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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.

Cytoplasmic male sterility (CMS)-restorer system is a useful tool to exploit heterosis in soybean. The major restorer gene for the M-type CMS is known as Rf-m, located in the 162.4-kb region on chromosome 16. Sequence analysis has revealed that the Rf-m locus in Glycine max consists of seven pentatricopeptide repeat (GmPPR) genes. The deduced amino acid sequences contain 8 to 14 PPR motifs, and a phylogenetic analysis grouped these GmPPR proteins into two PPR subfamilies: Glyma.16G161800 belongs to the PLS subfamily, and the P subfamily consists of Glyma.16G161900, Glyma.16G162000, Glyma.16G162100, Glyma.16G162700, Glyma.16G162800, and Glyma.16G163100. The phylogenetic analysis of seven GmPPR proteins and 27 other plant PPR proteins also showed that proteins in the same subfamilies cluster together. Comparative sequence analysis was conducted using the seven Rf-m candidate GmPPR genes from the sterile line W931A, the maintainer line W931B, and the restorer line WR016, the result showed that Glyma.16G161900 had higher polymorphism than the other candidate genes. Based on real-time quantitative RT-PCR data, all seven GmPPR genes were differentially expressed but showed constitutive expression in roots, stems, leaves, and pollen grains. Additionally, the expression level of Glyma.16G161900 in the sterile line W931A was significantly higher in all tissues than in the restorer line WR016. Taken together, these results suggest that Glyma.16G161900 is the most likely candidate for the restorer gene Rf-m. This study is the first report and analysis of candidate fertility restorer (Rf) genes encoding PPR proteins in soybean.

Chronic diabetes extensively complicates the glucose metabolism to onset and progress the complication. Concurrently, several contemporary medicines, especially organo-metallic formulations, are emerging to treat hyperglycemia. The current study aims to emphasize the gold nanoparticles (GNPs) potential for glucose metabolism regulation in Streptozotocin (STZ) induced diabetes. Quantitative real-time polymerase chain reaction (RT-PCR) was carried out to detect the mRNA expression of Glucose transporters 2 (GLUT2), Glucokinase (GK) and Glucose 6 Phosphatase (G-6-Pase). The study shows remarkable results such as the prognostic effect of GNPs in reinforcing the repression of enzyme complex G-6-Pase about 13.3-fold when compared to diabetes control. Also, molecular docking studies showed significant inhibition of G-6-Pase by the terpenoid ligands with alpha and beta amyrin from leaf extract of Couroupita guianensis. Thus the study explored the novel mechanism of G-6-Pase downregulated by GNPs intervention that majorly contributes to the regulation of circulatory glucose homeostasis during diabetes.

Rice (Oryza sativa L.) chalkiness greatly reduces the rice quality and the commercial value. In this study, qWCR4, a previously reported quantitative trait locus (QTL) of white-core rate (WCR), was confirmed by a BC5F2 segregation population and further fine mapped to a 35.26 kb region. In the qWCR4 region, LOC_Os04g50060 and LOC_Os04g50070 showed significant differences in expression level in endosperm between two NILs, whereas four other genes had no expression. Starch granules in the central endosperm of chalky grains from NIL(J23B) with higher WCR exhibited a typically round and loosely packed morphology. NIL(J23B) with higher WCR accompanied a higher seed filling speed. Moreover, qWCR4J23B (qWCR4 allele in J23B) increased WCR, grain numbers per plant, seed setting rate, grain width, and thousand-grain weight, contributing to a superior yield per plant. All in all, our research results not only lay a foundation for map-based cloning of qWCR4 but also provide new genetic resources for rice yield and quality breeding.

Almond [Prunus dulcis(Mill.) D.A. Webb] is an economically important nut crop susceptible to the genetic disorder, Non-infectious Bud Failure (NBF). Despite the severity of exhibition in several prominent almond cultivars, no causal mechanism has been identified underlying NBF development. The disorder is hypothesized to be associated with differential DNA methylation patterns based on patterns of inheritance (i.e., via sexual reproduction and clonal propagation) and previous work profiling methylation in affected trees. Peach (Prunus persicaL. Batsch) is a closely related species that readily hybridizes with almond; however, peach is not known to exhibit NBF. A cross between an NBF-exhibiting ‘Carmel’ cultivar and early flowering peach (‘40A17’) produced an F1where ∼50% of progeny showed signs of NBF, including canopy die-back, erratic branching patterns (known as “crazy-top”), and rough bark. In this study, whole-genome DNA methylation profiles were generated for three F1progenies exhibiting NBF and three progenies considered NBF-free. Subsequent alignment to both the almond and peach reference genomes showed an increase in genome-wide methylation levels in NBF hybrids in CG and CHG contexts compared to no-NBF hybrids when aligned to the almond genome but no difference in methylation levels when aligned to the peach genome. Significantly differentially methylated regions (DMRs) were identified by comparing methylation levels across the genome between NBF- and no-NBF hybrids in each methylation context. In total, 115,635 DMRs were identified based on alignment to the almond reference genome, and 126,800 DMRs were identified based on alignment to the peach reference genome. Nearby genes were identified as associated with the 39 most significant DMRs occurring either in the almond or peach alignments alone or occurring in both the almond and peach alignments. These DMR-associated genes include several uncharacterized proteins and transposable elements. Quantitative PCR was also performed to analyze the gene expression patterns of these identified gene targets to determine patterns of differential expression associated with differential DNA methylation. These DMR-associated genes, particularly those showing corresponding patterns of differential gene expression, represent key targets for almond breeding for future cultivars and mitigating the effects of NBF-exhibition in currently affected cultivars.

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.

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.

Tumor-infiltrating lymphocytes (TILs) represent cancer microenvironment. We previously reported TILs was prognosticators in pancreatic ductal adenocarcinoma (PDAC) patients by immunohistochemically measuring them in surgically-resected tissues. The aim of this study was to assess how best to evaluate TILs in PDAC tissue biopsies. First, we showed expression of CD3, CD4, or CD8 genes in PDAC tissue measured by quantitative RT-PCR (RT-qPCR) was prognostic using 241 surgically-resected specimens. We assessed whether the TILs in biopsied tissues can be effectively evaluated by comparing between immunohistochemistry and RT-qPCR. As a study model, we sampled twenty biopsies from surgically-resected PDAC specimen (n = 17). We investigated the variation levels of TILs in the different biopsies from the same specimen and evaluated using the intraclass correlation coefficient (ICC). The ICC value was 0.58 for CD3, 0.61 for CD4, and 0.46 for CD8, respectively; these ICC values meant correlations of "moderate" to "substantial" levels. To reach "near perfect", 3, 3, and 5 times biopsies were necessary for CD3, CD4, and CD8, respectively. When ICC values of immunolabeled TILs were of "low", ≥6 times biopsies were necessary to reach "moderate" levels. We found that TILs measured by RT-qPCR and repeated sampling increased reliability in TILs detected from biopsied PDAC tissues.

