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In quantitative reverse transcription polymerase chain reaction (qRT-PCR), normalizing target gene expression using a reference gene is an indispensable step to control the variability of RNA extraction yield, RNA integrity, reverse transcription efficiency, and PCR amplification. In the present study, we identified candidate reference genes with stable expression during grapes' flowering and berry development stages. Ten genes, including ACT, CYP5, RLI, TUB, UBC, UBC17, UBC60, UFD1, VAG, and ZNF with relatively stable expression, were selected based on RNAseq data generated earlier in grape hybrid 'ARI 516'. The expression of these candidate genes was tested at different stages of flowering and grape berry development. Five different algorithms such as RefFinder, geNorm, NormFinder, BestKeeper, and the comparative ΔCq method were used to test the expression stability of candidate genes. A comprehensive ranking obtained by RefFinder showed that UBC17, RLI, and ZNF were the most stable reference genes during flower and berry development stages. UBC17, RLI, and ZNF were calibrators to normalize the expression of VvAGL11 as a target gene to validate the worthiness of identified reference genes. The result demonstrated that newly identified reference genes could be successfully used to normalize the expression of the target gene accurately. These reference genes will provide more choices for selecting appropriate reference genes to normalize gene expression in grapes. [ABSTRACT FROM AUTHOR]

Identifying the type(s) of body fluid present in forensic casework exhibits can assist with scene reconstruction and indicate potential activity types. Confirmatory body fluid identification can be achieved using mRNA-based profiling assays. This commonly involves endpoint reverse-transcription PCR (RT-PCR) with capillary electrophoresis (CE) detection. In comparison, real-time quantitative RT-qPCR is more sensitive, quantitative, and does not require post-PCR processing. We have developed real-time RT-qPCR assays for forensic body fluid identification. This study compared the performance (sensitivity, PCR efficiency, precision) of five real-time RT-qPCR kits across circulatory blood, buccal, semen, and vaginal fluid samples with normal and extended (3-step) PCR cycling. An objective scoring system for the experimental performance parameters was considered along with other features of the commercial kits. Statistical analysis by ANOVA and post-hoc Tukey of the slope estimates, which relate to PCR efficiency, revealed that many observed differences were insignificant (p > 0.05). Sensitivity and precision were also similar across most kits and PCR cycling protocols. Using the scoring system, the five highest performing kit and cycling combinations were: TaqMan-extended, followed by the TaqPath-normal, QuantiNova-extended, QuantiNova-extended, then UltraPlex-normal. Based upon high performance, room temperature set-up, and multiplexing capability, the UltraPlex kit was selected for our body fluid identification assays. [ABSTRACT FROM AUTHOR]

Since the outbreak of the COVID-19 global pandemic in 2019, monitoring COVID-19 infection status and trend through wastewater, known as wastewater-based epidemiology (WBE), has been widely used in many countries and regions. WBE consists of five steps: wastewater sample collection, viral concentration, viral nucleic acid extraction, quantification of virus using quantitative RT-PCR, and dissemination of the wastewater surveillance results. This method could be used for early warning of COVID-19 outbreak in a population, monitoring COVID-19 distributions and epidemic trend, prediction of COVID-19 prevalence rate, understanding of temporal trend of SARS-COV-2 variants, and simultaneous surveillance of multiple pathogens. WBE and clinical surveillance can be used concurrently and the former is a good complement to the latter. [ABSTRACT FROM AUTHOR]

The expression level of the progesterone receptor (PGR) plays a crucial role in determining the biological characteristics of serous ovarian carcinoma. Low PGR expression is associated with chemoresistance and a poorer outcome. In this study, our objective was to explore the relationship between tumor progesterone receptor levels and RNA profiles (miRNAs, piwiRNAs, and mRNAs) to understand their biological characteristics and behavior. To achieve this, we employed next-generation sequencing of small non-coding RNAs, quantitative RT-PCR, and immunohistochemistry to analyze both FFPE and frozen tumor samples, as well as blood plasma from patients with benign cystadenoma (BSC), serous borderline tumor (SBT), low-grade serous ovarian carcinoma (LGSOC), and high-grade serous ovarian carcinoma (HGSOC). Our findings revealed significant upregulation of MMP7 and MUC16, along with downregulation of PGR, in LGSOC and HGSOC compared to BSC. We observed significant correlations of PGR expression levels in tumor tissue with the contents of miR-199a-5p, miR-214-3p, miR-424-3p, miR-424-5p, and miR-125b-5p, which potentially target MUC16, MMP7, and MMP9, as well as with the tissue content of miR-16-5p, miR-17-5p, miR-20a-5p, and miR-93-5p, which are associated with the epithelial–mesenchymal transition (EMT) of cells. The levels of EMT-associated miRNAs were significantly correlated with the content of hsa_piR_022437, hsa_piR_009295, hsa_piR_020813, hsa_piR_004307, and hsa_piR_019914 in tumor tissues. We developed two optimal logistic regression models using the quantitation of hsa_piR_020813, miR-16-5p, and hsa_piR_022437 or hsa_piR_004307, hsa_piR_019914, and miR-93-5p in the tumor tissue, which exhibited a significant ability to diagnose the PGR-negative tumor phenotype with 93% sensitivity. Of particular interest, the blood plasma levels of miR-16-5p and hsa_piR_022437 could be used to diagnose the PGR-negative tumor phenotype with 86% sensitivity even before surgery and chemotherapy. This knowledge can help in choosing the most effective treatment strategy for this aggressive type of ovarian cancer, such as neoadjuvant chemotherapy followed by cytoreduction in combination with hyperthermic intraperitoneal chemotherapy and targeted therapy, thus enhancing the treatment's effectiveness and the patient's longevity. [ABSTRACT FROM AUTHOR]

Within the successive waves that occurred during the SARS-CoV-2 pandemic, recommendations arose to test symptomatic and contact subjects by using rapid antigen devices directed against the viral nucleocapsid protein with the aim to isolate contagious patients without delay. The objective of this study was to evaluate the ability of four rapid lateral-flow tests (RLFT) that were commercially available on the French market in 2022 to recognize various strains of SARS-CoV-2. Series of five-fold dilutions of seven viral suspensions belonging to different lineages of SARS-CoV-2 (19A, 20A, Alpha, Beta, Gamma, Delta and Omicron) were used to evaluate the analytical sensitivity of four commercially available RLFTs (manufacturers: Abbott, AAZ, Becton-Dickinson and Biospeedia). Cell culture and quantitative RT-PCR were used as references. Excellent correlations were observed for each lineage strain between the viral titer obtained via cell culture and the number of RNA copies measured by quantitative RT-PCR. Although the four tests were able to recognize all the tested variants, significant differences in terms of sensitivity were observed between the four RLFTs. Despite the limitation represented by the small number of devices and clinical isolates that were tested, this study contributed by rapidly comparing the sensitivity of SARS-CoV-2 RLFTs in the Omicron era. [ABSTRACT FROM AUTHOR]

Recent studies have attempted to develop molecular signatures of epithelial ovarian cancer (EOC) based on the quantitation of protein-coding and non-coding RNAs to predict disease prognosis. Due to the heterogeneity of EOC, none of the developed prognostic signatures were directly applied in clinical practice. Our work focuses on high-grade serous ovarian carcinoma (HGSOC) due to the highest mortality rate relative to other types of EOC. Using deep sequencing of small non-coding RNAs in combination with quantitative real-time PCR, we confirm the dualistic classification of epithelial ovarian cancers based on the miRNA signature of HGSOC (type 2), which differs from benign cystadenoma and borderline cystadenoma—precursors of low-grade serous ovarian carcinoma (type 1)—and identified two subtypes of HGSOC, which significantly differ in the level of expression of the progesterone receptor in the tumor tissue, the secretion of miR-16-5p, miR-17-5p, miR-93-5p, miR-20a-5p, the level of serum CA125, tumor size, surgical outcome (optimal or suboptimal cytoreduction), and response to chemotherapy. It was found that the combined determination of the level of miR-16-5p, miR-17-5p, miR-20a-5p, and miR-93-5p circulating in blood plasma of patients with primary HGSOC tumors makes it possible to predict optimal cytoreduction with 80.1% sensitivity and 70% specificity (p = 0.022, TPR = 0.8, FPR = 0.3), as well as complete response to adjuvant chemotherapy with 77.8% sensitivity and 90.9% specificity (p = 0.001, TPR = 0.78, FPR = 0.09). After the additional verification of the obtained data in a larger HGSOC patient cohort, the combined quantification of these four miRNAs is proposed to be used as a criterion for selecting patients either for primary cytoreduction or neoadjuvant chemotherapy followed by interval cytoreduction. [ABSTRACT FROM AUTHOR]

