mRNA Transcripts Of Genes Research Articles

Modulation of redox and insulin signaling underlie the anti-hyperglycemic and antioxidant effects of diphenyl diselenide in zebrafish

The organic selenium compound diphenyl diselenide (DD) has been recognized as an antioxidant and neuroprotective agent, exerting an anti-hyperglycemic effect in experimental models of diabetes. However, the precise mechanisms involved in the protection are unclear. Using the zebrafish (Danio rerio) as a model organism, here we investigated biomarkers underlying the protective effects of DD against hyperglycemia, targeting in a transcriptional approach the redox and insulin-signaling pathway. Fish were fed on a diet containing DD (3 mg/kg) for 74 days. In the last 14 days, they were exposed to a 111 mM glucose solution to induce a hyperglycemic state. DD reduced blood glucose levels as well as normalized the brain mRNA transcription of four insulin receptors-coding genes (Insra1, Insra2, Insrb1, Insrb2), which were down-regulated by glucose. DD alone caused an up-regulation of relative mRNA transcription in both Insra receptors and glucose transporter 3 genes. DD counteracted hyperglycemia-induced lipid peroxidation, protein and thiol depletion. Along with the decreased activity of antioxidant enzymes SOD and GPx, the brain of hyperglycemic fish presented a reduction in mRNA transcription of FoxO3A, FoxO3B, Nrf2, GPx3A, SOD1, and SOD2 genes. Besides normalizing the transcriptional levels, DD caused an up-regulation of relative mRNAs that encode Nrf2, FoxO1A, FOXO3A, GPx4A, PTP1B, AKT and SelP. Collectively, our findings suggest that the antioxidant and anti-hyperglycemic actions of DD in a zebrafish diabetes model are likely associated with the regulation of the oxidative stress resistance and the insulin-signaling pathway and that could be related to the modulation at mRNA level of two important transcription factors, Nrf2 and FoxO.

Replacement of dietary fishmeal with Sargassum ilicifolium meal on growth, innate immunity and immune gene mRNA transcript abundance in Lates calcarifer juveniles

Replacement of dietary fishmeal with <i>Sargassum ilicifolium</i> meal on growth, innate immunity and immune gene mRNA transcript abundance in <i>Lates calcarifer</i> juveniles

Duck Enteritis Virus VP16 Antagonizes IFN-β-Mediated Antiviral Innate Immunity.

Duck enteritis virus (DEV) can successfully evade the host innate immune responses and establish a lifelong latent infection in the infected host. However, the study about how DEV escapes host innate immunity is still deficient up to now. In this study, for the first time, we identified a viral protein VP16 by which DEV can obviously downregulate the production of IFN-β in duck embryo fibroblast (DEF). Our results showed that ectopic expression of VP16 decreased duck IFN-β (duIFN-β) promoter activation and significantly inhibited the mRNA transcription of IFN-β. Further study showed that VP16 can also obviously inhibit the mRNA transcription of interferon-stimulated genes (ISGs), such as myxovirus resistance protein (Mx) and interferon-induced oligoadenylate synthetase-like (OASL). Furthermore, we found that this anti-interferon activity of VP16 depended on its N-terminus (aa1-200). Coexpression analysis revealed that VP16 selectively blocked duIFN-β promoter activity at the duIRF7 level rather than duIRF1. Based on the results of coimmunoprecipitation analysis (co-IP) and indirect immunofluorescence assay (IFA), VP16 was able to bind to duck IRF7 (duIRF7) directly, but did not interact with duck IRF1 (duIRF1) in vitro.

ADAR1-Dependent RNA Editing Promotes MET and iPSC Reprogramming by Alleviating ER Stress.

