Gene Expression Changes In Response Research Articles

Modeling Fetal Alcohol Spectrum Disorder: Validating an Ex Vivo Primary Hippocampal Cell Culture System.

Fetal alcohol spectrum disorder (FASD) is the leading nongenetic cause of mental retardation. There are no treatments for FASD to date. Preclinical invivo and invitro studies could help in identifying novel drug targets as for other diseases. Here, we describe an exvivo model that combines the physiological advantages of prenatal ethanol (EtOH) exposure invivo with the uniformity of primary fetal hippocampal culture to characterize the effects of prenatal EtOH. The insulin signaling pathways are known to be involved in hippocampal functions. Therefore, we compared the expression of insulin signaling pathway genes between fetal hippocampi (invivo) and primary hippocampal culture (exvivo). The similarity of prenatal EtOH effects in these 2 paradigms would deem the exvivo culture acceptable to screen possible treatments for FASD. Pregnant Sprague-Dawley rats received 1 of 3 diets: adlibitum standard laboratory chow (control-C), isocaloric pair-fed (nutritional control), and EtOH containing liquid diets from gestational day (GD) 8. Fetal male and female hippocampi were collected either on GD21 (invivo) or on GD18 for primary culture (exvivo). Transcript levels of Igf2, Igf2r, Insr, Grb10, Rasgrf1, and Zac1 were measured by reverse transcription quantitative polymerase chain reaction. Hippocampal transcript levels differed by prenatal treatment in both males and females with sex differences observed in the expression of Igf2 and Insr. The effect of prenatal EtOH on the hippocampal expression of the insulin pathway genes was parallel in the invivo and the exvivo conditions. The similarity of gene expression changes in response to prenatal EtOH between the invivo and the exvivo conditions ascertains that these effects are already set in the fetal hippocampus at GD18. This strengthens the feasibility of the exvivo primary hippocampal culture as a tool to test and screen candidate drug targets for FASD.

Persistent Alterations in Microglial Enhancers in a Model of Chronic Pain.

Chronic pain is a common and devastating condition that induces well-characterized changes in neurons and microglia. One major unanswered question is why these changes should persist long after the precipitating injury has healed. Here, we suggest that some of the longer-lasting consequences of nerve injury may be hidden in the epigenome. Cell sorting and sequencing techniques were used to characterize the spinal cord immune response in a mouse model of chronic neuropathic pain. Infiltration of peripheral myeloid cells was found to be absent, and RNA sequencing (RNA-seq) of central microglia revealed transient gene expression changes in response to nerve ligation. Conversely, examination of microglial enhancers revealed persistent, post-injury alterations in close proximity to transcriptionally regulated genes. Enhancers are regions of open chromatin that define a cell's transcription factor binding profile. We hypothesize that changes at enhancers may constitute a mechanism by which painful experiences are recorded at a molecular level.

Effects of a Closed Space Environment on Gene Expression in Hair Follicles of Astronauts in the International Space Station.

Adaptation to the space environment can sometimes pose physiological problems to International Space Station (ISS) astronauts after their return to earth. Therefore, it is important to develop healthcare technologies for astronauts. In this study, we examined the feasibility of using hair follicles, a readily obtained sample, to assess gene expression changes in response to spaceflight adaptation. In order to investigate the gene expression changes in human hair follicles during spaceflight, hair follicles of 10 astronauts were analyzed by microarray and real time qPCR analyses. We found that spaceflight alters human hair follicle gene expression. The degree of changes in gene expression was found to vary among individuals. In some astronauts, genes related to hair growth such as FGF18, ANGPTL7 and COMP were upregulated during flight, suggesting that spaceflight inhibits cell proliferation in hair follicles.

