Continuum theory of gene expression waves during vertebrate segmentation.

PubMed

Jörg, David J; Morelli, Luis G; Soroldoni, Daniele; Oates, Andrew C; Jülicher, Frank

2015-09-01

The segmentation of the vertebrate body plan during embryonic development is a rhythmic and sequential process governed by genetic oscillations. These genetic oscillations give rise to traveling waves of gene expression in the segmenting tissue. Here we present a minimal continuum theory of vertebrate segmentation that captures the key principles governing the dynamic patterns of gene expression including the effects of shortening of the oscillating tissue. We show that our theory can quantitatively account for the key features of segmentation observed in zebrafish, in particular the shape of the wave patterns, the period of segmentation and the segment length as a function of time.

Continuum theory of gene expression waves during vertebrate segmentation

PubMed Central

Jörg, David J; Morelli, Luis G; Soroldoni, Daniele; Oates, Andrew C; Jülicher, Frank

2015-01-01

Abstract The segmentation of the vertebrate body plan during embryonic development is a rhythmic and sequential process governed by genetic oscillations. These genetic oscillations give rise to traveling waves of gene expression in the segmenting tissue. Here we present a minimal continuum theory of vertebrate segmentation that captures the key principles governing the dynamic patterns of gene expression including the effects of shortening of the oscillating tissue. We show that our theory can quantitatively account for the key features of segmentation observed in zebrafish, in particular the shape of the wave patterns, the period of segmentation and the segment length as a function of time. PMID:28725158

Sequential inflammatory processes define human progression from M. tuberculosis infection to tuberculosis disease.

PubMed

Scriba, Thomas J; Penn-Nicholson, Adam; Shankar, Smitha; Hraha, Tom; Thompson, Ethan G; Sterling, David; Nemes, Elisa; Darboe, Fatoumatta; Suliman, Sara; Amon, Lynn M; Mahomed, Hassan; Erasmus, Mzwandile; Whatney, Wendy; Johnson, John L; Boom, W Henry; Hatherill, Mark; Valvo, Joe; De Groote, Mary Ann; Ochsner, Urs A; Aderem, Alan; Hanekom, Willem A; Zak, Daniel E

2017-11-01

Our understanding of mechanisms underlying progression from Mycobacterium tuberculosis infection to pulmonary tuberculosis disease in humans remains limited. To define such mechanisms, we followed M. tuberculosis-infected adolescents longitudinally. Blood samples from forty-four adolescents who ultimately developed tuberculosis disease (“progressors”) were compared with those from 106 matched controls, who remained healthy during two years of follow up. We performed longitudinal whole blood transcriptomic analyses by RNA sequencing and plasma proteome analyses using multiplexed slow off-rate modified DNA aptamers. Tuberculosis progression was associated with sequential modulation of immunological processes. Type I/II interferon signalling and complement cascade were elevated 18 months before tuberculosis disease diagnosis, while changes in myeloid inflammation, lymphoid, monocyte and neutrophil gene modules occurred more proximally to tuberculosis disease. Analysis of gene expression in purified T cells also revealed early suppression of Th17 responses in progressors, relative to M. tuberculosis-infected controls. This was confirmed in an independent adult cohort who received BCG re-vaccination; transcript expression of interferon response genes in blood prior to BCG administration was associated with suppression of IL-17 expression by BCG-specific CD4 T cells 3 weeks post-vaccination. Our findings provide a timeline to the different immunological stages of disease progression which comprise sequential inflammatory dynamics and immune alterations that precede disease manifestations and diagnosis of tuberculosis disease. These findings have important implications for developing diagnostics, vaccination and host-directed therapies for tuberculosis. Clincialtrials.gov, NCT01119521.

Evidence for the temporal regulation of insect segmentation by a conserved sequence of transcription factors

PubMed Central

2018-01-01

ABSTRACT Long-germ insects, such as the fruit fly Drosophila melanogaster, pattern their segments simultaneously, whereas short-germ insects, such as the beetle Tribolium castaneum, pattern their segments sequentially, from anterior to posterior. Although the two modes of segmentation at first appear quite distinct, much of this difference might simply reflect developmental heterochrony. We now show here that, in both Drosophila and Tribolium, segment patterning occurs within a common framework of sequential Caudal, Dichaete and Odd-paired expression. In Drosophila, these transcription factors are expressed like simple timers within the blastoderm, whereas in Tribolium they form wavefronts that sweep from anterior to posterior across the germband. In Drosophila, all three are known to regulate pair-rule gene expression and influence the temporal progression of segmentation. We propose that these regulatory roles are conserved in short-germ embryos, and that therefore the changing expression profiles of these genes across insects provide a mechanistic explanation for observed differences in the timing of segmentation. In support of this hypothesis, we demonstrate that Odd-paired is essential for segmentation in Tribolium, contrary to previous reports. PMID:29724758

Therapeutic effect for liver-metastasized tumor by sequential intravenous injection of anionic polymer and cationic lipoplex of siRNA.

PubMed

Hattori, Yoshiyuki; Arai, Shohei; Kikuchi, Takuto; Ozaki, Kei-Ichi; Kawano, Kumi; Yonemochi, Etsuo

2016-04-01

Previously, we developed a novel siRNA transfer method to the liver by sequential intravenous injection of anionic polymer and cationic liposome/siRNA complex (cationic lipoplex). In this study, we investigated whether siRNA delivered by this sequential injection could significantly suppress mRNA expression of the targeted gene in liver metastasis and inhibit tumor growth. When cationic lipoplex was intravenously injected into mice bearing liver metastasis of human breast tumor MCF-7 at 1 min after intravenous injection of chondroitin sulfate C (CS) or poly-l-glutamic acid (PGA), siRNA was accumulated in tumor-metastasized liver. In terms of a gene silencing effect, sequential injections of CS or PGA plus cationic lipoplex of luciferase siRNA could reduce luciferase activity in liver MCF-7-Luc metastasis. Regarding the side effects, sequential injections of CS plus cationic lipoplex did not exhibit hepatic damage or induction of inflammatory cytokines in serum after repeated injections, but sequential injections of PGA plus cationic lipoplex did. Finally, sequential injections of CS plus cationic lipoplex of protein kinase N3 siRNA could suppress tumor growth in the mice bearing liver metastasis. From these findings, sequential injection of CS and cationic lipoplex of siRNA might be a novel systemic method of delivering siRNA to liver metastasis.

Temporal course of gene expression during motor memory formation in primary motor cortex of rats.

PubMed

Hertler, B; Buitrago, M M; Luft, A R; Hosp, J A

2016-12-01

Motor learning is associated with plastic reorganization of neural networks in primary motor cortex (M1) that depends on changes in gene expression. Here, we investigate the temporal profile of these changes during motor memory formation in response to a skilled reaching task in rats. mRNA-levels were measured 1h, 7h and 24h after the end of a training session using microarray technique. To assure learning specificity, trained animals were compared to a control group. In response to motor learning, genes are sequentially regulated with high time-point specificity and a shift from initial suppression to later activation. The majority of regulated genes can be linked to learning-related plasticity. In the gene-expression cascade following motor learning, three different steps can be defined: (1) an initial suppression of genes influencing gene transcription. (2) Expression of genes that support translation of mRNA in defined compartments. (3) Expression of genes that immediately mediates plastic changes. Gene expression peaks after 24h - this is a much slower time-course when compared to hippocampus-dependent learning, where peaks of gene-expression can be observed 6-12h after training ended. Copyright © 2016 Elsevier Inc. All rights reserved.

Investigating the transcriptional control of cardiovascular development

PubMed Central

Kathiriya, Irfan S.; Nora, Elphege P.; Bruneau, Benoit G.

2015-01-01

Transcriptional regulation of thousands of genes instructs complex morphogenetic and molecular events for heart development. Cardiac transcription factors (TFs) choreograph gene expression at each stage of differentiation by interacting with co-factors, including chromatin-modifying enzymes, and by binding to a constellation of regulatory DNA elements. Here, we present salient examples relevant to cardiovascular development and heart disease and review techniques that can sharpen our understanding of cardiovascular biology. We discuss the interplay between cardiac TFs, cis-regulatory elements and chromatin as dynamic regulatory networks, to orchestrate sequential deployment of the cardiac gene expression program. PMID:25677518

Dynamics of wound healing signaling as a potential therapeutic target for radiation-induced tissue damage.

PubMed

Chung, Yih-Lin; Pui, Newman N M

2015-01-01

We hypothesized the histone deacetylase inhibitor phenylbutyrate (PB) has beneficial effects on radiation-induced injury by modulating the expression of DNA repair and wound healing genes. Hamsters received a radiosurgical dose of radiation (40 Gy) to the cheek and were treated with varying PB dosing regimens. Gross alteration of the irradiated cheeks, eating function, histological changes, and gene expression during the course of wound healing were compared between treatment groups. Pathological analysis showed decreased radiation-induced mucositis, facilitated epithelial cell growth, and preventing ulcerative wound formation, after short-term PB treatment, but not after vehicle or sustained PB. The radiation-induced wound healing gene expression profile exhibited a sequential transition from the inflammatory and DNA repair phases to the tissue remodeling phase in the vehicle group. Sustained PB treatment resulted in a prolonged wound healing gene expression profile and delayed the wound healing process. Short-term PB shortened the duration of inflammatory cytokine expression, triggered repeated pulsed expression of cell cycle and DNA repair-regulating genes, and promoted earlier oscillatory expression of tissue remodeling genes. Distinct gene expression patterns between sustained and short-term treatment suggest dynamic profiling of wound healing gene expression can be an important part of a biological therapeutic strategy to mitigate radiation-related tissue injury. © 2015 by the Wound Healing Society.

Culture medium composition affects the gene expression pattern and in vitro development potential of bovine somatic cell nuclear transfer (SCNT) embryos.

PubMed

Arias, María E; Ross, Pablo J; Felmer, Ricardo N

2013-01-01

Different culture systems have been studied that support development of somatic cell nuclear transfer (SCNT) embryos up to the blastocyst stage. However, the use of sequential and two-step culture systems has been less studied. The objective of the present study was to examine the developmental potential and quality of bovine SCNT embryos cultured in different two-step culture media based on KSOM, SOF and the macromolecules FBS and BSA (K-K/FBS, K-S/BSA and K-K/BSA, respectively). No differences were observed in the cleavage rate for any of the culture systems. However, there was a significant difference (P<0.01) in the rate of blastocyst development, with the K-K/ FBS culture system yielding a higher rate of blastocysts (28%) compared to other treatments (18 and 15%, for K-S/BSA and K-K/BSA, respectively). Although quality of embryos, as assessed by the total number of cells, was not different, the apoptosis index was significantly affected in the sequential culture system (K-S/BSA). Gene expression analysis showed alterations of DNMT1, IGF2, LIF, and PRDX6 genes in embryos cultured in K-S/FBS and of SOD2 in embryos cultured in K-K/BSA. In conclusion, we demonstrated that culture medium may affect not only the developmental potential of SCNT embryos but also, more importantly, the gene expression pattern and apoptotic index, presenting the possibility to manipulate the culture medium composition to modulate global gene expression and improve the overall efficiency of this technique.

Global analysis of gene expression in pulmonary fibrosis reveals distinct programs regulating lung inflammation and fibrosis

NASA Astrophysics Data System (ADS)

Kaminski, Naftali; Allard, John D.; Pittet, Jean F.; Zuo, Fengrong; Griffiths, Mark J. D.; Morris, David; Huang, Xiaozhu; Sheppard, Dean; Heller, Renu A.

2000-02-01

The molecular mechanisms of pulmonary fibrosis are poorly understood. We have used oligonucleotide arrays to analyze the gene expression programs that underlie pulmonary fibrosis in response to bleomycin, a drug that causes lung inflammation and fibrosis, in two strains of susceptible mice (129 and C57BL/6). We then compared the gene expression patterns in these mice with 129 mice carrying a null mutation in the epithelial-restricted integrin 6 subunit (6/-), which develop inflammation but are protected from pulmonary fibrosis. Cluster analysis identified two distinct groups of genes involved in the inflammatory and fibrotic responses. Analysis of gene expression at multiple time points after bleomycin administration revealed sequential induction of subsets of genes that characterize each response. The availability of this comprehensive data set should accelerate the development of more effective strategies for intervention at the various stages in the development of fibrotic diseases of the lungs and other organs.

Application of dynamic topic models to toxicogenomics data.

PubMed

Lee, Mikyung; Liu, Zhichao; Huang, Ruili; Tong, Weida

2016-10-06

All biological processes are inherently dynamic. Biological systems evolve transiently or sustainably according to sequential time points after perturbation by environment insults, drugs and chemicals. Investigating the temporal behavior of molecular events has been an important subject to understand the underlying mechanisms governing the biological system in response to, such as, drug treatment. The intrinsic complexity of time series data requires appropriate computational algorithms for data interpretation. In this study, we propose, for the first time, the application of dynamic topic models (DTM) for analyzing time-series gene expression data. A large time-series toxicogenomics dataset was studied. It contains over 3144 microarrays of gene expression data corresponding to rat livers treated with 131 compounds (most are drugs) at two doses (control and high dose) in a repeated schedule containing four separate time points (4-, 8-, 15- and 29-day). We analyzed, with DTM, the topics (consisting of a set of genes) and their biological interpretations over these four time points. We identified hidden patterns embedded in this time-series gene expression profiles. From the topic distribution for compound-time condition, a number of drugs were successfully clustered by their shared mode-of-action such as PPARɑ agonists and COX inhibitors. The biological meaning underlying each topic was interpreted using diverse sources of information such as functional analysis of the pathways and therapeutic uses of the drugs. Additionally, we found that sample clusters produced by DTM are much more coherent in terms of functional categories when compared to traditional clustering algorithms. We demonstrated that DTM, a text mining technique, can be a powerful computational approach for clustering time-series gene expression profiles with the probabilistic representation of their dynamic features along sequential time frames. The method offers an alternative way for uncovering hidden patterns embedded in time series gene expression profiles to gain enhanced understanding of dynamic behavior of gene regulation in the biological system.

[Chromosomal proteins: histones and acid proteins].

PubMed

Salvini, M; Gabrielli, F

1976-01-01

Experimental data about the chemistry and the biology of chromosomal proteins are reviewed. Paragraphs include: aminoacid sequential data and post-translational covalent modications of histones, histone chemical differences in different tissues of the same species and in homologous organs of different species, histone synthesis subcellular localization and its association with DNA synthesis, histone synthesis transcriptional and translational control, histone synthesis during meiosis, oogenesis and early embryogenesis. The possible role of histones as controllers of gene expression is discussed and a model of primary structure of chromatine is proposed. The "acidic proteins" data concern the high tissue eterogenity of these proteins and their role in the steroid-hormon-controlled gene expression. The possible role of acidic proteins as general controllers of gene expression in eucariotic cells is discussed.

HOXA9 gene expression in the chronic myeloid leukemia progression.

PubMed

Tedeschi, Fabian A; Zalazar, Fabian E

2006-11-01

In the present work we study the HOXA9 expression in sequential samples of patients with CML using RT-PCR. To obtain a semi-quantitative value, the HOXA9 expression was referred to the ABL gene in the same sample. The relative HOXA9 expression was higher in patients in the accelerated phase of the disease (p<0.005). Interestingly, a patient with poorer prognosis (high Sokal's score), showing the highest HOXA9/ABL ratio, quickly entered a blast crisis and died 5 months later. These first results could be considered as an evidence of an actual biological phenomenon that could provide additional information about the HOXA9 role in the CML progression.

Patterning in time and space: HoxB cluster gene expression in the developing chick embryo.

PubMed

Gouveia, Analuce; Marcelino, Hugo M; Gonçalves, Lisa; Palmeirim, Isabel; Andrade, Raquel P

2015-01-01

The developing embryo is a paradigmatic model to study molecular mechanisms of time control in Biology. Hox genes are key players in the specification of tissue identity during embryo development and their expression is under strict temporal regulation. However, the molecular mechanisms underlying timely Hox activation in the early embryo remain unknown. This is hindered by the lack of a rigorous temporal framework of sequential Hox expression within a single cluster. Herein, a thorough characterization of HoxB cluster gene expression was performed over time and space in the early chick embryo. Clear temporal collinearity of HoxB cluster gene expression activation was observed. Spatial collinearity of HoxB expression was evidenced in different stages of development and in multiple tissues. Using embryo explant cultures we showed that HoxB2 is cyclically expressed in the rostral presomitic mesoderm with the same periodicity as somite formation, suggesting a link between timely tissue specification and somite formation. We foresee that the molecular framework herein provided will facilitate experimental approaches aimed at identifying the regulatory mechanisms underlying Hox expression in Time and Space.

Patterning in time and space: HoxB cluster gene expression in the developing chick embryo

PubMed Central

Gouveia, Analuce; Marcelino, Hugo M; Gonçalves, Lisa; Palmeirim, Isabel; Andrade, Raquel P

2015-01-01

The developing embryo is a paradigmatic model to study molecular mechanisms of time control in Biology. Hox genes are key players in the specification of tissue identity during embryo development and their expression is under strict temporal regulation. However, the molecular mechanisms underlying timely Hox activation in the early embryo remain unknown. This is hindered by the lack of a rigorous temporal framework of sequential Hox expression within a single cluster. Herein, a thorough characterization of HoxB cluster gene expression was performed over time and space in the early chick embryo. Clear temporal collinearity of HoxB cluster gene expression activation was observed. Spatial collinearity of HoxB expression was evidenced in different stages of development and in multiple tissues. Using embryo explant cultures we showed that HoxB2 is cyclically expressed in the rostral presomitic mesoderm with the same periodicity as somite formation, suggesting a link between timely tissue specification and somite formation. We foresee that the molecular framework herein provided will facilitate experimental approaches aimed at identifying the regulatory mechanisms underlying Hox expression in Time and Space. PMID:25602523

Short-term administration of rhGH increases markers of cellular proliferation but not milk protein gene expression in normal lactating women

PubMed Central

Maningat, Patricia D.; Sen, Partha; Rijnkels, Monique; Hadsell, Darryl L.; Bray, Molly S.

2011-01-01

Growth hormone is one of few pharmacologic agents known to augment milk production in humans. We hypothesized that recombinant human GH (rhGH) increases the expression of cell proliferation and milk protein synthesis genes. Sequential milk and blood samples collected over four days were obtained from five normal lactating women. Following 24 h of baseline milk and blood sampling, rhGH (0.1 mg/kg/day) was administered subcutaneously once daily for 3 days. Gene expression changes were determined by microarray studies utilizing milk fat globule RNA isolated from each milk sample. Following rhGH administration, DNA synthesis and cell cycle genes were induced, while no significant changes were observed in the expression of milk synthesis genes. Expression of glycolysis and citric acid cycle genes were increased by day 4 compared with day 1, while lipid synthesis genes displayed a circadian-like pattern. Cell cycle gene upregulation occurred after a lag of ∼2 days, likely explaining the failure to increase milk production after only 3 days of rhGH treatment. We conclude that rhGH induces expression of cellular proliferation and metabolism genes but does not induce milk protein gene expression, as potential mechanisms for increasing milk production and could account for the known effect of rhGH to increase milk production following 7–10 days. PMID:21205870

Hox gene expression during postlarval development of the polychaete Alitta virens.

PubMed

Bakalenko, Nadezhda I; Novikova, Elena L; Nesterenko, Alexander Y; Kulakova, Milana A

2013-05-01

Hox genes are the family of transcription factors that play a key role in the patterning of the anterior-posterior axis of all bilaterian animals. These genes display clustered organization and colinear expression. Expression boundaries of individual Hox genes usually correspond with morphological boundaries of the body. Previously, we studied Hox gene expression during larval development of the polychaete Alitta virens (formerly Nereis virens) and discovered that Hox genes are expressed in nereid larva according to the spatial colinearity principle. Adult Alitta virens consist of multiple morphologically similar segments, which are formed sequentially in the growth zone. Since the worm grows for most of its life, postlarval segments constantly change their position along the anterior-posterior axis. We studied the expression dynamics of the Hox cluster during postlarval development of the nereid Alitta virens and found that 8 out of 11 Hox genes are transcribed as wide gene-specific gradients in the ventral nerve cord, ectoderm, and mesoderm. The expression domains constantly shift in accordance with the changing proportions of the growing worm, so expression domains of most Hox genes do not have stable anterior or/and posterior boundaries.In the course of our study, we revealed long antisense RNA (asRNA) for some Hox genes. Expression patterns of two of these genes were analyzed using whole-mount in-situ hybridization. This is the first discovery of antisense RNA for Hox genes in Lophotrochozoa. Hox gene expression in juvenile A. virens differs significantly from Hox gene expression patterns both in A. virens larva and in other Bilateria.We suppose that the postlarval function of the Hox genes in this polychaete is to establish and maintain positional coordinates in a constantly growing body, as opposed to creating morphological difference between segments.

Cooperative working of bacterial chromosome replication proteins generated by a reconstituted protein expression system

PubMed Central

Fujiwara, Kei; Katayama, Tsutomu; Nomura, Shin-ichiro M.

2013-01-01

Replication of all living cells relies on the multirounds flow of the central dogma. Especially, expression of DNA replication proteins is a key step to circulate the processes of the central dogma. Here we achieved the entire sequential transcription–translation–replication process by autonomous expression of chromosomal DNA replication machineries from a reconstituted transcription–translation system (PURE system). We found that low temperature is essential to express a complex protein, DNA polymerase III, in a single tube using the PURE system. Addition of the 13 genes, encoding initiator, DNA helicase, helicase loader, RNA primase and DNA polymerase III to the PURE system gave rise to a DNA replication system by a coupling manner. An artificial genetic circuit demonstrated that the DNA produced as a result of the replication is able to provide genetic information for proteins, indicating the in vitro central dogma can sequentially undergo two rounds. PMID:23737447

Sequential social experiences interact to modulate aggression but not brain gene expression in the honey bee (Apis mellifera).

PubMed

Rittschof, Clare C

2017-01-01

In highly structured societies, individuals behave flexibly and cooperatively in order to achieve a particular group-level outcome. However, even in social species, environmental inputs can have long lasting effects on individual behavior, and variable experiences can even result in consistent individual differences and constrained behavioral flexibility. Despite the fact that such constraints on behavior could have implications for behavioral optimization at the social group level, few studies have explored how social experiences accumulate over time, and the mechanistic basis of these effects. In the current study, I evaluate how sequential social experiences affect individual and group level aggressive phenotypes, and individual brain gene expression, in the highly social honey bee ( Apis mellifera ). To do this, I combine a whole colony chronic predator disturbance treatment with a lab-based manipulation of social group composition. Compared to the undisturbed control, chronically disturbed individuals show lower aggression levels overall, but also enhanced behavioral flexibility in the second, lab-based social context. Disturbed bees display aggression levels that decline with increasing numbers of more aggressive, undisturbed group members. However, group level aggressive phenotypes are similar regardless of the behavioral tendencies of the individuals that make up the group, suggesting a combination of underlying behavioral tendency and negative social feedback influences the aggressive behaviors displayed, particularly in the case of disturbed individuals. An analysis of brain gene expression showed that aggression related biomarker genes reflect an individual's disturbance history, but not subsequent social group experience or behavioral outcomes. In highly social animals with collective behavioral phenotypes, social context may mask underlying variation in individual behavioral tendencies. Moreover, gene expression patterns may reflect behavioral tendency, while behavioral outcomes are further regulated by social cues perceived in real-time.

Genome-Level Longitudinal Expression of Signaling Pathways and Gene Networks in Pediatric Septic Shock

PubMed Central

Shanley, Thomas P; Cvijanovich, Natalie; Lin, Richard; Allen, Geoffrey L; Thomas, Neal J; Doctor, Allan; Kalyanaraman, Meena; Tofil, Nancy M; Penfil, Scott; Monaco, Marie; Odoms, Kelli; Barnes, Michael; Sakthivel, Bhuvaneswari; Aronow, Bruce J; Wong, Hector R

2007-01-01

We have conducted longitudinal studies focused on the expression profiles of signaling pathways and gene networks in children with septic shock. Genome-level expression profiles were generated from whole blood-derived RNA of children with septic shock (n = 30) corresponding to day one and day three of septic shock, respectively. Based on sequential statistical and expression filters, day one and day three of septic shock were characterized by differential regulation of 2,142 and 2,504 gene probes, respectively, relative to controls (n = 15). Venn analysis demonstrated 239 unique genes in the day one dataset, 598 unique genes in the day three dataset, and 1,906 genes common to both datasets. Functional analyses demonstrated time-dependent, differential regulation of genes involved in multiple signaling pathways and gene networks primarily related to immunity and inflammation. Notably, multiple and distinct gene networks involving T cell- and MHC antigen-related biology were persistently downregulated on both day one and day three. Further analyses demonstrated large scale, persistent downregulation of genes corresponding to functional annotations related to zinc homeostasis. These data represent the largest reported cohort of patients with septic shock subjected to longitudinal genome-level expression profiling. The data further advance our genome-level understanding of pediatric septic shock and support novel hypotheses. PMID:17932561

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

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

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

2006-07-28

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

Dynamics of lineage commitment revealed by single-cell transcriptomics of differentiating embryonic stem cells.

PubMed

Semrau, Stefan; Goldmann, Johanna E; Soumillon, Magali; Mikkelsen, Tarjei S; Jaenisch, Rudolf; van Oudenaarden, Alexander

2017-10-23

Gene expression heterogeneity in the pluripotent state of mouse embryonic stem cells (mESCs) has been increasingly well-characterized. In contrast, exit from pluripotency and lineage commitment have not been studied systematically at the single-cell level. Here we measure the gene expression dynamics of retinoic acid driven mESC differentiation from pluripotency to lineage commitment, using an unbiased single-cell transcriptomics approach. We find that the exit from pluripotency marks the start of a lineage transition as well as a transient phase of increased susceptibility to lineage specifying signals. Our study reveals several transcriptional signatures of this phase, including a sharp increase of gene expression variability and sequential expression of two classes of transcriptional regulators. In summary, we provide a comprehensive analysis of the exit from pluripotency and lineage commitment at the single cell level, a potential stepping stone to improved lineage manipulation through timing of differentiation cues.

Transcriptional regulation of fetal to adult hemoglobin switching: new therapeutic opportunities

PubMed Central

Wilber, Andrew; Nienhuis, Arthur W.

2011-01-01

In humans, embryonic, fetal, and adult hemoglobins are sequentially expressed in developing erythroblasts during ontogeny. For the past 40 years, this process has been the subject of intensive study because of its value to enlighten the biology of developmental gene regulation and because fetal hemoglobin can significantly ameliorate the clinical manifestations of both sickle cell disease and β-thalassemia. Understanding the normal process of loss of fetal globin expression and activation of adult globin expression could potentially lead to new therapeutic approaches for these hemoglobin disorders. Herein, we briefly review the history of the study of hemoglobin switching and then focus on recent discoveries in the field that now make new therapeutic approaches seem feasible in the future. Erythroid-specific knockdown of fetal gene repressors or enforced expression of fetal gene activators may provide clinically applicable approaches for genetic treatment of hemoglobin disorders that would benefit from increased fetal hemoglobin levels. PMID:21321359

Small Laccase from "Streptomyces Coelicolor"--An Ideal Model Protein/Enzyme for Undergraduate Laboratory Experience

ERIC Educational Resources Information Center

Cook, Ryan; Hannon, Drew; Southard, Jonathan N.; Majumdar, Sudipta

2018-01-01

A one semester undergraduate biochemistry laboratory experience is described for an understanding of recombinant technology from gene cloning to protein characterization. An integrated experimental design includes three sequential modules: molecular cloning, protein expression and purification, and protein analysis and characterization. Students…

Short-term hyperglycaemia causes non-reversible changes in arterial gene expression in a fully 'switchable' in vivo mouse model of diabetes.

PubMed

Zervou, S; Wang, Y-F; Laiho, A; Gyenesei, A; Kytömäki, L; Hermann, R; Abouna, S; Epstein, D; Pelengaris, S; Khan, M

2010-12-01

Irreversible arterial damage due to early effects of hypo- or hyperglycaemia could account for the limited success of glucose-lowering treatments in preventing cardiovascular disease (CVD) events. We hypothesised that even brief hypo- or hyperglycaemia could adversely affect arterial gene expression and that these changes, moreover, might not be fully reversible. By controlled activation of a 'switchable' c-Myc transgene in beta cells, adult pIns-c-MycER(TAM) mice were rendered transiently hypo- and then hyperglycaemic, after which they were allowed to recover for up to 3 months. Immediate and sequential changes in aortic global gene expression from normal glycaemia through hypo- and hyperglycaemia to recovery were assessed. Gene expression was compared with that of normoglycaemic transgenic and tamoxifen-treated wild-type controls. Overall, expression of 95 genes was significantly affected by moderate hypoglycaemia (glucose down to 2.5 mmol/l), whereas over 769 genes were affected by hyperglycaemia. Genes and pathways activated included several involved in atherogenic processes, such as inflammation and arterial calcification. Although expression of many genes recovered to initial pre-exposure levels when hyperglycaemia was corrected (74.9%), in one in four genes this did not occur. Quantitative reverse transcriptase PCR and immunohistochemistry verified the gene expression patterns of key molecules, as shown by global gene arrays. Short-term exposure to hyperglycaemia can cause deleterious and persistent changes in arterial gene expression in vivo. Brief hypoglycaemia also adversely affects gene expression, although less substantially. Together, these results suggest that early correction of hyperglycaemia and avoidance of hypoglycaemia may both be necessary to avoid excess CVD risk in diabetes.

Sequential changes in the expression of Wnt- and Notch-related genes in the vagina and uterus of ovariectomized mice after estrogen exposure.

PubMed

Nakamura, Takeshi; Miyagawa, Shinichi; Katsu, Yoshinao; Sato, Tomomi; Iguchi, Taisen; Ohta, Yasuhiko

2012-01-01

Estrogen regulates morphological changes in reproductive organs, such as the vagina and uterus, during the estrous cycles in mice. Estrogen depletion by ovariectomy in adults results in atrophy accompanied by apoptosis in vaginal and uterine cells, while estrogen treatment following ovariectomy elicits cell proliferation in both organs. Sequential changes in mRNA expression of wingless-related MMTV integration site (Wnt) and Notch signaling genes were analyzed in the vagina and uterus of ovariectomized adult mice after a single injection of 17β-estradiol to provide understanding over the molecular basis of differences in response to estrogen in these organs. We found estrogen-dependent up-regulation of Wnt4, Wnt5a and p21 and down-regulation of Wnt11, hairy/enhancer-of-split related with YRPW motif-1 (Hey1) and delta-like 4 (Dll4) in the vagina, and up-regulation of Wnt4, Wnt5a, Hey1, Heyl, Dll1, p21 and p53 and down-regulation of Wnt11, Hey2 and Dll4 in the uterus. The expression of Wnt4, Hey1, Hey2, Heyl, Dll1 and p53 showed different patterns after the estrogen injection. Expression patterns for Wnt5a, Wnt11, Dll4 and p21 in the vagina and uterus were similar, suggesting that these genes are involved in the proliferation of cells in both those organs in mice.

SET1A/COMPASS and shadow enhancers in the regulation of homeotic gene expression

PubMed Central

Cao, Kaixiang; Collings, Clayton K.; Marshall, Stacy A.; Morgan, Marc A.; Rendleman, Emily J.; Wang, Lu; Sze, Christie C.; Sun, Tianjiao; Bartom, Elizabeth T.; Shilatifard, Ali

2017-01-01

The homeotic (Hox) genes are highly conserved in metazoans, where they are required for various processes in development, and misregulation of their expression is associated with human cancer. In the developing embryo, Hox genes are activated sequentially in time and space according to their genomic position within Hox gene clusters. Accumulating evidence implicates both enhancer elements and noncoding RNAs in controlling this spatiotemporal expression of Hox genes, but disentangling their relative contributions is challenging. Here, we identify two cis-regulatory elements (E1 and E2) functioning as shadow enhancers to regulate the early expression of the HoxA genes. Simultaneous deletion of these shadow enhancers in embryonic stem cells leads to impaired activation of HoxA genes upon differentiation, while knockdown of a long noncoding RNA overlapping E1 has no detectable effect on their expression. Although MLL/COMPASS (complex of proteins associated with Set1) family of histone methyltransferases is known to activate transcription of Hox genes in other contexts, we found that individual inactivation of the MLL1-4/COMPASS family members has little effect on early Hox gene activation. Instead, we demonstrate that SET1A/COMPASS is required for full transcriptional activation of multiple Hox genes but functions independently of the E1 and E2 cis-regulatory elements. Our results reveal multiple regulatory layers for Hox genes to fine-tune transcriptional programs essential for development. PMID:28487406

Unusual Gene Order and Organization of the Sea Urchin Hox Cluster

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

Cameron, R A; Rowen, L; Nesbitt, R

2005-10-11

The highly consistent gene order and axial colinear expression patterns found in vertebrate hox gene clusters are less well conserved across the rest of bilaterians. We report the first deuterostome instance of an intact hox cluster with a unique gene order where the paralog groups are not expressed in a sequential manner. The finished sequence from BAC clones from the genome of the sea urchin, Strongylocentrotus purpuratus, reveals a gene order wherein the anterior genes (Hox1, Hox2 and Hox3) lie nearest the posterior genes in the cluster such that the most 3 gene is Hox5. (The gene order is :more » 5-Hox1, 2, 3, 11/13c, 11/13b, 11/13a, 9/10, 8, 7, 6, 5 - 3). The finished sequence result is corroborated by restriction mapping evidence and BAC-end scaffold analyses. Comparisons with a putative ancestral deuterostome Hox gene cluster suggest that the rearrangements leading to the sea urchin gene order were many and complex.« less

Unusual Gene Order and Organization of the Sea Urchin HoxCluster

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

Richardson, Paul M.; Lucas, Susan; Cameron, R. Andrew

2005-05-10

The highly consistent gene order and axial colinear expression patterns found in vertebrate hox gene clusters are less well conserved across the rest of bilaterians. We report the first deuterostome instance of an intact hox cluster with a unique gene order where the paralog groups are not expressed in a sequential manner. The finished sequence from BAC clones from the genome of the sea urchin, Strongylocentrotus purpuratus, reveals a gene order wherein the anterior genes (Hox1, Hox2 and Hox3) lie nearest the posterior genes in the cluster such that the most 3' gene is Hox5. (The gene order is :more » 5'-Hox1,2, 3, 11/13c, 11/13b, '11/13a, 9/10, 8, 7, 6, 5 - 3)'. The finished sequence result is corroborated by restriction mapping evidence and BAC-end scaffold analyses. Comparisons with a putative ancestral deuterostome Hox gene cluster suggest that the rearrangements leading to the sea urchin gene order were many and complex.« less

Sequential and combinatorial roles of maf family genes define proper lens development.

PubMed

Reza, Hasan Mahmud; Urano, Atsuyo; Shimada, Naoko; Yasuda, Kunio

2007-01-16

Maf proteins have been shown to play pivotal roles in lens development in vertebrates. The developing chick lens expresses at least three large Maf proteins. However, the transcriptional relationship among the three large maf genes and their various roles in transactivating the downstream genes largely remain to be elucidated. Chick embryos were electroporated with wild-type L-maf, c-maf, and mafB by in ovo electroporation, and their effects on gene expression were determined by in situ hybridization using specific probes or by immunostaining. Endogenous gene expression was determined using nonelectroporated samples. A regulation mechanism exists among the members of maf family gene. An early-expressed member of this gene family typically stimulates the expression of later-expressed members. We also examined the regulation of various lens-expressing genes with a focus on the interaction between different Maf proteins. We found that the transcriptional ability of Maf proteins varies, even when the target is the same, in parallel with their discrete functions. L-Maf and c-Maf have no effect on E-cadherin expression, whereas MafB enhances its expression and thereby impedes lens vesicle formation. This study also revealed that Maf proteins can regulate the expression of gap junction genes, connexins, and their interacting partner, major intrinsic protein (MIP), during lens development. Misexpression of L-Maf and c-Maf induces ectopic expression of Cx43 and MIP; in contrast, MafB appears to have no effect on Cx43, but induces MIP significantly as evidenced from our gain-of-function experiments. Our results indicate that large Maf function is indispensable for chick lens initiation and development. In addition, L-Maf positively regulates most of the essential genes in this program and directs a series of molecular events leading to proper formation of the lens.

Predicting response to primary chemotherapy: gene expression profiling of paraffin-embedded core biopsy tissue.

PubMed

Mina, Lida; Soule, Sharon E; Badve, Sunil; Baehner, Fredrick L; Baker, Joffre; Cronin, Maureen; Watson, Drew; Liu, Mei-Lan; Sledge, George W; Shak, Steve; Miller, Kathy D

2007-06-01

Primary chemotherapy provides an ideal opportunity to correlate gene expression with response to treatment. We used paraffin-embedded core biopsies from a completed phase II trial to identify genes that correlate with response to primary chemotherapy. Patients with newly diagnosed stage II or III breast cancer were treated with sequential doxorubicin 75 mg/M2 q2 wks x 3 and docetaxel 40 mg/M2 weekly x 6; treatment order was randomly assigned. Pretreatment core biopsy samples were interrogated for genes that might correlate with pathologic complete response (pCR). In addition to the individual genes, the correlation of the Oncotype DX Recurrence Score with pCR was examined. Of 70 patients enrolled in the parent trial, core biopsies samples with sufficient RNA for gene analyses were available from 45 patients; 9 (20%) had inflammatory breast cancer (IBC). Six (14%) patients achieved a pCR. Twenty-two of the 274 candidate genes assessed correlated with pCR (p < 0.05). Genes correlating with pCR could be grouped into three large clusters: angiogenesis-related genes, proliferation related genes, and invasion-related genes. Expression of estrogen receptor (ER)-related genes and Recurrence Score did not correlate with pCR. In an exploratory analysis we compared gene expression in IBC to non-inflammatory breast cancer; twenty-four (9%) of the genes were differentially expressed (p < 0.05), 5 were upregulated and 19 were downregulated in IBC. Gene expression analysis on core biopsy samples is feasible and identifies candidate genes that correlate with pCR to primary chemotherapy. Gene expression in IBC differs significantly from noninflammatory breast cancer.

Molecular Dissection of a Major Gene Effect on a Quantitative Trait: The Level of Alcohol Dehydrogenase Expression in Drosophila Melanogaster

PubMed Central

Stam, L. F.; Laurie, C. C.

1996-01-01

A molecular mapping experiment shows that a major gene effect on a quantitative trait, the level of alcohol dehydrogenase expression in Drosophila melanogaster, is due to multiple polymorphisms within the Adh gene. These polymorphisms are located in an intron, the coding sequence, and the 3' untranslated region. Because of nonrandom associations among polymorphisms at different sites, the individual effects combine (in some cases epistatically) to produce ``superalleles'' with large effect. These results have implications for the interpretation of major gene effects detected by quantitative trait locus mapping methods. They show that large effects due to a single locus may be due to multiple associated polymorphisms (or sequential fixations in isolated populations) rather than individual mutations of large effect. PMID:8978044

Y-autosome translocation interferes with meiotic sex inactivation and expression of autosomal genes: a case study in the pig.

PubMed

Barasc, H; Mary, N; Letron, R; Calgaro, A; Dudez, A M; Bonnet, N; Lahbib-Mansais, Y; Yerle, M; Ducos, A; Pinton, A

2012-01-01

Y-autosome translocations are rare in humans and pigs. In both species, these rearrangements can be responsible for meiotic arrest and subsequent infertility. Chromosome pairing abnormalities on the SSCX, SSCY and SSC1 chromatin domains were identified by analyzing pachytene spermatocytes from a boar carrying a (Y;1) translocation by immunolocalization of specific meiotic protein combined with FISH. Disturbance of the meiotic sex chromosome inactivation (MSCI) was observed by Cot-RNA-FISH and analysis of ZFY gene expression by sequential RNA- and DNA-FISH on spermatocytes. We hypothesized that the meiotic arrest observed in this boar might be due to the silencing of critical autosomal genes and/or the reactivation of some sex chromosome genes. Copyright © 2011 S. Karger AG, Basel.

Developmental fate and lineage commitment of singled mouse blastomeres.

PubMed

Lorthongpanich, Chanchao; Doris, Tham Puay Yoke; Limviphuvadh, Vachiranee; Knowles, Barbara B; Solter, Davor

2012-10-01

The inside-outside model has been invoked to explain cell-fate specification of the pre-implantation mammalian embryo. Here, we investigate whether cell-cell interaction can influence the fate specification of embryonic blastomeres by sequentially separating the blastomeres in two-cell stage mouse embryos and continuing separation after each cell division throughout pre-implantation development. This procedure eliminates information provided by cell-cell interaction and cell positioning. Gene expression profiles, polarity protein localization and functional tests of these separated blastomeres reveal that cell interactions, through cell position, influence the fate of the blastomere. Blastomeres, in the absence of cell contact and inner-outer positional information, have a unique pattern of gene expression that is characteristic of neither inner cell mass nor trophectoderm, but overall they have a tendency towards a 'trophectoderm-like' gene expression pattern and preferentially contribute to the trophectoderm lineage.

Human IgG2- and IgG4-expressing memory B cells display enhanced molecular and phenotypic signs of maturity and accumulate with age.

PubMed

de Jong, Britt G; IJspeert, Hanna; Marques, Lemelinda; van der Burg, Mirjam; van Dongen, Jacques Jm; Loos, Bruno G; van Zelm, Menno C

2017-10-01

The mechanisms involved in sequential immunoglobulin G (IgG) class switching are still largely unknown. Sequential IG class switching is linked to higher levels of somatic hypermutation (SHM) in vivo, but it remains unclear if these are generated temporally during an immune response or upon activation in a secondary response. We here aimed to uncouple these processes and to distinguish memory B cells from primary and secondary immune responses. SHM levels and IgG subclasses were studied with 454 pyrosequencing on blood mononuclear cells from young children and adults as models for primary and secondary immunological memory. Additional sequencing and detailed immunophenotyping with IgG subclass-specific antibodies was performed on purified IgG + memory B-cell subsets. In both children and adults, SHM levels were higher in transcripts involving more downstream-located IGHG genes (esp. IGHG2 and IGHG4). In adults, SHM levels were significantly higher than in children, and downstream IGHG genes were more frequently utilized. This was associated with increased frequencies of CD27 + IgG + memory B cells, which contained higher levels of SHM, more IGHG2 usage, and higher expression levels of activation markers than CD27 - IgG + memory B cells. We conclude that secondary immunological memory accumulates with age and these memory B cells express CD27, high levels of activation markers, and carry high SHM levels and frequent usage of IGHG2. These new insights contribute to our understanding of sequential IgG subclass switching and show a potential relevance of using serum IgG2 levels or numbers of IgG2-expressing B cells as markers for efficient generation of memory responses.

UV radiation promotes melanoma dissemination mediated by the sequential reaction axis of cathepsins-TGF-β1-FAP-α.

PubMed

Wäster, Petra; Orfanidis, Kyriakos; Eriksson, Ida; Rosdahl, Inger; Seifert, Oliver; Öllinger, Karin

2017-08-08

Ultraviolet radiation (UVR) is the major risk factor for development of malignant melanoma. Fibroblast activation protein (FAP)-α is a serine protease expressed on the surface of activated fibroblasts, promoting tumour invasion through extracellular matrix (ECM) degradation. The signalling mechanism behind the upregulation of FAP-α is not yet completely revealed. Expression of FAP-α was analysed after UVR exposure in in vitro co-culture systems, gene expression arrays and artificial skin constructs. Cell migration and invasion was studied in relation to cathepsin activity and secretion of transforming growth factor (TGF)-β1. Fibroblast activation protein-α expression was induced by UVR in melanocytes of human skin. The FAP-α expression was regulated by UVR-induced release of TGF-β1 and cathepsin inhibitors prevented such secretion. In melanoma cell culture models and in a xenograft tumour model of zebrafish embryos, FAP-α mediated ECM degradation and facilitated tumour cell dissemination. Our results provide evidence for a sequential reaction axis from UVR via cathepsins, TGF-β1 and FAP-α expression, promoting cancer cell dissemination and melanoma metastatic spread.

Molecular cloning, polymorphisms, and expression analysis of the RERG gene in indigenous Chinese goats.

PubMed

Sui, M X; Wang, H H; Wang, Z W

2015-11-24

The current study aimed to investigate the coding sequence, polymorphisms, and expression of the RERG gene in indigenous Chinese goats. cDNA of RERG, obtained through reverse transcription PCR was analyzed using bioinformatic techniques. Polymorphisms in the exon regions of the RERG gene were identified and their associations with growth traits in three varieties of indigenous Chinese goats were investigated. Expression of the RERG gene in three goat breeds of the same age was detected using real-time quantitative PCR. The results revealed that the cDNA of RERG, which contained a complete open reading frame of 20-620 bp, was 629 bp in length. The associated accession numbers in GenBank are JN672576, JQ917222, and JN580309 for the QianBei Ma goat, the GuiZhou white goat, and the GuiZhou black goat, respectively. Four consistent SNP sites were found in the exon regions of the RERG gene for the three goat breeds. mRNA expression of the RERG gene differed between different tissues in adult goats of same age. The highest expression was observed in lung and spleen tissues, while the lowest expression was recorded in thymus gland tissue. In addition, the expression of the RERG gene in the muscle of Guizhou white goat, GuiZhou black goat, and QianBei Ma goat decreased sequentially. Our results lay the foundations for further investigation into the role of the RERG gene in goat growth traits.

Recrudescence Mechanisms and Gene Expression Profile of the Reproductive Tracts from Chickens during the Molting Period

PubMed Central

Ahn, Suzie E.; Lim, Chul-Hong; Lee, Jin-Young; Bae, Seung-Min; Kim, Jinyoung; Bazer, Fuller W.; Song, Gwonhwa

2013-01-01

The reproductive system of chickens undergoes dynamic morphological and functional tissue remodeling during the molting period. The present study identified global gene expression profiles following oviductal tissue regression and regeneration in laying hens in which molting was induced by feeding high levels of zinc in the diet. During the molting and recrudescence processes, progressive morphological and physiological changes included regression and re-growth of reproductive organs and fluctuations in concentrations of testosterone, progesterone, estradiol and corticosterone in blood. The cDNA microarray analysis of oviductal tissues revealed the biological significance of gene expression-based modulation in oviductal tissue during its remodeling. Based on the gene expression profiles, expression patterns of selected genes such as, TF, ANGPTL3, p20K, PTN, AvBD11 and SERPINB3 exhibited similar patterns in expression with gradual decreases during regression of the oviduct and sequential increases during resurrection of the functional oviduct. Also, miR-1689* inhibited expression of Sp1, while miR-17-3p, miR-22* and miR-1764 inhibited expression of STAT1. Similarly, chicken miR-1562 and miR-138 reduced the expression of ANGPTL3 and p20K, respectively. These results suggest that these differentially regulated genes are closely correlated with the molecular mechanism(s) for development and tissue remodeling of the avian female reproductive tract, and that miRNA-mediated regulation of key genes likely contributes to remodeling of the avian reproductive tract by controlling expression of those genes post-transcriptionally. The discovered global gene profiles provide new molecular candidates responsible for regulating morphological and functional recrudescence of the avian reproductive tract, and provide novel insights into understanding the remodeling process at the genomic and epigenomic levels. PMID:24098561

PSEN1 and PSEN2 gene expression in Alzheimer's disease brain: a new approach.

PubMed

Delabio, Roger; Rasmussen, Lucas; Mizumoto, Igor; Viani, Gustavo-Arruda; Chen, Elizabeth; Villares, João; Costa, Isabela-Bazzo; Turecki, Gustavo; Linde, Sandra Aparecido; Smith, Marilia Cardoso; Payão, Spencer-Luiz

2014-01-01

Presenilin 1 (PSEN1) and presenilin 2 (PSEN2) genes encode the major component of y-secretase, which is responsible for sequential proteolytic cleavages of amyloid precursor proteins and the subsequent formation of amyloid-β peptides. 150 RNA samples from the entorhinal cortex, auditory cortex and hippocampal regions of individuals with Alzheimer's disease (AD) and controls elderly subjects were analyzed with using real-time rtPCR. There were no differences between groups for PSEN1 expression. PSEN2 was significantly downregulated in the auditory cortex of AD patients when compared to controls and when compared to other brain regions of the patients. Alteration in PSEN2 expression may be a risk factor for AD.

The host-pathogen interaction between wheat and yellow rust induces temporally coordinated waves of gene expression.

PubMed

Dobon, Albor; Bunting, Daniel C E; Cabrera-Quio, Luis Enrique; Uauy, Cristobal; Saunders, Diane G O

2016-05-20

Understanding how plants and pathogens modulate gene expression during the host-pathogen interaction is key to uncovering the molecular mechanisms that regulate disease progression. Recent advances in sequencing technologies have provided new opportunities to decode the complexity of such interactions. In this study, we used an RNA-based sequencing approach (RNA-seq) to assess the global expression profiles of the wheat yellow rust pathogen Puccinia striiformis f. sp. tritici (PST) and its host during infection. We performed a detailed RNA-seq time-course for a susceptible and a resistant wheat host infected with PST. This study (i) defined the global gene expression profiles for PST and its wheat host, (ii) substantially improved the gene models for PST, (iii) evaluated the utility of several programmes for quantification of global gene expression for PST and wheat, and (iv) identified clusters of differentially expressed genes in the host and pathogen. By focusing on components of the defence response in susceptible and resistant hosts, we were able to visualise the effect of PST infection on the expression of various defence components and host immune receptors. Our data showed sequential, temporally coordinated activation and suppression of expression of a suite of immune-response regulators that varied between compatible and incompatible interactions. These findings provide the framework for a better understanding of how PST causes disease and support the idea that PST can suppress the expression of defence components in wheat to successfully colonize a susceptible host.

Roles of polypyrimidine tract binding proteins in major immediate-early gene expression and viral replication of human cytomegalovirus.

PubMed

Cosme, Ruth S Cruz; Yamamura, Yasuhiro; Tang, Qiyi

2009-04-01

Human cytomegalovirus (HCMV), a member of the beta subgroup of the family Herpesviridae, causes serious health problems worldwide. HCMV gene expression in host cells is a well-defined sequential process: immediate-early (IE) gene expression, early-gene expression, DNA replication, and late-gene expression. The most abundant IE gene, major IE (MIE) gene pre-mRNA, needs to be spliced before being exported to the cytoplasm for translation. In this study, the regulation of MIE gene splicing was investigated; in so doing, we found that polypyrimidine tract binding proteins (PTBs) strongly repressed MIE gene production in cotransfection assays. In addition, we discovered that the repressive effects of PTB could be rescued by splicing factor U2AF. Taken together, the results suggest that PTBs inhibit MIE gene splicing by competing with U2AF65 for binding to the polypyrimidine tract in pre-mRNA. In intron deletion mutation assays and RNA detection experiments (reverse transcription [RT]-PCR and real-time RT-PCR), we further observed that PTBs target all the introns of the MIE gene, especially intron 2, and affect gene splicing, which was reflected in the variation in the ratio of pre-mRNA to mRNA. Using transfection assays, we demonstrated that PTB knockdown cells induce a higher degree of MIE gene splicing/expression. Consistently, HCMV can produce more viral proteins and viral particles in PTB knockdown cells after infection. We conclude that PTB inhibits HCMV replication by interfering with MIE gene splicing through competition with U2AF for binding to the polypyrimidine tract in MIE gene introns.

Roles of Polypyrimidine Tract Binding Proteins in Major Immediate-Early Gene Expression and Viral Replication of Human Cytomegalovirus▿

PubMed Central

Cosme, Ruth S. Cruz; Yamamura, Yasuhiro; Tang, Qiyi

2009-01-01

Human cytomegalovirus (HCMV), a member of the β subgroup of the family Herpesviridae, causes serious health problems worldwide. HCMV gene expression in host cells is a well-defined sequential process: immediate-early (IE) gene expression, early-gene expression, DNA replication, and late-gene expression. The most abundant IE gene, major IE (MIE) gene pre-mRNA, needs to be spliced before being exported to the cytoplasm for translation. In this study, the regulation of MIE gene splicing was investigated; in so doing, we found that polypyrimidine tract binding proteins (PTBs) strongly repressed MIE gene production in cotransfection assays. In addition, we discovered that the repressive effects of PTB could be rescued by splicing factor U2AF. Taken together, the results suggest that PTBs inhibit MIE gene splicing by competing with U2AF65 for binding to the polypyrimidine tract in pre-mRNA. In intron deletion mutation assays and RNA detection experiments (reverse transcription [RT]-PCR and real-time RT-PCR), we further observed that PTBs target all the introns of the MIE gene, especially intron 2, and affect gene splicing, which was reflected in the variation in the ratio of pre-mRNA to mRNA. Using transfection assays, we demonstrated that PTB knockdown cells induce a higher degree of MIE gene splicing/expression. Consistently, HCMV can produce more viral proteins and viral particles in PTB knockdown cells after infection. We conclude that PTB inhibits HCMV replication by interfering with MIE gene splicing through competition with U2AF for binding to the polypyrimidine tract in MIE gene introns. PMID:19144709

A gene expression system offering multiple levels of regulation: the Dual Drug Control (DDC) system.

PubMed

Sudomoina, Marina; Latypova, Ekaterina; Favorova, Olga O; Golemis, Erica A; Serebriiskii, Ilya G

2004-04-29

Whether for cell culture studies of protein function, construction of mouse models to enable in vivo analysis of disease epidemiology, or ultimately gene therapy of human diseases, a critical enabling step is the ability to achieve finely controlled regulation of gene expression. Previous efforts to achieve this goal have explored inducible drug regulation of gene expression, and construction of synthetic promoters based on two-hybrid paradigms, among others. In this report, we describe the combination of dimerizer-regulated two-hybrid and tetracycline regulatory elements in an ordered cascade, placing expression of endpoint reporters under the control of two distinct drugs. In this Dual Drug Control (DDC) system, a first plasmid expresses fusion proteins to DBD and AD, which interact only in the presence of a small molecule dimerizer; a second plasmid encodes a cassette transcriptionally responsive to the first DBD, directing expression of the Tet-OFF protein; and a third plasmid encodes a reporter gene transcriptionally responsive to binding by Tet-OFF. We evaluate the dynamic range and specificity of this system in comparison to other available systems. This study demonstrates the feasibility of combining two discrete drug-regulated expression systems in a temporally sequential cascade, without loss of dynamic range of signal induction. The efficient layering of control levels allowed by this combination of elements provides the potential for the generation of complex control circuitry that may advance ability to regulate gene expression in vivo.

Assembly and multiple gene expression of thermophilic enzymes in Escherichia coli for in vitro metabolic engineering.

PubMed

Ninh, Pham Huynh; Honda, Kohsuke; Sakai, Takaaki; Okano, Kenji; Ohtake, Hisao

2015-01-01

In vitro reconstitution of an artificial metabolic pathway is an emerging approach for the biocatalytic production of industrial chemicals. However, several enzymes have to be separately prepared (and purified) for the construction of an in vitro metabolic pathway, thereby limiting the practical applicability of this approach. In this study, genes encoding the nine thermophilic enzymes involved in a non-ATP-forming chimeric glycolytic pathway were assembled in an artificial operon and co-expressed in a single recombinant Escherichia coli strain. Gene expression levels of the thermophilic enzymes were controlled by their sequential order in the artificial operon. The specific activities of the recombinant enzymes in the cell-free extract of the multiple-gene-expression E. coli were 5.0-1,370 times higher than those in an enzyme cocktail prepared from a mixture of single-gene-expression strains, in each of which a single one of the nine thermophilic enzymes was overproduced. Heat treatment of a crude extract of the multiple-gene-expression cells led to the denaturation of indigenous proteins and one-step preparation of an in vitro synthetic pathway comprising only a limited number of thermotolerant enzymes. Coupling this in vitro pathway with other thermophilic enzymes including the H2 O-forming NADH oxidase or the malate/lactate dehydrogenase facilitated one-pot conversion of glucose to pyruvate or lactate, respectively. © 2014 Wiley Periodicals, Inc.

Plant defences against ants provide a pathway to social parasitism in butterflies.

PubMed

Patricelli, Dario; Barbero, Francesca; Occhipinti, Andrea; Bertea, Cinzia M; Bonelli, Simona; Casacci, Luca P; Zebelo, Simon A; Crocoll, Christoph; Gershenzon, Jonathan; Maffei, Massimo E; Thomas, Jeremy A; Balletto, Emilio

2015-07-22

Understanding the chemical cues and gene expressions that mediate herbivore-host-plant and parasite-host interactions can elucidate the ecological costs and benefits accruing to different partners in tight-knit community modules, and may reveal unexpected complexities. We investigated the exploitation of sequential hosts by the phytophagous-predaceous butterfly Maculinea arion, whose larvae initially feed on Origanum vulgare flowerheads before switching to parasitize Myrmica ant colonies for their main period of growth. Gravid female butterflies were attracted to Origanum plants that emitted high levels of the monoterpenoid volatile carvacrol, a condition that occurred when ants disturbed their roots: we also found that Origanum expressed four genes involved in monoterpene formation when ants were present, accompanied by a significant induction of jasmonates. When exposed to carvacrol, Myrmica workers upregulated five genes whose products bind and detoxify this biocide, and their colonies were more tolerant of it than other common ant genera, consistent with an observed ability to occupy the competitor-free spaces surrounding Origanum. A cost is potential colony destruction by Ma. arion, which in turn may benefit infested Origanum plants by relieving their roots of further damage. Our results suggest a new pathway, whereby social parasites can detect successive resources by employing plant volatiles to simultaneously select their initial plant food and a suitable sequential host.

Plant defences against ants provide a pathway to social parasitism in butterflies

PubMed Central

Patricelli, Dario; Barbero, Francesca; Occhipinti, Andrea; Bertea, Cinzia M.; Bonelli, Simona; Casacci, Luca P.; Zebelo, Simon A.; Crocoll, Christoph; Gershenzon, Jonathan; Maffei, Massimo E.; Thomas, Jeremy A.; Balletto, Emilio

2015-01-01

Understanding the chemical cues and gene expressions that mediate herbivore–host-plant and parasite–host interactions can elucidate the ecological costs and benefits accruing to different partners in tight-knit community modules, and may reveal unexpected complexities. We investigated the exploitation of sequential hosts by the phytophagous–predaceous butterfly Maculinea arion, whose larvae initially feed on Origanum vulgare flowerheads before switching to parasitize Myrmica ant colonies for their main period of growth. Gravid female butterflies were attracted to Origanum plants that emitted high levels of the monoterpenoid volatile carvacrol, a condition that occurred when ants disturbed their roots: we also found that Origanum expressed four genes involved in monoterpene formation when ants were present, accompanied by a significant induction of jasmonates. When exposed to carvacrol, Myrmica workers upregulated five genes whose products bind and detoxify this biocide, and their colonies were more tolerant of it than other common ant genera, consistent with an observed ability to occupy the competitor-free spaces surrounding Origanum. A cost is potential colony destruction by Ma. arion, which in turn may benefit infested Origanum plants by relieving their roots of further damage. Our results suggest a new pathway, whereby social parasites can detect successive resources by employing plant volatiles to simultaneously select their initial plant food and a suitable sequential host. PMID:26156773

Reverse engineering gene regulatory networks from measurement with missing values.

PubMed

Ogundijo, Oyetunji E; Elmas, Abdulkadir; Wang, Xiaodong

2016-12-01

Gene expression time series data are usually in the form of high-dimensional arrays. Unfortunately, the data may sometimes contain missing values: for either the expression values of some genes at some time points or the entire expression values of a single time point or some sets of consecutive time points. This significantly affects the performance of many algorithms for gene expression analysis that take as an input, the complete matrix of gene expression measurement. For instance, previous works have shown that gene regulatory interactions can be estimated from the complete matrix of gene expression measurement. Yet, till date, few algorithms have been proposed for the inference of gene regulatory network from gene expression data with missing values. We describe a nonlinear dynamic stochastic model for the evolution of gene expression. The model captures the structural, dynamical, and the nonlinear natures of the underlying biomolecular systems. We present point-based Gaussian approximation (PBGA) filters for joint state and parameter estimation of the system with one-step or two-step missing measurements . The PBGA filters use Gaussian approximation and various quadrature rules, such as the unscented transform (UT), the third-degree cubature rule and the central difference rule for computing the related posteriors. The proposed algorithm is evaluated with satisfying results for synthetic networks, in silico networks released as a part of the DREAM project, and the real biological network, the in vivo reverse engineering and modeling assessment (IRMA) network of yeast Saccharomyces cerevisiae . PBGA filters are proposed to elucidate the underlying gene regulatory network (GRN) from time series gene expression data that contain missing values. In our state-space model, we proposed a measurement model that incorporates the effect of the missing data points into the sequential algorithm. This approach produces a better inference of the model parameters and hence, more accurate prediction of the underlying GRN compared to when using the conventional Gaussian approximation (GA) filters ignoring the missing data points.

Gene expression analysis of hypersensitivity to mosquito bite, chronic active EBV infection and NK/T-lymphoma/leukemia.

PubMed

Washio, Kana; Oka, Takashi; Abdalkader, Lamia; Muraoka, Michiko; Shimada, Akira; Oda, Megumi; Sato, Hiaki; Takata, Katsuyoshi; Kagami, Yoshitoyo; Shimizu, Norio; Kato, Seiichi; Kimura, Hiroshi; Nishizaki, Kazunori; Yoshino, Tadashi; Tsukahara, Hirokazu

2017-11-01

The human herpes virus, Epstein-Barr virus (EBV), is a known oncogenic virus and plays important roles in life-threatening T/NK-cell lymphoproliferative disorders (T/NK-cell LPD) such as hypersensitivity to mosquito bite (HMB), chronic active EBV infection (CAEBV), and NK/T-cell lymphoma/leukemia. During the clinical courses of HMB and CAEBV, patients frequently develop malignant lymphomas and the diseases passively progress sequentially. In the present study, gene expression of CD16 (-) CD56 (+) -, EBV (+) HMB, CAEBV, NK-lymphoma, and NK-leukemia cell lines, which were established from patients, was analyzed using oligonucleotide microarrays and compared to that of CD56 bright CD16 dim/- NK cells from healthy donors. Principal components analysis showed that CAEBV and NK-lymphoma cells were relatively closely located, indicating that they had similar expression profiles. Unsupervised hierarchal clustering analyses of microarray data and gene ontology analysis revealed specific gene clusters and identified several candidate genes responsible for disease that can be used to discriminate each category of NK-LPD and NK-cell lymphoma/leukemia.

Integration of multi-omics data for integrative gene regulatory network inference.

PubMed

Zarayeneh, Neda; Ko, Euiseong; Oh, Jung Hun; Suh, Sang; Liu, Chunyu; Gao, Jean; Kim, Donghyun; Kang, Mingon

2017-01-01

Gene regulatory networks provide comprehensive insights and indepth understanding of complex biological processes. The molecular interactions of gene regulatory networks are inferred from a single type of genomic data, e.g., gene expression data in most research. However, gene expression is a product of sequential interactions of multiple biological processes, such as DNA sequence variations, copy number variations, histone modifications, transcription factors, and DNA methylations. The recent rapid advances of high-throughput omics technologies enable one to measure multiple types of omics data, called 'multi-omics data', that represent the various biological processes. In this paper, we propose an Integrative Gene Regulatory Network inference method (iGRN) that incorporates multi-omics data and their interactions in gene regulatory networks. In addition to gene expressions, copy number variations and DNA methylations were considered for multi-omics data in this paper. The intensive experiments were carried out with simulation data, where iGRN's capability that infers the integrative gene regulatory network is assessed. Through the experiments, iGRN shows its better performance on model representation and interpretation than other integrative methods in gene regulatory network inference. iGRN was also applied to a human brain dataset of psychiatric disorders, and the biological network of psychiatric disorders was analysed.

Integration of multi-omics data for integrative gene regulatory network inference

PubMed Central

Zarayeneh, Neda; Ko, Euiseong; Oh, Jung Hun; Suh, Sang; Liu, Chunyu; Gao, Jean; Kim, Donghyun

2017-01-01

Gene regulatory networks provide comprehensive insights and indepth understanding of complex biological processes. The molecular interactions of gene regulatory networks are inferred from a single type of genomic data, e.g., gene expression data in most research. However, gene expression is a product of sequential interactions of multiple biological processes, such as DNA sequence variations, copy number variations, histone modifications, transcription factors, and DNA methylations. The recent rapid advances of high-throughput omics technologies enable one to measure multiple types of omics data, called ‘multi-omics data’, that represent the various biological processes. In this paper, we propose an Integrative Gene Regulatory Network inference method (iGRN) that incorporates multi-omics data and their interactions in gene regulatory networks. In addition to gene expressions, copy number variations and DNA methylations were considered for multi-omics data in this paper. The intensive experiments were carried out with simulation data, where iGRN’s capability that infers the integrative gene regulatory network is assessed. Through the experiments, iGRN shows its better performance on model representation and interpretation than other integrative methods in gene regulatory network inference. iGRN was also applied to a human brain dataset of psychiatric disorders, and the biological network of psychiatric disorders was analysed. PMID:29354189

Hybrid sterility and evolution in Hawaiian Drosophila: differential gene and allele-specific expression analysis of backcross males.

PubMed

Brill, E; Kang, L; Michalak, K; Michalak, P; Price, D K

2016-08-01

The Hawaiian Drosophila are an iconic example of sequential colonization, adaptive radiation and speciation on islands. Genetic and phenotypic analysis of closely related species pairs that exhibit incomplete reproductive isolation can provide insights into the mechanisms of speciation. Drosophila silvestris from Hawai'i Island and Drosophila planitibia from Maui are two closely related allopatric Hawaiian picture-winged Drosophila that produce sterile F1 males but fertile F1 females, a pattern consistent with Haldane's rule. Backcrossing F1 hybrid females between these two species to parental species gives rise to recombinant males with three distinct sperm phenotypes despite a similar genomic background: motile sperm, no sperm (sterile), and immotile sperm. We found that these three reproductive morphologies of backcross hybrid males produce divergent gene expression profiles in testes, as measured with RNA sequencing. There were a total of 71 genes significantly differentially expressed between backcross males with no sperm compared with those backcross males with motile sperm and immotile sperm, but no significant differential gene expression between backcross males with motile sperm and backcross males with immotile sperm. All of these genes were underexpressed in males with no sperm, including a number of genes with previously known activities in adult testis. An allele-specific expression analysis showed overwhelmingly more cis-divergent than trans-divergent genes, with no significant difference in the ratio of cis- and trans-divergent genes among the sperm phenotypes. Overall, the results indicate that the regulation of gene expression involved in sperm production likely diverged relatively rapidly between these two closely related species.

Phenotypic Checkpoints Regulate Neuronal Development

PubMed Central

Ben-Ari, Yehezkel; Spitzer, Nicholas C.

2010-01-01

Nervous system development proceeds by sequential gene expression mediated by cascades of transcription factors in parallel with sequences of patterned network activity driven by receptors and ion channels. These sequences are cell type- and developmental stage-dependent and modulated by paracrine actions of substances released by neurons and glia. How and to what extent these sequences interact to enable neuronal network development is not understood. Recent evidence demonstrates that CNS development requires intermediate stages of differentiation providing functional feedback that influences gene expression. We suggest that embryonic neuronal functions constitute a series of phenotypic checkpoint signatures; neurons failing to express these functions are delayed or developmentally arrested. Such checkpoints are likely to be a general feature of neuronal development and may constitute presymptomatic signatures of neurological disorders when they go awry. PMID:20864191

A C. elegans Hox gene switches on, off, on and off again to regulate proliferation, differentiation and morphogenesis.

PubMed

Salser, S J; Kenyon, C

1996-05-01

Hox genes establish body pattern throughout the animal kingdom, but the role these genes play at the cellular level to modify and shape parts of the body remains a mystery. We find that the C. elegans Antennapedia homolog, mab-5, sequentially programs many independent events within individual cell lineages. In one body region, mab-5 first switches ON in a lineage to stimulate proliferation, then OFF to specify epidermal structures, then ON in just one branch of the lineage to promote neuroblast formation, and finally OFF to permit proper sense organ morphology. In a neighboring lineage, continuous mab-5 expression leads to a different pattern of development. Thus, this Hox gene achieves much of its power to diversify the anteroposterior axis through fine spatiotemporal differences in expression coupled with a changing pattern of cellular response.

Gene Expression Analyses of Subchondral Bone in Early Experimental Osteoarthritis by Microarray

PubMed Central

Chen, YuXian; Shen, Jun; Lu, HuaDing; Zeng, Chun; Ren, JianHua; Zeng, Hua; Li, ZhiFu; Chen, ShaoMing; Cai, DaoZhang; Zhao, Qing

2012-01-01

Osteoarthritis (OA) is a degenerative joint disease that affects both cartilage and bone. A better understanding of the early molecular changes in subchondral bone may help elucidate the pathogenesis of OA. We used microarray technology to investigate the time course of molecular changes in the subchondral bone in the early stages of experimental osteoarthritis in a rat model. We identified 2,234 differentially expressed (DE) genes at 1 week, 1,944 at 2 weeks and 1,517 at 4 weeks post-surgery. Further analyses of the dysregulated genes indicated that the events underlying subchondral bone remodeling occurred sequentially and in a time-dependent manner at the gene expression level. Some of the identified dysregulated genes that were identified have suspected roles in bone development or remodeling; these genes include Alp, Igf1, Tgf β1, Postn, Mmp3, Tnfsf11, Acp5, Bmp5, Aspn and Ihh. The differences in the expression of these genes were confirmed by real-time PCR, and the results indicated that our microarray data accurately reflected gene expression patterns characteristic of early OA. To validate the results of our microarray analysis at the protein level, immunohistochemistry staining was used to investigate the expression of Mmp3 and Aspn protein in tissue sections. These analyses indicate that Mmp3 protein expression completely matched the results of both the microarray and real-time PCR analyses; however, Aspn protein expression was not observed to differ at any time. In summary, our study demonstrated a simple method of separation of subchondral bone sample from the knee joint of rat, which can effectively avoid bone RNA degradation. These findings also revealed the gene expression profiles of subchondral bone in the rat OA model at multiple time points post-surgery and identified important DE genes with known or suspected roles in bone development or remodeling. These genes may be novel diagnostic markers or therapeutic targets for OA. PMID:22384228

Homeobox genes in the rodent pineal gland: roles in development and phenotype maintenance.

PubMed

Rath, Martin F; Rohde, Kristian; Klein, David C; Møller, Morten

2013-06-01

The pineal gland is a neuroendocrine gland responsible for nocturnal synthesis of melatonin. During early development of the rodent pineal gland from the roof of the diencephalon, homeobox genes of the orthodenticle homeobox (Otx)- and paired box (Pax)-families are expressed and are essential for normal pineal development consistent with the well-established role that homeobox genes play in developmental processes. However, the pineal gland appears to be unusual because strong homeobox gene expression persists in the pineal gland of the adult brain. Accordingly, in addition to developmental functions, homeobox genes appear to be key regulators in postnatal phenotype maintenance in this tissue. In this paper, we review ontogenetic and phylogenetic aspects of pineal development and recent progress in understanding the involvement of homebox genes in rodent pineal development and adult function. A working model is proposed for understanding the sequential action of homeobox genes in controlling development and mature circadian function of the mammalian pinealocyte based on knowledge from detailed developmental and daily gene expression analyses in rats, the pineal phenotypes of homebox gene-deficient mice and studies on development of the retinal photoreceptor; the pinealocyte and retinal photoreceptor share features not seen in other tissues and are likely to have evolved from the same ancestral photodetector cell.

Homeobox genes in the rodent pineal gland: roles in development and phenotype maintenance

PubMed Central

Rath, Martin F.; Rohde, Kristian; Klein, David C.; Møller, Morten

2012-01-01

The pineal gland is a neuroendocrine gland responsible for nocturnal synthesis of melatonin. During early development of the rodent pineal gland from the roof of the diencephalon, homeobox genes of the orthodenticle homeobox (Otx)- and paired box (Pax)-families are expressed and are essential for normal pineal development consistent with the well-established role that homeobox genes play in developmental processes. However, the pineal gland appears to be unusual because strong homeobox gene expression persists in the pineal gland of the adult brain. Accordingly, in addition to developmental functions, homeobox genes appear to be key regulators in postnatal phenotype maintenance in this tissue. In this paper, we review ontogenetic and phylogenetic aspects of pineal development and recent progress in understanding the involvement of homebox genes in rodent pineal development and adult function. A working model is proposed for understanding the sequential action of homeobox genes in controlling development and mature circadian function of the mammalian pinealocyte based on knowledge from detailed developmental and daily gene expression analyses in rats, the pineal phenotypes of homebox gene-deficient mice and studies on development of the retinal photoreceptor; the pinealocyte and retinal photoreceptor share features not seen in other tissues and are likely to have evolved from the same ancestral photodetector cell. PMID:23076630

Cell wall invertase as a regulator in determining sequential development of endosperm and embryo through glucose signaling early in seed development.

PubMed

Wang, Lu; Liao, Shengjin; Ruan, Yong-Ling

2013-01-01

Seed development depends on coordination among embryo, endosperm and seed coat. Endosperm undergoes nuclear division soon after fertilization, whereas embryo remains quiescent for a while. Such a developmental sequence is of great importance for proper seed development. However, the underlying mechanism remains unclear. Recent results on the cellular domain- and stage-specific expression of invertase genes in cotton and Arabidopsis revealed that cell wall invertase may positively and specifically regulate nuclear division of endosperm after fertilization, thereby playing a role in determining the sequential development of endosperm and embryo, probably through glucose signaling.

Loss of Cytoplasmic CDK1 Predicts Poor Survival in Human Lung Cancer and Confers Chemotherapeutic Resistance

PubMed Central

Zhang, Chunyu; Elkahloun, Abdel G.; Robertson, Matthew; Gills, Joell J.; Tsurutani, Junji; Shih, Joanna H.; Fukuoka, Junya; Hollander, M. Christine; Harris, Curtis C.; Travis, William D.; Jen, Jin; Dennis, Phillip A.

2011-01-01

The dismal lethality of lung cancer is due to late stage at diagnosis and inherent therapeutic resistance. The incorporation of targeted therapies has modestly improved clinical outcomes, but the identification of new targets could further improve clinical outcomes by guiding stratification of poor-risk early stage patients and individualizing therapeutic choices. We hypothesized that a sequential, combined microarray approach would be valuable to identify and validate new targets in lung cancer. We profiled gene expression signatures during lung epithelial cell immortalization and transformation, and showed that genes involved in mitosis were progressively enhanced in carcinogenesis. 28 genes were validated by immunoblotting and 4 genes were further evaluated in non-small cell lung cancer tissue microarrays. Although CDK1 was highly expressed in tumor tissues, its loss from the cytoplasm unexpectedly predicted poor survival and conferred resistance to chemotherapy in multiple cell lines, especially microtubule-directed agents. An analysis of expression of CDK1 and CDK1-associated genes in the NCI60 cell line database confirmed the broad association of these genes with chemotherapeutic responsiveness. These results have implications for personalizing lung cancer therapy and highlight the potential of combined approaches for biomarker discovery. PMID:21887332

Tumor suppressor genes are larger than apoptosis-effector genes and have more regions of active chromatin: Connection to a stochastic paradigm for sequential gene expression programs.

PubMed

Garcia, Marlene; Mauro, James A; Ramsamooj, Michael; Blanck, George

2015-08-03

Apoptosis- and proliferation-effector genes are substantially regulated by the same transactivators, with E2F-1 and Oct-1 being notable examples. The larger proliferation-effector genes have more binding sites for the transactivators that regulate both sets of genes, and proliferation-effector genes have more regions of active chromatin, i.e, DNase I hypersensitive and histone 3, lysine-4 trimethylation sites. Thus, the size differences between the 2 classes of genes suggest a transcriptional regulation paradigm whereby the accumulation of transcription factors that regulate both sets of genes, merely as an aspect of stochastic behavior, accumulate first on the larger proliferation-effector gene "traps," and then accumulate on the apoptosis effector genes, thereby effecting sequential activation of the 2 different gene sets. As IRF-1 and p53 levels increase, tumor suppressor proteins are first activated, followed by the activation of apoptosis-effector genes, for example during S-phase pausing for DNA repair. Tumor suppressor genes are larger than apoptosis-effector genes and have more IRF-1 and p53 binding sites, thereby likewise suggesting a paradigm for transcription sequencing based on stochastic interactions of transcription factors with different gene classes. In this report, using the ENCODE database, we determined that tumor suppressor genes have a greater number of open chromatin regions and histone 3 lysine-4 trimethylation sites, consistent with the idea that a larger gene size can facilitate earlier transcriptional activation via the inclusion of more transactivator binding sites.

Sequential, Divergent, and Cooperative Requirements of Foxl2a and Foxl2b in Ovary Development and Maintenance of Zebrafish

PubMed Central

Yang, Yan-Jing; Wang, Yang; Li, Zhi; Zhou, Li; Gui, Jian-Fang

2017-01-01

Foxl2 is essential for mammalian ovary maintenance. Although sexually dimorphic expression of foxl2 was observed in many teleosts, its role and regulative mechanism in fish remained largely unclear. In this study, we first identified two transcript variants of foxl2a and its homologous gene foxl2b in zebrafish, and revealed their specific expression in follicular layer cells in a sequential and divergent fashion during ovary differentiation, maturation, and maintenance. Then, homozygous foxl2a mutants (foxl2a−/−) and foxl2b mutants (foxl2b−/−) were constructed and detailed comparisons, such as sex ratio, gonadal histological structure, transcriptome profiling, and dynamic expression of gonadal development-related genes, were carried out. Initial ovarian differentiation and oocyte development occur normally both in foxl2a−/− and foxl2b−/− mutants, but foxl2a and foxl2b disruptions result in premature ovarian failure and partial sex reversal, respectively, in adult females. In foxl2a−/− female mutants, sox9a-amh/cyp19a1a signaling was upregulated at 150 days postfertilization (dpf) and subsequently oocyte apoptosis was triggered after 180 dpf. In contrast, dmrt1 expression was greater at 105 dpf and increased several 100-fold in foxl2b−/− mutated ovaries at 270 dpf, along with other testis-related genes. Finally, homozygous foxl2a−/−/foxl2b−/− double mutants were constructed in which complete sex reversal occurs early and testis-differentiation genes robustly increase at 60 dpf. Given mutual compensation between foxl2a and foxl2b in foxl2b−/− and foxl2a−/− mutants, we proposed a model in which foxl2a and foxl2b cooperate to regulate zebrafish ovary development and maintenance, with foxl2b potentially having a dominant role in preventing the ovary from differentiating as testis, as compared to foxl2a. PMID:28193729

The Chromatin Protein DUET/MMD1 Controls Expression of the Meiotic Gene TDM1 during Male Meiosis in Arabidopsis

PubMed Central

Andreuzza, Sébastien; Nishal, Bindu; Singh, Aparna; Siddiqi, Imran

2015-01-01

Meiosis produces haploid cells essential for sexual reproduction. In yeast, entry into meiosis activates transcription factors which trigger a transcriptional cascade that results in sequential co-expression of early, middle and late meiotic genes. However, these factors are not conserved, and the factors and regulatory mechanisms that ensure proper meiotic gene expression in multicellular eukaryotes are poorly understood. Here, we report that DUET/MMD1, a PHD finger protein essential for Arabidopsis male meiosis, functions as a transcriptional regulator in plant meiosis. We find that DUET-PHD binds H3K4me2 in vitro, and show that this interaction is critical for function during meiosis. We also show that DUET is required for proper microtubule organization during meiosis II, independently of its function in meiosis I. Remarkably, DUET protein shows stage-specific expression, confined to diplotene. We identify two genes TDM1 and JAS with critical functions in cell cycle transitions and spindle organization in male meiosis, as DUET targets, with TDM1 being a direct target. Thus, DUET is required to regulate microtubule organization and cell cycle transitions during male meiosis, and functions as a direct transcription activator of the meiotic gene TDM1. Expression profiling showed reduced expression of a subset comprising about 12% of a known set of meiosis preferred genes in the duet mutant. Our results reveal the action of DUET as a transcriptional regulator during male meiosis in plants, and suggest that transcription of meiotic genes is under stagewise control in plants as in yeast. PMID:26348709

The Chromatin Protein DUET/MMD1 Controls Expression of the Meiotic Gene TDM1 during Male Meiosis in Arabidopsis.

PubMed

Andreuzza, Sébastien; Nishal, Bindu; Singh, Aparna; Siddiqi, Imran

2015-09-01

Meiosis produces haploid cells essential for sexual reproduction. In yeast, entry into meiosis activates transcription factors which trigger a transcriptional cascade that results in sequential co-expression of early, middle and late meiotic genes. However, these factors are not conserved, and the factors and regulatory mechanisms that ensure proper meiotic gene expression in multicellular eukaryotes are poorly understood. Here, we report that DUET/MMD1, a PHD finger protein essential for Arabidopsis male meiosis, functions as a transcriptional regulator in plant meiosis. We find that DUET-PHD binds H3K4me2 in vitro, and show that this interaction is critical for function during meiosis. We also show that DUET is required for proper microtubule organization during meiosis II, independently of its function in meiosis I. Remarkably, DUET protein shows stage-specific expression, confined to diplotene. We identify two genes TDM1 and JAS with critical functions in cell cycle transitions and spindle organization in male meiosis, as DUET targets, with TDM1 being a direct target. Thus, DUET is required to regulate microtubule organization and cell cycle transitions during male meiosis, and functions as a direct transcription activator of the meiotic gene TDM1. Expression profiling showed reduced expression of a subset comprising about 12% of a known set of meiosis preferred genes in the duet mutant. Our results reveal the action of DUET as a transcriptional regulator during male meiosis in plants, and suggest that transcription of meiotic genes is under stagewise control in plants as in yeast.

Measles virus minigenomes encoding two autofluorescent proteins reveal cell-to-cell variation in reporter expression dependent on viral sequences between the transcription units.

PubMed

Rennick, Linda J; Duprex, W Paul; Rima, Bert K

2007-10-01

Transcription from morbillivirus genomes commences at a single promoter in the 3' non-coding terminus, with the six genes being transcribed sequentially. The 3' and 5' untranslated regions (UTRs) of the genes (mRNA sense), together with the intergenic trinucleotide spacer, comprise the non-coding sequences (NCS) of the virus and contain the conserved gene end and gene start signals, respectively. Bicistronic minigenomes containing transcription units (TUs) encoding autofluorescent reporter proteins separated by measles virus (MV) NCS were used to give a direct estimation of gene expression in single, living cells by assessing the relative amounts of each fluorescent protein in each cell. Initially, five minigenomes containing each of the MV NCS were generated. Assays were developed to determine the amount of each fluorescent protein in cells at both cell population and single-cell levels. This revealed significant variations in gene expression between cells expressing the same NCS-containing minigenome. The minigenome containing the M/F NCS produced significantly lower amounts of fluorescent protein from the second TU (TU2), compared with the other minigenomes. A minigenome with a truncated F 5' UTR had increased expression from TU2. This UTR is 524 nt longer than the other MV 5' UTRs. Insertions into the 5' UTR of the enhanced green fluorescent protein gene in the minigenome containing the N/P NCS showed that specific sequences, rather than just the additional length of F 5' UTR, govern this decreased expression from TU2.

Transforming Growth Factor-β/SMAD Target Gene SKIL Is Negatively Regulated by the Transcriptional Cofactor Complex SNON-SMAD4*

PubMed Central

Tecalco-Cruz, Angeles C.; Sosa-Garrocho, Marcela; Vázquez-Victorio, Genaro; Ortiz-García, Layla; Domínguez-Hüttinger, Elisa; Macías-Silva, Marina

2012-01-01

The human SKI-like (SKIL) gene encodes the SMAD transcriptional corepressor SNON that antagonizes TGF-β signaling. SNON protein levels are tightly regulated by the TGF-β pathway: whereas a short stimulation with TGF-β decreases SNON levels by its degradation via the proteasome, longer TGF-β treatment increases SNON levels by inducing SKIL gene expression. Here, we investigated the molecular mechanisms involved in the self-regulation of SKIL gene expression by SNON. Bioinformatics analysis showed that the human SKIL gene proximal promoter contains a TGF-β response element (TRE) bearing four groups of SMAD-binding elements that are also conserved in mouse. Two regions of 408 and 648 bp of the human SKIL gene (∼2.4 kb upstream of the ATG initiation codon) containing the core promoter, transcription start site, and the TRE were cloned for functional analysis. Binding of SMAD and SNON proteins to the TRE region of the SKIL gene promoter after TGF-β treatment was demonstrated by ChIP and sequential ChIP assays. Interestingly, the SNON-SMAD4 complex negatively regulated basal SKIL gene expression through binding the promoter and recruiting histone deacetylases. In response to TGF-β signal, SNON is removed from the SKIL gene promoter, and then the activated SMAD complexes bind the promoter to induce SKIL gene expression. Subsequently, the up-regulated SNON protein in complex with SMAD4 represses its own expression as part of the negative feedback loop regulating the TGF-β pathway. Accordingly, when the SNON-SMAD4 complex is absent as in some cancer cells lacking SMAD4 the regulation of some TGF-β target genes is modified. PMID:22674574

Transforming growth factor-β/SMAD Target gene SKIL is negatively regulated by the transcriptional cofactor complex SNON-SMAD4.

PubMed

Tecalco-Cruz, Angeles C; Sosa-Garrocho, Marcela; Vázquez-Victorio, Genaro; Ortiz-García, Layla; Domínguez-Hüttinger, Elisa; Macías-Silva, Marina

2012-08-03

The human SKI-like (SKIL) gene encodes the SMAD transcriptional corepressor SNON that antagonizes TGF-β signaling. SNON protein levels are tightly regulated by the TGF-β pathway: whereas a short stimulation with TGF-β decreases SNON levels by its degradation via the proteasome, longer TGF-β treatment increases SNON levels by inducing SKIL gene expression. Here, we investigated the molecular mechanisms involved in the self-regulation of SKIL gene expression by SNON. Bioinformatics analysis showed that the human SKIL gene proximal promoter contains a TGF-β response element (TRE) bearing four groups of SMAD-binding elements that are also conserved in mouse. Two regions of 408 and 648 bp of the human SKIL gene (∼2.4 kb upstream of the ATG initiation codon) containing the core promoter, transcription start site, and the TRE were cloned for functional analysis. Binding of SMAD and SNON proteins to the TRE region of the SKIL gene promoter after TGF-β treatment was demonstrated by ChIP and sequential ChIP assays. Interestingly, the SNON-SMAD4 complex negatively regulated basal SKIL gene expression through binding the promoter and recruiting histone deacetylases. In response to TGF-β signal, SNON is removed from the SKIL gene promoter, and then the activated SMAD complexes bind the promoter to induce SKIL gene expression. Subsequently, the up-regulated SNON protein in complex with SMAD4 represses its own expression as part of the negative feedback loop regulating the TGF-β pathway. Accordingly, when the SNON-SMAD4 complex is absent as in some cancer cells lacking SMAD4 the regulation of some TGF-β target genes is modified.

Printing 2-dimentional droplet array for single-cell reverse transcription quantitative PCR assay with a microfluidic robot.

PubMed

Zhu, Ying; Zhang, Yun-Xia; Liu, Wen-Wen; Ma, Yan; Fang, Qun; Yao, Bo

2015-04-01

This paper describes a nanoliter droplet array-based single-cell reverse transcription quantitative PCR (RT-qPCR) assay method for quantifying gene expression in individual cells. By sequentially printing nanoliter-scale droplets on microchip using a microfluidic robot, all liquid-handling operations including cell encapsulation, lysis, reverse transcription, and quantitative PCR with real-time fluorescence detection, can be automatically achieved. The inhibition effect of cell suspension buffer on RT-PCR assay was comprehensively studied to achieve high-sensitivity gene quantification. The present system was applied in the quantitative measurement of expression level of mir-122 in single Huh-7 cells. A wide distribution of mir-122 expression in single cells from 3061 copies/cell to 79998 copies/cell was observed, showing a high level of cell heterogeneity. With the advantages of full-automation in liquid-handling, simple system structure, and flexibility in achieving multi-step operations, the present method provides a novel liquid-handling mode for single cell gene expression analysis, and has significant potentials in transcriptional identification and rare cell analysis.

Printing 2-Dimentional Droplet Array for Single-Cell Reverse Transcription Quantitative PCR Assay with a Microfluidic Robot

PubMed Central

Zhu, Ying; Zhang, Yun-Xia; Liu, Wen-Wen; Ma, Yan; Fang, Qun; Yao, Bo

2015-01-01

This paper describes a nanoliter droplet array-based single-cell reverse transcription quantitative PCR (RT-qPCR) assay method for quantifying gene expression in individual cells. By sequentially printing nanoliter-scale droplets on microchip using a microfluidic robot, all liquid-handling operations including cell encapsulation, lysis, reverse transcription, and quantitative PCR with real-time fluorescence detection, can be automatically achieved. The inhibition effect of cell suspension buffer on RT-PCR assay was comprehensively studied to achieve high-sensitivity gene quantification. The present system was applied in the quantitative measurement of expression level of mir-122 in single Huh-7 cells. A wide distribution of mir-122 expression in single cells from 3061 copies/cell to 79998 copies/cell was observed, showing a high level of cell heterogeneity. With the advantages of full-automation in liquid-handling, simple system structure, and flexibility in achieving multi-step operations, the present method provides a novel liquid-handling mode for single cell gene expression analysis, and has significant potentials in transcriptional identification and rare cell analysis. PMID:25828383

Uncovering the abilities of Agaricus bisporus to degrade plant biomass throughout its life cycle.

PubMed

Patyshakuliyeva, Aleksandrina; Post, Harm; Zhou, Miaomiao; Jurak, Edita; Heck, Albert J R; Hildén, Kristiina S; Kabel, Mirjam A; Mäkelä, Miia R; Altelaar, Maarten A F; de Vries, Ronald P

2015-08-01

The economically important edible basidiomycete mushroom Agaricus bisporus thrives on decaying plant material in forests and grasslands of North America and Europe. It degrades forest litter and contributes to global carbon recycling, depolymerizing (hemi-)cellulose and lignin in plant biomass. Relatively little is known about how A. bisporus grows in the controlled environment in commercial production facilities and utilizes its substrate. Using transcriptomics and proteomics, we showed that changes in plant biomass degradation by A. bisporus occur throughout its life cycle. Ligninolytic genes were only highly expressed during the spawning stage day 16. In contrast, (hemi-)cellulolytic genes were highly expressed at the first flush, whereas low expression was observed at the second flush. The essential role for many highly expressed plant biomass degrading genes was supported by exo-proteome analysis. Our data also support a model of sequential lignocellulose degradation by wood-decaying fungi proposed in previous studies, concluding that lignin is degraded at the initial stage of growth in compost and is not modified after the spawning stage. The observed differences in gene expression involved in (hemi-)cellulose degradation between the first and second flushes could partially explain the reduction in the number of mushrooms during the second flush. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

Steroid hormone induction of temporal gene expression in Drosophila brain neuroblasts generates neuronal and glial diversity.

PubMed

Syed, Mubarak Hussain; Mark, Brandon; Doe, Chris Q

2017-04-10

An important question in neuroscience is how stem cells generate neuronal diversity. During Drosophila embryonic development, neural stem cells (neuroblasts) sequentially express transcription factors that generate neuronal diversity; regulation of the embryonic temporal transcription factor cascade is lineage-intrinsic. In contrast, larval neuroblasts generate longer ~50 division lineages, and currently only one mid-larval molecular transition is known: Chinmo/Imp/Lin-28+ neuroblasts transition to Syncrip+ neuroblasts. Here we show that the hormone ecdysone is required to down-regulate Chinmo/Imp and activate Syncrip, plus two late neuroblast factors, Broad and E93. We show that Seven-up triggers Chinmo/Imp to Syncrip/Broad/E93 transition by inducing expression of the Ecdysone receptor in mid-larval neuroblasts, rendering them competent to respond to the systemic hormone ecdysone. Importantly, late temporal gene expression is essential for proper neuronal and glial cell type specification. This is the first example of hormonal regulation of temporal factor expression in Drosophila embryonic or larval neural progenitors.

Sequential expression of cyclooxygenase-2, glutamate receptor-2, and platelet activating factor receptor in rat hippocampal neurons after fluid percussion injury

PubMed Central

Li, Zhiqiang; Shu, Qingming; Li, Lingzhi; Ge, Maolin; Zhang, Yongliang

2014-01-01

Traumatic brain injury causes gene expression changes in different brain regions. Occurrence and development of traumatic brain injury are closely related, involving expression of three factors, namely cyclooxygenase-2, glutamate receptor-2, and platelet activating factor receptor. However, little is known about the correlation of these three factors and brain neuronal injury. In this study, primary cultured rat hippocampal neurons were subjected to fluid percussion injury according to Scott's method, with some modifications. RT-PCR and semi-quantitative immunocytochemical staining was used to measure the expression levels of cyclooxygenase-2, glutamate receptor-2, and platelet activating factor receptor. Our results found that cyclooxygenase-2 expression were firstly increased post-injury, and then decreased. Both mRNA and protein expression levels reached peaks at 8 and 12 hours post-injury, respectively. Similar sequential changes in glutamate receptor 2 were observed, with highest levels mRNA and protein expression at 8 and 12 hours post-injury respectively. On the contrary, the expressions of platelet activating factor receptor were firstly decreased post-injury, and then increased. Both mRNA and protein expression levels reached the lowest levels at 8 and 12 hours post-injury, respectively. Totally, our findings suggest that these three factors are involved in occurrence and development of hippocampal neuronal injury. PMID:25206921

A sequential EMT-MET mechanism drives the differentiation of human embryonic stem cells towards hepatocytes.

PubMed

Li, Qiuhong; Hutchins, Andrew P; Chen, Yong; Li, Shengbiao; Shan, Yongli; Liao, Baojian; Zheng, Dejin; Shi, Xi; Li, Yinxiong; Chan, Wai-Yee; Pan, Guangjin; Wei, Shicheng; Shu, Xiaodong; Pei, Duanqing

2017-05-03

Reprogramming has been shown to involve EMT-MET; however, its role in cell differentiation is unclear. We report here that in vitro differentiation of hESCs to hepatic lineage undergoes a sequential EMT-MET with an obligatory intermediate mesenchymal phase. Gene expression analysis reveals that Activin A-induced formation of definitive endoderm (DE) accompanies a synchronous EMT mediated by autocrine TGFβ signalling followed by a MET process. Pharmacological inhibition of TGFβ signalling blocks the EMT as well as DE formation. We then identify SNAI1 as the key EMT transcriptional factor required for the specification of DE. Genetic ablation of SNAI1 in hESCs does not affect the maintenance of pluripotency or neural differentiation, but completely disrupts the formation of DE. These results reveal a critical mesenchymal phase during the acquisition of DE, highlighting a role for sequential EMT-METs in both differentiation and reprogramming.

Comparison of gene expression profiles in primary and immortalized human pterygium fibroblast cells.

