Search

Aim: Influenza A virus (IAV) causes serious illness and is responsible for significant morbidity and mortality. To diagnose IAV infection in its early stages, a quick, sensitive, precise detection method is needed for effective clinical management. Materials & methods: In-house hydroxylnaphthol blue (HNB)-based RT-LAMP assay for early detection of IAV using the HA gene was compared with RT-PCR/multiplex-RT-PCR. Results: For the reference strains of IAV, (H1N1 (A/Texas/50/2012) and H3N2 (A/Malaysia/2089302/2009)) RT-LAMP and RT-PCR/M-RT-PCR exhibited a limit of detection of 10 and 100 fg/ml, respectively. Conclusion: HNB-based RT-LAMP is a rapid, sensitive, cost-effective diagnostic tool, and could be a point-of-care test for IAV patients during outbreaks. Influenza A virus (IAV) is responsible for severe disease worldwide and causes significant mortality and morbidity. A sensitive, specific tool for the detection of IAV infection in its early stages is needed, so we designed an HNB-based RT-LAMP assay for IAV detection. This is an easy, cost-effective, sensitive, and specific test that may become a point-of-care test for IAV detection during outbreaks. The limit of detection was found to be 10 fg/ml in a purified strain of H1N1/H3N2 by our LAMP assay, which was ten-times more sensitive than conventional RT-PCR/M-RT-PCR diagnostic tests. An LOD of 10 fg/ml was exhibited by both agarose and HNB-based RT-LAMP, while a LOD of 100 fg/ml was obtained by conventional RT-PCR/M-RT-PCR assay. [ABSTRACT FROM AUTHOR]

We used nasopharyngeal swab samples of patients with a symptomatic (n = 82) or asymptomatic (n = 20) coronavirus disease 2019 (COVID-19) diagnosis to assess the ability of antigen detection tests to infer active (potentially transmissible) or inactive (potentially non-transmissible) infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using the subgenomic RNA (sgRNA) as an active replication marker of SARS-CoV-2, 48 (76.2%), 56 (88.9%), and 63 (100%) of 63 samples with sgRNA positive results tested positive with the SD BIOSENSOR STANDARD Q COVID-19 Ag (Standard Q), the SD BIOSENSOR STANDARD F COVID-19 Ag FIA (Standard F), or the Fujirebio LUMIPULSE G SARS-CoV-2 Ag (Lumipulse) assay, respectively. Conversely, 37 (94.9%), 29 (74.4%), and 7 (17.9%) of 39 samples with sgRNA negative results tested negative with Standard Q, Standard F, or Lumipulse, respectively. Stratifying results by the number of days of symptoms before testing revealed that most antigen positive/sgRNA positive results were among samples tested at 2–7 days regardless of the assay used. Conversely, most antigen negative/sgRNA negative results were among samples tested at 16–30 days only when Standard Q or Standard F were used. In conclusion, based on our findings, a negative antigen test, especially with the Lumipulse assay, or a positive antigen test, especially with the Standard F assay, may suggest, respectively, the absence or presence of replication-competent SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Among 11 patients in Thailand infected with severe acute respiratory syndrome coronavirus 2, we detected viral RNA in upper respiratory specimens a median of 14 days after illness onset and 9 days after fever resolution. We identified viral co-infections and an asymptomatic person with detectable virus RNA in serial tests. We describe implications for surveillance.

Mesenchymal stem cells (MSCs) are multipotent adult stem cells with great potential in regenerative medicine. One method for stimulating proliferation and differentiation of MSCs is via electrical stimulation (ES). A valuable approach for evaluating the response of MSCs to ES is to assess changes in gene expression, relative to one or more reference genes. In a survey of 25 publications that used ES on cells, 70% selected GAPDH as the reference gene. We conducted a study to assess the suitability of six potential reference genes on an immortalized human MSC line following direct current ES at seeding densities of 5000 and 10,000 cells/cm2. We employed three methods to validate the most stable reference genes from qRT-PCR data. Our findings show that GAPDH and ACTB exhibit reduced stability when seeded at 5000 cell/cm2. In contrast, we found that the most stable genes across both plating densities and stimulation regimes were PPIA and YWHAZ. Thus, in ES gene expression studies in MSCs, we support the use of PPIA and YWHAZ as an optimal reference gene pair, and discourage the use of ACTB and GAPDH at lower seeding densities. However, it is strongly recommended that similar verification studies are carried out based on cell type and different ES conditions. [ABSTRACT FROM AUTHOR]

A novel strain of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) disease (COVID-19) has been recently identified as an infectious disease affecting the respiratory system of humans. This disease is caused by SARS-CoV-2 that was identified in Chinese patients having severe pneumonia and flu-like symptoms. COVID-19 is a contagious disease that spreads rapidly via droplet particles arising through sneezing and coughing action of an infected person. The reports of asymptomatic carriers changed the scenario of symptom based-diagnosis in COVID-19 and intensified the need for proper diagnosis of the majority of the population to combat the rapid transmission of virus. The diagnosis of positive cases is necessary to ensure prompt care to affected people and also to curb further spread of infection in the population. Collecting samples at the right time and from the exact anatomical site is crucial for proper molecular diagnosis. After the complete genome sequence was available, China formulated RT-PCR as a primary diagnostic procedure for detecting SARS-CoV-2. Many in-house and commercial diagnostic kits have been developed or are under development that have a potential to lower the burden of diagnosis on the primary diagnostic techniques like RT-PCR. Serological based diagnosis is another broad category of testing that can detect different serum antibodies like IgG, IgM, and IgA in an infected patient. PCR-based diagnostic procedures that are commonly used for pathogen detection need sophisticated machines and assistance of a technical expert. Despite their reliable accuracy, they are not cost-effective tests, which a common man can afford, so it becomes imperative to look for other diagnostic approaches, which could be cost effective, rapid, and sensitive with consistent accuracy. To make such diagnostics available to the common man, many techniques can be exploited among, which are Point of Care (POC), also known as bed side testing, which is developing as a portable and promising tool in pathogen diagnosis. Other lateral flow assay (LFA)-based techniques like SHERLOCK, CRISPR-Cas12a (AIOD-CRISPR), and FNCAS9 editor-limited uniform detection assay (FELUDA), etc. have shown promising results in rapid detection of pathogens. Diagnosis holds a critical importance in the pandemic situation when there is no potential drug for the pathogen available in the market. This review sums up the different diagnostic approaches designed or proposed to combat the crisis of widespread diagnosis due to the sudden outbreak of a novel pathogen, SARS-CoV-2 in 2019.

