Transcript Levels Of Target Genes Research Articles

Regulatory roles of introns in fungicide sensitivity of Fusarium graminearum.

Although the roles of introns have been much debated in eukaryotic organisms, none of them have been functionally characterized in Fusarium graminearum. In this study, we characterized the roles of introns in regulation of fungicide-sensitivity of F. graminearum. β2 tub, cyp51A and myosin-5 are important target genes of benzimidazoles, triazoles and cyanoacrylates respectively. To explore the sensitivity regulation functions of introns in target genes, several detailed deletion studies were completed on the intronic regions of β2 tub, cyp51A and myosin-5. Phenotypic analyses showed that deletion of the fourth intron from β2 tub gene (designated β2 Δi4), the sole intron from cyp51A gene (cyp51A-Δi) and the second intron from myosin-5 gene (myo5-Δi2) exhibited an increased sensitivity to corresponding fungicides. In contrast, deletion of the first or second intron from β2 tub gene exhibited a decreased sensitivity to carbendazim. qRT-PCR showed that the mRNA transcript levels of target genes were significantly downregulated in β2 Δi4, cyp51A-Δi and myo5-Δi2 respectively. Meanwhile, Western blot assays revealed that the protein expression levels of β2 tub was also dramatically reduced in β2 Δi4, but accumulated in β2 Δi1 and β2 Δi2. Overall, our results indicate that introns in target genes significantly regulate the fungicide-sensitivity by influencing expression of the corresponding resident genes in F. graminearum.

Double-Stranded RNA-Mediated Suppression of Trypsin-Like Serine Protease (t-SP) Triggers Over-Expression of Another t-SP Isoform in Helicoverpa armigera.

High diversity of digestive proteases is considered to be the key factor in the evolution of polyphagy in Helicoverpa armigera. Serine proteases (SPs) contribute ~85% of the dietary protein digestion in H. armigera. We investigated the dynamics of SP regulation in the polyphagous pest, H. armigera using RNA interference (RNAi). HaTry1, an isoform of SP, expressed irrespective of the composition of the diet, and its expression levels were directly proportional to the larval growth rate. Therefore, HaTry1 was silenced by delivering 10 and 20μg concentrations of double-stranded RNA through semi-synthetic diet. This led to a drastic reduction in the target gene transcript levels that manifested in a significant reduction in the larval weight initially, but the larvae recovered in later stages despite continuous dsRNA treatment. This was probably due to the compensatory effect by over-expression of HaTry13 (31-folds), another isoform of SP. Phylogenetic analysis of H. armigera SPs revealed that the over-expressed isoform was closely related to the target gene as compared to the other tested isoforms. Further, silencing of both the isoforms (HaTry1 and HaTry13) caused the highest reduction in the larval weight and there was no larval growth recovery. These findings provide a new evidence of the existence of compensatory effect to overcome the effect of silencing individual gene with RNAi. Hence, the study emphasizes the need for simultaneous silencing of multiple isoforms.

Molecular pathways related to the longevity promotion and cognitive improvement of Cistanche tubulosa in Drosophila

Background: The aging process, including physical dysfunction and age-related memory impairment (AMI), are considered to be correlated with cumulative oxidative damages and insulin/IGF-1 signaling pathway.Purpose: The present study was to elucidate the in vivo effects on delaying aging and ameliorating AMI and underlying molecular mechanisms of Cistanche tubulosa (CT), a herb used in traditional Chinese medicine to improve sexual function and treat kidney dysfunction.Methods: The flies, treated and untreated with CT, were observed for lifespan, resistance to oxidative stress with H2O2 or paraquat, starvation assay, cognitive behaviors with T-maze, and transcript levels of target genes with quantitative RT-PCR.Results: Administering CT extended the mean and maximum lifespan and increased resistance to oxidative stress in flies. CT supplementation also enhanced memory formation in young flies and suppressed AMI upon aging. Several genes and signaling pathways, including the mechanistic target of rapamycin (mTOR) and Notch networks, have been identified as causing these pharmacological effects and alterations in the gene expression of glutamate receptors.Conclusion: Our results indicate that CT supplementation may contribute to slowing aging phenotypes and alleviating cognitive behavioral decline in flies, indicating potential applicability for enhancing human health and reducing susceptibility to age-related disorders.