Dengue virus (DENV), a member of the family Flaviviridae, is a threat for global health as it infects more than 100 million people yearly. Approved antiviral therapies or vaccines for the treatment or prevention of DENV infections are not available. In the present study, natural compounds were screened for their antiviral activity against DENV by in vitro cell line-based assay. α-Mangostin, a xanthanoid, was observed to exert antiviral activity against DENV-2 under pre-, co- and post-treatment testing conditions. The antiviral activity was determined by foci forming unit (FFU) assay, quantitative RT-PCR and cell-based immunofluorescence assay (IFA). A complete inhibition of DENV-2 was observed at 8 µM under the co-treatment condition. The possible inhibitory mechanism of α-Mangostin was also determined by docking studies. The molecular docking experiments indicate that α-Mangostin can interact with multiple DENV protein targets such as the NS5 methyltransferase, NS2B-NS3 protease and the glycoprotein E. The in vitro and in silico findings suggest that α-Mangostin possesses the ability to suppress DENV-2 production at different stages of its replication cycle and might act as a prophylactic/therapeutic agent against DENV-2.

The emergence of SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19), represents a public health emergency of unprecedented proportion. The global containment efforts have been focused on testing, tracing of contacts and treatment (isolation) of those found COVID-19 positive. Since the whole genome sequences of a number of strains of this novel RNA virus were available in the public domain by early January 2020, a number of real-time polymerase chain reaction (RT-PCR) protocols were designed and used for diagnosis of this infection. Most RT-PCRs are designed for qualitative COVID-19 reporting (SARS-CoV-2 detected or not detected), but have been used for semi-quantitative estimation of viral load based on cycle threshold value. Our manuscript discusses the utility of quantitative PCR testing for COVID-19 and its patient management benefits.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) compromises the ability of military forces to fulfill missions. At the beginning of May 2020, 22 out of 70 Belgian soldiers deployed to a military education and training center in Maradi, Niger, developed mild COVID-19 compatible symptoms. Immediately upon their return to Belgium, and two weeks later, all seventy soldiers were tested for SARS-CoV-2 RNA (RT-qPCR) and antibodies (two immunoassays). Nine soldiers had at least one positive COVID-19 diagnostic test result. Five of them exhibited COVID-19 symptoms (mainly anosmia, ageusia, and fever), while four were asymptomatic. In four soldiers, SARS-CoV-2 viral load was detected and the genomes were sequenced. Conventional and genomic epidemiological data suggest that these genomes have an African most recent common ancestor and that the Belgian military service men were infected through contact with locals. The medical military command implemented testing of all Belgian soldiers for SARS-CoV-2 viral load and antibodies, two to three days before their departure on a mission abroad or on the high seas, and for specific missions immediately upon their return in Belgium. Some military operational settings (e.g., training camps in austere environments and ships) were also equipped with mobile infectious disease (COVID-19) testing capacity.

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.

The receptor activator of nuclear factor-kB ( RANK) gene is found in both human and murine mammary epithelial cells and in human cancer cell lines. We analyzed RANK expression in normal and proliferative canine mammary tissue samples ( n = 47) and cell lines ( n = 10), and identified its expression in epithelial cell populations. The correlation of RANK protein with clinicopathologic parameters was also studied. A double immunohistochemical method using RANK and p63 antibodies was applied to 33 tissue samples to analyze RANK protein expression and its possible co-expression with p63 protein, the latter used to identify myoepithelial (ME) cells (p63-positive) or luminal epithelial (LE) cells (p63-negative). RANK protein expression was found in ~75% of the tissue samples analyzed, at a similar level in all of the histologic types studied: dysplasias (4 of 4, 100%), malignant tumors (13 of 17, 76%), normal glands (12 of 17, 70%), and benign tumors (6 of 9, 67%). ME and LE cells expressed RANK protein at a similar level. A higher level of RANK protein expression was found in older animals (≥10 y, p = 0.027). Quantitative RT-PCR was applied to 6 ME (1 normal and 5 neoplastic) and 4 LE (1 normal and 3 neoplastic) primary cell lines. The RANK gene was found at similar expression levels in all canine mammary ME and LE cell lines studied. We found RANK expression in normal, dysplastic, and neoplastic canine mammary tissues and cell lines, in both ME and LE cell populations.

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 ?M Pb(NO3)2 and 25 ?M CdSO4 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

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.

Objective To investigate the antiviral property of a lead ligand, YK51 that was synthesized based on the flavanoid of a natural product toward dengue virus type-2 (DENV2) replication. Methods cRNA was isolated from HepG2 cells inoculated with 1 000 median tissue culture infective dose of DENV2 and treated with different doses of the ligand followed by RT-PCR to quantify the virus gene copies. Confocal microscopy of actin and tubulin redistribution was also performed. Results The quantitative RT-PCR result showed reduction of the DENV2 gene copies as the ligand concentration was increased. The confocal microscopy result showed increase in the tubulin intensity (79.6%) of infected BHK21 cells treated with the ligand, compared with the non-treated cells (54.8%). The 1.5-fold increase in the intensity of tubulin suggested that the ligand inhibitory effect stabilized the cellular microtubule structure. Conclusions The synthesized ligand YK51 reduced DENV2 viral load by inhibiting virus replication thus is highly potential to be developed as antiviral agent.

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.

Feeding is a complex behaviour comprised of satiety control, foraging, ingestion and subsequent digestion. Cichlids from the East African Great Lakes are renowned for their diverse trophic specializations, largely predicated on highly variable jaw morphologies. Thus, most research has focused on dissecting the genetic, morphological and regulatory basis of jaw and teeth development in these species. Here for the first time we explore another aspect of feeding, the regulation of appetite related genes that are expressed in the brain and control satiety in cichlid fishes. Using qPCR analysis, we first validate stably expressed reference genes in the brain of six haplochromine cichlid species at the end of larval development prior to foraging. We next evaluate the expression of 16 appetite related genes in herbivorous and carnivorous species from the parallel radiations of Lake Tanganyika, Malawi and Victoria. Interestingly, we find increased expression of two anorexigenic genes, cart and npy2r, in the brain of carnivorous species in all the lakes. This supports the notion that herbivory compared to carnivory requires stronger appetite stimulation in order to feed larger quantity of food and to compensate for the relatively poorer nutritional quality of a plant- and algae-based diet. Our study contributes to the limited body of knowledge on the neurological circuitry that controls feeding transitions and adaptations and in cichlids and other teleosts.