Mycobacterium tuberculosis Transcription Machinery: Ready To Respond to Host Attacks

Regulating responses to stress is critical for all bacteria, whether they are environmental, commensal, or pathogenic species. For pathogenic bacteria, successful colonization and survival in the host are dependent on adaptation to diverse conditions imposed by the host tissue architecture and the immune response. Once the bacterium senses a hostile environment, it must enact a change in physiology that contributes to the organism's survival strategy. Inappropriate responses have consequences; hence, the execution of the appropriate response is essential for survival of the bacterium in its niche. Stress responses are most often regulated at the level of gene expression and, more specifically, transcription. This minireview focuses on mechanisms of regulating transcription initiation that are required by Mycobacterium tuberculosis to respond to the arsenal of defenses imposed by the host during infection. In particular, we highlight how certain features of M. tuberculosis physiology allow this pathogen to respond swiftly and effectively to host defenses. By enacting highly integrated and coordinated gene expression changes in response to stress,M. tuberculosis is prepared for battle against the host defense and able to persist within the human population.

The Use of Real-Time Reverse Transcription-PCR for Assessing Estrogen Receptor and Estrogen-Responsive Gene Expression.

Real-time reverse transcription-polymerase chain reaction (RT-PCR), also known as quantitative RT-PCR (qRT-PCR), is a powerful tool for assessing gene transcription levels. The technique is especially useful for measuring estrogen receptor transcript levels as well as gene expression changes in response to estrogen stimulation as it is quick, accurate, robust, and allows the measurement of gene expression in a variety of tissues and cells. This chapter describes the protocols used for the real-time RT-PCR assay using hydrolysis (TaqMan-type) probes.

Overexpression of Forebrain CRH During Early Life Increases Trauma Susceptibility in Adulthood.

Although early-life stress is a significant risk factor for developing anxiety disorders, including posttraumatic stress disorder (PTSD), the underlying mechanisms are unclear. Corticotropin releasing hormone (CRH) is disrupted in individuals with PTSD and early-life stress and hence may mediate the effects of early-life stress on PTSD risk. We hypothesized that CRH hyper-signaling in the forebrain during early development is sufficient to increase response to trauma in adulthood. To test this hypothesis, we induced transient, forebrain-specific, CRH overexpression during early-life (pre-puberty, CRHOEdev) in double-mutant mice (Camk2a-rtta2 × tetO-Crh) and tested their behavioral and gene expression responses to the predator stress model of PTSD in adulthood. In one cohort of CRHOEdev exposed and unexposed mice, avoidance and arousal behaviors were examined 7-15 days after exposure to predator stress. In another cohort, gene expression changes in Crhr1, Crhr2, and Fkbp51 in forebrain of CRHOEdev exposed and unexposed mice were examined 7 days after predator stress. CRHOEdev induced robust increases in startle reactivity and reductions in startle inhibition independently of predator stress in both male and female mice. Avoidance behaviors after predator stress were highly dependent on sex and CRHOEdev exposure. Whereas stressed females exhibited robust avoidance responses that were not altered by CRHOEdev, males developed significant avoidance only when exposed to both CRHOEdev and stress. Quantitative real-time-PCR analysis indicated that CRHOEdev unexposed males exhibit significant changes in Crhr2 expression in the amygdala and bed nucleus stria terminalis in response to stress, whereas males exposed to CRHOEdev did not. Similar to CRHOEdev males, females exhibited no significant Crhr2 gene expression changes in response to stress. Cortical Fkbp51 expression was also significantly reduced by stress and CRHOEdev exposure in males, but not in females. These findings indicate that forebrain CRH hyper-signaling in early-life is sufficient to increase enduring effects of adult trauma and attenuate Crhr2 expression changes in response to stress in males. These data support growing evidence for significant sex differences in response to trauma, and support further study of CRHR2 as a candidate mechanism for PTSD risk.

269 - Comparative Analysis of Human Embryonic Stem Cell Global Gene Expression Changes in Response to Low, Medical Diagnostic Relevant, and High Doses of Ionizing Radiation Exposure

269 - Comparative Analysis of Human Embryonic Stem Cell Global Gene Expression Changes in Response to Low, Medical Diagnostic Relevant, and High Doses of Ionizing Radiation Exposure

Authentic Research Experience and "Big Data" Analysis in the Classroom: Maize Response to Abiotic Stress.