Background: G protein‐coupled receptors (GPCRs) play crucial roles in regulating islet function, with Gαs‐ and Gαq‐coupled receptors being linked to the stimulation of insulin secretion. We have quantified the mRNA expression of 384 non‐olfactory GPCRs in islets isolated from lean and obese organ donors to determine alterations in islet GPCR mRNA expression in obesity. Methods: RT‐qPCR was used to quantify GPCR mRNAs relative to five reference genes (ACTB, GAPDH, PPIA, TBP, and TFRC) in human islets isolated from lean (BMI = 22.6 ± 0.5) and obese (BMI = 32.0 ± 0.8) donors. Results: Overall, 197 and 256 GPCR mRNAs were detected above trace level in islets from lean and obese donors, respectively, with 191 GPCR mRNAs being common to the lean and obese groups. 40.9% (n = 157) and 27.1% (n = 104) of the mRNAs were expressed at trace level whilst 7.8% and 6.3% were absent in islets from lean and obese donors, respectively. Hundred and seventeen GPCR mRNAs were upregulated at least twofold in islets from obese donors, and there was >twofold downregulation of 21 GPCR mRNAs. Of particular interest, several receptors signalling via Gαs or Gαq showed significant mRNA upregulation in islets from obese donors (fold increase: PTH2R: 54.0 ± 14.6; MC2R: 34.3 ± 11.5; RXFP1: 8.5 ± 2.1; HTR2B: 6.0 ± 2.0; GPR110: 3.9 ± 1.2; PROKR2: 3.9 ± 0.7). Conclusions: Under conditions of obesity, human islets showed significant alterations in mRNAs encoding numerous GPCRs. The increased expression of Gαs‐ and Gαq‐coupled receptors that have not previously been investigated in β‐cells opens up possibilities of novel therapeutic candidates that may lead to the potentiation of insulin secretion and/or β‐cell mass to regulate glucose homeostasis. [ABSTRACT FROM AUTHOR]

The novel coronavirus pandemic incited unprecedented demand for assays that detect viral nucleic acids, viral proteins, and corresponding antibodies. The 320 molecular diagnostics in receipt of US Food and Drug Administration emergency use authorization mainly focus on viral detection; however, no currently approved test can be used to infer infectiousness, that is, the presence of replicable virus. As the number of tests conducted increased, persistent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA positivity by reverse-transcription polymerase chain reaction (RT-PCR) in some individuals led to concerns over quarantine guidelines. To this end, we attempted to design an assay that reduces the frequency of positive test results from individuals who do not shed culturable virus. We describe multiplex quantitative RT-PCR assays that detect genomic RNA (gRNA) and subgenomic RNA (sgRNA) species of SARS-CoV-2, including spike, nucleocapsid, membrane, envelope, and ORF8. Viral RNA abundances calculated from these assays were compared with antigen presence, self-reported symptoms, and culture outcome (virus isolation) using samples from a 14-day longitudinal household transmission study. By characterizing the clinical and molecular dynamics of infection, we show that sgRNA detection has higher predictive value for culture outcome compared to detection of gRNA alone. Our findings suggest that sgRNA presence correlates with active infection and may help identify individuals shedding culturable virus. [ABSTRACT FROM AUTHOR]

Objective To optimize the extraction method of total RNA from pancreatic cancer and adjacent tissues, and provide high-quality experimental samples for the pathogenesis of pancreatic-related diseases. Methods The pretreatment of pancreatic cancer and paracancerous tissues was accomplished through shredding, RNase-free washing and RNase inhibition. RNA was isolated by liquid nitrogen grinding and trizol-chloroform extraction. The integrity of RNA was identified by agarose gel electrophoresis and bioanalyzer. With the same amount of RNA as the reverse transcription template, oligo dT primers were used for reverse transcription of messenger RNA (mRNA); stem-loop structure primers were used to reverse transcription of microRNA (miRNA). The expression levels of mRNA and miRNA were detected by quantitative RT-PCR. Compared with conventional methods, RNA samples extracted by the optimized method had a lower degradation degree and higher integrity. Results Compared with pancreatic cancer tissue, RNA in adjacent tissue is more easily degraded, and the expression level of mRNA tends to decrease. However, there is no significant difference in miRNA expression between pancreatic cancer and adjacent tissue. Conclusions Pretreatment of pancreatic cancer and adjacent tissues could reduce the RNA degradation, and provide high-quality experimental samples for RNA investigation and high experimental accuracy. [ABSTRACT FROM AUTHOR]

Aims/Introduction: Several research groups have reported methods for quantifying pancreatic beta cell (β‐cell) injury by measuring β‐cell‐specific CpG unmethylation of the insulin gene in circulation using digital droplet PCR or next‐generation sequencing. However, these methods have certain disadvantages, such as the need to consider the background signal owing to the small number of target CpG sites and the need for unique equipment. Materials and Methods: We established a novel method for detecting four CpG unmethylations of the insulin gene using two‐step amplification refractory mutation system PCR. We applied it to type 1 diabetes (T1D) patients with a wide range of disease durations and to healthy adults. Results: The assay showed high linearity and could detect a single copy of unmethylated insulin DNA in experiments using methylated and unmethylated plasmid DNA. The unmethylated insulin DNA level in the type 1 diabetes group, whose β‐cell mass was considerably reduced, was similar to that of healthy adults. An inverse correlation was observed between copy number and disease duration in patients with unmethylated insulin DNA‐positive type 1 diabetes. Conclusions: We developed a novel method for detecting unmethylated insulin DNA in circulation that can be performed using a conventional real‐time PCR system. This method would be useful for analyzing dynamic profiles of β‐cells in human disease such as type 1 diabetes. [ABSTRACT FROM AUTHOR]

Photoperiodic responses are observed in many organisms living in the temperate zones. The circadian clock is involved in photoperiodic time measurement; however, the underlying molecular mechanism for detection of the day length remains unknown. We previously compared the expression profiles of the Cryptochrome(Cry) genes in the zebrafish eye and reported that Cry1ab has a double peak with variable expression duration depending on the photoperiod. In this study, to understand commonalities and differences in the photoperiodic responses of ocular Cry genes, we identified Cryptochrome genes in two other teleost species, goldfish and medaka, living in temperate zones, and measured ocular Cry mRNA levels in all of the three species, under different photoperiods (long-day [14 h light: 10 h dark] and short-day [10 h light: 14 h dark] and in constant darkness. Cry1ab mRNA levels did not show dual peaks in goldfish or medaka under the examined conditions; however, the mRNA expression profiles of many Crys were altered in all three species, depending on the day length and light condition. Based on their expression profiles, Cry mRNA peaks were classified into three groups that better synchronize to sunrise (light-on), midnight/midday (middle points of the dark/light periods), or sunset (light-off). These results suggest the presence of multiple oscillators that oscillate independently or a complex oscillator in which Cry expression cycles change in a photoperiod-dependent manner in the eye. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Quantitative RT- PCR is one of the most common methods to study gene expression in response to stress. Therefore, it is crucial to have suitable reference genes (RGs) for result normalization. Although several reports describe UV-B-modulated gene expression in Solanum lycopersicum , there are no suitable RGs identified until now. The aim of this work was to evaluate the suitability of seven traditional genes: actin (ACT), tubulin (TUB), ubiquitin (UBI), glyceraldehyde- 3 phosphate dehydrogenase (GAPDH), elongation factor 1α (EF1α), phosphatase 2A catalytic subunit (PP2A) and GAGA binding transcriptional activator (GAGA); and two non-traditional genes: thioredoxin h1 (TRX h1) and UV-B RESISTANCE LOCUS 8 (UVR8), as candidate RGs for their potential use as reliable internal controls in leaves, stems and roots of tomato seedlings exposed to acute and chronic UV-B. The stability of these genes expression was evaluated using five statistical algorithms: geNorm, NormFinder, BestKeeper, Delta Ct and ANOVA. Considering the comprehensive stability ranking, we recommend ACT + TUB as the best pair of RGs for leaves, PP2A + GAPDH + TRX h1 for stems and TUB + UVR8 for roots. The reliability of the selected RGs for each tissue was verified amplifying tomato chalcone synthase 1 (CHS1) and cyclobutane pyrimidine dimer (CPD) photolyase (PHR1-LIKE). Under UV-B treatment, CHS1 was upregulated in leaves, stems and roots whereas PHR1-LIKE was only upregulated in leaves and stems. This interpretation differs when the most and least stable RGs are chosen. This is the first report regarding suitable RGs selection for accurate normalization of gene expression in tomato seedlings exposed to UV-B irradiation. • Nine candidate reference genes (RGs) were validated in tomato seedlings exposed to UV-B • Four conditions and three tissues were analysed with five statistical algorithms. • ACT +(TUB/PP2A/TRX h1) were the suggested RG in leaves, PP2A + GAPDH + TRX h1 in stems and TUB + UVR8 in roots. • UV-B induced CHS1 and PHR1-LIKE mRNA in leaves and stems whereas CHS1 in roots. • This work will facilitate RT-qPCR studies of UV-B exposed tomato seedlings. [ABSTRACT FROM AUTHOR]

