Multiple tissue gene expression analyses in Japanese medaka (Oryzias latipes) exposed to hypoxia

Due in part to human population growth watersheds and coastal estuaries have been receiving increasing run-off of nutrients and genotoxins. As a consequence, the occurrences of nutrient-driven hypoxia in coastal waters appear to be increasing. Thus, understanding the molecular genetic response to hypoxia by model aquatic organisms is of interest both from environmental and physiological viewpoints. The major objectives of this study are to determine genome-wide gene expression profiles and to better understand how hypoxia influences global gene expression in medaka (Oryzias latipes), a well utilized aquatic model species. Herein we detail our development of a microarray containing 8046 medaka unigenes and describe our experimental results for measuring gene expression changes in the brain, gill, and liver of hypoxia exposed fish. Using conservative selection criteria, we determined that 501 genes in the brain, 442 in the gill, and 715 in the liver were differentially expressed in medaka exposed to hypoxia. These differentially expressed genes fell into a number of biological gene ontology groups related to general metabolism, catabolism, RNA and protein metabolism, etc. Two biological pathways, ubiquitin-proteasome and phosphatidylinositol signaling, were significantly dysregulated in medaka upon hypoxia exposure. Comparative genomics between medaka and human identified several human orthologies associated with known diseases.     

Altered gene expression in the brain and liver of female fathead minnows Pimephales promelas Rafinesque exposed to fadrozole

The fathead minnow Pimephales promelas is a small fish species widely used for ecotoxicology research and regulatory testing in North America. This study used a 2000 gene oligonucleotide microarray to evaluate the effects of the aromatase inhibitor, fadrozole, on gene expression in the liver and brain tissue of exposed females. Reproductive measures, plasma vitellogenin and gene expression data for the brain isoform of aromatase (cytP19B), vitellogenin precursors and transferrin provided evidence supporting the efficacy of the fadrozole exposure. Unsupervised analysis of the microarray results identified 20 genes in brain and 41 in liver as significantly up-regulated and seven genes in brain and around 45 in liver as significantly down-regulated. Differentially expressed genes were associated with a broad spectrum of biological functions, many with no obvious relationship to aromatase inhibition. However, in brain, fadrozole exposure elicited significant up-regulation of several genes involved in the cholesterol synthesis, suggesting it as a potentially affected pathway. Gene ontology-based analysis of expression changes in liver suggested overall down-regulation of protein biosynthesis. While real-time polymerase chain reaction analyses supported some of the microarray responses, others could not be verified. Overall, results of this study provide a foundation for developing novel hypotheses regarding the system-wide effects of fadrozole, and other chemical stressors with similar modes of action, on fish biology.     

Gene expression profiling of human epidermal keratinocytes in simulated microgravity and recovery cultures

Simulated microgravity （SMG） bioreactors and DNA microarray technology are powerful tools to identify ＂space genes＂ that play key roles in cellular response to microgravity. We applied these biotechnology tools to investigate SMG and post-SMG recovery effects on human epidermal keratinocytes by exposing cells to SMG for 3, 4, 9, and 10 d using the high aspect ratio vessel bioreactor followed by recovery culturing for 15, 50, and 60 d in normal gravity. As a result, we identified 162 differentially expressed genes, 32 of which were ＂center genes＂ that were most consistently affected in the time course experiments. Eleven of the center genes were from the integrated stress response pathways and were coordinately downregulated. Another seven of the center genes, which are all metallothionein MT-Ⅰ and MT-Ⅱ isoforms, were coordinately up-regulated. In addition, HLA-G, a key gene in cellular immune response suppression, was found to be significantly upregulated during the recovery phase. Overall, more than 80% of the differentially expressed genes from the shorter exposures （≤4 d） recovered in 15 d; for longer （≥9 d） exposures, more than 50 d were needed to recover to the impact level of shorter exposures. The data indicated that shorter SMG exposure duration would lead to quicker and more complete recovery from the microgravity effect.     