This research utilized the systematic biological and proteomics strategies to explore the regulatory mechanism of Danshen Yin Modified (DSYM) on atherosclerosis (AS) biological network. The traditional Chinese medicine database and HPLC was used to find the active compounds of DSYM, Pharmmapper database was used to predict potential targets, and OMIM database and GeneCards database were used to collect AS targets. String database was utilized to obtain the other protein of proteomics proteins and the protein‐protein interaction (PPI) data of DSYM targets, AS genes, proteomics proteins and other proteins. The Cytoscape 3.7.1 software was utilized to construct and analyse the network. The DAVID database is used to discover the biological processes and signalling pathways that these proteins aggregate. Finally, animal experiments and proteomics analysis were used to further verify the prediction results. The results showed that 140 active compounds, 405 DSYM targets and 590 AS genes were obtained, and 51 differentially expressed proteins were identified in the DSYM‐treated ApoE‐/‐ mouse AS model. A total of 4 major networks and a number of their derivative networks were constructed and analysed. The prediction results showed that DSYM can regulate AS‐related biological processes and signalling pathways. Animal experiments have also shown that DSYM has a therapeutic effect on ApoE‐/‐mouse AS model (P <.05). Therefore, this study proposed a new method based on systems biology, proteomics, and experimental pharmacology, and analysed the pharmacological mechanism of DSYM. DSYM may achieve therapeutic effects by regulating AS‐related signalling pathways and biological processes found in this research. [ABSTRACT FROM AUTHOR]

A novel strain of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) disease (COVID-19) has been recently identified as an infectious disease affecting the respiratory system of humans. This disease is caused by SARS-CoV-2 that was identified in Chinese patients having severe pneumonia and flu-like symptoms. COVID-19 is a contagious disease that spreads rapidly via droplet particles arising through sneezing and coughing action of an infected person. The reports of asymptomatic carriers changed the scenario of symptom based-diagnosis in COVID-19 and intensified the need for proper diagnosis of the majority of the population to combat the rapid transmission of virus. The diagnosis of positive cases is necessary to ensure prompt care to affected people and also to curb further spread of infection in the population. Collecting samples at the right time and from the exact anatomical site is crucial for proper molecular diagnosis. After the complete genome sequence was available, China formulated RT-PCR as a primary diagnostic procedure for detecting SARS-CoV-2. Many in-house and commercial diagnostic kits have been developed or are under development that have a potential to lower the burden of diagnosis on the primary diagnostic techniques like RT-PCR. Serological based diagnosis is another broad category of testing that can detect different serum antibodies like IgG, IgM, and IgA in an infected patient. PCR-based diagnostic procedures that are commonly used for pathogen detection need sophisticated machines and assistance of a technical expert. Despite their reliable accuracy, they are not cost-effective tests, which a common man can afford, so it becomes imperative to look for other diagnostic approaches, which could be cost effective, rapid, and sensitive with consistent accuracy. To make such diagnostics available to the common man, many techniques can be exploited among, which are Point of Care (POC), also known as bed side testing, which is developing as a portable and promising tool in pathogen diagnosis. Other lateral flow assay (LFA)-based techniques like SHERLOCK, CRISPR-Cas12a (AIOD-CRISPR), and FNCAS9 editor-limited uniform detection assay (FELUDA), etc. have shown promising results in rapid detection of pathogens. Diagnosis holds a critical importance in the pandemic situation when there is no potential drug for the pathogen available in the market. This review sums up the different diagnostic approaches designed or proposed to combat the crisis of widespread diagnosis due to the sudden outbreak of a novel pathogen, SARS-CoV-2 in 2019. [ABSTRACT FROM AUTHOR]

Since December 2019, an increasing number of cases associated with the 2019 novel coronavirus (2019-nCoV) have emerged in Wuhan, China, which has resulted in a rapid outbreak in China and worldwide. The present study aimed to describe the clinical, laboratory and radiological characteristics of 2019-nCoV pneumonia (NCP) in Zhejiang province, outside of Wuhan. A total of 74 patients with 2019-nCoV were continuously enrolled between January 22 and March 2, 2020 at Zhejiang Hospital. Diagnosis was confirmed at Zhejiang Hospital by reverse transcription-PCR (RT-PCR), which was approved by the Chinese government. Subsequently, the clinical features between positive- and negative-NCP patients in Zhejiang were compared. Among the 74 hospitalized patients with suspected 2019-NCP, six patients (one male and five female patients) were confirmed to be infected with 2019-nCoV by RT-PCR. The average age of the confirmed patients was 40±13 years. There were three family clusters among the confirmed cases, one patient from each of these families had travel history or contact with patients from Wuhan within 2 weeks. Compared with non-NCP patients, the most common symptoms at onset for patients with NCP were fever (5/6; 83.3%) and cough (5/6; 83.3%), followed by dyspnea/pharyngalgia (2/6; 33.3%), whereas myalgia (1/6; 16.7%) and fatigue (1/6; 16.7%) were less common. All 74 patients with suspected NCP exhibited abnormal computerized tomography (CT) images. In total, 2/6 (33.3%) patients with confirmed NCP presented with bilateral pneumonia, and 21/68 (30.9%) non-NCP patients exhibited bilateral pneumonia, with bilateral distribution of patchy shadows or ground glass opacity. The present study revealed that epidemiological history was critical to the diagnosis of 2019-nCoV in low epidemic regions outside Hubei province. It was also identified that chest CT could not replace nucleic acid testing due to similar radiological manifestations. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) are multipotent adult stem cells with great potential in regenerative medicine. One method for stimulating proliferation and differentiation of MSCs is via electrical stimulation (ES). A valuable approach for evaluating the response of MSCs to ES is to assess changes in gene expression, relative to one or more reference genes. In a survey of 25 publications that used ES on cells, 70% selected GAPDH as the reference gene. We conducted a study to assess the suitability of six potential reference genes on an immortalized human MSC line following direct current ES at seeding densities of 5000 and 10,000 cells/cm2. We employed three methods to validate the most stable reference genes from qRT-PCR data. Our findings show that GAPDH and ACTB exhibit reduced stability when seeded at 5000 cell/cm2. In contrast, we found that the most stable genes across both plating densities and stimulation regimes were PPIA and YWHAZ. Thus, in ES gene expression studies in MSCs, we support the use of PPIA and YWHAZ as an optimal reference gene pair, and discourage the use of ACTB and GAPDH at lower seeding densities. However, it is strongly recommended that similar verification studies are carried out based on cell type and different ES conditions.