RNA editing of adenosine to inosine (A to I) is catalyzed by ADAR1 and dramatically alters the cellular transcriptome, although its functional roles in somatic cell reprogramming are largely unexplored. Here, we show that loss of ADAR1-mediated A-to-I editing disrupts mesenchymal-to-epithelial transition (MET) during induced pluripotent stem cell (iPSC) reprogramming and impedes acquisition of induced pluripotency. Using chemical and genetic approaches, we show that absence of ADAR1-dependent RNA editinginducesaberrant innate immune responses through the double-stranded RNA (dsRNA) sensor MDA5, unleashing endoplasmic reticulum (ER) stress and hindering epithelial fate acquisition. We found that A-to-I editing impedes MDA5 sensing and sequestration of dsRNAs encoding membrane proteins, which promote ER homeostasis by activating the PERK-dependent unfolded protein response pathway to consequently facilitate MET. This study therefore establishes a critical role for ADAR1 and its A-to-I editing activity duringcell fate transitions and delineates a key regulatory layer underlying MET to control efficient reprogramming.

Diets supplemented with Saccharina latissima influence the expression of genes related to lipid metabolism and oxidative stress modulating rainbow trout (Oncorhynchus mykiss) fillet composition

This study aimed to evaluate the impact of diets including increasing amounts (1, 2 and 4%) of an iodine-rich macroalgae, Saccharina latissima, on gene expression and fillet composition of commercial-sized rainbow trout. Liver and muscle expression of genes related to growth, iodine, oxidative stress, and lipid metabolism, and the fillet content of fatty acids, cholesterol, and vitamin D3 were assessed. The highest kelp inclusion led to lower final body weight and HSI, without significant differences in mRNA transcription of genes involved in growth (ghr1, ghr2 and igf1) or iodine metabolism (dio1, thra, and thrb). A significant downregulation of an oxidative stress marker, gpx1b2, was observed in fish fed 2% S. latissima, which might suggest the need for less endogenous antioxidants. Dietary inclusion of kelp impacted lipid metabolism, with a downregulation of fatty acid synthase, accompanied by a general decrease of fatty acids in fillet. The present study demonstrated that supplementation of diets with 1 or 2% S. latissima can be achieved without detrimental effects on rainbow trout final weight. Evidence suggest a lipid-lowering effect of diets that did not compromise fillet EPA and DHA concentrations, being 3.7 times above the recommended levels for human consumption.

Infant Formula Feeding Changes the Proliferative Status in Piglet Neonatal Mammary Glands Independently of Estrogen Signaling

Background:Soy infant formula contains isoflavones, which are able to bind to and activate estrogen receptor (ER) pathways. The mammary gland is sensitive to estrogens, raising concern that the use of soy formulas may promote premature development. Objective:We aimed to determine if soy formula feeding increases mammary gland proliferation and differentiation in comparison to other infant postnatal diets. Methods:White-Dutch Landrace piglets aged 2 d received either sow milk (Sow), or were provided milk formula (Milk), soy formula (Soy), milk formula supplemented with 17-beta-estradiol (2 mg/(kgd); M + E2), or milk formula supplemented with genistein (84 mg/L of diet; M + G) until day 21. Mammary gland proliferation and differentiation was assessed by histology, and real-time RT-PCR confirmation of differentially expressed genes identified by microarray analysis. Results:Mammary terminal end bud numbers were 19–31% greater in the Milk, Soy, and M+G groups relative to the Sow and M+E2, P <0.05. Microarray analysis identified differentially expressed genes between each formula-fed group relative to the Sow (±1.7-fold, P <0.05). Real-time RT-PCR confirmed 2- to 4-fold increases in mRNA transcripts of genes involved in cell proliferation, insulin-like growth factor 1 (IGF1), fibroblast growth factor 10 (FGF10), and fibroblast growth factor 18 (FGF18), in all groups relative to the Sow, P <0.05. In contrast, genes involved in cell differentiation and ductal morphogenesis, angiotensin II receptor type 2 (AGTR2), microtubule associated protein 1b (MAP1B), and kinesin family member 26b (KIF26B), were significantly upregulated by 2-, 4-, and 13-fold, respectively, in the M+E2 group. Additionally, mRNA expression of ER-specific gene targets, progesterone receptor (PGR), was increased by 12-fold, and amphiregulin (AREG) and Ras-like estrogen regulated growth inhibitor (RERG) expression by 1.5-fold in the M+E2 group, P <0.05. In the soy and M+G groups, mRNA expressions of fatty acid synthesis genes were increased 2- to 4-fold. Conclusions:Our data indicate soy formula feeding does not promote ER-signaling in the piglet mammary gland. Infant formula feeding (milk- or soy-based) may initiate proliferative pathways independently of estrogenic signaling.

