Gel express: a novel frugal method quantifies gene relative expression in conventional RT-PCR

Background Real-time PCR system is a valuable scientific mainstream needed for quantifying specific gene expression. Nevertheless, compared with conventional PCR, the real-time PCR system is extremely expensive and not affordable for limited or mid-budget research laboratories. Here, a novel, doable and low-cost recipe (referred to as gel express) is developed to quantify gene expression using conventional RT-PCR assay. The novelty of the gel express method is based on replacing crossing point (CP) values with integrated density (IntDen) values of PCR amplicon bands in real-time PCR regular mathematical formulas. Results In this work, gene expression profiles of two different rice stress-marker genes (OsCYP94C2a and OsLOX8) were quantified in response to mechanical wounding at different time points (0, 30, 60, and 150 min). In the gel express method, the free software ImageJ was employed to measure integrated density (IntDen) values of PCR amplicon bands in agarose gel images. IntDen values were then used instead of crossing point (CP) values according to the following modified formula: [EIntDen(ref)/EIntDen(target)]sample ÷ [EIntDen(ref)/EIntDen(target)]control. Gene relative expression profiles (dynamic expression pattern) quantified by gel express method in both genes were highly comparable with real-time RT-PCR. R2 values were 0.9976 and 0.9975 in OsCYP94C2a and OsLOX, respectively. PCR amplification efficiency (E) for all studied genes could be calculated depending on IntDen values through experimentally designed calibration curves. PCR amplification efficiencies with all studied genes obtained by gel express were all in the accepted range. For better-visualized PCR amplicons thus detectable biological effects between treatments, the number of PCR cycles applied in gel express method (IntCyc) was experimentally estimated to be 29 cycles. Conclusions Gel express is a novel, cost-effective and feasible recipe for quantifying gene relative expression in conventional RT-PCR. The expression pattern quantified by gel express is highly comparable and fits the expression data revealed by the used real-time PCR system.

Genome-wide identification and functional analysis of circRNAs in Trichophyton rubrum conidial and mycelial stages

Background Circular RNAs (circRNAs) are a group of noncoding RNAs that participate in gene expression regulation in various pathways. The essential roles of circRNAs have been revealed in many species. However, knowledge of circRNAs in fungi is still not comprehensive. Results Trichophyton rubrum (T. rubrum) is considered a model organism of human pathogenic filamentous fungi and dermatophytes. In this study, we performed a genome-wide investigation of circRNAs in T. rubrum based on high-throughput sequencing and ultimately identified 4254 circRNAs. Most of these circRNAs were specific to the conidial or mycelial stage, revealing a developmental stage-specific expression pattern. In addition, 940 circRNAs were significantly differentially expressed between the conidial and mycelial stages. PCR experiments conducted on seven randomly selected differentially expressed (DE-) circRNAs confirmed the circularized structures and relative expression levels of these circRNAs. Based on their genome locations, most circRNAs originated from intergenic regions, unlike those in plants and animals. Furthermore, we constructed circRNA-miRNA-mRNA regulatory networks that included 661 DE-circRNAs targeting 140 miRNAs and further regulating 2753 mRNAs. The relative expression levels of two randomly selected circRNA-miRNA-mRNA axes were investigated by qRT-PCR, and the competing endogenous RNA (ceRNA) network theory was validated. Functional enrichment analysis of the target genes suggested that they were significantly involved in posttranscriptional processes and protein synthesis as well as some small-molecule metabolism processes. CircRNAs are relatively more conserved in closely related dermatophytes but rarely conserved in distantly related species. Tru_circ07138_001 is a highly conserved circRNA that was conserved in all ten dermatophytes analyzed in our study and three distantly related species. Its host gene TERG_07138 was also highly conserved in two of these distantly related species Gallus gallus and Caenorhabditis elegans. The specific role of this circRNA deserves further exploration. Conclusions Our study is the first to provide a global profile of circRNAs in T. rubrum as well as dermatophytes. These results could serve as valuable resources for research on circRNA regulatory mechanisms in fungi and reveal new insights for further investigation of the physical characteristics of these significant human fungal pathogens.