Promoter mutations of pleckstrin homology domain-containing S1 (PLEKHS1) are frequent in several cancer types. To evaluate the DNA mutations, the mRNA expression and prognostic value of PLEKHS1 was evaluated in bladder cancer. We investigated DNA mutations and mRNA expression of PLEKHS1 in a first series of 154 bladder tumors [71 non-muscle-invasive bladder cancer (NMIBC) and 83 muscle-invasive bladder cancers (MIBC)] from patients who underwent transurethral bladder resection or radical cystectomy between 2001 and 2006, and 20 normal bladder samples. Results were then validated in a second series of 181 bladder tumors (91 NMIBC and 90 MIBC). All patients have signed an informed consent form. DNA mutations were analysed by high-resolution melt analysis and sanger sequencing. The mRNA expression was measured by real-time reverse-transcriptase quantitative PCR. The results of the molecular analysis were compared with survival data. PLEKHS1 mutations occurred in 25.0 and 32.2% of NMIBC and MIBC, respectively in the first series. These results were confirmed in the second series (33.0 and 37.8% of NMIBC and MIBC, respectively). In MIBC, DNA mutations were significantly more frequent with the basal than non-basal phenotype (61.5 vs. 27.1%; P=0.0025). The PLEKHS1 mRNA level was increased in 22.5 and 27.7% of NMIBC and MIBC tumors but was not associated with DNA mutations. In NMIBC, PLEKHS1 mRNA overexpression was significantly associated with progression to muscle-invasive disease (P=0.0069) and remained an independent prognostic factor on multivariate analysis (P=0.034). DNA mutations of PLEKHS1 occurred in one-third of bladder tumors and was frequent in the basal MIBC phenotype. PLEKHS1 mRNA overexpression may be an independent prognostic factor of progression-free survival in NMIBC. [ABSTRACT FROM AUTHOR]

The potential of foot‐and‐mouth disease (FMD) to spread extensively means that rapid and accurate methods are needed for its diagnosis. Therefore, reverse transcription‐PCR (RT‐PCR) plays an important diagnostic role. Here, we designed the primer set FM8/9 to amplify 644 bases in the conserved 3D region of all seven serotypes of the FMD virus (FMDV). We compared the performance of RT‐PCR assays using FM8/9 with those using the primer set 1F/R, which targets the 5′‐UTR, and real‐time RT‐PCR (rRT‐PCR) assays described in the World Organization for Animal Health manual. Detection limits of the RT‐PCR assays were determined for 24 strains, representing all serotypes. The sensitivities of RT‐PCR assays using FM8/9 were 100.6‐ to 103.8‐fold higher than those of 1F/R assays for 21 strains. To assess the validity of the methods for analysing clinical samples, sera and saliva samples collected daily from pigs and cows infected with FMDV were analysed using the four PCR assays. FM8/9 assays detected FMDV from all infected pigs and cows for longer periods than 1F/R assays, indicating that FM8/9 assays have higher sensitivity for the clinical samples. Our results suggest that the FM8/9 RT‐PCR assay is highly sensitive and is therefore suitable for the diagnosis of FMD. [ABSTRACT FROM AUTHOR]

Objective To explore influence of artesunate on T-bet /GATA-3 equilibrium of peripheral blood in oral lichen planus.Methods 18 OLP patients were selected,and peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation. The cells were cultured and treated with different concentrations (0, 1, 100 μmol/L) of artesunate. Reverse transcription polymerase chain reaction (RT-PCR) and Western blot were applied to detect theT-bet and GATA-3 mRNA and protein expression respectively.Results The T-bet/GATA-3 mRNA and protein expressions in groups treated with artesunate (1 μmol/L & 100 μmol/L) were higher than that of control group (treated without artesunate) (P

Abstract: Aims: A molecular method for a rapid detection of viable Legionella pneumophila of all serogroups in tap water samples was developed as an alternative to the reference method (ISO). Legionellae are responsible for Legionnaires’ disease, a severe pneumonia in humans with high lethality. Methods and Results: The developed method is based on a nutritional stimulation and detection of an increase in precursor 16S rRNA as an indicator for viability. For quantification, DNA was detected by qPCR. This method was compared to the ISO method using water samples obtained from public sports facilities in Switzerland. The sensitivity and specificity were 91 and 97%, respectively, when testing samples for compliance with a microbiological criterion of 1000 cell equivalents per l. Conclusion: The new method is sensitive and specific for Leg. pneumophila and allows results to be obtained within 8 h upon arrival, compared to one week or more by the ISO method. Significance and Impact of the Study: The method represents a useful tool for a rapid detection of viable Leg. pneumophila of all serogroups in water by molecular biology. It can be used as an alternative to the ISO method for official water analysis for legionellae and particularly when a short test time is required. [ABSTRACT FROM AUTHOR]