A single transcription factor is known to coordinate expression of a set of metabolites in a biochemical pathway; its use therefore can be a functional strategy in generating plants with desired traits. Triticum aestivum Dof1 (TaDof1) transcription factor is mainly associated with improved nitrogen assimilation in plants. In the current research, the transgenic wheat overexpressing TaDof1 transcription factor, under a constitutive promoter, was developed by Agrobacterium-mediated transformation. The two elite wheat cultivars (Galaxy and Faisalabad-2008) were selected for transformation study. The T0 plants were subjected to screening using selection medium containing herbicide BASTA. PCR results confirmed that only 8 out of 31 plants possessed the complete TaDof1 cassette. A transformation efficiency of 0.46% for Galaxy and 0.08% for Faisalaad-2008 was obtained. The quantitative RT-PCR was performed on T1 plants grown under nitrogen-limiting conditions. A substantial rise in the expression of citrate synthase (CS), isocitrate dehydrogenase (ICDH), phosphoenolpyruvate carboxylase (PEPC), and pyruvate kinase (PK) genes regulated by TaDof1 was observed after 4 weeks of nitrogen stress in T1 plants. The maximum fold increase of 464 was recorded for ICDH. Our findings indicate a cooperative modification of nitrogen and carbon metabolisms since they are intimately linked together. Overexpression of TaDof1 in wheat resulted in a significant increase in various agronomic traits. Furthermore, various physiological and biochemical markers (chlorophyll, protein, and soluble sugar contents) exhibited a profound change in TaDof1 transgenic plants in comparison with wild type plants. The results clearly depict the merits of employing transcription factors in engineering plant metabolisms. [ABSTRACT FROM AUTHOR]

The quantitative Real-time Polymerase Chain Reaction (RT-qPCR) is the most common technique used to compare the expression levels of genes under different experimental or clinical conditions. Data should be normalized with housekeeping genes (HKGs) to obtain reliable results in RT-qPCR. Likewise, the use the most stable and suitable HKG for the study design improves the reliability of the study results. The selection and validation of a stable HKG according to the study design to obtain reliable data is an essential and integral step in expression -based studies. The several software programs have been developed for HKG selection. The most stable HKG to be selected for normalization needs to be carefully evaluated by experimental studies before selection. In this review, summarized Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE), characteristics of HKGs, selection and validation of most suitable and stable HKGs according to study design and, usage in RT-qPCR. [ABSTRACT FROM AUTHOR]

Background: Candida albicans is an opportunistic pathogen which is responsible for widespread nosocomial infections. It encompasses a fungus specific serine/threonine protein phosphatase gene, CaPPZ1 that is involved in cation transport, cell wall integrity, oxidative stress response, morphological transition, and virulence according to the phenotypes of the cappz1 deletion mutant. Results: We demonstrated that a short-term treatment with a sublethal concentration of tert-butyl hydroperoxide suppressed the growth of the fungal cells without affecting their viability, both in the cappz1 mutant and in the genetically matching QMY23 control strains. To reveal the gene expression changes behind the above observations we carried out a global transcriptome analysis. We used a pilot DNA microarray hybridization together with extensive RNA sequencing, and confirmed our results by quantitative RT-PCR. Novel functions of the CaPpz1 enzyme and oxidative stress mechanisms have been unraveled. The numbers of genes affected as well as the amplitudes of the transcript level changes indicated that the deletion of the phosphatase sensitized the response of C. albicans to oxidative stress conditions in important physiological functions like membrane transport, cell surface interactions, oxidation-reduction processes, translation and RNA metabolism. Conclusions: We conclude that in the wild type C. albicans CaPPZ1 has a protective role against oxidative damage. We suggest that the specific inhibition of this phosphatase combined with mild oxidative treatment could be a feasible approach to topical antifungal therapy. [ABSTRACT FROM AUTHOR]

Objective To observe the impact of serum miRNA-128a and miRNA-128b expression on early diagnosis and prognosis in patients with gastric cancer. Methods One hundred and eighteen patients with gastric cancer diagnosed in Huashan Hospital,Fudan University from Jan.2015 to Jan.2017 were selected as the gastric cancer group.Seventy-five patients with gastric polyps by pathological diagnosis and 45 healthy people were selected as gastric polyps group and healthy control.Quantitative RT-PCR was used to detect the expression levels of serum miRNA-128a and miRNA-128b in each group.The relation among serum miRNA-128a and miRNA-128b levels,clinic pathological parameters in gastric cancer patients and prognosis was observed,and their efficacy of diagnose and predicting death within 2 years was also observed. Results The serum miRNA-128a expression in gastric cancer group was significantly higher than that in gastric polyp group (q=26.388,P<0.01) and healthy control (q=32.195,P<0.01),the expression in gastric polyp group was significantly higher compared with the healthy control (q=7.198,P<0.01),and after operation the expression level was significantly lower than that before operation (t=14.321,P<0.01).The serum miRNA-128b expression in gastric cancer group was significantly lower than that in gastric polyp group (q=211.148,P<0.01) and healthy control group (q=249.984,P<0.01),the expression of those in gastric polyp group was also significantly lower compared healthy control group(q=38.232,P<0.01),and the expression was significantly higher after surgery than before surgery (t=15.139,P<0.01).The serum miRNA-128a and miRNA-128b expression had high sensitivity and specificity in the diagnosis of gastric cancer,the sensitivity of combined detection was 78.8%,the specificity was 97.3%,and the area under curve was significantly higher than miRNA-128a (Z=3.436,P<0.01) and miRNA-128b (Z=2.658,P<0.01).The expression of serum miRNA-128a and miRNA-128b in patients with gastric cancer were significantly correlated with tumor cell differentiation,TNM staging,lymph node metastasis and depth of invasion (P<0.01).After 2 years' follow-up,the serum miRNA-128a expression in survival group was significantly lower than that in death group (P<0.01).When its level was >98.47 fmol/L,the sensitivity of predicting death within 2 years was 89.5%,the specificity was 81.8%,and the curve area was 0.889.The serum miRNA-128b expression in survival group was significantly higher than that in death group (P<0.01),when its level was ≤ 94.86 fmol/L,the sensitivity of predicting death within 2 years was 94.7%,the specificity was 81.8%,and the curve area was 0.931. Conclusions Combined detection of serum miRNA-128a and miRNA-128b is helpful for diagnosis in patients with gastric cancer and has important reference value for predicting death in gastric cancer patients within 2 years. [ABSTRACT FROM AUTHOR]

l-Tyrosine serves as a common precursor for multiple valuable secondary metabolites. Synthesis of this aromatic amino acid in Bacillus licheniformis occurs via the shikimate pathway, but the underlying mechanisms involving metabolic regulation remain unclear. In this work, improved l-tyrosine accumulation was achieved in B. licheniformis via co-overexpression of aroGfbr and tyrAfbr from Escherichia coli to yield strain 45A12, and the l-tyrosine titer increased to 1005 mg/L with controlled glucose feeding. Quantitative RT-PCR results indicated that aroA, encoding DAHP synthase, and aroK, encoding shikimate kinase, were feedback-repressed by the end product l-tyrosine in the modified strain. Therefore, the native aroK was first expressed with multiple copies to yield strain 45A13, which could accumulate 1201 mg/L l-tyrosine. Compared with strain 45A12, the expression of aroB and aroF in strain 45A13 was upregulated by 21% and 27%, respectively, which may also have resulted in the improvement of l-tyrosine production. Furthermore, supplementation with 5 g/L shikimate enhanced the l-tyrosine titers of 45A12 and 45A13 by 29.1% and 24.0%, respectively. However, the yield of l-tyrosine per unit of shikimate decreased from 0.365 to 0.198 mol/mol after aroK overexpression in strain 45A12, which suggested that the gene product was also involved in uncharacterized pathways. This study provides a good starting point for further modification to achieve industrial-scale production of l-tyrosine using B. licheniformis, a generally recognized as safe workhorse. [ABSTRACT FROM AUTHOR]

p21 is a cyclin dependent kinase inhibitor, that plays an important role in cell growth, differentiation and apoptosis. The aim of present study was to analyze the mutation and expression of tumor suppressor gene ($21) in canine tumors. A total of 26 tumors (mammary tumors and mast cell tumors) and normal samples from different breed of dogs were analyzed by using direct DNA sequencing and quantitative RT-PCR for mutation and expression profiling of $21gene respectively. The coding exon of $21 gene (Exon2 and 3) did not show any polymorphism in studied samples while 69% tumors showed up-regulation of gene. Seventy five percent mammary tumors showed up-regulation of gene with highest fold change 8.69, 7.6, 5.63, 5.21 and 60% mast cell tumor showed up-regulation of gene with highest fold change 7.31. These results indicate that altered expression of tumor suppressor gene may be involved in malignant progression of tumors and might be helpful in the diagnosis and prognosis of canine tumors. [ABSTRACT FROM AUTHOR]

