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Coronary heart disease (CHD) is one of the leading causes of heart‐associated deaths worldwide. Conventional diagnostic techniques are ineffective and insufficient to diagnose CHD with higher accuracy. To use the circulating microRNAs (miRNAs) as non‐invasive, specific and sensitive biomarkers for diagnosing of CHD, 203 patients with CHD and 144 age‐matched controls (126 high‐risk controls and 18 healthy volunteers) were enrolled in this study. The direct S‐Poly(T)Plus method was used to identify novel miRNAs expression profile of CHD patients and to evaluate their clinical diagnostic value. This method is an RNA extraction‐free and robust quantification method, which simplifies procedures, reduces variations, in particular increases the accuracy. Twelve differentially expressed miRNAs between CHD patients and high‐risk controls were selected, and their performances were evaluated in validation set‐1 with 96 plasma samples. Finally, six (miR‐15b‐5p, miR‐29c‐3p, miR‐199a‐3p, miR‐320e, miR‐361‐5p and miR‐378b) of these 12 miRNAs were verified in validation set‐2 with a sensitivity of 92.8% and a specificity of 89.5%, and the AUC was 0.971 (95% confidence interval, 0.948‐0.993, P < .001) in a large cohort for CHD patients diagnosis. Plasma fractionation indicated that only a small amount of miRNAs were assembled into EVs. Direct S‐Poly(T)Plus method could be used for disease diagnosis and 12 unique miRNAs could be used for diagnosis of CHD.

Background Circular RNA_0001742 (circ_0001742) has been reported to be upregulated in tongue squamous cell carcinoma (TSCC) tissues/cells and regulate TSCC cell proliferation, migration, and invasion. This study aimed to further investigate the clinical significance of circ_0001742 in TSCC management. Methods Totally, 146 TSCC patients underwent surgical treatment were reviewed. Their fresh‐frozen tumor tissue and adjacent tissue were acquired for detecting circ_0001742 expression via reverse transcription‐quantitative polymerase chain reaction. According to circ_0001742 expression in tumor tissue, all patients were classified as tumor circ_0001742 low (0%‐50% percentile) and high (50%‐100% percentile) patients, the latter were further divided into the tumor circ_0001742 high+ (50%‐75% percentile), high++ (75%‐90% percentile), and high+++ (90%‐100% percentile) patients, respectively. Results Circ_0001742 expression was increased in TSCC tumor tissue compared with adjacent tissue, and it presented good value in discriminating tumor tissue from adjacent tissue (area under the curve (AUC): 0.870, 95% CI: 0.831‐0.910). Tumor high circ_0001742 expression was associated with higher T stage, N stage, and TNM stage, but not age, gender, or pathological grade. Furthermore, OS was reduced in tumor circ_0001742 high patients compared with tumor circ_0001742 low patients; moreover, OS was the shortest in tumor circ_0001742 high+++ patients, followed by tumor circ_0001742 high++ patients and tumor circ_0001742 high+ patients, and the longest in tumor circ_0001742 low patients. In addition, multivariate Cox's regression analysis revealed that higher tumor circ_0001742 expression was an independent predictive factor for decreased OS. Conclusion Circ_0001742 serves as a potential biomarker for advanced tumor stage and poor survival in TSCC patients.