Alteration in molecular markers of oocyte development and intersex condition in mullets impacted by wastewater treatment plant effluents

Wastewater Treatment Plant (WWTP) discharges are an important source of endocrine disrupting chemicals (EDCs) into the aquatic environment. Fish populations inhabiting downstream of WWTP effluents show alterations in gonad and gamete development such as intersex condition, together with xenoestrogenic effects such as vitellogenin up-regulation. However, the molecular mechanisms participating in the development of intersex condition in fish are not elucidated. The aim of this study was to assess the impact of two WWTPs effluents (Gernika and Bilbao-Galindo situated in the South East Bay of Biscay) with different contaminant loads, in thicklip grey mullet (Chelon labrosus) populations inhabiting downstream, examining the presence and severity of intersex condition, during two seasons. Molecular markers of xenoestrogenicity and oocyte differentiation and development (vtgAa, cyp19a1a, cyp19a1b, cyp11b, foxl2, dmrt1 and gtf3a) were also studied. Intersex mullets were identified downstream of both WWTPs and vtgAa was upregulated in intersex and non intersex males. Sex dependent differential transcription levels of target genes were detected in mullets from Galindo. However, no such pattern was observed in mullets from Gernika, suggesting an attenuating effect over studied genes caused by a higher presence of EDCs in this site, as indicated by the elevated prevalence of intersex mullets in this population. In conclusion, no direct association between xenoestrogenic responses and intersex condition was established. Mullets from Gernika showed signs of severe EDC exposure compared to those from Galindo, as demonstrated by the higher prevalence of intersex males and the reduction in transcription profile differences between sexes of gametogenic gene markers.

Enhanced 1-Butanol Production in Engineered Klebsiella pneumoniae by NADH Regeneration

1-Butanol, as a next-generation biofuel, is an important target product of biorefinery research. With the introduction of the CoA-dependent pathway or Ehrlich pathway, many engineered strains have been developed to produce 1-butanol, although cofactor imbalance occurred in engineered strains by the introduction of the 1-butanol synthesis pathway. Several studies have been performed to regenerate NADH by overexpressing NAD+-dependent enzymes. However, the significant role of cofactor regeneration in 1-butanol production and the transcription level of the target genes have seldom been studied. The 1-butanol producer, recombinant Klebsiella pneumoniae (KLA), was constructed by overexpressing the genes kivd, leuABCD, and adhE1 under the control of tac promoter in this study, and several NADH regeneration strategies were adopted to solve the problem of NADH imbalance, including the introduction of NAD+-dependent enzymes (formate dehydrogenase, pyridine nucleotide transhydrogenase, and glucose dehydrogenase) or elimination of the NADH competition pathway (1,3-propanediol synthesis). The resultant NADH/NAD+ ratio, 1-butanol production, and transcription levels have been significantly affected. In comparison to the wild-type strain, the NADH/NAD+ ratio in the reengineered strains was increased by 78–135%, and the transcript levels of target genes have been obviously interfered. Moreover, the resultant 1-butanol titer was increased by 83–114% in comparison to KLA.

Non-classical gluconeogenesis-dependent glucose metabolism in Rhipicephalus microplus embryonic cell line BME26.