• The Lwr gene of Schistosoma japonicum was used for bioinformatical analysis. • SjLwr protein was greatest in internal tissues and the surface layer on day 14 in female worms. • SjLwr functions in the growth, development, and reproduction of S. japonicum. The lesswright (lwr) gene and its products are essential molecules in mitosis, DNA repair, and embryo formation in many eukaryotes. In this study, immunohistochemical analysis revealed that the Lwr protein was located in the internal tissues and the surface layer of the adult Schistosoma japonicum (Sj) worms. The mRNA expression levels of SjLwr at different points were evaluated by quantitative real-time RT-PCR. The expression of SjLwr peaked at 14 days and then decreased thereafter. SjLwr expression was relatively more stable in male worms than in female worms. The functions of SjLwr were explored by siRNA-based gene silencing with a simple soaking method. The results showed that knockdown of the SjLwr gene impaired the growth and development of S. japonicum in mice, as well as survival, morphology, reproductive capacity, and egg vitality. These observations imply that SjLwr presents a novel target for the development of immuno- and/or small molecule-based therapeutics for the control and treatment of schistosome infections. [ABSTRACT FROM AUTHOR]

• The accuracy of 5 RT-qPCRs was assessed for their ability to quantify Europe's most frequently-detected viruses. • Honeybees collected in France (2012–2019) were tested to determine the frequency of each viral load. • Viral load thresholds were suggested to differentiate covert from overt infections. Acute bee paralysis virus (ABPV), Black queen cell virus (BQCV), Chronic bee paralysis virus (CBPV), Deformed wing virus (DWV), Sacbrood virus (SBV) and Varroa destructor virus 1 (VDV1) are the six main honeybee viruses reported in Europe. We assessed the accuracy (trueness and precision) of reverse transcriptase quantitative TaqMan® PCR methods (RT-qPCR) for quantifying ABPV, BQCV, DWV, VDV1 and SBV loads. Once the systematic bias in quantitative results had been corrected (overestimation in ABPV and BQCV quantification and underestimation in that of SBV and VDV1), measurements were taken to determine the viral load ranges for which quantification uncertainty was below ± 1 log 10 equivalent of genome copies per bee (hereafter reported as genome copies/bee). The accuracy range of RT-qPCR was found to be between 6.4 and 10.4 log 10 genome copies/bee for ABPV, between 3.0 and 10.0 log 10 genome copies/bee for BQCV, between 2.4 and 10.4 log 10 genome copies/bee for DWV and between 3.4 and 10.4 log 10 genome copies/bee for SBV. Outside these ranges, the results' uncertainty is higher. VDV1 RT-qPCR accuracy was outside accuracy limits for all viral loads. Using these RT-qPCR methods, we quantified viral loads in naturally-infected honeybees. The viral load distribution and clinical signs reported with the honeybee samples allowed us to define a threshold that could be used to differentiate between covert and overt infections. These methods will be useful in diagnosing the main viral infections impairing honeybee health. [ABSTRACT FROM AUTHOR]

We optimized a SYBR Green-based quantitative RT-PCR (qRT-PCR) assay technique to detect Potato virus A from potato leaf samples. Optimization was achieved by varying the components of PCR reaction mix and also by altering the PCR conditions specifically the annealing temperature of the primers where 60°C showed good amplification without any non-specific peak. To make the assay more feasible and economical for large scale detection of PVA in potato leaf samples, the final volume of the reaction mix was reduced from 50 to 20 µl without compromising its sensitivity. To evaluate the sensitivity and efficiency of the assay, a standard curve was generated by using known concentrations of 10-fold serially diluted pure RTPCR amplified coat protein gene of PVA which showed high efficiency of 93.419 % with high linearity (co-relation coefficient of R2 = 0.996). In our qRT-PCR analysis, the standard deviation of Ct values decreased with increase in DNA concentration and increased as concentration of DNA decreased. To overcome the problem of false results, elongation factor ef 1-a gene from potato was used which confirmed the reliability of the optimized assay. The assay was also validated by running across potato leaf samples from different geographical regions of India to detect PVA and found that it was sensitive and reliable as it showed consistent results. Hence, the optimized assay could be used in rapid screening of germplasm, large scale screening of planting material for virus freedom to ensure healthy seed potato production and in routine diagnostics laboratories as well. [ABSTRACT FROM AUTHOR]

Abstract The biological bases of recovery of two grapevine cultivars, Nebbiolo and Barbera, showing different susceptibility and recovery ability to "flavescence dorée" (FD) phytoplasma infection were investigated. The expression over one vegetative season, in FD-recovered and healthy grapevines, of 18 genes involved in defence, hydrogen peroxide and hormone production was verified at two time points. Difference (Δ) between the relative expressions of August and July were calculated for each target gene of both cultivars. The significance of differences among groups assessed by univariate and multivariate statistical methods, and sPLS-DA analyses of the Δ gene expression values, showed that control and recovered grapevines of both cultivars were clearly separated. The Barbera-specific deregulation of defence genes supports a stronger response of this variety, within a general frame of interactions among H 2 O 2 , jasmonate and ethylene metabolisms, common to both varieties. This may strengthen the hypothesis that FD-recovered Barbera grapevines modulate transcription of their genes to cope with potential damages associated to the alteration of their oxidative status. Nebbiolo variety would fit into this picture, although with a less intense response, in line with its lower degree of susceptibility and recovery incidence to FD, compared to Barbera. The results evidenced a scenario where plant response to phytoplasma infection is highly affected by climatic and edaphic conditions. Nevertheless, even after several years from the original FD infection, it was still possible to distinguish, at molecular level, control and recovered grapevines of both cultivars by analyzing their overall-season response, rather than that of a single time point. [ABSTRACT FROM AUTHOR]

Abstract Blueberries (Vaccinium ssp.) show relatively high resistance to pollution and have been reported to successfully colonize acid and heavy metal-contaminated soils. Blueberries were subjected to cadmium stress using a simulated pot-culture method. The intact CDS regions of VcCXIP4 and VcYSL6 were obtained, VcCXIP4 was located in the nucleus, while VcYSL6 was located in the chloroplast. Both genes were constructed into a modified plant expression vector pCambia1301 for tobacco transformation with agrobacterium infection methods. Results showed that VcCXIP4 did not function alone in regulating cadmium (Cd) transport. Cd content of Cd in the leaves of VcYSL6 transgenic tobacco by 15.57% under high Cd concentration. Both , VcCXIP4 and VcYSL6 genes were up-regulated under Cd stress. Blueberry primarily accumulated excess Cd in the root, but Cd content in the fruit was almost independent of Cd content in the soil. Further, the effect of soil Cd content on fruit Cd content was not significant. VcCXIP4 is likely to interact with other proteins to regulate excess Cd in blueberry, while VcYSL6 is a Cd transporter required for excess Cd detoxification in blueberry. Highlights • Blueberry trees can grow in heavy-metal contaminated soils. • VcCXIP4 and VcYSL6 expression levels varied with Cd concentration treatment. • Expression of VcCXIP4 was up-regulated with increasing Cd concentration. • Expression of VcYSL6 was up-regulated with increasing Cd concentration. • Cd content in fruit did not increase significantly with increasing soil Cd content. [ABSTRACT FROM AUTHOR]