Immunological responses of turbot (Psetta maxima) to nodavirus infection or polyriboinosinic polyribocytidylic acid (pIC) stimulation,using expressed sequence tags (ESTs) analysis and cDNA microarrays

To investigate the immunological responses of turbot to nodavirus infection or pIC stimulation, we constructed cDNA libraries from liver, kidney and gill tissues of nodavirus-infected fish and examined the differential gene expression within turbot kidney in response to nodavirus infection or pIC stimulation using a turbot cDNA microarray. Turbot were experimentally infected with nodavirus and samples of each tissue were collected at selected time points post-infection. Using equal amount of total RNA at each sampling time, we made three tissue-specific cDNA libraries. After sequencing 3230 clones we obtained 3173 (98.2%) high quality sequences from our liver, kidney and gill libraries. Of these 2568 (80.9%) were identified as known genes and 605 (19.1%) as unknown genes. A total of 768 unique genes were identified.The two largest groups resulting from the classification of ESTs according to function were the cell/organism defense genes (71 uni-genes) and apoptosis-related process (23 uni-genes). Using these clones, a 1920 element cDNA microarray was constructed and used to investigate the differential gene expression within turbot in response to experimental nodavirus infection or pIC stimulation. Kidney tissue was collected at selected times post-infection (HPI) or stimulation (HPS), and total RNA was isolated for microarray analysis. Of the 1920 genes studied on the microarray, we identified a total of 121 differentially expressed genes in the kidney: 94 genes from nodavirus-infected animals and 79 genes from those stimulated with pIC. Within the nodavirus-infected fish we observed the highest number of differentially expressed genes at 24 HPI. Our results indicate that certain genes in turbot have important roles in immune responses to nodavirus infection and dsRNA stimulation.     

Analysis of stress-induced hepatic gene expression in rainbow trout (Oncorhynchus mykiss) selected for high- and low-responsiveness to stress

The production and welfare of intensively reared fish would be improved by reducing stress responsiveness. One approach to achieving this goal is selective breeding utilising stress-responsive genes as direct genetic markers of the desirable trait. As a first step in this process, microarray analysis has been carried out on liver tissues of rainbow trout selectively bred for high (HR) or low (LR) responsiveness to a stressor. Microarray hybridizations provided gene expression profiles for pooled samples of fish confined for 6 h, 24 h and 168 h and for individual fish (168 h only). 161 genes were shown to be differentially regulated in HR and LR fish during confinement exposure and eight of these gene expression profiles were validated by quantitative PCR. Genes of particular interest included intelectin-2 precursor which showed greater than 100-fold higher expression in HR fish compared to LR fish irrespective of whether the fish were confined or not; interferon inducible transmembrane protein 3 which was differentially stress-induced between the two lines; and hepatic pro-opiomelanocortin B (POMC B) which was upregulated during stress in HR fish but downregulated in LR fish. All these offer potential as direct markers of low stress responsiveness in a marker-assisted selection scheme.     

Exposure to Cobalt Causes Transcriptomic and Proteomic Changes in Two Rat Liver Derived Cell Lines

Cobalt is a transition group metal present in trace amounts in the human diet, but in larger doses it can be acutely toxic or cause adverse health effects in chronic exposures. Its use in many industrial processes and alloys worldwide presents opportunities for occupational exposures, including military personnel. While the toxic effects of cobalt have been widely studied, the exact mechanisms of toxicity remain unclear. In order to further elucidate these mechanisms and identify potential biomarkers of exposure or effect, we exposed two rat liver-derived cell lines, H4-II-E-C3 and MH1C1, to two concentrations of cobalt chloride. We examined changes in gene expression using DNA microarrays in both cell lines and examined changes in cytoplasmic protein abundance in MH1C1 cells using mass spectrometry. We chose to closely examine differentially expressed genes and proteins changing in abundance in both cell lines in order to remove cell line specific effects. We identified enriched pathways, networks, and biological functions using commercial bioinformatic tools and manual annotation. Many of the genes, proteins, and pathways modulated by exposure to cobalt appear to be due to an induction of a hypoxic-like response and oxidative stress. Genes that may be differentially expressed due to a hypoxic-like response are involved in Hif-1α signaling, glycolysis, gluconeogenesis, and other energy metabolism related processes. Gene expression changes linked to oxidative stress are also known to be involved in the NRF2-mediated response, protein degradation, and glutathione production. Using microarray and mass spectrometry analysis, we were able to identify modulated genes and proteins, further elucidate the mechanisms of toxicity of cobalt, and identify biomarkers of exposure and effect in vitro, thus providing targets for focused in vivo studies.     