In this work we evaluated several genes involved in gluconeogenesis, glycolysis and glycogen metabolism, the major pathways for carbohydrate catabolism and anabolism, in the BME26 Rhipicephalus microplus embryonic cell line. Genetic and catalytic control of the genes and enzymes associated with these pathways are modulated by alterations in energy resource availability (primarily glucose). BME26 cells in media were investigated using three different glucose concentrations, and changes in the transcription levels of target genes in response to carbohydrate utilization were assessed. The results indicate that several genes, such as glycogen synthase (GS), glycogen synthase kinase 3 (GSK3), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6 phosphatase (GP) displayed mutual regulation in response to glucose treatment. Surprisingly, the transcription of gluconeogenic enzymes was found to increase alongside that of glycolytic enzymes, especially pyruvate kinase, with high glucose treatment. In addition, RNAi data from this study revealed that the transcription of gluconeogenic genes in BME26 cells is controlled by GSK-3. Collectively, these results improve our understanding of how glucose metabolism is regulated at the genetic level in tick cells.

Keeping qRT-PCR rigorous and biologically relevant.

Because of the importance of differential gene expression in the growth, development, and reproduction of organisms, assays of mRNA transcript levels are widely employed in biological research. The most frequently used assay combines reverse transcription (RT) with quantitative polymerase chain reaction (qPCR) and is known as qRT-PCR (or RT-qPCR). In practice, RT generates a single-stranded cDNA copy of each molecule in an mRNA population, after which a qPCR thermocycler is used in conjunction with gene-specific oligonucleotide primers to amplify selected target cDNAs (Bustin et al. 2009). Fluorescent probes allow progress to be monitored through three phases: an initial phase during which the increasing signal cannot be distinguished from background noise, a 2nd phase of observable exponential signal increase, and a third and final phase of slowing signal increase as reagents and other factors become rate limiting. Quantification of each target mRNA requires the determination of the number of cycles (known as Ct, Cq, Cp, or TOP) required to reach a pre-set fluorescence threshold during the second phase. When a transcript is twice as abundant in one sample as it is in another, the Ct will occur about one cycle earlier for the more abundant transcript. The varying transcript levels of target genes must then be normalized against the stable transcript levels of one or more reference genes present in the same mRNA population. Algorithms are available to validate the stability of these reference genes. With use of appropriate controls, standardization, and normalization, measurements of Ct can lead to reliable estimates of the relative amount of a target transcript in the input mRNA population (Bustin et al. 2009). Despite its complexity, qRT-PCR is rapid and cost effective, and, if sufficient care is taken with the design and execution of experiments, it is reliable and sensitive. There are, however, two trends of concern in the publication of qRT-PCR experiments. The first is that many papers do not contain enough information to judge whether the data are reliable. Bustin et al. (2009) addressed this question by defining ‘‘minimum information for publication of quantitative real-time PCR experiments’’ (MIQE). The second trend of concern is the increasing number of manuscripts that claim to have identified reference genes without comparing them to target genes in a well-defined and relevant biological context. We agree with Bustin et al. (2010), who considered it pointless and misleading to publish such ‘reference gene papers’ as stand-alone reports. In response to these trends, we propose that publishable qRT-PCR manuscripts should satisfy two criteria. First, the design, execution, and reporting of qRT-PCR experiments should address the items listed in the MIQE guidelines. Second, the selection and validation of reference genes should be reported in relation to target gene behavior in a well-defined biological context. Here we discuss the background to these criteria and their implementation relative to plant biology studies. Communicated by Neal Stewart.

Abstract A26: MYCN modifies p53 transcriptional responses to oppose apoptosis and activate cell cycle progression

