mRNA Transcript Levels Of Genes Research Articles

A method for reducing the concentrations of Fusarium graminearum trichothecenes in durum wheat grain with the use of Debaryomyces hansenii

Fusarium head blight (FHB), caused mainly by Fusarium graminearum, is one of the most dangerous diseases of durum wheat. This hemibiotrophic pathogen transitions from the biotrophic phase, during which it penetrates host tissues and secretes trichothecenes, to the necrotrophic phase which leads to the destruction of host tissues. Yeasts applied to spikes often reduce mycotoxin concentrations, but the underlying mechanisms have not been fully elucidated. Therefore, the aim of this study was to analyze the concentrations trichothecenes in durum wheat grain and changes in the F. graminearum transcriptome under the influence the Debaryomyces hansenii antagonistic yeast strain. Debaryomyces hansenii cells adhered to and formed cell aggregates/biofilm on the surface of spikes and pathogenic hyphae. Biological control suppressed the spread of F. graminearum by 90 % and decreased the content of deoxynivalenol (DON) in spikes by 31.2 %. Yeasts significantly reduced the expression of pathogen's genes encoding the rpaI subunit of RNA polymerase I and the activator of Hsp90 ATPase, but they had no effect on mRNA transcript levels of genes encoding the enzymes involved in the biosynthesis of trichothecenes. The yeast treatment reduced the number of F. graminearum operational taxonomic units (OTUs) nearly five-fold and increased the number of D. hansenii OTUs more than six-fold in the spike mycobiome. The mechanisms that suppress infections should be explored to develop effective biological methods for reducing the concentrations mycotoxins in wheat grain.

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.

Dose-response effects of light at night on the reproductive physiology of great tits (Parus major): Integrating morphological analyses with candidate gene expression.

Artificial light at night (ALAN) is increasingly recognized as a potential threat to wildlife and ecosystem health. Among the ecological effects of ALAN, changes in reproductive timing are frequently reported, but the mechanisms underlying this relationship are still poorly understood. Here, we experimentally investigated these mechanisms by assessing dose‐dependent photoperiodic responses to ALAN in the great tit (Parus major). We individually exposed photosensitive male birds to one of three nocturnal light levels (0.5, 1.5, and 5 lux), or to a dark control. Subsequent histological and molecular analyses on their testes indicated a dose‐dependent reproductive response to ALAN. Specifically, different stages of gonadal growth were activated after exposure to different levels of light at night. mRNA transcript levels of genes linked to the development of germ cells (stra8 and spo11) were increased under 0.5 lux compared to the dark control. The 0.5 and 1.5 lux groups showed slight increases in testis size and transcript levels associated with steroid synthesis (lhr and hsd3b1) and spermatogenesis (fshr, wt1, sox9, and cldn11), although spermatogenesis was not detected in histological analysis. In contrast, all birds under 5 lux had 10 to 30 times larger testes than birds in all other groups, with a parallel strong increase in mRNA transcript levels and clear signs of spermatogenesis. Across treatments, the volume of the testes was generally a good predictor of testicular transcript levels. Overall, our findings indicate that even small changes in nocturnal light intensity can increase, or decrease, effects on the reproductive physiology of wild organisms.

Dietary methionine supplementation alters the expression of genes involved in methionine metabolism in salmonids

The objective of the present study was to investigate the effect of dietary methionine (Met) concentration and alternating feeding strategies in methionine delivery, on the mRNA transcript levels of genes involved in Met resynthesis (betaine-homocysteine methyltransferase, BHMT; S-adenosylhomocysteine hydrolase, SAHH) and net Met loss (taurine synthesis) (cystathionine beta-synthase, CBS) in Atlantic salmon (Salmo salar) liver. Salmon alevins (265 ± 3 mg) were distributed into 24 tanks (50 fish per tank; 3 replicates). The experimental diets were supplemented with l-methionine at 0, 1.9, 5.8, and 17.4 g/kg (M0, M1/3, M1, and M3, respectively). The M3 diet without glycine was prepared to examine Met toxicity (M3-G). These diets were provided via “mono-feeding strategy,” meaning fish were fed a designated single diet. This experiment also included alternative feeding groups with “duo-feeding” strategy: AF1 (fish fed M0 for 2 days followed by M1 for 1 day), AF2 (fish fed M0 for 2 days followed by M3 for 1 day), and AF3 (fish fed two meals of M0 followed by one meal of M3). Salmon fed M0 diet had smaller weight compared to all other groups. There was no effect of alternative feeding on the growth except with the M0 group. The highest expression of CBS gene was found in the M0, M1/3, and AF-1 groups compared to the M3 and M3-G groups. The expression of CBS gene in the M1 group was lower compared to the AF-1 group. The expression of SAHH gene was the highest in the M3 group compared to the M1, M1/3, and AF-3 groups. The highest expression of BHMT gene was found in the M0, M1/3, and AF-1 groups, indicating enhanced re-methylation of homocysteine by betaine to Met. The lowest BHMT expression in M1, M3, M3-G, AF-2, and AF-3 groups compared to other treatments can be indicative of downregulation of remethylation in the liver. The alternate provision of methionine in a form of combination of low and high methionine containing diets seemed to support its efficient utilization in the salmon liver.