Cortisol inhibits the Escherichia coli-induced endometrial inflammatory response through NF-κB and MAPK pathways in postpartum goats

Glucocorticoids have been widely used as anti-inflammatory therapies. The mechanisms of cortisol action in goat does with endometritis, however, have not been reported. The aim of this study was to investigate the mechanism of cortisol in modulation of effects of E. coli-induced endometritis in the does. Does (n = 24) were assigned to four groups (n = 6): control, E. coli, cortisol, and E. coli + cortisol groups. Does in the cortisol and E. coli + cortisol group were treated with cortisol from 3 days before E. coli inoculations occurred to 36 days post-partum. Does in the E. coli and inoculation groups were administered via intrauterine infusion E. coli O55 (109 CFU/mL) at 0 h. Physical indicators, macroscopic and microscopic changes in the endometrium, uterine secretion cytology and bacteriology were evaluated before (0 h) and at 6, 12, 24, 48, and 72 h after E. coli inoculation. The TLR4 and pro-inflammatory cytokine mRNA transcripts were detected using qPCR. The activations of NF-κB and MAPK signaling pathways were detected using Western blot procedures. As a result, cortisol inhibited the inflammatory response of does by reducing the clinical symptoms, morphological endometrial damage, % PMN in uterine secretions, relative abundance of inflammatory gene mRNA transcripts in the endometrium of does. Cortisol inhibited NF-κB activity by reducing MyD88 and IκB phosphorylation. Treatment with cortisol suppressed the phosphorylation of ERK1/2, p38MAPK, and JNK. These results indicate the anti-inflammatory effect of cortisol in the endometrium of does may be regulated by NF-κB and MAPK pathways.

Microsatellite Instability and MMR Genes Abnormalities in Canine Mammary Gland Tumors.

Early diagnosis of mammary gland tumors is a challenging task in animals, especially in unspayed dogs. Hence, this study investigated the role of microsatellite instability (MSI), MMR gene mRNA transcript levels and SNPs of MMR genes in canine mammary gland tumors (CMT). A total of 77 microsatellite (MS) markers in 23 primary CMT were selected from four breeds of dogs. The results revealed that 11 out of 77 MS markers were unstable and showed MSI in all the tumors (at least at one locus), while the other markers were stable. Compared to the other markers, the ABC9TETRA, MEPIA, 9A5, SCNA11 and FJL25 markers showed higher frequencies of instability. All CMT demonstrated MSI, with eight tumors presenting MSI-H. The RT-qPCR results revealed significant upregulation of the mRNA levels of cMSH3, cMLH1, and cPMSI, but downregulation of cMSH2 compared to the levels in the control group. Moreover, single nucleotide polymorphisms (SNPs) were observed in the cMSH2 gene in four exons, i.e., 2, 6, 15, and 16. In conclusion, MSI, overexpression of MMR genes and SNPs in the MMR gene are associated with CMT and could be served as diagnostic biomarkers for CMT in the future.