PubMed

Hou, Aihua; Voorhoeve, P Mathijs; Lan, Wanwen; Tin, Minqi; Tong, Louis

2013-11-01

Pterygium is a fibrovascular growth on the ocular surface with corneal tissue destruction, matrix degradation and varying extents of chronic inflammation. To facilitate investigation of pterygium etiology, we immortalized pterygium fibroblast cells and profiled their global transcript levels compared to primary cultured cells. Fibroblast cells were cultured from surgically excised pterygium tissue using the explant method and propagated to passage number 2-4. We hypothesized that intervention with 3 critical molecular intermediates may be necessary to propage these cells. Primary fibroblast cells were immortalized sequentially by a retroviral construct containing the human telomerase reverse transcriptase gene and another retroviral expression vector expressing p53/p16 shRNAs. Primary and immortalized fibroblast cells were evaluated for differences in global gene transcript levels using an Agilent Genechip microarray. Light microscopic morphology of immortalized cells was similar to primary pterygium fibroblast at passage 2-4. Telomerase reverse transcriptase was expressed, and p53 and p16 levels were reduced in immortalized pterygium fibroblast cells. There were 3308 significantly dysregulated genes showing at least 2 fold changes in transcript levels between immortalized and primary cultured cells (2005 genes were up-regulated and 1303 genes were down-regulated). Overall, 13.58% (95% CI: 13.08-14.10) of transcripts in immortalized cells were differentially expressed by at least 2 folds compared to primary cells. Pterygium primary fibroblast cells were successfully immortalized to at least passage 11. Although a variety of genes are differentially expressed between immortalized and primary cells, only genes related to cell cycle are significantly changed, suggesting that the immortalized cells may be used as an in vitro model for pterygium pathology. © 2013 Elsevier Inc. All rights reserved.

Disruption of transforming growth factor-beta signaling by curcumin induces gene expression of peroxisome proliferator-activated receptor-gamma in rat hepatic stellate cells.

PubMed

Zheng, Shizhong; Chen, Anping

2007-01-01

Activation of hepatic stellate cells (HSC), the major effectors of hepatic fibrogenesis, is coupled with sequential alterations in gene expression, including an increase in receptors for transforming growth factor-beta (TGF-beta) and a dramatic reduction in the peroxisome proliferator-activated receptor-gamma (PPAR-gamma). The relationship between them remains obscure. We previously demonstrated that curcumin induced gene expression of PPAR-gamma in activated HSC, leading to reducing cell proliferation, inducing apoptosis and suppressing expression of extracellular matrix genes. The underlying molecular mechanisms are largely unknown. We recently observed that stimulation of PPAR-gamma activation suppressed gene expression of TGF-beta receptors in activated HSC, leading to the interruption of TGF-beta signaling. This observation supported our assumption of an antagonistic relationship between PPAR-gamma activation and TGF-beta signaling in HSC. In this study, we further hypothesize that TGF-beta signaling might negatively regulate gene expression of PPAR-gamma in activated HSC. The present report demonstrates that exogenous TGF-beta1 inhibits gene expression of PPAR-gamma in activated HSC, which is eliminated by the pretreatment with curcumin likely by interrupting TGF-beta signaling. Transfection assays further indicate that blocking TGF-beta signaling by dominant negative type II TGF-beta receptor increases the promoter activity of PPAR-gamma gene. Promoter deletion assays, site-directed mutageneses, and gel shift assays localize two Smad binding elements (SBEs) in the PPAR-gamma gene promoter, acting as curcumin response elements and negatively regulating the promoter activity in passaged HSC. The Smad3/4 protein complex specifically binds to the SBEs. Overexpression of Smad4 dose dependently eliminates the inhibitory effects of curcumin on the PPAR-gamma gene promoter and TGF-beta signaling. Taken together, these results demonstrate that the interruption of TGF-beta signaling by curcumin induces gene expression of PPAR-gamma in activated HSC in vitro. Our studies provide novel insights into the molecular mechanisms of curcumin in the induction of PPAR-gamma gene expression and in the inhibition of HSC activation.

Sequential establishment of stripe patterns in an expanding cell population.

PubMed

Liu, Chenli; Fu, Xiongfei; Liu, Lizhong; Ren, Xiaojing; Chau, Carlos K L; Li, Sihong; Xiang, Lu; Zeng, Hualing; Chen, Guanhua; Tang, Lei-Han; Lenz, Peter; Cui, Xiaodong; Huang, Wei; Hwa, Terence; Huang, Jian-Dong

2011-10-14

Periodic stripe patterns are ubiquitous in living organisms, yet the underlying developmental processes are complex and difficult to disentangle. We describe a synthetic genetic circuit that couples cell density and motility. This system enabled programmed Escherichia coli cells to form periodic stripes of high and low cell densities sequentially and autonomously. Theoretical and experimental analyses reveal that the spatial structure arises from a recurrent aggregation process at the front of the continuously expanding cell population. The number of stripes formed could be tuned by modulating the basal expression of a single gene. The results establish motility control as a simple route to establishing recurrent structures without requiring an extrinsic pacemaker.

miRNA863-3p sequentially targets negative regulators—atypical receptor-like Pseudokinases—and serrate, a positive regulator of plant immunity upon infection

USDA-ARS?s Scientific Manuscript database

Plant small RNAs (sRNAs) play important roles in regulating gene expression during pathogen infection. We identified miR863-3p, which is specifically induced by the avirulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000 carrying the effector avrRpt2. During early infection stages, miR86...

Unconventional function of an Achaete-Scute homolog as a terminal selector of nociceptive neuron identity

PubMed Central

Masoudi, Neda; Tavazoie, Saeed; Glenwinkel, Lori; Ryu, Leesun; Kim, Kyuhyung

2018-01-01

Proneural genes are among the most early-acting genes in nervous system development, instructing blast cells to commit to a neuronal fate. Drosophila Atonal and Achaete-Scute complex (AS-C) genes, as well as their vertebrate orthologs, are basic helix-loop-helix (bHLH) transcription factors with such proneural activity. We show here that a C. elegans AS-C homolog, hlh-4, functions in a fundamentally different manner. In the embryonic, larval, and adult nervous systems, hlh-4 is expressed exclusively in a single nociceptive neuron class, ADL, and its expression in ADL is maintained via transcriptional autoregulation throughout the life of the animal. However, in hlh-4 null mutants, the ADL neuron is generated and still appears neuronal in overall morphology and expression of panneuronal and pansensory features. Rather than acting as a proneural gene, we find that hlh-4 is required for the ADL neuron to function properly, to adopt its correct morphology, to express its unusually large repertoire of olfactory receptor–encoding genes, and to express other known features of terminal ADL identity, including neurotransmitter phenotype, neuropeptides, ion channels, and electrical synapse proteins. hlh-4 is sufficient to induce ADL identity features upon ectopic expression in other neuron types. The expression of ADL terminal identity features is directly controlled by HLH-4 via a phylogenetically conserved E-box motif, which, through bioinformatic analysis, we find to constitute a predictive feature of ADL-expressed terminal identity markers. The lineage that produces the ADL neuron was previously shown to require the conventional, transient proneural activity of another AS-C homolog, hlh-14, demonstrating sequential activities of distinct AS-C-type bHLH genes in neuronal specification. Taken together, we have defined here an unconventional function of an AS-C-type bHLH gene as a terminal selector of neuronal identity and we speculate that such function could be reflective of an ancestral function of an “ur-” bHLH gene. PMID:29672507

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

PubMed

Marco Antonio, David S; Hartfelder, Klaus

2017-01-01

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

A comprehensive analysis on preservation patterns of gene co-expression networks during Alzheimer's disease progression.

PubMed

Ray, Sumanta; Hossain, Sk Md Mosaddek; Khatun, Lutfunnesa; Mukhopadhyay, Anirban

2017-12-20

Alzheimer's disease (AD) is a chronic neuro-degenerative disruption of the brain which involves in large scale transcriptomic variation. The disease does not impact every regions of the brain at the same time, instead it progresses slowly involving somewhat sequential interaction with different regions. Analysis of the expression patterns of the genes in different regions of the brain influenced in AD surely contribute for a enhanced comprehension of AD pathogenesis and shed light on the early characterization of the disease. Here, we have proposed a framework to identify perturbation and preservation characteristics of gene expression patterns across six distinct regions of the brain ("EC", "HIP", "PC", "MTG", "SFG", and "VCX") affected in AD. Co-expression modules were discovered considering a couple of regions at once. These are then analyzed to know the preservation and perturbation characteristics. Different module preservation statistics and a rank aggregation mechanism have been adopted to detect the changes of expression patterns across brain regions. Gene ontology (GO) and pathway based analysis were also carried out to know the biological meaning of preserved and perturbed modules. In this article, we have extensively studied the preservation patterns of co-expressed modules in six distinct brain regions affected in AD. Some modules are emerged as the most preserved while some others are detected as perturbed between a pair of brain regions. Further investigation on the topological properties of preserved and non-preserved modules reveals a substantial association amongst "betweenness centrality" and "degree" of the involved genes. Our findings may render a deeper realization of the preservation characteristics of gene expression patterns in discrete brain regions affected by AD.

Nicotinamide N-methyltransferase expression in SH-SY5Y neuroblastoma and N27 mesencephalic neurones induces changes in cell morphology via ephrin-B2 and Akt signalling

PubMed Central

Thomas, M G; Saldanha, M; Mistry, R J; Dexter, D T; Ramsden, D B; Parsons, R B

2013-01-01

Nicotinamide N-methyltransferase (NNMT, E.C. 2.1.1.1) N-methylates nicotinamide to produce 1-methylnicotinamide (MeN). We have previously shown that NNMT expression protected against neurotoxin-mediated cell death by increasing Complex I (CxI) activity, resulting in increased ATP synthesis. This was mediated via protection of the NDUFS3 subunit of CxI from degradation by increased MeN production. In the present study, we have investigated the effects of NNMT expression on neurone morphology and differentiation. Expression of NNMT in SH-SY5Y human neuroblastoma and N27 rat mesencephalic dopaminergic neurones increased neurite branching, synaptophysin expression and dopamine accumulation and release. siRNA gene silencing of ephrin B2 (EFNB2), and inhibition of Akt phosphorylation using LY294002, demonstrated that their sequential activation was responsible for the increases observed. Incubation of SH-SY5Y with increasing concentrations of MeN also increased neurite branching, suggesting that the effects of NNMT may be mediated by MeN. NNMT had no significant effect on the expression of phenotypic and post-mitotic markers, suggesting that NNMT is not involved in determining phenotypic fate or differentiation status. These results demonstrate that NNMT expression regulates neurone morphology in vitro via the sequential activation of the EFNB2 and Akt cellular signalling pathways. PMID:23764850

Nicotinamide N-methyltransferase expression in SH-SY5Y neuroblastoma and N27 mesencephalic neurones induces changes in cell morphology via ephrin-B2 and Akt signalling.

PubMed

Thomas, M G; Saldanha, M; Mistry, R J; Dexter, D T; Ramsden, D B; Parsons, R B

2013-06-13

Nicotinamide N-methyltransferase (NNMT, E.C. 2.1.1.1) N-methylates nicotinamide to produce 1-methylnicotinamide (MeN). We have previously shown that NNMT expression protected against neurotoxin-mediated cell death by increasing Complex I (CxI) activity, resulting in increased ATP synthesis. This was mediated via protection of the NDUFS3 subunit of CxI from degradation by increased MeN production. In the present study, we have investigated the effects of NNMT expression on neurone morphology and differentiation. Expression of NNMT in SH-SY5Y human neuroblastoma and N27 rat mesencephalic dopaminergic neurones increased neurite branching, synaptophysin expression and dopamine accumulation and release. siRNA gene silencing of ephrin B2 (EFNB2), and inhibition of Akt phosphorylation using LY294002, demonstrated that their sequential activation was responsible for the increases observed. Incubation of SH-SY5Y with increasing concentrations of MeN also increased neurite branching, suggesting that the effects of NNMT may be mediated by MeN. NNMT had no significant effect on the expression of phenotypic and post-mitotic markers, suggesting that NNMT is not involved in determining phenotypic fate or differentiation status. These results demonstrate that NNMT expression regulates neurone morphology in vitro via the sequential activation of the EFNB2 and Akt cellular signalling pathways.

Decoding the Regulatory Network for Blood Development from Single-Cell Gene Expression Measurements

PubMed Central

Haghverdi, Laleh; Lilly, Andrew J.; Tanaka, Yosuke; Wilkinson, Adam C.; Buettner, Florian; Macaulay, Iain C.; Jawaid, Wajid; Diamanti, Evangelia; Nishikawa, Shin-Ichi; Piterman, Nir; Kouskoff, Valerie; Theis, Fabian J.; Fisher, Jasmin; Göttgens, Berthold

2015-01-01

Here we report the use of diffusion maps and network synthesis from state transition graphs to better understand developmental pathways from single cell gene expression profiling. We map the progression of mesoderm towards blood in the mouse by single-cell expression analysis of 3,934 cells, capturing cells with blood-forming potential at four sequential developmental stages. By adapting the diffusion plot methodology for dimensionality reduction to single-cell data, we reconstruct the developmental journey to blood at single-cell resolution. Using transitions between individual cellular states as input, we develop a single-cell network synthesis toolkit to generate a computationally executable transcriptional regulatory network model that recapitulates blood development. Model predictions were validated by showing that Sox7 inhibits primitive erythropoiesis, and that Sox and Hox factors control early expression of Erg. We therefore demonstrate that single-cell analysis of a developing organ coupled with computational approaches can reveal the transcriptional programs that control organogenesis. PMID:25664528

Thyroid hormone activation of retinoic acid synthesis in hypothalamic tanycytes.

PubMed

Stoney, Patrick N; Helfer, Gisela; Rodrigues, Diana; Morgan, Peter J; McCaffery, Peter

2016-03-01

Thyroid hormone (TH) is essential for adult brain function and its actions include several key roles in the hypothalamus. Although TH controls gene expression via specific TH receptors of the nuclear receptor class, surprisingly few genes have been demonstrated to be directly regulated by TH in the hypothalamus, or the adult brain as a whole. This study explored the rapid induction by TH of retinaldehyde dehydrogenase 1 (Raldh1), encoding a retinoic acid (RA)-synthesizing enzyme, as a gene specifically expressed in hypothalamic tanycytes, cells that mediate a number of actions of TH in the hypothalamus. The resulting increase in RA may then regulate gene expression via the RA receptors, also of the nuclear receptor class. In vivo exposure of the rat to TH led to a significant and rapid increase in hypothalamic Raldh1 within 4 hours. That this may lead to an in vivo increase in RA is suggested by the later induction by TH of the RA-responsive gene Cyp26b1. To explore the actions of RA in the hypothalamus as a potential mediator of TH control of gene regulation, an ex vivo hypothalamic rat slice culture method was developed in which the Raldh1-expressing tanycytes were maintained. These slice cultures confirmed that TH did not act on genes regulating energy balance but could induce Raldh1. RA has the potential to upregulate expression of genes involved in growth and appetite, Ghrh and Agrp. This regulation is acutely sensitive to epigenetic changes, as has been shown for TH action in vivo. These results indicate that sequential triggering of two nuclear receptor signalling systems has the capability to mediate some of the functions of TH in the hypothalamus. © 2015 Wiley Periodicals, Inc.

Simultaneous regulation of apoptotic gene silencing and angiogenic gene expression for myocardial infarction therapy: Single-carrier delivery of SHP-1 siRNA and VEGF-expressing pDNA.

PubMed

Kim, Dongkyu; Ku, Sook Hee; Kim, Hyosuk; Jeong, Ji Hoon; Lee, Minhyung; Kwon, Ick Chan; Choi, Donghoon; Kim, Sun Hwa

2016-12-10

Gene therapy is aimed at selectively knocking up or knocking down the target genes involved in the development of diseases. In many human diseases, dysregulation of disease-associated genes is occurred concurrently: some genes are abnormally turned up and some are turned down. In the field of non-viral gene therapy, plasmid DNA (pDNA) and small interfering RNA (siRNA) are suggested as representative regulation tools for activating and silencing the expression of genes of interest, representatively. Herein, we simultaneously loaded both siRNA (Src homology region 2 domain-containing tyrosine phosphatase-1 siRNA, siSHP-1) for anti-apoptosis and pDNA (hypoxia-inducible vascular endothelial growth factor expression vector, pHI-VEGF) for angiogenesis in a single polymeric nanocarrier and used to synergistically attenuate ischemia-reperfusion (IR)-induced myocardial infarction, which is mainly caused by dysregulating of cardiac apoptosis and angiogenesis. For dual-modality cardiac gene delivery, siSHP-1 and pHI-VEGF were sequentially incorporated into a stable nanocomplex by using deoxycholic acid-modified polyethylenimine (DA-PEI). The resulting DA-PEI/siSHP-1/pHI-VEGF complexes exhibited the high structural stability against polyanion competition and the improved resistance to digestion by nucleases. The cardiac administration of DA-PEI/siSHP-1/pHI-VEGF reduced cardiomyocyte apoptosis and enhanced cardiac microvessel formation, thereby reducing infarct size in rat ischemia-reperfusion model. The simultaneous anti-apoptotic and angiogenic gene therapies synergized the cardioprotective effects of each strategy; thus our dual-modal single-carrier gene delivery system can be considered as a promising candidate for treating ischemic heart diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of Type 1 Diabetes Mellitus in Nonobese Diabetic Mice Follows Changes in Thymocyte and Peripheral T Lymphocyte Transcriptional Activity

PubMed Central

Fornari, Thais A.; Donate, Paula B.; Macedo, Claudia; Sakamoto-Hojo, Elza T.; Donadi, Eduardo A.; Passos, Geraldo A.

2011-01-01

As early as one month of age, nonobese diabetic (NOD) mice feature pancreatic infiltration of autoreactive T lymphocytes, which destruct insulin-producing beta cells, producing autoimmune diabetes mellitus (T1D) within eight months. Thus, we hypothesized that during the development of T1D, the transcriptional modulation of immune reactivity genes may occur as thymocytes mature into peripheral T lymphocytes. The transcriptome of thymocytes and peripheral CD3+ T lymphocytes from prediabetic or diabetic mice analyzed through microarray hybridizations identified 2,771 differentially expressed genes. Hierarchical clustering grouped mice according to age/T1D onset and genes according to their transcription profiling. The transcriptional activity of thymocytes developing into peripheral T lymphocytes revealed sequential participation of genes involved with CD4+/CD8+ T-cell differentiation (Themis), tolerance induction by Tregs (Foxp3), and apoptosis (Fasl) soon after T-cell activation (IL4), while the emergence of T1D coincided with the expression of cytotoxicity (Crtam) and inflammatory response genes (Tlr) by peripheral T lymphocytes. PMID:21765850

Correction of gene expression data: Performance-dependency on inter-replicate and inter-treatment biases.

PubMed

Darbani, Behrooz; Stewart, C Neal; Noeparvar, Shahin; Borg, Søren

2014-10-20

This report investigates for the first time the potential inter-treatment bias source of cell number for gene expression studies. Cell-number bias can affect gene expression analysis when comparing samples with unequal total cellular RNA content or with different RNA extraction efficiencies. For maximal reliability of analysis, therefore, comparisons should be performed at the cellular level. This could be accomplished using an appropriate correction method that can detect and remove the inter-treatment bias for cell-number. Based on inter-treatment variations of reference genes, we introduce an analytical approach to examine the suitability of correction methods by considering the inter-treatment bias as well as the inter-replicate variance, which allows use of the best correction method with minimum residual bias. Analyses of RNA sequencing and microarray data showed that the efficiencies of correction methods are influenced by the inter-treatment bias as well as the inter-replicate variance. Therefore, we recommend inspecting both of the bias sources in order to apply the most efficient correction method. As an alternative correction strategy, sequential application of different correction approaches is also advised. Copyright © 2014 Elsevier B.V. All rights reserved.

Overexpression of Arabidopsis Molybdenum Cofactor Sulfurase Gene Confers Drought Tolerance in Maize (Zea mays L.)

PubMed Central

Zhang, Jiachang; Xiao, Yitao; Yue, Yuesen; Duan, Liusheng; Zhang, Mingcai; Li, Zhaohu

2013-01-01

Abscisic acid (ABA) is a key component of the signaling system that integrates plant adaptive responses to abiotic stress. Overexpression of Arabidopsis molybdenum cofactor sulfurase gene (LOS5) in maize markedly enhanced the expression of ZmAO and aldehyde oxidase (AO) activity, leading to ABA accumulation and increased drought tolerance. Transgenic maize (Zea mays L.) exhibited the expected reductions in stomatal aperture, which led to decreased water loss and maintenance of higher relative water content (RWC) and leaf water potential. Also, transgenic maize subjected to drought treatment exhibited lower leaf wilting, electrolyte leakage, malondialdehyde (MDA) and H2O2 content, and higher activities of antioxidative enzymes and proline content compared to wild-type (WT) maize. Moreover, overexpression of LOS5 enhanced the expression of stress-regulated genes such as Rad 17, NCED1, CAT1, and ZmP5CS1 under drought stress conditions, and increased root system development and biomass yield after re-watering. The increased drought tolerance in transgenic plants was associated with ABA accumulation via activated AO and expression of stress-related gene via ABA induction, which sequentially induced a set of favorable stress-related physiological and biochemical responses. PMID:23326325

Overexpression of Arabidopsis molybdenum cofactor sulfurase gene confers drought tolerance in maize (Zea mays L.).

PubMed

Lu, Yao; Li, Yajun; Zhang, Jiachang; Xiao, Yitao; Yue, Yuesen; Duan, Liusheng; Zhang, Mingcai; Li, Zhaohu

2013-01-01

Abscisic acid (ABA) is a key component of the signaling system that integrates plant adaptive responses to abiotic stress. Overexpression of Arabidopsis molybdenum cofactor sulfurase gene (LOS5) in maize markedly enhanced the expression of ZmAO and aldehyde oxidase (AO) activity, leading to ABA accumulation and increased drought tolerance. Transgenic maize (Zea mays L.) exhibited the expected reductions in stomatal aperture, which led to decreased water loss and maintenance of higher relative water content (RWC) and leaf water potential. Also, transgenic maize subjected to drought treatment exhibited lower leaf wilting, electrolyte leakage, malondialdehyde (MDA) and H(2)O(2) content, and higher activities of antioxidative enzymes and proline content compared to wild-type (WT) maize. Moreover, overexpression of LOS5 enhanced the expression of stress-regulated genes such as Rad 17, NCED1, CAT1, and ZmP5CS1 under drought stress conditions, and increased root system development and biomass yield after re-watering. The increased drought tolerance in transgenic plants was associated with ABA accumulation via activated AO and expression of stress-related gene via ABA induction, which sequentially induced a set of favorable stress-related physiological and biochemical responses.

Arabidopsis whole-transcriptome profiling defines the features of coordinated regulations that occur during secondary growth.

PubMed

Ko, Jae-Heung; Han, Kyung-Hwan

2004-05-01

Secondary growth in the inflorescence stems of Arabidopsis plants was induced by a combination of short-day and long-day treatments. The induced stems were divided into three different stem developmental stages (i.e., immature, intermediate, and mature) with regard to secondary growth. Whole transcriptome microarrays were used to examine the changes in global gene expression occurring at the different stem developmental stages. Over 70% of the Arabidopsis transcriptome was expressed in the stem tissues. In the mature stems with secondary growth, 567 genes were upregulated 5-fold or higher and 530 were downregulated, when compared to immature stems (with no secondary growth) and 10-day old seedlings (with no inflorescence stem). The transcription phenotypes obtained from the stems at different developmental stages largely confirm the existing insights into the biochemical processes involved in the sequential events that lead to wood formation. The major difference found between the stems undergoing secondary growth and only primary growth was in the expression profiles of transcriptional regulation-and signal transduction-related genes. An analysis of several shoot apical meristem (SAM) activity-related gene expression patterns in the stems indicated that the genetic control of secondary meristem activity might be governed by a different mechanism from that of SAM. The current study established the expression patterns of many unknown genes and identified candidate genes that are involved in the genetic regulation of secondary growth. The findings described in this report should improve our understanding of the molecular mechanisms that regulate the growth and development of the stem.

A transcriptome analysis of two grapevine populations segregating for tendril phyllotaxy

PubMed Central

Arro, Jie; Cuenca, Jose; Yang, Yingzhen; Liang, Zhenchang; Cousins, Peter; Zhong, Gan-Yuan

2017-01-01

The shoot structure of cultivated grapevine Vitis vinifera L. typically exhibits a three-node modular repetitive pattern, two sequential leaf-opposed tendrils followed by a tendril-free node. In this study, we investigated the molecular basis of this pattern by characterizing differentially expressed genes in 10 bulk samples of young tendril tissue from two grapevine populations showing segregation of mutant or wild-type shoot/tendril phyllotaxy. One population was the selfed progeny and the other one, an outcrossed progeny of a Vitis hybrid, ‘Roger’s Red’. We analyzed 13 375 expressed genes and carried out in-depth analyses of 324 of them, which were differentially expressed with a minimum of 1.5-fold changes between the mutant and wild-type bulk samples in both selfed and cross populations. A significant portion of these genes were direct cis-binding targets of 14 transcription factor families that were themselves differentially expressed. Network-based dependency analysis further revealed that most of the significantly rewired connections among the 10 most connected hub genes involved at least one transcription factor. TCP3 and MYB12, which were known important for plant-form development, were among these transcription factors. More importantly, TCP3 and MYB12 were found in this study to be involved in regulating the lignin gene PRX52, which is important to plant-form development. A further support evidence for the roles of TCP3-MYB12-PRX52 in contributing to tendril phyllotaxy was the findings of two other lignin-related genes uniquely expressed in the mutant phyllotaxy background. PMID:28713572

Speed regulation of genetic cascades allows for evolvability in the body plan specification of insects

PubMed Central

Zhu, Xin; Rudolf, Heike; Healey, Lucas; François, Paul; Brown, Susan J.; Klingler, Martin; El-Sherif, Ezzat

2017-01-01

During the anterior−posterior fate specification of insects, anterior fates arise in a nonelongating tissue (called the “blastoderm”), and posterior fates arise in an elongating tissue (called the “germband”). However, insects differ widely in the extent to which anterior−posterior fates are specified in the blastoderm versus the germband. Here we present a model in which patterning in both the blastoderm and germband of the beetle Tribolium castaneum is based on the same flexible mechanism: a gradient that modulates the speed of a genetic cascade of gap genes, resulting in the induction of sequential kinematic waves of gap gene expression. The mechanism is flexible and capable of patterning both elongating and nonelongating tissues, and hence converting blastodermal to germband fates and vice versa. Using RNAi perturbations, we found that blastodermal fates could be shifted to the germband, and germband fates could be generated in a blastoderm-like morphology. We also suggest a molecular mechanism underlying our model, in which gradient levels regulate the switch between two enhancers: One enhancer is responsible for sequential gene activation, and the other is responsible for freezing temporal rhythms into spatial patterns. This model is consistent with findings in Drosophila melanogaster, where gap genes were found to be regulated by two nonredundant “shadow” enhancers. PMID:28973882

Overproduction of Geranylgeraniol by Metabolically Engineered Saccharomyces cerevisiae▿

PubMed Central

Tokuhiro, Kenro; Muramatsu, Masayoshi; Ohto, Chikara; Kawaguchi, Toshiya; Obata, Shusei; Muramoto, Nobuhiko; Hirai, Masana; Takahashi, Haruo; Kondo, Akihiko; Sakuradani, Eiji; Shimizu, Sakayu

2009-01-01

(E, E, E)-Geranylgeraniol (GGOH) is a valuable starting material for perfumes and pharmaceutical products. In the yeast Saccharomyces cerevisiae, GGOH is synthesized from the end products of the mevalonate pathway through the sequential reactions of farnesyl diphosphate synthetase (encoded by the ERG20 gene), geranylgeranyl diphosphate synthase (the BTS1 gene), and some endogenous phosphatases. We demonstrated that overexpression of the diacylglycerol diphosphate phosphatase (DPP1) gene could promote GGOH production. We also found that overexpression of a BTS1-DPP1 fusion gene was more efficient for producing GGOH than coexpression of these genes separately. Overexpression of the hydroxymethylglutaryl-coenzyme A reductase (HMG1) gene, which encodes the major rate-limiting enzyme of the mevalonate pathway, resulted in overproduction of squalene (191.9 mg liter−1) rather than GGOH (0.2 mg liter−1) in test tube cultures. Coexpression of the BTS1-DPP1 fusion gene along with the HMG1 gene partially redirected the metabolic flux from squalene to GGOH. Additional expression of a BTS1-ERG20 fusion gene resulted in an almost complete shift of the flux to GGOH production (228.8 mg liter−1 GGOH and 6.5 mg liter−1 squalene). Finally, we constructed a diploid prototrophic strain coexpressing the HMG1, BTS1-DPP1, and BTS1-ERG20 genes from multicopy integration vectors. This strain attained 3.31 g liter−1 GGOH production in a 10-liter jar fermentor with gradual feeding of a mixed glucose and ethanol solution. The use of bifunctional fusion genes such as the BTS1-DPP1 and ERG20-BTS1 genes that code sequential enzymes in the metabolic pathway was an effective method for metabolic engineering. PMID:19592534

Transcriptional profiles discriminate patients with pulmonary tuberculosis from non-tuberculous individuals depending on the presence of non-insulin diabetes mellitus.

PubMed

Serrano, Carmen J; Cuevas-Córdoba, Betzaida; Macías-Segura, Noé; González-Curiel, Rosa Angélica; Martínez-Balderas, Víctor Yordani; Enciso-Moreno, Leonor; Small, Peter; Hernández-Pando, Rogelio; Enciso-Moreno, José Antonio

2016-01-01

Our objective was to identify transcriptional biomarkers in peripheral blood mononuclear cells (PBMC) that discriminate individuals with latent tuberculosis infection (LTBI) from those with pulmonary tuberculosis (PTB) in subjects with non-insulin-dependent diabetes mellitus (NIDDM) and in individuals without NIDDM. Using gene expression microarrays we identified differentially expressed genes from lungs of mice infected with Mycobacterium tuberculosis (Mtb) or a mutant (ΔsigH) representing a non-inflammatory model. Genes expressed in blood, with inflammatory related functions were evaluated in humans by RT-qPCR. NCF1 and ORM transcripts have the better discriminatory capacity to identify PTB subjects from LTBI and non-infected controls (NICs) independently of the presence of NIDDM. The sequential evaluation of the mRNA levels of NCF1 and ORM as multiple diagnostic tests showed 95% Sensitivity (Se) and 80% Specificity (Sp). In addition, FPR2 promises to be a good biomarker for the PTB detection in subjects with NIDDM (Se=100%; Sp=90%). Copyright © 2015 Elsevier Inc. All rights reserved.

Epigenomic Landscape of Human Fetal Brain, Heart, and Liver.

PubMed

Yan, Liying; Guo, Hongshan; Hu, Boqiang; Li, Rong; Yong, Jun; Zhao, Yangyu; Zhi, Xu; Fan, Xiaoying; Guo, Fan; Wang, Xiaoye; Wang, Wei; Wei, Yuan; Wang, Yan; Wen, Lu; Qiao, Jie; Tang, Fuchou

2016-02-26

The epigenetic regulation of spatiotemporal gene expression is crucial for human development. Here, we present whole-genome chromatin immunoprecipitation followed by high throughput DNA sequencing (ChIP-seq) analyses of a wide variety of histone markers in the brain, heart, and liver of early human embryos shortly after their formation. We identified 40,181 active enhancers, with a large portion showing tissue-specific and developmental stage-specific patterns, pointing to their roles in controlling the ordered spatiotemporal expression of the developmental genes in early human embryos. Moreover, using sequential ChIP-seq, we showed that all three organs have hundreds to thousands of bivalent domains that are marked by both H3K4me3 and H3K27me3, probably to keep the progenitor cells in these organs ready for immediate differentiation into diverse cell types during subsequent developmental processes. Our work illustrates the potentially critical roles of tissue-specific and developmental stage-specific epigenomes in regulating the spatiotemporal expression of developmental genes during early human embryonic development. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The evolutionary dynamics of variant antigen genes in Babesia reveal a history of genomic innovation underlying host–parasite interaction

PubMed Central

Jackson, Andrew P.; Otto, Thomas D.; Darby, Alistair; Ramaprasad, Abhinay; Xia, Dong; Echaide, Ignacio Eduardo; Farber, Marisa; Gahlot, Sunayna; Gamble, John; Gupta, Dinesh; Gupta, Yask; Jackson, Louise; Malandrin, Laurence; Malas, Tareq B.; Moussa, Ehab; Nair, Mridul; Reid, Adam J.; Sanders, Mandy; Sharma, Jyotsna; Tracey, Alan; Quail, Mike A.; Weir, William; Wastling, Jonathan M.; Hall, Neil; Willadsen, Peter; Lingelbach, Klaus; Shiels, Brian; Tait, Andy; Berriman, Matt; Allred, David R.; Pain, Arnab

2014-01-01

Babesia spp. are tick-borne, intraerythrocytic hemoparasites that use antigenic variation to resist host immunity, through sequential modification of the parasite-derived variant erythrocyte surface antigen (VESA) expressed on the infected red blood cell surface. We identified the genomic processes driving antigenic diversity in genes encoding VESA (ves1) through comparative analysis within and between three Babesia species, (B. bigemina, B. divergens and B. bovis). Ves1 structure diverges rapidly after speciation, notably through the evolution of shortened forms (ves2) from 5′ ends of canonical ves1 genes. Phylogenetic analyses show that ves1 genes are transposed between loci routinely, whereas ves2 genes are not. Similarly, analysis of sequence mosaicism shows that recombination drives variation in ves1 sequences, but less so for ves2, indicating the adoption of different mechanisms for variation of the two families. Proteomic analysis of the B. bigemina PR isolate shows that two dominant VESA1 proteins are expressed in the population, whereas numerous VESA2 proteins are co-expressed, consistent with differential transcriptional regulation of each family. Hence, VESA2 proteins are abundant and previously unrecognized elements of Babesia biology, with evolutionary dynamics consistently different to those of VESA1, suggesting that their functions are distinct. PMID:24799432

Gene expression and metabolism preceding soft scald, a chilling injury of 'Honeycrisp' apple fruit.

PubMed

Leisso, Rachel S; Gapper, Nigel E; Mattheis, James P; Sullivan, Nathanael L; Watkins, Christopher B; Giovannoni, James J; Schaffer, Robert J; Johnston, Jason W; Hanrahan, Ines; Hertog, Maarten L A T M; Nicolaï, Bart M; Rudell, David R

2016-10-12

'Honeycrisp' is an apple cultivar that is susceptible to soft scald, a chilling injury expressed as necrotic patches on the peel. Improved understanding of metabolism associated with the disorder would improve our understanding of soft scald and contribute to developing more effective management strategies for apple storage. It was expected that specific gene expression and specific metabolite levels in the peel would be linked with soft scald risk at harvest and/or specific time points during cold storage. Fruit from nine 'Honeycrisp' apple orchards that would eventually develop different incidences of soft scald between 4 and 8 weeks of cold air storage were used to contrast and determine differential transcriptomic and metabolomic changes during storage. Untargeted metabolic profiling revealed changes in a number of distinct pathways preceding and concurrent with soft scald symptom development, including elevated γ-aminobutryic acid (GABA), 1-hexanol, acylated steryl glycosides, and free p-coumaryl acyl esters. At harvest, levels of sesquiterpenoid and triterpenoid acyl esters were relatively higher in peel of fruit that did not later develop the disorder. RNA-seq driven gene expression profiling highlighted possible involvement of genes and associated metabolic processes with soft scald development. These included elevated expression of genes involved in lipid peroxidation and phenolic metabolism in fruit with soft scald, and isoprenoid/brassinosteroid metabolism in fruit that did not develop soft scald. Expression of other stress-related genes in fruit that developed soft scald included chlorophyll catabolism, cell wall loosening, and lipid transport while superoxide dismutases were up-regulated in fruit that did not develop the disorder. This study delineates the sequential transcriptomic and metabolomic changes preceding soft scald symptom development. Changes were differential depending on susceptibility of fruit to the disorder and could be attributed to key stress related and mediating pathways.

Steroid hormone induction of temporal gene expression in Drosophila brain neuroblasts generates neuronal and glial diversity

PubMed Central

Syed, Mubarak Hussain; Mark, Brandon; Doe, Chris Q

2017-01-01

An important question in neuroscience is how stem cells generate neuronal diversity. During Drosophila embryonic development, neural stem cells (neuroblasts) sequentially express transcription factors that generate neuronal diversity; regulation of the embryonic temporal transcription factor cascade is lineage-intrinsic. In contrast, larval neuroblasts generate longer ~50 division lineages, and currently only one mid-larval molecular transition is known: Chinmo/Imp/Lin-28+ neuroblasts transition to Syncrip+ neuroblasts. Here we show that the hormone ecdysone is required to down-regulate Chinmo/Imp and activate Syncrip, plus two late neuroblast factors, Broad and E93. We show that Seven-up triggers Chinmo/Imp to Syncrip/Broad/E93 transition by inducing expression of the Ecdysone receptor in mid-larval neuroblasts, rendering them competent to respond to the systemic hormone ecdysone. Importantly, late temporal gene expression is essential for proper neuronal and glial cell type specification. This is the first example of hormonal regulation of temporal factor expression in Drosophila embryonic or larval neural progenitors. DOI: http://dx.doi.org/10.7554/eLife.26287.001 PMID:28394252

Sequential intravenous injection of anionic polymer and cationic lipoplex of siRNA could effectively deliver siRNA to the liver.

PubMed

Hattori, Yoshiyuki; Arai, Shohei; Okamoto, Ryou; Hamada, Megumi; Kawano, Kumi; Yonemochi, Etsuo

2014-12-10

In this study, we developed novel siRNA transfer method to the liver by sequential intravenous injection of anionic polymer and cationic liposome/cholesterol-modified siRNA complex (cationic lipoplex). When cationic lipoplex was intravenously injected into mice, the accumulation of siRNA was mainly observed in the lungs. In contrast, when cationic lipoplex was intravenously injected at 1 min after intravenous injection of poly-L-glutamic acid (PGA) or chondroitin sulfate C (CS), siRNA was accumulated in the liver. In terms of suppression of gene expression in vivo, apolipoprotein B (ApoB) mRNA in the liver and low-density-lipoprotein (LDL) and very low-density-lipoprotein (VLDL) cholesterol level in serum were reduced at 48 h after single sequential injection of PGA or CS plus cationic lipoplex of cholesterol-modified ApoB siRNA. Furthermore, sequential injections of PGA plus cationic lipoplex of cholesterol-modified luciferase siRNA could reduce luciferase activity in tumor xenografts bearing liver metastasis of human breast tumor MCF-7-Luc. From these findings, sequential injection of anionic polymer and cationic lipoplex of siRNA might produce a systemic vector of siRNA to the liver. Copyright © 2014 Elsevier B.V. All rights reserved.

Integrative genomics identifies molecular alterations that challenge the linear model of melanoma progression.

PubMed

Rose, Amy E; Poliseno, Laura; Wang, Jinhua; Clark, Michael; Pearlman, Alexander; Wang, Guimin; Vega Y Saenz de Miera, Eleazar C; Medicherla, Ratna; Christos, Paul J; Shapiro, Richard; Pavlick, Anna; Darvishian, Farbod; Zavadil, Jiri; Polsky, David; Hernando, Eva; Ostrer, Harry; Osman, Iman

2011-04-01

Superficial spreading melanoma (SSM) and nodular melanoma (NM) are believed to represent sequential phases of linear progression from radial to vertical growth. Several lines of clinical, pathologic, and epidemiologic evidence suggest, however, that SSM and NM might be the result of independent pathways of tumor development. We utilized an integrative genomic approach that combines single nucleotide polymorphism array (6.0; Affymetrix) with gene expression array (U133A 2.0; Affymetrix) to examine molecular differences between SSM and NM. Pathway analysis of the most differentially expressed genes between SSM and NM (N = 114) revealed significant differences related to metabolic processes. We identified 8 genes (DIS3, FGFR1OP, G3BP2, GALNT7, MTAP, SEC23IP, USO1, and ZNF668) in which NM/SSM-specific copy number alterations correlated with differential gene expression (P < 0.05; Spearman's rank). SSM-specific genomic deletions in G3BP2, MTAP, and SEC23IP were independently verified in two external data sets. Forced overexpression of metabolism-related gene MTAP (methylthioadenosine phosphorylase) in SSM resulted in reduced cell growth. The differential expression of another metabolic-related gene, aldehyde dehydrogenase 7A1 (ALDH7A1), was validated at the protein level by using tissue microarrays of human melanoma. In addition, we show that the decreased ALDH7A1 expression in SSM may be the result of epigenetic modifications. Our data reveal recurrent genomic deletions in SSM not present in NM, which challenge the linear model of melanoma progression. Furthermore, our data suggest a role for altered regulation of metabolism-related genes as a possible cause of the different clinical behavior of SSM and NM.

Integrative genomics identifies molecular alterations that challenge the linear model of melanoma progression

PubMed Central

Rose, Amy E.; Poliseno, Laura; Wang, Jinhua; Clark, Michael; Pearlman, Alexander; Wang, Guimin; Vega y Saenz de Miera, Eleazar C.; Medicherla, Ratna; Christos, Paul J.; Shapiro, Richard; Pavlick, Anna; Darvishian, Farbod; Zavadil, Jiri; Polsky, David; Hernando, Eva; Ostrer, Harry; Osman, Iman

2011-01-01

Superficial spreading melanoma (SSM) and nodular melanoma (NM) are believed to represent sequential phases of linear progression from radial to vertical growth. Several lines of clinical, pathological and epidemiologic evidence suggest, however, that SSM and NM might be the result of independent pathways of tumor development. We utilized an integrative genomic approach that combines single nucleotide polymorphism array (SNP 6.0, Affymetrix) with gene expression array (U133A 2.0, Affymetrix) to examine molecular differences between SSM and NM. Pathway analysis of the most differentially expressed genes between SSM and NM (N=114) revealed significant differences related to metabolic processes. We identified 8 genes (DIS3, FGFR1OP, G3BP2, GALNT7, MTAP, SEC23IP, USO1, ZNF668) in which NM/SSM-specific copy number alterations correlated with differential gene expression (P<0.05, Spearman’s rank). SSM-specific genomic deletions in G3BP2, MTAP, and SEC23IP were independently verified in two external data sets. Forced overexpression of metabolism-related gene methylthioadenosine phosphorylase (MTAP) in SSM resulted in reduced cell growth. The differential expression of another metabolic related gene, aldehyde dehydrogenase 7A1 (ALDH7A1), was validated at the protein level using tissue microarrays of human melanoma. In addition, we show that the decreased ALDH7A1 expression in SSM may be the result of epigenetic modifications. Our data reveal recurrent genomic deletions in SSM not present in NM, which challenge the linear model of melanoma progression. Furthermore, our data suggest a role for altered regulation of metabolism-related genes as a possible cause of the different clinical behavior of SSM and NM. PMID:21343389

Triplication of a four-gene set during evolution of the goat beta-globin locus produced three genes now expressed differentially during development.

PubMed Central

Townes, T M; Fitzgerald, M C; Lingrel, J B

1984-01-01

Distinct hemoglobins are synthesized in goats at different stages of development, similar to humans. Embryonic hemoglobins (zeta 2 epsilon 2 and alpha 2 epsilon 2) are synthesized initially and are followed sequentially by fetal (alpha 2 beta F2), preadult (alpha 2 beta C2), and adult (alpha 2 beta A2) hemoglobins. To help understand the basis of these switches, the genes of the beta-globin locus have been cloned and their linkage arrangement has been determined by the isolation of lambda phage carrying overlapping inserts of genomic goat DNA. The locus extends over 120 kilobase pairs and consists of 12 genes arranged in the following order: epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-epsilon VI-psi beta Y-beta F. Comparison of the nucleotide sequence of the 12 genes shows that the locus is organized into three homologous four-gene sets that presumably evolved by the triplication of an ancestral set of four genes (epsilon-epsilon-psi beta-beta). Interestingly, the three genes (beta C, beta A, and beta F) located at the ends of the four-gene sets are expressed at different stages of development. Therefore, the goat beta F-, beta C-, and beta A-globin genes appear to have evolved by a mechanism that includes the triplication of 40-50 kilobase pairs of DNA and the recruitment of newly formed genes for expression in fetal, preadult, and adult life. PMID:6593719

Dual Luciferase Assay System for Rapid Assessment of Gene Expression in Saccharomyces cerevisiae

PubMed Central

McNabb, David S.; Reed, Robin; Marciniak, Robert A.

2005-01-01

A new reporter system has been developed for quantifying gene expression in the yeast Saccharomyces cerevisiae. The system relies on two different reporter genes, Renilla and firefly luciferase, to evaluate regulated gene expression. The gene encoding Renilla luciferase is fused to a constitutive promoter (PGK1 or SPT15) and integrated into the yeast genome at the CAN1 locus as a control for normalizing the assay. The firefly luciferase gene is fused to the test promoter and integrated into the yeast genome at the ura3 or leu2 locus. The dual luciferase assay is performed by sequentially measuring the firefly and Renilla luciferase activities of the same sample, with the results expressed as the ratio of firefly to Renilla luciferase activity (Fluc/Rluc). The yeast dual luciferase reporter (DLR) was characterized and shown to be very efficient, requiring approximately 1 minute to complete each assay, and has proven to yield data that accurately and reproducibly reflect promoter activity. A series of integrating plasmids were generated that contain either the firefly or Renilla luciferase gene preceded by a multicloning region in two different orientations and the three reading frames to make possible the generation of translational fusions. Additionally, each set of plasmids contains either the URA3 or LEU2 marker for genetic selection in yeast. A series of S288C-based yeast strains, including a two-hybrid strain, were developed to facilitate the use of the yeast DLR assay. This assay can be readily adapted to a high-throughput platform for studies requiring numerous measurements. PMID:16151247

Dynamics in the tomato root transcriptome on infection with the potato cyst nematode Globodera rostochiensis.

PubMed

Swiecicka, Magdalena; Filipecki, Marcin; Lont, Dieuwertje; Van Vliet, Joke; Qin, Ling; Goverse, Aska; Bakker, Jaap; Helder, Johannes

2009-07-01

Plant parasitic nematodes infect roots and trigger the formation of specialized feeding sites by substantial reprogramming of the developmental process of root cells. In this article, we describe the dynamic changes in the tomato root transcriptome during early interactions with the potato cyst nematode Globodera rostochiensis. Using amplified fragment length polymorphism-based mRNA fingerprinting (cDNA-AFLP), we monitored 17 600 transcript-derived fragments (TDFs) in infected and uninfected tomato roots, 1-14 days after inoculation with nematode larvae. Six hundred and twenty-four TDFs (3.5%) showed significant differential expression on nematode infection. We employed GenEST, a computer program which links gene expression profiles generated by cDNA-AFLP and databases of cDNA sequences, to identify 135 tomato sequences. These sequences were grouped into eight functional categories based on the presence of genes involved in hormone regulation, plant pathogen defence response, cell cycle and cytoskeleton regulation, cell wall modification, cellular signalling, transcriptional regulation, primary metabolism and allocation. The presence of unclassified genes was also taken into consideration. This article describes the responsiveness of numerous tomato genes hitherto uncharacterized during infection with endoparasitic cyst nematodes. The analysis of transcriptome profiles allowed the sequential order of expression to be dissected for many groups of genes and the genes to be connected with the biological processes involved in compatible interactions between the plant and nematode.

Effects of nutrient deprivation and differentiation on the expression of growth-arrest genes (gas and gadd) in F9 embryonal carcinoma cells.

PubMed Central

Fleming, J V; Hay, S M; Harries, D N; Rees, W D

1998-01-01

The growth-arrest genes (gas and gadd) are widely expressed during mammalian embryogenesis and may be useful as markers of nutritional stress in the embryo. F9 embryonal carcinoma cells have been used to characterize the effect of serum or amino acid deficiency on growth-arrest gene expression in a differentiating embryonic cell. The differentiation markers, homeobox B2 (HoxB2), collagen type IV and laminin B2, were not induced by growth arrest. Treatment with all-trans retinoic acid (RA) produced a dose-dependent increase in alkaline phosphatase activity, which was unchanged in lysine-deficient medium and reduced in low-serum medium. Low-serum medium also reduced HoxB2 expression. There was a transient 2-6-fold increase in mRNAs for C/EBP-beta, gadd153/CHOP-10 and gas5 genes 24 h after transfer to amino-acid-deficient media. The mRNAs for the gas2 and gas6 genes began to rise slowly by 5-10-fold after a delay of approx. 24 h. The transient increases did not occur in low-serum medium where there was a much smaller and slower increase. Differentiation caused 1-2-fold increases in gas2, gas3 and gas6 mRNA levels. The transient overexpression of gas5, gadd153/CHOP-10 and CCAAT-enhancer-binding protein-beta, and the later expression of gas6 mRNAs in response to amino acid deficiency, were not affected by differentiation. RA treatment increased the expression of gas3 and caused gas2 to be transiently overexpressed in amino-acid-deficient medium. Differentiation in serum-deficient medium did not significantly alter the levels of the growth-arrest gene mRNAs. These results show that in F9 cells the growth-arrest genes are expressed sequentially as a result of nutrient stress. PMID:9461558

Effects of nutrient deprivation and differentiation on the expression of growth-arrest genes (gas and gadd) in F9 embryonal carcinoma cells.

PubMed

Fleming, J V; Hay, S M; Harries, D N; Rees, W D

1998-02-15

The growth-arrest genes (gas and gadd) are widely expressed during mammalian embryogenesis and may be useful as markers of nutritional stress in the embryo. F9 embryonal carcinoma cells have been used to characterize the effect of serum or amino acid deficiency on growth-arrest gene expression in a differentiating embryonic cell. The differentiation markers, homeobox B2 (HoxB2), collagen type IV and laminin B2, were not induced by growth arrest. Treatment with all-trans retinoic acid (RA) produced a dose-dependent increase in alkaline phosphatase activity, which was unchanged in lysine-deficient medium and reduced in low-serum medium. Low-serum medium also reduced HoxB2 expression. There was a transient 2-6-fold increase in mRNAs for C/EBP-beta, gadd153/CHOP-10 and gas5 genes 24 h after transfer to amino-acid-deficient media. The mRNAs for the gas2 and gas6 genes began to rise slowly by 5-10-fold after a delay of approx. 24 h. The transient increases did not occur in low-serum medium where there was a much smaller and slower increase. Differentiation caused 1-2-fold increases in gas2, gas3 and gas6 mRNA levels. The transient overexpression of gas5, gadd153/CHOP-10 and CCAAT-enhancer-binding protein-beta, and the later expression of gas6 mRNAs in response to amino acid deficiency, were not affected by differentiation. RA treatment increased the expression of gas3 and caused gas2 to be transiently overexpressed in amino-acid-deficient medium. Differentiation in serum-deficient medium did not significantly alter the levels of the growth-arrest gene mRNAs. These results show that in F9 cells the growth-arrest genes are expressed sequentially as a result of nutrient stress.

Sequential Waves of Gene Expression in Patients with Clinically Defined Dengue Illnesses Reveal Subtle Disease Phases and Predict Disease Severity

PubMed Central

Sun, Peifang; García, Josefina; Comach, Guillermo; Vahey, Maryanne T.; Wang, Zhining; Forshey, Brett M.; Morrison, Amy C.; Sierra, Gloria; Bazan, Isabel; Rocha, Claudio; Vilcarromero, Stalin; Blair, Patrick J.; Scott, Thomas W.; Camacho, Daria E.; Ockenhouse, Christian F.; Halsey, Eric S.; Kochel, Tadeusz J.

2013-01-01

Background Dengue virus (DENV) infection can range in severity from mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Changes in host gene expression, temporally through the progression of DENV infection, especially during the early days, remains poorly characterized. Early diagnostic markers for DHF are also lacking. Methodology/Principal Findings In this study, we investigated host gene expression in a cohort of DENV-infected subjects clinically diagnosed as DF (n = 51) and DHF (n = 13) from Maracay, Venezuela. Blood specimens were collected daily from these subjects from enrollment to early defervescence and at one convalescent time-point. Using convalescent expression levels as baseline, two distinct groups of genes were identified: the “early” group, which included genes associated with innate immunity, type I interferon, cytokine-mediated signaling, chemotaxis, and complement activity peaked at day 0–1 and declined on day 3–4; the second “late” group, comprised of genes associated with cell cycle, emerged from day 4 and peaked at day 5–6. The up-regulation of innate immune response genes coincided with the down-regulation of genes associated with viral replication during day 0–3. Furthermore, DHF patients had lower expression of genes associated with antigen processing and presentation, MHC class II receptor, NK and T cell activities, compared to that of DF patients. These results suggested that the innate and adaptive immunity during the early days of the disease are vital in suppressing DENV replication and in affecting outcome of disease severity. Gene signatures of DHF were identified as early as day 1. Conclusions/Significance Our study reveals a broad and dynamic picture of host responses in DENV infected subjects. Host response to DENV infection can now be understood as two distinct phases with unique transcriptional markers. The DHF signatures identified during day 1–3 may have applications in developing early molecular diagnostics for DHF. PMID:23875036

Construction and characterization of VL-VH tail-parallel genetically engineered antibodies against staphylococcal enterotoxins.

PubMed

He, Xianzhi; Zhang, Lei; Liu, Pengchong; Liu, Li; Deng, Hui; Huang, Jinhai

2015-03-01

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus have increasingly given rise to human health and food safety. Genetically engineered small molecular antibody is a useful tool in immuno-detection and treatment for clinical illness caused by SEs. In this study, we constructed the V(L)-V(H) tail-parallel genetically engineered antibody against SEs by using the repertoire of rearranged germ-line immunoglobulin variable region genes. Total RNA were extracted from six hybridoma cell lines that stably express anti-SEs antibodies. The variable region genes of light chain (V(L)) and heavy chain (V(H)) were cloned by reverse transcription PCR, and their classical murine antibody structure and functional V(D)J gene rearrangement were analyzed. To construct the eukaryotic V(H)-V(L) tail-parallel co-expression vectors based on the "5'-V(H)-ivs-IRES-V(L)-3'" mode, the ivs-IRES fragment and V(L) genes were spliced by two-step overlap extension PCR, and then, the recombined gene fragment and V(H) genes were inserted into the pcDNA3.1(+) expression vector sequentially. And then the constructed eukaryotic expression clones termed as p2C2HILO and p5C12HILO were transfected into baby hamster kidney 21 cell line, respectively. Two clonal cell lines stably expressing V(L)-V(H) tail-parallel antibodies against SEs were obtained, and the antibodies that expressed intracytoplasma were evaluated by enzyme-linked immunosorbent assay, immunofluorescence assay, and flow cytometry. SEs can stimulate the expression of some chemokines and chemokine receptors in porcine IPEC-J2 cells; mRNA transcription level of four chemokines and chemokine receptors can be blocked by the recombinant SE antibody prepared in this study. Our results showed that it is possible to get functional V(L)-V(H) tail-parallel genetically engineered antibodies in same vector using eukaryotic expression system.

MicroRNA-320 family is downregulated in colorectal adenoma and affects tumor proliferation by targeting CDK6.

PubMed

Tadano, Toshihiro; Kakuta, Yoichi; Hamada, Shin; Shimodaira, Yosuke; Kuroha, Masatake; Kawakami, Yoko; Kimura, Tomoya; Shiga, Hisashi; Endo, Katsuya; Masamune, Atsushi; Takahashi, Seiichi; Kinouchi, Yoshitaka; Shimosegawa, Tooru

2016-07-15

To investigate the microRNA (miRNA) expression during histological progression from colorectal normal mucosa through adenoma to carcinoma within a lesion. Using microarray, the sequential changes in miRNA expression profiles were compared in colonic lesions from matched samples; histologically, non-neoplastic mucosa, adenoma, and submucosal invasive carcinoma were microdissected from a tissue sample. Cell proliferation assay was performed to observe the effect of miRNA, and its target genes were predicted using bioinformatics approaches and the expression profile of SW480 transfected with the miRNA mimics. mRNA and protein levels of the target gene in colon cancer cell lines with a mimic control or miRNA mimics were measured using qRT-PCR and Western blotting. The expression levels of miRNA and target gene in colorectal tissue samples were also measured. Microarray analysis identified that the miR-320 family, including miR-320a, miR-320b, miR-320c, miR-320d and miR-320e, were differentially expressed in adenoma and submucosal invasive carcinoma. The miR-320 family, which inhibits cell proliferation, is frequently downregulated in colorectal adenoma and submucosal invasive carcinoma tissues. Seven genes including CDK6 were identified to be common in the results of gene expression array and bioinformatics analyses performed to find the target gene of the miR-320 family. We confirmed that mRNA and protein levels of CDK6 were significantly suppressed in colon cancer cell lines with miR-320 family mimics. CDK6 expression was found to increase from non-neoplastic mucosa through adenoma to submucosal invasive carcinoma tissues and showed an inverse correlation with miR-320 family expression. MiR-320 family affects colorectal tumor proliferation by targeting CDK6, plays important role in its growth, and is considered to be a biomarker for its early detection.

MicroRNA-320 family is downregulated in colorectal adenoma and affects tumor proliferation by targeting CDK6

PubMed Central

Tadano, Toshihiro; Kakuta, Yoichi; Hamada, Shin; Shimodaira, Yosuke; Kuroha, Masatake; Kawakami, Yoko; Kimura, Tomoya; Shiga, Hisashi; Endo, Katsuya; Masamune, Atsushi; Takahashi, Seiichi; Kinouchi, Yoshitaka; Shimosegawa, Tooru

2016-01-01

AIM: To investigate the microRNA (miRNA) expression during histological progression from colorectal normal mucosa through adenoma to carcinoma within a lesion. METHODS: Using microarray, the sequential changes in miRNA expression profiles were compared in colonic lesions from matched samples; histologically, non-neoplastic mucosa, adenoma, and submucosal invasive carcinoma were microdissected from a tissue sample. Cell proliferation assay was performed to observe the effect of miRNA, and its target genes were predicted using bioinformatics approaches and the expression profile of SW480 transfected with the miRNA mimics. mRNA and protein levels of the target gene in colon cancer cell lines with a mimic control or miRNA mimics were measured using qRT-PCR and Western blotting. The expression levels of miRNA and target gene in colorectal tissue samples were also measured. RESULTS: Microarray analysis identified that the miR-320 family, including miR-320a, miR-320b, miR-320c, miR-320d and miR-320e, were differentially expressed in adenoma and submucosal invasive carcinoma. The miR-320 family, which inhibits cell proliferation, is frequently downregulated in colorectal adenoma and submucosal invasive carcinoma tissues. Seven genes including CDK6 were identified to be common in the results of gene expression array and bioinformatics analyses performed to find the target gene of the miR-320 family. We confirmed that mRNA and protein levels of CDK6 were significantly suppressed in colon cancer cell lines with miR-320 family mimics. CDK6 expression was found to increase from non-neoplastic mucosa through adenoma to submucosal invasive carcinoma tissues and showed an inverse correlation with miR-320 family expression. CONCLUSION: MiR-320 family affects colorectal tumor proliferation by targeting CDK6, plays important role in its growth, and is considered to be a biomarker for its early detection. PMID:27559432

Genome-wide co-localization of Polycomb orthologs and their effects on gene expression in human fibroblasts

PubMed Central

2014-01-01

Background Polycomb group proteins form multicomponent complexes that are important for establishing lineage-specific patterns of gene expression. Mammalian cells encode multiple permutations of the prototypic Polycomb repressive complex 1 (PRC1) with little evidence for functional specialization. An aim of this study is to determine whether the multiple orthologs that are co-expressed in human fibroblasts act on different target genes and whether their genomic location changes during cellular senescence. Results Deep sequencing of chromatin immunoprecipitated with antibodies against CBX6, CBX7, CBX8, RING1 and RING2 reveals that the orthologs co-localize at multiple sites. PCR-based validation at representative loci suggests that a further six PRC1 proteins have similar binding patterns. Importantly, sequential chromatin immunoprecipitation with antibodies against different orthologs implies that multiple variants of PRC1 associate with the same DNA. At many loci, the binding profiles have a distinctive architecture that is preserved in two different types of fibroblast. Conversely, there are several hundred loci at which PRC1 binding is cell type-specific and, contrary to expectations, the presence of PRC1 does not necessarily equate with transcriptional silencing. Interestingly, the PRC1 binding profiles are preserved in senescent cells despite changes in gene expression. Conclusions The multiple permutations of PRC1 in human fibroblasts congregate at common rather than specific sites in the genome and with overlapping but distinctive binding profiles in different fibroblasts. The data imply that the effects of PRC1 complexes on gene expression are more subtle than simply repressing the loci at which they bind. PMID:24485159

New Molecular Bridge between RelA/p65 and NF-κB Target Genes via Histone Acetyltransferase TIP60 Cofactor*

PubMed Central

Kim, Jung-Woong; Jang, Sang-Min; Kim, Chul-Hong; An, Joo-Hee; Kang, Eun-Jin; Choi, Kyung-Hee

2012-01-01

The nuclear factor-κB (NF-κB) family is involved in the expressions of numerous genes, in development, apoptosis, inflammatory responses, and oncogenesis. In this study we identified four NF-κB target genes that are modulated by TIP60. We also found that TIP60 interacts with the NF-κB RelA/p65 subunit and increases its transcriptional activity through protein-protein interaction. Although TIP60 binds with RelA/p65 using its histone acetyltransferase domain, TIP60 does not directly acetylate RelA/p65. However, TIP60 maintained acetylated Lys-310 RelA/p65 levels in the TNF-α-dependent NF-κB signaling pathway. In chromatin immunoprecipitation assay, TIP60 was primarily recruited to the IL-6, IL-8, C-IAP1, and XIAP promoters in TNF-α stimulation followed by acetylation of histones H3 and H4. Chromatin remodeling by TIP60 involved the sequential recruitment of acetyl-Lys-310 RelA/p65 to its target gene promoters. Furthermore, we showed that up-regulated TIP60 expression was correlated with acetyl-Lys-310 RelA/p65 expressions in hepatocarcinoma tissues. Taken together these results suggest that TIP60 is involved in the NF-κB pathway through protein interaction with RelA/p65 and that it modulates the transcriptional activity of RelA/p65 in NF-κB-dependent gene expression. PMID:22249179

The Ames dwarf gene, df, is required early in pituitary ontogeny for the extinction of Rpx transcription and initiation of lineage-specific cell proliferation.

PubMed

Gage, P J; Brinkmeier, M L; Scarlett, L M; Knapp, L T; Camper, S A; Mahon, K A

1996-12-01

Two nonallelic dwarfing mutations in mice define genes important for pituitary development and function. Mice homozygous for either the Ames (df) or Snell (Pit 1dw) dwarf mutations exhibit severe proportional dwarfism, hypothyroidism, and infertility due to the cytodifferentiation failure of three anterior pituitary cell types: thyrotropes, somatotropes, and lactotropes. Analysis of double heterozygotes and double mutants has provided evidence that the df and dw genes act sequentially in the same genetic pathway. Double heterozygotes had no reduction in growth rate or final adult size. Double homozygotes had essentially the same phenotype as the single mutants and were recovered at the predicted frequency, indicating that there are no previously unrecognized, redundant functions of the two genes. Several lines of evidence demonstrate that df acts earlier in the differentiation pathway than Pit1. The df mutants fail to extinguish expression of the homeobox gene Rpx on embryonic day 13.5 (e13.5), and the size of their nascent pituitary glands is reduced by e14.5. In contrast, Pit1dw mutants down-regulate Rpx appropriately and exhibit normal cell proliferation up to e14.5. The failure to extinguish Rpx and the concomitant hypocellularity of df pituitaries suggest the importance of Rpx repression in lineage-specific cell proliferation before the appearance of lineage-specific markers. Later, Pit-1 and hypothalamic neuropeptides act sequentially to regulate marker gene transcription and cell proliferation. These results establish the time of df action in a cascade of genes that regulate pituitary ontogeny.

Sequential Metabolic Phases as a Means to Optimize Cellular Output in a Constant Environment

PubMed Central

Bockmayr, Alexander; Holzhütter, Hermann-Georg

2015-01-01

Temporal changes of gene expression are a well-known regulatory feature of all cells, which is commonly perceived as a strategy to adapt the proteome to varying external conditions. However, temporal (rhythmic and non-rhythmic) changes of gene expression are also observed under virtually constant external conditions. Here we hypothesize that such changes are a means to render the synthesis of the metabolic output more efficient than under conditions of constant gene activities. In order to substantiate this hypothesis, we used a flux-balance model of the cellular metabolism. The total time span spent on the production of a given set of target metabolites was split into a series of shorter time intervals (metabolic phases) during which only selected groups of metabolic genes are active. The related flux distributions were calculated under the constraint that genes can be either active or inactive whereby the amount of protein related to an active gene is only controlled by the number of active genes: the lower the number of active genes the more protein can be allocated to the enzymes carrying non-zero fluxes. This concept of a predominantly protein-limited efficiency of gene expression clearly differs from other concepts resting on the assumption of an optimal gene regulation capable of allocating to all enzymes and transporters just that fraction of protein necessary to prevent rate limitation. Applying this concept to a simplified metabolic network of the central carbon metabolism with glucose or lactate as alternative substrates, we demonstrate that switching between optimally chosen stationary flux modes comprising different sets of active genes allows producing a demanded amount of target metabolites in a significantly shorter time than by a single optimal flux mode at fixed gene activities. Our model-based findings suggest that temporal expression of metabolic genes can be advantageous even under conditions of constant external substrate supply. PMID:25786979

Abscisic acid promotes proteasome-mediated degradation of the transcription coactivator NPR1 in Arabidopsis thaliana.

PubMed

Ding, Yezhang; Dommel, Matthew; Mou, Zhonglin

2016-04-01

Proteasome-mediated turnover of the transcription coactivator NPR1 is pivotal for efficient activation of the broad-spectrum plant immune responses known as localized acquired resistance (LAR) and systemic acquired resistance (SAR) in adjacent and systemic tissues, respectively, and requires the CUL3-based E3 ligase and its adaptor proteins, NPR3 and NPR4, which are receptors for the signaling molecule salicylic acid (SA). It has been shown that SA prevents NPR1 turnover under non-inducing and LAR/SAR-inducing conditions, but how cellular NPR1 homeostasis is maintained remains unclear. Here, we show that the phytohormone abscisic acid (ABA) and SA antagonistically influence cellular NPR1 protein levels. ABA promotes NPR1 degradation via the CUL3(NPR) (3/) (NPR) (4) complex-mediated proteasome pathway, whereas SA may protect NPR1 from ABA-promoted degradation through phosphorylation. Furthermore, we demonstrate that the timing and strength of SA and ABA signaling are critical in modulating NPR1 accumulation and target gene expression. Perturbing ABA or SA signaling in adjacent tissues alters the temporal dynamic pattern of NPR1 accumulation and target gene transcription. Finally, we show that sequential SA and ABA treatment leads to dynamic changes in NPR1 protein levels and target gene expression. Our results revealed a tight correlation between sequential SA and ABA signaling and dynamic changes in NPR1 protein levels and NPR1-dependent transcription in plant immune responses. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Evolution of the snake body form reveals homoplasy in amniote Hox gene function.

PubMed

Head, Jason J; Polly, P David

2015-04-02

Hox genes regulate regionalization of the axial skeleton in vertebrates, and changes in their expression have been proposed to be a fundamental mechanism driving the evolution of new body forms. The origin of the snake-like body form, with its deregionalized pre-cloacal axial skeleton, has been explained as either homogenization of Hox gene expression domains, or retention of standard vertebrate Hox domains with alteration of downstream expression that suppresses development of distinct regions. Both models assume a highly regionalized ancestor, but the extent of deregionalization of the primaxial domain (vertebrae, dorsal ribs) of the skeleton in snake-like body forms has never been analysed. Here we combine geometric morphometrics and maximum-likelihood analysis to show that the pre-cloacal primaxial domain of elongate, limb-reduced lizards and snakes is not deregionalized compared with limbed taxa, and that the phylogenetic structure of primaxial morphology in reptiles does not support a loss of regionalization in the evolution of snakes. We demonstrate that morphometric regional boundaries correspond to mapped gene expression domains in snakes, suggesting that their primaxial domain is patterned by a normally functional Hox code. Comparison of primaxial osteology in fossil and modern amniotes with Hox gene distributions within Amniota indicates that a functional, sequentially expressed Hox code patterned a subtle morphological gradient along the anterior-posterior axis in stem members of amniote clades and extant lizards, including snakes. The highly regionalized skeletons of extant archosaurs and mammals result from independent evolution in the Hox code and do not represent ancestral conditions for clades with snake-like body forms. The developmental origin of snakes is best explained by decoupling of the primaxial and abaxial domains and by increases in somite number, not by changes in the function of primaxial Hox genes.

MPIGeneNet: Parallel Calculation of Gene Co-Expression Networks on Multicore Clusters.

PubMed

Gonzalez-Dominguez, Jorge; Martin, Maria J

2017-10-10

In this work we present MPIGeneNet, a parallel tool that applies Pearson's correlation and Random Matrix Theory to construct gene co-expression networks. It is based on the state-of-the-art sequential tool RMTGeneNet, which provides networks with high robustness and sensitivity at the expenses of relatively long runtimes for large scale input datasets. MPIGeneNet returns the same results as RMTGeneNet but improves the memory management, reduces the I/O cost, and accelerates the two most computationally demanding steps of co-expression network construction by exploiting the compute capabilities of common multicore CPU clusters. Our performance evaluation on two different systems using three typical input datasets shows that MPIGeneNet is significantly faster than RMTGeneNet. As an example, our tool is up to 175.41 times faster on a cluster with eight nodes, each one containing two 12-core Intel Haswell processors. Source code of MPIGeneNet, as well as a reference manual, are available at https://sourceforge.net/projects/mpigenenet/.

Bromodomain and extraterminal inhibitors block the Epstein-Barr virus lytic cycle at two distinct steps.

PubMed

Keck, Kristin M; Moquin, Stephanie A; He, Amanda; Fernandez, Samantha G; Somberg, Jessica J; Liu, Stephanie M; Martinez, Delsy M; Miranda, Jj L

2017-08-11

Lytic infection by the Epstein-Barr virus (EBV) poses numerous health risks, such as infectious mononucleosis and lymphoproliferative disorder. Proteins in the bromodomain and extraterminal (BET) family regulate multiple stages of viral life cycles and provide promising intervention targets. Synthetic small molecules can bind to the bromodomains and disrupt function by preventing recognition of acetylated lysine substrates. We demonstrate that JQ1 and other BET inhibitors block two different steps in the sequential cascade of the EBV lytic cycle. BET inhibitors prevent expression of the viral immediate-early protein BZLF1. JQ1 alters transcription of genes controlled by the host protein BACH1, and BACH1 knockdown reduces BZLF1 expression. BET proteins also localize to the lytic origin of replication (OriLyt) genetic elements, and BET inhibitors prevent viral late gene expression. There JQ1 reduces BRD4 recruitment during reactivation to preclude replication initiation. This represents a rarely observed dual mode of action for drugs.

Distinct nuclear arrangement of active and inactive c-myc genes in control and differentiated colon carcinoma cells

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

Harnicarova, Andrea; Kozubek, Stanislav; Pachernik, Jiri

2006-12-10

Using sequential RNA-DNA fluorescence in situ hybridization, the nuclear arrangement of both the active and inactive c-myc gene as well as its transcription was investigated in colon cancer HT-29 cells induced to differentiate into enterocytes. Cytogenetic studies revealed the presence of two chromosomes 8 in HT-29 cells, of which the one containing c-myc gene amplicons was substantially larger and easily distinguished from the normal chromosome. This observation enabled detection of both activity and nuclear localization of c-myc genes in single cells and in individual chromosome territories. Similar transcriptional activity of the c-myc gene was observed in both the normal andmore » derivative chromosome 8 territories showing no influence of the amplification on the c-myc gene expression. Our experiments demonstrate strikingly specific nuclear and territorial arrangements of active genes as compared with inactive ones: on the periphery of their territories facing to the very central region of the cell nucleus. Nuclear arrangement of c-myc genes and transcripts was conserved during cell differentiation and, therefore, independent of the level of differentiation-specific c-myc gene expression. However, after the induction of differentiation, a more internal territorial location was found for the single copy c-myc gene of normal chromosome 8, while amplicons conserved their territorial topography.« less

A protocol for combined Photinus and Renilla luciferase quantification compatible with protein assays.

PubMed

Hampf, Mathias; Gossen, Manfred

2006-09-01

We established a quantitative reporter gene protocol, the P/Rluc assay system, allowing the sequential measurement of Photinus and Renilla luciferase activities from the same extract. Other than comparable commercial reporter assay systems and their noncommercial counterparts, the P/Rluc assay system was formulated under the aspect of full compatibility with standard methods for protein assays. This feature greatly expands the range of applications for assay systems quantifying the expression of multiple luciferase reporters.

Antigenic variation of Anaplasma marginale msp2 occurs by combinatorial gene conversion.

PubMed

Brayton, Kelly A; Palmer, Guy H; Lundgren, Anna; Yi, Jooyoung; Barbet, Anthony F

2002-03-01

The rickettsial pathogen Anaplasma marginale establishes lifelong persistent infection in the mammalian reservoir host, during which time immune escape variants continually arise in part because of variation in the expressed copy of the immunodominant outer membrane protein MSP2. A key question is how the small 1.2 Mb A. marginale genome generates sufficient variants to allow long-term persistence in an immunocompetent reservoir host. The recombination of whole pseudogenes into the single msp2 expression site has been previously identified as one method of generating variants, but is inadequate to generate the number of variants required for persistent infection. In the present study, we demonstrate that recombination of a whole pseudogene is followed by a second level of variation in which small segments of pseudogenes recombine into the expression site by gene conversion. Evidence for four short sequential changes in the hypervariable region of msp2 coupled with the identification of nine pseudogenes from a single strain of A. marginale provides for a combinatorial number of possible expressed MSP2 variants sufficient for lifelong persistence.

IL17 factors are early regulators in the gut epithelium during inflammatory response to Vibrio in the sea urchin larva

PubMed Central

Buckley, Katherine M; Ho, Eric Chun Hei; Hibino, Taku; Schrankel, Catherine S; Schuh, Nicholas W; Wang, Guizhi; Rast, Jonathan P

2017-01-01

IL17 cytokines are central mediators of mammalian immunity. In vertebrates, these factors derive from diverse cellular sources. Sea urchins share a molecular heritage with chordates that includes the IL17 system. Here, we characterize the role of epithelial expression of IL17 in the larval gut-associated immune response. The purple sea urchin genome encodes 10 IL17 subfamilies (35 genes) and 2 IL17 receptors. Most of these subfamilies are conserved throughout echinoderms. Two IL17 subfamilies are sequentially strongly upregulated and attenuated in the gut epithelium in response to bacterial disturbance. IL17R1 signal perturbation results in reduced expression of several response genes including an IL17 subtype, indicating a potential feedback. A third IL17 subfamily is activated in adult immune cells indicating that expression in immune cells and epithelia is divided among families. The larva provides a tractable model to investigate the regulation and consequences of gut epithelial IL17 expression across the organism. DOI: http://dx.doi.org/10.7554/eLife.23481.001 PMID:28447937

Prime-boost vaccination with plasmid and adenovirus gene vaccines control HER2/neu+ metastatic breast cancer in mice.

PubMed

Wang, Xiaoyan; Wang, Jian-Ping; Rao, Xiao-Mei; Price, Janet E; Zhou, Heshan S; Lachman, Lawrence B

2005-01-01

Once metastasis has occurred, the possibility of completely curing breast cancer is unlikely, particularly for the 30 to 40% of cancers overexpressing the gene for HER2/neu. A vaccine targeting p185, the protein product of the HER2/neu gene, could have therapeutic application by controlling the growth and metastasis of highly aggressive HER2/neu+ cells. The purpose of this study was to determine the effectiveness of two gene vaccines targeting HER2/neu in preventive and therapeutic tumor models. The mouse breast cancer cell line A2L2, which expresses the gene for rat HER2/neu and hence p185, was injected into the mammary fat pad of mice as a model of solid tumor growth or was injected intravenously as a model of lung metastasis. SINCP-neu, a plasmid containing Sindbis virus genes and the gene for rat HER2/neu, and Adeno-neu, an E1,E2a-deleted adenovirus also containing the gene for rat HER2/neu, were tested as preventive and therapeutic vaccines. Vaccination with SINCP-neu or Adeno-neu before tumor challenge with A2L2 cells significantly inhibited the growth of the cells injected into the mammary fat or intravenously. Vaccination 2 days after tumor challenge with either vaccine was ineffective in both tumor models. However, therapeutic vaccination in a prime-boost protocol with SINCP-neu followed by Adeno-neu significantly prolonged the overall survival rate of mice injected intravenously with the tumor cells. Naive mice vaccinated using the same prime-boost protocol demonstrated a strong serum immunoglobulin G response and p185-specific cellular immunity, as shown by the results of ELISPOT (enzyme-linked immunospot) analysis for IFNgamma. We report herein that vaccination of mice with a plasmid gene vaccine and an adenovirus gene vaccine, each containing the gene for HER2/neu, prevented growth of a HER2/neu-expressing breast cancer cell line injected into the mammary fat pad or intravenously. Sequential administration of the vaccines in a prime-boost protocol was therapeutically effective when tumor cells were injected intravenously before the vaccination. The vaccines induced high levels of both cellular and humoral immunity as determined by in vitro assessment. These findings indicate that clinical evaluation of these vaccines, particularly when used sequentially in a prime-boost protocol, is justified.

Improving cluster-based missing value estimation of DNA microarray data.

PubMed

Brás, Lígia P; Menezes, José C

2007-06-01

We present a modification of the weighted K-nearest neighbours imputation method (KNNimpute) for missing values (MVs) estimation in microarray data based on the reuse of estimated data. The method was called iterative KNN imputation (IKNNimpute) as the estimation is performed iteratively using the recently estimated values. The estimation efficiency of IKNNimpute was assessed under different conditions (data type, fraction and structure of missing data) by the normalized root mean squared error (NRMSE) and the correlation coefficients between estimated and true values, and compared with that of other cluster-based estimation methods (KNNimpute and sequential KNN). We further investigated the influence of imputation on the detection of differentially expressed genes using SAM by examining the differentially expressed genes that are lost after MV estimation. The performance measures give consistent results, indicating that the iterative procedure of IKNNimpute can enhance the prediction ability of cluster-based methods in the presence of high missing rates, in non-time series experiments and in data sets comprising both time series and non-time series data, because the information of the genes having MVs is used more efficiently and the iterative procedure allows refining the MV estimates. More importantly, IKNN has a smaller detrimental effect on the detection of differentially expressed genes.

Gingival tissue transcriptomes in experimental gingivitis

PubMed Central

Jönsson, Daniel; Ramberg, Per; Demmer, Ryan T.; Kebschull, Moritz; Dahlén, Gunnar; Papapanou, Panos N.

2012-01-01

Aims We investigated the sequential gene expression in the gingiva during the induction and resolution of experimental gingivitis. Methods Twenty periodontally and systemically healthy non-smoking volunteers participated in a 3-week experimental gingivitis protocol, followed by debridement and 2-week regular plaque control. We recorded clinical indices and harvested gingival tissue samples from 4 interproximal palatal sites in half of the participants at baseline, Day 7, 14 and 21 (‘induction phase’), and at day 21, 25, 30 and 35 in the other half (‘resolution phase’). RNA was extracted, amplified, reversed transcribed, amplified, labeled and hybridized with Affymetrix Human Genome U133Plus2.0 microarrays. Paired t-tests compared gene expression changes between consecutive time points. Gene ontology analyses summarized the expression patterns into biologically relevant categories. Results The median gingival index was 0 at baseline, 2 at Day 21 and 1 at Day 35. Differential gene regulation peaked during the third week of induction and the first four days of resolution. Leukocyte transmigration, cell adhesion and antigen processing/presentation were the top differentially regulated pathways. Conclusions Transcriptomic studies enhance our understanding of the pathobiology of the reversible inflammatory gingival lesion and provide a detailed account of the dynamic tissue responses during induction and resolution of experimental gingivitis. PMID:21501207

Discovery of error-tolerant biclusters from noisy gene expression data.

PubMed

Gupta, Rohit; Rao, Navneet; Kumar, Vipin

2011-11-24

An important analysis performed on microarray gene-expression data is to discover biclusters, which denote groups of genes that are coherently expressed for a subset of conditions. Various biclustering algorithms have been proposed to find different types of biclusters from these real-valued gene-expression data sets. However, these algorithms suffer from several limitations such as inability to explicitly handle errors/noise in the data; difficulty in discovering small bicliusters due to their top-down approach; inability of some of the approaches to find overlapping biclusters, which is crucial as many genes participate in multiple biological processes. Association pattern mining also produce biclusters as their result and can naturally address some of these limitations. However, traditional association mining only finds exact biclusters, which limits its applicability in real-life data sets where the biclusters may be fragmented due to random noise/errors. Moreover, as they only work with binary or boolean attributes, their application on gene-expression data require transforming real-valued attributes to binary attributes, which often results in loss of information. Many past approaches have tried to address the issue of noise and handling real-valued attributes independently but there is no systematic approach that addresses both of these issues together. In this paper, we first propose a novel error-tolerant biclustering model, 'ET-bicluster', and then propose a bottom-up heuristic-based mining algorithm to sequentially discover error-tolerant biclusters directly from real-valued gene-expression data. The efficacy of our proposed approach is illustrated by comparing it with a recent approach RAP in the context of two biological problems: discovery of functional modules and discovery of biomarkers. For the first problem, two real-valued S.Cerevisiae microarray gene-expression data sets are used to demonstrate that the biclusters obtained from ET-bicluster approach not only recover larger set of genes as compared to those obtained from RAP approach but also have higher functional coherence as evaluated using the GO-based functional enrichment analysis. The statistical significance of the discovered error-tolerant biclusters as estimated by using two randomization tests, reveal that they are indeed biologically meaningful and statistically significant. For the second problem of biomarker discovery, we used four real-valued Breast Cancer microarray gene-expression data sets and evaluate the biomarkers obtained using MSigDB gene sets. The results obtained for both the problems: functional module discovery and biomarkers discovery, clearly signifies the usefulness of the proposed ET-bicluster approach and illustrate the importance of explicitly incorporating noise/errors in discovering coherent groups of genes from gene-expression data.

The expression and regulation of enzymes mediating the biosynthesis of triglycerides and phospholipids in keratinocytes/epidermis

PubMed Central

Feingold, Kenneth R

2011-01-01

Triglycerides and phospholipids play an important role in epidermal permability barrier formation and function. They are synthesized de novo in the epidermis via the glycerol-3-phosphate pathway, catalyzed sequentially by a group of enzymes that have multiple isoforms including glycerol-3-phosphate acyltransferase (GPAT), 1-acylglycerol-3-phosphate acyltransferase (AGPAT), Lipin and diacylglycerol acyltransferase (DGAT). Here we review the current knowledge of GPAT, AGPAT, Lipin and DGAT enzymes in keratinocytes/epidermis focusing on the expression levels of the various isoforms and their localization in mouse epidermis. Additionally, the factors regulating their gene expression, including calcium induced differentiation, PPAR and LXR activators, and the effect of acute permeability barrier disruption will be discussed. PMID:21695015

The evolutionary dynamics of variant antigen genes in Babesia reveal a history of genomic innovation underlying host-parasite interaction.

PubMed

Jackson, Andrew P; Otto, Thomas D; Darby, Alistair; Ramaprasad, Abhinay; Xia, Dong; Echaide, Ignacio Eduardo; Farber, Marisa; Gahlot, Sunayna; Gamble, John; Gupta, Dinesh; Gupta, Yask; Jackson, Louise; Malandrin, Laurence; Malas, Tareq B; Moussa, Ehab; Nair, Mridul; Reid, Adam J; Sanders, Mandy; Sharma, Jyotsna; Tracey, Alan; Quail, Mike A; Weir, William; Wastling, Jonathan M; Hall, Neil; Willadsen, Peter; Lingelbach, Klaus; Shiels, Brian; Tait, Andy; Berriman, Matt; Allred, David R; Pain, Arnab

2014-06-01

Babesia spp. are tick-borne, intraerythrocytic hemoparasites that use antigenic variation to resist host immunity, through sequential modification of the parasite-derived variant erythrocyte surface antigen (VESA) expressed on the infected red blood cell surface. We identified the genomic processes driving antigenic diversity in genes encoding VESA (ves1) through comparative analysis within and between three Babesia species, (B. bigemina, B. divergens and B. bovis). Ves1 structure diverges rapidly after speciation, notably through the evolution of shortened forms (ves2) from 5' ends of canonical ves1 genes. Phylogenetic analyses show that ves1 genes are transposed between loci routinely, whereas ves2 genes are not. Similarly, analysis of sequence mosaicism shows that recombination drives variation in ves1 sequences, but less so for ves2, indicating the adoption of different mechanisms for variation of the two families. Proteomic analysis of the B. bigemina PR isolate shows that two dominant VESA1 proteins are expressed in the population, whereas numerous VESA2 proteins are co-expressed, consistent with differential transcriptional regulation of each family. Hence, VESA2 proteins are abundant and previously unrecognized elements of Babesia biology, with evolutionary dynamics consistently different to those of VESA1, suggesting that their functions are distinct. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Genome-wide expression analysis of human in vivo irritated epidermis: differential profiles induced by sodium lauryl sulfate and nonanoic acid.

PubMed

Clemmensen, Anders; Andersen, Klaus E; Clemmensen, Ole; Tan, Qihua; Petersen, Thomas K; Kruse, Torben A; Thomassen, Mads

2010-09-01

The pathogenesis of irritant contact dermatitis (ICD) is poorly understood, and genes participating in the epidermal response to chemical irritants are only partly known. It is commonly accepted that different irritants have different mechanisms of action in the development of ICD. To define the differential molecular events induced in the epidermis by different irritants, we collected sequential biopsies ((1/2), 4, and 24 hours after a single exposure and at day 11 after repeated exposure) from human volunteers exposed to either sodium lauryl sulfate (SLS) or nonanoic acid (NON). Gene expression analysis using high-density oligonucleotide microarrays (representing 47,000 transcripts) revealed essentially different pathway responses (1/2)hours after exposure: NON transiently induced the IL-6 pathway as well as a number of mitogen-activated signaling cascades including extracellular signal-regulated kinase and growth factor receptor signaling, whereas SLS transiently downregulated cellular energy metabolism pathways. Differential expression of the cyclooxygenase-2 and matrix metalloproteinase 3 transcripts was confirmed immunohistochemically. After cumulative exposure, 883 genes were differentially expressed, whereas we identified 23 suggested common biomarkers for ICD. In conclusion, we bring new insights into two hitherto less well-elucidated phases of skin irritancy: the very initial as well as the late phase after single and cumulative mild exposures, respectively.

From cytoskeletal dynamics to organ asymmetry: a nonlinear, regulative pathway underlies left-right patterning.

PubMed

McDowell, Gary; Rajadurai, Suvithan; Levin, Michael

2016-12-19

Consistent left-right (LR) asymmetry is a fundamental aspect of the bodyplan across phyla, and errors of laterality form an important class of human birth defects. Its molecular underpinning was first discovered as a sequential pathway of left- and right-sided gene expression that controlled positioning of the heart and visceral organs. Recent data have revised this picture in two important ways. First, the physical origin of chirality has been identified; cytoskeletal dynamics underlie the asymmetry of single-cell behaviour and patterning of the LR axis. Second, the pathway is not linear: early disruptions that alter the normal sidedness of upstream asymmetric genes do not necessarily induce defects in the laterality of the downstream genes or in organ situs Thus, the LR pathway is a unique example of two fascinating aspects of biology: the interplay of physics and genetics in establishing large-scale anatomy, and regulative (shape-homeostatic) pathways that correct molecular and anatomical errors over time. Here, we review aspects of asymmetry from its intracellular, cytoplasmic origins to the recently uncovered ability of the LR control circuitry to achieve correct gene expression and morphology despite reversals of key 'determinant' genes. We provide novel functional data, in Xenopus laevis, on conserved elements of the cytoskeleton that drive asymmetry, and comparatively analyse it together with previously published results in the field. Our new observations and meta-analysis demonstrate that despite aberrant expression of upstream regulatory genes, embryos can progressively normalize transcriptional cascades and anatomical outcomes. LR patterning can thus serve as a paradigm of how subcellular physics and gene expression cooperate to achieve developmental robustness of a body axis.This article is part of the themed issue 'Provocative questions in left-right asymmetry'. © 2016 The Author(s).

From cytoskeletal dynamics to organ asymmetry: a nonlinear, regulative pathway underlies left–right patterning

PubMed Central

Rajadurai, Suvithan

2016-01-01

Consistent left–right (LR) asymmetry is a fundamental aspect of the bodyplan across phyla, and errors of laterality form an important class of human birth defects. Its molecular underpinning was first discovered as a sequential pathway of left- and right-sided gene expression that controlled positioning of the heart and visceral organs. Recent data have revised this picture in two important ways. First, the physical origin of chirality has been identified; cytoskeletal dynamics underlie the asymmetry of single-cell behaviour and patterning of the LR axis. Second, the pathway is not linear: early disruptions that alter the normal sidedness of upstream asymmetric genes do not necessarily induce defects in the laterality of the downstream genes or in organ situs. Thus, the LR pathway is a unique example of two fascinating aspects of biology: the interplay of physics and genetics in establishing large-scale anatomy, and regulative (shape-homeostatic) pathways that correct molecular and anatomical errors over time. Here, we review aspects of asymmetry from its intracellular, cytoplasmic origins to the recently uncovered ability of the LR control circuitry to achieve correct gene expression and morphology despite reversals of key ‘determinant’ genes. We provide novel functional data, in Xenopus laevis, on conserved elements of the cytoskeleton that drive asymmetry, and comparatively analyse it together with previously published results in the field. Our new observations and meta-analysis demonstrate that despite aberrant expression of upstream regulatory genes, embryos can progressively normalize transcriptional cascades and anatomical outcomes. LR patterning can thus serve as a paradigm of how subcellular physics and gene expression cooperate to achieve developmental robustness of a body axis. This article is part of the themed issue ‘Provocative questions in left–right asymmetry’. PMID:27821521

Neurogenic gene regulatory pathways in the sea urchin embryo.

PubMed

Wei, Zheng; Angerer, Lynne M; Angerer, Robert C

2016-01-15

During embryogenesis the sea urchin early pluteus larva differentiates 40-50 neurons marked by expression of the pan-neural marker synaptotagmin B (SynB) that are distributed along the ciliary band, in the apical plate and pharyngeal endoderm, and 4-6 serotonergic neurons that are confined to the apical plate. Development of all neurons has been shown to depend on the function of Six3. Using a combination of molecular screens and tests of gene function by morpholino-mediated knockdown, we identified SoxC and Brn1/2/4, which function sequentially in the neurogenic regulatory pathway and are also required for the differentiation of all neurons. Misexpression of Brn1/2/4 at low dose caused an increase in the number of serotonin-expressing cells and at higher dose converted most of the embryo to a neurogenic epithelial sphere expressing the Hnf6 ciliary band marker. A third factor, Z167, was shown to work downstream of the Six3 and SoxC core factors and to define a branch specific for the differentiation of serotonergic neurons. These results provide a framework for building a gene regulatory network for neurogenesis in the sea urchin embryo. © 2016. Published by The Company of Biologists Ltd.

Oxalate-Metabolising Genes of the White-Rot Fungus Dichomitus squalens Are Differentially Induced on Wood and at High Proton Concentration

PubMed Central

de Vries, Ronald P.; Timonen, Sari; Hildén, Kristiina

2014-01-01

Oxalic acid is a prevalent fungal metabolite with versatile roles in growth and nutrition, including degradation of plant biomass. However, the toxicity of oxalic acid makes regulation of its intra- and extracellular concentration crucial. To increase the knowledge of fungal oxalate metabolism, a transcriptional level study on oxalate-catabolising genes was performed with an effective lignin-degrading white-rot fungus Dichomitus squalens, which has demonstrated particular abilities in production and degradation of oxalic acid. The expression of oxalic-acid decomposing oxalate decarboxylase (ODC) and formic-acid decomposing formate dehydrogenase (FDH) encoding genes was followed during the growth of D. squalens on its natural spruce wood substrate. The effect of high proton concentration on the regulation of the oxalate-catabolising genes was determined after addition of organic acid (oxalic acid) and inorganic acid (hydrochloric acid) to the liquid cultures of D. squalens. In order to evaluate the co-expression of oxalate-catabolising and manganese peroxidase (MnP) encoding genes, the expression of one MnP encoding gene, mnp1, of D. squalens was also surveyed in the solid state and liquid cultures. Sequential action of ODC and FDH encoding genes was detected in the studied cultivations. The odc1, fdh2 and fdh3 genes of D. squalens showed constitutive expression, whereas ODC2 and FHD1 most likely are the main responsible enzymes for detoxification of high concentrations of oxalic and formic acids. The results also confirmed the central role of ODC1 when D. squalens grows on coniferous wood. Phylogenetic analysis revealed that fungal ODCs have evolved from at least two gene copies whereas FDHs have a single ancestral gene. As a conclusion, the multiplicity of oxalate-catabolising genes and their differential regulation on wood and in acid-amended cultures of D. squalens point to divergent physiological roles for the corresponding enzymes. PMID:24505339

In vivo monitoring of transfected DNA, gene expression kinetics, and cellular immune responses in mice immunized with a human NIS gene-expressing plasmid.

PubMed

Son, Hye-Youn; Jeon, Yong-Hyun; Chung, June-Key; Kim, Chul-Woo

2016-12-01

In assessing the effectiveness of DNA vaccines, it is important to monitor: (1) the kinetics of target gene expression in vivo; and (2) the movement of cells that become transfected with the plasmid DNA used in the immunization of a subject. In this study, we used, as a visual imaging marker, expression of the transfected human sodium/iodide symporter (hNIS) gene, which enhances intracellular radio-pertechnetate (TcO4-) accumulation. After intradermal (i.d.) and systemic injection of mice with pcDNA-hNIS and radioactive Technetium-99m (Tc-99m), respectively, whole-body images were obtained by nuclear scintigraphy. The migration of mice cells transfected with the hNIS gene was monitored over a 2-week period by gamma-radioactivity counting of isolated cell populations and was demonstrated in peripheral lymphoid tissues, especially in the draining lymph nodes (dLNs). Beginning at 24 h after DNA inoculation and continuing for the 2-week monitoring period, hNIS-expressing cells were observed specifically in the T-cell-rich zones of the paracortical area of the dLNs. Over the same time period, high levels of INF-γ-secreting CD8 T-cells were found in the dLNs of the pcDNA-hNIS immunized mice. Tumor growth was also significantly retarded in the mice that received hNIS DNA immunization followed by inoculation with CT26 colorectal adenocarcinoma cells that had been transfected with the rat NIS gene (rNIS), which is 93% homologous to the hNIS gene. In conclusion, mouse cells transfected with hNIS DNA after i.d. immunization were found to traffic to the dLNs, and hNIS gene expression in these cells continued for at least 2 weeks post immunization. Furthermore, sequential presentation of NIS DNA to T-cells by migratory antigen presenting cells could induce NIS DNA-specific Th1 immune responses and thus retard the growth of NIS-expressing tumors. © The Author(s) 2016.

Three enhancer regions regulate gbx2 gene expression in the isthmic region during zebrafish development.

PubMed

Islam, Md Ekramul; Kikuta, Hiroshi; Inoue, Fumitaka; Kanai, Maiko; Kawakami, Atsushi; Parvin, Mst Shahnaj; Takeda, Hiroyuki; Yamasu, Kyo

2006-12-01

In vertebrate embryos, positioning of the boundary between the midbrain and hindbrain (MHB) and subsequent isthmus formation are dependent upon the interaction between the Otx2 and Gbx genes. In zebrafish, sequential expression of gbx1 and gbx2 in the anterior hindbrain contributes to this process, whereas in mouse embryos, a single Gbx gene (Gbx2) is responsible for MHB development. In the present study, to investigate the regulatory mechanism of gbx2 in the MHB/isthmic region of zebrafish embryos, we cloned the gene and showed that its organization is conserved among different vertebrates. Promoter analyses revealed three enhancers that direct reporter gene expression after the end of epiboly in the anterior-most hindbrain, which is a feature of the zebrafish gbx2 gene. One of the enhancers is located upstream of gbx2 (AMH1), while the other two enhancers are located downstream of gbx2 (AMH2 and AMH3). Detailed analysis of the AMH1 enhancer showed that it directs expression in the rhombomere 1 (r1) region and the dorsal thalamus, as has been shown for gbx2, whereas no expression was induced by the AMH1 enhancer in other embryonic regions in which gbx2 is expressed. The AMH1 enhancer is composed of multiple regulatory subregions that share the same spatial specificity. The most active of the regulatory subregions is a 291-bp region that contains at least two Pax2-binding sites, both of which are necessary for the function of the main component (PB1-A region) of the AMH1 enhancer. In accordance with these results, enhancer activity in the PB1-A region, as well as gbx2 expression in r1, was missing in no isthmus mutant embryos that lacked functional pax2a. In addition, we identified an upstream conserved sequence of 227bp that suppresses the enhancer activity of AMH1. Taken together, these findings suggest that gbx2 expression during the somitogenesis stage in zebrafish is regulated by a complex mechanism involving Pax2 as well as activators and suppressors in the regions flanking the gene.

