Quantitative Reverse transcription-PCR Method Research Articles

Gene Expression and Methylation Levels of PCSK9 Gene in Iraqi Patients with Coronary Artery Disease

The expression of the Proprotein Convertase Subtilisin/Kexin Type 9 gene (PCSK9) is inextricably related to lipid levels and a risk of atherosclerotic coronary artery disease (ASCAD). The present study aims to measure the quantity of PCSK9 gene expression and the effect of methylation on its expression level taking part in the pathogenesis of acute coronary artery disorder. A current study included 150 subjects from the Iraqi population, 100 ASCAD patients and 50 healthy controls. The concentration of PCSK9 in each serum sample was determined by the ELISA technique, the expression levels of the PCSK9 gene in whole blood were estimated by RT-qPCR – Quantitative Reverse Transcription PCR method, and DNA methylation level detection in PCSK9 gene by using High Resolution Melting Analysis (HRM) technique. The expression level of the PCSK9 gene was 6.06 ± 1.84 in ASCAD patients group compared with a control group 1.00 ±0.19. That indicates the up-regulation of the PCSK9 gene in patient with atherosclerosis. In addition, the PCSK9 concentration was higher in patient group in comparison to the healthy controls group P= < 0.01. However, PCSK9 methylation levels, a highly significant distinction between the two study groups p=0.002. In conclusion, serum PCSK9 levels are associated with coronary artery lesions as shown in receiver operating characteristic (ROC) analysis. Suggests that, serum PCSK9 might be a good indicator of coronary artery disease development.

Discovery and validation of circulating miRNAs for the clinical prognosis of severe dengue

BackgroundEarly prognostic markers of severe dengue may improve case management and reduce dengue-related mortalities. This study aimed to identify circulating microRNAs (miRNAs) as biomarkers for predicting severe dengue.MethodologySerum samples from dengue-infected patients were collected on the first day of admission. Patients were followed up for 14 days after admission to determine the final diagnosis. Participants were divided into non-severe and severe dengue, as defined by WHO 2009 criteria. Circulating microtranscriptome analysis was performed using NanoString miRNA Expression Assay. The expression level of candidate miRNAs were then validated by quantitative reverse transcription-PCR method.Principal findingsThe discovery cohort (N = 19) lead to the identification of 37 differentially expressed miRNAs between the two groups. Six up-regulated candidate miRNAs were selected and further validated in the larger cohort (N = 135). MiR574-5p and miR1246 displayed the highest diagnostic performance in discriminating between severe from non-severe dengue (ROC-AUC = 0.83). Additionally, miR574-5p and miR1246 had high sensitivity and high negative predictive value for detecting severe dengue. Multivariate analysis suggested that serum miR574-5p was an independent predictor of severe dengue (odds ratio 3.30, 95% CI 1.81–6.04; p<0.001).ConclusionOur study indicated that circulating miRNAs, especially miR-574-5p and miR-1246, might be a promising diagnostic and prognostic biomarker for severe dengue upon hospital admission, especially when using these biomarkers on days 1 to 2 before the onset of severe dengue complications.

WZB117 enhanced the anti-tumor effect of apatinib against melanoma via blocking STAT3/PKM2 axis.