Elucidation of The Effects and Underlying Mechanism of Aerobic Interval Training Combined With Liraglutide On Diabetic Cardiomyopathy

objective: This study intended to explore the hypoglycemic and cardioprotective effects of 8-week aerobic interval training combined with liraglutide and elucidate the underlying mechanisms.Method: Male Wistar rats were randomly divided into 5 groups - normal control (CON), diabetic cardiomyopathy (DCM), high-dose liraglutide (DH), low-dose liraglutide DL , and aerobic interval training combined with liraglutide (DLE). The cardiac function of rats ,the FBG the levels of fasting insulin (FIN), HbA1c, the total collagen content , AGEs, the mRNA expression of myocardial remodeling genes BNP, GSK3β, α-MHC, and β-MHC ,the expression of GLP-1 and GLP-1R proteins, Insulin resistance (HOMA-IR) and beta-cell function (HOMA-β) was analyze. Results: During the intervention, the FBG in each intervention group significantly decreased compared to the DCM group. After 8 weeks,the DH, DL, and DLE groups showed improved blood glucose-related indices and cleared the accumulated AGEs in the DCM groups. The heart function in the DLE groups was significantly improved than that in the DH and DL groups. The relative expression of BNP mRNA in the DH, DL, and DLE groups significantly reduced compared to the CON and the DCM group .Compared to the DCM group,the relative expression of α-MHC mRNA increased significantly and β-MHC mRNA decreased notably in the myocardium of the DH, DL, and the DLE group.The expression of GLP-1 in the myocardial tissue of rats in the DH group was higher than that in the DL and DLE groups. GLP-1R expression in the myocardial tissue in the DLE group was higher than that in the DH , DL and the DCM groups .Conclusion: Liraglutide combined with AIT intervention significantly reduced FBG and the fluctuations in FBG, alleviated myocardial fibrosis, improved cardiac function in DCM rats, supporting the efficacy of the combined pharmaceutical and physical intervention, and reduced the cost of treatment.

Effect of Xuezhikang Therapy on Expression of Pulmonary Hypertension Related miR-638 in Patients With Low HDL-C Levels

Objective: Low plasma level of high-density lipoprotein cholesterol (HDL-C) associated with poor outcomes in several cardiovascular diseases, including pulmonary arterial hypertension (PAH). Regulation of miR-638 have been proved to be associated with PAH. The aim of this study was to evaluate the expression of miR-638 after Xuezhikang (XZK) therapy in patients with low HDL-C.Methods: Plasma levels of miR-638 were quantified by real-time polymerase chain reactions in 20 patients with PAH and 30 healthy controls. A total of 40 subjects with low HDL-C were assigned to receive an XZK therapy for 6 months. The miR-638 expression profiles were detected in PAH patients, XZK-treated subjects and lovastatin treated pulmonary arterial smooth muscle cells (PA-SMCs).Results: The relative expression level of miR-638 in the plasma was lower in the PAH patients than that in the controls (p < 0.001). An increase of 11.2% from baseline in the HDL-C level was found after XZK therapy (p < 0.001). The relative expression of miR-638 was increased after XZK treatment (p < 0.01). The changes of miR-638 were inversely associated with baseline HDL-C levels. A significantly reduction in miR-638 expression were found in PDGF-BB-treated hPA-SMCs compared to the control cells, and the pre-treatment of the cells with lovastatin significantly re-gain the expression levels in miR-638.Conclusion: In patients with low HDL-C levels, XZK therapy raised the expression of miR-638, suggesting that the potential therapeutic effect of XZK in PAH patients with low serum HDL-C levels deserves further exploration.