Relative abundance of interferon-stimulated genes STAT1, OAS1, CXCL10 and MX1 in ovine lymph nodes during early pregnancy

Lymph nodes have functions in the adaptive immune response, and interferon-tau (IFNT), a primary pregnancy recognition signal in domestic ruminants has effects on immune regulation. It, however, is unclear whether early pregnancy induces an increase in the abundance of interferon-stimulated gene (ISG) mRNA transcripts and proteins in lymph nodes of sheep. In this study, lymph nodes were obtained on day 16 of the estrous cycle from non-pregnant ewes and days 13, 16 and 25 of gestation from pregnant ewes, and the abundance of ISG mRNA transcripts, including signal transducer and activator of transcription 1 (STAT1), phosphorylated STAT1 (p-STAT1), 2′,5′-oligoadenylate synthetase (OAS1), myxovirus resistance protein 1 (MX1) and C-X-C motif chemokine 10 (CXCL10), was analyzed using real-time quantitative PCR. Furthermore, Western blot and immunohistochemistry analysis was conducted to assess relative abundance of proteins encoded by these genes. The results indicated that there was a larger abundance of STAT1 mRNA transcript and protein, and p-STAT1 protein in the maternal lymph node at days 16 and 25 of gestation, and that abundances of OAS1, MX1 and CXCL10 mRNA transcripts and protein were greatest on day 16 of gestations. In addition, STAT1 protein was located in the subcapsular sinus, lymph sinuses, B cells and T cells. The larger relative abundances of STAT1, p-STAT1, OAS1, MX1 and CXCL10 mRNA transcripts and/or protein in the lymph nodes of ewes may be associated with maternal immunoregulation through blood circulation and lymph circulation during early pregnancy.

Effects of copper hydroxychloride on growth performance and abundance of genes involved in lipid metabolism of growing pigs.

An experiment was conducted to test the hypothesis that copper (Cu) hydroxychloride improves growth performance by upregulating the mRNA transcription of genes involved in lipid metabolism of pigs fed a diet based on corn, soybean meal (SBM), and distillers dried grains with solubles (DDGS). Thirty-two pigs (15.05 ± 0.98 kg) were allotted to 2 dietary treatments with 2 pigs per pen for a total of 8 replicate pens per treatment. Pigs were fed a corn-SBM-DDGS control diet that included Cu to meet the requirement. A second diet was formulated by adding 150 mg Cu/kg from copper hydroxychloride to the control diet. On the last day of the experiment, one pig per pen was sacrificed, and samples from liver, skeletal muscle, and subcutaneous adipose tissue were collected to analyze relative mRNA abundance of genes involved in lipid metabolism. Results indicated that overall ADG and G:F were greater (P < 0.05) for pigs fed the diet containing copper hydroxychloride compared with pigs fed the control diet. Pigs fed the diet supplemented with copper hydroxychloride also had increased (P < 0.05) abundance of cluster of differentiation 36 in the liver and increased (P < 0.05) abundance of fatty acid-binding protein 4 and lipoprotein lipase in subcutaneous adipose tissue. Inclusion of copper hydroxychloride also tended to increase (P < 0.10) the abundance of fatty acid-binding protein 1, peroxisome proliferator-activated receptor α, and carnitine palmitoyltransferase 1B in the liver, skeletal muscle, and subcutaneous adipose tissue, respectively. This indicates that dietary Cu may affect signaling pathways associated with lipid metabolism by improving the uptake, transport, and utilization of fatty acids. In conclusion, supplementation of copper hydroxychloride to the control diet improved growth performance and upregulated the abundance of some genes involved in postabsorptive metabolism of lipids.

Identification and Characterization of Thioredoxin H-Type Gene Family in Triticum turgidum ssp. durum in Response to Natural and Environmental Factor-Induced Oxidative Stress