Papaya sticky disease, caused by Papaya meleira virus (PMeV), results in complete crop loss in papaya orchards, because infected fruits are unacceptable for consumption. Little is known about sticky disease epidemiology, and the best available strategy for disease management is to rogue infected plants. Identification of alternative hosts for this virus is important for developing control strategies. In Mexico, papayas and watermelons are often cultivated in close proximity or overlapping cycles, which may favor virus interspecies transmission by putative insect vectors of PMeV. Watermelon seedlings (Citrullus lanatus Thunb.) were inoculated with latex from papayas infected with a Mexican variant of PMeV (PMeV-Mx), and infection was confirmed by reverse transcription-PCR (RT-PCR) and quantitative RT-PCR (RT-qPCR). Amplicons (491 bp) of the RNA-dependent RNA polymerase (RdRp) gene of PMeV-Mx were detected in axillary leaves of all inoculated seedlings seven days post-inoculation (dpi). Circa 10 to 12 dpi, necrotic lesions were observed on the edges of the leaves. Absolute quantification of PMeV-Mx-RNA by RT-qPCR showed an increase in viral load over time (from 0.51 to 586.15 pg/μL), three to 14 dpi. Molecular and experimental evidence demonstrate is also a host for PMeV-Mx and may be an alternative host. [ABSTRACT FROM AUTHOR]

Circulating tumor cells (CTCs) are established cancer biomarkers for the "liquid biopsy" of tumors. Molecular analysis of single CTCs, which recapitulate primary and metastatic tumor biology, remains challenging because current platforms have limited throughput, are expensive, and are not easily translatable to the clinic. Here, we report a massively parallel, multigene-profiling nanoplatform to compartmentalize and analyze hundreds of single CTCs. After high-efficiency magnetic collection of CTC from blood, a single-cell nanowell array performs CTC mutation profiling using modular gene panels. Using this approach, we demonstrated multigene expression profiling of individual CTCs from non-small-cell lung cancer (NSCLC) patients with remarkable sensitivity. Thus, we report a highthroughput, multiplexed strategy for single-cell mutation profiling of individual lung cancer CTCs toward minimally invasive cancer therapy prediction and disease monitoring. [ABSTRACT FROM AUTHOR]

This study examined the effects of Maillard reaction products reacted by casein and lactose (cMRP) and of cMRP fermented by Lactobacillus fermentum H9 (FcMRP) on hypolipidemic and antiinflammatory effects in rats fed a high-fat and high-cholesterol diet (HD). The HD-fed rats had significantly increased hepatic triglyceride concentrations compared with the rats fed a normal diet. It was shown that treatment with simvastatin, L. fermentum H9 (H9), cMRP, and F-cMRP decreased total triglycerides in the liver compared with the HD group. On histological analysis, a reduction of lipid accumulation in the liver and aortic tissues was observed in the cMRP, F-cMRP, and H9-fed rats. Also, F-cMRP and cMRP reduced intima-media thickness in the HD group. In addition, the H9, cMRP, and F-cMRP treatments significantly reduced the expression levels of ICAM-1 and VCAM-1, but not of MCP-1. In particular, the expressions of ICAM-1 and VCAM-1 were significantly decreased in the F-cMRP group compared with the HD group. These results of the present study suggest that cMRP and F-cMRP in dairy foods could potentially be used to prevent or treat cardiovascular diseases, especially atherosclerosis. [ABSTRACT FROM AUTHOR]

Concurrently, several porcine epidemic diarrhea virus (PEDV) variants are circulating in US swine farms, including the original US and the spike insertion-deletion (S-INDEL) strains. In this study, reverse transcription (RT)-PCR assays for the detection and differentiation of different US PEDV variants were developed based on the differences in the S1 domain of the spike (S) gene. This assay successfully differentiated three PEDV strains: PC22A (the original US virulent), Iowa106 (S-INDEL), and PC177 (S-197DEL) that was derived from cell culture adaptation and has a 197 amino acid-deletion in the S1 domain. The assays did not amplify the porcine deltacoronavirus OH-FD22 strain or transmissible gastroenteritis virus Miller strain. It is the first report on the development of RT-PCR assays allowing the detection and differentiation of all major types of US PEDV variants. [ABSTRACT FROM AUTHOR]

Twelve methylenedioxy-containing compounds including piperine and 10 piperine-like synthetic compounds were assessed to determine their antifungal and antiaflatoxigenic activities against Aspergillus flavus ATCC 22546 in terms of their structure-activity relationships. Piperonal and 1,3-benzodioxole had inhibitory effects against A. flavus mycelial growth and aflatoxin B1 production up to a concentration of 1000 μg/mL. Ten piperine-like synthetic compounds were synthesized that differed in terms of the carbon length in the hydrocarbon backbone and the presence of the methylenedioxy moiety. In particular, 1-(2-methylpiperidin-1-yl)-3-phenylprop-2-en-1-one had potent antifungal and antiaflatoxigenic effects against A. flavus up to a concentration of 1 μg/mL. This synthetic compound was remarkable because the positive control thiabendazole had no inhibitory effect at this concentration. Reverse transcription-PCR analysis showed that five genes involved in aflatoxin biosynthesis pathways were down-regulated in A. flavus, i.e., aflD, aflK, aflQ, aflR, and aflS; therefore, the synthetic compound inhibited aflatoxin production by down-regulating these genes. [ABSTRACT FROM AUTHOR]

Fusion genes are products of chromosomal translocations that generate either a dysregulated partner gene or a chimeric fusion protein with new properties, and contribute significantly to leukemia development and clinical risk stratification. However, simultaneous detection of several hundreds of fusion genes has always been a challenge in a clinical laboratory setting. In the present study, a total of 182 pediatric patients with leukemia were screened for fusion genes by employing a novel genomic DNA-, instead of RNA-, based next-generation sequencing (NGS) method. This involved the comparison of the multiply targeted capture sequencing method with a detection panel of 270 fusion genes (MTCS-270) with an RNA-based multiplex reverse transcription-PCR technique with a detection panel of 57 fusion genes (MRTP-57). MRTP-57 has been well established in the clinical lab at Beijing Hightrust Diagnostics, Co. (Beijing, China) for an up-front leukemia diagnosis and served as the control technique in the present study. In the series, MTCS-270 and MRTP-57 yielded a positive fusion gene detection rate of 50.0% (91/182) and 41.8% (76/182), respectively, indicating an advantage of MTCS-270 over MRTP-57 in overall detection sensitivity. Specifically, all the fusion genes detected by MRTP-57 were also identified by MTCS-270, clearly signifying the respectable detection accuracy of MTCS-270. Notably, across the patients screened, MTCS-270 identified more samples with fusion genes than MRTP-57, illustrating a broader fusion gene detection coverage by MTCS-270. The present study provides solid evidence that this DNA-based NGS approach can be used as a potential detection tool together with other well-established molecular cytogenetic methods for leukemia management, and to the best of our knowledge, represents the largest leukemia fusion gene identification analysis by genomic NGS. [ABSTRACT FROM AUTHOR]