Background: Melanoma is the most lethal skin malignant tumor with a short survival once stepping into the metastatic status and poses a therapeutic challenge. Apatinib (a tyrosine kinase inhibitor) is a promising antiangiogenic agent for the treatment of metastatic melanoma. However, antiangiogenic monotherapy is prone to acquired drug resistance and has a limited therapeutic effect. The persistence dependence of glycolytic metabolism in antiangiogenic therapy-resistant cells provides evidence that glycolysis inhibitors may enhance the effect of antiangiogenic therapy. So, this study aimed to investigate whether WZB117 (a specific GLUT1 inhibitor) could enhance the anti-tumor effect of apatinib against melanoma and its potential mechanisms. Methods: We investigated the anti-tumor effects of apatinib alone or in combination with WZB117 on human melanoma cell lines (A375 and SK-MEL-28). The MTT assay determined cell viability and the half-maximal inhibitory concentration (IC50). Multiple drug effect/combination indexes (CI) analysis was conducted to assess interactions between apatinib and WZB117. Signal transducer and activator of transcription 3 (STAT3) pathway measured by western blotting and immunofluorescence staining. RNA expression analyses were performed using the reverse transcription-quantitative PCR method. Results: Apatinib and WZB117 showed dose and time-dependent growth inhibitory effects in both melanoma cells. The IC50 of apatinib at 48 h in A375 and SK-MEL-28 cells was 62.58 and 59.61 μM, respectively, while the IC50 of WZB117 was 116.85 and 113.91 μM, respectively. The CI values of the two drugs were 0.538 and 0.544, respectively, indicating a synergistic effect of apatinib combined with WZB117. We also found that glucose consumption and lactate production were suppressed by apatinib plus WZB117 in a dose-dependent manner, paralleled by reducing glycolytic enzyme pyruvate kinase M2 (PKM2). The potential mechanism of the combination was to suppress the phosphorylation of STAT3. Knockdown of STAT3 by siRNA inhibited the expression of PKM2, while the activation of STAT3 by IL-6 increased the expression of PKM2. The effects of IL-6 were attenuated by apatinib combined with WZB117 treatment. Conclusion: WZB117 enhanced the anti-tumor effect of apatinib against melanoma via modulating glycolysis by blocking the STAT3/PKM2 axis, which suggested the combination of apatinib with WZB117 could be a potential therapeutic candidate for melanoma.

Optimization of quantitative reverse transcription PCR method for analysis of weakly expressed genes in crops based on rapeseed.

Rapeseed (Brassica napus) is an allopolyploid hybrid (AACC genome) of turnip rape (B. rapa, genome: AA) and vegetable cabbage (B. oleraceae, genome: CC). Rapeseed oil is one of the main vegetable oils used worldwide for food and other technical purposes. Therefore, breeding companies worldwide are interested in developing rapeseed varieties with high yields and increased adaptation to harsh climatic conditions such as heat and prolonged drought. One approach to studying the mechanism of the epigenetically regulated stress response is to analyze the transcriptional changes it causes. In addition, comparing the expression of certain genes between stress- and non-stress-tolerant varieties will help guide breeding in the desired direction. Quantitative reverse transcription PCR (RT-qPCR) has been intensively used for gene expression analysis for several decades. However, the transfer of this method from model plants to crop species has several limitations due to the high accumulation of secondary metabolites, the higher water content in some tissues and therefore problems with their grinding and other factors. For allopolyploid rapeseed, the presence of two genomes, often with different levels of expression of homeologous genes, must also be considered. In this study, we describe the optimization of transcriptional RT-qPCR analysis of low-expression epigenetic genes in rapeseed, using Kinetochore Null2 (KNL2), a regulator of kinetochore complex assembly, as an example. We demonstrated that a combination of various factors, such as tissue homogenization and RNA extraction with TRIzol, synthesis of cDNA with gene-specific primers, and RT-qPCR in white plates, significantly increased the sensitivity of RT-qPCR for the detection of BnKNL2A and BnKNL2C gene expression.

Non-coding RNAs transcribed from ultra-conserved regions (T-UCRs) are differentially expressed in dental follicle tissues of impacted mandibular third molars

IntroductionTranscribed ultra-conserved regions (T-UCRs) are a new class of long non-coding RNA molecules transcribed from ultra-conserved regions (UCRs) of the human genome. T-UCRs are extremely conserved in the human, rat, and mouse genomes. Deletions of genomic areas containing UCRs resulted in live mice that developed without distinguishable phenotypes, implying that T-UCRs are involved in developmental processes. In addition, there is increasing evidence that dental follicle tissues exhibit various cellular alterations involving deregulation of protein-coding genes and non-coding RNAs. Accordingly, the main objective of the present study was to determine the clinical significance and distinct expression signatures of non-coding RNA molecules transcribed from ultra-conserved regions in dental follicle tissues of impacted mandibular third molars. Materials and methodsFrom March 2021 to December 2021, a total of 42 patients who referred to clinic of oral and maxillofacial surgery department with the indications of impacted mandibular third molar extraction from 38th and 48th positions were enrolled for the study. For the analysis of T-UCR expression levels, real‐time quantitative reverse transcription PCR method was used. ResultsFindings of the present study indicated that T-UCRs are distinctly expressed in dental follicle tissues of impacted mandibular third molars. The expression of uc.38, uc.112, and uc.338 was found to be significantly increased in the dental follicles of impacted mandibular third molars, indicating a clinical significance of these molecules. In addition, no differences in T-UCR expression were found as a function of demographic characteristics. ConclusionsCollectively, transcribed ultra-conserved elements, such as uc.38, uc.112, and uc.338, are considerably deregulated in the dental follicle tissues of impacted mandibular third molars and might be responsible for the molecular changes acquired by dental follicle tissues of impacted mandibular third molars.