Social Regulation of Gene Expression in Threespine Sticklebacks

Identifying genes that are differentially expressed in response to social interactions is informative for understanding the molecular basis of social behavior. To address this question, we described changes in gene expression as a result of differences in the extent of social interactions. We housed threespine stickleback (Gasterosteus aculeatus) females in either group conditions or individually for one week, then measured levels of gene expression in three brain regions using RNA-sequencing. We found that numerous genes in the hindbrain/cerebellum had altered expression in response to group or individual housing. However, relatively few genes were differentially expressed in either the diencephalon or telencephalon. The list of genes upregulated in fish from social groups included many genes related to neural development and cell adhesion as well as genes with functions in sensory signaling, stress, and social and reproductive behavior. The list of genes expressed at higher levels in individually-housed fish included several genes previously identified as regulated by social interactions in other animals. The identified genes are interesting targets for future research on the molecular mechanisms of normal social interactions.     

Proteomic response to sublethal cadmium exposure in a sentinel fish species, Cottus gobio

The present study aimed at evaluating the toxicity of short-term cadmium (Cd) exposure in the European bullhead Cottus gobio, a candidate sentinel species. Several enzymatic activity assays (citrate synthase, cytochrome c oxidase, and lactate dehydrogenase) were carried out in liver and gills of fish exposed to 0.01, 0.05, 0.25, and 1 mg Cd/L for 4 days. Exposure to high Cd concentrations significantly altered the activity of these enzymes either in liver and/or in gills. Second, 2D-DIGE technique was used to identify proteins differentially expressed in tissues of fish exposed to either 0.01 or 1 mg Cd/L. Fifty-four hepatic protein spots and 37 branchial protein spots displayed significant changes in abundance in response to Cd exposure. A total of 26 and 12 different proteins were identified using nano LC-MS/MS in liver and gills, respectively. The identified differentially expressed proteins can be categorized into diverse functional classes, related to metabolic process, general stress response, protein fate, and cell structure for instance. This work provides new insights into the biochemical and molecular events in Cd-induced toxicity in fish and suggests that further studies on the identified proteins could provide crucial information to better understand the mechanisms of Cd toxicity in fish.     

Microarray gene expression analysis of Monochamus alternatus (Coleoptera: Cerambycidae) after treatment with a sublethal dose of chloramine phosphorus

Monochamus alternatus Hope (Coleoptera: cerambycidae), the Japanese pine sawyer beetle, is a serious pest management concern in pine stands. A new organophosphorus insecticide, chloramine phosphorus (CP), has been applied as an insecticide in China to control M. alternatus. In this study, we investigated gene expression changes in M. alternatus after 4 h of exposure to CP using a 60-mer oligonucleotide microarray. The results showed that 356 genes were differentially expressed, of which 76 were upregulated and 280 were downregulated. GO enrichment analysis of the differentially expressed genes indicated that those involved in structural molecule activity and transporter activity were among the most highly represented. Within the cellular component category of GO, cell and cell part were the most represented GO terms in upregulated genes. “Cellular process” was the most represented GO term in the biological process category among the downregulated genes. Molecular pathway analysis showed that a large portion of the differentially expressed genes were associated with metabolic pathways. This study sought to identify new biomarkers as nonacetylcholinesterase targets to assess the secondary effects of CP on M. alternatus and will provide a valuable resource for the scientific community to study the molecular toxicology of CP.     