Abstract Introduction: The MYCN transcription factor plays a central role in neuroblastoma pathogenesis. This aggressive pediatric malignancy is p53 wild type and repression of p53 functions is required for tumorigenesis. MYC family transcription factors activate and repress a larger number of genes involved in cell proliferation, metastasis and tumorigenesis through direct and indirect mechanisms. We hypothesized that MYCN promotes NB tumorigenesis through repression of p53 transcriptional function. Design: To define MYCN mediated alterations in p53 activity, we performed RNA sequencing on NB cells treated with the MDM2 inhibitor, Nutlin 3a, in the presence or absence of MYCN expression (conditional Tet-on system). MYCN3 cells were either uninduced or induced with doxycycline for 16 h followed by treatment for 8 hours with either Nutlin 3a or control. Total RNA was isolated and used for RNA-seq on the HiSeqTM platform (Illumina). Results: Between 6.2 × 107 to 7.6 × 107 sequence tags were obtained for each condition which uniquely mapped onto the human genome reference sequence (HG19) using the PASH algorithm. We normalized the data sets using a genomic distribution (FDR < 0.001). Next, determined fold changes in gene expression in response to p53 activation with high or low MYCN levels using stringent cutoffs of FKM ≥ 1 and log fold change ≥ 1.3. We found total 103 genes whose transcription was increased in response to Nutlin treatment and 161 genes whose transcription was repressed (p value of < 0.05). Comparison of p53 target gene transcript levels under these four conditions (low MYCN / low p53, low MYCN / high p53, high MYCN / low p53 and high MCYN / high p53) revealed that MYCN levels altered the transcriptional response to p53 activation with Nutlin. MYCN inhibited expression of 25 genes activated by p53 and promoted expression of 30 genes repressed by p53 (log fold change cutoff of ≤ 0.5 with a p value of < 0.01). Bioinformatic analysis using DAVID and GSEA further demonstrated that p53 response genes, altered by MYCN, are primarily involved in apoptotic pathways, cell cycle, and mitotic regulation. Next, we used MYCN ChIP-seq and p53 ChIP-seq datasets to evaluate gene promoters for presence of MYCN binding and p53 binding. Remarkably, we found that the promoters of p53 activated genes which were repressed by the presence of MYCN consistently bound both transcription factors, while the p53 repressed genes bound only MYCN and didn't display p53 binding. We have confirmed these ChIP-seq results with ChIP-qPCR for a subset of genes with central roles in tumor suppression and tumorigenicity (e.g. CDKN1A, CHEK1, AEN, MDM2, Notch1). In addition, a subset of these MYCN modulated p53 transcriptional targets is over expressed in tumors from patients with high-risk disease. Conclusions: Over all these data provide the first unbiased, genome-wide evaluation of how MYCN can modify p53 transcriptional function. Our data suggest a mechanism of direct interactions between MYCN and p53 on the promoters of p53 transcriptional targets, acting to inhibit p53 pro-apoptotic pathways. These data identify key components of the p53 transcriptional response altered by MYCN (i.e. apoptosis, mitotic checkpoints and cell cycle) and we propose that reactivation of elements provides a novel therapeutic strategy. Citation Format: Saurabh Agarwal, Kimal Rajapakshe, Cristian Coarfa, Jason Shohet. MYCN modifies p53 transcriptional responses to oppose apoptosis and activate cell cycle progression. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A26.

Dietary manipulation of oncogenic microRNA expression in human rectal mucosa: a randomized trial.

High red meat (HRM) intake is associated with increased colorectal cancer risk, while resistant starch is probably protective. Resistant starch fermentation produces butyrate, which can alter microRNA (miRNA) levels in colorectal cancer cells in vitro; effects of red meat and resistant starch on miRNA expression in vivo were unknown. This study examined whether a HRM diet altered miRNA expression in rectal mucosa tissue of healthy volunteers, and if supplementation with butyrylated resistant starch (HRM+HAMSB) modified this response. In a randomized cross-over design, 23 volunteers undertook four 4-week dietary interventions; an HRM diet (300 g/day lean red meat) and an HRM+HAMSB diet (HRM with 40 g/day butyrylated high amylose maize starch), preceded by an entry diet and separated by a washout. Fecal butyrate increased with the HRM+HAMSB diet. Levels of oncogenic mature miRNAs, including miR17-92 cluster miRNAs and miR21, increased in the rectal mucosa with the HRM diet, whereas the HRM+HAMSB diet restored miR17-92 miRNAs, but not miR21, to baseline levels. Elevated miR17-92 and miR21 in the HRM diet corresponded with increased cell proliferation, and a decrease in miR17-92 target gene transcript levels, including CDKN1A. The oncogenic miR17-92 cluster is differentially regulated by dietary factors that increase or decrease risk for colorectal cancer, and this may explain, at least in part, the respective risk profiles of HRM and resistant starch. These findings support increased resistant starch consumption as a means of reducing risk associated with an HRM diet.