Label-free and sensitive microRNA detection method based on the locked nucleic acid assisted fishing amplification strategy.

Herein, a label free and sensitive miRNA detection method with enhanced practical applicability was developed based on the locked nucleic acid (LNA) assisted repeated fishing amplification strategy. The working mechanism of the proposed method is as follows: 1) a DNA probe (i.e, L-DNA) with LNA bases is immobilized onto the surface of a gold foil. The L-DNA hybridizes with the 3' terminus of the first strands of complementary deoxyribonucleic acid (cDNA) of the target miRNA in the test samples; 2) The protruding 5' terminus of the cDNA serves as a 'fishhook' to repeatedly fish the products of a hybridization chain reaction (HCR) out from a 'reaction tube'; 3) The HCR products can be unloaded from the gold foil into a 'product tube' through temperature-controlled dehybridization; 4) The concentration of the target miRNA is determined based on the fluorescence intensity generated by the addition of SYBR-Green I (SG) into the 'product tube'. The proposed platform was applied to the detection of miRNA-122 in cell lysate samples and obtained quantitative results with accuracy comparable to the quantitative reverse transcription PCR method (qRT-PCR). It is worth pointing out that the proposed platform achieved a limit of detection value of 2.9fM for miRNA-122 by a simple but effective LNA-assisted repeated fishing amplification strategy instead of complicated enzyme-based amplification techniques. It is reasonable to expect that the proposed method provides a competitive alternative for designing practically applicable, cost-effective and label-free miRNA detection methods.

An improved RT-qPCR method for direct quantification of enveloped RNA viruses

Reverse transcription quantitative PCR (RT-qPCR) has emerged as the gold standard for virus detection and quantification, being utilized in numerous diagnostic and research applications. However, the direct detection of viruses has so far posed a challenge as the viral genome is often encapsidated by a proteinaceous layer surrounded by a lipid envelope. This necessitates an additional and undesired RNA extraction step prior to RT-qPCR amplification. To circumvent this limitation, we have developed a direct RT-qPCR method for the detection of RNA viruses. In our method, we provide a proof-of-concept using phage phi6, a safe-to-use proxy for pathogenic enveloped RNA viruses that is commonly utilized in e.g. aerosolization studies. First, the phage phi6 envelope is removed by 1% chloroform treatment and the virus is then directly quantified by RT-qPCR. To identify false negative results, firefly luciferase is included as a synthetic external control. Thanks to the duplex format, our direct RT-qPCR method reduces the reagents needed and provides an easy to implement and broadly applicable, fast, and cost-effective tool for the quantitative analysis of enveloped RNA viruses.•One-step direct RT-qPCR quantification of phage phi6 virus without prior RNA isolation.•Reduced reaction volume for sustainable and cost-effective analysis.

Development of a high-throughput RT-PCR based viral infectivity assay for monitoring the stability of a replicating recombinant Lymphocytic Choriomeningitis viral vector

Traditional virus infectivity titration methods for lymphocytic choriomeningitis virus (LCMV) are laborious, time-consuming, and low-throughput (e.g., focus forming unit (FFA) assay). In this report, we developed a high-throughput reverse transcription quantitative PCR (RT-qPCR)-based virus infectivity assay for relative quantitation of a live, recombinant replicating LCMV -based viral vector (TT1). This in vitro infectivity assay demonstrated a 4-log linear range for TT1 titer quantitation. A high positive Pearson correlation coefficient value (≥ 0.80) was obtained between the RT-qPCR vs. the “gold-standard” FFU assay when comparing the stability profiles of stressed TT1 vector samples. In addition to the RT-qPCR infectivity assay, the stability of the TT1 vector upon freeze-thaw stress was investigated further with complementary viral particle characterization techniques (e.g., TEM, NTA, MFI). Correlations between viral infectivity and particle measurements during forced degradation studies were observed to be specific to the TT1 vector and its various formulations and such results facilitated the rank-ordering of formulation conditions. Overall, this infectivity RT-qPCR method showed increased sample throughput and improved assay flexibility compared to traditional viral infectivity assays. These results are discussed in the context of enabling future TT1 vector formulation development work, and potential utilization as an in-process monitoring tool during TT1 vector manufacturing.