OBJECTIVE: H. pylori is the most important risk factor for gastric carcinoma. Ca-gA-positive H. pylori is associated with an increased risk for gastric cancer compared with negative strains. RUNX3 is a tumor suppressor gene, which is related to the genesis of gastric cancer. β-catenin is integrated with E-cadherin in the cell membrane, and aberrant expression of the complex was reported in gastric carcinoma. Aim of this paper is to determine of the relation between RUNX3, E-cadherin and β-catenin in chronic gastritis associated with cagA-positive H. pylori infection. PATIENTS AND METHODS: Retrospective study was done on formalin fixed paraffin embedded gastric biopsies blocks of 90 patients diagnosed as H. pylori associated chronic gastritis. H. pylori was detected using modified Giemsa stain. Nested PCR was used for detection of cagA, reverse transcription-PCR for detection of RUNX3 and immunohistochemistry for detection of E-cadherin and β-catenin. RESULTS: Fifty percent of cases were found to be cagA positive. CagA was significantly associated with the intensity of mononuclear inflammation, the intensity of neutrophilic inflammation, the degree of mucosal atrophy and loss of RUNX3 but not with the density of H. pylori, intestinal metaplasia, E-cadherin or β-catenin. There was significant relation between loss of RUNX3 and increasing density of H. pylori, intensity of neutrophilic inflammation, mucosal atrophy and intestinal metaplasia. RUNX3 was found to be significantly correlated with E-cadherin but not with β-catenin. E-cadherin showed decreased expression in 36.7% of biopsies while, β-catenin was decreased in 33% of biopsies. CONCLUSIONS: Loss of RUNX3, E-cadherin and β-catenin was considered early events in the cascade of gastric carcinoma development. Loss of RUNX3 but neither E-cadherin nor β-catenin was related to cagA positive H. pylori strains. [ABSTRACT FROM AUTHOR]

Of 322 stool specimens collected from children with diarrhea from October 2005 through September 2006 in Wuhan, China, group A rotavirus was identified in 101 (31.4%). The most prevalent group A rotavirus genotype was G3P[8] (62.6%), followed by G1P[8](17.6%), G1+G3P[8](8.8%), G3P[4](6.6%), G1P[4](2.2%), and G9P[8](2.2%). The G9 strains were first detected in Wuhan.

Normalization to a reference gene is the method of choice for quantitative reverse transcription-PCR (RT-qPCR) analysis. The stability of reference genes is critical for accurate experimental results and conclusions. We have evaluated the expression stability of eight commonly used reference genes found in four different human mesenchymal stem cells (MSC). Using geNorm, NormFinder and BestKeeper algorithms, we show that beta-2-microglobulin and peptidyl-prolylisomerase A were the optimal reference genes for normalizing RT-qPCR data obtained from MSC, whereas the TATA box binding protein was not suitable due to its extensive variability in expression. Our findings emphasize the significance of validating reference genes for qPCR analyses. We offer a short list of reference genes to use for normalization and recommend some commercially-available software programs as a rapid approach to validate reference genes. We also demonstrate that the two reference genes, β-actin and glyceraldehyde-3-phosphate dehydrogenase, are frequently used are not always successful in many cases. [ABSTRACT FROM AUTHOR]

Noroviruses are the most common viral agents of acute gastroenteritis in humans. Because noroviruses are very resistant to inactivation, they can lead to public health consequences via the contamination of foods (e.g. molluscs). The aim of this study was to evaluate the prevalence of noroviruses in marine bivalve molluscs sold as food. Standard and real-time reverse transcription-PCR (RT-PCR) procedures were used to monitor bivalve molluscs from the Granada (southern Spain) fish markets for this human enteric virus. Between February 2009 and October 2010 we collected a total of 329 samples of different types of bivalve molluscs (mussels and three types of clam). A total of 329 samples of four species of bivalves were analysed and 23% were positive for noroviruses. Given these results, we conclude that the current processing techniques for bivalve molluscs are ineffective at eliminating their viral load. [ABSTRACT FROM AUTHOR]

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to rapidly detect foot-and-mouth disease virus serotype C (FMDV C). By testing 10-fold serial dilutions of FMDV C samples, sensitivity of the FMDV C RT-LAMP was found to be 10 times higher than that of conventional reverse transcription-PCR (RT-PCR). No cross-reactivity with A, Asia 1, or O FMDV or swine vesicular disease virus (SVDV) indicated that FMDV C RT-LAMP may be an exciting novel method for detecting FMDV C. [ABSTRACT FROM AUTHOR]

Acute lymphoblastic leukemia represents a heterogeneous group of hematological malignancies, defined by clonal proliferation of lymphoid cells. Immunophenotyping by flow cytometry and molecular analysis for the detection of genetic anomalies are clinical standard procedures for diagnosis, sub-classification and post-therapeutic evaluation. Samples from 105 patients diagnosed with acute lymphoblastic leukemia were immunophenotyped at diagnosis and were investigated by molecular analysis in order to identify the occurrence of four fusion genes: MLL-AF4, TEL-AML-1, BCR-ABL-p190, E2A-PBX-1. There were no associations found between the immunophenotype and the presence of any fusion genes evaluated. Both methods in combination remain a prerequisite for an improved subclassification of hematological malignancies, therapeutic decision, and evaluation of treatment response. [ABSTRACT FROM AUTHOR]

The article presents a study which investigated the effects of sericin to hippocampal growth hormone/insulin-like growth factor 1 axis in diabetic rats. It says that sericin relieves hippocampal damage in diabetic rats by improving disorders of the growth hormone/insulin-like growth factor 1 axis. Other topics include streptozotocin, western blot assay, and reverse transcription polymerase chain reaction (PCR).