Hormonal control of GTP cyclohydrolase I gene expression and enzyme activity during color pattern development in wings of Precis coenia.

PubMed

Sawada, H; Nakagoshi, M; Reinhardt, R K; Ziegler, I; Koch, P B

2002-06-01

Color patterns of butterfly wings are composed of single color points represented by each scale. In the case of Precis coenia, at the end of pupal development, different types of pigments are synthesized sequentially in the differently colored scales beginning with white (pterins) followed by red (ommatins) and then black (melanin). In order to explain how formation of these different colors is regulated, we examined the expression of an mRNA-encoding guanosine triphosphate-cyclohydrolase I (GTP-CH I; EC 3.5.4.16), the first key enzyme in the biosynthesis of pteridines, during pigment formation in the wings of P. coenia. The strongest positive signal was recognized around pigment formation one day before butterfly emergence. This GTP-CH I gene expression is paralleled by GTP-CH I enzyme activity measured in wing extracts. We also investigated the effect of 20-hydroxyecdysone on the expression of GTP-CH I mRNA and the enzyme activity during color formation. The results strongly suggest that the onset and duration of the expression of a GTP-CH I mRNA is triggered by a declining ecdysteroid hormone titer during late pupal development.

Chronic Antibody-Mediated Rejection in Nonhuman Primate Renal Allografts: Validation of Human Histological and Molecular Phenotypes.

PubMed

Adam, B A; Smith, R N; Rosales, I A; Matsunami, M; Afzali, B; Oura, T; Cosimi, A B; Kawai, T; Colvin, R B; Mengel, M

2017-11-01

Molecular testing represents a promising adjunct for the diagnosis of antibody-mediated rejection (AMR). Here, we apply a novel gene expression platform in sequential formalin-fixed paraffin-embedded samples from nonhuman primate (NHP) renal transplants. We analyzed 34 previously described gene transcripts related to AMR in humans in 197 archival NHP samples, including 102 from recipients that developed chronic AMR, 80 from recipients without AMR, and 15 normal native nephrectomies. Three endothelial genes (VWF, DARC, and CAV1), derived from 10-fold cross-validation receiver operating characteristic curve analysis, demonstrated excellent discrimination between AMR and non-AMR samples (area under the curve = 0.92). This three-gene set correlated with classic features of AMR, including glomerulitis, capillaritis, glomerulopathy, C4d deposition, and DSAs (r = 0.39-0.63, p < 0.001). Principal component analysis confirmed the association between three-gene set expression and AMR and highlighted the ambiguity of v lesions and ptc lesions between AMR and T cell-mediated rejection (TCMR). Elevated three-gene set expression corresponded with the development of immunopathological evidence of rejection and often preceded it. Many recipients demonstrated mixed AMR and TCMR, suggesting that this represents the natural pattern of rejection. These data provide NHP animal model validation of recent updates to the Banff classification including the assessment of molecular markers for diagnosing AMR. © 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.

Defense to Sclerotinia sclerotiorum in oilseed rape is associated with the sequential activations of salicylic acid signaling and jasmonic acid signaling.

PubMed

Wang, Zheng; Tan, Xiaoli; Zhang, Zhiyan; Gu, Shoulai; Li, Guanying; Shi, Haifeng

2012-03-01

Signaling pathways mediated by salicylic acid (SA) and jasmonic acid (JA) are widely studied in various host-pathogen interactions. For oilseed rape (Brassica napus)-Sclerotinia sclerotiorum interaction, little information of the two signaling molecules has been described in detail. In this study, we showed that the level of SA and JA in B. napus leaves was increased with a distinct temporal profile, respectively, after S. sclerotiorum infection. The application of SA or methyl jasmonate enhanced the resistance to the pathogen. Furthermore, a set of SA and JA signaling marker genes were identified from B. napus and were used to monitor the signaling responses to S. sclerotiorum infection by examining the temporal expression profiles of these marker genes. The SA signaling was activated within 12h post inoculation (hpi) followed by the JA signaling which was activated around 24 hpi. In addition, SA-JA crosstalk genes were activated during this process. These results suggested that defense against S. sclerotiorum in oilseed rape is associated with a sequential activation of SA signaling and JA signaling, which provide important clues for designing strategies to curb diseases caused by S. sclerotioru. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Genome-Wide Identification, Evolutionary Expansion, and Expression Profile of Homeodomain-Leucine Zipper Gene Family in Poplar (Populus trichocarpa)

PubMed Central

Hu, Ruibo; Chi, Xiaoyuan; Chai, Guohua; Kong, Yingzhen; He, Guo; Wang, Xiaoyu; Shi, Dachuan; Zhang, Dongyuan; Zhou, Gongke

2012-01-01

Background Homeodomain-leucine zipper (HD-ZIP) proteins are plant-specific transcriptional factors known to play crucial roles in plant development. Although sequence phylogeny analysis of Populus HD-ZIPs was carried out in a previous study, no systematic analysis incorporating genome organization, gene structure, and expression compendium has been conducted in model tree species Populus thus far. Principal Findings In this study, a comprehensive analysis of Populus HD-ZIP gene family was performed. Sixty-three full-length HD-ZIP genes were found in Populus genome. These Populus HD-ZIP genes were phylogenetically clustered into four distinct subfamilies (HD-ZIP I–IV) and predominately distributed across 17 linkage groups (LG). Fifty genes from 25 Populus paralogous pairs were located in the duplicated blocks of Populus genome and then preferentially retained during the sequential evolutionary courses. Genomic organization analyses indicated that purifying selection has played a pivotal role in the retention and maintenance of Populus HD-ZIP gene family. Microarray analysis has shown that 21 Populus paralogous pairs have been differentially expressed across different tissues and under various stresses, with five paralogous pairs showing nearly identical expression patterns, 13 paralogous pairs being partially redundant and three paralogous pairs diversifying significantly. Quantitative real-time RT-PCR (qRT-PCR) analysis performed on 16 selected Populus HD-ZIP genes in different tissues and under both drought and salinity stresses confirms their tissue-specific and stress-inducible expression patterns. Conclusions Genomic organizations indicated that segmental duplications contributed significantly to the expansion of Populus HD-ZIP gene family. Exon/intron organization and conserved motif composition of Populus HD-ZIPs are highly conservative in the same subfamily, suggesting the members in the same subfamilies may also have conservative functionalities. Microarray and qRT-PCR analyses showed that 89% (56 out of 63) of Populus HD-ZIPs were duplicate genes that might have been retained by substantial subfunctionalization. Taken together, these observations may lay the foundation for future functional analysis of Populus HD-ZIP genes to unravel their biological roles. PMID:22359569

Coordinated metabolic transitions during Drosophila embryogenesis and the onset of aerobic glycolysis.

PubMed

Tennessen, Jason M; Bertagnolli, Nicolas M; Evans, Janelle; Sieber, Matt H; Cox, James; Thummel, Carl S

2014-03-12

Rapidly proliferating cells such as cancer cells and embryonic stem cells rely on a specialized metabolic program known as aerobic glycolysis, which supports biomass production from carbohydrates. The fruit fly Drosophila melanogaster also utilizes aerobic glycolysis to support the rapid growth that occurs during larval development. Here we use singular value decomposition analysis of modENCODE RNA-seq data combined with GC-MS-based metabolomic analysis to analyze the changes in gene expression and metabolism that occur during Drosophila embryogenesis, spanning the onset of aerobic glycolysis. Unexpectedly, we find that the most common pattern of co-expressed genes in embryos includes the global switch to glycolytic gene expression that occurs midway through embryogenesis. In contrast to the canonical aerobic glycolytic pathway, however, which is accompanied by reduced mitochondrial oxidative metabolism, the expression of genes involved in the tricarboxylic cycle (TCA cycle) and the electron transport chain are also upregulated at this time. Mitochondrial activity, however, appears to be attenuated, as embryos exhibit a block in the TCA cycle that results in elevated levels of citrate, isocitrate, and α-ketoglutarate. We also find that genes involved in lipid breakdown and β-oxidation are upregulated prior to the transcriptional initiation of glycolysis, but are downregulated before the onset of larval development, revealing coordinated use of lipids and carbohydrates during development. These observations demonstrate the efficient use of nutrient stores to support embryonic development, define sequential metabolic transitions during this stage, and demonstrate striking similarities between the metabolic state of late-stage fly embryos and tumor cells. Copyright © 2014 Tennessen et al.

Dnmts and Tet target memory-associated genes after appetitive olfactory training in honey bees

PubMed Central

Biergans, Stephanie D.; Giovanni Galizia, C.; Reinhard, Judith; Claudianos, Charles

2015-01-01

DNA methylation and demethylation are epigenetic mechanisms involved in memory formation. In honey bees DNA methyltransferase (Dnmt) function is necessary for long-term memory to be stimulus specific (i.e. to reduce generalization). So far, however, it remains elusive which genes are targeted and what the time-course of DNA methylation is during memory formation. Here, we analyse how DNA methylation affects memory retention, gene expression, and differential methylation in stimulus-specific olfactory long-term memory formation. Out of 30 memory-associated genes investigated here, 9 were upregulated following Dnmt inhibition in trained bees. These included Dnmt3 suggesting a negative feedback loop for DNA methylation. Within these genes also the DNA methylation pattern changed during the first 24 hours after training. Interestingly, this was accompanied by sequential activation of the DNA methylation machinery (i.e. Dnmts and Tet). In sum, memory formation involves a temporally complex epigenetic regulation of memory-associated genes that facilitates stimulus specific long-term memory in the honey bee. PMID:26531238

Characterization of the transcriptome profiles related to globin gene switching during in vitro erythroid maturation

PubMed Central

2012-01-01

Background The fetal and adult globin genes in the human β-globin cluster on chromosome 11 are sequentially expressed to achieve normal hemoglobin switching during human development. The pharmacological induction of fetal γ-globin (HBG) to replace abnormal adult sickle βS-globin is a successful strategy to treat sickle cell disease; however the molecular mechanism of γ-gene silencing after birth is not fully understood. Therefore, we performed global gene expression profiling using primary erythroid progenitors grown from human peripheral blood mononuclear cells to characterize gene expression patterns during the γ-globin to β-globin (γ/β) switch observed throughout in vitro erythroid differentiation. Results We confirmed erythroid maturation in our culture system using cell morphologic features defined by Giemsa staining and the γ/β-globin switch by reverse transcription-quantitative PCR (RT-qPCR) analysis. We observed maximal γ-globin expression at day 7 with a switch to a predominance of β-globin expression by day 28 and the γ/β-globin switch occurred around day 21. Expression patterns for transcription factors including GATA1, GATA2, KLF1 and NFE2 confirmed our system produced the expected pattern of expression based on the known function of these factors in globin gene regulation. Subsequent gene expression profiling was performed with RNA isolated from progenitors harvested at day 7, 14, 21, and 28 in culture. Three major gene profiles were generated by Principal Component Analysis (PCA). For profile-1 genes, where expression decreased from day 7 to day 28, we identified 2,102 genes down-regulated > 1.5-fold. Ingenuity pathway analysis (IPA) for profile-1 genes demonstrated involvement of the Cdc42, phospholipase C, NF-Kβ, Interleukin-4, and p38 mitogen activated protein kinase (MAPK) signaling pathways. Transcription factors known to be involved in γ-and β-globin regulation were identified. The same approach was used to generate profile-2 genes where expression was up-regulated over 28 days in culture. IPA for the 2,437 genes with > 1.5-fold induction identified the mitotic roles of polo-like kinase, aryl hydrocarbon receptor, cell cycle control, and ATM (Ataxia Telangiectasia Mutated Protein) signaling pathways; transcription factors identified included KLF1, GATA1 and NFE2 among others. Finally, profile-3 was generated from 1,579 genes with maximal expression at day 21, around the time of the γ/β-globin switch. IPA identified associations with cell cycle control, ATM, and aryl hydrocarbon receptor signaling pathways. Conclusions The transcriptome analysis completed with erythroid progenitors grown in vitro identified groups of genes with distinct expression profiles, which function in metabolic pathways associated with cell survival, hematopoiesis, blood cells activation, and inflammatory responses. This study represents the first report of a transcriptome analysis in human primary erythroid progenitors to identify transcription factors involved in hemoglobin switching. Our results also demonstrate that the in vitro liquid culture system is an excellent model to define mechanisms of global gene expression and the DNA-binding protein and signaling pathways involved in globin gene regulation. PMID:22537182

Lentivirus-mediated platelet gene therapy of murine hemophilia A with pre-existing anti-FVIII immunity

PubMed Central

Kuether, E. L.; Schroeder, J. A.; Fahs, S. A.; Cooley, B. C.; Chen, Y.; Montgomery, R. R.; Wilcox, D. A.; Shi, Q.

2012-01-01

Summary Background The development of inhibitory antibodies, referred to as inhibitors, against exogenous FVIII in a significant subset of patients with hemophilia A remains a persistent challenge to the efficacy of protein replacement therapy. Our previous studies using the transgenic approach provided proof-of-principle that platelet-specific expression could be successful for treating hemophilia A in the presence of inhibitory antibodies. Objective To investigate a clinically translatable approach for platelet gene therapy of hemophilia A with pre-existing inhibitors. Methods Platelet-FVIII expression in pre-immunized FVIIInull mice was introduced by transplantation of lentivirus-transduced bone marrow or enriched hematopoietic stem cells. FVIII expression was determined by a chromogenic assay. The transgene copy number per cell was quantitated by real time PCR. Inhibitor titer was measured by Bethesda assay. Phenotypic correction was assessed by the tail clipping assay and an electrolytic-induced venous injury model. Integration sites were analyzed by LAM-PCR. Results Therapeutic levels of platelet-FVIII expression were sustained long-term without evoking an anti-FVIII memory response in the transduced pre-immunized recipients. The tail clip survival test and the electrolytic injury model confirmed that hemostasis was improved in the treated animals. Sequential bone marrow transplants showed sustained platelet-FVIII expression resulting in phenotypic correction in pre-immunized secondary and tertiary recipients. Conclusions Lentivirus-mediated platelet-specific gene transfer improves hemostasis in hemophilic A mice with pre-existing inhibitors, indicating that this approach may be a promising strategy for gene therapy of hemophilia A even in the high-risk setting of pre-existing inhibitory antibodies. PMID:22632092

Human L-DOPA decarboxylase mRNA is a target of miR-145: A prediction to validation workflow.

PubMed

Papadopoulos, Emmanuel I; Fragoulis, Emmanuel G; Scorilas, Andreas

2015-01-10

l-DOPA decarboxylase (DDC) is a multiply-regulated gene which encodes the enzyme that catalyzes the biosynthesis of dopamine in humans. MicroRNAs comprise a novel class of endogenously transcribed small RNAs that can post-transcriptionally regulate the expression of various genes. Given that the mechanism of microRNA target recognition remains elusive, several genes, including DDC, have not yet been identified as microRNA targets. Nevertheless, a number of specifically designed bioinformatic algorithms provide candidate miRNAs for almost every gene, but still their results exhibit moderate accuracy and should be experimentally validated. Motivated by the above, we herein sought to discover a microRNA that regulates DDC expression. By using the current algorithms according to bibliographic recommendations we found that miR-145 could be predicted with high specificity as a candidate regulatory microRNA for DDC expression. Thus, a validation experiment followed by firstly transfecting an appropriate cell culture system with a synthetic miR-145 sequence and sequentially assessing the mRNA and protein levels of DDC via real-time PCR and Western blotting, respectively. Our analysis revealed that miR-145 had no significant impact on protein levels of DDC but managed to dramatically downregulate its mRNA expression. Overall, the experimental and bioinformatic analysis conducted herein indicate that miR-145 has the ability to regulate DDC mRNA expression and potentially this occurs by recognizing its mRNA as a target. Copyright © 2014. Published by Elsevier B.V.

Population structuring of multi-copy, antigen-encoding genes in Plasmodium falciparum

PubMed Central

Artzy-Randrup, Yael; Rorick, Mary M; Day, Karen; Chen, Donald; Dobson, Andrew P; Pascual, Mercedes

2012-01-01

The coexistence of multiple independently circulating strains in pathogen populations that undergo sexual recombination is a central question of epidemiology with profound implications for control. An agent-based model is developed that extends earlier ‘strain theory’ by addressing the var gene family of Plasmodium falciparum. The model explicitly considers the extensive diversity of multi-copy genes that undergo antigenic variation via sequential, mutually exclusive expression. It tracks the dynamics of all unique var repertoires in a population of hosts, and shows that even under high levels of sexual recombination, strain competition mediated through cross-immunity structures the parasite population into a subset of coexisting dominant repertoires of var genes whose degree of antigenic overlap depends on transmission intensity. Empirical comparison of patterns of genetic variation at antigenic and neutral sites supports this role for immune selection in structuring parasite diversity. DOI: http://dx.doi.org/10.7554/eLife.00093.001 PMID:23251784

CD38 expression and immunoglobulin variable region mutations are independent prognostic variables in chronic lymphocytic leukemia, but CD38 expression may vary during the course of the disease.

PubMed

Hamblin, Terry J; Orchard, Jenny A; Ibbotson, Rachel E; Davis, Zadie; Thomas, Peter W; Stevenson, Freda K; Oscier, David G

2002-02-01

Although the presence or absence of somatic mutations in the immunoglobulin variable region (IgV(H)) genes in chronic lymphocytic leukemia (B-CLL) identifies subtypes with very different prognoses, the assay is technically complex and unavailable to most laboratories. CD38 expression has been suggested as a surrogate marker for the 2 subtypes. IgV(H) mutations and CD38 expression in 145 patients with B-CLL with a long follow-up were compared. The 2 assays gave discordant results in 41 patients (28.3%). Multivariate analysis demonstrated that Binet stage, IgV(H) mutations and CD38 were independent prognostic indicators. Median survival time in patients whose cells had unmutated IgV(H) genes and expressed CD38 was 8 years; in those with mutated IgV(H) genes not expressing CD38, it was 26 years. For those with discordant results, median survival time was 15 years. Thus, although CD38 expression does not identify the same 2 subsets as IgV(H) mutations in CLL, it is an independent risk factor that can be used with IgV(H) mutations and clinical stage to select patients with B-CLL with the worst prognoses. Using cryopreserved cells taken at intervals during the course of the disease, however, changes of CD38 expression over time were demonstrated in 10 of 41 patients. Causes of the variation of CD38 expression require further study. Additional prospective studies are required for comparing CD38 expression with other prognostic factors and for taking sequential measurements during the course of the disease.

By Changing Dimensionality, Sequential Culturing of Midbrain Cells, rather than Two-Dimensional Culture, Generates a Neuron-Glia Ratio Closer to in vivo Adult Midbrain.

PubMed

Ganapathy, Kavina; Sowmithra, Sowmithra; Bhonde, Ramesh; Datta, Indrani

2016-07-16

The neuron-glia ratio is of prime importance for maintaining the physiological homeostasis of neuronal and glial cells, and especially crucial for dopaminergic neurons because a reduction in glial density has been reported in postmortem reports of brains affected by Parkinson's disease. We thus aimed at developing an in vitro midbrain culture which would replicate a similar neuron-glia ratio to that in in vivo adult midbrain while containing a similar number of dopaminergic neurons. A sequential culture technique was adopted to achieve this. Neural progenitors (NPs) were generated by the hanging-drop method and propagated as 3D neurospheres followed by the derivation of outgrowth from these neurospheres on a chosen extracellular matrix. The highest proliferation was observed in neurospheres from day in vitro (DIV) 5 through MTT and FACS analysis of Ki67 expression. FACS analysis using annexin/propidium iodide showed an increase in the apoptotic population from DIV 8. DIV 5 neurospheres were therefore selected for deriving the differentiated outgrowth of midbrain on a poly-L-lysine-coated surface. Quantitative RT-PCR showed comparable gene expressions of the mature neuronal marker β-tubulin III, glial marker GFAP and dopaminergic marker tyrosine hydroxylase (TH) as compared to in vivo adult rat midbrain. The FACS analysis showed a similar neuron-glia ratio obtained by the sequential culture in comparison to adult rat midbrain. The yield of β-tubulin III and TH was distinctly higher in the sequential culture in comparison to 2D culture, which showed a higher yield of GFAP immunopositive cells. Functional characterization indicated that both the constitutive and inducible (KCl and ATP) release of dopamine was distinctly higher in the sequential culture than the 2D culture. Thus, the sequential culture technique succeeded in the initial enrichment of NPs in 3D neurospheres, which in turn resulted in an optimal attainment of the neuron-glia ratio on outgrowth culture from these neurospheres. © 2016 S. Karger AG, Basel.

NRSF-dependent epigenetic mechanisms contribute to programming of stress-sensitive neurons by neonatal experience, promoting resilience.

PubMed

Singh-Taylor, A; Molet, J; Jiang, S; Korosi, A; Bolton, J L; Noam, Y; Simeone, K; Cope, J; Chen, Y; Mortazavi, A; Baram, T Z

2018-03-01

Resilience to stress-related emotional disorders is governed in part by early-life experiences. Here we demonstrate experience-dependent re-programming of stress-sensitive hypothalamic neurons, which takes place through modification of neuronal gene expression via epigenetic mechanisms. Specifically, we found that augmented maternal care reduced glutamatergic synapses onto stress-sensitive hypothalamic neurons and repressed expression of the stress-responsive gene, Crh. In hypothalamus in vitro, reduced glutamatergic neurotransmission recapitulated the repressive effects of augmented maternal care on Crh, and this required recruitment of the transcriptional repressor repressor element-1 silencing transcription factor/neuron restrictive silencing factor (NRSF). Increased NRSF binding to chromatin was accompanied by sequential repressive epigenetic changes which outlasted NRSF binding. chromatin immunoprecipitation-seq analyses of NRSF targets identified gene networks that, in addition to Crh, likely contributed to the augmented care-induced phenotype, including diminished depression-like and anxiety-like behaviors. Together, we believe these findings provide the first causal link between enriched neonatal experience, synaptic refinement and induction of epigenetic processes within specific neurons. They uncover a novel mechanistic pathway from neonatal environment to emotional resilience.

The Receptor Tyrosine Kinase EphA2 Is a Direct Target Gene of Hypermethylated in Cancer 1 (HIC1)*

PubMed Central

Foveau, Bénédicte; Boulay, Gaylor; Pinte, Sébastien; Van Rechem, Capucine; Rood, Brian R.; Leprince, Dominique

2012-01-01

The tumor suppressor gene hypermethylated in cancer 1 (HIC1), which encodes a transcriptional repressor, is epigenetically silenced in many human tumors. Here, we show that ectopic expression of HIC1 in the highly malignant MDA-MB-231 breast cancer cell line severely impairs cell proliferation, migration, and invasion in vitro. In parallel, infection of breast cancer cell lines with a retrovirus expressing HIC1 also induces decreased mRNA and protein expression of the tyrosine kinase receptor EphA2. Moreover, chromatin immunoprecipitation (ChIP) and sequential ChIP experiments demonstrate that endogenous HIC1 proteins are bound, together with the MTA1 corepressor, to the EphA2 promoter in WI38 cells. Taken together, our results identify EphA2 as a new direct target gene of HIC1. Finally, we observe that inactivation of endogenous HIC1 through RNA interference in normal breast epithelial cells results in the up-regulation of EphA2 and is correlated with increased cellular migration. To conclude, our results involve the tumor suppressor HIC1 in the transcriptional regulation of the tyrosine kinase receptor EphA2, whose ligand ephrin-A1 is also a HIC1 target gene. Thus, loss of the regulation of this Eph pathway through HIC1 epigenetic silencing could be an important mechanism in the pathogenesis of epithelial cancers. PMID:22184117

RD2-MolPack-Chim3, a packaging cell line for stable production of lentiviral vectors for anti-HIV gene therapy.

PubMed

Stornaiuolo, Anna; Piovani, Bianca Maria; Bossi, Sergio; Zucchelli, Eleonora; Corna, Stefano; Salvatori, Francesca; Mavilio, Fulvio; Bordignon, Claudio; Rizzardi, Gian Paolo; Bovolenta, Chiara

2013-08-01

Over the last two decades, several attempts to generate packaging cells for lentiviral vectors (LV) have been made. Despite different technologies, no packaging clone is currently employed in clinical trials. We developed a new strategy for LV stable production based on the HEK-293T progenitor cells; the sequential insertion of the viral genes by integrating vectors; the constitutive expression of the viral components; and the RD114-TR envelope pseudotyping. We generated the intermediate clone PK-7 expressing constitutively gag/pol and rev genes and, by adding tat and rd114-tr genes, the stable packaging cell line RD2-MolPack, which can produce LV carrying any transfer vector (TV). Finally, we obtained the RD2-MolPack-Chim3 producer clone by transducing RD2-MolPack cells with the TV expressing the anti-HIV transgene Chim3. Remarkably, RD114-TR pseudovirions have much higher potency when produced by stable compared with transient technology. Most importantly, comparable transduction efficiency in hematopoietic stem cells (HSC) is obtained with 2-logs less physical particles respect to VSV-G pseudovirions produced by transient transfection. Altogether, RD2-MolPack technology should be considered a valid option for large-scale production of LV to be used in gene therapy protocols employing HSC, resulting in the possibility of downsizing the manufacturing scale by about 10-fold in respect to transient technology.

Analyzing Kernel Matrices for the Identification of Differentially Expressed Genes

PubMed Central

Xia, Xiao-Lei; Xing, Huanlai; Liu, Xueqin

2013-01-01

One of the most important applications of microarray data is the class prediction of biological samples. For this purpose, statistical tests have often been applied to identify the differentially expressed genes (DEGs), followed by the employment of the state-of-the-art learning machines including the Support Vector Machines (SVM) in particular. The SVM is a typical sample-based classifier whose performance comes down to how discriminant samples are. However, DEGs identified by statistical tests are not guaranteed to result in a training dataset composed of discriminant samples. To tackle this problem, a novel gene ranking method namely the Kernel Matrix Gene Selection (KMGS) is proposed. The rationale of the method, which roots in the fundamental ideas of the SVM algorithm, is described. The notion of ''the separability of a sample'' which is estimated by performing -like statistics on each column of the kernel matrix, is first introduced. The separability of a classification problem is then measured, from which the significance of a specific gene is deduced. Also described is a method of Kernel Matrix Sequential Forward Selection (KMSFS) which shares the KMGS method's essential ideas but proceeds in a greedy manner. On three public microarray datasets, our proposed algorithms achieved noticeably competitive performance in terms of the B.632+ error rate. PMID:24349110

Effects of repeated potassium iodide administration on genes involved in synthesis and secretion of thyroid hormone in adult male rat.

PubMed

Lebsir, Dalila; Manens, Line; Grison, Stephane; Lestaevel, Philippe; Ebrahimian, Teni; Suhard, David; Phan, Guillaume; Dublineau, Isabelle; Tack, Karine; Benderitter, Marc; Pech, Annick; Jourdain, Jean-Rene; Souidi, Maâmar

2018-02-26

A single dose of potassium iodide (KI) is recommended to reduce the risk of thyroid cancer during nuclear accidents. However in case of prolonged radioiodine exposure, more than one dose of KI may be necessary. This work aims to evaluate the potential toxic effect of repeated administration of KI. Adult Wistar rats received an optimal dose of KI 1 mg/kg over a period of 1, 4 or 8 days. hormonal status (TSH, FT4) of treated rats was unaffected. Contrariwise, a sequential Wolff-Chaikoff effect was observed, resulting in a prompt decrease of NIS and MCT8 mRNA expression (-58% and -26% respectively), followed by a delayed decrease of TPO mRNA expression (-33%) in conjunction with a stimulation of PDS mRNA expression (+62%). we show for the first time that repeated administration of KI at 1 mg/kg/24h doesn't cause modification of thyroid hormones level, but leads to a reversible modification of the expression of genes involved in the synthesis and secretion of thyroid hormones. Copyright © 2018 Elsevier B.V. All rights reserved.

A cross-species bi-clustering approach to identifying conserved co-regulated genes.

PubMed

Sun, Jiangwen; Jiang, Zongliang; Tian, Xiuchun; Bi, Jinbo

2016-06-15

A growing number of studies have explored the process of pre-implantation embryonic development of multiple mammalian species. However, the conservation and variation among different species in their developmental programming are poorly defined due to the lack of effective computational methods for detecting co-regularized genes that are conserved across species. The most sophisticated method to date for identifying conserved co-regulated genes is a two-step approach. This approach first identifies gene clusters for each species by a cluster analysis of gene expression data, and subsequently computes the overlaps of clusters identified from different species to reveal common subgroups. This approach is ineffective to deal with the noise in the expression data introduced by the complicated procedures in quantifying gene expression. Furthermore, due to the sequential nature of the approach, the gene clusters identified in the first step may have little overlap among different species in the second step, thus difficult to detect conserved co-regulated genes. We propose a cross-species bi-clustering approach which first denoises the gene expression data of each species into a data matrix. The rows of the data matrices of different species represent the same set of genes that are characterized by their expression patterns over the developmental stages of each species as columns. A novel bi-clustering method is then developed to cluster genes into subgroups by a joint sparse rank-one factorization of all the data matrices. This method decomposes a data matrix into a product of a column vector and a row vector where the column vector is a consistent indicator across the matrices (species) to identify the same gene cluster and the row vector specifies for each species the developmental stages that the clustered genes co-regulate. Efficient optimization algorithm has been developed with convergence analysis. This approach was first validated on synthetic data and compared to the two-step method and several recent joint clustering methods. We then applied this approach to two real world datasets of gene expression during the pre-implantation embryonic development of the human and mouse. Co-regulated genes consistent between the human and mouse were identified, offering insights into conserved functions, as well as similarities and differences in genome activation timing between the human and mouse embryos. The R package containing the implementation of the proposed method in C ++ is available at: https://github.com/JavonSun/mvbc.git and also at the R platform https://www.r-project.org/ jinbo@engr.uconn.edu. © The Author 2016. Published by Oxford University Press.

Detection of Significant Pneumococcal Meningitis Biomarkers by Ego Network.

PubMed

Wang, Qian; Lou, Zhifeng; Zhai, Liansuo; Zhao, Haibin

2017-06-01

To identify significant biomarkers for detection of pneumococcal meningitis based on ego network. Based on the gene expression data of pneumococcal meningitis and global protein-protein interactions (PPIs) data recruited from open access databases, the authors constructed a differential co-expression network (DCN) to identify pneumococcal meningitis biomarkers in a network view. Here EgoNet algorithm was employed to screen the significant ego networks that could accurately distinguish pneumococcal meningitis from healthy controls, by sequentially seeking ego genes, searching candidate ego networks, refinement of candidate ego networks and significance analysis to identify ego networks. Finally, the functional inference of the ego networks was performed to identify significant pathways for pneumococcal meningitis. By differential co-expression analysis, the authors constructed the DCN that covered 1809 genes and 3689 interactions. From the DCN, a total of 90 ego genes were identified. Starting from these ego genes, three significant ego networks (Module 19, Module 70 and Module 71) that could predict clinical outcomes for pneumococcal meningitis were identified by EgoNet algorithm, and the corresponding ego genes were GMNN, MAD2L1 and TPX2, respectively. Pathway analysis showed that these three ego networks were related to CDT1 association with the CDC6:ORC:origin complex, inactivation of APC/C via direct inhibition of the APC/C complex pathway, and DNA strand elongation, respectively. The authors successfully screened three significant ego modules which could accurately predict the clinical outcomes for pneumococcal meningitis and might play important roles in host response to pathogen infection in pneumococcal meningitis.

CD4 memory T cells develop and acquire functional competence by sequential cognate interactions and stepwise gene regulation

PubMed Central

Kaji, Tomohiro; Hijikata, Atsushi; Ishige, Akiko; Kitami, Toshimori; Watanabe, Takashi; Ohara, Osamu; Yanaka, Noriyuki; Okada, Mariko; Shimoda, Michiko; Taniguchi, Masaru

2016-01-01

Memory CD4+ T cells promote protective humoral immunity; however, how memory T cells acquire this activity remains unclear. This study demonstrates that CD4+ T cells develop into antigen-specific memory T cells that can promote the terminal differentiation of memory B cells far more effectively than their naive T-cell counterparts. Memory T cell development requires the transcription factor B-cell lymphoma 6 (Bcl6), which is known to direct T-follicular helper (Tfh) cell differentiation. However, unlike Tfh cells, memory T cell development did not require germinal center B cells. Curiously, memory T cells that develop in the absence of cognate B cells cannot promote memory B-cell recall responses and this defect was accompanied by down-regulation of genes associated with homeostasis and activation and up-regulation of genes inhibitory for T-cell responses. Although memory T cells display phenotypic and genetic signatures distinct from Tfh cells, both had in common the expression of a group of genes associated with metabolic pathways. This gene expression profile was not shared to any great extent with naive T cells and was not influenced by the absence of cognate B cells during memory T cell development. These results suggest that memory T cell development is programmed by stepwise expression of gatekeeper genes through serial interactions with different types of antigen-presenting cells, first licensing the memory lineage pathway and subsequently facilitating the functional development of memory T cells. Finally, we identified Gdpd3 as a candidate genetic marker for memory T cells. PMID:26714588

Time-series resolution of gradual nitrogen starvation and its impact on photosynthesis in the cyanobacterium Synechocystis PCC 6803.

PubMed

Krasikov, Vladimir; Aguirre von Wobeser, Eneas; Dekker, Henk L; Huisman, Jef; Matthijs, Hans C P

2012-07-01

Sequential adaptation to nitrogen deprivation and ultimately to full starvation requires coordinated adjustment of cellular functions. We investigated changes in gene expression and cell physiology of the cyanobacterium Synechocystis PCC 6803 during 96 h of nitrogen starvation. During the first 6 h, the transcriptome showed activation of nitrogen uptake and assimilation systems and of the core nitrogen and carbon assimilation regulators. However, the nitrogen-deprived cells still grew at the same rate as the control and even showed transiently increased expression of phycobilisome genes. After 12 h, cell growth decreased and chlorosis started with degradation of the nitrogen-rich phycobilisomes. During this phase, the transcriptome showed suppression of genes for phycobilisomes, for carbon fixation and for de novo protein synthesis. Interestingly, photosynthetic activity of both photosystem I (PSI) and photosystem II was retained quite well. Excess electrons were quenched by the induction of terminal oxidase and hydrogenase genes, compensating for the diminished carbon fixation and nitrate reduction activity. After 48 h, the cells ceased most activities. A marked exception was the retained PSI gene transcription, possibly this supports the viability of Synechocystis cells and enables rapid recovery after relieving from nitrogen starvation. During early recovery, many genes changed expression, supporting the resumed cellular activity. In total, our results distinguished three phases during gradual nitrogen depletion: (1) an immediate response, (2) short-term acclimation and (3) long-term survival. This shows that cyanobacteria respond to nitrogen starvation by a cascade of physiological adaptations reflected by numerous changes in the transcriptome unfolding at different timescales. Copyright © Physiologia Plantarum 2012.

Removal of Heterologous Sequences from Plasmodium falciparum Mutants Using FLPe-Recombinase

PubMed Central

van Schaijk, Ben C. L.; Vos, Martijn W.; Janse, Chris J.; Sauerwein, Robert W.; Khan, Shahid M.

2010-01-01

Genetically-modified mutants are now indispensable Plasmodium gene-function reagents, which are also being pursued as genetically attenuated parasite vaccines. Currently, the generation of transgenic malaria-parasites requires the use of drug-resistance markers. Here we present the development of an FRT/FLP-recombinase system that enables the generation of transgenic parasites free of resistance genes. We demonstrate in the human malaria parasite, P. falciparum, the complete and efficient removal of the introduced resistance gene. We targeted two neighbouring genes, p52 and p36, using a construct that has a selectable marker cassette flanked by FRT-sequences. This permitted the subsequent removal of the selectable marker cassette by transient transfection of a plasmid that expressed a 37°C thermostable and enhanced FLP-recombinase. This method of removing heterologous DNA sequences from the genome opens up new possibilities in Plasmodium research to sequentially target multiple genes and for using genetically-modified parasites as live, attenuated malaria vaccines. PMID:21152048

Molecular mechanism of emotional stress-induced and catecholamine-induced heart attack.

PubMed

Ueyama, Takashi; Senba, Emiko; Kasamatsu, Ken; Hano, Takuzo; Yamamoto, Katsuhiro; Nishio, Ichiro; Tsuruo, Yoshihiro; Yoshida, Ken-ichi

2003-01-01

Emotional or physical stress triggers 'tako-tsubo' cardiomyopathy or 'transient left ventricular apical ballooning', but the pathogenesis is unclear. In response to the immobilization stress of rats, a useful model of emotional stress, rapid activation of p44/p42 mitogen-activated protein kinase was observed in the heart, followed by a transient upregulation of immediate early genes in the smooth muscle cells of coronary arteries, the endothelial cells and the myocardium. Heat shock protein 70 was induced in the aortic and coronary arterial smooth muscle cells and in the myocardium. Natriuretic peptide genes were also upregulated in the myocardium. Sequential gene expression can be considered as an adaptive response to emotional stress. Blocking of both alpha-adrenoceptors and beta-adrenoceptors eliminated the upregulation of immediate early genes induced by stress, while alpha-agonists and beta-agonists upregulated immediate early genes in the perfused heart. Activation of alpha-adrenoceptors and beta-adrenoceptors is the primary trigger of emotional stress-induced molecular changes in the heart.

Sequential pattern formation governed by signaling gradients

NASA Astrophysics Data System (ADS)

Jörg, David J.; Oates, Andrew C.; Jülicher, Frank

2016-10-01

Rhythmic and sequential segmentation of the embryonic body plan is a vital developmental patterning process in all vertebrate species. However, a theoretical framework capturing the emergence of dynamic patterns of gene expression from the interplay of cell oscillations with tissue elongation and shortening and with signaling gradients, is still missing. Here we show that a set of coupled genetic oscillators in an elongating tissue that is regulated by diffusing and advected signaling molecules can account for segmentation as a self-organized patterning process. This system can form a finite number of segments and the dynamics of segmentation and the total number of segments formed depend strongly on kinetic parameters describing tissue elongation and signaling molecules. The model accounts for existing experimental perturbations to signaling gradients, and makes testable predictions about novel perturbations. The variety of different patterns formed in our model can account for the variability of segmentation between different animal species.

Sunflower (Helianthus annuus) fatty acid synthase complex: β-hydroxyacyl-[acyl carrier protein] dehydratase genes.

PubMed

González-Thuillier, Irene; Venegas-Calerón, Mónica; Sánchez, Rosario; Garcés, Rafael; von Wettstein-Knowles, Penny; Martínez-Force, Enrique

2016-02-01

Two sunflower hydroxyacyl-[acyl carrier protein] dehydratases evolved into two different isoenzymes showing distinctive expression levels and kinetics' efficiencies. β-Hydroxyacyl-[acyl carrier protein (ACP)]-dehydratase (HAD) is a component of the type II fatty acid synthase complex involved in 'de novo' fatty acid biosynthesis in plants. This complex, formed by four intraplastidial proteins, is responsible for the sequential condensation of two-carbon units, leading to 16- and 18-C acyl-ACP. HAD dehydrates 3-hydroxyacyl-ACP generating trans-2-enoyl-ACP. With the aim of a further understanding of fatty acid biosynthesis in sunflower (Helianthus annuus) seeds, two β-hydroxyacyl-[ACP] dehydratase genes have been cloned from developing seeds, HaHAD1 (GenBank HM044767) and HaHAD2 (GenBank GU595454). Genomic DNA gel blot analyses suggest that both are single copy genes. Differences in their expression patterns across plant tissues were detected. Higher levels of HaHAD2 in the initial stages of seed development inferred its key role in seed storage fatty acid synthesis. That HaHAD1 expression levels remained constant across most tissues suggest a housekeeping function. Heterologous expression of these genes in E. coli confirmed both proteins were functional and able to interact with the bacterial complex 'in vivo'. The large increase of saturated fatty acids in cells expressing HaHAD1 and HaHAD2 supports the idea that these HAD genes are closely related to the E. coli FabZ gene. The proposed three-dimensional models of HaHAD1 and HaHAD2 revealed differences at the entrance to the catalytic tunnel attributable to Phe166/Val1159, respectively. HaHAD1 F166V was generated to study the function of this residue. The 'in vitro' enzymatic characterization of the three HAD proteins demonstrated all were active, with the mutant having intermediate K m and V max values to the wild-type proteins.

Single-Cell-Based Analysis Highlights a Surge in Cell-to-Cell Molecular Variability Preceding Irreversible Commitment in a Differentiation Process

PubMed Central

Boullu, Loïs; Morin, Valérie; Vallin, Elodie; Guillemin, Anissa; Papili Gao, Nan; Cosette, Jérémie; Arnaud, Ophélie; Kupiec, Jean-Jacques; Espinasse, Thibault

2016-01-01

In some recent studies, a view emerged that stochastic dynamics governing the switching of cells from one differentiation state to another could be characterized by a peak in gene expression variability at the point of fate commitment. We have tested this hypothesis at the single-cell level by analyzing primary chicken erythroid progenitors through their differentiation process and measuring the expression of selected genes at six sequential time-points after induction of differentiation. In contrast to population-based expression data, single-cell gene expression data revealed a high cell-to-cell variability, which was masked by averaging. We were able to show that the correlation network was a very dynamical entity and that a subgroup of genes tend to follow the predictions from the dynamical network biomarker (DNB) theory. In addition, we also identified a small group of functionally related genes encoding proteins involved in sterol synthesis that could act as the initial drivers of the differentiation. In order to assess quantitatively the cell-to-cell variability in gene expression and its evolution in time, we used Shannon entropy as a measure of the heterogeneity. Entropy values showed a significant increase in the first 8 h of the differentiation process, reaching a peak between 8 and 24 h, before decreasing to significantly lower values. Moreover, we observed that the previous point of maximum entropy precedes two paramount key points: an irreversible commitment to differentiation between 24 and 48 h followed by a significant increase in cell size variability at 48 h. In conclusion, when analyzed at the single cell level, the differentiation process looks very different from its classical population average view. New observables (like entropy) can be computed, the behavior of which is fully compatible with the idea that differentiation is not a “simple” program that all cells execute identically but results from the dynamical behavior of the underlying molecular network. PMID:28027290

Single-Cell-Based Analysis Highlights a Surge in Cell-to-Cell Molecular Variability Preceding Irreversible Commitment in a Differentiation Process.

PubMed

Richard, Angélique; Boullu, Loïs; Herbach, Ulysse; Bonnafoux, Arnaud; Morin, Valérie; Vallin, Elodie; Guillemin, Anissa; Papili Gao, Nan; Gunawan, Rudiyanto; Cosette, Jérémie; Arnaud, Ophélie; Kupiec, Jean-Jacques; Espinasse, Thibault; Gonin-Giraud, Sandrine; Gandrillon, Olivier

2016-12-01

In some recent studies, a view emerged that stochastic dynamics governing the switching of cells from one differentiation state to another could be characterized by a peak in gene expression variability at the point of fate commitment. We have tested this hypothesis at the single-cell level by analyzing primary chicken erythroid progenitors through their differentiation process and measuring the expression of selected genes at six sequential time-points after induction of differentiation. In contrast to population-based expression data, single-cell gene expression data revealed a high cell-to-cell variability, which was masked by averaging. We were able to show that the correlation network was a very dynamical entity and that a subgroup of genes tend to follow the predictions from the dynamical network biomarker (DNB) theory. In addition, we also identified a small group of functionally related genes encoding proteins involved in sterol synthesis that could act as the initial drivers of the differentiation. In order to assess quantitatively the cell-to-cell variability in gene expression and its evolution in time, we used Shannon entropy as a measure of the heterogeneity. Entropy values showed a significant increase in the first 8 h of the differentiation process, reaching a peak between 8 and 24 h, before decreasing to significantly lower values. Moreover, we observed that the previous point of maximum entropy precedes two paramount key points: an irreversible commitment to differentiation between 24 and 48 h followed by a significant increase in cell size variability at 48 h. In conclusion, when analyzed at the single cell level, the differentiation process looks very different from its classical population average view. New observables (like entropy) can be computed, the behavior of which is fully compatible with the idea that differentiation is not a "simple" program that all cells execute identically but results from the dynamical behavior of the underlying molecular network.

Intra-graft expression of genes involved in iron homeostasis predicts the development of operational tolerance in human liver transplantation

PubMed Central

Bohne, Felix; Martínez-Llordella, Marc; Lozano, Juan-José; Miquel, Rosa; Benítez, Carlos; Londoño, María-Carlota; Manzia, Tommaso-María; Angelico, Roberta; Swinkels, Dorine W.; Tjalsma, Harold; López, Marta; Abraldes, Juan G.; Bonaccorsi-Riani, Eliano; Jaeckel, Elmar; Taubert, Richard; Pirenne, Jacques; Rimola, Antoni; Tisone, Giuseppe; Sánchez-Fueyo, Alberto

2011-01-01

Following organ transplantation, lifelong immunosuppressive therapy is required to prevent the host immune system from destroying the allograft. This can cause severe side effects and increased recipient morbidity and mortality. Complete cessation of immunosuppressive drugs has been successfully accomplished in selected transplant recipients, providing proof of principle that operational allograft tolerance is attainable in clinical transplantation. The intra-graft molecular pathways associated with successful drug withdrawal, however, are not well defined. In this study, we analyzed sequential blood and liver tissue samples collected from liver transplant recipients enrolled in a prospective multicenter immunosuppressive drug withdrawal clinical trial. Before initiation of drug withdrawal, operationally tolerant and non-tolerant recipients differed in the intra-graft expression of genes involved in the regulation of iron homeostasis. Furthermore, as compared with non-tolerant recipients, operationally tolerant patients exhibited higher serum levels of hepcidin and ferritin and increased hepatocyte iron deposition. Finally, liver tissue gene expression measurements accurately predicted the outcome of immunosuppressive withdrawal in an independent set of patients. These results point to a critical role for iron metabolism in the regulation of intra-graft alloimmune responses in humans and provide a set of biomarkers to conduct drug-weaning trials in liver transplantation. PMID:22156196

Integrating multiple molecular sources into a clinical risk prediction signature by extracting complementary information.

PubMed

Hieke, Stefanie; Benner, Axel; Schlenl, Richard F; Schumacher, Martin; Bullinger, Lars; Binder, Harald

2016-08-30

High-throughput technology allows for genome-wide measurements at different molecular levels for the same patient, e.g. single nucleotide polymorphisms (SNPs) and gene expression. Correspondingly, it might be beneficial to also integrate complementary information from different molecular levels when building multivariable risk prediction models for a clinical endpoint, such as treatment response or survival. Unfortunately, such a high-dimensional modeling task will often be complicated by a limited overlap of molecular measurements at different levels between patients, i.e. measurements from all molecular levels are available only for a smaller proportion of patients. We propose a sequential strategy for building clinical risk prediction models that integrate genome-wide measurements from two molecular levels in a complementary way. To deal with partial overlap, we develop an imputation approach that allows us to use all available data. This approach is investigated in two acute myeloid leukemia applications combining gene expression with either SNP or DNA methylation data. After obtaining a sparse risk prediction signature e.g. from SNP data, an automatically selected set of prognostic SNPs, by componentwise likelihood-based boosting, imputation is performed for the corresponding linear predictor by a linking model that incorporates e.g. gene expression measurements. The imputed linear predictor is then used for adjustment when building a prognostic signature from the gene expression data. For evaluation, we consider stability, as quantified by inclusion frequencies across resampling data sets. Despite an extremely small overlap in the application example with gene expression and SNPs, several genes are seen to be more stably identified when taking the (imputed) linear predictor from the SNP data into account. In the application with gene expression and DNA methylation, prediction performance with respect to survival also indicates that the proposed approach might work well. We consider imputation of linear predictor values to be a feasible and sensible approach for dealing with partial overlap in complementary integrative analysis of molecular measurements at different levels. More generally, these results indicate that a complementary strategy for integrating different molecular levels can result in more stable risk prediction signatures, potentially providing a more reliable insight into the underlying biology.

Molecular changes in articular cartilage and subchondral bone in the rat anterior cruciate ligament transection and meniscectomized models of osteoarthritis.

PubMed

Pickarski, Maureen; Hayami, Tadashi; Zhuo, Ya; Duong, Le T

2011-08-24

Osteoarthritis (OA) is a debilitating, progressive joint disease. Similar to the disease progression in humans, sequential events of early cartilage degradation, subchondral osteopenia followed by sclerosis, and late osteophyte formation were demonstrated in the anterior cruciate ligament transection (ACLT) or ACLT with partial medial meniscectomy (ACLT + MMx) rat OA models. We describe a reliable and consistent method to examine the time dependent changes in the gene expression profiles in articular cartilage and subchondral bone. Local regulation of matrix degradation markers was demonstrated by a significant increase in mRNA levels of aggrecanase-1 and MMP-13 as early as the first week post-surgery, and expression remained elevated throughout the 10 week study. Immunohistochemistry confirmed MMP-13 expression in differentiated chondrocytes and synovial fibroblasts at week-2 and cells within osteophytes at week-10 in the surgically-modified-joints. Concomitant increases in chondrocyte differentiation markers, Col IIA and Sox 9, and vascular invasion markers, VEGF and CD31, peaked around week-2 to -4, and returned to Sham levels at later time points in both models. Indeed, VEGF-positive cells were found in the deep articular chondrocytes adjacent to subchondral bone. Osteoclastic bone resorption markers, cathepsin K and TRAP, were also elevated at week-2. Confirming bone resorption is an early local event in OA progression, cathepsin K positive osteoclasts were found invading the articular cartilage from the subchondral region at week 2. This was followed by late disease events, including subchondral sclerosis and osteophyte formation, as demonstrated by the upregulation of the osteoanabolic markers runx2 and osterix, toward week-4 to 6 post-surgery. In summary, this study demonstrated the temporal and cohesive gene expression changes in articular cartilage and subchondral bone using known markers of OA progression. The findings here support genome-wide profiling efforts to elucidate the sequential and complex regulation of the disease.

Molecular changes in articular cartilage and subchondral bone in the rat anterior cruciate ligament transection and meniscectomized models of osteoarthritis

PubMed Central

2011-01-01

Background Osteoarthritis (OA) is a debilitating, progressive joint disease. Methods Similar to the disease progression in humans, sequential events of early cartilage degradation, subchondral osteopenia followed by sclerosis, and late osteophyte formation were demonstrated in the anterior cruciate ligament transection (ACLT) or ACLT with partial medial meniscectomy (ACLT + MMx) rat OA models. We describe a reliable and consistent method to examine the time dependent changes in the gene expression profiles in articular cartilage and subchondral bone. Results Local regulation of matrix degradation markers was demonstrated by a significant increase in mRNA levels of aggrecanase-1 and MMP-13 as early as the first week post-surgery, and expression remained elevated throughout the 10 week study. Immunohistochemistry confirmed MMP-13 expression in differentiated chondrocytes and synovial fibroblasts at week-2 and cells within osteophytes at week-10 in the surgically-modified-joints. Concomitant increases in chondrocyte differentiation markers, Col IIA and Sox 9, and vascular invasion markers, VEGF and CD31, peaked around week-2 to -4, and returned to Sham levels at later time points in both models. Indeed, VEGF-positive cells were found in the deep articular chondrocytes adjacent to subchondral bone. Osteoclastic bone resorption markers, cathepsin K and TRAP, were also elevated at week-2. Confirming bone resorption is an early local event in OA progression, cathepsin K positive osteoclasts were found invading the articular cartilage from the subchondral region at week 2. This was followed by late disease events, including subchondral sclerosis and osteophyte formation, as demonstrated by the upregulation of the osteoanabolic markers runx2 and osterix, toward week-4 to 6 post-surgery. Conclusions In summary, this study demonstrated the temporal and cohesive gene expression changes in articular cartilage and subchondral bone using known markers of OA progression. The findings here support genome-wide profiling efforts to elucidate the sequential and complex regulation of the disease. PMID:21864409

Chronology of Islet Differentiation Revealed By Temporal Cell Labeling

PubMed Central

Miyatsuka, Takeshi; Li, Zhongmei; German, Michael S.

2009-01-01

OBJECTIVE Neurogenin 3 plays a pivotal role in pancreatic endocrine differentiation. Whereas mouse models expressing reporters such as eGFP or LacZ under the control of the Neurog3 gene enable us to label cells in the pancreatic endocrine lineage, the long half-life of most reporter proteins makes it difficult to distinguish cells actively expressing neurogenin 3 from differentiated cells that have stopped transcribing the gene. RESEARCH DESIGN AND METHODS In order to separate the transient neurogenin 3 –expressing endocrine progenitor cells from the differentiating endocrine cells, we developed a mouse model (Ngn3-Timer) in which DsRed-E5, a fluorescent protein that shifts its emission spectrum from green to red over time, was expressed transgenically from the NEUROG3 locus. RESULTS In the Ngn3-Timer embryos, green-dominant cells could be readily detected by microscopy or flow cytometry and distinguished from green/red double-positive cells. When fluorescent cells were sorted into three different populations by a fluorescence-activated cell sorter, placed in culture, and then reanalyzed by flow cytometry, green-dominant cells converted to green/red double-positive cells within 6 h. The sorted cell populations were then used to determine the temporal patterns of expression for 145 transcriptional regulators in the developing pancreas. CONCLUSIONS The precise temporal resolution of this model defines the narrow window of neurogenin 3 expression in islet progenitor cells and permits sequential analyses of sorted cells as well as the testing of gene regulatory models for the differentiation of pancreatic islet cells. PMID:19478145

Evaluation of Gaussia luciferase and foot-and-mouth disease virus 2A translational interrupter chimeras as polycistronic reporters for transgene expression.

PubMed

Puckette, Michael; Burrage, Thomas; Neilan, John G; Rasmussen, Max

2017-06-12

The Gaussia princeps luciferase is used as a stand-alone reporter of transgene expression for in vitro and in vivo expression systems due to the rapid and easy monitoring of luciferase activity. We sought to simultaneously quantitate production of other recombinant proteins by transcriptionally linking the Gaussia princeps luciferase gene to other genes of interest through the foot-and-mouth disease virus 2A translational interrupter sequence. We produced six plasmids, each encoding a single open reading frame, with the foot-and-mouth disease virus 2A sequence placed either N-terminal or C-terminal to the Gaussia princeps luciferase gene. Two plasmids included novel Gaussia princeps luciferase variants with the position 1 methionine deleted. Placing a foot-and-mouth disease virus 2A translational interrupter sequence on either the N- or C-terminus of the Gaussia princeps luciferase gene did not prevent the secretion or luminescence of resulting chimeric luciferase proteins. We also measured the ability of another polycistronic plasmid vector with a 2A-luciferase sequence placed downstream of the foot-and-mouth disease virus P1 and 3C protease genes to produce of foot-and-mouth disease virus-like particles and luciferase activity from transfected cells. Incorporation of the 2A-luciferase sequence into a transgene encoding foot-and-mouth disease virus structural proteins retained luciferase activity and the ability to form virus-like particles. We demonstrated a mechanism for the near real-time, sequential, non-destructive quantitative monitoring of transcriptionally-linked recombinant proteins and a valuable method for monitoring transgene expression in recombinant vaccine constructs.

Conceptual scoring of receptive and expressive vocabulary measures in simultaneous and sequential bilingual children.

PubMed

Gross, Megan; Buac, Milijana; Kaushanskaya, Margarita

2014-11-01

The authors examined the effects of conceptual scoring on the performance of simultaneous and sequential bilinguals on standardized receptive and expressive vocabulary measures in English and Spanish. Participants included 40 English-speaking monolingual children, 39 simultaneous Spanish-English bilingual children, and 19 sequential bilingual children, ages 5-7. The children completed standardized receptive and expressive vocabulary measures in English and also in Spanish for those who were bilingual. After the standardized administration, bilingual children were given the opportunity to respond to missed items in their other language to obtain a conceptual score. Controlling for group differences in socioeconomic status (SES), both simultaneous and sequential bilingual children scored significantly below monolingual children on single-language measures of English receptive and expressive vocabulary. Conceptual scoring removed the significant difference between monolingual and simultaneous bilingual children in the receptive modality but not in the expressive modality; differences remained between monolingual and sequential bilingual children in both modalities. However, in both bilingual groups, conceptual scoring increased the proportion of children with vocabulary scores within the average range. Conceptual scoring does not fully ameliorate the bias inherent in single-language standardized vocabulary measures for bilingual children, but the procedures employed here may assist in ruling out vocabulary deficits, particularly in typically developing simultaneous bilingual children.

Conceptual scoring of receptive and expressive vocabulary measures in simultaneous and sequential bilingual children

PubMed Central

Gross, Megan; Buac, Milijana; Kaushanskaya, Margarita

2014-01-01

Purpose This study examined the effects of conceptual scoring on the performance of simultaneous and sequential bilinguals on standardized receptive and expressive vocabulary measures in English and Spanish. Method Participants included 40 English-speaking monolingual children, 39 simultaneous Spanish-English bilingual children, and 19 sequential bilinguals, ages 5–7. The children completed standardized receptive and expressive vocabulary measures in English and also in Spanish for bilinguals. After the standardized administration, bilinguals were given the opportunity to respond to missed items in their other language to obtain a conceptual score. Results Controlling for group differences in socioeconomic status (SES), both simultaneous and sequential bilinguals scored significantly below monolinguals on single-language measures of English receptive and expressive vocabulary. Conceptual scoring removed the significant difference between monolinguals and simultaneous bilinguals in the receptive modality, but not in the expressive modality; differences remained between monolinguals and sequential bilinguals in both modalities. However, in both bilingual groups conceptual scoring increased the proportion of children with vocabulary scores within the average range. Conclusions Conceptual scoring does not fully ameliorate the bias inherent in single-language standardized vocabulary measures for bilinguals, but the procedures employed here may assist in ruling out vocabulary deficits, particularly in typically-developing simultaneous bilingual children. PMID:24811415

Global Expression Profiling of Globose Basal Cells and Neurogenic Progression Within the Olfactory Epithelium

PubMed Central

Krolewski, Richard C.; Packard, Adam; Schwob, James E.

2013-01-01

Ongoing, lifelong neurogenesis maintains the neuronal population of the olfactory epithelium in the face of piecemeal neuronal turnover and restores it following wholesale loss. The molecular phenotypes corresponding to different stages along the progression from multipotent globose basal cell (GBC) progenitor to differentiated olfactory sensory neuron are poorly characterized. We used the transgenic expression of enhanced green fluorescent protein (eGFP) and cell surface markers to FACS-isolate ΔSox2-eGFP(+) GBCs, Neurog1-eGFP(+) GBCs and immature neurons, and ΔOMP-eGFP(+) mature neurons from normal adult mice. In addition, the latter two populations were also collected 3 weeks after olfactory bulb ablation, a lesion that results in persistently elevated neurogenesis. Global profiling of mRNA from the populations indicates that all stages of neurogenesis share a cohort of >2,100 genes that are upregulated compared to sustentacular cells. A further cohort of >1,200 genes are specifically upregulated in GBCs as compared to sustentacular cells and differentiated neurons. The increased rate of neurogenesis caused by olfactory bulbectomy had little effect on the transcriptional profile of the Neurog1-eGFP(+) population. In contrast, the abbreviated lifespan of ΔOMP-eGFP(+) neurons born in the absence of the bulb correlated with substantial differences in gene expression as compared to the mature neurons of the normal epithelium. Detailed examination of the specific genes upregulated in the different progenitor populations revealed that the chromatin modifying complex proteins LSD1 and coREST were expressed sequentially in upstream ΔSox2-eGFP(+) GBCs and Neurog1-eGFP(+) GBCs/immature neurons. The expression patterns of these proteins are dynamically regulated after activation of the epithelium by methyl bromide lesion. PMID:22847514

ERK2-mediated C-terminal serine phosphorylation of p300 is vital to the regulation of epidermal growth factor-induced keratin 16 gene expression.

PubMed

Chen, Yun-Ju; Wang, Ying-Nai; Chang, Wen-Chang

2007-09-14

We previously reported that the epidermal growth factor (EGF) regulates the gene expression of keratin 16 by activating the extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling which in turn enhances the recruitment of p300 to the keratin 16 promoter. The recruited p300 functionally cooperates with Sp1 and c-Jun to regulate the gene expression of keratin 16. This study investigated in detail the molecular events incurred upon p300 whereby EGF caused an enhanced interaction between p300 and Sp1. EGF apparently induced time- and dose-dependent phosphorylation of p300, both in vitro and in vivo, through the activation of ERK2. The six potential ERK2 phosphorylation sites, including three threonine and three serine residues as revealed by sequential analysis, were first identified in vitro. Confirmation of these six sites in vivo indicated that these three serine residues (Ser-2279, Ser-2315, and Ser-2366) on the C terminus of p300 were the major signaling targets of EGF. Furthermore, the C-terminal serine phosphorylation of p300 stimulated its histone acetyltransferase activity and enhanced its interaction with Sp1. These serine phosphorylation sites on p300 controlled the p300 recruitment to the keratin 16 promoter. When all three serine residues on p300 were replaced by alanine, EGF could no longer induce the gene expression of keratin 16. Taken together, these results strongly suggested that the ERK2-mediated C-terminal serine phosphorylation of p300 was a key event in the regulation of EGF-induced keratin 16 expression. These results also constituted the first report identifying the unique p300 phosphorylation sites induced by ERK2 in vivo.

An Autoregulatory Pathway Establishes the Definitive Chromatin Conformation at the Pit-1 Locus

PubMed Central

Cooke, Nancy E.; Liebhaber, Stephen A.

2015-01-01

The transcription factor Pit-1 (POU1-F1) plays a dominant role in cell lineage expansion and differentiation in the anterior pituitary. Prior studies of the mouse Pit-1 (mPit-1) gene revealed that this master regulatory locus is activated at embryonic day 13.5 (E13.5) by an early enhancer (EE), whereas its subsequent expression throughout adult life is maintained by a more distal definitive enhancer (DE). Here, we demonstrate that the sequential actions of these two enhancers are linked to corresponding shifts in their proximities to the Pit-1 promoter. We further demonstrate that the looping of the definitive enhancer to the mPit-1 promoter is critically dependent on a self-sustaining autoregulatory mechanism mediated by the Pit-1 protein. These Pit-1-dependent actions are accompanied by localized recruitment of CBP and enrichment for H3K27 acetylation within the Pit-1 locus. These data support a model in which the sequential actions of two developmentally activated enhancers are linked to a corresponding shift in higher-order chromatin structures. This shift establishes an autoregulatory circuit that maintains durable expression of Pit-1 throughout adult life. PMID:25691665

Generalized bipartite quantum state discrimination problems with sequential measurements

NASA Astrophysics Data System (ADS)

Nakahira, Kenji; Kato, Kentaro; Usuda, Tsuyoshi Sasaki

2018-02-01

We investigate an optimization problem of finding quantum sequential measurements, which forms a wide class of state discrimination problems with the restriction that only local operations and one-way classical communication are allowed. Sequential measurements from Alice to Bob on a bipartite system are considered. Using the fact that the optimization problem can be formulated as a problem with only Alice's measurement and is convex programming, we derive its dual problem and necessary and sufficient conditions for an optimal solution. Our results are applicable to various practical optimization criteria, including the Bayes criterion, the Neyman-Pearson criterion, and the minimax criterion. In the setting of the problem of finding an optimal global measurement, its dual problem and necessary and sufficient conditions for an optimal solution have been widely used to obtain analytical and numerical expressions for optimal solutions. Similarly, our results are useful to obtain analytical and numerical expressions for optimal sequential measurements. Examples in which our results can be used to obtain an analytical expression for an optimal sequential measurement are provided.

FKBP5 DNA methylation does not mediate the association between childhood maltreatment and depression symptom severity in the Detroit Neighborhood Health Study.

PubMed

Bustamante, Angela C; Aiello, Allison E; Guffanti, Guia; Galea, Sandro; Wildman, Derek E; Uddin, Monica

2018-01-01

Exposure to childhood maltreatment increases the risk of developing mental illness later in life. Childhood maltreatment and depression have both been associated with dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis-a key regulator of the body's stress response. Additionally, HPA axis dysregulation has been implicated in the etiology of a range of mental illnesses. A substantial body of work has shown history of childhood maltreatment alters DNA methylation levels within key HPA axis genes. We therefore investigated whether one of these key genes, FKBP5 mediates the relationship between childhood maltreatment and depression, and assessed FKBP5 DNA methylation and gene expression within 112 adults from the Detroit Neighborhood Health Study (DNHS). DNA methylation was assessed in 4 regions, including the upstream promoter, downstream promoter, and two glucocorticoid response elements (GREs) via pyrosequencing using whole blood derived DNA; Taqman assays measured relative RNA expression from leukocytes. Mediation analyses were conducted using sequential linear regression. Childhood maltreatment was significantly associated with depression symptom severity (FDR < 0.006), but was not a significant predictor of DNA methylation in any of the four loci examined. FKBP5 showed elevated expression levels in participants with vs. without a history of depression (p < 0.001); no significant difference in gene expression levels was observed in relation to childhood maltreatment (p > 0.05). Our results suggest DNA methylation does not mediate the childhood maltreatment-depression association in the DNHS. Copyright © 2017 Elsevier Ltd. All rights reserved.

Transcriptomic and proteomic landscape of mitochondrial dysfunction reveals secondary coenzyme Q deficiency in mammals

PubMed Central

Atanassov, Ilian; Kuznetsova, Irina; Hinze, Yvonne; Mourier, Arnaud; Filipovska, Aleksandra

2017-01-01

Dysfunction of the oxidative phosphorylation (OXPHOS) system is a major cause of human disease and the cellular consequences are highly complex. Here, we present comparative analyses of mitochondrial proteomes, cellular transcriptomes and targeted metabolomics of five knockout mouse strains deficient in essential factors required for mitochondrial DNA gene expression, leading to OXPHOS dysfunction. Moreover, we describe sequential protein changes during post-natal development and progressive OXPHOS dysfunction in time course analyses in control mice and a middle lifespan knockout, respectively. Very unexpectedly, we identify a new response pathway to OXPHOS dysfunction in which the intra-mitochondrial synthesis of coenzyme Q (ubiquinone, Q) and Q levels are profoundly decreased, pointing towards novel possibilities for therapy. Our extensive omics analyses provide a high-quality resource of altered gene expression patterns under severe OXPHOS deficiency comparing several mouse models, that will deepen our understanding, open avenues for research and provide an important reference for diagnosis and treatment. PMID:29132502

Genomic Analysis Reveals Contrasting PIFq Contribution to Diurnal Rhythmic Gene Expression in PIF-Induced and -Repressed Genes.

PubMed

Martin, Guiomar; Soy, Judit; Monte, Elena

2016-01-01

Members of the PIF quartet (PIFq; PIF1, PIF3, PIF4, and PIF5) collectively contribute to induce growth in Arabidopsis seedlings under short day (SD) conditions, specifically promoting elongation at dawn. Their action involves the direct regulation of growth-related and hormone-associated genes. However, a comprehensive definition of the PIFq-regulated transcriptome under SD is still lacking. We have recently shown that SD and free-running (LL) conditions correspond to "growth" and "no growth" conditions, respectively, correlating with greater abundance of PIF protein in SD. Here, we present a genomic analysis whereby we first define SD-regulated genes at dawn compared to LL in the wild type, followed by identification of those SD-regulated genes whose expression depends on the presence of PIFq. By using this sequential strategy, we have identified 349 PIF/SD-regulated genes, approximately 55% induced and 42% repressed by both SD and PIFq. Comparison with available databases indicates that PIF/SD-induced and PIF/SD-repressed sets are differently phased at dawn and mid-morning, respectively. In addition, we found that whereas rhythmicity of the PIF/SD-induced gene set is lost in LL, most PIF/SD-repressed genes keep their rhythmicity in LL, suggesting differential regulation of both gene sets by the circadian clock. Moreover, we also uncovered distinct overrepresented functions in the induced and repressed gene sets, in accord with previous studies in other examined PIF-regulated processes. Interestingly, promoter analyses showed that, whereas PIF/SD-induced genes are enriched in direct PIF targets, PIF/SD-repressed genes are mostly indirectly regulated by the PIFs and might be more enriched in ABA-regulated genes.

Anti-oxidative assays as markers for anti-inflammatory activity of flavonoids.

PubMed

Chanput, Wasaporn; Krueyos, Narumol; Ritthiruangdej, Pitiporn

2016-11-01

The complexity of in vitro anti-inflammatory assays, the cost and time consumed, and the necessary skills can be a hurdle to apply to promising compounds in a high throughput setting. In this study, several antioxidative assays i.e. DPPH, ABTS, ORAC and xanthine oxidase (XO) were used to examine the antioxidative activity of three sub groups of flavonoids: (i) flavonol: quercetin, myricetin, (ii) flavanone: eriodictyol, naringenin (iii) flavone: luteolin, apigenin. A range of flavonoid concentrations was tested for their antioxidative activities and were found to be dose-dependent. However, the flavonoid concentrations over 50ppm were found to be toxic to the THP-1 monocytes. Therefore, 10, 20 and 50ppm of flavonoid concentrations were tested for their anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated THP-1 monocytes. Expression of inflammatory genes, IL-1β, IL-6, IL-8, IL-10 and TNF-α was found to be sequentially decreased when flavonoid concentration increased. Principle component analysis (PCA) was used to investigate the relationship between the data sets of antioxidative assays and the expression of inflammatory genes. The results showed that DPPH, ABTS and ORAC assays have an opposite correlation with the reduction of inflammatory genes. Pearson correlation exhibited a relationship between the ABTS assay and the expression of three out of five analyzed genes; IL-1β, IL-6 and IL-8. Our findings indicate that ABTS assay can potentially be an assay marker for anti-inflammatory activity of flavonoids. Copyright © 2016 Elsevier B.V. All rights reserved.

The combination of dimethoxycurcumin with DNA methylation inhibitor enhances gene re-expression of promoter-methylated genes and antagonizes their cytotoxic effect

PubMed Central

Hassan, Hazem E.; Keita, Jean-Arnaud; Narayan, Lawrence; Brady, Sean M.; Frederick, Richard; Carlson, Samuel; C. Glass, Karen; Natesan, Senthil; Buttolph, Thomm; Fandy, Tamer E.

2016-01-01

ABSTRACT Curcumin and its analogs exhibited antileukemic activity either as single agent or in combination therapy. Dimethoxycurcumin (DMC) is a more metabolically stable curcumin analog that was shown to induce the expression of promoter-methylated genes without reversing DNA methylation. Accordingly, co-treatment with DMC and DNA methyltransferase (DNMT) inhibitors could hypothetically enhance the re-expression of promoter-methylated tumor suppressor genes. In this study, we investigated the cytotoxic effects and epigenetic changes associated with the combination of DMC and the DNMT inhibitor decitabine (DAC) in primary leukemia samples and cell lines. The combination demonstrated antagonistic cytotoxic effects and was minimally cytotoxic to primary leukemia cells. The combination did not affect the metabolic stability of DMC. Although the combination enhanced the downregulation of nuclear DNMT proteins, the hypomethylating activity of the combination was not increased significantly compared to DAC alone. On the other hand, the combination significantly increased H3K27 acetylation (H3K27Ac) compared to the single agents near the promoter region of promoter-methylated genes. Furthermore, sequential chromatin immunoprecipitation (ChIP) and DNA pyrosequencing of the chromatin-enriched H3K27Ac did not show any significant decrease in DNA methylation compared to other regions. Consequently, the enhanced induction of promoter-methylated genes by the combination compared to DAC alone is mediated by a mechanism that involves increased histone acetylation and not through potentiation of the DNA hypomethylating activity of DAC. Collectively, our results provide the mechanistic basis for further characterization of this combination in leukemia animal models and early phase clinical trials. PMID:27588609

A distinct class of homeodomain proteins is encoded by two sequentially expressed Drosophila genes from the 93D/E cluster.

PubMed Central

Jagla, K; Stanceva, I; Dretzen, G; Bellard, F; Bellard, M

1994-01-01

Homeodomains appear to be one of the most frequently employed DNA-binding domains in a superfamily of transacting factors. It is likely that during evolution several sub-types of homeodomain have evolved from a common ancestral domain, resulting in distinct but closely related DNA-binding preferences. Here we describe the conservation of a distinct type of homeodomain encoded by the Drosophila lady-bird-late (lbl) gene, previously named nkch4 (1). Using degenerate PCR primers corresponding to the most divergent regions of the first and third helix of the Lbl homeodomain we have amplified, from genomic DNA of the fly, a lady-bird-like homeobox fragment. The Drosophila PCR products contained both the lbl (1) and a highly related homeobox sequence, which we named lady-bird-early (lbe). This new Drosophila gene resides directly upstream to lbl and together with tinman/NK4 (2, 3, 4, 5), bagpipe/NK3 (2, 4) S59/NK1 (4, 6) and 93Bal (7) compose the 93D/E homeobox gene cluster. Ibe and lbl are transcribed from the same strand and in a temporal order corresponding to their 5'-3' chromosomal location. Transcripts of both genes are found in the epiderm of Drosophila embryos, in cells known to express a segment polarity gene wingless (8), and their spatial and temporal colinearity of expression strongly suggests that they cooperate during segmentation. The amino-acid composition of both Lady-bird homeodomains differ from that of Antp-type at several positions involved in DNA recognition. These substitutions appear to modify DNA-binding preferences since Lbl homeodomain is unable to recognize the most common homeodomain binding TAAT motif in gel retardation experiments. Images PMID:7909370

Comprehensive analysis of coding-lncRNA gene co-expression network uncovers conserved functional lncRNAs in zebrafish.

PubMed

Chen, Wen; Zhang, Xuan; Li, Jing; Huang, Shulan; Xiang, Shuanglin; Hu, Xiang; Liu, Changning

2018-05-09

Zebrafish is a full-developed model system for studying development processes and human disease. Recent studies of deep sequencing had discovered a large number of long non-coding RNAs (lncRNAs) in zebrafish. However, only few of them had been functionally characterized. Therefore, how to take advantage of the mature zebrafish system to deeply investigate the lncRNAs' function and conservation is really intriguing. We systematically collected and analyzed a series of zebrafish RNA-seq data, then combined them with resources from known database and literatures. As a result, we obtained by far the most complete dataset of zebrafish lncRNAs, containing 13,604 lncRNA genes (21,128 transcripts) in total. Based on that, a co-expression network upon zebrafish coding and lncRNA genes was constructed and analyzed, and used to predict the Gene Ontology (GO) and the KEGG annotation of lncRNA. Meanwhile, we made a conservation analysis on zebrafish lncRNA, identifying 1828 conserved zebrafish lncRNA genes (1890 transcripts) that have their putative mammalian orthologs. We also found that zebrafish lncRNAs play important roles in regulation of the development and function of nervous system; these conserved lncRNAs present a significant sequential and functional conservation, with their mammalian counterparts. By integrative data analysis and construction of coding-lncRNA gene co-expression network, we gained the most comprehensive dataset of zebrafish lncRNAs up to present, as well as their systematic annotations and comprehensive analyses on function and conservation. Our study provides a reliable zebrafish-based platform to deeply explore lncRNA function and mechanism, as well as the lncRNA commonality between zebrafish and human.

Whole genome and global gene expression analyses of the model mushroom Flammulina velutipes reveal a high capacity for lignocellulose degradation.

PubMed

Park, Young-Jin; Baek, Jeong Hun; Lee, Seonwook; Kim, Changhoon; Rhee, Hwanseok; Kim, Hyungtae; Seo, Jeong-Sun; Park, Hae-Ran; Yoon, Dae-Eun; Nam, Jae-Young; Kim, Hong-Il; Kim, Jong-Guk; Yoon, Hyeokjun; Kang, Hee-Wan; Cho, Jae-Yong; Song, Eun-Sung; Sung, Gi-Ho; Yoo, Young-Bok; Lee, Chang-Soo; Lee, Byoung-Moo; Kong, Won-Sik

2014-01-01

Flammulina velutipes is a fungus with health and medicinal benefits that has been used for consumption and cultivation in East Asia. F. velutipes is also known to degrade lignocellulose and produce ethanol. The overlapping interests of mushroom production and wood bioconversion make F. velutipes an attractive new model for fungal wood related studies. Here, we present the complete sequence of the F. velutipes genome. This is the first sequenced genome for a commercially produced edible mushroom that also degrades wood. The 35.6-Mb genome contained 12,218 predicted protein-encoding genes and 287 tRNA genes assembled into 11 scaffolds corresponding with the 11 chromosomes of strain KACC42780. The 88.4-kb mitochondrial genome contained 35 genes. Well-developed wood degrading machinery with strong potential for lignin degradation (69 auxiliary activities, formerly FOLymes) and carbohydrate degradation (392 CAZymes), along with 58 alcohol dehydrogenase genes were highly expressed in the mycelium, demonstrating the potential application of this organism to bioethanol production. Thus, the newly uncovered wood degrading capacity and sequential nature of this process in F. velutipes, offer interesting possibilities for more detailed studies on either lignin or (hemi-) cellulose degradation in complex wood substrates. The mutual interest in wood degradation by the mushroom industry and (ligno-)cellulose biomass related industries further increase the significance of F. velutipes as a new model.

Whole Genome and Global Gene Expression Analyses of the Model Mushroom Flammulina velutipes Reveal a High Capacity for Lignocellulose Degradation

PubMed Central

Park, Young-Jin; Baek, Jeong Hun; Lee, Seonwook; Kim, Changhoon; Rhee, Hwanseok; Kim, Hyungtae; Seo, Jeong-Sun; Park, Hae-Ran; Yoon, Dae-Eun; Nam, Jae-Young; Kim, Hong-Il; Kim, Jong-Guk; Yoon, Hyeokjun; Kang, Hee-Wan; Cho, Jae-Yong; Song, Eun-Sung; Sung, Gi-Ho; Yoo, Young-Bok; Lee, Chang-Soo; Lee, Byoung-Moo; Kong, Won-Sik

2014-01-01

Flammulina velutipes is a fungus with health and medicinal benefits that has been used for consumption and cultivation in East Asia. F. velutipes is also known to degrade lignocellulose and produce ethanol. The overlapping interests of mushroom production and wood bioconversion make F. velutipes an attractive new model for fungal wood related studies. Here, we present the complete sequence of the F. velutipes genome. This is the first sequenced genome for a commercially produced edible mushroom that also degrades wood. The 35.6-Mb genome contained 12,218 predicted protein-encoding genes and 287 tRNA genes assembled into 11 scaffolds corresponding with the 11 chromosomes of strain KACC42780. The 88.4-kb mitochondrial genome contained 35 genes. Well-developed wood degrading machinery with strong potential for lignin degradation (69 auxiliary activities, formerly FOLymes) and carbohydrate degradation (392 CAZymes), along with 58 alcohol dehydrogenase genes were highly expressed in the mycelium, demonstrating the potential application of this organism to bioethanol production. Thus, the newly uncovered wood degrading capacity and sequential nature of this process in F. velutipes, offer interesting possibilities for more detailed studies on either lignin or (hemi-) cellulose degradation in complex wood substrates. The mutual interest in wood degradation by the mushroom industry and (ligno-)cellulose biomass related industries further increase the significance of F. velutipes as a new model. PMID:24714189

Responses to sugar and sugar receptor gene expression in different social roles of the honeybee (Apis mellifera).

PubMed

Değirmenci, Laura; Thamm, Markus; Scheiner, Ricarda

2018-04-01

Honeybees (Apis mellifera) are well-known for their sophisticated division of labor with each bee performing sequentially a series of social tasks. Colony organization is largely based on age-dependent division of labor. While bees perform several tasks inside the hive such as caring for brood ("nursing"), cleaning or sealing brood cells or producing honey, older bees leave to colony to collect pollen (proteins) and nectar (carbohydrates) as foragers. The most pronounced behavioral transition occurs when nurse bees become foragers. For both social roles, the detection and evaluation of sugars is decisive for optimal task performance. Nurse bees rely on their gustatory senses to prepare brood food, while foragers evaluate a nectar source before starting to collect food from it. To test whether social organization is related to differential sensing of sugars we compared the taste of nurse bees and foragers for different sugars. Searching for molecular correlates for differences in sugar perception, we further quantified expression of gustatory receptor genes in both behavioral groups. Our results demonstrate that nurse bees and foragers perceive and evaluate different sugars differently. Both groups, however, prefer sucrose over fructose. At least part of the taste differences between social roles could be related to a differential expression of taste receptors in the antennae and brain. Our results suggest that differential expression of sugar receptor genes might be involved in regulating division of labor through nutrition-related signaling pathways. Copyright © 2017 Elsevier Ltd. All rights reserved.

Sex Change in Clownfish: Molecular Insights from Transcriptome Analysis.

PubMed

Casas, Laura; Saborido-Rey, Fran; Ryu, Taewoo; Michell, Craig; Ravasi, Timothy; Irigoien, Xabier

2016-10-17

Sequential hermaphroditism is a unique reproductive strategy among teleosts that is displayed mainly in fish species living in the coral reef environment. The reproductive biology of hermaphrodites has long been intriguing; however, very little is known about the molecular pathways underlying their sex change. Here, we provide the first de novo transcriptome analyses of a hermaphrodite teleost´s undergoing sex change in its natural environment. Our study has examined relative gene expression across multiple groups-rather than just two contrasting conditions- and has allowed us to explore the differential expression patterns throughout the whole process. Our analysis has highlighted the rapid and complex genomic response of the brain associated with sex change, which is subsequently transmitted to the gonads, identifying a large number of candidate genes, some well-known and some novel, involved in the process. The present study provides strong evidence of the importance of the sex steroidogenic machinery during sex change in clownfish, with the aromatase gene playing a central role, both in the brain and the gonad. This work constitutes the first genome-wide study in a social sex-changing species and provides insights into the genetic mechanism governing social sex change and gonadal restructuring in protandrous hermaphrodites.

Sex Change in Clownfish: Molecular Insights from Transcriptome Analysis

PubMed Central

Casas, Laura; Saborido-Rey, Fran; Ryu, Taewoo; Michell, Craig; Ravasi, Timothy; Irigoien, Xabier

2016-01-01

Sequential hermaphroditism is a unique reproductive strategy among teleosts that is displayed mainly in fish species living in the coral reef environment. The reproductive biology of hermaphrodites has long been intriguing; however, very little is known about the molecular pathways underlying their sex change. Here, we provide the first de novo transcriptome analyses of a hermaphrodite teleost´s undergoing sex change in its natural environment. Our study has examined relative gene expression across multiple groups—rather than just two contrasting conditions— and has allowed us to explore the differential expression patterns throughout the whole process. Our analysis has highlighted the rapid and complex genomic response of the brain associated with sex change, which is subsequently transmitted to the gonads, identifying a large number of candidate genes, some well-known and some novel, involved in the process. The present study provides strong evidence of the importance of the sex steroidogenic machinery during sex change in clownfish, with the aromatase gene playing a central role, both in the brain and the gonad. This work constitutes the first genome-wide study in a social sex-changing species and provides insights into the genetic mechanism governing social sex change and gonadal restructuring in protandrous hermaphrodites. PMID:27748421

Network Analysis Reveals a Common Host-Pathogen Interaction Pattern in Arabidopsis Immune Responses.

PubMed

Li, Hong; Zhou, Yuan; Zhang, Ziding

2017-01-01

Many plant pathogens secrete virulence effectors into host cells to target important proteins in host cellular network. However, the dynamic interactions between effectors and host cellular network have not been fully understood. Here, an integrative network analysis was conducted by combining Arabidopsis thaliana protein-protein interaction network, known targets of Pseudomonas syringae and Hyaloperonospora arabidopsidis effectors, and gene expression profiles in the immune response. In particular, we focused on the characteristic network topology of the effector targets and differentially expressed genes (DEGs). We found that effectors tended to manipulate key network positions with higher betweenness centrality. The effector targets, especially those that are common targets of an individual effector, tended to be clustered together in the network. Moreover, the distances between the effector targets and DEGs increased over time during infection. In line with this observation, pathogen-susceptible mutants tended to have more DEGs surrounding the effector targets compared with resistant mutants. Our results suggest a common plant-pathogen interaction pattern at the cellular network level, where pathogens employ potent local impact mode to interfere with key positions in the host network, and plant organizes an in-depth defense by sequentially activating genes distal to the effector targets.

Cancer astrocytes have a more conserved molecular status in long recurrence free survival (RFS) IDH1 wild-type glioblastoma patients: new emerging cancer players

PubMed Central

Franceschi, Sara; Lessi, Francesca; Aretini, Paolo; Ortenzi, Valerio; Scatena, Cristian; Menicagli, Michele; La Ferla, Marco; Civita, Prospero; Zavaglia, Katia; Scopelliti, Claudia; Apollo, Alessandro; Carbone, Francesco Giovanni; Vannozzi, Riccardo; Bevilacqua, Generoso; Pasqualetti, Francesco; Naccarato, Antonio Giuseppe; Mazzanti, Chiara Maria

2018-01-01

Glioblastoma is a devastating disease that despite all the information gathered so far, its optimal management remains elusive due to the absence of validated targets from clinical studies. A better clarification of the molecular mechanisms is needed. In this study, having access to IDH1 wild-type glioblastoma of patients with exceptionally long recurrence free survival (RFS), we decided to compare their mutational and gene expression profile to groups of IDH1 wild-type glioblastoma of patients with shorter RFS, by using NGS technology. The exome analysis revealed that Long-RFS tumors have a lower mutational rate compared to the other groups. A total of 158 genes were found differentially expressed among the groups, 112 of which distinguished the two RFS extreme groups. Overall, the exome data suggests that shorter RFS tumors could be, chronologically, in a more advanced state in the muli-step tumor process of sequential accumulation of mutations. New players in this kind of cancer emerge from the analysis, confirmed at the RNA/DNA level, identifying, therefore, possible oncodrivers or tumor suppressor genes. PMID:29844869

Modeling a linkage between blood transcriptional expression and activity in brain regions to infer the phenotype of schizophrenia patients.

PubMed

Ibrahim, El Chérif; Guillemot, Vincent; Comte, Magali; Tenenhaus, Arthur; Zendjidjian, Xavier Yves; Cancel, Aida; Belzeaux, Raoul; Sauvanaud, Florence; Blin, Olivier; Frouin, Vincent; Fakra, Eric

2017-09-07

Hundreds of genetic loci participate to schizophrenia liability. It is also known that impaired cerebral connectivity is directly related to the cognitive and affective disturbances in schizophrenia. How genetic susceptibility and brain neural networks interact to specify a pathological phenotype in schizophrenia remains elusive. Imaging genetics, highlighting brain variations, has proven effective to establish links between vulnerability loci and associated clinical traits. As previous imaging genetics works in schizophrenia have essentially focused on structural DNA variants, these findings could be blurred by epigenetic mechanisms taking place during gene expression. We explored the meaningful links between genetic data from peripheral blood tissues on one hand, and regional brain reactivity to emotion task assayed by blood oxygen level-dependent functional magnetic resonance imaging on the other hand, in schizophrenia patients and matched healthy volunteers. We applied Sparse Generalized Canonical Correlation Analysis to identify joint signals between two blocks of variables: (i) the transcriptional expression of 33 candidate genes, and (ii) the blood oxygen level-dependent activity in 16 region of interest. Results suggested that peripheral transcriptional expression is related to brain imaging variations through a sequential pathway, ending with the schizophrenia phenotype. Generalization of such an approach to larger data sets should thus help in outlining the pathways involved in psychiatric illnesses such as schizophrenia. SEARCHING FOR LINKS TO AID DIAGNOSIS: Researchers explore links between the expression of genes associated with schizophrenia in blood cells and variations in brain activity during emotion processing. El Chérif Ibrahim and Eric Fakra at Aix-Marseille Université, France, and colleagues have developed a method to relate the expression levels of 33 schizophrenia susceptibility genes in blood cells and functional magnetic resonance imaging (fMRI) data obtained as individuals carry out a task that triggers emotional responses. Although they found no significant differences in the expression of genes between the 26 patients with schizophrenia and 26 healthy controls they examined, variations in activity in the superior temporal gyrus were strongly linked to schizophrenia-associated gene expression and presence of disease. Similar analyses of larger data sets will shed further light on the relationship between peripheral molecular changes and disease-related behaviors and ultimately, aid the diagnosis of neuropsychiatric disease.

Plasmidic Expression of nemA and yafC* Increased Resistance of Ethanologenic Escherichia coli LY180 to Nonvolatile Side Products from Dilute Acid Treatment of Sugarcane Bagasse and Artificial Hydrolysate

PubMed Central

Shi, Aiqin; Zheng, Huabao; Yomano, Lorraine P.; York, Sean W.; Shanmugam, Keelnatham T.

2016-01-01

Hydrolysate-resistant Escherichia coli SL100 was previously isolated from ethanologenic LY180 after sequential transfers in AM1 medium containing a dilute acid hydrolysate of sugarcane bagasse and was used as a source of resistance genes. Many genes that affect tolerance to furfural, the most abundant inhibitor, have been described previously. To identify genes associated with inhibitors other than furfural, plasmid clones were selected in an artificial hydrolysate that had been treated with a vacuum to remove furfural. Two new resistance genes were discovered from Sau3A1 libraries of SL100 genomic DNA: nemA (N-ethylmaleimide reductase) and a putative regulatory gene containing a mutation in the coding region, yafC*. The presence of these mutations in SL100 was confirmed by sequencing. A single mutation was found in the upstream regulatory region of nemR (nemRA operon) in SL100. This mutation increased nemA activity 20-fold over that of the parent organism (LY180) in AM1 medium without hydrolysate and increased nemA mRNA levels >200-fold. Addition of hydrolysates induced nemA expression (mRNA and activity), in agreement with transcriptional control. NemA activity was stable in cell extracts (9 h, 37°C), eliminating a role for proteinase in regulation. LY180 with a plasmid expressing nemA or yafC* was more resistant to a vacuum-treated sugarcane bagasse hydrolysate and to a vacuum-treated artificial hydrolysate than LY180 with an empty-vector control. Neither gene affected furfural tolerance. The vacuum-treated hydrolysates inhibited the reduction of N-ethylmaleimide by NemA while also serving as substrates. Expression of the nemA or yafC* plasmid in LY180 doubled the rate of ethanol production from the vacuum-treated sugarcane bagasse hydrolysate. PMID:26826228

Plasmidic Expression of nemA and yafC* Increased Resistance of Ethanologenic Escherichia coli LY180 to Nonvolatile Side Products from Dilute Acid Treatment of Sugarcane Bagasse and Artificial Hydrolysate.

PubMed

Shi, Aiqin; Zheng, Huabao; Yomano, Lorraine P; York, Sean W; Shanmugam, Keelnatham T; Ingram, Lonnie O

2016-01-29

Hydrolysate-resistant Escherichia coli SL100 was previously isolated from ethanologenic LY180 after sequential transfers in AM1 medium containing a dilute acid hydrolysate of sugarcane bagasse and was used as a source of resistance genes. Many genes that affect tolerance to furfural, the most abundant inhibitor, have been described previously. To identify genes associated with inhibitors other than furfural, plasmid clones were selected in an artificial hydrolysate that had been treated with a vacuum to remove furfural. Two new resistance genes were discovered from Sau3A1 libraries of SL100 genomic DNA: nemA (N-ethylmaleimide reductase) and a putative regulatory gene containing a mutation in the coding region, yafC*. The presence of these mutations in SL100 was confirmed by sequencing. A single mutation was found in the upstream regulatory region of nemR (nemRA operon) in SL100. This mutation increased nemA activity 20-fold over that of the parent organism (LY180) in AM1 medium without hydrolysate and increased nemA mRNA levels >200-fold. Addition of hydrolysates induced nemA expression (mRNA and activity), in agreement with transcriptional control. NemA activity was stable in cell extracts (9 h, 37°C), eliminating a role for proteinase in regulation. LY180 with a plasmid expressing nemA or yafC* was more resistant to a vacuum-treated sugarcane bagasse hydrolysate and to a vacuum-treated artificial hydrolysate than LY180 with an empty-vector control. Neither gene affected furfural tolerance. The vacuum-treated hydrolysates inhibited the reduction of N-ethylmaleimide by NemA while also serving as substrates. Expression of the nemA or yafC* plasmid in LY180 doubled the rate of ethanol production from the vacuum-treated sugarcane bagasse hydrolysate. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Insights into mechanisms of bacterial antigenic variation derived from the complete genome sequence of Anaplasma marginale.

PubMed

Palmer, Guy H; Futse, James E; Knowles, Donald P; Brayton, Kelly A

2006-10-01

Persistence of Anaplasma spp. in the animal reservoir host is required for efficient tick-borne transmission of these pathogens to animals and humans. Using A. marginale infection of its natural reservoir host as a model, persistent infection has been shown to reflect sequential cycles in which antigenic variants emerge, replicate, and are controlled by the immune system. Variation in the immunodominant outer-membrane protein MSP2 is generated by a process of gene conversion, in which unique hypervariable region sequences (HVRs) located in pseudogenes are recombined into a single operon-linked msp2 expression site. Although organisms expressing whole HVRs derived from pseudogenes emerge early in infection, long-term persistent infection is dependent on the generation of complex mosaics in which segments from different HVRs recombine into the expression site. The resulting combinatorial diversity generates the number of variants both predicted and shown to emerge during persistence.

SWATH-MS Quantitative Analysis of Proteins in the Rice Inferior and Superior Spikelets during Grain Filling

PubMed Central

Zhu, Fu-Yuan; Chen, Mo-Xian; Su, Yu-Wen; Xu, Xuezhong; Ye, Neng-Hui; Cao, Yun-Ying; Lin, Sheng; Liu, Tie-Yuan; Li, Hao-Xuan; Wang, Guan-Qun; Jin, Yu; Gu, Yong-Hai; Chan, Wai-Lung; Lo, Clive; Peng, Xinxiang; Zhu, Guohui; Zhang, Jianhua

2016-01-01

Modern rice cultivars have large panicle but their yield potential is often not fully achieved due to poor grain-filling of late-flowering inferior spikelets (IS). Our earlier work suggested a broad transcriptional reprogramming during grain filling and showed a difference in gene expression between IS and earlier-flowering superior spikelets (SS). However, the links between the abundances of transcripts and their corresponding proteins are unclear. In this study, a SWATH-MS (sequential window acquisition of all theoretical spectra-mass spectrometry) -based quantitative proteomic analysis has been applied to investigate SS and IS proteomes. A total of 304 proteins of widely differing functionality were observed to be differentially expressed between IS and SS. Detailed gene ontology analysis indicated that several biological processes including photosynthesis, protein metabolism, and energy metabolism are differentially regulated. Further correlation analysis revealed that abundances of most of the differentially expressed proteins are not correlated to the respective transcript levels, indicating that an extra layer of gene regulation which may exist during rice grain filling. Our findings raised an intriguing possibility that these candidate proteins may be crucial in determining the poor grain-filling of IS. Therefore, we hypothesize that the regulation of proteome changes not only occurs at the transcriptional, but also at the post-transcriptional level, during grain filling in rice. PMID:28066479

A Transcriptional Program for Arbuscule Degeneration during AM Symbiosis Is Regulated by MYB1.