International audience; We tested the hypothesis that mouse ATC1 and ATC7 cells, the first adrenocortical cell lines to exhibit a complete zona fasciculata (ZF) cell phenotype, respond to dynamic ACTH stimulation in a similar manner as the adrenal gland in vivo. Exploiting our previous in vivo observations that gene transcription within the steroidogenic pathway is dynamically regulated in response to a pulse of ACTH, we exposed ATC1 and ATC7 cells to various patterns of ACTH, including pulsatile and constant, and measured the transcriptional activation of this pathway. We show that pulses of ACTH administered to ATC7 cells can reliably stimulate a pulsatile pattern of transcriptional activity that is comparable to that observed in adrenal ZF cells in vivo. Hourly pulses of ACTH stimulate dynamic increases in CREB phosphorylation (pCREB) and transcription of genes involved in critical steps of steroidogenesis including signal transduction (e.g., MRAP), cholesterol delivery (e.g., StAR), and steroid biosynthesis (e.g., CYP11A1), as well as those relating to transcriptional regulation of steroidogenic factors (e.g., SF-1 and Nur-77). In contrast, constant ACTH stimulation results in a prolonged and exaggerated pCREB and steroidogenic gene transcriptional response. We also show that when a large dose of ACTH (100 nM) is applied after these treatment regimens, a significant increase in steroidogenic transcriptional responsiveness is achieved only in cells that have been exposed to pulsatile, rather than constant, ACTH. Our data support our in vivo observations that pulsatile ACTH is important for the optimal transcriptional responsiveness of the adrenal. Importantly, our data suggest that ATC7 cells respond to dynamic ACTH stimulation.

Sex determination and caste differentiation are two crucial processes for morphology building in honeybees. It is unclear whether there is an interaction between these two processes. Here, we investigated the expression of fem and Amdsx genes between female castes of honeybees. We found that the expression of fem and Amdsx is significantly higher in queens than in workers, and this expression was positively regulated by juvenile hormone (JH). Our results suggest that sex-determining genes fem and Amdsx are also involved in honeybee caste differentiation.

Background: Ascorbic acid (Asc) is one of the most abundant antioxidants and it serves as a major 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.

Background: MiRNA-30c was a tumor suppressor in several human cancers, however, its association with clinicopathological features and prognosis in colorectal cancer (CRC) is unclear. Materials and Methods: The expression level of miRNA-30c in 192 pairs of colorectal cancer and adjacent normal tissues was detected by Quantitative RT-PCR, the association between miRNA-30c expression and clinical characteristics and prognosis were statistically analyzed. Results: miRNA-30c was significantly lower in CRC tissues specimens compared with matched normal adjacent tissue (P Conclusion: miRNA-30c could predict the prognosis of colorectal cancer which is helpful to choose reasonable treatment measures.

// Hui Kong 1 , Ying Wu 1 , Mengou Zhu 2 , Changwen Zhai 3 , Jing Qian 1 , Xue Gao 4 , Shuyang Wang 1 , Yingyong Hou 3 , Shaohua Lu 3, * , Hongguang Zhu 1, 4, * 1 Department of Pathology, Shanghai Medical College, Fudan University, Shanghai, China 2 Harvard College, Harvard University, Cambridge, MA, USA 3 Department of Pathology, Zhongshan Hospital, Fudan University, Shanghai, China 4 Division of Surgical Pathology, Huashan Hospital, Fudan University, Shanghai, China * These authors have contributed equally to this work Correspondence to: Hongguang Zhu, email: hongguang_702@163.com Keywords: prognostic biomarker, long non-coding RNA, early stage colorectal cancer, liver metastasis, quantitative RT-PCR Received: December 06, 2015 Accepted: June 12, 2016 Published: July 06, 2016 ABSTRACT Liver metastasis is the primary cause of death for colorectal cancer (CRC) patients. To investigate the prognostic value of long non-coding RNAs (lncRNAs) on colorectal liver metastases, quantitative reverse-transcriptase PCR (quantitative RT-PCR) was performed on 15 lncRNAs in 51 stage IV CRC with liver metastases and 57 stage I/II CRC specimens. The expression levels of four lncRNAs (GAS5, H19, MEG3 and Yiya) were significantly different between liver metastases and primary tumors of stage IV CRC patients. Furthermore, the high expression levels of GAS5 and Yiya were significantly associated with future occurrence of liver metastases in early stage CRC patients. Kaplan-Meier analysis showed that the high expression levels of GAS5 or Yiya were correlated with poor prognosis of early stage CRC patients (p = 0.0206 and 0.0005 for GAS5 and Yiya, respectively). Yiya expression was proved to be an independent prognostic indicator of colorectal liver metastases in a multivariate analysis (relative risk = 10.7; p < 0.0001). Our study revealed that GAS5 and Yiya were promising prognostic biomarkers of liver metastases for early stage CRC patients.

Travellers' diarrhoea (TD) remains the most frequent health problem encountered by visitors to the (sub) tropics. Traditional stool culture identifies the pathogen in only 15% of cases. Exploiting PCR-based methods, we investigated TD pathogens with a focus on asymptomatic travellers and severity of symptoms. Pre- and post-travel stools of 382 travellers with no history of antibiotic use during travel were analysed with a multiplex quantitative PCR for Salmonella, Yersinia, Campylobacter, Shigella, Vibrio cholerae and five diarrhoeagenic Escherichia coli: enteroaggregative (EAEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), enterohaemorrhagic (EHEC) and enteroinvasive (EIEC). The participants were categorized by presence/absence of TD during travel and on return, and by severity of symptoms. A pathogen was indentified in 61% of the asymptomatic travellers, 83% of those with resolved TD, and 83% of those with ongoing TD; 25%, 43% and 53% had multiple pathogens, respectively. EPEC, EAEC, ETEC and Campylobacter associated especially with ongoing TD symptoms. EAEC and EPEC proved more common than ETEC. To conclude, modern methodology challenges our perception of stool pathogens: all pathogens were common both in asymptomatic and symptomatic travellers. TD has a multibacterial nature, but diarrhoeal symptoms mostly associate with EAEC, EPEC, ETEC and Campylobacter. (C) 2016 The Authors. Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases.