The article presents a study which examines the involvement of JNK3 pathway in nerve cell apoptosis and neurofunctional recovery of patients after traumatic brain injury. The study is investigated by reverse transcription-polymerase chain reaction (PCR), neurological function assessment, and Neurological Severity Scores. Results show that the downregulation of JNK3 expression can help in the recovery of neurological function.

The article offers information on a study that was designed to verify the properties of the stem cell of sheep amniotic epithelial cells and their capacity for neural differentiation. At 28 days after induction with serum-free neurobasal-A medium containing B-27, amniotic epithelial cells expressed β-III-tubulin, global fibrillary acidic protein, nestin and microtubule-associated protein-2.

MSCs (mesenchymal stem cells) are a stem cell source that can be easily obtained from bone marrow. Despite the increasing importance of the pig as a large animal model, little is known about foetal pMSCs (porcine MSCs). In this study, we observed the gene expression of pluripotent markers in foetal pMSCs and the capacity of pMSCs to differentiate into adipocytes, osteocytes and neural-like cells using quantitative RT-PCR (reverse transcription-PCR), normal histological staining and immunohistochemistry. Foetal pMSCs have either a spindle or a flattened shape, and flow cytometry revealed the expression of the MSC-related proteins CD44 and CD105 (endoglin) but not CD34 and CD45. pMSCs express pluripotent markers such as Oct4 (octamer-binding transcription factor 4) and Nanog at the protein and mRNA levels. qRT-PCR (quantitative real-time PCR) analyses revealed that pMSCs expressed nestin [for NSCs (neural stem cells)]. Immunocytochemical and RT-PCR data showed that 29% and 23% of pMSCs expressed MAP2 (microtubule-associated protein 2) for neurons and β-tubulin III (Tuj1) for immature neurons, respectively, after induction of neural differentiation. These findings demonstrate the plasticity of pMSCs and their potential for use in cellular replacement therapy for neural diseases. [ABSTRACT FROM AUTHOR]

The article presents a study which investigates the effect of electroacupuncture-regulated neurotrophic factor mRNA expression in Parkinson's disease rats' substantia nigra. The study employs RNA extraction, polymerase chain reaction (PCR) amplification, and reverse transcription. Results indicate that electroacupuncture treatment upregulated glial cell-line derived neurotrophic factor mRNA expression and brain-derived neurotrophic factor in the substantia nigra of rat models.

Abstract: Three cysteine protease genes, cathepsin B-like enzyme gene 1, 2 (AC-cathB-1, AC-cathB-2) and hemoglobin-type cysteine protease gene (AC-hem) were isolated and described from Angiostrongylus cantonensis adult. The deduced amino acid sequence of Ac-cathB-1 and AC-cathB-2 contain all of the conserved regions of cathepsin B. AC-cathB-2 is similar to a host intrusion-related cysteine protease B from Parelaphostrongylus tenuis, and the AC-hem shares high similarity to legumain from Haemonchus contortus. AC-cathB-1 was expressed significantly higher in L1 as compared with AC-hem, the AC-cathB-2 followed; AC-cathB-2 transcripts in L3 were found increased rapidly and obviously abundant, suggesting that AC-cathB-1 and AC-cathB-2 may play an important role in intermediate and final host invasion, separately. The cysteine protease genes were more or less expressed in adult stage excepted for AC-cathB-2. As the AC-cathB-1 and AC-hem highly expressed in adult worms, suggesting AC-hem may activate AC-cathB-1 which involved in the host invasion and feeding process. [Copyright &y& Elsevier]

The article discusses a study which investigated CD133+ cell infiltration in the brain of a rat following fluid percussion injury. The study used a rat model with traumatic brain injury and performed immunohistochemical staining and reverse transcription polymerase chain reaction (PCR). Results showed an increase in CD133 antigen expression in the rat brain and no change in CD133 mRNA expression in the injured brain tissue.

Abstract: Bcl-xL is a pro-survival member of the Bcl-2 family that plays indispensable roles in regulating cell survival and apoptosis. It is overexpressed in many malignant tumors including colorectal cancer (CRC). However, it is still unclear if Bcl-xL can be used as an independent molecular marker for predicting the prognosis of CRC patients. In this study, reverse transcription-PCR assay was performed to detect the expression of Bcl-xL mRNA in CRC and corresponding non-tumor colon tissues. Immunohistochemistry was performed to detect the immunolocalization of Bcl-xL protein in sixty-eight primary CRC tissue samples. The association between Bcl-xL protein expression and clinicopathological factors of CRC patients was analyzed and the survival was assessed by the Kaplan–Meier method and proportional hazards model. The averaged level of Bcl-xL mRNA expression in CRC tissues (0.85±0.13) was significantly higher than that in non-tumor colon tissues (0.08±0.02). Immunohistochemical staining showed that the Bcl-xL protein was mainly located in the cytoplasm of tumor cells. The level of Bcl-xL protein expression was closely correlated with tumor differentiation (P =0.002), lymph node metastasis (P =0.010), venous permeation (P =0.004), and Duke''s classification (P =0.021). Furthermore, patients with high Bcl-xL expression showed poorer overall survival than those with low Bcl-xL expression (P =0.016). Univariate and multivariate analysis indicated that the status of Bcl-xL protein expression might be an independent prognostic marker for CRC patients (P =0.032). Taken together, immunohistochemical assessment of status of Bcl-xL protein may offer a valuable approach for predicting survival after curative surgery for colorectal cancer. [Copyright &y& Elsevier]

Sugarcane yellow leaf virus (SCYLV) that causes yellow leaf disease (YLD) in sugarcane belongs to Polerovirus. The disease expression is commonly observed during 6 to 8 months stages of the crop and there is a need to diagnose the disease before the symptom appearance. We have standardized RT-PCR technique with a new set of primers to detect SCYLV in sugarcane in pre-symptomatic stages of YLD. During presymptomatic stage 33 of the 44 varieties studied tested positive to SCYLV in RT-PCR. During eighth month stage all the 44 varieties have shown characteristic disease symptoms and except one all of them tested positive to the virus in RT-PCR. The two RT-PCR assays performed separately during pre- and post-symptom expression stages in 44 varieties clearly revealed that most of the varieties have detectable level of the virus in asymptomatic stage. Expression of disease symptoms and presence of the virus in all the varieties except one very clearly indicated the severe virus infection in the tested varieties. The studies also proved the diagnostic efficiency of the new set of primers to detect SCYLV in asymptomatic plants. [ABSTRACT FROM AUTHOR]