Evaluation of candidate reference genes for gene expression studies in Cymbidium kanran

Quantitative real-time polymerase chain reaction (RT-qPCR) is a preferred method for rapid and reliable quantification of gene expression study. However the transcription levels of target genes may be misinterpreted due to differential expression of the reference gene which was thought to express stably. Thus, a systematic evaluation on the expression stability of nine frequently used reference genes across various tissues (including leaf, root, stem, sepal, peal, lip and gynostemium) and treatments (including phytohormone and stress treatments) in Cymbidium kanran was performed in this study. The results demonstrated that expression stability of the candidate genes varied greatly across different sample pools. Based on geNorm and NormFinder analysis, Tubulin (TUB), Actin (ACT) and Ribosomal protein S3 (RPS3) were considered to be the most suitable reference genes when all 25 samples were taken together. In addition, ACT and TUB were also the suitable internal controls in C. kanran with ABA, NaCl, cold and CuSO4 treatments. RPS3 and TUB exhibited the most stable expression pattern in NAA, 6-BA and GA treatments, while ACT and RNA polymerase beta (RpoB) could be used as the reliable internal control genes under PbAC stress. In different tissues, RPS3 and RpoB could be served as the reliable internal controls, while RpoB and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were ranked top in heat treatment. On the other hand, 18S ribosomal RNA (18S), ribosomal protein S19 (RPS19) and photosystem I P700 apoprotein B (PsaB) performed poorly in expression stability analysis across all sample pools, and they could not be used as internal controls for RT-qPCR studies in C. kanran.

Opposing Activities of DRM and MES-4 Tune Gene Expression and X-Chromosome Repression inCaenorhabditis elegansGerm Cells

During animal development, gene transcription is tuned to tissue-appropriate levels. Here we uncover antagonistic regulation of transcript levels in the germline of Caenorhabditis elegans hermaphrodites. The histone methyltransferase MES-4 (Maternal Effect Sterile-4) marks genes expressed in the germline with methylated lysine on histone H3 (H3K36me) and promotes their transcription; MES-4 also represses genes normally expressed in somatic cells and genes on the X chromosome. The DRM transcription factor complex, named for its Dp/E2F, Retinoblastoma-like, and MuvB subunits, affects germline gene expression and prevents excessive repression of X-chromosome genes. Using genome-scale analyses of germline tissue, we show that common germline-expressed genes are activated by MES-4 and repressed by DRM, and that MES-4 and DRM co-bind many germline-expressed genes. Reciprocally, MES-4 represses and DRM activates a set of autosomal soma-expressed genes and overall X-chromosome gene expression. Mutations in mes-4 and the DRM subunit lin-54 oppositely skew the transcript levels of their common targets and cause sterility. A double mutant restores target gene transcript levels closer to wild type, and the concomitant loss of lin-54 suppresses the severe germline proliferation defect observed in mes-4 single mutants. Together, “yin-yang” regulation by MES-4 and DRM ensures transcript levels appropriate for germ-cell function, elicits robust but not excessive dampening of X-chromosome-wide transcription, and may poise genes for future expression changes. Our study reveals that conserved transcriptional regulators implicated in development and cancer counteract each other to fine-tune transcript dosage.

Increased Expression Levels of Apoptosis Inhibitor BIRC5 Is Associated with Advanced Oropharyngeal Squamous Cell Carcinoma