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is widely used for molecular diagnostics and evaluating prognosis in cancer. The utility of mRNA expression biomarkers relies heavily on the accuracy and precision of quantification, which is still challenging for low abundance transcripts. The critical step for quantification is accurate estimation of efficiency needed for computing a relative qRT-PCR expression. We propose a new approach to estimating qRT-PCR efficiency based on modeling dynamics of polymerase chain reaction amplification. In contrast, only models for fluorescence intensity as a function of polymerase chain reaction cycle have been used so far for quantification. The dynamics of qRT-PCR efficiency is modeled using an ordinary differential equation model, and the fitted ordinary differential equation model is used to obtain effective polymerase chain reaction efficiency estimates needed for efficiency-adjusted quantification. The proposed new qRT-PCR efficiency estimates were used to quantify GUCY2C (Guanylate Cyclase 2C) mRNA expression in the blood of colorectal cancer patients. Time to recurrence and GUCY2C expression ratios were analyzed in a joint model for survival and longitudinal outcomes. The joint model with GUCY2C quantified using the proposed polymerase chain reaction efficiency estimates provided clinically meaningful results for association between time to recurrence and longitudinal trends in GUCY2C expression. [ABSTRACT FROM AUTHOR]

[Objectives] In present work,the characteristics of the sequence structure and expression pattern involving in pathogenesis-related protein gene(BcPR5)in non-heading Chinese cabbage(Brassica campestris ssp. chinensis),were investigated to explore its species-specific function in the tolerance of downy mildew defense.[Methods]The full length sequence of BcPR5 cDNA was cloned from the disease-resisted variety ‘Suzhouqing’ by performing the rapid amplification of cDNA ends(RACE)reactions. The RT-qPCR subsequently was done for investigating the expression pattern of BcPR5 gene under the induction of downy mildew and the treatment of plant hormones involving in MeJ,SA and ABA. Additionally,the prokaryotic expression of BcPR5 protein was performed by SDS-polyacrylamide gel electrophoresis(SDS-PAGE).[Results]The full length of BcPR5 cDNA sequence is 954 bp,of which,732 bp ORF is included which encodes a functional protein by 243 amino acid with the molecular weight of 26.1×103 and isoelectric point of 9.3. The phylogenetic analysis of amino acid homologous suggested that the BcPR5 from the non-heading Chinese cabbage has the highest similarity(100%)with the homologous in Chinese cabbage. The results of RT-qPCR showed that the expression level of BcPR5 gene increased at first and then decreased during infection by downy mildew and abiotic stresses(SA,MeJ and ABA)in the resistance variety ‘Suzhouqing’ and the susceptible variety ‘Aijiaohuang’,and the transcription at higher levels in resistance variety than that of in susceptible variety. Prokaryotic expression results showed that efficient expression of the protein could be realized after induced with 1.0 mmol·L-1 IPTG for 4 h.[Conclusions]These results suggest that BcPR5 plays an important role in response to the tolerance of downy mildew defense in non-heading Chinese cabbage,and will provide a theoretical basis for further functional verification for BcPR5 gene. [ABSTRACT FROM AUTHOR]

The source of small amounts of touch DNA, which is transferred from the skin to an object when it is handled or touched, could be an issue in the forensic analysis of criminal cases. Here, we performed an extended evaluation of skin- or sweat-characteristic mRNAs to investigate their usability to infer whether an object has been handled or touched by someone. First, we compared the expression levels of candidate genes between skin swabs and other body fluids by quantitative RT-PCR analysis. Among the analyzed genes, corneodesmosin ( CDSN ), late cornified envelope 1C ( LCE1C ), filaggrin ( FLG ), desmocollin 1, and dermcidin were selected for further analysis on the basis of their specificities and sensitivities. Then, we tried to detect these genes from mock casework samples. As a result, CDSN , LCE1C , and FLG could be good markers because of their detectability. Finally, we determined the correlation between the expression of these genes and DNA yield of skin swabs to assess their adaptability as a screening test for touch DNA samples. However, the detectability of these genes was not correlated with the DNA yield of skin swab samples. In conclusion, gene expression analysis of the skin- or sweat-characteristic mRNAs CDSN , LCE1C , and FLG could be useful for inferring the skin origin of touched contact traces, but the use of the expression levels of these mRNAs for the prediction of DNA yield is problematic. To develop a screening test for touch DNA samples, other markers that have a well-correlated sensitivity with DNA analysis should be investigated. [ABSTRACT FROM AUTHOR]

Background: Recently, microRNA-133b (miR-133b) dysregulation has been shown to play a key role in several human cancers, as well as glioma. In this study, we aimed to investigate the clinical significance and prognostic value of miR-133b in glioma. Methods: Real-time quantitative PCR was employed to measure the expression level of miR-133b in tissues. Survival analysis was carried out by using the log-rank test and Kaplan-Meier method. Prognostic factors for overall survival were identified by univariate and multivariate analyses using the Cox proportional hazards regression model. Results: The expression level of miR-133b was significantly lower in glioma tissues compared with matched non-cancerous brain tissues (p < .05). Its level was strongly correlated with Karnofsky Performance Scale score (p < .001) and WHO grade (p < .001). Kaplan-Meier survival and log-rank analysis indicated that the decreased expression of miR-133b was strongly correlated with shorter overall survival of patients with glioma (log-rank test, p = .03). Conclusions: The current investigation demonstrated that miR-133b level is useful for predicting the prognosis of patients with glioma. [ABSTRACT FROM AUTHOR]

The ascomycete fungus Beauveria bassiana is a natural pathogen of hundreds of insect species and is commercially produced as an environmentally friendly mycoinsecticide. Many genes involved in fungal insecticide infection have been identified but few have been further explored. In this study, we constructed three transcriptomes of B. bassiana at 24, 48 and 72 h post infection of insect pests (BbI) or control (BbC). There were 3148, 3613 and 4922 genes differentially expressed at 24, 48 and 72 h post BbI/BbC infection, respectively. A large number of genes and pathways involved in infection were identified. To further analyze those genes, expression patterns across different infection stages (0, 12, 24, 36, 48, 60, 72 and 84 h) were studied using quantitative RT-PCR. This analysis showed that the infection-related genes could be divided into four patterns: highly expressed throughout the whole infection process (thioredoxin 1); highly expressed during early stages of infection but lowly expressed after the insect death (adhesin protein Mad1); lowly expressed during early infection but highly expressed after insect death (cation transporter, OpS13); or lowly expressed across the entire infection process (catalase protein). The data provide novel insights into the insect-pathogen interaction and help to uncover the molecular mechanisms involved in fungal infection of insect pests. [ABSTRACT FROM AUTHOR]

Salmonella enterica serovar typhimurium is one of the important worldwide health issues. The hilA gene encodes a transcriptional activator that regulates expression of the majority of the genes responsible for the Salmonella invasive phenotype. In the present study, PCR was performed using our designed primers for amplification of upstream and downstream regions of Salmonella typhimurium hilA gene. Each DNA fragment was T/A-cloned into pGEM-T easy vector and then sub-cloned into pET32 expression vector together with Kanamycin resistance gene. The recombinant plasmid was transformed into bacteria (Salmonella typhimurium) using electroporation. The hilA-Knockout mutant was characterized to evaluate the predicted role of the hilA gene in virulence. Quantitative RT-PCR was carried out to study the impact of hilA knock out on the expression of downstream genes including invF and invA. We confirmed the successful preparation of hilA gene construct (pET32-up-kan-down) followed by the efficient electroporation of the construct to bacterial cells. The homologous recombination resulted in the hilA confirmed by PCR. We demonstrated that hilA knockout leads to attenuation of invA and invF genes expression. The hilA knockout strain may be useful for the development of efficient vaccines against the Salmonella typhimurium. [ABSTRACT FROM AUTHOR]

Although several ant species are important targets for the development of molecular control strategies, only a few studies focus on identifying and validating reference genes for quantitative reverse transcription polymerase chain reaction (RT-qPCR) data normalization. We provide here an extensive study to identify and validate suitable reference genes for gene expression analysis in the ant Atta sexdens, a threatening agricultural pest in South America. The optimal number of reference genes varies according to each sample and the result generated by RefFinder differed about which is the most suitable reference gene. Results suggest that the RPS16, NADH and SDHB genes were the best reference genes in the sample pool according to stability values. The SNF7 gene expression pattern was stable in all evaluated sample set. In contrast, when using less stable reference genes for normalization a large variability in SNF7 gene expression was recorded. There is no universal reference gene suitable for all conditions under analysis, since these genes can also participate in different cellular functions, thus requiring a systematic validation of possible reference genes for each specific condition. The choice of reference genes on SNF7 gene normalization confirmed that unstable reference genes might drastically change the expression profile analysis of target candidate genes. [ABSTRACT FROM AUTHOR]