Key message: Thioredoxin h-type isoforms are tissue-specific, differentially expressed in germinating seeds and under salinity stress and highly regulated by H2O2, suggestive of a role in germination and salinity adaptation in durum wheat through redox regulation. Thioredoxins (Trxs) are protein-disulfide reductases that perform multiple functions related to cellular redox homoeostasis. The most complex cluster in the family of plant Trxs is formed by h-type Trxs since their identity and functions are still largely unknown. Here, h-type Trxs from durum wheat (Triticum turgidum ssp. durum) (stated TdTrxh1, TdTrxh2, TdTrxh3, and TdTrxh9) were identified and characterized in response to oxidative stress either generated naturally (germination) or induced by salinity and hydrogen peroxide (H2O2). In silico expression analysis, based on RNA-seq data, revealed a tissue-specific expression for TdTrxh genes. Real-time q-PCR analysis showed differential expression patterns for TdTrxh genes between and within seed tissues during germination and a marked induction of TdTrxh2 and h3 ones. Moreover, the four TdTrxh isoforms were found to be differentially modulated by salinity in two durum wheat varieties contrasting in their salinity tolerance. Particularly, an upregulation of the four TdTrxh genes (mainly TdTrxh2 and h3) was noted in root tissues of the tolerant variety, while they were markedly downregulated in those of the sensitive one. This upregulation of TdTrxh genes in the tolerant variety coincided with an accumulation of H2O2 in root tissues. When exogenously applied, H2O2 increased mRNA transcripts of all TdTrxh genes in both varieties regardless of their salinity-tolerance degree. Collectively, our data suggest a non-redundant function for h-type Trxs besides to be potentially involved in salinity tolerance in durum wheat through redox regulation.

A Phase I/II Study to Investigate the Safety and Clinical Activity of the Protein Arginine Methyltransferase 5 Inhibitor GSK3326595 in Subjects with Myelodysplastic Syndrome and Acute Myeloid Leukemia

A Phase I/II Study to Investigate the Safety and Clinical Activity of the Protein Arginine Methyltransferase 5 Inhibitor GSK3326595 in Subjects with Myelodysplastic Syndrome and Acute Myeloid Leukemia

Effect of boar semen supplementation with recombinant heat shock proteins during summer

Artificial insemination (AI) in pigs is mainly performed with refrigerated boar semen. There is a marked negative seasonal effect on the quality of boar sperm, mainly due to relatively greater ambient temperatures; to counteract this thermal stress, sperm cells possess natural defensive mechanisms such as Heat Shock Proteins (HSPs) that prevent protein denaturation. Thus, the objective of this research was to improve the quality of commercial boar semen collected during the summer when ambient temperatures are greater using recombinant HSPs. For this purpose, different concentrations (0.1, 0.5 and 1 μg/ml) of recombinant heat shock proteins (HSPD1, HSPA8 or HSP86) were added to commercial boar semen and there was cooling for 48 h at 17 °C. After this storage period, sperm quality was assessed by analyzing sperm viability, mitochondrial membrane potential and plasma membrane lipid organization using flow cytometry; additionally, sperm motility was examined using a CASA system. Also, in vitro fertilization (IVF) using HSP-supplemented boar semen was performed and the quality of the embryos produced was evaluated using quantitative real-time polymerase chain reaction (qPCR) analyzing the relative abundance of mRNA transcripts for genes encoding for embryo quality-related proteins (BAX, TFAM, POLG and POG2). Sperm quality variables, blastocyst rates and the abundance of mRNA transcripts for the selected genes were not affected by the presence of recombinant HSPs at any concentration. These results indicate that the supplementation of commercial seminal doses with recombinant HSPs does not improve boar sperm quality or fertility during the summer months when ambient temperatures are greater.

Wheat F-box Protein TaFBA1 Positively Regulates Plant Drought Tolerance but Negatively Regulates Stomatal Closure.