Opium may affect coronary artery disease by inducing inflammation but not through the expression of CD9, CD36, and CD68

The molecular mechanisms of opium with regard to coronary artery disease (CAD) have not yet been determined. The aim of the present study was to evaluate the effect of opium...

Defence responses of grapevine cultivars to powdery mildew: Ontogenic resistance versus genetic resistance

AbstractThe most sustainable management of resistant varieties involves understanding which defence mechanisms the plant uses according to its resistance genes and stage of development and how effective these mechanisms are. Three grapevine varieties were compared for their susceptibility versus resistance to powdery mildew: Vitis vinifera ‘Cabernet Sauvignon’ (CS) and two hybrids, ‘Artaban’ and ‘Prior’. Four different leaf ages were collected from 2‐year‐old grafted grapevines planted in pot or in vineyard, and several variables were assessed for each leaf: (a) morphological and physiological indicators, (b) pathogenicity traits: spore germination, infection efficiency, mycelial growth and sporulation, and (c) gene expression using a high‐throughput quantitative reverse transcription PCR method to inform about plant defence status and functioning of primary metabolism. A significant decrease in germination and sporulation with increasing leaf age were observed for the three varieties: Prior showed a susceptibility similar to that of CS, whereas Artaban was fully resistant with no sporulation. Genes highlighted as markers of the variety were involved in defence (VvPR3, VvPR4 overexpressed in Artaban, VvPR4bis, VvAlli2 in Prior) or in primary metabolism (repression of VvCitS in Artaban, VvCHI in Prior, overexpression of VvCAD2, VvGST3 in Prior). After inoculation, more defence genes, especially PR proteins, were overexpressed in the oldest leaves as potential components of ontogenic resistance. PR proteins accounted for 25% of total disease variation. Phenylpropanoid and ethylene pathways were leaf age‐dependent. The statistical method used highlighted the contributions of the genotypic expression involved in genetic (cultivar) and ontogenic (leaf age) resistances and of the metabolic pathways to the disease.

Evaluation of the clinical performance of single-, dual-, and triple-target SARS-CoV-2 RT-qPCR methods

BackgroundThe coronavirus disease 2019 (COVID-19) has become a pandemic. Reverse transcription quantitative PCR (RT-qPCR) has played a vital role in the diagnosis of COVID-19, but the rates of false negatives is not ideal in dealing with this highly infectious virus. It is thus necessary to systematically evaluate the clinical performance of the single-, dual-, triple-target detection kits to guide the clinical diagnosis of this disease. MethodsA series of reference materials calibrated by droplet digital PCR (ddPCR) and 57 clinical samples were used to evaluate the clinical performance of six single-, dual-, triple-target SARS-CoV-2 nucleic acid detection kits based on RT-qPCR. ResultsThe dual-target kits, kit B and kit C had the highest and the lowest detection sensitivity, which was 125 copies/mL and 4000 copies/mL, respectively. Among the 57 clinical samples from patients with COVID-19, 47 were tested positive by the kit B, while 35, 29, 28, 30, and 29 were found positive by the kits A, C, D, E, and F, respectively. The number of targets in a detection kit is not a key factor affecting sensitivity, while the amount of sample loading may influence the performance of a detection kit. ConclusionsThis study provides a guide when choosing or developing a nucleic acid detection kit for the diagnosis of COVID-19. Also, the absolute-quantification feature and high-sensitivity performance of ddPCR, suggesting that it can be used to review clinically suspected samples.