Circulating extracellular microRNAs (miRNAs) in the serum or plasma has generated great interest as a promising biomarker for diverse pathological conditions - changes in miRNA expression in body fluids develop earlier than conventional biomarkers. Their expression in mother-newborn pairs may signal uterine exposure to environmental pollutants, including tobacco smoke (TS) and toxic metals, and the role of miRNAs in fetal programming remains largely unstudied. Assessment of extracellular miRNAs in plasma is limited and remains challenging due to their low amounts in circulation, small size and the lack of reference miRNA. Preamplification of the miRNA’s cDNA can be used to facilitate reliable quantification. Within our research, we assess daily exposure to main toxic metals and maternal susceptibility as factors of developmental origins of health and disease. Preliminary results on candidate miRNAs indicated aberrant expression of miR-21 and miR- 146a in maternal plasma related to TS exposure, whereas miR-1537 and miR-190b were not expressed or had a threshold cycle (Ct) value >35 in 95% and 50% of analyzed samples. We further optimized the protocol for circulating miRNA analysis (by Qiagen miRNA assays) and tested the preamplification step to increase the sensitivity of qPCR analysis and the number of detectable miRNAs in 19 paired maternal and umbilical cord blood plasma samples. By using the ∆∆Ct method of relative quantification, we analyzed fold change for miRNA expression in the samples from healthy smoking (n=10) and non-smoking (n=9) postpartum women collected immediately after delivery. Preamplification facilitated the detection of all assayed candidate miRNAs and yielded a mean Ct improvement of 6.7±1.1 (p0.9) for miR-16, miR-21 and miR- 146a (whose Ct values by direct qPCR were already

Transcriptomic analyses addressed to study phytoplasma gene expression may present few difficulties due to the uncultivable nature of these intracellular, obligate pathogens. While RNA extraction from insect vectors does not imply any particular adaptation of the protocols used in most commercial kits, RNA isolation from phytoplasma-infected plants can be a challenging task, given the high levels of polyphenol contents and accumulation of sucrose and starch in the different plant tissues. Here, we describe two different transcriptomic approaches, one focused on RNA phytoplasma sequencing and the other on phytoplasma quantitative gene expression in relation to pathogen load.

Background: Quantitative real-time reverse-transcription polymerase chain reaction is frequently used as research tool in experimental oncology. There are some studies of valid endogenous control genes in the field of human glioma research, which, however, only focus on the comparison between normal brain with tumor tissue and malignant transformation toward secondary glioblastomas. Aim of this study was to validate a more general reference gene also suitable for pre- and posttreatment analysis and other evaluations (eg, primary vs secondary glioblastoma). Methods: This quantitative polymerase chain reaction analysis was performed to test a panel of the 6 most suitable reference genes from other studies representing different physiological pathways (ACTB, GAPDH, POLR2A, RPL13A, SDHA, and TBP) in all common glioma groups, namely: diffuse astrocytoma World Health Organization II, anaplastic astrocytoma World Health Organization III, secondary glioblastoma World Health Organization IV with and without chemotherapy, primary glioblastoma, recurrent glioblastoma, and gliomas before and after radiation. Expression stability was tested during the longitudinal course of the disease in 8 single patients. Results: Evaluation of the expression levels of the 6 target genes showed that ACTB, GAPDH, and RPL13A show higher expression compared to SDHA, POLR2A, and TBP. ACTB, GAPDH, and RPL13A showed different expression levels between astrozytoma grade II and primary glioblastoma. Except for this difference, the candidate genes were not differentially expressed between primary and secondary glioblastomas and between the World Health Organization tumor grades. Furthermore, they remained stable before and after radiotherapy and/or chemotherapy. Therefore, they are adequate references for glioblastoma gene expression studies. The comparison of all tested genes resulted in SDHA and ACTB as most stable reference genes determined by the NormFinder software. Our data revealed lowest intragroup variation in the SDHA, highest in the RPL13A gene. Conclusions: All tested genes may be recommended as universal reference genes for data normalization in gene expression studies under different treatment regimens both in primary glioblastomas and astrocytomas of different grades (World Health Organization grades II-IV), respectively. In summary, ACTB and SDHA exhibited the best stability values and showed the lowest intergroup expression variability.

Rhizophora apiculatais a halophytic, small mangrove tree distributed along the coastal regions of the tropical and subtropical areas of the world. They are natural genetic reservoirs of salt adaptation genes and offer a unique system to explore adaptive mechanisms under salinity stress. However, there are no reliable studies available on selection and validation of reference genes for quantitative real-time polymerase chain reaction (qRT-PCR) inR. apiculataphysiological tissues and in salt stress conditions. The selection of appropriate candidate reference gene for normalization of qRT-PCR data is a crucial step towards relative analysis of gene expression. In the current study, seven genes such as elongation factor 1α (EF1α), Ubiquitin (UBQ), β-tubulin (β-TUB), Actin (ACT), Ribulose1,5-bisphosphate carboxylase/oxygenase (rbcL), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and 18S rRNA (18S) were selected and analyzed for their expression stability. Physiological tissues such as leaf, root, stem, and flower along with salt stress leaf samples were used for selection of candidate reference genes. The high-quality expression data was obtained from biological replicates and further analyzed using five different programs such as geNorm, NormFinder, BestKeeper, Delta Ct and RefFinder. All algorithms comprehensively rankedEF1α followed byACTas the most stable candidate reference genes inR. apiculataphysiological tissues. Moreover, β-TUBand 18S were ranked as moderately stable candidate reference genes, while GAPDH andrbcLwere least stable reference genes. Under salt stress,EF1α was comprehensively recommended top-ranked candidate reference gene followed byACTand 18S. In order to validate the identified most stable candidate reference genes,EF1α,ACT, 18S andUBQwere used for relative gene expression level of sodium/proton antiporter (NHX) gene under salt stress. The expression level ofNHXvaried according to the internal control which showed the importance of selection of appropriate reference gene. Taken together, this is the first ever systematic attempt of selection and validation of reference gene for qRT-PCR inR. apiculataphysiological tissues and in salt stress. This study would promote gene expression profiling of salt stress tolerance related genes inR. apiculata.

Pituitary-dependent hypercortisolism (PDH) is a common endocrinopathy in dogs, but the promotors and initiators of the tumourigenesis of corticotroph pituitary adenomas remain unknown. Based on human data, we investigated mRNA expression of pituitary tumour transforming gene 1 (PTTG1) with quantitative RT-PCR in canine corticotroph pituitary adenomas. PTTG1 was overexpressed in adenomas approximately 3-fold. A strong association was observed between PTTG1 expression and disease-free interval; dogs with high PTTG1 expression had a significantly (4 times; P=0.02) shorter disease-free interval than dogs with low PTTG1 expression. This paper shows that PTTG1 expression is a negative prognosticator in relation to disease-free interval and recurrence in dogs undergoing transsphenoidal hypophysectomy as treatment for PDH.