PubMed

Floss, Daniela S; Gomez, S Karen; Park, Hee-Jin; MacLean, Allyson M; Müller, Lena M; Bhattarai, Kishor K; Lévesque-Tremblay, Veronique; Maldonado-Mendoza, Ignacio E; Harrison, Maria J

2017-04-24

During the endosymbiosis formed between plants and arbuscular mycorrhizal (AM) fungi, the root cortical cells are colonized by branched hyphae called arbuscules, which function in nutrient exchange with the plant [1]. Despite their positive function, arbuscules are ephemeral structures, and their development is followed by a degeneration phase, in which the arbuscule and surrounding periarbuscular membrane and matrix gradually disappear from the root cell [2, 3]. Currently, the root cell's role in this process and the underlying regulatory mechanisms are unknown. Here, by using a Medicago truncatula pt4 mutant in which arbuscules degenerate prematurely [4], we identified arbuscule degeneration-associated genes, of which 38% are predicted to encode secreted hydrolases, suggesting a role in disassembly of the arbuscule and interface. Through RNAi and analysis of an insertion mutant, we identified a symbiosis-specific MYB-like transcription factor (MYB1) that suppresses arbuscule degeneration in mtpt4. In myb1, expression of several degeneration-associated genes is reduced. Conversely, in roots constitutively overexpressing MYB1, expression of degeneration-associated genes is increased and subsequent development of symbiosis is impaired. MYB1-regulated gene expression is enhanced by DELLA proteins and is dependent on NSP1 [5], but not NSP2 [6]. Furthermore, MYB1 interacts with DELLA and NSP1. Our data identify a transcriptional program for arbuscule degeneration and reveal that its regulators include MYB1 in association with two transcriptional regulators, NSP1 and DELLA, both of which function in preceding phases of the symbiosis. We propose that the combinatorial use of transcription factors enables the sequential expression of transcriptional programs for arbuscule development and degeneration. Copyright © 2017 Elsevier Ltd. All rights reserved.

Respiratory Nitrate Ammonification by Shewanella oneidensis MR-1▿

PubMed Central

Cruz-García, Claribel; Murray, Alison E.; Klappenbach, Joel A.; Stewart, Valley; Tiedje, James M.

2007-01-01

Anaerobic cultures of Shewanella oneidensis MR-1 grown with nitrate as the sole electron acceptor exhibited sequential reduction of nitrate to nitrite and then to ammonium. Little dinitrogen and nitrous oxide were detected, and no growth occurred on nitrous oxide. A mutant with the napA gene encoding periplasmic nitrate reductase deleted could not respire or assimilate nitrate and did not express nitrate reductase activity, confirming that the NapA enzyme is the sole nitrate reductase. Hence, S. oneidensis MR-1 conducts respiratory nitrate ammonification, also termed dissimilatory nitrate reduction to ammonium, but not respiratory denitrification. PMID:17098906

In vivo genome-wide analysis of multiple tissues identifies gene regulatory networks, novel functions and downstream regulatory genes for Bapx1 and its co-regulation with Sox9 in the mammalian vertebral column.

PubMed

Chatterjee, Sumantra; Sivakamasundari, V; Yap, Sook Peng; Kraus, Petra; Kumar, Vibhor; Xing, Xing; Lim, Siew Lan; Sng, Joel; Prabhakar, Shyam; Lufkin, Thomas

2014-12-05

Vertebrate organogenesis is a highly complex process involving sequential cascades of transcription factor activation or repression. Interestingly a single developmental control gene can occasionally be essential for the morphogenesis and differentiation of tissues and organs arising from vastly disparate embryological lineages. Here we elucidated the role of the mammalian homeobox gene Bapx1 during the embryogenesis of five distinct organs at E12.5 - vertebral column, spleen, gut, forelimb and hindlimb - using expression profiling of sorted wildtype and mutant cells combined with genome wide binding site analysis. Furthermore we analyzed the development of the vertebral column at the molecular level by combining transcriptional profiling and genome wide binding data for Bapx1 with similarly generated data sets for Sox9 to assemble a detailed gene regulatory network revealing genes previously not reported to be controlled by either of these two transcription factors. The gene regulatory network appears to control cell fate decisions and morphogenesis in the vertebral column along with the prevention of premature chondrocyte differentiation thus providing a detailed molecular view of vertebral column development.

Transforming growth factor-{beta} inhibits CCAAT/enhancer-binding protein expression and PPAR{gamma} activity in unloaded bone marrow stromal cells

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

Ahdjoudj, S.; Kaabeche, K.; Holy, X.

2005-02-01

The molecular mechanisms regulating the adipogenic differentiation of bone marrow stromal cells in vivo remain largely unknown. In this study, we investigated the regulatory effects of transforming growth factor beta-2 (TGF-{beta}2) on transcription factors involved in adipogenic differentiation induced by hind limb suspension in rat bone marrow stromal cells in vivo. Time course real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis of gene expression showed that skeletal unloading progressively increases the expression of CCAAT/enhancer-binding protein (C/EBP){alpha} and C/EBP{beta} {alpha} at 5 days in bone marrow stromal cells resulting in increased peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}2) transcripts at 7 days. TGF-{beta}2more » administration in unloaded rats corrected the rise in C/EBP{alpha} and C/EBP{beta} transcripts induced by unloading in bone marrow stromal cells. This resulted in inhibition of PPAR{gamma}2 expression that was associated with increased Runx2 expression. Additionally, the inhibition of C/EBP{alpha} and C/EBP{beta} expression by TGF-{beta}2 was associated with increased PPAR{gamma} serine phosphorylation in bone marrow stromal cells, a mechanism that inhibits PPAR{gamma} transactivating activity. The sequential inhibitory effect of TGF-{beta}2 on C/EBP{alpha}, C/EBP{beta}, and PPAR{gamma}2 resulted in reduced LPL expression and abolition of bone marrow stromal cell adipogenic differentiation, which contributed to prevent bone loss induced by skeletal unloading. We conclude that TGF-{beta}2 inhibits the excessive adipogenic differentiation of bone marrow stromal cells induced by skeletal unloading by inhibiting C/EBP{alpha}, C/EBP{beta}, and PPAR{gamma} expression and activity, which provides a sequential mechanism by which TGF-{beta}2 regulates adipogenic differentiation of bone marrow stromal cells in vivo.« less

Phenotype discovery by gene expression profiling: mapping of biological processes linked to BMP-2-mediated osteoblast differentiation.

PubMed

Balint, Eva; Lapointe, David; Drissi, Hicham; van der Meijden, Caroline; Young, Daniel W; van Wijnen, Andre J; Stein, Janet L; Stein, Gary S; Lian, Jane B

2003-05-15

Understanding physiological control of osteoblast differentiation necessitates characterization of the regulatory signals that initiate the events directing a cell to lineage commitment and establishing competency for bone formation. The bone morphogenetic protein, BMP-2, a member of the TGFbeta superfamily, induces osteoblast differentiation and functions through the Smad signal transduction pathway during in vivo bone formation. However, the molecular targets of BMP-mediated gene transcription during the process of osteoblast differentiation have not been comprehensively identified. In the present study, BMP-2 responsive factors involved in the early stages of commitment and differentiation to the osteoblast phenotype were analyzed by microarray gene expression profiling in samples ranging from 1 to 24 h following BMP-2 dependent differentiation of C2C12 premyoblasts into the osteogenic lineage. A total of 1,800 genes were responsive to BMP-2 and expression was modulated from 3- to 14-fold for less than 100 genes during the time course. Approximately 50% of these 100 genes are either up- or downregulated. Major events associated with phenotypic changes towards the osteogenic lineage were identified from hierarchical and functional clustering analyses. BMP-2 immediately responsive genes (1-4 h), which exhibited either transient or sustained expression, reflect activation and repression of non-osseous BMP-2 developmental systems. This initial response was followed by waves of expression of nuclear proteins and developmental regulatory factors including inhibitors of DNA binding, Runx2, C/EBP, Zn finger binding proteins, forkhead, and numerous homeobox proteins (e.g., CDP/cut, paired, distaless, Hox) which are expressed at characterized stages during osteoblast differentiation. A sequential profile of genes mediating changes in cell morphology, cell growth, and basement membrane formation is observed as a secondary transient early response (2-8 h). Commitment to the osteogenic phenotype is recognized by 8 h, reflected by downregulation of most myogenic-related genes and induction of a spectrum of signaling proteins and enzymes facilitating synthesis and assembly of an extracellular skeletal environment. These genes included collagens Type I and VI and the small leucine rich repeat family of proteoglycans (e.g., decorin, biglycan, osteomodulin, fibromodulin, and osteoadherin/osteoglycin) that reached peak expression at 24 h. With extracellular matrix development, the bone phenotype was further established from 16 to 24 h by induction of genes for cell adhesion and communication and enzymes that organize the bone ECM. Our microarray analysis resulted in the discovery of a class of genes, initially described in relation to differentiation of astrocytes and oligodendrocytes that are functionally coupled to signals for cellular extensions. They include nexin, neuropilin, latexin, neuroglian, neuron specific gene 1, and Ulip; suggesting novel roles for these genes in the bone microenvironment. This global analysis identified a multistage molecular and cellular cascade that supports BMP-2-mediated osteoblast differentiation. Copyright 2003 Wiley-Liss, Inc.

Oil body proteins sequentially accumulate throughout seed development in Brassica napus.

PubMed

Jolivet, Pascale; Boulard, Céline; Bellamy, Annick; Valot, Benoît; d'Andréa, Sabine; Zivy, Michel; Nesi, Nathalie; Chardot, Thierry

2011-11-15

Despite the importance of seed oil bodies (OBs) as enclosed compartments for oil storage, little is known about lipid and protein accumulation in OBs during seed formation. OBs from rapeseed (Brassica napus) consist of a triacylglycerol (TAG) core surrounded by a phospholipid monolayer embedded with integral proteins which confer high stability to OBs in the mature dry seed. In the present study, we investigated lipid and protein accumulation patterns throughout seed development (from 5 to 65 days after pollination [DAP]) both in the whole seed and in purified OBs. Deposition of the major proteins (oleosins, caleosins and steroleosins) into OBs was assessed through (i) gene expression pattern, (ii) proteomics analysis, and (iii) protein immunodetection. For the first time, a sequential deposition of integral OB proteins was established. Accumulation of oleosins and caleosins was observed starting from early stages of seed development (12-17 DAP), while steroleosins accumulated later (~25 DAP) onwards. Copyright © 2011 Elsevier GmbH. All rights reserved.

Six post-implantation lethal knockouts of genes for lipophilic MAPK pathway proteins are expressed in preimplantation mouse embryos and trophoblast stem cells.

PubMed

Xie, Yufen; Wang, Yingchun; Sun, Tong; Wang, Fangfei; Trostinskaia, Anna; Puscheck, Elizabeth; Rappolee, Daniel A

2005-05-01

Mitogen-activated protein kinase (MAPK) signaling pathways play an important role in controlling embryonic proliferation and differentiation. It has been demonstrated that sequential lipophilic signal transduction mediators that participate in the MAPK pathway are null post-implantation lethal. It is not clear why the lethality of these null mutants arises after implantation and not before. One hypothesis is that the gene product of these post-implantation lethal null mutants are not present before implantation in normal embryos and do not have function until after implantation. To test this hypothesis, we selected a set of lipophilic genes mediating MAPK signal transduction pathways whose null mutants result in early peri-implantation or placental lethality. These included FRS2alpha, GAB1, GRB2, SOS1, Raf-B, and Raf1. Products of these selected genes were detected and their locations and functions indicated by indirect immunocytochemistry and Western blotting for proteins and RT-polymerase chain reaction (PCR) for mRNA transcription. We report here that all six signal mediators are detected at the protein level in preimplantation mouse embryo, placental trophoblasts, and in cultured trophoblast stem cells (TSC). Proteins are all detected in E3.5 embryos at a time when the first known mitogenic intercellular communication has been documented. mRNA transcripts of two post-implantation null mutant genes are expressed in mouse preimplantation embryos and unfertilized eggs. These mRNA transcripts were detected as maternal mRNA in unfertilized eggs that could delay the lethality of null mutants. All of the proteins were detected in the cytoplasm or in the cell membrane. This study of spatial and temporal expression revealed that all of these six null mutants post-implantation genes in MAPK pathway are expressed and, where tested, phosphorylated/activated proteins are detected in the blastocyst. Studies on RNA expression using RT-PCR suggest that maternal RNA could play an important role in delaying the presence of the lethal phenotype of null mutations. Copyright (c) 2005 Wiley-Liss, Inc.

Genome Engineering in Bacillus anthracis Using Cre Recombinase

PubMed Central

Pomerantsev, Andrei P.; Sitaraman, Ramakrishnan; Galloway, Craig R.; Kivovich, Violetta; Leppla, Stephen H.

2006-01-01

Genome engineering is a powerful method for the study of bacterial virulence. With the availability of the complete genomic sequence of Bacillus anthracis, it is now possible to inactivate or delete selected genes of interest. However, many current methods for disrupting or deleting more than one gene require use of multiple antibiotic resistance determinants. In this report we used an approach that temporarily inserts an antibiotic resistance marker into a selected region of the genome and subsequently removes it, leaving the target region (a single gene or a larger genomic segment) permanently mutated. For this purpose, a spectinomycin resistance cassette flanked by bacteriophage P1 loxP sites oriented as direct repeats was inserted within a selected gene. After identification of strains having the spectinomycin cassette inserted by a double-crossover event, a thermo-sensitive plasmid expressing Cre recombinase was introduced at the permissive temperature. Cre recombinase action at the loxP sites excised the spectinomycin marker, leaving a single loxP site within the targeted gene or genomic segment. The Cre-expressing plasmid was then removed by growth at the restrictive temperature. The procedure could then be repeated to mutate additional genes. In this way, we sequentially mutated two pairs of genes: pepM and spo0A, and mcrB and mrr. Furthermore, loxP sites introduced at distant genes could be recombined by Cre recombinase to cause deletion of large intervening regions. In this way, we deleted the capBCAD region of the pXO2 plasmid and the entire 30 kb of chromosomal DNA between the mcrB and mrr genes, and in the latter case we found that the 32 intervening open reading frames were not essential to growth. PMID:16369025

Novel mouse model recapitulates genome and transcriptome alterations in human colorectal carcinomas.

PubMed

McNeil, Nicole E; Padilla-Nash, Hesed M; Buishand, Floryne O; Hue, Yue; Ried, Thomas

2017-03-01

Human colorectal carcinomas are defined by a nonrandom distribution of genomic imbalances that are characteristic for this disease. Often, these imbalances affect entire chromosomes. Understanding the role of these aneuploidies for carcinogenesis is of utmost importance. Currently, established transgenic mice do not recapitulate the pathognonomic genome aberration profile of human colorectal carcinomas. We have developed a novel model based on the spontaneous transformation of murine colon epithelial cells. During this process, cells progress through stages of pre-immortalization, immortalization and, finally, transformation, and result in tumors when injected into immunocompromised mice. We analyzed our model for genome and transcriptome alterations using ArrayCGH, spectral karyotyping (SKY), and array based gene expression profiling. ArrayCGH revealed a recurrent pattern of genomic imbalances. These results were confirmed by SKY. Comparing these imbalances with orthologous maps of human chromosomes revealed a remarkable overlap. We observed focal deletions of the tumor suppressor genes Trp53 and Cdkn2a/p16. High-level focal genomic amplification included the locus harboring the oncogene Mdm2, which was confirmed by FISH in the form of double minute chromosomes. Array-based global gene expression revealed distinct differences between the sequential steps of spontaneous transformation. Gene expression changes showed significant similarities with human colorectal carcinomas. Pathways most prominently affected included genes involved in chromosomal instability and in epithelial to mesenchymal transition. Our novel mouse model therefore recapitulates the most prominent genome and transcriptome alterations in human colorectal cancer, and might serve as a valuable tool for understanding the dynamic process of tumorigenesis, and for preclinical drug testing. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Functional Expression of Enterobacterial O-Polysaccharide Biosynthesis Enzymes in Bacillus subtilis

PubMed Central

Schäffer, Christina; Wugeditsch, Thomas; Messner, Paul; Whitfield, Chris

2002-01-01

The expression of heterologous bacterial glycosyltransferases is of interest for potential application in the emerging field of carbohydrate engineering in gram-positive organisms. To assess the feasibility of using enzymes from gram-negative bacteria, the functional expression of the genes wbaP (formerly rfbP), wecA (formerly rfe), and wbbO (formerly rfbF) from enterobacterial lipopolysaccharide O-polysaccharide biosynthesis pathways was examined in Bacillus subtilis. WbaP and WecA are initiation enzymes for O-polysaccharide formation, catalyzing the transfer of galactosyl 1-phosphate from UDP-galactose and N-acetylglucosaminyl 1-phosphate from UDP-N-acetylglucosamine, respectively, to undecaprenylphosphate. The WecA product (undecaprenylpyrophosphoryl GlcNAc) is used as an acceptor to which the bifunctional wbbO gene product sequentially adds a galactopyranose and a galactofuranose residue from the corresponding UDP sugars to form a lipid-linked trisaccharide. Genes were cloned into the shuttle vectors pRB374 and pAW10. In B. subtilis hosts, the genes were effectively transcribed under the vegII promoter control of pRB374, but the plasmids were susceptible to rearrangements and deletion. In contrast, pAW10-based constructs, in which genes were cloned downstream of the tet resistance cassette, were stable but yielded lower levels of enzyme activity. In vitro glycosyltransferase assays were performed in Escherichia coli and B. subtilis, using membrane preparations as sources of enzymes and endogenous undecaprenylphosphate as an acceptor. Incorporation of radioactivity from UDP-α-d-14C-sugar into reaction products verified the functionality of WbaP, WecA, and WbbO in either host. Enzyme activities in B. subtilis varied between 20 and 75% of those measured in E. coli. PMID:12324313

Nitrogen-responsive Regulation of GATA Protein Family Activators Gln3 and Gat1 Occurs by Two Distinct Pathways, One Inhibited by Rapamycin and the Other by Methionine Sulfoximine*

PubMed Central

Georis, Isabelle; Tate, Jennifer J.; Cooper, Terrance G.; Dubois, Evelyne

2011-01-01

Nitrogen availability regulates the transcription of genes required to degrade non-preferentially utilized nitrogen sources by governing the localization and function of transcription activators, Gln3 and Gat1. TorC1 inhibitor, rapamycin (Rap), and glutamine synthetase inhibitor, methionine sulfoximine (Msx), elicit responses grossly similar to those of limiting nitrogen, implicating both glutamine synthesis and TorC1 in the regulation of Gln3 and Gat1. To better understand this regulation, we compared Msx- versus Rap-elicited Gln3 and Gat1 localization, their DNA binding, nitrogen catabolite repression-sensitive gene expression, and the TorC1 pathway phosphatase requirements for these responses. Using this information we queried whether Rap and Msx inhibit sequential steps in a single, linear cascade connecting glutamine availability to Gln3 and Gat1 control as currently accepted or alternatively inhibit steps in two distinct parallel pathways. We find that Rap most strongly elicits nuclear Gat1 localization and expression of genes whose transcription is most Gat1-dependent. Msx, on the other hand, elicits nuclear Gln3 but not Gat1 localization and expression of genes that are most Gln3-dependent. Importantly, Rap-elicited nuclear Gln3 localization is absolutely Sit4-dependent, but that elicited by Msx is not. PP2A, although not always required for nuclear GATA factor localization, is highly required for GATA factor binding to nitrogen-responsive promoters and subsequent transcription irrespective of the gene GATA factor specificities. Collectively, our data support the existence of two different nitrogen-responsive regulatory pathways, one inhibited by Msx and the other by rapamycin. PMID:22039046

Nitrogen-responsive regulation of GATA protein family activators Gln3 and Gat1 occurs by two distinct pathways, one inhibited by rapamycin and the other by methionine sulfoximine.

PubMed

Georis, Isabelle; Tate, Jennifer J; Cooper, Terrance G; Dubois, Evelyne

2011-12-30

Nitrogen availability regulates the transcription of genes required to degrade non-preferentially utilized nitrogen sources by governing the localization and function of transcription activators, Gln3 and Gat1. TorC1 inhibitor, rapamycin (Rap), and glutamine synthetase inhibitor, methionine sulfoximine (Msx), elicit responses grossly similar to those of limiting nitrogen, implicating both glutamine synthesis and TorC1 in the regulation of Gln3 and Gat1. To better understand this regulation, we compared Msx- versus Rap-elicited Gln3 and Gat1 localization, their DNA binding, nitrogen catabolite repression-sensitive gene expression, and the TorC1 pathway phosphatase requirements for these responses. Using this information we queried whether Rap and Msx inhibit sequential steps in a single, linear cascade connecting glutamine availability to Gln3 and Gat1 control as currently accepted or alternatively inhibit steps in two distinct parallel pathways. We find that Rap most strongly elicits nuclear Gat1 localization and expression of genes whose transcription is most Gat1-dependent. Msx, on the other hand, elicits nuclear Gln3 but not Gat1 localization and expression of genes that are most Gln3-dependent. Importantly, Rap-elicited nuclear Gln3 localization is absolutely Sit4-dependent, but that elicited by Msx is not. PP2A, although not always required for nuclear GATA factor localization, is highly required for GATA factor binding to nitrogen-responsive promoters and subsequent transcription irrespective of the gene GATA factor specificities. Collectively, our data support the existence of two different nitrogen-responsive regulatory pathways, one inhibited by Msx and the other by rapamycin.

Dissection of Symbiosis and Organ Development by Integrated Transcriptome Analysis of Lotus japonicus Mutant and Wild-Type Plants

PubMed Central

Høgslund, Niels; Radutoiu, Simona; Krusell, Lene; Voroshilova, Vera; Hannah, Matthew A.; Goffard, Nicolas; Sanchez, Diego H.; Lippold, Felix; Ott, Thomas; Sato, Shusei; Tabata, Satoshi; Liboriussen, Poul; Lohmann, Gitte V.; Schauser, Leif; Weiller, Georg F.; Udvardi, Michael K.; Stougaard, Jens

2009-01-01

Genetic analyses of plant symbiotic mutants has led to the identification of key genes involved in Rhizobium-legume communication as well as in development and function of nitrogen fixing root nodules. However, the impact of these genes in coordinating the transcriptional programs of nodule development has only been studied in limited and isolated studies. Here, we present an integrated genome-wide analysis of transcriptome landscapes in Lotus japonicus wild-type and symbiotic mutant plants. Encompassing five different organs, five stages of the sequentially developed determinate Lotus root nodules, and eight mutants impaired at different stages of the symbiotic interaction, our data set integrates an unprecedented combination of organ- or tissue-specific profiles with mutant transcript profiles. In total, 38 different conditions sampled under the same well-defined growth regimes were included. This comprehensive analysis unravelled new and unexpected patterns of transcriptional regulation during symbiosis and organ development. Contrary to expectations, none of the previously characterized nodulins were among the 37 genes specifically expressed in nodules. Another surprise was the extensive transcriptional response in whole root compared to the susceptible root zone where the cellular response is most pronounced. A large number of transcripts predicted to encode transcriptional regulators, receptors and proteins involved in signal transduction, as well as many genes with unknown function, were found to be regulated during nodule organogenesis and rhizobial infection. Combining wild type and mutant profiles of these transcripts demonstrates the activation of a complex genetic program that delineates symbiotic nitrogen fixation. The complete data set was organized into an indexed expression directory that is accessible from a resource database, and here we present selected examples of biological questions that can be addressed with this comprehensive and powerful gene expression data set. PMID:19662091

A series of no isthmus (noi) alleles of the zebrafish pax2.1 gene reveals multiple signaling events in development of the midbrain-hindbrain boundary.

PubMed

Lun, K; Brand, M

1998-08-01

Generation of cell diversity in the vertebrate central nervous system starts during gastrulation stages in the ectodermal germ layer and involves specialized cell groups, such as the organizer located at the midbrain-hindbrain boundary (MHB). Mutations in the zebrafish no isthmus (noi) gene alter development of the MHB, and affect the pax2.1 gene (formerly pax(zf-b)). Analysis of the structure of pax2.1 reveals at least 12 normal splice variants. The noi alleles can be arranged, by molecular and phenotypic criteria, into a series of five alleles of differing strength, ranging from a null allele to weak alleles. In keeping with a role in development of the MHB organizer, gene expression is already affected in the MHB primordium of the gastrula neural ectoderm in noi mutants. eng3 activation is completely and eng2 activation is strongly dependent on noi function. In contrast, onset of wnt1, fgf8 and her5 expression occurs normally in the null mutants, but is eliminated later on. Our observations suggest that three signaling pathways, involving pax2.1, wnt1 and fgf8, are activated independently in early anterior-posterior patterning of this area. In addition, analysis of the allelic series unexpectedly suggests that noi activity is also required during dorsal-ventral patterning of the MHB in somitogenesis stages, and possibly in a later eng expression phase. We propose that noi/pax2.1 participates in sequential signaling processes as a key integrator of midbrain-hindbrain boundary development.

Loss of heterozygosity at D8S262: an early genetic event of hepatocarcinogenesis.

PubMed

Zhu, Qiao; Gong, Li; Liu, Xiaoyan; Wang, Jun; Ren, Pin; Zhang, Wendong; Yao, Li; Han, Xiujuan; Zhu, Shaojun; Lan, Miao; Li, Yanhong; Zhang, Wei

2015-06-16

Hepatocellular carcinoma (HCC) is a multi-factor, multi-step, multi-gene and complicated process resulting from the accumulation of sequential genetic and epigenetic alterations. An important change among them is from precancerous lesions to HCC. However, only few studies have been reported about the sequential genetic changes during hepatocarcinogenesis. We observed firstly molecular karyotypes of 10 matched HCC using Affymetrix single-nucleotide polymorphism (SNP) 6.0 arrays, and found chromosomal fragments with high incidence (more than 70%) of loss of heterozygosity (LOH). Then, we selected 28 microsatellite markers at some gene spanning these chromosomal fragments, and examined the frequency of LOH of 128 matched HCC and 43 matched precancerous lesions-dysplastic nodules (DN) by a PCR-based analysis. Finally, we investigated the expression of proteins encoded by these genes in HCC, DN and the surrounding hepatic tissues. The result of Affymetrix SNP6.0 arrays demonstrated that more than 70% (7/10) cases had chromosomal fragment deletion on 4q13.3-35.1, 8p23.2-21.2, 16q11.2-24.3, and 17p13.3-12. Among 28 microsatellite markers selected, LOH frequencies at D8S262 for DN and HCC were found to be the highest, 51.2% and 72.7%, respectively. Immunohistochemically, the positive rate of its adjacent gene CSMD1 in HCC, DN, and the surrounding hepatic tissues were 27.3% (35/128), 75% (33/44), and 82% (105/128), respectively. LOH at D8S262 may be associated with an early genetic event of hepatocarcinogenesis, and a predictor for the monitor and prevention of HCC. The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1557074981159099 .

The potential benefits of nicaraven to protect against radiation-induced injury in hematopoietic stem/progenitor cells with relative low dose exposures

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

Ali, Haytham; Department of Medical Physiology and Cell Biology, Qena Faculty of Medicine, South Valley University; Galal, Omima

Highlights: • Nicaraven mitigated the radiation-induced reduction of c-kit{sup +} stem cells. • Nicaraven enhanced the function of hematopoietic stem/progenitor cells. • Complex mechanisms involved in the protection of nicaraven to radiation injury. - Abstract: Nicaraven, a hydroxyl radical-specific scavenger has been demonstrated to attenuate radiation injury in hematopoietic stem cells with 5 Gy γ-ray exposures. We explored the effect and related mechanisms of nicaraven for protecting radiation injury induced by sequential exposures to a relatively lower dose γ-ray. C57BL/6 mice were given nicaraven or placebo within 30 min before exposure to 50 mGy γ-ray daily for 30 days inmore » sequences (cumulative dose of 1.5 Gy). Mice were victimized 24 h after the last radiation exposure, and the number, function and oxidative stress of hematopoietic stem cells were quantitatively estimated. We also compared the gene expression in these purified stem cells from mice received nicaraven and placebo treatment. Nicaraven increased the number of c-kit{sup +} stem/progenitor cells in bone marrow and peripheral blood, with a recovery rate around 60–90% of age-matched non-irradiated healthy mice. The potency of colony forming from hematopoietic stem/progenitor cells as indicator of function was completely protected with nicaraven treatment. Furthermore, nicaraven treatment changed the expression of many genes associated to DNA repair, inflammatory response, and immunomodulation in c-kit{sup +} stem/progenitor cells. Nicaraven effectively protected against damages of hematopoietic stem/progenitor cells induced by sequential exposures to a relatively low dose radiation, via complex mechanisms.« less

TRIP13 impairs mitotic checkpoint surveillance and is associated with poor prognosis in multiple myeloma

PubMed Central

Song, Dongliang; Hu, Liangning; Xie, Bingqian; Wang, Houcai; Gao, Lu; Gao, Minjie; Xu, Hongwei; Xu, Zhijian; Wu, Xiaosong; Zhang, Yiwen; Zhu, Weiliang; Zhan, Fenghuang; Shi, Jumei

2017-01-01

AAA-ATPase TRIP13 is one of the chromosome instability gene recently established in multiple myeloma (MM), the second most common and incurable hematological malignancy. However, the specific function of TRIP13 in MM is largely unknown. Using sequential gene expression profiling, we demonstrated that high TRIP13 expression levels were positively correlated with progression, disease relapse, and poor prognosis in MM patients. Overexpressing human TRIP13 in myeloma cells prompted cell growth and drug resistance, and overexpressing murine TRIP13, which shares 93% sequence identity with human TRIP13, led to colony formation of NIH/3T3 fibroblasts in vitro and tumor formation in vivo. Meanwhile, the knockdown of TRIP13 inhibited myeloma cell growth, induced cell apoptosis, and reduced tumor burden in xenograft MM mice. Mechanistically, we observed that the overexpression of TRIP13 abrogated the spindle checkpoint and induced proteasome-mediated degradation of MAD2 primarily through the Akt pathway. Thus, our results demonstrate that TRIP13 may serve as a biomarker for MM disease development and prognosis, making it a potential target for future therapies. PMID:28157697

Amino Acid Substitution in Trichophyton rubrum Squalene Epoxidase Associated with Resistance to Terbinafine

PubMed Central

Osborne, Colin S.; Leitner, Ingrid; Favre, Bertrand; Ryder, Neil S.

2005-01-01

There has only been one clinically confirmed case of terbinafine resistance in dermatophytes, where six sequential Trichophyton rubrum isolates from the same patient were found to be resistant to terbinafine and cross-resistant to other squalene epoxidase (SE) inhibitors. Microsomal SE activity from these resistant isolates was insensitive to terbinafine, suggesting a target-based mechanism of resistance (B. Favre, M. Ghannoum, and N. S. Ryder, Med. Mycol. 42:525-529, 2004). In this study, we have characterized at the molecular level the cause of the resistant phenotype of these clinical isolates. Cloning and sequencing of the SE gene and cDNA from T. rubrum revealed the presence of an intron in the gene and an open reading frame encoding a protein of 489 residues, with an equivalent similarity (57%) to both yeast and mammalian SEs. The nucleotide sequences of SE from two terbinafine-susceptible strains were identical whereas those of terbinafine-resistant strains, serially isolated from the same patient, each contained the same single missense introducing the amino acid substitution L393F. Introduction of the corresponding substitution in the Candida albicans SE gene (L398F) and expression of this gene in Saccharomyces cerevisiae conferred a resistant phenotype to the transformants when compared to those expressing the wild-type sequence. Terbinafine resistance in these T. rubrum clinical isolates appears to be due to a single amino acid substitution in SE. PMID:15980358

Microarray-Based Gene Expression Analysis for Veterinary Pathologists: A Review.

PubMed

Raddatz, Barbara B; Spitzbarth, Ingo; Matheis, Katja A; Kalkuhl, Arno; Deschl, Ulrich; Baumgärtner, Wolfgang; Ulrich, Reiner

2017-09-01

High-throughput, genome-wide transcriptome analysis is now commonly used in all fields of life science research and is on the cusp of medical and veterinary diagnostic application. Transcriptomic methods such as microarrays and next-generation sequencing generate enormous amounts of data. The pathogenetic expertise acquired from understanding of general pathology provides veterinary pathologists with a profound background, which is essential in translating transcriptomic data into meaningful biological knowledge, thereby leading to a better understanding of underlying disease mechanisms. The scientific literature concerning high-throughput data-mining techniques usually addresses mathematicians or computer scientists as the target audience. In contrast, the present review provides the reader with a clear and systematic basis from a veterinary pathologist's perspective. Therefore, the aims are (1) to introduce the reader to the necessary methodological background; (2) to introduce the sequential steps commonly performed in a microarray analysis including quality control, annotation, normalization, selection of differentially expressed genes, clustering, gene ontology and pathway analysis, analysis of manually selected genes, and biomarker discovery; and (3) to provide references to publically available and user-friendly software suites. In summary, the data analysis methods presented within this review will enable veterinary pathologists to analyze high-throughput transcriptome data obtained from their own experiments, supplemental data that accompany scientific publications, or public repositories in order to obtain a more in-depth insight into underlying disease mechanisms.

Protein Expression Profile of Rat Type Two Alveolar Epithelial Cells During Hyperoxic Stress and Recovery

NASA Astrophysics Data System (ADS)

Bhargava, Maneesh

Rationale: In rodent model systems, the sequential changes in lung morphology resulting from hyperoxic injury are well characterized, and are similar to changes in human acute respiratory distress syndrome (ARDS). In the injured lung, alveolar type two (AT2) epithelial cells play a critical role restoring the normal alveolar structure. Thus characterizing the changes in AT2 cells will provide insights into the mechanisms underpinning the recovery from lung injury. Methods: We applied an unbiased systems level proteomics approach to elucidate molecular mechanisms contributing to lung repair in a rat hyperoxic lung injury model. AT2 cells were isolated from rat lungs at predetermined intervals during hyperoxic injury and recovery. Protein expression profiles were determined by using iTRAQRTM with tandem mass spectrometry. Results: Of 959 distinct proteins identified, 183 significantly changed in abundance during the injury-recovery cycle. Gene Ontology enrichment analysis identified cell cycle, cell differentiation, cell metabolism, ion homeostasis, programmed cell death, ubiquitination, and cell migration to be significantly enriched by these proteins. Gene Set Enrichment Analysis of data acquired during lung repair revealed differential expression of gene sets that control multicellular organismal development, systems development, organ development, and chemical homeostasis. More detailed analysis identified activity in two regulatory pathways, JNK and miR 374. A Short Time-series Expression Miner (STEM) algorithm identified protein clusters with coherent changes during injury and repair. Conclusion: Coherent changes occur in the AT2 cell proteome in response to hyperoxic stress. These findings offer guidance regarding the specific molecular mechanisms governing repair of the injured lung.

Rearrangement of a large novel Pseudomonas aeruginosa gene island in strains isolated from a patient developing ventilator-associated pneumonia.

PubMed

Singh, G; Srinivasan, R; Cheng, J; Peng, Z; Fujimura, K; Baek, M S; Panzer, A R; Tringe, S G; Chen, F; Sorek, R; Weng, L; Bristow, J; Wiener-Kronish, J P; Lynch, S V

2014-07-01

Bacterial gene islands add to the genetic repertoire of opportunistic pathogens. Here, we perform comparative analyses of three Pseudomonas aeruginosa strains isolated sequentially over a 3-week period from a patient with ventilator-associated pneumonia (VAP) who received clindamycin and piperacillin-tazobactam as part of their treatment regime. While all three strains appeared to be clonal by standard pulsed-field gel electrophoresis, whole-genome sequencing revealed subtle alterations in the chromosomal organization of the last two strains; specifically, an inversion event within a novel 124-kb gene island (PAGI 12) composed of 137 open reading frames [ORFs]. Predicted ORFs in the island included metabolism and virulence genes. Overexpression of a gene island-borne putative β-lactamase gene was observed following piperacillin-tazobactam exposure and only in those strains that had undergone the inversion event, indicating altered gene regulation following genomic remodeling. Examination of a separate cohort of 76 patients with VAP for integration at this tRNA(lys) recombination site demonstrated that patients exhibiting evidence of integration at this site had significantly higher 28-day mortality. These findings provide evidence that P. aeruginosa can integrate, rapidly remodel, and express exogenous genes, which likely contributes to its fitness in a clinical setting. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Rearrangement of a Large Novel Pseudomonas aeruginosa Gene Island in Strains Isolated from a Patient Developing Ventilator-Associated Pneumonia

PubMed Central

Singh, G.; Srinivasan, R.; Cheng, J.; Peng, Z.; Fujimura, K.; Baek, M. S.; Panzer, A. R.; Tringe, S. G.; Chen, F.; Sorek, R.; Weng, L.; Bristow, J.; Wiener-Kronish, J. P.

2014-01-01

Bacterial gene islands add to the genetic repertoire of opportunistic pathogens. Here, we perform comparative analyses of three Pseudomonas aeruginosa strains isolated sequentially over a 3-week period from a patient with ventilator-associated pneumonia (VAP) who received clindamycin and piperacillin-tazobactam as part of their treatment regime. While all three strains appeared to be clonal by standard pulsed-field gel electrophoresis, whole-genome sequencing revealed subtle alterations in the chromosomal organization of the last two strains; specifically, an inversion event within a novel 124-kb gene island (PAGI 12) composed of 137 open reading frames [ORFs]. Predicted ORFs in the island included metabolism and virulence genes. Overexpression of a gene island-borne putative β-lactamase gene was observed following piperacillin-tazobactam exposure and only in those strains that had undergone the inversion event, indicating altered gene regulation following genomic remodeling. Examination of a separate cohort of 76 patients with VAP for integration at this tRNAlys recombination site demonstrated that patients exhibiting evidence of integration at this site had significantly higher 28-day mortality. These findings provide evidence that P. aeruginosa can integrate, rapidly remodel, and express exogenous genes, which likely contributes to its fitness in a clinical setting. PMID:24789195

[Changes of expression of miR-155 in colitis-associated colonic carcinogenesis].

PubMed

Li, Weiwei; Han, Wenxiao; Zhao, Xinhua; Wang, Hongying

2014-04-01

To investigate the changes of miR-155 and its target genes in colitis-associated carcinogenesis. Colitis-associated colon cancer was induced by azoxymethane (AOM) and dextran sulfate sodium (DSS) in C57BL/6 mice. Mice of three different stages during the development of colon cancer were obtained, named AD1, AD2 and AD3, respectively. A control group of mice without any treatment and a DSS only group representing chronic inflammation without cancer were set up as well. Colon tissue was collected and expression of miR-155 in the colon tissues was measured by real-time fluorescent quantitative PCR. TargetScan and PicTar were used to predict potential target genes of miR-155, which were then preliminarily screened with our gene expression microarray database of AOM-DSS mouse model. Regular PCR was used to confirm the changes of the expression of these potential target genes in AOM-DSS mouse model. Colitis-associated colon cancer was effectively induced by azoxymethane and dextran sulfate sodium in C57BL/6 mice. Histological examination revealed that the evolution process was sequentially from normal, mild dysplasia, moderate dysplasia, and severe dysplasia to adenocarcinoma in the AOM-DSS mouse model. The level of miR-155 was gradually elevated with the formation of colitis-associated colon cancer. There was no significant difference between the levels of miR-155 expression in the DSS group (0.005 6 ± 0.003 7) and control group (0.012 0 ± 0.005 1) (P > 0.05), but the level of miR-155 in the AD3 group (0.054 4 ± 0.027 0) was significantly higher than that of the DSS group (0.005 6 ± 0.003 7)(P < 0.01). No significant change of miR-155 expression was found in the DSS only group. The relative expression levels of miR-155 in the control group, DSS only group and AD3 group were 0.012 0 ± 0.005 1, 0.005 6 ± 0.003 7, 0.054 4 ± 0.027 0, respectively. Data analysis with the gene expression microarray showed that Tle4, Kcna1, Itk, Bcorl1, Cacna1c, Rspo2 and Foxo3 were potential target genes of miR-155 in the AOM-DSS mouse model. Changes of Kcna1 and Cacna1c in the AOM-DSS mouse model were validated to be consistent with the changes obtained with the gene expression microarray. The up-regulation of miR-155 is related to colitis-associated carcinogenesis, but is irrelevant to chronic inflammation in the mouse model.

Missing value imputation in DNA microarrays based on conjugate gradient method.

PubMed

Dorri, Fatemeh; Azmi, Paeiz; Dorri, Faezeh

2012-02-01

Analysis of gene expression profiles needs a complete matrix of gene array values; consequently, imputation methods have been suggested. In this paper, an algorithm that is based on conjugate gradient (CG) method is proposed to estimate missing values. k-nearest neighbors of the missed entry are first selected based on absolute values of their Pearson correlation coefficient. Then a subset of genes among the k-nearest neighbors is labeled as the best similar ones. CG algorithm with this subset as its input is then used to estimate the missing values. Our proposed CG based algorithm (CGimpute) is evaluated on different data sets. The results are compared with sequential local least squares (SLLSimpute), Bayesian principle component analysis (BPCAimpute), local least squares imputation (LLSimpute), iterated local least squares imputation (ILLSimpute) and adaptive k-nearest neighbors imputation (KNNKimpute) methods. The average of normalized root mean squares error (NRMSE) and relative NRMSE in different data sets with various missing rates shows CGimpute outperforms other methods. Copyright © 2011 Elsevier Ltd. All rights reserved.

Differentiation and Transplantation of Human Embryonic Stem Cell-Derived Hepatocytes

PubMed Central

Basma, Hesham; Soto-Gutiérrez, Alejandro; Yannam, Govardhana Rao; Liu, Liping; Ito, Ryotaro; Yamamoto, Toshiyuki; Ellis, Ewa; Carson, Steven D.; Sato, Shintaro; Chen, Yong; Muirhead, David; Navarro-Álvarez, Nalu; Wong, Ron; Roy-Chowdhury, Jayanta; Platt, Jeffrey L.; Mercer, David F.; Miller, John D.; Strom, Stephen C.; Kobayashi, Noaya; Fox, Ira J.

2009-01-01

Background & Aims The ability to obtain unlimited numbers of human hepatocytes would improve development of cell-based therapies for liver diseases, facilitate the study of liver biology and improve the early stages of drug discovery. Embryonic stem cells are pluripotent, can potentially differentiate into any cell type and could therefore be developed as a source of human hepatocytes. Methods To generate human hepatocytes, human embryonic stem cells were differentiated by sequential culture in fibroblast growth factor 2 and human Activin-A, hepatocyte growth factor, and dexamethasone. Functional hepatocytes were isolated by sorting for surface asialoglycoprotein receptor expression. Characterization was performed by real-time PCR, imunohistochemistry, immunoblot, functional assays and transplantation. Results Embryonic stem cell-derived hepatocytes expressed liver-specific genes but not genes representing other lineages, secreted functional human liver-specific proteins similar to those of primary human hepatocytes and demonstrated human hepatocyte cytochrome P450 metabolic activity. Serum from rodents given injections of embryonic stem cell-derived hepatocytes contained significant amounts of human albumin and alpha-1-antitrypsin. Colonies of cytokeratin-18 and human albumin-expressing cells were present in the livers of recipient animals. Conclusion Human embryonic stem cells can be differentiated into cells with many characteristics of primary human hepatocytes. Hepatocyte-like cells can be enriched and recovered based on asialoglycoprotein receptor expression and could potentially be used in drug discovery research and developed as therapeutics. PMID:19026649

Developmental transitions in C. elegans larval stages.

PubMed

Rougvie, Ann E; Moss, Eric G

2013-01-01

Molecular mechanisms control the timing, sequence, and synchrony of developmental events in multicellular organisms. In Caenorhabditis elegans, these mechanisms are revealed through the analysis of mutants with "heterochronic" defects: cell division or differentiation patterns that occur in the correct lineage, but simply at the wrong time. Subsets of cells in these mutants thus express temporal identities normally restricted to a different life stage. A seminal finding arising from studies of the heterochronic genes was the discovery of miRNAs; these tiny miRNAs are now a defining feature of the pathway. A series of sequentially expressed miRNAs guide larval transitions through stage-specific repression of key effector molecules. The wild-type lineage patterns are executed as discrete modules programmed between temporal borders imposed by the molting cycles. How these successive events are synchronized with the oscillatory molting cycle is just beginning to come to light. Progression through larval stages can be specifically, yet reversibly, halted in response to environmental cues, including nutrient availability. Here too, heterochronic genes and miRNAs play key roles. Remarkably, developmental arrest can, in some cases, either mask or reveal timing defects associated with mutations. In this chapter, we provide an overview of how the C. elegans heterochronic gene pathway guides developmental transitions during continuous and interrupted larval development. © 2013 Elsevier Inc. All rights reserved.

Gene targeting by TALEN-induced homologous recombination in goats directs production of β-lactoglobulin-free, high-human lactoferrin milk

PubMed Central

Cui, Chenchen; Song, Yujie; Liu, Jun; Ge, Hengtao; Li, Qian; Huang, Hui; Hu, Linyong; Zhu, Hongmei; Jin, Yaping; Zhang, Yong

2015-01-01

β-Lactoglobulin (BLG) is a major goat’s milk allergen that is absent in human milk. Engineered endonucleases, including transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases, enable targeted genetic modification in livestock. In this study, TALEN-mediated gene knockout followed by gene knock-in were used to generate BLG knockout goats as mammary gland bioreactors for large-scale production of human lactoferrin (hLF). We introduced precise genetic modifications in the goat genome at frequencies of approximately 13.6% and 6.09% for the first and second sequential targeting, respectively, by using targeting vectors that underwent TALEN-induced homologous recombination (HR). Analysis of milk from the cloned goats revealed large-scale hLF expression or/and decreased BLG levels in milk from heterozygous goats as well as the absence of BLG in milk from homozygous goats. Furthermore, the TALEN-mediated targeting events in somatic cells can be transmitted through the germline after SCNT. Our result suggests that gene targeting via TALEN-induced HR may expedite the production of genetically engineered livestock for agriculture and biomedicine. PMID:25994151

Gene targeting by TALEN-induced homologous recombination in goats directs production of β-lactoglobulin-free, high-human lactoferrin milk.

PubMed

Cui, Chenchen; Song, Yujie; Liu, Jun; Ge, Hengtao; Li, Qian; Huang, Hui; Hu, Linyong; Zhu, Hongmei; Jin, Yaping; Zhang, Yong

2015-05-21

β-Lactoglobulin (BLG) is a major goat's milk allergen that is absent in human milk. Engineered endonucleases, including transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases, enable targeted genetic modification in livestock. In this study, TALEN-mediated gene knockout followed by gene knock-in were used to generate BLG knockout goats as mammary gland bioreactors for large-scale production of human lactoferrin (hLF). We introduced precise genetic modifications in the goat genome at frequencies of approximately 13.6% and 6.09% for the first and second sequential targeting, respectively, by using targeting vectors that underwent TALEN-induced homologous recombination (HR). Analysis of milk from the cloned goats revealed large-scale hLF expression or/and decreased BLG levels in milk from heterozygous goats as well as the absence of BLG in milk from homozygous goats. Furthermore, the TALEN-mediated targeting events in somatic cells can be transmitted through the germline after SCNT. Our result suggests that gene targeting via TALEN-induced HR may expedite the production of genetically engineered livestock for agriculture and biomedicine.

Faster embryonic segmentation through elevated Delta-Notch signalling

PubMed Central

Liao, Bo-Kai; Jörg, David J.; Oates, Andrew C.

2016-01-01

An important step in understanding biological rhythms is the control of period. A multicellular, rhythmic patterning system termed the segmentation clock is thought to govern the sequential production of the vertebrate embryo's body segments, the somites. Several genetic loss-of-function conditions, including the Delta-Notch intercellular signalling mutants, result in slower segmentation. Here, we generate DeltaD transgenic zebrafish lines with a range of copy numbers and correspondingly increased signalling levels, and observe faster segmentation. The highest-expressing line shows an altered oscillating gene expression wave pattern and shortened segmentation period, producing embryos with more, shorter body segments. Our results reveal surprising differences in how Notch signalling strength is quantitatively interpreted in different organ systems, and suggest a role for intercellular communication in regulating the output period of the segmentation clock by altering its spatial pattern. PMID:27302627

Thiol synthetases of legumes: immunogold localization and differential gene regulation by phytohormones

PubMed Central

Clemente, Maria R.; Bustos-Sanmamed, Pilar; Loscos, Jorge; James, Euan K.; Pérez-Rontomé, Carmen; Navascués, Joaquín; Gay, Marina; Becana, Manuel

2012-01-01

In plants and other organisms, glutathione (GSH) biosynthesis is catalysed sequentially by γ-glutamylcysteine synthetase (γECS) and glutathione synthetase (GSHS). In legumes, homoglutathione (hGSH) can replace GSH and is synthesized by γECS and a specific homoglutathione synthetase (hGSHS). The subcellular localization of the enzymes was examined by electron microscopy in several legumes and gene expression was analysed in Lotus japonicus plants treated for 1–48 h with 50 μM of hormones. Immunogold localization studies revealed that γECS is confined to chloroplasts and plastids, whereas hGSHS is also in the cytosol. Addition of hormones caused differential expression of thiol synthetases in roots. After 24–48 h, abscisic and salicylic acids downregulated GSHS whereas jasmonic acid upregulated it. Cytokinins and polyamines activated GSHS but not γECS or hGSHS. Jasmonic acid elicited a coordinated response of the three genes and auxin induced both hGSHS expression and activity. Results show that the thiol biosynthetic pathway is compartmentalized in legumes. Moreover, the similar response profiles of the GSH and hGSH contents in roots of non-nodulated and nodulated plants to the various hormonal treatments indicate that thiol homeostasis is independent of the nitrogen source of the plants. The differential regulation of the three mRNA levels, hGSHS activity, and thiol contents by hormones indicates a fine control of thiol biosynthesis at multiple levels and strongly suggests that GSH and hGSH play distinct roles in plant development and stress responses. PMID:22442424

Nucleocytoplasmic Transport of RNAs and RNA-Protein Complexes.

PubMed

Sloan, Katherine E; Gleizes, Pierre-Emmanuel; Bohnsack, Markus T

2016-05-22

RNAs and ribonucleoprotein complexes (RNPs) play key roles in mediating and regulating gene expression. In eukaryotes, most RNAs are transcribed, processed and assembled with proteins in the nucleus and then either function in the cytoplasm or also undergo a cytoplasmic phase in their biogenesis. This compartmentalization ensures that sequential steps in gene expression and RNP production are performed in the correct order and it allows important quality control mechanisms that prevent the involvement of aberrant RNAs/RNPs in these cellular pathways. The selective exchange of RNAs/RNPs between the nucleus and cytoplasm is enabled by nuclear pore complexes, which function as gateways between these compartments. RNA/RNP transport is facilitated by a range of nuclear transport receptors and adaptors, which are specifically recruited to their cargos and mediate interactions with nucleoporins to allow directional translocation through nuclear pore complexes. While some transport factors are only responsible for the export/import of a certain class of RNA/RNP, others are multifunctional and, in the case of large RNPs, several export factors appear to work together to bring about export. Recent structural studies have revealed aspects of the mechanisms employed by transport receptors to enable specific cargo recognition, and genome-wide approaches have provided the first insights into the diverse composition of pre-mRNPs during export. Furthermore, the regulation of RNA/RNP export is emerging as an important means to modulate gene expression under stress conditions and in disease. Copyright © 2015. Published by Elsevier Ltd.

Thiol synthetases of legumes: immunogold localization and differential gene regulation by phytohormones.

PubMed

Clemente, Maria R; Bustos-Sanmamed, Pilar; Loscos, Jorge; James, Euan K; Pérez-Rontomé, Carmen; Navascués, Joaquín; Gay, Marina; Becana, Manuel

2012-06-01

In plants and other organisms, glutathione (GSH) biosynthesis is catalysed sequentially by γ-glutamylcysteine synthetase (γECS) and glutathione synthetase (GSHS). In legumes, homoglutathione (hGSH) can replace GSH and is synthesized by γECS and a specific homoglutathione synthetase (hGSHS). The subcellular localization of the enzymes was examined by electron microscopy in several legumes and gene expression was analysed in Lotus japonicus plants treated for 1-48 h with 50 μM of hormones. Immunogold localization studies revealed that γECS is confined to chloroplasts and plastids, whereas hGSHS is also in the cytosol. Addition of hormones caused differential expression of thiol synthetases in roots. After 24-48 h, abscisic and salicylic acids downregulated GSHS whereas jasmonic acid upregulated it. Cytokinins and polyamines activated GSHS but not γECS or hGSHS. Jasmonic acid elicited a coordinated response of the three genes and auxin induced both hGSHS expression and activity. Results show that the thiol biosynthetic pathway is compartmentalized in legumes. Moreover, the similar response profiles of the GSH and hGSH contents in roots of non-nodulated and nodulated plants to the various hormonal treatments indicate that thiol homeostasis is independent of the nitrogen source of the plants. The differential regulation of the three mRNA levels, hGSHS activity, and thiol contents by hormones indicates a fine control of thiol biosynthesis at multiple levels and strongly suggests that GSH and hGSH play distinct roles in plant development and stress responses.

Simulated Space Radiation: Murine Skeletal Responses During Recovery and with Mechanical Stimulation

NASA Technical Reports Server (NTRS)

Shirazi-Fard, Yasaman; Zaragoza, Josergio; Schreurs, Ann-Sofie; Truong, Tiffany; Tahimic, Candice; Alwood, Joshua S.; Castillo, Alesha B.; Globus, R. K.

2016-01-01

Simulated space radiation at doses similar to those of solar particle events or a round-trip sojourn to Mars (1-2Gy) may cause skeletal tissue degradation and deplete stem/progenitor cell pools throughout the body. We hypothesized that simulated space radiation (SSR) causes late, time-dependent deficits in bone structure and bone cell function reflected by changes in gene expression in response to anabolic stimuli. We used a unique sequential dual ion exposure (proton and iron) for SSR to investigate time-dependence of responses in gene expression, cell function, and microarchitecture with respect to radiation and an anabolic stimulus of axial loading (AL). Male 16-wk C57BL6/J mice (n=120 total) were exposed to 0Gy (Sham, n=10), 56Fe (2Gy, positive control dose, n=10), or sequential ions for SSR (1Gy 1H/56Fe/1H, n=10) by total body irradiation (IR), and the tissues were harvested 2 or 6 mo. later. Further, to assess the response to anabolic stimuli, we subjected additional Sham-AL (n=15) and SSR-AL (n=15) groups to rest-inserted tibial axial loading (AL) starting at 1 and 5 months post-IR (-9N, 60 cycles/day, 3 days/wk, 4 wks). Exposure to 56Fe caused a significant reduction in cancellous bone volume fraction (BV/TV) compared to Sham (-34%) and SSR (-20%) in the proximal tibia metaphysis at 2-months post-IR; however BV/TV for SSR group was not different than Sham. Both 56Fe and SSR caused significant reduction in trabecular number (Tb.N) compared to Sham (-33% and -16%, respectively). Further, Tb.N for 56Fe (2Gy) was significantly lower than SSR (-21%). Ex vivo culture of marrow cells to assess growth and differentiation of osteoblast lineage cells 6 months post-IR showed that both 56Fe and SSR exposures significantly impaired colony formation compared to Sham (-66% and -54%, respectively), as well as nodule mineralization (-90% and -51%, respectively). Two-way analysis of variance showed that both mechanical loading and radiation reduced BV/TV, mechanical loading reduced trabecular thickness (Tb.Th), and radiation reduced Tb.N, at both time points. To assess acute response to mechanical stimuli, samples were harvested from a subset of Sham-AL (n=5) and SSR-AL (n=5) to measure changes in gene expression levels. Preliminary results indicate that axial loading increased expression of the antioxidant response gene Nfe2l2 and the osteoprogenitor-associated marker Runx2 in the bone marrow cells, and there was an interaction effect between axial loading and radiation at 2-months post-IR. Additional analyses of gene expression levels in the mineralized tissue are in progress. Results indicate that SSR caused persistent impairment of osteoblast colony formation and nodule mineralization 6-mo post-IR. Contrary to our hypothesis, simulated space radiation did not impair the ability of cancellous bone to respond to a mechanical anabolic stimulus, consistent with our previous findings [1]. Hence, compressive loading may be a potential countermeasure against spaceflight-induced bone loss.

Simulated Space Radiation: Murine Skeletal Responses During Recovery and with Mechanical Stimulation

NASA Technical Reports Server (NTRS)

Shirazi-Fard, Yasaman; Zaragoza, Josergio; Schreurs, Ann-Sofie; Truong, Tiffany; Tahimic, Candice; Alwood, Joshua S.; Globus, R. K.

2016-01-01

Simulated space radiation at doses similar to those of solar particle events or a round-trip sojourn to Mars (1-2Gy) may cause skeletal tissue degradation and deplete stem/progenitor cell pools throughout the body. We hypothesized that simulated space radiation (SSR) causes late, time-dependent deficits in bone structure and bone cell function reflected by changes in gene expression in response to anabolic stimuli. We used a unique sequential dual ion exposure (proton and iron) for SSR to investigate time-dependence of responses in gene expression, cell function, and microarchitecture with respect to radiation and an anabolic stimulus of axial loading (AL). Male 16-wk C57BL6/J mice (n=120 total) were exposed to 0Gy (Sham, n=10), 56Fe (2Gy, positive control dose, n=10), or sequential ions for SSR (1Gy 1H/56Fe/1H, n=10) by total body irradiation (IR), and the tissues were harvested 2 or 6 mo. later. Further, to assess the response to anabolic stimuli, we subjected additional Sham-AL (n=15) and SSR-AL (n=15) groups to rest-inserted tibial axial loading (AL) starting at 1 and 5 months post-IR (-9N, 60 cycles/day, 3 days/wk, 4 wks). Exposure to 56Fe caused a significant reduction in cancellous bone volume fraction (BV/TV) compared to Sham (-34%) and SSR (-20%) in the proximal tibia metaphysis at 2-months post-IR; however BV/TV for SSR group was not different than Sham. Both 56Fe and SSR caused significant reduction in trabecular number (Tb.N) compared to Sham (-33% and -16%, respectively). Further, Tb.N for 56Fe (2Gy) was significantly lower than SSR (-21%). Ex vivo culture of marrow cells to assess growth and differentiation of osteoblast lineage cells 6 months post-IR showed that both 56Fe and SSR exposures significantly impaired colony formation compared to Sham (-66% and -54%, respectively), as well as nodule mineralization (-90% and -51%, respectively). Two-way analysis of variance showed that both mechanical loading and radiation reduced BV/TV, mechanical loading reduced trabecular thickness (Tb.Th), and radiation reduced Tb.N, at both time points. To assess acute response to mechanical stimuli, samples were harvested from a subset of Sham-AL (n=5) and SSR-AL (n=5) to measure changes in gene expression levels. Preliminary results indicate that axial loading increased expression of the antioxidant response gene Nfe2l2 and the osteoprogenitor-associated marker Runx2 in the bone marrow cells, and there was an interaction effect between axial loading and radiation at 2-months post-IR. Additional analyses of gene expression levels in the mineralized tissue are in progress. Results indicate that SSR caused persistent impairment of osteoblast colony formation and nodule mineralization 6-mo post-IR. Contrary to our hypothesis, simulated space radiation did not impair the ability of cancellous bone to respond to a mechanical anabolic stimulus, consistent with our previous findings. Hence, compressive loading may be a potential countermeasure against spaceflight-induced bone loss.

Expression and in vitro functional analyses of recombinant Gam1 protein

PubMed Central

Avila, Gustavo A.; Ramirez, Daniel H.; Hildenbrand, Zacariah L.; Jacquez, Pedro; Chiocca, Susanna; Sun, Jianjun; Rosas-Acosta, German; Xiao, Chuan

2014-01-01

Gam1, an early gene product of an avian adenovirus, is essential for viral replication. Gam1 is the first viral protein found to globally inhibit cellular SUMOylation, a critical posttranslational modification that alters the function and cellular localization of proteins. The interaction details at the interface between Gam1 and its cellular targets remain unclear due to the lack of structural information. Although Gam1 has been previously characterized, the purity of the protein was not suitable for structural investigations. In the present study, the gene of Gam1 was cloned and expressed in various bacterial expression systems to obtain pure and soluble recombinant Gam1 protein for in vitro functional and structural studies. While Gam1 was insoluble in most expression systems tested, it became soluble when it was expressed as a fusion protein with trigger factor (TF), a ribosome associated bacterial chaperone, under the control of a cold shock promoter. Careful optimization indicates that both low temperature induction and the chaperone function of TF play critical roles in increasing Gam1 solubility. Soluble Gam1 was purified to homogeneity through sequential chromatography techniques. Monomeric Gam1 was obtained via size exclusion chromatography and analyzed by dynamic light scattering. The SUMOylation inhibitory function of the purified Gam1 was confirmed in an in vitro assay. These results have built the foundation for further structural investigations that will broaden our understanding of Gam1’s roles in viral replication. PMID:25450237

Expression and in vitro functional analyses of recombinant Gam1 protein.

PubMed

Avila, Gustavo A; Ramirez, Daniel H; Hildenbrand, Zacariah L; Jacquez, Pedro; Chiocca, Susanna; Sun, Jianjun; Rosas-Acosta, German; Xiao, Chuan

2015-01-01

Gam1, an early gene product of an avian adenovirus, is essential for viral replication. Gam1 is the first viral protein found to globally inhibit cellular SUMOylation, a critical posttranslational modification that alters the function and cellular localization of proteins. The interaction details at the interface between Gam1 and its cellular targets remain unclear due to the lack of structural information. Although Gam1 has been previously characterized, the purity of the protein was not suitable for structural investigations. In the present study, the gene of Gam1 was cloned and expressed in various bacterial expression systems to obtain pure and soluble recombinant Gam1 protein for in vitro functional and structural studies. While Gam1 was insoluble in most expression systems tested, it became soluble when it was expressed as a fusion protein with trigger factor (TF), a ribosome associated bacterial chaperone, under the control of a cold shock promoter. Careful optimization indicates that both low temperature induction and the chaperone function of TF play critical roles in increasing Gam1 solubility. Soluble Gam1 was purified to homogeneity through sequential chromatography techniques. Monomeric Gam1 was obtained via size exclusion chromatography and analyzed by dynamic light scattering. The SUMOylation inhibitory function of the purified Gam1 was confirmed in an in vitro assay. These results have built the foundation for further structural investigations that will broaden our understanding of Gam1's roles in viral replication. Copyright © 2014 Elsevier Inc. All rights reserved.

Repeated exposure to two stressors in sequence demonstrates that corticosterone and paraventricular nucleus of the hypothalamus interleukin-1β responses habituate independently.

PubMed

Lovelock, D F; Deak, T

2017-09-01

A wide range of stress-related pathologies such as post-traumatic stress disorder are considered to arise from aberrant or maladaptive forms of stress adaptation. The hypothalamic-pituitary-adrenal (HPA) axis readily adapts to repeated stressor exposure, yet little is known about adaptation in neuroimmune responses to repeated or sequential stress challenges. In Experiment 1, rats were exposed to 10 days of restraint alone (60 minutes daily), forced swim alone (30 minutes daily) or daily sequential exposure to restraint (60 minutes) followed immediately by forced swim (30 minutes), termed sequential stress exposure. Habituation of the corticosterone (CORT) response occurred to restraint by 5 days and swim at 10 days, whereas rats exposed to sequential stress exposure failed to display habituation to the combined challenge. Experiment 2 compared 1 or 5 days of forced swim with sequential stress exposure and examined how each affected expression of several neuroimmune and cellular activation genes in the paraventricular nucleus of the hypothalamus (PVN), prefrontal cortex (PFC) and hippocampus (HPC). Sequential exposure to restraint and swim increased interleukin (IL)-1β in the PVN, an effect that was attenuated after 5 days. Sequential stress exposure also elicited IL-6 and tumour necrosis factor-α responses in the HPC and PFC, respectively, which did not habituate after 5 days. Experiment 3 tested whether prior habituation to restraint (5 days) would alter the IL-1β response evoked by swim exposure imposed immediately after the sixth day of restraint. Surprisingly, a history of repeated exposure to restraint attenuated the PVN IL-1β response after swim in comparison to acutely-exposed subjects despite an equivalent CORT response. Overall, these findings suggest that habituation of neuroimmune responses to stress proceeds: (i) independent of HPA axis habituation; (ii) likely requires more daily sessions of stress to develop; and (iii) IL-1β displays a greater tendency to habituate after repeated stress challenges compared to other stress-reactive cytokines. © 2017 British Society for Neuroendocrinology.

Mutations in spalt cause a severe but reversible neurodegenerative phenotype in the embryonic central nervous system of Drosophila melanogaster.

PubMed

Cantera, Rafael; Lüer, Karin; Rusten, Tor Erik; Barrio, Rosa; Kafatos, Fotis C; Technau, Gerhard M

2002-12-01

The gene spalt is expressed in the embryonic central nervous system of Drosophila melanogaster but its function in this tissue is still unknown. To investigate this question, we used a combination of techniques to analyse spalt mutant embryos. Electron microscopy showed that in the absence of spalt, the central nervous system cells are separated by enlarged extracellular spaces populated by membranous material at 60% of embryonic development. Surprisingly, the central nervous system from slightly older embryos (80% of development) exhibited almost wild-type morphology. An extensive survey by laser confocal microscopy revealed that the spalt mutant central nervous system has abnormal levels of particular cell adhesion and cytoskeletal proteins. Time-lapse analysis of neuronal differentiation in vitro, lineage analysis and transplantation experiments confirmed that the mutation causes cytoskeletal and adhesion defects. The data indicate that in the central nervous system, spalt operates within a regulatory pathway which influences the expression of the beta-catenin Armadillo, its ligand N-Cadherin, Notch, and the cell adhesion molecules Neuroglian, Fasciclin 2 and Fasciclin 3. Effects on the expression of these genes are persistent but many morphological aspects of the phenotype are transient, leading to the concept of sequential redundancy for stable organisation of the central nervous system.

Valproic Acid Induces Telomerase Reverse Transcriptase Expression during Cortical Development.

PubMed

Kim, Ki Chan; Choi, Chang Soon; Gonzales, Edson Luck T; Mabunga, Darine Froy N; Lee, Sung Hoon; Jeon, Se Jin; Hwangbo, Ram; Hong, Minha; Ryu, Jong Hoon; Han, Seol-Heui; Bahn, Geon Ho; Shin, Chan Young

2017-10-01

The valproic acid (VPA)-induced animal model is one of the most widely utilized environmental risk factor models of autism. Autism spectrum disorder (ASD) remains an insurmountable challenge among neurodevelopmental disorders due to its heterogeneity, unresolved pathological pathways and lack of treatment. We previously reported that VPA-exposed rats and cultured rat primary neurons have increased Pax6 expression during post-midterm embryonic development which led to the sequential upregulation of glutamatergic neuronal markers. In this study, we provide experimental evidence that telomerase reverse transcriptase (TERT), a protein component of ribonucleoproteins complex of telomerase, is involved in the abnormal components caused by VPA in addition to Pax6 and its downstream signals. In embryonic rat brains and cultured rat primary neural progenitor cells (NPCs), VPA induced the increased expression of TERT as revealed by Western blot, RT-PCR, and immunostainings. The HDAC inhibitor property of VPA is responsible for the TERT upregulation. Chromatin immunoprecipitation revealed that VPA increased the histone acetylation but blocked the HDAC1 binding to both Pax6 and Tert genes. Interestingly, the VPA-induced TERT overexpression resulted to sequential upregulations of glutamatergic markers such as Ngn2 and NeuroD1, and inter-synaptic markers such as PSD-95, α-CaMKII, vGluT1 and synaptophysin. Transfection of Tert siRNA reversed the effects of VPA in cultured NPCs confirming the direct involvement of TERT in the expression of those markers. This study suggests the involvement of TERT in the VPA-induced autistic phenotypes and has important implications for the role of TERT as a modulator of balanced neuronal development and transmission in the brain.

Development of Gene Therapeutics for Head and Neck Cancer in China: From Bench to Bedside.

PubMed

Guo, Wei; Song, Hao

2018-02-01

Head and neck cancer represents the seventh most common cancer worldwide. Although multidisciplinary sequential treatments have been used, there is still an urgent need for new treatment approaches that can effectively improve the outcomes of patients with advanced stages of head and neck cancer. Gene therapy is a rapidly evolving field in cancer therapy that has been shown to improve the efficacy of antitumor treatment. China is at the forefront in clinical trials and practice of gene therapy. Chinese researchers have mainly focused on gene therapeutics based on oncolytic virus and recombinant adenovirus expressing p53, antiangiogenesis factor or herpes simplex virus-thymidine kinase. Currently, two gene therapy drugs, Gendicine and Oncorine, have been marketed in China, and a number of upcoming gene therapy agents are under development for the treatment of head and neck cancer. Most gene therapy agents have demonstrated excellent tolerance. However, the therapeutic effects need further improvement. With current innovations in tumor biology and knowledge, gene therapy has great potential as a safe and effective anticancer treatment. In recent years, new gene therapy agents with promising effects have been incorporated into clinical trials in China. Thus, gene therapy may become an important part of anticancer therapy and is expected to improve the therapeutic effect of head and neck cancers in the near future.

Cloning and characterization of an 11S legumin, Car i 4, a major allergen in pecan.

PubMed

Sharma, Girdhari M; Irsigler, Andre; Dhanarajan, Pushparani; Ayuso, Rosalia; Bardina, Luda; Sampson, Hugh A; Roux, Kenneth H; Sathe, Shridhar K

2011-09-14

Among tree nut allergens, pecan allergens remain to be identified and characterized. The objective was to demonstrate the IgE-binding ability of pecan 11S legumin and characterize its sequential IgE-binding epitopes. The 11S legumin gene was amplified from a pecan cDNA library and expressed as a fusion protein in Escherichia coli. The native 11S legumin in pecan extract was identified by mass spectrometry/mass spectrometry (MS/MS). Sequential epitopes were determined by probing the overlapping peptides with three serum pools prepared from different patients' sera. A three-dimensional model was generated using almond legumin as a template and compared with known sequential epitopes on other allergenic tree nut homologues. Of 28 patients tested by dot blot, 16 (57%) bound to 11S legumin, designated Car i 4. MS/MS sequencing of native 11S legumin identified 33 kDa acidic and 20-22 kDa basic subunits. Both pecan and walnut seed protein extracts inhibited IgE binding to recombinant Car i 4, suggesting cross-reactivity with Jug r 4. Sequential epitope mapping results of Car i 4 revealed weak, moderate, and strong reactivity of serum pools against 10, 5, and 4 peptides, respectively. Seven peptides were recognized by all three serum pools, of which two were strongly reactive. The strongly reactive peptides were located in three discrete regions of the Car i 4 acidic subunit sequence (residues 118-132, 208-219, and 238-249). Homology modeling of Car i 4 revealed significant overlapping regions shared in common with other tree nut legumins.

SWI/SNF chromatin remodeling complex is critical for the expression of microphthalmia-associated transcription factor in melanoma cells

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

Vachtenheim, Jiri, E-mail: jivach@upn.anet.cz; Ondrusova, Lubica; Borovansky, Jan

2010-02-12

The microphthalmia-associated transcription factor (MITF) is required for melanocyte development, maintenance of the melanocyte-specific transcription, and survival of melanoma cells. MITF positively regulates expression of more than 25 genes in pigment cells. Recently, it has been demonstrated that expression of several MITF downstream targets requires the SWI/SNF chromatin remodeling complex, which contains one of the two catalytic subunits, Brm or Brg1. Here we show that the expression of MITF itself critically requires active SWI/SNF. In several Brm/Brg1-expressing melanoma cell lines, knockdown of Brg1 severely compromised MITF expression with a concomitant dowregulation of MITF targets and decreased cell proliferation. Although Brmmore » was able to substitute for Brg1 in maintaining MITF expression and melanoma cell proliferation, sequential knockdown of both Brm and Brg1 in 501mel cells abolished proliferation. In Brg1-null SK-MEL-5 melanoma cells, depletion of Brm alone was sufficient to abrogate MITF expression and cell proliferation. Chromatin immunoprecipitation confirmed the binding of Brg1 or Brm to the promoter of MITF. Together these results demonstrate the essential role of SWI/SNF for expression of MITF and suggest that SWI/SNF may be a promissing target in melanoma therapy.« less

Recombination and mutation of class II histocompatibility genes in wild mice.

PubMed

Wakeland, E K; Darby, B R

1983-12-01

We have compared the tryptic peptide fingerprints of the A alpha, A beta, E alpha, and E beta subunits encoded by four wild-derived H-2 complexes expressing A molecules closely related to Ak. The A molecules encoded by these Ak-related mice have A alpha and A beta subunits that differ from A alpha k and A beta k by less than 10% of their tryptic peptides. Comparisons among the four wild-derived A molecules suggested that these contemporary A alpha and A beta alleles arose by sequential mutational events from common ancestor A alpha and A beta alleles. These results suggest that A alpha and A beta may co-evolve as an A beta A alpha gene duplex in wild mice. Tryptic peptide fingerprint comparisons of the E beta gene linked to these Ak-related A beta A alpha gene duplexes indicate that two encode E beta d-like subunits, whereas another encodes an E beta s-like subunit. These results strongly suggest that the A beta A alpha duplex and E beta recombine in wild mouse populations. The significantly different evolutionary patterns exhibited by the class II genes encoding A vs E molecules are discussed.

Tissue Inhibitor of Metalloproteinase 1 Expression Associated with Gene Demethylation Confers Anoikis Resistance in Early Phases of Melanocyte Malignant Transformation1

PubMed Central

Ricca, Tatiana I; Liang, Gangning; Suenaga, Ana Paula M; Han, Sang W; Jones, Peter A; Jasiulionis, Miriam G

2009-01-01

Although anoikis resistance has been considered a hallmark of malignant phenotype, the causal relation between neoplastic transformation and anchorage-independent growth remains undefined. We developed an experimental model of murine melanocyte malignant transformation, where a melanocyte lineage (melan-a) was submitted to sequential cycles of anchorage blockade, resulting in progressive morphologic alterations, and malignant transformation. Throughout this process, cells corresponding to premalignant melanocytes and melanoma cell lines were established and show progressive anoikis resistance and increased expression of Timp1. In melan-a melanocytes, Timp1 expression is suppressed by DNA methylation as indicated by its reexpression after 5-aza-2′-deoxycytidine treatment. Methylation-sensitive single-nucleotide primer extension analysis showed increased demethylation in Timp1 in parallel with its expression along malignant transformation. Interestingly, TIMP1 expression has already been related with negative prognosis in some human cancers. Although described as a MMP inhibitor, this protein has been associated with apoptosis resistance in different cell types. Melan-a cells overexpressing Timp1 showed increased survival in suspension but were unable to form tumors in vivo, whereas Timp1-overexpressing melanoma cells showed reduced latency time for tumor appearance and increased metastatic potential. Here, we demonstrated for the first time an increment in Timp1 expression since the early phases of melanocyte malignant transformation, associated to a progressive gene demethylation, which confers anoikis resistance. In this way, Timp1 might be considered as a valued marker for melanocyte malignant transformation. PMID:19956395

Bioprospecting for Genes that Confer Biofuel Tolerance to Escherichia Coli Using a Genomic Library Approach

NASA Astrophysics Data System (ADS)

Tomko, Timothy

Microorganisms are capable of producing advanced biofuels that can be used as 'drop-in' alternatives to conventional liquid fuels. However, vital physiological processes and membrane properties are often disrupted by the presence of biofuel and limit the production yields. In order to make microbial biofuels a competitive fuel source, finding mechanisms for improving resistance to the toxic effects of biofuel production is vital. This investigation aims to identify resistance mechanisms from microorganisms that have evolved to withstand hydrocarbon-rich environments, such as those that thrive near natural oil seeps and in oil-polluted waters. First, using genomic DNA from Marinobacter aquaeolei, we constructed a transgenic library that we expressed in Escherichia coli. We exposed cells to inhibitory levels of pinene, a monoterpene that can serve as a jet fuel precursor with chemical properties similar to existing tactical fuels. Using a sequential strategy of a fosmid library followed by a plasmid library, we were able to isolate a region of DNA from the M. aquaeolei genome that conferred pinene tolerance when expressed in E. coli. We determined that a single gene, yceI, was responsible for the tolerance improvements. Overexpression of this gene placed no additional burden on the host. We also tested tolerance to other monoterpenes and showed that yceI selectively improves tolerance. Additionally, we used genomic DNA from Pseudomonas putida KT2440, which has innate solvent-tolerance properties, to create transgenic libraries in an E. coli host. We exposed cells containing the library to pinene, selecting for genes that improved tolerance. Importantly, we found that expressing the sigma factor RpoD from P. putida greatly expanded the diversity of tolerance genes recovered. With low expression of rpoDP. putida, we isolated a single pinene tolerance gene; with increased expression of the sigma factor our selection experiments returned multiple distinct tolerance mechanisms, including some that have been previously documented and also new mechanisms. Interestingly, high levels of rpoDP. putida, induction resulted in decreased diversity. We found that the tolerance levels provided by some genes are highly sensitive to the level of induction of rpoD P. putida,, while others provide tolerance across a wide range of rpoDP. putida, levels. This method for unlocking diversity in tolerance screening using heterologous sigma factor expression was applicable to both plasmid and fosmid-based transgenic libraries. These results suggest that by controlling the expression of appropriate heterologous sigma factors, we can greatly increase the searchable genomic space within transgenic libraries. This dissertation describes a method of effectively screening genomic DNA from multiple organisms for genes to mitigate biofuel stress and shows how tolerance genes can improve bacterial growth in the presence of toxic biofuel compounds. These identified genes can be targeted in future studies as candidates for use in biofuel production strains to increase biofuel yields.

Does the endometrial gene expression of fertile women vary within and between cycles?

PubMed

Evans, Gloria E; Phillipson, Gregory T M; Sykes, Peter H; McNoe, Les A; Print, Cristin G; Evans, John J

2018-01-23

Does gene expression of putative endometrial implantation markers vary in expression between menstrual cycles? In fertile women the expression of certain genes exhibits a pattern of stable regulation.which is not affected even when sampled twice in one cycle. Successful implantation occurs in a minority of IVF embryo transfers. In contrast to knowledge regarding the ovulatory process, there is a sparse understanding of endometrial genes critical to implantation. This lack of knowledge hinders progress in this field. Endometrial pipelle samples were collected based on blood endocrinological markers at 2 and 7 days post initial LH surge. Five samples were collected over four cycles where the interval between collections ranged from sequential months to three years. Six fertile women attending an IVF clinic for male factor infertility, had samples collected. Global gene expression profiles were obtained from laser-microdissected, endometrial glands and stroma. Nineteen potential proliferation, cytokine and adhesion markers based on previous validated reports were studied. There was a significant modification between LH+2 and LH+7 of expression for 23 genes-11 in 8 in glands and stroma, 4 in stroma only and 3 in glands only suggesting stable, controlled regulation. Nevertheless, genes exhibited individual characteristics, e.g MKI67 exhibited lower expression at LH+7 than LH+2 and CCL4 higher, whereas TRO expressed limited difference in both cell types. Stability between cycles was demonstrated for gene expression at both LH+2-more than 60% of genes had <25% variation and at LH+7-60% had <30% variation. Further, effects of prior collection of an LH+2 sample on gene expression at LH+7 were not detected. The range of mRNA expression suggested that a clinical/diagnostic sample at LH+2 and LH+7 is likely to be a better index of endometrial function than a single sample. The possibility of redundancy suggests a panel would be more informative than a single marker. Raw and normalized microarray data have been deposited with the EMBL's European Genome-Phenome Archive for collaborative analysis, reference ega-box-815 (Lappalainen I, Almeida-King J, Kumanduri V, Senf A, Spalding JD, Ur-Rehman S, Saunders G, Kandasamy J, Caccamo M, Leinonen R et al. The European Genome-phenome Archive of human data consented for biomedical research. Nat Genet 2015;47:692-695.) [https://www.ebi.ac.uk/ega/home]. This type of research has difficulties of recruitment of fertile women for multiple blood testing and repeat endometrial biopsies. Therefore, these data had decreased statistical power due to the overall participant numbers. However, the inclusion of four cycles for each participant permitted the aim of obtaining information on intercycle and intracycle variability to be achieved. Our results support the feasibility of a clinical means of identification of a functional receptive endometrium. The robustness of data from individual women suggests that samples from one cycle can generally be applied to subsequent cycles. Funding was granted from the Tertiary Education Commission of New Zealand, Contract I.D.:UOOX06007. There are no competing interests. © The Author(s) 2018. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Systematically labeling developmental stage-specific genes for the study of pancreatic β-cell differentiation from human embryonic stem cells.

PubMed

Liu, Haisong; Yang, Huan; Zhu, Dicong; Sui, Xin; Li, Juan; Liang, Zhen; Xu, Lei; Chen, Zeyu; Yao, Anzhi; Zhang, Long; Zhang, Xi; Yi, Xing; Liu, Meng; Xu, Shiqing; Zhang, Wenjian; Lin, Hua; Xie, Lan; Lou, Jinning; Zhang, Yong; Xi, Jianzhong; Deng, Hongkui

2014-10-01

The applications of human pluripotent stem cell (hPSC)-derived cells in regenerative medicine has encountered a long-standing challenge: how can we efficiently obtain mature cell types from hPSCs? Attempts to address this problem are hindered by the complexity of controlling cell fate commitment and the lack of sufficient developmental knowledge for guiding hPSC differentiation. Here, we developed a systematic strategy to study hPSC differentiation by labeling sequential developmental genes to encompass the major developmental stages, using the directed differentiation of pancreatic β cells from hPSCs as a model. We therefore generated a large panel of pancreas-specific mono- and dual-reporter cell lines. With this unique platform, we visualized the kinetics of the entire differentiation process in real time for the first time by monitoring the expression dynamics of the reporter genes, identified desired cell populations at each differentiation stage and demonstrated the ability to isolate these cell populations for further characterization. We further revealed the expression profiles of isolated NGN3-eGFP(+) cells by RNA sequencing and identified sushi domain-containing 2 (SUSD2) as a novel surface protein that enriches for pancreatic endocrine progenitors and early endocrine cells both in human embryonic stem cells (hESC)-derived pancreatic cells and in the developing human pancreas. Moreover, we captured a series of cell fate transition events in real time, identified multiple cell subpopulations and unveiled their distinct gene expression profiles, among heterogeneous progenitors for the first time using our dual reporter hESC lines. The exploration of this platform and our new findings will pave the way to obtain mature β cells in vitro.

Setting the Tempo in Development: An Investigation of the Zebrafish Somite Clock Mechanism

PubMed Central

Giudicelli, François; Özbudak, Ertuğrul M; Wright, Gavin J; Lewis, Julian

2007-01-01

The somites of the vertebrate embryo are clocked out sequentially from the presomitic mesoderm (PSM) at the tail end of the embryo. Formation of each somite corresponds to one cycle of oscillation of the somite segmentation clock—a system of genes whose expression switches on and off periodically in the cells of the PSM. We have previously proposed a simple mathematical model explaining how the oscillations, in zebrafish at least, may be generated by a delayed negative feedback loop in which the products of two Notch target genes, her1 and her7, directly inhibit their own transcription, as well as that of the gene for the Notch ligand DeltaC; Notch signalling via DeltaC keeps the oscillations of neighbouring cells in synchrony. Here we subject the model to quantitative tests. We show how to read temporal information from the spatial pattern of stripes of gene expression in the anterior PSM and in this way obtain values for the biosynthetic delays and molecular lifetimes on which the model critically depends. Using transgenic lines of zebrafish expressing her1 or her7 under heat-shock control, we confirm the regulatory relationships postulated by the model. From the timing of somite segmentation disturbances following a pulse of her7 misexpression, we deduce that although her7 continues to oscillate in the anterior half of the PSM, it governs the future somite segmentation behaviour of the cells only while they are in the posterior half. In general, the findings strongly support the mathematical model of how the somite clock works, but they do not exclude the possibility that other oscillator mechanisms may operate upstream from the her7/her1 oscillator or in parallel with it. PMID:17535112

Specific repression of β-globin promoter activity by nuclear ferritin

PubMed Central

Broyles, Robert H.; Belegu, Visar; DeWitt, Christina R.; Shah, Sandeep N.; Stewart, Charles A.; Pye, Quentin N.; Floyd, Robert A.

2001-01-01

Developmental hemoglobin switching involves sequential globin gene activations and repressions that are incompletely understood. Earlier observations, described herein, led us to hypothesize that nuclear ferritin is a repressor of the adult β-globin gene in embryonic erythroid cells. Our data show that a ferritin-family protein in K562 cell nuclear extracts binds specifically to a highly conserved CAGTGC motif in the β-globin promoter at −153 to −148 bp from the cap site, and mutation of the CAGTGC motif reduces binding 20-fold in competition gel-shift assays. Purified human ferritin that is enriched in ferritin-H chains also binds the CAGTGC promoter segment. Expression clones of ferritin-H markedly repress β-globin promoter-driven reporter gene expression in cotransfected CV-1 cells in which the β-promoter has been stimulated with the transcription activator erythroid Krüppel-like factor (EKLF). We have constructed chloramphenicol acetyltransferase reporter plasmids containing either a wild-type or mutant β-globin promoter for the −150 CAGTGC motif and have compared the constructs for susceptibility to repression by ferritin-H in cotransfection assays. We find that stimulation by cotransfected EKLF is retained with the mutant promoter, whereas repression by ferritin-H is lost. Thus, mutation of the −150 CAGTGC motif not only markedly reduces in vitro binding of nuclear ferritin but also abrogates the ability of expressed ferritin-H to repress this promoter in our cell transfection assay, providing a strong link between DNA binding and function, and strong support for our proposal that nuclear ferritin-H is a repressor of the human β-globin gene. Such a repressor could be helpful in treating sickle cell and other genetic diseases. PMID:11481480

Effects of cellular origin on differentiation of human induced pluripotent stem cell–derived endothelial cells

PubMed Central

Zhao, Ming-Tao; Jahanbani, Fereshteh; Lee, Won Hee; Snyder, Michael P.

2016-01-01

Human induced pluripotent stem cells (iPSCs) can be derived from various types of somatic cells by transient overexpression of 4 Yamanaka factors (OCT4, SOX2, C-MYC, and KLF4). Patient-specific iPSC derivatives (e.g., neuronal, cardiac, hepatic, muscular, and endothelial cells [ECs]) hold great promise in drug discovery and regenerative medicine. In this study, we aimed to evaluate whether the cellular origin can affect the differentiation, in vivo behavior, and single-cell gene expression signatures of human iPSC–derived ECs. We derived human iPSCs from 3 types of somatic cells of the same individuals: fibroblasts (FB-iPSCs), ECs (EC-iPSCs), and cardiac progenitor cells (CPC-iPSCs). We then differentiated them into ECs by sequential administration of Activin, BMP4, bFGF, and VEGF. EC-iPSCs at early passage (10 < P < 20) showed higher EC differentiation propensity and gene expression of EC-specific markers (PECAM1 and NOS3) than FB-iPSCs and CPC-iPSCs. In vivo transplanted EC-iPSC–ECs were recovered with a higher percentage of CD31+ population and expressed higher EC-specific gene expression markers (PECAM1, KDR, and ICAM) as revealed by microfluidic single-cell quantitative PCR (qPCR). In vitro EC-iPSC–ECs maintained a higher CD31+ population than FB-iPSC–ECs and CPC-iPSC–ECs with long-term culturing and passaging. These results indicate that cellular origin may influence lineage differentiation propensity of human iPSCs; hence, the somatic memory carried by early passage iPSCs should be carefully considered before clinical translation. PMID:27398408

A Strontium-Modified Titanium Surface Produced by a New Method and Its Biocompatibility In Vitro

PubMed Central

Liu, Chundong; Zhang, Yanli; Wang, Lichao; Zhang, Xinhua; Chen, Qiuyue; Wu, Buling

2015-01-01

Objective To present a new and effective method of producing titanium surfaces modified with strontium and to investigate the surface characteristics and in vitro biocompatibility of titanium (Ti) surfaces modified with strontium (Sr) for bone implant applications. Materials and Methods Sr-modified Ti surfaces were produced by sequential treatments with NaOH, strontium acetate, heat and water. The surface characteristics and the concentration of the Sr ions released from the samples were examined. Cell adhesion, morphology and growth were investigated using osteoblasts isolated from the calvaria of neonatal Sprague-Dawley rats. Expression of osteogenesis-related genes and proteins was examined to assess the effect of the Sr-modified Ti surfaces on osteoblasts. Results The modified titanium surface had a mesh structure with significantly greater porosity, and approximately5.37±0.35at.% of Sr was incorporated into the surface. The hydrophilicity was enhanced by the incorporation of Sr ions and water treatment. The average amounts of Sr released from the Sr-modified plates subjected to water treatment were slight higher than the plates without water treatment. Sr promoted cellular adhesion, spreading and growth compared with untreated Ti surfaces. The Sr-modified Ti plates also promoted expression of osteogenesis-related genes,and expression of OPN and COL-І by osteoblasts. Ti plates heat treated at 700°C showed increased bioactivity in comparison with those treated at 600°C. Water treatment upregulated the expression of osteogenesis-related genes. Conclusions These results show that Sr-modification of Ti surfaces may improve bioactivity in vitro. Water treatment has enhanced the response of osteoblasts. The Sr-modified Ti heat-treated at 700°C exhibited better bioactivity compared with that heated at 600°C. PMID:26529234

A Strontium-Modified Titanium Surface Produced by a New Method and Its Biocompatibility In Vitro.

PubMed

Liu, Chundong; Zhang, Yanli; Wang, Lichao; Zhang, Xinhua; Chen, Qiuyue; Wu, Buling

2015-01-01

To present a new and effective method of producing titanium surfaces modified with strontium and to investigate the surface characteristics and in vitro biocompatibility of titanium (Ti) surfaces modified with strontium (Sr) for bone implant applications. Sr-modified Ti surfaces were produced by sequential treatments with NaOH, strontium acetate, heat and water. The surface characteristics and the concentration of the Sr ions released from the samples were examined. Cell adhesion, morphology and growth were investigated using osteoblasts isolated from the calvaria of neonatal Sprague-Dawley rats. Expression of osteogenesis-related genes and proteins was examined to assess the effect of the Sr-modified Ti surfaces on osteoblasts. The modified titanium surface had a mesh structure with significantly greater porosity, and approximately5.37±0.35at.% of Sr was incorporated into the surface. The hydrophilicity was enhanced by the incorporation of Sr ions and water treatment. The average amounts of Sr released from the Sr-modified plates subjected to water treatment were slight higher than the plates without water treatment. Sr promoted cellular adhesion, spreading and growth compared with untreated Ti surfaces. The Sr-modified Ti plates also promoted expression of osteogenesis-related genes,and expression of OPN and COL-І by osteoblasts. Ti plates heat treated at 700°C showed increased bioactivity in comparison with those treated at 600°C. Water treatment upregulated the expression of osteogenesis-related genes. These results show that Sr-modification of Ti surfaces may improve bioactivity in vitro. Water treatment has enhanced the response of osteoblasts. The Sr-modified Ti heat-treated at 700°C exhibited better bioactivity compared with that heated at 600°C.

Gene expression and production of tumor necrosis factor alpha, interleukin 1, interleukin 6, and gamma interferon in C3H/HeN and C57BL/6N mice in acute Mycoplasma pulmonis disease.

PubMed Central

Faulkner, C B; Simecka, J W; Davidson, M K; Davis, J K; Schoeb, T R; Lindsey, J R; Everson, M P

1995-01-01

Studies were conducted to determine whether the production of various cytokines is associated with Mycoplasma pulmonis disease expression. Susceptible C3H/HeN and resistant C57BL/6N mice were inoculated intranasally with 10(7) CFU of virulent M. pulmonis UAB CT or avirulent M. pulmonis UAB T. Expression of genes for tumor necrosis factor alpha (TNF-alpha), interleukin 1 alpha (IL-1 alpha), IL-1 beta, IL-6, and gamma interferon (IFN-gamma) in whole lung tissue and TNF-alpha gene expression in bronchoalveolar lavage (BAL) cells was determined by reverse transcription-PCR using specific cytokine primers at various times postinoculation. In addition, concentrations of TNF-alpha, IL-1, IL-6, and IFN-gamma were determined in BAL fluid and serum samples at various times postinoculation. Our results showed that there was a sequential appearance of cytokines in the lungs of infected mice: TNF-alpha, produced primarily by BAL cells, appeared first, followed by IL-1 and IL-6, which were followed by IFN-gamma. Susceptible C3H/HeN mice had higher and more persistent concentrations of TNF-alpha and IL-6 in BAL fluid than did resistant C57BL/6N mice, indicating that TNF-alpha and possibly IL-6 are important factors in pathogenesis of acute M. pulmonis disease in mice. Serum concentrations of IL-6 were elevated in C3H/HeN mice, but not C57BL/6N mice, following infection with M. pulmonis, suggesting that IL-6 has both local and systemic effects in M. pulmonis disease. PMID:7558323

Spatiotemporal transcriptome provides insights into early fruit development of tomato (Solanum lycopersicum).

PubMed

Zhang, Shuaibin; Xu, Meng; Qiu, Zhengkun; Wang, Ketao; Du, Yongchen; Gu, Lianfeng; Cui, Xia

2016-03-18

Early fruit development is crucial for crop production in tomato. After fertilization, the ovary undergoes cell division and cell expansion before maturation. Although the roles of regulatory signals such as hormone and carbohydrate during early fruit development have been studied, the spatial distribution and the sequential initiation of these regulatory signals still need to be explored. Using the tomato cultivar 'Moneymaker', we analyzed the transcriptome of the ovule and the ovary wall/pericarp dissected from four different stages of the early developing fruits by stereoscope. These datasets give us the whole picture about the spatial and temporal signal distribution in early development of ovule and pericarp. Our results indicate that the hormone signal was initiated in both ovule and pericarp after fertilization. After that, different signals were activated in ovule and pericarp due to their distinct developmental processes. Our study provides spatiotemporal regulatory landscape of gene expression with sequential information which was not studied by previous work and further strengthens the comprehension of the regulatory and metabolic events controlling early fruit development.

Contrasting roles for MyoD in organizing myogenic promoter structures during embryonic skeletal muscle development.

PubMed

Cho, Ok Hyun; Mallappa, Chandrashekara; Hernández-Hernández, J Manuel; Rivera-Pérez, Jaime A; Imbalzano, Anthony N

2015-01-01

Among the complexities of skeletal muscle differentiation is a temporal distinction in the onset of expression of different lineage-specific genes. The lineage-determining factor MyoD is bound to myogenic genes at the onset of differentiation whether gene activation is immediate or delayed. How temporal regulation of differentiation-specific genes is established remains unclear. Using embryonic tissue, we addressed the molecular differences in the organization of the myogenin and muscle creatine kinase (MCK) gene promoters by examining regulatory factor binding as a function of both time and spatial organization during somitogenesis. At the myogenin promoter, binding of the homeodomain factor Pbx1 coincided with H3 hyperacetylation and was followed by binding of co-activators that modulate chromatin structure. MyoD and myogenin binding occurred subsequently, demonstrating that Pbx1 facilitates chromatin remodeling and modification before myogenic regulatory factor binding. At the same time, the MCK promoter was bound by HDAC2 and MyoD, and activating histone marks were largely absent. The association of HDAC2 and MyoD was confirmed by co-immunoprecipitation, proximity ligation assay (PLA), and sequential ChIP. MyoD differentially promotes activated and repressed chromatin structures at myogenic genes early after the onset of skeletal muscle differentiation in the developing mouse embryo. © 2014 Wiley Periodicals, Inc.