A Comparison of Two Standardized Quantitative RT-PCRs with CE IVD Kit for Digital PCR in CML Patients with Different Level of BCR-ABL1 Transcripts

The detection and quantification of BCR-ABL1 transcripts play a key role in therapy management of chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors (TKIs). Reverse transcription quantitative PCR (RT-qPCR) is considered the gold standard strategy for monitoring of minimal residual disease (MRD). Recently, digital PCR (dPCR) was introduced as a new quantification method based on determination of absolute target sequence quantity. In connection with the deep molecular response (MR) assessment and the option of TKIs treatment cessation, the dPCR has a potential of high sensitivity, robustness and accuracy of BCR-ABL1 transcripts detection. The aim of present work was to compare the results of two standardized RT-qPCR methods with RT-dPCR detection in clinical samples of CML patients with different BCR-ABL1 fusion transcripts level. In total, 62 peripheral blood samples of CML patients with the level of BCR-ABL1 transcripts (international scale, IS) between 100% and undetectable were enrolled. The samples were analyzed by three methods: (1) CE IVD Xpert BCR-ABL Ultra Kit (GeneXpert, Cepheid), (2) conventional RT-qPCR assay according to ELN guidelines and certified by EUTOS for the level of MR4.5 BCR-ABL1 detection (ABI7300, Applied Biosystems), and (3) RT-dPCR using CE IVD QXDx BCR-ABL %ID Kit (Bio-Rad) on QX200 Droplet Digital PCR System (Bio-Rad). All steps were performed according to manufacturer´s instructions. A linear regression analysis and an overall reporting bias analysis using the Bland-Altman test were used for comparison of the methods. Based on the results of GeneXpert as the determined routine method for BCR-ABL1 transcripts quantification, the samples were divided into two groups: 50/62 samples were scored as BCR-ABL1 positive (the first group), while 12/62 samples were scored as BCR-ABL1 undetectable (the second group). The first group of GeneXpert positive samples was analyzed using both conventional RT-qPCR and RT-dPCR (both in duplicates). The median of the sum of ABL copies per sample was 180,511 copies (range 56,466 - 536,453) by RT-qPCR vs. 39,960 (range 14,360 - 71,690) by RT-dPCR. Both conventional RT-qPCR and RT-dPCR did not detect any BCR-ABL1 transcript in 4/50 samples. In addition, RT-dPCR did not detect any BCR-ABL1 transcript in 5 other samples (Figure 1). Linear regression analysis showed good correlation between both sets of assays: GeneXpert vs. RT-dPCR (R2=0.965, N=41) and conventional RT-qPCR vs. RT-dPCR (R2=0.977, N=41). The slopes of regression curve were not significantly different from the value of 1 for both sets of the compared assays (1.02; 95% CI, range 0.95 - 1.08 and 1.02; 95% CI, range 0.97 - 1.08, respectively). In addition, an overall bias (Bland-Altman analyses) was -0.05 (GeneXpert vs. RT-dPCR) and -0.12 (conventional RT-qPCR vs. RT-dPCR) suggesting good concordance in % IS reporting between RT-dPCR and two standardized quantitative BCR-ABL1 assays. The evaluation of MR level was identical by all three methods in 26/50 samples. The comparison of two methods revealed consistent MR level by GeneXpert vs. RT-dPCR in 29/50 samples and by conventional RT-qPCR vs. RT-dPCR in 28/50 samples. The RT-dPCR scored 12/50 samples (GeneXpert vs. RT-dPCR) and 13/50 samples (conventional RT-qPCR vs. RT-qPCR) to the different level of MR compared to standardized RT-qPCR methods. The second group of samples (12/62) undetectable for BCR-ABL1 transcripts according to GeneXpert was analyzed using conventional RT-qPCR and RT-dPCR in tetraplicates to reach higher sensitivity. The BCR-ABL1 negativity was confirmed in 11/12 samples. One sample was BCR-ABL1 positive by both the conventional RT-qPCR (0.0008% IS) and RT-dPCR (0.0013% IS). We showed that the results of quantitative detection of BCR-ABL1 transcripts (% IS) obtained by the CE IVD RT-dPCR kit are in good correlation with the results of both standardized RT-qPCR methods and all three methods provide comparable sensitivity. RT-qPCR method yielded higher number of copies of control ABL gene per sample compared to RT-dPCR although the input amount of RNA into the RT reaction was the same. The MR level evaluation of RT-dPCR vs. both RT-qPCR methods was identical to the level MR3.0 (category &gt;0.01% IS), while for samples in deep MR (category ≤ 0.01% IS, MR4.0 and below) revealed partial difference in categorization into the individual MR levels. Supported by MH CZ - DRO (FNBr, 65269705). Disclosures Zackova: Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Angelini: Consultancy; Incyte: Consultancy. Mayer:AOP Orphan Pharmaceuticals AG: Research Funding.