Objectives: 1) Describe the role that BIRC5 and related activator genes (AURKB and CDCA8) play in various malignancies, and analyze the role that these genes play in oropharyngeal squamous cell carcinoma. 2) Implement innovative gene analysis technology (mRNA Seq) to determine gene expression and validate using established techniques (nCounter). 3) Analyze gene expression in the setting of human papillomavirus (HPV) status, smoking, and tumor stage. Methods: Tumor and adjacent normal oropharyngeal mucosal samples were obtained for validation studies from 24 patients undergoing surgical treatment of oropharyngeal squamous cell carcinoma (OPSCC) from 2010-2012. Transcriptome data (mRNA-Seq) were analyzed from patient matched tumor-normal tissues. Transcript levels of target genes were validated using the nCounter system. Expression levels were correlated with tumor stage, HPV infection status, or smoking status. Results: A statistically significant overexpression was noted in all 3 target genes in tumor versus normal tissue. Statistically significant associations were present between expression levels of target genes and advanced tumor stage. No significant association was found between the 3 genes and etiologic subgroupings (HPV latent versus active infections or smoking status). Conclusions: BIRC5 and related activator genes (AURKB and CDCA8) have an established role in the development and progression of other cancers. Expression of these 3 gene targets is related to tumor stage in oropharyngeal SCC. The significant associations between the expression of these genes and tumor staging suggest that these genes may play a role in tumor progression independent of HPV viral expression patterns.

Comparative analysis of the efficiencies of siRNA and amiRNA gene silencing of the tobacco lycopene cyclase gene (NsCrtL-1)

SummarySmall interfering RNAs (siRNA) and artificial microRNAs (amiRNA) are widely used for gene silencing in plants, but comparisons of their efficiencies at silencing the same gene have rarely been reported. Both RNAi methods were targeted at the lycopene cyclase gene (NsCrtL-1) in Nicotiana sylvestris. Two amiRNA vectors were constructed, one of which perfectly matched the target gene, while the other contained two nucleotide mismatches. Three vectors (one siRNA vector and two amiRNA vectors) were used to transform tobacco, and the abundances of NsCrtL-1 gene transcripts in the transgenic tobacco plants were determined by real-time reverse-transcription quantitative PCR (RT-qPCR). The results showed that siRNA had the greater silencing efficiency, with target gene transcript levels declining to 9.7% of those in non-transformed, wild-type plants. The gene silencing efficiencies of the two amiRNA vectors were similar. Target gene transcript levels in amiRNA-transgenic plants were reduced to approx. 20% of those in non-transformed, wild-type plants. HPLC was used to measure lycopene concentrations following NsCrtL-1 gene suppression in the three different transgenic plants.The mean lycopene concentration in wild-type control plants was 3.36 µg g–1 fresh weight (FW), while in siRNA-transgenic plants it increased to 7.37 µg g–1 FW. In the amiRNA-transgenic plants, mean lycopene concentrations increased to 5.23 µg g–1 FW and 5.82 µg g–1 FW for the amiRNA constructs with a perfect or an imperfect target sequence match, respectively. These results indicate a higher efficiency of gene silencing when the siRNA approach was used compared to the amiRNA approach. In the amiRNA constructs, two mismatches between the mature miRNA and the target gene sequence had no significant effect on the efficiency of gene silencing.

FHL2 Binds Calcineurin and Represses Pathological Cardiac Growth

Stress-induced hypertrophic growth of the heart predisposes the heart to arrhythmia, contractile dysfunction, and clinical heart failure. FHL2 (four-and-a-half LIM domain protein 2) is expressed predominantly in the heart, and inactivation of the gene coding for FHL2 leads to exaggerated responsiveness to adrenergic stress. Activation of calcineurin occurs downstream of β-adrenergic signaling and is required for isoproterenol-induced myocardial hypertrophy. Based on these facts, we hypothesized that FHL2 suppresses stress-induced activation of calcineurin. FHL2 is upregulated in mouse hearts exposed to isoproterenol, a β-adrenergic agonist, and isoproterenol-induced increases in the NFAT target genes RCAN1.4 and BNP were amplified significantly in FHL2 knockout (FHL2(-/-)) mice compared with levels in wild-type (WT) mice. To determine whether the effect of FHL2 on NFAT target gene transcript levels occurred at the level of transcription, HEK 293 cells and neonatal rat ventricular myocytes (NRVMs) were transfected with a luciferase reporter construct harboring the NFAT-dependent promoters of either RCAN1 or interleukin 2 (IL-2). Consistent with the in vivo data, small interfering RNA (siRNA) knockdown of FHL2 led to increased activation of these promoters by constitutively active calcineurin or the calcium ionophore ionomycin. Importantly, activation of the RCAN1 promoter by ionomycin, in control and FHL2 knockdown cells, was abolished by the calcineurin inhibitor cyclosporine, confirming the calcineurin dependence of the response. Overexpression of FHL2 inhibited activation of both NFAT reporter constructs. Furthermore, NRVMs overexpressing FHL2 exhibited reduced hypertrophic growth in response to constitutively active calcineurin, as measured by cell cross-sectional area and fetal gene expression. Finally, immunostaining in isolated adult cardiomyocytes revealed colocalization of FHL2 and calcineurin predominantly at the sarcomere and activation of calcineurin by endothelin-1-facilitated interaction between FHL2 and calcineurin. FHL2 is an endogenous, agonist-dependent suppressor of calcineurin.