Gene expression profiles in the liver and kidney of metallothionein-null mice

It has been reported that the expression of certain genes was altered in rodent cells lacking metallothioneins (MTs). To further explore the effects of MT deficiency, we screened genes differentially expressed in the liver and kidney of MT-null mice by cDNA microarray analysis. In the liver, 29 of 8737 genes analyzed were altered in their expression levels: 19 and 10 genes were up-regulated and down-regulated, respectively. Particularly, 14 of the 29 genes were related to energy metabolism, and some of these suggested that loss of MTs might lead to obesity and irregular ATP synthesis. In the kidney, 41 differentially expressed genes were observed: 27 and 14 genes were up-regulated and down-regulated, respectively. Eleven of the 41 genes were also related to energy metabolism. Microarray results were confirmed by Northern blot analysis for five of the energy metabolism-related genes.     

Differential gene expression in pancreatic tissues of streptozocin-induced diabetic rats and genetically-diabetic mice in response to hypoglycemic dipeptide cyclo (His-Pro) treatment

Diabetic studies are mostly interested in gene expression in the pancreas, the site of insulin secretion that regulates blood glucose levels. However, a single gene approach has been ruled out for many years in discovering new genes or the molecular networks involved in the induction process of diabetes. To understand the molecular mechanisms by which cyclo (His-Pro) (CHP) affects amelioration of diabetes mellitus, we performed gene expression profiling in the pancreatic tissues of two diabetic animal models, streptozocin (STZ)-induced diabetic rats (T1DM) and genetically-diabetic (C57BL/6J ob/ob) mice (T2DM). To understand the healing process of these diabetic rodents, we examined the effects of CHP on various gene expression in pancreatic tissues of both animal models. Our microarray analysis revealed that a total of 1,175 genes were down-regulated and 629 genes were up-regulated in response to STZ treatment, and the altered expression levels of numerous genes were restored to normal state upon CHP treatment. In particular, 476 genes showed significantly altered gene expression upon CHP treatment. In a functional classification, 7,198 genes were counted as differentially expressed in pancreatic tissues of STZ- and CHP-treated rats compared with control, whereas 1,534 genes were restored to normal states by CHP treatment. Microarray data demonstrated for the first time that overexpression of the genes encoding IL-1 receptor, lipid metabolic enzymes (e.g. Mte1, Ptdss1, and Sult2a1), myo-inositol oxygenase, glucagon, and somatostatin as well as down-regulation of olfactory receptor 984 and mitochondrial ribosomal protein, which are highly linked to T1DM etiology. In genetically-diabetic mice, 4,384 genes were altered in gene expression by more than 2-fold compared to the control mice, when counted differentially expressed. In genetically-diabetic mice, 4,384 genes altered in expression by higher than 2-fold were counted as differentially expressed genes in pancreatic tissues of CHP-treated mice. On the other hand, 2,140 genes were up-regulated and 2,244 genes were down-regulated by CHP treatment. The results of the microarray analysis revealed that up-regulation of IL-2, IL12a, and leptin receptor and down-regulation of PIK3 played important physiological roles in the onset of T2DM. In conclusion, we hypothesize that CHP accelerates alterations of gene expression in ameliorating diabetes and antagonizes those that induces the disease.     

Differential cellular gene expression induced by hepatitis B and C viruses

Hepatitis B virus (HBV) is a hepatotropic virus that causes acute and chronic hepatocellular injury and hepatocellular carcinoma. To clarify how HBV proteins regulate host cellular gene expression, we used our in-house cDNA microarray and HepG2.2.15 cells, which are derived from HepG2 cells and produce all HBV proteins. Of 2304 genes investigated, several genes were differentially expressed in HepG2.2.15 cells compared with HepG2 cells. These genes included insulin-like growth factor II and alpha-fetoprotein, consistent with previous reports. Furthermore, we previously performed similar microarray analyses to clarify the effects of hepatitis C virus (HCV) proteins on host cells, using a HepG2-derivative cell line, which produces all HCV proteins. Using these two microarray results, we compared the differences in cellular gene expression induced by HBV and HCV proteins. The expression of the majority of genes investigated differed only slightly between HBV and HCV protein-producing cells. However, HBV and HCV proteins clearly regulated several genes in a reciprocal manner. Combined, these microarray results shed new light on the effects of HBV proteins on cellular gene expression and on the differences in the pathogenic activities of these two hepatitis viruses.     