Assessing the Impacts of Experimentally Elevated Temperature on the Biological Composition and Molecular Chaperone Gene Expression of a Reef Coral

PubMed Central

Mayfield, Anderson B.; Wang, Li-Hsueh; Tang, Pei-Ciao; Fan, Tung-Yung; Hsiao, Yi-Yuong; Tsai, Ching-Lin; Chen, Chii-Shiarng

2011-01-01

Due to the potential for increasing ocean temperatures to detrimentally impact reef-building corals, there is an urgent need to better understand not only the coral thermal stress response, but also natural variation in their sub-cellular composition. To address this issue, while simultaneously developing a molecular platform for studying one of the most common Taiwanese reef corals, Seriatopora hystrix, 1,092 cDNA clones were sequenced and characterized. Subsequently, RNA, DNA and protein were extracted sequentially from colonies exposed to elevated (30°C) temperature for 48 hours. From the RNA phase, a heat shock protein-70 (hsp70)-like gene, deemed hsp/c, was identified in the coral host, and expression of this gene was measured with real-time quantitative PCR (qPCR) in both the host anthozoan and endosymbiotic dinoflagellates (genus Symbiodinium). While mRNA levels were not affected by temperature in either member, hsp/c expression was temporally variable in both and co-varied within biopsies. From the DNA phase, host and Symbiodinium hsp/c genome copy proportions (GCPs) were calculated to track changes in the biological composition of the holobiont during the experiment. While there was no temperature effect on either host or Symbiodinium GCP, both demonstrated significant temporal variation. Finally, total soluble protein was responsive to neither temperature nor exposure time, though the protein/DNA ratio varied significantly over time. Collectively, it appears that time, and not temperature, is a more important driver of the variation in these parameters, highlighting the need to consider natural variation in both gene expression and the molecular make-up of coral holobionts when conducting manipulative studies. This represents the first study to survey multiple macromolecules from both compartments of an endosymbiotic organism with methodologies that reflect their dual-compartmental nature, ideally generating a framework for assessing molecular-level changes within corals and other endosymbioses exposed to changes in their environment. PMID:22046302

Multi-Level Sequential Pattern Mining Based on Prime Encoding

NASA Astrophysics Data System (ADS)

Lianglei, Sun; Yun, Li; Jiang, Yin

Encoding is not only to express the hierarchical relationship, but also to facilitate the identification of the relationship between different levels, which will directly affect the efficiency of the algorithm in the area of mining the multi-level sequential pattern. In this paper, we prove that one step of division operation can decide the parent-child relationship between different levels by using prime encoding and present PMSM algorithm and CROSS-PMSM algorithm which are based on prime encoding for mining multi-level sequential pattern and cross-level sequential pattern respectively. Experimental results show that the algorithm can effectively extract multi-level and cross-level sequential pattern from the sequence database.

The spinocerebellar ataxias: order emerges from chaos.

PubMed

Margolis, Russell L

2002-09-01

In the past decade, the genetic etiologies accounting for most cases of adult-onset dominant cerebellar ataxia have been discovered. This group of disorders, generally referred to as the spinocerebellar ataxias (SCAs), can now be classified by a simple genetic nosology, essentially a sequential list in which each new SCA is given a number. However, recent advances in the elucidation of SCA pathogenesis provide the opportunity to subclassify the disorders into three discrete groups based on pathogenesis: 1) the polyglutamine disorders, SCAs 1, 2, 3, 7, and 17, which result from proteins with toxic stretches of polyglutamine; 2) the channelopathies, SCA6 and episodic ataxia types 1 and 2 (EA1 and EA2), which result from disruption of calcium or potassium channel function; and 3) the gene expression disorders, SCAs 8, 10, and 12, which result from repeat expansions outside of coding regions that may quantitatively alter gene expression. SCAs 4, 5, 9, 11, 13-16, 19, 21, and 22 are of unknown etiology, and may or may not fit into one of these three groups. At present, most diagnostic and therapeutic strategies apply equally to all of the SCAs. Therapy specific for individual diseases or types of diseases is a realistic goal in the foreseeable future.

A Clb/Cdk1-mediated regulation of Fkh2 synchronizes CLB expression in the budding yeast cell cycle.

PubMed

Linke, Christian; Chasapi, Anastasia; González-Novo, Alberto; Al Sawad, Istabrak; Tognetti, Silvia; Klipp, Edda; Loog, Mart; Krobitsch, Sylvia; Posas, Francesc; Xenarios, Ioannis; Barberis, Matteo

2017-01-01

Precise timing of cell division is achieved by coupling waves of cyclin-dependent kinase (Cdk) activity with a transcriptional oscillator throughout cell cycle progression. Although details of transcription of cyclin genes are known, it is unclear which is the transcriptional cascade that modulates their expression in a timely fashion. Here, we demonstrate that a Clb/Cdk1-mediated regulation of the Fkh2 transcription factor synchronizes the temporal mitotic CLB expression in budding yeast. A simplified kinetic model of the cyclin/Cdk network predicts a linear cascade where a Clb/Cdk1-mediated regulation of an activator molecule drives CLB3 and CLB2 expression. Experimental validation highlights Fkh2 as modulator of CLB3 transcript levels, besides its role in regulating CLB2 expression. A Boolean model based on the minimal number of interactions needed to capture the information flow of the Clb/Cdk1 network supports the role of an activator molecule in the sequential activation, and oscillatory behavior, of mitotic Clb cyclins. This work illustrates how transcription and phosphorylation networks can be coupled by a Clb/Cdk1-mediated regulation that synchronizes them.

Inhibition of GSK3β-mediated BACE1 expression reduces Alzheimer-associated phenotypes

PubMed Central

Ly, Philip T.T.; Wu, Yili; Zou, Haiyan; Wang, Ruitao; Zhou, Weihui; Kinoshita, Ayae; Zhang, Mingming; Yang, Yi; Cai, Fang; Woodgett, James; Song, Weihong

2012-01-01

Deposition of amyloid β protein (Aβ) to form neuritic plaques in the brain is the pathological hallmark of Alzheimer’s disease (AD). Aβ is generated from sequential cleavages of the β-amyloid precursor protein (APP) by the β- and γ-secretases, and β-site APP-cleaving enzyme 1 (BACE1) is the β-secretase essential for Aβ generation. Previous studies have indicated that glycogen synthase kinase 3 (GSK3) may play a role in APP processing by modulating γ-secretase activity, thereby facilitating Aβ production. There are two highly conserved isoforms of GSK3: GSK3α and GSK3β. We now report that specific inhibition of GSK3β, but not GSK3α, reduced BACE1-mediated cleavage of APP and Aβ production by decreasing BACE1 gene transcription and expression. The regulation of BACE1 gene expression by GSK3β was dependent on NF-κB signaling. Inhibition of GSK3 signaling markedly reduced Aβ deposition and neuritic plaque formation, and rescued memory deficits in the double transgenic AD model mice. These data provide evidence for regulation of BACE1 expression and AD pathogenesis by GSK3β and that inhibition of GSK3 signaling can reduce Aβ neuropathology and alleviate memory deficits in AD model mice. Our study suggests that interventions that specifically target the β-isoform of GSK3 may be a safe and effective approach for treating AD. PMID:23202730

Metabolite and transcriptome analysis during fasting suggest a role for the p53-Ddit4 axis in major metabolic tissues

PubMed Central

2013-01-01

Background Fasting induces specific molecular and metabolic adaptions in most organisms. In biomedical research fasting is used in metabolic studies to synchronize nutritional states of study subjects. Because there is a lack of standardization for this procedure, we need a deeper understanding of the dynamics and the molecular mechanisms in fasting. Results We investigated the dynamic changes of liver gene expression and serum parameters of mice at several time points during a 48 hour fasting experiment and then focused on the global gene expression changes in epididymal white adipose tissue (WAT) as well as on pathways common to WAT, liver, and skeletal muscle. This approach produced several intriguing insights: (i) rather than a sequential activation of biochemical pathways in fasted liver, as current knowledge dictates, our data indicates a concerted parallel response; (ii) this first characterization of the transcriptome signature of WAT of fasted mice reveals a remarkable activation of components of the transcription apparatus; (iii) most importantly, our bioinformatic analyses indicate p53 as central node in the regulation of fasting in major metabolic tissues; and (iv) forced expression of Ddit4, a fasting-regulated p53 target gene, is sufficient to augment lipolysis in cultured adipocytes. Conclusions In summary, this combination of focused and global profiling approaches provides a comprehensive molecular characterization of the processes operating during fasting in mice and suggests a role for p53, and its downstream target Ddit4, as novel components in the transcriptional response to food deprivation. PMID:24191950

Prolonged early G1 arrest by selective CDK4/CDK6 inhibition sensitizes myeloma cells to cytotoxic killing through cell cycle–coupled loss of IRF4

PubMed Central

Huang, Xiangao; Di Liberto, Maurizio; Jayabalan, David; Liang, Jun; Ely, Scott; Bretz, Jamieson; Shaffer, Arthur L.; Louie, Tracey; Chen, Isan; Randolph, Sophia; Hahn, William C.; Staudt, Louis M.; Niesvizky, Ruben; Moore, Malcolm A. S.

2012-01-01

Dysregulation of cyclin-dependent kinase 4 (CDK4) and CDK6 by gain of function or loss of inhibition is common in human cancer, including multiple myeloma, but success in targeting CDK with broad-spectrum inhibitors has been modest. By selective and reversible inhibition of CDK4/CDK6, we have developed a strategy to both inhibit proliferation and enhance cytotoxic killing of cancer cells. We show that induction of prolonged early-G1 arrest (pG1) by CDK4/CDK6 inhibition halts gene expression in early-G1 and prevents expression of genes programmed for other cell-cycle phases. Removal of the early-G1 block leads to S-phase synchronization (pG1-S) but fails to completely restore scheduled gene expression. Consequently, the IRF4 protein required to protect myeloma cells from apoptosis is markedly reduced in pG1 and further in pG1-S in response to cytotoxic agents, such as the proteasome inhibitor bortezomib. The coordinated loss of IRF4 and gain of Bim sensitize myeloma tumor cells to bortezomib-induced apoptosis in pG1 in the absence of Noxa and more profoundly in pG1-S in cooperation with Noxa in vitro. Induction of pG1 and pG1-S by reversible CDK4/CDK6 inhibition further augments tumor-specific bortezomib killing in myeloma xenografts. Reversible inhibition of CDK4/CDK6 in sequential combination therapy thus represents a novel mechanism-based cancer therapy. PMID:22718837

Prolonged early G(1) arrest by selective CDK4/CDK6 inhibition sensitizes myeloma cells to cytotoxic killing through cell cycle-coupled loss of IRF4.

PubMed

Huang, Xiangao; Di Liberto, Maurizio; Jayabalan, David; Liang, Jun; Ely, Scott; Bretz, Jamieson; Shaffer, Arthur L; Louie, Tracey; Chen, Isan; Randolph, Sophia; Hahn, William C; Staudt, Louis M; Niesvizky, Ruben; Moore, Malcolm A S; Chen-Kiang, Selina

2012-08-02

Dysregulation of cyclin-dependent kinase 4 (CDK4) and CDK6 by gain of function or loss of inhibition is common in human cancer, including multiple myeloma, but success in targeting CDK with broad-spectrum inhibitors has been modest. By selective and reversible inhibition of CDK4/CDK6, we have developed a strategy to both inhibit proliferation and enhance cytotoxic killing of cancer cells. We show that induction of prolonged early-G(1) arrest (pG1) by CDK4/CDK6 inhibition halts gene expression in early-G(1) and prevents expression of genes programmed for other cell-cycle phases. Removal of the early-G(1) block leads to S-phase synchronization (pG1-S) but fails to completely restore scheduled gene expression. Consequently, the IRF4 protein required to protect myeloma cells from apoptosis is markedly reduced in pG1 and further in pG1-S in response to cytotoxic agents, such as the proteasome inhibitor bortezomib. The coordinated loss of IRF4 and gain of Bim sensitize myeloma tumor cells to bortezomib-induced apoptosis in pG1 in the absence of Noxa and more profoundly in pG1-S in cooperation with Noxa in vitro. Induction of pG1 and pG1-S by reversible CDK4/CDK6 inhibition further augments tumor-specific bortezomib killing in myeloma xenografts. Reversible inhibition of CDK4/CDK6 in sequential combination therapy thus represents a novel mechanism-based cancer therapy.

Engineering improved bio-jet fuel tolerance in Escherichia coli using a transgenic library from the hydrocarbon-degrader Marinobacter aquaeolei.

PubMed

Tomko, Timothy A; Dunlop, Mary J

2015-01-01

Recent metabolic engineering efforts have generated microorganisms that can produce biofuels, including bio-jet fuels, however these fuels are often toxic to cells, limiting production yields. There are natural examples of microorganisms that have evolved mechanisms for tolerating hydrocarbon-rich environments, such as those that thrive near natural oil seeps and in oil-polluted waters. Using genomic DNA from the hydrocarbon-degrading microbe Marinobacter aquaeolei, we constructed a transgenic library that we expressed in Escherichia coli. We exposed cells to inhibitory levels of pinene, a monoterpene that can serve as a jet fuel precursor with chemical properties similar to existing tactical fuels. Using a sequential strategy with a fosmid library followed by a plasmid library, we were able to isolate a region of DNA from the M. aquaeolei genome that conferred pinene tolerance when expressed in E. coli. We determined that a single gene, yceI, was responsible for the tolerance improvements. Overexpression of this gene placed no additional burden on the host. We also tested tolerance to other monoterpenes and showed that yceI selectively improves tolerance. The genomes of hydrocarbon-tolerant microbes represent a rich resource for tolerance engineering. Using a transgenic library, we were able to identify a single gene that improves E. coli's tolerance to the bio-jet fuel precursor pinene.

Hierarchy of non-glucose sugars in Escherichia coli.

PubMed

Aidelberg, Guy; Towbin, Benjamin D; Rothschild, Daphna; Dekel, Erez; Bren, Anat; Alon, Uri

2014-12-24

Understanding how cells make decisions, and why they make the decisions they make, is of fundamental interest in systems biology. To address this, we study the decisions made by E. coli on which genes to express when presented with two different sugars. It is well-known that glucose, E. coli's preferred carbon source, represses the uptake of other sugars by means of global and gene-specific mechanisms. However, less is known about the utilization of glucose-free sugar mixtures which are found in the natural environment of E. coli and in biotechnology. Here, we combine experiment and theory to map the choices of E. coli among 6 different non-glucose carbon sources. We used robotic assays and fluorescence reporter strains to make precise measurements of promoter activity and growth rate in all pairs of these sugars. We find that the sugars can be ranked in a hierarchy: in a mixture of a higher and a lower sugar, the lower sugar system shows reduced promoter activity. The hierarchy corresponds to the growth rate supported by each sugar- the faster the growth rate, the higher the sugar on the hierarchy. The hierarchy is 'soft' in the sense that the lower sugar promoters are not completely repressed. Measurement of the activity of the master regulator CRP-cAMP shows that the hierarchy can be quantitatively explained based on differential activation of the promoters by CRP-cAMP. Comparing sugar system activation as a function of time in sugar pair mixtures at sub-saturating concentrations, we find cases of sequential activation, and also cases of simultaneous expression of both systems. Such simultaneous expression is not predicted by simple models of growth rate optimization, which predict only sequential activation. We extend these models by suggesting multi-objective optimization for both growing rapidly now and preparing the cell for future growth on the poorer sugar. We find a defined hierarchy of sugar utilization, which can be quantitatively explained by differential activation by the master regulator cAMP-CRP. The present approach can be used to understand cell decisions when presented with mixtures of conditions.

Distinct temporal roles for the promyelocytic leukaemia (PML) protein in the sequential regulation of intracellular host immunity to HSV-1 infection

PubMed Central

Alandijany, Thamir; Conn, Kristen L.; McFarlane, Steven; Orr, Anne

2018-01-01

Detection of viral nucleic acids plays a critical role in the induction of intracellular host immune defences. However, the temporal recruitment of immune regulators to infecting viral genomes remains poorly defined due to the technical difficulties associated with low genome copy-number detection. Here we utilize 5-Ethynyl-2’-deoxyuridine (EdU) labelling of herpes simplex virus 1 (HSV-1) DNA in combination with click chemistry to examine the sequential recruitment of host immune regulators to infecting viral genomes under low multiplicity of infection conditions. Following viral genome entry into the nucleus, PML-nuclear bodies (PML-NBs) rapidly entrapped viral DNA (vDNA) leading to a block in viral replication in the absence of the viral PML-NB antagonist ICP0. This pre-existing intrinsic host defence to infection occurred independently of the vDNA pathogen sensor IFI16 (Interferon Gamma Inducible Protein 16) and the induction of interferon stimulated gene (ISG) expression, demonstrating that vDNA entry into the nucleus alone is not sufficient to induce a robust innate immune response. Saturation of this pre-existing intrinsic host defence during HSV-1 ICP0-null mutant infection led to the stable recruitment of PML and IFI16 into vDNA complexes associated with ICP4, and led to the induction of ISG expression. This induced innate immune response occurred in a PML-, IFI16-, and Janus-Associated Kinase (JAK)-dependent manner and was restricted by phosphonoacetic acid, demonstrating that vDNA polymerase activity is required for the robust induction of ISG expression during HSV-1 infection. Our data identifies dual roles for PML in the sequential regulation of intrinsic and innate immunity to HSV-1 infection that are dependent on viral genome delivery to the nucleus and the onset of vDNA replication, respectively. These intracellular host defences are counteracted by ICP0, which targets PML for degradation from the outset of nuclear infection to promote vDNA release from PML-NBs and the onset of HSV-1 lytic replication. PMID:29309427

New genes in the evolution of the neural crest differentiation program

PubMed Central

2007-01-01

Background Development of the vertebrate head depends on the multipotency and migratory behavior of neural crest derivatives. This cell population is considered a vertebrate innovation and, accordingly, chordate ancestors lacked neural crest counterparts. The identification of neural crest specification genes expressed in the neural plate of basal chordates, in addition to the discovery of pigmented migratory cells in ascidians, has challenged this hypothesis. These new findings revive the debate on what is new and what is ancient in the genetic program that controls neural crest formation. Results To determine the origin of neural crest genes, we analyzed Phenotype Ontology annotations to select genes that control the development of this tissue. Using a sequential blast pipeline, we phylogenetically classified these genes, as well as those associated with other tissues, in order to define tissue-specific profiles of gene emergence. Of neural crest genes, 9% are vertebrate innovations. Our comparative analyses show that, among different tissues, the neural crest exhibits a particularly high rate of gene emergence during vertebrate evolution. A remarkable proportion of the new neural crest genes encode soluble ligands that control neural crest precursor specification into each cell lineage, including pigmented, neural, glial, and skeletal derivatives. Conclusion We propose that the evolution of the neural crest is linked not only to the recruitment of ancestral regulatory genes but also to the emergence of signaling peptides that control the increasingly complex lineage diversification of this plastic cell population. PMID:17352807

A damped oscillator imposes temporal order on posterior gap gene expression in Drosophila.

PubMed

Verd, Berta; Clark, Erik; Wotton, Karl R; Janssens, Hilde; Jiménez-Guri, Eva; Crombach, Anton; Jaeger, Johannes

2018-02-01

Insects determine their body segments in two different ways. Short-germband insects, such as the flour beetle Tribolium castaneum, use a molecular clock to establish segments sequentially. In contrast, long-germband insects, such as the vinegar fly Drosophila melanogaster, determine all segments simultaneously through a hierarchical cascade of gene regulation. Gap genes constitute the first layer of the Drosophila segmentation gene hierarchy, downstream of maternal gradients such as that of Caudal (Cad). We use data-driven mathematical modelling and phase space analysis to show that shifting gap domains in the posterior half of the Drosophila embryo are an emergent property of a robust damped oscillator mechanism, suggesting that the regulatory dynamics underlying long- and short-germband segmentation are much more similar than previously thought. In Tribolium, Cad has been proposed to modulate the frequency of the segmentation oscillator. Surprisingly, our simulations and experiments show that the shift rate of posterior gap domains is independent of maternal Cad levels in Drosophila. Our results suggest a novel evolutionary scenario for the short- to long-germband transition and help explain why this transition occurred convergently multiple times during the radiation of the holometabolan insects.

A damped oscillator imposes temporal order on posterior gap gene expression in Drosophila

PubMed Central

Verd, Berta; Clark, Erik; Wotton, Karl R.; Janssens, Hilde; Jiménez-Guri, Eva; Crombach, Anton

2018-01-01

Insects determine their body segments in two different ways. Short-germband insects, such as the flour beetle Tribolium castaneum, use a molecular clock to establish segments sequentially. In contrast, long-germband insects, such as the vinegar fly Drosophila melanogaster, determine all segments simultaneously through a hierarchical cascade of gene regulation. Gap genes constitute the first layer of the Drosophila segmentation gene hierarchy, downstream of maternal gradients such as that of Caudal (Cad). We use data-driven mathematical modelling and phase space analysis to show that shifting gap domains in the posterior half of the Drosophila embryo are an emergent property of a robust damped oscillator mechanism, suggesting that the regulatory dynamics underlying long- and short-germband segmentation are much more similar than previously thought. In Tribolium, Cad has been proposed to modulate the frequency of the segmentation oscillator. Surprisingly, our simulations and experiments show that the shift rate of posterior gap domains is independent of maternal Cad levels in Drosophila. Our results suggest a novel evolutionary scenario for the short- to long-germband transition and help explain why this transition occurred convergently multiple times during the radiation of the holometabolan insects. PMID:29451884

Identification of a novel long noncoding RNA that promotes osteoblast differentiation.

PubMed

Nardocci, Gino; Carrasco, Margarita E; Acevedo, Elvis; Hodar, Christian; Meneses, Claudio; Montecino, Martín

2018-05-28

Long noncoding RNAs (lncRNAs) are a heterogeneous class of transcripts, longer than 200 nucleotides, 5'-capped, polyadenylated, and poorly conserved among mammalian species. Several studies have shown the contribution of lncRNAs to different cellular processes, including regulation of the chromatin structure, control of messenger RNA translation, regulation of gene transcription, regulation of embryonic pluripotency, and differentiation. Although limited numbers of functional lncRNAs have been identified so far, the immense regulatory potential of these RNAs is already evident, indicating that a functional characterization of lncRNAs is needed. In this study, mouse preosteoblastic cells were induced to differentiate into osteoblasts. At 3 sequential differentiation stages, total RNA was isolated and libraries were constructed for Illumina sequencing. The resulting sequences were aligned and transcript abundances were determined. New lncRNA candidates that displayed differential expression patterns during osteoblast differentiation were identified by combining bioinformatics and reverse transcription polymerase chain reaction analyses. Among these, lncRNA-1 that exhibited increased expression during osteogenesis and was downregulated during myogenesis. Importantly, knockdown of lncRNA-1 expression in primary mouse preosteoblasts was found to inhibit osteogenic differentiation, reflected by a reduced transcription of the Runx2/p57 and Sp7 bone master genes. Together, our results indicate that lncRNA-1 represents a new regulatory RNA that plays a relevant role during the early stages of osteogenesis. © 2018 Wiley Periodicals, Inc.

Pea aphid infestation induces changes in flavonoids, antioxidative defence, soluble sugars and sugar transporter expression in leaves of pea seedlings.

PubMed

Morkunas, Iwona; Woźniak, Agnieszka; Formela, Magda; Mai, Van Chung; Marczak, Łukasz; Narożna, Dorota; Borowiak-Sobkowiak, Beata; Kühn, Christina; Grimm, Bernhard

2016-07-01

The perception of aphid infestation induces highly coordinated and sequential defensive reactions in plants at the cellular and molecular levels. The aim of the study was to explore kinetics of induced antioxidative defence responses in leaf cells of Pisum sativum L.cv. Cysterski upon infestation of the pea aphid Acyrthosiphon pisum at varying population sizes, including accumulation of flavonoids, changes of carbon metabolism, and expression of nuclear genes involved in sugar transport. Within the first 96 h, after A. pisum infestation, flavonoid accumulation and increased peroxidase activity were observed in leaves. The level of pisatin increased after 48 h of infestation and reached a maximum at 96 h. At this time point, a higher concentration of flavonols was observed in the infested tissue than in the control. Additionally, strong post-infestation accumulation of chalcone synthase (CHS) and isoflavone synthase (IFS) transcription products was also found. The levels of sucrose and fructose in 24-h leaves infested by 10, 20, and 30 aphids were significantly lower than in the control. Moreover, in leaves infested by 30 aphids, the reduced sucrose level observed up to 48 h was accompanied by a considerable increase in the expression level of the PsSUT1 gene encoding the sucrose transporter. In conclusion, A. pisum infestation on pea leads to stimulation of metabolic pathways associated with defence.

Human cytomegalovirus inhibits antigen presentation by a sequential multistep process.

PubMed Central

Ahn, K; Angulo, A; Ghazal, P; Peterson, P A; Yang, Y; Früh, K

1996-01-01

The human cytomegalovirus (HCMV) genomic unique short (US) region encodes a family of homologous genes essential for the inhibition of major histocompatibility complex (MHC) class I-mediated antigen presentation during viral infection. Here we show that US3, the only immediate early (IE) gene within the US region, encodes an endoplasmic reticulum-resident glycoprotein that prevents intracellular transport of MHC class I molecules. In contrast to the rapid degradation of newly synthesized MHC class I heavy chains mediated by the early gene product US11, we found that US3 retains stable MHC class I heterodimers in the endoplasmic reticulum that are loaded with peptides while retained in the ER. Consistent with the expression pattern of US3 and US11, MHC class I molecules are retained but not degraded during the IE period of infection. Our data identify the first nonregulatory role of an IE protein of HCMV and suggest that HCMV uses different T-cell escape strategies at different times during the infectious cycle. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8855296

Predicting ionizing radiation exposure using biochemically-inspired genomic machine learning.

PubMed

Zhao, Jonathan Z L; Mucaki, Eliseos J; Rogan, Peter K

2018-01-01

Background: Gene signatures derived from transcriptomic data using machine learning methods have shown promise for biodosimetry testing. These signatures may not be sufficiently robust for large scale testing, as their performance has not been adequately validated on external, independent datasets. The present study develops human and murine signatures with biochemically-inspired machine learning that are strictly validated using k-fold and traditional approaches. Methods: Gene Expression Omnibus (GEO) datasets of exposed human and murine lymphocytes were preprocessed via nearest neighbor imputation and expression of genes implicated in the literature to be responsive to radiation exposure (n=998) were then ranked by Minimum Redundancy Maximum Relevance (mRMR). Optimal signatures were derived by backward, complete, and forward sequential feature selection using Support Vector Machines (SVM), and validated using k-fold or traditional validation on independent datasets. Results: The best human signatures we derived exhibit k-fold validation accuracies of up to 98% ( DDB2 ,  PRKDC , TPP2 , PTPRE , and GADD45A ) when validated over 209 samples and traditional validation accuracies of up to 92% ( DDB2 ,  CD8A ,  TALDO1 ,  PCNA ,  EIF4G2 ,  LCN2 ,  CDKN1A ,  PRKCH ,  ENO1 ,  and PPM1D ) when validated over 85 samples. Some human signatures are specific enough to differentiate between chemotherapy and radiotherapy. Certain multi-class murine signatures have sufficient granularity in dose estimation to inform eligibility for cytokine therapy (assuming these signatures could be translated to humans). We compiled a list of the most frequently appearing genes in the top 20 human and mouse signatures. More frequently appearing genes among an ensemble of signatures may indicate greater impact of these genes on the performance of individual signatures. Several genes in the signatures we derived are present in previously proposed signatures. Conclusions: Gene signatures for ionizing radiation exposure derived by machine learning have low error rates in externally validated, independent datasets, and exhibit high specificity and granularity for dose estimation.

Precise Sequential DNA Ligation on A Solid Substrate: Solid-Based Rapid Sequential Ligation of Multiple DNA Molecules

PubMed Central

Takita, Eiji; Kohda, Katsunori; Tomatsu, Hajime; Hanano, Shigeru; Moriya, Kanami; Hosouchi, Tsutomu; Sakurai, Nozomu; Suzuki, Hideyuki; Shinmyo, Atsuhiko; Shibata, Daisuke

2013-01-01

Ligation, the joining of DNA fragments, is a fundamental procedure in molecular cloning and is indispensable to the production of genetically modified organisms that can be used for basic research, the applied biosciences, or both. Given that many genes cooperate in various pathways, incorporating multiple gene cassettes in tandem in a transgenic DNA construct for the purpose of genetic modification is often necessary when generating organisms that produce multiple foreign gene products. Here, we describe a novel method, designated PRESSO (precise sequential DNA ligation on a solid substrate), for the tandem ligation of multiple DNA fragments. We amplified donor DNA fragments with non-palindromic ends, and ligated the fragment to acceptor DNA fragments on solid beads. After the final donor DNA fragments, which included vector sequences, were joined to the construct that contained the array of fragments, the ligation product (the construct) was thereby released from the beads via digestion with a rare-cut meganuclease; the freed linear construct was circularized via an intra-molecular ligation. PRESSO allowed us to rapidly and efficiently join multiple genes in an optimized order and orientation. This method can overcome many technical challenges in functional genomics during the post-sequencing generation. PMID:23897972

Combinational treatment with retinoic acid derivatives in non-small cell lung carcinoma in vitro.

PubMed

Choi, Eun Jung; Whang, Young Mi; Kim, Seok Jin; Kim, Hyun Jin; Kim, Yeul Hong

2007-09-01

The growth inhibitory effects of four retinoic acid (RA) derivatives, 9-cis RA, 13-cis RA, N-(4-hydroxyphenyl) retinamide (4-HPR), and all-trans retinoic acid (ATRA) were compared. In addition, the effects of various combinations of these four agents were examined on non-small cell lung carcinoma (NSCLC) cell-lines, and on the expressions of retinoic acid receptors (RARs) and retinoid X receptors (RXRs) on these cells. At the clinically achievable concentration of 1 microM, only 4-HPR inhibited the growths of H1299 and H460 cells-lines. However, retinoic acid receptor beta(RAR beta) expression was up-regulated on H460 and H1299 cells treated with 1 microM of ATRA, 13-cis RA, or 9-cis RA. All NSCLC cell lines showed growth inhibition when exposed sequentially to 1 microM ATRA and 0.1 microM 4-HPR. In particular, sequential treatment with 1 microM ATRA or 13-cis RA and 4-HPR markedly inhibited H1703 cell growth; these cells exhibited no basal RAR beta expression and were refractory to 4-HPR. However, in NSCLC cell lines that expressed RAR beta, the expressional levels of RAR beta were up-regulated by ATRA alone and by sequential treatment with ATRA and 4-HPR. 4-HPR was found to be the most active of the four agents in terms of NSCLC growth-inhibition. Moreover, sequential treatments with ATRA or 13-cis RA followed by 4-HPR were found to have synergistic growth-inhibitory effects and to regulate RAR expression.

Genome-scale analysis and comparison of gene expression profiles in developing and germinated pollen in Oryza sativa

PubMed Central

2010-01-01

Background Pollen development from the microspore involves a series of coordinated cellular events, and the resulting mature pollen has a specialized function to quickly germinate, produce a polar-growth pollen tube derived from the vegetative cell, and deliver two sperm cells into the embryo sac for double fertilization. The gene expression profiles of developing and germinated pollen have been characterised by use of the eudicot model plant Arabidopsis. Rice, one of the most important cereal crops, has been used as an excellent monocot model. A comprehensive analysis of transcriptome profiles of developing and germinated pollen in rice is important to understand the conserved and diverse mechanism underlying pollen development and germination in eudicots and monocots. Results We used Affymetrix GeneChip® Rice Genome Array to comprehensively analyzed the dynamic changes in the transcriptomes of rice pollen at five sequential developmental stages from microspores to germinated pollen. Among the 51,279 transcripts on the array, we found 25,062 pollen-preferential transcripts, among which 2,203 were development stage-enriched. The diversity of transcripts decreased greatly from microspores to mature and germinated pollen, whereas the number of stage-enriched transcripts displayed a "U-type" change, with the lowest at the bicellular pollen stage; and a transition of overrepresented stage-enriched transcript groups associated with different functional categories, which indicates a shift in gene expression program at the bicellular pollen stage. About 54% of the now-annotated rice F-box protein genes were expressed preferentially in pollen. The transcriptome profile of germinated pollen was significantly and positively correlated with that of mature pollen. Analysis of expression profiles and coexpressed features of the pollen-preferential transcripts related to cell cycle, transcription, the ubiquitin/26S proteasome system, phytohormone signalling, the kinase system and defense/stress response revealed five expression patterns, which are compatible with changes in major cellular events during pollen development and germination. A comparison of pollen transcriptomes between rice and Arabidopsis revealed that 56.6% of the rice pollen preferential genes had homologs in Arabidopsis genome, but 63.4% of these homologs were expressed, with a small proportion being expressed preferentially, in Arabidopsis pollen. Rice and Arabidopsis pollen had non-conservative transcription factors each. Conclusions Our results demonstrated that rice pollen expressed a set of reduced but specific transcripts in comparison with vegetative tissues, and the number of stage-enriched transcripts displayed a "U-type" change during pollen development, with the lowest at the bicellular pollen stage. These features are conserved in rice and Arabidopsis. The shift in gene expression program at the bicellular pollen stage may be important to the transition from earlier cell division to later pollen maturity. Pollen at maturity pre-synthesized transcripts needed for germination and early pollen tube growth. The transcription regulation associated with pollen development would have divergence between the two species. Our results also provide novel insights into the molecular program and key components of the regulatory network regulating pollen development and germination. PMID:20507633

Ecdysteroid biosynthesis in varroa mites: identification of halloween genes from the biosynthetic pathway and their regulation during reproduction

USDA-ARS?s Scientific Manuscript database

Biosynthesis of ecdysteroids involves sequential enzymatic hydroxylations by microsomal enzymes and mitochondrial cytochrome P450’s. Enzymes of the pathway are collectively known as Halloween genes. Complete sequences for three Halloween genes, spook (Vdspo), disembodied (Vddib) and shade (Vdshd), w...

Hyper-activation of the TCP4 transcription factor in Arabidopsis thaliana accelerates multiple aspects of plant maturation.

PubMed

Sarvepalli, Kavitha; Nath, Utpal

2011-08-01

Plant organs are initiated as primordial outgrowths, and require controlled cell division and differentiation to achieve their final size and shape. Superimposed on this is another developmental program that orchestrates the switch from vegetative to reproductive to senescence stages in the life cycle. These require sequential function of heterochronic regulators. Little is known regarding the coordination between organ and organismal growth in plants. The TCP gene family encodes transcription factors that control diverse developmental traits, and a subgroup of class II TCP genes regulate leaf morphogenesis. Absence of these genes results in large, crinkly leaves due to excess division, mainly at margins. It has been suggested that these class II TCPs modulate the spatio-temporal control of differentiation in a growing leaf, rather than regulating cell proliferation per se. However, the link between class II TCP action and cell growth has not been established. As loss-of-function mutants of individual TCP genes in Arabidopsis are not very informative due to gene redundancy, we generated a transgenic line that expressed a hyper-activated form of TCP4 in its endogenous expression domain. This resulted in premature onset of maturation and decreased cell proliferation, leading to much smaller leaves, with cup-shaped lamina in extreme cases. Further, the transgenic line initiated leaves faster than wild-type and underwent precocious reproductive maturation due to a shortened adult vegetative phase. Early senescence and severe fertility defects were also observed. Thus, hyper-activation of TCP4 revealed its role in determining the timing of crucial developmental events, both at the organ and organism level. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

Direct biological effects of fractional ultrapulsed CO2 laser irradiation on keratinocytes and fibroblasts in human organotypic full-thickness 3D skin models.

PubMed

Schmitt, L; Huth, S; Amann, P M; Marquardt, Y; Heise, R; Fietkau, K; Huth, L; Steiner, T; Hölzle, F; Baron, J M

2018-05-01

Molecular effects of various ablative and non-ablative laser treatments on human skin cells-especially primary effects on epidermal keratinocytes and dermal fibroblasts-are not yet fully understood. We present the first study addressing molecular effects of fractional non-sequential ultrapulsed CO 2 laser treatment using a 3D skin model that allows standardized investigations of time-dependent molecular changes ex vivo. While histological examination was performed to assess morphological changes, we utilized gene expression profiling using microarray and qRT-PCR analyses to identify molecular effects of laser treatment. Irradiated models exhibited dose-dependent morphological changes resulting in an almost complete recovery of the epidermis 5 days after irradiation. On day 5 after laser injury with a laser fluence of 100 mJ/cm 2 , gene array analysis identified an upregulation of genes associated with tissue remodeling and wound healing (e.g., COL12A1 and FGF7), genes that are involved in the immune response (e.g., CXCL12 and CCL8) as well as members of the heat shock protein family (e.g., HSPB3). On the other hand, we detected a downregulation of matrix metalloproteinases (e.g., MMP3), differentiation markers (e.g., LOR and S100A7), and the pro-inflammatory cytokine IL1α.Overall, our findings substantiate the understanding of time-dependent molecular changes after CO 2 laser treatment. The utilized 3D skin model system proved to be a reliable, accurate, and reproducible tool to explore the effects of various laser settings both on skin morphology and gene expression during wound healing.

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

PubMed

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

1997-01-01

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

The peripheral sensory nervous system in the vertebrate head: a gene regulatory perspective.

PubMed

Grocott, Timothy; Tambalo, Monica; Streit, Andrea

2012-10-01

In the vertebrate head, crucial parts of the sense organs and sensory ganglia develop from special regions, the cranial placodes. Despite their cellular and functional diversity, they arise from a common field of multipotent progenitors and acquire distinct identity later under the influence of local signalling. Here we present the gene regulatory network that summarises our current understanding of how sensory cells are specified, how they become different from other ectodermal derivatives and how they begin to diversify to generate placodes with different identities. This analysis reveals how sequential activation of sets of transcription factors subdivides the ectoderm over time into smaller domains of progenitors for the central nervous system, neural crest, epidermis and sensory placodes. Within this hierarchy the timing of signalling and developmental history of each cell population is of critical importance to determine the ultimate outcome. A reoccurring theme is that local signals set up broad gene expression domains, which are further refined by mutual repression between different transcription factors. The Six and Eya network lies at the heart of sensory progenitor specification. In a positive feedback loop these factors perpetuate their own expression thus stabilising pre-placodal fate, while simultaneously repressing neural and neural crest specific factors. Downstream of the Six and Eya cassette, Pax genes in combination with other factors begin to impart regional identity to placode progenitors. While our review highlights the wealth of information available, it also points to the lack information on the cis-regulatory mechanisms that control placode specification and of how the repeated use of signalling input is integrated. Copyright © 2012. Published by Elsevier Inc.

Induction of intermediate mesoderm by retinoic acid receptor signaling from differentiating mouse embryonic stem cells.

PubMed

Oeda, Shiho; Hayashi, Yohei; Chan, Techuan; Takasato, Minoru; Aihara, Yuko; Okabayashi, Koji; Ohnuma, Kiyoshi; Asashima, Makoto

2013-01-01

Renal lineages including kidney are derived from intermediate mesoderm, which are differentiated from a subset of caudal undifferentiated mesoderm. The inductive mechanisms of mammalian intermediate mesoderm and renal lineages are still poorly understood. Mouse embryonic stem cells (mESCs) can be a good in vitro model to reconstitute the developmental pathway of renal lineages and to analyze the mechanisms of the sequential differentiation. We examined the effects of Activin A and retinoic acid (RA) on the induction of intermediate mesoderm from mESCs under defined, serum-free, adherent, monolayer culture conditions. We measured the expression level of intermediate mesodermal marker genes and examined the developmental potential of the differentiated cells into kidney using an ex vivo transplantation assay. Adding Activin A followed by RA to mESC cultures induced the expression of marker genes and proteins for intermediate mesoderm, odd-skipped related 1 (Osr1) and Wilm’s Tumor 1 (Wt1). These differentiated cells integrated into laminin-positive tubular cells and Pax2-positive renal cells in cultured embryonic kidney explants. We demonstrated that intermediate mesodermal marker expression was also induced by RA receptor (RAR) agonist, but not by retinoid X receptor (RXR) agonists. Furthermore, the expression of these markers was decreased by RAR antagonists. We directed the differentiation of mESCs into intermediate mesoderm using Activin A and RA and revealed the role of RAR signaling in this differentiation. These methods and findings will improve our understanding of renal lineage development and could contribute to the regenerative medicine of kidney.

Transcription factors define the neuroanatomical organization of the medullary reticular formation

PubMed Central

Gray, Paul A.

2013-01-01

The medullary reticular formation contains large populations of inadequately described, excitatory interneurons that have been implicated in multiple homeostatic behaviors including breathing, viserosensory processing, vascular tone, and pain. Many hindbrain nuclei show a highly stereotyped pattern of localization across vertebrates suggesting a strong underlying genetic organization. Whether this is true for neurons within the reticular regions of hindbrain is unknown. Hindbrain neurons are derived from distinct developmental progenitor domains each of which expresses distinct patterns of transcription factors (TFs). These neuronal populations have distinct characteristics such as transmitter identity, migration, and connectivity suggesting developmentally expressed TFs might identify unique subpopulations of neurons within the reticular formation. A fate-mapping strategy using perinatal expression of reporter genes within Atoh1, Dbx1, Lmx1b, and Ptf1a transgenic mice coupled with immunohistochemistry (IHC) and in situ hybridization (ISH) were used to address the developmental organization of a large subset of reticular formation glutamatergic neurons. All hindbrain lineages have relatively large populations that extend the entire length of the hindbrain. Importantly, the location of neurons within each lineage was highly constrained. Lmx1b- and Dbx1- derived populations were both present in partially overlapping stripes within the reticular formation extending from dorsal to ventral brain. Within each lineage, distinct patterns of gene expression and organization were localized to specific hindbrain regions. Rostro-caudally sub-populations differ sequentially corresponding to proposed pseudo-rhombomereic boundaries. Dorsal-ventrally, sub-populations correspond to specific migratory positions. Together these data suggests the reticular formation is organized by a highly stereotyped developmental logic. PMID:23717265

Transcription factors define the neuroanatomical organization of the medullary reticular formation.

PubMed

Gray, Paul A

2013-01-01

The medullary reticular formation contains large populations of inadequately described, excitatory interneurons that have been implicated in multiple homeostatic behaviors including breathing, viserosensory processing, vascular tone, and pain. Many hindbrain nuclei show a highly stereotyped pattern of localization across vertebrates suggesting a strong underlying genetic organization. Whether this is true for neurons within the reticular regions of hindbrain is unknown. Hindbrain neurons are derived from distinct developmental progenitor domains each of which expresses distinct patterns of transcription factors (TFs). These neuronal populations have distinct characteristics such as transmitter identity, migration, and connectivity suggesting developmentally expressed TFs might identify unique subpopulations of neurons within the reticular formation. A fate-mapping strategy using perinatal expression of reporter genes within Atoh1, Dbx1, Lmx1b, and Ptf1a transgenic mice coupled with immunohistochemistry (IHC) and in situ hybridization (ISH) were used to address the developmental organization of a large subset of reticular formation glutamatergic neurons. All hindbrain lineages have relatively large populations that extend the entire length of the hindbrain. Importantly, the location of neurons within each lineage was highly constrained. Lmx1b- and Dbx1- derived populations were both present in partially overlapping stripes within the reticular formation extending from dorsal to ventral brain. Within each lineage, distinct patterns of gene expression and organization were localized to specific hindbrain regions. Rostro-caudally sub-populations differ sequentially corresponding to proposed pseudo-rhombomereic boundaries. Dorsal-ventrally, sub-populations correspond to specific migratory positions. Together these data suggests the reticular formation is organized by a highly stereotyped developmental logic.

Exploiting the CRISPR/Cas9 System for Targeted Genome Mutagenesis in Petunia.

PubMed

Zhang, Bin; Yang, Xia; Yang, Chunping; Li, Mingyang; Guo, Yulong

2016-02-03

Recently, CRISPR/Cas9 technology has emerged as a powerful approach for targeted genome modification in eukaryotic organisms from yeast to human cell lines. Its successful application in several plant species promises enormous potential for basic and applied plant research. However, extensive studies are still needed to assess this system in other important plant species, to broaden its fields of application and to improve methods. Here we showed that the CRISPR/Cas9 system is efficient in petunia (Petunia hybrid), an important ornamental plant and a model for comparative research. When PDS was used as target gene, transgenic shoot lines with albino phenotype accounted for 55.6%-87.5% of the total regenerated T0 Basta-resistant lines. A homozygous deletion close to 1 kb in length can be readily generated and identified in the first generation. A sequential transformation strategy--introducing Cas9 and sgRNA expression cassettes sequentially into petunia--can be used to make targeted mutations with short indels or chromosomal fragment deletions. Our results present a new plant species amenable to CRIPR/Cas9 technology and provide an alternative procedure for its exploitation.

Integrated molecular analysis of tumor biopsies on sequential CTLA-4 and PD-1 blockade reveals markers of response and resistance

PubMed Central

Roh, Whijae; Chen, Pei-Ling; Reuben, Alexandre; Spencer, Christine N.; Prieto, Peter A.; Miller, John P.; Gopalakrishnan, Vancheswaran; Wang, Feng; Cooper, Zachary A.; Reddy, Sangeetha M.; Gumbs, Curtis; Little, Latasha; Chang, Qing; Chen, Wei-Shen; Wani, Khalida; Petaccia De Macedo, Mariana; Chen, Eveline; Austin-Breneman, Jacob L.; Jiang, Hong; Roszik, Jason; Tetzlaff, Michael T.; Davies, Michael A.; Gershenwald, Jeffrey E.; Tawbi, Hussein; Lazar, Alexander J.; Hwu, Patrick; Hwu, Wen-Jen; Diab, Adi; Glitza, Isabella C.; Patel, Sapna P.; Woodman, Scott E.; Amaria, Rodabe N.; Prieto, Victor G.; Hu, Jianhua; Sharma, Padmanee; Allison, James P.; Chin, Lynda; Zhang, Jianhua; Wargo, Jennifer A.; Futreal, P. Andrew

2018-01-01

Immune checkpoint blockade produces clinical benefit in many patients. However better biomarkers of response are still needed, and mechanisms of resistance remain incompletely understood. To address this, we recently studied a cohort of melanoma patients treated with sequential checkpoint blockade against cytotoxic T lymphocyte antigen-4 (CTLA-4) followed by programmed death receptor-1 (PD-1), and identified immune markers of response and resistance. Building on these studies, we performed deep molecular profiling including T-cell receptor sequencing (TCR-seq) and whole exome sequencing (WES) within the same cohort, and demonstrated that a more clonal T cell repertoire was predictive of response to PD-1 but not CTLA-4 blockade. Analysis of copy number alterations identified a higher burden of copy number loss in non-responders to CTLA-4 and PD-1 blockade and found that it was associated with decreased expression of genes in immune-related pathways. The effect of mutational load and burden of copy number loss on response was non-redundant, suggesting the potential utility of a combinatorial biomarker to optimize patient care with checkpoint blockade therapy. PMID:28251903

Exploiting the CRISPR/Cas9 System for Targeted Genome Mutagenesis in Petunia

PubMed Central

Zhang, Bin; Yang, Xia; Yang, Chunping; Li, Mingyang; Guo, Yulong

2016-01-01

Recently, CRISPR/Cas9 technology has emerged as a powerful approach for targeted genome modification in eukaryotic organisms from yeast to human cell lines. Its successful application in several plant species promises enormous potential for basic and applied plant research. However, extensive studies are still needed to assess this system in other important plant species, to broaden its fields of application and to improve methods. Here we showed that the CRISPR/Cas9 system is efficient in petunia (Petunia hybrid), an important ornamental plant and a model for comparative research. When PDS was used as target gene, transgenic shoot lines with albino phenotype accounted for 55.6%–87.5% of the total regenerated T0 Basta-resistant lines. A homozygous deletion close to 1 kb in length can be readily generated and identified in the first generation. A sequential transformation strategy—introducing Cas9 and sgRNA expression cassettes sequentially into petunia—can be used to make targeted mutations with short indels or chromosomal fragment deletions. Our results present a new plant species amenable to CRIPR/Cas9 technology and provide an alternative procedure for its exploitation. PMID:26837606

RIPiT-Seq: A high-throughput approach for footprinting RNA:protein complexes

PubMed Central

Singh, Guramrit; Ricci, Emiliano P.; Moore, Melissa J.

2013-01-01

Development of high-throughput approaches to map the RNA interaction sites of individual RNA binding proteins (RBPs) transcriptome-wide is rapidly transforming our understanding of post-transcriptional gene regulatory mechanisms. Here we describe a ribonucleoprotein (RNP) footprinting approach we recently developed for identifying occupancy sites of both individual RBPs and multi-subunit RNP complexes. RNA:protein immunoprecipitation in tandem (RIPiT) yields highly specific RNA footprints of cellular RNPs isolated via two sequential purifications; the resulting RNA footprints can then be identified by high-throughput sequencing (Seq). RIPiT-Seq is broadly applicable to all RBPs regardless of their RNA binding mode and thus provides a means to map the RNA binding sites of RBPs with poor inherent ultraviolet (UV) crosslinkability. Further, among current high-throughput approaches, RIPiT has the unique capacity to differentiate binding sites of RNPs with overlapping protein composition. It is therefore particularly suited for studying dynamic RNP assemblages whose composition evolves as gene expression proceeds. PMID:24096052

Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice.

PubMed

Mikami, Masafumi; Toki, Seiichi; Endo, Masaki

2015-08-01

The CRISPR/Cas9 system is an efficient tool used for genome editing in a variety of organisms. Despite several recent reports of successful targeted mutagenesis using the CRISPR/Cas9 system in plants, in each case the target gene of interest, the Cas9 expression system and guide-RNA (gRNA) used, and the tissues used for transformation and subsequent mutagenesis differed, hence the reported frequencies of targeted mutagenesis cannot be compared directly. Here, we evaluated mutation frequency in rice using different Cas9 and/or gRNA expression cassettes under standardized experimental conditions. We introduced Cas9 and gRNA expression cassettes separately or sequentially into rice calli, and assessed the frequency of mutagenesis at the same endogenous targeted sequences. Mutation frequencies differed significantly depending on the Cas9 expression cassette used. In addition, a gRNA driven by the OsU6 promoter was superior to one driven by the OsU3 promoter. Using an all-in-one expression vector harboring the best combined Cas9/gRNA expression cassette resulted in a much improved frequency of targeted mutagenesis in rice calli, and bi-allelic mutant plants were produced in the T0 generation. The approach presented here could be adapted to optimize the construction of Cas9/gRNA cassettes for genome editing in a variety of plants.

Rat embryo cells immortalized with transfected oncogenes are transformed by gamma irradiation.

PubMed

Endlich, B; Salavati, R; Sullivan, T; Ling, C C

1992-12-01

Cesium-137 gamma rays were used to transform rat embryo cells (REC) which were first transfected with activated c-myc or c-Ha-ras oncogenes to produce immortal cell lines (REC:myc and REC:ras). When exposed to 6 Gy of 137Cs gamma rays, some cells became morphologically transformed with focus formation frequencies of approximately 3 x 10(-4) for REC:myc and approximately 1 x 10(-4) for REC:ras, respectively. Cells isolated from foci of gamma-ray-transformed REC:myc (REC:myc:gamma) formed anchorage-independent colonies and were tumorigenic in nude mice, but foci from gamma-ray-transformed REC:ras (REC:ras:gamma) did not exhibit either of these criteria of transformation. Similar to the results with gamma irradiation, we observed a sequence-dependent phenomenon when myc and ras were transfected into REC, one at a time. REC immortalized by ras transfection were not converted to a tumorigenic phenotype by secondary transfection with myc, but REC transfected with myc were very susceptible to transformation by subsequent ras transfection. This suggests that myc-immortalized cells are more permissive to transformation via secondary treatments. In sequentially transfected REC, myc expression was high whether it was transfected first or second, whereas ras expression was highest when the ras gene was transfected secondarily into myc-containing REC. Molecular analysis of REC:ras:gamma transformants showed no alterations in structure of the transfected ras or of the endogenous ras, myc, p53, or fos genes. The expression of ras and p53 was increased in some isolates of REC:ras:gamma, but myc and fos expression were not affected. Similarly, REC:myc:gamma transformants did not demonstrate rearrangement or amplification of the transfected or the endogenous myc genes, or of the potentially cooperating Ha-, Ki-, or N-ras genes. Northern hybridization analysis revealed increased expression of N-ras in two isolates, REC:myc:gamma 33 and gamma 41, but no alterations in the expression of myc, raf, Ha-ras, or Ki-ras genes in any REC:myc transformant. DNA from several transformed REC:myc:gamma cell lines induced focus formation in recipient C3H 10T1/2 and NIH 3T3 cells. The NIH 3T3 foci tested positive when hybridized to a probe for rat repetitive DNA. A detailed analysis of the NIH 3T3 transformants generated from REC:myc:gamma 33 and gamma 41 DNA failed to detect Ha-ras, Ki-ras, raf, neu, trk, abl, fms, or src oncogenes of rat origin.(ABSTRACT TRUNCATED AT 400 WORDS)

Chordate evolution and the origin of craniates: an old brain in a new head.

PubMed

Butler, A B

2000-06-15

The earliest craniates achieved a unique condition among bilaterally symmetrical animals: they possessed enlarged, elaborated brains with paired sense organs and unique derivatives of neural crest and placodal tissues, including peripheral sensory ganglia, visceral arches, and head skeleton. The craniate sister taxon, cephalochordates, has rostral portions of the neuraxis that are homologous to some of the major divisions of craniate brains. Moreover, recent data indicate that many genes involved in patterning the nervous system are common to all bilaterally symmetrical animals and have been inherited from a common ancestor. Craniates, thus, have an "old" brain in a new head, due to re-expression of these anciently acquired genes. The transition to the craniate brain from a cephalochordate-like ancestral form may have involved a mediolateral shift in expression of the genes that specify nervous system development from various parts of the ectoderm. It is suggested here that the transition was sequential. The first step involved the presence of paired, lateral eyes, elaboration of the alar plate, and enhancement of the descending visual pathway to brainstem motor centers. Subsequently, this central visual pathway served as a template for the additional sensory systems that were elaborated and/or augmented with the "bloom" of migratory neural crest and placodes. This model accounts for the marked uniformity of pattern across central sensory pathways and for the lack of any neural crest-placode cranial nerve for either the diencephalon or mesencephalon. Anat Rec (New Anat) 261:111-125, 2000. Copyright 2000 Wiley-Liss, Inc.

The constitutive activation of Egr-1/C/EBPa mediates the development of type 2 diabetes mellitus by enhancing hepatic gluconeogenesis.

PubMed

Shen, Ning; Jiang, Shan; Lu, Jia-Ming; Yu, Xiao; Lai, Shan-Shan; Zhang, Jing-Zi; Zhang, Jin-Long; Tao, Wei-Wei; Wang, Xiu-Xing; Xu, Na; Xue, Bin; Li, Chao-Jun

2015-02-01

The sequential secretion of insulin and glucagon delicately maintains glucose homeostasis by inhibiting or enhancing hepatic gluconeogenesis during postprandial or fasting states, respectively. Increased glucagon/insulin ratio is believed to be a major cause of the hyperglycemia seen in type 2 diabetes. Herein, we reveal that the early growth response gene-1 (Egr-1) can be transiently activated by glucagon in hepatocytes, which mediates glucagon-regulated gluconeogenesis by increasing the expression of gluconeogenesis genes. Blockage of Egr-1 function in the liver of mice led to lower fasting blood glucose, better pyruvate tolerance, and higher hepatic glycogen content. The mechanism analysis demonstrated that Egr-1 can directly bind to the promoter of C/EBPa and regulate the expression of gluconeogenesis genes in the later phase of glucagon stimulation. The transient increase of Egr-1 by glucagon kept the glucose homeostasis after fasting for longer periods of time, whereas constitutive Egr-1 elevation found in the liver of db/db mice and high serum glucagon level overactivated the C/EBPa/gluconeogenesis pathway and resulted in hyperglycemia. Blockage of Egr-1 activation in prediabetic db/db mice was able to delay the progression of diabetes. Our results suggest that dysregulation of Egr-1/C/EBPa on glucagon stimulation may provide an alternative mechanistic explanation for type 2 diabetes. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Comparison of partial and full nitrification processes applied for treating high-strength nitrogen wastewaters: microbial ecology through nitrous oxide production.

PubMed

Ahn, Joon Ho; Kwan, Tiffany; Chandran, Kartik

2011-04-01

The goal of this study was to compare the microbial ecology, gene expression, biokinetics, and N2O emissions from a lab-scale bioreactor operated sequentially in full-nitrification and partial-nitrification modes. Based on sequencing of 16S rRNA and ammonia monooxygenase subunit A (amoA) genes, ammonia oxidizing bacteria (AOB) populations during full- and partial-nitrification modes were distinct from one another. The concentrations of AOB (XAOB) and their respiration rates during full- and partial-nitrification modes were statistically similar, whereas the concentrations of nitrite oxidizing bacteria (XNOB) and their respiration rates declined significantly after the switch from full- to partial-nitrification. The transition from full-nitrification to partial nitrification resulted in a protracted transient spike of nitrous oxide (N2O) and nitric oxide (NO) emissions, which later stabilized. The trends in N2O and NO emissions correlated well with trends in the expression of nirK and norB genes that code for the production of these gases in AOB. Both the transient and stabilized N2O and NO emissions during partial nitrification were statistically higher than those during steady-state full-nitrification. Based on these results, partial nitrification strategies for biological nitrogen removal, although attractive for their reduced operating costs and energy demand, may need to be optimized against the higher carbon foot-print attributed to their N2O emissions.

TNFα- and IKKβ-mediated TANK/I-TRAF phosphorylation: implications for interaction with NEMO/IKKγ and NF-κB activation

PubMed Central

Bonif, Marianne; Meuwis, Marie-Alice; Close, Pierre; Benoit, Valérie; Heyninck, Karen; Chapelle, Jean-Paul; Bours, Vincent; Merville, Marie-Paule; Piette, Jacques; Beyaert, Rudi; Chariot, Alain

2005-01-01

Pro-inflammatory cytokines trigger signalling cascades leading to NF-κB (nuclear factor-κB)-dependent gene expression through IKK [IκB (inhibitory κB) kinase]-dependent phosphorylation and subsequent degradation of the IκB proteins and via induced phosphorylation of p65. These signalling pathways rely on sequentially activated kinases which are assembled by essential and non-enzymatic scaffold proteins into functional complexes. Here, we show that the pro-inflammatory cytokine TNFα (tumour necrosis factor α) promotes TANK [TRAF (TNF receptor-associated factor) family member associated NF-κB activator] recruitment to the IKK complex via a newly characterized C-terminal zinc finger. Moreover, we show that TANK is phosphorylated by IKKβ upon TNFα stimulation and that this modification negatively regulates TANK binding to NEMO (NF-κB essential modulator). Interestingly, reduced TANK expression by RNA interference attenuates TNFα-mediated induction of a subset of NF-κB target genes through decreased p65 transactivation potential. Therefore the scaffold protein TANK is required for the cellular response to TNFα by connecting upstream signalling molecules to the IKKs and p65, and its subsequent IKKβ-mediated phosphorylation may be a mechanism to terminate the TANK-dependent wave of NF-κB activation. PMID:16336209

Sequential induction of three recombination directionality factors directs assembly of tripartite integrative and conjugative elements.

PubMed

Haskett, Timothy L; Terpolilli, Jason J; Ramachandran, Vinoy K; Verdonk, Callum J; Poole, Phillip S; O'Hara, Graham W; Ramsay, Joshua P

2018-03-01

Tripartite integrative and conjugative elements (ICE3) are a novel form of ICE that exist as three separate DNA regions integrated within the genomes of Mesorhizobium spp. Prior to conjugative transfer the three ICE3 regions of M. ciceri WSM1271 ICEMcSym1271 combine and excise to form a single circular element. This assembly requires three coordinated recombination events involving three site-specific recombinases IntS, IntG and IntM. Here, we demonstrate that three excisionases-or recombination directionality factors-RdfS, RdfG and RdfM are required for ICE3 excision. Transcriptome sequencing revealed that expression of ICE3 transfer and conjugation genes was induced by quorum sensing. Quorum sensing activated expression of rdfS, and in turn RdfS stimulated transcription of both rdfG and rdfM. Therefore, RdfS acts as a "master controller" of ICE3 assembly and excision. The dependence of all three excisive reactions on RdfS ensures that ICE3 excision occurs via a stepwise sequence of recombination events that avoids splitting the chromosome into a non-viable configuration. These discoveries expose a surprisingly simple control system guiding molecular assembly of these novel and complex mobile genetic elements and highlight the diverse and critical functions of excisionase proteins in control of horizontal gene transfer.

Algorithms for Hidden Markov Models Restricted to Occurrences of Regular Expressions

PubMed Central

Tataru, Paula; Sand, Andreas; Hobolth, Asger; Mailund, Thomas; Pedersen, Christian N. S.

2013-01-01

Hidden Markov Models (HMMs) are widely used probabilistic models, particularly for annotating sequential data with an underlying hidden structure. Patterns in the annotation are often more relevant to study than the hidden structure itself. A typical HMM analysis consists of annotating the observed data using a decoding algorithm and analyzing the annotation to study patterns of interest. For example, given an HMM modeling genes in DNA sequences, the focus is on occurrences of genes in the annotation. In this paper, we define a pattern through a regular expression and present a restriction of three classical algorithms to take the number of occurrences of the pattern in the hidden sequence into account. We present a new algorithm to compute the distribution of the number of pattern occurrences, and we extend the two most widely used existing decoding algorithms to employ information from this distribution. We show experimentally that the expectation of the distribution of the number of pattern occurrences gives a highly accurate estimate, while the typical procedure can be biased in the sense that the identified number of pattern occurrences does not correspond to the true number. We furthermore show that using this distribution in the decoding algorithms improves the predictive power of the model. PMID:24833225

Changes in the levels of mRNAs for GH/prolactin/somatolactin family and Pit-1/GHF-1 in the pituitaries of pre-spawning chum salmon.

PubMed

Taniyama, S; Kitahashi, T; Ando, H; Ban, M; Ueda, H; Urano, A

1999-10-01

Changes in the levels of pituitary mRNAs encoding GH, prolactin (PRL) and somatolactin (SL) were determined in pre-spawning chum salmon (Oncorhynchus keta) caught at a few key points along their homing pathway in 1994 and 1995. Furthermore, we analyzed relationships between expression of pituitary-specific POU homeodomain transcription factor (Pit-1/GHF-1) and GH/PRL/SL family genes. In 1994, seawater (SW) fish and matured fresh-water (FW) fish were sequentially captured at two points along their homing pathway, the coast and the hatchery. In addition to these two points, maturing FW fish were captured at the intermediate of the two points in 1995. The levels of hormonal mRNAs were determined by a quantitative dot blot analysis using single-stranded sense DNA as the standard. Relative levels of Pit-1/GHF-1 mRNAs were estimated by Northern blot analysis. In 1994, the levels of GH/PRL/SL family mRNAs except for PRL mRNA in the male FW fish were 1.8-4 times higher than those in the SW fish. In 1995, the level of PRL mRNA was somewhat sharply elevated in the maturing FW fish soon after entry into the FW environment, while that of SL mRNA was gradually increased during upstream migration from the coast to the hatchery. The levels of 3 kb Pit-1/GHF-1 mRNA in the FW fish were higher than those in the SW fish in both 1994 and 1995. The present results indicate that expression of genes for the GH/PRL/SL family and Pit-1/GHF-1 is coincidentally enhanced in homing chum salmon. Moreover, the present study suggests that expression of the SL gene is elevated with sexual maturation, whereas that of PRL gene is elevated with osmotic change during the final stages of spawning migration.

Identification and functional characterization of a flax UDP-glycosyltransferase glucosylating secoisolariciresinol (SECO) into secoisolariciresinol monoglucoside (SMG) and diglucoside (SDG).

PubMed

Ghose, Kaushik; Selvaraj, Kumarakurubaran; McCallum, Jason; Kirby, Chris W; Sweeney-Nixon, Marva; Cloutier, Sylvie J; Deyholos, Michael; Datla, Raju; Fofana, Bourlaye

2014-03-28

Lignans are a class of diphenolic nonsteroidal phytoestrogens often found glycosylated in planta. Flax seeds are a rich source of secoisolariciresinol diglucoside (SDG) lignans. Glycosylation is a process by which a glycosyl group is covalently attached to an aglycone substrate and is catalyzed by uridine diphosphate glycosyltransferases (UGTs). Until now, very little information was available on UGT genes that may play a role in flax SDG biosynthesis. Here we report on the identification, structural and functional characterization of 5 putative UGTs potentially involved in secoisolariciresinol (SECO) glucosylation in flax. Five UGT genes belonging to the glycosyltransferases' family 1 (EC 2.4.x.y) were cloned and characterized. They fall under four UGT families corresponding to five sub-families referred to as UGT74S1, UGT74T1, UGT89B3, UGT94H1, UGT712B1 that all display the characteristic plant secondary product glycosyltransferase (PSPG) conserved motif. However, diversity was observed within this 44 amino acid sequence, especially in the two peptide sequences WAPQV and HCGWNS known to play a key role in the recognition and binding of diverse aglycone substrates and in the sugar donor specificity. In developing flax seeds, UGT74S1 and UGT94H1 showed a coordinated gene expression with that of pinoresinol-lariciresinol reductase (PLR) and their gene expression patterns correlated with SDG biosynthesis. Enzyme assays of the five heterologously expressed UGTs identified UGT74S1 as the only one using SECO as substrate, forming SECO monoglucoside (SMG) and then SDG in a sequential manner. We have cloned and characterized five flax UGTs and provided evidence that UGT74S1 uses SECO as substrate to form SDG in vitro. This study allowed us to propose a model for the missing step in SDG lignan biosynthesis.

Identification and functional characterization of a flax UDP-glycosyltransferase glucosylating secoisolariciresinol (SECO) into secoisolariciresinol monoglucoside (SMG) and diglucoside (SDG)

PubMed Central

2014-01-01

Background Lignans are a class of diphenolic nonsteroidal phytoestrogens often found glycosylated in planta. Flax seeds are a rich source of secoisolariciresinol diglucoside (SDG) lignans. Glycosylation is a process by which a glycosyl group is covalently attached to an aglycone substrate and is catalyzed by uridine diphosphate glycosyltransferases (UGTs). Until now, very little information was available on UGT genes that may play a role in flax SDG biosynthesis. Here we report on the identification, structural and functional characterization of 5 putative UGTs potentially involved in secoisolariciresinol (SECO) glucosylation in flax. Results Five UGT genes belonging to the glycosyltransferases’ family 1 (EC 2.4.x.y) were cloned and characterized. They fall under four UGT families corresponding to five sub-families referred to as UGT74S1, UGT74T1, UGT89B3, UGT94H1, UGT712B1 that all display the characteristic plant secondary product glycosyltransferase (PSPG) conserved motif. However, diversity was observed within this 44 amino acid sequence, especially in the two peptide sequences WAPQV and HCGWNS known to play a key role in the recognition and binding of diverse aglycone substrates and in the sugar donor specificity. In developing flax seeds, UGT74S1 and UGT94H1 showed a coordinated gene expression with that of pinoresinol-lariciresinol reductase (PLR) and their gene expression patterns correlated with SDG biosynthesis. Enzyme assays of the five heterologously expressed UGTs identified UGT74S1 as the only one using SECO as substrate, forming SECO monoglucoside (SMG) and then SDG in a sequential manner. Conclusion We have cloned and characterized five flax UGTs and provided evidence that UGT74S1 uses SECO as substrate to form SDG in vitro. This study allowed us to propose a model for the missing step in SDG lignan biosynthesis. PMID:24678929

Collateral damage of flomoxef therapy: in vivo development of porin deficiency and acquisition of blaDHA-1 leading to ertapenem resistance in a clinical isolate of Klebsiella pneumoniae producing CTX-M-3 and SHV-5 beta-lactamases.

PubMed

Lee, Chen-Hsiang; Chu, Chishih; Liu, Jien-Wei; Chen, Yi-Shung; Chiu, Chiung-Jung; Su, Lin-Hui

2007-08-01

The study aimed to characterize the genetic basis of flomoxef and collateral ertapenem resistance in a clinical isolate of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBL-KP) after flomoxef exposure. Four ESBL-KP isolates (Lkp11-14) were recovered sequentially from four episodes of bacteraemia in an elderly patient. Laboratory investigations included genotyping by PFGE, resistance gene analysis by PCR and sequencing, and outer membrane protein analysis by SDS-PAGE. Plasmid analysis by DNA-DNA hybridization, electroporation and conjugation was also performed. Lkp14 was recovered after 21 days of flomoxef therapy. It demonstrated an indistinguishable PFGE pattern when compared with those produced by Lkp11-13. However, resistance to both flomoxef and ertapenem emerged in Lkp14. Molecular characterization revealed that, in addition to the pre-existing ESBL production (CTX-M-3 and SHV-5) and OmpK35 deficiency found in Lkp11-13, Lkp14 had acquired an extra plasmid-mediated AmpC beta-lactamase gene (blaDHA-1) and failed to express OmpK36, because of insertional inactivation by an insertion sequence IS5. Other resistance mechanisms, such as production of carbapenem-hydrolysing enzymes or expression of chromosomal efflux, were apparently not involved. Conjugational transfer of the plasmid-mediated blaDHA-1 gene into Lkp11 resulted in a significant increase in the MICs of cephamycins and beta-lactamase inhibitors, but not in those of carbapenems. Lkp14 was apparently derived from the previously flomoxef-susceptible isolates, Lkp11-13. After flomoxef exposure, the in vivo acquisition of the plasmid-mediated blaDHA-1 gene has led to flomoxef resistance in Lkp14, and the concomitant depletion of OmpK36 expression has resulted in a collateral effect of ertapenem resistance and diminished susceptibilities to imipenem and meropenem.

Maize Iranian mosaic virus shows a descending transcript accumulation order in plant and insect hosts.

PubMed

Hortamani, Mozhgan; Massah, Amir; Izadpanah, Keramat

2018-04-01

Maize Iranian mosaic virus (MIMV) is a distinct member of the genus Nucleorhabdovirus. In this study, expression of all MIMV genes in maize for four weeks after inoculation and in inoculative planthoppers was examined using a quantitative RT-PCR (RT-qPCR) assay. Accumulation of MIMV P, gene 3, M, G and L transcripts relative to N transcripts was measured and normalized to 18S rRNA in maize plants and to the ribosomal protein S13 gene (RPS13) in planthoppers using the comparative C T method. In plants, higher levels of MIMV N transcripts were found relative to other transcripts, while MIMV L transcripts were at the lowest levels. The highest accumulation of MIMV transcripts was found at 14 days postinoculation (dpi). At 21 dpi, we found the lowest transcript levels for all genes, which increased again at 28 dpi, although in lower amounts than at 14 dpi. In Laodelphax striatellus, MIMV M, G and L transcripts accumulated at lower levels than other transcripts. The gene 3 transcript level was high in both plants and planthoppers. Our results showed that transcript accumulation for the MIMV genes was similar in both hosts and followed the pattern of sequential transcriptional attenuation from the 3' to the 5' end of the genome, similar to vertebrate rhabdoviruses. These results indicate that the regulation of virus gene transcription for this plant-infecting rhabdovirus is similar to that of some vertebrate-infecting rhabdoviruses.

FOX and ETS family transcription factors regulate the pigment cell lineage in planarians.

PubMed

He, Xinwen; Lindsay-Mosher, Nicole; Li, Yan; Molinaro, Alyssa M; Pellettieri, Jason; Pearson, Bret J

2017-12-15

Many pigment cells acquire unique structural properties and gene expression profiles during animal development. The underlying differentiation pathways have been well characterized in cells formed during embryogenesis, such as the neural crest-derived melanocyte. However, much less is known about the developmental origins of pigment cells produced in adult organisms during tissue homeostasis and repair. Here we report a lineage analysis of ommochrome- and porphyrin-producing cells in the brown, freshwater planarian Schmidtea mediterranea Using an RNA-sequencing approach, we identified two classes of markers expressed in sequential fashion when new pigment cells are generated during regeneration or in response to pigment cell ablation. We also report roles for FOXF-1 and ETS-1 transcription factors, as well as for an FGFR-like molecule, in the specification and maintenance of this cell type. Together, our results provide insights into mechanisms of adult pigment cell development in the strikingly colorful Platyhelminthes phylum. © 2017. Published by The Company of Biologists Ltd.

A comprehensive gene expression analysis at sequential stages of in vitro cardiac differentiation from isolated MESP1-expressing-mesoderm progenitors

PubMed Central

den Hartogh, Sabine C.; Wolstencroft, Katherine; Mummery, Christine L.; Passier, Robert

2016-01-01

In vitro cardiac differentiation of human pluripotent stem cells (hPSCs) closely recapitulates in vivo embryonic heart development, and therefore, provides an excellent model to study human cardiac development. We recently generated the dual cardiac fluorescent reporter MESP1mCherry/wNKX2-5eGFP/w line in human embryonic stem cells (hESCs), allowing the visualization of pre-cardiac MESP1+ mesoderm and their further commitment towards the cardiac lineage, marked by activation of the cardiac transcription factor NKX2-5. Here, we performed a comprehensive whole genome based transcriptome analysis of MESP1-mCherry derived cardiac-committed cells. In addition to previously described cardiac-inducing signalling pathways, we identified novel transcriptional and signalling networks indicated by transient activation and interactive network analysis. Furthermore, we found a highly dynamic regulation of extracellular matrix components, suggesting the importance to create a versatile niche, adjusting to various stages of cardiac differentiation. Finally, we identified cell surface markers for cardiac progenitors, such as the Leucine-rich repeat-containing G-protein coupled receptor 4 (LGR4), belonging to the same subfamily of LGR5, and LGR6, established tissue/cancer stem cells markers. We provide a comprehensive gene expression analysis of cardiac derivatives from pre-cardiac MESP1-progenitors that will contribute to a better understanding of the key regulators, pathways and markers involved in human cardiac differentiation and development. PMID:26783251

Puzzles in modern biology. V. Why are genomes overwired?

PubMed

Frank, Steven A

2017-01-01

Many factors affect eukaryotic gene expression. Transcription factors, histone codes, DNA folding, and noncoding RNA modulate expression. Those factors interact in large, broadly connected regulatory control networks. An engineer following classical principles of control theory would design a simpler regulatory network. Why are genomes overwired? Neutrality or enhanced robustness may lead to the accumulation of additional factors that complicate network architecture. Dynamics progresses like a ratchet. New factors get added. Genomes adapt to the additional complexity. The newly added factors can no longer be removed without significant loss of fitness. Alternatively, highly wired genomes may be more malleable. In large networks, most genomic variants tend to have a relatively small effect on gene expression and trait values. Many small effects lead to a smooth gradient, in which traits may change steadily with respect to underlying regulatory changes. A smooth gradient may provide a continuous path from a starting point up to the highest peak of performance. A potential path of increasing performance promotes adaptability and learning. Genomes gain by the inductive process of natural selection, a trial and error learning algorithm that discovers general solutions for adapting to environmental challenge. Similarly, deeply and densely connected computational networks gain by various inductive trial and error learning procedures, in which the networks learn to reduce the errors in sequential trials. Overwiring alters the geometry of induction by smoothing the gradient along the inductive pathways of improving performance. Those overwiring benefits for induction apply to both natural biological networks and artificial deep learning networks.

Comprehensive gene expression profiling and immunohistochemical studies support application of immunophenotypic algorithm for molecular subtype classification in diffuse large B-cell lymphoma: A report from the International DLBCL Rituximab-CHOP Consortium Program Study

PubMed Central

Visco, Carlo; Li, Yan; Xu-Monette, Zijun Y.; Miranda, Roberto N.; Green, Tina M.; Li, Yong; Tzankov, Alexander; Wen, Wei; Liu, Wei-min; Kahl, Brad S.; d’Amore, Emanuele S. G.; Montes-Moreno, Santiago; Dybkær, Karen; Chiu, April; Tam, Wayne; Orazi, Attilio; Zu, Youli; Bhagat, Govind; Winter, Jane N.; Wang, Huan-You; O’Neill, Stacey; Dunphy, Cherie H.; Hsi, Eric D.; Zhao, X. Frank; Go, Ronald S.; Choi, William W. L.; Zhou, Fan; Czader, Magdalena; Tong, Jiefeng; Zhao, Xiaoying; van Krieken, J. Han; Huang, Qing; Ai, Weiyun; Etzell, Joan; Ponzoni, Maurilio; Ferreri, Andres J. M.; Piris, Miguel A.; Møller, Michael B.; Bueso-Ramos, Carlos E.; Medeiros, L. Jeffrey; Wu, Lin; Young, Ken H.

2013-01-01

Gene expression profiling (GEP) has stratified diffuse large B-cell lymphoma (DLBCL) into molecular subgroups that correspond to different stages of lymphocyte development - namely germinal center B-cell-like and activated B-cell-like. This classification has prognostic significance, but GEP is expensive and not readily applicable into daily practice, which has lead to immunohistochemical algorithms proposed as a surrogate for GEP analysis. We assembled tissue microarrays from 475 de novo DLBCL patients who were treated with rituximab-CHOP chemotherapy. All cases were successfully profiled by GEP on formalin-fixed, paraffin-embedded tissue samples. Sections were stained with antibodies reactive with CD10, GCET1, FOXP1, MUM1, and BCL6 and cases were classified following a rationale of sequential steps of differentiation of B-cells. Cutoffs for each marker were obtained using receiver operating characteristic curves, obviating the need for any arbitrary method. An algorithm based on the expression of CD10, FOXP1, and BCL6 was developed that had a simpler structure than other recently proposed algorithms and 92.6% concordance with GEP. In multivariate analysis, both the International Prognostic Index and our proposed algorithm were significant independent predictors of progression-free and overall survival. In conclusion, this algorithm effectively predicts prognosis of DLBCL patients matching GEP subgroups in the era of rituximab therapy. PMID:22437443

Murine bone cell lines as models for spaceflight induced effects on differentiation and gene expression

NASA Astrophysics Data System (ADS)

Lau, P.; Hellweg, C. E.; Baumstark-Khan, C.; Reitz, G.

Critical health factors for space crews especially on long-term missions are radiation exposure and the absence of gravity DNA double strand breaks DSB are presumed to be the most deleterious DNA lesions after radiation as they disrupt both DNA strands in close proximity Besides radiation risk the absence of gravity influences the complex skeletal apparatus concerning muscle and especially bone remodelling which results from mechanical forces exerting on the body Bone is a dynamic tissue which is life-long remodelled by cells from the osteoblast and osteoclast lineage Any imbalance of this system leads to pathological conditions such as osteoporosis or osteopetrosis Osteoblastic cells play a crucial role in bone matrix synthesis and differentiate either into bone-lining cells or into osteocytes Premature terminal differentiation has been reported to be induced by a number of DNA damaging or cell stress inducing agents including ionising and ultraviolet radiation as well as treatment with mitomycin C In the present study we compare the effects of sequential differentiation by adding osteoinductive substances ss -glycerophosphate and ascorbic acid Radiation-induced premature differentiation was investigated regarding the biosynthesis of specific osteogenic marker molecules and the differentiation dependent expression of marker genes The bone cell model established in our laboratory consists of the osteocyte cell line MLO-Y4 the osteoblast cell line OCT-1 and the subclones 4 and 24 of the osteoblast cell line MC3T3-E1 expressing several

Adenovirus-Associated Virus Vector–Mediated Gene Transfer in Hemophilia B

PubMed Central

Nathwani, Amit C.; Tuddenham, Edward G.D.; Rangarajan, Savita; Rosales, Cecilia; McIntosh, Jenny; Linch, David C.; Chowdary, Pratima; Riddell, Anne; Pie, Arnulfo Jaquilmac; Harrington, Chris; O’Beirne, James; Smith, Keith; Pasi, John; Glader, Bertil; Rustagi, Pradip; Ng, Catherine Y.C.; Kay, Mark A.; Zhou, Junfang; Spence, Yunyu; Morton, Christopher L.; Allay, James; Coleman, John; Sleep, Susan; Cunningham, John M.; Srivastava, Deokumar; Basner-Tschakarjan, Etiena; Mingozzi, Federico; High, Katherine A.; Gray, John T.; Reiss, Ulrike M.; Nienhuis, Arthur W.; Davidoff, Andrew M.

2012-01-01

BACKGROUND Hemophilia B, an X-linked disorder, is ideally suited for gene therapy. We investigated the use of a new gene therapy in patients with the disorder. METHODS We infused a single dose of a serotype-8–pseudotyped, self-complementary adenovirus-associated virus (AAV) vector expressing a codon-optimized human factor IX (FIX) transgene (scAAV2/8-LP1-hFIXco) in a peripheral vein in six patients with severe hemophilia B (FIX activity, <1% of normal values). Study participants were enrolled sequentially in one of three cohorts (given a high, intermediate, or low dose of vector), with two participants in each group. Vector was administered without immunosuppressive therapy, and participants were followed for 6 to 16 months. RESULTS AAV-mediated expression of FIX at 2 to 11% of normal levels was observed in all participants. Four of the six discontinued FIX prophylaxis and remained free of spontaneous hemorrhage; in the other two, the interval between prophylactic injections was increased. Of the two participants who received the high dose of vector, one had a transient, asymptomatic elevation of serum aminotransferase levels, which was associated with the detection of AAV8-capsid–specific T cells in the peripheral blood; the other had a slight increase in liver-enzyme levels, the cause of which was less clear. Each of these two participants received a short course of glucocorticoid therapy, which rapidly normalized aminotransferase levels and maintained FIX levels in the range of 3 to 11% of normal values. CONCLUSIONS Peripheral-vein infusion of scAAV2/8-LP1-hFIXco resulted in FIX transgene expression at levels sufficient to improve the bleeding phenotype, with few side effects. Although immune-mediated clearance of AAV-transduced hepatocytes remains a concern, this process may be controlled with a short course of glucocorticoids without loss of transgene expression. (Funded by the Medical Research Council and others; ClinicalTrials.gov number, NCT00979238.) PMID:22149959

Signal-transducing mechanisms of ketamine-caused inhibition of interleukin-1{beta} gene expression in lipopolysaccharide-stimulated murine macrophage-like Raw 264.7 cells

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

Chen, T.-L.; Chang, C.-C.; Lin, Y.-L.

2009-10-01

Ketamine may affect the host immunity. Interleukin-1{beta} (IL-1{beta}), IL-6, and tumor necrosis factor-{alpha} (TNF-{alpha}) are pivotal cytokines produced by macrophages. This study aimed to evaluate the effects of ketamine on the regulation of inflammatory cytokine gene expression, especially IL-1{beta}, in lipopolysaccharide (LPS)-activated murine macrophage-like Raw 264.7 cells and its possible signal-transducing mechanisms. Administration of Raw 264.7 cells with a therapeutic concentration of ketamine (100 {mu}M), LPS, or a combination of ketamine and LPS for 1, 6, and 24 h was not cytotoxic to macrophages. Exposure to 100 {mu}M ketamine decreased the binding affinity of LPS and LPS-binding protein but didmore » not affect LPS-induced RNA and protein synthesis of TLR4. Treatment with LPS significantly increased IL-1{beta}, IL-6, and TNF-{alpha} gene expressions in Raw 264.7 cells. Ketamine at a clinically relevant concentration did not affect the synthesis of these inflammatory cytokines, but significantly decreased LPS-caused increases in these cytokines. Immunoblot analyses, an electrophoretic mobility shift assay, and a reporter luciferase activity assay revealed that ketamine significantly decreased LPS-induced translocation and DNA binding activity of nuclear factor-kappa B (NF{kappa}B). Administration of LPS sequentially increased the phosphorylations of Ras, Raf, MEK1/2, ERK1/2, and IKK. However, a therapeutic concentration of ketamine alleviated such augmentations. Application of toll-like receptor 4 (TLR4) small interfering (si)RNA reduced cellular TLR4 amounts and ameliorated LPS-induced RAS activation and IL-1{beta} synthesis. Co-treatment with ketamine and TLR4 siRNA synergistically ameliorated LPS-caused enhancement of IL-1{beta} production. Results of this study show that a therapeutic concentration of ketamine can inhibit gene expression of IL-1{beta} possibly through suppressing TLR4-mediated signal-transducing phosphorylations of Ras, Raf, MEK1/2, ERK1/2, and IKK and subsequent translocation and transactivation of NF{kappa}B.« less

Comparative and evolutionary studies of vertebrate ALDH1A-like genes and proteins.

PubMed

Holmes, Roger S

2015-06-05

Vertebrate ALDH1A-like genes encode cytosolic enzymes capable of metabolizing all-trans-retinaldehyde to retinoic acid which is a molecular 'signal' guiding vertebrate development and adipogenesis. Bioinformatic analyses of vertebrate and invertebrate genomes were undertaken using known ALDH1A1, ALDH1A2 and ALDH1A3 amino acid sequences. Comparative analyses of the corresponding human genes provided evidence for distinct modes of gene regulation and expression with putative transcription factor binding sites (TFBS), CpG islands and micro-RNA binding sites identified for the human genes. ALDH1A-like sequences were identified for all mammalian, bird, lizard and frog genomes examined, whereas fish genomes displayed a more restricted distribution pattern for ALDH1A1 and ALDH1A3 genes. The ALDH1A1 gene was absent in many bony fish genomes examined, with the ALDH1A3 gene also absent in the medaka and tilapia genomes. Multiple ALDH1A1-like genes were identified in mouse, rat and marsupial genomes. Vertebrate ALDH1A1, ALDH1A2 and ALDH1A3 subunit sequences were highly conserved throughout vertebrate evolution. Comparative amino acid substitution rates showed that mammalian ALDH1A2 sequences were more highly conserved than for the ALDH1A1 and ALDH1A3 sequences. Phylogenetic studies supported an hypothesis for ALDH1A2 as a likely primordial gene originating in invertebrate genomes and undergoing sequential gene duplication to generate two additional genes, ALDH1A1 and ALDH1A3, in most vertebrate genomes. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

An image cytometric technique is a concise method to detect adenoviruses and host cell proteins and to monitor the infection and cellular responses induced.

PubMed

Morinaga, Takao; Nguyễn, Thảo Thi Thanh; Zhong, Boya; Hanazono, Michiko; Shingyoji, Masato; Sekine, Ikuo; Tada, Yuji; Tatsumi, Koichiro; Shimada, Hideaki; Hiroshima, Kenzo; Tagawa, Masatoshi

2017-11-10

Genetically modified adenoviruses (Ad) with preferential replications in tumor cells have been examined for a possible clinical applicability as an anti-cancer agent. A simple method to detect viral and cellular proteins is valuable to monitor the viral infections and to predict the Ad-mediated cytotoxicity. We used type 5 Ad in which the expression of E1A gene was activated by 5'-regulatory sequences of genes that were augmented in the expression in human tumors. The Ad were further modified to have the fiber-knob region replaced with that derived from type 35 Ad. We infected human mesothelioma cells with the fiber-replaced Ad, and sequentially examined cytotoxic processes together with an expression level of the viral E1A, hexon, and cellular cleaved caspase-3 with image cytometric and Western blot analyses. The replication-competent Ad produced cytotoxicity on mesothelioma cells. The infected cells expressed E1A and hexon 24 h after the infection and then showed cleavage of caspase-3, all of which were detected with image cytometry and Western blot analysis. Image cytometry furthermore demonstrated that increased Ad doses did not enhance an expression level of E1A and hexon in an individual cell and that caspase-3-cleaved cells were found more frequently in hexon-positive cells than in E1A-positive cells. Image cytometry thus detected these molecular changes in a sensitive manner and at a single cell level. We also showed that an image cytometric technique detected expression changes of other host cell proteins, cyclin-E and phosphorylated histone H3 at a single cell level. Image cytometry is a concise procedure to detect expression changes of Ad and host cell proteins at a single cell level, and is useful to analyze molecular events after the infection.

Sequential ChIP Protocol for Profiling Bivalent Epigenetic Modifications (ReChIP).

PubMed

Desvoyes, Bénédicte; Sequeira-Mendes, Joana; Vergara, Zaida; Madeira, Sofia; Gutierrez, Crisanto

2018-01-01

Identification of chromatin modifications, e.g., histone acetylation and methylation, among others, is widely carried out by using a chromatin immunoprecipitation (ChIP) strategy. The information obtained with these procedures is useful to gain an overall picture of modifications present in all cells of the population under study. It also serves as a basis to figure out the mechanisms of chromatin organization and gene regulation at the population level. However, the ultimate goal is to understand gene regulation at the level of single chromatin fibers. This requires the identification of chromatin modifications that occur at a given genomic location and within the same chromatin fiber. This is achieved by following a sequential ChIP strategy using two antibodies to distinguish different chromatin modifications. Here, we describe a sequential ChIP protocol (Re-ChIP), paying special attention to the controls needed and the required steps to obtain meaningful and reproducible results. The protocol is developed for young Arabidopsis seedlings but could be adapted to other plant materials.

Chromatin immunoprecipitation assays: application of ChIP-on-chip for defining dynamic transcriptional mechanisms in bone cells.

PubMed

van der Deen, Margaretha; Hassan, Mohammad Q; Pratap, Jitesh; Teplyuk, Nadiya M; Young, Daniel W; Javed, Amjad; Zaidi, Sayyed K; Lian, Jane B; Montecino, Martin; Stein, Janet L; Stein, Gary S; van Wijnen, Andre J

2008-01-01

Normal cell growth and differentiation of bone cells requires the sequential expression of cell type specific genes to permit lineage specification and development of cellular phenotypes. Transcriptional activation and repression of distinct sets of genes support the anabolic functions of osteoblasts and the catabolic properties of osteoclasts. Furthermore, metastasis of tumors to the bone environment is controlled by transcriptional mechanisms. Insights into the transcriptional regulation of genes in bone cells may provide a conceptual basis for improved therapeutic approaches to treat bone fractures, genetic osteopathologies, and/or cancer metastases to bone. Chromatin immunoprecipitation (ChIP) is a powerful technique to establish in vivo binding of transcription factors to the promoters of genes that are either activated or repressed in bone cells. Combining ChIP with genomic microarray analysis, colloquially referred to as "ChIP-on-chip," has become a valuable method for analysis of endogenous protein/DNA interactions. This technique permits assessment of chromosomal binding sites for transcription factors or the location of histone modifications at a genomic scale. This chapter discusses protocols for performing chromatin immunoprecipitation experiments, with a focus on ChIP-on-chip analysis. The information presented is based on the authors' experience with defining interactions of Runt-related (RUNX) transcription factors with bone-related genes within the context of the native nucleosomal organization of intact osteoblastic cells.

Phytochelatin synthases of the model legume Lotus japonicus. A small multigene family with differential response to cadmium and alternatively spliced variants.

PubMed

Ramos, Javier; Clemente, Maria R; Naya, Loreto; Loscos, Jorge; Pérez-Rontomé, Carmen; Sato, Shusei; Tabata, Satoshi; Becana, Manuel

2007-03-01

The biosynthesis of phytochelatins and homophytochelatins has been studied in nodulated plants of the model legume Lotus (Lotus japonicus). In the first 6 to 24 h of treatment with cadmium (Cd), roots started to synthesize elevated amounts of both polypeptides, with a concomitant increase of glutathione and a decrease of homoglutathione, indicating the presence of active phytochelatin synthase (PCS) genes. Screening of transformation-competent artificial chromosome libraries allowed identification of a cluster of three genes, LjPCS1, LjPCS2, and LjPCS3, which were mapped at 69.0 cM on chromosome 1. The genes differ in exon-intron composition and responsiveness to Cd. Gene structures and phylogenetic analysis of the three protein products, LjPCS1-8R, LjPCS2-7N, and LjPCS3-7N, are consistent with two sequential gene duplication events during evolution of vascular plants. Two sites for alternative splicing in the primary transcripts were identified. One of them, involving intron 2 of the LjPCS2 gene, was confirmed by the finding of the two predicted mRNAs, encoding LjPCS2-7R in roots and LjPCS2-7N in nodules. The amino acid sequences of LjPCS2-7R (or LjPCS2-7N) and LjPCS3-7N share 90% identity, but have only 43% to 59% identity with respect to the typical PCS1 enzymes of Lotus and other plants. The unusual LjPCS2-7N and LjPCS3-7N proteins conferred Cd tolerance when expressed in yeast (Saccharomyces cerevisiae) cells, whereas the alternatively spliced form, LjPCS2-7R, differing only in a five-amino acid motif (GRKWK) did not. These results unveil complex regulatory mechanisms of PCS expression in legume tissues in response to heavy metals and probably to other developmental and environmental factors.

ChIP-re-ChIP: Co-occupancy Analysis by Sequential Chromatin Immunoprecipitation.

PubMed

Beischlag, Timothy V; Prefontaine, Gratien G; Hankinson, Oliver

2018-01-01

Chromatin immunoprecipitation (ChIP) exploits the specific interactions between DNA and DNA-associated proteins. It can be used to examine a wide range of experimental parameters. A number of proteins bound at the same genomic location can identify a multi-protein chromatin complex where several proteins work together to regulate gene transcription or chromatin configuration. In many instances, this can be achieved using sequential ChIP; or simply, ChIP-re-ChIP. Whether it is for the examination of specific transcriptional or epigenetic regulators, or for the identification of cistromes, the ability to perform a sequential ChIP adds a higher level of power and definition to these analyses. In this chapter, we describe a simple and reliable method for the sequential ChIP assay.

Dynamic patterning by the Drosophila pair-rule network reconciles long-germ and short-germ segmentation

PubMed Central

2017-01-01

Drosophila segmentation is a well-established paradigm for developmental pattern formation. However, the later stages of segment patterning, regulated by the “pair-rule” genes, are still not well understood at the system level. Building on established genetic interactions, I construct a logical model of the Drosophila pair-rule system that takes into account the demonstrated stage-specific architecture of the pair-rule gene network. Simulation of this model can accurately recapitulate the observed spatiotemporal expression of the pair-rule genes, but only when the system is provided with dynamic “gap” inputs. This result suggests that dynamic shifts of pair-rule stripes are essential for segment patterning in the trunk and provides a functional role for observed posterior-to-anterior gap domain shifts that occur during cellularisation. The model also suggests revised patterning mechanisms for the parasegment boundaries and explains the aetiology of the even-skipped null mutant phenotype. Strikingly, a slightly modified version of the model is able to pattern segments in either simultaneous or sequential modes, depending only on initial conditions. This suggests that fundamentally similar mechanisms may underlie segmentation in short-germ and long-germ arthropods. PMID:28953896

Insulin-like growth factor-I induces CLU expression through Twist1 to promote prostate cancer growth.