Background/Aim: Variability in the severity and age at onset of autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI) may be associated with certain types of variants in the arginine vasopressin (AVP) gene. In this study, we aimed to describe a large family with an apparent predominant female occurrence of polyuria and polydipsia and to determine the underlying cause. Methods: The family members reported their family demography and symptoms. Two subjects were diagnosed by fluid deprivation and dDAVP challenge tests. Eight subjects were tested genetically. The identified variant along with 3 previously identified variants in the AVP gene were investigated by heterologous expression in a human neuronal cell line (SH-SY5Y). Results: Both subjects investigated clinically had a partial neurohypophyseal diabetes insipidus phenotype. A g.276_278delTCC variant in the AVP gene causing a Ser18del deletion in the signal peptide (SP) of the AVP preprohormone was perfectly co-segregating with the disease. When expressed in SH-SY5Y cells, the Ser18del variant along with 3 other SP variants (g.227G>A, Ser17Phe, and Ala19Thr) resulted in reduced AVP mRNA, impaired AVP secretion, and partial AVP prohormone degradation and retention in the endoplasmic reticulum. Impaired SP cleavage was demonstrated directly in cells expressing the Ser18del, g.227G>A, and Ala19Thr variants, using state-of-the-art mass spectrometry. Conclusion: Variants affecting the SP of the AVP preprohormone cause adFNDI with variable phenotypes by a mechanism that may involve impaired SP cleavage combined with effects at the mRNA, protein, and cellular level. [ABSTRACT FROM AUTHOR]

Tropane alkaloids (TAs), especially hyoscyamine and scopolamine, are important precursors for anticholinergic and antispasmodic drugs. Hyoscyamine and scopolamine are currently obtained at commercial scale from hybrid crosses of Duboisia myoporoides Duboisia leichhardtii plants. In this study, we present a global investigation of the localization and organization of TA biosynthesis in a Duboisia myoporoides R. Br. wild-type line. The tissue-specific spatial distribution of TAs within D. myoporoides is presented, including quantification of the TAs littorine, 6-hydroxy hyoscyamine, hyoscyamine, scopolamine and, additionally, hyoscyamine aldehyde as well as scopolamine glucoside. Scopolamine (14.77 ± 5.03 mg g-1), and to a lesser extent hyoscyamine (3.01 ± 1.54 mg g-1) as well as 6-hydroxy hyoscyamine (4.35 ± 1.18 mg g-1), are accumulated in leaves during plant development, with the highest concentration of total TAs detected in 6-month-old plants. Littorine, an early precursor in TA biosynthesis, was present only in the roots (0.46 ± 0.07 mg g-1). During development, the spatial distribution of all investigated alkaloids changed due to secondary growth in the roots. Transcripts of pmt, tr-I and cyp80f1 genes, involved in early stages of TA biosynthesis, were found to be most abundant in the roots. In contrast, the transcript encoding hyoscyamine 6β-hydroxylase (h6h) was highest in the leaves of 3-month-old plants. This investigation presents the spatial distribution of biochemical components as well as gene expression profiles of genetic factors known to participate in TA biosynthesis in D. myoporoides. The results of this investigation may aid in future breeding or genetic enhancement strategies aimed at increasing the yields of TAs in these medicinally valuable plant species. [ABSTRACT FROM AUTHOR]

The aim of the study was to evaluate the effect of inorganic and organic forms of Zn on the expression of cytokines (IL-2, TNF-α, IFN-γ, IL-12, IL-17, IL-4, IL-10, and TGF-β) and immunoglobulins (IgA and IgG) in the tissues of the small intestine (jejunum and ileum) of broiler chickens. In the experiment, 90 broiler chickens were divided into 4 experimental groups and a control group, with 18 birds each. The birds received Zn supplements in inorganic form with and without phytase (ZnSO4 and ZnSO4 + F), and in organic form with glycine, with and without phytase (Zn--Gly and Zn--Gly + F). The total rearing period was 42 days. Quantitative real-time (RT)- PCR was used to measure the expression of the cytokines and immunoglobulins. The differences between the results obtained for the control and experimental groups, between the groups receiving ZnSO4 and Zn- Gly, and between groups ZnSO4-F and Zn-Gly-F were analyzed statistically. High relative expression of IL-2 was observed for the chickens in the groups receiving ZnSO4-F, Zn-Gly, and Zn-Gly-F on d 42 in comparison to the control group. High relative expression of TNF-α, IL-12, and IL-17 was noted in the group that received ZnSO4 + F. High expression of IgG, IgA, IL-4, TGF-β, and IL-10 was noted in the groups of chickens that received feed supplemented with Zn--Gly and Zn--Gly + F chelates on d 42 of the study in comparison to the control group. In conclusion, supplementation with Zn--Gly chelates can ensure Th1 and Th2 balance during the immune response in the gut-associated lymphoid tissue (GALT), and, by increasing IgA and IgG expression, also can stimulate potentiation of the immune response involved in passive protection of the body from infection. In contrast, the use of inorganic forms of Zn, in the form of sulfates, can induce local inflammatory processes in the intestines, which, in the case of long-term supplementation, lead to the development of infections. [ABSTRACT FROM AUTHOR]

Flowering, an important agronomic trait for seed plants, represents the end of vegetative growth and begins to reproduce. In regulating the floral transition, FLOWERING LOCUS T (FT) encoding a mobile floral signal protein that belongs to the phosphatidylethanolamine-binding protein (PEBP) family acts as critical role. Here, an FT-like gene, DcFT, was isolated and cloned from European Daucus carota L. cultivar 'Nantes-H06' (GenBank accession number KY768910), and an alignment of the DcFT protein and other FT-homolog proteins showed that it shared 88.00% similarity with CsFT from Camellia sinensis. Phylogenetic tree analysis indicated that DcFT had the closest relationship with GpFT ( Gypsophila paniculata). Quantitative RT-PCR was analyzed to show that the expression pattern of DcFT in inflorescences sharply increased after 10DAA and then slowly increased reaching the maximum at 30DAA. Subcellular localization made clear that the DcFT protein was located in the nucleus and cytoplasm. Broadly speaking, DcFT is an FT-like homologous gene in carrot regulating the floral transition and could be a candidate gene for accelerating the process in carrot breeding. [ABSTRACT FROM AUTHOR]

Pleurotus ostreatus laccase gene poxc is transcriptionally induced by copper, and several putative MREs have been found and confirmed in its promoter region. However, the related transcription factors mediating copper response via MREs have not been reported. To isolate MRE binding proteins, we carried out yeast one-hybrid screens. Reporter genes containing two trimers of the cMRE2 and cMRE3 element were prepared and introduced into a yeast strain. The yeast was transformed with library cDNA that represents RNA isolated from CuSO 4 -treated fungi of P. ostreatus . From the screen of yeast transformants, we isolated ltf4 which encoded protein with HTH DNA binding domain. Electrophoretic mobility shift assay suggested direct and specific interaction of ltf4 with the MRE2 of poxc . Quantitative RT-PCR showed that the transcription of ltf4 was significantly up-regulated after the copper addition, and the expression trend was consistent with the poxc after copper introduction. The results indicated that ltf4 could interact with cMRE2 and thus participated in the regulation of copper mediated poxc transcription in P. ostreatus . [ABSTRACT FROM AUTHOR]

Clubroot disease is a serious threat to canola production in western Canada and many parts of the world. Rcr1 is a clubroot resistance (CR) gene identified recently and its molecular mechanisms in mediating CR have been studied using several omics approaches. The current study aimed to characterize the biochemical changes in the cell wall of canola roots connecting to key molecular mechanisms of this CR gene identified in prior studies using Fourier transform infrared (FTIR) spectroscopy. The expression of nine genes involved in phenylpropanoid metabolism was also studied using qPCR. Between susceptible (S) and resistance (R) samples, the most notable biochemical changes were related to an increased biosynthesis of lignin and phenolics. These results were supported by the transcription data on higher expression of BrPAL1. The up-regulation of PAL is indicative of an inducible defence response conferred by Rcr1; the activation of this basal defence gene via the phenylpropanoid pathway may contribute to clubroot resistance conferred by Rcr1. The data indicate that several cell-wall components, including lignin and pectin, may play a role in defence responses against clubroot. Principal components analysis of FTIR data separated non-inoculated samples from inoculated samples, but not so much between inoculated S and inoculated R samples. It is also shown that FTIR spectroscopy can be a useful tool in studying plant-pathogen interaction at cellular levels. [ABSTRACT FROM AUTHOR]