Integration of inquiry-based approaches into curriculum is transforming the way science is taught and studied in undergraduate classrooms. Incorporating quantitative reasoning and mathematical skills into authentic biology undergraduate research projects has been shown to benefit students in developing various skills necessary for future scientists and to attract students to science, technology, engineering, and mathematics disciplines. While large-scale data analysis became an essential part of modern biological research, students have few opportunities to engage in analysis of large biological data sets. RNA-seq analysis, a tool that allows precise measurement of the level of gene expression for all genes in a genome, revolutionized molecular biology and provides ample opportunities for engaging students in authentic research. We developed, implemented, and assessed a series of authentic research laboratory exercises incorporating a large data RNA-seq analysis into an introductory undergraduate classroom. Our laboratory series is focused on analyzing gene expression changes in response to abiotic stress in maize seedlings; however, it could be easily adapted to the analysis of any other biological system with available RNA-seq data. Objective and subjective assessment of student learning demonstrated gains in understanding important biological concepts and in skills related to the process of science.

Transcriptional profiling of Giardia intestinalis in response to oxidative stress

Giardia intestinalis is a microaerophilic parasite that infects the human upper small intestine, an environment that is fairly aerobic with reactive oxygen species being produced to fight off the parasite. It is quite perplexing how Giardia, lacking conventional eukaryotic antioxidant machinery (e.g. catalase, superoxide dismutase and glutathione peroxidase), can cope with the oxidative stress in this environment. We used transcriptomics (RNA sequencing and quantitative PCR) to study giardial gene expression changes in response to oxygen (O2; 1h) and hydrogen peroxide (H2O2; 150μM, 500μM and 1mM for 1h). The results showed phenotypic and transcriptional differences between Giardia isolates of different genotypes (WB, assemblage A and GS, assemblage B), with GS being more tolerant to H2O2 and exhibiting higher basic transcript levels of antioxidant genes (e.g. NADH oxidase lateral transfer candidate, peroxiredoxin 1 (Prx1) and thioredoxin (Trx)-like proteins). Cysteine is a major antioxidant in Giardia and its role in oxidative defense could be highlighted here by the up-regulation of gene transcripts encoding the cysteine-rich variable surface proteins (VSPs) and high cysteine membrane proteins (HCMPs). Genes in the thioredoxin system (Prx1, Trx and Trx reductase) occupied a central role in the gene expression response to oxidative stress, together with genes encoding metabolic (NADPH-producing enzymes, glutathione and glycerol biosynthetic enzymes) and O2-consuming nitric oxide detoxification enzymes (e.g. nitroreductase, flavohemoprotein and a flavodiiron protein). This study reveals the intricate network of genes associated with the oxidative stress response in Giardia, and provides a stepping-stone towards future studies at the protein level.

Abstract 3521: Combined targeting of DNA and histone methylation improves the efficacy and specificity of epigenetic therapy