The phytohormone abscisic acid (ABA) regulates plant growth and development, as well as responses to various stresses, such as salt and drought. The wheat TaFBA1 gene, which encodes an F-box protein, was previously identified in our laboratory by homologous cloning. We previously found that TaFBA1 expression was induced by ABA and drought stress. In this study, wild-type (WT), TaFBA1 over-expressing (OEs), TaFBA1 homologous gene mutants, and TaFBA1 recovery (Rs) Arabidopsis plants were used. We found that the germination rate, the cotyledon greening rate, the root length, and the photosynthetic performance of TaFBA1 OE plants were better than those of WT under drought and ABA conditions, but mutant plants showed the opposite trend, and overexpression of TaFBA1 in mutants can recover their phenotype. In addition, TaFBA1 was found to be a negative regulator of ABA-induced stoma movement; mRNA transcription of certain ABA signaling-related genes was lower in TaFBA1 OE plants than in WT plants following ABA treatment. Further, we found that TaFBA1 can interact with RCAR1 (an ABA receptor) and ABI5. BiFC assay showed that TaFBA1 may interact with RCAR1 in the plasma membrane. In addition, accumulation of ROS and MDA in TaFBA1 OE plants was lower than that in the WT plants after ABA and drought treatments. Based on these results, we suggest that TaFBA1-regulated ABA insensitivity may be dependent on regulating ABA-mediated gene expression through interacting with RCAR1 and ABI5. Increased antioxidant competence and decreased ROS accumulation may be an important mechanism that underlies improved drought tolerance in TaFBA1 OE plants.

Neutrophil β-defensin gene expression of postpartum dairy cows is altered by prepartum dietary cation-anion difference

The objective of this study was to evaluate expression of a cluster of genes encoding β-defensin antimicrobial peptides in neutrophils of postpartum cows in relation to prepartum dietary cation-anion difference (DCAD), vitamin D, and postpartum disease. Pregnant dry Holstein cows (28 nulliparous and 51 parous) at 255 d gestation were blocked by parity and randomly assigned to 4 prepartum diets of positive (+130 mEq/kg) or negative (-130 mEq/kg) DCAD and either 3 mg vitamin D3 or 3 mg of 25-hydroxyvitamin D3 per 11 kg of dry matter/d. Treatment diets were fed from 255 d of gestation until calving. Peripheral blood neutrophils of 35 parous cows were collected at 0 and 3 d after calving and stimulated with 0 or 100 ng/mL of lipopolysaccharide (LPS). Furthermore, serum Ca and incidences of postpartum diseases were recorded for all cows. The mRNA transcripts of β-defensin genes were quantified by real-time PCR, and data were analyzed with a general linear mixed model to test for fixed effects and interactions of day, level of DCAD, source of vitamin D, and incidence of disease. Effects of DCAD and vitamin D on neutrophil oxidative burst and phagocytosis were previously reported but were analyzed for effects of disease in the present study. Transcripts for DEFB1, DEFB3, DEFB4, DEFB5, DEFB7, DEFB10, and lingual antimicrobial peptide (LAP) in neutrophils were upregulated by LPS at 0 d but not at 3 d. Transcripts for DEFB4 and DEFB7 in LPS-stimulated neutrophils were greater in cows fed negative DCAD diets compared with positive DCAD. Source of vitamin D (vitamin D3 vs. 25-hydroxyvitamin D3) did not affect expression of β-defensins in neutrophils. Cows with postpartum subclinical hypocalcemia (serum Ca <2.0 mM) had decreased DEFB3, DEFB4, DEFB6, DEFB7, DEFB10, and LAP expression in LPS-stimulated neutrophils compared with cows that did not experience subclinical hypocalcemia. Likewise, DEFB4, DEFB6, DEFB7, DEFB10, and LAP in LPS-stimulated neutrophils at 3 d postpartum were positively associated with serum Ca at 0 d postpartum. Transcripts for DEFB7, DEFB10 and LAP also were less abundant in neutrophils from cows with metritis compared with healthy cows. In conclusion, feeding a prepartum negative DCAD to improve postpartum serum Ca resulted in greater neutrophil β-defensin expression, and greater neutrophil β-defensin expression was positively associated with postpartum health.