Craniofacial abnormalities and altered wnt and mmp mRNA expression in zebrafish embryos exposed to gasoline oxygenates ETBE and TAME

Gasoline additives ethyl tert butyl ether (ETBE) and tertiary amyl methyl ether (TAME) are used world wide, but the consequence of developmental exposure to these hydrophilic chemicals is unknown for aquatic vertebrates. The effect of ETBE and TAME on zebrafish embryos was determined following OECD 212 guidelines, and their toxicity was compared to structurally related methyl tert-butyl ether (MTBE), which is known to target developing vasculature. LC50s for ETBE and TAME were 14mM [95% CI=10–20] and 10mM [CI=8–12.5], respectively. Both chemicals caused dose dependent developmental lesions (0.625–10mM), which included pericardial edema, abnormal vascular development, whole body edema, and craniofacial abnormalities. The lesions were suggestive of a dysregulation of WNT ligands and matrix metalloproteinase (MMP) protein families based on their roles in development. Exposure to 5mM ETBE significantly (p≤0.05) decreased relative mRNA transcript levels of mmp-9 and wnt3a, while 2.5 and 5mM TAME significantly decreased wnt3a, and wnt8a. TAME also significantly decreased mmp-2 and -9 mRNA levels at 5mM. ETBE and TAME were less effective in altering the expression of vascular endothelial growth factor-a and -c, which were the only genes tested that were significantly decreased by MTBE. This is the first study to characterize the aquatic developmental toxicity following embryonic exposure to ETBE and TAME. Unlike MTBE, which specifically targets angiogenesis, ETBE and TAME disrupt multiple organ systems and significantly alter the mRNA transcript levels of genes required for general development.

Detection of ETV6 gene rearrangements in adult acute lymphoblastic leukemia

ETV6 is an important hematopoietic regulatory factor and ETV6 gene rearrangement is involved in a wide variety of hematological malignancies. In this study, we sought to investigate the incidence of ETV6-associated fusion genes in B- and T-lineage acute lymphoblastic leukemia (ALL) by multiplex-nested reverse transcription-polymerase chain reaction (RT-PCR) in 176 adult ALL patients. Total RNA was extracted from bone marrow samples of ALL patients including 136 B- and 40 T-lineage ALL, and ETV6 fusion genes were detected by multiplex-nested RT-PCR. Changes of ETV6 fusion gene mRNA transcript levels were examined by real-time RT-PCR. We detected a total of 15 ETV6 gene rearrangements with a positive rate of 8.5%, involving seven ETV6-associated fusion genes in 13 B-ALL (13/136, 9.6%) and 2 T-ALL patients (2/40, 5.0%). ETV6-RUNX1 were observed in six cases (3.4%), ETV6-JAK2 in three cases (1.7%), ETV6-ABL1 in two cases (1.1%), and ETV6-ABL2, ETV6-NCOA2, ETV6-SYK, and PAX5-ETV6 each in one case (0.6%). ETV6-JAK2 was found in both B-ALL and T-ALL patients. Furthermore, real-time quantitative RT-PCR assays showed that the ETV6-RUNX1 mRNA transcript levels decreased during conventional chemotherapy or hematopoietic stem cell transplantation. This study shows that multiplex-nested RT-PCR is an effective and accurate tool to identify ETV6 rearrangements in adult ALL, which provides some clues into the diagnosis and prognosis of ALL but also molecular markers for the detection of minimal residual disease in adult ALL.

IUGR decreases elastin mRNA expression in the developing rat lung and alters elastin content and lung compliance in the mature rat lung

Complications of intrauterine growth restriction (IUGR) include increased pulmonary morbidities and impaired alveolar development. Normal alveolar development depends upon elastin expression and processing, as well as the formation and deposition of elastic fibers. This is true of the human and rat. In this study, we hypothesized that uteroplacental insufficiency (UPI)-induced IUGR decreases mRNA levels of elastin and genes required for elastin fiber synthesis and assembly, at birth (prealveolarization) and postnatal day 7 (midalveolarization) in the rat. We further hypothesized that this would be accompanied by reduced elastic fiber deposition and increased static compliance at postnatal day 21 (mature lung). We used a well characterized rat model of IUGR to test these hypotheses. IUGR decreases mRNA transcript levels of genes essential for elastic fiber formation, including elastin, at birth and day 7. In the day 21 lung, IUGR decreases elastic fiber deposition and increases static lung compliance. We conclude that IUGR decreases mRNA transcript levels of elastic fiber synthesis genes, before and during alveolarization leading to a reduced elastic fiber density and increased static lung compliance in the mature lung. We speculate that the mechanism by which IUGR predisposes to pulmonary disease may be via decreased lung elastic fiber deposition.