Abstract Simultaneous treatment of cancer cells with DNA methyltransferase and HDAC inhibitors leads to synergistic increases of gene expression, indicating that certain genes require targeting of multiple modes of epigenetic regulation to be fully reactivated. However, a major obstacle facing the use of epigenetic therapies such as HDAC inhibitors in cancer is the lack of specificity in reactivating gene expression. Combined targeting of DNA and histone methylation is an unexplored and promising alternative to current therapies due to the role of both modifications in silencing of various tumor suppressor genes in cancer. To address this possibility, we performed a genome wide gene expression study of a DNA methyltransferase inhibitor (DAC) in combination with a LSD1 inhibitor (S2101), G9a inhibitor (UNC0638), EZH2 inhibitor (GSK343) or a HDAC inhibitor (Depsipeptide) in colon cancer (SW48) and breast cancer (MCF7) models. The trends of gene expression changes in response to the inhibitors were similar in both cancer models. S2101, UNC0638, and GSK343 treatment by themselves induced limited gene upregulation in comparison to DAC and especially Depsipeptide. Interestingly, UNC0638 along with DAC had a strong preference to upregulate genes that had high levels of DNA methylation in their promoters. DAC in combination with S2101, UNC0638, or GSK343 was able to synergistically or additively upregulate many DAC target genes compared to levels achieved by DAC alone, demonstrated limited overlap between the upregulated genes (only 4.42% of genes commonly upregulated in SW48, 0.32% in MCF7), and formed distinct clusters in a hierarchical cluster analysis. Each DAC+inhibitor combination also induced a significant number of novel genes that were only upregulated with the combination therapy and not with DAC or inhibitor alone (>490 genes for each inhibitor combination in both cancer models), and there was again limited overlap between the upregulated genes (only 7.64% of genes commonly upregulated in SW48, 7.31% in MCF7). IPA analysis demonstrated that genes induced uniquely by the inhibitor combinations are enriched in critical cancer regulation pathways such as cell proliferation, cell death, and cell movement. Overall, these results demonstrate that effectiveness of combined targeting of DNA and histone methylation is achieved through two mechanisms: 1) Increase effectiveness of DAC by further upregulation of DAC target genes, and 2) Reactivate novel target genes unable to be induced by DAC or the inhibitors alone. These results also demonstrate a promising ability to target specific, non-overlapping sets of genes through different inhibitor combinations, contrary to the nonspecific effect observed with traditional epigenetic therapies. Combined targeting of DNA and histone methylation is a promising novel epigenetic therapy that may allow for the targeting of specific tumor types based on their gene expression profiles. Citation Format: Takahiro Sato, Matteo Cesaroni, Jaroslav Jelinek, Jean-Pierre Issa. Combined targeting of DNA and histone methylation improves the efficacy and specificity of epigenetic therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3521. doi:10.1158/1538-7445.AM2015-3521

Nourishment level affects caste-related gene expression in Polistes wasps.

BackgroundSocial insects exhibit striking phenotypic plasticity in the form of distinct reproductive (queen) and non-reproductive (worker) castes, which are typically driven by differences in the environment during early development. Nutritional environment and nourishment during development has been shown to be broadly associated with caste determination across social insect taxa such as bees, wasps, and termites. In primitively social insects such as Polistes paper wasps, caste remains flexible throughout adulthood, but there is evidence that nourishment inequalities can bias caste development with workers receiving limited nourishment compared to queens. Dominance and vibrational signaling are behaviors that have also been linked to caste differences in paper wasps, suggesting that a combination of nourishment and social factors may drive caste determination. To better understand the molecular basis of nutritional effects on caste determination, we used RNA-sequencing to investigate the gene expression changes in response to proteinaceous nourishment deprivation in Polistes metricus larvae.ResultsWe identified 285 nourishment-responsive transcripts, many of which are related to lipid metabolism and oxidation-reduction activity. Via comparisons to previously identified caste-related genes, we found that nourishment restriction only partially biased wasp gene expression patterns toward worker caste-like traits, which supports the notion that nourishment, in conjunction with social environment, is a determinant of developmental caste bias. In addition, we conducted cross-species comparisons of nourishment-responsive genes, and uncovered largely lineage-specific gene expression changes, suggesting few shared nourishment-responsive genes across taxa.ConclusionOverall, the results from this study highlight the complex and multifactorial nature of environmental effects on the gene expression patterns underlying plastic phenotypes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1410-y) contains supplementary material, which is available to authorized users.

Gestational diabetes mellitus impairs fetal endothelial cell functions through a mechanism involving microRNA-101 and histone methyltransferase enhancer of zester homolog-2.