Differences of Viral Hemorrhagic Septicemia Virus Loads among Organs of Dead and Surviving Olive Flounder Infected by Intramuscular Injection and Immersion Challenge.

Viral hemorrhagic septicemia virus (VHSV) is an important viral pathogen in the culture of Olive Flounder Paralichthys olivaceus. Based on cumulative mortality, the virulence of VHSV was found to be highly different depending on challenge routes and exposure doses (using tissue culture infectious dose with 50% endpoint [TCID50]). Olive Flounder were injected with VHSV at 102.5 , 104.5 , 106.5 , and 108.5 TCID50/100μL/fish. A second group of fish was immersed at 103.5 , 105.5 , and 107.5 TCID50/mL at 10°C for 1h in this study. The cumulative mortality was observed at 15d postinfection. Immersion challenge at 103.5 TCID50/mL caused no mortality, while intramuscular injection challenge resulted in high levels of mortality with all VHSV exposure doses. Overall, Olive Flounder was susceptible to VHSV, with cumulative mortality of 90% or 100% in fish intramuscularly injected with high or low doses of VHSV. The cumulative mortality was 40% and 70% at 105.5 and 107.5 TCID50/mL, respectively, in the immersion challenge group. The VHSV titration and copy numbers were estimated by TCID50 and quantitative reverse transcription PCR methods. From dead Olive Flounder, VHSV titration was consistently detected in all tested organs, ranging from 105 to 109 TCID50/mL. The VHSV titration was under the detection limit from surviving Olive Flounder, but the VHSV N gene was detected.

Electrochemical Biosensor Based on Surface Imprinting for Zika Virus Detection in Serum.

Zika virus (ZIKV) is a flavivirus that was first identified in 1947. Initially, the virus was of little concern for health authorities given there were very few casualties among those suffering an infection. As such, only limited studies were performed on ZIKV. Recently, the viral infection has been linked to microcephaly in infants, which has prompted a dramatic increase in scientific interest in ZIKV research, including methods to allow for rapid virus identification. In this work we report the development of a new type of ZIKV electrochemical biosensor based on surface imprinted polymers and graphene oxide composites. The biosensor was used to detect ZIKV by measuring changes in the electrical signal with changing virus concentrations in buffer and serum using standard electrochemical techniques. The detection limit of our method is similar to the detection limit of the real-time quantitative reverse transcription PCR method.

Nitrate nutrition influences multiple factors in order to increase energy efficiency under hypoxia in Arabidopsis.

Nitrogen (N) levels vary between ecosystems, while the form of available N has a substantial impact on growth, development and perception of stress. Plants have the capacity to assimilate N in the form of either nitrate (NO3-) or ammonium (NH4+). Recent studies revealed that NO3- nutrition increases nitric oxide (NO) levels under hypoxia. When oxygen availability changes, plants need to generate energy to protect themselves against hypoxia-induced damage. As the effects of NO3- or NH4+ nutrition on energy production remain unresolved, this study was conducted to investigate the role of N source on group VII transcription factors, fermentative genes, energy metabolism and respiration under normoxic and hypoxic conditions. We used Arabidopsis plants grown on Hoagland medium with either NO3- or NH4+ as a source of N and exposed to 0.8 % oxygen environment. In both roots and seedlings, we investigated the phytoglobin-nitric oxide cycle and the pathways of fermentation and respiration; furthermore, NO levels were tested using a combination of techniques including diaminofluorescein fluorescence, the gas phase Griess reagent assay, respiration by using an oxygen sensor and gene expression analysis by real-time quantitative reverse transcription-PCR methods. Under NO3- nutrition, hypoxic stress leads to increases in nitrate reductase activity, NO production, class 1 phytoglobin transcript abundance and metphytoglobin reductase activity. In contrast, none of these processes responded to hypoxia under NH4+ nutrition. Under NO3- nutrition, a decreased total respiratory rate and increased alternative oxidase capacity and expression were observed during hypoxia. Data correlated with decreased reactive oxygen species and lipid peroxidation levels. Moreover, increased fermentation and NAD+ recycling as well as increased ATP production concomitant with the increased expression of transcription factor genes HRE1, HRE2, RAP2.2 and RAP2.12 were observed during hypoxia under NO3- nutrition. The results of this study collectively indicate that nitrate nutrition influences multiple factors in order to increase energy efficiency under hypoxia.