Exposure of aboriginals in British Columbia to methylmercury in freshwater fish: A comparison to reference doses and estimated thresholds

We have analyzed the methylmercury exposures of native women consuming fish from a reservoir and two lakes in British Columbia. Probability density functions representing methylmercury dose were generated using reasonable distributions for exposure parameters. Sensitivity analyses were performed to assess the impact of alternative parameter values on the exposure estimates. The effect of ignoring variability and uncertainty in exposure was also assessed. Calculated mean daily doses of methylmercury for the target populations were compared to the estimated average dose for the general population. We also determined the percentages of the native women populations with exposures exceeding current guidance values as well as published thresholds for neurological effects. The analysis demonstrates the importance of better characterizing the low dose effects of methylmercury, as the predicted doses fall in the range of recommended maximum daily doses but well below the higher estimates of the effect threshold.     

温度刺激对斑马鱼仔鱼基因转录表达的影响

许多优良鱼类养殖品种不耐低温或高温的特点给水产养殖业带来诸多限制和困难,这些鱼类在胚胎和仔鱼等早期阶段的抗寒和抗热能力比成体更差,育苗过程中很容易受到温度突然变化的影响。虽然目前利用基因芯片技术已研究了温度刺激对几种鱼类成体组织中基因表达的影响,但温度刺激对仔鱼基因转录表达的影响还未见报道。研究以斑马鱼受精后96h的出膜仔鱼为实验材料,分别在低温(16℃)和高温(34℃)条件下处理12h和24h,用基因芯片技术检测温度刺激对其基因表达的影响。与培养在28℃的对照相比,低温和高温处理后共有3633个基因发生差异表达,其中低温处理后差异表达基因数目多于高温处理,而且低温抑制基因数目多于诱导表达基因的数目。生物信息学分析结果表明,低温诱导基因主要参与RNA加工和核糖体生物发生等生物学过程,高温诱导基因则主要参与应激反应和未折叠蛋白结合。低温抑制基因主要参与蛋白质水解、视觉感知以及铁离子结合等生物学功能,高温抑制基因参与的生物学功能包括DNA复制、神经系统过程和类固醇激素生物合成等。除了已报道的温度刺激响应基因外,研究鉴定出了大量尚未报道与温度刺激相关的基因,如参与RNA加工的rnmtl1a和pus3基因,以及参与转录调控的twistnb和aebp2基因等。研究结果为进一步揭示鱼类冷或热适应的分子机理和培养耐寒或耐热的养殖新品种提供理论基础。     

Bioinformatics analysis of macrophages exposed to Porphyromonas gingivalis: implications in acute vs. chronic infections

Background

Periodontitis is the most common human infection affecting tooth-supporting structures. It was shown to play a role in aggravating atherosclerosis. To deepen our understanding of the pathogenesis of this disease, we exposed human macrophages to an oral bacteria, Porphyromonas gingivalis (P. gingivalis), either as live bacteria or its LPS or fimbria. Microarray data from treated macrophages or control cells were analyzed to define molecular signatures. Changes in genes identified in relevant pathways were validated by RT-PCR.

Methodology/Principal Findings

We focused our analysis on three important groups of genes. Group PG (genes differentially expressed by live bacteria only); Group LFG (genes differentially expressed in response to exposure to LPS and/or FimA); Group CG (core gene set jointly activated by all 3 stimulants). A total of 842 macrophage genes were differentially expressed in at least one of the three conditions compared to naïve cells. Using pathway analysis, we found that group CG activates the initial phagocytosis process and induces genes relevant to immune response, whereas group PG can de-activate the phagocytosis process associated with phagosome-lysosome fusion. LFG mostly affected RIG-I-like receptor signaling pathway.

Conclusion/Significance

In light of the fact that acute infections involve live bacteria while chronic infections involve a combination of live bacteria and their byproducts, group PG could represent acute P. gingivalis infection while group LFG could represent chronic P. gingivalis infection. Group CG may be associated with core immune pathways, triggered irrespective of the specific stimulants and indispensable to mount an appropriate immune response. Implications in acute vs. chronic infection are discussed.