Gestational diabetes mellitus (GDM) produces fetal hyperglycemia with increased lifelong risks for the exposed offspring of cardiovascular and other diseases. Epigenetic mechanisms induce long-term gene expression changes in response to in utero environmental perturbations. Moreover, microRNAs (miRs) control the function of endothelial cells (ECs) under physiological and pathological conditions and can target the epigenetic machinery. We investigated the functional and expressional effect of GDM on human fetal ECs of the umbilical cord vein (HUVECs). We focused on miR-101 and 1 of its targets, enhancer of zester homolog-2 (EZH2), which trimethylates the lysine 27 of histone 3, thus repressing gene transcription. EZH2 exists as isoforms α and β. HUVECs were prepared from GDM or healthy pregnancies and tested in apoptosis, migration, and Matrigel assays. GDM-HUVECs demonstrated decreased functional capacities, increased miR-101 expression, and reduced EZH2- β and trimethylation of histone H3 on lysine 27 levels. MiR-101 inhibition increased EZH2 expression and improved GDM-HUVEC function. Healthy HUVECs were exposed to high or normal d-glucose concentration for 48 hours and then tested for miR-101 and EZH2 expression. Similar to GDM, high glucose increased miR-101 expression. Chromatin immunoprecipitation using an antibody for EZH2 followed by polymerase chain reaction analyses for miR-101 gene promoter regions showed that both GDM and high glucose concentration reduced EZH2 binding to the miR-101 locus in HUVECs. Moreover, EZH2-β overexpression inhibited miR-101 promoter activity in HUVECs. GDM impairs HUVEC function via miR-101 upregulation. EZH2 is both a transcriptional inhibitor and a target gene of miR-101 in HUVECs, and it contributes to some of the miR-101-induced defects of GDM-HUVECs.

Gene expression profiles responses to aphid feeding in chrysanthemum (Chrysanthemum morifolium).

BackgroundChrysanthemum is an important ornamental plant all over the world. It is easily attacked by aphid, Macrosiphoniella sanbourni. The molecular mechanisms of plant defense responses to aphid are only partially understood. Here, we investigate the gene expression changes in response to aphid feeding in chrysanthemum leaf by RNA-Seq technology.ResultsThree libraries were generated from pooled leaf tissues of Chrysanthemum morifolium ‘nannongxunzhang’ that were collected at different time points with (Y) or without (CK) aphid infestations and mock puncture treatment (Z), and sequenced using an Illumina HiSeqTM 2000 platform. A total of 7,363,292, 7,215,860 and 7,319,841 clean reads were obtained in library CK, Y and Z, respectively. The proportion of clean reads was >97.29% in each library. Approximately 76.35% of the clean reads were mapped to a reference gene database including all known chrysanthemum unigene sequences. 1,157, 527 and 340 differentially expressed genes (DEGs) were identified in the comparison of CK-VS-Y, CK-VS-Z and Z-VS-Y, respectively. These DEGs were involved in phytohormone signaling, cell wall biosynthesis, photosynthesis, reactive oxygen species (ROS) pathway and transcription factor regulatory networks, and so on.ConclusionsChanges in gene expression induced by aphid feeding are shown to be multifaceted. There are various forms of crosstalk between different pathways those genes belonging to, which would allow plants to fine-tune its defense responses.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1050) contains supplementary material, which is available to authorized users.

Bacteria-triggered systemic immunity in barley is associated with WRKY and ETHYLENE RESPONSIVE FACTORs but not with salicylic acid.

Leaf-to-leaf systemic immune signaling known as systemic acquired resistance is poorly understood in monocotyledonous plants. Here, we characterize systemic immunity in barley (Hordeum vulgare) triggered after primary leaf infection with either Pseudomonas syringae pathovar japonica (Psj) or Xanthomonas translucens pathovar cerealis (Xtc). Both pathogens induced resistance in systemic, uninfected leaves against a subsequent challenge infection with Xtc. In contrast to systemic acquired resistance in Arabidopsis (Arabidopsis thaliana), systemic immunity in barley was not associated with NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 or the local or systemic accumulation of salicylic acid. Instead, we documented a moderate local but not systemic induction of abscisic acid after infection of leaves with Psj. In contrast to salicylic acid or its functional analog benzothiadiazole, local applications of the jasmonic acid methyl ester or abscisic acid triggered systemic immunity to Xtc. RNA sequencing analysis of local and systemic transcript accumulation revealed unique gene expression changes in response to both Psj and Xtc and a clear separation of local from systemic responses. The systemic response appeared relatively modest, and quantitative reverse transcription-polymerase chain reaction associated systemic immunity with the local and systemic induction of two WRKY and two ETHYLENE RESPONSIVE FACTOR (ERF)-like transcription factors. Systemic immunity against Xtc was further associated with transcriptional changes after a secondary/systemic Xtc challenge infection; these changes were dependent on the primary treatment. Taken together, bacteria-induced systemic immunity in barley may be mediated in part by WRKY and ERF-like transcription factors, possibly facilitating transcriptional reprogramming to potentiate immunity.