Frequent replanting causes biotic stress due to an altered soil and rhizosphere biome and results in apple replant disease (ARD). The disease is expressed by diminished growth and negatively affects fruit yield and quality. Recent studies aiming at understanding ARD on a molecular level showed that ARD affected plants suffer from oxidative stress. Genes involved in secondary metabolism reactions play an important role in the molecular ARD response of roots. Although the root system has to endure the biotic stress attack in the first place, severe symptoms of ARD can be visualized on aboveground plant parts. The objective of this study was to examine the transcriptomic response of leaves representing the metabolically active aboveground parts of ARD challenged apple plants and to compare results with existing data for roots to generate a more complete picture of ARD affected molecular reactions. For this, biotic stress response genes induced by ARD in roots were studied in RT-qPCR analyses using leaves of ARD sensitive ‘M26’ plants grown in two ARD soils, also in a time-dependent approach. Furthermore, an RNA sequencing approach employing MACE (massive analysis of cDNA ends) for transcriptome profiling was performed in order to identify further leaf specific candidate genes. RT-qPCR analyses did not reveal major differences in root candidate gene expression, but MACE indicated the upregulation of common biotic stress response genes. However, potential systemic oxidative stress occurred and ‘M26’ plants did not develop an effective defense response to ARD. [ABSTRACT FROM AUTHOR]

Heavy metals are significant inorganic environmental pollutants. Brassica juncea, frequently used as model for phytoremediation studies, can (hyper)accumulate some heavy metals with well-developed root system. Even though the elucidation of the differentially expressed genes in response to heavy metals is important for the improvement of phytoremediation capacity of plants, there has been limited number of reports about detailed functional characterization of these genes. In this study, 50 μM Pb(NO 3 ) 2 and 25 μM CdSO 4 were applied separately to 31 days-old Brassica juncea (var. P78) plants for 24 h to identify the putative genes associated with lead (Pb) and cadmium (Cd) stress. The metal content analyses indicated that B. juncea (var. P78) is lead and cadmium hyperaccumulator. To reveal molecular mechanisms responsible for metal metabolism, the differentially expressed genes in the roots of Pb-treated plants were investigated by using the microarray chips of Arabidopsis thaliana probes (Affymetrix-The GeneChip ® Arabidopsis ATH1 Genome Array). Out of 183 differentially expressed genes in response to Pb stress, only 20 of the up-regulated and 18 of the down-regulated genes were statistically significant at p < 0.05. Since most of 38 genes differentially expressed under Pb stress were interspecifically very well conserved, we concluded that the use of interspecific hybridization approaches for global gene expression profiling would be limited. The interspecific usability of the probes was further tested by determining the level of conservation between Arabidopsis microarray oligo and Brassica spp. sequences. The microarray results were validated by quantifying the level of expression of six important genes, which are likely to play a role in Pb metabolism, through quantitative RT-PCR. To further pinpoint the possible role of Pb stress-response genes, the expression of these genes at different tissues of the plants after Pb and Cd treatments were measured. In conclusion, the described genes are likely to be associated with Pb and Cd stress metabolism and provide potential targets for the improvement of phytoremediation capacity in plants. [ABSTRACT FROM AUTHOR]

Background: Invertases (INVs) are key enzymes regulating sucrose metabolism and are here revealed to be involved in responses to environmental stress in plants. To date, individual members of the invertase gene family and their expression patterns are unknown in sugarcane due to its complex genome despite their significance in sucrose metabolism. Results: In this study, based on comparative genomics, eleven cDNA and twelve DNA sequences belonging to 14 non-redundant members of the invertase gene family were successfully cloned from sugarcane. A comprehensive analysis of the invertase gene family was carried out, including gene structures, phylogenetic relationships, functional domains, conserved motifs of proteins. The results revealed that the 14 invertase members from sugarcane could be clustered into three subfamilies, including 6 neutral/alkaline invertases (ShN/AINVs), and 8 acid invertases (ShAINVs). Faster divergence occurred in acid INVs than in neutral/alkaline INVs after the split of sugarcane and sorghum. At least a one-time gene duplication event was observed to have occurred in the four groups of acid INVs, whereas ShN/AINV1 and ShN/AINV2 in the ß8 lineage were revealed to be the most recently duplicated genes among their paralogous genes in the ß group of N/AINVs. Furthermore, comprehensive expression analysis of these genes was performed in sugarcane seedlings subjected to five abiotic stresses (drought, low temperature, glucose, fructose, and sucrose) using Quantitative Real-time PCR. The results suggested a functional divergence of INVs and their potential role in response to the five different treatments. Enzymatic activity in sugarcane seedlings was detected under five abiotic stresses treatments, and showed that the activities of all INVs were significantly inhibited in response to five different abiotic stresses, and that the neutral/alkaline INVs played a more prominent role in abiotic stresses than the acid INVs. Conclusions: In this study, we determined the INV gene family members of sugarcane by PCR cloning using sorghum as a reference, providing the first study of the INV gene family in sugarcane. Combining existing INV gene data from 7 plants with a comparative approach including a series of comprehensive analyses to isolate and identify INV gene family members proved to be highly successful. Moreover, the expression levels of INV genes and the variation of enzymatic activities associated with drought, low temperature, glucose, fructose, and sucrose are reported in sugarcane for the first time. The results offered useful foundation and framework for future research for understanding the physiological roles of INVs for sucrose accumulation in sugarcane. [ABSTRACT FROM AUTHOR]

Ralstonia solanacearum, a soil-borne bacterium causes bacterial wilt, is a lethal disease of eggplant ( Solanum melongena L.). However, the first line of defense mechanism of R. solanacearum infection remains unclear. The present study focused on the role of induced HO, defense-related enzymes of ascorbate-glutathione pathway variations in resistant and susceptible cultivars of eggplant under biotic stress. Fifteen cultivars of eggplant were screened for bacterial wilt resistance, and the concentration of antioxidant enzymes were estimated upon infection with R. solanacearum. A quantitative real-time PCR was also carried out to study the expression of defense genes. The concentration of HO in the pathogen inoculated seedlings was two folds higher at 12 h after pathogen inoculation compared to control. Antioxidant enzymes of ascorbate-glutathione pathway were rapidly increased in resistant cultivars followed by susceptible and highly susceptible cultivars upon pathogen inoculation. The enzyme activity of ascorbate-glutathione pathway correlates by amplification of their defense genes along with pathogenesis-related protein-1a (PR-1a). The expressions of defense genes increased 2.5−3.5 folds in resistant eggplant cultivars after pathogen inoculation. The biochemical and molecular markers provided an insight to understand the first line of defense responses in eggplant cultivars upon inoculation with the pathogen. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is a food-borne pathogen of humans and other animals. The striking ability to survive several stresses usually used for food preservation makes L. monocytogenes one of the biggest concerns to the food industry. This ubiquity can be partly explained by the ability of the organism to grow and persist at very low temperatures, a consequence of its ability to accumulate cryoprotective compound called osmolytes. A quantitative RT-PCR assay was used to measure mRNA transcript accumulation for the stress response genes opuCA and betL (encoding carnitine and betaine transporters, respectively) and the housekeeping gene 16S rRNA. Assays were conducted on mid-exponential phase L. monocytogenes cells exposed to conditions reflecting cold and freezing stress, conditions usually used to preserve foods. We showed that expression of the two cold-adapted genes encoded the transporters of the cryoprotectants carnitine and betaine in ATCC 19115 and the food-isolated L. monocytogenes S1 is induced after cold and freezing stress exposure. Furthermore, transcriptional analysis of the genes encoding opuCA and betL revealed that each transporter is induced to different degrees upon cold shock of L. monocytogenes ATCC 19115 and S1. Our results confirm an increase in carnitine uptake at low temperatures more than in betaine after cold-shocked temperature compared to the non-stress control treatment. It was concluded the use of carnitine and betaine as cryoprotectants is essential for rapid induction of the tested stress response under conditions typically encountered during food preservation. [ABSTRACT FROM AUTHOR]