Hypoxia and Gut Neuroendocrine Cells: The Role of HIF-1α, NfκB and Serotonin Production From Enterochromaffin Cells in Intestinal Ischemia

(p<0.05). Western blot analysis demonstrated elevated protein levels of HIF-1α, pIKKα/β and pNFκB under hypoxic conditions. This was associated with elevated transcripts for Ki67, TPH1, HIF-1α, IKKβ as well as HIF-1α downstream targets Glut1, IGF2 and TFF3 (p<0.05). Targeting NFκB with curcumin (1μM) demonstrated a significant inhibitory effect on pNFκB as well as HIF-1α protein levels (p<0.05) accompanied by decreased transcript levels of target genes. Conclusion: Hypoxia stimulates intestinal neuroendocrine EC cells and increases their viability. Stimulation of both the NFκB and the HIF-1α pathways induces 5-HT production, effects which can be reversed by inhibition with curcumin. As 5-HT plays a pathogenic role in inflammatory conditions, shock and hypoperfusion, targeting the EC cell and 5-HT production may be a therapeutic target.

Transcription of reference genes used for quantitative RT-PCR in Atlantic salmon is affected by viral infection

Relative quantification using RT-qPCR is a widely used method for transcription profiling. Transcript levels of target genes in fish after experimental infection is often reported without documentation of stably transcribed reference genes. We present results demonstrating that transcription of typically used reference genes in Atlantic salmon is not stable during experimental infection with salmon pancreas disease virus (SPDV). Transcript levels 0 to 6 weeks after challenge revealed statistically significant changes between time-points that corresponded with a peak in viral load 3 weeks after challenge. The results emphasize the need for thorough method validation prior to transcriptional studies during viral infections.

The ATAC Acetyltransferase Complex Coordinates MAP Kinases to Regulate JNK Target Genes

In response to extracellular cues, signal transduction activates downstream transcription factors like c-Jun to induce expression of target genes. We demonstrate that the ATAC (Ada two A containing) histone acetyltransferase (HAT) complex serves as a transcriptional cofactor for c-Jun at the Jun N-terminal kinase (JNK) target genes Jra and chickadee. ATAC subunits are required for c-Jun occupancy of these genes and for H4K16 acetylation at the Jra enhancer, promoter, and transcribed sequences. Under conditions of osmotic stress, ATAC colocalizes with c-Jun, recruits the upstream kinases Misshapen, MKK4, and JNK, and suppresses further activation of JNK. Relocalization of these MAPKs and suppression of JNK activation by ATAC are dependent on the CG10238 subunit of ATAC. Thus, ATAC governs the transcriptional response to MAP kinase signaling by serving as both a coactivator of transcription and as a suppressor of upstream signaling.