Background The root extract of Rhodiola rosea has historically been used in Europe and Asia as an adaptogen, and similar to ginseng and Shisandra, shown to display numerous health benefits in humans, such as decreasing fatigue and anxiety while improving mood, memory, and stamina. A similar extract in the Rhodiola family, Rhodiola crenulata, has previously been shown to confer positive effects on the gut homeostasis of the fruit fly, Drosophila melanogaster. Although, R. rosea has been shown to extend lifespan of many organisms such as fruit flies, worms and yeast, its anti-aging mechanism remains uncertain. Using D. melanogaster as our model system, the purpose of this work was to examine whether the anti-aging properties of R. rosea are due to its impact on the microbial composition of the fly gut. Results Rhodiola rosea treatment significantly increased the abundance of Acetobacter, while subsequently decreasing the abundance of Lactobacillales of the fly gut at 10 and 40 days of age. Additionally, supplementation of the extract decreased the total culturable bacterial load of the fly gut, while increasing the overall quantifiable bacterial load. The extract did not display any antimicrobial activity when disk diffusion tests were performed on bacteria belonging to Microbacterium, Bacillus, and Lactococcus. Conclusions Under standard and conventional rearing conditions, supplementation of R. rosea significantly alters the microbial community of the fly gut, but without any general antibacterial activity. Further studies should investigate whether R. rosea impacts the gut immunity across multiple animal models and ages. Electronic supplementary material The online version of this article (10.1186/s13099-018-0239-8) contains supplementary material, which is available to authorized users.

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.

Pituitary-dependent hypercortisolism (PDH) is a common endocrinopathy in dogs, but the promotors and initiators of the tumourigenesis of corticotroph pituitary adenomas remain unknown. Based on human data, we investigated mRNA expression of pituitary tumour transforming gene 1 (PTTG1) with quantitative RT-PCR in canine corticotroph pituitary adenomas. PTTG1 was overexpressed in adenomas approximately 3-fold. A strong association was observed between PTTG1 expression and disease-free interval; dogs with high PTTG1 expression had a significantly (4 times; P=0.02) shorter disease-free interval than dogs with low PTTG1 expression. This paper shows that PTTG1 expression is a negative prognosticator in relation to disease-free interval and recurrence in dogs undergoing transsphenoidal hypophysectomy as treatment for PDH.

Background The root extract of Rhodiola rosea has historically been used in Europe and Asia as an adaptogen, and similar to ginseng and Shisandra, shown to display numerous health benefits in humans, such as decreasing fatigue and anxiety while improving mood, memory, and stamina. A similar extract in the Rhodiola family, Rhodiola crenulata, has previously been shown to confer positive effects on the gut homeostasis of the fruit fly, Drosophila melanogaster. Although, R. rosea has been shown to extend lifespan of many organisms such as fruit flies, worms and yeast, its anti-aging mechanism remains uncertain. Using D. melanogaster as our model system, the purpose of this work was to examine whether the anti-aging properties of R. rosea are due to its impact on the microbial composition of the fly gut. Results Rhodiola rosea treatment significantly increased the abundance of Acetobacter, while subsequently decreasing the abundance of Lactobacillales of the fly gut at 10 and 40 days of age. Additionally, supplementation of the extract decreased the total culturable bacterial load of the fly gut, while increasing the overall quantifiable bacterial load. The extract did not display any antimicrobial activity when disk diffusion tests were performed on bacteria belonging to Microbacterium, Bacillus, and Lactococcus. Conclusions Under standard and conventional rearing conditions, supplementation of R. rosea significantly alters the microbial community of the fly gut, but without any general antibacterial activity. Further studies should investigate whether R. rosea impacts the gut immunity across multiple animal models and ages.

Humanized mice are emerging as an alternative model system to well-established non-human primate (NHP) models for studying human immunodeficiency virus (HIV)-1 biology and pathogenesis. Although both NHP and humanized mice have their own strengths and could never truly reflect the complex human immune system and biology, there are several advantages of using the humanized mice in terms of using primary HIV-1 for infection instead of simian immunodeficiency virus or chimera simian/HIV. Several different types of humanized mice have been developed with varying levels of reconstitution of human CD45+ cells. In this study, we utilized humanized Rag1KO.IL2RγcKO.NOD mice expressing HLA class II (DR4) molecule (DRAG mice) infused with HLA-matched hematopoietic stem cells from umbilical cord blood to study early events after HIV-1 infection, since the mucosal tissues of these mice are highly enriched for human lymphocytes and express the receptors and coreceptors needed for HIV-1 entry. We examined the various tissues on days 4, 7, 14, and 21 after an intravaginal administration of a single dose of purified primary HIV-1. Plasma HIV-1 RNA was detected as early as day 7, with 100% of the animals becoming plasma RNA positive by day 21 post-infection. Single cells were isolated from lymph nodes, bone marrow, spleen, gut, female reproductive tissue, and brain and analyzed for gag RNA and strong stop DNA by quantitative (RT)-PCR. Our data demonstrated the presence of HIV-1 viral RNA and DNA in all of the tissues examined and that the virus was replication competent and spread rapidly. Bone marrow, gut, and lymph nodes were viral RNA positive by day 4 post-infection, while other tissues and plasma became positive typically between 7 and 14 days post-infection. Interestingly, the brain was the last tissue to become HIV-1 viral RNA and DNA positive by day 21 post-infection. These data support the notion that humanized DRAG mice could serve as an excellent model for studying the trafficking of HIV-1 to the various tissues, identification of cells harboring the virus, and thus could serve as a model system for HIV-1 pathogenesis and reservoir studies.

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.