PubMed

Takeuchi, Ario; Shiota, Masaki; Beraldi, Eliana; Thaper, Daksh; Takahara, Kiyoshi; Ibuki, Naokazu; Pollak, Michael; Cox, Michael E; Naito, Seiji; Gleave, Martin E; Zoubeidi, Amina

2014-03-25

Clusterin (CLU) is cytoprotective molecular chaperone that is highly expressed in castrate-resistant prostate cancer (CRPC). CRPC is also characterized by increased insulin-like growth factor (IGF)-I responsiveness which induces prostate cancer survival and CLU expression. However, how IGF-I induces CLU expression and whether CLU is required for IGF-mediated growth signaling remain unknown. Here we show that IGF-I induced CLU via STAT3-Twist1 signaling pathway. In response to IGF-I, STAT3 was phosphorylated, translocated to the nucleus and bound to the Twist1 promoter to activate Twist1 transcription. In turn, Twist1 bound to E-boxes on the CLU promoter and activated CLU transcription. Inversely, we demonstrated that knocking down Twist1 abrogated IGF-I induced CLU expression, indicating that Twist1 mediated IGF-I-induced CLU expression. When PTEN knockout mice were crossed with lit/lit mice, the resultant IGF-I deficiency suppressed Twist1 as well as CLU gene expression in mouse prostate glands. Moreover, both Twist1 and CLU knockdown suppressed prostate cancer growth accelerated by IGF-I, suggesting the relevance of this signaling not only in an in vitro, but also in an in vivo. Collectively, this study indicates that IGF-I induces CLU expression through sequential activation of STAT3 and Twist1, and suggests that this signaling cascade plays a critical role in prostate cancer pathogenesis. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Eicosapentaenoic acid induces DNA demethylation in carcinoma cells through a TET1-dependent mechanism.

PubMed

Ceccarelli, Veronica; Valentini, Virginia; Ronchetti, Simona; Cannarile, Lorenza; Billi, Monia; Riccardi, Carlo; Ottini, Laura; Talesa, Vincenzo Nicola; Grignani, Francesco; Vecchini, Alba

2018-05-14

In cancer cells, global genomic hypomethylation is found together with localized hypermethylation of CpG islands within the promoters and regulatory regions of silenced tumor suppressor genes. Demethylating agents may reverse hypermethylation, thus promoting gene re-expression. Unfortunately, demethylating strategies are not efficient in solid tumor cells. DNA demethylation is mediated by ten-eleven translocation enzymes (TETs). They sequentially convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which is associated with active transcription; 5-formylcytosine; and finally, 5-carboxylcytosine. Although α-linolenic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid, the major n-3 polyunsaturated fatty acids, have anti-cancer effects, their action, as DNA-demethylating agents, has never been investigated in solid tumor cells. Here, we report that EPA demethylates DNA in hepatocarcinoma cells. EPA rapidly increases 5hmC on DNA, inducing p21 Waf1/Cip1 gene expression, which slows cancer cell-cycle progression. We show that the underlying molecular mechanism involves TET1. EPA simultaneously binds peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor α (RXRα), thus promoting their heterodimer and inducing a PPARγ-TET1 interaction. They generate a TET1-PPARγ-RXRα protein complex, which binds to a hypermethylated CpG island on the p21 gene, where TET1 converts 5mC to 5hmC. In an apparent shuttling motion, PPARγ and RXRα leave the DNA, whereas TET1 associates stably. Overall, EPA directly regulates DNA methylation levels, permitting TET1 to exert its anti-tumoral function.-Ceccarelli, V., Valentini, V., Ronchetti, S., Cannarile, L., Billi, M., Riccardi, C., Ottini, L., Talesa, V. N., Grignani, F., Vecchini, A., Eicosapentaenoic acid induces DNA demethylation in carcinoma cells through a TET1-dependent mechanism.

Cyclase inhibitor tripropylamine significantly enhanced lycopene accumulation in Blakeslea trispora.

PubMed

Wang, Yanlong; Chen, Xiwen; Hong, Xiao; Du, Shipeng; Liu, Chunxiao; Gong, Wenfang; Chen, Defu

2016-11-01

Lycopene is a member of carotenoids that exhibits strong antioxidant activity. In this study, on the basis of screening suitable strain combination [ATCC 14271(+) and ATCC 14272(-)] and establishing the optimal inoculation proportion of mated culture (1/2, +/-, w/w) for carotenoid production, the efficiency of compounds, mainly tertiary amines, on enhancing the lycopene content of Blakeslea trispora was systematically assessed. Of these compounds, tripropylamine showed the best enhancing effect, and then sequentially followed by triethylamine, tributylamine, trimethylamine, diisopropylamine, and isopropylamine. After treated with 1.8 g/L tripropylamine for two days, the lycopene proportion was increased from 1.7% to 90.1%, while the β-carotene proportion was decreased from 91.1% to 6.4% of the total carotenoids. In this case, the lycopene and total carotenoid contents were increased to 83.2 and 92.4 mg/gDW, which were 315.8- and 5.9-fold of that of the untreated control, respectively; while the growth of mycelia was only decreased at 6.0 g/L tripropylamine. Gene expression analysis showed that all the tested genes, especially genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (hmgr) and isopentenyl pyrophosphate isomerase (ipi) in mevalonate pathway, as well as phytoene desaturase (carB) in carotenoid biosynthesis process were upregulated. Therefore, tripropylamine enhanced lycopene content of B. trispora by inhibiting the cyclase activity, and by upregulating the expression of genes associated with terpenoid biosynthesis. Besides, a possible association between the structure and the lycopene-enhancing capability of these compounds was also discussed. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Interaction of the androgen receptor, ETV1 and PTEN pathways in mouse prostate varies with pathological stage and predicts cancer progression

PubMed Central

Higgins, Jake; Brogley, Michele; Palanisamy, Nallasivam; Mehra, Rohit; Ittmann, Michael M.; Li, Jun Z.; Tomlins, Scott A.; Robins, Diane M.

2015-01-01

To examine the impact of common somatic mutations in prostate cancer (PCa) on androgen receptor (AR) signaling, mouse models were designed to perturb sequentially the AR, ETV1 and PTEN pathways. Mice with "humanized" AR (hAR) alleles that modified AR transcriptional strength by varying polyglutamine tract (Q-tract) length were crossed with mice expressing a prostate-specific, AR-responsive ETV1 transgene (ETV1Tg). While hAR allele did not grossly affect ETV1-induced neoplasia, ETV1 strongly antagonized global AR regulation and repressed critical androgen-induced differentiation and tumor suppressor genes, such as Nkx3-1 and Hoxb13. When Pten was varied to determine its impact on disease progression, mice lacking one Pten allele (Pten+/−) developed more frequent prostatic intraepithelial neoplasia (PIN). Yet only those with the ETV1 transgene progressed to invasive adenocarcinoma. Furthermore, progression was more frequent with the short Q-tract (stronger) AR, suggesting that the AR, ETV1 and PTEN pathways cooperate in aggressive disease. On the Pten+/− background, ETV1 had markedly less effect on AR target genes. However, a strong inflammatory gene expression signature, notably upregulation of Cxcl16, was induced by ETV1. Comparison of mouse and human patient data stratified by presence of ETS fusion genes highlighted additional factors, some not previously associated with prostate cancer but for which targeted therapies are in development for other diseases. In sum, concerted use of these mouse models illuminates the complex interplay of AR, ETV1 and PTEN pathways in pre-cancerous neoplasia and early tumorigenesis, disease stages difficult to analyze in man. PMID:25631336

Assisted Reproductive Technology affects developmental kinetics, H19 Imprinting Control Region methylation and H19 gene expression in individual mouse embryos

PubMed Central

Fauque, Patricia; Jouannet, Pierre; Lesaffre, Corinne; Ripoche, Marie-Anne; Dandolo, Luisa; Vaiman, Daniel; Jammes, Hélène

2007-01-01

Background In the last few years, an increase in imprinting anomalies has been reported in children born from Assisted Reproductive Technology (ART). Various clinical and experimental studies also suggest alterations of embryo development after ART. Therefore, there is a need for studying early epigenetic anomalies which could result from ART manipulations, especially on single embryos. In this study, we evaluated the impact of superovulation, in vitro fertilization (IVF) and embryo culture conditions on proper genomic imprinting and blastocyst development in single mouse embryos. In this study, different experimental groups were established to obtain embryos from superovulated and non-superovulated females, either from in vivo or in vitro fertilized oocytes, themselves grown in vitro or not. The embryos were cultured either in M16 medium or in G1.2/G2.2 sequential medium. The methylation status of H19 Imprinting Control Region (ICR) and H19 promoter was assessed, as well as the gene expression level of H19, in individual blastocysts. In parallel, we have evaluated embryo cleavage kinetics and recorded morphological data. Results We show that: 1. The culture medium influences early embryo development with faster cleavage kinetics for culture in G1.2/G2.2 medium compared to M16 medium. 2. Epigenetic alterations of the H19 ICR and H19 PP are influenced by the fertilization method since methylation anomalies were observed only in the in vitro fertilized subgroup, however to different degrees according to the culture medium. 3. Superovulation clearly disrupted H19 gene expression in individual blastocysts. Moreover, when embryos were cultured in vitro after either in vivo or in vitro fertilization, the percentage of blastocysts which expressed H19 was higher in G1.2/G2.2 medium compared to M16. Conclusion Compared to previous reports utilizing pools of embryos, our study enables us to emphasize a high individual variability of blastocysts in the H19 ICR and H19 promoter methylation and H19 gene expression, with a striking effect of each manipulation associated to ART practices. Our results suggest that H19 could be used as a sensor of the epigenetic disturbance of the utilized techniques. PMID:17949482

Assisted Reproductive Technology affects developmental kinetics, H19 Imprinting Control Region methylation and H19 gene expression in individual mouse embryos.

PubMed

Fauque, Patricia; Jouannet, Pierre; Lesaffre, Corinne; Ripoche, Marie-Anne; Dandolo, Luisa; Vaiman, Daniel; Jammes, Hélène

2007-10-18

In the last few years, an increase in imprinting anomalies has been reported in children born from Assisted Reproductive Technology (ART). Various clinical and experimental studies also suggest alterations of embryo development after ART. Therefore, there is a need for studying early epigenetic anomalies which could result from ART manipulations, especially on single embryos. In this study, we evaluated the impact of superovulation, in vitro fertilization (IVF) and embryo culture conditions on proper genomic imprinting and blastocyst development in single mouse embryos. In this study, different experimental groups were established to obtain embryos from superovulated and non-superovulated females, either from in vivo or in vitro fertilized oocytes, themselves grown in vitro or not. The embryos were cultured either in M16 medium or in G1.2/G2.2 sequential medium. The methylation status of H19 Imprinting Control Region (ICR) and H19 promoter was assessed, as well as the gene expression level of H19, in individual blastocysts. In parallel, we have evaluated embryo cleavage kinetics and recorded morphological data. We show that: 1. The culture medium influences early embryo development with faster cleavage kinetics for culture in G1.2/G2.2 medium compared to M16 medium. 2. Epigenetic alterations of the H19 ICR and H19 PP are influenced by the fertilization method since methylation anomalies were observed only in the in vitro fertilized subgroup, however to different degrees according to the culture medium. 3. Superovulation clearly disrupted H19 gene expression in individual blastocysts. Moreover, when embryos were cultured in vitro after either in vivo or in vitro fertilization, the percentage of blastocysts which expressed H19 was higher in G1.2/G2.2 medium compared to M16. Compared to previous reports utilizing pools of embryos, our study enables us to emphasize a high individual variability of blastocysts in the H19 ICR and H19 promoter methylation and H19 gene expression, with a striking effect of each manipulation associated to ART practices. Our results suggest that H19 could be used as a sensor of the epigenetic disturbance of the utilized techniques.

Frequency of Germline Mutations in 25 Cancer Susceptibility Genes in a Sequential Series of Patients With Breast Cancer

PubMed Central

Lin, Nancy U.; Kidd, John; Allen, Brian A.; Singh, Nanda; Wenstrup, Richard J.; Hartman, Anne-Renee; Winer, Eric P.; Garber, Judy E.

2016-01-01

Purpose Testing for germline mutations in BRCA1/2 is standard for select patients with breast cancer to guide clinical management. Next-generation sequencing (NGS) allows testing for mutations in additional breast cancer predisposition genes. The frequency of germline mutations detected by using NGS has been reported in patients with breast cancer who were referred for BRCA1/2 testing or with triple-negative breast cancer. We assessed the frequency and predictors of mutations in 25 cancer predisposition genes, including BRCA1/2, in a sequential series of patients with breast cancer at an academic institution to examine the utility of genetic testing in this population. Methods Patients with stages I to III breast cancer who were seen at a single cancer center between 2010 and 2012, and who agreed to participate in research DNA banking, were included (N = 488). Personal and family cancer histories were collected and germline DNA was sequenced with NGS to identify mutations. Results Deleterious mutations were identified in 10.7% of women, including 6.1% in BRCA1/2 (5.1% in non-Ashkenazi Jewish patients) and 4.6% in other breast/ovarian cancer predisposition genes including CHEK2 (n = 10), ATM (n = 4), BRIP1 (n = 4), and one each in PALB2, PTEN, NBN, RAD51C, RAD51D, MSH6, and PMS2. Whereas young age (P < .01), Ashkenazi Jewish ancestry (P < .01), triple-negative breast cancer (P = .01), and family history of breast/ovarian cancer (P = .01) predicted for BRCA1/2 mutations, no factors predicted for mutations in other breast cancer predisposition genes. Conclusion Among sequential patients with breast cancer, 10.7% were found to have a germline mutation in a gene that predisposes women to breast or ovarian cancer, using a panel of 25 predisposition genes. Factors that predict for BRCA1/2 mutations do not predict for mutations in other breast/ovarian cancer susceptibility genes when these genes are analyzed as a single group. Additional cohorts will be helpful to define individuals at higher risk of carrying mutations in genes other than BRCA1/2. PMID:26976419

Role of genetic mutations in folate-related enzyme genes on Male Infertility

PubMed Central

Liu, Kang; Zhao, Ruizhe; Shen, Min; Ye, Jiaxin; Li, Xiao; Huang, Yuan; Hua, Lixin; Wang, Zengjun; Li, Jie

2015-01-01

Several studies showed that the genetic mutations in the folate-related enzyme genes might be associated with male infertility; however, the results were still inconsistent. We performed a meta-analysis with trial sequential analysis to investigate the associations between the MTHFR C677T, MTHFR A1298C, MTR A2756G, MTRR A66G mutations and the MTHFR haplotype with the risk of male infertility. Overall, a total of 37 studies were selected. Our meta-analysis showed that the MTHFR C677T mutation was a risk factor for male infertility in both azoospermia and oligoasthenoteratozoospermia patients, especially in Asian population. Men carrying the MTHFR TC haplotype were most liable to suffer infertility while those with CC haplotype had lowest risk. On the other hand, the MTHFR A1298C mutation was not related to male infertility. MTR A2756G and MTRR A66G were potential candidates in the pathogenesis of male infertility, but more case-control studies were required to avoid false-positive outcomes. All of these results were confirmed by the trial sequential analysis. Finally, our meta-analysis with trial sequential analysis proved that the genetic mutations in the folate-related enzyme genes played a significant role in male infertility. PMID:26549413

CRISPR/Cas9 mediated sequential editing of genes critical for ookinete motility in Plasmodium yoelii.

PubMed

Zhang, Cui; Gao, Han; Yang, Zhenke; Jiang, Yuanyuan; Li, Zhenkui; Wang, Xu; Xiao, Bo; Su, Xin-Zhuan; Cui, Huiting; Yuan, Jing

2017-03-01

CRISPR/Cas9 has been successfully adapted for gene editing in malaria parasites including Plasmodium falciparum and Plasmodium yoelii. However, the reported methods were limited to editing one gene at a time. In practice, it is often desired to modify multiple genetic loci in a parasite genome. Here we describe a CRISPR/Cas9 mediated genome editing method that allows successive modification of more than one gene in the genome of P. yoelii using an improved single-vector system (pYCm) we developed previously. Drug resistant genes encoding human dihydrofolate reductase (hDHFR) and a yeast bifunctional protein (yFCU), with cytosine deaminase (CD) and uridyl phosphoribosyl transferase (UPRT) activities in the plasmid, allowed sequential positive (pyrimethamine, Pyr) and negative (5-fluorocytosine, 5FC) selections and generation of transgenic parasites free of the episomal plasmid after genetic modification. Using this system, we were able to efficiently tag a gene of interest (Pyp28) and subsequently disrupted two genes (Pyctrp and Pycdpk3) that are individually critical for ookinete motility. Disruption of the genes either eliminated (Pyctrp) or greatly reduced (Pycdpk3) ookinete forward motility in matrigel in vitro and completely blocked oocyst development in mosquito midgut. The method will greatly facilitate studies of parasite gene function, development, and disease pathogenesis. Copyright © 2016 Elsevier B.V. All rights reserved.

Stem cells and regenerative medicine for diabetes mellitus.

PubMed

Sumi, Shoichiro; Gu, Yuanjun; Hiura, Akihito; Inoue, Kazutomo

2004-10-01

A profound knowledge of the development and differentiation of pancreatic tissues, especially islets of Langerhans, is necessary for developing regenerative therapy for severe diabetes mellitus. A recent developmental study showed that PTF-1a is expressed in almost all parts of pancreatic tissues, in addition to PDX-1, a well-known transcription factor that is essential for pancreas development. Another study suggested that alpha cells and beta cells individually, but not sequentially, differentiated from neurogenin-3--expressing precursor cells. Under strong induction of pancreas regeneration, it is likely that pancreatic duct cells dedifferentiate to grow, express PDX-1, and re-differentiate toward other cell types including islet cells. Duct epithelium-like cells can be cultivated from crude pancreatic exocrine cells and can be induced to differentiate toward islet-like cell clusters under some culture conditions. These cell clusters made from murine pancreas have been shown to control hyperglycemia when transplanted into diabetic mice. Liver-derived oval cells and their putative precursor H-CFU-C have been shown to differentiate toward pancreatic cells. Furthermore, extrapancreatic cells contained in bone marrow and amniotic membrane are reported to become insulin-producing cells. However, their exact characterization and relationship between these cell types remain to be elucidated. Our recent study has shown that islet-like cell clusters can be differentiated from mouse embryonic stem cells. Transplantation of these clusters could ameliorate hyperglycemia of STZ-induced diabetic mice without forming teratomas. Interestingly, these cells expressed several genes specific to exocrine pancreatic tissue in addition to islet-related genes, suggesting that stable and efficient differentiation toward certain tissues can only be achieved through a process mimicking normal development of the tissue. Perhaps recent developments in these fields may rapidly lead to an established regenerative therapy for diabetes mellitus.

APP intracellular domain derived from amyloidogenic β- and γ-secretase cleavage regulates neprilysin expression

PubMed Central

Grimm, Marcus O. W.; Mett, Janine; Stahlmann, Christoph P.; Grösgen, Sven; Haupenthal, Viola J.; Blümel, Tamara; Hundsdörfer, Benjamin; Zimmer, Valerie C.; Mylonas, Nadine T.; Tanila, Heikki; Müller, Ulrike; Grimm, Heike S.; Hartmann, Tobias

2015-01-01

Alzheimer's disease (AD) is characterized by an accumulation of Amyloid-β (Aβ), released by sequential proteolytic processing of the amyloid precursor protein (APP) by β - and γ-secretase. Aβ peptides can aggregate, leading to toxic Aβ oligomers and amyloid plaque formation. Aβ accumulation is not only dependent on de novo synthesis but also on Aβ degradation. Neprilysin (NEP) is one of the major enzymes involved in Aβ degradation. Here we investigate the molecular mechanism of NEP regulation, which is up to now controversially discussed to be affected by APP processing itself. We found that NEP expression is highly dependent on the APP intracellular domain (AICD), released by APP processing. Mouse embryonic fibroblasts devoid of APP processing, either by the lack of the catalytically active subunit of the γ-secretase complex [presenilin (PS) 1/2] or by the lack of APP and the APP-like protein 2 (APLP2), showed a decreased NEP expression, activity and protein level. Similar results were obtained by utilizing cells lacking a functional AICD domain (APPΔCT15) or expressing mutations in the genes encoding for PS1. AICD supplementation or retransfection with an AICD encoding plasmid could rescue the down-regulation of NEP further strengthening the link between AICD and transcriptional NEP regulation, in which Fe65 acts as an important adaptor protein. Especially AICD generated by the amyloidogenic pathway seems to be more involved in the regulation of NEP expression. In line, analysis of NEP gene expression in vivo in six transgenic AD mouse models (APP and APLP2 single knock-outs, APP/APLP2 double knock-out, APP-swedish, APP-swedish/PS1Δexon9, and APPΔCT15) confirmed the results obtained in cell culture. In summary, in the present study we clearly demonstrate an AICD-dependent regulation of the Aβ-degrading enzyme NEP in vitro and in vivo and elucidate the underlying mechanisms that might be beneficial to develop new therapeutic strategies for the treatment of AD. PMID:26074811

The Receptive-Expressive Gap in the Vocabulary of Young Second-Language Learners: Robustness and Possible Mechanisms

ERIC Educational Resources Information Center

Gibson, Todd A.; Oller, D. Kimbrough; Jarmulowicz, Linda; Ethington, Corinna A.

2012-01-01

Adults and children learning a second language show difficulty accessing expressive vocabulary that appears accessible receptively in their first language (L1). We call this discrepancy the receptive-expressive gap. Kindergarten Spanish (L1)-English (L2) sequential bilinguals were given standardized tests of receptive and expressive vocabulary in…

Dynamic Cytology and Transcriptional Regulation of Rice Lamina Joint Development1[OPEN

PubMed Central

2017-01-01

Rice (Oryza sativa) leaf angle is determined by lamina joint and is an important agricultural trait determining leaf erectness and, hence, the photosynthesis efficiency and grain yield. Genetic studies reveal a complex regulatory network of lamina joint development; however, the morphological changes, cytological transitions, and underlying transcriptional programming remain to be elucidated. A systemic morphological and cytological study reveals a dynamic developmental process and suggests a common but distinct regulation of the lamina joint. Successive and sequential cell division and expansion, cell wall thickening, and programmed cell death at the adaxial or abaxial sides form the cytological basis of the lamina joint, and the increased leaf angle results from the asymmetric cell proliferation and elongation. Analysis of the gene expression profiles at four distinct developmental stages ranging from initiation to senescence showed that genes related to cell division and growth, hormone synthesis and signaling, transcription (transcription factors), and protein phosphorylation (protein kinases) exhibit distinct spatiotemporal patterns during lamina joint development. Phytohormones play crucial roles by promoting cell differentiation and growth at early stages or regulating the maturation and senescence at later stages, which is consistent with the quantitative analysis of hormones at different stages. Further comparison with the gene expression profile of leaf inclination1, a mutant with decreased auxin and increased leaf angle, indicates the coordinated effects of hormones in regulating lamina joint. These results reveal a dynamic cytology of rice lamina joint that is fine-regulated by multiple factors, providing informative clues for illustrating the regulatory mechanisms of leaf angle and plant architecture. PMID:28500269

Dynamic Cytology and Transcriptional Regulation of Rice Lamina Joint Development.

PubMed

Zhou, Li-Juan; Xiao, Lang-Tao; Xue, Hong-Wei

2017-07-01

Rice ( Oryza sativa ) leaf angle is determined by lamina joint and is an important agricultural trait determining leaf erectness and, hence, the photosynthesis efficiency and grain yield. Genetic studies reveal a complex regulatory network of lamina joint development; however, the morphological changes, cytological transitions, and underlying transcriptional programming remain to be elucidated. A systemic morphological and cytological study reveals a dynamic developmental process and suggests a common but distinct regulation of the lamina joint. Successive and sequential cell division and expansion, cell wall thickening, and programmed cell death at the adaxial or abaxial sides form the cytological basis of the lamina joint, and the increased leaf angle results from the asymmetric cell proliferation and elongation. Analysis of the gene expression profiles at four distinct developmental stages ranging from initiation to senescence showed that genes related to cell division and growth, hormone synthesis and signaling, transcription (transcription factors), and protein phosphorylation (protein kinases) exhibit distinct spatiotemporal patterns during lamina joint development. Phytohormones play crucial roles by promoting cell differentiation and growth at early stages or regulating the maturation and senescence at later stages, which is consistent with the quantitative analysis of hormones at different stages. Further comparison with the gene expression profile of leaf inclination1 , a mutant with decreased auxin and increased leaf angle, indicates the coordinated effects of hormones in regulating lamina joint. These results reveal a dynamic cytology of rice lamina joint that is fine-regulated by multiple factors, providing informative clues for illustrating the regulatory mechanisms of leaf angle and plant architecture. © 2017 American Society of Plant Biologists. All Rights Reserved.

An Intracellular Laccase Is Responsible for Epicatechin-Mediated Anthocyanin Degradation in Litchi Fruit Pericarp1[OPEN

PubMed Central

Fang, Fang; Zhang, Xue-lian; Gong, Yi-hui; Li, Wen-jun; Shi, Zhao-wan; He, Quan; Wu, Qing; Li, Lu; Jiang, Lin-lin; Cai, Zhi-gao; Oren-Shamir, Michal; Zhang, Zhao-qi

2015-01-01

In contrast to the detailed molecular knowledge available on anthocyanin synthesis, little is known about its catabolism in plants. Litchi (Litchi chinensis) fruit lose their attractive red color soon after harvest. The mechanism leading to quick degradation of anthocyanins in the pericarp is not well understood. An anthocyanin degradation enzyme (ADE) was purified to homogeneity by sequential column chromatography, using partially purified anthocyanins from litchi pericarp as a substrate. The purified ADE, of 116 kD by urea SDS-PAGE, was identified as a laccase (ADE/LAC). The full-length complementary DNA encoding ADE/LAC was obtained, and a polyclonal antibody raised against a deduced peptide of the gene recognized the ADE protein. The anthocyanin degradation function of the gene was confirmed by its transient expression in tobacco (Nicotiana benthamiana) leaves. The highest ADE/LAC transcript abundance was in the pericarp in comparison with other tissues, and was about 1,000-fold higher than the polyphenol oxidase gene in the pericarp. Epicatechin was found to be the favorable substrate for the ADE/LAC. The dependence of anthocyanin degradation by the enzyme on the presence of epicatechin suggests an ADE/LAC epicatechin-coupled oxidation model. This model was supported by a dramatic decrease in epicatechin content in the pericarp parallel to anthocyanin degradation. Immunogold labeling transmission electron microscopy suggested that ADE/LAC is located mainly in the vacuole, with essential phenolic substances. ADE/LAC vacuolar localization, high expression levels in the pericarp, and high epicatechin-dependent anthocyanin degradation support its central role in pigment breakdown during pericarp browning. PMID:26514808

Changes in ethylene signaling and MADS box gene expression are associated with banana finger drop.

PubMed

Hubert, O; Piral, G; Galas, C; Baurens, F-C; Mbéguié-A-Mbéguié, D

2014-06-01

Banana finger drop was examined in ripening banana harvested at immature (iMG), early (eMG) and late mature green (lMG) stages, with contrasting ripening rates and ethylene sensitivities. Concomitantly, 11 ethylene signal transduction components (ESTC) and 6 MADS box gene expressions were comparatively studied in median (control zone, CZ) and pedicel rupture (drop zone DZ) areas in peel tissue. iMG fruit did not ripen or develop finger drop while eMG and lMG fruits displayed a similar finger drop pattern. Several ESTC and MADS box gene mRNAs were differentially induced in DZ and CZ and sequentially in eMG and lMG fruits. MaESR2, 3 and MaEIL1, MaMADS2 and MaMADS5 had a higher mRNA level in eMG and acted earlier, whereas MaERS1, MaCTR1, MaEIL3/AB266319, MaEIL4/AB266320 and MaEIL5/AB266321, MaMADS4 and to a lesser extent MaMADS2 and 5 acted later in lMG. In this fruit, MaERS1 and 3, MaCTR1, MaEIL3, 4 and MaEIL5/AB266321, and MaMADS4 were enhanced by finger drop, suggesting their specific involvement in this process. MaEIL1, MaMADS1 and 3, induced at comparable levels in DZ and CZ, are probably related to the overall fruit ripening process. These findings led us to consider that developmental cues are the predominant finger drop regulation factor. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Transcriptomic profiling of adaptive responses to ocean acidification.

PubMed

Goncalves, Priscila; Jones, David B; Thompson, Emma L; Parker, Laura M; Ross, Pauline M; Raftos, David A

2017-11-01

Some populations of marine organisms appear to have inherent tolerance or the capacity for acclimation to stressful environmental conditions, including those associated with climate change. Sydney rock oysters from the B2 breeding line exhibit resilience to ocean acidification (OA) at the physiological level. To understand the molecular basis of this physiological resilience, we analysed the gill transcriptome of B2 oysters that had been exposed to near-future projected ocean pH over two consecutive generations. Our results suggest that the distinctive performance of B2 oysters in the face of OA is mediated by the selective expression of genes involved in multiple cellular processes. Subsequent high-throughput qPCR revealed that some of these transcriptional changes are exclusive to B2 oysters and so may be associated with their resilience to OA. The intracellular processes mediated by the differentially abundant genes primarily involve control of the cell cycle and maintenance of cellular homeostasis. These changes may enable B2 oysters to prevent apoptosis resulting from oxidative damage or to alleviate the effects of apoptosis through regulation of the cell cycle. Comparative analysis of the OA conditioning effects across sequential generations supported the contention that B2 and wild-type oysters have different trajectories of changing gene expression and responding to OA. Our findings reveal the broad set of molecular processes underlying transgenerational conditioning and potential resilience to OA in a marine calcifier. Identifying the mechanisms of stress resilience can uncover the intracellular basis for these organisms to survive and thrive in a rapidly changing ocean. © 2017 John Wiley & Sons Ltd.

Regulatory Shifts in Plastid Transcription Play a Key Role in Morphological Conversions of Plastids during Plant Development.

PubMed

Liebers, Monique; Grübler, Björn; Chevalier, Fabien; Lerbs-Mache, Silva; Merendino, Livia; Blanvillain, Robert; Pfannschmidt, Thomas

2017-01-01

Plastids display a high morphological and functional diversity. Starting from an undifferentiated small proplastid, these plant cell organelles can develop into four major forms: etioplasts in the dark, chloroplasts in green tissues, chromoplasts in colored flowers and fruits and amyloplasts in roots. The various forms are interconvertible into each other depending on tissue context and respective environmental condition. Research of the last two decades uncovered that each plastid type contains its own specific proteome that can be highly different from that of the other types. Composition of these proteomes largely defines the enzymatic functionality of the respective plastid. The vast majority of plastid proteins is encoded in the nucleus and must be imported from the cytosol. However, a subset of proteins of the photosynthetic and gene expression machineries are encoded on the plastid genome and are transcribed by a complex transcriptional apparatus consisting of phage-type nuclear-encoded RNA polymerases and a bacterial-type plastid-encoded RNA polymerase. Both types recognize specific sets of promoters and transcribe partly over-lapping as well as specific sets of genes. Here we summarize the current knowledge about the sequential activity of these plastid RNA polymerases and their relative activities in different types of plastids. Based on published plastid gene expression profiles we hypothesize that each conversion from one plastid type into another is either accompanied or even preceded by significant changes in plastid transcription suggesting that these changes represent important determinants of plastid morphology and protein composition and, hence, the plastid type.

Regulatory Shifts in Plastid Transcription Play a Key Role in Morphological Conversions of Plastids during Plant Development

PubMed Central

Liebers, Monique; Grübler, Björn; Chevalier, Fabien; Lerbs-Mache, Silva; Merendino, Livia; Blanvillain, Robert; Pfannschmidt, Thomas

2017-01-01

Plastids display a high morphological and functional diversity. Starting from an undifferentiated small proplastid, these plant cell organelles can develop into four major forms: etioplasts in the dark, chloroplasts in green tissues, chromoplasts in colored flowers and fruits and amyloplasts in roots. The various forms are interconvertible into each other depending on tissue context and respective environmental condition. Research of the last two decades uncovered that each plastid type contains its own specific proteome that can be highly different from that of the other types. Composition of these proteomes largely defines the enzymatic functionality of the respective plastid. The vast majority of plastid proteins is encoded in the nucleus and must be imported from the cytosol. However, a subset of proteins of the photosynthetic and gene expression machineries are encoded on the plastid genome and are transcribed by a complex transcriptional apparatus consisting of phage-type nuclear-encoded RNA polymerases and a bacterial-type plastid-encoded RNA polymerase. Both types recognize specific sets of promoters and transcribe partly over-lapping as well as specific sets of genes. Here we summarize the current knowledge about the sequential activity of these plastid RNA polymerases and their relative activities in different types of plastids. Based on published plastid gene expression profiles we hypothesize that each conversion from one plastid type into another is either accompanied or even preceded by significant changes in plastid transcription suggesting that these changes represent important determinants of plastid morphology and protein composition and, hence, the plastid type. PMID:28154576

Pax2/8 proteins coordinate sequential induction of otic and epibranchial placodes through differential regulation of foxi1, sox3 and fgf24

PubMed Central

Padanad, Mahesh S.; Riley, Bruce B.

2011-01-01

Vertebrate cranial placodes contribute vitally to development of sensory structures of the head. Amongst posterior placodes, the otic placode forms the inner ear whereas nearby epibranchial placodes produce sensory ganglia within branchial clefts. Though diverse in fate, these placodes show striking similarities in their early regulation. In zebrafish, both are initiated by localized Fgf signaling plus the ubiquitous competence factor Foxi1, and both express pax8 and sox3 in response. It has been suggested that Fgf initially induces a common otic/epibranchial field, which later subdivides in response to other signals. However, we find that otic and epibranchial placodes form at different times and by distinct mechanisms. Initially, Fgf from surrounding tissues induces otic expression of pax8 and sox3, which cooperate synergistically to establish otic fate. Subsequently, pax8 works with related genes pax2a/pax2b to downregulate otic expression of foxi1, a necessary step for further otic development. Additionally, pax2/8 activate otic expression of fgf24, which induces epibranchial expression of sox3. Knockdown of fgf24 or sox3 causes severe epibranchial deficiencies but has little effect on otic development. These findings clarify the roles of pax8 and sox3 and support a model whereby the otic placode forms first and induces epibranchial placodes through an Fgf-relay. PMID:21215261

A minimal murine Msx-1 gene promoter. Organization of its cis-regulatory motifs and their role in transcriptional activation in cells in culture and in transgenic mice.

PubMed

Takahashi, T; Guron, C; Shetty, S; Matsui, H; Raghow, R

1997-09-05

To dissect the cis-regulatory elements of the murine Msx-1 promoter, which lacks a conventional TATA element, a putative Msx-1 promoter DNA fragment (from -1282 to +106 base pairs (bp)) or its congeners containing site-specific alterations were fused to luciferase reporter and introduced into NIH3T3 and C2C12 cells, and the expression of luciferase was assessed in transient expression assays. The functional consequences of the sequential 5' deletions of the promotor revealed that multiple positive and negative regulatory elements participate in regulating transcription of the Msx-1 gene. Surprisingly, however, the optimal expression of Msx-1 promoter in either NIH3T3 or C2C12 cells required only 165 bp of the upstream sequence to warrant detailed examination of its structure. Therefore, the functional consequences of site-specific deletions and point mutations of the cis-acting elements of the minimal Msx-1 promoter were systematically examined. Concomitantly, potential transcriptional factor(s) interacting with the cis-acting elements of the minimal promoter were also studied by gel electrophoretic mobility shift assays and DNase I footprinting. Combined analyses of the minimal promoter by DNase I footprinting, electrophoretic mobility shift assays, and super shift assays with specific antibodies revealed that 5'-flanking regions from -161 to -154 and from -26 to -13 of the Msx-1 promoter contains an authentic E box (proximal E box), capable of binding a protein immunologically related to the upstream stimulating factor 1 (USF-1) and a GC-rich sequence motif which can bind to Sp1 (proximal Sp1), respectively. Additionally, we observed that the promoter activation was seriously hampered if the proximal E box was removed or mutated, and the promoter activity was eliminated completely if the proximal Sp1 site was similarly altered. Absolute dependence of the Msx-1 minimal promoter on Sp1 could be demonstrated by transient expression assays in the Sp1-deficient Drosophila cell line cotransfected with Msx-1-luciferase and an Sp1 expression vector pPacSp1. The transgenic mice embryos containing -165/106-bp Msx-1 promoter-LacZ DNA in their genomes abundantly expressed beta-galactosidase in maxillae and mandibles and in the cellular primordia involved in the formation of the meninges and the bones of the skull. Thus, the truncated murine Msx-1 promoter can target expression of a heterologous gene in the craniofacial tissues of transgenic embryos known for high level of expression of the endogenous Msx-1 gene and found to be severely defective in the Msx-1 knock-out mice.

Framework for Parallel Preprocessing of Microarray Data Using Hadoop

PubMed Central

2018-01-01

Nowadays, microarray technology has become one of the popular ways to study gene expression and diagnosis of disease. National Center for Biology Information (NCBI) hosts public databases containing large volumes of biological data required to be preprocessed, since they carry high levels of noise and bias. Robust Multiarray Average (RMA) is one of the standard and popular methods that is utilized to preprocess the data and remove the noises. Most of the preprocessing algorithms are time-consuming and not able to handle a large number of datasets with thousands of experiments. Parallel processing can be used to address the above-mentioned issues. Hadoop is a well-known and ideal distributed file system framework that provides a parallel environment to run the experiment. In this research, for the first time, the capability of Hadoop and statistical power of R have been leveraged to parallelize the available preprocessing algorithm called RMA to efficiently process microarray data. The experiment has been run on cluster containing 5 nodes, while each node has 16 cores and 16 GB memory. It compares efficiency and the performance of parallelized RMA using Hadoop with parallelized RMA using affyPara package as well as sequential RMA. The result shows the speed-up rate of the proposed approach outperforms the sequential approach and affyPara approach. PMID:29796018

UGT74S1 is the key player in controlling secoisolariciresinol diglucoside (SDG) formation in flax.

PubMed

Fofana, Bourlaye; Ghose, Kaushik; McCallum, Jason; You, Frank M; Cloutier, Sylvie

2017-02-02

Flax lignan, commonly known as secoisolariciresinol (SECO) diglucoside (SDG), has recently been reported with health-promoting activities, including its positive impact in metabolic diseases. However, not much was reported on the biosynthesis of SDG and its monoglucoside (SMG) until lately. Flax UGT74S1 was recently reported to sequentially glucosylate SECO into SMG and SDG in vitro. However, whether this gene is the only UGT achieving SECO glucosylation in flax was not known. Flax genome-wide mining for UGTs was performed. Phylogenetic and gene duplication analyses, heterologous gene expression and enzyme assays were conducted to identify family members closely related to UGT74S1 and to establish their roles in SECO glucosylation. A total of 299 different UGTs were identified, of which 241 (81%) were duplicated. Flax UGTs diverged 2.4-153.6 MYA and 71% were found to be under purifying selection pressure. UGT74S1, a single copy gene located on chromosome 7, displayed no evidence of duplication and was deemed to be under positive selection pressure. The phylogenetic analysis identified four main clusters where cluster 4, which included UGT74S1, was the most diverse. The duplicated UGT74S4 and UGT74S3, located on chromosomes 8 and 14, respectively, were the most closely related to UGT74S1 and were differentially expressed in different tissues. Heterologous expression levels of UGT74S1, UGT74S4 and UGT74S3 proteins were similar but UGT74S4 and UGT74S3 glucosylation activity towards SECO was seven fold less than UGT74S1. In addition, they both failed to produce SDG, suggesting neofunctionalization following their divergence from UGT74S1. We showed that UGT74S1 is closely related to two duplicated genes, UGT74S4 and UGT74S3 which, unlike UGT74S1, failed to glucosylate SMG into SDG. The study suggests that UGT74S1 may be the key player in controlling SECO glucosylation into SDG in flax although its closely related genes may also contribute to a minor extent in supplying the SMG precursor to UGT74S1.

Paracrine and autocrine signals promoting full chondrogenic differentiation of a mesoblastic cell line.

PubMed

Locker, Morgane; Kellermann, Odile; Boucquey, Marie; Khun, Huot; Huerre, Michel; Poliard, Anne

2004-01-01

The pluripotent mesoblastic C1 cell line was used under serum-free culture conditions to investigate how paracrine and autocrine signals cooperate to drive chondrogenesis. Sequential addition of two systemic hormones, dexamethasone and triiodothyronine, permits full chondrogenic differentiation. The cell intrinsic activation of the BMP signaling pathway and Sox9 expression occurring on mesoblastic condensation is insufficient for recruitment of the progenitors. Dexamethasone-dependent Sox9 upregulation is essential for chondrogenesis. Differentiation of lineage stem cells relies on cell autonomous regulations modulated by external signals. We used the pluripotent mesoblastic C1 cell line under serum-free culture conditions to investigate how paracrine and autocrine signals cooperate to induce differentiation of a precursor clone along the chondrogenic lineage. C1 cells, cultured as aggregates, were induced toward chondrogenesis by addition of 10(-7) M dexamethasone in serum-free medium. After 30 days, dexamethasone was replaced by 10 nM triiodothyronine to promote final hypertrophic conversion. Mature and hypertrophic phenotypes were characterized by immunocytochemistry using specific antibodies against types II and X collagens, respectively. Type II collagen, bone morphogenetic proteins (BMPs), BMP receptors, Smads, and Sox9 expression were monitored by reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blot, and/or Western blot analysis. Once C1 cells have formed nodules, sequential addition of two systemic hormones is sufficient to promote full chondrogenic differentiation. In response to dexamethasone, nearly 100% of the C1 precursors engage in chondrogenesis and convert within 30 days into mature chondrocytes, which triggers a typical cartilage matrix. On day 25, a switch in type II procollagen mRNA splicing acted as a limiting step in the acquisition of the mature chondrocyte phenotype. On day 30, substitution of dexamethasone with triiodothyronine triggers the final differentiation into hypertrophic chondrocytes within a further 15 days. The chondrogenic process is supported by intrinsic expression of Sox9 and BMP family genes. Similarly to the in vivo situation, activation of Sox9 expression and the BMP signaling pathway occurred on mesoblastic condensation. After induction, BMP-activated Smad nuclear translocation persisted throughout the process until the onset of hypertrophy. After dexamethasone addition, Sox9 expression was upregulated. Dexamethasone withdrawal reversed the increase in Sox9 expression and stopped differentiation. Thus, Sox9 seems to be a downstream mediator of dexamethasone action.

New tRNA contacts facilitate ligand binding in a Mycobacterium smegmatis T box riboswitch.

PubMed

Sherwood, Anna V; Frandsen, Jane K; Grundy, Frank J; Henkin, Tina M

2018-04-10

T box riboswitches are RNA regulatory elements widely used by organisms in the phyla Firmicutes and Actinobacteria to regulate expression of amino acid-related genes. Expression of T box family genes is down-regulated by transcription attenuation or inhibition of translation initiation in response to increased charging of the cognate tRNA. Three direct contacts with tRNA have been described; however, one of these contacts is absent in a subclass of T box RNAs and the roles of several structural domains conserved in most T box RNAs are unknown. In this study, structural elements of a Mycobacterium smegmatis ileS T box riboswitch variant with an Ultrashort (US) Stem I were sequentially deleted, which resulted in a progressive decrease in binding affinity for the tRNA Ile ligand. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) revealed structural changes in conserved riboswitch domains upon interaction with the tRNA ligand. Cross-linking and mutational analyses identified two interaction sites, one between the S-turn element in Stem II and the T arm of tRNA Ile and the other between the Stem IIA/B pseudoknot and the D loop of tRNA Ile These newly identified RNA contacts add information about tRNA recognition by the T box riboswitch and demonstrate a role for the S-turn and pseudoknot elements, which resemble structural elements that are common in many cellular RNAs.

PLETHORA transcription factors orchestrate de novo organ patterning during Arabidopsis lateral root outgrowth

PubMed Central

Du, Yujuan

2017-01-01

Plant development is characterized by repeated initiation of meristems, regions of dividing cells that give rise to new organs. During lateral root (LR) formation, new LR meristems are specified to support the outgrowth of LRs along a new axis. The determination of the sequential events required to form this new growth axis has been hampered by redundant activities of key transcription factors. Here, we characterize the effects of three PLETHORA (PLT) transcription factors, PLT3, PLT5, and PLT7, during LR outgrowth. In plt3plt5plt7 triple mutants, the morphology of lateral root primordia (LRP), the auxin response gradient, and the expression of meristem/tissue identity markers are impaired from the “symmetry-breaking” periclinal cell divisions during the transition between stage I and stage II, wherein cells first acquire different identities in the proximodistal and radial axes. Particularly, PLT1, PLT2, and PLT4 genes that are typically expressed later than PLT3, PLT5, and PLT7 during LR outgrowth are not induced in the mutant primordia, rendering “PLT-null” LRP. Reintroduction of any PLT clade member in the mutant primordia completely restores layer identities at stage II and rescues mutant defects in meristem and tissue establishment. Therefore, all PLT genes can activate the formative cell divisions that lead to de novo meristem establishment and tissue patterning associated with a new growth axis. PMID:29078398

Octopus-toolkit: a workflow to automate mining of public epigenomic and transcriptomic next-generation sequencing data

PubMed Central

Kim, Taemook; Seo, Hogyu David; Hennighausen, Lothar; Lee, Daeyoup

2018-01-01

Abstract Octopus-toolkit is a stand-alone application for retrieving and processing large sets of next-generation sequencing (NGS) data with a single step. Octopus-toolkit is an automated set-up-and-analysis pipeline utilizing the Aspera, SRA Toolkit, FastQC, Trimmomatic, HISAT2, STAR, Samtools, and HOMER applications. All the applications are installed on the user's computer when the program starts. Upon the installation, it can automatically retrieve original files of various epigenomic and transcriptomic data sets, including ChIP-seq, ATAC-seq, DNase-seq, MeDIP-seq, MNase-seq and RNA-seq, from the gene expression omnibus data repository. The downloaded files can then be sequentially processed to generate BAM and BigWig files, which are used for advanced analyses and visualization. Currently, it can process NGS data from popular model genomes such as, human (Homo sapiens), mouse (Mus musculus), dog (Canis lupus familiaris), plant (Arabidopsis thaliana), zebrafish (Danio rerio), fruit fly (Drosophila melanogaster), worm (Caenorhabditis elegans), and budding yeast (Saccharomyces cerevisiae) genomes. With the processed files from Octopus-toolkit, the meta-analysis of various data sets, motif searches for DNA-binding proteins, and the identification of differentially expressed genes and/or protein-binding sites can be easily conducted with few commands by users. Overall, Octopus-toolkit facilitates the systematic and integrative analysis of available epigenomic and transcriptomic NGS big data. PMID:29420797

The bHLH transcription factor BIS1 controls the iridoid branch of the monoterpenoid indole alkaloid pathway in Catharanthus roseus

PubMed Central

Van Moerkercke, Alex; Steensma, Priscille; Schweizer, Fabian; Pollier, Jacob; Gariboldi, Ivo; Payne, Richard; Vanden Bossche, Robin; Miettinen, Karel; Espoz, Javiera; Purnama, Purin Candra; Kellner, Franziska; Seppänen-Laakso, Tuulikki; O’Connor, Sarah E.; Rischer, Heiko; Memelink, Johan; Goossens, Alain

2015-01-01

Plants make specialized bioactive metabolites to defend themselves against attackers. The conserved control mechanisms are based on transcriptional activation of the respective plant species-specific biosynthetic pathways by the phytohormone jasmonate. Knowledge of the transcription factors involved, particularly in terpenoid biosynthesis, remains fragmentary. By transcriptome analysis and functional screens in the medicinal plant Catharanthus roseus (Madagascar periwinkle), the unique source of the monoterpenoid indole alkaloid (MIA)-type anticancer drugs vincristine and vinblastine, we identified a jasmonate-regulated basic helix–loop–helix (bHLH) transcription factor from clade IVa inducing the monoterpenoid branch of the MIA pathway. The bHLH iridoid synthesis 1 (BIS1) transcription factor transactivated the expression of all of the genes encoding the enzymes that catalyze the sequential conversion of the ubiquitous terpenoid precursor geranyl diphosphate to the iridoid loganic acid. BIS1 acted in a complementary manner to the previously characterized ethylene response factor Octadecanoid derivative-Responsive Catharanthus APETALA2-domain 3 (ORCA3) that transactivates the expression of several genes encoding the enzymes catalyzing the conversion of loganic acid to the downstream MIAs. In contrast to ORCA3, overexpression of BIS1 was sufficient to boost production of high-value iridoids and MIAs in C. roseus suspension cell cultures. Hence, BIS1 might be a metabolic engineering tool to produce sustainably high-value MIAs in C. roseus plants or cultures. PMID:26080427

Gene expression profiling of dendritic cells by microarray.

PubMed

Foti, Maria; Ricciardi-Castagnoli, Paola; Granucci, Francesca

2007-01-01

The immune system of vertebrate animals has evolved to respond to different types of perturbations (invading pathogens, stress signals), limiting self-tissue damage. The decision to activate an immune response is made by antigen-presenting cells (APCs) that are quiescent until they encounter a foreign microorganism or inflammatory stimuli. Early activated APCs trigger innate immune responses that represent the first line of reaction against invading pathogens to limit the infections. At later times, activated APCs acquire the ability to prime antigen-specific immune responses that clear the infections and give rise to memory. During the immune response self-tissue damage is limited and tolerance to self is maintained through life. Among the cells that constitute the immune system, dendritic cells (DC) play a central role. They are extremely versatile APCs involved in the initiation of both innate and adaptive immunity and also in the differentiation of regulatory T cells required for the maintenance of self-tolerance. How DC can mediate these diverse and almost contradictory functions has recently been investigated. The plasticity of these cells allows them to undergo a complete genetic reprogramming in response to external microbial stimuli with the sequential acquisition of different regulatory functions in innate and adaptive immunity. The specific genetic reprogramming DC undergo upon activation can be easily investigated by using microarrays to perform global gene expression analysis in different conditions.

Cloning, expression and mutation of a triazophos hydrolase gene from Burkholderia sp. SZL-1.

PubMed

Zhang, Hao; Li, Qiang; Guo, Su-Hui; Cheng, Ming-Gen; Zhao, Meng-Jun; Hong, Qing; Huang, Xing

2016-06-01

Triazophos is a broad-spectrum and highly effective insecticide, and the residues of triazophos have been frequently detected in the environment. A triazophos-degrading bacterium, Burkholderia sp. SZL-1, was isolated from a long-term triazophos-polluted soil. Strain SZL-1 could hydrolyze triazophos to 1-phenyl-3-hydroxy-1,2,4-triazole, which was further utilized as the carbon sources for growth. The triazophos hydrolase gene trhA, cloned from strain SZL-1, was expressed and homogenously purified using Ni-nitrilotriacetic acid affinity chromatography. TrhA is 55 kDa and displays maximum activity at 25°C, pH 8.0. This enzyme still has nearly 60% activity at the range of 15°C-50°C for 30 min. TrhA was mutated by sequential error prone PCR and screened for improved activity for triazophos degradation. One purified variant protein (Val89-Gly89) named TrhA-M1 showed up to 3-fold improvement in specific activity against triazophos, and the specificity constants of Kcat and Kcat/Km for TrhA-M1 were improved up to 2.3- and 8.28-fold, respectively, compared to the wild-type enzyme. The results in this paper provided potential material for the contaminated soil remediation and hydrolase genetic structure research. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Ex vivo-expanded bone marrow CD34(+) for acute myocardial infarction treatment: in vitro and in vivo studies.

PubMed

Gunetti, Monica; Noghero, Alessio; Molla, Fabiola; Staszewsky, Lidia Irene; de Angelis, Noeleen; Soldo, Annarita; Russo, Ilaria; Errichiello, Edoardo; Frasson, Chiara; Rustichelli, Deborah; Ferrero, Ivana; Gualandris, Anna; Berger, Massimo; Geuna, Massimo; Scacciatella, Paolo; Basso, Giuseppe; Marra, Sebastiano; Bussolino, Federico; Latini, Roberto; Fagioli, Franca

2011-10-01

Bone marrow (BM)-derived cells appear to be a promising therapeutic source for the treatment of acute myocardial infarction (AMI). However, the quantity and quality of the cells to be used, along with the appropriate time of administration, still need to be defined. We thus investigated the use of BM CD34(+)-derived cells as cells suitable for a cell therapy protocol (CTP) in the treatment of experimental AMI. The need for a large number of cells was satisfied by the use of a previously established protocol allowing the expansion of human CD34(+) cells isolated from neonatal and adult hematopoietic tissues. We evaluated gene expression, endothelial differentiation potential and cytokine release by BM-derived cells during in vitro culture. Basal and expanded CD34(+) cells were used as a delivery product in a murine AMI model consisting of a coronary artery ligation (CAL). Cardiac function recovery was evaluated after injecting basal or expanded cells. Gene expression analysis of in vitro-expanded cells revealed that endothelial markers were up-regulated during culture. Moreover, expanded cells generated a CD14(+) subpopulation able to differentiate efficiently into VE-cadherin-expressing cells. In vivo, we observed a cardiac function recovery in mice sequentially treated with basal and expanded cells injected 4 h and 7 days after CAL, respectively. Our data suggest that combining basal and expanded BM-derived CD34(+) cells in a specific temporal pattern of administration might represent a promising strategy for a successful cell-based therapy.

Genomics and expression profiles of the Hedgehog and Notch signaling pathways in sea urchin development.

PubMed

Walton, Katherine D; Croce, Jenifer C; Glenn, Thomas D; Wu, Shu-Yu; McClay, David R

2006-12-01

The Hedgehog (Hh) and Notch signal transduction pathways control a variety of developmental processes including cell fate choice, differentiation, proliferation, patterning and boundary formation. Because many components of these pathways are conserved, it was predicted and confirmed that pathway components are largely intact in the sea urchin genome. Spatial and temporal location of these pathways in the embryo, and their function in development offer added insight into their mechanistic contributions. Accordingly, all major components of both pathways were identified and annotated in the sea urchin Strongylocentrotus purpuratus genome and the embryonic expression of key components was explored. Relationships of the pathway components, and modifiers predicted from the annotation of S. purpuratus, were compared against cnidarians, arthropods, urochordates, and vertebrates. These analyses support the prediction that the pathways are highly conserved through metazoan evolution. Further, the location of these two pathways appears to be conserved among deuterostomes, and in the case of Notch at least, display similar capacities in endomesoderm gene regulatory networks. RNA expression profiles by quantitative PCR and RNA in situ hybridization reveal that Hedgehog is produced by the endoderm beginning just prior to invagination, and signals to the secondary mesenchyme-derived tissues at least until the pluteus larva stage. RNA in situ hybridization of Notch pathway members confirms that Notch functions sequentially in the vegetal-most secondary mesenchyme cells and later in the endoderm. Functional analyses in future studies will embed these pathways into the growing knowledge of gene regulatory networks that govern early specification and morphogenesis.

Drosophila histone locus bodies form by hierarchical recruitment of components

PubMed Central

White, Anne E.; Burch, Brandon D.; Yang, Xiao-cui; Gasdaska, Pamela Y.; Dominski, Zbigniew; Marzluff, William F.

2011-01-01

Nuclear bodies are protein- and RNA-containing structures that participate in a wide range of processes critical to genome function. Molecular self-organization is thought to drive nuclear body formation, but whether this occurs stochastically or via an ordered, hierarchical process is not fully understood. We addressed this question using RNAi and proteomic approaches in Drosophila melanogaster to identify and characterize novel components of the histone locus body (HLB), a nuclear body involved in the expression of replication-dependent histone genes. We identified the transcription elongation factor suppressor of Ty 6 (Spt6) and a homologue of mammalian nuclear protein of the ataxia telangiectasia–mutated locus that is encoded by the homeotic gene multisex combs (mxc) as novel HLB components. By combining genetic manipulation in both cell culture and embryos with cytological observations of Mxc, Spt6, and the known HLB components, FLICE-associated huge protein, Mute, U7 small nuclear ribonucleoprotein, and MPM-2 phosphoepitope, we demonstrated sequential recruitment and hierarchical dependency for localization of factors to HLBs during development, suggesting that ordered assembly can play a role in nuclear body formation. PMID:21576393

Transcriptomic and epigenomic characterization of the developing bat wing

PubMed Central

Eckalbar, Walter L.; Schlebusch, Stephen A.; Mason, Mandy K.; Gill, Zoe; Parker, Ash V.; Booker, Betty M.; Nishizaki, Sierra; Muswamba-Nday, Christiane; Terhune, Elizabeth; Nevonen, Kimberly; Makki, Nadja; Friedrich, Tara; VanderMeer, Julia E.; Pollard, Katherine S.; Carbone, Lucia; Wall, Jeff D.; Illing, Nicola; Ahituv, Nadav

2016-01-01

Bats are the only mammals capable of powered flight, but little is known about the genetic determinants that shape their wings. Here, we generated a genome for Miniopterus natalensis and performed RNA-seq and ChIP-seq (H3K27ac, H3K27me3) on its developing forelimb and hindlimb autopods at sequential embryonic stages to decipher the molecular events that underlie bat wing development. Over 7,000 genes and several lncRNAs, including Tbx5-as1 and Hottip, were differentially expressed between forelimb, hindlimb and different stages. ChIP-seq identified thousands of regions that are differentially modified in forelimb versus hindlimb. Comparative genomics found 2,796 bat-accelerated regions within H3K27ac peaks, several of which cluster near limb-associated genes. Pathway analyses revealed multiple ribosomal proteins and known limb patterning signaling pathways as differentially regulated, and implicated increased forelimb mesenchymal condensations with differential growth. Combined, our work outlines multiple genetic components that contribute to bat wing formation, providing a genomic blueprint for this morphological innovation. PMID:27019111

Wiring Together Synthetic Bacterial Consortia to Create a Biological Integrated Circuit.

PubMed

Perry, Nicolas; Nelson, Edward M; Timp, Gregory

2016-12-16

The promise of adapting biology to information processing will not be realized until engineered gene circuits, operating in different cell populations, can be wired together to express a predictable function. Here, elementary biological integrated circuits (BICs), consisting of two sets of transmitter and receiver gene circuit modules with embedded memory placed in separate cell populations, were meticulously assembled using live cell lithography and wired together by the mass transport of quorum-sensing (QS) signal molecules to form two isolated communication links (comlinks). The comlink dynamics were tested by broadcasting "clock" pulses of inducers into the networks and measuring the responses of functionally linked fluorescent reporters, and then modeled through simulations that realistically captured the protein production and molecular transport. These results show that the comlinks were isolated and each mimicked aspects of the synchronous, sequential networks used in digital computing. The observations about the flow conditions, derived from numerical simulations, and the biofilm architectures that foster or silence cell-to-cell communications have implications for everything from decontamination of drinking water to bacterial virulence.

Leber's hereditary optic neuropathy is associated with the mitochondrial ND6 T14484C mutation in three Chinese families

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

Sun Yanhong; Wei Qiping; Zhou Xiangtian

2006-08-18

We report here the clinical, genetic, and molecular characterization of three Chinese families with maternally transmitted Leber's hereditary optic neuropathy (LHON). Clinical and genetic evaluations revealed the variable severity and age-of-onset in visual impairment in these families. In the affected matrilineal relatives, the loss of central vision is bilateral, the fellow eye becoming affected either simultaneously (45%) or sequentially (55%). The penetrances of vision loss in these pedigrees were 27%, 50%, and 60%, respectively. The age-at-onset of vision loss in these families was 14, 19, and 24 years, respectively. Furthermore, the ratios between affected male and female matrilineal relatives weremore » 1:1, 1:1.2, and 1:2, respectively. Mutational analysis of mitochondrial DNA revealed the presence of homoplasmic ND6 T14484C mutation, which has been associated with LHON. The incomplete penetrance and phenotypic variability implicate the involvement of nuclear modifier gene(s), environmental factor(s) or mitochondrial haplotype(s) in the phenotypic expression of the LHON-associated T14484C mutation in these Chinese pedigrees.« less

A Novel Epigenetic Silencing Pathway Involving the Highly Conserved 5’-3’ Exoribonuclease Dhp1/Rat1/Xrn2 in Schizosaccharomyces pombe

PubMed Central

Tucker, James Franklin; Ohle, Corina; Schermann, Géza; Bendrin, Katja; Zhang, Wei; Fischer, Tamás; Zhang, Ke

2016-01-01

Epigenetic gene silencing plays a critical role in regulating gene expression and contributes to organismal development and cell fate acquisition in eukaryotes. In fission yeast, Schizosaccharomyces pombe, heterochromatin-associated gene silencing is known to be mediated by RNA processing pathways including RNA interference (RNAi) and a 3’-5’ exoribonuclease complex, the exosome. Here, we report a new RNA-processing pathway that contributes to epigenetic gene silencing and assembly of heterochromatin mediated by 5’-3’ exoribonuclease Dhp1/Rat1/Xrn2. Dhp1 mutation causes defective gene silencing both at peri-centromeric regions and at the silent mating type locus. Intriguingly, mutation in either of the two well-characterized Dhp1-interacting proteins, the Din1 pyrophosphohydrolase or the Rhn1 transcription termination factor, does not result in silencing defects at the main heterochromatic regions. We demonstrate that Dhp1 interacts with heterochromatic factors and is essential in the sequential steps of establishing silencing in a manner independent of both RNAi and the exosome. Genomic and genetic analyses suggest that Dhp1 is involved in post-transcriptional silencing of repetitive regions through its RNA processing activity. The results describe the unexpected role of Dhp1/Rat1/Xrn2 in chromatin-based silencing and elucidate how various RNA-processing pathways, acting together or independently, contribute to epigenetic regulation of the eukaryotic genome. PMID:26889830

Ligand-activated PPARα-dependent DNA demethylation regulates the fatty acid β-oxidation genes in the postnatal liver.

PubMed

Ehara, Tatsuya; Kamei, Yasutomi; Yuan, Xunmei; Takahashi, Mayumi; Kanai, Sayaka; Tamura, Erina; Tsujimoto, Kazutaka; Tamiya, Takashi; Nakagawa, Yoshimi; Shimano, Hitoshi; Takai-Igarashi, Takako; Hatada, Izuho; Suganami, Takayoshi; Hashimoto, Koshi; Ogawa, Yoshihiro

2015-03-01

The metabolic function of the liver changes sequentially during early life in mammals to adapt to the marked changes in nutritional environment. Accordingly, hepatic fatty acid β-oxidation is activated after birth to produce energy from breast milk lipids. However, how it is induced during the neonatal period is poorly understood. Here we show DNA demethylation and increased mRNA expression of the fatty acid β-oxidation genes in the postnatal mouse liver. The DNA demethylation does not occur in the fetal mouse liver under the physiologic condition, suggesting that it is specific to the neonatal period. Analysis of mice deficient in the nuclear receptor peroxisome proliferator-activated receptor α (PPARα) and maternal administration of a PPARα ligand during the gestation and lactation periods reveal that the DNA demethylation is PPARα dependent. We also find that DNA methylation of the fatty acid β-oxidation genes are reduced in the adult human liver relative to the fetal liver. This study represents the first demonstration that the ligand-activated PPARα-dependent DNA demethylation regulates the hepatic fatty acid β-oxidation genes during the neonatal period, thereby highlighting the role of a lipid-sensing nuclear receptor in the gene- and life-stage-specific DNA demethylation of a particular metabolic pathway. © 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

Simultaneous Breast Expression in Breastfeeding Women Is More Efficacious Than Sequential Breast Expression

PubMed Central

Garbin, Catherine P.; Hartmann, Peter E.; Kent, Jacqueline C.

2012-01-01

Abstract Introduction Simultaneous (SIM) breast expression saves mothers time compared with sequential (SEQ) expression, but it remains unclear whether the two methods differ in milk output efficiency and efficacy. Subjects and Methods The Showmilk device (Medela AG, Baar, Switzerland) was used to measure milk output and milk ejection during breast expression (electric pump) in 31 Australian breastfeeding mothers of term infants (median age, 19 weeks [interquartile range, 10–33 weeks]). The order of expression type (SIM/SEQ) and breast (left/right) was randomized. Results SIM expression yielded more milk ejections (p≤0.001) and greater amounts of milk at 2, 5, and 10 minutes (p≤0.01) and removed a greater total amount of milk (p≤0.01) and percentage of available milk (p<0.05) than SEQ expression. After SIM expression the cream content of both the overall (8.3% [p≤0.05]) and postexpression (12.6% [p≤0.001]) milk were greater. During SEQ expression, the breast expressed first had a shorter time to 50% and 80% of the total amount of milk than the breast expressed second (p≤0.05), but, overall, a similar percentage of available milk was removed from both breasts. Conclusions SIM expression stimulated more milk ejections and was a more efficient and efficacious method of expression, yielding milk with a higher energy content. PMID:23039397

Expression and functional analysis of citrus carotene hydroxylases: unravelling the xanthophyll biosynthesis in citrus fruits.

PubMed

Ma, Gang; Zhang, Lancui; Yungyuen, Witchulada; Tsukamoto, Issei; Iijima, Natsumi; Oikawa, Michiru; Yamawaki, Kazuki; Yahata, Masaki; Kato, Masaya

2016-06-29

Xanthophylls are oxygenated carotenoids and fulfill critical roles in plant growth and development. In plants, two different types of carotene hydroxylases, non-heme di-iron and heme-containing cytochrome P450, were reported to be involved in the biosynthesis of xanthophyll. Citrus fruits accumulate a high amount of xanthophylls, especially β,β-xanthophylls. To date, however, the roles of carotene hydroxylases in regulating xanthophyll content and composition have not been elucidated. In the present study, the roles of four carotene hydroxylase genes (CitHYb, CitCYP97A, CitCYP97B, and CitCYP97C) in the biosynthesis of xanthophyll in citrus fruits were investigated. Phylogenetic analysis showed that the four citrus carotene hydroxylases presented in four distinct clusters which have been identified in higher plants. CitHYb was a non-heme di-iron carotene hydroxylase, while CitCYP97A, CitCYP97B, and CitCYP97C were heme-containing cytochrome P450-type carotene hydroxylases. Gene expression results showed that the expression of CitHYb increased in the flavedo and juice sacs during the ripening process, which was well consistent with the accumulation of β,β-xanthophyll in citrus fruits. The expression of CitCYP97A and CitCYP97C increased with a peak in November, which might lead to an increase of lutein in the juice sacs during the ripening process. The expression level of CitCYP97B was much lower than that of CitHYb, CitCYP97A, and CitCYP97C in the juice sacs during the ripening process. Functional analysis showed that the CitHYb was able to catalyze the hydroxylation of the β-rings of β-carotene and α-carotene in Escherichia coli BL21 (DE3) cells. Meanwhile, when CitHYb was co-expressed with CitCYP97C, α-carotene was hydroxylated on the β-ring and ε-ring sequentially to produce lutein. CitHYb was a key gene for β,β-xanthophyll biosynthesis in citrus fruits. CitCYP97C functioned as an ε-ring hydroxylase to produce lutein using zeinoxanthin as a substrate. The results will contribute to elucidating xanthophyll biosynthesis in citrus fruits, and provide new strategies to improve the nutritional and commercial qualities of citrus fruits.

Gastrulation and pre-gastrulation morphogenesis, inductions, and gene expression: similarities and dissimilarities between urodelean and anuran embryos.

PubMed

Kaneda, Teruo; Motoki, Jun-ya Doi

2012-09-01

Studies of meso-endoderm and neural induction and subsequent body plan formation have been analyzed using mainly amphibians as the experimental model. Xenopus is currently the predominant model, because it best enables molecular analysis of these induction processes. However, much of the embryological information on these inductions (e.g., those of the Spemann-Mangold organizer), and on the morphogenetic movements of inductively interacting tissues, derives from research on non-model amphibians, especially urodeles. Although the final body pattern is strongly conserved in vertebrates, and although many of the same developmental genes are expressed, it has become evident that there are individually diverse modes of morphogenesis and timing of developmental events. Whether or not this diversity represents essential differences in the early induction processes remains unclear. The aim of this review is to compare the gastrulation process, induction processes, and gene expressions between a urodele, mainly Cynops pyrrhogaster, and an anura, Xenopus laevis, thereby to clarify conserved and diversified aspects. Cynops gastrulation differs significantly from that of Xenopus in that specification of the regions of the Xenopus dorsal marginal zone (DMZ) are specified before the onset of gastrulation, as marked by blastopore formation, whereas the equivalent state of specification does not occur in Cynops until the middle of gastrulation. Detailed comparison of the germ layer structure and morphogenetic movements during the pre-gastrula and gastrula stages shows that the entire gastrulation process should be divided into two phases of notochord induction and neural induction. Cynops undergoes these processes sequentially after the onset of gastrulation, whereas Xenopus undergoes notochord induction during a series of pre-gastrulation movements, and its traditionally defined period of gastrulation only includes the neural induction phase. Comparing the structure, fate, function and state of commitment of each domain of the DMZ of Xenopus and Cynops has revealed that the true form of the Spemann-Mangold organizer is suprablastoporal gsc-expressing endoderm that has notochord-inducing activity. Gsc-expressing deep endoderm and/or superficial endoderm in Xenopus is involved in inducing notochord during pre-gastrulation morphogenesis, rather than both gsc- and bra-expressing tissues being induced at the same time. Copyright © 2012 Elsevier Inc. All rights reserved.

Optimization of the primary recovery of human interferon alpha2b from Escherichia coli inclusion bodies.

PubMed

Valente, C A; Monteiro, G A; Cabral, J M S; Fevereiro, M; Prazeres, D M F

2006-01-01

The human interferon alpha2b (hu-IFNalpha2b) gene was cloned in Escherichia coli JM109(DE3) and the recombinant protein was expressed as cytoplasmic inclusion bodies (IB). The present work discusses the recovery of hu-IFNalpha2b IB from the E. coli cells. An optimized protocol is proposed based on the sequential evaluation of recovery steps and parameters: (i) cell disruption, (ii) IB recovery and separation from cell debris, (iii) IB washing, and (iv) IB solubilization. Parameters such as hu-IFNalpha2b purity and recovery yield were measured after each step. The optimized recovery protocol yielded 60% of hu-IFNalpha2b with a purity of up to 80%. The protein was renatured at high concentration after recovery and it was found to display biological activity.

Plant stimulation of soil microbial community succession: how sequential expression mediates soil carbon stabilization and turnover

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

Firestone, Mary

2015-03-31

It is now understood that most plant C is utilized or transformed by soil microorganisms en route to stabilization. Hence the composition of microbial communities that mediate decomposition and transformation of root C is critical, as are the metabolic capabilities of these communities. The change in composition and function of the C-transforming microbial communities over time in effect defines the biological component of soil C stabilization. Our research was designed to test 2 general hypotheses; the first two hypotheses are discussed first; H1: Root-exudate interactions with soil microbial populations results in the expression of enzymatic capacities for macromolecular, complex carbonmore » decomposition; and H2: Microbial communities surrounding roots undergo taxonomic succession linked to functional gene activities as roots grow, mature, and decompose in soil. Over the term of the project we made significant progress in 1) quantifying the temporal pattern of root interactions with the soil decomposing community and 2) characterizing the role of root exudates in mediating these interactions.« less

A Computational Algorithm for Functional Clustering of Proteome Dynamics During Development

PubMed Central

Wang, Yaqun; Wang, Ningtao; Hao, Han; Guo, Yunqian; Zhen, Yan; Shi, Jisen; Wu, Rongling

2014-01-01

Phenotypic traits, such as seed development, are a consequence of complex biochemical interactions among genes, proteins and metabolites, but the underlying mechanisms that operate in a coordinated and sequential manner remain elusive. Here, we address this issue by developing a computational algorithm to monitor proteome changes during the course of trait development. The algorithm is built within the mixture-model framework in which each mixture component is modeled by a specific group of proteins that display a similar temporal pattern of expression in trait development. A nonparametric approach based on Legendre orthogonal polynomials was used to fit dynamic changes of protein expression, increasing the power and flexibility of protein clustering. By analyzing a dataset of proteomic dynamics during early embryogenesis of the Chinese fir, the algorithm has successfully identified several distinct types of proteins that coordinate with each other to determine seed development in this forest tree commercially and environmentally important to China. The algorithm will find its immediate applications for the characterization of mechanistic underpinnings for any other biological processes in which protein abundance plays a key role. PMID:24955031

Calcium handling precedes cardiac differentiation to initiate the first heartbeat

PubMed Central

Tyser, Richard CV; Miranda, Antonio MA; Chen, Chiann-mun; Davidson, Sean M

2016-01-01

The mammalian heartbeat is thought to begin just prior to the linear heart tube stage of development. How the initial contractions are established and the downstream consequences of the earliest contractile function on cardiac differentiation and morphogenesis have not been described. Using high-resolution live imaging of mouse embryos, we observed randomly distributed spontaneous asynchronous Ca2+-oscillations (SACOs) in the forming cardiac crescent (stage E7.75) prior to overt beating. Nascent contraction initiated at around E8.0 and was associated with sarcomeric assembly and rapid Ca2+ transients, underpinned by sequential expression of the Na+-Ca2+ exchanger (NCX1) and L-type Ca2+ channel (LTCC). Pharmacological inhibition of NCX1 and LTCC revealed rapid development of Ca2+ handling in the early heart and an essential early role for NCX1 in establishing SACOs through to the initiation of beating. NCX1 blockade impacted on CaMKII signalling to down-regulate cardiac gene expression, leading to impaired differentiation and failed crescent maturation. DOI: http://dx.doi.org/10.7554/eLife.17113.001 PMID:27725084

Regulation of the nuclear factor (NF)-kappaB pathway by ISGylation.

PubMed

Minakawa, Miki; Sone, Takayuki; Takeuchi, Tomoharu; Yokosawa, Hideyoshi

2008-12-01

Post-translational modification with ISG15 (interferon-stimulated gene 15 kDa) (ISGylation) is mediated by a sequential reaction similar to ubiquitination, and various target proteins for ISGylation have been identified. We previously reported that ISGylation of the E2 ubiquitin-conjugating enzyme Ubc13 suppresses its E2 activity. Ubc13 forms a heterodimer with Uev1A, a ubiquitin-conjugating enzyme variant, and the Ubc13-Uev1A complex catalyzes the assembly of a Lys63-linked polyubiquitin chain, which plays a non-proteolytic role in the nuclear factor (NF)-kappaB pathway. In this study, we examined the effect of ISGylation on tumor necrosis factor receptor-associated factor (TRAF)-6/transforming growth factor beta-activated kinase (TAK)-1-dependent NF-kappaB activation. We found that expression of the ISGylation system suppresses NF-kappaB activation via TRAF6 and TAK1 and that the level of polyubiquitinated TRAF6 is reduced by expression of the ISGylation system. Taken together, the results suggest that the NF-kappaB pathway is negatively regulated by ISGylation.

Neurobiology of autism gene products: towards pathogenesis and drug targets.

PubMed

Kleijer, Kristel T E; Schmeisser, Michael J; Krueger, Dilja D; Boeckers, Tobias M; Scheiffele, Peter; Bourgeron, Thomas; Brose, Nils; Burbach, J Peter H

2014-03-01

The genetic heterogeneity of autism spectrum disorders (ASDs) is enormous, and the neurobiology of proteins encoded by genes associated with ASD is very diverse. Revealing the mechanisms on which different neurobiological pathways in ASD pathogenesis converge may lead to the identification of drug targets. The main objective is firstly to outline the main molecular networks and neuronal mechanisms in which ASD gene products participate and secondly to answer the question how these converge. Finally, we aim to pinpoint drug targets within these mechanisms. Literature review of the neurobiological properties of ASD gene products with a special focus on the developmental consequences of genetic defects and the possibility to reverse these by genetic or pharmacological interventions. The regulation of activity-dependent protein synthesis appears central in the pathogenesis of ASD. Through sequential consequences for axodendritic function, neuronal disabilities arise expressed as behavioral abnormalities and autistic symptoms in ASD patients. Several known ASD gene products have their effect on this central process by affecting protein synthesis intrinsically, e.g., through enhancing the mammalian target of rapamycin (mTOR) signal transduction pathway or through impairing synaptic function in general. These are interrelated processes and can be targeted by compounds from various directions: inhibition of protein synthesis through Lovastatin, mTOR inhibition using rapamycin, or mGluR-related modulation of synaptic activity. ASD gene products may all feed into a central process of translational control that is important for adequate glutamatergic regulation of dendritic properties. This process can be modulated by available compounds but may also be targeted by yet unexplored routes.

Characterization of Trichome-Expressed BAHD Acyltransferases in Petunia axillaris Reveals Distinct Acylsugar Assembly Mechanisms within the Solanaceae1[OPEN

PubMed Central

Uebler, Joseph B.; Liu, Xiaoxiao

2017-01-01

Acylsugars are synthesized in the glandular trichomes of the Solanaceae family and are implicated in protection against abiotic and biotic stress. Acylsugars are composed of either sucrose or glucose esterified with varying numbers of acyl chains of differing length. In tomato (Solanum lycopersicum), acylsugar assembly requires four acylsugar acyltransferases (ASATs) of the BAHD superfamily. Tomato ASATs catalyze the sequential esterification of acyl-coenzyme A thioesters to the R4, R3, R3ʹ, and R2 positions of sucrose, yielding a tetra-acylsucrose. Petunia spp. synthesize acylsugars that are structurally distinct from those of tomato. To explore the mechanisms underlying this chemical diversity, a Petunia axillaris transcriptome was mined for trichome preferentially expressed BAHDs. A combination of phylogenetic analyses, gene silencing, and biochemical analyses coupled with structural elucidation of metabolites revealed that acylsugar assembly is not conserved between tomato and petunia. In P. axillaris, tetra-acylsucrose assembly occurs through the action of four ASATs, which catalyze sequential addition of acyl groups to the R2, R4, R3, and R6 positions. Notably, in P. axillaris, PaxASAT1 and PaxASAT4 catalyze the acylation of the R2 and R6 positions of sucrose, respectively, and no clear orthologs exist in tomato. Similarly, petunia acylsugars lack an acyl group at the R3ʹ position, and congruently, an ortholog of SlASAT3, which catalyzes acylation at the R3ʹ position in tomato, is absent in P. axillaris. Furthermore, where putative orthologous relationships of ASATs are predicted between tomato and petunia, these are not supported by biochemical assays. Overall, these data demonstrate the considerable evolutionary plasticity of acylsugar biosynthesis. PMID:28701351

Characterization of Trichome-Expressed BAHD Acyltransferases in Petunia axillaris Reveals Distinct Acylsugar Assembly Mechanisms within the Solanaceae.

PubMed

Nadakuduti, Satya Swathi; Uebler, Joseph B; Liu, Xiaoxiao; Jones, A Daniel; Barry, Cornelius S

2017-09-01

Acylsugars are synthesized in the glandular trichomes of the Solanaceae family and are implicated in protection against abiotic and biotic stress. Acylsugars are composed of either sucrose or glucose esterified with varying numbers of acyl chains of differing length. In tomato ( Solanum lycopersicum ), acylsugar assembly requires four acylsugar acyltransferases (ASATs) of the BAHD superfamily. Tomato ASATs catalyze the sequential esterification of acyl-coenzyme A thioesters to the R4, R3, R3', and R2 positions of sucrose, yielding a tetra-acylsucrose. Petunia spp. synthesize acylsugars that are structurally distinct from those of tomato. To explore the mechanisms underlying this chemical diversity, a Petunia axillaris transcriptome was mined for trichome preferentially expressed BAHDs. A combination of phylogenetic analyses, gene silencing, and biochemical analyses coupled with structural elucidation of metabolites revealed that acylsugar assembly is not conserved between tomato and petunia. In P. axillaris , tetra-acylsucrose assembly occurs through the action of four ASATs, which catalyze sequential addition of acyl groups to the R2, R4, R3, and R6 positions. Notably, in P. axillaris , PaxASAT1 and PaxASAT4 catalyze the acylation of the R2 and R6 positions of sucrose, respectively, and no clear orthologs exist in tomato. Similarly, petunia acylsugars lack an acyl group at the R3' position, and congruently, an ortholog of SlASAT3, which catalyzes acylation at the R3' position in tomato, is absent in P. axillaris Furthermore, where putative orthologous relationships of ASATs are predicted between tomato and petunia, these are not supported by biochemical assays. Overall, these data demonstrate the considerable evolutionary plasticity of acylsugar biosynthesis. © 2017 American Society of Plant Biologists. All Rights Reserved.

Corrective recombination of mouse immunoglobulin kappa alleles in Abelson murine leukemia virus-transformed pre-B cells.

PubMed Central

Feddersen, R M; Van Ness, B G

1990-01-01

Previous characterization of mouse immunoglobulin kappa gene rearrangement products cloned from murine plasmacytomas has indicated that two recombination events can take place on a single kappa allele (R. M. Feddersen and B. G. Van Ness, Proc. Natl. Acad. Sci. USA 82:4792-4797, 1985; M. A. Shapiro and M. Weigert, J. Immunol. 139:3834-3839, 1987). To determine whether multiple recombinations on a single kappa allele can contribute to the formation of productive V-J genes through corrective recombinations, we have examined several Abelson murine leukemia virus-transformed pre-B-cell clones which rearrange the kappa locus during cell culture. Clonal cell lines which had rearranged one kappa allele nonproductively while maintaining the other allele in the germ line configuration were grown, and secondary subclones, which subsequently expressed kappa protein, were isolated and examined for further kappa rearrangement. A full spectrum of rearrangement patterns was observed in this sequential cloning, including productive and nonproductive recombinations of the germ line allele and secondary recombinations of the nonproductive allele. The results show that corrective V-J recombinations, with displacement of the nonproductive kappa gene, occur with a significant frequency (6 of 17 kappa-producing subclones). Both deletion and maintenance of the primary (nonfunctional) V-J join, as a reciprocal product, were observed. Images PMID:2153918

Heuristic Bayesian segmentation for discovery of coexpressed genes within genomic regions.

PubMed

Pehkonen, Petri; Wong, Garry; Törönen, Petri

2010-01-01

Segmentation aims to separate homogeneous areas from the sequential data, and plays a central role in data mining. It has applications ranging from finance to molecular biology, where bioinformatics tasks such as genome data analysis are active application fields. In this paper, we present a novel application of segmentation in locating genomic regions with coexpressed genes. We aim at automated discovery of such regions without requirement for user-given parameters. In order to perform the segmentation within a reasonable time, we use heuristics. Most of the heuristic segmentation algorithms require some decision on the number of segments. This is usually accomplished by using asymptotic model selection methods like the Bayesian information criterion. Such methods are based on some simplification, which can limit their usage. In this paper, we propose a Bayesian model selection to choose the most proper result from heuristic segmentation. Our Bayesian model presents a simple prior for the segmentation solutions with various segment numbers and a modified Dirichlet prior for modeling multinomial data. We show with various artificial data sets in our benchmark system that our model selection criterion has the best overall performance. The application of our method in yeast cell-cycle gene expression data reveals potential active and passive regions of the genome.

Transcriptome analysis on the exoskeleton formation in early developmetal stages and reconstruction scenario in growth-moulting in Litopenaeus vannamei.

PubMed

Gao, Yi; Wei, Jiankai; Yuan, Jianbo; Zhang, Xiaojun; Li, Fuhua; Xiang, Jianhai

2017-04-24

Exoskeleton construction is an important issue in shrimp. To better understand the molecular mechanism of exoskeleton formation, development and reconstruction, the transcriptome of the entire developmental process in Litopenaeus vannamei, including nine early developmental stages and eight adult-moulting stages, was sequenced and analysed using Illumina RNA-seq technology. A total of 117,539 unigenes were obtained, with 41.2% unigenes predicting the full-length coding sequence. Gene Ontology, Clusters of Orthologous Group (COG), the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and functional annotation of all unigenes gave a better understanding of the exoskeleton developmental process in L. vannamei. As a result, more than six hundred unigenes related to exoskeleton development were identified both in the early developmental stages and adult-moulting. A cascade of sequential expression events of exoskeleton-related genes were summarized, including exoskeleton formation, regulation, synthesis, degradation, mineral absorption/reabsorption, calcification and hardening. This new insight on major transcriptional events provide a deep understanding for exoskeleton formation and reconstruction in L. vannamei. In conclusion, this is the first study that characterized the integrated transcriptomic profiles cover the entire exoskeleton development from zygote to adult-moulting in a crustacean, and these findings will serve as significant references for exoskeleton developmental biology and aquaculture research.

Dietary Supplement Attenuates Radiation-Induced Osteoclastogenic and Oxidative Stress-Related Responses and Protects Adult Mice from Radiation-Induced Bone Loss

NASA Technical Reports Server (NTRS)

Globus, Ruth; Schreurs, Ann-Sofie; Tahimic, Candice; Shirazi-Fard, Yasaman; Alwood, Joshua; Shahnazari, Mohammed; Halloran, Bernard

2015-01-01

Our central hypothesis is that oxidative stress plays a key role in cell dysfunction and progressive bone loss caused by radiation exposure during spaceflight. In animal studies, excess free radical formation is associated with pathological changes in bone structure, enhanced bone resorption, reduced bone formation and decreased bone mineral density, which can lead to skeletal fragility. We previously reported that exposure to low or high-LET radiation rapidly increases expression levels of pro-osteoclastogenic and oxidative stress-related genes in bone and marrow, followed by pathological changes in skeletal structure. To screen various antioxidants for radioprotective effects on bone, 4 month old, male C57Bl6/J mice were treated with a dietary antioxidant cocktail, injectable alpha-lipoic acid, or a dried plum-enriched diet (DP). Mice were then exposed to 2Gy 137Cs total body radiation and one day later marrow cells were collected and the relevant genes analyzed for expression levels. Of the candidates tested, DP was most effective in reducing bone resorption-related gene expression. Microcomputed tomography revealed that DP also prevented the radiation-induced deterioration of skeletal microarchitecture, as indicated by percent bone volume, trabecular spacing and trabecular number. DP had similar protective effects on skeletal structure after sequential exposure to protons (0.5 Gy, 150MeV/n) and 56Fe 0.5Gy, 600 MeV/n). When cultured ex vivo under osteogenic conditions, bone marrow-derived cells from DP-fed animals exhibited increased colony numbers compared to control diet-fed animals. These findings suggest that DP exerted pro-osteogenic effects apart from previously identified anti-resorptive actions, which may contribute to radioprotection of skeletal tissue. In conclusion, a diet enriched in certain types of antioxidants and polyphenols such as DP may be useful as an intervention to protect tissues from degenerative effects of ionizing radiation.

Chemoresistance of CD133{sup +} colon cancer may be related with increased survivin expression

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

Lee, Mi-Ra; Ji, Sun-Young; Mia-Jan, Khalilullah

2015-07-31

CD133, putative cancer stem cell marker, deemed to aid chemoresistance. However, this claim has been challenged recently and we previously reported that patients with CD133{sup +} colon cancer have benefit from 5-fluorouracil (5-FU) chemotherapy incontrast to no benefit in patients with CD133{sup −} cancer. To elucidate the role of CD133 expression in chemoresistance, we silenced the CD133 expression in a colon cancer cell line and determined its effect on the biological characteristics downstream. We comparatively analyzed the sequential changes of MDR1, ABCG2, AKT1 and survivin expression and the result of proliferation assay (WST-1 assay) with 5-FU treatment in CD133{sup +}more » and siRNA-induced CD133{sup −} cells, derived from Caco-2 colon cancer cell line. 5-FU treatment induced significantly increase of the mRNA expression of MDR1, ABCG2 and AKT1genes, but not protein level. CD133 had little to no effect on the mRNA and protein expression of these genes. However, survivin expression at mRNA and protein level were significantly increased in CD133{sup +} cells compared with siRNA-induced CD133-cells and Mock (not sorted CD133{sup +} cells) at 96 h after siRNA transfection. The cytotoxicity assay demonstrated notable increase of chemoresistance to 5-FU treatment (10 μM) in CD133{sup +} cells at 96 h after siRNA transfection. From this study, we conclude that CD133{sup +} cells may have chemoresistance to 5-FU through the mechanism which is related with survivin expression, instead of MDR1, ABCG2 and AKT1 expression. Therefore a survivin inhibitor can be a new target for effective treatment of CD133{sup +} colon cancer. - Highlights: • We evaluate the role of CD133 in chemoresistance of colon cancer. • We compared the chemoresistance of CD133{sup +} cells and siRNA-induced CD133{sup −} cells. • CD133 had little to no effect on MDR1, ABCG2 and AKT1 expression. • Survivin expression and chemoresistance were increased in CD133{sup +} colon cancer cells.« less

Learning directed acyclic graphs from large-scale genomics data.

PubMed

Nikolay, Fabio; Pesavento, Marius; Kritikos, George; Typas, Nassos

2017-09-20

In this paper, we consider the problem of learning the genetic interaction map, i.e., the topology of a directed acyclic graph (DAG) of genetic interactions from noisy double-knockout (DK) data. Based on a set of well-established biological interaction models, we detect and classify the interactions between genes. We propose a novel linear integer optimization program called the Genetic-Interactions-Detector (GENIE) to identify the complex biological dependencies among genes and to compute the DAG topology that matches the DK measurements best. Furthermore, we extend the GENIE program by incorporating genetic interaction profile (GI-profile) data to further enhance the detection performance. In addition, we propose a sequential scalability technique for large sets of genes under study, in order to provide statistically significant results for real measurement data. Finally, we show via numeric simulations that the GENIE program and the GI-profile data extended GENIE (GI-GENIE) program clearly outperform the conventional techniques and present real data results for our proposed sequential scalability technique.

A generic motif discovery algorithm for sequential data.

PubMed

Jensen, Kyle L; Styczynski, Mark P; Rigoutsos, Isidore; Stephanopoulos, Gregory N

2006-01-01

Motif discovery in sequential data is a problem of great interest and with many applications. However, previous methods have been unable to combine exhaustive search with complex motif representations and are each typically only applicable to a certain class of problems. Here we present a generic motif discovery algorithm (Gemoda) for sequential data. Gemoda can be applied to any dataset with a sequential character, including both categorical and real-valued data. As we show, Gemoda deterministically discovers motifs that are maximal in composition and length. As well, the algorithm allows any choice of similarity metric for finding motifs. Finally, Gemoda's output motifs are representation-agnostic: they can be represented using regular expressions, position weight matrices or any number of other models for any type of sequential data. We demonstrate a number of applications of the algorithm, including the discovery of motifs in amino acids sequences, a new solution to the (l,d)-motif problem in DNA sequences and the discovery of conserved protein substructures. Gemoda is freely available at http://web.mit.edu/bamel/gemoda

Ketamine inhibits tumor necrosis factor-{alpha} and interleukin-6 gene expressions in lipopolysaccharide-stimulated macrophages through suppression of toll-like receptor 4-mediated c-Jun N-terminal kinase phosphorylation and activator protein-1 activation

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

Wu, G.-J.; Department of Anesthesiology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan; Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan

2008-04-01

Our previous study showed that ketamine, an intravenous anesthetic agent, has anti-inflammatory effects. In this study, we further evaluated the effects of ketamine on the regulation of tumor necrosis factor-{alpha} (TNF-{alpha}) and interlukin-6 (IL-6) gene expressions and its possible signal-transducing mechanisms in lipopolysaccharide (LPS)-activated macrophages. Exposure of macrophages to 1, 10, and 100 {mu}M ketamine, 100 ng/ml LPS, or a combination of ketamine and LPS for 1, 6, and 24 h was not cytotoxic to macrophages. A concentration of 1000 {mu}M of ketamine alone or in combined treatment with LPS caused significant cell death. Administration of LPS increased cellular TNF-{alpha}more » and IL-6 protein levels in concentration- and time-dependent manners. Meanwhile, treatment with ketamine concentration- and time-dependently alleviated the enhanced effects. LPS induced TNF-{alpha} and IL-6 mRNA syntheses. Administration of ketamine at a therapeutic concentration (100 {mu}M) significantly inhibited LPS-induced TNF-{alpha} and IL-6 mRNA expressions. Application of toll-like receptor 4 (TLR4) small interfering (si)RNA into macrophages decreased cellular TLR4 levels. Co-treatment of macrophages with ketamine and TLR4 siRNA decreased the LPS-induced TNF-{alpha} and IL-6 productions more than alone administration of TLR4 siRNA. LPS stimulated phosphorylation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos from the cytoplasm to nuclei. However, administration of ketamine significantly decreased LPS-induced activation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos. LPS increased the binding of nuclear extracts to activator protein-1 consensus DNA oligonucleotides. Administration of ketamine significantly ameliorated LPS-induced DNA binding activity of activator protein-1. Therefore, a clinically relevant concentration of ketamine can inhibit TNF-{alpha} and IL-6 gene expressions in LPS-activated macrophages. The suppressive mechanisms occur through suppression of TLR4-mediated sequential activations of c-Jun N-terminal kinase and activator protein-1.« less

Ketamine inhibits tumor necrosis factor-alpha and interleukin-6 gene expressions in lipopolysaccharide-stimulated macrophages through suppression of toll-like receptor 4-mediated c-Jun N-terminal kinase phosphorylation and activator protein-1 activation.

PubMed

Wu, Gone-Jhe; Chen, Ta-Liang; Ueng, Yune-Fang; Chen, Ruei-Ming

2008-04-01

Our previous study showed that ketamine, an intravenous anesthetic agent, has anti-inflammatory effects. In this study, we further evaluated the effects of ketamine on the regulation of tumor necrosis factor-alpha (TNF-alpha) and interlukin-6 (IL-6) gene expressions and its possible signal-transducing mechanisms in lipopolysaccharide (LPS)-activated macrophages. Exposure of macrophages to 1, 10, and 100 microM ketamine, 100 ng/ml LPS, or a combination of ketamine and LPS for 1, 6, and 24 h was not cytotoxic to macrophages. A concentration of 1000 microM of ketamine alone or in combined treatment with LPS caused significant cell death. Administration of LPS increased cellular TNF-alpha and IL-6 protein levels in concentration- and time-dependent manners. Meanwhile, treatment with ketamine concentration- and time-dependently alleviated the enhanced effects. LPS induced TNF-alpha and IL-6 mRNA syntheses. Administration of ketamine at a therapeutic concentration (100 microM) significantly inhibited LPS-induced TNF-alpha and IL-6 mRNA expressions. Application of toll-like receptor 4 (TLR4) small interfering (si)RNA into macrophages decreased cellular TLR4 levels. Co-treatment of macrophages with ketamine and TLR4 siRNA decreased the LPS-induced TNF-alpha and IL-6 productions more than alone administration of TLR4 siRNA. LPS stimulated phosphorylation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos from the cytoplasm to nuclei. However, administration of ketamine significantly decreased LPS-induced activation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos. LPS increased the binding of nuclear extracts to activator protein-1 consensus DNA oligonucleotides. Administration of ketamine significantly ameliorated LPS-induced DNA binding activity of activator protein-1. Therefore, a clinically relevant concentration of ketamine can inhibit TNF-alpha and IL-6 gene expressions in LPS-activated macrophages. The suppressive mechanisms occur through suppression of TLR4-mediated sequential activations of c-Jun N-terminal kinase and activator protein-1.

Increasing methylation of the calcitonin gene during disease progression in sequential samples from CML patients.

PubMed

Mills, K I; Guinn, B A; Walsh, V A; Burnett, A K

1996-09-01

In chronic myeloid leukaemia (CML), disease progression from the initial chronic phase to the acute phase or blast crisis has previously been shown to be correlated with progressive increases in hyper-methylation of the calcitonin gene, located at chromosome 11p15. However, sequential studies of individual patients were not performed in these investigations. We have analysed 44 samples from nine patients with typical Philadelphia chromosome positive CML throughout their disease progression to determine the methylation state of the calcitonin gene at these time points. Densitometry was used to quantitate the ratio of the normal 2.0 kb Hpa II fragments, indicating normal methylation status of the gene, compared to the intensity of the abnormal, hyper-methylated, 2.6-3.1 kb Hpa II fragments. We found a gradual increase in the ratio of methylated:unmethylated calcitonin gene during chronic phase with a dramatic rise at blast crisis. Further, the ratio of the abnormal hypermethylated 3.1 kb fragments to the methylated 2.6 kb fragment resulted in the identification of a clonal expansion of abnormally methylated cells. This expansion of cells with hypermethylation of the calcitonin gene during chronic phase was shown to coincide with the presence of a mutation in the p53 gene. The data presented in this study would suggest that an increased methylation status of the calcitonin gene during disease progression may indicate the expansion of abnormal blast cell populations and subsequent progression to blast crisis.