Identification and expression analysis of the E2F/DP genes under salt stress in Medicago truncatula

Cell cycle control in plants converges on the Rb/E2F/DP pathway, which is regulated by cyclin-dependent kinases. Plants can coordinate their cell cycles during salt stress to benefit their growth and development. However, the mechanism underlying cell cycle control under salt stress is still unclear. Here, we identified five predicted E2F/DP genes in the Medicago truncatula genome, including three E2Fs, one DP, and one DEL. The conserved domains of the E2F/DP proteins were relatively well conserved with those of Arabidopsis thaliana and Oryza sativa. Intron/exon organization analyses indicated that Medtr;E2Fc and Medtr;DPa gained/lost introns in the conserved domains during recent evolutionary process. Furthermore, an expression analysis showed that these genes were expressed with varied transcription levels in all of the tissues tested. Contrasting gene expression changes in response to salt treatment in salt-tolerant versus salt-sensitive genotypes indicated that Medtr;DPa might be a candidate gene underlying the salt tolerance. This study will contribute to the understanding of the E2F/DP transcription factors in M.truncatula and of the mechanism organizing cell cycle regulation and salt stress.

Identification of genes related to Paulownia witches' broom by AFLP and MSAP.

DNA methylation is believed to play important roles in regulating gene expression in plant growth and development. Paulownia witches’ broom (PaWB) infection has been reported to be related to gene expression changes in paulownia plantlets. To determine whether DNA methylation is associated with gene expression changes in response to phytoplasma, we investigated variations in genomic DNA sequence and methylation in PaWB plantlets treated with methyl methane sulfonate (MMS) using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MSAP) techniques, respectively. The results indicated that PaWB seedings recovered a normal morphology after treatment with more than 15 mg·L−1 MMS. PaWB infection did not cause changes of the paulownia DNA sequence at the AFLP level; However, DNA methylation levels and patterns were altered. Quantitative real-time PCR (qRT-PCR) showed that three of the methylated genes were up-regulated and three were down-regulated in the MMS-treated PaWB plantlets that had regained healthy morphology. These six genes might be involved in transcriptional regulation, plant defense, signal transduction and energy. The possible roles of these genes in PaWB are discussed. The results showed that changes of DNA methylation altered gene expression levels, and that MSAP might help identify genes related to PaWB.

Alfalfa Cellulose synthase gene expression under abiotic stress: a Hitchhiker's guide to RT-qPCR normalization.

Abiotic stress represents a serious threat affecting both plant fitness and productivity. One of the promptest responses that plants trigger following abiotic stress is the differential expression of key genes, which enable to face the adverse conditions. It is accepted and shown that the cell wall senses and broadcasts the stress signal to the interior of the cell, by triggering a cascade of reactions leading to resistance. Therefore the study of wall-related genes is particularly relevant to understand the metabolic remodeling triggered by plants in response to exogenous stresses. Despite the agricultural and economical relevance of alfalfa (Medicago sativa L.), no study, to our knowledge, has addressed specifically the wall-related gene expression changes in response to exogenous stresses in this important crop, by monitoring the dynamics of wall biosynthetic gene expression. We here identify and analyze the expression profiles of nine cellulose synthases, together with other wall-related genes, in stems of alfalfa plants subjected to different abiotic stresses (cold, heat, salt stress) at various time points (e.g. 0, 24, 72 and 96 h). We identify 2 main responses for specific groups of genes, i.e. a salt/heat-induced and a cold/heat-repressed group of genes. Prior to this analysis we identified appropriate reference genes for expression analyses in alfalfa, by evaluating the stability of 10 candidates across different tissues (namely leaves, stems, roots), under the different abiotic stresses and time points chosen. The results obtained confirm an active role played by the cell wall in response to exogenous stimuli and constitute a step forward in delineating the complex pathways regulating the response of plants to abiotic stresses.

Host responses to sepsis vary in different low-lethality murine models.