Up-regulation of TIMP-3 and RECK decrease the invasion and metastasis ability of colon cancer

Background and study aimsAlthough the function of microRNA21 (miR-21) in the invasion and metastasis of colon cancer has been extensively studied, the mechanisms of invasion and migration related pathways between and its targets are still not elucidated. This study explored the mechanisms of the pathway between miR-21 and the target genes in vitro and in vivo. Materials and methodsWe transfected pmiRZip21 or Leti3 into colon cancer cells. The levels of miR-21 expression, mRNA transcription and protein of target genes were analysed by TaqMan microRNA assays, RT-PCR and western blot, respectively. Scratch migration and trans-well assays were used to evaluate metastasis and invasion. To build a subcutaneous tumour animal model, detect the level of miR-21 and the target genes and then identify the mechanisms in vivo. ResultsMiR-21 expression levels in colon cancer cells transfected with pmiRZip21 in vivo or in vitro were decreased (P < 0.05). The mRNA and protein levels of TIMP-3 and RECK were up-regulated after inhibiting miR-21 in vitro and in vivo (P < 0.05), but those of BMPR-II and PCDH17 were not. In pmiRZip21-transfected colon cancer cells, invasion and migration were significantly decreased both in vitro and vivo (P < 0.05). ConclusionsUp-regulation of TIMP-3 and RECK, by inhibiting miR-21 expression can decrease tumour invasion and metastasis ability in vitro and in vivo, and has potential as a possible target site in anti-tumour therapy. More effects in vivo have to be investigated in further research.

Nuclear transcriptional changes in hypothalamus of Pomc enhancer knockout mice after excessive alcohol drinking.

Persistent alterations of proopiomelanocortin (Pomc) and mu-opioid receptor (Oprm1) activity and stress responses after alcohol are critically involved in vulnerability to alcohol dependency. Gene transcriptional regulation altered by alcohol may play important roles. Mice with genome-wide deletion of neuronal Pomc enhancer1 (nPE1-/- ), had hypothalamic-specific partial reductions of beta-endorphin and displayed lower alcohol consumption, compared to wildtype littermates (nPE1+/+ ). We used RNA-Seq to measure steady-state nuclear mRNA transcripts of opioid and stress genes in hypothalamus of nPE1+/+ and nPE1-/- mice after 1-day acute withdrawal from chronic excessive alcohol drinking or after water. nPE1-/- had lower basal Pomc and Pdyn (prodynorphin) levels compared to nPE1+/+ , coupled with increased basal Oprm1 and Oprk1 (kappa-opioid receptor) levels, and low alcohol drinking increased Pomc and Pdyn to the basal levels of nPE1+/+ in the water group, without significant effects on Oprm1 and Oprk1. In nPE1+/+ , excessive alcohol intake increased Pomc and Oprm1, with no effect on Pdyn or Oprk1. For stress genes, nPE1-/- had lowered basal Oxt (oxytocin) and Avp (arginine vasopressin) that were restored by low alcohol intake to basal levels of nPE1+/+ . In nPE1+/+ , excessive alcohol intake decreased Oxt and Avpi1 (AVP-induced protein1). Functionally examining the effect of pharmacological blockade of mu-opioid receptor, we found that naltrexone reduced excessive alcohol intake in nPE1+/+ , but not nPE1-/- . Our results provide evidence relevant to the transcriptional profiling of the critical genes in mouse hypothalamus: enhanced opioid and reduced stress gene transcripts after acute withdrawal from excessive alcohol may contribute to altered reward and stress responses.

Quantitative PCR primer design affects quantification of dsRNA-mediated gene knockdown.

RNA interference (RNAi) is a powerful tool for studying functions of candidate genes in both model and nonmodel organisms and a promising technique for therapeutic applications. Successful application of this technique relies on the accuracy and reliability of methods used to quantify gene knockdown. With the limitation in the availability of antibodies for detecting proteins, quantitative PCR (qPCR) remains the preferred method for quantifying target gene knockdown after dsRNA treatment. We evaluated how qPCR primer binding site and target gene expression levels affect quantification of intact mRNA transcripts following dsRNA‐mediated RNAi. The use of primer pairs targeting the mRNA sequence within the dsRNA target region failed to reveal a significant decrease in target mRNA transcripts for genes with low expression levels, but not for a highly expressed gene. By contrast, significant knockdown was detected in all cases with primer pairs targeting the mRNA sequence extending beyond the dsRNA target region, regardless of the expression levels of the target gene. Our results suggest that at least for genes with low expression levels, quantifying the efficiency of dsRNA‐mediated RNAi with primers amplifying sequences completely contained in the dsRNA target region should be avoided due to the risk of false‐negative results. Instead, primer pairs extending beyond the dsRNA target region of the mRNA transcript sequences should be used for accurate and reliable quantification of silencing efficiency.