Regulation of CcpA on the growth and organic acid production characteristics of ruminal Streptococcus bovis at different pH

Background Catabolite control protein A (CcpA) regulates the transcription of lactate dehydrogenase and pyruvate formate-lyase in Streptococcus bovis, but knowledge of its role in response to different pH is still limited. In this study, a ccpA-knockout strain of S. bovis S1 was constructed and then used to examine the effects of ccpA gene deletion on the growth and fermentation characteristics of S. bovis S1 at pH 5.5 or 6.5. Results There was a significant interaction between strain and pH for the maximum specific growth rate (μmax) and growth lag period (λ), which caused a lowest μmax and a longest λ in ccpA-knockout strain at pH 5.5. Deletion of ccpA decreased the concentration and molar percentage of lactic acid, while increased those of formic acid. Strains at pH 5.5 had decreased concentrations of lactic acid and formic acid compared to pH 6.5. The significant interaction between strain and pH caused the highest production of total organic acids and acetic acid in ccpA-knockout strain at pH 6.5. The activities of α-amylase and lactate dehydrogenase decreased in ccpA-knockout strain compared to the wild-type strain, and increased at pH 5.5 compared to pH 6.5. There was a significant interaction between strain and pH for the activity of acetate kinase, which was the highest in the ccpA-knockout strain at pH 6.5. The expression of pyruvate formate-lyase and acetate kinase was higher in the ccpA-knockout strain compared to wild-type strain. The lower pH improved the relative expression of pyruvate formate-lyase, while had no effect on the relative expression of acetate kinase. The strain × pH interaction was significant for the relative expression of lactate dehydrogenase and α-amylase, both of which were highest in the wild-type strain at pH 5.5 and lowest in the ccpA-knockout strain at pH 6.5. Conclusions Overall, low pH inhibited the growth of S. bovis S1, but did not affect the fermentation pattern. CcpA regulated S. bovis S1 growth and organic acid fermentation pattern. Moreover, there seemed to be an interaction effect between pH and ccpA deletion on regulating the growth and organic acids production of S. bovis S1.

Cord blood microRNA-376c and microRNA-1268a as biomarkers for neonatal hypoxic-ischaemic encephalopathy: a diagnostic accuracy study

BackgroundHypoxic-ischaemic encephalopathy (HIE) is one of the most common causes of morbidity and mortality among neonates. There is a critical need for non-invasive novel biomarkers to detect HIE early, predict its outcomes and monitor its progression. We conducted this observational study to assess the relative expression of miRNA-376c and miRNA-1268a in cord blood as potential diagnostic and prognostic biomarkers for HIE.MethodsA total of 100 neonates divided into two independent groups were included. The case group included 50 neonates with HIE, while the control group included 50 matched healthy neonates. Relative expressions of miRNA-376c and miRNA-1268a were measured in whole cord blood at birth using real-time PCR.ResultsCompared with the control group, patients with HIE had a significantly lower median level of miRNA-376c (0.168, IQR=0.011–0.411 vs 1, IQR=0.80–1.20) and a higher median level of miRNA-1268a (13.46, IQR=2.7–22.8 vs 1, IQR=0.4–1.6). Comparing neonates with HIE who survived versus those who did not survive, no statistically significant difference between the groups in terms of miRNA-376c and miRNA-1268a (p=0.124 and p=0.279) was elicited. Our diagnostic analysis showed that, at 0.90 points, miRNA-376c has a sensitivity and a specificity of 88% and 68.40%, with an area under the curve of 84%. At 2.70 points, miRNA-1268a has a sensitivity and a specificity of 76% and 100%, with an area under the curve of 96%.ConclusionThe relative expression of miRNA-376c and miRNA-1268a was altered in the cord blood of neonates with HIE. In addition, they have moderate diagnostic accuracy in detecting HIE.

The effect of titanium dioxide nanoparticles on the relative expression of catalase, P450, SOD, diTDS and WRKY genes of Vitex agnus-castus L.