International audience; Previous studies reported that the use of Metschnikowia pulcherrima in sequential culture fermentation with Saccharomyces cerevisiae mainly induced a reduction of volatile acidity in wine. The impact of the presence of this yeast on the metabolic pathway involved in pyruvate dehydrogenase (PDH) bypass and glycerol production in S. cerevisiae has never been investigated. In this work, we compared acetic acid and glycerol production kinetics between pure S. cerevisiae culture and its sequential culture with M. pulcherrima during alcoholic fermentation. In parallel, the expression levels of the principal genes involved in PDH bypass and glyceropyruvic fermentation in S. cerevisiae were investigated. A sequential culture of M. pulcherrima/S. cerevisiae at an inoculation ratio of 10: 1 produced 40% less acetic acid than pure S. cerevisiae culture and led to the enhancement of glycerol content (12% higher). High expression levels of pyruvate decarboxylase PDC1 and PDC5, acetaldehyde dehydrogenase ALD6, alcohol dehydrogenase ADH1 and glycerol-3-phosphate dehydrogenase PDC1 genes during the first 3 days of fermentation in sequential culture conditions are highlighted. Despite the complexity of correlating gene expression levels to acetic acid formation kinetics, we demonstrate that the acetic acid production pathway is altered by sequential culture conditions. Moreover, we show for the first time that the entire acetic acid and glycerol metabolic pathway can be modulated in S. cerevisiae by the presence of M. pulcherrima at the beginning of fermentation.

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 β8 lineage were revealed to be the most recently duplicated genes among their paralogous genes in the β 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. Electronic supplementary material The online version of this article (doi:10.1186/s12870-017-1052-0) contains supplementary material, which is available to authorized users.

While quantitative PCR (qPCR) is widely recognized as being among the most accurate methods for quantifying gene expression, it is highly dependent on the use of reliable, stably expressed reference genes. With the increased availability of high-throughput methods for measuring gene expression, whole-transcriptome approaches may be increasingly utilized for reference gene selection and validation. In this study, RNA-seq was used to identify a set of novel qPCR reference genes and evaluate a panel of traditional “housekeeping” reference genes in two species of the evolutionary model plant genus Mimulus. More broadly, the methods proposed in this study can be used to harness the power of transcriptomes to identify appropriate reference genes for qPCR in any study organism, including emerging and nonmodel systems. We find that RNA-seq accurately estimates gene expression means in comparison to qPCR, and that expression means are robust to moderate environmental and genetic variation. However, measures of expression variability were only in agreement with qPCR for samples obtained from a shared environment. This result, along with transcriptome-wide comparisons, suggests that environmental changes have greater impacts on expression variability than on expression means. We discuss how this issue can be addressed through experimental design, and suggest that the ever-expanding pool of published transcriptomes represents a rich and low-cost resource for developing better reference genes for qPCR.

Summary Extended darkness induces a transient increase in sugars and trehalose pathway gene expression., Energy resources in plants are managed in continuously changing environments, such as changes occurring during the day/night cycle. Shading is an environmental disruption that decreases photosynthesis, compromises energy status, and impacts on crop productivity. The trehalose pathway plays a central but not well-defined role in maintaining energy balance. Here, we characterized the maize trehalose pathway genes and deciphered the impacts of the diurnal cycle and disruption of the day/night cycle on trehalose pathway gene expression and sugar metabolism. The maize genome encodes 14 trehalose-6-phosphate synthase (TPS) genes, 11 trehalose-6-phosphate phosphatase (TPP) genes, and one trehalase gene. Transcript abundance of most of these genes was impacted by the day/night cycle and extended dark stress, as were sucrose, hexose sugars, starch, and trehalose-6-phosphate (T6P) levels. After extended darkness, T6P levels inversely followed class II TPS and sucrose non-fermenting-related protein kinase 1 (SnRK1) target gene expression. Most significantly, T6P no longer tracked sucrose levels after extended darkness. These results showed: (i) conservation of the trehalose pathway in maize; (ii) that sucrose, hexose, starch, T6P, and TPS/TPP transcripts respond to the diurnal cycle; and(iii) that extended darkness disrupts the correlation between T6P and sucrose/hexose pools and affects SnRK1 target gene expression. A model for the role of the trehalose pathway in sensing of sucrose and energy status in maize seedlings is proposed.

Reference genes are commonly used for normalization of target gene expression during RT-qPCR analysis. However, no housekeeping genes or reference genes have been identified to be stable across different tissue types or under different experimental conditions. To identify the most suitable reference genes for RT-qPCR analysis of target gene expression in the hepatopancreas of crucian carp (Carassius auratus) under various conditions (sex, age, water temperature, and drug treatments), seven reference genes, including beta actin (ACTB), beta-2 microglobulin (B2M), embryonic elongation factor-1 alpha (EEF1A), glyceraldehyde phosphate dehydrogenase (GAPDH), alpha tubulin (TUBA), ribosomal protein l8 (RPL8) and glucose-6-phosphate dehydrogenase (G6PDH), were evaluated in this study. The stability and ranking of gene expression were analyzed using three different statistical programs: GeNorm, Normfinder and Bestkeeper. The expression errors associated with selection of the genes were assessed by the relative quantity of CYP4T. The results indicated that all the seven genes exhibited variability under the experimental conditions of this research, and the combination of ACTB/TUBA/EEF1A or of ACTB/EEF1A was the best candidate that raised the accuracy of quantitative analysis of gene expression. The findings highlighted the importance of validation of housekeeping genes for research on gene expression under different conditions of experiment and species.

The human Igf-1 gene not only produces insulin‑like growth factor-I (IGF-I), but also different carboxy‑terminal extensions, known as E peptides, through alternative splicing. We and others have shown that human Eb peptide (hEb) derived from Igf-1 has intrinsic biological activity and is localized to nuclei of transfected cells. Since hEb actions can complement the activity of IGF-I itself, the aim of the present study was to compare IGF-I isoforms at the endogenous protein and transcript level in cancer cell lines, including HeLa, U2OS, HepG2 and K562 cells. Quantitative real-time PCR (qRT‑PCR) using Igf-1 isoform specific primers was performed to determine expression patterns, using β-actin as a reference gene. The overall relative Igf-1 transcript level was different across the cell lines, with ~80-fold higher expression in K562 (130.2±31.2) than in U2OS cells (1.7±1.1). The relative copy number of Igf-1b was the highest in HepG2 (69.9±28.6) and K562 cells (28.3±6.7), whereas the relative copy numbers of Igf-1a and Igf-1c were significantly higher in K562 cells compared to all other cell lines. Immunoblotting using total cell lysates, cytoplasmic and nuclear fractions were carried out to determine the level and distribution of IGF-I proteins. K562 cells exhibited the highest level of hEb in total cell lysates and nuclear fractions and no cell lines displayed hEb in the cytoplasmic fractions. In contrast, IGF-IA was the highest in HeLa cells and was enriched only in the cytoplasmic fraction. Since relatively low IGF-1A transcript level but relatively high pro‑IGF-1A protein level is plausible, we hypothesized that these transcripts could be processed with higher efficiency and/or the protein product may be stabilized by viral HPV oncogenes in HeLa cells. We assert that while it is important to analyze Igf-1 transcript level, it may be more relevant to determine the IGF isoforms at the protein level.

