    SciTech Connect

    Weston, B.H. )

    1990-01-01

    This book contains the following chapters: The Military and Alternative Security: New Missions for Stable Conventional Security; Technology and Alternative Security: A Cherished Myth Expires; Law and Alternative Security: Toward a Just World Peace; Politics and Alternative Security: Toward a More Democratic, Therefore More Peaceful, World; Economics and Alternative Security: Toward a Peacekeeping International Economy; Psychology and Alternative Security: Needs, Perceptions, and Misperceptions; Religion and Alternative Security: A Prophetic Vision; and Toward Post-Nuclear Global Security: An Overview.

  16. If-then contingencies and the differential effects of the availability of an attractive alternative on relationship maintenance for men and women.

    PubMed

    Lydon, John E; Menzies-Toman, Danielle; Burton, Kimberly; Bell, Chris

    2008-07-01

    In 7 experiments, the causal effects of the availability of an attractive alternative (AA) relationship partner on current relationship thoughts and intentions were tested using confederates, mental simulations, and virtual reality. Men behaved consistent with traditional relationship-commitment theories, showing decreased willingness to tolerate their partner's transgressions after the availability of an AA was made salient. However, consistent with a motivated cognition approach to commitment and work on relational self-construals, women increased their tolerance when presented with the relationship threat of an alternative. Word-fragment and lexical decision data suggested that AAs may activate threat for women, and their ability to dampen threat accessibility is associated with prorelationship responses. Finally, this "if relationship is threatened, then defend the relationship" contingency was induced in men with an implementation intention induction. PMID:18605851

  17. Genome-wide analysis reveals the differential regulations of mRNAs and miRNAs in Dorset and Small Tail Han sheep muscles.

    PubMed

    Miao, Xiangyang; Luo, Qingmiao; Qin, Xiaoyu

    2015-05-15

    Sheep are highly diverse species raised for meat and other agricultural products. The aim of the present study was to investigate the genetic regulators that could control muscle growth and development in different sheep breeds. The study showed that the differentially expressed genes are involved in various cellular activities, such as metabolic cascades, catalytic function and signaling pathway. Many signaling molecules are also found to be differentially expressed, suggesting important roles of signaling pathways contributing to genetic diversity and sheep development. Analysis of miRNAs suggested important roles of miRNAs in controlling muscle differences. This study provided a genome-wide resolution of mRNA and miRNA regulations in muscles from Dorset and Han sheep. PMID:25732516

  18. 18O-Tracer Metabolomics Reveals Protein Turnover and CDP-Choline Cycle Activity in Differentiating 3T3-L1 Pre-Adipocytes

    PubMed Central

    Kirkwood, Jay S.; Miranda, Cristobal L.; Bobe, Gerd; Maier, Claudia S.; Stevens, Jan F.

    2016-01-01

    The differentiation of precursor cells into mature adipocytes (adipogenesis) has been an area of increased focus, spurred by a rise in obesity rates. Though our understanding of adipogenesis and its regulation at the cellular level is growing, many questions remain, especially regarding the regulation of the metabolome. The 3T3-L1 cell line is the most well characterized cellular model of adipogenesis. Using a time course metabolomics approach, we show that the 3T3-L1 preadipocyte metabolome is greatly altered during the first 48 hours of differentiation, where cells go through about two rounds of cell division, a process known as mitotic clonal expansion. Short-chain peptides were among several small molecules that were increased during mitotic clonal expansion. Additional indicators of protein turnover were also increased, including bilirubin, a degradation product of heme-containing proteins, and 3-methylhistidine, a post-translationally modified amino acid that is not reutilized for protein synthesis. To study the origin of the peptides, we treated differentiating preadipocytes with 18O labeled water and found that 18O was incorporated into the short chain peptides, confirming them, at least in part, as products of hydrolysis. Inhibitors of the proteasome or matrix metalloproteinases affected the peptide levels during differentiation, but inhibitors of autophagy or peptidases did not. 18O was also incorporated into several choline metabolites including cytidine 5'-diphosphocholine (CDP-choline), glycerophosphocholine, and several phosphatidylcholine species, indicative of phosphatidylcholine synthesis/degradation and of flux through the CDP-choline cycle, a hallmark of proliferating cells. 18O-Tracer metabolomics further showed metabolic labeling of glutamate, suggestive of glutaminolysis, also characteristic of proliferating cells. Together, these results highlight the utility of 18O isotope labeling in combination with metabolomics to uncover changes in

  19. 18O-Tracer Metabolomics Reveals Protein Turnover and CDP-Choline Cycle Activity in Differentiating 3T3-L1 Pre-Adipocytes.

    PubMed

    Kirkwood, Jay S; Miranda, Cristobal L; Bobe, Gerd; Maier, Claudia S; Stevens, Jan F

    2016-01-01

    The differentiation of precursor cells into mature adipocytes (adipogenesis) has been an area of increased focus, spurred by a rise in obesity rates. Though our understanding of adipogenesis and its regulation at the cellular level is growing, many questions remain, especially regarding the regulation of the metabolome. The 3T3-L1 cell line is the most well characterized cellular model of adipogenesis. Using a time course metabolomics approach, we show that the 3T3-L1 preadipocyte metabolome is greatly altered during the first 48 hours of differentiation, where cells go through about two rounds of cell division, a process known as mitotic clonal expansion. Short-chain peptides were among several small molecules that were increased during mitotic clonal expansion. Additional indicators of protein turnover were also increased, including bilirubin, a degradation product of heme-containing proteins, and 3-methylhistidine, a post-translationally modified amino acid that is not reutilized for protein synthesis. To study the origin of the peptides, we treated differentiating preadipocytes with 18O labeled water and found that 18O was incorporated into the short chain peptides, confirming them, at least in part, as products of hydrolysis. Inhibitors of the proteasome or matrix metalloproteinases affected the peptide levels during differentiation, but inhibitors of autophagy or peptidases did not. 18O was also incorporated into several choline metabolites including cytidine