Background Long non‐coding RNA taurine‐upregulated gene 1 (lncRNA TUG1) is reported to be involved in the progression and development of several malignancies; however, its role in Philadelphia chromosome‐negative acute lymphoblastic leukemia (Ph−ALL) is unknown. The present study aimed to explore the correlation of lncRNA TUG1 with disease risk, disease condition, and prognosis of adult Ph−ALL. Methods Total 101 adult Ph− ALL patients and 40 bone marrow (BM) donors were included, followed by detection of BM monocyte cell lncRNA TUG1 expression by reverse transcription‐quantitative polymerase chain reaction. According to the quantiles of lncRNA TUG1 expression in Ph− ALL patients, these patients were divided into four tiers: tier 1 (ranked in 0%~25%), tier 2 (ranked in 25%~50%), tier 3 (ranked in 50%~75%), and tier 4 (ranked in 75%~100%). Results LncRNA TUG1 was upregulated in Ph− ALL patients compared with healthy donors. Further analysis indicated that in Ph− ALL patients, higher lncRNA TUG1 tier was correlated with the presence of central nervous system leukemia, increased white blood cell level, and bone marrow blasts. Furthermore, higher lncRNA TUG1 tier was negatively associated with complete remission (CR) within 4 weeks, total CR, and allogeneic hematopoietic stem cell transplant achievement. In addition, higher lncRNA TUG1 tier was associated with decreased disease‐free survival and overall survival, which was further verified to be an independent factor by Cox's regression analysis. Conclusion lncRNA TUG1 presents potential to be a novel biomarker for disease risk assessment and survival surveillance in Ph− ALL management. Long non‐coding RNA taurine‐upregulated gene 1 (lncRNA TUG1) is upregulated and correlates with poor treatment response and unfavrable survival profiles in Philadelphia chromosome‐negative Acute lymphoblastic leukemia (Ph‐ALL) patients, suggesting its potential as a candidate biomarker in ALL managment.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) has become a frequently used method in gene expression studies. The relative quantification method is an important and common method for the evaluation of RT-qPCR data. One of the key requirements of this method is to identify an applicable internal reference gene. However, to the best of our knowledge, no suitable reference genes have been identified for the genetic analysis of cholangiocarcinoma (CCA) in humans and cell lines. In the present study, screening was conducted using 12 common reference genes, which were selected in order to provide an experimental basis for the study of the gene expression in CCA patients and cell lines. Tumor samples and adjacent non-neoplastic tissues from 20 patients with CCA were obtained for the present study. The stability and applicability of the 12 reference gene candidates were validated using GeNorm, NormFinder and BestKeeper software. Based on a comparison of the results, the following reference genes are recommended for various tissue groups: Total sample group, ribosomal protein L29; CCA tissue group, TATA-box binding protein; CCA cell line and tissue group, actin-β; CCA cell line group, 18S ribosomal RNA; CCA tissue and adjacent non-neoplastic tissue group, 5′-aminolevulinate synthase 1; and adjacent non-neoplastic tissue group, hypoxanthine phosphoribosyltransferase 1. The results of the search also clearly reveal that a systematic study regarding the selection of suitable reference genes for studying the target gene profiling in CCA tissues and cell lines has not been previously published. The present study may provide useful information for future studies that examine the gene expression of CCA for choosing suitable reference genes.

Until now, no information has been available on the transcriptional response to the transduction of defense signals in brown seaweeds that leads to active resistance against pathogens or grazers. Using a polymerase chain reaction (PCR)-based, subtractive cDNA approach combined with filter arrays we demonstrated that Laminaria digitata exhibits a rapid response to oligoguluronate elicitors. The transcription levels of several genes were validated by quantitative real-time reverse-transcription PCR and further analysed using pharmacological approaches. Fifty upregulated genes were identified by differential screening in elicited algae over a 24-h time-course. These genes were related to oxidative stress responses, production of antimicrobial secondary compounds or cell wall strengthening. Moreover, pharmacological tests showed that intracellular signal transduction is likely to involve reactive oxygen species. A new oligoguluronate-inducible vanadium-dependent haloperoxidase (vHPO), specific to iodide was also characterized. The transcription of several vHPO genes was shown to be tightly regulated. Taken together, our data show that early transcriptional defense responses in L. digitata are similar to those in land plants but also include novel defense responses that involve tightly regulated iodine metabolism.

Aim: The aim of this study is to explore the prognostic significance and clinicopathological correlations of the Wnt pathway genes in a cohort of surgically treated patients with oral squamous cell carcinoma (OSCC) patients. Settings and Design: A prospective genetic study on patients with OSCC was carried out during the period from July 2014 to January 2016. Informed consent from patients and institutional ethical approval for the study was obtained and the guidelines were strictly followed for collection of samples. Subjects and Methods: Clinical data and mRNA expression analysis of ten genes in the canonical Wnt pathway were evaluated and their relationships with clinical and demographic variables were studied in 58 tissue samples. Wnt-3a, β-catenin, secreted frizzled-related proteins sFRP-1, sFRP-2, sFRP-4, sFRP-5, Wnt inhibitory factor 1, dickkopf-1, c-MYC, and cyclin-D1 from cancer (n = 29) and normal (n = 29) tissue samples were investigated using quantitative reverse transcription-polymerase chain reaction. Statistical Analysis: Descriptive statistics were used to summarize the sample characteristics and clinical variables. If the data were normal, then parametric tests were used; otherwise, nonparametric alternatives were used. All the analyses were carried out using SPSS version 23.0 (IBM SPSS Inc., USA). Results: Expression of sFRP-1, sFRP-2, and sFRP-5 in control samples and expression of c-MYC and cyclin D1 in cancer samples showed statistical significance. Significant expression of Wnt3A was observed among patients who had recurrence and were deceased. Conclusion: Wnt3A, β-catenin, and cyclin D1 are recognized as key components of Wnt/β-catenin signaling. However, in this study, there was no significant expression of all the three genes in OSCC. The proto-oncogene c-MYC showed statistically significant upregulation in cancer tissue samples suggesting that the OSCC among South Indian population is primarily not mediated by the canonical Wnt signaling pathway.