IntroductionAnimal models for the study of sepsis are being increasingly scrutinized, despite their essential role for early translational research. In particular, recent studies have suggested that at the level of the leukocyte transcriptome, murine models of burns, trauma and endotoxemia markedly differ from their human equivalents, and are only weakly similar amongst themselves. We compared the plasma cytokine and leukocyte transcriptome responses between two different low-lethality murine models of polymicrobial intra-abdominal sepsis.MethodsSix to ten week male C57BL/6j mice underwent either the ‘gold standard’ cecal ligation and puncture (CLP) model of intra-abdominal sepsis or administration of a cecal slurry (CS), where cecal contents are injected intraperitoneally. Surviving mice were euthanized at two hours, one or three days after sepsis.ResultsThe murine leukocyte transcriptomic response to the CLP and CS models of sepsis was surprisingly dissimilar at two hours, one, and three days after sepsis. The Pearson correlation coefficient for the maximum change in expression for the entire leukocyte transcriptome that changed significantly over time (n = 19,071) was R = 0.54 (R2 = 0.297). The CS model resulted in greater magnitude of early inflammatory gene expression changes in response to sepsis with associated increased production of inflammatory chemokines and cytokines. Two hours after sepsis, CLP had more significant expression of genes associated with IL-10 signaling pathways, whereas CS had greater expression of genes related to CD28, apoptosis, IL-1 and T-cell receptor signaling. By three days, the changes in gene expression in both sepsis models were returning to baseline in surviving animals.ConclusionThese analyses reveal that the murine blood leukocyte response to sepsis is highly dependent on which model of intra-abdominal sepsis is employed, despite their similar lethality. It may be difficult to extrapolate findings from one murine model to another, let alone to human sepsis.

Microarray analysis for the influence of CMV infection on tomato genomes (617.1)

Functional genomics is the study of gene function through parallel expression measurements of a genome. The most common tools used to carry out these measurements include complementary DNA microarrays, oligonucleotide microarrays or serial analysis of gene expression (SAGE)3. A custom‐designed Affymetrix tomato GeneChip array was used to study the gene expression changes in response to viral inoculation with a mild and severe CMV strains. The basic technique involves extraction of RNA from tomato leaf samples and then copied, while incorporating either fluorescent nucleotides or a tag that is later stained with fluorescence. Labeled RNA is then hybridized to a microarray for a period of time, then excess is washed off and the microarray is scanned under laser light. Different expression responses were resulted due to the type of viral infection.Total number of expressed genes were 3213, Out of this 1660 genes were down‐regulated and 1553 were up‐regulated. The response to CMV‐16 infection led to 1365 gene expression, 642 were down‐regulated and 723 genes were up‐regulated Specific seven different genes in tomato genome (Hsp70,ChMDH, Wif1, rccr, AAP2, PIIF & GAPDH endogenous gene used as a control) were analyzed and resulted in down regulation of Hsp70 and upper regulation of both ChMDH and PIIF. Gene expression results were confirmed with relative quantitative RT‐PCR technology.

Towards a molecular understanding of allostasis in free‐ranging marine mammals (703.6)

Chronic stress negatively affects reproduction and survival of pelagic predators, threatening ecosystem stability. However, our understanding of the stress response in marine mammals is limited and largely based on terrestrial model systems that do not reflect pelagic life history adaptations. We are examining the molecular signature of allostasis in northern elephant seals to develop species‐specific stress diagnostic tools and resolve interactions between the hypothalamic‐pituitary‐adrenal (HPA) axis and other physiological pathways. We conducted stress tests in juvenile elephant seals by intramuscular adrenocorticotropic hormone (ACTH) injection to analyze the magnitude and duration of the HPA axis response. To investigate global gene expression changes in response to a stress challenge, we sequenced RNA isolated from muscle tissue of animals before and after ACTH injection. De novo transcriptome assembly and analysis was performed using the Trinity platform and yielded a reference transcriptome for the northern elephant seal study system. We have identified a number of genes and pathways altered during the stress response in a free‐ranging marine mammal, including several candidate molecular markers of stress. Our data indicate that the magnitude of the stress response to ACTH stimulation provides key information about an animal’s physiological state.Grant Funding Source: Office of Naval Research