Plant microRNAs (miRNAs) play important roles in biological processes such as development and stress responses. Although the diverse functions of miRNAs in model organisms have been well studied, their function in wild rice is poorly understood. In this study, high-throughput small RNA sequencing was performed to characterize tissue-specific transcriptomes in Oryza longistaminata . A total of 603 miRNAs, 380 known rice miRNAs, 72 conserved plant miRNAs, and 151 predicted novel miRNAs were identified as being expressed in aerial shoots and rhizomes. Additionally, 99 and 79 miRNAs were expressed exclusively or differentially, respectively, in the two tissues, and 144 potential targets were predicted for the differentially expressed miRNAs in the rhizomes. Functional annotation of these targets suggested that transcription factors, including squamosa promoter binding proteins and auxin response factors, function in rhizome growth and development. The expression levels of several miRNAs and target genes in the rhizomes were quantified by RT-PCR, and the results indicated the existence of complex regulatory mechanisms between the miRNAs and their targets. Eight target cleavage sites were verified by RNA ligase-mediated rapid 5′ end amplification. These results provide valuable information on the composition, expression and function of miRNAs in O. longistaminata , and will aid in understanding the molecular mechanisms of rhizome development.

Highlights • Real-time quantitative PCR assays were developed for six different IBV types. • Rapid detection of IBV type is important for control. • Analytical sensitivity was evaluated using synthetic DNA standards. • Specificity was determined using clinical and biological specimens. • Linearity over a 5 log10 dynamic range and a limit of detection of ≤10 target copies was realized., Infectious bronchitis (IB) is a highly contagious upper respiratory tract disease of chickens caused by infectious bronchitis virus (IBV), which has various serotypes that do not cross-protect. Vaccine control strategies for this virus are only effective when designed around the currently circulating serotypes. It is essential to not only rapidly detect IBV but also to identify the type of virus causing disease. Six TaqMan™-based quantitative real-time RT-PCR assays (Universal, Ark, Mass, DE/GA98, GA07, GA08) were developed and examined the sensitivity and specificity for each assay. Assays were developed targeting the hypervariable region in the S1 gene subunit. The analytical sensitivity of TaqMan™-based quantitative real-time RT-PCR assays (qRT-PCR) assays was evaluated using synthetic DNA standards that were identical with the target sequence and specificity was further validated using clinical and biological specimens. All developed assays performed equivalently when using synthetic DNA templates as standard material, as it achieved linearity over a 5 log10 dynamic range with a reproducible limit of detection of ≤10 target copies per reaction, with high calculated amplification efficiencies ranging between 90%–115%. Further validation of specificity using clinical and biological specimens was also successful.

Highlights • LASV RT-qPCR diagnostic sensitivity was 96.4% & specificity 100%. • Limit of detection ranged from 103- 105copies/ml with lineages I-IV. • Assay can be used with whole blood & serum samples from humans and rodent tissues., Lassa fever is a severe viral hemorrhagic illness caused by Lassa virus. Based on estimates, the number of LASV infections ranges from 300,000 to 500,000 cases in endemic areas with a fatality rate of 1%. Development of fast and sensitive tools for the control and prevention of Lassa virus infection as well as for clinical diagnostics of Lassa fever are crucial. Here we reported development and evaluation of a one-step quantitative RT-qPCR assay for the Lassa virus detection – LASV-Fl. This assay is suitable for the detection of lineages I-IV of Lassa virus. The limit of detection of the assay ranged from 103 copies/ml to 105 copies/ml and has 96.4% diagnostic sensitivity, whereas analytical and diagnostic specificities both were 100%. Serum, whole blood and tissue are suitable for use with the assay. The assay contains all the necessary components to perform the analysis, including an armored positive control (ARC+) and an armored internal control (IC). The study was done during the mission of specialized anti-epidemic team of the Russian Federation (SAET) in the Republic of Guinea in 2015-2018. Based on sequencing data, LASV-specific assay was developed using synthetic MS2-phage-based armored RNA particles, RNA from Lassa virus strain Josiah, and further, evaluated in field conditions using samples from patients and Mastomys natalensis rodents.

We report two novel hypofibrinogenemias, Shizuoka III and Kanazawa II, which are caused by heterozygous mutations in FGG. Shizuoka III showed c.147delT and 147_149insACA in FGG exon 3 and a subsequent frameshift mutation, resulting in mature protein γ23X (native protein: γ49X), and Kanazawa II showed c.1205G>A in FGG exon 9, resulting in γ376X (native protein: γ402X). To determine whether the truncated γ-chains, γ23X and γ376X, were synthesized and participated in the assembly of fibrinogen, mutant-type cDNA vectors were transfected into Chinese hamster ovary (CHO) cells. Significant levels of mutant fibrinogen were not detected by ELISA in the culture media and cell lysates. Immunoblot analysis of cell lysates revealed that the mutant γ-chain of γ376X was observed but intact fibrinogen was not. On the other hand, mutant γ-chain was not observed in γ23X-expressing cells. To demonstrate the involvement of the mechanisms of nonsense-mediated mRNA decay (NMD), we cloned wild- and mutant-type mini-genes containing γ23 or γ376 codon and transfected these into CHO cell lines in the absence or presence of cycloheximide as an NMD inhibitor. mRNA levels were determined using real-time quantitative RT-PCR in CHO cells. In the absence of cycloheximide, levels of mRNAs transcribed from the mutant gene were lower than from the wild-type gene whereas, in the presence of cycloheximide, levels of mRNAs transcribed from the mutant gene increased dose-dependently. Finally, these results demonstrated that mRNAs containing γ23X or γ376X are degraded by the NMD system and translation of the truncated γ-chain polypeptide decrease in patients' hepatocytes, resulting in hypofibrinogenemias., Article, THROMBOSIS RESEARCH. 132(4):465-470 (2013)

microRNAs (miRNAs) are a class of newly identified, noncoding, small RNA molecules that negatively regulate gene expression. Many miRNAs are reportedly involved in plant growth, development and stress response processes. However, their roles in the sexual reproduction mechanisms in flowering plants remain unknown. Pollen development is an important process in the life cycle of a flowering plant, and it is closely related to the yield and quality of crop seeds. This study aimed to identify miRNAs involved in pollen development. A microarray assay was conducted using the known complementary sequences of plant miRNAs as probes on inflorescences of a sterile male line (Bcajh97-01A) and a fertile male line (Bcajh97-01B) of the Brassica campestris ssp. chinensis cv. ‘Aijiaohuang’ genic male sterility sister line system (Bcajh97-01A/B). The results showed that 44 miRNAs were differently expressed in the two lines. Of these, 15 had over 1.5-fold changes in their transcript levels, with 9 upregulated and 6 downregulated miRNAs in inflorescences of ‘Bcajh97-01A’ sterile line plants. We then focused on 3 of these 15 miRNAs (miR158, miR168 and miR172). Through computational methods, 13 family members were predicted for these 3 miRNAs and 22 genes were predicted to be their candidate target genes. By using 5’ modified RACE, 2 target genes of miR168 and 5 target genes of miR172 were identified. Then, qRT-PCR was applied to verify the existence and expression patterns of the 3 miRNAs in the flower buds at five developmental stages. The results were generally consistent with those of the microarray. Thus, this study may give a valuable clue for further exploring the miRNA group that may function during pollen development. Electronic Supplementary Material Supplementary material is available for this article at 10.2478/s11658-013-0097-9 and is accessible for authorized users.