Focal adhesion kinase (FAK) is critical in adhesion-dependent signaling, but its role in osteogenesis in vivo is ill defined. We deleted Fak in fibroblasts and osteoblasts in Floxed-Fak mice bred with those expressing Cre-recombinase driven by 3.6-kb a1(I)-collagen promoter. Compared with wild-type (WT), conditional FAK-knockout (CFKO) mice were shorter (2-fold; P < 0.0001) and had crooked, shorter tails (50%; P < 0.0001). Microcomputed tomography analysis showed reduced bone volume (4-fold in tails; P < 0.0001; 2-fold in mandibles; P < 0.0001), whereas bone surface area/bone volume increased (3-fold in tails; P < 0.0001; 2.5-fold in mandibles; P < 0.001). Collagen density and fiber alignment in periodontal ligament were reduced by 4-fold (P < 0.0001) and 30% (P < 0.05), respectively, in CFKO mice. In cultured CFKO osteoblasts, mineralization at d 7 and mineralizing colony-forming units at d 21 were 30% (P < 0.0001) and >3-fold less than WT, respectively. Disruptions of FAK function in osteoblasts by conditional knockout, siRNA-knockdown, or FAK inhibitor reduced mRNA and protein expression of Runx2 (>30%), Osterix (>25%), and collagen-1 (2-fold). Collagen synthesis was abrogated in WT osteoblasts with Runx2 knockdown and in Fak-null fibroblasts transfected with an FAK kinase domain mutant or a kinase-impaired mutant (Y397F). These data indicate that FAK regulates osteogenesis through transcription factors that regulate collagen synthesis.--Rajshankar, D., Wang, Y., McCulloch, C. A. Osteogenesis requires FAK-dependent collagen synthesis by fibroblasts and osteoblasts. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV) is etiologically linked to multiple acute, chronic and malignant diseases. Detection of EBV-RNA transcripts in tissues or biofluids besides EBV-DNA can help in diagnosing EBV related syndromes. Sensitive EBV transcription profiling yields new insights on its pathogenic role and may be useful for monitoring virus targeted therapy. Here we describe a multi-gene quantitative RT-PCR profiling method that simultaneously detects a broad spectrum (n = 16) of crucial latent and lytic EBV transcripts. These transcripts include (but are not restricted to), EBNA1, EBNA2, LMP1, LMP2, BARTs, EBER1, BARF1 and ZEBRA, Rta, BGLF4 (PK), BXLF1 (TK) and BFRF3 (VCAp18) all of which have been implicated in EBV-driven oncogenesis and viral replication. With this method we determine the amount of RNA copies per infected (tumor) cell in bulk populations of various origin. While we confirm the expected RNA profiles within classic EBV latency programs, this sensitive quantitative approach revealed the presence of rare cells undergoing lytic replication. Inducing lytic replication in EBV tumor cells supports apoptosis and is considered as therapeutic approach to treat EBV-driven malignancies. This sensitive multi-primed quantitative RT-PCR approach can provide broader understanding of transcriptional activity in latent and lytic EBV infection and is suitable for monitoring virus-specific therapy responses in patients with EBV associated cancers. [ABSTRACT FROM AUTHOR]

Biofilm architecture provides bacteria with enhanced antibiotic resistance, thus raising the need to search for alternative therapies that can inhibit the bacterial colonization. In the present study, we synthesized graphene oxide-silver nanocomposite (GO-Ag) by non-toxic and eco-friendly route using a floral extract of Legistromia speciosa (L.) Pers. The gas chromatography-mass spectrometry (GC-MS) analysis of plant extract revealed the presence of compounds which can simultaneously act as reducing and capping agents. The sub-inhibitory concentrations of synthesized GO-Ag reduced the biofilm formation in both gram-negative ( E. cloacae ) and gram-positive ( S. mutans ) bacterial models. Growth curve assay, membrane integrity assay, scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM) revealed different mechanisms of biofilm inhibition in E. cloacae and S. mutans. Moreover, quantitative RT-PCR (qRT-PCR) results suggested GO-Ag is acting on S. mutans biofilm formation cascade. Biofilm inhibitory concentrations GO-Ag were also found to be non-toxic against HEK-293 (human embryonic kidney cell line). The whole study highlights the therapeutic potential of GO-Ag to restrain the onset of biofilm formation in bacteria. [ABSTRACT FROM AUTHOR]

Background: Ascorbic acid (Asc) is one of the most abundant antioxidants and it serves as amajor contributor to protect plants against oxidative damage. Plants use two enzymes that participate in the metabolic recycling of Asc. One of these two enzymes is dehydroascorbate reductase (DHAR). It directly regenerates Asc from its oxidized state and thus prevents Asc from being irreversibly hydrolyzed to 2, 3-diketogulonic acid. This study aimed to examine whether over-expression of DHAR leads to an enhanced oxidative stress tolerance in tobacco plants. Results: In this study, we functionally characterized a novel JcDHAR gene from Jatropha curcas and found via quantitative RT-PCR analysis that JcDHAR can be induced with H2O2, salt and PEG stresses. The DHAR activities of transgenic tobacco plants increased from 2.0 to 5.3 fold compared to wild-type plants. As a result, the transgenic plants displayed enhanced tolerance to oxidative stress. Conclusions: Our results indicate that JcDHAR expression can effectively enhance the tolerance to oxidative stress in plants. [ABSTRACT FROM AUTHOR]

To evaluate the application of a DNA-based analysis of a microbial technique for oil and gas prospecting, the hydrocarbon potential predicted using the methanotrophic pmoA and propanotrophic prmA genes as targets was investigated in soil above a known oil reservoir. In total, 82 soil samples were collected at 50-cm depths above the Xiliu oil reservoir for quantitative real-time polymerase chain reaction (RT-PCR) analysis. The pmoA gene content ranged from 0 to 2.7 × 106 copies/g dw, whereas that of prmA varied from 0 to 1.6 × 106 copies/g dw. Microbial anomaly distribution maps of normalized values for the prmA gene as a main indicator suggested that the area with the highest potential of prospecting was an undeveloped location, and an area with a relatively large prmA background was identified at this oil-producing site. The areas of microbial anomaly exhibited different prmA/pmoA ratios (20, 1–5, 0.0n–0.25 and 0), and this ratio may be used as an indicator for predicting the properties of oil and gas reservoirs and biogenic methane or the influence of oil-producing activities. Our results suggest that a DNA-based analysis of a microbial technique is a powerful tool for oil and gas prospecting. This study provides a new microbial technique for the prospecting of oil and gas. [ABSTRACT FROM AUTHOR]

Interaction of living organisms with quantum dots (QDs) is certainly more focused on environment and occupational exposure associated with production and release or disposal. Here, the transcription of genes involved in mitochondrial organization and function in HepG2 cells exposed to cadmium sulphide (CdS) QDs has been profiled to highlight biomarkers of exposure and effect to be tested for other cadmium based QDs. At low concentrations, exposure to CdS QDs induced only minor damage to nuclear DNA, and none to mitochondrial DNA. However, the stress caused an increase in the production of reactive oxygen species (ROS), which triggered the mitochondria-mediated intrinsic apoptotic pathway involving a cascade of transcriptomic events, finally prompting the activation of a rescue pathway. The transcriptomic analysis confirmed the involvement in the response to CdS QDs of genes related to apoptosis ( AIFM2 and APAF1 ), oxidative stress response ( OXR1 and AOX1 ) and autophagy ( ATG3 and ATG7 ), as potential biomarkers. Other possible biomarkers specific for mitochondria function were LONP1 and HSPD1 . [ABSTRACT FROM AUTHOR]

Despite the rapid spread of Zika virus (ZIKV) infection and associated neurological complications in the America’s, prophylactic or therapeutic countermeasures are not currently available. This is mostly due to the fact that until recently there was no presumed need for medical intervention since there was no association between ZIKV infection and significant human morbidity. Consequently, there are currently no tools due mostly to the lack of sensitive cell based assays amenable for identification of ZIKV inhibitors. To address this unmet need we have developed a cell based virus yield assay suitable for testing antivirals against Zika virus. Using bioinformatics, several isolates of ZIKV from the Americas, Africa, and Asia were analyzed for sequence similarity. The alignment data were then used to design primers targeting a ZIKV genomic region that was highly conserved among all the ZIKV isolates. Subsequently, primers were used in a sensitive, quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay to detect ZIKV RNA. The qRT-PCR assay was found to be highly sensitive (lower limit of detection between-10-100 copies) and reproducible. Evaluation of the primers and probes used for ZIKV against another flavivirus (Dengue virus) demonstrated specificity of detection. To evaluate potential of qRT-PCR assay as an antiviral screening tool against ZIKV, Vero cells pretreated with Type I Interferons (IFN α) were infected with virus, followed by measurement of ZIKV RNA found in the cell culture supernatants using qRT-PCR assay. Dose-dependent antiviral activity of Type I Interferons and mycophenolic acid (MPA) against Zika virus in this cell culture system was confirmed using qRT-PCR. Due to reproducible assay performance, qPCR associated higher sensitivity and short duration of the assay time, this novel cell based assay will be very useful for confirming the activity of antivirals against ZIKV. [ABSTRACT FROM AUTHOR]