Modulations du génome exprimé dans le muscle squelettique avec l’entraînement physique

Aims Physical exercise leads to changes in contractile and biochemical properties. Muscle characteristics are mainly explained by the presence of several protein families, which exist as different isoforms. Each of these isoforms is encoded by specific genes. The aim of this paper is to present the current knowledge concerning alterations in muscle gene expression in response of physical exercise. In this paper, we stress the knowledge concerning the involvement of intracellular signaling pathways, which ensure the link between the molecular events related to physical exercise and changes in the transcription levels of target genes. Current knowledge Changes in contractile and metabolic properties of skeletal muscle observed during physical training are now well-known. In contrast, the molecular mechanisms at the origin of changes in gene expression remain to be extensively examined. It has been hypothesized that physical exercise leads to elementary signals that are distinctly sensed by skeletal muscle tissue. These signals will recruit specific intracellular pathways and then account for transcriptional adaptations that are seen in skeletal muscle. It has been suggested that transcriptional regulations during physical training result from the influence of many external and internal factors pooled into four categories. Neuronal factors recruit signaling pathways related to transient changes in intracellular calcium, such as calcineurin, calmodulin-dependent protein kinases and PKC pathways. Mechanical factors modulate specific transcriptional changes through mitrogen-activated protein kinase signaling pathways, especially p38 and extracellular signal-regulated kinase-1/2 pathways. Metabolic signals activate the mitochondrial biogenesis through PGC-1α or oxygen-sensitive genes through the hypoxia inducible factor complex. Hormonal signals will not be discussed in the present paper. Futur prospects Future studies are needed to estimate the role played by each of these signaling pathways to explain the phenotypic changes reported in skeletal according to the physical training characteristics. Moreover, the signaling pathways involved to explain muscle adaptation mostly interact; these interactions need to be examined during several forms of physical training.

Tlx acts as a proangiogenic switch by regulating extracellular assembly of fibronectin matrices in retinal astrocytes

In response to hypoxia, hypoxia-inducible factors act as the primary proangiogenic triggers by regulating transcription levels of target genes, including VEGF. However, little is known about the specific factors that control other components of the angiogenic process, particularly formation of matrix scaffolds that promote adhesion and migration of endothelial cells. We show that in the postnatal mouse retina, the orphan nuclear receptor tailless (Tlx) is strongly expressed in the proangiogenic astrocytes, which secrete VEGF and fibronectin. Tlx expression by retinal astrocytes is controlled by oxygen concentration and rapidly downregulated upon contact with blood vessels. In mice null for Tlx, retinal astrocytes maintain VEGF expression; however, the extracellular assembly of fibronectin matrices by astrocytes is severely impaired, leading to defective scaffold formation and a complete failure of normal retinal vascular development. This work identifies Tlx as an essential component of the molecular network involved in the hypoxia-inducible proangiogenic switch in retinal astrocytes.

Coordinated changes in mitochondrial function and biogenesis in healthy and diseased human skeletal muscle

We examined the transcriptional signaling cascade involved in the changes of mitochondrial biogenesis and mitochondrial function of skeletal muscle and of the exercise capacity of humans in response to long-term physical activity and chronic heart failure (CHF). Biopsy samples of vastus lateralis muscle were obtained from 18 healthy subjects with different fitness levels (assessed by maximal oxygen uptake, VO2 peak). We compared 9 sedentary subjects with 10 CHF patients undergoing transplantation. Muscle oxidative capacity was measured in permeabilized fibers (Vmax). Transcript levels of target genes were quantified by RT-PCR. In healthy subjects, VO2 peak was linearly related to Vmax (P<0.01) and to the gene expression of mitochondrial proteins and of the coactivator PGC-1alpha and its downstream transcription factors. A coordinate increase in PGC-1alpha and mRNA levels of proteins involved in degradation, fusion, and fission of mitochondria was observed associated with calcineurin activation. Despite decreased VO2 peak, in CHF patients skeletal muscles showed preserved Vmax in accordance with preserved markers and transcription factors of mitochondrial biogenesis and dynamics, with no calcineurin activation. The results provide strong support for a central role for PGC-1alpha and calcineurin activation in mitochondrial biogenesis in healthy and diseased human skeletal muscles.