In order to understand the gene regulation during berry development and examine the effect of abscisic acid (ABA) on gene expression related to berry maturation, we evaluated the validity of four housekeeping genes, elongation factor 1-α (EF1-α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin-conjugating enzyme (UBC), and 60S ribosomal protein L40-1 (VvUbiquitin1), as references by using ‘Aki Queen’ grapes with and without ABA treatment. The main contribution of this study is that a gene expression analysis using GAPDH as a reference gene will help to elucidate the berry development process and the physiological effects of ABA on berry maturation of ‘Aki Queen’ grapes., VITIS - Journal of Grapevine Research, Vol 55, No 4 (2016): Vitis

To date, a small number of major flowering time loci have been identified in the related Triticeae crops, bread wheat (Triticum aestivum), durum wheat (T. durum), and barley (Hordeum vulgare). Natural genetic variants at these loci result in major phenotypic changes which have adapted crops to the novel environments encountered during the spread of agriculture. The polyploid nature of bread and durum wheat means that major flowering time loci in which recessive alleles confer adaptive advantage in related diploid species have not been readily identified. One such example is the PPD-H2 flowering time locus encoded by FLOWERING LOCUS T 3 (HvFT3) in the diploid crop barley, for which recessive mutant alleles confer delayed flowering under short day (SD) photoperiods. In autumn-sown barley, such alleles aid the repression of flowering over the winter, which help prevent the development of cold-sensitive floral organs until the onset of inductive long day (LD) photoperiods the following spring. While the identification of orthologous loci in wheat could provide breeders with alternative mechanisms to fine tune flowering time, systematic identification of wheat orthologs of HvFT3 has not been reported. Here, we characterize the FT gene families in six Poaceae species, identifying novel members in all taxa investigated, as well as FT3 homoeologs from the A, B and D genomes of hexaploid (TaFT3) and tetraploid wheat. Sequence analysis shows TaFT3 homoeologs display high similarity to the HvFT3 coding region (95-96%) and predicted protein (96-97%), with conservation of intron/exon structure across the five cereal species investigated. Genetic mapping and comparative analyses in hexaploid and tetraploid wheat find TaFT3 homoeologs map to the long arms of the group 1 chromosomes, collinear to HvFT3 in barley and FT3 orthologs in rice, foxtail millet and brachypodium. Genome-specific expression analyses show FT3 homoeologs in tetraploid and hexaploid wheat are upregulated under SD photoperiods, but not under LDs, analogous to the expression of HvFT3. Collectively, these results indicate that functional wheat orthologs of HvFT3 have been identified. The molecular resources generated here provide the foundation for engineering a novel major flowering time locus in wheat using forward or reverse genetics approaches.

Background The incidence of and mortality from colorectal cancers (CRC) can be reduced by early detection. Currently there is a lack of established markers to detect early neoplastic changes. We aimed to identify the copy number variations (CNVs) and the associated genes which could be potential markers for the detection of neoplasia in both ulcerative colitis-associated neoplasia (UC-CRN) and sporadic colorectal neoplasia (S-CRN). Methods We employed array comparative genome hybridization (aCGH) to identify CNVs in tissue samples of UC nonprogressor, progressor and sporadic CRC. Select genes within these CNV regions as a panel of markers were validated using quantitative real time PCR (qRT-PCR) method along with the microsatellite instability (MSI) in an independent cohort of samples. Immunohistochemistry (IHC) analysis was also performed. Results Integrated analysis showed 10 overlapping CNV regions between UC-Progressor and S-CRN, with the 8q and 12p regions showing greater overlap. The qRT-PCR based panel of MYC, MYCN, CCND1, CCND2, EGFR and FNDC3A was successful in detecting neoplasia with an overall accuracy of 54 % in S-CRN compared to that of 29 % in UC neoplastic samples. IHC study showed that p53 and CCND1 were significantly overexpressed with an increasing frequency from pre-neoplastic to neoplastic stages. EGFR and AMACR were expressed only in the neoplastic conditions. Conclusion CNVs that are common and unique to both UC-associated and sporadic colorectal neoplasm could be the key players driving carcinogenesis. Comparative analysis of CNVs provides testable driver aberrations but needs further evaluation in larger cohorts of samples. These markers may help in developing more effective neoplasia-detection strategies during screening and surveillance programs. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2303-4) contains supplementary material, which is available to authorized users.

A quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) procedure using a general primer set and three TaqMan (R) MGB probes was developed for general and genotype-specific detection and quantitation of the genomic M segment of Tomato spotted wilt virus (TSWV). Standard curves using RNA transcripts homologous to the three probes allowed reproducible quantitative assays with a wide dynamic range (10(3)-10(10) TSWV M segment RNA copies/ng of total RNA) and high sensitivity. This protocol was assayed with a battery of TSWV isolates, covering the range of the present known genetic variation, in single and/or mix infections in three plant hosts, as well as in the thrips vector Frankliniella occidenialis. This quantitative detection assay will be a valuable tool for molecular biology and epidemiology studies, diagnosis and disease control., This work was supported by grants INIA RTA2008-00010-C03 and Generalitat Valenciana ACOMP/2009/103 and ACOMP/2010/085. D.E. Debreczeni is the recipient of a FPU pre-doctoral fellowship from MEC and B. Belliure was supported by a INIA-CCAA contract. We thank I. Ferriol, Dr. S. Ambros and Dr. A. Olmos for technical advice; Dolores Comin for technical assistance; Dr. D. Peters and Dr. J. Contreras for kindly providing the TSWV isolate BR01 and a thrips colony, respectively; and Daniel R. Pearce for English revision.