Schedule-dependent cytotoxic synergism of pemetrexed and erlotinib in BXPC-3 and PANC-1 human pancreatic cancer cells.

PubMed

Wang, Lin; Zhu, Zhi-Xia; Zhang, Wen-Ying; Zhang, Wei-Min

2011-09-01

Previous studies have shown that both pemetrexed, a cytotoxic drug, and erlotinib, an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), inhibit the cell growth of pancreatic cancer cells. However, whether they exert a synergistic antitumor effect on pancreatic cancer cells remains unknown. The present study aimed to assess the synergistic effect of erlotinib in combination with pemetrexed using different sequential administration schedules on the proliferation of human pancreatic cancer BXPC-3 and PANC-1 cells and to probe its cellular mechanism. The EGFR and K-ras gene mutation status was examined by quantitative PCR high-resolution melting (qPCR-HRM) analysis. BXPC-3 and PANC-1 cells were incubated with pemetrexed and erlotinib using different administration schedules. MTT assay was used to determine cytotoxicity, and cell cycle distribution was determined by flow cytometry. The expression and phosphorylation of EGFR, HER3, AKT and MET were determined using Western blotting. Both pemetrexed and erlotinib inhibited the proliferation of BXPC-3 and PANC-1 cells in a dose- and time-dependent manner in vitro. Synergistic effects on cell proliferation were observed when pemetrexed was used in combination with erlotinib. The degree of the synergistic effects depended on the administration sequence, which was most obvious when erlotinib was sequentially administered at 24-h interval following pemetrexed. Cell cycle studies revealed that pemetrexed induced S arrest and erlotinib induced G(0)/G(1) arrest. The sequential administration of erlotinib following pemetrexed induced S arrest. Western blot analyses showed that pemetrexed increased and erlotinib decreased the phosphorylation of EGFR, HER3 and AKT, respectively. However, both pemetrexed and erlotinib exerted no significant effects on the phosphorylation of c-MET. The phosphorylation of EGFR, HER3 and AKT was significantly suppressed by scheduled incubation with pemetrexed followed by erlotinib, but not by concomitant or sequential incubation with erlotinib followed by pemetrexed. In summary, our results demonstrated that the combined use of erlotinib and pemetrexed exhibited a strong synergism in BXPC-3 and PANC-1 cells. The inhibitory effects were strongest after sequential administration of pemetrexed followed by erlotinib. The synergistic effects may be related to activation of the EGFR/HER3/AKT pathway induced by pemetrexed.

A simple and reliable multi-gene transformation method for switchgrass.

PubMed

Ogawa, Yoichi; Shirakawa, Makoto; Koumoto, Yasuko; Honda, Masaho; Asami, Yuki; Kondo, Yasuhiro; Hara-Nishimura, Ikuko

2014-07-01

A simple and reliable Agrobacterium -mediated transformation method was developed for switchgrass. Using this method, many transgenic plants carrying multiple genes-of-interest could be produced without untransformed escape. Switchgrass (Panicum virgatum L.) is a promising biomass crop for bioenergy. To obtain transgenic switchgrass plants carrying a multi-gene trait in a simple manner, an Agrobacterium-mediated transformation method was established by constructing a Gateway-based binary vector, optimizing transformation conditions and developing a novel selection method. A MultiRound Gateway-compatible destination binary vector carrying the bar selectable marker gene, pHKGB110, was constructed to introduce multiple genes of interest in a single transformation. Two reporter gene expression cassettes, GUSPlus and gfp, were constructed independently on two entry vectors and then introduced into a single T-DNA region of pHKGB110 via sequential LR reactions. Agrobacterium tumefaciens EHA101 carrying the resultant binary vector pHKGB112 and caryopsis-derived compact embryogenic calli were used for transformation experiments. Prolonged cocultivation for 7 days followed by cultivation on media containing meropenem improved transformation efficiency without overgrowth of Agrobacterium, which was, however, not inhibited by cefotaxime or Timentin. In addition, untransformed escape shoots were completely eliminated during the rooting stage by direct dipping the putatively transformed shoots into the herbicide Basta solution for a few seconds, designated as the 'herbicide dipping method'. It was also demonstrated that more than 90 % of the bar-positive transformants carried both reporters delivered from pHKGB112. This simple and reliable transformation method, which incorporates a new selection technique and the use of a MultiRound Gateway-based binary vector, would be suitable for producing a large number of transgenic lines carrying multiple genes.

Transformation and radiosensitivity of human diploid skin fibroblasts transfected with activated ras oncogene and SV40 T-antigen.

PubMed

Su, L N; Little, J B

1992-08-01

Three normal human diploid cell strains were transfected with an activated Ha-ras oncogene (EJ ras) or SV40 T-antigen. Multiple clones were examined for morphological alterations, growth requirements, ability to grow under anchorage independent conditions, immortality and tumorigenicity in nude mice. Clones expressing SV40 T-antigen alone or in combination with ras protein p21 were significantly radioresistant as compared with their parent cells or clones transfected with the neo gene only. This radioresistant phenotype persisted in post-crisis, immortalized cell lines. Cells transfected with EJ ras alone showed no morphological alterations nor significant changes in radiosensitivity. Cell clones expressing ras and/or SV40 T-antigen showed a reduced requirement for serum supplements, an increase in aneuploidy and chromosomal aberrations, and enhanced growth in soft agar as an early cellular response to SV40 T-antigen expression. The sequential order of transfection with SV40 T-antigen and ras influenced radio-sensitivity but not the induction of morphological changes. These data suggest that expression of the SV40 T-antigen but not activated Ha-ras plays an important role in the radiosensitivity of human diploid cells. The radioresistant phenotype in SV40 T transfected cells was not related to the enhanced level of genetic instability seen in pre-crisis and newly immortalized cells, nor to the process of immortalization itself.

On the specificity of sequential congruency effects in implicit learning of motor and perceptual sequences.

PubMed

D'Angelo, Maria C; Jiménez, Luis; Milliken, Bruce; Lupiáñez, Juan

2013-01-01

Individuals experience less interference from conflicting information following events that contain conflicting information. Recently, Jiménez, Lupiáñez, and Vaquero (2009) demonstrated that such adaptations to conflict occur even when the source of conflict arises from implicit knowledge of sequences. There is accumulating evidence that momentary changes in adaptations made in response to conflicting information are conflict-type specific (e.g., Funes, Lupiáñez, & Humphreys, 2010a), suggesting that there are multiple modes of control. The current study examined whether conflict-specific sequential congruency effects occur when the 2 sources of conflict are implicitly learned. Participants implicitly learned a motor sequence while simultaneously learning a perceptual sequence. In a first experiment, after learning the 2 orthogonal sequences, participants expressed knowledge of the 2 sequences independently of each other in a transfer phase. In Experiments 2 and 3, within each sequence, the presence of a single control trial disrupted the expression of this specific type of learning on the following trial. There was no evidence of cross-conflict modulations in the expression of sequence learning. The results suggest that the mechanisms involved in transient shifts in conflict-specific control, as reflected in sequential congruency effects, are also engaged when the source of conflict is implicit. (c) 2013 APA, all rights reserved.

Morphological adaptation of sheep's rumen epithelium to high-grain diet entails alteration in the expression of genes involved in cell cycle regulation, cell proliferation and apoptosis.

PubMed

Xu, Lei; Wang, Yue; Liu, Junhua; Zhu, Weiyun; Mao, Shengyong

2018-01-01

The objectives of this study were to characterize changes in the relative mRNA expression of candidate genes and proteins involved in cell cycle regulation, cell proliferation and apoptosis in the ruminal epithelium (RE) of sheep during high-grain (HG) diet adaptation. Twenty sheep were assigned to four groups with five animals each. These animals were assigned to different periods of HG diet (containing 40% forage and 60% concentrate mix) feeding. The HG groups received an HG diet for 7 (G7, n  = 5), 14 (G14, n  = 5) and 28 d (G28, n  = 5), respectively. In contrast, the control group (CON, n  = 5) was fed the forage-based diet for 28 d. The results showed that HG feeding linearly decreased ( P  <  0.001) the ruminal pH, and increased the concentrations of ruminal total volatile fatty acid (linear, P  = 0.001), butyrate (linear, P  <  0.001), valerate (quadratic P  = 0.029) and the level of IGF-1 (quadratic, P  = 0.043) in plasma. The length (quadratic, P  = 0.004), width (cubic, P  = 0.015) and surface of the ruminal papillae (linear, P  = 0.003) were all enlarged after 14 d of HG diet feeding. HG feeding cubically increased the number of cell layers forming the stratum corneum (SC, P  <  0.001) and the thickness of the SC ( P  <  0.001) and stratum basale ( P  <  0.001). The proportion of basal layer cells in the RE decreased (linear, P  <  0.001) in the G0/G1-phase, but it increased linearly ( P  = 0.006) in the S-phase and cubically ( P  = 0.004) in the G2/M-phases. The proportion of apoptosis cells in G7, G14 and G28 was reduced compared to the CON (quadratic, P  < 0.001). HG diet feeding linearly decreased the mRNA expression of Cyclin E1 ( P  = 0.021) and CDK-2 ( P  = 0.001) and ( P  = 0.027) the protein expression of Cyclin E1. Feeding an HG diet linearly increased the mRNA expression of genes IGFBP-2 ( P  = 0.034) and IGFBP 5 ( P  < 0.009), while linearly decreasing ( P  < 0.001) the IGFBP 3 expression. The expression of cell apoptosis gene Caspase 8 decreased (quadratic, P  = 0.012), while Bad mRNA expression tended to decrease (cubic, P  = 0.053) after HG feeding. These results demonstrated sequential changes in rumen papillae size, cell cycle regulation and the genes involved in proliferation and apoptosis as time elapsed in feeding a high-grain diet to sheep.

An Intracellular Laccase Is Responsible for Epicatechin-Mediated Anthocyanin Degradation in Litchi Fruit Pericarp.

PubMed

Fang, Fang; Zhang, Xue-lian; Luo, Hong-hui; Zhou, Jia-jian; Gong, Yi-hui; Li, Wen-jun; Shi, Zhao-wan; He, Quan; Wu, Qing; Li, Lu; Jiang, Lin-lin; Cai, Zhi-gao; Oren-Shamir, Michal; Zhang, Zhao-qi; Pang, Xue-qun

2015-12-01

In contrast to the detailed molecular knowledge available on anthocyanin synthesis, little is known about its catabolism in plants. Litchi (Litchi chinensis) fruit lose their attractive red color soon after harvest. The mechanism leading to quick degradation of anthocyanins in the pericarp is not well understood. An anthocyanin degradation enzyme (ADE) was purified to homogeneity by sequential column chromatography, using partially purified anthocyanins from litchi pericarp as a substrate. The purified ADE, of 116 kD by urea SDS-PAGE, was identified as a laccase (ADE/LAC). The full-length complementary DNA encoding ADE/LAC was obtained, and a polyclonal antibody raised against a deduced peptide of the gene recognized the ADE protein. The anthocyanin degradation function of the gene was confirmed by its transient expression in tobacco (Nicotiana benthamiana) leaves. The highest ADE/LAC transcript abundance was in the pericarp in comparison with other tissues, and was about 1,000-fold higher than the polyphenol oxidase gene in the pericarp. Epicatechin was found to be the favorable substrate for the ADE/LAC. The dependence of anthocyanin degradation by the enzyme on the presence of epicatechin suggests an ADE/LAC epicatechin-coupled oxidation model. This model was supported by a dramatic decrease in epicatechin content in the pericarp parallel to anthocyanin degradation. Immunogold labeling transmission electron microscopy suggested that ADE/LAC is located mainly in the vacuole, with essential phenolic substances. ADE/LAC vacuolar localization, high expression levels in the pericarp, and high epicatechin-dependent anthocyanin degradation support its central role in pigment breakdown during pericarp browning. © 2015 American Society of Plant Biologists. All Rights Reserved.

Effects of abscisic acid on ethylene biosynthesis and perception in Hibiscus rosa-sinensis L. flower development

PubMed Central

Trivellini, Alice; Ferrante, Antonio; Vernieri, Paolo; Serra, Giovanni

2011-01-01

The effect of the complex relationship between ethylene and abscisic acid (ABA) on flower development and senescence in Hibiscus rosa-sinensis L. was investigated. Ethylene biosynthetic (HrsACS and HrsACO) and receptor (HrsETR and HrsERS) genes were isolated and their expression evaluated in three different floral tissues (petals, style–stigma plus stamens, and ovaries) of detached buds and open flowers. This was achieved through treatment with 0.1 mM 1-aminocyclopropane-1-carboxylic acid (ACC) solution, 500 nl l−1 methylcyclopropene (1-MCP), and 0.1 mM ABA solution. Treatment with ACC and 1-MCP confirmed that flower senescence in hibiscus is ethylene dependent, and treatment with exogenous ABA suggested that ABA may play a role in this process. The 1-MCP impeded petal in-rolling and decreased ABA content in detached open flowers after 9 h. This was preceded by an earlier and sequential increase in ABA content in 1-MCP-treated petals and style–stigma plus stamens between 1 h and 6 h. ACC treatment markedly accelerated flower senescence and increased ethylene production after 6 h and 9 h, particularly in style–stigma plus stamens. Ethylene evolution was positively correlated in these floral tissues with the induction of the gene expression of ethylene biosynthetic and receptor genes. Finally, ABA negatively affected the ethylene biosynthetic pathway and tissue sensitivity in all flower tissues. Transcript abundance of HrsACS, HrsACO, HrsETR, and HrsERS was reduced by exogenous ABA treatment. This research underlines the regulatory effect of ABA on the ethylene biosynthetic and perception machinery at a physiological and molecular level when inhibitors or promoters of senescence are exogenously applied. PMID:21841180

Enhancement of astaxanthin production in Xanthophyllomyces dendrorhous by efficient method for the complete deletion of genes.

PubMed

Yamamoto, Keisuke; Hara, Kiyotaka Y; Morita, Toshihiko; Nishimura, Akira; Sasaki, Daisuke; Ishii, Jun; Ogino, Chiaki; Kizaki, Noriyuki; Kondo, Akihiko

2016-09-13

Red yeast, Xanthophyllomyces dendrorhous is the only yeast known to produce astaxanthin, an anti-oxidant isoprenoid (carotenoid) widely used in the aquaculture, food, pharmaceutical and cosmetic industries. The potential of this microorganism as a platform cell factory for isoprenoid production has been recognized because of high flux through its native terpene pathway. Recently, we developed a multiple gene expression system in X. dendrorhous and enhanced the mevalonate synthetic pathway to increase astaxanthin production. In contrast, the mevalonate synthetic pathway is suppressed by ergosterol through feedback inhibition. Therefore, releasing the mevalonate synthetic pathway from this inhibition through the deletion of genes involved in ergosterol synthesis is a promising strategy to improve isoprenoid production. An efficient method for deleting diploid genes in X. dendrorhous, however, has not yet been developed. Xanthophyllomyces dendrorhous was cultivated under gradually increasing concentrations of antibiotics following the introduction of antibiotic resistant genes to be replaced with target genes. Using this method, double CYP61 genes encoding C-22 sterol desaturases relating to ergosterol biosynthesis were deleted sequentially. This double CYP61 deleted strain showed decreased ergosterol biosynthesis compared with the parental strain and single CYP61 disrupted strain. Additionally, this double deletion of CYP61 genes showed increased astaxanthin production compared with the parental strain and the single CYP61 knockout strain. Finally, astaxanthin production was enhanced by 1.4-fold compared with the parental strain, although astaxanthin production was not affected in the single CYP61 knockout strain. In this study, we developed a system to completely delete target diploid genes in X. dendrorhous. Using this method, we deleted diploid CYP61 genes involved in the synthesis of ergosterol that inhibits the pathway for mevalonate, which is a common substrate for isoprenoid biosynthesis. The resulting decrease in ergosterol biosynthesis increased astaxanthin production. The efficient method for deleting diploid genes developed in this study has the potential to improve industrial production of various isoprenoids in X. dendrorhous.

Pax2/8 proteins coordinate sequential induction of otic and epibranchial placodes through differential regulation of foxi1, sox3 and fgf24.

PubMed

Padanad, Mahesh S; Riley, Bruce B

2011-03-01

Vertebrate cranial placodes contribute vitally to development of sensory structures of the head. Amongst posterior placodes, the otic placode forms the inner ear whereas nearby epibranchial placodes produce sensory ganglia within branchial clefts. Though diverse in fate, these placodes show striking similarities in their early regulation. In zebrafish, both are initiated by localized Fgf signaling plus the ubiquitous competence factor Foxi1, and both express pax8 and sox3 in response. It has been suggested that Fgf initially induces a common otic/epibranchial field, which later subdivides in response to other signals. However, we find that otic and epibranchial placodes form at different times and by distinct mechanisms. Initially, Fgf from surrounding tissues induces otic expression of pax8 and sox3, which cooperate synergistically to establish otic fate. Subsequently, pax8 works with related genes pax2a/pax2b to downregulate otic expression of foxi1, a necessary step for further otic development. Additionally, pax2/8 activate otic expression of fgf24, which induces epibranchial expression of sox3. Knockdown of fgf24 or sox3 causes severe epibranchial deficiencies but has little effect on otic development. These findings clarify the roles of pax8 and sox3 and support a model whereby the otic placode forms first and induces epibranchial placodes through an Fgf-relay. Copyright © 2011 Elsevier Inc. All rights reserved.

Gestalt Imagery: A Critical Factor in Language Comprehension.

ERIC Educational Resources Information Center

Bell, Nanci

1991-01-01

Lack of gestalt imagery (the ability to create imaged wholes) can contribute to language comprehension disorder characterized by weak reading comprehension, weak oral language comprehension, weak oral language expression, weak written language expression, difficulty following directions, and a weak sense of humor. Sequential stimulation using an…

Anti-cancer effect of oncolytic adenovirus-armed shRNA targeting MYCN gene on doxorubicin-resistant neuroblastoma cells.

PubMed

Li, Yuan; Zhuo, Baobiao; Yin, Yiyu; Han, Tao; Li, Shixian; Li, Zhengwei; Wang, Jian

2017-09-09

Chemotherapy is one of the few effective choices for patients with neuroblastoma. However, the development of muti-drug resistance (MDR) to chemotherapy is a major obstacle to the effective treatment of advanced or recurrent neuroblastoma. The muti-drug resistance-associated protein (MRP), which encodes a transmembrane glycoprotein, is a key regulator of MDR. The expression of MRP is a close correlation with MYCN oncogene in neuroblastoma. We have recently shown ZD55-shMYCN (oncolytic virus armed with shRNA against MYCN) can down-regulate MYCN to inhibit tumor cells proliferation and induce apoptosis in neuroblastoma. Here we further report ZD55-shMYCN re-sensitized doxorubicin-resistant cells to doxorubicin (as shown by reduced proliferation, increased apoptosis, and inhibited cell migration), and reduced the in vivo growth rate of neuroblastoma xenografts by down-regulation of MRP expression. Sequential therapy with doxorubicin did not affect the replication of ZD55-shMYCN in doxorubicin-resistant neuroblastoma cells, but decreased the expression of Bcl-2, Bcl-X L , MMP-1. Thus, this synergistic effect of ZD55-shMYCN in combination with doxorubicin provides a novel therapy strategy for doxorubicin-resistant neuroblastoma, and is a promising approach for further clinical development. Copyright © 2017 Elsevier Inc. All rights reserved.

Neuronal Activation After Prolonged Immobilization: Do the Same or Different Neurons Respond to a Novel Stressor?

PubMed

Marín-Blasco, Ignacio; Muñoz-Abellán, Cristina; Andero, Raül; Nadal, Roser; Armario, Antonio

2018-04-01

Despite extensive research on the impact of emotional stressors on brain function using immediate-early genes (e.g., c-fos), there are still important questions that remain unanswered such as the reason for the progressive decline of c-fos expression in response to prolonged stress and the neuronal populations activated by different stressors. This study tackles these 2 questions by evaluating c-fos expression in response to 2 different emotional stressors applied sequentially, and performing a fluorescent double labeling of c-Fos protein and c-fos mRNA on stress-related brain areas. Results were complemented with the assessment of the hypothalamic-pituitary-adrenal axis activation. We showed that the progressive decline of c-fos expression could be related to 2 differing mechanisms involving either transcriptional repression or changes in stimulatory inputs. Moreover, the neuronal populations that respond to the different stressors appear to be predominantly separated in high-level processing areas (e.g., medial prefrontal cortex). However, in low-hierarchy areas (e.g., paraventricular nucleus of the hypothalamus) neuronal populations appear to respond unspecifically. The data suggest that the distinct physiological and behavioral consequences of emotional stressors, and their implication in the development of psychopathologies, are likely to be closely associated with neuronal populations specifically activated by each stressor.

Whole genome sequencing of an African American family highlights toll like receptor 6 variants in Kawasaki disease susceptibility.

PubMed

Kim, Jihoon; Shimizu, Chisato; Kingsmore, Stephen F; Veeraraghavan, Narayanan; Levy, Eric; Ribeiro Dos Santos, Andre M; Yang, Hai; Flatley, Jay; Hoang, Long Truong; Hibberd, Martin L; Tremoulet, Adriana H; Harismendy, Olivier; Ohno-Machado, Lucila; Burns, Jane C

2017-01-01

Kawasaki disease (KD) is the most common acquired pediatric heart disease. We analyzed Whole Genome Sequences (WGS) from a 6-member African American family in which KD affected two of four children. We sought rare, potentially causative genotypes by sequentially applying the following WGS filters: sequence quality scores, inheritance model (recessive homozygous and compound heterozygous), predicted deleteriousness, allele frequency, genes in KD-associated pathways or with significant associations in published KD genome-wide association studies (GWAS), and with differential expression in KD blood transcriptomes. Biologically plausible genotypes were identified in twelve variants in six genes in the two affected children. The affected siblings were compound heterozygous for the rare variants p.Leu194Pro and p.Arg247Lys in Toll-like receptor 6 (TLR6), which affect TLR6 signaling. The affected children were also homozygous for three common, linked (r2 = 1) intronic single nucleotide variants (SNVs) in TLR6 (rs56245262, rs56083757 and rs7669329), that have previously shown association with KD in cohorts of European descent. Using transcriptome data from pre-treatment whole blood of KD subjects (n = 146), expression quantitative trait loci (eQTL) analyses were performed. Subjects homozygous for the intronic risk allele (A allele of TLR6 rs56245262) had differential expression of Interleukin-6 (IL-6) as a function of genotype (p = 0.0007) and a higher erythrocyte sedimentation rate at diagnosis. TLR6 plays an important role in pathogen-associated molecular pattern recognition, and sequence variations may affect binding affinities that in turn influence KD susceptibility. This integrative genomic approach illustrates how the analysis of WGS in multiplex families with a complex genetic disease allows examination of both the common disease-common variant and common disease-rare variant hypotheses.

Differential muscle regulatory factor gene expression between larval and adult myogenesis in the frog Xenopus laevis: adult myogenic cell-specific myf5 upregulation and its relation to the notochord suppression of adult muscle differentiation.

PubMed

Yamane, Hitomi; Nishikawa, Akio

2013-08-01

During Xenopus laevis metamorphosis, larval-to-adult muscle conversion depends on the differential responses of adult and larval myogenic cells to thyroid hormone. Essential differences in cell growth, differentiation, and hormone-dependent life-or-death fate have been reported between cultured larval (tail) and adult (hindlimb) myogenic cells. A previous study revealed that tail notochord cells suppress terminal differentiation in adult (but not larval) myogenic cells. However, little is known about the differences in expression patterns of myogenic regulatory factors (MRF) and the satellite cell marker Pax7 between adult and larval myogenic cells. In the present study, we compared mRNA expression of these factors between the two types. At first, reverse transcription polymerase chain reaction analysis of hindlimb buds showed sequential upregulation of myf5, myogenin, myod, and mrf4 during stages 50-54, when limb buds elongate and muscles begin to form. By contrast, in the tail, there was no such increase during the same period. Secondary, these results were duplicated in vitro: adult myogenic cells upregulated myf5, myod, and pax7 in the early culture period, followed by myogenin upregulation and myotube differentiation, while larval myogenic cells did not upregulate these genes and precociously started myotube differentiation. Thirdly, myf5 upregulation and early-phase proliferation in adult myogenic cells were potently inhibited by the presence of notochord cells, suggesting that notochord cells suppress adult myogenesis through inhibiting the transition from Myf5(-) stem cells to Myf5(+) committed myoblasts. All of the data presented here suggest that myf5 upregulation can be a good criterion for the activation of adult myogenesis during X. laevis metamorphosis.

A novel visual hardware behavioral language

NASA Technical Reports Server (NTRS)

Li, Xueqin; Cheng, H. D.

1992-01-01

Most hardware behavioral languages just use texts to describe the behavior of the desired hardware design. This is inconvenient for VLSI designers who enjoy using the schematic approach. The proposed visual hardware behavioral language has the ability to graphically express design information using visual parallel models (blocks), visual sequential models (processes) and visual data flow graphs (which consist of primitive operational icons, control icons, and Data and Synchro links). Thus, the proposed visual hardware behavioral language can not only specify hardware concurrent and sequential functionality, but can also visually expose parallelism, sequentiality, and disjointness (mutually exclusive operations) for the hardware designers. That would make the hardware designers capture the design ideas easily and explicitly using this visual hardware behavioral language.

Generation of Five Human Lactoferrin Transgenic Cloned Goats Using Fibroblast Cells and Their Methylation Status of Putative Differential Methylation Regions of IGF2R and H19 Imprinted Genes

PubMed Central

Sun, Yanyan; Zhang, Yanli; Wang, Ziyu; Song, Yang; Wang, Feng

2013-01-01

Background Somatic cell nuclear transfer (SCNT) is a promising technique to produce transgenic cloned mammalian, including transgenic goats which may produce Human Lactoferrin (hLF). However, success percentage of SCNT is low, because of gestational and neonatal failure of transgenic embryos. According to the studies on cattle and mice, DNA methylation of some imprinted genes, which plays a vital role in the reprogramming of embryo in NT maybe an underlying mechanism. Methodology/Principal Findings Fibroblast cells were derived from the ear of a two-month-old goat. The vector expressing hLF was constructed and transfected into fibroblasts. G418 selection, EGFP expression, PCR, and cell cycle distribution were applied sequentially to select transgenic cells clones. After NT and embryo transfer, five transgenic cloned goats were obtained from 240 cloned transgenic embryos. These transgenic goats were identified by 8 microsatellites genotyping and southern blot. Of the five transgenic goats, 3 were lived after birth, while 2 were dead during gestation. We compared differential methylation regions (DMR) pattern of two paternally imprinted genes (H19 and IGF2R) of the ear tissues from the lived transgenic goats, dead transgenic goats, and control goats from natural reproduction. Hyper-methylation pattern appeared in cloned aborted goats, while methylation status was relatively normal in cloned lived goats compared with normal goats. Conclusions/Significance In this study, we generated five hLF transgenic cloned goats by SCNT. This is the first time the DNA methylation of lived and dead transgenic cloned goats was compared. The results demonstrated that the methylation status of DMRs of H19 and IGF2R were different in lived and dead transgenic goats and therefore this may be potentially used to assess the reprogramming status of transgenic cloned goats. Understanding the pattern of gene imprinting may be useful to improve cloning techniques in future. PMID:24204972

Kupffer cell ablation attenuates cyclooxygenase-2 expression after trauma and sepsis.

PubMed

Keller, Steve A; Paxian, Marcus; Lee, Sun M; Clemens, Mark G; Huynh, Toan

2005-03-01

Prostaglandins, synthesized by cyclooxygenase (COX), play an important role in the pathophysiology of inflammation. Severe injuries result in immunosuppression, mediated, in part, by maladaptive changes in macrophages. Herein, we assessed Kupffer cell-mediated cyclooxygenase-2 (COX-2) expression on liver function and damage after trauma and sepsis. To ablate Kupffer cells, Sprague Dawley rats were treated with gadolinium chloride (GdCl3) 48 and 24 h before experimentation. Animals then underwent femur fracture (FFx) followed 48 h later by cecal ligation and puncture (CLP). Controls received sham operations. After 24 h, liver samples were obtained, and mRNA and protein expression were determined by PCR, Western blot, and immunohistochemistry. Indocyanine-Green (ICG) clearance and plasma alanine aminotransferase (ALT) levels were determined to assess liver function and damage, respectively. One-way analysis of variance (ANOVA) with Student-Newman-Keuls test was used to assess statistical significance. After CLP alone, FFx+CLP, and GdCl3+FFx+CLP, clearance of ICG decreased. Plasma ALT levels increased in parallel with severity of injury. Kupffer cell depletion attenuated the increased ALT levels after FFx+CLP. Femur fracture alone did not alter COX-2 protein compared with sham. By contrast, COX-2 protein increased after CLP and was potentiated by sequential stress. Again, Kupffer cell depletion abrogated the increase in COX-2 after sequential stress. Immunohistochemical data confirmed COX-2 positive cells to be Kupffer cells. In this study, sequential stress increased hepatic COX-2 protein. Depletion of Kupffer cells reduced COX-2 and attenuated hepatocellular injuries. Our data suggest that Kupffer cell-dependent pathways may contribute to the inflammatory response leading to increased mortality after sequential stress.

Molecular characterization of the fatty alcohol oxidation pathway for wax-ester mobilization in germinated jojoba seeds.

PubMed

Rajangam, Alex S; Gidda, Satinder K; Craddock, Christian; Mullen, Robert T; Dyer, John M; Eastmond, Peter J

2013-01-01

Jojoba (Simmondsia chinensis) is the only plant species known to use liquid wax esters (WEs) as a primary seed storage reserve. Upon germination, WE hydrolysis releases very-long-chain fatty alcohols, which must be oxidized to fatty acids by the sequential action of a fatty alcohol oxidase (FAO) and a fatty aldehyde dehydrogenase (FADH) before they can be β-oxidized. Here, we describe the cloning and characterization of genes for each of these two activities. Jojoba FAO and FADH are 52% and 68% identical to Arabidopsis (Arabidopsis thaliana) FAO3 and ALDH3H1, respectively. The genes are expressed most strongly in the cotyledons of jojoba seedlings following germination, but transcripts can also be detected in vegetative tissues. Proteomic analysis indicated that the FAO and FADH proteins can be detected on wax bodies, but they localized to the endoplasmic reticulum when they were expressed as amino-terminal green fluorescent protein fusions in tobacco (Nicotiana tabacum) leaves. Recombinant jojoba FAO and FADH proteins are active on very-long-chain fatty alcohol and fatty aldehyde substrates, respectively, and have biochemical properties consistent with those previously reported in jojoba cotyledons. Coexpression of jojoba FAO and FADH in Arabidopsis enhanced the in vivo rate of fatty alcohol oxidation more than 4-fold. Taken together, our data suggest that jojoba FAO and FADH constitute the very-long-chain fatty alcohol oxidation pathway that is likely to be necessary for efficient WE mobilization following seed germination.

Molecular Characterization of the Fatty Alcohol Oxidation Pathway for Wax-Ester Mobilization in Germinated Jojoba Seeds1[W

PubMed Central

Rajangam, Alex S.; Gidda, Satinder K.; Craddock, Christian; Mullen, Robert T.; Dyer, John M.; Eastmond, Peter J.

2013-01-01

Jojoba (Simmondsia chinensis) is the only plant species known to use liquid wax esters (WEs) as a primary seed storage reserve. Upon germination, WE hydrolysis releases very-long-chain fatty alcohols, which must be oxidized to fatty acids by the sequential action of a fatty alcohol oxidase (FAO) and a fatty aldehyde dehydrogenase (FADH) before they can be β-oxidized. Here, we describe the cloning and characterization of genes for each of these two activities. Jojoba FAO and FADH are 52% and 68% identical to Arabidopsis (Arabidopsis thaliana) FAO3 and ALDH3H1, respectively. The genes are expressed most strongly in the cotyledons of jojoba seedlings following germination, but transcripts can also be detected in vegetative tissues. Proteomic analysis indicated that the FAO and FADH proteins can be detected on wax bodies, but they localized to the endoplasmic reticulum when they were expressed as amino-terminal green fluorescent protein fusions in tobacco (Nicotiana tabacum) leaves. Recombinant jojoba FAO and FADH proteins are active on very-long-chain fatty alcohol and fatty aldehyde substrates, respectively, and have biochemical properties consistent with those previously reported in jojoba cotyledons. Coexpression of jojoba FAO and FADH in Arabidopsis enhanced the in vivo rate of fatty alcohol oxidation more than 4-fold. Taken together, our data suggest that jojoba FAO and FADH constitute the very-long-chain fatty alcohol oxidation pathway that is likely to be necessary for efficient WE mobilization following seed germination. PMID:23166353

Embryotrophic factor-3 from human oviductal cells enhances proliferation, suppresses apoptosis and stimulates the expression of the beta1 subunit of sodium-potassium ATPase in mouse embryos.

PubMed

Xu, J S; Lee, Y L; Lee, K F; Kwok, K L; Lee, W M; Luk, J M; Yeung, W S B

2004-12-01

Embrytrophic factor-3 (ETF-3) from human oviductal cells enhanced the development of mouse preimplantation embryos. This report studied the embryotrophic mechanisms of the molecule. Mouse embryos were incubated with ETF-3 for 24 h at different stages of development. ETF-3 treatment between 96 and 120 h post-HCG increased the cell count of blastocysts, whilst treatment between 72 and 96 h post-HCG enhanced the expansion and hatching of the blastocysts. ETF-3 increased the cell number of the embryos by suppressing apoptosis and increasing proliferation as determined by TUNEL and bromodeoxyuridine uptake assays, respectively. Real-time quantitative PCR showed that the in vivo developed and ETF-3-treated blastocysts had a significantly higher mRNA copy number of Na/K-ATPase-beta1, but not of hepsin, than that of blastocysts cultured in medium alone. The former gene was associated with cavitation of blastocysts while the latter was related to hatching of blastocyst. The beneficial effect of ETF-3 on blastocyst hatching was also seen when ETF-3-supplemented commercially available sequential culture medium for human embryo culture was used to culture mouse embryos. ETF-3 improves embryo development by enhancing proliferation, suppressing apoptosis and stimulating expression of genes related to blastocyst cavitation. Supplementating human embryo culture medium with ETF-3 may improve the success rate in clinical assisted reproduction.

The Evolution of Gene Regulatory Networks that Define Arthropod Body Plans.

PubMed

Auman, Tzach; Chipman, Ariel D

2017-09-01

Our understanding of the genetics of arthropod body plan development originally stems from work on Drosophila melanogaster from the late 1970s and onward. In Drosophila, there is a relatively detailed model for the network of gene interactions that proceeds in a sequential-hierarchical fashion to define the main features of the body plan. Over the years, we have a growing understanding of the networks involved in defining the body plan in an increasing number of arthropod species. It is now becoming possible to tease out the conserved aspects of these networks and to try to reconstruct their evolution. In this contribution, we focus on several key nodes of these networks, starting from early patterning in which the main axes are determined and the broad morphological domains of the embryo are defined, and on to later stage wherein the growth zone network is active in sequential addition of posterior segments. The pattern of conservation of networks is very patchy, with some key aspects being highly conserved in all arthropods and others being very labile. Many aspects of early axis patterning are highly conserved, as are some aspects of sequential segment generation. In contrast, regional patterning varies among different taxa, and some networks, such as the terminal patterning network, are only found in a limited range of taxa. The growth zone segmentation network is ancient and is probably plesiomorphic to all arthropods. In some insects, it has undergone significant modification to give rise to a more hardwired network that generates individual segments separately. In other insects and in most arthropods, the sequential segmentation network has undergone a significant amount of systems drift, wherein many of the genes have changed. However, it maintains a conserved underlying logic and function. © The Author 2017. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.

Molecular Cytogenetic Identification of a New Wheat-Rye 6R Chromosome Disomic Addition Line with Powdery Mildew Resistance

PubMed Central

An, Diaoguo; Zheng, Qi; Luo, Qiaoling; Ma, Pengtao; Zhang, Hongxia; Li, Lihui; Han, Fangpu; Xu, Hongxing; Xu, Yunfeng; Zhang, Xiaotian; Zhou, Yilin

2015-01-01

Rye (Secale cereale L.) possesses many valuable genes that can be used for improving disease resistance, yield and environment adaptation of wheat (Triticum aestivum L.). However, the documented resistance stocks derived from rye is faced severe challenge due to the variation of virulent isolates in the pathogen populations. Therefore, it is necessary to develop desirable germplasm and search for novel resistance gene sources against constantly accumulated variation of the virulent isolates. In the present study, a new wheat-rye line designated as WR49-1 was produced through distant hybridization and chromosome engineering protocols between common wheat cultivar Xiaoyan 6 and rye cultivar German White. Using sequential GISH (genomic in situ hybridization), mc-FISH (multicolor fluorescence in situ hybridization), mc-GISH (multicolor GISH) and EST (expressed sequence tag)-based marker analysis, WR49-1 was proved to be a new wheat-rye 6R disomic addition line. As expected, WR49-1 showed high levels of resistance to wheat powdery mildew (Blumeria graminis f. sp. tritici, Bgt) pathogens prevalent in China at the adult growth stage and 19 of 23 Bgt isolates tested at the seedling stage. According to its reaction pattern to different Bgt isolates, WR49-1 may possess new resistance gene(s) for powdery mildew, which differed from the documented powdery mildew gene, including Pm20 on chromosome arm 6RL of rye. Additionally, WR49-1 was cytologically stable, had improved agronomic characteristics and therefore could serve as an important bridge for wheat breeding and chromosome engineering. PMID:26237413

Intergenic Transcriptional Interference Is Blocked by RNA Polymerase III Transcription Factor TFIIIB in Saccharomyces cerevisiae

PubMed Central

Korde, Asawari; Rosselot, Jessica M.; Donze, David

2014-01-01

The major function of eukaryotic RNA polymerase III is to transcribe transfer RNA, 5S ribosomal RNA, and other small non-protein-coding RNA molecules. Assembly of the RNA polymerase III complex on chromosomal DNA requires the sequential binding of transcription factor complexes TFIIIC and TFIIIB. Recent evidence has suggested that in addition to producing RNA transcripts, chromatin-assembled RNA polymerase III complexes may mediate additional nuclear functions that include chromatin boundary, nucleosome phasing, and general genome organization activities. This study provides evidence of another such “extratranscriptional” activity of assembled RNA polymerase III complexes, which is the ability to block progression of intergenic RNA polymerase II transcription. We demonstrate that the RNA polymerase III complex bound to the tRNA gene upstream of the Saccharomyces cerevisiae ATG31 gene protects the ATG31 promoter against readthrough transcriptional interference from the upstream noncoding intergenic SUT467 transcription unit. This protection is predominately mediated by binding of the TFIIIB complex. When TFIIIB binding to this tRNA gene is weakened, an extended SUT467–ATG31 readthrough transcript is produced, resulting in compromised ATG31 translation. Since the ATG31 gene product is required for autophagy, strains expressing the readthrough transcript exhibit defective autophagy induction and reduced fitness under autophagy-inducing nitrogen starvation conditions. Given the recent discovery of widespread pervasive transcription in all forms of life, protection of neighboring genes from intergenic transcriptional interference may be a key extratranscriptional function of assembled RNA polymerase III complexes and possibly other DNA binding proteins. PMID:24336746

Global Microarray Analysis of Carbohydrate Use in Alkaliphilic Hemicellulolytic Bacterium Bacillus sp. N16-5

PubMed Central

Song, Yajian; Xue, Yanfen; Ma, Yanhe

2013-01-01

The alkaliphilic hemicellulolytic bacterium Bacillus sp. N16-5 has a broad substrate spectrum and exhibits the capacity to utilize complex carbohydrates such as galactomannan, xylan, and pectin. In the monosaccharide mixture, sequential utilization by Bacillus sp. N16-5 was observed. Glucose appeared to be its preferential monosaccharide, followed by fructose, mannose, arabinose, xylose, and galactose. Global transcription profiles of the strain were determined separately for growth on six monosaccharides (glucose, fructose, mannose, galactose, arabinose, and xylose) and four polysaccharides (galactomannan, xylan, pectin, and sodium carboxymethylcellulose) using one-color microarrays. Numerous genes potentially related to polysaccharide degradation, sugar transport, and monosaccharide metabolism were found to respond to a specific substrate. Putative gene clusters for different carbohydrates were identified according to transcriptional patterns and genome annotation. Identification and analysis of these gene clusters contributed to pathway reconstruction for carbohydrate utilization in Bacillus sp. N16-5. Several genes encoding putative sugar transporters were highly expressed during growth on specific sugars, suggesting their functional roles. Two phosphoenolpyruvate-dependent phosphotransferase systems were identified as candidate transporters for mannose and fructose, and a major facilitator superfamily transporter was identified as a candidate transporter for arabinose and xylose. Five carbohydrate uptake transporter 1 family ATP-binding cassette transporters were predicted to participate in the uptake of hemicellulose and pectin degradation products. Collectively, microarray data improved the pathway reconstruction involved in carbohydrate utilization of Bacillus sp. N16-5 and revealed that the organism precisely regulates gene transcription in response to fluctuations in energy resources. PMID:23326578

Neuron-derived orphan receptor 1 transduces survival signals in neuronal cells in response to hypoxia-induced apoptotic insults.

PubMed

Chio, Chung-Ching; Wei, Li; Chen, Tyng Guey; Lin, Chien-Min; Shieh, Ja-Ping; Yeh, Poh-Shiow; Chen, Ruei-Ming

2016-06-01

OBJECT Hypoxia can induce cell death or trigger adaptive mechanisms to guarantee cell survival. Neuron-derived orphan receptor 1 (NOR-1) works as an early-response protein in response to a variety of environmental stresses. In this study, the authors evaluated the roles of NOR-1 in hypoxia-induced neuronal insults. METHODS Neuro-2a cells were exposed to oxygen/glucose deprivation (OGD). Cell viability, cell morphology, cas-pase-3 activity, DNA fragmentation, and cell apoptosis were assayed to determine the mechanisms of OGD-induced neuronal insults. RNA and protein analyses were carried out to evaluate the effects of OGD on expressions of NOR-1, cAMP response element-binding (CREB), and cellular inhibitor of apoptosis protein 2 (cIAP2) genes. Translations of these gene expressions were knocked down using RNA interference. Mice subjected to traumatic brain injury (TBI) and NOR-1 was immunodetected. RESULTS Exposure of neuro-2a cells to OGD decreased cell viability in a time-dependent manner. Additionally, OGD led to cell shrinkage, DNA fragmentation, and cell apoptosis. In parallel, treatment of neuro-2a cells with OGD time dependently increased cellular NOR-1 mRNA and protein expressions. Interestingly, administration of TBI also augmented NOR-1 levels in the impacted regions of mice. As to the mechanism, exposure to OGD increased nuclear levels of the transcription factor CREB protein. Downregulating CREB expression using RNA interference simultaneously inhibited OGD-induced NOR-1 mRNA expression. Also, levels of cIAP2 mRNA and protein in neuro-2a cells were augmented by OGD. After reducing cIAP2 translation, OGD-induced cell death was reduced. Sequentially, application of NOR-1 small interfering RNA to neuro-2a cells significantly inhibited OGD-induced cIAP2 mRNA expression and concurrently alleviated hypoxia-induced alterations in cell viability, caspase-3 activation, DNA damage, and cell apoptosis. CONCLUSIONS This study shows that NOR-1 can transduce survival signals in neuronal cells responsible for hypoxiainduced apoptotic insults through activation of a CREB/cIAP2-dependent mechanism.

CaSPIAN: A Causal Compressive Sensing Algorithm for Discovering Directed Interactions in Gene Networks

PubMed Central

Emad, Amin; Milenkovic, Olgica

2014-01-01

We introduce a novel algorithm for inference of causal gene interactions, termed CaSPIAN (Causal Subspace Pursuit for Inference and Analysis of Networks), which is based on coupling compressive sensing and Granger causality techniques. The core of the approach is to discover sparse linear dependencies between shifted time series of gene expressions using a sequential list-version of the subspace pursuit reconstruction algorithm and to estimate the direction of gene interactions via Granger-type elimination. The method is conceptually simple and computationally efficient, and it allows for dealing with noisy measurements. Its performance as a stand-alone platform without biological side-information was tested on simulated networks, on the synthetic IRMA network in Saccharomyces cerevisiae, and on data pertaining to the human HeLa cell network and the SOS network in E. coli. The results produced by CaSPIAN are compared to the results of several related algorithms, demonstrating significant improvements in inference accuracy of documented interactions. These findings highlight the importance of Granger causality techniques for reducing the number of false-positives, as well as the influence of noise and sampling period on the accuracy of the estimates. In addition, the performance of the method was tested in conjunction with biological side information of the form of sparse “scaffold networks”, to which new edges were added using available RNA-seq or microarray data. These biological priors aid in increasing the sensitivity and precision of the algorithm in the small sample regime. PMID:24622336

Production of cattle lacking prion protein

PubMed Central

Richt, Jürgen A; Kasinathan, Poothappillai; Hamir, Amir N; Castilla, Joaquin; Sathiyaseelan, Thillai; Vargas, Francisco; Sathiyaseelan, Janaki; Wu, Hua; Matsushita, Hiroaki; Koster, Julie; Kato, Shinichiro; Ishida, Isao; Soto, Claudio; Robl, James M; Kuroiwa, Yoshimi

2010-01-01

Prion diseases are caused by propagation of misfolded forms of the normal cellular prion protein PrPC, such as PrPBSE in bovine spongiform encephalopathy (BSE) in cattle and PrPCJD in Creutzfeldt-Jakob disease (CJD) in humans1. Disruption of PrPC expression in mice, a species that does not naturally contract prion diseases, results in no apparent developmental abnormalities2–5. However, the impact of ablating PrPC function in natural host species of prion diseases is unknown. Here we report the generation and characterization of PrPC-deficient cattle produced by a sequential gene-targeting system6. At over 20 months of age, the cattle are clinically, physiologically, histopathologically, immunologically and reproductively normal. Brain tissue homogenates are resistant to prion propagation in vitro as assessed by protein misfolding cyclic amplification7. PrPC-deficient cattle may be a useful model for prion research and could provide industrial bovine products free of prion proteins. PMID:17195841

Neural Stem Cells: Historical Perspective and Future Prospects

PubMed Central

Breunig, Joshua J.; Haydar, Tarik F.; Rakic, Pasko

2011-01-01

How a single fertilized cell generates diverse neuronal populations has been a fundamental biological problem since the 19th century. Classical histological methods revealed that post-mitotic neurons are produced in a precise temporal and spatial order from germinal cells lining the cerebral ventricles. In the 20th century DNA labeling and histo- and immuno-histochemistry helped to distinguish the subtypes of dividing cells and delineate their locations in the ventricular and subventricular zones. Recently, genetic and cell biological methods have provided insights into sequential gene expression and molecular and cellular interactions that generate heterogeneous populations of NSCs leading to specific neuronal classes. This precisely regulated developmental process does not tolerate significant in vivo deviation, making replacement of adult neurons by NSCs during pathology a colossal challenge. In contrast, utilizing the trophic factors emanating from the NSC or their derivatives to slow down deterioration or prevent death of degenerating neurons may be a more feasible strategy. PMID:21609820

The sunburn response in human skin is characterized by sequential eicosanoid profiles that may mediate its early and late phases

PubMed Central

Rhodes, Lesley E.; Gledhill, Karl; Masoodi, Mojgan; Haylett, Ann K.; Brownrigg, Margaret; Thody, Anthony J.; Tobin, Desmond J.; Nicolaou, Anna

2009-01-01

Sunburn is a commonly occurring acute inflammatory process, with dermal vasodilatation and leukocyte infiltration as central features. Ultraviolet (UV) B-induced hydrolysis of membrane phospholipids releases polyunsaturated fatty acids, and their subsequent metabolism by cyclooxygenases (COXs) and lipoxygenases (LOXs) may produce potent eicosanoid mediators modulating different stages of the inflammation. Our objective was to identify candidate eicosanoids formed during the sunburn reaction in relation to its clinical and histological course. We exposed skin of healthy humans (n=32) to UVB and, for 72 h, examined expression of proinflammatory and anti-inflammatory eicosanoids using LC/ESI-MS/MS, and examined immunohistochemical expression of COX-2, 12-LOX, 15-LOX, and leukocyte markers, while quantifying clinical erythema. We show that vasodilatory prostaglandins (PGs) PGE2, PGF2α, and PGE3 accompany the erythema in the first 24–48 h, associated with increased COX-2 expression at 24 h. Novel, potent leukocyte chemoattractants 11-, 12-, and 8-monohydroxy-eicosatetraenoic acid (HETE) are elevated from 4 to 72 h, in association with peak dermal neutrophil influx at 24 h, and increased dermal CD3+ lymphocytes and 12- and 15-LOX expression from 24 to 72 h. Anti-inflammatory metabolite 15-HETE shows later expression, peaking at 72 h. Sunburn is characterized by overlapping sequential profiles of increases in COX products followed by LOX products that may regulate subsequent events and ultimately its resolution.—Rhodes, L. E., Gledhill, K., Masoodi, M., Haylett, A. K., Brownrigg, M., Thody, A. J., Tobin, D. J., Nicolaou, A. The sunburn response in human skin is characterized by sequential eicosanoid profiles that may mediate its early and late phases. PMID:19584301

Thermodynamically balanced inside-out (TBIO) PCR-based gene synthesis: a novel method of primer design for high-fidelity assembly of longer gene sequences

PubMed Central

Gao, Xinxin; Yo, Peggy; Keith, Andrew; Ragan, Timothy J.; Harris, Thomas K.

2003-01-01

A novel thermodynamically-balanced inside-out (TBIO) method of primer design was developed and compared with a thermodynamically-balanced conventional (TBC) method of primer design for PCR-based gene synthesis of codon-optimized gene sequences for the human protein kinase B-2 (PKB2; 1494 bp), p70 ribosomal S6 subunit protein kinase-1 (S6K1; 1622 bp) and phosphoinositide-dependent protein kinase-1 (PDK1; 1712 bp). Each of the 60mer TBIO primers coded for identical nucleotide regions that the 60mer TBC primers covered, except that half of the TBIO primers were reverse complement sequences. In addition, the TBIO and TBC primers contained identical regions of temperature- optimized primer overlaps. The TBC method was optimized to generate sequential overlapping fragments (∼0.4–0.5 kb) for each of the gene sequences, and simultaneous and sequential combinations of overlapping fragments were tested for their ability to be assembled under an array of PCR conditions. However, no fully synthesized gene sequences could be obtained by this approach. In contrast, the TBIO method generated an initial central fragment (∼0.4–0.5 kb), which could be gel purified and used for further inside-out bidirectional elongation by additional increments of 0.4–0.5 kb. By using the newly developed TBIO method of PCR-based gene synthesis, error-free synthetic genes for the human protein kinases PKB2, S6K1 and PDK1 were obtained with little or no corrective mutagenesis. PMID:14602936

Sequential Infection in Ferrets with Antigenically Distinct Seasonal H1N1 Influenza Viruses Boosts Hemagglutinin Stalk-Specific Antibodies

PubMed Central

Kirchenbaum, Greg A.; Carter, Donald M.

2015-01-01

ABSTRACT Broadly reactive antibodies targeting the conserved hemagglutinin (HA) stalk region are elicited following sequential infection or vaccination with influenza viruses belonging to divergent subtypes and/or expressing antigenically distinct HA globular head domains. Here, we demonstrate, through the use of novel chimeric HA proteins and competitive binding assays, that sequential infection of ferrets with antigenically distinct seasonal H1N1 (sH1N1) influenza virus isolates induced an HA stalk-specific antibody response. Additionally, stalk-specific antibody titers were boosted following sequential infection with antigenically distinct sH1N1 isolates in spite of preexisting, cross-reactive, HA-specific antibody titers. Despite a decline in stalk-specific serum antibody titers, sequential sH1N1 influenza virus-infected ferrets were protected from challenge with a novel H1N1 influenza virus (A/California/07/2009), and these ferrets poorly transmitted the virus to naive contacts. Collectively, these findings indicate that HA stalk-specific antibodies are commonly elicited in ferrets following sequential infection with antigenically distinct sH1N1 influenza virus isolates lacking HA receptor-binding site cross-reactivity and can protect ferrets against a pathogenic novel H1N1 virus. IMPORTANCE The influenza virus hemagglutinin (HA) is a major target of the humoral immune response following infection and/or seasonal vaccination. While antibodies targeting the receptor-binding pocket of HA possess strong neutralization capacities, these antibodies are largely strain specific and do not confer protection against antigenic drift variant or novel HA subtype-expressing viruses. In contrast, antibodies targeting the conserved stalk region of HA exhibit broader reactivity among viruses within and among influenza virus subtypes. Here, we show that sequential infection of ferrets with antigenically distinct seasonal H1N1 influenza viruses boosts the antibody responses directed at the HA stalk region. Moreover, ferrets possessing HA stalk-specific antibody were protected against novel H1N1 virus infection and did not transmit the virus to naive contacts. PMID:26559834

Development of a Markerless Genetic Exchange System in Desulfovibrio vulgaris Hildenborough and Its Use in Generating a Strain with Increased Transformation Efficiency

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

Keller, Kimberly L.; Bender, Kelly S.; Wall, Judy D.

2009-07-21

In recent years, the genetic manipulation of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough has seen enormous progress. In spite of this progress, the current marker exchange deletion method does not allow for easy selection of multiple sequential gene deletions in a single strain because of the limited number of selectable markers available in D. vulgaris. To broaden the repertoire of genetic tools for manipulation, an in-frame, markerless deletion system has been developed. The counterselectable marker that makes this deletion system possible is the pyrimidine salvage enzyme, uracil phosphoribosyltransferase, encoded by upp. In wild-type D. vulgaris, growth was shown to bemore » inhibited by the toxic pyrimidine analog 5-fluorouracil (5-FU); whereas, a mutant bearing a deletion of the upp gene was resistant to 5-FU. When a plasmid containing the wild-type upp gene expressed constitutively from the aph(3')-II promoter (promoter for the kanamycin resistance gene in Tn5) was introduced into the upp deletion strain, sensitivity to 5-FU was restored. This observation allowed us to develop a two-step integration and excision strategy for the deletion of genes of interest. Since this inframe deletion strategy does not retain an antibiotic cassette, multiple deletions can be generated in a single strain without the accumulation of genes conferring antibiotic resistances. We used this strategy to generate a deletion strain lacking the endonuclease (hsdR, DVU1703) of a type I restriction-modification system, that we designated JW7035. The transformation efficiency of the JW7035 strain was found to be 100 to 1000 times greater than that of the wild-type strain when stable plasmids were introduced via electroporation.« less

[FOXP2 and the molecular biology of language: new evidence. II. Molecular aspects and implications for the ontogenesis and phylogeny of language].

PubMed

Benítez-Burraco, A

FOXP2 is the first gene linked to a hereditary variant of specific language impairment and seems to code for a transcriptional repressor that intervenes in the regulation of the development and the functioning of certain thalamic-cortical-striatal circuits. In the last three years, significant progress has been made in the determination of the structural and functional properties of the gene. These advances essentially have to do with the precise analysis of the most important structural motifs of the protein that it codes for and the main parameters that determine its interaction with DNA. They also concern the determination of the functional and behavioural properties in vivo of the main isoforms of the FOXP2 protein, the exact determination of the pattern of expression of new orthologues of the gene, and the identification of the different target genes for factor FOXP2. This new evidence suggests that protein FOXP2 protein has a high degree of versatility in vivo when it comes to binding to DNA; that its different isoforms are biologically functional; and that the FOXP2 gene is functional during embryonic development and during the adult phase. It also suggests that it is involved in the development and/or functioning of the thalamic-cortical-striatal circuits associated to motor planning, sequential behaviour and procedural learning (a significant saving in developmental terms of the regulatory mechanism in which the gene is involved), as well as the accuracy of the models of linguistic processing that consider language to be, to a large extent, the result of an interaction between certain cortical and subcortical structures.

GEM-TREND: a web tool for gene expression data mining toward relevant network discovery

PubMed Central

Feng, Chunlai; Araki, Michihiro; Kunimoto, Ryo; Tamon, Akiko; Makiguchi, Hiroki; Niijima, Satoshi; Tsujimoto, Gozoh; Okuno, Yasushi

2009-01-01

Background DNA microarray technology provides us with a first step toward the goal of uncovering gene functions on a genomic scale. In recent years, vast amounts of gene expression data have been collected, much of which are available in public databases, such as the Gene Expression Omnibus (GEO). To date, most researchers have been manually retrieving data from databases through web browsers using accession numbers (IDs) or keywords, but gene-expression patterns are not considered when retrieving such data. The Connectivity Map was recently introduced to compare gene expression data by introducing gene-expression signatures (represented by a set of genes with up- or down-regulated labels according to their biological states) and is available as a web tool for detecting similar gene-expression signatures from a limited data set (approximately 7,000 expression profiles representing 1,309 compounds). In order to support researchers to utilize the public gene expression data more effectively, we developed a web tool for finding similar gene expression data and generating its co-expression networks from a publicly available database. Results GEM-TREND, a web tool for searching gene expression data, allows users to search data from GEO using gene-expression signatures or gene expression ratio data as a query and retrieve gene expression data by comparing gene-expression pattern between the query and GEO gene expression data. The comparison methods are based on the nonparametric, rank-based pattern matching approach of Lamb et al. (Science 2006) with the additional calculation of statistical significance. The web tool was tested using gene expression ratio data randomly extracted from the GEO and with in-house microarray data, respectively. The results validated the ability of GEM-TREND to retrieve gene expression entries biologically related to a query from GEO. For further analysis, a network visualization interface is also provided, whereby genes and gene annotations are dynamically linked to external data repositories. Conclusion GEM-TREND was developed to retrieve gene expression data by comparing query gene-expression pattern with those of GEO gene expression data. It could be a very useful resource for finding similar gene expression profiles and constructing its gene co-expression networks from a publicly available database. GEM-TREND was designed to be user-friendly and is expected to support knowledge discovery. GEM-TREND is freely available at . PMID:19728865

GEM-TREND: a web tool for gene expression data mining toward relevant network discovery.

PubMed

Feng, Chunlai; Araki, Michihiro; Kunimoto, Ryo; Tamon, Akiko; Makiguchi, Hiroki; Niijima, Satoshi; Tsujimoto, Gozoh; Okuno, Yasushi

2009-09-03

DNA microarray technology provides us with a first step toward the goal of uncovering gene functions on a genomic scale. In recent years, vast amounts of gene expression data have been collected, much of which are available in public databases, such as the Gene Expression Omnibus (GEO). To date, most researchers have been manually retrieving data from databases through web browsers using accession numbers (IDs) or keywords, but gene-expression patterns are not considered when retrieving such data. The Connectivity Map was recently introduced to compare gene expression data by introducing gene-expression signatures (represented by a set of genes with up- or down-regulated labels according to their biological states) and is available as a web tool for detecting similar gene-expression signatures from a limited data set (approximately 7,000 expression profiles representing 1,309 compounds). In order to support researchers to utilize the public gene expression data more effectively, we developed a web tool for finding similar gene expression data and generating its co-expression networks from a publicly available database. GEM-TREND, a web tool for searching gene expression data, allows users to search data from GEO using gene-expression signatures or gene expression ratio data as a query and retrieve gene expression data by comparing gene-expression pattern between the query and GEO gene expression data. The comparison methods are based on the nonparametric, rank-based pattern matching approach of Lamb et al. (Science 2006) with the additional calculation of statistical significance. The web tool was tested using gene expression ratio data randomly extracted from the GEO and with in-house microarray data, respectively. The results validated the ability of GEM-TREND to retrieve gene expression entries biologically related to a query from GEO. For further analysis, a network visualization interface is also provided, whereby genes and gene annotations are dynamically linked to external data repositories. GEM-TREND was developed to retrieve gene expression data by comparing query gene-expression pattern with those of GEO gene expression data. It could be a very useful resource for finding similar gene expression profiles and constructing its gene co-expression networks from a publicly available database. GEM-TREND was designed to be user-friendly and is expected to support knowledge discovery. GEM-TREND is freely available at http://cgs.pharm.kyoto-u.ac.jp/services/network.

Graphics Processing Unit–Enhanced Genetic Algorithms for Solving the Temporal Dynamics of Gene Regulatory Networks

PubMed Central

García-Calvo, Raúl; Guisado, JL; Diaz-del-Rio, Fernando; Córdoba, Antonio; Jiménez-Morales, Francisco

2018-01-01

Understanding the regulation of gene expression is one of the key problems in current biology. A promising method for that purpose is the determination of the temporal dynamics between known initial and ending network states, by using simple acting rules. The huge amount of rule combinations and the nonlinear inherent nature of the problem make genetic algorithms an excellent candidate for finding optimal solutions. As this is a computationally intensive problem that needs long runtimes in conventional architectures for realistic network sizes, it is fundamental to accelerate this task. In this article, we study how to develop efficient parallel implementations of this method for the fine-grained parallel architecture of graphics processing units (GPUs) using the compute unified device architecture (CUDA) platform. An exhaustive and methodical study of various parallel genetic algorithm schemes—master-slave, island, cellular, and hybrid models, and various individual selection methods (roulette, elitist)—is carried out for this problem. Several procedures that optimize the use of the GPU’s resources are presented. We conclude that the implementation that produces better results (both from the performance and the genetic algorithm fitness perspectives) is simulating a few thousands of individuals grouped in a few islands using elitist selection. This model comprises 2 mighty factors for discovering the best solutions: finding good individuals in a short number of generations, and introducing genetic diversity via a relatively frequent and numerous migration. As a result, we have even found the optimal solution for the analyzed gene regulatory network (GRN). In addition, a comparative study of the performance obtained by the different parallel implementations on GPU versus a sequential application on CPU is carried out. In our tests, a multifold speedup was obtained for our optimized parallel implementation of the method on medium class GPU over an equivalent sequential single-core implementation running on a recent Intel i7 CPU. This work can provide useful guidance to researchers in biology, medicine, or bioinformatics in how to take advantage of the parallelization on massively parallel devices and GPUs to apply novel metaheuristic algorithms powered by nature for real-world applications (like the method to solve the temporal dynamics of GRNs). PMID:29662297

Graphics Processing Unit-Enhanced Genetic Algorithms for Solving the Temporal Dynamics of Gene Regulatory Networks.

PubMed

García-Calvo, Raúl; Guisado, J L; Diaz-Del-Rio, Fernando; Córdoba, Antonio; Jiménez-Morales, Francisco

2018-01-01

Understanding the regulation of gene expression is one of the key problems in current biology. A promising method for that purpose is the determination of the temporal dynamics between known initial and ending network states, by using simple acting rules. The huge amount of rule combinations and the nonlinear inherent nature of the problem make genetic algorithms an excellent candidate for finding optimal solutions. As this is a computationally intensive problem that needs long runtimes in conventional architectures for realistic network sizes, it is fundamental to accelerate this task. In this article, we study how to develop efficient parallel implementations of this method for the fine-grained parallel architecture of graphics processing units (GPUs) using the compute unified device architecture (CUDA) platform. An exhaustive and methodical study of various parallel genetic algorithm schemes-master-slave, island, cellular, and hybrid models, and various individual selection methods (roulette, elitist)-is carried out for this problem. Several procedures that optimize the use of the GPU's resources are presented. We conclude that the implementation that produces better results (both from the performance and the genetic algorithm fitness perspectives) is simulating a few thousands of individuals grouped in a few islands using elitist selection. This model comprises 2 mighty factors for discovering the best solutions: finding good individuals in a short number of generations, and introducing genetic diversity via a relatively frequent and numerous migration. As a result, we have even found the optimal solution for the analyzed gene regulatory network (GRN). In addition, a comparative study of the performance obtained by the different parallel implementations on GPU versus a sequential application on CPU is carried out. In our tests, a multifold speedup was obtained for our optimized parallel implementation of the method on medium class GPU over an equivalent sequential single-core implementation running on a recent Intel i7 CPU. This work can provide useful guidance to researchers in biology, medicine, or bioinformatics in how to take advantage of the parallelization on massively parallel devices and GPUs to apply novel metaheuristic algorithms powered by nature for real-world applications (like the method to solve the temporal dynamics of GRNs).

Neighboring Genes Show Correlated Evolution in Gene Expression

PubMed Central

Ghanbarian, Avazeh T.; Hurst, Laurence D.

2015-01-01

When considering the evolution of a gene’s expression profile, we commonly assume that this is unaffected by its genomic neighborhood. This is, however, in contrast to what we know about the lack of autonomy between neighboring genes in gene expression profiles in extant taxa. Indeed, in all eukaryotic genomes genes of similar expression-profile tend to cluster, reflecting chromatin level dynamics. Does it follow that if a gene increases expression in a particular lineage then the genomic neighbors will also increase in their expression or is gene expression evolution autonomous? To address this here we consider evolution of human gene expression since the human-chimp common ancestor, allowing for both variation in estimation of current expression level and error in Bayesian estimation of the ancestral state. We find that in all tissues and both sexes, the change in gene expression of a focal gene on average predicts the change in gene expression of neighbors. The effect is highly pronounced in the immediate vicinity (<100 kb) but extends much further. Sex-specific expression change is also genomically clustered. As genes increasing their expression in humans tend to avoid nuclear lamina domains and be enriched for the gene activator 5-hydroxymethylcytosine, we conclude that, most probably owing to chromatin level control of gene expression, a change in gene expression of one gene likely affects the expression evolution of neighbors, what we term expression piggybacking, an analog of hitchhiking. PMID:25743543

Leukotriene B4—leukotriene B4 receptor axis promotes oxazolone-induced contact dermatitis by directing skin homing of neutrophils and CD8+ T cells

PubMed Central

Lv, Jiaoyan; Zou, Linlin; Zhao, Lina; Yang, Wei; Xiong, Yingluo; Li, Bingji; He, Rui

2015-01-01

Leukotriene B4 (LTB4) is a lipid mediator that is rapidly generated in inflammatory sites, and its functional receptor, BLT1, is mostly expressed on immune cells. Contact dermatitis is a common inflammatory skin disease characterized by skin oedema and abundant inflammatory infiltrates, primarily including neutrophils and CD8+ T cells. The role of the LTB4–BLT1 axis in contact dermatitis remains largely unknown. In this study, we found up-regulated gene expression of 5-lipoxygenase and leukotriene A4 hydrolase, two critical enzymes for LTB4 synthesis, BLT1 and elevated LTB4 levels in skin lesions of oxazolone (OXA)-induced contact dermatitis. BLT1 deficiency or blockade of LTB4 and BLT1 by the antagonists, bestatin and U-75302, respectively, in the elicitation phase caused significant decreases in ear swelling and skin-infiltrating neutrophils and CD8+ T cells, which was accompanied by significantly reduced skin expression of CXCL1, CXCL2, interferon-γ and interleukin-1β. Furthermore, neutrophil depletion during the elicitation phase of OXA-induced contact dermatitis also caused significant decreases in ear swelling and CD8+ T-cell infiltration accompanied by significantly decreased LTB4 synthesis and gene expression of CXCL2, interferon-γ and interleukin-1β. Importantly, subcutaneous injection of exogenous LTB4 restored the skin infiltration of CD8+ T cells in neutrophil-depleted mice following OXA challenge. Collectively, our results demonstrate that the LTB4–BLT1 axis contributes to OXA-induced contact dermatitis by mediating skin recruitment of neutrophils, which are a major source of LTB4 that sequentially direct CD8+ T-cell homing to OXA-challenged skin. Hence, LTB4 and BLT1 could be potential therapeutic targets for the treatment of contact dermatitis. PMID:25959240

Leukotriene B₄-leukotriene B₄ receptor axis promotes oxazolone-induced contact dermatitis by directing skin homing of neutrophils and CD8⁺ T cells.

PubMed

Lv, Jiaoyan; Zou, Linlin; Zhao, Lina; Yang, Wei; Xiong, Yingluo; Li, Bingji; He, Rui

2015-09-01

Leukotriene B4 (LTB4 ) is a lipid mediator that is rapidly generated in inflammatory sites, and its functional receptor, BLT1, is mostly expressed on immune cells. Contact dermatitis is a common inflammatory skin disease characterized by skin oedema and abundant inflammatory infiltrates, primarily including neutrophils and CD8(+) T cells. The role of the LTB4 -BLT1 axis in contact dermatitis remains largely unknown. In this study, we found up-regulated gene expression of 5-lipoxygenase and leukotriene A4 hydrolase, two critical enzymes for LTB4 synthesis, BLT1 and elevated LTB4 levels in skin lesions of oxazolone (OXA)-induced contact dermatitis. BLT1 deficiency or blockade of LTB4 and BLT1 by the antagonists, bestatin and U-75302, respectively, in the elicitation phase caused significant decreases in ear swelling and skin-infiltrating neutrophils and CD8(+) T cells, which was accompanied by significantly reduced skin expression of CXCL1, CXCL2, interferon-γ and interleukin-1β. Furthermore, neutrophil depletion during the elicitation phase of OXA-induced contact dermatitis also caused significant decreases in ear swelling and CD8(+) T-cell infiltration accompanied by significantly decreased LTB4 synthesis and gene expression of CXCL2, interferon-γ and interleukin-1β. Importantly, subcutaneous injection of exogenous LTB4 restored the skin infiltration of CD8(+) T cells in neutrophil-depleted mice following OXA challenge. Collectively, our results demonstrate that the LTB4 -BLT1 axis contributes to OXA-induced contact dermatitis by mediating skin recruitment of neutrophils, which are a major source of LTB4 that sequentially direct CD8(+) T-cell homing to OXA-challenged skin. Hence, LTB4 and BLT1 could be potential therapeutic targets for the treatment of contact dermatitis. © 2015 John Wiley & Sons Ltd.

A Promising Trigene Recombinant Human Adenovirus Vaccine Against Classical Swine Fever Virus.

PubMed

Li, Helin; Gao, Rui; Zhang, Yanming

2016-05-01

Classical swine fever (CSF) vaccine based on HAdV-5 had achieved an efficient protection in swine. Both classical swine fever virus (CSFV) E0 glycoprotein and E2 glycoprotein were the targets for neutralizing antibodies and related to immune protection against CSF. Interleukin-2 (IL2), as an adjuvant, also had been used in CSF vaccine research. In this study, coexpression of the CSFV E0, E2, and IL2 genes by HAdV-5 (rAdV-E0-E2-IL2) was constructed and immunized to evaluate its efficacy. Three expressed genes had been sequentially connected with foot-and-mouth disease virus 2A (FMDV 2A). The vaccine was administered by intramuscular inoculation to CSFV-free pigs (10(8) TCID50) twice at triweekly intervals. No adverse clinical signs were observed in any of the pigs after vaccination. The vaccine induced strong humoral and cellular responses that led to complete protection against clinical signs of lethal CSFV infection, viremia, and shedding of challenge virus. The rAdV-E0-E2-IL2 is a promising, efficient, and safe marker vaccine candidate against CSFV.

Identification of Human HK Genes and Gene Expression Regulation Study in Cancer from Transcriptomics Data Analysis

PubMed Central

Zhang, Zhang; Liu, Jingxing; Wu, Jiayan; Yu, Jun

2013-01-01

The regulation of gene expression is essential for eukaryotes, as it drives the processes of cellular differentiation and morphogenesis, leading to the creation of different cell types in multicellular organisms. RNA-Sequencing (RNA-Seq) provides researchers with a powerful toolbox for characterization and quantification of transcriptome. Many different human tissue/cell transcriptome datasets coming from RNA-Seq technology are available on public data resource. The fundamental issue here is how to develop an effective analysis method to estimate expression pattern similarities between different tumor tissues and their corresponding normal tissues. We define the gene expression pattern from three directions: 1) expression breadth, which reflects gene expression on/off status, and mainly concerns ubiquitously expressed genes; 2) low/high or constant/variable expression genes, based on gene expression level and variation; and 3) the regulation of gene expression at the gene structure level. The cluster analysis indicates that gene expression pattern is higher related to physiological condition rather than tissue spatial distance. Two sets of human housekeeping (HK) genes are defined according to cell/tissue types, respectively. To characterize the gene expression pattern in gene expression level and variation, we firstly apply improved K-means algorithm and a gene expression variance model. We find that cancer-associated HK genes (a HK gene is specific in cancer group, while not in normal group) are expressed higher and more variable in cancer condition than in normal condition. Cancer-associated HK genes prefer to AT-rich genes, and they are enriched in cell cycle regulation related functions and constitute some cancer signatures. The expression of large genes is also avoided in cancer group. These studies will help us understand which cell type-specific patterns of gene expression differ among different cell types, and particularly for cancer. PMID:23382867

Cre/lox-based multiple markerless gene disruption in the genome of the extreme thermophile Thermus thermophilus.

PubMed

Togawa, Yoichiro; Nunoshiba, Tatsuo; Hiratsu, Keiichiro

2018-02-01

Markerless gene-disruption technology is particularly useful for effective genetic analyses of Thermus thermophilus (T. thermophilus), which have a limited number of selectable markers. In an attempt to develop a novel system for the markerless disruption of genes in T. thermophilus, we applied a Cre/lox system to construct a triple gene disruptant. To achieve this, we constructed two genetic tools, a loxP-htk-loxP cassette and cre-expressing plasmid, pSH-Cre, for gene disruption and removal of the selectable marker by Cre-mediated recombination. We found that the Cre/lox system was compatible with the proliferation of the T. thermophilus HB27 strain at the lowest growth temperature (50 °C), and thus succeeded in establishing a triple gene disruptant, the (∆TTC1454::loxP, ∆TTC1535KpnI::loxP, ∆TTC1576::loxP) strain, without leaving behind a selectable marker. During the process of the sequential disruption of multiple genes, we observed the undesired deletion and inversion of the chromosomal region between multiple loxP sites that were induced by Cre-mediated recombination. Therefore, we examined the effects of a lox66-htk-lox71 cassette by exploiting the mutant lox sites, lox66 and lox71, instead of native loxP sites. We successfully constructed a (∆TTC1535::lox72, ∆TTC1537::lox72) double gene disruptant without inducing the undesired deletion of the 0.7-kbp region between the two directly oriented lox72 sites created by the Cre-mediated recombination of the lox66-htk-lox71 cassette. This is the first demonstration of a Cre/lox system being applicable to extreme thermophiles in a genetic manipulation. Our results indicate that this system is a powerful tool for multiple markerless gene disruption in T. thermophilus.

Neighboring Genes Show Correlated Evolution in Gene Expression.

PubMed

Ghanbarian, Avazeh T; Hurst, Laurence D

2015-07-01

When considering the evolution of a gene's expression profile, we commonly assume that this is unaffected by its genomic neighborhood. This is, however, in contrast to what we know about the lack of autonomy between neighboring genes in gene expression profiles in extant taxa. Indeed, in all eukaryotic genomes genes of similar expression-profile tend to cluster, reflecting chromatin level dynamics. Does it follow that if a gene increases expression in a particular lineage then the genomic neighbors will also increase in their expression or is gene expression evolution autonomous? To address this here we consider evolution of human gene expression since the human-chimp common ancestor, allowing for both variation in estimation of current expression level and error in Bayesian estimation of the ancestral state. We find that in all tissues and both sexes, the change in gene expression of a focal gene on average predicts the change in gene expression of neighbors. The effect is highly pronounced in the immediate vicinity (<100 kb) but extends much further. Sex-specific expression change is also genomically clustered. As genes increasing their expression in humans tend to avoid nuclear lamina domains and be enriched for the gene activator 5-hydroxymethylcytosine, we conclude that, most probably owing to chromatin level control of gene expression, a change in gene expression of one gene likely affects the expression evolution of neighbors, what we term expression piggybacking, an analog of hitchhiking. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Statistical characteristics of the sequential detection of signals in correlated noise

NASA Astrophysics Data System (ADS)

Averochkin, V. A.; Baranov, P. E.

1985-10-01

A solution is given to the problem of determining the distribution of the duration of the sequential two-threshold Wald rule for the time-discrete detection of determinate and Gaussian correlated signals on a background of Gaussian correlated noise. Expressions are obtained for the joint probability densities of the likelihood ratio logarithms, and an analysis is made of the effect of correlation and SNR on the duration distribution and the detection efficiency. Comparison is made with Neumann-Pearson detection.

Application of community phylogenetic approaches to understand gene expression: differential exploration of venom gene space in predatory marine gastropods.

PubMed

Chang, Dan; Duda, Thomas F

2014-06-05

Predatory marine gastropods of the genus Conus exhibit substantial variation in venom composition both within and among species. Apart from mechanisms associated with extensive turnover of gene families and rapid evolution of genes that encode venom components ('conotoxins'), the evolution of distinct conotoxin expression patterns is an additional source of variation that may drive interspecific differences in the utilization of species' 'venom gene space'. To determine the evolution of expression patterns of venom genes of Conus species, we evaluated the expression of A-superfamily conotoxin genes of a set of closely related Conus species by comparing recovered transcripts of A-superfamily genes that were previously identified from the genomes of these species. We modified community phylogenetics approaches to incorporate phylogenetic history and disparity of genes and their expression profiles to determine patterns of venom gene space utilization. Less than half of the A-superfamily gene repertoire of these species is expressed, and only a few orthologous genes are coexpressed among species. Species exhibit substantially distinct expression strategies, with some expressing sets of closely related loci ('under-dispersed' expression of available genes) while others express sets of more disparate genes ('over-dispersed' expression). In addition, expressed genes show higher dN/dS values than either unexpressed or ancestral genes; this implies that expression exposes genes to selection and facilitates rapid evolution of these genes. Few recent lineage-specific gene duplicates are expressed simultaneously, suggesting that expression divergence among redundant gene copies may be established shortly after gene duplication. Our study demonstrates that venom gene space is explored differentially by Conus species, a process that effectively permits the independent and rapid evolution of venoms in these species.

Gq protein mediates UVB-induced cyclooxygenase-2 expression by stimulating HB-EGF secretion from HaCaT human keratinocytes

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

Seo, MiRan; Juhnn, Yong-Sung, E-mail: juhnn@snu.ac.kr

Ultraviolet (UV) radiation induces cyclooxygenase-2 expression to produce cellular responses including aging and carcinogenesis in skin. We hypothesised that heterotrimeric G proteins mediate UV-induced COX-2 expression by stimulating secretion of soluble HB-EGF (sHB-EGF). In this study, we aimed to elucidate the role and underlying mechanism of the {alpha} subunit of Gq protein (G{alpha}q) in UVB-induced HB-EGF secretion and COX-2 induction. We found that expression of constitutively active G{alpha}q (G{alpha}qQL) augmented UVB-induced HB-EGF secretion, which was abolished by knockdown of G{alpha}q with shRNA in HaCaT human keratinocytes. G{alpha}q was found to mediate the UVB-induced HB-EGF secretion by sequential activation of phospholipasemore » C (PLC), protein kinase C{delta} (PKC{delta}), and matrix metaloprotease-2 (MMP-2). Moreover, G{alpha}qQL mediated UVB-induced COX-2 expression in an HB-EGF-, EGFR-, and p38-dependent manner. From these results, we concluded that G{alpha}q mediates UV-induced COX-2 expression through activation of EGFR by HB-EGF, of which ectodomain shedding was stimulated through sequential activation of PLC, PKC{delta} and MMP-2 in HaCaT cells.« less

"Maxillary lateral incisor partial anodontia sequence": a clinical entity with epigenetic origin.

PubMed

Consolaro, Alberto; Cardoso, Maurício Almeida; Consolaro, Renata Bianco

2017-01-01

The relationship between maxillary lateral incisor anodontia and the palatal displacement of unerupted maxillary canines cannot be considered as a multiple tooth abnormality with defined genetic etiology in order to be regarded as a "syndrome". Neither were the involved genes identified and located in the human genome, nor was it presumed on which chromosome the responsible gene would be located. The palatal maxillary canine displacement in cases of partial anodontia of the maxillary lateral incisor is potentially associated with environmental changes caused by its absence in its place of formation and eruption, which would characterize an epigenetic etiology. The lack of the maxillary lateral incisor in the canine region means removing one of the reference guides for the eruptive trajectory of the maxillary canine, which would therefore, not erupt and /or impact on the palate. Consequently, and in sequence, it would lead to malocclusion, maxillary atresia, transposition, prolonged retention of the deciduous canine and resorption in the neighboring teeth. Thus, we can say that we are dealing with a set of anomalies and multiple sequential changes known as sequential development anomalies or, simply, sequence. Once the epigenetics and sequential condition is accepted for this clinical picture, it could be called "Maxillary Lateral Incisor Partial Anodontia Sequence."

“Maxillary lateral incisor partial anodontia sequence”: a clinical entity with epigenetic origin

PubMed Central

Consolaro, Alberto; Cardoso, Maurício Almeida; Consolaro, Renata Bianco

2017-01-01

ABSTRACT The relationship between maxillary lateral incisor anodontia and the palatal displacement of unerupted maxillary canines cannot be considered as a multiple tooth abnormality with defined genetic etiology in order to be regarded as a “syndrome”. Neither were the involved genes identified and located in the human genome, nor was it presumed on which chromosome the responsible gene would be located. The palatal maxillary canine displacement in cases of partial anodontia of the maxillary lateral incisor is potentially associated with environmental changes caused by its absence in its place of formation and eruption, which would characterize an epigenetic etiology. The lack of the maxillary lateral incisor in the canine region means removing one of the reference guides for the eruptive trajectory of the maxillary canine, which would therefore, not erupt and /or impact on the palate. Consequently, and in sequence, it would lead to malocclusion, maxillary atresia, transposition, prolonged retention of the deciduous canine and resorption in the neighboring teeth. Thus, we can say that we are dealing with a set of anomalies and multiple sequential changes known as sequential development anomalies or, simply, sequence. Once the epigenetics and sequential condition is accepted for this clinical picture, it could be called “Maxillary Lateral Incisor Partial Anodontia Sequence.” PMID:29364376

Expression and kinetic properties of a recombinant 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase isoenzyme of human liver.

PubMed

Deyashiki, Y; Tamada, Y; Miyabe, Y; Nakanishi, M; Matsuura, K; Hara, A

1995-08-01

Human liver cytosol contains multiple forms of 3 alpha-hydroxysteroid dehydrogenase and dihydrodiol dehydrogenase with hydroxysteroid dehydrogenase activity, and multiple cDNAs for the enzymes have been cloned from human liver cDNA libraries. To understand the relationship of the multiple enzyme froms to the genes, a cDNA, which has been reported to code for an isoenzyme of human liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase, was expressed in Escherichia coli. The recombinant enzyme showed structural and functional properties almost identical to those of the isoenzyme purified from human liver. In addition, the recombinant isoenzyme efficiently reduced 5 alpha-dihydrotestosterone and 5 beta-dihydrocortisone, the known substrates of human liver 3 alpha-hydroxysteroid dehydrogenase and chlordecone reductase previously purified, which suggests that these human liver enzymes are identical. Furthermore, the steady-state kinetic data for NADP(+)-linked (S)-1-indanol oxidation by the recombinant isoenzyme were consistent with a sequential ordered mechanism in which NADP+ binds first. Phenolphthalein inhibited this isoenzyme much more potently than it did the other human liver dihydrodiol dehydrogenases, and was a competitive inhibitor (Ki = 20 nM) that bound to the enzyme-NADP+ complex.

Sequential changes in chromatin structure during transcriptional activation in the beta globin LCR and its target gene.

PubMed

Kim, Kihoon; Kim, AeRi

2010-09-01

Chromatin structure is modulated during transcriptional activation. The changes include the association of transcriptional activators, formation of hypersensitive sites and covalent modifications of histones. To understand the order of the various changes accompanying transcriptional activation, we analyzed the mouse beta globin gene, which is transcriptionally inducible in erythroid MEL cells over a time course of HMBA treatment. Transcription of the globin genes requires the locus control region (LCR) consisting of several hypersensitive sites (HSs). Erythroid specific transcriptional activators such as NF-E2, GATA-1, TAL1 and EKLF were associated with the LCR in the uninduced state before transcriptional activation. The HSs of the LCR were formed in this state as revealed by high sensitivity to DNase I and MNase attack. However the binding of transcriptional activators and the depletion of histones were observed in the promoter of the beta globin gene only after transcriptional activation. In addition, various covalent histone modifications were sequentially detected in lysine residues of histone H3 during the activation. Acetylation of K9, K36 and K27 was notable in both LCR HSs and gene after induction but before transcriptional initiation. Inactive histone marks such as K9me2, K36me2 and K27me2 were removed coincident with transcriptional initiation in the gene region. Taken together, these results indicate that LCR has a substantially active structure in the uninduced state while transcriptional activation serially adds active marks, including histone modifications, and removes inactive marks in the target gene of the LCR. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

FARO server: Meta-analysis of gene expression by matching gene expression signatures to a compendium of public gene expression data.

PubMed

Manijak, Mieszko P; Nielsen, Henrik B

2011-06-11

Although, systematic analysis of gene annotation is a powerful tool for interpreting gene expression data, it sometimes is blurred by incomplete gene annotation, missing expression response of key genes and secondary gene expression responses. These shortcomings may be partially circumvented by instead matching gene expression signatures to signatures of other experiments. To facilitate this we present the Functional Association Response by Overlap (FARO) server, that match input signatures to a compendium of 242 gene expression signatures, extracted from more than 1700 Arabidopsis microarray experiments. Hereby we present a publicly available tool for robust characterization of Arabidopsis gene expression experiments which can point to similar experimental factors in other experiments. The server is available at http://www.cbs.dtu.dk/services/faro/.

Digital gene expression analysis of the zebra finch genome

PubMed Central

2010-01-01

Background In order to understand patterns of adaptation and molecular evolution it is important to quantify both variation in gene expression and nucleotide sequence divergence. Gene expression profiling in non-model organisms has recently been facilitated by the advent of massively parallel sequencing technology. Here we investigate tissue specific gene expression patterns in the zebra finch (Taeniopygia guttata) with special emphasis on the genes of the major histocompatibility complex (MHC). Results Almost 2 million 454-sequencing reads from cDNA of six different tissues were assembled and analysed. A total of 11,793 zebra finch transcripts were represented in this EST data, indicating a transcriptome coverage of about 65%. There was a positive correlation between the tissue specificity of gene expression and non-synonymous to synonymous nucleotide substitution ratio of genes, suggesting that genes with a specialised function are evolving at a higher rate (or with less constraint) than genes with a more general function. In line with this, there was also a negative correlation between overall expression levels and expression specificity of contigs. We found evidence for expression of 10 different genes related to the MHC. MHC genes showed relatively tissue specific expression levels and were in general primarily expressed in spleen. Several MHC genes, including MHC class I also showed expression in brain. Furthermore, for all genes with highest levels of expression in spleen there was an overrepresentation of several gene ontology terms related to immune function. Conclusions Our study highlights the usefulness of next-generation sequence data for quantifying gene expression in the genome as a whole as well as in specific candidate genes. Overall, the data show predicted patterns of gene expression profiles and molecular evolution in the zebra finch genome. Expression of MHC genes in particular, corresponds well with expression patterns in other vertebrates. PMID:20359325

Inferring species divergence times using pairwise sequential Markovian coalescent modelling and low-coverage genomic data.

PubMed

Cahill, James A; Soares, André E R; Green, Richard E; Shapiro, Beth

2016-07-19

Understanding when species diverged aids in identifying the drivers of speciation, but the end of gene flow between populations can be difficult to ascertain from genetic data. We explore the use of pairwise sequential Markovian coalescent (PSMC) modelling to infer the timing of divergence between species and populations. PSMC plots generated using artificial hybrid genomes show rapid increases in effective population size at the time when the two parent lineages diverge, and this approach has been used previously to infer divergence between human lineages. We show that, even without high coverage or phased input data, PSMC can detect the end of significant gene flow between populations by comparing the PSMC output from artificial hybrids to the output of simulations with known demographic histories. We then apply PSMC to detect divergence times among lineages within two real datasets: great apes and bears within the genus Ursus Our results confirm most previously proposed divergence times for these lineages, and suggest that gene flow between recently diverged lineages may have been common among bears and great apes, including up to one million years of continued gene flow between chimpanzees and bonobos after the formation of the Congo River.This article is part of the themed issue 'Dating species divergences using rocks and clocks'. © 2016 The Author(s).

Alx4 relays sequential FGF signaling to induce lacrimal gland morphogenesis

PubMed Central

Garg, Ankur; Gotoh, Noriko; Feng, Gen-Sheng; Zhong, Jian; Wang, Fen; Kariminejad, Ariana; Brooks, Steven

2017-01-01

The sequential use of signaling pathways is essential for the guidance of pluripotent progenitors into diverse cell fates. Here, we show that Shp2 exclusively mediates FGF but not PDGF signaling in the neural crest to control lacrimal gland development. In addition to preventing p53-independent apoptosis and promoting the migration of Sox10-expressing neural crests, Shp2 is also required for expression of the homeodomain transcription factor Alx4, which directly controls Fgf10 expression in the periocular mesenchyme that is necessary for lacrimal gland induction. We show that Alx4 binds an Fgf10 intronic element conserved in terrestrial but not aquatic animals, underlying the evolutionary emergence of the lacrimal gland system in response to an airy environment. Inactivation of ALX4/Alx4 causes lacrimal gland aplasia in both human and mouse. These results reveal a key role of Alx4 in mediating FGF-Shp2-FGF signaling in the neural crest for lacrimal gland development. PMID:29028795

Alx4 relays sequential FGF signaling to induce lacrimal gland morphogenesis.

PubMed

Garg, Ankur; Bansal, Mukesh; Gotoh, Noriko; Feng, Gen-Sheng; Zhong, Jian; Wang, Fen; Kariminejad, Ariana; Brooks, Steven; Zhang, Xin

2017-10-01

The sequential use of signaling pathways is essential for the guidance of pluripotent progenitors into diverse cell fates. Here, we show that Shp2 exclusively mediates FGF but not PDGF signaling in the neural crest to control lacrimal gland development. In addition to preventing p53-independent apoptosis and promoting the migration of Sox10-expressing neural crests, Shp2 is also required for expression of the homeodomain transcription factor Alx4, which directly controls Fgf10 expression in the periocular mesenchyme that is necessary for lacrimal gland induction. We show that Alx4 binds an Fgf10 intronic element conserved in terrestrial but not aquatic animals, underlying the evolutionary emergence of the lacrimal gland system in response to an airy environment. Inactivation of ALX4/Alx4 causes lacrimal gland aplasia in both human and mouse. These results reveal a key role of Alx4 in mediating FGF-Shp2-FGF signaling in the neural crest for lacrimal gland development.

High Yields of 2,3-Butanediol and Mannitol in Lactococcus lactis through Engineering of NAD+ Cofactor Recycling ▿ †

PubMed Central

Gaspar, Paula; Neves, Ana Rute; Gasson, Michael J.; Shearman, Claire A.; Santos, Helena

2011-01-01

Manipulation of NADH-dependent steps, and particularly disruption of the las-located lactate dehydrogenase (ldh) gene in Lactococcus lactis, is common to engineering strategies envisaging the accumulation of reduced end products other than lactate. Reverse transcription-PCR experiments revealed that three out of the four genes assigned to lactate dehydrogenase in the genome of L. lactis, i.e., the ldh, ldhB, and ldhX genes, were expressed in the parental strain MG1363. Given that genetic redundancy is often a major cause of metabolic instability in engineered strains, we set out to develop a genetically stable lactococcal host tuned for the production of reduced compounds. Therefore, the ldhB and ldhX genes were sequentially deleted in L. lactis FI10089, a strain with a deletion of the ldh gene. The single, double, and triple mutants, FI10089, FI10089ΔldhB, and FI10089ΔldhBΔldhX, showed similar growth profiles and displayed mixed-acid fermentation, ethanol being the main reduced end product. Hence, the alcohol dehydrogenase-encoding gene, the adhE gene, was inactivated in FI10089, but the resulting strain reverted to homolactic fermentation due to induction of the ldhB gene. The three lactate dehydrogenase-deficient mutants were selected as a background for the production of mannitol and 2,3-butanediol. Pathways for the biosynthesis of these compounds were overexpressed under the control of a nisin promoter, and the constructs were analyzed with respect to growth parameters and product yields under anaerobiosis. Glucose was efficiently channeled to mannitol (maximal yield, 42%) or to 2,3-butanediol (maximal yield, 67%). The theoretical yield for 2,3-butanediol was achieved. We show that FI10089ΔldhB is a valuable basis for engineering strategies aiming at the production of reduced compounds. PMID:21841021

A novel approach for human whole transcriptome analysis based on absolute gene expression of microarray data.

PubMed

Bikel, Shirley; Jacobo-Albavera, Leonor; Sánchez-Muñoz, Fausto; Cornejo-Granados, Fernanda; Canizales-Quinteros, Samuel; Soberón, Xavier; Sotelo-Mundo, Rogerio R; Del Río-Navarro, Blanca E; Mendoza-Vargas, Alfredo; Sánchez, Filiberto; Ochoa-Leyva, Adrian

2017-01-01

In spite of the emergence of RNA sequencing (RNA-seq), microarrays remain in widespread use for gene expression analysis in the clinic. There are over 767,000 RNA microarrays from human samples in public repositories, which are an invaluable resource for biomedical research and personalized medicine. The absolute gene expression analysis allows the transcriptome profiling of all expressed genes under a specific biological condition without the need of a reference sample. However, the background fluorescence represents a challenge to determine the absolute gene expression in microarrays. Given that the Y chromosome is absent in female subjects, we used it as a new approach for absolute gene expression analysis in which the fluorescence of the Y chromosome genes of female subjects was used as the background fluorescence for all the probes in the microarray. This fluorescence was used to establish an absolute gene expression threshold, allowing the differentiation between expressed and non-expressed genes in microarrays. We extracted the RNA from 16 children leukocyte samples (nine males and seven females, ages 6-10 years). An Affymetrix Gene Chip Human Gene 1.0 ST Array was carried out for each sample and the fluorescence of 124 genes of the Y chromosome was used to calculate the absolute gene expression threshold. After that, several expressed and non-expressed genes according to our absolute gene expression threshold were compared against the expression obtained using real-time quantitative polymerase chain reaction (RT-qPCR). From the 124 genes of the Y chromosome, three genes (DDX3Y, TXLNG2P and EIF1AY) that displayed significant differences between sexes were used to calculate the absolute gene expression threshold. Using this threshold, we selected 13 expressed and non-expressed genes and confirmed their expression level by RT-qPCR. Then, we selected the top 5% most expressed genes and found that several KEGG pathways were significantly enriched. Interestingly, these pathways were related to the typical functions of leukocytes cells, such as antigen processing and presentation and natural killer cell mediated cytotoxicity. We also applied this method to obtain the absolute gene expression threshold in already published microarray data of liver cells, where the top 5% expressed genes showed an enrichment of typical KEGG pathways for liver cells. Our results suggest that the three selected genes of the Y chromosome can be used to calculate an absolute gene expression threshold, allowing a transcriptome profiling of microarray data without the need of an additional reference experiment. Our approach based on the establishment of a threshold for absolute gene expression analysis will allow a new way to analyze thousands of microarrays from public databases. This allows the study of different human diseases without the need of having additional samples for relative expression experiments.

Differential gene expression analysis in glioblastoma cells and normal human brain cells based on GEO database.