A large set of nucleic acid detection methods with good sensitivity and specificity are now available for the detection of pathogens in clinical, food and environmental samples. Given increasing demand, many efforts have been made to combine these methods to assess viability. Genomic DNA PCR amplification has been shown to be inappropriate for distinguishing viable from dead bacteria owing to DNA stability. Many authors have tried to bypass this difficulty by switching to RNA amplification methods such as reverse transcription-PCR and nucleic acid sequence-based amplification. More recently, researchers have developed methods combining specific sample pretreatment with nucleic acid detection methods, notably ethidium or propidium monoazide pretreatment coupled with PCR DNA detection or direct viable count methods and subsequent fluorescent in situ hybridization of 16S rRNA. This review evaluates the performance of these different methods for viability assessment. [ABSTRACT FROM AUTHOR]

The genome of Natronomonas pharaonis encodes genes annotated as alcohol dehydrogenase (ADH; EC 1.1.1.1) and aldehyde dehydrogenase (ALDH; EC 1.2.1.3), enzymes involved in alcohol metabolism. These genes ( adh and aldH2) occur in a single copy on the chromosome. We have studied the role of these genes in ethanol metabolism in N. pharaonis. Reverse transcription-PCR analysis showed that the aldH2 gene was inducible by ethanol, but the adh gene was transcribed both in the presence and absence of ethanol. The gene encoding for ALDH of N. pharaonis ( NpALDH) was cloned into a pET41a vector containing a glutathione S-transferase tag, expressed in Escherichia coli and purified by glutathione sepharose affinity chromatography. The GST- NpALDH fusion protein was cleaved by bovine enterokinase and the target enzyme showed a molecular mass of approximately 60 kDa by SDS-PAGE. The enzyme was thermophilic and alkaliphilic, the optimal temperature and pH being 60°C and 8.0, respectively. NpALDH was salt independent, being most active at 0.25 M NaCl or KCl. [ABSTRACT FROM AUTHOR]

Abstract: Enteric viruses are important agents of foodborne disease. Unfortunately, robust, quantitative methods for sampling and analysis of enteric and other viruses in processed or complex foods are not well-established. As a result, epidemiologically determined etiologies or pathogen sources in foodborne outbreaks are rarely confirmed by virological analysis. In this study, an acid-adsorption elution concentration (AEC) method previously used to monitor virus occurrence and investigate enteric virus outbreaks in shellfish was adapted for examination of processed food items, namely tomato sauce and blended strawberries. Hepatitis A virus (HAV), poliovirus, and coliphage MS2 (MS2) were seeded in 10 or 30 g samples of tomato sauce or blended strawberries, recovered by AEC, and quantified by cell culture infectivity assay. In addition, nested reverse transcription-polymerase chain reaction (RT-PCR) and TaqMan RT-PCR assays were used to detect HAV RNA. Viruses were efficiently adsorbed to foods as an initial concentration step, with infectious HAV and MS2 adsorption of 67% and 93%, respectively, to tomato sauce, and 89% and 99%, respectively, to blended strawberries. Forty-three to 65% of HAV and poliovirus were subsequently eluted and recovered from tomato sauce using 0.5 M threonine, pH 7.2. The lower limits of HAV detection were at initial seeding levels of 14 PFU/g of tomato sauce and 33 PFU/g of blended strawberries. Unlike TaqMan RT-PCR, nested RT-PCR was not inhibited by undiluted final RNA extracts of tomato sauce or blended strawberries. The successful adaptation of the AEC method for enteric and other virus recovery, quantitation and detection in processed foods demonstrates its potential for use in the investigation of foodborne outbreaks of viral etiology and for validation of virus disinfection and sanitary processing procedures used by the food industry. [Copyright &y& Elsevier]

In this report we describe the proof of principle of a reverse transcription polymerase chain reaction (RT-PCR) but on-chip, with immobilized specific primers using a transcriptome of mouse-muscle fibroblasts for detection of muscle-specific expression products of these cells. The isolated total mRNA was directly incubated on an array of immobilized and solubilized specific primers, which allow the amplification of certain muscle-specific RNAs via its immobilized cDNAs. In contrast to others, the immobilized cDNA-products were directly synthesized on the chip by applying covalently bound specific primers. The products were detected by the incorporated and fluorophore-modified specific primers of the subsequently synthezised second strand. In addition, this second-strand served as a further template (like the basically used mRNA) in the subsequent solid-phase-PCR to amplify first-strand cDNA copies at the remaining immobilized specific primer-probes. This is the intrinsic factor of the amplification of certain signals of this application. The specific cDNA templates of genes coding for subunits of the mouse muscle acetylcholine receptor (Chrna1, Chrnb1, Chrnd) and the genes coding for myogenin (Myog), muscle creatine kinase (Ckmm), and ATPase (Atp2a2) were amplified on a biochip by RT-PCR directly from freshly isolated mRNA. The resulting procedure allows the detection of mRNA sequences from less than 5 pg of total RNA preparations. [ABSTRACT FROM AUTHOR]

A novel cinnamate 4-hydroxylase (C4H) gene (designated as SmC4H) involved in the rosmarinic acid biosynthesis pathway is cloned from Salvia miltiorrhiza. The full-length cDNA of SmC4H is 1800 bp long with an open reading frame of 1512 bp encoding a polypeptide of 504 amino acid residues. Like other C4Hs, the predicted SmC4H polypeptide includes three domains: a heme-binding domain, a proline-rich region, and a P450 (E)EFRPER-motif region, which represents a typical structure of plant C4Hs. Analysis of SmC4H genomic DNA reveals that it contains 3 exons, 2 introns, the length of exons being highly conserved. Semiquantitative RT-PCR analysis revealed that the constitutive expression of SmC4H in the root or stem was much higher than in the leaf. Further expression analysis revealed that the signaling components of defense/stress pathways, such as methyl jasmonate, abscisic acid, and ultraviolet-B radiation, up-regulated the SmC4H transcript levels over the control. Calcium chloride and hydrogen peroxide, nevertheless, had no significant effect on SmC4H expression. [ABSTRACT FROM AUTHOR]