Maintenance of translational elongation rate underlies the survival of Escherichia coli during oxidative stress.

To cope with harsh circumstances, bacterial cells must initiate cellular stress response programs, which demands the de novo synthesis of many stress defense proteins. Reactive oxygen species (ROS) is a universal environmental stressor for both prokaryotic cells and eukaryotic cells. However, the physiological burden that limits the survival of bacterial cells during oxidative stress remains elusive. Here we quantitatively characterize the cell growth and translational elongation rate of Escherichia coli cells treated with different doses of hydrogen peroxide. Cell growth is immediately arrested by low to moderate levels of hydrogen peroxide, but completely recovers after a certain lag time. The lag time depends positively on the dose of hydrogen peroxide. During the lag time, translational elongation rate drops by as much as ∼90% at initial stage and recovers to its normal state later, a phenomenon resulting from the dramatic alteration in cellular tRNA pools during oxidative stress. However, translational elongation is completely stalled at a certain threshold-level of hydrogen peroxide, at which cells ultimately fail to resume growth. Although the mRNA transcription of oxidative defense genes in oxyR regulon is dramatically induced upon hydrogen peroxide treatment, the extreme slow-down of translational elongation during high levels of hydrogen peroxide has severely compromised the timely synthesis of those oxidative defense proteins. Our study demonstrates that the tRNA-limited translational elongation is a key physiological bottleneck that the bacteria must overcome to counteract ROS, and the maintenance of translational elongation rate for timely synthesis of stress defense proteins is crucial for cells to smoothly get over the oxidative stress.

Optimum salinity for Nile tilapia (Oreochromis niloticus) growth and mRNA transcripts of ion-regulation, inflammatory, stress- and immune-related genes.

We aim to study the optimum salinity concentration for Nile tilapia, through the assessment of its growth performance and the expression of its related genes (Gh and Igf-1), as well as its salinity adaptation and immune status through the assessment of the gene expression of ion-regulation genes (Na+/K+-ATPase α-1a and α-1b), stress-related genes (GST, HSP27, and HSP70), inflammatory-related genes (IL1, IL8, CC, and CXC chemokine), and immune-related genes (IgMH TLR7, MHC, and MX) at the osmoregulatory organs (gills, liver, and kidney). Based on the least mortality percentage and the physical appearance of the fish, three salt concentrations (6, 16, and 20ppt) were chosen following a 6-month preliminary study using serial salt concentrations ranged from 6 to 36ppt, which were obtained by rearing the fish in gradual elevated pond salinity through daily addition of 0.5ppt saline water. The fish size was 10.2-12cm and weight was 25.5-26.15g. No significant differences in the fish weight gain were observed among the studied groups. The group reared at 16-ppt salt showed better performance than that of 20ppt, as they have lower morality % and higher expression of ion-regulated gene (Na+/K+-ATPase α1-b), stress-related genes (GST, HSP27, and HSP70) of the gills and also GST, inflammatory-related genes (IL-1β and IL8), and TLR in the liver tissue. Higher expression of kidney-immune-related genes at 20-ppt salt may indicate that higher salinity predispose to fish infection and increased mortality. We concluded that 16-ppt salinity concentration is suitable for rearing O. niloticus as the fish are more adaptive to salinity condition without changes in their growth rate. Also, we indicate the use of immune stimulant feed additive to overcome the immune suppressive effect of hyper-salinity. Additionally, the survival of some fish at higher salinity concentrations (30-34ppt) increase the chance for selection for salinity resistance in the Nile tilapia.