Using biomarker signature patterns for an mRNA molecular diagnostic of mouse embryonic stem cell differentiation state

BackgroundThe pluripotency and self-renewal capabilities, which define the "stemness" state, of mouse embryonic stem (ES) cells, are usually investigated by functional assays or quantitative measurements of the expression levels of known ES cell markers. Strong correlations between these expression levels and functional assays, particularly at the early stage of cell differentiation, have usually not been observed. An effective molecular diagnostic to properly identify the differentiation state of mouse ES cells, prior to further experimentation, is needed.ResultsA novel molecular pattern recognition procedure has been developed to diagnose the differentiation state of ES cells. This is based on mRNA transcript levels of genes differentially expressed between ES cells and their differentiating progeny. Large publicly available ES cell data sets from various platforms were used to develop and test the diagnostic model. Signature patterns consisting of five gene expression levels achieved high accuracy at determining the cell state (sensitivity and specificity > 97%).ConclusionThe effective ES cell state diagnostic scheme described here can be implemented easily to assist researchers in identifying the differentiation state of their cultures. It also provides a step towards standardization of experiments relying on cells being in the stem cell or differentiating state.

Fluid flow decreases preproendothelin mRNA levels and suppresses endothelin-1 peptide release in cultured human endothelial cells

Endothelin-1, a 21-amino acid peptide secreted by endothelial cells, has constrictor and mitogenic activity for vascular smooth muscle cells, and its mitogenic activity is synergistic with that of platelet-derived growth factor. Endothelial cell-derived endothelin-1 might therefore contribute to intimal hyperplasia in reendothelialized segments of vascular grafts or of endarterectomy and angioplasty sites. Because intimal hyperplasia occurs most often at sites with disordered flow patterns and lower fluid shear stress, we tested the effects of static culture versus high laminar shear stress (25 dyne/cm2) on endothelin-1 precursor (preproendothelin) gene mRNA transcript levels and endothelin-1 peptide release in cultured human endothelial cells. Primary cultures of human umbilical vein endothelial cells were subjected to controlled levels of shear stress in parallel plate flow chambers for 24 hours. To detect preproendothelin mRNA we applied a linked reverse transcriptase-polymerase chain reaction (RT/PCR) to RNA extracted from cultures. Southern blots of RT/PCR reaction products were hybridized with radioactive phosphorous (32P) labeled probes for the amplified preproendothelin complementary deoxyribonucleic acid (cDNA). Detection by RT/PCR of mRNA for glyceraldehyde 3-phosphate dehydrogenase was used to measure a constitutively expressed control signal. Endothelin-1 release into culture medium was measured by radioimmunoassay. Application of 25 dyne/cm2 of shear stress for 24 hours sharply reduced endothelial cell levels of precursor preproendothelin mRNA. High shear stress also decreased endothelin-1 peptide release rates into culture medium from 0.55 ± 0.02 and 0.49 ± 0.03 ng/106 endothelial cells/hour (n = 3 replicate cultures in each experiment) (static conditions) to values of 0.13 ± 0.03 and 0.28 ± 0.06 ng/106 cells/hour (shear stress) (p < 0.01). The flow-induced suppression of preproendothelin gene expression suggests that endothelin-1 release from endothelial cells might help mediate the inverse relationship between local fluid shear rates and the severity of neointimal hyperplasia.

Down-regulation of cystic fibrosis gene mRNA transcript levels and induction of the cystic fibrosis chloride secretory phenotype in epithelial cells by phorbol ester

To evaluate the hypothesis that phorbol myristate acetate (PMA) might modulate the expression of the cystic fibrosis (CF) gene in epithelial cells, we examined the effect of PMA on CF mRNA levels and regulation of Cl- secretion. Strikingly, PMA down-regulated CF mRNA transcript numbers in a dose- and time-dependent manner. Importantly, in parallel with the reduction of CF mRNA levels, PMA-treated cells were unable to up-regulate Cl- secretion in a normal fashion in response to forskolin, an effect which was also dose- and time-dependent. Thus, PMA is capable of modulating expression of the CF gene and induces T84 cells to adopt the "CF phenotype" in regard to regulation of Cl- ion transport.