Overexpression of eis without a mutation in promoter region of amikacin- and kanamycin-resistant Mycobacterium tuberculosis clinical strain

BackgroundAminoglycosides such as amikacin and kanamycin are effective injectable second-line drugs for treatment of multidrug-resistant tuberculosis. Molecular mechanisms underlying aminoglycoside resistance are not well understood. We have previously identified the amikacin- and kanamycin-resistant M. tuberculosis MT433 clinical strain, of which all known mutations related to resistance have not been found. Drug efflux pump is one of reported resistance mechanisms that might play a role in aminoglycoside resistance.MethodsThe expression levels of sixteen putative efflux pump genes, including eis and one regulator gene, whiB7, of MT433 in the presence of kanamycin were determined using the reverse transcription-quantitative PCR method. The effects of upregulated genes on amikacin and kanamycin resistance were investigated by overexpression in M. tuberculosis H37Ra strain.ResultsUpon kanamycin exposure, other than whiB7 and eis that were found extremely overexpressed, two drug efflux pump genes, namely Rv1877 and Rv2846c, showed specifically high-level of expression in M. tuberculosis MT433 strain. However, direct effect of overexpressed Rv1877 and Rv2846c on amikacin and kanamycin resistance could not be demonstrated in M. tuberculosis H37Ra overexpressed strain.ConclusionsOur finding demonstrated that overexpression of eis could occur without any mutations in the promoter region and be detectable in clinical isolate. This might be a consequence of overexpressed whiB7, resulting in amikacin and kanamycin resistance in M. tuberculosis MT433 strain.

Circulating and Adipose Tissue mRNA Levels of Zinc-α2-Glycoprotein, Leptin, High-Molecular-Weight Adiponectin, and Tumor Necrosis Factor-Alpha in Colorectal Cancer Patients With or Without Obesity.

To explore zinc-α2-glycoprotein (ZAG), leptin, high-molecular-weight adiponectin (HMW-ADPN), and tumor necrosis factor-alpha (TNF-α) levels in serum and subcutaneous and visceral white adipose tissue (sWAT and vWAT) among normal weight (NW) and overweight/obese (OW/OB) patients with colorectal cancer (CRC). A total of 76 Chinese CRC patients (42 NW + CRC, 34 OW/OB + CRC) and 40 healthy controls were recruited. Serum levels of the adipokines of interest were measured by an enzyme-linked immunosorbent assay method, and their mRNA levels in sWAT and vWAT were determined by reverse transcription quantitative PCR methods. Serum ZAG levels in the NW + CRC group were significantly increased by 11.7% compared with the healthy controls. Serum leptin levels in the OW/OB + CRC group were found to be increased by 57.7%, while HMW-ADPN levels were decreased by 23.5% when compared with the NW + CRC group of CRC patients. Additionally, ZAG mRNA levels in sWAT were significantly reduced by 78.8% in OB + CRC in comparison with NW + CRC patients. ZAG mRNA levels were negatively associated with body mass index (BMI) in sWAT but positively correlated with BMI in vWAT. TNF-α mRNA levels in vWAT of OB + CRC patients were significantly increased by 2.8-fold when compared with NW + CRC patients. In particular, CRC was independently associated with serum ZAG levels. The risk of CRC in participants with high tertile serum ZAG levels was 5.84-fold higher than in those with low tertile ZAG levels after adjusting for age, gender, and other confounders [odds ratio (OR) = 6.84, 95% confidence interval (CI) 1.70-27.54, P = 0.03]. The CRC risk in participants with high tertile leptin levels was only 10.7% of those with low tertile leptin levels (OR = 0.11, 95% CI 0.01-0.89, P = 0.04). The area under the receiver operating characteristic (ROC) curve of ZAG was 0.66 (95% CI 0.54-0.77, P < 0.05). At the cutoff value of 1.42 µg/mL serum ZAG, the sensitivity and specificity for differentiating patients with CRC from controls were 62.2 and 69.2%, respectively. Serum ZAG levels were significantly increased in CRC patients. Subjects with higher circulating ZAG and lower leptin levels were more likely to have CRC than those with lower ZAG and higher leptin levels. Serum ZAG might be a potential diagnostic biomarker for CRC in the Chinese population.