Chromatin regulation ensures stable repression of stress-inducible genes under non-stress conditions and transcriptional activation and memory of stress-related genes after stress exposure. However, there is only limited knowledge on how chromatin genes are regulated at the transcriptional and post-transcriptional level upon stress exposure and relief from stress. We reveal that the repressive modification histone H3 lysine 27 trimethylation (H3K27me3) targets genes which are quickly activated upon cold exposure, however, H3K27me3 is not necessarily lost during a longer time in the cold. In addition, we have set-up a quantitative reverse transcription polymerase chain reaction-based platform for high-throughput transcriptional profiling of a large set of chromatin genes. We find that the expression of many of these genes is regulated by cold. In addition, we reveal an induction of several DNA and histone demethylase genes and certain histone variants after plants have been shifted back to ambient temperature (deacclimation), suggesting a role in the memory of cold acclimation. We also re-analyze large scale transcriptomic datasets for transcriptional regulation and alternative splicing (AS) of chromatin genes, uncovering an unexpected level of regulation of these genes, particularly at the splicing level. This includes several vernalization regulating genes whose AS may result in cold-regulated protein diversity. Overall, we provide a profiling platform for the analysis of chromatin regulatory genes and integrative analyses of their regulation, suggesting a dynamic regulation of key chromatin genes in response to low temperature stress.

Environmental compounds with estrogen- or antiestrogen-like activity can enter rivers from multiple sources, including municipal wastewater and agricultural runoff. We used longnose dace (Rhinichthys cataractae) to investigate exposure to compounds with estrogen-like activity, which we measured in water at multiple sites in the Oldman and Bow rivers (AB, Canada). We evaluated changes in vitellogenin mRNA with quantitative reverse transcription-polymerase chain reaction, then compared vitellogenin levels to sex ratios and fish performance indices to assess how exposure to compounds with estrogen-like activity affects longnose dace populations. Vitellogenin levels were elevated at least 59 to 110 km downstream of municipalities. In the Oldman River, increased vitellogenin expression and female-biased sex ratios suggest severe endocrine disruption, likely resulting from the combined impacts of municipal wastewater, agriculture, and large cattle operations within the basin. In the Bow River, municipal wastewater may be the major source of compounds with estrogen-like activity that affect longnose dace. The sex ratios were not heavily skewed, as in the more agriculturally influenced Oldman River. We detected organic contaminants in river samples at every site, but the highest concentrations were found downstream of municipalities and in areas with intense agriculture. Vitellogenin levels and sex ratios of longnose dace suggest basin-wide exposure to compounds with estrogen-like activity. Our results demonstrate that it is important to assess rivers at large spatial scales to detect fully the impacts of municipal wastewater and agriculture on fish populations.

Background Reverse Transcription quantitative polymerase chain reaction (RT-qPCR) is a sensitive and reliable method for mRNA quantification and rapid analysis of gene expression from a large number of starting templates. It is based on the statistical significance of the beginning of exponential phase in real-time PCR kinetics, reflecting quantitative cycle of the initial target quantity and the efficiency of the PCR reaction (the fold increase of product per cycle). Results We used the large clinical biomarker dataset and 94-replicates-4-dilutions set which was published previously as research tools, then proposed a new qPCR curve analysis method——C q MAN, to determine the position of quantitative cycle as well as the efficiency of the PCR reaction and applied in the calculations. To verify algorithm performance, 20 genes from biomarker and partial data with concentration gradients from 94-replicates-4-dilutions set of MYCN gene were used to compare our method with various publicly available methods and established a suitable evaluation index system. Conclusions The results show that C q MAN method is comparable to other methods and can be a feasible method which applied to our self-developed qPCR data processing and analysis software, providing a simple tool for qPCR analysis.

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), can cause direct damage to citrus trees and is the main vector for the devastating disease, citrus greening disease or huanglongbing. Most molecular studies on this important insect pest use real-time reverse-transcription quantitative polymerase chain reaction (RT-qPCR) to quantify gene expression, including analyzing molecular basis for insecticide resistance in field populations. One critical factor to cause inaccuracy in RT-qPCR results is the lack of appropriate internal reference genes for optimal data normalization. In this study, the expression levels of 10 selected reference genes were evaluated in different tissue samples of psyllid adults and in the insects treated with different temperatures and insecticides. Data were analyzed using different computational algorithms, including Delta Ct, BestKeeper, NormFinder, geNorm, and RefFinder. According to our results, at least two reference genes should be used for the normalization of RT-qPCR data in this insect. The best choices of reference genes for different samples are as follows: ACT1 and Ferritin for different tissue samples, RPS20 and Ferritin for samples treated with different temperatures, TBP and EF1α for samples treated with imidacloprid, and Ferritin and TBP for samples treated with beta-cypermethrin. The reference genes identified in this study should be useful for future studies to analyze the expression patterns of target genes, especially for genes linked with temperature adaptability and insecticide resistance in this insect species in the future.