PubMed

Wang, Anping; Zhang, Guibin

2017-11-01

The differentially expressed genes between glioblastoma (GBM) cells and normal human brain cells were investigated to performed pathway analysis and protein interaction network analysis for the differentially expressed genes. GSE12657 and GSE42656 gene chips, which contain gene expression profile of GBM were obtained from Gene Expression Omniub (GEO) database of National Center for Biotechnology Information (NCBI). The 'limma' data packet in 'R' software was used to analyze the differentially expressed genes in the two gene chips, and gene integration was performed using 'RobustRankAggreg' package. Finally, pheatmap software was used for heatmap analysis and Cytoscape, DAVID, STRING and KOBAS were used for protein-protein interaction, Gene Ontology (GO) and KEGG analyses. As results: i) 702 differentially expressed genes were identified in GSE12657, among those genes, 548 were significantly upregulated and 154 were significantly downregulated (p<0.01, fold-change >1), and 1,854 differentially expressed genes were identified in GSE42656, among the genes, 1,068 were significantly upregulated and 786 were significantly downregulated (p<0.01, fold-change >1). A total of 167 differentially expressed genes including 100 upregulated genes and 67 downregulated genes were identified after gene integration, and the genes showed significantly different expression levels in GBM compared with normal human brain cells (p<0.05). ii) Interactions between the protein products of 101 differentially expressed genes were identified using STRING and expression network was established. A key gene, called CALM3, was identified by Cytoscape software. iii) GO enrichment analysis showed that differentially expressed genes were mainly enriched in 'neurotransmitter:sodium symporter activity' and 'neurotransmitter transporter activity', which can affect the activity of neurotransmitter transportation. KEGG pathway analysis showed that the differentially expressed genes were mainly enriched in 'protein processing in endoplasmic reticulum', which can affect protein processing in endoplasmic reticulum. The results showed that: i) 167 differentially expressed genes were identified from two gene chips after integration; and ii) protein interaction network was established, and GO and KEGG pathway analyses were successfully performed to identify and annotate the key gene, which provide new insights for the studies on GBN at gene level.

Discovery and validation of a glioblastoma co-expressed gene module

PubMed Central

Dunwoodie, Leland J.; Poehlman, William L.; Ficklin, Stephen P.; Feltus, Frank Alexander

2018-01-01

Tumors exhibit complex patterns of aberrant gene expression. Using a knowledge-independent, noise-reducing gene co-expression network construction software called KINC, we created multiple RNAseq-based gene co-expression networks relevant to brain and glioblastoma biology. In this report, we describe the discovery and validation of a glioblastoma-specific gene module that contains 22 co-expressed genes. The genes are upregulated in glioblastoma relative to normal brain and lower grade glioma samples; they are also hypo-methylated in glioblastoma relative to lower grade glioma tumors. Among the proneural, neural, mesenchymal, and classical glioblastoma subtypes, these genes are most-highly expressed in the mesenchymal subtype. Furthermore, high expression of these genes is associated with decreased survival across each glioblastoma subtype. These genes are of interest to glioblastoma biology and our gene interaction discovery and validation workflow can be used to discover and validate co-expressed gene modules derived from any co-expression network. PMID:29541392

Discovery and validation of a glioblastoma co-expressed gene module.

PubMed

Dunwoodie, Leland J; Poehlman, William L; Ficklin, Stephen P; Feltus, Frank Alexander

2018-02-16

Tumors exhibit complex patterns of aberrant gene expression. Using a knowledge-independent, noise-reducing gene co-expression network construction software called KINC, we created multiple RNAseq-based gene co-expression networks relevant to brain and glioblastoma biology. In this report, we describe the discovery and validation of a glioblastoma-specific gene module that contains 22 co-expressed genes. The genes are upregulated in glioblastoma relative to normal brain and lower grade glioma samples; they are also hypo-methylated in glioblastoma relative to lower grade glioma tumors. Among the proneural, neural, mesenchymal, and classical glioblastoma subtypes, these genes are most-highly expressed in the mesenchymal subtype. Furthermore, high expression of these genes is associated with decreased survival across each glioblastoma subtype. These genes are of interest to glioblastoma biology and our gene interaction discovery and validation workflow can be used to discover and validate co-expressed gene modules derived from any co-expression network.

Modified SSCP method using sequential electrophoresis of multiple nucleic acid segments

DOEpatents

Gatti, Richard A.

2002-10-01

The present invention is directed to a method of screening large, complex, polyexonic eukaryotic genes such as the ATM gene for mutations and polymorphisms by an improved version of single strand conformation polymorphism (SSCP) electrophoresis that allows electrophoresis of two or three amplified segments in a single lane. The present invention also is directed to new mutations and polymorphisms in the ATM gene that are useful in performing more accurate screening of human DNA samples for mutations and in distinguishing mutations from polymorphisms, thereby improving the efficiency of automated screening methods.

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

PubMed Central

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

2011-01-01

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

The Long Adventurous Journey of Rhombic Lip Cells in Jawed Vertebrates: A Comparative Developmental Analysis

PubMed Central

Wullimann, Mario F.; Mueller, Thomas; Distel, Martin; Babaryka, Andreas; Grothe, Benedikt; Köster, Reinhard W.

2011-01-01

This review summarizes vertebrate rhombic lip and early cerebellar development covering classic approaches up to modern developmental genetics which identifies the relevant differential gene expression domains and their progeny. Most of this information is derived from amniotes. However, progress in anamniotes, particularly in the zebrafish, has recently been made. The current picture suggests that rhombic lip and cerebellar development in jawed vertebrates (gnathostomes) share many characteristics. Regarding cerebellar development, these include a ptf1a expressing ventral cerebellar proliferation (VCP) giving rise to Purkinje cells and other inhibitory cerebellar cell types, and an atoh1 expressing upper rhombic lip giving rise to an external granular layer (EGL, i.e., excitatory granule cells) and an early ventral migration into the anterior rhombencephalon (cholinergic nuclei). As for the lower rhombic lip (LRL), gnathostome commonalities likely include the formation of precerebellar nuclei (mossy fiber origins) and partially primary auditory nuclei (likely convergently evolved) from the atoh1 expressing dorsal zone. The fate of the ptf1a expressing ventral LRL zone which gives rise to (excitatory cells of) the inferior olive (climbing fiber origin) and (inhibitory cells of ) cochlear nuclei in amniotes, has not been determined in anamniotes. Special for the zebrafish in comparison to amniotes is the predominant origin of anamniote excitatory deep cerebellar nuclei homologs (i.e., eurydendroid cells) from ptf1a expressing VCP cells, the sequential activity of various atoh1 paralogs and the incomplete coverage of the subpial cerebellar plate with proliferative EGL cells. Nevertheless, the conclusion that a rhombic lip and its major derivatives evolved with gnathostome vertebrates only and are thus not an ancestral craniate character complex is supported by the absence of a cerebellum (and likely absence of its afferent and efferent nuclei) in jawless fishes PMID:21559349

Interleukin-7-induced Stat-5 acts in synergy with Flt-3 signaling to stimulate expansion of hematopoietic progenitor cells.

PubMed

Åhsberg, Josefine; Tsapogas, Panagiotis; Qian, Hong; Zetterblad, Jenny; Zandi, Sasan; Månsson, Robert; Jönsson, Jan-Ingvar; Sigvardsson, Mikael

2010-11-19

The development of lymphoid cells from bone marrow progenitors is dictated by interplay between internal cues such as transcription factors and external signals like the cytokines Flt-3 ligand and Il-7. These proteins are both of large importance for normal lymphoid development; however, it is unclear if they act in direct synergy to expand a transient Il-7R(+)Flt-3(+) population or if the collaboration is created through sequential activities. We report here that Flt-3L and Il-7 synergistically stimulated the expansion of primary Il-7R(+)Flt-3(+) progenitor cells and a hematopoietic progenitor cell line ectopically expressing the receptors. The stimulation resulted in a reduced expression of pro-apoptotic genes and also mediated survival of primary progenitor cells in vitro. However, functional analysis of single cells suggested that the anti-apoptotic effect was additive indicating that the synergy observed mainly depends on stimulation of proliferation. Analysis of downstream signaling events suggested that although Il-7 induced Stat-5 phosphorylation, Flt-3L caused activation of the ERK and AKT signaling pathways. Flt-3L could also drive proliferation in synergy with ectopically expressed constitutively active Stat-5. This synergy could be inhibited with either receptor tyrosine kinase or MAPK inhibitors suggesting that Flt-3L and Il-7 act in synergy by activation of independent signaling pathways to expand early hematopoietic progenitors.

Restoration of Tamoxifen Sensitivity in Estrogen Receptor–Negative Breast Cancer Cells: Tamoxifen-Bound Reactivated ER Recruits Distinctive Corepressor Complexes

PubMed Central

Sharma, Dipali; Saxena, Neeraj K.; Davidson, Nancy E.; Vertino, Paula M.

2010-01-01

Breast tumors expressing estrogen receptor-α (ER) respond well to therapeutic strategies using selective ER modulators, such as tamoxifen. However, ~ 30% of invasive breast cancers are hormone independent because they lack ER expression due to hypermethylation of ER promoter. Treatment of ER-negative breast cancer cells with demethylating agents [5-aza-2′-deoxycytidine (5-aza-dC)] and histone deacetylase (HDAC) inhibitors (trichostatin A) leads to expression of ER mRNA and functional protein. Here, we examined whether epigenetically reactivated ER is a target for tamoxifen therapy. Following treatment with trichostatin A and 5-aza-dC, the formerly unresponsive ER-negative MDA-MB-231 breast cancer cells became responsive to tamoxifen. Tamoxifen-mediated inhibition of cell growth in these cells is mediated at least in part by the tamoxifen-bound ER. Tamoxifen-bound reactivated ER induces transcriptional repression at estrogen-responsive genes by ordered recruitment of multiple distinct chromatin-modifying complexes. Using chromatin immunoprecipitation, we show recruitment of two different corepressor complexes to ER-responsive promoters in a mutually exclusive and sequential manner: the nuclear receptor corepressor-HDAC3 complex followed by nucleosome remodeling and histone deacetylation complex. The mechanistic insight provided by this study might help in designing therapeutic strategies directed toward epigenetic mechanisms in the prevention or treatment of breast cancer. PMID:16778215

Gene expression variability in human hepatic drug metabolizing enzymes and transporters.

PubMed

Yang, Lun; Price, Elvin T; Chang, Ching-Wei; Li, Yan; Huang, Ying; Guo, Li-Wu; Guo, Yongli; Kaput, Jim; Shi, Leming; Ning, Baitang

2013-01-01

Interindividual variability in the expression of drug-metabolizing enzymes and transporters (DMETs) in human liver may contribute to interindividual differences in drug efficacy and adverse reactions. Published studies that analyzed variability in the expression of DMET genes were limited by sample sizes and the number of genes profiled. We systematically analyzed the expression of 374 DMETs from a microarray data set consisting of gene expression profiles derived from 427 human liver samples. The standard deviation of interindividual expression for DMET genes was much higher than that for non-DMET genes. The 20 DMET genes with the largest variability in the expression provided examples of the interindividual variation. Gene expression data were also analyzed using network analysis methods, which delineates the similarities of biological functionalities and regulation mechanisms for these highly variable DMET genes. Expression variability of human hepatic DMET genes may affect drug-gene interactions and disease susceptibility, with concomitant clinical implications.

Hippocampal and Cognitive Aging across the Lifespan: A Bioenergetic Shift Precedes and Increased Cholesterol Trafficking Parallels Memory Impairment

PubMed Central

Kadish, I; Thibault, O; Blalock, EM; Chen, K-C; Gant, JC; Porter, NM; Landfield, PW

2009-01-01

Multiple hippocampal processes and cognitive functions change with aging or Alzheimer’s disease, but the potential triggers of these aging cascades are not well understood. Here, we quantified hippocampal expression profiles and behavior across the adult lifespan, to identify early aging changes and changes that coincide with subsequent onset of cognitive impairment. Well-powered microarray analyses (N=49 arrays), immunohistochemistry and Morris spatial maze learning were used to study male F344 rats at 5 age points. Genes that changed with aging (by ANOVA) were assigned to one-of-four onset-age ranges based upon template pattern matching; functional pathways represented by these genes were identified statistically (Genome Ontology). In the earliest onset-age range (3–6 months-old), upregulation began for genes in lipid/protein catabolic and lysosomal pathways, indicating a shift in metabolic substrates, whereas downregulation began for lipid synthesis, GTP/ATP-dependent signaling and neural development genes. By 6–9 months-of-age, upregulation of immune/inflammatory cytokines was pronounced. Cognitive impairment first appeared in the Midlife range (9–12 months), and coincided and correlated primarily with midlife upregulation of genes associated with cholesterol trafficking (apolipoprotein E), myelinogenic and proteolytic/MHC antigen-presenting pathways. Immunolabeling revealed that cholesterol trafficking proteins were substantially increased in astrocytes, and that myelination increased with aging. Together, our data suggest a novel sequential model in which an early-adult metabolic shift, favoring lipid/ketone body oxidation, triggers inflammatory degradation of myelin and resultant excess cholesterol that, by midlife, activates cholesterol transport from astrocytes to remyelinating oligodendrocytes. These processes may damage structure and compete with neuronal pathways for bioenergetic resources, thereby impairing cognitive function. PMID:19211887

Proteomic analyses reveal the key roles of BrlA and AbaA in biogenesis of gliotoxin in Aspergillus fumigatus

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

Shin, Kwang-Soo, E-mail: shinks@dju.kr; Kim, Young Hwan; Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, 305-764

2015-07-31

The opportunistic human pathogenic fungus Aspergillus fumigatus primarily reproduces by forming a large number of asexual spores (conidia). Sequential activation of the central regulators BrlA, AbaA and WetA is necessary for the fungus to undergo asexual development. In this study, to address the presumed roles of these key developmental regulators during proliferation of the fungus, we analyzed and compared the proteomes of vegetative cells of wild type (WT) and individual mutant strains. Approximately 1300 protein spots were detectable from 2-D electrophoresis gels. Among these, 13 proteins exhibiting significantly altered accumulation levels were further identified by ESI-MS/MS. Markedly, we found thatmore » the GliM and GliT proteins associated with gliotoxin (GT) biosynthesis and self-protection of the fungus from GT were significantly down-regulated in the ΔabaA and ΔbrlA mutants. Moreover, mRNA levels of other GT biosynthetic genes including gliM, gliP, gliT, and gliZ were significantly reduced in both mutant strains, and no and low levels of GT were detectable in the ΔbrlA and ΔabaA mutant strains, respectively. As GliT is required for the protection of the fungus from GT, growth of the ΔbrlA mutant with reduced levels of GliT was severely impaired by exogenous GT. Our studies demonstrate that AbaA and BrlA positively regulate expression of the GT biosynthetic gene cluster in actively growing vegetative cells, and likely bridge morphological and chemical development during the life-cycle of A. fumigatus. - Highlights: • Proteome analyses of WT and mutants reveal 13 differentially expressed proteins. • The GliT and GliM proteins are significantly down-regulated by ΔabaA and ΔbrlA. • Expression of other gliotoxin biosynthetic genes is lowered by ΔabaA and ΔbrlA. • Growth of ΔbrlA strain lacking GliT is completely inhibited by exogenous gliotoxin. • BrlA and AbaA play key roles in biogenesis of gliotoxin in Aspergillus fumigatus.« less

Amplification and propagation of interleukin-1β signaling by murine brain endothelial and glial cells.

PubMed

Krasnow, Stephanie M; Knoll, J Gabriel; Verghese, Santhosh Chakkaramakkil; Levasseur, Peter R; Marks, Daniel L

2017-07-01

During acute infections and chronic illnesses, the pro-inflammatory cytokine interleukin-1β (IL-1β) acts within the brain to elicit metabolic derangements and sickness behaviors. It is unknown which cells in the brain are the proximal targets for IL-1β with respect to the generation of these illness responses. We performed a series of in vitro experiments to (1) investigate which brain cell populations exhibit inflammatory responses to IL-1β and (2) examine the interactions between different IL-1β-responsive cell types in various co-culture combinations. We treated primary cultures of murine brain microvessel endothelial cells (BMEC), astrocytes, and microglia with PBS or IL-1β, and then performed qPCR to measure inflammatory gene expression or immunocytochemistry to evaluate nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation. To evaluate whether astrocytes and/or BMEC propagate inflammatory signals to microglia, we exposed microglia to astrocyte-conditioned media and co-cultured endothelial cells and glia in transwells. Treatment groups were compared by Student's t tests or by ANOVA followed by Bonferroni-corrected t tests. IL-1β increased inflammatory gene expression and NF-κB activation in primary murine-mixed glia, enriched astrocyte, and BMEC cultures. Although IL-1β elicited minimal changes in inflammatory gene expression and did not induce the nuclear translocation of NF-κB in isolated microglia, these cells were more robustly activated by IL-1β when co-cultured with astrocytes and/or BMEC. We observed a polarized endothelial response to IL-1β, because the application of IL-1β to the abluminal endothelial surface produced a more complex microglial inflammatory response than that which occurred following luminal IL-1β exposure. Inflammatory signals are detected, amplified, and propagated through the CNS via a sequential and reverberating signaling cascade involving communication between brain endothelial cells and glia. We propose that the brain's innate immune response differs depending upon which side of the blood-brain barrier the inflammatory stimulus arises, thus allowing the brain to respond differently to central vs. peripheral inflammatory insults.

Alternative splicing produces transcripts coding for alpha and beta chains of a hetero-dimeric phosphagen kinase.

PubMed

Ellington, W Ross; Yamashita, Daisuke; Suzuki, Tomohiko

2004-06-09

Glycocyamine kinase (GK) catalyzes the reversible phosphorylation of glycocyamine (guanidinoacetate), a reaction central to cellular energy homeostasis in certain animals. GK is a member of the phosphagen kinase enzyme family and appears to have evolved from creatine kinase (CK) early in the evolution of multi-cellular animals. Prior work has shown that GK from the polychaete Neanthes (Nereis) diversicolor exits as a hetero-dimer in vivo and that the two polypeptide chains (termed alpha and beta) are coded for by unique transcripts. In the present study, we demonstrate that the GK from a congener Nereis virens is also hetero-dimeric and is coded for by alpha and beta transcripts, which are virtually identical to the corresponding forms in N. diversicolor. The GK gene from N. diversicolor was amplified by PCR. Sequencing of the PCR products showed that the alpha and beta chains are the result of alternative splicing of the GK primary mRNA transcript. These results also strongly suggest that this gene underwent an early tandem exon duplication event. Full-length cDNAs for N. virens GKalpha and GKbeta were individually ligated into expression vectors and the resulting constructs used to transform Escherichia coli expression hosts. Regardless of expression conditions, minimal GK activity was observed in both GKalpha and GKbeta constructs. Inclusion bodies for both were harvested, unfolded in urea and alpha chains, beta chains and mixtures of alpha and beta chains were refolded by sequential dialysis. Only modest amounts of GK activity were observed when alpha and beta were refolded individually. In contrast, when refolded the alpha and beta mixture yielded highly active hetero-dimers, as validated by size exclusion chromatography, electrophoresis and mass spectrometry, with a specific activity comparable to that of natural GK. The above evidence suggests that there is a preference for hetero-dimer formation in the GKs from these two polychaetes. The evolution of the alternate splicing and an additional exon in these GKs, producing alpha and beta transcripts, can be viewed as a possible compensation for a mutation(s) in the original gene, which most likely coded for a homo-dimeric protein.

A novel approach for human whole transcriptome analysis based on absolute gene expression of microarray data

PubMed Central

Bikel, Shirley; Jacobo-Albavera, Leonor; Sánchez-Muñoz, Fausto; Cornejo-Granados, Fernanda; Canizales-Quinteros, Samuel; Soberón, Xavier; Sotelo-Mundo, Rogerio R.; del Río-Navarro, Blanca E.; Mendoza-Vargas, Alfredo; Sánchez, Filiberto

2017-01-01

Background In spite of the emergence of RNA sequencing (RNA-seq), microarrays remain in widespread use for gene expression analysis in the clinic. There are over 767,000 RNA microarrays from human samples in public repositories, which are an invaluable resource for biomedical research and personalized medicine. The absolute gene expression analysis allows the transcriptome profiling of all expressed genes under a specific biological condition without the need of a reference sample. However, the background fluorescence represents a challenge to determine the absolute gene expression in microarrays. Given that the Y chromosome is absent in female subjects, we used it as a new approach for absolute gene expression analysis in which the fluorescence of the Y chromosome genes of female subjects was used as the background fluorescence for all the probes in the microarray. This fluorescence was used to establish an absolute gene expression threshold, allowing the differentiation between expressed and non-expressed genes in microarrays. Methods We extracted the RNA from 16 children leukocyte samples (nine males and seven females, ages 6–10 years). An Affymetrix Gene Chip Human Gene 1.0 ST Array was carried out for each sample and the fluorescence of 124 genes of the Y chromosome was used to calculate the absolute gene expression threshold. After that, several expressed and non-expressed genes according to our absolute gene expression threshold were compared against the expression obtained using real-time quantitative polymerase chain reaction (RT-qPCR). Results From the 124 genes of the Y chromosome, three genes (DDX3Y, TXLNG2P and EIF1AY) that displayed significant differences between sexes were used to calculate the absolute gene expression threshold. Using this threshold, we selected 13 expressed and non-expressed genes and confirmed their expression level by RT-qPCR. Then, we selected the top 5% most expressed genes and found that several KEGG pathways were significantly enriched. Interestingly, these pathways were related to the typical functions of leukocytes cells, such as antigen processing and presentation and natural killer cell mediated cytotoxicity. We also applied this method to obtain the absolute gene expression threshold in already published microarray data of liver cells, where the top 5% expressed genes showed an enrichment of typical KEGG pathways for liver cells. Our results suggest that the three selected genes of the Y chromosome can be used to calculate an absolute gene expression threshold, allowing a transcriptome profiling of microarray data without the need of an additional reference experiment. Discussion Our approach based on the establishment of a threshold for absolute gene expression analysis will allow a new way to analyze thousands of microarrays from public databases. This allows the study of different human diseases without the need of having additional samples for relative expression experiments. PMID:29230367

CoCr wear particles generated from CoCr alloy metal-on-metal hip replacements, and cobalt ions stimulate apoptosis and expression of general toxicology-related genes in monocyte-like U937 cells

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

Posada, Olga M., E-mail: O.M.PosadaEstefan@leeds.ac.uk; Gilmour, Denise; Tate, Rothwelle J., E-mail: r.j.tate@strath.ac.uk

Cobalt-chromium (CoCr) particles in the nanometre size range and their concomitant release of Co and Cr ions into the patients' circulation are produced by wear at the articulating surfaces of metal-on-metal (MoM) implants. This process is associated with inflammation, bone loss and implant loosening and led to the withdrawal from the market of the DePuy ASR™ MoM hip replacements in 2010. Ions released from CoCr particles derived from a resurfacing implant in vitro and their subsequent cellular up-take were measured by ICP-MS. Moreover, the ability of such metal debris and Co ions to induce both apoptosis was evaluated with bothmore » FACS and immunoblotting. qRT-PCR was used to assess the effects on the expression of lymphotoxin alpha (LTA), BCL2-associated athanogene (BAG1), nitric oxide synthase 2 inducible (NOS2), FBJ murine osteosarcoma viral oncogene homolog (FOS), growth arrest and DNA-damage-inducible alpha (GADD45A). ICP-MS showed that the wear debris released significant (p < 0.05) amounts of Co and Cr ions into the culture medium, and significant (p < 0.05) cellular uptake of both ions. There was also an increase (p < 0.05) in apoptosis after a 48 h exposure to wear debris. Analysis of qRT-PCR results found significant up-regulation (p < 0.05) particularly of NOS2 and BAG1 in Co pre-treated cells which were subsequently exposed to Co ions + debris. Metal debris was more effective as an inducer of apoptosis and gene expression when cells had been pre-treated with Co ions. This suggests that if a patient receives sequential bilateral CoCr implants, the second implant may be more likely to produce adverse effects than the first one. - Highlights: • Effects of CoCr nanoparticles and Co ions on U937 cells were investigated. • Ions released from wear debris play an important role in cellular response, • Toxicity of Co ions could be related to NO metabolic processes and apoptosis. • CoCr particles were a more effective inducer of apoptosis after cell priming. • CoCr particles were a more effective inducer of gene expression after cell priming.« less

Dried Plum Protects From Radiation-Induced Bone Loss by Attenuating Pro-Osteoclastic and Oxidative Stress Responses

NASA Technical Reports Server (NTRS)

Globus, Ruth

2015-01-01

Future space explorations beyond the earths magnetosphere will increase human exposure to space radiation and associated risks to skeletal health. We hypothesize that oxidative stress resulting from radiation exposure plays a major role in progressive bone loss and dysfunction in associated tissue. In animal studies, increased free radical formation is associated with pathological changes in bone structure, enhanced bone resorption, reduced bone formation and decreased bone mineral density, which can lead to skeletal fragility. Our long-term goals are to define the mechanisms and risk of bone loss in the spaceflight environment and to facilitate the development of effective countermeasures. We had previously reported that exposure to low or high-LET radiation correlates with an acute increase in the expression of pro-osteoclastic and oxidative stress genes in bone during the early response to radiation followed by pathological changes in skeletal structure. We then conducted systematic screening for potential countermeasures against bone loss where we tested the ability of various antioxidants to mitigate the radiation-induced increase in expression of these markers. For the screen, 16-week old C57Bl6J mice were treated with a dietary antioxidant cocktail, injectable DHLA or a dried plum-enriched diet (DP). Mice were then exposed to 2Gy 137Cs radiation and one day later, marrow cells were collected and the relevant genes analyzed for expression levels. Among the candidate countermeasures tested, DP was most effective in reducing the expression of genes associated with bone loss. Furthermore, analysis of skeletal structure by microcomputed tomography (microCT) revealed that DP also prevents the radiation-induced deterioration in skeletal microarchitecture as indicated by parameters such as percent bone volume (BVTV), trabecular spacing and trabecular number. We also found that DP has similar protective effects on skeletal structure in a follow-up study using 1 Gy of sequential proton and iron, radiation species relevant to spaceflight. When cultured ex vivo under osteogenic conditions, bone marrow-derived cells from DP-fed animals exhibited increased colony numbers compared to control diet-fed animals. These findings suggest that DP exerts pro-osteogenic effects apart from its previously demonstrated anti-resorptive action, which may be one of the mechanisms underlying its radioprotective effect on bone. In conclusion, a diet enriched in certain types of antioxidants may be useful as an intervention for radiation-induced bone loss.

Rabbit model for human EBV-associated hemophagocytic syndrome (HPS): sequential autopsy analysis and characterization of IL-2-dependent cell lines established from herpesvirus papio-induced fatal rabbit lymphoproliferative diseases with HPS.

PubMed

Hayashi, Kazuhiko; Jin, Zaishun; Onoda, Sachiyo; Joko, Hiromasa; Teramoto, Norihiro; Ohara, Nobuya; Oda, Wakako; Tanaka, Takehiro; Liu, Yi-Xuan; Koirala, Tirtha Raj; Oka, Takashi; Kondo, Eisaku; Yoshino, Tadashi; Takahashi, Kiyoshi; Akagi, Tadaatsu

2003-05-01

Epstein-Barr virus-associated hemophagocytic syndrome (EBV-AHS) is often associated with fatal infectious mononucleosis or T-cell lymphoproliferative diseases (LPD). To elucidate the true nature of fatal LPD observed in Herpesvirus papio (HVP)-induced rabbit hemophagocytosis, reactive or neoplastic, we analyzed sequential development of HVP-induced rabbit LPD and their cell lines. All of the seven Japanese White rabbits inoculated intravenously with HVP died of fatal LPD 18 to 27 days after inoculation. LPD was also accompanied by hemophagocytic syndrome (HPS) in five of these seven rabbits. Sequential autopsy revealed splenomegaly and swollen lymph nodes, often accompanied by bleeding, which developed in the last week. Atypical lymphoid cells infiltrated many organs with a "starry sky" pattern, frequently involving the spleen, lymph nodes, and liver. HVP-small RNA-1 expression in these lymphoid cells was clearly demonstrated by a newly developed in situ hybridization (ISH) system. HVP-ISH of immunomagnetically purified lymphoid cells from spleen or lymph nodes revealed HVP-EBER1+ cells in each CD4+, CD8+, or CD79a+ fraction. Hemophagocytic histiocytosis was observed in the lymph nodes, spleen, bone marrow, and thymus. HVP-DNA was detected in the tissues and peripheral blood from the infected rabbits by PCR or Southern blot analysis. Clonality analysis of HVP-induced LPD by Southern blotting with TCR gene probe revealed polyclonal bands, suggesting polyclonal proliferation. Six IL-2-dependent rabbit T-cell lines were established from transplanted scid mouse tumors from LPD. These showed latency type I/II HVP infection and had normal karyotypes except for one line, and three of them showed tumorigenicity in nude mice. These data suggest that HVP-induced fatal LPD in rabbits is reactive polyclonally in nature.

Priming of Wheat with the Green Leaf Volatile Z-3-Hexenyl Acetate Enhances Defense against Fusarium graminearum But Boosts Deoxynivalenol Production1

PubMed Central

Ameye, Maarten; Audenaert, Kris; De Zutter, Nathalie; Steppe, Kathy; Van Meulebroek, Lieven; Vanhaecke, Lynn; De Vleesschauwer, David; Haesaert, Geert; Smagghe, Guy

2015-01-01

Priming refers to a mechanism whereby plants are sensitized to respond faster and/or more strongly to future pathogen attack. Here, we demonstrate that preexposure to the green leaf volatile Z-3-hexenyl acetate (Z-3-HAC) primed wheat (Triticum aestivum) for enhanced defense against subsequent infection with the hemibiotrophic fungus Fusarium graminearum. Bioassays showed that, after priming with Z-3-HAC, wheat ears accumulated up to 40% fewer necrotic spikelets. Furthermore, leaves of seedlings showed significantly smaller necrotic lesions compared with nonprimed plants, coinciding with strongly reduced fungal growth in planta. Additionally, we found that F. graminearum produced more deoxynivalenol, a mycotoxin, in the primed treatment. Expression analysis of salicylic acid (SA) and jasmonic acid (JA) biosynthesis genes and exogenous methyl salicylate and methyl jasmonate applications showed that plant defense against F. graminearum is sequentially regulated by SA and JA during the early and later stages of infection, respectively. Interestingly, analysis of the effect of Z-3-HAC pretreatment on SA- and JA-responsive gene expression in hormone-treated and pathogen-inoculated seedlings revealed that Z-3-HAC boosts JA-dependent defenses during the necrotrophic infection stage of F. graminearum but suppresses SA-regulated defense during its biotrophic phase. Together, these findings highlight the importance of temporally separated hormone changes in molding plant health and disease and support a scenario whereby the green leaf volatile Z-3-HAC protects wheat against Fusarium head blight by priming for enhanced JA-dependent defenses during the necrotrophic stages of infection. PMID:25713338

Electrospun biomimetic scaffold of hydroxyapatite/chitosan supports enhanced osteogenic differentiation of mMSCs

NASA Astrophysics Data System (ADS)

Peng, Hongju; Yin, Zi; Liu, Huanhuan; Chen, Xiao; Feng, Bei; Yuan, Huihua; Su, Bo; Ouyang, Hongwei; Zhang, Yanzhong

2012-12-01

Engaging functional biomaterial scaffolds to regulate stem cell differentiation has drawn a great deal of attention in the tissue engineering and regenerative medicine community. In this study, biomimetic composite nanofibrous scaffolds of hydroxyapatite/chitosan (HAp/CTS) were prepared to investigate their capacity for inducing murine mesenchymal stem cells (mMSCs) to differentiate into the osteogenic lineage, in the absence and presence of an osteogenic supplementation (i.e., ascorbic acid, β-glycerol phosphate, and dexamethasone), respectively. Using electrospun chitosan (CTS) nanofibrous scaffolds as the control, cell morphology, growth, specific osteogenic genes expression, and quantified proteins secretion on the HAp/CTS scaffolds were sequentially examined and assessed. It appeared that the HAp/CTS scaffolds supported better attachment and proliferation of the mMSCs. Most noteworthy was that in the absence of the osteogenic supplementation, expression of osteogenic genes including collagen I (Col I), runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteocalcin (OCN) were significantly upregulated in mMSCs cultured on the HAp/CTS nanofibrous scaffolds. Also increased secretion of the osteogenesis protein markers of alkaline phosphatase and collagen confirmed that the HAp/CTS nanofibrous scaffold markedly promoted the osteogenic commitment in the mMSCs. Moreover, the presence of osteogenic supplementation proved an enhanced efficacy of mMSC osteogenesis on the HAp/CTS nanofibrous scaffolds. Collectively, this study demonstrated that the biomimetic nanofibrous HAp/CTS scaffolds could support and enhance the adhesion, proliferation, and particularly osteogenic differentiation of the mMSCs. It also substantiated the potential of using biomimetic nanofibrous scaffolds of HAp/CTS for functional bone repair and regeneration applications.

Deep analysis of wild Vitis flower transcriptome reveals unexplored genome regions associated with sex specification.

PubMed

Ramos, Miguel Jesus Nunes; Coito, João Lucas; Fino, Joana; Cunha, Jorge; Silva, Helena; de Almeida, Patrícia Gomes; Costa, Maria Manuela Ribeiro; Amâncio, Sara; Paulo, Octávio S; Rocheta, Margarida

2017-01-01

RNA-seq of Vitis during early stages of bud development, in male, female and hermaphrodite flowers, identified new loci outside of annotated gene models, suggesting their involvement in sex establishment. The molecular mechanisms responsible for flower sex specification remain unclear for most plant species. In the case of V. vinifera ssp. vinifera, it is not fully understood what determines hermaphroditism in the domesticated subspecies and male or female flowers in wild dioecious relatives (Vitis vinifera ssp. sylvestris). Here, we describe a de novo assembly of the transcriptome of three flower developmental stages from the three Vitis vinifera flower types. The validation of de novo assembly showed a correlation of 0.825. The main goals of this work were the identification of V. v. sylvestris exclusive transcripts and the characterization of differential gene expression during flower development. RNA from several flower developmental stages was used previously to generate Illumina sequence reads. Through a sequential de novo assembly strategy one comprehensive transcriptome comprising 95,516 non-redundant transcripts was assembled. From this dataset 81,064 transcripts were annotated to V. v. vinifera reference transcriptome and 11,084 were annotated against V. v. vinifera reference genome. Moreover, we found 3368 transcripts that could not be mapped to Vitis reference genome. From all the non-redundant transcripts that were assembled, bioinformatics analysis identified 133 specific of V. v. sylvestris and 516 transcripts differentially expressed among the three flower types. The detection of transcription from areas of the genome not currently annotated suggests active transcription of previously unannotated genomic loci during early stages of bud development.

CpG island methylator phenotype is associated with the efficacy of sequential oxaliplatin- and irinotecan-based chemotherapy and EGFR-related gene mutation in Japanese patients with metastatic colorectal cancer.

PubMed

Zhang, Xiaofei; Shimodaira, Hideki; Soeda, Hiroshi; Komine, Keigo; Takahashi, Hidekazu; Ouchi, Kota; Inoue, Masahiro; Takahashi, Masanobu; Takahashi, Shin; Ishioka, Chikashi

2016-12-01

The CpG island methylator phenotype (CIMP) with multiple promoter methylated loci has been observed in a subset of human colorectal cancer (CRC) cases. CIMP status, which is closely associated with specific clinicopathological and molecular characteristics, is considered a potential predictive biomarker for efficacy of cancer treatment. However, the relationship between the effect of standard chemotherapy, including cytotoxic drugs and anti-epidermal growth factor receptor (EGFR) antibodies, and CIMP status has not been elucidated. In 125 metastatic colorectal cancer (mCRC) patients, we investigated how clinical outcome of chemotherapy was related to CIMP status as detected by methylation-specific PCR (MSP) and to genetic status in five EGFR-related genes (KRAS, BRAF, PIK3CA, NRAS, and AKT1) as detected by direct sequencing. CIMP-positive status was significantly associated with proximal tumor location and peritoneum metastasis (all P values <0.05). The progression-free survival of patients with CIMP-positive tumors receiving sequential therapy with FOLFOX as the first-line treatment followed by irinotecan-based therapy as the second-line treatment (median = 6.6 months) was inferior to that of such patients receiving the reverse sequence (median = 15.2 months; P = 0.043). Furthermore, CIMP-positive tumors showed higher mutation frequencies for the five EGFR-related genes (74.1 %) than the CIMP-negative tumors did (50.0 %). Among the KRAS wild-type tumors, CIMP-positive tumors were associated with a worse clinical outcome than CIMP-negative tumors following anti-EGFR antibody therapy. Sequential FOLFOX followed by an irinotecan-based regimen is unfavorable in patients with CIMP-positive tumors. High frequencies of mutation in EGFR-related genes in CIMP-positive tumors may cause the lower response to anti-EGFR antibody therapy seen in patients with wild-type KRAS and CIMP-positive tumors.

Wt1a, Foxc1a, and the Notch mediator Rbpj Physically Interact and Regulate the Formation of Podocytes in Zebrafish

PubMed Central

O’Brien, Lori L.; Grimaldi, Michael; Kostun, Zachary; Wingert, Rebecca A.; Selleck, Rori; Davidson, Alan J.

2011-01-01

Podocytes help form the glomerular blood filtration barrier in the kidney and their injury or loss leads to renal disease. The Wilms’ tumor suppressor-1 (Wt1) and the FoxC1/2 transcription factors, as well as Notch signaling, have been implicated as important regulators of podocyte fate. It is not known whether these factors work in parallel or sequentially on different gene targets, or as higher-order transcriptional complexes on common genes. Here, we use the zebrafish to demonstrate that embryos treated with morpholinos against wt1a, foxc1a, or the Notch transcriptional mediator rbpj develop fewer podocytes, as determined by wt1b, hey1 and nephrin expression, while embryos deficient in any two of these factors completely lack podocytes. From GST-pull-downs and co-immunoprecipitation experiments we show that Wt1a, Foxc1a, and Rbpj can physically interact with each other, whereas only Rbpj binds to the Notch intracellular domain (NICD). In transactivation assays, combinations of Wt1, FoxC1/2, and NICD synergistically induce the Hey1 promoter, and have additive or repressive effects on the Podocalyxin promoter, depending on dosage. Taken together, these data suggest that Wt1, FoxC1/2, and Notch signaling converge on common target genes where they physically interact to regulate a podocyte-specific gene program. These findings further our understanding of the transcriptional circuitry responsible for podocyte formation and differentiation during kidney development. PMID:21871448

MAAMD: a workflow to standardize meta-analyses and comparison of affymetrix microarray data

PubMed Central

2014-01-01

Background Mandatory deposit of raw microarray data files for public access, prior to study publication, provides significant opportunities to conduct new bioinformatics analyses within and across multiple datasets. Analysis of raw microarray data files (e.g. Affymetrix CEL files) can be time consuming, complex, and requires fundamental computational and bioinformatics skills. The development of analytical workflows to automate these tasks simplifies the processing of, improves the efficiency of, and serves to standardize multiple and sequential analyses. Once installed, workflows facilitate the tedious steps required to run rapid intra- and inter-dataset comparisons. Results We developed a workflow to facilitate and standardize Meta-Analysis of Affymetrix Microarray Data analysis (MAAMD) in Kepler. Two freely available stand-alone software tools, R and AltAnalyze were embedded in MAAMD. The inputs of MAAMD are user-editable csv files, which contain sample information and parameters describing the locations of input files and required tools. MAAMD was tested by analyzing 4 different GEO datasets from mice and drosophila. MAAMD automates data downloading, data organization, data quality control assesment, differential gene expression analysis, clustering analysis, pathway visualization, gene-set enrichment analysis, and cross-species orthologous-gene comparisons. MAAMD was utilized to identify gene orthologues responding to hypoxia or hyperoxia in both mice and drosophila. The entire set of analyses for 4 datasets (34 total microarrays) finished in ~ one hour. Conclusions MAAMD saves time, minimizes the required computer skills, and offers a standardized procedure for users to analyze microarray datasets and make new intra- and inter-dataset comparisons. PMID:24621103

Accelerated Evolution of Developmentally Biased Genes in the Tetraphenic Ant Cardiocondyla obscurior.

PubMed

Schrader, Lukas; Helanterä, Heikki; Oettler, Jan

2017-03-01

Plastic gene expression underlies phenotypic plasticity and plastically expressed genes evolve under different selection regimes compared with ubiquitously expressed genes. Social insects are well-suited models to elucidate the evolutionary dynamics of plastic genes for their genetically and environmentally induced discrete polymorphisms. Here, we study the evolution of plastically expressed genes in the ant Cardiocondyla obscurior-a species that produces two discrete male morphs in addition to the typical female polymorphism of workers and queens. Based on individual-level gene expression data from 28 early third instar larvae, we test whether the same evolutionary dynamics that pertain to plastically expressed genes in adults also pertain to genes with plastic expression during development. In order to quantify plasticity of gene expression over multiple contrasts, we develop a novel geometric measure. For genes expressed during development, we show that plasticity of expression is positively correlated with evolutionary rates. We furthermore find a strong correlation between expression plasticity and expression variation within morphs, suggesting a close link between active and passive plasticity of gene expression. Our results support the notion of relaxed selection and neutral processes as important drivers in the evolution of adaptive plasticity. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

α2 Integrin, extracellular matrix metalloproteinase inducer, and matrix metalloproteinase-3 act sequentially to induce differentiation of mouse embryonic stem cells into odontoblast-like cells

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

Ozeki, Nobuaki; Kawai, Rie; Hase, Naoko

We previously reported that interleukin 1β acts via matrix metalloproteinase (MMP)-3 to regulate cell proliferation and suppress apoptosis in α2 integrin-positive odontoblast-like cells differentiated from mouse embryonic stem (ES) cells. Here we characterize the signal cascade underpinning odontoblastic differentiation in mouse ES cells. The expression of α2 integrin, extracellular matrix metalloproteinase inducer (Emmprin), and MMP-3 mRNA and protein were all potently increased during odontoblastic differentiation. Small interfering RNA (siRNA) disruption of the expression of these effectors potently suppressed the expression of the odontoblastic biomarkers dentin sialophosphoprotein, dentin matrix protein-1 and alkaline phosphatase, and blocked odontoblast calcification. Our siRNA, western blotmore » and blocking antibody analyses revealed a unique sequential cascade involving α2 integrin, Emmprin and MMP-3 that drives ES cell differentiation into odontoblasts. This cascade requires the interaction between α2 integrin and Emmprin and is potentiated by exogenous MMP-3. Finally, although odontoblast-like cells potently express α2, α6, αV, β1, and β3, integrins, we confirmed that β1 integrin acts as the trigger for ES cell differentiation, apparently in complex with α2 integrin. These results demonstrate a unique and unanticipated role for an α2 integrin-, Emmprin-, and MMP-3-mediated signaling cascade in driving mouse ES cell differentiation into odontoblast-like cells. - Highlights: • Odontoblast differentiation requires activation of α2 integrin, Emmprin and MMP-3. • α2 integrin, Emmprin and MMP-3 form a sequential signaling cascade. • β1 integrin acts a specific trigger for odontoblast differentiation. • The role of these effectors is highly novel and unanticipated.« less

Parallel solution of closely coupled systems

NASA Technical Reports Server (NTRS)

Utku, S.; Salama, M.

1986-01-01

The odd-even permutation and associated unitary transformations for reordering the matrix coefficient A are employed as means of breaking the strong seriality which is characteristic of closely coupled systems. The nested dissection technique is also reviewed, and the equivalence between reordering A and dissecting its network is established. The effect of transforming A with odd-even permutation on its topology and the topology of its Cholesky factors is discussed. This leads to the construction of directed graphs showing the computational steps required for factoring A, their precedence relationships and their sequential and concurrent assignment to the available processors. Expressions for the speed-up and efficiency of using N processors in parallel relative to the sequential use of a single processor are derived from the directed graph. Similar expressions are also derived when the number of available processors is fewer than required.

Analysis of multiplex gene expression maps obtained by voxelation.

PubMed

An, Li; Xie, Hongbo; Chin, Mark H; Obradovic, Zoran; Smith, Desmond J; Megalooikonomou, Vasileios

2009-04-29

Gene expression signatures in the mammalian brain hold the key to understanding neural development and neurological disease. Researchers have previously used voxelation in combination with microarrays for acquisition of genome-wide atlases of expression patterns in the mouse brain. On the other hand, some work has been performed on studying gene functions, without taking into account the location information of a gene's expression in a mouse brain. In this paper, we present an approach for identifying the relation between gene expression maps obtained by voxelation and gene functions. To analyze the dataset, we chose typical genes as queries and aimed at discovering similar gene groups. Gene similarity was determined by using the wavelet features extracted from the left and right hemispheres averaged gene expression maps, and by the Euclidean distance between each pair of feature vectors. We also performed a multiple clustering approach on the gene expression maps, combined with hierarchical clustering. Among each group of similar genes and clusters, the gene function similarity was measured by calculating the average gene function distances in the gene ontology structure. By applying our methodology to find similar genes to certain target genes we were able to improve our understanding of gene expression patterns and gene functions. By applying the clustering analysis method, we obtained significant clusters, which have both very similar gene expression maps and very similar gene functions respectively to their corresponding gene ontologies. The cellular component ontology resulted in prominent clusters expressed in cortex and corpus callosum. The molecular function ontology gave prominent clusters in cortex, corpus callosum and hypothalamus. The biological process ontology resulted in clusters in cortex, hypothalamus and choroid plexus. Clusters from all three ontologies combined were most prominently expressed in cortex and corpus callosum. The experimental results confirm the hypothesis that genes with similar gene expression maps might have similar gene functions. The voxelation data takes into account the location information of gene expression level in mouse brain, which is novel in related research. The proposed approach can potentially be used to predict gene functions and provide helpful suggestions to biologists.

Sequential treatment with aurora B inhibitors enhances cisplatin-mediated apoptosis via c-Myc.

PubMed

Ma, Yaxi; Cao, Handi; Lou, Siyue; Shao, Xuejing; Lv, Wen; Qi, Xiaotian; Liu, Yujia; Ying, Meidan; He, Qiaojun; Yang, Xiaochun

2015-04-01

Platinum compound such as cisplatin is the first-line chemotherapy of choice in most patients with ovarian carcinoma. However, patients with inherent or acquired cisplatin resistance often experience relapse. Therefore, novel therapies are urgently required to treat drug-resistant ovarian carcinoma. Here, we showed that compared to the non-functional traditional simultaneous treatment, sequential combination of Aurora B inhibitors followed by cisplatin synergistically enhanced apoptotic response in cisplatin-resistant OVCAR-8 cells. This effect was accompanied by the induction of polyploidy in a c-Myc-dependent manner, as c-Myc knockdown reduced the efficacy of the combination by suppressing the expression of Aurora B and impairing cellular response to Aurora B inhibitor, as indicated by the decreased polyploidy and hyperphosphorylation of histone H1. In c-Myc-deficient SKOV3 cells, c-Myc overexpression restored Aurora B expression, induced polyploidy after inhibition of Aurora B, and sensitized cells to this combination therapy. Thus, our report reveals for the first time that sequential treatment of Aurora B inhibitors and cisplatin is essential to inhibit ovarian carcinoma by inducing polyploidy and downregulating c-Myc and that c-Myc is identified as a predictive biomarker to select cells responsive to chemotherapeutical combinations targeting Aurora B. Collectively, these studies provide novel approaches to overcoming cisplatin chemotherapy resistance in ovarian cancer. Pretreatment of Aurora B inhibitors augment apoptotic effects of cisplatin. The synergy of Aurora B inhibitor with cisplatin is dependent on c-Myc expression. c-Myc-dependent induction of polyploidy sensitizes cells to cisplatin.

Sequential immunization with V3 peptides from primary human immunodeficiency virus type 1 produces cross-neutralizing antibodies against primary isolates with a matching narrow-neutralization sequence motif.

PubMed

Eda, Yasuyuki; Takizawa, Mari; Murakami, Toshio; Maeda, Hiroaki; Kimachi, Kazuhiko; Yonemura, Hiroshi; Koyanagi, Satoshi; Shiosaki, Kouichi; Higuchi, Hirofumi; Makizumi, Keiichi; Nakashima, Toshihiro; Osatomi, Kiyoshi; Tokiyoshi, Sachio; Matsushita, Shuzo; Yamamoto, Naoki; Honda, Mitsuo

2006-06-01

An antibody response capable of neutralizing not only homologous but also heterologous forms of the CXCR4-tropic human immunodeficiency virus type 1 (HIV-1) MNp and CCR5-tropic primary isolate HIV-1 JR-CSF was achieved through sequential immunization with a combination of synthetic peptides representing HIV-1 Env V3 sequences from field and laboratory HIV-1 clade B isolates. In contrast, repeated immunization with a single V3 peptide generated antibodies that neutralized only type-specific laboratory-adapted homologous viruses. To determine whether the cross-neutralization response could be attributed to a cross-reactive antibody in the immunized animals, we isolated a monoclonal antibody, C25, which neutralized the heterologous primary viruses of HIV-1 clade B. Furthermore, we generated a humanized monoclonal antibody, KD-247, by transferring the genes of the complementary determining region of C25 into genes of the human V region of the antibody. KD-247 bound with high affinity to the "PGR" motif within the HIV-1 Env V3 tip region, and, among the established reference antibodies, it most effectively neutralized primary HIV-1 field isolates possessing the matching neutralization sequence motif, suggesting its promise for clinical applications involving passive immunizations. These results demonstrate that sequential immunization with B-cell epitope peptides may contribute to a humoral immune-based HIV vaccine strategy. Indeed, they help lay the groundwork for the development of HIV-1 vaccine strategies that use sequential immunization with biologically relevant peptides to overcome difficulties associated with otherwise poorly immunogenic epitopes.

Sequential Immunization with V3 Peptides from Primary Human Immunodeficiency Virus Type 1 Produces Cross-Neutralizing Antibodies against Primary Isolates with a Matching Narrow-Neutralization Sequence Motif

PubMed Central

Eda, Yasuyuki; Takizawa, Mari; Murakami, Toshio; Maeda, Hiroaki; Kimachi, Kazuhiko; Yonemura, Hiroshi; Koyanagi, Satoshi; Shiosaki, Kouichi; Higuchi, Hirofumi; Makizumi, Keiichi; Nakashima, Toshihiro; Osatomi, Kiyoshi; Tokiyoshi, Sachio; Matsushita, Shuzo; Yamamoto, Naoki; Honda, Mitsuo

2006-01-01

An antibody response capable of neutralizing not only homologous but also heterologous forms of the CXCR4-tropic human immunodeficiency virus type 1 (HIV-1) MNp and CCR5-tropic primary isolate HIV-1 JR-CSF was achieved through sequential immunization with a combination of synthetic peptides representing HIV-1 Env V3 sequences from field and laboratory HIV-1 clade B isolates. In contrast, repeated immunization with a single V3 peptide generated antibodies that neutralized only type-specific laboratory-adapted homologous viruses. To determine whether the cross-neutralization response could be attributed to a cross-reactive antibody in the immunized animals, we isolated a monoclonal antibody, C25, which neutralized the heterologous primary viruses of HIV-1 clade B. Furthermore, we generated a humanized monoclonal antibody, KD-247, by transferring the genes of the complementary determining region of C25 into genes of the human V region of the antibody. KD-247 bound with high affinity to the “PGR” motif within the HIV-1 Env V3 tip region, and, among the established reference antibodies, it most effectively neutralized primary HIV-1 field isolates possessing the matching neutralization sequence motif, suggesting its promise for clinical applications involving passive immunizations. These results demonstrate that sequential immunization with B-cell epitope peptides may contribute to a humoral immune-based HIV vaccine strategy. Indeed, they help lay the groundwork for the development of HIV-1 vaccine strategies that use sequential immunization with biologically relevant peptides to overcome difficulties associated with otherwise poorly immunogenic epitopes. PMID:16699036

Co-expression network analysis of duplicate genes in maize (Zea mays L.) reveals no subgenome bias.

PubMed

Li, Lin; Briskine, Roman; Schaefer, Robert; Schnable, Patrick S; Myers, Chad L; Flagel, Lex E; Springer, Nathan M; Muehlbauer, Gary J

2016-11-04

Gene duplication is prevalent in many species and can result in coding and regulatory divergence. Gene duplications can be classified as whole genome duplication (WGD), tandem and inserted (non-syntenic). In maize, WGD resulted in the subgenomes maize1 and maize2, of which maize1 is considered the dominant subgenome. However, the landscape of co-expression network divergence of duplicate genes in maize is still largely uncharacterized. To address the consequence of gene duplication on co-expression network divergence, we developed a gene co-expression network from RNA-seq data derived from 64 different tissues/stages of the maize reference inbred-B73. WGD, tandem and inserted gene duplications exhibited distinct regulatory divergence. Inserted duplicate genes were more likely to be singletons in the co-expression networks, while WGD duplicate genes were likely to be co-expressed with other genes. Tandem duplicate genes were enriched in the co-expression pattern where co-expressed genes were nearly identical for the duplicates in the network. Older gene duplications exhibit more extensive co-expression variation than younger duplications. Overall, non-syntenic genes primarily from inserted duplications show more co-expression divergence. Also, such enlarged co-expression divergence is significantly related to duplication age. Moreover, subgenome dominance was not observed in the co-expression networks - maize1 and maize2 exhibit similar levels of intra subgenome correlations. Intriguingly, the level of inter subgenome co-expression was similar to the level of intra subgenome correlations, and genes from specific subgenomes were not likely to be the enriched in co-expression network modules and the hub genes were not predominantly from any specific subgenomes in maize. Our work provides a comprehensive analysis of maize co-expression network divergence for three different types of gene duplications and identifies potential relationships between duplication types, duplication ages and co-expression consequences.

Tissue Non-Specific Genes and Pathways Associated with Diabetes: An Expression Meta-Analysis.

PubMed

Mei, Hao; Li, Lianna; Liu, Shijian; Jiang, Fan; Griswold, Michael; Mosley, Thomas

2017-01-21

We performed expression studies to identify tissue non-specific genes and pathways of diabetes by meta-analysis. We searched curated datasets of the Gene Expression Omnibus (GEO) database and identified 13 and five expression studies of diabetes and insulin responses at various tissues, respectively. We tested differential gene expression by empirical Bayes-based linear method and investigated gene set expression association by knowledge-based enrichment analysis. Meta-analysis by different methods was applied to identify tissue non-specific genes and gene sets. We also proposed pathway mapping analysis to infer functions of the identified gene sets, and correlation and independent analysis to evaluate expression association profile of genes and gene sets between studies and tissues. Our analysis showed that PGRMC1 and HADH genes were significant over diabetes studies, while IRS1 and MPST genes were significant over insulin response studies, and joint analysis showed that HADH and MPST genes were significant over all combined data sets. The pathway analysis identified six significant gene sets over all studies. The KEGG pathway mapping indicated that the significant gene sets are related to diabetes pathogenesis. The results also presented that 12.8% and 59.0% pairwise studies had significantly correlated expression association for genes and gene sets, respectively; moreover, 12.8% pairwise studies had independent expression association for genes, but no studies were observed significantly different for expression association of gene sets. Our analysis indicated that there are both tissue specific and non-specific genes and pathways associated with diabetes pathogenesis. Compared to the gene expression, pathway association tends to be tissue non-specific, and a common pathway influencing diabetes development is activated through different genes at different tissues.

Spatiotemporal expression dynamics of selectins govern the sequential extravasation of neutrophils and monocytes in the acute inflammatory response.

PubMed

Zuchtriegel, Gabriele; Uhl, Bernd; Hessenauer, Maximilian E T; Kurz, Angela R M; Rehberg, Markus; Lauber, Kirsten; Krombach, Fritz; Reichel, Christoph A

2015-04-01

Leukocyte recruitment to the site of inflammation is a key event in a variety of cardiovascular pathologies. Infiltrating neutrophils constitute the first line of defense that precedes a second wave of emigrating monocytes reinforcing the inflammatory reaction. The mechanisms initiating this sequential process remained largely obscure. Using advanced in vivo microscopy and in vitro/ex vivo techniques, we identified individual spatiotemporal expression patterns of selectins and their principal interaction partners on neutrophils, resident/inflammatory monocytes, and endothelial cells. Coordinating the intraluminal trafficking of neutrophils and inflammatory monocytes to common sites of extravasation, selectins assign different sites to these immune cells for their initial interactions with the microvascular endothelium. Whereas constitutively expressed leukocyte L-selectin/CD62L and endothelial P-selectin/CD62P together with CD44 and P-selectin glycoprotein ligand-1/CD162 initiate the emigration of neutrophils, de novo synthesis of endothelial E-selectin/CD62E launches the delayed secondary recruitment of inflammatory monocytes. In this context, P-selectin/CD62P and L-selectin/CD62L together with P-selectin glycoprotein ligand-1/CD162 and CD44 were found to regulate the flux of rolling neutrophils and inflammatory monocytes, whereas E-selectin/CD62E selectively adjusts the rolling velocity of inflammatory monocytes. Moreover, selectins and their interaction partners P-selectin glycoprotein ligand-1/CD162 and CD44 differentially control the intraluminal crawling behavior of neutrophils and inflammatory monocytes collectively enabling the sequential extravasation of these immune cells to inflamed tissue. Our findings provide novel insights into the mechanisms initiating the sequential infiltration of the perivascular tissue by neutrophils and monocytes in the acute inflammatory response and might thereby contribute to the development of targeted therapeutic strategies for prevention and treatment of cardiovascular diseases. © 2015 American Heart Association, Inc.

Large scale analysis of signal reachability.

PubMed

Todor, Andrei; Gabr, Haitham; Dobra, Alin; Kahveci, Tamer

2014-06-15

Major disorders, such as leukemia, have been shown to alter the transcription of genes. Understanding how gene regulation is affected by such aberrations is of utmost importance. One promising strategy toward this objective is to compute whether signals can reach to the transcription factors through the transcription regulatory network (TRN). Due to the uncertainty of the regulatory interactions, this is a #P-complete problem and thus solving it for very large TRNs remains to be a challenge. We develop a novel and scalable method to compute the probability that a signal originating at any given set of source genes can arrive at any given set of target genes (i.e., transcription factors) when the topology of the underlying signaling network is uncertain. Our method tackles this problem for large networks while providing a provably accurate result. Our method follows a divide-and-conquer strategy. We break down the given network into a sequence of non-overlapping subnetworks such that reachability can be computed autonomously and sequentially on each subnetwork. We represent each interaction using a small polynomial. The product of these polynomials express different scenarios when a signal can or cannot reach to target genes from the source genes. We introduce polynomial collapsing operators for each subnetwork. These operators reduce the size of the resulting polynomial and thus the computational complexity dramatically. We show that our method scales to entire human regulatory networks in only seconds, while the existing methods fail beyond a few tens of genes and interactions. We demonstrate that our method can successfully characterize key reachability characteristics of the entire transcriptions regulatory networks of patients affected by eight different subtypes of leukemia, as well as those from healthy control samples. All the datasets and code used in this article are available at bioinformatics.cise.ufl.edu/PReach/scalable.htm. © The Author 2014. Published by Oxford University Press.

The Constrained Maximal Expression Level Owing to Haploidy Shapes Gene Content on the Mammalian X Chromosome.

PubMed

Hurst, Laurence D; Ghanbarian, Avazeh T; Forrest, Alistair R R; Huminiecki, Lukasz

2015-12-01

X chromosomes are unusual in many regards, not least of which is their nonrandom gene content. The causes of this bias are commonly discussed in the context of sexual antagonism and the avoidance of activity in the male germline. Here, we examine the notion that, at least in some taxa, functionally biased gene content may more profoundly be shaped by limits imposed on gene expression owing to haploid expression of the X chromosome. Notably, if the X, as in primates, is transcribed at rates comparable to the ancestral rate (per promoter) prior to the X chromosome formation, then the X is not a tolerable environment for genes with very high maximal net levels of expression, owing to transcriptional traffic jams. We test this hypothesis using The Encyclopedia of DNA Elements (ENCODE) and data from the Functional Annotation of the Mammalian Genome (FANTOM5) project. As predicted, the maximal expression of human X-linked genes is much lower than that of genes on autosomes: on average, maximal expression is three times lower on the X chromosome than on autosomes. Similarly, autosome-to-X retroposition events are associated with lower maximal expression of retrogenes on the X than seen for X-to-autosome retrogenes on autosomes. Also as expected, X-linked genes have a lesser degree of increase in gene expression than autosomal ones (compared to the human/Chimpanzee common ancestor) if highly expressed, but not if lowly expressed. The traffic jam model also explains the known lower breadth of expression for genes on the X (and the Z of birds), as genes with broad expression are, on average, those with high maximal expression. As then further predicted, highly expressed tissue-specific genes are also rare on the X and broadly expressed genes on the X tend to be lowly expressed, both indicating that the trend is shaped by the maximal expression level not the breadth of expression per se. Importantly, a limit to the maximal expression level explains biased tissue of expression profiles of X-linked genes. Tissues whose tissue-specific genes are very highly expressed (e.g., secretory tissues, tissues abundant in structural proteins) are also tissues in which gene expression is relatively rare on the X chromosome. These trends cannot be fully accounted for in terms of alternative models of biased expression. In conclusion, the notion that it is hard for genes on the Therian X to be highly expressed, owing to transcriptional traffic jams, provides a simple yet robustly supported rationale of many peculiar features of X's gene content, gene expression, and evolution.

The Constrained Maximal Expression Level Owing to Haploidy Shapes Gene Content on the Mammalian X Chromosome

PubMed Central

Hurst, Laurence D.; Ghanbarian, Avazeh T.; Forrest, Alistair R. R.; Huminiecki, Lukasz

2015-01-01

X chromosomes are unusual in many regards, not least of which is their nonrandom gene content. The causes of this bias are commonly discussed in the context of sexual antagonism and the avoidance of activity in the male germline. Here, we examine the notion that, at least in some taxa, functionally biased gene content may more profoundly be shaped by limits imposed on gene expression owing to haploid expression of the X chromosome. Notably, if the X, as in primates, is transcribed at rates comparable to the ancestral rate (per promoter) prior to the X chromosome formation, then the X is not a tolerable environment for genes with very high maximal net levels of expression, owing to transcriptional traffic jams. We test this hypothesis using The Encyclopedia of DNA Elements (ENCODE) and data from the Functional Annotation of the Mammalian Genome (FANTOM5) project. As predicted, the maximal expression of human X-linked genes is much lower than that of genes on autosomes: on average, maximal expression is three times lower on the X chromosome than on autosomes. Similarly, autosome-to-X retroposition events are associated with lower maximal expression of retrogenes on the X than seen for X-to-autosome retrogenes on autosomes. Also as expected, X-linked genes have a lesser degree of increase in gene expression than autosomal ones (compared to the human/Chimpanzee common ancestor) if highly expressed, but not if lowly expressed. The traffic jam model also explains the known lower breadth of expression for genes on the X (and the Z of birds), as genes with broad expression are, on average, those with high maximal expression. As then further predicted, highly expressed tissue-specific genes are also rare on the X and broadly expressed genes on the X tend to be lowly expressed, both indicating that the trend is shaped by the maximal expression level not the breadth of expression per se. Importantly, a limit to the maximal expression level explains biased tissue of expression profiles of X-linked genes. Tissues whose tissue-specific genes are very highly expressed (e.g., secretory tissues, tissues abundant in structural proteins) are also tissues in which gene expression is relatively rare on the X chromosome. These trends cannot be fully accounted for in terms of alternative models of biased expression. In conclusion, the notion that it is hard for genes on the Therian X to be highly expressed, owing to transcriptional traffic jams, provides a simple yet robustly supported rationale of many peculiar features of X’s gene content, gene expression, and evolution. PMID:26685068

Human leucocyte antigen (HLA-DR) gene expression is reduced in sepsis and correlates with impaired TNFα response: A diagnostic tool for immunosuppression?

PubMed Central

Winkler, Martin Sebastian; Rissiek, Anne; Priefler, Marion; Schwedhelm, Edzard; Robbe, Linda; Bauer, Antonia; Zahrte, Corinne; Zoellner, Christian; Kluge, Stefan; Nierhaus, Axel

2017-01-01

Background Sepsis is defined as a dysregulated immune response to infection. Impaired immune response in sepsis, often described as endotoxin tolerance, is characterized by unresponsiveness of monocytes on lipopolysaccharide (LPS) stimulation to release tumor necrosis factor α (TNFα). Furthermore, decreased monocyte surface protein expression of human leucocyte antigen DR (HLA-DR) is a marker for changes of the innate immune response during sepsis. Quantitative polymerase chain reaction (qPCR) and flow-cytometry (FACS) have been used to measure protein or gene expression of HLA-DR. We aimed to determine whether changes in mRNA expression of HLA-DR are associated with impaired TNFα response in human sepsis. Methods Surface protein together with mRNA expression of HLA-DR were measured by FACS and qPCR in a cohort of 9 sepsis patients and compared to 10 pre-operative control patients in a prospective study. In addition, 20 patients with post-surgical inflammation, 20 patients with sepsis or septic shock were included and TNFα was determined following ex vivo stimulation of whole blood with 500 pg/mL LPS. Total RNA was prepared from whole blood and subjected to qPCR analysis for expression analysis of HLA-DR alpha (HLA-DRA) to correlate TNFα response with HLA-DRA expression. Results Patients with sepsis presented higher numbers of monocytes in peripheral blood (P<0.001) but decreased surface protein and mRNA HLA-DR levels when compared to controls. In all patients mRNA expression of HLA-DRA was decreased by approximately 70% compared to controls (P<0.01) and was lowest in patients with sepsis or septic shock (P<0.01). TNFα response to LPS was decreased in all patients (median 319 pg/mL versus controls 1256 pg/mL; P<0.01) and lowest in patients with sepsis or septic shock (median 128 pg/mL; P<0.01). HLA-DRA correlated positively with TNFα response in all study participants (r +0.60, P<0.001) and within patients (r +0.67, P<0.001). The TNFα:HLA-DRA ratio correlated negatively with severity and the Sequential Organ Failure Assessment (SOFA) score (Spearman’s rho -0.59, P<0.001) Conclusion In this study, HLA-DRA expression was associated with a functional assay of the innate immune response. Future interventional studies aimed at the immune response during sepsis could make use of these methods for optimizing target groups based on biological plausibility and intervention effectiveness. PMID:28771573

Human leucocyte antigen (HLA-DR) gene expression is reduced in sepsis and correlates with impaired TNFα response: A diagnostic tool for immunosuppression?

PubMed

Winkler, Martin Sebastian; Rissiek, Anne; Priefler, Marion; Schwedhelm, Edzard; Robbe, Linda; Bauer, Antonia; Zahrte, Corinne; Zoellner, Christian; Kluge, Stefan; Nierhaus, Axel

2017-01-01

Sepsis is defined as a dysregulated immune response to infection. Impaired immune response in sepsis, often described as endotoxin tolerance, is characterized by unresponsiveness of monocytes on lipopolysaccharide (LPS) stimulation to release tumor necrosis factor α (TNFα). Furthermore, decreased monocyte surface protein expression of human leucocyte antigen DR (HLA-DR) is a marker for changes of the innate immune response during sepsis. Quantitative polymerase chain reaction (qPCR) and flow-cytometry (FACS) have been used to measure protein or gene expression of HLA-DR. We aimed to determine whether changes in mRNA expression of HLA-DR are associated with impaired TNFα response in human sepsis. Surface protein together with mRNA expression of HLA-DR were measured by FACS and qPCR in a cohort of 9 sepsis patients and compared to 10 pre-operative control patients in a prospective study. In addition, 20 patients with post-surgical inflammation, 20 patients with sepsis or septic shock were included and TNFα was determined following ex vivo stimulation of whole blood with 500 pg/mL LPS. Total RNA was prepared from whole blood and subjected to qPCR analysis for expression analysis of HLA-DR alpha (HLA-DRA) to correlate TNFα response with HLA-DRA expression. Patients with sepsis presented higher numbers of monocytes in peripheral blood (P<0.001) but decreased surface protein and mRNA HLA-DR levels when compared to controls. In all patients mRNA expression of HLA-DRA was decreased by approximately 70% compared to controls (P<0.01) and was lowest in patients with sepsis or septic shock (P<0.01). TNFα response to LPS was decreased in all patients (median 319 pg/mL versus controls 1256 pg/mL; P<0.01) and lowest in patients with sepsis or septic shock (median 128 pg/mL; P<0.01). HLA-DRA correlated positively with TNFα response in all study participants (r +0.60, P<0.001) and within patients (r +0.67, P<0.001). The TNFα:HLA-DRA ratio correlated negatively with severity and the Sequential Organ Failure Assessment (SOFA) score (Spearman's rho -0.59, P<0.001). In this study, HLA-DRA expression was associated with a functional assay of the innate immune response. Future interventional studies aimed at the immune response during sepsis could make use of these methods for optimizing target groups based on biological plausibility and intervention effectiveness.

Three-Dimensionality as an Effective Mode of Representation for Expressing Sequential Time Perception

ERIC Educational Resources Information Center

Eden, Sigal; Passig, David

2007-01-01

The process of developing concepts of time continues from age 5 to 11 years (Zakay, 1998). This study sought the representation mode in which children could best express time concepts, especially the proper arrangement of events in a logical and temporal order. Usually, temporal order is examined and taught by 2D (2-dimensional) pictorial scripts.…

Validating internal controls for quantitative plant gene expression studies.

PubMed

Brunner, Amy M; Yakovlev, Igor A; Strauss, Steven H

2004-08-18

Real-time reverse transcription PCR (RT-PCR) has greatly improved the ease and sensitivity of quantitative gene expression studies. However, accurate measurement of gene expression with this method relies on the choice of a valid reference for data normalization. Studies rarely verify that gene expression levels for reference genes are adequately consistent among the samples used, nor compare alternative genes to assess which are most reliable for the experimental conditions analyzed. Using real-time RT-PCR to study the expression of 10 poplar (genus Populus) housekeeping genes, we demonstrate a simple method for determining the degree of stability of gene expression over a set of experimental conditions. Based on a traditional method for analyzing the stability of varieties in plant breeding, it defines measures of gene expression stability from analysis of variance (ANOVA) and linear regression. We found that the potential internal control genes differed widely in their expression stability over the different tissues, developmental stages and environmental conditions studied. Our results support that quantitative comparisons of candidate reference genes are an important part of real-time RT-PCR studies that seek to precisely evaluate variation in gene expression. The method we demonstrated facilitates statistical and graphical evaluation of gene expression stability. Selection of the best reference gene for a given set of experimental conditions should enable detection of biologically significant changes in gene expression that are too small to be revealed by less precise methods, or when highly variable reference genes are unknowingly used in real-time RT-PCR experiments.

General statistics of stochastic process of gene expression in eukaryotic cells.

PubMed Central

Kuznetsov, V A; Knott, G D; Bonner, R F

2002-01-01

Thousands of genes are expressed at such very low levels (< or =1 copy per cell) that global gene expression analysis of rarer transcripts remains problematic. Ambiguity in identification of rarer transcripts creates considerable uncertainty in fundamental questions such as the total number of genes expressed in an organism and the biological significance of rarer transcripts. Knowing the distribution of the true number of genes expressed at each level and the corresponding gene expression level probability function (GELPF) could help resolve these uncertainties. We found that all observed large-scale gene expression data sets in yeast, mouse, and human cells follow a Pareto-like distribution model skewed by many low-abundance transcripts. A novel stochastic model of the gene expression process predicts the universality of the GELPF both across different cell types within a multicellular organism and across different organisms. This model allows us to predict the frequency distribution of all gene expression levels within a single cell and to estimate the number of expressed genes in a single cell and in a population of cells. A random "basal" transcription mechanism for protein-coding genes in all or almost all eukaryotic cell types is predicted. This fundamental mechanism might enhance the expression of rarely expressed genes and, thus, provide a basic level of phenotypic diversity, adaptability, and random monoallelic expression in cell populations. PMID:12136033

The changes of gene expression profiling between segmental vitiligo, generalized vitiligo and healthy individual.

PubMed

Wang, Ping; Li, Yong; Nie, Huiqiong; Zhang, Xiaoyan; Shao, Qiongyan; Hou, Xiuli; Xu, Wen; Hong, Weisong; Xu, Aie

2016-10-01

Vitiligo is a common acquired depigmentation skin disease characterized by loss or dysfunction of melanocytes within the skin lesion, but its pathologenesis is far from lucid. The gene expression profiling of segmental vitiligo (SV) and generalized vitiligo (GV) need further investigation. To better understanding the common and distinct factors, especially in the view of gene expression profile, which were involved in the diseases development and maintenance of segmental vitiligo (SV) and generalized vitiligo (GV). Peripheral bloods were collected from SV, GV and healthy individual (HI), followed by leukocytes separation and total RNA extraction. The high-throughput whole genome expression microarrays were used to assay the gene expression profiles between HI, SV and GV. Bioinformatics tools were employed to annotated the biological function of differently expressed genes. Quantitative PCR assay was used to validate the gene expression of array. Compared to HI, 239 over-expressed genes and 175 down-expressed genes detected in SV, 688 over-expressed genes and 560 down-expressed genes were found in GV, following the criteria of log2 (fold change)≥0.585 and P value<0.05. In these differently expressed genes, 60 over-expressed genes and 60 down-expressed genes had similar tendency in SV and GV. Compared to SV, 223 genes were up regulated and 129 genes were down regulated in GV. In the SV with HI as control, the differently expressed genes were mainly involved in the adaptive immune response, cytokine-cytokine receptor interaction, chemokine signaling, focal adhesion and sphingolipid metabolism. The differently expressed genes between GV and HI were mainly involved in the innate immune, autophagy, apoptosis, melanocyte biology, ubiquitin mediated proteolysis and tyrosine metabolism, which was different from SV. While the differently expressed genes between SV and GV were mainly involved in the metabolism pathway of purine, pyrimidine, glycolysis and sphingolipid. Above results suggested that they not only shared part bio-process and signal pathway, but more important, they utilized different biological mechanism in their pathogenesis and maintenance. Our results provide a comprehensive view on the gene expression profiling change between SV and GV especially in the side of leukocytes, and may facilitate the future study on their molecular mechanism and theraputic targets. Copyright © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Sequential CD4-Coreceptor Interactions in Human Immunodeficiency Virus Type 1 Env Function: Soluble CD4 Activates Env for Coreceptor-Dependent Fusion and Reveals Blocking Activities of Antibodies against Cryptic Conserved Epitopes on gp120

PubMed Central

Salzwedel, Karl; Smith, Erica D.; Dey, Barna; Berger, Edward A.

2000-01-01

We devised an experimental system to examine sequential events by which the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) interacts with CD4 and coreceptor to induce membrane fusion. Recombinant soluble CD4 (sCD4) activated fusion between effector cells expressing Env and target cells expressing coreceptor (CCR5 or CXCR4) but lacking CD4. sCD4-activated fusion was dose dependent, occurred comparably with two- and four-domain proteins, and demonstrated Env-coreceptor specificities parallel to those reported in conventional fusion and infectivity systems. Fusion activation occurred upon sCD4 preincubation and washing of the Env-expressing effector cells but not the coreceptor-bearing target cells, thereby demonstrating that sCD4 exerts its effects by acting on Env. These findings provide direct functional evidence for a sequential two-step model of Env-receptor interactions, whereby gp120 binds first to CD4 and becomes activated for subsequent functional interaction with coreceptor, leading to membrane fusion. We used the sCD4-activated system to explore neutralization by the anti-gp120 human monoclonal antibodies 17b and 48d. These antibodies reportedly bind conserved CD4-induced epitopes involved in coreceptor interactions but neutralize HIV-1 infection only weakly. We found that 17b and 48d had minimal effects in the standard cell fusion system using target cells expressing both CD4 and coreceptor but potently blocked sCD4-activated fusion with target cells expressing coreceptor alone. Both antibodies strongly inhibited sCD4-activated fusion by Envs from genetically diverse HIV-1 isolates. Thus, the sCD4-activated system reveals conserved Env-blocking epitopes that are masked in native Env and hence not readily detected by conventional systems. PMID:10590121

Identification of reference genes in human myelomonocytic cells for gene expression studies in altered gravity.

PubMed

Thiel, Cora S; Hauschild, Swantje; Tauber, Svantje; Paulsen, Katrin; Raig, Christiane; Raem, Arnold; Biskup, Josefine; Gutewort, Annett; Hürlimann, Eva; Unverdorben, Felix; Buttron, Isabell; Lauber, Beatrice; Philpot, Claudia; Lier, Hartwin; Engelmann, Frank; Layer, Liliana E; Ullrich, Oliver

2015-01-01

Gene expression studies are indispensable for investigation and elucidation of molecular mechanisms. For the process of normalization, reference genes ("housekeeping genes") are essential to verify gene expression analysis. Thus, it is assumed that these reference genes demonstrate similar expression levels over all experimental conditions. However, common recommendations about reference genes were established during 1 g conditions and therefore their applicability in studies with altered gravity has not been demonstrated yet. The microarray technology is frequently used to generate expression profiles under defined conditions and to determine the relative difference in expression levels between two or more different states. In our study, we searched for potential reference genes with stable expression during different gravitational conditions (microgravity, normogravity, and hypergravity) which are additionally not altered in different hardware systems. We were able to identify eight genes (ALB, B4GALT6, GAPDH, HMBS, YWHAZ, ABCA5, ABCA9, and ABCC1) which demonstrated no altered gene expression levels in all tested conditions and therefore represent good candidates for the standardization of gene expression studies in altered gravity.

Adaptation of Musca domestica L. Field Population to Laboratory Breeding Causes Transcriptional Alterations

PubMed Central

Højland, Dorte H.; Jensen, Karl-Martin Vagn; Kristensen, Michael

2014-01-01

Background The housefly, Musca domestica, has developed resistance to most insecticides applied for its control. Expression of genes coding for detoxification enzymes play a role in the response of the housefly when encountered by a xenobiotic. The highest level of constitutive gene expression of nine P450 genes was previously found in a newly-collected susceptible field population in comparison to three insecticide-resistant laboratory strains and a laboratory reference strain. Results We compared gene expression of five P450s by qPCR as well as global gene expression by RNAseq in the newly-acquired field population (845b) in generation F1, F13 and F29 to test how gene expression changes following laboratory adaption. Four (CYP6A1, CYP6A36, CYP6D3, CYP6G4) of five investigated P450 genes adapted to breeding by decreasing expression. CYP6D1 showed higher female expression in F29 than in F1. For males, about half of the genes accessed in the global gene expression were up-regulated in F13 and F29 in comparison with the F1 population. In females, 60% of the genes were up-regulated in F13 in comparison with F1, while 33% were up-regulated in F29. Forty potential P450 genes were identified. In most cases, P450 gene expression was decreased in F13 flies in comparison with F1. Gene expression then increased from F13 to F29 in males and decreased further in females. Conclusion The global gene expression changes massively during adaptation to laboratory breeding. In general, global expression decreased as a result of laboratory adaption in males, while female expression was not unidirectional. Expression of P450 genes was in general down-regulated as a result of laboratory adaption. Expression of hexamerin, coding for a storage protein was increased, while gene expression of genes coding for amylases decreased. This suggests a major impact of the surrounding environment on gene response to xenobiotics and genetic composition of housefly strains. PMID:24489682

An RNA-Seq based gene expression atlas of the common bean.

PubMed

O'Rourke, Jamie A; Iniguez, Luis P; Fu, Fengli; Bucciarelli, Bruna; Miller, Susan S; Jackson, Scott A; McClean, Philip E; Li, Jun; Dai, Xinbin; Zhao, Patrick X; Hernandez, Georgina; Vance, Carroll P

2014-10-06

Common bean (Phaseolus vulgaris) is grown throughout the world and comprises roughly 50% of the grain legumes consumed worldwide. Despite this, genetic resources for common beans have been lacking. Next generation sequencing, has facilitated our investigation of the gene expression profiles associated with biologically important traits in common bean. An increased understanding of gene expression in common bean will improve our understanding of gene expression patterns in other legume species. Combining recently developed genomic resources for Phaseolus vulgaris, including predicted gene calls, with RNA-Seq technology, we measured the gene expression patterns from 24 samples collected from seven tissues at developmentally important stages and from three nitrogen treatments. Gene expression patterns throughout the plant were analyzed to better understand changes due to nodulation, seed development, and nitrogen utilization. We have identified 11,010 genes differentially expressed with a fold change ≥ 2 and a P-value < 0.05 between different tissues at the same time point, 15,752 genes differentially expressed within a tissue due to changes in development, and 2,315 genes expressed only in a single tissue. These analyses identified 2,970 genes with expression patterns that appear to be directly dependent on the source of available nitrogen. Finally, we have assembled this data in a publicly available database, The Phaseolus vulgaris Gene Expression Atlas (Pv GEA), http://plantgrn.noble.org/PvGEA/ . Using the website, researchers can query gene expression profiles of their gene of interest, search for genes expressed in different tissues, or download the dataset in a tabular form. These data provide the basis for a gene expression atlas, which will facilitate functional genomic studies in common bean. Analysis of this dataset has identified genes important in regulating seed composition and has increased our understanding of nodulation and impact of the nitrogen source on assimilation and distribution throughout the plant.

Sequential changes in luminal microflora and mucosal cytokine expression during developing of colitis in HLA-B27/beta2-microglobulin transgenic rats.

PubMed

Hata, K; Andoh, A; Sato, H; Araki, Y; Tanaka, M; Tsujikawa, T; Fujiyama, Y; Bamba, T

2001-11-01

Transgenic rats expressing HLA-B27 and human beta2-microglobulin (HLA-B27 rats) spontaneously develop chronic colitis resembling human inflammatory bowel disease. We investigated the sequential changes in the luminal bacterial flora and mucosal cytokine mRNA expression in this model. HLA-B27 rats were maintained in a specific pathogen-free environment, and luminal microflora was evaluated by standard bacterial culture technique. The expression of mucosal cytokine mRNA was analysed by RT-PCR methods. Clinical symptoms of colitis appeared at 8 weeks of age. The total number of obligate anaerobes was higher than those of facultative anaerobes during the experimental period. At 6 weeks of age, the colonization of Bacteroides spp., Bifidobacterium spp. and Lactobacillus spp. was already detectable at high concentrations, whereas Clostridium spp. and Eubacterium spp. were not detected. The expression of proinflammatory cytokines (IL-Ibeta, IL-8 and TNF-alpha) appeared at 8 weeks of age, and these were detectable until 17 weeks. A similar pattern was observed in the expression of Th1 cytokines (IL-2, IL-12 and IFN-gamma). On the other hand, the expression of Th2 cytokines (IL-4, IL-10 and TGF-beta) was weak. IL-4 mRNA expression was weakly detectable only at 6 and 8 weeks of age. The expression of IL-10 and TGF-beta mRNA was scarcely detectable throughout the experimental period. The development of colitis may be mediated by both the predominant expression of Th1 cytokines and the weakness of Th2 cytokine expression in the mucosa. The colonization of anaerobic bacteria, especially Bacteroides spp., may be initiating and promoting these cytokine responses.

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

PubMed Central

2015-01-01

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

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

PubMed

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

2014-12-23

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

Vascular Endothelial Growth Factor Secretion by Nonmyocytes Modulates Connexin-43 Levels in Cardiac Organoids

PubMed Central

Iyer, Rohin K.; Odedra, Devang; Chiu, Loraine L.Y.; Vunjak-Novakovic, Gordana

2012-01-01

We previously showed that the sequential, but not simultaneous, culture of endothelial cells (ECs), fibroblasts (FBs), and cardiomyocytes (CMs) resulted in elongated, beating cardiac organoids. We hypothesized that the expression of Cx43 and contractile function are mediated by vascular endothelial growth factor (VEGF) released by nonmyocytes during the preculture period. Cardiac organoids (∼200 μm diameter) were cultivated in microchannels to enable rapid screening. Three experimental groups were formed: (i) Simultaneous Preculture (ECs+FBs for 48 h, followed by CMs), (ii) Sequential Preculture (ECs for 24 h, FBs for 24 h, followed by CMs), and (iii) Simultaneous Triculture (ECs+FBs+CMs). Controls included CMs only, FBs only, and ECs only groups, and preculture with ECs only or FBs only. The highest VEGF levels were found in the Preculture groups [Simultaneous Preculture, 8.9±2.7 ng/(mL·h−1); Sequential Preculture, 16.6±3.4 ng/(mL·h−1)], as compared with Simultaneous Triculture where VEGF was not detectable, as shown by enzyme-linked immunosorbent assay. Analytical flow cytometry showed that VEGFR2 was expressed by ECs (86%±2 VEGFR2+), FBs (44%±1 VEGFR2+), and CMs (49%±2 VEGFR2+), showing that all three cell types were capable of responding to changes in VEGF. Addition of anti-VEGF neutralizing IgG (0.4 μg/mL) to Simultaneous Preculture resulted in 3-fold decrease in Cx43 mRNA and 1.5-fold decrease in Cx43 protein, while Simultaneous Triculture supplemented with VEGF ligand (30 ng/mL) had a threefold increase in Cx43 mRNA and a twofold increase in Cx43 protein. Addition of a small molecule inhibitor of the VEGFR2 receptor (19.4 nM) to Sequential Preculture caused a 1.4-fold decrease in Cx43 mRNA and a 4.1-fold decrease in Cx43 protein. Cx43 was localized within CMs, and not within FBs or ECs. Enriched CM organoids and Sequential Preculture organoids grown in the presence of VEGFR2 inhibitor displayed low levels of Cx43 and poor functional properties. Taken together, these results suggest that endogenous VEGF-VEGFR2 signaling enhanced Cx43 expression and cardiac function in engineered cardiac organoids. PMID:22519405

Analysis of bHLH coding genes using gene co-expression network approach.

PubMed

Srivastava, Swati; Sanchita; Singh, Garima; Singh, Noopur; Srivastava, Gaurava; Sharma, Ashok

2016-07-01

Network analysis provides a powerful framework for the interpretation of data. It uses novel reference network-based metrices for module evolution. These could be used to identify module of highly connected genes showing variation in co-expression network. In this study, a co-expression network-based approach was used for analyzing the genes from microarray data. Our approach consists of a simple but robust rank-based network construction. The publicly available gene expression data of Solanum tuberosum under cold and heat stresses were considered to create and analyze a gene co-expression network. The analysis provide highly co-expressed module of bHLH coding genes based on correlation values. Our approach was to analyze the variation of genes expression, according to the time period of stress through co-expression network approach. As the result, the seed genes were identified showing multiple connections with other genes in the same cluster. Seed genes were found to be vary in different time periods of stress. These analyzed seed genes may be utilized further as marker genes for developing the stress tolerant plant species.

Sequential growth factor application in bone marrow stromal cell ligament engineering.

PubMed

Moreau, Jodie E; Chen, Jingsong; Horan, Rebecca L; Kaplan, David L; Altman, Gregory H

2005-01-01

In vitro bone marrow stromal cell (BMSC) growth may be enhanced through culture medium supplementation, mimicking the biochemical environment in which cells optimally proliferate and differentiate. We hypothesize that the sequential administration of growth factors to first proliferate and then differentiate BMSCs cultured on silk fiber matrices will support the enhanced development of ligament tissue in vitro. Confluent second passage (P2) BMSCs obtained from purified bone marrow aspirates were seeded on RGD-modified silk matrices. Seeded matrices were divided into three groups for 5 days of static culture, with medium supplement of basic fibroblast growth factor (B) (1 ng/mL), epidermal growth factor (E; 1 ng/mL), or growth factor-free control (C). After day 5, medium supplementation was changed to transforming growth factor-beta1 (T; 5 ng/mL) or C for an additional 9 days of culture. Real-time RT-PCR, SEM, MTT, histology, and ELISA for collagen type I of all sample groups were performed. Results indicated that BT supported the greatest cell ingrowth after 14 days of culture in addition to the greatest cumulative collagen type I expression measured by ELISA. Sequential growth factor application promoted significant increases in collagen type I transcript expression from day 5 of culture to day 14, for five of six groups tested. All T-supplemented samples surpassed their respective control samples in both cell ingrowth and collagen deposition. All samples supported spindle-shaped, fibroblast cell morphology, aligning with the direction of silk fibers. These findings indicate significant in vitro ligament development after only 14 days of culture when using a sequential growth factor approach.

Validating internal controls for quantitative plant gene expression studies

PubMed Central

Brunner, Amy M; Yakovlev, Igor A; Strauss, Steven H

2004-01-01

Background Real-time reverse transcription PCR (RT-PCR) has greatly improved the ease and sensitivity of quantitative gene expression studies. However, accurate measurement of gene expression with this method relies on the choice of a valid reference for data normalization. Studies rarely verify that gene expression levels for reference genes are adequately consistent among the samples used, nor compare alternative genes to assess which are most reliable for the experimental conditions analyzed. Results Using real-time RT-PCR to study the expression of 10 poplar (genus Populus) housekeeping genes, we demonstrate a simple method for determining the degree of stability of gene expression over a set of experimental conditions. Based on a traditional method for analyzing the stability of varieties in plant breeding, it defines measures of gene expression stability from analysis of variance (ANOVA) and linear regression. We found that the potential internal control genes differed widely in their expression stability over the different tissues, developmental stages and environmental conditions studied. Conclusion Our results support that quantitative comparisons of candidate reference genes are an important part of real-time RT-PCR studies that seek to precisely evaluate variation in gene expression. The method we demonstrated facilitates statistical and graphical evaluation of gene expression stability. Selection of the best reference gene for a given set of experimental conditions should enable detection of biologically significant changes in gene expression that are too small to be revealed by less precise methods, or when highly variable reference genes are unknowingly used in real-time RT-PCR experiments. PMID:15317655

Characterization of basal gene expression trends over a diurnal cycle in Xiphophorus maculatus skin, brain and liver.

PubMed

Lu, Yuan; Reyes, Jose; Walter, Sean; Gonzalez, Trevor; Medrano, Geraldo; Boswell, Mikki; Boswell, William; Savage, Markita; Walter, Ronald

2018-06-01

Evolutionarily conserved diurnal circadian mechanisms maintain oscillating patterns of gene expression based on the day-night cycle. Xiphophorus fish have been used to evaluate transcriptional responses after exposure to various light sources and it was determined that each source incites distinct genetic responses in skin tissue. However, basal expression levels of genes that show oscillating expression patterns in day-night cycle, may affect the outcomes of such experiments, since basal gene expression levels at each point in the circadian path may influence the profile of identified light responsive genes. Lack of knowledge regarding diurnal fluctuations in basal gene expression patterns may confound the understanding of genetic responses to external stimuli (e.g., light) since the dynamic nature of gene expression implies animals subjected to stimuli at different times may be at very different stages within the continuum of genetic homeostasis. We assessed basal gene expression changes over a 24-hour period in 200 select Xiphophorus gene targets known to transcriptionally respond to various types of light exposure. We identified 22 genes in skin, 36 genes in brain and 28 genes in liver that exhibit basal oscillation of expression patterns. These genes, including known circadian regulators, produced the expected expression patterns over a 24-hour cycle when compared to circadian regulatory genes identified in other species, especially human and other vertebrate animal models. Our results suggest the regulatory network governing diurnal oscillating gene expression is similar between Xiphophorus and other vertebrates for the three Xiphophorus organs tested. In addition, we were able to categorize light responsive gene sets in Xiphophorus that do, and do not, exhibit circadian based oscillating expression patterns. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of Reference Genes in Human Myelomonocytic Cells for Gene Expression Studies in Altered Gravity

PubMed Central

Thiel, Cora S.; Hauschild, Swantje; Tauber, Svantje; Paulsen, Katrin; Raig, Christiane; Raem, Arnold; Biskup, Josefine; Gutewort, Annett; Hürlimann, Eva; Philpot, Claudia; Lier, Hartwin; Engelmann, Frank; Layer, Liliana E.

2015-01-01

Gene expression studies are indispensable for investigation and elucidation of molecular mechanisms. For the process of normalization, reference genes (“housekeeping genes”) are essential to verify gene expression analysis. Thus, it is assumed that these reference genes demonstrate similar expression levels over all experimental conditions. However, common recommendations about reference genes were established during 1 g conditions and therefore their applicability in studies with altered gravity has not been demonstrated yet. The microarray technology is frequently used to generate expression profiles under defined conditions and to determine the relative difference in expression levels between two or more different states. In our study, we searched for potential reference genes with stable expression during different gravitational conditions (microgravity, normogravity, and hypergravity) which are additionally not altered in different hardware systems. We were able to identify eight genes (ALB, B4GALT6, GAPDH, HMBS, YWHAZ, ABCA5, ABCA9, and ABCC1) which demonstrated no altered gene expression levels in all tested conditions and therefore represent good candidates for the standardization of gene expression studies in altered gravity. PMID:25654098

Life-cycle and growth-phase-dependent regulation of the ubiquitin genes of Trypanosoma cruzi.

PubMed

Manning-Cela, Rebeca; Jaishankar, Sobha; Swindle, John

2006-07-01

Trypanosoma cruzi, the causative agent of Chagas disease, exhibits a complex life cycle that is accompanied by the stage-specific gene expression. At the molecular level, very little is known about gene regulation in trypanosomes. Complex gene organizations coupled with polycistronic transcription units make the analysis of regulated gene expression difficult in trypanosomes. The ubiquitin genes of T. cruzi are a good example of this complexity. They are organized as a single cluster containing five ubiquitin fusion (FUS) and five polyubiquitin (PUB) genes that are polycistronically transcribed but expressed differently in response to developmental and environmental changes. Gene replacements were used to study FUS and PUB gene expression at different stages of growth and at different points in the life cycle of T. cruzi. Based on the levels of reporter gene expression, it was determined that FUS1 expression was downregulated as the parasites approached stationary phase, whereas PUB12.5 polyubiquitin gene expression increased. Conversely, FUS1 expression increases when epimastigotes and amastigotes differentiate into trypomastigotes, whereas the expression of PUB12.5 decreases when epimastigotes differentiate into amastigotes and trypomastigotes. Although the level of CAT activity in logarithmic growing epimastigotes is six- to seven-fold higher when the gene was expressed from the FUS1 locus than when expressed from the PUB12.5 locus, the rate of transcription from the two loci was the same implying that post-transcriptional mechanisms play a dominant role in the regulation of gene expression.

Chromosome engineering of Escherichia coli for constitutive production of salvianic acid A.

PubMed

Zhou, Liang; Ding, Qi; Jiang, Guo-Zhen; Liu, Zhen-Ning; Wang, Hai-Yan; Zhao, Guang-Rong

2017-05-16

Salvianic acid A (SAA), a valuable natural product from herbal plant Salvia miltiorrhiza, exhibits excellent antioxidant activities on food industries and efficacious therapeutic potential on cardiovascular diseases. Recently, production of SAA in engineered Escherichia coli was established via the artificial biosynthetic pathway of SAA on the multiple plasmids in our previous work. However, the plasmid-mediated system required to supplement expensive inducers and antibiotics during the fermentation process, restricting scale-up production of SAA. Microbial cell factory would be an attractive approach for constitutive production of SAA by chromosome engineering. The limited enzymatic reactions in SAA biosynthetic pathway from glucose were grouped into three modules, which were sequentially integrated into chromosome of engineered E. coli by λ Red homologous recombination method. With starting strain E. coli BAK5, in which the ptsG, pykF, pykA, pheA and tyrR genes were previously deleted, chassis strain BAK11 was constructed for constitutive production of precursor L-tyrosine by replacing the 17.7-kb mao-paa cluster with module 1 (P lacUV5 -aroG fbr -tyrA fbr -aroE) and the lacI gene with module 2 (P trc -glk-tktA-ppsA). The synthetic 5tacs promoter demonstrated the optimal strength to drive the expression of hpaBC-d-ldh Y52A in module 3, which then was inserted at the position between nupG and speC on the chromosome of strain BAK11. The final strain BKD13 produced 5.6 g/L of SAA by fed-batch fermentation in 60 h from glucose without any antibiotics and inducers supplemented. The plasmid-free and inducer-free strain for SAA production was developed by targeted integration of the constitutive expression of SAA biosynthetic genes into E. coli chromosome. Our work provides the industrial potential for constitutive production of SAA by the indel microbial cell factory and also sets an example of further producing other valuable natural and unnatural products.