Establishment of a real time quantitative reverse-transcription PCR method for detection of hepatitis E virus based on standard plasmid

Objective To establish a real-time quantitative reverse-transcription PCR (qRT-PCR) method for detection of hepatitis E virus (HEV) of different genotypes based on standard HEV DNA plasmid in order to promote its application in clinical laboratory. Methods Specific primers and probe of HEV were designed based on the conserved open reading frame 3 (ORF3) regions. HEV DNA plasmids were constructed and 10-fold serial dilutions of the plasmids were prepared and used as standards to establish one-step qRT-PCR. The established method was compared with HEV antigen, antibody and RT-nPCR assays. Some positive samples were sequenced and analyzed by evolutionary tree. Results The one-step qRT-PCR method for HEV detection in serum or feces samples was successfully establish. It could reach a sensitivity of 25 copies/test and 77.8% of its results were consistent with those by HEV antigen assay. Nine patients were infected with HEV of genotypes 4a, 4d or 4n as indicated by evolutionary tree. Conclusion The HEV qRT-PCR method based on its standard plasmid is successfully established, which paves the way for commercialization of clinical applications. Key words: HEV; Standard plasmid; qRT-PCR; RT-nPCR

Reverse-transcription quantitative PCR directly from cells without RNA extraction and without isothermal reverse-transcription: a 'zero-step' RT-qPCR protocol.

We describe an ultra-rapid and sensitive method to quantify gene expression levels in cultured cells. The procedure is based on reverse-transcription quantitative PCR (RT-qPCR) directly from cells, without RNA extraction and without an isothermal reverse-transcription step. Human neurons (Lund human mesencephalic cells) were lysed at different stages of differentiation, and the lysates were used directly as template for the combined RT-qPCR reaction. We detected a down-regulation of a proliferation marker and an up-regulation of neuronal dopaminergic genes expression. We were able to detect the reference gene target from as few as a single cell, demonstrating the application of the method for efficient amplification from small cell numbers. The data were fully in line with those obtained by the standard two-step RT-qPCR from the extracted total RNA. Our ‘zero-step’ RT-qPCR method proved to be simple and reliable with a total time from cell lysis to the end of the qPCR as short as 1.5 h. It is therefore particularly suitable for RT-qPCRs where large numbers of samples must be handled, or where data are required within short time.

Molecular cloning and characterization of porcine interferon-induced protein with tetratricopeptide repeats (IFIT) 5

Zhang, J., Shao, S.-y., Li, L.-z., Liu, D. and Yang, X.-q. 2015. Molecular cloning and characterization of porcine interferon-induced protein with tetratricopeptide repeats (IFIT) 5. Can. J. Anim. Sci. 95: 551–556. Interferon-induced protein with tetratricopeptide repeats (IFIT) family members play important roles in host defense against viral infection. In the present study, the complete coding sequence (CDS) of porcine IFIT5 gene was cloned using molecular biology techniques, and the genomic structure was determined using the bioinformatic method. The porcine IFIT5 is located on chromosome 14 containing 2 exons. Quantitative real-time PCR revealed that the transcripts of IFIT5 gene were unevenly distributed in all tissues studied, including heart, bladder, liver, large intestine, spleen, small intestine, lung, kidney, stomach, muscle, and lymph. Only one synonymous single nucleotide polymorphism was found in the complete CDS except for the first five nucleotides. IFIT5 is induced by poly(I:C) in a dose- and time-dependent manner, as revealed by using dual-luciferase analysis and reverse transcription-quantitative PCR methods. Furthermore, ectopic expression of porcine IFIT5 had no effect on the activation of interferon regulatory factor 3 (IRF3) significantly (P&gt;0.05), suggesting it might not be a regulator of IRF3 signaling pathway.