Each environmental factor is able to change the way genes are expressed. Application of nanoparticles also affects the expression of different genes in plants. The aim of this study was to investigate the effect of three different concentration of titanium dioxide nanoparticles, TiO2 (zero, 200 and 800 micrograms per milliliter) on the relative expression of catalase, P450, SOD, diTDS and WRKY genes in Vitex plant leaf tissue using qRT- PCR. Plant cultivation was carried out in 2018 in the greenhouse of Islamic Azad University of Mashhad. The experiment was arranged as completely random design with 5 replications. XRD measurements showed that applied TiO2 nanoparticles were in the form of anatase. Statistical analysis of gene expression in treated leaves of Vitex plant with TiO2 nanoparticles showed that this nanoparticle significantly affected the expression of catalase, P450, SOD, diTPS and WRKY genes. A concentration of 800 micrograms per milliliter of TiO2 nanoparticle increased the expression of catalase, P450, SOD and WRKY genes and decreased the expression of diTPS gene. In contrast, concentrations of 200 micrograms per milliliter only increased the expression of catalase and WRKY genes. The expression of the diTPS gene under treatments of 200 and 800 micrograms per liter of TiO2, compared with control, decreased by 2.1 and 0.46, respectively. Overall, the nanoparticle was able to influence the expression of genes in the biosynthetic pathway of terpenoids, as well as the plant's antioxidant enzymes, depending on the concentration of nanoparticles.

Lactobacillus fermentum ZS40 Ameliorates Inflammation in Mice With Ulcerative Colitis Induced by Dextran Sulfate Sodium

Ulcerative colitis is an inflammatory disease of the intestine caused by many reasons, and it may even develop into colon cancer. Probiotics are normal bacteria that exist in the human body and have been proven to regulate the balance of intestinal flora and alleviate inflammation. The current study aimed to study the effect of Lactobacillus fermentum ZS40 (ZS40) on dextran sulfate sodium (DSS)-induced ulcerative colitis mice. The length and weight of the colon were measured, and the histopathological morphological changes of colon tissue were observed to evaluate the effects of ZS40 on colitis. Biochemical kits, ELISA kits, real-time quantitative PCR (RT-qPCR), and western blot were also used to detect the effects of ZS40 on serum and colon tissue related oxidative indicators and pro-inflammatory and anti-inflammatory cytokines. We found that ZS40 could reduce colonic inflammatory cell infiltration and goblet cell necrosis, increase total superoxide dismutase and catalase in mouse serum, and reduce myeloperoxidase and malondialdehyde levels. ZS40 could down-regulate the level of proinflammatory cytokines and up-regulate the level of anti-inflammatory cytokines. More importantly, ZS40 down-regulated the relative expression of nuclear factor-κB p65 (NF-κBp65), IL-6, and TNF-α mRNA and protein, up-regulated the relative expression of inhibitor kapa B alpha (IκB-α). By regulating the NF-κB and MAPK pathways to down-regulated the relative expression of p38 and JNK1/2 mRNA and p38, p-p38, JNK1/2, and p-JNK1/2 proteins. Our study suggested that ZS40 may serve as a potential therapeutical strategy for ulcerative colitis.

Evaluation of level of expression of microRNA-21 and let-7g in serum and stool of patients with colorectal cancer

Background MicroRNAs (miRs) are involved in the pathogenesis of various malignancies such as colorectal cancer through regulating multiple cellular processes, including cell proliferation, cell cycle, apoptosis and migrationMiR-21 and let-7 are two important genes that have confirmed in this pathway. The role of the let-7 gene as a gene tumor process in various cancers and the role of miR-21 in the development and progression of cancer has been conclusively identified also this gene has an oncogenic role in various cancers. In this study, the expression patterns of miR-21 and let-7 in serum and stool samples of colorectal cancer patients were evaluated. Materials and Methods During the present study, 120 samples including 40 serum samples of CRC and 40 stool samples from the same patients and 40 healthy samples were collected. After total RNA extraction, real-time PCR was used to measure changes in genes expression. Statistical analysis of data was performed with GraphPad Prism statistical software (Version 6.0) with a significance level of 5%. Results The relative expression level of miR-21 in the serum samples of CRC increased compared to the healthy group, which was statistically significant. On the other hand, the relative expression level of let-7g in the serum samples of CRC showed a significant decrease compared to the healthy sample. In stool samples, the expression changes of either of the two genes were not significant. Conclusion Our findings indicate that the relative expression of miR-21 and let-7g genes can be used as a diagnostic or predictive biomarker in colorectal cancer serum samples. While, this is not the case in stool samples. Moreover, further investigations at the protein level should be performed.