Abstract: The basic helix-loop-helix/Per-Arnt-Sim (bHLH-PAS) homology protein family is an important class of transcriptional regulators that are involved in a wide variety of biological processes. In a previous study, we characterized a novel bHLH-PAS factor ‘NXF’ as a new member of the mammalian bHLH-PAS family. If model animals have ortholog genes for NXF, they might have advantages for characterization of its function and regulation in vivo. Here we document cDNA cloning and characterization of putative NXF homolog genes (for NXF-like-factors) in Drosophila and C. elegans. Some conservation of the exon–intron gene architecture was noted in the bHLH-PAS domains among NXF-like-factors and human NXF. The NXF-like-factors selectively interacted with an Arnt ortholog in vitro assay. Moreover, they selectively associated with the mammalian NXF:Arnt2 binding element in the presence of the Arnt ortholog, and synergistically activated a mammalian NXF:Arnt-responsive promoter. These data strongly suggest that the Drosophila and C. elegans NXF-like-factors reported here are homologs of mammalian NXF. Further elucidation of whether they are true orthologs with the same function in vivo is now necessary. [Copyright &y& Elsevier]

Abstract: In our present study we used preparations from Xenopus laevis lungs to perform electrophysiological Ussing chamber measurements, unidirectional flux measurements, and employed molecular approaches to elucidate the presence and function of a cystic fibrosis transmembrane conductance regulator (CFTR) homolog in this tissue. Application of different CFTR blockers (NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid), niflumic acid (NFA), glibenclamide, lonidamine, CFTRinh-172) to the apical side of the tissues was able to significantly decrease the measured short circuit current (I SC) indicating a Cl− secretion due to luminal located CFTR channels. This was further supported by a net 36Cl− secretion determined by radioactive tracer flux experiments. Further, Xenopus pulmonary epithelia responded to apical chlorzoxazone exposure – a CFTR activator – and this activated current was inhibited by CFTRinh-172. We performed reverse transcription-PCR (RT-PCR) and Western blot analysis and with both approaches we found characteristic signals indicating the presence of a CFTR homolog in Xenopus lung. In addition, we were able to detect CFTR in apical membranes of Xenopus lung slices with immunohistological techniques. We conclude that Xenopus lung epithelium exhibits functional CFTR channels and that this tissue represents a valuable model for the investigation of ion transport properties in pulmonary epithelia. [Copyright &y& Elsevier]

Abstract: Background: Human respiratory coronavirus (hCoV) HKU1 infections were reported for the first time in 2005 in Hong Kong. Objective: To investigate epidemiological, clinical, and diagnostic features of HKU1 infections. Study design: Longitudinal, prospective study from November 2005 through May 2006 in a hospitalised patient population. Results: Overall, 48/426 (11.3%) patients were found to be infected by hCoV acute respiratory tract infections (ARTI). Of these, 10 (19.2%) were caused by HKU1 (6 single infections and 4 coinfections) during the period January–May 2006. Diagnosis was made by using RT-PCR for all four hCoVs, and in parallel, in-house developed group-specific monoclonal antibodies (MAbs) for HKU1 and 229E. HKU1-specific MAb was able to retrospectively identify 8 of 10 HKU1 strains detected by RT-PCR. Phylogenetic analysis showed that four HKU1 strains were genotype A and six genotype B. In HKU1-infected patients, the predominant clinical symptom was rhinorrhea (nine patients). Within group II hCoV, HKU1-infected patients had a significantly lower rate of lower ARTI compared to OC43-infected patients. Conclusion: HKU1 hCoV strains circulated in northern Italy during the winter–spring season 2005–2006. Both HKU1 genotypes were detected. HKU1-specific MAb may contribute to the rapid diagnosis of HKU1 infections currently performed by RT-PCR. [Copyright &y& Elsevier]

Aims: The aim of this study was to detect the main pathogenic human RNA enteric viruses able to persist in the environment such as astrovirus, enterovirus, norovirus and hepatitis A virus (HAV) in shellfish collected from two locations in northern Tunisia. Methods and Results: Viruses were eluted from digestive tissues and concentrated by polyethylene glycol precipitation before nucleic acid extraction and purification. After checking for inhibitors, all viruses were detected by reverse transcription-polymerase chain reaction (RT-PCR) and confirmed by hybridization. Overall, 83% of the samples were found positive for at least one virus. Astrovirus was detected in 61% of the samples, norovirus in 35% and HAV in 26%. Surprisingly, only one sample was found positive for enterovirus. Conclusions: The mean number of positive samples found in this study is in accordance with the data found in the literature, indicating that no real difference exists in this respect among countries studied. A notable exception is HAV, which reflects the epidemiological status of the population. Significance and Impact of the Study: This study highlights the interest to analyse shellfish samples from different production areas. These data will be helpful to understand virus circulation and to improve shellfish safety. The results, which confirm contamination, necessitate the development of appropriate studies and monitoring in all shellfish-producing countries. [ABSTRACT FROM AUTHOR]

Abstract: Recombinant antigens that have been cloned from Taenia solium and Taenia ovis have been shown to be highly effective when used as vaccines against cysticercosis in the intermediate hosts. This study investigated the presence of mRNA encoding the TSOL18 and TSOL16 antigens in different life-cycle stages of T. solium, and their related homologues in T. ovis. Reverse transcription-PCR and Southern blotting demonstrated that the antigens are stage-specifically expressed in the oncosphere. The apparent absence of expression of TSOL18 in the metacestode life-cycle stage suggests that the vaccine based on this antigen targets exclusively the early stages in the development of the parasite. [Copyright &y& Elsevier]